Water Extraction Process of Chinese Herbal Compound Man Gan Ning

Chunli TANG2, Jiangcun WEI1,*, Zhen XIE, Meiyan QIU1, Jiabao MA2, Fengxian ZHAO, Hongxia CHEN

1. Guangxi International Zhuang Medical Hospital, Nanning 530201, China; 2. The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530023, China; 3. Guangxi University of Chinese Medicine, Nanning 530200, China

Abstract [Objectives] To optimize the water extraction process of Chinese Herbal Compound Man Gan Ning and establish a method for its extraction and content determination. [Methods] The L9(34) orthogonal test was carried out, the comprehensive scores of ginsenoside Rb1 content, notoginsenoside R1 content, tanshinone I content and saikosaponin A content in the compound extract were used as evaluation indicators to explore the effects of water addition, the water addition, decoction time and decoction times on the water extraction process, and verification tests were carried out. [Results] The optimized water extraction process was as follows: adding 9 times of water in the first extraction, and extracting for 90 min; adding 7 times of water in the second extraction, and extracting for 60 min; adding 7 times of water in the third extraction, and extracting for 60 min. [Conclusions] The optimized water extraction process is stable and feasible, and can be used for the extraction of various herbs in Compound Man Gan Ning. It can also provide experimental basis for the formulation development of Compound Man Gan Ning sustained release tablet.

Key words Man Gan Ning, Orthogonal test, Extraction process, Ginsenoside Rb1, Notoginsenoside R1, Tanshinone I, Saikosaponin A

1 Introduction

Compound Man Gan Ning has the functions of invigorating the blood circulation and dispelling the fatigue, strengthening and benefiting qi, and can be used for treating the chronic hepatitis. Previous studies have found that patients with chronic hepatitis and cirrhosis often have varying degrees of endocrine hormone disorders[1-4]. The application of Chinese Herbal Man Gan Ning can nourish the liver and tonify the kidney, restore liver function and promote the repair and regeneration of liver cells[5]. For patients with different degrees of fibrosis or early cirrhosis confirmed by liver perforation, through analyzing the clinical and pathological data before and after treatment, it was found that this preparation can significantly improve clinical symptoms and liver function. Many cases of liver pathological examination showed the mitigation of hepatitis activity and fibrosis[6]. Despite different functions and structures, the liver and the kidney are of the same origin. And their physiological lesions are interrelated. Thus, it will have a good effect on hepatitis B using the principle of the same origin of liver and kidney[7-8]. In this study, we mainly summarized the progress in the study of Compound Man Gan Ning in the treatment of liver disease, to lay a foundation for the future clinical application of traditional Chinese medicine and its compound treatment for liver disease, it is expected to bring benefits to human health. Chronic viral hepatitis is a common infectious disease threatening people’s health. Thus, it is of great significance to find effective drug treatment methods and to study the traditional Chinese medicine compound for treating liver diseases.

On the basis of previous studies, we carried out the orthogonal experiment. Using the traditional water extraction method, we selected the optimal extraction process for effective components including ginsenoside Rb1 content, notoginsenoside R1 content, tanshinone I in Compound Man Gan Ning, to fully use active components of the medicine, bring into play their adequate functions, and provide a theoretical basis for the study of Chinese herbal compound products.

2 Instruments and reagents

2.1 InstrumentsWaters 2695 high-performance liquid chromatography and chromatography workstation (American Waters Corporation); SQP electric balance (Sartorius Sartorius Scientific Instruments (Beijing) Co., Ltd.); LGL-16G high-speed desktop centrifuge (Shanghai Anting Scientific Instrument Factory); 101A-3E electric air blowing dryer (Shanghai Laboratory Instrument Works Co., Ltd.); KQ5200B ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); EYELA-N-1100E rotary evaporator (Shanghai Ailang Instrument Co.,Ltd.); HWS-26 electroheating thermostatic water bath (Shanghai Bluepard Instruments Co., Ltd.); 98-1-B electronic temperature-adjusting heating mantle (Tianjin Taisite Instrument Co., Ltd.); SHB-III circulating water type multi-purpose vacuum pump (Zhengzhou Great Wall Scientific Industrial & Trade Co., Ltd.).

2.2 Reagents and medicinal materialsNotoginsenoside R1 (batch No.80419-24-1, purity>98%); ginsenoside Rb1 (batch No.41752-43-8, purity>98%); saikosaponin A (batch No.20736-09-9, purity>98%); tanshinone I (batch No.568-73-1, purity>98%); all were purchased from Chengdu Must Bio-Technology Co., Ltd.; chromatographic acetonitrile was provided by American Thermo Fisher Scientific; methanol, ethanol, and phosphate were analytical reagents, and experimental water was ultrapure water.

Panaxnotoginseng(batch No.20180418) is Araliaceae herbaceous perennial plants, and Zhibijia is a Chinese herbal medicine, it was decocted for 25 min, and taking after cooking is preferred (batch No.20150419);SalviamiltiorrhizaBunge (batch No.20180416);BupleurumchanenseDC orB.scorzonerifoliumWi11d (batch No.20180416);Astragalusmembranaceus(Fisch.) Bge. var. mongholicus (Bge.) Hsiao orAstragalusmembranaceus(Fisch.) Bge. (batch No.20180416);Cordycepssanensas(Berk.) Sacc.(batch No.20180416);Ganodermalucidum(Leyss.exFr.) Karst.(batch No.20180216);EupolyphagasinensisWalker or Steleophaga plancyi (Boleny) (batch No.20180315);AtractylodisMacrocephalaeRhizoma; Polygonatum sibiricum (batch No.20180412). All were purchased from Liuzhou City Chinese Herbal Medicine Co., Ltd.

3 Methods and results

3.1 Chromatographic conditionsThe chromatographic column was Agilent ZORBAX SB-C18(5 μm, 4.6 mm×250 mm), mobile phase: acetonitrile-0.05% phosphoric acid aqueous solution, gradient elution, the elution procedure is shown in Table 1, detection wavelength: 203, 204 and 275 nm, Column temperature: 30 ℃, flow rate: 1 mL/min, injection volume 10 μL, set ginsenoside Rb1, notoginsenoside R1 detection wavelength at 203 nm; tanshinone I detection wavelength at 275 nm; saikosaponin A detection wavelength at 204 nm. The theoretical plate number should be not less than 4 000 calculated as the peak of ginsenoside Rb1, and not less than 10 000 calculated by the peak of saikosaponin A. The four active ingredients in Compound Man Gan Ning water extract could be well separated. The order of the peaks was notoginsenoside R1, ginsenoside Rb1, saikosaponin A and tanshinone I. The other components in the sample did no have interference on the measured components; according to the above conditions, the separation of each component was good.

Table 1 Gradient elution procedure

TimeminAcetonitrile%0.05% (V/V) phosphoricacid solution∥%Wavelengthnm01895203122488203183080203324564203334564204455548204586840275658518275

3.2 Preparation of reference substance solutionPrecisely weighed 12. 6 mg of ginsenoside Rb: 1, 12.21 mg of notoginsenoside R1 , 14.28 mg of tanshinone I, and 10.39 mg of saikosaponin A. Placed in four 25-mL volumetric flasks, added methanol to dissolve and fixed to the desired volume, obtained the reference stock, namely, 0.498 4 mg/mL of ginsenoside Rb1, and 0.488 4 mg/mL of notoginsenoside R1, 0.572 1 mg/mL of tanshinone I, and 0.415 6 mg/mL of saikosaponin A. Preparation of mixed reference solution: precisely weighed proper volume of the reference substance stock and placed in the same 10-mL volumetric flask, added methanol to fix the volume and mixed well, then obtained the mixed reference solution.

3.3 Preparation of sample solutionThe prescription consists of Notoginseng Radix Et Rhizoma, Bijia, Salviae Miltiorrhizae Radix Et Rhizoma, Astragali Radix, Cordyceps, Ganoderma, Eupolyphaga Steleophaga, Atractylodis Macrocephalae Rhizoma, and Polygonati Rhizoma. Added proper volume of pure water to the medicinal material, heated and extracted for a certain period of time, then filtered with 4 layers of gauze, concentrated under reduced pressure (60 ℃) to close to 200 mL, placed in a 200-mL volumetric flask, added water to fix the volume to the desired value and shook well. After standing for a period of time, precisely weighed 4 mL of the supernatant, added to a 10-mL volumetric flask, added methanol to fix the volume, shook well, centrifuged for 10 min at 13 000 r/min, to obtain the sample solution.

3.4 Linear relationship testSeparately and precisely absorbed certain volume of the above reference solution, mixed in a brown volumetric flask, and added the methanol to fix the volume to desired value, prepared into 7 different concentrations of the Compound Man Gan Ning mixed reference solution, the injection volume was 10 μL, samples were injected separately, measured by liquid chromatography, and determined the peak area. Taking the concentration (μg/mL) as the abscissa and the peak area as the ordinate, the linear equation chart was plotted, and calculated the linear equation, as shown in Table 2.

Gradually diluted the mixed reference solution to obtain a series of mixed reference solutions, and then operated using the method in Section3.1, and measured the signal to noise ratio (in terms of peak height), set the concentration when S/N=3 as the limit of detection (LOD), and set the concentration when S/N=10 as the limit of quantitation (LOQ). The results indicate that the linear relationship of components was good within the respective content range, as shown in Table 2.

3.5 Precision testIPrecisely weighed proper volume of mixed reference solution, continuously injected 6 times under the conditions in Section3.1, separately made records of the respective peak areas, and calculated theRSD(%) of the peak area of ginsenoside Rb1, notoginsenoside R1, tanshinone I, and saikosaponin A. The results indicate that theRSDof the peak area of ginsenoside Rb1, notoginsenoside R1, tanshinone I, and saikosaponin A was 0.64%, 1.44%, 1.02% and 0.99%, all were less than 3.0%, indicating that the precision of the liquid chromatograph used is high.

Table 2 Linear regression equation

ComponentLinear regression equationCorrelationcoefficient∥R2Linearrange∥μg/mLLimit ofdetection (LOD)∥ngLimit ofquantitation (LOQ)∥ngGinsenoside Rb1Y=19 732X+23 9300.999 219.94-149.523.632.5Notoginsenoside R1Y=4 893X+76 4600.999 17.81-78.145.842.8Tanshinone IY=15 488X+2 0490.999 422.90-22.884.536.4Saikosaponin AY=15 135X-4 2380.999 72.49-20.786.244.9

3.6 Stability testWeighed Compound Man Gan Ning powder according to the ratio, precisely weighed, and prepared the sample solution according to the preparation method of the sample solution in Section3.3, and determined the peak area of sample in 0, 2, 4, 8, 12, 24 h after preparation of the test solution using the method in Section3.1. The results indicate that theRSDof ginsenoside Rb1, notoginsenoside R1, tanshinone I and saikosaponin A in Compound Man Gan Ning was 1.62%, 1.89%, 2.26% and 1.67%, respectively. AllRSDs were less than 3.0%, indicating that the test solution has high stability within 24 h.

3.7 Reproducibility testIIPrecisely weighed 6 pieces of Compound Man Gan Ning powder according to the ratio, prepared the sample solution according to the preparation method of the sample solution in Section3.3, determined the peak area of sample using the method in Section3.1. Then, calculated the average mass concentration of ginsenoside Rb1, notoginsenoside R1, tanshinone, and saikosaponin A in Compound Man Gan Ning and calculated theRSDof the mass concentration of the corresponding component, the results indicate that the mass concentrations of ginsenoside Rb1, notoginsenoside R1, tanshinone I and saikosaponin A in Compound Man Gan Ning were 135.60, 54.56, 21.49 and 15.31 μg/mL, respectively. The results indicate that theRSDof the mass concentration of ginsenoside Rb1, notoginsenoside R1, tanshinone I, and saikosaponin A in Compound Man Gan Ning was 2.64%, 2.48%, 1.47% and 1.50%, all were less than 3.0%, indicating that this method has high reproducibility.

3.8 Sample recovery testPrecisely weighed half of 9 pieces of Compound Man Gan Ning powder, divided into 3 groups, namely, low, medium, and high dose groups (80%, 100%, and 120% of extract of half Compound Man Gan Ning); each group separately was added with mixed reference solution (ginsenoside Rb1, notoginsenoside R1, tanshinone I, and saikosaponin A), prepared the sample solution using the method in Section3.3, and determined under the conditions in Section3.1, calculated the average recovery rate andRSDof ginsenoside Rb1, notoginsenoside R1, tanshinone I, and saikosaponin A in low, medium, and high dose groups of Compound Man Gan Ning extract. The results are listed in Table 3, indicating that this method has high accuracy.

Table 3 Results of sample recovery test of Compound Man Gan Ning(n=6)

ComponentNo.Mass concentrationμg/mLSample addedμg/mLSample measuredμg/mLRecoveryrate∥%Average recoveryrate∥%RSD∥%Ginsenoside Rb1154.24 53.83 108.71 101.1998.962.24254.24 53.83 106.32 96.75354.24 53.83 107.50 98.94467.80 67.78 135.31 99.6097.751.85567.80 67.78 132.86 95.99667.80 67.78 133.99 97.65781.36 81.74 161.92 98.5598.962.42881.36 81.74 160.48 96.80981.36 81.74 164.35 101.53Notoginsenoside R1121.82 21.49 43.50 100.8799.862.59221.82 21.49 42.65 96.92321.82 21.49 43.70 101.80427.28 27.35 54.56 99.74100.002.19527.28 27.35 55.26 102.31627.28 27.35 54.07 97.95732.74 32.23 64.78 99.40100.052.55832.74 32.23 65.89 102.86932.74 32.23 64.29 97.88Tanshinone I 18.60 8.58 17.16 99.8199.952.3828.60 8.58 17.39 102.4038.60 8.58 16.98 97.65410.75 10.30 20.96 99.1299.062.99510.75 10.30 21.26 102.00

(To be continued)

(Continued)

ComponentNo.Mass concentrationμg/mLSample addedμg/mLSample measuredμg/mLRecoveryrate∥%Average recoveryrate∥%RSD∥%610.75 10.30 20.65 96.07712.89 12.59 25.45 99.7398.852.37812.89 12.59 25.00 96.19912.89 12.59 25.56 100.62Saikosaponin A16.12 5.82 12.00 100.9899.441.6326.12 5.82 11.92 99.5936.12 5.82 11.81 97.7547.66 7.48 15.04 98.7099.222.1657.66 7.48 14.94 97.3867.66 7.48 15.26 101.5779.19 9.14 18.36 100.3799.832.4889.19 9.14 18.07 97.1299.19 9.14 18.51 101.99

3.9 Orthogonal experiment

3.9.1Evaluation indicator of water extraction process. In the prescription, Notoginseng Radix Et Rhizoma, Bijia, Salviae Miltiorrhizae Radix Et Rhizoma, Bupleuri Radix, Astragali Radix, Cordyceps, Ganoderma, Eupolyphaga Steleophaga, Atractylodis Macrocephalae Rhizoma, and Polygonati Rhizoma were extracted by the traditional water extraction method, taking the yield of ginsenoside Rb1, notoginsenoside R1, tanshinone I, saikosaponin A as evaluation indicators. Notoginseng Radix Et Rhizoma is the sovereign medicinal, insenoside Rb1 and notoginsenoside R1 are its main active components; tanshinone I and saikosaponin A are main active components of ministerial medicinal Salviae Miltiorrhizae Radix Et Rhizoma and Bupleuri Radix. Through detection of these four components at the same time can make effective control of their quality. They are main indicators for the extraction indicators. For the same medicinal materials, the higher the extraction rate, the better the extraction effect. Thus, it will have a high reference value for the extraction process. Therefore, the weight coefficients of ginsenoside Rb1, notoginsenoside R1, tanshinone I, and saikosaponin A were set to 0.25, that is, the comprehensive score=[(ginsenoside Rb1 content×0.25)+(notoginsenoside R1 content×0.25)+(tanshinone I content×0.25)+(saikosaponin A content×0.25)]×100%.

3.9.2Design of orthogonal experiment. According to the pre-experimental results, it was found that the amount of water added, the extraction time and the extraction times had a great influence on the dissolution of the active components and the yield of the extract. Therefore, the water addition amount (A), the extraction time (B) and the extraction times (C) were selected as the factors to be investigated, and three different levels in each factor were taken, and the comprehensive scores of ginsenoside Rb1 content, notoginsenoside R1 content, tanshinone I content and saikosaponin A content were used as evaluation indicators, weighed 9 pieces of Notoginseng Radix Et Rhizoma, Bijia, Salviae Miltiorrhizae Radix Et Rhizoma, Bupleuri Radix, Astragali Radix, Cordyceps, Ganoderma, Eupolyphaga Steleophaga, Atractylodis Macrocephalae Rhizoma, and Polygonati Rhizoma, and experiment was arranged according to L9(34) orthogonal table, to screen the optimal water extraction process. The design factor level is shown in Table 4. The results of orthogonal experiment are shown in Table 5. The variance analysis is shown in Table 6.

Table 4 Orthogonal design factor level

LevelWater addition(A)∥timesExtraction time(B)∥minExtraction times(C)1740129602312903

Table 5 Results of orthogonal experiment for Compound Man Gan Ning extraction process

No.ABCDGinsenosideRb1∥μg/gNotoginsenosideR1∥μg/gTanshinoneI∥μg/gSaikosaponinA∥μg/gComprehensivescore11111131.39 54.74 20.91 14.16 55.30 21222131.94 55.40 21.47 14.66 55.87 31333143.06 59.51 23.42 15.92 60.48 42123134.04 55.99 22.57 14.56 56.79 52231140.08 59.25 24.82 16.58 60.18 62312132.86 55.45 22.10 14.23 56.16 73132148.41 53.91 22.17 17.19 60.42 83213134.32 57.83 19.87 14.73 56.69 93321135.80 54.45 20.45 14.94 56.41 K157.21757.50356.05057.297K257.71057.58056.35757.483K357.84057.68360.36057.987R0.6230.1804.3100.690

Table 6 Variance analysis of orthogonal experiment for Compound Man Gan Ning extraction process

FactorSum of square of deviationsDegree of freedomF ratioF critical valueSignificanceWater addition (A)0.64920.84919>0.05Extraction time (B)0.04920.06419>0.05Extraction times (C)34.697245.41519<0.05Error (D)0.7602119>0.05

3.9.3Results and analysis. According to Table 5, the order of the extreme differenceRvalue of the water addition, the extraction time and the extraction times was as follows: extraction times (C)>water addition (A)>extraction time (B), where A factorK3>K2>K1, B factorK3>K2>K1, C factorK3>K2>K1, thus the optimal extraction process is A3B3C3, namely, 12 times of water, 90 min of extraction time, and 3 extraction times.

From Table 6, it can be known that the water addition (A) and extraction time (B) were not statistically significant. Combining visual analysis results and the variance analysis results of Table 5 and Table 6, it can be found that the factor C, namely, the extraction times, had the largest influence of the experimental results, while the water addition (A) and extraction time (B) had little effect on the results. To save energy and resources, we optimized the selected process and obtained the optimal extraction process.

The improved factors were water addition (A) and extraction time (B). The comprehensive scores of ginsenoside Rb1 content, notoginsenoside R1 content, tanshinone I content and saikosaponin A content were used as evaluation indicators. The improvement scheme is as follows: in the first extraction, added 9 times of water and extracted for 90 min; in the second extraction, added 7 times of water and extracted for 60 min; in the third extraction, added 7 times of water and extracted for 60 min.

3.9.4Verification test of water extraction process. The varification test of the content of ginsenoside Rb1, the content of notoginsenoside R1, the content of tanshinone I and the content of saikosaponin A in the prescription was carried out in three parallels under the optimized Compound Man Gan Ning water extraction conditions. The content of ginsenoside Rb1, notoginsenoside R1, tanshinone I and saikosaponin A was calculated, and the 140.95, 55.25, 23.17, 16.05 μg/mL, respectively, and theRSDvalue was 1.60%, 2.92%, 2.64% and 2.43%, respectively, (n=3). The verification test indicates that the optimized components of ginsenoside Rb1, notoginsenoside R1, tanshinone I and saikosaponin A were in good condition and the process was feasible.

4 Content determination

On the basis of the optimized extraction process, precisely weighed 6 pieces of the medicine, and extracted 3 pieces, and then determined the contents of ginsenoside Rb1, notoginsenoside R1, tanshinone I and saikosaponin A in the prescription. The results are listed in Table 7.

From the above results, it can be seen that the content of ginsenoside Rb1, notoginsenoside R1, tanshinone I and saikosaponin A in the optimal extraction process of this experiment is basically stable, and has good reproducibility and operability. Therefore, the optimized water extraction process is as follows: adding 9 times of water in the first extraction, and extracting for 90 min; adding 7 times of water in the second extraction, and extracting for 60 min; adding 7 times of water in the third extraction, and extracting for 60 min.

Table 7 Results of verification test for Compound Man Gan Ning water extraction process

ExperimentNo.GinsenosideRb1∥μg/mLNotoginsenosideR1∥μg/mLTanshinoneI∥μg/gSaikosaponinA∥μg/mL1138.99 56.78 24.14 15.812144.61 55.96 23.08 16.313143.06 58.49 23.42 15.924139.11 55.99 23.86 15.395140.08 59.25 24.82 16.416145.53 55.45 23.71 15.32Mean141.9056.99 23.84 15.86RSD∥%2.032.702.522.85

5 Conclusions

According to the experimental results, when Compound Man Gan Ning is extracted by the traditional water extraction method, the extraction time should not be too long, and some active components are not heat-resistant, but more than two extractions have been proved to improve the dissolution of the active components. Other medicines in the prescription were extracted by traditional water extraction process. The water addition, the extraction time and the extraction times were taken as the investigation factors. Three levels were selected for each investigation factor, namely, ginsenoside Rb1, notoginsenoside R1, tanshinone I, and saikosaponin A were taken as the comprehensive score evaluation indicators, and the orthogonal test was carried out to optimize the extraction process: adding 12 times of water, extracting 90 min, and extracting three times. Through analysis, it is found that the water addition and extraction time were not statistically significant. Thus, the optimized water extraction process was as follows: adding 9 times of water in the first extraction, and extracting for 90 min; adding 7 times of water in the second extraction, and extracting for 60 min; adding 7 times of water in the third extraction, and extracting for 60 min. Therefore, the optimized method is better than the original method, so the optimized method is finally selected as the water extraction process of the medicinal material.

For the selection of the detection wavelength in this experiment, the ultraviolet absorption spectrum of the four main active components in Compound Man Gan Ning was comprehensively considered, so that the tested active components have good UV absorption. By comparison, it is found that ginsenoside Rb1, III The saponin R1 has a large UV absorption at a wavelength of 203 nm, and the tanshinone I has a good UV absorption at a wavelength of 275 nm, and the saikosaponin A has a large ultraviolet absorption at a wavelength of 204 nm. Therefore, the three-wavelength switching method was selected to determine the content of four active components in Compound Man Gan Ning. This experiment method is simple, rapid and sensitive, and has certain reference value for the quality control of Compound Man Gan Ning, and is worthy of application to the quality control of other traditional Chinese medicine prescriptions.

We investigated the effects of elution system such as methanol-0.1% glacial acetic acid, methanol-0.3% glacial acetic acid, methanol-0.1% phosphoric acid, methanol-0.05% phosphoric acid, acetonitrile-0.1% phosphoric acid, and acetonitrile-0.05% phosphoric acid on the peak time, resolution, and baseline. The results indicate that using the acetonitrile-0.05% phosphoric acid system, the chromatographic peak resolution was better, the baseline was relatively stable, and the appropriate gradient elution procedure can achieve good separation of the peaks and the baseline was balanced, so the acetonitrile-0.05% phosphoric acid system was used to conduct gradient elution in this experiment.

This experiment investigated the effects of 20, 25, 30 and 35 ℃ on resolution, peak time, and baseline stability. The experimental results show that the column temperature has little effect, so the column temperature of this experiment was set at 30 ℃. Besides, this experiment also investigated the effects of three different flow rates (1.2, 1.0, and 0.8 mL/min) on the resolution, and the results indicate that the separation was better at 1.0 mL/min and the baseline was also balanced. Therefore, the flow rate used in this experiment was 1.0 mL/min.