Evaluation on Quality and Utilization Value of Bupleurum falcatum Linne

Risha WEIZE, Yisong LI, Yuan LIU, Zhifeng ZHANG, Hua QIU, Kedu MUGA, Yuming GAO, Ying LI,*

1. College of Pharmacy, Southwest Minzu University, Chengdu 610041, China; 2. Ethnic Medicine Institute, Southwest Minzu University, Chengdu 610041, China; 3. Ganluo County Kangyuan Traditional Chinese Medicine Farmers’ Cooperative, Chengdu 616850, China; 4. Ganluo County Veteran Scientific Workers Association, Chengdu 616850, China; 5. Ziyang Center for Food and Drug Control, Ziyang 512000, China

Abstract [Objectives] This study aimed to evaluate the quality of Bupleurum falcatum Linne interplanted with walnut in Ganluo and study the development and utilization value of other parts. [Methods] The contents of saikosaponins a and d were determined with RP-HPLC, and the contents of 7 kinds of harmful elements were determined by ICP-MS. The quality of B. falcatum Linne was compared with that of other Bupleurum species. [Results]The total saikosaponin content in Radix Bupleurum Falcatum (15.894 mg/g) was higher than that in Radix Bupleuri of other origins (8.748 mg/g). The total saikosaponin content in leaves of B. falcatum Linne (7.518 mg/g) is more than twice the limit promulgated by Chinese Pharmacopoeia. The contents of Al, Cr, Cu, As, Cd, Pb and Hg in B. falcatum Linne were all lower than the limits promulgated by the pharmacopoeia. In short, the quality of Radix Bupleurum Falcatum was better than that of Radix Bupleurum Marginatum (S3, S4) and Radix Bupleurum Sachalinense (S5). The leaves of B. falcatum Linne contained more saikosaponins and less harmful elements. [Conclusions] The method and technology of interplanting B. falcatum Linne with walnut in Ganluo are mature. The quality of the medicinal materials produced is superior, and the leaves are also rich in saikosaponins a and d, and can be used as the raw material for extracting saikosaponins a and d. This study provides a basis for further in-depth research on the cultivation of B. falcatum Linne in the domestic market.

Key words RI-HPLC, ICP-MS, Bupleurum falcatum Linne, Saikosaponin a, Saikosaponin d, Heavy metal, Harmful element

1 Introduction

Radix Bupleurum Falcatum, also known as Chuandao Chaihu and No.31 Chaihu, is the dried root ofBupleurumfalcatumLinne (Umbelliferae)[1]. It is the main raw material of the Kampo preparation Xiaochaihu decoction, which clears heat and detoxifies and is effective in treating fever and is widely used in Japan[2]. In Ganluo, interplantingB.falcatumLinne with walnut has been for ten years. This economic model has achieved initial results. The core area has reached more than 66.67 ha, driving a contiguous area of more than 266.67 ha. Generally, the roots ofB.falcatumLinne are used as medicine, and they are acquired by purchasers every year, while other parts are directly discharged. The comparison of the quality betweenB.falcatumLinne and other Bupleurum species commonly used in China and the utilization value of other parts need to be studied. If the market supply problem is resolved, the income of growers will be increased, and the protection of the resource will be promoted as well.

InChinesePharmacopoeia, Radix Bupleurum refers to the dried roots ofBupleurumchinenseDC. orBupleurumscorzonerifoLium Willd[3]. But at present, other species ofBupleurumsuch asBupleurummarginatumandBupleurumfalcatumare also used as raw material of Radix Bupleurum[4-5]. In order to comprehensively evaluate the quality ofB.falcatumLinne, it was compared with other 9 batches ofBupleurumsamples in this study. Referring toChinesePharmacopoeia[3], the contents of saikosaponins a and d inB.falcatumLinne were determined with RI-HPLC, and the contents of 7 harmful elements of Al, Cr, Cu, As, Hg, Cd and Pb were determined by ICP-MS.

2 Instruments and drugs

2.1 InstrumentsThe instruments and equipment used mainly included electronic balance (accurate to 0.000 01, ISO 9001, Sartorius Scientific Instruments Co., Ltd.), electronic balance (accurate to 0.000 1, ESJ200-4, Shenyang Longteng Electronics Co., Ltd.), rotary steamer (RV10 C S96, Zhengzhou Greatwall Scientific Industrial and Trade Co., Ltd.), ultrasonic cleaner (HU20500D, Kunshan Ultrasonic Instrument Co., Ltd.), ultrapure water system (Q-POD, Merck Millipore, USA), HPLC [DIONEX UltiMate 3000, Thermo Fisher Scientific (China) Co., Ltd.], digital display stainless steel electric heating plate (DB-6A, Changzhou Shenguang Instrument Co., Ltd.), Youpu ultrapure water machine (Sichuan Youpu Ultrapure Technology Company), inductively coupled plasma-mass spectrometer (Nex ION2000, PerkinElmer, USA) and teflon digestion tank.

2.2 Reagents and drugsSaikosaponin a (18120402, HPLC≥98%) and Saikosaponin d (1901208, HPLC≥98%) were purchased from Chengdu Pufei De Biotech Co., Ltd. Acetonitrile was chromatographically pure; nitric acid, perchloric acid and hydrofluoric acid were all excellent pure; and other organic solvents were analytical pure. Al (BWJ4224-2016), Cr (BWB2142-2016), Cu (BWB2048-2016), As (BWJ4223-2016), Cd (BWB2077-2016), Hg (BWB2127-2016) and Pb (BWZ6637-2016) standard liquids were produced by Beijing Shiji Aoke Biotech Co., Ltd.B.falcatumLinne samples were collected at different times at the Walnut-Chinese Medicinal Material Interplanting Technology Research Base (29.201 10° N, 102.791 00° E; 2 030 m asl.; Group 4, Panzeluo Village, Heima Township, Ganluo County, Liangshan Yi Autonomous Prefecture, Sichuan Province) of Kangyuan Traditional Chinese Medicine Farmers’ Professional Cooperative in Ganluo County. Other Bupleurum medicinal materials were purchased from Weite (Yiguanmiao) Pharmacy of Sichuan Xinglin Pharmaceutical Chain Co., Ltd. and Chengdu Haitian (Yiguanmiao) Pharmacy (Table 1).

3 Methods and results

3.1 Determination of saikosaponin

3.1.1Chromatographic conditions. Column: Agilent 5 HC-C18chromatographic column (250 mm×4.6 mm); mobile phase: acetonitrile (A)-water (B); gradient elution (0-15 min, 25% A→40% A; 15-25 min, 40% A; 25-30 min, 45% A→55% A; 30-35 min, 45% A→50% A; 35-40 min, 50%A); flow rate: 1 mL/min; injection volume: 10 μL; column temperature: 35 ℃. Under these chromatographic conditions, the separation effect of saikosaponins a and d was good, and their retention times were 26.2 and 39.5 min, respectively (Fig.1).

Note: 1. Saikosaponin a; 2. Saikosaponin d.

3.1.2Preparation of reference solution. Accurate amounts of saikosaponin a standard and saikosaponin d standard were weighed, mixed together, and dissolved in methanol to 25 mL to obtain the mixed reference solution (saikosaponin a, 0.040 88 mg/mL; saikosaponin d, 0.032 48 mg/mL).

3.1.3Preparation of sample solution. Accurate amount (0.5 g) of coarse powder of each sample was weighed, added with 5% concentrated ammonia-methanol solution according to a solid to liquid ratio of 1∶50, ultrasonicated for 30 min and filtered. The process above was repeated three times. The filtrate was blended and evaporated to dryness. The residue obtained was dissolved in chromatographic methanol to 5 mL as sample solution.

3.1.4Methodological investigation. (i) Investigation of linear relationship. Different volume (0.5, 1, 2, 4, 6, 8 and 10 μL) of the reference solution prepared in Section3.1.2was diluted to 10 mL and then detected under the chromatographic conditions described in Section3.1.1, respectively. Taking the peak area as the ordinate (Y) and the concentration of the corresponding reference solution as the abscissa (X, μg), linear regression was performed. The results show that saikosaponin a and saikosaponin d had a good linear relationship in the range of 0.511-10.220 μg (R2=0.998 8) and 0.406-8.120 μg (R2=0.999 5), respectively. The regression equations arey=3 365.7x-27.093 andy=3 243.4x-8.752 2.

(ii) Precision test. Under the conditions in Section3.1.1, the reference solutions of saikosaponins a and d were detected five times, respectively to obtain the peak areas. The result show that theRSDvalues of peak areas of saikosaponins a and d (n=5) were 0.55% and 0.78%, respectively, indicating that the precision of the instrument is good.

(iii) Reproducibility test. Five portions of the coarse powder of sample S1, about 0.5 g for each were weighed, prepared into solutions according to the method in Section3.1.3, and detected under the conditions described in Section3.1.1, respectively. The results show that theRSDvalues of saikosaponins a and d (n=5) were 0.41% and 0.33%, respectively, indicating good reproducibility.

(iv) Stability test. A certain amount (about 0.5 g) of the coarse powder of sample F1 was weighed, and prepared into solution in line with the method in Section3.1.3. Six portions of the test solution of sample F1 were sampled, and detected 0, 4, 8, 16, 24 and 32 h after the preparation, respectively under the chromatographic conditions described in Section3.1.1. TheRSDvalues of saikosaponins a and d were calculated to be 1.04% and 0.73% (n=6), respectively, indicating that the sample solution has good stability.

(v) Determination of detection limit and quantification limit. The reference solution prepared in Section3.1.2was diluted gradiently until S/N=3 (lower limit of detection, LOD) and S/N=10 (lower limit of quantification, LOQ) when detected under the chromatographic conditions in Section3.1.1. The detection limits of saikosaponin a and saikosaponin d (S/N=3) were 1.53 and 1.22 ng, respectively, and their limits of quantification (S/N=10) were 5.11 and 4.06 ng, respectively.

(vi) Sample recovery test. Six portions of the coarse powder of sample F1 with known contents of saikosaponins a and d, about 0.2 g for each, were weighed accurately, added with saikosaponins a and d standards according to the ratio close to 1∶1 between the added amount and the sample content, prepared into solutions according to the method in Section3.1.3, and detected under the chromatographic conditions described in Section3.1.1, respectively. The measured contents and recovery rates were calculated by the external standard method, and the results are shown in Table 2.

Table 2 Results of recovery test (n=6)

3.1.5Sample determination. An accurate amount of each sample was weighed, prepared into test solution according to the method in Section3.1.3, and detected under the chromatographic conditions in Section3.1.1, respectively. The contents of saikosaponins a and d were calculated by external standard method, and the results are shown in Table 3. The contents of total saikosaponins in the roots, stems and leaves ofB.falcatumLinne were 10.723-21.983, 0.576-0.894 and 5.910-8.886 mg/g, respectively, and that in the other Radix Bupleurum pieces was 1.406-20.048 mg/g. The content of saikosaponins in Radix Bupleurum Falcatum (15.894 mg/g) was higher than those in the other origins of Radix Bupleurum (10.906 mg/g). The content of saikosaponins in the leaves ofB.falcatumLinne (7.518 mg/g) was more than twice the limit (3.0 mg/g) promulgated by theChinesePharmacopoeia(2015 Edition). The contents of saikosaponins in the stems ofB.falcatumLinne and the sample S3 were all lower than the limit.

Table 3 Contents of saikosaponins a and d in the samples (n=3, mg/g)

3.2 Determination of harmful elements

3.2.1Preparation of reference solution. (i) Preparation of multi-element standard solution. Accurate volumes of the standard solution of each element, 1 mL for each, were diluted with deionized water to 100 mL to prepare into a multi-element mixed reference solution with a mass concentration of 10 μg/mL. Different volume (0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5 and 10 mL) of the mixed reference solution was diluted to 100 mL, respectively to obtain the mixed reference solutions in which the mass concentrations of Al, Cr, Cu, Cd, As and Pb were 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500 and 1 000 μg/L.

(ii) Preparation of Hg reference solution. An accurate volume (1 mL) of Hg reference solution was added with 1 mL of Au reference solution and then diluted with 2% nitric acid to 100 mL (concentration of 10 μg/mL). Different volume (0.005, 0.01, 0.02, 0.05 and 0.1 mL) of the diluent was diluted to 100 mL, respectively (mass concentrations of 0.5, 1, 2, 5 and 10 μg/L).

(iii) Preparation of mixed internal standard solution. An accurate volume (0.2 mL) of Bi, Sc and Y with a mass concentration of 10 μg/mL was diluted to 100 mL, respectively (concentration of 20 μg/mL). Sc was used as internal standard to determine Al; Y was used as internal standard to determine Cu and As; and Bi was used as internal standard to determine Cd, Hg and Pb.

3.2.2Preparation of sample solution. Electric heating plate digestion was used. A certain amount (around 0.5 g) of the coarse powder of each sample was weighed accurately, poured into a 50 mL digestion tank, added with 25mL of mixed acid (nitric acid∶perchloric acid=4∶1), shaken well, let stand overnight, evaporated to dryness at 130 ℃ using an electric heating plate, added with 10 mL of mixed acid, evaporated to dryness at 150 ℃, added with 10 mL of mixed acid, evaporated to dryness at 160 ℃, added with 10 mL of mixed acid, evaporated to dryness at 180 ℃. The process above was repeated until complete digestion. The residue obtained was dissolved in 2% nitric acid and diluted to 25 mL as test solution. In the preparation of Hg test solution, an appropriate amount of Au standard solution was added.

3.2.3ICP-MS conditions. Power: 1 600 W; cooling air flow rate, 20 L/min; atomizing gas volume flow rate: 0.9 L/min; auxiliary gas flow rate: 2 L/min; lift amount: 1.2 mL/min; number of determinations: 3; scanning mode: peak jumping; Measurement method: standard curve method; reading method: peak intensity method.

3.2.4Methodological investigation. (i) Investigation of linear relationship. According to the element to be tested in the sample, the reference solution was prepared. The reference solutions of the 7 kinds of inorganic elements were determined sequentially. Taking the mass concentration of the reference solution as the abscissa (x) and the peak intensity as the ordinate (y), the standard curve was plotted. The regression equation, correlation coefficient and linear range of each element are shown in Table 4.

Table 4 Regression equations for content determination of inorganic elements

(ii) Precision test. The reference solutions prepared in Section3.2.1were determined six times under the conditions described in Section3.2.3. TheRSDvalues were 0.2%-1.6% (Table 5), indicating that the instrument has good precision.

(iii) Reproducibility test. Six portions of coarse powder of sample S1, about 0.5 g for each, were weighed accurately, prepared into test solutions according to the description in Section3.2.2, and determined under the conditions in Section3.2.3. TheRSDvalues were calculated to be 0.4%-3.1% (Table 5), indicating good reproducibility.

(iv) Stability test. A certain amount (about 0.5 g) of the coarse powder of sample S1 was prepared into test solution in line with the method in Section3.2.2. The test solution prepared was detected 0, 2, 4, 8, 16, 24 and 32 h after the preparation (n=7), respectively. TheRSDvalues were calculated to be 1.1%-3.0% (Table 5), indicating that the test solution prepared is stable within 32 h.

(v) Sample recovery test. Three portions the coarse powder of sample S1 with known contents of heavy metals, about 0.5 g for each, were weighed. They were added with the reference solution of each element in a ratio of 1∶1 between the added amount and the amount contained, prepared into test solutions according to the description in Section3.2.2, and detected under the conditions described in Section3.2.3. The measured contents and recovery rates of the elements were calculated, and the results are shown in Table 5.

(vi) Detection limit. Under the conditions in Section3.2.3, the blank solution was determined 11 times to determine the standard deviation SA of the instrument response value of each element. The mass concentration corresponding to three times of SA is the detection limit. The results show that the detection limits of each element are in the range of 0.001-0.194 μg/L (Table 5), indicating that the detection limit of each element meets the analysis requirements.

3.2.5Sample determination. A certain amount (around 0.5 g) of each sample was weighed accurately, prepared into test solution in line with the method in Section3.2.2, and detected under the conditions described in Section3.2.3, respectively. The content was calculated. As shown in Table 6, the Hg element did not reach the detection limit, and the contents of Al, Cr, Cu, As, Cd and Pb elements were 9.707-130.658, 0.872-1.942, 1.667-5.483, 0.218-0.288, 0.011-0.070 and 0.044-0.121 μg/g, respectively, all lower than the limits promulgated by theChinesePharmacopoeia(2015 edition)[3], indicating thatB.falcatumLinne interplanted with walnut has safety guarantee.

Table 5 Results of methodological investigation of element determination

Table 6 Contents of harmful elements in Bupleurum falcatum Linne (n=3)

3.3 Statistical analysisIn order to comprehensively and accurately analyze and evaluate the quality ofB.falcatumLinne, the total saikosaponins content of Radix Bupleurum Falcatum and other Radix Bupleurum pieces were compared through a two-dimensional column chart; the total saikosaponins content in different parts ofB.falcatumLinne were compared through univariate variance analysis, multiple comparison of average values and two-dimensional line graph, and the contents of harmful elements in different parts ofB.falcatumLinne were analyzed intuitively through fingerprint chromatogram (Al reduced by 100 times, Cr and Cu reduced by 10 times). The results are shown in Table 7-8, Fig.2-4. According to Fig.2, it can be seen intuitively that the quality ofB.falcatumLinne was better than that of Radix Bupleurum pieces (S3, S4, S5). As shown in Table 7-8 and Fig.3, there were significant differences in saikosaponins content among the roots, stems and leaves ofB.falcatumLinne (P<0.05,P<0.01), and in different parts, the saikosaponins content ranked as roots>stems>leaves. The influence of harvest time on the content of saikosaponins inB.falcatumLinne is shown intuitively from the two-dimensional line chart. Fig.4 shows that the contents of harmful elements in each sample were generally low, and among different parts, they were in the order as roots>stems>leaves.

In short, the quality of Radix Bupleurum Falcatum is better. The leaves ofB.falcatumLinne have a higher saikosaponins content and lower harmful elements content.

Table 7 Analysis of variance of contents of total saikosaponins in different parts of Bupleurum falcatum Linne

Table 8 Multiple comparisons of means among different treatments (LSD method)

Fig.2 Comparison of total saikosaponins content among bupleurum samples

Fig.3 Comparison of total saikosaponins content among different parts of Bupleurum falcatum Linne

Fig.4 Content of harmful elements in different parts of Bupleurum falcatum Linne

4 Discussion

Radix Bupleurum is a commonly used Chinese medicinal material and is in great demand in the Chinese medicine market. In Japan, the annual output of artificial planting only meets 10%-20% of the market demand, and its supply is in short throughout the year[2].B.falcatumLinne interplanted with walnut in Ganluo has an excellent quality, and its contents of heavy metals and harmful elements are low, but a large amount of them are exported to foreign markets every year. At the same time, out of the protection of resources and the environment, the discovery of the usefulness of its leaf part is of great significance.

The sample preparation and chromatographic conditions developed by this study for content determination of total saikosaponins inB.falcatumLinne by referring to theChinesePharmacopoeiaare stable and reliable, and the methodological inspection indicators all meet the requirements. Under the conditions of ICP-MS, the performance of all the indicators meets the requirements. Considering that Hg element has the properties of adsorption and volatilization during sample preparation and detection, appropriate amount of Au is added in sample preparation and standard solution preparation. In addition, the sample solution is further diluted to a low concentration for determination, in order to prevent the adsorption and volatilization of Hg element. The method is simple to operate and the effect is ideal. Based on the determination results, the quality of Radix Bupleurum Falcatum and other Radix Bupleurum pieces was compared through univariate analysis of variance, two-dimensional histogram comparison and element fingerprint analysis. The contents of total saikosaponins and harmful elements in different parts ofB.falcatumLinne were compared. The analysis method is simple to operate, relatively comprehensive, accurate, reliable, and more intuitive. In summary,B.falcatumLinne interplanted with walnut in Ganluo has a better quality, and its leaves are also rich in saikosaponins a and d.B.falcatumLinne can be used as the raw material for extracting saikosaponins a and d. This study can provide suggestions for the cultivation and purchase ofB.falcatumLinne in the domestic market and provide a basis for further in-depth research on its leaf position.