Medicinal component detection method of traditional Chinese medicine composition UPLC-MS / MS

Abstract

The invention provides a method for detecting medicinal components of a traditional Chinese medicine composition UPLC-MS / MS. The detection method can be used for identifying 14 medicinal components of the traditional Chinese medicine composition, namely saikoside A, saikoside D, naringin, paeoniflorin, hesperidin, chicoric acid, chlorogenic acid, salvianolic acid B, narcissoside, kaempferitrin, lobetyolin, amygdalin, hyperoside and rutin. And a reference is provided for evaluating the internal quality of the traditional Chinese medicine composition.

Description

Technical Field

[0001] The invention relates to a UPLC-MS / MS method for detecting effective components of a traditional Chinese medicine composition, and belongs to the field of traditional Chinese medicine component detection methods. Background Art

[0002] The Chinese medicine composition of the present invention is Ganshuang granules, which are produced and marketed by Baoding Tianhao Pharmaceutical Co., Ltd. The medicine is prepared from 13 medicinal materials including Codonopsis pilosula, Bupleurum chinense, White Peony Root, Angelica Root, Poria, Atractylodes macrocephala, Fructus Aurantii Immaturus, Herba Taraxaci, Polygonum cuspidatum, Herba Prunellae, Salvia miltiorrhizae, Semen Persicae, and Carapax Trionycis. In recent years, studies have found that it has a significant anti-hepatic fibrosis effect, but current research is mostly focused on clinical practice, and research on its components is less, and the anti-hepatic fibrosis pharmacological substances are still unclear. Modern research shows that it is closely related to the anti-hepatic fibrosis effect of the drug. As a Chinese medicine oral preparation, Ganshuang granules inevitably interact with intestinal flora after taking, and this interaction will affect the structure of Chinese medicine components and the corresponding efficacy.

[0003] This paper systematically combs through the existing technical methods for the detection method of the traditional Chinese medicine composition. It can be found that: in the quality standard research, Han Dong established the fingerprint of Ganshuang Granules and identified 10 components (ferulic acid, polydatin, chicoric acid, naringin, hesperidin, rosmarinic acid, neohesperidin, salvianolic acid B, rhamnolide, and rhamnolide methyl ether) in 18 batches of samples; Wang Guofeng identified 8 components (rosmarinic acid, ferulic acid, peony root glycosides, polydatin, naringin, neohesperidin, and rhamnolide methyl ether) in 14 batches of samples. =Phenolic acid B, tanshinone ⅡA); chemometrics can quickly and comprehensively identify and classify a large amount of sample data. Han Dong's cluster analysis and principal component analysis (PCA) basically divided 18 batches of Ganshuang granules into 3 categories, that is, batches of similar years were divided into one category each; Wang Guofeng clustered 14 batches of samples into 2 categories; Han Dong used orthogonal partial least squares method-discriminant analysis (OPLS-DA) to screen chicoric acid, neohesperidin, ferulic acid, naringin, and rosmarinic acid as differential markers of Ganshuang granules. Wang Guofeng obtained salvianolic acid B, tanshinone ⅡA, ferulic acid, rosmarinic acid, and peony root glycosides as differential markers of Ganshuang granules. Salvianolic acid B, rosmarinic acid, and ferulic acid were the common differential components screened out, indicating that phenolic acid compounds may be the main differential substances in Ganshuang granules. In the content determination study, Li Liang established a UHPLC method to determine the content of paeoniflorin, paeoniflorin, naringin, polydatin, and tanshinone ⅡA in Ganshuang Granules. The methodological validation was well combined and can provide a basis for the quality control of Ganshuang Granules. Yang Wen established a high-performance liquid chromatography-one-test-multiple-evaluation method (HPLC-QAMS) to determine polydatin, resveratrol, oxidized paeoniflorin, paeoniflorin, and saikosaponin a, b1, and d in Ganshuang Granules. The results obtained by the one-test-multiple-evaluation method for each component in Ganshuang Granules were not significantly different from those obtained by the external standard method. In the analysis of inorganic elements, it was found that the inorganic elements in Ganshuang Granules were mainly K, Mg, Na, Ca, Al, Fe, and Mn. And the limits of the five heavy metal elements Cu, As, Cd, Hg, and Pb are in line with the relevant limits of the Chinese Pharmacopoeia. Judging from the fingerprint similarity, cluster analysis and PCA results, the quality uniformity of inorganic elements in Ganshuang Granules is good, and the main characteristic elements are Mg, K, Ca, Mo, Tl, Cd, Se, and Zn, which can provide a reference for the quality uniformity and safety evaluation of Ganshuang Granules.

[0004] The current standard for Ganshuang Granules only uses TLC identification for qualitative analysis of paeoniflorin, rhein and angelica, and HPLC for quantitative analysis of paeoniflorin. The number of medicinal materials controlled by the quality standard is less than one-third of the total number of medicinal materials in the prescription, which makes it difficult to reflect the overall characteristics and intrinsic quality of the preparation. Moreover, the current research on this drug is mainly focused on clinical research, and there is little research on the identification of active substances and their quality standards. There is an urgent need for relevant research to clarify its active ingredients. Therefore, it is very necessary to develop a detection method that can simultaneously detect its multiple intrinsic active index components. Summary of the invention

[0005] The present invention provides a UPLC-MS / MS method for detecting the active ingredients of a traditional Chinese medicine composition, and the method can identify 14 active ingredients 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 traditional Chinese medicine composition of the present invention. Through methodological investigation, the determination method is accurate and reliable, with good stability, and provides a new reference for the intrinsic quality evaluation of the traditional Chinese medicine of the present invention.

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

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

[0008] ⑴. Preparation of mixed reference solution: Weigh saikosaponin A, saikosaponin D, naringin, peony root, hesperidin, chicoric acid, chlorogenic acid, salvianolic acid B, narcissus glycosides, kaempferol, codonopsis pilosula, amygdalin, hyperoside, and rutin reference substances respectively, weigh, add methanol, dilute to the scale, shake well, and obtain;

[0009] ⑵. Preparation of test solution: Take the Chinese medicine composition, grind it into fine powder, accurately weigh it, add methanol, ultrasonicate it, centrifuge it, take the supernatant, filter it, and obtain it;

[0010] ⑶、Chromatographic conditions: ultra-high performance liquid chromatography, C 18 Chromatographic column; column temperature: 38-42°C; flow rate: 0.1-0.5 mL min -1 ; Mobile phase: A: 0.1% formic acid water; Mobile phase: B: acetonitrile; Gradient elution (0-5min, 95-90% A; 5-10min, 90%-65% A; 10-15min, 65% A; 15-20min, 65%-60% A; 20-25min, 60-35% A; 25-25.1min, 35%-95% A; 25.1-26min, 95% A);

[0011] (4) Mass spectrometry conditions: ionization mode: electrospray ionization; ion source temperature: 150°C; capillary voltage: 3.0 kV; cone gas flow rate: 50 L / h; desolvation temperature: 350°C; desolvation gas flow rate: 1000 L / h; nebulizer gas pressure: 7×105 Pa; parent ion scanning range: m / z 80-1200;

[0012] Preferably, in step (1) of the detection method, the concentration of saikosaponin A in the test solution is 50-120 μg / mL, the concentration of saikosaponin D is 150-120 μg / mL, the concentration of naringin is 80-100 μg / mL, the concentration of paeoniflorin is 50-70 μg / mL, the concentration of hesperidin is 50-70 μg / mL, the concentration of chicoric acid is 100-125 μg / mL, and the concentration of chlorogenic acid is The concentration of tannic acid B is 170-190μg / mL, the concentration of narcissin is 85-100μg / mL, the concentration of narcissin is 105-120μg / mL, the concentration of kaempferol is 80-90μg / mL, the concentration of codonopsis pilosula glycoside is 90-115μg / mL, the concentration of amygdalin is 55-65μg / mL, the concentration of hyperoside is 60-70μg / mL, and the concentration of rutin is 60-70g / mL.

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

[0014] Preferably, in the preparation of the test solution in step (2) of the detection method, the rotation speed of the centrifugal treatment 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 in step (3) of the detection method are: 18 The specifications of the chromatographic column are 2.1×50 mm, 1.7 μm.

[0017] The chromatographic detection conditions in step (3) of the detection method are as follows: the column temperature is 40° C.; the flow rate is 0.3 mL·min -1 .

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

[0019]

[0020]

[0021] In order to reflect the innovativeness of the technical solution of the present invention, we have organized some experimental processes of the optimization of the chromatogram of the drug efficacy detection component of the present 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, ethanol) and their solid-liquid ratios (1:60, 1:100, 1:150, 1:200 times) on sample extraction. The results showed that the number of chromatographic peaks in methanol extract was large, and when the solid-liquid ratio was 1:60, the peak height and peak area of ​​each chromatographic peak were ideal. In addition, the effects of different ultrasonic extraction times (30, 45, 60 min) on the extraction effect were also investigated. The results showed that with the increase of extraction time, the number of chromatographic peaks and their absorption intensity did not change significantly. Therefore, it was finally determined to use methanol as the solvent, the solid-liquid ratio was 1:60, and the ultrasonic extraction was 30 min.

[0024] 1.2 Optimization of chromatographic conditions

[0025] This experiment focused on the types of mobile phases (methanol-water, methanol-0.1% phosphoric acid, acetonitrile-0.1% phosphoric acid) and their proportions. When the mobile phase was methanol-water, the chromatographic peaks were broad and flat with tailing; when the mobile phase was acetonitrile-0.1% phosphoric acid, the chromatographic peaks appeared quickly, but the separation was poor; when the mobile phase was methanol-0.1% phosphoric acid, the baseline was stable, the chromatographic peaks had a good shape, and the separation was good. The target component, saikosaponin D, has a low polarity and a slow peak. In order to avoid solvent interference, its retention time was delayed, so the mobile phase ratio was 60% to 35% of 0.1% formic acid water, and the time was 0 to 25 minutes. Since Ganshuang granules contain a large number of flavonoid compounds with similar polarity, in order to achieve a better separation effect for each flavonoid component, a gradient elution method was used, and the final elution time was determined to be 0-5 min, 95-90% 0.1% formic acid water; 5-10 min, 90%-65% 0.1% formic acid water; 10-15 min, 65% 0.1% formic acid water; 15-20 min, 65%-60% 0.1% formic acid water.

[0026] In addition, the present invention also investigates 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°C, 30°C, 35°C) and different injection volumes (1μL, 2μL, 3μL, 4μL, 5μL). The results show that when the Agilent HP-5C18 (2.1×50mm, 1.7μm) chromatographic column; column temperature: 40°C; flow rate: 0.3mL / min; injection volume: 2μL, the number of chromatographic peaks is large, and the peak shape and separation of each chromatographic peak are good.

[0027] 1.3 Selection of efficacy indicator ingredients

[0028] In this study, the main Chinese compounds of Ganshuang Granules that can be significantly degraded by intestinal flora, have poor ADME properties, and have anti-liver fibrosis effects were selected as the active ingredients by UPLC-MS / MS technology under MRM. The results showed that 14 compounds, including saikosaponin A, saikosaponin D, naringin, paeoniflorin, hesperidin, chicoric acid, chlorogenic acid, salvianolic acid B, narcissin, kaempferol, codonopsis pilosula, amygdalin, hyperoside, and rutin, met the above criteria, so the above 14 compounds were identified as the active ingredients of Ganshuang Granules.

[0029] Beneficial effects of the UPLC-MS / MS detection method of the present invention:

[0030] ⑴. The present invention establishes a UPLC-MS / MS method to detect and identify 14 active ingredients of the traditional Chinese medicine composition of the present invention, including saikosaponin A, saikosaponin D, naringin, paeoniflorin, hesperidin, chicoric acid, chlorogenic acid, salvianolic acid B, narcissin, kaempferol, codonopsis pilosula glycoside, amygdalin, hyperoside and rutin, which provides a reference for the intrinsic quality evaluation of the traditional Chinese medicine of the present invention.

[0031] ⑵. The detection method of the present invention also combines in vitro intestinal microbiota, everted intestinal sac model, liver fibrosis cell model and liquid chromatography-mass spectrometry technology to establish quality standards for the effective ingredients through the difference in the absorption of compounds and efficacy of the fore and aft intestines mediated by the flora. 31 components that changed before and after the flora incubation were identified, of which 14 compounds such as saikosaponin A, saikosaponin D, and rutin were identified as potential effective ingredients of Ganshuang granules. The detection method of the present invention also detects the above-mentioned effective substance components. The experimental results show that the ratio of saikosaponin A, saikosaponin D, naringin, paeoniflorin, hesperidin, chicoric acid, chlorogenic acid, salvianolic acid B, narcissus glycoside, kaempferol glycoside, codonopsis pilosula glycoside, amygdalin, hyperoside and rutin in the original prescription is 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] ⑶. The results of the system applicability test of the detection method of the present invention show that: ①. The results of the recovery test show that the retention time of saikosaponin A is 18.21-18.55 min, and the recovery rate of the peak area is 109.68%. The retention time of saikosaponin D is 24.33-24.55 min, and the recovery rate of the peak area is 105.8%. The retention time of naringin is 9.73-9.91 min, and the recovery rate of the peak area is 108.2%. The retention time of paeoniflorin is 8.30-9.36 min, and the recovery rate of the peak area is 96.59%. The retention time of hesperidin is 9.83-10.05 min, and the recovery rate of the peak area is 97.21%. The retention time of chicoric acid is 10.01-10.87 min, and the recovery rate of the peak area is 106.99%. The retention time of chlorogenic acid is 5.32-5.55 min. , the recovery rate of the peak area was 107.9%, the retention time of salvianolic acid B was 10.45-10.64 min, and the recovery rate of the peak area was 109.28%, the retention time of narcissusin was 9.43-9.60 min, and the recovery rate of the peak area was 109.88%, the retention time of kaempferol was 9.09-9.26 min, and the recovery rate of the peak area was 94.37%, the retention time of codonopsis pilosula glycoside was 9.59-9.81 min, and the recovery rate of the peak area was 107.89%, the retention time of amygdalin was 6.34-6.58 min, and the recovery rate of the peak area was 90.90%, the retention time of hyperoside was 9.03-9.20 min, and the recovery rate of the peak area was 98.21%, and the retention time of rutin was 8.82-8.97 min, and the recovery rate of the peak area was 110.00%. It can be seen that the above content determination method is accurate, reliable, and has good stability. It can be applied to the quality component detection of the Chinese medicine composition of the present invention and has a very broad market application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The drawings described herein are used to provide further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

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

[0035] Figure 3 Total ion current in intestinal bacteria incubation solution under negative ion mode;

[0036] Figure 4 - Diagram of the difference in intestinal absorption of Ganshuang granules;

[0037] Figure 5 - Screening of medicinal ingredients in Ganshuang 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 compared with the normal group, there is a statistical difference; *P<0.05, ## P<0.01, which was statistically significant compared with the model group. DETAILED DESCRIPTION

[0039] In order to more fully understand the implementation of the present invention, an experimental example is listed below, and the present invention is further described below through typical embodiments.

[0040] Example 1 Quantitative analysis of potential active ingredients of Ganshuang granules based on UPLC-MS / MS 1.1 Instruments and reagents

[0041] 1.1.1 Test drugs and reagents

[0042] Ganshuang granules (Baoding Tianhao Pharmaceutical Co., Ltd., specification: 3g per bag, batch number: 201314), acetonitrile and methanol are UPLC-MS grade (USA) . Sodium formate, leucine-enkephalin Provided by Waters Corporation (USA). Mass spectrometry water is ultrapure water, produced by Milli-Q water system. Other reagents are analytical grade, and the drugs are shown in Table 5.

[0043] Table 1 - Information of experimental reference substances of the present invention

[0044]

[0045]

[0046] 1.1.2 Instruments

[0047] Binary pump and sample manager (Waters Company, USA), quadrupole time-of-flight mass spectrometer (Waters Company, USA), 1 / 100,000 electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.), ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), ultrapure water meter (Merck Chemical Technology (Shanghai) Co., Ltd.), high-speed refrigerated centrifuge (Anhui Zhongke Zhongjia Scientific Instrument Co., Ltd.), vortex oscillator (Changzhou Guohua Electric Co., Ltd.), nitrogen blowdown instrument (Beijing Yousheng United Technology Co., Ltd.), biochemical culture instrument (Shanghai Kuntian Laboratory Instrument Co., Ltd.), digital display gas 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 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, peony indole, hesperidin, chicoric acid, chlorogenic acid, salvianolic acid B, narcissin, kaempferol, codonopsis pilosula, amygdalin, hyperoside, and rutin reference substances, respectively, with precision. Weigh, place in a 2 ml volumetric flask, add chromatographic methanol, dilute to the scale, shake well to obtain mother solutions of reference substances (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 4.11, 8.00, 3.59, 2.37, 2.39, 4.98, 7.75, 4.04, 4.76, 3.37, 4.45, 2.46, 2.67, and 2.84 mg of saikosaponin A, saikosaponin D, naringin, peony indole, hesperidin, chicoric acid, chlorogenic acid, salvianolic acid B, narcissin, kaempferol, codonopsis pilosula, amygdalin, hyperoside, and rutin reference substances, respectively, with precision. Weigh, place in a 2 ml volumetric flask, add chromatographic methanol, dilute to the scale, shake well to obtain mother solutions of reference substances (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 in the reference solution stock solution 1, place it in the same 2 mL volumetric flask, make up to volume with chromatographic methanol, and shake well to obtain mixed reference substances 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, respectively.

[0056] (4) Mixed reference solution 2

[0057] Take 100 μL of each compound in the reference solution stock solution 2, place it in the same 2 mL volumetric flask, make up to volume with chromatographic methanol, and shake well to obtain mixed reference substances 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, respectively.

[0058] 1.2.2 Preparation of test samples

[0059] Grind the Ganshuang granules into fine powder, take 1.0000g of Ganshuang granule powder, accurately weigh it, put it in a stoppered conical flask, add 20mL of methanol, weigh it, ultrasonicate it for 30min, centrifuge it (12000rpm, 10min, centrifugal radius 10cm), take the supernatant, and filter it through a 0.22μm microporous filter membrane to obtain the product.

[0060] 1.2.3 Chromatographic conditions

[0061] Waters Xevo G2-XS Q-TOF ultra-high performance liquid chromatograph, chromatographic column (2.1×50 mm, 1.7 μm); column temperature: 40°C; flow rate: 0.3 mL·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°C; capillary voltage: 3.0 kV; cone gas flow rate: 50 L / h; desolvation temperature: 350°C; desolvation gas flow rate: 1000 L / h; nebulizer gas pressure: 7×105 Pa; parent ion scanning range: m / z 80-1200. The 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 suitability investigation

[0068] After the system is fully balanced, take the mixed reference solution 1 under item 2.1 and inject it continuously for 5 injections, and the mixed reference solution 2 and inject it continuously for 2 injections; saikosaponin A, saikosaponin D, naringin, paeoniflorin, hesperidin, chicoric acid, chlorogenic acid, salvianolic acid B, narcissin, kaempferol, codonopsis pilosula, amygdalin, hyperoside, rutin). In the chromatogram of each reference solution in the system suitability investigation, the separation between the peak to be measured and the adjacent peak is less than 1.5: the number of theoretical plates is greater than 5000; the reference solution 1 is injected continuously for 5 injections, and the relative standard deviation (RSD) of the peak area to be measured does not exceed 10.0%; the relative standard deviation of retention time does not exceed 2.0%, and the recovery rate of reference solution 2 should be between 90.0% and 110.0%. In the linear relationship investigation, R>9.88 for each compound. It shows that the analytical system (including method, operation and instrument) meets the requirements of the determination.

[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 Ganshuang granules test solution under "1.2.2" and inject and measure according to the test conditions under "1.2.3 and 1.2.4". Compare the known standard concentration and peak area with the peak area of ​​the compound in the test sample, and calculate the proportion of the test varieties of husaponin A, bupleuronidin, naringin, peony indole, 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 Pharmacodynamic Components of Ganshuang 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 methods

[0080] Same as Example 1

[0081] 2.3 Experimental Results

[0082] 2.3.1 Information on compounds with poor ADME properties in Ganshuang granules

[0083] The molecular weights of saikosaponin A, saikosaponin D, naringin, paeoniflorin, hesperidin, chicoric acid, chlorogenic acid, salvianolic acid B, narcissusin, kaempferol glycoside, codonopsis pilosula glycoside, amygdalin, hyperoside and rutin are all greater than 300, the number of polar functional groups and the degree of unsaturation of benzene rings are relatively high, the plasma protein binding rate is low, they are non-hepatic drug 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 compounds with poor ADME properties after bacterial incubation

[0085] This compound can be detected in the Ganshuang granules test sample by UPLC-MS / MS, indicating that it is a compound in the Ganshuang granules. It can be seen from the table that the peak area of ​​the compound decreases after bacterial incubation, 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] Bupleurosaponin A, Bupleurosaponin D, naringin, paeoniflorin, hesperidin, chicoric acid, chlorogenic acid, salvianolic acid B, narcissus glycoside, kaempferol glycoside, codonopsis pilosula, amygdalin, hyperoside and rutin are compounds in Ganshuang Granules, and their ADME properties are poor and their contents are significantly reduced after incubation with bacteria. These 14 compounds are compounds contained in the monarch drug Bupleurum of Ganshuang Granules, chlorogenic acid and codonopsis pilosula are compounds in the monarch drug Codonopsis pilosula, salvianolic acid B, naringin, hesperidin, peoniflorin, chicoric acid and amygdalin are the main compounds in the ministerial and adjuvant compounds, and according to literature research, these 14 compounds have anti-liver fibrosis effects, so the above 14 compounds are identified as potential active ingredients of Ganshuang Granules.

[0091] Example 3

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

[0093] 3.1 Materials

[0094] 3.1.1 Instruments

[0095] Binary pump and sample manager (Waters, USA); quadrupole time-of-flight mass spectrometer (Waters, USA); 1 / 100,000 electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.); ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); ultrapure water instrument (Merck Chemical Technology (Shanghai) Co., Ltd.); high-speed refrigerated centrifuge (Anhui Zhongke Zhongjia Scientific Instrument Co., Ltd.); vortex oscillator (Changzhou Guohua Electric Co., Ltd.); nitrogen blowdown instrument (Beijing Yousheng United Technology Co., Ltd.); biochemical culture instrument (Shanghai Kuntian Laboratory Instrument Co., Ltd.).

[0096] 3.1.2 Drugs and reagents

[0097] 3.1.2 Drugs and reagents

[0098] Ganshuang granule extract powder was purchased from Buchang Baoding Tianhao Pharmaceutical Co., Ltd. (production date 2023.5.27, extract yield 23.65%), and reference substances such as saikosaponin A, saikosaponin D, and naringin were purchased from China Food and Drug Inspection Institutes.

[0099] 3.1.3 Animals

[0100] SD male 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 Chinese Medicine (approval number: SVCMDL20230214002).

[0101] 3.2 Methods

[0102] 3.2.3 Preparation of anaerobic culture medium

[0103] Weigh 49.0 g of GAM medium, heat and dissolve in 1000 mL of distilled water, sterilize under high pressure at 121°C for 15 min, cool to about 50°C, add 1 mL of sterile 0.1% vitamin K1 solution and 1 mL of hemin (5 mg / mL) per 1000 mL 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 normal saline, and rotate at 5000r / min -1 , 10 minutes, take the supernatant and add 9 times the amount of anaerobic culture medium.

[0106] Preparation of reference solution

[0107] Weigh about 2 mg of standard products such as tanshinone ⅡA, ferulic acid, emodin, quercetin, emodin methyl ether, kaempferol, codonopsis pilosula glycoside, hesperetin, paeoniflorin, neohesperidin, tanshinone Ⅰ, chicoric acid, saikosaponin D, polydatin, baicalin, naringin, luteolin, paeoniflorin, rosmarinic acid, baicalin, oxidized paeoniflorin, gallic acid, aloe-emodin, chrysophanol, isorhamnetin, rhein, and rutin, add 70% methanol for ultrasonic dissolution, and pass through a 0.22 μm filter membrane.

[0108] 3.2.5 Experimental grouping and incubation

[0109] The experiment set up a blank group, a control group, and a drug group. The blank group was 10mL of bacterial incubation solution, and the control group was 25mg of Ganshuang granule extract powder and 10mL of anaerobic culture solution. The drug group was 25mg of Ganshuang granule extract powder and 10mL of bacterial incubation solution. Three parallel experiments were set up in each group. After vortexing for 2 minutes, the culture bags containing anaerobic gas production bags were placed in a culture bag at 37℃ for 24 hours.

[0110] 3.2.6 Intestinal flora incubation sample processing

[0111] Take 1 mL of the blank group, control group and drug-treated group solution in item "3.2.5" and add 1 mL of chromatographic methanol to terminate the reaction. Centrifuge at 8000 r / min. -1 Centrifuge for 10 min, take the supernatant, blow dry with nitrogen, and then dilute with methanol to 15 mg ml -1 , shake well and filter through a 0.22μm filter membrane, take the filtrate into a liquid phase vial as the test sample, and perform sample injection and analysis 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-treated group solution in 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, spin dry it on a rotary evaporator, and add 20 mL of benchtop solution to reconstitute it.

[0114] 3.2.8 Preparation of everted intestinal sac model

[0115] Take rats that have fasted for 12 hours, kill them by dislocation, cut the skin and muscles along the midline of the abdomen and quickly remove the small intestine, divide the small intestine into three sections, put the intestinal sections into 0℃ Tyrode solution, carefully flip them with silicone hoses and hang them in the isolated intestinal perfusion experimental device, keep the temperature at 37℃, and pass 95% O2 and 5% CO2 mixed gas. There is 2mL blank Tyrode solution in each intestinal tube, and the blank group, control group, and drug group intestinal absorption test solution in "2.2.7" are respectively outside the intestinal tube. All the liquid in the intestinal sac of each group is collected after 1 hour. One part is used for qualitative analysis, and the other part is used for pharmacodynamic research.

[0116] 3.2.9 Intestinal absorption sample processing

[0117] Take 1 ml of the intestinal absorption liquid under "3.2.8" and centrifuge it at 8000 r / min. -1 , centrifuge for 10 minutes, take the supernatant, blow dry with nitrogen, dilute 1000 times with chromatographic methanol, filter through a 0.22μm filter membrane, take the filtrate and place it in a liquid phase vial as a test sample for intestinal absorption, and perform sampling and analysis 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, chromatographic column (2.1×50 mm, 1.7 μm); column temperature: 40°C; flow rate: 0.3 mL·min -1 ; Injection volume: 2μL; Mobile phase: A: 0.1% formic acid water; Mobile phase: B: acetonitrile; Gradient elution (0-5min, 4-8% B; 5-10min, 8%-13% B; 10-15min, 13% B; 15-18min, 13%-15% B; 18-19min, 15-20% B; 19-24min, 20% B; 24-28min, 20%-40% B; 28-33min, 40% B; 33-35min, 40-50% B; 35-40min, 5% B).

[0120] 3.2.11 Mass spectrometry conditions

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

[0122] 3.3 Results

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

[0124] The total ion current of the intestinal absorption fluid after incubation with intestinal flora of the compounds measured by UPLC-Q-TOF / MS technology is shown in the attached Figure 3 After normalization, the intestinal absorption difference diagram is shown in Figure 4 As can be seen from the figure, the number of compound peaks increased after bacterial incubation, and the relative intensity was higher than before incubation.

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

[0126] 4.1 Instruments

[0127] 4.1 Test drugs and reagents

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

[0129] 4.2 Methods

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

[0131] The blank group, control group and drug group were intestinal absorbed liquid nitrogen and dried, dissolved in 1 ml DMSO, and diluted with DMEM to 1·103, 1·10 4 , 1.10 5 , 1.10 6 , 1.10 7 Store at 4℃.

[0132] 4.2.2 Screening of safe drug concentration

[0133] Cells in the logarithmic growth phase were collected and HSC-T6 cells were plated at 5×103·100 μL per well. -1 Inoculate into 96-well plate, incubate for 24h and dilute 1 · 10 3 , 1.10 4 , 1.10 5 , 1.10 6 , 1.10 7The blank group intestinal absorption solution was added to each well at 10ul and continued to be cultured. After 24 hours, 10μL of CCK-8 reagent was added to each well. After 2 hours, the absorbance at 450nm was measured with an enzyme marker to screen the safe dosage. The experiment also set up a control well (without drug), 3 secondary wells for each group, and each experiment was performed at least 3 times independently.

[0134] 4.2.3 Cell modeling and drug intervention

[0135] Cells in the logarithmic growth phase were collected, and HSC-T6 cells were inoculated into 96-well plates at 5×103·100μL-1 per well. After 24h, the cells were washed once with PBS, and fresh DMEM without PBS was added to the culture dish to keep the cells in the G0 phase and starved for 12h. Then, the intestinal absorption fluid of the control group and the drug group at the safe drug concentration was diluted in the culture medium containing 10ng·mL-1TGF-β1 and added to each well. After incubation for 48h, CCK-810μl was added, and the culture was continued at 37℃ for 2h. The absorbance (A) value at a wavelength of 450nm was measured by an enzyme marker. The experiment also set up normal control wells (containing cells, culture medium and CCK8 reagent) and model wells (containing cells, culture medium, TGF-β1 and CCK8 reagent), and 3 sub-wells were set for each group. Each experiment was performed at least 3 times independently.

[0136] 4.3 Results

[0137] 4.3.1 Screening of safe drug concentration in intestinal absorption solution based on CCK8 method

[0138] Dilution 1·10 3 , 1.10 4 , 1.10 5 Blank enteric absorption solution inhibited the growth of hepatic stellate cells and was cytotoxic, while the 1·10 dilution 6 and 1.10 7 The blank intestinal absorption solution had no toxic effect on hepatic stellate cells, so the dilution of 1·10 6 ~1.10 7 The intestinal absorption solution was used as the safe dosing concentration to investigate the effects of the compounds on hepatic stellate cells before and after incubation with the flora (see Figure 5 ).

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

[0140] Compared with the blank group, the number of surviving HSC-T6 cells in the model group increased significantly (P<0.01); compared with the model group, the number of surviving HSC-T6 cells in the model group increased significantly (P<0.01); 6 , 4.10 6 , 6.10 6 ,8.10 6The intestinal absorption fluid of the control group and the drug group acted on the cells, and it was found that the intestinal absorption fluid before and after bacterial incubation could inhibit the proliferation of HSC-T6 cells (P<0.01), and the inhibition rate of intestinal absorption fluid on hepatic stellate cells after bacterial incubation was significantly higher than that of the control group, indicating that the metabolites produced after bacterial incubation can increase intestinal absorption and thus increase the efficacy of drugs ( Figure 6 ).

[0141] 4.4 Experimental Conclusion

[0142] Fourteen compounds including saikosaponin A, saikosaponin D, and rutin were found in Ganshuang granules with poor ADME properties and could be degraded by intestinal flora. The number of compounds in the intestinal absorption fluid increased after incubation with flora, and the inhibition rate of liver fibrosis cells was stronger than that of the non-incubated group.

[0143] Finally, it should be noted that the present invention is not limited to the above specific embodiments, which are merely illustrative and instructive, rather than restrictive. Under the guidance of this specification, any changes, equivalent substitutions and improvements made by a person skilled in the art within the spirit and substance of the present invention are within the protection scope of the present invention.

Claims

1. A method for detecting the active ingredients of a traditional Chinese medicine composition by UPLC-MS / MS, characterized in that: The detection method comprises the following steps: ⑴. Preparation of mixed reference solution: Weigh saikosaponin A, saikosaponin D, naringin, peony root, hesperidin, chicoric acid, chlorogenic acid, salvianolic acid B, narcissus glycosides, kaempferol, codonopsis pilosula, amygdalin, hyperoside, and rutin reference substances respectively, weigh, add methanol, dilute to the scale, shake well, and obtain; ⑵. Preparation of test solution: Take the Chinese medicine composition, grind it into fine powder, accurately weigh it, add methanol, ultrasonicate it, centrifuge it, take the supernatant, filter it, and obtain it; ⑶、Chromatographic conditions: ultra-high performance liquid chromatography, C 18 Chromatographic column; column temperature: 38-42°C; flow rate: 0.1-0.5 mL min -1 ; Mobile phase: A: 0.1% formic acid water; Mobile phase: B: acetonitrile; Gradient elution (0-5min, 95-90% A; 5-10min, 90%-65% A; 10-15min, 65% A; 15-20min, 65%-60% A; 20-25min, 60-35% A; 25-25.1min, 35%-95% A; 25.1-26min, 95% A); (4) Mass spectrometry conditions were as follows: ionization mode: electrospray ionization; ion source temperature: 150°C; capillary voltage: 3.0 kV; cone gas flow rate: 50 L / h; desolvation air temperature: 350°C; desolvation gas flow rate: 1000 L / h; nebulizer gas pressure: 7×105 Pa; parent ion scanning range: m / z 80-1200.

2. The detection method according to claim 1, characterized in that: In step (1) of the detection method, the concentration of saikosaponin A in the test solution is 50-120 μg / mL, the concentration of saikosaponin D is 150-120 μg / mL, the concentration of naringin is 80-100 μg / mL, the concentration of paeoniflorin is 50-70 μg / mL, the concentration of hesperidin is 50-70 μg / mL, the concentration of chicoric acid is 100-125 μg / mL, and the concentration of chlorogenic acid is 17 0-190μg / mL, the concentration of salvianolic acid B is 85-100μg / mL, the concentration of narcissin is 105-120μg / mL, the concentration of kaempferol is 80-90μg / mL, the concentration of codonopsis pilosula glycoside is 90-115μg / mL, the concentration of amygdalin is 55-65μg / mL, the concentration of hyperoside is 60-70μg / mL, and the concentration of rutin is 60-70g / mL.

3. The detection method according to claim 1, characterized in that: In the preparation of the test solution in step (2) of the detection method, the ultrasonic treatment time is 20 to 40 minutes.

4. The detection method according to claim 1, characterized in that: In the preparation of the test solution in step (2) of the detection method, the rotation speed of the centrifugal treatment is 11000-14000 rpm and the centrifugation time is 8-12 min.

5. The detection method according to claim 1, characterized in that: 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.

6. The detection method according to claim 1, characterized in that: The chromatographic detection conditions in step (3) of the detection method are: 18 The specifications of the chromatographic column are 2.1×50 mm, 1.7 μm.

7. The detection method according to claim 1, characterized in that: The chromatographic detection conditions in step (3) of the detection method are as follows: column temperature: 40° C.; flow rate: 0.3 mL·min -1 .

8. The detection method according to claim 1, characterized in that: The main mass spectrometry parameters of the components to be tested in the mass spectrometry conditions of step (4) are as follows:

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