A hemp seed enzymatic hydrolysis extract and its preparation method

Abstract

This invention relates to an enzymatic extract obtained from hemp seeds, and its uses in medical fields such as lowering blood sugar, lowering blood lipids, protecting the liver, protecting the kidneys, and regulating intestinal flora.

Description

[0001] This application is a divisional application of the invention patent application with the application date of October 17, 2022, the application number of 202211267911.1, and the invention name of "Hemp seed enzymatic extract and preparation method thereof". TECHNICAL FIELD

[0002] The present application relates to the field of biology, and particularly relates to a hemp seed enzymatic extract and the use thereof in reducing blood sugar, reducing blood lipids, protecting liver, protecting kidney and the like. BACKGROUND

[0003] Hemp seed is the dried mature seed of Cannabis sativa L. in the family Moraceae, has the property of being both medicine and food, is cultivated in various places in China, and also has semi-wild ones, and is widely distributed in Northeast China, North China, East China, Central South China and the like. Hemp seed is neutral in nature and sweet in taste, has the effect of moistening the intestines and promoting defecation, and contains rich proteins, vitamins, lecithin, volatile oil, and trace elements such as calcium and magnesium. As a traditional Chinese medicinal material and food material, hemp seed has multiple physiological effects, but its research is not in-depth, and its potential application in the fields of medicine, health products and food has not been fully developed.

[0004] Polypeptide substances are intermediate products and functionally active fragments of protein hydrolysis, have a molecular weight of less than 10,000 daltons, and can pass through a semi-permeable membrane. Research has found that after protein is enzymatically hydrolyzed in the digestive tract, it is mainly absorbed in the form of peptides, and polypeptides from protein hydrolysis have better solubility, complete absorbability and biological activity than original proteins, can not only meet the needs of the body for essential amino acid nutrition supplementation, but also promote the metabolism, growth and development of the body and regulate the physiology and immunity of the body.

[0005] In recent years, bioactive peptides of natural origin have become a research hotspot in the development of drugs or functional foods due to their mild action, clear function, high safety and small side effects. Preparing hemp active peptides from hemp seed as raw material and developing active peptide products with medical and health care and nutritional uses have broad application prospects. SUMMARY

[0006] In view of this, the purpose of the present application is to provide a proteinase hydrolysis extract derived from hemp seed and the use thereof.

[0007] Specifically, the present application provides a hemp seed enzymatic extract, which comprises 25-90% of a polypeptide component with a molecular weight of less than or equal to 1000 Da, 15-50% of a polypeptide component with a molecular weight of 1000-3000 Da, and 1-50% of a component with a molecular weight of greater than 3000 Da, in terms of the mass percentage in the hemp seed enzymatic extract.

[0008] Further, the protein is 70-100%, preferably 75-85% by mass percentage in the enzymatic extract of Cannabis sativa L. seeds.

[0009] Further, the enzymatic extract of Cannabis sativa L. seeds is prepared by a method comprising the following steps:

[0010] defatting the Cannabis sativa L. seed raw material powder with an organic solvent to obtain defatted Cannabis sativa L. seed powder,

[0011] extracting Cannabis sativa L. seed protein from the defatted Cannabis sativa L. seed powder,

[0012] adding a protease to the Cannabis sativa L. seed protein to perform enzymatic hydrolysis,

[0013] filtering and centrifuging the enzymatic hydrolysate to obtain the enzymatic extract of Cannabis sativa L. seeds.

[0014] Further, the organic solvent is selected from pure petroleum ether, n-hexane, n-butanol, anhydrous ethanol, 5:2 petroleum ether / n-butanol, 5:2 n-hexane / n-butanol, 5:2 petroleum ether / ethanol, or 5:2 n-hexane / ethanol, preferably anhydrous ethanol.

[0015] Further, the method for extracting Cannabis sativa L. seed protein is selected from ultrasonic-assisted organic extraction, ultrasonic-assisted alkali extraction and acid precipitation, alkali extraction and acid precipitation, or salt extraction, preferably salt extraction.

[0016] Further, the protease is selected from pepsin, trypsin, a-chymotrypsin, papain, flavourzyme, proteinase K, neutral protease, alkaline protease, thermolysin, pepsin / trypsin complex, or chymotrypsin / trypsin complex, preferably thermolysin, flavourzyme, or proteinase K, further preferably thermolysin.

[0017] Further, the filtration has a molecular weight cut-off of ≤3000 Da.

[0018] The present application also provides a method for preparing an enzymatic extract of Cannabis sativa L. seeds, comprising:

[0019] defatting the Cannabis sativa L. seed raw material powder with an organic solvent to obtain defatted Cannabis sativa L. seed powder,

[0020] extracting Cannabis sativa L. seed protein from the defatted Cannabis sativa L. seed powder,

[0021] adding a protease to the Cannabis sativa L. seed protein to perform enzymatic hydrolysis,

[0022] filtering and centrifuging the enzymatic hydrolysate to obtain the enzymatic extract of Cannabis sativa L. seeds.

[0023] The organic solvent is selected from pure petroleum ether, n-hexane, n-butanol, anhydrous ethanol, 5:2 petroleum ether / n-butanol, 5:2 n-hexane / n-butanol, 5:2 petroleum ether / ethanol or 5:2 n-hexane / ethanol;

[0024] The method for extracting protein is selected from ultrasonic-assisted organic extraction, ultrasonic-assisted alkali extraction and acid precipitation, alkali extraction and acid precipitation or salt extraction.

[0025] The protease is selected from pepsin, trypsin, α-chymotrypsin, papain, flavourzyme, proteinase K, neutral protease, alkaline protease, thermolysin, pepsin / pancreatin complex or chymotrypsin / pancreatin complex.

[0026] Further, the organic solvent is anhydrous ethanol, and the hemp seed raw material powder is mixed with anhydrous ethanol at a ratio of 1:10-1:20, followed by stirring, centrifugation, and discarding the supernatant to obtain the defatted hemp seed powder.

[0027] Further, the method for extracting protein is salt extraction, which comprises dissolving the defatted hemp seed powder in a 0.8M NaCl solution at a ratio of 1:10-1:20, followed by stirring, centrifugation, taking the supernatant, adjusting the pH value to ≤5 to obtain hemp seed protein.

[0028] Further, the protease is thermolysin, flavourzyme or proteinase K, preferably thermolysin.

[0029] Further, thermolysin is added to the hemp seed protein at a concentration of 8000-10000U / g, and enzymolysis is carried out at a pH value of 6.0-8.5 to obtain the enzymolysis product.

[0030] Further, the filtration has a molecular weight cut-off of ≤3000Da.

[0031] The application also provides the use of the hemp seed enzymolysis extract or the hemp seed enzymolysis extract prepared by the method provided in the application in the preparation of a medicine, special medical purpose food and health food with the functions of reducing fat and regulating metabolism.

[0032] The application also provides the use of the hemp seed enzymolysis extract or the hemp seed enzymolysis extract prepared by the method provided in the application in the preparation of a medicine, special medical purpose food and health food for preventing, treating or improving hyperglycemia and human diseases caused by hyperglycemia.

[0033] The application also provides the use of the hemp seed enzymatic extract or the hemp seed enzymatic extract prepared by the method provided in the application in the preparation of a medicine, a special medical purpose food and a health food with the functions of protecting the liver, reducing glutamic-pyruvic transaminase or improving liver injury function.

[0034] The application also provides the use of the hemp seed enzymatic extract or the hemp seed enzymatic extract prepared by the method provided in the application in the preparation of a medicine, a special medical purpose food and a health food with the functions of protecting the kidney, improving kidney injury function.

[0035] The application also provides the use of the hemp seed enzymatic extract or the hemp seed enzymatic extract prepared by the method provided in the application in the preparation of a medicine, a special medical purpose food and a health food with the functions of improving intestinal flora disorder.

[0036] Inventive Effects

[0037] 1. The hemp seed protein enzymatic extract provided in the application can reduce the concentration of TC, TG and FFA in blood plasma, increase the concentration of PYY, and has a certain fat-reducing effect.

[0038] 2. The hemp seed enzymatic extract can increase the concentration of GLP-1 in blood plasma, reduce fasting blood glucose, increase oral glucose tolerance, increase the concentration of ADPN in blood plasma and insulin sensitivity, and promote blood glucose metabolism balance.

[0039] 3. The hemp seed enzymatic extract can reduce the concentration of ALT, AST, ALP and TP II in blood plasma, and has a certain liver protection function.

[0040] 4. The hemp seed enzymatic extract can reduce the concentration of creatinine (Crea) and urea (Urea) in blood plasma, and has a certain kidney protection effect.

[0041] 5. The hemp seed enzymatic extract can effectively increase the abundance of beneficial bacteria including Lactobacillus, Akkermansia, Bacteroides, etc., reduce the abundance of harmful bacteria Prevotella, increase the ratio of Bacteroidetes / Firmicutes, and increase the alpha diversity of intestinal flora, and has a certain function of improving intestinal flora disorder.

[0042] 6. The hemp seed enzymatic extract provided in the application is derived from the seed kernel (hemp seed) of the seed of the homoeopathic crop hemp after shelling, has almost no side effects, is safer for the human body, and can achieve the effect of reducing blood sugar by oral administration, and is safer and more convenient to take. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 Figure 3 shows the DPP IV inhibitory activity in the cell experiment of the synthetic polypeptide of Example 6.

[0044] Figure 2 Figure 4 shows the GLP-1 concentration in the cell experiment of the synthetic polypeptide of Example 6.

[0045] Figure 3 Figure 5 shows the insulin concentration in the cell experiment of the synthetic polypeptide of Example 6.

[0046] Figure 4 Figure 6 shows the water intake of mice in the animal experiment of Example 7.

[0047] Figure 5 Figure 7 shows the food intake of mice in the animal experiment of Example 7.

[0048] Figure 6 Figure 8 shows the energy intake of mice in the animal experiment of Example 7.

[0049] Figure 7 Figure 9 shows the body weight change of mice in the animal experiment of Example 7.

[0050] Figure 8 Figure 10 shows the plasma PYY concentration of mice in the animal experiment of Example 7.

[0051] Figure 9 Figure 11 shows the plasma TG content of mice in the animal experiment of Example 7.

[0052] Figure 10 Figure 12 shows the plasma TC content of mice in the animal experiment of Example 7.

[0053] Figure 11 Figure 13 shows the plasma FFA content of mice in the animal experiment of Example 7.

[0054] Figure 12 Figure 14 shows the blood glucose change of mice during gavage in the animal experiment of Example 7.

[0055] Figure 13 Figure 15 shows the glucose concentration change (left) and the area under the curve (right) during the oral glucose tolerance test in the animal experiment of Example 7.

[0056] Figure 14 Figure 16 shows the plasma GLP-1 concentration in the animal experiment of Example 7.

[0057] Figure 15 Figure 17 shows the plasma insulin concentration and HOMA-IR in the animal experiment of Example 7.

[0058] Figure 16 Figure 7 shows the plasma GHb concentration profile in the animal experiment of Example 7.

[0059] Figure 17 Figure 8 shows the plasma ADPN concentration profile in the animal experiment of Example 7.

[0060] Figure 18 Figure 9 shows the intestinal flora alpha diversity profile in the animal experiment of Example 7.

[0061] Figure 19 Figure 10 shows the Lactobacillus, Akkermansia, Bacteroides, Prevotella abundance and Bacteroidetes / Firmicutes ratio profile in the animal experiment of Example 7.

[0062] Figure 20 Figure 11 shows the DPP IV inhibitory activity profile in the cell experiment of enzymatic extract of Example 8.

[0063] Figure 21 Figure 12 shows the GLP-1 concentration profile in the cell experiment of enzymatic extract of Example 8.

[0064] Figure 22 Figure 13 shows the insulin concentration profile in the cell experiment of enzymatic extract of Example 8.

[0065] Figure 23 Figure 14 shows the water intake profile of mice in the animal experiment of Example 9

[0066] Figure 24 Figure 15 shows the diet profile of mice in the animal experiment of Example 9.

[0067] Figure 25 Figure 16 shows the energy intake profile of mice in the animal experiment of Example 9.

[0068] Figure 26 Figure 17 shows the body weight change profile of mice in the animal experiment of Example 9.

[0069] Figure 27 Figure 18 shows the plasma PYY concentration profile of mice in the animal experiment of Example 9.

[0070] Figure 28 Figure 19 shows the plasma TG content profile of mice in the animal experiment of Example 9.

[0071] Figure 29 Figure 20 shows the plasma TC content profile of mice in the animal experiment of Example 9.

[0072] Figure 30 Figure 21 shows the plasma FFA content profile of mice in the animal experiment of Example 9.

[0073] Figure 31 Fig. 9 shows the blood glucose level of mice during gavage in the animal experiment of Example 9.

[0074] Figure 32 Fig. 10 shows the glucose concentration (left) and the area under the curve (right) during oral glucose tolerance test in the animal experiment of Example 9.

[0075] Figure 33 Fig. 11 shows the plasma GLP-1 concentration of mice in the animal experiment of Example 9.

[0076] Figure 34 Fig. 12 shows the plasma insulin concentration and HOMA-IR of mice in the animal experiment of Example 9.

[0077] Figure 35 Fig. 13 shows the plasma GHb concentration of mice in the animal experiment of Example 9.

[0078] Figure 36 Fig. 14 shows the plasma ADPN concentration of mice in the animal experiment of Example 9.

[0079] Figure 37 Fig. 15 shows the intestinal flora alpha diversity of mice in the animal experiment of Example 9.

[0080] Figure 38 Fig. 16 shows the abundance of Lactobacillus, Akkermansia, Bacteroides, Prevotella and the ratio of Bacteroidetes / Firmicutes in the animal experiment of Example 9. DETAILED DESCRIPTION

[0081] The application will be further described below in conjunction with specific embodiments, which are presented to enable a full and complete understanding of the application. The embodiments are presented to enable a full and complete understanding of the application, and to enable the full scope of the application to be communicated to those skilled in the art.

[0082] It should be noted that certain terms are used throughout the specification and claims which have particular meanings as set forth below. Those skilled in the art will appreciate that the same terms can be used to describe different components, methods, or operations having similar or identical functions. The specification and claims are not limited to any particular terminology. Rather, the specification and claims are to be interpreted to cover all components, methods, or operations which carry out the same functions as those described in the specification and claims. The terms “comprise” or “include” are used in the specification and claims as open-ended terms, and thus should be interpreted as “comprising but not limited to.” The description that follows is intended to be a best mode of carrying out the application, and thus should be interpreted as such. The scope of the application is to be determined by the appended claims.

[0083] The present application provides a hemp seed enzymatic extract extracted from hemp seed protein, wherein the hemp seed enzymatic extract comprises 25-90% of polypeptide components with a molecular weight less than or equal to 1000 Da, 15-50% of polypeptide components with a molecular weight of 1000-3000 Da, and 1-50% of components with a molecular weight greater than 3000 Da.

[0084] For example, the hemp seed enzymatic extract comprises 77% of polypeptide components with a molecular weight less than or equal to 1000 Da, 14% of polypeptide components with a molecular weight of 1000-3000 Da, and 9% of polypeptide components with a molecular weight greater than 3000 Da, in terms of the mass percentage in the hemp seed enzymatic extract.

[0085] In the present application, for the polypeptide with a molecular weight of 1000-3000 Da, it refers to the polypeptide with a molecular weight greater than 1000 Da and a molecular weight less than or equal to 3000 Da.

[0086] In the present application, for the determination method of the different content of polypeptide with a molecular weight in the hemp seed enzymatic extract, the present application does not make any limitation, which can be determined according to the conventional method in the art, for example, the molecular weight distribution of the hemp seed enzymatic extract is analyzed by high performance liquid chromatography. In some embodiments, the content of different peptides in the hemp seed enzymatic extract is determined by using standard preparation standard curve.

[0087] In some embodiments, the protein is 70-100%, preferably 75-85%, in terms of the mass percentage in the hemp seed enzymatic extract. For example, the protein is 70%, 75%, 80%, 85%, 90%, 95%, or 100%, in terms of the mass percentage in the hemp seed enzymatic extract.

[0088] In the present application, for the determination of the content of protein in the hemp seed polypeptide mixture, the present application does not make any limitation, which can be determined according to the conventional method in the art, for example, the method for determining protein in the first method of national standard GB 5009.5-2016 is used.

[0089] In the embodiments provided in the present application, the hemp seed enzymatic extract is prepared by a method comprising the following steps:

[0090] The hemp seed raw material powder is subjected to defatting treatment with an organic solvent to obtain defatted hemp seed powder,

[0091] The hemp seed protein is extracted from the defatted hemp seed powder,

[0092] The hemp seed protein is subjected to enzymatic hydrolysis by adding protease,

[0093] The enzymatic extract of Cannabis sativa L. seeds is obtained by filtering and centrifuging the enzymatic product.

[0094] In the present application, the preparation method of the Cannabis sativa L. seed raw material powder is not limited, and can be prepared according to the conventional method in the art, including but not limited to using the method of grinding.

[0095] In the present application, the Cannabis sativa L. seed raw material powder is added to the extraction reagent, stirred and centrifuged to remove the oil in the Cannabis sativa L. seed powder. The extraction reagent is an organic solvent, and in a specific embodiment, the organic solvent is selected from one or more of pure petroleum ether, n-hexane, n-butanol, and anhydrous ethanol. In some specific embodiments, the organic solvent is petroleum ether / n-butanol, n-hexane / n-butanol, petroleum ether / ethanol, or n-hexane / ethanol mixed in a certain ratio. In some specific embodiments, the organic solvent is petroleum ether / n-butanol 5:2, n-hexane / n-butanol 5:2, petroleum ether / ethanol 5:2, or n-hexane / ethanol 5:2. In a preferred embodiment, the organic solvent is anhydrous ethanol.

[0096] In a specific embodiment of the present application, the Cannabis sativa L. seed raw material powder is mixed with the organic solvent at a solid-liquid ratio of 1:10 to 1:20 (g / mL), stirred, centrifuged, and the supernatant is discarded to obtain the defatted Cannabis sativa L. seed powder.

[0097] For example, the mixing ratio (g / mL) of the Cannabis sativa L. seed raw material powder and the organic solvent can be 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, etc.

[0098] In a preferred embodiment, the Cannabis sativa L. seed raw material powder is mixed with anhydrous ethanol at a solid-liquid ratio of 1:20 (g / mL).

[0099] In the present application, the method for extracting the oil in the Cannabis sativa L. seed powder is not limited, and can be performed according to the conventional method in the art, and in some specific embodiments, the static stirring or ultrasonic extraction method can be used.

[0100] In a specific embodiment, the static stirring method is used for oil extraction: the mixture of the Cannabis sativa L. seed raw material powder and the extraction reagent is stirred at room temperature of 20-25°C for 12h, centrifuged to remove the supernatant, the precipitated material is again added with the extraction reagent and stirred at room temperature of 20-25°C for 6h, centrifuged to remove the supernatant, and the precipitated material is first dried in a water bath at about 60°C until there is no organic solvent, and then dried and cooled to obtain the defatted Cannabis sativa L. seed powder.

[0101] In another specific embodiment, the oil extraction is carried out by ultrasonic extraction method: the mixture of the raw material powder of Cannabis sativa L. seeds and the extraction reagent is stirred at room temperature of 20-25°C for 10 min, ultrasonic extraction (100 kHz) is carried out for 10 min, the supernatant is removed by centrifugation, the precipitate is added with the extraction reagent again, and the stirring and ultrasonic extraction are repeated, the supernatant is removed by centrifugation, and the precipitate is first dried in a water bath at about 60°C until no organic substance is left, and then dried and cooled to obtain the defatted Cannabis sativa L. seed powder.

[0102] In a preferred embodiment of the present application, the oil in the Cannabis sativa L. seed powder is extracted by static stirring method.

[0103] In the present application, the extraction method of the Cannabis sativa L. seed protein is not limited, and can be carried out according to the conventional method in the art. In some specific embodiments, the method for extracting the Cannabis sativa L. seed protein is selected from ultrasonic-assisted organic extraction method, ultrasonic-assisted alkali extraction and acid precipitation extraction method, alkali extraction and acid precipitation extraction method, or salt extraction method.

[0104] In one specific embodiment, the Cannabis sativa L. seed protein is extracted by ultrasonic-assisted organic extraction method: the defatted Cannabis sativa L. seed powder is mixed with n-hexane, the supernatant is taken after centrifugation under ultrasonic, and the remaining substance can be repeated for several times, the combined supernatant is dried until no organic substance is left, and then dried to obtain the product.

[0105] Preferably, the defatted Cannabis sativa L. seed powder is mixed with n-hexane at a ratio of 1:30-1:50 (g / mL); preferably, the ultrasonic frequency is 2 kHz; preferably, the ultrasonic time is 20-40 min each time; preferably, the drying is carried out in a water bath at 55-65°C; and preferably, the drying is carried out in an oven at 55-65°C.

[0106] In another specific embodiment, the Cannabis sativa L. seed protein is extracted by ultrasonic-assisted alkali extraction and acid precipitation extraction method: the defatted Cannabis sativa L. seed powder is mixed with ultrapure water (pH 8.5), and the supernatant is taken after ultrasonic extraction (200 W) and centrifugation, and the remaining substance can be repeated for several times, the combined supernatant is adjusted to pH 4.5 to precipitate the protein, washed with water, adjusted to pH 7.0, and then dried to obtain the product.

[0107] Preferably, the defatted Cannabis sativa L. seed powder is mixed with ultrapure water at a ratio of 1:10-1:30 (g / mL); preferably, the ultrasonic frequency is 100-300 W; preferably, the ultrasonic treatment temperature is 20-25°C; preferably, the ultrasonic time is 20-40 min each time; and preferably, the drying is carried out in an oven at 55-65°C.

[0108] In another specific embodiment, the hemp seed protein is extracted by alkali extraction and acid precipitation: defatted hemp seed powder is mixed with ultrapure water (pH 10.0), stirred and centrifuged to obtain the supernatant, and the remaining material can be repeated several times. The supernatants are combined and adjusted to pH 5.0 to precipitate the protein. After washing with water, the pH is adjusted to 7.0, and drying or freeze-drying is performed to obtain the protein.

[0109] Preferably, the defatted hemp seed powder is mixed with ultrapure water at a ratio of 1:5 to 1:20 (g / mL); preferably, the stirring treatment temperature is 30-40°C; preferably, the stirring time is 1-3 h each time; and preferably, drying is performed using an oven at 55-65°C.

[0110] In another specific embodiment, the hemp seed protein is extracted by salt extraction: defatted hemp seed powder is mixed with a NaCl solution (pH 7.0), stirred and centrifuged to obtain the supernatant, and the remaining material can be repeated several times. The remaining material is then mixed with ultrapure water (pH 10.0), stirred and centrifuged to obtain the supernatant. The supernatants are combined and adjusted to pH 4.5 to precipitate the protein. After washing with water, the pH is adjusted to 7.0, and drying or freeze-drying is performed to obtain the protein.

[0111] Preferably, the NaCl solution has a concentration of 0.5-1.0 M; preferably, the defatted hemp seed powder is mixed with the NaCl solution at a ratio of 1:5 to 1:20 (g / mL); preferably, the stirring treatment temperature is 30-40°C; preferably, the stirring time is 1-3 h each time; and preferably, drying is performed using an oven at 55-65°C.

[0112] In a preferred embodiment of the present application, the hemp seed protein is extracted by salt extraction.

[0113] In the present application, the hemp seed protein is subjected to protease enzymolysis. In some embodiments, the protease is selected from the group consisting of pepsin, trypsin, α-chymotrypsin, papain, flavourzyme, proteinase K, neutral protease, alkaline protease, thermolysin, or a complex enzyme such as pepsin / trypsin complex, chymotrypsin / trypsin complex.

[0114] In some preferred embodiments, the protease is selected from the group consisting of thermolysin, flavourzyme, or proteinase K. Further preferably, the protease is thermolysin.

[0115] In some embodiments, the hemp seed protein is prepared into a protein solution with a certain mass concentration, and the protease is added at a concentration of 8000-10000 U / g for enzymolysis. Preferably, the protease enzymolysis is performed at 35-60°C; preferably, the protease enzymolysis is performed at pH 7-11; and preferably, the enzymolysis time is 3-6 h.

[0116] For example, the protease enzyme hydrolysis can be carried out at 35℃, 40℃, 42℃, 45℃, 48℃, 50℃, 52℃, 55℃, 58℃, 60℃, 65℃, 70℃, etc.

[0117] The protease enzyme hydrolysis can be carried out at pH 6, 7, 8, 9, 10, 11, etc.

[0118] The enzyme hydrolysis time can be 3h, 4h, 5h, 6h, 7h, etc.

[0119] In the present application, the product after protease enzyme hydrolysis is filtered, and further centrifuged, freeze-dried to obtain the hemp seed enzyme hydrolysate.

[0120] In a specific embodiment, the enzyme hydrolysate with a molecular weight of ≤3000Da is collected, for example, a 3kDa ultrafiltration tube is selected to fractionate the enzyme hydrolysate.

[0121] Further, the enzyme hydrolysate with a molecular weight of ≤1000Da is collected, for example, a 1kDa ultrafiltration tube is selected to fractionate the enzyme hydrolysate.

[0122] The polypeptide amino acid sequence of <3kDda is about 23, which belongs to short peptides and is more easily used by oral administration, so <3kDa short peptides are preferred in the present application, and more preferably polypeptides less than 1000Da

[0123] The present application provides the use of the above-mentioned hemp seed enzyme hydrolysate and the hemp seed enzyme hydrolysate prepared by the above-mentioned method in the preparation of drugs, special medical purpose foods and health foods with fat reduction and metabolism regulation functions.

[0124] Obesity is a chronic metabolic disease, mainly characterized by excessive accumulation of fat and overweight. Existing drugs mainly achieve fat reduction function by delaying, reducing fat absorption or promoting fat decomposition and metabolism in the body.

[0125] In some specific embodiments, the hemp seed enzyme hydrolysate provided by the present application can reduce energy intake, slow down weight gain, and reduce the concentration of TC, TG and FFA in plasma, increase the concentration of PYY, and can be applied to the development of drugs, special medical purpose foods and health foods related to fat reduction and metabolism regulation.

[0126] The present application provides the use of the above-mentioned hemp seed enzyme hydrolysate and the hemp seed enzyme hydrolysate prepared by the above-mentioned method in the preparation of drugs, special medical purpose foods and health foods for preventing, treating or improving hyperglycemia and human diseases caused by hyperglycemia.

[0127] Hyperglycemia is a metabolic disorder caused by a significant increase in blood glucose. The related diseases caused by hyperglycemia include chronic diseases caused by long-term hyperglycemia, such as diabetes, retinopathy, kidney disease, diabetic nephropathy, diabetic retinopathy, atherosclerosis, peripheral neuropathy, autonomic neuropathy, etc. The acute severe metabolic disorders caused by acute hyperglycemia include diabetic ketoacidosis, hyperosmolar hyperglycemic syndrome, etc.

[0128] In some specific embodiments, the hemp seed enzymatic extract provided by the present application can inhibit DPP-IV activity, increase GLP-1 level in vivo, produce blood glucose regulation effect, and can be applied to the development of drugs, special medical purpose foods and health foods for hyperglycemia and related diseases caused by hyperglycemia.

[0129] The present application provides the use of the hemp seed enzymatic extract and the hemp seed enzymatic extract prepared by the above-mentioned method in the preparation of drugs, special medical purpose foods and health foods with the functions of protecting liver, reducing the concentration of plasma glutathione and glutathione transaminase or improving liver damage function.

[0130] The present application provides the use of the hemp seed enzymatic extract and the hemp seed enzymatic extract prepared by the above-mentioned method in the preparation of drugs, special medical purpose foods and health foods with the functions of protecting kidney, improving kidney damage function.

[0131] The present application provides the use of the hemp seed enzymatic extract and the hemp seed enzymatic extract prepared by the above-mentioned method in the preparation of drugs, special medical purpose foods and health foods with the functions of intestinal flora regulation.

[0132] In the drugs, special medical purpose foods and health foods provided by the present application, a pharmaceutically acceptable carrier or excipient can be further included.

[0133] Specifically, examples of the above-mentioned pharmaceutically acceptable carrier include excipients, binders, buffers, antioxidants, solubilizers, thickening agents, lubricants, disintegrating agents, diluents, stabilizers, preservatives, coloring agents, flavoring agents, dissolution aids, emulsifiers, isotonic agents and the like.

[0134] The excipient can be selected from, but not limited to, starch, lactose, sucrose, calcium carbonate, calcium phosphate; the binder can be selected from, but not limited to, starch, gum arabic, carboxymethyl cellulose, hydroxypropyl cellulose, crystalline cellulose, alginic acid, gelatin, polyvinylpyrrolidone; the buffering agent can be selected from, but not limited to, citrate buffer solution, acetate buffer solution, phosphate buffer solution, borate buffer solution, tris salt buffer solution and the like; the antioxidant can be selected from, but not limited to, butylated hydroxytoluene, butylated hydroxyanisole, sodium sulfite, sodium bisulfite, sodium metabisulfite, ascorbic acid, cysteine hydrochloride, cystine, thioctic acid, thioglycerol and the like; the lubricant can be selected from, but not limited to, magnesium stearate, calcium stearate, talc; the thickening agent can be selected from, but not limited to, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, amorphous cellulose, polysaccharides (including starch derivatives), polyvinyl alcohol and polyvinylpyrrolidone or mixtures thereof; the disintegrating agent can be selected from, but not limited to, carboxymethyl cellulose calcium, talc; the diluent can be selected from, but not limited to, water for injection, saline; the preservative can be selected from, but not limited to, sodium bisulfate, sodium bisulfite, sodium trisulfate, benzalkonium chloride, chlorobutanol, thiomersal, phenylmercuric acetate, methyl paraben, propyl paraben and phenylethyl alcohol; the isotonic agent can be selected from, but not limited to, chloride and sugar.

[0135] The medicine containing the hemp seed enzymatic extract according to the present application can be prepared in the form of mixing the polypeptide and a pharmaceutically acceptable carrier, for example, to obtain oral preparations such as tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, oral disintegrating tablets), capsules (including soft capsules, microcapsules), granules, powders, lozenges, syrups, emulsions, suspensions, films (for example, oral disintegrating films) and the like, parenteral preparations such as injections (for example, subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, drip infusions), external preparations (for example, skin preparations, ointments), suppositories (for example, rectal suppositories, vaginal suppositories), pills, nasal drops, respiratory preparations (inhalants), eye drops and the like. In addition, these preparations can be controlled release preparations (for example, sustained release microcapsules), such as immediate release preparations, sustained release preparations and the like. Such preparations can be obtained by preparation methods conventionally used in the technical field.

[0136] The medicine, special medical purpose food and health food containing the hemp seed enzymatic extract according to the present application can be administered to mammals (for example, humans, mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys). The administration mode can be oral or parenteral administration (for example, intravenous, intramuscular, subcutaneous, intracerebral, intranasal, intradermal, drip, intracerebral, rectal, vaginal, intraperitoneal and the like).

[0137] The amount of the polypeptide of the present application administered to a subject varies depending on the route of administration, symptoms, patient age, and the like, and can be actually determined by a clinician.

[0138] The drugs, special medical purpose foods, and health foods involved in the present application can also be used together with other existing known drugs for treating hyperglycemia, type 2 diabetes, obesity, metabolic abnormality diseases, and drugs having the functions of protecting liver and kidney and regulating intestinal flora. When used together, there is no limitation on the administration time of the respective drugs, and two or more different drugs can be administered simultaneously or at different times. The dosage of the known drugs can be determined according to the amount of administration used clinically, and can be appropriately selected depending on the patient to be administered, the route of administration, and the like.

[0139] Examples

[0140] The hemp seeds used in the following examples of the present application are derived from Guangxi Bama and protease from thermophilic bacteria, which are purchased from Shanghai Yuanye Biotechnology Co., Ltd. Other materials, reagents, and the like, unless otherwise specified, can be obtained through commercial channels.

[0141] Example 1 Optimization of Enzymatic Extraction Process and Method of Hemp Seeds

[0142] The hemp seed enzymatic extraction method disclosed in this example includes the following specific steps: removing oil from hemp seeds using an organic solvent; extracting protein from defatted hemp seeds; adding protease to hemp seed protein for enzymatic hydrolysis; centrifuging the enzymatic hydrolysate to collect the supernatant to obtain the hemp seed enzymatic extract. The process conditions in each step of oil extraction, protein extraction, and enzymatic hydrolysis are screened through the following experiments to obtain a better preparation method of the hemp seed enzymatic extract.

[0143] (1) Screening of hemp seed oil extraction method

[0144] Pure petroleum ether, n-hexane, n-butanol, ethanol, 5:2 petroleum ether / n-butanol, 5:2 n-hexane / n-butanol, 5:2 petroleum ether / ethanol, and 5:2 n-hexane / ethanol were selected as extraction reagents to extract oil from hemp seeds at a solid-liquid ratio of 1:20. The extraction methods were static stirring and ultrasonic extraction. The detailed process parameters are as follows:

[0145] Static stirring method: weigh hemp seeds (~ 2 g) → add extraction reagent at a solid-liquid ratio of 1:20 (w / v, g / mL) → stir at room temperature (20-25°C) for 12 h → centrifuge and discard the supernatant → add extraction reagent at a solid-liquid ratio of 1:20 (w / v, g / mL) to the precipitate again → stir at room temperature (20-25°C) for 6 h → centrifuge and discard the supernatant → dry the precipitate in a water bath (60°C) until there is no organic solvent → dry in an oven (60°C) for 30 min → cool to room temperature and weigh;

[0146] Ultrasonic extraction method: take hemp seeds (~ 2g) → add extraction reagent with solid-liquid ratio of 1:20 (w / v) → stir at room temperature (20-25℃) for 10 min → ultrasonic (100kHZ) at room temperature (20-25℃) for 10 min → centrifugal to remove supernatant → add extraction reagent with solid-liquid ratio of 1:20 (w / v, g / mL) to the precipitate again → stir at room temperature (20-25℃) for 10 min → ultrasonic (100kHZ) at room temperature (20-25℃) for 10 min → centrifugal, discard supernatant → the precipitate is first dried in a water bath (60℃) until there is no organic → oven drying (60℃) for 30 min → cool to room temperature and weigh.

[0147] The yield of the final raw material using the above reagents and extraction methods is shown in Table 1. The recovery rate of oil extraction by stirring extraction with n-butanol is the lowest, indicating that it has the best oil removal effect. The second is ethanol stirring extraction and petroleum ether / n-butanol ultrasonic extraction. However, it was found that the hemp seed residue after extraction with n-butanol or petroleum ether has an unpleasant irritating taste, and it was also found that the color of the residue obtained by ethanol extraction is lighter than that of other methods. Therefore, the ethanol stirring extraction method is the best.

[0148] Table 1 Recovery rate of hemp seed oil removal by different reagents and different extraction methods (n=3)

[0149]

[0150] (2) Optimization of hemp seed protein extraction process

[0151] Ultrasonic-assisted organic extraction, ultrasonic-assisted alkali extraction and acid precipitation extraction, alkali extraction and acid precipitation extraction, and salt extraction were used to extract hemp seed protein, and the extraction process was optimized. The specific operation process of each method is as follows:

[0152] Ultrasonic-assisted organic extraction: take hemp seeds (~ 5g) → add n-hexane with solid-liquid ratio of 1:40 (w / v, g / mL) → 30℃, 2kHZ ultrasonic for 30 min → centrifugal to take supernatant → add n-hexane with solid-liquid ratio of 1:40 (w / v, g / mL) to the remaining material again → 30℃, 2kHZ ultrasonic for 30 min → centrifugal, take supernatant → combine the two supernatants and dry them in a water bath (60℃) until there is no organic → oven drying (60℃).

[0153] Ultrasonic assisted alkali extraction and acid precipitation extraction method: take hemp seed (~ 5g) → add ultrapure water (pH 8.5) with a solid-liquid ratio of 1:20 (w / v, g / mL) → ultrasonic (200W) for 30min at room temperature (20-25℃) → centrifugal take supernatant → add ultrapure water (pH 8.5) to the remaining material with a solid-liquid ratio of 1:10 (w / v, g / mL) → ultrasonic (200W) for 30min at room temperature (20-25℃) → centrifugal take supernatant → combine the supernatant of the two times and adjust the pH to 4.5 to precipitate the protein → water wash 3 times → adjust the pH to 7.0 → oven drying.

[0154] Alkali extraction and acid precipitation extraction method: take hemp seed (~ 5g) → add ultrapure water (pH 10.0) with a solid-liquid ratio of 1:10 (w / v, g / mL) → stirring extraction for 2h at 35℃ → centrifugal take supernatant → add ultrapure water (pH 10.0) to the remaining material with a solid-liquid ratio of 1:10 (w / v, g / mL) → stirring extraction for 2h at 35℃ → centrifugal take supernatant → combine the supernatant of the two times and adjust the pH to 5.0 to precipitate the protein → water wash 3 times → adjust the pH to 7.0 → oven drying / freeze drying preservation.

[0155] Salt extraction method: take hemp seed (~ 5g) → add 0.8M NaCl solution (pH 7.0) with a solid-liquid ratio of 1:10 (w / v, g / mL) → stirring extraction for 2h at 35℃ → centrifugal take supernatant → add ultrapure water (pH 10.0) to the remaining material with a solid-liquid ratio of 1:10 (w / v, g / mL) → stirring extraction for 2h at 35℃ → centrifugal take supernatant → combine the supernatant of the two times and adjust the pH to 4.5 to precipitate the protein → water wash 3 times → adjust the pH to 7.0 → oven drying / freeze drying preservation.

[0156] The final protein yield using the above four methods is shown in Table 3. As shown in Table 2, the protein yield obtained by salt extraction and freeze drying method is as high as 24.15%, and it is found by comparison that the color of the protein obtained by salt extraction method is significantly lighter than that obtained by alkali extraction method. Therefore, in terms of comprehensive evaluation, the salt extraction method is the best for protein extraction.

[0157] Table 2 Protein yield of different extraction methods (n=3)

[0158]

[0159] (3) Enzymatic hydrolysis process optimization of hemp seed protein

[0160] a Enzymatic hydrolysis of hemp seed protein

[0161] Pepsin, trypsin, chymotrypsin, papain, flavourzyme, proteinase K, neutral protease, alcalase, thermolysin, trypsin / pepsin, chymotrypsin / pepsin were used to hydrolyze the hemp seed protein, and the specific method was as follows: 0.1% mass concentration of protein solution was prepared, and 10000 U / g (8000 U / g for thermolysin) of protease was added to the protein sample, and the enzyme was hydrolyzed for 5 hours according to the optimal conditions of each enzyme reported in the literature. After hydrolysis, centrifugation was performed at 5000 rpm for 20 min, and the supernatant was collected and freeze-dried to obtain the hemp seed enzyme hydrolysate. The optimal conditions of each enzyme hydrolysis are shown in Table 3.

[0162] Table 3: Types of enzymes and their hydrolysis conditions

[0163]

[0164] b. Enzymatic hydrolysate ultrafiltration fractionation

[0165] A 3 kDa ultrafiltration tube was selected for fractionation of the enzymatic hydrolysate, and the centrifugation conditions were 4200 rpm / min, and the centrifugation was repeated for 25 min each time until no filtrate was collected. The filtrate was combined and used as needed.

[0166] c. Determination of protein / polypeptide concentration of enzymatic hydrolysate

[0167] The bicinchoninic acid method was used to determine the protein / polypeptide concentration of the fractionated liquid, and the specific steps were as follows: 200 μL of each enzymatic hydrolysate / standard solution (5 mg / mL bovine serum albumin) was taken, 1 mL of color developing agent was added and mixed, and then left to stand for 15 min. Then 200 μL of the reaction liquid was taken to a 96-well plate, and the absorbance was measured at 540 nm. The protein / polypeptide concentration calculation formula of the test solution was as follows:

[0168] C = 5 * (A1-A0) / (A2-A0)

[0169] Where A0 is the absorbance of the blank, i.e. the sample solvent hole, A1 is the absorbance of the sample to be tested, and A2 is the absorbance of the standard. The protein / polypeptide concentration of each fraction was calculated according to the formula and is shown in Table 4.

[0170] Table 4: Concentration and proportion of each fraction after enzyme digestion

[0171]

[0172]

[0173] From Table 5, it can be seen that the enzymolysis effect of thermolysin, flavor protease and protease K is better, among which, especially the thermolysin, the content of <3 kDa enzymolysis component in the enzymolysis component is greater than 86.31%.

[0174] Example 2 Preparation of enzymolysis extract of Cannabis sativa L. seed

[0175] According to the optimized process obtained from the above experiment, the preparation of the enzymolysis extract of Cannabis sativa L. seed is carried out by the following steps and conditions:

[0176] The Cannabis sativa L. seed raw material is crushed by a grinding instrument, a certain amount of Cannabis sativa L. seed powder is mixed with anhydrous ethanol at a solid-liquid ratio of 1:20 (w / v, g / mL), stirred at 20-25°C for 12h, then centrifuged at 5000 rpm for 10 min to discard the supernatant and collect the precipitate, and the above mixing, stirring and centrifugation steps are repeated for 3 times, then all the precipitate is combined and dried by ethanol in a 60°C water bath to obtain oil-removed Cannabis sativa L. seed powder;

[0177] A certain amount of the above Cannabis sativa L. seed powder is weighed, 0.8M NaCl solution (pH 7.0) is added at a solid-liquid ratio of 1:10 (w / v, g / mL), stirred at 35°C for 2h, centrifuged at 8000 rpm for 10 min to take the supernatant, and the remaining material is again added with ultrapure water (pH 10.0) at a solid-liquid ratio of 1:10 (w / v, g / mL), stirred at 35°C for 2h, centrifuged to take the supernatant, and the supernatants from the two times of adjustment are combined and adjusted to pH 4.5 to precipitate the protein, washed with water for 3 times, adjusted to pH 7.0, and freeze-dried to obtain Cannabis sativa L. seed protein powder;

[0178] The above Cannabis sativa L. seed protein powder is added with ultrapure water to prepare a 0.1% mass concentration protein solution, thermolysin is added at a concentration of 8000 U / g protein sample, the pH value is adjusted to 7.0, and enzymolysis is carried out at 55°C for 5h, after enzymolysis, centrifugation is carried out at 5000 rpm for 20 min, the supernatant is collected, and freeze-drying is carried out to obtain the enzymolysis extract of Cannabis sativa L. seed.

[0179] Example 3 Identification of polypeptides in the enzymolysis extract of Cannabis sativa L. seed

[0180] The enzymatic extract of Cannabis sativa L. obtained in Example 2 was prepared into a solution of 5 mg / mL with ultrapure water, then desalted with a desalting column, followed by lyophilization and re-dissolution with acetonitrile aqueous solution. The polypeptide concentration was quantified by NannoDrop, and was uniformly prepared into 2 mg / mL. Finally, it was filtered through a 0.22 μM membrane and detected by a QExactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer of Thermo Scientific. The data was analyzed by Maxquant, and finally the polypeptide composition of the enzymatic extract of Cannabis sativa L. was obtained. The top ten polypeptides with the highest relative abundance were LLY, VFTPQ (Seq ID No. 21), VADW (Seq ID No. 1), YNLP (Seq ID No. 14), LNAP (Seq ID No. 13), VAMP (Seq ID No. 2), NYLP (Seq ID No. 15), FNPRG (Seq ID No. 16), IEQMPQRS (Seq ID No. 23), and PQNH (Seq ID No. 20), as shown in Table 5.

[0181] Table 5: Polypeptides with the highest relative abundance identified by mass spectrometry

[0182]

[0183]

[0184] The ten polypeptides were artificially synthesized for subsequent in vitro DPP-IV inhibitory activity verification.

[0185] Example 4: Screening of DPP-IV inhibitory activity polypeptides based on molecular docking technology

[0186] The A chain of DPP-IV pretreated with 5YP3 crystal structure was used as the receptor, the identified hemp seed polypeptide library was used as the receptor, and the MOE software was used for molecular docking. Then, the hemp seed polypeptide sequence with the strongest potential DPP-IV inhibitory activity was screened according to the size of the Docking score. Finally, YGDQ (Seq ID No. 6), LTTVASY (Seq ID No. 26), WIAVK (Seq ID No. 12), YSYA (Seq ID No. 10), WNVN (Seq ID No. 11), FNVDSE (Seq ID No. 24), PSSQQTR (Seq ID No. 25), PQNHA (Seq ID No. 22), WDSY (Seq ID No. 19), YTGD (Seq ID No. 7), YQLM (Seq ID No. 3), FSPSSQQ (Seq ID No. 5), FDGEL (Seq ID No. 18), YTPHW (Seq ID No. 17), PSSQQ (Seq ID No. 9), YQL, FPQS (Seq ID No. 4), FLQ, YNL, FQL, WLE, VVDNNGRS (Seq ID No. 8) were selected for subsequent in vitro DPP-IV inhibitory activity verification. The specific screening results are shown in Table 6.

[0187] Table 6 Molecular docking results

[0188]

[0189]

[0190] Example 5 DPP IV in vitro inhibitory activity determination of polypeptides

[0191] The 32 polypeptides obtained in Example 3 and Example 4 were artificially synthesized, and the IC 50 values of the polypeptides for DPP-IV inhibition were determined to evaluate their inhibitory effect on DPP IV.

[0192] Experimental method: 1.0, 0.4, 0.08, 0.016 and 0.0032 mg / mL five sample concentration gradients were prepared for each polypeptide. The following 100 μL reaction system was constructed in a 96-well transparent microplate: 1) 60 μL of the above polypeptide solution of different concentrations was first added, 2) 20 μL of 2.0 mM Gly-Pro-pNA solution (final concentration 0.40 mM) was added and mixed well using a plate shaker for 1 minute, 3) 20 μL of rhDPP-IV solution was added to make the final rhDPP-IV activity 0.025 Unit / mL. After the addition of rhDPP-IV, it was immediately incubated in an enzyme marker at 37°C for 60 min, and the absorbance value at 405 nm was measured every 10 min during the incubation. For any sample, two points with changing absorbance value in the linear range were selected to calculate the change rate of absorbance with time S = (Abs2 - Abs1) / (t2 - t1), and the DPP-IV inhibition rate was calculated by the following formula (2-1):

[0193]

[0194] In formula (2-1), IRsample represents the inhibition rate of the sample on rhDPP-IV, SNC represents the change rate (slope) of absorbance of the negative control (solvent group) in the detection system, and Ssample represents the change rate of absorbance of the sample group. All samples and control groups need to be detected in triplicate, and the standard deviation is calculated. The experimental results are shown in Table 7. According to the determination results, the IC 50 of polypeptides VAMP, YQLM, FPQS, FSPSSQQ, YGDQ and WLE is less than 1.0 mmol / L, especially the IC 50 of polypeptide VAMP is 1.0 μmol / L; the IC 50 of polypeptides YTGD, VVDNNGRS, PSSQQ, VADW, YSYA, WNVN, WIAVK, LNAP is less than 10 mmol / L; the IC 50 of polypeptides YNLP, FLQ, NYLP, LLY is less than 100 mmol / L; and the IC 50 of polypeptides FNVDSE, PSSQQTR, FQL, LTTVNSY, YNL and YQL is relatively high.

[0195] Table 7 IC 50

[0196]

[0197]

[0198] Example 6 Evaluation of the hypoglycemic activity of polypeptides by cell experiment

[0199] The 32 polypeptides obtained in Example 3 and Example 4 were artificially synthesized, and Caco2 cell DPP-IV inhibitory activity, GLP-1 concentration and insulin concentration determination experiments were performed to evaluate their hypoglycemic activity.

[0200] Experimental method:

[0201] (1) 1 x 10 5 cells / mL were inoculated in a 24-well plate, and then after 24 h of culture in an incubator, the culture medium was replaced with a culture medium containing 2 mg / mL of each synthetic polypeptide or 5 mg / mL of a proteolytic solution for 24 h. Then the supernatant was taken out and the DPP IV activity and GLP-1 concentration in the supernatant were determined. The DPP IV inhibitory activity was determined using DPPIV-Glo TM Protease Assay (Promega), and the GLP-1 concentration was determined using a Solerio GLP-1 ELSA reagent, and the determination results are shown in Figure 1 and Figure 2 .

[0202] (2) 1000 μL of 1 x 10 5 cells / mL INS-1 cells were inoculated in a 24-well plate, and then after 24 h of culture in an incubator, the culture medium was replaced with a culture medium containing 2 mg / mL of each synthetic polypeptide for 24 h. Then, after washing once with KRBH buffer (Krebs-Ringer Bicarbonate HEPES Buffer), 1000 μL of KRBH buffer was added to the 24-well plate and incubated in an incubator for 1 hour. Then the KRBH buffer was removed and KRBH buffer containing 16.7 mM glucose solution was added and incubated in an incubator for 2 hours. The supernatant was taken out and the insulin concentration in the supernatant was determined using a Solerio Insulin kit, and the determination results are shown in Figure 3 .

[0203] From the analysis results, it can be seen that all the synthetic polypeptides can inhibit DPP IV activity and promote insulin secretion to some extent. Among them, polypeptides VADW, WDSY, VAMP, WIAVK and FNPRG have the best effect in inhibiting DPP IV activity; polypeptides FPQS, VFTPQ, FNVDSE, YTGD and YQLM have the best effect in promoting GLP-1 secretion; and polypeptides YSYA, PSSQQTR, VFTPQ, PQNHA and VADW have the best effect in promoting insulin secretion.

[0204] Example 7 Animal experiment for evaluating the hypoglycemic, fat-reducing, liver-protecting, kidney-protecting and intestinal flora regulating effects of polypeptides

[0205] The relative abundance and IC50 of the comprehensive mass spectrometry identification 50 Based on the results of value and cellular activity evaluation, three peptides, VADW, VAMP, and YQLM, were selected for in vivo activity verification. The specific experimental protocol is as follows:

[0206] Sixty male C57BL / 6J mice (6 weeks old, weighing 18-22g) were selected (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.). After one week of acclimatization, the mice were used to establish the model and were administered the mice by gavage.

[0207] The experiment was divided into 6 groups, with 10 mice in each group. The animals were distinguished by ear tagging. The groups were: normal group (WideType, WT), model group (High-fat-diet, HFD), positive drug group (Sitagliptin, PC), and 3 peptide sample groups (VADW, VAMP, and YQLM). The mice were administered the drugs by gavage.

[0208] During the experiment, all mice except the normal group were fed a high-fat diet (20 kcal of protein, 20 kcal of carbohydrates, and 60 kcal of fat) to induce the model. No other special treatments were administered. All groups were gavaged once daily at 0.2 mL / 20 g mouse body weight. The normal group (Wide Type, WT) and the model group (High-fat-diet, HFD) were gavaged daily with water (sample solvent). The positive control group was gavaged daily with 1.25 mg / kg of sitagliptin phosphate (purchased from Sigma). The VADW, VAMP, and YQLM peptide sample groups were gavaged with 50 mg / kg of their respective samples. The experiment was terminated when the body weight and fasting blood glucose levels of the model group mice were more than 20% higher than those of the normal group.

[0209] During the gavage period, the mice's diet, water intake, blood glucose, and body weight were measured and statistically analyzed weekly. The results showed that after 8 weeks of gavage, the body weight and fasting blood glucose levels of the model group mice were 20% higher than those of the normal group. Therefore, the entire animal experiment was terminated after 8 weeks of gavage.

[0210] (1) Evaluation of the efficacy of peptides in reducing fat

[0211] After 8 weeks of intragastric administration, the mice were treated and the mouse blood was collected. After centrifugation, the mouse plasma was obtained, and the total cholesterol (TC) and total triglycerides (TG) in the plasma were analyzed by using an animal automatic biochemical analyzer (chemistry analyzer), and the non-esterified fatty acid (FFA) and tyrosine peptide (PPY) in the plasma were determined by using an Elasa kit (Elabscience). The statistical results are shown in Table 1. Figures 4-11

[0212] After analyzing the results, it was found that intragastric administration of the three polypeptide samples could significantly reduce the diet, water intake and energy intake of the mice, and slow down the increase of the body weight of the mice. At the same time, intragastric administration of the three polypeptides could also significantly reduce the concentration of TC, TG and FFA in the plasma of the mice, and increase the concentration of PYY, indicating that several polypeptides had a fat-reducing effect.

[0213] (2) Evaluation of the hypoglycemic effect of polypeptides

[0214] During the experiment, the fasting blood glucose of the mice was determined every week, and the oral glucose tolerance level of the mice was evaluated at the eighth week. The specific operation was as follows: the fasting blood glucose of the mice was determined at 0 min, then the mice were intragastrically administered with 2 g / kg of glucose, then the blood glucose of the mice was determined at 15 min, 30 min, 60 min, 90 min and 120 min, and the area under the curve (Area under the curve) was calculated, and the concentration of insulin, glucagon-like peptide 1 (GLP-1), glycosylated hemoglobin (GHb) and adiponectin in the plasma of the mice was determined by using an Elasa kit (Elabscience), and the insulin resistance index (HOMA-IR) was calculated according to the following formula to evaluate the insulin resistance of the mice. The experimental results are shown in Table 2. Figures 12-17

[0215] HOMA-IR = C(Insulin) * C(FBS) / 22.5

[0216] Wherein, C(Insulin): concentration of plasma insulin; C(FBS): fasting glucose concentration

[0217] ​​After analyzing the results, it was found that intragastric administration of VADW, VAMP and YQLM could significantly increase the concentration of GLP-1 in the plasma, reduce the fasting blood glucose of mice, increase the oral glucose tolerance of mice, increase the concentration of ADPN in the plasma, and thus increase the insulin sensitivity of mice. It was also found that the concentration of GHb in the plasma of mice was reduced, indicating that intragastric administration of VADW, VAMP and YQLM polypeptides could promote the blood glucose metabolism balance of mice.

[0218] (3) Evaluation of the protective effect of polypeptides on liver and kidney

[0219] After 8 weeks of intragastric administration, the T-bil-D-II in the plasma of mice was measured, which is direct bilirubin, and an increase indicates that liver cells are damaged. The contents of glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminase (AST), alkaline phosphatase (ALP), total protein (TP II) and albumin (ALB II) were measured to evaluate the liver protection effect of VADW, VAMP and YQLM polypeptides. The concentrations of creatinine and urea in the plasma were also measured to evaluate the kidney protection effect of the polypeptides, and the specific results are shown in Table 8.

[0220] Table 8 Changes in blood biochemical indicators after polypeptide intervention (n = 6)

[0221]

[0222] Note: Different letters indicate significant differences, p < 0.05. T-bil-D-II: direct bilirubin; ALT: glutamic-pyruvic transaminase; AST: glutamic-oxalacetic transaminase; ALP: alkaline phosphatase; TP II: total protein; ALB II: albumin;

[0223] Creas: creatinine; Urea: urea.

[0224] After analysis, it was found that intragastric administration of VADW, VAMP and YQLM could significantly reduce the concentrations of ALT, AST, ALP and TP II in the plasma, indicating that VADW, VAMP and YQLM polypeptides had certain liver protection effects. It was also found that the concentrations of creatinine (Creas) and urea (Urea) in the plasma of the three polypeptide intragastric administration groups were also significantly lower than those of the model group, indicating that VADW, VAMP and YQLM polypeptides had certain kidney protection effects.

[0225] (4) Evaluation of the efficacy of polypeptide regulation of intestinal flora

[0226] After 8 weeks of intragastric administration, the colon contents of mice were taken and the composition of intestinal microorganisms in the colon contents of mice was analyzed and measured using 16sRNA sequencing technology, and the specific results are shown in Table 9. Figure 18 、 19 Figure 18 ​Results show that intragastric administration of several polypeptides can significantly increase the alpha diversity of colonic intestinal flora, including increasing Chao1 and Observed_species index, and reducing Goods_coverage index; at the same time, intragastric administration of hemp seed polypeptides can effectively increase the abundance of beneficial bacteria genera including Lactobacillus, Akkermansia, Bacteroides, etc., reduce the abundance of harmful bacteria genus Prevotella, and increase the ratio of Bacteroidetes / Firmicutes Figure 19 ), indicating that intragastric administration of hemp seed polypeptides has a function of improving intestinal flora disorder.

[0227] Example 8 Evaluation of the hypoglycemic activity of hemp seed enzymatic extract (cell experiment)

[0228] Experimental method: 1x10 5 6 / mL of Caco2 cells were inoculated in a 24-well plate, then after 24h of incubation in an incubator, the culture medium was replaced with a culture medium containing 5mg / mL of each hemp seed proteinase enzymatic component and incubated for 24h. Then the supernatant was taken out and the activity of DPP IV and the concentration of GLP-1 in the supernatant were determined. The concentration of DPP IV was determined using Elasa kit of Elabscience, and the concentration of GLP-1 was determined using GLP-1 ELSA kit of Solabio, and the results are shown in Figure 20 , 21 .

[0229] 1000μL of 1x10 5 6 / mL of INS-1 cells were inoculated in a 24-well plate, then after 24h of incubation in an incubator, the culture medium was replaced with a culture medium containing 5mg / mL of each hemp seed proteinase enzymatic component and incubated for 24h. Then washed once with KRBH buffer (Krebs-Ringer Bicarbonate HEPES Buffer), then 1000μL of KRBH buffer was added to the 24-well plate and incubated in an incubator for 1 hour. Then the KRBH buffer was taken out and 16.7mM glucose solution was added to the KRBH buffer and incubated in an incubator for 2 hours. The supernatant was taken out and the concentration of insulin in the supernatant was determined using Solabio Insulin kit, and the results are shown in Figure 22 .

[0230] In the above experiment, each hemp seed proteinase is 11 proteinases in Table 3 of Example 1. A control group is set as Sitagliptin (10μM).

[0231] Comprehensive evaluation of the results of cell experiments can show that each protease enzyme hydrolysis component can reduce the concentration of DPPIV to some extent, increase the concentration of GLP-1 and insulin, and have potential hypoglycemic effect. Among them, the enzyme hydrolysis product obtained by protease enzyme hydrolysis of hemp seed protein has the best potential hypoglycemic effect, which is better than the effect of the commonly used DPP IV inhibitor class of hypoglycemic drugs Sitagliptin in the prior art.

[0232] Animal experiment for evaluating the fat-reducing, blood sugar-lowering, liver-protecting, kidney-protecting, and intestinal flora regulating effects of hemp seed enzyme hydrolysis extract

[0233] The hemp seed protein and enzyme hydrolysis extract obtained in Example 2 were taken as the research objects, and animal experiments were carried out to evaluate their effects in fat reduction, blood sugar reduction, liver protection, kidney protection, intestinal flora regulation, etc. The experimental method is as follows:

[0234] 6-week-old (body weight 18-22 g) C57BL / 6J male mice (purchased from Zhejiang Vantong Lihua Experimental Animal Technology Co., Ltd.) were selected, and after adaptive feeding for one week, modeling and gavage were started.

[0235] The experiment was divided into 7 groups, 10 mice in each group, and the animals were distinguished by ear tags, which were normal group (Wide Type, WT), model group (High-fat-diet, HFD), positive drug group (Sitagliptin, PC), 3 hemp seed enzyme hydrolysis extract sample groups (low-dose group, TPH-L; medium-dose group, TPH-M; high-dose group, TPH-H) and hemp seed protein group (Pro), which were carried out by gavage.

[0236] During the experiment, all groups were gavaged according to 0.2 mL / 20 g mouse body weight once a day, except for the normal group, which was fed with high-fat feed (in high-fat feed, protein 20 kcal%, carbohydrate 20 kcal%; fat 60 kcal%) for modeling, and no other special treatment was given. Among them, the normal group (Wide Type, WT) and the model group (High-fat-diet, HFD) were gavaged with water (sample solvent) every day, the positive drug group was gavaged with 1.25 mg / kg positive drug Sitagliptin (purchased from sigma) every day, the hemp seed enzyme hydrolysis extract low-dose group (TPH-L), medium-dose group (TPH-M) and high-dose group (TPH-H) were gavaged with 80, 160 and 320 mg / kg hemp seed enzyme hydrolysis extract respectively, and the hemp seed protein group (Pro) was gavaged with 320 mg / kg unhydrolyzed hemp seed protein. Gavage for 8 weeks, during which the diet, drinking water and body weight were measured and counted every week. The experiment was ended when the body weight and fasting blood glucose value of the model group mice were more than 20% higher than those of the normal group.

[0237] The diet, drinking water, blood glucose and body weight of the mice were measured and statistically analyzed every week during the gavage. The results showed that after 8 weeks of gavage, the body weight and fasting blood glucose value of the model group mice were 20% higher than those of the normal group, so the entire animal experiment ended after 8 weeks of gavage.

[0238] (1) Fat-reducing efficacy evaluation of hemp seed enzymatic extract

[0239] After 8 weeks of gavage, the mice were treated and the mouse blood was collected. After centrifugation, mouse plasma was obtained, and the total cholesterol (TC) and total triglycerides (TG) in the plasma were analyzed using an animal full-automatic biochemical analyzer (chemistry analyzer). At the same time, the concentration of non-esterified fatty acid (FFA) and tyrosine peptide (PYY) in the plasma was determined using an Elasa kit (Elabscience). The statistical results are shown in Table 1. Figures 23-30

[0240] After analyzing the results, it was found that the hemp seed enzymatic extract of the three doses could reduce the diet, drinking water and energy intake of the mice, slow down the increase of the body weight of the mice, and also significantly reduce the concentration of TC, TG and FFA in the plasma of the mice, and increase the concentration of PYY, indicating that the hemp seed enzymatic extract has a fat-reducing effect.

[0241] (2) Fat-reducing efficacy evaluation of hemp seed enzymatic extract

[0242] During the experiment, the fasting blood glucose of the mice was determined every week, and the oral glucose tolerance level of the mice was evaluated at the eighth week. The specific operation was as follows: the fasting blood glucose of the mice was determined at 0 min, then the mice were gavaged with 2 g / kg of glucose, then the blood glucose of the mice was determined at 15 min, 30 min, 60 min, 90 min and 120 min, and the area under the curve (Area under the curve) was calculated, and the concentration of insulin, glucagon-like peptide 1 (GLP-1), glycosylated hemoglobin (GHb) and adiponectin in the plasma of the mice was determined using an Elasa kit (Elabscience), and the insulin resistance index (HOMA-IR) was calculated according to the following formula to evaluate the insulin resistance of the mice. The experimental results are shown in Table 2. Figures 31-36

[0243] HOMA-IR = C(Insulin) * C(FBS) / 22.5

[0244] ​​Wherein, C(Insulin): the concentration of plasma insulin; C(FBS): the concentration of fasting blood glucose

[0245] After analyzing the results, it was found that the intragastric administration of the enzymatic extract of Cannabis sativa L. could significantly increase the concentration of GLP-1 in the plasma, reduce the fasting blood glucose of mice, increase the oral glucose tolerance of mice, increase the concentration of ADPN in the plasma and thus increase the insulin sensitivity of mice. It was also found that the concentration of GHb in the plasma of mice was reduced, indicating that the intragastric administration of the enzymatic extract of Cannabis sativa L. could promote the blood glucose metabolism balance of mice.

[0246] (3) Evaluation of the protective effect of the enzymatic extract of Cannabis sativa L. on liver and kidney

[0247] After 8 weeks of intragastric administration, the T-bil-D-II: direct bilirubin in the plasma of mice was measured to evaluate the liver protection effect of the enzymatic extract of Cannabis sativa L. The contents of glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminase (AST), alkaline phosphatase (ALP), total protein (TP II), and albumin (ALB II) were also measured. The concentrations of creatinine and urea in the plasma were also measured to evaluate the kidney protection effect of the enzymatic extract of Cannabis sativa L. The specific results are shown in Table 9.

[0248] Table 9 Changes in blood biochemical indicators after intervention of protein and protein enzymatic extract (n=6)

[0249]

[0250]

[0251] Note: Different letters indicate significant differences, p<0.05. T-bil-D-II: direct bilirubin; ALT: glutamic-pyruvic transaminase; AST: glutamic-oxalacetic transaminase; ALP: alkaline phosphatase; TP II: total protein; ALB II: albumin;

[0252] Creas: creatinine; Urea: urea.

[0253] After analysis, it was found that the intragastric administration of the enzymatic extract of Cannabis sativa L. could significantly reduce the concentrations of ALT, AST, ALP, and TP II in the plasma, indicating that the intragastric administration of the enzymatic extract of Cannabis sativa L. had a certain liver protection effect. It was also found that the concentrations of creatinine (Creas) and urea (Urea) in the plasma of the intragastric administration group of the enzymatic extract of Cannabis sativa L. were significantly lower than those of the model group, indicating that the intragastric administration of the enzymatic extract of Cannabis sativa L. had a certain kidney protection effect.

[0254] (4) Evaluation of the intestinal flora regulation effect of the enzymatic extract of Cannabis sativa L.

[0255] After an 8-week gavage experiment, colonic contents were collected from mice, and the intestinal microbiota composition was analyzed using 16S rRNA sequencing. Specific results are as follows: Figure 37 , 38 As shown. By Figure 37 The results showed that gavage administration of hemp seed enzymatic hydrolysate significantly increased the α-diversity of the colonic gut microbiota, including increasing the Chao1 and Observed_species indices (higher values ​​indicate higher microbiota richness) and decreasing the Goods_coverage index (indicating higher sample richness). Simultaneously, gavage administration of hemp seed enzymatic hydrolysate effectively increased the abundance of beneficial bacteria genera including Lactobacillus, Akkermansia, and Bacteroides, decreased the abundance of harmful bacteria genus Prevotella, and increased the Bacteroides / Firmicutes ratio (Bacteroides / Firmicutes, which, according to literature, is inversely correlated with obesity). Figure 38 The results indicate that oral administration of hemp seed enzymatic hydrolysate has a certain function in improving intestinal flora imbalance.

[0256] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application, without departing from the scope of the technical solution of this application, shall still fall within the protection scope of this application.

Claims

1. Use of an enzymatic extract of Cannabis sativa L. seeds in the preparation of a medicament and a health food for improving hyperglycemia, wherein, the content of enzymatic components with a molecular weight less than 3 kDa in the enzymatic extract of Cannabis sativa L. seeds is greater than 86.31% by mass percentage in the enzymatic extract of Cannabis sativa L. seeds, the protein content is 75-85% by mass percentage in the enzymatic extract of Cannabis sativa L. seeds, the enzymatic extract of Cannabis sativa L. seeds is prepared by a method comprising the following steps: defatting the Cannabis sativa L. seed powder with an organic solvent to obtain defatted Cannabis sativa L. seed powder, extracting Cannabis sativa L. protein from the defatted Cannabis sativa L. seed powder, adding protease to the Cannabis sativa L. protein for enzymatic hydrolysis, filtering and centrifuging the enzymatic hydrolysate to obtain the enzymatic extract of Cannabis sativa L. seeds wherein the organic solvent is anhydrous ethanol, the defatting method is static stirring, the method for extracting Cannabis sativa L. protein is salt extraction, and the protease is thermolysin.

2. The use according to claim 1, wherein, The filtration has a molecular weight cut-off of ≤3000 Da.

3. The use according to claim 1, wherein, The enzymatic hydrolysis is performed by adding the thermolysin to the Cannabis sativa L. protein at a concentration of 8000-10000 U / g, the pH value of the enzymatic hydrolysis is 6.0-8.5, the filtration has a molecular weight cut-off of ≤3000 Da.

4. The use according to claim 1, wherein, The defatted Cannabis sativa L. seed powder is obtained by mixing the Cannabis sativa L. seed powder with anhydrous ethanol at a ratio of 1:10-1:20, followed by stirring, centrifugation, and discarding the supernatant.

5. The use according to claim 1, wherein, The salt extraction method comprises dissolving the defatted Cannabis sativa L. seed powder in a 0.8 M NaCl solution at a ratio of 1:10-1:20, followed by stirring, centrifugation, taking the supernatant, adjusting the pH value to ≤5, and obtaining the Cannabis sativa L. protein.

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