Endringer et al 2010 Evaluation of Brazilian Plants on Cancer Chemoprevention Targets In Vitro

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SHORT COMMUNICATION
Evaluation of Brazilian Plants on Cancer 
Chemoprevention Targets In Vitro
Denise C. Endringer1, Ydia M. Valadares1, Priscilla R. V. Campana1, Jussara J. Campos1, 
Keller G. Guimarães1, John M. Pezzuto2, Fernão C. Braga1*
1Faculdade de Farmácia, Universidade Federal de Minas Gerais. Av. Antônio Carlos, 6627; CEP 31270-901, Belo Horizonte, Brazil
2College of Pharmacy, University of Hawaii at Hilo, Hilo, Hawaii, USA 96720
Cancer is a leading cause of death worldwide. Cancer chemoprevention is one of the promising strategies to 
decrease its incidence and both plant extracts and natural products may constitute sources of new chemopre-
vention agents. Some Brazilian species popularly used to treat infl ammatory conditions were selected for evalu-
ation for cancer chemoprevention. A total of 32 extracts/fractions from Hancornia speciosa, Davilla elliptica, 
Jacaranda caroba, Mansoa hirsuta, Remija ferrugina, Solanum paniculatum and Xyris pterygoblephara, 
along with a mixture of ursolic and oleanolic acids obtained from J. caroba and a dihydroisocoumarin isolated 
from aerial parts of X. pterygoblephara were tested for their cancer chemoprevention activity [inhibition of 
12-O-tetradecanoyl-13-acetate (TPA)-mediated NF-κB activation, ornithine decarboxylase (ODC) and cyclo-
oxygenase-1 (COX-1); induction of antioxidant response element (ARE)]. Several extracts / fractions were 
active in more than one assay and those from H. speciosa, M. hirsuta and J. caroba mediated strong responses 
with NF-κB, COX-1 and ARE, respectively. Copyright © 2009 John Wiley & Sons, Ltd.
Keywords: cancer chemoprevention; inhibition of NF-κB; Jacaranda caroba; Solanum paniculatum; Hancornia speciosa; 
dihydroisocoumarin.
INTRODUCTION
Cancer is a leading cause of death worldwide. The 
World Health Organization (WHO) estimates that 7.6 
million people died of cancer in 2005 and 84 million are 
expected to die in the next 10 years if no effective prog-
ress is achieved (WHO, 2007). Based on these devastat-
ing statistics, WHO has given priority to research on 
cancer prevention, early detection, and management 
strategies, including the use of traditional medicines 
and therapies, for either treatment or palliative care 
(WHO, 2007).
Cancer chemoprevention is described as the use of 
pharmaceutical agents, natural or synthetic, or the 
ingestion of dietary components, with the goal of pre-
venting, delaying or reversing the process of carcino-
genesis (Pezzuto et al., 2005). Chemopreventive agents 
are grouped into two classes: (1) blocking agents, 
capable of inhibiting the initiation step by preventing 
carcinogen activation; and (2) suppressing agents, which 
hinder malignant cell proliferation (Pezzuto et al., 2005). 
A main objective for the chemoprevention of cancer 
remains the discovery of new effective agents with low 
side effects and no toxicity (Pezzuto et al., 2005).
Carcinogenesis is divided into three stages, which fre-
quently overlap, namely initiation, progression and pro-
motion (Pezzuto et al., 2005). For this study, a panel of 
short-term in vitro bioassays designated to monitor inhi-
bition of carcinogenesis at various stages has been 
developed (Pezzuto et al., 2005). These assays constitute 
an appropriate platform to screen natural products 
possessing potential anticarcinogenic activity (Pezzuto 
et al., 2005).
Keeping in mind that infl ammatory processes are 
involved in the progression and promotion of carcino-
genesis (Pezzuto et al., 2005), some Brazilian species 
popularly used to treat infl ammatory conditions were 
selected to be evaluated for cancer chemoprevention 
(Table 1). Other ethomedical uses reported for the 
selected species are summarized in Table 1.
The chemistry of some of the selected species has 
been previously investigated. Flavonoids and polyphe-
nols/tannins were detected in Davila elliptica (Rodrigues 
et al., 2008). High performance liquid chromatography 
(HPLC) analyses of an ethanol extract from Hancornia 
speciosa leaves indicated rutin as a major constituent 
(Ferreira et al., 2007), whereas proanthocyanidins, rutin, 
quinic acid and l-bornesitol were also isolated from this 
species (Endringer et al., 2007; 2009; Rodrigues et al., 
2007). Alkanols and alkanodiols were identifi ed in low 
polar fractions of Mansoa hirsuta (Rocha et al., 2004), 
and proanthocyanidins were described for the ethanol 
extract of its leaves (Campana et al., 2009). Several 
steroidal saponins and alkaloids were obtained from 
Solanum paniculatum (Ripperger and Schreiber, 1968; 
Blankemeyer et al., 1998). The isolation of the new 
(3R,4R)-(−)-6-methoxy-1-oxo-3-pentyl-3,4-dihydro-
1H-isochromen-4-yl acetate (compound 1; fi gure 1) 
from Xyris pterygoblephara aerial parts has been 
recently reported by our group (Guimarães et al., 2008). 
To the best of our knowledge, the chemical composi-
Received 27 April 2009
Revised 09 September 2009
Copyright © 2009 John Wiley & Sons, Ltd. Accepted 11 September 2009
PHYTOTHERAPY RESEARCH
Phytother. Res. 24: 928–933 (2010)
Published online 2 December 2009 in Wiley InterScience
(www.interscience.wiley.com) DOI: 10.1002/ptr.3050
* Correspondence to: Fernão C. Braga, Faculdade de Farmácia, Universi-
dade Federal de Minas Gerais. Av. Antônio Carlos, 6627; CEP 31270-901, 
Belo Horizonte, Brazil.
E-mail: fernao@netuno.lcc.ufmg.br
 CANCER CHEMOPREVENTION OF BRAZILIAN PLANTS 929
Copyright © 2009 John Wiley & Sons, Ltd. Phytother. Res. 24: 928–933 (2010)
DOI: 10.1002/ptr
Table 1. Ethomedical uses of the screened plants and voucher numbers of the specimens
Plant species Family Voucher number Ethnomedical uses Reference
Davilla elliptica St. Hill. Dilleniaceae BHCB 57175 anti-infl ammatory, 
antiulcer, purgative, 
aphrodisiac
Pio Corrêa, 1984
Hancornia speciosa Gomes Apocynaceae BHCB 49895 hypertension and 
anti-infl ammatory
Gavilanes and Brandão, 1992; 
Hirschmann and Arias, 1990
Jacaranda caroba D.C. Bignoniaceae BHCB 51460 as bitter, astringent, 
diuretic, antiulcer and 
anti-syphilitic
Gavilanes and Brandão, 1992; 
Hirschmann and Arias, 1990;
Di Stasi and Hiruma-Lima, 2002
Mansoa hirsuta D.C. Bignoniaceae BHCB 23862 sore throats and diabetes Chaves and Reinhard, 2003
Remijia ferrugina D.C. Rubiaceae BHCB 3833 as bitter, to treat 
hepatitis, intestinal 
fevers, fever and as 
antiulcer
Pio Corrêa, 1978
Solanum paniculatum L. Solanaceae BHCB 69902 bronchitis and cough; 
arthritis, anemia, as 
bitter, to treat hepatitis 
and intestinal fevers 
Pio Corrêa, 1978
Xyris pterygoblephara Steud. Xyridaceae BHZB 2496 eczemas and dermatitis Pio Corrêa and Penna, 1969
tions of Jacaranda caroba and Remijia ferruginea have 
not yet been established in the literature.
Aiming at the discovery of new cancer chemopreven-
tive agents from plants, 32 extracts and/or fractions 
from the selected Brazilian plants, along with a mixture 
of oleanolic and ursolic acids from J. caroba, compound 
1 from X. pterygoblephara, were evaluated in a battery 
of in vitro tests [inhibition of 12-O-tetradecanoyl-13-
acetate (TPA)-mediated NF-kB activation, ornithine 
descarboxilase (ODC) and cyclooxygenase-1 (COX-1); 
induction of antioxidant response element (ARE)]. In 
this study, their potential chemopreventive activity is 
described.
MATERIALS AND METHODS
Chemicals and cell lines. All chemicals, unless specifi ed 
otherwise, were purchased from Sigma Chemical Co. or 
Aldrich (St Louis, MO, USA). Cell culture media and 
supplements were obtained from Gibco. Wild-type 
HepG2 and HepG2 transfected with ARE-luciferase 
plasmid, human hepatoma cells, were supplied by Dr 
Hong-Jie Zhang (University of Illinois at Chicago, 
Chicago, IL, USA); 293/NF-κB, human kidney embri-
onary cells, transfected with NF-κB-luciferase plasmid 
and pHigromicina (RC0014) were purchased from 
Panomics (Fremont, CA, USA). All media contained 
10% fetal bovine serum (FBS) containing 100 U/mL 
penicillin and 0.1 mg/mL streptomycin. Cells were incu-
bated at 37°C with 95%air and 5% CO2. All cells were 
maintained below passage 20 and used in experiments 
during the linear phase of growth. COX-1 enzyme was 
purchased from Animal Technologies Inc. (Tyler, 
Texas, USA). For the assays, the compounds were dis-
solved in dimethyl sulfoxide (DMSO) and diluted to 
their fi nal concentrations. An equivalent volume of 
DMSO was added to control wells and showed no mea-
surable effect on the assayed culture cells or enzymes.
Plant material. The plant species (Table 1) were col-
lected in the state of Minas Gerais, Brazil, and were 
identifi ed either by botanists from the Department of 
Botany, Universidade Federal de Minas Gerais, Belo 
Horizonte, Brazil, or from the Fundação Zoo-Botânica, 
Belo Horizonte, Brazil, where voucher specimens are 
deposited.
Tested material. After separately drying the distinct 
parts of the plants at 40°C, during 72 h, the following 
materials were powdered and percolated with 96% 
EtOH (Table 2): D. elliptica (aerial parts, 713.5 g), H. 
speciosa (leaves, 251.8 g), J. caroba (aerial parts, 
6,800.0 g), M. hirsuta (leaves, 40.0 g), R. ferruginea 
(stems and bark, 616.0 g), S. paniculatum (leaves, 
1,510.6 g) and X. pterygoblephara (capitula and scapes, 
48.0 g). The solvent was removed under reduced pres-
sure, at 50°C, furnishing the corresponding extracts: 
D. elliptica (139.6 g), H. speciosa (69.1 g), J. caroba 
(1585.1 g), M. hirsuta (11.6 g), R. ferruginea (131.77 g), 
S. paniculatum (254.63 g) and X. pterygoblephara 
(7.1 g). H. speciosa extrat (69.1 g) was subjected to 
column chromatography over silica gel (Merck, 
0.2–5 mm mesh) eluted with solvents of increasing 
polarity, yielding the n-hexane (1.2 g), CH2Cl2 (7.2 g), 
CH2Cl2 : EtOAc (1 : 1; 1.7 g), EtOAc (500 mg), 
EtOAc : MeOH (1 : 1; 31.4 g), MeOH (4.3 g) and 
MeOH : H2O (1 : 1; 2.0 g) fractions. J. caroba (580.0 g), 
S. paniculatum (75.0 g) and R. ferruginea were fraction-
ated over silica gel (Merck, 0.2–5 mm mesh) giving the 
n-hexane (46.02 g), CH2Cl2 (202.85 g), CH2Cl2 : EtOAc 
(97.5 : 2.5; 24.67 g), CH2Cl2: EtOAc (1 : 1; 211.59 g), 
EtOAc (85.17 g) and MeOH (149.54 g) fractions from 
J. caroba; the n-hexane (20.15 g), CH2Cl2 (21.81 g), 
EtOAc (26.44 g) and MeOH (4.15 g) fractions from S. 
paniculatum; and the n-hexane (0.08 g), CH2Cl2: EtOAc 
(1 : 1; 6.76 g) and MeOH (23.70 g) fractions from R. fer-
ruginea. The extract of M. hirsuta (9.0 g) was submitted 
to silica gel column chromatography (Merck, 0.2–5 mm 
mesh), eluting with EtOAc : MeOH 95 : 5 yielding M1 
(543 mg), M2 (560 mg) and M3 (2037 mg) fractions, 
and then with MeOH to give M4 fraction (200 mg). The 
isolation of compound 1 [(3R,4R)-(−)-6-methoxy-1-
oxo-3-pentyl-3,4-dihydro-1H-isochromen-4-yl acetate] 
Copyright © 2009 John Wiley & Sons, Ltd. Phytother. Res. 24: 928–933 (2010)
DOI: 10.1002/ptr
930 D. C. ENDRINGER ET AL.
from the EtOH extract of X. pterygoblephara aerial 
parts has been previously described (Guimarães et al., 
2008). The extract of D. elliptica (130 g) was fraction-
ated over silica gel (Merck, 0.2–5 mm mesh) to afford 
the n-hexane (3.1 g), CH2Cl2 (4.64 g), EtOAc (7.65 g) 
and MeOH (92.77 g) fractions. The n-hexane fraction 
(1.0 g) was further chromatographed on a silica gel 
column employing a gradient of n-hexane : CH2Cl2 
(3 : 7), CH2Cl2 and CH2Cl2 : EtOAc (8 : 2) to give nH1 
(452.9 mg).
Luciferase assays. For the determination of NF-κB 
inhibition and ARE induction, luciferase assays were 
conducted as previously described (Kang et al., 2009). 
Briefl y, transfected cells were incubated at an initial 
density of 1.5 × 105 cells per well for 48 h in 96-well 
plates. After 6 h incubation with TPA (100 nM) and 
test compounds, cells were analyzed for their luciferase 
activity. Cells were washed with PBS and lysed using 
50 μL 1X Reporter Lysis Buffer (Promega, Madison, 
WI, USA) for 10 min, and the luciferase determination 
was performed according to the manufacturer’s proto-
col. IC50 and EC50 were calculated only for samples 
showing at least 50% of inhibition or induction at the 
assayed concentration (20 μg/mL). Data for ARE 
induction (EC50 values) were depicted as the concen-
tration of the compound that provoked activation 
halfway between baseline (DMSO control) and 
maximum response at a concentration of 20 μg/mL. 
Data for NF-κB constructs were expressed as IC50 
values (the concentration required to inhibit TPA-
activated NF-κB activity by 50%). For ARE induction, 
sulforaphane (EC50 4–6 μM) was used as a standard 
inducer. Resveratrol was used as a standard TPA-
Table 2. Cancer chemopreventive activity of the studied species
Samples IC50 (μg/mL)
Plant Part *Extract / fraction NF-kB Inhibition ARE induction ODC inhibition COX-1 inhibition 
D. elliptica aerial parts EtOH* >20 >20 > 20# >10#
n-Hexane >20# >20 >20 >10#
CH2Cl2 >20 >20 >20 >10
EtOAc >20 >20 >20 >10
MeOH >20 >20 >20 >10
nH1 14.2 ± 0.8 >20 >20 8.5 ± 2.1
H. speciosa leaves EtOH* 17.4 ± 5.8 19.7 ± 0.1 >20 >10
n-Hexane 19.7 ± 0.4 >20 >20 >10
CH2Cl2 >20 >20# >20 >10
CH2Cl2: EtOAc (1 : 1) >20 >20# >20 >10
EtOAc 12.9 ± 4.8 >20 >20 >10
EtOAc: MeOH (1 : 1) 1.1 ± 0.0 17.8 ± 1.7 >20 >10#
MeOH 8.8 ± 3.8 17.4 ± 2.7 >20 >10#
MeOH: H2O (1 : 1) 0.5 ± 0.1 4.5 ± 0.4 >20 >10
M. hirsuta leaves EtOH* >20 >20 >20 >10#
M1 (EtOAc : MeOH 95 : 05) >20 >20 >20# >10#
M2 (EtOAc : MeOH 95 : 05) >20 >20 >20# >10#
M3 (EtOAc : MeOH 95 : 05) >20 >20 >20 >10
MeOH >20 >20 >20 0.3 ± 0.1
J. caroba aerial parts EtOH* >20# >20# >20# >10
n-Hexane >20 >20 >20 >10
CH2Cl2 >20# 0.3 ± 0.0 19.9 ± 0.1 >10
CH2Cl2 : EtOAc (97.5 : 2.5)a 10.1 ± 2.4 17.9 ± 1.1 14.5 ± 0.8 >10
CH2Cl2: EtOAc (1 : 1) >20 14.3 ± 4.2 >20 >10
MeOH 19.3 ± 0.7 >20 >20 >10
R. ferruginea stems and 
 bark
EtOH* >20 >20 >20 >10
n-Hexane >20 >20 >20# >10
CH2Cl2: EtOAc (1 : 1) >20 >20 >20 >10
MeOH >20 >20 >20 >10
S. paniculatum leaves EtOH* >20 >20 >20# >10
n-Hexane >20 >20 >20 >10
CH2Cl2 2.2 ± 0.2 >20 7.9 ± 0.1 >10
EtOAc >20 11.3 ± 3.9 >20 >10
MeOH >20 >20 >20 >10
X. pterygoblephara capitula and 
 scapes
EtOH* >20 14.5 ± 6.0 >20 >10
Compound 1† >20 35.1 ± 0.3 >20 >10
a Mixture of ursolic and oleanolic acids. 
* The symbol indicates the assayed crude extracts.
# The symbol indicates the assayed samples which elicited dose-independent activity.
† Compound 1 = (3R,4R)-(−)-6-methoxy-1-oxo-3-pentyl-3,4-dihydro-1H-isochromen-4-yl acetate.
 CANCER CHEMOPREVENTION OF BRAZILIAN PLANTS 931
Copyright © 2009 John Wiley & Sons, Ltd. Phytother. Res. 24: 928–933 (2010)
DOI: 10.1002/ptr
activated NF-κB activity inhibitor (IC50 = 25.0 ± 4.4 μM). 
IC50 values were generated from the results of four 
serial dilutions of the active samples tested in duplicate 
(Tablecurve 2D, AISN Software, USA, 1996). No signs 
of overt cellular toxicity were observed under the 
employed experimental conditions.
Assay for inhibition of COX-1 activity. The effect of 
test compounds on cyclooxygenase-1 (COX-1) was 
determined by measuring PGE2 production as previ-
ously described (Kang et al., 2009). Reaction mixtures 
were prepared in 100 mM Tris-HCl buffer, pH 8.0, con-
taining 1 μM heme, 500 μM phenol, 300 μM epineph-
rine, suffi cient amounts of COX-1 to generate 150 ng of 
PGE2/mL, and various concentrations of test samples. 
The reaction was initiated by the addition of arachi-
donic acid (fi nal concentration, 10 μM) and incubated 
for 10 min at room temperature (fi nal volume, 200 μL). 
Subsequently, the reaction was terminated by adding 
20 μL of the reaction mixture to 180 μL of 27.8 μM 
indomethacin, and PGE2 was quantitated by an ELISA 
method. Samples were diluted to the desired concentra-
tion with 100 mM potassium phosphate buffer (pH 7.4) 
containing 2.34% NaCl, 0.1% bovine serum albumin, 
0.01% sodium azide, and 0.9 mM Na4EDTA. After a 
transfer to a 96-well plate (Nunc-Immuno Plate Maxi-
sorp, Fisher) coated with a goat anti-mouse IgG 
(Jackson Immuno Research Laboratories), the tracer 
(PGE2-acetylcholinesterase; Cayman Chemical, Ann 
Arbor, MI, USA) and primary antibody (mouse antiPGE2; Monsanto, St Louis, MO, USA) were added. 
Plates were then incubated at room temperature over-
night, reaction mixtures were removed, and wells were 
washed using a solution of 10 mM potassium phosphate 
buffer (pH 7.4) containing 0.01% sodium azide and 
0.05% Tween 20. Ellman’s reagent (200 μL) was added 
to each well, and the plate was incubated at 37 °C for 
3–5 h, until the control wells yielded OD 0.5–1.0 at 
412 nm. A standard curve with PGE2 (Cayman Chemi-
cal, Ann Arbor, MI, USA) was generated on the same 
plate, which was used to quantify the PGE2 levels pro-
duced in the presence of test samples. Results were 
expressed as a percentage, relative to control (solvent-
treated) samples. IC50 was determined only for samples 
showing at least 50% of inhibition in the concentration 
used for the assay (10 μg/mL). Indomethacin was used 
as a positive control, yielding IC50 values between 0.05 
and 0.1 observed with COX-1.
Determination of TPA-induced ODC activity with T24 
cells. Determination of ODC activity was performed as 
previously described (Gerhäuser et al., 1997). In brief, 
T24 cells were plated at an initial density of 2 × 105 cells 
per well in 24-well plates. After pre-incubation for 18 h, 
the sample solution in DMSO was added in duplicate 
(20 μg/mL, 0.5% fi nal concentration), before the induc-
tion of ODC activity with TPA (200 nM, fi nal concen-
tration). After additional 6 h incubation, plates were 
washed twice with PBS and kept at −85 °C until they 
were analyzed. ODC activity was directly assayed by 
measuring the release of [14C]CO2 from L-[1-14C]-orni-
thine hydrochloride in the presence of 190 μM nonra-
dioactive ornithine hydrochloride. The amount of 
radioactivity captured in NaOH-impregnated fi lter discs 
was determined by scintillation counting in 24-well 
plates using a Beckman CouterTM LS 6500 multipurpose 
scintallation counter. Deguelin (IC50 0.1 μM), apigenin 
(IC50 6 μM) and menadione (IC50 8.3 μM) were used as 
positive controls. IC50 was determined only for samples 
showing at least 50% of inhibition at the assayed con-
centration (20 μg/mL).
RESULTS AND DISCUSSION
Analysis of the RP-HPLC fi ngerprints obtained for the 
extracts (data not shown) combined with data previ-
ously reported on their chemical composition directed 
the fractionation over silica gel, using different elution 
conditions for each species. The CH2Cl2 : EtOAc 
(97.5 : 2.5) fraction from J. caroba was characterized as 
a mixture of ursolic and oleanolic acids by comparison 
with authentic samples in TLC and RP-HPLC analysis, 
as well as by comparison of 13C and 1H NMR spectra 
with literature records (Mahato and Kundu, 1994; See-
bacher et al., 2003).
Samples were evaluated for potential chemopreven-
tive activity using a battery of short-term in vitro bioas-
says (Pezzuto et al., 2005), being considered active when 
showing at least 50% of inhibition or induction at a 
concentration of 10 or 20 μg/ml depending on the assay. 
However, some of these samples showed a dose-
independent activity during the IC50 determination. 
Those samples were subsequently regarded as inactive 
(Table 2).
Activation of NF-κB has been demonstrated to par-
ticipate in infl ammation, cell proliferation and onco-
genic processes (Nakanishi and Toi, 2005). The EtOAc, 
EtOAc : MeOH (1 : 1), MeOH, and MeOH: H2O (1 : 1) 
fractions of H. speciosa leaves showed potent NF-κB 
inhibitory activity, as well as the nH1 fraction of D. 
eliptica, the mixture of ursolic and oleanolic acids, the 
MeOH fraction of J. caroba and the CH2Cl2 fraction of 
S. paniculatum (Table 2). Several studies have shown 
that proanthocyanidins exert anti-cancer effects through 
the suppression of NF-κB (Mantena and Katiyar, 2006; 
Nandakumar et al., 2008). Constituents of this class have 
been isolated from the bark of H. speciosa (Rodrigues 
et al., 2007). In addition, HPLC analyses of the assayed 
extract of H. speciosa showed the predominance of 
polar compounds (data not shown), the UV spectra of 
which recorded on line by DAD detector are com-
patible with proanthocyanidins (Revilla et al., 2005). 
Recently, quinic acid, l-(+)-bornesitol and rutin iso-
lated from H. speciosa showed moderate to high inhibi-
tory activity on TPA-induced NF-κB inhibition assay 
(Endringer et al., 2009). Therefore, we can suggest that 
proanthocyanidins and cyclitols found in the polar 
O
O
H3CO
O
O
Figure 1. Chemical structure of dihydroisocoumarin (1) [(3R,4R)-
(−)-6-methoxy-1-oxo-3-pentyl-3,4-dihydro-1H-isochromen-4-yl 
acetate].
Copyright © 2009 John Wiley & Sons, Ltd. Phytother. Res. 24: 928–933 (2010)
DOI: 10.1002/ptr
932 D. C. ENDRINGER ET AL.
fractions of H. speciosa might be responsible for the 
observed NF-κB inhibition. On the other hand, ursolic 
acid, a triterpene identifi ed as one of the major consti-
tuents of the CH2Cl2 : EtOAc (97.5 : 2.5) fraction of J. 
caroba could contribute for its NF-κB inhibition activ-
ity. Several reports have shown that triterpenes can sup-
press tumorigenesis and carcinogenesis by inhibition of 
NF-κB (Homhual et al., 2006; Shishodia et al., 2003; 
Pezzuto et al., 2005). Shishoda et al. (2003) have dem-
onstrated that ursolic acid can suppress NF-κB activa-
tion induced by various carcinogens, infl ammatory 
agents, and tumor promoters and those effects were not 
specifi c for a cell type.
ARE is a cis-acting regulatory enhancer sequence 
found in the promoter regions of a battery of genes 
encoding protective proteins (Nguyen et al., 2003). 
Those include phase II detoxifi cation enzymes such as 
NAD(P)H: quinone oxidoreductase and anti-oxidant 
enzymes like glutathione (GSH) S-transferases (GST) 
(Nguyen et al., 2003). Flavonoids and proanthocyani-
dins have been demonstrated to regulate ARE-medi-
ated expression in cell lines (Kweon et al., 2006; Bahia 
et al., 2008). In the present work, ARE induction activ-
ity was observed for EtOH extract, EtOAc : MeOH 
(1 : 1), MeOH, MeOH : H2O (1 : 1) fractions of H. spe-
ciosa, CH2Cl2, CH2Cl2 : EtOAc (1 : 1) fractions and a 
mixture of ursolic and oleanolic acids from J. caroba, 
EtOAc fraction of S. paniculatum and EtOH extract of 
X. pterygoblephara. The CH2Cl2 fraction of J. caroba 
showed potent activity in this assay, whereas the mixture 
of ursolic and oleanolic acids was moderately active 
(11.3 ± 0.3 μM). Compound 1 (fi gure 1) elicited moder-
ate induction of ARE (35.1 ± 0.3 μM or 10.7 ± 0.1 g/mL) 
and might account for the effect of X. pterygoblephara 
(14.5 ± 0.1 g/mL) extract. In a previous work, we dem-
onstrated signifi cant aromatase inhibitory activity for 
compound 1 (IC50 1.6 ± 0.1 μM) (Endringer et al., 2008). 
Taking these results together, this may indicate some 
specifi city of this compound as a cancer chemopreven-
tive agent.
The increase of amino-acid-derived polyamines such 
as putrescine, spermidine and spermine, synthesized 
from ornithine by ODC, has long been associated with 
cell growth and cancer (Gerner and Meyskens, 2004). 
The majority of the evaluated species were inactive in 
the ODC inhibition assay, with the exception of the 
CH2Cl2 extract of S. paniculatum and the mixture of 
ursolic and oleanolic acids from of J. caroba, which 
presented moderate and weak activity, respectively.
The enzymes COX-1 and COX-2 catalyze the fi rst 
two steps in the synthesis of all vasoactive prostaglan-
dins (PGs). Non-steroidal anti-infl ammatory drugs are 
known as potent inhibitors of cyclooxygenases. Some 
studies have suggested a possible involvement of COX-1 
in the genesis of cancer (Rotondo et al., 2004). Further-
more, recent studies have shown low doses of aspirin or 
ibuprofen, both known as non-selective COX inhibi-
tors, are equipotent as selective COX-2 inhibitors in the 
reduction of tumor growth (Yao et al., 2005; Li et al., 
2008). All evaluated samples were inactive on the 
COX-1 inhibition assay, except of the MeOH fraction 
from M. hirsuta leaves, which demonstrated a strong 
inhibitory response, andnH1, a moderate active frac-
tion from D. elliptica.
The data obtained in this study indicate that several 
Brazilian plants are promising sources of cancer chemo-
prevention agents, specially H. speciosa, J. caroba, M. 
hirsuta and S. paniculatum. However, potent responses 
were observed only in some assays, which may at the 
same time indicate some selectivity. Further work is 
required to identify the active principles.
Acknowledgments
CNPq/Brazil (Conselho Nacional de Desenvolvimento Científi co e 
Tecnológico) is acknowledged for a research fellowship (FCB) and 
CAPES/Brazil (Coordenação de Aperfeiçoamento de Pessoal de 
Nível Superior) for a PhD fellowship (DCE). This work was sup-
ported by program project P01 CA48112 awarded by the National 
Cancer Institute and with funds from CNPq and FAPEMIG (Funda-
ção de Amparo à Pesquisa do Estado de Minas Gerais), Brazil.
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