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Journal of Food Processing and Preservation ISSN 1745-4549

WATER EXTRACTION OF ANTHOCYANINS FROM BLACK RICE


AND PURIFICATION USING MEMBRANE SEPARATION AND
RESIN ADSORPTION
SHUDONG HE, QIUYAN LOU, JUAN SHI, HANJU SUN1, MANLI ZHANG and QIAN LI
College of Food Science and Engineering, Hefei University of Technology, Hefei, Anhui 230009, China

1
Corresponding author. ABSTRACT
TEL: 186-551-62901505;
FAX: 186-551-62901516; A simple and efficient method to extract and purify anthocyanins from black rice
EMAIL: sunhanjv@163.com was developed in this work. After pretreatment of black rice, anthocyanins were
extracted by buffer solutions, and were purified using membrane separation and
Shudong He and Qiuyan Lou are co-first resin adsorption. The single-factor experiments and orthogonal design were
authors.
applied. The results showed that extraction ratio could be 0.99 6 0.01% under the
Received for Publication January 8, 2016
optimal condition of liquid–solid ratio 12:1, temperature 50C, time 80 min and
Accepted for Publication April 29, 2016 pH 3.2. The 3 kDa molecular weight cut-off (MWCO) was used to remove
proteins and polysaccharides, and the DM301 resin was found to have both
doi:10.1111/jfpp.13091 good adsorptivity and desorptivity for anthocyanins. After the elution using 85%
(vol/vol) ethanol solution, the purified anthocyanins were obtained by retention
with the membrane of 200 Da MWCO. The recovery and purity of the anthocya-
nins finally obtained was 0.86% and 95.93%, respectively. The results are benefi-
cial to the large-scale extraction of anthocyanins from black rice.

PRACTICAL APPLICATIONS
Due to the growing demand for natural pigments, the natural anthocyanins have
drawn great attentions. However, the immaturity of the large-scale purification
procedures has limited the application of anthocyanins in functional food and
pharmaceutical industries. Since the black rice is the rich source of anthocyanins,
the large-scale water extraction and macroporous absorbent resins purification
parameters were systematically investigated in the current study. About 0.86 g
anthocyanins could be obtained from per 100 g black rice based on the large-scale
method, which has a good recovery, and the purity could reach 95.93%. Mean-
while, the whole procedure was green and non-pollution as the primary solvents
were water and ethanol. Furthermore, the results also would be conducive to the
processing and utilization of black rice.

INTRODUCTION Hence, natural pigments have attracted increasing attention


Because of bright colors, good stability and low cost, chemo- because of safety and non-toxicity.
synthetic pigments have been widely used in foods, medi- As one kind of water-soluble natural pigments, anthocya-
cines and cosmetics in order to diversify product colors for nins are the polyphenolic compounds. They are the most
decades. Nevertheless, most of them are harmful to human abundant flavonoid glycosides (Wu et al. 2015) and charac-
health and may cause asthma, rhinitis, diarrhea and even terized by a C6-C3-C6 skeleton (Patras et al. 2010). They
cancers (Deubert 1978; Mazza and Brouillard 1987; Giusti ubiquitously exist in many kinds of plants, such as sweet
and Wrolstad 1996). With the improvement of living stand- potato, red radish, red cabbage, grape, strawberry, haw-
ards and social progress, people are increasingly concerned thorn, red raspberry, black rice and black soybean (Teh and
about health and prefer natural food and consumer goods. Francis 1988; Murai and Wilkins 1990; Rommel et al. 1990;

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PURIFICATION OF ANTHOCYANINS FROM BLACK RICE S. HE ET AL.

TABLE 1. PHYSICAL PROPERTIES OF MACROPOROUS RESINS Black rice, which is a peculiar kind of rice and popular in
Average the Asian, African and American countries, has a long cultiva-
Surface Particle pore tion history in China. It is rich in proteins, starches, vitamins,
area diameter diameter fatty acids and mineral elements that are necessary to the
Name (m2/g) (mm) (nm) Polarity human body. Moreover, it also contains significant health-
D101 450–500 0.30–1.25 12–16 Non-polar promoting functional elements, such as anthocyanins, pheno-
AB-8 480–520 0.30–1.25 13–14 Weak-polar lic acids and flavonoids (Zhang et al. 2015). It has been con-
DM301 330–380 0.30–1.25 13–17 Moderate-polar
firmed that there were about 2% anthocyanins in black rice
which is higher than that of other plants (Ryu et al. 1998).
Di Carlo et al. 1999; McDougall et al. 2007), and the antho- However, so far, information about extraction and purifica-
cyanins are responsible for the vivid red, purple, blue and tion of anthocyanins from black rice is rare. The main aim of
violet colors of most plants (Jing et al. 2012). Meanwhile, the present work is to develop a simple and high-efficient
anthocyanins possess strong antioxidant activity, which is method to extract and purify anthocyanins from black rice on
much better than that of vitamins C and E (Motohashi and a large scale, which would be beneficial for the development
Sakagami 2008), and also have multifariously potential health of functional foods and bio-pharmaceutical industries.
benefits, such as lowering the incidence of coronary heart dis-
eases, age-related diseases and neurodegenerative diseases,
MATERIALS AND METHODS
improving visual acuity, dermal health and brain function,
and exhibiting anti-aging, anti-cancer, anti-microbial, anti-
Chemicals and Reagents
inflammatory and anti-diabetes activities (Kamei et al. 1995;
Wang et al. 1997; Nam et al. 2006; Stoia and Oancea 2012). Black rice (Oryza sativa L. indica) used as experimental mate-
Therefore, anthocyanins could be applied as natural antioxi- rial was cultured at the suburb of Hefei city, Anhui Province,
dants, coloring foodstuffs or natural colorants. Therefore, a China and harvested in September 2014. It contained water
lot of food sources, such as grape seeds and skins as well as (13.50 6 1.20%, wt/wt), protein (8.49 6 1.27%, wt/wt), total
the black carrots have been successfully explored to extract carbohydrate (74.41 6 2.37%, wt/wt), fat (2.86 6 0.25%,
anthocyanins (Kamiloglu et al. 2016). Anthocyanins are wt/wt) and ash (1.16 6 0.08%, wt/wt) determined according
unstable and easily oxidized or decomposed when exposed to to the AOAC official methods. Ethanol, disodium hydrogen
high temperature, oxygen, enzymes, light and metal ions phosphate, hydrochloric acid and citric acid were purchased
(Tiwari et al. 2009). Especially, the low contents in most from Shanghai Chemical Reagents Company (Shanghai,
plants make the anthocyanins, particularly of high purity, China). An anthocyanin standard (Cyanidin 3-glucoside,
scarce and expensive in market. purity > 97%) was purchased from Ronghe Medical Technol-
The new separation techniques, such as membranes and ogy Corp (Shanghai, China). Three types of macroporous
macroporous adsorption resins, have been applied to sepa- resins (AB-8, D101 and DM301) were purchased from Sam-
rate and purify anthocyanins (Woo et al. 1980; Lee et al. sung Resin Technology Co. (Anhui, China) and their physical
1982; Chung et al. 1986). Compared with other separation properties are presented in Table 1. Two membranes of 0.2
and concentration methods such as distillation and vacuum and 3 kDa MWCO were provided by Hefei Huakang Mem-
drying, membrane separation has advantages of simple brane Co. (Hefei, China) and their specifications were pre-
equipment, ambient operating temperature, high selectivity, sented in Table 2. Milli-Q water was used through the whole
low energy consumption and no phase transition or chemi- experiments. All reagents were of analytical grade.
cal changes. Thus, it has been widely used in food and phar-
macy industries (Teramoto et al. 2005). On the other hand,
macroporous adsorption resins are also an efficient method
Preparation of Black Rice
to concentrate and purify polyphenols by means of van der After manual removal of visible impurities, black rice was
Waals forces, with merits of simple process, low energy con- crushed with a high speed grinder (HX-200, Xian Suqing
sumption and easy regeneration (Buran et al. 2014). Since Co., China) at 2000 rpm for 5 min until the particle sizes
the sugars and salts mixed in polyphenols are not absorbed were smaller than 150 lm. The powder was collected for the
by the resins and could easily be eluted by water, the poly- subsequent experiments.
phenols absorbed by resins can be purified by the elution
using methanol or ethanol. Hence, macroporous adsorption
Extraction of Anthocyanins
resins have also been extensively applied in purification of
natural products on a large scale (Gao et al. 2007; Zhang Buffer solutions of pH values varying from 2.0 to 6.2 at 0.6
et al. 2008). intervals were prepared with 0.2 M disodium hydrogen

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S. HE ET AL. PURIFICATION OF ANTHOCYANINS FROM BLACK RICE

TABLE 2. SPECIFICATION OF MEMBRANES Purification of Anthocyanins


Membrane type Parameters
The crude extract of anthocyanin was filtered with 3 kDa
Membrane material Polyether sulphone membrane at room temperature under the operation pres-
Membrane configuration Roll film sure of 1 bar in order to hold back most of impurity proteins
Operation pressure (bar) 0–1
and polysaccharides. The anthocyanins were filtrated out,
Operating temperature (C) 0–40
Molecular weight cut-off (kDa) 3 and 0.2
and the feed tank was supplemented with deionized water
Effectives surface area (m2) 0.3 to the original volume till no anthocyanins were detected in
pH value range 2–11 the trapped fluid (Sato et al. 1996). Finally, the filtrate was
Resistance (Pa s/m) 5.40 3 1011 and 2.43 3 1011 collected for further tests.
for 3 and 0.2 kDa A glass column (20 mm 3 1000 mm) was prepared using
membranes, respectively.
pretreated resin with a resin bed volume of 300 mL. After
the concentrated filtrate (50 mL) was subjected, the column
was eluted with deionized water (500 mL) to remove impur-
phosphate and 0.1 M citric acid using a pH meter (pHS-2C, ities such as monoses and inorganic salts (Gilewicz-Łukasik
Precise Scientific Instrument Co., Shanghai, China). et al. 2007; Patil and Raghavarao 2007). Then, the anthocya-
According to the previous literatures (Deubert 1978; Ryu nins adsorbed by the resins were desorbed with 65–95%
et al. 1998), black rice powder (500 g) was finely ground in a (vol/vol) ethanol solutions (500 mL) at a flow rate of
paste mill (FDM-100, Nanfang Corp., Zhenjiang, China) 1.0 mL/min. The eluates were collected with an automatic
after mixing with 4–18 volumes of the buffer solutions. Sub- fraction collector and were further separated with the mem-
sequently, the mixing solutions held in dark bottles were brane of 200 Da MWCO at room temperature under opera-
incubated at temperatures from 20 to 70C at 10C intervals, tion pressure of 1 bar until most of ethanol (>95%) were
and the extraction time was in the range from 20 to 160 min filtered out. Finally, the retentate was freeze-dried by a vac-
at 20 min intervals. Then the crude extract of anthocyanins uum freeze drier (LGJ-12, Beijing Songyuanhuaxing Co.,
(supernatant) were obtained after the centrifugation (ST16R, China) until the water content was < 5%.
Thermo Fisher Scientific, Waltham, MA) at 4000 rpm (equiv- The pretreatments for macroporous adsorption resins
alent 3078 g) for 5 min. (AB-8, D101 and DM301) were carried out as previously
An orthogonal (L9(34)) test (Table 3) was deliberately described (Sun et al. 2009). Static adsorption experiments
employed to investigate the optimal extraction conditions, were adopted to screen optimal macroporous adsorption
according to a previous description (Sun et al. 2012a) with resins. Ten grams of pre-treated resins was mixed with
minor modifications. Liquid–solid ratio (A), temperature 50 mL of the crude extract in a conical flask, accompanied
(B), pH (C) and time (D) were selected as independent vari- by incubation at 25C with shaking at 2 s intervals for 24 h in
ables, and the ranges of the levels were based on the results a constant temperature shaker (HZQ-D, Jintan Xinnuo
of the experiments. Experimental Apparatus Co., China). Then, the saturated
The extraction ratio (E) of the anthocyanins was calcu- resins were filtrated with a filtering bag of 200 mesh before
lated according to Eq. (1). the resulting solutions were analyzed for the adsorptivity
and desorptivity of anthocyanins.
CV Equation 2 was used to quantify the adsorptivity of each
Eð%Þ5 3100 (1)
M type of resins.
where, C and V referred to the anthocyanin concentration C0 V0 2C1 V1
(mg/mL) and volume (mL) of the supernatant, respectively, Adsorptivityð%Þ5 3100 (2)
C0 V0
while M referred to the mass (mg) of black rice.
where, C0 and C1 referred to the anthocyanin concentrations
(mg/mL) of the crude extract and solutions after adsorp-
TABLE 3. FACTORS AND LEVELS FOR ORTHOGONAL
tion, respectively, while V0 and V1 referred to the volumes
TEST OF ANTHOCYANIN EXTRACTION
(mL) of the crude extract and solutions after adsorption.
Levels
The desorptivity was calculated according to Eq. (3).
Variables 1 2 3
C2 V2
(A) Liquid-solid ratio 12:1 14:1 16:1 Desorptivity ð%Þ5 3100 (3)
C0 V0 2C1 V1
(B) Extraction temperature (C) 40 50 60
(C) pH value 2.6 3.2 3.8
where, C2 and V2 referred to the anthocyanin concentration
(D) Extraction time (min) 60 80 100
(mg/mL) and volume (mL) of the eluent, respectively.

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PURIFICATION OF ANTHOCYANINS FROM BLACK RICE S. HE ET AL.

Anthocyanin Recovery and Anthocyanin sis of variance (ANOVA) was carried out to further evaluate
Purity the significant difference (P < 0.05) between treatments
using SAS 9.1.3 software (SAS Institute Inc., Cary, NC).
Anthocyanin recovery was defined as the ratio of the mass
(Mpowder) of the resulting anthocyanin powder to the mass
(M) of black rice and calculated according to Eq. (4). RESULTS AND DISCUSSION
Mpowder The Extraction of Anthocyanins
Recovery ð%Þ5 3100 (4)
M
The effect of liquid–solid ratio on the anthocyanin extrac-
where, Mpowder referred to the mass (mg) of the resulting tion is presented in Fig. 1a. As expected, it initially increased
anthocyanin powder obtained after the extraction and purifica- quickly with the increase of liquid–solid ratio and reached
tion procedures and M referred to the mass (mg) of black rice. an extremum (0.86%) at the liquid–solid ratio of 14:1. This
Anthocyanin purity was defined as the ratio of the mass might be due to the fact that the increasing liquid–solid
of the anthocyanins (Manthocyanins) in the power to the mass ratio favored the mass transfer and diffusion of the antho-
of power (Mpowder) and calculated according to Eq. (5). cyanins into the solvent. Subsequently, it decreased as liq-
uid–solid ratio extended from 14:1 to 18:1. The increase of
Manthocyanins liquid–solid ratio meant that there would be more oxygen in
Purity ð%Þ5 3100 (5)
Mpowder the solution, which could accelerate the anthocyanin oxida-
tion (Wang et al. 1997). Moreover, further increase of liq-
where, Manthocyanins referred to the mass (mg) of the antho-
uid–solid ratio was also accompanied by dissolution of
cyanins in the power determined by the UPLC analysis.
some impurities such as proteins and starches, which
impeded subsequent purification steps (Deubert 1978; Patil
Identification and Measurement of and Raghavarao 2007). Finally, the increase of liquid–solid
Anthocyanins ratio meant increases of water and labor consumption,
which would raise the operation cost. Comprehensively
Anthocyanins were measured in an Acquity UPLC H-class
considering the above factors, the optimal liquid–solid ratio
system (Waters Co., MA) equipped with a column of ACQ-
was 14:1.
UITY BEH Shield RP18 column (50 mm 3 2.1 mm ID, 1.7
Figure 1b shows the extraction ratio as a function of feed
lm, Waters Co., MA) and a UV–vis programmable detector.
pH ranging from 2.0 to 6.2. The maximum (0.91%) was
Since cyaniding 3-glucoside has been proven to be the major
achieved at pH 3.2, followed by a dramatic decrease as pH
anthocyanin (> 91%) present in black rice (Hou et al. 2013),
further increased. The anthocyanin molecules keep dynamic
a series of anthocyanin standard (Cyanidin 3-glucoside) solu-
equilibrium that exist as a number of different molecular
tions with different concentrations (50, 100, 250, 400, 500,
forms in acidic solutions (pH < 7.0), and its phenolic
600 and 750 lg/mL) were used to prepare a calibration curve
hydroxyl groups might be damaged at alkaline or highly
in order to evaluate the purity of the anthocyanin. One
acidic condition (pH < 2.0), while anthocyanins became
microliter of the solution was injected into the column, of
more and more stable as pH increased from 2.0 to 3.2,
which temperature was set at 30C, and the mobile phase was
resulting in a dramatic increase of extraction ratio (McGhie
the mixture of acetonitrile and water, as well as the acetoni-
and Walton 2007). Since anthocyanins stably existed in the
trile content linearly increased from 10 to 90% in 5 min at a
form of red flavylium cation, the highest extraction ratio
flow rate of 0.8 mL/min with the detection at 530 nm, while
(0.91%) was fulfilled at pH 3.2. However, due to nucleo-
the column decreased to initial condition in 0.5 min and was
equilibrated for 3 min prior to the next injection (Braun et al. philic reaction with hydroxyl group, a transformation from
2001; Sun et al. 2009; Ludwig et al. 2012). A linear regression red flavylium cation to colorless chalcone would occur at
equation of the concentrations of anthocyanin (Y, lg/mL) vs. pH > 3.2, leading to the remarkable drop of extraction ratio
response areas (X) was set up according to Eq. 6. (Figueiredo et al. 1996). Similarly, Coutinho et al. (2015)
reported that anthocyanins tended to fade in a short time

Y 5 6:32X11236:70 R 2 5 0:9968 (6) due to hydration competing reactions in mildly acidic aque-
ous condition. Hence, the optimal feed pH was set at 3.2.
In order to explore temperature on the anthocyanin
extraction, temperatures were adjusted in the range of 20–
Statistical Analysis
70C at 10C intervals, followed by incubation at liquid–solid
All experiments were performed and analyzed in triplicates, ratio of 14:1 and pH 3.2 for 100 min (Fig. 1c). Extraction
the data were expressed as mean 6 SD. And one-way analy- ratio increased slowly with the increasing temperatures in

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S. HE ET AL. PURIFICATION OF ANTHOCYANINS FROM BLACK RICE

FIG. 1. EFFECT OF
EXTRACTION CONDITIONS ON
ANTHOCYANIN EXTRACTION
(a) Liquid–solid ratio, (b) feed
pH, (c) extraction temperature
and (d) extraction time.

the range of 20–50C, and reached a maximum (0.82%) at gated structure, which belongs to the conjugated aromatic
50C, followed by a tremendous decrease from 50 to 70C. structure, and has high biological activity. In the molecule
Temperature played a significant role on the anthocyanin of anthocyanin, there are several hydroxyl groups on A, B
extraction that it provided energy for anthocyanin molecule and C rings of the flavonoid skeleton. And the activity of the
motions at first (Sun et al. 2012b), which facilitated the hydrogen atoms is strengthened significantly after the con-
anthocyanin dissolution and improved extraction efficiency. jugated system has formed. However, once an anthocyanin
And high temperatures promoted proteins and nucleic acids contacted with oxygen for a long time, its phenolic hydroxyl
to be easily denatured and destroyed. In this case, the per- groups would be partly oxidized (Coutinho et al. 2015).
meability of the cytomembranes was improved so that Therefore, the optimal time was 80 min.
anthocyanins were easily transferred into the solvent. How- On the basis of the single-factor experiments, the extrac-
ever, extremely high temperatures would accelerate antho- tion conditions were optimized using an orthogonal test
cyanins to be oxidized (Figueiredo et al. 1996). The results (L9(34)). According to Table 4, the maximal extraction ratio
suggested that the anthocyanins were gradually oxidized as was 0.99% using the combination condition of A1B2C2D2.
temperatures exceeded 50C. Patras et al. (2009) reported Based on the analysis of extreme deviation, the relationship
that the level pelargonidin-3-glucoside (a kind of anthocya- between the primary and secondary factors was determined
nin) in strawberry purees were reduced significantly because in the order of B > C > D > A by the comparison of the R
of thermal degradation after subjected to conventional ther- values, and the appropriate combination condition might
mal treatment (70C, 2 min). More importantly, high tem- be A2B2C2D3 through the calculation analysis. Since the
perature also increased energy dissipation. Based on the combination condition of A2B2C2D3 was not in the list of
results above, it was reasonable to choose 50C as the optimal orthogonal test, the validation tests were then carried out,
extraction temperature. and the extraction ratio was found to be 0.96 6 0.02%.
The plot of extraction time vs. extraction ratio is shown Because of the longer extraction time, the extraction ratio of
in Fig. 1d. Clearly, the extraction ratio was not only time- A2B2C2D3 was lower than the maximum listed in Table 4;
dependent, but also nearly increased in first-order linear in therefore, the combination condition of A1B2C2D2 was
the range of 20–80 min. The inflection point occurred at 80 employed in the study. Then, the optimal condition should
min, and the extraction ratio afterward decreased sharply. be liquid–solid ratio 12:1, temperature 50C, time 80 min
Generally, an anthocyanin molecule keeps a tricyclic conju- and pH 3.2.

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PURIFICATION OF ANTHOCYANINS FROM BLACK RICE S. HE ET AL.

TABLE 4. ANALYSIS OF ORTHOGONAL EXPERIMENT RESULTS In terms of polarity, macroporous adsorption resins are
OF ANTHOCYANIN EXTRACTION generally divided into non-polarity, weakly polarity, moder-
A: Liquid– C: ately polarity, polarity and strongly polarity. As showed in
solid ratio Temperature D: Time Extraction Table 1, D101 resin is non-polar, AB-8 is weakly polar and
Number (mL/g) B: pH (C) (min) ratio (%) DM301 is moderately polar. Generally, substances of similar
1 1(12:1) 1(2.6) 1(40) 1(60) 0.76 6 0.04 polarity are easily miscible. Polar resins preferably absorb
2 1 2(3.2) 2(50) 2(80) 0.99 6 0.01 polar molecules, moderate-polar resins moderately absorb
3 1 3(3.8) 3(60) 3(100) 0.92 6 0.03 both non-polar and polar molecules and non-polar resins
4 2(14:1) 2 1 3 0.94 6 0.04
preferably absorb non-polar molecules. Since anthocyanin
5 2 3 2 1 0.90 6 0.02
6 2 1 3 2 0.92 6 0.03
molecules are of moderate polarity, as they have both polar
7 3(16:1) 3 1 2 0.78 6 0.03 and non-polar groups (McGhie and Walton 2007; Buran
8 3 1 2 3 0.91 6 0.02 et al. 2014), DM301 of moderate polarity was superior to
9 3 2 3 1 0.93 6 0.04 the other resins that could strongly adsorb anthocyanins.
k1 0.89 0.83 0.86 0.86
k2 0.92 0.93 0.95 0.89
k3 0.87 0.92 0.87 0.92 Effect of Ethanol Concentrations
Range 0.05 0.10 0.09 0.06 on Dynamic Recovery and Purity
of Anthocyanins
In order to desorb the anthocyanins from DM301, ethanol sol-
Effect of Resin Types on Static Adsorptivity utions of different concentrations (65–95%) were employed as
and Desorptivity of Anthocyanins eluents, and four kinds of anthocyanins were identified based
on the UPLC spectra according to the previous studies (Hou
The effect of resin types on the adsorptivity and desorptivity
et al. 2013), while the relative contents of cyanidin 3,5-digluco-
of the anthocyanins are presented in Table 5. Apparently,
side, cyanidin 3-glucoside, cyanidin 3-rutinoside and peonidin
D101 had the lowest adsorptivity (82.63%) and desorptivity
3-glucoside were 1.02%, 95.37%, 2.98% and 0.63%, respec-
(76.17%), while DM301 had the highest adsorptivity
tively. Then the effects of ethanol concentrations on the
(96.18%) and desorptivity (90.06%). Two effects (adsorp-
dynamic recovery and purity of the anthocyanins were ana-
tion and desorption) are involved when a macroporous
lyzed by the characterization of cyaniding-3-diglucoside. As
adsorption resin is chosen to separate and purify substances.
presented in Table 6, in general, different ethanol concentra-
Theoretically, macroporous adsorption resins are non-ionic
tions had distinct effects on the anthocyanin elution, and it
polymer adsorbents, which have mesh-hole structures
revealed that both the recovery and purity were quickly
formed by polymerization of divinylbenzene, styrene, meth-
improved with increasing ethanol concentrations from 65 to
acrylate, etc. They could adsorb a vast variety of compounds
85% (vol/vol). It was found that the recovery of anthocyanin
from solutions by means of van der Waals and hydrogen
was 0.86% at an ethanol concentration of 85% (vol/vol), indi-
bond forces, and screen compounds of different molecular
cated the anthocyanin was well recycled, while the purity of
sizes through their porous-mesh structures. On the con-
anthocyanin also arrived at maximum (95.93%). As the etha-
trary, these molecules adsorbed by the resins could be fur-
nol concentrations further increased, both the recovery and
ther eluted with ethanol, methanol and saline solutions. As
purity decreased slightly. Comprehensively considering cost
the binding forces combining the adsorbed molecules with
and efficiency, 85% (vol/vol) ethanol solution was the optimal
eluents are greater than those between the molecules and
eluent for elution of the anthocyanins.
resins, the molecules adsorbed by the resins would inevita-
bly be selectively eluted (Gao et al. 2007; Sun et al. 2011,
TABLE 6. EFFECT OF ETHANOL CONCENTRATION ON DYNAMIC
2012b). RECOVERY AND PURITY OF ANTHOCYANINS
Ethanol
concentration Recovery Purity
TABLE 5. STATIC ADSORPTIVE CAPABILITY OF THREE RESINS
(%, vol/vol) (%) (%)
Resin type Adsorptivity (%) Desorptivity (%)
65.0 0.65 6 0.01d 86.66 6 0.23c
D101 82.63 6 0.12c 76.17 6 0.32c 75.0 0.77 6 0.04c 92.41 6 0.19b
AB-8 88.36 6 0.28b 82.73 6 0.24b 85.0 0.86 6 0.01a 95.93 6 0.14a
DM301 96.18 6 0.17a 90.06 6 0.13a 95.0 0.83 6 0.02b 95.50 6 0.15a

The data (mean 6 standard deviation) related to three replicates. The data (mean 6 standard deviation) related to three replicates.
The difference between the means with the same superscript letter in The difference between the means with the same superscript letter in
the column was significant at 95% confidence interval. the column was significant at 95% confidence interval.

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S. HE ET AL. PURIFICATION OF ANTHOCYANINS FROM BLACK RICE

CONCLUSIONS GAO, M., HUANG, W. and LIU, C.Z. 2007. Separation of


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to obtain anthocyanins of high purity and recovery from L.L. 2012. Anthocyanin and glucosinolate occurrences in the
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