罗智敏，Elisa linares Navarro，陶燕铎，邵赟，梅丽娟

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

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

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。

血满草;HPLC-DAD;熊果酸;齐墩果酸

Introduction

Sambucus adnata Wall is a tibetan medicine for thetreatment of acute and chronic nephritis，rubella，fracturing 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 distributedinTibet，Qinghai，Ningxia，Gansu，Sichuan，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 subtropical regions of the world．China has about 5 species including S．adnata Wall．ex DC，S．chinensis Lindl．，S．williamsii Hance，S．siniricaNakaiandS．nigra Linn［5］．And there are many reports about their pharmacologyfunctionincludinganti-visusandantigerm［6-8］，anti-inflammatory and antinociceptive［9］，antioxidant［10，11］，antidiabetics［11］，immunity activity［12］，antiosteoporoticactivity［13］，anti-HIV［14］，antitumour［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 establish 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 researchers 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 chromatography［quaternionicpump，DADdetector］; 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 phosphoric acid(analyze grade);Ultra pure water with a resistance 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 comprised a binary high-pressure pump，an online vacuum degasser，an auto-sampler，a thermostated column compartment and a photodiode array detector using a maximum plot in the range 190-800 nm，was used for the chromatographic analysis．All separations were carried out on a fusion-RP C18column(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 volumetric 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 methanol，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 Millipore filter membrane and was injected for the HPLC analysis．

Validation

Linearity

Acceptable linearity was observed over the range of inpouring from 6 to14 μL，using ursolic acid as spike standard and the typical calibration curves had a regression equation of y=37．31x+314．125，r= 0.9999．And acceptable linearity was observed over therange 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，respectively，after chromatographic conditions in the above-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 stable solution in 21 h．

Precision and accuracy

The precision of the assay was determined from the samples by replicate analyses of five concentration levels 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 replicate 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，respectively，and following 3．3 to prepare the sample solution．

Table 1 Recovery of ursolic acid and oleanolic acid from S．adnata

Determination of samples

Detecting the solution of samples and substance references after chromatographic conditions in the abovementioned sample，and got the integral of peak areas， and calculated the contents of ursolic acid and oleanolic 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 WallA:oleanolic acid;B:ursolic acid;C:the sample of Sambucus adnataWall 1．oleanolic acid 2．Ursolic acid

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 methanol was added，Microwave-assisted extraction(1 h，80 mA)，refluxing extraction(1 h，80℃)，soxhlet extraction(4 h，80℃)，soaking extraction(8 h，ordinary temperature)and ultrasonic extraction(1 h，ordinary temperature)were used．Afrer cooling，the mixture was filtered，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 Millipore 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 conditions 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:According 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

Choosing the best chromatography conditions:ursolic acid and oleanolic acid are isomeride of triterpenic acid，and they have the similar structure，so it is different to be separated using HPLC．methanol/water and methanol/0．2%phosphoric acid solution(85∶15，87∶13，90∶10)were compared，and GimininC18column (4．6 mm×250 mm，5 μm)and fusion-RP C18column (4．6 mm×250 mm，4 μm)were compared．The result is that methanol/0．2%phosphoric acid solution(90∶10)and fusion-RP C18column 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

Conclusion

The content of ursolic acid and ooleanolic acid of S．adnata Wall were determined in this paper．The method which we built has high sensitivity，reproduction，stability and validity，and the speed of determination is fast．So this paper provided a fast，accurate and effective analysis method for the quality control and evaluation of S．adnata．

Acknowledgement We are grateful to Zhang Xingwang，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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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-juan11Northwest 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

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 water(90∶10)as the mobile phase and UV detection was at 210 nm．In addition，five different extraction methods including 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 HPLCDAD 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．

Sambucus adnata Wall;HPLC-DAD;ursolic acid;oleanolic acid

January 27，2010;Accepted June 12，2010

This study was supported by the National Key Tech

book=2011,ebook=6

R284.1

A

1001-6880(2011)06-1095-05

nologies R＆D Program of China(2007BAI45B00)．

*Corresponding author E-mail:taoyanduo@163．com