Liu 2020 - Artigo Extração, Identificação, Purificação de Antocianinas

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LWT - Food Science and Technology
journal homepage: www.elsevier.com/locate/lwt
Isolation, purification, identification, and stability of anthocyanins from
Lycium ruthenicum Murr
Pei Liua, Wanrong Lia, Zhongze Hua, Xinguang Qina,∗∗, Gang Liua,b,c,∗
aWuhan Polytechnic University, College of Food Science and Engineering, Wuhan, China
b Key Laboratory for Deep Processing of Major Grain and Oil (Wuhan Polytechnic University), Ministry of Education, China
cHubei Key Laboratory for Processing and Transformation of Agricultural Products (Wuhan Polytechnic University), Wuhan, China
A R T I C L E I N F O
Keywords:
Lycium ruthenicum Murr
Anthocyanins
Response surface methodology
HPLC-MS2
A B S T R A C T
Response surface methodology was used to investigate the ultrasonic-assisted extraction of anthocyanins from
Lycium ruthenicum Murr. A Box-Behnken design of three-level-three-factor was used to optimize extractive fac-
tors, including ethanol concentration (A), ultrasonic extraction time (B), and liquid-solid ratio (C) and to achieve
high extraction yield of the anthocyanins. The three parameters were optimized at 70% (v/v), 28min, 33: 1 (mL/
g), respectively, the best extraction yield of anthocyanins was 7.12 ± 0.093%. The AB-8 resin was further to be
chosen for purification the anthocyanins. Anthocyanin composition was analyzed by HPLC-MS2, the possible
structures can be speculated as petunidin-3-O-(glucosyl-p-coumaroyl)-rutinoside-5-O-glucoside, pentuidin-3-O-
(caffeoyl)-rutinoside-5-O-glucoside, delphinidin-3-O-(p-coumaroyl)-rutinoside-5-O-glucoside, petunidin-3-O-(p-
coumaroyl)-rutinoside-5-O-glucoside, malvidin-3-O-(p-coumaroyl)-rutinoside-5-O-glucoside, cyanidin-3-O-ruti-
noside, and petunidin-3-O-(p-coumaroyl)-rutinoside. The thermal stability indicated the high purity of antho-
cyanins, and purified anthocyanins combined with whey protein isolate (WPI) can enhance the thermal stability
of anthocyanins. The thermal stability of the purified anthocyanins was higher than that of the unpurified
anthocyanins; whereas the thermal stability of the purified anthocyanin-WPI synthesis was greater than that of
the purified anthocyanins. The thermal stable anthocyanins and anthocyanin-protein synthesis has the potential
application in the functional food industry.
1. Introduction
Lycium ruthenicum Murr is a unique species of Lycium that belongs
to the family of Solanaceae and is a functional food mainly distributed
in Xinjiang, Ningxia, Tibet, and Qinghai China (Chen et al., 2018; Liu
et al., 2019a). The fruits of L. ruthenicum Murr are nearly spherical,
bluish violet in color when ripe, and rich in a variety of amino acids and
vitamins and anthocyanins. Hence, their health value is extremely high.
Anthocyanins extracted from L. ruthenicum Murr exhibit great stability,
strong coloring power, and no toxic side effects and can be used in
medicine, food industry, and light textile industry. L. ruthenicum Murr is
abundant in anthocyanins, which hence attracted great attention be-
cause of their potential therapeutic benefit (Wang et al., 2018a; Zheng
et al., 2011). This research aims to explore the good extraction condi-
tion of anthocyanins from L. ruthenicum Murr and identify the main
components of anthocyanins.
Anthocyanins are a class of flavonoids abundantly found in plant
cell fluids and are widely distributed in nature, safe, and not noxious.
These substances have strong antioxidant activities, mainly for anti-
inflammatory, cardiovascular protection, and anti-tumor (Heinonen
et al., 2016; Keppler & Humpf, 2005; Moldovan, David, Chisbora, &
Cimpoiu, 2012). With the improvement of food safety requirements and
the development of biology and medicine, natural pigments have gra-
dually replaced artificial synthetic pigments in the development of food
coloring industry (Zhao et al., 2017). Anthocyanins aid in the stabili-
zation of foodstuffs and increase food shelf life because of their anti-
oxidation properties and antimicrobial potential (Silva, Costa, Calhau,
Morais, & Pintado, 2017). These natural water-soluble pigments (Yang,
Yuan, Xu, & Yu, 2015) belong to polyphenols (Zheng et al., 2011).
Anthocyanins are usually obtained from flowers, fruits, and vegetables
through conventional solid-liquid extraction, ultrasound-assisted ex-
traction (Romero-Diez et al., 2019), enzymatic assisted extraction (Silva
https://doi.org/10.1016/j.lwt.2020.109334
Received 25 October 2019; Received in revised form 23 March 2020; Accepted 24 March 2020
∗ Corresponding author. College of Food Science and Engineering, Wuhan Polytechnic University, No. 68 Xue Fu South Road, Changqing Garden, 430023, Wuhan,
China.
∗∗ Corresponding author.,
E-mail addresses: 76516589@qq.com (X. Qin), lg820823@163.com (G. Liu).
LWT - Food Science and Technology 126 (2020) 109334
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et al., 2017), and High Pressure Pulse Electric assisted extraction (Chen
et al., 2018; Heinonen et al., 2016). Ultrasonic-assisted extraction is
convenient and highly efficient because of the presence of cavitation
bubbles during mass transfers. The use of aqueous ethanol can also
increase the diffusion of compounds. Therefore, this work studied the
ultrasonic-assisted extraction time and the concentration of aqueous
ethanol.
Response surface methodology (RSM) (Iyyappan, Bharathiraja,
Baskar, & Kamalanaban, 2019; Liu, Wei, & Liao, 2013) and orthogonal
test analysis (Li, Pan, Cui, & Duan, 2010) are used to optimize the
conditions after completion of the single factor test. RSM has ad-
vantages of achieving good extraction within few experiments and is a
widely used experimental optimization method. However, the use of
RSM to optimize ultrasound-assisted extraction and increase the ex-
traction rate of anthocyanins in L. ruthenicum Murr has been rarely
reported.
Anthocyanins have potential applications in foods; however, their
thermal stability remains a problem. Numerous studies combined pro-
teins with biologically active ingredients to increase the stability of
anthocyanins while allowing proteins to be loaded with natural active
ingredients. However, few studies focused on the difference in the
thermal stability of anthocyanins and anthocyanins–protein synthesis.
Whey protein isolate (WPI) is a by-product in the cheese industry that
has received attention because it contains all essential amino acids and
has great functional properties (Liu et al., 2019b). Therefore, the
combination of anthocyanins and WPI for thermal stability can serve as
a basis for the experimental application of anthocyanins in foods.
This study aims to: i) obtain optimum extraction conditions for
anthocyanins using Box-Behnken design (BBD) that is one mode of RSM
to explore different independent variables, consisting of ethanol con-
centration (A), extraction time (B), liquid-solid ratio (C), and their in-
teractions; ii) investigate the composition using high performance li-
quid chromatography coupled with a diode array detector (HPLC-DAD)
and high performance liquid chromatography equipped with a QE-Plus
hybrid quadrupole orbitrap mass spectrometry (HPLC-MS2); and iii)
evaluate the thermal stability of anthocyanins and anthocyanin-WPI.
2. Materials and methods
2.1. Samples, chemicals, and standard
The L. ruthenicumMurr were bought in Xinjiang, China and stored in
the laboratory at room temperature.
The AB-8 macroporous resin was purchase from Tianjin Guangfu
Fine Chemical Research Institute, China. Formic acid (Sinopharm
Chemical Reagent Company Limited, China) and acetonitrile (Fisher
Chemical Company Limited,
USA) of chromatographic grade were used
for HPLC analysis. Cyanidin-3-glucoside (94% purity) was purchased
from Shanghai Yuanye Bio-technology Company Limited, China. Whey
protein isolate (product 9020) was provided by Hilmar Ingredients,
USA. And other chemicals were analytical reagent. Ultra-pure water
was produced using Milli-Q system (Millipore Lab equipment Company
Limited, USA).
2.2. Extraction of anthocyanins
Anthocyanins of L. ruthenicum Murr were extracted by ultrasonic-
assisted treatment using an ultrasonic generator (300 W, 45 kHz, SB-
5200DTS, Ningbo Scientz Biotechnology Company Limited, China). The
dry L. ruthenicum Murr was powdered with a pulverizer (XL-10 B,
Guangzhou Xulang Equipment Company Limited, China), subsequently
make it across the 80 mesh sifter. The powder was degreased with
petroleum benzin, and the L. ruthenicum Murr sample was stored at a
low temperature. The powder (0.2 g) was placed in a beaker and used
for each case. Three factors were selected based on the article written
by Liu et al. (2013) Putting the powder in an ultrasonic generator and
extracting anthocyanins at different ethanol concentrations, ultrasonic
extraction times, and in individual liquid-solid ratio.
2.3. Measurement of anthocyanin yield
The absorbance values of the anthocyanins were measured wave-
length at 525 nm using multifunction microplate spectrophotometer
(SpectraMax M2e, Molecular Devices, USA). The extraction yield of
anthocyanin (using cyanidin-3-glucoside as the equivalents) (%) was
calculated using
= + × × ×TA A V n m[( 0.15) ]/(30.38 ),525 (1)
where TA is the total anthocyanin extraction yield expressed as cya-
nidin-3-glicoside equivalents (mg/g), A525 represents the absorbance at
525 nm, V means the volume of solvent (mL), n means the dilution
factor, and m represents the weight of the raw material (g).
2.4. Optimization using Box-Behnken design
The following test schemes and results were obtained by RSM.
Based upon the preliminary results of single factor test, a Box-Behnken
design of three-factor-three-level was employed to identify the great
condition of the variables for anthocyanins. The following three in-
dependent factors were considered: ethanol concentration (A), extrac-
tion time (B), and liquid-solid ratio (C), and the level and code of in-
dependent factors are displayed in Table 1. The experimental plan of
extraction yield (Y) of anthocyanins is shown in Table 2.
2.5. Purification procedure by AK-8 macroporous resins
Guided by the dynamic adsorption and desorption reported by
Zheng et al. (2015), we slowly added 200 mL extract of anthocyanins to
a pretreatment AB-8 macroporous resin column (400 mL) with a flow
rate of 3–4 BV/h. The column that has absorbed anthocyanins was
washed with ultrapure water at a flow rate of 3–4 BV/h to remove
proteins, sugars, and polar compounds. Then, 70% (v/v) ethanol of pH
3 was used to elute at a flow rate of 1–2 BV/h to obtain an anthocyanin-
rich solution. The solution was collected, used the technique of vacuum
freeze-drying, and stored the purified anthocyanins at 4 °C.
2.6. The analysis of anthocyanins by HPLC-DAD
The purified anthocyanins were resolved using HPLC-DAD (SSI
Series 1500, Science System Incorporated, USA) equipped with
Lichrospher RP-C18 column (250 mm × 4.6 mm, 5 μm particle size;
Merck & Company Incorporated, Germany). The temperature of column
maintained at 30 °C, injection amount was 20 μl, wavelength was set to
525 nm, and total flow rate of 0.7 mL/min. The solvent A contained
water/formic acid (9:1, v/v), and the solvent B contained water/acet-
onitrile/acetonitrile (6:3:1, v/v/v). Gradient elution: 10–25% B
(0–10 min); 25–30% B (10–15 min); 30–90% B (15–30 min); 90% B
(30–32 min); 90–30% B (32–35 min).
2.7. Identification of anthocyanins by HPLC-MS2
A Thermo U3000 high-performance liquid chromatography
Table 1
Independent variables and their levels for Box-Behnken design.
Independent variables Levels
−1 0 1
Ethanol concentration (A) (%, v/v) 70 75 80
Extraction time (B) (min) 20 30 40
Liquid-solid ratio (C) (mL/g) 20:1 30:1 40:1
P. Liu, et al. LWT - Food Science and Technology 126 (2020) 109334
2
equipment equipped with a QE-plus hybrid quadrupole orbitrap mass
spectrometer (Thermo Fisher Scientific Incorporated, USA) and a
ACQUITY HSS T3 column (100 mm × 2.1 mm, 1.8 μm particle size;
Waters Corporation, USA) was used to identify the anthocyanin ex-
tracted from L. ruthenicum Murr. The temperature of column main-
tained at 30 °C, injection amount was set to 20 μl, and wavelength was
set to 525 nm. Electrospray ionization was set to positive mode, the
scan range of 80 and 1200 m/z. Nitrogen served as drying and neb-
ulizing gas. The flow velocity of dry gas was 20.0 l/min, the tempera-
ture of dry gas was 270 °C, and capillary voltage was 26 V. The solvent
A contained water/formic acid (9:1, v/v), and the solvent B contained
water/acetonitrile/acetonitrile (6:3:1, v/v/v). Gradient elution:
10–25% B (0–10 min); 25–30% B (10–15 min); 30–90% B (15–30 min);
90% B (30–32 min); 90–30% B (32–35 min).
2.8. Thermal stability studies
The effect of temperature on anthocyanins was determined fol-
lowing the method obtained from Swer and Chauhan (2019) and Chen
et al. (2018) with minimal modifications. Combining proteins and an-
thocyanins can improve the stability of some biologically active sub-
stances (Liu et al., 2019b, 2020; Wang et al., 2019). This experiment
aimed to explore this phenomenon. Unpurified and purified anthocya-
nins were dissolved in WPI solutions (1:1, v/v). The mixed solution was
stirred for 12 h at room temperature for further tests. Unpurified and
purified anthocyanins-WPI samples were used for thermal stability
tests. The unpurified and purified anthocyanins-WPI synthesis, un-
purified and purified anthocyanins solution were individually subjected
to heat treatment in water bath at 80 °C for 0, 30, 60, 90, 120, 180, 240,
and 300 min to test their thermal stability and were then rapidly cooled
down to room temperature. The same solvent was used as a blank. The
absorbance of anthocyanins was plotted on a vertical axis, and the
heating time was plotted on the horizontal axis to map the anthocya-
nins thermal stability curve (Jiang et al., 2019; Wu, Yang, & Chiang,
2018).
2.9. Statistical analysis
All data of this study were obtained in three times, and the results
were reported as average ± standard deviation of replicates. The data
was analyzed for Analysis of Variance (ANOVA). The regression ana-
lysis was carried out using Design Expert software version 8.0.6.
3. Results and discussion
3.1. The standard curve of anthocyanins
The different concentration of reference substance (using cyanindin-
3-glucosideas) as abscissa, the absorbance values which were measured
wavelength at 525 nm as vertical axis, and then drawing the standard
curve. The standard curve was shown in Fig. 1.
3.2. Influence of ethanol concentration on the extraction yield of
anthocyanins
The extraction was conducted at diverse ethanol concentrations of
65%, 70%, 75%, 80%, and 85% (v/v). The other arguments were set as
extraction time of 30 min, and the liquid-solid ratio of 40:1 (mL/g).
Fig. 2a shows that the extraction yield of anthocyanins was affected by
ethanol concentration. As the ethanol concentration increased, the
growth of the extraction yield was slow, reached a peak at 75% (v/v),
and then decreased with the increase in ethanol concentration. Ethanol
concentration of 75% was used to extract anthocyanins in L. ruthenicum
Murr because it is the most suitable according to the theory that the
solvent polarity and the solubility of extract is related (Liu et al., 2013).
These results may be connected to the concentration of ethanol and the
dissolvability of anthocyanins in L. ruthenicum Murr. Thus, an ethanol
concentration of 75% was found to be favorable for anthocyanin ex-
traction.
3.3. Influence of extraction time on the extraction yield of anthocyanins
The extraction times was set
to 10, 20, 30, 40, and 50 min. The
other arguments were set to ethanol concentration of 75% (v/v), and
liquid-solid ratio of 40:1 (mL/g). The extraction yield of anthocyanins
was affected by extraction time as displayed in Fig. 2b. The value firstly
increased as a function of extraction time, reached the highest at
30 min, and then decreased with prolonged extraction time. This phe-
nomenon is probably caused by the extended time for ultrasonic-as-
sisted extraction and the increase in the temperature of the reaction
system, which leads to the destruction and decomposition of antho-
cyanins (Liu et al., 2013). Therefore, 30 min was found to be favorable
for anthocyanin extraction.
3.4. Influence of the liquid-solid ratio on the extraction yield of
anthocyanins
The extraction was conducted at an aqueous ethanol to raw material
ratio of 20:1, 30:1, 40:1, 50:1, and 60:1 (mL/g). The other arguments
were fixed at the ethanol concentration of 75% (v/v), the extraction
time of 30 min. The extraction yield affected by the liquid–solid ratio is
displayed in Fig. 2c. When the liquid-solid ratio increased, the
Table 2
Box-Behnken design for independent variables and their extraction yield.
Run A(ethanol
concentration, %)
B (extraction
time, min)
C (liquid-
solid ratio,
mL/g)
Y (Extraction
yield, %)
1 −1 0 1 6.4110
2 0 0 0 6.8590
3 1 −1 0 5.1625
4 0 0 0 6.9358
5 1 1 0 5.2520
6 −1 −1 0 3.6595
7 0 1 −1 5.6335
8 1 0 −1 5.2425
9 0 −1 −1 4.5840
10 0 0 0 7.3405
11 −1 1 0 6.9035
12 −1 0 −1 5.5515
13 0 0 0 6.9620
14 0 1 1 6.8425
15 0 −1 1 4.1450
16 1 0 1 6.1670
17 0 0 0 6.9390
Fig. 1. The standard curve of cyanidin-3-glucoside.
P. Liu, et al. LWT - Food Science and Technology 126 (2020) 109334
3
extraction yield of anthocyanins accomplished a highest at 30:1 (mL/g).
Lower liquid–solid ultrasonic energy density in the extraction solutions
showed predominance and a passive effect on the extraction yield. A
similar tendency for this parameter was also observed during poly-
saccharide extraction from Euryale ferox seed shells (Liu et al., 2013;
Wang et al., 2018b). Therefore, the aqueous ethanol to raw material
ratio of 30:1 (mL/g) is appropriate for anthocyanin extraction.
3.5. Optimization of the extraction conditions for anthocyanins
The experimental plans and data of 17 runs embracing five center
points per block was displayed in Table 2. On the base of multiple re-
gression analysis, the regression equation would be obtained:
Y = 7.00–0.24A-0.39B + 0.85C + 0.17AB+0.059AC+0.14BC-
0.049A2 -1.07B2-1.18C2 (2)
Using Design-Expert 8.0.6 software analyzed the data in Table 2,
and the ANOVA results are displayed in Table 3. The significance of
each parameter was also determined by adopting p-value and F-value.
Low p-value and high F-value indicated that the relevant factors are
greatly significant. If the p-value is below 0.01, then the model is sig-
nificant and can promote the extraction factors (Iyyappan et al., 2019).
The relationship among the above regression equation description and
the response surface value was significant (p = 0.0005 < 0.05), but
the lack of fit was not significant (p = 0.0595 > 0.05). R2 = 0.9589
indicated that the model was well fitted with the experimental data and
thus is a suitable mathematical design for the extraction yield of an-
thocyanins. Hence, regression equation can be adopted to determine
the optimal extraction of anthocyanins from L. ruthenicum Murr.
Two independent factors (B, C) and two quadratic terms (B2, C2)
influenced the extraction yield and were significant within 95% con-
fidence interval according to the following ANOVA: p-valueA = 0.0843,
p-valueB = 0.0149, and p-valueC = 0.002. The sequence of the influ-
ence of each element on anthocyanin yield is as follows: liquid–solid
ratio > extraction time > ethanol concentration. Other factors were
not significant probably because those that were selected were un-
suitable. The response surface analysis diagram visually shows the in-
teraction between various elements in the extraction process and ex-
amines the effect of the interaction of the other two elements on the
extraction rate when two factors are fixed at the central value (Liu
et al., 2013). The figure of 3D response surface and the figure of 2D
contour plots were plotted according to the regression equation. The
interaction of some factors is shown in Fig. 3. The 3D response surface
can visually reveal the influence of the interaction of all kinds of factors
on anthocyanin yield. A steep 3D response surface indicates the great
influence of this element on the extraction yield, and the interaction of
the two factors is highly significant. In the 2D contour, an oval contour
reveals that the interaction between the two factors is significant, and
the circular contours indicate that the interaction is insignificant.
Fig. 3e and f shows that the liquid–solid ratio exhibits the greatest in-
fluence on extraction yield, and extraction time is only within seconds.
The regression analysis results in Table 3 are also consistent with the
above phenomenon that the p-value of the liquid-solid ratio and ex-
traction time are both less than 0.05 (significant).
Under the selected optimum conditions, the model equation which
Fig. 2. Effect of different extraction variables on extraction yield: (a) ethanol
concentration; (b) extraction time; (c) liquid-solid ratio.
Table 3
Analysis of variance for fitted quadratic model of extraction of phenolic compounds.
Source Sum of squares df Mean square F-value p-value (Prob > F)
Model 19.01 9 2.12 18.15 0.0005 Significant
A 0.48 1 0.48 4.07 0.0834
B 1.20 1 1.20 10.31 0.0149
C 5.78 1 5.78 49.41 0.0002
AB 0.12 1 0.12 1.02 0.3453
AC 0.014 1 0.014 0.12 0.7396
BC 0.081 1 0.081 0.69 0.4340
A2 0.009988 1 0.009988 0.085 0.7785
B2 4.85 1 4.85 41.52 0.0004
C2 5.85 1 5.85 50.07 0.0002
Residual 0.82 7 0.12
Lack of fit 0.67 3 0.22 5.91 0.0595 Not significant
Pure error 0.15 4 0.038
Cor Total 19.92 16
R2 = 0.9589; R2adj = 0.9061; R2pred = 0.4519; C·V.% = 5.78; Adequate precisior = 13.852.
P. Liu, et al. LWT - Food Science and Technology 126 (2020) 109334
4
was suitable for forecasting the optimum response values was affirmed.
The most suitable experimental conditions are as below: ethanol con-
centration (A), 70.00%; extraction time (B), 27.60 min; and liquid-solid
ratio (C), 33.21:1 (mL/g), and the forecasted extraction yield was
7.39%. But taking into consideration the maneuverability in practical
manufacture, the suitable parameters were revised as below: ethanol
concentration (A), 70%; extraction time (B), 28 min; and liquid-solid
ratio (C), 33:1 (mL/g). The experimental extraction yield was
7.12 ± 0.093%, which agree with the forecasted extraction yield and
indicates that the model of RSM is content and precise.
3.6. Comparison of the anthocyanins content from L. ruthenicum Murr after
purification
The total anthocyanin extraction yield of the unpurified anthocya-
nins and purified anthocyanins can be calculated from Formula (1).
Cyanidin-3-glucoside (94% purity) was used as standard. According to
the total anthocyanin extraction yield and purify of standard, the purity
of the unpurified anthocyanins was 52.10%, and that of purified an-
thocyanins was increased to 77.62% after purification by AB-8 mac-
roporous resins.
Fig. 3. 3D response surface plots showing interactions between (a) ethanol concentration and extraction time, (c) ethanol concentration and liquid-solid ratio, (e)
extraction time and liquid-solid ratio, contour plots showing interactions between (b) ethanol concentration and extraction time, (d) ethanol concentration and
liquid-solid ratio, (f) extraction time and liquid-solid ratio.
P. Liu, et al. LWT - Food Science and Technology 126 (2020) 109334
5
3.7. Identification of anthocyanins from L. ruthenicum Murr
The chromatographic profile of anthocyanins from L. ruthenicum
Murr at 525 nm obtained using HPLC-DAD is displayed (Zheng et al.,
2011) in Fig. 4. In this HPLC-DAD analysis, seven peaks were
visible,
and the gradient procedure was used to obtain the maximal separation.
The individual anthocyanins were identified mostly by elution order,
retention time, and compared the data of MS spectra according to
previously reported (Wang et al., 2018a).
The chromatograph obtained using HPLC-MS2 was used to de-
termine the compositions of anthocyanins from L. ruthenicum Murr
(Grajeda-Iglesias, Salas, Barouh, Barea, & Figueroa-Espinoza, 2017).
The identification process was built by comparing the mass of mole-
cular ions and fragment ions with the results that had been reported
(Chen et al., 2018; Kim, Kim, Lee, Jang, & Kim, 2017; Sang et al., 2018;
Wang et al., 2018a; Wu & Ronald, 2005; Zheng et al., 2011) in litera-
ture. The HPLC-DAD chromatogram at 525 nm was displayed in Fig. 4,
and the data obtained using the molecular ion, fragment ion, and re-
tention time of the anthocyanin peaks in HPLC-MS2 analysis were
shown in Table 4.
The fragments determined using HPLC-MS2 revealed that only four
aglycones, including cyanidin (m/z 287), delphinidin (m/z 303), pet-
unidin (m/z 317), and malvidin (m/z 331) were found in L. ruthenicum
Murr. The structures of individual anthocyanins from L. ruthenicum
Murr were identified based on the mass fragmentation patterns of ru-
tinoside (m/z 308), glucoside (m/z 162), p-coumaric acid (m/z 164),
and caffeic acid (m/z 180). These anthocyanins possessed a basic
structure of 3-rutinoside or 3-O-rutinoside-5-O-glucoside. The specific
MS data of predicted anthocyanins are summarized in Table 4, and the
mass spectrums are shown in Fig. 5.
3.8. Protection effect of WPI on the thermal stability of anthocyanins from
L. ruthenicum Murr
Anthocyanin and protein interact through noncovalent bonds,
which mostly contain hydrogen bonds, hydrophobic interactions, and
van der Waals forces (Liu et al., 2019b). Therefore protein often be used
to load anthocyanins, and usually anthocyanins-protein synthesis ap-
plied to food industry. Thermal sterilization is a common treatment in
the food industry, and thermal stability of substance is important. Fig. 6
shows that the thermal stability of the unpurified anthocyanins is in-
ferior to that of the purified anthocyanins-WPI synthesis. The thermal
stability of the synthesis and anthocyanins greatly differs before heating
at 60 min. With prolonged heating time of 60–120 min, the thermal
stability increases, but the thermal stability curve is relatively flat. This
phenomenon indicates that the thermal stability of anthocyanins is
stable during this period. When heated for 120–300 min, the antho-
cyanins show a steep thermal stability curve, whereas that of the an-
thocyanin-WPI synthesis has improved.
The thermal stability curve of the unpurified anthocyanin-WPI
synthesis decreases sharply within 0–60 min, and the thermal stability
of the unpurified anthocyanins is slower than that of the unpurified
anthocyanins-WPI synthesis. The purified anthocyanins and antho-
cyanin-WPI synthesis exhibit a similar stationary phase at 60–120 min,
and the thermal stability of the unpurified anthocyanins is slower than
that of the unpurified anthocyanins-WPI synthesis at 120–300 min.
The results show that the thermal stability of the purified antho-
cyanins is higher than that of the unpurified anthocyanins; whereas the
thermal stability of the purified anthocyanin-WPI synthesis is greater
than that of the purified anthocyanins. These facts indicate that high-
purity anthocyanins can achieve a high degree of thermal stability, and
the stability of purified anthocyanins-WPI is improved. The anthocya-
nins-WPI synthesis and the improvement of their thermal stability are
consistent with the previous research (Qin et al., 2018). Therefore, the
present experiment can serve as a basis for the application for antho-
cyanin-protein synthesis in the food industry.
4. Conclusions
This article explores the L. ruthenicum Murr extract conditions for
anthocyanins optimized by RSM. Comparing the results from RSM and
single factor test can obtain an optimum extract condition. Purification
of anthocyanins using AB-8 resin produced a purified anthocyanin with
purity of 77.62%. Many kinds of anthocyanins are found in L. ruthe-
nicum Murr by HPLC. The seven possible structures can be speculated
through HPLC-MS2. The thermal stability indicated the high purity of
anthocyanins, and purified anthocyanins combined with WPI can en-
hance the thermal stability of anthocyanins.
CRediT authorship contribution statement
Pei Liu: Formal analysis, Writing - original draft. Wanrong Li:
Methodology, Validation. Zhongze Hu: Supervision. Xinguang Qin:
Conceptualization, Methodology, Validation. Gang Liu:
Conceptualization, Writing - review & editing.
Fig. 4. HPLC chromatograms at 525 nm of anthocyanins from L. ruthenicum
Murr.
Table 4
Anthocyanins identified in the extract of L. ruthenicum Murr.
Peak No. RT (min) M+ (m/z) Fragment (m/z) Anthocyanin
1 22.933 1095 933, 479, 317 Petunidin-3-O-(glucosyl-p-coumaroyl)-rutinoside-5-O-glucoside
2 23.383 949 787, 479, 317 Pentuidin-3-O-(caffeoyl)-rutinoside-5-O-glucoside
3 24.410 919 757, 465, 303 Delphinidin-3-O-(p-coumaroyl)-rutinoside-5-O-glucoside
4 25.128 933 771, 479, 317 Petunidin-3-O-(p-coumaroyl)- rutinoside-5-O-glucoside
5 26.158 947 785, 493, 331 Malvidin-3-O-(p-coumaroyl)-rutinoside-5-O-glucoside
6 26.675 595 287 Cyanidin-3-O-rutinoside
7 28.243 771 317 Petunidin-3-O-(p-coumaroyl)-rutinoside
P. Liu, et al. LWT - Food Science and Technology 126 (2020) 109334
6
Fig. 5. MS spectra of anthocyanins in L. ruthenicum Murr. The number of each spectrum corresponds to the peak number in Table 4 and Fig. 4.
P. Liu, et al. LWT - Food Science and Technology 126 (2020) 109334
7
Fig. 5. (continued)
P. Liu, et al. LWT - Food Science and Technology 126 (2020) 109334
8
Declaration of competing interest
The authors declare that there are no conflicts of interest.
Acknowledgment
This research received funding from the Chinese National Natural
Science Foundation of China[grant number 31771925]. Also, it was
supported by the Research and Innovation Initiatives of Wuhan
Polytechnic University [grant number 2020J01].
References
Chen, S., Zeng, Z., Hu, N., Bai, B., Wang, H., & Suo, Y. (2018). Simultaneous optimization
of the ultrasound-assisted extraction for phenolic compounds content and antioxidant
activity of Lycium ruthenicum Murr. fruit using response surface methodology. Food
Chemistry, 242, 1–8.
Grajeda-Iglesias, C., Salas, E., Barouh, N., Barea, B., & Figueroa-Espinoza, M. C. (2017).
Lipophilization and MS characterization of the main anthocyanins purified from hi-
biscus flowers. Food Chemistry, 230, 189–194.
Heinonen, J., Farahmandazad, H., Vuorinen, A., Kallio, H., Yang, B., & Sainio, T. (2016).
Extraction and purification of anthocyanins from purple-fleshed potato. Food and
Bioproducts Processing, 99, 136–146.
Iyyappan, J., Bharathiraja, B., Baskar, G., & Kamalanaban, E. (2019). Process optimiza-
tion and kinetic analysis of malic acid production from crude glycerol using
Aspergillus Niger. Bioresource Technology, 281, 18–25.
Jiang, T., Mao, Y., Sui, L., Yang, N., Li, S., Zhu, Z., et al. (2019). Degradation of antho-
cyanins and polymeric color formation during heat treatment of purple sweet potato
extract at different pH. Food Chemistry, 274, 460–470.
Keppler, K., & Humpf, H. U. (2005). Metabolism of anthocyanins and their phenolic
degradation products by the intestinal microflora. Bioorganic & Medicinal Chemistry,
13(17), 5195–5205.
Kim, H. W., Kim, S. R., Lee, Y. M., Jang, H. H., & Kim, J. B. (2017). Analysis of variation in
anthocyanin composition in Korean coloured potato cultivars by LC-DAD-ESI-MS and
PLS-DA. Potato Research, 61(1), 1–17.
Li, Z., Pan, Q. H., Cui, X. Y., & Duan, C. Q. (2010). Optimization on anthocyanins ex-
traction from wine grape skins using orthogonal test design. Food Sci. Biotechnol.
19(4), 1047–1053.
Liu, G., Li, W., Qin, X., & Zhong, Q. (2020). Pickering emulsions stabilized
by amphiphilic
anisotropic nanofibrils of glycated whey proteins. Food Hydrocolloids, 101, 105503.
Liu, Z. G., Liu, B. L., Kang, H. L., Yue, H. L., Chen, C., Jiang, L., et al. (2019a). Subcritical
fluid extraction of Lycium ruthenicum seeds oil and its antioxidant activity.
International Journal of Food Science and Technology, 54(1), 161–169.
Liu, G., Wang, Q., Hu, Z., Cai, J., & Qin, X. (2019b). Maillard-reacted whey protein iso-
lates and epigallocatechin gallate complex enhance the thermal stability of the
pickering emulsion delivery of curcumin. Journal of Agricultural and Food Chemistry,
67(18), 5212–5220.
Liu, Y., Wei, S. L., & Liao, M. C. (2013). Optimization of ultrasonic extraction of phenolic
compounds from Euryale ferox seed shells using response surface methodology.
Industrial Crops and Products, 49, 837–843.
Moldovan, B., David, L., Chisbora, C., & Cimpoiu, C. (2012). Degradation kinetics of
anthocyanins from European cranberrybush (Viburnum opulus L.) fruit extracts.
Effects of temperature, pH and storage solvent. Molecules, 17(10), 11655–11666.
Qin, X., Yuan, D., Wang, Q., Hu, Z., Wu, Y., Cai, J., et al. (2018). Maillard-reacted whey
protein isolates enhance thermal stability of anthocyanins over a wide pH range.
Journal of Agricultural and Food Chemistry, 66(36), 9556–9564.
Romero-Diez, R., Matos, M., Rodrigues, L., Bronze, M. R., Rodriguez-Rojo, S., Cocero, M.
J., et al. (2019). Microwave and ultrasound pre-treatments to enhance anthocyanins
extraction from different wine lees. Food Chemistry, 272, 258–266.
Sang, J., Li, B., Huang, Y. Y., Ma, Q., Liu, K., & Li, C. Q. (2018). Deep eutectic solvent-
based extraction coupled with green two-dimensional HPLC-DAD-ESI-MS/MS for the
determination of anthocyanins from Lycium ruthenicum Murr. fruit. Anal. Method,
10(10), 1247–1257.
Silva, S., Costa, E. M., Calhau, C., Morais, R. M., & Pintado, M. E. (2017). Anthocyanin
extraction from plant tissues: A review. Critical Reviews in Food Science and Nutrition,
57(14), 3072–3083.
Swer, T. L., & Chauhan, K. (2019). Stability studies of enzyme aided anthocyanin extracts
from Prunus nepalensis L. Lebensmittel-Wissenschaft und -Technologie- Food Science and
Technology, 102, 181–189.
Wang, Q., Li, W., Liu, P., Hu, Z., Qin, X., & Liu, G. (2019). A glycated whey protein isolate-
epigallocatechin gallate nanocomplex enhances the stability of emulsion delivery of
beta-carotene during simulated digestion. Food Funct. 10(10), 6829–6839.
Wang, Y. W., Luan, G. X., Zhou, W., Meng, J., Wang, H. L., Hu, N., et al. (2018a).
Subcritical water extraction, UPLC-Triple-TOF/MS analysis and antioxidant activity
of anthocyanins from Lycium ruthenicum Murr. Food Chemistry, 249, 119–126.
Wang, Q., Qin, X., Liang, Z., Li, S., Cai, J., Zhu, Z., et al. (2018b). HPLC–DAD–ESI–MS2
analysis of phytochemicals from Sichuan red orange peel using ultrasound-assisted
extraction. Food Bioscience, 25, 15–20.
Wu, X. L., & Ronald, L. P. (2005). Systematic identification and characterization of an-
thocyanins by HPLC-ESI-MS/MS in common foods in the United States: Fruits and
berries. Journal of Agricultural and Food Chemistry, 53(7), 2589–2599.
Wu, H. Y., Yang, K. M., & Chiang, P. Y. (2018). Roselle anthocyanins: Antioxidant
properties and stability to heat and pH. Molecules, 23(6), 1357.
Yang, Y., Yuan, X. H., Xu, Y. Q., & Yu, Z. Y. (2015). Purification of anthocyanins from
extracts of red raspberry using macroporous resin. International Journal of Food
Properties, 18(5), 1046–1058.
Zhao, Z., Wu, M., Zhan, Y., Zhan, K., Chang, X., Yang, H., et al. (2017). Characterization
and purification of anthocyanins from black peanut (Arachis hypogaea L.) skin by
combined column chromatography. Journal of Chromatography A, 1519, 74–82.
Zheng, J., Ding, C. X., Wang, L. S., Li, G. L., Shi, J. Y., Li, H., et al. (2011). Anthocyanins
composition and antioxidant activity of wild Lycium ruthenicum Murr. from Qinghai-
Tibet Plateau. Food Chemistry, 126(3), 859–865.
Zheng, X. Z., Zhang, Z. G., Jin, C. J., Mu, Y. Q., Liu, C. H., Chen, Z. Y., et al. (2015).
Purification characteristics and parameters optimization of anthocyanin extracted
from blueberry. International Journal of Agricultural and Biological Engineering, 8(2),
135–144.
Fig. 6. Thermal stability of anthocyanins and anthocyanins-WPI synthesis.
P. Liu, et al. LWT - Food Science and Technology 126 (2020) 109334
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	Isolation, purification, identification, and stability of anthocyanins from Lycium ruthenicum Murr
	Introduction
	Materials and methods
	Samples, chemicals, and standard
	Extraction of anthocyanins
	Measurement of anthocyanin yield
	Optimization using Box-Behnken design
	Purification procedure by AK-8 macroporous resins
	The analysis of anthocyanins by HPLC-DAD
	Identification of anthocyanins by HPLC-MS2
	Thermal stability studies
	Statistical analysis
	Results and discussion
	The standard curve of anthocyanins
	Influence of ethanol concentration on the extraction yield of anthocyanins
	Influence of extraction time on the extraction yield of anthocyanins
	Influence of the liquid-solid ratio on the extraction yield of anthocyanins
	Optimization of the extraction conditions for anthocyanins
	Comparison of the anthocyanins content from L. ruthenicum Murr after purification
	Identification of anthocyanins from L. ruthenicum Murr
	Protection effect of WPI on the thermal stability of anthocyanins from L. ruthenicum Murr
	Conclusions
	CRediT authorship contribution statement
	Declaration of competing interest
	Acknowledgment
	References