Composition for treating chronic kidney disease as well as preparation method and application thereof

Abstract

The invention provides a composition for treating chronic kidney diseases and a preparation method and application thereof, and belongs to the technical field of traditional Chinese medicine compositions. In the preparation process, firstly, leech and stiff silkworm are crushed, so that effective components can be better dissolved out; secondly, the traditional Chinese medicine powder is fermented by adopting mixed strains, so that effective components are better separated out, the medicine effect is improved, and the treatment effect on patients is remarkably improved; beta-cyclodextrin is added in the preparation process of the composition, and the effective components of the medicine can be clathrated by adding the beta-cyclodextrin, so that the stability of the medicine is improved, the taste is covered, and the compliance of a patient is improved.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine composition technology, specifically relating to a composition for treating chronic kidney disease, its preparation method, and its application. Background Technology

[0002] Chronic kidney disease (CHD) is a chronic disorder of kidney structure and function (with a history of kidney damage longer than 3 months) caused by various factors, culminating in end-stage renal disease (uremia). It is characterized by high incidence, long course, complex pathogenesis, and poor prognosis, and currently there is no specific treatment. Clinically, treatment mainly involves traditional nutritional therapy and management of complications and comorbidities. End-stage treatment includes hemodialysis and kidney transplantation. However, nutritional therapy has a long treatment cycle, slow effects, and can only delay progression to a limited extent; replacement therapies such as hemodialysis and peritoneal dialysis have significant side effects and high costs; kidney transplantation faces challenges such as a shortage of donors, high surgical costs, postoperative rejection, and the use of large amounts of immunosuppressants leading to weakened immunity, placing a heavy economic and social burden on patients' families and society.

[0003] Traditional Chinese medicine (TCM) theory classifies chronic kidney disease (CKD) as a condition of deficiency of vital energy and excess of pathogenic factors. The deficiency primarily involves depletion of Qi, blood, Yin, and Yang, as well as weakness of the spleen and kidneys. The excess pathogenic factors include dampness, blood stasis, water retention, and external pathogens, which are significant factors in the development and progression of CKD. Therefore, accurately identifying the main causes of CKD and employing a treatment method that combines tonifying the body's vital energy with eliminating pathogenic factors is crucial for treatment. Tonifying the body's vital energy involves adjusting the balance of Qi, blood, Yin, and Yang, restoring organ function, protecting kidney tissue, and enhancing the body's resistance to disease. This approach focuses on tonifying the kidneys, often combined with methods to strengthen the spleen and nourish the liver. Specific methods include strengthening the spleen and tonifying the kidneys, nourishing the kidneys and liver, tonifying Qi and nourishing Yin (blood), and tonifying both Yin and Yang. The principles for medication are to avoid stagnation, to warm without causing dryness, and to nourish without being cloying. Eliminating pathogenic factors involves removing external pathogens and water retention, blood stasis, and toxins from the body. Common methods for eliminating pathogenic factors include promoting diuresis and removing dampness, clearing heat and detoxifying, purging the bowels and eliminating turbidity, and promoting blood circulation and removing blood stasis. However, current Chinese medicine has had very limited effectiveness in treating chronic kidney disease.

[0004] The inventors discovered in previous research that a combination of leeches, silkworm pupae, rhubarb, and astragalus has therapeutic effects on chronic kidney disease (CKD). They applied for a patent to protect this formulation, publication number CN107582648A. This patent discloses a traditional Chinese medicine composition for treating CKD, consisting of a first extract component, a second extract component, and a powdering component. The first extract component includes 5-7 parts by weight of leeches and 5-7 parts by weight of silkworm pupae; the second extract component includes 6-8 parts by weight of rhubarb; and the powdering component includes 45-55 parts by weight of astragalus and 2.5-3.5 parts by weight of rhubarb. The patent also relates to a method for preparing a traditional Chinese medicine preparation for treating CKD, and the traditional Chinese medicine preparation prepared by the method, which can be formulated as a kidney failure pill. This traditional Chinese medicine preparation has a simpler process and is easier to operate. The kidney failure pill has been used clinically at Wuhan Union Hospital for 10 years, with few side effects and definite efficacy, and is widely accepted by patients. Clinical studies have shown that Shenshui Pills can significantly reduce serum creatinine and blood urea nitrogen levels in patients with chronic kidney disease (CKD) and slow the progression of CKD.

[0005] However, subsequent studies have found that the stability of the kidney failure pills prepared by the existing methods is poor, especially due to their high hygroscopicity, which leads to the loss of the active ingredients. Therefore, it is necessary to develop a composition with high stability, good taste, and good efficacy for chronic kidney disease, as well as its preparation method. Summary of the Invention

[0006] Based on the shortcomings of existing technologies, this invention improves the preparation method, significantly enhancing the stability of the composition, especially by significantly reducing hygroscopicity, thereby making the content of active ingredients in the composition more stable. This provides a composition with high stability, good taste, and good therapeutic effect on chronic kidney disease, as well as its preparation method.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing a composition for treating chronic kidney disease includes the following steps: (1) Add water to leeches and silkworms and crush them to obtain a solid mixture slurry; concentrate the solid mixture slurry and let it stand to obtain a precipitate, and dry the precipitate for later use; (2) Rhubarb and Astragalus are crushed separately, the powders are mixed and extracted with ethanol, filtered, and the filtrate is concentrated to a density of 1.12-1.15 to obtain extract. The filter residue is dried and used for later use. (3) Mix the precipitate in step (1) and the filter residue in step (2) to obtain a mixture; soak the mixture in hot water for 5-10 hours, and when the water temperature drops below 35℃, add Bacillus subtilis and Aspergillus oryzae for fermentation to obtain the fermentation product, then distill, collect the distillate, dry, and obtain the drug powder; (4) The extract from step (2) and the medicinal powder and β-cyclodextrin from step (3) are mixed to obtain the composition.

[0008] The amount of water added in step (1) is 10-20 times; the concentration is carried out by using an ultrafiltration machine.

[0009] The particle size of the pulverized material in step (2) is 1-3 mm.

[0010] The volume concentration of ethanol in step (2) is 80-90%, the volume-to-mass ratio of ethanol to powder is 10-25 mL: 1 g, the number of extractions is 1-2, the extraction temperature is 70℃-80℃, and the extraction time is 8-12 hours.

[0011] The hot water temperature mentioned in step (3) is 90-100℃.

[0012] The mass ratio of Bacillus subtilis and Aspergillus oryzae in step (3) is 1:3-5; preferably 1:4.

[0013] The inoculation amount of Bacillus subtilis and Aspergillus oryzae in step (3) is 8-12%; preferably 10%.

[0014] The volume-to-mass ratio of hot water to the mixture in step (3) is 8-12 mL: 1 g; preferably 10 mL: 1 g.

[0015] The mass ratio of β-cyclodextrin to extract in step (3) is 1:5-10; preferably 1:6-8; and even more preferably 1:8.

[0016] The drying process described in step (3) is spray drying.

[0017] As some preferred embodiments, the method for preparing the composition for treating chronic kidney disease includes the following steps: (1) Add 10-20 times the amount of water to the leeches and silkworms and then crush them to obtain a solid mixture slurry; concentrate the solid mixture slurry and let it stand to obtain a precipitate, and dry the precipitate for later use; (2) Rhubarb and Astragalus are pulverized to 1-3 mm respectively. After mixing the powders, they are extracted with 80-90% ethanol. The volume-to-mass ratio of ethanol to powder is 10-25 mL: 1 g. The extraction is performed 1-2 times, at a temperature of 70℃-80℃, for 8-12 hours. After extraction, the mixture is filtered and the filtrate is concentrated to a density of 1.12-1.15 to obtain an extract. The filter residue is dried and stored for later use. (3) Mix the precipitate in step (1) and the filter residue in step (2) to obtain a mixture; soak the mixture in hot water at 90-100℃ for 5-10 hours, with the volume-to-mass ratio of hot water to mixture being 8-12mL:1g. When the water temperature drops below 35℃, add Bacillus subtilis and Aspergillus oryzae at a mass ratio of 1:3-5 for fermentation, with an inoculum amount of 8-12%, to obtain the fermentation product. Then distill, collect the distillate, dry, and obtain the drug powder. (4) The extract from step (2) and the medicinal powder and β-cyclodextrin from step (3) are mixed to obtain the composition.

[0018] First, the composition provided by the present invention first crushes the leeches and silkworm pupae during the preparation process, so that the active ingredients can be better dissolved and the utilization of the medicinal materials can be improved.

[0019] Secondly, the composition provided by the present invention uses Bacillus subtilis and Aspergillus oryzae to ferment the Chinese medicine powder during the preparation process. The bacteria will produce a variety of proteases in the process of metabolism, which can lyse the cell wall of the Chinese medicine, reduce the encapsulation effect of polysaccharides on the effective ingredients, and release more effective ingredients such as alkaloids, flavonoids, glycosides, organic acids and terpenes, thereby improving the efficacy. Furthermore, during the implementation of this invention, it was discovered that using Bacillus subtilis and Aspergillus oryzae in a mass ratio of 1:3-5 to ferment the components of traditional Chinese medicine can improve the dissolution of the active ingredients and significantly reduce the hygroscopicity of the resulting drug composition.

[0020] Furthermore, this invention incorporates β-cyclodextrin during the preparation of the composition. The addition of β-cyclodextrin can encapsulate the active pharmaceutical ingredient, thereby improving the stability of the drug, masking the taste, and improving patient compliance. The amount of β-cyclodextrin added is 10%-15% of the total content of leeches, silkworm pupae, astragalus, and rhubarb, preferably 10%.

[0021] The composition for treating chronic kidney disease comprises, by weight, the following components: Leech 5-10 parts, silkworm 5-10 parts, astragalus 40-50 parts and rhubarb 5-15 parts.

[0022] Preferably, the composition for treating chronic kidney disease comprises, by weight, the following components: Leech 6-9 parts, silkworm 6-9 parts, astragalus 42-48 parts and rhubarb 8-12 parts.

[0023] More preferably, the composition for treating chronic kidney disease comprises, by weight, the following components: Leech 8 parts, silkworm 8 parts, astragalus 45 parts and rhubarb 10 parts.

[0024] On the other hand, the present invention also provides the use of the above-mentioned composition for treating chronic kidney disease in the preparation of products for treating chronic kidney disease; wherein the dosage form of the product is tablets, pills or granules.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The composition prepared by the method provided by the present invention has smaller molecules, which are easier to absorb, thereby improving the bioavailability of the composition.

[0026] (2) First, the composition provided by the present invention first crushes the leeches and silkworms during the preparation process, so that the active ingredients can be better dissolved and the utilization of the medicinal materials is improved; Secondly, the composition provided by this invention uses Bacillus subtilis and Aspergillus oryzae to ferment the traditional Chinese medicine powder during the preparation process. The bacteria produce a variety of proteases during metabolism, which can lyse the cell walls of the traditional Chinese medicine, reduce the encapsulation effect of polysaccharides on the effective components, and release more effective components such as alkaloids, flavonoids, glycosides, organic acids and terpenes, thereby improving the efficacy and significantly improving the treatment effect for patients.

[0027] (3) In the process of implementation, it was found that fermenting the Chinese medicine ingredients with Bacillus subtilis and Aspergillus oryzae in a mass ratio of 1:3-5 can improve the dissolution of the active ingredients and significantly reduce the hygroscopicity of the resulting drug composition. Furthermore, β-cyclodextrin was added during the preparation of the composition. The addition of β-cyclodextrin can encapsulate the active ingredients of the drug, thereby improving the stability of the drug, masking the taste, and improving patient compliance. Attached Figure Description

[0028] Figure 1 Images of mouse kidneys stained with hematoxylin and eosin (HE) before and after drug administration; Figure 2 Data on collagen fiber deposition in mouse kidneys before and after drug administration; Figure 3 Data on the mRNA levels of α-SMA in the kidneys of mice before and after drug administration; Figure 4 This data represents the mRNA levels of type I collagen in the kidneys of mice before and after drug administration. Detailed Implementation

[0029] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0030] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0031] In the following examples, Bacillus subtilis and Aspergillus oryzae were purchased from Beijing Kezhan Biotechnology Co., Ltd., with product numbers BNCC185269 and BNCC185853, respectively.

[0032] The present invention will be further described below by way of specific embodiments.

[0033] Example 1: A method for preparing a composition for treating chronic kidney disease It comprises the following components by weight: Leech 5 parts, silkworm 5 parts, astragalus 40 parts and rhubarb 5 parts.

[0034] The preparation method includes the following steps: (1) Add 10 times the amount of water to leeches and silkworms and crush them to obtain a solid mixture slurry; concentrate the solid mixture slurry and let it stand to obtain a precipitate, and dry the precipitate for later use; (2) Rhubarb and Astragalus are pulverized to 1-3 mm respectively. After mixing the powders, they are extracted with 85% ethanol at a volume concentration of 20 mL: 1 g. The extraction is performed twice at a temperature of 80 °C for 8 hours. After extraction, the mixture is filtered and the filtrate is concentrated to a density of 1.12-1.15 to obtain an extract. The residue is dried and stored for later use. (3) Mix the precipitate in step (1) and the filter residue in step (2) to obtain a mixture; soak the mixture in 90°C hot water for 8 hours, with a volume-to-mass ratio of 8mL:1g for hot water to mixture. When the water temperature drops below 35°C, add Bacillus subtilis and Aspergillus oryzae in a mass ratio of 1:3 for fermentation, with an inoculum of 8%. Ferment for 30 hours to obtain the fermentation product, then distill, collect the distillate, dry, and obtain the drug powder. (4) Mix the extract from step (2) with the medicinal powder from step (3) and β-cyclodextrin, pass through a 10-mesh sieve, and granulate to obtain the final product; the amount of β-cyclodextrin added is 10% of the total content of leeches, silkworm, astragalus and rhubarb.

[0035] Example 2: Preparation method of a composition for treating chronic kidney disease It comprises the following components by weight: Leech 10 parts, silkworm 10 parts, astragalus 50 parts and rhubarb 15 parts.

[0036] The preparation method includes the following steps: The preparation method includes the following steps: (1) Add 20 times the amount of water to leeches and silkworms and then crush them to obtain a solid mixture slurry; concentrate the solid mixture slurry and let it stand to obtain a precipitate, and dry the precipitate for later use; (2) Rhubarb and Astragalus were pulverized to 1-3 mm respectively. The powders were mixed and extracted with 90% ethanol. The volume-to-mass ratio of ethanol to powder was 25 mL: 1 g. The extraction was performed twice at 80 °C for 12 hours. After extraction, the mixture was filtered and the filtrate was concentrated to a density of 1.12-1.15 to obtain an extract. The residue was dried and stored for later use. (3) Mix the precipitate in step (1) and the filter residue in step (2) to obtain a mixture; soak the mixture in 100°C hot water for 10 hours, with a volume-to-mass ratio of 12mL:1g. When the water temperature drops below 35°C, add Bacillus subtilis and Aspergillus oryzae in a mass ratio of 1:5 for fermentation, with an inoculum of 12%, to obtain the fermentation product. Then distill, collect the distillate, dry, and obtain the drug powder. (4) Mix the extract from step (2) with the medicinal powder from step (3) and β-cyclodextrin, pass through a 10-mesh sieve, and granulate to obtain the product. The amount of β-cyclodextrin added is 10% of the total content of leeches, silkworms, astragalus and rhubarb.

[0037] Example 3: Preparation method of a composition for treating chronic kidney disease It comprises the following components by weight: Leech 8 parts, silkworm 8 parts, astragalus 45 parts and rhubarb 10 parts.

[0038] The preparation method includes the following steps: (1) Add 15 times the amount of water to leeches and silkworms and then crush them to obtain a solid mixture slurry; concentrate the solid mixture slurry and let it stand to obtain a precipitate, and dry the precipitate for later use; (2) Rhubarb and Astragalus are pulverized to 1-3 mm respectively. After mixing the powders, they are extracted with 85% ethanol at a volume concentration of 20 mL: 1 g. The extraction is performed twice at a temperature of 80 °C for 10 hours. After extraction, the mixture is filtered and the filtrate is concentrated to a density of 1.12-1.15 to obtain an extract. The residue is dried and stored for later use. (3) Mix the precipitate in step (1) and the filter residue in step (2) to obtain a mixture; soak the mixture in 100°C hot water for 8 hours, with a volume-to-mass ratio of hot water to mixture of 10mL:1g. When the water temperature drops to below 35°C, add Bacillus subtilis and Aspergillus oryzae in a mass ratio of 1:4 for fermentation, with an inoculum of 10%, to obtain the fermentation product. Then distill, collect the distillate, dry, and obtain the drug powder. (4) Mix the extract from step (2) with the medicinal powder from step (3) and β-cyclodextrin, pass through a 10-mesh sieve, and granulate to obtain the product. The amount of β-cyclodextrin added is 10% of the total content of leeches, silkworms, astragalus and rhubarb.

[0039] Comparative Example 1 Prepared using the method described in patent CN107582648A, namely: Grind the astragalus and rhubarb (for powdering) into powder and pass them through an 80-mesh sieve. Boil leeches and silkworm pupae in 200mL of water for 45-60 minutes, filter, and collect the supernatant. Add the rhubarb (for extraction) to the residue, add another 200mL of water, boil, and simmer for 45-60 minutes. Filter, collect the supernatant, combine the filtrates, and concentrate to a certain volume (density 1.12-1.15). Then add the astragalus powder and rhubarb powder, pass through a 10-mesh sieve, and granulate.

[0040] Comparative Example 2 The difference from Example 3 is that only Bacillus subtilis is used for fermentation, while the rest is the same as Example 3.

[0041] Comparative Example 3 The difference from Example 3 is that only Aspergillus oryzae is used for fermentation, while the rest is the same as Example 3.

[0042] Comparative Example 4 The difference from Example 3 is that the mass ratio of Bacillus subtilis and Aspergillus oryzae is 1:1, while the rest is the same as Example 3.

[0043] Comparative Example 5 The difference from Example 3 is that the volume-to-mass ratio of hot water to the mixture in step (3) is 20 mL: 1 g, while the rest is the same as in Example 3.

[0044] Results data: 1. Detection of active ingredient content High performance liquid chromatography was used to simultaneously determine the active ingredients verbascoflavonoid glucoside, emodin, and emodin methyl ether in Astragalus membranaceus and Rheum palmatum, specifically referring to the content determination methods for Rheum palmatum and Astragalus membranaceus in the 2015 edition of the Chinese Pharmacopoeia.

[0045] The details are as follows: (1) Chromatographic conditions and system suitability: An Agilent TC-C18 column (4.6 mm × 250 mm, 5 μm) was used, with a mobile phase of acetonitrile (A) and 0.2% formic acid solution (B) gradient elution (0–20 min, 20%–40% A; 20–30 min, 40% A; 30–35 min, 90% A; 35–40 min, 20% A), at a flow rate of 1.0 mL / min. -1The column temperature was 30℃, the injection volume was 10 μL, and the detection wavelength was 260 nm. The theoretical plate number, calculated based on the isoflavone glucoside peak, should be no less than 3000; calculated based on the emodin peak, no less than 4000; and calculated based on the emodin methyl ether peak, no less than 3000.

[0046] Preparation of mixed reference solution (2) Preparation of reference stock solution: Take about 2 mg of verbascoside glucoside reference standard, accurately weigh it into a 10 mL volumetric flask, dissolve and dilute it to the mark with methanol, shake well, and use it as stock solution A1. Weigh approximately 3.6 mg of emodin reference standard into a 10 mL volumetric flask, dissolve and dilute to the mark with methanol, and shake well to obtain stock solution A2. Weigh approximately 2 mg of emodin methyl ether reference standard into a 50 mL volumetric flask, dissolve and dilute to the mark with methanol, and shake well to obtain stock solution A3.

[0047] Preparation of mixed reference solution: Take 1 mL each of stock solutions A1 and A2, and 7 mL of stock solution A3 into a 10 mL volumetric flask. Dissolve and dilute to the mark with methanol, and shake well to prepare mixed reference solution B1, in which the concentrations of verbascoside glucoside, emodin, and emodin methyl ether are 20.2 μg / mL, 36.1 μg / mL, and 30.8 μg / mL, respectively. Take 5 mL of mixed reference solution B1 into a 10 mL volumetric flask, dissolve and dilute to the mark with methanol, and shake well to prepare mixed reference solution B2, in which the concentrations of verbascoside glucoside, emodin, and emodin methyl ether are 10.1 μg / mL, 18.05 μg / mL, and 15.4 μg / mL, respectively.

[0048] (3) Preparation of the test solution Take approximately 2g of the powdered compositions prepared in Examples 1-3 and Comparative Examples 1-5 respectively, weigh accurately, place them in a stoppered conical flask, add 25 mL of methanol accurately, weigh, heat under reflux for 1 hour, cool, weigh again, replenish the lost weight with methanol, shake well, filter, and collect the filtrate to obtain the final product.

[0049] Each sample was tested in parallel three times, and the average value was taken and retained to two decimal places. The test results are shown in Table 1 below.

[0050] Table 1

[0051] According to the test results in Table 1 above, the present invention uses Bacillus subtilis and Aspergillus oryzae to ferment the Chinese medicine powder during the preparation process. The bacteria will produce a variety of proteases in their metabolism, which can lyse the cell wall of the Chinese medicine and reduce the encapsulation effect of polysaccharides on the effective ingredients, thereby significantly improving the dissolution rate of the effective ingredients and significantly increasing the content of the effective ingredients in Astragalus membranaceus and Rheum palmatum, such as isoflavone glucoside, emodin and emodin methyl ether. In Comparative Example 1, a traditional water extraction method was used, and some Astragalus membranaceus and Rheum palmatum were added to the composition in powder form without extraction. This significantly reduced the content of the active ingredients. Furthermore, the powder form resulted in poor bioavailability for patients and an unpleasant taste.

[0052] In Comparative Examples 2 and 3, only one strain of bacteria was used for fermentation. According to the test results in Table 1, the contents of verbascoside, emodin, and emodin methyl ether in the compositions obtained by fermentation with only one strain were significantly reduced compared with Examples 1-3. The predicted reason may be that the protease produced by one strain is relatively simple, so it cannot better cleave the cell walls of various Chinese medicines, thus affecting the dissolution of active ingredients and reducing the content of active ingredients.

[0053] In Comparative Example 3, the mass ratio of Bacillus subtilis and Aspergillus oryzae was changed to 1:1, which is not within the scope of protection of this invention. The content of the effective ingredients in the resulting composition was also lower than that in Examples 1-3. It is predicted that the reason may be that when two equal amounts of bacteria act on the Chinese medicine ingredients, the bacteria will compete with each other, thereby affecting the fermentation effect, which will affect the dissolution of the effective ingredients and thus reduce the content of the effective ingredients.

[0054] In Comparative Example 4, changing the volume-to-mass ratio of hot water to the mixture to 20 mL: 1 g increased the content of hot water, which also reduced the content of verbascoside, emodin, and emodin methyl ether. The reason is likely that the increased water content will affect the reproduction of the strain, thereby affecting the fermentation effect and thus affecting the dissolution of the active ingredients, resulting in a decrease in the content of the active ingredients.

[0055] In summary, only by using the preparation method provided by this invention can the content of the active ingredients in the composition be significantly increased, thereby improving the efficacy and thus the therapeutic effect.

[0056] 2. Hygroscopicity test Test samples: Compositions prepared in Examples 1-3 and Comparative Examples 1-4.

[0057] Test method: Place the test sample in a stoppered glass weighing bottle (size: outer diameter 30mm, height 10mm) that has already been weighed (m1), weigh it again and record it as m2. Place the weighing bottle open in a constant temperature desiccator at 25℃ and 55% relative humidity for 48h. Then close the weighing bottle lid, weigh it again and record it as m3. Calculate the moisture absorption rate (%).

[0058] The formula is as follows: Moisture absorption rate (%) = (m3 - m2) / (m2 - m1) × 100% The test results are shown in Table 2 below.

[0059] Table 2

[0060] According to the test results in Table 2 above, the moisture absorption rate of the compositions prepared in Examples 1-3 of this invention is less than 0.35%, which is relatively low. The reasons for this are speculated to be: First, this invention uses two strains of bacteria to ferment the Chinese medicine components. The fermented product molecules are relatively small and have a small surface area, thus reducing the moisture absorption. Second, this invention uses β-cyclodextrin to encapsulate the composition, thereby reducing the basis of the effective components and external moisture, further reducing the moisture absorption of the composition.

[0061] Comparative Example 1 used a traditional water extraction method, and some Astragalus membranaceus and Rheum palmatum were added to the composition in powder form without extraction. The addition of drug powder significantly increased the hygroscopicity of the composition, thereby affecting the stability of the composition.

[0062] In Comparative Examples 2-3 and 4, changing the type or ratio of bacterial strains will affect the fermentation effect of the Chinese medicine components, thereby affecting the rupture of cell walls, etc., and thus affecting the moisture absorption effect of the composition, resulting in an increase in moisture absorption rate compared to Examples 1-3.

[0063] 3. Efficacy test Eight-week-old male C57BL / 6 mice (Shulaibao Biotechnology, Wuhan, China) were selected and housed at the Laboratory Animal Center of Tongji Medical College, following the guidelines of the National Institutes of Health (NIH) for the use and care of laboratory animals. All mice were housed at a temperature of 23±2℃ and a humidity range of 30%-70%, with a 12-hour dark / light cycle and an adequate supply of food and water.

[0064] Before unilateral ureteral ligation (UUO) surgery, mice were fasted for 6-12 hours. Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital. Abdominal hair was shaved, and the surgical area was disinfected with povidone-iodine. The skin was incised along the midline of the abdomen (approximately 2-3 cm), and the muscle layer was separated to expose the abdominal cavity. The intestines were gently pushed to the right to expose the left kidney and ureter. Right-side manipulation was avoided to preserve a control. The ureter was lifted with sterile forceps and double-ligated with 4-0 silk suture approximately 0.5 cm below the renal pelvis to ensure complete cessation of urine flow. Slight swelling of the ureter above the ligation point could be observed. The muscle layer and skin were sutured layer by layer, and the incision was disinfected again. Two weeks post-surgery, the treatment group mice were treated by gavage with the composition prepared in Example 3 at a dose of 1.5 g / kg / day. Ten weeks after treatment, the animals were sacrificed, and test samples were collected for further analysis.

[0065] 3.1 HE staining (hematoxylin-eosin staining) 3.1.1 Paraffin-embedded sections: Mouse kidney tissue was fixed with 4% formalin phosphate and then embedded in paraffin. The section thickness was 4 μm. 3.1.2 Dewaxing to water: The sections were dewaxed sequentially by passing them through xylene I (10-15 minutes), xylene II (10-15 minutes), anhydrous ethanol I (2-5 minutes), anhydrous ethanol II (2-5 minutes), 95% ethanol (2-5 minutes), 80% ethanol (2-5 minutes), and 70% ethanol (2-5 minutes), and then rinsed with distilled water for 2 minutes. 3.1.3 Hematoxylin staining: Immerse in hematoxylin staining solution for 5-10 minutes, then rinse with tap water for 1-2 minutes to remove excess dye; 3.1.4 Differentiation: Differentiate with 1% hydrochloric acid alcohol for a few seconds to tens of seconds, observe under a microscope until the cell nuclei are clear and the background is colorless, and rinse with tap water for 1 minute; 3.1.5 Blue Reversion: Rinse with running water for 5-15 minutes, and observe under a microscope that the cell nuclei change from red to blue-purple; 3.1.6 Eosin staining: Stain with eosin solution for 30 seconds to 2 minutes, then rinse with distilled water for a few seconds; 3.1.7 Dehydration and Transparency: The product is dehydrated sequentially by passing it through 80% ethanol (30 seconds, then quickly rinsed with water), 95% ethanol I (1-2 minutes), 95% ethanol II (1-2 minutes), anhydrous ethanol I (2 minutes), anhydrous ethanol II (2 minutes), xylene I (5 minutes), and xylene II (5 minutes). 3.1.8 Mounting: Add an appropriate amount of neutral resin, cover with a coverslip, and avoid air bubbles.

[0066] 3.2 Masson's trichrome staining (modified method) 3.2.1 Embedded sections and dewaxed sections were then stained with hematoxylin and eosin (HE) in water. 3.2.2 Removal of fixative residue: Immerse in Lugol's iodine solution for 5 minutes, rinse with running water for 5 minutes; deiodinate with 5% sodium thiosulfate for 5 minutes, rinse with running water for 5 minutes; 3.3.2 Nuclear staining: Regaud hematoxylin staining for 5-10 minutes to enhance nuclear contrast; 3.2.4 Staining with acidic dye: Stain with Ponceau S acid fuchsin dye for 5-10 minutes, then rinse with distilled water for a few seconds; 3.2.5 Phosphomolybdic acid differentiation: Immerse in 1% phosphomolybdic acid aqueous solution for 5-10 minutes to remove the red color from muscle fibers; do not wash with water. 3.2.6 Collagen fiber staining: Stain with aniline blue solution for 5 minutes to make the collagen fibers blue, then rinse with distilled water for a few seconds; 3.2.7 Color separation and transparency: Immerse in 1% glacial acetic acid aqueous solution for 1 minute to reduce non-specific background staining; 3.2.8 Dehydration and Transparency: The product is sequentially dehydrated by passing it through 95% ethanol (1 minute), anhydrous ethanol I (1 minute), anhydrous ethanol II (1 minute), xylene I (5 minutes), and xylene II (5 minutes). 3.2.9 Sealing: Add a drop of neutral resin to seal the slide, and gently press the coverslip to remove bubbles.

[0067] 3.3 Detection of renal fibrosis markers Total RNA was extracted from collected mouse kidney tissue using TRIzol reagent (Servicebio, Wuhan, China), and its concentration and purity were measured by spectrophotometer.

[0068] The extracted RNA was reverse transcribed into cDNA using the Prime Script RT Replication Kit (Vazyme, Nanjing, China). Quantitative PCR analysis was performed using the SYBR Green Mixture (Vazyme, Nanjing, China) according to the manufacturer's instructions.

[0069] according to Figure 1-4 The test results showed that HE staining indicated that the composition prepared in Example 3 could improve renal tubular atrophy and cast formation in mice with renal interstitial fibrosis (see Appendix). Figure 1 Masson staining and quantitative analysis showed that the composition prepared in Example 3 significantly reduced collagen fiber deposition in the kidneys of mice with a renal interstitial fibrosis model (see Appendix). Figure 2 PCR results also showed that the mRNA levels of type I collagen and α-SMA, fibrosis markers, were significantly reduced in the kidneys of mice with renal interstitial fibrosis model after using the composition prepared in Example 3 (see Appendix). Figure 3-4 ).

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a composition for treating chronic kidney disease, characterized in that: Includes the following steps: (1) Add water to leeches and silkworms and crush them to obtain a solid mixture slurry; concentrate the solid mixture slurry and let it stand to obtain a precipitate, and dry the precipitate for later use; (2) Rhubarb and Astragalus are crushed separately, the powders are mixed and extracted with ethanol, filtered, and the filtrate is concentrated to a density of 1.12-1.15 to obtain extract. The filter residue is dried and used for later use. (3) Mix the precipitate in step (1) and the filter residue in step (2) to obtain a mixture; soak the mixture in hot water for 5-10 hours, and when the water temperature drops below 35℃, add Bacillus subtilis and Aspergillus oryzae for fermentation to obtain the fermentation product, then distill, collect the distillate, dry, and obtain the drug powder; (4) The extract from step (2) and the medicinal powder and β-cyclodextrin from step (3) are mixed to obtain the composition described above; The mass ratio of Bacillus subtilis and Aspergillus oryzae in step (3) is 1:3-5.

2. The preparation method according to claim 1, characterized in that: The amount of water added in step (1) is 10-20 times; the particle size of the pulverized material in step (2) is 1-3 mm.

3. The preparation method according to claim 1, characterized in that: The volume concentration of ethanol in step (2) is 80-90%, the volume-to-mass ratio of ethanol to powder is 10-25 mL: 1 g, the number of extractions is 1-2, the extraction temperature is 70℃-80℃, and the extraction time is 8-12 hours.

4. The preparation method according to claim 1, characterized in that: The mass ratio of Bacillus subtilis and Aspergillus oryzae in step (3) is 1:

4.

5. The preparation method according to claim 1, characterized in that: The inoculation amount of Bacillus subtilis and Aspergillus oryzae in step (3) is 8-12%.

6. The preparation method according to claim 1, characterized in that: The volume-to-mass ratio of the hot water to the mixture in step (3) is 8-12 mL: 1 g.

7. The preparation method according to claim 1, characterized in that: The mass ratio of β-cyclodextrin to extract in step (3) is 1:5-10.

8. The preparation method according to any one of claims 1-7, characterized in that: Includes the following steps: (1) Add 15 times the amount of water to leeches and silkworms and then crush them to obtain a solid mixture slurry; concentrate the solid mixture slurry and let it stand to obtain a precipitate, and dry the precipitate for later use; (2) Rhubarb and Astragalus are pulverized to 1-3 mm respectively. After mixing the powders, they are extracted with 85% ethanol at a volume concentration of 20 mL: 1 g. The extraction is performed twice at a temperature of 80 °C for 10 hours. After extraction, the mixture is filtered and the filtrate is concentrated to a density of 1.12-1.15 to obtain an extract. The residue is dried and stored for later use. (3) Mix the precipitate in step (1) and the filter residue in step (2) to obtain a mixture; soak the mixture in 100°C hot water for 8 hours, with a volume-to-mass ratio of 10mL:1g for hot water to mixture; when the water temperature drops below 35°C, add Bacillus subtilis and Aspergillus oryzae in a mass ratio of 1:4 for fermentation, with an inoculum of 10% to obtain the fermentation product; then distill, collect the fraction, dry, and obtain the drug powder. (4) Mix the extract from step (2) with the medicinal powder and β-cyclodextrin from step (3), pass through a 10-mesh sieve, and granulate to obtain the final product.

9. The preparation method according to any one of claims 1-7, characterized in that: The composition for treating chronic kidney disease comprises, by weight, the following components: Leech 5-10 parts, silkworm 5-10 parts, astragalus 40-50 parts and rhubarb 5-15 parts.

10. The use of the composition prepared by the method according to any one of claims 1-9 in the preparation of products for treating chronic kidney disease, characterized in that: The product dosage form is tablet, pill, or granule.

Images