全 文 ：Received:May 18，2012 Accepted October 11，2012
Foundation Item:Hunan Provincial Natural Science Foundation
(11JJ2042) ;Science of Pharmaceutical Analysis of Twelfth Five-Year
Key Discipline Projects of Hunan University of Chinese Medicine;In-
novation Platform and Open Foundation Project of Higher Colleges of
Hunan Province(11K048) ;Changsha City Science and Technology
Bureau Key Projects(K1104104-21)
* Corresponding author Tel:86-731-88458240;E-mail:lxq0001cn @
163． com
天然产物研究与开发 Nat Prod Res Dev 2013，25:71-75
文章编号:1001-6880(2013)1-0071-05
RP-HPLC法同时测定十种五加属植物叶
中三萜 Chiisanoside和 Chiisanogenin
刘向前1，2* ，戴 玲1，戴秀珍2
1湖南省中药现代化研究重点实验室 湖南中医药大学药学院，长沙 410208;2中南大学制药工程系，长沙 410083
摘 要:建立反相高效液相色谱法(RP-HPLC)测定 chiisanoside和 chiisanogenin的定量分析方法，利用最优色谱
条件对十种中韩五加属植物叶中的三萜 chiisanoside和 chiisanogenin进行定量分析。研究结果表明，最佳色谱条
件为:ODS-C18色谱柱(250 mm × 4． 6 mm，5 μm) ;流动相:乙腈-水(55∶ 45) ;检测波长:205 nm;柱温:40 ℃;流
速:1． 0 mL /min。Chiisanoside和 chiisanogenin线性范围和回归方程分别为 16． 5 ～ 181． 5 μg /mL，Y = 5498356X-
9738 (r = 0． 9995)和 4． 88 ～ 78． 0 μg /mL，Y = 5753131X-8289 (r = 0． 9979) ，加样回收率分别为 98． 87%(标准偏
差为 1． 14%)和 98． 83%(标准偏差为 0． 72%)。
关键词:五加属;chiisanoside;chiisanogenin;反相高效液相色谱法
中图分类号:R284. 1 文献标识码:A
Simultaneous Determination of Chiisanoside and Chiisanogenin from Leaves
of Ten Kinds of Acanthopanax Miq． Plants by RP-HPLC
LIU Xiang-qian 1，2* ，DAI Ling 1，DAI Xiu-zhen 2
1Hunan Key Laboratory of Traditional Chinese Medicine modernization，School of Pharmacy，Hunan University of Chinese Medicine，
Changsha 410208，China;2School of Chemistry and Chemical Engineering，Central South University，Changsha 410083，China
Abstract:A RP-HPLC method for simultaneous quantitative analysis of chiisanoside and chiisanogenin was developed
and optimized． Chiisanoside and chiisanogenin from leaves of ten kinds of Acanthopanax Miq． plants were quantitatively
analyzed by the developed method． The best condition was established as follows:the separation was performed with an
ODS-C18 column (250 mm × 4． 6 mm，5 μm)at 40 ℃，and the mobile phase was CH3CN-H2O (55∶ 45)． Chiisanoside
and chiisanogenin were detected at 205 nm，the flow rate was 1 mL /min． The linear ranges of chiisanoside and chiisano-
genin were 16． 5-181． 5 μg /mL and 4． 88-78． 0 μg /mL，and gave correlations (r)of 0． 9995 (Y = 5498356X-9738)
and 0． 9979 (Y = 5753131X-8289) ，respectively． The recoveries of chiisanoside and chiisanogenin were 98. 87%
(RSD was 1． 14%)and 98． 83% (RSD was 0． 72%) ，respectively．
Key words:Acanthopanax Miq．;chiisanoside;chiisanogenin;RP-HPLC
Introduction
The plants of Acanthopanax Miq． are richly distributed
in Asia，growing about 26 kinds and 18 varieties in
China，11 kinds and 3 varieties in Korea，and 9 kinds
in Japan． A large number of Acanthopanax Miq． plants
have shown some medical values，including nourishing，
anti-rheumatic， anti-stress， anti-fatigue， and anti-
tumor［1，2］． Chiisanoside was reported to have anti-canc-
er，anti-hepatotoxic and anti-diabetic activities ［6］． It
had been isolated from A． chiisanensis，A． divaricatus
var allbeofructus and other Acanthopanax genus ［3-5］．
While，chiisanogenin，a metabolite of chiisanoside by
human intestinal bacteria ［7］，also showed some biologi-
cal activities such as protection of ulcer of stomach and
liver damage ［8-10］． In order to further develop and uti-
lize of these plants，a RP-HPLC method for quantitative
analysis of chiisanoside and chiisanogenin was devel-
DOI:10.16333/j.1001-6880.2013.01.018
oped and optimized in this manuscript［10，11］． This meth-
od was validated for its precision，stability and repeat-
ability． In addition，chiisanoside and chiisanogenin from
leaves of ten kinds of Acanthopanax Miq． plants were
simultaneously determined and quantified to provide a
basis for the follow-up study of Acanthopanax Miq．
plants．
Materials and Methods
Chemicals and reagents
Reference standards of chiisanoside and chiisanogenin
(Fig． 1)were internally prepared in the laboratory
(purities and structures were determined by HPLC，
NMR，MS，etc;and their purities were above 98． 5%)．
Acetonitrile (CH3CN，HPLC grade) and methanol
(CH3OH，HPLC grade) were both purchased from
Tianjin Kermel Chemical Reagent Co． Ltd;double-dis-
tilled water for the chromatography was purified by u-
sing the automatic double pure water distillatory． The
solvents were filtered through 0． 45 μm membranes and
degassed in an ultrasonic bath before use．
Fig. 1 Chemical structures of chiianoside (A)and chi-
isanogenin (B)
Ten plant materials were A． gracilistylus W． W． Smith
(Ningxiang county，Hunan Province) ;A． senticosus
(Fusong county，Jilin province) ;A． henryi (Oliv．)
Harms and A． sessiliflorus (Xinhua county，Hunan
Province) ;A． seboldianus Makino，A． koreanum，A． di-
varicatus var． albeofructus，A． divaricatus f． distimatis，
A． divarcatus f． inermis and A． chiisanesis，which were
all collected from the herb garden of Kyung Hee Uni-
versity，Korea． All samples were randomly divided into
three batches and were identified by one of authors，
Professor Liu Xiang-qian． The voucher specimens of
these samples were kept in Herbarium of Hunan Uni-
versity of Chinese Medicine．
The ten kinds of A． Miq． plants chosen in this study
were distributed widely and richly in China and Korea．
Among them，the dried roots and stem barks of A． gra-
cilistylus W． W． Smith and A． senticosus were listed offi-
cially in Chinese Pharmacopoeia as Acanthopanacis
Cortex (Wujiapi)and Acanthopanacis Senticosi Radix
et Rhizoma seu caulis (Ciwujia) ，respectively ［12］． Mo-
reover，A． sessilifluous was examined and approved as a
new resource of food． A． henryi (Oliv．)Harms was col-
lected into the local standards of Hunan Province．
Instrumental and chromatographic conditions
A HPLC system (Shimadzu，Japan，LC-10AT)consis-
ted of a SPD-10A UV-Vis detector and a CBM-102
chromatography workstation was used for the quantita-
tive analyses． Chromatographic separation was achieved
with an ODS-C18column (250 mm × 4． 6 mm，5 μm)
at 40 ℃，and CH3CN-H2O (55∶ 45)was employed as
the mobile system． The flow rate was kept constantly at
1． 0 mL /min． The UV detection wavelength was select-
ed as 205 nm． The injection volume was 10 μL．
Preparation of standard solution
Standard stock solutions were prepared by dissolving an
appropriate amount of chiisanoside and chiisanogenin
in methanol to give a final concentration of 330 μg /mL
and 195 μg /mL． A serial of mixed standard solutions
were then prepared at concentrations of 16． 5，49． 5，
82． 5，115． 5，148． 5，181． 5 μg /mL and 4． 88，19． 5，
34． 1，48． 8，63． 4，78． 0 μg /mL for chiisanoside and
chiisanogenin，respectively．
Preparation of sample solution
All of the samples were powdered that passed through
40 mesh s sieve． Took about 4 g of each sample，and
weighed precisely before extraction． Then，reflux extrac-
tion was carried out two times using methanol for 4
hours at 65 ℃，and filtered． Finally，after evaporation of
the methanol under vacuum，the residue was diluted to
50 mL with methanol and then filtered with 0． 45 μm
membrane filter．
Method validation
Linearity
The linearity of the method was established by injecting
the series of standard mixtures of chiisanoside and chi-
27 Nat Prod Res Dev Vol. 25
isanogenin． The data of peak areas versus concentra-
tions were treated by linear least square regression a-
nalysis．
Precision
The precision of the method was determined by injec-
ting one sample with five replicates． In this manuscript，
five applications of A． divarcatus f． inermis were assayed
and the peak areas were recorded．
Stability
The stability study of the sample solution of A． divarca-
tus f． inermis was carried out over a period of 12 h at 25
℃ (room temperature under laboratory light)． The var-
iability was assayed at the same concentration for every
4 hours，during 12 hours．
Repeatability
The repeatability of the method was detected by extrac-
ting one sample of A． divarcatus f． inermis five times，
and the areas of the peaks were recorded．
Recovery
To test the extraction recovery，quantified samples of A．
divarcatus f． inermis were added with low，middle and
high concentrations standards before extraction． The fol-
low-up extractions and HPLC analyses were accom-
plished in the same manner as detailed above． The re-
covery was compared with the theoretical concentra-
tion．
Results and Discussion
Method development
To develop a suitable method for the quantification of
chiisanoside and chiisanogenin，different mobile phases
were employed to achieve the best separation and reso-
lution． The method development was initiated with u-
sing a mobile phase of acetonitrile and water at various
ratios (50∶ 50，55∶ 45，60∶ 40，65∶ 35)． Acetonitrile and
sodium dihydrogen phosphate buffer at different pH
were also investigated in this study． However，the pH
values of mobile phase were found to have no effect on
the separation of chiisanoside and chiisanogenin． Final-
ly，the mobile phase consisting of acetonitrile and water
(55∶ 45)was found to be appropriate allowing good
separation of compounds at a flow rate of 1 mL /min． In
addition，in order to obtain a satisfactory and full detec-
tion for this new method，UV-Vis spectra of chiisano-
side and chiisanogenin standards were obtained． Based
on the maximum UV absorbance of chiisanoside and
chiisanogenin，205 nm was selected as the optical de-
tection wavelength．
Method validation
Calibration curves
The linearity of the detector responses was investigated
for each standard mixture solution by plotting peak are-
as against concentrations． Good correlation between the
peak areas and concentrations at the range of 16． 5-
181． 5 μg /mL for chiianoside and 4． 88-78． 0 μg /mL
for chiisanogenin was achieved． The regression equa-
tions and correlation coefficients determined were Y =
5498356X-9738 (r = 0． 9995)for chiianoside and Y =
5753131X-8289 (r = 0． 9979) for chiisanogenin，re-
spectively．
Precision
The RSDs of the peak areas were 0． 22% and 0． 44%
for chiianoside and chiisanogenin respectively，which
indicated that the developed and optimized method was
precise．
Stability
The RSDs values of the peak areas were 1． 04% and
1． 17% for chiianoside and chiisanogenin，respectively．
It indicated that sample solutions were stable for 12 h
at room temperature．
Repeatability
37
Vol. 25 LIU Xiang-qian，et al:Simultaneous Determination of Chiisanoside and Chiisanogenin
from Leaves of Ten Kinds of Acanthopanax Miq． Plants by RP-HPLC
The RSDs values of the peak areas were 0． 66% for
chiianoside and 1． 11% for chiisanogenin，which indi-
cated that the proposed method was repeatable．
Recovery test
The average recovery rates were 98． 87 % (RSD was
1． 14%)for chiianoside and 98． 83 % (RSD was 0． 72
%) for chiisanogenin． The results of recovery tests
were shown in Table 1．
Table 1 Recoveries of chiisanoside and chiiisanogenin
Sample No． The amountof sample (g)
Initial amount
(mg)
Spiked
amount (mg)
Detected
amount (mg)
Recovery
(%)
Average
(%)
RSD
(%)
chiisanoside 10． 0061 8． 4851 4． 0304 12． 1388 96． 99 98． 87 1． 14
10． 0020 8． 4817 4． 0354 12． 2530 97． 89
10． 0059 8． 4851 8． 0707 16． 5028 99． 68
10． 0130 8． 4910 8． 0721 16． 4985 99． 61
10． 0162 8． 4936 12． 1061 20． 5091 99． 56
10． 0203 8． 4970 12． 1075 20． 4974 99． 48
chiiisanogenin 10． 0104 3． 9061 1． 8521 5． 6471 98． 07 98． 83 0． 72
10． 0099 3． 9059 1． 8527 5． 6405 97． 95
10． 0108 3． 9062 3． 9143 7． 8009 99． 75
10． 0116 3． 9065 3． 9150 7． 7581 99． 19
10． 0113 3． 9064 5． 4514 9． 2380 98． 72
10． 0109 3． 9060 5． 4512 9． 2926 99． 31
Quantitative determination
The validated method was employed to determine the
two ingredients in each sample of the ten kinds of Ac-
anthopanax Miq． plants，and every batch of sample was
concurrently determined three times． The quantification
results of the ingredients were shown in Table 2．
Table 2 Concentrations of chiisanoside and chiisanogenin detected in the ten kinds of Acanthopanax Miq．(n = 3)
Samples
chiisanoside chiisanogenin
Average peak area RSD(%) Content(mg /g)Average peak area RSD(%) Content(mg /g)
A． sessilifluous 200867． 2 0． 43 0． 4614 26830． 9 0． 68 0． 0616
A． henryi(Oliv．)Harms 781245． 7 0． 63 1． 7946 229218． 0 0． 66 0． 5267
A． divaricatus f． distigmatis 689215． 0 0． 30 1． 5832 72393． 7 0． 19 0． 1663
A． senticosus 107334． 4 0． 33 0． 2466 455701． 9 0． 75 1． 0470
A． divaricatus var． albeofructus 986051． 1 0． 53 2． 2651 44588． 1 0． 49 0． 1024
A． divaricatus f． inermis 369158． 6 0． 46 0． 8480 169848． 0 0． 36 0． 3902
A． chiisanesis 566951． 2 0． 48 1． 3024 144600． 6 0． 25 0． 3322
A． seboldianus Makino 2732． 4 0． 68 0． 0063 44100． 7 0． 35 0． 1013
A． gracilistylus W． W． Smith － －
A． koreanum － －
* “ －”means not detected
Conclusion
A simple and rapid method was successfully developed
for simultaneous determination of chiianoside and chi-
isanogenin from leaves of Acanthopanax Miq． plants．
The proposed method was optimized and validated for
the various parameters． The results demonstrated the
method was highly specific，accurate and precise，which
is promising of being used in quality control of Acan-
thopanax Miq． ． The concentrations of chiianoside and
47 Nat Prod Res Dev Vol. 25
chiisanogenin of ten kinds of Acanthopanax Miq． plants
were quantified using the developed method． The re-
sults indicated that the two ingredients were not detec-
ted in A． gracilistylus W． W． Smith and A． koreanum，
while A． divaricatus var． albeofructus had the highest
concentration of chiianoside，A． senticosus had the high-
est concentration of chiisanogenin．
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Vol. 25 LIU Xiang-qian，et al:Simultaneous Determination of Chiisanoside and Chiisanogenin
from Leaves of Ten Kinds of Acanthopanax Miq． Plants by RP-HPLC