Simultaneous determination of Saikosaponin a, c and d indifferent parts of wild bupleurumfrom Shanxi Province
Rui Wang*, Ximei Yuan, Boya Ju, Rui Liu, Hongxia Yuan
School of Traditional Chinese Materia Medica, Shanxi University of Traditional Chinese Medicine, Jinzhong 030619, China
 
 
Abstract: Asa traditional Chinese herbal medicine exhibiting analgesic, fever-reducing and anti-inflammatory effects, Radix Bupleuri (Chai-Hu) is commonly used for the treatment of influenza, which is derived from the dried roots of Bupleurum chinense DC. and Bupleurum scorzonerifolium Willd. Among of diverse chemical components, saikosaponins are the key active components of the herb medicine. In the present study, we established a method of high performance liquid chromatography (HPLC) coupled with evaporative light scattering detection (ELSD) for simultaneous determination of saikosaponin a, c and d in root, stem, leaf and flower of Bupleurumchinense (B chinense) collected from different areas of Shanxi Province, China. The results from 16 samples of root, stem, leaf and flower of B chinense demonstrated that the total contents of the three saikosaponins in the root of B chinense collected from Dongshan Taiyuan, Xishan, Tianlongshan and Pangquangou were 4.26 mg/g, 3.22 mg/g, 4.23 mg/g and 3.05 mg/g, respectively. However, there was scarcely any saikosaponins in the stem, leaf and flower of B chinensecollected from above-mentioned areas. The method of HPLC coupled with ELSD was suitable for quality control of Radix Bupleuri. The result also confirmed that the root of B chinense was the best medicinal part. 
Keywords: Bupleurum chinense DC.; Different parts; Saikosaponins; Content analysis  
CLC number: R284                Document code: A                 Article ID: 10031057(2018)1070308 
 
 
Abstract: Asa traditional Chinese herbal medicine exhibiting analgesic, fever-reducing and anti-inflammatory effects, Radix Bupleuri (Chai-Hu) is commonly used for the treatment of influenza, which is derived from the dried roots of Bupleurum chinense DC. and Bupleurum scorzonerifolium Willd. Among of diverse chemical components, saikosaponins are the key active components of the herb medicine. In the present study, we established a method of high performance liquid chromatography (HPLC) coupled with evaporative light scattering detection (ELSD) for simultaneous determination of saikosaponin a, c and d in root, stem, leaf and flower of Bupleurumchinense (B chinense) collected from different areas of Shanxi Province, China. The results from 16 samples of root, stem, leaf and flower of B chinense demonstrated that the total contents of the three saikosaponins in the root of B chinense collected from Dongshan Taiyuan, Xishan, Tianlongshan and Pangquangou were 4.26 mg/g, 3.22 mg/g, 4.23 mg/g and 3.05 mg/g, respectively. However, there was scarcely any saikosaponins in the stem, leaf and flower of B chinensecollected from above-mentioned areas. The method of HPLC coupled with ELSD was suitable for quality control of Radix Bupleuri. The result also confirmed that the root of B chinense was the best medicinal part. 
Keywords: Bupleurum chinense DC.; Different parts; Saikosaponins; Content analysis  
CLC number: R284                Document code: A                 Article ID: 10031057(2018)1070308
 
 
1. Introduction
The Chinese herbal medicine Radix bupleuri (Chai-Hu in Chinese) was first recorded in Shen Nong Ben Cao Jing, and it has analgesic, fever-reducing and anti-inflammatoryeffects. It is commonly used for treatments of respiratory infections, bronchitis and pneumonia[1,2]. Radix bupleuri is derived from the dried roots of Bupleurum chinense DC. and Bupleurum scorzonerifolium Willd, named as Bei-Chai-Hu and Nan-Chai-Hu in Chinese, respectively. Chemical studies have demonstrated that saikosaponins, volatile oil, flavonoids and polysaccharides are the main constituents of the herb[3–5]. The current investigations on Radix bupleuri primarily focus on pharmacological actions[6–13], pharmacokinetics[14–19] and determination of saikosaponins a, c and d[20–26] (Fig. 1). Little information has been achieved about whether other parts (stem, leaf and flower) of B chinense DC. or B scorzonerifolium Willd contain similar chemical constituents as roots. Therefore, in the present study, we established a method of HPLC coupled with evaporative light scattering detection (ELSD) and determined contents of saikosaponins a, c and d in roots, stems, leaves and flowers of B chinense DC collected from Shanxi Province in China. 
 
 
 
Figure 1. Chemical structures of saikosaponins a, c and d. 
 
2. Experimental section
2.1. Materials and reagents
Saikosaponin a, c and d were purchased from Shanghai Winherb Medical S&T Development Co., Ltd. (Batch No. 081026; purity>98%). B Chinense DC. was  collected from Tianlongshan, Taiyuan (2011.10.8), Dongshan,Taiyuan (2011.10.9), Xishan, Taiyuan (2011.10.16), and Pongquangou, Lvliang (2011.9.25), Shanxi Province,China, and these specimens were identified as B chinenseDC. by Prof. XiangpingPei, School of Traditional ChineseMateria Medica, Shanxi University of Traditional ChineseMedicine. Aqueous ammonia and methanol used for HPLC were of analytical grade and obtained from Tianjin Kemiou Chemical Reagent Co., Ltd. Acetonitrile was of HPLC-grade and provided by DiKMA.
2.2. HPLC instrumentation and chromatographic conditions
Waters 2996 HPLC system (Waters, USA) and Diamonsil RP-C18 column (4.6 mm×250 mm, 5 μm) were used in the experiments. Mobile phase contained acetonitrile and water. The gradient elution was performed as shown in Table 1. Flow rate was set at 1.0 mL?min–1, and column temperature was maintained at 30 ºC. ELSD was used for monitoring.  
 
Table 1. HPLC gradient elution program.
  
 
2.3. Preparations of reference solution and sample solution
Saikosaponin a, c and d were accurately weighed (10.42 mg, 10.00 mg and 10.15 mg, respectively) and placed in 1-mL volumetric flask, respectively, followed by complete dissolution with 1 mL methanol. Above-mentioned solutions (0.5 mL) were taken into 5-mL volumetric flask and completely dissolved with 5 mL methanol to obtain a mixed reference stock solution with concentrations of saikosaponin a, c and d at 1.042, 1.000 and 1.015 mg?mL–1 respectively.
Roots, stems, leaves and flowers of B chinense DC. were placed in the shade to dry, powdered and passed through 80-mesh screen to obtain sample powder. About 0.5 g of the samples was placed in 150-mL flask, extracted with 25 mL ammonia–methanol solution (1:9, v/v) on ultrasonic apparatus for 40 min and then filtered. The filtrate was evaporated in vacuum at 60 ºC. The residue was completely dissolved in volumetric flask for subsequent detection.
3. Results and discussion
3.1. Sample pretreatment method
L9 (33) orthogonal assay was used for evaluation of extract method for the root of B Chinense collected from Dongshan Taiyuan at various volume ratios of ammonia to methanol, volumes of extracting solution and sonication times. The factors and levels of orthogonal test were shown in Table 2, and the results and analysis of orthogonal test were shown in Table 3. 
 
Table 2. Factors and levels of orthogonal test.
 
 
 
Table 3. Results and analysis of orthogonal test.
   
 
Table 2 and Table 3 demonstrate that A3B3C3 was the optimal scheme of orthogonal test. However, the addition of the solvent had less influence, which was the secondary factor. Therefore, the optimal method was confirmed to be sonication with 25 mL 10% ammonia–methanol solution for 40 min (A3B2C3).
3.2. Linearity
The mixed reference stock solution (0.05 mL, 0.1 mL,0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL and 1.0 mL) was added into 1-mL volumetric flasks and completely dissolved with methanol, respectively. The concentrations of saikosaponin a of the mixed reference working solutions were 0.0521 mg/mL, 0.1042 mg/mL, 0.2084 mg/mL, 0.4168 mg/mL, 0.6252 mg/mL, 0.8336 mg/mL and 1.042 mg/mL, respectively.Those of saikosaponin c were 0.050 mg/mL, 0.100 mg/mL, 0.200 mg/mL, 0.400 mg/mL, 0.600 mg/mL, 0.800 mg/mL and 1.000 mg/mL, and those of saikosaponin d were0.0575 mg/mL, 0.1015 mg/mL, 0.203 mg/mL, 0.406 mg/mL, 0.609 mg/mL, 0.812 mg/mL and 1.015mg/mL, respectively. In addition, 20 μL of each of the reference working solution was injected into the column for separation. Linear regression analysis was made between the logarithm of peak area Y and the logarithm of amount X injected. The correlation coefficients of all the calibration curves were higher than 0.9968 (Table 4).  
 
 
Table 4. Statistical analysis of the linear regression equation employed in the determination of Saikosaponin a, c and d. 
 
aIn the regression equation y = ax + b; y refers to the log-transformed peak area; x to the log-transformed concentration of the reference saikosaponins (μg).
 
 
3.3. Limits of detection (LOD) and limits of quantification (LOQ)
LOD and LOQ under the present chromatographic conditions were determined on the basis of response and slope of each regression equation at a signal-to-noise ratio (S/N) of 3 and 10, respectively. The LOD and LOQ ranged within 0.26–0.51 μg and 1.00–1.02 μg, respectively (Table 4).
3.4. Precision
The precision of the developed assay was determinedfor intra and inter-day variations. The intra-day variation was determined by analyzing the same mixed standardsolution for three times within 1 day. While for inter-day variability test, solution was examined in triplicate for three consecutive days. The R.S.D.s of retention times and peak areas were taken as a measure tool, which were less than 4.62% for all three compounds (Table 5). 
 
Table 5. Precision and recovery of the analytical method for the three saikosaponins.
 
aThe concentration (mg/mL) of each compounds was: saikosaponin c (0.200); saikosaponin a (0.2084); saikosaponin d (0.203). bRetention time. cPeak area. 
 
3.5. Recovery
In order to examine the recovery of the developed method, the standards of three saikosaponins were added to samples at three concentration levels (approximatelyequivalent to 0.5, 1.0 and 1.5 times of the concentration of the matrix) with three parallels at each level. The solutions were prepared in accordance with the sample preparation procedure, and the mean recovery was calculated for three assays of the standard. The results showed that the recovery of these spiked standards ranged from 91.3% to 92.7%, showing satisfactory recovery (Table 5).
3.6. Determination of three saikosaponins in different parts of B chinense
The chromatogram of the mixed reference standards was made on 5 μL and 15 μL injections of the mixed reference stock solution described in 3.2 (the concentrations of saikosaponin a, c, and d were 0.2084 mg/mL, 0.200 mg/mL and 0.203 mg/mL, respectively). The retention times of saikosaponin a, c, and d were 18.8 min, 14.4 min and 24.8 min, respectively (Fig. 2). 
 
 
Figure 2. Typical chromatograms of mixed reference standards of saikosapinin a, c, and d (A) and the root of B chinense collected from Dongshan, Taiyuan (B). 1: saikosaponin a, 2: saikosaponin c, 3: saikosaponin d. 
 
About 0.5 g powdered samples of the root, stem, leaf and flower of wild B chinense collected from different locations of Shanxi were accurately weighed, and samplesolutions were prepared according to the method described in 2.3. The contents of saikosaponins were determined as shown in Table 6. 
 
Table 6. The contents of saikosaponins in different parts of B chinense. 
  
aAverage of triplicates; bUnder detection limit; cUnder quantification limit. 
 
The contents of the three saikosaponins were differentamong different samples of different collection locationsas shown in Table 6. The total contents of the saikosaponins were more than 3 mg/g, and the content of saikosaponin c was the least in all samples. Although saikosaponin a, c and d were detected in the leaves of samples collected from Pangquangou, Lvliang, they were not detected in the stems, leaves and flowers of samples collected from Dongshan, Taiyuan, Xishan, Taiyuan and Tianlongshan, Taiyuan.  
Although saikosaponin a, c and d were well separated by using gradient elution in the preliminary test, the drifting baselines in chromatography owing to gradient elution made them hard for quantification as shown in Figure 3. This problem could be effectively solved by the present validated HPLC-ELSD method. 
 
 
 
Figure 3. Chromatogram of the root of B chinense collected from Dongshan, Taiyuan by HPLC-UV. 1. saikosaponin a, 2. saokosaponin c, 3. saikosaponin d. 
 
  
The favorable factors were all appeared to be the maximum level in first orthogonal experiment. Then in a second orthogonal experiment, the volume ratio of ammonia and methanol was increased to 15%, the volume of extracting solution was 25 mL, and the extraction time was 50 min. There was no significant difference between the total contents of saikosaponin a, c and d detected by the increased level method and those detected by the present validated method. Therefore, the present validated method was established as the optimal method.
The result showed that this validated HPLC-ELSD method was simple and easy to operate with good accuracy, precision and satisfied separation. Therefore, it could be applied as a determination method for saikosaponin a, c and d. Meanwhile, the result also showed that small amounts of saikosaponin a, c andd were detected in the leaves of B chinense of Pangquangou, Lvliang, which might be associated with the collection time and local climate.
4. Conclusions
In conclusion, a simple and accurate HPLC-ELSD method was developed for simultaneous determination of three major saikosaponins in Bchinense, and the method was suitable for quality control of the species of Bupleurum. Our findings also proved that the medicinal part of the herb was the root.
Acknowledgements
This work was supported by Shanxi Educational Committee (Grant No. 20111113) and Shanxi Science and Technology Department (Grant No. 2016ZD0201).
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山西野生柴胡不同部位柴胡皂苷acd的含量测定
王瑞*, 闫玺镁, 巨博雅, 刘瑞, 原红霞
山西中医药大学中药学院, 山西 晋中 030619       
摘要: 柴胡为伞形科植物柴胡或狭叶柴胡的干燥根, 具有镇痛、解热、抗炎之功效, 常用于流感的治疗。柴胡皂苷是柴胡的主要活性成分, 本研究采用高效液相色谱法, 以蒸发光散射检测器进行检测, 对山西不同产地野生柴胡(北柴胡)的根、茎、叶、花中柴胡皂苷acd进行了含量测定, 结果表明, 东山、西山、天龙山、庞泉沟采集的柴胡根中3种柴胡总含量分别为4.26 mg/g, 3.22 mg/g, 4.23 mg/g3.05 mg/g。然而, 上述地区的柴胡茎, 叶和花中几乎不含有柴胡皂苷。高效液相色谱-ELSD法适用于柴胡药材的质量控制, 本研究的结果也验证了北柴胡以根入药的传统入药习惯。 
关键词: 野生柴胡; 不同部位; 皂苷类成分; 含量分析
  
  
Received: 2018-06-18, Revised: 2018-07-20, Accepted: 2018-08-16.
Foundation items: Shanxi Educational Committee (Grant No. 20111113)and Shanxi Science and Technology Department (Grant No. 2016ZD0201).
*Corresponding author. Tel.: +86-0351-3179903, E-mail: wrgreentea@163.com    
  

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