全 文 ：277Journal of Chinese Pharmaceutical Sciences
Fingerprint analysis of different Panax herbal species
by HPLC-UV method
Feng Zeng1, 2, Xiao-Ming Wang 2, Min Yang 2, Zhi-Qiang Lu 2 and De-An Guo1, 2*
1. School of Pharmaceutical Sciences, Peking University, Beijing 100083, China;
2. Shanghai Research Center for Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, Shanghai 201203, China
Abstract: Aim To establish a method for differentiating commercial samples of Panax species including notoginseng, cultivated
ginseng (Chinese ginseng and Korean ginseng), wild ginseng, red ginseng, three types of American ginsengs, and one Ameri-
can ginseng preparation with their HPLC fingerprints for assnrning the quality of different commercial samples of Panax species.
Methods HPLC-UV method was used to establish their fingerprints, Zorbax Extend C18 (250 mm × 4.6 mm, 5μm) was used as
the analytical column, and acetonitrile/KH2PO4 aqueous solution was used as the mobile phase with gradient elution. Results The
fingerprints of different commercial samples of Panax species varied in their holistic chromatograms and some specific
constituents. Conclusion This method is reliable, reproducible and simple. It could be applied in the routine authentication of dif-
ferent commercial samples of Panax species
Keywords: Ginseng; Panax species; HPLC fingerprint
CLC number: R917 Document code: A Article ID: 1003–1057(2007)4–277–05
Introduction
Panax ginseng C. A. Mey is probably the most fa-
mous herbal medicine in the world with a long his-
tory in traditional Chinese medicine (TCM). There
also existed a number of herbs similar to ginseng
which come from the same category of Panax
Genus, such as Panax notoginseng (Burk.) F. H.
Chen and Panax quinquefolium L. Ginseng was
usually classified into Chinese ginseng, Korean gin-
seng and their processed samples such as red gin-
seng and their preparations. American ginseng has
been reported to have three types in terms of their
differences in the concentrations of ginsenoside Rg1
and Re, and in the ratios of ginsenoside Rg1 to Re.
These results indicated that the ratios of ginsenoside
Rg1 to Re in American ginseng roots varies by geo-
graphical origin more than environmental factors[1].
Some west researchers usually think that ginseng,
American ginseng and notoginseng have the same or
similar pharmacological activities. However, based
on the theory of TCM, Ginseng, American ginseng
and notoginseng possess very different functions.
Ginseng is regarded as an invigorant used to be re-
storative with a character of nourishing the Yang,
while American ginseng is also considered as an
Received date: 2007-04-24.
Foundation item: Program for Changjiang Scholar and Innovative
Team in University (Grant No. 985-2-063-112), and National
Supporting Program for TCM from Ministry of Science and
Technology of China (Grant No. 2006BAI08B03-03).
*Corresponding author. Tel./fax: 86-10-82802700;
e-mail: gda@bjmu.edu.cn
invigorant with a comparable character of nourishing
the Yin. Although notoginseng also has similar
ginsenoside profile as ginseng, it is usually used to
stanch bleeding and invigorate the circulation of
flood[2]. For the different processing products, dried
ginseng is good at nourishing the Yin and Qi, and red
ginseng is good at the Yang and Qi. There are still
some differences between the cultivated ginseng and
wild ginseng samples. Immunochemical and genetic
methods have been developed in order to distinguish
P. notoginseng, P. quinquefolius and P. japonicus[3].
It is necessary to establish a simple, quick and reli-
able method to discriminate these Panax species,
which would be very important for insuring the safe
use of ginseng or related herbal medicines. Among
these analytical methods, HPLC method is the most
widely used one and can be well-suited for most
analytical laboratories. The present study aimed to
establish a simple HPLC method to discriminate
these different samples of Panax species.
Experimental
Chemicals and materials
Methanol and KH2PO4 were of analytical grade and
purchased from Sinopharm Chemical Reagent Co.,
Ltd (China). Acetonitrile was of HPLC grade and
purchased from Honeywell International Inc. (USA).
Distilled water was prepared with a Millipore-Q wa-
ter purification system (Millipore, Bedford, MA,
USA). The standards of ginsenosides Rg1, Re, Rf,
Rb1, Rh1, Rc, Rb2, Rb3, and Rd, and ginseng con-
trol sample were purchased from National Institute
for the Control of Pharmaceutical and Biological
Products (Beijing, China). Wild ginseng samples
were kindly provided by Professor Tu-Fu Fang, Cu-
rator of ginseng museum in Hangzhou city, China.
Korean ginseng samples were purchased from stores
in Seoul, Korea; American ginseng samples, culti-
vated Chinese ginseng samples and red ginseng
samples were purchased from the different drug
stores in China.
Preparation of samples
The dried powdered sample (0.3 g) was extracted
with 30 mL 70% (V/V) methanol aqueous solution in
an ultrasonic water bath for 1 h. The extracted solu-
tion was filtrated through analytical filter paper and
then the filtered solution was evaporated at 40 °C to
dryness in vacuum. The dry extract was dissolved
in 5 mL 70% (V/V) methanol aqueous solution and
filtrated through a 0.45 mm membrane filter unit.
Then 10 mL of each sample solution was analyzed
by HPLC.
HPLC conditions
For all experiments, an Angilent HPLC 1100 series
(Angilent technologies, USA) equipped with a qua-
druplet pump, auto sampler, column oven, and diode
array detector was used. The column used for sepa-
ration was Extend C18 (250 mm × 4.6 mm, 5.0 µm,
Angilent technologies, USA) with a pre-column of
Extend C18 (20 mm ´ 4 mm, 5 mm, Angilent
technologies, USA). Gradient elution was used with
a mobile phase consisting of (A) 0.01 mol·L–1
KH2PO4 aqueous solution, and (B) acetonitrile. The
initial condition was set at 18% of B; to keep con-
sistent for 10 min, then, gradient up to 30% of B in
35 min and up to 33% in next 35 min. Detection
was set at 203 nm. The initial oven temperature was
set at 15 °C; keep consistent for 30 min, then
gradient up to 35 °C in the next 15 min, and then
keep consistent up to 80 min. The flow rate was
set at 0.8 mL·min–1.
Results and discussion
HPLC method validation
Method reproducibility and repeatability were eval-
uated by the analysis of seven injections of sample
solution. Precision of retention times and peak areas
were evaluated by the computer aided similarity
evaluation system, the similarity of HPLC finger-
prints of seven injections was more than 98%, and
the RSD of the retention times was less than 2%
and the RSD of peak area was less than 5%,
respectively.
HPLC fingerprints of Panax species
Altogether 47 Panax samples were analyzed includ-
ing 10 cultivated Chinese ginseng samples, 10 culti-
vated Korean ginseng samples, 7 wild ginsengs, 10
red ginseng samples and 10 high Re/Low Rg1
chemo-type American ginseng samples. These samples
were collected from a variety of sources and condi-
tions. In order to express the exact distribution of
ginsenosides in the chromatograms, 9 ginsenoside
markers were used to identify the ginsenosides in
samples. The results indicated that 8 ginsenosides
were detected in cultivated Chinese ginseng and cul-
tivated Korean ginseng, 9 ginsenosides were detected
in red ginseng, and 7 ginsenosides were detected in
American ginseng in terms of retention times consis-
tent with the ginsenoside markers.
Differentiation of different Panax herbal samples
Different Panax herbal samples could be well distin-
guished from each other by their HPLC fingerprints.
American ginseng (High Re/Low Rg1 chemo-type)
and ginseng have similar HPLC fingerprints, except
for the presence of ginsenoside Rf in ginseng and
some difference of the peak area ratio of Rg1 to Re.
Usually, the peak area ratio of Rg1 to Re (High
Re/Low Rg1 chemo-type) was obviously less than
that of ginseng, which could be easily discriminated
from each other by their HPLC fingerprints. Red
ginseng, as ginseng’s processed materials, had the
thick colour than crude ginseng materials and this
could be a simple parameter used for differentiat-
ing ginseng from red ginseng. Rh1 was gradually
generated in the process of steaming and drying,
so there could be more Rh1 detected in red ginseng.
Figure1. HPLC chromatograms of standard mixture. (1) Rg1,
(2) Re, (3) Rb1, (4) Rc, (5) Rb2, (6) Rb3, (7) Rd, (8) Rf,
(9) Rh1.
278 F. Zeng et al. / Journal of Chinese Pharmaceutical Sciences 16 (2007) 277–281
Figure 2. HPLC fingerprints of cultivated ginseng (n = 10).
Figure 3. HPLC fingerprints of wild ginseng (n = 7).
Figure 4. HPLC fingerprints of American ginseng (high Re/High Rg1 chemo-type, n = 10).
Figure 5. HPLC fingerprints of red ginseng (n = 10).
279F. Zeng et al. / Journal of Chinese Pharmaceutical Sciences 16 (2007) 277–281
Figure 7. HPLC fingerprints of different chemo-types of
American ginseng and its preparation. A. Wild American
ginseng; B. American ginseng (High Rg1/Low Re chemo-type);
C. American ginseng (high Re/Low Rg1 chemo-type); D. American
ginseng tablet.
Figure 8. HPLC fingerprints of different ginsengs. A. Cultivated
Chinese ginseng (ginseng control sample); B. Cultivated Korean
ginseng; C. Wild ginseng.
Notoginseng could be easily distinguished from gin-
seng and American ginseng by their HPLC finger-
prints for it is short of many peaks contrasted to
ginseng and American ginseng. However, our study
were not conclusive for differentiation of cultivated
Chinese ginseng and cultivated Korean ginseng,
which suggested that cultivated Chinese ginseng and
cultivated Korean ginseng could be used equally for
they belong to the same species and have similar
growth environment.
Discrimination of three chemo-types of American
ginseng
It has been reported that using Fourier transform-in-
frared spectroscopy (FT-IR) and two dimensional
correlation infrared spectroscopy (2D-IR) could dis-
criminate and identify the American ginseng of dif-
ferent geographical regions[4]. However, in the previ-
ous HPLC study of American ginseng, it was diffi-
cult to get a good separation of Rg1 and Re[5] and
this may lead to a wrong identification of different
chemo-types of American ginseng. In most herbal
markets and drug stores of China, the high Re/Low
Rg1 chemo-type American ginseng took a dominant
position based on literature studies and typical Ameri-
can ginseng is thought to have Rg1 to Re ratios of
less than 1.0[6]. In the present study, the American
ginseng samples were randomly collected from dif-
ferent drug stores and one American ginseng of the
high Rg1/Low Re chemo-type and one wild Ameri-
can ginseng were obtained. These three chemo-types
of American ginsengs could be well discriminated
from each other by their HPLC fingerprints. From
these results, it is suggested that the three chemo-types
of American ginseng should be considered to be used
separately in terms of the different physiological and
medicinal activities of Rg1 and Re.
In order to investigate the exact difference in the
ratios of Rg1 to Re in different chemo-types of
American ginseng, quantification of Rg1 and Re was
developed. The results showed that the content ratios of
Rg1 to Re were 1.09 in wild American ginseng (n = 1),
Figure 6. HPLC fingerprint of notoginseng (n = 1).
280 F. Zeng et al. / Journal of Chinese Pharmaceutical Sciences 16 (2007) 277–281
0.05 – 0.13 (n = 10) in American ginseng of high
Re/Low Rg1 chemo-type, 25.5 (n = 1) in American
ginseng of High Rg1/Low Re chemo-type, and 0.15
(n = 1) in American ginseng tablet, respectively. The
HPLC fingerprints showed that the American ginseng
tablet has fewer chemical constituent peaks, which
indicated that the processing of American ginseng
preparation may lead to the loss of some specific
chemical constituents.
Distinction of wild ginseng from cultivated ginseng
As shown in Figure 4, one specific chemical constituent
peak was detected in wild ginseng referred to peak
10, and this need some further study in order to
identify the specific chemical constituent. Except for
this special peak, the other peaks were both detected
in wild ginseng and cultivated ginseng, while the
HPLC fingerprints visually showed that the peak
areas in wild ginseng were much higher than that of
cultivated ginseng, which indicated that wild ginseng
could accumulate more constituents than cultivated
ginseng.
Conclusion
Panax herbs take an important position in TCM.
There are some similarities and differences both in
morphological characters and the chemical contents
as a result of genetic difference and the processing
methods, which will lead to the similarity and difference
of their HPLC fingerprints. The current developed
HPLC fingerprints could differentiate ginseng from
American ginseng by the absence of Rf in American
ginseng. Red ginseng could be distinguished from
ginseng by the presence of Rh1 in red ginseng.
Notoginseng could be easily discriminated from the
others since it has much less peaks and high contents
of ginsenosie Re and Rb1. This method was proved
to be simple, reliable and reproducible. Therefore it
can be applied in the routine authentication of different
commercial samples of Panax species.
Acknowledgements
This work was supported by Program for Changjiang
Scholars and Innovative Team in University (985-2-
063-112), and National Supporting Program for
TCM from Ministry of Science and Technology of
China (2006BAI08B03-03).
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高效液相色谱法对不同种人参属药用植物的指纹图谱分析
曾凤1, 2, 王晓明2, 杨敏2, 卢志强2, 果德安1, 2
(1.北京大学药学院, 北京 100083; 2.中国科学院上海药物研究所 上海中药现代化研究中心 上海 201203)
摘要: 目的 建立高效液相指纹图谱方法用于区分人参属不同商品药材包括三七, 圆参(种植国产人参和种植高丽参) ,
野山参, 红参以及三种不同化学分型的西洋参, 保证人参属药材的安全, 正确使用。方法 采用Zorbax Extend C18 (250 mm ×
4.6 mm, 5μm) 色谱柱, 乙腈和 10 mmol·L–1 KH 2PO4水溶液为洗脱溶剂, 建立人参属药材的液相指纹图谱方法。结果 根据人
参属不同商品药材液相指纹图谱的整体和一些特殊成分的差异, 可以区分人参属不同商品药材。结论 高效液相色谱指纹
图谱方法简单, 有效, 可重复, 可以用于人参属不同商品药材的常规鉴别。
关键词 : 人参; 人参属药材; 高效液相指纹图谱
281F. Zeng et al. / Journal of Chinese Pharmaceutical Sciences 16 (2007) 277–281