A detection method for under-forest ginseng bao yuan decoction and application thereof

Abstract

The present application relates to the technical field of medicine detection, in particular to a detection method of under-forest ginseng Bao Yuan decoction and application thereof. The pretreatment process is as follows: methanol is added to the under-forest ginseng Bao Yuan decoction and mixed uniformly, then the supernatant is taken after standing, and the supernatant is evaporated to dryness and redissolved with water, then the supernatant is extracted with chloroform, water-saturated n-butanol in turn or directly extracted with water-saturated n-butanol, the n-butanol extract is evaporated to dryness and dissolved with methanol, and the filtrate after filtration is the test sample; wherein, the test sample obtained by directly extracting with water-saturated n-butanol is test sample 1, and the test sample obtained by extracting with chloroform, water-saturated n-butanol in turn is test sample 2; in the process of establishing the characteristic spectrum, the detection wavelength of test sample 1 is 250-260 nm, and the detection wavelength of test sample 2 is 200-210 nm. The present application can improve the overall quality control level of under-forest ginseng Bao Yuan decoction products.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of drug detection, and particularly relates to a detection method of Linxia Shenshen Baoyuan Decoction and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing an understanding of the background of the present application and should not be taken as an acknowledgement or any form of suggestion that this information forms prior art with respect to the present application.

[0003] Baoyuan Decoction is from Ming Dynasty Sun Zihong's Simple Medical Recipes, which is composed of five medicines, i.e., ginseng, astragalus, licorice, cassia bark and ginger. Ginseng is the monarch medicine, which can tonify primordial qi, tonify the spleen and lung. Astragalus is the minister medicine, which can tonify the lung and consolidate the exterior. Licorice is the auxiliary medicine, which can tonify the spleen and lung, and harmonize other medicines. Cassia bark is the assistant medicine, which can warm and activate blood and kidney, and assist yang. Ginger is the minister medicine, which can warm the middle and assist cassia bark to warm yang. The whole prescription has the functions of tonifying qi and warming yang, and is used for treating deficiency of primordial qi and qi deficiency.

[0004] In recent years, the State Bureau has successively promulgated the Key Information Table of Ancient Classical Famous Formulas, the Technical Guidelines for Pharmaceutical Research of Traditional Chinese Medicine Compound Preparations Managed According to the Catalogue of Ancient Classical Famous Formulas (for Trial Implementation) and other relevant regulations, which are beneficial to the research and development of classical famous formulas. It is particularly crucial to develop reasonable quality standards and establish a whole-process quality control system for the research and development of classical famous formulas. At present, there are many quality detection methods of Baoyuan Decoction, mainly including thin layer identification, content determination, fingerprint (characteristic) spectrum. Since the specificity of thin layer identification is not strong and the repeatability is poor, advanced technical means such as ultra-high performance liquid chromatography, fingerprint (characteristic) spectrum and multi-component content determination are widely used.

[0005] The document "Establishment of UPLC-PDA characteristic spectrum of Baoyuan Decoction material basis and identification of chemical components" (Chinese Journal of Experimental Traditional Medical Formulae, 2021, 27(07) 16-23), "Study on the material basis of the classic prescription Baoyuan Decoction" (Yang Jingyao. Changchun University of Chinese Medicine, 2020), "Characteristic spectrum of material basis and multi-index quantitative value transmission of classic prescription Baoyuan Decoction" (Journal of Pharmaceutical Analysis, 2021, 41(02): 345-357) established the detection method of UPLC-PDA double wavelength characteristic spectrum of Baoyuan Decoction material basis freeze-dried powder, but the inventors found that the properties of freeze-dried powder do not completely conform to traditional decoction, which may be due to the inconsistency of the chemical substance basis of Baoyuan Decoction standard decoction, and the response value of ginsenosides in the 203 nm spectrum is small and the reproducibility is poor; "Study on UPLC characteristic spectrum and multi-index component content determination of classic prescription Baoyuan Decoction" (World Science and Technology-Chinese Medicine Modernization, 2021, 23(08): 2643-2650) established the characteristic spectrum of Baoyuan Decoction at 203 nm, but the inventors found that the identified chromatographic peaks do not cover the chemical components of the five medicinal ingredients in the prescription, and the comprehensiveness of the characteristic spectrum needs to be strengthened.

[0006] Patent "A detection method of HPLC characteristic spectrum of Baoyuan Decoction" (Application No. 201911323091.1), "Composition for preparing Baoyuan Decoction, Baoyuan Decoction product, and its characteristic spectrum determination and quality detection method" (Application No. CN202010003443.1), "Preparation method and detection method of Baoyuan medicinal composition" (Application No. 202011521544.4) established the characteristic spectrum, however, according to the inventors' research, the addition of ammonia solution during the processing of the test sample will remove the effective components with obvious acidity such as glycyrrhizic acid and cinnamic acid in Baoyuan Decoction, and the preparation method of the test sample is not appropriate. At the same time, the inventors also found that the response of ginsenoside Rg1 and 6-gingerol in the characteristic spectrum established by "HPLC characteristic spectrum of Baoyuan Decoction preparation and its construction method" (Application No. 202110140726.5) is not obvious, therefore the preparation method of the test sample needs to be improved, and the analysis time is too long; "Establishment method of double wavelength characteristic spectrum of Baoyuan Decoction and its standard characteristic spectrum" (Application No. 202111514128.6) uses solid phase extraction method to prepare the test sample solution, which is complicated and has poor reproducibility, and the saponin components of the monarch drug ginseng are less identified, and the analysis time is too long, therefore the detection method still needs to be improved. SUMMARY

[0007] In order to solve the problems of the prior art, the present application aims to provide a detection method of under-forest ginseng Baoyuan Decoction and application thereof, which is based on the traditional preparation method of Baoyuan Decoction and the compatibility principle of monarch, minister, assistant and guide, establishes the characteristic map of under-forest ginseng Baoyuan Decoction by dual-wavelength detection, and can determine the content of four ginsenosides, thereby serving as a comprehensive quality control method of under-forest ginseng Baoyuan Decoction and improving the overall quality control level of the product.

[0008] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0009] In one aspect, the present application provides a detection method of under-forest ginseng Baoyuan Decoction, which comprises a pretreatment process of under-forest ginseng Baoyuan Decoction and a characteristic map establishment process.

[0010] The pretreatment process of under-forest ginseng Baoyuan Decoction comprises the following steps: adding methanol into the decoction of under-forest ginseng Baoyuan Decoction and mixing uniformly, taking the supernatant after standing, evaporating to dryness and then redissolving in water, and then performing vibration extraction with chloroform, water-saturated n-butanol in sequence or directly performing vibration extraction with water-saturated n-butanol, dissolving the n-butanol extract in methanol after evaporation to dryness, and filtering the solution to obtain the test sample; wherein the test sample obtained by directly performing vibration extraction with water-saturated n-butanol is test sample 1, and the test sample obtained by performing vibration extraction with chloroform and water-saturated n-butanol in sequence is test sample 2.

[0011] The characteristic map establishment process comprises the following steps: performing high-performance liquid chromatography on the test samples of under-forest ginseng Baoyuan Decoction after pretreatment to obtain a map comprising characteristic peaks of ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, ginsenoside Rb2, ginsenoside Rf, ginsenoside Rc, ginsenoside Rd, calycosin-7-glucoside, calycosin, formononetin, ongracillin, glycyrrhizin, isoglycyrrhizin, glyyrrhetinic acid, 6-shogaol, and constructing a characteristic map of under-forest ginseng Baoyuan Decoction according to the relative retention time of the characteristic peaks; wherein the detection wavelength of test sample 1 is 250-260 nm, and the detection wavelength of test sample 2 is 200-210 nm.

[0012] Preferably, in the high-performance liquid chromatography, the detection wavelength is 200-210 nm and 250-260 nm, the mobile phase A is acetonitrile, the mobile phase B is a phosphoric acid aqueous solution with a volume fraction of 0.02-0.08 %, the chromatographic column filler is octadecylsilane bonded silica gel with a particle size of 1.6-2.1 μm, the length of the chromatographic column is 50-150 mm, and the diameter of the chromatographic column is 1.8-2.5 mm.

[0013] In another aspect, the detection method of under-forest ginseng Baoyuan Decoction is applied to the quality control of industrial production of under-forest ginseng Baoyuan Decoction.

[0014] The present application has the following advantages:

[0015] (1) The present application takes the decoction of undergrowth ginseng Baoyuan decoction prepared in a traditional way as an analysis sample, and the established characteristic map reflects the quality of the undergrowth ginseng Baoyuan decoction standard decoction, thereby providing a reference value for the research of the undergrowth ginseng Baoyuan decoction reference sample.

[0016] (2) The present application adopts a reasonable test sample preparation method, removes the polysaccharide, protein, tannin and other components in the undergrowth ginseng Baoyuan decoction, retains the pharmacodynamic material basis components, avoids the interference of the miscellaneous peaks in the chromatogram, and makes the separation degree of most common peaks better.

[0017] (3) The present application separates more ginsenoside components in the undergrowth ginseng Baoyuan decoction through the selection of the mobile phase and the cooperation of the chromatographic column, and simultaneously detects the characteristic map of the undergrowth ginseng Baoyuan decoction and determines the content of four ginsenosides.

[0018] (4) The present application selects 200-210 nm and 250-260 nm as the detection wavelength of the characteristic map of the undergrowth ginseng Baoyuan decoction, the ginsenosides have the maximum absorption at 200-210 nm, and the characteristic map including the common peaks of ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, ginsenoside Rd, ginsenoside Rb2, ginsenoside Rf and ginsenoside Rc can be obtained; at 250-260 nm, the chemical components of astragalus, licorice, cinnamon and ginger have obvious absorption, and the separation degree is better, and the characteristic map including calycosin-7-glucoside, calycosin, formononetin, calycosin-7-glucoside, glycyrrhizin, isoglycyrrhizin, glycyrrhizin, glycyrrhizic acid and 6-shogaol can be obtained.

[0019] (6) The present application can shorten the detection time of a single sample to 65 min or less, and improve the inspection efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0020] The drawings accompanying the specification of the present application form a part thereof, serve to provide further understanding of the present application, and together with the specification of the present application and the explanation thereof, explain the present application, and do not constitute an improper limitation of the present application.

[0021] Figure 1 The high performance liquid chromatogram of the undergrowth ginseng Baoyuan decoction test sample and the control sample in the present application is detected at a detection wavelength of 203 nm, 1, ginsenoside Rg1, 2, ginsenoside Re, 3, ginsenoside Rf, 4, ginsenoside Rb1, 5, ginsenoside Rc, 6, ginsenoside Rb2, 7, ginsenoside Rd;

[0022] Figure 2For the comparative graph of the test sample and the control sample of the under-forest ginseng Baoyuan Decoction in the embodiments of the present application, the high performance liquid chromatography at the detection wavelength of 254 nm is as follows: 1, calycosin-7-glucoside, 2, glycyrrhizin, 3, ononin, 4, isoglycyrrhizin, 5, glyasperin, 6, calycosin, 7, formononetin, 8, glycyrrhizic acid, and 9, 6-shogaol;

[0023] Figure 3 The high performance liquid chromatogram of the experimental example 1 of the present application is as follows:

[0024] Figure 4 The high performance liquid chromatogram (203 nm) of the experimental example 2 of the present application is as follows:

[0025] Figure 5 The high performance liquid chromatogram (254 nm) of the experimental example 2 of the present application is as follows:

[0026] Figure 6 The high performance liquid chromatogram of the experimental example 3 of the present application is as follows:

[0027] Figure 7 The high performance liquid chromatogram of the experimental example 4 of the present application is as follows:

[0028] Figure 8 The high performance liquid chromatogram of the experimental example 5 of the present application is as follows:

[0029] Figure 9 The high performance liquid chromatogram of the experimental example 6 of the present application is as follows:

[0030] Figure 10 The characteristic chromatogram (wavelength 203 nm) generated by the determination chromatogram of the best embodiment of the present application is as follows.

[0031] Figure 11 The characteristic chromatogram (wavelength 254 nm) generated by the determination chromatogram of the best embodiment of the present application is as follows. DETAILED DESCRIPTION

[0032] It should be noted that the following detailed description is merely exemplary in nature and is intended to provide further description of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0033] It is to be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments consistent with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0034] In view of the fact that the current detection method for Baoyuan Decoction of Undergrowth Ginseng is difficult to efficiently and comprehensively reflect the ginsenoside chemical components, and is difficult to effectively control the internal quality of the monarch drug undergrowth ginseng, the present application provides a detection method for Baoyuan Decoction of Undergrowth Ginseng and application thereof.

[0035] In a typical embodiment of the present application, a detection method for Baoyuan Decoction of Undergrowth Ginseng is provided, which comprises a pretreatment process of Baoyuan Decoction of Undergrowth Ginseng and a characteristic spectrum establishment process.

[0036] The pretreatment process of Baoyuan Decoction of Undergrowth Ginseng comprises the following steps: adding methanol into the decoction of Baoyuan Decoction of Undergrowth Ginseng and mixing uniformly, taking the supernatant after standing, evaporating to dryness and redissolving with water, then sequentially performing vibration extraction with chloroform, water-saturated n-butanol or directly performing vibration extraction with water-saturated n-butanol, dissolving the n-butanol extract in methanol after evaporation to dryness, and taking the filtrate after filtration as the test sample; wherein the test sample obtained by directly performing vibration extraction with water-saturated n-butanol is test sample 1, and the test sample obtained by sequentially performing vibration extraction with chloroform, water-saturated n-butanol is test sample 2.

[0037] The characteristic spectrum establishment process comprises the following steps: performing high performance liquid chromatography detection on the pretreated test samples of Baoyuan Decoction of Undergrowth Ginseng from several batches to obtain a spectrum containing characteristic peaks of ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, ginsenoside Rb2, ginsenoside Rf, ginsenoside Rc, ginsenoside Rd, scutellarein glucoside, scutellarein, formononetin, acteoside, glycyrrhizin, isoglycyrrhizin, glycyrrhetic acid, 6-shogaol, and constructing a characteristic spectrum of Baoyuan Decoction of Undergrowth Ginseng according to the relative retention time of the characteristic peaks; wherein the detection wavelength of test sample 1 is 250-260 nm, and the detection wavelength of test sample 2 is 200-210 nm.

[0038] In the present application, methanol is added to Baoyuan Decoction of Undergrowth Ginseng for pretreatment, which can remove the interfering components such as polysaccharides, proteins and tannins in Baoyuan Decoction, then the supernatant is evaporated to dryness and redissolved, which can concentrate and enrich the effective components of Baoyuan Decoction, improve the response value of each component in the determination process, and reduce the error. In the present application, chloroform is used for vibration extraction, which can remove the interfering components of ginsenoside determination, increase the separation degree of ginsenoside, and increase the accuracy of content determination.

[0039] In some embodiments, the identification process of the characteristic peaks is as follows: preparing control sample solutions of ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, ginsenoside Rb2, ginsenoside Rf, ginsenoside Rc, ginsenoside Rd, calycosin-7-glucoside, calycosin, formononetin, aglyconin, glyyrrhizinin, glyyrrhizin, glyyrrhetinic acid, and 6-shogaol at a set concentration, respectively, and detecting the undergrowth ginseng Baoyuan decoction and the control sample solutions by high performance liquid chromatography, and determining the corresponding characteristic peaks of the undergrowth ginseng Baoyuan decoction high performance liquid chromatography according to the peak retention time of the control sample high performance liquid chromatography.

[0040] In one or more embodiments, the undergrowth ginseng Baoyuan decoction is prepared as follows: taking undergrowth ginseng 3.73 g, astragalus 7.46 g, licorice 1.87 g, cassia 0.75 g, and ginger 3.00 g, and placing them in a 700 ml decoction kettle, and decocting twice, adding 600 ml of water for the first decoction, decocting for 150 min, filtering, adding 450 ml of water to the residue, decocting again for 100 min, filtering, and combining the two decoctions to obtain the undergrowth ginseng Baoyuan decoction.

[0041] In one or more embodiments, the solvent of the control sample solution is a methanol aqueous solution. The methanol aqueous solution, especially the methanol aqueous solution with a volume fraction of 75-85%, has good solubility for each component, is conducive to the dispersion of each compound, is the same as the solvent of the test sample solution, reduces the matrix effect, and improves the accuracy of establishing the characteristic spectrum.

[0042] In some embodiments, in the high performance liquid chromatography detection, the detection wavelength is 200-210 nm and 250-260 nm, the mobile phase A is acetonitrile, the mobile phase B is a 0.02-0.08% phosphoric acid aqueous solution, the chromatographic column filler is 1.6-2.1 μm octadecylsilane bonded silica gel, the chromatographic column length is 50-150 mm, and the chromatographic column diameter is 1.8-2.5 mm. The detection column temperature is 25-35 °C.

[0043] In one or more embodiments, the mobile phase B is a 0.045-0.055% phosphoric acid solution. At 203 nm, this mobile phase system can avoid the influence of ultraviolet absorption of the mobile phase on the baseline, and improve the reproducibility of the content determination.

[0044] In one or more embodiments, in the high performance liquid chromatography detection, the flow rate of the mobile phase is 0.10-0.25 mL / min, and the injection amount is 3-6 μl.

[0045] In one or more embodiments, in the high performance liquid chromatography detection process, the gradient elution program is as follows:

[0046] 0-12 min, 18% mobile phase A, the rest mobile phase B;

[0047] 12-13 min, 18%-23% mobile phase A, the rest mobile phase B;

[0048] 13-25 min, 23% mobile phase A, the rest mobile phase B;

[0049] 25-30 min, 23%-25% mobile phase A, the rest mobile phase B;

[0050] 30-40 min, 25%-40% mobile phase A, the rest mobile phase B;

[0051] 40-65 min, 40%-46% mobile phase A, the rest mobile phase B.

[0052] According to the elution sequence of the concentration gradient, the separation degree and detection sensitivity between components can be improved, and the stability is good and the precision is high.

[0053] In some embodiments, the test sample characteristic map is compared with the reference peak of the control sample, 14 characteristic peaks are present in the test sample characteristic map at 203 nm wavelength, and 18 characteristic peaks are present in the test sample characteristic map at 254 nm wavelength.

[0054] In some embodiments, under the condition of 203 nm wavelength, the relative retention time of the calycosin-7-glucoside characteristic peak is 0.2±10%, the relative retention time of the glycyrrhizin characteristic peak is 0.24±10%, the relative retention time of the ononin characteristic peak is 0.52±10%, the relative retention time of the glycyurin characteristic peak is 0.54±10%, the relative retention time of the ginsenoside Rg1 characteristic peak is 0.61±10%, the relative retention time of the ginsenoside Re characteristic peak is 0.63±10%, the relative retention time of the ginsenoside Rf characteristic peak is 0.96±10%, and the relative retention time of the ginsenoside Rb2 characteristic peak is 1.03±10%; under 254 nm, the relative retention time of the calycosin-7-glucoside characteristic peak is 0.85±10%, the relative retention time of the ononin characteristic peak is 2.25±10%, the relative retention time of the isoglycyrrhizin characteristic peak is 2.38±10%, the relative retention time of the glycyurin characteristic peak is 2.77±10%, the relative retention time of the calycosin characteristic peak is 3.23±10%, the relative retention time of the ononin characteristic peak is 4.41±10%, the relative retention time of the glycyrrhizinic acid characteristic peak is 4.57±10%, and the relative retention time of the 6-shogaol characteristic peak is 4.76±10%.

[0055] In some embodiments, different batches of the Baoyuan Decoction of Undergrowth Gynostemma Pentaphyllum are detected by high performance liquid chromatography at detection wavelengths of 200-210 nm and 250-260 nm. The ginsenoside chemical components have obvious absorption at 200-210 nm, which can reflect the quality of the undergrowth Gynostemma Pentaphyllum in the Baoyuan Decoction of Undergrowth Gynostemma Pentaphyllum; and the characteristic components of other medicinal materials can be reflected at 250-260 nm, and the multi-wavelength detection comprehensively reflects the quality of the medicine.

[0056] In some embodiments, the test sample characteristic map is compared with the reference peak of the control sample, and it is determined that 14 characteristic peaks are present in the characteristic map of the test sample at 203 nm, and 18 characteristic peaks are present in the characteristic map at 254 nm.

[0057] In some embodiments, the detection process of the Baoyuan Decoction of Undergrowth Gynostemma Pentaphyllum further comprises: detecting the to-be-detected Baoyuan Decoction of Undergrowth Gynostemma Pentaphyllum by high performance liquid chromatography, obtaining the chromatogram of the to-be-detected Baoyuan Decoction of Undergrowth Gynostemma Pentaphyllum, and comparing the chromatogram of the to-be-detected Baoyuan Decoction of Undergrowth Gynostemma Pentaphyllum with the characteristic map.

[0058] In one or more embodiments, according to the characteristic peaks determined after comparison, the peak areas of each characteristic peak are obtained, and then the contents of ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1 and ginsenoside Rb2 are calculated according to the peak areas. Meanwhile, the contents of ginsenoside Rf, ginsenoside Rc and ginsenoside Rd can also be calculated according to the peak areas.

[0059] In one or more embodiments, the conditions of the high performance liquid chromatography in the detection process of the Baoyuan Decoction of Undergrowth Gynostemma Pentaphyllum are the same as the conditions of the high performance liquid chromatography in the process of establishing the characteristic map. The conditions can ensure the accuracy of the detection results.

[0060] In another embodiment of the present application, the detection method of the above-mentioned Baoyuan Decoction of Undergrowth Gynostemma Pentaphyllum is applied to quality monitoring in industrial production of the Baoyuan Decoction of Undergrowth Gynostemma Pentaphyllum.

[0061] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in combination with specific experimental examples.

[0062] Experimental Example 1

[0063] Instruments: Waters H-class ultra-high performance liquid chromatograph; ten-millionth electronic balance, Mettler-Toledo Instrument (Shanghai) Co., Ltd.; KQ-300DE type numerical control ultrasonic cleaner.

[0064] Reagents: Ginsenoside Rg1, Ginsenoside Rb1, Ginsenoside Re, Ginsenoside Rb2, Ginsenoside Rf, Calycosin-7-glucoside, Calycosin, Formononetin, Comfirmatone B, Liquiritin, Isoliquiritigenin, Glycyrrhizin, Glycyrrhizic acid, 6-shogaol. The control samples were purchased from China Institute of Food and Drug Control. The Linxia Shenshengbaoyuan Decoction was self-made in the laboratory. Acetonitrile was chromatographically pure; phosphoric acid was chromatographically pure; water was ultrapure water.

[0065] Chromatographic conditions: Waters ACQUITY UPLC HSS T3 column (2.1 x 100 mm, 1.8 μm); mobile phase acetonitrile (A)-0.05% phosphoric acid (B), gradient elution sequence: 0-12 min, 18% A; 12-13 min, 18-23% A; 13-25 min, 23% A; 25-30 min, 23-25% A; 30-40 min, 25-40% A; 40-65 min, 40-46% A; column temperature 30°C; flow rate 0.15 ml / min; detection wavelength 254 nm.

[0066] Preparation of test solution: 20 ml of Linxia Shenshengbaoyuan Decoction water decoction liquid was taken and placed in a 100 ml volumetric flask, methanol was added to the mark, shaken well, and placed for 1 h. The supernatant was taken and filtered with a 0.22 μm microporous filter to obtain the test solution.

[0067] The response values of each component in the test sample chromatogram were too low, as shown in Table 1, indicating that the concentration of the test sample was not suitable and needed to be concentrated. Figure 3

[0068] Experimental Example 2

[0069] Instruments: Waters H-class ultra-high performance liquid chromatograph; ten-millionth electronic balance, Mettler-Toledo Instruments (Shanghai) Co., Ltd.; KQ-300DE type digital ultrasonic cleaner.

[0070] Reagents: Ginsenoside Rg1, Ginsenoside Rb1, Ginsenoside Re, Ginsenoside Rb2, Ginsenoside Rf, Ginsenoside Rd, Calycosin-7-glucoside, Calycosin, Formononetin, Comfirmatone B, Liquiritin, Isoliquiritigenin, Glycyrrhizin, Glycyrrhizic acid, 6-shogaol. The control samples were purchased from China Institute of Food and Drug Control. The Linxia Shenshengbaoyuan Decoction was self-made in the laboratory. Acetonitrile was chromatographically pure; phosphoric acid was chromatographically pure; water was ultrapure water.

[0071] ​Chromatographic conditions: Column Waters ACQUITY UPLC HSS T3 (2.1 x 100 mm, 1.8 μm); mobile phase acetonitrile (A) - 0.05% phosphoric acid (B), gradient elution sequence: 0-12 min, 18% A; 12-13 min, 18-23% A; 13-25 min, 23% A; 25-30 min, 23-25% A; 30-40 min, 25-40% A; 40-65 min, 40-46% A; column temperature 30 °C; flow rate 0.15 ml / min; detection wavelength 203 nm, 254 nm.

[0072] Preparation of test solution: Take 20 ml of Linxiashenbaoyuantang decoction, add methanol to 100 ml volumetric flask, shake well, stand for 1 h, take 50 ml of supernatant, evaporate to dryness, dissolve the residue with 80% methanol, transfer to 5 ml volumetric flask, filter the supernatant with 0.22 μm microporous filter, take the filtrate, and obtain.

[0073] The test sample has interfering chromatographic peaks near the retention time of ginsenoside Rf and ginsenoside Rd in the chromatogram at 203 nm, as shown in Figure 4 The separation degree of formononetin is poor at 254 nm, and the test sample needs to be further purified, as shown in Figure 5 .

[0074] Experimental Example 3

[0075] Instruments: Waters H-class ultra-high performance liquid chromatograph; ten-millionth electronic balance, Mettler-Toledo Instruments (Shanghai) Co., Ltd.; KQ-300DE type numerical control ultrasonic cleaner.

[0076] Reagents: ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, ginsenoside Rb2, ginsenoside Rf, calycosin-7-glucoside, calycosin, formononetin, calycosin-7-glucoside, glycyrrhizin, isoglycyrrhizin, glycyrrhizin, glycyrrhizic acid, 6-shogaol. The control samples were purchased from China Institute for Food and Drug Control, and Linxiashenbaoyuantang was self-made in the laboratory. Acetonitrile was chromatographically pure; phosphoric acid was chromatographically pure; water was ultrapure water.

[0077] Chromatographic conditions: Column Waters ACQUITY UPLC HSS T3 (2.1 x 100 mm, 1.8 μm); mobile phase acetonitrile (A) - 0.05% phosphoric acid (B), gradient elution sequence: 0-12 min, 18% A; 12-13 min, 18-23% A; 13-25 min, 23% A; 25-30 min, 23-25% A; 30-40 min, 25-40% A; 40-65 min, 40-46% A; column temperature 30 °C; flow rate 0.15 ml / min; detection wavelength 254 nm.

[0078] Preparation of test solution: 20 ml of the decoction of Baoyuan Tang was taken and placed in a 100 ml volumetric flask. Methanol was added to the mark, shaken well, and left to stand for 1 h. 50 ml of supernatant was taken, evaporated to dryness, dissolved in 10 ml of water, and extracted with water-saturated n-butanol for 3 times, 20 ml each time. The n-butanol extract was combined, washed with ammonia test solution for 3 times, 20 ml each time, and discarded. The n-butanol extract was evaporated to dryness, and the residue was transferred to a 5 ml volumetric flask. The supernatant was filtered with a 0.22 um microporous filter, and the filtrate was taken, i.e. the test solution was obtained.

[0079] After the Baoyuan Tang was treated with ammonia test solution, no glycyrrhizic acid chromatographic peak appeared, as shown in FIG. 1, indicating that the effective components with obvious acidity in the Baoyuan Tang were removed by the ammonia test solution, and thus the Baoyuan Tang was not suitable for use. Figure 6

[0080] Experimental Example 4

[0081] Instruments: Waters H-class ultra-high performance liquid chromatograph; ten-millionth electronic balance, Mettler-Toledo Instruments (Shanghai) Co., Ltd.; KQ-300DE type digital ultrasonic cleaner.

[0082] Reagents: Ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, ginsenoside Rb2, ginsenoside Rf, calycosin-7-glucoside, calycosin, formononetin, calycosin-7-glucoside, glycyrrhizin, isoglycyrrhizin, glycyrrhizinic acid, 6-shogaol. The control samples were purchased from China Institute for Food and Drug Control, and the Baoyuan Tang of wild ginseng was prepared in the laboratory. Acetonitrile was chromatographically pure; phosphoric acid was chromatographically pure; water was ultrapure water.

[0083] Chromatographic conditions: chromatographic column Waters ACQUITY UPLC HSS T3 (2.1 x 100 mm, 1.8 um); mobile phase acetonitrile (A)-0.1% formic acid (B), gradient elution sequence: 0-12 min, 20% A; 12-13 min, 20-23% A; 13-25 min, 23% A; 25-30 min, 23-25% A; 30-40 min, 25-40% A; 40-65 min, 40-46% A; column temperature 30°C; flow rate 0.15 ml / min; detection wavelength 203 nm.

[0084] Preparation of test solution: 20 ml of the decoction of Baoyuan Tang was taken and placed in a 100 ml volumetric flask. Methanol was added to the mark, shaken well, and left to stand for 1 h. 50 ml of supernatant was taken, evaporated to dryness, dissolved in 10 ml of water, and extracted with water-saturated n-butanol for 3 times, 20 ml each time. The n-butanol extract was combined, washed with ammonia test solution for 3 times, 20 ml each time, and discarded. The n-butanol extract was evaporated to dryness, and the residue was transferred to a 5 ml volumetric flask. The supernatant was filtered with a 0.22 um microporous filter, and the filtrate was taken, i.e. the test solution was obtained. ​

[0085] With acetonitrile-formic acid system as the mobile phase, the baseline of the chromatogram at 203 nm drifts, as shown in Figure 7 , affecting the accuracy and repeatability of the test, and therefore is not suitable for selection.

[0086] Experimental Example 5

[0087] Instrument: Waters H-class ultra-high performance liquid chromatograph; ten-millionth electronic balance, Mettler Toledo Instrument (Shanghai) Co., Ltd.; KQ-300DE type numerical control ultrasonic cleaner.

[0088] Reagents: Ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, ginsenoside Rb2, ginsenoside Rf, calycosin-7-glucoside, calycosin, formononetin, calycosin-7-glucoside, glycyrrhizin, isoglycyrrhizin, glycyrrhizin, glycyrrhizic acid, 6-shogaol. The control samples were purchased from China Institute for Food and Drug Control, and the Linxiashan Shenbaoyuantang was self-made by the laboratory. Acetonitrile was chromatographically pure; phosphoric acid was chromatographically pure; water was ultrapure water.

[0089] Chromatographic conditions: chromatographic column Waters ACQUITY UPLC HSS T3 (2.1 x 100 mm, 1.8 μm); mobile phase acetonitrile (A)-0.1% phosphoric acid (B), gradient elution sequence: 0-10 min, 16% A; 10-15 min, 16-23% A; 15-25 min, 23% A; 25-28 min, 23-29% A; 28-40 min, 29-35% A; 40-50 min, 35-55% A; 50-55 min, 55-90% A; 40-50 min, 35-55% A, column temperature 30°C; flow rate 0.15 ml / min; detection wavelength 203 nm.

[0090] Preparation of test solution: accurately take 20 ml of Linxiashan Shenbaoyuantang, add methanol to dilute to the mark in a 100 ml volumetric flask, shake well, stand for 1 h, filter, take 50 ml of the filtrate, evaporate to dryness, dissolve the residue in 10 ml of water, extract with chloroform for 3 times, 15 ml each time, discard the chloroform liquid, extract with water-saturated n-butanol for 3 times, 15 ml each time, combine the n-butanol extract, evaporate to dryness, dissolve the residue in methanol, transfer to a 5 ml volumetric flask, add methanol to the mark, shake well, filter, take the filtrate, and it is obtained.

[0091] Gradient peaks appear in the chromatogram at 28-32 min, as shown in Figure 8 , and this gradient condition is not suitable for the separation of ginsenoside components.

[0092] Experimental Example 6

[0093] Instrument: Waters H-class ultra-high performance liquid chromatograph; ten millionth electronic balance, Mettler-Toledo Instruments (Shanghai) Co., Ltd.; KQ-300DE type numerical control ultrasonic cleaner.

[0094] Reagents: Ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, ginsenoside Rb2, ginsenoside Rf, calycosin-7-glucoside, calycosin, formononetin, calycosin-7-glucoside, glycyrrhizin, isoglycyrrhizin, glycyrrhizin, glycyrrhizic acid, 6-shogaol. The control samples were purchased from China Institute for Drug Control, and the Linxiashan Shenshuangbaoyuantang was prepared by the laboratory. Acetonitrile was chromatographically pure; phosphoric acid was chromatographically pure; water was ultrapure water.

[0095] Chromatographic conditions: chromatographic column Waters ACQUITY UPLC HSS T3 (2.1 x 100 mm, 1.8 μm); mobile phase acetonitrile (A)-0.05% phosphoric acid (B), gradient elution sequence: 0-12 min, 18% A; 12-13 min, 18-23% A; 13-25 min, 23% A; 25-30 min, 23-25% A; 30-40 min, 25-40% A; 40-65 min, 40-46% A; column temperature 30°C; flow rate 0.3 ml / min; detection wavelength 254 nm.

[0096] Preparation of test solution: accurately take 20 ml of Linxiashan Shenshuangbaoyuantang, add methanol to 100 ml volumetric flask, shake well, stand for 1 h, filter, take 50 ml of the filtrate, evaporate to dryness, add 20 ml of water to dissolve, extract with water-saturated n-butanol for 3 times, 25 ml each time, combine the n-butanol extract, evaporate to dryness, dissolve the residue with methanol, transfer to a 5 ml volumetric flask, add methanol to the mark, shake well, filter, take the filtrate, and it is obtained.

[0097] Increasing the flow rate causes poor separation of common peaks such as glycyrrhizin, as shown in Figure 9 .

[0098] Best implementation method:

[0099] Instrument: Agilent 1260 high performance liquid chromatograph; ten millionth electronic balance, Mettler-Toledo Instruments (Shanghai) Co., Ltd.; KQ-300DE type numerical control ultrasonic cleaner.

[0100] Re, ginsenoside Rb2, ginsenoside Rf, ginsenoside Rd, ginsenoside Rc, calycosin-7-glucoside, calycosin, formononetin, calycosin-7-glucoside, glycyrrhizin, isoglycyrrhizin, glycyrrhetic acid, 6-shogaol. The control samples were purchased from China Institute of Food and Drug Control, and the Linxiashan Shenshuai Baoyuan Decoction was prepared in the laboratory. Acetonitrile was chromatographically pure; phosphoric acid was chromatographically pure; water was ultrapure water.

[0101] Chromatographic conditions: Waters ACQUITY UPLC HSS T3 column (2.1 x 100 mm, 1.8 μm); mobile phase acetonitrile (A)-0.05% phosphoric acid (B), gradient elution sequence: 0-12 min, 18% A; 12-13 min, 18-23% A; 13-25 min, 23% A; 25-30 min, 23-25% A; 30-40 min, 25-40% A; 40-65 min, 40-46% A; column temperature 30°C; flow rate 0.15 ml / min; detection wavelength 254 nm.

[0102] Preparation of test solution:

[0103] Precisely take 20 ml of Linxiashan Shenshuai Baoyuan Decoction, dilute to the mark in a 100 ml volumetric flask with methanol, shake well, stand for 1 h, filter, take 50 ml of the filtrate, evaporate to dryness, dissolve the residue in 20 ml of water, extract with water-saturated n-butanol for 3 times, 25 ml each time, combine the n-butanol extracts, evaporate to dryness, dissolve the residue in methanol, transfer to a 5 ml volumetric flask, add methanol to the mark, shake well, filter, take the filtrate for the test sample for determination at a wavelength of 254 nm.

[0104] Precisely take 20 ml of water decoction of Linxiashan Shenshuai Baoyuan Decoction, dilute to the mark in a 100 ml volumetric flask with methanol, shake well, stand for 1 h, filter, take 50 ml of the filtrate, evaporate to dryness, dissolve the residue in 10 ml of water, first extract with chloroform for 2 times, 15 ml each time, then extract with water-saturated n-butanol for 3 times, 15 ml each time, combine the n-butanol extracts, evaporate to dryness, dissolve the residue in methanol, transfer to a 5 ml volumetric flask, add methanol to the mark, shake well, filter, take the filtrate for the test sample for determination at a wavelength of 203 nm.

[0105] Preparation of control solution: take ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, ginsenoside Rc, ginsenoside Rd, ginsenoside Rb2, ginsenoside Rf, accurately weigh and determine, add methanol to prepare ginsenoside mixed control solution containing ginsenoside Rg1 0.104 mg / ml, ginsenoside Re 0.112 mg / ml, ginsenoside Rb1 0.124 mg / ml, ginsenoside Rc 0.106 mg / ml, ginsenoside Rd 0.110 mg / ml, ginsenoside Rb2 0.108 mg / ml, ginsenoside Rf 0.102 mg / ml. Take calycosin-7-glucoside, calycosin, formononetin, calycosin-7-glucoside, glycyrrhizin, isoglycyrrhizin, glycyrrhizin, glycyrrhizic acid, 6-shogaol, accurately weigh and determine, add methanol to prepare mixed control solution containing calycosin-7-glucoside 0.054 mg / ml, calycosin 0.051 mg / ml, formononetin 0.063 mg / ml, calycosin-7-glucoside 0.045 mg / ml, glycyrrhizin 0.082 mg / ml, isoglycyrrhizin 0.034 mg / ml, glycyrrhizin 0.047 mg / ml, glycyrrhizic acid 0.091 mg / ml.

[0106] Accurately take 5 μl of the test sample and control solution, inject into the liquid chromatograph, determine, and the obtained chromatogram is shown in Figures 1-2

[0107] Establishment of characteristic chromatogram of Linxiashenbaoyuantang:

[0108] Common peak calibration: 10 batches of Linxiashenbaoyuantang decoction liquid samples were determined according to the chromatographic conditions in the technical scheme, and the determination chromatograms at 203 nm and 254 nm were imported into the "Chinese medicine chromatographic characteristic chromatogram similarity evaluation system". The S1 sample with more chemical composition information was set as the reference chromatogram, and the characteristic chromatogram common mode was generated as shown in Figures 10-11 The 203 nm chromatogram was calibrated with 14 common peaks, and the 254 nm chromatogram was calibrated with 18 common peaks. The chromatographic peaks with moderate peak area and good separation degree were selected as the reference peaks S at each wavelength, and the relative retention time of other common peaks and S peaks was calculated as shown in Tables 1-2.

[0109] Table 1 Relative retention time (203 nm)

[0110]

[0111]

[0112] Table 2 Relative retention time (254 nm)

[0113]

[0114] ​

[0115] Correlation between the Baoyuan Decoction of Under-forest Ginseng and each medicinal material:

[0116] Characteristic chromatograms of the Baoyuan Decoction of Under-forest Ginseng, the Baoyuan Decoction of Under-forest Ginseng minus the flavoring medicinal materials, Under-forest Ginseng, Huangqi, Gancao, Ruxiang, Shengjiang were detected. In the 203 nm chromatogram, common peaks 1 and 5 were from Huangqi; 1, 2, 3, 4, 5, 6 were from Gancao; 7, 8, 9, 10, 11, 12, 13 were from Under-forest Ginseng. By comparison with the control solution, peak 7 was identified as ginsenoside Rg1; peak 8 was ginsenoside Re; peak 9 was ginsenoside Rf; peak 10 was ginsenoside Rb1; peak 11 was ginsenoside Rc; peak 12 was ginsenoside Rb2; peak 13 was ginsenoside Rd. In the 254 nm chromatogram, common peaks 1, 5, 8, 9, 10, 12 were from Huangqi; 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 13, 14, 15, 17, 18 were from Gancao; 9 was from Ruxiang; 16 was from Shengjiang. By comparison with the mixed control solution, peak 1 was identified as calycosin-7-glucoside; peak 3 was glycyrrhizin; peak 6 was isoglycyrrhizin; peak 8 was glycyrrhetic acid; peak 10 was calycosin; peak 12 was formononetin; peak 13 was glycyrrhizinic acid; peak 16 was 6-shogaol.

[0117] Sample content determination:

[0118] Ten batches of the Baoyuan Decoction of Under-forest Ginseng were prepared, and the contents of ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, and ginsenoside Rb2 were determined by the above method. The results are shown in Table 4.

[0119] According to DB21 / T2932-2018, the content of ginsenoside Rb1 in Under-forest Ginseng should not be less than 0.41%. According to the determination results, the content of ginsenoside Rb1 in the Baoyuan Decoction of Under-forest Ginseng was greater than 0.05 mg / ml.

[0120] Table 4 Determination results of sample content (mg / ml)

[0121]

[0122]

[0123] Methodology investigation:

[0124] Precision of characteristic chromatogram:

[0125] Take 20 ml of the Baoyuan Decoction of Under-forest Ginseng, and determine by the above method. The characteristic chromatogram was recorded by continuous sampling for 6 times. The peaks of ginsenoside Rb1 (203 nm chromatogram peak) and calycosin-7-glucoside (254 nm chromatogram peak) were used as the reference peaks. Figure 10 Figure 5 ​The relative retention time of each common peak was calculated with the retention time of ginsenoside Rbl (203 nm chromatogram peak No. 1) and delphinidin 3-O- rutinoside (254 nm chromatogram peak No. 1) as the reference (see Tables 5-6). The relative retention time of each common peak was less than 3%, indicating good precision.

[0126] Table 5 Relative retention time (203 nm)

[0127]

[0128] Table 6 Relative retention time (254 nm)

[0129]

[0130]

[0131] Characteristic pattern repeatability:

[0132] Take 20 ml of Linxiashenbaoyuantang, prepare 6 samples in parallel according to the above method, and record the characteristic pattern. Take the retention time of ginsenoside Rbl (203 nm chromatogram peak No. 1) and delphinidin 3-O-rutinoside (254 nm chromatogram peak No. 1) as the reference, and calculate the relative retention time of each common peak (see Tables 7-8). The relative retention time of each common peak is less than 3%, indicating good repeatability. Figure 10 Figure 5 Take 20 ml of Linxiashenbaoyuantang, prepare 6 samples in parallel according to the above method, and record the characteristic pattern. Take the retention time of ginsenoside Rbl (203 nm chromatogram peak No. 1) and delphinidin 3-O-rutinoside (254 nm chromatogram peak No. 1) as the reference, and calculate the relative retention time of each common peak (see Tables 7-8). The relative retention time of each common peak is less than 3%, indicating good repeatability.

[0133] Table 7 Relative retention time (203 nm)

[0134]

[0135] Table 8 Relative retention time (254 nm)

[0136]

[0137]

[0138] Characteristic pattern stability:

[0139] Take 20 ml of Linxiashenbaoyuantang, prepare 6 samples in parallel according to the above method, and record the characteristic pattern. Take the retention time of ginsenoside Rbl (203 nm chromatogram peak No. 1) and delphinidin 3-O-rutinoside (254 nm chromatogram peak No. 1) as the reference, and calculate the relative retention time of each common peak (see Tables 7-8). The relative retention time of each common peak is less than 3%, indicating good repeatability. Figure 10 Figure 5 Take 20 ml of Linxiashenbaoyuantang, prepare 6 samples in parallel according to the above method, and record the characteristic pattern. Take the retention time of ginsenoside Rbl (203 nm chromatogram peak No. 1) and delphinidin 3-O-rutinoside (254 nm chromatogram peak No. 1) as the reference, and calculate the relative retention time of each common peak (see Tables 7-8). The relative retention time of each common peak is less than 3%, indicating good repeatability.

[0140] Table 9 Relative retention time (203 nm)

[0141] ​​

[0142] Table 10 Relative retention time (254 nm)

[0143]

[0144] Linearity of assay:

[0145] Take ginsenoside Rg1 control amount, add 80% methanol to dissolve, prepare 398.21 μg / ml control solution, then add methanol to dilute to concentrations of 199.12, 99.55, 49.78, 24.89 μg / ml control diluent.

[0146] Take ginsenoside Re control amount, add 80% methanol to dissolve, prepare 191.82 μg / ml control solution, then add methanol to dilute to concentrations of 95.91, 47.96, 23.98, 11.99 μg / ml control diluent.

[0147] Take ginsenoside Rb1 control amount, add 80% methanol to dissolve, prepare 462.93 μg / ml control solution, then add methanol to dilute to concentrations of 231.47, 115.73, 57.87, 28.93 μg / ml control diluent.

[0148] Take ginsenoside Rb2 control amount, add 80% methanol to dissolve, prepare 484.21 μg / ml control solution, then add methanol to dilute to concentrations of 242.11, 121.05, 60.53, 30.26 μg / ml control diluent.

[0149] Take 5 μl of each control solution, measure according to the above chromatographic method, linear regression of each control concentration and peak area, get the regression equation, see Table 11. Linear correlation coefficient r of 6 components is greater than 0.999, indicating good linear relationship in the determination range.

[0150] Table 11 Regression equation

[0151]

[0152] Precision of assay

[0153] Take ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, ginsenoside Rb2 four component mixed control solution, according to the above method, measure 6 times continuously, measure the peak area of 6 components. The results are shown in Table 12, the RSD of the peak area of 4 components is less than 3%, indicating good precision.

[0154] Table 12 Peak area of 4 components

[0155] SEQ ID NO Ginsenoside Re Ginsenoside Rb1 Ginsenoside Rg1 Ginsenoside Rb2 1 152474 759910 274281 223617 2 151728 759832 275273 226371 3 153192 751283 272418 228129 4 151657 753215 273283 225281 5 153128 758923 274928 223019 6 151823 751829 272987 222049 Mean 152333.667 755832.000 273861.667 224744.333 RSD 0.462 0.548 0.416 1.013

[0156] Content determination repeatability

[0157] Take 20 ml of the forest-grown ginseng decoction and prepare 6 parallel samples according to the above method. Measure the peak area of ​​the four components and calculate their content (mg / ml). The results are shown in Table 13. The RSD of the peak area of ​​the four components is less than 3%, indicating good repeatability.

[0158] Table 13 Repeatability results of content determination of the four components

[0159] SEQ ID NO Ginsenoside Re Ginsenoside Rb1 Ginsenoside Rg1 Ginsenoside Rb2 1 152355 761144 275564 222406 2 152345 761066 276556 225160 3 153456 752517 273701 226918 4 152136 754449 274566 224070 5 152146 760157 276211 221808 6 152918 753063 274270 220838 Mean 152559.333 757066.000 275144.667 223533.333 RSD 0.343 0.547 0.414 1.018

[0160] Content determination stability

[0161] Take 20ml of the Linxiashan ginseng Baoyuan decoction and prepare it according to the above method. Inject the sample at 0h, 2h, 4h, 8h, 12h and 24h after sample preparation and measure the peak area of ​​the four components. The RSD of the peak area of ​​the four components at different measurement times is less than 3%, indicating that the test solution has good stability within 24h, as shown in Table 14.

[0162] Table 14. Stability results of the content determination of the four components.

[0163] SEQ ID NO Ginsenoside Re Ginsenoside Rb1 Ginsenoside Rg1 Ginsenoside Rb2 1 151109 762438 277393 749879 2 151099 762360 278385 749801 3 152210 753811 275530 741252 4 150890 755743 276395 743184 5 150900 761451 278040 748892 6 151672 754357 276099 741798 Mean 151313.333 758360.000 276973.667 745801.000 RSD 0.346 0.546 0.411 0.555

[0164] Content determination accuracy

[0165] Take 6 portions of the forest-grown ginseng decoction (10 ml each), and add reference standards with approximately the same content of ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, and ginsenoside Rb2 as in the sample. Determine the chromatographic peak areas of the four compounds using the method described above, calculate the content of the four compounds, and calculate the recovery rate and RSD for each compound. The results are shown in Tables 15-18. The average recovery rate and RSD of the four components meet the requirements, indicating that the method is accurate.

[0166] Table 15 Results of Ginsenoside Rg1 Recovery Rate

[0167]

[0168] Table 16 Results of Ginsenoside Re Recovery Rate

[0169]

[0170] Table 17 Results of Ginsenoside Rb1 Recovery Rate

[0171]

[0172] Table 18 Results of Ginsenoside Rb2 Recovery Rate

[0173]

[0174] The above descriptions are only the preferred embodiments of the present application, not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application should be included in the scope of the present application.

Claims

1. A method for detecting *Ginseng Baoyuan Decoction* from forest-grown ginseng, characterized in that... This includes the pretreatment process of the forest-grown ginseng and the establishment of its characteristic map. The pretreatment process of the *Linxiashan Shenbao Yuan Tang* decoction is as follows: methanol is added to the *Linxiashan Shenbao Yuan Tang* decoction and mixed evenly. After standing, the supernatant is evaporated to dryness and reconstituted with water. Then, it is extracted sequentially by shaking with chloroform and water-saturated n-butanol, or directly by shaking with water-saturated n-butanol. The n-butanol extract is evaporated to dryness and dissolved in methanol. The filtered filtrate is the test sample. The test sample obtained by shaking extraction directly with water-saturated n-butanol is test sample 1, and the test sample obtained by shaking extraction sequentially with chloroform and water-saturated n-butanol is test sample 2. The process of establishing the characteristic chromatogram is as follows: several batches of pre-treated forest ginseng Baoyuan Decoction samples were subjected to ultra-high performance liquid chromatography (UHPLC) to obtain chromatograms containing characteristic peaks of ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, ginsenoside Rb2, ginsenoside Rf, ginsenoside Rc, ginsenoside Rd, verrucoside glucoside, verrucoside, gentianin, gentianin, glycyrrhizin, isoglycyrrhizin, glycyrrhizin, glycyrrhizic acid, and 6-gingerol. The characteristic chromatogram of forest ginseng Baoyuan Decoction was constructed based on the relative retention time of the characteristic peaks. Among them, the detection wavelength of sample 1 was 250~260nm, and the detection wavelength of sample 2 was 200~210nm. In ultra-high performance liquid chromatography (UHPLC), mobile phase A was acetonitrile, and mobile phase B was a 0.02–0.08% (v / v) aqueous solution of phosphoric acid; the chromatographic column was a Waters ACQUITY UPLC HSS T3, 2.1 × 100 mm, 1.8 μm; the flow rate of the mobile phase was 0.10–0.15 mL / min; the gradient elution program was as follows: 0~12min, 18% mobile phase A, balance mobile phase B; 12-13 min, 18%-23% mobile phase A, balance mobile phase B; 13 ~ 25 min, 23% mobile phase A, balance mobile phase B; 25-30 min, 23%-25% mobile phase A, balance mobile phase B; 30-40 min, 25%-40% mobile phase A, balance mobile phase B; 40 ~ 65 min, 40% ~ 46% mobile phase A, balance mobile phase B.

2. The detection method for *Ginseng Baoyuan Decoction* under forest cover as described in claim 1, characterized in that, The identification process for the characteristic peaks is as follows: Ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, ginsenoside Rb2, ginsenoside Rf, ginsenoside Rc, ginsenoside Rd, verrucoside glucoside, verrucoside, gentianin, gentianin, glycyrrhizin, isoglycyrrhizin, glycyrrhizin, glycyrrhizic acid, and 6-gingerol are prepared into reference solutions of a set concentration. Ultra-high performance liquid chromatography (UHPLC) is used to detect both the *Linxia Shanshen Baoyuan Tang* and the reference solutions. The corresponding characteristic peaks of the *Linxia Shanshen Baoyuan Tang* are determined based on the peak retention times of the reference solutions in UHPLC.

3. The detection method for *Ginseng Baoyuan Decoction* under forest cover as described in claim 2, characterized in that, The solvent for the reference solution is an aqueous methanol solution.

4. The detection method for *Ginseng Baoyuan Decoction* under forest cover as described in claim 3, characterized in that, The volume fraction of the methanol-water solution is 75-85%.

5. The detection method for *Ginseng Baoyuan Decoction* under forest cover as described in claim 1, characterized in that, In ultra-high performance liquid chromatography (UHPLC) detection, mobile phase B is a 0.045~0.055% phosphoric acid solution.

6. The detection method for *Ginseng Baoyuan Decoction* under forest cover as described in claim 1, characterized in that, During ultra-high performance liquid chromatography (UHPLC) detection, the injection volume is 3~6 μl.

7. The detection method for *Ginseng Baoyuan Decoction* under forest cover as described in claim 1, characterized in that, The characteristic spectrum of the test sample was compared with the peaks of the reference standard. At a wavelength of 203 nm, the characteristic spectrum of the test sample showed 14 characteristic peaks, and at a wavelength of 254 nm, the characteristic spectrum of the test sample showed 18 characteristic peaks. Alternatively, at a wavelength of 203 nm, with ginsenoside Rb1 as the S peak, the relative retention times of the characteristic peaks of verrucoside glucoside, glycyrrhizin, ginsenoside Rf, and ginsenoside Rb2 were 0.2±10%, 0.24±10%, 0.52±10%, 0.54±10%, 0.61±10%, 0.63±10%, and 0.96±10%, respectively. 1.03±10%; At 254 nm, with glycyrrhizin as the S peak, the relative retention times of the characteristic peaks of verbascoside glucoside, argentin, isoglycyrrhizin, glycyrrhizin, isoglycyrrhizin, isoglycyrrhizin, isoglycyrrhizin, isoglycyrrhizin, isoglycyrrhizin, isoglycyrrhizin, isoglycyrrhizin, isoglycyrrhizin, isoglycyrrhizin, isoglycyrrhizin, isoglycyrrhizin, isoglycyrrhizin, isoglycyrrhizin, isoglycyrrhizin, isoglycyrrhizin, isoglycyrrhizin, isoglycyrrhizin, isoglycyrrhizin, isoglycyrrhizin, isoglycyrrhizin, isoglycyrrhizin, and 6-gingerol are 4.76±10%.

8. The detection method for *Ginseng Baoyuan Decoction* under forest cover as described in claim 1, characterized in that, It also includes the process of detecting the forest-grown ginseng and ginseng soup, which is as follows: ultra-high performance liquid chromatography is used to detect the sample to be tested, the chromatogram of the sample to be tested is obtained, and the chromatogram of the sample to be tested is compared with the characteristic spectrum.

9. The detection method for *Ginseng Baoyuan Decoction* under forest cover as described in claim 8, characterized in that, Based on the characteristic peaks determined by comparison, the peak area of ​​each characteristic peak is obtained, and then the contents of ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, and ginsenoside Rb2 are calculated based on the peak area, or the contents of ginsenoside Rf, ginsenoside Rc, and ginsenoside Rd are calculated based on the peak area.

10. The detection method for *Ginseng Baoyuan Decoction* under forest cover as described in claim 8, characterized in that, The ultra-high performance liquid chromatography (UHPLC) conditions used in the detection of Linxiashan ginseng and Baoyuan decoction were the same as those used in the establishment of the characteristic chromatogram.

11. The application of the detection method of forest ginseng Baoyuan Decoction according to any one of claims 1 to 10 in the quality control of forest ginseng Baoyuan Decoction in industrial production.

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