A natural solvent extraction and membrane separation process of astragalus glycitein and its application

Abstract

The invention discloses a natural solvent extraction and membrane separation process and application of astragalus glycitein, which relates to the fields of biochemistry and health food technology. The key points of the technical solution are: including preparing a natural low eutectic solvent; under preset conditions, using the natural low eutectic solvent to ultrasonically extract the dry sample powder of astragalus to obtain a crude extract; introducing the crude extract into a preset membrane separation device, filtering to obtain a retained liquid; vacuum drying the obtained retained liquid to obtain crude astragalus glycitein. The invention prepares a natural low eutectic solvent system, extracts glycitein from astragalus under the action of microwave assistance, and finally separates and enriches glycitein by combining membrane separation technology, and compares the product purity with the traditional separation and purification process of glycitein, thereby obtaining glycitein with higher purity than the traditional process, and at the same time solves the environmental pollution problems of traditional organic solvent extraction, separation and purification, organic solvent residues and low extraction and separation efficiency.

Description

Technical Field

[0001] The invention relates to the technical field of biochemistry and health food, and more specifically to a natural solvent extraction and membrane separation process of astragalus glycitein and its application. Background Art

[0002] Astragalus is the dried rhizome of Astragalus mongolica or Astragalus capsulae, a plant of the Leguminosae family. It is sweet and slightly warm in taste and belongs to the lung and spleen meridians. It has the effects of replenishing qi and raising yang, replenishing qi and strengthening the exterior, promoting diuresis and relieving swelling, relieving stagnation and relieving arthritis, removing toxins and draining pus, and healing sores and promoting tissue regeneration. Traditional Chinese medicine often uses it to treat symptoms caused by deficiency, such as fatigue caused by qi deficiency, poor appetite and loose stools caused by spleen deficiency, spontaneous sweating due to exterior deficiency, and sores that do not heal for a long time after ulceration.

[0003] Astragalus is a plant that is both a medicine and food, and contains a variety of ingredients such as polysaccharides, saponins, flavonoids, amino acids, alkaloids, etc. Combining modern pharmacological research and clinical applications, it is found that Astragalus has significant effects in anti-tumor, protecting cardiovascular and cerebrovascular vessels, improving the body's immune function, protecting metabolic (liver and kidney) tissues, regulating blood pressure, and preventing and treating osteoporosis. Astragalus glycitein has strong antioxidant properties that can prevent tissue damage caused by free radicals, and is beneficial for protecting the cardiovascular system and promoting metabolism. It is considered to be a powerful phytochemical for preventing metabolic diseases.

[0004] In the traditional extraction and separation of glycitein, organic solvents or pure water are usually used to extract plant samples. This process has the disadvantages of long process, high cost of organic solvents or low extraction rate, and toxic solvent residues in the product. Natural low eutectic solvents are a kind of low eutectic solvents composed of natural substances with good biocompatibility as hydrogen bond donors and acceptors, such as glycerol, amino acids and sugars. They have the advantages of low cost, easy preparation and environmental friendliness, and have been widely used in many fields. Due to its good biocompatibility and relatively safe use, it is particularly suitable for the food and medical fields. The separation process of ultrafiltration membranes is relatively simple, there is no phase conversion, and the composition can be kept stable to a great extent. Therefore, it has been widely used in the field of modern Chinese medicine production. In addition, the separation and extraction process of ultrafiltration membrane is a purely physical process, without adding chemical reagents, which has little effect on the effective ingredients of the drug, can maintain most of the efficacy, and also reduce pollution to the environment; finally, the ultrafiltration membrane can filter unstable micelles, macromolecules and other impurities, pyrogens, tannins in the solution, and improve the clarity of the liquid preparation; its application operation is simple, easy to realize automatic operation, saving labor while being easy to maintain, and safe and stable operation. With the continuous improvement of modern Chinese medicine extraction, separation and purification technology, ultrafiltration membrane has many successful cases in the field of Chinese medicine. And the application of ultrafiltration membrane has obvious advantages over traditional separation and extraction methods, which is conducive to preserving the physiological activity and physical and chemical stability of Chinese medicine, and improving the taste and quality of Chinese medicine preparations.

[0005] Therefore, the present invention provides a natural solvent extraction and membrane separation process and application of astragalus glycitein. By jointly using a natural low eutectic solvent and a membrane separation system, a method for separating and enriching glycitein from astragalus is explored, and an in vitro enzyme inhibition test, a hyperuricemia mouse model and a clinical trial are designed to analyze and verify the difference in hyperuricemia intervention effect of the glycitein obtained under the new process conditions compared with that obtained by the traditional method. Summary of the invention

[0006] The purpose of the present invention is to provide a natural solvent extraction and membrane separation process of astragalus glycitein and its application. The present invention prepares a natural low eutectic solvent system, extracts glycitein in astragalus under the action of microwave assistance, finally separates and enriches glycitein in combination with membrane separation technology, and compares the product purity with that of a traditional process for separating and purifying glycitein, thereby obtaining glycitein with higher purity than that of the traditional process, and at the same time solves the problems of environmental pollution, organic solvent residue and low extraction and separation efficiency of traditional organic solvent extraction, separation and purification.

[0007] The above technical purpose of the present invention is achieved through the following technical scheme: a natural solvent extraction and membrane separation process of astragalus glycitein, comprising the following steps:

[0008] S1. preparing a natural deep eutectic solvent;

[0009] S2. Under preset conditions, using a natural low eutectic solvent to ultrasonically extract the Astragalus root dry sample powder to obtain a crude extract;

[0010] S3, introducing the crude extract into a preset membrane separation device, filtering to obtain a retained liquid;

[0011] S4. The obtained retained solution is vacuum dried to obtain crude astragalus glycitein.

[0012] The present invention is further configured as follows: the preparation process of the natural low eutectic solvent in step S1 is: mixing the hydrogen bond donor raw material and the hydrogen bond acceptor raw material in a water bath at 80-90° C. according to a specific molar ratio, and stirring until a uniform transparent liquid is formed to obtain a natural low eutectic solvent.

[0013] The present invention is further configured as follows: the natural low eutectic solvent comprises lactic acid-glycine, citric acid-betaine, glycerol-menthol and glycerol-lysine, and the molar ratios of the hydrogen bond donor raw materials and the hydrogen bond acceptor raw materials of the lactic acid-glycine, citric acid-betaine, glycerol-menthol and glycerol-lysine are 2:1, 2:1, 1:1 and 1:1 respectively.

[0014] The present invention is further configured as follows: the preset conditions in step S2 are: at 85° C., the ultrasonic power is 70 W, the frequency is 50 Hz, the ratio of the astragalus dry sample powder to the natural low eutectic solvent is 1:20, and 1 volume of water is added, and ultrasonic extraction is performed for 0.5 h to obtain a crude extract.

[0015] The present invention is further configured as follows: the membrane separation equipment preset in step S3 includes setting the membrane filtration temperature, membrane material and separation pressure, wherein the membrane separation equipment pumps the crude extract solution through the membrane, so that smaller molecules pass through the pores and larger molecules are retained, and the retained liquid is obtained by filtration.

[0016] The present invention also provides an application of a natural solvent extraction and membrane separation process of astragalus glycitein in the efficient extraction, separation and enrichment of astragalus glycitein.

[0017] In summary, the present invention has the following beneficial effects:

[0018] 1. The natural deep eutectic solvent prepared in the present invention is a solvent composed of natural substances with good biocompatibility as hydrogen bond donors and acceptors, and has the advantages of low cost, easy preparation and environmental friendliness, and can be widely used in many fields. Since the prepared natural deep eutectic solvent has good biocompatibility, it is safe to use and is also suitable for food and medicine. In addition, the present invention can adjust the components of the natural deep eutectic solvent according to the properties of the target extract to achieve targeted extraction to increase the yield of the target component, and the separation process of the ultrafiltration membrane is relatively simple, there is no phase conversion, and the components can be kept stable to a great extent; the process of separation and extraction by ultrafiltration membrane in the present invention is a purely physical process, no chemical reagents are added, and the properties of the target components are little affected, most of the activity can be maintained, and the pollution to the environment is also reduced.

[0019] 2. The present invention adopts a natural low eutectic solvent agent combined with an ultrafiltration membrane to extract, separate and enrich glycitein in astragalus. Compared with traditional ultrasonic-assisted extraction, the extraction method has a significant effect on improving the yield of glycitein, and the purity of the glycitein in astragalus obtained by extracting, separating and enriching the glycitein in the natural low eutectic solvent agent combined with an ultrafiltration membrane is significantly higher than that of the glycitein enriched by ultrasonic-assisted combined macroporous resin. At the same time, in the process of extracting, separating and enriching using a natural low eutectic solvent combined with an ultrafiltration membrane, since natural edible substances are used in the final extraction and enrichment process, and no organic solvent is introduced, the problem of avoiding organic solvent residues can be solved.

[0020] 3. The natural low eutectic solvent system used in the present invention is a stable polymer nanosystem, which can effectively avoid the environmental pollution problems caused by the volatilization of organic solvents. At the same time, the experiments of the present invention effectively demonstrate that the uric acid-lowering effect of the combined separation and enrichment of glycitein on hyperuricemia mice is close to or even better than that of the allopurinol-positive drug, and the uric acid-lowering effect is better than that of the glycitein product prepared by traditional extraction, separation and enrichment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a schematic flow chart of a natural solvent extraction and membrane separation process of astragalus glycitein in an embodiment of the present invention;

[0022] Figure 2 is a bar graph showing the effect of different extraction solvents on the yield of glycitein in the embodiments of the present invention;

[0023] Figure 3 1. is a curve diagram showing the influence of each single factor on the concentration of the purified glycitein in the embodiment of the present invention;

[0024] Figure 4 This is a schematic diagram of the results of BBD optimizing the total content of glycitein in an embodiment of the present invention;

[0025] Figure 5 Schematic diagram of the inhibitory effect of the purified glycitein on xanthine oxidase in an embodiment of the present invention;

[0026] Figure 6 Schematic diagram of the effect of purified glycitein on the expression levels of uric acid reabsorption protein and excretion protein in an embodiment of the present invention. DETAILED DESCRIPTION

[0027] The following is combined with Figure 1-6 The present invention is described in further detail.

[0028] The instruments and equipment used in this embodiment are as follows:

[0029] HPLC analysis was performed on an Agilent 1200 system (Agilent Technologies, USA). The Agilent 1200 system was equipped with a G1354A solvent delivery unit, a G1315B UV-visible photodiode array detector, a G1316A column thermostat, a G1313A autosampler, an Agilent eclipse-xdb C18 (250×4.6 mm, 5 μm) analytical column, and an Agilent HPLC workstation. Membrane separation was performed on a small multifunctional membrane separation device. Ultrafiltration membranes were purchased from GE Healthcare.

[0030] In this embodiment, the HPLC determination conditions are to use an Agilent Eclipse-XDB C18 column (250×4.6 mm, 5 μm). The mobile phase is water (solvent A) and methanol (solvent B). The gradient elution program is: 0-2 min, 20% B; 2-3 min, 40% B; 3-5 min, 55% B; 5-10 min, 90% B; 10-20 min, 20% B. The flow rate is 1.0 mL / min, the column temperature is 25°C, and the detection wavelength is 270 nm.

[0031] The inhibition of xanthine oxidase (XOD) activity was determined as follows: First, 50 μL of 0.1 unit (U) XOD was added to the buffer, 100 μL of the test extract or compound (glycitein) was added to DMSO, mixed at 37°C for 15 min, and 50 μL of 0.6 mM xanthine was added to double distilled water to start the reaction. Incubate at room temperature and immediately measure the absorbance at 295 nm. Allopurinol was used instead of the extract to obtain a positive control result. Each sample was repeated 3 times. The percentage inhibition (%) was calculated with reference to the existing data. For competitive inhibition, the data were analyzed according to the classic Lineweaver-Burk plot.

[0032]

[0033] Where f is the XOD rate, V s and V n Represents the XOD activity (production of uric acid per minute) of the sample and negative control reactions, respectively.

[0034] Example 1: A natural solvent extraction and membrane separation process for astragalus glycitein

[0035] 1) Prepare natural low eutectic solvents, using hydrogen bond donors (HBD) and hydrogen bond acceptors (HBA) as raw materials. All natural low eutectic solvents (NADES) include lactic acid-glycine, citric acid-betaine, glycerol-menthol, and glycerol-lysine. Prepare NADES with different molar ratios of hydrogen bond HBD and HBA. Mix the two components in a water bath at 80-90°C and stir for about 2 hours until a uniform and transparent liquid is formed to obtain a eutectic mixture. NADES preparation and grouping are shown in Table 1:

[0036] Table 1 NADES preparation and grouping

[0037] HBD-HBA Molar Ratio Lactate-Glycine 2:1 Citric Acid-Betaine 2:1 Glycerin-Menthol 1:1 Glycerol-Lysine 1:1

[0038] 2) Screening the best extraction solvent and standard preparation, the dried Astragalus root sample (10g) was ground into powder, and ultrasonic (70W, 50HZ) extraction was performed at 85°C with different types of low eutectic solvents, the solid-liquid ratio was 1:20, and the extraction time was 0.5h. All filtrates were mixed and 1 volume of water was added to obtain a crude extract, and the yield of glycitein was determined by HPLC to determine the best extraction solvent.

[0039] Dissolve 5.0 mg of glycitein in 50 mL of 30% ethanol to prepare a standard solution. All solutions were stored at 4°C for future use. The glycitein yield (Y) was used to evaluate the extraction effect. The calculation formula is as follows:

[0040]

[0041] Among them, m is the total amount of glycitein, m e is the amount of glycitein extracted from Astragalus.

[0042] 3) Membrane separation process and single factor experimental design, 100mL of crude astragalus glycitein extract (glycitein content 0.3±0.1mg / mL) was introduced into the membrane separation equipment. Under the selected pressure and membrane material, the solution was pumped through the membrane; smaller molecules passed through the pores, while larger molecules were retained. Finally, the retained solution was vacuum dried to obtain crude astragalus glycitein.

[0043] Different membrane filtration temperatures (10, 20, 30 and 40°C), membrane materials (UE050, UE005, NF3 and RO2) and separation pressures (0.1, 0.15, 0.2 and 0.3 MPa) were selected to study their effects on the glycitein content in Astragalus.

[0044] 4) Optimization of membrane separation process The response surface methodology was used to optimize the process conditions for separation and preparation of astragalus glycitein. The variables used were: separation pressure (A), temperature (B), membrane material (C), and the total content of astragalus glycitein was used as the response value, as shown in Table 2:

[0045] Table 2 Box-Benhnken design independent variables and levels

[0046]

[0047] The various groupings can be seen in Table 2. The experiments were randomized to minimize the effects of unexplained variability in the observed responses due to systematic errors.

[0048] The least squares multiple regression method was used to investigate the relationship between the independent and dependent variables. After selecting the most accurate model, the analysis of variance test was used, and Fisher's f test was performed at the 95% confidence level to test the statistical significance of the regression coefficients. The surface maps derived from the selected model were used to observe the interaction of the factors.

[0049] Experimental data:

[0050] In this example, four different natural deep eutectic solvents were used to extract glycitein from Astragalus. Figure 2 As shown, the yield of glycitein in glycerol-menthol is the highest at 3.06 mg / g. This is because the polarity of flavonoids is similar to that of the extract. Compared with other NADES, glycitein has a high solubility efficiency in organic NADES, and the yield of glycitein is significantly higher than that of traditional 75% ethanol. Therefore, the glycerol-menthol solvent system is identified as the optimal extraction solvent.

[0051] like Figure 3 As shown, (a) is the effect of different membrane materials on the permeability of glycitein membrane, (b) is the effect of different membrane materials on the concentration of purified glycitein, (c) is the effect of pressure on the permeability of glycitein membrane, (d) is the effect of pressure on the concentration of purified glycitein, (e) is the effect of temperature on the permeability of glycitein membrane, and (f) is the effect of temperature on the concentration of purified glycitein. The effect of membrane material on the total content of glycitein is shown in Figure 3 (a) and Figure 3 As shown in (b), as the membrane type changes from reverse osmosis membrane to nanofiltration membrane to ultrafiltration membrane (the membrane pore size increases successively), the flux of glycitein in Astragalus increases. When treated with ultrafiltration membrane UE050, glycitein is basically passed through the membrane. The concentration of glycitein in the filtrate is 0.282 mg / mL, and its recovery rate is (81.09±0.315)%.

[0052] Effect of separation pressure on total content of glycitein Figure 3 (c) and Figure 3 As shown in (d), the membrane flux increases significantly with the increase of pressure, and when the pressure exceeds 0.25MPa, the membrane flux tends to be stable. At this time, the total glycitein concentration in the filtrate increases significantly until the pressure reaches 0.3MPa. The highest concentration of glycitein in the filtrate is 3.03mg / mL, and its recovery rate is (87.17±0.19)%. The phenomenon shows that the concentration on the membrane surface increases with the increase of pressure, and at the same time, the reverse diffusion flux increases. If the pressure difference continues to increase in a stable state, when the membrane surface concentration reaches a certain value, a gel layer is formed on the membrane surface. After the gel layer is formed, the concentration of the gel no longer changes (given that the conditions remain unchanged), the membrane flux no longer changes significantly with the increase of pressure, and the amount of glycitein passing through does not change significantly.

[0053] Effect of temperature on the total flavonoid content Figure 3 (e) and Figure 3 As shown in (f), when the working temperature increases from 10℃ to 30℃, the membrane flux increases by almost 8 times, and the total glycitein concentration in the filtrate increases significantly with the increase in temperature. After 30℃, the effect of temperature on membrane flux is no longer significant, and the glycitein concentration in the filtrate does not change significantly. This shows that a certain temperature range (30-45℃) is conducive to reducing the viscosity of the liquid to be separated, increasing the redispersion rate of the substance, making it easier for the active ingredient to pass through the ultrafiltration membrane to obtain a better separation and purification effect. In the range of 30-45℃, the concentration of glycitein in the filtrate is 0.259±0.01mg / mL, and its recovery rate is (74.48±0.32)%.

[0054] Example 2: Extraction, separation and purification process experiment

[0055] The optimal ultrasonic-assisted conditions for flavonoid extraction (Luo et al. 2020) were adopted, pure water, solid-liquid ratio of 1:10 (g / mL), ultrasonic power of 150 W, extraction temperature of 60 °C, and extraction time of 60 min. Glycitein in 10 g of astragalus powder was extracted to obtain a crude extract of glycitein. The solvent was rotary evaporated to obtain a concentrate and dried. The calculation formula for the glycitein yield (Y) was the same as formula (1).

[0056] Based on the existing data, a resin (AB-8) and purification conditions suitable for the adsorption and desorption of soybean yellow compounds were selected and modified.

[0057] The crude extract obtained by water extraction was adjusted to a concentration of glycitein between 0.3±0.1mg / mL, and 100mL was loaded onto the resin column at a flow rate of 2mL / min; the adsorption temperature was 35°C and the adsorption time was 4h. The column was first washed with distilled water, and then desorbed with 2BV 80% ethanol, the elution time was 2h, and the elution temperature was 50°C. The flavonoid content in each extract and eluate was analyzed by HPLC. The flavonoid recovery rate and relative content calculation formula are:

[0058]

[0059] Among them, m1 is the amount of glycitein obtained after resin purification, m 总 W is the amount of glycitein in the water extract concentrate; e and W represent the total glycitein content and dry residue content, respectively.

[0060] In this embodiment, BBD was used to optimize the experimental parameters, as shown in Table 3:

[0061] Table 3 Response surface experimental design and results

[0062] Test No. A B C Glycitein concentration (mg / mL) 1 0 0 0 2.321 2 -1 0 1 1.524 3 0 1 1 1.788 4 0 0 0 2.321 5 -1 -1 0 1.6 6 0 -1 1 1.578 7 0 0 0 2.321 8 0 -1 -1 1.783 9 1 1 0 1.732 10 1 0 1 1.59 11 0 0 0 2.321 12 -1 0 -1 1.566 13 1 0 -1 2.55 14 1 -1 0 1.652 15 0 1 -1 1.692 16 -1 1 0 1.69

[0063] Table 3 shows the experimental condition design of the response surface methodology to optimize the extract. The concentration of glycitein (Y) was used as the dependent variable, and pressure (A), temperature (B), and membrane material (C) were used as independent variables. The fitting equation for separating glycitein was Y = 2.32-0.1430A + 0.0361B-0.1389C-0.0025AB-0.2295AC + 0.0752BC-0.2776A 2 -0.3749B 2 -0.2359C 2 . And it was found that significant (p ≤ 0.05) model F values ​​were observed for all responses, but there was no significant lack of fit. This shows that the model is meaningful for evaluating the optimal extraction conditions of glycitein, and it is feasible to use the fitting equation to evaluate the true value. In addition, the determination coefficient R 2 =0.91, adjusted Adj.R 2 It is considered to be an important parameter in determining the adequacy of the model, and Adj.R 2 =0.89. This indicates that the model fitting results have a certain degree of fit with the actual experimental results, and a preliminary prediction can be made through the fitting test results. In the model terms, the linear term AC and the quadratic term (A 2 , B 2 and C 2 ) were all less than 0.05, indicating that they had a significant effect on the extraction of glycitein. The prediction results showed that when the pressure was 0.2MPa, the temperature was 20℃, and the membrane material was UE005, the highest glycitein concentration of 2.321mg / mL would be obtained.

[0064] In this example, the effects of different separation and purification processes on the concentration of glycitein were compared. The results were analyzed using the Design Expert 8.0.6 software. Figure 4 As shown, (a) is the effect of the interaction between temperature and pressure on the purification of glycitein, (b) is the effect of the interaction between temperature and membrane material on the purification of glycitein, and (c) is the effect of the interaction between membrane material and pressure on the purification of glycitein. The optimal conditions for membrane separation and extraction of glycitein from Astragalus are obtained, pressure 0.2MPa (A), temperature (B) 20℃, membrane material (C) UE005. The glycitein concentration obtained under these conditions is 2.321mg / mL. In order to verify the credibility of this process optimization experiment and consider the feasibility of the operation, the separation was repeated three times under the optimal conditions, and the actual separated glycitein concentration was 2.51mg / mL.

[0065] Comparison of the effects of different separation and purification processes on glycitein concentration, the results are shown in Table 4:

[0066] Table 4 Results of separation and purification of astragalus glycitein by ultrasonic water extraction combined with AB-8 macroporous resin

[0067]

[0068] From the results in Table 4, it can be seen that the yield of glycitein in Astragalus by NADES extraction is significantly higher than that by ultrasound-assisted extraction; the membrane separation method increases the purity of glycitein from 3.06% to 30.43%; the traditional macroporous resin only improves the purification rate of the sample by 17.72%. Therefore, NADES is helpful in extracting glycitein from Astragalus, and combined with membrane separation, the purity of glycitein can be improved to a greater extent. Compared with traditional separation and purification methods, NADES can be selected according to the properties of the extraction object to increase the yield of the extraction object, and NADES components are usually green and edible substances, which can reduce environmental pollution and resource waste. Further, by selecting a membrane with a suitable pore size, the separation and enrichment of glycitein can be further improved.

[0069] The xanthine oxidase inhibition effects of purified astragalus glycitein obtained under different process conditions were compared. Figure 5 As shown, (a) shows the effect of glycitein purification obtained under different process conditions on the inhibition rate of xanthine oxidase, (b) shows the effect of allopurinol on the inhibition rate of xanthine oxidase, and (c) shows the inhibition type of membrane-separated glycitein purification on xanthine oxidase. The in vitro XOD inhibition experiment found that the inhibitory effect of astragalus glycitein purification on XOD activity was dose-dependent, with an IC50 of 5.85±0.52μg / mL ( Figure 5 a), but lower than allopurinol, with an IC50 of 1±0.17μg / mL ( Figure 5 b).

[0070] The Lineweaver-Burk diagram was performed using the Michaelis-Menten equation to investigate the inhibition type of XOD by purified glycitein. The slope of the curve is proportional to Km, and the y-intercept represents the 1 / Vmax value. Figure 5 As shown in Figure c, the fitting lines with different slopes and intercepts are all in the third quadrant. The slope and y-intercept of the curve increase with the increase of the amount of purified astragalus glycitein, indicating that Km increases and Vmax decreases. The results show that purified astragalus glycitein is a good mixed inhibitor of XOD.

[0071] Example 3: Animal model and group experiments

[0072] Thirty SPF male SD rats were randomly divided into a control group (M1 group), a potassium oxalate model group (M2 group), a potassium oxalate (PO) combined with allopurinol group (M3 group), a PO combined with membrane-purified glycitein group (M4 group), and a PO combined with resin-purified glycitein group (M5 group), with 6 rats in each group. M2, M3, M4, and M5 were gavaged with 750 mg / kg body weight of PO (dissolved in 0.5% NaCl) every day, and the M1 group was gavaged with the same volume of solvent (0.5% NaCl) for 3 consecutive weeks. Afterwards, the M1 and M2 groups were fed with ordinary feed, the M3 group was fed with allopurinol, the M4 group was fed with membrane-purified glycitein (50 mg / kg), and the M5 group was fed with resin-purified glycitein (50 mg / kg). Blood was drawn from the inner corner of the eye every 7 days. Tissue samples were taken after 21 days. ELISA kits were used to detect mouse serum indicators (UA, CRE, BUN, IL-6, IL-1β, TNF-α).

[0073] The kidney tissue was fixed with formaldehyde for 3 days, embedded in paraffin, and stained with H&E. The photos were taken under a clear field of view with a 200x magnifying glass.

[0074] The detailed grouping is shown in Table 5:

[0075] Table 5 Trial grouping and dosage design

[0076] Group Dosage (mg / kg) Concentration (mg / mL) Number of animals Normal control group (M1) - - 6 Model control group (M2) - - 6 Allopurinol group (M3) 130 6.5 6 Membrane purified glycitein group (M4) 50 4.0 6 Resin-purified glycitein group (M5) 50 8.0 6

[0077] The detection index in this embodiment is: after the last administration, blood is collected from the mouse orbit, centrifuged at 3000 rpm for 10 min, and the supernatant is collected. The serum UA content is detected using an automatic biochemical analyzer according to the instructions of the UA kit.

[0078] Western Blot assay for related protein expression levels was performed as follows: about 100 mg of organ (liver or kidney) was dissolved in 1 mL of radioimmunoprecipitation assay (RIPA) buffer and then homogenized in an ice bath. The organ proteins of each group were obtained in the form of supernatant, which was obtained by centrifugation at 12,000 rpm for 10 min at 4 °C. The protein (1 μg / μL, 15 μL) was heated in a buffer at 100 °C for 5 min and then separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After wet transmembrane, the polyvinylidene fluoride (PVDF) membrane was sealed with 5% skim milk and washed three times with TBST buffer for 10 min each. The resulting PVDF membrane was incubated overnight at 4 °C with the corresponding primary antibodies for GLUT9 (1:1000), URAT1 (1:1000), OAT1 (1:1000), ABCG2 (1:1000) and GAPDH (1:3000). After incubation, the membrane was washed and incubated with goat anti-rabbit secondary antibody for 2 hours. The formed membrane was detected with TANON-5200 (Shanghai TANON Technology Co., Ltd.) and analyzed with ImageJ software (Bio-Rad, Richmond, CA, USA), with GAPDH as the control.

[0079] like Figure 6 As shown, (a) is the effect of purified glycitein on the expression level of ABCG2, a uric acid excretion protein in rat kidney, (b) is the effect of purified glycitein on the expression level of OAT1, a uric acid excretion protein in rat kidney, (c) is the effect of purified glycitein on the expression level of GLUT9, a uric acid reabsorption protein in rat kidney, and (d) is the effect of purified glycitein on the expression level of URAT1, a uric acid reabsorption protein in rat kidney. The resin-purified astragalus glycitein and the membrane-purified glycitein both significantly increased the expression of PDZK1 and ABCG2 and reduced the expression of URAT1 in PO-induced hyperuricemia mice. This suggests that purified astragalus glycitein can regulate UA transport by activating PDZK1. Although the resin-purified astragalus glycitein is better than the membrane-purified glycitein in promoting the expression of ABCG2, the membrane-purified glycitein showed better effects in inhibiting the expression of URAT1 and promoting the expression of PDZK1.

[0080] Overexpression of GLUT9 leads to decreased cellular UA transport dynamics, and UA accumulation leads to renal damage, while OAT1 mainly regulates urine volume by promoting the transfer of uric acid from the interrenal station to the renal tubular epithelial cells. In PO-induced hyperuricemia mice, two purified astragalus glycitein products significantly reduced the expression of GLUT9 (P<0.05) and increased the expression of OAT1 (P<0.01) in a dose-dependent manner, indicating that purified astragalus glycitein can promote UA excretion by regulating UA transporters GLUT9 and OAT1. After the treatment of HUA mice with purified astragalus glycitein, the expression of XOD in the liver also seemed to be greatly upregulated. Among them, the membrane-purified glycitein showed better effects than the purified astragalus glycitein resin in inhibiting GLUT9 and XOD and promoting the expression of OAT1.

[0081] Example 4: Clinical trial design experiment

[0082] In this example, 70 patients with hyperuricemia who met the inclusion and exclusion criteria were selected from the Department of Endocrinology of China Resources Wuhan Iron and Steel General Hospital from March 2022 to July 2022. The inclusion criteria were (1) aged between 18 and 70 years old, regardless of gender; (2) blood uric acid level ≥ 480 μmol / L and no acute gouty arthritis attack; (3) blood uric acid level ≥ 480 μmol / L and acute gouty arthritis attack; (4) body weight remained relatively stable, with a body weight change of ≤ 10% in at least 3 months before screening; (5) voluntarily participated in this clinical trial and signed the informed consent form.

[0083] Exclusion criteria were: (1) patients who did not meet the above inclusion criteria; (2) patients who were pregnant, breastfeeding, or intended to become pregnant; (3) patients who had participated in other clinical trials within 3 months before enrollment; (4) patients who had long-term or recent use of drugs that affect serum uric acid; (5) patients with a clear diagnosis of secondary hyperuricemia caused by endocrine diseases, immune diseases, radiotherapy (chemotherapy), drugs, etc.; (6) patients with concomitant diseases of the heart, brain, kidney, liver, etc., who needed to take drugs that could affect the test results; (7) patients undergoing hemodialysis or peritoneal dialysis with severe kidney or liver complications; (8) patients diagnosed with heart failure and severe heart or lung diseases, such as decompensated heart failure (NYHA stage III and IV). ; Unstable angina pectoris; History of myocardial infarction within the past 12 months; (9) Patients are in a state of severe stress, such as severe infection, major surgery, acute cardiovascular and cerebrovascular disease, and during radiotherapy and chemotherapy for malignant tumors; (10) Pancreatic disease or malignant tumors 3 months ago; (11) Use of antibiotics in the past month; (12) Those with allergic constitution, intolerance or aversion to the components of Astragalus products; (13) Those who do not use the medicine as prescribed, whose efficacy cannot be judged, or whose data are incomplete and affect the judgment of efficacy or safety; (14) Those who are unwilling to sign the informed consent form; (15) Those who have other potential factors that may be unfavorable to the trial and are considered unsuitable to participate in the trial by the researchers.

[0084] Clinical trial grouping and dietary intervention: 70 patients (serum uric acid level ≥ 480 μmol / L and no acute gouty arthritis attack) were randomly divided into two groups according to the ratio of 1:1 between the experimental group and the control group, with 35 cases in the experimental group and 35 cases in the placebo group. General treatment: Both groups restricted the intake of high-purine foods such as seafood, red meat, animal offal, beer, etc., ensured 2000 ml of water per day, quit smoking and limit alcohol, avoid strong tea and greasy food intake, and insist on 30 minutes of soothing exercise every day. Placebo group: given a placebo similar to the experimental group for general treatment (Group A). ​​Experimental group 1: a group containing membrane-separated purified astragalus glycitein (Group B); experimental group 2: a group containing resin-separated purified astragalus glycitein (Group C), each person took 2 pieces per day, washed down with warm water, for 8 consecutive weeks. The serum uric acid level was monitored once every 0, 2, 4, and 8 weeks.

[0085] Sampling: Blood pressure was measured in the left upper arm using a cuffless upper arm blood pressure monitor (Panasonic) in a quiet room at normal room temperature. Participants rested in a seated position for 15 minutes before blood pressure recording. In each assessment, 3 blood pressure readings were recorded continuously with an interval of 5 minutes. The average of these measurements was used to evaluate the health indicators of participants.

[0086] Venous blood samples were collected into blood collection tubes containing sodium fluoride / potassium oxalate (Greiner BioOne) according to the standard venipuncture protocol. Blood samples were immediately centrifuged at 3000 g for 10 min at 4°C and stored in aliquots at -80°C until analysis.

[0087] Determination method: Determine the uric acid content in blood based on the uric acid determination kit.

[0088] Statistical analysis: The significant figures of the data in this embodiment were rounded off and statistical analysis was performed in accordance with the regulations of this center. The statistical software used was SPSS22.0. The measurement data were expressed as mean ± standard deviation (x ± s), and the Leven's test method was used to test normality and homogeneity of variance. If there was no statistical significance (P>0.05), one-way analysis of variance (ANOVA) was used for statistical analysis. If ANOVA was statistically significant (P≤0.05), LSDtest (parametric method) was used for comparative analysis. If the variance was not homogeneous (P≤0.05), Kruskal-Wallis test was used. If the Kruskal-Wallis test was statistically significant (P≤0.05), Dunntt's Test (non-parametric method) was used for comparative analysis. The statistical results were tested with a=0.05 as the test limit, where P≤0.05 indicated statistical significance, and P≤0.01 indicated that the difference tested was very significant.

[0089] In this example, 70 volunteers were recruited and maintained according to the lifestyle questionnaire. No significant lifestyle or drug changes occurred during the study, and no adverse events were reported after receiving drugs and products or placebos, as shown in Table 6:

[0090] Table 6 Serum uric acid levels (μmol / L)

[0091]

[0092]

[0093] In this embodiment, the plasma uric acid level remained unchanged throughout the illness period, and after 8 weeks of general treatment, the uric acid level was significantly reduced by nearly 0.16%. During the intervention period of the product prepared by membrane separation and purification of astragalus glycitein (experimental group 1), the plasma uric acid level gradually decreased over time. From baseline to 2 weeks, the average plasma uric acid level showed a downward trend (27.59%; P = 0.005); from baseline to 4 weeks, the average plasma uric acid level showed a downward trend (32.05%; P = 0.007); from baseline to 8 weeks, the average plasma uric acid level decreased significantly (32.98%; P = 0.002); during the intervention period of the product prepared by water extraction and purification of astragalus glycitein (experimental group 2), the average plasma uric acid level decreased by 24.67% after 8 weeks.

[0094] in conclusion:

[0095] The present invention compares and studies the effects of different extraction solvents on the extraction yield of glycitein in Astragalus, wherein the natural deep eutectic solvent prepared by glycerol-menthol can extract glycitein more efficiently, and compared with the traditional water extraction, the yield of glycitein extracted by the natural deep eutectic solvent is more than 2.5 times higher than the traditional water extraction yield. Continue to use membrane separation technology and macroporous resin separation to enrich the glycitein extracted by the natural deep eutectic solvent and the glycitein extracted by water, and find that membrane separation can purify glycitein more efficiently than macroporous resin, and the purity improvement rate is significantly higher than the macroporous resin purification technology (membrane separation increases the purity of glycitein by 27.39%; the traditional macroporous resin purification rate of the sample is only 19.72%). Animal experimental studies have found that the astragalus glycitein purified by membrane separation is more effective than the glycitein purified by macroporous resin in inhibiting the activity of XOD and reducing the production of UA, and regulating the expression of uric acid transporters (OAT1, ABCG2, URAT1 and GLUT9) to increase the excretion of UA. Human clinical trials have also verified that the two glycitein products can significantly reduce and restore normal uric acid levels, and the uric acid-lowering effect of the membrane-separated glycitein product is better than that of the resin-separated and purified glycitein product.

[0096] The present invention combines a natural low eutectic solvent with membrane separation for the first time to study the extraction, separation and purification of astragalus glycitein. The system is cheap, easy to operate, and has a high degree of enrichment of active ingredients, solving the problems of low traditional water extraction rate and low separation and purification efficiency.

[0097] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A natural solvent extraction and membrane separation process for astragalus glycitein, characterized in that: The following steps are involved: S1. preparing a natural deep eutectic solvent; S2. Under preset conditions, using a natural low eutectic solvent to ultrasonically extract the Astragalus root dry sample powder to obtain a crude extract; S3, introducing the crude extract into a preset membrane separation device, filtering to obtain a retained liquid; S4. The obtained retained solution is vacuum dried to obtain crude astragalus glycitein.

2. The natural solvent extraction and membrane separation process of astragalus glycitein according to claim 1, characterized in that: The preparation process of the natural deep eutectic solvent in step S1 is as follows: mixing the hydrogen bond donor raw material and the hydrogen bond acceptor raw material in a water bath at 80-90° C. according to a specific molar ratio, and stirring until a uniform transparent liquid is formed to obtain the natural deep eutectic solvent.

3. The natural solvent extraction and membrane separation process of astragalus glycitein according to claim 2, characterized in that: The natural low eutectic solvent comprises lactic acid-glycine, citric acid-betaine, glycerol-menthol and glycerol-lysine, and the molar ratios of hydrogen bond donor raw materials and hydrogen bond acceptor raw materials of lactic acid-glycine, citric acid-betaine, glycerol-menthol and glycerol-lysine are 2:1, 2:1, 1:1 and 1:1 respectively.

4. The natural solvent extraction and membrane separation process of astragalus glycitein according to claim 1, characterized in that: The preset conditions in step S2 are: at 85° C., the ultrasonic power is 70 W, the frequency is 50 Hz, the ratio of the dry astragalus powder to the natural low eutectic solvent is 1:20, and 1 volume of water is added, and ultrasonic extraction is performed for 0.5 h to obtain a crude extract.

5. The natural solvent extraction and membrane separation process of astragalus glycitein according to claim 1, characterized in that: The membrane separation device preset in step S3 includes setting the membrane filtration temperature, membrane material and separation pressure, wherein the membrane separation device pumps the crude extract solution through the membrane so that smaller molecules pass through the pores and larger molecules are retained, and the retained liquid is obtained by filtration.

6. Application of a natural solvent extraction and membrane separation process of astragalus glycitein according to any one of claims 1 to 5 in efficient extraction, separation and enrichment of astragalus glycitein.

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