CN107050116B

CN107050116B - Traditional Chinese medicine pill for treating diabetic nephropathy and preparation method and application thereof

Info

Publication number: CN107050116B
Application number: CN201611046921.7A
Authority: CN
Inventors: 王汝上; 朱荃; 郑兆广; 胡琴; 何宝; 段婷婷; 黄晓玲; 黄寿义; 高俊飞; 范丽霞; 顾斐; 杨琳琳; 黄艳霞
Original assignee: Guangzhou Consun Medicine R & D Co ltd; Kangchen Pharmaceutical Inner Mongolia Co ltd
Current assignee: GUANGZHOU CONSUN MEDICINE R & D Co.,Ltd.; Kangchen Pharmaceutical (Inner Mongolia) Co.,Ltd.
Priority date: 2016-11-23
Filing date: 2016-11-23
Publication date: 2020-10-30
Anticipated expiration: 2036-11-23
Also published as: CN107050116A

Abstract

The invention relates to a traditional Chinese medicine pill for treating diabetic nephropathy and a preparation method and application thereof. The active ingredients of the traditional Chinese medicine pill are traditional Chinese medicine composition prepared from astragalus, kudzu root and cortex mori radicis, microcrystalline cellulose is selected as a diluent, and talcum powder is selected as an anti-sticking agent, so that the traditional Chinese medicine pill is high in drug loading, high in product yield and high in coating efficiency. The active ingredients of the traditional Chinese medicine pill have obvious effects of resisting cell oxidation, resisting cell apoptosis and resisting reduction of negative charges of cell membranes, have obvious dose-effect relationship, can effectively protect structural barriers and charge barriers of kidney cells, and have obvious effect of protecting the kidney.

Description

Traditional Chinese medicine pill for treating diabetic nephropathy and preparation method and application thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to a traditional Chinese medicine pill for treating diabetic nephropathy, and a preparation method and application thereof.

Background

Diabetic nephropathy (DKD) is one of the most prominent microvascular complications of Diabetes Mellitus (DM), the leading cause of end-stage renal disease (ESRD). The microangiopathy in diabetic nephropathy refers to glomerulosclerosis, nodular, exudative and diffuse, and the three may exist alone or in combination. Statistically, the number of DM patients worldwide is 4.15 billion, which is expected to increase to 6.42 billion in 2040 years, with 20% to 45% of DM patients developing DKD. DM patients in China already reach 1.1 hundred million, and the incidence rate rises year by year. In 2012, 19% of ESRD patients in China are caused by DKD, and the proportion is as high as 38% -45.2% in developed countries such as Japan, Korea, United states, Australia and the like. DKD has become a major social problem threatening human health, placing a huge economic burden on society and individuals.

Clinically, diabetic nephropathy is manifested as proteinuria, hypertension and progressive renal function impairment. The pathological manifestations of the disease are the proliferation of glomerular mesangial cells, the increase of oxidative stress of glomerular capillaries, apoptosis and other symptoms. Among them, Advanced Glycation End products (AGEs) participate in the pathogenesis of diabetic nephropathy, and are significant pathogenic factors.

The treatment of DKD mainly comprises the control of blood sugar, the control of blood pressure and the reduction of urine protein in western medicine, and also comprises the lifestyle intervention, the correction of lipid metabolism disorder, the treatment of complications of renal insufficiency, dialysis treatment and the like. The western medicine treatment method solves the clinical problems of blood pressure elevation, proteinuria and the like of DKD patients to a certain extent, but cannot effectively prevent disease progression and even cause drug-induced nephropathy, application of part of medicines is controversial, side effects such as hyperkalemia and hypercreatinine are easy to occur, and the levels of blood potassium and serum creatinine need to be strictly monitored during use, so that the clinical use of the medicine is limited.

Disclosure of Invention

Based on the above, the invention aims to provide a traditional Chinese medicine pill for treating diabetic nephropathy and a preparation method and application thereof.

The specific technical scheme for realizing the purpose is as follows:

a Chinese medicinal pill for treating diabetic nephropathy comprises active ingredients, diluent 20-70 wt%, anti-adhesive 0-6 wt%, and wetting agent; the anti-sticking agent is microcrystalline cellulose, the anti-sticking agent is talcum powder, the wetting agent is water, the active ingredient is a traditional Chinese medicine composition, and the active ingredients of the traditional Chinese medicine composition are prepared from the following raw materials, by mass, 0.5-1.5 parts of astragalus membranaceus, 1.5-2.5 parts of kudzu roots and 0.5-1.5 parts of cortex mori radicis.

In one embodiment, the microcrystalline cellulose accounts for 38-60% of the total mass, and the talcum powder accounts for 2-5% of the total mass.

In one embodiment, the microcrystalline cellulose accounts for 38-42% of the total mass.

A preparation method of a traditional Chinese medicine pill for treating diabetic nephropathy comprises the following steps:

s1, weighing 0.5-1.5 parts of astragalus, 1.5-2.5 parts of kudzu root and 0.5-1.5 parts of white mulberry root-bark, adding 1-15 times of solvent for reflux extraction, and filtering to obtain a crude filtrate;

s2, filtering the crude filtrate by a ceramic membrane, collecting the filtrate, recovering the solvent and concentrating to obtain an extract;

s3, mixing the extract, microcrystalline cellulose, talcum powder and a proper amount of water uniformly to prepare a soft material, extruding and rolling the soft material into pellets, drying and screening;

s4, coating the screened pellets with a film and drying to obtain the finished product.

In one embodiment, the microcrystalline cellulose accounts for 20-70% of the total mass, and the talcum powder accounts for 0-6% of the total mass.

In one embodiment, in step S1, the solvent is 5-95% ethanol water, and the reflux extraction is performed 1-5 times, each time for 1-5 hours;

in step S2, the aperture of the ceramic membrane is 20-300 nm;

in the step S3, the extrusion rounding process parameters include an extrusion speed of 10-50 rpm, a rounding time of 2-10 min and a rounding speed of 600-1500 rpm, and the drying is carried out at 50-80 ℃ for 2-6 hours;

in step S4, the process parameters of the film coating are 800-2000 rpm of air inlet frequency, 800-2000 rpm of air outlet frequency, 0.2-0.5 Mpa of atomization pressure, 2-8 rpm of coating liquid flow rate and 40-65 ℃ of material temperature.

In one embodiment, in step S1, the concentration of the ethanol aqueous solution is 60-80%, and the reflux extraction is performed for 2-3 times, each time for 2-3 hours;

in step S2, the aperture of the ceramic membrane is 20-100 nm;

in the step S3, the extrusion rounding process parameters include extrusion speed of 20-40 rpm, rounding time of 5-8 min and rounding speed of 800-1200 rpm, and drying is carried out at 50-70 ℃ for 2-4 hours;

in step S4, the process parameters of the film coating are 1000-2000 rpm of inlet air frequency, 1000-2000 rpm of outlet air frequency, 0.2-0.4 Mpa of atomization pressure, 3-6 rpm of coating liquid flow rate and 40-50 ℃ of material temperature.

The traditional Chinese medicine pill for treating diabetic nephropathy is applied to preparing the medicine for treating diabetic nephropathy.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a traditional Chinese medicine pill for treating diabetic nephropathy, which is prepared from active ingredients, a diluent, an anti-sticking agent and a wetting agent, wherein the active ingredients are traditional Chinese medicine compositions prepared from astragalus, kudzu roots and cortex mori radicis, microcrystalline cellulose is selected as the diluent, and talcum powder is selected as the anti-sticking agent, so that the traditional Chinese medicine pill is high in medicine loading amount, high in product yield and high in coating efficiency. Especially when the dosage of microcrystalline cellulose is 38-60% and the dosage of talcum powder is 2-5%, the yield of the pellet is 92.1-97.5%, the coating efficiency is 95.7-97.6%, the particle size is 0.5-2mm, and the product quality is uniform, stable and controllable. The research through cell tests finds that: in a cell test of a diabetic nephropathy model prepared from AGEs (AGEs), namely glycosylation end products, the active ingredient has obvious effects of resisting cell oxidation, resisting cell apoptosis and resisting reduction of negative charges of cell membranes, has obvious dose-effect relationship, and can effectively protect structural barriers and charge barriers of kidney cells. In an animal model test of the diabetic nephropathy induced by combination of STZ-CFA and high lipid, the traditional Chinese medicine pill can obviously reduce the total urine protein, urine microalbumin, urine inosine, urea nitrogen level and creatinine clearance rate of a model animal, can obviously reduce the MDA, CHOL, LDL, AGEs, IL-6 and TNF-alpha level in the serum of the model animal, can increase the SOD and serum creatinine level, can reduce the pathological score of the kidney staining HE in the animal model of the diabetic nephropathy, obviously improves the positive percentage of PAS staining, and has the obvious effect of protecting the kidney.

The preparation method of the traditional Chinese medicine pill provided by the invention can enrich effective components by adopting an ultrafiltration technology, and the extract of the traditional Chinese medicine composition is directly matched with microcrystalline cellulose and talcum powder to be extruded, rounded, granulated and coated with a film coating, so that the medicine loading amount is high, the product yield is high and the coating efficiency is high. The preparation method has the advantages of simple operation and stable process.

Drawings

FIG. 1 is a graph of the effect of compositions on AGEs-induced oxidative stress of HUVEC cells, wherein A: percent positive staining; b: cytogram (Dihydroethidium staining, 20-fold objective) a: blank group, b: model group, c: group of positive drugs, d-h: composition group (concentration is respectively 10, 1, 0.1, 0.01, 1x10^-3g/L)；

FIG. 2 is a graph of the effect of compositions on AGEs-induced apoptosis of HUVEC cells, wherein A: percent positive staining; b: cell photograph (AO-EB staining, 20-fold objective) a: blank group, b: model group, c: group of positive drugs, d-h: composition group (concentration is respectively 10, 1, 0.1, 0.01, 1x10^-3g/L)；

FIG. 3 is a graph of the effect of compositions on AGEs-induced reduction of negative charge in HUVEC cell membranes, where A: percent positive staining; b: cytogram (AB stain, 20-fold objective) a: blank group, b: model group, c: group of positive drugs, d-h: composition group (concentration is respectively 10, 1, 0.1, 0.01, 1x10^-3g/L)；

Fig. 4 is a photograph (HE staining, 200-fold) of the effect of renal pathology in STZ-CFA combined induced diabetic nephropathy model rats, wherein a: normal group, b: model group, c: irbesartan group, d-e: a composition group;

fig. 5 is a photograph of the effect of renal pathology in a STZ-CFA combination-induced diabetic nephropathy model rat (PAS staining, 200-fold), wherein a: normal group, b: model group, c: irbesartan group, d-e: composition group.

Detailed Description

The invention is further illustrated with reference to the following specific examples.

Preparation of Chinese medicine pill

1 test Material

1.1 instruments

A multifunctional extraction, reflux and concentration unit for Chinese medicinal materials (Hunan Jinyi Sai pharmaceutical equipment, Ltd., RZZ Xiang-082-06).

Multifunctional pill coating machine (Shenzhen Xinyite science and technology Limited, Mini-250).

1.2 raw and auxiliary materials

Radix astragali (Bozhou city huikang herbal pieces Limited);

kudzu root (mn city huikang chinese herbal pieces limited);

cortex Mori (Bozhou city huikang Chinese medicinal decoction pieces Co., Ltd.);

ethanol (manufactured by Guangdong Shunhuan gas solvent Co., Ltd.);

microcrystalline cellulose (produced by Anhui mountain river pharmaceutic adjuvant, Inc.);

starch (produced by Anhui mountain river pharmaceutic adjuvant, Inc.);

dextrin (produced by Anhui mountain river pharmaceutic adjuvant, Inc.);

talc (produced by Guangxi Longshenghuamei talc Co., Ltd.);

coating liquid (produced by Shanghai Kalekang coating technology Co., Ltd.).

Example 1

Taking 2.5kg of astragalus, 5.0kg of kudzu root and 2.5kg of white mulberry root-bark, adding 8 times of 70% ethanol for reflux extraction twice, each time for 3 hours, filtering, combining the filtrates, filtering by a 50nm ceramic membrane, collecting the filtrate, recovering the ethanol, and concentrating (the temperature is 70 ℃, the air pressure is-0.8 to-0.6 MPa) to the relative density of 1.32-1.35 (70 ℃), and collecting the paste for later use. The same method is adopted to prepare three parts: extract 1, extract 2 and extract 3.

Taking the three batches of extractum, respectively adding microcrystalline cellulose, starch and dextrin accounting for 40-60% of the total amount, respectively adding 5% of talcum powder serving as an anti-sticking agent, uniformly mixing, adding a proper amount of water to prepare a soft material, extruding and rounding to form pellets, wherein the process parameters of the step are as follows: extrusion speed 35rpm, spheronization time 4min, spheronization speed 1000 rpm. Drying at 60 + -10 deg.C for 3 hr, sieving with 18-24 mesh sieve, collecting micropellets which can pass through 18 mesh sieve but not 24 mesh sieve, and coating with film. The technological parameters of the film coating are as follows: the air inlet frequency is 1500rpm, the air outlet frequency is 1500rpm, the atomization pressure is 0.3Mpa, the coating liquid flow rate is 4rpm, and the material temperature is 45 ℃. Drying, sieving with 16 and 24 mesh sieves, collecting micropellets which pass through 16 mesh sieves but not pass through 24 mesh sieves, testing quality, and packaging to obtain the final product.

As a result: when the three diluents are independently selected to prepare the pellets respectively, the maximum drug-loading rate of the microcrystalline cellulose is 60 percent; the maximum drug loading of the starch is 40 percent; the maximum drug loading of dextrin is 38%. Wherein the highest drug loading refers to the amount of the Chinese medicinal extract which can be allowed to be added by selecting the auxiliary materials on the basis of ensuring that the Chinese medicinal extract can be prepared into pills.

Example 2

Taking the three batches of extractum, respectively investigating the influence of microcrystalline cellulose, starch and dextrin on the yield of the pellet, adding 5 percent of talcum powder serving as an anti-sticking agent respectively to ensure that the drug-loading rate is 50 percent, uniformly mixing, adding a proper amount of water to prepare a soft material, extruding and rounding to form the pellet, wherein the process parameters of the step are as follows: extrusion speed 35rpm, spheronization time 4min, spheronization speed 1000 rpm. Drying at 60 + -10 deg.C for 3 hr, sieving with 18-24 mesh sieve, collecting micropellets which can pass through 18 mesh sieve but not 24 mesh sieve, and coating with film. The technological parameters of the film coating are as follows: the air inlet frequency is 1500rpm, the air outlet frequency is 1500rpm, the atomization pressure is 0.3Mpa, the coating liquid flow rate is 4rpm, and the material temperature is 45 ℃. Drying, sieving with 16 and 24 mesh sieves, collecting micropellets which pass through 16 mesh sieves but not pass through 24 mesh sieves, testing quality, and packaging to obtain the final product.

As a result: the yield of the micro-pill of the microcrystalline cellulose is 94 plus or minus 5 percent; neither starch nor dextrin can be shaped.

Example 3

Taking 2.5kg of astragalus, 5.0kg of kudzu root and 2.5kg of white mulberry root-bark, adding 8 times of 70% ethanol for reflux extraction twice, each time for 3 hours, filtering, combining the filtrates, filtering by a 50nm ceramic membrane, collecting the filtrate, recovering the ethanol, and concentrating (the temperature is 70 ℃, the air pressure is-0.8 to-0.6 MPa) to the relative density of 1.32-1.35 (70 ℃), and collecting the paste for later use.

Taking the extract, adding microcrystalline cellulose accounting for 40% of the total amount and talcum powder accounting for 5% of the total amount, uniformly mixing, adding a proper amount of water to prepare a soft material, extruding and rolling into pellets, wherein the process parameters of the step are as follows: extrusion speed 35rpm, spheronization time 4min, spheronization speed 1000 rpm. Drying at 60 + -10 deg.C for 3 hr, sieving with 18-24 mesh sieve, collecting micropellets which can pass through 18 mesh sieve but not 24 mesh sieve, and coating with film. The technological parameters of the film coating are as follows: the air inlet frequency is 1500rpm, the air outlet frequency is 1500rpm, the atomization pressure is 0.3Mpa, the coating liquid flow rate is 4rpm, and the material temperature is 45 ℃. Drying, sieving with 16 and 24 mesh sieves, collecting micropellets which pass through 16 mesh sieves but not pass through 24 mesh sieves, testing quality, and packaging to obtain the final product.

As a result: the particle size of the round pellet is 1mm, the pellet yield is 97.5%, the coating weight is increased by 3%, the coating efficiency is 97.6%, and the finished product is qualified through inspection.

Example 4

Taking 2.5kg of astragalus, 5.0kg of kudzu root and 2.5kg of white mulberry root-bark, adding 1 time of 5% ethanol for reflux extraction for 5 hours, filtering, combining the filtrates, filtering by a 300nm ceramic membrane, collecting the filtrate, recovering the ethanol and concentrating (the temperature is 70 ℃, the air pressure is-0.8 to-0.6 MPa) to the relative density is 1.35 to 1.40(70 ℃), and collecting the paste for later use.

Taking the extract, adding 65% of microcrystalline cellulose, uniformly mixing, adding a proper amount of water to prepare a soft material, extruding and rolling into pellets, wherein the process parameters of the step are as follows: extrusion speed 10rpm, spheronization time 10min, spheronization speed 600 rpm. Drying at 60 + -10 deg.C for 3 hr, sieving with 18-24 mesh sieve, and coating with film coating to obtain micropellets which can pass through 18 mesh sieve but not 24 mesh sieve. The technological parameters of the film coating are as follows: the air inlet frequency is 800rpm, the air outlet frequency is 800rpm, the atomization pressure is 0.2Mpa, the coating liquid flow rate is 2rpm, and the material temperature is 45 ℃. Drying, sieving with 16 and 24 mesh sieves, collecting micropellets which pass through 16 mesh sieves but not pass through 24 mesh sieves, testing quality, and packaging to obtain the final product.

As a result: the particle size of the round pellets is 0.5mm, the pellet yield is 77.0%, the coating weight is increased by 3%, the coating efficiency is 95.4%, and the finished product is qualified through inspection.

Example 5

Taking 2.5kg of astragalus, 5.0kg of kudzu root and 2.5kg of white mulberry root-bark, adding 15 times of 95% ethanol for reflux extraction for 5 times, 1 hour each time, filtering, combining the filtrates, filtering by a 20nm ceramic membrane, collecting the filtrate, recovering the ethanol, and concentrating (the temperature is 70 ℃, the air pressure is-0.8 to-0.6 MPa) to the relative density of 1.25-1.30 (70 ℃), and collecting the paste for later use.

Mixing the above extracts with 25% microcrystalline cellulose and 5% pulvis Talci, adding appropriate amount of water, making soft mass, extruding, and making into pellet. The technological parameters of the step are as follows: extrusion speed 50rpm, spheronization time 10min and spheronization speed 1500 rpm. Drying at 60 + -10 deg.C for 3 hr, sieving with 18-24 mesh sieve, and coating with film coating to obtain micropellets which can pass through 18 mesh sieve but not 24 mesh sieve. The technological parameters of the film coating are as follows: the air inlet frequency is 2000rpm, the air outlet frequency is 2000rpm, the atomization pressure is 0.5Mpa, the coating liquid flow rate is 8rpm, and the material temperature is 65 ℃. Drying, sieving with 16 and 24 mesh sieves, collecting micropellets which pass through 16 mesh sieves but not pass through 24 mesh sieves, testing quality, and packaging to obtain the final product.

As a result: the particle size of the round pellet is 5mm, the pellet yield is 90.3%, the coating weight is increased by 5%, the coating efficiency is 96.1%, and the finished product is qualified through inspection.

Example 6

Taking 2.5kg of astragalus, 5.0kg of kudzu root and 2.5kg of white mulberry root-bark, adding 5 times of 60% ethanol for reflux extraction for 3 times, each time for 2 hours, filtering, combining the filtrates, filtering by a 20nm ceramic membrane, collecting the filtrate, recovering the ethanol, and concentrating (the temperature is 70 ℃, the air pressure is-0.8 to-0.6 MPa) to the relative density of 1.30-1.35 (70 ℃), and collecting the paste for later use.

Taking the extract, adding 60% of microcrystalline cellulose and 5% of talcum powder, mixing uniformly, adding a proper amount of water to prepare a soft material, extruding and rolling into pellets, wherein the process parameters of the step are as follows: extrusion speed 20rpm, spheronization time 5min and spheronization speed 1200 rpm. Drying at 60 + -10 deg.C for 2 hr, sieving with 18-24 mesh sieve, collecting micropellets which can pass through 18 mesh sieve but not 24 mesh sieve, and coating with film. The technological parameters of the film coating are as follows: the air inlet frequency is 1000rpm, the air outlet frequency is 1000rpm, the atomization pressure is 0.2Mpa, the coating liquid flow rate is 3rpm, and the material temperature is 40 ℃. Drying, sieving with 16 and 24 mesh sieves, collecting micropellets which pass through 16 mesh sieves but not pass through 24 mesh sieves, testing quality, and packaging to obtain the final product.

As a result: the particle size of the round pellets is 0.5mm, the pellet yield is 92.1%, the coating weight is increased by 2%, the coating efficiency is 95.7%, and the finished product is qualified through inspection.

Example 7

Taking 2.5kg of astragalus, 5.0kg of kudzu root and 2.5kg of white mulberry root-bark, adding 10 times of 80% ethanol for extraction for 2 times, each time for 3 hours, filtering, combining the filtrates, filtering by a 100nm ceramic membrane, collecting the filtrate, recovering the ethanol, and concentrating (the temperature is 70 ℃, the air pressure is-0.8 to-0.6 MPa) to the relative density of 1.35-1.40 (70 ℃), and collecting the paste for later use.

Taking the extract, adding microcrystalline cellulose 40% and talcum powder 2% of the total weight, mixing uniformly, adding a proper amount of water to prepare a soft material, extruding and rolling into pellets, wherein the process parameters of the step are as follows: extrusion speed 40rpm, spheronization time 8min and spheronization speed 800 rpm. Drying at 60 + -10 deg.C for 4 hr, sieving, collecting qualified pellet, and coating with film. The technological parameters of the film coating are as follows: the air inlet frequency is 2000rpm, the air outlet frequency is 2000rpm, the atomization pressure is 0.4Mpa, the coating liquid flow rate is 6rpm, and the material temperature is 50 ℃. Drying, sieving, collecting qualified pellet, inspecting quality, and packaging.

As a result: the particle size of the round pellet is 2mm, the pellet yield is 93.5%, the coating weight is increased by 5%, the coating efficiency is 96.3%, and the finished product is qualified through inspection.

In the above embodiment, the microcrystalline cellulose, the talcum powder and the extract of the traditional Chinese medicine composition are used for preparing the traditional Chinese medicine pill for treating diabetic nephropathy. The preparation method adopts ceramic membrane ultrafiltration technology to enrich effective components, and the obtained extract of the Chinese medicinal composition is directly matched with microcrystalline cellulose and talcum powder for extrusion, spheronization, granulation and film coating, so that the medicine loading is high, the product yield is high and the coating efficiency is high. Meanwhile, the traditional Chinese medicine pill prepared by the preparation method is convenient to take, good in compliance and high in bioavailability, and the pellets are coated with a film so as to be light-proof, moisture-proof and antioxidant, and the stability of the medicine is improved.

Preferably, when the dosage of the microcrystalline cellulose is 40-60% and the dosage of the talcum powder is 2-5%, the yield of the pellet is 92.1-97.5%, the particle size is 0.5-2mm, and the quality is uniform, stable and controllable. Particularly, when 8 times of 70% ethanol is added for reflux extraction twice, each time lasts for 3 hours, the filtration is carried out, the filtrates are combined, 50nm ceramic membrane filtration is carried out, the filtrate is collected, the ethanol is recovered and concentrated (the temperature is 70 ℃, the air pressure is-0.8 to-0.6 MPa) to the relative density of 1.32 to 1.35(70 ℃), and the extract is obtained. And adding microcrystalline cellulose accounting for 40% of the total amount and talcum powder accounting for 5% of the total amount into the obtained extract, and rolling to obtain the pellet with the particle size of 1mm, wherein the yield of the pellet is up to 97.5%, and the coating efficiency is 97.6%.

(II) Effect test

Example 8

Oxidative stress, apoptosis and cell membrane charge barrier disruption are important manifestations of diabetic nephropathy. Cell membrane charge barrier disruption is mainly manifested in reduced negative charge in diabetic nephropathy. This example is based on the effect of advanced glycation end products (AGEs) on oxidative stress, apoptosis, and cell membrane charge barrier.

1 materials of the experiment

1.1 instruments

IX71, IX81 microscope, olympus japan;

LDZ4-1.2 Low speed centrifuge, Beijing R centrifuge, Inc.;

sartorius BS224S/CB-25A electronic analytical balance, beijing sidoris instruments systems ltd;

CP-ST100A CO²incubator, changsha changjin science and technology limited;

24-well cell culture plates, Corning USA.

1.2 reagents and drugs

Low-sugar DMEM medium, Gibco;

fetal bovine serum, by biotechnology limited, hang in Zhejiang;

acridine Orange (AO), guangzhou jetvia biotechnology limited;

ethidium Bromide (EB), dimethyl sulfoxide (DMSO), hematoxylin, Bovine Serum Albumin (BSA), D-glucose, Sigma;

alcian Blue (Alcain Blue, AB), Biosharp;

superoxide anion fluorescent probe (Dihydroethidium), the institute of biotechnology in cloudy days;

vitamin E (VitE), Alfa Aesar;

penicillin sodium, north china pharmaceutical company;

streptomycin sulfate, Dalian Meiluo Dayao pharmaceutical factory.

The other reagents are all commercial domestic analytical purifiers.

Composition extract, prepared from example 1.

1.3 cell lines

Human umbilical vein endothelial cell line HUVEC, from American Type Culture Collection (ATCC).

2 method of experiment

2.1 preparation of reagents

Advanced glycation end products (AGEs) preparation: dissolving 5g BSA and 9g D-glucose in 100mL PBS, filtering, sterilizing, packaging, and placing at 37 deg.C CO²Culturing in incubator for 90 days, and extensively dialyzing to remove unreacted protein and glucose.

Preparing Dihydroxyethylium: dissolving appropriate amount of Dihydroethidium and DMSO into 10mM mother liquor, subpackaging, and refrigerating at-20 deg.C in dark for use. Before use, the solution was diluted to 5. mu.M in PBS and used.

Preparing an AO-EB dye solution: 1mg of AO and EB were taken and dissolved in 5mL of PBS, respectively, to obtain 200. mu.g/mL stock solutions, which were then refrigerated at 4 ℃ for further use. Mixing in equal volume before use.

Preparing an AB dye solution: 0.05g of alcian blue is taken and dissolved in 100mL of 3% acetic acid, and 0.5g/L of AB dye solution is obtained.

Preparing a Harris hematoxylin staining solution: 5.0g of hematoxylin is taken and put into 50mL of absolute ethyl alcohol, and stirred until the hematoxylin is completely dissolved. Adding 44g of aluminum potassium sulfate into 1000mL of distilled water, heating to dissolve, then adding hematoxylin anhydrous ethanol solution, boiling the solution as soon as possible, and then leaving the flame. The solution was stirred and 5g of mercuric oxide was slowly added when the temperature dropped to 91 ℃. Stirring until no yellow mercury oxide can be observed, quickly placing in cold water, and placing onto the bottle mouth with gauze. The next day, filtering, adding 4g citric acid, and stirring.

VitE preparation: mu.L of VitE 91.0 is added with 410 mu.L of absolute ethyl alcohol to prepare a mother liquor of 400mM, and the mother liquor is refrigerated at 4 ℃ for standby and diluted to the required concentration by PBS when in use.

Preparing a test medicine: taking the composition extract, diluting with water to the concentration of 100g/L, and diluting with 10 times of water to 10 g/L-10^-2g/L, and refrigerating at 4 ℃ for standby.

2.2 grouping

The experiment was divided into blank control group, model group (AGEs, final concentration of 100. mu.g/L), positive drug group (VitE, final concentration of 200. mu. mol/L), and each concentration group of composition (final concentrations of 10, 1, 0.1, 0.01, 1 × 10 respectively)^-3g/L)。

2.3 cell culture and administration

HUVEC cells, routinely cultured in low-sugar DMEM containing 5% FBS. Cells in logarithmic growth phase were prepared into single cell suspension, and 1 mL/well of the single cell suspension was inoculated into a 24-well plate containing a cover slip pretreated with polylysine, the number of cells being 1X10⁵Pore, 5% CO at 37 ℃²After 24h of culture, the cells were synchronized.

Cell supernatants were discarded, and low-sugar DMEM containing 5% FBS and each drug (or PBS) were added to make the total volume 1.5 mL. PBS is added into a normal control group, PBS and AGEs are added into a model group, VitE and AGEs are added into a positive group, and medicines with the matched concentrations and AGEs are added into each test medicine group respectively.

2.4 cellular oxidative stress assay

After culturing the cells for 48h, removing the culture medium, washing with PBS for 2 times, adding 50 μ L of Dihydroethidium working solution into each hole, and reacting for 30min at 37 ℃ in a dark place. The cell slide was removed, washed with PBS, blotted with filter paper, placed on a slide with the cell side down, viewed with the microscope WIB fluorescence channel, and photographed. The obtained pictures are subjected to statistics of positive staining integrated optical density (namely, the part with the integrated optical density of more than 5000) and total cell staining area (namely, the part with the integrated optical density of more than 100) by using IPP image analysis software, and the positive staining percentage is calculated according to the formula positive staining percentage which is the positive staining integrated optical density/total cell staining area x 100%. All the above experiments were repeated 3 times.

2.5 apoptosis assay

After 48h of cell culture, the cell slide was taken out, washed with PBS, and blotted with filter paper for use. And (3) dripping 5 mu L of AO-EB staining solution on a glass slide, covering the AO-EB staining solution with a cell surface of a cell slide facing downwards, observing the cell slide by using a WIB fluorescence channel of a microscope after 30 seconds, and shooting the cell slide. The obtained pictures were analyzed by IPP image analysis software to calculate the integrated optical density of red and orange parts and the total area of cell staining (i.e. the total area of red + orange + green parts), and the percentage of apoptosis was calculated according to the formula integrated optical density of red and orange parts/total area of cell staining x 100%. All the above experiments were repeated 3 times.

2.6 detection of negative charges in cell membranes

After culturing the cells for 48h, taking out the cell slide, washing with PBS, drying with filter paper, and fixing with 10% formaldehyde for 20 min; dyeing with AB dye solution for 5min, and washing with tap water for 5 min; counterstaining with hematoxylin for 10s, and washing with tap water for 5 min; dehydrating 76%, 90%, 96% and 100% ethanol twice in sequence for 10min each time; xylene transparent, sealing with neutral gum, and performing microscopic examination and photography. The obtained pictures were counted by using IPP image analysis software for positive staining area (i.e., light blue staining area of cell membrane) and total cell area, and the positive staining percentage was calculated according to the formula positive staining percentage (positive staining area/total cell area × 100%). All the above experiments were repeated 3 times.

3 data processing

Data were summarized using Excel, expressed as mean ± variance. The data were tested by student's t-test, with p <0.05 indicating statistical significance.

4 results of the experiment

4.1 Effect on cellular oxidative stress

As can be seen from Table 1 and FIG. 1, the percentage of positive staining was significantly increased in the cells of the model group as compared with the normal group (p)<0.01). Compared with the model group, the positive medicine group and the mass concentration are 1 to 1x10^-3The composition between g/L can obviously reduce the increase of positive staining percentage of cells caused by AGEs (p)<0.05, 0.01), which shows that the composition can prevent the excessive occurrence of the oxidative stress of cells in the concentration range and has obvious antioxidation. When the mass concentration of the composition is 10g/L, the increase of the percentage of positive staining of cells by AGEs cannot be reduced.

TABLE 1 antioxidant Effect of compositions on AGEs-induced HUVEC cells

vs Control，##p<0.01；vs Model，*p<0.05，**p<0.01

4.2 Effect on apoptosis

As can be seen from Table 2 and FIG. 2, the percentage of apoptosis was significantly increased in the model group as compared with the normal group, and the percentage of apoptosis was significantly different from that in the normal group (p)<0.05). Compared with the model group, the apoptosis rate of the group in which the positive medicine group and the composition with the mass concentration of 0.1g/L are positioned is obviously reduced, and the significant difference (p) is provided<0.05) in optimal dosage. When the mass concentration of the composition is between 10 and 0.01g/L, the percentage of apoptosis can be obviously reduced. But when the mass concentration of the composition is low, e.g. 1X 10-³And at g/L, the apoptosis of model group cells cannot be effectively inhibited.

TABLE 2 Effect of compositions on AGEs-induced apoptosis of HUVEC cells

vs Control，#p<0.05；vs Model，*p<0.05

4.3 Effect on negative cell Membrane charges

As can be seen from table 3 and fig. 3, the HUVEC cell membranes of the model group cells were significantly reduced in the amount of binding to alcian blue (p <0.01) compared to the normal group. Compared with the model group, the combination amount of the positive drug group and the alcian blue is obviously increased, and the statistical significance is achieved (p is less than 0.05); the composition with the mass concentration of 0.1g/L can effectively resist the reduction of negative charges of cell membranes (p is less than 0.01), and the composition with the mass concentration of more than or less than the mass concentration can resist the reduction of negative charges of the cell membranes to a certain extent, which indicates that the composition has the function of protecting the charge barrier of the kidney.

TABLE 3 Effect of compositions on AGES-induced reduction of negative charges in HUVEC cell membranes

vs Control，^##p<0.01；vs Model，*p<0.05，**p<0.01

Conclusion of the experiment

As can be seen from tables 1 to 3 and fig. 1 to 3, in the cell model test of diabetic nephropathy induced by AGEs, the composition extract has significant effects of resisting cell oxidation, resisting cell apoptosis and resisting reduction of negative charges of cell membranes within a certain mass concentration range, and the dose-effect relationship is obvious, which indicates that the composition extract has a structural barrier and a charge barrier for protecting kidney cells, and plays a role in protecting the kidney.

Example 9

This example explores the effect of traditional Chinese medicine pills in treating STZ-CFA combination-induced diabetic nephropathy model rats.

1 materials of the experiment

1.1 animals

SPF grade male Wistar rats 140, weight 180-: SCXK (Yue) 2011-.

1.2 medicine

The test drugs are: the composition pellet is provided by a Guangzhou Kangchen drug research Co., Ltd, and the batch number is as follows: 20131012, respectively;

positive drugs: the Irbesartan capsule is provided by Zhejiang Honghong pharmaceutical industry Co., Ltd, and has the following batch number: 671321, respectively;

streptazocin, Sigma, lot number SLBB 7526V;

complete Freund's adjivant, Chondrex corporation, lot number 120383;

glucose assay kit, product of roche diagnostics, germany, lot number: 673318, respectively;

urine total protein detection kit, product of roche diagnostics, germany, lot number: 675595, respectively;

creatinine assay kit, product of roche diagnostics, germany, lot number: 673318, 688815;

urea/urea nitrogen detection kit, product of roche diagnostics, germany, lot No.: 686545, respectively;

cholesterol assay kit, product of roche diagnostics, germany, lot number: 682310, respectively;

low density lipoprotein cholesterol assay kit, roche diagnostics, germany, lot number: 674550, respectively;

rat microalbumin Elisa test kit, product of Wuhan Huamei bioengineering Co., Ltd, batch number: u05018355;

rat SOD Elisa detection kit, product of Wuhan Huamei bioengineering Co., Ltd, batch number: v25017660;

rat MDA Elisa detection kit, product of wuhan huamei bioengineering limited company, lot number: w09017654;

rat AGEs Elisa detection kit, product of Wuhan Huamei bioengineering Co., Ltd, batch number: v25017656;

rat IL-6Elisa test kit, product of Wuhan Huamei bioengineering Co., Ltd, batch number: v18017652;

rat TNF-alpha Elisa detection kit, product of Wuhan Huamei bioengineering Co., Ltd, batch number: t30017658;

microalbumin assay kit, zhejiang nipag biotechnology limited, lot number 20130508, 20140314;

creatinine assay kit, Nanjing, established science and technology Co., Ltd, lot number 20131101;

a urea nitrogen determination kit, Nanjing, Kangji science and technology Co., Ltd., batch No. 20131025;

total protein assay kit, Shanghai Biyuntian Biotechnology Co., Ltd., lot number 20130521;

SOD reagent box, Nanjing institute of bioengineering product, batch number: 20140107, respectively;

MDA determination kit, Nanjing institute of bioengineering product, batch number: 20140106, respectively;

common reagents such as citric acid, sodium citrate and the like are all domestic analytical purifiers.

1.3 Main instruments

Roche glucometer and test paper, roche diagnostics china company;

SPECTRA MAX 190, Molecular Devices, USA;

CODA noninvasive blood pressure monitor, Kent corporation, usa;

BS224S type electronic balance, SARTORIUS, germany;

roche P800 Biochemical apparatus, product of Roche diagnostics, Germany;

sunrise full-automatic enzyme marking apparatus, product of Tecan, Switzerland.

2 method of experiment

2.1 reagent and pharmaceutical formulation

0.1mol/L citric acid-sodium citrate buffer: 1.1976g of citric acid and 1.2646g of sodium citrate are precisely weighed and prepared into 100ml of solution by using sterilized water for injection for later use.

And (3) STZ preparation: taking 1g of Streptozocin, preparing a 44mg/ml solution by using 0.1mol/L citric acid-sodium citrate buffer solution, filtering and sterilizing by using a 0.22 mu m filter membrane, and preparing the solution for use.

Preparing pills: taking appropriate amount of the Chinese medicinal pill, adding ultrapure water to the concentration of 2.4 and 1.2g/ml, respectively, and refrigerating at 4 deg.C for use.

Preparing irbesartan: taking the capsule produced by Zhejiang Hongyao pharmaceutical Co Ltd, taking out the content, adding ultrapure water to the content of 6mg/ml, and storing at normal temperature once every 2 days.

2.2 Molding method

Animals can drink and eat water freely. The rats were divided into 90 groups, 10 normal control groups and 80 model groups. The model group is used for carrying out intraperitoneal injection on STZ (total circulating fluid) at the rate of 30mg/kg, and carrying out intraperitoneal injection on CFA (total circulating fluid) at the rate of 0.1 ml/piece once a week on the next day; the control group was injected with a corresponding volume of citrate buffer. The above procedure was repeated for 3 consecutive weeks and the injection site was sterilized with an alcohol cotton swab. The building block is fed with high-fat feed, and the normal block is fed conventionally. Keeping the animal breeding environment clear: dark time 12: for 12 hours.

2.3 blood glucose determination

Blood is taken from the tip of the rat tail by a Roche glucometer matched with a blood taking needle, the blood sugar value is directly measured, and the blood taking part is disinfected by an alcohol cotton ball. Before the animal is given the model-making medicine, the normal blood sugar is measured once under the condition of free drinking water and eating, and the blood sugar is measured once per week within three weeks of the model-making medicine.

2.4 animal grouping and administration

And (3) determining the blood sugar of the molded animals after 3 weeks, taking the animals with the blood sugar value of 16-30 mmol/L as qualified animals, grouping the qualified animals according to the blood sugar, wherein each group comprises 15 animals, and the blood sugar value of each group of animals has no obvious difference among the groups. The grouping situation is as follows: model group, high (5.4 g/kg dose), low (2.7 g/kg dose) and erxatan (13.5 mg/kg dose). The normal control group, 10 rats, remained unchanged.

The administration groups are respectively administered with corresponding drugs, the volume is 0.5mL/220g, and the methods of intragastric administration are adopted; normal control group and model group were given the same volume of physiological saline. Dosing was 6 days per week for 11 weeks.

2.5 animal daily management and Observation

During the molding and administration period, each rat is fed with 30g of fresh cold boiled water every day, the normal group is fed with common feed, and the other groups are fed with high-fat feed; the padding was changed daily. The animal status was observed daily.

2.6 index detection

The dosing period weighed 1 body weight per week and urine volume was measured 1 time per 2 weeks. And collecting urine for 24 hours after the 5 th week of administration, and detecting the total protein, microalbumin and creatinine contents in the urine.

After the administration, urine is collected for 24 hours, and the total protein, microalbumin and creatinine contents in the urine are measured. Animal serum was collected, blood glucose, total Cholesterol (CHOL), Malondialdehyde (MDA), blood creatinine, blood urea nitrogen, advanced glycation end products (AGEs), Low Density Lipoprotein (LDL), superoxide dismutase (SOD), tumor necrosis factor (TNF-alpha), interleukin 6 (IL-6) were measured, and creatinine clearance was calculated. And (3) taking kidney formalin at one side for storage, slicing, observing pathological changes of the kidney by HE and PAS staining, carrying out pathological scoring on the HE staining, carrying out image analysis on the PAS staining, and calculating the positive percentage.

HE staining pathology scoring criteria:

1) glomeruli: firstly, membrane cell proliferation: none, light, medium and heavy are respectively counted for 0, 1, 2 and 3 points; matrix broadening: normal 0, slight change, no obvious influence of the capillary loop 1, diffuse broadening of moderate change, occlusion of the capillary loop with stenosis below 50%, diffuse broadening of severe change, occlusion of the capillary loop with stenosis above 50% 3; ③ hardening change: normal 0, focal segmental distribution, hardened glomeruli < 30% 1, 30-60% 2, > 60% 3; fourthly, the glomerular capsule. 2) Renal tubules: denaturation, necrosis, atrophy, no 0, small range 1, piece range 2, and diffuse 3; ② interstitial inflammatory cell infiltration is not 0, the small focus is 1, the piece focus is 2, the diffuse is 3; ③ interstitial fibrosis, no 0, small range 1, piece range 2 and diffuse 3. The pellet and the tubule are respectively graded as 0 grade and 0 grade according to the total score; grade 1, 1-4 points; grade 2, 5-8 min; grade 3, 9-12.

3 data processing

Mean ± sd of each group

Showing that differences between groups were compared using one-way analysis of variance.

4 results of the experiment

4.1 Effect on body weight

As can be seen from Table 4, the body weight of rats in the model group was significantly reduced (p <0.01) compared to the normal group. Compared with the model group, the weight of the rats in the positive drug group and the high and low amount groups of the traditional Chinese medicine pill slowly rises, but is still lower than the average weight of the rats in the normal group, and the weight is reduced, which shows that the active ingredients of the traditional Chinese medicine pill can relieve the symptoms of weight loss of the model animals.

TABLE 4 Effect on STZ-CFA combination induced diabetic nephropathy model rat body weight

vs is normal ## p <0.01

4.2 effects on 24-hour urine volume, urine biochemical indicators such as urine microalbumin, serum biochemical indicators such as blood creatinine, and creatinine clearance

As can be seen from Table 5, the 24-hour urine volume was significantly increased in the model group (p <0.01) compared to the normal group. The bolus low dose group was able to improve urine volume at different times compared to the model group.

TABLE 5 Effect on STZ-CFA combination induced diabetic nephropathy model rat 24h urine volume

vs is normal, # p <0.05, # p < 0.01; vs model p <0.05, p <0.01

As can be seen from Table 6, the amount of urinary microalbumin in 24 urine of the model group rats was significantly increased as compared with that of the normal group. Both the bolus high and low dose groups reduced the amount of urinary microalbumin in urine compared to the model group.

TABLE 6 Effect on STZ-CFA Combined induced diabetic nephropathy model rat 24h urine microalbumin

vs is normal, # p <0.05, # p < 0.01; vs model p <0.05, p <0.01

As can be seen from table 7, total urine protein, urinary inosine and urinary urea nitrogen were significantly increased in urine of rats in the model group and creatinine clearance was significantly increased compared to the normal group. Compared with the model group, the Irersartan group and the high-low dose pill group can effectively reduce the total urine protein, the urinary inosine and the urinary urea nitrogen content in the urine of rats in the model group, and reduce the creatinine clearance rate.

TABLE 7 STZ-CFA Combined induced Effect of Total urinary protein, Urea, urinary creatinine and creatinine clearance in rats in diabetic nephropathy model

vs is normal, # p <0.05, # p < 0.01; vs model p <0.05, p < 0.01;

as can be seen from Table 8, the relevant indexes in the model group all changed significantly compared to the normal group. Compared with a model group, the irbesartan has the obvious effect of improving or inhibiting the change of related indexes (p is less than 0.05, and p is less than 0.01) on MDA, LDL, AGEs, IL-6 and TNF-alpha; the pill high and low dose groups can obviously improve or inhibit the change of related indexes (p <0.05, p < 0.01).

TABLE 8 STZ-CFA combination induced changes in SOD, MDA, CHOL, LDL, AGEs, IL-6 and TNF-alpha in diabetic nephropathy model rats

vs is normal, # p <0.05, # p < 0.01; vs model p <0.05, p <0.01

4.3 Effect on Kidney Pathology

As can be seen from table 9, both HE staining pathology scores and PAS staining positive percentage were significantly increased in the model group compared to the normal control group (p < 0.01). Both the bolus high and low dose group and the irlsartan group reduced HE staining pathology score values and PAS positive percentage (p <0.01) compared to the model group. In addition, as can be seen from fig. 4, the glomeruli and tubules of the model group had many lesions, and the degree of lesions was reduced after administration of the drug. As can be seen from fig. 5, the positive staining of the model group was significantly increased, and the percentage of positive staining was significantly different from that of the model group in each administration group.

TABLE 9 Effect on STZ-CFA combination induced diabetic nephropathy model rat Kidney Pathology

vs is normal ## p < 0.01; vs model:. about.p <0.01

5. Conclusion

The traditional Chinese medicine pill has obvious curative effect or improvement effect on multiple indexes of diabetic nephropathy model animals induced by STZ-CFA combination, and the effect of multiple ways and multiple links of traditional Chinese medicine compound for treating diabetic nephropathy is also met. The total urine protein, urine microalbumin, urine inosine, urine urea nitrogen level and creatinine clearance rate of the model animal can be obviously reduced by the pill high-low dose group; obviously reduces the levels of MDA, CHOL, LDL, AGEs, IL-6 and TNF-alpha in serum of model animals, increases the levels of SOD and serum creatinine in serum, reduces the pathological score of HE staining of kidney, obviously improves the positive percentage of PAS staining and has the function of protecting kidney.

In conclusion, the invention provides a traditional Chinese medicine pill for treating diabetic nephropathy, which is prepared from active ingredients, a diluent, an anti-sticking agent and a wetting agent, wherein the active ingredients are a traditional Chinese medicine composition prepared from astragalus membranaceus, radix puerariae and cortex mori radicis, microcrystalline cellulose is selected as the diluent, and talcum powder is selected as the anti-sticking agent, so that the drug loading capacity is high, the product yield is high, and the coating efficiency is high. The research through cell tests finds that: in a cell test of a diabetic nephropathy model prepared from AGEs (AGEs), namely glycosylation end products, the active ingredient has obvious effects of resisting cell oxidation, resisting cell apoptosis and resisting reduction of negative charges of cell membranes, has obvious dose-effect relationship, and can effectively protect structural barriers and charge barriers of kidney cells. In an animal model test of the diabetic nephropathy induced by combination of STZ-CFA and high lipid, the traditional Chinese medicine pill can obviously reduce the total urine protein, urine microalbumin, urine inosine, urea nitrogen level and creatinine clearance rate of a model animal, can obviously reduce the MDA, CHOL, LDL, AGEs, IL-6 and TNF-alpha level in the serum of the model animal, can increase the SOD and serum creatinine level, can reduce the pathological score of the kidney staining HE in the animal model of the diabetic nephropathy, obviously improves the positive percentage of PAS staining, and has the obvious effect of protecting the kidney.

The preparation method of the traditional Chinese medicine pill provided by the invention can enrich effective components by adopting an ultrafiltration technology, and the extract of the traditional Chinese medicine composition is directly matched with microcrystalline cellulose and talcum powder to be extruded, rounded, granulated and coated with a film coating, so that the medicine loading amount is high, the product yield is high and the coating efficiency is high. The preparation method has the advantages of simple operation, stable process and good reproducibility. Especially, when 60-80% ethanol is used for extraction for 2-3 times, active ingredients are obtained by filtering through a ceramic membrane with the thickness of 20-100nm, and the traditional Chinese medicine micro-pills are prepared by matching with microcrystalline cellulose with the dosage of 40-60% and talcum powder with the dosage of 2-5%, the yield of the micro-pills is 92.1-97.5%, the particle size is 0.5-2mm, and the quality is uniform, stable and controllable.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A traditional Chinese medicine pill for treating diabetic nephropathy is characterized by being prepared from active ingredients, a diluent accounting for 38-60% of the total mass, an anti-adhesion agent accounting for 2-5% of the total mass and a wetting agent; the anti-sticking agent is microcrystalline cellulose, the anti-sticking agent is talcum powder, the wetting agent is water, the active ingredients are traditional Chinese medicine compositions, and the active ingredients of the traditional Chinese medicine compositions are prepared from the following raw materials in parts by mass: 0.5-1.5 parts of astragalus root, 1.5-2.5 parts of kudzu root and 0.5-1.5 parts of white mulberry root-bark;

the traditional Chinese medicine pill for treating diabetic nephropathy is prepared by the following method:

s1, weighing 0.5-1.5 parts of astragalus, 1.5-2.5 parts of kudzu root and 0.5-1.5 parts of white mulberry root-bark, adding 60-80% ethanol, refluxing and extracting for 2-3 times, and filtering to obtain rough filtrate;

s2, filtering the crude filtrate through a 20-100nm ceramic membrane, collecting the filtrate, recovering the solvent and concentrating to obtain an extract;

2. The traditional Chinese medicine pill for treating diabetic nephropathy of claim 1, wherein the microcrystalline cellulose accounts for 40% by mass of the total amount, and the talc accounts for 5% by mass of the total amount.

3. The traditional Chinese medicine pill for treating diabetic nephropathy of claim 1, wherein the microcrystalline cellulose accounts for 38-42% by mass of the total amount.

4. A preparation method of a traditional Chinese medicine pill for treating diabetic nephropathy is characterized by comprising the following steps:

s3, mixing the extract, microcrystalline cellulose, talcum powder and a proper amount of water uniformly to prepare a soft material, extruding and rolling the soft material into pellets, drying and screening; the microcrystalline cellulose accounts for 38-60% of the total mass, and the talcum powder accounts for 2-5% of the total mass;

5. The method for preparing a traditional Chinese medicine pill for treating diabetic nephropathy of claim 4, wherein the microcrystalline cellulose accounts for 40% by mass of the total amount, and the talc accounts for 5% by mass of the total amount.

6. The method for preparing a traditional Chinese medicine pill for treating diabetic nephropathy of claim 5, wherein the microcrystalline cellulose accounts for 38-42% by mass of the total amount.

7. The method for preparing a Chinese medicinal pill for treating diabetic nephropathy of claim 4, wherein in step S1, the extraction is performed for 1-5 hours each time.

8. The method for preparing a Chinese medicinal pill for treating diabetic nephropathy of claim 7,

9. The method for preparing a Chinese medicinal pill for treating diabetic nephropathy of claim 8,

10. Use of the Chinese medicinal pill for treating diabetic nephropathy of any one of claims 1 to 3 in the preparation of a medicament for treating diabetic nephropathy.