One-pot β-cyclodextrin-assisted extraction of active ingredients from Xue-Zhi-Ning basing its encapsulated ability

Article abstract of DOI:10.1016/j.carbpol.2015.06.072

Abstract Xue-Zhi-Ning (XZN) is a traditional Chinese medicine formula, containing active ingredients with poor solubility in water, which has been demonstrated to be helpful for patients with hyperlipidemia. One-pot β-cyclodextrin (β-CD)-assisted extracti

Full text of DOI:10.1016/j.carbpol.2015.06.072

Carbohydrate Polymers 132 (2015) 437–443
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
One-pot ␤-cyclodextrin-assisted extraction of active ingredients from
Xue–Zhi–Ning basing its encapsulated ability
Hui-Jie Zhang, Ya-Nan Liu, Meng Wang, Yue-Fei Wang, Yan-Ru Deng, Ming-Lei Cui,
∗
Xiao-Liang Ren , Ai-Di Qi
Tianjin University of Traditional Chinese Medicine, Tianjin 300193, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 April 2015
Received in revised form 10 June 2015
Accepted 15 June 2015
Available online 25 June 2015
Xue–Zhi–Ning (XZN) is a traditional Chinese medicine formula, containing active ingredients with poor
solubility in water, which has been demonstrated to be helpful for patients with hyperlipidemia. One-pot
␤
-cyclodextrin (␤-CD)-assisted extraction of active ingredients from XZN has been carried out to develop
an efficient and eco-friendly extraction process. Five active compounds—rubrofusarin gentiobioside,
ꢀ
2
,3,5,4 -tetrahydroxy-stilbene-2-O-␤-D-glucoside, emodin, nuciferine and quercetin—were identified
by UPLC/DAD/MS and used as indexes to evaluate the process optimized by an orthogonal test. The
results showed that addition of ␤-CD significantly enhanced the extraction ratios of all five components.
The enhancement of extraction ratios was positively correlated with the apparent formation constants
between ␤-CD and the compounds. The study also showed that the stabilities and dissolution rates of
the active ingredients were improved in the presence of ␤-CD. This one-pot ␤-cyclodextrin-assisted
extraction has the potential to be applied in pharmaceutical preparations directly.
Chemical compounds studied in this article:
Rubrofusarin gentiobioside (PubChem CID:
5
2
03733)
ꢀ
,3,5,4 -tetrahydroxy-stilbene-2-O-␤-d-
glucoside (PubChem CID:
1632914)
9
Emodin (PubChem CID: 3220)
Nuciferine (PubChem CID: 10146)
Quercetin (PubChem CID: 5280343)
© 2015 Elsevier Ltd. All rights reserved.
Keywords:
␤
-Cyclodextrin
Xue–Zhi–Ning
Extraction process
Apparent formation constant
Stability
Dissolution rate
1
. Introduction
Traditional Chinese medicine (TCM) is famous for satisfactory
liquids, granules or capsules, prepared using modern technology
after organic solvent extraction. Xue–Zhi–Ning (XZN) is a TCM for-
mula recorded in the Pharmacopeia of the People’s Republic of
China (China, 2010a). It is composed of four food homology Chinese
herbs, Semen Cassiae, Radix Polygoni Multiflori Preparata, Folium
Nelumbinis and Fructus Crataegi, at a ratio of 3:2:1.5:1. The com-
bination of multiple herbs in XZN has been clinically applied in the
treatment of hyperlipidemia (Chen et al., 2011; Du et al., 2010; Ko
et al., 2011; Wang, Zhao, Wang, Mao, & Yu, 2012; Xie, Zhao, & Du,
2012).
treatment with few side effects. The most widely used forms of
TCM are those involving multiple herbs combined under the guid-
ance of TCM theories, known as formulae. It is widely accepted
that the therapeutic effects of TCM are based on the synergistic
effects of multiple herbs, each of which contains active chemical
constituents affecting multiple targets (Jiang, 2005; Normile, 2003).
Traditionally, patients drink an aqueous solution of decocted herbs.
Modern forms of TCM have been developed including pills, oral
Naturally occurring cyclodextrins (CDs) are cyclic oligosaccha-
rides containing six, seven, or eight d-(+)-glucopyranose units
(
(
␣-, ␤-, and ␥-cyclodextrin) connected by ␣-1,4 glycosidic bonds
Harada, 1997; Liu, Han, Qi, & Chen, 1997; Tafazzoli & Ghiasi, 2009).
Abbreviations: ␤-CD, ␤-cyclodextrin; TCM, traditional Chinese medicine; XZN,
Xue–Zhi–Ning; RG, rubrofusarin gentiobioside; TSG, 2,3,5,4 -tetrahydroxy-stilbene-
-O-␤-d-glucoside; OAD, orthogonal array design; AHP, analytic hierarchy process.
∗ Corresponding author. Tel.: +86 22 59596221; fax: +86 22 59596221.
E-mail address: xiaoliang ren@sina.com (X.-L. Ren).
CDs have a hydrophilic exterior and an internal hydrophobic cavity
and can act as host molecules that form inclusion complexes with
lipophilic guest molecules (Irie & Uekama, 1997). ␤-Cyclodextrin
(␤-CD) has been used to form inclusion complexes with
ꢀ
2
http://dx.doi.org/10.1016/j.carbpol.2015.06.072
144-8617/© 2015 Elsevier Ltd. All rights reserved.
0

4
38
H.-J. Zhang et al. / Carbohydrate Polymers 132 (2015) 437–443
Fig. 1. (A) Structures of ␤-CD, RG, TSG, emodin, nuciferine and quercetin; (B) schematic of ␤-CD-assisted extraction.
bioactive compounds, which can improve the solubility, stability
and bioavailability of the guest molecule. As a consequence, the
use of ␤-CD in the food and pharmaceutical industries is increas-
ingly common (Pinho, Grootveld, Soares, & Henriques, 2014; Szente
optimized using an orthogonal experiment design. The apparent
formation constants and stoichiometries of inclusion complexes
formed between ␤-CD and each active ingredient have been deter-
mined, in addition to the stability and dissolution rate of extract
in vitro (Fig. 1).
&
Szejtli, 2004). Recently, studies have demonstrated that herbal
extracts containing multiple ingredients can be complexes with
CDs to enhance their solubility and bioactivity (Hsu, Yu, Tsai, & Tsai,
2
013). Of particular interest, other scholars studies have reported
2. Experimental
that ␤-CD aqueous solution can be used to extract polyphenols from
grape pomace, apple pomace or Polygonum cuspidatum (Mantegna
et al., 2012; Parmar, Sharma, & Rupasinghe, 2014; Ratnasooriya &
Rupasinghe, 2012). Instead of organic reagents, this method is a
way to use water as a solvent in the process of extraction, which is
safer and more environmentally friendly.
2.1. Materials
␤-CD was provided by Huaxing Biological Chemical Co.,
Ltd. (Mengzhou, China). Deionized water was supplied by
Hangzhou WAHAHA Group Co., Ltd. (Hangzhou, China). 2,3,5,4 -
ꢀ
To the best of our knowledge, ␤-CD has never been applied to
the extraction of formulae. In this study, the encapsulation abil-
ity of ␤-CD has been used to assist the extraction of bioactive
ingredients. Our previous research showed that the hypolipi-
demic effect of a ␤-CD extract in rats was better than that of
an aqueous extract (Yang et al., 2015). In this work, the sys-
tem of ultra performance liquid chromatography coupled with
diode-array detector-tandem mass spectrometry (UPLC/DAD/MS)
has been used to identify the active ingredients of XZN and a
UPLC–DAD analysis method was established for the determina-
tion of active ingredients. The process of ␤-CD extraction was
Tetrahydroxy-stilbene-2-O-␤-d-glucoside (TSG), emodin, nucifer-
ine and quercetin reference substances were purchased from the
National Institutes for Food and Drug Control (Beijing, China).
Rubrofusarin gentiobioside (RG) was purchased from Tuohai Bio-
logical Technology Co., Ltd. (Nanjing, China). Methanol (HPLC
grade) and formic acid (HPLC grade) were purchased from
Sigma–Aldrich (USA). Dried Semen Cassiae, Radix Polygoni Multiflori
Preparata, Folium Nelumbinis and Fructus Crataegi were purchased
from a Chinese medicine store (Chang’an, Anguo) and authen-
ticated by pharmacognosists at Tianjin University of Traditional
Chinese Medicine.

H.-J. Zhang et al. / Carbohydrate Polymers 132 (2015) 437–443
439
2.2. Materials and sample preparation
Extraction ratios were calculated using Eq. (2):
cv
R =
%
(2)
For the calibration curves of RG, TSG, emodin, nuciferine and
mw
quercetin, standard solutions at different concentrations were pre-
pared in methanol and a 3 ␮L sample of each was injected for
UPLC/DAD analysis. The linear ranges were: 0.21–13.40 ␮g/mL
for RG, 1.59–101.50 ␮g/mL for TSG, 0.04–4.92 ␮g/mL for emodin,
where R is the extraction ratio of the component; c (mg/mL) is the
concentration of the component determined by UPLC–DAD analysis
in the orthogonal tests; v (mL) is the volume of the sample in the
orthogonal test; m (g) is the mass of the medicinal materials that
the component is obtained from; w (mg/g) is the content of the
component in the medicinal material it is obtained from, measured
and calculated according to the China Pharmacopeia (China, 2010b)
and related reports (Dong, Zhang, & Li, 2010; Sun et al., 2009). The
process derived from the orthogonal experiment was compared
with the traditional extraction method that uses distilled water as
the extraction solvent.
0.07–4.20 ␮g/mL for nuciferine and 0.10–3.14 ␮g/mL for quercetin.
A 1 mL ␤-CD extract or aqueous extract at room temperature
was diluted with methanol in a 10 mL volumetric flask (final vol-
ume 10 mL) and the solution subjected to ultrasound irradiation
for 30 min to precipitate ␤-CD. A 2 mL sample was filtered using
0.22 ␮m nylon filters and 3 ␮L aliquots of the filtrate were used for
UPLC analysis.
2.3. UPLC/DAD/MS analysis
2.5. Apparent formation constants and stoichiometries of ˇ-CD
for each active ingredient
Chromatographic analysis was performed on a Waters ACQUITY
TM
UPLC
system (Waters Co., USA) equipped with binary sol-
2.5.1. UV–vis spectrophotometry
vent manager, sample manager, column oven and diode array
detector (DAD). The software used was Mass Lynx V4.1. The col-
UV–vis spectrophotometry was used to determine the stoichio-
metric ratios and binding constants of inclusion complexes formed
between ␤-CD and all five ingredients. Absorption spectra were
recorded on a TU-1901 spectrophotometer (Beijing Purkinje Gen-
eral Instrument Co., Ltd). A fixed concentration of guest was added
into a series of aqueous solutions containing increasing amounts
umn used for analyses was an ACQUITY UPLC BEH shield RP18
◦
(
2.1 mm × 100 mm, 1.7 ␮m; Waters Co., USA) at 45 C. For the
mobile phase, 0.1% aqueous formic acid (A) and methanol (B)
were used. The gradient program was 10–25% B from 0 to 2 min,
2
4
5–35% B from 2 to 9 min, 35–40% B from 9 to 12 min, and
0–90% B from 12 to 40 min. The flow rate was 2 ␮L/min. Detection
wavelengths were changed at different time intervals as follows:
.00–4.00 min, 270 nm; 8.01–10.50 min, 320 nm; 10.51–19.00 min,
◦
of ␤-CD. The mixtures were treated with ultrasound at 25 C and
protected from light. The absorbance of each solution was mea-
sured at the maximum absorption wavelength of the guest against
a reagent blank that was prepared using an identical reagent con-
centration but without guest. The wavelengths used for UV–visible
analysis were: 277 nm for RG, 319 nm for TSG, 296 nm for emodin,
0
2
77 nm; 19.01–30.00 min, 360 nm; 30.01–40.00 min, 254 nm.
TM
Waters LC-MS/MS (Waters ACQUITY UPLC
system tandem
Waters Quattro Premier XE MS, software version: Mass Lynx V4.1)
was used for the qualitative analysis. The conditions were the same
as those used by Zhang et al. (2012).
2
70 nm for nuciferine and 254 nm for quercetin. The stoichiometric
ratios and the apparent formation constants (K) for the complexes
were calculated using the Benesi–Hildebrand method (Benesi &
Hildebrand, 1949). If the stoichiometric ratio of the inclusion com-
plex between host and guest is 1:1, a good linear relationship will
be obtained between 1/ꢀA and 1/[CD]0 according to the following
equation:
2
.4. Optimization of extraction by orthogonal array design (OAD)
OAD is a systematic method for optimizing experimental
parameters (Zhang et al., 2013; Ai et al., 2013). In our work, an
1
1
1
4
L9(3) orthogonal matrix was applied to optimize four factors: the
=
+
ꢀ␧[G] K[CD]0
0
(3)
ꢀA
ꢀ␧[G]0
ratio of ␤-CD to XZN (%), time (h), number of extractions and the
ratio of solid to liquid. Each of these factors was varied at three
levels: ␤-CD to XZN ratios of 5%, 10% and 15%; times of 1, 1.5,
and 2 h; number of extractions of 1, 2 and 3; solid/liquid ratios
of 1/15, 1/20 and 1/25. All nine experimental runs were carried
out in round-bottomed flasks, and the reflux extraction was con-
ducted after XZN had soaked for 1 h. If the extraction was processed
more than once, the aqueous extracts were combined and filtered.
Filtrates were prepared as described in Section 2.2 and analyzed
using UPLC–DAD. Using the extraction ratios of RG, TSG, emodin,
nuciferine and quercetin as indexes, the weighting coefficients of
these components were determined by an analytic hierarchy pro-
cess (AHP) (Ren, Lu, Tian, & He, 2008; Xie et al., 2014). For evaluation
of the OAD, the following weighting coefficients of the compo-
nents were used: RG 0.3544, TSG 0.3544, emodin 0.1441, nuciferine
where [G]0 is the initial concentration of the guest, [CD]0 is the
initial concentration of ␤-CD, ꢀA is the change in the absorbance
of the guest after addition of ␤-CD, ꢀ␧ is the difference in the molar
absorptivities of the complex and free guest, and K is the binding
constant, calculated from the slope of the equation.
2.5.2. Phase solubility analysis
Phase solubility studies were conducted in distilled water at
◦
2
5 C based on the method reported by Higuchi and Connors (1965).
An excess of emodin or quercetin was added to 10 mL of distilled
water containing increasing concentrations of ␤-CD. The suspen-
sions were equilibrated in a thermostatic shaking water bath for
◦
2
4 h at 25 C protected from light. After equilibrium was achieved,
the supernatants were filtered through 0.45 ␮m nylon filters. The
concentration of dissolved emodin or quercetin was assayed using
HPLC (Waters, USA). Phase solubility diagrams were plotted using
CD concentration as the X-axis and guest concentration as the Y-
axis. Apparent formation constants were calculated from the phase
solubility diagram using the Higuchi–Connors equation:
0.0898 and quercetin 0.0574. Composite scores were calculated
using Eq. (1):
ꢀ
ꢁ
Ai
B
C
D
E
i
i
i
i
Y= 0.3544 + 0.3544 B + 0.1441 C + 0.0898 D + 0.0574 E
%
A
(
1)
slope
s0(1 − slope)
K =
(4)
where Y is the composite score; A , B , C , D , and E are the extraction
i
i
i
i
i
ratios of RG, TSG, emodin, nuciferine and quercetin, respectively; A,
B, C, D, and E are the maximum extraction ratios of RG, TSG, emodin,
nuciferine and quercetin, respectively.
where K is the apparent formation constant and s0 is the solubility
of guest in the absence of ␤-CD.

4
40
H.-J. Zhang et al. / Carbohydrate Polymers 132 (2015) 437–443
Table 1
2
.6. In vitro stability of the active ingredients in ˇ-CD and
Factors and levels for the orthogonal test.
aqueous extracts
Variable
Levels
1
XZN was extracted with water or ␤-CD solution using the pro-
cess established in the orthogonal test (ratio of ␤-CD to XZN,
extraction time, number of extractions and ratio of solid/liquid).
The aqueous extracts were concentrated by rotary evaporation and
dried under vacuum. Equal masses of each dried, powdered sam-
ple were added to distilled water in 10 mL brown volumetric flasks
2
3
(
(
(
A) Ratio of ␤-CD to XZN (%)
B) Extraction time (h)
C) Number of extractions (n)
5
1
1
1/15
10
1.5
2
15
2
3
(D) Ratio of solid to liquid (n)
1/20
1/25
(
9
final volume of 10 mL), then placed in water baths at 25, 37, and
0 C. Separately, equal masses of each dried, powdered sample
◦
3.2. Optimization of the extraction process by OAD
were added to buffer solution at varying pH values (1.2, 6.8, and
.3) in 10 mL brown volumetric flasks (final volume of 10 mL), then
8
Various parameters play a significant role in the development
of a solvent extraction method. In this work, single factor experi-
ments were conducted to establish the factors and their levels for
OAD. The ratio of solid to liquid, extraction method, extraction time
(h), number of extractions and the ratio of ␤-CD to XZN (%) were
studied in single factor experiments. The results indicated that a
reflux process should be used, and four factors needed to be opti-
mized in the orthogonal test as described in Section 2.4. Details
of the single factor experiments are presented in the supplemen-
tary materials (Liang, 2008). The investigated levels of independent
◦
placed in a water bath at 25 C. Finally, powdered extracts were
added to distilled water in transparent volumetric flasks (final vol-
ume of 10 mL) and stored at room temperature under natural light.
At predetermined time intervals, aliquots (1 mL) were processed as
described in Section 2.2 for UPLC determination of RG, TSG, emodin,
nuciferine and quercetin (Ren, Wang, Wang, Ou-Yang, & Qi, 2011).
2.7. In vitrodissolution rates
variables are listed in Table 1. All selected factors were explored
4
using an orthogonal L (3) test design. The total evaluation index
Dissolution rate studies were carried out using the disso-
9
was used for statistical analysis and the results of the orthogonal
test and extreme difference analysis are presented in Table 2. The
extraction ratios of the five active ingredients were predetermined
and the composite score for each sample was calculated accord-
ing to the methods discussed in Section 2.4. The results indicated a
maximum score of 97.34 and suggested that a further orthogonal
analysis was warranted. Statistical software was employed to cal-
culate the values of K and R as shown in Table 2. As can be seen
from the results, the influence of factors on the composite scores
decreased in the order C > B > D > A, according to the R values. The
maximum score was obtained when the ratio of ␤-CD to XZN (%),
time (h), number of extractions and ratio of solid to liquid were
lution test method II described in the China Pharmacopoeia
(
China, 2010c). Dissolution flasks were immersed in a water bath
◦
at 37.0 ± 0.5 C. Phosphate buffer (pH 6.8, 900 mL) or aqueous
hydrochloric acid (pH 1.2, 900 mL) were used as the dissolution
media and the stirring rate was set at 100 rpm. At the beginning
of the tests, extracts were added to the media. Samples (5 mL)
were withdrawn and replaced with 5 mL fresh medium after 5, 10,
2
0, 40, 60 and 90 min. The dissolution samples were processed as
described in Section 2.2 then analyzed using UPLC–DAD. The dis-
solution rate was determined by plotting the cumulative amount
of dissolved solute against time. Rates were calculated according to
Eq. (5).
1
5%, 2 h, 3 and 1/25, respectively. According to the R value, time
ꢂ
(h), number of extractions and ratio of solid to liquid were more
important than the ratio of ␤-CD to XZN (%). To minimize cost of
production and avoid excess extraction agent, the optimum pro-
cess was selected as follows: ␤-CD to XZN (%), time (h), number
of extractions and ratio of solid to liquid of 5%, 2 h, 3 and 1/25,
respectively.
A confirmatory experiment was conducted to verify the pro-
cess suggested by the orthogonal experiment and demonstrate
whether the addition of ␤-CD could increase the extraction ratio
of active ingredients or not. Time (h), number of extractions and
ratio of solid to liquid were set at 5%, 2 h, 3 and 1/25, respec-
tively, and run in the presence or absence of ␤-CD. ␤-CD-assisted
and aqueous extractions were both performed in triplicate and the
n−1
CnVn + Vn
Cn
i=1
D =
×100%
(5)
M
where D is the dissolution rate, Cn is the concentration of ingre-
dients at each time interval, Vn is the sample volume at each time
interval, and M is the content of the active ingredient in the powder
extract determined by UPLC/DAD.
3
. Results and discussion
3
.1. UPLC/DAD/MS analysis
In recent years, the major constituents in each herb of XZN have
Table 2
been studied. The results suggest that RG, TSG, emodin, nuciferine
and quercetin, as analyzed in this work, are the active components
of XZN (Jung et al., 2010; Lee, Jang, Lee, Kim, & Kim, 2006; Lin, Kuo,
Lin, & Chiang, 2009; Lin et al., 2015; Wang et al., 2012, 2014).
The UPLC/MS analyses of aqueous and ␤-CD extracts are
shown in Fig. S1, in which nuciferine (m/z = 296, reten-
tion time = 5.86 min), TSG (m/z = 407, retention time = 10.49 min),
RG (m/z = 597, retention time = 12.63 min), quercetin (m/z = 303,
retention time = 21.27 min) and emodin (m/z = 271, retention
time = 33.71 min) are marked. It can be seen that RG, TSG, emodin,
nuciferine and quercetin are all present in both extracts. Consider-
ing the maximum absorption wavelength of each component, the
detection wavelengths were changed at different time intervals as
described in Section 2.3.
4
Analysis of L9(3) test results.
No.
A
B
C
D
Composite scores
1
2
3
5
5
5
1
1.5
2
1
2
3
2
3
1
3
1
2
1:15
39.19
77.85
97.34
71.96
85.57
62.44
75.50
57.22
87.65
1:20
1:25
1:25
1:15
1:20
1:20
1:25
1:15
70.80
71.93
75.51
4.70
4
10
1
5
6
10
10
1.5
2
7
15
1
8
9
15
15
1.5
2
K1
K2
K3
R
71.46
73.33
73.46
2.00
62.22
73.55
82.48
20.26
52.95
79.15
86.14
33.19

H.-J. Zhang et al. / Carbohydrate Polymers 132 (2015) 437–443
441
A
B
Fig. 2. (A) Effect of ␤-CD on the extraction ratios of active compounds. Significance was evaluated using Student’s t-test (**P < 0.01); (B) Linear correlation between the
enhancement of extraction ratio and apparent formation constant.
results are shown in Fig. 2(A) (details are reported in Table S1).
In the confirmatory test, a high composite score of 94.48 ± 0.52
was obtained. The extraction ratios of the active ingredients in
constants of inclusion complexes between ␤-CD and RG, TSG,
emodin, nuciferine and quercetin, which had been determined
using the same method.
␤
-CD-assisted and aqueous extractions were calculated. Statisti-
cal analysis was performed using ANOVA and Student’s t-test. A
least significant difference (LSD) test with a confidence interval of
3
.4. In vitro stability of active ingredients in ˇ-CD and aqueous
9
9% was used to compare the means. As shown in Fig. 2(A), ␤-CD-
extracts
assisted extraction significantly enhanced the extraction ratios of
RG, TSG, emodin, nuciferine and quercetin, compared with aqueous
extraction (P < 0.01), particularly those of TSG and emodin.
Thermal stabilities of RG, TSG, emodin, nuciferine and quercetin
◦
◦
were conducted at 25, 37 and 90 C. At 25 C, the compounds
from both ␤-CD and aqueous extracts were mostly unchanged
after 120 h. The addition of ␤-CD increased in vitro stability of TSG
(aqueous extract = 69.44% vs. ␤-CD extract = 86.60%) and nucifer-
ine (aqueous extract = 39.76% vs. ␤-CD extract = 84.63%) after 120 h
3.3. Apparent formation constants and stoichiometries of ˇ-CD
complexes
◦
The assisted extraction effect of ␤-CD may be mainly due to
at 37 C (Fig. S9). The same effect of ␤-CD was observed for RG
association of ␤-CD with the compound, so apparent formation
constants for the formation of ␤-CD inclusion complexes were
determined by UV–vis spectrophotometry or phase solubility anal-
ysis. Both methods are widely accepted for the determination of
binding constants in CD complexation. The changes of absorbance
value indicate that the formation of inclusion complex between ␤-
CD and guest. Binding constants were calculated as described in
Section 2.5.1, and are listed in Table 3. Because of the poor solubil-
ities of emodin and quercetin, phase solubility experiments have
been carried out. In the phase solubility experiments, the solubil-
ities of emodin and quercetin increased linearly with increasing
(aqueous extract = 66.91% vs. ␤-CD extract = 74.83%), TSG (aque-
ous extract = 84.28% vs. ␤-CD extract = 91.81%), nuciferine (aqueous
extract = 60.01% vs. ␤-CD extract = 76.74%) and quercetin (aque-
◦
ous extract = 66.90% vs. ␤-CD extract = 84.01%) after 6 h at 90 C
(Fig. S10). The results show that addition of ␤-CD can improve the
stability of the active ingredients at high temperature, reducing
losses during the reflux extraction process. We speculate that this
is another reason for the increased extraction ratios of the active
ingredients in ␤-CD-assisted extraction.
At pH 1.2, 6.8 and 8.3 (simulated gastric juice, small intestine and
colon pH environments, respectively), there were different degrees
of degradation after 120 h, and stabilities were improved by ␤-
CD: RG (pH1.2, aqueous extract = 84.73% vs. ␤-CD extract = 87.46%;
pH 6.8, aqueous extract = 92.38% vs. ␤-CD extract = 99.46%; pH 8.3,
aqueous extract = 38.45% vs. ␤-CD extract = 50.32%), TSG(pH1.2,
aqueous extract = 89.30% vs. ␤-CD extract = 91.88%; pH 6.8, aque-
ous extract = 79.85% vs. ␤-CD extract = 88.13%; pH 8.3, aqueous
extract = 10.71% vs. ␤-CD extract = 19.67%), emodin (pH1.2, aque-
ous extract = 61.17% vs. ␤-CD extract = 74.37%; pH 6.8, aqueous
extract = 99.71% vs. ␤-CD extract = 100.00%; pH 8.3, aqueous
extract = 99.95% vs. ␤-CD extract = 99.38%), nuciferine (pH1.2,
aqueous extract = 79.09% vs. ␤-CD extract = 89.54%; pH 6.8, aque-
ous extract = 76.96% vs. ␤-CD extract = 83.87%; pH 8.3, aqueous
extract = 86.85% vs. ␤-CD extract = 89.74%) and quercetin (pH1.2,
aqueous extract = 99.59% vs. ␤-CD extract = 98.85%; pH 6.8, aque-
ous extract = 100.00% vs. ␤-CD extract = 100.00%; pH 8.3, aqueous
extract = 100.00% vs. ␤-CD extract = 99.20%) (Figs. S11–S13).
Cumulative light exposure was measured at the same time in the
light stability test. ␤-CD in the extract also increased the light stabil-
ity of emodin (aqueous extract = 18.65% vs. ␤-CD extract = 44.19%)
␤
-CD concentration, giving AL-type phase-solubility diagrams with
the formation of 1:1 complexes (Higuchi & Connors, 1965) (Fig.
S8). The binding constants of the complexes were calculated as
described in Section 2.5.2, and are presented in Table 3.
In general, the range of K values for CDs or their derivatives with
most drugs is 100–20,000 L/mol (Stella & Rajewski, 1997). Higher
values of K indicate enhanced complexation between CDs and
guest drugs, resulting in higher solubility (Banerjee, Chakraborty, &
Sarkar, 2004). The results for the five active ingredients show that
they form inclusion complexes with ␤-CD at a molar ratio of 1:1.
We suspect that this is one of the reasons that their extraction ratios
were significantly increased in the presence of ␤-CD. Table 3 shows
that the binding constants for complexes with RG, TSG, emodin
and quercetin were distinctly higher than that for nuciferine, con-
sistent with the observed improvements in extraction ratios. The
enhancement of extraction ratios by ␤-CD was positively corre-
lated with apparent formation constants as shown in Fig. 2(B). The
linear equation was y = 0.0078x −2.7821, and the correlation coef-
ficient was 0.9803. The curve was fitted on the apparent formation

4
42
H.-J. Zhang et al. / Carbohydrate Polymers 132 (2015) 437–443
Table 3
Apparent formation constants and stoichiometries of inclusion complexes.
Compound
Method
Stoichiometry
Apparent formation constant (L/mol)
Coefficient of determination (R2)
RG
TSG
Nuciferine
Quercetin
UV–vis spectrophotometry
UV–vis spectrophotometry
UV–vis spectrophotometry
UV–vis spectrophotometry
Phase solubility analysis
UV–vis spectrophotometry
Phase solubility analysis
1:1
1:1
1:1
1:1
1:1
1:1
1:1
1005
3591
376
1502
1033
1003
1362
0.9972
0.9895
0.9981
0.9976
0.9939
0.9896
0.9959
Emodin
A
B
C
D
E
Fig. 3. Dissolution curves of RG (A), TSG (B), emodin (C), nuciferine (D) and quercetin (E).
and quercetin (aqueous extract = 80.44% vs. ␤-CD extract = 91.23%)
extraction can also improve dissolution of some ingredients in vivo
after 37,079 lux h (Fig. S14).
to some extent.
3.5. In vitro dissolution rates
4. Conclusion
The dissolution profiles of ␤-CD and aqueous extracts were
In this study, a new eco-friendly extraction method has
efficiently been applied to the extraction of a variety of active ingre-
dients from XZN, a formula of TCM, by using OAD. UPLC/DAD/MS
results showed that both aqueous and ␤-CD extracts contained the
active ingredients, RG, TSG, emodin, nuciferine and quercetin. ␤-
CD-assisted extraction significantly enhanced the extraction ratios
of RG, TSG, emodin, nuciferine and quercetin, compared with aque-
ous extraction (P < 0.01). Apparent formation constants showed
that ␤-CD could form inclusion complexes with the active ingre-
dients. A speculation that the enhancement of extraction ratios
by ␤-CD was positively correlated with the apparent formation
constants between ␤-CD and the compounds was proposed. In
addition, the presence of ␤-CD in the final extract may improve the
stability and dissolution of the active ingredients. In conclusion, ␤-
CD can assist the extraction process of Chinese medicine formulae
without organic solvents and the powder extract containing ␤-CD
can be used in pharmaceutical preparations directly. This ␤-CD-
assisted extraction technology could provide a new eco-friendly
and efficient alternative to traditional extraction methods for TCM
formulae.
studied by UPLC/DAD. The profiles for RG, TSG, emodin, nucifer-
ine and quercetin in both ␤-CD and aqueous extracts are shown
in Fig. 3. After 90 min, the amounts of dissolved active ingredients
were determined as follows: RG (aqueous extract = 75.85% vs. ␤-
CD extract = 78.70% at pH 1.2; aqueous extract = 84.87% vs. ␤-CD
extract = 89.96% at pH 6.8), TSG (aqueous extract = 94.81% vs. ␤-
CD extract = 95.55% at pH 1.2; aqueous extract = 76.32% vs. ␤-CD
extract = 89.50% at pH 6.8), emodin (aqueous extract = 57.64% vs.
-CD extract = 65.27% at pH 1.2; aqueous extract = 47.75% vs. ␤-CD
extract = 51.54% at pH 6.8), nuciferine (aqueous extract = 73.60% vs.
-CD extract = 85.72% at pH 1.2; aqueous extract = 76.16% vs. ␤-CD
extract = 90.66% at pH 6.8) and quercetin (aqueous extract = 82.77%
vs. ␤-CD extract = 87.73% at pH 1.2; aqueous extract = 63.86% vs.
␤
␤
␤
-CD extract = 70.00% at pH 6.8). It was evident that emodin and
nuciferine in the ␤-CD extracts exhibited higher dissolution rates
than the aqueous extracts at pH 1.2. The total dissolution rates
for RG, TSG, nuciferine and quercetin from the ␤-CD extracts were
higher at pH 6.8. Cyclodextrin extraction improves the dissolution
of some of the active ingredients. We presume that ␤-CD-assisted

H.-J. Zhang et al. / Carbohydrate Polymers 132 (2015) 437–443
Liang, R. J. (2008). Orthogonal test design for optimization of the extraction of
443
Acknowledgments
polysaccharides from Phascolosoma esulenta and evaluation of its immunity
activity. Carbohydrate Polymers, 73, 558–563.
This work was financially supported by the National Natural Sci-
ence Foundation of China (No. 81473543), the Specialized Research
Fund for the Doctoral Program of Higher Education of China (No.
Lin, H. Y., Kuo, Y. H., Lin, Y. L., & Chiang, W. (2009). Antioxidative effect and active
components from leaves of Lotus (Nelumbo nucifera). Journal of Agricultural
Food Chemistry, 57, 6623–6629.
Lin, L. F., Ni, B. R., Lin, H. M., Zhang, M., Li, X. C., Yin, X. B., et al. (2015). Traditional
usages, botany, phytochemistry, pharmacology and toxicology of Polygonum
multiflorum Thunb.: A review. Journal of Ethnopharmacology, 159, 158–183.
Liu, Y., Han, B. H., Qi, A. D., & Chen, R. T. (1997). Molecular recognition study of a
supramolecular system. Bioorganic Chemistry, 25, 155–162.
2
0111210110010).
Appendix A. Supplementary data
Mantegna, S., Binello, A., Boffa, L., Giorgis, M., Cena, C., & Cravotto, G. (2012). A
one-pot ultrasound-assisted water extraction/cyclodextrin encapsulation of
resveratrol from Polygonum cuspidatum. Food Chemistry, 130, 746–750.
Normile, D. (2003). Asian medicine. The new face of traditional Chinese medicine.
Science, 299, 188–190.
Supplementary material related to this article can be found, in
the online version, at doi:10.1016/j.carbpol.2015.06.072
Parmar, I., Sharma, S., & Rupasinghe, H. P. (2014). Optimization of
References
␤-cyclodextrin-based flavonol extraction from apple pomace using response
surface methodology. Journal of Food Science Technology, http://dx.doi.org/10.
1007/s13197-014-1282-1
Pinho, E., Grootveld, M., Soares, G., & Henriques, M. (2014). Cyclodextrins as
encapsulation agents for plant bioactive compounds. Carbohydrate Polymers,
101, 121–135.
Ratnasooriya, C. C., & Rupasinghe, H. P. (2012). Extraction of phenolic compounds
from grapes and their pomace using ␤-cyclodextrin. Food Chemistry, 134,
625–631.
Ren, A. N., Lu, A. L., Tian, Y. Z., & He, S. F. (2008). AHP application to study of
weighted coefficient on multicriteria optimization of extraction technology
about Chinese traditional compound drugs. China Journal of Chinese Materia
Medica, 33, 372–374.
Ai, S. T., Fan, X. D., Fan, L. F., Sun, Q., Liu, Y., Tao, X. F., et al. (2013). Extraction and
chemical characterization of Angelica sinensis polysaccharides and its
antioxidant activity. Carbohydrate Polymers, 94, 731–736.
Banerjee, R., Chakraborty, H., & Sarkar, M. (2004). Host–guest complexation of
oxicam NSAIDs with ␤-cyclodextrin. Biopolymers, 75, 355–365.
Benesi, H. A., & Hildebrand, J. H. (1949). A spectrophotometric investigation of the
interaction of iodine with aromatic hydrocarbons. Journal of the American
Chemical Society, 71, 2703–2707.
Chen, J. X., Zhao, H. H., Yang, Y., Liu, B., Ni, J., & Wang, W. (2011). Lipid-lowering
and antioxidant activities of Jiang–Zhi–Ning in Traditional Chinese Medicine.
Journal of Ethnopharmacology, 134, 919–930.
Chinese Pharmacopoeia Commission. (2010a). (2010, pp. 696). Pharmacopoeia of
the People’s Republic of China (Vol. 1) Beijing: Medicine Science and Technology
Press of China.
Chinese Pharmacopoeia Commission. (2010b). (2010, pp. 135–136). Pharmacopoeia
of the People’s Republic of China (Vol. 1) Beijing: Medicine Science and
Technology Press of China., 165, 258
Chinese Pharmacopoeia Commission. (2010c). (2010, pp. 86–87). Pharmacopoeia of
the People’s Republic of China (Vol. 1) Beijing: Medicine Science and Technology
Press of China. Appendix.
Dong, C. Y., Zhang, X. L., & Li, H. F. (2010). Effect of processing on the contents of
nuciferine and quercetin in Nelumbo nucifera Gaertn. Chinese Traditional Patent
Medicine, 32, 973–976.
Ren, X. L., Wang, G. F., Wang, M., Ou-Yang, H. Z., & Qi, A. D. (2011). Kinetics and
ꢀ
mechanism of 2,3,5,4 -tetrahydroxystilbene-2-O-␤-d-glycoside (THSG)
degradation in aqueous solutions. Journal of Pharmaceutical and Biomedical
Analysis, 55, 211–215.
Stella, V. J., & Rajewski, R. A. (1997). Cyclodextrins: Their future in drug
formulation and delivery. Pharmaceutical Research, 14, 556–567.
Sun, Y. X., Li, Y. J., Li, M. Q., Ton, H. B., Yang, X. D., & Liu, J. C. (2009). Optimization of
extraction technology of the Anemone raddeana polysaccharides (ARP) by
orthogonal test design and evaluation of its anti-tumor activity. Carbohydrate
Polymers, 75, 575–579.
Szente, L., & Szejtli, J. (2004). Cyclodextrins as food ingredients. Trends in Food
Science and Technology, 15, 137–142.
Du, H., You, J. S., Zhao, X., Park, J. Y., Kim, S. H., & Chang, K. J. (2010). Antiobesity and
hypolipidemic effects of lotus leaf hot water extract with taurine
supplementation in rats fed a high fat diet. Journal of Biomedical Science, 17, S42.
Harada, A. (1997). Construction of supramolecular structures from cyclodextrins
and polymers. Carbohydrate Polymers, 34, 183–188.
Higuchi, T., & Connors, K. A. (1965). Phase solubility techniques. Advances in
Analytical Chemistry and Instrumentation, 4, 117–212.
Hsu, C. M., Yu, S. C., Tsai, F. J., & Tsai, Y. (2013). Enhancement of rhubarb extract
solubility and bioactivity by 2-hydroxypropyl-␤-cyclodextrin. Carbohydrate
Polymers, 98, 1422–1429.
Tafazzoli, M., & Ghiasi, M. (2009). Structure and conformation of ␣-, ␤- and
␥-cyclodextrin in solution: Theoretical approaches and experimental
validation. Carbohydrate Polymers, 78, 10–15.
Wang, W. G., He, Y. R., Lin, P., Li, Y. F., Sun, R. F., Gu, W., et al. (2014). In vitro effects
of active components of Polygonum Multiflorum Radix on enzymes involved in
the lipid metabolism. Journal of Ethnopharmacology, 153, 763–770.
Wang, M. J., Zhao, R. H., Wang, W. G., Mao, X. J., & Yu, J. (2012). Lipid regulation
effects of Polygoni Multiflori Radix, its processed products and its major
substances on steatosis human liver cell line L02. Journal of
Ethnopharmacology, 139, 287–293.
Irie, T., & Uekama, K. (1997). Pharmaceutical applications of cyclodextrins. III.
Toxicological issues and safety evaluation. Journal of Pharmaceutical Sciences,
Xie, R. F., Shi, Z. N., Li, Z. C., Chen, P. P., Li, Y. M., & Zhou, X. (2014). Optimization of
high pressure machine decocting process for Dachengqi Tang using HPLC
finger-prints combined with Box-Behnken experimental design. Journal of
Pharmaceutical Analysis, http://dx.doi.org/10.1016/j.jpha.2014.07.001
Xie, W. D., Zhao, Y. N., & Du, L. (2012). Emerging approaches of traditional Chinese
medicine formulas for the treatment of hyperlipidemia. Journal of
Ethnopharmacology, 140, 345–367.
Yang, J. W., Liu, Y. N., Gao, J., Chen, L. N., Ren, X. L., & Qi, A. D. (2015). The influence
of xuezhining-␤-cyclodextrin extract on hyperlipidemia and fatty
degeneration of liver in Rats. Chinese Traditional Patent Medicine, 37, 649–653.
Zhang, Q. A., Fan, X. H., Zhang, Z. Q., Li, T., Zhu, C. P., Zhang, X. R., et al. (2013).
Extraction, antioxidant capacity and identification of Semen Astragali
Complanati (Astragalus complanatus R. Br.) phenolics. Food Chemistry, 141,
1295–1300.
86, 147–162.
Jiang, W. Y. (2005). Therapeutic wisdom in traditional Chinese medicine: A
perspective from modern science. Trends in Pharmacological Sciences, 26,
5
58–563.
Jung, D. H., Kim, Y. S., Kim, N. H., Lee, J., Jang, D. S., & Kim, J. S. (2010). Extract of
Cassiae Semen and its major compound inhibit S100b-induced TGF-beta1 and
fibronectin expression in mouse glomerular mesangial cells. European Journal
of Pharmacology, 641, 7–14.
Ko, C. N., Park, S. U., Chang, G. T., Jung, W. S., Moon, S. K., Park, J. M., et al. (2011).
Antihyperlipidemic and antioxidant effects of the mixture of Ginseng radixand
Crataegi fructus: Experimental study and preliminary clinical results. Journal of
Ginseng Research, 35, 162–169.
Lee, G. Y., Jang, D. S., Lee, Y. M., Kim, J. M., & Kim, J. S. (2006). Naphthopyrone
glucosides from the seeds of Cassia tora with inhibitory activity on advanced
glycation end products (AGEs) formation. Archives of Pharmacal Research, 29,
Zhang, L., Zhu, L., Wang, Y. F., Jiang, Z. Z., Chai, X., Zhua, Y., et al. (2012).
Characterization and quantification of major constituents of Xue Fu Zhu Yu by
UPLC–DAD–MS/MS. Journal of Pharmaceutical and Biomedical Analysis, 62,
203–209.
5
87–590.