New application and method of asiaticoside

Abstract

The invention provides novel application and a method of centella total glycosides. The extraction method of the centella total glycosides comprises the following steps: S1, extraction: taking a centella medicinal material, performing coarse crushing, adding ethanol, performing reflux extraction, performing filtration, and combining filtrate for standby application; s2, concentrating: concentrating the ethanol extracting solution under reduced pressure until no alcohol smell exists, centrifuging, and reserving supernate for later use; s3, carrying out D101 column chromatography; s4, alumina column chromatography; s5, performing concentration; s6, drying; the invention provides application of centella asiatica total glycosides to treatment of hyperuricemia, and particularly, the new application comprises reduction of uric acid (UA), urea (UREA), creatinine (CREZ), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglyceride (TRIG) and total cholesterol (CHOL) in serum of a hyperuricemia model SD rat, and reduction of damage of hyperuricemia to kidney. And the purity of the total asiaticoside extracted by the method is high.

Description

Technical Field

[0001] The present invention relates to a new application and method of asiaticoside, belonging to the technical field of biomedicine. Background Art

[0002] Centella asiatica (L.) Urban, also known as Iron Lamp, Qianchi Grass, Copper Coin Grass, Horse Hoof Grass, Thunder God Root, etc., is a perennial herb of the genus Centella in the family Apiaceae (Apiaceae). Centella asiatica has the effects of clearing away heat and dampness, promoting blood circulation and stopping bleeding, and detoxifying and detumescence. It is mainly used to treat fever, cough, sore throat, enteritis, dysentery, etc.; it is tender in texture and good in palatability, and can be eaten raw. In southern China, it is often used as a herbal tea by the people. In Southeast Asia, India, Pakistan, Sri Lanka and South America, it is used as a vegetable; the leaves of Centella asiatica are beautiful in shape, evergreen all year round, resistant to trampling, and have a good effect of preventing soil erosion. It is an excellent lawn plant; its extract has a significant promoting effect on the healing of wounds and ulcers, so it is also often used in the cosmetics industry.

[0003] Asiaticoside, a proprietary Chinese medicine name, is composed of Centella asiatica. It has the effect of promoting wound healing and is used to treat wounds with quick results.

[0004] It is of great significance to extract high-purity asiaticoside and further study its new applications. Summary of the Invention

[0005] The present invention provides a new application and method of asiaticoside. Asiaticoside can be used to treat hyperuricemia. Specifically, it can reduce uric acid (UA), urea (UREA), creatinine (CREZ), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglyceride (TRIG) and total cholesterol (CHOL) in the serum of SD rats with hyperuricemia model, and can also reduce the damage of hyperuricemia to the kidneys. Moreover, the asiaticoside extracted by the method of the present invention has high purity.

[0006] To achieve this purpose, the present invention provides the following technical solutions: In the first aspect of the present invention, there is provided the application of asiaticoside in the preparation of a drug for treating hyperuricemia.

[0007] Preferably, the hyperuricemia includes hyperuricemia induced by a high-purine diet.

[0008] Preferably, the treatment includes reducing uric acid, urea, creatinine, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglyceride and total cholesterol.

[0009] Preferably, the treatment includes reducing the damage of hyperuricemia to the kidneys.

[0010] The second aspect of the present invention provides a method for extracting total glycosides of Centella asiatica, comprising the following steps: S1. Extraction: Crush the Centella asiatica root material, add ethanol to reflux for extraction, filter, combine the filtrate and set aside; S2, concentration: the ethanol extract is concentrated under reduced pressure until there is no alcohol taste, centrifuged, and the supernatant is reserved; S3, D101 column chromatography; S4, alumina column chromatography; S5, concentration; S6. Drying.

[0011] Preferably, in step S3, V / M of medicinal material / D101 macroporous resin is 1 / (2.5-3).

[0012] Preferably, step S3 comprises: taking the supernatant of step S2 and loading it onto D101 macroporous resin for adsorption, after loading, firstly eluting it with 5-10BV of water until it is colorless, then eluting it with 6-10BV of ethanol, recovering the ethanol eluate under reduced pressure, and concentrating it into small bodies.

[0013] Preferably, in step S3, the concentration of ethanol used for elution is 65-75%.

[0014] Preferably, step S4 comprises: taking the above-mentioned concentrated solution and adding 95% ethanol to an alcohol concentration of more than 90%, stirring while adding, centrifuging, dissolving the precipitate with 50 times 95% ethanol, centrifuging, combining the two supernatants and loading them on a neutral alumina column, and after loading, eluting with 5BV ethanol and collecting.

[0015] Preferably, in step S4, the concentration of ethanol used for elution is 80-95%.

[0016] Preferably, a method for extracting total glycosides of Centella asiatica comprises the following steps: Step 1: Extraction: Crush the Centella asiatica root material, add 8 times 70% ethanol and reflux extract for 3 times, each time for 1 hour, filter, combine the filtrate and set aside; Step 2, concentration: the above ethanol extract is concentrated under reduced pressure until there is no alcohol taste, the vacuum degree is -0.06~-0.08mpa, the temperature is 60-70℃, the concentrate is 10 times the amount of the medicinal material, centrifuged at 3600r / min, and the supernatant is reserved; Step 3: D101 column chromatography: Load the above-mentioned supernatant onto D101 macroporous resin (medicinal material / resin (V / M, 1 / 2.5)) for adsorption, control the flow rate at 1.0 BV / h. After the loading is completed, first elute with 5 BV of water until colorless, control the flow rate at 1.5 BV / h, and then elute with 6 BV of 65% ethanol, control the flow rate at 1.0% BV / min; Recover the 65% ethanol eluate under reduced pressure and concentrate it to a small volume; Step 4: Alumina column chromatography: Add 95% ethanol to the above-mentioned concentrated solution until the ethanol concentration is above 90%, stir while adding, centrifuge, dissolve the precipitate in 50 times of 95% ethanol again, centrifuge, combine the supernatants of the two times and load them onto a neutral alumina column (medicinal material / alumina, 1.3 / 1). After the loading is completed, elute and collect with 5 BV of 95% ethanol and 6 BV of 80% ethanol in sequence; Step 5: Concentration: Monitor the content of the target substance in the above-mentioned eluate and concentrate to recover ethanol; Concentrate the 80% ethanol eluate to a specific gravity of 1.10 ± 0.02 (60 - 70 °C), the vacuum degree is -0.06 to -0.08 mpa, and the temperature is 60 - 70 °C; Step 6: Drying: Take the above-mentioned concentrated solution and dry it at a vacuum degree of -0.06 to -0.08 mpa and a temperature of 60 - 70 °C for 4 h.

[0017] In the present invention, the extracted asiaticoside can be used to treat hyperuricemia. Specifically, it can reduce uric acid (UA), urea (UREA), creatinine (CREZ), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglyceride (TRIG) and total cholesterol (CHOL) in the serum of SD rats with hyperuricemia model, and can also reduce the damage of hyperuricemia to the kidneys.

[0018] The second aspect of the present invention provides an asiaticoside, which is prepared by the method described in the present invention.

[0019] Compared with the prior art, the beneficial effects and significant progress of applying the technical solution of the present invention are as follows: The present invention provides a new application and method of asiaticoside. Asiaticoside can be used to treat hyperuricemia. Specifically, it can reduce uric acid (UA), urea (UREA), creatinine (CREZ), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglyceride (TRIG) and total cholesterol (CHOL) in the serum of SD rats with hyperuricemia model, and can also reduce the damage of hyperuricemia to the kidneys. Moreover, the asiaticoside extracted by the method of the present invention has high purity. Brief Description of the Drawings

[0020] To more clearly illustrate the technical solution of the present invention, the drawings used in the embodiments of the present invention will be briefly introduced below.

[0021] Figure 1 is the chromatogram of total asiaticosides in Example 1; Figure 2 is the change diagram of the content of uric acid (UA) in the serum of SD rats during the treatment in Example 5; Figure 3 is the change diagram of the content of urea (UREA) in the serum of SD rats during the treatment in Example 5; Figure 4 is the change diagram of the content of creatinine (CREZ) in the serum of SD rats during the treatment in Example 5; Figure 5 is the change diagram of the content of low density lipoprotein cholesterol (LDL-C) in the serum of SD rats after the treatment in Example 5; Figure 6 is the change diagram of the content of high density lipoprotein cholesterol (HDL-C) in the serum of SD rats after the treatment in Example 5; Figure 7 is the change diagram of the content of triglyceride (TRIG) in the serum of SD rats after the treatment in Example 5; Figure 8 is the change diagram of the content of total cholesterol (CHOL) in the serum of SD rats after the treatment in Example 5; Figure 9 is the HE staining diagram of the kidney tissue of SD rats after the treatment in Example 6; Figure 10 is the change diagram of the pathological tubulointerstitial injury score of the kidney tissue of SD rats after the treatment in Example 6; Figure 11 is the change diagram of the pathological renal inflammatory cell infiltration score of the kidney tissue of SD rats after the treatment in Example 6; Figure 12 is the Gomori hexamine silver staining diagram of the kidney tissue of SD rats after the treatment in Example 6; Figure 13 is the change diagram of the pathological urate crystal score of the kidney tissue of SD rats after the treatment in Example 6; Figure 14 is the change diagram of the kidney coefficient of SD rats after the treatment in Example 7; Figure 15 is the change diagram of the adrenal coefficient of SD rats after the treatment in Example 7. Detailed implementation manners

[0022] To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0023] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which the application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present application.

[0024] Example 1 Preparation of Total Glucosides of Centella asiatica In this example, total glycosides of Centella asiatica were prepared according to the following method.

[0025] 1.1. Extraction: Crush the Centella asiatica root, add 8 times 70% ethanol and reflux extract for 3 times, 1 hour each time, filter, combine the filtrate and set aside; 1.2 Concentration: The above ethanol extract is concentrated under reduced pressure until there is no alcohol taste, the vacuum degree is -0.06~-0.08mpa, the temperature is 60-70℃, the concentrate is 10 times the amount of the medicinal material, centrifuged at 3600r / min, and the supernatant is reserved; 1.3, D101 column chromatography: take the above supernatant and load it on D101 macroporous resin (medicinal material / resin (V / M, 1 / 2.5)) for adsorption, the flow rate is controlled at 1.0 BV / h, after loading, first elute with 5BV of water until colorless, the flow rate is controlled at 1.5 BV / h, and then elute with 6BV of 65% ethanol, the flow rate is controlled at 1.0%BV / min; the 65% ethanol eluate is recovered under reduced pressure and concentrated to a small volume; 1.4. Alumina column chromatography: Take the above concentrated solution and add 95% ethanol to an alcohol concentration of more than 90%, stir while adding, centrifuge, and continue to dissolve the precipitate with 50 times 95% ethanol, centrifuge, combine the two supernatants and load them on a neutral alumina column (medicinal material / alumina, 1.3 / 1). After loading, elute with 5BV95% ethanol and 6BV80% ethanol in turn and collect; 1.5. Concentration: The above eluent is used to monitor the target content and to recover ethanol through concentration; the 80% ethanol eluent is concentrated to a specific gravity of 1.10±0.02 (60~70℃), a vacuum degree of -0.06~-0.08mpa, and a temperature of 60-70℃; 1.6. Drying: Take the above concentrated solution and dry it at a vacuum degree of -0.06~-0.08mpa and a temperature of 60-70℃ for 4h.

[0026] The total asiaticosides obtained were subjected to liquid chromatography. The liquid chromatography conditions were as follows: chromatographic column: ZORBAX SB-C18 (4.6×250mm 5-Micron); flow rate: 1ml / min; column temperature: 30°C; mobile phase: A: acetonitrile, B: 2mmol / L β-cyclodextrin; injection volume: 10μl; wavelength: 205nm; mobile phase gradient is shown in Table 1 below.

[0027] Table 1 The chromatogram of the total asiaticosides obtained is as Figure 1 shown.

[0028] Example 2 Resin Screening Experiment Through single-factor process and orthogonal experiments, the extraction process of total asiaticosides was determined to be reflux extraction with 70% ethanol for 1h, and extracted 3 times, with a high extraction yield; then through the study of various resin process parameters, the purification effect of resin D101 was good (as shown in Table 2 below), and 6BV of 65% ethanol was used for elution, and the content of the total glycoside extract reached 29%.

[0029] Table 2 Example 3 Alumina Purification Experiment Take the neutral alumina of the resin-purified sample, elute it successively with 95% ethanol and 80% ethanol, collect the eluate, detect the elution situation by liquid phase, concentrate and dry it, and detect the content of glycoside components. The influence of the alumina purification process on the yield of asiaticoside components is shown in Table 3 below.

[0030] Table 3 In summary, after the total asiaticosides were purified by D101 macroporous resin, the content was about 29%, and then columned on neutral alumina for purification to a content of about 50%.

[0031] Example 4 4.1. The information of the experimental mice is shown in Table 4 below.

[0032] Table 4 This example strictly complies with all applicable guidelines for the care, management, and use of laboratory animals. It refers to The Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Academy Press, Washington, D.C., 2011. To ensure compliance with the principles related to animal welfare, this experimental protocol was submitted to the Institutional Animal Care and Use Committee (IACUC) for review before the laboratory animals were received or transferred to this research institution.

[0033] Before the establishment of the model, animals were screened based on comprehensive indicators such as body weight and clinical observation. The random grouping method was used, and the animals were grouped according to their body weight. After grouping, the body weight of the animals should not exceed ±20% of the average body weight of the corresponding group. The average body weight of each group of animals should have no statistical difference at the 5.0% significance level.

[0034] 4.2, Hyperuricemia Model Mice Male Sprague-Dawley (SD) rats were adaptively raised for 6 days. The SD rats were randomly grouped into a normal group and a model group according to their body weight. The SD rats in the normal group were not given any intervention. The SD rats in the 5 model groups were gavaged with adenine 50 mg / kg + potassium oxonate 1.5 g / kg once a day according to the reference method in the literature. After 1 week of gavage with the modeling drug, the contents of uric acid (UA), urea (UREA), and creatinine (CREZ) in the serum of the SD rats were detected by biochemical methods. According to the UA value, the SD rats with successful modeling were screened and randomly grouped for drug administration. During the drug administration treatment, the modeling with adenine 100 mg / kg + potassium oxonate 1.5 g / kg was continued, once every 2 days, and continuous gavage was performed for 30 days. Except for the blank control group, the experimental animals after modeling were screened according to the modeling indicators, and the experimental animals that met the model criteria were selected to participate in the subsequent experiments. 4.3, Grouping and Drug Administration The animals in the hyperuricemia model group were randomly divided into 12 groups, including a blank control group, with a total of 13 groups. There were 11 animals in the blank control group and 10 animals in the hyperuricemia model group. The first group, the blank control group, was given an equal amount of distilled water once a day for 30 consecutive days; the second group, the model + vehicle group, was given an equal amount of vehicle once a day for 30 consecutive days; the third group, the model + allopurinol (10 mg / kg) group, was given 10 mg / kg of allopurinol once a day for 30 consecutive days; the fourth group, the model + EZY-E (8 mg / kg) group, was given 8 mg / kg of EZY-E once a day for 30 consecutive days; the fifth group, the model + EZY-E (16 mg / kg) group, was given 16 mg / kg of EZY-E once a day for 30 consecutive days. The specific drug administration information is shown in Table 5 below.

[0035] Table 5 4.4 In-vivo detection indicators and detection (1) Body weight measurement: The body weight of the rats was measured once a week.

[0036] (2) General observation: The appearance signs, behavioral activities, fecal characteristics, etc. of the rats were observed once a day.

[0037] According to the change graph of the body weight data of SD rats, it can be seen that the body weight of the rats in each group increased evenly during the whole experiment (the overall body weight of the rats decreased at the end of the experiment due to fasting the previous night), and no abnormalities were found. No abnormal clinical manifestations were found in each drug administration group during the treatment process.

[0038] Example 5 Biochemical detection Once a week, the content changes of uric acid (UA), urea (UREA), creatinine (CREZ), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in the serum of the rats in Example 2 were detected biochemically. At the end of the treatment, the serum of the rats was taken to detect the content changes of uric acid (UA), urea (UREA), serum creatinine (CREZ), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglyceride (TRIG), and total cholesterol (CHOL) in the serum.

[0039] Specific operation steps: ⑴ Loading reagent blank calibrator: ① Preparing blank calibrator: Prepare the blank calibrator according to the requirements of the analysis method (generally use deionized water or normal saline), and place it in the sample cup. ② Loading: Load the blank calibrator at the specified position on the blue sample rack. The reagent blank of the serum sample is placed in the first position of the blue rack. When calibrating with the calibrator, the blank sample calibration measurement must be performed simultaneously. ⑵ Loading calibrator: ① Calibrator preparation: Prepare the calibrator according to the operating procedures of the analysis item. Generally, use the supporting calibration serum, and reconstitute, store and use it strictly according to the requirements of the calibration serum instruction manual. ② Loading: Place the calibrator of this item at the corresponding position on the yellow calibration rack according to the position of the calibrator set in the "calibration parameters". You can click "Show sample cup settings" to view the position of the calibrator when editing the calibration worksheet. ⑶ Loading the calibration sample rack: ① Loading the blank calibration sample rack (blue): Place the blue sample rack at the first position on the left side of the sample transfer track, with the barcode sticker facing forward (left). ② Loading the calibrator rack (yellow): The yellow calibrator rack is placed closely following the blue rack, with the barcode sticker facing forward (left). ⑷ Compiling the worksheet for the routine specimen measurement procedure Click "Reset" - Select "Sample rack application" - Select "Test application" in "Samples": ① Single sample programming: Click "Reset" - Select "Sample rack application" - Select "Test application" in "Samples" - Confirm the sample number to be edited currently in "Sample number" - After confirmation, click "Start login" and then select the item - Click "Login" to confirm - If you want to edit the next sample, continue to select the item and then click "Login" - After completing the item programming, click "Exit" - Place the corresponding samples on the white rack in the sample injection area in sequence - Click "Start" to start running. ② Batch sample programming: Click "Reset" - Select "Sample rack application" - Select "Test application" in "Samples" - Confirm the sample number to be edited currently in "Sample number" - After confirmation, click "Start login" and then select the item - Click "Batch input" - Select the number of samples to be programmed batchwise in "Number of samples" - Click "OK" and then confirm again at "Sample number" - After confirmation, click "Exit" - Place the corresponding samples on the white rack in the sample injection area in sequence - Click "Start" to start running. ③ In the barcode mode, there is no need for the above complex programming. Just place the sample with the barcode directly on the sample rack. However, the barcode mode must develop a "Laboratory Information System" (LIS system) that matches the Beckman Coulter AU series. Different laboratories can set the corresponding operation SOP according to the specific situation of the laboratory.

[0040] Experimental results The results of the contents of uric acid (UA), urea (UREA), and creatinine (CREZ) in the rat serum are as Figures 2 - 4 shown.

[0041] After 1 week of intragastric administration of the modeling drug, the content of uric acid (UA) in the serum of SD rats in the model group was significantly higher than that in the blank control group (p<0.01), indicating that the model of hyperuricemia in SD rats was successfully established.

[0042] After the successful establishment of the model, each treatment group was given the corresponding dose of the test drug for treatment. The contents of uric acid (UA), urea (UREA), and creatinine (CREZ) in the serum of SD rats on Day7, Day14, Day21, and Day30 were detected by biochemical methods. The test results showed that: As Figure 2 shown, compared with the blank control group, the content of uric acid (UA) in the serum of SD rats in the solvent group was significantly increased (P<0.01); compared with the solvent group, the contents of uric acid (UA) in the serum of SD rats in the drug groups (allopurinol and total triterpenoids of Centella asiatica) were significantly decreased after treatment (P<0.01).

[0043] As Figure 3 shown, compared with the normal group, the content of urea (UREA) in the serum of SD rats in the solvent group was significantly increased (P<0.01); compared with the solvent group, the contents of urea (UREA) in the serum of SD rats in the drug groups (allopurinol and total triterpenoids of Centella asiatica) were significantly decreased after treatment (P<0.01).

[0044] As Figure 4 shown, compared with the normal group, the content of creatinine (CREZ) in the serum of SD rats in the solvent group was significantly increased (P<0.01); compared with the solvent group, the contents of creatinine (CREZ) in the serum of SD rats in the drug groups (allopurinol and total triterpenoids of Centella asiatica) were significantly decreased after treatment (P<0.01).

[0045] The results of the contents of low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglyceride (TRIG), and total cholesterol (CHOL) in the serum of rats were as Figures 5 - 8 shown.

[0046] The contents of low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglyceride (TRIG), and total cholesterol (CHOL) in the serum of SD rats were detected by biochemical methods. The test results showed that: As Figure 5 shown, compared with the blank control group, the content of low-density lipoprotein cholesterol (LDL-C) in the serum of SD rats in the solvent group was significantly increased (P<0.01); compared with the solvent group, the contents of low-density lipoprotein cholesterol (LDL-C) in the serum of SD rats in the drug groups (allopurinol and total triterpenoids of Centella asiatica) were significantly decreased after treatment (P<0.01).

[0047] As Figure 6As shown, compared with the blank control group, the content of high-density lipoprotein cholesterol (HDL-C) in the serum of SD rats in the solvent group was significantly increased (P<0.01); compared with the solvent group, EZY-E (8 mg / kg) could not significantly reduce the content of high-density lipoprotein cholesterol (HDL-C) in the serum of SD rats.

[0048] As Figure 7 shown, compared with the blank control group, the content of triglyceride (TRIG) in the serum of SD rats in the solvent group was increased; compared with the solvent group, the content of triglyceride (TRIG) in the serum of SD rats could be reduced after treatment in the allopurinol (10 mg / kg) group and the EZY-E (8 mg / kg and 16 mg / kg) groups.

[0049] As Figure 8 shown, compared with the blank control group, the content of total cholesterol (CHOL) in the serum of SD rats in the solvent group was significantly increased (P<0.01); compared with the solvent group, the content of total cholesterol (CHOL) in the serum of SD rats could be significantly reduced after treatment in the drug groups (allopurinol and total saponins of Centella asiatica) (P<0.05).

[0050] Example 6 In vitro Detection Indexes and Detection Methods At the end of the experiment, observe the pathological changes of the heart, liver, spleen, lung, thymus, kidney, adrenal gland, pancreas, stomach, duodenum, jejunum, ileum, rectum, colon, cecum, etc., and weigh the heart, liver, spleen, lung, thymus, and kidney; Take the left and right kidney tissues of rats for pathological examination (HE staining and Gomori hexamine silver staining).

[0051] HE staining detection method: Place the sample in 10% neutral formalin solution at room temperature for 4 h, take out the left and right kidney tissues of the rat and rinse with running water for several hours, dehydrate with 70%, 80%, and 90% ethanol solutions, 15 min in a mixture of absolute alcohol and xylene in equal amounts, 15 min in xylene I and 15 min in xylene II (until transparent). Place in a mixture of xylene and paraffin for 15 min, and then place in paraffin I and paraffin II for infiltration for 50 - 60 min each. Embed in paraffin and section. Bake the paraffin sections, then dewax and hydrate. Place the sections that have been put into distilled water into hematoxylin aqueous solution for staining for 3 min, differentiate with hydrochloric acid ethanol differentiation solution for 15 s, wash slightly with water, blue with blueing solution for 15 s, rinse with running water, stain with eosin for 3 min, rinse with running water, dehydrate, clear, mount, and examine under the microscope.

[0052] Gomori silver hexamine staining detection method: 1. Tissue fixation: Fix in absolute ethanol for 16 h or overnight. Then soak in absolute ethanol 3 times, 30 min each time. 2. Soak in xylene 2 times, 20 min each time, and embed in paraffin by conventional wax infiltration. 3. Cut sections with a thickness of 5 μm, and dewax the sections with xylene to absolute ethanol. 4. Prepare Gomori silver hexamine solution in advance and use it up within 2 h. Drop Gomori silver hexamine solution on the sections and incubate in a constant temperature incubator at 58 - 60 °C in the dark for 30 min (ensure sufficient staining solution to avoid drying out due to evaporation). If urate is present, the sections will be black. Wash slightly with distilled water. 5. Drop gold chloride solution and treat for 1 min. Wash slightly with tap water. 6. Drop hypo solution and treat for 5 min. Rinse with tap water for 5 min. 7. Drop eosin staining solution and lightly stain for 30 s. Wash slightly with tap water. 8. Dehydrate and clear by conventional methods, and seal with neutral balsam.

[0053] Staining results: Urate crystals: black. Background: light red.

[0054] Negative control: After dewaxing the consecutive sections, first incubate with Gomori control solution for 5 min, wash with absolute ethanol 2 times, and then put into Gomori silver hexamine solution. The remaining steps are the same as above, and calcium salts are negative.

[0055] Data statistical analysis: Experimental data are all expressed as mean ± standard deviation (Mean ± SE), and Graphpad Prism or SPSS Statistics is used.

[0056] Experimental results The HE pathological staining results are as Figures 9 - 11 shown.

[0057] As Figure 9 shown, renal injury was observed in all animals in the model solvent group, manifested as a large number of renal tubular degeneration, necrosis, urate deposition in the renal tubules, renal tubular dilation, inflammatory cell infiltration in some renal tubules, and cellular casts in a small number of renal tubules. After treatment with each drug group (allopurinol and total triterpenoids of Centella asiatica), a certain degree of repair of renal injury was observed, and the degree of injury was lighter than that in the model solvent group, manifested as partial renal tubular degeneration, obvious improvement in renal tubular dilation and urate deposition, and only a small amount of urate deposition in a small number of renal tubules.

[0058] Compared with the blank control group, the scores of renal tubular interstitial injury ( Figure 10 ) and the scores of renal inflammatory cell infiltration ( Figure 11) Significantly increased (P<0.01); compared with the solvent group, both the drug groups (allopurinol and total saponins of Centella asiatica) could significantly reduce the scores of renal tubulointerstitial injury and the scores of infiltration of renal inflammatory cells in the renal tissues of SD rats after treatment (P<0.01).

[0059] The results of Gomori hexamine silver staining were as Figures 12 - 13 shown.

[0060] As Figure 12 shown, urate deposition in renal tubules was observed in all animals of the model control group. After treatment with the drug groups (allopurinol and total saponins of Centella asiatica), the urate deposition was significantly improved, and only a small amount of urate deposition was seen in a few renal tubules.

[0061] As Figure 13 shown, compared with the blank control group, the urate crystal score in the renal tissues of SD rats in the solvent group was significantly increased (P<0.01); compared with the solvent group, both the drug groups (allopurinol and total saponins of Centella asiatica) could significantly reduce the urate crystal score in the renal tissues of SD rats after treatment (P<0.01).

[0062] Example 7 In the efficacy test, the kidney and adrenal gland indices of the test animals were analyzed respectively.

[0063] The results were as Figure 14 and 15 shown. After modeling, the kidney and adrenal gland indices of the solvent group were significantly increased compared with the blank control group (P<0.01); compared with the solvent group, each treatment group could significantly reduce the kidney and adrenal gland indices (P<0.01), indicating that each treatment drug could better inhibit and repair kidney damage.

[0064] The applicant declares that in the description process of the above specification: Descriptions of terms such as "this example", "the embodiments of the present invention", "as shown in...", "further", "further improved technical sub-schemes", etc. mean that the specific features, structures, materials or characteristics described in this example or example are included in at least one embodiment or example of the present invention; in this specification, the schematic expressions of the above terms are not necessarily directed to the same embodiment or example, and moreover, the specific features, structures, materials or characteristics described can be combined or combined in a suitable manner in any one or more embodiments or examples; in addition, on the premise of no contradiction, those of ordinary skill in the art can combine or combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0065] Finally, it should be noted that: The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features. These modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. Non-essential improvements, adjustments or replacements made by those skilled in the art according to the content of this specification all fall within the scope claimed by the present invention.

Claims

1. Use of asiaticosides in the preparation of a medicament for treating hyperuricemia.

2. The application according to claim 1, characterized in that, The hyperuricemia includes hyperuricemia induced by a high-purine diet.

3. The application according to claim 1, characterized in that, The treatment includes reducing uric acid, urea, creatinine, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglyceride or total cholesterol.

4. The application according to claim 1, characterized in that, The treatment includes reducing the damage of hyperuricemia to the kidneys.

5. A method for extracting asiaticoside total glycosides, characterized in that, It includes the following steps: S1. Extraction: Coarsely crush the centella asiatica medicinal material, reflux and extract with ethanol, filter, combine the filtrates, and set aside. S2. Concentration: Concentrate the ethanol extract under reduced pressure until there is no alcohol smell, centrifuge, and set aside the supernatant. S3. D101 column chromatography; S4. Alumina column chromatography; S5. Concentration; S6. Drying.

6. The extraction method of centella asiatica total glycosides according to claim 5, characterized in that, In step S3, the V / M of the medicinal material to D101 macroporous resin is 1 / (2.5 - 3).

7. The extraction method of asiaticoside total glycosides according to claim 5 or 6, characterized in that, Step S3 includes: Loading the supernatant of step S2 onto D101 macroporous resin for adsorption. After the column loading is completed, first wash with 5 - 10 BV of water until colorless, then elute with 6 - 10 BV of ethanol. The ethanol eluate is recovered under reduced pressure and concentrated to a small volume.

8. The extraction method of asiaticoside total glycosides according to claim 7, characterized in that, In step S3, the concentration of ethanol for elution is 65 - 75%.

9. The extraction method of centella asiatica total glycosides according to claim 5, characterized in that, Step S4 includes: Adding ethanol to the above concentrated solution to make the ethanol concentration above 90%, stirring while adding, centrifuging. The precipitate is continuously dissolved with 50 times the amount of ethanol, centrifuged, and the supernatants from the two times are combined and loaded onto a neutral alumina column. After the column loading is completed, elute with 5 BV of ethanol and collect.

10. The extraction method of centella asiatica total glycosides according to claim 9, characterized in that, In step S4, the concentration of ethanol for elution is 80 - 95%.

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