Novel application and method of asiatic acid

Abstract

The invention provides a novel application and method of asiatic acid. The method comprises the following steps: S1, extracting; s2, performing concentration; s3, carrying out D101 column chromatography; s4, alumina column chromatography; s5, performing concentration; s6, drying; s7, dissolving with methanol, adding water, standing, and monitoring clear liquid and crystallization conditions in a liquid phase; s8, filtering after crystallization is finished, washing with ethanol, drying crystals under the conditions that the vacuum degree is-0.06 to-0.08 mpa and the temperature is 60-70 DEG C, and crushing and sieving dry paste to obtain asiaticoside; s9, taking asiaticoside, and hydrolyzing in a sodium hydroxide ethanol water solution; and S10, after complete hydrolysis, adjusting to be neutral by using acetic acid, concentrating until no alcohol smell exists, continuously adding acetic acid to adjust the pH value to be 3-4, standing, filtering, collecting a solid, and drying to obtain asiatic acid. 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) and total cholesterol (CHOL) in serum of a hyperuricemia model SD rat, and reduction of damage of hyperuricemia to kidney.

Description

Technical Field

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

[0002] Centella asiatica (L.) Urban, also known as iron lantern, money-tooth grass, copper coin grass, horse hoof grass, and Gotu Kola, is a perennial herbaceous plant in the genus Centella, family Apiaceae. It has the properties of clearing heat and dampness, promoting blood circulation and stopping bleeding, and detoxifying and reducing swelling. It is used to treat fever, cough, asthma, sore throat, enteritis, and dysentery. Its tender texture and palatability make it edible raw. It is commonly used as an herbal tea in southern China and as a vegetable in Southeast Asia, India, Pakistan, Sri Lanka, and South America. Its beautiful, evergreen leaves are resistant to trampling and are effective in preventing soil erosion, making it an excellent lawn plant. Its extract significantly promotes the healing of wounds and ulcers, making it a popular choice in the cosmetics industry.

[0003] Asiatic acid, also known as Asian asiatic acid or Asian asiatic acid, is a needle-shaped crystal with the molecular formula C30H48O5. It is derived from the resin and volatile oil of the plant Dipterocarpa arborescens, also known as Centella asiatica. It can also be obtained by hydrolysis of Centella asiatica, also known as Centella asiatica. It is a component of Centella asiatica preparations. These preparations promote wound healing, stimulate granulation growth, promote epidermal keratinization, and aid in the production of new connective tissue. It is used to treat various skin lesions (including leprosy and tuberculosis) and burns.

[0004] It is of great significance to extract asiatic acid and further study its new applications. Summary of the Invention

[0005] The present invention provides a new application and method of Centella asiatica acid. Centella asiatica 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) and total cholesterol (CHOL) in the serum of hyperuricemia model SD rats, and can also reduce the damage of hyperuricemia to the kidneys.

[0006] To achieve this object, the present invention provides the following technical solutions: The first aspect of the present invention provides the use of asiatic acid in the preparation of a medicament for treating hyperuricemia.

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

[0008] Preferably, the treatment comprises lowering uric acid, urea, creatinine, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol or total cholesterol; further preferably, the treatment comprises lowering low-density lipoprotein cholesterol, high-density lipoprotein cholesterol or total cholesterol.

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

[0010] Preferably, the effective dose of asiatic acid is 5-20 mg / kg; further preferably, the effective dose of asiatic acid is 16 mg / kg.

[0011] The second aspect of the present invention provides a method for extracting asiatic acid, comprising the following steps: S1. Extraction: Crush the Centella asiatica root, extract with ethanol under reflux, filter, and combine the filtrates for later use; S2. Concentration: The ethanol extract is concentrated under reduced pressure until there is no alcohol smell, centrifuged, and the supernatant is reserved; S3, D101 column chromatography; S4, alumina column chromatography; S5, concentration; S6, drying; S7. Dissolve with methanol, add water and let stand, and monitor the clear liquid and crystallization of the liquid phase; S8. After the crystallization is completed, the crystals are filtered and washed with ethanol. The crystals are dried at a vacuum degree of -0.06 to -0.08 MPa and a temperature of 60-70° C. The dry paste is crushed and sieved to obtain asiaticoside; S9, taking Centella asiatica glycoside, hydrolyzing it in a sodium hydroxide ethanol aqueous solution, and monitoring the hydrolysis status by liquid phase; S10. After the hydrolysis is complete, adjust the solution to neutrality with acetic acid, concentrate until there is no alcohol taste, continue to add acetic acid to adjust the pH to 3-4, let it stand, filter, collect the solid and dry it to obtain Centella asiatica.

[0012] Preferably, in step S3, the V / M ratio of Centella asiatica / D101 macroporous resin is 1 / (2.5-3).

[0013] Preferably, step S4 comprises: taking the above-mentioned concentrated solution and adding ethanol to an alcohol concentration of more than 90%, stirring while adding, centrifuging, dissolving the precipitate with 50 times ethanol, centrifuging, combining the two supernatants and loading them onto a neutral alumina column, and after loading, eluting with 5BV ethanol and collecting.

[0014] Preferably, step S10 further comprises: if the precipitate is dark in color after filtration, it can be dissolved in 95% ethanol and then decolorized with activated carbon.

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

[0016] Preferably, step S3 comprises: taking the supernatant of step S2 and loading it onto D101 macroporous resin for adsorption. After loading, the supernatant is first eluted with 5-10 BV of water until colorless, and then eluted with 6-10 BV of ethanol. The ethanol eluate is recovered under reduced pressure and concentrated to a small volume.

[0017] Preferably, in step S3, the concentration of ethanol used for elution is 65-75%.

[0018] Preferably, a method for extracting asiatic acid comprises the following steps: Step 1: Extraction: Crush the Centella asiatica root, add 8 times 70% ethanol and reflux extract 3 times, each time for 1 hour, filter, and combine the filtrates for later use; Step 2: Concentrate the ethanol extract under reduced pressure until there is no alcohol smell. The vacuum degree is -0.06~-0.08 MPa and the temperature is 60-70°C. The concentrate is 10 times the amount of the medicinal material. Centrifuge at 3600 r / min and the supernatant is reserved. Step 3, D101 column chromatography: The supernatant was loaded onto D101 macroporous resin (medicinal material / resin (V / M, 1 / 2.5)) for adsorption at a flow rate of 1.0 BV / h. After loading, the supernatant was first eluted with 5 BV of water until colorless at a flow rate of 1.5 BV / h, and then eluted with 6 BV of 65% ethanol at a flow rate of 1.0% BV / min. The 65% ethanol eluate was recovered under reduced pressure and concentrated to a small volume. Step 4, alumina column chromatography: take the above concentrate and add 95% ethanol to an alcohol concentration of more than 90%, stir while adding, centrifuge, dissolve the precipitate in 50 times 95% ethanol, centrifuge, combine the two supernatants and load them on a neutral alumina column (medicinal material / alumina, 1.3 / 1). After the loading is completed, elute with 5BV 95% ethanol and 6BV 80% ethanol in sequence and collect; Step 5: Concentration: Monitor the target compound content in the eluate and concentrate to recover ethanol; the 80% ethanol eluate is concentrated to a specific gravity of 1.10 ± 0.02 (60-70°C), with a vacuum degree of -0.06-0.08 MPa and a temperature of 60-70°C; Step 6: Drying: The concentrated solution was dried at a vacuum degree of -0.06 to -0.08 MPa and a temperature of 60-70°C for 4 hours to obtain total glycosides of Centella asiatica. Step 7: Take the total glycosides of Centella asiatica, dissolve them in 5 times methanol, add water to a methanol volume fraction of 35%, stir while adding, let it stand for more than 24 hours, and monitor the clear liquid and crystallization of the liquid phase; Step 8: After the crystallization is completed, the mixture is filtered and washed with a small amount of 95% ethanol. The crystals are dried at a vacuum degree of -0.06 to -0.08 MPa and a temperature of 60-70°C for 4 hours. The dry paste is crushed and passed through a 100-mesh sieve to obtain asiaticoside; Step 9: Take a sample of Centella asiatica and hydrolyze it in 50 times 1 mol / L sodium hydroxide ethanol (70%) aqueous solution at 60°C. The hydrolysis is monitored by liquid phase. The liquid phase detection results show that the hydrolysis is complete after 2.0 hours. Step 10: After the reaction is completed, adjust to neutrality with acetic acid, concentrate until there is no alcohol smell, continue to add acetic acid to adjust the pH to 4, let it stand for 3 hours, filter, if the precipitate is dark in color, dissolve it in 95% ethanol and then decolorize it with activated carbon, collect the solid and dry it to obtain the Centella asiatica sample.

[0019] Compared with the existing technology, the beneficial effects and significant improvements of the technical solution of the present invention are: the Centella asiatica provided by the present invention 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), and total cholesterol (CHOL) in the serum of hyperuricemia SD rats, and can also reduce the damage to the kidneys caused by hyperuricemia. Furthermore, the effect of Centella asiatica acid in treating hyperuricemia is superior to that of total Centella asiatica glycosides. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solution of the present invention, the following briefly introduces the drawings used in the embodiments of the present invention.

[0021] Figure 1 is the asiatic acid chromatogram of Example 1; Figure 2 is a graph showing changes in serum uric acid (UA) levels in SD rats during the treatment process of Example 3; Figure 3 is a graph showing changes in urea (UREA) content in the serum of SD rats during the treatment process of Example 3; Figure 4 is a graph showing changes in serum creatinine (CREZ) levels in SD rats during the treatment process of Example 3; Figure 5 is a graph showing changes in serum low-density lipoprotein cholesterol (LDL-C) levels in SD rats after treatment in Example 3; Figure 6 is a graph showing changes in high-density lipoprotein cholesterol (HDL-C) levels in the serum of SD rats after treatment in Example 3; Figure 7 is a graph showing changes in total cholesterol (CHOL) content in serum of SD rats after treatment in Example 3; Figure 8 This is a HE staining image of SD rat kidney tissue after treatment in Example 4; Figure 9 This is a graph showing changes in renal tubulointerstitial injury scores in SD rat kidney tissue pathology after treatment in Example 4; Figure 10 This is a graph showing changes in renal inflammatory cell infiltration scores in renal tissue pathology of SD rats after treatment in Example 4; Figure 11 is a Gomori hexamine silver staining image of SD rat kidney tissue after treatment in Example 4; Figure 12 This is a graph showing changes in urate crystal scores in the kidney tissue pathology of SD rats after treatment in Example 4; Figure 13 This is a graph showing changes in the kidney coefficient of SD rats after treatment in Example 5; Figure 14 This is a graph showing changes in the adrenal gland coefficient of SD rats after treatment in Example 5. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the present invention will be 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, for which specific conditions are not specified, are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. The experimental materials and reagents used in the following examples can be obtained from commercial sources 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 this 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 this application.

[0024] Example 1 Preparation of Asiatic Acid In this example, asiatic acid was prepared according to the following method.

[0025] 1.1 Extraction: Crush the Centella asiatica root into small pieces, add 8 times 70% ethanol and reflux extract 3 times, each time for 1 hour, filter, and combine the filtrates for later use; 1.2 Concentration: The above ethanol extract is concentrated under reduced pressure until there is no alcohol smell, the vacuum degree is -0.06~-0.08mpa, the temperature is 60-70℃, the concentrate is 10 times the amount of medicinal materials, centrifuged at 3600r / min, and the supernatant is reserved; 1.3. D101 column chromatography: The supernatant was loaded onto D101 macroporous resin (medicinal material / resin (V / M, 1 / 2.5)) for adsorption at a flow rate of 1.0 BV / h. After loading, the supernatant was first eluted with 5 BV of water until colorless at a flow rate of 1.5 BV / h, and then eluted with 6 BV of 65% ethanol at a flow rate of 1.0% BV / min. The 65% ethanol eluate was recovered under reduced pressure and concentrated to a small volume. 1.4. Alumina column chromatography: Take the above concentrate and add 95% ethanol to an alcohol concentration of more than 90%, stirring while adding, centrifuge, and dissolve the precipitate in 50 times 95% ethanol, centrifuge, combine the two supernatants and load them onto a neutral alumina column (medicinal material / alumina, 1.3 / 1). After loading, elute with 5BV 95% ethanol and 6BV 80% ethanol in sequence and collect; 1.5. Concentration: Monitor the target compound content in the eluate and concentrate to recover ethanol; the 80% ethanol eluate is concentrated to a specific gravity of 1.10±0.02 (60-70°C), with a vacuum degree of -0.06-0.08 MPa and a temperature of 60-70°C; 1.6. Drying: Take the above concentrate and dry it at a vacuum degree of -0.06~-0.08 MPa and a temperature of 60-70°C for 4 hours to obtain total Centella asiatica glycosides; 1.7. Dissolve the total glycosides of Centella asiatica in 5 times methanol, add water to a methanol volume fraction of 35%, stir while adding, and let it stand for more than 24 hours. Monitor the clear solution and crystallization of the liquid phase; 1.8. After the crystallization is completed, filter and wash with a small amount of 95% ethanol. Dry the crystals at a vacuum degree of -0.06~-0.08 MPa and a temperature of 60-70°C for 4 hours. Grind the dry paste and pass it through a 100-mesh sieve to obtain asiaticoside; 1.9: Take a sample of asiaticaoside and hydrolyze it in 50 times 1 mol / L sodium hydroxide ethanol (70%) aqueous solution at 60°C. Monitor the hydrolysis by liquid chromatography (the liquid chromatography test results show that the hydrolysis is complete after 2.0 h). 1.10. After the reaction is completed, adjust to neutrality with acetic acid, concentrate until there is no alcohol smell, continue to add acetic acid to adjust the pH to 4, let it stand for 3 hours, filter, if the precipitate is dark in color, dissolve it in 95% ethanol and then decolorize it with activated carbon, collect the solid and dry it to obtain the Centella asiatica acid sample.

[0026] The prepared asiatic acid was subjected to liquid chromatography. The liquid chromatography conditions were: 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-cyclopentane; injection volume: 10μl; wavelength: 205nm; mobile phase gradient is shown in Table 1.

[0027] Table 1 The chromatogram of the prepared Centella asiatica acid is as follows Figure 1 shown.

[0028] Example 2 2.1. The information of experimental mice is shown in Table 2 below.

[0029] Table 2 This example strictly adhered to all applicable guidelines for the care and use of laboratory animals. Reference was made to "The Guide for the Care and Use of Laboratory Animals," Institute of Laboratory Animal Resources, National Academy Press, Washington, DC, 2011. To ensure adherence to animal welfare principles, this experimental protocol was reviewed by the Institutional Animal Care and Use Committee (IACUC) before animals were received or transferred to this research institution.

[0030] Before modeling, animals were screened based on weight, clinical observations, and other indicators. Animals were randomly assigned to groups based on weight. After grouping, animal weight should not exceed ±20% of the group mean. The mean weight of each group should show no statistically significant difference at a 5.0% detection level.

[0031] 2.2 Hyperuricemia Model Mice Male SD rats were adapted for 6 days and randomly divided into normal group and model group according to body weight. The SD rats in the normal group did not receive any intervention. The SD rats in the model group 5 were gavaged with adenine 50 mg / kg + potassium oxonate 1.5 g / kg once a day according to the reference method. The uric acid (UA), urea (UREA) and creatinine (CREZ) levels in the serum of SD rats were biochemically detected one week after gavage of modeling drugs. According to the uric acid (UA) value, the SD rats with successful modeling were screened and randomly divided into groups for drug administration. During the treatment period, the modeling was maintained with adenine 100 mg / kg + potassium oxonate 1.5 g / kg, once every 2 days, and for 30 consecutive 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 standards were selected to participate in subsequent experiments. 2.3 Grouping and Dosing The animals in the hyperuricemia model group were randomly divided into 12 groups, including a blank control group with 11 animals in the blank control group and 10 animals in the hyperuricemia model group. 1) The blank control group was given an equal amount of distilled water once a day for 30 consecutive days; 2) The model + vehicle group was given an equal amount of vehicle once a day for 30 consecutive days; 3) The model + allopurinol (10 mg / kg) group was given 10 mg / kg of allopurinol once a day for 30 consecutive days; 4) The model + EZY-C (8 mg / kg) group was given 8 mg / kg of EZY-C once a day for 30 consecutive days; 5) The model + EZY-C (16 mg / kg) group was given 16 mg / kg of EZY-C once a day for 30 consecutive days; 6) The model + EZY-E (8 mg / kg) group was given 8 mg / kg of EZY-E once a day for 30 consecutive days; 7) The model + EZY-E (16 mg / kg) group was given 16 mg / kg of EZY-E, once a day, for 30 consecutive days. Specific dosing information is shown in Table 3 below. Both asiaticoside and asiatic acid were prepared using the method of Example 1.

[0032] Table 3 2.4 In vivo detection indicators and testing (1) Body weight: Weigh the rats once a week.

[0033] (2) General observation: Observe the rats’ appearance, physical signs, behavioral activities, and fecal characteristics once a day.

[0034] According to the SD rat weight data change chart, the weight gain of rats in each group was balanced throughout the entire experiment (due to fasting the night before, the overall weight of rats decreased at the end of the experiment) and no abnormalities were observed. No abnormal clinical manifestations were found in any of the drug-treated groups during the treatment process.

[0035] Example 3 Biochemical Detection Biochemical tests were performed weekly to measure changes in the serum levels of uric acid (UA), urea (UREA), creatinine (CREZ), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in the rats of Example 2. At the endpoint of treatment, serum samples were collected from the rats to measure changes in the serum levels of uric acid (UA), urea (UREA), creatinine (CREZ), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and total cholesterol (CHOL).

[0036] Specific steps: (1) Loading Reagent Blank Calibrators: ① Prepare Blank Calibrators: Prepare blank calibrators according to the analytical method requirements (generally, deionized water or saline) and place them in the sample cup. ② Loading: Load the blank calibrator into the designated position on the blue sample rack. The reagent blank for the serum sample should be placed in the first position on the blue rack. Calibration with calibrators must be performed simultaneously with blank sample calibration. (2) Loading Calibrators: ① Prepare Calibrators: Prepare calibrators according to the analytical project's operating procedures. Typically, the accompanying calibration serum should be used. Reconstitution, storage, and use procedures should be strictly followed according to the calibration serum's instructions. ② Loading: Place the calibrator for that project in the corresponding position on the yellow calibration rack, according to the calibrator position specified in "Calibration Parameters." The calibrator position can be viewed by clicking "Show Sample Cup Settings" when editing the calibration worksheet. (3) Loading the Calibration Sample Rack: ① Load the Blank Calibration Sample Rack (blue): Place the blue sample rack in the first position on the left side of the sample conveyor track, with the barcode end facing forward (left). ② Load the calibrator rack (yellow): Place the yellow calibrator rack immediately following the blue rack, with the barcode facing forward (left). ⑷ Routine Sample Measurement Programming Worksheet: Click "Reset" - Select "Sample Rack Request" - Select "Test Request" under "Sample": ① Programming a single sample: Click "Reset" - Select "Sample Rack Request" - Select "Test Request" under "Sample" - Confirm the sample number to be edited in "Sample Number" - After confirmation, click "Start Login" and select the project - Click "Login" to confirm - To edit the next sample, continue selecting the project and click "Login" - After completing project programming, click "Exit" - Place the corresponding samples in the white rack in the sample injection area in order - Click "Start" to begin the run. ② Batch sample programming: Click "Reset" - Select "Sample Rack Application" - Select "Test Application" in "Sample" - Confirm the current sample number to be edited in "Sample Number" - After confirmation, click "Start Login" and select the project - Click "Batch Input" - Select "Number of Samples" and enter the number of samples to be batch programmed - Click "OK" and confirm the "Sample Number" again - After confirmation, click "Exit" - Place the corresponding samples in order on the white rack in the sample injection area - Click "Start" to start the run. ③ Using the barcode mode does not require the above complex programming, and the barcoded samples can be directly placed in the sample rack. However, the barcode mode must develop a "Laboratory Information System" or LIS system that matches the BeckmanCoulter AU series. Different laboratories can set corresponding operating SOPs according to the specific conditions of the laboratory.

[0037] Experimental results The results of uric acid (UA), urea (UREA), and creatinine (CREZ) levels in rat serum are as follows: Figure 2-4 shown.

[0038] One week after oral administration of the modeling drug, the serum uric acid (UA) content of SD rats in the model group was significantly higher than that in the blank control group (p<0.01), indicating that the hyperuricemia model of SD rats was successfully established.

[0039] After successful modeling, each treatment group was given the corresponding dose of the test drug for treatment. The levels of uric acid (UA), urea (UREA), and creatinine (CREZ) in the serum of SD rats on Day 7, Day 14, Day 21, and Day 30 were detected by biochemical methods. The test results showed that: like Figure 2 As shown in the data, compared with the blank control group, the uric acid (UA) content in the serum of SD rats in the vehicle group was significantly increased (P<0.01); compared with the vehicle group, the uric acid (UA) content in the serum of SD rats in the drug groups (allopurinol, asiaticoside and asiatic acid) were significantly reduced after treatment (P<0.01).

[0040] like Figure 3 As shown, compared with the normal group, the serum urea (UREA) level in SD rats in the vehicle group was significantly increased (P < 0.01). Compared with the vehicle group, treatment with allopurinol, asiaticoside, and asiatic acid significantly reduced the serum urea (UREA) level in SD rats (P < 0.01). Asiatic acid was the most effective treatment, with 16 mg / kg of asiatic acid being more effective than allopurinol.

[0041] like Figure 4 As shown in the data, compared with the normal group, the serum creatinine (CREZ) content of SD rats in the vehicle group was significantly increased (P<0.01); compared with the vehicle group, the drug groups (allopurinol, asiaticoside and asiatic acid) could significantly reduce the serum creatinine (CREZ) content of SD rats after treatment (P<0.01).

[0042] The results of the low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and total cholesterol (CHOL) levels in rat serum were as follows: Figure 5-7 shown.

[0043] Biochemical methods were used to detect the levels of low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and total cholesterol (CHOL) in the serum of SD rats. The test results showed that: like Figure 5As shown in the results, compared with the blank control group, the serum low-density lipoprotein cholesterol (LDL-C) level of SD rats in the vehicle group was significantly increased (P<0.01). Compared with the vehicle group, the drug groups (allopurinol, asiaticoside, and asiatic acid) all significantly reduced the serum low-density lipoprotein cholesterol (LDL-C) level of SD rats after treatment (P<0.01). Furthermore, asiatic acid at a dose of 16 mg / kg was more effective than asiaticoside and allopurinol in reducing the serum low-density lipoprotein cholesterol (LDL-C) level of SD rats.

[0044] like Figure 6 As shown in the results, compared with the blank control group, the serum high-density lipoprotein cholesterol (HDL-C) level of SD rats in the vehicle group was significantly increased (P<0.01). Compared with the vehicle group, the drug groups (allopurinol, asiaticoside, and asiatic acid) reduced the serum HDL-C level of SD rats after treatment. Furthermore, asiaticoside (16 mg / kg) was more effective than asiaticoside and allopurinol in reducing the serum HDL-C level of SD rats.

[0045] like Figure 7 As shown in the results, compared with the blank control group, the total cholesterol (CHOL) content in the serum of SD rats in the vehicle group was significantly increased (P<0.01). Compared with the vehicle group, the drug groups (allopurinol, asiaticoside, and asiatic acid) all reduced the total cholesterol (CHOL) content in the serum of SD rats after treatment (P<0.05). In particular, asiatic acid at a dose of 16 mg / kg was more effective than asiaticoside and allopurinol in reducing the total cholesterol (CHOL) content in the serum of SD rats.

[0046] Example 4 In vitro detection indicators and detection methods The rats of Example 2 were observed for pathological changes in the heart, liver, spleen, lung, thymus, kidney, adrenal gland, pancreas, stomach, duodenum, jejunum, ileum, rectum, colon, cecum, etc., and the heart, liver, spleen, lung, thymus, kidney, and adrenal gland were weighed; The left and right kidney tissues of the rats were collected for pathological examination (HE staining and Gomori hexamine silver staining).

[0047] HE staining method: Samples were fixed in 10% neutral formalin at room temperature for 4 hours. The rat kidney tissue was removed and rinsed with running water for several hours. The tissue was then dehydrated with 70%, 80%, and 90% ethanol solutions, followed by a mixture of equal parts pure alcohol and xylene for 15 minutes, followed by xylene I and II for 15 minutes (until transparent). The tissue was then placed in a mixture of 50% xylene and 50% paraffin for 15 minutes, followed by paraffinization in paraffin I and II for 50-60 minutes each. The tissue was then paraffin-embedded and sectioned. The paraffin sections were baked, dewaxed, and hydrated. After distilled water exposure, the sections were stained with hematoxylin-water solution for 3 minutes, differentiated in hydrochloric acid-ethanol solution for 15 seconds, briefly washed with water, and then blued with bluing solution for 15 seconds. The sections were then rinsed with running water and stained with eosin for 3 minutes. The sections were then rinsed with running water, dehydrated, transparentized, mounted, and examined under a microscope.

[0048] Gomori silver hexamine staining method: 1. Tissue fixation: Fix in anhydrous ethanol for 16 hours or overnight. Then, soak in anhydrous ethanol three times, 30 minutes each time. 2. Soak in xylene twice, 20 minutes each time, and embed in conventional wax. 3. Sections are cut to 5 μm thickness and deparaffinized in xylene and then in anhydrous ethanol. 4. Prepare the Gomori silver hexamine solution in advance and use within 2 hours. Add the Gomori silver hexamine solution dropwise to the sections and incubate in a 58-60°C incubator in the dark for 30 minutes (ensure sufficient dye solution to prevent evaporation and drying of the sections). The presence of urate will cause the sections to appear black. Rinse briefly with distilled water. 5. Add gold chloride solution dropwise for 1 minute. Rinse briefly with tap water. 6. Add Hypo solution dropwise for 5 minutes. Rinse with tap water for 5 minutes. 7. Add eosin solution dropwise for 30 seconds. Rinse briefly with tap water. 8. Conventional dehydration and transparency, neutral gum sealing.

[0049] Staining results: Urate crystals: black. Background: light red.

[0050] Negative control: Dewax serial sections and incubate them in Gomori control solution for 5 minutes, rinse twice with anhydrous ethanol, and then place them in Gomori silver hexamine solution. The remaining steps are the same as before. Calcium salts are negative.

[0051] Data statistical analysis: Experimental data are expressed as mean ± standard deviation (Mean ± SE) using Graphpad.Prism or SPSS Statistics Experimental results HE pathological staining results Figure 8-10 shown.

[0052] like Figure 8As shown, all animals in the vehicle group showed renal damage, manifested by extensive tubular degeneration and necrosis, tubular urate deposition, tubular dilatation, inflammatory cell infiltration in some tubules, and cellular casts in a small number of tubules. Treatment with all drugs (allopurinol, asiaticoside, and asiatic acid) showed some degree of renal repair, with the severity of the damage being milder than in the vehicle group. This was manifested by significant improvement in partial tubular degeneration, tubular dilatation, and urate deposition, with only a small amount of urate deposits present in the tubules.

[0053] Compared with the blank control group, the tubulointerstitial injury score of SD rats in the vehicle group ( Figure 9 ) and renal inflammatory cell infiltration score of renal tissue ( Figure 10 ) significantly increased (P<0.01). Compared with the vehicle group, treatment with the drug groups (allopurinol, asiaticoside, and asiatic acid) significantly reduced the tubulointerstitial injury score and renal inflammatory cell infiltration score in the kidneys of SD rats (P<0.01). The therapeutic effects of asiaticoside and asiatic acid were concentration-dependent, with 16 mg / kg of asiatic acid and allopurinol being the most effective.

[0054] Gomori hexamine silver staining results are as follows Figure 11-12 shown.

[0055] like Figure 11 As shown in the figure, all animals in the model control group showed renal tubular urate deposition. After treatment with allopurinol, asiaticoside, and asiatic acid, urate deposition was significantly improved, with only a small amount of urate deposits in the renal tubules.

[0056] like Figure 12 As shown, compared with the blank control group, the urate crystal score in the renal tissue of SD rats in the vehicle group was significantly increased (P < 0.01). Compared with the vehicle group, treatment with the drug groups (allopurinol, asiaticoside, and asiatic acid) significantly reduced the urate crystal score in the renal tissue of SD rats (P < 0.01). The therapeutic effects of asiaticoside and asiatic acid were concentration-dependent, with 16 mg / kg of asiatic acid being the most effective.

[0057] Example 5 In the efficacy test, the kidney and adrenal gland indexes of the rats of Example 2 were analyzed respectively.

[0058] The results are as follows Figure 13 and 14 As shown in the results, after modeling, the kidney and adrenal indexes of the vehicle group were significantly higher than those of the blank control group (P<0.01); compared with the vehicle group, the kidney and adrenal indexes of each treatment group were significantly reduced (P<0.01), indicating that each therapeutic drug can effectively inhibit and repair kidney damage.

[0059] The applicant declares that, in the description of the above specification: The descriptions of terms such as "this embodiment", "an embodiment of the present invention", "as shown in...", "further", "a further improved technical sub-scheme", etc., mean that the specific features, structures, materials or characteristics described in the embodiment 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 do not necessarily refer to the same embodiment or example, and the specific features, structures, materials or characteristics described may be combined or combined in an appropriate manner in any one or more embodiments or examples; in addition, a person of ordinary skill in the art may combine or combine different embodiments or examples and features of different embodiments or examples described in this specification without causing any contradiction.

[0060] 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 the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein, and 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 based on the contents of this specification are all within the scope of protection required by the present invention.

Claims

1. Application of asiatic acid in the preparation of medicines for treating hyperuricemia.

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

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

4. The use according to claim 1, wherein The treatment involves reducing the damage to the kidneys caused by hyperuricemia.

5. The use according to claim 1, characterized in that The effective dose of Centella asiatica is 5-20 mg / kg.

6. The use according to claim 5, characterized in that The effective dose of asiatic acid is 16 mg / kg.

7. A method for extracting asiatic acid, characterized in that: The steps include: S1. Extraction: Crush the Centella asiatica root, extract with ethanol under reflux, filter, and combine the filtrates for later use; S2. Concentration: The ethanol extract is concentrated under reduced pressure until there is no alcohol smell, centrifuged, and the supernatant is reserved; S3, D101 column chromatography; S4, alumina column chromatography; S5, concentration; S6, drying; S7. Dissolve with methanol, add water and let stand, and monitor the clear liquid and crystallization of the liquid phase; S8. After the crystallization is completed, the crystals are filtered and washed with ethanol. The crystals are dried at a vacuum degree of -0.06 to -0.08 MPa and a temperature of 60-70° C. The dry paste is crushed and sieved to obtain asiaticoside; S9, taking Centella asiatica glycoside, hydrolyzing it in a sodium hydroxide ethanol aqueous solution, and monitoring the hydrolysis status by liquid phase; S10. After the hydrolysis is complete, adjust the solution to neutrality with acetic acid, concentrate until there is no alcohol taste, continue to add acetic acid to adjust the pH to 3-4, let it stand, filter, collect the solid and dry it to obtain Centella asiatica.

8. The method for extracting asiatic acid according to claim 7, wherein: In step S3, the V / M ratio of Centella asiatica / D101 macroporous resin is 1 / (2.5-3).

9. The method for extracting asiatic acid according to claim 7, wherein: Step S4 includes: taking the above concentrated solution and adding ethanol to an alcohol concentration of more than 90%, stirring while adding, centrifuging, dissolving the precipitate with 50 times ethanol, centrifuging, combining the two supernatants and loading them onto a neutral alumina column. After loading, eluting with 5BV ethanol and collecting.

10. The method for extracting asiatic acid according to claim 7, wherein: Step S10 also includes: after filtering, if the precipitate is dark in color, it can be dissolved in 95% ethanol and then decolorized with activated carbon.

Images