New application and method of asiaticoside

Abstract

The invention provides novel application and a method of asiaticoside. The method comprises the following steps: S1, extraction: taking a centella asiatica 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; 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; specifically, 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. And the yield of asiaticoside extracted by the method is high and is about 84%.

Description

Technical Field

[0001] The present invention relates to a new application and method of asiaticoside, and belongs 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. 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; the leaves of Centella asiatica are beautiful in shape, evergreen all year round, resistant to trampling, and have a good effect on preventing soil erosion, so it is an excellent lawn plant; its extract has a significant promoting effect on the healing of wounds and ulcers, so it is also commonly used in the cosmetics industry.

[0003] Asiaticoside is an organic compound with the molecular formula C 48 H 78 O 19 . The pure product is white needle-shaped crystals, and the industrial product is a powder from light yellow to light brownish yellow; odorless, bitter taste, slightly hygroscopic. It is easily soluble in water and ethanol, and insoluble in ether and chloroform. It is mainly extracted from the dried whole herb of the umbelliferous plant Centella aslatica (L.) Urb. It has the effect of promoting wound healing. It is used to treat trauma, surgical wounds, burns, keloid and scleroderma.

[0004] It is of great significance to extract 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) 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 a high yield, about 84%.

[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 use of asiaticoside in the preparation of a drug for treating hyperuricemia.

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

[0008] Preferably, the treatment includes reducing uric acid, urea, creatinine, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, or total cholesterol; more preferably, the treatment includes reducing 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 asiaticoside is 5 - 20 mg / kg; more preferably, the effective dose of asiaticoside is 16 mg / kg.

[0011] In the second aspect of the present invention, a method for extracting asiaticoside is provided, which includes the following steps: S1. Extraction: Coarsely crush the centella asiatica medicinal material, add ethanol for reflux extraction, 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; S7. Dissolve with methanol, add water and let it stand, and monitor the supernatant and crystallization by liquid phase. S8. After crystallization, filter, wash with ethanol, dry the crystals under a vacuum of -0.06 to -0.08 mpa at a temperature of 60 - 70 °C, and pulverize and sieve the dry paste to obtain asiaticoside.

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

[0013] Preferably, step S4 includes: adding ethanol to the above concentrated solution until the alcohol concentration is above 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, elute with 5 BV ethanol and collect.

[0014] Preferably, in step S4, the concentration of the ethanol for elution is 80 - 95%.

[0015] Preferably, step S3 includes: loading the supernatant of step S2 onto D101 macroporous resin for adsorption. After loading, first elute 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.

[0016] Preferably, in step S3, the concentration of the ethanol for elution is 65 - 75%.

[0017] Preferably, a method for extracting asiaticoside includes the following steps: Step 1, extraction: Coarsely crush the centella asiatica herb, add 8 times of 70% ethanol, reflux and extract 3 times, each time for 1 h, filter, combine the filtrates, and set aside for later use; Step 2, concentration: Concentrate the above ethanol extract under reduced pressure until the alcohol smell disappears, with a vacuum degree of -0.06 to -0.08 mpa and a temperature of 60 - 70 °C, concentrate the extract to 10 times the amount of the herb, centrifuge at 3600 r / min, and set aside the supernatant for later use; Step 3, D101 column chromatography: Load the above supernatant onto D101 macroporous resin (herb / resin (V / M, 1 / 2.5)) for adsorption, control the flow rate at 1.0 BV / h. After the loading is completed, first wash with 5 BV of water until colorless, control the flow rate at 1.5 BV / h, 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 concentrated solution until the alcohol concentration is above 90%, stir while adding, centrifuge, dissolve the precipitate with 50 times of 95% ethanol again, centrifuge, combine the supernatants of the two times and load them onto a neutral alumina column (herb / alumina, 1.3 / 1). After the loading is completed, elute successively with 5 BV of 95% ethanol and 6 BV of 80% ethanol and collect the eluates; Step 5, concentration: Monitor the content of the target substance in the above eluate, concentrate and recover ethanol; Concentrate the 80% ethanol eluate to a specific gravity of 1.10 ± 0.02 (60 - 70 °C), with a vacuum degree of -0.06 to -0.08 mpa and a temperature of 60 - 70 °C; Step 6, drying: Take the above concentrated solution, dry it at a vacuum degree of -0.06 to -0.08 mpa and a temperature of 60 - 70 °C for 4 h to obtain total centella glycosides; Step 7, Take the total centella glycosides, dissolve them with 5 times of methanol, add water until the volume fraction of methanol is 35%, stir while adding, and let it stand for more than 24 h, monitor the clear liquid and crystallization by liquid phase; Step 8, After crystallization, filter, wash with a small amount of 95% ethanol, dry the crystals at a vacuum degree of -0.06 to -0.08 mpa and a temperature of 60 - 70 °C for 4 h, pulverize the dry paste and pass it through a 100-mesh sieve to obtain asiaticoside.

[0018] 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), and total cholesterol (CHOL) in the serum of SD rats with hyperuricemia model, and can also reduce the damage of hyperuricemia to the kidneys. In particular, the effects of reducing low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and total cholesterol (CHOL) are better than those of total asiaticosides.

[0019] In the third aspect of the present invention, there is provided an asiaticoside prepared by the method described in the present invention.

[0020] Compared with the prior art, the beneficial effects and remarkable progress of applying the technical solution of the present invention are as follows: 1. The new application of asiaticoside provided by the present invention is that 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), and total cholesterol (CHOL) in the serum of SD rats with hyperuricemia model, and can also reduce the damage of hyperuricemia to the kidneys. In particular, the effects of reducing low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and total cholesterol (CHOL) are better than those of total asiaticosides.

[0021] 2. The asiaticoside extracted by the method of the present invention has a high yield, about 84%. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] For a clearer description of the technical solution of the present invention, the drawings used in the embodiments of the present invention will be briefly introduced below.

[0023] Figure 1 It is the chromatogram of asiaticoside in Example 1; Figure 2 It 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 It is the change diagram of the content of urea (UREA) in the serum of SD rats during the treatment in Example 5; Figure 4 It is the change diagram of the content of creatinine (CREZ) in the serum of SD rats during the treatment in Example 5; Figure 5 It is the change diagram of the content of low-density lipoprotein cholesterol (LDL-C) in the serum of SD rats after treatment in Example 5; Figure 6It is a graph showing the change in the content of high-density lipoprotein cholesterol (HDL-C) in the serum of SD rats after treatment in Example 5; Figure 7 It is a graph showing the change in the content of total cholesterol (CHOL) in the serum of SD rats after treatment in Example 5; Figure 8 It is a HE staining map of the kidney tissue of SD rats after treatment in Example 6; Figure 9 It is a graph showing the change in the pathological tubulointerstitial injury score of the kidney tissue of SD rats after treatment in Example 6; Figure 10 It is a graph showing the change in the pathological kidney inflammatory cell infiltration score of the kidney tissue of SD rats after treatment in Example 6; Figure 11 It is a Gomori hexamine silver staining map of the kidney tissue of SD rats after treatment in Example 6; Figure 12 It is a graph showing the change in the pathological urate crystal score of the kidney tissue of SD rats after treatment in Example 6; Figure 13 It is a graph showing the change in the kidney coefficient of SD rats after treatment in Example 7; Figure 14 It is a graph showing the change in the adrenal coefficient of SD rats after treatment in Example 7. Detailed implementation manners

[0024] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturers. Unless otherwise stated, percentages and parts are weight percentages and weight parts. The experimental materials and reagents used in the following embodiments can be obtained from commercial channels without special instructions.

[0025] Unless otherwise specified, the technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the technical field to which this application belongs. It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary embodiments of this application.

[0026] Example 1 Preparation of asiaticoside Asiaticoside was prepared according to the following method in this example.

[0027] 1.1 Extraction: Take the crude Centella asiatica herbs, add 8 times of 70% ethanol and reflux for extraction 3 times, 1 hour each time, filter, combine the filtrates, and reserve; 1.2 Concentration: The above ethanol extract is concentrated under reduced pressure until there is no alcohol smell, with a vacuum degree of -0.06 to -0.08 mpa and a temperature of 60 - 70 °C. The concentrated solution is up to 10 times the amount of the medicinal material, centrifuged at 3600 r / min, and the supernatant is reserved for use; 1.3 D101 column chromatography: The above supernatant is loaded onto D101 macroporous resin (medicinal material / resin (V / M, 1 / 2.5)) for adsorption, with the flow rate controlled at 1.0 BV / h. After the loading is completed, first wash with 5 BV of water until colorless, with the flow rate controlled at 1.5 BV / h, and then elute with 6 BV of 65% ethanol, with the flow rate 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: Add 95% ethanol to the above concentrated solution until the alcohol concentration is above 90%, stir while adding, centrifuge, dissolve the precipitate continuously with 50 times of 95% ethanol, centrifuge, and 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 successively with 5 BV of 95% ethanol and 6 BV of 80% ethanol; 1.5 Concentration: Monitor the content of the target substance in the above 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 to -0.08 mpa and a temperature of 60 - 70 °C; 1.6 Drying: Take the above concentrated solution, dry it at a vacuum degree of -0.06 to -0.08 mpa and a temperature of 60 - 70 °C for 4 h to obtain asiaticoside total glycosides; 1.7 Take asiaticoside total glycosides, dissolve them with 5 times of methanol, add water until the volume fraction of methanol is 35%, stir while adding, and let it stand for more than 24 h, and monitor the clear liquid and crystallization by liquid phase; 1.8 After crystallization, filter, wash with a small amount of 95% ethanol, dry the crystals at a vacuum degree of -0.06 to -0.08 mpa and a temperature of 60 - 70 °C for 4 h, and pulverize the dry paste through a 100-mesh sieve to obtain asiaticoside.

[0028] The prepared asiaticoside is subjected to liquid chromatography. The liquid chromatography conditions are as follows: Chromatographic column: ZORBAX SB-C18 (4.6×250mm 5-Micron); Flow rate: 1 ml / min; Column temperature: 30 °C; Mobile phase: A: Acetonitrile, B: 2 mmol / L β-cyclodextrin; Injection volume: 10 μl; Wavelength: 205 nm; The mobile phase gradient is as shown in Table 1 below.

[0029] Table 1 The chromatogram of the prepared asiaticoside is as Figure 1 shown.

[0030] Example 2 Resin Screening Experiment Through single-factor process and orthogonal experiment, the extraction process of asiaticoside was determined as reflux extraction with 70% ethanol for 1 h, three times, with a high extraction yield. Then, through the study of various resin process parameters, resin D101 had good purification effect (as shown in Table 2 below). Using 65% ethanol to elute 6 BV, the refined asiaticoside content reached 60.35%.

[0031] Table 2 Example 3 Alumina Purification Experiment Take 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 as shown in Table 3 below.

[0032] Table 3 In summary, after purification by D101 macroporous resin, the content of asiaticoside is about 60%. After column loading on neutral alumina, the content is purified to about 84%.

[0033] Example 4 4.1. Information of experimental mice is as shown in Table 4 below.

[0034] Table 4 This example strictly complies with all applicable laboratory animal care and use guidelines. Referenced 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 experimental animals were received or transferred to this research institution.

[0035] Before the start of modeling, animals were screened based on comprehensive indicators such as animal body weight and clinical observation. The random grouping method was used, and the animals were grouped according to their body weight. After grouping, the animal body weight 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.

[0036] 4.2. Hyperuricemia Model Mice Male Sprague-Dawley (SD) rats were adaptively raised for 6 days. The SD rats were randomly divided into a normal group and a model group according to their body weights. The SD rats in the normal group were not given any intervention, while the SD rats in the model group were intragastrically administered adenine (50 mg / kg) + potassium oxonate (1.5 g / kg) once a day according to the reference method. After 1 week of intragastric administration of the modeling drug, the levels of uric acid (UA), urea (UREA), and creatinine (CREZ) in the serum of the SD rats were detected by biochemical tests. According to the UA values, the SD rats with successful modeling were screened and randomly divided into groups 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, with continuous intragastric administration 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 meeting the model criteria were selected to participate in the subsequent experiments.

[0037] 4.3. Grouping and Drug Administration The animals in the hyperuricemia model group were randomly divided into 12 groups, including a blank control group, for 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 (blank control group) was given an equal volume of distilled water once a day for 30 consecutive days; the second group (model + vehicle group) was given an equal volume of vehicle once a day for 30 consecutive days; the third group (model + allopurinol (10 mg / kg) group) was given 10 mg / kg of allopurinol once a day for 30 consecutive days; the fourth group (model + EZY-A (8 mg / kg) group) was given 8 mg / kg of EZY-A once a day for 30 consecutive days; the fifth group (model + EZY-A (16 mg / kg) group) was given 16 mg / kg of EZY-A once a day for 30 consecutive days; the sixth group (model + EZY-E (8 mg / kg) group) was given 8 mg / kg of EZY-E once a day for 30 consecutive days; the seventh group (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.

[0038] Table 5 2.4. In Vivo Detection Indexes and Detection (1) Body weight measurement: The body weight of the rats was measured once a week.

[0039] (2) General observation: The appearance signs, behavioral activities, fecal characteristics, etc. of the rats were observed once a day.

[0040] According to the change graph of the body weight data of the 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 at the end of the experiment decreased 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.

[0041] 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 rat serum of Example 2 were detected biochemically. At the end of the treatment, the content changes of uric acid (UA), urea (UREA), serum creatinine (CREZ), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and total cholesterol (CHOL) were detected in the rat serum.

[0042] Specific operation steps: ⑴ Loading reagent blank calibrator: ① Preparing the blank calibrator: Prepare the blank calibrator according to the requirements of the analysis method (usually deionized water or normal saline is used), and place it in the sample cup. ② Loading: Load the blank calibrator at the designated 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 carried out simultaneously. ⑵ Loading the calibrator: ① Calibrator preparation: Prepare the calibrator according to the operating procedures of the analysis item. Generally, a matching calibrator serum is used, and it is reconstituted, stored and used strictly in accordance with the requirements of the calibrator serum instruction manual. ② Loading: Place the calibrator for 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 determination 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 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 batch programmed 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 injection area in sequence - Click "Start" to start running. ③ In the barcode mode, the above complex programming is not required. 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 operating SOP according to the specific situation of the laboratory.

[0043] 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.

[0044] 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 hyperuricemia model of SD rats was successfully constructed.

[0045] After successful modeling, 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 content of uric acid (UA) in the serum of SD rats could be significantly reduced after treatment in the drug groups (allopurinol, total asiaticoside, and asiaticoside) (P<0.01). And there was no obvious difference in the treatment effects of allopurinol, total asiaticoside, and asiaticoside.

[0046] 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 content of urea (UREA) in the serum of SD rats could be significantly reduced after treatment in the drug groups (allopurinol, total asiaticoside, and asiaticoside) (P<0.01). And there was no obvious difference in the treatment effects of allopurinol, total asiaticoside, and asiaticoside.

[0047] 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 content of creatinine (CREZ) in the serum of SD rats could be significantly reduced after treatment in the drug groups (allopurinol, total asiaticoside, and asiaticoside) (P<0.01). And there was no obvious difference in the treatment effects of allopurinol, total asiaticoside, and asiaticoside.

[0048] The results of the contents of low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and total cholesterol (CHOL) in the serum of rats were as Figures 5-7 shown.

[0049] The contents of low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and total cholesterol (CHOL) in the serum of SD rats were detected by biochemical methods. The test results showed that: As Figure 5As 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, after treatment, the drug groups (allopurinol, total asiaticoside, and madecassoside) could significantly reduce the content of low-density lipoprotein cholesterol (LDL-C) in the serum of SD rats (P<0.01). And the effect of madecassoside in reducing the content of low-density lipoprotein cholesterol (LDL-C) in the serum of SD rats was better than that of total asiaticoside and allopurinol.

[0050] As Figure 6 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, after treatment, the drug groups (allopurinol, total asiaticoside, and madecassoside) could reduce the content of high-density lipoprotein cholesterol (HDL-C) in the serum of SD rats. And the effect of madecassoside in reducing the content of high-density lipoprotein cholesterol (HDL-C) in the serum of SD rats was better than that of total asiaticoside and allopurinol.

[0051] As Figure 7 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, after treatment, the drug groups (allopurinol, total asiaticoside, and madecassoside) could reduce the content of total cholesterol (CHOL) in the serum of SD rats (P<0.05). And the effect of madecassoside in reducing the content of total cholesterol (CHOL) in the serum of SD rats was better than that of total asiaticoside and allopurinol.

[0052] 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 the rats for pathological examination (HE staining and Gomori hexamine silver staining).

[0053] 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 them with running water for several hours. Dehydrate them with 70%, 80%, and 90% ethanol solutions successively, soak them in an equal mixture of absolute alcohol and xylene for 15 min, and soak them in xylene I for 15 min and xylene II for 15 min (until transparent). Place them in a mixture of xylene and paraffin (half and half) for 15 min, and then place them in paraffin I and paraffin II for infiltration for 50 - 60 min each. Embed them in paraffin and section them. Bake the paraffin sections, then dewax and hydrate them. Place the sections that have been put into distilled water into hematoxylin aqueous solution for staining for 3 min, differentiate them with hydrochloric acid ethanol differentiation solution for 15 s, wash them slightly with water, blue them with bluing solution for 15 s, rinse them with running water, stain them with eosin for 3 min, rinse them with running water, dehydrate them, make them transparent, mount them, and examine them under a microscope.

[0054] Gomori hexamine silver staining detection method: 1. Tissue fixation: Fix it in absolute ethanol for 16 h or overnight. Then soak it in absolute ethanol 3 times, 30 min each time. 2. Soak it in xylene 2 times, 20 min each time, and embed it in paraffin by conventional infiltration. 3. Cut the sections with a thickness of 5 μm, dewax them with xylene to absolute ethanol. 4. Prepare the Gomori hexamine silver solution in advance and use it up within 2 h. Drop the Gomori hexamine silver solution on the sections and incubate them in a constant temperature incubator at 58 - 60 °C in the dark for 30 min (ensure that there is enough staining solution to avoid drying out the sections due to evaporation). If urate is present, the sections will turn black. Wash them slightly with distilled water. 5. Drop the gold chloride solution and treat them for 1 min. Wash them slightly with tap water. 6. Drop the hypo solution and treat them for 5 min. Rinse them with tap water for 5 min. 7. Drop the eosin staining solution and lightly stain them for 30 s. Wash them slightly with tap water. 8. Dehydrate and make them transparent conventionally, and seal them with neutral balsam.

[0055] Staining results: Urate crystals: black. Background: light red.

[0056] Negative control: After dewaxing the consecutive sections, first incubate them with Gomori control solution for 5 min, wash them with absolute ethanol 2 times, and then put them into Gomori hexamine silver solution. The remaining steps are the same as above, and calcium salts are negative.

[0057] Data statistical analysis: The experimental data are all expressed as mean ± standard deviation (Mean ± SE), and Graphpad Prism or SPSS Statistics is used.

[0058] Experimental results The HE pathological staining results are as Figures 8-10 shown.

[0059] As Figure 8As shown, renal injury was observed in all animals in the model solvent group, manifested as extensive degeneration and necrosis of renal tubules, urate deposition in renal tubules, dilation of renal tubules, infiltration of inflammatory cells in some renal tubules, and cellular casts in a small number of renal tubules. After treatment with each drug group (allopurinol, total triterpenoids of Centella asiatica, and asiaticoside), 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 degeneration of renal tubules, significant improvement in the dilation of renal tubules and urate deposition, and only a small amount of urate deposition was seen in a small number of renal tubules.

[0060] Compared with the blank control group, the scores of renal tubulointerstitial injury ( Figure 9 ) and the scores of infiltration of renal inflammatory cells ( Figure 10 ) in the renal tissues of SD rats in the solvent group were significantly increased (P < 0.01); compared with the solvent group, after treatment with the drug groups (allopurinol, total triterpenoids of Centella asiatica, and asiaticoside), the scores of renal tubulointerstitial injury and the scores of infiltration of renal inflammatory cells in the renal tissues of SD rats could be significantly decreased (P < 0.01). Moreover, the therapeutic effects of total triterpenoids of Centella asiatica and asiaticoside were concentration-dependent. Asiaticoside at 16 mg / kg had the best effect.

[0061] The results of Gomori's hexamine silver staining were as Figures 11-12 shown.

[0062] As Figure 11 shown, urate deposition in renal tubules was observed in all animals in the model control group. After treatment with the drug groups (allopurinol, total triterpenoids of Centella asiatica, and asiaticoside), the urate deposition was significantly improved, and only a small amount of urate deposition was seen in a small number of renal tubules.

[0063] As Figure 12 shown, compared with the blank control group, the score of urate crystal in the renal tissues of SD rats in the solvent group was significantly increased (P < 0.01); compared with the solvent group, after treatment with the drug groups (allopurinol, total triterpenoids of Centella asiatica, and asiaticoside), the score of urate crystal in the renal tissues of SD rats could be significantly decreased (P < 0.01). Moreover, the therapeutic effects of total triterpenoids of Centella asiatica and asiaticoside were concentration-dependent. Asiaticoside at 16 mg / kg had the best effect.

[0064] Example 7 In the efficacy test, the kidney and adrenal gland indices of the test animals were analyzed respectively.

[0065] The results were as Figure 13 and 14 shown. After modeling, the kidney and adrenal gland indices of the solvent group were significantly higher than those of the blank control group (P < 0.01); compared with the solvent group, each treatment group could significantly decrease the kidney and adrenal gland indices (P < 0.01), indicating that each therapeutic drug could better inhibit and repair renal injury.

[0066] The applicant declares that during the description in the above specification: Descriptions of terms such as "this embodiment", "embodiment of the present invention", "as shown in...", "further", "further improved technical solution", 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 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 not generating contradictions, 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.

[0067] 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 them; 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, 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 according to the content of this specification all fall within the scope of protection required by the present invention.

Claims

1. Application of asiaticoside 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 or total cholesterol.

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

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

6. The application according to claim 5, characterized in that, The effective dose of asiaticoside is 16 mg / kg.

7. A method for extracting asiaticoside, characterized in that, It includes the following steps: S1. Extraction: Coarsely crush the asiatic plantain herb, reflux and extract with ethanol, filter, combine the filtrates, and set aside for later use; S2. Concentration: Concentrate the ethanol extract under reduced pressure until it has no alcohol smell, centrifuge, and set aside the supernatant for later use; S3. D101 column chromatography; S4. Alumina column chromatography; S5. Concentration; S6. Drying; S7. Dissolve with methanol, add water and let it stand, monitor the supernatant and crystallization by liquid phase; S8. After crystallization, filter, wash with ethanol, dry the crystals under a vacuum degree of -0.06 to -0.08 mpa and at a temperature of 60 - 70 °C, crush the dry paste and sieve to obtain asiaticoside.

8. The extraction method of asiaticoside according to claim 7, wherein, In step S3, the V / M of the asiatic plantain herb / D101 macroporous resin is 1 / (2.5 - 3).

9. The extraction method of asiaticoside according to claim 7, wherein, Step S4 includes: Add ethanol to the above concentrated solution until the alcohol concentration is above 90%, stir while adding, centrifuge, dissolve the precipitate with 50 times ethanol again, centrifuge, combine the supernatants of the two times and load them onto a neutral alumina column. After loading, elute with 5BV ethanol and collect.

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

Images