Method for enhancing xanthine oxidase activity inhibition of tartary buckwheat flavone extract

Abstract

The invention discloses a method for enhancing xanthine oxidase activity inhibition of a tartary buckwheat flavone extract. The method comprises the following steps: mixing choline chloride and citric acid according to a molar ratio of (1-1.5): 1, heating and stirring to form clear and transparent natural ionic liquid, and adding ultrapure water to dilute the natural ionic liquid to form reaction liquid; dissolving the tartary buckwheat flavone extract in the reaction solution, and uniformly mixing to prepare a mixed solution; putting the mixed solution into a pulsed electric field for treatment; and adding ultrapure water into the mixed solution treated by the pulsed electric field, refrigerating, centrifugally separating the obtained precipitate, washing, and freeze-drying. According to the method, flavone glycoside in the tartary buckwheat flavone extract can be converted into flavone aglycone in a green and efficient mode within a short time so as to enhance the activity of the tartary buckwheat flavone extract for inhibiting xanthine oxidase, and after treatment is finished, the natural ionic liquid can be recycled and reused, and no waste liquid is generated.

Description

Technical Field

[0001] The invention relates to the modification of tartary buckwheat flavonoids extract, and in particular to a method for enhancing the xanthine oxidase inhibition activity of the tartary buckwheat flavonoids extract. Background Art

[0002] Gout is usually a joint disease caused by abnormal purine and uric acid metabolism. Severe gout can form uric acid stones in the joints, causing joint deformation and posing a serious threat to human health. Xanthine oxidase (XOD) is mainly responsible for purine metabolism in the human body and plays an important role in maintaining uric acid balance in the body. Studies have shown that by inhibiting the activity of xanthine oxidase, the synthesis of uric acid in the body can be significantly reduced, thereby achieving the purpose of preventing and alleviating hyperuricemia. Currently, drugs such as allopurinol and febuxostat are used on the market to inhibit XOD activity, but long-term use of these drugs can cause serious side effects and affect the patient's physical health. Therefore, the development of natural XOD inhibitors has aroused the interest of researchers.

[0003] Flavonoids have a variety of biological activities. In recent years, studies have also shown that flavonoids can inhibit XOD activity and have the potential to be used as natural XOD inhibitors. Tartary buckwheat (Fagopyrum tataricum (L.) Gaertn.) flavonoid extract is a common flavonoid extract with antioxidant, anti-inflammatory, antiviral and cancer cell proliferation inhibition functions. Since the main component of tartary buckwheat flavonoid extract is the macromolecular flavonoid glycoside rutin, its ability to bind to XOD is weak, thereby limiting its ability to inhibit XOD activity. Therefore, the development of a method that can enhance the inhibitory activity of tartary buckwheat flavonoid extracts in XOD is of great significance for expanding the application scenarios of tartary buckwheat flavonoid extracts such as flavonoid glycosides as the main component.

[0004] Chinese invention patent application CN116965538A discloses a method for enhancing the inhibitory activity of Sophora japonica seed on xanthine oxidase by acid-heat treatment, wherein the flavonoid glycosides in Sophora japonica seed are degraded and converted into flavonoid aglycones with better activity by acid-heat treatment (10% oxalic acid solution), thereby enhancing the ability of Sophora japonica seed to inhibit XOD. However, the method has complicated steps and needs to be dried with high temperature hot air for a long time (200°C, 90min), which has high energy consumption. In addition, the acid solution used in the method cannot be recycled, which does not meet the concept of green production. Summary of the invention

[0005] In order to overcome the deficiencies and defects of the prior art, the purpose of the present invention is to provide a method for enhancing the inhibition of xanthine oxidase activity of tartary buckwheat flavonoid extract in an efficient and green manner, so as to provide a reference for expanding the application scenarios of tartary buckwheat flavonoid extract.

[0006] The purpose of the present invention is achieved by the following measures:

[0007] A method for enhancing the inhibition of xanthine oxidase activity by tartary buckwheat flavonoid extract, comprising the following steps:

[0008] 1) mixing choline chloride and citric acid in a molar ratio of (1-1.5):1, heating and stirring until a clear and transparent natural ionic liquid is formed, and adding ultrapure water to dilute the natural ionic liquid to form a reaction solution;

[0009] 2) dissolving the tartary buckwheat flavonoid extract in the reaction solution and mixing well to prepare a mixed solution;

[0010] 3) placing the mixed solution in a pulsed electric field for treatment;

[0011] 4) adding ultrapure water to the mixed solution treated with the pulsed electric field, refrigerating, centrifuging the obtained precipitate, washing and freeze-drying, thereby obtaining a tartary buckwheat flavonoid extract having enhanced inhibition of xanthine oxidase activity.

[0012] To further achieve the purpose of the present invention, preferably, in step 1), the heating temperature is 90-95°C.

[0013] Preferably, in step 1), the heating is water bath heating.

[0014] Preferably, in step 1), the mass concentration of the reaction solution is 95-100%.

[0015] Preferably, in step 2), the concentration of the tartary buckwheat flavonoid extract in the mixed solution is 10-50 mg / mL.

[0016] Preferably, in step 3), the pulse electric field treatment conditions are: electric field strength is 1.0-1.5 kV / cm, frequency is 20-30 Hz, pulse width is 20-40 μs, and pulse number is 1600-2400 times.

[0017] Preferably, in step 2), the amount of ultrapure water added is 6-8 times the amount of the mixed solution after the pulse electric field treatment.

[0018] Preferably, in step 4), the refrigeration is refrigeration at a low temperature of 4-8° C. for 1-3 hours; and the washing is washing with ultrapure water for 2-4 times.

[0019] Preferably, the liquid after centrifugal separation is collected and poured into a rotary evaporator to separate the water in the mixed liquid by rotary evaporation; after all the water in the mixed liquid is evaporated and separated, the collected high-viscosity component is the successfully recovered natural ionic liquid component.

[0020] Preferably, in step 4), the rotary evaporation temperature is 45-55°C.

[0021] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0022] 1) The present invention can enhance the ability of the tartary buckwheat flavonoid extract to inhibit xanthine oxidase by directional regulation of the ratio of flavonoid glycosides and aglycones in the tartary buckwheat flavonoid extract, thereby expanding the application scenarios of the tartary buckwheat flavonoid extract.

[0023] 2) The present invention utilizes a pulsed electric field combined with a natural ionic liquid method to efficiently and greenly convert flavonoid glycosides in the buckwheat flavonoid extract into flavonoid aglycones. Compared with the acid-heat treatment method reported in existing patent literature, the entire process takes only a few minutes, does not require long-term high-temperature treatment, and is more energy-efficient. In addition, the natural ionic liquid used in the present method can be recovered after treatment, and no acidic waste liquid is generated. DETAILED DESCRIPTION

[0024] In order to better understand the present invention, the present invention is further described below in conjunction with embodiments, but the embodiments of the present invention are not limited thereto.

[0025] Natural ionic liquid is an ionic liquid formed by hydrogen bond donors and hydrogen bond acceptors. The ionic liquid has low preparation cost, is non-toxic and recyclable, and is a new type of green solvent. By adjusting the preparation ratio, the natural ionic liquid formed by choline chloride and citric acid has a strong hydrogen supply capacity, and can replace strong acid as a reaction solvent for converting flavonoid glycosides in tartary buckwheat flavonoid extracts into flavonoid aglycones. The present invention finds that the natural ionic liquid dissolved in the tartary buckwheat flavonoid extract is placed in an electric field for treatment. Under the action of the electric field, the glycosidic bonds of the flavonoid glycosides in the tartary buckwheat flavonoid extract absorb the electric field energy and vibrate, reducing the energy required for the glycosidic bond breaking, and significantly promoting the conversion of flavonoid glycosides in the tartary buckwheat extract into flavonoid aglycones; at the same time, the particle diffusion coefficient in the reaction system is also increased under the action of the electric field, so that the hydrogen ions in the system are more likely to contact the glycosidic bonds of the flavonoid glycosides, significantly promoting the cleavage of the glycosidic bonds, and increasing the proportion of flavonoid aglycones in the tartary buckwheat extract. After the electric field treatment and conversion are completed, water is added to destroy the hydrogen bonds of the ionic liquid, reducing the solubility, and the flavonoid extract with poor water solubility will precipitate at the bottom for easy centrifugal separation. After the water in the ionic liquid is removed, the ionic liquid can be recovered. Therefore, the proportion of flavonoid aglycones in the tartary buckwheat flavonoid extract after the electric field synergistic natural ionic liquid conversion increases, and its ability to bind to XOD is stronger, which can more effectively inhibit the activity of XOD, thereby achieving the purpose of enhancing the inhibition of XOD activity of the tartary buckwheat flavonoid extract.

[0026] In view of the shortcomings of the prior art, the present invention uses natural ionic liquids in combination with pulsed electric fields to provide an efficient, green and environmentally friendly method to enhance the ability of tartary buckwheat flavonoid extracts, such as extracts with flavonoid glycosides as the main component, to inhibit XOD; specifically, a method of the present invention for enhancing the inhibition of xanthine oxidase activity of tartary buckwheat flavonoid extracts comprises the following steps:

[0027] 1) mixing choline chloride and citric acid in a molar ratio of (1-1.5):1, heating and stirring until a clear and transparent natural ionic liquid is formed, and adding ultrapure water to dilute the natural ionic liquid to form a reaction solution;

[0028] 2) dissolving the tartary buckwheat flavonoid extract in the reaction solution and mixing well to prepare a mixed solution;

[0029] 3) placing the mixed solution in a pulsed electric field for treatment;

[0030] 4) adding ultrapure water to the mixed solution treated with the pulsed electric field, refrigerating, centrifuging the obtained precipitate, washing and freeze-drying, thereby obtaining a tartary buckwheat flavonoid extract having enhanced inhibition of xanthine oxidase activity.

[0031] Among the technical measures, the core measure is to form a natural ionic liquid with choline chloride and citric acid in a suitable molar ratio, and the purpose of the invention is achieved by cooperating with the action of a pulse electric field. The specific treatment process in the pulse electric field can be obtained by combining the purpose of the invention with a general pulse electric field process; as for the dosage ratio of buckwheat flavonoid extract and natural ionic liquid, the amount of ultrapure water added to the mixed liquid after pulse electric field treatment, and the processes of refrigeration, centrifugation, freeze-drying, etc., they can be obtained by combining the purpose of the invention with relevant practices in the prior art.

[0032] In the embodiment, the pulse electric field device used is the PEF-EX-19002 pulse electric field device produced by Guangzhou Xin'an Food Technology Co., Ltd.

[0033] In the embodiment, the preparation method of the bitter buckwheat extract is as follows: bitter buckwheat powder is subjected to 80% ethanol 80°C hydrothermal extraction for 3 hours and then filtered to obtain a crude liquid, the crude liquid is purified by an AB-8 purification column, the ethanol is removed by rotary evaporation, and then freeze-dried to obtain a bitter buckwheat extract. According to high performance liquid chromatography analysis, the main components of the bitter buckwheat extract are flavonoid glycoside rutin and flavonoid aglycone quercetin.

[0034] It should be noted that choline chloride and oxalic acid are solid when they exist alone. Only when the two are mixed and heated in a certain molar ratio will an ionic liquid be formed. At this time, the mixture changes from solid to liquid, and no water needs to be added in this process.

[0035] Example 1

[0036] A method for enhancing the inhibition of xanthine oxidase activity by tartary buckwheat flavonoid extract, comprising the following steps:

[0037] Preparation of natural ionic liquid: Choline chloride and oxalic acid are mixed in a molar ratio of 1:1, and then stirred in a 90°C water bath until a clear and transparent natural ionic liquid is formed. The natural ionic liquid is configured with ultrapure water to a reaction solution with a concentration of 95% for later use.

[0038] Sample preparation: Dissolve 100 mg of tartary buckwheat flavonoid extract in 10 ml of the prepared natural ionic liquid and stir evenly to prepare a 10 mg / mL sample solution for later use.

[0039] Pulse electric field treatment: The prepared sample is placed in a pulse treatment chamber, and the treatment conditions are: electric field strength is 1.0 kV / cm, frequency is 20 Hz, pulse width is 20 μs, and pulse number is 1600 times.

[0040] Separation of samples: Add 6 times the volume of ultrapure water to the natural ionic liquid sample treated with the pulsed electric field, and then refrigerate at 4°C for 1 hour. Centrifuge the precipitate obtained after refrigeration, wash it twice with ultrapure water, and then freeze-dry it to obtain the treated buckwheat flavonoid extract.

[0041] Recycling natural ionic liquid: Collect the liquid after centrifugation, pour it into a rotary evaporator, and separate the water in the mixed liquid by rotary evaporation at 45°C. After all the water in the mixed liquid is evaporated and separated, the collected high-viscosity component is the successfully recovered natural ionic liquid component.

[0042] Example 2

[0043] A method for enhancing the inhibition of xanthine oxidase activity by tartary buckwheat flavonoid extract, comprising the following steps:

[0044] Preparation of natural ionic liquid: Choline chloride and oxalic acid were mixed in a molar ratio of 1.25:1, and then stirred in a 93°C water bath until a clear and transparent natural ionic liquid was formed. The natural ionic liquid was prepared with ultrapure water into a reaction solution with a concentration of 98% for later use.

[0045] Sample preparation: Dissolve 250 mg of tartary buckwheat flavonoid extract in 10 mL of the previously prepared natural ionic liquid and stir evenly to prepare a 25 mg / mL sample solution for use.

[0046] Pulsed electric field treatment: The prepared sample was placed in a pulse treatment chamber, and the treatment conditions were an electric field strength of 1.25 kV / cm, a frequency of 25 Hz, a pulse width of 30 μs, and a pulse number of 2000 times.

[0047] Separation of samples: Add 7 times the volume of ultrapure water to the natural ionic liquid sample treated with the pulsed electric field, and then refrigerate at 6°C for 2 hours. Centrifuge the precipitate, wash it with ultrapure water 3 times, and then freeze-dry it to obtain the treated buckwheat flavonoid extract.

[0048] Recycling natural ionic liquid: Collect the liquid after centrifugation, pour it into a rotary evaporator, and separate the water in the mixed liquid by rotary evaporation at 50°C. After all the water in the mixed liquid is evaporated and separated, the collected high-viscosity component is the successfully recovered natural ionic liquid component.

[0049] Example 3

[0050] A method for enhancing the inhibition of xanthine oxidase activity by tartary buckwheat flavonoid extract, comprising the following steps:

[0051] Preparation of natural ionic liquid: Choline chloride and oxalic acid were mixed in a molar ratio of 1.5:1, and then stirred in a 95°C water bath until a clear and transparent natural ionic liquid was formed. The natural ionic liquid was configured into a reaction solution with a concentration of 100% for later use.

[0052] Sample preparation: Dissolve 500 mg of tartary buckwheat flavonoids extract in 10 mL of the previously prepared natural ionic liquid and stir evenly to prepare a 50 mg / mL sample solution for later use.

[0053] Pulsed electric field treatment: The prepared sample was placed in a pulse treatment chamber, and the treatment conditions were as follows: electric field strength of 1.50 kV / cm, frequency of 30 Hz, pulse width of 40 μs, and pulse number of 2400 times.

[0054] Separation of samples: Add 8 times the volume of ultrapure water to the natural ionic liquid sample treated with the pulsed electric field, and then refrigerate at 8°C for 3 hours. Centrifuge the precipitate, wash it with ultrapure water 4 times, and then freeze-dry it to obtain the treated buckwheat flavonoid extract.

[0055] Recycling natural ionic liquid: Collect the liquid after centrifugation, pour it into a rotary evaporator, and separate the water in the mixed liquid by rotary evaporation at 55°C. After all the water in the mixed liquid is evaporated and separated, the collected high-viscosity component is the successfully recovered natural ionic liquid component.

[0056] Comparative Example 1

[0057] Unprocessed flavonoid extract of Tartary Buckwheat.

[0058] Comparative Example 2

[0059] Preparation of natural ionic liquid: Choline chloride and oxalic acid are mixed in a molar ratio of 1:1, and then stirred in a 90°C water bath until a clear and transparent natural ionic liquid is formed. The natural ionic liquid is configured with ultrapure water to a reaction solution with a concentration of 95% for later use.

[0060] Sample preparation: Dissolve 100 mg of tartary buckwheat flavonoid extract in 10 ml of the prepared natural ionic liquid and stir evenly to prepare a 10 mg / mL sample solution for later use.

[0061] Sample processing: Place the prepared samples at room temperature and let them stand for 60 minutes to complete the processing.

[0062] Separation of samples: Add 6 times the volume of ultrapure water to the treated natural ionic liquid sample, and then refrigerate at 4°C for 1 hour. Centrifuge the precipitate, wash it twice with ultrapure water, and then freeze-dry it to obtain the treated buckwheat flavonoid extract.

[0063] Recycling natural ionic liquid: Collect the liquid after centrifugation, pour it into a rotary evaporator, and separate the water in the mixed liquid by rotary evaporation at 45°C. After all the water in the mixed liquid is evaporated and separated, the collected high-viscosity component is the successfully recovered natural ionic liquid component.

[0064] Comparative Example 3

[0065] Preparation of natural ionic liquid: Choline chloride and oxalic acid were mixed in a molar ratio of 1.25:1, and then stirred in a 93°C water bath until a clear and transparent natural ionic liquid was formed. The natural ionic liquid was prepared with ultrapure water into a reaction solution with a concentration of 98% for later use.

[0066] Sample preparation: Dissolve 250 mg of tartary buckwheat flavonoid extract in 10 mL of the previously prepared natural ionic liquid and stir evenly to prepare a 25 mg / mL sample solution for use.

[0067] Sample processing: Place the prepared samples at room temperature and let them stand for 60 minutes to complete the processing.

[0068] Separation of samples: Add 7 times the volume of ultrapure water to the treated natural ionic liquid sample, and then refrigerate at 6°C for 2 hours. Centrifuge the precipitate, wash it with ultrapure water 3 times, and then freeze-dry it to obtain the treated buckwheat flavonoid extract.

[0069] Recycling natural ionic liquid: Collect the liquid after centrifugation, pour it into a rotary evaporator, and separate the water in the mixed liquid by rotary evaporation at 50°C. After all the water in the mixed liquid is evaporated and separated, the collected high-viscosity component is the successfully recovered natural ionic liquid component.

[0070] Comparative Example 4

[0071] Preparation of natural ionic liquid: Choline chloride and oxalic acid were mixed in a molar ratio of 1.5:1, and then stirred in a 95°C water bath until a clear and transparent natural ionic liquid was formed. The natural ionic liquid was configured into a reaction solution with a concentration of 100% for later use.

[0072] Sample preparation: Dissolve 500 mg of tartary buckwheat flavonoids extract in 10 mL of the previously prepared natural ionic liquid and stir evenly to prepare a 50 mg / mL sample solution for later use.

[0073] Sample processing: Place the prepared samples at room temperature and let them stand for 60 minutes to complete the processing.

[0074] Separation of samples: Add 8 times the volume of ultrapure water to the treated natural ionic liquid sample, and then refrigerate at 8°C for 3 hours. Centrifuge the precipitate, wash it with ultrapure water 4 times, and then freeze-dry it to obtain the treated buckwheat flavonoid extract.

[0075] Recycling natural ionic liquid: Collect the liquid after centrifugation, pour it into a rotary evaporator, and separate the water in the mixed liquid by rotary evaporation at 55°C. After all the water in the mixed liquid is evaporated and separated, the collected high-viscosity component is the successfully recovered natural ionic liquid component.

[0076] Comparative Example 5

[0077] Pour the untreated buckwheat flavonoid extract into 10% oxalic acid solution and mix well to prepare a reaction solution with a flavonoid extract concentration of 10 mg / mL. After soaking for 120 minutes, filter and separate the solution. Then transfer the extract to an oven for hot air drying at 160°C for 60 minutes, and then continue drying at 180°C for 60 minutes to obtain the treated buckwheat flavonoid extract.

[0078] Comparative Example 6

[0079] Pour the untreated buckwheat flavonoid extract into 10% oxalic acid solution and mix well to prepare a reaction solution with a flavonoid extract concentration of 25 mg / mL. After soaking for 120 minutes, filter and separate the solution. Then transfer the extract to an oven for hot air drying at 160°C for 60 minutes, and then continue drying at 180°C for 60 minutes to obtain the treated buckwheat flavonoid extract.

[0080] Comparative Example 7

[0081] Pour the untreated buckwheat flavonoid extract into 10% oxalic acid solution and mix well to prepare a reaction solution with a flavonoid extract concentration of 50 mg / mL. After soaking for 120 minutes, filter and separate the solution. Then transfer the extract to an oven for hot air drying at 160°C for 60 minutes, and then continue drying at 180°C for 60 minutes to obtain the treated buckwheat flavonoid extract.

[0082] The flavonoid composition and content of Examples 1-3 and Comparative Examples 1-7 were detected by high performance liquid chromatography. The sample was dissolved in chromatography grade methanol and mixed, and then passed through a 0.45 μm nylon membrane for use. The detection equipment was an Agilent 1260 Infinity high performance liquid chromatograph, the chromatographic column model was Thermo Accucore XL C18 (250 mm × 4.6 mm, 4 μm), and 0.2% acetic acid water and acetonitrile were used as the detection mobile phase. The detection procedure was: 0.00-2.00 min, B: 5-5%, 2.00-10.00 min, B: 5-30%, 10.00-12.00 min, B: 30-100%, 12.00-18.00 min, B: 100-100%, 18.00-20.00 min, B: 100-5%, 20.00-25.00 min, B: 5-5%. The flow rate was 0.8 mL / min, the detection wavelength was 210 nm, and the detection temperature was 25°C.

[0083] The ability of Examples 1-3 and Comparative Examples 1-7 to inhibit XOD activity was determined by the following method. The specific steps are as follows: xanthine oxidase was dissolved in Tris-HCl buffer at a pH of 7.4 to prepare a xanthine oxidase standby solution, and then the enzyme solution was incubated at a constant temperature of 37°C for 30 minutes before use. The sample was dissolved in anhydrous ethanol and prepared into different concentrations for use. 2.65 mL of Tris-HCl buffer was added to a 5 mL centrifuge tube and incubated in a 37°C water bath until the buffer temperature reached 37°C. Then 50 μL of enzyme solution and 200 μL of flavonoid samples of different concentrations were added to the centrifuge tube and incubated for another 30 minutes. After the incubation was completed, 400 μL of xanthine solution was added to start the reaction. The reaction time was 190 seconds, and the change in absorbance was recorded every 10 seconds. In the experiment, the concentration of xanthine oxidase in the entire reaction system was 2.5×10 -6 mol / L, and the xanthine concentration was 5.0×10 -4 mol / L. In addition, in order to reduce the effect of sample solvent on xanthine oxidase, the ethanol concentration in the reaction system was controlled not to exceed 1%. Each experiment was repeated three times, and the relative activity of xanthine oxidase was calculated by the following formula:

[0084]

[0085] Wherein, AC0 is the rate of change of the absorbance of the reaction system with time when the inhibitor is added; AC1 refers to the rate of change of the absorbance of the reaction system with time when no inhibitor is added.

[0086] The binding capacity of the samples of Examples 1-3 and Comparative Examples 1-7 to XOD was determined by fluorescence spectroscopy. The fluorescence quenching experiment was used to determine the binding capacity of the samples to xanthine oxidase. An appropriate amount of xanthine oxidase solution was drawn into a 3 cm fluorescent cuvette, and the XOD concentration in the whole system was controlled to be 2.5×10 -6 mol / L. Then gradually add the sample of different concentrations, stir and mix, let stand for 5 minutes, and measure the fluorescence spectrum change of the reaction system in the wavelength range of 300-500nm. During the measurement, the excitation wavelength is set to 280nm, and the excitation and emission slits are 2.5nm.

[0087] The test results of the flavonoid contents of the samples of Examples 1-3 and Comparative Examples 1-7 are shown in Table 1.

[0088] Table 1. Molar ratio of flavonoid glycoside (rutin) and flavonoid aglycone (quercetin) in samples

[0089]

[0090]

[0091] As shown in Table 1, the bitter buckwheat flavonoid extract mainly contains two flavonoid compounds, rutin and quercetin. In the untreated sample (Comparative Example 1), the flavonoid glycoside rutin is the main component, and the flavonoid aglycone quercetin is the secondary component. Through detection and calculation, the molar ratio of these two flavonoid compounds in the untreated bitter buckwheat flavonoid extract is 98%:2% (in percentage). After pulse electric field synergistic natural ionic liquid treatment, natural ionic liquid treatment and acid-heat treatment, the flavonoid glycosides in the bitter buckwheat flavonoid extract are degraded, and part of the rutin is converted into quercetin. As can be seen from Table 1, the proportion of quercetin in the samples of Examples 1-3 is significantly higher than that in the samples of Comparative Examples 2-4, which shows that the use of natural ionic liquid treatment alone can slowly degrade the rutin in the bitter buckwheat extract, but the synergistic treatment of the electric field can significantly improve the efficiency of natural ionic liquid degradation of rutin. In addition, the proportion of quercetin in the samples of Examples 1-3 is also significantly higher than that in Comparative Examples 5-7. Within a treatment time of 80 seconds, the pulsed electric field synergistically with natural ionic liquid can increase the proportion of quercetin in the buckwheat flavonoid extract by more than 40 times, while the acid-heat treatment can only increase the proportion of quercetin in the buckwheat flavonoid extract by 9-15 times within a treatment time of 240 minutes. This indicates that the efficiency of pulsed electric field synergistically with natural ionic liquid treatment in converting rutin into quercetin is much higher than that of acid-heat treatment, and this method does not require long-term high-temperature treatment, and is more green and energy-saving.

[0092] Table 2. IC50 values ​​of samples for inhibition of XOD activity

[0093]

[0094]

[0095] The results of the determination of the ability of Examples 1-3 and Comparative Examples 1-7 to inhibit XOD activity are shown in Table 2. As can be seen from Table 2, the untreated buckwheat flavonoid extract did not show XOD inhibitory activity within the test concentration, and the treated samples all showed XOD inhibitory activity. The buckwheat flavonoid extract treated with pulsed electric field and natural ionic liquid in the tested samples had a lower IC50 value, which was more than 8 times lower than the samples treated with natural ionic liquid alone and acid-heat treatment. The results show that the buckwheat flavonoid extract treated with pulsed electric field and natural ionic liquid has a stronger ability to inhibit XOD activity. This may be because the treatment with pulsed electric field and natural ionic liquid can convert more rutin in the buckwheat flavonoid extract into quercetin with higher activity and stronger binding ability.

[0096] The binding ability of Examples 1-3 and Comparative Examples 1-7 with XOD was determined by fluorescence quenching experiment, and the results are shown in Table 3. As shown in Table 3, at the same concentration (0.3 μg / mL), the untreated tartary buckwheat flavonoid extract is basically unable to quench the XOD fluorescence, while the treated tartary buckwheat flavonoid extract can reduce the fluorescence intensity of XOD, which indicates that the untreated tartary buckwheat flavonoid extract is basically unable to bind to XOD. After treatment, the macromolecular rutin in the tartary buckwheat flavonoid extract is converted into small molecule quercetin, which also increases the binding ability of the extract with XOD, resulting in a decrease in the fluorescence intensity of XOD. The results in Table 3 show that the tartary buckwheat flavonoid extract treated with pulsed electric field and natural ionic liquid is much stronger than the XOD activity of the tartary buckwheat flavonoid extract treated with ionic liquid alone and acid-heat. Because in Examples 1-3, the XOD fluorescence inhibition rates were 77.16%, 76.38% and 76.54%, respectively, which were significantly higher than those in Comparative Examples 2-7 (16.04%, 16.66%, 16.35%, 33.02%, 33.33% and 20.98%). This is because Examples 1-3 contain more small molecule flavonoids quercetin, which can better bind to XOD and enter its active center to inhibit activity compared to rutin, resulting in the buckwheat flavonoid extract samples of Examples 1-3 having stronger XOD activity inhibition ability.

[0097] Table 3. Sample fluorescence quenching experimental results

[0098]

[0099] The implementation methods of the present invention are not limited to the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. A method for enhancing the inhibition of xanthine oxidase activity by tartary buckwheat flavonoid extract, characterized in that The steps include: 1) mixing choline chloride and citric acid in a molar ratio of (1-1.5):1, heating and stirring until a clear and transparent natural ionic liquid is formed, and adding ultrapure water to dilute the natural ionic liquid to form a reaction solution; 2) dissolving the tartary buckwheat flavonoid extract in the reaction solution and mixing well to prepare a mixed solution; 3) placing the mixed solution in a pulsed electric field for treatment; 4) adding ultrapure water to the mixed solution treated with the pulse electric field, refrigerating, centrifuging the obtained precipitate, washing and freeze-drying, thereby obtaining a tartary buckwheat flavonoid extract with enhanced inhibition of xanthine oxidase activity.

2. The method for enhancing the inhibition of xanthine oxidase activity of tartary buckwheat flavonoid extract according to claim 1, characterized in that: In step 1), the heating temperature is 90-95°C.

3. The method for enhancing the inhibition of xanthine oxidase activity of tartary buckwheat flavonoid extract according to claim 2 or 3, characterized in that: In step 1), the heating is water bath heating.

4. The method for enhancing the inhibition of xanthine oxidase activity of tartary buckwheat flavonoid extract according to claim 1, characterized in that: In step 1), the mass concentration of the reaction solution is 95-100%.

5. The method for enhancing the inhibition of xanthine oxidase activity of tartary buckwheat flavonoid extract according to claim 1, characterized in that: In step 2), the concentration of the tartary buckwheat flavonoid extract in the mixed solution is 10-50 mg / mL.

6. The method for enhancing the inhibition of xanthine oxidase activity of tartary buckwheat flavonoid extract according to claim 1, characterized in that: In step 3), the pulse electric field treatment conditions are: electric field strength is 1.0-1.5 kV / cm, frequency is 20-30 Hz, pulse width is 20-40 μs, and pulse number is 1600-2400 times.

7. The method for enhancing the inhibitory effect of tartary buckwheat flavonoid extract on xanthine oxidase activity according to claim 1, characterized in that: In step 2), the amount of ultrapure water added is 6-8 times that of the mixed solution after the pulse electric field treatment.

8. The method for enhancing the inhibitory effect of tartary buckwheat flavonoid extract on xanthine oxidase activity according to claim 1, characterized in that: In step 4), the refrigeration is refrigeration at a low temperature of 4-8° C. for 1-3 hours; and the washing is washing with ultrapure water for 2-4 times.

9. The method for enhancing the inhibition of xanthine oxidase activity of tartary buckwheat flavonoid extract according to claim 1, characterized in that: The liquid after centrifugal separation is collected and poured into a rotary evaporator to separate the water in the mixed liquid by rotary evaporation; after all the water in the mixed liquid is evaporated and separated, the collected high-viscosity component is the successfully recovered natural ionic liquid component.

10. The method for enhancing the inhibitory effect of tartary buckwheat flavonoid extract on xanthine oxidase activity according to claim 9, characterized in that: In step 4), the rotary evaporation temperature is 45-55°C.

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