A method for detecting pharmacodynamic components of a traditional Chinese medicine composition by UPLC-MS / MS

Abstract

The application provides a method for detecting the pharmacodynamic components of a traditional Chinese medicine composition by UPLC-MS / MS. The method can identify 14 pharmacodynamic components of the traditional Chinese medicine composition, including saikosaponin A, saikosaponin D, naringin, paeoniflorin, hesperidin, chicoric acid, chlorogenic acid, salvianolic acid B, zephyranthine, kaempferitrin, and codariusrhizome glycoside, amargin, hyperoside and rutin, thereby providing a reference for evaluating the intrinsic quality of the traditional Chinese medicine composition.

Description

Technical Field

[0001] This invention relates to a method for detecting the active ingredients in a traditional Chinese medicine composition using UPLC-MS / MS, belonging to the field of methods for detecting traditional Chinese medicine ingredients. Background Technology

[0002] The traditional Chinese medicine composition described in this invention is Ganshuang Granules, produced and marketed by Baoding Tianhao Pharmaceutical Co., Ltd. This medicine is prepared from 13 medicinal materials: Codonopsis pilosula, Bupleurum chinense, Paeonia lactiflora, Angelica sinensis, Poria cocos, Atractylodes macrocephala, Citrus aurantium, Taraxacum mongolicum, Polygonum cuspidatum, Prunella vulgaris, Salvia miltiorrhiza, Prunus persica, and Carapax Trionycis. In recent years, studies have found that it has a significant anti-liver fibrosis effect; however, current research is mostly focused on clinical applications, with limited research on its components, and the active substances responsible for its anti-liver fibrosis effect remain unclear. Modern research shows that this is closely related to the drug's anti-liver fibrosis effect. As an oral preparation of traditional Chinese medicine, Ganshuang Granules inevitably interact with the intestinal flora after administration, and this interaction affects the structure of the medicinal components and their corresponding efficacy.

[0003] This article systematically reviews existing technical methods for detecting this traditional Chinese medicine composition. It reveals that in quality standard research, Han Dong established a fingerprint spectrum for Gan Shuang granules, identifying 10 components (ferulic acid, polygalactoside, chicoric acid, naringin, hesperidin, rosmarinic acid, neohesperidin, salvianolic acid B, emodin, and emodin methyl ether) in 18 batches of samples; Wang Guofeng identified 8 components (rosmarinic acid, ferulic acid, paeoniflorin, polygalactoside, naringin, neohesperidin, salvianolic acid B, emodin, and emodin methyl ether) in 14 batches of samples. Phenolic acid B and tanshinone IIA were used to identify and classify large amounts of sample data. Han Dong's cluster analysis and principal component analysis (PCA) divided 18 batches of Gan Shuang granules into three categories: batches of similar production years were grouped into one category each. Wang Guofeng clustered 14 batches of samples into two categories. Han Dong used orthogonal partial least squares-discriminant analysis (OPLS-DA) to screen for chicoric acid, neohesperidin, ferulic acid, naringin, and rosmarinic acid as differential markers for Gan Shuang granules. Wang Guofeng identified phenolic acid B, tanshinone IIA, ferulic acid, rosmarinic acid, and paeoniflorin as differential markers for Gan Shuang granules. The presence of phenolic acid B, rosmarinic acid, and ferulic acid as the commonly identified differential components suggests that phenolic acid compounds may be the main differential substances in Gan Shuang granules. In the content determination study, Li Liang established a UHPLC method to determine the contents of paeoniflorin, paeoniflorin, naringin, polygalactoside, and tanshinone IIA in Ganshuang granules. The method validation was good and can provide a basis for the quality control of Ganshuang granules. Yang Wen established a high-performance liquid chromatography-quantitative analysis (HPLC-QAMS) method to determine polygalactoside, resveratrol, oxidized paeoniflorin, paeoniflorin, and saikosaponins a, b1, and d in Ganshuang granules. The results obtained by the quantitative analysis method for each component in Ganshuang granules showed no significant difference from the results of the external standard method. In the inorganic elemental analysis, it was found that the main inorganic elements in Ganshuang granules were K, Mg, Na, Ca, Al, Fe, and Mn. The limits of the five heavy metal elements Cu, As, Cd, Hg, and Pb met the relevant limits stipulated in the Chinese Pharmacopoeia. Based on fingerprint similarity, cluster analysis, and PCA results, the inorganic elements in Ganshuang granules exhibit good uniformity. The main characteristic elements are Mg, K, Ca, Mo, Tl, Cd, Se, and Zn, which can provide a reference for evaluating the uniformity and safety of Ganshuang granules.

[0004] The current standards for Gan Shuang Granules only use TLC for qualitative analysis of paeoniflorin, emodin, and angelica root, and HPLC for quantitative analysis of paeoniflorin. The quality standard controls less than one-third of the total number of medicinal materials in the prescription, making it difficult to reflect the overall characteristics and intrinsic quality of the preparation. Furthermore, current research on this drug mainly focuses on clinical trials, with limited research on the identification of its active ingredients and their quality standards. There is an urgent need for further research to clarify its active components. Therefore, developing a detection method capable of simultaneously detecting multiple intrinsic active ingredient markers is highly necessary. Summary of the Invention

[0005] This invention provides a method for detecting the pharmacodynamic components of a traditional Chinese medicine composition using UPLC-MS / MS. This method identifies 14 pharmacodynamic components in the traditional Chinese medicine composition of this invention, including saikosaponin A, saikosaponin D, naringin, paeoniflorin, hesperidin, chicoric acid, chlorogenic acid, salvianolic acid B, narcissin, kaempferol, codonopsis pilosula, amygdalin, hyperoside, and rutin. Methodological investigations have shown that the method is accurate, reliable, and stable, providing a new reference for evaluating the intrinsic quality of the traditional Chinese medicine of this invention.

[0006] The technical solution provided by this invention is as follows:

[0007] A method for detecting the active ingredients in a traditional Chinese medicine composition by UPLC-MS / MS, the method comprising the following steps:

[0008] (1) Preparation of mixed reference solution: Weigh out the reference standards of saikosaponin A, saikosaponin D, naringin, paeoniflorin, hesperidin, chicoric acid, chlorogenic acid, salvianolic acid B, narcissin, kaempferol, codonopsis pilosula, amygdalin, hyperoside, and rutin respectively, add methanol, dilute to the mark, shake well, and the solution is ready.

[0009] (2) Preparation of test solution: Take the Chinese herbal medicine composition, grind it into a fine powder, weigh it accurately, add methanol, sonicate, centrifuge, take the supernatant, filter it, and the test solution is obtained.

[0010] (3) Chromatographic conditions: Ultra-high performance liquid chromatograph, C 18 Chromatographic column; column temperature: 38–42℃; flow rate: 0.1–0.5 mL / min -1 Mobile phase: A: 0.1% formic acid in water; Mobile phase: B: acetonitrile; Gradient elution (0–5 min, 95–90% A; 5–10 min, 90%–65% A; 10–15 min, 65% A; 15–20 min, 65%–60% A; 20–25 min, 60–35% A; 25–25.1 min, 35%–95% A; 25.1–26 min, 95% A);

[0011] (4) Mass spectrometry conditions: Ionization mode: electrospray ionization; Ion source temperature: 150℃; Capillary voltage: 3.0kV; Cone gas flow rate: 50L / h; Desolvation gas temperature: 350℃; Desolvation gas flow rate: 1000L / h; Nebulizer gas pressure: 7×105Pa; Precursor ion scan range: m / z 80~1200;

[0012] Preferably, in step (1) of the detection method, the concentrations of saikosaponin A, saikosaponin D, naringin, paeoniflorin, hesperidin, chicoric acid, and chlorogenic acid in the test solution are 50–120 μg / mL, 150–120 μg / mL, 80–100 μg / mL, 50–70 μg / mL, 50–70 μg / mL, 100–125 μg / mL, and 100–125 μg / mL, respectively. The concentrations of the following compounds are: 170–190 μg / mL, 85–100 μg / mL of salvianolic acid B, 105–120 μg / mL of narcissin, 80–90 μg / mL of kaempferol, 90–115 μg / mL of codonopsis pilosula, 55–65 μg / mL of amygdalin, 60–70 μg / mL of hyperoside, and 60–70 μg / mL of rutin.

[0013] Preferably, in step (2) of the detection method, the ultrasonic treatment time is 20-40 min during the preparation of the test solution.

[0014] Preferably, in step (2) of the detection method, the centrifugation speed is 11000-14000 rpm and the centrifugation time is 8-12 min.

[0015] Preferably, the chromatographic detection conditions in step (3) of the detection method are as follows: the model of the ultra-high performance liquid chromatograph is: Waters Xevo G2-XS Q-TOF.

[0016] The chromatographic detection conditions for step (3) of the detection method are as follows: The C 18 The chromatographic column has a specification of 2.1×50mm and a diameter of 1.7μm.

[0017] The chromatographic detection conditions for step (3) of the detection method are as follows: column temperature: 40℃; flow rate: 0.3 mL / min. -1 .

[0018] Preferably, the main mass spectrometry parameters of the analyte in step (4) mass spectrometry conditions are as follows:

[0019]

[0020]

[0021] To demonstrate the innovativeness of the technical solution of this invention, we have summarized part of the experimental process for optimizing the chromatography of the drug efficacy detection components of this invention as follows:

[0022] 1.1 Investigation of the preparation method of the test solution

[0023] This experiment investigated the effects of different extraction solvents (DMSO, ultrapure water, methanol, and ethanol) and their solid-liquid ratios (1:60, 1:100, 1:150, and 1:200) on the extraction efficiency. The results showed that the methanol extract produced a greater number of chromatographic peaks, and the peak heights and areas were most ideal at a solid-liquid ratio of 1:60. Furthermore, the effect of different ultrasonic extraction times (30, 45, and 60 min) on the extraction efficiency was also investigated. The results showed that the number of chromatographic peaks and their absorption intensities did not change significantly with increasing extraction time. Therefore, methanol was ultimately selected as the solvent, with a solid-liquid ratio of 1:60 and ultrasonic extraction for 30 min.

[0024] 1.2 Optimization of chromatographic conditions

[0025] This experiment focused on investigating the types (methanol-water, methanol-0.1% phosphoric acid, acetonitrile-0.1% phosphoric acid) and their proportions of mobile phases. When the mobile phase was methanol-water, the chromatographic peaks were broad, flat, and tailed. When the mobile phase was acetonitrile-0.1% phosphoric acid, the peaks eluted quickly, but the resolution was poor. When the mobile phase was methanol-0.1% phosphoric acid, the baseline was stable, the peak shapes were good, and the resolution was excellent. The target component, saikosaponin D, has low polarity and elutes slowly. To avoid solvent interference, its retention time was prolonged, therefore, the mobile phase ratio was determined to be 60%–35% 0.1% formic acid-water for 0–25 min. Because Ganshuang granules contain a large number of flavonoids with similar polarities, a gradient elution method was used to achieve better separation of the various flavonoid components. The final elution methods were: 0–5 min, 95–90% 0.1% formic acid solution; 5–10 min, 90%–65% 0.1% formic acid solution; 10–15 min, 65% 0.1% formic acid solution; 15–20 min, 65%–60% 0.1% formic acid solution.

[0026] In addition, this invention also investigated different chromatographic columns (Agilent HP-5C18 (2.1×50mm, 1.7μm), XBridge BEH C18 (2.1×50mm, 1.7μm), Acquity BEH C18 (2.1×50mm, 1.7μm)), different column temperatures (25℃, 30℃, 35℃), and different injection volumes (1μL, 2μL, 3μL, 4μL, 5μL). The results showed that with the Agilent HP-5C18 (2.1×50mm, 1.7μm) column; column temperature: 40℃; flow rate: 0.3mL / min; injection volume: 2μL, there were more chromatographic peaks, and the peak shapes and resolutions were better.

[0027] 1.3 Selection of efficacy indicator components

[0028] This study selected the main compounds of Ganshuang granules, which showed significant degradation by intestinal flora, poor ADME properties, and anti-liver fibrosis effects, as the active ingredients, based on quantitative analysis using UPLC-MS / MS under MRM. The results showed that 14 compounds—Saikosaponin A, Saikosaponin D, Naringin, Paeoniflorin, Hesperidin, Chicoric acid, Chlorogenic acid, Tanshinone B, Narcissin, Kaempferol, Codonopsis pilosula, Amygdalin, Hyperoside, and Rutin—met the above criteria, and therefore these 14 compounds were identified as the active ingredients of Ganshuang granules.

[0029] The beneficial effects of the UPLC-MS / MS detection method of this invention are as follows:

[0030] (1) This invention establishes a UPLC-MS / MS method to detect and identify 14 pharmacodynamic components in the traditional Chinese medicine composition of this invention, including saikosaponin A, saikosaponin D, naringin, paeoniflorin, hesperidin, chicoric acid, chlorogenic acid, salvianolic acid B, narcissin, kaempferol, codonopsis alkaloid, amygdalin, hyperoside, and rutin, providing a reference for the intrinsic quality evaluation of the traditional Chinese medicine of this invention.

[0031] (2) The detection method of this invention also combines in vitro intestinal microbiota, everted intestinal sac model, liver fibrosis cell model, and liquid chromatography-mass spectrometry (LC-MS) technology to explore the differences in the absorption of compounds and efficacy of drugs before and after intestinal incubation mediated by the microbiota, and to establish quality standards for the active pharmaceutical ingredients. Thirty-one components that changed before and after incubation with the microbiota were identified, among which 14 compounds, including saikosaponin A, saikosaponin D, and rutin, were identified as potential active pharmaceutical ingredients of Gan Shuang granules. The detection method of this invention also detects the above-mentioned pharmacologically active ingredients. The experimental results show that the proportions of saikosaponin A, saikosaponin D, naringin, paeoniflorin, hesperidin, chicoric acid, chlorogenic acid, salvianolic acid B, narcissin, kaempferol, codonopsis pilosula, amygdalin, hyperoside, and rutin in the original prescription are 100:94.17:76.48:71.07:47.53:21.89:19.74:17.06:12.54:11.35:9.58:4.67:0.07:0.01.

[0032] (3) The results of the applicability test of the detection method system of the present invention show that: ① The recovery rate test results are as follows: the retention time of saikosaponin A is 18.21-18.55 min and the recovery rate of peak area is 109.68%; the retention time of saikosaponin D is 24.33-24.55 min and the recovery rate of peak area is 105.8%; the retention time of naringin is 9.73-9.91 min and the recovery rate of peak area is 108.2%; the retention time of paeoniflorin is 8.30-9.36 min and the recovery rate of peak area is 96.59%; the retention time of hesperidin is 9.83-10.05 min and the recovery rate of peak area is 97.21%; the retention time of chicoric acid is 10.01-10.87 min and the recovery rate of peak area is 106.99%; and the retention time of chlorogenic acid is 5.32-5.55 min. The recovery rates of the peak areas were as follows: 107.9% for salvianolic acid B (retention time 10.45–10.64 min, peak area recovery rate 109.28%); 109.88% for narcissin (retention time 9.43–9.60 min, peak area recovery rate 94.37%); 107.89% for kaempferol (retention time 9.09–9.26 min, peak area recovery rate 94.37%); 107.89% for codonopsis pilosula (retention time 9.59–9.81 min, peak area recovery rate 90.90%); 98.21% for hyperoside (retention time 9.03–9.20 min, peak area recovery rate 98.21%); and 110.00% for rutin (retention time 8.82–8.97 min, peak area recovery rate 110.00%). Therefore, the above-mentioned content determination method is accurate, reliable, and stable, and can be applied to the quality component detection of the traditional Chinese medicine composition of the present invention, and has a very broad market application prospect. Attached Figure Description

[0033] The accompanying drawings, which are provided to further illustrate the invention and constitute a part of this invention, are illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention.

[0034] Figures 1-2 - Peak area before and after incubation of mixed standard bacterial flora;

[0035] Figure 3 Total ion chromatogram of intestinal bacteria incubation medium under negative ion mode;

[0036] Figure 4 -Graph showing the difference in intestinal absorption of Liver-Soothing Granules;

[0037] Figure 5 - Screening of active ingredients in Gan Shuang Granules;

[0038] Figure 6 - Screening of the active ingredients of Ganshuang granules; Note: A, Screening of safe concentration dosage; B, Inhibitory effect of intestinal absorption fluid on hepatic stellate cells before and after bacterial incubation; **P<0.01 indicates a statistically significant difference compared with the normal group; *P<0.05, ## P<0.01, indicating a statistically significant difference compared to the model group. Detailed Implementation

[0039] To better understand the implementation of this invention, an experimental example is given below, and the invention will be further explained through typical embodiments.

[0040] Example 1 Quantitative analysis of potential active ingredients of Gan Shuang Granules based on UPLC-MS / MS of the present invention 1.1 Instruments and reagents

[0041] 1.1.1 Reagents and Tests

[0042] Liver-Soothing Granules (Baoding Tianhao Pharmaceutical Co., Ltd., specification: 3g per bag, batch number: 201314), acetonitrile, and methanol are all UPLC-MS grade (USA). Sodium formate, leucine-enkephalin Provided by Waters Corporation (USA). The water used for mass spectrometry was ultrapure water, produced using a Milli-Q water purification system. Other reagents were of analytical grade, as shown in Table 5.

[0043] Table 1 - Information on experimental control standards of the present invention

[0044]

[0045]

[0046] 1.1.2 Instruments

[0047] Binary pump and sample manager (Waters Corporation, USA), quadrupole time-of-flight mass spectrometer (Waters Corporation, USA), 0.0001 ppm electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.), ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.), ultrapure water system (Merck Chemical Technology (Shanghai) Co., Ltd.), high-speed refrigerated centrifuge (Anhui Zhongke Zhongjia Scientific Instruments Co., Ltd.), vortex oscillator (Changzhou Guohua Electric Co., Ltd.), nitrogen blower (Beijing Yousheng United Technology Co., Ltd.), biochemical culture instruments (Shanghai Kuntian Laboratory Instruments Co., Ltd.), digital display air bath constant temperature oscillator (Jintan Keheng Experimental Equipment Factory).

[0048] 1.2 Methods

[0049] 1.2.1 Preparation of reference solution

[0050] (1) Reference solution stock solution 1

[0051] Weigh out 3.09, 6.85, 3.55, 2.63, 2.81, 4.26, 7.32, 3.61, 4.49, 3.52, 3.81, 2.29, 2.78, and 2.80 mg of saikosaponin A, saikosaponin D, naringin, paeoniflorin, hesperidin, chicoric acid, chlorogenic acid, salvianolic acid B, naringin, kaempferol, codonopsis pilosula, amygdalin, hyperoside, and rutin reference standards, respectively. Weigh the sample and place it in a 2ml volumetric flask. Add chromatographic methanol, dilute to the mark, and shake well to obtain the reference standard stock solution (after purification) with concentrations of 1.5141, 3.3565, 1.7111, 1.2532, 1.3769, 2.1172, 3.5868, 1.7689, 2.2001, 1.7248, 1.8669, 1.0797, 1.3622, and 1.3949 mg / ml.

[0052] (2) Reference solution stock solution 2

[0053] Weigh out 4.11 mg of each of the following reference standards: saikosaponin A, saikosaponin D, naringin, paeoniflorin, hesperidin, chicoric acid, chlorogenic acid, salvianolic acid B, narcissin, kaempferol, codonopsis pilosula, amygdalin, hyperoside, and rutin. Weigh the sample and place it in a 2 ml volumetric flask. Add chromatographic methanol, dilute to the mark, and shake well to obtain the reference standard stock solution (after purification) with concentrations of 2.0139, 3.9200, 1.7304, 1.1293, 1.1711, 2.4751, 3.7975, 1.9796, 2.3324, 1.6513, 2.1805, 1.1599, 1.3083, and 1.4149 mg / ml.

[0054] (3) Mixed reference solution 1

[0055] Take 100 μL of each compound from the reference solution stock solution 1 and place them in the same 2 mL volumetric flask. Dilute to volume with methanol and shake well to obtain a mixed reference standard with concentrations of 0.0757, 0.1678, 0.0856, 0.0627, 0.0688, 0.1059, 0.1793, 0.0884, 0.1100, 0.0862, 0.0933, 0.05399, 0.0681, and 0.0697 mg / mL.

[0056] (4) Mixed reference solution 2

[0057] Take 100 μL of each compound from the reference solution stock solution 2 and place them in the same 2 mL volumetric flask. Dilute to volume with methanol and shake well to obtain a mixed reference standard with concentrations of 0.1007, 0.1960, 0.0865, 0.0565, 0.0586, 0.1238, 0.1899, 0.0989, 0.1166, 0.0826, 0.1090, 0.0579, 0.0654, and 0.0707 mg / mL.

[0058] 1.2.2 Preparation of test sample

[0059] Grind the liver-soothing granules into a fine powder. Take 1.0000g of the liver-soothing granule powder, weigh it accurately, place it in a stoppered conical flask, add 20mL of methanol, weigh it, sonicate for 30min, centrifuge (12000rpm, 10min, centrifugation radius 10cm), take the supernatant, filter it through a 0.22μm microporous membrane, and you will get the product.

[0060] 1.2.3 Chromatographic conditions

[0061] Waters Xevo G2-XS Q-TOF ultra-high performance liquid chromatograph, column (2.1×50mm, 1.7μm); column temperature: 40℃; flow rate: 0.3mL·min -1 Injection volume: 2 μL; Mobile phase: A: 0.1% formic acid water; Mobile phase: B: acetonitrile; Gradient elution (0–5 min, 95–90% A; 5–10 min, 90%–65% A; 10–15 min, 65% A; 15–20 min, 65%–60% A; 20–30 min, 60–35% A; 30–30.1 min, 35%–95% A; 30.1–35 min, 95% A).

[0062] 1.2.4 Mass Spectrometry Conditions

[0063] Ionization mode: electrospray ionization; ion source temperature: 150℃; capillary voltage: 3.0kV; cone gas flow rate: 50L / h; desolvation gas temperature: 350℃; desolvation gas flow rate: 1000L / h; nebulizer gas pressure: 7×10⁵Pa; precursor ion scan range: m / z 80~1200. Main mass spectrometry parameters are shown in Table 2.

[0064] Table 2 Main mass spectrometry parameters

[0065]

[0066] 1.3 Experimental Results

[0067] 1.3.1 System Applicability Assessment

[0068] After the system was fully equilibrated, five consecutive injections were performed on mixed reference solution one (section 2.1), and two consecutive injections were performed on mixed reference solution two (containing saikosaponin A, saikosaponin D, naringin, paeoniflorin, hesperidin, chicoric acid, chlorogenic acid, salvianolic acid B, narcissin, kaempferol, codonopsis pilosula, amygdalin, hyperoside, and rutin). In the system suitability study, the resolution between the analyte peak and adjacent peaks in the chromatograms of each reference solution was less than 1.5; the theoretical plate number was greater than 5000; for reference solution one, after five consecutive injections, the relative standard deviation (RSD) of the analyte peak area did not exceed 10.0%; the relative standard deviation of the retention time did not exceed 2.0%; the recovery rate of reference solution two should be between 90.0% and 110.0%; and in the linearity study, R > 9.88 for each compound. This indicates that the analytical system (including methods, operations, and instruments) meets the requirements for the assay.

[0069] Table 3 System Suitability Test Results

[0070]

[0071]

[0072]

[0073]

[0074] 1.3.3 Content Determination

[0075] After the system is fully balanced, take the test solution of Ganshuang Granules under section "1.2.2" and inject it for determination according to the test conditions under sections "1.2.3 and 1.2.4". Compare the known standard concentrations and peak areas with the peak areas of compounds in the test sample, and calculate the proportions of the test compounds rutin A, saikosaponin D, naringin, paeoniflorin, hesperidin, chicoric acid, chlorogenic acid, salvianolic acid B, narcissin, kaempferol, codonopsis pilosula, amygdalin, hyperoside, and rutin in Ganshuang Granules as 100:94.17:76.48:71.07:47.53:21.89:19.74:17.06:12.54:11.35:9.58:4.67:0.07:0.01.

[0076] Example 2: Screening of potential active ingredients in Gan Shuang granules based on UPLC-MS / MS technology and ADME properties of compounds

[0077] 2.1 Instruments and Reagents

[0078] Same as Example 1

[0079] 2.2.2 Detection Method

[0080] Same as Example 1

[0081] 2.3 Experimental Results

[0082] 2.3.1 Information on poor-quality compounds in Ganshuang Granules (ADME)

[0083] Saikosaponin A, Saikosaponin D, Naringin, Paeoniflorin, Hesperidin, Chicoric acid, Chlorogenic acid, Tanshinone B, Narcisin, Kaempferol, Codonopsis pilosula, Amygdalin, Hyperoside, and Rutin all have a molecular weight greater than 300. They have a relatively high number of polar functional groups and a high degree of unsaturation of benzene rings. They have a low plasma protein binding rate, are not hepatic enzyme inhibitors, and are soluble in water at 25°C, but have poor intestinal permeability.

[0084] 2.3.2 Changes in peak area of ​​some ADME-containing compounds after incubation with bacterial communities

[0085] The compound was detected in the Ganshuang Granules test sample by UPLC-MS / MS, indicating that it is a compound in Ganshuang Granules. As shown in the table, the peak area of ​​the compound decreased after incubation with intestinal flora, indicating that the intestinal flora can degrade the compound.

[0086] Table 4 Peak area of ​​mixed standard bacterial flora before and after incubation

[0087]

[0088]

[0089] 2.4 Experimental Results

[0090] Saikosaponin A, Saikosaponin D, Naringin, Paeoniflorin, Hesperidin, Chicoric acid, Chlorogenic acid, Tanshinone B, Narcisin, Kaempferol, Codonopsis pilosula, Amygdalin, Hypericin, and Rutin are compounds found in Ganshuang granules. These compounds exhibit poor ADME properties and their content significantly decreases after incubation with microbial flora. These 14 compounds are contained in Bupleurum chinense, the principal ingredient of Ganshuang granules. Chlorogenic acid and Codonopsis pilosula are compounds found in Codonopsis pilosula, the principal ingredient. Tanshinone B, Naringin, Hesperidin, Paeoniflorin, Chicoric acid, and Amygdalin are the main compounds in the adjuvant and assistant ingredients. Furthermore, literature studies have shown that these 14 compounds have anti-liver fibrosis effects; therefore, these 14 compounds are identified as potential active ingredients in Ganshuang granules.

[0091] Example 3

[0092] Study on the difference in chemical composition of intestinal absorption fluid before and after bacterial colony incubation using UPLC-Q-TOF-MS / MS technology

[0093] 3.1 Materials

[0094] 3.1.1 Instruments

[0095] Binary pump and sample manager (Waters Corporation, USA); Quadrupole time-of-flight mass spectrometer (Waters Corporation, USA); 0.0001 g electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.); Ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); Ultrapure water system (Merck Chemical Technology (Shanghai) Co., Ltd.); High-speed refrigerated centrifuge (Anhui Zhongke Zhongjia Scientific Instruments Co., Ltd.); Vortex oscillator (Changzhou Guohua Electric Co., Ltd.); Nitrogen blower (Beijing Yousheng United Technology Co., Ltd.); Biochemical culture instruments (Shanghai Kuntian Laboratory Instruments Co., Ltd.).

[0096] 3.1.2 Medicines and Reagents

[0097] 3.1.2 Medicines and Reagents

[0098] The liver-soothing granules extract powder was purchased from Buchang Baoding Tianhao Pharmaceutical Co., Ltd. (production date: May 27, 2023, yield: 23.65%). Reference standards such as saikosaponin A, saikosaponin D, and naringin were purchased from the China National Institutes for Food and Drug Control.

[0099] 3.1.3 Animals

[0100] Male SD rats, weighing 250–300 g, were purchased from Chengdu Dashuo Co., Ltd. The animal experiments and protocols were approved by the Experimental Animal Ethics Committee of Shaanxi University of Traditional Chinese Medicine (Approval No.: SVCMDL20230214002).

[0101] 3.2 Methods

[0102] 3.2.3 Preparation of anaerobic culture medium

[0103] Weigh 49.0g of GAM medium, heat and dissolve it in 1000mL of distilled water, autoclave at 121℃ for 15min, and when cooled to about 50℃, add 1mL of sterile 0.1% vitamin K1 solution and 1mL of heme chloride (5mg / mL) to every 1000mL of medium, mix well and set aside.

[0104] 3.2.4 Preparation of in vitro intestinal flora incubation medium

[0105] Take 2.5g of rat cecal contents, add 4 times the amount of physiological saline, and incubate at 5000 rpm. -1 After 10 minutes, take the supernatant and add 9 times the amount of anaerobic culture medium to obtain the final product.

[0106] Preparation of reference solution

[0107] Weigh approximately 2 mg of the following standards: tanshinone IIA, ferulic acid, emodin, quercetin, emodin methyl ether, kaempferol, codonopsis glycoside, hesperidin, paeoniflorin, neohesperidin, tanshinone I, chicoric acid, saikosaponin D, polygalactoside, baicalin, naringin, luteolin, paeoniflorin lactone, rosmarinic acid, baicalin, oxypaeoniflorin, gallic acid, aloe-emodin, rhein, isorhamnetin, rhein, and rutin. Dissolve in 70% methanol by sonication and filter through a 0.22 μm filter membrane.

[0108] 3.2.5 Experimental Grouping and Incubation

[0109] The experiment included a blank control group, a control group, and a drug-treated group. The blank control group received 10 mL of bacterial incubation medium, the control group received 25 mg of Gan Shuang granules extract powder and 10 mL of anaerobic culture medium, and the drug-treated group received 25 mg of Gan Shuang granules extract powder and 10 mL of bacterial incubation medium. Each group was divided into three parallel experiments. After vortexing for 2 min, the mixture was placed in a culture bag containing an anaerobic gas-generating bag and incubated at 37℃ for 24 h.

[0110] 3.2.6 Sample processing for intestinal flora incubation

[0111] Take 1 mL each of the blank group, control group, and drug treatment group solutions from item "3.2.5", add 1 mL of chromatographic methanol to each to terminate the reaction, and centrifuge at 8000 r·min. -1 Centrifuge for 10 min, collect the supernatant, dry it under nitrogen, and then dilute it with methanol to 15 mg / ml. -1 After shaking well, filter through a 0.22 μm filter membrane, and take the filtrate into a liquid chromatography vial as the test sample. Inject and analyze according to the conditions under "3.2.10" and "3.2.11".

[0112] 3.2.7 Preparation of intestinal absorption test samples

[0113] Take 19 mL of the blank group, control group, and drug treatment group solution from item "3.2.5", add 19 mL of chromatographic methanol to each group to terminate the reaction, and centrifuge at 8000 r·min. -1 Centrifuge for 10 minutes, take the supernatant, evaporate to dryness using a rotary evaporator, and reconstitute with 20 mL of Benzoate solution.

[0114] 3.2.8 Preparation of the everted intestinal sac model

[0115] Rats fasted for 12 hours were euthanized by dislocation. The skin and muscle were quickly cut along the midline of the abdomen to remove the small intestine, which was then divided into three segments. Each segment was placed in a 0°C Tyrode's solution and carefully inverted using a silicone tube before being suspended in an isolated intestinal perfusion apparatus. The apparatus was kept at a constant temperature of 37°C and purged with a mixture of 95% O2 and 5% CO2. Each segment contained 2 mL of blank Tyrode's solution, and the external contents were the intestinal absorption test solution for the blank group, control group, and drug treatment group (as per section 2.2.7). After 1 hour, all fluid from the intestinal pouches of each group was collected. Part of the fluid was used for qualitative analysis, and the rest for pharmacodynamic studies.

[0116] 3.2.9 Processing of intestinal absorption samples

[0117] Take 1 ml of the intestinal absorption fluid from section "3.2.8" and centrifuge at 8000 r·min. -1 Centrifuge for 10 min, collect the supernatant, dry it with nitrogen, dilute it 1000 times with chromatographic methanol, filter it through a 0.22 μm filter membrane, and take the filtrate into a liquid chromatography vial as an intestinal absorption test sample. Inject and analyze it according to the conditions under "3.2.10" and "3.2.11".

[0118] 3.2.10 Chromatographic conditions

[0119] Waters Xevo G2-XS Q-TOF ultra-high performance liquid chromatograph, column (2.1×50mm, 1.7μm); column temperature: 40℃; flow rate: 0.3mL·min -1 Injection volume: 2 μL; Mobile phase: A: 0.1% formic acid water; Mobile phase: B: acetonitrile; Gradient elution (0–5 min, 4–8% B; 5–10 min, 8%–13% B; 10–15 min, 13% B; 15–18 min, 13%–15% B; 18–19 min, 15–20% B; 19–24 min, 20% B; 24–28 min, 20%–40% B; 28–33 min, 40% B; 33–35 min, 40–50% B; 35–40 min, 5% B).

[0120] 3.2.11 Mass Spectrometry Conditions

[0121] Electrospray ionization source: negative ion mode scanning; ion source temperature: 120℃; cone voltage: 40V; capillary voltage: 3.0KV; cone gas flow rate: 50L·h -1 Argon flow rate: 0.15 mL·min⁻¹; Desolventizing gas: nitrogen; Desolventizing gas temperature: 300℃; Scan range: m / z 50~950.

[0122] 3.3 Results

[0123] 3.3.2 Study on the difference in intestinal absorption before and after microbial incubation based on UPLC-Q-TOF-MS / MS technology

[0124] The total ion chromatogram of the intestinal absorption fluid after incubation with gut microbiota, as determined by UPLC-Q-TOF / MS, is attached. Figure 3 After normalization, the intestinal absorption difference graph is shown below. Figure 4 As shown in the figure, the number of compound peaks increased after incubation, and the relative intensity also increased compared to before incubation.

[0125] Example 4: Identification of the active ingredients of Ganshuang Granules based on a liver fibrosis cell model

[0126] 4.1 Instruments

[0127] 4.1 Reagents and Tests

[0128] The Cell Counting Kit-8 (CCK8; C0038) was purchased from Beyotime Biotechnology Co., Ltd. The human hepatic stellate cell line HSC-T6 was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. Penicillin-streptomycin (PS), phosphate-buffered saline (PBS), and 0.25% trypsin were purchased from Corning Incorporated (USA), and DMEM culture medium was purchased from Sigma (USA).

[0129] 4.2 Methods

[0130] 4.2.1 Preparation of drug-containing intestinal absorption solution

[0131] After the intestinal absorption fluid of the blank group, control group, and drug treatment group was dried by blowing with nitrogen, it was dissolved in 1 ml of DMSO, and then diluted with DMEM to 1.10³ and 1.10³, respectively. 4 1.10 5 1.10 6 1.10 7 Store at 4°C for multiples of the original volume.

[0132] 4.2.2 Screening of safe dosing concentrations

[0133] Collect cells in the logarithmic growth phase and add HSC-T6 at a ratio of 5 × 10³ × 10⁰ μL per well. -1 Inoculate into 96-well plates and incubate for 24 hours, then dilute to 1 · 10 3 1.10 4 1.10 5 1.10 6 1.10 7A blank control group of intestinal absorption solution was added to each well at 10 μL for further culture. After 24 hours, 10 μL of CCK-8 reagent was added to each well. Two hours later, the absorbance at 450 nm was measured using a microplate reader to screen for safe dosage. Control wells (without drug) were also included in the experiment. Three auxiliary wells were set up for each group, and each experiment was performed independently at least three times.

[0134] 4.2.3 Cell modeling and drug administration intervention

[0135] Cells in logarithmic growth phase were collected, and HSC-T6 cells were seeded at a rate of 5 × 10³ × 100 μL⁻¹ into 96-well plates. After 24 h, the cells were washed once with PBS, and fresh DMEM (without PBS) was added to the culture dish to induce G0 phase and starve the cells for 12 h. Then, the intestinal absorption solutions of the control group and the treated group at the safe drug concentration were diluted in medium containing 10 ng·mL⁻¹ TGF-β1 and added to each well. After incubation for 48 h, 10 μL of CCK-8 was added, and the cells were cultured at 37 °C for another 2 h. The absorbance (A) was measured at 450 nm using a microplate reader. The experiment also included normal control wells (containing cells, medium, and CCK-8 reagent) and model wells (containing cells, medium, TGF-β1, and CCK-8 reagent), with three auxiliary wells for each group. Each experiment was performed independently at least three times.

[0136] 4.3 Results

[0137] 4.3.1 Screening of safe drug concentrations in intestinal absorption fluids based on the CCK8 method

[0138] Dilute 1.10 3 1.10 4 1.10 5 Blank intestinal absorption solution can inhibit the growth of hepatic stellate cells and has cytotoxic effects, while a dilution of 1.10... 6 and 1.10 7 At that time, the blank intestinal absorption solution had no toxic effect on hepatic stellate cells, so a dilution of 1.10 was selected. 6 ~1.10 7 The effect of compounds on hepatic stellate cells before and after incubation with intestinal absorption fluid was investigated to determine the safe dosage concentration (see [link to study]. Figure 5 ).

[0139] 4.3.2 Effects of intestinal absorption fluid on hepatic stellate cells before and after bacterial incubation

[0140] Compared with the control group, the number of surviving HSC-T6 cells in the model group was significantly increased (P<0.01); compared with the model group, the number of surviving cells at a dilution of 2.10 was significantly increased. 6 4.10 6 6·10 6 8·10 6The intestinal absorption fluids of both the control and treatment groups were applied to cells. It was found that both intestinal absorption fluids before and after bacterial incubation inhibited the proliferation of HSC-T6 cells (P<0.01). Furthermore, the inhibitory rate of the intestinal absorption fluid on hepatic stellate cells after bacterial incubation was significantly greater than that in the control group, indicating that the metabolites produced after bacterial incubation can increase intestinal absorption, thereby enhancing the efficacy of the drug. Figure 6 ).

[0141] 4.4 Experimental Conclusions

[0142] Fourteen compounds, including saikosaponin A, saikosaponin D, and rutin, are compounds with poor ADME properties in Ganshuang granules and can be degraded by intestinal flora. After incubation with flora, the number of compounds in the intestinal absorption fluid increased and their inhibitory rate on liver fibrosis cells was stronger than that of the unincubated group.

[0143] Finally, it should be noted that this invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and instructive, not restrictive. Any changes, equivalent substitutions, and improvements made by those skilled in the art under the guidance of this specification, within the spirit and scope of this invention, are within the protection scope of this invention.

Claims

1. A method for detecting the active pharmaceutical ingredients of Gan Shuang granules by UPLC-MS / MS, characterized in that, The detection method includes the following steps: (1) Preparation of mixed reference solution: Weigh out the reference standards of saikosaponin A, saikosaponin D, naringin, paeoniflorin, hesperidin, chicoric acid, chlorogenic acid, salvianolic acid B, narcissin, kaempferol, codonopsis pilosula, amygdalin, hyperoside, and rutin respectively, add methanol, dilute to the mark, and shake well to obtain the solution. (2) Preparation of test solution: Take liver-soothing granules, grind them into fine powder, weigh them accurately, add methanol, sonicate, centrifuge, take the supernatant, filter, and the solution is obtained. (3) Chromatographic conditions: Ultra-high performance liquid chromatograph, C 18 Chromatographic column; column temperature: 38–42℃; flow rate: 0.1–0.5 mL / min -1 Mobile phase: A: 0.1% formic acid in water; Mobile phase: B: acetonitrile; Gradient elution: 0–5 min, 95–90% A; 5–10 min, 90%–65% A; 10–15 min, 65% A; 15–20 min, 65%–60% A; 20–25 min, 60–35% A; 25–25.1 min, 35%–95% A; 25.1–26 min, 95% A; (4) Mass spectrometry conditions: Ionization mode: electrospray ionization; Ion source temperature: 150℃; Capillary voltage: 3.0kV; Cone gas flow rate: 50L / h; Desolvation gas temperature: 350℃; Desolvation gas flow rate: 1000L / h; Nebulizer gas pressure: 7×10 5 Pa; Precipitated ion scan range: m / z 80~1200, The main mass spectrometry parameters of the analyte in step (4) mass spectrometry conditions are as follows:

2. The detection method as described in claim 1, characterized in that, In step (1) of the detection method, the concentrations of the mixed reference solution are as follows: saikosaponin A: 50–120 μg / mL; saikosaponin D: 120–150 μg / mL; naringin: 80–100 μg / mL; paeoniflorin: 50–70 μg / mL; hesperidin: 50–70 μg / mL; chicoric acid: 100–125 μg / mL; and chlorogenic acid: 17 μg / mL. The concentrations of the following compounds are: 0–190 μg / mL, 85–100 μg / mL of salvianolic acid B, 105–120 μg / mL of narcissin, 80–90 μg / mL of kaempferol, 90–115 μg / mL of codonopsis pilosula, 55–65 μg / mL of amygdalin, 60–70 μg / mL of hyperoside, and 60–70 μg / mL of rutin.

3. The detection method as described in claim 1, characterized in that, In step (2) of the detection method, during the preparation of the test solution, the ultrasonic treatment time is 20–40 min.

4. The detection method as described in claim 1, characterized in that, In step (2) of the detection method, the preparation of the test solution involves centrifugation at a speed of 11000–14000 rpm for 8–12 min.

5. The detection method as described in claim 1, characterized in that, The chromatographic detection conditions for step (3) of the detection method are as follows: the model of the ultra-high performance liquid chromatograph is: Waters Xevo G2-XS Q-TOF.

6. The detection method as described in claim 1, characterized in that, The chromatographic detection conditions for step (3) of the detection method are as follows: The C 18 The chromatographic column has a specification of 2.1×50mm and a diameter of 1.7μm.

7. The detection method as described in claim 1, characterized in that, The chromatographic detection conditions for step (3) of the detection method are as follows: column temperature: 40℃; flow rate: 0.3 mL / min. -1 .

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