A composition for treating chronic kidney disease, and a preparation method and application thereof

Abstract

The application provides a composition for treating chronic kidney disease and a preparation method and application thereof, and belongs to the technical field of traditional Chinese medicine compositions. In the preparation process, first, leeches and cantharides are crushed, so that effective components can be better dissolved; second, mixed strains are used to ferment traditional Chinese medicine powder, so that effective components are better precipitated, the efficacy is improved, and the treatment effect of patients is significantly improved; and in the preparation process of the composition, beta-cyclodextrin is added, the addition of the beta-cyclodextrin can complex the effective components of the medicine, so that the stability of the medicine is improved, the taste is covered, and the compliance of patients is improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of traditional Chinese medicine composition, and particularly relates to a composition for treating chronic kidney disease and a preparation method and application thereof. BACKGROUND

[0002] Chronic kidney disease is a disease caused by various reasons, which is chronic kidney structure and function disorder (kidney damage history is more than 3 months), and end-stage is uremia. It has the characteristics of high incidence, long course, complex pathogenesis and poor prognosis, and there is no specific treatment method at present. The traditional nutritional therapy, treatment of complications and comorbidities are mainly used in clinic. The end-stage is blood purification treatment and kidney transplantation treatment. However, the nutritional therapy has a long cycle, slow treatment effect, and can only delay the progression in a limited range; the replacement therapy such as hemodialysis and peritoneal dialysis has large side effects and high cost; kidney transplantation has problems such as lack of kidney source and high surgical cost, postoperative rejection, and application of a large number of immunosuppressive agents to make patients have low immunity, which brings serious economic and social burden to patients' families and society.

[0003] According to the theory of traditional Chinese medicine in China, chronic kidney disease belongs to the syndrome of deficiency of vital qi and excess of pathogenic factors. Deficiency of vital qi is mainly deficiency of qi, blood, yin and yang and deficiency of spleen and kidney. Excess of pathogenic factors including dampness, blood stasis, water and external pathogens, is an important factor for the occurrence and development of chronic kidney disease. Therefore, accurately grasping the main causes of chronic kidney disease and using the treatment method of combination of strengthening healthy qi and eliminating pathogenic factors are the key to treat the disease. Strengthening healthy qi is to adjust the balance of qi, blood, yin and yang of the body, restore the function of viscera, protect the kidney tissue and improve the disease resistance of the body. The method of strengthening healthy qi is mainly to benefit the kidney, and is often combined with methods of invigorating the spleen and nourishing the liver. The specific methods include invigorating the spleen and kidney, nourishing the kidney and liver, tonifying qi and nourishing yin (blood), and supplementing yin and yang. The principle of drug use is to avoid stagnation, to be warm but not dry, and to be nourishing but not greasy. Eliminating pathogenic factors is to eliminate external pathogens and water, blood stasis and turbidity in the body. The common methods for eliminating pathogenic factors include removing water and dampness, clearing heat and resolving toxin, purging the bowels and removing turbidity, promoting blood circulation and removing blood stasis, etc. However, the current traditional Chinese medicine has little effect on the treatment of chronic kidney disease.

[0004] The inventors found in previous studies that the combination of leeches, silkworms, rhubarb and astragalus has the efficacy of treating chronic kidney disease, and a patent has been applied for protection, with the publication number CN107582648A. The patent discloses a traditional Chinese medicine composition for treating chronic kidney disease (CKD), which is composed of a first extraction component, a second extraction component and a powder component. The first extraction component includes 5-7 parts by weight of leeches and 5-7 parts by weight of silkworms; the second extraction component includes 6-8 parts by weight of rhubarb; and the powder 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 the traditional Chinese medicine preparation for treating CKD, and the traditional Chinese medicine preparation prepared by the method, which can be prepared into kidney failure pills. The traditional Chinese medicine preparation process is more concise and simple to operate. The kidney failure pills have been used in Wuhan Union Hospital for 10 years, have small toxic and side effects, have definite curative effect, and are widely accepted by patients. Clinical studies have shown that the kidney failure pills can significantly reduce the blood creatinine and urea nitrogen levels of CKD and delay the progression of CKD.

[0005] However, in subsequent studies, it was found that the kidney failure pills prepared by the existing method have poor stability, especially high hygroscopicity, which can cause the loss of effective ingredients of the medicine. Therefore, it is necessary to develop a composition with high stability, good taste and good efficacy for chronic kidney disease and a preparation method thereof. SUMMARY

[0006] Based on the deficiencies of the prior art, the stability of the composition is significantly improved by improving the preparation method, especially the hygroscopicity is significantly reduced, so that the content of the effective ingredients in the composition is more stable, thereby providing a composition with high stability, good taste and good efficacy for chronic kidney disease and a preparation method thereof.

[0007] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0008] A preparation method of a composition for treating chronic kidney disease, comprising the following steps:

[0009] (1) crushing leeches and silkworms after adding water to obtain a solid mixture slurry; concentrating the solid mixture slurry and then standing to obtain a precipitate, which is dried for standby use;

[0010] (2) respectively crushing rhubarb and astragalus, mixing the powders and then extracting with ethanol, filtering, concentrating the filtrate to a density of 1.12-1.15 to obtain an extract, and drying the filter residue for standby use;

[0011] (3) mixing the precipitate in step (1) and the residue in step (2) to obtain a mixture; soaking the mixture in hot water for 5-10 hours, adding Bacillus subtilis and Aspergillus oryzae when the water temperature drops to below 35℃ to obtain a fermentation product, then distilling, collecting the distillate and drying to obtain a medicinal powder;

[0012] (4) mixing the extract in step (2) and the medicinal powder in step (3) and β-cyclodextrin to obtain the composition.

[0013] The amount of water in step (1) is 10-20 times; the concentration is performed by using an ultrafiltration machine.

[0014] The particle size of the powder in step (2) is 1-3 mm.

[0015] The volume concentration of ethanol in step (2) is 80-90%, the volume-mass ratio of ethanol to powder is 10-25 mL:1 g, the extraction times is 1-2 times, the extraction temperature is 70-80℃, and the time is 8-12 hours.

[0016] The temperature of the hot water in step (3) is 90-100℃.

[0017] The mass ratio of Bacillus subtilis to Aspergillus oryzae in step (3) is 1:3-5; preferably 1:4.

[0018] The inoculation amount of Bacillus subtilis and Aspergillus oryzae in step (3) is 8-12%; preferably 10%.

[0019] The volume-mass ratio of hot water to the mixture in step (3) is 8-12 mL:1 g; preferably 10 mL:1 g.

[0020] The mass ratio of β-cyclodextrin to the extract in step (3) is 1:5-10; preferably 1:6-8; more preferably 1:8.

[0021] The drying in step (3) is spray drying.

[0022] As some preferred embodiments, the preparation method of the composition for treating chronic kidney disease comprises the following steps:

[0023] (1) crushing Leech and Bombyx Batryticatus in 10-20 times of water to obtain a solid mixture slurry; concentrating the solid mixture slurry and then standing to obtain a precipitate, which is dried for later use;

[0024] (2) respectively, the rhubarb and Astragalus root is crushed to 1-3mm, powder mixture is carried out after using the volume concentration of 80-90% ethanol, the volume mass ratio of ethanol and powder is 10-25mL: 1g, the extraction frequency is 1-2 times, the extraction temperature is 70-80 DEG C, the time is 8-12 hours, after filtering, the filtrate is concentrated to the density of 1.12-1.15, the extract is obtained, the filter residue is dried and reserved;

[0025] (3) the precipitate in step (1) and the filter residue in step (2) are mixed to obtain a mixture; the mixture is soaked in hot water at 90-100 DEG C for 5-10 hours, the volume mass ratio of hot water and the mixture is 8-12mL: 1g, when the water temperature is reduced to below 35 DEG C, Bacillus subtilis and Aspergillus oryzae with a mass ratio of 1:3-5 are added for fermentation, the inoculation amount is 8-12%, a fermentation product is obtained, then distillation is carried out, the distillate is collected and dried to obtain a medicinal powder;

[0026] (4) the extract of step (2) and the medicinal powder of step (3) and β-cyclodextrin are mixed to obtain the composition.

[0027] Firstly, in the preparation process of the composition, water leech and silkworm chrysalis are first broken, so that the effective components can be better dissolved out, and the utilization of medicinal materials is improved.

[0028] Secondly, in the preparation process of the composition, Bacillus subtilis and Aspergillus oryzae are used for fermentation of traditional Chinese medicine powder, and various proteases are produced in the metabolism of the strains, which can lyse the cell wall of traditional Chinese medicine, reduce the wrapping effect of polysaccharides and other effective components, and precipitate more alkaloids, flavones, glycosides, organic acids and terpenes, so as to improve the efficacy.

[0029] Thirdly, in the implementation process of the present application, it is found that the use of Bacillus subtilis and Aspergillus oryzae with a mass ratio of 1:3-5 for fermentation of traditional Chinese medicine components can improve the dissolution of effective components, and can significantly reduce the hygroscopicity of the obtained medicinal composition.

[0030] Fourthly, β-cyclodextrin is added in the preparation process of the composition, which can complex the effective components of the medicine, thereby improving the stability of the medicine and masking the taste, and improving the patient compliance. The addition amount of β-cyclodextrin is 10%-15% of the total content of water leech, silkworm chrysalis, Astragalus root and rhubarb, preferably 10%.

[0031] The composition for treating chronic kidney disease comprises the following components by weight fraction:

[0032] Water leech 5-10 parts, silkworm chrysalis 5-10 parts, Astragalus root 40-50 parts and rhubarb 5-15 parts.

[0033] Preferably, the composition for treating chronic kidney disease comprises the following components by weight fraction:

[0034] Leech 6-9 parts, Can 6-9 parts, Huangqi 42-48 parts and Dahuang 8-12 parts.

[0035] More preferably, the composition for treating chronic kidney disease comprises the following components by weight fraction:

[0036] Leech 8 parts, Can 8 parts, Huangqi 45 parts and Dahuang 10 parts.

[0037] In another aspect, the present application also provides the use of the above-mentioned composition for treating chronic kidney disease in the preparation of a product for treating chronic kidney disease; the product dosage form is a tablet, a pill or a granule.

[0038] Compared with the prior art, the present application has the beneficial effects that:

[0039] (1) The composition prepared by the method provided by the present application has smaller molecules, is easy to absorb, and thus improves the bioavailability of the composition.

[0040] (2) First, the composition provided by the present application is first crushed during preparation to make the effective components better dissolved, thus improving the utilization of medicinal materials.

[0041] Secondly, the composition provided by the present application is fermented by Bacillus subtilis and Aspergillus oryzae during preparation, and the strains produce various proteases in metabolism, which can lyse the cell walls of traditional Chinese medicines, reduce the wrapping effect of polysaccharides and other effective components, and precipitate more effective components such as alkaloids, flavonoids, glycosides, organic acids and terpenes, thus improving the efficacy and significantly improving the treatment effect of patients.

[0042] (3) It is found in the implementation of the present application that the fermentation of traditional Chinese medicine components by Bacillus subtilis and Aspergillus oryzae with a mass ratio of 1:3-5 can improve the dissolution of effective components, and the obtained pharmaceutical composition has significantly reduced hygroscopicity; and β-cyclodextrin is added during the preparation of the composition, which can complex the effective components of the medicine, thus improving the stability of the medicine and masking the taste, and improving the patient compliance. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 is the HE staining chart of the kidney of a mouse before and after taking the medicine;

[0044] Figure 2 is the data of the collagen fiber deposition index in the kidney of a mouse before and after taking the medicine;

[0045] Figure 3The mRNA level of α-SMA in the kidney of the mice before and after the administration of the composition is taken as an index.

[0046] Figure 4 The mRNA level of collagen type I in the kidney of the mice before and after the administration of the composition is taken as an index. DETAILED DESCRIPTION

[0047] The following non-limiting examples can make those skilled in the art more fully understand the present application, but do not limit the present application in any way. The following content is only an exemplary description of the scope of the present application, and those skilled in the art can make various changes and modifications to the present application based on the disclosed content, and it should also belong to the scope of the present application.

[0048] When the examples give numerical ranges, it should be understood that, unless otherwise specified by the present application, each numerical range of two endpoints and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as generally understood by those skilled in the art to which the present application belongs.

[0049] In the following examples, Bacillus subtilis and Aspergillus oryzae were purchased from Beijing Kaye Biological Technology Co., Ltd., product numbers BNCC185269 and BNCC185853, respectively.

[0050] The present application is further described below in the form of specific examples.

[0051] Example 1 Preparation method of a composition for treating chronic kidney disease

[0052] The following components are included by weight fraction:

[0053] Leech 5 parts, Can 5 parts, Huangqi 40 parts and Dahuang 5 parts.

[0054] The preparation method includes the following steps:

[0055] (1) Leech and Can are crushed after adding 10 times water to obtain a solid mixture slurry; the solid mixture slurry is concentrated and then left to stand to obtain a precipitate, which is dried for later use;

[0056] (2) Dahuang and Huangqi are respectively crushed to 1-3 mm, the powders are mixed and then extracted with 85% ethanol by volume concentration, the volume to mass ratio of ethanol to powder is 20 mL:1 g, the extraction is performed twice, the extraction temperature is 80°C, the time is 8 hours, after the end of extraction, filtration is performed, the filtrate is concentrated to a density of 1.12-1.15 to obtain an extract, and the filter residue is dried for later use;

[0057] (3) mixing the precipitate in step (1) and the residue in step (2) to obtain a mixture; soaking the mixture in 90℃ hot water for 8 hours, the volume to mass ratio of the hot water to the mixture being 8mL:1g, and then adding Bacillus subtilis and Aspergillus oryzae with a mass ratio of 1:3 to the mixture for fermentation after the water temperature drops to below 35℃, the inoculation amount being 8%, and obtaining a fermentation product after fermentation for 30 hours, then distilling, collecting the distillate, and drying to obtain a medicinal powder;

[0058] (4) mixing the extract in step (2) and the medicinal powder in step (3) and β-cyclodextrin, passing through a 10-mesh sieve, and granulating, to obtain the product, the addition amount of β-cyclodextrin being 10% of the total content of the water leech, the silkworm chrysalis, the astragalus root, and the rhubarb.

[0059] Example 2: a preparation method of a composition for treating chronic kidney disease

[0060] The composition comprises the following components by weight:

[0061] 10 parts of water leech, 10 parts of silkworm chrysalis, 50 parts of astragalus root, and 15 parts of rhubarb.

[0062] The preparation method comprises the following steps:

[0063] The preparation method comprises the following steps:

[0064] (1) crushing the water leech and the silkworm chrysalis after adding 20 times water to obtain a solid mixture slurry; concentrating the solid mixture slurry and then standing to obtain a precipitate, which is dried for later use;

[0065] (2) crushing the rhubarb and the astragalus root to 1-3mm respectively, mixing the powders, and then extracting with 90% ethanol, the volume to mass ratio of the ethanol to the powders being 25mL:1g, the extraction being performed twice, the extraction temperature being 80℃, and the extraction time being 12 hours, then filtering, concentrating the filtrate to a density of 1.12-1.15 to obtain an extract, and drying the residue for later use;

[0066] (3) mixing the precipitate in step (1) and the residue in step (2) to obtain a mixture; soaking the mixture in 100℃ hot water for 10 hours, the volume to mass ratio of the hot water to the mixture being 12mL:1g, and then adding Bacillus subtilis and Aspergillus oryzae with a mass ratio of 1:5 to the mixture for fermentation after the water temperature drops to below 35℃, the inoculation amount being 12%, and obtaining a fermentation product after fermentation for 30 hours, then distilling, collecting the distillate, and drying to obtain a medicinal powder;

[0067] (4) mixing the extract in step (2) and the medicinal powder in step (3) and β-cyclodextrin, passing through a 10-mesh sieve, and granulating, to obtain the product, the addition amount of β-cyclodextrin being 10% of the total content of the water leech, the silkworm chrysalis, the astragalus root, and the rhubarb.

[0068] Example 3 A method for preparing a composition for treating chronic kidney disease

[0069] The following components are included by weight parts:

[0070] Leech 8 parts, can 8 parts, radix astragali 45 parts and rhubarb 10 parts.

[0071] The preparation method comprises the following steps:

[0072] (1) Leech and can are crushed after adding 15 times water, and a solid mixture slurry is obtained. The solid mixture slurry is concentrated and then left to stand, and a precipitate is obtained. The precipitate is dried and reserved for later use;

[0073] (2) Rhubarb and radix astragali are respectively crushed to 1-3 mm. The powders are mixed and then extracted with 85% ethanol by volume concentration. The volume to mass ratio of ethanol to powder is 20 mL:1 g. The extraction is performed twice. The extraction temperature is 80°C. The time is 10 hours. After the extraction is completed, filtration is performed. The filtrate is concentrated to a density of 1.12-1.15. The extract is obtained. The filter residue is dried and reserved for later use;

[0074] (3) The precipitate in step (1) and the filter residue in step (2) are mixed to obtain a mixture. The mixture is soaked in hot water at 100°C for 8 hours. The volume to mass ratio of hot water to the mixture is 10 mL:1 g. When the water temperature drops to below 35°C, Bacillus subtilis and Aspergillus oryzae with a mass ratio of 1:4 are added for fermentation. The inoculation amount is 10%. The fermentation product is obtained. Then, distillation is performed. The distillate is collected and dried to obtain medicinal powder;

[0075] (4) The extract of step (2), the medicinal powder of step (3) and β-cyclodextrin are mixed and passed through a 10-mesh sieve. The whole particle is obtained. The addition amount of β-cyclodextrin is 10% of the total content of leech, can, radix astragali and rhubarb.

[0076] Comparative Example 1

[0077] The method in patent CN107582648A is used for preparation, that is:

[0078] Rhubarb for powdering and radix astragali are crushed and passed through an 80-mesh sieve and reserved for later use. Leech and can are decocted in water 200 mL for 45-60 min. Filtration is performed. The supernatant is obtained. Rhubarb for extraction is added to the filter residue. Water 200 mL is added. Decoction is performed for 45-60 min. Filtration is performed. The supernatant is obtained. The filtrates are combined. Concentration is performed to a certain volume (the density is detected to be 1.12-1.15). Then, radix astragali powder and rhubarb powder are added. The mixture is passed through a 10-mesh sieve. The whole particle is obtained.

[0079] Comparative Example 2

[0080] The difference from Example 3 is that only Bacillus subtilis is used for fermentation. The other steps are the same as those in Example 3.

[0081] Comparative Example 3

[0082] The difference from Example 3 is that only Aspergillus oryzae is used for fermentation, and the others are the same as Example 3.

[0083] Comparative Example 4

[0084] The difference from Example 3 is that the mass ratio of Bacillus subtilis and Aspergillus oryzae is 1:1, and the others are the same as Example 3.

[0085] Comparative Example 5

[0086] The difference from Example 3 is that in step (3), the volume-to-mass ratio of hot water to the mixture is 20 mL:1 g, and the others are the same as Example 3.

[0087] Effect data:

[0088] 1. Active ingredient content detection

[0089] The active ingredients in Astragalus and Rheum, calycosin-7-glucoside, emodin and physcion, were simultaneously determined by high performance liquid chromatography, and the content determination method of Rheum and Astragalus in the 2015 edition of Chinese Pharmacopoeia was referred to.

[0090] Specifically as follows:

[0091] (1) Chromatographic conditions and system suitability:

[0092] Agilent TC-C18 chromatographic column (4.6 mm x 250 mm, 5 μm), mobile phase acetonitrile (A)-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), flow rate 1.0 mL·min -1 , column temperature 30℃, injection volume 10 μL, detection wavelength 260 nm. The theoretical plate number is not less than 3000 according to the peak of calycosin-7-glucoside, not less than 4000 according to the peak of emodin, and not less than 3000 according to the peak of physcion.

[0093] Preparation of mixed reference solution

[0094] (2) Preparation of reference stock solution:

[0095] Take about 2 mg of calycosin-7-glucoside reference substance, accurately weigh into a 10 mL volumetric flask, dissolve and dilute to the mark with methanol, shake well, and use as stock solution A1;

[0096] Take about 3.6 mg of emodin reference substance, accurately weigh into a 10 mL volumetric flask, dissolve and dilute to the mark with methanol, shake well, as stock solution A2;

[0097] Take about 2 mg of emodin methyl ether reference substance, accurately weigh into a 50 mL volumetric flask, dissolve and dilute to the mark with methanol, shake well, as stock solution A3.

[0098] The preparation of the mixed reference solution: take 1 mL of each of stock solutions A1 and A2, take 7 mL of stock solution A3 into a 10 mL volumetric flask, dissolve and dilute to the mark with methanol, shake well, to prepare mixed reference solution B1, wherein the concentrations of calycosin-7-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, shake well, to prepare mixed reference solution B2, wherein the concentrations of calycosin-7-glucoside, emodin and emodin methyl ether are 10.1 μg / mL, 18.05 μg / mL and 15.4 μg / mL respectively.

[0099] (3) Preparation of test sample solution

[0100] Take about 2 g of the composition powder prepared in Examples 1-3 and Comparative Examples 1-5 respectively, accurately weigh, place in a conical flask with a stopper, accurately add 25 mL of methanol, weigh, heat to reflux for 1 hour, cool, weigh again, make up the weight loss with methanol, shake well, filter, take the filtrate, and obtain.

[0101] Each sample is detected in parallel for 3 times, take the average value, retain 2 decimal places, and the detection results are shown in Table 1 below.

[0102] Table 1

[0103]

[0104] According to the detection results in Table 1 above, in the preparation process, the traditional water extraction method is used in Comparative Example 1, and part of the Astragalus and rhubarb is not extracted, but added to the composition in the form of powder, so that the content of the effective components is obviously reduced, and the bioavailability is not good when the patient takes it, and the taste is not good.

[0105] In Comparative Example 1, the traditional water extraction method is used, and part of the Astragalus and rhubarb is not extracted, but added to the composition in the form of powder, so that the content of the effective components is obviously reduced, and the bioavailability is not good when the patient takes it, and the taste is not good.

[0106] The fermentation in Comparative Example 2 and Comparative Example 3 is carried out by using only one kind of bacteria, and according to the detection results in Table 1, it can be seen that the contents of calycosin-7-glucoside, emodin and physcion in the composition obtained by using one kind of bacteria for fermentation are also obviously reduced compared with those in Example 1-3, and the predicted reason is that the kind of protease produced by one kind of bacteria is relatively single, thus the cell walls of the multiple traditional Chinese medicines cannot be betterly lysed, thus the dissolution of the effective components is affected, and thus the contents of the effective components are reduced.

[0107] In Comparative Example 3, the mass ratio of Bacillus subtilis and Aspergillus oryzae is changed to 1:1, which is not within the protection scope of the present application, and the contents of the effective components in the obtained composition are also reduced compared with those in Example 1-3, and the predicted reason is that when the two kinds of bacteria act on the traditional Chinese medicine components, competition between the bacteria occurs, thus the fermentation effect is affected, and thus the dissolution of the effective components is affected, and thus the contents of the effective components are reduced.

[0108] In Comparative Example 4, the volume-mass ratio of hot water to the mixture is changed to 20 mL:1 g, and the content of hot water is increased, which also reduces the contents of calycosin-7-glucoside, emodin and physcion, and the expected reason is that the increase of the water content affects the propagation of the bacteria, thus the fermentation effect is affected, and thus the dissolution of the effective components is affected, and thus the contents of the effective components are reduced.

[0109] In summary, only by using the preparation method provided in the present application can the contents of the effective components in the composition be obviously increased, thus the drug efficacy is improved, and thus the treatment effect is improved.

[0110] 2. Hygroscopicity detection

[0111] Test sample: the composition prepared in Example 1-3 and Comparative Example 1-4.

[0112] Test method: the test sample is placed in a weighed (m1) glass weighing bottle with a stopper (the size is: outer diameter 30 mm, height 10 mm), weighed again, recorded as m2, and placed in a constant temperature dryer with a temperature of 25℃ and a relative humidity of 55% with the weighing bottle open, placed for 48 h, covered with the weighing bottle cover, weighed, recorded as m3, and the hygroscopicity (%) is calculated.

[0113] The formula is as follows: hygroscopicity (%) = (m3-m2) / (m2-m1) x 100%

[0114] The detection results are shown in Table 2 below.

[0115] Table 2

[0116]

[0117] According to the detection results in Table 2, the moisture absorption rate of the composition prepared in the present application examples 1-3 is less than 0.35%, which has a lower moisture absorption rate. It is speculated that the reason is as follows: firstly, two kinds of bacteria are used to ferment the traditional Chinese medicine components in the present application, and the product after fermentation has smaller molecules and smaller surface area, thereby reducing the moisture absorption; secondly, the composition is wrapped by β-cyclodextrin, thereby reducing the basis of the effective components and external moisture, and further reducing the moisture absorption of the composition.

[0118] The comparative example 1 uses the traditional water extraction method, and part of the radix astragali and rhubarb is not extracted, but added in the form of powder to the composition. Since the addition of the drug powder significantly increases the moisture absorption of the composition, the stability of the composition is affected.

[0119] In the comparative examples 2-3 and the comparative example 4, the type or ratio of the bacteria is changed, which will affect the fermentation effect of the traditional Chinese medicine components, thereby affecting the rupture of the cell wall, and further affecting the moisture absorption effect of the composition, so that the moisture absorption rate is increased compared with the examples 1-3.

[0120] 3. Pharmacodynamic test

[0121] 8-week-old male C57BL / 6 mice (Shulaibao Biotechnology, Wuhan, China) were used in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All mice were housed under a 12-hour light / dark cycle at 23 ± 2 °C with a humidity range of 30-70% and had free access to food and water.

[0122] Before unilateral ureteral ligation (UUO) surgery, the mice were fasted for 6-12 hours. The mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital, the abdominal hair was shaved, and the surgical area was disinfected with iodophor. The skin was incised along the abdominal midline (about 2-3 cm), and the muscle layer was separated to expose the abdominal cavity. The intestinal tube was gently pushed to the right side to expose the left kidney and ureter, and the right side was avoided to keep the control. The ureter was lifted with a sterile forceps, and a 4-0 silk thread was double-ligated about 0.5 cm below the renal pelvis to completely block the urine flow. The ureter above the ligation point was observed to be slightly swollen. The muscle layer and skin were sutured layer by layer, and the incision was disinfected again. Two weeks after the operation, the mice in the treatment group were treated with the composition prepared in example 3 at a dose of 1.5 g / kg / d by gavage. Ten weeks after the treatment, the animals were sacrificed, and the test samples were collected for further analysis.

[0123] 3.1 HE staining (hematoxylin-eosin staining)

[0124] 3.1.1 Paraffin-embedded section: the kidney tissue of the mouse was fixed with 4% paraformaldehyde, then embedded with paraffin, and the section thickness was 4 μm;

[0125] 3.1.2 Dewaxing to water: Dewaxing the sections through xylene I (10-15 min), xylene II (10-15 min), absolute ethanol I (2-5 min), absolute ethanol II (2-5 min), 95% ethanol (2-5 min), 80% ethanol (2-5 min), 70% ethanol (2-5 min), and then rinsing with distilled water for 2 min;

[0126] 3.1.3 Hematoxylin staining: immersing in hematoxylin staining solution for 5-10 min, and then rinsing with tap water for 1-2 min to remove the background color;

[0127] 3.1.4 Differentiation: differentiating with 1% hydrochloric acid alcohol for several seconds to tens of seconds, and then observing under a microscope until the cell nucleus is clear and the background is colorless, and then rinsing with tap water for 1 min;

[0128] 3.1.5 Counterstaining: rinsing with running water for 5-15 min, and then observing under a microscope until the cell nucleus changes from red to blue-violet;

[0129] 3.1.6 Eosin staining: staining with eosin staining solution for 30 s-2 min, and then rinsing with distilled water for several seconds;

[0130] 3.1.7 Dehydration and transparency: dehydrating through 80% ethanol (30 s, quickly rinsing with water), 95% ethanol I (1-2 min), 95% ethanol II (1-2 min), absolute ethanol I (2 min), absolute ethanol II (2 min), xylene I (5 min), and xylene II (5 min) in sequence;

[0131] 3.1.8 Mounting: adding an appropriate amount of neutral balsam, and then covering with a cover glass to avoid air bubbles.

[0132] 3.2 Masson's trichrome staining (improved method)

[0133] 3.2.1 Embedding, sectioning, and dewaxing to water are the same as HE staining;

[0134] 3.2.2 Removing residual fixing solution: immersing in Lugol's iodine solution for 5 min, and then rinsing with running water for 5 min; removing iodine with 5% sodium thiosulfate for 5 min, and then rinsing with running water for 5 min;

[0135] 3.3.2 Nucleus staining: Regaud hematoxylin staining for 5-10 min to enhance the contrast of the nucleus;

[0136] 3.2.4 Acidic dye solution staining: staining with ponceau acid fuchsin staining solution for 5-10 min, and then rinsing with distilled water for several seconds;

[0137] 3.2.5 Phosphomolybdic acid differentiation: immersing in 1% phosphomolybdic acid aqueous solution for 5-10 min to remove the red color of muscle fibers, and then not rinsing with water;

[0138] 3.2.6 Collagen fiber staining: Stain with aniline blue staining solution for 5 minutes to make collagen fibers blue, and rinse with distilled water for a few seconds;

[0139] 3.2.7 Distinguish transparent: immerse in 1% aqueous solution of glacial acetic acid for 1 minute to reduce non-specific background staining;

[0140] 3.2.8 Dehydration transparent: sequentially pass through 95% ethanol (1 minute), anhydrous ethanol I (1 minute), anhydrous ethanol II (1 minute), xylene I (5 minutes), xylene II (5 minutes) for dehydration;

[0141] 3.2.9 Mounting: add neutral gum mounting, and press the cover glass to remove bubbles.

[0142] 3.3 Detection of renal fibrosis indicators

[0143] Total RNA was extracted from the collected mouse kidney tissues using TRIzol reagent (Servicebio, Wuhan, China), and its concentration and purity were measured by spectrophotometer.

[0144] The extracted RNA was reversely transcribed into cDNA using Prime Script RT Replication Kit (Vazyme, Nanjing, China). According to the manufacturer's instructions, quantitative PCR analysis was performed using SYBR Green mixture (Vazyme, Nanjing, China).

[0145] According to the detection results of Figures 1-4 , it can be known that HE staining shows that the composition prepared in Example 3 can improve the tubular atrophy and cast formation of the kidney of the renal interstitial fibrosis model mouse (see attached Figure 1 ); Masson staining and quantitative analysis show that the composition prepared in Example 3 can significantly reduce the deposition of collagen fibers in the kidney of the renal interstitial fibrosis model mouse (see attached Figure 2 ); and the PCR results also show that the mRNA levels of fibrosis indicators collagen type I and a-SMA in the kidney of the renal interstitial fibrosis model mouse after using the composition prepared in Example 3 are significantly reduced (see attached Figures 3-4 ).

[0146] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method of preparing a composition for treating chronic kidney disease, characterized by: It comprises the following steps: (1) grind the leeches and the silkworm chrysalis after adding water, to obtain a solid mixture slurry; concentrate the solid mixture slurry and then stand it, to obtain a precipitate, dry the precipitate and reserve it for later use; (2) grind the rhubarb and the astragalus separately, mix the powders, and then extract them with ethanol, filter, concentrate the filtrate to a density of 1.12-1.15, to obtain an extract, dry the residue and reserve it for later use; (3) mix the precipitate in step (1) and the residue in step (2), to obtain a mixture; soak the mixture in hot water for 5-10 hours, then add bacillus subtilis and aspergillus oryzae to ferment it when the water temperature drops below 35℃, to obtain a fermentation product, then distill it, collect the distillate, and dry it, to obtain a medicinal powder; (4) mix the extract in step (2) and the medicinal powder in step (3) with β-cyclodextrin, to obtain the composition. The mass ratio of the bacillus subtilis and the aspergillus oryzae in step (3) is 1:3-5. The amount of water added in step (1) is 10-20 times. The volume concentration of the ethanol in step (2) is 80-90%, the volume-mass ratio of the ethanol to the powder is 10-25 mL:1 g, the extraction is performed 1-2 times, the extraction temperature is 70-80℃, and the extraction time is 8-12 hours. The mass ratio of the β-cyclodextrin to the extract in step (3) is 1:5-10, the volume-mass ratio of the hot water to the mixture is 8-12 mL:1 g, and the inoculation amount of the bacillus subtilis and the aspergillus oryzae is 8-12%.

2. The method of claim 1, wherein: The particle size of the ground powder in step (2) is 1-3 mm.

3. The method of claim 1, wherein: The mass ratio of the bacillus subtilis to the aspergillus oryzae in step (3) is 1:

4.

4. The method of any one of claims 1-3, wherein: It comprises the following steps: (1) grind the leeches and the silkworm chrysalis after adding 15 times of water, to obtain a solid mixture slurry; concentrate the solid mixture slurry and then stand it, to obtain a precipitate, dry the precipitate and reserve it for later use; (2) grind the rhubarb and the astragalus to 1-3 mm, mix the powders, and then extract them with 85% ethanol, the volume-mass ratio of the ethanol to the powder is 20 mL:1 g, the extraction is performed 2 times, the extraction temperature is 80℃, and the extraction time is 10 hours, then filter the extract, concentrate the filtrate to a density of 1.12-1.15, to obtain an extract, dry the residue and reserve it for later use; (3) mix the precipitate in step (1) and the residue in step (2), to obtain a mixture; soak the mixture in 100℃ hot water for 8 hours, the volume-mass ratio of the hot water to the mixture is 10 mL:1 g, then add bacillus subtilis and aspergillus oryzae with a mass ratio of 1:4 to ferment it when the water temperature drops below 35℃, the inoculation amount is 10%, to obtain a fermentation product, then distill it, collect the distillate, and dry it, to obtain a medicinal powder; (4) mix the extract in step (2) and the medicinal powder in step (3) with β-cyclodextrin, pass them through a 10-mesh sieve, and then sieve them, to obtain the product.

5. The method of claim 4, wherein: The composition for treating chronic kidney disease comprises the following components by weight: Leeches 5-10 parts, silkworm chrysalis 5-10 parts, astragalus 40-50 parts, and rhubarb 5-15 parts.

6. Use of a composition produced by the process according to any one of claims 1 to 5 for the manufacture of a product for the treatment of chronic kidney disease, characterized in that: The product dosage form is a tablet, a pill, or a granule.

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