UPLC (Ultra Performance Liquid Chromatography) specific chromatogram of Kaixin powder, detection method and application thereof

Abstract

The invention provides a Kaixin powder preparation UPLC specific chromatogram, a detection method and application thereof.The UPLC specific chromatogram comprises 11 characteristic peaks, the number of characteristic peaks belonging to the medicine flavor of polygala tenuifolia is 5, and the number of characteristic peaks belonging to the medicine flavor of ginseng, the number of characteristic peaks belonging to the medicine flavor of poria cocos and the number of characteristic peaks belonging to the medicine flavor of rhizoma acori graminei are 2. Meanwhile, the detection method of the UPLC characteristic spectrum is good in precision, repeatability and stability, the quality difference of the Kaixin powder can be truly reflected, and a Kaixin powder quality control system is perfected.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine component analysis, and in particular to a UPLC characteristic spectrum of Kaixin powder, a detection method and application thereof. Background Art

[0002] Kaixin Powder was first seen in the Collection of Effective Prescriptions by Yao Sengyuan of the Northern Zhou Dynasty, but the book has been lost. Later, it was circulated through the Medical Prescriptions, which said, "Kaixin Powder is a recipe that makes people never forget. Three liang of calamus and poria, two liang of ginseng, and four liang of polygala". Later versions of Kaixin Powder were mostly copied from the Prescriptions for Emergencies Worth a Thousand Gold Pieces written by Sun Simiao in the Tang Dynasty. "Kaixin Powder is a recipe for forgetfulness: four fen of polygala and ginseng, two liang of poria, and one liang of calamus. The above four ingredients are sieved and taken three times a day." Kaixin Powder "treats forgetfulness". The drug dosages are converted by weights and measures. The dosages of polygala, ginseng, poria and calamus are 13.80g, 13.80g, 27.60g, and 13.80g, respectively, with a ratio of 1:1:2:1. This is consistent with the "Key Information Table of Ancient Classic Prescriptions (25 Prescriptions)". Later generations of doctors expanded the treatment scope of Kaixinsan to treat palpitations and spermatorrhea caused by insufficient heart qi and heat in the gallbladder. Kaixinsan is mainly composed of Polygala, Ginseng, Poria, and Acorus calamus, and is a basic prescription for Kaixinsan. In the Kaixinsan prescription, the main drug is Polygala, which can calm the mind and improve intelligence, connect the heart and kidney, and eliminate phlegm and reduce swelling; the minister drug is Ginseng, which can greatly replenish the vital energy, promote fluid and nourish blood, calm the mind and improve intelligence, and is supplemented by Poria, which can promote diuresis and eliminate dampness, calm the mind and calm the mind; and Acorus calamus can open the mind and eliminate phlegm, wake up the mind and improve intelligence. The combination of the four medicines has the effects of calming the mind, replenishing qi, and removing dampness and turbidity, and is mainly used to treat forgetfulness. Modern studies have shown that Kaixinsan has significant therapeutic effects in the treatment of Alzheimer's disease, depression, forgetfulness and other diseases. Kaixinsan also has certain pharmacological effects in anti-oxidation, anti-fatigue, and anti-aging.

[0003] In order to improve the quality of traditional Chinese medicine and ensure the therapeutic efficacy of traditional Chinese medicine, the analysis of traditional Chinese medicine ingredients has become a key link. In the research and development process of traditional Chinese medicine, the analysis of traditional Chinese medicine ingredients occupies a very important position. The ingredients of traditional Chinese medicine are complex and it is difficult to conduct a comprehensive quality analysis. The establishment of a characteristic spectrum of the hallmark ingredients of Kaixinsan is an urgent problem to be solved. Through literature and patent consultation, the current detection and analysis methods cannot fully cover the hallmark ingredients of Kaixinsan and have not established an overall quality control method for its multi-index ingredients related to efficacy. For example, patent CN115825272A uses two sets of HPLC characteristic spectra to measure the Kaixinsan benchmark sample (Polygala tenuifolia Ketone III, 3,6-dimesone acyl sucrose, β-asarone, α-asarone, polioic acid C, ginsenoside Rg1, Re, Rb1, Rb2, Rf, Rd), the detection method uses two sets of characteristic spectrum analysis, the analysis method is cumbersome and complicated; such as patent CN115267005B, which protects a method for detecting the content of multiple index components in Kaixin powder, including Polygala tenuifolia Five components, namely ketone Ⅲ, 3,6'-di-sinapoyl sucrose, ginsenoside Rg1, pachymic acid, and dehydro-pachymic acid, were involved, but the determination of the contents of the main pharmacodynamic components β-asarone and α-asarone was not carried out. This detection method analyzed and detected the contents of the five components at four wavelengths. The analysis method was cumbersome, and the sample components were not fully extracted and detected, so it could not truly reflect the components and contents in KaiXinSan, and had limitations. Summary of the Invention

[0004] In view of the problems existing in the above-mentioned prior art, the purpose of the present invention is to provide a UPLC characteristic fingerprint, a detection method and its application of KaiXinSan. By optimizing the pretreatment conditions and ultra-high performance liquid chromatography detection conditions of KaiXinSan, the UPLC characteristic fingerprint of KaiXinSan was established, comprehensively characterizing the characteristic peaks of the active components of the four medicinal flavors in KaiXinSan, improving the quality control system of KaiXinSan, and providing a reference for the research of KaiXinSan.

[0005] KaiXinSan is composed of four traditional Chinese medicines: ginseng, polygala root, poria cocos, and acorus tatarinowii. Ginseng has been a good tonic since ancient times. Its main components include saponins, polysaccharides, volatile oils, polyacetylenes, trace elements, etc. Ginsenosides, as the active components in ginseng, mainly exist in plants of the genus Panax. Ginsenoside Rg1 and ginsenoside Re are suitable as markers for measuring ginsenosides; Polygala root, as the monarch drug in KaiXinSan, mainly contains triterpenoid saponins, ketones, glycolipids, alkaloids, sterols, simple phenolic acids, etc. Among them, tenuifoliside A5, tenuifoliside A6, 3,6'-di-sinapoyl sucrose, and polygala ketone Ⅲ, and tenuifolin A represent the oligosaccharide esters, ketones, and triterpenoid saponins of polygala root; The most abundant compounds in poria cocos are triterpenoids, which have various biological activities, such as anti-inflammatory, anti-apoptotic, and anti-immune rejection reactions. Pachymic acid C and dehydro-pachymic acid are representative triterpenoids in poria cocos; The main components of acorus tatarinowii are volatile oils, which mainly include β-asarone and α-asarone, etc., and have significant activity in the treatment of central nervous system diseases. Therefore, in the present invention, tenuifoliside A5, tenuifoliside A6, polygala ketone Ⅲ, 3,6'-di-sinapoyl sucrose, tenuifolin A, ginsenoside Rg1, ginsenoside Re, β-asarone, α-asarone, pachymic acid C, and dehydro-pachymic acid are used as the index components of KaiXinSan.

[0006] On the one hand, the present invention provides a UPLC characteristic fingerprint of KaiXinSan, which includes the characteristic peaks of tenuifoliside A5, tenuifoliside A6, polygala ketone Ⅲ, 3,6'-di-sinapoyl sucrose, tenuifolin A, ginsenoside Rg1, ginsenoside Re, β-asarone, α-asarone, pachymic acid C, and dehydro-pachymic acid.

[0007] Furthermore, the UPLC characteristic fingerprint of the Kāixīn Sàn of the present invention has characteristic peaks substantially as shown in Figure 1 shown.

[0008] Furthermore, the UPLC characteristic fingerprint of the Kāixīn Sàn contains 11 common characteristic peaks. Taking the 8th peak as the reference peak (S peak, relative retention time is 1.00), the specified values of the relative retention times of the other 10 common characteristic peaks are successively the 1st peak (0.17), the 2nd peak (0.18), the 3rd peak (0.36), the 4th peak (0.50), the 5th peak (0.59), the 6th peak (0.63), the 7th peak (0.67), the 9th peak (1.11), the 10th peak (1.58), and the 11th peak (1.65). Except for the 8th peak, the RSD% of the specified values of the relative retention times of the remaining 10 common characteristic peaks are all ≤ 3.0%. On the one hand, the present invention provides a method for detecting the UPLC characteristic fingerprint of Kāixīn Sàn, which includes the following steps: preparation of the reference solution, preparation of the test solution, detection using an ultra-high performance liquid chromatograph, and identification of the characteristic peaks according to the retention times of the reference solution and the test solution, so as to obtain the characteristic fingerprint of the test sample;

[0009] wherein, the reference substance includes polygala tenuifolia glycoside A5, polygala tenuifolia glycoside A6, polygala ketone III, 3,6'-di-O-sinapoyl-sucrose, tenuifoliside A, ginsenoside Rg1, ginsenoside Re, β-asarone, α-asarone, polyporenic acid C, and dehydrotumulosic acid;

[0010] the test sample is Kāixīn Sàn;

[0011] The preparation of the reference solution includes adding samples of each reference substance to a solvent and dissolving to obtain a reference solution containing specific concentrations of polygala tenuifolia glycoside A5, polygala tenuifolia glycoside A6, polygala ketone III, 3,6'-di-O-sinapoyl-sucrose, tenuifoliside A, ginsenoside Rg1, ginsenoside Re, β-asarone, α-asarone, polyporenic acid C, and dehydrotumulosic acid;

[0012] The preparation of the test solution includes adding the test sample to a solvent, dissolving, ultrasonically treating, shaking well, filtering, and taking the subsequent filtrate to obtain the test solution;

[0013] The characteristic peaks of the following substances are included in the characteristic fingerprint of the test sample: polygala tenuifolia glycoside A5, polygala tenuifolia glycoside A6, polygala ketone III, 3,6'-di-O-sinapoyl-sucrose, tenuifoliside A, ginsenoside Rg1, ginsenoside Re, β-asarone, α-asarone, polyporenic acid C, and dehydrotumulosic acid.

[0014] Preferably, the solvent is 50% to 100% methanol, more preferably 75% methanol.

[0015] Preferably, the ratio of the added mass g of the test sample to the volume mL dissolved by the solvent is 2:25 to 2:50, more preferably 2:25.

[0016] Preferably, the ultrasonic time is 30 min to 60 min, more preferably 30 min.

[0017] Preferably, the power of the ultrasonic extraction is 300 W and the frequency of the ultrasonic extraction is 40 kHz.

[0018] Preferably, the concentrations of the substances in the reference substance solution are as follows: polygala tenuifolia glycoside A5 25 μg / mL, polygala tenuifolia glycoside A6 20 μg / mL, tenuifolin 15 μg / mL, 3,6'-di-O-sinapoyl-sucrose 90 μg / mL, tenuifoliside A 45 μg / mL, ginsenoside Rg1 45 μg / mL, ginsenoside Re 45 μg / mL, β-asarone 200 μg / mL, α-asarone 100 μg / mL, polyporenic acid C 8 μg / mL, dehydrotumulosic acid 15 μg / mL.

[0019] Preferably, the chromatographic column for detection by the ultra-high performance liquid chromatograph is a C14 chromatographic column. More preferably, the ultra-high performance liquid chromatographic column is Agilent ZORBAX BOUNS-RP.

[0020] Preferably, the wavelength mode used for detection by the ultra-high performance liquid chromatograph is dual-wavelength switching detection, and the dual wavelengths are 203 nm and 245 nm.

[0021] Further, the specific wavelengths used for detection by the ultra-high performance liquid chromatograph are: 0 - 39 min: 245 nm, 39 - 44 min: 203 nm, 44 - 110 min: 245 nm;

[0022] Among them, when the retention time is 0 - 39 min and the wavelength is 245 nm, polygala tenuifolia glycoside A5, polygala tenuifolia glycoside A6, tenuifolin 15 μg / mL, 3,6'-di-O-sinapoyl-sucrose, tenuifoliside A can be detected; when the retention time is 39 - 44 min and the wavelength is 203 nm, ginsenoside Rg1 and ginsenoside Re can be detected; when the retention time is 44 - 110 min and the wavelength is 245 nm, β-asarone, α-asarone, polyporenic acid C, dehydrotumulosic acid can be detected.

[0023] Preferably, the column temperature during the detection by the ultra - performance liquid chromatograph is 30 - 40 °C. More preferably, the column temperature in the ultra - performance liquid chromatography method is 35 - 40 °C.

[0024] Preferably, the flow rate during the detection by the ultra - performance liquid chromatograph is 0.30 - 0.32 mL / min.

[0025] Preferably, the injection volume during the detection by the ultra - performance liquid chromatograph is 2 - 4 μL. More preferably, the injection volume in the ultra - performance liquid chromatography method is 2 μL.

[0026] Preferably, during the detection by the ultra - performance liquid chromatograph, the mobile phase is acetonitrile - 0.05 - 0.1% phosphoric acid aqueous solution, where phase A is acetonitrile and phase B is 0.05 - 0.1% phosphoric acid aqueous solution; the analysis time is 110 min; gradient elution is used.

[0027] More preferably, in the ultra - performance liquid chromatography method, the mobile phase is acetonitrile - 0.1% phosphoric acid aqueous solution, where phase A is acetonitrile and phase B is 0.1% phosphoric acid aqueous solution; the analysis time is 110 min; gradient elution is used.

[0028] Further preferably, the specific program of the gradient elution is as follows:

[0029] Time Mobile phase 0 - 5 min Volume ratio of phase A to phase B is 5:95 - 10:90 5 - 25 min Volume ratio of phase A to phase B is 10:90 - 19:81 25 - 40 min Volume ratio of phase A to phase B is 19:81 - 20:80 40 - 50 min Volume ratio of phase A to phase B is 20:80 - 27:73 50 - 70 min Volume ratio of phase A to phase B is 27:73 - 28:72 70 - 85 min Volume ratio of phase A to phase B is 28:72 - 50:50 85 - 95 min Volume ratio of phase A to phase B is 50:50 - 60:40 95 - 105 min Volume ratio of phase A to phase B is 60:40 - 85:15 105 - 110 min Volume ratio of phase A to phase B is 85:15

[0030] On the one hand, the present invention provides an application of the UPLC characteristic fingerprint of Kāixīn Sǎn and its detection method in the quality research, inspection, and control of related drugs or medicinal materials, where the drugs or medicinal materials are Kāixīn Sǎn or drugs or medicinal materials containing components similar to Kāixīn Sǎn.

[0031] On the other hand, the present invention provides a method for constructing the UPLC characteristic fingerprint of Kāixīn Sǎn, including: detecting multiple batches of Kāixīn Sǎn samples respectively by the above - mentioned detection method of the UPLC characteristic fingerprint of Kāixīn Sǎn to obtain multiple characteristic fingerprints, generating a common reference characteristic fingerprint, and after selecting the reference peaks, calculating the relative retention times of all common characteristic peaks, thereby establishing the standard characteristic fingerprint of the Kāixīn Sǎn preparation.

[0032] Preferably, the method for establishing the UPLC characteristic fingerprint of Kāixīn Sǎn adopts the average method. The average method is a type of common mode.

[0033] Preferably, the standard characteristic chromatogram of the Kāixīn Sàn comprises 11 common characteristic peaks. Taking the 8th peak as the reference peak (S peak, relative retention time is 1.00), the specified values of the relative retention times of the other 10 common characteristic peaks are in sequence: the 1st peak (0.17), the 2nd peak (0.18), the 3rd peak (0.36), the 4th peak (0.50), the 5th peak (0.59), the 6th peak (0.63), the 7th peak (0.67), the 9th peak (1.11), the 10th peak (1.58), the 11th peak (1.65). The RSD% of the specified values of the relative retention times of the 10 common characteristic peaks except the 8th peak is ≤ 3.0%.

[0034] Preferably, the similarity of the characteristic chromatograms of the multi-batch reference samples of the Kāixīn Sàn is between 0.994 and 1.000.

[0035] Preferably, after comparing the standard characteristic chromatogram of the Kāixīn Sàn sample with the characteristic chromatogram of the reference substance solution, it is found that: the common pattern contains 11 common characteristic peaks, which are respectively Peak 1: Polygalacin A5, Peak 2: Polygalacin A6, Peak 3: Senegenin III, Peak 4: 3,6'-Di-O-sinapoyl-sucrose, Peak 5: Tenuifoliside A, Peak 6: Ginsenoside Rg1, Peak 7: Ginsenoside Re, Peak 8: β-Asarone, Peak 9: α-Asarone, Peak 10: Polyporenic acid C, Peak 11: Dehydrotumulosic acid.

[0036] In addition, the present invention provides a method for locating the characteristic peaks of the pharmacodynamic components of the four medicinal flavors in the Kāixīn Sàn, comprising the following steps:

[0037] A) Preparation of the sample solution without a certain ingredient: Remove any one of the four medicinal materials, namely Polygala tenuifolia, Panax ginseng, Poria cocos, and Acorus tatarinowii, from the sample preparation of the sample preparation containing the four medicinal materials of Polygala tenuifolia, Panax ginseng, Poria cocos, and Acorus tatarinowii, and prepare according to the same steps as those for preparing the test solution in the detection method of the characteristic chromatogram of the Kāixīn Sàn to obtain 4 sample solutions without a certain ingredient: the Polygala tenuifolia negative solution, the Panax ginseng negative solution, the Poria cocos negative solution, and the Acorus tatarinowii negative solution;

[0038] B) Preparation of the reference substance solution: Prepare the single-herb samples of Polygala tenuifolia, Panax ginseng, Poria cocos, and Acorus tatarinowii according to the same steps as those for preparing the test solution in the detection method of the characteristic chromatogram of the Kāixīn Sàn to obtain 4 reference substance solutions: the Polygala tenuifolia solution, the Panax ginseng solution, the Poria cocos solution, and the Acorus tatarinowii solution;

[0039] C) Determination: Use ultra-high performance liquid chromatography (UPLC) with the same chromatographic conditions as those in the detection method of the characteristic chromatogram of the test solution to separately determine the 4 sample solutions without a certain ingredient and the 4 reference substance solutions, and obtain the characteristic chromatograms of the 4 sample solutions without a certain ingredient and the characteristic chromatograms of the 4 reference substance solutions respectively;

[0040] D) Quality inspection: Compare the characteristic spectra of 4 kinds of sample-deficient negative solutions and the characteristic spectra of 4 kinds of reference substance solutions with the standard characteristic spectrum established by the construction method of the standard characteristic spectrum of the test sample. Through the relative retention time, identify the common characteristic peaks of the corresponding single herbs in the 4 kinds of sample-deficient negative solutions in the standard characteristic spectrum of the pharmaceutical preparation, so as to attribute and locate the characteristic peaks in the characteristic spectra of the corresponding single herbs in the 4 kinds of sample-deficient negative solutions.

[0041] Preferably, in step D), in the standard characteristic spectrum of the test sample, after comparing the relative retention time, the attribution and location of the common characteristic peaks are as follows: the peaks numbered 1, 2, 3, 4, and 5 are derived from Polygala tenuifolia Willd.; the peaks numbered 6 and 7 are derived from Panax ginseng C. A. Mey.; the peaks numbered 8 and 9 are mainly derived from Acorus tatarinowii Schott; the peaks numbered 10 and 11 are derived from Poria cocos (Schw.) Wolf.

[0042] In the present invention, the test sample is the Kāixīn Sǎn sample, and the experimental water is all self-made ultrapure water. In the present invention, UPLC refers to ultra-high performance liquid chromatography; multiple times in the present invention refers to a limited number of integers about once; basic in the present invention refers to within the error range generally recognized by those skilled in the art, such as within the range where the RSD% of the specified value of the relative retention time of the characteristic peak ≤ 3.0%.

[0043] The present invention provides a method for detecting the UPLC characteristic spectrum of Kāixīn Sǎn and its application. By optimizing the pretreatment conditions of Kāixīn Sǎn and using the ultra-high performance liquid chromatography detection method to qualitatively detect the components of the four medicinal flavor components in Kāixīn Sǎn, and at the same time using relevant methods to detect and analyze multiple batches of Kāixīn Sǎn samples, it is finally confirmed that the UPLC characteristic spectrum of Kāixīn Sǎn of the present invention has 11 characteristic peaks, specifically including sibiricose A5, sibiricose A6, tenuifolin ketone III, 3,6'-di-O-sinapoyl-sucrose, tenuifoliside A, ginsenoside Rg1, ginsenoside Re, β-asarone, α-asarone, polyporenic acid C, dehydrotumulosic acid. The UPLC characteristic spectrum of the present invention has important significance for the quality research of traditional Chinese medicines or Chinese patent medicines of Kāixīn Sǎn.

[0044] The present invention was verified by an ultra-high performance liquid chromatography (UPLC) detection method. For the same test sample solution, six consecutive injections were made. The relative standard deviation (RSD) of the relative retention time of each chromatographic peak of the 11 characteristic peaks was less than 0.1%, and the relative peak area was less than 3.9%. The results met the requirements, indicating that the method had good precision. For six parallel test sample solutions, one injection was made for each. The RSD of the relative retention time of each chromatographic peak was less than 0.1%, and the RSD of the relative peak area of each chromatographic peak was less than 2.0%. The results met the requirements, indicating that the method had good repeatability. The same test sample was injected at 0 h, 10.9 h, 23.9 h, 34.8 h, and 47.8 h respectively. The RSD of the relative retention time of each chromatographic peak was ≤0.1%, and the RSD of the relative peak area was ≤4.8%. The results met the requirements, indicating that the method had good stability.

[0045] After the precision, repeatability, and stability tests of the detection method of the present invention, all parameters were within the acceptable range. It was applicable to the detection of the characteristic chromatogram of the KaiXinSan sample, and could provide a reference basis for the quality control and determination of related drugs, medicinal materials, foods, or health products containing Polygala tenuifolia, Panax ginseng, Acorus tatarinowii, and Poria cocos.

[0046] The present invention provides a UPLC characteristic chromatogram, a detection method, and its application for a KaiXinSan preparation. The UPLC characteristic chromatogram of the present invention contains 11 characteristic peaks, among which 5 characteristic peaks belong to the medicinal flavor of Polygala tenuifolia, and 2 characteristic peaks each belong to the medicinal flavors of Panax ginseng, Poria cocos, and Acorus tatarinowii. The UPLC characteristic chromatogram of the present invention can comprehensively characterize the component characteristics of the four medicinal flavors in the KaiXinSan preparation. At the same time, the detection method of the UPLC characteristic chromatogram of the present invention has good precision, good repeatability, and good stability, can truly reflect the quality differences of KaiXinSan, and improve the quality control system of KaiXinSan. Description of the Drawings

[0047] Figure 1 It is the UPLC characteristic chromatogram of the KaiXinSan in Example 1 of the present invention, where Peak 1: Polygalacin A5, Peak 2: Polygalacin A6, Peak 3: Polygalaxanthone III Ketone III, Peak 4: 3,6'-Di-O-sinapoyl-sucrose, Peak 5: Tenuifoliside A, Peak 6: Ginsenoside Rg1, Peak 7: Ginsenoside Re, Peak 8: β-Asarone, Peak 9: α-Asarone, Peak 10: Polyporenic acid C, Peak 11: Dehydrotumulosic acid.

[0048] Figure 2 It is the investigation chromatogram of different extraction solvents for the KaiXinSan preparation in Example 2 of the present invention.

[0049] Figure 3 It is the investigation chromatogram of different ultrasonic times for the KaiXinSan preparation in Example 2 of the present invention.

[0050] Figure 4It is the inspection chromatogram of different solute ratios of the Kāixīn Sǎn preparation in Example 2 of the present invention.

[0051] Figure 5 It is the inspection chromatogram of different sample injection volumes of the Kāixīn Sǎn preparation in Example 2 of the present invention.

[0052] Figure 6 It is the inspection chromatogram of different column temperatures of the Kāixīn Sǎn preparation in Example 2 of the present invention.

[0053] Figure 7 It is the inspection chromatogram of different flow rates of the Kāixīn Sǎn preparation in Example 2 of the present invention.

[0054] Figure 8 It is the inspection chromatogram of different mobile phases of the Kāixīn Sǎn preparation in Example 2 of the present invention.

[0055] Figure 9 It is the inspection chromatogram of different chromatographic columns of the Kāixīn Sǎn preparation in Example 2 of the present invention.

[0056] Figure 10 It is the inspection chromatogram for establishing the standard characteristic chromatogram of the Kāixīn Sǎn preparation in Example 3 of the present invention. Among them, the 8th peak is used as the reference peak (S peak, relative retention time is 1.00), the 1st peak is the characteristic peak of sibiricose A5, the 2nd peak is the characteristic peak of sibiricose A6, the 3rd peak is the characteristic peak of onjisaponin III, the 4th peak is the characteristic peak of 3,6'-di-O-sinapoyl-sucrose, the 5th peak is the characteristic peak of tenuifoliside A, the 6th peak is the characteristic peak of ginsenoside Rg1, the 7th peak is the characteristic peak of ginsenoside Re, the 8th peak is the characteristic peak of β-asarone, the 9th peak is the characteristic peak of α-asarone, the 10th peak is the characteristic peak of polyporenic acid C, and the 11th peak is the characteristic peak of dehydrotumulosic acid.

[0057] Figure 11 is the finger-printing and localization chromatogram of the components of 4 herbs in Kāixīn Sǎn in Example 4 of the present invention, where Figure 11 - A chromatographic peaks 1 to 5 all come from the Radix Polygalae decoction pieces; Figure 11 - B chromatographic peaks 6 and 7 all come from the Radix Ginseng medicinal flavor; Figure 11 - C chromatographic peaks 10 and 11 all come from the Poria medicinal flavor; Figure 11 - D chromatographic peaks 8 and 9 mainly come from the Rhizoma Acori Tatarinowii medicinal flavor. Detailed implementation manners

[0058] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the protection scope of the present invention. The following illustrates the implementation manners of the present invention through specific specific embodiments, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0059] The reagents and instruments used in the following examples are as follows:

[0060] 1. Preparation of test samples

[0061] Take the Polygala tenuifolia Willd. medicinal material, remove impurities, wash slightly, moisten thoroughly, cut into sections, and dry to obtain Polygala tenuifolia Willd. cut pieces.

[0062] Take the Panax ginseng C. A. Mey. medicinal material, remove impurities, and powder to obtain Panax ginseng C. A. Mey. cut pieces.

[0063] Take the Poria cocos (Schw.) Wolf medicinal material, remove impurities, and dry to obtain Poria cocos (Schw.) Wolf cut pieces.

[0064] Take the Acorus tatarinowii Schott medicinal material, remove impurities, wash, moisten thoroughly, cut into thick slices, and dry to obtain Acorus tatarinowii Schott cut pieces.

[0065] Weigh the Polygala tenuifolia Willd. cut pieces, Panax ginseng C. A. Mey. cut pieces, Poria cocos (Schw.) Wolf cut pieces, and Acorus tatarinowii Schott cut pieces according to the ratio of 1:1:2:1, mix and powder, and pass through a No. 6 pharmacopoeia sieve to obtain the fine powder of KaiXinSan, batches S1 - S20 of samples.

[0066] Table 1 Source of samples

[0067] Raw material Source Radix Polygalae medicinal material Shanxi Zhendong Authentic Medicinal Materials Development Co., Ltd. Radix Ginseng medicinal material Bozhou Shuzhong Pharmaceutical Co., Ltd. Poria medicinal material Guoyao Health Bozhou Pharmaceutical Co., Ltd. Rhizoma Acori Tatarinowii medicinal material Guoyao Health Bozhou Pharmaceutical Co., Ltd.

[0068] 2. Instruments and reagents

[0069] Table 2 Summary of instruments and reagents

[0070]

[0071] Example 1 Establishment of characteristic fingerprint method

[0072] 1. Preparation of reference substance solution

[0073] Take sibiricose A5, sibiricose A6, and Polygala tenuifolia Willd. Reference substances of ketone Ⅲ, 3,6'-di-sinapoyl sucrose, tenuifoliside A, ginsenoside Rg1, ginsenoside Re, β-asarone, α-asarone, polyporenic acid C, and dehydrotumulosic acid were accurately weighed and dissolved in 75% methanol to prepare a reference solution with the mass concentrations of the above substances being 25, 20, 15, 90, 45, 45, 45, 200, 100, 8, and 15 μg / mL in sequence.

[0074] 2. Preparation of test solution

[0075] Approximately 2.0 g of the Kaixin Powder sample was accurately weighed and placed in a conical flask. 25 mL of 75% methanol was accurately added, and the weight was determined. It was sonicated (frequency: 40 kHz, power: 300 W) for 30 minutes, cooled, and the weight was determined again. The lost weight was made up with 75% methanol. It was shaken well and filtered, and the subsequent filtrate was taken to obtain the test solution.

[0076] 3. Determination

[0077] The reference solution and the test solution were detected by ultra-high performance liquid chromatography (UPLC). 2 μL of the reference solution and the test solution were accurately pipetted and injected into the liquid chromatograph. Among them, the ultra-high performance liquid chromatography (UPLC) included the following detection conditions: the chromatographic column was Agilent ZORBAX BOUNS-RP (2.1 * 150 mm, 1.8 μm); the detector was a diode array detector (DAD); the detection wavelengths were 245 nm from 0 to 39 min, 203 nm from 39 to 44 min, and 245 nm from 44 to 110 min; the column temperature was 35 - 40 °C; the flow rate was 0.30 mL / min - 0.32 mL / min; the injection volume was 2 μL; the mobile phase was acetonitrile - 0.1% phosphoric acid aqueous solution, where phase A was acetonitrile and phase B was 0.1% phosphoric acid aqueous solution; the analysis time was 110 min; gradient elution. The specific program of gradient elution was as shown in Table 3:

[0078] Table 3 Gradient elution program

[0079] Time Mobile phase 0 - 5 min Volume ratio of phase A to phase B is 5:95 - 10:90 5 - 25 min Volume ratio of phase A to phase B is 10:90 - 19:81 25 - 40 min Volume ratio of phase A to phase B is 19:81 - 20:80 40 - 50 min Volume ratio of phase A to phase B is 20:80 - 27:73 50 - 70 min Volume ratio of phase A to phase B is 27:73 - 28:72 70 - 85 min Volume ratio of phase A to phase B is 28:72 - 50:50 85 - 95 min Volume ratio of phase A to phase B is 50:50 - 60:40 95 - 105 min Volume ratio of phase A to phase B is 60:40 - 85:15 105 - 110 min Volume ratio of phase A to phase B is 85:15

[0080] The characteristic chromatograms of the reference solution and the test solution were obtained by determination. As shown in, the characteristic chromatograms of the reference solution and the test solution were compared by retention time for qualitative analysis, and the peaks in the characteristic chromatogram of the test solution were identified and located. 11 characteristic peaks in the characteristic chromatogram of Kaixin Powder were confirmed, namely peak 1: sibiricose A5, peak 2: sibiricose A6, peak 3: polygala tenuifolia Figure 1 as follows. The characteristic chromatograms of the reference solution and the test solution were compared by retention time for qualitative analysis, and the peaks in the characteristic chromatogram of the test solution were identified and located. 11 characteristic peaks in the characteristic chromatogram of Kaixin Powder were confirmed, namely peak 1: sibiricose A5, peak 2: sibiricose A6, peak 3: polygala tenuifolia Ketone Ⅲ, Peak 4: 3,6'-di-sinapoyl sucrose, Peak 5: tenuifoliside A, Peak 6: ginsenoside Rg1, Peak 7: ginsenoside Re, Peak 8: β-asarone, Peak 9: α-asarone, Peak 10: polyporenic acid C, Peak 11: dehydrotumulosic acid.

[0081] Investigation of the Pretreatment Method and Chromatographic Conditions in Example 2

[0082] 2.1 Investigation of Different Extraction Solvents

[0083] Take the same test sample, prepare the test solution according to step 2) in Example 1. After dissolving the sample with methanol, 75% methanol, and 50% methanol extraction solvents respectively, carry out the determination according to the chromatographic conditions in step 3) of Example 1. It can be seen from Figure 2 that when methanol is used as the extraction solvent, the peak shapes of sibiricose A5 and A6 are not good; when 50% methanol is used as the extraction solvent, polyporenic acid C and dehydrotumulosic acid are not completely extracted or not extracted; when 75% methanol is used as the extraction solvent, the extraction of each characteristic component is relatively sufficient, and the peak shapes of the characteristic peaks in the chromatogram are good, and the response values are appropriate. Therefore, the preferred extraction solvent is 75% methanol.

[0084] 2.2 Investigation of Different Ultrasonic Times

[0085] Take the same test sample, prepare the test solution according to step 2) in Example 1. After adding the solvent to the test sample, ultrasonicate for 30 min, 40 min, and 60 min respectively, and then carry out the determination according to the chromatographic conditions in step 3) of Example 1. It can be seen from Figure 3 that when the ultrasonic times are 30 min, 40 min, and 60 min respectively, there is no obvious difference in the response values of the characteristic peaks among the chromatograms. Considering the simplicity and efficiency of the experiment, the preferred ultrasonic time is 30 min.

[0086] 2.3 Investigation of the Solute Ratio

[0087] Take the same test sample and prepare test solutions with different solute ratios at solute ratios of 2:50 and 2:25 (g / mL) according to step 2) in Example 1, and carry out the determination according to the chromatographic conditions in step 3) of Example 1. It can be seen from Figure 4 that when the solute ratio is 50:2, components with low response values such as ginsenoside Re are not easily detected. When the solute ratio is 2:25 (g / mL), the responses of each component are moderate. Therefore, the preferred solute ratio is 2:25 (g / mL).

[0088] 2.4 Investigation of Different Injection Volumes

[0089] Take the same test sample, prepare the test solution according to step 2) in Example 1, and carry out the determination at injection volumes of 2 μL and 4 μL respectively according to the chromatographic conditions in step 3) of Example 1. It can be seen fromFigure 5 It can be seen that: when the injection volume is 4 μL, Siberian Polygala sugar A5 and Siberian Polygala sugar A6 may show split peaks and poor peak shape due to excessive injection volume and chromatographic column overload. When the injection volume is 2 μL, the response of each component is moderate and the chromatographic peaks are well separated, so the preferred injection volume is 2 μL.

[0090] 2.5 Investigation of different column temperatures

[0091] Take the same test sample, prepare the test solution according to step 2) in Example 1, and measure it at 30°C, 35°C, and 40°C according to the chromatographic conditions of step 3) in Example 1. Figure 6 It can be seen that when the column temperature is 30℃, The chromatographic peak of ketone III was attached to its front peak, and ginsenoside Re was not baseline-separated from its adjacent peak. When the column temperature was 35°C and 40°C, the separation effect of the 11 characteristic peaks was better, so the preferred column temperature was 35°C-40°C.

[0092] 2.6 Investigation of different flow rates

[0093] Take the same test sample and prepare the test solution according to step 2) in Example 1. According to the chromatographic conditions of step 3) in Example 1, the changes of chromatographic peaks at different flow rates of 0.28 mL / min, 0.30 mL / min, and 0.32 mL / min are compared. Figure 7 It can be seen that when the flow rate is 0.28mL / min, the characteristic peaks are well separated, and ginsenoside Re peaks after the wavelength conversion and does not appear in the spectrum. When the flow rate is 0.30mL / min and 0.32mL / min, each characteristic peak is baseline separated from its adjacent peak. Therefore, the preferred flow rate is 0.30mL / min to 0.32mL / min.

[0094] 2.7 Investigation of different mobile phases

[0095] The same test sample was taken and the test solution was prepared according to step 2) in Example 1. The chromatographic conditions were followed according to step 3) in Example 1, and the separation effects after treatment with different mobile phases: acetonitrile-water, acetonitrile-0.05% phosphoric acid, and acetonitrile-0.1% phosphoric acid were compared. Figure 8 It can be seen that when acetonitrile-water is used as the mobile phase, the The separation effect of ketone III and its adjacent chromatographic peaks is not good; when acetonitrile-0.05% phosphoric acid is used as the mobile phase, the separation effect of ginsenoside Re and its adjacent chromatographic peaks is not good; when acetonitrile-0.1% phosphoric acid is used as the mobile phase, the separation of each characteristic peak is better, so acetonitrile-0.1% phosphoric acid is the preferred mobile phase.

[0096] 2.8 Investigation of different chromatographic columns

[0097] Take the same test sample, prepare the test solution according to step 2) in Example 1, and perform injection analysis under the chromatographic conditions in step 3) of Example 1 to compare the analytical performance of Agilent SB-AQ (2.1*150 mm, 1.8 μm), Waters ACQUITY UPLC HSS T3 (2.1*150 mm, 1.8 μm), and Agilent ZORBAX BONDS-RP (2.1*150 mm, 1.8 μm). It can be seen from Figure 9 that: The chromatographic column ③ Agilent ZORBAX BONDS-RP (2.1*150 mm, 1.8 μm) has excellent separation effects on 11 characteristic peaks, and the chromatographic peaks are evenly distributed, which is suitable for characteristic fingerprint analysis. Therefore, Agilent ZORBAX BONDS-RP (2.1*150 mm, 1.8 μm) is preferably used for the characteristic fingerprint analysis of the Kaixin San sample.

[0098] Establishment of the standard characteristic fingerprint in Example 3

[0099] Take 20 batches of Kaixin San samples (S1-S20) for detection. Prepare the test solution according to step 2) in Example 1, and obtain the UPLC characteristic fingerprints of 20 batches of samples under the chromatographic conditions in step 3) of Example 1. Import the obtained characteristic fingerprints into the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition)", set the fingerprint of Kaixin San sample S1 within 5-110 min as the reference fingerprint, set the time window width to 0.1, perform multi-point correction and Mark peak matching, generate the reference characteristic fingerprint R by the average method, and use the chromatographic peaks that exist in all the fingerprints as the common characteristic peaks to establish the standard characteristic fingerprint of the Kaixin San sample. The specific results are shown in Figure 10 . Calculate the similarity, and the results are shown in Table 4. The similarities of the characteristic fingerprints of 20 batches of Kaixin San reference samples are between 0.994 and 1.000.

[0100] As Figure 10As shown, the standard characteristic chromatogram of the Kāixīn Sǎn sample includes 11 common characteristic peaks. Using the 8th peak (β-asarone) as the reference peak (S peak, relative retention time is 1.00), the relative retention times of the other 10 common characteristic peaks are calculated, and the results are shown in Table 5. The specified values (averages) of the relative retention times of the other 10 common characteristic peaks are in sequence: the 1st peak (0.17), the 2nd peak (0.18), the 3rd peak (0.36), the 4th peak (0.50), the 5th peak (0.59), the 6th peak (0.63), the 7th peak (0.67), the 9th peak (1.11), the 10th peak (1.58), and the 11th peak (1.65). Except for the 8th peak, the RSD% of the specified values of the relative retention times of the remaining 10 common characteristic peaks are all ≤ 0.3%. The above-mentioned 1st peak is the characteristic peak of polygalacin A5, the 2nd peak is the characteristic peak of polygalacin A6, and the 3rd peak is the characteristic peak of onjisaponin III. The 4th peak is the characteristic peak of 3,6'-di-O-sinapoyl-sucrose, the 5th peak is the characteristic peak of tenuifoliside A, the 6th peak is the characteristic peak of ginsenoside Rg1, the 7th peak is the characteristic peak of ginsenoside Re, the 8th peak is the characteristic peak of β-asarone, the 9th peak is the characteristic peak of α-asarone, the 10th peak is the characteristic peak of polyporenic acid C, and the 11th peak is the characteristic peak of dehydrotumulosic acid.

[0101] Table 4 Similarity Results

[0102] S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 R S1 1.000 0.995 0.996 0.997 0.998 0.996 1.000 0.995 1.000 0.999 0.999 1.000 0.994 0.999 0.995 0.997 0.997 0.996 0.997 0.996 0.999 S2 0.995 1.000 1.000 0.999 0.998 1.000 0.994 0.998 0.996 0.998 0.998 0.997 0.998 0.997 0.993 0.998 0.988 0.990 0.993 0.998 0.998 S3 0.996 1.000 1.000 0.999 0.998 1.000 0.995 0.998 0.997 0.999 0.998 0.997 0.999 0.997 0.993 0.999 0.987 0.989 0.994 0.998 0.998 S4 0.997 0.999 0.999 1.000 1.000 0.999 0.996 0.996 0.997 0.999 0.999 0.999 0.995 0.997 0.992 0.997 0.991 0.995 0.993 0.996 0.999 S5 0.998 0.998 0.998 1.000 1.000 0.998 0.996 0.994 0.997 0.998 0.999 0.999 0.994 0.997 0.991 0.996 0.993 0.996 0.992 0.995 0.998 S6 0.996 1.000 1.000 0.999 0.998 1.000 0.995 0.997 0.997 0.998 0.998 0.997 0.998 0.996 0.992 0.998 0.987 0.990 0.993 0.997 0.998 S7 1.000 0.994 0.995 0.996 0.996 0.995 1.000 0.995 1.000 0.998 0.999 0.999 0.994 0.998 0.996 0.996 0.996 0.996 0.997 0.996 0.999 S8 0.995 0.998 0.998 0.996 0.994 0.997 0.995 1.000 0.997 0.998 0.997 0.995 1.000 0.998 0.998 1.000 0.989 0.986 0.998 1.000 0.998 S9 1.000 0.996 0.997 0.997 0.997 0.997 1.000 0.997 1.000 0.999 0.999 0.999 0.996 0.999 0.997 0.998 0.996 0.995 0.998 0.998 0.999 S10 0.999 0.998 0.999 0.999 0.998 0.998 0.998 0.998 0.999 1.000 1.000 0.999 0.998 0.999 0.996 0.999 0.994 0.993 0.997 0.999 1.000 S11 0.999 0.998 0.998 0.999 0.999 0.998 0.999 0.997 0.999 1.000 1.000 1.000 0.996 0.999 0.995 0.998 0.995 0.996 0.996 0.997 1.000 S12 1.000 0.997 0.997 0.999 0.999 0.997 0.999 0.995 0.999 0.999 1.000 1.000 0.994 0.998 0.994 0.997 0.995 0.997 0.995 0.996 0.999 S13 0.994 0.998 0.999 0.995 0.994 0.998 0.994 1.000 0.996 0.998 0.996 0.994 1.000 0.997 0.997 0.999 0.986 0.985 0.996 1.000 0.998 S14 0.999 0.997 0.997 0.997 0.997 0.996 0.998 0.998 0.999 0.999 0.999 0.998 0.997 1.000 0.998 0.999 0.996 0.993 0.999 0.999 1.000 S15 0.995 0.993 0.993 0.992 0.991 0.992 0.996 0.998 0.997 0.996 0.995 0.994 0.997 0.998 1.000 0.998 0.993 0.987 1.000 0.998 0.997 S16 0.997 0.998 0.999 0.997 0.996 0.998 0.996 1.000 0.998 0.999 0.998 0.997 0.999 0.999 0.998 1.000 0.992 0.990 0.998 1.000 0.999 S17 0.997 0.988 0.987 0.991 0.993 0.987 0.996 0.989 0.996 0.994 0.995 0.995 0.986 0.996 0.993 0.992 1.000 0.997 0.995 0.990 0.994 S18 0.996 0.990 0.989 0.995 0.996 0.990 0.996 0.986 0.995 0.993 0.996 0.997 0.985 0.993 0.987 0.990 0.997 1.000 0.990 0.988 0.994 S19 0.997 0.993 0.994 0.993 0.992 0.993 0.997 0.998 0.998 0.997 0.996 0.995 0.996 0.999 1.000 0.998 0.995 0.990 1.000 0.998 0.998 S20 0.996 0.998 0.998 0.996 0.995 0.997 0.996 1.000 0.998 0.999 0.997 0.996 1.000 0.999 0.998 1.000 0.990 0.988 0.998 1.000 0.999 R 0.999 0.998 0.998 0.999 0.998 0.998 0.999 0.998 0.999 1.000 1.000 0.999 0.998 1.000 0.997 0.999 0.994 0.994 0.998 0.999 1.000

[0103] Table 5 Relative Retention Time Results

[0104]

[0105] Example 4 Peak Attribution and Location

[0106] Take the test sample (S20) and prepare the test solution as in Example 1. At the same time, prepare the reference solution samples according to step 2) in Example 1, which are Polygala tenuifolia, Panax ginseng, Poria cocos, and Acorus tatarinowii. Prepare the missing sample negative solutions by removing any one of the four herbs in the Kāixīn Sǎn preparation sample containing Polygala tenuifolia, Panax ginseng, Poria cocos, and Acorus tatarinowii according to step 1) in Example 1: the Polygala tenuifolia negative sample, the Panax ginseng negative sample, the Poria cocos negative sample, and the Acorus tatarinowii negative sample.

[0107] Using the same ultra - performance liquid chromatography (UPLC) method as in step 3) of Example 1, the test solution, 4 reference solution, and 4 sample - lacking negative sample solutions were respectively determined to obtain the characteristic chromatograms of the test solution, the characteristic chromatograms of the 4 reference solution, and the characteristic chromatograms of the 4 sample - lacking negative sample solutions. By relative retention time, the common characteristic peaks of the corresponding single herbs in the 4 sample - lacking negative solutions in the standard characteristic chromatogram of the test sample preparation were identified, so as to attribute and locate the characteristic peaks in the characteristic chromatograms of the corresponding single herbs in the 4 sample - lacking negative solutions.

[0108] As shown in Figure 11, from Figure 11 - A the chromatogram, it can be seen that chromatographic peaks 1 - 5 all come from the Radix Polygalae slices; from Figure 11 - B the chromatogram, it can be seen that chromatographic peaks 6 and 7 all come from the Ginseng flavor; from Figure 11 - C the chromatogram, it can be seen that chromatographic peaks 10 and 11 all come from the Poria flavor; from Figure 11 - D the chromatogram, it can be seen that chromatographic peaks 8 and 9 mainly come from the Acori Tatarinowii Rhizoma flavor.

[0109] Method Validation of Example 5

[0110] The detection method of the UPLC characteristic chromatogram of the Kāixīn Sǎn sample of the present invention was subjected to methodological validation, and the results of its performance indicators are as follows.

[0111] 5.1 Peak Identification

[0112] Taking the same batch of test samples (S16) and reference samples, the test solution and reference solution were prepared according to steps 2) and 1) in Example 1. The test solution and reference solution were respectively determined using the same ultra - performance liquid chromatography (UPLC) method as in step 3) of Example 1 to obtain the characteristic chromatograms of the test solution and reference solution, and a blank solution control group was set up at the same time. By comparing the retention times, the attribution and location of the characteristic peaks in the test solution were identified, as shown in Table 6:

[0113] Table 6 Results of Peak Identification Test

[0114]

[0115]

[0116] Results: The blank solution had no interference at the elution time of the characteristic peaks. The retention times of sibiricose A5, sibiricose A6, polygalaxanthone III, 3,6'-di - sinapoyl - sucrose, tenuifoliside A, ginsenoside Rg1, ginsenoside Re, β - asarone, α -asarone, polyporenic acid C, and dehydrotumulosic acid in the test solution were consistent with the corresponding characteristic peaks in the reference solution. ​

[0117] 5.2 Precision

[0118] Take the same batch of test samples (S16), prepare the test sample solution according to step 2) in Example 1, and under the chromatographic conditions of step 3) in Example 1. Inject the sample continuously 6 times on the same day. Using β-asarone as the reference peak (S peak), calculate the relative retention times of 11 characteristic peaks and obtain the average relative retention times of 11 characteristic peaks. The results are shown in Tables 7 and 8.

[0119] Table 7 Results of precision test (relative retention time)

[0120]

[0121] Table 8 Results of precision test (relative peak area)

[0122]

[0123]

[0124] As shown in Tables 7 and 8: When the same test sample solution is injected continuously 6 times, the RSDs of the relative retention times of each chromatographic peak are all less than 0.1%, and the relative peak areas are all less than 3.9%. The results meet the requirements, indicating that the precision of this method is good.

[0125] 5.3 Repeatability

[0126] Take the same batch of test samples (S16), prepare the test sample solution according to step 2) in Example 1, and under the chromatographic conditions of step 3) in Example 1. Inject 1 injection for each of 6 parallel test sample solutions. Using β-asarone as the reference peak (S peak), calculate the relative retention times of 11 characteristic peaks and obtain the average relative retention times of 11 characteristic peaks. The results are shown in Tables 9 and 10.

[0127] Table 9 Results of repeatability test (relative retention time)

[0128]

[0129] Table 10 Results of repeatability test (relative peak area)

[0130]

[0131]

[0132] As shown in Tables 9 and 10: When 1 injection is made for each of 6 parallel test sample solutions, the RSDs of the relative retention times of each chromatographic peak are all less than 0.1%, and the RSDs of the relative peak areas of each chromatographic peak are all less than 2.0%. The results meet the requirements, indicating that the repeatability of this method is good.

[0133] 5.4 Stability

[0134] Take the same batch of test preparation (S16), prepare the test solution according to step 2) in Example 1, and inject samples at 0 h, 10.9 h, 23.9 h, 34.8 h, and 47.8 h respectively under the chromatographic conditions in step 3) of Example 1. Using β-asarone as the reference peak (S peak), calculate the relative retention times of 11 characteristic peaks and obtain the average relative retention time of 11 characteristic peaks. The results are shown in Tables 11 and 12.

[0135] Table 11 Results of solution stability test (relative retention time)

[0136]

[0137] Table 12 Results of solution stability test (relative peak area)

[0138]

[0139]

[0140] As shown in Tables 11 and 12: When the same test sample is injected at 0 h, 10.9 h, 23.9 h, 34.8 h, and 47.8 h respectively, the RSD of the relative retention time of each chromatographic peak is ≤ 0.1%, and the RSD of the relative peak area is ≤ 4.8%. The results meet the requirements. It shows that the test solution is stable after being placed at room temperature for 47.8 h.

[0141] After peak identification, precision, and repeatability tests of the detection method of the present invention, all parameters are within the acceptable range; through the solution stability test, it is shown that the test solution can still be very stable after being placed at room temperature for 47.8 h. The above results indicate that this method is applicable to the detection of the characteristic chromatogram of Kaixin San samples.

[0142] The above embodiments merely illustrate the principles and effects of the present invention, rather than limiting the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A UPLC characteristic spectrum of Kaixinsan, characterized in that Including Siberian Polygala A5, Siberian Polygala A6, The characteristic peaks of ketone III, 3,6'-diesinaroylsucrose, polygala tenuifolia glycoside A, ginsenoside Rg1, ginsenoside Re, β-asarone, α-asarone, policolic acid C, and dehydropachymic acid.

2. The UPLC characteristic spectrum of Kaixinsan according to claim 1, characterized in that: The UPLC characteristic spectrum has characteristic peaks substantially as shown in FIG1 .

3. The UPLC characteristic spectrum of Kaixinsan as claimed in claim 1 or 2, characterized in that: The UPLC characteristic spectrum of Kaixinsan comprises 11 common characteristic peaks, with peak 8 as the reference peak (S peak, relative retention time is 1.00), and the specified values ​​of relative retention time of the other 10 common characteristic peaks are peak 1 (0.17), peak 2 (0.18), peak 3 (0.36), peak 4 (0.50), peak 5 (0.59), peak 6 (0.63), peak 7 (0.67), peak 9 (1.11), peak 10 (1.58), and peak 11 (1.65), and except for peak 8, the RSD% of the specified values ​​of relative retention time of the other 10 common characteristic peaks are all ≤3.0%.

4. The method for detecting the UPLC characteristic spectrum of Kaixinsan according to any one of claims 1 to 3, characterized in that: The method comprises: Preparation of reference solution, preparation of test solution, detection by ultra-high performance liquid chromatography, identification of characteristic peaks according to the retention times of reference solution and test solution, so as to obtain the characteristic spectrum of the test sample; Wherein, the reference material includes Siberian Polygala A5, Siberian Polygala A6, Ketone III, 3,6'-dienasinoylsucrose, polygala tenuifolia glycoside A, ginsenoside Rg1, ginsenoside Re, β-asarone, α-asarone, policolic acid C, and dehydropachymic acid; The test sample is Kaixin powder; The preparation of the reference solution comprises adding each sample of each reference to a solvent, dissolving the sample to obtain a solution containing Siberian Polygala sugar A5, Siberian Polygala sugar A6, and Polygala sugar A7. A mixed reference solution of ketone III, 3,6'-dienasinoylsucrose, polygala tenuifolia glycoside A, ginsenoside Rg1, ginsenoside Re, β-asarone, α-asarone, policolic acid C, and dehydropachymic acid; The preparation of the test solution comprises adding the test sample to a solvent, dissolving the test sample, ultrasonicating, shaking, filtering, and taking a filtrate to obtain a test solution; The characteristic spectrum of the test sample contains characteristic peaks of the following substances: Siberian Polygala A5, Siberian Polygala A6, Ketone III, 3,6'-dienasinoylsucrose, polygala tenuifolia glycoside A, ginsenoside Rg1, ginsenoside Re, β-asarone, α-asarone, policolic acid C, and dehydropachymic acid.

5. The detection method of the UPLC characteristic spectrum of Kaixinsan as claimed in claim 4, characterized in that, The solvent is 50% to 100% methanol, more preferably 75% methanol.

6. The detection method of the UPLC characteristic spectrum of Kaixinsan as described in claim 4 or 5, characterized in that, The ratio of the added mass g of the test sample to the volume mL of the solvent dissolved is 2:25 to 2:50, more preferably 2:

25.

7. The detection method of the UPLC characteristic spectrum of Kaixinsan according to any one of claims 4 to 6, characterized in that: The ultrasonic time is 30 min to 60 min, more preferably 30 min; the power of the ultrasonic extraction is 300 W, and the frequency of the ultrasonic extraction is 40 kHz.

8. The detection method of the UPLC characteristic spectrum of Kaixinsan according to any one of claims 4 to 7, characterized in that: The solubility of each substance in the reference solution is as follows: Siberian Polygala A5 25 μg / mL, Siberian Polygala A6 20 μg / mL, Ketone III 15μg / mL, 3,6'-dienasinoylsucrose 90μg / mL, polygala tenuifolia glycoside A 45μg / mL, ginsenoside Rg1 45μg / mL, ginsenoside Re 45μg / mL, β-asarone 200μg / mL, α-asarone 100μg / mL, policolic acid C 8μg / mL, and dehydropachymic acid 15μg / mL.

9. The detection method of the UPLC characteristic spectrum of Kaixinsan according to any one of claims 4 to 8, characterized in that: The chromatographic column used for detection by the ultra-high performance liquid chromatograph is a C14 chromatographic column. Preferably, the ultra-high performance liquid chromatographic column is Agilent ZORBAX BOUNS-RP.

10. The method for detecting the UPLC characteristic spectrum of Kaixinsan according to any one of claims 4 to 9, characterized in that: The wavelength mode used for detection by ultra-high performance liquid chromatography is dual-wavelength switching detection, and the dual wavelengths are 203nm and 245nm.

11. The method for detecting the UPLC characteristic spectrum of Kaixinsan as claimed in claim 10, characterized in that: The specific wavelengths used for detection by the ultra-high performance liquid chromatograph are: 0-39min: 245nm, 39-44min: 203nm, 44-110min: 245nm.

12. The method for detecting the UPLC characteristic spectrum of Kaixinsan according to any one of claims 4 to 11, characterized in that: The column temperature during detection by the ultra-high performance liquid chromatograph is 30-40°C, and more preferably, the column temperature during detection by the ultra-high performance liquid chromatography is 35-40°C.

13. The method for detecting the UPLC characteristic spectrum of Kaixinsan according to any one of claims 4 to 12, characterized in that: The flow rate of the ultra-high performance liquid chromatograph during detection is 0.30 to 0.32 mL / min.

14. The method for detecting the UPLC characteristic spectrum of Kaixinsan according to any one of claims 4 to 13, characterized in that: The injection volume in the detection by the ultra-high performance liquid chromatography is 2 to 4 μL, and preferably, the injection volume in the ultra-high performance liquid chromatography is 2 μL.

15. The method for detecting the UPLC characteristic spectrum of Kaixinsan according to any one of claims 4 to 14, characterized in that: In the detection by the ultra-high performance liquid chromatograph, the mobile phase is acetonitrile-0.05-0.1% phosphoric acid aqueous solution, wherein phase A is acetonitrile and phase B is 0.05-0.1% phosphoric acid aqueous solution; the analysis time is 110 min; gradient elution; preferably, in the ultra-high performance liquid chromatography, the mobile phase is acetonitrile-0.1% phosphoric acid aqueous solution, wherein phase A is acetonitrile and phase B is 0.1% phosphoric acid aqueous solution; the analysis time is 110 min; gradient elution; preferably, the specific procedure of the gradient elution is:

16. Use of the UPLC characteristic spectrum of Kaixinsan according to any one of claims 1 to 3 or the detection method described in claims 4 to 15 in the quality research, detection and control of related drugs or medicinal materials, wherein the drug or medicinal material is Kaixinsan or a drug or medicinal material containing components similar to Kaixinsan.

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