Method for determining content of index components in Kaixin powder reference sample and establishment of fingerprint spectrum methodology

Abstract

The invention relates to the technical field of medicine detection, in particular to a method for determining the content of index components of a Kaixin powder reference sample and establishment of fingerprint methodology, and used detection indexes are as follows: polygala xanthone III, 3, 6 '-dimustard acyl sucrose, ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 are used as index components. The invention provides a method for determining the content of five index components in the intermediate process of the Kaixin powder preparation, and the method is accurate, reliable, simple and convenient; through content determination of a reference sample, a quantity value transfer relation of processes of medicinal materials, decoction pieces, reference substances, powder and the like can be established, and meanwhile, methodology of a Kaixin powder fingerprint spectrum is established, and the method can reflect characteristics of various medicinal materials of a Kaixin powder compound preparation. Therefore, a reference basis is provided for comprehensively controlling the quality stability of the traditional Chinese medicine compound preparation.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical detection technology, and in particular to a method for determining the content of index components of a Kaixinsan reference sample and the establishment of a fingerprint methodology. Background Art

[0002] Kaixinsan comes from "Prescriptions for Emergencies Worth a Thousand Gold Pieces" by Sun Simiao in the Tang Dynasty and has been included in the "Catalogue of Ancient Classic Prescriptions". Kaixinsan is composed of four parts of Polygala tenuifolia, four parts of Panax ginseng, one liang of Acorus calamus and two liang of Poria cocos. Among the four medicinal materials, Polygala tenuifolia is the monarch drug, and the indicative ingredients are Polygala tenuifolia xanthinone III, 3,6'-dianisinoylsucrose, and Polygala tenuifolia saponins; Ginseng is the ministerial drug, and the indicative ingredients are ginsenoside Rg1, ginsenoside Rb1, and ginsenoside Re; Acorus calamus is the adjuvant drug, and its indicative ingredients are the volatile components in the volatile oil; Poria cocos is the guiding drug, and its indicative ingredients are mainly triterpenoid saponins and Poria cocos polysaccharides.

[0003] Traditional Chinese medicine compound can act on multiple targets and pathways, with multiple indicator components working synergistically. Selecting only a single component is not representative. However, selecting all the components of the compound preparation for testing and analysis will cost a lot of manpower and material resources, causing unnecessary losses, and it takes a long time, the pretreatment process is cumbersome, and the nature of some components is unstable and the content is small. Whether they play a pharmacological role and mechanism is unknown. Therefore, at present, combining the "Q-marks" principle of traditional Chinese medicine and the prediction of Kaixinsan's network pharmacology, the indicator components of Kaixinsan are selected, and the components whose pharmacological effects have been proven by current studies and whose components are stable and controllable are finally selected. The chemical components of Polygala root, including Polygala xanthinone III and 3,6'-bisinapoylsucrose, and ginsenoside Rg1, ginsenoside Rb1, and ginsenoside Re in the adjuvant, have been used to establish a method for determining the content of the main pharmacological effects in the compound preparation of Kaixinsan. It has high feasibility and low requirements for the precision of instruments and chromatographic columns. It has greatly avoided the defects of some content determination methods that are short in time but require high instruments. Through a large number of experimental explorations, the pretreatment method has been determined, which can extract the effective components of Kaixinsan to the greatest extent, bringing great convenience to the content determination of Kaixinsan compound preparations and providing ideas and guidance for the content determination of other compound preparations. With the relevant application of modern technology, establishing fingerprints is an effective way to identify the key information of compound preparations. Its types include TLC identification, liquid fingerprints, GC fingerprints, GC-MS fingerprints, and infrared fingerprints. The methods and types are diverse, liquid fingerprints are widely used, and the technology is relatively mature. However, the disadvantages of the existing technology are that the liquid phase methodology processing process that can cover the determination of all index components is relatively complicated, requires high conditions for chromatographic columns and instruments, and the liquid phase operation time is too long. Other methods measure a single content, which only includes the content of a single medicinal material and cannot fully cover the overall characteristics of the Kaixinsan compound preparation. Summary of the invention

[0004] In view of the deficiencies of the above-mentioned technologies, the present invention has established a method for determining the content of indicative components of Kaixin Powder benchmark samples. The fingerprint spectrum methodology is used to completely identify the characteristics of Kaixin Powder compound preparations, thereby overcoming the limitations of existing detection methods and providing a basis for comprehensive quality control of subsequent Kaixin Powder preparations and product stability.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for determining the content of index components of Kaixinsan reference sample and establishing fingerprint methodology, comprising:

[0006] Part I: A method for determining the content of index components in a Kaixinsan reference sample, including:

[0007] The test is to determine the index components in Kaixinsan preparations by high performance liquid chromatography, and the analysis is carried out by measuring the test solution and the reference solution;

[0008] The indicative ingredients include polygala tenuifolia xanthophyll III, 3,6'-dienasinoylsucrose, ginsenoside Rg1, ginsenoside Rb1, and ginsenoside Re.

[0009] As a preferred solution of the present invention, preparing the test solution includes the following steps:

[0010] S1: Take the Xinxin powder test sample and put it in a container;

[0011] S2: Add 70% methanol into the container, shake the container to mix Kaixinsan and 70% methanol evenly, let it stand and cool, and then weigh it;

[0012] S3: placing the container into an ultrasonic cleaning apparatus for ultrasonic treatment;

[0013] S4: taking the container out of the ultrasonic cleaner and weighing it, adding methanol to the container so that the overall weight is consistent with the weight in step S2;

[0014] S5: placing the container in a centrifuge to centrifuge the solution;

[0015] S6: Filter the solution through a PTFE filter membrane and take the filtrate, which is the test solution.

[0016] As a preferred solution of the present invention, preparing the reference solution comprises the following steps:

[0017] S7: Aspiration Ketone III, 3,6'-dienasinoylsucrose, ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 are placed in a container;

[0018] S8: Add methanol into the container to prepare a mixed solution.

[0019] As a preferred scheme of the present invention, the chromatographic conditions are as follows:

[0020] Octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A; 0.05% phosphoric acid water was used as mobile phase B; the detection wavelength was 203 nm, the column temperature was 25°C; the flow rate was 0.6 ml / min; the elution procedure was as follows: 0-35 min, 20% A, 35-65 min, 20% A-29% A, 65-100 min, 29% A-40% A, 100-101 min, 40% A-85% A, 101-114 min, 85% A-85% A.

[0021] As a preferred solution of the present invention, the weight of Kaixin powder in step S1 is 5g;

[0022] The concentration of methanol in step S2 is 70%, and the amount added is 25 ml; the concentration of methanol in step S4 is 70%;

[0023] In step S3, the ultrasonic treatment time is 40 min and the temperature is 60° C.;

[0024] In step S5, the centrifugal speed is 5000 r / min and the time is 5 min; in step S6, the pore size of the PTFE filter membrane is 0.22 μm.

[0025] As a preferred embodiment of the present invention, the contents of polygala tenuifolia III reference substance, 3,6'-dicerinoylsucrose reference substance, ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 in the reference solution are 0.15 mg / ml, 0.55 mg / ml, 0.1 mg / ml, 0.1 mg / ml and 0.15 mg / ml respectively.

[0026] The second part is to establish a fingerprint methodology for the index components of Kaixinsan reference sample, characterized in that the preparation of the test solution includes the following steps:

[0027] S9: Take 1 g of the Kaixinsan test sample and place it in a container;

[0028] S10: Accurately add 25 ml of 70% methanol into the container and weigh accurately;

[0029] S11: Place the container in an ultrasonic cleaning apparatus for 40 minutes, take it out, and let it cool;

[0030] S12: Use solvent to make up the lost weight and weigh accurately again;

[0031] S13: Filter with a 0.22 um filter membrane to obtain.

[0032] As a preferred solution of the present invention, the preparation of single-flavor decoction pieces and negative control solution comprises the following steps:

[0033] S14: Accurately weigh 1g of the negative samples of Polygala, Ginseng, Poria, Acorus calamus, and each single herbal medicine into a container;

[0034] S15: Accurately add 25 ml of 70% methanol into the container and weigh accurately;

[0035] S16: placing the container in an ultrasonic cleaning apparatus for 40 minutes, taking it out, and letting it cool;

[0036] S17: Use solvent to make up the lost weight and weigh accurately again;

[0037] S18: Shake well and filter with a 0.22um filter membrane.

[0038] As a preferred scheme of the present invention, the chromatographic conditions are as follows:

[0039] Octadecylsilane bonded silica gel was used as filler; acetonitrile mobile phase A; 0.05% phosphoric acid water was used as mobile phase B; the detection wavelength was 203nm, and the column temperature was 30℃. The flow rate was 0.8ml / min for 0-68min, 1.0ml / min for 70-109min, and the theoretical plate number of mobile phase A should not be less than 3000 according to the peak of 3,6'-dicorinoylsucrose. The elution procedure was as follows: 0-5min, 16%A-18%A, 5-20min, 18%A-23%A, 20-38min, 23%A-40%A, 38-68min, 40%A-52%A, 68-70min, 52%A-73%A, 70-83min, 73%A-80%A, 83-93min, 80%A, 93-98min, 80%A-95%A.

[0040] As a preferred solution of the present invention, the established content determination method is characterized in that, during the detection, the mixed reference solution and the test solution are respectively taken into the high performance liquid chromatograph for detection according to the law, and the peak areas Ax reference and Ax test of each index component in the reference solution and the test solution are obtained from each chromatogram. The concentration C of the reference solution of each index component is known. 对照品 , the concentration of each index component in the test solution was calculated by external standard method, formula: C 供试品 =C 对照品 / A 对照品 ×A 供试品 , and then the content of each index component is obtained by the sample weight and dilution multiple.

[0041] In the established fingerprint method, the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition) was finally used for calculation, and the reference peak was selected. After peak matching and multi-point correction, the indicative components with higher response values, more stable properties, and more indicative components were selected as characteristic peaks. The median method was used to generate the corresponding control spectrum, match the data, and calibrate the relative retention time. Finally, the similarity was calculated. If it was greater than 0.95, the samples were considered to have high similarity.

[0042] The beneficial effects of the present invention are:

[0043] (1) The present invention provides a method for accurately measuring the contents of five index components of the intermediate (reference substance) of Kaixin Powder preparations. The processing method is simple, thereby establishing the quantity transfer relationship of each process such as medicinal materials, decoction pieces, reference substances, powders, etc., and studying the changes in the transfer rate, so as to comprehensively control the quality of Kaixin Powder.

[0044] (2) Through a large number of preliminary experiments, it was found that if a method needs to take into account the index components of all medicinal materials in the prescription, it will take a long time and have high requirements on instruments and chromatographic columns, and the actual operation process has certain limitations. This patent invention solves the above problems, and the extraction method studied in the present invention has high extraction efficiency and is simple and easy to operate.

[0045] (3) The fingerprint provided by the present invention can reflect the index components of all single medicinal materials of Kaixinsan compound preparation. The method is simple and easy to operate. Compared with previous studies, the time is shortened and the overall characteristics of Kaixinsan compound preparation can be reflected as a whole. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 It is a chromatogram of a mixed reference solution used in the content determination method of the present invention;

[0047] Figure 2 It is a chromatogram of a test solution used in the content determination method of the present invention;

[0048] Figure 3 , 4 It is the reference solution chromatogram used in the fingerprint methodology of the present invention;

[0049] Figure 5 It is the common peak attribution chromatogram of the test solution used in the fingerprint methodology of the present invention;

[0050] Figure 6 This is a comparison chart of the whole prescription and single medicinal materials used for fingerprint spectrum of the present invention. A is Poria cocos, B is Acorus calamus, C is Ginseng, D is Polygala tenuifolia, and E is the reference sample;

[0051] Figure 7 This is a comparison diagram of the whole prescription and negative medicinal materials used for fingerprint spectrum of the present invention; A is negative Poria cocos, B is negative Acorus calamus, C is negative ginseng, D is negative Polygala tenuifolia, and E is a negative reference sample;

[0052] Figure 8 It is a similarity diagram of traditional Chinese medicine fingerprints of 15 batches of benchmark sample fingerprints of the present invention. DETAILED DESCRIPTION

[0053] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0054] A method for determining the content of index components of Kaixinsan reference samples and the establishment of fingerprint methodology, including:

[0055] Part I: A method for determining the content of index components in a Kaixinsan reference sample, including:

[0056] The test is to determine the index components in Kaixinsan preparations by high performance liquid chromatography, and the analysis is carried out by measuring the test solution and the reference solution;

[0057] The indicative ingredients include polygala tenuifolia xanthophyll III, 3,6'-dienasinoylsucrose, ginsenoside Rg1, ginsenoside Rb1, and ginsenoside Re.

[0058] As a preferred solution of the present invention, preparing the test solution includes the following steps:

[0059] S1: Take the Xinxin powder test sample and put it in a container;

[0060] S2: Add 70% methanol into the container, shake the container to mix Kaixinsan and 70% methanol evenly, let it stand and cool, and then weigh it;

[0061] S3: placing the container into an ultrasonic cleaning apparatus for ultrasonic treatment;

[0062] S4: taking the container out of the ultrasonic cleaner and weighing it, adding 70% methanol to the container so that the overall weight is consistent with the weight in step S2;

[0063] S5: placing the container in a centrifuge to centrifuge the solution;

[0064] S6: Filter the solution through a PTFE filter membrane and take the filtrate, which is the test solution.

[0065] As a preferred solution of the present invention, preparing the reference solution comprises the following steps:

[0066] S7: Aspiration Ketone III, 3,6'-dienasinoylsucrose, ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 are placed in a container;

[0067] S8: Add methanol into the container to prepare a mixed solution.

[0068] As a preferred scheme of the present invention, the chromatographic conditions are as follows:

[0069] Octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A; 0.05% phosphoric acid water was used as mobile phase B; the detection wavelength was 203 nm, the column temperature was 25°C; the flow rate was 0.6 ml / min; the elution procedure was as follows: 0-35 min, 20% A, 35-65 min, 20% A-29% A, 65-100 min, 29% A-40% A, 100-101 min, 40% A-85% A, 101-114 min, 85% A-85% A.

[0070] As a preferred solution of the present invention, the weight of Kaixin powder in step S1 is 5g;

[0071] The concentration of methanol in step S2 is 70%, and the amount added is 25 ml; the concentration of methanol in step S4 is 70%;

[0072] In step S3, the ultrasonic treatment time is 60 min and the temperature is 70° C.;

[0073] In step S5, the centrifugal speed is 5000 r / min and the time is 5 min; in step S6, the pore size of the PTFE filter membrane is 0.22 μm.

[0074] As a preferred embodiment of the present invention, the contents of polygala tenuifolia III reference substance, 3,6'-dicerinoylsucrose reference substance, ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 in the reference solution are 0.15 mg / ml, 0.55 mg / ml, 0.1 mg / ml, 0.1 mg / ml and 0.15 mg / ml respectively.

[0075] The second part is to establish a fingerprint methodology for the index components of Kaixinsan reference sample, characterized in that the preparation of the test solution includes the following steps:

[0076] S9: Take 1 g of the Kaixinsan test sample and place it in a container;

[0077] S10: Accurately add 25 ml of 70% methanol into the container and weigh accurately;

[0078] S11: Place the container in an ultrasonic cleaning apparatus for 40 minutes, take it out, and let it cool;

[0079] S12: Use solvent to make up the lost weight and weigh accurately again;

[0080] S13: Filter with a 0.22 um filter membrane to obtain.

[0081] As a preferred solution of the present invention, the preparation of single-flavor decoction pieces and negative control solution comprises the following steps:

[0082] S14: Accurately weigh 1g of the negative samples of Polygala, Ginseng, Poria, Acorus calamus, and each single herbal medicine into a container;

[0083] S15: Accurately add 25 ml of 70% methanol into the container and weigh accurately;

[0084] S16: placing the container in an ultrasonic cleaning apparatus for 40 minutes, taking it out, and letting it cool;

[0085] S17: Use solvent to make up the lost weight and weigh accurately again;

[0086] S18: Shake well and filter with a 0.22um filter membrane.

[0087] As a preferred scheme of the present invention, the chromatographic conditions are as follows:

[0088] Octadecylsilane bonded silica gel was used as filler; acetonitrile mobile phase A; 0.05% phosphoric acid water was used as mobile phase B; the detection wavelength was 203nm, and the column temperature was 30℃. The flow rate was 0.8ml / min for 0-68min, 1.0ml / min for 70-109min, and the theoretical plate number of mobile phase A should not be less than 3000 according to the peak of 3,6'-dicorinoylsucrose. The elution procedure was as follows: 0-5min, 16%A-18%A, 5-20min, 18%A-23%A, 20-38min, 23%A-40%A, 38-68min, 40%A-52%A, 68-70min, 52%A-73%A, 70-83min, 73%A-80%A, 83-93min, 80%A, 93-98min, 80%A-95%A.

[0089] As a preferred solution of the present invention, during the detection, the mixed reference solution and the test solution are respectively taken into the high performance liquid chromatograph for detection according to the law, and the peak areas Ax reference and Ax test of each index component in the reference solution and the test solution are obtained from each chromatogram. The concentration C of the reference solution of each index component is known. 对照品 , the concentration of each index component in the test solution was calculated by external standard method, formula: C 供试品 =C 对照品 / A 对照品 ×A 供试品 ,Then the content of each index component is obtained by the sample weight and dilution multiple of the test sample. In the established fingerprint method, the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) is finally used for calculation, and the reference peak is selected. After peak matching and multi-point correction, the index component with higher response value, more stable properties is selected as the characteristic peak. The corresponding control spectrum is generated by the median method, the data is matched, the relative retention time is checked, and the similarity is calculated. If it is greater than 0.95, the similarity between samples is considered high.

[0090] Example:

[0091] At present, there is no quality standard for Kaixin Powder, nor is there a method for quickly identifying the authenticity of Kaixin Powder. The ingredients of Kaixin Powder are the main ingredient Polygala tenuifolia, the assistant ingredient Ginseng, the adjuvant Acorus calamus, and the guiding ingredient Poria, which are mixed in a certain proportion, sieved, and combined into a compound preparation to exert the effects of invigorating qi and nourishing the heart, calming the nerves and settling the mind. The present invention combines the technical guiding principles of classic prescriptions such as the "Chinese Pharmacopoeia" with scientific basis as ideological guidance, and then uses the Q-market principle, one test and multiple evaluation method, and network pharmacology prediction of effective ingredients proposed by modern Chinese medicine concepts as a guide, and finally determines and determines 5 index components in the main ingredient and assistant ingredient in Kaixin Powder. The extraction method investigates the effects of different extraction methods, different temperatures, different concentrations of solvents, and different extraction times. The extraction method finally determined can maximize the extraction rate of these 5 index components with the highest efficiency, accurately determine the content of effective ingredients in Kaixin Powder, and quickly and efficiently identify other medicinal flavors in Kaixin Powder.

[0092] The present invention uses HPLC to determine the content of polygala tenuifolia xanthoside III, 3,6'-diesinapoylsucrose in Kaixin Powder, ginsenoside Rg1, ginsenoside Rb1 and ginsenoside Re in the auxiliary medicine, investigates the influence of different parameters on the content determination, and at the same time invents and establishes a liquid fingerprint spectrum methodology that can comprehensively reflect the characteristics of Kaixin Powder compound preparations. The fingerprint spectrum has a large amount of information, strong overall characteristic, and can intuitively characterize the quality characteristics and identity information of traditional Chinese medicine, and finally obtains the following optimization:

[0093] The instruments, reagents and reagents used in the following examples are as follows:

[0094] (1) Instruments: Thermo Ultimate 3000 HPLC (Thermo Fisher Scientific); Phenom C18 column (4.6x150mm, 3μm); Thermo Acclaim TM 120C18 column (4.6x250mm, 5pum); Kromasi1 100-5-C18 (4.6x250mm, 5um); Waters Xbridge C18 (4.6x250mm, 5pum), Agilent ZORBAX SB-C18 (4.6x250mm, 5pum), Thermo Hypersil GOLD (4.6x250mm, 5pum); TG16 high-speed centrifuge (Shanghai Luxiangyi Centrifuge Instrument Co., Ltd.); JP-100S ultrasonic cleaner (Shumei); constant temperature water bath (Changzhou Jintan Liangyou); XPR36 Delta Range 1 / 100000 electronic analytical balance (Switzerland Mettler-Toledo International Trading (Shanghai) Co., Ltd.); SQP 1 / 100000 electronic analytical balance (Germany Sartorius); RE-201D rotary evaporator (Shanghai Kexing Instrument Co., Ltd.)

[0095] (2) Reagents: methanol (chromatographic grade), acetonitrile (chromatographic grade), phosphoric acid (analytical grade).

[0096] (3) Test drugs: Polygala tenuifolia sterol III (batch number 111850-202207, China Food and Drug Inspection Institute), 3,6-dicerinoylsucrose (batch number 111848-202307, China Food and Drug Inspection Institute), ginsenoside Rg1 (batch number 110703-202235, China Food and Drug Inspection Institute), ginsenoside Re (batch number 110754-202330, China Food and Drug Inspection Institute) Food and Drug Inspection Institute), Ginsenoside Rb1 (Batch No. 110754-202330, China Food and Drug Inspection Institute), β-Asarone (Batch No. JB268993, Shanghai Yuanye), α-Asarone (Batch No. 100298-201203, China Food and Drug Inspection Institute), Pachymic acid (Batch No. JB242338, Shanghai Yuanye), Dehydrogenated tert-butyl ester (Batch No. A28IB214159, Shanghai Yuanye)

[0097] Kaixinsan benchmark sample, prescription: Polygala 200g, ginseng 200g, Poria 400g, Acorus calamus 200g, the above four medicinal materials are mixed in a ratio of 1:1:2:1 and passed through a No. 6 sieve to obtain 15 different batches of Kaixinsan benchmark samples, named S1-S15.

[0098] Negative herbs are composed of herbs that lack the medicinal flavor of the prescription.

[0099] Example 1: Determination of the content of Kaixinsan, the method is as follows:

[0100] 1. Chromatographic conditions

[0101] 1.1 Selection of detection wavelength

[0102] Under the detection of DAD detector, this method combines the detection wavelength when determining the medicinal materials with the formula and the taste. The maximum absorption wavelength of ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 in Kaixin Powder determined by this method is 203nm, with no obvious absorption at high wavelength. The maximum absorption wavelengths of polygala tenuifolia ketone III and 3,6-dicansinoylsucrose under DAD detector are 259nm and 330nm respectively. Taking the absorption of all components into consideration, it was finally decided to use 203nm as the detection wavelength for this method.

[0103] 1.2 Selection of mobile phase and chromatographic column

[0104] This method investigates two mobile phases, (methanol: phosphoric acid water) and (acetonitrile: phosphoric acid water). The latter has lower instrument pressure and better effect during operation. Finally, it is decided to use acetonitrile: phosphoric acid water as the mobile phase of this method. The instrument pressure is lower during operation. Then, the effects of different concentrations of phosphoric acid water methanol: 0.01% phosphoric acid water, 0.05% phosphoric acid water, and 0.1% phosphoric acid aqueous solution are considered. It is found that the above three different concentrations of phosphoric acid water have little effect on the overall test sample. Considering the peak time and the applicability of the chromatographic column, this method compares the three chromatographic columns of ThermoAccalim 120, Kromail C18, and AglientXbridge C18. It is proposed that ThermoAccalim 120 will have better effect. The durable pH value range of the chromatographic column is 2-8. Finally, 0.05% phosphoric acid water: acetonitrile solution is used as the mobile phase.

[0105] 1.3 Development and optimization of elution procedures

[0106] Among them, through a lot of condition exploration and research, the chromatographic conditions are as follows: octadecylsilane bonded silica gel as filler; acetonitrile mobile phase A; 0.05% phosphoric acid water as mobile phase B; detection wavelength is 203nm, column temperature is 25℃. Flow rate is 0.6ml / min, and elution is performed according to the following procedure. The separation degree of ginsenoside Rg1 and ginsenoside Re shall not be less than 1.5, and the theoretical plate number of ginsenoside Rg1 shall not be less than 6000. The elution procedure is as follows: 0~35min, 20%A, 35~65min, 20%A~29%A, 65~100min, 29%A~40%A, 100~101min, 40%A~85%A, 101~114min, 85%A~85%A.

[0107] 1.4 Selection and determination of index components

[0108] Kaixinsan is a traditional Chinese medicine compound preparation with complex medicinal flavors. Kaixinsan contains many active ingredients, including saponin compounds, triterpene acid compounds, ketone compounds, oligosaccharide ester compounds and other compounds. At present, some people use network pharmacology and experimental verification to exert effects on AD through multi-target, multi-component and multi-pathway mechanisms. After multiple investigations, it was found that Polygala tenuifolia has the effects of calming the mind and improving intelligence, connecting the heart and kidney, and removing phlegm and opening the orifices. It is a commonly used tranquilizer in clinical practice, which is consistent with the clinical main effect of Kaixinsan. It is the main drug. Polygala tenuifolia can increase the content of serotonin in the human body and thus play an antidepressant role. Polygala tenuifolia oligosaccharides and saponin components are the main active ingredients of Polygala tenuifolia. However, it was found through investigation that the extraction process of saponin components is relatively cumbersome, and the saponin components cannot be completely extracted by ultrasonic method alone. Most of the blood-entering components of Kaixinsan come from Polygala tenuifolia. Ginsenosides in ginseng are active ingredients in the formula, and ginsenoside Rg1 has been clinically reported to have potential therapeutic effects on depression. Experiments have shown that the content of other active ingredients, such as triterpenoids such as pachymic acid, is small and the differences between batches are large. Although the content of volatile oil components a-asarone and β-asarone in Acorus calamus is high, the properties of the compounds are unstable. Combined with traditional medicinal properties, traditional efficacy, new drug properties, measurability of chemical components, and compound compatibility, we finally screened and selected polygalaconone III and 3,6-dicerinoylsucrose in Polygala tenuifolia, and ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 in ginseng as assistant drugs and quality markers of Kaixinsan compound medicine.

[0109] 2. Preparation of test samples

[0110] 2.1 Investigation of extraction methods

[0111] Take an appropriate amount of the reference sample of Example 1 of Xin San, add an equal amount of the same extraction solvent 70% methanol, weigh accurately, and use two different extraction treatment methods of ultrasonic extraction and reflux extraction for determination.

[0112] 2.2 Investigation of solvent concentration

[0113] Take an appropriate amount of the reference sample of Kaixin Powder Example 1, add an equal amount of the same extraction solvent 45% methanol, 70% methanol, and methanol, accurately weigh, and perform ultrasonic treatment for 30 minutes respectively. Take out and cool, add solvent to return to the original weight, shake well, filter with a microporous filter membrane (0.22μm), and use it as the test solution for detection.

[0114] 2.3 Investigation of extraction temperature

[0115] Take an appropriate amount of the reference sample of Example 1 of Xin San, add an equal amount of the same extraction solvent 70% methanol, accurately weigh, place in an ultrasonic cleaner at 25°C, 40°C, 55°C, and 70°C for ultrasonic treatment for 30 minutes, take out and cool, add solvent to return to the original weight, shake well, filter with a microporous filter membrane (0.22μm), and use it as the test solution for detection.

[0116] 2.4 Investigation of extraction times

[0117] Take an appropriate amount of the reference sample of Kaixin Powder Example 1, add an equal amount of the same extraction solvent 70% methanol, accurately weigh, and reflux extraction for 30 minutes, respectively, and extract 1, 2, 3, and 4 times. Take out and cool, add solvent to return to the original weight, shake well, filter with a microporous filter membrane (0.22μm), and use it as the test solution for detection.

[0118] 2.5 Investigation of extraction time

[0119] Take an appropriate amount of the reference sample of Kaixin Powder Example 1, add an equal amount of the same extraction solvent 70% methanol, accurately weigh, and perform ultrasound and reflux (at different temperatures) for 30 minutes, 45 minutes, 60 minutes, and 1.5 hours, respectively, and take out by ultrasound and let cool, add solvent to return to the original weight, shake well, filter with a microporous filter membrane (0.22μm), and use it as the test solution for detection.

[0120] 2.6 Investigation of the extraction solid-liquid ratio

[0121] Take an appropriate amount of the reference sample of Kaixin Powder Example 1, add the corresponding solvent 70% methanol according to the solid-liquid ratio of 1:3, 1:5, 1:7, 1:9, 1:11, accurately weigh, reflux for 45 minutes, make up to the original weight with the solvent, shake well, filter with a microporous filter membrane (0.22μm), and use this as the test solution for detection.

[0122] 2.7 Results

[0123] After the samples were injected in sequence according to the above conditions, the relevant values ​​measured were as follows. The measurement results of the above specific parameters are shown in Tables 1 to 5 below.

[0124] Table 1 Results of different extraction solvent concentrations

[0125]

[0126] Table 2 Results of different extraction temperatures

[0127]

[0128]

[0129] Table 3 Results of different extraction times

[0130]

[0131] Table 4 Results of different extraction times

[0132]

[0133]

[0134] Table 5 Results of different material-liquid ratios

[0135]

[0136]

[0137] As shown in Table 1, in the ultrasonic extraction method, the contents of the five index components are the highest overall when the four solvents with different concentrations are extracted: 40% methanol, 55% methanol, 70% methanol, and 70% methanol. Therefore, 70% methanol is selected as the concentration of the extraction solvent.

[0138] As can be seen from Table 2, in the ultrasonic extraction method, when the ultrasonic temperature is 70°C, the contents of the five index components are the highest overall when the ultrasonic temperature is 70°C. Therefore, when the ultrasonic temperature is set to 70°C, the contents of the five index components are the highest overall, because the increase in temperature will enhance the thermal motion of molecules, thereby increasing the content of the index components in the extraction solution.

[0139] As can be seen from Table 3, the extraction times of 1, 2, 3 and 4 were investigated respectively, and it was found that with the increase of the extraction times, the content of the index components did not increase significantly. Among them, the content of some index components decreased slightly. Taking into account the actual situation and economic efficiency, the final extraction time was determined to be 1.

[0140] It can be seen from Table 4 that the extraction rates of the index components at different times are different. In the ultrasonic extraction group and the reflux extraction group, with the increase of ultrasonic time, from 30min, 45min, 60min, and 1.5h, the index components all increased to a certain extent, but the increase was slow after 60min, so the final time was set to 60min. The principle of the ultrasonic method is to destroy the cell wall, the extraction method is more efficient, and the experimental operability is enhanced. There is no significant difference between the two extraction methods, and the final extraction method selected is ultrasonic extraction.

[0141] As can be seen from Table 4, the material-liquid ratios examined were 1:3, 1:5, 1:7, 1:9, and 1:11, respectively. When the material-liquid ratio was 1:3, the content of the index component was not high and it might not have been extracted. However, when the material-liquid ratio was 1:9 and 1:11, the content of the index component decreased significantly, and finally the material-liquid ratio was set at 1:5.

[0142] Through the investigation of the above extraction parameters, the extraction method was finally confirmed as: ultrasonic extraction at 70°C for 1h, and the solvent was 70% methanol.

[0143] 3. Preparation of reference solution

[0144] Take appropriate amounts of reference substances such as Polygala tenuifolia III, 3,6'-dicerinoylsucrose, ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 and put them into a container, accurately weigh them, and add 70% methanol to make a mixed solution containing 0.15 mg Polygala tenuifolia III, 0.55 mg 3,6'-dicerinoylsucrose, 0.1 mg ginsenoside Rg1, 0.10 mg ginsenoside Re, and 0.15 mg ginsenoside Rb1 per 1 ml.

[0145] 4. Methodological verification of Kaixinsan content determination

[0146] 4.1 Exclusivity

[0147] Under this condition, the solvent, reference solution, and test solution are tested according to the law, as shown in Table 6. Figure 1-2 As shown, the blank solvent has no interference with the determination of the contents of the five active ingredients in Kaixin Powder and is specific.

[0148] Table 6 Specificity test results 1

[0149]

[0150] 4.2 Repeatability

[0151] Six different test sample solutions were prepared in parallel, injected sequentially, and the peak area changes of the corresponding index components of the six test samples were calculated. The corresponding peak area RSDs were all less than 5%, indicating that this method had good repeatability.

[0152] Table 7 Assay repeatability results

[0153]

[0154]

[0155] 4.3 Accuracy

[0156] Weigh 6 test samples from the same batch separately, add appropriate amount of polygala tenuifolia xanthophyll III, 3,6'-dienasinoylsucrose, ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 reference substances, and prepare them into 70%, 100%, and 130% of the corresponding components, respectively. Prepare 2 portions in parallel for each concentration, test according to the law, and then analyze and calculate the half-spiked recovery rate. The recoveries of the corresponding components are shown in Tables 8 to 12. Therefore, the recoveries of the components are within the limit and meet the requirements.

[0157] Table 8 Recovery of spiked xanthones III in Polygala tenuifolia

[0158]

[0159] Table 9 Recovery of spiked 3,6-dicercosyl sucrose

[0160]

[0161]

[0162] Table 10 Recovery of ginsenoside Rg1 spiked

[0163]

[0164] Table 11 Recovery of ginsenoside Re spiked

[0165]

[0166] Table 12 Recovery of ginsenoside Rb1 spiked

[0167]

[0168] The calculation formula of spike recovery in this table is as follows:

[0169] Spiked recovery rate = (measured amount - background amount) / added amount * 100

[0170] The recoveries of the five index components measured above were all within the specified range and in compliance with regulations. This method can accurately determine the contents of the five index components in Kaixin Powder.

[0171] 4.4 Durability

[0172] Different chromatographic conditions including different column temperatures (25°C, 30°C, 35°C), phosphoric acid aqueous concentrations (0.01%, 0.05%, 0.1%), flow rates (0.55ml / min, 0.60ml / min, 0.65ml / min), and different types of chromatographic columns (Agilent ZORBAX SB-C18, WatersXbridge C18) were investigated. The specific range of variation is shown in Tables 13-1 to 13-4. The reference solution and the test solution were injected in turn, and the changes in the peak areas of the index components of the reference solution and the test solution were recorded. The peak areas of the index components after different temperatures, phosphoric acid aqueous concentrations, and flow rates were recorded. If the relevant peak area RD is less than 1%, and the retention time RD of different chromatographic columns is less than 1%, it means that this method has good durability.

[0173] Table 13 Content determination parameters

[0174] condition Specified value Range of change Column temperature 30℃ ±5℃ Flow rate 0.6ml / min ±0.05ml / min Phosphoric acid concentration in mobile phase A 0.05％ ±0.05％ Chromatographic column-1 ThermoAccalim Agilent ZORBAX Chromatographic column-2 ThermoAccalim WatersXbridge

[0175] Table 13-1 Column temperature durability results for content determination

[0176]

[0177] Table 13-2 Content Determination Flow Rate Durability Results

[0178]

[0179]

[0180] Table 13-3 Durability results of phosphoric acid water concentration determination

[0181]

[0182] Table 13-4 Durability results of different types of chromatographic columns for content determination

[0183]

[0184] 5. Content determination of Kaixinsan preparation

[0185] The Kaixin powder test samples of 15 batches of the prepared preparations were prepared and the content was determined according to the law. The results were calculated. The results of the content determination of each indicator component of the 15 batches are shown in Table 14 below.

[0186] Table 14 Batch benchmark sample content determination values

[0187]

[0188]

[0189] Example 2: The fingerprint detection method of the Kaixinsan reference sample is as follows:

[0190] 1. Chromatographic conditions

[0191] The chromatographic parameters and flow rate were the same as those in Example 1, Item 2.1 and 2.2. The absorption wavelength was selected to be 203 nm and the chromatographic column was ThermoAccalim 120.

[0192] 2. Elution program parameters

[0193] After a lot of condition exploration, the elution was carried out according to the following procedure. Acetonitrile was used as mobile phase A; 0.05% phosphoric acid water was used as mobile phase B; the detection wavelength was 203nm, and the column temperature was 30℃. The flow rate was 0.8ml / min for 0-68min, 1.0ml / min for 70-109min, and the theoretical plate number of mobile phase A should be no less than 3000 according to the 3,6'-dicorinoylsucrose peak. The elution procedure was as follows: 0-5min, 16%A-18%A, 5-20min, 18%A-23%A, 20-38min, 23%A-40%A, 38-68min, 40%A-52%A, 68-70min, 52%A-73%A, 70-83min, 73%A-80%A, 83-93min, 80%A, 93-98min, 80%A-95%A.

[0194] 3. Reference solution: Take appropriate amounts of reference substances such as Polygala tenuifolia xanthomonone III, 3,6'-dicorinoylsucrose, ginsenoside Rb1, a-asarone, β-asarone, and pachymic acid, accurately weigh them, and add methanol to prepare a mixed solution containing 0.15 mg of Polygala tenuifolia xanthomonone III, 0.55 mg of 3,6'-dicorinoylsucrose, 0.1 mg of ginsenoside Rb1, 18 ug of a-asarone, 35 ug of β-asarone, and 74 ug of pachymic acid per 1 ml.

[0195] 4. Test solution

[0196] Accurately weigh 1g of the sample powder in a container, then accurately add 25ml of 70% methanol and accurately weigh. Place in an ultrasonic cleaner for 40 minutes, take out, cool, make up the lost weight with solvent, and accurately weigh again. Shake well, filter with a 0.22um filter membrane, and you are done.

[0197] 5. Single herbal medicine slices and negative control solution

[0198] Weigh 1g of the negative samples of Polygala, Ginseng, Poria, Acorus calamus, and each single herbal medicine accurately in a container. The preparation method is the same as that of the test sample. Then accurately add 25ml of 70% methanol and weigh accurately. Place in an ultrasonic cleaner for 40 minutes, take out, cool, make up the lost weight with solvent, and weigh accurately again. Shake well and filter with a 0.22um filter membrane to obtain.

[0199] According to the above chromatographic conditions, the reference solution, the test solution, the single-flavor decoction piece, and the negative control solution were injected in sequence for measurement to obtain liquid chromatograms of the reference solution, the test solution, the single-flavor decoction piece, and the negative control solution.

[0200] 6. Establishment of standard fingerprint and similarity calculation

[0201] Take the prepared reference solution, test solution, single-flavor decoction piece, and negative control solution, and inject and measure. The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) is used for calculation, with S1 as the reference peak. After peak matching and multi-point correction, the components with higher response value, more stable properties, and indicative components are selected as characteristic peaks. The median method is used to generate the corresponding reference spectrum, match the data, calibrate the relative retention time, and finally calculate the similarity. The similarity range of 15 batches of benchmark samples is , with a total of 23 common characteristic peaks. The specific similarity results are shown in Table 15.

[0202] Table 15 Calculation results of fingerprint similarity of 15 batches of Kaixinsan reference samples

[0203]

[0204]

[0205] 7. Methodological validation of fingerprint

[0206] The specific solvent, reference solution, and test solution are injected in sequence. For details, see Table 16. Figure 3-4 As shown, the solvent has no interference with the test sample and is specific.

[0207] Table 16 Specificity test results

[0208]

[0209] System precision: According to Example 2, the reference solution was injected 5 times in succession, and the relative retention time of each index component was recorded respectively. The RSD was calculated. The RSD was less than 5%, indicating that the instrument precision was good. See Table 17 for details. The RSDs were 3.5, 4.0, and 0.9 respectively, indicating that the system precision was good.

[0210] Table 17 System precision results

[0211]

[0212]

[0213] Repeatability: Six different test solutions were prepared in parallel, injected sequentially, and the fingerprints were determined. The similarity was calculated according to the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition). The results are shown in Table 18. The similarity result was not less than 0.95, indicating that the method had good repeatability.

[0214] Table 18 Repeatability results

[0215]

[0216] stability

[0217] The reference solution and the test solution were injected at 0h, 8h, 16h and 24h respectively, and the changes in the peak areas of the index components of the reference solution and the test solution were recorded respectively. If the RSD of the peak areas of polygala tenuifolia ketone III, 3,6-dicerinoylsucrose and ginsenoside Rb1 was less than 5%, it means that the reference and test samples had good stability. The specific test results are shown in Tables 19 to 20.

[0218] Table 19 Reference solution stability test results

[0219] Storage time (h) Polygala tenuifolia Xanthones III 3,6-Dierucyl Sucrose Ginsenoside Rb1 0 118.7521 153.9242 12.0661 8 120.1342 155.7209 12.1091 16 116.9287 150.9496 12.0402 24 119.2231 153.9571 12.0900 RSD(%) 1.2 1.3 0.3

[0220] Table 20 Test solution stability test results

[0221] Storage time (h) Polygala tenuifolia Xanthones III 3,6-Dierucyl Sucrose Ginsenoside Rb1 0 25.3953 58.7816 2.0885 8 25.5728 58.8913 2.2846 16 25.3193 58.6595 2.0969 24 25.4467 58.8278 2.1779 RSD(%) 0.5 0.2 4.3

[0222] After the reference solution was left at room temperature for 24 hours, the RSD of the peak area of ​​Polygala tenuifolia xanthones III was 1.2%, the RSD of 3,6-dicansinoylsucrose was 1.3%, the RSD of the peak area of ​​ginsenoside Rb1 was 0.3%, and the solution was stable; after the test solution was left at room temperature for 24 hours, the RSD of the peak area of ​​Polygala tenuifolia xanthones III was 0.5%, the RSD of 3,6-dicansinoylsucrose was 0.2%, the RSD of the peak area of ​​ginsenoside Rb1 was 4.3%, and the solution was stable.

[0223] Durability: Take the test solution in Example 2, and examine the column temperature, flow rate, and concentration of phosphoric acid in the mobile phase in the chromatographic conditions. The corresponding parameters that are changed are shown in Table 21. Chromatographic columns of different models and brands are replaced, namely WatersXbridge C18, Agilent ZORBAX SB-C18, and ThermoHypersil GOLD. The reference solution and the test solution are injected in turn, and the liquid phase determination is carried out. The similarity is calculated according to the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition). The results are shown in Tables 22-25.

[0224] Table 21 Stability test results under different chromatographic conditions

[0225] condition Specified value Range of change Column temperature 30 ±5℃ Flow rate 0.8 ±0.05ml / min Phosphoric acid concentration 0.05％ ±0.05％ Different columns Accalim 120 Waters Xbridge C18 Chromatographic column-2 Accalim 120 Agilent ZORBAX SB-C18 Chromatographic column-3 Accalim 120 ThermoHypersil GOLD

[0226] Table 22 Fingerprint column temperature durability results

[0227] Column temperature 25℃ 30℃ 35℃ Comparison fingerprint 25℃ 1.000 1.000 1.000 1.000 30℃ 1.000 1.000 1.000 1.000 35℃ 1.000 1.000 1.000 1.000 Comparison fingerprint 1.000 1.000 1.000 1.000

[0228] Table 23 Fingerprint flow rate durability results

[0229] Flow rate 0.75ml / min 0.80ml / min 0.85ml / min Comparison fingerprint 0.75ml / min 1.000 0.999 1.000 1.000 0.80ml / min 1.000 1.000 0.999 1.000 0.85ml / min 1.000 0.999 1.000 1.000 Comparison fingerprint 1.000 1.000 1.000 1.000

[0230] Table 24 Fingerprint spectrum phosphoric acid water concentration durability results

[0231] Phosphoric acid water concentration 0.01％ 0.05％ 0.1％ Comparison fingerprint 0.01％ 1.000 1.000 0.999 1.000 0.05％ 1.000 1.000 1.000 1.000 0.1％ 0.999 1.000 1.000 1.000 Comparison fingerprint 1.000 1.000 1.000 1.000

[0232] Table 25 Fingerprint durability results of different chromatographic column models

[0233]

[0234] 8. Characteristic peak attribution and identification

[0235] The reference solution, test solution, single-ingredient medicinal slices, and negative control solution were measured to obtain the corresponding liquid chromatograms. There were 23 common peaks in total, and all the common peaks were assigned. Among them, peak 3 was xanthonone III from Polygala tenuifolia, peak 4 was 3,6-diesinapoylsucrose peak, peak 14 was ginsenoside Rb1, peak 20β-asarone, peak 21 was a-asarone, and peak 22 was pachymic acid. Among them, peaks 5, 6, 8, 9, 10, 11, 14, 16, 17, 18, 22, and 23 were derived from Polygala tenuifolia, peaks 7, 13, and 23 were derived from ginseng, peaks 2, 19, and 20 were derived from Acorus tatarinowii, and peaks 21 and 22 were derived from Poria. The specific attribution of common peaks and attribution of single medicinal materials are shown in the figure below. Figure 5-7 shown.

[0236] 9.15 Batch Kaixinsan Benchmark Sample Similarity Evaluation

[0237] Take the prepared reference solution, test solution, single-flavor decoction piece, and negative control solution, and inject and measure. The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) is used for calculation, with S1 as the reference peak. After peak matching and multi-point calibration, the index component with higher response value, more stable properties and characteristic peak is selected. The corresponding reference spectrum is generated by the median method, the data is matched, the relative retention time is checked, and finally the similarity is calculated. The results are shown in Table 15. The similarity diagram of the fingerprint spectrum of the 15 batches of benchmark samples is shown in Figure 8 .

[0238] 10.15 batches of Kaixinsan benchmark samples similarity calculation results

[0239] As shown in Table 15, the similarity calculation results of the fingerprint spectra of 15 batches of Kaixin Powder are between 0.993 and 1.000, all greater than 0.99, indicating that the quality of the 15 batches of Kaixin Powder is basically the same and the properties are similar. It also shows that the process for preparing Kaixin Powder is stable.

[0240] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for determining the content of index components of a Kaixinsan reference sample, characterized in that: include: The test is to determine the index components in Kaixinsan preparations by high performance liquid chromatography, and the analysis is carried out by measuring the test solution and the reference solution; The indicative ingredients include polygala tenuifolia xanthophyll III, 3,6'-dienasinoylsucrose, ginsenoside Rg1, ginsenoside Rb1, and ginsenoside Re.

2. The method for determining the content of indicative components of a Kaixinsan reference sample according to claim 1, characterized in that: The preparation of the test solution includes the following steps: S1: Take the Xinxin powder test sample and put it in a container; S2: Add 70% methanol into the container, shake the container to mix Kaixinsan and 70% methanol evenly, let it stand and cool, and then weigh it; S3: placing the container into an ultrasonic cleaning apparatus for ultrasonic treatment; S4: taking the container out of the ultrasonic cleaner and weighing it, adding 70% methanol to the container so that the overall weight is consistent with the weight in step S2; S5: placing the container in a centrifuge to centrifuge the solution; S6: Filter the solution through a PTFE filter membrane and take the filtrate, which is the test solution.

3. The method for determining the content of indicative components of a Kaixinsan reference sample according to claim 1, characterized in that: The preparation of the reference solution includes the following steps: S7: Aspiration Ketone III, 3,6'-dienasinoylsucrose, ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 are placed in a container; S8: Add methanol into the container to prepare a mixed solution.

4. The method for determining the content of indicative components of a Kaixinsan reference sample according to claim 1, characterized in that: The chromatographic conditions are as follows: Octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A; 0.05% phosphoric acid water was used as mobile phase B; the detection wavelength was 203 nm, the column temperature was 25°C; the flow rate was 0.6 ml / min; the elution procedure was as follows: 0-35 min, 20% A, 35-65 min, 20% A-29% A, 65-100 min, 29% A-40% A, 100-101 min, 40% A-85% A, 101-114 min, 85% A-85% A.

5. The method for determining the content of indicative components of a Kaixinsan reference sample according to claim 2, characterized in that: The weight of Kaixin powder in step S1 is 5g; The concentration of methanol in step S2 is 70%, and the amount added is 25 ml; the concentration of methanol in step S4 is 70%; In step S3, the ultrasonic treatment time is 60 min and the temperature is 70° C.; In step S5, the centrifugal speed is 5000 r / min and the time is 5 min; in step S6, the pore size of the PTFE filter membrane is 0.22 μm.

6. The method for determining the content of indicative components of a Kaixinsan reference sample according to claim 3, characterized in that: The contents of polygala tenuifolia III reference substance, 3,6'-dicerinoylsucrose reference substance, ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 in the reference solution were 0.15 mg / ml, 0.55 mg / ml, 0.1 mg / ml, 0.1 mg / ml and 0.15 mg / ml, respectively.

7. The fingerprint method of the index component of the Kaixinsan reference sample according to claims 1-6 is established, characterized in that: The preparation of the test solution includes the following steps: S10: Take 1 g of the test sample of Xinxin Powder and put it in a container; S11: Accurately add 25 ml of 70% methanol into the container and weigh accurately; S12: placing the container in an ultrasonic cleaning apparatus for 40 minutes, taking it out, and letting it cool; S13: Use solvent to make up the lost weight and weigh accurately again; S14: Filter with a 0.22um filter membrane to obtain.

8. The method for determining the index component fingerprint of a Kaixinsan reference sample according to claim 7, characterized in that: The preparation of single-flavor decoction pieces and negative control solutions includes the following steps: S15: Accurately weigh 1g of the negative samples of Polygala, Ginseng, Poria, Acorus calamus, and each single herbal medicine into a container; S16: Accurately add 25 ml of 70% methanol into the container and weigh accurately; S17: placing the container in an ultrasonic cleaning apparatus for 40 minutes, taking it out, and cooling it; S18: Use solvent to make up the lost weight and weigh accurately again; S19: Shake well and filter with a 0.22um filter membrane.

9. The method for determining the fingerprint spectrum of a Kaixinsan reference sample according to claim 7, characterized in that: The chromatographic conditions are as follows: Octadecylsilane bonded silica gel was used as filler; acetonitrile mobile phase A; 0.05% phosphoric acid water was used as mobile phase B; the detection wavelength was 203nm, and the column temperature was 30℃. The flow rate was 0.8ml / min for 0-68min, 1.0ml / min for 70-109min, and the theoretical plate number of mobile phase A should not be less than 3000 according to the peak of 3,6'-dicorinoylsucrose. The elution procedure was as follows: 0-5min, 16%A-18%A, 5-20min, 18%A-23%A, 20-38min, 23%A-40%A, 38-68min, 40%A-52%A, 68-70min, 52%A-73%A, 70-83min, 73%A-80%A, 83-93min, 80%A, 93-98min, 80%A-95%A.

10. The method for determining the content of index components of a Kaixinsan reference sample according to claim 7, characterized in that: During the test, take the mixed reference solution and the test solution and enter the high performance liquid chromatograph for test according to the law. From each chromatogram, obtain the peak area Ax reference and Ax test of each index component in the reference solution and the test solution. The concentration C of the reference solution of each index component is known. 对照品 , the concentration of each index component in the test solution was calculated by external standard method, formula: C 供试品 =C 对照品 / A 对照品 ×A 供试品 , and then the content of each index component is obtained by the sample weight and dilution multiple. The established fingerprint method is used as the preferred scheme of the present invention. After detection, the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) is finally used for calculation, and the reference peak is selected. After peak matching and multi-point correction, the indicative component with higher response value, more stable property and characteristic peak is selected. The median method is used to generate the corresponding control spectrum, match the data, calibrate the relative retention time, and finally calculate the similarity. If it is greater than 0.95, it is considered that the similarity between samples is high.

Images