Kudzuvine root non-starch polysaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the field of natural active substance extraction, and particularly relates to radix puerariae non-starch polysaccharide as well as a preparation method and application thereof. The preparation method of the pueraria lobata non-starch polysaccharide comprises the following steps: drying a pueraria lobata raw material, sequentially performing supercritical carbon dioxide fluid extraction treatment, cerium chloride synergistic ultrasonic extraction, enzymolysis and alcohol precipitation to obtain pueraria lobata crude polysaccharide, and separating and purifying the pueraria lobata crude polysaccharide to obtain the pueraria lobata non-starch polysaccharide. According to the preparation method provided by the invention, non-starch polysaccharide in radix puerariae can be efficiently extracted, the content of a target product is remarkably increased, the polysaccharide content reaches 90% or above, impurities can be effectively removed, and no organic reagent is left. Meanwhile, the obtained pueraria lobata non-starch polysaccharide has remarkable hypoglycemic activity and has important application value and market potential in the field of hypoglycemic drug development.

Description

Technical Field

[0001] The present invention belongs to the field of extraction of natural active substances, and particularly relates to a pueraria non-starch polysaccharide, a preparation method thereof, and an application thereof. Background Art

[0002] Pueraria is the hypertrophied root of a perennial deciduous vine of the genus Pueraria in the legume family ( Pueraria. DC ) There are nearly 18 species of Pueraria plants globally, mainly distributed in subtropical and temperate regions. There are about 9 species and 2 varieties in China, concentrated in Yunnan and its neighboring provinces. Pueraria is recorded in the Chinese Pharmacopoeia as having the effects of relieving muscle fever, promoting fluid production to quench thirst, rash promotion, and raising yang to stop diarrhea. Modern pharmacology has proven that Pueraria has effects such as antipyretic, anti-inflammatory, anti-infective, blood pressure lowering, blood sugar lowering, blood lipid lowering, improving cardiovascular and cerebrovascular circulation, protecting the liver, and regulating immunity.

[0003] Domestic and foreign research on Pueraria extracts mainly focuses on components such as Pueraria starch, Pueraria flavonoids, and isoflavones, while research on Pueraria non-starch polysaccharides is relatively less. Although Pueraria non-starch polysaccharides have potential biological activities such as antioxidant and liver protection. However, existing extraction methods have deficiencies such as low enzymatic hydrolysis efficiency of starch, high starch and protein content in the extract, low polysaccharide content, and cumbersome protein removal steps by the Sevag method and organic reagent residues, resulting in low polysaccharide extraction rate, insufficient product purity, and organic reagent residue problems, which greatly limit its function verification and industrial application. Therefore, developing a high-efficiency and highly selective preparation technology for Pueraria non-starch polysaccharides is of great significance for deeply exploring its medicinal value and expanding the high-value utilization of Pueraria resources. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a pueraria non-starch polysaccharide, a preparation method thereof, and an application thereof. The preparation method provided by the present invention can efficiently extract non-starch polysaccharides from Pueraria, significantly improving the quality and content of Pueraria non-starch polysaccharides. At the same time, the obtained pueraria non-starch polysaccharide of the present invention has a significant blood sugar lowering effect and can be used for developing blood sugar lowering drugs, with broad application prospects.

[0005] In the first aspect, the present invention provides a preparation method of a pueraria non-starch polysaccharide, and the preparation method includes the following steps: Step 1: Wash, peel, dry, pulverize, and sieve the Pueraria raw material to obtain crude Pueraria powder; Step 2: Perform supercritical carbon dioxide fluid extraction treatment on the crude Pueraria powder obtained in Step 1, and the residue obtained by extraction is dried to obtain Pueraria powder; Step 3: After mixing the kudzu root powder described in Step 2 with water, a suspension is obtained; cerium chloride is added to the suspension and ultrasonic treatment is carried out; after the ultrasonic treatment is completed, α-amylase and glucoamylase are added to the suspension and enzymatic hydrolysis is carried out until the iodine color reaction of the suspension is colorless or light yellow, obtaining an enzymatic hydrolysate; Step 4: Hydrogen peroxide is added to the enzymatic hydrolysate obtained in Step 3, stirred evenly, then ultrasonic treatment is carried out, and then the enzyme is inactivated and hydrogen peroxide is removed; solid-liquid separation is carried out on the treated enzymatic hydrolysate, the supernatant is taken for alcohol precipitation, and the precipitate is collected; the ethanol residue in the precipitate is removed, and after drying, crude kudzu root polysaccharide is obtained; Step 5: The crude kudzu root polysaccharide described in Step 4 is obtained as kudzu root non-starch polysaccharide after separation and purification.

[0006] The preparation method of kudzu root non-starch polysaccharide provided by the present invention significantly improves the purity of kudzu root non-starch polysaccharide, reduces the content of impurities such as starch and protein, and the polysaccharide content in the obtained extract reaches more than 90%. This method first performs supercritical carbon dioxide fluid extraction on crude kudzu root powder to remove impurities of alcohol-soluble substances (such as pigments, lipids) therein, and then adds cerium chloride to the kudzu root powder suspension for ultrasonic treatment. Cerium chloride is used to destroy the cell wall structure of kudzu root, making it easier for polysaccharides to be released from the cells. At the same time, its antioxidant properties are used to protect the structure and activity of polysaccharides. Then, cerium chloride is used as a catalyst to carry out enzymatic hydrolysis treatment in combination with α-amylase and glucoamylase to efficiently remove starch, obtaining an enzymatic hydrolysate; on this basis, hydrogen peroxide is added to the enzymatic hydrolysate for ultrasonic treatment, and hydrogen peroxide is used to oxidize some amino acid residues in proteins to make them denatured, so that proteins and polysaccharides are separated, and then crude kudzu root polysaccharide is obtained by alcohol precipitation; the target polysaccharide is obtained after further separation and purification of the crude kudzu root polysaccharide. This method realizes the high-purity extraction of kudzu root non-starch polysaccharide by optimizing the extraction and purification processes, and at the same time, the obtained kudzu root non-starch polysaccharide has the effect of lowering blood sugar.

[0007] The drying and powdering of kudzu root in Step 1 is helpful for subsequent extraction, and the crude kudzu root powder can pass through a 40-100 mesh sieve.

[0008] The drying methods in Steps 1, 2, 5 and 6 can adopt direct drying, concentration first and then drying, or reduced-pressure vacuum drying, etc., and it can be dried to a constant weight.

[0009] The end point of enzymatic hydrolysis in Step 3 is that no starch can be detected in the suspension, and the enzymatic hydrolysis time is controlled based on this standard. The present invention uses the iodine color reaction to represent the starch content in the suspension corresponding to the end point of enzymatic hydrolysis, but it does not mean that the iodine color reaction must be carried out in this step, nor does it mean that the detection method of starch content is limited to the iodine color reaction.

[0010] In Step 4, solid-liquid separation can be carried out by filtration or centrifugation, and the corresponding filtrate or supernatant is collected, which is the enzymatic hydrolysate. In the alcohol precipitation process, ethanol is slowly added to the supernatant and left standing until the precipitation no longer increases, generally for 8 - 12 h.

[0011] Preferably, in Step 2, the crude kudzu root powder is subjected to the supercritical carbon dioxide fluid extraction treatment in an extraction kettle, the carbon dioxide flow rate is 30 - 50 L / h, and the extraction is carried out at a pressure of 30 - 50 MPa and a temperature of 30 - 50 °C for 3 - 5 hours. The remaining kudzu root solid residue in the extraction kettle is dried by microwave vacuum drying to obtain kudzu root powder.

[0012] Preferably, in Step 3, the material-liquid ratio of the kudzu root powder to water is 1:15 - 20 (g / mL); the addition amount of cerium chloride is 1 - 2 g / 100 g of kudzu root powder; the power of the ultrasonic treatment is 200 - 400 W, the temperature is 30 - 50 °C, and the duration is 20 - 40 min; after the ultrasonic treatment, thermostable α-amylase is added to the suspension, and the addition amount is 1 - 2 g / 100 g of kudzu root powder, and the enzymatic hydrolysis is carried out at pH = 6 - 6.5 and 90 - 95 °C for 1 - 2 h; then glucoamylase is added, and the addition amount of glucoamylase is 1 g / 100 g of kudzu root powder, and the enzymatic hydrolysis is carried out at pH = 4 - 4.5 and 55 - 65 °C until the iodine color reaction of the suspension is colorless or yellow to obtain the enzymatic hydrolysate.

[0013] Thermostable α-amylase is mainly used for the rapid liquefaction of starch, and glucoamylase is used to further decompose the liquefied starch into glucose. Through the synergistic effect of thermostable α-amylase and glucoamylase, the starch in kudzu root can be efficiently decomposed and removed, thus providing favorable conditions for the extraction and purification of non-starch polysaccharides in kudzu root.

[0014] More preferably, in Step 3, the material-liquid ratio of the kudzu root powder to water is 1:15 (g / mL); the addition amount of cerium chloride is 2 g / 100 g of kudzu root powder; the power of the ultrasonic treatment is 200 W, the temperature is 30 °C, and the duration is 40 min; the pH of the enzymatic hydrolysis is 5.5, and the enzymatic hydrolysis is carried out at 55 °C for 1 - 2 h. The addition amount of thermostable α-amylase is 2 g / 100 g of kudzu root powder, and the enzymatic hydrolysis is carried out at pH 6.5 and 95 °C for 1 h; the addition amount of glucoamylase is 1 g / 100 g of kudzu root powder, and the enzymatic hydrolysis is carried out at pH 4 and 55 °C.

[0015] Preferably, in Step 4, the final concentration of hydrogen peroxide added to the enzymatic hydrolysate in the solution is 1% - 3%; the power of the ultrasonic treatment is 250 - 350 W, the temperature is 40 - 60 °C, and the duration is 20 - 40 min.

[0016] Preferably, in the alcohol precipitation process, ethanol is added to make the mass percentage content of ethanol in the solution reach 70% - 80%.

[0017] More preferably, the final concentration of hydrogen peroxide added to the enzymolysis solution in step 4 in the solution is 3%; the power of the ultrasonic treatment is 250 W, the temperature is 40 °C, and the duration is 40 min.

[0018] More preferably, during the alcohol precipitation process, ethanol is added to make the mass percentage of ethanol in the solution reach 70%.

[0019] Preferably, the separation and purification of crude pueraria polysaccharide in step 5 includes: dissolving the crude pueraria polysaccharide in water to prepare a polysaccharide solution; performing column chromatography on the polysaccharide solution, using a D101 macroporous resin column as the stationary phase and distilled water as the elution solvent. When the molish reaction of the eluate is negative, stop the elution. The collected eluate is concentrated and dried to obtain crude pueraria polysaccharide II. The obtained crude pueraria polysaccharide II is used to prepare a pueraria non-starch polysaccharide solution. The filtrate obtained after filtration is subjected to gel filtration chromatography. The gel filtration chromatography uses a Sephacryl S-400 HR gel filtration column, and the elution solvent is 0.1 M sodium chloride solution. Collect the eluate corresponding to the polysaccharide peak in the absorbance curve at 490 nm; the eluate is dialyzed through a dialysis bag with a molecular weight cut-off of 8000 - 12000 Da, and the dialysis retentate is dried to obtain pueraria non-starch polysaccharide.

[0020] During column chromatography, the elution end point is that no polysaccharide substance can be detected in the eluate, and the elution time is controlled based on this standard. The present invention uses the molish reaction to represent the polysaccharide content in the eluate at the elution end point, but it does not mean that the molish reaction must be carried out in this step, nor does it mean that the detection method of polysaccharide in the eluate is limited to the molish reaction.

[0021] More preferably, the concentration of the pueraria non-starch polysaccharide solution is 5 - 10 mg / mL; the flow rate of the elution solvent is controlled to be 0.5 - 1.0 mL / min.

[0022] More preferably, the concentration of the pueraria non-starch polysaccharide solution is 10 mg / mL; the flow rate is 0.8 mL / min, and the eluate with an elution time between 13.9 - 15.7 min is collected.

[0023] In a second aspect, the present invention also provides pueraria non-starch polysaccharide prepared by the above preparation method.

[0024] Preferably, the kudzu non-starch polysaccharide is a pyranose polysaccharide with an α-configuration, and is composed of (0.17±0.25)% fucose, (20.79±0.56)% rhamnose, (3.28±0.54)% arabinose, (38.97±0.21)% galactose, (22.09±0.41)% glucose, (3.9±0.34)% xylose, (4.72±0.3)% mannose, (3.49±0.25)% galacturonic acid and (2.59±0.72)% glucuronic acid, and has a molecular weight of 95210 Da.

[0025] In a third aspect, the present invention also provides the use of the above-mentioned kudzu non-starch polysaccharide, including: Use in hypoglycemic drugs; Use in drugs for relieving insulin resistance; Use in drugs for reducing the level of glycosylated serum proteins.

[0026] The kudzu non-starch polysaccharide provided by the present invention shows significant hypoglycemic effects in both in vitro and in vivo hypoglycemic experiments. The results of in vitro experiments show that the polysaccharide has good inhibitory effects on the activities of α-amylase and α-glucosidase, thereby exerting hypoglycemic effects. In vivo experiments further verify its hypoglycemic efficacy: in diabetic model mice induced by high-fat and high-sugar diet combined with low-dose streptozotocin (STZ), the kudzu non-starch polysaccharide can significantly reduce the blood glucose content in mice, restore the body weight of mice, and significantly reduce the areas under the glucose tolerance and insulin tolerance curves. In addition, the polysaccharide can also significantly reduce the level of glycosylated serum proteins, the concentration of mouse serum insulin and the insulin resistance index. These results indicate that the kudzu non-starch polysaccharide can effectively inhibit the abnormal increase of blood glucose level through a multi-target action mechanism, and overall improve the metabolic status of diabetic mice, providing a new potential strategy for the treatment of diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0028] Figure 1 It is the high performance gel permeation chromatogram of the kudzu non-starch polysaccharide provided by the present invention; Figure 2 It is the monosaccharide composition result of the kudzu non-starch polysaccharide provided by the present invention; Figure 3 It is the infrared spectrogram of the kudzu non-starch polysaccharide provided by the present invention; Figure 4 The inhibitory effect of the pueraria non-starch polysaccharide provided by the present invention on α-amylase; Figure 5 The inhibitory effect of the pueraria non-starch polysaccharide provided by the present invention on α-glucosidase; Figure 6 The change of fasting blood glucose in mice in Example 5; Figure 7 The change of fasting body weight in mice in Example 5; Figure 8 The fasting oral glucose tolerance in mice in Example 5; Figure 9 The insulin tolerance in mice in Example 5; Figure 10 The content of glycosylated serum protein in mice in Example 5; Figure 11 The serum insulin concentration and insulin resistance index in mice in Example 5; Figure 12 The comparison of intestinal flora at the phylum level among groups of mice in Example 5. Specific Embodiments

[0029] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0030] The high-temperature resistant α-amylase is purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and the model is A109182. The glucoamylase is purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and the model is A107823.

[0031] Example 1 This example provides an extract of pueraria non-starch polysaccharide, and the specific preparation method is as follows: Step 1: Take fresh pueraria medicinal materials, wash them clean, cut them into blocks with a volume of 2 cm 2 after oven drying, crush them with a pulverizer, and pass through a 60-mesh sieve to obtain crude pueraria powder.

[0032] Step 2: Load the crude kudzu root powder into the extraction kettle until it reaches 70% of the kettle volume. Use carbon dioxide with a purity higher than 99% as the extraction agent, control the flow rate at 30 L / h, and extract for 3 hours at a pressure of 40 MPa and a temperature of 50°C. After the extraction is completed, precipitate the impurities, pigments and other alcohol-soluble substances carried by CO2 in the separator. Perform microwave vacuum drying on the remaining kudzu root solid residue in the extraction kettle at a pressure of 10 KPa, a temperature of 30°C, and a power of 500 W until it is dried to a constant weight to obtain kudzu root powder.

[0033] Step 3: Add distilled water to the dried kudzu root powder according to a solid-liquid ratio of 1:15 (g / mL), mix well to prepare a suspension. Add cerium chloride to the suspension, with an addition amount of 10 g / 100 g of kudzu root powder. After ultrasonic extraction at a power of 400 W and a temperature of 50°C for 20 minutes, add thermotolerant α-amylase and glucoamylase for enzymatic hydrolysis. The addition amount of thermotolerant α-amylase is 1 g / 100 g of kudzu root powder, and perform enzymatic hydrolysis at pH 6.5 and 95°C for 2 h; the addition amount of glucoamylase is 1 g / 100 g of kudzu root powder, and perform enzymatic hydrolysis at pH 4.5 and 65°C for 1 h to obtain an enzymatic hydrolysate.

[0034] Step 4: Add hydrogen peroxide to the enzymatic hydrolysate to make the final concentration of hydrogen peroxide in the solution 1% (v / v), stir evenly, ultrasonic at a power of 350 W and a temperature of 60°C for 20 minutes, then heat in a water bath at 100°C for 20 minutes to inactivate the enzyme and remove hydrogen peroxide. After the solution cools, use a refrigerated centrifuge to centrifuge at a speed of 8000 rpm and a temperature of 4°C for 15 minutes, and take the supernatant. Add ethanol to the supernatant to make the final mass percentage of ethanol in the solution 70%, stir for 1 h, then let it stand for 12 h, and centrifuge at a speed of 5500 rpm at room temperature for 15 minutes to collect the precipitate. The precipitate is redissolved with distilled water, stirred until completely dissolved, and the ethanol residue is removed using a rotary evaporator (temperature is 60 - 65°C, pressure is 60 - 65 mbar, and the rotation speed of the rotating shaft is 60 - 65 rpm). After concentration, it is dried in an oven to obtain crude kudzu polysaccharide I.

[0035] Step 5: Dissolve crude kudzu polysaccharide I in water to prepare a polysaccharide solution with a concentration of 20 mg / mL. Load it onto a D101 macroporous resin column (2.5×50 cm) at a sample loading volume of 15 mL. After adsorption for 12 h, elute with distilled water and collect the eluate. Stop elution when the molish reaction is negative. The collected eluate is concentrated by reduced pressure and dried to obtain crude kudzu polysaccharide II.

[0036] Step 6: Dissolve crude kudzu polysaccharide II in water to prepare a 10 mg / mL kudzu non-starch polysaccharide solution. Filter the obtained filtrate and perform gel filtration chromatography on it. The gel filtration chromatography uses a Sephacryl S-400 HR gel filtration column. The sample loading volume is 10 mL, the elution solvent is 0.1 M sodium chloride solution, the flow rate is 0.8 mL / min, and collect the eluate with an elution time between 13.9 - 15.7 min; Dialyze the collected eluate using a dialysis bag with a molecular weight cut-off of 8000 Da. After concentrating the dialysis retentate on a rotary evaporator under reduced pressure, freeze-dry it using a vacuum freeze dryer to obtain kudzu non-starch polysaccharide extract 1.

[0037] Example 2 This example provides a kudzu non-starch polysaccharide extract, and the specific preparation method is as follows: Step 1: Take fresh kudzu medicinal materials, wash them clean, cut them into blocks with a volume of 2 cm 2 in size after peeling, dry them in an oven and then crush them with a pulverizer, and pass through a 60-mesh sieve to obtain crude kudzu powder.

[0038] Step 2: Load the crude kudzu powder into the extraction kettle until it reaches 70% of the kettle volume. Use carbon dioxide with a purity higher than 99% as the extractant, control the flow rate at 40 L / h, and extract at a pressure of 50 MPa and a temperature of 40°C for 4 hours; After the extraction is completed, precipitate the impurities and pigment and other alcohol-soluble substances carried by CO2 in the separator, and perform microwave vacuum drying on the remaining kudzu solid residue in the extraction kettle at a pressure of 10 KPa, a temperature of 30°C, and a power of 600 W until it is dried to a constant weight to obtain kudzu powder.

[0039] Step 3: Add distilled water to the dried kudzu powder according to a solid-liquid ratio of 1:20 (g / mL), mix well to prepare a suspension; Add cerium chloride to the suspension, with an addition amount of 1.5 g / 100 g of kudzu powder. After ultrasonic extraction at a power of 300 W and a temperature of 40°C for 30 minutes, add thermostable α-amylase and glucoamylase for enzymatic hydrolysis. The addition amount of thermostable α-amylase is 2 g / 100 g of kudzu powder, and perform enzymatic hydrolysis at pH 6 and 90°C for 2 h; The addition amount of glucoamylase is 1 g / 100 g of kudzu powder, and perform enzymatic hydrolysis at pH 4 and 60°C for 2 h to obtain an enzymatic hydrolysate.

[0040] Step 4: Add hydrogen peroxide to the enzymatic hydrolyzate to make the final concentration of hydrogen peroxide in the solution 2% (v / v), stir evenly, ultrasonicate at 300 W ultrasonic power and 50°C for 30 minutes, then inactivate the enzyme and remove hydrogen peroxide in a water bath at 80°C for 30 minutes; after the solution is cooled, centrifuge it at 8000 rpm and 4°C for 15 minutes in a refrigerated centrifuge to take the supernatant; add ethanol to the supernatant to make the final mass percentage of ethanol in the solution 70%, stir for 1 hour, let it stand for 12 hours, and centrifuge it at 5500 rpm at room temperature for 15 minutes to collect the precipitate; the precipitate is re-dissolved with distilled water, dialyzed for 48 hours, and then freeze-dried to obtain Pueraria lobata crude polysaccharide 1.

[0041] Step 5: Dissolve the Pueraria lobata crude polysaccharide 1 in water to prepare a 20 mg / mL polysaccharide solution, load 15 mL on a D101 macroporous resin column (2.5×50 cm), adsorb for 12 h, elute with distilled water and collect the eluate. When the molish reaction is negative, stop the elution. The collected eluate is concentrated and dried under reduced pressure to obtain Pueraria lobata crude polysaccharide 2.

[0042] Step 6: Dissolve the crude kudzu root polysaccharide II in water to prepare a 10 mg / mL kudzu root non-starch polysaccharide solution. The filtrate obtained after filtration is subjected to gel filtration chromatography. The gel filtration chromatography adopts a Sephacryl S-400 HR gel filtration column, the sample volume is 10 mL, the elution solvent is 0.1 M sodium chloride solution, the flow rate is 0.8 mL / min, and the eluate with an elution time between 13.9 and 15.7 min is collected; the collected eluate is dialyzed with a 12000 Da dialysis bag, and the dialyzate is concentrated on a reduced pressure vacuum rotary evaporator, and then freeze-dried with a vacuum freeze dryer to obtain the kudzu root non-starch polysaccharide extract 2.

[0043] Example 3 This embodiment provides a non-starch polysaccharide extract of Pueraria lobata, and the specific preparation method is as follows: Step 1: Take fresh kudzu root, clean it, peel it and cut it into 2 cm pieces 2 The lumps of volume are dried in an oven and crushed with a powder grinder, and passed through a 60-mesh sieve to obtain coarse kudzu root powder.

[0044] Step 2: The crude kudzu root powder is loaded into the extraction kettle to reach 70% of the volume of the kettle, and carbon dioxide with a purity higher than 99% is used as the extractant. The flow rate is controlled to be 50 L / h, and the extraction is carried out at a pressure of 30 MPa and a temperature of 30°C for 5 hours. After the extraction is completed, the impurities and alcohol-soluble substances such as pigments carried by CO2 in the separator are precipitated, and the remaining kudzu root solid residue in the extraction kettle is subjected to microwave vacuum drying at a pressure of 5 KPa, a temperature of 40°C, and a power of 700 W to a constant weight to obtain kudzu root powder.

[0045] Step 3: Add the dried kudzu root powder to distilled water at a ratio of 1:15 (g / mL), mix well to prepare a suspension; add cerium chloride to the suspension, with an addition amount of 2 g / 100 g of kudzu root powder. After ultrasonic extraction at 200 W ultrasonic power and 30 °C for 40 minutes, add thermostable α-amylase and glucoamylase for enzymatic hydrolysis. The addition amount of thermostable α-amylase is 2 g / 100 g of kudzu root powder, and enzymatic hydrolysis is carried out at pH 6.5 and 95 °C for 1 h; the addition amount of glucoamylase is 1 g / 100 g of kudzu root powder, and enzymatic hydrolysis is carried out at pH 4 and 55 °C for 2 h to obtain an enzymatic hydrolysate.

[0046] Step 4: Add hydrogen peroxide to the enzymatic hydrolysate to make the final concentration of hydrogen peroxide in the solution 3% (v / v), stir evenly, and after ultrasonic treatment at 350 W ultrasonic power and 40 °C for 40 minutes, heat in a water bath at 100 °C for 20 minutes to inactivate the enzyme and remove hydrogen peroxide; after the solution cools, use a refrigerated centrifuge to centrifuge at a rotation speed of 8000 rpm and a temperature of 4 °C for 15 minutes, and take the supernatant; add ethanol to the supernatant to make the final mass percentage of ethanol in the solution 70%, stir for 1 h, then let stand for 12 h, and centrifuge at a rotation speed of 5500 rpm at room temperature for 15 minutes to collect the precipitate; dissolve the precipitate in distilled water, dialyze for 48 h, and then freeze-dry to obtain crude kudzu polysaccharide I.

[0047] Step 5: Dissolve crude kudzu polysaccharide I in water to prepare a polysaccharide solution with a concentration of 20 mg / mL, load it onto a D101 macroporous resin column (2.5×50 cm) with a sample loading volume of 15 mL, adsorb for 12 h, then elute with distilled water and collect the eluate. When the molish reaction is negative, stop elution. The collected eluate is concentrated and dried under reduced pressure to obtain crude kudzu polysaccharide II.

[0048] Step 6: Dissolve crude kudzu polysaccharide II in water to prepare a kudzu non-starch polysaccharide solution with a concentration of 10 mg / mL. After filtering the obtained filtrate, perform gel filtration chromatography. The gel filtration chromatography uses a Sephacryl S-400 HR gel filtration column, with a sample loading volume of 10 mL, an elution solvent of 0.1 M sodium chloride solution, and a flow rate of 0.8 mL / min. Collect the eluate with an elution time between 13.9 - 15.7 min; dialyze the collected eluate with a dialysis bag with a molecular weight cut-off of 12000 Da. After concentrating the dialysis retentate on a rotary evaporator under reduced pressure, freeze-dry it with a vacuum freeze dryer to obtain the kudzu non-starch polysaccharide extract 3.

[0049] Comparative Example 1 This comparative example provides a kudzu non-starch polysaccharide extract, and the specific preparation method is as follows: Step 1: Take fresh kudzu root medicinal materials, wash them clean, cut them into 2 cm after peeling 2The massive material of the corresponding volume is oven-dried and then crushed by a powder mill, and sieved through a 60-mesh sieve to obtain crude kudzu root powder.

[0050] Step 2: Add distilled water to the crude kudzu root powder according to the ratio of solid to liquid of 1:15 (g / mL), and mix well to prepare a suspension; take 1 L of the suspension, add 1 g of cerium chloride, and perform ultrasonic extraction at a ultrasonic power of 200 W and 30 °C for 40 minutes, then add thermotolerant α-amylase and glucoamylase for enzymatic hydrolysis. The addition amount of thermotolerant α-amylase is 2 g / 100 g of kudzu root powder, and enzymatic hydrolysis is carried out at pH 6.5 and 95 °C for 1 h; the addition amount of glucoamylase is 1 g / 100 g of kudzu root powder, and enzymatic hydrolysis is carried out at pH 4 and 55 °C for 2 h to obtain an enzymatic hydrolysate. The enzymatic hydrolysate is centrifuged at a rotation speed of 8000 rpm and a temperature of 4 °C for 15 minutes, and the supernatant is taken; ethanol is added to the supernatant, and the final mass percentage content of ethanol is 70%. After stirring for 1 h, it is left standing for 12 h, and then centrifuged at a rotation speed of 5500 rpm at room temperature for 15 minutes to collect the precipitate; the precipitate is redissolved with distilled water, dialyzed for 48 h and then freeze-dried to obtain crude kudzu polysaccharide I.

[0051] Step 3: Dissolve crude kudzu polysaccharide I in water to prepare a polysaccharide solution with a concentration of 20 mg / mL, and load 15 mL of the sample onto a D101 macroporous resin column (2.5×50 cm). After adsorption for 12 h, distilled water is used for elution and the eluate is collected. When the molish reaction is negative, the elution is stopped. The collected eluate is concentrated and dried under reduced pressure to obtain crude kudzu polysaccharide II.

[0052] Step 4: Dissolve crude kudzu polysaccharide II in water to prepare a kudzu non-starch polysaccharide solution with a concentration of 10 mg / mL. The filtrate obtained after filtration is subjected to gel filtration chromatography. The gel filtration chromatography uses a Sephacryl S-400 HR gel filtration column, the sample loading volume is 10 mL, the elution solvent is 0.1 M sodium chloride solution, and the flow rate is 0.8 mL / min. The eluate with an elution time between 13.9 - 15.7 min is collected; the collected eluate is dialyzed with a dialysis bag with a molecular weight cut-off of 12000 Da. After the dialysis retentate is concentrated on a vacuum rotary evaporator under reduced pressure, it is freeze-dried with a vacuum freeze dryer to obtain kudzu non-starch polysaccharide extract 4.

[0053] Comparative Example 2 This comparative example provides a kudzu non-starch polysaccharide extract, and the specific preparation method is as follows: Step 1: Take fresh kudzu root medicinal materials, wash them clean, cut them into blocks with a volume of 2 cm 2 The massive material of the corresponding volume is oven-dried and then crushed by a powder mill, and sieved through a 60-mesh sieve to obtain crude kudzu root powder.

[0054] Step 2: Load the crude kudzu root powder into the extraction kettle until it reaches 70% of the kettle volume. Use carbon dioxide with a purity higher than 99% as the extractant, control the flow rate at 50 L / h, and extract at a pressure of 30 MPa and a temperature of 30 °C for 5 hours. After extraction, precipitate the impurities, pigments and other alcohol-soluble substances carried by CO2 in the separator. Perform microwave vacuum drying on the remaining kudzu root solid residue in the extraction kettle at a pressure of 5 KPa, a temperature of 40 °C, and a power of 500 W until its moisture content is about 4% to obtain kudzu root powder.

[0055] Step 3: Add distilled water to the dried kudzu root powder according to the ratio of solid to liquid of 1:15 (g / mL), mix well to prepare a suspension. The suspension is refluxed and extracted at 90 °C for 4 h, and the supernatant is combined. It is concentrated to 1 / 2 of the original suspension volume using a vacuum rotary evaporator (the temperature of the rotary evaporator is 60 °C, the pressure is 60 mbar, and the rotation speed of the rotating shaft is 60 rpm). Add thermotolerant α-amylase with an addition amount of 2 g / 100 g of kudzu root powder, and enzymatically hydrolyze at pH 6.5 and 95 °C for 1 h. Then adjust the pH of the suspension to 4, raise the temperature to 55 °C, add glucoamylase with an addition amount of 1 g / 100 g of kudzu root powder, and enzymatically hydrolyze for 1 h. Then adjust the pH of the suspension to 6, raise the temperature to 65 °C, add papain with an addition amount of 1 g / 100 g of kudzu root powder, and enzymatically hydrolyze for 1.5 h to obtain an enzymatic hydrolysate.

[0056] Step 4: Add hydrogen peroxide to the enzymatic hydrolysate to make the final concentration of hydrogen peroxide in the solution 3% (v / v), stir evenly, ultrasonicate at a power of 350 W and a temperature of 40 °C for 40 minutes, and then heat in a water bath at 100 °C for 20 minutes to inactivate the enzyme and remove hydrogen peroxide. After the solution cools, centrifuge it at a speed of 8000 rpm and a temperature of 4 °C for 15 minutes, and take the supernatant. Add ethanol to the supernatant so that the final mass percentage of ethanol is 70%, stir for 1 h, then let it stand for 12 h, and centrifuge at a speed of 5500 rpm at room temperature for 15 minutes to collect the precipitate. The precipitate is redissolved with distilled water, dialyzed for 48 h, and then freeze-dried to obtain crude kudzu polysaccharide I.

[0057] Step 5: Dissolve crude kudzu polysaccharide I in water to prepare a polysaccharide solution with a concentration of 20 mg / mL. Load it onto a D101 macroporous resin column (2.5×50 cm) with a sample loading volume of 15 mL. After adsorption for 12 h, elute with distilled water and collect the eluate. Stop elution when the molish reaction is negative. The collected eluate is concentrated and dried under reduced pressure to obtain crude kudzu polysaccharide II.

[0058] Step 6: Dissolve crude pueraria polysaccharide II in water to prepare a 10 mg / mL pueraria non-starch polysaccharide solution. The filtrate obtained after filtration is subjected to gel filtration chromatography. The gel filtration chromatography uses a Sephacryl S-400 HR gel filtration column, with a sample loading volume of 10 mL. The elution solvent is 0.1 M sodium chloride solution, and the flow rate is 0.8 mL / min. Collect the eluate with an elution time between 13.9 - 15.7 min; Dialyze the collected eluate with a dialysis bag of 12,000 Da. After concentrating the dialysis retentate on a vacuum rotary evaporator under reduced pressure, lyophilize it with a vacuum freeze dryer to obtain pueraria non-starch polysaccharide extract 5.

[0059] Measure the polysaccharide, protein, and starch contents in the pueraria non-starch polysaccharide extracts of Examples 1 - 3 and Comparative Examples 1 - 2. The results are shown in Table 1. Among them, the neutral sugar content is determined by the phenol-sulfuric acid method, the acidic sugar content is determined by the carbazole-ethanol method, the starch content is determined by the BC0700 starch content determination kit (Beijing Solarbio Science & Technology Co., Ltd.), and the protein content is determined by the Coomassie Brilliant Blue method.

[0060] Table 1 Component contents in pueraria non-starch polysaccharide

[0061] The pueraria non-starch polysaccharide extracted by the preparation method provided by the present invention has a non-starch polysaccharide content of over 90%, with low contents of impurities such as starch and protein. The extract presents as a white flocculent solid.

[0062] In contrast, the non-starch polysaccharide content in the polysaccharide extract prepared in Comparative Example 1 decreased significantly, the protein content increased, and the color was darker. It shows that the addition of the supercritical carbon dioxide fluid extraction step effectively removed alcohol-soluble substances such as pigments and lipids. At the same time, the removal of lipids avoided their encapsulation of proteins or interference with the subsequent separation process, contributing to the release and removal of proteins and the extraction of non-starch polysaccharides. Further, through hydrogen peroxide ultrasonic treatment, the amino acid residues in the oxidized polysaccharide-protein complex were oxidized, promoting protein denaturation and destroying its interaction with polysaccharides, thereby improving the purity of non-starch polysaccharides and reducing the protein content.

[0063] The non-starch polysaccharide content in the polysaccharide extract prepared in Comparative Example 2 decreased significantly, and the starch content increased significantly. It shows that the method of combining cerium chloride mixed ultrasonic extraction with enzymatic hydrolysis can destroy the cell wall, promote the release of sugars, and efficiently degrade starch, thereby achieving the removal of starch and increasing the non-starch polysaccharide content in the extract.

[0064] Verification Example 1 Characterization of pueraria non-starch polysaccharide 1. Determination of polysaccharide molecular weight The relative molecular mass of Pueraria lobata non-starch polysaccharide (PLPS, provided by Example 3) was determined by high performance gel permeation chromatography, and the results are as Figure 1 shown. The chromatographic conditions are as follows: Agilent 1260 high performance liquid chromatograph (Agilent), the chromatographic column is Ultra hydrogel TM linear gel column (7.8 mm×300 mm, Waters), the guard column is Ultra hydrogel guard column (6 mm×40 mm, Waters), the detector is a differential detector (G1362A, Agilent), the column oven temperature is 35 ºC, the mobile phase is 0.1 M NaNO3 (containing 0.02% NaN3, w / w), the flow rate is 0.6 mL / min, the injection volume is 20 μL, and the data acquisition time is 30 min. The sample polysaccharide and dextran series standard products (glucose, T-10, T-40, T-70, T-500 and T-2000) were respectively prepared into solutions with a concentration of 1.0 mg / mL with the mobile phase, and were injected after passing through a 0.22 μm water-based filter membrane.

[0065] It can be seen from Figure 1 that the chromatographic peak of Pueraria lobata non-starch polysaccharide PLPS is a single peak and shows a normal distribution, indicating that the PLPS obtained by gel column chromatography separation and purification is a relatively homogeneous component, and its weight average molecular weight Mw is 95210 Da.

[0066] 2. Determination of monosaccharide composition The proportions of various monosaccharides in Pueraria lobata non-starch polysaccharide (PLPS, provided by Example 3) were quantitatively analyzed by HPAEC-PAD method. The results are shown in Table 2 and Figure 2 .

[0067] Weigh 5 mg of Pueraria lobata non-starch polysaccharide into a test tube, add 0.5 mL of 12 mol / L concentrated sulfuric acid under ice bath and stir for 30 minutes, then add 2.5 mL of ultrapure water and stir for 1 minute, and then transfer it to a 120 °C oil bath and stir for 1 hour. After volume fixation to a 50 mL volumetric flask and dilution, it was passed through a 0.22 μm water-based filter membrane and analyzed by Dionex ICS 6000 ion chromatograph. At the same time, monosaccharide standard products with different concentrations (fucose, rhamnose, galactose, arabinose, xylose, glucose, mannose, fructose, ribose, galacturonic acid and glucuronic acid) were configured for chromatographic analysis to determine the types and contents of monosaccharides in the sample. All experiments were repeated three times.

[0068] The chromatographic conditions are as follows: the chromatographic column is CarboPac TMPA20 (150 mm×3 mm) anion exchange column, eluent flow rate is 0.5 mL / min, mobile phase A is ultrapure water, mobile phase B is 0.25 mol / L NaOH solution, mobile phase C is 1 mol / L sodium acetate, elute according to the method in Table 3, detection method is pulsed amperometric detection, working electrode is Au, reference electrode is Ag / AgCl, injection volume is 10 μL, column temperature is 30ºC.

[0069] Table 2 Monosaccharide composition and proportion of Pueraria non-starch polysaccharide

[0070] Table 3 Ion exchange chromatography elution time

[0071] Analysis of the monosaccharide composition of Pueraria non-starch polysaccharide PLPS shows that it is mainly composed of three monosaccharides: galactose (Gal), glucose (Glc) and rhamnose (Rha), and other monosaccharides such as arabinose (Ara), xylose (Xyl), mannose (Man), etc. also exist in smaller proportions. In addition, PLPS also contains uronic acid derivatives, including galacturonic acid (GalA) and glucuronic acid (GlcA), which may endow PLPS with specific biological activities.

[0072] 3. Determination of polysaccharide linkage sequence and glycosidic bond type Take 1-2 mg of Pueraria non-starch polysaccharide (PLPS, provided by Example 3), mix it with potassium bromide powder and grind it into powder, and use the solid tablet method to scan with a Thermo Nicolet 5700 Fourier transform infrared spectrometer. The scanning range is set to 400 - 4000 cm -1 , and the resolution is 4 cm -1 . The infrared spectrum of Pueraria non-starch polysaccharide PLPS is as Figure 3 shown.

[0073] As Figure 3 can be seen, the absorption peak of PLPS at 593.97 cm -1 belongs to the bending vibration of β-type C-H bond, and the characteristic peak at 1076.10 cm -1 corresponds to the C1-O-C5 oxygen bridge vibration of pyranose ring sugar. Combining the C-O-C and C-O-H stretching vibrations at 1315.24 cm -1 and 1243.88 cm -1 indicates that its sugar ring is in pyranose configuration; the peak at 1403.95 cm -1 suggests the presence of uronic acid (such as galacturonic acid or glucuronic acid) in the sugar chain, while the peak at 1544.95 cm -1The -CONH vibration at [location] indicates trace protein residue. Through the analysis of the main chain structure, it can be seen that PLPS forms a backbone with alternating →4)-β-D-galactopyranose (1→) and →6)-α-D-glucopyranose (1→), and the side chains are connected to the O-3 or O-4 sites of the main chain through →2)-α-L-rhamnopyranose (1→) and →3)-α-L-arabinofuranose (1→). The embedding of uronic acid units (such as →4)-α-D-galacturonic acid (1→)) imparts acidic properties to the polysaccharide, and its C=O stretching vibration (1656.58 cm -1 ), together with the carboxylic acid group (1403.95 cm -1 ), supports this conclusion. Combining with the O-H stretching vibration (3399.94 cm -1 ), and the C-H stretching peak (2923.60 cm -1 ), the structure of PLPS can be further confirmed as an α-configuration pyran polysaccharide.

[0074] Example 4 In vitro hypoglycemic experiment of Pueraria non-starch polysaccharide extract By measuring the inhibition rates of Pueraria non-starch polysaccharide (PLPS, prepared in Example 3) on the activities of α-amylase and α-glucosidase, its potential hypoglycemic effect was evaluated, and acarbose was used as a positive control.

[0075] 1. Inhibitory effect of Pueraria non-starch polysaccharide on α-amylase (1) Solution preparation: Accurately weigh 0.5 g of starch, add an appropriate amount of phosphate buffer solution, shake well and heat in a boiling water bath until completely dissolved. Cool to room temperature and make up to 50 mL to prepare a 1% starch solution for standby. Accurately weigh α-amylase powder, dissolve it with phosphate buffer solution and dilute it to 0.1 U / mL to prepare a 0.1 U / mL α-amylase solution for standby. Weigh an appropriate amount of Pueraria non-starch polysaccharide sample, completely dissolve it with distilled water, and dilute it to prepare a series of PLPS solutions with concentrations of 0.1, 0.25, 0.5, 1, 2, 4, and 8 mg / mL for standby. Weigh acarbose, dissolve it with distilled water and dilute it to prepare a series of positive control solutions with concentrations of 0.1, 0.25, 0.5, 1, 2, 4, and 8 mg / mL for standby.

[0076] (2) Experimental grouping: The α-amylase inhibition detection of each PLPS solution was divided into a drug reaction group, a drug control group, a blank reaction group, and a blank control group, with 3 replicates in each group. The additives and addition amounts of each group are shown in Table 4.

[0077] Table 4 Additive amounts of each group solution for α-amylase inhibition rate

[0078] (3) Experimental procedure: Add 300 μL of α-amylase solution or buffer, 100 μL of PLPS solutions with different mass concentrations (0.1, 0.25, 0.5, 1, 2, 4, 8 mg / mL) or distilled water into a test tube, and incubate in a water bath at 37 °C for 5 min; then add 400 μL of 1% starch solution, mix well and incubate in a water bath at 37 °C for 5 min; finally, add 200 μL of DNS reagent to terminate the reaction, take out after color development in a boiling water bath for 5 min, cool to room temperature, dilute the solution 25 times with distilled water and then add the sample to a 96-well plate, 200 μL per well, set 3 replicates, measure the absorbance at a wavelength of 540 nm. Replace the PLPS solution with acarbose solution, measure the absorbance of acarbose according to the above method, and calculate the inhibition rate of α-amylase (the results are shown in Figure 4 ), and the calculation formula is as follows: Formula (1): Inhibition rate = (A 空 - A 样 ) / A 空 × 100%; A 空 = A 空白反应组 - A 空白对照组 ; A 样 = A 药物反应组 - A 药物对照组 .

[0079] 2. Inhibitory effect of Pueraria non-starch polysaccharides on α-glucosidase (1) Solution preparation: Accurately weigh α-glucosidase powder, dissolve and dilute it with phosphate buffer to 1 U / mL to obtain 1 U / mL α-glucosidase solution, and store it at -20 °C for later use; accurately weigh 0.3765 g of Pueraria non-starch polysaccharide sample, add an appropriate amount of phosphate buffer, stir well to completely dissolve it, then transfer it to a 50 mL volumetric flask, and make up the volume to the mark with phosphate buffer, shake well to prepare 25 mmol / L PNPG solution; weigh 0.53 g of Na2CO3 powder, dissolve it with distilled water and make up the volume to 50 mL to prepare 0.1 mol / L Na2CO3 reaction termination solution, and store it at 4 °C for later use; weigh an appropriate amount of Pueraria non-starch polysaccharide sample, completely dissolve it with distilled water, and dilute it to prepare a series of PLPS solutions with concentrations of 0.1, 0.25, 0.5, 1, 2, 4, 8 mg / mL for later use. Weigh acarbose, dissolve it with distilled water and dilute it to prepare a series of positive control solutions with concentrations of 0.1, 0.25, 0.5, 1, 2, 4, 8 mg / mL for later use.

[0080] (2) Experimental grouping: The α-glucosidase inhibition detection of each PLPS solution was evenly divided into a drug response group, a drug control group, a blank response group, and a blank control group, with 3 replicates in each group. The additives and their dosages in each group are shown in Table 5.

[0081] Table 5 Additive amounts of each group of solutions for α-glucosidase inhibition rate

[0082] (3) Experimental procedure: Add 40 μL of α-glucosidase solution or phosphate buffer, and 40 μL of PLPS solutions with different mass concentrations (0.1, 0.25, 0.5, 1, 2, 4, 8 mg / mL) or distilled water into a test tube. Take it out after reacting at 37 °C for 15 min; add PNPG solution and react at 37 °C for 25 min; finally add Na2CO3 solution to terminate the reaction. Use acarbose as the positive control, measure the absorbance at a wavelength of 405 nm, replace the PLPS solution with the acarbose solution, measure the absorbance of acarbose according to the above method, and calculate the α-glucosidase inhibition rate (the results are shown in Figure 5 ), and the calculation formula is the same as formula (1).

[0083] It can be seen from Figure 4 , 5 that the pueraria non-starch polysaccharide (PLPS) provided by the present invention shows significant inhibitory effects on α-amylase and α-glucosidase, and this inhibitory effect has an obvious concentration dependence. At a concentration of 8 mg / mL, the inhibition rates of PLPS on α-amylase and α-glucosidase are 82.85% and 87.93% respectively, indicating its potential hypoglycemic ability.

[0084] Example 5 Evaluation of the hypoglycemic effect of the pueraria non-starch polysaccharide extract in vivo By measuring the effects of pueraria non-starch polysaccharide (PLPS, prepared in Example 3) on the body weight, blood glucose level, glucose tolerance, insulin tolerance, glycated serum protein level, serum insulin concentration, and insulin resistance index of diabetic model mice, the hypoglycemic effect of pueraria non-starch polysaccharide in vivo was evaluated.

[0085] 1. Establishment of diabetic mouse models (1) Adaptation period: Select 36 male C57BL / 6 mice with a body weight of 22 - 26 g and raise them individually under controlled conditions of 25 ± 2 °C, 50 ± 5% relative humidity, and a 12 / 12 h day / night cycle. During this period, the mice can drink water and eat freely.

[0086] (2) High-fat and high-sugar diet modeling period: After a one-week adaptation period, the mice were randomly divided into a standard diet group (maintenance feed) and a high-fat and high-sugar diet group (D 12492 feed), and this stage lasted for 6 weeks.

[0087] (3)Type 2 diabetes mellitus (T2DM) model establishment period: After the modeling period, the mice were fasted for 12 hours and then intraperitoneally injected with low-temperature streptozotocin (STZ, dosage of 75 mg / kg body weight) for 5 consecutive days; One week after STZ injection, the fasting blood glucose index (FBG) was measured. If FBG ≥ 11.1 mmol / L and was stably maintained for more than 2 weeks, the modeling was successful. Mice on a standard diet were given an equal amount of 0.5 mol / L citrate buffer solution.

[0088] 2. Group feeding The diabetic mice were randomly divided into 3 groups with 12 mice in each group: diabetic model group (DC), metformin positive control group (Met, Met dosage of 200 mg / kg body weight), pueraria non-starch polysaccharide group (PLPS, PLPS dosage of 200 mg / kg body weight), and mice on a standard diet were used as the control group (NC). Dimethylbiguanide solution and pueraria non-starch polysaccharide solution with a concentration of 20 mg / mL were prepared. During the feeding period, all mice were allowed to freely eat and drink water. At 9:00 am every day, the mice in the Met group and the PLPS group were respectively gavaged with the corresponding solvents, and the mice in the DC group and the NC group were gavaged with an equal amount of pure water for 5 weeks. After the feeding ended, all mice were fasted for 12 h and then sacrificed.

[0089] The fasting blood glucose of the mice in each group was recorded once a week (the results are shown in Figure 6 ), and the body weight was recorded once a week (the results are shown in Figure 7 ). Method for measuring the fasting blood glucose of mice: After the mice were fasted for 12 h, the tail root was wiped from the tail root end to the tail tip with an alcohol cotton two to three times to ensure vasodilation; The remaining alcohol was wiped off with dry absorbent cotton; A blood collection needle was inserted about 3 - 5 mm at the tail end, and the first 1 - 2 drops of blood were wiped off with absorbent cotton; The tail root of the mouse was gently pinched to the tail tip to make the blood gather at the tail tip, and the blood glucose test strip was used to sample by the principle of siphon, and the blood glucose value was read; After hemostasis with iodophor, the mouse was returned to the cage.

[0090] Oral glucose tolerance test (OGTT) was performed 1 week before the end of feeding. After the mice were fasted for 12 hours, they were gavaged with glucose for mice, with a dosage of 2 g / kg body weight. The blood glucose levels of the mice were measured at 0, 15, 30, 60, and 120 min after gavage with glucose, and the area under the blood glucose change curve (AUC) was calculated to evaluate the oral glucose tolerance ability of the mice. The results are shown in Figure 8 .

[0091] Insulin tolerance test (ITT) was performed 3 days before the end of feeding. After the mice were fasted for 6 hours, insulin was then injected at a dose of 1.0 U / kg body weight. The blood glucose levels of the mice were measured at 0, 15, 30, 60, and 120 min after insulin injection, and the area under the blood glucose change curve (AUC) was calculated to evaluate the insulin tolerance of the mice. The results are shown in Figure 9 .

[0092] After the end of feeding, the levels of glycated proteins (GSP), insulin, and intestinal flora in the mice were measured. Blood was collected from the retroorbital venous plexus of the mice, and mouse serum was collected. ELISA kits were used to measure the GSP and insulin levels in the mouse serum, and the insulin resistance index (HOMA-IR) was calculated according to the following formula (2). The results are shown in Figure 10 , 11 . The intestinal flora of the mice was measured by 16S rRNA gene sequencing technology based on high-throughput sequencing technology. The results are shown in Figure 12 .

[0093] Formula (2): HOMA-IR = [serum insulin concentration (μIU / mL) × fasting blood glucose concentration (mmol / L)] / 22.5.

[0094] According to Figures 6 - 12 the in vivo hypoglycemic experimental results shown, the pueraria non-starch polysaccharide provided by the present invention shows a significant hypoglycemic effect on diabetic model mice. Pueraria non-starch polysaccharide can effectively reduce the blood glucose level of diabetic model mice, restore the body weight of mice, significantly reduce the glucose tolerance and insulin tolerance of mice, significantly reduce the level of glycated serum proteins, the serum insulin concentration and insulin resistance index of mice, and effectively regulate the composition of the intestinal flora of mice. Pueraria non-starch polysaccharide may inhibit the abnormal increase of blood glucose by improving the balance of intestinal flora and alleviating insulin resistance.

[0095] In summary, the preparation method provided by the present invention can significantly improve the extraction efficiency and purity of pueraria non-starch polysaccharide, and effectively solve the problems existing in the traditional process, such as low efficiency of enzymatic hydrolysis of starch, high impurity content in the extract, low polysaccharide yield, and residual organic reagents. The pueraria non-starch polysaccharide prepared by the present invention has excellent hypoglycemic activity and contributes an efficient raw material choice for the development of a new generation of hypoglycemic drugs.

[0096] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A preparation method of pueraria non-starch polysaccharide, characterized in that The preparation method comprises the following steps: Step 1: Wash, peel, dry, pulverize and sieve the Pueraria lobata raw material to obtain crude Pueraria lobata powder; Step 2: Perform supercritical carbon dioxide fluid extraction on the crude Pueraria lobata powder obtained in Step 1, and dry the residue obtained by extraction to obtain Pueraria lobata powder; Step 3: After mixing the Pueraria lobata powder described in Step 2 with water to obtain a suspension; add cerium chloride to the suspension and perform ultrasonic treatment; after the ultrasonic treatment is completed, add α-amylase and glucoamylase to the suspension and perform enzymatic hydrolysis until the iodine color reaction of the suspension is colorless or light yellow to obtain an enzymatic hydrolysate; Step 4: Add hydrogen peroxide to the enzymatic hydrolysate obtained in Step 3, stir evenly, perform ultrasonic treatment, then inactivate the enzyme and remove hydrogen peroxide; perform solid-liquid separation on the treated enzymatic hydrolysate, take the supernatant for alcohol precipitation, and collect the precipitate; remove the ethanol residue in the precipitate and dry to obtain crude Pueraria lobata polysaccharide; Step 5: The crude Pueraria lobata polysaccharide described in Step 4 is subjected to separation and purification to obtain Pueraria lobata non-starch polysaccharide.

2. The preparation method according to claim 1, characterized in that, The crude Pueraria lobata powder described in Step 2 is subjected to the supercritical carbon dioxide fluid extraction treatment in an extraction kettle, the carbon dioxide flow rate is 30-50 L / h, and the extraction is carried out for 3-5 hours at a pressure of 30-50 MPa and a temperature of 30-50 °C. The remaining Pueraria lobata solid residue in the extraction kettle is dried by microwave vacuum to obtain the Pueraria lobata powder.

3. The preparation method according to claim 1, characterized in that, In Step 3, the material-liquid ratio of the Pueraria lobata powder to water is 1:15-20 (g / mL); the addition amount of the cerium chloride is 1-2 g / 100 g of Pueraria lobata powder; the power of the ultrasonic treatment is 200-400 W, the temperature is 30-50 °C, and the duration is 20-40 min; after the ultrasonic treatment is completed, add thermostable α-amylase to the suspension, the addition amount is 1-2 g / 100 g of Pueraria lobata powder, and perform enzymatic hydrolysis at pH = 6-6.5 and 90-95 °C for 1-2 h; then add glucoamylase, the addition amount of glucoamylase is 1 g / 100 g of Pueraria lobata powder, and perform enzymatic hydrolysis at pH = 4-4.5 and 55-65 °C until the iodine color reaction of the suspension is colorless or light yellow to obtain an enzymatic hydrolysate.

4. The preparation method according to claim 3, wherein In Step 3, the material-liquid ratio of the Pueraria lobata powder to water is 1:15 (g / mL); the addition amount of the cerium chloride is 2 g / 100 g of Pueraria lobata powder; the power of the ultrasonic treatment is 200 W, the temperature is 30 °C, and the duration is 40 min; the addition amount of the thermostable α-amylase is 2 g / 100 g of Pueraria lobata powder, and perform enzymatic hydrolysis at pH 6.5 and 95 °C for 1 h; the addition amount of glucoamylase is 1 g / 100 g of Pueraria lobata powder, and perform enzymatic hydrolysis at pH 4 and 55 °C.

5. The preparation method according to claim 1, characterized in that, In Step 4, the final concentration of hydrogen peroxide added to the enzymatic hydrolysate in the solution is 1%-3%; the power of the ultrasonic treatment is 250-350 W, the temperature is 40-60 °C, and the duration is 20-40 min; and / or During the alcohol precipitation process, add ethanol to make the mass percentage of ethanol in the solution reach 70%-80%.

6. The preparation method according to claim 5, wherein The final concentration of hydrogen peroxide added to the enzymatic hydrolysate described in step 4 in the solution is 3%; the power of the ultrasonic treatment is 250 W, the temperature is 40 °C, and the duration is 40 min; and / or During the ethanol precipitation process, ethanol is added to make the mass percentage of ethanol in the solution reach 70%.

7. The preparation method according to claim 1, characterized in that, The separation and purification treatment of crude pueraria polysaccharide in step 5 includes: dissolving the crude pueraria polysaccharide in water to prepare a polysaccharide solution; performing column chromatography on the polysaccharide solution, using a D101 macroporous resin column as the stationary phase and distilled water as the elution solvent, and stopping the elution when the molish reaction of the eluate is negative. The collected eluate is concentrated and dried to obtain crude pueraria polysaccharide II; The obtained crude pueraria polysaccharide II is used to prepare a pueraria non-starch polysaccharide solution, and the filtrate obtained after filtration is subjected to gel filtration chromatography. The gel filtration chromatography uses a Sephacryl S-400 HR gel filtration column, and the elution solvent is 0.1 M sodium chloride solution. The eluate corresponding to the polysaccharide peak in the absorbance curve at 490 nm is collected; the eluate is dialyzed through a dialysis bag with a molecular weight cut-off of 8000 - 12000 Da, and the dialysis retentate is dried to obtain pueraria non-starch polysaccharide; preferably, the concentration of the pueraria non-starch polysaccharide solution is 5 - 10 mg / mL; the flow rate of the elution solvent is controlled at 0.5 - 1.0 mL / min.

8. Pueraria non-starch polysaccharide prepared by the preparation method according to any one of claims 1 - 7.

9. The pueraria non-starch polysaccharide according to claim 8, characterized in that, The pueraria non-starch polysaccharide is a pyranose polysaccharide with an α-configuration, and is composed of (0.17 ± 0.25)% fucose, (20.79 ± 0.56)% rhamnose, (3.28 ± 0.54)% arabinose, (38.97 ± 0.21)% galactose, (22.09 ± 0.41)% glucose, (3.9 ± 0.34)% xylose, (4.72 ± 0.3)% mannose, (3.49 ± 0.25)% galacturonic acid, and (2.59 ± 0.72)% glucuronic acid, with a molecular weight of 95210 Da.

10. Use of the pueraria non-starch polysaccharide according to claim 9, characterized in that, Including: Application in hypoglycemic drugs; Application in drugs for relieving insulin resistance; Application in drugs for reducing the level of glycosylated serum protein.

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