Tetrastigma hemsleyanum Diels et Gilg extract for reducing uric acid, traditional Chinese medicine composition and application thereof

Abstract

The invention provides a radix tetrastigme extract for reducing uric acid, a traditional Chinese medicine composition and application of the radix tetrastigme extract. Specifically, the invention discloses application of the radix tetrastigme extract in preparation of drugs, food or health care products for reducing uric acid. The invention further discloses a radix tetrastigme extract for reducing uric acid, and the radix tetrastigme extract is obtained by extracting radix tetrastigme powder by adopting a solvent and an ultrasonic extraction method. The invention further discloses a traditional Chinese medicine composition which comprises a radix tetrastigme extract, an ampelopsis grossedentata extract, a fiveleaf gynostemma herb extract and a chicory extract. An in-vitro xanthine oxidase inhibition test and an in-vivo uric acid reduction animal test show that the radix tetrastigme extract and the traditional Chinese medicine composition have relatively strong inhibition activity on xanthine oxidase, and the traditional Chinese medicine composition has a synergistic effect. The traditional Chinese medicine composition can reduce the contents of serum uric acid (UA), urea nitrogen (BUN) and creatinine (Cr) of a hyperuricemia animal model, and has the effect of reducing uric acid.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and health care products. Specifically, the present invention relates to a use of a clover extract and a traditional Chinese medicine composition and its use. Background Art

[0002] Tetrastigma hemsleyanum Diels et Gilg is a traditional Chinese medicinal material belonging to the genus Tetrastigma of the grape family. It contains a variety of medicinal ingredients such as flavonoids, phenylpropanoids, polysaccharides, etc., and has anti-inflammatory, anti-cancer, analgesic and detoxification effects. It is widely used in the clinic to treat a variety of diseases, including pneumonia, hepatitis, dysentery, high fever convulsions in children, edema and snake bites.

[0003] The invention patents of Sanyeqing mainly focus on extraction methods, preparation and application of preparations. For example, He Yi et al. provided a preparation process of Sanyeqing freeze-dried powder (CN201810815195.3), aiming to retain more active ingredients and improve the effect of taking.

[0004] Li Qingpo et al. provided a three-leaf green spray film for wound healing and its preparation method (CN202111281786.5). Through a specific formula and process, an easy-to-use film with protective and therapeutic effects was prepared.

[0005] Ding Zhishan provides a method for preparing trifoliate green polysaccharide particles with anti-tumor effects (CN201610149782.4). The preparation uses trifoliate green polysaccharide as an active ingredient and has good anti-tumor and immune-enhancing effects in vivo and in vitro.

[0006] Hyperuricemia is a purine metabolism disorder, mainly due to excessive accumulation of uric acid in the body or impaired excretion, which leads to excessive uric acid in the blood. When the uric acid level in the human body is maintained at a high level for a long time, it will cause gout physiologically. Xanthine oxidase is the key rate-limiting enzyme in the uric acid metabolism pathway in the human body. Therefore, the main way to clinically treat hyperuricemia or gout is to reduce uric acid levels by inhibiting the activity of xanthine oxidase, thereby alleviating or treating hyperuricemia and gout.

[0007] At present, allopurinol, colchicine, febuxostat and other chemical drugs are usually used clinically to control high uric acid levels and treat hyperuricemia, but their adverse reactions are unacceptable. Traditional Chinese medicine in my country believes that hyperuricemia belongs to the category of "uric acid turbidity". The accumulation of uric acid in the blood is mainly due to the generation of dampness and turbidity in the body, which causes poor circulation of qi and blood, accumulation of turbidity, and deposition of dampness and stagnation in the tendons, bones and joints, resulting in pain and swelling.

[0008] Based on the practical experience of using traditional Chinese medicine or Chinese herbal medicine, finding an efficient, low-toxic, safe and healthy functional ingredient to replace clinical drugs to alleviate the health problems caused by hyperuricemia has become a research and development hotspot. Summary of the invention

[0009] The object of the present invention is to provide a use of a clover extract in preparing a medicine, food or health product for lowering uric acid.

[0010] Another object of the present invention is to provide a Tripterygium wilfordii extract for reducing uric acid.

[0011] The present invention also aims to provide a Chinese medicine composition which can effectively, safely and healthily relieve hyperuricemia with low toxicity.

[0012] The above object of the present invention is achieved through the following solutions:

[0013] In a first aspect, the present invention provides a use of a clover extract in the preparation of a uric acid-lowering medicine, food or health product. The extract can be used to lower uric acid, relieve and / or treat hyperuricemia, gout, and inhibit xanthine oxidase.

[0014] In the present invention, the extract of Tripterygium wilfordii can be a commercial product, or can be prepared by conventional traditional Chinese medicine extraction methods in the art.

[0015] The inventors surprisingly found that the extract of Tripterygium wilfordii can be used to relieve and / or treat hyperuricemia, gout, and inhibit xanthine oxidase. The inventors also surprisingly found that the extract of Tripterygium wilfordii, the extract of Rhizoma Angustifolia, the extract of Gynostemma pentaphyllum, and the extract of Cichorium indica can reduce the serum uric acid (UA), urea nitrogen (BUN), and creatinine (Cr) content of hyperuricemia animal models, and have a uric acid-lowering effect. The present invention provides the use of the extract of Tripterygium wilfordii as a xanthine oxidase inhibitory active substance in the preparation of a drug, food, or health product with a uric acid-lowering effect.

[0016] Preferably, in the use of the cloverleaf green extract of the present invention, the cloverleaf green extract is prepared by a method comprising the following steps: extracting cloverleaf green powder by ultrasonic extraction using a solvent to obtain the cloverleaf green extract.

[0017] Preferably, in the use of the Tripterygium wilfordii extract of the present invention, the solvent is an organic solvent.

[0018] Preferably, in the use of the Tripterygium wilfordii extract of the present invention, the organic solvent comprises ethanol.

[0019] Preferably, in the use of the Tripterygium wilfordii extract described in the present invention, the solvent is an aqueous solution of ethanol with a mass percentage of 30-45%.

[0020] Preferably, in the use of the clover extract of the present invention, the usage ratio of the clover to the solvent is clover: solvent = 1 g: (15-30) mL.

[0021] Preferably, in the use of the Tripterygium wilfordii extract of the present invention, the Tripterygium wilfordii powder is obtained by drying and crushing the Tripterygium wilfordii.

[0022] Preferably, in the use of the Tripterygium wilfordii extract of the present invention, the extraction is carried out under heating conditions.

[0023] Preferably, in the use of the Tripterygium wilfordii extract of the present invention, the extraction is carried out at 40-55°C.

[0024] Preferably, in the use of the Tripterygium wilfordii extract of the present invention, the ultrasound is performed for 30-50 min.

[0025] Preferably, in the use of the clover extract of the present invention, the method for preparing the clover extract also includes the following steps: filtering and removing residues from the mixed solution after ultrasound, and vacuum concentrating and drying the filtrate after filtration.

[0026] In a second aspect, the present invention provides a clover extract for lowering uric acid, which is obtained by extracting clover powder using a solvent and an ultrasonic extraction method.

[0027] Preferably, in the Tripterygium wilfordii extract for lowering uric acid of the present invention, the solvent is an organic solvent.

[0028] Preferably, in the Tripterygium wilfordii extract for lowering uric acid of the present invention, the organic solvent comprises ethanol.

[0029] Preferably, in the Tripterygium wilfordii extract for lowering uric acid of the present invention, the solvent is an aqueous solution of ethanol with a mass percentage of 30-45%.

[0030] Preferably, in the clover extract for lowering uric acid of the present invention, the usage ratio of the clover to the solvent is clover: solvent = 1 g: (15-30) mL.

[0031] Preferably, in the Tripterygium wilfordii extract for lowering uric acid of the present invention, the Tripterygium wilfordii powder is obtained by drying and pulverizing the Tripterygium wilfordii.

[0032] Preferably, in the Tripterygium wilfordii extract for lowering uric acid of the present invention, the extraction is carried out under heating conditions.

[0033] Preferably, in the Tripterygium wilfordii extract for lowering uric acid of the present invention, the extraction is carried out at 40-55°C.

[0034] Preferably, in the Tripterygium wilfordii extract for lowering uric acid of the present invention, the ultrasonication is performed for 30-50 min.

[0035] Preferably, in the clover extract for lowering uric acid of the present invention, the method for preparing the clover extract also includes the following steps: filtering and removing residues from the mixed solution after ultrasound, and vacuum concentrating and drying the filtered filtrate.

[0036] In a third aspect, the present invention provides a traditional Chinese medicine composition comprising a clover extract, a rattan tea extract, a gynostemma pentaphyllum extract and a chicory extract and optional pharmaceutically acceptable excipients.

[0037] The pharmaceutically acceptable excipients of the present invention may be gum arabic, β-cyclodextrin, microcrystalline cellulose, hydroxypropyl cellulose, edible starch, and the like.

[0038] In the present invention, the clover extract, rattan tea extract, gynostemma pentaphyllum extract and chicory extract can be commercially available products, or can be prepared using conventional traditional Chinese medicine extraction methods in the art.

[0039] Preferably, in the traditional Chinese medicine composition of the present invention, the cloverleaf green extract is prepared by a method comprising the following steps: extracting cloverleaf green powder by ultrasonic extraction using a solvent to obtain the cloverleaf green extract.

[0040] Preferably, in the traditional Chinese medicine composition of the present invention, the solvent is an organic solvent.

[0041] Preferably, in the traditional Chinese medicine composition of the present invention, the organic solvent comprises ethanol.

[0042] Preferably, in the traditional Chinese medicine composition of the present invention, the solvent is an aqueous solution of ethanol with a mass percentage of 30-45%.

[0043] Preferably, in the traditional Chinese medicine composition of the present invention, the usage ratio of the cloverleaf green to the solvent is cloverleaf green: solvent = 1g: (15-30) mL.

[0044] Preferably, in the traditional Chinese medicine composition of the present invention, the Tripterygium wilfordii powder is obtained by drying and crushing Tripterygium wilfordii.

[0045] Preferably, in the traditional Chinese medicine composition of the present invention, the extraction is carried out under heating conditions.

[0046] Preferably, in the traditional Chinese medicine composition of the present invention, the extraction is carried out at 40-55°C.

[0047] Preferably, in the traditional Chinese medicine composition of the present invention, the ultrasound is performed for 30-50 min.

[0048] Preferably, in the Chinese medicine composition described in the present invention, the method for preparing the clover extract further comprises the following steps: filtering and removing residues from the mixed solution after ultrasonication, and vacuum concentrating and drying the filtered filtrate.

[0049] Preferably, in the traditional Chinese medicine composition described in the present invention, the weight ratio of the rattan tea extract, clover extract, gynostemma extract and chicory extract is rattan tea extract:clover extract:gynostemma extract:chicory extract = (10-15): (50-70): (1-5): (50-60).

[0050] Preferably, in the traditional Chinese medicine composition described in the present invention, the weight ratio of the rattan tea extract, clover extract, gynostemma extract and chicory extract is rattan tea extract:clover extract:gynostemma extract:chicory extract = 12:64:1:60.

[0051] In a fourth aspect, the present invention provides use of the Chinese medicine composition of the present invention in the preparation of a medicine, food or health product for lowering uric acid.

[0052] The Chinese medicine composition of the present invention can be used for alleviating and / or treating hyperuricemia, gout, and inhibiting xanthine oxidase.

[0053] The present invention provides the use of the traditional Chinese medicine composition of the present invention as a xanthine oxidase inhibitory active substance in the preparation of medicines, foods or health products with uric acid lowering effects.

[0054] In vitro xanthine oxidase inhibition tests and in vivo uric acid lowering animal tests show that the clover extract and the Chinese medicine composition of the present invention have strong inhibitory activity on xanthine oxidase, and the Chinese medicine composition produces a synergistic effect. The Chinese medicine composition can reduce the serum uric acid (UA), urea nitrogen (BUN) and creatinine (Cr) contents of hyperuricemia animal models, and has a uric acid lowering effect.

[0055] Therefore, the clover extract and the composition can be used to prepare xanthine oxidase inhibitors, uric acid lowering health products or biomedical drugs.

[0056] Compared with the prior art, the present invention has the following advantages:

[0057] The raw materials used in the present invention are all derived from natural plants. Rhizoma Cibotii, Rhizoma Cibotii, Rhizoma Cibotii and Rhizoma Cichorii are all traditional Chinese medicinal materials with a long history of application. Compared with chemical synthetic drugs, it may have the advantages of better safety and lower side effects. The Rhizoma Cibotii extract of the present invention can effectively, safely and healthily relieve hyperuricemia with low toxicity.

[0058] The extracts and compositions can be made into oral liquids, capsules or tablets, etc., which are convenient for patients to take, have natural herbal flavors, and may have a better taste, thereby improving patient compliance. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0060] Figure 1 The inhibitory effect of the rattan tea extract of Example 1 of the present invention on xanthine oxidase is shown;

[0061] Figure 2 The figure shows the inhibitory effect of the clover extract of Example 2 of the present invention on xanthine oxidase;

[0062] Figure 3 The figure shows the inhibitory effect of the Gynostemma pentaphyllum extract of Example 3 of the present invention on xanthine oxidase;

[0063] Figure 4 The results show the inhibitory effect of the chicory extract of Example 4 of the present invention on xanthine oxidase. DETAILED DESCRIPTION

[0064] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but not for limiting the scope of the present invention.

[0065] The present invention is further described in detail below in conjunction with specific examples. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are also reagents and materials available from commercial channels unless otherwise specified.

[0066] Several selected plant extracts were examined for their inhibitory activity on xanthine oxidase alone or in combination. The plant extracts selected in the present invention are vine tea extract, clover extract, gynostemma pentaphyllum extract and chicory extract. The vine tea extract, gynostemma pentaphyllum extract and chicory extract can be prepared by conventional Chinese medicine extraction methods in the art. Depending on the nature of the raw materials, the greening, defatting and decolorization treatments were performed before extraction, as follows:

[0067] Example 1

[0068] Preparation method of rattan tea extract

[0069] The rattan tea was treated by killing green, and placed in a 60℃ constant temperature drying oven for 4 hours. After being crushed with a traditional Chinese medicine grinder, it was passed through an 80-mesh sieve to obtain the rattan tea raw material. Weigh an appropriate amount of rattan tea powder and add it to water at a solid-liquid ratio of 1:20, shake it well so that the material is completely immersed in water; ultrasonic-assisted extraction is performed at a temperature of 50℃ for 30 minutes, centrifuged or filtered after the extraction, and the supernatant is collected; the filter residue is added to water again at a solid-liquid ratio of 1:10, and ultrasonic extraction is repeated once under the above conditions. After the obtained extract is centrifuged or filtered, the filter residue is discarded, and the supernatants obtained twice are combined, and concentrated by vacuum decompression at 45℃, and the concentrate is vacuum freeze-dried to obtain the rattan tea extract.

[0070] Example 2

[0071] Preparation method of clover extract

[0072] The three-leaf green was treated with greening, and placed in a 60℃ constant temperature drying oven for 4 hours. After being crushed with a traditional Chinese medicine grinder, it was passed through an 80-mesh sieve to obtain the three-leaf green raw material. Weigh an appropriate amount of three-leaf green powder and add it to a 45% ethanol aqueous solution at a solid-liquid ratio of 1:20, shake it well so that the material is completely immersed in the ethanol aqueous solution; ultrasonic-assisted extraction is performed at a temperature of 50℃ for 30 minutes, centrifuged or filtered after the extraction, and the supernatant is collected; the filter residue is added to a 45% ethanol aqueous solution at a solid-liquid ratio of 1:10 again, and the ultrasonic extraction is repeated once under the above conditions. After the obtained extract is centrifuged or filtered, the filter residue is discarded, and the supernatants obtained twice are combined, and the ethanol is removed by vacuum decompression concentration at 45℃, and the concentrate is vacuum freeze-dried to obtain the three-leaf green extract.

[0073] Example 3

[0074] Preparation method of Gynostemma pentaphyllum extract

[0075] The Gynostemma pentaphyllum was treated with greening, and placed in a 60℃ constant temperature drying oven for 4 hours. After being crushed with a traditional Chinese medicine grinder, it was passed through an 80-mesh sieve to obtain the raw material of Sanyeqing. An appropriate amount of Gynostemma pentaphyllum powder was weighed and added to a 45% ethanol aqueous solution at a solid-liquid ratio of 1:20, and the material was shaken to completely immerse the material in the ethanol aqueous solution; ultrasonic-assisted extraction was performed at a temperature of 50℃ for 30 minutes, and after the extraction was completed, the supernatant was collected by centrifugation or filtration; the filter residue was added to a 45% ethanol aqueous solution at a solid-liquid ratio of 1:10 again, and the ultrasonic extraction was repeated once under the above conditions. After the obtained extract was centrifuged or filtered, the filter residue was discarded, and the supernatants obtained twice were combined, and the ethanol was removed by vacuum decompression concentration at 45℃, and the concentrate was vacuum freeze-dried to obtain the Gynostemma pentaphyllum extract.

[0076] Example 4

[0077] Preparation method of chicory extract

[0078] Place the chicory root in a 60℃ constant temperature drying oven and dry it for 4 hours. After crushing it with a traditional Chinese medicine grinder, pass it through an 80-mesh sieve to obtain the chicory raw material. Weigh an appropriate amount of chicory powder and add it to water at a solid-liquid ratio of 1:20, shake it well so that the material is completely immersed in water; perform ultrasonic-assisted extraction at a temperature of 50℃ for 30 minutes, centrifuge or filter it after the extraction, and collect the supernatant; add water to the residue again at a solid-liquid ratio of 1:10, repeat the ultrasonic extraction once under the above conditions, centrifuge or filter the obtained extract, discard the residue, combine the supernatants obtained twice, concentrate it under vacuum at 45℃, and vacuum freeze-dry the concentrate to obtain the chicory extract.

[0079] Example 5

[0080] In vitro xanthine oxidase inhibition assay

[0081] 1.1 Solution preparation

[0082] (1) PBS buffer (pH 7.73): Weigh 2.27 g of potassium dihydrogen phosphate and dissolve it in ultrapure water to make a 1 / 15 mol / L potassium dihydrogen phosphate solution (Solution A). Then weigh 23.88 g of disodium hydrogen phosphate and dissolve it in ultrapure water to make a 1 / 15 mol / L disodium hydrogen phosphate solution (Solution B). Mix the two solutions in a ratio of A:B = 1:9 to obtain a PBS buffer solution with a pH of 7.73.

[0083] (2) Xanthine substrate solution (2.25 mmol / L, pH 7.5): Weigh 10.27 mg of xanthine, dissolve it with an appropriate amount of NaOH solution, then dilute it with 30 mL of PBS buffer, and finally adjust the pH to 7.5 with phosphoric acid to obtain 2.25 mmol / L pH 7.5 xanthine substrate solution.

[0084] (3) Xanthine oxidase solution: Weigh 1 mg of xanthine oxidase (50 U / mg) and add it to 10 mL of PBS to dissolve it to obtain a 5 U / mL XO stock solution, which is retained for later use. Take 1 mL of the stock solution and dilute it with 9 mL of PBS buffer to obtain a 0.5 U / mL xanthine oxidase solution.

[0085] 1.2 Sample dissolution

[0086] Weigh 20 mg of plant extract sample, add PBS buffer or DMSO solvent, dissolve by ultrasonication, prepare a 10 mg / mL sample solution, and dilute the sample solution into a series of concentrations to be measured using the multiple dilution method.

[0087] 1.3 Experimental methods

[0088] Since the uric acid generated by xanthine oxidase (XO) catalyzing the substrate xanthine has a characteristic absorption peak at a wavelength of 290nm, the activity intensity of XO can be reflected by measuring the absorbance value at this wavelength per unit time, that is, the rate of uric acid generation. The absorbance was measured every 15s using the kinetic / time software of the UV spectrophotometer for a total of 20 times. During this period, the absorbance increased linearly with time, and its slope was the reaction rate of the enzyme. The larger the slope, the stronger the activity of the enzyme.

[0089] The entire enzyme inhibition experiment was carried out in a 2mL centrifuge tube, and the kinetic method was used to measure the enzyme inhibition on a spectrophotometer using a cuvette. 200μL of xanthine oxidase solution and 200μL of different sample solutions were added to the reaction wells of the experimental group in turn, and the sample series gradient concentrations to be tested were set respectively, and the samples were incubated at a constant temperature of 37°C for 5min. Before the start of the assay, 400μL of xanthine substrate solution was quickly added to the reaction well to start the reaction. The blank control wells were tested with 200μL PBS buffer solution instead of the sample solution. According to XO relative activity (%) = R / R0×100%, the relative activity of XO in the reaction system containing different concentrations and different samples was calculated. Among them, R0 and R represent the slopes of the "absorbance-time" fitting equation straight line of the reaction system without inhibitor and when adding different concentrations of sample inhibitors, respectively. The relative enzyme activity of the blank control well without inhibitor was defined as 100%. SPSS22.0 was used to calculate the IC50 values ​​of different samples.

[0090] 1.4 Experimental Results

[0091] The results showed that the extracts of rattan tea, clover extract, gynostemma pentaphyllum extract and chicory extract all had a certain degree of inhibitory effect on xanthine oxidase, and the inhibition rate increased with the increase of sample concentration, and the inhibition was concentration-dependent.

[0092] Table 1 IC inhibitory activity of vine tea extract, clover extract, Gynostemma pentaphyllum extract and chicory extract on xanthine oxidase 50 value

[0093] Measurement samples <![CDATA[IC 50 Value (mg / mL)]]> Rattan Tea Extract 0.83±0.03 Clover Extract 2.04±0.08 Gynostemma pentaphyllum extract 8.24±0.08 Chicory Extract 6.89±0.4

[0094] Example 6

[0095] Determination of the ratio of Chinese herbal medicine composition and its inhibitory effect on xanthine oxidase by uniform design method

[0096] 1.1 Solution preparation

[0097] (1) PBS buffer (pH 7.73): Weigh 2.27 g of potassium dihydrogen phosphate and dissolve it in ultrapure water to make a 1 / 15 mol / L potassium dihydrogen phosphate solution (Solution A). Then weigh 23.88 g of disodium hydrogen phosphate and dissolve it in ultrapure water to make a 1 / 15 mol / L disodium hydrogen phosphate solution (Solution B). Mix the two solutions in a ratio of A:B = 1:9 to obtain a PBS buffer solution with a pH of 7.73.

[0098] (2) Xanthine substrate solution (2.25 mmol / L, pH 7.5): Weigh 10.27 mg of xanthine, dissolve it with an appropriate amount of NaOH solution, then dilute it with 30 mL of PBS buffer, and finally adjust the pH to 7.5 with phosphoric acid to obtain 2.25 mmol / L pH 7.5 xanthine substrate solution.

[0099] (3) Xanthine oxidase solution: Weigh 1 mg of xanthine oxidase (50 U / mg) and add it to 10 mL of PBS to dissolve it to obtain a 5 U / mL XO stock solution, which is retained for later use. Take 1 mL of the stock solution and dilute it with 9 mL of PBS buffer to obtain a 0.5 U / mL xanthine oxidase solution.

[0100] 1.2. Sample dissolution

[0101] Weigh 20 mg of plant extract sample, add PBS buffer or DMSO solvent, dissolve by ultrasonication, prepare a 10 mg / mL sample solution, and dilute the sample solution into a series of concentrations to be measured using the multiple dilution method.

[0102] 1.3 Experimental methods

[0103] According to the inhibitory effects of rattan tea extract, clover extract, Gynostemma pentaphyllum extract and chicory extract on XO, the upper and lower limits of the concentration of each extract were set for the uniform design experiment. The upper limits of rattan tea extract, clover extract, Gynostemma pentaphyllum extract and chicory extract were 1 mg / mL, 2 mg / mL, 8 mg / mL and 8 mg / mL, respectively. All of them were diluted by 2 times to design a series of gradient concentrations.

[0104] According to the principle of uniform design with no less than 2 factors, 4 factors and 9 levels [U9*(9 4 )] The experiment was designed, and the inhibitory effect on XOD was used as the evaluation index. The DPS software was used to perform quadratic polynomial stepwise regression analysis on the experimental data to obtain the recommended optimal parameters. The in vitro xanthine oxidase inhibition activity experiment was verified, and the formula ratio of the extract composition was selected through comprehensive analysis.

[0105] 1.4 Experimental Results

[0106] The uniform design experimental data were analyzed by quadratic polynomial stepwise regression using DPS software, and the multiple regression equation of the four factors was obtained: Y=958.790946+8.955161553×X1+3.0540842414×X2+0.8860189979×X4-3.205010588×X1×X1+0.04510143768×X3×X3-15433.902997×X2×X3-0.24008244980×X2×X4 (P=0.0052, p<0.01, significant). The equation was extremely significant, indicating that the model had a good degree of fit. The optimal composition ratio predicted by the equation model is: 0.3733 mg / mL of rattan tea extract, 2 mg / mL of clover extract, 0.0313 mg / mL of gynostemma pentaphyllum extract, and 1.8595 mg / mL of chicory extract, and the inhibition rate of the combination reaches 91.6%. Converted into percentage, the weight ratio of the four extracts of rattan tea extract, clover extract, gynostemma pentaphyllum extract, and chicory extract is 12:64:1:60.

[0107] Table 2 The combined inhibitory effect of vine tea extract, clover extract, Gynostemma pentaphyllum extract and chicory extract on xanthine oxidase

[0108]

[0109] Note: X1 is rattan tea extract, X2 is clover extract, X3 is Gynostemma pentaphyllum extract, X4 is chicory extract

[0110] To further verify the optimal ratio of the composition obtained by the uniform design method: 0.3733 mg / mL of rattan tea extract, 2 mg / mL of clover extract, 0.0313 mg / mL of Gynostemma pentaphyllum extract, and 1.8595 mg / mL of chicory extract, an in vitro xanthine oxidase inhibition activity experiment was performed. The inhibition rate determined by the experimental results was 90.3%, which was basically consistent with the inhibition rate predicted by the equation model of 91.6%. The combination index (CI) between the four drugs in the composition was calculated using CompuSyn software to evaluate the synergistic effect of the drugs. The result was that the CI value of the combination index was 0.486. According to the CI judgment standard, it was less than 1, indicating that the four extracts of the composition had a synergistic effect.

[0111] Table 3 Uniform design experimental verification and joint index CI analysis

[0112]

[0113] Example 7

[0114] Experimental study on the evaluation of uric acid lowering effect in mice

[0115] 1.1 Experimental methods

[0116] 1.1.1 Experiments with mice

[0117] The three-leaf green composition was prepared according to the weight ratio of four extracts of vine tea extract, three-leaf green extract, Jiaogulan extract and chicory extract of 12:64:1:60. Fifty Kunming male mice were adaptively raised for 5 to 7 days, and the animals were free to eat and drink water. The bedding was changed every two days. Then the mice were randomly divided into 5 groups, namely: normal group, model group, positive control group, three-leaf green extract group and three-leaf green composition experimental group, with 10 mice in each group. Under the condition of ensuring normal diet and drinking water, all mice were fasted at 8 am every day, and gavage began after 1 hour of food deprivation. The mice in the normal group were gavaged with 0.5% CMC Na solution, and all the other groups except the normal group were gavaged with potassium oxonate suspension for modeling. The mice were gavaged for 21 consecutive days at a dose of 250 mg / kg·d to establish a mouse hyperuricemia animal model. One hour after the intragastric administration of potassium oxonate suspension, the mice in the positive control group were intragastrically administered with 5 mg / kg of allopurinol suspension solution, the mice in the experimental group were intragastrically administered with 250 mg / kg of Tripterygium wilfordii extract and combination solution, and the mice in the normal group and the model group were intragastrically administered with distilled water. After the intragastric administration of mice on the 20th day, they were fasted for more than 12 hours but not water, so that blood and organ samples could be extracted from the mice after the intragastric administration on the 21st day.

[0118] 1.1.2 Determination of mouse organ index

[0119] On the 21st day, after the mice were gavaged, their eyeballs were removed and blood was collected from the eye sockets. The blood samples were naturally coagulated for 1 hour at room temperature and then quickly centrifuged to separate the serum to avoid hemolysis. After the eyeballs were blooded, the mice were killed by breaking the cervical vertebrae. The dead mice were fixed on the dissection board, and their liver and kidney tissues were separated. The obtained mouse organ tissues were rinsed in 4°C pre-cooled saline to remove blood and adherent connective tissue, and then wiped dry with filter paper and weighed to calculate the organ index.

[0120] 1.1.3 Determination of serum uric acid (UA), urea nitrogen (BUN) and creatinine (Cr) levels in mice

[0121] After the collected mouse blood samples were naturally coagulated for 1 hour at room temperature, they were placed at 4°C and centrifuged at 3000r / min for 10 minutes, and the supernatant was taken as the experimental serum sample. After the samples were divided and stored at 20°C for use in the kit test, the uric acid (UA) test kit, urea nitrogen (BUN) test kit and creatinine (Cr) test kit were used to determine the contents in mouse serum.

[0122] 1.1.4 Determination of xanthine oxidase (XO) activity in mouse liver

[0123] Select fresh liver tissue samples from mice, accurately weigh 0.50g of tissue, add 4℃ pre-cooled saline at a mass ratio of 1:9, and perform mechanical homogenization under ice bath conditions. The homogenate is placed at 4℃ and centrifuged at 3500r / min for 10min. After gently aspirating the surface fat tissue, slowly aspirate the supernatant, which is the 10% liver tissue homogenate sample for the experiment. After aliquoting, store it at 20℃ for testing. The total protein content in the liver tissue homogenate was determined using a total protein (TP) test kit, and the xanthine oxidase (XO) test kit was used to determine the activity of XO in the mouse liver homogenate.

[0124] 1.2 Experimental Results

[0125] 1.2.1 Effects of each experimental group on the body weight, liver, kidney and spleen weights and organ indexes of hyperuricemia mice

[0126] After 21 days of oral gavage on mice, the difference analysis of organ weights and organ indexes of each group showed that there were no significant differences in body weight, liver, kidney and spleen weights and organ indexes of the mice among the groups (p>0.05), indicating that the sample solutions did not cause damage to the mouse organs.

[0127] 1.2.2 Effects of each experimental group on uric acid-lowering in hyperuricemia mice

[0128] Table 4 Effects of the three-leaf green composition on serum UA levels in mice with hyperuricemia (n=10)

[0129] Measurement group UA value (μmol / L) Normal group 85.16±5.58** Model Group 483.8±8.1 Positive control group 225.6±4.91* Clover extract group 198.9±10.89** Three leaf green combination group 134.3±13.35**

[0130] Compared with the model group, *P<0.05, **P<0.01.

[0131] 1.2.3 Effects of each experimental group on improving renal function in hyperuricemia mice

[0132] Table 5 Effects of the three-leaf green composition on serum BUN and Cr values ​​in mice with hyperuricemia (n=10)

[0133] Measurement group BUN value (mmol / L) Cr value (μmol / L) Normal group 6.82±1.03** 10.88±4.02** Model Group 16.24±1.44 28.47±1.84 Positive control group 8.02±0.92** 17.00±6.31* Clover extract group 12.08±0.86* 15.88±3.82** Three leaf green combination group 9.53±0.49** 9.61±3.97**

[0134] Compared with the model group, *P<0.05, **P<0.01.

[0135] 1.2.4 Inhibitory effects of each experimental group on xanthine oxidase in the liver of hyperuricemia mice

[0136] Table 6 Effects of the three-leaf green composition on liver XO in hyperuricemia mice (n=10)

[0137] Measurement group XO activity value (U / g prot) Normal group 0.58±0.04** Model Group 1.47±0.22 Positive control group 0.68±0.10** Clover extract group 1.19±0.07* Three leaf green combination group 0.76±0.09**

[0138] Compared with the model group, *P<0.05, **P<0.01.

[0139] From the above experiments, it can be concluded that the three-leaf green composite can reduce the serum uric acid (UA), urea nitrogen (BUN) and creatinine (Cr) levels in the hyperuricemia animal model and inhibit the activity of liver xanthine oxidase, indicating that it can also play a uric acid-lowering role in the body.

Claims

1. Use of the extract of Tripterygium wilfordii in the preparation of medicines, foods or health products for lowering uric acid.

2. The use according to claim 1, wherein The cloverleaf green extract is prepared by a method comprising the following steps: extracting cloverleaf green powder by using a solvent through an ultrasonic extraction method to obtain the cloverleaf green extract.

3. The use according to claim 2, wherein The solvent is an organic solvent; Preferably, the organic solvent comprises ethanol; Preferably, the solvent is an aqueous solution of ethanol with a mass percentage of 30-45%.

4. The use according to claim 2, wherein The cloverleaf green powder is obtained by drying and crushing the cloverleaf green.

5. The use according to claim 2, wherein The extraction is carried out under heating conditions; Preferably, the extraction is carried out at 40-55°C; Preferably, the ultrasonication is performed for 30-50 min.

6. The use according to claim 2, wherein: The method for preparing the clover extract also includes the following steps: filtering and removing residues from the mixed solution after ultrasonication, and vacuum concentrating and drying the filtered filtrate.

7. A clover extract for lowering uric acid, which is obtained by extracting clover powder using a solvent and an ultrasonic extraction method.

8. A Chinese medicine composition comprising a clover extract, a rattan tea extract, a gynostemma pentaphyllum extract and a chicory extract and optional pharmaceutically acceptable excipients.

9. The Chinese medicine composition according to claim 8, wherein The clover extract is prepared by a method comprising the following steps: extracting clover powder by ultrasonic extraction using a solvent to obtain the clover extract; Preferably, the solvent is an organic solvent; Preferably, the organic solvent comprises ethanol; Preferably, the solvent is an aqueous solution of ethanol with a mass percentage of 30-45%; Preferably, the cloverleaf green powder is obtained by drying and crushing cloverleaf green; Preferably, the extraction is carried out under heating conditions; Preferably, the extraction is carried out at 40-55°C; Preferably, the ultrasound is performed for 30-50 min; Preferably, the method for preparing the extract of Tripterygium wilfordii further comprises the following steps: filtering and removing residues from the mixed solution after ultrasonication, and vacuum concentrating and drying the filtered filtrate; Preferably, the weight ratio of the vine tea extract, the clover extract, the gynostemma pentaphyllum extract and the chicory extract is vine tea extract:clover extract:gynostemma pentaphyllum extract:chicory extract = (10-15): (50-70): (1-5): (50-60); More preferably, the weight ratio of the rattan tea extract, clover extract, gynostemma extract and chicory extract is rattan tea extract:clover extract:gynostemma extract:chicory extract=12:64:1:

60.

10. Use of the Chinese medicine composition according to claim 8 or 9 in the preparation of medicines, foods or health products for lowering uric acid.

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