Fingerprint spectrum construction method of ancient-Chinese health-preserving essence oral liquid and multi-index component detection method

Abstract

The invention provides a fingerprint spectrum construction method of an ancient-Chinese health-preserving essence oral liquid and a multi-index component detection method, and relates to the technical field of analysis and detection. The fingerprint spectrum of the ancient-Chinese health-preserving essence oral liquid constructed by adopting a high-performance liquid chromatography has 20-50 common peaks, 9 chemical components are determined, ginsenoside Rb1 is a component in a monarch drug ginseng, calycosin-7-glucoside is a component in a monarch drug astragalus membranaceus, specnuezhenide is a component in a ministerial drug glossy privet fruit, and the fingerprint spectrum of the ancient-Chinese health-preserving essence oral liquid is a component in a ministerial drug glossy privet fruit. Epimedin A, Epimedin B, Epimedin C and icariin are all components in a ministerial drug of herba epimedii, paeoniflorin is a component in an adjuvant drug of radix paeoniae alba, and liquiritin is a component in a conductant drug of liquorice. According to the fingerprint spectrum of the ancient-Chinese health-preserving essence oral liquid, a plurality of substances in monarch, minister, assistant and guide are taken as quality indexes of the ancient-Chinese health-preserving essence oral liquid, so that accurate and comprehensive quality control on the quality of the ancient-Chinese health-preserving essence oral liquid can be realized, and too simple currently executed quality standards of the ancient-Chinese health-preserving essence oral liquid are perfected.

Description

Technical Field

[0001] The present invention relates to the field of analysis and detection technology, and in particular to a fingerprint construction method of Gu Han Health Essence Oral Liquid and a multi-index component detection method. Background Art

[0002] Gu Han Yangsheng Jing is a traditional Chinese herbal compound preparation derived from ancient health-preserving formulas unearthed from the Mawangdui Han Dynasty Tomb and developed in conjunction with modern TCM theories. It is composed of 11 herbs, including ginseng, roasted astragalus root, Rosa laevigata fruit, wolfberry fruit, processed Ligustrum lucidum fruit, Cuscuta seed, and Epimedium. It tonifies Qi, nourishes the kidneys, and improves essence. It is used to treat dizziness, palpitations, vertigo, tinnitus, forgetfulness, insomnia, impotence, spermatorrhea, fatigue, and weakness caused by Qi and Yin deficiency and kidney essence deficiency.

[0003] In this formula, ginseng significantly replenishes vital energy, strengthening the spleen and lungs; astragalus nourishes qi and raises yang, strengthening the defensive system and consolidating the exterior. These two herbs serve as the main ingredients, invigorating the spleen and stomach and strengthening the acquired foundation. Polygonatum sibiricum and wolfberry nourish the liver and kidneys, replenishing essence and filling the marrow, and protecting the innate foundation. Tonifying both the spleen and kidneys, this promotes the active production of qi and blood, enriching the essence and qi. Epimedium warms and nourishes kidney yang, while Ligustrum lucidum nourishes yin and nourishes the liver, tonifying yang without dryness and yin without greasiness. Rosa canal fruit astringes and consolidates essence, while Cuscuta chinensis nourishes the liver and kidneys, achieving a balanced and restrained tonification, preventing the dissipation of essence and qi. These six herbs serve as the auxiliary ingredients. When combined with ginseng and astragalus, essence can be transformed into qi, and qi can be generated into essence. With abundant essence and qi, the mind is naturally nourished. Since the liver and kidney share a common origin, and essence and blood are mutually generated, it is therefore combined with white peony root to nourish blood and soften the liver. Malt aids digestion and strengthens the stomach, and when combined with ginseng and astragalus, it strengthens the spleen and stomach. These two herbs serve as the adjuvant ingredients. Licorice harmonizes the other herbs and serves as an adjuvant. When used together, these herbs tonify Qi, nourish the kidneys, and improve essence. As a classic Chinese herbal compound, Gu Han Yangsheng Jing Oral Liquid boasts significant clinical efficacy and broad application prospects.

[0004] The related technology discloses a UPLC-MS method for simultaneously determining the contents of five chemical components (icariin, astragaloside IV, ginsenosides Rb1, Re and Rg1) in Gu Han Yangsheng Jing oral liquid. The chromatographic conditions used are as follows: Waters Acquity UPLCC18 chromatographic column (2.1 mm × 50 mm, 1.7 μm), column temperature 30 ° C; mobile phase acetonitrile 0.1% formic acid aqueous solution (15:85) isocratic elution, flow rate 0.2 mL / min, injection volume 4 μL; the mass spectrometry conditions used are as follows: electrospray ionization (ESI) source, capillary voltage 3.0 kV, ion source temperature 120 ° C, desolvation gas temperature 350 ° C, desolvation gas flow rate 800 L / hr, cone gas flow rate 50 L / hr; detection method positive ion multiple reaction monitoring (MRM). However, the aforementioned testing method only covers the detection of four components of the main herb (astragaloside IV, ginsenosides Rb1, Re, and Rg1) and one component of the adjuvant (icariin) in Gu Han Yang Sheng Jing Oral Liquid. However, traditional Chinese medicine compounds are characterized by complex ingredients, multi-target synergistic effects, and the potential for new active substances to be produced after drug metabolism in the body. Quantitative analysis of the main herb and adjuvant components alone cannot fully reflect their overall quality, thus establishing an accurate and comprehensive quality control method is particularly important. Summary of the Invention

[0005] In view of this, the present invention aims to provide a method for constructing a fingerprint of Gu Han Yang Sheng Jing Oral Liquid and a method for detecting multiple components. The fingerprint of Gu Han Yang Sheng Jing Oral Liquid provided by the present invention uses multiple substances in the main, secondary, auxiliary and guiding ingredients as quality indicators for Gu Han Yang Sheng Jing Oral Liquid, enabling accurate and comprehensive quality control of Gu Han Yang Sheng Jing Oral Liquid.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for constructing a fingerprint spectrum of Gu Han Health Essence Oral Liquid, comprising the following steps:

[0008] Mixing the Gu Han Health Essence Oral Liquid with an extractant to obtain a test solution; the extractant includes a lower alcohol or a lower alcohol aqueous solution;

[0009] The test solution and the mixed reference solution are respectively subjected to high performance liquid chromatography to obtain a test sample liquid phase spectrum and a reference liquid phase spectrum, respectively; and a fingerprint spectrum of Gu Han Yang Sheng Jing oral liquid is obtained based on the test sample liquid phase spectrum and the reference liquid phase spectrum; the characteristic peaks of the fingerprint spectrum of Gu Han Yang Sheng Jing oral liquid include characteristic peaks of paeoniflorin, calycosin isoflavone glucoside, liquiritin, privet glycoside, epimedium components and ginsenoside Rb1; the epimedium components include at least one of epimedin A, epimedin B, epimedin C and icariin;

[0010] The conditions for high performance liquid chromatography detection include: a mobile phase system including mobile phase A and mobile phase B, wherein the mobile phase A is acetonitrile, and the mobile phase B is a formic acid aqueous solution, wherein the volume fraction of formic acid in the formic acid aqueous solution is 0.05-0.2%; an elution mode is gradient elution, and the gradient elution program includes: from 0 to 18 minutes, the volume fraction of the mobile phase A linearly increases from 5% to 20%; from 18 to 33 minutes, the volume fraction of the mobile phase A is 20%; from 33 to 43 minutes, the volume fraction of the mobile phase A linearly increases from 20% to 23%; and from 43 to 65 minutes, the volume fraction of the mobile phase A linearly increases from 23% to 45%.

[0011] Preferably, the volume ratio of the Gu Han Health Essence Oral Liquid to the extractant is 1:5-9;

[0012] The volume fraction of the lower alcohol in the extractant is 50-100%, and the lower alcohol includes methanol.

[0013] Preferably, the mixing includes ultrasonic mixing; the power of the ultrasonic mixing is 100 to 500 W, and the time is 25 to 35 minutes.

[0014] Preferably, the reference substances in the mixed reference solution include paeoniflorin, calycosin glucoside, liquiritin, privet glycoside, epimedium components and ginsenosides; the epimedium components include at least one of epimedin A, epimedin B, epimedin C and icariin; the concentration of a single reference substance in the mixed reference solution is 0.02 to 0.13 mg / mL; the solvent in the mixed reference solution includes lower alcohol or lower alcohol aqueous solution.

[0015] Preferably, the chromatographic column used in the high performance liquid chromatography detection includes a C18 chromatographic column, the mobile phase flow rate is 0.9 to 1.1 mL / min, the detection wavelength is 203 to 270 nm, the column temperature is 25 to 40° C., and the injection volume is 5 to 15 μL.

[0016] The present invention also provides a method for detecting multiple index components of Gu Han Health Essence Oral Liquid, comprising the following steps:

[0017] Mixing the Gu Han Health Essence oral liquid to be tested with an extractant to obtain a sample liquid to be tested; the extractant includes a lower alcohol or a lower alcohol aqueous solution;

[0018] The sample solution is subjected to high performance liquid chromatography to obtain a multi-index component detection result of the Gu Han Health Essence Oral Liquid; the multi-index components include at least two of ginsenoside Rb1, calycosin isoflavone glucoside, privet glycoside, epimedin A, epimedin B, epimedin C, icariin, paeoniflorin and liquiritin;

[0019] The conditions for high performance liquid chromatography detection include: a mobile phase system including mobile phase A and mobile phase B, wherein the mobile phase A is acetonitrile, and the mobile phase B is a formic acid aqueous solution, wherein the volume fraction of formic acid in the formic acid aqueous solution is 0.05-0.2%; an elution mode is gradient elution, and the gradient elution program includes: from 0 to 18 minutes, the volume fraction of the mobile phase A linearly increases from 5% to 20%; from 18 to 33 minutes, the volume fraction of the mobile phase A is 20%; from 33 to 43 minutes, the volume fraction of the mobile phase A linearly increases from 20% to 23%; and from 43 to 65 minutes, the volume fraction of the mobile phase A linearly increases from 23% to 45%.

[0020] Preferably, the volume ratio of the Gu Han Health Essence oral liquid to be tested to the extractant is 1:5-9;

[0021] The volume fraction of the lower alcohol in the extractant is 50-100%, and the lower alcohol includes methanol.

[0022] Preferably, the mixing includes ultrasonic mixing; the power of the ultrasonic mixing is 100 to 500 W, and the time is 25 to 35 minutes.

[0023] Preferably, the chromatographic column used in the high performance liquid chromatography detection includes a C18 chromatographic column, the mobile phase flow rate is 0.9 to 1.1 mL / min, the detection wavelength is 203 to 270 nm, the column temperature is 25 to 40° C., and the injection volume is 5 to 15 μL.

[0024] Preferably, the test results include quantitative test results and / or qualitative test results; the qualitative test results are obtained by comparing the liquid phase spectrum of the sample liquid to be tested with the fingerprint spectrum of Gu Han Health Essence Oral Liquid obtained by the fingerprint spectrum construction method described in the above technical solution.

[0025] The present invention adopts a high performance liquid chromatography (HPLC) method to construct a fingerprint of Guhan Health Essence Oral Liquid. The fingerprint has 20 to 50 common peaks, among which 24 common peaks have good peak shapes. Nine chemical components, including ginsenoside Rb1, calycosin isoflavone glucoside, terglucosidin, epimedin A, epimedin B, epimedin C, icariin, paeoniflorin and liquiritin, are determined. Among them, ginsenoside Rb1 is a component of the monarch drug Panax ginseng, calycosin isoflavone glucoside is a component of the monarch drug Astragalus, terglucosidin is a component of the auxiliary drug Ligustrum lucidum fruit, epimedin A, epimedin B, epimedin C and icariin are all components of the auxiliary drug Epimedium, paeoniflorin is a component of the adjuvant drug White Peony Root, and liquiritin is a component of the guiding drug Liquorice. The fingerprint of Gu Han Yang Sheng Jing Oral Liquid constructed by the present invention uses multiple substances in the monarch, minister, assistant and envoy components as quality indicators of Gu Han Yang Sheng Jing Oral Liquid. This can more scientifically evaluate the quality of Gu Han Yang Sheng Jing Oral Liquid and achieve accurate and comprehensive quality control of the quality of Gu Han Yang Sheng Jing Oral Liquid, thereby better ensuring the quality stability, consistency and controllability of Gu Han Yang Sheng Jing Oral Liquid. This improves the overly simple current quality standards for Gu Han Yang Sheng Jing Oral Liquid and provides a basis for the quality evaluation and clinical application of Gu Han Yang Sheng Jing Oral Liquid. Moreover, compared with UPLC-MS, the present invention adopts HPLC, which has strong universality, can capture components without UV absorption or low response (such as some saponins), and has lower detection costs.

[0026] The present invention establishes a multi-index component synchronous detection method for Gu Han Health Essence Oral Liquid. The method has the advantages of strong specificity and simple operation, and has good stability, precision, reproducibility and sample recovery rate. The methodological investigation test results are good. The method can comprehensively detect the contents of up to 9 main active ingredients in Gu Han Health Essence Oral Liquid, including paeoniflorin, calycosin isoflavone glucoside, liquiritin, privet glycoside, epimedin A, epimedin B, epimedin C, icariin and ginsenoside Rb1, and can be used as marker components for evaluating the quality of Gu Han Health Essence Oral Liquid medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 HPLC chromatograms of Gu Han Yang Sheng Jing oral liquid in Example 1 under different chromatographic columns;

[0028] Figure 2 HPLC chromatograms of Gu Han Yang Sheng Jing oral liquid in Example 1 under different mobile phase systems;

[0029] Figure 3 This is a 3D isovalue diagram of the chemical components of Gu Han Yang Sheng Jing oral liquid in Example 1;

[0030] Figure 4 This is the HPLC chromatogram of the ingredient specificity test of Gu Han Yang Sheng Jing Oral Liquid in Example 1;

[0031] Figure 5HPLC chromatogram of the mixed reference solution in Example 2;

[0032] Figure 6 The HPLC superimposed patterns (S1-S10) and the reference pattern (R) of 10 batches of Gu Han Yang Sheng Jing Oral Liquid in Example 2 are shown;

[0033] Figure 7 This is the PCA score graph of 10 batches of Gu Han Health Essence Oral Liquid in Example 2;

[0034] Figure 8 This is the OPLS-DA score graph for 10 batches of Gu Han Health Essence Oral Liquid in Example 2;

[0035] Figure 9 This is the permutation test diagram of the OPLS-DA model for 10 batches of Gu Han Health Essence Oral Liquid in Example 2;

[0036] Figure 10 This is a graph showing the VIP scores for the common peaks of the 10 batches of Gu Han Yang Sheng Jing Oral Liquid in Example 2. DETAILED DESCRIPTION

[0037] The present invention provides a method for constructing a fingerprint spectrum of Gu Han Health Essence Oral Liquid, comprising the following steps:

[0038] Mixing the Gu Han Health Essence Oral Liquid with an extractant to obtain a test solution; the extractant includes a lower alcohol or a lower alcohol aqueous solution;

[0039] The test solution and the mixed reference solution are respectively subjected to high performance liquid chromatography to obtain a test sample liquid phase spectrum and a reference liquid phase spectrum, respectively; and a fingerprint spectrum of Gu Han Yang Sheng Jing oral liquid is obtained based on the test sample liquid phase spectrum and the reference liquid phase spectrum; the fingerprint spectrum of Gu Han Yang Sheng Jing oral liquid has characteristic peaks of paeoniflorin, calycosin isoflavone glucoside, liquiritin, privet glycoside, epimedium components and ginsenoside Rb1; the epimedium components include at least one of epimedin A, epimedin B, epimedin C and icariin;

[0040] The conditions for high performance liquid chromatography detection include: a mobile phase system including mobile phase A and mobile phase B, wherein the mobile phase A is acetonitrile, and the mobile phase B is a formic acid aqueous solution, wherein the volume fraction of formic acid in the formic acid aqueous solution is 0.05-0.2%; an elution mode is gradient elution, and the gradient elution program includes: from 0 to 18 minutes, the volume fraction of the mobile phase A linearly increases from 5% to 20%; from 18 to 33 minutes, the volume fraction of the mobile phase A is 20%; from 33 to 43 minutes, the volume fraction of the mobile phase A linearly increases from 20% to 23%; and from 43 to 65 minutes, the volume fraction of the mobile phase A linearly increases from 23% to 45%.

[0041] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.

[0042] The invention mixes the Guhan health-preserving essence oral liquid with an extractant to obtain a test solution.

[0043] In the present invention, the extractant includes a lower alcohol or a lower alcohol aqueous solution; the volume fraction of the lower alcohol in the extractant is preferably 50-100%, and can be specifically 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%; the lower alcohol includes methanol.

[0044] In the present invention, the volume ratio of the Gu Han Health Essence Oral Liquid to the extractant is 1:5-9, and can be specifically 1:5, 1:6, 1:7, 1:8 or 1:9.

[0045] In the present invention, the mixing preferably includes ultrasonic mixing, and the temperature of the ultrasonic mixing is preferably 30-60°C, and can be specifically 30°C, 35°C, 40°C, 45°C, 50°C, 55°C or 60°C; the power of the ultrasonic mixing is preferably 100-500W, and can be specifically 100W, 200W, 300W, 400W or 500W; the time of the ultrasonic mixing is preferably 25-35min, and can be specifically 25min, 28min, 30min, 32min or 35min.

[0046] After the mixing, the present invention preferably further comprises cooling the obtained mixed system to room temperature and filtering to obtain a test solution. In the present invention, the filtration preferably comprises filtration with a 0.22 μm filter membrane.

[0047] After obtaining the test sample solution, the present invention performs high performance liquid chromatography on the test sample solution and the mixed reference solution, respectively, to obtain a test sample liquid phase spectrum and a reference liquid phase spectrum, respectively, and obtains a fingerprint spectrum of the Gu Han Health Essence Oral Liquid based on the test sample liquid phase spectrum and the reference liquid phase spectrum; the characteristic peaks of the fingerprint spectrum of the Gu Han Health Essence Oral Liquid include characteristic peaks of paeoniflorin, calycosin isoflavone glucoside, liquiritin, privet glycoside, epimedium components and ginsenoside Rb1; the epimedium components include at least one of epimedin A, epimedin B, epimedin C and icariin.

[0048] In the present invention, the reference substances in the mixed reference solution include paeoniflorin, calycosin glucoside, liquiritin, terunguside, an epimedium component, and ginsenoside Rb1; the epimedium component includes at least one of epimedin A, epimedin B, epimedin C, and icariin. In the present invention, the concentration of each reference substance in the mixed reference solution is preferably 0.02 to 0.13 mg / mL, and can specifically be 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.11 mg / mL, 0.12 mg / mL, or 0.13 mg / mL. In the present invention, the solvent in the mixed reference solution preferably includes a lower alcohol or a lower alcohol aqueous solution; the volume fraction of the lower alcohol in the extractant is preferably 50-100%, and can be specifically 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%; the lower alcohol includes methanol.

[0049] In the present invention, the high performance liquid chromatography detection conditions include: the chromatographic column includes a C18 chromatographic column, which can be specifically Agilent Eclipse Plus C18, the specifications of the chromatographic column are preferably 4.6mm×250mm, and the filler particle size is preferably 5μm; the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, and the mobile phase B is a formic acid aqueous solution, and the volume fraction of formic acid in the formic acid aqueous solution is 0.05-0.2%, which can be specifically 0.05%, 0.08%, 0.1%, 0.12%, 0.15%, 0.18% or 0.2%; the elution mode is gradient elution, and the program of the gradient elution is shown in Table 1; the mobile phase flow rate is preferably 0.9-1.1mL / min, which can be specifically 0.9mL / min, 1mL / min in or 1.1 mL / min, the detection wavelength is preferably 203-270 nm, specifically 203 nm, 204 nm, 205 nm, 206 nm, 207 nm, 208 nm, 209 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm or 270 nm, more preferably 204 nm; the column temperature is preferably 25-40 ° C, specifically 25 ° C, 28 ° C, 30 ° C, 32 ° C, 35 ° C, 38 ° C or 40 ° C; the injection volume is preferably 5-15 μ L, specifically 5 μ L, 8 μ L, 10 μ L, 12 μ L or 15 μ L.

[0050] Table 1 Gradient elution program

[0051] Time / min Volume fraction of mobile phase A / % Volume fraction of mobile phase B / % 0 5 95 18 20 80 33 20 80 43 23 77 65 45 55

[0052] The present invention preferably introduces the liquid phase spectrum of the test sample into the traditional Chinese medicine chromatographic characteristic spectrum similarity evaluation system, adopts the characteristic spectrum common mode, and obtains the fingerprint spectrum of Gu Han Yang Sheng Jing oral liquid, wherein the fingerprint spectrum of Gu Han Yang Sheng Jing oral liquid has 20 to 50 common peaks, among which the peak shapes of 24 common peaks are better; by comparing the retention time of the fingerprint spectrum of Gu Han Yang Sheng Jing oral liquid with the liquid phase spectrum of the reference substance, the characteristic peaks of the fingerprint spectrum of Gu Han Yang Sheng Jing oral liquid are obtained, among which peak 6 is paeoniflorin, peak 7 is calycosin isoflavone glucoside, peak 8 is liquiritin, peak 10 is teruncifloside, peak 18 is epimedin A, peak 19 is epimedin B, peak 20 is epimedin C, peak 22 is icariin, and peak 23 is ginsenoside Rb1.

[0053] In the present invention, the liquid phase spectrum of the test sample is preferably calculated by the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software to establish the fingerprint spectrum of Gu Han Health Essence Oral Liquid. In the present invention, the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software is preferably "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System Software (2012 Edition)". The present invention preferably uses the median method for similarity analysis, the time window width is preferably set to 0.1min, and peak matching is performed by multi-point correction, and a total of 24 common peaks are calibrated. In an embodiment of the present invention, 10 batches of Gu Han Health Essence Oral Liquid are specifically subjected to high performance liquid chromatography analysis, and the obtained test sample liquid phase spectrum is introduced into the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System for similarity analysis. The similarity of each batch of Gu Han Health Essence Oral Liquid is ≥0.993, specifically between 0.993 and 0.999.

[0054] The present invention also provides a method for detecting multiple index components of Gu Han Health Essence Oral Liquid, comprising the following steps:

[0055] Mixing the Gu Han Health Essence oral liquid to be tested with an extractant to obtain a sample liquid to be tested; the extractant includes a lower alcohol;

[0056] The sample solution is subjected to high performance liquid chromatography to obtain a multi-index component detection result of the Gu Han Health Essence Oral Liquid; the multi-index components include at least two of ginsenoside Rb1, calycosin isoflavone glucoside, privet glycoside, epimedin A, epimedin B, epimedin C, icariin, paeoniflorin and liquiritin;

[0057] The conditions for high performance liquid chromatography detection include: a mobile phase system including mobile phase A and mobile phase B, wherein the mobile phase A is acetonitrile, and the mobile phase B is a formic acid aqueous solution, wherein the volume fraction of formic acid in the formic acid aqueous solution is 0.05-0.2%; an elution mode is gradient elution, and the gradient elution program includes: from 0 to 18 minutes, the volume fraction of the mobile phase A linearly increases from 5% to 20%; from 18 to 33 minutes, the volume fraction of the mobile phase A is 20%; from 33 to 43 minutes, the volume fraction of the mobile phase A linearly increases from 20% to 23%; and from 43 to 65 minutes, the volume fraction of the mobile phase A linearly increases from 23% to 45%.

[0058] In the present invention, the preparation method of the sample liquid to be tested is preferably the same as the preparation method of the test solution; the conditions of the high performance liquid chromatography detection are the same as the conditions of the high performance liquid chromatography detection in the fingerprint spectrum construction method of the Biaoguhan Health Essence Oral Liquid, and will not be repeated here.

[0059] In the present invention, the multi-index components preferably include 2, 3, 4, 5, 6, 7, 8 or 9 of ginsenoside Rb1, calycosin glucoside, privet glycoside, epimedin A, epimedin B, epimedin C, icariin, paeoniflorin and liquiritin. In the present invention, the multi-index components can also include at least two of the monarch drug components, ministerial drug components, adjuvant drug components and envoy drug components, which can be specifically 2, 3 or 4 types; the monarch drug component includes ginsenoside Rb1 and / or calycosin isoflavone glucoside; the ministerial drug component includes privet glycoside and / or epimedium component, the epimedium component includes at least one of epimedin A, epimedin B, epimedin C and icariin, which can be specifically 1, 2, 3, 4, 4 or 5 types; the adjuvant component includes paeoniflorin; the envoy drug component includes liquiritin.

[0060] In the present invention, the test results preferably include quantitative test results and / or qualitative test results; the qualitative test results are obtained by comparing the liquid phase spectrum of the sample liquid to be tested with the fingerprint spectrum of Gu Han Health Essence Oral Liquid obtained by the fingerprint spectrum construction method described in the above technical solution.

[0061] The present invention has no particular limitation on the method for obtaining the quantitative detection results, and a quantitative method of high performance liquid chromatography detection well known to those skilled in the art may be used, such as a standard curve method.

[0062] To further illustrate the present invention, the fingerprint of the Gu Han Health Essence Oral Liquid, its construction method, and the multi-index component detection method provided by the present invention are described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present invention.

[0063] In the following examples, the instruments and materials are as follows:

[0064] Instruments: U3000 Series high performance liquid chromatograph (Thermo Fisher Scientific, USA); Cubis II laboratory ultramicrobalance (Sartorius, Germany); KQ-300DE digitally controlled ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

[0065] Chemometrics of (multidimensional) fingerprints: Select wavelengths and detectors to obtain a certain number of signal peaks while ensuring resolution. The retention time and peak area of ​​each chemical component are collected for similarity evaluation and principal component analysis. Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines; Thermo Fisher Scientific (U3000 Series, USA).

[0066] Reference substances: salidroside (lot number: PRF23082101), paeoniflorin (lot number: PRF23041743), calycoside (lot number: PRF23031601), liquiritin (lot number: PRF22051342), hyperoside (lot number: PRF22062401), teruncleside (lot number: PRF23103022), glycyrrhizic acid (lot number: PRF23112701), epimedin A (lot number: PRF24013141), epimedin The reference substances of Huoding B (batch number: PRF24090641) and Chaohuoding C (batch number: PRF24121103) were purchased from Chengdu Purifa Technology Development Co., Ltd.; the reference substances of ginsenoside Rg1 (batch number: 23070712), ginsenoside Re (batch number: 23071802), icariin (batch number: 23011004), ginsenoside Rb1 (batch number: 21101405), and astragaloside IV (batch number: 23052303) were purchased from Chengdu Pufeide Biotechnology Co., Ltd.

[0067] Solvents: Methanol, acetonitrile, and formic acid were all of chromatographic grade; water was ultrapure water.

[0068] Test samples: 10 batches of Gu Han Health Essence Oral Liquid (batch number 23064 was recorded as S1, batch number 23067 was recorded as S2, batch number 23070 was recorded as S3, batch number 23072 was recorded as S4, batch number 23074 was recorded as S5, batch number 23077 was recorded as S6, batch number 23080 was recorded as S7, batch number 23083 was recorded as S8, batch number 23086 was recorded as S9, batch number 23088 was recorded as S10, Gu Han Chinese Medicine Co., Ltd.

[0069] HPLC detection conditions: chromatographic column: Agilent Eclipse Plus C18 (4.6 mm × 250 mm, 5 μm); mobile phase A: acetonitrile, mobile phase B: 0.1 v / v% formic acid in water; gradient elution program: see Table 1; column temperature: 30°C; mobile phase flow rate: 1 mL / min; injection volume: 10 μL; detection wavelength: 204 nm.

[0070] Example 1

[0071] Preparation of test samples and screening of HPLC detection conditions

[0072] 1. Screening of detection wavelength

[0073] Gu Han Yang Sheng Jing (Gu Han Yang Sheng Jing) is composed of 11 herbs. In terms of its efficacy in "tonifying Qi, nourishing the kidneys, and improving essence," in addition to Epimedium, the Qi-tonifying herbs ginseng and astragalus are the main ingredients. This is because the main components of ginseng and astragalus are terminally absorbed triterpenoid saponins.

[0074] The test results of the index components of the traditional Chinese medicines related to Gu Han Yang Sheng Jing are shown in Table 2. It can be seen that ginseng, astragalus and licorice mainly contain saponins, whose structures do not have or have weak UV absorption; Rosa laevigata, Lycium barbarum and Polygonatum sibiricum mainly contain polysaccharides, which often need to be hydrolyzed before determination; Ligustrum lucidum, Cuscuta australis and Epimedium mainly contain flavonoids, which have strong UV absorption. Paeoniflorin, calycosin glucoside, liquiritin, terglucosidin, epimedin A, epimedin B, epimedin C, icariin and ginsenoside Rb1

[0075] Table 2 Indicative components of traditional Chinese medicines related to Gu Han Yang Sheng Jing

[0076]

[0077] Note: UV-Vis refers to ultraviolet-visible spectrophotometer.

[0078] Samples of Gu Han Yang Sheng Jing oral liquid were taken for HPLC detection at the maximum absorption wavelengths of medicinal materials such as ginseng, white peony root, and epimedium. It was found that short wavelength analysis could obtain more compound absorption peak information and the absorption value of the compound was larger; at the same time, substances that can produce signal responses at 220nm, 270nm, and 360nm can still obtain larger absorption values ​​at 204nm, so 204nm was selected for the establishment and analysis of the Gu Han Yang Sheng Jing fingerprint.

[0079] 2. Screening of chromatographic columns

[0080] The Gu Han Health Essence Oral Liquid was tested by HPLC using Agilent Eclipse Plus C18, Agilent TC-C18, ZORBAX 300SB-C18 and ZORBAX SB-C18, all with specifications of 4.6 mm × 250 mm, 5 μm. The results are shown in Figure 1 As can be seen, at a wavelength of 204 nm, the chemical components of Gu Han Yang Sheng Jing Oral Liquid were largely eluted and peaked within 0 to 30 minutes. Based on the peak resolution and compound retention, the Agilent Eclipse Plus C18 was initially selected for subsequent experiments.

[0081] 3. Screening of mobile phase

[0082] The Gu Han Health Essence Oral Liquid was tested by HPLC under different mobile phase systems (acetonitrile-water, methanol-0.1 v / v% formic acid aqueous solution, acetonitrile-0.1 v / v% formic acid aqueous solution). The results are shown in Figure 2 It can be seen that the chromatographic information obtained under the acetonitrile-0.1v / v% formic acid aqueous solution mobile phase system is significantly more than that under the methanol-0.1v / v% formic acid aqueous solution and acetonitrile-water mobile phase systems. Therefore, selecting acetonitrile-0.1v / v% formic acid aqueous solution as the mobile phase system has the best chromatographic peak separation effect.

[0083] 4. Selection of fingerprint wavelength

[0084] In order to obtain as much UV absorption information as possible, it is necessary to determine the optimal wavelength for subsequent experiments. Figure 3 ), the experiment selected data at wavelengths of 204nm, 220nm, 270nm and 360nm for comparison.

[0085] like Figure 3 As shown, selecting a short wavelength for analysis yields more compound absorption peak information and greater absorption values. Furthermore, substances that generate signal responses at 270nm and 360nm also exhibit significant absorption values ​​at 204nm and 220nm. Compared to 270nm and 360nm, 204nm and 220nm are more suitable for establishing and analyzing the fingerprint of Gu Han Yang Sheng Jing.

[0086] 5. Screening of test solution preparation conditions

[0087] Preparation of the test solution: Accurately pipette 1 mL of Gu Han Health Essence Oral Liquid into a 15 mL centrifuge tube, add 7 mL of the extractant, and ultrasonically treat at 50°C and 300W for 30 minutes. Cool to room temperature to obtain the test solution. Different concentrations of methanol (50 v / v% methanol-water, 60 v / v% methanol-water, 70 v / v% methanol-water, 80 v / v% methanol-water, 90 v / v% methanol-water, and anhydrous), acetonitrile, and water were used as extractants. It was found that when high concentration methanol (80-100 v / v% methanol-water) was used as the extractant, a brown precipitate formed at the bottom of the extract after cooling to room temperature, and the content of each indicator compound was not much different.

[0088] 6. Specificity test of ingredients in Gu Han Health Essence Oral Liquid

[0089] Specificity / selectivity can be verified by demonstrating that the identification and / or quantification of the analyte is not affected by interference from other coexisting substances such as impurities, degradation products, related substances, matrix, or other components present in the operating environment. For example, when using a negative sample (a simulated formulation sample without the analyte or without the analyte), a negative sample without the analyte should be tested under the same conditions as the test solution and compared to confirm the presence of interference.

[0090] Preparation of test solution: Accurately pipette 1 mL of Gu Han Health Essence Oral Liquid (S1-S10) into a 15 mL centrifuge tube, add 7 mL of methanol, ultrasonically treat at 50°C and 300 W for 30 min, cool to room temperature, add methanol to the scale, filter with a 0.22 μm filter membrane, and the filtrate is the test solution.

[0091] Negative samples: Place 1 mL of Gu Han Yang Sheng Jing oral liquid lacking certain medicinal ingredients in a 15 mL centrifuge tube, add 7 mL of methanol, and ultrasonically treat at 50°C and 300W for 30 minutes. Cool to room temperature, add methanol to the mark, and filter through a 0.22 μm filter. The filtrate is a negative sample. The Gu Han Yang Sheng Jing oral liquid lacking certain medicinal ingredients is Gu Han Yang Sheng Jing oral liquid lacking ginseng, roasted astragalus root, Rosa laevigata fruit, wolfberry fruit, processed Ligustrum lucidum fruit, Cuscuta australis seed, and Epimedium.

[0092] Preparation of mixed reference solution: Accurately weigh salidroside, paeoniflorin, calycosin glucoside, liquiritin, hyperoside, privet glycoside, ginsenoside Rg1, ginsenoside Re, epimedium A, epimedium B, epimedium C, icariin, ginsenoside Rb1, astragaloside IV, and glycyrrhizic acid reference substances, respectively, dissolve them in methanol and make up to volume to obtain mother solutions of each reference substance with a concentration of 1 mg / mL. Accurately measure 10 μL of each mother solution of each reference substance and place it in the same volumetric flask, add 50 μL of methanol and mix evenly to obtain a mixed reference solution with a concentration of 0.05 mg / mL for each reference substance.

[0093] Each solution was tested by HPLC. The results are shown in Figure 4 and Table 3, Figure 4 In the table, A is a mixed control, C is lacking Ligustrum lucidum fruit, D is lacking white peony root, E is lacking Astragalus root, F is lacking Glycyrrhiza uralensis, G is lacking Cuscuta seed, H is lacking Panax ginseng, and I is lacking Epimedium.

[0094] Depend on Figure 4 As shown in Table 3, paeoniflorin, calycosin glucoside, liquiritin, privet glycoside, epimedin A, epimedin B, epimedin C, icariin and ginsenoside Rb1 had no interference. Therefore, the above control compounds were selected as the control substances for the detection of Gu Han Health Essence Oral Liquid.

[0095] Table 3 Retention time and interference of components

[0096]

[0097]

[0098] Example 2

[0099] 1. Constructing fingerprint

[0100] Preparation of test solution: Accurately pipette 1 mL of Gu Han Yang Sheng Jing Oral Liquid into a 15 mL centrifuge tube. Add 7 mL of methanol and sonicate at 50°C, 300W for 30 min. Cool to room temperature, add methanol to the mark, and filter through a 0.22 μm filter. The filtrate is the test solution. A total of 10 batches of Gu Han Yang Sheng Jing Oral Liquid test solutions were prepared.

[0101] Preparation of mixed reference solution: Accurately weigh paeoniflorin, calycosin glucoside, liquiritin, terunoside, epimedin A, epimedin B, epimedin C, icariin, and ginsenoside Rb1 reference substances, add methanol to dissolve them, and obtain reference substance mother solutions with a concentration of 1 mg / mL. Accurately measure 10 μL of each reference substance mother solution and place it in the same volumetric flask, add methanol for quantitative dilution, and shake well to obtain a mixed reference substance solution with a concentration of 0.05 mg / mL for each reference substance.

[0102] The mixed reference solution was subjected to HPLC detection to obtain the reference liquid phase spectrum. Figure 5 .

[0103] Ten batches of test sample solutions were tested by HPLC, and the test sample liquid phase spectra were recorded. The test sample liquid phase spectra were imported into the Chinese Pharmacopoeia Committee's "Chinese Herbal Chromatographic Fingerprint Similarity Software Evaluation System (2012 Edition)". Sample S3 was used as a reference spectrum, and the median method was used with a time window width of 0.1 min. Peak matching was performed through multi-point calibration to generate the overlay of the fingerprint spectrum of Gu Han Yang Sheng Jing Oral Liquid and its reference fingerprint spectrum (R). The results are shown in Figure 6A total of 24 common peaks were calibrated and compared with the reference liquid phase spectrum ( Figure 5 ) were compared, and peaks 6-8, 10, 18-20, 22, and 23 were determined to be paeoniflorin, calycosin glucoside, liquiritin, terglucosidin, epimedin A, epimedin B, epimedin C, icariin, and ginsenoside Rb1, respectively. The similarities between the fingerprints of 10 batches of Gu Han Health Essence Oral Liquid samples (S1-S10) and their control fingerprints (S, R) were 0.995, 0.996, 0.997, 0.993, 0.999, 0.996, 0.999, 0.998, 0.997, and 0.998, respectively. Figures 4-6 It can be seen that chromatographic peaks 4, 14, 15, and 23 come from ginseng; chromatographic peak 7 comes from astragalus; chromatographic peaks 17 to 22 come from epimedium; chromatographic peaks 4 and 10 come from Ligustrum lucidum fruit; chromatographic peak 9 comes from Cuscuta chinensis; chromatographic peaks 8 and 24 come from liquorice; and chromatographic peak 6 comes from white peony root.

[0104] 2. Chemometric Analysis

[0105] 2.1 Principal Component Analysis (PCA)

[0106] The peak areas of 24 common peaks in 10 batches of Gu Han Health Essence Oral Liquid were used as raw data and imported into SIMCA 14.1 software for CA. The results are shown in Figure 7 The 10 batches of Gu Han Yang Sheng Jing oral liquid can be divided into two groups, samples S1 to S4 as one group, and samples S5 to S10 as one group. The peak areas of the 24 common peaks in the 10 batches of Gu Han Yang Sheng Jing oral liquid were imported into SPSS 20.0 software, the data were standardized, and PCA was performed. The results are shown in Tables 4 and 5. A total of 6 principal components were extracted, with a cumulative variance contribution rate of 91.029%. Therefore, these 6 principal components can basically reflect most of the information of different batches of Gu Han Yang Sheng Jing oral liquid. According to the analysis of the component matrix results after rotation, based on the absolute value of the loading exceeding 0.8, principal component 1 reflects the information of peaks 3 and 19 (epimedin B), principal component 2 reflects the information of peaks 5 and 6 (peoniflorin), principal component 3 reflects the information of peaks 20 (epimedin C), 22 (icariin), and 23 (ginsenoside Rb1), principal component 4 reflects the information of peaks 11 and 16, and principal component 6 reflects the information of peak 15. It shows that the quality of Gu Han Yangsheng Jing oral liquid is affected by multiple ingredients, such as ginseng, epimedium and white peony root.

[0107] Table 4 PCA eigenvalues ​​and variance contribution rates of Gu Han Health Essence Oral Liquid

[0108] Element Eigenvalue Variance contribution rate / % Cumulative variance contribution rate / % 1 6.119 25.494 25.494 2 5.557 23.156 48.650 3 3.718 15.491 64.141 4 2.623 10.929 75.070 5 1.979 8.246 83.316 6 1.851 7.712 91.029

[0109] Table 5 PCA rotation factor loading matrix of Gu Han Health Essence Oral Liquid

[0110]

[0111]

[0112] 2.2 Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA)

[0113] The 24 common peak areas in the fingerprints of 10 batches of Gu Han Health Essence Oral Liquid were imported into SIMCA14.1 software, and the model was established in the supervised mode to obtain the OPLS-DA graph. Figure 8 The results showed that the 10 batches of Gu Han Health Essence Oral Liquid could be divided into two groups, S1 to S4 as one group and S5 to S10 as the other group, which was consistent with the PCA results. Figure 9 After all random permutations, the R2 and Q2 of the right model are higher than those on the left, and the slopes of the R2 and Q2 regression lines are positive, indicating that the model does not overfit. The variable importance projection diagram can be used to determine the influence and explanatory power of each characteristic peak on the sample classification and discriminant analysis, which is beneficial for the screening of quality difference markers. Figure 10 Screening of ingredients using a VIP value > 1 revealed six common peaks meeting this requirement. The order of differential component peaks was 11, 2, 4, 19 (epimedium B), 20 (epimedium C), and 22 (icariin). These peaks were primarily attributed to Epimedium, Panax ginseng, and Ligustrum lucidum fruit. Therefore, the quality differences among these three ingredients are key factors affecting the quality stability of Gu Han Yangsheng Jing Oral Liquid.

[0114] The present invention adopts HPLC method to establish the fingerprint of Gu Han Yang Sheng Jing oral liquid, and performs multi-pattern recognition and content determination of 9 components on it. It also performs chemometric analysis by principal component analysis, orthogonal partial least squares discriminant analysis, etc. to determine the content of multiple index components of Gu Han Yang Sheng Jing oral liquid.

[0115] The present invention uses high-performance liquid chromatography combined with multivariate statistical analysis technology to establish its fingerprint and simultaneously determine the contents of nine key active ingredients. This method can systematically characterize the overall chemical characteristics of the compound and, combined with pattern recognition technology, conduct consistency evaluation of different batches of samples, thereby providing a more comprehensive and reliable reference basis for the quality control of Gu Han Health Essence Oral Liquid and promoting its standardized production and clinical application.

[0116] Example 3

[0117] Methodological validation

[0118] 1. Precision test

[0119] The test solution (S4) was prepared according to Example 2 and tested by HPLC six times. Chromatographic peak No. 12 (icariin) was selected as the reference peak. The RSDs of the relative retention times of the common peaks were all <0.30%, and the RSDs of the relative peak areas were all <3.92%, indicating that the instrument had good precision.

[0120] 2. Stability test

[0121] The test solution (S4) was prepared according to Example 2, and HPLC detection was performed at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h after preparation. Chromatographic peak No. 12 (icariin) was used as the reference peak. The RSD of the relative retention time of each common peak was <0.23%, and the RSD of the relative peak area was <3.70%, indicating that the test solution had good stability within 24 h.

[0122] 3. Repeatability test

[0123] Six test solutions (S4) were prepared in parallel according to Example 2 and subjected to HPLC detection. Using chromatographic peak No. 12 (icariin) as the reference peak, the RSDs of the relative retention times of the common peaks were <0.25%, and the RSDs of the relative peak areas were <3.76%, indicating that the method had good reproducibility.

[0124] Example 4

[0125] Determination of the content of multiple components in Guhan Yangshengjing oral liquid

[0126] 1. Solution Preparation

[0127] Preparation of mixed reference solution: Accurately weigh paeoniflorin, calycosin glucoside, liquiritin, privet glycoside, epimedin A, epimedin B, epimedin C, icariin, and ginsenoside Rb1 reference substances, and add methanol to prepare a reference substance stock solution with a concentration of 1 mg / mL. Accurately measure 100 μL each of the reference stock solutions of calycosin isoflavone glucoside, liquiritin, epimedin A, epimedin B, epimedin C, and ginsenoside Rb1, 110 μL of the reference stock solution of peonyside, 60 μL of the privet glycoside reference stock solution, and 130 μL of the icariin reference stock solution, and add 100 μL of methanol to prepare a mixed reference solution containing 0.1 mg / mL each of calycosin isoflavone glucoside, liquiritin, epimedin A, epimedin B, epimedin C, and ginsenoside Rb1 reference solutions, 0.11 mg / mL of peonyside reference solution, 0.06 mg / mL of privet glycoside reference solution, and 0.13 mg / mL of icariin reference solution.

[0128] Preparation of test solution: same as in Example 2.

[0129] The detection wavelengths are 204nm, 220nm, and 270nm respectively.

[0130] 2. Linear relationship investigation

[0131] Accurately pipette each reference substance mother solution into the same volumetric flask. Add methanol to dilute each component at 1x, 2x, 3.3x, 5x, 10x, 20x, 33x, 50x, and 100x dilutions to create linear mixed reference substance solutions of varying mass concentrations. HPLC analysis was performed separately, and the peak area of ​​each component was determined. A standard curve was plotted with reference substance mass concentration as the abscissa (x) and peak area as the ordinate (y). The linear regression equation was calculated. The results are shown in Table 6.

[0132] Table 6 Linearity of 9 chemical components in Gu Han Yang Sheng Jing Oral Liquid

[0133]

[0134]

[0135] 3. Precision test

[0136] Each reference substance mother solution was sampled and placed in the same volumetric flask, diluted with methanol and fixed to volume to obtain a mixed reference substance solution with a concentration of 50 ppm. The mixed reference substance solution was repeatedly subjected to HPLC detection six times, and the RSDs of the peak areas of paeoniflorin, calycosin glucoside, liquiritin, terunoside, epimedin A, epimedin B, epimedin C, icariin, and ginsenoside Rb1 were 1.11%, 0.76%, 0.61%, 2.23%, 1.32%, 1.26%, 0.86%, 0.96%, and 2.87%, respectively. The results indicate that the instrument has good precision.

[0137] 4. Stability test

[0138] The test solution (S4) was prepared according to Example 2, and HPLC detection was performed at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h after preparation. The RSDs of the peak areas of paeoniflorin, calycosin glucoside, liquiritin, epimedin A, epimedin B, epimedin C, icariin, and ginsenoside Rb1 were 1.40%, 3.19%, 2.73%, 2.53%, 0.77%, 1.22%, 1.33%, 1.12%, and 2.69%, respectively, indicating that the test solution had good stability within 24 h.

[0139] 5. Repeatability test

[0140] According to Example 2, 6 test solutions (S4) were prepared in parallel and subjected to HPLC detection respectively. The RSDs of the peak areas of paeoniflorin, calycosin glucoside, liquiritin, terunoside, epimedin A, epimedin B, epimedin C, icariin and ginsenoside Rb1 were 2.12%, 2.54%, 2.16%, 1.30%, 2.95%, 2.52%, 2.62%, 2.47% and 3.71%, respectively, indicating that the method has good reproducibility.

[0141] 6. Sample recovery test

[0142] Take 6 portions of the measured Gu Han Health Essence Oral Liquid sample (S4), accurately aspirate 1 mL, and accurately add the same content of paeoniflorin, calycosin glucoside, liquiritin, epimedin A, epimedin B, epimedin C, icariin, and ginsenoside Rb1 as the sample's mother solution, place in a 15 mL centrifuge tube, add 7 mL of methanol, ultrasonically treat at 50 ° C and 300 W for 30 min, cool to room temperature, add methanol to the scale, filter with 0.22 μm filter membrane, and the filtrate is the spiked test solution (the spiked concentration is the same as the sample concentration).

[0143] The spiked test solutions were subjected to HPLC detection. The average recoveries of paeoniflorin, calycosin glucoside, liquiritin, teruncinol, epimedin A, epimedin B, epimedin C, icariin, and ginsenoside Rb1 were 102.07%, 101.54%, 98.91%, 103.27%, 104.57%, 102.16%, 103.49%, 102.63%, and 100.38%, respectively. The RSDs were 1.08%, 0.93%, 0.31%, 1.48%, 1.79%, 1.96%, 1.76%, 0.50%, and 0.26%, respectively.

[0144] 7. Sample Content Determination

[0145] Ten batches of Gu Han Health Essence Oral Liquid samples (S1-S10) were taken to prepare test solutions according to Example 2, and HPLC detection was performed respectively to calculate the contents of the above 9 components in the samples. The results are shown in Table 7.

[0146] Table 7 Determination results of the contents of 9 chemical components in Gu Han Yang Sheng Jing oral liquid (μg / mL)

[0147]

[0148] Discussion and Conclusion: Regarding the preparation of the test solution and the selection of chromatographic conditions, various concentrations of methanol, acetonitrile, and water were initially used as extraction solvents. High methanol concentrations resulted in a brown precipitate at the bottom of the extract after cooling to room temperature, with similar concentrations of the various index compounds. Given the high concentration of carbohydrates in the Gu Han Health Essence Oral Liquid samples, the precipitate was hypothesized to be a non-UV-absorbing carbohydrate. Mobile phases tested included acetonitrile-0.1 v / v% formic acid in water, methanol-0.1 v / v% formic acid in water, acetonitrile-water, and methanol-water. Acetonitrile-0.1 v / v% formic acid in water yielded the best separation of the chromatographic peaks.

[0149] Fingerprint and multi-pattern recognition analysis: Fingerprints for 10 batches of Gu Han Yang Sheng Jing oral liquid were generated, demonstrating high similarity, indicating good overall quality consistency. Furthermore, comprehensive analysis was conducted using chemical pattern recognition techniques such as PCA and OPLS-DA. The results of these two methods complemented and validated each other. The OPLS-DA results, combined with the PCA component matrix, revealed that six components had a VIP value greater than 1, among which epimedium components epimedium B, epimedium C, and icariin were among the most prominent, representing important indicators for evaluating the quality of traditional Chinese medicine compounds. Principal component analysis revealed that the 10 batches of Gu Han Yang Sheng Jing oral liquid fell into two categories, indicating differences between samples from different batches, presumably due to differences in raw material quality and batch-to-batch quality.

[0150] Regarding the selection of indicator ingredients, Gu Han Yang Sheng Jing oral liquid is composed of 11 traditional Chinese medicinal herbs, including ginseng, roasted astragalus root, processed Ligustrum lucidum fruit, and epimedium. It contains saponins, flavonoids, and polysaccharides. Ginsenoside Rb1 is the primary active ingredient in Gu Han Yang Sheng Jing oral liquid, exhibiting neuroprotective effects such as antioxidant, anti-inflammatory, and anti-apoptotic properties. Isoflavone glucosides in astragalus root have antioxidant, antiviral, and immunosuppressive properties. Astragaloside IV can enhance immunity and regulate immune balance, but it is difficult to detect using UV detectors and was therefore not included as an indicator. The ingredients in epimedium, epimedium B, epimedium C, and icariin, are all flavonoids and show the highest variance in OPLS-DA analysis. They are widely used to treat a variety of conditions, including sexual dysfunction, osteoporosis, cancer, rheumatoid arthritis, and brain diseases. At the same time, paeoniflorin in the adjuvant drug white peony root and liquiritin in the envoy drug liquorice were included in the quantitative analysis. According to the principle of "monarch, minister, assistant and envoy", the index components for content determination were finally determined to be paeoniflorin, calycosin glucoside, liquiritin, privet glycoside, epimedin A, epimedin B, epimedin C, icariin and ginsenoside Rb1. A multi-component content determination method covering both pharmacological activity and characteristic components was established.

[0151] The HPLC fingerprint of Gu Han Yang Sheng Jing oral liquid established by the present invention calibrated 24 common peaks and identified 9 components. The content of 9 of these components was determined, and each component showed good linearity within a certain concentration range. Samples were clustered into two categories, with epimedin B, epimedin C, and icariin as the main differential components, which can reflect the quality fluctuations between batches of Gu Han Yang Sheng Jing oral liquid. The fingerprint of Gu Han Yang Sheng Jing oral liquid and the multi-index component detection method provided by the present invention are stable and can provide a basis for the quality evaluation, quality control, and clinical application of Gu Han Yang Sheng Jing oral liquid.

[0152] The present invention uses HPLC-UV instead of UPLC-MS. The method has strong universality, can capture components with no UV absorption or low response (such as some saponins), and has lower detection costs.

[0153] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for constructing a fingerprint spectrum of Gu Han Health Essence Oral Liquid, characterized in that: The following steps are involved: Mixing the Guhan Health Essence Oral Liquid with an extractant to obtain a test solution; the extractant includes a lower alcohol or a lower alcohol aqueous solution; The test solution and the mixed reference solution are respectively subjected to high performance liquid chromatography to obtain a test sample liquid phase spectrum and a reference liquid phase spectrum, respectively; and a fingerprint spectrum of Gu Han Yang Sheng Jing oral liquid is obtained based on the test sample liquid phase spectrum and the reference liquid phase spectrum; the characteristic peaks of the fingerprint spectrum of Gu Han Yang Sheng Jing oral liquid include characteristic peaks of paeoniflorin, calycosin isoflavone glucoside, liquiritin, privet glycoside, epimedium components and ginsenoside Rb1; the epimedium components include at least one of epimedin A, epimedin B, epimedin C and icariin; The conditions for high performance liquid chromatography detection include: a mobile phase system including mobile phase A and mobile phase B, wherein the mobile phase A is acetonitrile, and the mobile phase B is a formic acid aqueous solution, wherein the volume fraction of formic acid in the formic acid aqueous solution is 0.05-0.2%; an elution mode is gradient elution, and the gradient elution program includes: from 0 to 18 minutes, the volume fraction of the mobile phase A linearly increases from 5% to 20%; from 18 to 33 minutes, the volume fraction of the mobile phase A is 20%; from 33 to 43 minutes, the volume fraction of the mobile phase A linearly increases from 20% to 23%; and from 43 to 65 minutes, the volume fraction of the mobile phase A linearly increases from 23% to 45%.

2. The fingerprint construction method according to claim 1, characterized in that: The volume ratio of the Gu Han Health Essence Oral Liquid to the extractant is 1:5-9; The volume fraction of the lower alcohol in the extractant is 50-100%, and the lower alcohol includes methanol.

3. The fingerprint construction method according to claim 1 or 2, characterized in that: The mixing includes ultrasonic mixing; the power of the ultrasonic mixing is 100 to 500 W, and the time is 25 to 35 minutes.

4. The fingerprint construction method according to claim 1, characterized in that: The reference substances in the mixed reference solution include paeoniflorin, calycosin glucoside, liquiritin, privet glycoside, epimedium components and ginsenosides; the epimedium components include at least one of epimedin A, epimedin B, epimedin C and icariin; the concentration of a single reference substance in the mixed reference solution is 0.02 to 0.13 mg / mL; the solvent in the mixed reference solution includes lower alcohol or lower alcohol aqueous solution.

5. The fingerprint construction method according to claim 1, characterized in that: The chromatographic column used in the high performance liquid chromatography detection includes a C18 chromatographic column, a mobile phase flow rate of 0.9 to 1.1 mL / min, a detection wavelength of 203 to 270 nm, a column temperature of 25 to 40° C., and an injection volume of 5 to 15 μL.

6. A multi-index component detection method for Gu Han Health Essence Oral Liquid, characterized in that: The following steps are involved: Mixing the Gu Han Health Essence oral liquid to be tested with an extractant to obtain a sample liquid to be tested; the extractant includes a lower alcohol or a lower alcohol aqueous solution; The sample solution is subjected to high performance liquid chromatography to obtain a multi-index component detection result of the Gu Han Health Essence Oral Liquid; the multi-index components include at least two of ginsenoside Rb1, calycosin isoflavone glucoside, privet glycoside, epimedin A, epimedin B, epimedin C, icariin, paeoniflorin and liquiritin; The conditions for high performance liquid chromatography detection include: a mobile phase system including mobile phase A and mobile phase B, wherein the mobile phase A is acetonitrile, and the mobile phase B is a formic acid aqueous solution, wherein the volume fraction of formic acid in the formic acid aqueous solution is 0.05-0.2%; an elution mode is gradient elution, and the gradient elution program includes: from 0 to 18 minutes, the volume fraction of the mobile phase A linearly increases from 5% to 20%; from 18 to 33 minutes, the volume fraction of the mobile phase A is 20%; from 33 to 43 minutes, the volume fraction of the mobile phase A linearly increases from 20% to 23%; and from 43 to 65 minutes, the volume fraction of the mobile phase A linearly increases from 23% to 45%.

7. The multi-index component detection method according to claim 6, characterized in that: The volume ratio of the tested Gu Han Health Essence oral solution to the extractant is 1:5-9; The volume fraction of the lower alcohol in the extractant is 50-100%, and the lower alcohol includes methanol.

8. The multi-index component detection method according to claim 6 or 7, characterized in that: The mixing includes ultrasonic mixing; the power of the ultrasonic mixing is 100 to 500 W, and the time is 25 to 35 minutes.

9. The multi-index component detection method according to claim 6, characterized in that: The chromatographic column used in the high performance liquid chromatography detection includes a C18 chromatographic column, a mobile phase flow rate of 0.9 to 1.1 mL / min, a detection wavelength of 203 to 270 nm, a column temperature of 25 to 40° C., and an injection volume of 5 to 15 μL.

10. The multi-index component detection method according to claim 6 or 9, characterized in that: The test results include quantitative test results and / or qualitative test results; the qualitative test results are obtained by comparing the liquid phase spectrum of the sample liquid to be tested with the fingerprint spectrum of Gu Han Health Essence Oral Liquid obtained by the fingerprint spectrum construction method according to any one of claims 1 to 5.

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