Professor Dr. Hildebert Wagner, Dr. Sabine Bladt, Eva Maria Zgainski (auth.) Plant Drug Analysis A Thin Layer Chromatography Atlas Springer Berlin Heidelberg (1984)

Prévia do material em texto

H. Wagner S. Bladt E.M. Zgainski 
Plant Drug 
Analysis 
A Thin Layer Chromatography Atlas 
Translated by Th.A. Scott 
With 170 Colored Photographs 
Springer-Verlag Berlin Heidelberg GmbH 1984 
Professor Dr. Hildebert Wagner 
Dr. Sabine Bladt 
Eva Maria Zgainski (Fachphotographin) 
Institut für Pharmazeutische Biologie 
der Universität München, 
Karlstraße 29, 
D-8000 München 2 
Translator: 
Dr. Thomas A. Scott 
Department of Biochemistry 
University of Leeds 
GB-Leeds LS2 9JT 
Translation of the German edition' Drogenanalyse ' 
© Springer-Verlag Berlin Heide1berg 1983 
ISBN 978-3-662-02400-3 ISBN 978-3-662-02398-3 (eBook) 
DOI 10.1007/978-3-662-02398-3 
Library of Congress Cataloging in Publication Data 
Wagner, H. (Hildebert), 1929-
Plant drug analysis. 
Includes bibliographical references and index. 
1. Materia medica, Vegetable-Analysis. 2. Drugs-Analysis. 3. Thin layer chromatography. I. 
Bladt, S. (Sabine), 1945- . H. Zgainski, E.M. (Eva Maria), 1928- . III. Title. 
RS190.P55W3313 1984 615'.32 84-5348 
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Under § 54 of the German Copyright Law where copies are made for other than private 
use, a fee is payable to "Verwertungsgesellschaft Wort", Munich. 
© by Springer-Verlag Berlin Heidelberg 1984 
Originally published by Springer-Verlag Berlin Heidelberg New York in 1984. 
Softcover reprint of the hardcover 1 st edition 1984 
The use of registered names, trademarks, etc. in this publication does not imply, even in 
the absence of a specific statement, that such names are exempt from the relevant protective 
laws and regulations and therefore for general use. 
Product Liability: The publisher can give no guarantee for information ab out drug dosage 
and application thereof contained in this book. In every individual case the respective user 
must check its accuracy by consulting other pharmaceuticalliterature. 
Reproduction of the figures : Gebrüder Czech, D-8000 München 
2131/3130-543210 
Preface 
Thin layer chromatography is the most widely used of all the simple chromatographie 
methods for the analysis of mixtures. On account of its extreme rapidity and ease 
of visual evaluation, thin layer chromatography has become the most ideal analytical 
method for plant drugs and for preparations that contain drug extracts or pure 
drug constituents. 
One disadvantage of the technique was the lack of a satisfactory method for 
the permanent recording of results. Analyses were recorded simply by verbal descrip-
tion of the chromatogram (e.g., in the pharmacopoeias), by schematic drawings, 
or by idealized colored diagrams. All these methods are c1early makeshift solutions 
to the problem of documentation. In order to make the documentation as realistic 
as possible, we have therefore attempted to make faithful photographic reproduc-
tions of thin layer chromatographie separations of drugs in visible and UV-light. 
The chief difficulty that we encountered in the preparation of this TLC atlas was 
knowing how to take into account the natural qualitative and quantitative variations 
that occur in the composition of individual drugs. We believe we have solved this 
problem by selecting, from more than 10 years of photo graphie recording, those 
pictures that are the most representative and are photographically the best reproduc-
tions of chromatograms of standard drugs. In choosing solvent systems, we have 
relied primarilyon the pharmacopoeias, but we have also made proposals for im-
provements and for future standardization. In view of the extent of the present-day 
world drug market, analyses of many commercial, non-official drugs have also been 
documented. Emphasis was placed primarily on commercial European drugs. 
We hope that this chromatographie drug atlas will help those who are learning 
the technique, and serve as an aid for the evaluation of thin layer chromatograms. 
Furthermore, we believe that this thin layer chromatographie drug atlas represents 
an aid to the standardization of industrial plant drug preparations. It will enable 
the systematization of the identification and purity control of plant drugs. 
The authors are especially grateful to Mrs. J. DUFOSSE of Agfa photographie 
services, Munieh, for the painstaking and faithful reproduction of the color pictures. 
We thank Miss MAHN and Miss SCHUCKER for technical assistance, and Mrs. 
ANDRAE for drawing the formulae. Thanks are due to Springer-Verlag, and especially 
to Mrs. DEIGMÖLLER, for their helpful co operation to our wishes with regard to 
the reproduction of the chromatograms and the overall layout of the book. 
HILDEBER T WAGNER 
SABINE BLADT 
EVA MARIA ZGAINSKI 
Contents 
Introduction 
Essential Oil Drugs (Aetherolea) 
Extraction and TLC . . . . . . . . 
List of Essential Oil Drugs . . . . . 
Formulae of Constituents of Essential Oils 
Reference Compounds 
Cinnamoni cortex 
Calami rhizoma 
Anisi fructus 
F oeniculi fructus 
Sassafras lignum 
· Figs. 1, 2 
· Figs. 3, 4 
Basilici herba ............. Figs. 5, 6 
Petroselini fructus 
Myristicae semen 
Macis 
Caryophylli flos ............. Figs. 7, 8 
Carvi fructus 
Coriandri fructus 
Juniperi fructus 
Rosmarini folium 
Matricariae flos 
Anthemidis flos 
Lavandulae flos 
Cinae flos ... 
Menthae piperitae folium 
Menthae crispae folium 
Salviae officinalis folium 
Salviae trilobae folium 
· Figs. 9,10 
. Figs. 11, 12 
· Figs. 13, 14 
. Figs. 15, 16 
Eucalypti folium . . . . . . . . . . Figs. 17, 18 
Thymi herba 
Serpylli herba 
Ajowani fructus 
Melissae folium . Figs. 19,20 
Curcumae rhizoma · Figs. 21, 22 
5 
5 
9 
19 
22 
24 
26 
28 
30 
32 
34 
36 
38 
40 
42 
VII 
VIII 
Ci tri pericarpium 
Aurantii pericarpium . . . . . . . . . . . Figs. 23, 24 
Terebinthinae aeth. 
Pini aetherolea 
Myrrha . . . .. ........... Figs. 25, 26 
Benzoin 
Balsamum tolutanum 
Balsamum peruvianum .......... Figs. 27, 28 
Alkaloid Drugs 
Extraction and TLC 
List of Alkaloid Drugs 
Formulae of Constituents of Alkaloid Drugs 
Reference Compounds 
Y ohimbe cortex 
Quebracho cortex 
Rauwolfiae radix 
Strychni semen 
Ignatii semen 
Secale cornutum 
Chinae cortex 
Ipecacuanhae radix 
Opium 
Berberidis radix 
Colombo radix 
Hydrastis rhizoma 
Chelidonii herba 
Colchici ·semen 
Aconiti tuber (herba) 
Sabadillae semen 
Lobeliae herba 
Jaborandi folium 
Boldo folium 
Cacao semen 
Coffeae semen 
Nicotianae folium 
Ephedrae herba 
· Figs 1, 2 
· Figs. 3, 4 
· Figs. 5, 6 
. Fig. 7 
. Fig. 8 
· Figs. 9, 10 
.Figs. 11, 12 
· Figs. 13, 14 
. Figs. 15, 16 
· Fig. 17 
· Fig. 18 
· Figs. 19, 20 
· Fig. 21 
44 
46 
48 
51 
51 
55 
61 
66 
68 
70 
72 
72 
74 
76 
78 
80 
82 
82 
84 
86 
Spartii herba .. ............ Fig. 22 . . . . . . . . . . 86 
Belladonnae folium 
Hyoscyami folium 
Stramonii folium . 
Belladonnae folium/semen/radix 
Hyoscyami mutici/nigri folium 
Stramonii folium/semen 
. . . . . . Figs. 23, 24 
Scopoliae radix . . . . . . . . . . . . . . Figs. 25, 26 
88 
90 
Drugs Containing Anthracene Derivatives 
Extraction and TLC . . . . . . . . . . . . . 
List of Drugs Containing Anthracene Glycosides 
Formulae of Anthracene Derivatives Identified as Drug Constituents 
Aloe resina 
Rhamni purshiani cortex 
TLC-Synopsis 
Frangulae cortex 
Oreoherzogiae cortex 
Frangulae fructus 
Rhamni cathartici fructus 
Rhei radix .. 
Sennae folium 
Sennae fructus 
Hyperici herba 
Circular TLC 
Sennae foliumjfructus 
Rhei radix ..... 
Arbutin Drugs 
Extractionand TLC 
List of Drugs Containing Arbutin 
Formulae ...... . 
Uvae ursi folium 
Vitis ideae folium 
Myrtilli folium . 
Viburni prunifolii cortex 
Viburni opuli cortex 
Bitter Principle Drugs 
Extraction and TLC . . . 
List of Bitter Principle Drugs 
Formulae of Constituents 
of Bitter Principle Drugs 
Aurantii pericarpium 
Harpagophythi radix 
Gentianae radix 
Centaurii herba 
Condurango cortex 
. Figs. 1,2 
. Fig. 3 
. Fig. 4 
· Figs. 5, 6 
· Figs. 7, 8 
. Figs. 9,10 
. Figs. 11, 12 
· Figs. 13, 14 
· Figs. 1,2 
· Figs. 3,4 
93 
93 
97 
100 
102 
104 
104 
" 106 
. 108 
110 
112 
114 
117 
117 
119 
119 
120 
122 
125 
125 
127 
130 
Menyanthidis folium ........... Figs. 1,2 . . . . . . . . . 132 
Cnici herba 
Marrubii herba 
Absinthii herba 
Quassiae lignum .. . . . . . . . . . . . Figs. 3, 4 . . . . . . . . . 134 
IX 
x 
Gentianae radix 
Plantaginis herba 
Oleae folium/fructus 
Bryoniae radix 
Cucurbitae semen 
Humuli strobuli 
Salviae folium 
Rosmarini folium 
Cynarae herba . 
Coumarin Drugs 
Extraction and TLC 
List of Coumarin Drugs 
Formulae of Constituents of Coumarin Drugs 
Reference compounds 
Pimpinellae radix 
Heraclei radix 
Angelicae radix 
Levistici radix 
Imperatoriae radix 
· Figs. 5, 6 
· Figs. 7, 8 
· Figs. 9, 10 
· Fig. 11 
· Fig. 12 
. Fig. 1 
. Fig. 2 
136 
138 
140 
142 
142 
145 
145 
147 
150 
152 
152 
Scopoliae radix. ............ Figs. 3, 4 . . . . . . . . . 154 
Herniariae herba 
Meliloti herba 
Asperulae herba 
Abrotani herba 
Rutae herba. ............. Figs. 5, 6 . . . . . . . . . 156 
Mezerei cortex 
Fraxini cortex 
Asa foetida 
Ammi majoris fructus 
Ammi visnagae fructus 
Flavonoid Drugs 
Extraction and TLC 
List of Flavonoid Drugs 
· Figs. 7, 8 
· Figs. 9, 10 
Formulae of Constituents of Flavonoid Drugs 
Reference Compounds . Figs. 1,2 
TLC Synopsis (flos) . . . . . . . . . . . . Figs. 3, 4 
Arnicae flos 
Calendulae flos 
Cacti flos 
Farfarae flos 
Primulae flos 
Herniariae herba 
Crataegi flos/folium/fructus 
Sambuci flos 
Stoechados flos 
........ Figs. 5, 6 
158 
160 
163 
163 
165 
170 
172 
174 
......... 176 
Verbasci flos . . . . . . . . . . . . . . . Figs. 7, 8 . . . . . . . . . 178 
Acaciae flos 
Pruni spinosae flos 
Spireaae flos 
Robiniae flos 
Sambuci flos 
Tiliae flos . . . . . . . . . . . . . . Figs. 9, 10 
Betulae folium 
Juglandis folium 
Anthemidis flos 
Matricariae flos 
TLC Synopsis (Herba) 
Anserinae herba 
Equiseti herba 
Leonuri herba 
Rutae herba 
Sarothamni herba 
Sophorae gemma 
Veronicae herba 
Violae tricoloris herba 
Citri pericarpium 
Aurantii pericarpium 
Eriodictyonis herba 
Orthosiphonis folium 
Cardui mariae (Silybi) fructus 
Farfarae folium 
Petasites folium 
Cardiac Glycoside Drugs 
Extraction and TLC . . . . 
List of Cardiac Glycoside Drugs 
Formulae of Constituents 
of Cardiac Glycoside Drugs 
Reference compounds . . 
Digitalis lanatae folium 
Digitalis purpureae folium 
Nerii (oleandri) folium 
TLC Comparison 
Strophanthi semen 
Adonidis herba 
Convallariae herba 
Strophanthi grati semen 
Strophanthi kombe semen 
Adonidis herba 
Convallariae herb'it 
· Figs. 11, 12 
. Fig. 13 
· Fig. 14 
. Figs. 15, 16 
· Fig. 17 
· Fig. 18 
· Figs. 19,20 
· Figs. 21, 22 
Figs. 1,2 
· Figs. 3, 4 
· Figs. 5-8 
· Figs. 9, 10 
· Figs. 11, 12 
· Figs. 13, 14 
· Figs. 15, 16 
........ 180 
182 
184 
184 
186 
188 
188 
190 
192 
195 
195 
198 
200 
204 
206 
208 
212 
214 
216 
218 
XI 
XII 
Uzarae (Xysmalobii) radix 
Helleborii radix 
Scillae bulbus 
var. alba/var. rubra 
Saponin Drugs 
Extraction and TLC 
List of Saponin Drugs 
Formulae of Constituents of Saponin Drugs 
TLC Synopsis 
Primulae radix 
Saponariae radix 
Ginseng radix 
Eleutherococci radix 
Liquiritiae radix 
A venae herba 
Hederae folium 
Hippocastani semen 
TLC-Analysis of Saponins 
Drugs Containing Pungent Principles 
Extraction and TLC . . . . . . . . . . 
List of Drugs Containing Pungent Principles 
Formulae ........... . 
Capsici fructus 
. Fig. 17 
· Fig. 18 
· Figs. 19,20 
· Figs. 1, 2 
. Fig. 3 
. Fig. 4 
· Figs. 5, 6 
· Figs. 7, 8 
. Fig. 9 
· Fig. 10 
. Figs. 11, 12 
Cubebae fructus 
Piperis fructus . . . . Figs. 1, 2 
Mustard Oil Drugs and Allium 
Extraction and TLC 
List of Drugs . . . 
Formulae 
Sinapis albae (Erucae) semen 
Sinapis nigri semen 
Thiourea derivatives 
Allium sativum . . 
N arcotic Drugs . 
Extraction and TLC 
List of Drugs . 
Formulae 
Cannabis herba 
. Fig. 3 
. Fig. 4 
Hashish . . . . . . . . . . . . . . . . . Figs. 1, 2 
220 
220 
222 
225 
225 
227 
230 
234 
236 
236 
238 
240 
242 
242 
244 
247 
247 
248 
249 
......... 250 
253 
253 
255 
255 
256 
256 
259 
259 
259 
260 
260 
Drugs Containing Valepotriates 
Extraction and TLC 
List of Drugs . 
Forrnulae 
Valerianae radix .... . . . . . . . . . Figs. 1, 2 
Drugs Containing Pigments 
Extraction and TLC 
List of Drugs 
Forrnulae 
Cyani flos 
Hibisci flos 
263 
263 
264 
265 
266 
269 
269 
270 
271 
Malvae flos ............... Figs. 1, 2 . . . . . . . . . 272 
Hibisci flos 
Paeoniae flos 
Croci stigma . . . . . . . . . . . . . . . Figs. 3, 4 . . . . . . . . . 274 
Drugs with Miscellaneous Constituents 
Extraction and TLC . . . 
List of Miscellaneous Drugs 
Forrnulae ...... . 
Salicis cortex . . . . . . . . . . . . . . . Fig. 1 
Hamamelidis folium 
Filicis rhizoma 
Pyrethri flos . . 
Lichen islandicus 
Podophylli resina 
Visci albi herba 
Amino acids . . 
. Fig. 2 
. Fig. 3 
. Fig. 4 
. Figs. 5, 6 
. Figs. 7, 8 
TLC Screening of an Unknown Commercial Drug 
Extraction and TLC . . . . . . . . . 
Scheme of Separation and Identification 
TLC Analysis of Herbai Drug Mixtures 
Spray Reagents . . . . . . 
Abbreviations and Definitions 
References . 
Subject Index 
277 
277 
278 
280 
282 
282 
284 
284 
286 
288 
291 
291 
294 
296 
299 
305 
307 
309 
XIII 
Introduction 
I. Thin Layer Chromatographie-Analysis (TLC) of Drugs 
Of the many chromatographie methods presently available, thin layer chromatogra-
phy has become widely adopted for the rapid and positive analysis of drugs and 
drug preparations. 
There are several reasons for the popularity of this method: 
- The time required for the demonstration of most of the characteristic constitutents 
of a drug by TLC is very short. 
- In addition to qualitative detection, TLC also provides semi-quantitative informa-
tion on the chief active constituents of a drug or drug preparation, thus enabling 
an assessment of drug quality. 
- TLC provides a chromatographie drug fingerprint. It is therefore suitable for 
monitoring the identity and purity of drugs, and for detecting adulterations and 
substitutions. 
- With the aid of appropriate separation procedures, TLC can be used to analyse 
drug combinations and phytochemical preparations. 
- Thin layer chromatograms can be documented. 
11. Doeumentation of Thin Layer Chromatograms 
Various methods of documentation are possible: 
- Description of the Rf values and colours of the characteristic main zones, with 
reference to a standard substance or test mixture. This method has been adopted 
in the 8th edition of the German pharmacopoeia, the European pharmacopoeia, 
USP XX and others. 
- Construction of ascale diagram of the thin layer separation, showing migration 
distances and intensities of the characteristic zones. The zones observed in visible 
light (vis.) in UV-254 nm and UV-365 nm are described.- Colour photography in daylight or UV-light, under conditions that give the most 
authentie reproduction of the colours and intensities of the separated zones. 
- Densitometry or fluorometry of the chromatogram at certain wavelengths. Under 
favourable conditions, this procedure also yields a drug fingerprint, and enables 
the quantification of certain chief constituents. It suffers from the disadvantage 
that a calibration graph constructed at one wavelength is applicable to only some 
of the constituents. 
111. Photo graphie Reeord of Thin Layer Separations of Drug 
Extraets (A Photographie TLC Drug Atlas) 
- A photographie TLC atlas fulfils the same function and purpose as a catalogue 
of spectra. The identity or non-identity of an official drug can be established 
by comparison with the chromatogram of the 'standard drug'. 
2 
- Unknown commercial drugs can be more easily identified by comparison with 
the visual record in the TLC atlas. 
- The photographic drug atlas is an aid to the routine identification and purity 
testing of drugs in control laboratories, and it can be used without previous 
pharmacognostic training. 
- Photographic reproduction ofthin layer separations has a large didactic advantage 
over mere graphic representation. The TLC photo-drug atlas has an immediate 
clarity of representation that facilitates the learning of TLC drug analysis for 
the student. 
IV. Compilation of a TLC Drug Atlas 
Compilation of a TLC drug atlas was governed by certain preconditions, related 
to the source of the drugs, the TLC technique in general, and the photographic 
reproduction of the thin layer chromatograms. 
1. Source of the drugs 
The drugs used in the compilation of a drug atlas must meet the standards of 
the official pharmacopoeia, and they must originate from a clearly identified botani-
cal source. 
Slight variations in the chromatographic picture, due to botanical varieties, or 
differences in cultivation, climatic conditions, time of harvesting, drying and extrac-
tion methods are normal. 
2. Extraction conditions 
The chosen extraction procedures are the best available in the light ofpresent scientif-
ic knowledge. As far as possible they have been adopted unchanged from the phar-
macopoeias, and modified only when new substances and separation problems have 
been encountered. 
3. TLC 
Reproducible TLC separations can be guaranteed only if standardized adsorption 
layers are used. Commercially available TLC plates were therefore used: Si/ica gel 
60F254 pre-coated TLC platesfrom Merck, Darmstadt. Since special chromatography 
rooms are not always available, all TLC separations were performed at room temper-
ature, i.e. 18-22° C. Details ofthe TLC technique are to be found in pharmacopoeias 
and books on methodology (see list on page 307). 
Generally a distance of 15 cm is used for the development of a chromatogram. 
The pictures shown in the book are approx: 1/3 of the original size of the TLC-plate. 
4. Chromatography solvents 
In choosing suitable solvent systems, preference has been given to those specified 
in the pharmacopoeias. These often represent a compromise, and where necessary 
they have been modified. In most cases, the resulting improvement in separation 
has been documented with chromatograms in both systems. Where possible, an 
attempt has been made to standardize the systems. Systems were chosen for their 
minimal temperature sensitivity. 
5. Concentration of substances for TLC 
In order to obtain sharply resolved zones, the quantity of material applied to the 
chromatogram should be as small as possible. Rather large sample volumes are, 
however, often necessary for the detection (by colour reactions) of substances that 
are present in low concentration. This inevitably results in broadening and overlap-
ping of zones. 
6. Detection methods 
- For the detection of the main, characteristic compounds of a drug, methods were 
chosen that give the most striking colours. 
- The active principles of a group of drugs may be very sirnilar (e.g. drugs from 
Solanaceae or saponin drugs), so that differentiation and identification are difficult 
or impossible on the basis of the active principles alone. In such cases, other 
c1asses of compounds have been exploited for the purposes of differentiation. 
- For drugs that contain unknown or incompletely known activeprinciples, identifi-
cation has been based on those constituents that can be regarded as "guide sub-
stances". 
7. Photography 
The developed chromatograms were photographed on Agfachrome 50L or 50S Pro-
fessional. To achieve authentie colour reproduction, each picture needs a specific 
technic of exposure and deve1opment. 
3 
Essential Oil Drugs (Aetherolea), 
Gums and Resins 
Essential oils are mixtures of many substances, predominantly terpenes (ca. 90%) 
and phenylpropane derivatives. Other components inc1ude simple phenols, sulphur-
containing compounds (mustard oils), methyl anthranilate and coumarins. 
I. Extraction of Essential OUs 
1. Official method 
2. 
Steam distillation with a modified distillation apparatus according to Cocking and 
Middleton (Ph. Eur. III, p. 62) 
Principle. The quantity of drug used is sufficient to yield 0.1-0.3 ml essential oil. 
The sample weight is 10 to 50 g, depending on the type of drug (DAB 8, Ph. Eur.), 
and the rate of distillation is no greater than 2-3 ml per minute. When ebullition 
has become steady, distillation is continued for between 11/ 2 and 4 hours. Xylene 
(1 ml) is placed in the distillation flask, so a blank" xylene value" must be determined 
in aseparate distillation in the absence of the drug. 
Table 1 shows the essential oil drugs of the DAB 8 and Ph. Eur., with sample 
weight and distillation conditions. 
Abridged official method 
If a quantitative determination of the oil content is not required, a sample of essential 
oil suitable for investigation by TLC can be obtained by reducing the distillation 
Table 1 
Drug Content of Sampie Water Time Rate 
essential oil weight (mI) (hr) (ml/min) 
(ml/lOO g) (g) 
Absinthii herba 0.3 50 300 3 2-3 
Anisi fructus 2.0 25 200 2 2-3 
Anthernidis flos 0.7 30 300 3 3-5.5 
Aurantii percarpium 1.0 20 250 1.5 2-3 
Carvi fructus 4.0 10 200 1.5 2-3 
Curcumae rhizoina 3.5 10 200 3 3--4 
Foeniculi fructus 4.0 10 200 2 2-3 
Juniperi fructus 1.0 20 200 1.5 3--4 
Matricariae flos 0.4 50 500 a 4 3--4 
Melissae folium 0.05 40 400 2 2-3 
Menthae folium 1.2 50 500 2 3-3.5 
Salviae offic. folium 1.5 50 500 1.5 2-3 
Salviae trilobae folium 1.8 50 500 1.5 2-3 
Thyrni herba 1.2 20 300 2 2-3 
a distilled from 1 % NaCI solution. 
5 
6 
period to one ho ur. With the exception of drugs containing e.g. eugenol, the distilla-
tion is then performed without xylene. The resulting oil is diluted 1: 10 with toluene. 
Eugenol-containing oils, obtained by distillation in the presence of xylene (Ph. 
Eur. III), can be applied directly to chromatograms. If the concentration of oil 
is still too high, the xylene solution should be diluted 1: 5 with toluene. 
3. Micromethods 
a) Micro steam distillation after Luckner 
1 g powdered drug and 10 ml water are placed in a 50 ml Erlenmeyer flask. A 
glass U-tube (10-15 cm long; 0 ca. 5 mm) is placed between the distillation flask 
and the receiver (test tube). The contents of the flask are heated to boiling (boiling 
stones) and distillation via the U-tube performed slowly until about 1 ml of distillate 
has collected in the test tube. The distillate is extracted by shaking with 1 ml pentane, 
the pentane solution removed with a pipette, and 20-100111 of this solution are 
used for TLC. A range of different sam pie concentrations is used for the TLC 
separation. 
Remarks: This rapid method gives only a guide to the composition of the essential oil. 
b) Thermomicrodistillationafter Stahl (T AS-method) 
With the aid of a so-called TAS-oven (Desaga), substances that volatilize at fairly 
high temperatures can be distilled from the drug onto the TLC plate. 
The tapered end of a T AS-glass cartridge is cIosed with a packing of quartz 
wool, followed by about 50 mg powdered drug and about 50 mg starch. The cartridge 
is sealed with a cIamp and placed in the oven block of the T AS-apparatus, which 
is heated to about 220° C. The point of the cartridge is directed onto the surface 
of the TLC plate. Substances that are volatile at the given temperature distil onto 
the starting zone of the TLC plate in about 90 sec. 
Remarks: All components of essential oils and other volatile compounds, e.g. coumarins, 
are obtained by this method. 
c) Extraction with methylene chloride (dichloromethane (DCM)-extract) 
1 g powdered drug is extracted by shaking for 15 min with 10 ml methylene chloride. 
The suspension is filtered and the cIear filtrate evaporated to dryness. The residue 
is dissolved in 1 ml toluene, and 50-iOO 111 are applied for TLC. 
Remarks: This method also extracts other, interfering lipophilic substances. 
d) Extraction with methanol (MeOH-extract) 
a) Curcumae rhizoma (cinnamoyl pigments). 1 g powdered drug is extracted by 
shaking for 5 min with 5 ml MeOH at about 60° C. 10111 of the cIear filtrate are 
applied for TLC. 
ß) Gum resins (e.g. Myrrha). 0.5 g powdered drug is extracted by shaking for 5 min 
with 5 ml 96% ethanol. 20 111 of the supernatant or cIear filtrate are applied for 
TLC. 
y) Oleo-resins (Balsamum peruvianum, B. tolutanum). 0.5 g peru balsam is dissolved 
in 10 ml ethyl acetate, and 10 111 of this solution are applied for TLC. 
For tolu balsam, 10111 of a 1: 10 dilution in toluene are applied for TLC. 
11. Thin Layer Chromatography 
1. Reference solutions 
Solutions of each of the following compounds are prepared in toluene (1: 30). 
Alcohols: borneol, geraniol, linalool, menthol 
Phenols: thymol, carvacrol 
Aldehydes: anisaldehyde, citral, citronellal 
Ketones: carvone, fenchone, menthone/isomenthone, piperitone, thujone 
Oxides: 1.8-cineole 
Phenylpropane derivatives: anethole, apiol, allyltetramethoxybenzene, eugenol, myr-
isticin, safrol 
2. Adsorbent 
Silica gel 60Fz54 pre-coated TLC plates (Merck, Darmstadt) 
3. Sampie concentration 
5 111 of a 1 : 10 dilution of the essential oil in toluene are applied to the TLC plate. 
3 111 of each reference solution are used. 
When applied in quantities of about 100 I1g/3 111, all the reference compounds 
can be detected by treatment ofthe chromatogram with VS-reagent (No. 38, p. 304). 
Thymol and anethole are detectable in quantities down to 5 I1g. 
4. Chromatography solvents 
A-l Toluene-ethyl acetate (93: 7) 
This system is suitable for the analysis and direct comparison of.all impor-
tant essential oils. 
The DAB 8 and Ph. Eur. describe different solvent systems for individual 
essential oils: 
A-2 
A-3 
A-4 
A-5 
A-6 
A-7 
A-8 
Solvent system 
Benzenea 
Chloroform 
Methylene chloride 
Benzenea-ethyl acetate (90: 10) 
Benzenea-ethyl acetate (95: 5) 
Chloroform-benzenea (75: 25) 
Chloroform-ethanol-glacial 
acetic acid (94: 5: 1) 
Drug or essential oil 
Anisi fructus 
Curcumae xanth. rhizoma, Melissae folium 
Anisi aeth., Carvi fructus, Carvi aeth., Caryo-
phylli aeth., Foeniculi aeth., Juniperi fructus, La-
vandulae aeth., Rosmarini aeth., Salviae off. and 
S. trilobae folium 
Eucalypti aeth. 
Menthae piperitae aeth. 
Absinthii herba, Matricariae flos, Menthae 
piperitae folium, Thyrni herba 
For the separation of cinnamoyl pigments 
from Curcumae rhizoma 
a Benzene is carcinogenic and should be replaced by toluene. 
III. Detection 
1. Without chemical treatment 
UV-254 nm 
All compounds containing at least two conjugated double bonds quench fluorescence 
and appear as dark zones against the light green fluorescent background of the 
TLC plate. Phenylpropane derivatives have this property, e.g. anethole, safrol, apiol, 
myristicin, eugenol, asarone, methyl chavicol. Other compounds that quench fluores-
cence are cinnamic aldehyde, anisaldehyde, thymol and piperitone. 
UV-365 nm 
An intense blue fluorescence is given by e.g. methyl anthranilate. 
7 
8 
2. Spray reagents 
a) Anisaldehyde-sulphuric acid (AS No. 2, p. 299) 
In the visible, the components of essential oils show strong blue, green, red and 
brown colouration. Some compounds also fluoresce under UV-365 nm. 
b) Vanillin-sulphuric acid (VS No. 38, p. 304) 
Visible colourations are very similar to those obtained with the AS-reagent. 
Exception : Thujone shows weak red with the AS-reagent, and only very weak blue 
with the VS-reagent when viewed in the visible. 
c) Phosphomolybdic acid (PMA No. 27, p. 303) 
With the exception of anisaldehyde and fenchone, the constituents of essential oils 
show uniform blue on a yellow background when viewed in the visible. 
rx) Anisaldehyde shows blue with PMA-reagent only when present in concentrations higher 
than 100 J..tg. At lower concentrations, its colour response varies from whitish to pale green 
in visible light. 
When sprayed with conc. H ZS04 and heated at about 1000 C for 5 min, anis aldehyde 
gives a red zone in the visible. 
ß) Fenchone. After treatment of the TLC plate with PMA-reagent as described above, it 
is then sprayed with a solution of 0.5 g potassium permanganate in 5 ml conc. sulphuric 
acid. After heating for 5 min at 1000 C, fenchone appears as a dark blue zone in the visible. 
These conditions are optimal for the detection of fenchone, but great care is advisable in 
the preparation and use of the reagent. 
Larger quantities (>100 J..tg) of fenchone, when heated with conc. H ZS04 , appear as a 
yellow zone in visible light. 
IV. List of Essential on Drugs, Gums and Resins 
Chromatograms (Figs. 3-28) are reproduced on pp. 24-49. 
THC = Terpene hydrocarbon(s) 
Fig. 
3 
4 
5,6 
5,6 
5,6 
Drug/Plant source/Family/Phar-
macopoeia 
Cinnamomi Cortex 
Cinnamon bark 
Cinnamomum zeylanicum BLUME 
Ceylon cinnamon 
Cinnamomum aromaticum NEES. 
(c. cassia BLUME) 
Chinese or cassia cinnamon 
Lauraceae 
NF XV, ÖAB, Helv. VI 
Calami Rhizoma (Radix) 
Sweet Flag rhizome 
Acorus calamus L. 
Araceae 
ÖAB, Helv. VI 
Anisi Fructus 
Anise 
Pimpinella anisum L. 
Apiaceae 
NF XV (oil), Ph. Eur. III, 2. AB-
DDR (oil) 
Anisi stellati Fructus 
Star anise 
Illicium verum HOOK. 
Illiciaceae 
ÖAB 
Foeniculi Fructus 
Fennel 
Foeniculum vulgare MILL. 
Apiaceae 
ÖAB, Helv. VI, DAB 8,2. AB-
DDR, NF XV (oil) 
Content of essential oil 
Main constituents 
Ceylon cinnamon: 1-1.5% ess. oil. 
Cinnamic aldehyde (67-75%), eugenol 
(4--10%) and THC (e.g. caryophyllene, oc-pin-
ene). 
Chinese cinnamon: 1-2% ess. oil. 
Cinnamic aldehyde (75-90%); eugenol absent. 
In addition, the bark contains unsubst. cou-
marin. 
Triploid European race: up to 3 % ess. oil with 
variable content of IX-, p- and y-asarones 
(1-99%, average 50--60%). 
Diploid races: 2.7-5% ess. oil, asarone absent; 
containing ca. 30 compounds, e.g. isoeugenol, 
isoeugenol methyl ether, acaromone, asarylal-
dehyde and artefacts formed during distilla-
tion. 
2-6% ess. oil (Ph. Eur. III specifies not less 
than 2%). 
Anethole (80--90%), methyl chavicol and 
anisaldehyde. 
Adulteration : Illicium anisatum L. (poisonous 
shikirni fruits I), mostly Safrol; Anethol is 
absent. 
5-8% ess. oil (ÖAB specifies not 1ess than 
5%) 
Anethole (85-90%), terpineol, phellandrene. 
F. vulgare var. dulce (French sweet or Roman 
Fennei): 2-5% ess. oil. 
Anethole (50--60%), methyl chavicol (=estra-
goi), safrol, anisaldehyde and fenchone 
(0.4--0.8%). 
F. vulgare var. vulgare (French bitter Fennei) : 
5-7% ess. oil (DAB 8 specifies not less than 
4%) 
Anethole (60--80%),methyl chavicol, anisal-
dehyde andfenchone (12-22%). 
9 
Fig. 
5 
5 
7A 
8A 
8B/C 
9A 
10 
Drug/Plant source/Family/Phar-
macopoeia 
Basilici Herba 
Basil 
Ocimum basilicum L. 
Lamiaceae 
Sassafras Lignum 
Sassafras wood 
Sassafras albidum (NUTT.) NEES. 
vaL molle (RAF.) FERN. (syn. S. of-
ficinale NEES et EBERM.) 
Lauraceae 
Petroselini Fructus 
Parsley fruits 
Petroselinum crispum (MILL.) 
NYM. ex hort. KEW (syn. P. hor-
tense HOFFM.) vaL foliosum 
(ALEF.) THELL. Leafparsley 
vaL tuberosum (BERNH.) THELL. 
Root parsley 
Apiaceae 
2. AB-DDR (oil) 
Myristicae Semen 
Nutmeg 
Myristica fragrans HOUTT. 
M yristicaceae 
Helv. VI, NF XV (oil) 
Myristicae arillus 
Mace 
Myristica fragrans HOUTT. 
Myristicaceae 
Caryophylli Flos 
Cloves 
Syzygium aromaticum MERR. et 
PERRY 
Myrtaceae 
NF XV (oil), Helv. VI, ÖAB, 
DAB 8,2. AB-DDR, ÖAB (oil) 
Carvi Fructus 
Caraway fruits 
Carum carvi L. 
Apiaceae 
NF XV (oil), DAB 8, Helv. VI, 
ÖAB, 2. AB-DDR 
Content of essential oil 
Main constituents 
0.1-0.45% ess. oi!. 
Methylchavicol (ca. 55%) and linaloo!. 
1-2% ess. oi!. 
Safrol (ca. 80%) and eugenol (ca. 0.5%). 
3-6% ess. oi!. 
Phenylpropane derivatives: apiol, myristicin 
and allyltetramethoxybenzene. 
Apiol race: 60-80% apio!. 
Myristicin race: 55-75% myristicin. 
Allyltetramethoxybenzene race: 50-60% allyl-
tetramethoxybenzene. 
Remarks: Petroselini radix (2. AB-DDR) con-
tains 0.2-0.3% ess. oil with apiol and myristi-
cin, as weil as thefuranocoumarins, bergapten 
and isoimperatorin. 
6-10% ess. oil (Helv. VI specifies not less than 
6.5%). 
Phenylpropane derivatives: myristicin (ca. 
8%), safrol, eugenol, elemicin and TBC (IX-
pinene, limonene, p-cymene) and low concen-
trations of the terpene alcohols, geraniol, bor-
neol, linalool and terpineo!. 
4-12% ess. oil, with the same qualitative com-
position as the seed oi!. 
14-20% ess. oil (ÖAB specifies not less than 
16%). 
Eugenol (4-allyl-2-methoxyphenol) (72-90%), 
aceteugenol (10-15%), ß-caryophyllene 
(3-12%) and epoxidihydrocaryophyllene. 
Remarks: Clove stalks contain only 5-6% ess. 
oi!. Mother cloves or anthophylli (an adulter-
ation) contain 2-9% ess. oi!. 
2.5-7% ess. oil (DAB 8 specifies not less than 
4%). 
D-Carvone (50-85%), with small amounts of 
carveol, dihydrocarveol, limonene and perillyl 
alcoho!. 
Fig. 
9B 
9C 
10A/B 
10C 
Drug/Plant souree/Family /Phar-
macopoeia 
Coriandri Fructus 
Coriander fruits 
Coriandrum sativum L. var. 
vulgare ALEF. 
Large Indian coriander 
var. microcarpum DC. 
Small Russian coriander 
Apiaceae 
NF XV (oi!), ÖAB 
Cardamomi Fructus 
Cardamoms 
Elletaria cardamomum (L.) WHITE 
etMAsoN 
Zingiberaceae 
USP XX (seeds) 
Juniperi Fructus 
Juniper berries 
Juniperus communis L. 
Cupressaceae 
USP XX (tar), DAB 8,2. AB-
DDR, ÖAB, Helv. VI (fruit and 
oi!) 
Rosmarini Folium 
Rosemary leaves 
Rosmarinus officinalis L. 
Lamiaceae 
DAB 8,2. AB-DDR, Helv. VI, 
ÖAB (oil) 
11 A, 12 MatricarJae Flos 
(Chamomillae flos) Chamomile 
flowers 
Chamomilla recutita (L.) RAUSCH 
Asteraceae 
Ph. Eur. III, Helv. VI, ÖAB, 
2. AB-DDR (0.1-0.16% matricin) 
11 B Anthemidis Flos 
Roman chamomile flowers 
Chamaemelum nobile (L.) ALL. 
Asteraceae 
Ph. Eur. III, ÖAB 
Content of essential oil 
Main constituents 
0.2% ess. oil (Indian eoriander) 0.8-1 % ess. 
oil (Russian eoriander) (ÖAB speeifies not 
less than 0.5%). 
Linalool (50-70%) with small amounts of 
geraniol and geranyl acetate, borneol and 
citronellol, ca. 20% THC (ß-pinene, cx-terpin-
ene, myrcene). 
3-7% ess. oil (fruits), 4-9% ess. oil (seeds), 
0.5-1 % ess. oil (pericarp). 
'X.-Terpinyl acetate and 1,8-cineole (ca. 50%) 
are the chief constituents, with small amounts 
of borneol, cx-terpineol and limonene. 
0.2-2% ess. oil (DAB 8 specifies not less than 
1 %). 
Varying composition of terpinene-4-01, caryo-
phyllene, epoxydihydrocaryophyllene, ter-
pinyl acetate, camphor and the THC, cx-, ß-
pinene, myrcene and camphene. 
1-2% ess. oi!. 
1,8-Cineole (15-30%), borneol (10-20%), bor-
nyl acetate, camphene (5-10%) and cx- and 
ß-pinene. 
0.5-1.5% ess. oil (Ph. Eur. specifies not less 
than 0.4%; 2. AB-DDR 1.2-1.8%). 
Chamazulene (0-15%), bisabolol (10-25%), 
bisabolol oxide A and B (10-25%), polyines 
(cis- and trans-ene-ine-dicycloether, 1-40%) 
and farnesene (15%). 
0.6-2.4% ess. oil (Ph. Eur. III specifies not 
less than 0.7%), with a high proportion of 
esters of angelie, methacrylic, tiglie and isobu-
tyric acids with aliphatic alcohols; cis and 
trans-dehydromatriearia acid, polyines. 
Flavonoids: Apigenin, A.-7-glucoside, A.-7-
apiosylglucoside, luteolin, luteolin-7 -gluco-
side, quercitrin (see chapter on Flavonoids, 
p. 182, Fig. 11/12). 
11 
Fig. 
13 
14 
15j16 
12 
DrugjPlant sourcejFamily jPhar-
macopoeia 
Lavandulae Flos 
Lavender flowers 
Lavandula angustifolia MILL. 
Lamiaceae 
USP XX (oil), DAB 8,2. AB-
DDR, ÖAB (oil) 
Lavandula latifolia MED. 
Lavandula hybrida, e.g. L. latifolia 
+ L. offic. 
Cinae Flos 
Wormseed 
Artemisia cina O.C. BERG et C.F. 
SCHMIDT 
Asteraceae 
Menthae piperitae Folium 
Peppermint leaves 
Mentha piperita L. 
Lamiaceae 
NF XV (oil), Ph. Eur. III, ÖAB, 
2. AB-DDR, Helv. VI 
M. arvensis L. var. piperascens 
HOLMES ex CHRISTY 
NF XV (oil) 
Lamiaceae 
Mentha pulegium L. (an adultera-
tion ofM. piperita and M. arvenis) 
Lamiaceae 
Menthae crispae Folium 
Spearmint leaves 
Mentha spicata L. emend. L. var. 
crispa (BENTH.) DANERT 
NF XV (oil) 
Lamiaceae 
Content of essential oil 
Main constituents 
1-3% ess. oil (DAB 8 and ÖAB specify not 
less than 35% esters). 
Linalyl acetate (30-50%), ünalool (10-15%), 
with small quantities ofnerol, borneol, geran-
iol, cineole and caryophyllene. 
Oil 0/ spike: esters are low or absent; chief 
constituents linalool and cineole. 
"Lavandin oils": 20-24% or 30-32% linalyl 
acetate, linalool, terpene hydrocarbons and 
terpene alcohols, as in L. angustifolia. 
2-3% ess. oil. 
1.8-Cineole (ca. 80%) with small amounts of 
a-terpineol, carvacrol and sesquiterpene hy-
drocarbons. 
Bitter principles: up to 6% L-rz.-santonin and 
rz.-hydroxy-santonin (artemisin). 
1.3-2.1 % ess. oil. 
Menthol (50-78%), (- )menthone (10-30%), 
menthyl acetate (5-20%), menthofuran 
(2.5-5%) with small amounts ofisomenthone, 
pulegone, piperitone, cineole, limonene, jas-
mone (0.1 %). 
Ph. Eur. Irr specifies not less than 1.2 % ess. 
oil, containing 4.5-10% esters (calculated as 
menthyl acetate), at least 44% alcohols (cal-
culated as menthol) and 15-32% ketones (cal-
culated as menthonejisomenthone). 
0.2-0.3% ess. oil; approx. same qualitative 
composition as the oil from M. piperita, but 
menthofuran and cineole are absent. 
Cornmint oil (DAB 8): 3-17% esters (calc. as 
menthyl acetate), at least 42% alcohols (calc. 
as menthol), at least 25-40% ketones (calc. 
as menthone). 
1-2% ess. oil. 
Pulegone (80-95%) with small amounts of pi-
peritone, menthol and THC. 
1-2% ess. oil. 
L-Carvone (42-67%), acetates of dihydrocar-
veol and dihydrocuminyl alcohol, THC (pin-
ene, limonene, phellandrene). 
Fig. 
17/18 
18 
19 
Drug/Plant source/Family/Phar-
macopoeia 
Salviae Folium 
Sageleaves 
Salvia officinalis L. 
S. officinalis ssp. rninor (GMEL.) 
GAMS. S. officinalis ssp. officinalis. 
Dalmatian sage. 
S. officinalis ssp. lavandulifolia 
(V AHL) GAMS, 
Spanish sage 
Salviae trilobae Folium 
Greeksage 
Salvia trilo ba L. fil. 
Lamiaceae 
DAB8 
Eucalypti Folium 
Eucalyptus leaves 
Eucalyptus globulus LABILL., 
E. fruticetorum F. v. MUELLER, 
E. smithii R.T. BAKER 
Myrtaceae 
NF XV (oil), Ph. Eur. III, ÖAB, 
2. AB-DDR, Helv. VI (oil) 
ThymiHerba 
Thyme 
Thymus vulgaris L. 
Lamiaceae 
DAB 8,2.AB-DDR, Helv. VI (oil) 
Thymus zygis L. 
Spanish thyme 
Lamiaceae 
DAB8 
Content of essential oil 
Main constituents 
1.3-2.6% ess. oil (DAB 8 specifies not less 
than 1.5%). 
Composition varies, depending on origin: 
Thujone 35-50% (S. offic. ssp. minor/major), 
cineole ca. 14%, camphor 7-8% 
Thujone absent (S. offic. ssp. lavandulifolia), 
cineoie ca. 30%,camphor 30% 
Also present are terpene alcohols (borneol 
5-8%) and THC (pinene, camphor), 
Rosmarinic acid (2-3%), depside of caffeic 
and IX-hydroxydihydrocaffeic acids. 
Diterpene bitter principles: picrosalvin ( = car-
nosol) in Dalmatian sage (ca. 0.35%). 
Bitter principles (see p. 142, Fig. 11). 
Up to 3% ess. oil (DAB 8 specifies not less 
than 1.8%). 
1,8-Cineole (60-70%), thujone (ca. 5%) and 
borneol (ca. 0.35%), bornyl acetate, THC 
(pinene, camphene). 
Bitter principle: picrosalvin (=carnosol, 
0.2-0.3%) (see Bitter principle drugs, p. 142, 
Fig.11). 
Flavone: salvigenin (=8-hydroxy-6,7,4'-tri-
methoxyllavone ). 
1-3% ess. oil. 
1,8-Cineole (=eucalyptol, at least 70%) with 
small amts. of piperitone, phellandrene, alde-
hydes*. 
Non-official oils sometimes contain high lev-
els ofpiperitone and/or phellandrene, e.g. Eu-
calyptus dives SCHAUER 
* Non-rectified oils contain, e.g. butyralde-
hyde and capronaldehyde, which cause 
bronchial irritation. 
0.75-6.3% ess. oil (DAB 8 specifies not less 
than 1.2%). 
Thymollcarvacrol (20-60%) with small 
amounts of 1.8-cineole, borneol, geraniol, lin-
alool, bornyl and linalyl acetate, thymol 
methyl ether and IX-pinene. 
Content and composition of ess. oil corre-
spond with those of Th. vulgaris, but propor-
tion of carvacrol is higher than that of thy-
mol. 
13 
Fig. 
20 
21/22 
14 
Drug/Plant source/Family/Phar-
macopoeia 
Serpylli Herba 
Wild thyme 
Thymus serpyllum L. 
Lamiaceae 
Helv. VI 
Ajowani Fructus 
Ajowan fruits 
Trachyspermum am mi (L.) 
SPRAGUE 
Apiaceae 
Melissae Folium 
Melissa or Lemon balm leaves 
Melissa officinalis L. 
Lamiaceae 
DAB 8,2. AB-DDR, ÖAB, 
Helv. VI (oil) 
Curcumae Rhizoma 
Turmeric 
Curcuma xanthorrhiza RoXB. 
Round turmeric 
Zingi beraceae 
DAB 8,2. AB-DDR 
Curcuma longa L. (syn. C. domes-
tica VAHL.) 
Finger or Long turmeric 
Zingiberaceae 
Content of essential oil 
Main constituents 
Composition of ess. oil similar to that of 
Thymi Herba, with lower contents of thymol 
and carvacrol, and higher contents ofp-cymol 
and linalool, together with terpene esters. 
2.6--4.5% ess. oil. 
Thymol (35-60%) with sm all amts. of carvac-
rol and THC. 
0.01-0.25% ess. oil (DAB 8 specifies not less 
than 0.05%). 
Citronellal (ca. 39%), citral (ca. 30%), citro-
nellol, linalool, geraniol and THC (caryophyl-
lene). 
M etissa oU substitutes: 
Java Citronella oil, Cymbopogon nardus (L.) 
W. WATS., Poaceae. 0.5-1.2% ess. oil, con-
taining citronellal (25-54%) and geraniol 
(16-45%). 
Lemon grass oil, Cymbopogon flexuosus 
(NEES et STEUD) W. WATS., Poaceae. 
53-83% Citral (West Indian type) with farne-
sol, geraniol and linalool. 
70-85% Citral (East Indian type). 
80-84% Citral (Angola type; odourless!). 
6-11 % ess. oil (DAB 8 specifies not less than 
3.5%). 
L-Cycloisoprenmyrcene (ca. 85%), xanthor-
rhizol (phenolic sesquiterpene), tolylmethyl-
carbinol (ca. 5%, an artefact), camphor 
(1-5%). 
0.3-5% ess. oil. 
Sesquiterpenes ca. 65% (e.g. turmerone), 
zingiberene (ca. 25%), phellandrene, sabin-
ene, borneol and cineole. 
Curcumins: non-steam volatile diferuloyl- and 
dicinnamoyl-methane compounds. 
C. xanth.: 1.2-2% curcumin and monode-
methoxy-curcumin. 
C. tonga: 3-4% curcumin, monodemethoxy-
curcumin and bisdemethoxycurcumin. 
Fig. 
23/24 
23/24 
Pine Oils 
Drug/Plant source/Family/Phar-
macopoeia 
Aurantü Pericarpium 
SeviIIe orange peeI 
Citrus aurantium L. ssp. aurantium 
Rutaceae 
NF XV (oil), DAB 8,2. AB-DDR, 
ÖAB, Helv. VI 
Aurantii Flos 
Orange flowers 
Citrus aurantium L. ssp. amara 
ENGL. (syn. C. aurantium ssp. sin-
ensis) 
Rutaceae 
NF XV (oil), ÖAB, Helv. VI 
Citri Pericarpium 
Lemonpeel 
Citrus limon (L.) BURM. 
Rutaceae 
USP XX (oil) 
Citrus aurantium (L.) ssp. berga-
mia (RISSO et POlT) ENGL. 
Rutaceae 
Content of essential oil 
Main constituents 
0.6-2.2% ess. oil (DAB 8 specifies not less 
than 1 %). 
(+ )-Limonene (90%) with terpene alcohols 
and aldehydes. 
F/avonoids: rutin, eriocitrin, naringin, neohes-
peridin (see section on Flavonoids, p. 189, 
Fig. 17). 
Methyl anthranilate, coumarins. 
0.1-0.6% ess. oil (ÖAB specifies not less than 
0.2%). 
Linalyl acetate (8-25%), linalool (ca. 30%), 
farnesol, limonene, jasmone. 
(Oil of Neroli) 
0.1-6% ess. oiI. 
(+ )-Limonene (90%), citral (3.5-5%) with 
smaII amts. of terpineol, linalyl and geranyl 
acetate. 
Coumarins: geranylmethoxycoumarin, citrop-
ten, bergamottin. 
Flavonoids: rutin, eriocitrin, neo hesperidin 
(see section on Flavonoids, p. 189, Fig. 17) . 
.. Bergamot oil" (fruit peel oil): chiefly linalyl 
acetate, with a dihydrocumin alcohol and 
linalooI. 
Coumarin: hergapten (ca. 5%). 
"Oil 0/ Petit Grain" (leaf oil) contains chiefly 
linalyl acetate and a terpene alcohoI. 
These are essential oils from the needles and branch tips of Abies, Picea and Pinus spp. 
(family Pinaceae). 
25 Pini pumilionis Aeth. 
Mountain pine oil 
Pinus mugo TURRA ssp. mugo agg. 
Pinus mugo ssp. pumilio (HAENKE) 
FRANCO 
DAB 7-DDR, ÖAB, Helv. VI, 
NF XV (oil) 
Pini silvestris Aeth. 
Scots pine needle oil 
Pinus sylvestris L. 
10% ess. oiI. 
3-10% esters, calc. as hornyl acetate (DAB 7-
DDR). Chief terpenes: IJ(- and ß-pheIIandrene 
(ca. 60%) and IJ(- and ß-pinene (10-20%). 
15 
16 
Fig. 
25 
25 
Drug/Plant source/Family/Phar-
macopoeia 
Picea spp. (parent plant not de-
fined) 
USP XX (oil) 
Pine needle oils 
Pini sibirici Aeth. 
Siberian spruce oil 
Abies sibirica LEDEB. (Erg. Bd. 6) 
Abies pictinatae Aeth. 
Silver fir oil 
Abies alba MILLER (Erg. Bd. 6) 
Terebinthinae Aeth. 
Terebinthinae rectificatum aeth. 
Turpentine oil, rectified oil of tur-
pentine 
Pinus ssp., Pinus palustris MILLER, 
Pinus pinaster AITON et al. 
DAB 8,2. AB-DDR, ÖAB, 
Helv. VI 
Gums and Resins 
26 Myrrha 
Myrrh 
Commiphora molmol ENGL. 
Commiphora spp. 
Burseraceae 
DAB 8,2. AB-DDR, ÖAB, 
Helv. VI 
Benzresins and Balsams 
Content of essential oil 
Main constituents 
Oils of Pine, Spruce and Silver fir all have 
similar terpene compositions. Siberian spruce 
needle oils have a markedly higher content 
of bornyl acetate and terpineol. 
Distillate of turpentine (Terebinthinae Balsa-
mum) from various Pinus spp. 
80-90% TBC (iX-, ß-pinene, limonene, phel-
landrene). Autoxidation produces iX-pinene 
peroxides and subsequently verbenol and pinol 
hydrate ( = sorbenol). 
2-10% ess. oil. 
Cinnamic aldehyde, cuminaldehyde, eugenol, 
m-cresol, sesquiterpenes. 
25-40% Ethanol-soluble resin fraction, con-
taining diterpene acids, e.g. iX-, ß- and y-com-
miphoric acids and esters. 
Main constituents are cinnamic acid, ferulic acid and coniferyl a1cohol and their esters. 
27 Benzoe tonkinensis 
Siam benzoin 
Styrax tonkinensis (PIERRE) CRAIB 
exHARTWICH 
Styracaceae 
USP XX, Ph. Eur. III, ÖAB. 
Helv. VI 
Benzoe Sumatra 
Sumatra benzoin 
Styrax benzoin DRYANDER 
Styracaceae 
At least 25% free or combined acids, deter-
mined as benzoic acid (Ph. Eur. III). 
Coniferyl benzoate (60-80%), cinnamoyl ben-
zoate (ca. 2%), benzoic acid (10-20%), vanil-
lin (ca. 0.3%), iX-siaresinolic acid (19-hydro-
xyoleanolic acid). 
Coniferyl cinnamate and coniferyl benzoate 
(70-80%), cinnamic acid esters, styracin, cin-
namic acid (ca. 10%), cinnamic acid phenyl-
propyl ester (ca. 1 %), vanillin (ca. 1 %), su-
maresinol (6-hydroxyoleanolic acid). 
Fig. 
28 
Drug/Plantsource/Family/Phar-
macopoeia 
Tolutanum Balsamum 
Tolu balsam 
Myroxylon balsamum (L.) HARMS 
var. balsamum 
Fabaceae 
USP XX (tinct.), He1v. VI, ÖAB 
Peruvianum Balsamum 
Peru balsam 
Myroxylon balsamum (L.) HARMS 
var. pereira (ROYLE) HARMS 
Fabaceae 
DAB 8, Helv. VI, ÖAB, 2. AB-
DDR 
Content of essential oil 
Main constituents 
ca. 7.5% "cinnamein ", a mixture of benzoyl 
benzoate and cinnamoyl benzoate (2: 1); ca. 
80% resin (mostly cinnamic esters of toluresi-
tannol), cinnamic acid, benzoic acid, vanillin, 
eugenol. 
At least 50-70% "cinnamein" (DAB 8), con-
sisting ofbenzoyl benzoate (25-40%) and cin-
namoyl benzoate (10-25%) in ratio 2.8:1, 
sometimes other proportions in the range 2: 1 
to 4: 1. 
20-28% resin (mostly cinnamic esters ofpere-
sitannol), cinnamic acid (ca. 10%), benzoic 
acid, dihydrobenzoic acid and 0(- and ß-neroli-
dol (3-5%). 
17 
v. Formulae of Constituents of Essential Oils 
Cl-Pinene ß-Pinene 
Geraniol 
(trans) 
Carene Cl-Phellandrene Limonene Cl-Caryophyllene 
Nerol 
(cis) 
Carveol Terpinen-4-ol Linalool 
ß-Caryophyllene 
Cl-Terpineol: R = H 
Terpinylacetate: 
R=CHaCO 
tr°H &OCOCHJ ~OCOCHJ cD ~OH ~OH 
Borneol Bornylacetate Linalylacetate 1,8-Cineole Thymol Carvacrol 
(Eucalyptol) 
I ~O I 5!0 ctr (it ~90 S:90 ~O I 'H I 'H 
Citral Citronellal Carvone Piperitone Thujone ( + )-Fenchone Camphor 
O~ 
o 
Turmerone Xanthorrh izol Cl-Santonin 
19 
20 
OR 
~ ~ C~~ COOH 
Cinnamic 
aldehyde 
trans-
Cinnamic 
acid 
Eugenol: R = H trans-Anethole Methyl- An isaldehyde Safrol 
Aceteugenol: chavicol 
R=CH3 CO 
Apiol Myristicin Allyltetramethoxybenzene Elemicin 
Benzyl benzoate 
OCH3 
Cinnamein Ö rA;O OH 
~o~ ~O~ o Coniferyl benzoate 
o 
Benzyl cinnamate 
R, R2 
Isoeugenol methyl ether - CH = CH - CH3 (trans)H 
y-Asarone - CH2 - CH = CH 2 OCH 3 
cis-Asarone (ß-Asarone) - CH = CH - CH3 (cis) OCH3 
trans-Asarone (cx-Asarone) - CH = CH - CH3 (trans)OCH3 
Acaramone - CH2 - CO - CH3 OCH3 
Asarylaldehyde - CHO OCH3 
HO 
D(-)-Menthol (-)-Menthone Menthofuran 
eH3 
OCOCH, ~ 
eH3 rH2 
eH3 
Jasmone 
Proazulene 
(Matricin) 
Chamazulene cis(trans)-ene-ine-Dicycloether 
(l---bl 
/V O><'OH O? 
OH 
(-)-a-Bisabolol (-)-a-Bisabolol oxide A (-)-a-Bisabolol oxide B (-)-a-Bisabolone oxide A 
21 
TLC Synopsis 
Terpene and Phenylpropane Reference Compounds 
Fig.l 
Fig.2 
Fig.1 
Fig.2 
Solvent 
system 
Detection 
22 
Compounds applied in order of increasing Rf-value and decreasing polarity 
Monoterpene alcohols and their esters 
Track Reference compound Rf-value Colour 
1= borneol 0.24 violet-blue 
2=linalool 0.30 blue 
3 = piperitone 0.35 orange 
4=cineole 0.40 blue 
5=citral 0.42 blue-violet 
6=carvone 0.46 red-violet 
7=eugenol 0.47 yellow-brown 
8=thymol 0.52 red-violet 
9= citronellal 0.65 blue 
10=apiol 0.65 brown-red 
11 = myristicin (Macis aeth.) 0.75 red-brown 
12 = anethole 0.85 red-brown 
13= safrol 0.87 red-brown 
14 = geraniol 0.25 grey-blue 
15 = geranyl acetate 0.73 grey-blue 
16=nerol 0.26 grey-blue 
17 = neryl acetate 0.74 grey-blue 
18=linalool (~2/Fig. 1) 0.36 grey-blue 
19=1inalyl acetate 0.75 grey-blue 
20 = trans-sabinene hydrate 0.25 violet 
21 = terpineol 0.25 blue-violet 
22 = phytol 0.35 violet 
23 = farnesol (impure ) 0.30 blue 
A-1: toluene-ethyl acetate (93: 7) 
Vanillin-sulphuric acid (VS No. 38, p. 304); vis. 
The reference compounds can be divided into four main groups on the basis of 
their characteristic colour reactions: 
brown-red/violet phenylpropane derivatives: safrol, anethole, 
myristicin, apiol and eugenol. 
orange to red-violet 
blue / blue-violet 
grey-blue 
carvone, thymol, piperitone. 
citral, citronellal, cineole. 
most monoterpene alcohols and their esters 
(geraniol, nerol, linalool, borneol; cf. menthol, 
menthyl acetate, Fig. 15, p. 37). 
Remarks: Commercially available reference substances often show weak extra zones at lower 
Rf-values and sometimes also at higher Rf-values. These are partly due to resinification, decom-
position products and incompletely removed impurities. 
Fig.l 
'--____ 4 ___ 7 11 
Tl-13 
Fig.2 
T 14 - 23 
-FRONT 
Rf 
-0.5 
-START 
-FRONT 
Rf 
-0.5 
-START 
23 
Cinnamomi Aetheroleum, Calami Aetheroleum 
Tracks 
Tests 
1 = Cinnamomi ceylanici aeth. (Ceylon cinnamon oil) 
2 = Cinnamomi aromatici aeth. (Cassia cinnamon oil) 
3,4= Cinnamomi aeth. commercial oils I, II 
5 -11 = Calami aeth. (various sourees) 
Tl = cinnamic aldehyde 
T2 = coumarin 
T3=asarone 
T4=eugenol 
Solvent A-1 : toluene-ethyl acetate (93: 7) Fig. 3A; 4A 
Fig.4B system toluene-ethyl acetate (97: 3) modified 
A-4: dichloromethane Fig. 3B; 3C 
Detection Vanillin-sulphuric acid (VS No. 38, p. 304) 
Potassium hydroxide (KOH No. 21, p. 302) 
vis. Fig. 3A, C; 4A, B 
UV-365 nm Fig.3B 
For description of drugs see p. 9. Formulae p. 20. 
Chromatogram 
3 Cinnamomi aetherolea. The grey-brown zone of cinnamic aldehyde at Rf 0.5, ob-
tained after treatment with VS-reagent (cf. Tl), characterizes the TLC picture of 
the cinnamic oils 1-4. 
3 A 1 Ceylon cinnamon oil shows an additional violet-red zone directiy above the cinnamic 
aldehyde. In the region of the terpene alcohols (RfO.15-0.3) are four weak zones 
(linalool Rf ca. 0.3), and the THC (a-pinene, caryophyllene) run with the solvent 
front. 
3 A 2 Cassia cinnamon oil. In the visible this differs from official oil of cinnamon only 
with respect to the secondary zones. A dear differentiation is achieved (DCM-
extract, p. 6) in UV-365 nm after treatment with KOH-reagent. The strongly blue 
fluorescent zone of unsubstituted coumarin (cf. T2) appears at RfO.4. Only traces 
of this coumarin are detectable in Ceylon cinnamon oil. 
4 5-11 Calami aeth. After treatment with VS-reagent, chromatograms of Sweet Flag oil 
24 
are characterized by aseries of violet, blue and brown-violet zones extending from 
Rf 0.1 to the solvent front. Asarone appears at Rf ca. 0.4 (cf. T3) as a red-violet 
zone. The concentration varies greatly, depending on the origin and chemical race 
of the drug. The oils in tracks 5 and 6 are from asarone-poor or asarone-free diploid 
races of American origin. 
The oils in tracks 7-11 have relatively high asarone contents and are derived 
from triploid or tetraploid races. 
At the Rf of the reference substance, eugenol (T4), all the oils show only a 
weak zone. Above this are 3-4 blue to blue-violet zones, due to various concentra-
tions of sesquiterpenes. The lower Rf region contains e.g. asaraldehyde. 
Fig.3 
Tl 2 T2 2 
A B 
Fig.4 
T3 5 6 7 T4 8 9 
3 4 Tl 
10 11 T3 
-FRONT 
Rf 
-0.5 
-START 
-FRONT 
Rf 
-0.5 
-START 
25 
Anisi, Foeniculi, Basilici, Sassafras Aetherolea, Anisi Fructus 
Essential oils with anethole, methylchavicol or safrol as the main constituents 
Tracks 
Tests 
Solvent 
system 
Detection 
5 = Basilici aeth. 
6 = Sassafras acth. 
1 = Anisi aeth. (Offic. anise) 
2=Anisi stellati aeth. (Star anise) 
3 = F oeniculi aeth. (Bitter fennel) 
4 = Foeniculi aeth. (Sweet fennei) 
7= Anisi stellati fructus (DCM-extract) 
8 = Anisi fructus (DCM -extract) 
T1 = anethole T3 = eugenol 
T2 = safrol T 4 = fenchone 
A-l : toluene-ethyl acetate (93: 7) 
A-2: toluene 
Vanillin-sulphuric acid reag. (VS No. 38, p. 304) 
Phosphomolybdic acid reag. (PMA No. 27, p. 303) 
PMA-potassium permanganate reag. (PMA-PM No. 22, 
p.302) 
conc. Sulphuric acid (H 2S04 No. 34C, p. 303) 
For description of drugs see p. 9-10. Formulae p. 20. 
Fig. 5; 6A, B 
Fig.6C 
VlS. Fig. 5A, B 
vis. Fig.6C 
VlS Fig.6B 
vis. Fig.6A 
Chromatogram 
5 A Anisi and Foeniculi aetherolea. In oils 1-4, treatment with VS-reagent reveals the 
characteristic,main red-brown zone (Rf ca. 0.95) of the anethole-methy1chavicol 
mixture (cf. Tl). 
1 Offic. anise oU shows practically no terpenoid zones. 
2 Star anise oil shows an additional 5-6 blue-violet zones of low intensity in the Rf 
range 0.2-0.6. 
6C 7 A DCM-extract of Star anise fruits developed in toluene according to Ph. Eur. III. 
After treatment with PMA reagent, a weak blue zone (er T2) immediately above 
the anethole zone (er Tl) can be seen. The triglycerides present in the seeds and 
fruits show as a blue zone at Rf ca. 0.3. 
6A, B 3,4 Fennel oil also contains fenchone (er T4, bitter fennel 12-22%, sweet fennel 
0.4-0.8%) in addition to anethole (cf. Tl). 
Fenchone cannot be detected with VS-reagent. In quantities greater than lOOllg 
it gives an ochre-yellow zone with conc. H 2 S04 . At lower concentrations the PMA-
PM reagent gives a more reliable detection. 
In bitter fennel (3) fenchone is c1early seen as a dark blue zone; the traces 
present in sweet fennel (4) are recognizable as a whitish zone. Fenchone is not 
detectable in Anise oil (1). 
Anise and fennel oils contain anisaldehyde, which can be detected in the Rf 
region below fenchone (violet-red zone with conc. H 2S04 , or a whitish zone with 
PMA-PM, both in the vis.). Anisaldehyde shows a prominent fluorescence quenching 
when observed directly in UV-254 nm. 
5 A 5 Basilici aeth. Basil oil shows methylchavicol as a red-violet zone, and three intense 
blue zones in the Rf range 0.2-0.5. The zone at Rf ca. 0.3 corresponds to linalool. 
5 B 6 Sassafras aeth. Sassafras wood oil is characterized by the zone of safrol at Rf ca. 
26 
0.95 (er T2). There are two weaker zones in the Rf region of the standard eugenol 
(er T3), and two stronger zones in the lower Rf range (terpene a1cohols). 
A 
Fig.5 
Tl 2 3 4 5 
A c 
Fig.6 
3 T4 T4 3 4 
B 
T2 6 T3 
7 8 Tl 
-FRONT 
Rf 
-0.5 
- START 
-FRONT 
Rf 
-0.5 
-START 
27 
Petroselini, Myristicae, Caryophylli Aetherolea 
Tracks 
Tests 
1-5 = Petroselini aeth. (P. fructus, steam distillate) 
6 = Myristicae aeth. (M. semen, steam distillate) 
7 = Macis aeth. 
8= Caryophylli aeth. (e. flos, steam distillate) 
Tl =eugenol 
T2=apiol 
T3 = allyltetramethoxybenzene 
T4 = aceteugenol 
Solvent A-l : toluene-ethyl acetate (93: 7) Fig. 7 A, B; 8A, B 
Fig.8C system A-2: toluene 
Detection Vanillin-sulphuric acid-reag. (VS No. 38, p. 304) vis. Fig. 7-8 
For description of drugs see p. 10. Formulae p. 20. 
Chromatogram 
7 A 1-5 Petroselini aeth. After treatment with VS-reagent, oils 2-5 show the two main brown-
violet zones of apiol (T2, Rf ca. 0.75) and myristicin (Rf ca. 0.8) in the upper 
Rf range, together with weaker violet-brown zones of eugenol (Tl) or eugenol methyl-
ether and allyltetramethoxybenzene (cf. T3) in the intermediate Rf range. Oi11 is 
apiol-free. 
7 B When applied in fairly small quantities (2 111), myristicin and apio! (cf. T2) show 
decreased Rf values. 
There are "chemical races" of Petroselini fructus, in which the predominant constituent of 
the fruit oil is myristicin (= oil 1) or apiol (= oil 2) or more rarely allyltetramethoxybenzene. 
Most oils (tracks 3, 4, 5) contain approximately equal concentrations of the two main constitu-
ents. Allyltetramethoxybenzene and eugenol are present in lower concentrations. Occasionally, 
as in oil 4, safrol can be detected directly above myristicin. 
8 A 6, 7 Myristicae aeth., Macis aeth. Both oils give similar chromatograms after treatment 
with VS-reagent; there are about 8 predominantly brown to brown-vio!et zones 
in the Rf range 0.2 to the solvent front. 
The main zone of myristicin (Rf ca. 0.8) is more prominent in mace oil. Safrol 
(Rf ca. 0.9), like eugenol (cf. Tl), is present only as a weak brown zone. 
At low concentrations, terpene a1cohols (linalool, geraniol and terpineol) can be detected 
as weak violet-brown zones in the Rfrange 0.25--0.4. The THC, pinene, limonene and p-cymene, 
mi grate as a single violet-brown, unresolved zone at the solvent front. 
8 B 8 Caryophylli aeth. Treatment with VS-reagent reveals (in the vis.) the main orange-
brown zone of eugenol (cf. Tl), and the strong red-violet zone ofthe THC (humulene 
and caryophyllene) at the solvent front. 
8 C A satisfactory separation of eugenol (cf. Tl) and aceteugenol (cf. T 4) can only 
be achieved with the solvent system toluene. Epoxydihydrocaryophyllene is seen 
as a red-violet zone directly below aceteugenol. 
28 
A 
Fig.7 
Tl 2 3 4 5 
A 
Fig.8 
6 7 Tl 8 T4 
B 
T2 5 TJ 
c 
Tl 8 T4 
-FRONT 
Rf 
-0.5 
~START 
FRONT 
Rf 
-0.5 
-START 
29 
Carvi, Coriandri, Cardamomi, J uniperi, Rosmarini Aetherolea 
Tracks 
Tests 
1 = Carvi aeth. (C. fructus, steam distillate) 
2 = Carvi aeth. (commercial oil) 
3=Coriandri aeth. (C. fructus, steam distillate) 
4 = Coriandri aeth. (e. semen, steam distillate) 
5 = Cardamomi aeth. (C. fructus, steam distillate) 
6 = J uniperi aeth. (J. fructus, steam distillate) 
7 = Rosmarini aeth. (official rosemary oil) 
8= Rosmarini aeth. (oil from R. hispidus) 
T1 =carvone 
T2 = linalool 
T3=cineole 
T4=borneol 
T5 = bornyl acetate 
T6 = a-terpineol (Rf ca. 0.25) and 
a-terpinyl acetate (Rf ca. 0.75) 
Solvent A-1: toluene-ethyl acetate (93: 7) Fig. 9A-C; lOB, C 
Fig.lOA system A-4: dichloromethane, DAB 8 
Detection Vanillin-sulphuric acid (VS No. 38, p. 304) 
Anisaldehyde-sulphuric acid (AS No. 2, p. 299) 
VlS. Fig.9A-C 
vis. Fig. lOA-C 
F or description of drugs see p. 10-11. F ormulae p. 19. 
Chromatogram 
9A 1,2 Carvi aeth. Caraway oils are characterized by the intense, raspberry-red zone of 
carvone (T1) at Rf ca. 0.5. The terpene a1cohols, carveol and perillyl alcohol, migrate 
together and are seen as a weakly defined, blue zone at Rf ca. 0.2. 
9B 3,4 Coriandri aeth. The characteristic main zone of the oil from coriander fruits or 
9C 
10A, B 
30 
seeds is due to linalool (T2; Rf ca. 0.35) 
Seed oils have a markedly higher linalool content. In addition, geraniol (Rf ca. 0.2) and 
geranyl acetate (Rf ca. 0.7) are detectable as grey-blue zones. 
5 Cardamomi aeth. Cardamom oils produce 5-6 strong blue zones: a.-terpinyl acetate 
(cf. T6; Rf ca. 0.75), cineole (cf. T3; Rf ca. 0.5) and less intense zones of the 
terpene a1cohols, linalool (Rf ca. 0.35), borneol and terpineol (cf. T6; Rf 0.2-0.25). 
The THC, limonene, appears as a weak violet zone at the solvent front. 
6 Juniperi aeth. In solvent systems A-1 or A-4 (DAB 8) juniper oil exhibits about 6 
red to red-violet zones in the Rf range 0.2-0.5, with terpinene-4-o1 (ab out the same 
Rf as standard cineoie ; cf. T3) and a striking red zone direcdy above it, presumably 
due to epoxydihydrocaryophyllene. 
Some of the zones in the lower Rf range are degradation products of THC that were initially 
present in the oi!. 
7, 8 Rosmarini aeth. Rosemary oil yields at least 9 red-violet or blue-violet zones over 
practically the whole range of Rf values. Offiäal rosemary oi! (7) has a low content 
of cineole (cf. T3), and increased levels of terpene a1cohols (borneal: Rf ca. 0.25; 
cf. T4); there is a litde bornyl acetate (Rf ca. 0.7; cf. T5) and the THC (a- and 
ß-pinene) mi grate with the solvent front. 
The commercial oil (8) shows the characteristic main zones in the intermediate Rf range 
0.35-0.5, with a predominance of cineole (Rf ca. 0.45; cf. T3) and a low proportion of terpene 
alcohols (Rf range 0.2-0.3), terpene esters (Rf ca. 0.7) and THC (solvent front). 
Remarks: Solvent system A-1 and A-4 (DAB 8) show practically the same separation properties 
for Juniperi and Rosmarini aeth. 
A B 
Fig.9 
Tl 2 3 
A B c 
Fig.10 
6 T3 6 T3 
c 
T2 4 T3 5 
T4 T3 7 8 
T6 
T5 
-FRONT 
Rf 
-0.5 
-START 
-FRONT 
Rf 
-0.5 
-START 
31Matricariae (Chamomillae), Anthemidis Aetherolea 
Tracks 
Tests 
1-13 = Matricariae aeth. (Matricariae flos, various origins, steam distillate) 
14= Anthemidis aeth. (steam distillate) 
15 = Matricariae flos (DCM-extract) 
16= Matricariae aeth. (steam distillate) 
Tl = bisabolol oxide A (Rf ca. 0.2); bisabolol (Rf ca. 0.35) 
T2 = linalool 
T3 = bisabolol oxide A 
T4 = bisabolol oxide A and B (Rf ca. 0.1--0.2); bisabolol (Rf ca. 0.35); 
chamazulene (Rf ca. 0.85) 
T5 = borneol (Rf ca. 0.2); bornyl acetate (Rf ca. 0.55) (Ph. Eur.) 
T6 = linalool (Rf ca. 0.25); linalyl acetate (Rf ca. 0.55) 
Solvent A-1 : toluene-ethyl acetate (93: 7) Fig. 11 A, B; 12A, B 
Fig.12C system A-7:chloroform-benzene (75:25) 
Detection Vanillin-sulphuric acid-reag. (VS No. 28, p. 303) 
Anisaldehyde-sulphuric acid-reag. (AS No. 2, p. 299) 
VIS. Fig. 11A, B; 12A, B 
vis. Fig. 12 C 
For description of drugs see p. 11. Formulae 21. 
Chromatogram 
11j12 Matricariae aeth. After treatment with VS-reagent, oils obtained by distillation and 
DCM-extracts are characterized by the following main zones: 
Zone Rf Colour Assignment 
I ca. 0.2 yellow-green bisabolol oxide AlB 
II ca. 0.25 violet terpene alcohol 
III ca. 0.35 violet bisabolol 
IV ca. 0.5-0.6 brown cis I trans-ene-ine-dicycloether 
V ca. 0.95 red-violet azulene (non present in DCM-extract) 
VI ca. 0.99 blue-violet farnesene 
Compounds I-IV lie in the Rf range between borneoljlinalool and bornyl acetatej 
linalyl acetate (cf. T5jT6). 
11 B 14 Anthemidis aeth. shows none of the compounds I-V. The oil is characterized by 
the ester zone in the upper Rf range. 
11 1-13 Oil distillates of commercial chamomile flowers. 
1,6 Azulene-containing oils of official quality show relatively high contents of THC, azulene, 
polyines and bisabolol oxides. 
2-5,7,13 Azulene-poor or azulene-free oils ofEgyptian, Bulgarian or Jugoslavian origin. The characteris-
tic oil constituents are present in very low concentrations. Oil 13 shows a high polyine content. 
8, 9 Polyine-poor oils with relatively high contents of azulene and bisabolol oxides. 
10,11,12 Oils with medium concentrations of azulene and variable contents of bisabolol. 
12AjB 15 Dichloromethane extracts of commercial chamomile flowers 
32 
The chromatogram of a DCM-extract (15) differs from that of a steam distillate (16) primarily 
by the absence of azulene; DCM only extracts proazulene (=matricin 1), which is located 
above the start. 
With slight shifts of Rf values in the lower range, solvent systems A-l and A-7 (Ph. Eur.) 
give the same sequence of zones. VS-reag. produces red-violet and brown, while matricin 1 
in particular produces a red-violet zone with AS-reagent. 
1 Matricin can be specifically detected with EP-reagent (No. 15, p. 301). 
A 
Fig.11 
A 
Fig.12 
VI 
V 
IV { 
ttI 
11 
Tl 
15 
1-13 
B c 
T3 T4 16 15 
B 
14 T2 
T5 T6 16 
Ph .Eur.III 
-FRONT 
Rf 
-0.5 
-START 
-FRONT 
Rf 
-0.5 
-START 
33 
Lavandulae Aetherolea. Cinae Flos 
Tracks 
Tests 
1 = Lavandulae aeth. (Lavandin oil) 
2 = Lavandulae aeth. (Barreme oil) 
3 = Lavandulae aeth. (Mont Blanc oil) 
4 = Lavandulae aeth. (oil of spike; L. latifolia) 
5 = Lavandulae aeth. (L. angustif., steam distillate) 
6 = Citri aurantii aeth. (oil of petit grain) 
7 = Cinae fios (DCM extract) 
Tl = mixture of linalool (Rf ca. 0.35) and linalyl acetate (Rf ca. 0.7) 
T2=cineole 
T3 = o:-santonin 
Solvent A-l : toluene-ethyl acetate (93: 7) Fig. 13A, B; 14A, B 
Fig.14C system A-2: dichloromethane 
Detection Vanillin-sulphuric acid-reag. (VS No. 38, p. 304) 
Phosphomolybdic acid-reag. (PMA No. 27, p. 303) 
VlS. Fig. 13A, B; 14A 
vis. Fig. 14B, C 
For description of drugs see p. 12. Formulae p. 19. 
Chromatogram 
13A Lavandulae aeth. After treatment with VS-reagent, chromatograms of the various 
lavender oils (tracks 1-5) show intense blue zones of linalyl acetate (cf. Tl; Rf 
ca. 0.7) and linalool (cf. Tl; Rf ca. 0.35), the striking, red-violet zone of epoxydihydro-
caryophyllene (Rf ca. 0.5, directly above the blue zone of cineole), and weak zones 
of terpene alcohols (e.g. geraniol, borneol and nerol) between the start and Rf ca. 
0.3. 
13B 
14A, B 
14C 
34 
1 Lavandin oil (commercial) contains all the chief terpenes in approximately equal concentrations, 
with a slight predominance of linalool. 
2 Freneh oil (Barreme) (commercial) is very similar to lavandin oil, with high concentrations 
of linalool and linalyl acetate. 
3 Freneh oil (Mont Blane) (commercial) contains about the same concentrations of linalool 
and linalyl acetate as oill and 2, but only traces of cineole and epoxydihydrocaryophyllene. 
4 Spike oil (commercial) contains high concentrations of cineole and linalool, and is free of 
linalyl acetate. 
5 The steam distillate of official lavender jlowers is similar to lavandin oil with respect to the 
concentrations of the terpene alcohols, linalool, geraniol and borneol, but with a somewhat 
lower concentration of linalyl acetate. Only traces of cineole are present. 
6 Petit grain oil contains only linalyl acetate (up to 90% of the essential oil) and a low concentra-
tion of a terpene alcohol at Rf ca. 0.2. This oil is regarded as an adulterant. 
Remarks: The blue zones produced with VS-reagent fade or change to green-blue 
or brown with time. Borneol is then seen as a prominent brown zone. 
7 Cinae jlos. After treatment with VS-reagent, the main zones of cineole (cf. T2; 
Rf ca. 0.45) and et.-santonin (cf. T3; Rf ca. 0.1) are stained blue and grey (vis.), 
respectively. With PMA-reagent, they appear as dark blue, main zones. 
Dichloromethane (solvent system A-2) gives a better separation of IX-santonin (Rf 
ca. 0.4). 
A 
Fig.13 
Tl 2 3 4 5 6 
Fig.14 
7 T2 T3 7 Tl T3 
B 
T2 2 
T2 7 
3 
T3 
-FRONT 
Rf 
NT 
-START 
35 
Menthae Aetherolea 
Tracks 
Solvent 
system 
Detection 
1 = Menthae piperitae aeth. (steam distillate) 
2 = Menthae arvensis aeth. (commercial oil) 
3= Menthae crispae aeth. (steam distillate) 
Tests Tl = menthol 
4 = Carvi aeth. (commercialoil) 
5 = Menthae aeth. (commercial oil, batch I) 
6 = Menthae aeth. (commercial oil, batch 11) 
A-l : toluene-ethyl acetate (93: 7) 
A -4: dichloromethane 
Vanillin-sulphuric acid-reag. (VS No. 38, p. 304) 
Anisaldehyde-sulphuric acid-reag. (AS No. 2, p. 299) 
Phosphomolybdic acid-reag. (PMA No. 27, p. 303) 
For description of drugs see p. 12. Formulae p. 21. 
T2 = menthone/isomenthone 
T3 = menthyl acetate 
T4 = menthofuran 
Fig. lSA, B, C; 16C 
Fig. 16A, B 
VlS. Fig. lSA, C; 16C 
vis. Fig. 16A 
VlS. Fig. lSB; 16B 
Chromatogram 
15 A 1 Menthae piperitae aeth. After treatment with VS-reagent, the TLC of official pepper-
mint oil is characterized by the following terpenes: 
No. Rf value Terpene Colour 
I 0.3 menthol blue 
11 0.35 piperitone orange 
III 0.4 cineole bIue 
IV 0.48 not identified blue 
V 0.55 isomenthone blue-green 
VI 0.70 menthone blue-green 
VII 0.75 menthyl acetate blue 
15B,16B After treatment with PMA-reagent, all zones are blue-black on a yellow-green back-
ground. Menthyl acetate, menthone, isomenthone and THC are more prominently 
stained than after treatment with VS-reagent. 
16A 2 Menthae arvensis aeth. With respect to the chief constituents, Menthae arv. aeth. 
and Menthae pip. aeth show only quantitative differences. In corn mint oil, menthyl 
acetate and menthol are present in fairly high concentrations. In contrast to freshly 
distilled peppermint oil, however, corn mint oil contains no menthofuran (cf. T4). 
The zones of THC and menthofuran have similar Rf values, and they frequently 
overlap. 
Remarks: Separation with dichloromethane (Ph. Eur. III) and detection with AS-reagent give 
a similar chromatographicpicture to that obtained with solvent system A-l and detection 
with VS. The Rf values of menthyl acetate, menthone/isomenthone are shifted slightly to 
lower values. Menthone and isomenthone appear yellow-brown in the visible. The advantage 
of the AS-reagent is that it permits the detection of some terpenes under UV -365 nm: menthol 
gives a red fluorescence, cineole green-brown, menthyl acetate blue-red, menthone yellow-
brown. 
15C 3 Menthae crispae aeth. This oil is characterized by the main red-violet zone due to carvone 
at Rf ca. 0.5 (cf. Carvi aeth. (4)), and the dark blue zones of dihydrocumin alcohol (Rf ca. 
0.25) and dehydrocarveol acetate (Rf ca. 0.7). 
16C 5,6 In addition to the blue zone of menthol (Rf ca. 0.3), the two commercial oils contain little 
36 
menthone and menthyl acetate (Rf 0.65-0.75). Piperitone is c1early seen as an orange zone, 
while epoxydihydrocaryophyllene or pule gone (Rf region 0.5) give distinct brown-red zones. 
This type of terpene composition indicates an adulteration with the oil of Pulegii herba. 
A 
Fig.15 
T1 
A 
Fig.16 
2 
VII 
VI 
V 
IV 
T2 T3 Tl 
• 
T3 T4 2 
c 
T2 T3 3 4 
C 
T3 T4 5 6 
-FRONT 
Rf 
-0.5 
-START 
-FRONT 
Rf 
-START 
37 
Salviae Aetherolea, Eucalypti Aetheroleum 
Tracks 
Tests 
Solvent 
system 
1, 2 = Salviae aeth. (Dalmatian oil 1/11) 
3 = Salviae aeth. (Greek oil, DAB 8) 
4 = Salviae aeth. (commercial oil of sage) 
5 = Salviae aeth. (Spanish oil) 
6, 7 = Salviae aeth. (Greek oil 1/11) 
8= Eucalypti aeth. 
Tl = rx-, ß-thujone « - )-thujone/( + )-isothujone 35%: 65%) 
T2=cineole 
A-1 : toluene-ethyl acetate (93: 7) 
Detection Vanillin-sulphuric acid-reag. (VS No. 38, p. 304) VIS. Fig. 17 A, C; 18C 
VIS. Fig. 17B, D; 18A, B Phosphomolybdic acid-reag. (PMA No. 27, p. 303) 
For description of drugs see p. 13. Formulae p. 19. 
Chromatogram 
17 A, C 1-7 Salviae aetherolea. After treatment with VS-reagent, the TLC of sage oils show 
a main blue (vis.) zone at Rf ca. 0.5 due to cineole (cf. T2), 2-3 blue to blue-violet 
zones of terpene alcohols in the Rf range 0.2-0.35 (borneol Rf ca. 0.2), and a red-
violet zone of epoxydihydrocaryophyllene directly above cineoie. This is followed 
by the thujone-isothujone mixt ure (= a, cf. Tl) and immediately above that hornyl 
acetate (= b) at Rf 0.6-0.7. The TBC form a strong violet zone at the solvent 
front. 
17 B, D; 18 A After treatment with PMA reagent the thujone mixture is seen as a strong blue-black 
zone. All other terpenoids give a uniform blue-black colouration. 
18B, C 
38 
1-7 Differentiation ofvarious sage oils 
The oils can be classified according to their contents of thujone, cineole and bornyl acetate. 
1,2 Dalmatian sage oil is characterized, after treatment with PMA-reagent, by a prominent thujone 
zone and a relatively weak zone of cineole, and two terpene alcohols in the Rf range 0.2-0.3. 
3 Greek oils show cineole as the main zone, together with a little epoxydihydrocaryophyllene, 
bornyl acetate (= b) and three terpene alcohols in the Rf range 0.2-0.4. After treatment with 
PMA-reagent, thujone ( = a) can only be detected in oil 6 and not in oil 7. 
5 Spanish oil can be differentiated from Greek oil by its low content of cineole, and from 
Dalmatian oil by the absence of thujone. The zone of bornyl acetate (= b) and four zones 
in the Rf range of the terpene alcohols are prominent. 
4 Many commercial sage oils or drugs cannot be unequivocally assigned to one species of sage. 
Thujone and cineole are present in approximately equal concentrations. 
8 Eucalypti aeth. is characterized by one main zone due to cineole. In addition there 
are only two weak blue zones in the region of the terpene alcohols (Rf ca. 0.2-0.3), 
two more weak blue zones in the ester region (Rf 0.6-0.7), and THC at the solvent 
front. Thujone is absent. 
A B c 
Fig.17 
Tl 2 2 3 
A 
Fig.18 
Tl T2 4 5 6 
D 
3 Tl T2 
B c 
7 8 8 
FRONT 
Rf 
-START 
-FRONT 
Rf 
-0.5 
-START 
39 
Thymi, Serpylli, Ajowani Aetherolea Fig.19 
Tracks 1, 2 = Thymi aeth. (Thymi herba, steam distillate) 
4,5, 7 = Thymi aeth. (official commercial oils from various sources) 
3,8= Serpylli aeth. (Serpylli herba, steam distillate) 
6 = Ajowani aeth. (Ajowani fructus, steam distillate) 
Tests Tl = carvacrol 
T2=thymol 
Melissae Aetherolea and substitutes Fig.20 
Tracks 9,10,11 = Melissae aeth. (Melissae folium, steam distillate) 
12 = Citronellae aeth. 
Tests T3 = lemon grass oil (for citral reference) 
T 4 = citronellal 
Solvent A-l : toluene-ethyl acetate (93: 7) Fig. 19A, B; 20B 
system A-3: chloroform (DAB 8) Fig.20A 
Detection Vanillin-sulphuric acid-reag. (VS No. 38, p. 304) VIS. Fig. 19A, B; 20B 
Anisaldehyde-sulphuric acid-reag. (AS No. 2, p. 299) vis. Fig.20A 
For description of drugs see p. 13-14. Formulae p. 19. 
Chromatogram 
19 Thymi aeth. After treatment with VS-reagent, TLC of oils from the Thymus species, 
Th. vulgaris, Th. zygis (1, 2, 4, 7) and Th. serpyllum (3, 8) show a characteristic 
main zone in the Rf range 0.5-0.55, due to a mixture of thymol and carvacrol 
(cf. Tl/T2). 
There are also lower concentrations of the terpene alcohols, borneol, geraniol and linalool 
(grey zones, Rf 0.1-0.3), the terpene esters, bornyl and linalyl acetate (blue zones, Rf 0.55-0.8), 
and THC at the solvent front. 
3, 8 Thymi serpylli aeth. shows one or two additional ester zones at Rf ca. 0.6. 
5 Thymi aeth. (rectified commercial oil) from an unknown species of thyme shows additional 
zones in the Rf ranges 0.3-0.4 and 0.6-0.95, which give the characteristic red reaction of 
thymol derivatives. 
6 Ajowani aeth. The oil from Ajowani fructus contains practically only thymol (cf. T2), with 
small quantities of terpene alcohols. 
Remarks: The thymol-carvacrol mixture is efficiently separated by two-dimensional TLC: 
first dimension A-l, second dimension toluene-carbon tetrachloride-o-nitrotoluene (1 : 1: 1). 
20A 9-11 Melissae aeth. Chromatograms of official melissa aeth., after treatment with VS-
20ß 
40 
or AS-reagent, show a characteristic main blue zone, due to citronellal (cf. T4). 
Also present are the weak grey to blue zones of citral (cf. T3) and the terpene 
alcohols, geraniol, linalool and citronellol in the Rf range 0.2-0.4. 
12 Melissa oil substitutes: 
Ceylon or Java lemon grass oil (12) (Citronellae aeth.) resembles official Melissa oil (10) 
in its high content of citronellal, and its chromatographic picture is also largely similar. 
Rf values in A-3 (DAß 8) are somewhat lower than those in A-l for the same 
substances. 
A 
Fig.19 
Tl 2 3 4 5 
A 
Fig.20 
T3 9 10 11 12 
DAS8 
T2 T2 6 7 
8 
T4 12 10 
8 
T3 
-FRONT 
Rf 
-0.5 
-START 
-FRONT 
Rf 
-0.5 
-START 
41 
Curcumae Aetherolea 
Tracks 
Tests 
Solvent 
system 
Detection 
1 = Curcumae aeth. (e. longae rhizoma, steam distillate) 
2-4 = Curcumae aeth. (e. xanthorrhizae rhizoma, steam distillate) 
5 = Curcumae longae rhizoma (MeOH-extract 1 g/5 ml/5 min, 60° C, p. 6) 
6=Curcumae xanthorrhizae rhizoma (MeOH-extract 1 g/5 ml/5 min, 60° C) 
Tl = thymol 
T2 = curcumin 
T3 = fluorescein 
A-1 : toluene-ethyl acetate (93: 7) 
A-8: chloroform-ethanol-glacial acetic acid (94: 5: 1) 
Vanillin-sulphuric acid-reag. (VS No. 38, p. 304) VlS. 
Fast blue salt reagent + NH 3 vapour (FBS No. 12, p. 301) vis. 
Without chemical treatment UV-365 nm 
For description of drugs see p. 14. Formulae p. 19. 
Fig. 21 A, 22A 
Fig. 21 B, 22B 
Fig.21A 
Fig.22A 
Fig. 21B, 22B 
Chromatogram 
21 A 1-4 Curcumae aeth. VS-reagent gives about 8 red-violet zones in the Rf range 0.3 to 
the solvent front. The zones at Rf ca. 0.8 and ca. 0.95 are especially concentrated. 
Xanthorrhizol and alicyc1ic or aromatic(ar) turmerone show higher Rf values than 
the standard thymol, and sesquiterpene hydrocarhons (e.g. zingiberene) are located 
below the solvent front. 
22A After treatment with FBS re agent, xanthorrhizol, a phenolic sesquiterpene, in particu-
lar gives an intense red. 
21 B; 22B 
42 
Xanthorrhizol is a characteristic constituent of C. xanthorrhiza. Traces are also visible in 
C. longa (1), where, according to the literature, it should be absent. Commercial drugs are 
sometimes mixtures of both types of turmeric rhizome. 
These two drugs are weil distinguished on the basis of their different cinnamoyl 
compounds (methanol extract). The pigment zones appear yellow in the vis., and 
form yellow-white fluorescent zones in UV-365 nm. 
6 Curcuma xanthorrhiza. The main compound is curcumin (cf. T2) at Rf ca. 0.6. De-
methoxycurcumin is present, in lower concentration, direct1y beneath (Rf ca. 0.5) 
the zone of curcumin. 
5 Curcuma longa also contains hisdemethoxycurcumin in the Rf region above the stan-
dard fluorescein (cf. T3). 
Remarks: The colours can be intensified with the boron-oxalic acid reagent (DAB 8) (rubrocur-
cumines). 
A 
Fig.21 
2 3 4 Tl 
A 
Fig.22 
2 3 4 Tl 
5 6 T2 
5 6 T3 
-FRONT 
Rf 
-0.5 
-START 
43 
Aurantii, Citri Aetherolea, Aurantii, Citri Pericarpium 
Tracks 1 = Aurantii pericarpium (steam distillate) 
2 = Aurantii pericarpium (pressed oil; bitter) 
3 = Aurantii pericarpium (pressed oil; sweet) 
4=Citri pericarpium (steam distillate) 
5 = Citri aeth. (pressed oil, DAB 7) 
6 = Citri aeth. (Messina oil) 
7 = Aurantii flos aeth. (Neroli oil) 
8 = Citri var. bergamiae aeth. (bergamot oil) 
9 = Citri var. bergamiae aeth. (petit grain oil) 
lO=Citri peric. (see p. 163) (MeOH-extract) 
11 = Aurantii peric. (flavonoids, see p. 188) 
Test Tl = ci tral 
Solvent A-1 : toluene-ethyl acetate (93: 7) Fig. 23A, B; 24A 
system F-7: ethyl acetate-formic acid-water (67:7:26; upper phase) Fig.24B 
Detection Vanillin-sulphuric acid-reag. (VS No. 38, p. 304) vis. Fig. 23A, B 
Without chemical treatment UV-365 nm Fig.24A 
Natural products-polyethylene glycol reagent 
(NPjPEG No. 28, p. 303) UV-365 nm Fig.24B 
For description of drugs see p. 15. Formulae p. 19. 
Chromatogram 
Aurantii and eitri pericarpium oils 
23A 1,4 Steam distillation oils. After treatment with VS-reagent, the chromatograms ofboth 
oils show at least 10 grey to red-violet zones (terpenes) in the Rf range 0-0.75, 
and a THC zone at the solvent front. 
Aurantii aeth. (1) shows about 6 main zones in the Rf range 0.15-0.4, while lemon 
oil (4) shows four zones at RfO.15, 0.25, 0.4 and 0.6. 
Distilled oils of C. aurantium are considered to be inferior. 
2,3,5,6 Pressed oils. The commercial oils obtained from Aurantii pericarpium (2, 3) and 
Citri peric. (5) by pressing procedures always have a characteristically high terpene 
content. Citrat (cf. Tl) is prominent in oil5. 
24A 5,6 Additional coumarin compounds, e.g. bergamottin (a), geranyl methoxycoumarin (b), 
citropten (c) and a psoralene derivative (d) are characteristic components of lemon 
oils. 
In C. aurantium oils (1-4), the weak blue zones, seen in the Rf region of geranyl 
methoxycoumarin (b), at the start and at the solvent front, originate partly from 
methyl anthranilate and from blue-fluorescing flavonoids, such as sinensetin. 
23 B 7 Neroli oil. The oil can be obtained from fresh orange blossoms by distillation, extrac-
tion, or the process of enfleurage. It contains a high amount of linalyl acetate 
(Rf ca. 0.6), linalool (Rf ca. 0.25), and other a1cohols (terpineol, d-nerolidol, geraniol) 
that migrate directly below linalool. 
8, 9 Petit grain oil and hergamot oil (adulterants of neroli oil). Bergamot oil contains 
lower concentrations of linalyl acetate and linalool than does neroli oil. Linalool 
is complete1y absent from petit grain oil. 
24 B 10, 11 MeO H-extracts of eitri and Aurantii pericarpium 
44 
The two drugs can also be differentiated on the basis of the flavonoid glycosides, 
rutin, eriocitrin, naringin and neohesperidin (see section on Flavonoid drugs p. 188, 
Figs. 17 and 18). 
A 
Fig.23 
2 3 4 5 6 
Fig.24 
2 3 4 5 6 
• 
Tl 7 8 
Tl 10 
9 
11 
-FRONT 
Rf 
- START 
45 
Pi ni Aetherolea, Myrrha 
Tracks 
Tests 
Solvent 
system 
1-4 = Terebinthinae aeth. (commercial) 
5-9= Pi ni aeth. (commercial) 
5 = "Scots pine oil " 
6, 7 =" Spruce oil" 
8 = "Silver fir oil" 
9 = "Siberian spruce oil" 
10= Myrrha 
Tl = terpineol 
T2=cineole 
T3 = bornyl acetate 
A-1 : toluene-ethyl acetate (93: 7) 
Detection Phosphomolybdic acid-reag. (PMA No. 27, p. 303) vis. Fig. 25 A 
Vanillin-sulphuric acid-reag. (VS No. 38, p. 304) 
Anisaldehyde-sulphuric acid-reag. (AS No. 2, p. 299) 
Vanillin-hydrochloric acid-reag. (VHA No. 37, p. 304) 
For description of drugs see p. 15-16. Formulae p. 19. 
vis. Fig. 25B, C; 26B 
VlS. Fig.26A 
vis. Fig.26C 
Chromatogram 
25 A, B 1-4 Terebinthinae aeth. Rectified turpentine oil (1) is characterized by a high content 
of THC (a-, ß-pinene, a-, ß-phellandrene, limonene). With PMA- or VS-reagent, 
these appear as blue or blue-violet zones, respectively, at the solvent front. Terpene 
a1cohols and other additional zones are present only in low concentrations between 
the standards of terpineol and cineole (cf. T1 and T2). 
25 C In stored oils (2-4) the THC content decreases due to autoxidation. The chromato-
gram then shows increasingly blue to red-violet zones in the Rf range 0.1-0.5 (e.g. 
pinene oxides, pinene hydrate, verbenol). 
26A 5-9 Pine oils. This term refers to the essential oils from species of Pinus, Abies and 
Picea; the source plants are rarely stated. After treatment with AS-reagent, the 
chief feature of chromatograms of "Scots pine oil" (5) and "Silver fir oil" (8) 
is a group of zones between the start and Rf ca. 0.45, with a low content of THC 
in the area of the solvent front. In "Spruce oil" (6, 7), the main zones are due 
to THC, with only weak zones on the rest of the chromatogram. Oils 5-8 show 
a marked red zone at Rf ca. 0.4. "Siberian spruce oil" (9) is characterized by high 
contents of bornyl acetate (cf. T3) and terpineol (cf. T1), and a low content of 
THC. 
26B, C 10 Myrrha. The chromatogram ofthe ethanolic resin fraction is characterized by intense 
46 
blue, blue-violet and red zones distributed over the wh oIe area between the start 
and the solvent front (VS- or VSL-reagent). Red zones in the Rf range 0.4 and 
0.6-0.75 (commiphoric acid esters?) are typical. Sesquiterpene hydrocarbons of the 
essential oil fraction are found in the region of the solvent front. 
B c 
Fig.25 
Tl T2 Tl T2 
A 
Fig.26 
Tl T3 5 6 7 8 
2 3 4 
B c 
9 10 10 
-FRONT 
Rf 
-0.5 
-START 
-FRONT 
Rf 
-0.5 
-START 
47 
Resins and Balsams 
Tracks 
Tests 
Solvent 
system 
1 = benzoin (Sumatra benzoin) 
2 = benzoin (Siam benzoin) 
3 = Balsamum tolutanum 
4 = Balsamum peruvianum 
Tl = benzoic acid 
T2=eugenol 
A-l : toluene-ethyl acetate (93: 7) 
Detection Without chemical treatment UV-254 nm Fig. 27 A, 28A 
Anisaldehyde-sulphuric acid-reag. (AS No. 2, p. 299) VlS. Fig. 27B, 28B 
Phosphomolybdic acid-reag. (PMA No. 27, p. 303) VlS. Fig.28C 
Description of benzoins and balsams, p. 16-17. F ormulae p. 20. 
Chromatogram 
27 A 1,2 Benzoins. Visualization in UV-254 nm shows pronounced quenching of fluorescence 
at Rf ca. 0-0.15 (benzoic or cinnamic acid, cf. Tl), Rf ca. 0.25-0.4 (coniferyl benzoate) 
and RfO.7-0.75 (main zone with a small accompanying zone: cinnamoyl cinnamate, 
propyl cinnamate, cinnamoyl benzoate) 
27 B The fluorescence-quenching zones are stained blue to violet in the vis. by treatment 
with AS-reagent. 
1 Sumatl'a benzoin is characterized by about 4 equallyintense zones in UV-254 nm. 
These are benzoic or cinnamic acid in the lower Rf range, coniferyl benzoate at 
an intermediate Rf, and a prominent ester zone (cinnamoyl cinnamate, propyl cinna-
mate) at Rf ca. 0.7. 
2 Siam benzoin pro duces zones primarily at lower and intermediate Rf values. The 
main zone, due to coniferyl benzoate, is at Rf ca. 0.4. Only a very weak zone 
is seen at Rf ca. 0.7. 
28 3,4 Bahams 
48 
3 Tolu balsam. Direct evaluation in UV-254 nm shows the fluorescence-quenching 
zones of benzoyl benzoate and benzoyl cinnamate (" cinnamein " mixture). The ratio 
of the two esters is ca. 1: 2. Above the start are seen the zones of benzoic acid 
(cf. Tl, Fig. 27 A) and cinnamic acid. In the intermediate Rf range are weak zones, 
due in part to eugenol (cf. T2) and vanillin. After treatment with PMA-reagent, 
the fluorescence-quenching zones give a prominent blue in the vis. In addition, 
this reagent shows a zone ofTHC at the solvent front (cf. also AS-reagent, Fig. 28 B). 
4 Peru balsam contains a much higher concentration of the "cinnamein" mixture, 
with different relative proportions of the esters (ratio 2.5: 1). 
Nerolidol, which is especially characteristic of Peru balsam, is made visible at Rf 
ca. 0.3 by treatment with PMA- or AS-reagent. 
Fig.27 
Tl T2 2 
• 
Fig.28 
3 4 T2 3 
T2 2 
4 3 4 
-FRONT 
RI 
-0.5 
-START 
FRONT 
RI 
-0.5 
-START 
49 
Alkaloid Drugs 
Most plant alkaloids are derivatives oftertiary amines, while others contain primary, 
secondary or quaternary nitrogen. The basicity of individual alkaloids varies greatly, 
depending on which of the four types is represented. The pKB-values (dissociation 
constants) lie in the range pR 10--12 for very weak bases (e.g. purines), pR 7-10 
for weak bases (e.g. Cinchona alkaloids) and pR 3-7 for medium strength bases 
(e.g. opium alkaloids). 
I. Preparation of Drug Extracts for TLC 
1. Alkaloid drugs with high and medium alkaloid contents (~ 1 % ) 
Powdered drug (1 g) is mixed thoroughly with 1 ml of 10% ammonia soln. or 10% 
Na2C03 soln., then extracted by shaking for about 5 min with 5 ml methanol at 
60° C (water bath). The filtrate is cooled and concentrated so that 100111 (the maxi-
mal quantity that should be applied to the TLC plate) contains 50--100 Ilg alkaloids 
(see II, 1.) 
2. Alkaloid drugs with low alkaloid contents ( < 1 %) 
a) Enrichment using an aluminium oxide column 
Powdered drug (2 g) is ground in a mortar for about 1 min with 2 ml of 10% 
ammonia soln., then mixed with 7 g basic aluminium oxide (activity stage I). The 
whole mixture is packed loosely into a glass column (1.5 cm diarn., 20 cm long). 
Alkaloid bases are eluted with about 10 ml CRCI3 . The first 5 ml of eluate are 
collected, evaporated to 1 ml, then used for chromatography. 
This method is suitable for, e.g. the Solanaceae drugs, e.g. Belladonnae or Scopo-
liae radix and Stramonii semen. Seed drugs must first be defatted by extraction 
with light petroleum. 
Leaf extracts contain chlorophyll, which can interfere with the TLC separation. 
In such cases the official pharmacopoeia method is recommended (see b). 
b) Enrichment by extraction according to DAß 8 
Powdered drug (1 or 2 g) is shaken for 5 min with 10.0 ml of 0.05 M sulphuric 
acid, then filtered. To the filtrate is added 1.0 ml conc. ammonia soln. The mixture 
is diluted to 10 ml with water, and extracted by shaking with 10 ml peroxide-free 
ether. The ether phase is dried over anhydrous sodium sulphate, filtered, evaporated 
to dryness on the water bath (fume cup board), and the residue dissolved in 0.25 ml 
methanol. 
This is the preferred method for Belladonnae and Stramonii folium (1 gof each) 
and for Ryoscyami folium (2 15). 
11. Sampie Quantities for TLC 
1. Drug extracts 
The sampIe applied to the TLC plate should contain 50--1OOllg total alkaloids. 
The size of the sampIe can be ca1culated from the average alkaloid content of the 
drug. 
51 
52 
Example: powdered drug (1 g) with a total alkaloid content of 0.3%, extracted 
by method 1., will yield 3 mg in 5 ml methanolic soln., containing ca. 60 Ilg total 
alkaloids per 100 111. 
2. Reference substances from proprietary pharmaceuticals (see table) 
Alkaloids can be obtained from pharmaceutical products by extraction with metha-
nol. Since pharmaceutical products contain a wide range of different alkaloid concen-
trations, the method used for the preparation of each test solution depends on 
the alkaloid content. 
The sample applied to the TLC plate should contain 50-100 Ilg alkaloid. 
Alkaloid content 10-250 mg per tablet or dragee: One powdered tablet or dragee 
is mixed with 1 ml methanol per 10 mg alkaloid and shaken for about 5 min at 
60° C. After filtration or centrifugation, the extract is applied directly; 10 111 corre-
spond to 100 Ilg alkaloid. 
Alkaloid content 0.075-1.0 mg per tablet or dragee: Ten powdered tablets or dragees 
are mixed with 5 ml methanol, shaken for about 5 min at 60° C, and the c1ear 
filtrate evaporated to dryness. The residue is dissolved in 1 ml methanol, and, if 
necessary, the solution c1eared by centrifugation. This solution is applied to the 
TLC plate; 10111 contain 100llg alkaloid (1.0 mgjtablet), or 100111 contain 751lg 
alkaloid (0.075 mgjtablet). 
Reference substances from pharmaceutical preparations for the TLC identification 
of alkaloid drugs 
Drug and major 
alkaloids 
Aconiti tuber 
Aconitine 
Chinae cortex 
Quinine 
Quinidine 
Cinchonine 
Cinchonidine 
Caffeine drugs 
Caffeine 
Theobromine 
Theophylline 
Colchici semen 
Co1chicine 
Ipecacuanhae radix 
Cephaeline 
Emetine 
Lobeliae herba 
Lobeline 
Pharmaceutical a 
Aconitysat (drops) 
Chinin Compr. 
Chinidin Compr. 
Sedovegan 
Sedovegan 
Coffein Compr. 
Theo-Miroton 
Theo-Miroton 
Co1chicum Dispert 
Dicton 
Lobelin Amp. 
Drug and chief 
alkaloids 
Opium 
Codeine 
Morphine 
Noscapine (Narcotine) 
Papaverine 
Rauwolfia spp. 
Ajmaline 
Raubasine 
Rescinnamine 
Raupine 
Reserpine 
Secale cornutum 
Ergobasine 
Ergocristine 
Ergotamine 
Solanaceae drugs 
Atropine 
Scopolamine 
Strychni semen 
Strychnine 
Pharmaceutical a 
Codein phos. Compr. 
Noflu 
Papaverin Tab!. 
Gilurytmal 
Triraupin 
Triraupin 
Rauwopur 
Sedaraupin 
Ergotren 
Ergotren 
Gynergen 
Atropin sulf. Compr. 
Boro-Scopol (drops) 
Dysurgal 
Remarks: Some pharmaceutical preparations contain a mixture of different alkaloids, e.g. 
Rauwopur dragees: 1 dragee contains 0.1 mg reserpine-HCI, 0.25 mg rescinnamine, 0.01 mg 
raupine, 0.19 mg ajmaline, 0.6 mg yohimbine. Thus, in reference solutions prepared for the 
detection of raupine, the concentration of yohimbine will be too high. 
a Pharmaceutical preparations on the German drug market. Instead of these, other prepara-
tions, available under other names, with the same constituents can be used as references. 
3. Reference substances 
a) These are usually prepared in 1 % a1coholic soln., and 10111 are applied for TLC, 
e.g. atropine, brucine, strychnine, morphine. 
b) Rauwolfia alkaloids. Reserpine, rescinnamine, rauwolscine, ajmaline and serpentine 
are each prepared in 0.5% a1colholic soln., and 10111 are applied for TLC. 
c) Colchicine is prepared as a 0.5% soln. in 70% ethanol, and 10111 are applied for 
TLC (DAC). 
4. Test mixtures from the pharmacopoeias 
a) Cinchona alkaloid test mixture, Ph. Eur., for the TLC identification of Chinae cortex. 
A mixture of 17.5 mg quinine, 0.5 mg quinidine, 10 mg cinchonine and 10 mg cin-
chonidine is dissolved in 5 ml ethanol, and 5 111 of this soln. are applied for TLC. 
b) Test mixture for Ipecacuanhae radix, Ph. Eur. 
4.6 mg emetine and 5.7 mg cephaeline are dissolved in 20 ml methanol, and 5 111 
of this soln. are applied for TLC. 
c) Test mixture forSolanaceae drugs, Ph. Eur. 
24 mg atropine sulphate dissolved in 9 ml methanol. 7.5 mg scopolamine hydrobro-
mide dissolved in 10 ml methanol. 
For Belladonnaefolium: 1 ml scopolamine soln. is added to 9 ml atropine sulphate 
soln. 20 111 are used for TLC. 
For Hyoscyami folium: 3 ml atropine sulphate soln. mixed with 0.4 ml scopolamine 
hydrobromide soln., and diluted to 10 ml with methanol. 20111 are used for TLC. 
For Stramonii folium: 5 ml atropine sulphate soln. mixed with 3 ml scopolamine 
hydro bromide soln., and diluted to 10 ml with methanol. 20111 are used for TLC. 
5. Adsorbent 
Silica gel 60F2S4 pre-coated TLC plates (Merck, Darmstadt). 
Most of the alkaloids can be separated on silicic acid. 
Aluminium oxide (see Fig. 16B, p. 80) is more suitable for the separation ofberberine, 
columbamine and jateorhizin. 
53 
54 
6. Chromatography solvents 
Solvent system 
AL-l Toluene-ethyl acetate-diethylamine 
(70: 20: 10) 
AL-2 Chloroform-diethylamine 
(90: 10) 
AL-3 Toluene-acetone-ethanol-conc. ammonia 
(40 :40: 6: 2) 
AL-4 Acetone-water-conc. ammonia 
(90: 7: 3) 
AN-l Ethyl acetate-methanol-water 
(100: 13.5: 10) 
AL-5 Toluene-chloroform-ethanol 
(28.5: 57: 14.5) 
AL-6 n-Heptane-ethylmethyl ketone-methanol 
(58: 34: 8) 
AL-7 Chloroform-methanol 
(85: 15) 
AL-8 Toluene-methanol 
(86: 14) 
AL-9 n-Propanol-formic acid-water 
(90: 1: 9) 
AL-l0 Cyc1ohexane-chloroform-glacial 
acetic acid (45: 45: 10) 
111. Detection 
1. Without chemical treatment 
UV-254 nm 
Drug; Alkaloids 
Screening system, suitable for the 
major alkaloids of most drugs. 
Chinae cortex; Cinchona alkaloids 
(Ph. Eur. III) 
Opium; opium alkaloids (DAB 8) 
Solanaceae drugs (atropine, 
hyoscyamine) according to Ph. Eur. I 
Screening system, suitable for Rau-
wolfia alkaloids, xanthine derivatives 
(Caffeine drugs), Colchicum alkaloids. 
Secale alkaloids 
Rauwolfia alkaloids (DAB 8) 
Isoquinoline alkaloids 
Ipecacuanhae radix (Ph. Eur.) 
Colchici semen (DAC). Solvent run 
twice over 15 cm. 
Berberidis cortex, Hydrastis rhizoma, 
Colombo radix and Chelidonii herba. 
Berberine-, and protoberberine-type 
alkaloids 
A lot of alkaloids show a pronounced quenching of fluorescence in UV -254 nm 
(e.g. strychnine, brucine, purines). 
UV-365 nm 
Some alkaloids (Fig. 2, p. 67) fluoresce blue or yellow in UV -365 nm. 
2. Spray reagents 
a) Dragendorffreagent (DRG No. 11 A-11 E, p. 300-301) 
Brown or orange (vis.) zones appear immediatelyon spraying. The colours are 
not stable. 
Remarks: The alkaloid zones can be made more distinct by spraying first with 
Dragendorff re agent and then with 5% sodium nitrite soln. or 5% ethanolic sul-
phuric acid. 
b) Iodoplatinate reagent (IP No. 19, p. 302) 
Directly after spraying, alkaloid zones appear brown, blue or whitish (vis.) on the 
blue-grey background of the TLC plate. 
IV. List of Alkaloid Drugs 
Chromatograms (Figs. 1-26) are reproduced on pp. 66--91. 
Fig. 
4-6 
7,8 
DrugjPlant source 
F amily jPharrnacopoeia 
A. Indole alkaloids 
Rauvolfiae Radix 
Rauwolfia root 
Rauvolfia serpentina (L.) BENTHAM 
ex KURZ 
Rauvolfia vomitoria AFZEL 
Apocynaceae 
DAB 8 (R. serp.) 
USPXX 
Y ohimbe Cortex 
Y ohimbehe bark 
Pausinystalia yohimba PIERRE 
Rubiaceae 
DAC (0.5-1 % ca1c. as yohimbine) 
Quebracho Cortex 
Aspidosperma bark 
Aspidosperma quebracho blanco 
SCHLECHT 
Apocynaceae 
Secale Cornutum 
Ergot 
Claviceps purpurea (FRIES.) Tu-
LASNE 
Clavicipitaceae (Ascomycetes) 
ÖAB 
Total alkaloid content (TA) 
Major alkaloids 
TA 0.6-1.5% (R. serpentina) 
1.3-3% (R. vomitoria) 
DAB 8 specifies not less than 1 %, ca1c. as 
reserpine; about 50 indole alkaloids, mostly 
yohimbane derivatives. 
Main alkaloids: reserpine, rescinnamine (tert. 
indole alkaloids); rauwolscine (R. vomitoria 
only); ajmaline (tert. indole alk.); serpentine 
(quaternary base). 
Minor alkaloids: raubasine (corynantheine 
type), raupine (sarpagine type). 
TA 2.3-5.9% (not less than 1.5% ca1c. as yo-
himbine) 
Yohimbine is the main alkaloid. (1.- and ß-yo-
himbane, pseudoyohimbine and coryantheine 
are the most important minor alkaloids. 
TA 0.3-1.5% 
Main alkaloids: yohimbine, pseudoyohim-
bine, aspidospermine, aspidosperrnatine, que-
brachamine, hypoquebrachamine, quebra-
chocidine. 
TA 0.2-1% 
Lysergic acid alkaloids 
Amide alkaloids (ergometrine = ergobasine, 
"water soluble") and peptide alkaloids (ergo-
tamine) or alkaloids of the ergotoxin group 
("water insoluble "). 
55 
Fig. 
9, 10 
11, 12 
13,14 
56 
Drug/Plant source 
Family /Pharmacopoeia 
Strychni Semen 
Nux vomica seeds 
Strychnos nux vomica L. 
Loganiaceae 
2. AB-DDR, Helv. VI, ÖAB 
Ignatii Semen 
Ignatius beans 
Strychnos ignatii BERG 
Loganiaceae 
B. Quinoline and isoquinoline alkaloids 
Alkaloids of the morphinane type 
(phenanthrene type) 
Chinae Cortex 
Cinchonae cortex 
Cinchona bark 
Cinchona pubescens V AHL (syn. 
C. succirubra PAVON) 
Ph. Eur. III, ÖAB, 2. AB-DDR, 
Helv. VI 
Cinchona ledgeriana MOENS. 
YeIIow Cinchona bark 
Rubiaceae 
Ipecacuanhae Radix 
Ipecacuanha root 
Cephaelis ipecacuanha 
(BROT.) RICH. (Rio and Matto-
Grosso drugs) 
Cephaelis acuminata KARSTEN 
(Cartagena, Panama and Costa-
Rica drugs) 
Rubiaceae 
Ph. Eur. 1,2. AB-DDR, USP XX 
Opium 
Opium 
Papaver somniferum L. subsp. 
somniferum and varieties 
Papaveraceae 
DAB 8,2. AB-DDR, Helv. VI, 
ÖAB, USPXX 
Total alkaloid content (TA) 
Major alkaloids 
TA 2-3% 
ca. 1 % strychnine and 1.5% brucine. Minor 
alkaloids 0(- and ß-colubrine. 
TA 2.5-3% 
Strychnine (45-50% oftotal) and brucine 
TA 4-12% 
(Ph. Eur. specifies not less than 6.5%; 2. AB-
DDR specifies 7-10% quinine/cinchonine) 
Main alkaloids are the diastereomeric pairs 
quininel quinidine and cinchoninel cinchonidine. 
Official Cinchona bark contains ca. 20 known 
alkaloids (== 100%), consisting of ca. 25% 
quinine, ca. 45% cinchonine and ca. 5% quin-
idine. YeIIow Cinchona bark contains up to 
90% quinine. 
TA 1.8-6% 
(Ph. Eur. specifies not Iess than 2%; 2. AB-
DDR not less than 2%, with emetine as 60% 
of total) 
Main alkaloids are emetine and cephaeline, 
and the corresponding dehydro-compounds, 
O-methylpsychotrine and psychotrine. 
Rio drug contains 3: 1 - 1: 1 ratio of emetine: 
cephaeline. 
Panama drug contains mostly 1 : 1 ratio eme-
tine: cephaeline. 
Drugs contain about 0.05% minor alkaloids. 
TA (raw opium) 20-29% with ca. 30 alka-
loids. 
Main alkaloids of the phenanthrene type: 
morphine (3-23%), codeine (0.3-3%), thebaine 
(0.1-3%). 
Benzylisoquinoline type: papaverine (0.1-2%), 
noscapine (narcotine) (2-12%), narceine 
(0.1-2%) 
Opium DAB 8 (dried latex containing at least 
9.5% morphine) 
Opium 2. AB-DDR (at least 12% morphine) 
Opium titratum DAB 8 (standardized at 
9.8% morphine content) 
Fig. 
15-18 
DrugjPlant source 
Family jPharmacopoeia 
C. Miscellaneous classes or alkaloids 
Chelidonii Herba 
Greater celandine 
Chelidonium majus L. 
Papaveraceae 
DAß 8, 2. AB-DDR 
ßerberidis Radicis Cortex 
ßarberry bark 
ßerberis vulgaris L. 
ßerberidaceae 
Hydrastis Rhizoma 
Golden seal rhizoma 
Hydrastis canadensis L. 
Ranunculaceae 
Colombo Radix 
Colombo root 
Jateorhiza palmata (LAM.) MIERS 
Menispermaceae 
Colchici Semen 
Co1chicum seeds 
Co1chicum autumnale L. 
Liliaceae 
DAC 
19,20,22 Aconiti Tuber 
Aconite root 
Aconitum napellus L. 
Ranunculaceae 
Helv. VI (Aconitinum: Helv. VI, 
ÖAB) 
Boldo Folium 
ßoldo 1eaves 
Peumus boldus J.1. MOLINA 
Monimiaceae 
Helv. VI 
Ephedrae Herba 
Ephedra (Ma-huang) 
Ephedra distachya L. and other 
species 
Ephedraceae (Ephedrine DAB 8) 
Total alkaloid content (TA) 
Major alkaloids 
Opii extractum DAß 8 (dried extract from 
raw opium; morphine content 19.6--20.4%) 
Opii tincturaDAß 8 (from raw opium; mor-
phine content 0.95-1.05%) 
TA 0.35-0.9% (DAß 8 specifies not less than 
0.6%; 2. Aß-DDR 0.4-0.8%). About 20 al-
kaloids are present. 
ßenzophenanthridine type: chelidonine, che-
lerythrine and sanguinarine. 
Protoberberine type: berberine. 
Protopine type: protopine, OC-, ß-allocrypto-
pine. 
TA 0.95-3% 
Main alkaloids berberine, jateorhizine and 
palmatine (protoberberine type). 
TA 2.5--6%, comprising ca. 3 % berberine, ca. 
1 % tetrahydroberberine (canadine) and ca. 
1.5-4% hydrastine (a phthalidisoquinoline al-
kaloid). 
TA 1-2% 
with the protoberberine alkaloids, palmatine, 
jateorhizin and columbamine. 
TA 0.5-1% 
ca. 20 alkaloids. Main alkaloid colchicine; mi-
nor alkaloid demeco1cine. 
TA 0.3-1.5% (Helv. VI specifies not less than 
0.6% ether-soluble alkaloids, ca1culated as 
aconitine) 
Ester alkaloids (alkamine esters), the main 
one being aconitine. Hydrolytic cleavage 
products, benzoylaconine and aconine, are 
present. 
TA not less than 0.1% (Helv. VI) 
Aporphine alkaloid, boldine. 
TA up to 3.3% 
ca. 75% L-ephedrine and 25% ( + )-pseudoephedrine and nor-pseudoephe-
drine. The pseudoephedrines are diastereo-
isomers of ephedrine. 
57 
Fig. 
21 
58 
DrugjPlant source 
F amily jPharmacopoeia 
Jaborandi Folium 
Jaborandi leaves 
Pilocarpus jaborandi HOLMES (Per-
nambuco jaborandi) 
Pilocarpus pennatifolius LEMAIRE 
(Paraguay jaborandi) and other 
species 
Rutaceae 
Helv. VI; (Pilocarpine-HCI, 
DAB 8 and other pharmacopoeias) 
Lobeliae Herba 
Lobelia 
Lo belia infla ta L. 
Lobeliaceae 
2. AB-DDR, ÖAB 
Sabadillae Semen 
Sabadilla seeds 
Schoenocaulon officinale ASAGRA Y 
Liliaceae 
Veratri Rhizoma 
White hellebore rhizome 
Veratrum album subsp. album, and 
V. lobelianum BERHN. 
Liliaceae 
2. AB-DDR, Helv. VI 
Sarothamni (Spartii) scoparii Herba 
Broom 
Sarothamnus scoparius (L.) WIM-
MER ex KOCH 
Fabaceae 
Nicotinae Folium 
Tobacco leaves 
Nicotiana tabacum L., N. rustica 
L. and other varieties 
Solanaceae 
D. Purine alkaloids 
CacaoSemen 
Cacao seeds 
Theobroma cacao L. 
Sterculiaceae 
Coffeae Semen 
Coffee seeds 
Coffeae arabica L. and other spe-
cies 
Rubiaceae 
Total alkaloid content (TA) 
Major alkaloids 
TA 0.5-7% (Helv. VI specifies not less than 
0.5%). The imidazole alkaloids, pilocarpine 
and isopilocarpine, may represent 25-50% of 
the total alkaloids. 
TA not less than 6.2% (2. AB-DDR) 
Main alkaloid lobeline (piperidine ring sys-
tem). 
Minor alkaloid isolobinine (dehydropiperi-
dine ring system) 
GA 1-5% 
Steroid alkaloids (" Veratrinum alkaloid mix-
ture") with C-nor-C-homo-cholestane struc-
ture. 
TA not less than 1 % (Helv. VI) 
Tetraesters of protoverine, mainly Protover-
ine A and B, together with free alkaloids. 
TA 0.8--1.5% 
Main alkaloid sparteine (a tetracyclic quinoli-
zidine alkaloid) 
Minor alkaloid IX-isosparteine (gentisine) 
Flavonoids (see p. 184, Fig. 14, chapter on fla-
vonoids) 
TA variable 
L-Nicotine (0.05-10%), nornicotine, anaba-
sine and nicotyrine. 
0.2-0.5% caffeine 
1-2% theobromine 
0.3--2.5% caffeine 
(traces of theophylline) 
Chlorogenic acid 
Fig. 
23-26 
Drug/Plant source 
F amily /Pharmacopoeia 
ColaeSemen 
Colaseeds 
Cola nidita SCHOTT et ENDL. 
Cola acuminata SCHOTT et ENDL. 
Sterculiaceae 
Helv. VI, ÖAB 
Mate Folium 
Mate 
Ilex paraguariensis St. HILAIRE 
Aquifoliaceae 
Thea Folium 
Tea 
Camellia sinensis (L.) KUNTZE, and 
other varieties 
Theaceae 
E. Tropine alkaloids 
Belladonnae Folium 
Belladonna leaves 
USPXX 
Ph. Eur. I, ÖAB, 
2. AB-DDR, He1v. VI 
Belladonnae Radix 
Belladonna root 
Atropa belladonna L. 
Solanaceae 
2. AB-DDR, ÖAB 
Scopoliae Radix 
Scopolia root 
Scopolia carniolica JACQ. 
Solanaceae 
Hyoscyami Folium 
Henbane leaves 
Hyoscyamus niger L. 
Ph. Eur. I, Helv. VI, 2. AB-DDR 
Hyoscyami mutici Folium 
Hyoscyamus muticus L. 
Solanaceae 
Total alkaloid content ( TA) 
Major alkaloids 
0.6-3% caffeine 
ca. 0.1 % theobromine 
0.5-1.5% caffeine 
ca. 0.05% theophylline 
ca. 0.2-0.45% theobromine 
Chlorogenic acid 
2.5-4.5% caffeine 
0.02-0.05% theophylline 
0.05% theobromine 
TA 0.2-0.5% (Ph. Eur. I specifies not less 
than 0.3%) 
Main alkaloids ( - )-hyoscyamine/atropine 
and scopolamine in ratio ca. 3: 1. 
TA 0.3-0.8% (2. AB-DDR specifies not less 
than 0.4%) 
Main alkaloids ( - )-hyoscyamine and scopola-
mine. 
Minor alkaloids apoatropine, belladonnine, 
cuskhygrine, norhyoscyamine, noratropine 
and meteloidine. 
TA 0.4-0.95% 
Alkaloids similar to those of Belladonnae ra-
dix, differentiation on basis of coumarin pat-
tern (see p. 90, Fig. 26) 
TA 0.04-0.17% (Ph. Eur. specifies not less 
than 0.05%) 
(- )-Hyoscyamine/atropine and scopolamine 
in ratio ca. 1.2: 1. 
TA not less than 0.8% (0.8-1.4%) 
Main alkaloids ( - )-hyoscyamine/atropine 
and scopolamine. 
Minor alkaloids apoatropine and belladon-
nine. 
59 
60 
Fig. Drug/Plant source 
F amily /Pharmacopoeia 
Stramonii Folium 
Thornapple leaves 
Datura stramonium L. 
Solanaceae 
Ph. Eur. I, Helv. VI, 2. AB-DDR, 
ÖAB 
Total alkaloid content (TA) 
Major alkaloids 
TA 0.1-0.6% (Ph. Eur. specifies not less than 
0.25%) 
Main alkaloids ( - )-hyoscyamine and scopola-
mine in ratio ca. 2: 1. 
Minor alkaloid atropamine. 
AU Solanaceae leaf drugs also contain flavonoids, and to some extent coumarins and plant 
acids (see Fig. 26, p. 90) 
v. Fonnulae of Constituents of Alkaloid Drugs 
(- )-Emetine: R = CHa __ ---=2'-'-H_~~ O-Methylpsychotrine 
Cephaline: R = H - 2H ~ Psychotrine 
o 
~OH 
JL~Jl HOOe 0 eOOH 
Morphine: R, = R2 = H 
Codeine: R, = H; R2 = CHa 
Thebaine: R, = R2 = CHa, with 
additional double bonds 
at 6/7 and 8/14 
Papaverine 
R 
( - ) Quinine: R = OCHa 
Cinchonidine: R = H 
Meconic acid 
R 
OH 
Boldine 
Noscapine 
(= Narcotine) 
H 
(+) Quinidine: R = OCHa 
Cinchonine: R = H 
61 
ß-Carboline 
~ 
~ChinoliZidine 
N~ '\ R, 
Yohimbane: R, = R2 = H 
Yohimbine: R, = CH 300C; R2 = OH 
OH 
: OR2 
6CH3 
Reserpine: R, = OCH3 ; 
R2 = 3,4,5-Trimethoxybenzoyl 
Rescinnamine: R, = OCH3 ; 
R2 = 3,4,5-Trimethoxycinnamoyl 
Raubasine: 
(= Ajmalicine) 
HO 
Ajmaline Sarpagine: R = H Serpentine 
Strychnine: 
Brucine: 
cr-Colubrine: 
ß-Colubrine: 
62 
Raupine: R = CH 3 
ROC 
R = R = H 
R; = R: = OCH3 
R, = H; R2 = OCH 3 
R, = OCH3 ; R2 = H 
L YSERGYL RESIDUE 
Alkaloids R, 
1. Ergotamine group 
Ergotamine CH 3 
2. Ergotoxine group 
Ergocristine CH(CH 3 )2 
AMIDE ALKALOIDS 
Ergometrine (Ergobasine) R = NH-CH-CH20H 
I 
CH 3 
PEPTIDE ALKALOIDS 
1 
:~ R, i OH 
H 1/0,/--1 
-N···C I C N 
1 1-1-----1----
-::/C--: N" /C~ 
o : /C'" 0 
---------: H 'R2 
Proline 
I 
Amino acids R2 Amino acids 
cr-Hyd roxy- CH2-<Q) Phenylalanine alanine 
cr-Hyd roxy- CH2-<Q) Phenylalanine valine 
Berberine: R,; R2 = -CH 2 -
Palmatine: R, = R2 = CH3 
Jateorh izi ne: R, = H; R2 = CH 3 
o 
\-0 
Chelidonine 
L-Hyoscyamine 
I 
Chelerythrine: R, = R2 = CH3 
Sanguinarine: R,; R2 = CH2 -
D-Hyoscyam i ne 
I 
Atropine (DL-Hyoscyamine) 
6,7-Epoxidation 
L-Scopolamine 
!-H20 
T 
6 
I 
C=O 2Mol IQ\-b Apoatropine ~II 
CH2 
Apoatropine Belladonnine 
T = Tropyl residue 
Tropine 
(Tropane-3-IX-ol) 
Cuskhygrine 
63 
64 
~ 
HO-C-H 
I 
H CHN~C-H 
3 I 
CH3 
L-Ephedrine 
Acontine 
enant. 
~ 
H-C-OH 
I 
H-C-NHCH3 
I 
CH3 
D-Ephedrine 
L------v 
erythro 
CH3 
I 
R-C-H 
I 
Colchicine: 
Demecolci ne: 
R= COCH3 
R=CH3 
OH 045 ~ 
I H c.· 2 N 6 " CH2-c-Q R-CH- 2 I 11 
CH3 0 
(-) Lobeline: R= -© 
Isolobinine: R = C2 H. 4,5= 
oR N R H3C-~-OH 3 :: OH ••••• 0'6': 7 •• OHO! -C-CH-CH2-CH3 
. . I 
Nicotine:. R:: ~H3 
Nornicotme. R-
C-O 
11 
o 
: H: '0 11 CH 6H 6 ' 0 3 l~etYI 
. A"R-H Protoveratr!ne : = OH 
Protoveratnne B. R-
d50 
H 
Pilocarpine Sparteine 
R, R2 Ra 
Theobromine H CH3 CH3 
Theophylline CHa CH3 H 
Caffeine CH3 CH3 CH 3 
65 
TLC Synopsis of the Most Important Alkaloids 
Alkaloids I - reference compounds detected with Dragendorff reagent 
Tracks 
Solvent 
system 
1 = colchicine 12 = scopolamine 
2 = boldine 13 = strychnine 
3 = morphine 14 = yohimbine 
4 = pilocarpine 15 = physostigmine 
5 = quinine 16 = nicotine 
6 = brucine 17 = veratrine 
7 = cephaeline 18 = emetine 
8= Quinidine 19= papaverine 
9= atropine 20= lobeline 
10= co deine 21 = aconitine 
11 = cinchonine 22 = noscapine (narcotine) 
AL-1 : toluene-ethyl acetate-diethylamine (70: 20: 10) 
Detection Dragendorff reagent (No. 11 C, p. 300) vis. Fig. 1 A 
Dragendorff reagent with sodium nitrite (No. 11 F, p. 301) vis. Fig. 1B; 2A 
Chromato- With Dragendorff reagent alkaloids give spontaneously an orange-brown, usually 
gram stable, colour in the visible. With some alkaloids, e.g. boldine (2), morphine (3) 
1 A and nicotine (16), the colour fades rapidly. 
1 B, 2A The colour can be intensified by spraying afterwards with somum nitrite reagent. 
The zones then appear dark brown (e.g. morphine (3)) or violet-brown (e.g. atropine 
(9)). The colours of pilocarpine (4) and nicotine (16) are unstable. 
Alkaloids 11 - reference compounds that fluoresce in UV-365 nm 
Tracks 
Solvent 
system 
23 = serpentine 29 = emetine 
24=quinine 30=yohimbine 
25 = cinchonine 31 = noscapine (narcotine) 
26 = quinidine 32 = hydrastine 
27 = cinchonidine 33 = berberine 
28 = cephaeline 34 = sanguinarine 
AL-1 : toluene-ethyl acetate-diethylamine (70: 20: 10) 
Detection: Sulphuric acid reagent (5%) (No. 34, p. 303) UV-365 nm Fig.2B 
Chromato- Fluorescence of these alkaloids is predominantly blue, and is intensified by treatment 
gram with 5% sulphuric acid. 
2 B In the case of the quinine alkaloids, the initial weak blue fluorescence of quinine 
66 
and quinidine becomes a radiant blue (this appears white in the illustration), and 
cinchonine and cinchonidine show a deep violet fluorescence. 
Berberine and sanguinarine are exceptional in showing a yellow fluorescence. 
Remarks: The commercial alkaloid reference compounds (e.g. hydrastine) frequently show 
zones of minor alkaloids. Some alkaloids form degradation products in solution or during 
development of the TLC plate. 
Fig.l 
Tl - 21 
A 
Fig.2 
T12345679 T23-34 
n 
Tl3-22 
-FRONT 
Rf 
-0.5 
START 
FRONT 
Rf 
START 
67 
Alkaloids 111 "Rauwolfia alkaloids" 
Quebracho and Yohimbe Cortex 
Tracks 
Tests 
Solvent 
system 
Detection 
1 = Quebracho Cortex 
2 = Y ohimbe Cortex 
Tl = serpentine 
T2 = ajmaline 
T3 = yohimbine 
T4 = reserpine 
T5 = rescinnamine 
AL-l : toluene-ethyl acetate-diethylamine (70: 20: 10) 
AL-6: n-heptane-ethylmethylketone-methanol (58: 34: 8) 
AN-1: ethyl acetate-methanol-water (100: 13.5: 10) 
Without chemical treatment UV -365 nm 
Iodoplatinate re agent (IP No. 19, p. 302) UV-365 nm 
Dragendorffreagent (No. IlF, p. 301) vis. 
For description of drugs see p. 55. Formulae p. 62. 
Fig. 3A; 4A 
Fig. 3B; 4B 
Fig. 3C; 4C 
Fig. 3B, C; 4A, B 
Fig.3A 
Fig.4C 
Chromato- Rauwolfia alkaloids (reference compounds). Alkaloids of the yohimbine and corynan-
gram theine type (" Rauwolfia alkaloids" Tl-5) give an intense blue fluorescence in UV-
3 365 nm. Ajmaline is characterized by strong fluorescence quenching in UV-254 nm, 
and by only a weak blue fluorescence in UV-365 nm, which can, however, be intensi-
fied by treatment with iodoplatinate reagent (Fig. 3 A). 
3A-C AL-I Of the three solvent systems, AL-I gives the best separation of ajmaline and serpen-
tine, which remain in the starting region in other systems. 
AL-6 shows the separation of reserpine and rescinnamine. 
AN-I shows the best separation of yohimbine and reserpine/rescinnamine mixtures. 
Remarks: In drug extracts containing alkaloids of the yohimbine/corynantheine type, which 
occur in a variety of structural variants, it is necessary to use different solvent systems for 
the analysis (see Rauwolfiae radix Fig. 5/6, p. 70). 
4A 1,2 Quebracho and Yohimbe cortex. In the basic solvent system, AL-I, extracts of Quebra-
cho and Yohimbe bark show many blue fluorescent zones distributed over the whole 
Rf range; the main compound is yohimbine (cf. T3) in the intermediate Rf region. 
4B In the neutral system, AL-6 (cf. DAB 8/Rauwolfiae radix), most compounds are 
found in the lower Rf region. The strongly fluorescent zone at Rf ca. 0.7 can be 
used to differentiate between Quebracho and Yohimbe cortex. 
4C AN-I Treatment with Dragendorff reagent gives brown, rapidly fading zones in the visible. 
68 
Compared with Y ohimbe cortex, Quebracho cortex shows more stable and stronger 
zones in the lower Rf range and in the Rf region of standard yohimbine. Y ohimbe 
cortex shows an additional zone at Rf ca. 0.8. 
Fig.3 
T1 T2 T3 T4 T5 T3 
Fig.4 
T3 2 T3 
T4 T5 T3 T4 T4T5 
2 T3 2 
FRONT 
Rf 
69 
Rauwolfiae Radix 
Tracks 1 = Rauwolfiae radix (R. serpentina - "Siam drug") 
2 = Rauwolfiae radix (R. vomitoria - " African drug") 
3 = Rauwolfiae radix (R. serpentina - "J ndian drug") 
Tests Tl = serpentine T4 = reserpinejrescinnamine test mixture 
T2 = ajmaline T5 = rauwolscine (with reserpine) 
T3 = reserpine T6 = Rauwopur® (see p. 52 for composition) 
Solvent AL-l : toluene-ethyl acetate-diethylamine (70: 20: 10) Fig. 5 
system AL-6: n-heptane-ethylmethylketone-methanol (58:34:8) Fig.6 
Detection Without chemical treatment UV-365 nm Fig. 5A; 6A 
Dragendorff reagent (No. 11 F, p. 301) vis. Fig. 5B; 6B 
For description of drugs see p. 55. Formulae p. 62. 
Chromato- Rauwoljia root extracts, after chromatography in solvent systems AL-l and AL-6, 
gram show many light blue fluorescing zones from the starting point to Rf ca. 0.8, when 
viewed in UV-365 nm. 
5 1 Rauwoljiae serpentinae radix (Siam drug). In solvent system AL-l the following 
6 
70 
pattern is obtained in order of increasing Rf: 
Above the dark blue fluorescing starting zone, serpentine is weakly visible (cf. 
Tl) at Rf ca. 0.1. Two pronounced blue zones are seen at Rf 0.2 and 0.25, followed 
by the darker blue fluorescent zone of ajmaline (cf. T2) at Rf ca. 0.35. A further 
blue fluorescent zone at Rf ca. 0.4 is followed by the hardly separated zones of 
reserpine and rescinnamine (cf. T3jT4). The upper Rf range is occupied chiefly by 
weakly concentrated zones, and the more prominent zone of rauhasine at Rf ca. 
0.75. 
3 Rauwoljiae serpentinae radix (Indian drug). The concentration of reserpine/rescinna-
mine is similar to that of the Siam drug, but the Indian drug shows a higher content 
of serpentine (cf. Tl). The zones up to ajmaline (cf. T2) and the zones in the upper 
Rf range are more pronounced than in the Siam drug. 
2 Rauwoljiae vomitoriae radix (African drug). This drug, which is not admitted by 
DAB 8, shows markedly high er contents of ajmaline, reserpine and rescinnamine. 
In addition the TLC reveals rauwolscine (cf. T5) at Rf ca. 0.45, and other zones 
directly above it. 
Solvent system AL-6 (DAB 8) gives a similar range of separation for the main 
alkaloids, but at lower Rf values. Serpentine and ajmaline remain at the start (cf. 
TljT2). Reserpine and rescinnamine (cf. T3jT4) can be assigned to the two zones 
at Rf ca. 0.3-0.35. In contrast to its behaviour in the basic solvent system, rauwolscine 
mi grates with a higher Rf than reserpine. 
Remarks: With Dragendorff reagent all Rauwolfia alkaloids show brown colour in the visible. 
Ajmaline shows a prominent fluorescence quenching in UV-254 and can be made visible by 
treatment with conc. HN03 (red). 
Fig.5 
Tl T2 2 3 
Fig.6 
Tl T2 2 3 
DABS 
T3 T4 T5 T6 T2 Tl 
T3 T4 T5 T6 
-FRONT 
Rf 
-0.5 
-START 
-FRONT 
Rf 
-0.5 
-START 
71 
Strychni, Ignatii Semen 
Tracks 
Solvent 
system 
1 = Ignatii semen 
2 = Strychni semen 
AL-l: toluene-ethyl acetate-diethylamine (70: 20: 10) 
AL-2: chloroform-diethylamine (90: 10) 
Tests Tl = brucine 
T2 = strychnine 
Fig. 7 A, B 
Fig.7C 
Detection Dragendorff reagent (No. 11 B) 
Chromato-
gram 
with sodium nitrite (No. 11 F, p. 301) 
Conc. nitric acid 
For description of drugs see p. 56. Formulae p. 62. 
vis. 
VIS. 
Fig. 7 A, C 
Fig.7B 
7 A After TLC separation in solvent system AL-l, followed by treatment with Dragen-
dorff reagent, both drug extracts show 4-6 orange-brown zones of differing concen-
trations in the Rfrange 0.15-0.4. 
7B 
7C 
1 Ignatii semen contains hrucine (cf. Tl) and strychnine (cf. T2) in the ratio ca. 1: 3. 
2 Strychni semen has a lower content of total alkaloids, with approximately equal 
proportions of strychnine and hrucine. Minor alkaloids, e.g. oc- and ß-colubrine and 
pseudo-strychnine, tend to mi grate ahead of the strychnine zone. 
Remarks: The strychnine zone is initially prominent, but fades rapidly. 
Brucine (cf. Tl; Rf ca. 0.15) is typically red in the visible when treated with conc. 
HN0 3 • Strychnine does not react with this colour. 
In solvent system AL-2, the main alkaloids, brucine and strychnine, migrate in 
the higher Rf region (cf. Tl/T2). 
Secale cornutum 
Track 3 = Secale cornutum Tests T3 = ergometrine 
T 4 = ergotamine 
T5 = ergocristine 
Solvent AL-l : toluene-ethyl acetate-diethylamine (70: 20: 10) Fig.8A 
Fig.8B system AL-5: toluene-chloroform-ethanol (28.5: 57: 14.5) 
Detection Van URK reagent (No. 39, p. 304) VIS. Fig. 8A, B 
Chromato-
gram 
8A, B 
For description of drugs see p. 55. Formulae 'po 62. 
After treatment with van Urk reagent chromatograms of Secale extracts show 
5-6 blue-violet alkaloid zones in the Rf range 0.05-0.25 (solvent system AL-l) or 
0.05-0.6 (solvent system AL-5). 
The zone at Rf 0.05 represents ergometrine1 , at Rf ca. 0.03 ergotamine 1 , and 
at Rf ca. 0.45 ergocristine1 (solvent system AL-5). 
TLC separations of commercially available references (T3-T5) show a second, usually weak zone. This 
is also seen in Secale extracts, and is due to degradation products (Iumi-compounds or similar products). 
72 
A B 
Fig.7 
Tl 2 T2 Tl 
A B 
Fig.8 
T3 3 T4 T5 
c 
2 Tl 2 
T3 3 T4 
T2 
T5 
-FRONT 
Rf 
-0.5 
-START 
-FRONT 
Rf 
-0.5 
-START 
73 
Chinae Cortex 
Tracks 
Tests 
Solvent 
system 
Detection 
Chromato-
gram 
1 = Chinae (e. ledgeriana) cortex 
2 = Chinae (C. succirubra) cortex 
TG = standard mixture of quinine alkaloids, Ph. Eur. III (for composition, see p. 53) 
Ti =quinine 
T2 = cinchonidine 
T3 = quinidine 
T 4 = cinchonine 
AL-2: chloroform-diethylamine (90: 10) Ph. Eur. III 
AL-1 : toluene-ethyl acetate-diethylamine (70: 20: 10) 
Sulphuric acid (No. 34, p. 303) UV-365 nm 
Sulphuric acid-iodoplatinate (No. 19, p. 302) VIS. 
For description of drugs see p. 56. Formulae p. 61. 
Fig. 9; lOA 
Fig. lOB, C 
Fig. 9; lOC 
Fig. lOA, B 
9 1,2 
After treatment with sulphuric acid, chromatograms of both cinchona drugs in sol-
vent system AL-2 (Ph. Eur.) reveal at least 6 intense light blue and 5-6 weaker, 
dark violet fluorescent zones in UV -365 nm, distributed from the start to the solvent 
front. 
10A 
10B, C 
74 
After sulphuric acid treatment, the main alkaloids, quinine (cf. Ti) and quinidine 
(cf. T3), show an intense blue fluorescence, and cinchonidine (cf. T2) and cinchonine 
(cf. T4) show a dark violet fluorescence in UV-365 nm. In cinchona extracts cinchoni-
dine is overlapped by the strongly blue fluorescing quinidine. 
Treatment afterwards with iodoplatinate reagent gives a violet colouration in the 
visible. The grey-violet of cinchonidine is distinct from the violet-brown of quinine, 
quinidine and cinchonine. 
In separations with the screening system for alkaloids (solvent system AL-I), the 
four main alkaloids mi grate only in the Rf range 0-0.2. 
Remarks: In addition to the four main alkaloids, further cinchona alkaloids and epiquinine 
bases can also be detected. The epibases (threo-compounds) of quinine and quinidine migrate 
in the lower Rf range. The dihydrocompounds of quinine, quinidine, cinchonine and cinchoni-
dine show no fluorescence in UV-365 nm. Certain yellow-green fluorescing zones may represent 
quinonoid structures. 
The two cinchona extracts can be dijJerentiated on the basis of their quinine contents. 
2 Cinchonae succirubrae cortex contains the 4 main cinchona alkaloids in approximate-
Iy the same proportions as in the standard mixture TG (Fig. 9j10A). 
1 In extracts of C. ledgerianae cortex, quinine is the predominant alkaloid (Fig. 9). 
Fig.9 
2 TG Tl T2 
A 
Fig.10 
TG 2 2 TG 
T3 T4 
2 TG , 
-FRONT 
Rf 
0 .5 
-START 
FRONT 
Rf 
START 
75 
Ipecacuanhae Radix 
Tracks 
Test 
Solvent 
system 
1 = Ipecacuanhae radix (CephaeIis acuminata "Cartagena/Panama drug") 
2 = Ipecacuanhae radix (e. ipecacuanha "Rio/Matto-Grosso drug") 
3 = Ipecacuanhae radix (commercial; plant of unspecified origin) 
4= Ipeeaeuanhae radix (eommercial "Jahore") 
TG = standard mixture of cephaeline (Rf ea. 0.2) and emetine (Rf ca. 004) Ph. Eur. 
(see p. 53) 
T1 = eephaeline 
T2=emetine 
AL-1 : toluene-ethyl acetate-diethylamine (70: 20: 10) 
AL-1: with chamber saturation 
AL-7: chloroform-methanol (85: 15) Ph. Eur. 
Fig. 11 A, B, C 
Fig.12A 
Fig.12B 
Detection Iodine-chloroform-reag. UV-365 nm Fig. 11A; 12A, 12B 
VIS. Fig. 11 B (I/CHCI3 No. 17, p. 301) 
Dragendorff re agent (No. 11 B, p. 300) VIS. Fig. 11 C 
For description of drugs see p. 56. Formulae p. 61. 
Chromato-
gram 
Ipecacuanhae radix. Without prior chemical treatment, the main alkaloids, emetine 
and cephaeline (cf. TG), appear in UV-365 nm as uniformly blue fluorescing zones. 
After treatment with iodine reagent and brief warming, they show a characteristic 
yellow-white or light blue, respectively, in UV-365 nm (Fig. 11 A; 12A, B) and red-
11A 1-4 
12A, B 
brown or yellowish zones, respeetively, in the visible (Fig. 11 B). 
1,2 Cartagena and Rio drugs both show the intense light blue zone of cephaeline (cf. 
T1) and the yellow-white zone of emetine (cf. T2). In addition there are weaker 
blue or yellow fluorescent zones in the starting region and direetly above and be10w 
the zone of emetine (cf. T2). The minor alkaloid, O-methylpsychotrine (in the Rf 
region of emetine) also fluoresees yellow. In contrast, psychotrine, whieh is found 
below cephaeline, gives a blue fluorescence. 
11 C Dragendorff reagent deteets the main alkaloids as red-brown zones in the visible. 
The minor alkaloids are only partly detectable. 
Commercial ipecacuanha drugs can be differentiated on the basis of the eephaeline: 
emetine ratio. 
1 The Cartagena (Panama or Costa Riea) drug from Cephaelis acuminata contains 
almost equal proportions of emetine and cephaeline. 
2 In the Rio (Matto-Grosso) drug from C. ipecacuanha the emetine: cephae1ine ratio 
is 2-3:1. 
3,4 Ipecaeuanha drugs from other sourees (e.g. Jahore drug 4) tend to have a slightly 
higher eontent of emetine than cephaeline. 
12 B In the solvent system of Ph. Eur. (AL-7), with chromatographie development over 
15 em, emetine and cephaeline (cf. T6) remain poorly separated in the lower Rf 
range; psychotrine stays ne ar the start and methylpsychotrine mi grates above emetine. 
Double development gives slightly higher Rf values for emetine and cephaeline. 
76 
Fig.11 
TG 2 TG 
Fig.12 
Tl 3 4 T2 
c 
2 TG 
TG 2 
Ph . Eur I 
2 
-FRONT 
Rf-0.5 
_&:10 '"'""T 
77 
Opium 
Tracks 
Solvent 
system 
1 = Opium DAB 8 
2=Opium 
3=Opii tinctura DAB 8 
Tests Ti = morphine 
T2=codeine 
T3 = papaverine 
T4 = noscapine 
AL-3: toluene-acetone-ethanol-conc. ammonia (40: 40: 6: 2) 
AL-1 : toluene-ethyl acetate-diethylamine (70: 20: 10) 
Fig. 13A, B 
Fig. 14A, B 
Detection Dragendorff re agent with NaNOz reagent (No. 11 CjF, p. 300) 
Chromato-
gram 
BA 1,2,3 
Marquis reagent (No. 25, p. 302) 
Natural products-polyethyleneglycol re agent 
(NP/PEG No. 28, p. 303) 
For description of drugs see p. 56. Formulae p. 61. 
VIS. Fig. 13A; 14B 
VIS. Fig. 13B 
UV-365 nm Fig. 14A 
14B After treatment with Dragendorff-NaNOz, opium extracts show six orange-brown 
main zones from the start to Rf ca. 0.85. Narceine remains at the origin; in order 
of increasing Rf, it is followed by morphine (cf. Ti), codeine (cf. T2), thebaine and 
minor alkaloids (RfO.3-0.5 in solvent system AL-3, and 0.4-0.5 in AL-1),papave";ne 
(cf. T3) and noscapine (narcotine) (cf. T4). 
13 B With Marquis reagent, morphine and codeine are immediately stained violet. A weak 
violet zone of thebaine, and orange-brown zones of minor alkaloids are seen in 
the intermediate Rf region. Papaverine and noscapine give violet and brown zones, 
respectively, with Marquis reagent. 
14 An analogous separation is seen in solvent system AL-1 (screening system), with 
the difference that morphine and codeine migrate with slightly higher Rf values. 
14A In UV-365 nm, without prior chemical treatment, the TLC of an opium extract 
(1) shows pale blue fluorescent zones over the whole Rf range. After treatment 
with NP/PEG reagent, the fluorescence is intensified. Morphine (cf. Ti) fluoresces 
pale blue, papaverine (cf. T3) pale green, and noscapine (cf. T4) intense blue. Codeine 
gives no j1uorescence. 
14B Brown to brown-red zones are obtained with Dragendorffreagent. 
78 
A B 
Fig.13 
Tl T2 T3 T4 Tl 
DAß B 
Fig. 14 
Tl T2 T3 T4 Tl 
T2 T3 T4 2 
T2 T3 T4 3 
-FRONT 
Rf 
-0.5 
-START 
-FRONT 
Rf 
-0.5 
-START 
79 
Berberidis, Colombo Radix Hydrastis Rhizoma 
Tracks 1 = Berberidis radix 
2 = Hydrastis rhizoma 
3 = Colombo radix 
Tests Tl = berberine T5 = jateorhizine 
T6 = columbamine 
T7 = palmatine 
T2 = palmatine-jateorhizine mixture 
T3 = hydrastine (Rf ca. 0.05, Fig. 15 C) 
T 4 = sanguinarine 
Adsorbent Silica gel 60F 254 pre-coated TLC plates 
Aluminium oxide TLC sheets 
Fig. 15A, B, C; 16A 
Fig.16B 
Solvent 
system 
Detection 
Chromato-
gram 1,2,3 
AL-9: n-propanol-formic acid-water (90: 1 : 9) 
AL-l: toluene-ethyl acetate-diethylamine (70: 20: 10) 
AL-I0: cyc1ohexane-chloroform-glacial acetic acid 
(45: 45: 10) for aluminium oxide adsorbent 
Without chemical treatment visjUV-365 nm 
Dragendorff reagent (No. 11, p. 300) vis. 
For description of drugs see p. 57. Formulae p. 63. 
Fig. 15 A, B, C 
Fig.16A 
Fig.16B 
Fig. 15A, C; 16A, B 
Fig.15B 
15 A The three drug extracts characteristically show yellow fluorescent alkaloids in UV-
15B 365 nm. 
15 C 1 Berberidis radix. Berberine appears as a light yellow zone (vis.) on untreated chro-
matograms, and as a lemon-yellow fluorescent zone in UV-365 nm. With Dragen-
dorff reagent it gives a strong brown-red zone (cf. Tl). Weaker concentrations of 
blue fluorescent alkaloids are seen in the lower Rf range. 
15 C 2 Hydrastis rhizoma. The main zone is again berberine (cf. Tl) (yellow fluorescence 
in UV-365 nm). This drug extract can be differentiated from that of Berberidis 
radix by the presence of the blue-white fluorescent zone of hydrastine (cf. T3) at 
the start. A second, light blue fluorescent zone, also present in the standard hydras-
tine (T3) is present at Rf ca. 0.9. 
15 C 3 Colombo radix. In system AL-9, the main yellow fluorescent zone (UV -365 nm) 
is a mixture ofjateorhizine-palmatine-columbamine (cf. T2). 
16A In screening system AL-l, the yellow fluorescent zone of berberine (cf. Tl) migrates in the 
middle of the chromatogram, while the blue fluorescent hydrastine zone has a higher Rf value 
(cf. T3), thus serving for the simple differentiation of Hydrastis and Berberis extracts. Colombo 
radix gives a mixt ure of yellow fluorescing alkaloids in the lower Rf range. 
16B On aluminium oxide in solvent system AL-10, the alkaloids (cf. T5, T6, Ti, T7) show marked 
differences in Rf values. 
80 
Remarks: Sanguinarine (cf. T4, Fig. 15C), another yellow fluorescing alkaloid, can be found 
in extracts of Chelidonii herba (see Fig. 17). 
A B 
Fig.15 
Tl Tl Tl 2 
Fig. 16 
T3 2 Tl 3 
T2 3 T3 T4 
T5 T6 Tl T7 
FRONT 
Rf 
START 
- FRONT 
Rf 
-START 
81 
Chelidonii Herba 
Tracks 
Tests 
1 = Chelidonii herba 
Tl = berberine 
T2 = sanguinarine 
T3 = papaverine 
T 4 = colchicine 
Colchici Semen 
2 = Colchici semen 
Solvent AL-9: n-propanol-formic acid-water (90: 1 : 9) 
system AN-1: ethyl acetate-methanol-water (100: 13.5: 10) 
AL-8: toluene-methanol (86: 14) 2 x 15 cm; DAC 
Fig. 17 A, B 
Fig. 18A, B 
Fig.18C 
Detection Without chemical treatment Fig. 17 A; 18A 
Fig. 17B; 18B 
Fig.18C 
Draggendorff-NaN02 reagent (No. 11 CjF, p. 300-301) 
10% Ethanolic HCl (DAC) 
For description of drugs see p. 57. Formulae p. 63-64. 
Chromatogram 
17 A 1 Chelidonii herba. In UV -365 nm, without treatment, the chromatogram usually 
shows two to three powerfully fluorescent yellow zones in the Rf range 0.1-0.4 
and three weaker, blue and yellow-green fluorescent zones in the Rfrange 0.75-0.9. 
There is a prominent yellow fluorescent zone at Rf ca. 0.2; directly above this 
is seen the narrow yellow-green zone of ehelerythrine, followed by the tailing yellow 
zone of sanguinarine (cf. T2). The pale white fluorescent zone of protropine is located 
in the same position as standard berberine (cf. Tl); this is usually overlapped by 
sanguinarine. 
17B After treatment with Dragendorff-NaN0 2 , ehelidonine (Rf ca. 0.6) and the other 
alkaloids form rapidly fading brown (vis.) zones. Papaverine (cf. T3) can be used 
for the comparison of Rf values. 
18 2 Colehici semen. In UV-365 nm many blue or yellow-green fluorescent zones are 
18 A seen between the origin and the solvent front. There are two prominent yellow 
zones in the Rf region of standard colchicine (cf. T4). Colehicine and the minor 
alkaloids, colchiceine, N-acetyldemecolcine, 1-ethyl-2-demethylcolchiceine and cor-
nigerine, give a yellowish fluorescence in UV-365 nm. 
Blue fluorescent zones are given by O-benzoylcolchiceine, N-formyl-deacetyl-
colchicine, colchicoside and N-methyldemecolcine. 
18 B Colehieine forms a brown (vis.) zone with DragendorJJ reagent and a lemon-yellow 
18 C (vis.) zone with ethanolie hydroehlorie acid. In solvent system AL-8 (DAC) colchicine 
(cf. T4) migrates at Rf ca. 0.15. 
82 
Remarks: Commercial sampies of standard colchicine contain an impurity that migrates in 
the higher Rf range and stains violet with Dragendorff-NaNOz; the same substance is present 
in low concentration in the extract. 
Fig.17 
Tl T2 Tl 
c 
Fig.18 
T4 2 T4 2 
T2 T3 
T4 2 
-FRONT 
Rf 
-0.5 
-START 
-FRONT 
Rf 
-0.5 
-START 
83 
Aconiti Tuber/Herba Sabadillae Semen 
J aborandi, Boldo Folium 
Lobeliae Herba 
Tracks 
Tests 
Solvent 
system 
1 = Aconiti tuber 
2 = Aconiti herba 
3 = Sabadillae semen 
Tl = aconitine (alkaloid mixture) 
T2=veratrine (alkaloid mixture) 
T3 = 10 beline 
4 = Lobeliae herba 
5=Jaborandi folium 
6 = Boldo folium 
T 4 = pilocarpine 
T5= boldine 
AL-1 : toluene-ethyl acetate-diethylamine (70: 20: 10) 
Detection Dragendorffreagent (DRG No. 11 C/F, p. 300-301) vis. Fig. 19A, B; 20B 
VlS. Fig.20A Iodoplatinate reagent (IPL No. 19, p. 302) 
For description of drugssee p. 57-58. Forrnulae p. 61, 64, 65. 
Chromato- Treatment of chromatograms of extracts of Aconiti tuber or herba, Sabadillae semen 
gram and Lobeliae herba with Dragendorff reagent shows that, in each case, the main 
19 A alkaloid zones migrate in the Rf range 0.6-0.75. 
1,2 Aconiti tuber or herba. Both drug extracts show different concentrations of the 
same two alkaloid zones at Rf ca. 0.6-0.65, which correspond to the aconitine alka-
loid mixture (cf. Tl). 
3 Sabadillae semen. Chromatograms of this drug show two main alkaloid zones at 
Rf 0.5-0.7 ("veratrine alkaloid mixture", cf. T2), two weaker zones at Rf ca. 0.8 
and 0.4, and a more prominent zone at Rf ca. 0.1. 
4 Lobeliae herba. The extract is characterized by the presence of the main alkaloid 
lobeline (cf. T3), which migrates at Rf ca. 0.65. 
20A With iodoplatinate reagent all the alkaloids give an immediate brown-violet colour 
(vis.), which later becomes a stable white zone on a grey-blue background. 
This reagent permits a better evaluation of the drug extract, because it also reveals 
the minor alkaloids. 
19B 5 laborandifolium. The main alkaloid ispilocarpine (cf. T4) at Rfca. 0.1. 
20B 6 Boldo folium. In system AL-1, the main alkaloid, boldine (cf. TS), mi grates at Rf 
84 
ca. 0.25. On untreated chromatograms, boldine fluoresces dark blue in UV-365 nm; 
it gives a pronounced brown (vis.) with Dragendorff reagent. 
A 
Fig.19 
Tl 2 T2 3 T3 
Fig.20 
Tl 2 T2 3 T3 
B 
4 T4 5 
B 
4 6 T5 
-FRONT 
Rf 
-0.5 
-START 
-FRONT 
Rf 
-0.5 
-START 
85 
Purine-containing and Miscellaneous Drugs 
Tracks 
Tests 
Solvent 
system 
1 = Cacao semen 
2 = Coffeae semen 
T1 = trigonelline 
T2 = theobromine 
T3 = caffeine 
T4 = theophylline 
3 = Nicotianae folium 
4 = Ephedrae herba 
5 = Spartii herba 
T5 = chlorogenic acid 
T6 = nicotine 
AN-i: ethyl acetate-methanol-water (100: 13.5: 10) 
AL-i: toluene-ethyl acetate-diethylamine (70: 20: 10) 
T7 = ephedrine 
T8 = sparteine 
Fig. 21A, B 
Fig.22A-C 
Detection Iodine-potassium iodide-HCI-reag. (I/HCI No. 20, p. 302) vis. Fig.21A 
Natural products-polyethyleneglycol reagent (NP/PEG no. 28, p. 303) 
UV-365 nm Fig.21B 
Dragendorffreagent with NaN02 (No. 11 C/F, p. 300-301) VIS. Fig.22A 
Ninhydrin reagent (NIH No. 29, p. 303) VIS. Fig.22B 
Iodoplatinate reagent (IPL No. 19, p. 302) vis. Fig. 22C, D 
For description of drugs see p. 57-59. Formulae p. 64-65. 
Chromatogram 
21 Al, 2 Caeao semen-Coffeae semen 
In solvent system AN-i, both drug extracts show the brown zone of eaffeine (cf. 
T3) at Rf ca. 0.5 after treatment with I/HCI reagent. 
1 Caeao semen. Theobromine (cf. T2) can also be detected below eaffeine. 
2 Coffeae semen. In addition to eaffeine, the alkaloid trigonelfine (cf. T1) is also visible 
at the start. 
21 B After treatment with NP/pEG reagent and visualization in UV-365 nm, chromato-
grams of Coffeae semen extracts reveal chlorogenie acid at Rf ca. 0.1 and caffeic 
acid at Rf ca. 0.75; this distinguishes the drug from other caffeine-containing drugs. 
22A 
22B/C 
22D 
86 
Remarks: Owing to the small amount of theophylline in Coffeae semen, its presence in the 
drug can only be demonstrated after enrichement of the extract. 
In addition to caffeine, other purine-containing drugs, e.g. Colae semen, Theae 
folium and Mate folium, usually contain detectable quantities of theobromine and 
theophylline (for % see Drug List on p. 58-59). 
3 Nieotianaefolium. The main alkaloid, nicotine (cf. T6), shows a pronounced quench-
ing of fluorescence in UV-254 nm. With Dragendorff reagent (solvent system AL-i) 
it gives only an unstable colour in the visible. In non-concentrated extracts, nornico-
tine and anabasine are only seen as weak zones in the middle and lower Rf ranges. 
4 Ephedrae herba. With ninhydrin reagent ephedrine appears with a light colour against 
a red-violet background of the TLC-plate; or it becomes more violet, depending 
on the extent of heating. Other reagents, like Dragendorff or iodoplatinate (Fig. 22 C) 
produce only faint zones in the visible. 
The isomers, pseudo-ephedrine and nor-pseudo-ephedrine, are not separated 
from ephedrine in these systems. 
5 Sarothamni (Spartii) seoparii herba. Sparteine (cf. T8) is the characteristic main 
alkaloid of the drug extract. With IPL reagent it gives a deep blue-violet (vis.) 
on a red-violet background (Flavonoids, cf. Fig. 14, p. 184). 
A 
Fig.21 
Tl T2 T3 
A c 
Fig.22 
T6 3 T7 4 
2 T4 2 
T7 4 T8 
TS 
5 
FRONT 
Rf 
FRONT 
Rf 
TART 
87 
Solanaceae Drugs I 
Tracks 
Tests 
1 = Belladonnae folium 
2 = Hyoscyami folium 
3 = Stramonii folium 
Tl = Ph. Eur. standard solution (for Belladonnae folium), see p. 53 
T2 = Ph. Eur. standard solution (for Hyoscyami folium), see p. 53 
T3 = Ph. Eur. standard solution (for Stramonii folium), see p. 53 
T4=atropine 100 llg 
T5 = atropine 50 llg 
T6 = atropine 25 llg 
Solvent AL-l : toluene-ethyl acetate-diethylamine (70: 20: 10) Fig. 23A, B 
Fig. 24A, B system AL-4: acetone-water-conc. ammonia (90: 7: 3) 
Detection Dragendorff reagent (No. 11 C) vis. Fig. 23A 
Dragendorff-NaN02 reagent (No. 11 CjF, p. 300-301) vis. Fig. 23B; 24A, B 
For description of drugs see p. 59-60. Formulae p. 63. 
Chromatogram 
23 A Belladonnae, Hyoscyami and Stramonii folium 
(- )-Hyoscyamine (or atropine) is the main alkaloid in all three Solanaceae drugs. 
The TLC differentiation of these drugs is based on the hyoscyamine: scopolamine 
ratio, and to a limited extent on the contents of the minor alkaloids, belladonnine, 
atropamine and cuskhygrine. Detection of the minor alkaloids, however, requires 
special enrichment prior to chromatography. 
For identification and determination of alkaloid content, Ph. Eur. describes a 
TLC comparison with alkaloid mixtures containing defined ratios of atropine-S04 
to scopolamine-HBr (cf. T1-T3). Identification of the drug is then based on the 
similarity of colour intensity and zone size between the standard solutions and 
drug extracts. 
1 Belladonnae folium. The ratio hyoscyaminejatropine (Rf ca. 0.25) to scopolamine 
(Rf ca. 0.4) corresponds to that of Tl. 
2 Hyoscyami folium. The hyoscyaminejatropine: scopolamine ratio of standard solu-
tion T2 is about 3: 1. The total alkaloid content of the drug does not meet the 
quality requirements of the pharmacopoeia, it is generally too low in the stored 
commercial drugs. 
3 Stramonii foiium. The typical hyoscyaminejatropine: scopolamine ratio for this drug 
is ab out 2: 1. The total alkaloid content of this sampie is higher than that demanded 
by the pharmacopoeia. The concentration of scopolamine in the drug extract is 
higher than that in T3. 
24A In solvent system AL-4 (Ph. Eur.) hyoscyaminejatropine (Rf ca. 0.1-0.15) mi grates 
in the lower Rf range, while scopolamine (Rf ca. 0.8) is found in the upper region 
of the chromatogram. 
88 
Remarks: Dragendorff reagent, specified by Ph. Eur. (orange zones; vis.), suffers from the 
dis advantage that it gives unstable colours with tropane alkaloids. Subsequent treatment with 
sodium nitrite intensifies the colour and increases the stability of the colour of hyoscyamine/ 
atropine, but not of scopolamine (Fig. 23 B, 24A). 
Fig.23 
Tl 2 T2 3 T3 
Fig.24 
Tl 2 T2 3 T3 
T2 3 T3 
• 
T4 T5 T6 
-FRONT 
Rf 
-0.5 
-START 
-FRONT 
Rf 
-0.5 
-START 
89 
Solanaceae Drugs 11 
Tracks 1 = Belladonnae folium 8 = Scopoliae radix 
Tests 
2 = Stramonii folium 
3 = Stramonii semen 
4 = Hyoscyami mutici folium 
5 = Hyoscyami nigri folium 
6 = Belladonnae semen 
7 = Belladonnae radix 
(1-7: concentrated chloro-
form extracts; 150 Ilg) 
T1 = atropine (Rf ca. 0.25) 
scopolamine (Rf ca. 0.35) 
T2 = atropine 
9= Belladonnaeradix 
10= Belladonnae folium 
11 = Stramonii folium 
12 = Hyoscyami nigri folium 
13 = Hyoscyami mutici folium 
(8-13: methanol extract (see 
p. 163); 20 111 applied to 
chromatogram) 
T3 = rutin (Rf ca. 0.35), chlorogenic acid 
(Rf ca. 0.45), hyperoside (Rf ca. 0.6) 
T4=caffeic acid 
T5 = scopoletin 
Solvent 
system 
AL-1 : toluene-ethyl acetate-diethylamine (70: 20: 10) 
F-1: ethyl acetate-formic acid-glacial acetic 
Fig. 25A, B 
Fig.26 
acid-water (100: 11 : 11 : 27) 
Detection Dragendorffreagent (No. 11 C) with NaN0 2 (No. 11 F, p. 301) VIS. Fig.25 
Natural products-polyethyleneglycol reagent 
(NP/PEG No. 28, p. 303) UV-365 nm Fig.26 
For description of drugs see p. 59. Formulae p. 63. 
Chromato- Tropine alkaloids. The chromatogram in Fig. 25A was obtained by applying rather 
large quantities of the extracts (ca. 150llg, cf. p. 89). It then permits the following 
differentiation: 
gram 
25A, B 
2, 3 The scopolamine content of Stramonii semen is higher than that of Stramonii folium. 
4, 5 The hyoscyamine and scopolamine contents of the industrial drug, Hyoscyamus muticus, (total 
ca. 1 % according to the literature) are much higher than those of the official drug. 
1,6,7 Belladonnae folium contains a higher amount of hyoscyamine than Belladonnae radix or 
semen. 
Remarks: It is difficult to identify unequivocally Solanaceae drug extracts only on the basis of 
the alkaloid content. The flavonoid or coumarin patterns are a more certain guide to identitiy. 
26 8-13 Flavonoids, coumarins 
90 
8 Scopoliae radix gives five intensely blue fluorescent zones in the Rf range 0.1-0.5, with a 
main zone at Rf 0.2. Scopoletin (cf. T5) with traces of caffeie acid (cf. T4) are responsible 
for blue fluorescence in the region of the solvent front. 
9 Belladonnae radix. The main zone is that of scopoletin (T5; blue fluorescence in the region 
of the solvent front). There are also four less intense zones between the start and Rf 0.5, 
which fluoresce dark blue or blue-green. 
10-13 The TLC of all lea! drugs reveals chlorophyll (red fluorescence, UV-365 nm) at 
the solvent front. 
10,12 On chromatograms of Belladonnae and Hyoscyami nigrifolium, the main zones are rutin (Rf 
ca. 0.4; orange fluorescence) and chlorogenie acid (Rf ca. 0.45; blue fluorescence). In Hyoscyami 
nigri folium, these are the only two detectable zones, whereas Belladonnae folium shows addi-
tional blue, yellow-green and orange fluorescent zones in the Rf range 0.05--0.1 (7-glucosyl-3-
rhamnogalactosides of kampferol and quercetin). 
11 Stramonii folium is characterized by five orange fluorescent zones (quercetin glyeosides) in 
the Rf range 0.03-0.25. The absence of rutin and chi orogenie acid c1early distinguishes this 
drug from Belladonnae and Hyoscyami folium. 
13 Hyoscyami mutici folium has only a low total flavonoid content, and the presence of other 
compounds causes adepression of Rf values. 
A 
Fig.25 
Tl 2 3 4 5 
Fig.26 
T3 T4 T5 8 9 10 
B 
6 7 
11 12 13 
T2 
-FRONT 
Rf 
-0.5 
-START 
FRONT 
Rf 
START 
91 
Drugs Containing Anthracene Derivatives 
The characteristic constituents of this drug group are anthraquinones and their 
reduced derivatives, oxanthrones, anthranols and anthrones. The anthraquinones, 
which have purgative properties, possess phenolic groups on C-1 and C-8, and 
keto groups on C-9 and C-10. In the anthrones and anthranols, only C-9 carries 
an oxygen function. In addition, a methyl, oxymethyl or carboxyl group may be 
present on C-3, and an hydroxy or methoxy group on C-6. 
Most compounds in this group are present in the plant as O-glycosides. The 
glycoside linkage is usually at C-1, C-8 or C-6. C-Glycosides are also present: these 
occur in the anthrone form only, with the C-C bond always at C-10. 
In the 0- and C-glycosides, the only sugars found so far are glucose, rhamnose 
and apiose. 
I. Extraction of Drugs and Pharmaceutical Preparations for TLC 
1. Drugs 
Powdered drug (0.5 g) is extracted by warrning for 5 min on the water bath with 
5 ml of methanol. The dear filtrate is used directly for TLC. 
Exception: Sennae folium or fructus are extracted with 50% methanol. 
2. Pharmaceutical preparations 
Three finely powdered dragees or tablets are extracted on a water bath with 5 ml 
of methanol. The dear filtrate is used directly for TLC. 
3. Hydrolysis of Rhei radix, according to DAß 8 
A sampie (0.5 g) of the drug is heated under reflux for 15 min with 25 ml of 7.5% 
hydrochloric acid. After cooling, the mixture is extracted by shaking with 20 ml 
of ether. The ether phase is concentrated by evaporation and applied to the TLC 
plate (see DAB 8). 
11. Thin Layer Chromatography 
1. Reference solutions 
Aloin, frangulin AlB, glucofrangulin AlB and the aglycones, rhein, Aloe-emodin and 
Frangula-emodin, are applied to the TLC plate as 0.1 % methanolic solutions. 
The mixture of sennosides A and Bis prepared as a 0.1 % solution in n-propanol-
ethyl acetate-water (4:4:3). 
The stilbene glucoside, rhaponticin (= rhaponticoside) is applied as a 0.1 % metha-
nolic solution. Commercial "rhaponticin" also contains deoxyrhaponticin. 
2. Adsorbent 
Chromatography is perforrned on silica gel 60F 254 pre-coated plates (Merck, Darm-
stadt) (see also Circular TLC IV, p. 95). 
93 
94 
3. Sampie concentration 
Aloe extracts, 5 111. 
Rheum, Frangula, Cascara and Senna extracts, 10111. 
Reference solutions, 10 111. 
4. Chromatography solvents 
With the exception of Senna preparations, the solvent system suitable for the chro-
matography of all drug extracts is ethyl acetate-methanol-water (100: 17: 13; DAB 8 
and Ph. Eur.). 
AN-1: ethyl acetate-methanol-water (100:17:13) 
AN-2: ethyl acetate-methanol-water (100: 13.5: 10) 
This slightly different ratio of components prevents the "wave shaped" sepa-
ration, which is often observed with AN-i. On the other hand, AN-2 is 
also suitable for other types of constitutents, such as cardiac glycosides, 
bitter principles and some alkaloids. 
AN-3: n-propanol-ethyl acetate-water (40: 40: 30) 
Sennae folium and fructus (Ph. Eur. I) 
AN-4: toluene-ethyl formiate-formic acid (50: 40: 10) 
Hypericine from Hyperiei herba (DAC) 
AN-5: light petroleum-ethyl aeetate-formie acid (75: 25: 1) 
Anthraquinone aglyeons (Rhei radix, DAB 8) 
111. Detection 
1. Without chemical treatment 
UV-254 nm 
All anthraquinone derivatives queneh fluoreseence 
UV-365 nm 
All anthraquinone derivatives give yellow or red-brown fluoreseenee 
2. Spray reagents 
a) Potassium hydroxide (KOH No. 21, p. 302: Bornträger reaetion) 
After spraying with 5% or 10% ethanolie KOH, anthraquinones show red in the 
visible, and red fluoreseence in UV-365 nm. Anthrones and anthranols show yellow 
in the visible, and yellow fluoreseence in UV-365 nm. 
For detection of dianthrones, see 2e. 
b) Natural produets-polyethyleneglyeol reagent (NP/PEG No. 28, p. 303) 
Anthrones and anthranols give an intense yellow fluoreseenee in UV -365 nm. 
e) Sennoside deteetion 
Nitrie acid reagent (HN0 3 No. 33, p. 303) with potassium hydroxide reagent (KOH 
No. 21, p. 302) Ph. Eur. 
The TLC plate is sprayed with eone. HN03 , then heated for 10 min at 1200 C. 
It is then sprayed with 5% ethanolic KOH. After further heating, sennosides appear 
as yellow zones in UV-365 nm, and red-brown zones in the visible. 
Remarks: Sennosides can also be detected with a 1 % soln. of sodium metaperiodate in 10% 
ethanolic KOR. After spraying and heating (ca. 5 min), yellow-brown zones are obtained 
in UV-365 nm. 
d) Phosphomolybdate-H2 S04 reagent (PMA-H 2S04 No. 27, p. 303) 
Rhaponticin and deoxyrhapontiein give intense blue zones in the visible. 
e) Hypericin detection (DAC) 
A 10% soln. of pyridine in acetone intensifies the red fluorescenee of hyperiein 
in UV-365 nm.IV. TLC Methods 
Anthraquinone derivatives ean be separated by the usual aseending TLC method 
or by eireular TLC. 
Circular thin layer chromatography 
1. Method. The solvent mi grates circularly from the point of application, so that the 
zones form ares. 
2. Adsorbent. Silica gel 60F 254 pre-eoated plates, 20 x 20 cm (Merek, Darmstadt). 
3. Solvent systems. The same solvent systems ean be used as for ascending TLC. 
4. Method of application. Two diagonal pencil lines are drawn from the corners of 
the TLC plate. The eentre point of the plate is marked and a eirc1e is drawn around 
it with a diameter of ca. 2 cm. 
The circ1e is thus divided into fOUf segments by the diagonals. The perimeter 
of each segment serves for the application of drug extracts or reference solutions 
(Fig. 1, below). 
5. Development. 100 ml of solvent are placed in a round, straight sided chamber (glass 
trough, 10 cm high, 20 cm diameter). A glass funnel is loosely packed with cotton, 
which extends as a wick through the tube of the funnel. The funnel is placed in 
the solvent system, so that the solvent soaks into the cotton. With the loaded side 
facing downwards, the TLC plate is plaeed over the top of the trough, so that 
the wick from the funne1 makes contact exaetly at the marked centre. 
The solvent spreads as a circ1e over the TLC plate, and the zones ofthe separating 
substanees form ares, whieh inerease in length from the starting point to the peri-
phery of the spreading solvent. 
6. Detection. All the same deteetion methods can be used as for ascending TLC. 
7. Application. This method is especially useful for drug extracts containing a high 
proportion of ballast substances, e.g. mucilages from Sennae folium. Good separa-
tions are obtained with solvent migrations of 5-6 cm. 
~/s~~ "". = .. =:,=-==-==-=-, /-Y .;: '.' ~.\.--= '='='::-:" .=;:::::=-.1 
• / .. "' . I\. • ~.- .. 
Fig.l Glass trough 
Funne l 
+ wick 
Solvent 
system 
95 
v. List of Drugs Containing Anthracene Glycosides 
Chromatograms (Figs. 1-14) are reproduced on pp. 102-114. 
Fig. 
1,2 
3 
Drug/Plant source 
Farnily /Pharmacopoeia 
Aloe extractum 
Aloe extract 
Various types of Aloes are de-
scribed by the pharmacopoeias 
(Cape Aloes and Curacao Aloes), 
and as commercial drugs (Uganda 
Aloes, Kenya Aloes and Indian 
Aloes); 
see below 
Aloe barbadensis 
Curacao aloes 
Aloe barbadensis MILLER 
Liliaceae 
USP XX, Ph. Eur. III, Helv. VI 
Aloe capensis 
Cape aloes 
Aloe ferox MILLER and hybrids 
Liliaceae 
USP XX, Ph. Eur. III, Helv. VI, 
ÖAB 
2. AB-DDR 
Aloe perryi 
Socotrine aloes 
Aloe perryi BAKER 
Liliaceae 
Main constituents (anthraquinone deriva-
tives) % content and identity 
Dried extract (aqueous) of Aloe spp., contain-
ing 
Aloin (10-C-ß-D-glucopyranoside of Aloe-
emodin-anthrone), in most cases as a mixture 
of the cx- and ß-stereoisomers 
Aloinoside A and B (stereoisomers of aloin-
11-cx-L-rhamnoside) 
Aloesine A and B (" Aloe resin" with no pur-
gative action) 
Aloesine B (chromone-C-glucoside) 
Aloesine A (p-coumaric acid ester of aloe-
sine B) 
Aglycone: Aloe-emodin 
Not less than 28% hydroxyanthracene deriva-
tives ca1culated as aloin (Ph. Eur.) 
Aloin 
Aloesine A and B (" Aloe resin ") 
Not less than 18% hydroxyanthracene deriva-
tives ca1culated as aloin (Ph. Eur.) 
Aloin, aloinosides AlB, aloesines AlB (type 
Cape A) 
Aloin, aloesines AlB (type Cape B) 
ca. 14% hydroxyanthracene derivatives ca1cu-
lated as aloin 
Aloin, aloinosides AlB, aleosines AlB 
IsoharhaÜJin test (after Klunge) for the differentiation of Aloe capensis and Aloe 
barbadensis. 
One drop of saturated CuS04 soln., 1 g NaCI and 10 ml of 90% ethanol are added 
to 20 ml of an aqueous soln. of Aloe barbadensis (Curacao aloes) (1: 200); a wine 
red colour is produced, which is stable for at least 12 h. Solutions of Aloe capensis 
fade rapidly to yellow. 
Rhamni purshiani Cortex 
Cascarae sagradae cortex 
Cascara sagrada bark 
Sacred bark 
Chittem bark 
Rhamnus purshianus DE CANDOLLE 
Rhamnaceae 
USP XX, Ph. Eur. II, ÖAB 
Not less than 8% hydroxyanthracene deriva-
tives, of which at least 60% are cascarosides 
ca1c. as cascaroside A (Ph. Eur. II) 
Cascarosides A and B (diastereoisomers of 
aloin-8-0-ß-D-glucoside) 
Cascarosides C and D (diastereoisomers of 
deoxyaloin-8-0-ß-D-glucoside) 
Aloin and deoxyaloin 
97 
98 
Fig. 
5,6 
7,8 
13,14 
DrugjPlant source 
Family jPharmacopoeia 
Frangulae Cortex 
Rhamni frangulae cortex 
Alder buckthorn bark 
Rhamnus frangula L. 
Rhamnaceae 
Ph. Eur. H, 2. AB-DDR, Helv. VI, 
ÖAB 
Frangulae Fructus 
Alder buckthorn fruits 
Rhamnus frangula L. 
Rhamnaceae 
Oreoherzogiae Cortex 
Rhamni faIIaci cortex 
FaIIax bark 
Rhamnus faIIax (Boiss.) VENT. 
Rhamnaceae 
Rhamni cathartici Fructus 
Buckthorn berries 
Rhamnus catharticus L. 
Rhamnaceae 
Main constituents (anthraquinone deriva-
tives) % content and identity 
Not less than 6% anthraquinone glycosides 
(Ph. Eur. H) 
The content of anthranol derivatives should 
not exceed 30% (Helv. VI) 
Main glycosides: Glucofrangulin A and B 
(emodin -6-0-ex-L-rhamnosyl -8-0-ß-D-gluco-
side and emodin-6-0-ex-L-apiosyl-8-0-ß-D-glu-
coside) 
Frangulin A and B (emodin-6-0-ex-L-rhamno-
side and emodin-6-0-ex-L-apioside) 
Mono- and diglucosides of emodin, glycosides 
of physcione and chrysophanol, and free agly-
cones. 
Low concentrations of anthraquinone agly-
cones and traces of anthraquinone glycosides 
are detectable. 
1-3% Hydroxyanthracene derivatives. 
Falax bark is an adulterant of alder buck-
thorn bark. 
Low contents of frangulin and glucofrangulin. 
Higher concentrations of the flavonol glyco-
sides: catharticin (rhamnocitrin-3-0-ß-rham-
ninoside), xanthorhamin (= 7-methyl-querce-
tin; rhamninoside of rhamnetin) and rhamna-
zin (3,7' -dimethylquercetin), kaempferol-3-0-
ß-D-rhamninoside. 
Remarks: Rhamni cathartici cortex contains naphthalide glycosides (ex-sorigenin glu-
coside and primveroside, and ß-sorigenin primveroside) and a low concentration 
of anthraquinone derivatives; it may be found as an adulterant of Frangulae cortex. 
Rhei Radix 
Rhubarb rhizome 
Rheum palmatum L. 
Rheum officinale BAILLON (and hy-
brids) 
Polygonaceae 
DAB 8, ÖAB, Helv. VI, 
2. AB-DDR 
3-7.5% Hydroxyanthracene derivatives 
(DAB 8 specifies not less than 3% caIc. as 
rhein) 
Main glycosides: mono- and di-glucosides of 
physcione, chrysophanol and rhein. 
Aglycones: rhein, physcione, chrysophanol, 
emodin. 
Anthrones: rheidine, sennidine. 
Adulterant: Rhei rhapontici radix (R. rhapon-
ticum L.), which contains the stilbene deriva-
tives, rhapontin (rhapontizin) and deoxyrha-
pontin. 
Fig. 
9, 10 
11,12 
10 
DrugjPlant source 
F arnily jPharrnacopoeia 
Sennae Folium 
Senna leaves 
Cassia senna L. (Alexandrian 
senna) 
Cassia angustifolia V AHL (Tinne-
velly senna) 
Sennae Fructus 
Senna pods 
Cassia senna L. 
Cassia angustifolia V AHL 
Caesalpiniaceae 
USP XX, Ph. Eur. I ÖAB, 
He1v. VI, 2.AB-DDR 
Hyperici Herba 
St. Iohn's wort 
Hypericum perforatum L. 
Hypericaceae 
DAC 
Main constituents (anthraquinone deriva-
tives) % content and identity 
Both drugs contain 2-3.5% dianthrone glyco-
sides. 
Sennae folium: Ph. Eur. I specifies not less 
than 2.5% ca1c. as sennoside B. 
Sennae acutifoliae frnctus (Alexandrian senna 
pods): not less than 3.6% (Ph. Eur. I) 
Sennae angustifoliae fructus (Tinnevelly senna 
pods): not less than 2.5% ca1c. as sennoside B 
(Ph. Eur. I). 
The chief compounds are the dianthrone gly-
cosides, sennoside A and B. Sennoside C and 
D are also present, 
Mono- and diglycosides of emodin and rhein. 
Rhein is found especially in Sennae folium. 
Of the flavonoids present in Sennae fructus 
and folium, rntin can be detected only in Sen-
nae folium.Main compounds: 
Dehydrodianthrone compounds (no purgative 
action): hypericin, isohypericin, protohyperi-
cin. 
Flavonoids: rutin, hyperoside. 
Chlorogenie acid. 
99 
VI. Formulae of Anthraquinone Derivatives Identitied as Drug Constituents 
OH 
RO 
OH 0 OH 
Aloin A 
Aloinoside A 
o OH 
o 
Gluc-O 0 OH 
OH 0 OH 
Aloin B 
Aloinoside B 
@R 
H Gluc 
100 
R, R2 
CH3 OH Rheum 
(= Frangula)-Emodin 
CH 3 OCH3 Physcione 
CH 3 H Chrysophanol 
CH2 0H H Aloe-Emodin 
COOH H Rhein 
R R, 
Glucosyl Rhamnosyl 
Glucosyl Apiosyl 
Glucofrangulin A 
Glucofrangulin B 
Frangulin A 
Frangulin B 
H Rhamnosyl 
H Apiosyl 
R 
CH20R H Aloin A, B 
Rhamnosyl Aloinoside A, B 
R 
CH 20H Cascaroside A, B 
CH3 Cascaroside C, 0 
Gluc-O 
Gluc-O 
0 OH 
Sennoside A: R, R, = COOH( + )-form 
R Sennoside B: R, R, = COOH meso-form 
0 OH 
HO 
HO 
R, Sennoside C: R=COOH 
Sennoside D: R=COOH 
HO 0 OH 
Hypericin 
HO OH 
P-CH=CH-60CH, 
R-O 
R = GI ucosyl : Rhaponticoside 
R= H: Rhapontigenin 
R, = CH 20H( + )-form 
R, = CH 20H meso-form 
101 
Aloe Resina 
Tracks 1 = Aloe capensis (Cape type A) 
2=Aloe capensis (Cape type B) 
3=Aloe barbadensis (Curacao aloes) 
4 = Aloe perryi (Socotrine aloes) 
5 = aloes (Kenyan drug) 
6 = aloes (U gandan drug) 
7 = aloes (Indian drug) 
Test T = aloin (Rf 0.45-0.50), Aloe-emodin (solvent front) 
Solvent AN-2: ethyl acetate-methanol-water (100:13.5:10) 
system 
Detection Potassium hydroxide (KOH No. 21, p. 302) 
Natural products-polyethyleneglycol reagent 
(NP/PEG No. 28, p. 303) 
UV-365 nm Fig. 1 
UV-365 nm Fig.2 
For description of drugs see p. 97. Formulae p. 100. 
Chromato- After treatment of chromatograms with KOH reagent, the aloe resins (1-7) are 
gram seen to contain different amounts of aloin (Rf ca. 0.4). They can be distinguished 
1 mainly by the presence or absence of aloinosides A and B (RfO.2-0.25) and aloesines. 
Aloesine Amigrates directly above aloin, and aloesine B dose to and below the 
aloinosides. 
In UV -365 nm, aloin and aloinosides A and B show the typical yellow-orange 
fluorescence of anthrones. The aloesines gives strong light blue fluorescent zones. 
The red fluorescent zone of aloe-emodin lies at the solvent front. 
The upper and lower Rf ranges contain further blue fluorescent zones, which 
can be used for differentiation (e.g. RfO.7=p-Coumaric acid methylester). 
2 NP/PEG reagent intensifies the yellow fluorescence of aloin and the aloinosides 
in UV-365 nm. Under these conditions the aloesines fluoresce only slightly blue. 
102 
Differentiation of aloe resins 
Aloe resin Aloin Aloinosides Aloesines AlB Rernarks 
1 Cape aloes type A ++ ++! ++ 
2 Cape aloes type B ++ ++ 
3 Curacao aloes ++ ++ 1. 2 zone X 
4 Socotrine aloes ++ ++! ++ 2 zone X 
5 Kenya aloes ++ + (+) 
6 Uganda aloes ++ (+) 
7 Indian aloes (+ ) +++ (+) 
1 Curacao and Cape aloes can be differentiated by the isoharhaloin test of Klunge (see p. 97), 
in which they give a wine red or yellow colour, respectively. 
2 Zone X appears as a dark zone in UV-365 nrn and as a red-brown zone in the visible, 
direcdy below aloin (Curacao aloe: X = 5-Hydroxyaloin). 
Aloinosides are present in Cape aloes type A, but not in Cape aloes type B. 
Socotrine aloes show aloinosides and an additional dark brown zone (zone X) below 
aloin. 
In contrast to Socotrine aloes, Curacao aloes have no aloinosides. 
Aloes 5-7 contain only traces of aloesines. 
Indian aloes show only aloinosides, and no aloin. 
Fig.l 
T 2 3 4 5 
Fig.2 
T 2 3 4 
6 7 
5 6 7 
FRONT 
Rf 
START 
FRONT 
Rf 
START 
103 
Rhamni purshiani Cortex 
Tracks 
Test 
Solvent 
system 
1-4 = Rhamni purshiani cortex (Cascarae cortex) 
(commercial drugs from various sources) 
Tl =aloin (Rf ca. 0.5), aloinoside A (Rf ca. 0.45) 
AN-l: ethyl acetate-methanol-water (100: 17: 13) 
Detection Natural products-polyethyleneglycol reagent 
Chromato-
gram 1-4 
3 
(NPjPEG No. 28, p. 303) UV-365 nm Fig.3 
For description of drugs see p. 97. Formulae p. 100. 
Cascarae cortex. After treatment with NPjPEG reagent, chromatograms of Cascarae 
cortex extract show two pairs of strikingly yellow fluorescent zones in UV-365 nm; 
these are due to cascarosides A and B (Rf 0.10-0.15) and cascarosides C and D 
(Rf 0.20-0.25). Weak yellow zones appear at Rf ca. 0.5 (aloin A, B), Rf ca. 0.6 
(deoxyaloin). The predominant cascarosides are normally A and B. 
The red fluorescent zones at the solvent front are due to the aglycones, emodin, 
aloe-emodin and chrysophanol. 
Additional blue and green-blue fluorescent zones are seen in the Rf range 
0.35-0.75. All drug sampies show 4-5 blue fluorescent zones below the standard 
aloin (presumably naphthalide derivatives). 
TLC Synopsis 
Tracks 
Test 
Solvent 
system 
5 = Aloe capensis 
6 = Aloe barbadensis 
7 = Rhamni purshiani cortex 
8 = Frangulae cortex 
T2 = aloin (Rf ca. 0.5), frangulin A (Rf ca. 0.85) 
AN-l: ethyl acetate-methanol-water (100: 17: 13) 
Detection Potassium hydroxide reagent (KOR No. 21, p. 302) 
Chromato-
gram 5-8 
4 
104 
UV-365 nm Fig.4 
For description of drugs see p. 97-98. Formulae p. 100. 
Chromatograms of the four official drugs, Cape aloes, curacao aloes, american and 
official alder huckthorn hark, can be differentiated after treatment with KOR reagent 
and inspection in UV -365 nm, by the presence of characteristic yellow or red fluores-
cent zones at defined Rf values. 
Yellow fluorescent zones are due to the an throne glycosides, aloin and deoxyaloin, 
and to aloinosides and cascarosides (see p. 102-104). 
Red fluorescent zones are given by frangulins A and B, glucofrangulins A and B 
and their aglycones (see p. 106). 
Remarks: AN-l has a higher methanol and water content than AN-2, and it separates aloin 
and aloinosides in a higher Rf range (cf. aloin Rf ca. 0.4, AN-2, Fig. 1, p. 102). The higher 
methanol and water content often results in the formation of wavy zones (see (Fig. 3/4). 
Fig.3 
Tl 2 
Fig.4 
T2 5 6 
3 4 
7 8 
FRONT 
Rf 
START 
FRONT 
Rf 
105 
Frangulae Cortex and aduIterant. Rhamni cath. Fructus 
Tracks 
Tests 
Solvent 
system 
1 = Frangulae cortex 
2 = Oreoherzogiae cortex 
3 = Frangulae fructus 
4 = Rhamni cath. fructus 
Tl = glucofrangulins A and B (Rf ca. 0.25-0.3), aloin (Rf ca. 0.45), 
frangulin A (Rf ca. 0.75), frangula-emodin (solvent front) 
T2 = rutin (Rf ca. 0.4), chlorogenic acid (Rf ca. 0.45), 
hyperoside (Rf ca. 0.6) 
AN-2: ethyl acetate-methanol-water (100:13.5:10) Fig. 5A, B; 6A 
F-l: ethyl acetate-forrnic acid-glacial acetic 
acid-water (100: 11 : 11: 27) Fig. 6B 
Detection Potassium hydroxide reagent (KOH No. 21, p. 302) 
Chromato-
gram 1 
5A, B 
5A, B 
5B 
6A/B 
5B 
6A 
6B 
106 
vis./UV-365 nm Fig. 5A, B 
Natural products-polyethyleneglycol re agent 
(NP/PEG No. 28, p. 303) 
For description of drugs see p. 98. Formulae p. 100. 
UV-365 nm Fig. 6A, B 
Frangulae cortex. The KOH-treated chromatogram is characterized by four red (vis.), 
or red fluorescent (UV-365 nm) zones; these are Jrangulins A and B (Rf 0.75-0.85) 
and glucoJrangulins A and B (Rf 0.25-0.35). The aglycones,frangula-emodin, physcion 
and chrysophanol, are not separated and migrate with the solvent front. 
Additional anthraquinones are detected above the glucofrangulin zones, 
especially at Rf ca. 0.5 (emodin monoglycosides). 
The yellow zone directly below the glucofrangulins is due to a flavonol glycoside 
(see also Fig. 6A, UV-365 nm). 
2 Oreoherzogiae cortex. KOH treatment shows the red zones (vis. and UV-365 nm) 
of the aglycones (solvent front), anthraquinone monoglycosides (Rf ca. 0.5) and digly-
cosides (Rf 0.2-0.35), and glucoJrangulins A and B (Rf ca. 0.25). Onlytraces of 
frangulins are present. In contrast to official alder buckthorn bark, four characteris-
tic red zones of the glycosides of emodin and physcione are seen above the zone 
of the glucofrangulins. As in Frangulae cortex, a flavonol glycoside is detectable 
below the zone of the glucofrangulins. 
3 Frangulae Jructus. After KOH treatment the chromatogram shows only one red 
zone (vis. and UV-365 nm) in the aglycone region. 
After NP/PEG treatment, yellow-green fluorescent zones (UV-365 nm) are detect-
able at Rf 0.15 and 0.4-0.45, and at the solvent front. The lowest fluorescent zone 
(see also solvent system F-l, Fig. 6B) is due to the flavonol glycoside, catharticin. 
4 Rhamni cathartici Jructus. After KOH treatment only glucoJrangulin A, Jrangulin A 
and emodin are detectable (vis. and UV-365 nm) (cf. Tl). 
NP/PEG reagent produces several yellow, and green-orange zones (UV-365 nm) 
in the Rf ranges 0-0.25, and 0.7 to the solvent front, respectively (solvent system 
AN-2). 
Overlapping yellow and orange fluorescent zones are seen in the lower Rf range 
(catharticin, "xanthorhamnin" and kaempJerol-3-0-rhamninoside). The yellow fluo-
rescent zone of catharticin is found in extracts of Frangulae and Rhamni cath. 
fructus. 
Fig.5 
Tl 2 Tl 2 
Fig.6 
3 4 T2 
3 4 
4 3 
-FRONT 
Rf 
-0.5 
-START 
107 
Rhei Radix 
Tracks 
Tests 
Solvent 
system 
Detection 
1 = Rhei rhapontici radix 
2 = Rhei palmati radix 
3 = Rhei radix hydrolysate (see p. 93) 
Tl = rhein 
T2 = rhapontizin/deoxyrhapontizin mixture (Rf 0.45-0.55) 
AN-2: ethyl acetate-methanol-water (100: 13.5: 10) 
AN-5: light petroleum-ethyl acetate-formic acid (75: 25: 1) 
Potassium hydroxide reagent (KOH No. 21, p. 302) 
UV-365 nm/vis. 
Natural products-polyethyleneglycol reagent 
(NP/PEG No. 28, p. 303) UV-365 nm 
Phosphomolybdic acid-H2S04 reagent (PMA No. 27, p. 303) 
vis. 
For description of drugs see p. 98. Formulae p. 100. 
Fig. 7 A, B; 8A, B 
Fig.8C 
Fig. 7 A, B; 8C 
Fig.8A 
Fig.8B 
Chromato- 2 Rhei radix. After treatment with KOH, chromatograms of the official drug are 
gram characterized by zones at Rf 0.45 and 0.55, which are orange-red in UV-365 nm 
7 A, Band red in the visible. The lower zone is due to rhein (cf. Tl). The broad upper 
zone represents a mixture of 8-0-monoglucosides of emodin, physcione and chryso-
phanol. The corresponding diglycosides are present in lower concentrations in the 
Rf range 0.05-0.3. The aglycones mi grate as an orange-red zone at the solvent 
front. 
8A 1 Rhei rhapontici radix (adulterant). The pattern of anthraquinones corresponds closely 
to that of the official Rheum drug. 
In additon, especially after NP/PEG treatment, the intermediate Rf range con-
tains a broad light blue fluorescent zone (UV-365 nm) between rhein and the mono-
glycoside mixture; this zone contains the stilbene derivatives, rhaponticin (rhaponti-
coside) and deoxyrhaponticin. 
8B With PMA-H2S04 reagent, the stilbene glycosides give powerfully dark blue zones 
in the visible. 
8C 3 Rhei radix (aglycones). Hydrochloric acid hydrolysis (see p. 93) of Rheum extract 
108 
pro duces a mixture of aglycones. Aloe-emodin and rhein migrate at Rf 0.2-0.25, 
emodin at Rf ca. 0.35, chrysophanol and physcione at Rf 0.6-0.7. 
In UV 365 nm, all the aglycones fluoresce uniformly yellow, or orange-red if 
the chromatogram is treated with KOH (no Fig.). 
If the TLC plate is heated after KOH treatment (Fig. 8C), frangula(rheum)-
emodin appears as a dark zone in UV 365 nm. 
Remarks: see also Circular TLC Fig. 13/14, p. 114. 
Fig.7 
Tl T2 2 Tl 
Fig.8 
Tl T2 2 2 
T2 2 
T2 3 
-FRONT 
Rf 
-0.5 
-START 
-FRONT 
Rf 
109 
Dianthrones Dehydrodianthrones 
Sennae Folium, Fructus Hyperici Herba 
3 = Hyperici herba Tracks 
Tests 
Solvent 
system 
1 = Sennae folium 
2 = Sennae fructus 
T1 = rhein 
T2 = sennoside A 
T3 = sennoside B 
TA = rutin (Rf ca. 0.4), chlorogenie acid 
(Rf ca. 0.45), hyperoside (Rf ca. 0.6) 
AN-3: n-propanol-ethyl acetate-water (40:40:30) 
AN-4: toluene-ethyl formate-formic acid (50:40:10) 
F-l: ethyl acetate-formic acid-glacial acetic acid-water 
(100: 11: 11 :27) 
Fig. 9A, B 
Fig.10C 
Fig. lOA, B 
Detection Nitric acid-KOH reagent (No. 33, p. 303) 
Natural products-polyethyleneglycol reagent 
(NP/PEG No. 28, p. 303) 
vis./UV-365 nm Fig. 9A, B 
UV-365 nm Fig. lOA, B 
UV-365 nm Fig. lOC Pyridine (10% in ethanol) 
For description of drugs see p. 99. Formulae p. 101. 
Chromato- 1 Sennaefolium. After treatment with HN0 3 /KOH reagent, 7-9 red-brown (vis.) zones 
are seen between the start and the solvent front. In UV-365 nm the same zones 
fluoresce lemon yellow or light blue. The main zones are due to sennoside A (Rf 
0.4, cf. T2) and sennoside B (Rf 0.2, cf. T3). Sennoside Dappears at Rf 0.5--0.55, 
and directly above the rhein-8-0-glueoside is found. Sennoside Cis seen as a weakly 
coloured zone at Rf ca. 0.7, and rhein as an orange-red zone (cf. Tl) at Rf ca. 
0.8. The other aglycones migrate together at the solvent front. 
gram 
9A, B 
10A 
9A, B 
10A 
10C 
lOB 
110 
NP/PEG reagent (solvent system Fl) reveals at least 8 yellow to yellow-green fluores-
cent (UV-365 nm) jlavonoid zones. The main zone corresponds to rutin (cf. TA). 
Two further zones are found above and below rutin, two weak zones above the 
hyperoside standard (cf. TA, Rf ca. 0.6), and a zone of flavonoid aglycones that 
mi grates with the solvent front. 
2 Sennae fruetus. Compared with Sennae folium, the concentrations of sennosides A, 
B, C, D and rhein are somewhat lower. 
The jlavonoid pattern shows two main zones below rutin, two zones above hyperoside 
and a zone at the solvent front. Rutin is not present. 
3 Hyperici herba. In solvent system AN-4, the hypericins mi grate in the Rf range 
0.45-0.5. In UV 365 nm they appear as reddish zones (untreated chromatograms) 
or orange-red zones (after pyridine treatment). 
Chromatography in solvent system Fl, followed by NP/PEG treatment, shows the 
flavonoids, rutin and hyperoside, at Rf 0.35 and 0.6 (cf. TA), other weaker flavonoid 
zones at Rf ca. 0.7-0.8, and aglycones on the solvent front. Chlorogenie and neo-
ehlorogenie acid (Rf 0.45-0.55, cf. TA) give a light blue fluorescence, and the hyperi-
eins (Rf 0.9) show a red fluorescence. 
Fig.9 
Tl T2 TJ 2 Tl T2 
Fig. 10 
TA 2 TA 
TJ 2 
3 3 
FRONT 
Rf 
START 
FRONT 
Rf 
START 
111 
Sennae Folium, Fructus Circular TLC 
Segment 
Solvent 
system 
Sennae folium (upper segment) 
Sennae fructus (lower segment) 
sennoside-A=Test 1 +rhein=Test 4 
sennoside B = Test 2 + aloin = Test 5 
n-propanol-ethyl acetate-water (40: 40: 30) 
Detection HN03-KOH reagent (No. 33, p. 303) vis. Fig.11 
UV -365 nm Fig. 12 
For description of drugs see p. 99. Formulae p. 101. 
For description of circular TLC, see p. 95. 
Chromato- Sennae folium. The two yellow (vis.) zones of sennoside B (Test 2) and sennoside A 
gram (Test 1) have intermediate Rf values. Above these lie the weak red-violet zone of 
11 rhein-8-0-monoglucoside and the yellow zones of sennosides D and C on either side 
of the monoglucoside zone (cf. 3). 
12 
112 
Rhein is overlapped by yellow zones (Test 4). Weak zones of anthraquinone agly-
cones and flavonoids are found at the solvent front, just ahead of the aloin standard 
(cf. 5). 
Sennae fructus. As in Sennae folium, sennosides Band A (cf. Tests 2 and 1) are 
followed by the red zone of rhein-8-0-glucoside and rhein. 
In UV-365 nm sennoside B appears in both drug preparations in approximately the 
same concentration [yellow fluorescent zone (cf. standard 2)]. Sennoside A is more 
concentrated in Sennae folium than in Sennae fructus. 
Rhein-8-0-monoglucoside and rhein are more distinct in the visible than inUV-
365 nm, where they show only a weak orange-red fluorescence above Test 4. 
Sennae folium can be clearly distinguished from Sennae fructus by the presence 
of a pink fluorescent zone just above the aloin standard (not present in the extract). 
.... SENNAE FOLIUM 
4 
ENNAE FRUCTUS 
Fig.11 
.... SENNAE FOLIUM 
.... SENNAE FRUCTUS 
Fig.12 
113 
Rhei Radix Comparison of Circular TLC and Ascending TLC 
Tracks 
Tests 
Solvent 
system 
1= Rhei rhapontici radix 
2 = Rhei palmati radix 
3 = rhaponticin/deoxyrhaponticin mixture 
4= rhein 
AN-2: ethyl acetate-methanol-water (100: 13.5: 10) 
Detection Potassium hydroxide reagent (KOR No. 21, p. 302) vis. Fig. 13A, B 
UV-365 nm Fig. 14A, B 
For description of drugs see p. 98. Formulae p. 100. 
Chromato- Rhei radix 
gram Ascending TLC 
114 
1,2 The two red (vis.) or red-orange fluorescent (UV-365 nm) anthraquinone zones are 
due to rhein (Rf ca. 0.5) and to a mixture of 8-0-monoglucosides of emodin, physcione 
and chrysophanol (Rf ca. 0.6). 
(For further data, see Fig. 7/8, p. 108) 
Circular TLC 
With a development distance of 5-6 cm, the following zones can be identified in 
the visible: 
- aglycone mixture (rheum-emodin, physcione, chrysophanol) as a red zone on the 
periphery of the chromatogram. Then moving inwards towards the centre: 
- a weak orange zone 
- the main orange zone (mixture of the monoglucosides of the three aglycones) 
- a weak zone of rhein 
In UV-365 nm: 
- a pronounced orange fluorescent zone on the periphery (aglycone mixture). Then 
moving inwards towards the centre: 
- a dark zone between two blue fluorescent zones 
- the orange zone of the monoglucoside mixture 
- rhein, partly overlapped by blue fluorescent zones. 
In Rhei rhapontici radix, the strikingly light blue fluorescent zone of rhaponticin 
(rhaponticoside) is easily recognisable (cf. T3). 
2 
3 4 
Fig.13 
c-oc 
Fig.14 
c- OC 
2 
2 
FRONT 
Rf 
START 
FRONT 
Rf 
START 
115 
Arbutin Drugs 
The main constituents ofthe arbutin drugs are arbutin (hydroquinone-ß-O-glucoside) 
and methylarbutin, with small amounts of 2-0-galloyl-arbutin, 6-0-acetyl-arbutin 
and free hydroquinone. The presence of gallo- and ellagotannins is also characteristic 
of these drugs. 
I. Preparation of Extracts of Drugs and Pharmaceutical 
Preparations for TLC 
1. Drug extracts (for the detection of arbutin) 
The powdered drug (2 g) and calcium carbonate (0.2 g) are mixed, then extracted 
for 15 min under reflux (water bath) with 20 ml of 50% methanol. The mixture 
is filtered and the residue washed with 50% methanol to give a total filtrate volume 
of 20 ml. To remove tannins, 10 ml of this solution are treated with 0.5 g basic 
lead acetate, shaken, then filtered. The pale yellow filtrate (30 ~l) is applied direct1y 
to the chromatogram. 
2. Extraction of bearberry leaves according to DAß 8 
The powdered drug (5 g), is extracted with 75% methanol (50 ml) under reflux 
for 30 min, then filtered. The filtrate is evaporated to about 12 ml and transferred 
to a separating funnel together with 50 ml water. This solution is extracted twice 
with 30 ml ether and the ether extracts are discarded. It is then extracted three 
times with 50 ml ethyl acetate, the combined ethyl acetate extracts evaporated to 
dryness and the residue dissolved in 10 ml methanol: 20 ~l are applied to the chro-
matogram. This extract is suitable for the TLC investigation of phenol glucosides 
and flavonoids. 
3. Extract of Viburni prunifolii and V. opuli cortex 
To remove interfering resins and lipids, the powdered drug (2 g) is extracted under 
reflux for about 15 min with 50 ml light petroleum. The drug residue is extracted 
by boiling for about 20 min with 20 ml methanol, then filtered. The filtrate is evapor-
ated to about 1 ml, and 50 ~l are applied to the chromatogram. 
Remarks: Turbidity or sediment which may appear in the concentrated filtrate, can be ignored. 
4. Extracts of pharmaceutical preparations 
Five powdered tablets or dragees are mixed with calcium carbonate (0.1 g) and 
extracted with 25 ml of 50% methanol for about 10 min on a water bath. To the 
first 10 ml of filtrate are added 200 mg basic lead acetate. This is again filtered 
and the filtrate is used direct1y for TLC. 
117 
118 
11. Thin Layer Chromatography 
1. Reference solutions 
Tl: 25 mg arbutin and 25 mg hydroquinone are dissolved in 10 ml of 50% methanol, 
and 10 111 of this soln. are applied to the chromatogram. 
T2: 25 mg hydroquinone, 25 mg gallic acid and 25 mg arbutin are dissolved in 
10 ml methanol, and 10 111 of this soln. are applied to the chromatogram (DAB 8). 
Scopoletin, amentoflavone and catechin/epicatechin are prepared separately as 0.1 % 
methanolic solns., and 10111 of each are applied for chromatography. 
2. Adsorbent 
Silica gel 60F 254 pre-coated TLC plates (Merck, Darmstadt). 
3. Chromatography solvents 
AN 2: ethyl acetate-methanol-water (100: 13.5: 10) 
DAß 8: ethyl acetate-formic acid-water (88:6:6) 
F-3: chloroform-acetone-formic acid (75: 16.5: 8.5) 
111. Detection 
1. Without chemical treatment 
Arbutin and other phenolic compounds show a pronounced quenching of fluores-
cence in UV -254 nm. 
2. Spray reagents 
a) Berlin blue reaction (BB No. 5, p. 299) 
Arbutin, methylarbutin, hydroquinone and methylhydroquinone appear as blue 
zones (vis.). 
b) Millons reagent (ML No. 26, p. 303) 
Arbutin, methylarbutin, hydroquinone and methylhydroquinone appear as yellow 
zones (vis.). 
c) ReagentsJrom DAB 8 
a) Jor arbutin and hydroquinones: 1 % 2,6-dichloroquinonechloroimide in methanol 
(DCC No. 8, p. 300). The chromatogram is sprayed with the reagent then exposed 
to ammonia vapour. Arbutin appears as a violet (vis.) zone. 
b) flavonoids: saturated methanolic aluminium chloride shows flavonoids as orange 
or blue fluorescent zones in UV-365 nm. 
d) Natural products-polyethyleneglycol reagent (NP/PEG No. 28, p. 303) 
Intense orange or blue fluorescent zones are seen in UV-365 nm. Amentoflavone 
gives a yellow-brown fluorescence. 
e) Potassium hydroxide reagent (KOH No. 21, p. 302) 
After spraying with 5% ethanolic KOH, arnentoflavone gives a yellow-green fluorescence, 
and cournarins and phenol carboxylic acids give a light blue fluorescence in UV-365 nrn. 
J) Fast blue salt reagent (FBS/KOH No. 12, p. 301) 
Phenolic compounds appear as red-brown zones (vis.). 
Figs. 1,2 
Figs. 3, 4 
IV. Important Arbutin Drugs 
Drug Plant of origin Total Pharmaco-
Family hydroquinone poeia 
content 
Uvae ursi folium Arctostaphylos 4-12% DAB 8 (not 
Bearberry leaves uva-ursi (L.) SPRENGEL less than 
Ericaceae 6%), ÖAB, 
Helv. VI. 
Vitis idaeae folium Vaccinium vitis idaea L. 5.5-7% 
Cowberry leaves Ericaceae 
Myrtilli folium Vaccinium myrtillus L. 0.4-1.5% 
Bilberry leaves Ericaceae 
Ericae (Callunae) herba Calluna vulgaris (L.) HULL. 0.6-0.68% 
Heather Ericaceae 
Pyri communis folium Pyrus communis L. up to 4.7% 
Pear leaves Rosaceae (young leaves) 
Viburni prunifolii cortex Viburnum prunifolium L. 0.5% 
Black haw bark Caprifoliaceae 
Bergeniae crassifoliae folium Bergenia crassifolia ca. 12% 
Bergenia (L.) FRITSCH (tannins 
Saxifragaceae 15-25% !) 
Remarks: Other constituents such as flavonoids or coumarins can also be used 
for the identification of arbutin drugs: 
Uvae ursi folium contains the flavonoids, quercitrin, isoquercitrin, myricitrin and 
quercetin-3-ß-D-6-0-galloyl-galactoside. 
Viburni prunifoli cortex is characterized by the biflavone, amentoflavone (see p. 170) 
and the coumarins, scopoletin and scopoline. 
~ 
OH ~ O-Gluc 
Hydroquinone Arbutin R=H 
Methylarbutin: R = eHa 
119 
Arbutin Drugs 
Tracks 1 = Uvae ursi folium 
2 = Vitis ideae folium 
3 = Myrtilli folium 
Tests Tl = arbutin (Rf ca. 0.4),hydroquinone (Rf ca. 0.95) 
T2 = arbutin (Rf ca. 0.25), gallic acid + hydroquinone (Rf 0.9-0.95) 
Solvent AN-2: ethyl acetate-methanol-water (100: 13.5: 10) Fig. 1 
system DAB 8: ethyl acetate-forrnic acid-water (88: 6: 6) Fig. 2 
Detection Berlin blue reagent (BB No. 5, p. 299) VIS. Fig.1A 
Chromato-
gram 1-3 
1A, B 
Millons reagent (ML No. 26, p. 303) vis. Fig. 1 B 
DichlorpquinonechioroimidejNH3 reagent (DCC No. 8, p. 300) VIS. Fig.2A 
Natural products-polyethyleneglycol reagent 
(NPjPEG No. 28, p. 303) UV-365 nm Fig.2B 
For description of drugs see p. 119. Formulae p. 119. 
Chromatograms of Uvae ursi folium and Vitis ideae folium show a distinct blue 
(BB) or yellow (ML) zone (Rf ca. 0.4) of arbutin. Myrtilli folium gives onIy a very 
weak zone of arbutin; above and below this zone are several weak blue (BB) or 
yellow (ML) zones. All three extracts contain only traces of free hydroquinone (Rf 
ca. 0.95). BIue zones (BB) at and directly above the start are due to tannins. 
2A 1 After treatment with DCC reagent, chromatograms of Uvae ursi folium show a 
strong vioiet (vis.) zone (Rf ca. 0.25; cf. T2) of arbutin. Six further zones (flavones 
and acids) are seen between arbutin and Rf ca. 0.75, and three brown-violet zones 
(gallic acid and hydroquinone; cf. T2) are present below the solvent front. 
2B Uvae ursi folium shows four orange fluorescent (NP/PEG reagent; UV-365 nm) 
zones of flavone glycosides in the Rf range 0.2-0.4: quercetin-3-ß-D-6-0-galloyl-
galactoside, isoquercitrin, quercitrin and myricitrin. Pale blue fluorescent zones in 
the Rf range 0.5-0.8 are typical, e.g. of cinnamic acid derivatives. Gallic acid gives 
an intense blue fluorescence at Rf ca. 0.9 (cf. T3). 
Remarks: Arbutin does not fluoresce in UV-365 nm. 
120 
Fig.l 
Tl 2 3 
A 
Fig.2 
T2 
DAS 8 
Tl 2 3 
T2 
FRONT 
Rf 
START 
121 
Vihurni Cortex 
Tracks 
Tests 
Solvent 
system 
1 = Viburni prunifolii cortex 
2 = Viburni opuli cortex 
Tl = scopoletin 
T2 = amentoflavone 
T3 = catechin/epicatechin mixture 
F-3: chloroform-acetone-formic acid (75: 16.5: 8.5) 
Detection Potassium hydroxide reagent (KOH No. 21, p. 302) UV-365 nm Fig.3A 
Chromato-
gram 
3A 
1 
4 
2 
3B 
122 
Fast blue salt reagent (FBS No. 12; followed by KOH, p. 301) VlS. Fig.3B 
Natural products-polyethyleneglycol reagent 
(NP/PEG No. 28, p. 303) UV-365 nm Fig.4 
For description of drugs see p. 119. Formulae p. 150 and 170. 
Viburni prunifolii cortex and Viburni opuli cortex. After KOH treatment both extracts 
show at least seven blue fluorescent zones (UV -365 nm) between the start and Rf 
ca. 0.7 and a blue or orange fluorescent zone at the solvent front. 
Viburni prunifolii cortex is characterized by the prominent main zones (light blue 
fluorescence) of the coumarins, scopoletin (cf. Tl) and scopoline (near the start), 
and by the presence of the biflavone, amentojlavone (cf. T2; green fluorescence 
with KOH, yellow-green fluorescence with NP/PEG). Other blue fluorescent zones 
are due to plant acids, e.g. caffeic acid. 
Viburni opuli cortex shows blue fluorescent zones in more or less the same Rf regions 
as in V. prunifolium; scopoletin is less pronounced and amentoflavone is completely 
absent. 
The two extracts can also be differentiated by treatment of chromatograms with 
FBS-KOH. This reveals the catechin/epicatechin mixture, which is present only in 
V. opulus, as a brown-red zone (vis.) at RfO.l-0.2 (cfT3). 
Remarks: Arbutin (ca. 0.5%) is not detectable under the above conditions. Extracts must 
be specially concentrated prior to chromatography for the detection of arbutin. 
Fig.3 
Tl T2 2 
Fig.4 
Tl T2 
T3 T2 
2 
2 
F 
RI 
5 
123 
Bitter Principle Drugs 
Most ofthe bitter principles in important official drugs possess a terpenoid structure, 
representing derivatives of mono terpenes (secoiridoids), sesquiterpenes, diterpenes 
and triterpenes. 
I. Preparation of Drug Extracts for TLC 
Powdered drug (1 g) is extracted for 10 min with 10 ml methanol at 60° C on the 
water bath. The mixture is filtered and the filtrate is evaporated to a volume of 
ca. 2 ml. 
Exceptions: 
1. Humuli lupuli strobulus 
a) The drug (1 g) is extracted for 24 h with 15 ml of cold ether. The filtrate is 
allowed to stand for 12 hin the refrigerator, precipitated waxy materials are removed 
by filtration, and the filtrate is evaporated to dryness at room temperature. The 
residue is dissolved in 1 ml methanol, and this soln. is used for chromatography. 
b) The drug (1 g) is extracted for 3 h at room temperature with 10 ml dichlorometh-
ane. The filtrate is evaporated to about 3 ml, and used for chromatography. 
2. Salviae folium and Rosmarini folium 
Coarsely powdered leaves (3 g) are extracted under reflux (water bath) for 1 h with 
100 ml ether. The filtrate is evaporated to about 3 ml, and this soln. is used for 
chromatography. 
11. Thin Layer Chromatography 
1. Reference solutions 
All standard substances are prepared as 1 % methanolic solutions. 
2. Adsorbent 
Silica gel 60F 254 pre-coated TLC plates (Merck, Darmstadt). 
3. Sampie concentration 
Drug extracts 40 111 
Reference solutions 20 111 
125 
126 
4. Chromatography solvents 
B-1 
B-2 
B-3 
B-4 
B-5 
B-6 
B-7 
B-8 
B-9 
Solvent system 
ethyl acetate-methanol-water (77: 15: 8) 
acetone-chloroform-water (70: 30: 2) 
chloroform-methanol (95: 5) 
chloroform-methanol (97: 3) 
acetone-chloroform (30: 40) 
n-propanol-toluene-glacial acetic acid-water 
(25 :20: 10: 10) 
ethyl acetae-dioxan-water (30: 10: 0.3) 
iso-octane-iso-propanol-formic acid } 
(83.5: 16.5 :0.5) 
n-heptane-iso-propanol-formic acid 
(90: 15:0.5) 
B-10 chloroform-ethanol (95: 5) 
111. Detection 
1. Without chemical treatment 
UV-254 nm 
For detection of: 
"Screening system" 
amarogentin/Gentianae radix 
absinthin/ Absinthii herba 
quassin/Quassiae lignum 
carnosolic acid, carnosol/ 
Rosmarini and Salviae folium 
cnicin/Cardui benedicti herba 
aucubine/Plantaginis herba 
oleuropein/Oleae folium 
humulone and lupulone (bitter 
acids)/Humulus lupulus 
cucurbitacins/Bryoniae radix 
Substances with conjugated double bond systems show fluorescence quenching (e.g. 
quassin). 
UV-365 nm 
Mostly unspecific fluorescence, with the exception of e.g. the flavonoid compounds 
in Aurantii pericarpium. 
2. Spray reagents 
a) Vanillin-sulphuric acid (VS No. 38, p. 304) 
Visualization after ca. 10 min at 1000 C: 
neo hesperidin, naringin, harpagoside 
gentiopicroside, swertiamarin 
condurangins 
foliamenthin, menthiafolin, quassin 
marrubiin, absinthin, cnicin 
red-violet 
brown-red 
blue-green 
blue 
blue 
b) Anisaldehyde-sulphuric acid (AS No. 2, p. 299) 
Similar colours (vis.) to those obtained with VS re agent. 
c) Liebermann-Burchard reagent (LB No. 16, p. 301) 
The TLC plate is sprayed with freshly prepared soln., heated for 10 min at 1000 C, 
and inspected in UV-365 nm or vis. 
Absinthin: sand colour in UV-365 nm; dark brown in vis. 
Cnicin: light grey in UV-365 nm; weak grey in vis. 
d) Fast red salt B (FRS No. 13, p. 301) 
For reducing substances and phenolic compounds, chromatograms are inspected 
in visible light 
Amarogentin: orange 
Gentiopicroside: red 
e) Benzidine reagent (BZ No. 4, p. 299) 
Aucubine: brown-black (vis.) 
f) FeCl3 (10% FeCl3 soin. No. 14, p. 301) 
The TLC plate is inspected immediately after spraying. Oleuropein, carnosolic acid 
(carnosol) and hop bitter principles show yellow-brown to yellow-green (vis.). 
g) Vanillin-phosphoric acid reagent (VPA No. 36B, p. 304) 
Cucurbitacins: blue or red-violet (vis.); bIue, pink, yellow and green fluorescence 
in UV-365 nm. 
h) Natural products-polyethyleneglycol-reagent (NPjPEG No. 28, p. 303) 
Dicaffeoylquinic acids(e.g. cynarinjCynarae herba) show intense blue to bIue-green 
fluorescence in UV-365 nm. 
IV. List of Bitter Principle Drugs 
Chromatograms (Figs. 1-12) are reproduced on pp. 13i-143. 
Fig. 
1,2 
DrugjPlant source 
Family jPharmacopoeia 
Main constituents 
Bitterness index (BI) 
Drugs containing terpenoid bitter principles 
Centaurii Herba 
Centaury 
Centaurium minus MOENCH 
Gentianaceae 
DAB 8, Helv. VI, ÖAB 
Gentianae Radix 
Gentian root 
Gentiana lutea L. 
Gentianaceae 
Ph. Eur. 1,2. AB-DDR, 
Helv. VI, ÖAB 
Harpagophyti Radix 
Grapple plant root 
Grapple plant 
Harpagophytum procumbens 
(BRUCH) DC ex MEISSEN 
Pedaliaceae 
Scrophulariae Herba 
Figwort 
Scrophularia nodosa L. 
Scrophulariaceae 
1. Monoterpenes (C-10) 
Secoiridoid bitter principles: 
swertiamarin, gentiopicroside (= gentiopic-
rin); traces of amarogentin. 
BI plant: at least 2000 (10,000 Helv. VI) 
BI flowers: ca. 12,000 
Secoiridoid bitter principles: 
gentiopicrin (2-3.5%; BI 12,000) 
amarogentin (0.05%; BI 58 x 106). 
Trisaccharide bitter principle: 
gentianose (BI 120). 
BI ofthe drug 10,000-30,000. 
Iridoid bitter principles: 
harpagoside, isoharpagoside, procumbid. 
BI of the drug 600-2,000. 
Substitute drug for Harpagophyti radix with 
the same qualitative composition of bitter 
principles, but about half the content of har-
pagoside. 
127 
Fig. 
6 
5 
3 
4 
11 
128 
Drug/Plant source 
F amily /Pharmacopoeia 
Menyanthidis Folium 
Trifolii fibrini folium 
Buckbean leaf 
Menyanthes trifoliata L. 
Menyanthaceae 
Helv. VI, ÖAB 
Oleae Folium 
Olive leaf 
Olea europea L. ssp. 
silvestris 
Oleaceae 
Plantaginis Folium 
Ribwort leaf 
Plantago lanceolata L. 
Plantaginaceae 
Helv. VI, ÖAB (Folium), 
2. AB-DDR (Herba) 
Cardui benedicti Herba 
Cnici herba 
Blessed thistle 
Cnicus benedictus L. 
Asteraceae 
DAC, Helv. VI, ÖAB 
Absinthii Herba 
Wormwood 
Artemisia absinthium L. 
Asteraceae 
DAB 8,2. AB-DDR, Helv. VI, 
ÖAB 
Marrubii Herba 
White Horehound 
Marrubium vulgare L. 
Lamiaceae 
ÖAB 
Salviae Folium 
Sage leaves 
Rosmarini Folium 
Rosemary leaves 
For further details see section on 
Essential oil drugs, pp. 11 and 13. 
Main constituents 
Bitterness index (BI) 
Secoiridoid bitter principles: 
foliamenthin, menthiafolin, T,8'-dihydrofo-
liamenthin and sweroside. 
Verbenalin type bitter principle: 
loganin. 
Monoterpene alkaloids: 
gentianin, gentianidine. 
BI of the drug 4,000-10,000 
Iridoid bitter principles: 
gentianin type: ca. 0.75% oleuropein in fresh 
leaves. On storage, this is c1eaved to 3,4-di-
hydroxyphenylethyl a1cohol and a dicarb-
oxylic acid methyl ester. 
Olives contain up to 2% oleuropein. 
Iridoid bitter principle: 
aucubine (1.9-2.4%) 
Remarks: aucubin is also present in Aucuba 
japonica and Vitex agnus castus. 
2. Sesquiterpenes (C-15) 
Germacran type bitter principles (ca. 0.25%), 
with cnicin, an unsaturated sesquiterpene di-
hydrolactone, and artemisiifolin (a sesquiter-
pene lactone). 
BI ofthe drug 800-1,800. 
Bitter principle content ca. 0.3% in leaves, 
0.15% in flowers. 
Sesquiterpene lactones: absinthin (ca. 0.2%) 
and its isomer, anabsinthin. 
Artabsin can be detected only in freshly har-
vested plants. 
BI of the drug 10,000-25,000 
BI of absinthin ca. 12,700,000. 
3. Diterpenes (C-20) 
Bitter principle content 0.3-1 %. Marrubiin 
(BI 65,000) is the main constituent. Marru-
biol, marrubenol and its half acetal are not 
bitter. 
The bitter principle of both drugs is picrosal-
vin (=carnosol). During extraction picrosal-
vin is converted into the non-bitter, opened 
ring form, carnosolic acid. 
Fig. 
7 
8 
3 
9, 10 
12 
Drug/Plant source 
Family /Pharmacopoeia 
Bryoniae Radix 
Bryonyroot 
Bryonia alba L. and Bryonia cre-
tica ssp. dioica PLANCH. 
Cucurbitaceae 
Cucurbitae pepo Semen 
Melon pumpkin seeds 
Cucurbita pepo L. 
Cucurbitaceae 
Quassiae Lignum 
Quassia wood 
Quassia amara L. 
Picrasma excelsa PLANCH. 
Simarubaceae 
Condurango Cortex 
Condurango bark 
Marsdenia condurango 
REICHB. 
Asc1epidiaceae 
Helv. VI, ÖAB 
Main constituents 
Bitterness index (BI) 
4. Triterpenes (C-30) 
Tetracyclic triterpene bitter principles: Cucur-
bitacins B, D, E, I, J, K, Land dihydrocucurbi-
tacins E and B possess the same ring system, 
but differ with respect to substitution in 
ring A. 
Bryonia alba and dioica show qualitatively 
similar contents of bitter principles, but the 
content of B. dioica is about 10 times higher. 
Main compounds are sterols besides amines 
e.g. cucurbitine. 
Secotriterpenes ca. 0.25%: 
quassin (0.1-0.15%), neoquassin and 18-hy-
droxy-quassin. 
BI of the drug 40,000-50,000 
BI of quassin/neoquassin 17 x 106 • 
5. Pregnane type (steroids) 
Bitter principles ca. 1-2%. 
Condurangins are ester glycosides of a pre-
gnane derivative of the digitanol group. Con-
duragenin A is linked to a pentasaccharide, 
and esterified with cinnamic and acetic acid. 
BI of the drug ca. 15,000. 
Drugs containing non-terpenoid bitter principles 
Aurantü Pericarpium 
Seville orange peel 
Citrus aurantium L. ssp. aurantium 
Rutaceae 
DAB 8, Helv. VI, ÖAB 
(see sections on flavonoids, p. 169 
and essential oils, p. 15) 
HlUßuli lupuli Strobuli 
Hops 
Humulus lupulus L. 
Moraceae 
ÖAB 
Cynarae Herba 
Artichoke 
Cynara scolymus L. 
Asteraceae 
Bitter principles: 
FIavanone glycosides, neo hesperidin and nar-
ingin. 
The triterpene bitter principle, limonin (BI 
106) is found chiefly in the seeds. In the fresh 
plant it is present in a non-bitter form. 
BI of the flavanone glycosides ca. 500,000 
BI ofthe drug 600-1,500. 
Bitter principles: 
Chief compounds are the prenylated phloro-
glucinol derivatives, iso-humulone and lupu-
lone. These are very unstable, and are trans-
formed into the so-called bitter acids. 
Sesquiterpene lactones: 
e.g. cynaropicrin, grossheimin 
Phenol carboxylic acids: 
e.g. 1,3-dicaffeoylquinic acid (cynarin) and 
chlorogenic acid. 
129 
130 
v. Formulae of Constituents of Bitter Principle Drugs 
MONOTERPENES 
~o ~ -....;:: 
o 
IH 
O-Gluc 
Gentiopicrin 
o OH 
(= Gentiopicroside) 
Amarogenti n: R, = H, R2 = OH 
Amaroswerin: R, = OH, R2 = OH 
Amaropanin: R,=H, R2 =H 
R R R 
CO ~ c : CH,OH 
Gentianin 
f;l 
:" 
0 ~CH~ OH R-0;i5 IA : O-Gluc 
Foliamenthin Dihydrofolia- Menthiafolin 
menthin 
HO 150 OCH3 ~oy- __ -....;:: 
HO 0 0 
~ 
O-Gluc 
R = OH Procumbid Oleuropein (= Oleuropaeoside) Aucubin 
R= H Harpagid 
SESQUITERPENES 
DITERPENES 
TRITERPENES 
Cnicin 
OH 
Carnosol 
(Picrosalvin) 
Quassin 
OTHER BITTER PRINCIPLES 
Humulone 
Neohesperidose 
Naringin: R, R, = H 
Neohesperidin: R=CH 3 ; R,=OH 
~ 
o 
Artabsin Absinthin 
HO 
o 
Marrubiin 
Cucurbitacin E: R=COCH3 Cucurbitacin I: R = H 
OR 
Condurangin A R, = Cinnamoyl 
R2 = Pentaglycosyl 
Limonin 
131 
Drugs with Bitter Principles 
TLC Synopsis 
Tracks 
Test 
Solvent 
system 
1 = Aurantii pericarpium 
2 = Harpagophyti radix 
3 = Gentianae radix 
4 = Centaurii herba 
5 = Condurango cortex 
6 = Menyanthidis folium 
T = neo hesperidin 
B-l: ethyl acetate-methanol-water (77: 15: 8) 
F-l: ethyl acetate-formic acid-glacial acetic acid-water (100: 11: 11 :26) 
Fig.l 
Fig.2 
Detection Vanillin-sulphuric acid reagent (VS No. 38, p. 304) 
Anisaldehyde-sulphuric acid re agent (AS No. 2, p. 299) 
vis. Fig. 1 
vis. Fig.2 
For description of drugs see p. 127. Formulae p. 130-131 
Chromato-
gram 1 Aurantii pericarpium. The TLC shows two characteristic intense zones, which are 
1,2 red-orange (VS) or brown-violet (AS), in the Rfrange 0045-0.5 (naringin and neohe-
speridin). 
132 
The non-bitter flavonoids, rutin, eriocitrin andchlorogenie acid, can be located 
with the NP/PEG re agent (see Flavonoids, p. 189, Fig. 17 AlB), which produces fluo-
rescent zones (orange, violet-red and blue-green, respective1y) in UV-365 nm. 
2 Harpagophyti radix is characterized by two prominent violet zones at Rf ca. 0.5 
and 0.1-0.2 (harpagoside and procumbid or harpagid). 
Remarks: Scrophulariae herba, which contains about 50% less harpagoside, counts as an 
acceptable substitute. 
3 Gentianae radix shows gentiopicrin (= gentiopicroside) as a brown-violet main zone 
at Rf ca. 0045 (B-l) or 0.35 (F-l). Immediately below there is a weak brown zone 
due to swertiamarin, the main constituent of Centaurii herba (cf. 4). 
Amarogentin, which quenches fluorescence in UV-254 nm (Rf 0.8), can be better 
located in the visible with the fast red reagent (see p. 136 Fig. 5 A). 
4 Centaurii herba is characterized by 4 yellow or orange-brown main zones in the 
Rf range 0.25-0.5 (B-1) or 0.2-004 (F-l). The main zone at Rf ca. 004 (B-1) or 
0.3 (F-1) is due to swertiamarin. The weak brown zone directly above is due to 
gentiopicrin (cf. 3, Gentianae radix). 
5 Condurango cortex shows several intense, black-green zones in the Rfrange 0045-0.5, 
due to condurangins. 
6 Menyanthidis folium. Treatment with VS re agent produces one typical dark blue 
zone at Rf ca. 0.6, and two in the Rf range 0.8-0.85 (B-1). These correspond to 
foliamenthin, menthiafolin and dehydrofoliamenthin. With AS reagent and solvent 
system F -1, these zones are not c1early defined. 
Remarks: Chromatograms 1-6 show dark blue zones of essential oils and aromatic acids 
below the solvent front. The black or brown zones at and above the start originate partly 
from sugars. 
Fig.l 
T 2 3 4 
Fig.2 
T 2 3 4 
5 6 
5 6 
FRONT 
Rf 
-0.5 
-START 
FRONT 
Rf 
-0.5 
-START 
133 
Cnici, Absinthi, Marrubii Herba Quassiae Lignum 
Tracks 
Tests 
Solvent 
system 
Detection 
Chromato-
1 = Gnici herba 
2 = Absinthii herba 
3 = Quassiae lignum 
4 = Marrubii herba 
Tl =cnicin 
T2 = absinthin (anabsinthin) 
T3=quassin 
T4 = marrubiin 
B-3: chloroform-methanol (95: 5) 
B-5: acetone-chloroform (30: 40) 
Liebermann-Burchard reagent (LB No. 16, p. 301) 
Vanillin-sulphuric acid reagent (VS No. 38, p. 304) 
Fig. 3A, B; 4C 
Fig. 4A, B 
UV 365 nm Fig. 3A; 4A 
vis. Fig. 3B; 4B, C 
For description of drugs see p. 128-129·. Formulae p. 131 
gram 1 Cnici herba. Treatment with LB reagent, followed by observation in UV-365 nm, 
reveals at least 14 fluorescent zones (mainly light blue, red-brown or yellow green) 
between the start and the solvent front. In UV -365 nm cnicin shows yellow-green 
3A 
4A, B 
fluorescence (Rf ca. 0.05). 
In solvent system B-5 cnicin migrates at Rf ca. 0.4 (cf. Tl). LB and VS reagents 
give only weak, grey-violet colours in the visible. 
3 A 2 Absinthii herba. LB reagent reveals two main zones with intense ochre fluorescence 
3A, B 
3B 
4C 
134 
in UV-365 nm (absinthinfanabsinthin, Rf 0.3-0.4), together with at least ten other 
mainly blue or green fluorescent zones. After VS reagent, absinthin and anabsinthin 
appear as dark blue and violet zones (vis.), respectively (cf. T2-3, Fig. 4B). 
Remarks: Treatment with 50% sulphuric acid also produces ochre fluorescent zones (in UV-
365 nm) of absinthin and anabsinthin. 
3 Quassiae lignum. After treatment with LB reagent, about 11 weak blue or blue-green 
fluorescent zones (in UV-365 nm) are seen between Rf 0.1 and the solvent front 
(partly alkaloids and coumarins). 
The bitter principle, quassin, shows a distinct quenching of fluorescence in UV-
254 nm, but does not itself fluoresce in UV -365 nm. The weak blue and green fluores-
cent zones (UV-365 nm) accompanying the quassin standard (T3) are due to impuri-
ties of alkaloids and coumarins. 
After treatment with VS reagent, the quassin (cf. T3) appears as a violet (vis.) 
zone. In solvent system B-5 quassin migrates at Rfca. 0.9 (cf. Fig. 4B). 
4 Marrubii herba. Treatment with VS reagent pro duces violet zones, in particular 
three pronounced violet (vis.) zones in the Rf range 0.5-0.9. The zone at Rf ca. 
0.8 is due to marrubiin (cf. T4); the zone at Rfca. 0.5 is presumably due to premarru-
biin. 
Remarks: Visualization in UV-365 nm without chemical treatment reveals only non-specific 
fluorescent zones. 
Fig.3 
Tl T2 2 T3 3 T3 
c 
Fig.4 
Tl T4 
3 
4 
-FRONT 
Rf 
-0.5 
START 
-FRONT 
- Rf 
=0.5 
-START 
135 
Gentianae Radix/amarogentin 
Track 
Solvent 
system 
Detection 
1 = Gentianae radix (G.lutea) Test T1 = amarogentin 
B-1: ethyl acetate-methanol-water (77: 15: 8) 
B-2: acetone-chloroform-water (70: 30: 2) 
Fast red salt reagent (FRS No. 13, p. 301)+NaOH VlS. 
or+25% NH 3 vis. 
Fig.5A 
Fig.5B 
Fig.5A 
Fig.5B 
Gentianae radix contains 0.01-0.04% amarogentin; only traces are present in Centaurii herba. 
Chromato-
gram 
5A, B 
With fast red salt reagent, amarogentin shows orange ( + KOlI) or red-violet (+ NH3 ) 
colour (vis.). It migrates at Rf ca. 0.8 (solvent system B-1) or ca. 0.45 (B-2). 
Gentianae radix also shows a strong zone of the xanthone, gentisin, at the solvent 
front. 
Chromatograms of root extracts of G. pannonica, purpurea and punctata differ from 
those of G. lutea by the presence of the additional bitter principles, amaropanin and amaro-
swerin. These compounds migrate direct1y above and below amarogentin, respectively, and 
they also give various reds with FRS reagent. 
Plantaginis Herba/aucubine 
Track 
Solvent 
system 
2 = Plantaginis herba (Plantago lanceolata L.) Test T2 = aucubine 
B-6: n-propanol-toluene-glacial acetic acid-water (25: 20: 10: 10) 
Detection Benzidine reagent (BZ No. 4, p. 299) VlS. Fig.5C 
Chromato- After treatment with benzidine reagent, aucubine becomes visible as a brown zone 
gram at Rf ca. 0.35. Plantaginis herba shows additional yellow zones between the start 
5 C and Rf 0.3, and another brown zone at the solvent front. 
Oleae Folium, Fructus/oleuropein 
Tracks 
Solvent 
system 
3 = Oleae folium Test T3 = oleuropein (Rf ca. 0.25) 
4 = Oleae fructus (fresh) 
5 = Oleae fructus (stored) 
B-7: ethyl acetate-dioxan-water (30: 10: 0.3) 
Detection Without chemical treatment vis. Fig.6A 
vis. Fig.6B 10% FeCl3 soln. 
Oleuropein is easily detectable in freshly harvested olive leaves and fruits. During storage it is 
cleaved into 3,4-dihydroxyphenylethyl alcohol and an iridoid carboxylic acid. 
Chromato-
gram 
6A,B 
136 
Without treatment of the chromatogram, oleuropein is visible as a weak brown 
zone at Rf 0.25-0.35. Treatment with FeCl3 produces a stronger brown (vis.). A 
zone below oleuropein becomes dark brown. Breakdown products appear as grey-
green zones near the solvent front and at the start. 
A 
Fig.5 
Tl Tl 
A • 
Fig.6 
T3 3 4 T3 
c 
T2 2 
3 4 5 
-FRONT 
Rf 
-0.5 
-START 
-FRONT 
Rf 
-0.5 
-START 
137 
Bryoniae Radix Cucurbitae Semen 
Tracks 
Tests 
Solvent 
system 
1 = Bryoniae dioicae radix} . I d 
2 B . d·· d· commerCIa rugs = ryomae lOIcae ra IX 
3 = Bryoniae albae radix 
4 = Cucurbitae semen 
T1 = 25-0-acetylbryomarid 
T2 = cucurbitacin D 
T3 = dihydrocucurbitacin D 
T 4 = cucur bitacin I 
T5 = cucurbitacin L 
B-l0: chloroform-ethanol (95:5) 
Detection Vanillin-phosphoric acid reag. (VPA No. 36, p. 304) 
UV-365 nm Fig. 7 A, 8B 
vis. Fig. 7B, 8A, C 
For description of drugs see p. 129. Formulae p. 131 
Chromato-
gram 1,2 
7A, B 
Bryoniae dioicae radix. After treatment with VPA reagent, chromatograms of Bryo-
niae dioicae radix extracts show at least 12 blue, bIue-green or orange fluorescent 
zones (UV-365 nm) between the start and the solvent front. The same zones appear 
violet or brown violet in vis. 
Cucurbitacins B, D, E, I,K, Land the corresponding dihydrocucurbitacins migrate 
in the Rf range 0.2-0.4. Bryomarid (at the start) and acetylbryomarid (directly above 
the start) (cf. T1) appear as pale blue fluorescent zones in UV-365 nm, and as 
violet zones in vis. 
3 Bryoniae albae radix has alm ost the same qualitative pattern of cucurbitacins as 
B. dioicae radix, but shows normally a lower content of bitter principles. 
The specific source plant of any commercial preparation of Bryoniae radix cannot 
be unequivocally identified. Moreover, the cucurbitacin content depends on the 
time of harvesting and extraction method. 
8B, C 4 Cucurbitae semen. In UV-365 nm the chromatogram shows four characteristic, 
138 
strong orange-brown fluorescent zones at Rf ca. 0.4, 0.6, 0.85 and at the solvent 
front. After 5-10 min the fluorescence changes to blue. The same zones appear 
violet in vis. Most of these zones are due to sterols, Cucurbitacins are not present. 
4,1 After treatment with VPA reagent and visualization in UV-365 nm, chromatograms 
of Cucurbita and Bryonia extracts appear somewhat similar; but a five-fold greater 
quantity of Cucurbitae semen extract must be applied for the detection of the zones 
in the Rf range 0.1-0.3. 
Remarks: Solvent system B-I0 is temperature-dependent, resulting in shifts in the Rf values 
ofthe zones (cf. Fig. SA/SC). Shorter solvent migrations are therefore recommended, as shown 
in Fig. SC. 
Fig.7 
Tl T2 T3 2 3 T4 T5 
A 
Fig.8 
Tl 2 3 T2 T3 T4 T5 
2 3 T4 T5 
4 4 
-0.5 
-5 
F 
Rf 
139 
Humuli Strobuli (Glandulae) 
Tracks 
Tests 
Solvent 
system 
Detection 
Chromato-
1 = Humuli strobuli DCM extract (commercial pattern I) 
2 = Humuli strobuli ether extract (commercial pattern II) 
3 = H umuli stro buli methanol extract (commercial pattern I) 
4 = Humuli strobuli (fresh hops pattern I) 
5 = Humuli strobuli (fresh hops pattern II) 
Tl = lupulone with degradation products 
T2 = humulone with degradation products 
TG = flavonoid mixture : rutin (Rf ca. 0.35), chlorogenic acid (Rf ca. 0.5), hyperoside 
(Rf ca. 0.6). 
B-8: iso-octane-isopropanol-formic acid 
(83.5:16.5:0.5) developed over 15 cm 
B-9: n-heptane-isopropanol-formic acid 
(90: 15: 0.5) developed twice over 15 cm 
F-1: ethyl acetate-formic acid-glacial acetic 
acid-water (100: 11 : 11 : 27) 
Fast blue salt-KOH (FBSjKOH No. 12, p. 301) 
Without chemical treatment 
FeCl 3 reagent (No. 14, p. 301) 
Natural products-polyethyleneglycol reagent 
(NPjPEG No. 28, p. 303) 
Fig. 9A, B 
Fig. 10A, B, C 
Fig.9C 
vis. Fig.9A 
UV-365 nm Fig. 9B; lOC 
VlS. Fig. 10A, B 
UV-365 nm Fig.9C 
For description of drugs see p. 129. Formulae p. 131 
gram 1,2 Commercial drugs. The phloroglucinol derivatives, lupulone and humulone, are unsta-
9 A, B ble. The commercial drugs are therefore found to contain mostly breakdown or 
decomposition products, known as bitter acids. 
After treatment with FBS re agent, chromatograms of DCM or ether extracts 
show a broad, red-brown (vis.) zone of bitter acids at Rf 0.4-0.45. In the same 
Rfrange, visualization in UV-365 nm reveals weak blue or reddish fluorescent zones. 
10 A 1 Double development of the TLC plate over 15 cm in solvent system B-9 gives better 
resolution of the individual zones between Rf 0.3-0.6. 
lOB, C 4,5 Freshly harvested, Jreeze-dried drugs. Ether extracts of hops (patterns land II) still 
contain lupulone (Rf ca. 0.5) andjor humulone (Rf ca. 0.75). Track 4, however, shows 
that an extensive breakdown to bitter acids has already occurred (Rf ca. 0.4 and 
0.4-0.6). 
9 C 3 Flavonoids and ehlorogenie aeid: 
140 
After treatment with NP/PEG reagent and visualization in UV-365 nm, chromato-
grams of methanolic drug extracts mainly show yellow-green fluorescent zones of 
rutin (Rf ca. 0.35), hyperoside (Rf ca. 0.55), ehlorogenie acid (Rf ca. 0.45) (cf. TG), 
and an additional flavonoid monoglycoside (Rf ca. 0.7). 
A 
Fig.9 
Tl 2 Tl 
A B 
Fig. 10 
Tl Tl 4 5 T2 
2 TG 3 
Tl 4 5 T2 
FRONT 
Rf 
START 
-FRONT 
Rf 
0 .5 
START 
141 
Salviae, Rosmarini FoliumjCarnosolic acid (Carnosol) Cynarae Herba 
Tracks 
Tests 
Solvent 
system 
1 = Salviae folium 
2 = Rosmarini folium 
T = carnosol (picrosalvin) 
Tl = rutin 
T2 = chlorogenie acid 
T3 = luteolin-7-0-glucoside 
T4=cynarin 
3 = Cynarae herba (fresh plant) 
4 = Cynarae herba (dried drug) 
T5 = caffeic acid (Rf ca. 0.9), isochlorogenie acid (Rf ca. 0.8), 
chlorogenie acid (Rf ca. 0.4) 
B-4: chloroform-methanol (97: 3) 
F-1: ethyl acetate-formic acid-glacial 
acetic acid-water (100: 11: 11 :27) 
Detection Without chemical treatment 
Fig.11 
Fig.12 
VlS. Fig. 11 A 
vis. Fig. 11 B 
VlS. Fig. 11 C 
Chromato- 1 
gram 2 
11A, B 
11C 
FeCl3 reagent (FeCI 3 No. 14, p. 301) 
Vanillin-sulphuric acid reagent (VS No. 38, p. 304) 
Natural products-polyethyleneglycol-reagent 
(NP/PEG No. 28, p. 303) UV-365 nm Fig.12 
For description ofdrugs see p. 128-129. Formulae p.131 and 171 
Salviae folium 
Rosmarini folium 
The bitter principle, earnosol, is present in both drugs, but it undergoes ring opening 
to earnosolie acid during extraction, and is detected as such on the chromatogram. 
Without chemical treatment, carnosolic acid is seen as a yellow-brown zone at Rf 
ca. 0.2. Treatment with FeCl3 reagent converts this to a green-brown (vis.) zone. 
In Rosmarini folium a second strong zone is seen at Rf ca. 0.4. 
VS-reagent pro duces violet (vis.) zones of the terpenoid compounds of the essential 
oil fraction (see p. 30, Fig. 10; p. 38, Fig. 17). 
Remarks: Rosmarini folium also shows blue fluorescent zones (UV-365 nm) of rosmarinic 
acid and the jlavonoid, sinensetin (Rf 0.8-0.85). 
12 3,4 Cynarae herba. The detection ofthe bitter principles, eynaropierin, dehydroeynaropie-
142 
rin and grossheimin is only possible after enrichment of the extract. Identification 
is therefore more conveniently based on the presence of eynarin 1 (an hepatoactive 
compound), a 1,3-dicaffeoylquinic acid, other phenol carboxylic acids and flavon-
oids. 
3 In fresh plant extracts1 , treatment of chromatograms with NPjPEG reagent shows 
eynarin as a blue fluorescent zone (UV-365 nm) at Rf 0.6-0.65 (cf. T4). The other 
acids appear at RfO.4-0.55 (cf. T2; ehlorogenie and neoehlorogenie acid) and between 
Rf 0.7 and the solvent front (isoehlorogenie aeid and eajJeie acid, cf. T5). 
Luteolin-7-0-glueoside (cf. T3) is seen as a yellow fluorescent, main zone at 
Rf ca. 0.6. Luteolin-3-0-rutinoside is located in the same region as the rutin standard 
(cf. Tl), with the weak zone of luteolin-7-0-rutinosyl-4'-O-glueoside below it. 
1 Remarks: Cynarin easily isomerizes to 1,5-dicaffeoylquinic acid during extraction. Extracts 
of stored drugs (= 4) show chiefly caffeic acid (Rf ca. 0.9) and isomerization products 
(Rf 0.75-0.85), and little cynarin (T4). 
A 
Fig.11 
2 T 
Fig.12 
Tl T2 3 4 
c 
2 
T3 T4 T5 
-FRONT 
Rf 
-0.5 
-START 
143 
Coumarin Drugs 
The active principles of coumarin drugs are benzo-a.-pyrones. They can be further 
c1assified as folIows: 
1. Simple coumarins 
Most of the compounds in this series are substituted with OH or OCH 3 at positions 
C-6 and C-7. Substitution at C-5 and C-8 is less common. 
Drugs: 
Angelicae, Imperatoriae, Levisticae, Pimpinellae and Herac1ei radix, 
e.g. umbelliferone. 
Scopoliae radix, e.g. scopoletin. 
Abrotani herba, e.g. scopoletin and umbelliferone. 
Fraxini cortex, e.g. fraxidin, isofraxidin and fraxetin. 
Herniariae herba, e.g. herniarin. 
2. C-prenylated coumarins 
Drugs: 
Rutae herba, e.g. rutamarin. 
Angelicae radix, e.g. umbelliprenin. 
Imperatoriae radix, e.g. ostruthin. 
3. "Condensed coumarins" 
a) Furanocoumarins 
A furan ring is fused at C-6 and C-7 (psoralen-type)or C-7 and C-8 (angelicin-type) 
of the coumarin ring system. 
Drugs: 
Angelicae, Imperatoriae, Levisticae, Pimpinellae and Herac1ei radix, e.g. bergapten, 
angelicin, imperatorin. 
Ammi majoris fructus, e.g. bergapten, xanthotoxin. 
Rutae herba, e.g. bergapten, psoralene. 
b) Pyranocoumarins 
A pyran ring is fused at C-7 and C-8 ofthe coumarin ring system (seselin-type). 
Drug: 
Amrni majoris fructus, e.g. visnadin, sarnidin. 
4. Dimeric coumarins 
Drugs: 
Daphne mezerei cortex, e.g. daphnoretin. 
5. Ammi visnagae fructus occupies a special position, because it contains both benzo-a.-
pyrones and the isomerie benzo-y-pyrones or furanochromones. 
145 
146 
I. Preparation of Drug Extracts and Pharmaceuticals for TLC 
1. Drugs 
Powdered drug (1 g) is extracted by shaking with 10 ml methanol for 30 min on 
the water bath. The clear filtrate is evaporated to about 1 ml, and 20 J.lI are applied 
to TLC. 
DAB 8: Ammeos visnagae fructus: The drug (0.5 g) is extracted by shaking with 
10 ml of 60% ethanol for 30 min on a water bath. The filtrate is evaporated to 
about 5 ml, and 20 J.lI are applied to TLC. 
2. Pharmaceutical preparations 
Carduben35®: 1 dragee contains 35 mg visnadin. 
One dragee is powdered and extracted by shaking with 5 ml methanol for 5 min 
on the water bath. Five microlitres of the clear filtrate are applied to the chromato-
gram. 
Application of 20 J.lg ensures the chromatographie detection of all coumarins. 
11. Thin Layer Chromatography 
1. Reference solutions 
All coumarin standards are prepared as 1 % methanolic solutions. 
2. Adsorbent 
Silica gel 60F 254 pre-coated TLC plates (Merck, Darmstadt). 
3. SampIe size 
The following quantities are applied to TLC: drug extracts (20 J.lI), standard solutions 
(5 or 10 J.ll), extracts of pharmaceuticals (5 J.ll). 
4. Chromatography solvents 
C-1 toluene-ether (1: 1, saturated with 10% acetic acid) 
Toluene (50 ml) and ether (50 ml) are shaken 5 min with 50 ml of 10% acetic acid 
in a separating funnel. The lower phase is discarded, and the toluene-ether mixture 
is used for TLC. 
C-l is a universally applicable TLC solvent for coumarin aglycones; it must 
be freshly prepared. 
C-2 ethyl acetate 
Ammeos visnagae fructus extracts (DAB 8). 
C-2 separates polar coumarins in a relatively higher Rf range in comparison with 
C-1. 
Remarks: Polar coumarins from Rutae herba extracts can also be separated in the solvent 
system, ethyl acetate-forrnic acid-glacial acetic acid-water (100: 11: 11: 27) (see Flavonoids, 
p. 164). 
III. Detection 
1. Without chemical treatment 
UV-254 nm 
All coumarins show a distinct fluorescence quenching. 
UV-365 nm 
All simple coumarins show intense blue or blue-green fluorescence. Furanocouma-
rins sometimes show yellow, brown or blue fluorescence (detection limit ca. 1 J.lg). 
Non-substituted coumarin fluoresces yellow-green in UV-365 nm only after treat-
ment with KOH-reagent. 
Chromones show less intense fluorescence (e.g. visnagin, pale blue; khellin, weak 
yellow-brown). 
2. Spray reagents 
a) Potassium hydroxide (KOR No. 21, p. 302) 
Blue fluorescent zones are intensified by spraying with 5% ethanolic KOR. Ammo-
nia vapour has the same effect. 
b) Natural products-polyethyleneglycol-reagent (NPjPEG No. 28, p. 303) 
This reagent intensifies and stabilizes the existing fluorescence of the native couma-
nns. 
c) Antimony III chloride (SbCI 3 No. 3, p. 299) 
Visnagin gives alernon yellow fluorescence in UV-365 nm after treatment with this 
reagent. 
IV. List of Coumarin Drugs 
Chromatograms (Figs. 1-10) are reproduced on pp. 152-161. 
Fig. Drug/Plant source 
F amily /Pharmacopoeia 
A. Drugs from the family Apiaceae 
2--4 Angelicae Radix 
Angelica root 
Angelica archangelica L. 
ÖAB, 2. AB-DDR (essential oils) 
Angelicae silvestris radix 
Wild angelica 
Angelica sylvestris 
Imperatoriae Radix 
Master-wort 
Peucedanum ostruthium L. 
Levistici Radix 
Lovage 
Levisticum officinale KOCH 
ÖAB, 2. AB-DDR (essential oils) 
Main constituents 
Coumarins: 0.001 %--0.008%, consisting of 
angelicin (2' ,3': 7 ,8-furanocoumarin); bergap-
ten (5-methoxy-(2',3':7,6-FC)); imperatorin 
(8-oxy-y-y-dimethyl-allyl-2' ,3': 7,6-FC); os-
thenol (7 -oxy-8-(8,8-dimethylallyl)-coumarin) 
and its methyl ester, osthol; oxypeucedanin 
hydrate (hydrate of 5-oxy (3,3' -dimethyl-2,3-
epoxy-propano-2' ,3': 7,6-FC); xanthotoxin 
(8-methoxy-2' ,3': 7,6-FC); xanthotoxol 
(8-oxy-2' ,3': 7,6-FC); umbelliferone (7-hy-
droxycoumarin); umbelliprenin (farnesyl 
ether of umbelliferone). 
This adulterant of Angelica archang. contains 
only umbelliferone, isoimperatorin (5-oxy-(y-
y-dimethyl-allyl-2',3': 7,6-FC)), oxypeuce-
danin and its hydrate. 
Coumarins: oxypeucedanin, oxypeucedanin 
hydrate, imperatorin, isoimperatorin, osthol 
(cf. Angelica root); also ostruthin (6-(3-
methyl-6-dimethyl-2,5-hexene )-7 -oxycou-
marin), and ostruthol (angelic acid ester of 
oxypeucedanin hydrate). 
Coumarins: The total coumarin content is low 
compared with that of Angelicae and Impera-
toriae radix; bergapten, umbelliferone. 
Phthalide: 3-butylidenephtalide (ligusticum 
lacton). 
147 
148 
Fig. 
9, 10 
7,8 
DrugjPlant source 
F amily jPharmacopoeia 
Pimpinellae Radix 
Burnet root 
Pimpinella saxifraga L. 
Pimpinella major (L.) HUDS. 
Helv. VI 
Heraclei Radix 
Hogweed root 
Herac1eum sphondylium L. 
Pastinacae Radix 
Wild parsnip root 
Pastinaca sativa L. 
Ammi majoris Fructus 
Ammi fruit 
Ammi majus L. 
Ammi visnagae Fructus 
Ammeos visnagae fructus 
Ammi visnaga fruits 
Ammi visnaga (L.) LAMARCK 
DAB8 
Asa foetida 
Asafetida 
Ferula assa-foetida L. 
B. Drugs from other plant ramilies 
5,6 Asperulae Herba 
Woodruff 
Galium odoratum (L.) ScoP. 
Rubiaceae 
Meliloti Herba 
Tall melilot 
Melilotus officinalis LAM. em. THUILL. 
Fabaceae 
Herniariae Herba 
Rupture-wort 
Herniaria glabra L. 
Herniaria hirsuta L. 
Caryophyllaceae 
ÖAB 
Main constituents 
Coumarins (in both species): 
bergapten, umbelliferone, umbelliprenin, sco-
poletin (6-methoxy-7-oxy-coumarin); sphon-
din (6-methoxy-(2',3': 7,8-FC)); isobergapten 
(5-methoxy-(2' ,3': 7,8-FC); pimpinellin (5,6-
dimethoxy-(2' ,3': 7,8-FC)); isopimpinellin 
(5,8-dimethoxy-(2' ,3': 7,6-FC)). 
The drug is an acceptable substitute for Pim-
pinellae radix. The coumarin content is ab out 
20 times higher than that ofPimpinellae radix. 
An adulterant of Pimpinellae radix with a low 
coumarin content. Bergapten, imperatorin 
and osthol are present. 
Coumarins: bergapten, imperatorin, xantho-
toxin (see Angelicae radix), ammajiin, a glu-
coside of marmesin. 
Coumarins: visnagan group, with samidin, di-
hydrosamidin and visnadin. 
Furanochromones: (2-4%; DAB 8 specifies 
not less than 1%): khellin (5,8-dimethoxy-2-
methylfuranochromone) is the major constit-
uent (0.3-1%), accompanied by visnagin, 
khellinol, khellol, khellol glucoside, ammiol 
and visammiol. 
A gum resin containing 25-65% asaresin. This 
in turn contains about 60% ferulic acid esters, 
asaresitannol, and about 1.3% free ferulic 
acid, which is converted to umbelliferone on 
dry distillation. Umbelliferone derivatives are 
also presen t. 
Unsubstituted coumarin. 
Unsubstituted coumarin, melilotoside. 
Remarks: Bacterial action on the damp drug 
produces dicoumarol (3,3' -methylene-bis-4-
hydroxy-coumarin). 
Coumarins: herniarin, umbelliferone. 
Flavonoids; rutin, narcissin (see section on 
Flavonoids, Fig. 6, p. 176). 
Saponins: Herniaria saponins land II. 
Fig. Drug/Plant source 
F amily /Pharmacopoeia 
Rutae Herba 
Rue 
Ruta graveolens L. 
Rutaceae 
Abrotani Herba 
Southernwood 
Artemisia abrotanum L. 
Asteraceae 
7, 8 Fraxini Cortex 
Ash bark 
Fraxinus excelsior L. 
Fraxinus ornus L. 
Oleaceae 
Mezerei CortexMezereon bark 
Daphne mezereum L. 
Thyme1aeaceae 
4 Scopoliae Radix 
Scopolia rhizome 
Scopolia carniolica L. 
Solanaceae 
Main constituents 
Coumarins: scopoletin, umbe1liferone, ber-
gapten, isoimperatorin, psoralene, xantho-
toxin, rutacultin, rutamarin, daphnoretin, 
daphnoretin methyl ether. 
Flavonoids: rutin (see Flavonoids, Fig. 16, 
p.186). 
Coumarins: umbeIliferone, scopoletin. 
Coumarins: fraxidin (0.6%), isofraxidin 
(0.12%), fraxetin, fraxin (fraxetin glucoside), 
fraxinol (6-hydroxy-5, 7 -dimethoxycoumarin; 
ca. 0.45%). 
Coumarins: daphnetin, daphnin (7,8-dihy-
droxy-coumarin-7 -O-glucoside), umbellife-
rone, scopoletin (traces). 
Coumarins: scopoletin, scopoline (scopoletin 
glucoside). 
Alkaloids: hyoscyamine/atropine, scopol-
amine (see also Fig. 26, p. 90). 
149 
150 
v. Formulae of Constituents of Coumarin Drugs 
SIMPLE COUMARINS 
R, R2 Rs R. 
H H OH H 
H H OCHs H 
H OH OH H 
H H OH OH 
H OCHs OH H 
H OCHs OH OCHs 
OCHs OH OCHs H 
C-PRENYLATED COUMARINS 
R, R2 R3 R4 
H H X H Umbelliprenin 
H X OH H Ostruthin 
Umbellifefone 
(7-Hydroxycoumarin) 
Herniarin 
(7-Methoxycoumarin) 
Aesculetin 
(6,7-Hydroxycoumarin) 
Daphnoretin 
(7,8-Hydroxycoumarin) 
Scopoletin 
(6-Methoxy-7-hydroxycoumarin) 
Isofraxidin 
(7-Hydroxy-6,8-methoxycoumarin) 
Fraxinol 
(6-Hyd roxy-5, 7 -methoxycou marin) 
CH3 CH3 \ I 
X = C(=CH-CH2-CH2-Ch=CH-CH20-
CHi 
Rutacultin 
Rutamarin 
DIMERIC COUMARIN ~ 
H3CO~O 0 0 Daphnoretin HoMo~o 
FURANOCOUMARINS R1 R2 Furanocoumarin 
H H Psoralen 
7,6-Furanocoumarins OCH3 H Bergapten 
H OCH3 Xanthotoxin 
oQo H OH Xanthotoxol OCH3 OCH3 Isopimpineliin /CH3 Imperatorin H -OCH2-CH=C 1 0 7 0 0 "'CH3 /CH3 
R2 -OCH2 -CH=C H Isoimperatorin ~CH3 
/0 /CH3 
-OCH2-CH-C H Oxypeucedanin 
'CH3 
___ CH3 
H Oxypeucedanin hydrate -OCH2-CH-CH 
I I-CH3 OH OH 
7,8-Furanocoumarins 
R290 R1 R2 Furanocoumarin H H Angelicin OCH3 H Isobergapten 
o 8 0 0 H OCH3 Sphondin OCH3 OCH3 Pimpineliin 
PYRANOCOUMARINS (VISNAGAN GROUP) 
R = -CO-CH=C-CH3 
tH3 
Samidin 
o R = -CO-CH2-CH-CH3 Dihydrosamidin 
I 
CH3 
R = -CO-CH-CH2-CH3 Visnadin 
I 
CH3 
FURANOCHROMONES 
R, 0 R1 R2 R3 Chromone 
~ OCH3 OCH3 CH3 Khellin OH OCH3 CH3 Kheliinol o 0 R3 OCH3 H CH3 Visnagin OCH3 H CH20H Kheliol R2 OCH3 OCH3 CH20H Ammiol 
151 
Coumarins - Chromatographie Standards 
Tests 
Solvent 
system 
Tl = daphnoretin 
T2 = scopoletin 
T3 = isofraxidin 
T4 = umbelliferone 
T5 = herniarin 
T6 = xanthotoxin 
T7 = imperatorin 
T8 = ferulic acid * 
T 9 = caffeic acid * 
T10 = isopimpinellin 
T11 = isobergapten 
T12 = oxypeucedanin 
C-l: toluene-ether (1: 1, saturated with 10% acetic acid) 
Detection Without chemical treatment UV-365 nm Fig.l 
Chromato-
gram 
1 
UV-365 nm: 
light blue: 
violet: 
light green: 
yellow-green: 
daphnoretin, scopoletin, isofraxidin, umbelliferone. 
herniarin. 
xanthotoxin, isobergapten, oxypeucedanin. 
isopimpinellin. 
* Remarks: Coumarin drugs often contain plant acids, e.g. ferulic acid and caffeic acid, which 
also give blue fluorescence. In methanolic extracts, caffeic acid (T9) always produces two 
zones, corresponding to the free acid and its faster migrating methyl ester. 
Pimpinellae Radix Heraclei Radix 
Tracks 
Tests 
Solvent 
system 
1 = Pimpinellae radix (P . saxifraga) 
2=Pimpinellae radix (P. major) 
3 = Heraclei radix 
TGl = scopoletin (Rf ca. 0.3), umbelliferone (Rf ca. 0.45), imperatorin (Rf ca. 0.6) 
TG2 = umbelliferone (Rf ca. 0.45), xanthotoxin (Rf ca. 0.55) 
C-l: toluene-ether (1: 1, saturated with 10% acetic acid) 
Detection Without chemical treatment UV-365 nm Fig.2 
Chromato-
gram For description of drugs see p. 148. Formulae p. 150-151 
2 1,2 Pimpinellae radix: a strong blue fluorescent zone at the start in UV-365 nm, and 
152 
5 weaker blue fluorescent zones in the Rf range 0.1-0.55. 
Scopoletin (TG1) and sphondin (direcdy below the xanthotoxin test) can be identi-
fied. 
Remarks: Other coumarins e.g. isobergapten or isopimpinellin as described in the literature, 
can be detected only in enriched extracts of the drug. 
3 Heraclei radix: at least 10 blue or green-blue fluorescent zones in the Rf range 
0.1-0.8. 
Between the start and Rf 0.5, the chromatographie features are very similar 
to those obtained with Pimpinella extracts; sphondin (main zone Rf ca. 0.5) shows 
a distinct light blue fluorescence; a green-blue fluorescent zone of isopimpinellin 
(Rf ca. 0.55), and a blue fluorescent zone of isobergapten (Rf ca. 0.75) are also 
detectable and between these two zones lies pimpinellin, overlapped by bergapten. 
The violet fluorescent zone at Rf ca. 0.8 is due to umbelliprenin. 
Fig.l 
Fig.2 
153 
Angelicae, Levistici, Imperatoriae, Scopoliae Radix 
Tracks 
Tests 
Solvent 
system 
1 = Angelicae radix 
2 = Levistici radix 
3 = Imperatoriae radix 
Tl = xanthotoxin 
4 = Mei athamantici radix 
5 = Scopoliae radix 
T2 = umbelliferone (Rf ca. 0.4), imperatorin (Rf ca. 0.6) 
T3 = scopoletin 
C-l: toluene-ether (1: 1, saturated with 10% acetic acid) Fig. 3, 4 
Detection Without chemical treatment UV-365 nm Fig.3 
UV-365 nm Fig.4 
Chromato-
gram 
3,4 
154 
KOR reagent (No. 21, p. 302) 
For description of drugs see p. 147-149. Formulae p. 150-151 
Angelicae (1) and Imperatoriae (3) radix contain many structurally similar coumarins, 
which partly overlap on the chromatogram picture. Levistici radix (2) shows a lower 
coumarin content. 
Coumarins 1 
U mbelliprenin x 
Bergapten x 
Ostruthin 
Xanthotoxin x 
Imperatorin x 
Angelicin x 
Umbelliferone x 
Scopoletin x 
Oxypeucedanin hydrate x 
Plant acids x 
2 
x 
x 
x 
x 
3 
x 
x 
x 
x 
x 
: } 
Rf-range (approx.) 
0.8 
0.6 
I 
0.5 
0.45 
0.25 
0.2-0.1 
1 Angelicae radix: at least 15 light blue, dark blue or yellow-green fluorescent zones 
between the start and Rf 0.85. The prominent zones are in the Rf range 0.5-0.75; 
these are the partly overlapping zones of angelicin, imperatorin, xanthotoxin, hergap-
ten and osthenol (see Table). 
KOH treatment intensifies the fluorescence, especially in the Rfrange 0.7-0.75. 
2 Levistici radix: 5-7 mostly very weak light or dark blue fluorescent zones in the 
Rf range 0.25-0.9. Bergapten (Rf ca. 0.6, very elose to the imperatorin test) and 
umhelliferone (cf. T2) can be identified. The main zone at Rf ca. 0.9 is due to 
3-butylidenephtalide (ligusticum lactone). 
3 Imperatoriae radix: the chromatogram is similar to that of Angelicae radix, but 
differs by the presence of four especially strong, light blue fluorescent zones in 
the Rf range 0.3-0.6. The strong zone above the umbelliferone test (cf. T2) is due 
to ostruthin. Imperatorin (cf. T2) gives a green-blue fluorescence after KOR treatment 
(Fig.4). 
4 Mei athamantici radix: the single strong blue fluorescent zone is due to ligustilide. 
Mei atham. radix can be used as a test standard for Levistici radix. 
5 Scopoliae radix: the chromatogram is characterized by the presence of scopoletin 
(T3, Rf ca. 0.25) and scopoline (scopoletin glucoside), which remains near the start 
(see also Alkaloids, p. 90, Fig. 26). 
Fig.3 
Tl 2 3 
Fig.4 
Tl 2 3 
T2 2 4 
T2 5 T3 
FRONT 
Rf 
START 
155 
Herniariae, Meliloti, Asperulae, Abrotani, Rutae Herba 
Tracks 
Tests 
Solvent 
system 
1 = Herniariae herba 
2 = Meliloti herba 
3 = Asperulae herba 
4 = Abrotani herba 
5 = Rutae herba 
Tl = herniarin 
T2 = coumarin 
T3 = scopoletin (Rf ca. 0.25), umbelliferone (Rf ca. 0.4) 
C-1: toluene-ether (1: 1, saturated with 10% acetic acid) 
Detection Without chemical treatment 
KOH reagent (No. 21, p. 302) 
UV-365 nm Fig.5 
UV-365 nm Fig.6 
Chromato-
gram 
5, 6 
156 
For descriptionof drugs see p. 149. Formulae p. 150-151 
In drugs 1-4, the red fluorescent zones are due to various chlorophyll derivatives. 
1 Herniariae herba is characterized in UV-365 nm by the intense violet fluorescent 
zone of herniarin (cf T1), and two weak blue-violet fluorescent zones at Rf 0.35-0.4. 
Umbelliferone migrates at Rf ca. 0.4. 
The fluorescence of herniarin and umbelliferone is intensified by KOH treatment 
(Fig. 6) (see also Flavonoids, p. 176). 
2 Meliloti herba and 
3 Asperulae herba 
A large number of red fluorescent zones are seen in UV-365 nm. Otherwise there 
are only 4 weak blue or violet fluorescent zones in the Rf range 0.25--0.5. Coumarin 
(fluorescence quenching in UV-254 nm!) is clearly detectable only after KOH treat-
ment, when it forms an intense green-yellow main zone at Rf ca. 0.65 (Fig. 6, cf. 
T2). Both drugs also show weak zones of scopoletin and umbelliferone (cf. T3). 
Meliloti herba can be differentiated from Asperulae herba by the presence of 
an additional blue fluorescent zone at Rf ca. 0.5. 
After KO H treatment, only the chromatogram of Asperulae herba shows a strong 
blue fluorescence at the start. 
4 Abrotani herba is characterized by strong blue fluorescent zones, which correspond 
to scopoletin and umbelliferone in the test mixture T3. Directly below scopoletin 
is another, equally strong, blue fluorescent zone. 
5 Rutae herba shows at least 12 blue fluorescent zones between the start and the 
solvent front. The furanocoumarins, xanthotoxin, psoralene, bergapten und iso-
imperatorin (cf. Apiaceae) migrate in the upper half of the chromatogram (Rf 
0.5-0.8). 
Scopoletin and umbelliferone (Rf 0.25-0.4, cf. T4), rutaretin, daphnoretin and 
daphnoretin methyl ether can be identified in the lower half of the chromatogram. 
A TLC separation of Ruta-Flavonoids is described in the section on Flavonoids, 
p. 186, Fig. 16. 
Fig.5 
Tl 2 3 T2 4 
Fig.6 
Tl 2 3 T2 4 
T3 5 
T3 5 
FRONT 
Rf 
5 
FRONT 
Rf 
START 
157 
Mezerei, Fraxini Cortex Asa foetida 
Tracks 
Tests 
Solvent 
system 
1 = Mezerei cortex 
2 = Fraxini cortex 
3 = Asa foetida 
Tl = scopoletin 
T2 = umbelliferone 
C-l: toluene-ether (1: 1, saturated with 10% acetic acid) 
Detection Without chemical treatment UV-365 nm Fig.7 
Natural products-polyethyleneglycol reagent 
(NP/PEG No. 28, p. 303) UV-365 nm Fig.8 
For description of drugs see p. 148-149. Formulae p. 150-151 
Chromato- 1 Mezerei cortex is characterized in UV-365 nm by about 5 blue fluorescent zones 
in the Rf range 0.2-0.75. gram 
7, 8 
158 
NP/PEG reagent intensifies the fluorescence, which becomes light blue; in addi-
tion, yellow-green fluorescent zones appear at Rf 0.3 and above the start. Coumarin 
glucosides, e.g. daphnetin glucoside, remain at the start. After NP/PEG treatment, 
the zones at RF 0.35-0.45 are intensified, which is typical of ferulic and caffeic 
acid. 
2 Fraxini cortex shows about 6 blue fluorescent zones in the lower and intermediate 
Rf range. The four c10sely neighbouring zones at Rf 0.25-0.4 are due to fraxetin, 
fraxidin, isofraxidin and fraxinol, all of which are coumarins with very similar pat-
terns of substitution. Coumarin glycosides (e.g. fraxin) remain at or ne ar the start. 
Intensification of fluorescence in the lower Rf range, observed after treatment 
with NP/PEG reagent, is typical of this drug 
3 Asa foetida. In UV-365 nm, the chromatogram shows a characteristic series of at 
least 10 blue or violet fluorescent zones between the start and Rf 0.8. The main 
zone corresponds chromatographically with the umbelliferone test (T2), and it con-
sists of umhelliferone plus ferulic acid. The other fluorescent zones are due to umbelli-
ferone derivatives. 
Fig.7 
Fig.8 
159 
Ammi (Ammeos) Fructus 
Tracks 
Tests 
Solvent 
system 
Detection 
1 = Ammi fructus (A. major) 
2 = Ammi fructus (A. visnaga) 
Tl =visnadin (extracted and concentrated from Carduben®) 
Rf ca. 0.6 (solvent C-l), Rf ca. 0.9 (solvent C-2) 
T2=khellin 
T3 = visnagin 
C-l: toluene-ether (1: 1, saturated with 10% acetic acid) 
C-2: ethyl acetate 
Without chemical treatment 
KOR reagent (No. 21, p. 302) 
SbCl3 reagent (No. 3, p. 299) 
UV-365 nm 
UV-365 nm 
UV-365 nm 
For description of drugs see p. 148. Formulae p. 151 
Fig. 9A, B 
Fig. lOA, B 
Fig.9A 
Fig. 9B; lOA 
Fig. lOB 
Chromato- 1 Ammi majoris fructus. In UV-365 nm the chromatogram is characterized by aseries 
gram of at least 12 intense, light blue fluoreszent zones between the start and Rf ca. 
Solvent C-I 0.7. 
9 A, B The fluorescence is intensified by KO H treatment (Fig. 9 B). 
The furanocoumarins, bergapten, xanthotoxin, isopimpinellin and imperatorin 
(cf. tests, Fig. 1, p. 152) migrate above RfO.5, overlapping to some extent. According 
to the literature, the zones below Rf 0.5 are due to marmesin, marmesinin and 
ammaJllll. 
2 Ammi (Ammeos) visnagae fructus. In UV-365 nm the chromatogram shows only 
a few weak violet and pale blue fluorescent zones at and near the start, and at 
Rf 0.4-0.6 with visnadin as the main one. The zones also show a distinct fluorescence 
quenching in UV-254 nm. 
KOH treatment intensifies the fluorescence, especially in the Rf range 0.4-0.6. 
Visnagin and khellin (cf. T3 and T2) show prominent fluorescence quenching 
in UV-254 nm, and light blue (visnagin) and weak green-blue (khellin) fluorescence 
in UV-365 nm, which is hardly intensified by treatment with KOR reagent. 
In solvent C-2, ethyl acetate, all coumarins and chromones have rather high Rf 
values. 
10A 1 Ammi majoris fructus. There are many overlapping coumarin zones, especially in 
the Rf range 0.7-0.9. 
2 Ammi visnagae fructus shows a better separation of khellin and visnagin in the 
solvent system C-2. 
10 B After treatment with SbCI3 , visnagin gives a typical yellow-green fluorescence. Khel-
lin, however, appears black-brown and is no longer c1early defined against the back-
ground. Khellol glucoside, khellol and khellinol mi grate at Rf 0.1-0.25 as blue 
fluorescent zones. Visnadin (Tl) forms a blue-violet fluorescent zone be10w the zones 
of samidin and dihydrosamidin at the solvent front. 
160 
Fig.9 
Tl 2 Tl 
Fig.10 
T 2 T2 T3 
2 T2 
Tl 2 
T3 
FRONT 
Rf 
START 
161 
Flavonoid Drugs 
The main eonstituents of flavonoid drugs are 2-phenyl-y-benzopyrones (2-phenyl-
ehromones) or strueturally related, mostly phenolie, eompounds. 
The various types of flavonoid strueture differ in the degree of oxidation of 
ring C, and in the pattern of substitution in the A and/or B rings (see formulae, 
p. 170). 
Most of these eompounds are present in the drugs as mono-or diglyeosides. 
I. Preparation of Drug Extracts for TLC 
Powdered drug (1 g) is extraeted with 10 ml methanol for 5 min on a water bath 
at about 60° C. The clear filtrate is used for ehromatography. 
This rapid method also extraets both lipophilie and hydrophilie flavonoids. 
Exeeptions: 
Cardui mariae fructus (Silybi fruetus): powdered drug (1 g) is first defatted by heating 
under reflux for 30 min with 50 ml light petroleum. The petroleum extraet is dis-
earded and the drug residue is heated under reflux for 15 min with 10 ml methanol. 
The filtrate is eoneentrated to 5 ml, and 30 111 are used for ehromatography. 
Orthosiphonisfolium, DAB 8: powdered drug (1 g) is extraeted by shaking for 15 min 
with 10 ml diehloromethane. The clear filtrate (30 111) is used for ehromatography. 
Farfarae folium, Petasites folium (test for petasins): powdered drug (2 g) is extraeted 
by heating under reflux for about 20 min with about 40 ml light petroleum on 
a water bath. The clear filtrate is eoneentrated to about 1 ml, and 30 111 are used 
for ehromatography. 
11. Thin Layer Chromatography 
1. Reference solutions 
Standard eompoundsare prepared as 0.05% solutions in methanol, and about 10 f..ll 
are used for ehromatography. The average deteetion limit for flavonoids is 5~10 f..lg. 
Unless otherwise stated, a mixture of standards (Tl) of rutin, ehlorogenie aeid and 
hyperoside is used. 
2. Adsorbent 
Siliea gel 60F 254 pre-eoated TLC plates (Merek, Darmstadt). 
3. Sampie concentration 
Provided the flavonoid eontent ofthe drug is between 0.5 and 1.5%, 25~30 f..ll extraet 
are suffieient. For pharmaeeutical preparations, the quantity of extract used for 
TLC must be adjusted in proportion to the flavonoid eontent. 
163 
164 
4. Chromatography solvents 
F -1 Ethyl acetate-formic acid-glacial acetic acid-water (100: 11: 11: 27) 
The ethyl acetate, formic acid and glacial acetic acid are mixed first, and the 
water is added gradually with vigorous shaking. If the ethyl acetate is technical 
grade, the composition 100: 11 : 11 : 26 should be used; separations with this 
mixture show very slightly different Rf values, but no change in the order 
of separation. F -1 is suitable as a screening system for the TLC investigation 
of flavonoid glycosides. 
Solvent systems specified in the official methods are variations of similar 
system of ethyl acetate-glacial acetic acid-water: 
Arnicae flos, DAB 8 (66: 15:20) 
Tiliae flos, DAB 8 (67: 13: 20) 
Aurantii pericarpium, DAB 8 (67: 7: 26) 
Crataegi folium cum flore, DAB 8 (67: 7: 26) 
F-2 Ethyl acetate-formic acid-glacial acetic acid-ethylmethyl ketone-water 
(50:7:3:30:10) 
Certain separation problems have been overcome by addition of ethylmethyl 
ketone to F-1, e.g. for the constituents of Crataegi folium or flos (see p. 179, 
Fig.8). 
F-3 Chloroform-acetone-formic acid (75: 16.5: 8.5) 
For the separation of the flavanolignans of Cardui mariae fructus. 
F-4 Chloroform-ethyl acetate (60:40) 
For the separation of the flavonoid aglycones of Orthosiphonis folium. 
Remarks: Flavonoid aglycones can also be separated in benzene-pyridine-formic 
acid (72: 18: 10) or in toluene-ethyl formate-formic acid (50: 40: 10). 
F-5 n-Butanol-glacial acetic acid-water (40: 10: 50) (upper phase). 
For the separation of flavonoid glycosides on cellulose plates according to 
DAB8. 
F-6 Analytical grade chloroform (with chamber saturation) 
For the detection of petasins. Petasites folium is an adulterant of Farfarae 
folium. 
IH. Detection 
Residual solvent (acids) must be thoroughly removed from the silica gel layer with 
the aid of a hot air blower. 
1. Without chemical treatment 
UV-254 nm: All flavonoids cause fluorescence quenching, which is seen as dark 
blue zones on the yellow background of the TLC plate. 
UV-365 nm: Depending on the structural type, flavonoids fluoresce yellow, blue 
or green. 
Intensification and greater differentiation of fluorescence in UV-365 nm can be 
achieved by the use of various spray reagents. 
Flavonoid extracts often contain other materials, such as plant acids and couma-
rins, which form blue fluorescent zones (e.g. Rutae herba). 
2. Spray reagents 
a. Natural products reagent (NP/PEG No. 28, p. 303) 
Typical intense fluorescent colours in UV-365 nm are produced immediatelyon 
spraying, or after about 15 min. Addition of PEG lowers the detection limit from 
10 Ilg to 0.5 Ilg· 
Fluorescence behaviour is structure-dependent: 
Flavonols: 
Glycosides of quercetin and myricetin -> orange 
Glycosides of kaempferol and isorhamnetin -> yellow-green 
Flavones: 
Glycosides of luteolin -> orange 
Glycosides of apigenin -> yellow-green 
b) Fast blue saft B (FBS No. 12, p. 301) 
Blue or blue-violet (vis.) azo-dyes are formed in daylight. To some extent, these 
can be intensified by further spraying with 0.1 M sodium hydroxide or 10% potassi-
um hydroxide. 
IV. List of Flavonoid Drugs 
Chromatograms (Figs. 3-22) are reproduced on pp. 174-193. 
Grouping of drug chromatograms according to plant parts: 
Flos: Figs. 3-10 
Folium! Herba: Figs. 11-16, 22 
Gemma! Pericarpium: Figs. 15, 17 
Drugs containing predominantly flavonoid aglycones: Figs. 18-20 
Fig. 
5 
10 
11 
12 
13 
6 
. Drug/Plant source 
F amily /Pharmacopoeia 
Arnicae Flos 
Arnica flowers 
Arnica montana L. 
Asteraceae 
DAB 8, Helv. VI, ÖAB 
Arnica chamissonis LESS. 
2. AB-DDR 
Acaciae Flos 
Acacia flowers 
Robinia pseudoacacia L. 
Fabaceae 
Anthemidis Flos 
Chamomile flowers 
Chamaemelum nobile (L.) 
ALLIONI (syn. Anthemis 
nobilis L.) 
Asteraceae 
Ph. Eur. III, ÖAB 
CactiFlos 
Night-blooming Cereus 
Cereus grandiflorus MILL. 
Cactaceae 
Main constituents 
Adulterants 
Quercetin-3-0-glucoside, 
Q-3-0-glucogalacturonide, 
luteolin-7-0-glucoside, 
kaempferol-3-0-glucoside. 
Adulterants : Calendulae flos, Heterothecae 
inuloidis flos, Farfarae flos, Taraxaci flos. 
Kaempferol-3-0-rhamnosylgalactosyl-7-
rhamnoside (robinin), acacetin-7-0-rutino-
side, acaciin. 
Adulterant: Pruni spinosae flos 
Apigenin-7-0-glucoside, luteolin-7-0-gluco-
side. 
Essential oil (see Essential Oil Drugs, Fig. 11, 
p.32) 
Isorhamnetin glycosides: 
1-3-0-galactoside (cacticin), 
1-3-0-galactosyl-rutinoside, 
I-3-0-rutinoside (nareissin). 
Rutin 
165 
Fig. 
5, 6 
7,8 
5 
21,22 
13 
6 
166 
Drug/Plant source 
Family /Pharmacopoeia 
Calendulae Flos 
Marigold flowers 
Calendula officinalis L. 
Asteraceae 
2. AB-DDR 
Crataegi Flos 
Hawthorn flowers 
Crataegi Folium c. Flore 
Hawthorn leaves with flowers 
DAB8 
Crataegi Folium 
Hawthorn leaves 
Helv. VI 
Crataegi Fructus 
Hawthorn fruits 
Crataegus monogyna JAQUIN 
emend. LIND MANN 
Crataegus pentagyna, C. nigra, 
C. azarolus L. 
Rosaceae 
Farfarae Flos 
Coltsfoot flowers 
Farfarae Folium 
Coltsfoot leaves 
Tussilago farfara L. 
Asteraceae 
DAB 8 (leaves), Helv. VI (flowers) 
Matricariae Flos 
(Chamomillae Flos) 
German chamomlIe flowers 
Chamomilla recutita (L.) St. RAu-
SCHERT 
(syn. Matricaria chamomilla L.) 
Asteraceae 
Ph. Eur. III, 2. AB-DDR, 
Helv. VI, ÖAB 
Primulae FIos 
Official primrose flowers 
Primula veris L. 
Primula elatior (L.) HILL. 
Primulaceae 
Main constituents 
Adulterants 
Isorhamnetin glycosides: 
I -3-0-glucoside, 
I-3-0-rutinoside (narcissin), 
I -3-0-rutinorhamnoside. 
Quercetin-3-0-glucoside and Q-3-0-gluco-
rhamnoside, 
Adulterant: Arnicae flos, Anthemis tinctoria L., 
Inula spp. 
Glycosides of quercetin and apigenin 
Hyperoside, rutin, quercetin rhamnogalacto-
side, vitexin, vitexin-2" -O-rhamnoside and 
other flavone-C-glycosides in varying concen-
trations in all parts of the drug. 
Adulterant: Acaciae flos 
Quercetin glycosides: rutin, hyperoside and 
isoquercetin in varying concentrations in both 
drug parts. 
Adulterants: Petasites folium (Petasites hybri-
dus, P. albus, P. paradoxus): petasin, isopeta-
sin in varying concentrations. 
0.5--3% total flavonoids: quercimeritrin, api-
genin-7-glucoside, luteolin-7-0-glucoside, pa-
tuletin-7-0-glucoside and more than 7 agly-
cones. 
Adulterant: Anthemidis flos. 
Essential oil (see Essential Oi! Drugs, Figs. 11 
& 12, p. 32). 
Glycosides of quercetin or gossypetin, kaemp-
ferol-dirhamnoside (Primula-flavonoside) and 
K -3-0-gentiotrioside. 
Adulterant: Verbasci flos. 
Remarks: for Primulae radix, see under Sa-
ponin drugs, Fig. 3, p. 236. 
Fig. 
9 
7 
9 
7 
10 
7 
11 
Drug/Plant source 
F amily /Pharrnacopoeia 
Pruni spinosae Flos 
Blackthorn flowers 
Prunus spinosa L. 
Rosaceae 
Sambuci Flos 
Eider flowers 
Sambucus nigra L. 
Caprifoliaceae 
2. AB-DDR, Helv. VI, ÖAB 
Spiraeae Flos 
Meadow-sweet flowers 
Filipendula ulmaria L. 
Rosaceae 
Stoechados Flos 
(syn. Helichrysi flos) 
Cat's-foot flowers 
Helichrysum arenarium (L.) DC 
Asteraceae 
Helv. VI 
Tiliae Flos 
Lime flowers 
Tilia cordata MILL. 
Tilia platyphyllaScoP. 
Tiliaceae 
DAB 8, Helv. VI, ÖAB, 
2. AB-DDR 
Verbasci Flos 
Mullein flowers 
Verbascum phlomoides L. 
V. thapsiforrne SCHRADER 
Scrophulariaceae 
Helv. VI, ÖAB 
Betulae Folium 
Birch leaves 
Betula pendula ROTH. 
B. pubescens ERHART 
Betulaceae 
DAB 8, Helv. VI, ÖAB, 
2. AB-DDR 
Main constituents 
Adulterants 
Quercetin glycosides: rutin, avicularin (Q-3-
O-arabinoside ). 
Kaempferol glycosides: K-3,7-0-dirhamno-
side, K-3-0-rhamnoside, K-3-arabinoside. 
Adulterant: Robiniae flos. 
Quercetin glycosides 1.5-3%: hyperoside, 
isoquercitrin, rutin. 
Spiraeoside (quercetin-4' -O-glucoside), hyper-
oside, avicularin (quercetin-3-0-arabinoside). 
Adulterant: Sambuci flos. 
N aringenin-5-monoglucoside (salipurposide), 
kaempferol-3-0-glucoside, K-3-0-digluco-
side, apigenin-7 -glucoside, luteolin-7 -O-glu-
coside. 
Quercetin glycosides: Q-3-0-g1ucoside, Q-3-
O-rhamnoside, Q-3-0-glucosyl-7-0-rhamno-
side. 
Kaempferol glycosides: K-3-0-glucoside, K-3-
O-rhamnoside, K -3-0-glucosyl-7 -O-rhamno-
side, K-3,7-0-dirhamnoside, K-p-coumaroyl-
glucoside (tiliroside). 
Myricetin glycosides: M-3-0-glucoside, M-3-
O-rhamnoside. 
Adulterant: T. argentea. 
2-4% total flavonoids. The main flavonoids 
are rutin, hesperidin and other flavonol glyco-
sides. 
Adulterants: Primulae flos, and other species 
ofVerbascum and Genista 
About 1.5% Quercetin glycosides: 
Q-3-0-arabinoside, Q-3-0-rhamnoside (quer-
citrin), Q-3-0-galactoside (hyperoside), Q-3-
O-rutinoside (rutin). 
Myricetin-3-digalactoside 
Kaempferol-3-0-glucoside, 
K-3-0-rhamnoside, Hesperidin. 
167 
Fig. 
12 
15 
16 
6 
16 
16 
15 
16 
14 
15 
168 
Drug/Plant source 
F amily /Pharrnacopoeia 
Juglandis Folium 
Walnut leaves 
J uglans regia L. 
Juglandaceae 
Anserinae Herba 
Silverweed 
Potentilla anserina L. 
Rosaceae 
Equiseti Herba 
Common horsetail 
Equisetum arvense L. 
Equisetaceae 
DAB 8, Helv. VI, ÖAB, 
2. AB-DDR 
Herniariae Herba 
See drug list on p. 148. 
Leonuri Herba 
Motherwort 
Leonurus cardiaca L. 
Lamiaceae 
Rutae Herba 
See drug list on p. 149. 
Sarothamni scop. Herba 
Broom 
Sarothamnus scoparius (L.) WIM-
MER 
Fabaceae 
DAC 
Veronicae Herba 
Common speedweIl 
Veronica officinalis L. 
Scrophulariaceae 
Vigaureae Herba 
(Solidaginis virgaureae herba) 
Golden-rod 
Solidago virgaurea L. 
Asteraceae 
Violae tricoloris Herba 
Wildpansy 
Viola tricolor L. 
Violaceae, ÖAB 
Main constituents 
Adulterants 
Hyperoside (ca. 0.2%) and other flavonol gly-
cosides. 
Quercetin-3-0-glucoside, Q-3-0-rhamnoside. 
Myricetin and myricetin rhamnoside. 
Flavonoids: Luteolin-5-0-glucoside (galuteo-
lin), isoquercitrin, kaempferol-7-0-digluco-
side (equisetrin). 
Saponins: Equisetonin (HI 660). 
Flavonoids: Rutin, narcissin. 
Coumarins: see Figs. 5 & 6, p. 156. 
Rutin 
Adulterant: Leonurus glaucescens. 
Rutin 
Coumarins: see Figs. 5 & 6, p. 156. 
Scoparin (3' -O-methyl-orientin), vitexin. 
Adulterant: Spartium junceum. 
Alkaloids (see p. 86, sparteine). 
Luteolin, L-7-0-glucoside, rutin. 
Adulterant: Stachys alpina. 
Quercetin glycosides: 
Q-3-0-rutinoside (rutin), 
Q-3-0-rhamnoside (quercitrin), 
Q-3-0-glucoside (isoquercitrin). 
Kaempferol-3-0-glucoside (astragalin). 
Adulterants: S. canadensis L., S. gigantea L. 
Quercetin glycosides: high content of rutin 
(" Viola-quercitrin "). 
Adulterant: V. tricolor var. vulgo 
Fig. 
15 
17 
18 
19, 20 
Drug/Plant source 
F amily /Pharmacopoeia 
Sophorae Gemma 
Sophora buds 
Sophora japonica L. 
Fabaceae 
Aurantii Pericarpium 
Seville orange peel 
Citrus aurantium L. ssp. aurantium 
Rutaceae 
DAB 8,2. AB-DDR, ÖAB, 
Helv. VI 
Citri pericarpium 
Lemonpeel 
Citrus media L. 
Rutaceae 
Main constituents 
Adulterants 
Rutin (ca. 20%) and other flavonol glycosides. 
Eriocitrin, rutin, naringenin, naringin, hesperi-
din, neohesperidin, sinensetin. 
Adulterant: Aurantii albedo. 
Bitter principles: see Fig. p. 132. 
Essential oiIs: see Fig. p. 44. 
Eriocitrin (eriodictyol-7 -O-rutinoside), rutin, 
naringenin -7 -0-hesperoside, neohesperidin, 
hesperidin, apigenin-C-glucoside; aureusidin, 
Au-6-glucoside, Au-6-rhamnoglucoside; iso-
rhamentin-3-arabinoglucoside; limocitrol, L-
3-glucoside, L-3-arabinoglucoside; limocitrin, 
L-3-g1ucoside, L-3-arabinoglucoside; luteo-
lin-7-rutinoside. 
Bitter principles and essential oils: see p. 132 
and p. 44. 
Drugs containing predominantly flavonoid aglycones: 
Eriodictyonis Herba 
Yerba Santa (herba) 
Eriodictyon glutinosum BENTH. 
Hydrophyllaceae 
Orthosiphonis Folium 
Orthosiphon Ieaves 
Orthosiphon spicatus (THUNB.) 
Bak. 
Lamiaceae 
DAB 8, Helv. VI 
Cardni mariae Fructus 
Milk-thistle fruits 
Silybum marianum GAERTNER 
Asteraceae 
DAB8 
Homoeriodictyol ( = eriodictyone), eriodictyol, 
chrysoeriodictyol, xanthoeriodictyol. 
Adulterant: Eriodictyon crassifolium Benth. 
About 0.2% flavonoids: 
sinensetin (3',4',5,6,7 -pentamethoxyflavone), 
scutellarein tetramethyl ether and eupatorin 
(3' ,5-dihydroxy-4' ,6, 7 -trimethoxyflavone). 
Flavanolignans: 
silybin, silychristin and silydianin. 
Adulterant: other Silybum spp. 
169 
v. Formulae of Constituents of Flavonoid Drugs 
FLAVONES R R 
OH H 
HO OH 
OH 0 
FLAVONOLS R, 
OH OH 
HO 
R2 H 
OH 
OCHa 
OH 0 
FLAVANON(OL)S R, 
R3 H 
HO H 
H 
H 
OH 0 
OH 
HO 
Amentoflavone 
170 
Aglycones Glycosides 
Apigenin Apigenin 8-C-glucoside 
(=Vitexin) 
Vitexi n-2" -O-rhamnoside 
Luteolin Luteolin-8-C-glucoside 
R2 
H 
H 
OH 
H 
R2 
H 
OH 
OCHa 
OH 
OH 
OH 
(= Orientin) 
Aglycones Glycosides 
Quercetin Q-3-0-galactoside (Hyperoside) 
Q-3-0-glucoside (Isoquercitrin) 
Q-3-0-rhamnoside (Quercitrin) 
Q-3-0-rutinoside (Rutin) 
Kaempferol K-3-0-glucoside (Astragalin) 
Myricetin M-3-0-digalactoside 
Iso- 1-3-0-rutinoside (Narcissin) 
rhamnetin 
Ra Aglycones Glycosides 
OH Naringe- Nari ngeni n-7 -O-neohespe-
nin ridoside (Naringin) 
OH Eriodic- Eriodictyol-7 -O-ruti no-
tyol side (Eriocitrin) 
OH Homoerio-
dictyol 
OCHa Hespere- Hespereti n-7 -O-neohes-
tin peridoside (Neohesperidin) 
Hespereti n-7 -O-ruti noside 
(Hesperidin) 
OH Taxifolin 
rAr0 .CH20H HOuO'(~OCH' 
~OH OH 
HO 0 
Silybin 
Sinensetin 
~OH 
H°ItY°i···OOH ~OH 
OH 0 H 
Taxifolin Coniferyl alcohol 
Caffeic acid Quinic acid PHENOL CARBOXYLIC ACIDS 
.-______ ~A~ _____ , ,--__ --'A\.-__ ----, 
HO~ R : ;-l!}0H 
H0-O--CH=CH-C+-O~ 
PETASINS 
Petasin 
S-Petasin 
HO OH 
Chlorogenic acid 
~""OR 'O~ 
o CH3 
11 I (I)R=C-C=CH 
I 
o 
11 
CH3 
(11) R =C-CH=CHSCH3 
( !!!) R = H (Petasol) 
Chlorogenic acid 
Neochlorogenic acid 
Isochlorogenic 
acid 
Cynarin (native) 
Cynarin (isoL) 
m OR 
o CH3 
11 I 
3-Caffeoylquinic acid 
5-Caffeoylquinic acid 
4-Caffeoylquinic acid 
1-Caffeoylquinic acid j1,3-DicaffeOYlqUinic aicd 3,5-Dicaffeoylquinic acid 
3,4-Dicaffeoylquinic acid 
4,5-Dicaffeoylquinic acid 
1 ,3-Dicaffeoylqu inic acid 
1 ,5-Dicaffeoylquinic acid 
(IV) R= C-C=CH Isopetasin 
I 
CH3 
(V) R = H (Isopetasol) 
171 
Reference Compounds Flavones, Flavonols, Flavanones, Phenol carboxylic acids 
Test series A: 
Tracks 1 = quercetin-3-0-gentiobioside 
2 = kaempferol-3-0-gentiobioside 
Fig. 1 3 = quercetin-3-0-rutinoside (rutin) 
4 = vitexin-2" -O-rhamnoside 
5 = naringin and neo hesperidin 
6 = chlorogenic acid (Rf ca. OA5) 
7=orientin 
8=vitexin 
9= isorhamnetin-3-0-glucoside (with isoquercitrin) 
10= chlorogenic acid, isochlorogenic acid (Rf ca. 0.8), caffeic acid 
(Rf ca. 0.9) 
11 = isorhamnetin-3-0-galactoside 
12 = quercetin-3-0-rhamnoside (with traces of astragalin)13 = kaempferol-7-0-rhamnoside 
14 = caffeic acid and ferulic acid (Rf 0.9-0.95) 
15 = rutin (Rf ca. OA), chlorogenic acid (Rf ca. OA5), hyperoside (Rf ca. 0.6) 
(standard mixture Tl; these are the three main compounds 
for standardization of flavonoid chromatograms) 
Test series B: 
Tracks 
Fig.2 
Solvent 
system 
1 = quercetin-3-0-gentiobioside 
2 = quercetin-3-0-sophoroside 
3 = quercetin-3-0-galactosyl-7-0-rhamnoside 
4 = kaempferol-3-0-gentiobioside 
5 = quercetin-3-0-rutinoside (rutin) 
6 = kaempferol-3-0-rhamnoglucoside 
7 = quercetin-3-0-glucuronide 
8 = quercetin-3-0-galactoside (hyperoside) 
9 = quercetin-3-0-glucoside (isoquercitrin) 
10= kaempferol-3, 7-0-dirhamnoside 
11 = quercetin-3-0-rhamnoside (quercitrin) 
12 = kaempferol-3-0-arabinoside 
13 = quercetin 
14 = kaempferol 
15=mixture of 1-14 
F-l: ethyl acetate-formic acid-glacial acetic acid-water 
(100:11:11:27) 
Detection Natural products-polyethyleneglycol reagent 
(NPjPEG No. 28, p. 303) UV-365 nm Fig. 1,2 
Fig.l 
Fig.2 
172 
shows glycosides of flavones, flavonols and flavanones, which fluoresce orange, 
yellow-green and dark green in UV-365 nm after treatment with NP/PEG reagent. 
Phenol carhoxylic acids, which frequently occur in flavonoid drugs, appear as 
intense, light blue zones. 
shows flavonol glycosides of the aglycones, quercetin and kaempferol. 
Orange or yellow-green fluorescence in UV-365 nm, following NP/PEG treat-
ment, is re1ated to the specific substitution pattern in ring B: 
Two adjacent hydroxyl groups in ring B (e.g. quercetin) give rise to orange fluo-
rescence, whereas a single free hydroxyl group (e.g. kaempferol) results in yellow-
green fluorescence. 
Fig.l 
2 3 4 5 6 7 8 9 10 11 1213 14 
- .. 
• • 
e • 
. t . , 
• 
• • 
• • I • 
Fig.2 
1 2 3 4 5 6 7 8 9 1011 12 13 14 15 
-FRONT 
Rf 
START 
15 TestreiheA 
Test series A 
• • 
. . 
TestreiheB 
Test series B 
173 
Flower Drugs I TLC Synopsis 
Tracks 
Solvent 
system 
1 = Arnicae flos 
2 = Stoechados flos 
3 = Sam buci flos 
4 = Pruni spinosae flos 
5 = Tiliae flos 
6 = Verbasci flos 
7 = Calendulae flos 
8 = Cacti flos 
9= Primulae flos 
F-1: ethyl acetate-formic acid-glacial acetic acid-water 
(100:11:11:27) 
Detection Natural products reagent (NP No. 28, p. 303) 
Natural products-polyethyleneglycol reagent 
(NP/PEG No. 28, p. 303) 
UV -365 nm Fig. 3 
UV-365 nm Fig.4 
Chromato-
gram 
3,4 
174 
For description of drugs see pp. 165-167. Formulae p. 170 
Each drug shows a characteristic order and number of yellow, green or blue fluores-
cent zones. 
In drugs 1-6, flavonoids are absent in the lower Rf-range of the chromatogram. 
The main flavonoids and acids are found between Rf 0.35 and the solvent front. 
Drugs 7-9 show flavonoid glycosides especially at Rf 0.1-0.4. Drugs 7 and 8, 
but not 9, also show a few weaker zones in the higher Rf range. 
The numerous blue fluorescent zones of phenol carhoxylic acids are characteristic 
of drugs 1-4; these are almost absent from drugs 5, 8 and 9, and only weakly 
represented in drugs 6 and 7. 
Figs. 3 and 4 show pronounced differences in the fluorescence colours of the 
flavonoids. These gradations of colour amongst yellow, green and orange become 
more distinct after treatment with NP/PEG reagent. 
Characterization of these individual drugs 1-9, with identification of the main 
flavonoids, is described on pp. 176 and 180, Figs. 5-10. 
Fig.3 
2 3 4 5 6 7 8 9 
-: -, -- -
-. · .. -tr 
: _ 111 • -
- .. --- -_ ... -
-
Fig.4 
2 3 4 5 6 7 8 9 
-FRONT 
Rf 
0 .5 
START 
-FRONT 
Rf 
-0.5 
START 
175 
Flower Drugs 11 
Tracks 
Tests 
Solvent 
system 
1 = Arnicae flos 
2 = F arfarae flos 
3 = Calendulae flos (patterns land 11) 
4 = Herniariae herba 
5 = Cacti flos 
6 = Primulae flos 
Tl = rutin (Rf ca. 0.3), chlorogenic acid (Rf ca. 0.4), hyperoside (Rf ca. 0.55) 
F-l: ethyl acetate-formic acid-glacial acetic acid-water (100: 11: 11 :27) 
Detection Natural products-polyethyleneglycol reagent 
(NPjPEG No. 28, p. 303) UV -365 nm Fig. 5, 6 
For description of drugs see p. 165-168. Formulae p. 170 
Chromato- 1 Arnieae jlos 
gram In UV -265 nm, three orange fluorescent zones are seen at Rf 0.45-0.6: isoquercitrin 
5 directly above hyperoside test, luteolin-7-0-glueoside at the same Rf as for the stan-
dard hyperoside test, and a third flavonol glycoside at Rf 0.45 (cf. Tl). Also present 
are the weak, green fluorescent zone of a kaempferol monoglycoside (Rf ca. 0.7) 
and two intense, blue fluorescent zones due to ehlorogenie acid (Rf ca. 0.4, cf. 
Tl) and eajJeie acid (Rf ca. 0.9). 
2 Farfarae jlos 
This drug is characterized by three orange fluorescent zones. The main zone is 
due to rutin (Rf ca. 0.3, cf. Tl); the other two, which migrate at Rf 0.6-0.7 (above 
the hyperoside test), are due to flavonol monoglycosides. The blue fluorescent zones 
are due to ehlorogenie acid (Rf ca. 0.4, cf. Tl), isoehlorogenie aeid (Rf ca. 0.75) 
and cajJeie acid (Rf ca. 0.9). 
3 Calendulae jlos 
The chromatogram shows orange and yellow-green fluorescent zones in UV-365 nm, 
which are most intense between Rf 0.15 and 0.4. Rutin (cf. Tl) lies between two 
yellow-green fluorescent zones, which are due to narcissin (isorhamnetin-3-0-rutino-
side) (above rutin) and isorhamnetin rutinorhamnoside (below rutin). In addition, 
there is a weak, green fluorescent zone of isorhamnetin-3-0-glueoside (Rf ca. 0.7) 
and a weak, blue fluorescent zone in the same Rf region as eh/orogenie acid (cf. 
Tl). 
Differentiation 
The main zone of rutin and nareissin in Calendulae jlos, and the zones of rutin 
and isoehlorogenie acid in Farfarae jlos are easily recognized, even in mixtures with 
Arnicae jlos. 
6 4, 5, 6 Herniariae herba, Caeti jlos, Primulae jlos 
176 
Extracts of Herniariae herba and Caeti jlos, like those of Calendulae jlos (3), show 
the typical zone of rutin and narcissin at Rf 0.3-0.35. 
The orange fluorescent zones from Primulae jlos are due to glycosides of querce-
tin and gossypetin; the green fluorescent zones are due to kaempferol diglycosides. 
In the lower Rf region a high concentration of flavonol triglycosides is evident. 
Fig.5 
2 3 
Fig.6 
4 5 6 
FRONT 
Rf 
FRONT 
Rf 
177 
Flower Drugs 111 Crataegi Folium, Fructus, Flos 
Tracks 
Tests 
1 = Stoechados flos 
2 = Sambuci flos 
3 = Crataegi flos 
4 = Verbasci flos 
5 = Crataegi folium 
6 = Crataegi fructus 
Tl = rutin (Rf ca. 0.35), chlorogenic acid (Rf ca. 0.45), hyperoside (Rf ca. 0.55) 
T2 = vitexin-2" -O-rhamnoside (Rf ca. 0.35), vitexin (Rf ca. 0.7) 
Solvent F -1: ethyl acetate-formic acid-glacial acetic acid-water 
system (100 : 11 : 11 : 27) Fig. 7 
F-2: ethyl acetate-formic acid-glacial acetic acid-
ethylmethyl ketone-water (50:7:3:30:10) Fig.8 
Detection Natural products-polyethyleneglycol 
reagent (NP/PEG No. 28, p. 303) UV- 365 nm Fig. 7, 8 
For description of drugs see p. 166-167. Formulae p. 170 
Chromato- 1 Stoeehados flos 
gram The flavanone glycoside, ( - ) or ( + ) naringenin-5-0-glueoside (salipurposide), which 
7 is characteristic of this drug extract, appears as a black-brown zone at Rf ca. 0.8 
(cf. Fig. 3, p. 175). Apigenin-7-0-glueoside (green-yellow fluorescence) and luteolin-7-
O-glueoside (orange fluorescence) migrate above the hyperoside test (cf. Tl) at Rf 
0.6-0.7. The green-yellow fluorescent zone of kaempferol-3-0-glueoside lies directly 
below the intense, blue fluorescent zone of eaffeie acid (Rf ca. 0.9). Other blue 
fluorescent zones are due to ehlorogenie aeid (cf. Tl) or isoehlorogenie aeid (Rf 
ca. 0.7). 
2 Samhuci flos 
The chromatogram shows two orange fluorescent zones of about equal intensity, 
due to rutin (Rf ca. 0.35) and isoquercitrin (Rf ca. 0.6). Each is accompaniedby 
a weak, yellow-green fluorescent zone directly above it. Chlorogenie acid (cf. T1) 
and eaffeie acid are present, as in drugs 1 and 3. 
3 Crataegi flos 
The chromatogram characteristically shows two strong, orange fluorescent zones 
in the region of the hyperoside test due to hyperoside and a flavonol monoglycoside, 
two strong, blue fluorescent zones in the region of the chlorogenic acid test (cf. 
T1) (due to ehlorogenie aeid and neoehlorogenie aeid), a weak orange zone of rutin 
(cf. Tl) and a blue fluorescent zone of eaffeie acid (Rf ca. 0.9). Rutin and vitexin-2"-
O-rhamnoside can be separated in solvent system F-2 (see Fig. 8). 
4 J7erhaseiflos 
There are three orange fluorescent flavone glycosides in the Rf region of the rutin 
test, and above and below the hyperoside test. Phenol carboxylic acids are absent, 
and an intensive yellow zone (flavonoid aglycones) is seen at the solvent front. 
The yellow-green zone at Rf ca. 0.6 is due to hesperidin. 
8 5,6 Crataegifolium (eumflore), Crataegifruetus 
178 
Using solvent system F-2 (a modified system, based on the addition of ethyl methyl 
ketone), vitexin and vitexin-2"-O-rhamnoside (cf. T2) are separated from hyperoside 
and rutin, respectively. Crataegi folium and flos contain weil detectable quantities 
of these two compounds, whereas little or none is present in Crataegi fruetus, which 
has a relatively low total flavonoid content. Chlorogenie acid and other phenol 
carboxylic acids are present in all parts of the drug. 
Fig.7 
2 3 
Fig.8 
5 6 
4 
3 
FRONT 
Rf 
FRONT 
Rf 
TART 
179 
Flower Drugs IV, with Their Most Common Adulterants 
Tracks 
Tests 
Solvent 
system 
1 = Pruni spinosae flos 
2 = Sambuci flos 
3 = Spiraeae flos 
4 = Robiniae (Acaciae) flos 
5 = Acaciae verticil. flos 
6-10= Tiliae flos (patterns from official commercial 
drugs I-V) 
T1 = rutin (Rf ca. 004), chlorogenic acid (Rf ca. 0.5), hyperoside 
(Rf ca. 0.6) 
F-l: ethyl acetate-formic acid-glacial acetic acid-water 
(100: 11 : 11: 27) 
Detection Natural products-polyethyleneglycol reagent 
(NP/PEG No. 28, p. 303) UV-365 nm Fig. 9, 10 
For description of drugs see p. 165-167. Formulae p. 170 
Chromato-
gram 1, 4 Pruni spinosae flos, A eaciae flos 
1 Pruni flos' chromatogram is similar to that of Tiliae flos (Fig. 10) m showing 
9 at least 8 strong, orange fluorescent zones in the Rf range 0.4-0.9: 
rutin 
kaempferol diglycoside 
isoquercitrin (Q-3-0-glucoside) 
kaempferol-3,7-dirhamnoside 
avicularin (Q-3-0-arabinoside) 
kaempferol-3-0-arabinoside 
Rf ca. 0.35 
ca. 0.4 
ca. 0.55 
ca. 0.6 
ca. 0.85 
ca. 0.9 
In addition, there are two powerful, blue fluorescent zones in the same Rf range as the chIoro-
genie acid test. 
4 Rohiniae (Aeaciae)flos 
Predominantly green-yellow fluorescent zones in the lower third of the chromato-
gram. The main compound is rohinin (kaempferol-3-0-rhamnosyl-galactosyl-7-
rhamnoside) at Rf ca. 0.2. Aeaeetin-7-0-rutinoside migrates directly above rutin. 
5 Extracts of flowers of Acacia verticil. show additional orange zones at Rf 0.6-0.75. 
2, 3 Samhuci flos, Spiraeae flos 
2 Samhuci flos is characterized by the presence of rutin, ehlorogenie acid (cf. T1) and 
isoquercitrin (Rf ca. 0.65) (cf. Fig. 7, p. 178). . 
3 Spiraeae flos is characterized chiefly by two main blue fluorescent zones in the 
Rf region of the hyperoside test. The upper zone overlaps the green fluorescent 
zone of spiraeoside (quercetin-4' -O-glucoside). 
10 6-10 Tiliae flos 
180 
The flavonoid pattern of Tiliae flos extracts consists of at least 8 glycosides of 
quercetin, myrieetin and kaempferol: 
Rf ca. 0.9 
ca. 0.8 
ca. 0.7 
tiliroside 
Q-3-0-rhamnoside 
{ Q-3-0-glucoside Q-3,7 -dirhamnoside 
ca. 0.4 rutin 
M-3-0-rhamnoside 
M-3-0-glucoside 
K-3-0-rhamnoside 
K-3-0-glucoside 
K-3,7-dirhamnoside 
Tiliae flos eommercial drugs show qualitative differences in flavonoid composition. 
The flavone zone in the Rf range of the chlorogenie acid test is occasionally absent. 
The adulterant, Tilia argentea, is recognizable by the presence of an additional 
zone below the standard rutin test, and by the absence of rutin itself. 
Fig.9 
2 3 
Fig.1O 
7 8 
4 5 
9 10 
FRONT 
Rf 
FRONT 
Rf 
181 
Betulae, J uglandis Folium Anthemidis Flos 
Tracks 
Tests 
1 = Betulae folium 
2 = Juglandis folium 
3 = Anthemidis flos 
Tl = rutin (Rf ca. OA), chlorogenie acid (Rf ca. 0.5), hyperoside (Rf ca. 0.6), 
quercitrin (Rf ca. 0.8), kaempferol arabinoside (Rf ca. 0.9) 
T2 = luteolin-7-0-glucoside 
T3 = apigenin-7-0-glucoside 
T4 = isochlorogenie acid (Rf ca. 0.75), chlorogenie acid, 
caffeic acid (Rf ca. 0.85) 
T5 = rutin (Rf ca. OA), chlorogenie acid (Rf ca. 0.5), hyperoside (Rf ca. 0.6) 
T6 = rutin, hyperoside 
T7 = rutin, hyperoside, caffeic acid 
Adsorbent Silica gel 60F 254 pre-coated plates (Merck, Darmstadt) 
Cellulose F 254 pre-coated plates (Merck, Darmstadt) 
Fig. HA, B 
Fig. 12A, B, C, D 
Solvent F -1: ethyl acetate-formic acid-glacial acetic 
system acid-water(100:11:11:27) Fig. HA, B 
F -7: n-butanol-glacial acetic acid-water 
(40: 10: 50), upper phase 
Detection Natural products-polyethyleneglycol 
reagent (NP/PEG No. 28, p. 303) 
Fig. 12A, B, C, D 
UV-365 nm Fig. HA, B; 12B, D 
VlS. Fig. 12A, C 
For description of drugs see p. 167-168. Formulae p. 170 
Chromato- Betulae folium, Juglandis folium 
gram Both drug extracts show a very similar pattern of flavonoids in the intermediate 
11 A and upper Rf range. 
1 Betulae folium shows hyperoside, quercitrin (cf. Tl), quereetin-3-0-arabinoside (Rf 
ca. 0.9), traces of rutin, and a blue fluorescent zone of chlorogenie acid (cf. Tl). 
There are also two further orange zones, one above and one below quercitrin. 
11 B 2 Juglandis folium shows, in addition, a yellow-green zone at Rf 0.95, and an orange 
zone above hyperoside. Rutin and chi orogenie acid are absent, but the neochloro-
genie acid is present. 
12A, B The TLC separation of Betulae folium extract on cellulose, as specified by DAB 8, 
shows main orange (vis.) zones in the Rf range 0.4-0.85. 
3 Anthemidis flos 
The chromatogram is characterized by the intense, light blue fluorescent zones of 
"isochlorogenic acid" and chlorogenie acid (cf. T4 and T5), the intense, yellow-green 
fluorescent zone of apigenin-7-0-glueoside (Rf ca. 0.7, cf. T3), the orange zone of 
luteolin-7-0-glueoside (cf. T2) and a strong, yellow fluorescent zone of flavonoid 
aglyeones at the solvent front (cf. also Fig. 13, p. 184). Apiin (apigenin apiosyl gluco-
side), a constituent reported in the literature, may be present in traces (Rf ca. OA5). 
12C, D The TLC separation on cellulose, as specified by DAB 8, shows two orange (vis.) 
zones and one yellowish (vis.) zone in the upper Rf range. In UV-365 nm, these 
appear as almost white or dark blue fluorescent zones, respectively. 
182 
A e 
-
~ 
• 
-
.. 
• -~ 
• 
-
... 
..... 
- , 
• 
..... 
Fig.11 
Tl 2 
A B c 
Fig.12 
T6 T6 
.... 
• 
• I 
: 
T2 3 
T7 3 
• 11 
-t' 
• • 
.. 
T3 T4 T5 
D 
T7 3 
-FRONT 
Rf 
5 
TARl 
-FRONT 
RI 
-0.5 
-START 
183 
Matricariae Flos "Herba Drugs" TLC Synopsis 
Tracks 
Test 
Solvent 
system 
1-3 = Matricariae flos (commercial pattern) 
4 = Anthemidis flos 
5 = Anserinae herba 
6 = Leonuri herba 
7 = Virgaureae herba 
8 = Sarothamni scopariae herba 
9= Veronicae herba 
10= Violae tricoloris herba 
T1 = rutin (Rf ca. 0.4), chlorogenie acid (Rf ca. 0.45), 
hyperoside (Rf ca. 0.55), caffeic/ferulic acid (Rf 0.9-0.95) 
Fig.13 
Fig.14 
T2 = rutin (Rf ca. 0.4), chi orogenie acid (Rf ca. 0.5), hyperoside (Rf ca. 0.6), 
"isochlorogenie acid" (Rf 0.75-0.85), caffeic acid (Rf ca. 0.9) 
F-1: ethylacetate-formic acid-glacial acetic acid-water 
(100: 11: 11:27) 
Detection Natural products-polyethyleneglycol reagent 
(NP/PEG No. 28, p. 303) UV-365 nm Fig. 13, 14 
For description of drugs see p. 166-168. Formulae p. 170 
Chromato-
gram 1-3 Matricariae (Chamomillae)flos 
13 Quercimeritrin, luteolin- and patuletin-7-0-glucoside (constituents reported in the Iit-
erature) migrate at Rf ca. 0.6 in the same region as for the hyperoside test. A 
weak, green fluorescent zone at Rf ca. 0.7 shows the same Rf as apigenin-7-0-
glucoside (cf. Anthemidis flos, Fig. 11, p. 183). A narrow, orange-yellow zone of 
flavonoid agIycones is found at the solvent front. 
Commercial drugs show variations in the concentrations of constituents in the 
Rf-range 0.4 and 0.6. 
The blue fluorescent zones are due to phenol carboxylic acids (chlorogenie, neo-
chlorogenie, isochlorogenie, caffeic and ferulic acid respectively). 
4 Anthemidis flos can be distinguished from Matricariae flos, because of the high amount of 
flavonoid aglycones (solvent front) and apigenin-7-0-glucoside (Rf ca. 0.7), and the presence 
of luteolin-7-0-glucoside (below apigenin-7-0-glucoside). Both extracts have a similar pattern 
of "acids", but Anthemidis flos also shows two very distinct blue fluorescent zones, one 
directly above chlorogenie acid and the other at Rf ca. 0.8 (cf. Essential Oils, Figs. 11 and 
12, p. 32). 
14 5-10 TLC synopsis 0/" Herba dl'ugs" (see p. 186, Figs. 15 and 16) 
184 
Extracts of Anserinae herba (5) and Virgaureae herba (7) show similar patterns of 
flavonoids, consisting of orange fluorescent quercetin monoglycosides (Rf 0.6-0.8) 
and diglycosides (Rf 0.35-0.45). 
Violae tricol. herba (10) is characterized by a high content of flavonoid di-, and 
triglycosides at Rf 0.1-0.5, and by the absence of flavonoids in the upper Rf range. 
Leonuri herba (6) and Veronicae herba (9) show similar patterns of "acids" at Rf 
0.1-0.9. 
Sal'othamni (Spartii) herba (8) is characterized by four yellow-green fluorescent zones 
at Rf 0.6-0.85. 
Fig.13 
Tl 2 
Fig. 14 
T2 5 6 7 
3 4 
8 9 10 
FRONT 
Rf 
START 
FRONT 
Rf 
START 
185 
Herba Drugs 
Tracks 
Tests 
Solvent 
system 
1 = Anserinae herba 
2 = Violae tricoloris herba 
3 = Sarothamni herba 
4 = Sophorae gemma 
Ti = rutin, chlorogenie acid, hyperoside 
5 = Leonuri herba 
6 = Veronicae herba 
7= Rutae herba 
8 = Equiseti herba 
T2 = rutin (Rf ca. 0.35), chI orogenie acid (Rf ca. 0.45), hyperoside (Rf ca. 0.6), 
isochlorogenie acid (Rf ca. 0.8), caffeic acid (Rf ca. 0.95) 
F-1: ethyl acetate-formic acid-glacial acetic acid-water 
(100: 11 : 11: 27) 
Detection Natural products-polyethyleneglycol reagent 
(NPjPEG No. 28, p. 303) UV-365 nm Fig. 15, 16 
For description of drugs see p. 168-169. Formulae p. 170 
Chromato- 1 Anserinae herba 
gram Extracts show 8 strong fluorescent orange zones, due to glycosides of quercetin 
15 and myricetin, in the Rf range 0.3-0.9. Quercetin-3-0-glucoside (Rf ca. 0.6) and 
myricetin- and quercetin-3-0-rhamnoside (Rf 0.65--0.7) mi grate above the hyperoside 
test (T1), while the corresponding diglycosides migrate in the same region as the 
rutin test (Ti). 
2, 4 Violae herba, Sophorae gemma 
Both drugs are characterized by a high content of rutin (cf. Ti), and by aseries 
of predominantly orange fluorescent flavonol di-, and triglycosides in the Rf range 
0.1-0.4. 
3 Sarothamni scopariae (Spartii) herba 
Chromatograms characteristically show a main yellow-green fluorescent zone of 
scoparin (Rf ca. 0.7) and a green-yellow fluorescent zone of vitexin (Rf range of 
the hyperoside test, cf. T1), together with three other weak, green fluorescent zones 
(Rf 0.7-0.9) and 4-5 weak, blue fluorescent zones (Rf 0.2-0.4). 
16 5,6 Leonuri herba, Veronicae herba 
186 
The chromatograms of these drug extracts show similar patterns of predominantly 
blue fluorescent zones. Rutin is only detectable as a weak zone (cf. T2), but the 
orange fluorescent zone of aglycones at the solvent front is more pronounced. 
The blue fluorescent zones are due partly to phenol carboxylic acids (cf. T2, chloro-
genie and isochlorogenic acid). 
7 Rutae herba 
The drug is characterized by the orange main zone of rutin, and the blue-violet 
fluorescent zones of coumarins (see also Coumarin drugs, Figs. 5 and 6, p. 156). 
8 Equiseti herba 
The orange fluorescent, main zone is due to isoquercitrin (Rf ca. 0.7). Galuteolin 
(luteolin-5-0-glucoside), ferulic acid and caffeic acid appear as blue fluorescent zones 
in the upper Rf range. About six more very weak blue or blue-green fluorescent 
zones are present in the lower and intermediate Rf range. 
Remarks: Red fluorescent zones at the solvent front are due to the chlorophyll fraction. 
Fig.15 
2 3 
Fig.16 
T2 5 6 7 
4 
8 
FRONT 
RI 
TART 
FRONT 
RI 
187 
Citri, Aurantii Pericarpium 
Eriodictyonis Herba, Orthosiphonis Folium 
Tracks 
Tests 
Solvent 
system 
Detection 
Chromato-
gram 1,2 
17A, B 
1 = Citri pericarpium 
2 = Aurantii pericarpium 
3 = Eriodictyonis herba 
4, 5 = Orthosiphonis folium 
Tl = rutin T3 = eriodictyol 
T2 = homoeriodictyol T 4 = sinensetin 
F-l: ethyl acetate-formic acid-glacial acetic acid-water 
(100: 11: 11 :27) 
F-3: chloroform-acetone-formic acid 
(75: 16.5: 8.5) 
F-4: chloroform-ethyl acetate (60:40) 
with and without chamber saturation 
Natural products-polyethyleneglycol reagent 
(NP/PEG No. 28, p. 303) 
Without chemical treatment 
For description of drugs see p. 169. Formulae p. 170 
Citri pericarpium, Aurantii pericarpium 
UV-365 nm 
VlS. 
UV-365 nm 
Fig.17A, B 
Fig.18A 
Fig. 18B, C 
Fig. 17 A; 18A 
Fig.17B 
Fig. 18B, C 
After treatment with NPjPEG reagent, observation in UV-365 nm reveals the red-
orange fluorescent zone of eriocitrin (Rf ca. 0.4) and the yellow zone of rutin (cf. 
Tl). 
The broad zone of naringin, neohesperidin and hesperidin (dark green fluorescence 
in UV-365 nm; ochre in vis.), which migrates directly above the eriocitrin zone, 
can be used to distinguish between these two drugs; Citri pericarp. contains only 
traces of neo hesperidin and hesperidin. The higher proportion of flavanones in 
Aurantii pericarp. is indicated by two powerful, ochre (vis.) zones directly above 
the violet zone of eriocitrin. 
Aurantii pericarp. also shows additional, blue fluorescent zones in the Rf range 
0.5-0.9; these are due to -methyl anthranilate, flavonoids (e.g. sinensetin) and couma-
rins. 
Remarks: The eriocitrin zone becomes intensely red after 15 min exposure to UV-365 nm. 
18A 3 Eriodictyonis herba (Yerba Santa) 
After NP/PEG treatment and observation in UV-365 nm, the chromatogram (run 
in solvent system F-3) characteristically shows yellow-green (homoeriodictyol (cf. 
T2) and chrysoeriodictyol) and yellow-orange (xanthoeriodictyol) fluorescent zones 
in the Rf range 0.55-0.75, and the zone of eriodictyol (cf. T3) at Rf ca. 0.4. 
18 B 4 Orthosiphonis folium 
U sing solvent system F -4, observation of the chromatogram in UV -365 nm without 
chemical treatment shows 3-4 light blue fluorescent zones of flavone aglycones: 
sinensetin (cf. T4) is the main zone directly below scutellarein methyl ether. Eupatorin 
and 3'-hydroxy-5,6,7,4'-tetramethoxyflavone are found at Rf 0.3-0.4. 
18 C 5 Without chamber solvent saturation, the characteristic flavones of Orthosiph. folium 
migrate at lower Rf values (Rf 0.1-0.4). 
The chlorophyll fraction of the fresh drug (5) fluoresces red in UV-365 nm. 
188 
Fig.17 
Tl 2 Tl 
Fig.18 
T2 3 13 T4 4 
2 
5 
FRONT 
Rf 
-0.5 
-START 
FRONT 
RI 
START 
189 
Cardui mariae (Silybi) Fructus 
Tracks 
Tests 
Solvent 
system 
1-4 = Cardui mariae fructus 
Tl = taxifolin (Rf ca. 0.4), silybin (Rf ca. 0.6) 
T2= silychristin 
F-3: chloroform-acetone-formic acid (75: 16.5: 8.5) 
Detection Natural products-polyethyleneglycol reagent 
Chromato-
gram 1-4 
19 
20 
190 
(NPjPEG No. 28, p. 303) 
Fast blue salt reagent (FBS No. 12, p. 301) 
For description of drugs see p. 169. Formulae p. 170-171 
Cardui mariae fructus 
UV-365 nm Fig.19 
VIS. Fig.20 
After treatment with NP/PEG reagent, chromatograms are characterized in UV-
365 nm by the two intense, light yellow fluorescent zones of silybinjisosilybin (Rf 
ca. 0.6, cf. Tl) and silychristin (Rf ca. 0.35, cf. T2), and by the yellow-orange 
fluorescent zone of taxifolin (Rf ca. 0.4, cf. Tl). A third, pale yellow fluorescent 
zone of silydianin, between taxifolin and silybin, is not always present in commercial 
drugs. 1 The fluorescent zones above silybin are due to dehydro-derivatives of silybin 
and isosilybin. 
The same main zones become red-brown (vis.) after treatment with FBS/KOH re-
agent. 
1 Remarks: There are chemical races of Silybum marianum, e.g. with high content of silydianin 
and relatively low content of silybin and silychristin. 
Fig.19 
Tl T2 2 3 
Fig.20 
2 3 
4 
4 
FRONT 
RI 
-FRONT 
RI 
-0.5 
-START 
191 
Farfarae Folium Petasites Folium 
Tracks 
Tests 
Solvent 
system 
1 = Farfarae folium (light petroleum extract, p. 163) 
2 = Petasites hybridi folium (light petroleum extract, p. 163) 
3 = Petasites albi folium (light petroleum extract, p. 163) 
4 = Petasites paradoxi folium (light petroleum extract, p. 163) 
5 = Farfarae folium c. flore (MeOH extract, p. 163) 
6 = Farfarae folium (MeOH extract, p. 163) 
7 = Petasites folium (MeOH extract, p. 163) 
Tl =isopetasin (Rfca. 0.47) 
T2=petasin 
T3 = rutin (Rf ca. 0.4), chlorogenic acid (Rf ca. 0.5), hyperoside (Rf ca. 0.6) 
T4 = chlorogenic acid, isochlorogenic acid (Rf ca. 0.8), caffeic acid (Rf ca. 0.9) 
F-6: chloroform p.A. Fig. 21, 22A 
F-l: ethyl acetate-formic acid-glacial acetic acid-water Fig.22B 
(100: 11: 11 :27) 
Detection Anisaldehyde-acetic acid reagent (AA No. 1, p. 299) UV-365 nm Fig.21A 
vis. Fig. 21 B 
UV-365 nm Fig.22A 
Chromato-
gram 
21A, B 
22A 
Sulphuric acid (H 2S04 conc. No. 34C, p. 303) 
Natural products-polyethyleneglycol re agent 
(NP/PEG No. 28, p. 303) UV-365 nm Fig.22B 
For description of drugs see p. 166. Formulae p. 170-171 
Farfarae folium, Petasites folium 
After treatment with AA reagent or H 2S04 and observation in UV-365 nm, petro-
leum extracts of the leaf drugs of Tussilago farfara (1) and Petasites hybridus, 
P. albus and P. paradoxus (2, 3, 4) show similar chromatographic patterns, except 
in the Rf range 0.35-0.45. 
Petasin and isopetasin (cf. T2 and Tl) cause a distinct quenching of fluorescence 
in UV-254 nm before chemical treatment. After treatment, they show green fluores-
cence in UV-365 nm, and a weak grey in the vis. Petasins are absent from Farfarae 
folium, but present in Petasites in species-dependent concentrations: 
2 Petasites hybridus: medium concentration of petasin and isopetasin. 
3 Petasites albus: practically no petasin; albopetasin * is present. 
4 Petasites paradoxus: rather high content of petasin and isopetasin. 
* Albopetasin forms a reddish fluorescent zone (Rf 0.75-0.8) in UV-365 nm after treatment 
with SbCl3 reagent (No. 3, p. 299). 
22B To a limited extent, Farfarae folium and Petasites spp. can also be distinguished 
by the differentJlavone and acid pattern of their methanol extracts. 
192 
5,6,7 Flavonoids: Rutin is clearly demonstrable in Farfarae folium only when flower heads 
are present in the drug (5). Petasites drugs show yellow to yellow-orange zones 
(e.g. isoquercitrin) in or above the Rf-region of standard hyperoside; these may 
also be present in lower concentration in Farfarae folium extracts. These zones 
become more distinct when larger quantities (ca. 40 J.lI) of extract are applied to 
the TLC; under these conditions, rutin also becomes detectable in both Farfarae 
folium and Petasites folium (cf. Fig. 5, p. 176). 
Phenol carboxylic acids: Farfarae folium and Petasites hybr. folium show practically 
identical patterns of "acids" (chlorogenic, isochlorogenic, caffeic, ete.). Extracts 
of Petasites paradoxus show an additional zone (Rf ca. 0.8) directly above that 
of chlorogenic acid; this zone may be absent from other Petasites spp. 
Fig.21 
Tl T2 2 3 4 
Fig.22 
T2 2 3 4 T3 
T2 3 4 
5 6 7 
-FRONT 
Rf 
-0.5 
START 
T4 
193 
Cardiac Glycoside Drugs 
These drugs contain steroid glycosides which specifically affect the dynamics and 
rhythm of the insufficient heart musc1e. 
The steroids are structurally derived from the tetracyclic 10,13-dimethylcyclopen-
tanoperhydrophenanthrene ring system. They posses a y-lactone ring (cardenolides) 
or a J-lactone ring (bufadienolides) attached in the ß-position at C-17. The sugar 
residues are typically derived from deoxy-, and/or C-3-0-methylated sugars, and 
they are linked glycosidically via the C-3-0H groups of the steroid aglycones. 
I. Preparation of Drug-extracts for TLC 
Powdered drug (1 g) is extracted by heating for 15 min under reflux with 20 ml 
of 50% ethanol, with the addition of 10 ml of 10% lead(II) acetate soln. After 
cooling and filtration, the c1ear soln. is treated with a small quantity of acetic acid, 
then extracted by shaking with three 15 ml quantities of dichloromethane; shaking 
must be gentle to avoid emulsion formation. 
The combined lower phases are filtered over anhydrous sodium sulphate and 
finally evaporated to dryness. The residue is dissolved in 1 ml dichloromethane/ 
ethanol (1: 1), and this soln. is used for chromatography. 
All cardiac glycoside drugs can be extracted by this method. Since Strophanthi 
semen has a high cardenolide content, a simplified extraction procedure can be 
applied to this drug. 
Strophanthi semen 
Finely ground seeds (2 g) are defatted by heating for 1 h under reflux with light 
petroleum. The defatted and dried seed powder (1 g) is extracted for 5 min with 
10 ml ethanol at ca. 60° C. The filtered soln. is used directly for chromatography. 
11. Thin Layer Chromatography 
1. Reference solutions 
a) Pure reference substances 
Convallatoxin: 30 mg are dissolved, in 10 ml of 80% ethanol on a water bath. 
Gitoxin: 10 mg are shaken with 0.1 ml pyridine, then brought completely into solu-
tion by adding 2 ml methanol at 60° C. 
Digoxin, lanatoside A, B, C, oleandrin, g- and k-strophanthin: separate solutions 
of each compound are made by dissolving 5 mg in 2 ml methanol at 60° C. 
b) Standard compounds from proprietary pharmaceuticals 
Digitalis glycosides 
Ten tablets or dragees (see Table I) are finely powdered in a mortar, than extracted 
by heafing in a flask at 60° C for 5 min with 5-15 ml (depending on the weight 
of powder) dichloromethane/ethanol (1: 1). The c1ear filtrate is evaporated to ca. 
2 ml, and 20 1-11 of tbis soln. is used for chromatography. 
195 
196 
Table 1 
Constituent 
digitoxin 
pen taacety 19i toxin 
acetyldigitoxin 
acetyldigoxin 
methyldigoxin 
lanatoside A, B, C 
digoxin 
lanatoside C 
Strophanthus glycosides 
Examples of appropriate 
pharmaceuticals ® 
(*) see remark p. 52) 
Digimerck 
Cordoval 
Carnacid 
Acylanid 
Novodigal 
Lanitop 
Digilanid 
Lanicor 
Cedilanid 
Allocor 
mg per tablet 
or dragee 
0.1 
0.4 
0.2 
0.2 
0.1 
0.25 
0.25 
0.25 
0.2 
Six tablets of Strophoral®, or 10 tablets of Purostrophan®, or 12 tablets of Stro-
phanon ® are finely powdered and extracted with 10 ml methanol for 5 min on 
the water bath. 20 fJ.l of each filtrate are used for chromatography. 
Scilla glycosides 
Twenty dragees of Talusin ®, Sandoscill ®, Scilloral ®, or Scillaren ® are finely pow-
dered and extracted with 10 ml methanol for 5 min at ca. 60° C. 20 fJ.l ofeach 
c1ear filtrate are used for chromatography. 
Uzara and Thevetia glycosides 
Five dragees of Uzara ® (total glycosides) or Encordin ® (peruvoside) are fine1y pow-
dered and extracted with 10 ml methanol for 5 min at 60° C. 20 fJ.l of each c1ear 
filtrate are used for chromatography. 
2. Adsorbent 
Si li ca gel 60F 254 pre-coated plates (Merck, Darmstadt) 
3. Sampie concentration 
Depending on the total cardenolide or bufadienolide concentration, 30-50 fJ.l of 
drug extracts are applied to the chromatogram. Solutions of test substances prepared 
from the individual compounds: 5 fJ.1. 
Reference solutions prepared from pharmaceuticals: 20 fJ.l 
4. Chromatography solvents 
H-1 Ethyl acetate-methanol-water (100: 13.5: 10) ~ (81: 11 :8). 
A generally applicable solvent system for cardiac glycosides. 
H-2 Ethyl acetate-methanol-ethanol-water (81: 11: 4: 8). The addition of ethanol in-
creases the Rf values of strongly polar compounds, e.g. k-strophanthoside. 
H-3 Methylethyl ketone-toluene-water-glacial acetic acid (40: 5: 3: 2.5: 1). Separa-
tions in this system are similar to those in H-1 and H-2. It is suitable for 
the separation of Scilla glycosides. 
H-4 Chloroform-methanol-water (65: 35: 10); lower phase. For chromatography of 
Hellebori radix extracts. 
III. Detection 
1. Without chemical treatment 
Fluorescence quenching by cardenolides is only very weak in UV-265 nm, but more 
distinct zones of fluorescence quenching are produced by bufadienolides. 
Cardiac glycosides do not fluoresce in UV-365 nm. 
2. Spray reagents 
a) Specific detection oJ the y-Iactone ring (cardenolides) 
Kedde reagent (No. 23, p. 302) 
Immediatelyon spraying, cardenolides form a pink or blue-violet (vis.) colour. Bufa-
dienolides do not react. 
The colour fades after a few minutes, but can be regained by repeated spraying 
even after several days. 
Remarks: 
LEGAL reagent (alkali ne sodium nitroprusside soln.) 
BALlET reagent (alkaline picric acid soln.) 
RAYMOND reagent (alkali ne m-dinitrobenzene soln.) 
They give red, red-orange or violet (vis.) colours with cardenolides. 
b) General detection methods Jor cardenolides and buJadienolides 
IX) Antimony (III) chloride reagent (SbCl3 No. 3, p. 299) 
The TLC plate is sprayed with at least 10 ml SbCl3 reagent, heated at 100° C for 
about 6 min, then observed immediately in UV-365 nm (see Table 11). Changes are 
observed in the fluorescence response, if the sprayed plate is allowed to stand for 
a longer time. In visible light, the zones appear mainly violet or brown. 
Table 2 
Cardiac glycoside 
K- and g-strophanthidine derivatives 
K-strophantoside, k-strophanthidin-ß } 
Cymarin, helveticoside, erysimoside, 
G-strophanthin, convallatoxin 
Digitalis glycosides 
Digitoxin, acetyldigitoxin } 
Purpurea glycoside A, lanatoside A 
Gitoxin, digoxin } 
Purpurea glycoside B, lanatoside BjC 
Oleander glycosides 
Bufadienoüdes 
Proscillaridin, scillaren A, glucoscillaren 
Scilliroside, glucoscilliroside 
Hellebrin, helleborogenone 
Fluorescence in UV -365 nm 
(after SbCI 3 and 100° C) 
orange, pale brown or 
yellow-green 
dark blue or dark brown 
light blue 
light blue 
yellow-brown 
pale green 
yellow 
ß) Chloramine-trichloroacetic acid reagent (CT AN o. 7, p. 300) 
Blue, yellow or yellow-green fluorescent zones are observed in UV -365 nm, similar 
to those obtained with SbCl3 reagent. Only weak, unspecific colours are seen in 
visible light. 
y) Sulphuric acid reagent (conc. H 2S04 No. 34, p. 303) 
The TLC plate is sprayed with ca. 5 ml of reagent, then heated for 3-5 min at 
100° C under observation. 
Blue, brown, green and yellowish fluorescent zones are seen in UV-365 nm; 
the same zones appear brown or blue in daylight. 
Remarks: Liebermann-Burchard reagent (LB No. 16, p. 301) can also be used for detection 
of cardiac glycosides. 
197 
198 
IV. List of Cardiac Glycoside Drugs 
Chromatograms (Figs. 5-20) are reproduced on pp. 208-223. 
Fig. Drug/Plant source 
F amily /Pharmacopoeia 
5,6, 7, 8 Digitalis lanatae Folium 
White foxglove leaves 
9, 10 
11,12 
15, 16 
Digitalis lanata ERHARD 
Scrophulariaceae 
USP XX, DAB 8, ÖAB 
DAB 8: Digitalis lanata powder 
standardized at ca. 0.5% digoxin 
activity 
Digitalis purpureae Folium 
Red foxglove leaves 
Digitalis purpurea L. 
Scrophulariaceae 
Ph. Eur. III, Helv. VI, ÖAB 
DAB 8: Digitalis purpurea powder 
standardized at ca. 1 % digitoxin 
activity 
Nerii (oleandri) Folium 
Oleander leaves 
Nerium oleander L. 
Apocynaceae 
DAC 
Adonidis Herba 
Adonis 
Adonis vernalis L. 
Ranunculaceae 
DAB8 
DAB 8: Adonis powder standard-
ized at ca. 0.2% cymarin activity 
Convallariae Herba 
Lily of the valley 
Convallaria majalis L. 
Liliaceae 
DAB8 
DAB 8: lily of the valley powder 
standardized at ca. 0.2% convalla-
toxin activity 
Main constituents 
Total cardenolide content ca. 1 %, comprising 
over 60 different compounds. 
Lanatosides A and C constitute ca. 50% of 
total cardenolides. Lanatosides B, D, E, di-
goxin and digitoxin are present only in lower 
concentrations. 
Total cardenolide content 0.15-0.4%. 
(Ph. Eur. III specifies not less than 0.3% re-
lated to digitoxin); ab out 30 glycosides are 
present. 
Purpurea-glycosides A and B constitute ca. 
60% of the mixt ure ; digitoxin ca. 12 %; gi-
toxin and gitaloxin 10% each. 
The major cardenolides of both species are 
derivatives of digitoxigenin, gitoxigenin and 
digoxigenin. 
Total cardenolide content 1-1.5% 
Odoroside A and H (derived from the agly-
cone, digitoxigenin) ; oleandrin, oleandrin 
monoglucoside (glucosyl oleandrin), gentio-
biosyl oleandrin and nerigoside (all derived 
from the aglycone, oleandrigenin). 
Oleasides A and E are glycosides of the olea-
genin series. Adynerin is derived from adyneri-
genin. 
Total cardenolide content ca. 0.25%, compris-
ing about 20 glycosides. 
Adonitoxin (adonitoxigenin-3-0-rhamnoside) 
(0.07%) is one of the main glycosides, accom-
panied by k-strophanthidin glycosides (e.g. 
cymarin, 0.02%) 
Flavonoid: adonivernith (a flavone C-glyco-
side) 
Total cardenolide content 0.2-0.3%, compris-
ing about 20 glycosides. 
The major glycosides, convallatoxin, convallo-
side and glucoconvalloside are derived from 
k-strophanthidin (=convallatoxigenin). Con-
vallatoxol, convallatoxoloside and glucocon-
vallatoxoloside are derived from k-strophan-
thidol. 
Convallatoxin is the main glycoside in drugs 
of western and northern European origin and 
represents 40-45% of the glycosides. In mid-
dIe European drugs, lokundjoside predomi-
nates. 
Fig. 
11,12 
13,14 
17 
18 
19,20 
DrugjPlant source 
F amily jPharmacopoeia 
Strophanthi grati Semen 
Strophanthus seeds 
Strophanthus gratus (WALL et 
HOOK.) FRANCHET 
Apocynaceae 
Strophanthi kombe Semen 
Strophanthus seeds 
Strophanthus kombe Oliver 
Apocynaceae 
Xysmalobii Radix 
U zarae radix 
Uzara root 
Xysmalobium undulatum 
R.BROWN 
Asc1epidiaceae 
Hellebori Radix 
Hellebore root 
Helleborus niger L. 
Helleborus odorus W ALDST. et KIT. 
Helleborus viridis L. and other 
Helleborus spp. 
Ranunculaceae 
Scillae Bulbus 
White squill (var. alba) 
Red squill (var. rubra) 
Urginea maritima L. 
var. alba BAKER 
Liliaceae 
DAB 8, Helv. VI 
DAB: squill powder standardized 
at ca. 0.5% proscillaridin activity 
Main constituents 
Total cardenolide content 4-7%, with 90-95% 
g-strophanthin, together with small quantities 
of sarmentosides A, D, E. 
5--10% cardenolides, derived from k-stro-
phanthidin. 
The glycoside mixture, called "k-strophan-
tin", consists of 80% k-strophanthoside and 
k-strophanthin-ß, and 10-15% erysimoside 
and cymarin. 
Minor glycosides are cymarol, helveticosol 
and periplocymarin. 
The main glycosides are the mono- and diglu-
cosides of the aglycones,xysmalogenin (5,6-
dehydrodigitoxin) and uzarigenin. The diglu-
cosides, uzarin and xysmalorin, are the main 
compounds. Uzarigenin differs from digitoxin 
in possessing a trans linkage of rings A and 
B. 
The bufadienolide content and glycoside pat-
tern depend on the origin of the drug. 
The main glycoside in H. viridis and H. odor-
us is hellebrin (ca. 0.5%), which is a gluco-
rhamnoside of hellebrigenin. 
Other Helleborus spp. are very often free of 
hellebrin. 
White variety: 0.2-0.4% bufadienolides, com-
prising about 15 glycosides derived from scil-
larenin. 
Main glycosides are proscillaridin 
(0.005--0.05%), scillaren A (ca. 0.06%) and 
glucoscillarin (ca. 0.005%). Scilliglaucoside, 
scillaphaeoside and scillacyanoside are minor 
constituents. 
Red variety: 0.04-0.1 % bufadienolides. 
Main glycosides are scilliroside and glucoscil-
liroside, which are derived from scillirosidin. 
As in the white variety, proscillaridin and scil-
laren Aare present but in low concentration. 
199 
200 
v. Formulae of Constituents of Cardiac Glycoside Drugs 
HO H 
Cardenolide: Digitoxigenin 
DIGITALIS GL YCOSIDES 
Purpureaglycoside A Digitoxigenin 
Lanatoside A Digitoxigenin 
Digitoxin Digitoxigenin 
ex/ß-Acetyldigitoxin Digitoxigenin 
Purpureaglycoside B Gitoxigenin 
Lanatoside B Gitoxigenin 
Gitoxin Gitoxigenin 
ex/ß-Acetylgitoxin Gitoxigenin 
Lantoside C 
ex/ß-Acetyldigoxi n 
Digoxin 
Dox = Digitoxose 
Digoxigenin 
Digoxigenin 
Digoxigenin 
Dox-Ac = Acetyldigitoxose 
Purpureaglycoside A: R, = R2 = H 
Purpureaglycoside B: R, = OH; R2 = H 
o 
HO H 
Bufadienolide: Bufalin 
-Dox-Dox-Dox-G I ucose 
-Dox-Dox-(Dox-Ac)-Glucose 
-Dox-Dox-Dox 
-Dox-Dox-(Dox-Ac) 
-Dox-Dox-Dox-Glucose 
-Dox-Dox-(Dox-Ac)-Glucose 
-Dox-Dox-Dox 
-Dox-Dox-(Dox-Ac) 
-Dox-Dox-(Dox-Ac )-G I ucose 
-Dox-Dox-(Dox-Ac) 
-Dox-Dox-Dox 
Lanatoside A: R, = H; R2 = Acetyl 
Lanatoside B: R, = OH; R2 = Acetyl 
ILanatoside AI IPurpureaglycoside AI I Purpureaglycoside 8 I ILanatoside 81 
t t t t 
I Acetyl-Digitoxin 1--1 Digitoxin I IGitoxinl- I Acetyl-Gitoxinl 
~ t 
I Digitoxigenin I 
.-----'----.t ~ 
IGitoxigenin I 
o 
HO HO 
I Lanatoside cl IGlucogitaloxinl 
t 
IAcetyl-Digoxin I I Gitaloxi nl 
t 
IDi90xigenini 
t 
I Gitaloxigeninl 
o 
HO HO 
201 
Oleandrosyl-O 
H 
Oleandrin 
o 
HO 
Rha-O OH 
g-Strophanthin 
Rha-O 
Adonitoxin 
202 
o o 
OCOCH3 
o H I 
Thevetosyl 
Peruvoside 
o 
p 
K-Strophanthidin: R = -C~ 
H 
Strophanthidol: R = -CH20H 
OH Periplogenin: R = - CH3 
R, R2 
Convalloside CHO H 
Convallatoxin CHO H 
Convallatoxol CH,oH H 
Convallatoxoloside CH,oH H 
Lokundjoside CH3 OH 
R3 
Rhamnosyl-Glucosyl 
Rhamnosyl 
Rhamnosyl 
Rhamnosyl-Glucosyl 
Rhamnosyl 
Gluc- Gluc-Rha-O 
1Gluc -1Gluc I Glucoscillaren AI ---- Scillaren A -
o 
Gluc-O 
-- Proscillaridin A ~ Scillarenin GI "11""d -1GIUC ls "li" " I 1Gluc UCOSCI Irosl e --- CI Iroslde ---- SClllirosidin 
Gluc-Rha-O 
OH 
Hellebrin 
o 
RO RO 
Uzarigenin: R = H Xysmalogenin: R = H 
Uzarin: R = Gluc - Gluc Xysmalorin: R = Gluc - Gluc 
203 
TLC Synopsis of Cardiac Glycosides 11 
Tracks 1 = g-strophanthin 
2 =" k-strophanthin" 
3 = convallatoxin 
4=cymarin 
5 = lanatoside A 
6 = lanatoside B 
7 = lanatoside C 
8=digoxin 
9= gitoxin 
10=digitoxin 
11 =cymarol 
12 = peruvoside 
13 = oleandrin 
Solvent 
system 
H-1: ethyl acetate-methanol-water (81: 11 : 8) 
Detection Kedde reagent (No. 23, p. 302) 
Chloramine-trichloroacetic acid reagent 
(CTA No. 7, p. 300) 
vis. Fig. 1 
UV-365 nm Fig.2 
Chromato- Kedde reagent (vis.) 
gram Immediately after spraying, cardiac glycosides 1-13 form blue to red-violet colours. 
1 With the exception of peruvoside, these colours are fairly stable. 
2 
204 
Digitalis glycosides 
Digoxin and lanatoside C, red-violet. 
Gitoxin and Ianatoside B, blue-violet. 
Digitoxin and lanatoside A, blue. 
The colour range is indicative of the structural type. 
CTA reagent (UV-365 nm) 
All cardiac gIycosides give light blue, blue-green or yellow-green fluorescent zones. 
Blue-green fluorescence is given by the Strophanthus, Convallaria and Thevetia glyco-
sides: g- and k-strophanthin (for TLC analysis ofthis gIycoside mixture, see Fig. 14, 
p. 216), cymarin, cymaroI, convallatoxin and peruvoside. 
Intense, light blue fluorescence is given by the Digitalis and Oleander glycosides, 
with the exception of digitoxin, which fluoresces yellow-green. 
After CT A treatment, chromatograms of some standard substances show additional zones 
in UV-365 nm, due to degradation products and impurities. 
Fig.l 
2 3 4 5 6 7 8 9 10 11 12 13 
Fig.2 
2 3 4 567 8 9 10 111213 
FRONT 
Rf 
-0.5 
205 
TLC Synopsis of Cardiac Glycosides U 1 
Tracks 
Solvent 
system 
1 = g-strophanthin 
2 =" k -strophanthin" 
3 = convallatoxin (Rf ca. 0.30) 
4=cymarin 
5 = lanatoside A 
6 = lanatoside B 
7 = lanatoside C 
8=digoxin 
9=gitoxin 
lO=digitoxin 
l1=cymarol 
12 = peruvoside 
13 = oleandrin (Rf ca. 0.9) 
14 = hellebrin (Rf ca. 0.15) 
15 = proscillaridin 
H -1: ethyl acetate-methanol-water (81 : 11 : 8) 
Detection Sulphuric acid reagent (conc. H 2S04 No. 34, p. 303) vis. Fig.3 
UV-365 nm Fig.4 
Chromato- 1 g-Strophanthin is immediately recognizable in UV-365 nm as a distinct, yellow-brown 
gram fluorescent zone. In visible light a weak brown appears if the TLC plate is exposed 
3, 4 for 15 min to the air. 
206 
2,4 Strophanthus glycosides, and convallatoxin (3) show only weak, brown in vis., but 
intense, blue or yellow-green fluorescent zones in UV-365 nm. 
5-10 The Digitalis glycosides give violet or brown in vis., and light to dark blue fluores-
cence in UV-365 nm (see also Fig. 2). 
12 Peruvoside gives a fading brown in vis. and blue fluorescence in UV-365 nm. 
13 Oleandrin shows brown in vis., and a radiant blue fluorescence in UV-365 nm. 
14 Hellehrin gives brown in vis., and a green-brown fluorescence in UV-365 nm. 
15 Proscillaridin shows violet in vis., and a yellow fluorescence in UV-365 nm. 
Remarks: Treatment with chloramine-trichloroacetic acid reagent (eTA No. 7, p. 300) pro-
duces similar fluorescent zones in UV-365 nm, but only weak colours in visible light. 
After treatment with conc. H 2S04 , additional zones appear in UV-365 nm, due to impurities 
and degradation products. 
Fig.3 
Fig.4 
n 
, .... 
.. . -
2345678 91011 12131415 
2 3 4 56 7 8 9 10 11 12 13 1415 
-FRONT 
Rf 
-0.5 
-START 
FRONT 
Rf 
START 
207 
Digitalis Folium 
Tracks 
Tests 
Solvent 
system 
Detection 
1-3 = Digitalis lanatae folium 
(various commercial patterns) 
Tl =lanatoside A T4=digoxin 
T2 = lanatoside B T5 = gitoxin 
T3 = lanatoside C T6 = digitoxin 
4-6 = Digitalis purpureae folium 
(various commercial patterns) 
T = mixture of gitoxin 
and digitoxin 
H-l: ethyl acetate-methanol-water (81: 11: 8) 
Chloramine-trichloroacetic acid reagent 
(CTA No. 7, p. 300) 
Kedde reagent (No. 23, p. 302) 
Antimony(III) chloride reagent (SbC13 No. 3, p. 299) 
UV-365 nm Fig.5A 
vis. Fig. 5B, 6A, C 
vis. Fig.6B 
For description of drugs see p. 198. Formulae p. 200-201 
Chromato- 1 Digitalis lanatae folium 
gram After CT A treatment, ab out 9 predominantly blue and blue-green fluorescent zones 
5A (UV-365 nm) are seen in the Rf range 0.25-0.75. Weak, blue fluorescent zones of 
gitoxin (T5) and digitoxin (T6), and stronger, blue or blue-green zones of lanatosides 
A, B, C (cf. Tl-T3) are present. Lanatoside A forms a major zone, while lanatosides B 
and C show lower concentrations. 
In addition, a green fluorescent zone is present at the origin, and two fluorescent 
zones, one green and one orange, are seen at the solvent front. 
5B1 With Kedde reagent, these cardenolides form blue or red-violet (vis.) zones. Lanatosi-
de A is again a major zone. 
The orange zones in the region ofthe solvent front are flavones or anthraquinones 
(e.g. digitolutein); to some extent, they overlap the genins (red-violet zone). 
5 A 4 Digitalis purpureae folium 
After CT A treatment the chromatogram is similar to that of Digitalis lanatae folium 
in the Rf range 0.45 to 0.75, except that gitoxin and digitoxin (T4 and T5) are 
more prominent. Purpurea-glycosides A and Bare present at Rf 0.2-0.25, the first 
migrating as a main zone at about the same Rf as the lanatoside C test (T3). 
5B 4 With Kedde reagent, Purpurea-glycoside A appears as a distinct, blue-violet (vis.) 
6A,C 
6B 
208 
main zone. 
Digitalis extracts 2 and 5 originate from commercial drugs with relatively high con-
tents of primary glycosides. Digitalis extracts 3 and 6 are from drugs that have 
been stored for a rather long time, so that the primary glycosides have become 
largely degraded (see Figs. 7 and 8, and accompanying explanations). 
The primary glycosides, lanatosides A, B, C (cf. Tl, T2, T3) mi grate at Rf 0.2-0.25, 
while the secondary glycosides, gitoxin, digoxin and digitoxin are found at Rf 
0.5-0.7. 
After treatment with SbC13 reagent, they form grey-blue (vis.) zones (cf. Figs. 7 
and 8, p. 210). 
Fig.5 
Tl-2-3 4 T T6 Tl-2-3 4 
A 
Fig.6 
T1·2-3 2 T5 3 Tl-2-3 T4 T5 T6 5 
T5 T6 
T6 6 
-FRONT 
Rf 
-0.5 
START 
-FRONT 
Rf 
-0 .5 
-START 
209 
Digitalis lanatae Folium Digitalis purpureae Folium 
TLC comparison 
Tracks 1 = Digitalis lanatae folium (commercial drug, DAB 8 quality) 
2= Digitalis lanatae folium (commercial drug, stored, fermented) 
3= Digitalis purpureae folium (commercial drug, DAB 8 quality) 
4 = Digitalis purpureae folium (commercial drug, stored, fermen ted) 
Tests Tl-3 = lanatosides A, B, C 
T4=digoxin 
T5=gitoxin 
Solvent H-l: ethyl acetate-methanol-water (81: 11: 8) 
system 
Detection Antimony (III) chloride reagent (SbCI 3 No. 3, p. 299) UV-365 nm Fig.7 
vis. Fig.8 
For description of drugs see p. 198. Formulae pp. 200-201 
Chromato- As seen in Figs. 5 and 6, commercial drugs show very variable contents of primary 
and secondary glycosides. This is due to different rates of c1eavage of primary into 
secondary glycosides (e.g. Purpurea-glycoside A into digitoxin). 
gram 
7,8 
210 
During storage of the drugs, plant enzymes (digipurpidase and digilanidase) preferentially 
remove the terminal glucose residues of Purpurea-glycosides. The lanatosides are more stable, 
due to the presence of the acetyl group. 
TLC comparison of 
1, 2 Digitalis lanatae folium and 
3,4 Digitalis purpureae folium 
After SbCl3 treatment and inspection in UV-365 nm or vis., the differences between 
standardized official drugs (1 and 3) and stored and/or fermented drugs (2 and 
4) are very apparent. In Digitalis purpurea, the primary glycosides are less stable 
than in Digitalis lanata, as evidenced by the strong zones of digitoxin and gitoxin 
(cf. T5). 
After 2 hours maceration of Digitalis purpureae folium powder in water, the 
primary glycosides are c1eaved into the secondary glycosides. If this mixture is ex-
tracted with DCM (see p. 195), a glycoside mixture is obtained, which gives the 
TLC pattern shown on track 4. 
Fig.7 
Tl-2 - 3 2 3 
Fig.8 
Tl - 2- 3 2 3 
4 T4 T5 
4 T4 T5 
-FRONT 
Rf 
-0.5 
-START 
211 
Nerii (oleandri) Folium 
Tracks 
Tests 
Solvent 
system 
1 = Nerii oleandri folium 
Tl = oleaside E 
T2 = gentiobiosyloleandrin (partially purified reference substance)1 
T3 = glucosylnerigoside (partially purified reference substance)1 
T4 = glucosyloleandrin (Rf ca. 0.4)1 
T5 = odoroside H 
T6 = odoroside A 
T7 = oleaside A 
T8 = nerigoside (Rf ca. 0.7) and adynerin (Rf ca. 0.8) 
T9 = oleandrin 
Tl 0 = mixture of Oleander glycosides (from Roth Co.) 
Tll = adynerin 
H-l: ethyl acetate-methanol-water (81 : 11: 8) 
Detection Sulphuric acid reagent (conc. H ZS04 No. 34, p. 303) vis. Fig.9 
VlS. Fig. IOA 
Chromato- 1 
gram 
9,10 
9 
212 
Kedde reagent (No. 23, p. 302) 
Chloramine-trichloroacetic acid reagent 
(CTA No. 7, p. 300) 
For description of drug see p. 198. Formulae p. 202 
UV-365 nm Fig. lOB 
Nerii/olium extracts produce 13-15 zones ofcardenolides in the Rfrange 0.1-0.95. 
Oleander extracts normally show a high cardenolide concentration. A large 
number ofindividual compounds have been found which occur in varying concentra-
tions, depending on the origin ofthe drug. Oleandrin and adynerin are major constitu-
ents. Freshly harvested drug also contains high concentrations of primary glycosides 
(e.g. glucosyloleandrin). 
Kedde reagent produces very stable, intense, blue to red-violet (vis.) zones. With 
er Areagent, the same compounds form mostly light blue, with some yellow-green, 
fluorescent zones in UV-365 nm; and with sulphuric acid reagent, they form zones 
with colour gradations of red or brown-green (vis.) (see list below, and chromato-
gram in Fig. 9). 
Treatment with sulphuric acid reagent pro duces the following coloured (vis.) zones: 
Glycosides %leandrigenin (red): 
oleandrin Rf ca. 0.9 
nerigoside Rf ca. 0.7 
glucosyloleandrin Rf ca. 0.1 
glucosylnerigoside Rf ca. 0.4 
Glycosides 0/ digoxigenin (brown): 
odoroside A Rf ca. 0.8 
odoroside H Rf ca. 0.55 
(T9)} . (T8) monoglycosldes 
(T2) } d' I 'd (T3) 19 YCOSI es 
(T6) 
(T5) 
Glycosides %leagenin (green-brown): 
oleaside E Rf ca. 0.1 (Tl) 
oleaside A Rf ca. 0.75 (T7) 
Glycosides 0/ adynerigenin (red-violet): 
adynerin Rfca.0.8 (Tl 1) 
1 Some of the test compounds were isolated from the drug and only partially purified. 
Fig.9 
Tl T2 T3 T4 T5 T6 T7 T8 
A 
Fig. 10 
T9 T 10 T 11 T 10 T9 
-FRONT 
Rf 
-0.5 
-START 
FRONT 
Rf 
START 
213 
Strophanthi Semen 
Adonidis and Convallariae Herba TLC Comparison 
Tracks 
Tests 
Solvent 
system 
1 = Strophanthi grati semen 
2 = Strophanthi kombe semen 
3 = Adonidis herba 
4 = Convallariae herba 
Tl = g-strophanthin 
T2 = "k -strophanthin" 
T3 = convallatoxin 
H -1: ethyl acetate-methanol-water (81 : 11 : 8) 
Detection Sulphuric acid reagent (cone. H 2S04 No. 34, p. 303) VIS. Fig.11 
UV-365 nm Fig.12 
Chromato-
gram 
11, 12 
214 
For description of drugs see p. 198-199. Formulae p. 202 
The fOUf drug extracts can be quickly differentiated by chromatography in solvent 
system H-1, treatment with sulphuric acid reagent, and inspection in UV-365 nm 
or vis: 
1 Strophanthi grati semen is characterized by g-strophanthin (cf. Ti) at Rf ca. 0.05 
(pale fluorescent zone in UV-365 nm; brown zone in vis.). 
2 Strophanthi kombe semen shows the three characteristic, blue-green fluorescent (UV-
365 nm) zones of" k-strophantin" mixture (cf. T2) in the lower Rf range. The glyco-
sides, cymarin and helveticoside, migrate ahead of "k-strophantin" at intermediate 
Rfvalues (see also Figs. 13 and 14, p. 216). 
These major glycosides form brown zones in vis. 
3 Adonidis herba is characterized in UV-365 nm by at least 10 predominantly blue 
fluorescent zones between Rf 0.3 and the solvent front. The same zones appear 
blue, red-violet and brown in vis. 
4 Convallariae herba shows green-blue fluorescent zones in the Rf range 0.2-0.35; 
these inc1ude the major zone of convallatoxin (cf. T3; Rf ca. 0.3), which also appears 
brown in vis. 
Apreeise chromatographie identijication and characterization of the individual drugs 
is given in Figs. 13 and 14, p. 216 (Strophanthi semen), and in Figs. 15 and 16, 
p. 218 (Adonidis and Convallariae herba); 
, r ·n,..."0.., T 
0.5 
-START 
Fig.11 
Tl 2 
•• 
e ... .. .... 
-
.-
... _ .... 
-
,.. .. 
I • 
-
--~ 
.... 
• 
.....- .. 
Fig.12 
215 
Strophanthi Semen 
Tracks 
Tests 
Solvent 
system 
1 = Strophanthi grati semen 
2 = Strophanthi kombi: semen 
Tl = g-strophanthin 
T2 =" k-strophanthin" 
T3 = k-strophanthoside 
T4 = k-strophanthin-ß 
T5 = erysimoside 
T6 = helveticoside 
T7=cymarin 
H-l: ethyl acetate-methanol-water (81 : 11 : 8) 
Detection Kedde reagent (No. 23, p. 302) VIS. Fig. 13A 
Chloramine-trichloroacetic acid reagent 
(CTA No. 7, p. 300) 
Sulphuric acid reagent (cone. H 2S04 No. 34, p. 303) 
SbCI 3 re agent (No. 3, p. 299) 
For description of drugs see p. 199. Formulae p. 202 
UV-365 nm Fig.13B 
vis. Fig. 14A 
UV-365 nm Fig.14B 
Chromato- 1 Strophanthi grati semen 
gram Kedde reagent produces a powerful violet (vis.) zone at Rf ca. 0.1, due to g-strophan-
13A thin (cf. Tl). Ahead of g-strophanthin, up to Rf ca. 0.45, are 4-5 weaker zones, 
due partly to sarmentosides. 
13B After CTA treatment, the only distinct blue-green fluorescent zone in UV-365 nm 
is that of g-strophanthin. A blue fluorescent zone and a broad, greenish fluorescent 
zone (lipids) are present below the solvent front. 
14B SbCl3 treatment pro duces two powerfully yellow fluorescent (UV-365 nm) zones, 
identical with those from standard g-strophanthin (Tl), and ab out 5 other yellow 
zones up to Rf ca. 0.4 (cf. Fig. 13A). 
13 A 2 Strophanthi komM semen 
Treatment with Kedde reagent reveals k-strophanthoside direct1y above the start, 
and the closely juxtaposed zones of k-strophanthin-p and erysimoside at Rf ca. 0.2. 
The intermediate Rf range contains four weaker zones, the upper two being 
helveticoside and cymarin (cf. Fig. 14B). 
14A Treatment with conc. H 2S04 reveals a similar pattern (vis.) to that obtained with 
Kedde reagent. 
13 B After treatment with CT Areagent, the glycosides are revealed as light green or 
yellowish fluorescent zones in UV-365 nm. The zones ne ar the solvent front are 
due to the lipid fraction of the seeds. 
14B With SbCl3 reagent, cardiac glycosides are revealed as intense, yellow fluorescent 
zones in UV-365 nm (cf. standards T3-T7). 
216 
Remarks: The presence of Strophanthus sarmentosus is recognizable by the detection of very 
high concentrations of sarmentosides at Rf 0.25--0.4, sarmentoside A at Rf ca. 0.4, and of 
sarmentocymarin and saveroside at RfO.7-0.9. 
Fig.13 
Tl 2 T2 
A 
Fig.14 
2 Tl 2 T 3 T4 T5 
Tl 2 T2 
Tb T7 12 
FRONT 
Rf 
0 .5 
FRONT 
Rf 
START 
217 
Adonidis Herba Convallariae Herba 
Tracks l=Adonidis herba (DCM extract) Fig.15A, B; 16A, B 
(MeOH extract; Flavonoids, p. 163) Fig.16C 
2=Convallariae herba (DCM extract) Fig. 15C; 16A, B 
(MeOH extract; Flavonoids, p. 163) Fig. 15D 
Tests TS =" k-strophanthin" T3 = hyperoside 
Tl =cymarin T4 = rutin 
T2 = convallatoxin (Rf ca. 0.35) T5 = adonivernith 
Solvent H-l: ethyl acetate-methanol-water (81: 11: 8) Fig. 15A-C; 16A, B 
system F -1: ethyl acetate-formic acid-glacial acetic acid-water 
(100: 11: 11: 27) Fig. 15D; 16C 
Detection Kedde reagent (No. 23, p. 302) vis. Fig. 15A, C 
Chloramine-trichloroacetic acid reagent 
(CTA No. 7, p. 300) vis. Fig.15B 
SbCl3 reagent (No. 3, p. 299) vis. Fig. 16A 
UV-365 nm Fig. 16B 
Natural products-polyethyleneglycol reagent 
(NP/PEG No. 28, p. 303) UV-365 nm Fig. 15D; 16C 
For description of drugs see p. 198. Formulae p. 202 
Chromato- 1 Adonidis herba 
gram Kedde reagent gives 4-5 weak, violet zones in the Rf range 0.25-0.6, which corre-
15A spond partly with the glycoside mixture "k-strophanthin" (cf. TS). The upper zone 
is cymarin (cf. Tl). Adonitoxin is seen at a similar Rf to that of the convallatoxin 
test (cf. T2). 
16B 
16A, B 
15C 
CTA reagent produces blue or blue-green fluorescent zones (in UV-365 nm) between 
Rf 0.2 and the solvent front. Cymarin (cf. Tl) and adonitoxin (Rf ca. 0.3) fluoresce 
green-blue. 
SbCl3 reagent gives blue (vis.) zones at Rf 0.25-0.35 and at Rf 0.75-0.85 (16A), 
which are characteristic of the drug extract, but are not due to cardiac glycosides. 
Inspection in UV-365 nm (16B) shows conspicuous light blue fluorescent zones, 
mainly in the intermediate Rf range. 
2 Convallariae herba 
Kedde reagent gives a distinct violet (vis.) main zone of convallatoxin. In addition, 
there are two weak, violet zones (also present in the standard convallatoxin test; 
cf. T2): one above (Rf ca. 0.65) and the other be10w the main zone. 
16A, B After treatment with SbCl3 reagent, only weak, uncharacteristic zones are seen in 
vis. (16A), but inspection in UV-365 nm (16B) reveals numerous, predominantly 
yellow-brown and light blue fluorescent zones. Convalloside, convallatoxoloside and 
glucoconvallatoxoloside (in order of decreasing mobility) mi grate below convalla-
toxin (Rf ca. 0.3; T2). Deglucocheirotoxin migrates directly ahead of convallatoxin. 
15 D, 16 C Flavonoids 
218 
1 Adonidis herba is characterized by the yellow fluorescent (UV-365 nm) flavone-C-
glycoside, adonivernith (cf. T5), together with three other intensely yellow fluorescent 
flavonoid zones at Rf 0.2-0.6. 
2 Convallariae herba. Using the same sampie size, this drug shows only weak green 
or orange fluorescent zones (in UV-365 nm) of kaempferol and quercetin triglyco-
sides at Rf 0.2-0.25. 
Fig.15 
TS Tl Tl 2 
Fig.16 
2 2 
T2 T3 2 
T2 
T4 
T5 
FRONT 
Rf 
219 
U zarae (Xysmalobii) Radix 
Tracks 
Tests 
Solvent 
system 
1 = Uzarone extract 
Tl = xysmalorin 
T2=uzarin 
T3 = uzarigenin 
T4 = 3-0-acetyl-xysmalogenin 
T5 = lanatoside B (with traces of lanatosides A and C) 
H-l: ethyl acetate-methanol-water (81 : 11: 8) 
Detection Chloramine-trichloroacetic acid reagent 
Chromato-
gram 
17A, B 
(CTA No. 7, p. 300) 
SbCl3 reagent (No. 3, p. 299) 
For description of drug see p. 199. Formulae p. 203 
UV-365 nm Fig. 17 A 
VIS. Fig. 17B 
One dragee of the proprietary pharmaceutical Uzara® contains 50 mg of a dried 
extract of Xysmalobium undulatum with ab out 30% total glycosides ("Uzarone 
extract "). 
After treatment with CT Areagent, the chromatogram shows at least 7 light yellow 
to blue-green fluorescent zones in UV-365 nm. The major zone at Rf ca. 0.1 contains 
the diglucosides, uzarin (T2) and xysmalorin (Tl), while the prominent zone at Rf 
ca. 0.3 is due to the corresponding monoglucosides, uzarigenin monoglucoside and 
xysmalogenin monoglucoside. The latter migrate at about the same Rf value as for 
the lanatoside test (cf. T5), which are not present in this drug. The upper part 
of the chromatogram shows uzarigenin and 3-0-acetylxysmalogenin (cf. T3 and T4). 
Treatment with SbC/3 re agent reveals mainly the blue-violet (vis.) zone of uzarin 
and xysmalorin. 
Hellebori Radix 
Tracks 
Tests 
Solvent 
system 
Detection 
Chromato-
gram 
18A, B 
18C 
220 
2 = Hellebori radix (H. viridis) 
3 = Hellebori radix (commercial drug of unknown botanical source) 
T6 = hellebrin 
T7 = g-strophanthin 
H-l: ethyl acetate-methanol-water (81: 11 : 8) 
H-4: chloroform-methanol-water (64:35:10) (lower phase) 
SbCl3 reagent (No. 3), p. 299) UV-365 nm 
VIS. 
Anisaldehyde-sulphuric acid reagent (AS No. 2, p. 299) VIS. 
For description of drug see p. 199. Formulae p. 203 
Fig. 18A, B 
Fig.18C 
Fig.18A 
Fig.18B 
Fig.18C 
Chromatography in solvent system H-l, followed by treatment with SbC/3 reagent 
and inspection in UV -365 nm, reveals at least 13 light yellow fluorescent zones be-
tween the start and the solvent front (18A). The zone of hellebrin (Rf ca. 0.1; 
cf. T6) fluoresces intense light yellow; with AS reagent it produces a dark blue 
(vis.) colour (18B). 
In solvent system H-4, hellebdn migrates at Rf ca. 0.35. g-Strophanthin, which 
is chromatographed alongside for comparison, migrates somewhat higher. 
Remarks: Hellebrin, the main glycoside of Helleborus viridisand H. odorus may be absent 
from drugs derived from other Helleborus species. 
Fig.17 
Fig.18 
Tl T2 
a 
• I 
.. 
~ 
..... 
- I· .. ~ 
T6 2 T6 
T3 T4 T5 
c 
2 3 T7 
r- FRONT 
Rf 
START 
FRONT 
Rf 
0 .5 
START 
221 
Scillae Bulbus 
Tracks 
Tests 
Solvent 
system 
1 = Scillae bulb. var. rubra 
2 = Scillae bulb. var. rubra (standarized extract) 
3 = Scillae bulb. var. alba (commercial pattern) 
4 = Scillae bulb. var. alba (commercial pattern) 
5 = Scillae bulb. var. alba (standardized extract) 
T = proscillaridin 
H-1: ethyl acetate-methanol-water (81 : 11 : 8) 
Detection SbCI3 reagent (No. 3, p. 299) vis. Fig. 19 
UV-365 nm Fig.20 
Chromato-
gram 1-5 
19,20 
1-2 
3,4,5 
222 
For description of drugs see p. 199. Formulae p. 203 
After SbCl3 treatment, chromatograms of Scilla extracts produce predominantly 
blue (vis.) zones, or intense, light yellow, yellow-brown or light blue fluorescent 
zones in UV-365 nm. The standardized extracts (2, 5) contain higher concentrations 
of primary glycosides. 
Scillae bulbus var. rubra 
The drug extract produces only a few weak blue (vis.) zones in the middle and 
upper Rf range. The test extract (2) gives more blue zones in the middle and lower 
Rfrange. 
In UV-365 nm, at least 10 intense, yellow-green or blue fluorescent zones appear, 
especially at Rf 0.4 and in the Rf range 0.8-0.9 (aglycones). 
Scillirosidin migrates at Rf ca. 0.8, its monoglycoside, scilliroside, at Rf ca. 0.4; 
these zones fluoresce light green in UV-365 nm (almost white in the reproduced 
chromatogram, Fig. 20), and they appear green-yellow in vis. The corresponding 
diglycoside, glucoscilliroside, is detectable only in the test extract (light green fluores-
cence at Rf ca. 0.2). Proscillaridin (cf. T5) migrates at Rf ca. 0.65, and it is present 
in especially high concentration in the test extract. Scillaren A is found directly 
above scilliroside (see Sc. var. alba 3, 4, 5) as a blue zone in vis. 
Scillae bulbus var. alba 
The TLC of the test extract (5) is characterized in particular by the blue (vis.) 
or light yellow fluorescent (UV-365 nm) zones of proscillaridin (Rf ca. 0.65), scilla-
ren A (Rf ca. 0.4) and glucoscillaren (Rf ca. 0.2). The main glycoside in this extract 
is scillaren A. Commercial drugs 3 and 4 have high contents of proscillaridin, and 
contain only small quantities of scillaren A; the zones in the upper part of the 
chromatogram are also more intense. 
Fig.19 
T 2 
Fig.20 
FRONT 
Rf 
0 .5 
-START 
223 
Saponin Drugs 
The saponins in official saponin drugs are mainly triterpene derivatives, with a smaller 
number of steroids. 
Sugar residues may be linked via a single OH-group (usually C-3-0H) of the 
aglycone (monodesmoside saponins) or more rarely via two OH-groups or a single 
OH-group and a carboxyl group (bis-desmoside saponins). 
Triterpene saponins 
These saponins possess the oleanane ring system, or more rarely ursane or dammarane 
systems. Many are acidic, due to the presence of one or two carboxyl groups in 
the aglycone and/or sugar moiety. Other oxygen-containing groups mayaiso be 
present in the sapogenin, i.e. -OH, -CH20H or -CHO. 
The carbohydrate group usually contains 1-6 mono saccharide units, the most 
common of these being glucose, galactose, rhamnose, ara bi no se, fucose, xylose, 
glucuronic acid and galacturonic acid. The horse chestnut saponins also contain 
various esterified aliphatic acids. 
All triterpene saponins possess haemolytic activity, which varies from strong 
to weak, depending on the type of substitution. 
Steroid saponins 
The sapogenins ofthe steroid saponins are mostly spirostanols. Furostanol derivatives 
are usually converted into spirostanols during isolation procedures: these sapogenins 
do not carry carboxyl groups. Steroid saponins possess fewer sugar units than the 
tri terpene saponins. In contrast to the monodesmosides, the bis-desmoside furostanol 
glycosides have no haemolytic activity. 
I. Preparation of Drug Extracts for TLC 
Powdered drug (2 g) is extracted by heating for 10 min under reflux with 10 ml 
of 70% ethanol. The clear filtrate is evaporated to ca. 5 ml, and 25-40 111 of this 
soln. are applied for chromatography. 
Exceptions: 
Ginseng radix is extracted under similar conditions with 90% ethanol. 
Liquiritiae radix, according to Ph. Eur. 11. 
Powdered drug (1 g) is shaken for 15 min with 20 ml chloroform, then filtered. 
The filtrate is evaporated to dryness, and the residue is dissolved in 2.0 ml chloro-
form/methanol (1: 1) (CHCI3 extract I). 
The extracted drug is then heated under reflux for 1 h with 30 ml of 0.5 M 
sulphuric acid. After cooling, the unfiltered mixture is shaken twice with 20 ml 
quantities of chloroform. The combined chloroform extracts are dried over anhy-
drous sodium sulphate, filtered and evaporated to dryness. The residue is dissolved 
in 2.0 ml chloroform/methanol (1: 1) (CHCI3 extract 11 after hydrolysis). 10111 of 
this soln. are applied for chromatography. 
225 
226 
11. Thin Layer Chromatography 
1. Reference solutions 
Each test compound is prepared as a 0.1 % solution in methanol, and 10111 are 
applied for chromatography. 
2. Adsorbent 
Silica gel 60F 254 pre-coated TLC plates (Merck, Darmstadt). 
3. Sampie concentration 
Drug extracts 25-40 111. 
Test solutions 10 111. 
4. Chromatography solvents 
SP-l Chloroform-methanol-water (64: 50: 10) 
SP-1 is suitable for the separation of all saponin mixtures from drugs. How-
ever, the mixture must be prepared exactly: analytical grade CHCI3 must 
be used (technical grade contains ethanol) and chromatography must be per-
formed at 20° C after 30 min chamber saturation. At higher temperatures, 
all zones are shifted to the upper Rf range of the chromatogram. 
SP-2 n-Butanol-glacial acetic acid-water (50: 10:40); upper phase. This system (spe-
cified by DAB 8) is less temperature-sensitive than SP-1. The disadvantages 
are that it separates the main saponins of official drugs in the low Rf range, 
and the developing time is 5-6 h on TLC plates. 
SP-3 Chloroform-methanol-water (70: 30: 4) 
SP-3 is especially suitable for the separation of ginsenosides from Ginseng 
radix, and the eleutherosides of Eleutherococci radix. 
SP-4 Chloroform-methanol (95: 5) 
The solvent system is specified by Ph. Eur. II far separation of glycyrrhetic 
acid. 
III. Detection 
1. Without chemie al treatment 
With the exception of glycyrrhetic acid (from Liquiritiae radix), no sapomns are 
detectable by exposure to UV-254 nm or UV-365 nm. 
2. Spray reagents 
a) Blood reagent (BL No. 6, p. 299) 
Haemolytic saponins are detected as white zones on a reddish background. Haemoly-
sis may occur immediately, or after allowing the TLC plate to stand, or after drying 
the plate in warm air. 
b) Vanillin-sulphuric acid reagent (VS No. 38, p. 304) 
With this reagent, saponins form mainly blue or blue-violet and sometimes yellowish 
zones (vis.). 
c) Anisaldehyde-sulphuric acid reagent (AS No. 2, p. 299) 
Colours are similar to those with VS re agent. 
d) Antimony( III) chloride reagent (SbCI3 No. 3, p. 299) 
Red-violet colours in vis. Mostly red-violet, blue and green fluorescence in UV-
365 nm. 
e) Vanillin-phosphoric acid reagent (VPA No. 36A, p. 304) 
Ginsenosides give red-violet colours in vis., and reddish or blue fluorescence in UV-
365 nm. 
The main constituents of Eleutherococcus root form weak, violet zones in vis., 
and intense, yellow, pale blue and orange fluorescent zones in UV-365 nm. 
f) Komarowsky reagent (KOM No. 24, p. 302) 
The sprayed TLC plate is heated for 5-10 min at 100° C in a drying cabinet with 
constant observation. Saponins form blue, yellow and red (vis.) zones. 
IV. List of Saponin Drugs 
Chromatograms (Figs. 1-12)are reproduced on pp. 234-245. 
Fig. 
1,2 
5, 6 
6 
9 
1,2 
10 
Drug/Plant source 
F amily /Pharmacopoeia 
TLC synopsis 
Ginseng Radix 
Ginseng root 
Panax ginseng MEYER 
and other Panax spp. 
Araliaceae 
Eleutherococci Radix 
Siberian ginseng 
Acanthopanax senticosus 
(Rupp. et MAXIM. ex MAXIM.) 
Harms 
Araliaceae 
Hederae Folium 
Ivy leaves 
Hedera helix L. 
Araliaceae 
2. AB-DDR 
TLC synopsis 
Hippocastani Semen 
Horse chestnut seeds 
Aesculus hippocastanum L. 
Hippocastanaceae 
DAB 8, 2. AB-DDR 
Main constituents 
Haemolytic index (HI) 
2-3% of a saponin mixture, contammg at 
least 10 glycosides, known as "ginsenosides" 
Rx (x = 0, a, b 1, b z, C, d, e, f, gl' h). These 
are present in the form of neutral bis-desmo-
sides. The tri terpene sapogenins, 20-S-proto-
panaxadiol and 20-S-proto-panaxatriol have 
a dammarane ring system. The aglycone, 
oleanolic acid, is found only in ginsenoside 
Ro. The glycosides contain glucose, arabinose, 
rhamnose and glucuronic acid. 
HI (drug) < 100 
Oleanolic acid glycosides (eleutherosides I-
M), related to Hedera saponins. 
Lignane compounds (eleutheroside E, sy-
ringaresinol). 
Coumarins (e1eutheroside B1 =isofraxidin glu-
coside) and chlorogenic acid. 
5% of a tri terpene glycoside mixture ; The 
main saponin is hederacoside H, accompanied 
by the monodesmosides, ß-hederin, hederaco-
side C (derivatives of oleanolic acid) and Ot:-he-
derin (derived from hederagenin). 
HI (drug) 1,000-1,500, HI (ß-hederin) 15,000. 
At least 3% of an ester saponin mixture, 
known as "aescin" (DAB 8), containing de-
rivatives of protoaescigenin and barringto-
genol C. Both sapogenins are linked to 1 mol 
glucuronic acid and 2 mol glucose, and esteri-
fied with angelica, tiglic, a-butyric or isobu-
tyric, and acetic acids. 
HI (drug) ca. 6,000 
HI (aescin) 9,500-12,500 
227 
228 
Fig. Drug(Plant source 
F amily (Pharmacopoeia 
1, 2 TLC synopsis 
7,8 Liquiritiae Radix 
Licorice root 
(peeled(unpeeled) 
Glycyrrhiza glabra L. 
Fabaceae 
DAB 8, Ph. Eur. H, 2. AB-DDR 
(3-5.5% glycyrrhizin), Helv. VI, 
ÖAB 
1,2,11,12 TLC synopsis 
3 Primulae Radix 
Primrose root 
Primula veris L. 
Primula elatior (L.) HILL. 
Primulaceae 
DAB 8,2. AB-DDR (HI at not less 
than 2,500), ÖAB (HI at not less 
than 3,000) 
1,2,11, 12 TLC synopsis 
Quillajae Cortex 
Quillaja bark 
Soap bark 
Quillaja saponaria MOLINA 
Rosaceae 
Helv. VI, ÖAB (HI not less than 
3,000), DAC 
1, 2 TLC synopsis 
4 Saponariae Radix 
S. rubrae Radix 
Red soapwort root 
Saponaria officinalis L. 
Caryophyllaceae 
2.AB-DDR(HI 1,200-1,800),DAC 
S. albae Radix 
White soapwort root 
Gypsophila spp. (G. paniculata L., 
G. arrostii Guss.) 
Caryophyllaceae 
Main constituents 
Haemolytic index (HI) 
Saponins: 8-12% glycyrrhizin, which is pres-
ent as the calcium and(or potassium salt of 
glycyrrhizic acid. It does not show haemolytic 
activity. The aglycone, glycyrrhetic acid, does 
possess haemolytic activity. 
Flavonoids: 1-1.5% of a flavonoid mixture. 
The main constituent is liquiritin (4',7-dihy-
droxyflavanone-7-0-glucoside), accompanied 
by liquiritoside (liquiritigenin-gluc-rham) and 
the corresponding chalcones. 
HI (drug) 250-300. 
5-10% of a saponin mixture saponin is Pri-
mula acid A, which is derived from protopri-
mulagenin. 
Primula veris has a higher saponin content 
than P. elatior, and contains more individual 
saponins. P. elatior saponins consist of 90% 
Primula acid; those from P. veris also com-
prise glycosides derived from the genins, pri-
mulagenin A, dehydroprimulagenin A and 
primverogenins A and B. 
Adulterant: Vincetoxicum hirundinaria Med. 
(=Cynanchum vincetoxicum (L.) Persoon), 
containing the steroid glycoside mixture, 
" vincetoxin " . 
10% saponin mixture "Quillaja saponin", 
with Quillaja acid as the aglycone, and glucu-
ronic or galacturonic acid as the sugar moiety. 
HI 3,500-4,500 
S. rubrae radix: 2-5% saponin mixture, main 
constituents saponasides A and D. Gypso-
genin is the aglycone of these acidic bis-des-
mosides. 
HI 1,200-2,000 
S. albae radix: up to 20% saponin with Gyp-
soside A (a bis-desmoside glycoside) as a main 
compound. This saponin mixture serves as 
standard saponin for the determination of hae-
molytic activity. 
HI 2,600-3,900 
Fig. DrugjPlant source 
F amily jPharmacopoeia 
Main constituents 
Haemolytic index (HI) 
1, 2, 11, 12 TLC synopses 
Sarsaparillae Radix 
Sarsaparilla 
1.8-2.4% steroid saponins, comprising paril-
lin, pariglin and others. 
9 
Smilax spp. (S. regelü KILIP et 
MORTON, Honduras drug) (S. med-
ica SCHL. et CHAM, Veracruz drug) 
Liliaceae 
Senegae Radix 
Polygalae radix 
Milkwort root 
Polygala senega L. and other 
Polygala spp. 
Polygalaceae 
Helv. VI, ÖAB 
A venae sativae Herba 
(Fructus) 
Oats 
Avena sativa L. 
Avena orientalis Schreb. 
Poaceae 
The aglycones are sarsasapogenin (= pari-
genin) and its isomer, smilagenin. 
HI 3,500-4,200 
8-10% mixed saponins, comprising at least 
8 glycosides. Main saponin is senegin. The ag-
lycone is presenegenin, with glucose, galac-
tose, rhamnose and fucose as sugar residues. 
The fucose is esterified with 3,4-dimethoxy-
cinnamic acid. 
HI 2,500-4,500 
Steroid saponins: avenacosides A and B. The 
aglycone is nuatigenin, with glucose and 
rhamnose (ratio 3: 1 or 4: 1) as the sugar resi-
dues. 
Triterpene saponins: e.g. avenacin. 
Saponin drugs that possess little or no haemolytic activity andjor are better identified 
on the basis of components other than saponins (relevant chromatograms are repro-
duced in the sections on Flavonoid and Coumarin Drugs). 
Betulae Folium Saponin (ca. 3%): betulin (derived from betu-
Birch leaves linie acid). 
Betula pendula ROTH. Flavonoids (1.5-3%): main components are 
Betula pubescens ERH. hyperoside and myricetin digalactoside (see 
Betulaceae Flavonoid drugs, Figs. 11 and 12, p. 182). 
2. AB-DDR, ÖAB, Helv. VI, 
DAB8 
Eqniseti Herba 
Common horsetail 
Equisetum arvense L. 
Equisetaceae 
2. AB-DDR, DAB 8, Helv. VI, 
ÖAB 
Herniariae Herba 
Rupture-wort 
Herniaria glabra L. 
Herniaria hirsuta L. 
Caryophyllaceae 
ÖAB 
Verbasci Flos 
Mullein flowers 
Verbascum densiflorum 
BERTOLONI 
Scrophulariaceae 
Helv. VI, ÖAB 
Triterpene saponin: equisetonin. 
Flavonoids: isoquercitrin, galuteolin (see Fla-
vonoid Drugs, Fig. 16, p. 186). 
Up to 3% saponin mixture (Herniaria saponins 
land 11), derived from medicagenic acid. 
Flavonoids: glycosides of quercetin and iso-
rhamnetin (rutin, narcissin) (see Flavonoid 
Drugs, Fig. 6, p. 177). 
Coumarins: herniarin (see Coumarin Drugs, 
Figs. 5 and 6, p. 156). 
Saponins without haemolytic activity. 
Flavonoids (up to 3.8%): main compounds ru-
tin and hesperidin (see Flavonoid Drugs, Fig. 7, 
p. 178). 
229 
230 
V. Formulae of Constituents of Saponin Drugs 
Oleanolic acid: Rl = H; R2 = CH3 
Quillaja acid: Rl = OH; R2 = C~ 
Gypsogenin: Rl = H; R2 = C~~ 
Hederagenin: Rl = H; R2 = CH20H 
HO 
HO 
HO 
HOOe 
Primulagenin A 
Presenegenin: R = CH20H 
Hippocastani semen: 
COOH 
~o OH 
Gluc-O 
O-Gluc 
Medicagenic acid: R = H 
~--------~v~----------~ 
Protoaescigenin: Rl = R2 = R3 = H; R4 = OH 
Barringtogenol: Rl = R2 = R3 = R4 = H 
Aescin: R, = Tiglyl, Angelyl, Isobutyryl, cr-Methylbutyryl residues 
R2 = acetyl; R3 = H; R4 = H or OH 
Kryptoaescin: R, = Tiglyl, Angelyl, cr-Methylbutyryl residues 
R2 =H; R3 =acetyl; R4 =H or OH 
Ginseng radix: 
RO 
HO 
OR 
Eleutherococci radix: 
MeO 
R0-9-CH=CH-CH20H 
MeO 
Eleutheroside 8 R=Glucosyl 
Sinapyl alcohol R = H 
R 
(20 S-Protopanaxadiol) H 
Ginsenoside Rb, I ~-D-Gq 1 ~ 21 ~-D-Gq 
Gi nsenoside Rb2 1 ~-D-G111 ~ 21 ~-D-GII 
Ginsenoside Re 1 ~-D-Gq 1 ~ 21 ~-D-Gq 
Ginsenoside Rd 1i3-D-G!J 1 ~21 ~-D-Gq 
R 
(20 S-Protopanaxatrioll H 
Ginsenoside Re la-L-Rh 11 ~ 21 ~-D-Gq 
Ginsenoside Rg, 1 ~-D-GII 
Ginsenoside R9 2 la-L-RhI1 ~ 21 i3-D-Gq 
MeO 
RO 
OR 
OMe 
Eleutheroside E R = Glucosyl 
Syringaresinol R = H 
R' 
H 
I ~-D-GI11 ~ 61 ~-D-GII 
la-L-Ar 11 --. 61 i3-D-Gq 
I a-L-ArfI1 --. 61 i3-D-G1 1 
I i3-D-G11 
R' 
H 
1i3-D-GI! 
!i3-D-G1! 
H 
Eleutheroside 8, 
231 
232 
Liquiritiae radix: OH 
HO HO 
Liquiritin .. 
Liquiritigenin Isoliquiritigenin 
Glr 1-2Glr-O 
Glycyrrhizic acid 
Primulae radix: 
la-L-Rha 11 '-.,. 
....... mlIß-O-Gl:r:J 1-3IProto-Primu1agenin AI 
) B-0-GlI 1-31 ß-O-Ga 11 
Primula acid A 
Senegae radix: 
\ß-0-G111_3)Presenegeni n128-11 0-FuI2_1) L-Rha 14-1) D-Xy 14+-1)B-D-Gal 
4 
t 
Dimethoxycinnamic acid 
Senegin 
Hederae folium: 
la-L-RhaI1-2Ia-L-ArI1-310Ieanolic acid 128-11 8-0-GlI6+-1IB-0-GlI4+-1Ia-L-Rhal 
Hederacoside B 
OH 
STEROID-SAPOGENINE 
H 
Sarsasapogenin: R1 = H; R2 = H; 5~. 25~ 
Smilagenin: R1 = H; R2 = H; 5~. 25a 
Sarsaparillae radix: 
Gluc~ 
Rha14Gluc-O 
G!uc~ 
Avenae herba: 
Ar = Arabinose 
Fu = Fucose 
Ga = Galactose 
GI =Glucose 
Glr = Glucuronic acid 
Rh = Rhamnose 
Xy =Xylose 
H 
HO 
HO 
Sarsaparilloside 
Nuatigenin 
H 
Hecogenin: 5a. 20~. 25a 
O-gluc 
233 
Saponin Drugs TLC Synopsis 
Tracks 
Tests 
Solvent 
system 
1 = Senegae radix 
2 = Sarsaparillae radix 
3 = Ginseng radix 
4 = Hippocastani semen 
5 = Primulae radix 
6 = Saponariae albae radix 
7 = Liquiritiae radix 
8 = Quillajae cortex 
T = mixture of saponins from Senegae radix 
SP-1: chloroform-methanol-water (65: 50: 10) 
Detection Vanillin-sulphuric acid reagent (VS No. 38, p. 304) vis. Fig. 1 
vis. Fig.2 Blood reagent (No. 6, p. 299) 
For description of drugs see p. 227-229. Formulae p. 230-233. 
Chromato- 1 
gram 
Senegae radix. Immediately after spraying with VS reagent, strong red zones are 
formed in the Rfrange 0.3-0.55; the colour fades on heating (cf. T, "Senega saponin 
mixture "). After heating, 7-8 additional violet zones appear in the upper part of 
the chromatogram; only some of these are due to saponins. The main components 
of Senega saponin mixt ure have strong haemolytic activity. 
1,2 
234 
2 Sarsaparillae radix. After treatment with VS reagent, the chromatogram is character-
ized by at least 8 yellow or yellow-brown zones in the Rf range 0.2-0.75. The zones 
between Rf 0.55 and 0.75, and directly below the solvent front, show haemolytic 
activity. 
3 Ginseng radix produces at least 10 intense, dark violet, main zones (ginsenosides) 
in the Rf range 0.35-0.75. The main haemolysis zones are in the intermediate Rf 
range (for identification, see Fig. 5, p. 238). 
4 Hippocastani semen is characterized by the broad violet zone of "aescin" at Rf 
0.5-0.6, which shows distinct haemolytic activity (for identification, see Fig. 10, 
p.242). 
5 Primulae radix shows the brown zone of " Primula acid" (a mixture of three compo-
nents) at Rf 0.3-0.4. Like the other zones in the upper Rf region (0.6--0.75 and 
0.95), Primula acid shows only weak haemolytic activity. (See Fig. 3, p. 236 for 
further details). 
6 Saponariae radix. After treatment with VS reagent, the chromatogram shows several 
intense, dark brown zones in the same region (Rf 0.1-0.3) as the saponin test. The 
same zones are also strongly haemolytic. There are also several weaker, violet zones 
between Rf 0.7 and the solvent front (the most intensive one of these is at Rf 
0.75), which also possess haemolytic activity (for identification, see Fig. 4, p. 236). 
7 Liquiritiae radix is especially characterized by intense, yellow-brown zones in the 
Rfrange 0.6--0.75 (flavonoids). Haemolytic activity is found only in the region above 
Rf 0.75. The triterpene saponin, glycyrrhizin, is detected as glycyrrhetic acid after 
hydrolysis (for furt her details and identification of zones, see Figs. 7 and 8, p. 240). 
8 Quillajae cortex produces about 6 dark brown zones in the Rfrange 0.15-0.4, which 
represent the saponin mixture of this drug. Strongly haemolytic zones are found 
between RfO.1 and 0.7 (see also Fig. llA, p. 244). 
Remarks: All these drug extracts show strongly haemolytic zones directly below the solvent 
front (sapogenins and some sterols). 
Fig.l 
T 2 3 4 
Fig.2 
T 2 3 4 
5 6 7 
5 6 7 
8 
8 
F 
Rf 
5 
235 
Primulae Radix Saponariae Radix 
Tracks 
Tests 
Solvent 
system 
Detection 
Chromato-
gram 1,2 
3 
1 = Prim ulae radix (P. veris) 
2 = Primulae radix (P. elatior) 
3 = Saponariae albae radix 
4 = Saponariae rubrae radix 
Tl =Primula acid 
T2 = standard saponin from Gypsophila spp. 
SP-l: chloroform-methanol-water (64: 50: 10) 
Blood reagent (No. 6, p. 299) 
Antimony(III) chloride reagent (SbCI 3 No. 3, p. 299) 
vis. 
vis. 
UV-365 nm 
For description of drugs see p. 228. Formulae pp. 230-233. 
Primulae radix 
Fig. 3A; 4A 
Fig. 3B; 4B 
Fig. 3C; 4C 
After treatment with ShCl3 reagent, the standard "Primula acid" (cf. Tl) gives 
three violet zones in vis., and light brown fluorescence in UV-365 nm. 
In Primula veris, "Primula acid" appears as two main zones with a minor zone. 
In Primula elatior, the saponins are present in lower concentration. Extracts of 
Primula veris also show a violet (vis.) zone at Rf ca. 0.75, which gives a greenish 
fluorescence in UV-365 nm; this zone is absent from P. elatior (see also Fig. 1, 
p. 234; VS reagent). Only one zone of Primula acid gives an immediate haemolytic 
response with Blood reagent; the other zones show positive, but slower haemolysis. 
Remarks: Primulaverin is present in high concentration in Primula veris, and is absent from 
P. elatior. This phenol glycoside can therefore be used to differentiate between the two roots. 
By enzymic c1eavage, it yields methyl methoxysalicylate, which has a characteristic odour. 
The root of Cynanchum vincetoxicum, which may be encountered as an adulterant, contains 
steroid glycosides. These can be extracted with toluene. If the toluene extract is completely 
evaporated, redissolved in ethanol and treated with conc. H 2S04 and Feel3 , a violet coloura-
tion is produced due to the presence of vincetoxin. 
4 3, 4 Saponariae radix 
236 
After ShCl3 treatment, both Saponaria extracts show a large number of overlapping 
zones in the Rf range 0.05-0.5; these are violet or black-violet in vis., and fluoresce 
green-blue in UV-365 nm (cf. also Fig. 1, track 6, TLC synopsis p. 234). 
The saponin test mixture shows two main zones (cf. T2; RfO.25-0.4). The immedi-
ate appearance of powerful haemolysis zones in the Rf range 0.2-0.45 is typical 
for Saponariae radix. 
It can be seen from the chromatogram that S. albae radix has a markedly higher 
saponin content than S. rubrae radix (S. alba 6-30%; S. rubra 2-5%). 
c 
~' 
....... 
~ 
... 
~ 
.... ,Vr ... 1 
Fig.3 
Tl 2 
Fig.4 
3 3 T2 4 3 
....,.........r . 
11 
Tl 
T2 
...... 
-
\.", 
y.l~'--~' 
-
2 
4 
-F 
Rf 
s 
237 
Ginseng, Eleutherococci Radix 
Tracks 
Tests 
Solvent 
system 
Detection 
1 = Ginseng radix 
2 = Eleutherococci radix 
ginsenosides: Re, Rb2 , Rb 1 , Re, Rd, Rg', Rh'. 
SP-1: chloroform-methanol-water (64: 50: 10) 
SP-3: chloroform-methanol-water (70: 30: 4) 
Antimony(III) chloride (SbCI3 No. 3, p. 299) 
Blood re agent (No. 6, p. 299) 
Vanillin-phosphoric acid (VPA No. 36A, p. 304) 
Fig. 5A, B, C 
Fig. 5D; 6A, B 
UV-365 nm Fig.5A 
VIS. Fig.5B 
VIS. Fig.5C 
VIS. Fig. 5D; 6A 
UV -365 nm Fig. 6B 
For description of drugs see p. 227. Formulae p. 231. 
Chromato- 1 Ginseng radix. In both solvent systems, in vis. and in UV-365 nm, the chromatograms 
gram show about 10 zones between the originand the solvent front. 
5,6 
5A, B 
5C 
5D 
6A, B 
238 
Treatment with SbCl3 and inspection in UV-365 nm reveals pale green fluorescent 
zones (ginsenosides), which are especially conspicuous in the Rf range 0.25-0.6. 
Weaker fluorescent zones occur in the upper Rf range. In vis. the ginsenoside zones 
appear violet-blue. The prominent zone at Rf ca. 0.2 is due to sugars. 
Treatment with Blood reagent reveals weak haemolytic zones at Rf 0.6-0.65 and 
astronger zone at Rf ca. 0.9. 
VPA reagent gives 8-10 distinct red-violet (vis.) zones in the Rf range 0.05-0.4 
(e.g. ginsenosides Re, Rb2 , Rb 1 , Re, Rd, Rg'), and a few weaker zones in the upper 
Rf range. In UV-365 nm the ginsenosides give a distinct red-violet fluorescence 
(Fig.6B). 
Remarks: The ginsenosides show higher Rf values in SP-I than in SP-3; solvent system SP-l 
is more suitable for the TLC comparison of Ginseng and Eleutherococcus extracts. 
2 Eleutheroeoeeus root extraet. After treatment with VPA reagent, chromatograms 
are characterized by three violet-red (vis.), main zones at Rf 0.3-0.5. In UV-365 nm 
the two upper zones give characteristic yellow and orange fluorescence, while the 
lower zone gives a dull, dark green fluorescence (= E). An additional pale yellow 
fluorescent zone appears above the orange zone. The weak blue fluorescence at 
Rf ca. 0.1 is due to ehlorogenie acid. 
Eleutheroside E is seen as a dull, dark green zone (E at Rf ca. 0.4) directly 
below the yellow fluorescent zone, and at about the same Rf as ginsenoside Rg'. 
Ginseng radix, on the other hand, shows the characteristic, red-violet fluorescent 
zones of ginsenosides mainly between the start and Rf ca. 0.45. 
Fig.5 
Re Rb2 Rb' 
Fig.6 
2 Re Rg ' 2 
Re Rd Rg' Rh' 
Re Rg ' 
F 
Rf 
0 .5 
5 
-F 
Rf 
0.5 
239 
Liquiritiae Radix 
Tracks 
Test 
Solvent 
system 
Detection 
1 = Liquiritiae radix (EtOH extract, see p. 225) 
2 = Liquiritiae radix (hydrolysate, see p. 225) 
3 = Liquiritiae radix (CHCI 3 extract I, see p. 225) 
Tl = hyperoside (Rf ca. 0.6), rutin (Rf ca. 0.35) 
T2 = standard saponin 
T3 = glycyrrhetic acid 
F-l: ethyl acetate-formic acid-glacial acetic acid-water 
(100: 11: 11 :27) 
SP-l: chloroform-methanol-water (64: 50: 10) 
SP-4: chloroform-methanol (95: 5) 
Natural products-polyethyleneglycol reagent 
(NPjPEG No. 28, p. 303) 
Sulphuric acid (H 2S04 50%, No. 34B, p. 303) 
Blood reagent (No. 6, p. 299) 
Antimony(I1I) chloride reag. (SbCI3 No. 3, p. 299) 
UV-365 nm 
UV-365 nm 
vis. 
VlS. 
VlS. 
UV-365 nm 
Anisaldehyde-sulphuric acid reag. (AS No. 2, p. 299) vis. 
For description of drugs see p. 228. Formulae p. 232. 
Fig. 7A, B, C 
Fig. 8A, B, C 
Fig.8D 
Fig. 7 A 
Fig.7B 
Fig.7C 
Fig.8A 
Fig.8B 
Fig.8C 
Fig.8D 
Chromato- Unlike other saponin drugs, licorice is better identified and characterized on the 
gram basis of its flavonoid fingerprint. 
7 A, B, C Flavonoids 
Chromatography in F-1, followed by treatment with NP/PEG or sulphuric acid 
reagent and inspection in UV -365 nm, reveals at least 12 yellow-green or light blue 
fluorescent zones, particularly in the Rf range 0.2-0.8. Two characteristic, strongly 
yellow-brown (vis.) flavone zones migrate between the test zones of the rutin and 
hyperoside. The upper of these two zones (Rf ca. 0.45) is due to the flavanone 
glycoside, liquiritin, and its cha1cone form. 
8A-C Saponins 
The characteristic saponin of Liquiritiae radix is glycyrrhizin, which is present in 
the drug in salt form and only partially extractable with EtOH (see p. 225). Glycyr-
rhizin however is easily detectable in aqueous extracts. In SP-1, there is a distinct 
quenching of fluorescence in UV -254 nm at Rf ca. 0.2. 
8 BjC After treatment with SbCI3 , two violet (vis.) zones are seen at Rf ca. 0.2-0.3 (glycyr-
rhizin and a sugar), and a red-brown jlavonoid zone appears at Rf 0.6-0.75. In 
UV-365 nm, these zones give partly dark, and partly light blue fluorescence. 
8 D 2, 3 Glycyrrhizin is detected as glycyrrhetic acid following acid hydrolysis (T3). It migrates 
240 
at Rf ca. 0.25. A CHCI 3 extract from licorice, prepared for comparison, contains 
practically no glycyrrhetic acid (detection according to Ph. Eur. II). 
Fig.7 
Tl Tl Tl 
Fig.8 
T2 2 T3 3 
-FRONT 
Rf 
-0.5 
-STAIH 
-FRONT 
Rf 
-0.5 
-START 
241 
A venae Herba, Hederae Folium, Hippocastani Semen 
Tracks 1 = Avenae herba (MeOH - or BuOH extract) 
2 = Hederae folium 
3 = Hippocastani semen 
Fig. 9A, B 
Fig. 9C, D 
Fig.10A-D 
Tests T = vanillin glucoside 
Tl =avenacoside B 
T2 = ß-hederin 
T3=aescin 
Solvent SP-l: chloroform-methanol-water (64: 50: 10) Fig. 9A-D; lOA-C 
system SP-2: n-butanol-glacial acetic acid-water 
(50: 10 :40); upper phase Fig. lOD 
Detection Vanillin-sulphuric acid reag. (VS No. 38, p. 304) vis. Fig. 9A, B 
Blood reagent (No. 6, p. 299) VlS. Fig. 9C; lOA 
Vanillin-phosphoric acid reag. (VPA No. 36, p. 304) VlS. Fig.9D 
Antimony(III) chloride reag. (SbCl3 ) No. 3, p. 299) vis. Fig. lOB 
UV-365 nm Fig.10C 
Anisaldehyde-sulphuric acid reag. (AS No. 2, p. 299) VlS. Fig. lOD 
For description of drugs see p. 227-229. Formulae p. 230-233. 
Chromato- 1 Avenae herba 
gram After treatment with VS reagent, methanol extracts show about 16 predominantly 
9A, B grey to red-violet (vis.) zones over the whole Rf range (9B). If the plate is heated 
for a longer period, the zones become more distinct, but uniformly red-brown (9 A). 
In the butanol extract (to the right ofTl) the saponin zones are the most conspicuous. 
Avenacoside (cf. T1) appears at Rf ca. 0.2, vanillin glucoside at Rf ca. 0.55 (cf. T). 
9C,D 
1OA-D 
242 
2 Hederae folium 
VPA reagent reveals the characteristic violet or intense blue-black (vis.) zone of 
the hederacoside mixture in the Rf range ca. 0.4-0.6. This zone and the zone of 
ß-hederin (cf. T2) show only weak haemolytic activity. 
3 Hippocastani semen 
Treatment with SbCl3 reveals the broad zone of the saponin mixture, "aescin ", 
at Rf ca. 0.5. This appears violet in vis., and gives an intense, green-grey fluorescence 
in UV-365 nm. Aescin shows pronounced haemolysis (cf. T3). 
Three other violet (vis.) or black zones (in UV-365 nm) (Fig. lOB; treated with 
SbCl3 ) in the Rf range 0.05-0.2 are due to sugars; with AS reagent they form 
brown (vis.) zones (Fig. 10D). In the solvent system, specified by DAß 8 (SP-2; 
detection with AS reagent), aescin migrates with a much lower Rf value (cf. 
Fig.l0D). 
8 
Fig.9 
T Tl T2 
Fig. 10 
T3 3 T3 3 
2 2 T2 
T3 3 T3 
-0.5 
-5 
-F 
Rf 
-0.5 
-5 
243 
TLC-Analysis of Saponins, Using Different Solvent Systems 
and Spray Reagents 
Tracks 
Tests 
Solvent 
system 
1 = Quillajae cortex 
2 = Senegae radix 
3 = Ginseng radix 
Tl = Senega saponin mixture 
4 = Primulae radix 
5 = Hippocastani semen 
6 = Sarsaparillae radix 
T2 = Primula acid T3=aescin 
SP-l: chloroform-methanol-water (64: 50: 10) 
SP-2: n-butanol-glacial acetic acid-water 
Fig. 11A, B, D; 12A, D, E 
Fi~11C,E,F;12B,C,F 
(50: 10:40); upper phase 
Detection Komarowsky reagent (KOM No. 24, p. 302) 
Anisaldehyde-sulphuric acid 
VIS. Fig. 11B, D; 12A, D, E 
vis. Fig. 11 C, E, F; 12 B, C, F 
(AS No. 2, p. 299) 
Blood re agent (No. 6, p. 299) VIS. Fig. 11 A 
General: The chromatograms shown in Figs. 11 and 12 were developed in solvent systems 
SP-1 and SP-2 and treated with KOM and AS reagents. 
1. Solvent systems 
SP-2 is less temperature-dependent than SP-1, but it has the disadvantage that 
the development over 15 cm requires 5-6 h. Furthermore, the main saponins migrate 
at low Rf values; for example, the differentiation of aescin and Primula acid is 
difficult. 
2. Detection 
Sulphuric acid reagents with anisaldehyde, vanillin or p-hydroxybenzaldehydepro-
duce colours not only with saponins, but also with other compounds (e.g. sugars). 
Specific detection of saponins is possible with Blood re agent only (Fig. 5). 
Chromato- 1 Quillajae cortex (cf. TLC synopsis Fig. 1, p. 234) 
gram A: SP-1: the main haemolysis zones lie in the region RfO.l-0.4. 
11 B: SP-1: three brown zones are obtained with KOM reagent. The start zone is stained 
intense red. 
C: SP-2: AS reagent produces main brown zones in lower Rf region. 
11 2,3 Senegae radix, Ginseng radix (cf. TLC synopsis Fig. 1, p. 234) 
D: SP-1: with KOM reagent, Senegae radix gives stable, intense, red zones; Ginseng 
radix gives black-violet colours. 
E, F: SP-2: the saponin zones of Senega (cf. Tl) lie at Rf ca. 0.2, while the main zones 
of Ginseng radix appear between Rf 0.2 and 0.65. 
12 4,5 Primulae radix, Hippocastani semen 
A, D: SP-1: with KOM re agent, the saponin mixtures "Primula acid" (T2) and "aescin" 
(T3) form distinct violet zones at Rf 0.2-0.3 and 0.5, respectively. 
B, C: SP-2: Primula acid and aescin show rather similar Rf values. 
12 6 Sarsaparillae radix (cf. TLC synopsis Fig. 1, p. 234) 
244 
E: SP-1: treatment with KOM re agent pro duces at least 8 typical yellow or yellow-
brown zones over the Rf range 0.25-0.75. 
F: SP-2: the main zones appear mainly at Rf 0.1-0.2. 
Fig.11 
2 3 
Fig.12 
T2 4 T2 4 5 T3 5 
T1 2 3 
T3 6 6 
f 
Rf 
0.5 
5 
f 
Rf 
0 .5 
245 
Drugs Containing Pungent Principles 
The sharp-tasting constituents of these drugs belong mainly to one of the following 
types: 
Amides 
e.g. piperine from Piperis fructus. 
e.g. capsaicin from Capsici fructus. 
o-Methoxyphenols and phenylpropanes 
e.g. eugenol in Caryophylli flos and Myristicae semen; gingerols in Zingiberis rhi-
zoma; elemicin and asarone in Calami and Asari rhizoma. 
Phenolic sesquiterpenes 
e.g. xanthorrhizol in Curcumae rhizoma. 
I. Preparation of Drug Extracts for TLC 
Piperis fructus, Cubebae fructus 
Powdered drug (1 g) is extracted by heating under reflux for 10 min with 10 ml 
methanol. The filtrate is evaporated to 3 ml and this soln. is applied for chromatogra-
phy. 
Capsici fructus 
Powdered drug (1 g) is extracted by heating under reflux for 10 min with 10 ml 
CHCI3 • The filtrate is used for TLC. 
11. Thin Layer Chromatography 
1. Reference solutions 
5 mg standard compound (capsaicin, piperine or cubebin) are dissolved in 5 ml 
MeOH. 
2. Adsorbent 
Silica gel 60F 254 pre-coated TLC plates (Merck, Darmstadt). 
3. Sampie concentration 
Drug extracts 5 111 or 10 Ill. 
Piperis fructus (ca. 3 % soln.): 5 111 (~ca. 50 Ilg piperine) 
Capsici fructus (ca. 0.4% soln.): 10 111 (~ca. 14 Ilg capsaicin) 
Solutions of standards: 5 111 (~ca. 5 Ilg standard substance) 
4. Chromatography solvents 
S-l toluene-ethyl acetate (70: 30): Piperis, Cubebae, Capsici fructus. 
S-2 toluene-diethyl ether-dioxan (62.5: 21.5: 16): Piperis fructus. 
S-3 diethyl ether: Capsici fructus. 
247 
248 
III. Detection 
1. Without chemical treatment 
UV-254 nm: capsaicin shows fluorescence quenching only at high concentrations. 
Cubebin and piperine cause distinct fluorescence quenching. 
UV-365 nm: piperine gives dark blue, piperyline light blue fluorescence. 
2. Spray reagents 
a) Vanillin-sulphuric acid (VS No. 38, p. 304) 
After spraying, the plate is heated for 10 min at 100° C then inspected in visible 
light. Piperine gives alemon yellow (vis.); cubebin gives a red-violet (vis.). 
b) Conc. sulphuric acid (No. 34C, p. 303) 
Piperine: dark brown (vis.). 
Cubebin: red-violet (vis.) dark red fluorescence after ca. 10 min in UV-365 nm (gen-
eral reaction of lignanes) 
c) Capsaicin colour reaction (DCC No. 8, p. 300) 
Capsaicin and other capsaicinoids give a blue (vis.) zone; detection limit 0.1 Ilg. 
IV. List of Drugs Containing Pungent Principles 
Fig. Drug/Plant source 
F amily /Pharmacopoeia 
1, 2 1. Amides 
Piperis Fructus 
Black pepper 
Piper nigrum L. 
Piperaceae 
ÖAB 
Cubebae Fructus 
Cubebs 
Piper cubeba L. 
Piperaceae 
Capsici Fructus 
Capsicums 
Capsicum annum var. longum L. 
Capsici acris Fructus 
Cayenne pepper 
Chillies 
Capsicum frutescens L. 
Solanaceae 
DAB 8 (Cayenne pepper), 
Helv. VI, ÖAB, 2. AB-DDR 
Main constituents 
4-7% sharp tasting substances, with 2-5% 
trans-piperine (pungency index 1: 2,000,000). 
The accompanying compounds, piperettin, pi-
peranin, piperaesthin A and piperyline, con-
tribute only ca. 5% to the pungency of the 
drug. 
Up to 0.1 % piperine. 
Ca. 2.5% cubebin (a non-pungent pepper lig-
nane). 
Capsaicinoids 0.1-0.5% (C. annum), 
0.6-0.9% (C. frutescens), comprising 70% 
capsaicin (the vanillylamide of an 8-methyl-
(trans)-non-6-enoic acid) and the capsaici-
noids, homocapsaicin, dihydrocapsaicin, ho-
modihydrocapsaicin and nordihydrocapsai-
ein. Pungency index of capsaicin is 1: 2 mil-
lion. 1: 5,000 Dilutions of the drug extract 
should be still sharp tasting. 
DAB 8 specifies not less than 0.4% capsaicin. 
The capsaicin content of stored drugs is often 
less than 0.4%. 
Fig. Drug(Plant source 
Family (Pharmacopoeia 
Main constituents 
2. o-Methoxyphenols and other compounds 
Pungent principles present in the essential oil: 
Calami rhizoma: 3-5% essential oil, containing 0-95% asarone. 
Caryophylli Oos: 14-20% essential oil, containing ca. 90% eugenol. 
Myristicae semen: up to 16% essential oil, containing ca. 8% myristicin and eugenol. 
Pungent principles present in the resin: e.g. in Galangae and Zingiberis rr.izoma 
(galangoi and gingerol). 
Curcumae zanthorrhizae rhizoma: xanthorrhizol 
For description of the drugs, their constituents, formulae of constituents and TLC 
separations, see Essential Oi! Drugs. 
v. Formulae 
<O~(CH2)2~CO-NH-iSO-ButYI 
o Piperaesthin A 
R: -CO-(CH2)4-CH=CH-CH(CH3)2 Capsaicin 
R: -CO-(CH2)6-CH(CH3)2 Dihydrocapsaicin 
Piperettin 
o 
R: -CO-(CH2h- CH(CH3l2 Homodihydrocapsaicin Cubebin' 
Ginger 
o HO H H3CO~(CH2)n/CH3 
HO Gingerols 
n:4.6.8 
(For fonnulae of eugenol, asarone and xanthorrhizol, see p. 19-20) 
o 
H 
OH 
The lignane derivative, cubebin, does not have a sharp taste, but it serves for the differentia-
tion of Piperis nigri and Piperis cubebae fructus. 
249 
Piperis, Cubebae, Capsici Fructus 
Tracks 1 = Piperis nigri fructus 
2 = Piperis albi fructus 
3 = Capsici fructus 
4 = Capsici acris fructus 
5 = Cubebae fructus 
Tests Ti = piperettin 
T2 = dipiperine (with traces of piperine) 
T3 = piperine 
T 4 = capsaicin 
T5=cubebin 
T6 = piperine and dipiperine 
Solvent SC-i: toluene-ethyl acetate (70: 30) Fig. lA; 2A, B 
Fig.2C 
Fig.1B 
system SC-2: toluene-diethyl ether-dioxan (62.5:21.5:16) 
SC-3: diethyl ether 
Detection Vanillin-sulphuric acid reagent (VS No. 38, p. 304) 
Dichloroquinone-chloroimide re agent (DCC No. 8, p. 300) 
Sulphuric acid reagent (H2S04 conc. No. 34, p. 303) 
Fig. lA; 2A, C 
Fig.1B 
Fig.2B 
Chromato-
gram 1,2 
1A 
2A 
2C 
For description of drugs see p. 248. Formulae p. 249. 
Piperis Jructus 
After treatment with VS reagent, the chromatogram is characterized by 3-4lemon 
yellow (vis.) zones in the lower and intermediate Rf range (piperine and piperine 
derivatives), a blue-violet (vis.) zone at Rf ca. 0.6 and 1-2 further blue-violet zones 
below the solvent front. 
Zone 
Piperyline 
Piperine } 
Piperettin 
Piperine isomers 
Dipiperine 
Piperaesthin A 
Approx. Rf in SC-l (Fig. 1 A) 
0.1 
0.25 
0.35 
0.4 
Approx. Rf in SC-2 (Fig. 2 C) 
0.15 
0.50 
0.55 
0.55 
0.63 
0.70 
Black and white pepper show the same qualitative composition of pungent principles, 
but differ especially with respect to the quantities of minor constituents. White 
pepper has a somewhat lower content of pungent principles. 
2A, B 5 Cuhehae Jructus 
Aftertreatment with conc. H 2S04 , the chromatogram shows the red-violet (vis.) 
zone of cuhehin (cf. T5), which is characteristic of this drug. The weaker, brown 
to red-violet zones in the upper Rf range are components of the essential oil fraction 
(e.g. cadinen, cadinol), which are stained strongly violet with VS reagent (Fig. 2A). 
Remarks: Cubebae fructus contains only traces of piperine; without further enrichment it 
is not detectable on TLC. Piperis fructus contains traces of cubebin. 
1 B 3,4 CapsiciJructus 
250 
Treatment with DCC reagent produces the characteristic blue (vis.) zone of the 
capsaicin at Rf ca. 0.4 (cf. T4). Extracts of Capsici acris fructus show a pronounced 
carotene zone at the solvent front. An exact differentiation between the two drugs 
can only be made on the basis of a quantitative capsaicin determination. 
• 
Fig.l 
Tl T2 TJ 
A B 
Fig.2 
T6 5 T5 T6 5 T5 
J T4 4 
c 
T6 2 
-FRONT 
Rf 
-0.5 
-START 
-FRONT 
Rf 
-0.5 
-START 
251 
Mustard on Drugs 
and Allium (Garlic) 
The mustard oils (isothiocyanates) are always present in the drug as glucosinolates 
(S-glucosides). These are split by the enzyme myrosinase (ß-thioglucosidase) when 
plant tissues are damaged, or by steam distillation. 
I. Preparation of the Drug Extract, and TLC Methods 
A. TLC investigation of the mustard oil glycosides 
1. Drug extraction (Sinapis semen) 
Ground seeds (10 g) are added to 50 ml boiling methanol, boiled for 5 min, then 
allowed to stand 1 h with occasional shaking. The filtrate is evaporated to 5 ml, 
then applied to a column (length ca. 20 cm, diam. ca. 1 cm) containing 5 g cellulose 
powder (cellulose MN 100, Machery & Nagel, Düren). The column is eluted with 
methanol and the first 20 ml of eluate are discarded. The next 100 ml are collected 
and evaporated to ca. 1 ml at 20--300 C under reduced pressure. 25 J.l.I of this soln. 
are applied for chromatography. 
2. Thin layer chromatography, and detection methods 
Adsorbent: Silica gel 60F 254 pre-coated TLC plates (Merck, Darmstadt). 
Chromatography solvent: 
n-butanol-n-propanol-glacial acetic acid-water (30: 10: 10: 10). 
The developed TLC plate is dried and sprayed with 25% trichloroacetic acid in 
chloroform. After heating for 10 min at 1400 C, the plate is sprayed with a 1: 1 
mixture of 1% aqueous potassium hexacyanoferrate and 5% aqueous FeCl3 (TPF 
No. 35, p. 304). 
Mustard oil glycosides give intense blue zones on a yellow background. The detection 
limit is about 5 J.l.g glucoside. 
B. TLC of mustard oils as their thiourea derivatives 
1. Drug extraction (Armoraciae radix) 
Ground roots (200 g) are mixed with 1 I water, allowed to stand for 2 h, and finally 
submitted to steam distillation. 
F or the preparation of crystalline thiourea derivatives from horse radish oil 
(the procedure is similar for other mustard oils), 0.3 g oil is dissolved in 1 ml of 
95% ethanol, and an equal volume of 25% ammonia soln. is added. The mixture 
is heated for about 20 min on the water bath until the beginning of the exothermic 
reaction. After standing 12 h, the crystals are removed by filtration and recrystallized 
from warm ethanol. 
2. Thin layer chromatography, and delection methods 
Using the upper phase from ethyl acetate-chloroform (analytical grade)-water 
(30: 30: 40), chromatography is performed on Silica gel60F 254 pre-coated TLC plates 
(Merck, Darmstadt) over a distance of 15 cm. 
Inspection in UV-254 nm, without chemical treatment, reveals thiourea deriva-
tives as violet-black zones on a yellow background. 
253 
254 
C. TLC investigation of garlic 
1. Isolation of the amino acid mixture of garlic 
Finely chopped, fresh garlic cloves (25 g) are frozen with solid carbon dioxide, 
and shaken twice with separate 100 ml quantities of 80% methanol. The combined, 
filtered extracts are completely evaporated. The residue (ca. 2 g) is dissolved in 
90 ml water, and about 60 ml ethanol are added drop-wise over aperiod of 10 min. 
After allowing the mixture to stand 12 h in the refrigerator, the clear supernatant 
is decanted and completely evaporated. The light yellow residue (ca. 1 g; hygro-
scopic) is extracted with ab out 15 ml ice-cold methanol for about 2 hat 0° C. After 
filtration, the residue is washed with ice-cold methanol, then with anhydrous diethyl 
ether, and finally dried at 150° C for 2 h. The product (ca. 0.2 g), which is no 
longer hygroscopic, is dissolved in ca. 1 ml of 40% ethanol, and fractionated on 
a column (length 20 cm, diam. 1 cm) of silica gel. Each 5 ml fraction is tested with 
ninhydrin for the presence of amino acids. Fractions containing amino acids are 
combined and evaporated to dryness (yield ca. 50 mg). Asoln. containing 50 mg 
in 1 ml of 40% ethanol is used for chromatography. 
2. Reference substances 
Sulphoxycysteine derivatives can be prepared as follows: 
e.g. S-allyl-L-cysteine sulphoxide 
S-allyl-cysteine (2 g) is dissolved in 35 ml glacial acetic acid. After the addition 
of 1.6 ml of 30% H 2 0 2 , the solution is allowed to stand for 8 h at 10--12° C. Animal 
charcoal is added and the solution is filtered. The filtrate is evaporated to dryness 
at about 50° C, and the residue recrystallized from acetone. 
3. Thin layer chromatography, and detection methods 
Adsorbent: Silica gel 60F 254 pre-coated TLC plates (Merck, Darmstadt) 
Chromatography solvent: n-butanol-n-propanol-glacial acetic acid-water 
(30: 10: 10: 10). 
Delection: modified ninhydrin reagent (NIH No. 29, p. 303) 
Sulphoxy derivatives give orange colours (vis.). 
Alkylcysteines give violet colours (vis.). 
11. Drug List 
Drug/Plant source 
Family 
Sinapis nigrae Semen 
Black mustard seeds 
Brassica nigra (L.) KOCH 
Brassicaceae 
Sinapis albae Semen 
(Erucae semen) 
White mustard seeds 
Sinapis alba L. 
Brassicaceae 
Armoracia rusticana 
MEY. ex SCHREB. 
Horse raddish 
Brassicaceae 
Allii sativi Bulbus 
Garlic 
Allium sativum L. 
Liliaceae 
111. Formulae 
HO-fi-CH -C" S-Glucose ~ 2 ~N-O-S030 
Sinalbin 
Sinigrin 
Glucosinolates 
Sinigrin 
Sinalhin 
Gluconasturtiin 
+ 
Sinigrin 
M ustard oils 
Allyliso thiocyana te 
p-Hydroxybenylisothiocyanate 
Phenylethylisothiocyanate 
Allylisothiocyanate 
Cysteine, cystine, methionine, methionine sulphoxide, cycloalliin, 
S-allyl-cysteine sulphoxide (= alliin) and S-allyl-cysteine (= 
deoxyalliin) are present, together with other S-containing amino 
acids and cysteine sulphoxides. 
By oxidation in the air, or under alkaline conditions, alliin is 
converted into allicin (allylthiousulphinic acid allyl ester) and 
other diallyl polysulphides, mainly diallyl disulphide. 
Allicin 
255 
Mustard on Drugs 
Tracks 
Tests 
Solvent 
system 
1 = Sinapis albae semen (Erucae semen) 
2 = Sinapis nigri semen 
Tl = sinalbin test mixt ure 
T2 = sinigrin 
SC-4: n-butanol-n-propanol-glacial acetic acid-water (30: 10: 10: 10) 
Detection Trichloroacetic acid-hexacyanoferrate-FeCI3 reagent vis. Fig. 1 A, B 
(TPF No. 35, p. 304) 
For description of drugs see p. 255. Formulae p. 255. 
Chromato- Sinapis semen 
gram 1,2 After treatment with TPF reagent, extracts show at least five blue (vis.) zones. 
1A, B 1 Sinapis albae semen is characterized by the two main zones of " sinalbin " (cf. Tl), 
and the weaker zone between them. 
2 Sinapis nigri semen contains sinigrin (cf. T2, Rf ca. 0.35). The zone directly above 
this, which is characteristic of Sinapis alba, is absent. 
Remarks: If larger quantities (50 111) of Sinapis albae semen extract are used, the chromatogram 
shows a large number of weak zones in the upper Rf range. 
Thiourea derivatives (TU Derivatives) - Chromatogram 2A 
Tracks 
Solvent 
system 
1 = methyl-TU 
2= ethyl-TU 
3 = isopropyl- TU 
4= allyl-TU 
5 = sec.n-butyl- TU 
6 =ß-phenyl; TU 
ethyl acetate-chloroform-water (30: 30: 40, upper phase) 
7= benzyl-TU 
8= phenyl-TU 
Detection Without chemical treatment in UV -254 nm 
The naturally occurring mustard oils can also be analysed as their thiourea deriva-
tives. 
Allium sativum - Chromatogram 2B 
Tracks 
Solvent 
system 
G=amino acid mixture from Alii bulbus 
9-14 = S-alkyl-cysteine sulphoxides: 
9 = methyl- 12 = n-propyl-
10= ethyl- J3=n-butyl-
11 = iso-propyl- 14 = allyl-
15-20 = S-alkyl-cysteines: 
15 = methyl- 18=iso-propyl-
16= ethyl- 19=n-propyl-
17= allyl- 20=n-butyl-
n-butanol-n-propanol-glacial acetic acid-water (30: 10: 10: 10) 
Detection Modified ninhydrin reagent (NIH No. 29, p. 303) vis. 
256 
The following compounds can be detected in Allii sativi bulbus: 
9-14 Cysteine sulphoxides (orange, vis.) 
S-methyl-, S-ethyl-, S-n-butyl-cysteine sulphoxide, and S-allyl-cysteine sulphoxide 
(= alliin) 
15-20 Cysteines (violet, vis.) 
S-ally1cysteine (= deoxyalliin) and S-n-methy1cysteine. 
A 
Fig.l 
Tl 
A B 
• 
-
-
-~ 0 
-
~ 
-
0 
-
0 
• 
Fig.2 
1- 8 G 
8 
Tl 2 T2 
-
-,J-
-
&' 
~ 
<D 
9-14 15-20 
-FRONT 
Rf 
-0.5 
-START 
FRONT 
Rf 
0.5 
START 
257 
N arcotic Drugs Cannabis sativa var. indica L., Cannabaceae 
Marihuana: the flowering or seed carrying, dried branch tips of the female plant. 
Hashish: the resin exuded from the leaves and flower stalks of the female plant. 
The constituents of cannabis, the cannabinoids, are benzopyran derivatives. Of the 
various cannabinoids, only L19,lO-tetrahydro-cannabinol (THC) has hallucinogenic 
activity. The type and quantity ofthe various constituents depends on the geographi-
calorigin of the drug, c1imatic conditions of growth, time of harvesting and storage 
conditions. 
I. Preparation of Drug Extracts and Reference Solutions 
Powdered drug (1 g) is extracted by shaking at room temperature for 10 rnin with 
10 ml methanol. The filtrate is evaporated complete1y, and the residue dissolved 
in 1 ml toluene. This toluene soln. (5~50 111, depending on the cannabinoid concentra-
tion) is applied to the chromatogram. 
11. Thin Layer Chromatography 
1. Adsorbent 
Silica gel 60F 25" pre-coated TLC plates (Merck, Darmstadt) 
2. Chromatography solvents 
CA-i: n-hexane-diethyl ether (80:20) 
CA-2: n-hexane-dioxan (90:10) 
CA-3: cyc1ohexane; the plate is impregnated with N,N-dimethylformamide by run-
ning unloaded in a mixture of N,N-dimethylformamide and carbon tetrachloride 
(6:4). After evaporation of the carbon tetrachloride (room temp., 2 h), the extract 
is applied to the origin, and chromatography performed in cyc1ohexane. 
3. Reference solutions 
Thymol (0.1 % soln. in toluene), 5111. 
THC (synthetic productjl mg in 5 ml CHC13), 3 ,.11. 
111. Detection 
1. Without chemical treatment 
In UV-254 nm, the cannabinoids show fluorescence quenching. 
2. Fast blue salt reagent (FBS No. 12, p. 301) 
Fast blue salt B (0.5 g) is dissolved in 100 ml water. The developed TLC plate is 
sprayed, dried in a warm air stream, then immediately sprayed with 0.1 M NaOH. 
Cannabinoids form violet-red, orange-red or carmine (vis.); standard thymol gives 
orange. 
The dried plate mayaiso be sprayed with a solution of fast blue salt (15 mg) 
in 0.1 M NaOH (20 ml), but this reagent is not stable, and must be used immediately. 
259 
Cannabis Herba Hashish 
Tracks 1 = Hashish (Turkish, 1980) 
2 = Hashish (Irani an, 1980) 
3 = Hashish cigarette (1979) 
4,5,6= Cannabis herba (drug collection of the Institute) 
7, 8 = Hashish (unknown origin) 
Tests T=thymol 
THC = tetrahydrocannabinol (synthetic) 
Solvent CA-1: n-hexane-diethyl ether (80:20) Fig. 1A, B 
Fig.2A system CA-2: cyc10hexane (after impregnation, see p. 259) 
CA-3: n-hexane-dioxan (90: 10) 
(double development: 1 x 10 cm and 1 x 15 cm) Fig. 2B, C 
Detection Fast blue salt reagent (FBS No. 12, p. 301), 
followed by treatment with KOH VlS. Fig. 1, 2 
Chromato-
gram 
1A, B 
2A 
2B, C 
260 
Treatment with FBS-KOB produces intense red-violet to red-orange (vis.) zones. 
Between the start and Rf 0.2 are three to fOUf red zones, due to cannabidiol acid 
and other polar cannabinoids. Cannabinol (CBN, Rf ca. 0.45) migrates direct1y above 
the thymol standard (cf. T1), followed by tetrahydrocannabinol (THC, Rf ca. 0.5) 
and cannabidiol (CBD, Rf ca. 0.55). 
The intensities of the COIOUfS depend strongly on the quantity of applied material 
and on the subsequent KOH treatment. COIOUfS vary from red-violet (Fig. 1 A) 
to red-orange (Fig. 1 B). 
TBC is c1early evident only in sampie 2. 
The separation in solvent system CA-2 on the impregnated TLC plate approximates 
to that in CA-I, but with a better separation of zones in the lower Rf range. Sampie 7 
contains TBC. 
After double development in CA-3, the zones are found in a very narrow Rf range 
between Rf 0.4-0.6. The two main zones at the same Rf as the thymol test are 
due to CBN and CBD. In this system, TBC migrates above or in the same Rf 
range as CBD. 
Remarks: The Rfvalues of cannabinoids in solvent systems CA 1-3 are dependent on tempera-
ture and chamber saturation (cf. Figs. 1 AlB and 2B/c). 
8 
Cannabidiol acid (CBDA) Cannabidiol (CBD) 
8 
Cannabinol (CBN) LI 9, 10-Tetrahydrocannabinol (THC) 
A 
Fig.l 
2 3 4 5 
A 8 
Fig.2 
7 8 3 3 
6 T THC 
2 T THC 
T 
T 
-0.5 
5 
-F 
Rf 
-5 
261 
Drugs Containing Valepotriates 
The main active constituents of these drugs, the valepotriates, can best be detected 
infreshly harvested and carefully dried roots. 
Valepotriates are triesters of a terpenoid, trihydric alcohol. This alcohol has 
the structure of an iridoid cyclopenta-(c)-pyran with an attached epoxide ring. The 
following acids are fou~d: isovaleric, acetoxyisovaleric, isovaleroxy-hydroxyiso-
valeric, acetic and isocaproic acids. 
Valepotriates with conjugated diene structure (valtrate and acevaltrate) form 
blue (vis.) salts with acetic acid-HCl reagent; didrovaltrate which lack this diene 
structure, give yellow-brown colour. 
I. Preparation of Extract for TLC from Drugs and from Pharmaceuticals 
Drug extracts 
Powdered drug (0.2 g) is extracted with 5 ml dichloromethane for 5 min at ca. 60° C 
with occasional shaking. After filtration, the drug residue is washed with a further 
2 ml dichloromethane. The combined extracts are evaporated to dryness, and the 
residue is dissolved in 0.2 ml ethyl acetate. 10 111 of this soln. are applied for chroma-
tography. 
Pharmaceutical preparations 
One or two dragees are extracted by shaking with 5 ml dichloromethane for 10 min. 
The clear filtrate (15-20 111) is used directly for chromatography. 
11. Thin Layer Chromatography 
1. Reference solutions 
Solutions ofvaltrate, isovaltrate, didrovaltrate or acevaltrate are prepared by dissolv-
ing 5 mg in 5 ml methanol. 10111 of each solution are applied to the chromatogram. 
2. Adsorbent 
Silica gel 60F 254 pre-coated TLC plates (Merck, Darmstadt). 
3. Sampie concentration 
For good quality drugs, 10111 of extract is adequate for the chromatographie detec-
tion of the main constituents. Commercial valerian roots, however, often have a 
low valtrate content, so that 50111 of extract are recommended for the TLC investiga-
tion. 
4. Chromatography solvents 
V-I toluene-ethyl acetate (75: 25); single development over 15 Cll. 
V-2 n-hexane-methylethyl ketone (80:20); DAB 8, double development. 
263 
264 
III. Detection 
1. Without chemical treatment 
UV-254 nm: acevaltrate and valtrathydrines. 
UV-365 nm: yellow fluorescence, e.g. baldrinal and homobaldrinal. 
2. Spray reagents 
a) Hydrochloric acid-acetic acid reagent (No. 32, p. 303) 
This is for the specific detection of valtrate and acevaltrate, which giveblue zones 
(vis.) (halazuchrome reaction): didrovaltrate shows brown (vis.). 
b) Dinitrophenylhydrazine reagent, DAB 8 (DNP No. 10, p. 300) 
After heating, green-grey or blue zones (vis.) appear; but all zones become uniformly 
brown-yellow (vis.) if the heating is excessive. 
IV. Drug List 
Drug/Plant source 
F amily /Pharmacopoeia 
Valerianae Radix 
Valerian rhizome 
Valeriana officinalis L. 
Valerianaceae 
Ph. Eur. III, 2. AB-DDR (not less than 1-2% 
valepotriates ), 
Helv. VI, ÖAB 
Indian valerian 
Valeriana wallichii DEcANDoLLE 
Mexican valerian 
Valeriana edulis ssp. procera MEYER 
Table 
Main constituents 
Content 
lridoids (valepotriates): 
valtrate, isovaltrate, IVHD-valtrate, didroval-
trate, acevaltrate; for distribution and con-
tents, see Table below. 
Essentialoi/: ca. 0.25% in fresh Valerian rhi-
zome, containing valerenal with some vale-
ranone and valerenic acid. 
The essential oil represents one third of the 
total activity of the drug. 
Main constituent Valeriana Valeriana Valeriana wallichi 
Valtrate / isovaltrate 
Didrovaltrate 
Acevaltrate 
Other valepotriates 
ojJicinalis 
(ca. %) 
0.4-2 
0.1 
0.1 
0.1 
edulis 
(ca. %) 
3.5 
1.5-4 
0.1 
1.0 
valtrate / acevaltrate- didrovaltrate-
race (ca. %) race (ca. %) 
2 0.7 
0.1 1.5-3 
0.4 0.4 
0.1 0.1 
v. Formulae 
Valtrate: R1, R2 = Isovalerianyl 
R3 =Acetyl 
Isolvatrate: R1, R3 = Isovalerianyl 
R2 =Acetyl 
Homovaltrate: R1 = Isovalerianyl 
R2 = Isocapronyl 
R3 = Acetyl 
Acevaltrate: R1 od. R2 = Isovalerianyl 
R1 od. R2 = Acetoxy-
isovalerianyl 
HO 
H3ChH, HO-~X_§ CH20 - Gluc 
Isovalerianyl -0 0 I 
Valeriosidate 
Didrovaltrate: R1, R3 = Isovalerianyl 
R2 = Acetyl, R4 = H 
Homodidrovaltrate: R1 = Isocapronyl 
R2 = Acetyl 
R3 = Isovalerianyl, R4 = H 
IVHD-Valtrate: R1 od. R2 od. R3 = Isovalerianyl 
R1 od. R2 od. R3 = Isovaleroxy-hydroxy-
isovalerianyl 
R1 od. R2 od. R3 = Acetyl, R4 = OH 
Baldrinal: R = Acetyl 
Homobaldrinal: R = Isovalerianyl 
265 
Valerianae Radix 
Tracks 1 = Valerianae radix (Mexican Valerian) 
2 = V. radix (Indian Valerian; didrovaltrate race) 
3,4,5= V. radix (drugs with degraded valepotriates) 
6= V. radix (freshly harvested root) 
7 = V. radix (tincture of Valerian from a dispensary) 
8= V. radix (extract from pharmaceutical prep. I) 
9= V. radix (extract from pharmaceutical prep. 11) 
Tests Tl = valtrate 
T2 = acevaltrate 
T3 = didrovaltrate 
Solvent V-l: toluene-ethyl acetate (75:25) Fig. lA, B 
system V -2: hexane-methyl ethyl ketone (80: 20); 
double development over 15 cm Fig; 2A, B 
vis. Fig. 1 A, B 
vis. Fig. 2A, B 
Detection Conc. HCI-glacial acetic acid (2:8) (No. 32, p. 303) 
Dinitrophenylhydrazine reagent (DNPH No. 10, p. 300) 
Chromato-
gram 
lA, B 
For description of drugs see p. 264. Formulae p. 265. 
Valerianae radix 
The chromatogram shows the blue (vis.) zone of the valtrate-isolvaltrate mixture 
(cf. Tl), the brown zone of didrovaltrate (cf. T3), the blue zone of IVDH-valtrate 
and degradation products of valepotriates (valtrathydrins). The type and quality 
of the drug determines which of these zones is predominant. The degradation pro-
ducts of isovaltrate (homobaldrinal) and valtrate (baldrinal) (Rf ca. 0.5 and DA, 
respectively) give an intense yellow fluorescence in UV-365 nm. Yellow zones are 
also formed in vis. (Fig. 1 B, track 9). 
Approx. Rf Compounds UV-254 nm HCI-acetic acid 
(solvent (Fig. 1 A, B) reagent * vis. 
system V-I) 
0.7-0.75 lsovaltratefvaltrate + blue * Colours are blue to 
0.65 Didrovaltrate brown black-blue, or light 
0.55 Acevaltrate + blue to dark brown, de-
0.4 IVDH-valtrate blue pending on the de-
1 Valtrathydrins + blue gree of heating. 
Start Degradation products (+) brown/blue 
1 A, B 1 Mexican Valerian: high content of isovaltrate/valtrate; medium content of didroval-
trate; low conte nt of acevaltrate. 
2 Indian Valerian: predominantly didrovaltrate, with low concentration of isovaltrate/ 
valtrate and IVDH-valtrate. 
3,4,5 Official Valerian: commercial drugs with little or no valtrate, and containing chiefly 
degradation products of valepotriates. 
6 Official Valerian root (freshly harvested): ca. 90% valtrate. 
7 Tincture of Valerian (commercial product): low concentration of valtrate, with medi-
um concentrations of acevaltrate, IVHD-valtrate and valtrathydrins. 
8 Pharmaceutical: high content of valtrate (V. officinalis). 
9 Pharmaceutical (5 years oId): degradation products of isovaltrate (homobaldrinal) 
and valtrate (baldrinal). 
2A In solvent system V-2, the order of separation is the same, but the Rf values are lower (0-0.6), 
and separation is less satisfactory. The double development often leads to variable Rf values 
(see chromatograms 2A and 2B). 
2B Treatment with DNPH re agent guarantees the detection of all the constituents, but if the 
plate is heated too strongly a11 zones become uniformly ye11ow-brown (vis.). 
266 
Fig.l s 
2 3 4 5 6 Tl T2 T3 7 8 9 
Fig.2 
2 3 4 5 6 Tl T2 T3 7 8 9 
267 
Drugs Containing Pigments 
A. Drugs containing anthocyanins (flavylium derivatives) 
Anthocyanins are responsible for the red, violet and blue colours of flowers and 
other plant parts. They are present in the plant as glycosides of hydroxylated 2-phe-
nylbenzopyrylium salts (flavylium salts). Cleavage by acid hydrolysis produces the 
corresponding free flavylium salt. 
B. Crocus 
I. Preparation of Drug Extracts for TLC 
1. Cyan i jlos, Hibisci jlos, Malvae jlos, Paeoniae jlos (anthocyanins) 
Powdered drug (1 g) is extracted by shaking for 15 min with 6 ml methanol/HCl 
(9 parts methanol: 1 part 25% HCl). 25111 of the filtrate are used for chromatogra-
phy. 
2. Croci stigma 
Four or five crushed stigmas are moistened with one drop of water. After about 
3 min, ca. 1 ml methanol is added and extraction continued for ab out 20 min in 
the dark, with occasional shaking. 10111 of the supernatant or filtrate are used 
for chromatography. 
11. Thin Layer Chromatography 
1. Reference solutions 
a) Anthocyanins: 1 mg standard compound dissolved in 1 ml methanol; sampie 5 111. 
b) Methylene blue: 5 mg dissolved in 10 ml methanol; sampie 10111. 
c) Naphthol yellow/Sudan red: 5 mg naphthol yellow in 5 ml methanol, and 5 mg Sudan 
red in 5 ml chloroform. The two solutions are mixed, and 5 111 are used for chroma-
tography. 
2. Adsorbents 
Silica gel 60F 254 pre-coated TLC plates (Merck, Darmstadt). 
Cellulose pre-coated TLC plates (Merck, Darmstadt). 
Silica gel plates are used for TLC of Croci stigma extracts. 
Chromatography of flower pigments (anthocyanins) is performed on both silica 
gel and cellulose plates. 
3. Chromatography solvents 
a) Anthocyanins 
n-Butanol-glacial acetic acid-water (40: 10: 20) (for silica ge1 and cellulose plates) 
b) Croci stigma 
Ethyl acetate-isopropanol-water (65: 25: 10) 
269 
270 
111. Detection 
1. Withaut chemical treatment 
Anthacyanins form red to blue-violet (vis.) zones. The constituents of Craci stigma 
are yellow. 
2. Anisaldehyde-sulphuric acid reagent (AS No. 2, p. 299) 
Craci stigma 
After spraying, picrocrocin gives a red-violet (vis.) zone, while crocin gives a blue-
violet (vis.) zone. 
IV. Drug List 
Fig. 
1,2 
1,2 
3 
1,2 
3 
4 
Drug/Plant source 
F amily /Pharmacopoeia 
Cyani Flos 
Cornflowers 
Centaurea cyanus L. 
Asteraceac 
Hibisci Flos 
Hibiscus flowers 
Hibiscus sabdariffa L. 
Malvaceac 
DAB8 
MalvaeFlos 
Common mallow flowers 
Malva sylvestris (L.) MILL. 
Malva sylvestris L. ssp. mauritania 
(L.) ASCHERSON et GRAEBNER 
("Mauretanian mallow, dark violet 
mallow") 
Malvaceae 
Malvae (arboreae) Flos 
Hollyhock 
Althaea rosea (L.) CAV. var. nigra 
HORT. 
Malvaceae 
Paeoniae Flos 
Peony 
Paeonia spp. 
PaeoniaceaeCroci Stigma 
Saffron (Crocus) 
Crocus sativus L. 
Iridaceae 
Ph. Eur. II1, Helv. VI, ÖAB 
Main compounds 
Anthocyanins 
Cyanidin-3,5-diglucoside (cyan in) 
Pelargonidin-3, 5-diglucoside (pelargonin) 
Delphinidin-3-xylosyl-glucoside (hibiscin) 
Malvidin-3,5-diglucoside (malvin) 
Delphinidin -3-glucoside 
Malvidin-3-glucoside 
(A mixture of these two compounds is known 
as althaein) 
Paeonidin-3,5-diglucoside 
1.9-15% crocin (digentiobiose ester of croce-
tin) 
2.7-12.9% picrocrocin (ß-hydroxycyclocitral 
glucoside) 
ß-Hydroxycyclocitral and safranal (dehydro-ß-
cyclocitral) are formed from picrocrocin dur-
ing storage or steam distillation. Carotene gly-
cosides are also present. 
v. Formulae 
HO 
OH 
OH 
R, 
H 
OH 
OCH3 
OH 
OCH3 
OCH3 
R2 
H 
H 
H 
OH 
OCH3 
OH 
Aglycones 
Pelargonidin 
Cyanidin 
Peonidin 
Delphinidin 
Malvidin 
Petunidin 
Crocin 
Pigment 
CH3 CH3 
____ HOOC AA A A ~ "",,-~ .... COOH 
, ~ ~ ~ ..." TC "" T + Gentiobiose 
-;:::::-0 
NC'H 
Gluc -O~ 
Picrocrocin 
Bitter principle 
H3 CH3 
Crocetin 
?yC~S 
HO~ 
~_HYdö:rOXycYclO~ÖI 
C'H 
~I 
Safranal 
Odour constituents 
+ Glucose 
+ Glucose 
271 
Flower Pigments, Anthocyanins 
Tracks 
Tests 
1 = Cyani flos 
2 = Malvae sylvestris flos 
3 = Malvae arboreae flos 
4 = Hibisci flos 
Tl = naphthol yellow and Sudan red 
T2 = paeonidin mono glucoside 
T3 = petunidin-3,5-diglucoside 
T4 = delphinidin-3,5-diglucoside and monoglucoside 
T5 = malvidin monoglucoside 
T6 = cyanidin-3,5-diglucoside 
T7 = methylene blue 
Adsorbent Silica ge1 60F 254 pre-coated TLC plates 
Cellulose pre-coated TLC plates 
Fig.1 
Fig.2 
Solvent 
system 
n-butanol-glacial acetic acid-water (40: 10: 20); developed over 10 cm 
Detection Without chemical treatment in vis. Fig. 1,2 
Chromato-
gram 
1,2 
272 
For description of drugs see p. 271. Formulae p. 271. 
On silica gel the zones are less sharply defined, but show more distinct colour 
differences than on cellulose. On cellulose plates the Rf values are in the lower 
range. 
TLC separations 0/ anthocyanins 
J Cyanijlos 
2 Malvae sylvestris jlos 
3 Malvae arhoreae jlos 
4 Hihisci jlos 
On silica gel 
Approx. Rf 
main zone 0.3 
secondary zone 0.5 
main zone 0.4 
2 secondary zones 
0.45 and 0.7 
2 main zones 0.25-0.55 
3 secondary zones 
0.6-0.8-0.9 
2 main zones 
2 secondary zones 
0.25-0.5 
On cellulose 
Approx. Rf 
1 main zone 0.15 
2 secondary zones 0.25-0.3 
1 main zone 0.15 
2 secondary zones 0.25-0.3 
Closely neighbouring zones 
of about equal intensity 
0.1-0.25 
2 main zones 
2 secondary zones 
0.15-0.3 
Remarks: A better differentiation and identijication of anthocyanins can be achieved by ascend-
ing or descending paper chromatography, using Ion ger migration distances and solvents based 
on mixtures of butanol, acetic acid and water. 
Fig.l 
Tl - T 7 2 3 
Fig.2 
Tl - T 7 2 3 
4 
4 
-FRONT 
Rf 
-0.5 
-START 
-FRONT 
-0.5 
-START 
273 
Flower Pigments, Anthocyanins Croci Stigma 
Tracks 
Tests 
Solvent 
system 
Adsorbent 
Detection 
Chromato- 1 
gram 
3A, B 
1 = Hibisci flos 
2 = Paeoniae flos 
3 = Croci stigma 
T1 = methylene blue 
T2 = naphthol yellow (Rf ca. 0.2) and Sudan red (Rf ca. 0.95) 
n-butanol-glacial acetic acid-water (40: 10: 20) 
ethyl acetate-isopropanol-water (65: 25: 10) 
Silica gel 60F 254 pre-coated TLC plates 
Without chemical treatment VIS. 
Without chemical treatment UV-365 nm 
Without chemical treatment UV-254 nm 
Anisaldehyde-sulphuric acid reagent (AS No. 2, p. 299) vis. 
For description of drugs see p. 270. Formulae p. 271. 
Hihisci jlos 
Fig. 3A, B 
Fig. 4A, B, C 
Fig. 3A, 4B 
Fig.3B 
Fig.4A 
Fig.4C 
The extract is characterized by two main, blue-violet zones in the Rf region of 
the methylene blue standard. In UV-365 nm two zones appear in this region, one 
dark and the other light blue fluorescent. 
2 Paeoniae jlos 
4A-C 
274 
A red-violet (vis.) or orange fluorescent (in UV-365 nm) zone is present in approxi-
mately the same Rf region as the methylene blue test (cf. Tl). A weaker, blue-violet 
zone is also present below the main zone. 
3 Croci stigma 
Four zones of strong fluorescence quenching are seen in UV -254 nm. In vis., 3-4 yel-
low zones are seen in the Rf range 0.05-0.45. The main zone above the start is 
due to crocin, which shows grey-green with AS reagent. 
Picrocrocin (Rf ca. 0.55) shows distinct fluorescence quenching in UV-254 nm, and 
violet (vis.) with AS reagent. 
The fluorescence-quenching zones of 4-hydroxycyclocitral and safranal, in the Rf 
region of the Sudan red test (Rf 0.8-0.95), are not always present. 
Fig.3 
Tl 2 Tl 
A • 
Fig.4 
T2 3 T2 
Tl 2 
3 T2 3 
Tl 
FRONT 
Rf 
-0.5 
-START 
275 
Drugs with Miscellaneous Constituents 
These drugs cannot be assigned to any of the foregoing drug groups. They contain 
a wide variety of constituents, some of which do not occur in other drugs. 
I. Preparation of Drug Extracts for TLC 
1. Salicis cortex 
Powdered drug (2 g) is extracted by heating under reflux for 10 min with 10 ml 
methanol. The filtrate is evaporated to ca. 3 ml, and 20 f.tl are used for chromatogra-
phy. Extracts may be similarly prepared in CHCl3 or ethyl acetate. Alternatively, 
the concentrated methanol extract may be extracted by shaking with two separate 
10 ml quantities of CHCl3 or ethyl acetate; combined extracts are evaporated to 
ca. 3 ml, and 20 f.tl are used for chromatography. 
2. Pyrethri jlos 
Pyrethrins are extracted from the flowers with methanol (1 g drug, 5 ml methanol, 
10 min extraction). This extract is used directly for chromatography (15 or 30 f.tl, 
depending on the pyrethrin concentration). 
3. Filicis rhizoma 
Powdered drug (1 g) is mixed with 15 ml saturated barium hydroxide and shaken 
for ca. 30 min. The filtrate is adjusted to pH 3-5 with dilute HCI, then extracted 
with three separate 10 ml quantities of diethyl ether. The combined ether extracts 
are dried over anhydrous sodium sulphate, then completely evaporated. The residue 
is dissolved in ca. 1 ml CHCI3 , and 10 f.tl are used for chromatography. 
4. Hamamelidis folium/cortex 
Powdered drug (1 g) is extracted by heating under reflux for 10 min with 10 ml 
methanol. The filtrate is evaporated to ca. 5 ml, and 10 f.tl are used for chromatogra-
phy. 
5. Lichen islandicus 
Powdered drug (2 g) is heated for 5 min with 5 ml methanol and 10 f.tl of the c1ear 
filtrate are used for chromatography. 
6. Podophylli resina/rhizoma 
Resin (0.5 g) is extracted by heating under reflux for 30 min with 5 ml CHCI3 ; 
20 f.tl of the c1ear filtrate are used for chromatography. 
7. Visci albi herba (folium) 
Powdered drug (1 g) is extracted by heating under reflux for 20 min with 20 ml 
of 50% methanol. The c1ear filtrate is evaporated to ca. 5 ml. 
For the investigation ofjlavonoids, 20 f.tl are applied to the chromatogram. Alter-
natively, 2.5 ml of the methanol extract can be extracted with 10 ml ethyl acetate, 
the ethyl acetate phase evaporated to ca. 2 ml, and 25 f.tl of this soln. used for 
chromatography. 
For the investigation of amino acids, 30 f.tl of the concentrated methanol extract 
are applied to the chromatogram. 
277 
278 
H. Thin Layer Chromatography 
1. Reference solutions 
Salicis cortex Salicin, pieein, triandrin: 1 mg dissolved with warming in 1 ml 
methanol; sampie volume 15111. 
Pyrethri flos 
Filicis rhizoma 
Thymol: 5 mg in 5 ml methanol; sam pie volume 5 111. 
Resorcinol, phloroglucinol: 5 mg in 5 ml methanol; sam pie vol-
urne 511l. 
Hamamelidis folium Hamamelitannin: 10 mg in 5 ml methanol; sampie volume 
10 111. 
Lichen islandicus 
Podophylli rhizoma(resina) 
Caffeie acid: 5 mg in 5 ml methanol; sampie volume 10111. 
Fluoreseein: 5 mg in 10 ml methanol; sampie volume 10111. 
Podophyllotoxin: 5 mg in 5 ml methanol; sam pie volume 10 111. 
Visci albi herba 
(folium) 
Chlorogenie acid: 5 mg in 5 ml methanol; sampie volume 10 111. 
Amino acids: 5 mg of each amino acid in 5 ml methanol; sam-
pIe volume 10111. 
2. Adsorbent 
Silica gel 60F 254 pre-coated TLC plates (Merck, Darmstadt). 
3. Chromatography solvents and detection methods 
See the corresponding chromatograms (Figs. 1-8, pp. 283-287). 
IH. List of Miscellaneous Drugs 
Chromatograms (Figs. 1-8) are reproduced on pp. 283-287. 
Fig. 
2A 
Drug/Plant source 
Family jPharmacopoeia 
Salicis Cortex 
Willow bark 
Salix spp., e.g. 
Salix alba L., 
White willow 
Salix viminalis L. 
Common osier 
Salix cinerea L. 
Grey willow 
Salix fragilis L. 
Crack willow 
Salicaceae 
Hamamelidis Folium 
Witch-hazelleaves 
Hamamelis virginia L. 
Hamamelidaceae 
2. AB-DDR, Helv. VI 
Main constituents 
ca. 4--5% phenol glycosides in varying propor-
tions: 
Salicin (salicyl alcohol-ß-D-glucopyranoside), 
0.5% in Salix alba; 1-3% in Salix fragilis. 
Triandrin (3-(-4-hydroxyphenol-2-propen-l-
ol)-1-ß-D-glucopyranoside, up to 6% in Salix 
viminalis L. 
Picein (4-hydroxyacetophenone-ß-D-gluco-
side), ca. 2% in Salix cinerea L. 
Esters of salicyclic acid and salicyl alcohol. 
Acetylated salicin, salicortin and salireposide 
mayaiso OCCUf. 
Tannins 
Not less than 8% tannins (Helv. VI), with ß-
hamamelitannin (formed from gallic acid and 
hamamelose = 2-C-hydroxymethyl-D-ribose) 
Fig. 
2B 
2C 
3A 
3B 
3C 
4A 
4B 
4C 
5, 6 
Drug/Plant source 
F amily /Pharmacopoeia 
Filicis Rhizoma 
(Polypodii filicis maris radix) 
Male fern rhizome 
Dryopteris filix-mas (L.) SCOTT 
Polypodiaceae 
Helv. VI 
Pyrethri Flos 
Insect flowers 
Chrysanthemum cinerariifolium 
(TREVISAN) VISANI 
Dalmatian insect flowers 
Chry. marschellii ACHERSON 
Chry. occineum WILLD. 
Caucasian insect flowers 
Asteraceae 
ÖAB 
Lichen Islandicus 
Ice1and moss 
Cetraria islandica (L.) ACHARIUS 
Parmeliaceae 
Helv. VI (the 2. AB-DDR alsoad-
mits C. tenuifolia (RETz) HOWE 
Podophylli Rhizoma 
May-apple root 
Podophyllum peltatum L. 
Berberidaceae 
Podophyllinum 
ÖAB, Helv. VI 
Visci albi Herba (Folium) 
Mistletoe leaves 
Viscum album L. ssp. album 
Deciduous mistletoe (on practically 
all European deciduous trees, ex-
cept beech) 
ssp. abies (WIESB.) 
Abromeit 
Silver fir mistletoe (onlyon silver 
fir; not on Scots pine or deciduous 
trees) 
ssp. austriacum (WIESB.) 
VOLLMANN 
Seots pine mistletoe (on Pinus spp.; 
rarelyon spruce) 
Loranthaceae 
Main constituents 
Not less than 1.5% crude filicin, and 20% 
flavaspidic acid (Helv. VI). 
Main compounds are the hutanone-phloroglu-
eides. 
Dimeric phloroglucides: albaspidin, flavas-
pidic acid and others. 
Trimeric phloroglucides: filixie acid, trifla-
vaspidic acid and others. 
Tetramerie phloroglucides: methylene-bis-
nor-flavaspidic acid. 
Not less than 1 % total pyrethrins, containing 
50% pyrethrin I (ÖAB). 
Main compounds are pyrethrins land II, and 
einerins land II. They are converted into per-
oxides and lumi-compounds on exposure to 
air and light. 
Main compounds are polysaccharides (ca. 
50%): liehen in and isoliehenin. 
Also present are 2-3% bitter tasting depsi-
dones (lichen acids): fumarprotocetraric aeid, 
protocetraric aeid and cetraric aeid (1-2%), 
with protolichesteric acid and lichesteric acid. 
Main compounds are lignanes (podophyllo-
toxin, (X- and ß-peltatin) and flavonoids. 
Podophylli resina contains ca. 50% podo-
phyllotoxin. 
Main compounds in branches and leaves: 
Flavonoids: 
Quercetin-3-0-rhamnoside, Q-3-0-arabino-
side; in V. alb. ssp. coloratum: 
Flavoyadorinin A, Flavoyadorinin B (7,3'-di-
O-methyl-luteolin-4' -O-monoglucoside) and 
homoflavoyadorinin B (sugar residue D-glu-
cosylapiose ). 
Plant acids: chlorogenie acid, caffeic acid, sin-
apic acid. 
Fresh leaves contain high concentration of 
amino acids (ca. 0.43%), e.g. arginine, aspara-
gine, proline, lysine, serine, alanine, threo-
nme. 
"Viscotoxin": a polypeptide with 46 amino 
acid residues (Mol. Wt. 5000). 
7, 8 Remarks: TLC separation of amino aeids; see Figs. 7 and 8 for reference compounds 
for Visci albi herba. 
279 
IV. Formulae 
OH Q 0 CH,OH CH=CH-CH2-0~ HO 
Gallic acid 
(3,4,5-Trihydroxy-
benzoic acid) 
280 
OH 
Triandrin OH 
Salicortin 
Salireposide 
Galloyl -O-~:O-GaIlOYI 
OH OH 
~-Hamamelitannin 
Salicin 
CH3 
I 
C=O Q CH,OH 
~ 
HO 
HO 
OH 
OH 
Picein 
OH 
OH 
Catechin 
OH 
CH3 
~oc~ 
o OH 
Aspidinol 
Chrysanthemum-mono-
and dicarbonic acid 
Pyrethrolone, 
Cinerolone 
Pyrethrin I: R1 = CH3 ; R2 = -CH2-CH=CH-CH=CH2 
Pyrethrin 11: R1 = COOCH3 ; R2 = -CH2-CH=CH-CH=CH2 
Cinerin I: R1 = CH3 ; R2 = -CH2-CH=CH-CH3 
Cinerin 11: R1 = COOCH3 ; R2 = -CH2-CH=CH-CH3 
Filixic acid 
R= -CO-CH=CH-COOH 
Fumarprotocetraric acid 
R= -C2H. 
Cetraric acid 
R=-H 
Protocetraric acid 
Podophyllotoxin: R1 = H; R2 = OH; R3 = CH3 
a-Peltatin: R1 = OH; R2 = H; R3 = H 
~-Peltatin: R1 = OH; R2 = H; R3 = CH3 
281 
Salicis Cortex, 
Filicis Rhizoma, 
Hamamelidis Folium 
Pyrethri Flos 
Tracks 
Tests 
1 = Salicis cortex (MeOH total extract) 
2 = Salicis cortex (CHC1 3 extract) 
3 = Salicis cortex (ethyl acetate extract) 
4 = Hamamelidis folium (MeOH extract) 
5 = Filicis rhizoma (diethyl ether extract) 
6 = Pyrethri flos (MeO H extract) 
Tl = salicin 
T2 = triandrin 
T3=picein 
T4 = phloroglucinol 
T5 = catechin 
T6 = hamamelitannin 
T7 = resorcinol 
T8 = phloroglucinol 
T9 = thymol 
Solvent AN-1: ethyl acetate-methanol-water (100: 13.5: 10) Fig. lA, B 
Fig.2A 
Fig.2B 
Fig.2C 
system E-1: ethyl formate-formic acid-water (80: 10: 10) 
E-2: chloroform-methanol (85: 15) 
E-3: hexane-ethyl acetate (90: 10) 
Detection Fast blue salt reagent (FBS No. 12, p. 301) 
FBS, followed by 5% KOH 
Fig.1A 
Fig. lB, 2B 
Fig.2A 
Fig.2C 
Chromato-
gram 1,2,3 
1A, B 
Iron (III) chloride (FeCI 3 No. 14, p. 301) 
Phosphomolybdic acid reagent (PMA No. 27, p. 303) 
For description of drugs see p. 278-279. Formulae p. 280-281. 
Salicis cortex (commercial drugs, botanically not defined). FBS reagent produces 
3-4 red or yellow-brown (vis.) zones in the intermediate Rf range. The colours 
are intensified by KOH treatment. 
The phenol glycosides that are characteristic of individual Salix species mi grate 
at Rf 0.4-0.6. In some species, salicin is the main compound. The glycosides are 
partially c1eaved during extraction. The main zones in the upper Rf range, which 
also stain red with FBSjKOH, are due to catechin, p-coumaric acid, saligenin and 
salicylic acid (yellow-brown) and salicyl alcohol. Some Salix species show only a 
low content of salicin. 
2A 4 Hamamelidisfolium 
The zones of catechin (cf. T5), phloroglucinol (cf. T4) and hamamelitannin (cf. T6) 
show varying intensities of blue-black (vis.) by FeCI3 . 
2 B 5 Filicis rhizoma 
FBS treatment reveals 7-8 red (vis.) zones of the butanone phloroglucides. There 
are also two zones in the region of the resorcinol test (the lower of these is filixic 
acid), one weaker zone in the region of the phloroglucinol test (= jlavaspidic acid) 
and major zones at Rf ca. 0.15 and the solvent front (=aspidinol) 
2 C 6 Pyrethri jlos 
282 
The pyrethrins are stained blue (vis.) by PMA reagent. The main compounds, pyreth-
rins land 11, and cinerins land 11 become rapidly converted into lumi-compounds 
or peroxides on exposure to light and air. 
A satisfactory detection of pyrethrins can only be performed with fresh flowers. 
The chromatogram shown, was made with an extract of commercially available