Application of composition in preparation of medicine for preventing and treating diabetic nephropathy

Abstract

The invention relates to the technical field of traditional Chinese medicines, in particular to application of a composition in preparation of a medicine for preventing and treating diabetic nephropathy. Research finds that after alisol A, alisol A-23-acetate and alisol A-24-acetate are compounded according to the ratio of 1: (0.05-0.3): (0.05-0.5), the fasting blood-glucose level can be remarkably improved, the weight of DN animals is increased, the kidney index is reduced, and the content of serum urea nitrogen and creatinine is reduced, and it is proved that the composition has a good treatment effect on diabetic nephropathy.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine compositions, and in particular to application of the compositions in preparing medicines for preventing and treating diabetic nephropathy. Background Art

[0002] Diabetes nephropathy (DN) is relatively common in patients with diabetes mellitus (DM), with an incidence rate of up to 20% to 40%. Some patients with poorly controlled disease may even deteriorate into end-stage renal disease, leading to uremia, renal failure, etc., seriously endangering the patient's life safety.

[0003] For the treatment of DN, modern medicine mainly allows patients to take drugs such as angiotensin converting enzyme inhibitors / angiotensin II receptor inhibitors that have the effect of controlling blood sugar, blood pressure and correcting lipid metabolism disorders, with the control of the progression of the disease as an indicator of improvement, but the treatment effect has certain limitations. With the continuous deepening of multi-angle research of traditional Chinese and Western medicine, experimental research on the treatment of DN with traditional Chinese medicine is constantly developing and deepening. Summary of the invention

[0004] In view of this, the object of the present invention is to provide an application of the composition in the preparation of a medicament for preventing and treating diabetic nephropathy.

[0005] The present invention provides the use of the composition in preparing a medicine for preventing and treating diabetic nephropathy;

[0006] The composition comprises alisol A, alisol A-23-acetate and alisol A-24-acetate; the mass ratio of alisol A, alisol A-23-acetate and alisol A-24-acetate is 1:(0.05-0.3):(0.05-0.5).

[0007] In the application of the present invention, the mass ratio of the alisol A, alisol A-23-acetate and alisol A-24-acetate is 1:0.3:0.5, 1:0.1:0.1 or 1:0.05:0.05.

[0008] In the application of the present invention, the raw materials of the composition, alisamol A, alisamol A-23-acetate and alisamol A-24-acetate can be obtained from the market, or can be extracts or other forms prepared from alisamol medicinal materials by conventional means. In some embodiments, alisamol A, alisamol A-23-acetate and alisamol A-24-acetate in the composition are prepared by the following method:

[0009] (1) Extract the medicinal material of Alisma orientalis using method 1 or method 2

[0010] Method 1: Take 1 kg of dried tubers of Alisma orientalis, make them into medicinal slices, and extract them with ethyl acetate reflux twice, each time for 0.5 h; combine the extracts, recover the solvent under reduced pressure, and obtain a dark brown extract for later use.

[0011] Method 2: Take the dried tubers of Alisma orientalis, crush them into coarse particles, percolate with 70% ethanol, combine the percolated liquid, recover the solvent under reduced pressure, and obtain a dark brown extract for later use.

[0012] (2) Preparation of alisol A, alisol A-23-acetate and alisol A-24-acetate

[0013] The preparation of alismatol A includes method a or method b:

[0014] Method a: The extract obtained in method 2 was loaded onto a silica gel column and eluted with petroleum ether-ethyl acetate (3:1), with 500 ml as one fraction. The fraction was detected by HPLC, and the target fraction was collected to obtain alisol A solid.

[0015] Method b: The extract prepared by method 1 was applied to D101 macroporous adsorption resin, and after eluting with 20% ethanol for 10 column volumes, it was eluted with 70% ethanol for 5 column volumes, the 70% ethanol eluate was collected, and the solvent was recovered under reduced pressure to obtain a yellow oil; the yellow oil was dissolved in 65% methanol, and the target compound was separated by preparative HPLC under the following separation conditions: C18 column, 65% methanol as mobile phase, flow rate of 50ml / min, detection wavelength of 208nm, to obtain alisma alcohol A.

[0016] The preparation of alisol A-24-acetate includes method c or method d:

[0017] Method c: Weigh the extract obtained in method 2, apply it to a silica gel column, elute with petroleum ether-ethyl acetate (5:1), 300 ml as one fraction, detect by HPLC, collect the target fraction, and distill under reduced pressure to obtain alisol A-24-acetate solid.

[0018] Method d: Weigh the extract obtained in method 1, apply it to AB-8 macroporous adsorption resin, elute with 30% ethanol for 10 column volumes, then elute with 80% ethanol for 5 column volumes, collect the 80% ethanol eluate, and recover the solvent under reduced pressure to obtain a yellow oily substance; dissolve the yellow oily substance in 68% methanol, and separate the target compound by preparative HPLC. The separation conditions are: C18 column, mobile phase is 68% methanol, flow rate is 50ml / min, and detection wavelength is 208nm. Alismatol A24-acetate is obtained.

[0019] Preparation of alismatol A-23-acetate:

[0020] Method e: Weigh the extract obtained in method 1, add sulfuric acid, and reflux in a water bath for 15 minutes. Adjust the pH to 5-6 with 20% sodium hydroxide, add deionized water to adjust the ethanol concentration to 40%, pass through a DM130 macroporous adsorption resin column (diameter-to-height ratio 1:5) at a flow rate of 0.5 BV / h, then elute impurities with 40% ethanol, and then elute the target component with 55% ethanol. Determine the content of the main component (alismulol A23-acetate) in the eluate by HPLC, determine the elution endpoint, combine the eluates, concentrate under reduced pressure, and dry. The macroporous adsorption resin purified product was dissolved twice in 10 times the amount of a mixed solvent of petroleum ether-ethyl acetate (5:2), the residue was dissolved in ethanol and then adsorbed on silica gel, the solvent was evaporated, and the sample was loaded by dry method. The petroleum ether-ethyl acetate mixed solution containing the sample was subjected to silica gel column chromatography (300-400 mesh), and impurities were continuously eluted with petroleum ether-ethyl acetate (5:2), and then the target component was eluted with petroleum ether-ethyl acetate (2:3), and the end point was detected by HPLC, the eluate was collected, concentrated under reduced pressure to near dryness, and dried under reduced pressure at 70°C for 72 hours to obtain a 23-acetate solid of alismatol.

[0021] In the application of the present invention, the prevention and treatment includes at least one of the following:

[0022] Reduce fasting blood sugar levels, reduce urine protein levels, increase body weight, reduce renal index, and reduce urea nitrogen and creatinine levels.

[0023] In some embodiments, the urea nitrogen is serum urea, and the creatinine is blood creatinine.

[0024] In the above application of the present invention, the effective dose of the composition is 1.428 g of crude drug / d.

[0025] The present invention also provides a drug for preventing and treating diabetic nephropathy, comprising alismatol A, alismatol A-23-acetate, alismatol A-24-acetate and pharmaceutically acceptable excipients;

[0026] The mass ratio of alisol A, alisol A-23-acetate and alisol A-24-acetate is 1:0.05-0.3:0.05-0.5.

[0027] In the present invention, the pharmaceutically acceptable excipients include one or more of fillers, disintegrants, lubricants, suspending agents, adhesives, sweeteners, flavoring agents, preservatives, and bases, including but not limited to these. Suitable excipients can be selected as needed, and conventional dosage forms acceptable to pharmacy can be prepared according to conventional production methods in the pharmaceutical field. The dosage forms include oral dosage forms, injection dosage forms, or external dosage forms, specifically including but not limited to decoctions, granules, capsules, tablets, oral liquids, pills, tinctures, syrups, suppositories, gels, sprays, injections, and the like.

[0028] The present invention provides an application of a composition in the preparation of a medicine for preventing and treating diabetic nephropathy. The present invention has found that after alisol A, alisol A-23-acetate and alisol A-24-acetate are compounded at a ratio of 1:0.05-0.3:0.05-0.5, fasting blood glucose levels can be significantly improved, the body weight of DN animals can be increased, the renal index can be reduced, and the serum urea nitrogen and creatinine content can be reduced, which confirms that the composition has a good therapeutic effect on diabetic nephropathy. DETAILED DESCRIPTION

[0029] The present invention provides an application of a Chinese medicine composition in the preparation of a drug for preventing and treating diabetic nephropathy. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0030] As mentioned above, the present invention aims to provide an application of a Chinese medicine composition in preparing a medicine for preventing and treating diabetic nephropathy.

[0031] The following experiments, if no specific conditions are specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The APIs or excipients, reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially. Unless otherwise specified, all percentages, ratios, proportions or parts are by weight.

[0032] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention.

[0033] The present invention will be further described below in conjunction with embodiments:

[0034] Example 1 Extraction of medicinal materials

[0035] Method 1: Take 1 kg of dried tubers of Aliams orientalis (Sam.) Juzep. of the Alismataceae family, prepare them into medicinal slices, and extract them twice with 7000 mL of ethyl acetate under reflux, each time for 0.5 h. Combine the extracts, recover the solvent under reduced pressure, and obtain 35 g of dark brown extract.

[0036] Method 2: Take 3 kg of dried tubers of Aliams orientalis (Sam.) Juzep. of the Alismataceae family, grind them into coarse particles, percolate with 70% ethanol, combine the percolate, recover the solvent under reduced pressure, and obtain 115 g of dark brown extract.

[0037] Example 2 Preparation of alismatol A

[0038] Method a: Weigh 50 g of the extract of method 2 of Example 1, put it on a silica gel column (450 g), and elute it with petroleum ether-ethyl acetate (3:1), with 500 ml as one fraction. Detect by HPLC, collect the target fraction, and distill under reduced pressure to obtain 0.4 g of solid. The purity was determined by the area normalization method and was greater than 50% (weight).

[0039] Method b: Weigh 10g of the extract obtained in method 1 of Example 1, apply it to D101 macroporous adsorption resin, elute with 20% ethanol for 10 column volumes, then elute with 70% ethanol for 5 column volumes, collect the 70% ethanol eluate, and recover the solvent under reduced pressure to obtain 2g of yellow oily substance; dissolve 2g of the yellow oily substance in 65% methanol, and separate the target compound by preparative HPLC. Separation conditions are: C18 column, mobile phase is 65% methanol, flow rate is 50ml / min, and detection wavelength is 208nm. 100mg of alismatol A with a purity of 95% (weight) is obtained.

[0040] Example 3 Preparation of alismatol A24-acetate

[0041] Method c: Weigh 50 g of the extract prepared by method 2 of Example 1, apply it to a silica gel column (450 g), and elute with petroleum ether-ethyl acetate (5:1), with 300 ml as one fraction. Detect by HPLC, collect the target fraction, and distill under reduced pressure to obtain 0.4 g of alismatol A24-acetate solid, which was determined by the area normalization method and had a purity of more than 55% (by weight).

[0042] Method d: Weigh 20g of the extract prepared by method 1 of Example 1, apply it to AB-8 macroporous adsorption resin, elute with 30% ethanol for 10 column volumes, then elute with 80% ethanol for 5 column volumes, collect the 80% ethanol eluate, and recover the solvent under reduced pressure to obtain 1.5g of yellow oil; dissolve 1.5g of yellow oil in 68% methanol, and separate the target compound by preparative HPLC. Separation conditions are: C18 column, mobile phase is 68% methanol, flow rate is 50ml / min, and detection wavelength is 208nm. 60mg of alismatol A24-acetate with a purity of (95% by weight) is obtained.

[0043] Example 4 Preparation of alismatol A23-acetate

[0044] Method e: Weigh 10 g of the extract obtained by method 1 of Example 1, add 4 mL of sulfuric acid, and reflux in a water bath for 15 min. Adjust the pH to 5-6 with 20% sodium hydroxide, add deionized water to adjust the ethanol concentration to 40%, pass through a DM130 macroporous adsorption resin column (diameter-to-height ratio 1:5) at a flow rate of 0.5 BV / h, then elute impurities with 40% ethanol, and then elute the target component with 55% ethanol, determine the content of the main component (alismulol A23-acetate) in the eluate by HPLC, determine the elution endpoint, combine the eluates, concentrate under reduced pressure, and dry. The purified product of macroporous adsorption resin was dissolved twice with 10 times the amount of petroleum ether-ethyl acetate (5:2) mixed solvent, the residue was dissolved in ethanol and then adsorbed on silica gel, the solvent was evaporated, the sample was loaded by dry method, and the petroleum ether-ethyl acetate mixed solution containing the sample was subjected to silica gel column chromatography (300-400 mesh), and impurities were continuously eluted with petroleum ether-ethyl acetate (5:2), and then the target component was eluted with petroleum ether-ethyl acetate (2:3), the end point was detected by HPLC, the eluate was collected, concentrated under reduced pressure to near dryness, and dried under reduced pressure at 70°C for 72 hours to obtain alisol A23-acetate solid.

[0045] Example 5 Preparation of the composition of the present invention

[0046] Alisma alcohol A prepared by method b of Example 2, Alisma alcohol A23-acetate prepared by method b of Example 4 and Alisma alcohol A24-acetate prepared by method d of Example 3 were mixed in mass ratios of 1:0.3:0.5, 1:0.1:0.1 and 1:0.05:0.05, respectively, to obtain composition group A (1:0.05:0.05, weight ratio), composition group B (1:0.1:0.1, weight ratio) and composition group C (1:0.3:0.5, weight ratio), respectively.

[0047] Preparation of Comparative Examples 1 to 3 Compositions

[0048] Alisma alcohol A prepared by method b of Example 2, Alisma alcohol A23-acetate prepared by method b of Example 4 and Alisma alcohol A24-acetate prepared by method d of Example 3 were mixed in a mass ratio of 1:0.5:1 and 1:0.01:1, respectively, to obtain composition group D (1:0.01:0.01, weight ratio), composition group E (1:0.01:1, weight ratio) and composition group F (1:0.5:1, weight ratio), respectively.

[0049] Experimental Example 1 Therapeutic effect of Chinese medicine composition on streptozotocin-induced diabetic nephropathy (DN) model in rats

[0050] 1. Experimental Materials

[0051] Animals: 81 male SD rats, SPF grade, weighing 200-220 g, purchased from Sibeifu (Suzhou) Biotechnology Co., Ltd.

[0052] The experimental mouse maintenance feed was purchased from Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd. and complies with GB14924.1 "General Quality Standard for Laboratory Animal Formula Feed", production license number: Su Feed Certificate (2019) 01003.

[0053] Drug: Metformin, purchased from Beijing Jingfeng Pharmaceutical Group Co., Ltd., batch number: 191113, specification: 250 mg / tablet.

[0054] Test drug: Chinese medicine composition capsule prepared in Preparation Example, provided by Jiangsu Kangyuan Pharmaceutical Co., Ltd.

[0055] Reagents: Streptozotocin (STZ) was purchased from Sigma, USA, batch number: WXBD1402V; urine protein was purchased from Nanjing Jiancheng Biotechnology Research Institute, item number: C035-2-1; urea nitrogen (BUN) was purchased from Nanjing Jiancheng Biotechnology Research Institute, item number: C013-2-1; creatinine (Cr) kit was purchased from Nanjing Jiancheng Biotechnology Research Institute, item number: C011-2-1.

[0056] Instruments: Blood glucose meter and supporting test strips, Johnson & Johnson (China) Medical Devices Co., Ltd., model: OneTouchUltraEasy; animal weighing scale, Shanghai Xiangchuan Electronic Scale Co., Ltd., model: ACX-SC-DA; electronic balance, Shanghai Zhuojing Electronics Co., Ltd., model: BSM220.4; high-speed refrigerated centrifuge, Eppendorf, Germany, model: Centrifuge5810R; multi-function microplate reader, MD Company, USA, model: Flexstation3.

[0057] (II) Experimental methods

[0058] After 1 week of adaptive feeding, SD rats were fasted for 12 hours and randomly divided into a normal group of 9 and a model group of 72. A streptozotocin solution (STZ dissolved in sodium citrate buffer, pH adjusted to 4) was prepared. The model group rats were intraperitoneally injected with 55 mg / kg STZ to induce a diabetes model within 10 minutes, and the normal group was given an equal amount of sodium citrate solution. After 72 hours, the fasting blood glucose of the rats was detected by blood sampling from the tail vein. Blood glucose ≥16.7mmol / L was considered to have successfully established a diabetes model. After 4 weeks of modeling of diabetic rats, 24h urine was collected and urine protein was measured. If >30mg / 24h, diabetic nephropathy (DN) was considered to have been successfully modeled.

[0059] 64 rats with successful DN modeling were randomly divided into 8 groups, including model group, metformin group, Chinese medicine composition group A (1:0.05:0.05, weight ratio), group B (1:0.1:0.1, weight ratio), group C (1:0.3:0.5, weight ratio), group D (1:0.01:0.01, weight ratio), group E (1:0.01:1, weight ratio), and group F (1:0.5:1, weight ratio). Except for the 9 animals in the normal group, there were 8 model animals in each group. The rats in the Chinese medicine composition A, B, C, D, E, and F groups were given 0.15g crude drug / kg Chinese medicine composition by gavage every morning; the rats in the metformin group were given 135mg / kg metformin by gavage; the normal group and DN model group were given the same amount of pure water by gavage. 1 time / d, for 4 consecutive weeks.

[0060] After the drug intervention was completed, the rats in each group were fasted for 12 hours and fasting blood glucose was tested. The rats' 24-hour urine was collected, and the 24-hour urine protein content was tested according to the instructions of the urine protein kit. The rats were weighed, anesthetized, and blood was collected from the abdominal aorta and centrifuged. The serum BUN and creatinine (Cr) index contents were tested according to the instructions of the BUN and Cr kits. After the blood was collected from the rats, the rats were killed, and the kidney tissues of both rats were weighed to calculate the renal index.

[0061] Detection indicators: 1. Fasting blood glucose (FPG), urine protein. 2. Serum urea nitrogen (BUN), creatinine (Cr). 3. Model animal body weight, kidney index.

[0062] (III) Experimental results

[0063] The results showed that compared with the normal group, the fasting blood glucose of the diabetic model group rats increased significantly 72 hours after modeling, and was maintained above 16.7mmol / L during the experiment. After 6 and 8 weeks of administration, compared with the diabetic nephropathy (DN) model group, the fasting blood glucose of the rats in the metformin group and the Chinese medicine composition A, B, C and F groups decreased significantly (P<0.05, P<0.01, P<0.001); there was no significant difference between the D group and the E group after 8 weeks of administration and the model group (P>0.051), indicating that the hypoglycemic effect of the Chinese medicine composition D and E groups was significantly inferior to that of the other groups. See Table 1.

[0064] Compared with the normal group, the urine protein of rats in the STZ modeling group at 4 weeks was significantly increased and greater than 30 mg / 24 h, indicating that the DN modeling was successful. After 8 weeks of administration, the urine protein of rats in each administration group decreased compared with the DN model group, among which the urine protein of rats in the metformin group and the Chinese medicine composition A, B, and C groups decreased significantly (P<0.05, P<0.01, P<0.001). See Table 2.

[0065] Compared with the normal group, the serum BUN and Cr levels of the rats in the DN model group were significantly increased; compared with the DN model group, the serum BUN and Cr levels of the rats in the metformin group and the Chinese medicine composition group A, group B and group C were significantly reduced (P<0.001). See Table 3.

[0066] Compared with the normal group, the rats in each model group lost weight before administration, and the difference was significant. After administration, compared with the DN model group, the rats in the metformin group and the Chinese medicine combination groups A, B, and C had significantly increased weight (P<0.001), and the renal index was significantly lower than that in the DN model group (P<0.001). The weight increase and renal index decrease of the rats in groups D to E were also significantly different from those in the DN model group (P<0.05, P<0.01,), but the difference was not as significant as that in groups A to C. See Table 4.

[0067] Table 1 Comparison of fasting blood glucose (FPG) of rats in different treatment groups (mmol / L)

[0068] Group 2w 4w 6w 8w Normal group 5.54±0.62 5.27±0.58 5.34±0.60 5.27±0.61 Model Group <![CDATA[28.21±4.53 ### ]]> <![CDATA[26.99±4.47 ### ]]> <![CDATA[28.76±2.84 ### ]]> <![CDATA[27.61±3.90 ### ]]> Metformin group 27.83±3.95 26.51±5.20 <![CDATA[17.24±2.68 *** ]]> <![CDATA[13.45±2.87 *** ]]> Group A 28.45±4.33 27.04±5.71 <![CDATA[25.25±2.96 * ]]> <![CDATA[20.93±2.83 ** ]]> Group B 28.36±3.45 27.38±5.28 <![CDATA[20.96±4.60 ** ]]> <![CDATA[17.67±2.82 *** ]]> Group C 29.66±4.05 27.31±4.78 <![CDATA[17.57±3.15 *** ]]> <![CDATA[13.65±3.30 *** ]]> Group D 29.47±4.56 27.95±8.03 27.42±3.13 25.52±2.73 Group E 28.87±4.69 27.35±6.11 <![CDATA[25.19±2.76 * ]]> 24.44±2.25 Group F 27.06±6.05 26.89±6.26 <![CDATA[24.77±3.11 * ]]> <![CDATA[23.34±2.03 * ]]>

[0069] Note: Compared with the normal group, ### P<0.001, compared with the model group, * P<0.05, ** P<0.01, *** P<0.001.

[0070] Table 2 Comparison of urine protein in rats in different treatment groups (X±S, n=8,9)

[0071]

[0072]

[0073] Note: Compared with the normal group, ### P<0.001, compared with the model group, * P<0.05, *** P<0.001.

[0074] Table 3 Comparison of serum urea nitrogen and creatinine in rats in different treatment groups (X±S, n=8,9)

[0075]

[0076] Note: Compared with the normal group, ### P<0.001, compared with the model group, * P<0.05, ** P<0.01, *** P<0.001.

[0077] Table 4 Comparison of body weight and renal index of rats in different treatment groups (X±S, n=8,9)

[0078]

[0079]

[0080] Note: Compared with the normal group, ### P<0.001, compared with the model group, * P<0.05, ** P<0.01, *** P<0.001. Experimental Example 2 Therapeutic effect of the composition on the diabetic nephropathy model of mice induced by streptozotocin (STZ)

[0081] 1. Experimental Materials

[0082] Animals: C57BL / 6J mice, SPF grade, male, 100 mice, weighing 18-20 g, purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.

[0083] The experimental mouse maintenance feed and high-sugar and high-fat feed were purchased from Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd., which complies with GB14924.1 "General Quality Standard for Laboratory Animal Formula Feed", and the production license number is Su Feed Certificate (2019) 01003.

[0084] Drug: Metformin, purchased from Beijing Jingfeng Pharmaceutical Group Co., Ltd., batch number: 191113, specification: 250 mg / tablet.

[0085] Test drug: Chinese medicine composition capsule prepared in Preparation Example, provided by Jiangsu Kangyuan Pharmaceutical Co., Ltd.

[0086] Reagents: Streptozotocin (STZ) was purchased from Sigma, USA, batch number: WXBD1402V; urine protein was purchased from Nanjing Jiancheng Biotechnology Research Institute, item number: C035-2-1; urea nitrogen (BUN) was purchased from Nanjing Jiancheng Biotechnology Research Institute, item number: C013-2-1; creatinine (Cr) kit was purchased from Nanjing Jiancheng Biotechnology Research Institute, item number: C011-2-1.

[0087] Instruments: Blood glucose meter and supporting test strips, Johnson & Johnson (China) Medical Devices Co., Ltd., model: OneTouchUltraEasy; animal weighing scale, Shanghai Xiangchuan Electronic Scale Co., Ltd., model: ACX-SC-DA; electronic balance, Shanghai Zhuojing Electronics Co., Ltd., model: BSM220.4; high-speed refrigerated centrifuge, Eppendorf, Germany, model: Centrifuge5810R; multi-function microplate reader, MD Company, USA, model: Flexstation3.

[0088] (II) Experimental methods

[0089] 100 healthy C57BL / 6J mice, SPF grade, male, weighing 18-20g, were randomly selected as the normal group after adaptive feeding for 1 week, and the remaining 90 were included in the modeling group. After the modeling group mice were fed with a high-sugar and high-fat diet for 4 weeks, they were fasted for 12 hours and intraperitoneally injected with 50mg / kg STZ solution (once a day for 4 consecutive days). The mice in the normal group were fed with ordinary feed and intraperitoneally injected with an equal volume of sodium citrate buffer. After 7 days of STZ injection, they were fasted for 12 hours and fasting blood glucose (FPG) was measured. FPG>11.1mmol / L was considered to be a successful modeling of the diabetes model. The mice with successful modeling continued to be fed with high-sugar and high-fat for 1 week, and the 24h urine of the modeling mice was collected to detect the urine protein content. The diabetic nephropathy (DN) model was successfully prepared when the urine protein was>30mg / 24h.

[0090] A total of 80 mice with successful modeling were selected and randomly divided into model group, metformin group, Chinese medicine composition A, B, C, D, E, F groups, with 10 mice in each group. The mice in Chinese medicine composition A, B, C, D, E, and F groups were given 0.217g crude drug / kg Chinese medicine composition by gavage every morning; the mice in metformin group were given 195mg / kg metformin by gavage; the normal group and DN model group were given the same amount of pure water by gavage. 1 time / d, for 8 consecutive weeks. All groups except the normal group were fed with high-fat diet.

[0091] After the administration, fasting but not water restriction was performed, and urine of each group of mice was collected for 24 hours. After the urine was collected, the mice were weighed, anesthetized, and blood was collected by removing the eyeballs. The serum was separated by centrifugation for urea nitrogen and creatinine detection. Both kidneys were quickly removed, weighed, and the kidney index was calculated. Detection indicators: 1. Fasting blood glucose (FPG), urine protein. 2. Serum urea nitrogen (BUN), creatinine (Cr). 3. Model animal weight and kidney index.

[0092] (III) Experimental results

[0093] The results showed that compared with the normal group, the fasting blood glucose of mice in the diabetic model group increased significantly after high-sugar and high-fat diet combined with STZ injection modeling, and was maintained above 11.1mmol / L during the experiment. After 4 weeks of administration, the fasting blood glucose of mice in the metformin group and the Chinese medicine composition A, B, C, D, E, and F groups was significantly decreased compared with the DN model group. After 8 weeks of administration, the fasting blood glucose of mice in the metformin group and the Chinese medicine composition A, B, C, D, E, and F groups was significantly decreased compared with the DN model group. See Table 5.

[0094] Compared with the normal group, the urine protein of mice in the high-sugar and high-fat diet combined with STZ injection modeling group increased significantly and was greater than 30 mg / 24h, indicating that the DN modeling was successful. After 8 weeks of administration, the urine protein of mice in the metformin group and the Chinese medicine composition A, B, C, D, E, and F groups was significantly lower than that in the DN model group. Among them, the urine protein of the metformin group and the Chinese medicine composition C group basically returned to normal levels. See Table 6.

[0095] Compared with the normal group, after 8 weeks of administration, the serum BUN and Cr levels of mice in the DN model group were significantly increased (P<0.001); compared with the DN model group, the serum BUN levels of mice in the metformin group and the Chinese medicine composition A, B, C, and F groups were significantly reduced (P<0.01, P<0.001), and the serum Cr levels of mice in the metformin group and the Chinese medicine composition A, B, C, E, and F groups were also significantly reduced (P<0.05, P<0.01,). See Table 7.

[0096] Compared with the normal group, the mice in each model group lost weight, and the weight loss was significant (P<0.001). After administration, except for the model group, the weight of mice in the metformin group and the Chinese medicine composition B, C, and F groups increased significantly compared with the DN model group (P<0.05, P<0.001). In addition, the renal index of mice in the metformin group and the Chinese medicine composition A, B, C, and F groups was also significantly lower than that in the DN model group (P<0.05, P<0.01, P<0.001). See Table 8.

[0097] Table 5 Effects on fasting blood glucose in diabetic nephropathy mice (X±S, n=10)

[0098]

[0099] Note: Compared with the normal group, ### P<0.001, compared with the model group, *** P<0.001.

[0100] Table 6 Effect on 24h urine protein in diabetic nephropathy mice (X±S, n=10)

[0101]

[0102]

[0103] Note: Compared with the normal group, ### P<0.001, compared with the model group, *** P<0.001.

[0104] Table 7 Effects on urea nitrogen and creatinine in diabetic nephropathy mice (X±S, n=10)

[0105]

[0106] Note: Compared with the normal group, ### P<0.001, compared with the model group, * P<0.05, ** P<0.01, *** P<0.001.

[0107] Table 8 Effects on body weight and renal index of diabetic nephropathy mice (X±S, n=10)

[0108]

[0109] Note: Compared with the normal group, ### P<0.001, compared with the model group, * P<0.05, ** P<0.01, *** P<0.001.

[0110] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of the composition in the preparation of drugs for preventing and treating diabetic nephropathy; The composition comprises alisol A, alisol A-23-acetate and alisol A-24-acetate; the mass ratio of alisol A, alisol A-23-acetate and alisol A-24-acetate is 1:(0.05-0.3):(0.05-0.5).

2. The use according to claim 1, characterized in that: The mass ratio of the alisol A, alisol A-23-acetate and alisol A-24-acetate is 1:0.3:0.5, 1:0.1:0.1 or 1:0.05:0.

05.

3. The use according to claim 1, characterized in that: The control comprises at least one of the following: Reduce fasting blood sugar levels, reduce urine protein levels, increase body weight, reduce renal index, and reduce urea nitrogen and creatinine levels.

4. The use according to claim 3, characterized in that: The urea nitrogen is the urea in serum, and the creatinine is the creatinine in blood.

5. The use according to any one of claims 1 to 4, characterized in that: The effective dosage of the composition is 1.428 g of crude drug per day.

6. A drug for preventing and treating diabetic nephropathy, characterized in that: It includes alisol A, alisol A-23-acetate, alisol A-24-acetate and pharmaceutically acceptable excipients; The mass ratio of alisol A, alisol A-23-acetate and alisol A-24-acetate is 1:0.05-0.3:0.05-0.

5.

7. The drug according to claim 6, characterized in that The pharmaceutically acceptable agent includes at least one of a filler, a disintegrant, a lubricant, a suspending agent, a binder, a sweetener, a flavoring agent, a preservative or a matrix.

8. The drug according to claim 7, characterized in that The dosage form of the drug includes decoction, granules, capsules, tablets, oral liquid, pills, tincture, syrup, suppository, gel, spray or injection.

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