Application of tetrastigma planicaule water extract in preparation of medicine for preventing or treating hyperuricemia and related metabolic diseases

Abstract

The invention discloses a novel application of a tetrastigma planicaule water extract in preparation of a medicine for preventing or treating hyperuricemia and related metabolic diseases, the treatment effect and action mechanism of the tetrastigma planicaule water extract on HUA are discussed by establishing a PO and HX induced rat HUA model, and the result shows that the tetrastigma planicaule water extract can be used for preparing a medicine for preventing or treating hyperuricemia and related metabolic diseases. The tetrastigma planicaule water extract can reduce the uric acid level in a rat body by adjusting uric acid transporter protein, promote uric acid excretion, remarkably improve liver and kidney injury and inflammatory cell infiltration induced by PO and HX, relieve a series of reactions caused by hyperuricemia and improve the curative effect of hyperuricemia. And a theoretical basis and a clinical basis are provided for new medicines for clinically treating hyperuricemia and related metabolic diseases thereof.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicines, and in particular to application of a water extract of Herba Eupatorii in preparing a medicine for preventing or treating hyperuricemia and related metabolic diseases. Background Art

[0002] Hyperuricemia (HUA) is a common metabolic disease, clinically characterized by excessively elevated serum uric acid concentrations. Hyperuricemia is a long-term, slowly progressive process, with the main clinical feature being a long-term increase in uric acid levels in the blood. As the course of the disease progresses, uric acid can form urate in the body, which is gradually deposited in joints, kidneys and other parts, causing complications such as gouty arthritis, tophi, uric acid nephropathy, and cognitive impairment. Xanthine oxidase (XOD) and adenosine deaminase (ADA) are two important enzymes in the body that play a key role in the decomposition and metabolism of purine nucleotides. Therefore, the functions and effects of XOD and ADA cannot be ignored when studying uric acid metabolism and related diseases. Currently, the drugs used in clinical treatment of HUA mainly include xanthine oxidase inhibitors, urate oxidase and urate transporter inhibitors, which can effectively reduce serum uric acid levels, but are often accompanied by a series of adverse reactions, such as renal dysfunction and liver damage.

[0003] The main medicine of the stems and roots of the spatholobi vine is that it is mainly distributed in Fujian, Guangdong, Guangxi, Guizhou, Yunnan, Tibet and other places in China. It is one of the 108 commonly used old medicines in Yao medicine and is often used to dispel wind and dampness, traumatic injuries and other diseases. According to relevant literature reports, the quality control research of the spatholobi vine mostly stays on the identification of properties, microscopic identification, thin layer identification and chemical composition research, and its medicinal material basis and quality evaluation need to be further studied. The pharmacological research of the spatholobi vine mainly focuses on its antioxidant and anti-liver damage effects. Studies have shown that compared with the reference substances ascorbic acid and tea polyphenols, the Fe of the flavonoid-rich spatholobi vine is 3+ Restore antioxidant capacity and scavenge ABTS + .The ability is greater than that of tea polyphenols. In addition, the alcohol extract of Herba Lycopodii has a certain protective effect on acute liver injury caused by CCl4 in mice. However, the effect and mechanism of Herba Lycopodii in treating hyperuricemia and its related metabolic diseases have not been reported. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a new application of the water extract of Herba Eupatorii in the preparation of drugs for preventing or treating hyperuricemia and its related metabolic disorders, in order to provide a theoretical basis and clinical foundation for the research of new drugs for preventing or treating hyperuricemia and its related metabolic disorders.

[0005] Tetrastigma planicaule (Hook.) Gagnep. is a large woody vine of the genus Tetrastigma in the family Vitaceae.

[0006] The hyperuricemia and its related metabolic disorders described in the present invention include gout or gout complications caused by hyperuricemia. The gout includes acute gout or chronic gout; the gout complications include gouty arthritis, gout attacks, gouty nephropathy or uric acid nephrolithiasis.

[0007] The preparation method of the water extract of the Herba Eupatorii of the present invention comprises the following steps:

[0008] Soak the rhizomes of Herba Lycopodiellae in water for 20-40 minutes, add 5-10 times the amount of water, heat and boil twice, each time for 20-40 minutes, combine the two decoctions, and concentrate under reduced pressure to obtain the Herba Lycopodiellae water extract.

[0009] The medicine of the present invention comprises an effective amount of the water extract of the Herba Eupatorii and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier includes but is not limited to a filler, a disintegrant, a sweetener, a lubricant, a suspending agent, a preservative, a binder, etc., and the amount thereof is the conventional amount in the art.

[0010] The drug of the present invention can be prepared into a suitable dosage form by conventional methods in the art. Preferably, the dosage form of the drug is tablets, capsules, powders, injections, powders, syrups, pills, mixtures or granules.

[0011] Compared with the prior art, the present invention has the following excellent effects:

[0012] The present invention provides a new use of a water extract of the Herba Epimedii in preparing a drug for preventing or treating hyperuricemia and related metabolic diseases. By establishing a PO and HX-induced HUA model in rats, the therapeutic effect and mechanism of the water extract of the Herba Epimedii on HUA are explored. The results show that the water extract of the Herba Epimedii can reduce the uric acid level in rats by regulating uric acid transporters and promote uric acid excretion. It also significantly improves liver and kidney damage and inflammatory cell infiltration induced by PO and HX, and alleviates a series of reactions caused by hyperuricemia, thus providing a theoretical basis and clinical foundation for new drugs for the clinical treatment of hyperuricemia and related metabolic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The effect of BDT water extract on the pathological changes of heart, liver, spleen, lung and kidney in rats (×200);

[0014] Figure 2 The effect of BDT on the renal appearance and pathological changes of HUA rats (×200);

[0015] Figure 3 The effect of BDT on the expression of OAT1 and ABCG2 proteins in the kidney of HUA rats (*** indicates P<0.001 compared with the normal control group, ### indicates P<0.001 compared with the model control group, # indicates P<0.05 compared with the model control group);

[0016] Figure 4 The effect of BDT on liver pathological changes in HUA rats (×200);

[0017] Figure 5 This is the effect of BDT on the expression of XDH and HPRT1 proteins in the liver of HUA rats. DETAILED DESCRIPTION

[0018] The present invention is further described below by means of specific implementation methods. The following examples are specific implementation methods of the present invention, but the implementation methods of the present invention are not limited to the following examples.

[0019] The reagents or drugs used in the embodiments of the present invention are as follows:

[0020] Benzbromarone (batch number: J20180561, purchased from Hermann Pharmaceuticals, Germany);

[0021] Colchicine (Batch No. 220417, purchased from Yunnan Botanical Pharmaceutical Co., Ltd.);

[0022] Potassium oxonate (batch number: C15784656, purchased from Shanghai MacLean Biochemical Technology Co., Ltd.);

[0023] Sodium urate (Batch No.: C15085256, purchased from Shanghai MacLean Biochemical Technology Co., Ltd.);

[0024] The dosage of BDT was 2.5 g / kg, 5 g / kg and 10 g / kg, and the administration dosage was 10 ml / kg.

[0025] The Herba Lycopodii used in this experiment was the rhizome of the dried Herba Lycopodii. The preparation method of the Herba Lycopodii water extract was as follows: 600 g of Herba Lycopodii was taken, 3 L of water was added, and the mixture was soaked for 0.5 hours, decocted for 30 minutes and then poured out, 3 L of water was added again and decocted for 30 minutes, the two decoctions were combined, and the decoctions were concentrated under reduced pressure to a concentration of 1 g / mL.

[0026] The HUA model is induced by PO and HX.

[0027] The PO preparation method is as follows: weigh 3 g of PO and dilute it into 100 mL of normal saline to make 3% PO, and the dosage is 300 mg / kg.

[0028] The preparation method of HX is as follows: weigh 3 g of HX and dilute it to 100 mL of pure water to make 3% HX, and the dosage is 300 mg / kg.

[0029] Benzbromarone and colchicine were used as positive drug groups, with dosages of 0.0045 mg / kg and 0.27 mg / kg, respectively.

[0030] The following experiments demonstrate the technical effects of the present invention.

[0031] Example 1: Protective effect of the aqueous extract of Psoralea corylifolia in a hyperuricemia model

[0032] 1 Experimental Materials

[0033] Clean-grade male SD rats, weighing 260±20 g, were housed in cages and allowed free access to food and water for 7 days to allow them to adapt to the environment.

[0034] 2 Experimental methods

[0035] In this study, a rat model of hyperuricemia was established by potassium oxonate (PO) and hypoxanthine (HX) to study the therapeutic effect of the aqueous extract of Herba Eupatorii on the model rats.

[0036] 2.1 Experimental Grouping

[0037] Forty rats were randomly divided into normal control group (CON), non-modeling group (NM-BDT) group treated with 10 g / kg aqueous extract of Ulmus pumila (MOD), model group (MOD), benzbromarone group (BEN), colchicine group (COL), group treated with 2.5 g / kg aqueous extract of Ulmus pumila (BDT-2.5), group treated with 5 g / kg aqueous extract of Ulmus pumila (BDT-5), and group treated with 10 g / kg aqueous extract of Ulmus pumila (BDT-10).

[0038] 2.2 Administration

[0039] Each group was given intraperitoneal injection of 3% PO and oral gavage of 3% HX to prepare hyperuricemia model every day. One hour after modeling, the positive drug group and the drug group were given corresponding drugs by oral gavage at 10ml / kg, once a day; the CON group and MOD group were given the same volume of normal saline. Once in the morning and evening, for 7 consecutive days. The urine of 24 hours was collected in a metabolic cage. The rats were killed 1 hour after weighing and drug administration on the 8th day for sampling. Blood was collected from the abdominal aorta to prepare serum, and the right kidney and liver lobule were fixed in 4% paraformaldehyde for later use.

[0040] 2.3 Detection indicators

[0041] 2.3.1 Determination of serum, plasma and urine indicators

[0042] Blood was collected from the abdominal aorta of rats and allowed to stand for 2 hours before centrifugation at 3500 rpm / min for 15 minutes. The supernatant was collected and packaged to prepare serum and plasma. The levels of uric acid (UA), urea nitrogen (BUN), and creatinine (CR) in serum and urine of each group were detected according to the instructions of the kit.

[0043] 2.3.2 Histopathological observation

[0044] The right kidney and liver lobule of rats in each treatment group were fixed with 4% paraformaldehyde, embedded in paraffin, sliced ​​at 4-5 μm, dewaxed with xylene, dehydrated with gradient ethanol, stained with conventional HE, dehydrated with ethanol, transparentized with xylene, sealed with resin, and observed under a microscope. The same operation was performed, and the heart, liver, spleen, lung, and kidney of rats in the NM-BDT group were stained with HE and observed under a microscope to determine the biological safety of BDT.

[0045] 2.3.3 Western-blot analysis

[0046] Prepare 10% separation gel and concentrated gel; the protein loading amount is 50μg, and the protein is separated by SDS-PAGE electrophoresis at a voltage of 60V. When bromophenol blue runs to the bottom, stop electrophoresis; quickly transfer it to a PVDF membrane activated with methanol in advance, and transfer the membrane under a constant current of 250mA for 2h; after the time is up, wash it once with 1×TBST, 5min / time, and then block it with 5% skim milk powder for 90min, wash it 3 times with 1×TBST, 5min / time, and then incubate the corresponding primary antibody (OAT1, ABCG2, HPRT1, XDH) at 4℃ overnight; the next day, recover the primary antibody, wash it 3 times with 1×TBST, and incubate it with rabbit secondary antibody / mouse secondary antibody at room temperature for 90min; after the time is up, recover the secondary antibody, wash it 3 times with 1×TBST, and develop it with a chemiluminescence instrument. Finally, use ImageJ software to process and analyze the gray value of the band.

[0047] 3 Data analysis

[0048] SPSS26.0 software was used for data processing, and one-way analysis of variance was selected as the statistical method. Non-parametric tests were used for those that did not conform to normal distribution.

[0049] 4 Experimental results

[0050] 4.1 Pathological histological observation of 10g / kg BDT on the heart, liver, spleen, lung and kidney of rats

[0051] from Figure 1The H&E staining results showed that the myocardial cell fibers were arranged in an orderly manner, the horizontal stripes were clearly visible, the cell nucleus was centered, no necrotic tissue was observed, and no fibrous scar hyperplasia was observed. The rat liver lobule structure was intact, the central vein was not congested, and the hepatocytes were neatly arranged. The spleen corpuscles were not significantly enlarged, the blood sinusoids and lymphoid follicles were not significantly different, and the hematopoietic erythrocyte islands did not proliferate or decrease. The lung tissue structure was intact, the alveoli were clear, and no abnormalities were observed. The glomeruli were normal in size and shape, and the structure was intact. The renal tubular epithelial cells were intact and clear. This shows that 10g / kg BDT has good biosafety and can be used for subsequent related experimental studies.

[0052] 4.2BDT reduces serum uric acid in rats and promotes uric acid excretion

[0053] As shown in Tables 1 and 2, the UA, CR, and BUN in the serum and plasma of the MOD group increased significantly, and the UA in the urine decreased significantly, indicating that the synthesis of uric acid in the MOD group increased and the excretion decreased. The experiment successfully established a hyperuricemia rat model. After BDT administration, the UA, CR, and BUN levels in the serum of rats decreased significantly, and BDT-10 administration increased the levels of UA, CR, and BUN in the urine, indicating that it can reduce the level of uric acid in the body, promote uric acid excretion, and alleviate the renal function damage caused by modeling to a certain extent.

[0054] Table 1 Effects of BDT on blood biochemical parameters in hyperuricemia rats ( n=5)

[0055]

[0056] Table 2 Effects of BDT on urine biochemical indices in hyperuricemia rats ( n=5)

[0057]

[0058] ## indicates P<0.01 compared with the normal control group.

[0059] ** indicates P < 0.01 compared with the model control group, * indicates P < 0.05 compared with the model control group.

[0060] ### indicates P<0.001 compared with the normal control group.

[0061] *** indicates P < 0.001 compared with the model control group, and * indicates P < 0.05 compared with the model control group.

[0062] 4.3 Effects of BDT on renal appearance and pathological changes in HUA rats

[0063] To further evaluate the protective effect of BDT on the kidneys, Figure 2The kidney appearance and kidney index show that the model group has a larger kidney volume, a rougher surface, and more serious lesions than the control group. After BDT administration, the degree of lesions was alleviated, and BDT-5 and BDT-10 significantly reduced the kidney index. And from the H&E staining results, it was observed that the glomeruli of the rats in the model group were severely atrophied and deformed (black arrows), and the renal tubules were significantly dilated (red arrows), which was related to the severe inflammatory cell infiltration in the renal tubules. After treatment with different doses of BDT, these pathological changes in the kidneys were improved, indicating that BDT can play a renal protective role.

[0064] Effect of BDT on renal uric acid transporter in HUA rats

[0065] Based on the results of the previous indicators, it was found that BDT reduced UA, BUN, and CR levels in a dose-dependent manner, and played a significant role in the treatment of HUA. Therefore, BDT-10 was selected for Western-Blot detection. Figure 3 As shown in the data, compared with the CON group, the protein expressions of OAT1 and ABCG2 in the MOD group were significantly increased (P < 0.01); compared with the MOD group, BDT-10 could significantly up-regulate the protein expressions of OAT1 and ABCG2.

[0066] Effects of 4.5BDT on liver pathological changes in HUA rats

[0067] Depend on Figure 4 The results of liver HE staining showed that the liver lobule structure of rats in the normal group was intact, the central vein was not congested, and the hepatocytes were neatly arranged. The liver lobule structure of rats in the model group was deformed, the central vein of the liver lobule was congested, and there were a lot of inflammatory infiltrations around it (red arrows). The central vein of the liver lobule of rats in the BDT group was not congested, the structure was normal, and the inflammatory infiltration was greatly reduced, alleviating the pathological changes of the liver.

[0068] Effect of 4.6BDT on uric acid synthesis protein in the liver of HUA rats

[0069] The liver is the main site for uric acid synthesis, and xanthine oxidase (XDH) is the key enzyme in the body that catalyzes xanthine and hypoxanthine to produce uric acid. After oral administration of hypoxanthine, XDH increases in the body, promoting the increase of uric acid synthesis in rats and the conversion of hypoxanthine to xanthine. Guanine phosphoribosyltransferase (HPRT1) deficiency will decompose excess purine into uric acid and excrete it outside the cell, which is an important reason for the increase of blood uric acid. Figure 5 The results showed that BDT administration significantly downregulated the expression level of XDH protein, upregulated the expression level of HPRT1, and inhibited the synthesis of uric acid.

[0070] In summary, BDT significantly reduces serum uric acid in a dose-dependent manner. It reduces the production of uric acid and promotes the excretion of uric acid by regulating uric acid transporters. It also has a certain protective effect on the liver and kidneys, reducing liver and kidney damage caused by hyperuricemia.

Claims

1. Use of the water extract of Herba Eupatorii in the preparation of a medicament for preventing or treating hyperuricemia and its related metabolic disorders.

2. The use according to claim 1, characterized in that: The hyperuricemia and its related metabolic disorders include gout or gout complications caused by hyperuricemia.

3. The use according to claim 2, characterized in that: The gout includes acute gout or chronic gout; the gout complications include gouty arthritis, gout attacks, gouty nephropathy or uric acid nephrolithiasis.

4. The use according to claim 1, characterized in that: The preparation method of the water extract of Caulis Spatholobi comprises the following steps: Soak the rhizomes of Herba Lycopodiellae in water for 20-40 minutes, add 5-10 times the amount of water, heat and boil twice, each time for 20-40 minutes, combine the two decoctions, and concentrate under reduced pressure to obtain the Herba Lycopodiellae water extract.

5. The use according to claim 1, characterized in that: The medicine comprises an effective amount of the water extract of the Herba Eupatorii and a pharmaceutically acceptable carrier.

6. The use according to claim 5, characterized in that: The dosage form of the medicine is tablet, capsule, powder, injection, powder, syrup, pill, mixture or granule.

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