A kind of bitter melon bioactive peptide, preparation method and application thereof

Abstract

The present invention relates to a bitter melon bioactive peptide, a preparation method and an application thereof. The bitter melon bioactive peptide comprises a peptide chain 1, a peptide chain 2 and a peptide chain 3, wherein the peptide chain 1 comprises an amino acid sequence shown in SEQ ID NO: 1, the peptide chain 2 comprises an amino acid sequence shown in SEQ ID NO: 2, and the peptide chain 3 comprises an amino acid sequence shown in SEQ ID NO: 3. The bitter melon bioactive peptide provided by the present invention is obtained by extracting from the whole bitter melon plant, has the advantages of no toxic side effects, can be taken orally, does not require a special administration method, is suitable for large-scale synthesis, is easy to separate and purify, etc., has biological activities such as regulating abnormal glucose metabolism, anti-diabetes, and anti-tumor, and can be used to prepare drugs for preventing and treating diabetes and anti-tumor.

Description

Technical Field

[0001] The invention belongs to the field of pharmacy and medical technology, and in particular relates to a bitter melon bioactive peptide, a preparation method and application thereof. Background Art

[0002] Diabetes is a common and frequently occurring disease, and a global public health problem that seriously threatens human health. Currently, the prevalence and incidence of diabetes are rising sharply worldwide, and more significantly, they are becoming younger. Over the past 50 years, the prevalence of diabetes has continued to rise, with an estimated 425,000 new cases occurring each year. According to current forecasts, the growth of diabetes will exceed 50% between 2017 and 2045, resulting in approximately 693 million people suffering from diabetes. In 2019, the number of people with diabetes worldwide reached 463 million, and the number of people with diabetes in China in 2019 was approximately 116.4 million.

[0003] Glucose in human blood, referred to as blood sugar, is an important source of energy to maintain the normal functioning of human tissues and organs. However, long-term high blood sugar levels will endanger human health, and the body's ability to regulate blood sugar will be damaged and cause illness. Diabetes is a metabolic disease characterized by high blood sugar. Under normal circumstances, the pancreas releases insulin to help the body store and utilize sugar and fat in food. When the pancreas secretes less insulin, or the body's sensitivity to insulin decreases, it will lead to diabetes. According to the standards of the World Health Organization, if there are clinical manifestations of diabetes symptoms such as thirst, polydipsia, and weight loss, fasting blood sugar ≥7mmol / L and / or blood sugar ≥11.1mmol / L 2 hours after a meal, it can be diagnosed as diabetes.

[0004] Diabetes includes retinopathy, diabetic nephropathy leading to renal failure and peripheral neuropathy. Peripheral neuropathy is the most easily ignored, but the consequences may be serious. In mild cases, the skin may be burned, and in severe cases, the patient may fall due to unsteady gait. Acute complications: such as diabetic ketoacidosis, hyperglycemic hyperosmolar syndrome are usually more dangerous and can easily endanger the life safety of diabetic patients. The typical symptoms of diabetes are "three more and one less". In addition, there will be a series of atypical symptoms such as numbness of hands and feet, lack of energy, decreased vision, constipation, diarrhea, etc. The complications of diabetes can be extremely harmful to the human body and are an important cause of disability or death for patients. If diabetes is not well controlled, systemic complications such as "heart, brain, eyes, kidneys, nerves" will also occur, and severe cases may even cause blindness and disability. Therefore, prevention is more important. Once diagnosed with diabetes, active treatment is required.

[0005] The pathogenesis of type 2 diabetes has not been clearly studied so far. According to existing research reports, its pathogenesis is mainly related to multiple factors such as genetics, environmental changes, insulin resistance and insulin secretion defects. Insufficient insulin secretion and persistent hyperglycemia caused by insulin resistance are the main causes of type 2 diabetes. Insulin resistance and cell function defects are characteristic pathophysiological changes of type 2 diabetes, among which insulin resistance is an important link in the pathogenesis of type 2 diabetes. One of the main manifestations of insulin resistance is reduced glucose uptake in insulin-sensitive tissues (muscle, adipose tissue).

[0006] Diabetes is a non-infectious chronic disease that requires lifelong prevention and treatment. Currently, the commonly used treatment approaches in clinical practice are mainly hypoglycemic drugs. Oral hypoglycemic drugs for diabetes include: ① Insulin secretagogues, which include long-acting secretagogues and short-acting secretagogues. Their main function is to promote the secretion of insulin by pancreatic β cells, including amaryl and tangshipin. ② Non-insulin secretagogues include biguanides and glycosidase inhibitors, namely metformin and glycopyrrolate. Biguanide hypoglycemic drugs achieve the effect of hypoglycemia by inhibiting the output of liver glycogen and increasing the sensitivity of peripheral tissues to insulin. Glucopyrex can delay the absorption of glucose. ③ Insulin sensitizers, pioglitazone has the effect of expanding insulin to lower blood sugar. ④ DPP-4 inhibitors promote insulin secretion and inhibit glucagon secretion. The two-way action makes the blood sugar drop more stable and is not easy to produce hypoglycemia. There is sitagliptin. ⑤ Dapagliflozin, a drug that delays the reabsorption of glucose by the renal tubules.

[0007] The side effects of therapeutic drugs cannot be ignored. Hypoglycemic drugs: ① Glucobay and Amaryl can control blood sugar before and after meals. The main side effect is gastrointestinal symptoms, which can also cause hypotension. A small number of patients taking Amaryl may experience hepatitis, elevated liver enzymes or cholestasis and jaundice (although they will be relieved after stopping the drug, they are likely to cause psychological problems in patients); ② The mechanisms of action of Glucobay and metformin are different. Glucobay is an α-glucosidase inhibitor that can inhibit α-glucosidase and affect the absorption of carbohydrates. Its hypoglycemic effect is relatively weak, and it can only delay the absorption of carbohydrates and better control postprandial blood sugar. The main side effect of Glucobay is abdominal distension; ③ Metformin is a drug that increases insulin sensitivity. It is the most basic hypoglycemic drug for the treatment of type 2 diabetes. The side effects of metformin include symptoms of physical fatigue or dizziness, and a few people will experience nausea and vomiting after taking it. Long-term use will irritate the gastrointestinal tract and cause diarrhea. Studies have shown that long-term use of metformin can cause a decrease in vitamin B12 levels; ③ Pioglitazone hydrochloride tablets are oral hypoglycemic drugs of the thiazolidinedione class, and their side effects mainly include the following aspects: edema, patients with congestive heart failure are prohibited from using; osteoporosis, women who have already menopause, patients with a history of osteoporosis or a history of fractures are prohibited from using; the use of pioglitazone hydrochloride tablets may increase the incidence of bladder cancer, so patients with a history of bladder cancer, family history, or unexplained hematuria are prohibited from using; ④ Dapagliflozin: dry mouth, polyuria, urinary and reproductive tract infections may occur; ⑤ Injectable insulin is effective, but it is inconvenient and long-term use can lead to local skin infections. Insulin is a commonly used hypoglycemic drug in the treatment of diabetes. Patients with type 1 diabetes must use insulin for treatment, and patients with type 2 diabetes also need to use insulin for treatment when their blood sugar is relatively high. Insulin is a double-edged sword in the treatment of diabetes. It can effectively control blood sugar, but it also has certain side effects, mainly in the following aspects: the main side effect is hypoglycemia, especially in the case of irregular diet or increased exercise, which is more likely to induce hypoglycemia. Therefore, diabetic patients who use insulin should carry sugar cubes when exercising or traveling, so that they can take sugar cubes orally to relieve symptoms when hypoglycemia occurs; a common side effect of insulin is weight gain. After the injection of insulin, it can stimulate appetite, and the patient's food intake increases, so the weight will also increase. Insulin can also cause water and sodium retention, which can easily lead to lower limb edema.

[0008] Epidemiological studies have shown that compared with non-diabetic patients, tumors are more common in diabetic patients, and there is a certain correlation between diabetes and tumors. Compared with healthy adults, the risk of liver cancer in male diabetic patients increased by 2.24 times, kidney cancer increased by 1.92 times, and pancreatic cancer increased by 1.85 times. In female diabetic patients, the risk of liver cancer was 1.94 times and that of gastric cancer was 1.61 times. The cause and pathogenesis of diabetes-induced tumors are still unclear. At present, it is mainly believed to be related to factors such as hyperglycemia, insulin resistance, immune dysfunction, trace element deficiency, vascular endothelial growth factor, and obesity. At present, anti-tumor drugs still have the problem of "not distinguishing between friend and foe" in treatment, which leads to further decline in the body's immune function and produces mild side effects such as nerves, gastrointestinal, and hair loss. Therefore, exploring new preparations or new approaches with low toxicity and side effects, strong targeting, and good therapeutic effects is a hot topic in research worldwide.

[0009] In summary, patients with diabetes and cancer need to take medication for a long time, and various drugs bring adverse reactions during treatment. Therefore, it is urgent to find green preparations that can not only regulate blood sugar and achieve the goal of lowering blood sugar, but also prevent the occurrence of diabetes, inhibit tumor cell proliferation or enhance immunity, but have no toxic side effects, which is also the most ideal choice at present.

[0010] As the development of new drugs with completely new chemical structures becomes increasingly difficult and the investment costs increase year by year, scientists have gradually turned their attention to the development of active peptides and protein drugs. Bioactive peptides are intermediate products of protein hydrolysis and are composed of three or more amino acid molecules. Peptides have the effects of immunomodulation and promoting metabolism, and have the functions of transporting nutrients and physiological regulation. It has four major advantages: ① unique hypoglycemic mechanism; ② protection of pancreatic islets; ③ high safety; ④ good synergy. It can activate "inert" insulin that cannot function normally, restore its activity, and participate in sugar metabolism normally, thereby improving the body's own ability to utilize and convert blood sugar and steadily lower blood sugar. Protect pancreatic islet cells and inhibit pancreatic islet failure. Reduce dependence on chemical drugs. The pancreatic islets are rested, their functions are gradually restored, and their ability to regulate blood sugar is enhanced, which correspondingly reduces the body's dependence on drugs. On this basis, patients can reduce the amount and variety of chemical drugs to reduce drug damage to organs. Correct the three major metabolic disorders and prevent the occurrence of complications. Improving the ability to convert and utilize blood sugar can not only effectively improve sugar metabolism disorders, but also play a positive role in regulating fat and protein metabolism disorders. Existing bioactive peptides have the following defects: they are easy to degrade, have a short half-life, have poor bioavailability, cannot be taken orally, are generally injections, and require the development of appropriate administration methods; and are difficult to synthesize on a large scale and to separate and purify.

[0011] Bitter melon (Momordica charantia) is a common vegetable in Asia. It has a unique bitter taste and is both a medicine and food. Bitter melon is rich in essential substances for the human body and is known as a valuable plant that is beneficial to health. The chemical substances in bitter melon include: protein, peptide, polysaccharide, flavonoids, triterpenes, saponin, ascorbic acid and steroids. Among them, protein and peptide are the main active ingredients of bitter melon. Bitter melon peptides usually have the effects of diuresis, blood circulation, anti-inflammatory and fever-reducing, and clearing the mind and improving eyesight. Summary of the invention

[0012] In view of the problems existing in the prior art, the present invention provides a bitter melon bioactive peptide, a preparation method and an application thereof, which has the advantages of no toxic side effects, can be taken orally, does not require a special administration method, is suitable for large-scale synthesis, is easy to separate and purify, etc., can prevent and treat diabetes and has an anti-tumor effect.

[0013] The technical solution of the present invention to solve the above technical problems is as follows:

[0014] The invention provides a bitter melon bioactive peptide, comprising a peptide chain 1, a peptide chain 2 and a peptide chain 3, wherein the peptide chain 1 comprises an amino acid sequence shown in SEQ ID NO: 1, the peptide chain 2 comprises an amino acid sequence shown in SEQ ID NO: 2, and the peptide chain 3 comprises an amino acid sequence shown in SEQ ID NO: 3.

[0015] The bitter melon bioactive peptide provided by the present invention is extracted from bitter melon which can be used as medicine and food. The active peptide is a mixture of three peptide chains and is an important substance related to various cell functions in the human body. It has the advantages of no toxic side effects, can be taken orally, does not require a special administration method, is suitable for large-scale synthesis, and is easy to separate and purify. It has the functions of regulating abnormal glucose metabolism, anti-diabetes (preventing and treating diabetes), anti-tumor, regulating inflammatory response, anti-oxidation, improving body immunity, effectively inhibiting cancer cell secretion, preventing the growth of malignant tumors, synthesizing cells, and regulating cell function activities. It can promote human metabolism, regulate abnormal glucose metabolism and other biological activities, and can be used to prepare drugs for preventing and treating diabetes and anti-tumor.

[0016] The active peptide has no additives during the preparation process, so it is a green product with no toxic side effects.

[0017] The bitter melon bioactive peptide provided by the invention is obtained by extraction from the whole fruit of bitter melon.

[0018] The present invention provides a preparation, comprising the above-mentioned bitter melon bioactive peptide. The present invention has no particular limitation on the dosage form of the preparation, and it can be any conventional dosage form in the art. For example, the dosage form of the preparation can be selected from at least one of the following: oral liquid, ordinary tablet, sustained-release tablet, microporous controlled-release tablet, sublingual tablet, enteric-coated tablet, granule, enteric-coated capsule, timed / positioned release tablet, ordinary capsule, sustained-release capsule, controlled-release capsule, film or patch preparation, and other ingredients can be included in addition to the bitter melon bioactive peptide.

[0019] The preparation provided by the present invention has a main biologically active component of bitter melon bioactive peptides, which are extracted from the whole bitter melon plant, which is used as medicine and food, and contain three peptide chains. They are important substances involved in various cell functions in the human body. They have the functions of synthesizing cells and regulating cell function activities, and can promote human metabolism, regulate abnormal sugar metabolism, prevent and treat diabetes, resist diabetes, resist tumors, regulate inflammatory reactions, resist oxidation, improve body immunity, effectively inhibit cancer cell secretion, and prevent the growth of malignant tumors. The active peptide has no additives during the preparation process, so it is a green product without toxic side effects.

[0020] The invention provides a method for preparing the above-mentioned bitter melon bioactive peptide, which is characterized by comprising the following steps: enzymolysis of bitter melon homogenate with neutral protease once, and enzyme inactivation once; enzymolysis with acid protease twice, and enzyme inactivation twice; enzymolysis with trypsin three times, and enzyme inactivation three times.

[0021] The beneficial effects of adopting the above technical scheme: the present invention utilizes modern high-tech means to separate bioactive peptides from plants, which have the advantages of being not easily degraded, having a long half-life, having good bioavailability, being able to be taken orally, having a simple preparation method, and being able to be synthesized on a large scale.

[0022] Neutral proteases have many benefits for human health, such as anti-inflammation, relieving swelling and a series of other effects. By selecting appropriate proteases to enzymatically hydrolyze the peptide bonds in plant proteins, large protein molecules can be hydrolyzed into small fragments of peptides and amino acids. The enzymatic method has many advantages in producing active peptides, such as: the enzymatic hydrolysis process and results are highly controllable and reproducible; the enzymatic protein hydrolysis conditions are mild and safe; the enzymatic hydrolysis reaction has no side reactions and does not reduce the nutritional value of the protein, and can reduce the soluble sugar content. The above advantages have laid the foundation for its standardized production. Enzyme inactivation after enzymatic hydrolysis can prevent the extracted peptides from being degraded.

[0023] Furthermore, after the three inactivations, the method further includes the steps of primary filtration, alkaline protein removal, secondary filtration, ultrafiltration and fine filtration.

[0024] The beneficial effects of adopting the above technical solution include: the above method can prevent the extracted peptides from being degraded and achieve the sterilization goal.

[0025] Furthermore, after fine filtration, the method further comprises the step of preparing bitter melon bioactive peptide dry powder and / or bitter melon bioactive peptide oral liquid.

[0026] Furthermore, in the first enzymatic hydrolysis, the amount of neutral protease used was 3‰ of the net weight of bitter melon, and the temperature was kept at 50°C for 3 h; in the second enzymatic hydrolysis, under the condition of pH 3.0, the amount of acidic protease used was 3‰ of the net weight of bitter melon, and the temperature was kept at 50°C for 2 h; in the third enzymatic hydrolysis, under the condition of pH 8.0, the amount of trypsin used was 3‰ of the net weight of bitter melon, and the temperature was kept at 40°C for 2 h.

[0027] The beneficial effects of adopting the above technical solution include: the macromolecular substances in the original solution can be converted into small molecular substances through enzymatic hydrolysis; under the above conditions, the total amount of free amino acids in the treated solution after enzymatic hydrolysis shows a significant increase.

[0028] Furthermore, in the primary enzyme inactivation, the temperature was kept at 80°C for 10 min and then cooled to 50°C; in the secondary enzyme inactivation, the temperature was kept at 80°C for 10 min and then cooled to 40°C; in the tertiary enzyme inactivation, the temperature was kept at 70°C for 10 min.

[0029] The beneficial effects of adopting the above technical solution include: the above conditions are conducive to stopping the enzymatic hydrolysis and avoiding the degradation of the extracted peptides.

[0030] The present invention provides the above-mentioned bitter melon bioactive peptide or preparation which can be used for preparing food, medicine and the like.

[0031] For example, the present invention provides the use of the above-mentioned bitter melon bioactive peptide in any one or more of (1) to (7),

[0032] (1) In the preparation of special medical foods and drugs for the prevention of diabetes;

[0033] (2) In the preparation of special medical foods and drugs for the treatment of diabetes;

[0034] (3) In the preparation of anti-tumor special medical foods and drugs;

[0035] (4) In the preparation of anti-inflammatory special medical foods and drugs;

[0036] (5) In the preparation of special medical foods and drugs for immunomodulation;

[0037] (6) In the preparation of special medical foods and drugs for regulating blood sugar;

[0038] (7) In the preparation of special medical foods and drugs for preventing complications of diabetes.

[0039] The present invention provides the use of the above-mentioned preparation in any one or more of (1) to (7),

[0040] (1) In the preparation of special medical foods and drugs for the prevention of diabetes;

[0041] (2) In the preparation of special medical foods and drugs for the treatment of diabetes;

[0042] (3) In the preparation of anti-tumor special medical foods and drugs;

[0043] (4) In the preparation of anti-inflammatory special medical foods and drugs;

[0044] (5) In the preparation of special medical foods and drugs for immunomodulation;

[0045] (6) In the preparation of special medical foods and drugs for regulating blood sugar;

[0046] (7) In the preparation of special medical foods and drugs for preventing complications of diabetes.

[0047] The present invention also relates to the use of bitter melon bioactive peptides or preparations containing bitter melon bioactive peptides in the preparation and treatment of diabetes and anti-inflammatory. The bitter melon bioactive peptides and preparations provided by the present invention can effectively regulate blood sugar, have obvious beneficial effects in preventing and treating diabetes, and have anti-tumor, anti-inflammatory and other functions. The bitter melon bioactive peptides and preparations provided by the present invention for preventing and treating diabetes and anti-tumor functions contain three peptide chains, which come from the whole bitter melon plant for both medicinal and edible purposes. It is a preventive and therapeutic dose of bioactive peptides, a green bioactive peptide without any additives and toxic side effects, and the bioactive peptide can be used as a carrier. The bioactive peptide has outstanding effects in preventing and treating diabetes and anti-tumor, without producing toxic side effects, and is a green therapy. During the application process, a suitable dose can be designed. For example, the dose of the bioactive peptide can be 4.57 mg / ml per tube. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The present invention is a process flow chart for preparing bitter melon polypeptide.

[0049] Figure 2 The results of mass spectrometry analysis of bitter melon bioactive peptides.

[0050] Figure 3 These are the results of toxicity analysis of bitter melon bioactive peptides.

[0051] Figure 4 Effects of Momordica charantia polypeptide on glucose consumption in insulin-resistant C2C12 cells (Note: n = 6; compared with the normal control group, #P < 0.05, ##P < 0.01, compared with the model control group, *P < 0.05, **P < 0.01; Nor = normal group, Con = model control group.)

[0052] Figure 5 These are the changes in myofibrils in the model group in Example 3.

[0053] Figure 6 The results are the changes in myofibrils in the momordica charantia polypeptide treatment group in Example 3.

[0054] Figure 7 These are the changes in myofibrils of the blank control group in Example 3.

[0055] Figure 8 These are the animal experiment results for verifying the anti-tumor effect in Example 4.

[0056] Fig. 9 These are the detection results of the inflammatory factors INFα and INFγ in Example 4; the left figure is the detection result of INFγ, and the right figure is the detection result of INFα. DETAILED DESCRIPTION

[0057] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0058] The present invention aims at the problems existing in the current oral and injection insulin treatment of diabetes, and provides a green, non-toxic and side-effect bioactive peptide and preparation, so as to explore a new and effective treatment approach that does not damage other organs and has no adverse reactions. The bioactive peptides involved in the present invention are derived from the whole bitter melon plant, which contains three amino acid sequences and is an effective anti-diabetic preparation for the purpose of regulating blood sugar to prevent and treat diabetes. The bioactive peptides provided by the present invention have a good effect on preventing diabetic complications on the basis of the effective effects of regulating blood sugar, preventing and treating diabetes. Epidemiological studies have shown that compared with non-diabetic patients, tumors are more common in diabetic patients. Therefore, it is of great significance to provide a drug that has both anti-tumor and anti-diabetic effects. The bioactive peptides provided by the present invention have both anti-tumor and anti-diabetic effects, and can be used as and prepare anti-tumor drugs and anti-diabetic drugs. At the same time, the bioactive peptides provided by the present invention are derived from the whole bitter melon plant that is both medicinal and edible, have no toxic side effects, can be taken orally, do not require a special administration method, are suitable for large-scale synthesis, are easy to separate and purify, have a simple preparation process, have clear efficacy, are suitable for industrialization, etc. Therefore, the bioactive peptides provided by the present invention are new bioactive peptides with good application prospects.

[0059] The inventors further analyzed the sequence of the bioactive peptide and found for the first time that the bioactive peptide includes three peptide chains: (1) GGEQQDQEGEEER; (2) EINERDRQSPA; (3) SDEEHHFESK. It was unexpectedly found in the study that the bioactive peptide prepared by the present invention has a good therapeutic effect on diabetes, can prevent diabetic complications, and has the effect of preventing the occurrence of diseases. It regulates blood sugar, thereby reducing the occurrence of diabetes, treating diabetes, reducing the dosage of chemical drugs or stopping the use of chemical hypoglycemic drugs, and reducing the occurrence of complications, including reducing the occurrence of tumors. The bioactive bitter melon whole plant-derived bioactive peptide composed of three amino acid sequences not only has a therapeutic effect, but also has a good preventive effect and a certain effect of preventing complications. It also has good immunomodulatory, anti-tumor and anti-inflammatory effects.

[0060] In the present invention, neutral protease was purchased from Shaanxi Chenming Biotechnology Co., Ltd., acidic protease and trypsin were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Trypsin was purchased from Promega; acetonitrile was purchased from Fisher Chemical; formic acid was purchased from Fluka; ammonium bicarbonate (NH4HCO3) was purchased from Sigma-Aldrich; dithiothreitol was purchased from Sigma; iodoacetamide was purchased from Sigma; ultrapure water (H2O), methanol, and acetonitrile were purchased from Fisher Chemical. C2C12 cells were purchased from Xiehe Cell Bank. Acetic acid was purchased from Hangzhou Gaojing Fine Chemicals; ammonium acetate was purchased from Sinopharm Group. INFαKit and INFγKit were purchased from Ekosai.

[0061] The mass spectrometer was purchased from Thermo Scientific TMQ Exactive Plus.

[0062] Unless otherwise specified, the reagents of the present invention are all conventional reagents in the art and can be obtained commercially. Unless otherwise specified, the experimental methods used in the present invention are all conventional experimental methods in the art.

[0063] Example 1

[0064] like Figure 1 As shown, the preparation method of bitter melon bioactive peptide comprises the following steps:

[0065] (1) Raw material processing: all fresh bitter melon fruits are washed with pure water to remove mud and sand, etc., and the water is controlled to dry, sliced ​​by an automatic vegetable cutter to cut the fibers, minced by a commercial meat grinder, and homogenized by a flash extractor to obtain bitter melon homogenate;

[0066] (2) water preparation: pour the bitter melon homogenate into a reaction tank, add 0.3 times the net weight of the bitter melon with pure water, stir evenly, and obtain a mixture;

[0067] (3) Heating: The mixture after adding water is heated to 50°C under stirring;

[0068] (4) Primary enzymatic hydrolysis: Weigh 3‰ of the net weight of bitter melon with neutral protease, dissolve it with a small amount of water, slowly pour it into the reaction tank under stirring, stir evenly, and keep warm at 50°C for 3 h. During this period, stir intermittently for 3-5 min each time to obtain a primary enzymatic hydrolyzate;

[0069] (5) Primary enzyme inactivation: heating the primary enzymatic hydrolysate to 80°C under constant stirring, stirring and keeping warm for 10 min, and cooling to 50°C to obtain a primary inactivated product;

[0070] (6) Secondary enzymatic hydrolysis: adjust the pH to 3.0 with hydrochloric acid HCl, weigh 3‰ of the net weight of bitter melon with acidic protease, dissolve it with a small amount of pure water, slowly pour it into the reaction tank under stirring, stir evenly, and keep warm at 50°C for 2h. During this period, stir intermittently for 3-5min each time to obtain a secondary enzymatic hydrolyzate;

[0071] (7) Secondary enzyme inactivation: heating the secondary enzymatic hydrolysate to 80°C under continuous stirring, stirring and keeping warm for 10 min, and cooling to 40°C to obtain a secondary inactivated product;

[0072] (8) Tertiary enzymatic hydrolysis: adjust the pH to 8.0 with NaOH solution, weigh 3‰ of the net weight of bitter melon with trypsin, dissolve it with a small amount of pure water, slowly pour it into the reaction tank under stirring, stir evenly, and keep warm at 40°C for 2 h. During this period, stir intermittently for 3-5 min each time to obtain tertiary enzymatic hydrolysate;

[0073] (9) Tertiary enzyme inactivation: The tertiary enzymatic hydrolyzate was heated to 70°C under constant stirring, and stirred and kept warm for 10 min to obtain a tertiary inactivated product;

[0074] (10) Primary filtration: Filter the tertiary inactivated material while hot using a plate and frame filter to obtain a filtrate;

[0075] (11) Removing alkaline proteins: The filtrate of step (10) was adjusted to pH 8.0 with NaOH solution, 90° C., stirred and kept warm for 10 min to obtain a polypeptide solution from which alkaline proteins had been removed;

[0076] (12) Secondary filtration: The polypeptide solution after removing the basic protein is filtered while hot using a plate and frame filter to obtain a filtrate, which is then cooled to 40° C.;

[0077] (13) Ultrafiltration: The filtrate of step (12) is ultrafiltered using a membrane package with a molecular weight cutoff of 6 kDa, and the ultrafiltrate is collected;

[0078] (14) Fine filtration: The ultrafiltrate is finely filtered through 0.45 μm and 0.1 μm sterilizing filters respectively to separate into two parts for subsequent process treatment;

[0079] (15) Preparation of pilot products

[0080] Taking the preparation of bitter melon polypeptide dry powder as an example, the preparation method includes the following steps: spray drying the solution after 0.45 μm fine filtration, controlling the air inlet temperature at 80° C., controlling the discharge temperature at 45° C., and collecting the dry powder;

[0081] Taking the preparation of bitter melon polypeptide oral liquid as an example, the preparation method includes the following steps: the concentrated liquid after 0.1 μm fine filtration is diluted into a 4.5 mg / ml diluent, and then packaged into 10 ml / bottle oral liquid for standby use.

[0082] (16) Product inspection: including inspection of physical and chemical form, content, microbiological indicators, etc.

[0083] The dry powder product prepared by the present invention was tested to be a light yellow powder with a maximum absorption of 265 nm and a pH value of 7.0. No heavy metals were detected, the total colony count was less than 3000 CFU / g, and Escherichia coli and Salmonella were not detected.

[0084] Example 2 Mass Spectrometry Analysis of Momordica charantia Bioactive Peptides

[0085] 1. Collecting peptides, including the following steps: taking the product prepared in Example 1 as a sample, transferring the sample to an ultrafiltration tube, centrifuging at room temperature for 20 minutes at 12000g to recover the peptides, and then recovering the peptides once with ultrapure water, and combining the two peptide solutions. Desalting: After the peptide solution is frozen and drained, it is dissolved with 0.5% acetic acid. The SCX-tip column is first rinsed with methanol, then balanced with 0.5% acetic acid solution, the sample is loaded, and washed twice with 0.5% acetic acid solution and 80% acetonitrile aqueous solution containing 0.5% acetic acid, respectively, and finally eluted with 50mM ammonium acetate, 50mM ammonium bicarbonate and 0.1% ammonia water, respectively, and the eluents are combined and then freeze-dried in vacuum to obtain peptides, and the peptides are used for mass spectrometry analysis.

[0086] 2. Liquid chromatography-mass spectrometry analysis

[0087] The peptides prepared above were dissolved in liquid chromatography mobile phase A and separated using the EASY-nLC 1200 ultra-high performance liquid phase system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an aqueous solution containing 0.1% formic acid and 90% acetonitrile. Liquid phase gradient settings: 0-62min, 4%-21% B; 62-82min, 21%-32% B; 82-86min, 32%-80% B; 86-90min, 80% B, and the flow rate was maintained at 500nL / min. After the peptides were separated by the ultra-high performance liquid phase system, they were injected into the NSI ion source for ionization and then subjected to Q Exactive TM HF-X mass spectrometry was used for analysis. The ion source voltage was set to 2.1 kV, and the peptide parent ions and their secondary fragments were detected and analyzed using a high-resolution Orbitrap. The primary mass spectrometry scan range was set to 350-1800 m / z, and the scan resolution was set to 120,000; the secondary scan resolution was set to 15,000. The data acquisition mode used a data-dependent scan (DDA) program, that is, after the primary scan, the top 10 peptide parent ions with the highest signal intensity were selected to enter the HCD collision cell in turn and use 28% fragmentation energy for fragmentation, and the secondary mass spectrometry analysis was also performed in turn. In order to improve the effective utilization of the mass spectrometer, the automatic gain control (AGC) was set to 5E4, the signal threshold was set to 2.5E5 ions / s, the maximum injection time was set to 40 ms, and the dynamic exclusion time of the tandem mass spectrometry scan was set to 30 s to avoid repeated scanning of the parent ion.

[0088] Through mass spectrometry analysis of bitter melon bioactive peptides, it was found that the bitter melon bioactive peptides prepared by the present invention include peptide chain 1, peptide chain 2 and peptide chain 3, peptide chain 1 includes the amino acid sequence shown in SEQ ID NO: 1, peptide chain 2 includes the amino acid sequence shown in SEQ ID NO: 2, and peptide chain 3 includes the amino acid sequence shown in SEQ ID NO: 3. Specifically, peptide chain 1: GGEQQDQEGEEER (SEQ ID NO: 1); peptide chain 2: EINERDRQSPA (SEQ ID NO: 2); peptide chain 3: SDEEHHFESK (SEQ ID NO: 3). Taking the EINERDRQSPA peptide segment as an example, the experimental results are as follows: Figure 2 shown. Figure 2 In the figure, y represents the amino acid residue whose charge is retained at the C-terminus, b represents the amino acid residue whose charge is retained at the N-terminus, and the subscript indicates the sequence number of the amino acid residue in the fragment. The specific mass-to-nuclear ratio m / z can specifically represent a certain amino acid fragment. Scan number is the total number of amino acid residues scanned in the fragment, FTMS is an ion cyclotron resonance mass spectrometer, and the box part is the comparison information between the peptide sequence and the ion peak.

[0089] Example 3 Verification of the anti-diabetic effect of Momordica charantia polypeptide

[0090] Insulin is involved in regulating glucose metabolism and controlling blood sugar balance. Insulin resistance refers to the fact that various reasons cause insulin to promote glucose uptake and utilization efficiency to decrease, and the body compensatory secretion of excessive insulin produces hyperinsulinemia to maintain blood sugar stability. Skeletal muscle is one of the important tissues involved in glucose metabolism. Whether its metabolism is normal or not is directly related to the body's exercise capacity and blood sugar level. Therefore, promoting muscle cell glucose uptake has become one of the important indicators for studying hypoglycemic drugs. Therefore, this embodiment selects the myoblast cell line C2C12, which is a precursor cell found in adult skeletal muscle tissue that rebuilds muscle tissue after trauma and has good differentiation ability. C2C12 myoblasts are often used as in vitro systems to study muscle development and differentiation, and are commonly used cell lines for studying insulin resistance. Therefore, the present invention uses C2C12 cells to establish a cell model of insulin resistance.

[0091] (1) Cytotoxic effects of bitter melon peptides on C2C12 cells

[0092] The present invention firstly observes the toxic effect of Momordica charantia polypeptide on C2C12 cells.

[0093] Cell culture: After routine recovery, C2C12 cells were inoculated into a 100 ml culture bottle. Routine in vitro culture of C2C12 mouse myoblasts: DMEM (containing high glucose) medium plus 10% fetal bovine serum. Cultured in an incubator at 37°C and 5% CO2 saturated humidity. When 80% of the cells were confluent, they were digested and passaged with 0.25% trypsin.

[0094] In order to verify the cytotoxic effect of different concentrations of bitter melon polypeptide on C2C12 cells, the final experimental concentrations of bitter melon polypeptide were set to 1000, 500, 250, 125, 62.5, 31.25, and 15.625 μg mL -1 Thiazolyl blue assay showed that all concentrations tested had no significant effect on the proliferation of C2C12 cells. Figure 3 As shown, from Figure 3 It can be seen that all the concentrations tested had no obvious toxic side effects on C2C12 cells.

[0095] (2) Effect of Momordica charantia polypeptide on glucose consumption in insulin-resistant (IR) C2C12 cells

[0096] The experimental method includes the following steps: C2C12 cells were divided into a normal group (referred to as Nor), a model group, and a bitter melon polypeptide group. The model group was cultured with 0.25mmol / L palmitic acid and 1% bovine serum albumin for 16 hours to induce IR (insulin resistance); the bitter melon polypeptide group was cultured with 1000, 500, 250, 125, and 62.5μg·mL on the basis of the model group.-1 The cells were intervened with different concentrations of bitter melon polypeptide for 48 hours. The glucose consumption was reflected by detecting the glucose content in the culture medium. The CCK8 kit and light microscope were used to observe the effects of different concentrations of bitter melon polypeptide on the activity and morphology of the two cells.

[0097] Figure 4 Effects of Momordica charantia polypeptide on glucose consumption in insulin-resistant C2C12 cells (Note: n = 6; compared with the normal control group, #P < 0.05, ##P < 0.01, compared with the model control group, *P < 0.05, **P < 0.01;

[0098] from Figure 4 Analysis showed that bitter melon peptides at high concentrations (1000, 500, 250, 125 μg mL -1 ) has a significant effect of promoting glucose consumption in IR-C2C12 cells. This indicates that the bitter melon bioactive peptide prepared by the present invention has a significant blood sugar regulating effect on induced IR (insulin resistance) model mice.

[0099] (3) Changes in myofibrils after bitter melon peptides act on cells

[0100] The experimental results of the changes in myofibrils after bitter melon peptides acted on cells are as follows Figures 5 to 7 shown. Figures 5 to 7 They were model group, bitter melon peptide treatment group and blank control group. The results showed that the active peptide treatment had an improving effect on myofibrils of myoblasts in mice with insulin resistance.

[0101] Figure 5 The results of the changes in myofibrils in the model group in Example 3. Left: The myofibrils are arranged slightly irregularly, and small focal myofibril dissolution can be seen. Mitochondria (M) between myofibrils are mostly long rod-shaped, and some of the inner ridges are blurred. The inner ridges of a few mitochondria are broken, and the sarcoplasmic reticulum (ER) is dilated to varying degrees. Right: Enlarged image.

[0102] Figure 6 The results of the changes in myofibrils in the Momordica charantia polypeptide treatment group in Example 3. Left: The myofibrils are arranged slightly irregularly, and small focal myofibril dissolution can be seen. However, the mitochondria (M) between myofibrils are mostly oval, slightly swollen, some of the inner ridges are blurred, and some of the mitochondrial inner ridges are broken. Right: A partial enlarged view

[0103] Figure 7 The results are the changes of myofibrils in the blank control group in Example 3. Left: Myofibrils are arranged regularly, and the mitochondria (M) between myofibrils are long rod-shaped or oval, with complete structure and clear inner ridges.

[0104] Example 4 Verification of the anti-tumor effect of Momordica charantia polypeptide

[0105] (1) Anti-tumor effect in animal experiments

[0106] The experimental method includes the following steps: MFC cells are purchased from the Concord Cell Bank, and the cells are cultured normally in T25 bottles. After taking out, the cell status is observed under a microscope, and after strict disinfection and drying, the cells are placed in an incubator for buffering for several hours, and then the waste liquid is discarded according to the instructions, and fresh complete culture medium (sigama) is added, and the cells are placed in an incubator and cultured at 37°C. The medium is changed normally the next day. When the cell density reaches 70%-80%, the old cell culture medium in the culture dish is discarded, the cells are washed twice with PBS, 0.5 ml of 0.25% trypsin is added, and the cells are observed under a microscope. When the cells become round and fall off from the bottom of the culture dish, most of the trypsin is discarded. Add 1-2mL of fresh complete culture medium, blow the cells to suspend and disperse them, and divide them equally into 2 or 3 culture dishes at a ratio of 1:3. Fill each 10cm culture dish with 8mL of complete culture medium (1640 liquid culture medium + 10% FBS), and culture in an incubator at 37°C and 5% CO2. Check the cell status the next day. If there are many dead cells, replace with fresh complete culture medium. If there are almost no dead cells, add culture medium to 10mL and culture normally. When the cells are in the logarithmic growth phase, digest the cells, centrifuge, wash twice with serum-free culture medium, and then resuspend the cell pellet with about 1mL of serum-free culture medium. After counting with a counter, subcutaneous inoculation of C57 mice is performed. The inoculation amount for each mouse is 1*10E 7 The injection volume was 100 μl / cell. About 7 days after injection, tumor formation was observed. When the tumor grew to 100 mm 3 At the same time, the tumor-bearing mice were treated with drugs by gavage once a day. Three groups were set up, namely: H drug treatment group (H group for short), Vehicle group and BG drug treatment group (BG group for short). Vehicle group, as the blank control group, used normal saline as the drug; H group was the positive control, and BG group was the bitter melon polypeptide group. The drug dosage was carried out as required. Tumor measurements were performed every three days, and the mice were weighed. After the experiment, the mice were anesthetized with pentobarbital, and the tumors were removed and weighed.

[0107] Animal experimental results to verify the anti-tumor effect are as follows Figure 8 shown.

[0108] Figure 8In the figure, A, photos of the tumors of the control group, H drug treatment group and BG drug treatment group after the mouse tumors were removed; B, the tumors were measured every three days after medication, and the tumor proliferation curve analysis showed that both H drug and BG drug treatment could significantly inhibit the proliferation ability of the tumor. **, p<0.01; ***, p<0.001. C, tumor weight analysis. The analysis showed that both H drug and BG drug treatment could significantly reduce the weight of the tumor. Ng, no statistical significance; *, p<0.05. D, body weight analysis showed that H drug and BG drug had no significant effect on the body weight of mice.

[0109] The analysis results suggest that the drugs in group H and group BG can significantly inhibit the proliferation of tumors derived from MFC cells. And they have no significant effect on the weight of mice, indicating that the drugs in group H and group BG have less toxicity.

[0110] (2) In vivo animal experiments to detect inflammatory factors INFα and INFγ

[0111] The serum of each mouse in each group was collected and the expression levels of INF-α and INF-γ were detected by ELISA. The detection method includes the following steps:

[0112] ① Take out the strips needed for the test from the sealed bag that has been equilibrated to room temperature. Put the unused strips and desiccant back into the aluminum foil bag and seal it at 4℃.

[0113] ② Leave a blank well (if dual wavelength plate reader is used, blank wells can be omitted).

[0114] ③Prepare samples, standards and biotinylated antibody working solution in advance.

[0115] ④ Add the specimens or standards of different concentrations (0pg / ml well plus reagent diluent) to the corresponding wells (100μl / well). If the sample volume is small, this kit can be used for micro-volume detection, i.e. 40μl / Well for standards and 40μl / Well for samples; then add 50μl of biotinylated antibody working solution to the sample and standard wells, and seal the reaction wells with sealing tape.

[0116] ⑤ Incubate at room temperature (20-25°C) for 120 minutes using a micro-oscillator (frequency, 300 rpm).

[0117] ⑥ Prepare the enzyme conjugate working solution 30 minutes in advance. Place at room temperature away from light. Wash the plate 5 times.

[0118] ⑦Except for the blank wells, add enzyme conjugate working solution (100 μl / well) and seal the reaction wells with sealing tape.

[0119] ⑧ Incubate at room temperature (20-25°C) for 60 minutes. Use a micro-oscillator (frequency, 300 rpm). Wash the plate 5 times.

[0120] ⑨Add 100 μl / well of colorimetric substrate (including blank wells) and incubate at room temperature (20-25°C) in the dark for 15 minutes.

[0121] ⑩Add 100 μl / well of stop solution (including blank wells), mix well and measure OD immediately 450 value (within 10 minutes).

[0122] The experimental results are Fig. 9 As shown. Fig. 9 It can be seen that BG drug treatment can significantly reduce the amount of INF-α (*, p<0.05).

[0123] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A bitter melon bioactive peptide composition, characterized in that: It includes peptide chain 1, peptide chain 2 and peptide chain 3, peptide chain 1 is the amino acid sequence shown in SEQ ID NO:1, peptide chain 2 is the amino acid sequence shown in SEQ ID NO:2, and peptide chain 3 is the amino acid sequence shown in SEQ ID NO:

3.

2. A preparation, characterized in that The invention comprises the bitter melon bioactive peptide composition as claimed in claim 1.

3. The preparation according to claim 2, characterized in that The dosage form of the preparation is selected from at least one of the following: oral solution, ordinary tablets, sustained-release tablets, microporous controlled-release tablets, sublingual tablets, enteric-coated tablets, granules, enteric-coated capsules, timed / positioned release tablets, ordinary capsules, sustained-release capsules, controlled-release capsules, films or patches.

4. The method for preparing the bitter melon bioactive peptide composition according to claim 1, characterized in that: The following steps are involved: The bitter melon homogenate is enzymatically hydrolyzed by neutral protease for one time, and the enzyme is inactivated for one time; and the bitter melon homogenate is enzymatically hydrolyzed by acidic protease for two times, and the enzyme is inactivated for two times; Trypsin was used for three times of enzymatic digestion and three times of enzyme inactivation.

5. The method for preparing the bitter melon bioactive peptide composition according to claim 4, characterized in that: After the three inactivations, the method further includes the steps of primary filtration, alkaline protein removal, secondary filtration, ultrafiltration and fine filtration.

6. The method for preparing the bitter melon bioactive peptide composition according to claim 5, characterized in that: After fine filtration, the method further comprises the step of preparing bitter melon bioactive peptide dry powder and / or bitter melon bioactive peptide oral liquid.

7. The method for preparing the bitter melon bioactive peptide composition according to any one of claims 4 to 6, characterized in that: In the first enzymatic hydrolysis, the amount of neutral protease used was 3‰ of the net weight of bitter melon, and the temperature was kept at 50℃ for 3h; in the second enzymatic hydrolysis, under the condition of pH 3.0, the amount of acidic protease used was 3‰ of the net weight of bitter melon, and the temperature was kept at 50℃ for 2h; in the third enzymatic hydrolysis, under the condition of pH 8.0, the amount of trypsin used was 3‰ of the net weight of bitter melon, and the temperature was kept at 40℃ for 2h.

8. The method for preparing the bitter melon bioactive peptide composition according to any one of claims 4 to 6, characterized in that: In the primary enzyme inactivation, keep warm at 80°C for 10 min and then cool to 50°C; in the secondary enzyme inactivation, keep warm at 80°C for 10 min and then cool to 40°C; in the tertiary enzyme inactivation, keep warm at 70°C for 10 min.

9. Use of the bitter melon bioactive peptide composition of claim 1 in any one or more of (1) to (7), (1) In the preparation of drugs for preventing diabetes; (2) In the preparation of drugs for the treatment of diabetes; (3) In the preparation of drugs for treating gastric cancer; (4) In the preparation of anti-inflammatory drugs; (5) In the preparation of drugs for regulating blood sugar, inhibiting the proliferation of MFC cell-derived tumors, and inhibiting the expression of INF-α inflammatory factors; (6) In the preparation of drugs for regulating blood sugar; (7) In the preparation of drugs for preventing complications of diabetes.

10. Use of the preparation according to claim 2 or 3 in any one or more of (1) to (7), (1) In the preparation of drugs for preventing diabetes; (2) In the preparation of drugs for the treatment of diabetes; (3) In the preparation of drugs for treating gastric cancer; (4) In the preparation of anti-inflammatory drugs; (5) In the preparation of drugs for regulating blood sugar, inhibiting the proliferation of MFC cell-derived tumors, and inhibiting the expression of INF-α inflammatory factors; (6) In the preparation of drugs for regulating blood sugar; (7) In the preparation of drugs for preventing complications of diabetes.

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