Saponin extract with uric acid regulating and antioxidant activity and application
A saponin extract from Holly leaves, prepared through ethanol extraction and resin purification, effectively inhibits xanthine oxidase and exhibits strong antioxidant properties, addressing the limitations of current treatments for hyperuricemia and providing a natural alternative for uric acid regulation and gout relief.
Patent Information
- Application Number
- CN202311091649.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-08-29
- Publication Date
- 2024-12-20
AI Technical Summary
Current treatments for hyperuricemia, such as xanthine oxidase inhibitors like allopurinol, have adverse reactions and toxic side effects, while natural medicines for uric acid regulation and antioxidant activity from Holly (Ilex cornuta Lindl. et Paxt.) leaves have limited research on xanthine oxidase inhibition and uric acid reduction.
A saponin extract is prepared from Holly leaves using ethanol extraction, purification, and vacuum freeze-drying, followed by further purification with AB-8 macroporous adsorption resin, to obtain a crude extract with high total saponin content and xanthine oxidase inhibitory and antioxidant activity.
The crude extract demonstrates concentration-dependent inhibitory activity against xanthine oxidase and strong antioxidant capacity, making it suitable for health products to lower uric acid and treat gout, as well as antioxidant products.
Smart Images
Figure CN119157915A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the field of biomedical technology, and more specifically, to a saponin extract having uric acid regulating and antioxidant activities and its application. Background Art
[0002] Hyperuricemia is a major metabolic disease in my country, which refers to excessive production and / or insufficient excretion of uric acid in the body under normal dietary conditions. The symptom of hyperuricemia is high uric acid concentration in the blood. About 5%-12% of hyperuricemia patients eventually develop gout.
[0003] Current treatments for hyperuricemia are mainly aimed at reducing the production of uric acid and promoting its excretion. A class of drugs currently used in the clinical treatment of hyperuricemia, xanthine oxidase (XOD) inhibitors, achieve the effect of reducing blood uric acid by inhibiting the activity of XOD, a key oxidase in the production of uric acid. Allopurinol is a representative drug among XOD inhibitors, which can inhibit XOD from converting xanthine and purine to produce uric acid. However, XOD inhibitors represented by allopurinol have various adverse reactions and toxic side effects, such as skin diseases, gastrointestinal irritation, headaches, liver and kidney damage, etc.
[0004] Natural medicines are gaining more and more attention and favor due to their high safety and low toxicity and side effects. Screening effective ingredients for regulating uric acid and relieving gout from natural active substances has become a hot topic in current research.
[0005] Holly (Ilex cornuta Lindl. et Paxt.) is a plant of the genus Ilex of the Aquifoliaceae family, mainly distributed in Zhejiang, Jiangsu, Anhui, Jiangxi and other places in my country. Its leaves clear away heat, nourish yin, and tonify the liver and kidneys. They are often drunk as tea. Traditional doctors use them to treat yin deficiency, hot flashes, coughing and coughing up blood, dizziness, soreness of the waist and knees, and rheumatic pain. Modern pharmacological studies have shown that holly has the effects of lowering blood lipids, lowering blood sugar, anti-oxidation, anti-fertility, antibacterial, dilating coronary arteries, immunosuppression, and anti-tumor. However, there are few reports on the research related to the inhibition of xanthine oxidase and in vitro uric acid reduction in holly leaves. Based on the above statements, the present application provides a saponin extract with uric acid regulation and antioxidant activity and its application. Summary of the invention
[0006] Based on the above technical problems, the present application provides a saponin extract and application with uric acid regulating and antioxidant activities.
[0007] In the first aspect, the present application provides a saponin extract having uric acid regulating and antioxidant activities, using the following technical scheme:
[0008] A saponin extract with uric acid regulating and antioxidant activities is prepared by the following method:
[0009] S1. According to the solid-liquid ratio of 1:12-18, crush the holly leaves, add ethanol solution, stir and mix evenly, heat and reflux to extract 2-3 times, filter and combine the filtrate to obtain a mixed extract;
[0010] S2. The mixed extract in step S1 is concentrated under reduced pressure, extracted with petroleum ether to remove impurities, and then freeze-dried in vacuum and stored in the dark to obtain a crude extract of holly leaf saponin.
[0011] Preferably, the volume fraction of the ethanol solution in step S1 is 75-85%.
[0012] Preferably, in step S1, the extraction temperature is 70-75° C., and the extraction time is 1.5-1.8 h.
[0013] Preferably, the filtering in step S1 refers to: centrifuging at a speed of 8000-10000 r / min for 10-15 min.
[0014] Preferably, the reduced pressure concentration in step S2 refers to: concentrating the mixed extract until there is no alcohol taste and the relative density is 1.02-1.20 g / cm at room temperature under the conditions of a temperature of 50-60°C and a pressure of -0.1-0 MPa. 3 .
[0015] Preferably, in step S2, extracting and removing impurities with petroleum ether means that the volume ratio of the concentrate to petroleum ether is 1:0.3-0.4, and the extraction is performed 2-3 times.
[0016] Preferably, the vacuum freeze drying in step S2 refers to freeze drying to a water content of no more than 8% under the conditions of a pressure of -0.1-0 MPa and a temperature of -35 to -40°C.
[0017] Preferably, step S2 also includes a further purification process.
[0018] Preferably, the further purification process specifically comprises the following steps:
[0019] A1: Take AB-8 macroporous adsorption resin, soak it in 95% ethanol for 24 hours, and then install it in a column by wet method. Wash the column with 95% ethanol until the effluent is no longer white and turbid, then wash the column with pure water until there is no alcohol smell, pass 4% HCl through the resin column, soak it for 6 hours, wash it with water until pH = 7, then pass 1.0 mol / L NaOH through the resin column, soak it for 6 hours, wash it with water until pH = 7, and set aside;
[0020] A2: Take the crude extract of holly leaf saponin, suspend it with distilled water, adjust the pH to 4, adsorb it on AB-8 resin column chromatography for 5 hours, first elute it with pure water, then elute it with 80% ethanol until it is almost colorless, collect the eluate and dry it to obtain the crude extract of holly leaf saponin.
[0021] In a second aspect, the present application provides a method for preparing a saponin extract having uric acid regulating and antioxidant activities, using the following technical scheme:
[0022] The invention discloses an application of a saponin extract having uric acid regulating and antioxidant activities, an application of the saponin extract in preparing products for preventing and treating xanthine oxidase-related diseases, and an application of the saponin extract in preparing antioxidant and anti-aging products.
[0023] In summary, this application has the following beneficial effects:
[0024] 1. This application uses holly leaves as raw materials, adds ethanol solution to extract and purify to obtain holly leaf saponin crude extract, in which the total saponin content is as high as 41.1 mg / g; through further purification process, column saponin crude extract is purified, in which the total saponin content is as high as 86.1 mg / g.
[0025] 2. The crude extracts of holly leaf saponins and holly leaf column saponins obtained in this application both have concentration-dependent inhibitory activities on xanthine oxidase in vitro, and their IC 50 They were 239 μg / mL and 1667 μg / mL respectively, indicating that they both have certain in vitro inhibitory activity against xanthine oxidase, among which the crude extract of holly leaf saponin has more potential. Therefore, this crude extract of holly leaf saponin can be used as a health product for lowering uric acid and relieving gout.
[0026] 3. The crude extracts of holly leaf saponins and holly leaf column saponins obtained in this application showed concentration-dependent ABTS free radical scavenging rates, and their IC 50 The results showed that the saponin extracts from hollyhock leaves had a certain antioxidant effect compared with the positive control.
[0027] 4. The crude extracts of holly leaf saponins and holly leaf column saponins obtained in this application showed concentration-dependent scavenging rates for DPPH free radicals, and their IC 50 The results showed that the extract had good scavenging ability and the scavenging rate of crude extract of hollyhock leaf saponin was better than that of crude extract of hollyhock leaf column saponin, and both of them had potential antioxidant capacity.
[0028] In summary, the crude extract of holly leaf saponin and the crude extract of holly leaf column saponin obtained in the present application both have certain xanthine oxidase inhibition ability and antioxidant activity, among which the crude extract of holly leaf saponin has better xanthine oxidase inhibition ability and higher potential for lowering uric acid; and both have good antioxidant capacity. Therefore, the crude extract of holly leaf saponin can be used to prepare health products for preventing / treating diseases related to xanthine oxidase; the crude extract of holly leaf saponin and the crude extract of holly leaf column saponin can both be used in the development of antioxidant products. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1 It is the in vitro half-inhibitory XOD concentration of the crude extract of holly leaf saponins obtained in Example 1.
[0030] Figure 2 It is the in vitro half-inhibitory XOD concentration of the crude extract of hollyhock leaf saponins obtained in Example 2.
[0031] Figure 3 is the concentration of allopurinol that half inhibits XOD in vitro.
[0032] Figure 4 is the ABTS free radical scavenging rate IC of the crude extract of holly leaf saponin obtained in Example 1 50 .
[0033] Figure 5 is the ABTS free radical scavenging rate IC of the crude extract of hollyhock saponin obtained in Example 2 50 .
[0034] Figure 6 IC for free radical scavenging rate of ABTS of vitamin C 50 .
[0035] Figure 7 is the DPPH free radical scavenging rate IC of the crude extract of holly leaf saponin obtained in Example 1 50 .
[0036] Figure 8 is the DPPH free radical scavenging rate IC of the crude extract of hollyhock saponin obtained in Example 2 50 .
[0037] Fig. 9 IC for DPPH free radical scavenging rate of vitamin C 50 . DETAILED DESCRIPTION
[0038] The present application is further described in detail below with reference to the embodiments.
[0039] In the following examples of the present application, the leaves of holly (Ilex cornuta Lindl. et Paxt.) were collected from Xiaying Village, Dingtang Town, Xiangshan County, Ningbo City, Zhejiang Province.
[0040] The main reagents are shown in Table 1, and all reagents are of analytical grade.
[0041] Table 1 Main reagents
[0042]
[0043] Example 1
[0044] The crude extract of hollyhock leaf saponin was prepared by the following method:
[0045] S1. Crush 50 g of holly leaves and add 80% ethanol solution in a round-bottom flask at a solid-liquid ratio of 1:15. Heat and reflux at 75°C for extraction twice, each time for 1.5 h. Centrifuge at a speed of 10,000 r / min for 15 min for filtration, and combine the filtrates to obtain a mixed extract;
[0046] S2, the mixed extract in step S1 is concentrated under the conditions of temperature of 50°C and pressure of -0.09Mpa until the mixed extract has no alcohol taste and the relative density is 1.12g / cm at room temperature. 3 , add 1 / 3 volume of petroleum ether of the concentrate to extract and remove impurities such as lipids and pigments, extract 3 times, vacuum freeze-dry to a water content of 5% under the conditions of pressure of -0.09Mpa and temperature of -40°C, and then store in the dark to obtain a crude extract of holly leaf saponin.
[0047] Example 2
[0048] The crude extract of hollyhock leaf saponin is prepared by the following method:
[0049] Pretreatment: Take 250g AB-8 macroporous adsorption resin, soak it in 95% ethanol for 24h, and then install it in a wet column. Wash the column with 95% ethanol until the effluent is no longer white and turbid, then wash the column with pure water until there is no alcohol taste, pass 4% HCl through the resin column, soak it for 6h, wash it with water until pH=7, then pass 1.0mol / LNaOH through the resin column, soak it for 6h, wash it with water until pH=7, and set aside;
[0050] Loading: Take 2 g of the crude extract of holly leaf saponin obtained in Example 1, suspend it with distilled water, adjust the pH to 4, and adsorb it on an AB-8 resin column chromatography for 5 h. First, elute it with 300 mL of pure water, and then elute it with 80% ethanol until it is almost colorless. Collect the eluate and dry it to obtain the crude extract of holly leaf saponin.
[0051] 1. Determination of total saponin content
[0052] The crude extract of hollyhock leaf saponins obtained in Example 1 and the crude extract of hollyhock column saponins obtained in Example 2 were both tested for total saponin content using a total saponin content test kit. The results are shown in Table 2.
[0053] Table 2 Total saponin content of crude extracts of holly leaf saponin and crude extracts of holly leaf column saponin
[0054]
[0055] It can be seen from Table 2 above that the total saponin content of the crude extract of hollyhock leaf saponin determined by the kit is 41.1 mg / g, and the total saponin content of the crude extract of hollyhock column saponin is 86.1 mg / g.
[0056] 2. Determination of the in vitro inhibition rate of crude extracts of holly leaf saponins and crude extracts of holly leaf column saponins on xanthine oxidase
[0057] 1. Solution preparation
[0058] ① Phosphate buffer solution: Accurately weigh 0.9652 g of potassium dihydrogen phosphate, 6.9424 g of dipotassium hydrogen phosphate and 0.02812 g of EDTA, and make up to 500 mL to obtain phosphate buffer solution (PBS, containing 75 mmol / L phosphate ions and 200 μmol / L EDTA) with a pH of 7.4.
[0059] ②Substrate solution: Accurately weigh 0.01702 g of xanthine into a 100 mL volumetric flask, first dissolve it with 1 mL of 1 mol / L NaOH, and then make up to volume with PBS to obtain a 1.0 mmol / L substrate solution, which is dispensed into 15 mL centrifuge tubes and stored at -20°C.
[0060] ③ Enzyme solution: Take 1 bottle of 100U XOD and dissolve it with 25mL PBS to obtain a XOD stock solution with a concentration of 4U / mL; divide the above stock solution into 200μL per tube and store it in a -20℃ refrigerator. Before each use, take an appropriate amount of the above stock solution and dilute it with PBS to obtain a XOD solution with a concentration of 0.2U / mL.
[0061] ④ Control solution: Accurately weigh an appropriate amount of allopurinol, dissolve it in DMSO, and dilute it with an appropriate amount of PBS to prepare an allopurinol stock solution with a mass concentration of 1000μg / mL for standby use. Use the two-fold dilution method to dilute the above stock solution step by step with PBS containing DMSO as the solvent to prepare control solutions with mass concentrations of 1000, 500, 250, 125, and 62.5μg / mL, respectively, with a DMSO content of less than 2%.
[0062] ⑤ Sample solution: Accurately weigh the crude extracts of holly leaf saponins and holly leaf column saponins, dissolve them with a small amount of DMSO, add PBS to prepare a stock solution with a concentration of 4 mg / mL, and use the two-fold dilution method to dilute the above-mentioned mother solution step by step with PBS containing DMSO as the solvent to prepare sample solutions with mass concentrations of 4000, 2000, 1000, 500, and 250 μg / mL, respectively, and the DMSO content is less than 2%.
[0063] 2. Detection of XOD inhibitory activity
[0064] Under appropriate conditions, xanthine will be catalyzed by XOD to produce uric acid, which has a characteristic absorption peak at 290nm. Based on this, an ELISA reader was used to detect XOD inhibition activity.
[0065] After mixing 200 μL of sample solution and 200 μL of control solution with 200 μL of enzyme solution, incubate at 30°C for 15 min, add 1000 μL of substrate solution to start the reaction, incubate at 30°C for 20 min, and then add 200 μL of 1 mol / L HCl solution to terminate the reaction. Take 200 μL of the above reaction solution, use a microplate reader to detect the absorbance (OD) of each reaction solution at 290 nm, and take the difference between this value and the OD value at 0 min of incubation as the detection result. Each sample is operated in parallel 3 times. The OD value of the blank sample (PBS solution containing 2% DMSO to determine the maximum reaction activity of the enzyme) is detected by the same method, and the inhibition rate is calculated, inhibition rate (%) = (1-average OD value of the test sample / average OD value of the blank sample) × 100%.
[0066] 3. IC 50 Calculation
[0067] Graphpadprism6.0 software was used for data analysis. The (mass) concentration (c) of crude extract of holly leaf saponin and crude extract of holly leaf column saponin (or allopurinol) was used as the horizontal axis, and the corresponding inhibition rate was used as the vertical axis for linear fitting to calculate the drug concentration when half of XOD was inhibited, that is, IC 50 , the specific measurement results can be found in Figure 1-3 .
[0068] Depend on Figure 1-3 It can be seen that within the concentration range, the crude extracts of holly leaf saponins and holly leaf column saponins have a concentration-dependent inhibitory activity on xanthine oxidase in vitro, and their IC 50 They were 239 μg / mL and 1667 μg / mL respectively, indicating that they both have certain in vitro inhibitory activity against xanthine oxidase, among which the crude extract of holly leaf saponin has more potential. Therefore, this crude extract of holly leaf saponin can be used as a health product for lowering uric acid and relieving gout.
[0069] 3. Determination of ABTS free radical scavenging rate
[0070] The antioxidant capacity of crude extracts of holly leaf saponins and crude extracts of holly leaf column saponins was determined using the ABTS free radical scavenging ability test kit. The specific test results are shown in Figure 4-6 .
[0071] Depend on Figure 4-6 It can be seen that within the concentration range, the crude extracts of holly leaf saponins and holly leaf column saponins showed concentration-dependent ABTS free radical scavenging rates, and their IC 50 The concentrations of glycosides in the leaves of hollyhock were 306.2 μg / mL and 287.6 μg / mL, respectively. Compared with the positive control (vitamin C), it had a certain scavenging ability, reflecting that the hollyhock leaf saponin extract had a potential antioxidant effect.
[0072] 4. DPPH free radical scavenging rate determination
[0073] 1. Solution preparation
[0074] ①DPPH solution: Accurately weigh 0.00200g DPPH, add 5.072ml anhydrous ethanol, shake to dissolve, and prepare 1mmol / L DPPH solution, which is prepared before use.
[0075] ②Control solution: Accurately weigh an appropriate amount of vitamin C, add pure water and ultrasonically dissolve it to prepare a vitamin C mother solution with a mass concentration of 4000μg / mL. Use the two-fold dilution method to dilute the above mother solution step by step to prepare control solutions with mass concentrations of 4000, 2000, 1000, 500, 250, 125, 62.5, 31.25, and 15.625μg / mL.
[0076] ③Sample solution: Accurately weigh the crude extracts of holly leaf saponins and holly leaf column saponins, dissolve them in pure water to prepare a stock solution with a concentration of 4000 μg / mL. Use the two-fold dilution method to dilute the above-mentioned mother solution step by step to prepare sample solutions with mass concentrations of 4000, 2000, 1000, 500, 250, 125, and 62.5 μg / mL, respectively.
[0077] 2. Determination of DPPH free radical scavenging rate
[0078] Take 300 μl of sample solution, add 300 μl of 1 mmol / L DPPH solution, mix well and let stand for 30 min, and measure the absorbance (A) at 517 nm. 样品 ). At the same time, take 300 μl of sample solution, add 300 μl of anhydrous ethanol, and measure the absorbance at 517 nm (A 对照 ). Mix 300 μl of pure water with 300 μl of 1 mmol / L DPPH solution and measure the absorbance at 517 nm (A 空白). The DPPH removal rate of the sample solution was calculated according to the following formula:
[0079] DPPH clearance rate (%) = [A 空白 -(A 样品 -A 对照 )]÷A 空白 ×100%
[0080] 3. IC 50 Calculation
[0081] Graphpadprism6.0 software was used for data analysis. Linear fitting was performed with the (mass) concentration (c) of crude extract of holly leaf saponin and crude extract of holly leaf column saponin (or vitamin C) as the horizontal axis and the corresponding clearance rate as the vertical axis to calculate the drug concentration at which half of DPPH was inhibited, i.e., IC 50 , the specific measurement results can be found in Figure 7-9 .
[0082] Depend on Figure 7-9 It can be seen that within the concentration range, the crude extracts of holly leaf saponins and holly leaf column saponins showed concentration-dependent scavenging rates for DPPH free radicals, and their IC 50 The results showed that the extract had good scavenging ability and the scavenging rate of crude extract of hollyhock leaf saponin was better than that of crude extract of hollyhock leaf column saponin, and both of them had potential antioxidant capacity.
[0083] The present invention studies the in vitro inhibitory activity of crude extracts of holly leaf saponins and crude extracts of holly leaf column saponins on xanthine oxidase, as well as the scavenging ability of ABTS and DPPH free radicals, and finds that both have certain xanthine oxidase inhibitory ability and antioxidant activity, among which crude extracts of holly leaf saponins have better xanthine oxidase inhibitory ability and higher potential for lowering uric acid; and both have good antioxidant capacity. Therefore, crude extracts of holly leaf saponins can be used to prepare health products for preventing / treating diseases related to xanthine oxidase; crude extracts of holly leaf saponins and crude extracts of holly leaf column saponins can both be used in the development of antioxidant products.
[0084] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.
Claims
1. A saponin extract having uric acid regulating and antioxidant activities, characterized in that: Prepared by the following method: S1. According to the solid-liquid ratio of 1:12-18, crush the holly leaves, add the ethanol solution, stir and mix evenly, heat and reflux to extract 2-3 times, filter and combine the filtrate to obtain a mixed extract; S2. The mixed extract in step S1 is concentrated under reduced pressure, and the lipid and pigment impurities are removed by petroleum ether extraction. The mixture is freeze-dried in vacuum and stored away from light to obtain a crude extract of holly leaf saponin.
2. The saponin extract having uric acid regulating and antioxidant activities according to claim 1, characterized in that The volume fraction of the ethanol solution in step S1 is 75-85%.
3. The saponin extract having uric acid regulating and antioxidant activities according to claim 1, characterized in that In step S1, the extraction temperature is 70-75° C., and the extraction time is 1.5-1.8 h.
4. The saponin extract having uric acid regulating and antioxidant activities according to claim 1, characterized in that The filtering in step S1 refers to: centrifuging at a speed of 8000-10000 r / min for 10-15 min.
5. The saponin extract having uric acid regulating and antioxidant activities according to claim 4, characterized in that: The reduced pressure concentration in step S2 refers to: under the conditions of a temperature of 50-60°C and a pressure of -0.1-0 MPa, concentrating the mixed extract until there is no alcohol taste and the relative density is 1.02-1.20 g / cm at room temperature. 3 .
6. The saponin extract having uric acid regulating and antioxidant activities according to claim 4, characterized in that In step S2, the extraction with petroleum ether to remove impurities means that the volume ratio of the concentrate to petroleum ether is 1:0.3-0.4, and the extraction is performed 2-3 times.
7. The saponin extract having uric acid regulating and antioxidant activities according to claim 1, characterized in that: The vacuum freeze drying in step S2 refers to freeze drying to a water content of no more than 8% under the conditions of a pressure of -0.1-0 MPa and a temperature of -35 to -40°C.
8. The saponin extract having uric acid regulating and antioxidant activities according to claim 1, characterized in that The step S2 also includes a further purification process.
9. The saponin extract having uric acid regulating and antioxidant activities according to claim 8, characterized in that The further purification step is specifically The following steps are involved: A1: Take AB-8 macroporous adsorption resin, soak it in 95% ethanol for 24 hours, and then install it in a column by wet method. Wash the column with 95% ethanol until the effluent is no longer white and turbid, then wash the column with pure water until there is no alcohol smell, pass 4% HCl through the resin column, soak it for 6 hours, wash it with water until pH = 7, then pass 1.0 mol / L NaOH through the resin column, soak it for 6 hours, wash it with water until pH = 7, and set aside; A2: Take the crude extract of holly leaf saponin, suspend it with distilled water, adjust the pH to 4, adsorb it on AB-8 resin column chromatography for 5 hours, first elute it with pure water, then elute it with 80% ethanol until it is almost colorless, collect the eluate and dry it to obtain the crude extract of holly leaf saponin.
10. Use of a saponin extract having uric acid regulating and antioxidant activities according to any one of claims 1 to 9, characterized in that: The saponin extract is used in preparing products for preventing and treating xanthine oxidase-related diseases; and is used in preparing anti-oxidation and anti-aging products.