A composition and its applications

Abstract

The present invention relates to new uses of natural medicines, and particularly to a composition and its application, that is, the application of a pharmaceutical composition in the preparation of a drug for improving or preventing renal fibrosis in diabetic nephropathy, characterized in that the pharmaceutical composition comprises asiatic acid and astragalin. The active components of Cyclocarya paliurus in the present invention, asiatic acid and astragalin, can synergistically balance the TGF-β1 / BMP7 pathway through combined application, block the accumulation of extracellular matrix in renal tubules, and inhibit the process of renal fibrosis in diabetic nephropathy, and the effect is superior to that of the positive drug dapagliflozin group.

Description

Technical Field

[0001] The present invention relates to natural medicines, and in particular to a composition and its application, that is, the use of active ingredients asiatic acid and astragalin of Cyclocarya paliurus in improving renal fibrosis of diabetic nephropathy by balancing TGF-β1 / BMP7. Background Art

[0002] Diabetic nephropathy (DN) is one of the most common microvascular complications of diabetes. The latest epidemiological results show that currently 537 million adults aged 20-79 have diabetes. This accounts for 10.5% of the global population in this age group. It is estimated that by 2030, the total number will rise to 643 million (11.3%). The incidence of diabetic nephropathy among global diabetes patients is about 30-40%. At present, due to progressive renal fibrosis, DN patients have a great probability of developing into end-stage renal disease (ESRD) clinically. Once developed into ESRD, patients can only use long-term dialysis or kidney transplantation for treatment, which causes a heavy burden on their living standards and social and economic pressures.

[0003] The progression of DN occurs in several stages. The early changes in the kidneys of diabetic patients include obvious glomerular ultrafiltration and hypertrophy, followed by damage to the glomerular filtration barrier, increased urinary albumin excretion, mesangial matrix accumulation, hypertrophy, nodular glomerulosclerosis, and tubulointerstitial fibrosis, which are the keys to the progression of ESRD and renal failure. Renal fibrosis plays a key role in the pathogenesis of DN. Currently, the drugs for treating diabetic nephropathy clinically are generally angiotensin enzyme inhibitor antihypertensive drugs such as captopril, or hypoglycemic drugs such as metformin. These drugs cannot effectively improve or block the development of renal fibrosis, and have obvious limitations in the treatment of diabetic nephropathy. Therefore, developing drugs with significant effects on renal fibrosis is a feasible treatment strategy for improving diabetic nephropathy.

[0004]

[0005] Asiatic acid (AA) is a pentacyclic triterpenoid compound with an oleanane-type skeleton isolated from the plant Centella asiatica of the Umbelliferae family. Since it was discovered in 1971 that asiatic acid can treat skin wounds, a large number of studies have been carried out subsequently, and it has been found that asiatic acid also has various pharmacological activities such as antibacterial, anti-tumor, anti-inflammatory, liver-protecting, improving neurocognitive impairment, and hypoglycemic. In particular, its anti-tumor activity has been extensively studied. Currently, it has been found that asiatic acid can effectively inhibit the proliferation of a variety of cancer cells including liver cancer, breast cancer, human tongue squamous cell carcinoma, ovarian cancer, and melanoma.

[0006] Astragalin is a natural flavonoid compound widely present in medicinal plants, with anti-inflammatory, antioxidant, cardiotonic, analgesic, antibacterial, anti-allergic, and anti-hepatotoxic effects, and can enhance the body's resistance, stimulate the production of interferon, anti-arrhythmia, dilate blood vessels, protect the myocardium, etc.

[0007] Therefore, there is an urgent need for more effective treatment strategies to prevent the occurrence and development of DN. Different from the single target of synthetic small molecule drugs, traditional Chinese medicines and natural products have the advantages of multi-target and low toxicity, and have a multi-path synergistic effect on the complex lesions of metabolic diseases, and may be a better choice to relieve the process of DN. Summary of the Invention

[0008] In order to solve the problem of ineffective treatment of renal fibrosis in diabetic nephropathy patients in current clinical practice, the present invention provides an application of the active ingredients of Cyclocarya paliurus in combination with asiatic acid (AA) and astragalin (AG) for the preparation of a drug for improving or preventing renal fibrosis in diabetic nephropathy.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention discloses a new use of Cyclocarya paliurus. The present invention first discovers that the combined use of the active ingredients of Cyclocarya paliurus with asiatic acid and astragalin has a synergistic effect on improving renal fibrosis in diabetic nephropathy. There is no literature report on the effect of astragalin on renal fibrosis in nephropathy.

[0010] An application of a pharmaceutical composition in the preparation of a drug for improving or preventing renal fibrosis in diabetic nephropathy, characterized in that the pharmaceutical composition comprises asiatic acid and astragalin.

[0011] The application as described above, characterized in that the pharmaceutical composition is composed of asiatic acid, astragalin and pharmaceutically acceptable excipients.

[0012] An ethanol extract of Cyclocarya paliurus, characterized in that it is prepared by the following method:

[0013] Take 49.59 kg of dried Cyclocarya paliurus leaves, pulverize them, add 80% ethanol according to the material-liquid ratio of 1:6, extract 3 times by the impregnation method for 7 days each time, combine the extracts and concentrate them under reduced pressure until the alcohol smell disappears to obtain the ethanol extract of Cyclocarya paliurus.

[0014] The ethanol extract of Cyclocarya paliurus as described above, characterized in that its active ingredients are asiatic acid and astragalin.

[0015] The ethanol extract of Cyclocarya paliurus as described above, characterized in that the mass ratio of asiatic acid to astragalin is 1:1.

[0016] The application of the ethanol extract of Cyclocarya paliurus in the preparation of a drug for improving or preventing renal fibrosis in diabetic nephropathy.

[0017] Beneficial effects:

[0018] 1. The present invention verifies that the ethanol extract of Cyclocarya paliurus (the active ingredients thereof are asiatic acid and astragalin) and the mixture of asiatic acid and astragalin monomer compounds can synergistically balance the TGF–β / BMP7 pathway through combined application, block the accumulation of extracellular matrix in renal tubular cells, and inhibit the process of renal fibrosis in diabetic nephropathy, and the effect is better than that of the positive drug dapagliflozin group.

[0019] 2. The application of the active ingredients of Cyclocarya paliurus in the combined use of asiatic acid and astragalin for the preparation of a drug for improving or preventing renal fibrosis in diabetic nephropathy.

[0020] 3. As a new food resource in China, Cyclocarya paliurus has a long application history and high safety. Description of the drawings

[0021] Figure 1 : HPLC chromatograms of the total ethanol extract of Cyclocarya paliurus (CPE), AA, and AG.

[0022] Figure 2 : Graphs of the body weight (A) and blood glucose changes (B) of diabetic nephropathy mice intervened by AA and AG.

[0023] Figure 3 : A is the graph of the urinary albumin change of diabetic nephropathy mice intervened by AA and AG; B is the graph of the blood urea nitrogen change; C is the graph of the creatinine change.

[0024] Figure 4 : Immunohistochemical results of the morphological changes of the renal tissues of diabetic mice by AA and AG.

[0025] Figure 5 : Schematic diagrams of Masson staining of collagen deposition and immunohistochemistry of fibronectin in the kidneys of diabetic mice by AA and AG.

[0026] Figure 6 : Graph of the change in the survival ratio of renal tubular epithelial cells induced by advanced glycation end products (AGEs) by AA and AG in Example 2, where A is AA, B is AG, and C is AA:AG = 1:1.

[0027] Figure 7 : Schematic diagram of the ELISA results of fibronectin in renal tubular epithelial cells induced by advanced glycation end products (AGEs) by AA and AG in Example 2, where A is AA, B is AG, C is AA:AG = 1:1, D is Fa, E is Log(Fa / Fu), F is CI, and G is Log(CI).

[0028] Figure 8: Western-blot figure of the cell protein blotting method in Example 2; A is the protein blotting figure; B is the protein gray scale integral figure.

[0029] Figure 9 : Western-blotting figure of the mouse kidney tissue protein blotting method in Example 3. A is the extracellular matrix protein blotting figure; B is the extracellular matrix protein gray scale integral figure; C is the TGF-β1 / BMP7 / Smads pathway protein blotting figure; D-F are the TGF-β1 / BMP7 / Smads pathway protein gray scale integral figures, specifically, D is the BMP7 and TGF-β1 gray scale integral figure, E is the Smad1, Smad3 and Smad4 gray scale integral figure, and F is the TIMP1 and TIMP2 gray scale integral figure.

[0030] Figure 10 : Co-inmunoprecipitationWestern-blotting figure of the mouse kidney tissue in Example 3. A is the immunoprecipitation protein blotting figure; B is the immunoprecipitation positive control protein blotting figure; C is the Smad4-binding protein gray scale integral figure, D is the Smad4-binding Smad3 protein gray scale integral figure, and E is the Smad4-binding Smad1 protein gray scale integral figure. Detailed implementation manners

[0031] The following specifically introduces the substantial content of the present invention in combination with the drawings and embodiments. However, the present invention is not limited to the following embodiments. The experimental methods in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used in the following embodiments can be obtained from public commercial channels unless otherwise specified.

[0032] Asiatic acid and astragalin can synergistically improve renal fibrosis and reduce the accumulation of extracellular matrix in diabetic nephropathy.

[0033] To find the mechanism by which asiatic acid and astragalin can improve renal fibrosis in diabetic nephropathy, in vitro cell and in vivo animal experiments were carried out, and the expression of proteins in the key fibrosis pathway TGF-β1 / BMP7 pathway was detected. Western Blotting and immunoprecipitation experiments were used to verify that asiatic acid and astragalin improve renal fibrosis in diabetic nephropathy by synergistically balancing the TGF-β1 / BMP7 pathway.

[0034] In a preferred embodiment of the present invention, the experimental animals are SPF-grade C57BL / 6J mice, and the kidney cells are renal tubular epithelial cells HK-2. The experimental results of the specific implementation method show that at the animal level, asiatic acid and astragalin can synergistically improve the levels of urinary albumin, creatinine, and blood urea nitrogen in streptozotocin (STZ)-stimulated C57BL / 6J mice, restore the imbalance of the TGF-β1 / BMP7 pathway, and thus reduce the accumulation of extracellular matrix and improve renal fibrosis in diabetic nephropathy. At the cellular level, asiatic acid and astragalin can synergistically improve the accumulation of extracellular matrix in AGEs-induced renal tubular cells, achieving the purpose of improving renal fibrosis in diabetic nephropathy.

[0035] Example 1 Asiatic acid and astragalin can synergistically improve renal function injury in diabetic nephropathy mice

[0036] Seventy-five male C57BL / 6J mice with an SPF grade and a body weight of 21 ± 3.2 g were taken. After 1 week of adaptive feeding, they were fasted for 12 h. STZ was dissolved in 0.1 mmol·L -1 citrate buffer (pH = 4.2), and 60 randomly selected mice were intraperitoneally injected with a dose of 55 mg·kg -1 ·d -1 for 5 consecutive days at a volume of 10 mL·kg -1 ·d -1 The remaining 15 mice were injected with citrate buffer at the same dose. After 1 week, the blood glucose level was measured, and the blood glucose level was greater than 16.7 mmol·L -1 . Then, the 60 mice were randomly divided into 5 groups, with 15 mice in each group, namely the model group (STZ group), asiatic acid group (AA group), astragalin group (AG group), combined application group of asiatic acid and astragalin (AA + AG group), Cyclocarya paliurus ethanol extract group (CPE group), and positive drug dapagliflozin group (DAPA group). Each group was intragastrically administered CMC-Na solution, 40 mg·kg -1 ·d -1 asiatic acid, 40 mg·kg -1 ·d -1 astragalin, 40 mg·kg -1 ·d -1 a mixture of asiatic acid and 40 mg·kg -1 ·d -1 astragalin, 240 mg·kg -1 ·d -1 Cyclocarya paliurus ethanol extract, 5 mg·kg -1 ·d -1Dapagliflozin was used for continuous intervention for 18 weeks. Body weight was measured once a week, and fasting blood glucose was measured once every three weeks. After the experiment ended, the mice were sacrificed, serum was collected, and the levels of blood glucose, urinary albumin, creatinine, and urea nitrogen were detected. Kidney tissues were taken for HE staining to observe the morphological changes of the kidney tissues.

[0037] Kidney tissues of mice in each group were fixed in 4% paraformaldehyde fixative for 48 hours, then paraffin-embedded, cut into 5-μm-thick sections, and subjected to Masson staining and immunohistochemical staining of fibronectin, and then observed and photographed under an upright optical microscope.

[0038] Results: It was found that AA + AG could play a synergistic role in protecting the kidney. The results of the effects on body weight and blood glucose are as Figure 2 shown. AA + AG could synergistically reduce the body weight of the diabetic nephropathy mouse model, AG could improve the blood glucose of the diabetic nephropathy mouse model while AA could not. The blood glucose-lowering effect of the CPE group was better than that of AA + AG. As Figure 1 known, the ethanol extract group of Cyclocarya paliurus contains other components in addition to AA and AG, and its administration dose is also high (240 mg·kg-1·d-1); the results of the effects on urinary albumin, urea nitrogen, and creatinine are as Figure 3 shown. AA + AG could synergistically improve the renal function injury of the diabetic nephropathy mouse model. The results of the effects on the morphological changes of the mouse kidney tissues are as Figure 4 shown. AA + AG could synergistically improve tubular atrophy (blue arrow), cell shedding (red arrow), and glomerulosclerosis (black arrow). The results of the effects on the morphological changes of the mouse kidney tissues are as Figure 5 shown. AA + AG could synergistically improve the collagen deposition and fibronectin deposition in the kidneys of diabetic nephropathy mice.

[0039] Example 2 AA + AG can synergistically improve the extracellular matrix accumulation in renal tubular epithelial cells

[0040] Human renal tubular epithelial cells (HK-2) were divided into 5 groups, with three parallel wells in each group. Each well was inoculated with 5*10 3Cells were seeded into 96-well plates and cultured in high-glucose DMEM medium containing 10% fetal bovine serum in a 37°C incubator with 5% CO2. After seeding the cells, the three groups of cells were treated as follows: The first group was the blank group, the second group was the high-glucose group with an AGEs concentration of 800 mg / mL, the third and fourth groups were the AA group (20 μM) and the AG group (20 μM) respectively, with an AGEs concentration of 800 mg / mL, and the fifth group was the combined application group of AA and AG (AA + AG = 10 μM + 10 μM). All 5 groups were treated for 72 h. After the above 5 groups of cells were treated as described, the medium was discarded, thiazolyl blue (MTT) was added, and DMSO was added 4 h later for the MTT assay. The absorbance at 490 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader, and cell viability was calculated. The results are shown in Figure 6 as follows. At the same time, HK-2 cells were seeded at 10×10 4 cells per well into 48-well plates. After treatment as the above 4 groups of cells, the medium was discarded, and cell samples were taken with a cell scraper. After washing 3 times with PBS, the samples were lysed by repeated freezing and thawing at -80°C 3 times and detected by ELISA. Proteins were quantified by the BCA method and the results were normalized. The results are shown in Figure 7 as follows.

[0041] Results: Figure 6 and Figure 7 , especially Figure 7 The combination index CI of F-G indicated that AA + AG had a synergistic effect, and it improved the extracellular matrix accumulation of renal tubular epithelial cells induced by AGEs.

[0042] After sampling, the above 5 groups of cells were added with RIPA lysis buffer containing protease inhibitor, ground with a tissue grinder, lysed on ice for 30 min, centrifuged at 12,000 rpm for 15 min, and the supernatant protein samples were taken. The protein concentration was measured by the BCA method and Western Blotting was performed. Protein samples were separated by 8% SDS-PAGE and transferred to a nitrocellulose (NC) membrane. At room temperature, the membrane was blocked in 5% BSA prepared in TBST buffer for 1 h. Then, the membrane was incubated with the first antibody of extracellular matrix protein overnight at 4°C. The next day, the membrane was incubated with the relevant second antibody for 1 h at room temperature and the protein expression was detected by chemiluminescence with ECL solution, with GAPDH as the normalization internal reference protein.

[0043] Results: Figure 8It is the ratio of extracellular matrix proteins to reference proteins in animals for Western Blotting. Fibronectin, Laminin 1, Collagen I, and Collagen IV were all significantly increased in the AGEs group, while they were significantly decreased in the AA group, AG group, and AA + AG group, and the combined effect was better than that of AA and AG alone. The above indicates that the combination of AA and AG has a synergistic improvement effect on the accumulation of extracellular matrix in diabetic renal tubules.

[0044] Example 3 Synergistic balance effect of AA + AG on TGF-β1 / BMP7 / Smads pathway

[0045] For the kidney tissue in Example 1, wash it, cut it into pieces, weigh 20 mg of the tissue, add it to RIPA lysis buffer containing protease inhibitor, grind it with a tissue grinder, then lyse it on ice for 30 min, centrifuge at 12,000 rpm for 15 min, take the supernatant protein sample, measure the protein concentration by the BCA method, and perform Western Blotting experiment (protein blotting method). Use 8% SDS-PAGE to separate the protein sample and transfer it to a nitrocellulose (NC) membrane. At room temperature, place the membrane in 5% BSA prepared in TBST buffer and block it for 1 h. Then, incubate the membrane with the first antibodies of extracellular matrix proteins and TGF-β1 / BMP7 / Smads pathway proteins overnight at 4°C. The next day, incubate the membrane with the relevant second antibodies at room temperature for 1 h, and detect the protein expression level by luminescence with ECL solution, where GAPDH is used as the normalized reference protein.

[0046] Figure 9It is the ratio of extracellular matrix proteins, TGF-β1 / BMP7 / Smads pathway proteins and internal reference proteins in animals for Western Blotting. Western Blotting results showed that AA and AG, either alone or in combination, could significantly improve the accumulation of extracellular matrix in the kidneys of DN mice. The effect of CPE on renal fibrosis in DN mice was not significantly different from that of the combination of AA and AG, indicating that AA and AG are the main synergistic effect factors for CPE to treat renal fibrosis in DN mice. The expression of TGF-β1 in DN mice was significantly increased, while the combination or single use of AA and AG could reduce the expression of TGF-β1, but the inhibitory effect of the combination of AA+AG was significantly better than that of single use. AG could significantly increase the expression of BMP7, while AA could inhibit the expression of BMP7 by activating Smad7. The combination of AA+AG could simultaneously increase the expression of Smad7 and BMP7, thus playing a better role in reducing TGF-β1. The excitatory effect of CPE on BMP7 and Smad7 was basically the same as that of the combination of AA+AG, while DAPA had basically no excitatory effect on them. The expression of Smad1 and Smad3 was basically not affected by drug administration, while the expression of Smad4 was decreased in DN mice, indicating an obvious imbalance in the TGF family balance in DN mice, and drug administration could restore this balance state.

[0047] Wash and cut the kidney tissues in Example 1 into small pieces. Weigh 20 mg of the tissues and add them to RIPA lysis buffer containing protease inhibitors. Grind them with a tissue grinder and then lyse them on ice for 30 min. Centrifuge at 12,000 rpm for 15 min. Take the supernatant protein sample, measure the protein concentration by the BCA method, normalize it to a unified concentration, and then perform protein quantification using the Enhanced BCA Protein Assay Kit. Take an equal amount of protein (1 mg) and perform immunoprecipitation reactions using the rProtein A / G Magnetic IP / Co-IP Kit, mouse-derived Smad1, Smad3, and Smad4. After denaturation, detect them by Western Blotting using rabbit-derived Smad1, Smad3, and Smad4.

[0048] Figure 10The binding of Smad2 / 3, Smad1 / 5 / 8 and Smad4 pathway proteins in co-immunoprecipitation Western Blotting of animal kidney tissues. The results showed that the binding of Smad3 and Smad4 in DN mice was significantly increased, while administration of drugs could significantly reduce their binding, and the inhibitory effect of AA on their binding was better than that of AG, and the combined use was better than AA alone. The binding rate of Smad1 and Smad4 in the kidneys of DN mice was decreased, administration of AG could significantly increase their binding, while AA could not, and the effect of the combined use group was slightly lower than that of AG alone. The above results indicate that the combined use of AA and AG can synergistically restore the balance of the TGF-β1 / BMP7 / Smads pathway.

[0049] Obviously, the role of the above embodiments is to clearly and specifically illustrate the content of the present invention, rather than a limitation on the implementation mode. For those skilled in the art to which the present invention pertains, other different forms of changes can be made based on the content of the present invention. It is unnecessary and impossible for the present invention to enumerate all implementation modes. Therefore, the obvious changes or variations derived therefrom are still within the protection scope of the present invention.

Claims

1. Use of a pharmaceutical composition in the preparation of a medicament for improving or preventing renal fibrosis in diabetic nephropathy, characterized in that, The pharmaceutical composition comprises asiatic acid and astragalin, wherein the mass ratio of asiatic acid to astragalin is 1:

1.

2. Use of an ethanol extract of Cyclocarya paliurus in the preparation of a drug for improving or preventing renal fibrosis in diabetic nephropathy, characterized in that, The ethanol extract of Cyclocarya paliurus is prepared by the following steps: taking 49.59 kg of dried Cyclocarya paliurus leaves, pulverizing them, adding 80% ethanol according to a material-liquid ratio of 1:6, extracting 3 times by the impregnation method, combining the extraction solutions, and concentrating them under reduced pressure until the alcohol smell disappears to obtain the ethanol extract of Cyclocarya paliurus; the mass ratio of asiatic acid to astragalin in the ethanol extract of Cyclocarya paliurus is 1:

1.

3. The application according to claim 1 or 2, characterized in that, The pharmaceutical composition or the ethanol extract of Cyclocarya paliurus further contains pharmaceutically acceptable excipients.

4. The application according to claim 2, characterized in that The active ingredients of the ethanol extract of Cyclocarya paliurus are asiatic acid and astragalin.

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