全 文 ：天然产物研究与开发 Nat Prod Res Dev 2011，23:1095-1098，1138
文章编号:1001-6880(2011)06-1095-05
Received January 27，2010;Accepted June 12，2010
Foundation Item:This study was supported by the National Key Tech-
nologies R ＆ D Program of China (2007BAI45B00)．
* Corresponding author E-mail:taoyanduo@ 163． com
HPLC-DAD法测定血满草中熊果酸和齐墩果酸的含量
罗智敏1，2，Elisa linares Navarro3，陶燕铎1* ，邵 赟1，梅丽娟1
1中国科学院西北高原生物研究所，西宁 810008;2 中国科学院研究生院，北京 100049;
3巴伦西亚大学，C /塞尔皮斯，N°6，6，巴伦西亚，西班牙
摘 要:建立了 HPLC-DAD法测定血满草中熊果酸和齐墩果酸含量，并进行方法学考察。采用 HPLC-DAD进行
分析，fusion-RP C18柱(4． 6 mm × 250 mm，4 μm) ，甲醇-0． 2%磷酸水溶液(90∶ 10)为流动相，检测波长 210 nm，体
积流量 1． 0 mL /min。同时采用微波辅助提取、回流提取、索氏提取、冷浸提取、超声提取五种方法对血满草中熊
果酸和齐墩果酸含量进行测定并比较不同方法所得结果的差异，还比较了血满草不同部位中熊果酸和齐墩果
酸的含量差异。测定结果表明熊果酸进样量在 3． 6 ～ 8． 4 μg范围内，齐墩果酸进样量在 3． 2 ～ 16 μg范围内，呈
良好线性关系。血满草中熊果酸和齐墩果酸平均回收率分别为 98． 3%和 101． 4%(n = 5) ，相对标准偏差分别为
1． 13%和 0． 72%(n = 5)。五种方法比较得出索氏提取得熊果酸和齐墩果酸含量最高;血满草花中熊果酸和齐
墩果酸含量最高，而根中含量最低。该方法使血满草中熊果酸和齐墩果酸达到基线分离，操作简便，结果稳定
可靠。
关键词:血满草;HPLC-DAD;熊果酸;齐墩果酸
中图分类号:R284. 1 文献标识码:A
Determination of Ursolic Acid and Oleanolic Acid in
Sambucus adnata Wall by HPLC-DAD
LUO Zhi-min1，2，Elisa linares Navarro3，TAO Yan-duo1* ，SHAO Yun1，MEI Li-juan1
1Northwest Plateau Institute of Biology，Chinese Academy of Sciences，Xining 810008，China;2Graduate University
of Chinese Academy of Sciences，Beijing 100049，China;3Valencia university C / Serpis N°6，6，Valencia，Spanish
Abstract:The study aimed to determine the content of ursolic acid and oleanolic acid in Sambucus adnata Wall using
HPLC-DAD． By this method，fusion-RP C18(4． 6 mm × 250 mm，4 μm)was used with methanol-0． 2% phosphate in wa-
ter (90∶ 10)as the mobile phase and UV detection was at 210 nm． In addition，five different extraction methods inclu-
ding microwave-assisted extraction，reflexing extraction，soxhlet extraction，soaking extraction，and ultrasonic extraction
were used for determine the content of ursolic acid and oleanolic acid in different parts of S． adnata Wall and the results
were compared． Within the injection volume range between 3． 6 and 8． 4 μg for ursolic acid and between 3． 2 and 16 μg
for oleanolic acid，the results had good linearity． The average recovery was 98． 3% (RSD = 1． 13%，n = 5)for ursolic
acid and 101． 4% (n = 5) (RSD = 0． 72%，n = 5)for oleanolic acid． The highest content of ursolic acid and oleanolic
acid was obtained by soxhlet extraction methods comparing with other methods． In addition，the flower of S． adnata Wall
had the highest content of ursolic acid and oleanolic acid and the root had the lowest amount． Conclusively，the HPLC-
DAD is a stable and credible method for determining the content of ursolic acid and oleanolic acid in S． adnata Wall with
satisfactory baseline separation for these two kinds of acids．
Key words:Sambucus adnata Wall;HPLC-DAD;ursolic acid;oleanolic acid
Introduction
Sambucus adnata Wall is a tibetan medicine for the
treatment of acute and chronic nephritis，rubella，fractu-
ring of the bones，bruise，rheumatoid arthritis，the pain
of waist and leg，bleeding and some other injuries［1，2］．
And it was firstly written down in Illustrated Catalogue
of Plants in A． D． 1848［3］． So it is one of the precious
and unique herbs and has a long history． It is distribu-
ted in Tibet，Qinghai，Ningxia，Gansu，Sichuan，
DOI:10.16333/j.1001-6880.2011.06.022
Guizhou，Yunnan and Shanxi province in China［4］，and
grows under the forests and shrubs beside rivers at the
altitude of 1600-3600 m． S． adnata wall is belong to the
family of Caprifoliaceae． Sambucus has about 20 species
which always are distributed in temperate and subtropi-
cal regions of the world． China has about 5 species in-
cluding S． adnata Wall． ex DC，S． chinensis Lindl．，S．
williamsii Hance，S． sinirica Nakai and S． nigra
Linn［5］． And there are many reports about their phar-
macology function including anti-visus and anti-
germ［6-8］，anti-inflammatory and antinociceptive ［9］，an-
tioxidant［10，11］，antidiabetics［11］，immunity activity［12］，
antiosteoporotic activity［13］， anti-HIV［14］， antitu-
mour［15］，etc． So this genus of plants are commonly
used． However，there is no research about the quality
control of S． adnata Wall． For the full development and
scientific utilization of this plant，it is necessary to es-
tablish a standard of quantitative analysis．
Some reseachers studied on the chemical constituents of
S． adnata［16，17］，and ursolic acid (UA) ，oleanolic acid
(OA) ，lariciresinol p-hydroxybenzoic and some other
compounds were isolated and identificated． Many re-
searchers reported about the important pharmacological
effects of OA and UA，so we chose UA and OA for
quantitative analysis to provide a basis for its quality
control．
Materials and Reagents
Apparatus:Agilent1200 high performance liquid chro-
matography ［quaternionic pump，DAD detector］;
AG204 electronic analytical balance．
Reagents:standard of ursolic acid and oleanolic acid
were obtained from the National Institute for the Control
of Pharmaceuticals and Biological Products，Beijing，
China;Methanol(HPLC grade) ;Ethanol and phosphor-
ic acid(analyze grade) ;Ultra pure water with a resist-
ance greater than 18 M?was collected from a certified
Millipore Milli-Q system．
Materials:The whole herb samples of S． adnata were
collected from Xunhua county in Qinghai province and
authenticated by Mei Li-juan，a senior engineer of
Northwest Institute of Plateau Biology．
Materials and Methods
Chromatography
The HPLC system HP 1200 series，equipped with the
ChemStation software(Agilent Technologies)and com-
prised a binary high-pressure pump，an online vacuum
degasser，an auto-sampler，a thermostated column com-
partment and a photodiode array detector using a maxi-
mum plot in the range 190-800 nm，was used for the
chromatographic analysis． All separations were carried
out on a fusion-RP C18 column(4． 6 mm × 250 mm，4
μm particle size)． The mobile phase was methanol and
0． 2% phosphate solution(methanol /phosphate solution
= 90 /10，v /v)． The flow rate of mobile phase was set
at 1． 0 mL /min． The injection volume was 10 μL，and
the column temperature was maintained at 25 ℃ ．
Preparation of standard solutions
6． 0 mg of ursolic acid and 16． 0 mg of oleanolic acid
were accurately weighed and placed in 10 mL volumet-
ric flask respectively，and added methanol to the scales
and shaked the flask to mixing;0． 6 mg /mL ursolic
acid standard reference and 1． 6 mg /mL oleanolic acid
standard reference were prepared．
Preparation of sample solutions
Samples were powdered by an electrical blender and
sieved through a 60-mesh sieve． 3． 0 g powder was
transferred into a 250 mL flask and added 125 mL
methanol into it． The soxhlet method was used and the
extracting time was 4 h． Afrer cooling，the mixture was
filtered，and the residue was washed with 10 mL metha-
nol，mixed with the filtrate． The filtrate was dried and
left about 20 mL by Rotary Evaporator，and metered
volume to a 25 mL volumetric flask． An aliquot of 10
μL of the final solution filtered through a 4． 5 μm Mil-
lipore filter membrane and was injected for the HPLC
analysis．
Validation
Linearity
Acceptable linearity was observed over the range of in-
pouring from 6 to14 μL，using ursolic acid as spike
standard and the typical calibration curves had a re-
gression equation of y = 37． 31x + 314． 125，r =
0. 9999． And acceptable linearity was observed over the
6901 Nat Prod Res Dev Vol. 23
range of inpouring from 2 to10 μL，using oleanolic acid
as spike standard and the typical calibration curves had
a regression equation of y = 731． 94x + 27． 74，r =
0. 9999．
Stability
Detecting the solution of S． adnata，0，7，14，21 h，re-
spectively，after chromatographic conditions in the a-
bove-mentioned sample，the results of ursolic for peak
area RSD was 0． 62%，oleanolic acid for peak area
RSD was 2． 61%，indicating that the sample was a sta-
ble solution in 21 h．
Precision and accuracy
The precision of the assay was determined from the
samples by replicate analyses of five concentration lev-
els of ursolic acid(0． 36，0． 48，0． 60，0． 72 and 0． 84
mg /mL)and oleanolic acid(0． 32，0． 64，0． 96，1． 28，
1． 60 mg /mL) ，and the results of ursolic for peak area
RSD was 0． 97%，oleanolic acid for peak area RSD was
1． 03%，indicating that the assay was precise．
reproducibility
The reproducibility of the assay was determined by rep-
licate analyses of five extraction of the same sample，
and the results of ursolic for peak area RSD was
1. 84%，oleanolic acid for peak area RSD was 2． 21%，
indicating that the assay had a good reproduce．
Extraction recovery
Table 1 showed that the extraction recoveries of ursolic
acid and oleanolic acid were determined． Recoveries
were calculated by comparing the contents obtained
from five extracted samples with the contents from five
adding ration standard solutions into the samples，re-
spectively，and following 3． 3 to prepare the sample so-
lution．
Table 1 Recovery of ursolic acid and oleanolic acid from S． adnata
Reference Content of sample
(mg)
Adding
(mg)
Detected
(mg)
Recovery
(%)
Average
(%)
RSD
(%)
Ursolic acid 12． 45 1． 20 13． 21 96． 8 98． 3 1． 13
12． 33 13． 29 98． 2
12． 51 13． 44 98． 0
12． 39 13． 57 99． 9
12． 41 13． 49 98． 5
Oleanolic acid 6． 26 3． 20 9． 64 101． 9 101． 4 0． 72
6． 31 9． 58 100． 7
6． 34 9． 59 100． 5
6． 29 9． 67 101． 9
6． 40 9． 79 102． 0
Determination of samples
Detecting the solution of samples and substance refer-
ences after chromatographic conditions in the above-
mentioned sample，and got the integral of peak areas，
and calculated the contents of ursolic acid and oleanol-
ic acid in the samples using external reference method
(Fig． 1)．
Fig. 1 HPLC Chromatograms of reference substances(A，B)and the sample of Sambucus adnata Wall
A:oleanolic acid;B:ursolic acid;C:the sample of Sambucus adnataWall 1． oleanolic acid 2． Ursolic acid
7901Vol. 23 LUO Zhi-min，et al:Determination of Ursolic Acid and Oleanolic Acid in Sambucus adnata Wall by HPLC-DAD
Comparision of five different extraction methods
5 powders were weighed and each 3． 0 g，each of them
was transferred into a 100 mL flask and 60 mL metha-
nol was added，Microwave-assisted extraction(1 h，80
mA) ，refluxing extraction(1 h，80 ℃) ，soxhlet extrac-
tion(4 h，80 ℃) ，soaking extraction(8 h，ordinary tem-
perature)and ultrasonic extraction(1 h，ordinary tem-
perature)were used． Afrer cooling，the mixture was fil-
tered，and the residue was washed with 10 mL methanol
and mixed with the filtrate． The filtrate was dried and
left about 20 mL by Rotary Evaporator，and metered
volume to a 25 mL volumetric flask． An aliquot of 10
μL of the final solution filtered through a 4． 5 μm Mil-
lipore filter membrane and was injected for the HPLC
analysis，respectively．
Comparision of different part of Sambucus adnata
Wall
The powder of root，stem，leaves，flower were weighed，
each of them was 3． 0 g，after chromatographic condi-
tions in the above-mentioned sample，recorded the peak
area，and calculated the ratio using external reference
method．
Result and Discussion
Optimization of the extraction method and solvent:Ac-
cording the solubility of ursolic acid and oleanolic
acid，methanol and ethanol were choosed as the solvent
(60% methanol，80% methanol，absolute methanol，
absolute ethanol) ，and five different extraction method
were compared． The result is that absolute methanol is
the best solvent，and soxhleting extraction is better than
other methods (Table 2)．
Table 2 The result of five different extraction methods
Extraction method UA content /% OA content /%
Microwave-assisted 0． 33 0． 18
Refluxing 0． 22 0． 14
Soxhlet 0． 42 0． 21
Soaking 0． 37 0． 18
Ultrasonic 0． 27 0． 15
Choosing the best chromatography conditions:ursolic
acid and oleanolic acid are isomeride of triterpenic
acid，and they have the similar structure，so it is differ-
ent to be separated using HPLC． methanol /water and
methanol /0． 2% phosphoric acid solution (85 ∶ 15，87
∶ 13，90 ∶ 10)were compared，and GimininC18 column
(4． 6 mm ×250 mm，5 μm)and fusion-RP C18 column
(4． 6 mm ×250 mm，4 μm)were compared． The result
is that methanol /0． 2% phosphoric acid solution (90∶
10)and fusion-RP C18 column is better．
Comparing the different part of samples:The content of
ursolic acid and oleanolic acid is getting higher and
higher from the table 3，and this result will provide a
useful reference for fully using the different part of
samples．
Table 3 the determination of different part of Sambucus
adnata Wall
Samples UA content /% OA content /%
Root 0． 03 0． 05
Stem 0． 22 0． 11
Leaves 0． 47 0． 27
Flower 0． 73 0． 44
Whole plant 0． 42 0． 21
Conclusion
The content of ursolic acid and ooleanolic acid of S． ad-
nata Wall were determined in this paper． The method
which we built has high sensitivity，reproduction，stabil-
ity and validity，and the speed of determination is fast．
So this paper provided a fast，accurate and effective a-
nalysis method for the quality control and evaluation of
S． adnata．
Acknowledgement We are grateful to Zhang Xing-
wang，Chen Chen and Chi Xiaofeng，for assistance with
plant material collection． We also thank Sun Jianwen
for the useful comments and help in improving the
manuscript． This study was supported by the National
Key Technologies R＆D Program of China (2007BAI45B00)．
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