Traditional Chinese medicine compound composition for treating middle and advanced diabetic nephropathy as well as preparation method and quality detection method of traditional Chinese medicine compound composition

Abstract

The invention discloses a traditional Chinese medicine compound composition for treating middle and advanced diabetic nephropathy as well as a quality detection method and application of the traditional Chinese medicine compound composition. The traditional Chinese medicine compound composition is prepared from fructus rosae laevigatae, radix astragali, fructus corni, radix cyathulae, cortex eucommiae, poria cocos, roasted rhizoma atractylodis macrocephalae, herba leonuri, rhizoma alismatis, herba cistanche and rhizoma imperatae. A quality detection method is screened through a large number of experiments, and finally qualitative and quantitative detection is performed by adopting high performance liquid chromatography, so that the compound components can be analyzed comprehensively and objectively. According to the present invention, the formula is optimized according to the pathogenesis characteristics of diabetic nephropathy and the traditional Chinese medicine syndrome differentiation and treatment theory, and the clinical research results show that the compound composition can significantly improve the renal hypofunction caused by diabetes mellitus, and the clinical medication is safe.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicines, and in particular to a traditional Chinese medicine compound composition for treating middle and late stage diabetic nephropathy, a preparation method thereof, a quality detection method thereof and an application thereof. Background Art

[0002] In recent years, the prevalence of diabetes in my country has increased significantly, and the number of patients with microvascular complications has increased year by year. Epidemiological surveys show that the vast majority of diabetic patients in my country have type 2 diabetes. Type 2 diabetic nephropathy (T2DKD) is the most important chronic microvascular complication of type 2 diabetes, which is clinically characterized by persistent albuminuria and progressive decline in glomerular filtration rate. Continuous proteinuria continues to cause kidney damage, and renal dysfunction in the middle and late stages becomes a difficulty in clinical treatment. At present, Western medicine has limited treatment methods, mainly through lifestyle intervention and control of blood pressure, blood sugar, blood lipids, etc. to delay the progression of kidney disease. Commonly used drugs such as angiotensin-converting enzyme inhibitors (ACEI) have been proven to have a renal protective effect, but may have a series of adverse reactions such as urogenital infection and ketoacidosis. This situation has led us to focus our research on further exploration of traditional Chinese medicine treatment, hoping to find a breakthrough treatment method. Summary of the invention

[0003] Purpose of the invention: The purpose of the present invention is to provide a quality detection method and application of a Chinese medicine compound composition for treating mid-to-late stage diabetic nephropathy.

[0004] The present invention screened the compound composition through a large number of experiments. The Chinese medicine compound composition consists of eleven Chinese medicinal materials including Rosa laevigatae, Astragalus, Cornus officinalis, Cyathula officinalis, Eucommia ulmoides, Poria, stir-fried Atractylodes macrocephala, Leonurus japonicus, Alisma orientalis, Cistanche deserticola and Imperata cylindrica. Clinical studies have shown that the Chinese medicine compound composition has the effect of significantly improving the renal function impairment in patients with middle and late stage T2DKD.

[0005] The chemical composition of Jinqi Yishen Fang is complex, the origin of the ingredients is widely distributed, and there are few related quality control methods. In order to ensure the effectiveness and safety of the medication, the present invention establishes a high-performance liquid chromatography detection method to provide a reference for comprehensive evaluation and control of the quality and clinical application of Jinqi Yishen Fang.

[0006] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0007] A Chinese medicinal compound composition for treating mid-to-late stage diabetic nephropathy, characterized in that it is composed of Rosa laevigata, Astragalus, Fructus Corni, Cyathula officinalis, Eucommia ulmoides, Poria, stir-fried Atractylodes macrocephala, Leonurus japonicus, Rhizoma Alismatis, Cistanche deserticola, and Imperata cylindrica.

[0008] As a preferred embodiment, the above-mentioned Chinese herbal compound composition for treating diabetic nephropathy is composed of 15-45 parts of Rosea Laevigata, 20-60 parts of Astragalus, 20-60 parts of Cornus Officinalis, 15-45 parts of Cyathulae Bidentatae, 10-30 parts of Eucommia ulmoides, 20-60 parts of Poria, 15-45 parts of stir-fried Atractylodes macrocephala, 20-60 parts of Leonurus japonicus, 10-30 parts of Alisma Orientalis, 10-30 parts of Cistanche, and 20-60 parts of Imperatae Rhizoma.

[0009] As a preferred embodiment, the above-mentioned Chinese herbal compound composition for treating middle and late stage diabetic nephropathy is composed of 15 parts of Rosea Laevigata, 20 parts of raw Astragalus, 20 parts of Cornus Officinalis, 15 parts of Cyathula Bidentatae, 10 parts of Eucommia ulmoides, 20 parts of Poria, 15 parts of stir-fried Atractylodes macrocephala, 20 parts of Leonurus japonicus, 10 parts of Alisma Orientalis, 10 parts of Cistanche deserticola, and 20 parts of Imperata cylindrica.

[0010] The preparation method of the Chinese medicine compound composition for treating middle and late stage diabetic nephropathy of the present invention comprises the following steps: taking Rosa laevigata, Astragalus, Fructus Corni, Cyathula officinalis, Eucommia ulmoides, Poria, stir-fried Atractylodes macrocephala, Leonurus japonicus, Rhizoma Alismatis, Cistanche deserticola and Imperatae cylindrica according to weight proportions, adding water or ethanol for extraction, combining the extracts, filtering to obtain a filtrate, taking the filtrate and concentrating it under reduced pressure, then centrifuging it in a straight tube high-speed centrifuge, taking the supernatant and concentrating it to obtain the compound.

[0011] As a preferred embodiment, in the method for preparing the Chinese herbal compound composition for treating mid-to-late stage diabetic nephropathy, the extraction solvent is water or ethanol, and the extraction method is decoction, ultrasound, reflux or continuous reflux extraction.

[0012] As a preferred embodiment, the method for preparing the Chinese medicine compound composition for treating mid-to-late stage diabetic nephropathy of the present invention comprises the following steps:

[0013] The above 11 medicinal pieces were decocted twice. The first time, 12 times the amount of water was added, soaked for 30 minutes, decocted for 30 minutes, and filtered; the second time, 10 times the amount of water was added, decocted for 30 minutes, and filtered. The filtrate was concentrated under reduced pressure to a relative density of about 0.5 (60°C), and centrifuged in a straight tube high-speed centrifuge (speed: 16000r·min -1 , flow rate: 4~6L·min -1 ), take the supernatant and get the medicinal solution.

[0014] The quality detection method of the Chinese medicinal compound composition for treating diabetic nephropathy of the present invention adopts high performance liquid chromatography for detection.

[0015] As a preferred embodiment, the high performance liquid phase quantitative detection comprises the following steps:

[0016] (1) Preparation of mixed reference solution

[0017] Accurately weigh gallic acid, 5-hydroxymethylfurfural, geniposide, neochlorogenic acid, loganin, leonurine hydrochloride, echinacoside, calycosin and amaranthol and place them in 10mL volumetric flasks, add appropriate amount of methanol to dissolve, treat with ultrasound (280W, 40kHz) for 15min, cool and dilute to scale with methanol to obtain each reference substance stock solution. Accurately draw appropriate amount of each reference substance stock solution, place it in the same 10mL volumetric flask, make up to volume with methanol, shake well, and obtain a mixed reference substance solution (the mass concentration of each component is 0.474, 2.00, 0.194, 0.106, 0.698, 0.225, 0.137, 0.169, 0.156mg·mL -1 );

[0018] (2) Preparation of test solution

[0019] Accurately measure 5 mL of the Chinese medicine compound extract into a 10 mL volumetric flask, add an appropriate amount of methanol, and treat with ultrasound (280 W, 40 kHz) for 15 min. After cooling, dilute with methanol to the scale, and the solution is heated at 14000 r / min. -1 Centrifuge for 10 minutes and take the supernatant to obtain the test solution;

[0020] (3) Establishment of standard curve

[0021] Take the mixed reference solution of step (1), and place 0.1, 0.2, 0.5, 1.0, 2.0, and 3.0 mL of the mixed reference solution in 5 mL volumetric flasks, dilute to the mark with methanol, and obtain mixed reference solutions of a series of concentrations. Inject and measure, and record the peak area. Perform linear regression with mass concentration (X) as the abscissa and corresponding chromatographic peak area (Y) as the ordinate, and obtain the regression equation of gallic acid as Y=2.57×10 6 X+1.17×10 4 , r = 0.9998; the regression equation of 5-hydroxymethylfurfural is Y = 7.64 × 10 6 X+3.16×10 3 , r = 0.9998; the regression equation of geniposide acid is Y = 8.52 × 10 6 X+1.20×10 4 , r = 0.9998; the regression equation of neochlorogenic acid is Y = 1.10 × 10 7 X-5.54×10 3 , r = 0.9996; the regression equation of loganin is Y = 9.41 × 10 6 X-1.67×10 4 , r = 0.9997; the regression equation of leonurine hydrochloride is Y = 9.37 × 10 6 X-3.47×10 3, r = 0.9999; the regression equation of echinacea glycoside is Y = 3.99 × 10 6 X-1.55×10 3 , r = 0.9999; the regression equation of calycosin isoflavone glycosides is Y = 4.58 × 10 6 X-5.95×10 3 , r = 0.9997; the regression equation of cyathasterol is Y = 5.88 × 10 6 X-7.32×10 3 , r = 0.9999;

[0022] (4) Content determination

[0023] Accurately pipette the test solution of step (1) and the reference solution of step (2) into the liquid chromatograph, substitute the peak area into the standard curve equation of step (3), and calculate the contents of gallic acid, 5-hydroxymethylfurfural, geniposide, neochlorogenic acid, loganin, leonurine hydrochloride, echinacoside, calycosin and amaranthol in the test sample.

[0024] As a preferred embodiment, in the above-mentioned method for detecting the quality of the Chinese herbal compound composition for treating diabetic nephropathy, the chromatographic conditions of step (3) and step (4) are:

[0025] Tianjin Zhefen Acutfex PW-C 18 Chromatographic column (250×4.6 mm, 5 μm), mobile phase: acetonitrile (A)-0.1% phosphoric acid solution (B), gradient elution (0-12 min, 3%→5% A; 12-27 min, 5%→8% A; 27-40 min, 8%→12% A; 40-56 min, 12%→14% A; 56-77 min, 14%→23% A; 77-86 min, 23%→85% A; 86-90 min, 85% A), detection wavelength: 240 nm (geniposide, loganin, calixasterol), 310 nm (gallic acid, 5-HMF, neochlorogenic acid, leonurine hydrochloride, echinacoside, calycosin), volume flow rate: 1.0 mL min -1 , column temperature 30°C, injection volume 5 μL.

[0026] The beneficial effects of the present invention are:

[0027] 1. The present invention selects and forms a prescription according to the characteristics of the onset of middle and late stage diabetic nephropathy and the principle of syndrome differentiation and treatment of traditional Chinese medicine. In the prescription, Cherry Fructus can nourish the kidney and consolidate essence, Astragalus can strengthen the spleen and replenish qi, and they are all monarch drugs; Poria can strengthen the spleen and promote diuresis and eliminate dampness, Stir-fried Atractylodes can replenish qi, strengthen the spleen and promote diuresis, Cornus can nourish the liver and kidney, and Alisma can promote diuresis and reduce turbidity, and they are all minister drugs; Eucommia can nourish the liver and kidney, Cistanche can nourish the kidney and promote yang and relieve constipation, and the three are adjuvant drugs; Cyathula can promote blood circulation, remove blood stasis and reduce turbidity, Leonurus can clear heat, promote blood circulation and promote diuresis, and Imperata cylindrica can clear heat, cool blood and promote diuresis, and they are all guide drugs. Looking at the whole prescription, tonifying the kidney and strengthening the spleen, promoting blood circulation and reducing turbidity can improve the pathogenesis of "blood stasis and damage to collaterals" in the middle and late stages of T2DKD. The best raw material composition and dosage ratio are screened out through a large number of experiments, and the Chinese medicine compound obtained by combination can effectively alleviate the renal function impairment of patients in the middle and late stages of T2DKD, and significantly improve the clinical efficacy of treating diabetic nephropathy.

[0028] The Chinese medicine compound disclosed in the patent ZL2023108283120 previously authorized by the applicant is significantly different from the Chinese medicine compound of the present invention, as follows: (1) Efficacy and indications. The main efficacy of patent ZL2023108283120 is "removing dampness and removing blood stasis, unblocking collaterals and tonifying the kidney". It is mainly used for patients with diabetic nephropathy who are diagnosed as "dampness and blood stasis blocking collaterals" in traditional Chinese medicine, and is mainly used to treat diabetic nephropathy proteinuria; while the main efficacy of the present invention is "tonifying the kidney and strengthening the spleen, activating blood circulation and reducing turbidity". It is mainly used for patients with diabetic nephropathy who are diagnosed as "blood stasis and damage to collaterals" in traditional Chinese medicine, and is mainly used to treat diabetic nephropathy renal insufficiency. (2) Applicable population. Patent ZL2023108283120 is suitable for patients with mid-stage diabetic nephropathy (the main clinical manifestation is massive proteinuria); while the present invention is suitable for patients with late-stage diabetic nephropathy (the main clinical manifestation is severe renal dysfunction). (3) Clinical efficacy. Qi patent ZL2023108283120 is used to reduce the level of proteinuria (urine microalbumin creatinine ratio, UACR) in patients with diabetic nephropathy; while the present invention is used to reduce the serum creatinine level and delay the decline of renal function (glomerular filtration rate, GFR) in patients with diabetic nephropathy. Therefore, the two are significantly different in terms of treatment principles and types of treatment.

[0029] 2. The present invention uses a large number of experimental screening detection methods and adopts high performance liquid chromatography for quantitative detection, which can comprehensively and objectively detect and analyze the content of active ingredients in the other party, and methodological tests show that the high performance liquid chromatography provided by the present invention has high precision, good repeatability and stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a schematic diagram of the chromatogram of the mixed reference substance at 240 nm, in which 1. gallic acid 2.5-HMF 3. geniposide 4. neochlorogenic acid 5. loganin 6. leonurine hydrochloride 7. echinacoside 8. calycosin glycosides 9. cyperasterol.

[0031] Figure 2Schematic diagram of the chromatogram of the test sample at 240 nm.

[0032] Figure 3 Schematic diagram of the chromatogram of the mixed reference substance at 310 nm.

[0033] Figure 4 Schematic diagram of the chromatogram of the test sample at 310 nm.

[0034] Figure 5 Schematic diagram of the chromatogram at 240nm for a negative sample lacking Cornus officinalis.

[0035] Figure 6 Schematic diagram of the chromatogram at 310 nm for a negative sample lacking Cornus officinalis.

[0036] Figure 7 Schematic diagram of the chromatogram at 240nm for negative samples lacking Eucommia ulmoides and Cistanche deserticola.

[0037] Figure 8 This is a schematic diagram of the chromatogram at 310 nm for a negative sample lacking Eucommia ulmoides and Imperata cylindrica.

[0038] Fig. 9 This is a schematic diagram of the chromatogram at 310nm for a negative sample lacking Leonurus japonicus.

[0039] Fig.10 Schematic diagram of the chromatogram at 310 nm for a negative sample lacking Cistanche deserticola.

[0040] Fig.11 Schematic diagram of the chromatogram at 310 nm for a negative sample lacking Astragalus.

[0041] Fig.12 This is a schematic diagram of the chromatogram at 240nm for a negative sample lacking Cyathula Bidentata. DETAILED DESCRIPTION

[0042] The embodiments of the present invention will be described in detail below in conjunction with the examples. If no specific conditions are specified in the examples, they are carried out under conventional conditions or conditions recommended by the manufacturer. After reading the present invention, modifications of various equivalent forms of the present invention by those skilled in the art all fall within the scope defined by the claims attached to this application. All other embodiments obtained by those of ordinary skill in the art without creative work based on the embodiments in the present invention belong to the scope of protection of the present invention.

[0043] Example 1

[0044] 1. A Chinese medicinal compound composition for treating middle and late stage diabetic nephropathy, which is composed of 15g of Rosea Laevigata, 20g of raw Astragalus, 20g of Cornus Officinalis, 15g of Cyathula officinalis, 10g of Eucommia ulmoides, 20g of Poria, 15g of stir-fried Atractylodes macrocephala, 20g of Leonurus japonicus, 10g of Alisma orientalis, 10g of Cistanche deserticola, and 20g of Imperata cylindrica.

[0045] 2. The specific steps of the preparation method of the Chinese medicine compound composition are:

[0046] Take 15g of Rosa Laevigatae, 20g of Raw Astragalus, 20g of Cornus officinalis, 15g of Cyathula officinalis, 10g of Eucommia ulmoides, 20g of Poria, 15g of Stir-fried Atractylodes macrocephala, 20g of Leonurus japonicus, 10g of Alisma orientalis, 10g of Cistanche deserticola, and 20g of Imperata cylindrica. Add 12 times the amount of water for the first time, soak for 30min, decoct for 30min, and filter; add 10 times the amount of water for the second time, decoct for 30min, and filter. Concentrate the filtrate under reduced pressure to a relative density of about 0.5 (60℃), and centrifuge it in a straight tube high-speed centrifuge (speed: 16000r·min -1 , flow rate: 4~6L·min -1 ), take the supernatant and get the medicinal solution.

[0047] Example 2

[0048] 1. A Chinese medicinal compound composition for treating middle and late stage diabetic nephropathy, which is composed of 45g of Rosea Laevigata, 60g of raw Astragalus, 60g of Cornus Officinalis, 45g of Cyathula, 30g of Eucommia, 60g of Poria, 45g of stir-fried Atractylodes, 60g of Leonurus japonicus, 30g of Alisma Orientalis, 30g of Cistanche, and 60g of Imperatae Rhizoma.

[0049] 2. The specific steps of the preparation method of the Chinese medicine compound composition are:

[0050] Take 45g of Rosa Laevigatae, 60g of Raw Astragalus, 60g of Cornus officinalis, 45g of Cyathula officinalis, 30g of Eucommia ulmoides, 60g of Poria, 45g of Stir-fried Atractylodes macrocephala, 60g of Leonurus japonicus, 30g of Alisma orientalis, 30g of Cistanche deserticola, and 60g of Imperata cylindrica. Add 10 times the amount of ethanol for the first time, soak for 30min, reflux extract for 60min, and filter; add 8 times the amount of ethanol for the second time, reflux extract for 30min, and filter. The filtrate is concentrated under reduced pressure to a relative density of about 0.5 (60℃), and centrifuged in a straight tube high-speed centrifuge (speed: 16000r·min -1 , flow rate: 4~6L·min -1 ), take the supernatant and get the medicinal solution.

[0051] Example 3

[0052] The liquid phase detection method of the Chinese medicine compound composition specifically comprises the following steps:

[0053] (1) Chromatographic conditions

[0054] Tianjin Zhefen Acutfex PW-C 18Chromatographic column (250×4.6 mm, 5 μm), mobile phase: acetonitrile (A)-0.1% phosphoric acid solution (B), gradient elution (0-12 min, 3%→5% A; 12-27 min, 5%→8% A; 27-40 min, 8%→12% A; 40-56 min, 12%→14% A; 56-77 min, 14%→23% A; 77-86 min, 23%→85% A; 86-90 min, 85% A), detection wavelength: 240 nm (geniposide, loganin, calixasterol), 310 nm (gallic acid, 5-HMF, neochlorogenic acid, leonurine hydrochloride, echinacoside, calycosin), volume flow rate: 1.0 mL min -1 , column temperature 30°C, injection volume 5 μL.

[0055] (2) Preparation of mixed reference solution

[0056] Accurately weigh the reference substances gallic acid, 5-hydroxymethylfurfural, geniposide, neochlorogenic acid, loganin, leonurine hydrochloride, echinacoside, calycosin and amaranthol, and place them in 10 mL volumetric flasks, add appropriate amount of methanol to dissolve, treat with ultrasound (280W, 40kHz) for 15 min, cool and dilute to the mark with methanol to obtain the reference substance stock solution. Accurately draw appropriate amount of each reference substance stock solution, place it in the same 10 mL volumetric flask, make up to volume with methanol, shake well, and obtain the mixed reference substance solution (the mass concentration of each component is 0.474, 2.00, 0.194, 0.106, 0.698, 0.225, 0.137, 0.169, 0.156 mg·mL -1 ).

[0057] (3) Preparation of test solution

[0058] Accurately measure 5 mL of the solution in Example 1 into a 10 mL volumetric flask, add an appropriate amount of methanol, and treat with ultrasound (280 W, 40 kHz) for 15 min. After cooling, dilute with methanol to the scale, and rotate the solution at 14000 r / min. -1 Centrifuge for 10 minutes and take the supernatant to obtain the test solution;

[0059] (4) Preparation of negative sample solution

[0060] According to the prescription and extraction process of Example 1, negative samples lacking Cornus officinalis (gallic acid, 5-hydroxymethylfurfural, loganin), lacking Eucommia ulmoides and Cistanche deserticola (geniposide), lacking Eucommia ulmoides and Imperata cylindrica (neochlorogenic acid), lacking Leonurus japonicus (leonurine hydrochloride), lacking Cistanche deserticola (echinacoside), lacking Astragalus membranaceus (calycosin isoflavone glycosides), and lacking Cyathula capitata (amaranthus sterol) were prepared respectively, and treated according to the method in (3) to obtain negative samples.

[0061] (5) Specificity test

[0062] Take the mixed reference solution, test solution and each negative sample solution and inject them under the chromatographic conditions of item (1) and record the chromatogram. Figure 1 The results showed that the chromatographic peaks of the components to be tested in the test solution had good separation, the retention time was consistent with the chromatographic peaks in the mixed reference solution, and there was no interference in the negative.

[0063] (6) Linear relationship investigation

[0064] Take 0.1, 0.2, 0.5, 1.0, 2.0, and 3.0 mL of the mixed reference solution prepared in step (1) and place them in 5 mL volumetric flasks, respectively. Dilute to the mark with methanol to obtain mixed reference solutions of different concentrations. Inject and measure, and record the peak area. Linear regression was performed with mass concentration (X) as the abscissa and the corresponding chromatographic peak area (Y) as the ordinate. The regression equation of gallic acid was Y=2.57×106X+1.17×104, r=0.9998; the regression equation of 5-hydroxymethylfurfural was Y=7.64×106X+3.16×103, r=0.9998; the regression equation of geniposide was Y=8.52×106X+1.20×104, r=0.9998; the regression equation of neochlorogenic acid was Y=1.10×107X-5.54×103, r=0.9996; The regression equation of qiansu glycoside is Y=9.41×106X-1.67×104, r=0.9997; the regression equation of leonurine hydrochloride is Y=9.37×106X-3.47×103, r=0.9999; the regression equation of echinacoside is Y=3.99×106X-1.55×103, r=0.9999; the regression equation of calycosin isoflavone glycoside is Y=4.58×106X-5.95×103, r=0.9997; the regression equation of cyperasterol is Y=5.88×106X-7.32×103, r=0.9999;

[0065] (7) Precision test

[0066] The test solution under item (3) was taken and sampled and measured six times continuously under the chromatographic conditions of item (1). The RSDs of the peak areas of gallic acid, 5-hydroxymethylfurfural, geniposide, neochlorogenic acid, loganin, leonurine hydrochloride, echinacoside, calycosin and cyperasterol were 0.76%, 0.12%, 0.78%, 0.83%, 0.16%, 0.27%, 1.4%, 1.6% and 1.0%, respectively, indicating that the instrument had good precision.

[0067] (8) Stability test

[0068] The test solution under item (3) was placed at room temperature for 0, 2, 4, 8, 16 and 24 h, respectively. Then, the sample was injected and measured under the chromatographic conditions of item (1). The RSDs of the peak areas of gallic acid, 5-hydroxymethylfurfural, geniposide, neochlorogenic acid, loganin, leonurine hydrochloride, echinacoside, calycosin and calix sterol were 1.4%, 0.83%, 0.56%, 1.2%, 1.0%, 2.3%, 2.2%, 1.6% and 1.1%, respectively, indicating that the solution had good stability within 24 h.

[0069] (9) Repeatability test

[0070] From the same batch of water extracts, 6 test solutions were prepared in parallel according to the method in item (3). The samples were injected and determined under the chromatographic conditions in item (1). The average contents of gallic acid, 5-hydroxymethylfurfural, geniposide, neochlorogenic acid, loganin, leonurine hydrochloride, echinacoside, calycosin and calixasterol were 0.368, 0.486, 0.059, 0.026, 0.552, 0.091, 0.048, 0.059 and 0.063 mg·g, respectively. -1 , RSDs were 2.7%, 0.9%, 2.0%, 2.8%, 2.1%, 1.1%, 2.9%, 1.5%, and 2.3%, respectively, indicating that the method had good repeatability.

[0071] (10) Sample recovery test

[0072] Accurately pipette 2.5 mL of the aqueous extract of each component with known content, add the mixed reference solution (the mass concentrations of gallic acid, 5-hydroxymethylfurfural, geniposide, neochlorogenic acid, loganin, leonurine hydrochloride, echinacoside, calycosin and calix sterol are 94.8, 400, 38.9, 21.2, 140, 45.0, 27.5, 33.8 and 31.1 μg mL -1 )2.5mL, dilute to 10mL volumetric flask with methanol, prepare 6 sample recovery solutions in parallel according to the method under (3), inject and measure under the chromatographic conditions of (1), calculate the recovery rate, and the results are shown in Table 1.

[0073] Table 1 Sample recovery test results (n = 6)

[0074]

[0075]

[0076] (11) Sample content determination

[0077] Prepare three test sample solutions in parallel according to the method in item (3), inject and measure under the chromatographic conditions in item (1), calculate the component content and average value, and the results are shown in Table 2.

[0078] Table 2 Determination results of 9 components (mg·g -1 , n=3)

[0079] Element 1 2 3 average value gallic acid 0.356 0.359 0.362 0.359 5-Hydroxymethylfurfural 0.486 0.487 0.485 0.486 Geniposidic acid 0.059 0.060 0.058 0.059 Neochlorogenic Acid 0.026 0.025 0.026 0.026 Loganin 0.551 0.559 0.559 0.556 Leonurine Hydrochloride 0.090 0.092 0.091 0.091 Echinacoside 0.047 0.048 0.049 0.048 Isoflavone glycosides 0.059 0.059 0.059 0.059 Cyasterol 0.063 0.062 0.062 0.062 .

[0080] Example 4 Clinical Study

[0081] 1. Case inclusion criteria

[0082] (1) Meet the diagnostic criteria for type 2 diabetes; (2) Meet the diagnostic criteria for type 2 diabetic nephropathy stage A1-3G3-4, and exclude other causes of renal impairment, such as primary and other secondary glomerular diseases, hypertensive kidney damage, acute kidney injury, etc.; (3) Aged 30 to 75 years, regardless of gender; (4) Sign the informed consent form.

[0083] 2. Case Exclusion Criteria

[0084] (1) FBG ≥ 13.9 mmol / L or acute complications of diabetes within the past 3 months, such as ketoacidosis or diabetic hyperosmolar state; (2) Other kidney diseases not caused by diabetes, such as hypertensive nephropathy, membranous nephropathy and secondary nephritis. (3) Patients with liver failure and or a history of hepatic encephalopathy; acute severe infection; acute cardiovascular and cerebrovascular disease; malignant tumors or other malignant diseases that affect prognosis; (4) Patients who have taken glucocorticoids, immunosuppressants and other Chinese medicine decoctions within 1 month; (5) Patients who are pregnant, breastfeeding, mentally ill or other conditions that may lead to decreased compliance.

[0085] 3. Case exclusion criteria

[0086] (1) Those who do not take medication regularly; (2) Those who have serious adverse events during medication and are not suitable for continued treatment; Those who use other drugs during the observation period that may affect the experimental judgment of this study; (3) Those who participate in other clinical research at the same time during the trial.

[0087] 4. Sample size estimation

[0088] This study was a randomized controlled trial. The treatment group was the Jinqi Yishen prescription treatment group, and the control group was the basic treatment control group. The eGFR of the subjects was the observed outcome indicator. According to the literature review and pre-experimental results, the mean eGFR of the control group was 46.36±13.93ml / min / 1.73m 2 (σ=13.93), the mean difference of eGFR between the treatment group and the control group is expected to be 10 (δ=10), assuming α=0.05, bilateral Z α =1.96, the confidence level is 90%, Z β =1.28. According to the sample size calculation formula:

[0089]

[0090] Calculation shows that n=82 cases, considering 1:1 randomization, that is, 41 subjects are needed in the treatment group and 41 subjects in the control group. Taking into account 10% loss to follow-up and refusal to be interviewed, the final number of subjects required is at least 45 in the treatment group and 45 in the control group, for a total of at least 90 subjects.

[0091] 5. Study Design

[0092] This experiment adopts a randomized controlled study. Patients who meet the inclusion criteria and visit the outpatient and inpatient departments of the Endocrinology Department of Jiangsu Provincial Hospital of Traditional Chinese Medicine were selected and randomly divided into a treatment group and a control group. Both groups were given basic Western medicine treatment, and the treatment group was treated with the decoction of Example 1 (named Jinqi Yishen Fang) at the same time. All patients were followed up once every 4 weeks for 12 consecutive weeks. The changes in indicators such as eGFR and glucose and lipid metabolism in the two groups were observed and compared at the time of enrollment, after the 0th and 12th week.

[0093] 6. Statistical Analysis

[0094] Excel was used to establish a data information database, and SPSS25.0 statistical software was used to conduct statistical analysis on the collected case data. The t test was used to express the difference between the two groups. The median and interquartile range of non-normal distribution quantitative data were calculated using the rank sum test. The chi-square test was used to analyze the categorical data, and P < 0.05 was considered statistically significant.

[0095] 7. Research Results

[0096] Baseline data showed that there were no significant differences in gender, age, disease course, HbA1c, urine albumin to creatinine ratio (UACR) and estimated glomerular filtration rate (eGFR) between the control group and the treatment group in patients with T2DKD in the middle and late stages, and they were clinically comparable, as shown in Table 3. Preliminary clinical studies have shown that Jinqi Yishen Recipe, which has the effect of "tonifying the kidney, activating blood circulation and reducing turbidity" and has been taken orally for 12 consecutive weeks, is significantly better than the control group in improving the decline in eGFR in patients with T2DKD in the middle and late stages, and has achieved good clinical efficacy in treating renal impairment in patients with T2DKD, as shown in Table 4.

[0097] Table 3 Comparison of baseline data between the treatment group and the control group

[0098]

[0099] Table 4 Comparison of eGFR between the treatment group and the control group after treatment

[0100]

[0101] The present invention adopts the theory of syndrome differentiation and treatment to formulate the prescription according to the theory of traditional Chinese medicine. The above experimental results show that the Chinese medicine compound provided by the present invention has the effect of improving the renal function impairment of patients with T2DKD in the middle and late stages, and the clinical experimental results show that the Chinese medicine compound provided by the present invention has no adverse reactions after long-term use, and the medication is safer.

[0102] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Chinese medicinal compound composition for treating mid- to late-stage diabetic nephropathy, characterized in that: It is composed of Rosa laevigata, Astragalus, Cornus officinalis, Cyathula officinalis, Eucommia ulmoides, Poria cocos, stir-fried Atractylodes macrocephala, Leonurus japonicus, Alisma orientalis, Cistanche deserticola, and Imperata cylindrica.

2. The Chinese medicinal compound composition for treating mid- to late-stage diabetic nephropathy according to claim 1, characterized in that: It consists of 15-45 parts of Rosea Laevigatae, 20-60 parts of Astragalus, 20-60 parts of Cornus Officinalis, 15-45 parts of Cyathulae, 10-30 parts of Eucommia, 20-60 parts of Poria, 15-45 parts of stir-fried Atractylodes, 20-60 parts of Leonurus japonicus, 10-30 parts of Alisma Orientalis, 10-30 parts of Cistanche, and 20-60 parts of Imperatae Rhizoma.

3. The Chinese medicinal compound composition for treating mid- to late-stage diabetic nephropathy according to claim 2, characterized in that: It consists of 15 parts of golden cherry fruit, 20 parts of raw astragalus, 20 parts of cornus fruit, 15 parts of Sichuan cyathula, 10 parts of eucommia, 20 parts of tuckahoe, 15 parts of stir-fried atractylodes, 20 parts of motherwort, 10 parts of oriental water plantain, 10 parts of cistanche and 20 parts of imperata root.

4. The method for preparing the Chinese medicinal compound composition for treating mid- to late-stage diabetic nephropathy according to any one of claims 1 to 3, characterized in that: Take Rosa Laevigatae, Astragalus, Cornus officinalis, Cyathula officinalis, Eucommia ulmoides, Poria, stir-fried Atractylodes macrocephala, Leonurus japonicus, Alisma orientalis, Cistanche deserticola, Imperata cylindrica by weight, add water or ethanol to extract, combine the extracts, filter to obtain a filtrate, take the filtrate and concentrate it under reduced pressure, then centrifuge it in a straight tube high-speed centrifuge, take the supernatant and concentrate it to obtain the product.

5. The method for preparing the Chinese medicinal compound composition for treating mid- to late-stage diabetic nephropathy according to claim 4, characterized in that: The extraction solvent is water or ethanol, and the extraction method is decoction, ultrasound, reflux or continuous reflux extraction.

6. The quality detection method of the Chinese medicinal compound composition for treating mid-to-late stage diabetic nephropathy according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Preparation of mixed reference solution Accurately weigh gallic acid, 5-hydroxymethylfurfural, geniposide, neochlorogenic acid, loganin, leonurine hydrochloride, echinacoside, calycosin and calix sterol and place them in 10 mL volumetric flasks, add appropriate amount of methanol to dissolve, treat with ultrasound (280 W, 40 kHz) for 15 min, cool and dilute to the mark with methanol to obtain the stock solutions of each reference substance. Accurately pipette appropriate amount of each reference substance stock solution and place it in the same 10 mL volumetric flask, dilute to volume with methanol, shake well, and obtain the mixed reference substance solution; (2) Preparation of test solution Accurately measure the extract of the Chinese medicine composition prepared in claim 4 into a volumetric flask, add an appropriate amount of methanol, perform ultrasonic treatment, cool, dilute to scale with methanol, centrifuge, and take the supernatant to obtain the test solution; (3) Establishment of standard curve Take 0.1, 0.2, 0.5, 1.0, 2.0, and 3.0 mL of the mixed reference solution prepared in step (1) and place them in volumetric flasks, respectively, and dilute to the mark with methanol to obtain mixed reference solutions of a series of concentrations. Inject the solution for measurement and record the peak area. Perform linear regression with mass concentration X as the abscissa and corresponding chromatographic peak area Y as the ordinate. (4) Content determination Accurately pipette the test solution of step (1) and the reference solution of step (2) into the liquid chromatograph, substitute the peak area into the standard curve equation of step 3), and calculate the contents of gallic acid, 5-hydroxymethylfurfural, geniposide, neochlorogenic acid, loganin, leonurine hydrochloride, echinacoside, calycosin and amaranthol in the test sample.

7. The quality detection method of the Chinese medicinal compound composition for treating mid-to-late stage diabetic nephropathy according to claim 6, characterized in that: The mass concentrations of the components of the mixed reference solution are 0.474, 2.00, 0.194, 0.106, 0.698, 0.225, 0.137, 0.169, and 0.156 mg•mL -1 .

8. The quality detection method of the Chinese medicinal compound composition for treating mid-to-late stage diabetic nephropathy according to claim 6, characterized in that: The regression equation of gallic acid obtained in step (3) is Y = 2.57×106X + 1.17×104, r = 0.999 8; the regression equation of 5-hydroxymethylfurfural is Y = 7.64×106X + 3.16×103, r = 0.999 8; the regression equation of geniposide is Y = 8.52×106X + 1.20×104, r = 0.999 8; the regression equation of neochlorogenic acid is Y = 1.10×107X - 5.54×103, r = 0.999 6; the regression equation of loganin is Y = 9.41×106X - 1.67×104, r = 0.999 7; the regression equation of leonurine hydrochloride is Y = 9.37×106X - 3.47×103, r=0.999 9; the regression equation of echinacoside is Y = 3.99×106X - 1.55×103, r=0.999 9; the regression equation of calycosin isoflavone glycoside is Y = 4.58×106X - 5.95×103, r=0.999 7; the regression equation of amaranthosterol is Y = 5.88×106X -7.32×103, r=0.999 9.

9. The quality detection method of the Chinese medicinal compound composition for treating mid-to-late stage diabetic nephropathy according to claim 6, characterized in that: The chromatographic conditions of step (3) and step (4) are: Tianjin Zhefen Acutfex PW-C 18 The chromatographic column was 250×4.6 mm, 5 μm. The mobile phase was acetonitrile A-0.1% phosphoric acid solution B. The gradient elution was as follows: 0-12 min, 3%→5%A; 12-27 min, 5%→8%A; 27-40 min, 8%→12%A; 40-56 min, 12%→14%A; 56-77 min, 14%→23%A; 77-86 min, 23%→85%A; 86-90 min, 85%A. The detection wavelengths of geniposide, loganin, and calixasterol were 240 nm, and those of gallic acid, 5-HMF, neochlorogenic acid, leonurine hydrochloride, echinacoside, and calycosin were 310 nm. The volume flow rate was 1.0 mL·min -1 , column temperature 30 °C, injection volume 5 μL.

10. Use of the Chinese medicine compound composition according to any one of claims 1 to 3 in the preparation of a medicament for treating diabetic nephropathy or mid-to-late stage diabetic nephropathy.

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