Establishment of a Method for Determination of Anemoside B4 Content in Pulsatilla Water Extract

Jianfang WANG

Beijing Key Laboratory of TCVM,Beijing University of Agriculture,Beijing 102206,China

Establishment of a Method for Determination of Anemoside B4 Content in Pulsatilla Water Extract

Jianfang WANG*

Beijing Key Laboratory of TCVM,Beijing University of Agriculture,Beijing 102206,China

[Objective]This study aimed to establish a new method for determination of anemoside B4 content in pulsatilla water extract.[Method]Using acetonitrile-water (28:72)as the mobile phase,the high performance liquid chromatography,equipped with UV detector,was used to determine the anemoside B4 content in pulsatilla water extract. [Result]In the concentration range of 300-800 μg/ml,anemoside B4 content showed a good linear relationship with peak area.The average recovery of anemoside B4 was 98.12%(n=6;RSD=1.37%).[Conclusion]The established method meets the requirements by methodology,and it can be used to determine the anemoside B4 content in pulsatilla water extract.

Pulsatilla;Anemoside B4;High performanceliquid chromatography; Method establishment

P ulsatillareferstothedried roots ofPulsatilla chinensis (Bge.)Regel.It is dug out in spring and autumn.After removing the sediment on the surface,the pulsatilla is dried.Pulsatilla has functions of clearing heat, detoxifying, cooling blood and curing dysentery,so it is often used to cure heat toxin,bloody dysentery,pruitus vulvae and morbid leucorrhea[1-2].In this study,the anemoside B4 content in pulsatilla water extract was determined by high performance liquid chromatography,which is a determination method with strong specificity and reproducibility.

Materials and Methods

Materials

The used equipment and reagents included high performance liquid chromatography(Waters Technology(Shanghai)Co.,Ltd.),ultrapure water(Shanghai Lixin Instrument Co., Ltd.),microporous membrane(Tianjin Keyilong Experimental Equipment Co., Ltd.),anemoside B4 (Batch Number, E-0675;Shanghai Tongtian Biotechnology Co.,Ltd.),pulsatilla(Beijing Tongrentang Co.,Ltd.;Origin,Hebei), pulsatilla water extract(prepared by the Beijing Key Laboratory of TCVM), acetonitrile and methanol(of chromatographic grade;Tianjin Guangfu Institute of Fine Chemicals).

Methods

Chromatography conditionsThe chromatography conditions were as follows:column,Bonna-Agela (5 μm, 150 mm×4.6 mm);mobile phase, acetonitrile-water (28∶72);flow rate, 1.0 ml/min;detection wavelength,205 nm;column temperature,25℃[3].

Preparation of standard solutions

A certain amount(25 mg)of anemoside B4 standard was weighed accurately and dissolved in methanol to 25 ml.The standard solution of 1 mg/ml was prepared.Certain amounts(1.5, 2.0,2.5,3.0,3.5 and 4.0 ml)of previously prepared standard solution(1 mg/ml)were diluted with methanol to 5 ml,respectively.Thus,the standard solutions of 300,400,500,600,700 and 800 μg/ml were prepared.

Preparation of pulsatilla solutionsA certain amount (4.0 g)ofdried anemoside B4 sample was dissolvedin 100 ml of distilled water,and then heated to reflux for 60 min.The solution was cooled and filtered,and finally,90 ml of sample solution was obtained.

Preparation of negative control solutionA certain volume(2 ml)of ultrapure water was filtered and treated as the negative control solution.

Results and Analysis

Methodological analysis

System suitabilityCertain amounts (10 μl for each)of negative control solution,anemoside B4 standard solution and anemoside B4 sample solution were automatically injected into the high performance liquid chromatography.As shown in Fig.1,the peak of anemoside B4 appeared around the retention time of 8.5 min,and it was also shown on the chromatogram of anemoside B4 sample solution,but was not shown on the chromatogram of negative control solution.Under above chromatography conditions,the anemoside B4 could be separated with other components in the sample solution with a resolution of 5.87.According to the peak of anemoside B4,the theoretical plate number was calculated as 4 150.

LinearrelationshipCertain amounts(10 μl for each)of anemoside B4 standard solutions with concentrations of 300,400,500,600,700 and 800 μg/ml were loaded automatically into the high performance liquid chromatography.After the processing,the peak area of anemoside B4 for each concentration was calculated.Treating anemoside B4 concentration(μg/ml) as the abscissa and peak area of anemoside B4 as the ordinate,the regression equation was deduced:

y=2 426.1x+3 345.9,R2=0.999 7 (r=0.999 8).

The results showed that within the concentration range of 300-800 μg/ml, the peak area of anemoside B4 had a good linear relationship with anemoside B4 concentration(Fig.2).

PrecisionThe anemoside B4 concentration in the anemoside B4 standard solution was determined 6 times under the same chromatography conditions.The relative standard deviation (RSD)ofobtainedpeakareasof anemoside B4 was 0.21%,indicating the HPLC instrument has a good precision.

ReproducibilityThe anemoside B4 concentration in the same-batch pulsatilla water extract was determined 6 times under the same chromatography conditions.The results showed that the RSD of obtained peak areas of anemoside B4 was 0.93%,indicating that the established method has a good reproducibility.

Solution stabilityThe anemoside B4 concentration in the same-batch pulsatilla water extract was determined 0,1,4,8,12 and 24 h after the preparation respectively. The results showed that within 24 h,the RSD of peak areas of anemoside B4 was 1.03%,indicating that the prepared sample solution was stable within 24 h.SamplingrecoveryA certain amount(9 ml)of pulsatilla water extract,in which the anemoside B4 concentration was known,was mixed with a certain amount(1 ml)of anemoside B4 standard solution.The anemoside B4 concentration in the new solution was determined 6 times.The results showed that the average recovery of the 6 samples was 98.12%,and RSD was 1.37%,indicating that the established method has a good sampling recovery.

Determination of samples

The anemoside B4 concentration in the prepared sample solution was determined under the samechromatography conditions.The anemo-side B4 content in the sample solution was calculated by one point external standard method.The results showed that the anemoside B4 concentration in the sample solution was 579.3 μg/ml,and in the dried anemoside B4 sample was 13.03 mg/g.

Conclusions and Discussion

In the experiment,it was found that the column temperature showed great effect on peak flowing out of anemoside B4.At the temperature of 25℃,the retention time of anemoside B4 peak was 8.544 min,while at the temperature of 30℃,the retention time was changed to 19.301 min.In conclusion,the established method meets the requirements by methodology,and it can be used to determine the anemoside B4 content in pulsatilla water extract.

[1]State Pharmacopoeia Commission of China(国家药典委员会).Pharmacopoeia of the People’s Republic of China(中华人民共和国药典(一部))[S]. Beijing:China Medical Science Press (北京:中国医药科技出版社),2010.

[2]WU FY(吴凤英),YOU BG(游本刚), TANG LH(唐丽华),et al.Determination of pulchinenoside B4 from Pulsatillae radix(白头翁药材中白头翁皂苷B4的测定 )[J].Chinese TraditionalPatent Medicine(中成药),2011,33(6):1021-1024.

[3]QU LM(曲龙妹),ZHAO CJ(赵春杰),LI D (李丹),et al.Determination of pulchinenoside B4 in Pulsatillae decoction by RP-HPLC(RP-HPLC法测定白头翁汤中白头翁皂苷 B4的含量)[J].Journal of Shenyang PharmaceuticalUniversity (沈阳药科大学学报),2009,26(1):45-47.

Responsible editor:Tingting XU

Responsible proofreader:Xiaoyan WU

白头翁水提液中白头翁皂苷B4含量检测方法的建立

王建舫*
（北京农学院兽医学（中医药）北京市重点实验室，北京102206）

[目的]建立白头翁水提液中白头翁皂苷B4含量检测的方法。[方法]以乙腈-水（28∶72）为流动相，采用高压液相色谱仪和紫外检测器进行测定。[结果]在300～800 μg/ml范围内，白头翁皂苷B4浓度和峰面积的线性关系良好，白头翁皂苷B4的平均回收率为98.12%（n=6），RSD为1.37%。[结论]该方法符合方法学考察的规定，可作为白头翁水提液中白头翁皂苷B4含量测定的方法。

白头翁；白头翁皂苷B4；高效液相色谱；方法建立

北京市教育委员会面上项目（KM201410020007)。

王建舫（1969-），女，河北肃宁人，实验师，从事中兽药防治动物疾病的研究，E-mail：wjfhlx@126.com。*通讯作者。

2015-06-05

修回日期 2015-07-13

Supported by GeneralProgram ofBeijing MunicipalEducation Commission (KM201410020007).

*Corresponding author.E-mail:wjfhlx@126.com

Received:June 5,2015 Accepted:July 13,2015