Traditional Chinese medicine composition as well as preparation method and application thereof

Abstract

The invention relates to the technical field of traditional Chinese medicine, in particular to a traditional Chinese medicine composition and a preparation method and application thereof. The invention provides a traditional Chinese medicine composition, which is prepared from the following medicinal components in parts by weight: 15 to 40 parts of herba taxilli; 6 to 30 parts of mulberry; 6-30 parts of mantis egg-case; 6-30 parts of mulberry twigs; 6-30 parts of white mulberry root-bark; 6-30 parts of bighead atractylodes rhizome; and 3-20 parts of divaricate saposhnikovia root. The traditional Chinese medicine composition disclosed by the invention can be used for treating diabetic nephropathy, remarkably reducing the blood sugar level and reducing microalbuminuria, and shows the effect of improving the renal function.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition, a preparation method thereof, and an application thereof. Background Art

[0002] Diabetic nephropathy (DN) is one of the serious complications of diabetes, mainly caused by type 2 diabetes (T2DM), and can develop into chronic kidney disease (CKD) and end-stage renal disease (ESRD). Many clinical practices and experimental data show that renal fibrosis is an important part of DN-related kidney injury, and its pathological features are myofibroblast activation, deposition of extracellular matrix (ECM) components, and kidney inflammation. High glucose levels can stimulate renal dysfunction, oxidative stress, fibrosis, and inflammatory responses in diabetic mice. A limited number of drugs have been proven to block the progression of DN. Developing drugs with fewer side effects and costs to prevent or mitigate the progression of DN is an urgent issue. In recent years, traditional Chinese herbs, with their rich historical uses and significant multi-target effects, are expected to improve kidney injury and fibrosis caused by DN. Summary of the Invention

[0003] In view of the above-mentioned technical problems of high danger and low product purity in the existing reaction process, the purpose of the present invention is to provide a method for the selective oxidation of cyanoaldehyde to prepare cyanoacid and a method for the preparation of aliphatic diacid. Compared with chemical drugs, traditional Chinese medicine compounds have the advantages of low toxicity and side effects and multiple targets, and are suitable for early intervention and preventive medication for early DN. Currently, the method of replenishing qi and nourishing yin is often used for the treatment of early DN; the method of promoting diuresis, removing stasis, and dredging collaterals is often used in the middle stage; if it progresses to the late stage, the method of tonifying the kidney, promoting blood circulation, and reducing turbidity is adopted. Wusangyin is a traditional Chinese medicine prescription with the functions of invigorating the spleen and kidney and dispelling wind and dampness. The treatment method is novel and unique, and can reduce proteinuria in patients with diabetic nephropathy and improve renal function.

[0004] To achieve the above object and other related objects, the first aspect of the present invention provides a traditional Chinese medicine composition, and its active ingredients, by weight, include the following components

[0005]

[0006]

[0007] Preferably, by weight, it includes the following components

[0008]

[0009] In the second aspect of the present invention, a traditional Chinese medicine preparation is provided, and the traditional Chinese medicine preparation comprises an effective amount of the above-mentioned traditional Chinese medicine composition.

[0010] Preferably, the traditional Chinese medicine preparation is one or more of a traditional Chinese medicine decoction, a traditional Chinese medicine tablet, a traditional Chinese medicine granule, a traditional Chinese medicine capsule, a traditional Chinese medicine oral liquid, and a traditional Chinese medicine syrup.

[0011] In the third aspect of the present invention, a preparation method of a traditional Chinese medicine granule is provided. The above-mentioned traditional Chinese medicine composition is subjected to first decoction and first filtration in sequence to obtain a first decoction liquid, then second decoction and second filtration are carried out to obtain a second filtrate. The first filtrate and the second filtrate are combined, first concentration and second concentration are carried out to obtain a fluid extract, and granulation is carried out to obtain the traditional Chinese medicine granule.

[0012] Preferably, A1) the time of the first decoction is 30 to 90 min, for example, it can be 30 to 40 min, 40 to 50 min, 50 to 60 min, 60 to 70 min, 70 to 80 min, 80 to 90 min, etc.

[0013] Preferably, A2) the mass ratio of the water added in the first decoction to the mass of the traditional Chinese medicine composition is (10 to 15):1, for example, it can be (10 to 11):1, (11 to 12):1, (12 to 13):1, (13 to 14):1, (14 to 15):1, etc.

[0014] Preferably, A3) the time of the second decoction is 30 to 90 min, for example, it can be 30 to 40 min, 40 to 50 min, 50 to 60 min, 60 to 70 min, 70 to 80 min, 80 to 90 min, etc.

[0015] Preferably, A4) the mass ratio of the water added in the second decoction to the mass of the residue after the first decoction and filtration is (6 to 10):1, for example, it can be (6 to 7):1, (7 to 8):1, (8 to 9):1, (9 to 10):1, etc.

[0016] Preferably, A5) the first concentration is carried out to a relative density of 1.05 to 1.06 kg / m 3 (70 °C);

[0017] Preferably, A6) after the first concentration, ethanol is further added and left standing for third filtration; preferably, ethanol is added such that the mass fraction of ethanol is 50 to 60%, for example, it can be 50 to 52%, 52 to 54%, 54 to 56%, 56 to 58%, 58 to 60%, etc.

[0018] Preferably, A7) the second concentration is carried out to a relative density of 1.12 to 1.16 kg / m 3 (60 °C);

[0019] Preferably, in A8), the granulation raw materials include liquid extract, inclusion compound, excipient and fine powder of traditional Chinese medicine composition. The inclusion compound includes one or more of cyclodextrin and its derivatives, cholic acid, starch, cellulose, protein or nucleic acid; the excipient includes one or more of calcium oxide, microcrystalline cellulose, sodium carboxymethylcellulose or sodium stearate.

[0020] The fourth aspect of the present invention provides a traditional Chinese medicine granule prepared by the above method.

[0021] The fifth aspect of the present invention provides a preparation method of a traditional Chinese medicine capsule, wherein the above traditional Chinese medicine granule is filled into a capsule to obtain the traditional Chinese medicine capsule.

[0022] The sixth aspect of the present invention provides a preparation method of a traditional Chinese medicine tablet, wherein the above traditional Chinese medicine granule is tabletted and film-coated to obtain the traditional Chinese medicine tablet.

[0023] The seventh aspect of the present invention provides a preparation method of a traditional Chinese medicine oral liquid or traditional Chinese medicine syrup. The above traditional Chinese medicine composition is subjected to the third decoction and the third filtration to obtain a third decoction liquid, then subjected to the fourth decoction and the fourth filtration to obtain a fourth filtrate. The third filtrate and the fourth filtrate are combined, and then subjected to the third concentration, the fourth concentration and the fifth concentration, and excipients are added to obtain the traditional Chinese medicine oral liquid or traditional Chinese medicine syrup.

[0024] Preferably, in B1), the time of the third decoction is 2-4 h, for example, it can be 2-3 h, 3-4 h, etc.

[0025] Preferably, in B2), the mass ratio of the water added in the third decoction to the mass of the traditional Chinese medicine composition is (8-10):1, for example, it can be (8-9):1, (9-10):1, etc.

[0026] Preferably, in B3), the time of the fourth decoction is 1-2 h;

[0027] Preferably, in B4), the mass ratio of the water added in the fourth decoction to the mass of the residue after the third decoction and filtration is (5-9):1, for example, it can be (5-6):1, (6-7):1, (7-8):1, (8-9):1, etc.

[0028] Preferably, in B5), ethanol is added during the fourth concentration. Preferably, the mass of the ethanol is 1.5-3 times that of the third concentrated liquid, for example, it can be 1.5-1.7 times, 1.7-1.9 times, 1.9-2.1 times, 2.1-2.3 times, 2.3-2.5 times, 2.5-2.7 times, 2.7-3 times, etc.

[0029] Preferably, in B6), the fifth concentration is to a thick extract.

[0030] The eighth aspect of the present invention provides the above-mentioned traditional Chinese medicine composition and the above-mentioned traditional Chinese medicine preparation for use in the preparation of a medicament for treating diabetic nephropathy.

[0031] The above technical solutions have at least one of the following beneficial effects:

[0032] 1) The decoction of the traditional Chinese medicine composition in the present invention can significantly reduce the blood glucose, blood lipids, serum creatinine, blood urea nitrogen, 24h UAER and 24 h UTP in diabetic patients when taken orally.

[0033] 2) The traditional Chinese medicine composition in the present invention can treat diabetic nephropathy, significantly reduce the blood glucose level, reduce urinary microalbumin, and show the effect of improving renal function.

[0034] 3) The traditional Chinese medicine composition in the present invention can effectively improve the quality of life of patients with diabetic nephropathy and has good clinical value and development prospects.

[0035] 4) The traditional Chinese medicine composition in the present invention can significantly reduce the renal function damage in patients with diabetic nephropathy and play a role in protecting renal function.

[0036] 5) The traditional Chinese medicine composition in the present invention can reduce the excretion of proteinuria in patients with diabetic nephropathy, improve the histopathological morphology of the kidney tissue and cell apoptosis, and reduce renal fibrosis and oxidative stress by enhancing SIRT1 expression and activating the Nrf2 signaling pathway. Description of the Drawings

[0037] Figure 1 It is a diagram showing the general conditions, blood glucose and blood lipid changes in mice in Example 11. The three groups are the control group (Control), the diabetes group (DM) and the Wusangyin group (WSY). (A) Body weight (BW) of mice, (B) kidney weight (KW), (C) kidney hypertrophy index (KW / BW), (D) blood glucose (mmol / L), (E) HbA1c (%), (F) triglycerides and (G) total cholesterol. Data are expressed as mean ± SD (n = 8 for each group). Compared with the Control group, **P < 0.01, ***P < 0.001; compared with the DM group, #P < 0.05, ##P < 0.01, P < 0.001.

[0038] Figure 2Illustration of the renal function changes in the mice of Example 11. (A) Serum creatinine (Scr) of mice in each group; (B) Blood urea nitrogen (BUN); (C) 24-hour urinary albumin excretion rate (24h UAER); (D) 24-hour urinary albumin quantification (24hUTP). Data are expressed as mean ± SD (n = 8 per group). Compared with the Control group, ***P < 0.001; compared with the DM group, P < 0.001.

[0039] Figure 3 Observation of the histological changes in the renal tissues of each group by (A) HE and (B) PAS staining in Example 11. Magnification: 400×.

[0040] Figure 4 Illustration that the traditional Chinese medicine composition can inhibit renal fibrosis in diabetic mice in Example 11. (A) Renal tissue images by Masson trichrome staining. (B) Evaluation of fibrosis by the percentage of renal fibrosis area. (C) Detection of the mRNA expression levels of three fibrosis markers (α-SMA, collagen type I, and collagen type III) by RT-qPCR. (D) Detection of the protein bands of α-SMA, collagen type I, and collagen type III by Western blot. (E) Protein expression quantification of α-SMA, collagen type I, and collagen type III with GAPDH as the reference standard. Data are expressed as mean ± SD (n = 8 per group). Compared with the Control group, ***P < 0.001; compared with the DM group, P < 0.001.

[0041] Figure 5 Illustration that the traditional Chinese medicine composition inhibits apoptosis of renal tissue cells in diabetic mice in Example 11. (A) Representative renal images by TUNEL staining. (B) Quantification of TUNEL-positive cells in the renal tissues. (C) Detection of the mRNA levels of BAX and BCL-2 in the renal tissues by RT-qPCR with GAPDH as the reference standard. (D) Detection of the protein bands of BAX and BCL-2 in the renal tissues by Western blot. (E) Protein expression quantification of BAX and BCL-2 with GAPDH as the reference standard. Data are expressed as mean ± SD (n = 8 per group). Compared with the Control group, ***P < 0.001; compared with the DM group, P < 0.001.

[0042] Figure 6Illustration of the inhibitory effect of the traditional Chinese medicine composition in Example 11 on oxidative stress in the kidney tissues of diabetic mice. (A) Kidney superoxide images stained by DHE. (B) Quantitative analysis of the relative fluorescence intensity of DHE in kidney tissues. Quantitative analysis of (C) MDA, (D) SOD, and (E) CAT in renal tissue lysates. Data are expressed as mean ± SD (n = 8 per group). Compared with the Control group, ***P < 0.001; compared with the DM group, P < 0.001.

[0043] Figure 7 shows the illustration that the traditional Chinese medicine composition in Example 11 can enhance the protein expression of SIRT1 and Nrf2 in the renal tissues of diabetic mice. (A) Protein expression of SIRT1 and Nrf2 was detected by Western blot, and the pictures are typical protein bands. (B) Compared with the mice in the Control group, the SIRT1 protein in the DM group of mice was significantly decreased, and the Nrf2 protein was significantly increased. Compared with the mice in the DM group, both the SIRT1 protein and the Nrf2 protein were significantly increased after treatment in the WSY group. The expression of the SIRT1 protein was normalized to GAPDH, and the expression of the Nrf2 protein was normalized to PCNA. Data are expressed as mean ± SD (n = 3 per group). Compared with the Control group, ***P < 0.001; compared with the DM group, P < 0.001. Detailed implementation mode

[0044] The technical solutions of the present invention are illustrated by specific specific examples below. It should be understood that one or more method steps mentioned in the present invention do not exclude the existence of other method steps before and after the combined steps or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. Moreover, unless otherwise specified, the numbers of each method step are only convenient tools for identifying each method step, rather than limiting the arrangement order of each method step or the scope in which the present invention can be implemented. The change or adjustment of their relative relationship, without substantial change in the technical content, should also be regarded as the scope in which the present invention can be implemented.

[0045] Example 1

[0046] A traditional Chinese medicine composition, the medicinal components of which are calculated by weight and include the following components: 30 parts of Taxillus chinensis; 15 parts of mulberry fruits; 15 parts of ootheca mantidis; 15 parts of mulberry branches; 15 parts of mulberry bark; 15 parts of Atractylodes macrocephala; 10 parts of Saposhnikovia divaricata.

[0047] Using the conventional method for making traditional Chinese medicine decoctions, the above traditional Chinese medicine composition is decocted with water into a decoction.

[0048] Example 2

[0049] A traditional Chinese medicine composition, the medicinal effective components of which, calculated by weight parts, include the following components: 30 parts of Taxillus sutchuenensis; 15 parts of mulberries; 15 parts of ootheca mantidis; 15 parts of twigs of mulberry; 15 parts of cortex mori; 15 parts of bighead atractylodes rhizome; 10 parts of ledebouriella root.

[0050] Decoct the above traditional Chinese medicine composition with water twice, each time for 1 h. Add 12 times the amount of water for the first time and 8 times the amount of water for the second time. Filter the medicinal residues, combine the filtrates of the two times, concentrate the combined aqueous solution to a relative density of 1.05 - 1.06 (70 °C), add ethanol to make the alcohol content reach 55%, let it stand, filter, recover the ethanol from the filtrate and concentrate it to a fluid extract with a relative density of 1.12 - 1.16 (60 °C); mix the fine powder of the above medicinal residues, inclusion compound, fluid extract, and appropriate excipients evenly and granulate in one step, then press tablets and coat them with a film coating to obtain the tablets of the traditional Chinese medicine composition.

[0051] Example 3

[0052] A traditional Chinese medicine composition, the medicinal effective components of which, calculated by weight parts, include the following components: 30 parts of Taxillus sutchuenensis; 15 parts of mulberries; 15 parts of ootheca mantidis; 15 parts of twigs of mulberry; 15 parts of cortex mori; 15 parts of bighead atractylodes rhizome; 10 parts of ledebouriella root.

[0053] Decoct the above traditional Chinese medicine composition with water twice, each time for 1 h. Add 12 times the amount of water for the first time and 8 times the amount of water for the second time. Filter the medicinal residues, combine the filtrates of the two times, concentrate the combined aqueous solution to a relative density of 1.05 - 1.06 (70 °C), add ethanol to make the alcohol content reach 55%, let it stand, filter, recover the ethanol from the filtrate and concentrate it to a fluid extract with a relative density of 1.12 - 1.16 (60 °C); mix the fine powder of the above medicine, inclusion compound, fluid extract, and appropriate excipients evenly and granulate in one step to obtain the granules of the traditional Chinese medicine composition.

[0054] Example 4

[0055] A traditional Chinese medicine composition, the medicinal effective components of which, calculated by weight parts, include the following components: 30 parts of Taxillus sutchuenensis; 15 parts of mulberries; 15 parts of ootheca mantidis; 15 parts of twigs of mulberry; 15 parts of cortex mori; 15 parts of bighead atractylodes rhizome; 10 parts of ledebouriella root.

[0056] Decoct the above traditional Chinese medicine composition with water twice, each time for 1 h. Add 12 times the amount of water for the first time and 8 times the amount of water for the second time. Filter the medicinal residues, combine the filtrates of the two times, concentrate the combined aqueous solution to a relative density of 1.05 - 1.06 (70 °C), add ethanol to make the alcohol content reach 55%, let it stand, filter, recover the ethanol from the filtrate and concentrate it to a fluid extract with a relative density of 1.12 - 1.16 (60 °C); mix the fine powder of the above medicine, inclusion compound, fluid extract, and appropriate excipients evenly, fill them into capsules to obtain the capsule of the above traditional Chinese medicine composition.

[0057] Example 5

[0058] A traditional Chinese medicine composition, the active ingredients of which, by weight, include the following components: 30 parts of Taxillus sutchuenensis; 15 parts of mulberries; 15 parts of ootheca mantidis; 15 parts of twigs of mulberry; 15 parts of cortex mori; 15 parts of atractylodes macrocephala; 10 parts of ledebouriella seseloides.

[0059] Add 8 - 10 times the amount of water to the above traditional Chinese medicine composition, decoct for 3 hours, and filter out the medicinal juice. Then add 8 times the amount of water, decoct for 2 hours, filter out the medicinal juice, combine the two decoctions, let it stand, filter to obtain the supernatant, concentrate, cool, add 2 times the amount of alcohol of the concentrated solution, stir and precipitate overnight. Take the supernatant and concentrate it to a thick extract; add appropriate pharmaceutical excipients to make the mixture, oral liquid or syrup of this traditional Chinese medicine composition.

[0060] Example 6

[0061] A traditional Chinese medicine composition, the active ingredients of which, by weight, include the following components: 20 parts of Taxillus sutchuenensis; 12 parts of mulberries; 12 parts of ootheca mantidis; 15 parts of twigs of mulberry; 10 parts of cortex mori; 15 parts of atractylodes macrocephala; 10 parts of ledebouriella seseloides.

[0062] Using the conventional method for making traditional Chinese medicine decoction, decoct the above traditional Chinese medicine composition with water to make a decoction.

[0063] Example 7

[0064] A traditional Chinese medicine composition, the active ingredients of which, by weight, include the following components: 40 parts of Taxillus sutchuenensis; 25 parts of mulberries; 25 parts of ootheca mantidis; 20 parts of twigs of mulberry; 20 parts of cortex mori; 15 parts of atractylodes macrocephala; 10 parts of ledebouriella seseloides.

[0065] Decoct the above traditional Chinese medicine composition with water 3 times, each time for 2 hours. Add 10 times the amount of water for the first time and 6 times the amount of water for the second time. Filter the medicinal residues, combine the two filtrates, concentrate the aqueous solution after combination to a relative density of 1.06 - 1.11 (70 °C), add ethanol to make the alcohol content reach 60%, let it stand, filter, recover the ethanol from the filtrate and concentrate it to a fluid extract with a relative density of 1.13 - 1.22 (60 °C); mix the fine powder of the above medicinal residues, inclusion compound, fluid extract, and appropriate excipients evenly and granulate in one step, then press into tablets and coat with a film coating to obtain the tablets of this traditional Chinese medicine composition.

[0066] Example 8

[0067] A traditional Chinese medicine composition, the active ingredients of which, by weight, include the following components: 30 parts of Taxillus sutchuenensis; 25 parts of mulberries; 15 parts of ootheca mantidis; 15 parts of twigs of mulberry; 20 parts of cortex mori; 15 parts of atractylodes macrocephala; 10 parts of ledebouriella seseloides.

[0068] The above traditional Chinese medicine composition is decocted with water three times, each time for 2 hours. The first time, 10 times the amount of water is added, and the second time, 8 times the amount of water is added. The medicinal residues are filtered, and the two filtrates are combined. After being combined with the above aqueous solution, it is concentrated to a relative density of 1.06 - 1.11 (70 °C), ethanol is added to make the alcohol content reach 60%, allowed to stand, filtered, and the filtrate is concentrated to a flowing extract with a relative density of 1.13 - 1.22 (60 °C) after recovering ethanol; the above fine powder of the medicine, inclusion compound, flowing extract, and appropriate excipients are mixed evenly and granulated in one step to obtain the granules of this traditional Chinese medicine composition.

[0069] Example 9

[0070] A traditional Chinese medicine composition, the active ingredients of which are calculated by weight and include the following components: 20 parts of Taxillus chinensis; 25 parts of mulberries; 20 parts of Ootheca Mantidis; 20 parts of Ramulus Mori; 20 parts of Cortex Mori; 15 parts of Atractylodes macrocephala; 10 parts of Saposhnikovia divaricata.

[0071] The above traditional Chinese medicine composition is decocted with water three times, each time for 2 hours. The first time, 10 times the amount of water is added, and the second time, 8 times the amount of water is added. The medicinal residues are filtered, and the two filtrates are combined. After being combined with the above aqueous solution, it is concentrated to a relative density of 1.06 - 1.11 (70 °C), ethanol is added to make the alcohol content reach 60%, allowed to stand, filtered, and the filtrate is concentrated to a flowing extract with a relative density of 1.13 - 1.22 (60 °C) after recovering ethanol; the above fine powder of the medicine, inclusion compound, flowing extract, and appropriate excipients are mixed evenly and filled into capsules to obtain the capsule of the above traditional Chinese medicine composition.

[0072] Example 10

[0073] A traditional Chinese medicine composition, the active ingredients of which are calculated by weight and include the following components: 20 parts of Taxillus chinensis; 20 parts of mulberries; 25 parts of Ootheca Mantidis; 25 parts of Ramulus Mori; 20 parts of Cortex Mori; 10 parts of Atractylodes macrocephala; 10 parts of Saposhnikovia divaricata.

[0074] The above traditional Chinese medicine composition is added with 10 times the amount of water and decocted for 3 hours, and the medicinal juice is filtered out. Then, 2 times the amount of water is added and decocted for 3 hours, and the medicinal juice is filtered out. The two decoctions are combined, allowed to stand, the supernatant is filtered and taken, concentrated, cooled, 2 times the amount of alcohol of the concentrated solution is added, and stirred to precipitate overnight. The supernatant is taken and concentrated to a thick extract; appropriate pharmaceutical excipients are added to make the mixture, oral liquid or syrup of this traditional Chinese medicine composition.

[0075] Example 11

[0076] 1) Animals

[0077] Twenty-four healthy specific pathogen-free male C57BL / 6 mice (6 - 7 weeks old, weighing 22 - 25 g) were purchased from Shanghai Slac Laboratory Animal Co., Ltd. The mice were housed in a temperature- and humidity-controlled room 7 days before the experiment, maintained at a constant temperature of 20 - 24 °C, 35 - 45% humidity, and a 12-hour light / dark cycle, and had free access to standard specific pathogen-free feed and water. All experimental protocols were approved by the Animal Ethics Committee of our hospital and were conducted in accordance with the research protocols for experimental animals.

[0078] 2) Experimental materials and reagents

[0079] Streptozotocin (STZ) was purchased from Sigma. Mouse creatinine (SCr) ELISA kit (ml037726), urea nitrogen (BUN) test kit (ml076479), mouse urinary microalbumin (UAER) ELISA kit (ml063626), and mouse urinary protein (UTP) ELISA kit (ml037585) were purchased from Enzyme-linked Biotechnology. Hematoxylin and eosin (HE) staining kit (C0105S) and periodic acid-Schiff (PAS) staining kit (C0142S) were purchased from Beyotime. Oxidative stress kits MDA (S0131S), SOD (S0109), and CAT (S0051) were purchased from Beyotime. PrimeScript TM RT reagent Kit (RR047Q) and TB Premix Ex Taq TM II FAST qPCR (CN830S) was purchased from Takara. Rabbit anti-SIRT1 monoclonal antibody was purchased from Abcam (ab189494); rabbit anti-Nrf2 polyclonal antibody was purchased from Abcam (ab92946); rabbit anti-GAPDH polyclonal antibody was purchased from Abcam (ab9485), and rabbit anti-PCNA polyclonal antibody was purchased from Abcam (ab18197).

[0080] 3) Establishment of a diabetic nephropathy mouse model

[0081] Twenty-four mice were randomly divided into a control group (Control group), a diabetes group (DM group), and a Wusangyin group (WSY group), with 8 mice in each group. Mice in the DM group and the WSY group were fed with a high-fat and high-sugar diet for 4 weeks, fasted for 12 hours without water deprivation, and intraperitoneally injected with streptozotocin (STZ, 50 mg / kg) once a day for 5 consecutive days. Three days after the end of the intraperitoneal injection, the mice were tail-pricked for blood sampling to measure blood glucose. Diabetic mice were successfully modeled when the random fasting blood glucose was ≥16.7 mmol / L for more than 3 days. After STZ injection, 24-hour urine specimens were collected once a week from the diabetic mouse model, and a kit was used to measure the level of 24-hour urine total protein (UTP). When 24-hour UTP ≥ 30 mg, it indicated the successful establishment of a diabetic nephropathy mouse model.

[0082] 4) Preparation of the decoction of the traditional Chinese medicine composition

[0083] The decoction juice of the traditional Chinese medicine composition in Example 1 was concentrated to about 100 mL (each milliliter contained 1.25 grams of crude drug). It was stored in a 4°C refrigerator for later use. The adult clinical dosage of the crude drug of the traditional Chinese medicine composition was 115 g / day (based on a standard body weight of 60 kg, the dosage was 1.912 g / kg). Referring to the animal dosage conversion formula in the "Fourth Edition of Pharmacological Experiment Methodology", the dosage of mice was 12 times that of humans. The equivalent dosage of mice was obtained as 23 g / kg, and 46 g / kg, which was 2 times of it, was taken as the intragastric administration dosage of traditional Chinese medicine for mice.

[0084] 5) Grouping and intervention of experimental animals

[0085] After successful modeling, the diabetic mice were randomly divided into 2 groups according to different intervention factors: (1) DM group: After successful modeling, they were intragastrically administered with the same volume of distilled water every day; (2) WSY group: They were intragastrically administered with the concentrated juice of the decoction of the traditional Chinese medicine composition (46 g / kg); Another 8 mice were selected as the Control group: They were intragastrically administered with the same volume of distilled water every day. All three groups of mice were fed with a normal diet. The drug was administered once a day for 8 weeks.

[0086] 6) Specimen collection

[0087] The mice in the DM group, the WSY group, and the Control group were placed in a metabolic cage to collect 24-hour urine, record the urine volume, separate the supernatant after centrifugation, and aliquot and store it in an -80°C refrigerator. The mice were fasted for 12 hours without water deprivation, anesthetized by inhalation (3% isoflurane), and blood was taken from the femoral artery. After separating the serum, it was aliquot and stored at -80°C. The mice were sacrificed by cervical dislocation, and the kidneys were taken. The kidneys were divided into two parts. One part was fixed in 4% paraformaldehyde for histological detection, and the other part was frozen in an -80°C refrigerator for mRNA and protein expression detection.

[0088] Note: All data in the following examples are expressed as mean ± standard deviation. The SPSS 20.0 software package was used to analyze the data. One-way analysis of variance (ANOVA) was used for between-group comparison, and LSD (L) analysis was used for pairwise comparison. P < 0.05 was considered statistically significant.

[0089] 7) Detection of blood and urine biochemical indexes

[0090] The fasting blood glucose (FBG) of mice in the Control group, DM group, and WSY group was detected by the glucose oxidase method, the glycosylated hemoglobin (HbA1c) in mouse serum was determined by ELISA, the triglyceride (TG) and total cholesterol (TC) in blood were detected by enzymatic analysis (GPO-PAP colorimetric method), the serum creatinine (Scr) was measured by ELISA, the blood urea nitrogen (BUN) was detected by colorimetry, and the urinary albumin excretion rate (UAER) and urinary total protein (UTP) were measured by ELISA.

[0091] As Figure 1 shown, the fasting blood glucose (FBG), glycosylated hemoglobin (HbA1c), triglyceride (TG), and total cholesterol (TC) in the DM group of mice were significantly higher than those in the Control group (P < 0.001). Compared with the DM group, the FBG, HbA1c, TG, and TC in the WSY group were significantly decreased (P < 0.001) ( Figure 1 D, 1E, 1F, 1G).

[0092] As Figure 2 shown, administration of the decoction of the traditional Chinese medicine composition for 8 weeks significantly reduced the levels of serum creatinine (Scr), blood urea nitrogen (BUN), 24-hour urinary albumin excretion rate (24h UAER), and 24-hour urinary protein quantification (24 h UTP) in mice (P < 0.001). ( Figure 2 A, 2B, 2C, 2D).

[0093] 8) Effects of the traditional Chinese medicine composition on the body weight, kidney weight, and kidney hypertrophy index of mice

[0094] The body weights (BW) of mice in the Control group, DM group, and WSY group were weighed respectively. After cutting off the kidneys, the kidney weights (KW) were weighed, and the kidney hypertrophy index (KW / BW) was obtained from the ratio of KW (mg) to BW (g).

[0095] As Figure 1As shown, after 8 weeks of administration of the traditional Chinese medicine composition, the mice were weighed. Compared with the Control group, the body weight of the mice in the DM group decreased (P<0.01), the kidney weight increased (P<0.001), and the KW / BW increased significantly (P<0.001). Compared with the DM group, the body weight of the mice in the WSY group increased significantly (P<0.05), the kidney weight decreased significantly (P<0.01), and the KW / BW decreased significantly (P<0.01)( Figure 1 A,1B,1C).

[0096] 9) Biochemical analysis of kidney tissue

[0097] The kidney tissues of the mice in the Control group, DM group and WSY group were respectively prepared into tissue homogenates (10%, w / v), and commercial kits were used to measure the MDA content, SOD and CAT activities.

[0098] As Figure 6 shown, the kidney tissue sections of the mice were stained with DHE. Compared with the Control group, the red fluorescence in the kidney tissue of the mice in the DM group was significantly enhanced, but this increase was attenuated in the mice treated with the traditional Chinese medicine composition( Figure 6 A). Quantitative analysis showed that the traditional Chinese medicine composition significantly reduced the relative intensity of DHE fluorescence in the kidney tissue of the mice( Figure 6 B). The key biomarkers of oxidative stress, including MDA, SOD and CAT, were measured in the kidney tissue lysate. Treatment with the traditional Chinese medicine composition reduced the MDA content in the kidney tissue of the mice and increased the activities of SOD and CAT( Figure 6 C,6D,6E).

[0099] 10) Morphological analysis of kidney tissue

[0100] Part of the kidney tissues of the Control group, DM group and WSY group were fixed in 4% paraformaldehyde, embedded and fixed, made into paraffin sections 3 μm thick, dewaxed, and then stained with HE, PAS and Masson, and the morphological changes of the kidney tissues of each group were observed under an ordinary optical microscope.

[0101] As Figure 3 shown, the glomeruli and renal tubules of the mice in the Control group had clear structures, the epithelial cells were arranged neatly, and the basement membrane was intact; the glomeruli of the mice in the DM group were hypertrophied, the mesangial extracellular matrix increased, the basement membrane thickened, the renal tubule lumen dilated, and there was infiltration of inflammatory cells around the renal tubules and renal interstitium; compared with the DM group, the kidney lesions of the mice in the WSY group were alleviated, and the renal tissue damage improved to varying degrees( Figure 3 A,3B).

[0102] As Figure 4 shown, compared with the control group, there was deposition of blue collagen fibers in the renal tubules and renal interstitium of the diabetic group mice, and the deposition of blue collagen fibers decreased significantly after treatment with WusangyinFigure 4 A). Quantitative analysis showed that the renal fibrosis area in diabetic mice increased significantly, while WSY significantly attenuated this effect ( Figure 4 B).

[0103] 11) Immunofluorescence

[0104] Kidney tissues of mice in the Control group, DM group, and WSY group were obtained from frozen sections. The sections were stained with TUNEL (C1086, Beyotime) and DAPI (S0063, Beyotime), and observed under an inverted fluorescence microscope (IX51, Olympus, Japan) after washing three times in phosphate-buffered saline (PBS).

[0105] As Figure 5 shown, the number of TUNEL+ cells in the kidney tissues of diabetic mice increased significantly (green fluorescence), but this increase was significantly attenuated in mice treated with the traditional Chinese medicine composition ( Figure 5 A, 5B). RT-qPCR was used to analyze the expression of apoptosis-related genes. Compared with diabetic mice, treatment with the traditional Chinese medicine composition significantly decreased BAX mRNA expression and increased BCL-2 mRNA expression ( Figure 5 C, 5D).

[0106] 12) Quantitative PCR

[0107] Respectively, take 0.1 g of the renal tissue cortex of mice in the Control group, DM group, and WSY group, add 1 mL of TRIzol to extract the RNA of mouse renal tissue, and extract total RNA. Use reverse transcriptase for cDNA synthesis and a green PCR kit for quantitative PCR. Design primers for α-SMA, Collagen I, Collagen III, BAX, BCL-2, and GAPDH using DNAMAN software and synthesize them by Shanghai Bioengineering Co., Ltd. Primer sequences: α-SMA: forward: 5'-TCC TGA CGC TGA AGT ATC CGA TA-3', reverse: 5'-GGC CAC ACG AAG CTC GTT AT-3'; Collagen I: forward: 5'-GCT CCT CTT AGG GGC CACT-3', reverse: 5'-CCA CGT CTC ACC ATT GGG G-3'; Collagen III: forward: 5'-TCC CCT GGA ATC TGT GAA TC-3', reverse: 5'-TGA GTC GAA TTG GGG AGA AT-3'; BAX: forward: 5'-GCC TCC TCT CCT ACT TCG G-3', reverse: 5'-AAA AAT GCC TTT CCC CTT C-3'; BCL-2, forward: 5'-CTCGTC GCT ACC GTC GTG ACT TCG-3', reverse: 5'-CAG ATG CCG GTT CAG GTA CTC AGT C-3'; GADPH: forward: 5'-ACT CCA CTC ACG GCA AAT TC-3', reverse: 5'-TCT CCA TGG TGG TGA AGA CA-3'. The relative mRNA expression levels of podocyte autophagy-related genes LC3 II and p62 were collected and analyzed by the 2-ΔΔCt method. Using GAPDH as an internal reference, the relative expression levels of the genes to be measured were analyzed by the 2-ΔΔCt method.

[0108] As Figure 4 shown, the mRNA expressions of three fibrosis markers in mouse renal tissue were measured by RT-qPCR. The mRNA levels of α-SMA, collagen type I, and collagen type III in the renal tissue of DM group mice increased significantly, but the WSY group reduced these changes ( Figure 4 C). The expressions of these fibrosis-related proteins were analyzed by Western blot, showing that the decoction of the traditional Chinese medicine composition reduced the expressions of α-SMA, Collagen I, and Collagen III in renal tissue ( Figure 4 D, 4E).

[0109] 13) Western blot

[0110] Take 0.1 g of the renal tissue cortex of mice in the Control group, DM group, and WSY group respectively, add 1 mL of RIPA tissue protein lysate, grind thoroughly and centrifuge to obtain the supernatant. After quantification, perform SDS-PAGE electrophoresis and transfer to a nitrocellulose membrane. Block with 5% skim milk in TBST solution for 2 h, and then incubate with specific primary antibodies against SIRT1, Nrf2, GAPDH, and PCNA. After incubating with the secondary antibody (1:1000) overnight at 4°C, detect the protein bands by enhanced chemiluminescence (ECL) method. The bands are scanned and the gray values are calculated using gel analysis software.

[0111] As Figure 7 shown, Western blot was applied to analyze the protein expression levels of the SIRT1 / Nrf2 pathway in the renal tissues of mice in each group ( Figure 7 A). Compared with the mice in the Control group, the SIRT1 protein in the DM group mice was significantly decreased, and the Nrf2 protein was significantly increased. Compared with the DM group mice, both the SIRT1 protein and Nrf2 protein in the renal tissues of the WSY group mice were significantly increased ( Figure 7 B).

[0112] The above embodiments are only illustrative of the principles and effects of the present invention and are not used to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A traditional Chinese medicine composition, characterized in that, Its medicinal ingredients, by weight parts, include the following components, 2. The traditional Chinese medicine composition according to claim 1, wherein By weight parts, include the following components, 3. A traditional Chinese medicine preparation, characterized in that, The traditional Chinese medicine preparation includes an effective amount of the traditional Chinese medicine composition as described in claim 1 or 2.

4. The traditional Chinese medicine preparation according to claim 3, wherein The traditional Chinese medicine preparation is one or more of a traditional Chinese medicine decoction, traditional Chinese medicine tablets, traditional Chinese medicine granules, traditional Chinese medicine capsules, traditional Chinese medicine oral liquids, and traditional Chinese medicine syrups.

5. A preparation method of traditional Chinese medicine granules, characterized in that, The traditional Chinese medicine composition as described in claim 1 or 2 is subjected to first decoction and first filtration in sequence to obtain a first decoction liquid, then second decoction and second filtration are carried out to obtain a second filtrate, the first filtrate and the second filtrate are combined, first concentration and second concentration are carried out to obtain a fluid extract, and granulation is carried out to obtain the traditional Chinese medicine granules.

6. The preparation method of the traditional Chinese medicine granules according to claim 5, characterized in that, Includes at least one of the following technical features: A1) The time of the first decoction is 30 - 90 min; A2) The mass ratio of the water added in the first decoction to the mass of the traditional Chinese medicine composition is (10 - 15):1; A3) The time of the second decoction is 30 - 90 min; A4) The mass ratio of the water added in the second decoction to the mass of the residue after the first decoction filtration is (6 - 10):1; A5) The first concentration is to a relative density of 1.05 - 1.06; A6) After the first concentration, ethanol is further added and left standing for third filtration; preferably, ethanol is added such that the mass fraction of ethanol is 50 - 60%; A7) The second concentration is to a relative density of 1.12 - 1.16; A8) The granulation raw materials include a fluid extract, an inclusion compound, auxiliary materials, and fine powder of the traditional Chinese medicine composition.

7. A traditional Chinese medicine granule obtained by the method as described in claim 5 or 6.

8. A preparation method of a traditional Chinese medicine capsule, characterized in that, The traditional Chinese medicine granule as described in claim 7 is filled into capsules to obtain the traditional Chinese medicine capsules.

9. A preparation method of a traditional Chinese medicine tablet, characterized in that, The traditional Chinese medicine granule as described in claim 7 is tabletted and film-coated to obtain the traditional Chinese medicine tablets.

10. A method for preparing a traditional Chinese medicine oral liquid or traditional Chinese medicine syrup, characterized in that, The traditional Chinese medicine composition as described in claim 1 or 2 is subjected to third decoction and third filtration to obtain a third decoction liquid, then fourth decoction and fourth filtration are carried out to obtain a fourth filtrate, the third filtrate and the fourth filtrate are combined, third concentration, fourth concentration, and fifth concentration are carried out, and auxiliary materials are added to obtain a traditional Chinese medicine oral liquid or a traditional Chinese medicine syrup.

11. The preparation method of the traditional Chinese medicine oral liquid or traditional Chinese medicine syrup according to claim 10, characterized in that, Includes at least one of the following technical features: B1) The time of the third decoction is 2 - 4 h; B2) The mass ratio of the water added in the third decoction to the mass of the traditional Chinese medicine composition is (8 - 10):1; B3) The time of the fourth decoction is 1 - 2 h; B4) The mass ratio of the water added in the fourth decoction to the mass of the residue after the third decoction filtration is (5 - 9):1; B5) Ethanol is added during the fourth concentration, preferably, the mass of the ethanol is 1.5 - 3 times that of the third concentrated liquid; B6) The fifth concentration is to a thick extract.

12. Use of the traditional Chinese medicine composition as described in claim 1 or 2 and the traditional Chinese medicine preparation as described in claim 3 or 4 in the preparation of a drug for treating diabetic nephropathy.

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