A method for detecting active ingredients of sanjianji fibrosis of the kidney

Abstract

The application discloses an anti-renal interstitial fibrosis Sanhuang formula and a metabolomics research method thereof, the Sanhuang formula is composed of Astragalus membranaceus, Abelmoschus flower and rhubarb, and the application screens optimal chromatographic conditions and mass spectrometric conditions through a large number of experiments to analyze active ingredients and metabolites of the Sanhuang formula by LC-Q-TOF / MS, and by combining chemical group analysis with metabolomics analysis, active ingredients of the Sanhuang formula and metabolites with significant differences before and after intervention on renal interstitial fibrosis rats can be relatively comprehensively, effectively and reliably revealed, and this has important significance for exploring a potential action mechanism of the Sanhuang formula against renal interstitial fibrosis and evaluating safety and effectiveness of the Sanhuang formula.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine detection technology, specifically relating to a method for studying the active ingredients of Sanhuang formula and their metabolomics. Background Technology

[0002] The Sanhuang formula is composed of astragalus, hibiscus flower, and rhubarb. Clinical trial results show that it has a good anti-renal interstitial fibrosis effect. Currently, there are no reports on the quality testing methods and metabolomics methods for the Sanhuang formula.

[0003] Metabolomics analysis uses biological samples as the testing object, with the main purpose of detecting and screening metabolites of significant biological importance and statistically significant differences, and elucidating the metabolic processes and mechanisms of change in organisms. By screening differentially expressed metabolites and analyzing metabolic pathways, the functions of metabolites in the samples are predicted and analyzed.

[0004] This invention, based on metabolomics and LC-Q-TOF / MS, screened the active ingredients of Sanhuang Formula and identified metabolites with significant differences in animal models of renal interstitial fibrosis. This is of great significance for exploring the active ingredients of Sanhuang Formula and its potential mechanism of action in animal models of renal interstitial fibrosis, and provides a scientific basis for the use of this formula in clinical patients with renal interstitial fibrosis. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to overcome the shortcomings of the prior art and develop a method for detecting the active ingredients of Sanhuang formula and a metabolomics research method. The method of this invention can screen out the active ingredients of Sanhuang formula and screen out metabolites that show significant differences before and after Sanhuang formula intervention in animal models of renal interstitial fibrosis. This is of great significance for exploring the potential mechanism of Sanhuang formula in treating renal interstitial fibrosis and evaluating its safety and efficacy.

[0006] Technical solution: To achieve the above objectives, the technical solution adopted by this invention is as follows:

[0007] A traditional Chinese medicine composition for treating renal interstitial fibrosis, comprising Astragalus membranaceus, Hibiscus syriacus, and Rheum palmatum.

[0008] As a preferred embodiment, the traditional Chinese medicine composition for resisting renal interstitial fibrosis is characterized by comprising 30-90 parts of Astragalus membranaceus, 30-90 parts of Hibiscus syriacus, and 10-30 parts of Rheum palmatum.

[0009] A method for detecting the active ingredients of a new Sanhuang formula, comprising the following steps:

[0010] (1) Preparation of the extract of Xin Sanhuang Fang:

[0011] Take Astragalus membranaceus, Hibiscus syriacus flowers and rhubarb in the weight ratio, add water and decoct to extract, take the decoction, filter and collect the decoction extract, combine the decoction extracts, concentrate to obtain the new Sanhuang formula extract;

[0012] (2) Preparation of mixed standard solutions

[0013] Gallic acid, epicatechin, verbascoside glucose, hyperoside, hyoscyamine, ferulic acid, quercetin, kaempferol, aloe-emodin, rhein, astragaloside A, emodin, chrysophanol, and emodin methyl ether were weighed separately to prepare single reference solutions.

[0014] Then, take the reference solutions of gallic acid, epicatechin, verbascoside glucose, hyperoside, scopolamine, ferulic acid, quercetin, kaempferol, aloe-emodin, rhein, astragaloside A, emodin, chrysophanol and emodin methyl ether, mix them evenly and dilute them to obtain a mixed standard solution.

[0015] (3) Component analysis of the extract of Xin Sanhuang Formula

[0016] Take the extract of the new Sanhuang formula from step (1), centrifuge, and inject the centrifuged liquid into a liquid chromatography-mass spectrometry (LC-Q-TOF / MS) instrument for analysis.

[0017] A metabolomics research method for a new Sanhuang formula includes the following steps:

[0018] (1) Preparation of the extract of Xin Sanhuang Fang:

[0019] Take Astragalus membranaceus, Hibiscus syriacus flowers and Rheum palmatum according to the weight ratio, add water and decoct to extract, take the decoction, filter and collect the decoction extract, combine the decoction extracts, concentrate to obtain the new Sanhuang formula extract;

[0020] (2) Preparation of mixed standard solutions

[0021] Gallic acid, epicatechin, verbascoside glucose, hyperoside, hyoscyamine, ferulic acid, quercetin, kaempferol, aloe-emodin, rhein, astragaloside A, emodin, chrysophanol, and emodin methyl ether were weighed separately to prepare single reference solutions.

[0022] Then, take the reference solutions of gallic acid, epicatechin, verbascoside glucose, hyperoside, scopolamine, ferulic acid, quercetin, kaempferol, aloe-emodin, rhein, astragaloside A, emodin, chrysophanol and emodin methyl ether, mix them evenly and dilute them to obtain a mixed standard solution.

[0023] (3) Animal models and serum preparation:

[0024] Male SD rats were randomly divided into a normal control group and a renal interstitial fibrosis animal model group 14 days after modeling. Both the normal and model groups were administered the Xin Sanhuang Fang extract prepared in step (1) by gavage. Blood was collected from the orbital cavity of the rats by gavage. The plasma was then processed to obtain serum sample solution.

[0025] (4) Metabolic component analysis of the new Sanhuang formula

[0026] Take the mixed standard solution from step (2) and the serum sample from step (3) and inject them into a liquid chromatography-mass spectrometry (LC-Q-TOF / MS) instrument for analysis;

[0027] (5) The mass spectrometry data of the Sanhuangfang extract and plasma samples collected by LC-Q-TOF / MS were processed, and the metabolites of Sanhuangfang were identified and characterized by comparison and analysis with reference standards and database.

[0028] As a preferred option, the method described above, step (1) is as follows: take Astragalus membranaceus, Hibiscus syriacus and rhubarb in a weight ratio of 3:3:1, add 8 to 16 times the volume of water and decoct twice, each time for 30 to 120 minutes, filter and collect the decoction extract, combine the decoction extracts, concentrate and obtain the new Sanhuang formula extract.

[0029] As a preferred embodiment, the method for preparing the mixed standard solution in step (2) of the above-described method is as follows:

[0030] Weigh out astragaloside A, dilute with 50% ethanol containing 30% DMSO to obtain a stock solution of astragaloside A with a concentration of 2 mg / mL, and dilute with 50% methanol to a concentration of 100 μg / mL for later use.

[0031] Weigh out verbascoside, dilute with 50% ethanol to obtain a verbascoside stock solution with a concentration of 1 mg / mL, and dilute with 50% methanol to a concentration of 100 μg / mL for later use.

[0032] Weigh ferulic acid and dilute it with methanol to obtain a ferulic acid stock solution with a concentration of 1 mg / mL. Dilute it with 50% methanol to a concentration of 100 μg / mL for later use.

[0033] Weigh out kaempferol and dilute it with methanol to obtain a kaempferol stock solution with a concentration of 1 mg / mL. Dilute it with 50% methanol to a concentration of 100 μg / mL for later use.

[0034] Weigh out quercetin, dilute it with methanol to obtain a quercetin stock solution with a concentration of 2 mg / mL, and then dilute it with 50% methanol to a concentration of 100 μg / mL for later use.

[0035] Weigh out Hypericum and dilute it with 50% methanol to obtain a hyperoside stock solution with a concentration of 1 mg / mL. Dilute it with 50% methanol to a concentration of 100 μg / mL for later use.

[0036] Weigh out aloe-emodin and dilute it with ethanol and DMSO at a volume ratio of 1:1 to obtain an aloe-emodin stock solution with a concentration of 2 mg / mL. Dilute it with 50% methanol to a concentration of 100 μg / mL for later use.

[0037] Weigh out rhein and dilute it with ethanol:DMSO at a volume ratio of 1:1 to obtain a stock solution of rhein with a concentration of 1 mg / mL. Dilute it with 50% methanol to a concentration of 100 μg / mL for later use.

[0038] Take emodin and dilute it with ethanol:DMSO at a volume ratio of 1:1 to obtain a stock solution of emodin with a concentration of 1 mg / mL. Then dilute it with 50% methanol to a concentration of 100 μg / mL for later use.

[0039] Weigh out rhein and dilute it with ethanol:DMSO at a volume ratio of 5:13 to obtain a rhein stock solution with a concentration of 2.00 mg / mL. Dilute it with 50% methanol to a concentration of 100 μg / mL for later use.

[0040] Weigh out emodin methyl ether and dilute it with a mixture of ethanol and DMSO at a volume ratio of 5:13 to obtain a stock solution of emodin methyl ether with a concentration of 0.572 mg / mL. Dilute it with 50% methanol to a concentration of 100 μg / mL for later use.

[0041] Weigh out gallic acid and dilute it with 50% methanol to obtain a gallic acid stock solution with a concentration of 1 mg / mL. Dilute it with 50% methanol to a concentration of 100 μg / mL for later use.

[0042] Weigh epicatechin and dilute it with methanol to obtain a stock solution of epicatechin with a concentration of 1 mg / mL. Dilute it with 50% methanol to a concentration of 100 μg / mL for later use.

[0043] Weigh out hyoscyamine, dilute with 50% ethanol to obtain a hyoscyamine stock solution with a concentration of 1 mg / mL, and dilute with 50% methanol to a concentration of 100 μg / mL for later use;

[0044] Preparation of mixed standard solutions

[0045] Take individual standard solutions of astragaloside A, verbascoside, ferulic acid, kaempferol, quercetin, hyperoside, aloe-emodin, rhein, emodin, chrysophanol, emodin methyl ether, gallic acid, epicatechin, and hyoscyamine, each with a concentration of 100 μg / mL, mix them thoroughly, and dilute with 50% methanol to obtain a mixed standard solution with a concentration of 2.00 μg / mL for each compound.

[0046] As a preferred option, the metabolomics research method of the above-described Sanhuang formula, step (3) animal model and serum preparation method are as follows:

[0047] Male SD rats were randomly divided into a normal control group and a renal interstitial fibrosis animal model group. Fourteen days after modeling, both normal and model rats were administered the Xin Sanhuang Fang extract prepared in step (1) by gavage at a rate of 7.25 g / kg. Blood was collected from the orbital cavity of the rats at 0 h, 0.5 h, 1 h, 2 h and 4 h after administration.

[0048] Take 200 μL of plasma, add 800 μL of methanol, vortex for 1 min, centrifuge at 12000 rpm at 4℃ for 10 min, take 900 μL of supernatant, dry at 40℃, add 100 μL of 50% methanol to the residue to reconstitute, vortex for 1 min, centrifuge at 12000 rpm at 4℃ for 10 min, take 80 μL of supernatant, centrifuge at 12000 rpm at 4℃ for 10 min to obtain serum sample solution.

[0049] As a preferred embodiment, the liquid chromatography conditions for LC-Q-TOF / MS analysis in step 4 of the above-described method are as follows:

[0050] Chromatographic column: Agilent SB C18, 4.6 mm × 100 mm, 1.8 μm; mobile phase: Phase A: 0.1% formic acid aqueous solution, Phase B: methanol and acetonitrile (v / v); column temperature: 40 °C; gradient elution; gradient elution program as follows:

[0051] 0 0.40 85 15 2.5 0.40 80 20 7.0 0.40 75 25 12.0 0.40 50 50 19.0 0.40 10 90 21.0 0.40 10 90 28.0 0.55 40 60 31.0 0.55 85 15 35.0 0.40 85 15 .

[0052] The mass spectrometry conditions for the LC-Q-TOF / MS analysis are as follows:

[0053] Q-TOF / MS analysis employed ESI source positive and negative ion scanning modes. The negative ion source voltage was -4500V, the ion source temperature was 550℃, and the nebulizer gas was N2. The primary mass spectrometry scanning range was m / z 100–1000, DP: 80V; GS1: 50, GS2: 60, CUR: 20, CE: 10eV. Secondary mass spectrometry data were acquired using IDA mode, with an acquisition range of 90–1000, DP: 100V, CE: 25eV; CES: 15. The positive ion source voltage was 5500V, the ion source temperature was 550℃, and the nebulizer gas was N2. The primary mass spectrometry scanning range was: m / z 100~1000, DP: 100V; GS1: 50, GS2: 60, CUR: 20, CE: 10eV; Secondary mass spectrometry data were acquired using IDA mode, acquisition range: 90~1000, DP: 100V, CE: 30eV;

[0054] CES: 15; IDA settings: 10 peaks with response signal intensity > 100 cps were used for secondary mass spectrometry scanning, with dynamic background subtraction enabled.

[0055] The beneficial effects are as follows: This invention screened out the Sanhuang formula (Astragalus membranaceus, Hibiscus syriacus, and Rheum palmatum) with anti-renal interstitial fibrosis properties through extensive experiments. Furthermore, the optimal chromatographic and mass spectrometric conditions were determined through extensive experimentation, and LC-Q-TOF / MS analysis was performed to analyze the active ingredients and metabolites of the Sanhuang formula. The combination of chemomics and metabolomics analysis can comprehensively, effectively, and reliably reveal the active ingredients of the Sanhuang formula and the metabolites that showed significant differences before and after administration to animal models of renal interstitial fibrosis. This is of great significance for exploring the potential mechanism of action of the Sanhuang formula in treating renal interstitial fibrosis in animals and for evaluating its safety and efficacy. Attached Figure Description

[0056] Figure 1 The BPC diagram of the LC-Q-TOF / MS standard of the new Sanhuangfang formula is shown.

[0057] Figure 2 The TIC chromatograms of the new Sanhuang formula by LC-Q-TOF / MS are shown (top image shows positive ions, bottom image shows negative ions).

[0058] Figure 3 The image shows the XIC plot of the new Sanhuangfang LC-Q-TOF / MS in positive ion mode (the top image is the normal plot, and the bottom image is a magnified view).

[0059] Figure 4 The XIC spectra of the new Sanhuang formula under negative ion mode (LC-Q-TOF / MS: top image is the normal image, bottom image is a magnified view).

[0060] Figure 5 Plasma LC-Q-TOF / MS TIC plot (positive ion) of normal rats after gavage administration of Xin Sanhuang Fang (dark blue, purple-red, red, green, and light blue represent blank and plasma at 0.5h, 1h, 2h, and 4h after administration, respectively).

[0061] Figure 6 TIC plot of plasma LC-Q-TOF / MS in model rats after gavage administration of Xin Sanhuang Fang (positive ion) (dark blue, purple-red, red, green, and light blue represent blank and plasma at 0.5h, 1h, 2h, and 4h after administration, respectively).

[0062] Figure 7 TIC plot of plasma LC-Q-TOF / MS in normal rats after gavage administration of Xin Sanhuang Fang (negative ions) (dark blue, purple-red, red, green, and light blue represent blank and plasma at 0.5h, 1h, 2h, and 4h after administration, respectively).

[0063] Figure 8TIC plot of plasma LC-Q-TOF / MS in model rats after gavage administration of Xin Sanhuang Fang (negative ions) (dark blue, purple-red, red, green, and light blue represent blank and plasma at 0.5h, 1h, 2h, and 4h after administration, respectively).

[0064] Figure 9 Statistical graph of plasma migration components (original + original metabolites) in rats after gavage administration of Xin Sanhuang Fang.

[0065] Figure 10 Changes in liver and kidney function in UUO rats after intervention with Xin Sanhuang Fang extract.

[0066] Figure 11 Effect of Xin Sanhuang Formula Extract on Renal Fibrosis in UUO Rats 14 Days After Modeling.

[0067] Figure 12 Effects of the freeze-dried powder of Xin Sanhuang Fang on HK-2 cells.

[0068] Figure 13 The new Sanhuang formula reduces ROS generation and has antioxidant effects. Detailed Implementation

[0069] The embodiments of the present invention will be described in detail below with reference to the examples. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0070] The reagents and samples used in the following examples were astragaloside A, kaempferol, gallic acid, hyperoside, scopolamine, verbascoside, ferulic acid, quercetin, epicatechin, emodin, emodin methyl ether, rhein, and chrysophanol, purchased from Shanghai Yuanye Biotechnology Co., Ltd. Methanol and acetonitrile (chromatographic grade, Merck); formic acid (chromatographic grade, Shanghai Chemical Reagent Co., Ltd.); and ultrapure water.

[0071] Experimental instruments: AB SCIEX Triple TOF 5600 mass spectrometer; Sartorius CPA225D electronic balance (Sartorius GmbH, Germany); Biofuge PrimoR refrigerated high-speed centrifuge (Heraeus GmbH, Germany); Millipore Drict-Q ultrapure water system (Millipore GmbH, France); KQ 3200E ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); Centri Vap centrifuge concentrator (Labconco); WH-2 miniature vortex mixer (Shanghai Huxi Analytical Instrument Factory).

[0072] Example 1

[0073] A metabolomics research method for a new Sanhuang formula includes the following steps:

[0074] (1) Preparation of the extract of Xin Sanhuang Fang:

[0075] Take 30g of Astragalus membranaceus, 30g of Hibiscus syriacus, and 10g of Rheum palmatum. Add water at a ratio of 8:1 and decoct twice for 40 minutes each time. After filtration, collect the decoction extracts and combine them. Concentrate the extracts using a rotary evaporator at 50℃ and 200 rpm to obtain a new Sanhuang formula extract with a concentration of 5g / ml.

[0076] (2) Preparation of mixed standard solutions

[0077] Weigh out astragaloside A, dilute with 50% ethanol containing 30% DMSO to obtain a stock solution of astragaloside A with a concentration of 2 mg / mL, and dilute with 50% methanol to a concentration of 100 μg / mL for later use.

[0078] Weigh out verbascoside, dilute with 50% ethanol to obtain a verbascoside stock solution with a concentration of 1 mg / mL, and dilute with 50% methanol to a concentration of 100 μg / mL for later use.

[0079] Weigh ferulic acid and dilute it with methanol to obtain a ferulic acid stock solution with a concentration of 1 mg / mL. Dilute it with 50% methanol to a concentration of 100 μg / mL for later use.

[0080] Weigh out kaempferol and dilute it with methanol to obtain a kaempferol stock solution with a concentration of 1 mg / mL. Dilute it with 50% methanol to a concentration of 100 μg / mL for later use.

[0081] Weigh out quercetin, dilute it with methanol to obtain a quercetin stock solution with a concentration of 2 mg / mL, and then dilute it with 50% methanol to a concentration of 100 μg / mL for later use.

[0082] Weigh out Hypericum and dilute it with 50% methanol to obtain a hyperoside stock solution with a concentration of 1 mg / mL. Dilute it with 50% methanol to a concentration of 100 μg / mL for later use.

[0083] Weigh out aloe-emodin and dilute it with ethanol and DMSO at a volume ratio of 1:1 to obtain an aloe-emodin stock solution with a concentration of 2 mg / mL. Dilute it with 50% methanol to a concentration of 100 μg / mL for later use.

[0084] Weigh out rhein and dilute it with ethanol:DMSO at a volume ratio of 1:1 to obtain a stock solution of rhein with a concentration of 1 mg / mL. Dilute it with 50% methanol to a concentration of 100 μg / mL for later use.

[0085] Take emodin and dilute it with ethanol:DMSO at a volume ratio of 1:1 to obtain a stock solution of emodin with a concentration of 1 mg / mL. Then dilute it with 50% methanol to a concentration of 100 μg / mL for later use.

[0086] Weigh out rhein and dilute it with ethanol:DMSO at a volume ratio of 5:13 to obtain a rhein stock solution with a concentration of 2.00 mg / mL. Dilute it with 50% methanol to a concentration of 100 μg / mL for later use.

[0087] Weigh out emodin methyl ether and dilute it with a mixture of ethanol and DMSO at a volume ratio of 5:13 to obtain a stock solution of emodin methyl ether with a concentration of 0.572 mg / mL. Dilute it with 50% methanol to a concentration of 100 μg / mL for later use.

[0088] Weigh out gallic acid and dilute it with 50% methanol to obtain a gallic acid stock solution with a concentration of 1 mg / mL. Dilute it with 50% methanol to a concentration of 100 μg / mL for later use.

[0089] Weigh epicatechin and dilute it with methanol to obtain a stock solution of epicatechin with a concentration of 1 mg / mL. Dilute it with 50% methanol to a concentration of 100 μg / mL for later use.

[0090] Weigh out hyoscyamine, dilute with 50% ethanol to obtain a hyoscyamine stock solution with a concentration of 1 mg / mL, and dilute with 50% methanol to a concentration of 100 μg / mL for later use;

[0091] Preparation of mixed standard solutions

[0092] Take individual standard solutions of astragaloside A, verbascoside, ferulic acid, kaempferol, quercetin, hyperoside, aloe-emodin, rhein, emodin, chrysophanol, emodin methyl ether, gallic acid, epicatechin, and hyoscyamine, each with a concentration of 100 μg / mL, mix them thoroughly, and dilute with 50% methanol to obtain a mixed standard solution with a concentration of 2.00 μg / mL for each compound.

[0093] (3) Animal models and serum preparation:

[0094] Male SD rats were randomly divided into a normal control group and a renal interstitial fibrosis (UUO) model group. (The UUO modeling method was as follows: after fasting for 12 hours, the rats were weighed, and isoflurane was used to induce anesthesia at a concentration of 2-3%, and 1.5-2% was used for maintenance. The limbs were fixed. Routine disinfection was performed, a 2cm incision was made in the left lower abdomen, the left ureter was freed, the ureter was ligated twice along the lower pole of the left kidney, and the ureter was cut in the middle.) 14 days after modeling, both normal and model rats were given the Xin Sanhuang Fang extract prepared in step (1) by gavage at a concentration of 7.25g / kg. Blood was collected from the rats' orbital sinuses at 0h, 0.5h, 1h, 2h and 4h after administration.

[0095] Take 200 μL of plasma, add 800 μL of methanol, vortex for 1 min, centrifuge at 12000 rpm at 4℃ for 10 min, take 900 μL of supernatant, dry at 40℃, add 100 μL of 50% methanol to the residue to reconstitute, vortex for 1 min, centrifuge at 12000 rpm at 4℃ for 10 min, take 80 μL of supernatant, centrifuge at 12000 rpm at 4℃ for 10 min to obtain serum sample solution.

[0096] (4) Metabolic component analysis of the new Sanhuang formula

[0097] Take the decoction from step (1) and let it cool to room temperature. Shake and mix well. Take 100 μL of the decoction, add 900 μL of 50% methanol, shake and mix well, sonicate for 20 min, centrifuge at 12000 rpm and 4℃ for 10 min, and inject 5 μL of the supernatant decoction sample into a liquid chromatography-mass spectrometry (LC-Q-TOF / MS) instrument for analysis.

[0098] In addition, 5 μL of the mixed standard solution from step (2) and the serum sample from step (3) were injected into the liquid chromatography-mass spectrometry (LC-Q-TOF / MS) instrument for analysis.

[0099] (5) The mass spectrometry data of the Sanhuangfang extract and plasma samples collected by LC-Q-TOF / MS were processed, and the metabolites of Sanhuangfang were identified and characterized by comparison and analysis with reference standards and database.

[0100] The liquid chromatography conditions for the LC-Q-TOF / MS analysis are as follows:

[0101] Chromatographic column: Agilent SB C18, 4.6 mm × 100 mm, 1.8 μm; mobile phase: Phase A: 0.1% formic acid aqueous solution, Phase B: methanol and acetonitrile (volume ratio 1:1); column temperature: 40 °C; gradient elution; gradient elution program is shown in Table 1 below:

[0102] Table 1

[0103] 0 0.40 85 15 2.5 0.40 80 20 7.0 0.40 75 25 12.0 0.40 50 50 19.0 0.40 10 90 21.0 0.40 10 90 28.0 0.55 40 60 31.0 0.55 85 15 35.0 0.40 85 15 .

[0104] The mass spectrometry conditions for the LC-Q-TOF / MS analysis are as follows:

[0105] R-TOF / MS analysis was performed using ESI source positive and negative ion scanning modes. The negative ion source voltage was -4500V, the ion source temperature was 550℃, and the nebulizer gas was N2. The primary mass spectrometry scanning range was 100–1000 m / z, with DP: 80V; GS1: 50, GS2: 60, CUR: 20, and CE: 10 eV. Secondary mass spectrometry data were acquired using IDA mode, with an acquisition range of 90–1000 m / z, DP: 100V, CE: 25 eV, and CES: 15. The positive ion source voltage was 5500V, the ion source temperature was 550℃, and the nebulizer gas was N2. The primary mass spectrometry scanning range was: m / z 100~1000, DP: 100V; GS1: 50, GS2: 60, CUR: 20, CE: 10eV; Secondary mass spectrometry data were acquired using IDA mode, acquisition range: 90~1000, DP: 100V, CE: 30eV;

[0106] CES: 15; IDA settings: 10 peaks with response signal intensity > 100 cps were used for secondary mass spectrometry scanning, with dynamic background subtraction enabled.

[0107] (6) Analysis Results

[0108] (6.1) Chromatographic behavior of each standard

[0109] Under the above-mentioned chromatographic and mass spectrometric conditions, the retention times of gallic acid, epicatechin, verbascoside glucose, hyperoside, scopolamine, ferulic acid, quercetin, kaempferol, aloe-emodin, rhein, astragaloside A, emodin, chrysophanol, and emodin methyl ether were approximately 4.0, 9.0, 12.2, 12.5, 12.7, 13.0, 15.8, 17.3, 19.5, 20.2, 21.0, 21.8, 22.6, and 23.4 min, respectively. (See [reference needed]). Figure 1 And Table 2.

[0110] Table 2. LC-Q-TOF / MS data of various standards of Xin Sanhuang Fang.

[0111]

[0112] 2.2 Construction of the New Sanhuang Formula Compound Library

[0113] Literature reviews and the TCMSP database were used to construct compound libraries for Abelmoschus manihot, Astragalus membranaceus, and Rheum palmatum. Abelmoschus manihot contained 59 compounds, Astragalus membranaceus contained 153 compounds, and Rheum palmatum contained 307 compounds. After deduplication, a total of 468 compounds were identified. The structural formulas of each compound were plotted for identification and characterization of compounds in the Sanhuang formula and plasma samples.

[0114] 2.3 Identification and Characterization of the Chemical Components of the New Sanhuang Formula

[0115] Through comparison with standard samples, literature, and chemical fragment information, a total of 67 compounds were identified from the new Sanhuang formula.

[0116] The compounds included 7 phenolic acids, 3 nucleotides, 32 flavonoids (glycosides), 5 chromogens (glycosides), 10 anthraquinones, 5 amino acids, and 5 other compounds; 14 were derived from *Abelmoschus manihot*, 23 from *Astragalus membranaceus*, and 25 from *Rheum palmatum*. Four components were shared by *Astragalus membranaceus* and *Abelmoschus manihot*, and one component was shared by *Astragalus membranaceus* and *Rheum palmatum*. The LC-Q-TOF / MS chromatograms of the Sanhuang formula (including the total ion current TIC chromatogram and the extracted ion current XIC chromatograms of all identified compounds in positive and negative ion modes) are shown below. Figures 2-4 Data from LC-Q-TOF / MS and identification results for each compound are shown in Tables 3 to 8 (partial).

[0117] Table 3. LC-Q-TOF / MS data of the Xin Sanhuang Fangfang sample and identification results of each compound.

[0118]

[0119] Table 4

[0120]

[0121] Table 5

[0122]

[0123] Table 6

[0124]

[0125] Table 7

[0126]

[0127] Table 8

[0128]

[0129] Table 9

[0130]

[0131] Note: * indicates comparison with standard sample.

[0132] By comparing the component library of the aqueous decoction of Xin San Huang Formula and references, a total of 7 characteristic prototype components from the formula (excluding common components in aqueous decoctions and medicinal materials such as nucleotides, amino acids and vitamins), namely gallic acid, 2-(2'-hydroxypropyl)-5-methyl-7-hydroxychromone-7-O-β-D-glucopyranoside, formononetin, 6-methylrhein, aloe-emodin, rhein and emodin, and 27 potential metabolites including quercetin glucuronide conjugate and gossypetin / myricetin glucuronide conjugate were identified from the plasma of rats intragastrically administered with Xin San Huang Formula. The results are shown in Tables 10 to 12.

[0133] Table 10

[0134]

[0135] Table 11

[0136]

[0137]

[0138] Table 12

[0139]

[0140] Note: ND means not detected; Glu stands for glucuronic acid conjugate; SO3 stands for sulfate conjugate

[0141] The LC-Q-TOF / MS chromatogram (TIC diagrams in positive and negative ion modes) of plasma samples after intragastric administration of Xin San Huang Formula to rats is shown in Figures 5-8 . The transitional components in plasma of rats after intragastric administration of Xin San Huang Formula are shown in Figure 9 .

[0142] Example 2 Efficacy experiment of San Huang Formula

[0143] 1. Experimental animals and cells

[0144] Male SD rats aged 6-8 weeks (Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) with a body weight of 250±50 g were selected and housed in the Basic Pharmacology Laboratory of the Affiliated Hospital of Nanjing University of Chinese Medicine (Nanjing, Jiangsu). License No.: SYXK (Su) 2022-0070. All animals had free access to water and food, and were randomly divided into groups for experiment after 1 week of adaptive feeding. The feeding conditions were 50±5% humidity, 20°C room temperature, and 12 h alternating lighting. Human renal proximal tubular cells (human renal proximal tubular cells, HK-2) cell line was provided by the Cell Bank of the Chinese Academy of Sciences.

[0145] 2. Experimental methods

[0146] 2.1 Animal experiment

[0147] 2.1.1 Thirty-five SD rats were randomly divided into five groups (n=7 per group) after one week of acclimatization: a normal control group (saline by gavage); a renal interstitial fibrosis (UUO) model group; a UUO + low-dose Xin Sanhuang Fang group (Xin Sanhuang Fang decoction 5.42 g / kg / d by gavage); a UUO + high-dose Xin Sanhuang Fang group (Xin Sanhuang Fang decoction 10.85 g / kg / d by gavage); and a UUO + metformin group (metformin 65 mg / kg / d). All groups received Xin Sanhuang Fang by gavage for four weeks starting from the beginning of the modeling process. No antibiotics were used in any group. Gavage began one week before modeling, with each group receiving 10 ml / kg once daily at a fixed time.

[0148] Dosage calculation: New Sanhuang Formula (Astragalus membranaceus 30g, Hibiscus syriacus 30g, Rheum palmatum 10g). Unless otherwise specified, all Rheum palmatum mentioned in this invention is processed Rheum palmatum.

[0149] The dosage of Xin Sanhuang Formula in the low-dose group = clinical low dose (35g) / 60kg / day × rat equivalent area dose coefficient (6.3); the dosage of Xin Sanhuang Formula in the high-dose group = clinical low dose (770g) / 60kg / day × rat equivalent area dose coefficient (6.3).

[0150] New Three-Yellow Formula Decoction: Take 30g of Astragalus membranaceus, 30g of Hibiscus syriacus flower, and 10g of Rheum palmatum. Add 8 times the amount of water and decoct for 40 minutes. Filter and collect the filtrate. Add 8 times the amount of water again and decoct for 40 minutes. Filter and collect the filtrate. Mix the two filtrates and filter with medical gauze. Then concentrate in a rotary evaporator at 50℃ and 200 rpm. Store the concentrate at -80℃. After 24 hours, place it in a freeze dryer under vacuum mode at 0.25 mBar and -50℃ to freeze-dry into powder. Store in a sealed container at -20℃. Weigh an appropriate amount of the freeze-dried powder, dissolve it in PBS to form a prescription aqueous extract mother liquor (1g / mL), and then filter it through a 0.22μm filter. Aliquot the filtered mother liquor and store at -20℃.

[0151] 2.1.2 Specimen Collection

[0152] Four weeks after the start of modeling, rats were fasted for 12 hours. After weighing, they were anesthetized with isoflurane, and the abdominal cavity was opened. Blood samples were collected from the abdominal aorta. After exposing the kidneys, the left kidney tissue was excised, the capsule was removed, and the kidneys were washed with PBS, photographed, and their size, wet weight, and recorded. The kidneys were cut into two parts. One part was fixed in 4% paraformaldehyde for paraffin section preparation, followed by immunohistochemical staining and HE staining. The other part was placed in a cryovial, rapidly frozen in liquid nitrogen overnight, and then stored at -80°C.

[0153] 2.1.3 Detection Indicators

[0154] ① Use a biochemical analyzer to detect liver and kidney function indicators (Cr, BUN, ALT, and AST, etc.) in blood samples.

[0155] ②Western-blot detection of α-SMA, Fibronectin, E-cadherin, SOD2 and VDAC2-related proteins.

[0156] ③ Fresh kidney tissue was stained with DHE to observe changes in ROS expression in the kidney tissue; after the kidney tissue was embedded in paraffin blocks, HE and MASSON staining were performed, and α-SMA, Fibronectin, and E-cadherin tissue fluorescence staining were performed.

[0157] 2.2 In vivo experiments

[0158] 2.2.1 Cell grouping and model preparation:

[0159] HK-2 cells were cultured in DMEM / F12 medium supplemented with 10% Gibberish fetal bovine serum and 1% penicillin-streptomycin in a 37°C, 5% CO2 incubator. P4–P10 HK-2 cells in the logarithmic growth phase and in good growth condition were selected and cultured at 1 × 10⁶ cells per well. 5 Cells were seeded into 6-well plates and cultured at 37°C with 5% CO2 and saturated humidity. When the cells reached 50%–60% confluence, they were grouped according to the experimental objectives.

[0160] The effects of TGF-β stimulation on HK-2 cells were investigated: ① Normal group: cultured in DMEM / F12 medium; ② TGF-β group: TGF-β final concentration 10 ng / ml added to the culture medium; ③ TGF-β + Xin Sanhuang Fang lyophilized powder: TGF-β + Xin Sanhuang Fang lyophilized powder (0.1, 0.5, ...

[0161] Cells were cultured in 1.0, 2.5, 5.0, 7.5 and 10 mg / L medium; ④ TGF-β + metformin: Cells were cultured in TGF-β + metformin (1 mmol / L) medium. Note: Cells were treated with TGF-β and Xin Sanhuang Fang lyophilized powder for 24 h.

[0162] 2.2.2 Detection Indicators

[0163] ① Cellular TGF-β and drug intervention 24 hours later, the cells were photographed and preserved using an optical microscope.

[0164] ② Extract cell proteins from each group and detect α-SMA, Fibronectin, E-cadherin, AMPK and p-AMPK, SOD2 and VDAC2-related proteins by Western blot; find the optimal concentration for drug intervention.

[0165] ③ Cells were cultured in 35mm dishes and divided into groups: normal group, TGF-β, TGF+Xin Sanhuang Fang lyophilized powder, and TGF-β+metformin. After 24h of intervention, mitochondrial ROS were stained and photographed under a confocal microscope. Cells were also stained with fibrosis antibodies (Fibronectin, E-cadherin and α-SMA) and photographed under a confocal microscope.

[0166] 2.3 Data Statistics and Analysis

[0167] SPSS 26.0 statistical software was used. Quantitative data were described as mean ± standard deviation (±s). The t-test was used for comparisons between two groups, one-way ANOVA was used for comparisons among multiple groups, and Tukey or Dunnett's test was used for pairwise comparisons. An α = 0.05 level was used; a p-value < 0.05 was considered statistically significant.

[0168] 3. Experimental Results

[0169] 3.1 Changes in liver and kidney function after intervention with Xin Sanhuang Fang extract in UUO rats

[0170] like Figure 10 In UUO rats, blood urea nitrogen and creatinine levels were significantly elevated compared to the normal group. Gavage administration of Xin Sanhuang Fang decoction, regardless of the low or high dose, alleviated the deterioration of renal function in the rats. Simultaneously, administration of Xin Sanhuang Fang did not show significant abnormalities in liver function (alanine aminotransferase and aspartate aminotransferase) in the rats.

[0171] 3.2 Effect of Xin Sanhuang Formula Extract on Renal Fibrosis in UUO Rats 14 Days After Modeling

[0172] like Figure 11 As shown. Fourteen days after UUO modeling, the rat kidneys were visibly enlarged and the cortex thinned. HE and Masson staining results indicated significant dilation of the renal tubules, disappearance of tubular epithelial villi, widening of the renal interstitium, and aggravation of interstitial fibrosis. After intervention with Xin Sanhuang Fang, the above morphological changes were alleviated, and with the increase of Xin Sanhuang Fang drug dosage, renal interstitial fibrosis was further reduced. Immunofluorescence and Western blot analysis of renal tissue showed that compared with the normal group, the expression levels of fibrosis markers (Fbironectin, E-cadherin, and α-SMA) were significantly increased. After treatment with Xin Sanhuang Fang, the expression levels of the above two proteins gradually decreased with the increase of drug dosage. The expression level of the renal tubular epithelial marker protein E-cadherin was significantly decreased in the UUO group. After treatment with Xin Sanhuang Fang, the expression gradually increased.

[0173] 3.3 Effects of the freeze-dried powder of Xin Sanhuang Fang on HK-2 cells

[0174] like Figure 12 Under light microscopy, normal cells exhibited a cobblestone-like morphology. After TGF-β stimulation, the cells became spindle-shaped. Following treatment with Xin Sanhuang Fang lyophilized powder, the cells gradually changed from spindle-shaped to cobblestone-like with increasing drug concentration. At a drug concentration of 5 mg / L, the cells retained the cobblestone-like morphology. Simultaneously, Western blot analysis showed that the protein expression levels of fibrosis markers (Fbironectin and α-SMA) significantly increased after TGF-β stimulation. After intervention with Xin Sanhuang Fang lyophilized powder, the expression levels of these two proteins gradually decreased with increasing drug concentration, reaching their lowest values ​​at a Xin Sanhuang Fang lyophilized powder concentration of 5 mg / L. The expression of the renal tubular epithelial cell marker protein E-cadherin showed the opposite trend to the above two markers. After TGF-β stimulation, intervention with 5 mg / L of Xin Sanhuang Fang lyophilized powder showed a significant increase in the expression of Fbironectin and α-SMA in the cytoplasm via immunofluorescence. After further intervention with Xin Sanhuang Fang lyophilized powder, the distribution of these two proteins in the cytoplasm was significantly reduced. Simultaneously, E-cadherin expression showed the opposite trend.

[0175] 3.4 The effects of the new Sanhuang formula in reducing ROS generation and its antioxidant properties

[0176] like Figure 13 Fourteen days after UUO modeling, renal ROS production in rats significantly increased, while in rats administered Xin Sanhuang Fang by gavage, renal cortical ROS production significantly decreased, with higher doses showing a more significant reduction compared to lower doses. Western blot analysis revealed significantly reduced expression of renal superoxide dismutase type 2 (SOD 2) and mitochondrial porin 1 (VDAC 1) in UUO rats, indicating impaired antioxidant capacity. Intervention with Xin Sanhuang Fang significantly increased the expression of these two proteins, with higher doses showing better results than lower doses. In cell experiments, after TGF-β stimulation, Western blot analysis showed significantly reduced expression of SOD 2 and VDAC 1. Intervention with Xin Sanhuang Fang lyophilized powder showed a significant increase in the expression of these two proteins with increasing drug concentration, reaching a peak expression at a concentration of 5 mg / L. In mitochondrial membrane potential detection, normal cells showed high expression of membrane potential, exhibiting red fluorescence. Following TGF-β stimulation, mitochondria in renal tubular epithelial cells were damaged, resulting in a decrease in mitochondrial membrane potential and a greenish fluorescence. However, after intervention with Xin Sanhuang Fang (a traditional Chinese medicine formula), the fluorescence gradually increased to a reddish hue, indicating a significant reduction in damaged mitochondria. DHE staining and mitochondrial ROS staining both showed that TGF-β stimulation significantly increased ROS production in renal tubular epithelial cells, while intervention with Xin Sanhuang Fang significantly reduced ROS production, suggesting that Xin Sanhuang Fang possesses antioxidant capabilities.

Claims

1. A method for detecting the active ingredients of a new Sanhuang formula, characterized in that, The new Sanhuang formula consists of 30-90 parts of Astragalus membranaceus, 30-90 parts of Hibiscus syriacus, and 10-30 parts of Rheum palmatum. The method for detecting active ingredients includes the following steps: (1) Preparation of the extract of Xin Sanhuang Formula: Take Astragalus membranaceus, Hibiscus syriacus flowers and Rheum palmatum according to the weight ratio, add water and decoct to extract, take the decoction, filter and collect the decoction extract, combine the decoction extracts, concentrate to obtain the new Sanhuang formula extract; (2) Preparation of mixed standard solutions Gallic acid, epicatechin, verbascoside glucose, hyperoside, hyoscyamine, ferulic acid, quercetin, kaempferol, aloe-emodin, rhein, astragaloside A, emodin, chrysophanol, and emodin methyl ether were weighed separately to prepare single reference solutions. Then, take the reference solutions of gallic acid, epicatechin, verbascoside glucose, hyperoside, scopolamine, ferulic acid, quercetin, kaempferol, aloe-emodin, rhein, astragaloside A, emodin, chrysophanol and emodin methyl ether, mix them evenly and dilute them to obtain a mixed standard solution. (3) Component analysis of the extract of Xin Sanhuang Formula Take the extract of the new Sanhuang formula from step (1), centrifuge, and take the centrifuged liquid. Also take the mixed standard solution from step (2) and inject them into the liquid chromatography-mass spectrometry (LC-Q-TOF / MS) instrument for analysis. The liquid chromatography conditions for the LC-Q-TOF / MS analysis are as follows: Chromatographic column: Agilent SB C18, 4.6 mm × 100 mm, 1.8 μm; mobile phase: Phase A: 0.1% formic acid aqueous solution, Phase B: methanol and acetonitrile (v / v); column temperature: 40 °C; gradient elution; gradient elution program as follows: The mass spectrometry conditions for the LC-Q-TOF / MS analysis are as follows: Q-TOF / MS analysis was performed using ESI source positive and negative ion scanning modes. The negative ion source voltage was -4500V, the ion source temperature was 550℃, and the nebulizer gas was N2. The primary mass spectrometry scanning range was m / z 100~1000, DP: 80 V; GS1: 50, GS2: 60, CUR: 20, CE: 10 eV. Secondary mass spectrometry data were acquired using IDA mode, with an acquisition range of 90~1000, DP: 100V, CE: 25 eV; CES:

15. The positive ion source voltage was 5500V, the ion source temperature was 550℃, and the nebulizer gas was N2. The primary mass spectrometry scanning range was m / z 100~1000, DP: 100 V; GS1: 50, GS2: 60, CUR: 20, CE: 10 eV. IDA mode was used. The mode acquires secondary mass spectrometry data, with an acquisition range of 90~1000, DP: 100V, CE: 30 eV; CES: 15; IDA is set to scan the 10 highest peaks with response signal intensity >100 cps for secondary mass spectrometry, and dynamic background subtraction is enabled.

2. The method according to claim 1, characterized in that, Step (1): Take Astragalus membranaceus, Hibiscus syriacus flowers and Rheum palmatum in a weight ratio of 3:3:1, add 8-16 times the volume of water and decoct twice, each time for 30-120 minutes. After filtration, collect the decoction extract, combine the decoction extracts, concentrate and obtain the new Sanhuang formula extract.

3. The method according to claim 1, characterized in that, Step (2) Preparation of mixed standard solution: Weigh astragaloside A, dilute with 50% ethanol containing 30% DMSO to obtain astragaloside A stock solution with a concentration of 2 mg / mL, and dilute with 50% methanol to a concentration of 100 μg / mL for later use; Weigh out verbascoside, dilute with 50% ethanol to obtain a verbascoside stock solution with a concentration of 1 mg / mL, and dilute with 50% methanol to a concentration of 100 μg / mL for later use. Weigh ferulic acid and dilute it with methanol to obtain a ferulic acid stock solution with a concentration of 1 mg / mL. Dilute it with 50% methanol to a concentration of 100 μg / mL for later use. Weigh out kaempferol and dilute it with methanol to obtain a kaempferol stock solution with a concentration of 1 mg / mL. Dilute it with 50% methanol to a concentration of 100 μg / mL for later use. Weigh out quercetin and dilute it with methanol to obtain a quercetin stock solution with a concentration of 2 mg / mL. Dilute it with 50% methanol to a concentration of 100 μg / mL for later use. Weigh out Hypericum and dilute it with 50% methanol to obtain a hyperoside stock solution with a concentration of 1 mg / mL. Dilute it with 50% methanol to a concentration of 100 μg / mL for later use. Weigh out aloe-emodin and dilute it with ethanol and DMSO at a volume ratio of 1:1 to obtain an aloe-emodin stock solution with a concentration of 2 mg / mL. Dilute it with 50% methanol to a concentration of 100 μg / mL for later use. Weigh out rhein and dilute it with ethanol:DMSO at a volume ratio of 1:1 to obtain a stock solution of rhein with a concentration of 1 mg / mL. Dilute it with 50% methanol to a concentration of 100 μg / mL for later use. Take emodin and dilute it with ethanol:DMSO at a volume ratio of 1:1 to obtain an emodin stock solution with a concentration of 1 mg / mL. Then dilute it with 50% methanol to a concentration of 100 μg / mL for later use. Weigh out rhein and dilute it with ethanol:DMSO at a volume ratio of 5:13 to obtain a rhein stock solution with a concentration of 2.00 mg / mL. Dilute it with 50% methanol to a concentration of 100 μg / mL for later use. Weigh out emodin methyl ether and dilute it with a mixture of ethanol and DMSO at a volume ratio of 5:13 to obtain a stock solution of emodin methyl ether with a concentration of 0.572 mg / mL. Dilute it with 50% methanol to a concentration of 100 μg / mL for later use. Weigh out gallic acid and dilute it with 50% methanol to obtain a gallic acid stock solution with a concentration of 1 mg / mL. Dilute it with 50% methanol to a concentration of 100 μg / mL for later use. Weigh epicatechin and dilute it with methanol to obtain a stock solution of epicatechin with a concentration of 1 mg / mL. Dilute it with 50% methanol to a concentration of 100 μg / mL for later use. Weigh out hyoscyamine, dilute it with 50% ethanol to obtain a hyoscyamine stock solution with a concentration of 1 mg / mL, and dilute it with 50% methanol to a concentration of 100 μg / mL for later use. Preparation of mixed standard solutions Take individual standard solutions of astragaloside A, verbascoside, ferulic acid, kaempferol, quercetin, hyperoside, aloe-emodin, rhein, emodin, chrysophanol, emodin methyl ether, gallic acid, epicatechin, and hyoscyamine, each with a concentration of 100 μg / mL, mix them thoroughly, and dilute with 50% methanol to obtain a mixed standard solution with a concentration of 2.00 μg / mL for each compound.

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