全 文 ：1 Introduction
Paederia scandens (Lour.) Merri., a climbing plant,
belonging to the family Rubiaceae, is popularly
known as “Ji Shi Teng”in Chinese and widely
grows in India, Vietnam, China, Japan, Philippines
and USA [1] . It has been traditionally used as folk
medicine and food in Southeast of Asia for thou-
sands of years. The leaves of the plant are used as
an ingredient in various foods in Vietnam.
Recently, it has been reported that the iridoid
glycosides paederoside, asperuloside, paederosidic
acid, methyl paederosidate, deacetylasperuloside and
scandoside were isolated from the MeOH extract
from the stems and roots of P. scandens [2]. These
chemical constituents of P. scandens have biological
activities such as anti-virus, anti-tumor, anti-inflam-
mation and anti -microbial activities. In folklore
medicine, the roots, stems, leaves, barks and fruits of
P. scandens have been used to treat toothache, chest
pain, piles, inflammation of the spleen, emesis,
rheumatic arthritis and bacillary dysentery in China,
Japan, Vietnam and other countries in the south east
Asia for thousands of years.
2 Experiments
2.1 Chemical reagents and samples
Acetonitrile was of HPLC grade from Merck
(Darmstadt, Germany); distilled water was further
purified by a Mili-Q system (Milipore, MA, USA);
the other chemicals were of analytical grade. All
solvents and samples were filtered through
Foundation Item the Key Program of Natural Science Foundation
of Jiangsu province(SBK200930526)
Corresponding Author Wang Yong-bing Tel: 0512-62956097,
E-mail: ybwang2000@126.com
Received 2011-02-21 Accepted 2011-03-17
**
*
HPLC Analysis of Iridoid Glycosides from the
Stem of Paederia scandens*
WANG Yong-bing**， LI Quan， JIANG Yi， LIU Guang-yao
Suzhou Yihua Biomedical Technology Co.,Ltd, Suzhou，Jiangsu 215123, China
ABSTRACT Objective: To establish a quantitative analysis method with HPLC-DAD on
simultaneous determination of iridoid glycosides in the stem of Paederia scandens from different
place of production for quality control. Methods: A rapid and accurate HPLC-DAD method was
developed and validated for the quantitative determination of three iridoid glycosides: paederosidic
acid (1), paederoside (2) and methyl paederosidate (3). The subsequent HPLC separation and
quantification was achieved in 45minutes using a Shim -pack VP -ODS C18 column and at
240nm. The mobile phase comprised solvent A（0.1% formic acid aqueous, V/V) and solvent B
(acetonitrile) and a gradient elution mode was applied. This established method was therefore
applied to determining the amounts of iridoid glycosides in eight samples collected from different
regions in China. Results: The linear regression analytical data for the calibration plots showed
a good linear relationship with r2>0.9999 within the tested ranges. The average recoveries were
from 98.47% to 104.85% indicating good accuracy. It was successfully applied to quantifying the
three iridoid glycosides in different samples. Conclusion: It is the first time to establish a
quantitative method to investigate the contents of iridoid glycosides in Paederia scandens from
different places of production, and this method can be applied to the quality control of raw
material Paederia scandens.
KEY WORDS HPLC-DAD; Paederia scandens; Iridoid glycosides; Quantitative analysis
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研 究
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DOI:10.13664/j.cnki.pcr.2011.02.004
a 0.45μm micropore filters before they were inject-
ed for HPLC.
The three reference compounds paederosidic
acid (1), paederoside (2) and methyl paederosidate
(3) were isolated previously from Paederia scandens
(Lour.) Merri. in our laboratory, and the structures
of them were confirmed based on spectroscopic ex-
periments(1HNMR, 13CNMR, ESI-MS) and by com-
parison with literature dat a [1]. The purity of each
compound was determined to be higher than 98%
by normalization of the peak area detected by
HPLC-DAD, and the chemicals in aqueous solution
were very stable during our experimental period.
Eight batches of Paederia scandens(Lour.) Merri.
samples were collected from different provinces in
China and the voucher specimens of these collec-
tions were deposited at our company.
2.2 Preparation of Paederia scandens extracts
The stems of P. scandens collected from
different areas were powdered by an electrical
blender and sieved through a 40-mesh sieve. The
powder was quantitatively extracted by methanol
(1g to 100mL) through refluxing for 2hrs twice.
The combined extracts were concentrated on a
rotary evaporator. The residues were then
transferred into a 10 mL volumetric flask, and
brought to volume with water. The sample solutions
were filtered through a 0.45 μm micropore filter
before HPLC analysis.
2.3 Preparation of standard solutions
The stock mixed solution of the three
standards which were separated from the crude
extract samples was prepared by dissolving
accurately weighted portions of the standards in
water. The concentrations of the three reference
compounds paederosidic acid(1), paederoside(2) and
methyl paederosidate (3) were 1.047, 0.556 and
0.494 mg·mL -1, respectively. The solution was
further diluted to a series of concentrations with
water for gain of calibration curve, limits of
detection(LOD) and quantification(LOQ).
2.4 Instrumentation and chromatographic
conditions
An Agilent 1200 series HPLC system (Agilent
Technologies, USA) and Agilent Chem Stations
software were used for the chromatographic
analysis. The separation was carried out on a
Shim-pack VP-ODS C18 column(250 mm×4.6 mm, 5
μm). The mobile phase was composed of solvent A
(0.1% formic acid aqueous, V/V) and solvent B
(acetonitrile). A linear gradient program at a flow
rate of 1.0 mL·min-1 was performed as follows: 0~5
min,15.0%B; 5 ~30 min, linear gradient 15.0%B~
20.0%B; 30~45 min, linear gradient 20.0%B~45.0%
B. The injection volume, column temperature, and
UV wave length were set at 20 μL, 25°C and 240
nm, respectively. The typical HPLC -DAD
chromatograms of the three reference compounds
and samples were shown in Figure 1.
Fig.1 Typical HPLC-DAD chromatograms
(A)standard mixture solution; (B)extract of P. scandens;
(1) paederosidic acid; (2) paederoside; (3) methyl paederosidate
2.5 Calibration curve, limits of detection and
quantification
The standard stock solution containing three
iridoid glucosides was prepared and diluted to ap-
2011
Apr;19(2) HPLC Analysis of Iridoid Glycosides from the Stem of Paederia scandens
116
propriate concentrations for the plotting of calibra-
tion curves. Each analyte solution of five concen-
trations covering the concentration range according
to the level expected in the plant samples was an-
alyzed in triplicate, and then the calibration curves
were constructed by plotting the peak areas versus
the concentration(μg·mL-1) of each analyte.
Dilutions and injections of the stock standards
were made until an HPLC chromatogram showed
that the iridoid glycosides peak height reached an
S/N of approximately 10︰1 and 3︰1 for LOQ and
LOD respectively.
2..6 Precision, stability, repeatability, and accuracy
The precisions were determined by analyzing
standard solutions of three iridoid glycosides in six
replicates during a single day. The relative stan-
dard devi ation (RSD) of peak area of each com-
pound were calculated. Stability of sample was
tested separately at 0, 2, 4, 8, 24, and 48hrs,
within 2days. The sample solution was kept at 4℃
before analysis. To confirm the repeatability, six
replicates of sample solutions describes as above
were prepared and analyzed, the RSD values of
the peak areas were calculated.
Method accuracy was calculated by spiking six
samples of the extract of P. scandens with standard
stock solution of standard compounds of 1~3. The
theoretical amount of analyte in the sample prepa-
rations and the average percentage analyte recov-
ered in the spiked solutions were calculated.
3 Result and discussion
3.1 Optimization of extraction procedure
The optimization of extraction was targeted on
iridoid glucosides. In order to obtain quantitative
extraction of iridoid glycosides from P. scandens,
the variables involved in the extraction were opti-
mized, such as solvent and extraction method. Both
methanol and ethanol were tested for their effi-
ciency as extraction solvent. Methanol was found to
be methanol, which allowed a complete extraction
of the iridoid glucosides as tested. In order to find
the best extraction better ultrasonic processing for
30 min, refluxing for twice were tested and com-
pared with various other conditions. The results
suggested that refluxing for 2hrs twice was simpler
and more effective in extracting the iridoid glucosides.
3.2 Optimization of chromatographic condi-
tions
Studies were carried out in order to validate
an efficient method for the analysis of the iridoid
glycosides. Parameters, such as detection wave-
length, compositions and percentages of the mobile
phases, temperatures and flow rates of the chro-
matographic columns were all studied.
Different settings of mobile phase (organic sol-
vent, acid percentage and gradient programming)
were tested. Since acid is known to achieve better
separation for paederosidic acid by reducing the
tailing of the peaks, various mixtures of water and
acetonitrile in combination with formic acid were
tried. As a result, a good separation of the iridoid
glucoside peaks was achieved with the mobile
phase comprising solvent A (0.1% aqueous formic
acid, V/V) and solvent B (acetonitrile) with a gradient
elution mode. The chromatographic conditions were
then validated to be applicable for the analysis of
the content of iridoid glucosides of P. scandens.
UV/DAD spectra were recorded within a range
of 200~400 nm to select a suitable wavelength and
to maximize the detection of the standard com-
pounds. The spectra illustrated that the wavelength
of 240 nm was determined to be the most appro-
priate for the simultaneous analyses of the three
iridoid glucosides present in P. scandens.
The flow rate and column temperature were
also optimized to obtain suitable resolution. At
temperature 25°C and flow rate 1.0 mL·min-1, the
method achieved the best separation.
3.3 Method validation
The typical HPLC-DAD chromatograms of the
three reference compounds was shown in Figure 1
(A). As a result, a baseline separation was
achieved within 45min under the optimized condi-
tions, with symmetrical, sharp and well -resolved
peaks for all the three analytes. The linear regres-
sion analytical data for the calibration plots showed
a good linear relationship with r2 >0.9999 wit hin
the tested ranges. The LOD and LOQ for all the
compounds were less than 52 ng·mL-1 and 170 ng·
mL-1, respectively. These values were described in
Table 1. They were sufficient for the analyses of
药学与临床研究
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药 学
研 究
117
the extracts and met the precision and accuracy
criteria. The variations of precision were less than
2.08% for the analytes. The standards in sample
extracted from P. scandens remained very stable in
aqueous solution (RSD <2.00% ) at 4° C over the
tested period. The recovery studies were carried
out at six samples, and the developed analytical
method has an excellent accuracy with a re-
covery rates from 98% to 105% for the three
iridoid glycosides (Table 2). Therefore, the HPLC–
DAD method was precise , accurate and sensi-
tive enough for simultaneous quantitative evalua-
tion of the three iridoid glycosides in the stem
of P. scandens .
Table1 Calibration curves,LOD and LOQ for the standards
Standard Retention time(min) Calibration curve Correlation coefficient(r2) Linear range(μg·mL-1) LOD(ng·mL-1) LOQ(ng·mL-1)
1 14.347 A=28.965 C+77.331 0.99994 26.12-1047.00 37.15 115.21
2
3
22.859
31.679
A=20.278 C+19.346
A=27.228 C+74.583
0.99995
0.99991
13.90-556.00
12.35-494.00
51.20
39.21
165.10
123.33
Table2 Recoveries of the three iridoid glycosides in P. scandens (n=6)
Standard Background (mg) Added (mg) Found (mg) Recovery (%) RSD (%)
1 2.4040 2.2460 4.579 98.47 1.89
2 0.6035 0.5270 1.165 103.03 1.88
3 0.1470 0.1567 0.318 104.85 2.15
Table3 Contents of the three iridoid glycosides in P. scandens collected from different provinces in China (mg.g-1)
Sample Origin
Analytes
1 2 3 sum
100324 Chongqing 3.16 0.19 0.33 3.68
100222 Yunnan 16.30 1.90 2.60 20.90
090621 Hubei 10.09 1.48 7.87 19.44
090707 Hunan 9.04 1.71 0.31 11.06
090811 Nanning, Guangxi 4.81 1.21 0.29 6.31
090824 Nanning, Guangxi 9.23 1.03 1.84 12.10
090924 Guizhou 3.65 0.97 0.20 4.82
091009 Nanning, Guangxi 7.29 3.15 1.65 12.09
3.4 Sample analysis
The suitability of the developed analytical method
was demonstrated by analyzing P. scandens extracts
from cultivated or wild samples from different place of
China and the results were summarized. The concen-
tration ranges of the iridoid glycosides were very wide in
samples from different places of China, as shown in
Table 3, the contents of the three glycosides were 3~16
mg·g-1 (paederosidic acid), 0.1~2 mg·g-1(paederoside),
and 0.2~7 mg·g-1(methyl paederosidate), respectively.
4 Conclusion
It is the first time to establish a quantitative
method to investigate the content of iridoid gluco-
sides of in Paederia scandens. Among the three
glucosides, paederosidic acid was commonly dis-
tributed, and was found to be the most abundant
one, which can be applied to the quality control of
Paederia scanden. This method can meet the re-
quirements of quantitative analysis of the samples.
Reference
[1] Quang DN, Hashimoto T, Tanaka M, et al. Iridoid glu-
cosides from roots of Vietnamese Paederia scandens [J].
Phytochemistry. 2002, 60(5): 505-14.
[2] Zou X, Peng S, Liu X, et al. Sulfur-containing iridoid
glucosides from Paederia scandens[J]. Fitoterapia, 2006,
77(5): 374-7.
2011
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118
HPLC 法测定鸡矢藤药材中环烯醚萜苷含量﹡
王永兵 **，李 全，蒋 毅，刘光耀
苏州颐华生物医药技术有限公司，江苏 苏州，215123
摘 要 目的: 采用高效液相色谱和二极管阵列检测器法（HPLC-DAD）同时测定鸡矢藤药材中 3
种主要环烯醚萜苷类成分的含量，研究不同产地鸡矢藤药材中环烯醚萜苷的含量。 方法: 色谱分析采用
Shim-pack VP-ODS C18 分析柱，0.1%的甲酸（A）和乙腈（B）为流动相进行梯度洗脱，检测波长：240 nm。
结果: 3个环烯醚萜苷[鸡矢藤苷酸（1）、鸡矢藤苷（2）、鸡矢藤苷酸甲酯（3）]在标准曲线内均有较好的线性
（r2>0.9999），且该方法具有良好的精密度，灵敏度，重复性和准确度；应用所建立的方法同时测定了 8 批鸡
矢藤药材中三个环烯醚萜苷的含量。 结论: 本文首次报道建立 HPLC-DAD方法同时测定鸡矢藤中主要
环烯醚萜苷类成分含量，该方法简单、快速、可靠，适用于鸡矢藤药材及其制剂的质量控制。
关键词 鸡矢藤；环烯醚萜苷；HPLC-DAD；含量测定
中图分类号 R927.2 文献标志码 A 文章编号 1673-7806（2011）02-115-05
作者简介 王敏，女，硕士生
E-mail: wangmin0106@hotmail com
通讯作者 马世平，男，教授，博士生导师
E-mail: shipingma@hotmail.com
收稿日期 2010-11-01 修回日期 2011-02-28
*
抑肝散抗小鼠脑缺血/再灌注损伤的作用研究(Ⅰ)
王 敏，傅 强，马占强，马世平 *
中国药科大学中药药理教研室，南京 211198
摘 要 目的：观察抑肝散对小鼠大脑中动脉缺血再灌注的影响。方法：小鼠连续灌胃 7d，末
次给药 1h 后通过线栓法建立小鼠大脑缺血再灌注模型，测定模型鼠的神经功能变化、脑梗塞范
围、脑含水量、脑指数、脑组织形态、血浆中 NO、总 NOS、iNOS含量。 结果：抑肝散明显改善小鼠
脑缺血后的行为状态， 缩小缺血范围， 使缺血组织病变程度减轻， 降低因脑缺血造成 NO、总
NOS、iNOS的升高。 结论：抑肝散对小鼠脑缺血再灌注损伤有明显的保护作用。
关键词 抑肝散；缺血再灌注；一氧化氮；脑组织；总 NOS；iNOS
中图分类号 R965.1 文献标志码 A 文章编号 1673-7806（2011）02-119-04
缺血性脑血管病是一种常见病和多发病，是目
前严重危害人类健康与生命的主要疾病之一，因具
高发病率、高死亡率、高致残率和高复发率等特点
而引起国内外医学界的广泛关注与高度重视 [1]。 抑
肝散出自于《保缨撮要》，由当归、钩藤、茯苓、白术、
川芎、柴胡及甘草等组成，主治神经症、不眠症、小
儿夜啼等。 近年来，中日两国学者应用该方治疗血
管性痴呆、急性缺血性脑病和中风后遗症，疗效显
著 [2-4]。 本文用小鼠缺血再灌注模型模拟人类脑梗
死，观察抑肝散对脑缺血的保护作用。
1 材料与方法
1.1 药品和试剂
抑肝散方中各药材均购于南京市金陵大药房并
经本校秦民坚教授鉴定： 当归为 Angelica sinensis
(Oliv.) Diels 的根， 钩藤为 Unearia rhynchophy lia
(Miq.) Jacks 的干燥带钩茎枝 ， 川芎为 Ligusticum
药学与临床研究
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研 究
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