Gastrodia elata protein polypeptide with effects of reducing blood sugar and blood fat and managing body weight as well as preparation method and application of gastrodia elata protein polypeptide

Abstract

The invention belongs to the technical field of medical technology and deep processing of food-borne protein or polypeptide, and discloses gastrodia elata protein polypeptide with the effects of reducing blood sugar and blood fat and managing body weight, and the gastrodia elata protein polypeptide is obtained by extracting gastrodia elata to obtain crude protein and then carrying out food-borne enzyme hydrolysis, enzyme deactivation, centrifugation and ultrafiltration; the protein polypeptide is obtained by concentrating a stock solution with the molecular weight cutoff of 500-5000Da; the food-borne enzyme is a single enzyme of food-borne protease or a compound enzyme mixed with the food-borne protease. The protein production process is green and environment-friendly, the product safety is good, the protein content is high, and the protein has good alpha-glucosidase activity and blood fat reducing activity. Therefore, the gastrodia elata polypeptide provided by the invention has good effects of reducing blood sugar and blood fat, and has application prospects in the aspects of special medical foods, health foods, functional foods and medicines.

Description

Technical Field

[0001] The present invention belongs to the technical fields of pharmaceutical technology and deep processing technology of food-derived proteins or polypeptides, and in particular relates to a gastrodia protein polypeptide with the functions of reducing blood sugar, reducing blood lipids and weight management, a preparation method thereof, and an application thereof. Background Art

[0002] Gastrodiae rhizoma is a plant of the genus Gastrodia in the Orchidaceae family, also known as divine herb, red arrow, white dragon skin, mountain sweet potato, etc. Gastrodiae rhizoma has a clinical application history of more than two thousand years. It is recorded in "Shennong Ben Cao Jing" that Gastrodiae rhizoma has the miraculous effect of "eliminating poisons and evil qi, prolonging life with qi and strength, nourishing yin and promoting health, and lightening the body and increasing lifespan"; it is recorded in "Mingyi Bielu" that Gastrodiae rhizoma can "reduce swelling and pain, relieve lower abdominal fullness, (treat) cold hernia with bloody stools"; it is recorded in "Rihuazi Bencao" that Gastrodiae rhizoma can "boost yang qi, tonify the five overstrains and seven injuries, promote blood circulation, and open the orifices"; it is recorded in "Kaibao Bencao" that Gastrodiae rhizoma can "treat various wind-damp arthralgia, limb contracture, infantile epilepsy, frightened qi, benefit the waist and knees, and strengthen the tendons and bones". It is recorded in "Chinese Pharmacopoeia" that Gastrodiae rhizoma has the effects of calming endogenous wind and relieving convulsions, and is used for dizziness, limb numbness, infantile convulsions, epilepsy, hypertension, and otogenic vertigo.

[0003] Gastrodiae rhizoma has extremely high nutritional value, rich in gastrodin, vanillin, protein, amino acids, trace elements, etc. In November 2023, Gastrodiae rhizoma was included in the list of "substances that are both foods and traditional Chinese medicines according to tradition". At present, there are relatively many reports on gastrodin and gastrodia polysaccharide, but there are few reports on the protein polypeptide in Gastrodiae rhizoma, and there is no report on the activity in reducing blood sugar and blood lipids.

[0004] α-Glucosidase can hydrolyze the α-1,4-glycosidic bond of starch and can slowly hydrolyze the α-1,6-glycosidic bond, converting starch into glucose, thereby increasing blood sugar in the human body. Inhibiting α-glucosidase becomes one of the treatment means for controlling postprandial hyperglycemia in type 2 diabetes by delaying the digestion and absorption of carbohydrates, prolonging the digestion time, and reducing the absorption rate of glucose. At present, clinically used α-glucosidase inhibitors include acarbose, miglitol, voglibose, etc., but they are prone to cause gastrointestinal adverse reactions, such as side effects like diarrhea and flatulence. Therefore, it is of great research significance and practical application value to discover new, highly efficient and low-side-effect food-derived α-glucosidase inhibitors.

[0005] Pancreatic lipase is the most important enzyme for hydrolyzing dietary fat. It acts on the emulsified particles of triglycerides, mainly converting the latter into free fatty acids and 2-monoacylglycerol, etc., which are then absorbed by the human body. If a large amount of the absorbed fat is stored in the human body, it will cause people to become obese. Lipase inhibitors can effectively inhibit the fat-hydrolyzing effect of lipase, thereby preventing the human body from absorbing fat, controlling blood lipids, and treating obesity. Statins are common drugs for inhibiting cholesterol synthesis mechanisms, but long-term use can also cause adverse reactions in the human body, such as muscle weakness and muscle pain, etc., and at the same time increase the risk of diabetes. Orlistat is a gastrointestinal lipase inhibitor that can inhibit the absorption of triglycerides, thereby reducing calorie intake, controlling weight, and further reducing obesity and its complications, but there are also side effects such as gastrointestinal discomfort. Therefore, discovering new, highly efficient, and low-side-effect food-derived pancreatic lipase inhibitors also has important research significance and practical application value.

[0006] In today's society, obesity has become a serious public health problem globally. According to the data of the World Obesity Atlas (2024 Edition), 5 million people die globally every year from diseases related to overweight or obesity. Obesity is significantly associated with 5% of cancer deaths, 17% of stroke deaths, 19% of coronary heart disease deaths, and 42% of type 2 diabetes deaths. Therefore, discovering functional foods with the characteristics of weight control, safety, effectiveness, and suitability for long-term use has great market application prospects.

[0007] The currently clinically used new weight-loss drug semaglutide has achieved great commercial success. It is a GLP-1 receptor agonist used to treat obesity and diabetes. Although it is significantly effective in controlling blood sugar and weight loss, there are obvious side effects, such as gastrointestinal reactions like nausea, vomiting, diarrhea, constipation, etc., as well as decreased appetite, headache, fatigue, etc. In addition, semaglutide is a prescription drug and needs to be injected, so there are certain limitations in its ease of use. Orlistat is a pancreatic lipase inhibitor and an oral drug currently used for weight loss, and it also has obvious gastrointestinal reactions, such as oily stools, a sense of urgency to defecate, steatorrhea, abdominal pain or discomfort, increased flatulence, etc., as well as headache, upper respiratory tract infection, etc. And it will affect the absorption of fat-soluble vitamins (A, D, E, K). In this invention, the raw material of Gastrodia elata is a plant with both medicinal and edible uses, which has a long history of consumption, high safety, and is suitable for long-term use. In addition, Gastrodia elata polypeptide has both blood sugar-lowering and blood lipid-lowering effects and can also provide nutrition. Therefore, discovering food-derived, safe, and effective ingredients with both blood sugar-lowering and blood lipid-lowering effects is the motivation of this invention, which can make up for the deficiencies of the existing technology.

[0008] Through retrieval, the following several publicly available documents related to this invention patent application were found:

[0009] 1. Patent Publication Document 1: Li Changqing, et al., A Gastrodia elata Protein Peptide Effervescent Tablet and Its Preparation Method. Application No. 201610864954.6. It discloses a preparation method of an effervescent tablet with Gastrodia elata protein peptide as the main raw material. Through technologies such as enzyme, membrane coupling, and freeze-drying, Gastrodia elata protein peptide is prepared, and various excipients are added to prepare an effervescent tablet, which has a certain health care effect. The protein peptide prepared by this invention has not been reported to have hypoglycemic and hypolipidemic activities.

[0010] 2. Patent Publication Document 2: Lu Jiahui, et al., A Gastrodia elata Polypeptide and Its Preparation Method and Medical Use in Antibacterial and Antiviral. Application No. 201510534793.X. It discloses a Gastrodia elata polypeptide and its preparation method and medical use in antibacterial and antiviral. It is obtained by extracting, filtering and concentrating, enzymolyzing, and ultrafiltration separation of Gastrodia elata protein, and then mixing with appropriate excipients in proportion according to the use and application field to prepare corresponding preparations. This peptide is an acidic polypeptide with a molecular weight range of 5.8 - 7.8 KD and has antibacterial and antiviral abilities. It is different from the basic characteristics such as the molecular weight of the polypeptide in this invention, and the activities are also different.

[0011] 3. Patent Publication Document 3: Yuan Guangxin, et al., A Gastrodia elata Small Peptide and Its Preparation Method and Application, Application No. 202410933731.5. This patent discloses a Gastrodia elata small peptide and its preparation method and use in anti-aging and relieving cognitive dysfunction.

[0012] 4. Journal 1: Study on the Chemical Constituents and Hypoglycemic Activity of Gastrodia elata from Zhaotong Yiliang, Journal of Chinese Medicinal Materials, 2024, 47(2): 357 - 364. This report mainly separates and purifies small molecule components from the acetone extract of Gastrodia elata from Zhaotong Yiliang, and the target substance is essentially different from this invention.

[0013] 5. Journal 2: Effects and Molecular Mechanisms of Gastrodin Injection on High Glucose-Induced Proliferation, Migration and Epithelial-Mesenchymal Transition of Podocytes, Laboratory Medicine and Clinic, 2025, 22(01): 18 - 23. This report explores the effects of Gastrodin injection (GI), which takes gastrodin, the main component of Gastrodia elata extract, on the proliferation, migration and epithelial-mesenchymal transition (EMT) process of human glomerular podocytes (HG-PC), and analyzes whether GI plays a role by regulating the JAK2 / STAT3 signaling pathway.

[0014] 6. Journal 3: Study on the Chemical Constituents of Gastrodia elata and Their Antioxidant and α-Glucosidase Inhibitory Activities. Chinese Traditional and Herbal Drugs, 2024, 55(19): 6474-6481. This study first separated and purified the 80% ethanol extract of the dried rhizome of Gastrodia elata, isolated 10 compounds from it, and determined the chemical constituents of the rhizome of Gastrodia elata and their antioxidant and α-glucosidase inhibitory activities. The compounds reported in this literature are small molecule compounds, which are essentially different from the present invention.

[0015] 7. Journal 4: Constituents of Gastrodia elata and their neuroprotective effects in HT22 hippocampal neuronal, R28 retinal cells, and BV2 microglial cells. Plants, 2020, 9(8): 1051. This study isolated two new phenolic compounds (1 and 2) and a new tripeptide (3), as well as 16 known compounds (4-19) from the rhizome of the plant G. elata, and evaluated the ability of all the obtained compounds to protect neuronal cells from neurotoxicity and neuroinflammation. It is also essentially different from the present invention.

[0016] By comparison, the present invention is essentially different from the above-mentioned published literatures. First, the enzymes used in the enzymatic hydrolysis method are different. The polypeptide structures obtained by hydrolysis with different enzymes vary greatly, and their activities cannot be predicted. In addition to using single enzymatic hydrolysis, the present invention also adopts a composite enzyme cascade enzymatic hydrolysis method, and the obtained polypeptides have novel activities. Second, the molecular weights of the peptides retained by ultrafiltration are different. Peptides with different molecular weights have greatly different structures, and their activities will also be unpredictable. Third, in addition to inhibiting α-glucosidase, the present invention also has pancreatic lipase inhibitory activity, and the protein polypeptides with both blood glucose lowering and blood lipid lowering effects have high novelty. Therefore, the new application of obtaining novel Gastrodia elata active protein peptides by different enzymatic hydrolysis techniques is the motivation of the technical solution of the present invention. Summary of the Invention

[0017] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a Gastrodia elata protein polypeptide with the functions of blood glucose lowering, blood lipid lowering and weight management, a preparation method thereof and an application thereof.

[0018] The technical solution adopted by the present invention to solve its technical problems is:

[0019] A Gastrodia elata protein polypeptide with the functions of blood glucose lowering, blood lipid lowering and weight management, wherein the protein polypeptide is obtained by extracting crude protein from Gastrodia elata, followed by food-derived enzyme hydrolysis, enzyme inactivation, centrifugation, and ultrafiltration.

[0020] The protein polypeptide is obtained by concentrating the stock solution with a molecular weight cut-off of 500 to 5000 Da;

[0021] The food-derived enzyme is a single enzyme of food-derived protease or a composite enzyme used in combination.

[0022] Furthermore, the food-derived protease is one of flavor protease, acidic protease, neutral protease, alkaline protease or papain, or a composite enzyme used in combination of several of them.

[0023] The method for preparing gastrodia protein polypeptide as described above includes the following steps:

[0024] (1) Extraction of gastrodia crude protein: Mix the dried tubers of gastrodia with water at a feeding ratio of g:mL of 1:2 to 30, add 0.1 to 10 mol / L NaOH aqueous solution to adjust the pH to 8.0 to 11.0, stir at a temperature of 0 to 80 °C, and keep stirring for 1 to 10 h; cool to room temperature, centrifuge at 3500 to 9000 rpm for 10 to 20 min, take the supernatant, adjust the pH to 2 to 5 with 0.1 to 1 mol / L HCl, then centrifuge this liquid at 3500 to 9000 rpm for 10 to 20 min, take the precipitate, and freeze-dry to obtain gastrodia crude protein, with a yield of 10 to 20%;

[0025] (2) Preparation of gastrodia protein polypeptide: Take the gastrodia crude protein prepared in step (1), mix it with water at a feeding ratio of g:mL of 1:50 to 500, add a food-derived enzyme for enzymatic hydrolysis, with an enzyme activity of 10 to 1000 KU / g, the pH of the enzymatic hydrolysis reaction is 3 to 9, the reaction time is 2 to 4 h, the reaction temperature is 30 to 50 °C. After the enzymatic hydrolysis is completed, inactivate the enzyme at high temperature for 10 to 30 min, cool to room temperature, centrifuge at 7000 to 9000 r / min for 10 to 30 min, take the supernatant, ultrafilter to collect the supernatant with a molecular weight below 5 KDa, and dry to obtain gastrodia protein polypeptide.

[0026] Furthermore, the drying method is vacuum drying, spray drying, freeze-drying or atmospheric heating drying.

[0027] Furthermore, for every 1 part by weight of gastrodia crude protein, add 0.01 to 10 parts by weight of acidic protease, or add 0.01 to 10 parts by weight of neutral protease, or add 0.01 to 10 parts by weight of alkaline protease, or add 0.01 to 10 parts by weight of flavor protease, or add 0.01 to 10 parts by weight of papain, or use a composite enzyme in combination according to the above parts by weight.

[0028] The preparation obtained by using the gastrodia protein polypeptide as described above.

[0029] Furthermore, the preparation includes powder, granule, powder injection, tablet, capsule or liquid preparation.

[0030] Use of the Gastrodia elata protein polypeptide as described above in the preparation of a medicament having both blood glucose lowering, blood lipid lowering and weight management effects.

[0031] Use of the Gastrodia elata protein polypeptide as described above in the preparation of a food having both blood glucose lowering, blood lipid lowering, weight management and nutrition providing effects.

[0032] Use of the Gastrodia elata protein polypeptide as described above in special medical purpose foods and / or health foods and / or functional foods and / or medicaments.

[0033] The advantages and positive effects achieved by the present invention are as follows:

[0034] 1. The protein production process of the present invention is green and environmentally friendly, the product has good safety, high protein content (>70%), and has good α-glucosidase activity (similar to the clinically used α-glucosidase inhibitor acarbose), and has blood lipid lowering activity. Therefore, the Gastrodia elata polypeptide provided by the present invention has both good blood glucose lowering and blood lipid lowering effects, and has application prospects in special medical purpose foods, health foods, functional foods, and medicaments.

[0035] 2. The Gastrodia elata raw material of the present invention belongs to the products in the agricultural field, can improve the added value of agricultural products, increase farmers' income, and meet the national strategic needs.

[0036] 3. The present invention discloses a new method and new application of a novel protein peptide having both blood glucose lowering and blood lipid lowering activities using the medicine and food homologous Gastrodia elata as a raw material. In the enzymatic hydrolysis method, both single enzyme hydrolysis and composite enzyme cascade hydrolysis are used, and polypeptides with a certain molecular weight (<5000) are intercepted by ultrafiltration, obtaining polypeptides with good purity and high activity.

[0037] 4. The Gastrodia elata short peptide protein of the present invention has a uniform color, is white to light yellow, has a small molecular weight, and is easier to be absorbed and utilized by the human body compared with proteins. The preparation process is simple, the quality and efficacy of the product are good, the production cost is low, and the investment is small.

[0038] 5. The raw material Gastrodia elata used in the protein of the present invention is a medicine and food dual-purpose raw material; the enzymes used are all food-derived enzymes, which are safe and reliable; the main solvent used in the production process is water, without the emission of organic solvents, green and environmentally friendly, and suitable for the requirements of green and environmentally friendly industrial production.

[0039] 6. The Gastrodia elata short peptide protein of the present invention can provide nutrients for the human body as a protein, and has both blood glucose regulating and blood lipid lowering activities. It can be applied to fields such as ordinary foods, special diets, health foods, food additives, special medical purpose foods, and drugs. It has a wide range of uses and good market prospects. There is no report on the Gastrodia elata protein polypeptide having both blood glucose lowering and blood lipid lowering activities at present, and it can be applied in the preparation of treating diabetes, hyperlipidemia and their complications. Brief Description of the Drawings

[0040] Figure 1 This is an inhibition curve of Gastrodia elata polypeptide obtained by hydrolysis with acid protease against α-glucosidase in the present invention. Detailed Embodiments

[0041] The present invention will be further described below in conjunction with embodiments. The following embodiments are narrative and not restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0042] All kinds of experimental operations involved in the specific embodiments are conventional techniques in the art. For parts not specifically noted in this article, those of ordinary skill in the art can refer to various commonly used reference books, scientific and technological literatures, or relevant specifications, manuals, etc. before the filing date of this invention application for implementation.

[0043] A Gastrodia elata protein polypeptide with the functions of reducing blood sugar, reducing blood lipids and weight management. The protein polypeptide is obtained by extracting crude protein from Gastrodia elata, and then through food-derived enzyme hydrolysis, enzyme inactivation, centrifugation, and ultrafiltration;

[0044] The protein polypeptide is obtained by concentrating the stock solution with a molecular weight cut-off of 500 - 5000 Da;

[0045] The food-derived enzyme is a single enzyme of food-derived protease or a composite enzyme used in combination.

[0046] Preferably, the food-derived protease is one of flavor protease, acid protease, neutral protease, alkaline protease or papain, or a composite enzyme used in combination of several of them.

[0047] The preparation method of the Gastrodia elata protein polypeptide as described above includes the following steps:

[0048] (1) Extraction of Gastrodia elata crude protein: Mix the dried tuber of Gastrodia elata with water at a feeding ratio of g:mL of 1:2 - 30, add 0.1 - 10 mol / L NaOH aqueous solution to adjust the pH = 8.0 - 11.0, stir at a temperature of 0 - 80 °C, and keep stirring for 1 - 10 h; cool to room temperature, centrifuge at 3500 - 9000 rpm for 10 - 20 min, take the supernatant, adjust the pH = 2 - 5 with 0.1 - 1 mol / L HCl, then centrifuge this liquid at 3500 - 9000 rpm for 10 - 20 min, take the precipitate, and freeze-dry to obtain Gastrodia elata crude protein, with a yield of 10 - 20%;

[0049] (2) Preparation of Gastrodia elata protein polypeptide: Take the crude Gastrodia elata protein prepared in step (1), mix it with water at a feeding ratio of g:mL of 1:50 - 500, add food-derived enzymes for enzymatic hydrolysis, with the enzyme activity of 10 - 1000 KU / g, the pH of the enzymatic hydrolysis reaction being 3 - 9, the reaction time being 2 - 4 h, the reaction temperature being 30 - 50 °C. After the enzymatic hydrolysis is completed, inactivate the enzyme at high temperature for 10 - 30 min, cool to room temperature, centrifuge at 7000 - 9000 r / min for 10 - 30 min, take the supernatant, ultrafilter and collect the supernatant with a molecular weight below 5 KDa, and dry it to obtain Gastrodia elata protein polypeptide.

[0050] Preferably, the drying method is vacuum drying, spray drying, freeze drying or atmospheric heating drying.

[0051] Preferably, for every 1 part by weight of the crude Gastrodia elata protein, add 0.01 - 10 parts by weight of acidic protease, or add 0.01 - 10 parts by weight of neutral protease, or add 0.01 - 10 parts by weight of alkaline protease, or add 0.01 - 10 parts by weight of flavor protease, or add 0.01 - 10 parts by weight of papain, or use a compound enzyme by mixing according to the above parts by weight.

[0052] A preparation obtained by using the Gastrodia elata protein polypeptide as described above.

[0053] Preferably, the preparation includes powder, granule, powder injection, tablet, capsule or liquid preparation.

[0054] Application of the Gastrodia elata protein polypeptide as described above in the preparation of drugs with functions of reducing blood sugar, reducing blood lipid and weight management.

[0055] Application of the Gastrodia elata protein polypeptide as described above in the preparation of foods with functions of reducing blood sugar, reducing blood lipid, weight management and providing nutrition.

[0056] Application of the Gastrodia elata protein polypeptide as described above in special medical foods and / or health foods and / or functional foods and / or drugs.

[0057] Specifically, the relevant preparation and detection are as follows:

[0058] Example 1

[0059] A preparation method of Gastrodia elata short peptide protein, and the preparation steps are as follows:

[0060] S1: Take the dried gastrodia elata tuber, crush it, mix 20 g of the crushed gastrodia elata material with 240 mL of water, adjust the pH to 10 with 1 mol / L NaOH aqueous solution, stir at 60 °C for 4 h. After stirring, cool it to room temperature, centrifuge at 8000 rpm for 12 min, take the supernatant, adjust the pH to 3 with 1 mol / L HCl, then centrifuge this liquid at 8000 rpm for 15 min, take the precipitate, and freeze-dry to obtain crude gastrodia elata protein, with a yield of approximately 12%.

[0061] S2: Take 400 mg of the crude gastrodia elata protein prepared in S1, add 50 mL of water, stir at 55 °C for 15 min, adjust the pH to 7.5 with 1 mol / L NaOH aqueous solution, add 100 mg of flavor protease according to the enzyme addition amount of 10000 μ / g, with an enzyme activity unit of 40 KU / g. At 55 °C, the enzymatic hydrolysis time is 2.5 h. After the enzymatic hydrolysis, raise the temperature to 90 °C to inactivate the enzyme for 20 min. After cooling to room temperature, centrifuge at 9000 rpm for 12 min, take the supernatant, filter the supernatant with an ultrafiltration centrifugal tube, collect the supernatant below 3 KDa, and vacuum freeze-dry to obtain oligopeptides of gastrodia elata protein hydrolyzed by flavor protease.

[0062] Example 2

[0063] A preparation method of gastrodia elata short peptide protein, the preparation steps are as follows:

[0064] The preparation of crude gastrodia elata protein is the same as that in Example 1. Take 400 mg of the crude gastrodia elata protein, add 50 mL of water, stir at 55 °C for 15 min, adjust the pH to 7.5 with 1 mol / L NaOH aqueous solution, add 5 mg of papain according to the enzyme addition amount of 10000 μ / g, with an enzyme activity unit of 800 KU / g. At 55 °C, the enzymatic hydrolysis time is 2.5 h. After the enzymatic hydrolysis, raise the temperature to 90 °C to inactivate the enzyme for 20 min. After cooling to room temperature, centrifuge at 9000 rpm for 12 min, take the supernatant, filter the supernatant with an ultrafiltration centrifugal tube, collect the supernatant below 3 KDa, and vacuum freeze-dry to obtain oligopeptides of gastrodia elata protein hydrolyzed by papain.

[0065] Example 3

[0066] A preparation method of gastrodia elata short peptide protein, the preparation steps are as follows:

[0067] The preparation of gastrodia elata crude protein was the same as in Example 1. 400 mg of gastrodia elata crude protein was taken, added with 50 mL of water, stirred at 45 °C for 15 min, adjusted to pH = 7.0 with 1 mol / L NaOH aqueous solution, added 20 mg of neutral protease according to the enzyme addition amount of 10000 μ / g, the enzyme activity unit was 200 KU / g, at 45 °C, the enzymatic hydrolysis time was 2.5 h. After the enzymatic hydrolysis was completed, the temperature was raised to 90 °C to inactivate the enzyme for 20 min. After cooling to room temperature, it was centrifuged at 9000 rpm for 12 min, and the supernatant was taken. The supernatant was filtered with an ultrafiltration centrifuge tube, and the supernatant below 3 KDa was collected and freeze-dried under vacuum to obtain gastrodia elata protein oligopeptides hydrolyzed by neutral protease.

[0068] Example 4

[0069] A preparation method of gastrodia elata short peptide protein is as follows:

[0070] The preparation of gastrodia elata crude protein was the same as in Example 1. 400 mg of gastrodia elata crude protein was taken, added with 50 mL of water, stirred at 50 °C for 15 min, adjusted to pH = 10.0 with 1 mol / L NaOH aqueous solution, added 20 mg of alkaline protease according to the enzyme addition amount of 10000 μ / g, the enzyme activity unit was 200 KU / g, at 50 °C, the enzymatic hydrolysis time was 2.5 h. After the enzymatic hydrolysis was completed, the temperature was raised to 90 °C to inactivate the enzyme for 20 min. After cooling to room temperature, it was centrifuged at 9000 rpm for 12 min, and the supernatant was taken. The supernatant was filtered with an ultrafiltration centrifuge tube, and the supernatant below 3 KDa was collected and freeze-dried under vacuum to obtain gastrodia elata protein oligopeptides hydrolyzed by alkaline protease.

[0071] Example 5

[0072] A preparation method of gastrodia elata short peptide protein is as follows:

[0073] The preparation of gastrodia elata crude protein was the same as in Example 1. 400 mg of gastrodia elata crude protein was taken, added with 50 mL of water, stirred at 50 °C for 15 min, adjusted to pH = 3.5 with 1 mol / L HCl aqueous solution, added 17 mg of acidic protease according to the enzyme addition amount of 10000 μ / g, the enzyme activity unit was 240 KU / g, at 50 °C, the enzymatic hydrolysis time was 2.5 h. After the enzymatic hydrolysis was completed, the temperature was raised to 90 °C to inactivate the enzyme for 20 min. After cooling to room temperature, it was centrifuged at 9000 rpm for 12 min, and the supernatant was taken. The supernatant was filtered with an ultrafiltration centrifuge tube, and the supernatant below 3 KDa was collected and freeze-dried under vacuum to obtain gastrodia elata protein oligopeptides hydrolyzed by acidic protease.

[0074] α-glucosidase inhibition experiment of gastrodia elata short peptide protein in Example 6

[0075] Weigh potassium dihydrogen phosphate and dipotassium hydrogen phosphate and prepare a buffer solution with a concentration of 50 mM and a pH of 6.8. Dissolve α-glucosidase in the above buffer solution to prepare an α-glucosidase solution with a concentration of 0.04 U / mL. Then add the same buffer solution to the substrate 4-nitrophenyl-α-D-glucopyranoside (p-NPG) to prepare a 0.5 mM p-NPG substrate solution. The test protein peptide and the positive control are both prepared with the buffer solution to the concentrations required for the experiment.

[0076] This test system is divided into a blank group, a control group, a solvent blank group, and a compound test group. The specific grouping is as follows:

[0077] Table 1 α-Glucosidase reaction system

[0078] Reagent Blank group (μL) Control group (μL) Solvent blank group (μL) Compound test group (μL) Buffer solution 170 150 160 140 α-Glucosidase - 20 - 20 Protein polypeptide - - 10 10 Substrate 30 30 30 30

[0079] In the above experimental steps, add the above reagents to a 96-well microplate in sequence, incubate at 37 °C for 5 min, add the substrate after the incubation ends, and continue to incubate for 30 min. Measure the absorbance value (OD) with a microplate reader at 405 nm. The calculation method of the inhibition rate is as follows in formula (1):

[0080]

[0081] Among them, OD 1 is the absorbance value of the blank group which is the buffer solution, OD 2 is the absorbance value of the control group which is only the buffer solution added, OD 3 is the absorbance value of the test compound, OD 4 is the absorbance value after the reaction with the compound added. Table 2 shows the results of the inhibition of α-glucosidase activity by gastrodia protein polypeptide.

[0082] Table 2 Inhibitory activity of gastrodia protein polypeptide on α-glucosidase

[0083]

[0084] Note: a All results of α-glucosidase are the average value ± SD of three experiments; b Acarbose is used as the positive control.

[0085] The results in Table 2 show that at a test concentration of 2 mg / mL, the gastrodia protein polypeptides obtained by hydrolysis with papain, neutral protease, and acidic protease all have good inhibitory activity on α-glucosidase. Among them, the gastrodia protein polypeptide obtained by hydrolysis with acidic protease has the strongest inhibitory activity, with an inhibitory activity close to 100%. After dilution by 10 times, it still has certain inhibitory activity. For the polypeptide obtained by hydrolysis with acidic protease, the half-maximal inhibitory concentration (IC 50 ) was further tested, and its IC 50= 0.56 mg / mL, and its curve is shown in Figure 1 . Therefore, gastrodia protein peptide has good α-glucosidase inhibitory activity and has the potential to be developed into an α-glucosidase inhibitor.

[0086] Example 7 Preparation of Gastrodia Short Peptide Proteins by Complex Enzymatic Hydrolysis

[0087] Select the neutral protease and acidic protease with the best activity as the complex enzyme to enzymatically hydrolyze gastrodia. The preparation of gastrodia crude protein is the same as in Example 1, and the specific different operations are as follows:

[0088] 1. Take 400 mg of gastrodia crude protein, add 50 mL of water, stir at 50 °C for 15 min, adjust the pH to 5 with 1 mol / L HCl aqueous solution. At 50 °C, add 9 mg of acidic protease with an enzyme activity unit of 240 KU / g and 10 mg of neutral protease with an enzyme activity unit of 200 KU / g at the same time. The enzymatic hydrolysis time is 2.5 h. After the enzymatic hydrolysis is completed, raise the temperature to 90 °C to inactivate the enzyme for 20 min. After cooling to room temperature, centrifuge at 9000 rpm for 12 min, take the supernatant, filter the supernatant with an ultrafiltration centrifugal tube, collect the supernatant below 3 KDa, and vacuum freeze-dry to obtain the gastrodia protein oligopeptide hydrolyzed by acidic protease and neutral protease simultaneously.

[0089] 2. Take 400 mg of gastrodia crude protein, add 50 mL of water, stir at 50 °C for 15 min, adjust the pH to 5 with 1 mol / L HCl aqueous solution. At 50 °C, first add 9 mg of acidic protease with an enzyme activity unit of 240 KU / g and enzymatically hydrolyze for 1.5 h, then add 10 mg of neutral protease with an enzyme activity unit of 200 KU / g, and the enzymatic hydrolysis time is 1.5 h. After the enzymatic hydrolysis is completed, raise the temperature to 90 °C to inactivate the enzyme for 20 min. After cooling to room temperature, centrifuge at 9000 rpm for 12 min, take the supernatant, filter the supernatant with an ultrafiltration centrifugal tube, collect the supernatant below 3 KDa, and vacuum freeze-dry to obtain the gastrodia protein oligopeptide hydrolyzed by acidic protease first and then neutral protease.

[0090] 3. Take 400 mg of gastrodia crude protein, add 50 mL of water, stir at 50 °C for 15 min, adjust the pH to 5 with 1 mol / L HCl aqueous solution. At 50 °C, first add 10 mg of neutral protease with an enzyme activity unit of 200 KU / g and enzymatically hydrolyze for 1.5 h, then add 9 mg of acidic protease with an enzyme activity unit of 240 KU / g, and the enzymatic hydrolysis time is 1.5 h. After the enzymatic hydrolysis is completed, raise the temperature to 90 °C to inactivate the enzyme for 20 min. After cooling to room temperature, centrifuge at 9000 rpm for 12 min, take the supernatant, filter the supernatant with an ultrafiltration centrifugal tube, collect the supernatant below 3 KDa, and vacuum freeze-dry to obtain the gastrodia protein oligopeptide hydrolyzed by neutral protease first and then acidic protease.

[0091] The oligopeptides of Gastrodia elata protein obtained by three compound enzymatic hydrolysis methods were further tested for α-glucosidase inhibitory activity, and the results are shown in Table 3.

[0092] Table 3 Inhibitory activity of Gastrodia elata protein polypeptides against α-glucosidase

[0093]

[0094]

[0095] Note: a All results of α-glucosidase were the average value of three experiments ± SD; b Acarbose was used as the positive control.

[0096] As can be seen from Table 3, at a concentration of 0.2 mg / mL, the combination of neutral protease followed by acidic protease increased the activity by more than 2-fold compared with a single enzyme, showing significance.

[0097] As shown in Table 3, the α-glucosidase inhibitory activities of the three Gastrodia elata protein polypeptides obtained by compound enzymatic hydrolysis were improved. At a test concentration of 0.2 mg / mL, the sequential cascade enzymatic hydrolysis of acidic protease and neutral protease, that is, adding one enzyme for hydrolysis first and then adding another enzyme for continuous hydrolysis, resulted in better activity of the obtained protein peptides. The activity was comparable to that of the positive control acarbose, reaching an inhibition rate of about 50%. Therefore, the Gastrodia elata protein polypeptides obtained by single enzyme (acidic protease) and the sequential enzymatic hydrolysis of acidic protease and neutral protease have good α-glucosidase inhibitory activity.

[0098] Example 8 Pancreatic lipase inhibitory experiment of Gastrodia elata protein polypeptides

[0099] Preparation of Tris-HCl buffer: Weigh the powder of tris(hydroxymethyl)aminomethane, add distilled water to prepare a 13 mM buffer, and adjust the pH to 8.0.

[0100] Preparation of sodium citrate reaction termination solution: Weigh the powders of citric acid and sodium citrate, and prepare 0.1 mol / L solutions with distilled water respectively. Slowly add the citric acid solution to the sodium citrate solution and adjust the pH to 4.2 to obtain the reaction termination solution.

[0101] Weigh the pancreatic lipase powder, add Tris-HCl buffer to prepare an enzyme reaction solution of 1 mg / mL; dissolve the substrate 4-methylumbelliferyl oleate with dimethyl sulfoxide (DMSO) to prepare a 5 mM stock solution, and then dilute it with Tris-HCl buffer to a concentration of 0.1 mM for use to obtain the lipase reaction solution; dissolve the Gastrodia elata protein peptide and the positive control orlistat with DMSO to the test concentration.

[0102] The test system is as follows: Blank background group (A1): Tris-HCl buffer (50 μL) + substrate (50 μL) + sodium citrate termination solution (100 μL); Blank group (A2): Tris-HCl buffer (25 μL) + substrate (50 μL) + pancreatic lipase reaction solution (25 μL) + sodium citrate termination solution (100 μL); Compound background group (A3): Tris-HCl buffer (40 μL) + substrate (50 μL) + compound (10 μL) + sodium citrate termination solution (100 μL); Compound test group (A4): Tris-HCl buffer (15 μL) + substrate (50 μL) + compound (10 μL) + pancreatic lipase reaction solution (25 μL) + sodium citrate termination solution (100 μL).

[0103] Add the above various solutions into a 96-well plate in sequence and mix the liquids evenly. After adding the enzyme solution and reacting at 25 °C for another 30 min, add 100 μL of sodium citrate reaction termination solution to each well. Measure the OD value at an excitation wavelength of 360 ± 20 nm and an emission wavelength of 460 ± 20 nm. The calculation formula for the inhibition rate of the compound on pancreatic lipase is as follows: Pancreatic lipase inhibition rate % = ((A2 - A1) - (A4 - A3)) / (A2 - A1) * 100

[0104] The results of the pancreatic lipase inhibitory activity of gastrodin protein peptide are shown in Table 4.

[0105] Table 4 Test results of the pancreatic lipase activity of gastrodin protein peptide

[0106]

[0107] Note: a The results of pancreatic lipase inhibitory activity are the average value ± SD of three experiments; b Orlistat is used as a positive control, and the inhibitory activity is at a concentration of 80 nM.

[0108] As can be seen from the results in Table 4, at a test concentration of 2 mg / mL, flavor protease, papain, alkaline protease, acid protease, and three gastrodin protein peptides obtained by hydrolysis with complex enzymes have good pancreatic lipase inhibitory activity. Among them, acid protease and three gastrodin protein polypeptides obtained by hydrolysis with complex enzymes have the strongest inhibitory activity, and the inhibitory activity reaches more than 80%. Among them, the three gastrodin protein polypeptides obtained by hydrolysis with complex enzymes also have certain inhibitory activity at a test concentration of 0.2 mg / mL. Therefore, gastrodin protein peptide also has the potential to be developed into a pancreatic lipase inhibitor.

[0109] Example 8 Determination of protein content in Example 5

[0110] The Kjeldahl method was adopted to determine the protein content with reference to "GB 5009.5—2016". Three protein polypeptides obtained by compound enzymes from Example 5 with the best single enzymatic hydrolysis activity and Example 7 were selected, and the protein content of the protein polypeptides obtained by enzymatic hydrolysis was measured as shown in Table 5.

[0111] Table 5 Results of protein content determination

[0112] Number Enzymolysis method Protein content (%) Example 5 Acid protease 71.5 Example 7 Acid protease and neutral protease simultaneously 82.1 Example 7 Acid protease first and then neutral protease 84.5 Example 7 Neutral protease first and then acid protease 85.1

[0113] The enzyme amount selected for the compound enzyme was 50% of that for single enzymatic hydrolysis, excluding the influence of exogenous added enzymes on the protein content. As shown in Table 5, after dialysis, the obtained Gastrodia elata protein polypeptides had a relatively high protein content. The protein content of the Gastrodia elata protein peptide obtained by single enzymatic hydrolysis with acid protease was 71.5%, and the protein content of the three Gastrodia elata protein polypeptides obtained by compound enzyme hydrolysis reached over 80%.

[0114] In the prior art, there are marketed products containing Gastrodia elata. For example, the Tuokang Gastrodia elata original pulp drink produced by Tianda Pharmaceutical Co., Ltd. selects Gastrodia elata from Xiaocaoba in Yunnan. Its main ingredient is Gastrodia elata extract, which has the effects of calming endogenous wind, expelling wind and dredging collaterals, improving sleep, relieving headache, enhancing immunity, etc., and has a certain improvement effect on dizziness, limb numbness, insomnia and dreaminess, etc. However, due to factors such as raw material quality and extraction process, the content of effective components of Gastrodia elata in the drink may be unstable, and its ingredients are different from those of the present invention. The storage conditions of this product are strict, usually requiring low temperature, light avoidance, etc. Improper storage is likely to cause deterioration and affect the effect. In addition, there is also Compound Gastrodia elata Armillariella mellea and Glycyrrhiza glabra and Astragalus membranaceus and Angelica sinensis tablets, which is a compound preparation composed of Armillariella mellea extract of Gastrodia elata, Astragalus membranaceus and Angelica sinensis extracts. It can stop dizziness, tonify qi and blood, dredge blood vessels, and relax muscles and activate blood circulation. It can be used for symptoms such as dizziness, head distension, headache, dizziness, limb numbness caused by various diseases such as hypertension, cerebral thrombosis, and cerebral arteriosclerosis, and has a comprehensive effect on improving cardiovascular and cerebrovascular circulation and nervous system function. Through clinical verification, it has a good curative effect on diseases such as vertebrobasilar artery insufficiency and cerebral infarction, can improve the hemorheological indexes of patients, increase cerebral blood flow, reduce vascular resistance, and promote nerve function recovery. It is also essentially different from the present invention.

[0115] The present invention adopts vacuum freeze-drying technology, and the obtained polypeptide is solid, so it has many advantages, such as: (1) Stable nutritional components: Drying is carried out in a low-temperature and vacuum environment, which can maximize the retention of nutrients such as proteins, vitamins, and minerals in the product and reduce nutrient loss caused by factors such as high temperature; (2) Convenient for transportation and storage: Solid raw materials are convenient for transportation and storage, reducing logistics costs and storage space requirements; no refrigeration or special storage conditions are required, and the shelf life is long. (3) Multiple application forms: The polypeptide of the present invention can be added as a raw material to various other solid or liquid products, or can be directly used as a product to prepare solid or liquid products.

[0116] In summary, there is no report in the prior art that gastrodia protein peptide has both hypoglycemic and hypolipidemic activities. The present invention provides a gastrodia protein peptide, a preparation method thereof and an application. The present invention discovers for the first time that gastrodia protein peptide has good in vitro α-glucosidase inhibitory activity and hypolipidemic activity, and has a unique effect on the treatment of comprehensive disorders of glucose and lipid metabolism. In addition, as a raw material for both medicine and food, the hydrolytic enzymes of gastrodia are all food-derived enzymes, the solvent used is water, and the obtained protein peptide has high safety. Therefore, gastrodia protein peptide has good practical application value and market value as a safe and reliable food-derived hypoglycemic and hypolipidemic substance.

[0117] Although embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art can understand that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the content disclosed in the embodiments.

Claims

1. A Gastrodia protein polypeptide having the effects of lowering blood sugar, lowering blood lipids and managing weight, characterized in that: The protein polypeptide is obtained by extracting crude protein from Gastrodia elata, and then hydrolyzing it with food-derived enzymes, inactivating the enzymes, centrifuging it, and ultrafiltrating it; The protein polypeptide is obtained by concentrating a stock solution with a molecular weight cutoff of 500 to 5000Da; The food-derived enzyme is a single enzyme of food-derived protease or a composite enzyme used in combination.

2. The Gastrodia elata protein polypeptide according to claim 1, characterized in that: The food-derived protease is one of flavor protease, acid protease, neutral protease, alkaline protease or papain, or a composite enzyme of several of them.

3. The method for preparing the Gastrodia elata protein polypeptide according to claim 1 or 2, characterized in that: The steps include: (1) Extraction of crude protein from Gastrodia elata: Mix dried tubers of Gastrodia elata with water at a ratio of 1:2 to 30 in terms of g:mL, add 0.1 to 10 mol / L NaOH aqueous solution to adjust the pH to 8.0 to 11.0, stir at 0 to 80°C, and keep stirring at the temperature for 1 to 10 hours; cool to room temperature, centrifuge at 3500 to 9000 rpm for 10 to 20 minutes, take the supernatant, adjust the pH to 2 to 5 with 0.1 to 1 mol / L HCl, centrifuge the liquid at 3500 to 9000 rpm for 10 to 20 minutes, take the precipitate, and freeze-dry to obtain crude protein from Gastrodia elata with a yield of 10 to 20%; (2) Preparation of Gastrodia elata protein polypeptide: Take the Gastrodia elata crude protein prepared in step (1), mix it with water at a feed ratio of g:mL of 1:50-500, add food-derived enzymes for enzymatic hydrolysis, the enzyme activity is 10-1000 KU / g, the enzymatic hydrolysis reaction pH is 3-9, the reaction time is 2-4 h, the reaction temperature is 30-50°C, after the enzymatic hydrolysis is completed, inactivate the enzyme at high temperature for 10-30 min, cool to room temperature, centrifuge at 7000-9000 r / min for 10-30 min, take the supernatant, collect the supernatant below 5 KDa by ultrafiltration, and dry to obtain Gastrodia elata protein polypeptide.

4. The preparation method according to claim 3, characterized in that: The drying method is vacuum drying, spray drying, freeze drying or normal pressure heating drying.

5. The preparation method according to claim 3 or 4, characterized in that: For every 1 weight part of crude Gastrodia elata protein, 0.01-10 weight parts of acidic protease, or 0.01-10 weight parts of neutral protease, or 0.01-10 weight parts of alkaline protease, or 0.01-10 weight parts of flavor protease, or 0.01-10 weight parts of papain are added, or a combination of the above weight parts is used.

6. A preparation prepared using the Gastrodia elata protein polypeptide as claimed in claim 1 or 2.

7. The preparation according to claim 6, characterized in that: The preparations include powders, granules, powder injections, tablets, capsules or liquid preparations.

8. Use of the Gastrodia elata protein polypeptide as claimed in claim 1 or 2 in the preparation of medicines for lowering blood sugar, lowering blood lipids and managing body weight.

9. Use of the Gastrodia elata protein polypeptide as claimed in claim 1 or 2 in the preparation of food having the functions of lowering blood sugar, lowering blood lipids, managing body weight and providing nutrition.

10. Use of the Gastrodia elata protein polypeptide as claimed in claim 1 or 2 in special medical foods and / or health foods and / or functional foods and / or medicines.

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