全 文 ： 2012 年 3 月 第 10 卷 第 2 期 Chin J Nat Med Mar. 2012 Vol. 10 No. 2 119

Chinese Journal of Natural Medicines 2012, 10(2): 0119−0124
doi: 10.3724/SP.J.1009.2012.00119
Chinese
Journal of
Natural
Medicines







Enrichment and purification of flavones from rhizomes of
Abacopteris penangiana by macroporous resins
WEI Han, RUAN Jin-Lan*, LEI Yong-Fang, YANG Chun
Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation of Hubei Province, College of Pharmacy, Tongji Medical
Center, Huazhong University of Science and Technology, Wuhan 430030, China
Available online 20 Mar. 2012
[ABSTRACT] AIM: To investigate the enrichment and purification of flavones from the rhizomes of Abacopteris penangiana (RAP)
by macroporous resins. METHODS: Static adsorption and desorption tests were performed to select the appropriate resin. The kinetic
adsorption and desorption experiments were carried out on selected HPD500 resin to optimize the separation process of flavones.
Additionally, the effects of four parameters including adsorption flow rate, elute flow rate, volume of water and ethanol solution for
elution were explored by a L4/3 orthogonal experiment. Finally, the ABTS (2, 2′-azinobis-3-ethylbenzothiazoline-6-sulphonic acid)
radical scavenging activities of samples before and after being treated by HPD500 were compared. RESULTS: The results showed that
the optimal parameters were initial concentration of 2.86 mg·mL−1, elute solution of 70% ethanol, absorb flow rate of 1 mL·min−1, elute
flow rate of 2 mL·min−1, 5 BV of water for elution and 5 BV of ethanol solution for elution. CONCLUSION: The content of flavones
is above 60% in RAP after being treated by HPD500, indicating that macroporous resins could be successfully applied to enrich and
purify flavones in RAP.
[KEY WORDS] Abacopteris penangiana; Flavones; Macroporous resins
[CLC Number] R284.2 [Document code] A [Article ID] 1672-3651(2012)02-0119-06

1 Introduction
The rhizomes of Abacopteris penangiana (Hook.) Ching
(RAP), called jixuelian, is a folk medicine used by Tujia
people to promote blood circulation and remove blood stasis
in the western region of Hubei Province, China [1]. Previous
phytochemistry researches suggested that novel flavan-4-ol
compounds were the main kind of constituents in RAP [2-4].
Moreover, those compounds were proved to present powerful
antioxidant and neuroprotective activities [2, 5].
Conventional methods including liquid–liquid extraction
and gel chromatography are not effective to obtain these
compounds concerning regents, labor, energy consumption
and environmental protection. Alternatively, growing interest

[Received on] 30-Mar.-2011
[Research funding] This project was supported by the National
Natural Science Foundation of China (Nos. 30973864 and 81173065)
and Ph.D. Programs Foundation of the Ministry of Education of
China (No. 20090142110021).
[*Corresponding author] RUAN Jin-Lan: Prof., Tel: 86-27-
83692311, Fax: 86-27-83692762, E-mail: jinlan8152@163.com
These authors have no any conflict of interest to declare.
has been focused on employing macroporous resins to enrich
and purify bioactive constitutes from traditional Chinese
herbs [6-14]. Macroporous resins can be used to selectively
absorb targeted phytochemicals because of their unique
properties, including ideal pore structure and various surface
functional groups available, low operation expense, less sol-
vent consumption and easy regeneration [10].
In the present study, various macroporous resins with
different chemical and physical properties were employed to
investigate the adsorption and desorption process and to de-
velop a convenient and efficient method for enrichment and
separation of antioxidant flavones from RAP with the optimal
resin. Various parameters influencing the adsorption and
desorption processes were optimized.
2 Materials and Methods
2.1 Chemicals and regents
The dried rhizomes of Abacopteris penangiana were
collected in June 2009 from Jiujiang, Jiangxi province, China
and authenticated by Prof. TAN Ce-Ming, Jiujiang Forest
Plants Specimen Mansion. The voucher specimen (PZX0311)
has been deposited in College of Pharmacy, Tongji Medical
Center, Huazhong University of Science and Technology. All
WEI Han, et al. /Chinese Journal of Natural Medicines 2012, 10(2): 119−124
120 Chin J Nat Med Mar. 2012 Vol. 10 No. 2 2012 年 3 月 第 10 卷 第 2 期

chemicals and regents used in the experiments were of ana-
lytical or chromatographic grade. Abacopterin I, triphyllin A,
eruberin B and 6′′-O-acetyl eruberin B were isolated from
RAP in our laboratory and purity of each compound was
more than 98% by HPLC.
2.2 Absorbents
Macroporous resins including HPD100, HPD400,
HPD450, HPD500, HPD600, HPD722 and D101 were pur-
chased from Bonherb Technology Company (Hebei, China).
These resins were soaked with 95% ethanol for 24 h to swell
adequately. Subsequently the resins were eluted by 95%
ethanol until white casse disappeared when the eluting re-
agent was mixed with pure water (1 : 5, V/V), and then the
resins were washed with pure water until the liquor had no
alcoholic odor. The pre-treated resins were then placed in the
drying oven at 70 °C over 24 h until constant.
Table 1 The parameters tested in the orthogonal experiment
Absorb flow
rate/mL·min−1
Elute flow
rate/ mL·min−1
Volume of
water for elu-
tion/BV
Volume of ethanol
solution for elution
/BV
1 2 3 3
2 3 5 5
4 4 7 10

2.3 Preparation of sample solutions of RAP
Three hundred grams of dry and minced RAP was ex-
tracted with 1 800 mL of ethanol–water (80 : 20, V/V) solu-
tion by hot reflux extraction for 90 min. Extract of RAP was
obtained by concentrating the extracting solution to dryness
by removing the ethanol solvent in a rotary evaporator
(Shanghai SENCO Science & Technology Co., Ltd., China)
at 60 °C. Pure water was added into the extract, and then the
liquor was centrifuged by a centrifugal machine (Jintan
Xinkang instrument factory, China) with 2 500 r·min−1 for 15
min to obtain the sample solutions.
2.4 Determination of flavones content by UV spectropho-
tometry
On account of no chromogenic structure of flavones,
content of flavones-4-ol compounds in RAP cannot be de-
termined based on the conventional color reaction of rutin.
Alternatively, concerning the flavones-4-ol compounds in
RAP have same skeleton, abacopterin I isolated from RAP
was employed as a standard compound to determine the con-
tent of flavones by UV spectrophotometry in this study. The
working curve of abacopterin I was: A = 3.820 3 C + 0.138 2
(r = 0.999 8, 0.017 1−0.220 mg·mL−1, detective wavelength
of 276 nm), where A is absorbance of the tested sample and C
is concentration of the tested sample.
2.5 Static absorption and desorption tests
The static absorption test for screening the best resins
were performed as follows: pre-weighted quantities of hy-
drated resins (equal to 250 mg in dry weight) were added into
250 mL Erlenmeyer flasks containing 50 mL of sample solu-
tions of RAP obtained in Section 2.3. The Erlenmeyer flasks
were shaken at 120 r·min−1 for 12 h at room temperature; and
then standing for 12 h. Subsequently, the content of flavones
in sample solution was analyzed by UV spectrophotometry.
The static desorption experiments were performed on
three types of resins which had largest absorption capacity by
placing the resins that had completed the absorption process
described previously in 250 mL Erlenmeyer flasks containing
50 mL of 95% ethanol. The Erlenmeyer flasks were shaken at
120 r min−1 for 12 h at room temperature; and then standing
for 12 h. The total concentration of flavones in desorbed
solution was determined.
2.6 Absorption and desorption kinetics
All kinetic adsorption and desorption experiments were
carried out in a glass column (20 mm × 300 mm) wet-packed
with the selected HPD500 resin. The bed volume (BV) of the
resin was 23 mL. All kinetic adsorption and desorption ex-
periments were performed at room temperature.
The Kinetic-Tandem assay was employed to investigate
kinetic adsorption capacity of HPD500 resins. The Ki-
netic-Tandem assay was performed as follows: 350 mL ex-
tract sample solution (the content of flavones was 1.467 8
mg·mL−1) flowed through the glass column at the flow rate of
3 mL·min−1. The content of flavones (C1) in elution (E1) was
detected by UV analysis when the sample solution drained
away through the column. Subsequently the elution flowed
through the column again at the same flow rate. Concentra-
tion of flavones (C2) in elution (E2) was analyzed. Repeated
the above steps until Cn+1 changed little comprised with Cn.
At this moment the kinetic absorption can be seen reaching
the equilibrium point. Then the leached fraction and kinetic
absorption mass of HPD500 were calculated.
Five sample solutions with different initial concentra-
tions (0.53, 1.47, 2.86, 4.29 and 6.70 mg·mL−1) were em-
ployed to investigate effect of the initial concentration of
sample solution on the enrichment and purification process.
These five samples were dealt with same parameters includ-
ing loading mass of extract, absorb flow rate, elute flow rate
and so on. The kinetic absorption capacities were compared
when the absorb processes were finished.
The resin column that reached absorb equilibrium was
used to do kinetic desorption tests based on a gradient elution
program. The column was eluted by pure water, 30% ethanol
(V/V), 50% ethanol (V/V), 70% ethanol (V/V), 80% ethanol
(V/V) for 5 BV, respectively, at a flow rate of 1.5 mL·min−1.
The concentration of flavones in desorption solution col-
lected at 1 BV internals was monitored.
2.7 L4/3 orthogonal experiment
In order to fully study the best parameters in process of
purification of flavones by macroporous resin, a L4/3 or-
thogonal experiment was performed in a glass column (20
mm × 500 mm) wet-packed with HPD500 resin. The bed
volume (BV) of the resin was 50 mL. The parameters to be
tested and their values are listed in Table 1. After each test
WEI Han, et al. /Chinese Journal of Natural Medicines 2012, 10(2): 119−124
2012 年 3 月 第 10 卷 第 2 期 Chin J Nat Med Mar. 2012 Vol. 10 No. 2 121

was finished, the yield mass of extract, yield mass of flavones,
yield ratio and content of flavones were investigated. The
L4/3 orthogonal experiment was performed at room tem-
perature.
Table 2 Results of the orthogonal experiment
Order
Yield mass
of extract
/mg
Yield mass
of flavones
/mg
Loading
mass of
extract/mg
Yield ratio
/%
Content of
flavones /%
1 231.8 138.78 981.5 14.14 59.87
2 215.1 132.35 1 022.4 12.95 61.53
3 295.1 148.17 1 022.4 14.49 50.21
4 251.0 144.70 1 034.0 13.99 57.65
5 232.7 113.81 1 034.0 11.01 48.91
6 223.9 125.12 1 034.0 12.10 55.88
7 264.5 131.32 1 034.0 12.70 49.65
8 218.7 118.80 1 022.4 11.62 54.32
9 263.0 137.65 1 034.0 13.31 52.34

2.8 Enrich and purify flavones by HPD500 with optimized
parameters and HPLC analysis of flavones in RAP
The Enrichment and purification process with optimized
parameters was performed in a 37 mm × 300 mm column
with the parameters optimized in the experiments above and
content of flavones in the obtained sample was determined.
Subsequently, Chromatography of obtained flavones was
achieved on a Hitachi pump L-2130 equipped with a Hitachi
UV Detector L-2400 (Hitachi, Japan) with a detective wave-
length of 276 nm. The chromatographic separation was per-
formed on an amethyst C18-p column (Sepax-tech, 4.6 mm ×
200 mm, 5 μm) with column temperature of 25 °C and flow
rate of 1 mL·min−1. The injection volume of each sample was
20 μL. The solvents were filtered through a 0.45 μm nylon
filter membrane and degassed in a sonicator bath prior to use.
The solvents were water containing 0.1% phosphate acid
(V/V, solvent A) and acetonitrile (solvent B). The gradient
elution program was follow: 0.00 min, 82.0% A (18.0% B);
3.00 min, 82.0% A (18.0% B); 15.00 min, 79.0% A (21.0%
B); 50.00 min, 55.0% A (45.0% B); 60.00 min, 55.0% A
(45.0% B).
2.9 ABTS radical scavenging activity
To assess the ABTS scavenging activity, an improved
ABTS method [15] was used. Briefly, ABTS•+ was produced
by mixing ABTS (7 mmol·L−1; Sigma–Aldrich, St.Louis, MO,
USA) and ammonium persulphate (2.45 m mol·L−1), the
mixture was kept in the dark at room temperature for 16 h
before use. The ABTS•+ solution was diluted with ethanol (1 :
12, V/V) to obtain an absorbance of 0.7 ± 0.02 at 734 nm.
Sample solution (100 µL, 0.04−2.27 mg·mL−1) in ethanol
was added to 3.9 mL ABTS•+ solution. Absorbance at 734 nm
was measured after reaction of 6 min. Trolox (Sigma–Aldrich,
St.Louis, MO, USA; purity ≥ 98%) was used as a positive
control. All measurements were made in triplicate and aver-
aged.
3 Results and Discussion
3.1 Absorption capacity and ratio of desorption
The capacity of adsorption and desorption ratios are cal-
culated as follows.
Adsorption capacity: 0 0( )eC C VQ
W
−=
where Q is the adsorption capacity, which represents the
mass of adsorbate adsorbed on 1 g of dry resin at adsorption
equilibrium; C0 and Ce is the initial and equilibrium concen-
tration of flavones in the sample solutions, respectively; V0 is
the initial volume of sample solution, and W is the weight of
the dry resin.
Desorption ratio:
0 0( )
d d
e
C VD
C C V
= −
where D is the desorption ratio (%); Cd is the concentration
of flavones in the desorption solutions (mg/ml); Vd is the
volume of the desorption solution; C0, Ce and V0 are the same
as those defined above.
Seven macroporous resins with different physical prop-
erties were employed to enrich and purify flavones in RAP,
and the results were shown in Figure 1. The absorption ca-
pacity and desorption ratio of flavones on HPD500 resins
were higher than other resins. Hence, HPD500 was employed
in the process of enrichment and purification of flavones in
the following study.

Fig. 1 (A) The result of static absorption tests on seven
macroporous resins. (B) The result of static desorption tests
on three macroporous resins
3.2 Absorption and desorption kinetics
The leached fraction is calculated as follows.
WEI Han, et al. /Chinese Journal of Natural Medicines 2012, 10(2): 119−124
122 Chin J Nat Med Mar. 2012 Vol. 10 No. 2 2012 年 3 月 第 10 卷 第 2 期

Leached fraction (LF): 1n
n
CLF
C
+= (Cn and Cn+1 are de-
fined in 2.6)
The kinetic absorption process is different with the static
absorption and may be influenced with several factors in-
cluding absorb flow rate, the initial concentration of load
solution and the size of columns. In our study, the ki-
netic-Tandem absorption test was carried out to determine the
maximum absorption capacity when the selected resins
reached the equilibrium point in kinetic process. As shown in
Figure 2, the leached fraction raised rapidly when the second
tandem absorption was performed. After sixth tandem ab-
sorption process, the leached fraction of resins almost
reached 100% (97.9%), meaning the resins reached its equi-
librium point in kinetic absorption process. According to the
dates shown in Fig. 2, the maximum absorption capacity was
calculated as 18.46 mg/(1 mL weighted resins), which can be
used to determine the weight of macroporous resins on the
basis of the mass of sample.

Fig. 2 The curves of leached fraction and absorption capac-
ity in the Kinetic-Tandem assay
In order to investigate the initial concentration of loading
sample solution, five concentrations (0.53, 1.47, 2.86, 4.29
and 6.70 mg·mL−1) of sample solution were selected. Shown
in Fig. 3, when initial concentration is above 2.86 mg·mL−1,
the absorption capacities did not increase significantly. On
the contrary, the mass of flavones not be absorbed increased
inevitably. Due to these reasons, 2.86 mg·mL−1 was chosen to
be initial concentration of the sample solution.

Fig. 3 The effect of initial concentration of loading sample
solution on kinetic absorption capacity

Fig. 4 The kinetic desorption curve based on the gradient
elution program
The kinetic desorption curve on HPD500 was obtained
based on a gradient elution program. As depicted in figure 4,
the flavones absorbed by HPD500 were desorbed almost
completely after eluted by 30%, 50% and 70% ethanol.
Hence, the appropriate and economic ethanol concentration
of elution solution can be optimized as 70%.
3.3 The optimization of parameters by orthogonal experiment
According table 2 and 3 the parameters of absorb flow
rate, elute flow rate, volume of water for elution and volume
of ethanol solution for elution were optimized as 1 mL·min−1,
2 mL·min−1, 5 BV and 5 BV, respectively. Furthermore,
among these four parameters, volume of ethanol solution for
elution and absorb flow rate were important factors in the
enrichment and purification process of flavones in RAP ac-
cording to Rj.
Table 3 Analysis of the orthogonal experiment on the basis
of content of flavones in RAP

Absorb
flow rate/
(mL·min−1)
Elute flow
rate/
(mL·min−1)
Volume of water
for elution/BV
Volume of ethanol
solution for elu-
tion/BV
Kj.1 57.203 55.723 53.707 56.690
Kj.2 54.147 54.920 55.687 57.173
Kj.3 52.103 52.810 54.060 49.590
Rj 5.100 2.913 1.980 7.583

3.4 The optimal enrich and purify process and HPLC
analysis of flavones in RAP
After dealt by the optimal enrich and purify process on
HPD500, the content of flavones in the sample is increased
from 21.85% to 63.12%. The chromatograms of the tested
samples before and after treatment with HPD500 resin were
shown in Fig. 5. By comparison, it can be seen that some
impurities in the crude extracts were removed and the relative
peak area of three typical falvone-4-ol compounds, triphyllin
A, eruberin B and 6′′-O-acetyl eruberin B, increased signifi-
cantly after treated by HPD500 resins.
WEI Han, et al. /Chinese Journal of Natural Medicines 2012, 10(2): 119−124
2012 年 3 月 第 10 卷 第 2 期 Chin J Nat Med Mar. 2012 Vol. 10 No. 2 123



Fig. 5 The HPLC comparation of samples before and after treated by HPD500
3.5 ABTS radical scavenging activity of samples before and
after treated by HPD500
As can be seen from Figure 6, sample after treated by

Fig. 6 The ABTS radical scavenging activities of samples
before and after treated by HPD500
HPD500 was found to be a very effective scavenger against
ABTS radical compared with the sample without the treat-
ment, and its activity increased in a concentration-dependent
manner. The antioxidant compound, trolox, was acted as the
reference. The ABTS radical scavenging activity of sample
treated by HPD500 was moderately higher than that of trolox,
indicating the sample presented powerful antioxidant activity.
4 Conclusion
In our study, the enrichment and purification process of
flavones of RAP with macroporous resin HPD500 has been
successfully developed. HPD500 was selected because of the
best performance in static absorption and desorption tests.
Additionally, the effects of several factors were investigated
to make optimization of the adsorption and desorption condi-
tions. Our study showed that under the optimized conditions,
i.e. initial concentration of 2.86 mg·mL−1, elution solution of
70% ethanol, absorb flow rate of 1 m L·min−1, elute flow rate
of 2 mL·min−1, 5 BV of water for elution and 5 BV of ethanol
for elution, content of flavones in RAP was increased sig-
nificantly after dealt by HPD500 according to HPLC com-
parison. In conclusion, the result of our study suggested that
macroporous resin adsorption method was applied success-
fully to enrich and purify flavones in RAP.
References
[1] Administration Bureau of National Chinese Traditional Medi-
cine. Chinese Herbal [M]. Shanghai Scientific and Technical
Publisher, 1998: 164.
[2] Zhao ZX, Ruan JL, Jin J, et al. Flavan-4-ol glycosides from the
rhizomes of Abacopteris penangiana [J]. J Nat Prod, 2006,
69(2): 265-268.
[3] Zhao ZX, Ruan JL, Jin J, et al. A novel anthocyanidin gly-
cosides from the rhizomes of Abacopteris penangiana [J]. Fi-
toterapia, 2010, 81(8): 1171-1175.
[4] Zhao ZX, Ruan JL, Jin J, et al. Two new acetylated flavan
glycosides from the rhizomes of Abacopteris penangiana [J]. J
Asian Nat Prod Res, 2010, 12(12): 1015-1019.
[5] Lei YF, Fu W, Chen JL, et al. Neuroprotective effects of Aba-
copteris E from Abacopteris penangiana against oxidant
stress-induced neurotoxicity [J]. J Ethnopharmacol, 2011,
134(2): 275-280.
[6] Ouyang F, Liu Y, Li R, et al. Five lignans and an iridoid from
Sambucus williamsii [J]. Chin J Nat Med, 2011, 9(1): 26-29.
[7] Xu QP, Zhu GC. Macroporous resin separation process of
WEI Han, et al. /Chinese Journal of Natural Medicines 2012, 10(2): 119−124
124 Chin J Nat Med Mar. 2012 Vol. 10 No. 2 2012 年 3 月 第 10 卷 第 2 期

flavone from lonicera japonica Thunb [J]. Food Eng, 2008,
vol?(4): 35-37.
[8] Gao M, Huang W, Liu CZ. Separation of scutellarin from crude
extracts of Erigeron breviscapus (vant.) Hand. Mazz. by
macroporous resins [J]. J Chromatogr B, 2007, 858(1-2):
22-26.
[9] Jia GT, Lu XY. Enrichment and purification of madecassoside
and asiaticoside from Centella asiatica extracts with macro-
porous resins [J]. J Chromatogr A, 2008, 1193(1-2): 136-141.
[10] Fu BQ, Liu J, Li H, et al. The application of macroporous
resins in the separation of licorice flavonoids and glycyrrhizic
acid [J]. J Chromatogr A, 2005, 1089(1-2): 18-24.
[11] Liu W, Zhang S, Zu YG, et al. Preliminary enrichment and
separation of genistein and apigenin from extracts of pigeon
pea roots by macroporous resins [J]. Bioresour Technol, 2010,
101(12): 4667–4675.
[12] Zhang Y, Li SF, Wu XW, et al. Macroporous resin adsorption
for purification of flavonoids in Houttuynia cordatu Thunb [J].
Chin J Chem Eng, 2007, 15(6): 872-876.
[13] Zhang B, Yang RY, Zhao Y, et al. Separation of chlorogenic
acid from honeysuckle crude extracts by macroporous resins [J].
J Chromatogr B, 2009, 867(2): 253-258.
[14] Zhou YF, Qi J, Zhu DN, et al. Homoisoflavonoids from
Ophiopogon japonicus and its oxygen free radicals (OFRs)
scavenging effects [J]. Chin J Nat Med, 2008, 6(3): 201-204.
[15] Re R, Pellegrini N, Proteggente A, et al. Antioxidant activity
applying an improved ABTS radical cation decolorization As-
say [J]. Free Radical Biol Med, 1999, 26(9-10): 1231- 1237.

大孔树脂富集纯化披针新月蕨(Abacopteris penangiana)中的
黄酮类成分
魏 涵, 阮金兰*, 雷永芳, 杨 春
华中科技大学同济医学院药学院 湖北省天然药物化学与资源评价重点实验室, 武汉 430030
【摘 要】 目的：研究使用大孔树脂富集纯化披针新月蕨根茎中的黄酮类成分。方法：通过静态吸附及解吸附试验筛选最
优型号树脂; 动态吸附、解吸附试验和正交试验优化树脂最大上样量、最佳上样浓度、最佳洗脱醇浓度、吸附流速、解析流速、
水洗体积和醇洗体积等工艺参数; 采用最佳参数富集纯化黄酮类成分后, HPLC 分析和 ABTS (2, 2′-azinobis-3-ethylbenzothia-
zoline-6-sulphonic acid)抗氧化试验比较样品经树脂处理前后的差别。结果：优化大孔树脂富集纯化披针新月蕨黄酮类成分的条
件为：上样浓度 2.86 mg·mL−1, 醇洗浓度 70%, 吸附流速 1 mL·min−1, 解吸流速 2 mL·min−1, 水洗体积和醇洗体积均为 5 倍柱体
积。样品经大孔树脂处理后黄烷-4-醇类黄酮含量明显增加。结论: 样品经大孔树脂处理后黄酮含量由 21.85%增加到 63.12 %, 表
明大孔树脂可以应用于披针新月蕨中黄酮成分的富集纯化过程。
【关键词】 披针新月蕨; 黄酮; 大孔树脂

【基金项目】 国家自然科学基金(Nos. 30973864, 81173065)及教育部博士点专项基金(No. 20090142110021)资助项目