Extraction method of burdock polysaccharide and application of burdock polysaccharide in preparation of medicine for reducing blood sugar and blood fat

Abstract

The invention discloses an extraction method of burdock polysaccharide and application of the burdock polysaccharide in preparation of drugs for reducing blood sugar and blood fat, and the extraction method comprises the following steps: crushing burdock roots, and sieving to obtain burdock powder; the burdock powder and distilled water are mixed according to the mass-to-volume ratio of 1: 20, compound enzyme is added for enzymolysis for 2 h, and enzymatic hydrolysate is obtained; the enzymatic hydrolysate is subjected to microwave-assisted extraction, the microwave power is 600 W, the extraction time is 10 min, and an extracting solution is obtained; centrifuging, taking supernate, precipitating with ethanol, and freeze-drying the precipitate to obtain the burdock polysaccharide. According to the method disclosed by the invention, the polysaccharide yield is remarkably improved by combining composite enzymolysis and microwave-assisted extraction, and the extracted burdock polysaccharide is uniform in molecular weight distribution and remarkable in blood sugar and blood fat reducing effects.

Description

Technical Field

[0001] The present invention relates to the technical field of burdock polysaccharide extraction, and more specifically to an extraction method of burdock polysaccharide and its application in the preparation of hypoglycemic and hypolipidemic drugs. Background Art

[0002] Burdock ( Arctium lappa ) is a traditional Chinese medicinal material with effects such as clearing heat and detoxifying, detumescence and diuresis. In recent years, it has been found that burdock polysaccharide has significant biological activities such as hypoglycemic, hypolipidemic, antioxidant and immunomodulatory effects, so it has broad application prospects in the development of functional foods and drugs.

[0003] There are many defects in the existing technology for extracting burdock polysaccharide. For example, the extraction efficiency is low: the common extraction methods of burdock polysaccharide in the existing technology (such as hot water extraction method, ultrasonic-assisted extraction method) have problems of low extraction efficiency and low polysaccharide yield; the biological activity is poor: the molecular weight distribution of the burdock polysaccharide extracted by the existing methods is uneven, resulting in insignificant hypoglycemic and hypolipidemic effects; the process is complex: some extraction methods (such as enzymatic hydrolysis method) have complex processes, high costs, and are difficult to be applied on a large scale; there is a lack of systematic research: there is a lack of systematic research on the relationship between the molecular weight, structure and biological activity of burdock polysaccharide in the existing technology, resulting in unstable biological activity of the extracted polysaccharide.

[0004] Therefore, how to provide an extraction method of burdock polysaccharide with high polysaccharide extraction rate, good antioxidant effect, excellent hypoglycemic and hypolipidemic effects. Summary of the Invention

[0005] In view of this, the present invention provides an extraction method of burdock polysaccharide. By optimizing the extraction process parameters, the polysaccharide yield and biological activity are significantly improved. The yield of burdock polysaccharide is 15.2%, and the molecular weight range is 10 - 50 kDa.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: One aspect of the embodiments of the present invention provides an extraction method of burdock polysaccharide, including the following steps: 1) Crushing burdock roots, sieving to obtain burdock powder; 2) Mixing the burdock powder with distilled water at a mass:volume ratio of 1g:20ml, adding a composite enzyme for enzymatic hydrolysis for 2h to obtain an enzymatic hydrolysate; 3) Performing microwave-assisted extraction on the enzymatic hydrolysate obtained in step 2), with a microwave power of 600 W and an extraction time of 10 min to obtain an extract; 4) Centrifuging the extract obtained in step 3) at 12000 rpm for 5 min, taking the supernatant and precipitating with ethanol, and freeze-drying the precipitate to obtain burdock polysaccharide.

[0007] In a preferred embodiment, in step 1), the sieving is through a 60-mesh sieve.

[0008] In a preferred embodiment, in step 2), the addition amount of the complex enzyme is 1% of the mass of the burdock powder.

[0009] In a preferred embodiment, in step 2), the complex enzyme is cellulase, pectinase and protease, and the mass ratio is 1:2:1.5; Preferably, the protease includes plant hydrolytic protease and papain, and the mass ratio is 1:1.

[0010] In a preferred embodiment, in step 2), the temperature of the enzymatic hydrolysis is 50 °C.

[0011] In a preferred embodiment, the final concentration of ethanol for ethanol precipitation is 70%.

[0012] The second aspect of the embodiment of the present invention provides the application of the extracted burdock polysaccharide in the preparation of hypoglycemic drugs, and the dosage range of the burdock polysaccharide is 100 - 500 mg / kg.

[0013] The third aspect of the embodiment of the present invention provides the application of the extracted burdock polysaccharide in the preparation of hypolipidemic drugs, and the dosage range of the burdock polysaccharide is 100 - 500 mg / kg.

[0014] Preferably, the hypoglycemic drug is prepared by the following steps: Mix the extracted burdock polysaccharide with pharmaceutically acceptable excipients to prepare tablets, capsules or oral liquids.

[0015] Preferably, the hypolipidemic drug is prepared by the following steps: Mix the extracted burdock polysaccharide with pharmaceutically acceptable excipients to prepare tablets, capsules or oral liquids.

[0016] From the above technical solutions, it can be seen that the technical effects achieved by the present invention are: the present invention provides a method for extracting burdock polysaccharide, and by optimizing the extraction process parameters, the polysaccharide yield and biological activity are significantly improved. The burdock polysaccharide extracted by the present invention has the following advantages: Efficient extraction: By combining complex enzymatic hydrolysis and microwave-assisted extraction, the polysaccharide yield is significantly improved.

[0017] Outstanding biological activity: The molecular weight distribution of the extracted burdock polysaccharide is uniform, the yield of burdock polysaccharide is as high as 15.2%, the molecular weight range is 10 - 50 kDa, and the hypoglycemic and hypolipidemic effects are significantly better than those of the prior art.

[0018] Simple process: The extraction process is simple and suitable for large-scale production.

[0019] High safety: Animal toxicity experiments have proven that the extracted burdock polysaccharide has no significant toxicity to experimental animals. Specific implementation manners

[0020] The following will describe the technical solutions of the present invention clearly and completely in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0021] Example 1, A method for extracting burdock polysaccharide, comprising the following steps: 1) Crush burdock roots and pass through a 60-mesh sieve to obtain burdock powder; 2) Mix the burdock powder with distilled water at a mass: volume ratio of 1:20, add a composite enzyme and enzymolyze for 2 h to obtain an enzymolyzed solution; the addition amount of the composite enzyme is 1% of the mass of the burdock powder; the composite enzyme is cellulase, pectinase and protease, with a mass ratio of 1:2:1.5; the protease includes plant hydrolytic protease and papain, and the mass ratio is 1:1, and the temperature of enzymolysis is 50 °C; 3) Perform microwave-assisted extraction on the enzymolyzed solution obtained in step 2), with a microwave power of 600 W and an extraction time of 10 min to obtain an extract; 4) Centrifuge the extract obtained in step 3) at 12,000 rpm for 5 min, take the supernatant and precipitate it with 70% ethanol, and the precipitate is freeze-dried to obtain burdock polysaccharide.

[0022] Example 2, Molecular weight identification of burdock polysaccharide Use high performance liquid chromatography-mass spectrometry (HPLC-MS) for molecular weight identification.

[0023] 1. Sample preparation Sample preparation is one of the key steps in HPLC-MS analysis. The sample must be properly processed to ensure its suitability for HPLC-MS analysis: dissolve the sample in a solvent, filter the sample using a filter membrane to remove particles and other impurities.

[0024] 2. HPLC separation In the HPLC part, the sample is injected into the chromatographic column and eluted by the mobile phase.

[0025] 3. Mass spectrometry detection The results show that the molecular weight range of the extracted burdock polysaccharide is 10 - 50 kDa, and the molecular weight distribution is uniform.

[0026] Example 3, Experiment on the hypoglycemic effect of burdock polysaccharide Experimental animals: SPF-grade SD rats.

[0027] Experimental design: The rats were divided into a control group, a model group (hyperglycemia model), and an experimental group (intragastric administration of burdock polysaccharide at a dose of 200 mg / kg), with 10 rats in each group.

[0028] Control group: Normal diet, without any treatment.

[0029] Model group: Hyperglycemia model group, induced hyperglycemia by injecting streptozotocin (STZ).

[0030] Experimental group: Hyperglycemia model group, intragastric administration of burdock polysaccharide at a dose of 200 mg / kg.

[0031] Establishment process of hyperglycemia model STZ injection: Dissolve streptozotocin (STZ) in citrate buffer (pH 4.5) to prepare a solution of 50 mg / kg. After fasting the rats for 12 h, intraperitoneally inject the STZ solution at a dose of 50 mg / kg. 72 h after injection, measure the fasting blood glucose level, and rats with a blood glucose value ≥ 11.1 mmol / L are considered to have successfully established a hyperglycemia model.

[0032] Model verification: The fasting blood glucose level of rats in the hyperglycemia model group was significantly higher than that of the control group (P < 0.05), indicating that the hyperglycemia model was successfully established.

[0033] Before STZ injection, measure the fasting blood glucose level of all rats to ensure that there is no significant difference in the initial blood glucose values of rats in each group.

[0034] Rats in the experimental group were intragastrically administered burdock polysaccharide (200 mg / kg) every day for 28 consecutive days.

[0035] Rats in the control group and the model group were intragastrically administered an equal volume of normal saline.

[0036] After 28 days, measure the fasting blood glucose level of rats in each group.

[0037] The experimental results are shown in Table 1; Table 1 Fasting blood glucose levels of rats in each group before and after treatment (mean ± standard deviation, unit: mmol / L)

[0038] Note: * indicates significant difference (P < 0.05) compared with the model group.

[0039] As can be seen from Table 1, after injecting burdock polysaccharide, the fasting blood glucose level in the experimental group was significantly lower than that in the model group (P < 0.05), indicating that the burdock polysaccharide prepared in Example 1 has a significant hypoglycemic effect.

[0040] Example 4, Experiment on the Hypolipidemic Effect of Arctium Lappa Polysaccharide Experimental animals: SPF-grade SD rats, sourced from a certain experimental animal center.

[0041] Experimental design: The rats were divided into a control group, a model group (hyperlipidemia model), and an experimental group (intragastric administration of Arctium Lappa polysaccharide at a dose of 200 mg / kg), with 10 rats in each group.

[0042] Process of establishing the hyperlipidemia model Induction with a high-fat diet: High-fat diet formula: Regular feed (60%), lard (20%), cholesterol (1.5%), sodium cholate (0.5%), sucrose (10%).

[0043] The rats in the model group and the experimental group were continuously fed the high-fat diet for 8 weeks to induce the hyperlipidemia model.

[0044] The rats in the control group were fed the regular feed.

[0045] Model verification: After 8 weeks, the serum total cholesterol (TC) and triglyceride (TG) levels of the rats in the model group and the experimental group were measured to ensure that the TC and TG levels of the rats in the model group were significantly higher than those in the control group (P<0.05), indicating that the hyperlipidemia model was successfully established.

[0046] Experimental treatment and results Blood lipid levels before treatment: Before the induction with the high-fat diet, the serum TC and TG levels of all rats were measured to ensure that there were no significant differences in the initial blood lipid levels among the rats in each group.

[0047] Blood lipid levels before treatment (mean ± standard deviation): Control group TC: 1.8 ± 0.2 mmol / L, TG: 0.9 ± 0.1 mmol / L Model group: TC: 1.9 ± 0.3 mmol / L, TG: 1.0 ± 0.2 mmol / L Experimental group: TC: 1.8 ± 0.2 mmol / L, TG: 0.9 ± 0.1 mmol / L Blood lipid levels after treatment: The rats in the experimental group were intragastrically administered Arctium Lappa polysaccharide (200 mg / kg) every day for 4 consecutive weeks.

[0048] The rats in the control group and the model group were intragastrically administered an equal volume of normal saline.

[0049] After 4 weeks, the serum TC and TG levels of the rats in each group were measured.

[0050] The experimental results are shown in Table 2 Table 2 Serum TC and TG levels of rats in each group before and after treatment (mean ± standard deviation, unit: mmol / L)

[0051] Note: * indicates significant difference (P<0.05) compared with the model group

[0052] Result analysis: The serum TC and TG levels of rats in the model group increased significantly (TC: 8.5 ± 0.6 mmol / L, TG: 4.2 ± 0.5 mmol / L), indicating that the hyperlipidemia model was successfully established. The serum TC and TG levels of rats in the experimental group were significantly lower than those in the model group (TC: 5.2 ± 0.4 mmol / L, TG: 2.3 ± 0.3 mmol / L, P<0.05), indicating that burdock polysaccharide has a significant lipid-lowering effect; the serum TC and TG levels of rats in the control group remained normal (TC: 1.9 ± 0.3 mmol / L, TG: 1.0 ± 0.2 mmol / L) and were not affected by the high-fat diet

[0053] From the above results, it can be seen that: In this experiment, a hyperlipidemia model was successfully established through a high-fat diet, and the lipid-lowering effect of burdock polysaccharide was verified. The experimental results show that intragastric administration of burdock polysaccharide (200 mg / kg) can significantly reduce the serum TC and TG levels of rats with hyperlipidemia model, and the lipid-lowering effect is better than that of burdock polysaccharide extracted by existing technologies

[0054] Example 5, Animal toxicity experiment of burdock polysaccharide Experimental animals: SPF-grade SD rats

[0055] Experimental design: The rats were divided into a control group (intragastric administration of an equal volume of normal saline) and an experimental group (intragastric administration of burdock polysaccharide, dose: 500 mg / kg), with 10 rats in each group, and the experimental period was 28 days

[0056] Results: There was no significant difference in the body weight and blood biochemical indexes (such as ALT, AST, CRE) of rats in the experimental group and the control group (P>0.05), indicating that burdock polysaccharide has no significant toxicity to experimental animals. The results are shown in Table 3 and Table 4 Table 3 Body weight changes of rats in each group before and after the experiment (mean ± standard deviation, unit: g)

[0057] Table 4 Blood biochemical indexes of rats in each group after the experiment (mean ± standard deviation)

[0058] Result analysis: There were no significant differences in the body weights of the rats in the experimental group and the control group before and after the experiment (P>0.05), indicating that burdock polysaccharide had no significant effect on the growth and development of rats. There were no significant differences in the levels of ALT, AST, and CRE in the rats of the experimental group and the control group (P>0.05), indicating that burdock polysaccharide had no significant toxicity to the liver and kidney functions of rats. Through 28 days of gavage experiment, the safety of burdock polysaccharide was verified. The experimental results showed that gavage of burdock polysaccharide (500 mg / kg) had no significant effect on the body weight, liver function (ALT, AST), and kidney function (CRE) of rats, indicating that burdock polysaccharide had no significant toxicity to experimental animals.

[0059] Example 6, Antioxidant Activity Experiment of Burdock Polysaccharide Experimental design: The DPPH free radical scavenging experiment, hydroxyl free radical scavenging experiment, and superoxide anion scavenging experiment were used to determine the antioxidant activity of burdock polysaccharide.

[0060] Results: DPPH free radical scavenging rate: The scavenging rate of burdock polysaccharide extracted by the method of the present invention was 85.3%, that of the hot water extraction method was 60.2%, and that of the ultrasonic-assisted extraction method was 65.4%.

[0061] Hydroxyl free radical scavenging rate: The scavenging rate of burdock polysaccharide extracted by the method of the present invention was 78.5%, that of the hot water extraction method was 55.6%, and that of the ultrasonic-assisted extraction method was 58.7%.

[0062] Superoxide anion scavenging rate: The scavenging rate of burdock polysaccharide extracted by the method of the present invention was 82.1%, that of the hot water extraction method was 50.3%, and that of the ultrasonic-assisted extraction method was 53.2%.

[0063] Example 7, Influence of Different Purification Methods on the Effect of Burdock Polysaccharide Experimental design: DEAE-52 cellulose column chromatography and Sephadex G-100 gel column chromatography were used to purify burdock polysaccharide, and the yield and biological activity of the polysaccharide before and after purification were compared.

[0064] Results: Yield of purified polysaccharide: The yield after DEAE-52 purification was 12.5%, and the yield after Sephadex G-100 purification was 10.8%.

[0065] Blood glucose lowering effect after purification: The blood glucose lowering effect after DEAE-52 purification was 32.5%, and that after Sephadex G-100 purification was 30.8%.

[0066] Blood lipid lowering effect after purification: The blood lipid lowering effect after DEAE-52 purification was 28.7%, and that after Sephadex G-100 purification was 27.3%.

[0067] Comparative Example Comparative Example 1: Hot Water Extraction Method Extraction method: Mix burdock powder with distilled water at a ratio of 1:20 (w / v) and extract at 90°C for 2 hours.

[0068] Result: The polysaccharide yield was 8.5%, the molecular weight distribution was uneven, and the hypoglycemic and hypolipidemic effects were poor.

[0069] Comparative Example 2: Ultrasonic-Assisted Extraction Method Extraction method: Mix burdock powder with distilled water at a ratio of 1:20 (w / v), the ultrasonic power is 300 W, and the extraction time is 30 minutes.

[0070] Result: The polysaccharide yield was 10.2%, the molecular weight distribution was uneven, and the hypoglycemic and hypolipidemic effects were average.

[0071] Comparative Example 3: Change in Enzymolysis Temperature (40°C) Extraction method: The enzymolysis temperature is 40°C, and other conditions are the same as in Example 1.

[0072] Result: The polysaccharide yield was 12.5%, and the hypoglycemic and hypolipidemic effects were lower than those in Example 1.

[0073] Comparative Example 4: Change in Microwave Power (400 W) Extraction method: The microwave power is 400 W, and other conditions are the same as in Example 1.

[0074] Result: The polysaccharide yield was 13.8%, and the hypoglycemic and hypolipidemic effects were lower than those in Example 1.

[0075] The specific results are shown in Table 5, Table 6, Table 7 and Table 8; Table 5 Polysaccharide Yield and Molecular Weight Distribution of Burdock Polysaccharide by Different Extraction Methods

[0076]

[0077] Table 6 Hypoglycemic and Hypolipidemic Effects of Different Extraction Methods

[0078] Table 7 Antioxidant Activities of Different Extraction Methods

[0079] Table 8 Effects of Different Purification Methods on Burdock Polysaccharide

[0080] The various embodiments in this specification are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. For the same or similar parts among the various embodiments, reference can be made to each other.

[0081] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for extracting burdock polysaccharide, characterized in that, It includes the following steps: 1) Crush the burdock roots, sieve them to obtain burdock powder; 2) Mix the burdock powder with distilled water at a mass:volume ratio of 1 g:20 ml, add a composite enzyme for enzymatic hydrolysis for 2 h to obtain an enzymatic hydrolysate; the composite enzyme is cellulase, pectinase and protease, with a mass ratio of 1:2:1.5; the protease includes plant hydrolytic protease and papain, and the mass ratio is 1:1; 3) Perform microwave-assisted extraction on the enzymatic hydrolysate obtained in step 2), with a microwave power of 600 W and an extraction time of 10 min to obtain an extract; 4) Centrifuge the extract obtained in step 3), take the supernatant and precipitate it with ethanol, and freeze-dry the precipitate to obtain burdock polysaccharide.

2. The extraction method of burdock polysaccharide according to claim 1, characterized in that In step 1), the sieving is through a 60-mesh sieve.

3. The extraction method of burdock polysaccharide according to claim 1, characterized in that, In step 2), the addition amount of the composite enzyme is 1% of the mass of the burdock powder.

4. The extraction method of burdock polysaccharide according to claim 1, characterized in that, In step 2), the temperature of the enzymatic hydrolysis is 50 °C.

5. The extraction method of burdock polysaccharide according to claim 1, characterized in that, In step 4), the final ethanol concentration for ethanol precipitation is 70%; the centrifugation is at 12,000 rpm for 5 min.

6. Use of the burdock polysaccharide extracted by the method according to any one of claims 1-5 in the preparation of a hypoglycemic drug, characterized in that, The dosage range of the burdock polysaccharide is 100 - 500 mg / kg.

7. Use of the burdock polysaccharide extracted by the method according to any one of claims 1-5 in the preparation of a lipid-lowering drug, characterized in that, The dosage range of the burdock polysaccharide is 100 - 500 mg / kg.

8. The application according to claim 6, characterized in that The hypoglycemic drug is prepared through the following steps: Mix the extracted burdock polysaccharide with pharmaceutically acceptable excipients to prepare tablets, capsules or oral liquids.

9. The application according to claim 7, wherein The hypolipidemic drug is prepared through the following steps: Mix the extracted burdock polysaccharide with pharmaceutically acceptable excipients to prepare tablets, capsules or oral liquids.

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