WO2018188565A1 - 用于治疗代谢系统疾病的多肽及其组合物 - Google Patents

用于治疗代谢系统疾病的多肽及其组合物 Download PDF

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WO2018188565A1
WO2018188565A1 PCT/CN2018/082401 CN2018082401W WO2018188565A1 WO 2018188565 A1 WO2018188565 A1 WO 2018188565A1 CN 2018082401 W CN2018082401 W CN 2018082401W WO 2018188565 A1 WO2018188565 A1 WO 2018188565A1
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group
polypeptide
composition
mammal
diabetes
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PCT/CN2018/082401
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English (en)
French (fr)
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张宇飞
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武汉格睿特科技有限公司
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Priority to JP2019556236A priority Critical patent/JP7242060B2/ja
Priority to EP18784747.0A priority patent/EP3653639B1/en
Publication of WO2018188565A1 publication Critical patent/WO2018188565A1/zh
Priority to US16/598,713 priority patent/US20200038478A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/185—Vegetable proteins
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
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    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/0056—Mouth soluble or dispersible forms; Suckable, eatable, chewable coherent forms; Forms rapidly disintegrating in the mouth; Lozenges; Lollipops; Bite capsules; Baked products; Baits or other oral forms for animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins

Definitions

  • the invention belongs to the field of biomedicine, relates to a polypeptide and a composition thereof, and particularly to a polypeptide for treating diseases of a metabolic system and a composition thereof.
  • Diabetes is a comprehensive metabolic system disorder in which the insulin secretion in the body is relatively or absolutely insufficient, insulin does not play a normal physiological role in the target cells, and/or the glucagon is excessively secreted, and the metabolism of sugar, protein and fat is disordered.
  • the basic feature is long-term hyperglycemia in patients.
  • diabetes complications Long-term diabetes can damage multiple systems of the whole body, especially the cardiovascular system, leading to chronic progressive lesions of the eyes, kidneys, nerves, heart, blood vessels and other tissues, causing functional defects and failure, such as Cardiovascular and cerebral arteriosclerosis, retinal and renal microangiopathy, neuropathy and gangrene of lower limbs.
  • Diabetic retinopathy (DR) and diabetic nephropathy (DN) are common microvascular complications of diabetes.
  • diabetes is the leading cause of blindness, kidney failure, heart attack, stroke and lower extremity amputation. At present, diabetes has become the third leading cause of death in addition to heart disease and cancer among the elderly.
  • the drugs for treating diabetes are now mainly insulin, oral hypoglycemic drugs, and incretin.
  • the existing drugs have obvious disadvantages, such as metformin easily lead to lactic acidemia, thiazolidinediones have hepatotoxicity and bone loss and other side effects, long-term use of sulfonylurea drugs can cause hypoglycemia, and long-term use DD-IV inhibitors are prone to cough, GLP-1 is easy to cause nausea and vomiting in patients, and insulin has problems such as inability to be taken orally, inconvenient to store and use, patients with hypoglycemia, long-term use of insulin resistance and subcutaneous fat atrophy.
  • hypoglycemic drugs have various defects, and can not cure diabetes, patients need lifelong medication, therefore, it is urgent to develop safe and effective new hypoglycemic drugs, especially convenient oral drugs to meet the increasing global The needs of people with diabetes.
  • diabetes is a metabolic disease caused by the absolute or insufficient insulin secretion, which causes three major metabolic disorders of sugar, fat and protein. Diabetes patients are often accompanied by hyperlipidemia, which is characterized by cholesterol, triglycerides and low density. High lipoprotein content or low HDL content.
  • Diabetes is closely related to hyperlipidemia.
  • the activity of lipase in diabetic patients is also lowered, and blood lipids are elevated.
  • diabetes itself is accompanied by disorders of fat, protein, water and dielectric, and free fatty acids are often released from fat stores.
  • increase the concentration of triglycerides and free fatty acids in the blood and people with type 2 diabetes eat more, which promotes the increase of lipid synthesis in the body and also leads to an increase in blood lipids.
  • obesity with hyperlipidemia due to the relative decrease in the number of insulin receptors, resulting in insulin resistance, which in turn is likely to induce diabetes.
  • Hyperlipidemia is a secondary disease of diabetes, which in turn can easily cause cardiac and cerebrovascular complications. According to statistics, about 40% of diabetic patients have hyperlipidemia, and diabetes with hyperlipidemia is more likely to cause stroke, coronary heart disease, limb necrosis, fundus lesions, kidney disease, neuropathy and so on. Therefore, there is a need to develop a new hypoglycemic and lipid-lowering drug that can lower blood sugar in diabetic patients while also lowering blood lipids.
  • the invention provides a polypeptide and a composition thereof, which have excellent hypoglycemic and hypolipidemic activities, and are resistant to pepsin and trypsin enzymatic hydrolysis, thereby being capable of being administered by injection or orally, and also having protection. It can repair the activity of pancreatic ⁇ -cell function, and it will not cause hypoglycemia and insulin resistance in patients, without any side effects. It is convenient and safe to use, and can be stored stably for a long time. It is a new type of hypoglycemic and lipid-lowering drugs.
  • the inventors of the present invention have found that natural polypeptides composed of a plurality of amino acids extracted from legumes and polypeptides having similar structures have excellent hypoglycemic activity, and further studies have found that such polypeptides also have excellent hypolipidemic properties. Activity, recent studies have also found that these polypeptides are capable of achieving better hypoglycemic and hypolipidemic effects when used in combination with certain amino acids, thus completing the present invention.
  • the invention relates to a method for reducing blood sugar and/or blood lipids in a mammal, characterized in that a therapeutically effective amount of one of the polypeptides of the sequence of the following formula (I) is administered thereto , a mixture of two or more of any ratio:
  • X 1 -X 11 are independently selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, M ox .
  • the present invention relates to a method for preventing and/or treating diabetes and/or hyperlipidemia in a mammal, characterized in that a therapeutically effective amount thereof is administered as shown in the general formula (I) A mixture of one, two or more of the polypeptides of the sequence in any ratio.
  • the invention relates to a method for reducing blood glucose and/or blood lipids in a mammal, characterized in that a therapeutically effective amount of one of the polypeptides of the sequence of the formula (I) is administered thereto , a mixture of two or more of any ratio, and other hypoglycemic agents and / or lipid-lowering drugs.
  • the present invention relates to a method for preventing and/or treating diabetes and/or hyperlipidemia in a mammal, characterized in that a therapeutically effective amount thereof is administered as shown in the general formula (I) One or a mixture of two or more of the polypeptides of the sequence, and other hypoglycemic agents and/or lipid lowering agents.
  • the present invention relates to a mixture of one, two or more of the polypeptides of the sequence of the formula (I) in a preparation for reducing blood sugar and/or blood lipids in a mammal.
  • a preparation for reducing blood sugar and/or blood lipids in a mammal Use in pharmaceutical, food, health food or formula for special medical uses.
  • the present invention relates to a mixture of one, two or more of the polypeptides of the sequence of the formula (I) in the preparation for the prevention and/or treatment of diabetes in a mammal and / or use in drugs for hyperlipidemia.
  • the present invention relates to a mixture of one, two or more of the polypeptides of the sequence of the formula (I), which is prepared for use in combination with other hypoglycemic agents and/or The use of a lipid combination in a pharmaceutical, food, health food or formula for a particular medical use that reduces blood sugar and/or blood lipids in a mammal.
  • the present invention relates to a mixture of one, two or more of the polypeptides of the sequence of the formula (I), which is prepared for use in combination with other hypoglycemic agents and/or Use of a lipid combination in the prevention and/or treatment of a medicament for diabetes and/or hyperlipidemia in a mammal.
  • the present invention relates to a pharmaceutical composition or preparation for oral administration, a food, a health food or a special medical use formula, which comprises one of the polypeptides of the sequence of the formula (I) A mixture of two, or more, in any ratio, and a pharmaceutically acceptable carrier.
  • the present invention relates to a pharmaceutical composition or preparation for intravenous, intramuscular or subcutaneous injection comprising one or two of the polypeptides of the sequence of the formula (I) Or a mixture of any of a plurality of ratios, and a pharmaceutically acceptable carrier.
  • the present invention relates to a composition
  • a composition comprising one or a mixture of two or more of the polypeptides of the sequence of the formula (I), and at least one amino acid.
  • the invention relates to a method of reducing blood glucose and/or blood lipids in a mammal, characterized in that a therapeutically effective amount of the above composition is administered thereto.
  • the invention relates to a method for the prevention and/or treatment of diabetes and/or hyperlipidemia in a mammal, characterized in that a therapeutically effective amount of the above composition is administered thereto.
  • the invention relates to a method of reducing blood glucose and/or blood lipids in a mammal, characterized by administering a therapeutically effective amount of the above composition, and other hypoglycemic agents and/or lipid lowering agents thereto. medicine.
  • the invention relates to a method for the prevention and/or treatment of diabetes and/or hyperlipidemia in a mammal, characterized in that a therapeutically effective amount of the above composition, and other hypoglycemic agents are administered thereto Medicine and / or lipid-lowering drugs.
  • the invention relates to the use of a composition as described above for the preparation of a medicament, food, health food or special medical use formula for reducing blood sugar and/or blood lipids in a mammal.
  • the invention relates to the use of a composition as described above for the manufacture of a medicament for the prevention and/or treatment of diabetes and/or hyperlipidemia in a mammal.
  • the invention relates to a pharmaceutical, food, health food or special medical use formulation of the above composition for the preparation of a medicament for reducing blood sugar and/or blood lipids in a mammal in combination with other hypoglycemic agents and/or lipid lowering agents Use in food.
  • the invention relates to the use of the above composition for the manufacture of a medicament for the prevention and/or treatment of diabetes and/or hyperlipidemia in a mammal in combination with other hypoglycemic agents and/or lipid lowering agents .
  • a mammal such as a diabetic, glycosylated hemoglobin >7.0%, preferably >7.5% prior to administration of the polypeptide of the invention or composition thereof, glycosylated hemoglobin ⁇ 7.5%, preferably ⁇ 7.0 after administration of the polypeptide of the invention or composition thereof %.
  • the present invention relates to a pharmaceutical preparation for oral administration, a food, a health food or a special medical use formula comprising the above composition and a pharmaceutically acceptable carrier.
  • the invention relates to a pharmaceutical composition or formulation for intravenous, intramuscular or subcutaneous injection comprising the above composition together with a pharmaceutically acceptable carrier.
  • the polypeptide of the invention has the sequence of formula (I).
  • X 1 is a hydrophobic amino acid selected from the group consisting of A, L, V, I, P, F, M, W, and M ox ;
  • X 2 is an acidic amino acid selected from E and D; and
  • X 3 is a hydrophobic amino acid.
  • X 4 is a polar neutral amino acid other than cysteine, selected from G, S, T, Y, N, Q
  • X 5 is a neutral amino acid other than cysteine, selected from G, S, T, Y, N, Q, A, L, V, I, P, F, M, W;
  • X 6 is hydrophobic Amino acid, selected from A, L, V, I, P, F, M, W, M ox ;
  • X 7 is a neutral amino acid other than cysteine, selected from G, S, T, Y, N, Q , A, L, V, I, P, F, M, W;
  • X 8 is a hydrophobic amino acid selected from the group consisting of A, L, V, I, P, F, M, W, M ox ;
  • X 9 is hydrophobic Amino acid, selected from A, L, V, I, P, F, M, W,
  • X 2 X 3 is selected from one of DI, EI, DM, EM, DM ox , EM ox ;
  • X 4 X 5 X 6 is selected from the group consisting of SPL, SPA, SSL, SSA, TPL, TPA, TSL, One of TSA;
  • X 8 X 9 is selected from one of VI, VV, FI, FV, LI, LV.
  • the polypeptide of the invention is selected from a mixture of one, two or more of the following sequence polypeptides:
  • SEQ ID No. 1 ASCNGVCSPFEMPPCGTSACRCIPVGLVVGYCRNPSG
  • SEQ ID No. 2 ASCNGVCSPFEM ox PPCGTSACRCIPVGLVVGYCRNPSG
  • SEQ ID No. 3 ASCNGVCSPFEMPPCGTSACRCIPVGLVIGYCRNPSG
  • SEQ ID No. 4 ASCNGVCSPFEM ox PPCGTSACRCIPVGLVIGYCRNPSG
  • SEQ ID No. 5 ASCNGVCSPFEMPPCGSSACRCIPVGLLIGYCRNPSG
  • SEQ ID No. 7 ASCNGVCSPFEMPPCGTSACRCIPVGLFIGYCRNPSG
  • SEQ ID No. 8 ASCNGVCSPFEM ox PPCGTSACRCIPVGLFIGYCRNPSG
  • SEQ ID No. 9 ASCNGVCSPFEMPPCGTSACRCIPYGLFIGYCRNPSG
  • SEQ ID No. 10 ASCNGVCSPFDIPPCGSPLCRCIPVGLVIGKCRNPYG,
  • SEQ ID No. 12 VSCNGVCSPFDIPPCGSPLCRCIPAGLVIGKCRNPYG,
  • SEQ ID No. 13 ASCNGVCSPFDIPPCGTPLCRCIPVGLVIGNCRNPYG,
  • SEQ ID No. 14 ASCNGVCSPFDMPPCGSSACRCIPVGLFIGNCRNPYG
  • SEQ ID No. 15 ASCNGVCSPFDMPPCGSSACRCIPVGLVIGYCRNPYG
  • SEQ ID No. 16 ASCNGVCSPFDMPPCGSSLCRCIPAGLVVGYCRNPSG
  • SEQ ID No. 17 ASCNGVCSPFDMPPCGTSACRCIPAGLFIGKCRNPYG,
  • SEQ ID No. 18 ASCNGVCSPFDMPPCGTSACRCIPVGLVVGYCRNPSG
  • SEQ ID No. 19 ASCNGVCSPFDMPPCGTSLCRCIPAGLFIGYCRNPSG
  • SEQ ID No. 20 ASCNGVCSPFDMPPCGTSLCRCIPAGLVIGYCRNPYG,
  • SEQ ID No. 21 ASCNGVCSPFEIPPCGSPLCRCIPVGLVIGNCRNPYG,
  • SEQ ID No. 22 ASCNGVCSPFEIPPCGTPLCRCIPAGLVIGKCRNPYG,
  • SEQ ID No. 24 ASCNGVCSPFEMPPCGSSACRCIPAGLFIGKCRNPYG,
  • SEQ ID No. 25 ASCNGVCSPFEMPPCGSSACRCIPVGLFVGYCRNPYG
  • SEQ ID No. 26 ASCNGVCSPFEMPPCGSSACRCIPVGLVVGYCRNPSG
  • SEQ ID No. 27 ASCNGVCSPFEMPPCGSSLCRCIPAGLFIGYCRNPSG
  • SEQ ID No. 28 ASCNGVCSPFEMPPCGSSLCRCIPAGLVIGYCRNPSG,
  • SEQ ID No. 29 ASCNGVCSPFEMPPCGTSACRCIPAGLFIGNCRNPYG,
  • SEQ ID No. 30 ASCNGVCSPFEMPPCGTSACRCIPAGLVIGYCRNPYG,
  • SEQ ID No. 36 ISCNGVCSPFDIPPCGTPLCRCIPAGLFVGKCRNPYG,
  • SEQ ID No. 39 ISCNGVCSPFDMPPCGTSACRCIPAGLVIGYCRNPSG,
  • SEQ ID No. 40 ISCNGVCSPFEIPPCGSPLCRCIPAGLFIGKCRNPSG,
  • SEQ ID No. 42 ISCNGVCSPFEIPPCGTPACRCIPAGLVIGKCRNPYG,
  • SEQ ID No. 48 ISCNGVCSPFEMPPCGSSACRCIPAGLVIGYCRNPSG,
  • SEQ ID No. 58 VSCNGVCSPFDMPPCGSSACRCIPAGLVIGYCRNPYG,
  • SEQ ID No. 60 VSCNGVCSPFDMPPCGSSACRCIPVGLFIGYCRNPSG
  • SEQ ID No. 61 VSCNGVCSPFDMPPCGTSACRCIPAGLFIGYCRNPSG,
  • SEQ ID No. 68 VSCNGVCSPFEMPPCGSSACRCIPYGLVVGNCRNPSG
  • SEQ ID No. 69 VSCNGVCSPFEMPPCGTPLCRCIPYGLLIGKCRNPYG,
  • SEQ ID No. 70 VSCNGVCSPFEMPPCGTSACRCIPAGLVIGYCRNPSG
  • SEQ ID No. 71 VSCNGVCSPFEMPPCGTSACRCIPAGLFIGYCRNPSG.
  • polypeptide of the present invention is selected from a mixture of one, two or more of the polypeptides having SEQ ID No. 1 to SEQ ID No. 12 as described above in any ratio.
  • the polypeptides of the present invention all contain six cysteines at positions 3, 7, 15, 20, 22 and 32, respectively, forming three pairs of disulfide bonds in the peptide chain, respectively Cys3-Cys20, Cys7-Cys22 and Cys15-Cys32, which is a "cystine knot motif" in which a disulfide bond between Cys3-Cys20 and Cys7-Cys22 forms a skeleton, and a disulfide bond between Cys15-Cys32 penetrates the skeleton.
  • ICK inhibitor cystine structure
  • the polypeptide of the present invention further contains 14 to 16 hydrophobic amino acids, wherein the amino acids at positions 1, 6, 9, 10, 12, 13, 14, 19, 23, 24, 27, 28, 29 and 35 are Hydrophobic amino acids, these hydrophobic amino acids are highly aligned in the amino acid sequence.
  • the above structural features enable the polypeptide of the present invention to be resistant to enzymatic hydrolysis by pepsin and trypsin, and have excellent hypoglycemic and lipid-lowering activities, and have hypoglycemic and hypothmic effects whether administered orally or subcutaneously, intramuscularly or intravenously. Lipid activity.
  • the polypeptides of the invention and mixtures thereof are derived from the seeds of legumes or their processing by-product, the extract of soybean meal.
  • Legumes include, but are not limited to, soybeans, broad beans, peas, red beans, mung beans, cowpeas, green beans, lentils, etc., and mixtures of one or two or more thereof in any ratio; preferably peas, soybeans, mung beans or one of them a mixture of two, or more, in any ratio.
  • the extract contains the polypeptides of SEQ ID No. 1 to SEQ ID No. 12 as described above.
  • the total content of the 12 polypeptides is at least 5%, preferably at least 8%, further preferably at least 10%, further preferably at least 20%, further preferably at least 30%, further preferably at least 50%, further preferably at least 80%, It is still more preferably at least 90%, still more preferably at least 95%, still more preferably at least 97%, and most preferably at least 98%.
  • the present inventors have found through research that the extract has good hypoglycemic and lipid-lowering activity even in the case of low purity, for example, but not limited to, a total content of 12 polypeptides of 10%.
  • the extract is an organic solvent extract selected from the group consisting of n-butanol, isopropanol, ethanol, methanol, formic acid, acetic acid, and any ratio of two or more organic solvents. a mixture; preferably n-butanol.
  • the extraction method comprises the following steps:
  • the seeds of the pulverized legumes or the processing by-products thereof are degreased and degreased before being extracted with the organic solvent in the step (1).
  • the mixture is degreased and degreased with a mixture of any one selected from the group consisting of chloroform, cyclohexane, petroleum ether, and two or more organic solvents; chloroform is preferably used for decolorization and degreasing.
  • the extraction temperature is from 0 to 30 ° C, most preferably 10 ° C.
  • the extraction time is from 1 to 40 hours, most preferably from 20 to 30 hours.
  • the amino acid is selected from one of methionine, histidine, tyrosine, a mixture of two or more in any ratio. It will be understood by those skilled in the art that methionine, histidine, and tyrosine may be D-form, L-form or DL-type, preferably L-form.
  • the weight ratio of the polypeptide to the amino acid is from 100:1 to 1:100, more preferably, the weight ratio is from 50:1 to 1:50, and more preferably, the weight ratio is from 50:1 to 1 More preferably, the weight ratio is from 20:1 to 10:1.
  • the composition containing the polypeptide of the present invention and the composition containing the polypeptide of the present invention and the amino acid may be a liquid composition or a solid composition.
  • the composition comprising the polypeptide of the present invention and the composition comprising the polypeptide of the present invention and the amino acid may further comprise recombinant human serum albumin and/or bovine serum albumin for enhancing the stability of the polypeptide of the present invention.
  • a liquid composition in an amount ranging from 0.5 to 50 mg/ml (in a liquid composition), more preferably from 1 to 20 mg/ml, more preferably from 5 to 10 mg/ml, or from 0.5 to 50 mg/g (in a solid composition), It is preferably 1 to 20 mg/g, more preferably 5 to 10 mg/g.
  • the composition containing the polypeptide of the present invention and the composition containing the polypeptide of the present invention and the amino acid may further comprise sodium chloride for increasing ionic strength and preventing aggregation of the polypeptide, and the content thereof is in the range of 1 to 20 mg. /ml (in liquid composition), more preferably 5 to 15 mg/ml, more preferably 8 to 12 mg/ml, or 1 to 20 mg/g (in solid composition), more preferably 5 to 15 mg/g, more preferably 8 ⁇ 12mg / g.
  • the composition containing the polypeptide of the present invention and the composition containing the polypeptide of the present invention and the amino acid may further comprise disodium edetate for complexing metal ions to prevent oxidation of the polypeptide.
  • the range is from 0.05 to 0.1 mg/ml (in liquid composition) or from 0.05 to 0.2 mg/g (in solid composition).
  • the composition comprising the polypeptide of the present invention and the composition comprising the polypeptide of the present invention and the amino acid may further comprise sodium acetate, acetic acid for adjusting pH and ionic strength, and increasing the solubility of the polypeptide in water.
  • the content of sodium acetate is in the range of 0.5 to 3 mg/ml (in the liquid composition), more preferably 1 to 2.5 mg/ml, still more preferably 1.5 to 2 mg/ml.
  • the composition containing the polypeptide of the present invention and the composition containing the polypeptide of the present invention and the amino acid may further comprise one, two or more kinds of glycerin, mannitol, and sorbitol, and are used as And increasing the stability of the polypeptide of the present invention in an amount ranging from 0.5 to 100 mg/ml (in a liquid composition), more preferably from 1 to 50 mg/ml, more preferably from 8 to 15 mg/ml, or from 0.5 to 100 mg/g. (in the solid composition), more preferably 1 to 50 mg/g, still more preferably 8 to 15 mg/g.
  • the pH is from 3.5 to 6.5, more preferably from 4 to 5, more preferably 4.5; preferably, when present
  • the pH of the 5% solution is from 3.5 to 6.5, more preferably from 4 to 5, still more preferably 4.5.
  • composition comprising a polypeptide of the invention and an amino acid is a liquid composition comprising:
  • composition comprising a polypeptide of the invention and an amino acid is a solid composition comprising:
  • Disodium edetate Preferably, it further comprises sodium chloride.
  • the mammal is a human.
  • polypeptides of the invention and compositions thereof are capable of reducing blood glucose and/or elevated blood lipids caused by various causes including, but not limited to, type I and/or type II diabetes, preferably type II diabetes, with elevated blood lipids including but not Limited to hyperlipidemia.
  • polypeptide of the present invention or a composition thereof can be formulated into a pharmaceutical composition or preparation, a food, a health food or a formula for a special medical use.
  • a therapeutically effective amount of a polypeptide of the invention or a composition thereof can determine a therapeutically effective amount of a polypeptide of the invention or a composition thereof by a limited number of routine experiments.
  • a therapeutically effective amount of a polypeptide of the present invention or a mixture thereof is, for example, 1 to 20 mg/(kg.d); and a therapeutically effective amount of a composition comprising the polypeptide of the present invention and an amino acid is, for example, 1 to 30 mg/(kg.d).
  • hypoglycemic agents include, but are not limited to, insulin and its derivatives such as long acting insulin, oral hypoglycemic agents, incretin, and the like, and combinations thereof.
  • other oral hypoglycemic agents can be used in the present invention, including but not limited to biguanides such as metformin, sulfonylurea secretagogues, thiazolidinediones such as glitazone, alpha-glycosides.
  • Enzyme inhibitors such as acarbose and the like, and combinations thereof.
  • other lipid lowering agents include, but are not limited to, lovastatin, simvastatin, pravastatin, fenofibrate, gemfibrozil, bezafibrate, sodium alginate, and the like.
  • the polypeptide of the present invention or a composition thereof and other hypoglycemic agents and/or lipid-lowering drugs may be administered simultaneously as a single composition, or may be administered simultaneously in separate compositions, or may be separated.
  • sequence administration in the compositions will be understood by those skilled in the art, and the order of administration of the sequential administrations does not affect the therapeutic effect of the polypeptides of the invention or compositions thereof and other hypoglycemic agents and/or lipid lowering agents.
  • the polypeptide of the present invention or a composition thereof may be formulated into various dosage forms suitable for oral, intravenous, intramuscular or subcutaneous injection, including, but not limited to, tablets suitable for oral administration (including but not limited to various coated tablets).
  • sustained release or controlled release tablets sustained release or controlled release tablets
  • troches capsules (including soft and hard capsules), granules, dispersible powders, aqueous or oily suspensions, emulsions, elixirs or syrups, etc.; Injections for intravenous, intramuscular or subcutaneous injection, powder injections for injection, and the like.
  • the polypeptide of the present invention or a composition thereof may further contain a pharmaceutically acceptable carrier including, but not limited to, various organic or inorganic pharmaceutical carriers such as excipients, lubricants, binders, disintegration.
  • a pharmaceutically acceptable carrier including, but not limited to, various organic or inorganic pharmaceutical carriers such as excipients, lubricants, binders, disintegration.
  • Agent water-soluble polymer, inorganic salt, solvent, dissolution aid, suspending agent, isotonic agent, buffer, preservative, antioxidant, coloring agent, sweetener, sour agent, foaming agent and flavoring agent, etc. Wait.
  • polypeptides of the present invention are commercially available or can be obtained according to various preparation methods known in the art including, but not limited to, extraction from legumes, chemical synthesis (e.g., solid phase synthesis), and genetic recombination.
  • the method of obtaining a recombinant protein or the like is preferably extracted from a legume.
  • compositions, pharmaceutical formulations, foods, health foods or specialty medical use formulas of the present invention can be prepared by a variety of methods known in the art.
  • the polypeptide of the invention, or a composition, pharmaceutical preparation, food, health food or special medical use formula is stored under the protection of an inert gas, preferably under the protection of nitrogen.
  • A represents an alanine in the amino acid sequence
  • C represents a cysteine
  • D represents aspartic acid
  • E represents glutamic acid
  • F represents phenylalanine.
  • G stands for glycine
  • H stands for histidine
  • I stands for isoleucine
  • K stands for lysine
  • L stands for leucine
  • M stands for methionine
  • N stands for asparagine
  • P stands for proline
  • Q stands for Glutamine
  • R stands for arginine
  • S stands for serine
  • T stands for threonine
  • V stands for valine
  • W stands for tryptophan
  • Y stands for tyrosine.
  • M ox represents methionine sulfoxide.
  • the present invention has the following advantages and benefits:
  • the polypeptide of the invention has excellent hypoglycemic and hypolipidemic activities, and when combined with amino acids, not only improves stability, but also increases blood sugar lowering and blood fat lowering activity.
  • the polypeptide of the present invention and the composition thereof and the amino acid are resistant to pepsin and trypsin, so that they can be administered orally, intravenously, intramuscularly or subcutaneously, and are rare in all four modes of administration.
  • blood lipid-lowering drugs flexible in the way of administration, and also have the activity of protecting and repairing the function of pancreatic ⁇ -cells, and will not cause hypoglycemia and insulin resistance, no drug side effects, convenient and safe medication, and stable storage for a long time. It can effectively prevent and treat type 2 diabetes and hyperlipidemia.
  • Figure 1 HPLC chromatogram of a polypeptide of the invention extracted from a leguminous plant
  • Example 1 Extracting a polypeptide of the invention from a legume
  • the polypeptide of the present invention can be extracted from the seeds or processing by-products of legumes.
  • the specific extraction process is as follows:
  • the above-mentioned decolorized and degreased soybean meal is mixed with 100 liters of n-butanol, homogenized for 30 minutes, and the working temperature of the freezer is controlled at 10 ° C, then transferred to a jacketed extraction tank, stirred at 10 ° C for 24 hours, and centrifuged to obtain The supernatant was washed with n-butanol, and the supernatant was collected and combined.
  • the supernatant was concentrated by distillation under reduced pressure at 50 ° C for 10 times, and the concentrate was freeze-dried to obtain 95.6 g of a crude polypeptide.
  • the above crude polypeptide was separated and purified by reverse phase liquid chromatography.
  • the appropriate amount of the crude polypeptide was dissolved in chromatography A (water + 0.1% trifluoroacetic acid), filtered through a 0.22 micron filter, and the filtrate was loaded.
  • SEQ ID No. 1 ASCNGVCSPFEMPPCGTSACRCIPVGLVVGYCRNPSG
  • SEQ ID No. 2 ASCNGVCSPFEM ox PPCGTSACRCIPVGLVVGYCRNPSG
  • SEQ ID No. 3 ASCNGVCSPFEMPPCGTSACRCIPVGLVIGYCRNPSG
  • SEQ ID No. 4 ASCNGVCSPFEM ox PPCGTSACRCIPVGLVIGYCRNPSG
  • SEQ ID No. 5 ASCNGVCSPFEMPPCGSSACRCIPVGLLIGYCRNPSG
  • SEQ ID No. 7 ASCNGVCSPFEMPPCGTSACRCIPVGLFIGYCRNPSG
  • SEQ ID No. 8 ASCNGVCSPFEM ox PPCGTSACRCIPVGLFIGYCRNPSG
  • SEQ ID No. 9 ASCNGVCSPFEMPPCGTSACRCIPYGLFIGYCRNPSG
  • SEQ ID No. 10 ASCNGVCSPFDIPPCGSPLCRCIPVGLVIGKCRNPYG,
  • SEQ ID No. 12 VSCNGVCSPFDIPPCGSPLCRCIPAGLVIGKCRNPYG,
  • SEQ ID No. 72 ASCNGVCSPFEMPPCGSSACRCIPVGLVVGYCRHPSG.
  • polypeptide of the present invention can also be synthesized by a solid phase polypeptide synthesis method, and the specific process is as follows:
  • the groups used to protect the amino acid side chain are: tert-butyl with Ser, Thr and Tyr (tBu) ), Asp, Glu with tert-butyl ester group, Asn, Gln and Cys with trityl (Trt), Arg with 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonate Acyl (Pbf), Lys with 2-chlorobenzyloxycarbonyl.
  • the synthesis scale is 0.5 mmol, the protected amino acid and the condensing agent are 10 times in excess, and the process is automatically completed after the setting.
  • the resin was washed three times with DCM, vacuum-dried, and a mixture of TFA/TIS/H 2 O/Phenol (the addition of phenol to avoid oxidation of cysteine to form a disulfide bond) was added to the peptide solid phase reactor.
  • polypeptide having the following amino acid sequence is synthesized by the above solid phase polypeptide synthesis method:
  • SEQ ID No. 1 ASCNGVCSPFEMPPCGTSACRCIPVGLVVGYCRNPSG
  • SEQ ID No. 13 ASCNGVCSPFDIPPCGTPLCRCIPVGLVIGNCRNPYG,
  • SEQ ID No. 17 ASCNGVCSPFDMPPCGTSACRCIPAGLFIGKCRNPYG,
  • SEQ ID No. 21 ASCNGVCSPFEIPPCGSPLCRCIPVGLVIGNCRNPYG,
  • SEQ ID No. 26 ASCNGVCSPFEMPPCGSSACRCIPVGLVVGYCRNPSG
  • SEQ ID No. 36 ISCNGVCSPFDIPPCGTPLCRCIPAGLFVGKCRNPYG,
  • SEQ ID No. 61 VSCNGVCSPFDMPPCGTSACRCIPAGLFIGYCRNPSG,
  • SEQ ID No. 68 VSCNGVCSPFEMPPCGSSACRCIPYGLVVGNCRNPSG.
  • Peptide the polypeptide of SEQ ID No. 7 in Example 1, the average molecular weight is 3789, the amino acid sequence is ASCNGVCSPFEMPPCGTSACRCIPVGLFIGYCRNPSG (see Fig. 2), the HPLC purity is ⁇ 98%, and the solution is prepared by using physiological saline to prepare a solution of 1 mg/ml;
  • Kunming mice 40 health and epidemic prevention stations in Hubei province, weighing 20 ⁇ 2g, male;
  • mice were fasted for 4 hours (cannot be forbidden) and were randomly divided into 4 groups, 10 in each group, each of which was labeled with picric acid, and blood glucose was measured by tail vein blood.
  • the control group was given a saline injection of 0.05 ml/10 g of the tail vein of the rats once.
  • Group A high-dose hypoglycemic group of the peptide was injected with 1 mg/ml of the peptide once per 0.10 ml/10 g of the tail vein.
  • Group B dose in the peptide
  • group C low-dose hypoglycemic group of the polypeptide
  • Blood glucose changes were measured by blood glucose meter at 15 minutes, 30 minutes, 45 minutes, and 60 minutes after injection. The blood glucose level was mmol/L.
  • Peptide the polypeptide of SEQ ID No. 8 in Example 1, the average molecular weight of 3805, the amino acid sequence is ASCNGVCSPFEM ox PPCGTSACRCIPVGLFIGYCRNPSG (see FIG. 3), the HPLC purity is ⁇ 98%, and the solution is prepared by using physiological saline to prepare a solution of 1 mg/ml;
  • Kunming mice 40 health and epidemic prevention stations in Hubei province, weighing 20 ⁇ 2g, male;
  • mice were fasted for 4 hours (cannot be forbidden) and were randomly divided into 4 groups, 10 in each group, each of which was stained with picric acid, and blood glucose was measured by tail vein blood.
  • group A high-dose hypoglycemic group of polypeptide
  • Group B dose hypoglycemic in polypeptide
  • group C low-dose hypoglycemic group of polypeptide
  • Blood glucose changes were measured by blood glucose meter at 15 minutes, 30 minutes, 45 minutes, and 60 minutes after injection. The blood glucose level was mmol/L.
  • Polypeptide the polypeptide of SEQ ID No. 72 in Example 1, having an average molecular weight of 3736 and an amino acid sequence of ASCNGVCSPFEMPPCGSSACRCIPVGLVVGYCRHPSG, HPLC purity ⁇ 98%, prepared by using a physiological saline solution to prepare a solution of 1 mg/ml;
  • the polypeptide of SEQ ID No. 26 in Example 2 has an average molecular weight of 3716 and an amino acid sequence of ASCNGVCSPFEMPPCGSSACRCIPVGLVVGYCRNPSG, HPLC purity ⁇ 98%, and is prepared by using a physiological saline solution to prepare a solution of 1 mg/ml;
  • Kunming mice 30 health and epidemic prevention stations in Hubei province, weighing 20 ⁇ 2g, male;
  • mice were fasted for 4 hours (not allowed for water) and were randomly divided into 3 groups, 10 in each group, each of which was labeled with picric acid, and blood glucose was measured by tail vein blood.
  • the control group was given a subcutaneous injection of 0.05 ml/10 g of physiological saline once a day.
  • Group A polypeptide of SEQ ID No. 72
  • group B SEQ ID No.
  • the polypeptide of 26 was subcutaneously injected with 1 mg/ml polypeptide once per 0.05 ml/10 g of the back.
  • Blood glucose changes were measured by blood glucose meter at 15 minutes, 30 minutes, 45 minutes, and 60 minutes after injection. The blood glucose level was mmol/L.
  • Subcutaneous injection of a peptide having an amino acid sequence of ASCNGVCSPFEMPPCGSSACRCIPVGLVVGYCRNPSG can lower blood glucose and have hypoglycemic activity in mice.
  • Subcutaneous injection of a peptide having an amino acid sequence of ASCNGVCSPFEMPPCGSSACRCIPVGLVVGYCRHPSG increased blood glucose in mice, and had the opposite effect to the former.
  • the structural difference between the two is only reflected in the amino acid at position 34, the former being the neutral amino acid-asparagine (Asn) and the latter being the basic amino acid-histidine (His).
  • the possible reason for the difference in biological activity between the two is that the latter is highly hydrophilic, which is confirmed by the shortest retention time of C18 reverse phase chromatography.
  • the polypeptide of SEQ ID No. 72 is the peak No. 1 in Figure 1, and the retention time is 16.9 min.
  • histidine is protonated and becomes more hydrophilic, thereby changing the spatial conformation of the polypeptide and the manner of binding to the protein, thereby not having hypoglycemic activity.
  • the amino acid at position 34 of the polypeptide is a key site affecting the hypoglycemic activity, and if it is H (histidine), the corresponding polypeptide has no hypoglycemic activity.
  • Example 6 Comparative experiment of hypoglycemic effect of natural extract polypeptide and solid phase synthetic polypeptide of the present invention injected subcutaneously into normal mice
  • Peptide A the polypeptide of SEQ ID No. 1 in Example 1, having an average molecular weight of 3727, an amino acid sequence of ASCNGVCSPFEMPPCGTSACRCIPVGLVVGYCRNPSG, HPLC purity ⁇ 98%, and preparing a solution of 1 mg/ml with physiological saline for use;
  • Peptide B the polypeptide of SEQ ID No. 1 in Example 2, having an average molecular weight of 3727, an amino acid sequence of ASCNGVCSPFEMPPCGTSACRCIPVGLVVGYCRNPSG, HPLC purity ⁇ 98%, and preparing a solution of 1 mg/ml with physiological saline for use;
  • Kunming mice 30 health and epidemic prevention stations in Hubei province, weighing 20 ⁇ 2g, male;
  • mice were fasted for 4 hours (not allowed for water) and were randomly divided into 3 groups, 10 in each group, each of which was labeled with picric acid, and blood glucose was measured by tail vein blood.
  • the control group was given a subcutaneous injection of 0.05 ml/10 g of normal saline once a day.
  • Group A polypeptide A
  • group B polypeptide B
  • a 1 mg/ml polypeptide was injected subcutaneously once.
  • Blood glucose changes were measured by blood glucose meter at 15 minutes, 30 minutes, 45 minutes, and 60 minutes after injection. The blood glucose level was mmol/L.
  • Example 7 Comparative experiment of hypoglycemic effect of the polypeptide of the present invention by tail vein injection, subcutaneous injection and intramuscular injection of normal mice
  • Peptide the polypeptide of SEQ ID No. 3 in Example 1, the average molecular weight of 3741, the amino acid sequence of ASCNGVCSPFEMPPCGTSACRCIPVGLVIGYCRNPSG (see Figure 4), HPLC purity ⁇ 98%, formulated with physiological saline to prepare a solution of 1 mg / ml;
  • Kunming mice 40 health and epidemic prevention stations in Hubei province, weighing 20 ⁇ 2g, male;
  • mice were fasted for 4 hours (cannot be forbidden) and were randomly divided into 4 groups, 10 in each group, each of which was labeled with picric acid, and blood glucose was measured by tail vein blood.
  • 0.05 ml/10 g of normal saline was administered subcutaneously for 1 time.
  • Group A was injected with 1 mg/ml of peptide in the tail vein of 0.05 ml/10 g
  • group B was injected subcutaneously with 1 mg/ml of peptide in the back of 0.05 ml/10 g.
  • group C was injected with 1 mg/ml polypeptide once per 0.05 ml/10 g of the lateral thigh muscle.
  • Blood glucose changes were measured by blood glucose meter at 15 minutes, 30 minutes, 45 minutes, and 60 minutes after injection. The blood glucose level was mmol/L.
  • the polypeptide has significant hypoglycemic effect in three modes of administration (intravenous, subcutaneous and intramuscular injection), and there is no significant difference in the hypoglycemic effect. Since the subcutaneous injection operation is convenient, subcutaneous injection can be used as a screening method for hypoglycemic activity screening of the polypeptide of the present invention and its composition.
  • Example 8 Subcutaneous injection of the polypeptide of the present invention for hypoglycemic effect test of normal mice
  • Polypeptide 12 of the polypeptides of SEQ ID No. 13, 17, 21, 26, 36, 38, 44, 50, 52, 61, 63, 68 in Example 2, HPLC purity ⁇ 98%, the above 12
  • the polypeptide is prepared into a 1 mg/ml solution with physiological saline, respectively;
  • rats were randomly divided into 13 groups, 10 in each group, each of which was labeled with picric acid, and blood glucose was measured by tail vein blood.
  • 0.05 ml/10 g of physiological saline was administered subcutaneously once, and the other groups were subcutaneously injected with 1 mg/ml 12 polypeptides once per 0.05 ml/10 g.
  • Blood glucose changes were measured by blood glucose meter at 15 minutes, 30 minutes, 45 minutes, and 60 minutes after injection. The blood glucose level was mmol/L.
  • X 1 is A, V, I, and X 2 X 3 is EI, EM, DI, DM X 4 X 5 X 6 is SPA, SPL, SSA, SSL, TPA, TPL, TSA, TSL, X 7 is A, V, Y, and X 8 X 9 is FI, LI, VI, FV, LV, VV, X 10 is K, N, and Y, and X 11 is S and Y, and all of the polypeptides arranged in this combination have a clear hypoglycemic activity. Therefore, polypeptides having the above general structure have hypoglycemic activity.
  • Example 9 Comparative experiment of hypoglycemic effect of the polypeptide of the present invention and insulin in the tail vein of normal mice
  • Peptide the polypeptide of SEQ ID No. 11 in Example 1, the average molecular weight is 3788, the amino acid sequence is ISCNGVCSPFDIPPCGSPLCRCIPAGLVIGNCRNPYG, the HPLC purity is ⁇ 98%, and a solution of 2 mg/ml is prepared by using physiological saline;
  • Kunming mice 30 health and epidemic prevention stations in Hubei province, weighing 20 ⁇ 2g, male;
  • mice were fasted for 4 hours (not allowed for water) and were randomly divided into 3 groups, 10 in each group, each of which was labeled with picric acid, and blood glucose was measured by tail vein blood.
  • the control group was given a saline injection of 0.05 ml/10 g once in the tail vein.
  • Group A polypeptide hypoglycemic group
  • group B insulin hypoglycemic control group
  • 0.05 ml/10 g of tail vein was injected with 0.5 U/ml insulin once.
  • Blood glucose changes were measured by blood glucose meter at 15 minutes, 30 minutes, 45 minutes, and 60 minutes after injection. The blood glucose level was mmol/L.
  • the normal mouse tail vein injection of the polypeptide of the present invention with the sequence of SEQ ID No. 11 can be hypoglycemic as the insulin injection, and for the mouse, the injection of the polypeptide of 10 mg/kg body weight is Injection of 2.5 U/kg body weight of insulin, the two hypoglycemic effects are equivalent. Therefore, the peptide has a definite hypoglycemic activity.
  • Peptide the polypeptide of SEQ ID No. 10 in Example 1, the average molecular weight is 3788, the amino acid sequence is ASCNGVCSPFDIPPCGSPLCRCIPVGLVIGKCRNPYG (see Figure 5), the HPLC purity is ⁇ 98%, and the solution of 1 mg/ml is prepared by using physiological saline and 10% glucose solution, respectively. spare;
  • Kunming mice 30 health and epidemic prevention stations in Hubei province, weighing 20 ⁇ 2g, male;
  • mice were fasted for 4 hours (not allowed for water) and were randomly divided into 3 groups, 10 in each group, each of which was labeled with picric acid, and blood glucose was measured by tail vein blood.
  • Group A was given 10% glucose solution 0.10ml/10g by gavage
  • group B polypeptide group
  • group C polypeptide + glucose group
  • group C was administered with 0.10ml/10g. 1 mg/ml polypeptide glucose (10%) solution.
  • Blood glucose changes were measured by blood glucose meter at 15 minutes, 30 minutes, 45 minutes, and 60 minutes after intragastric administration. The blood glucose level was mmol/L.
  • Example 11 Subcutaneous injection of hypoglycemic activity of a polypeptide mixture of the present invention in normal mice
  • polypeptide mixture A the polypeptide of SEQ ID No. 3 in Example 1 and the polypeptide of SEQ ID No. 12 in Example 1 are uniformly mixed in a ratio of 1:1 by mass to obtain a polypeptide mixture A;
  • the polypeptide mixture B the polypeptide of SEQ ID No. 1 in Example 1 and the polypeptide of SEQ ID No. 9 in Example 1 are uniformly mixed in a ratio of mass ratio of 8:1 to obtain a polypeptide mixture B;
  • polypeptide mixture C the polypeptide of SEQ ID No. 36 in Example 2 and the polypeptide of SEQ ID No. 68 in Example 2 are uniformly mixed at a mass ratio of 1:6 to obtain a polypeptide mixture C;
  • Peptide mixture D The peak between 17.5 and 25 min in Example 1 was collected and lyophilized to obtain a polypeptide mixture D, wherein the total content of 12 polypeptides of SEQ ID No. 1 - SEQ ID No. 12 was ⁇ 98%;
  • mice were fasted for 4 hours (not allowed to drink water) and were randomly divided into 5 groups, 10 in each group, each of which was labeled with picric acid, and blood glucose was measured by tail vein blood.
  • the control group was given a subcutaneous injection of 0.05 ml/10 g of normal saline once a day.
  • Group A polypeptide mixture A
  • group B polypeptide mixture B
  • polypeptide of the present invention in any proportion of the composition, also has excellent hypoglycemic activity, and the compounding does not affect the hypoglycemic activity of the polypeptide.
  • the naturally extracted polypeptide mixture also has significant hypoglycemic activity, which means that the mixture of polypeptides extracted from the leguminous plants (collections of 17.5 to 25 min in HPLC) can be directly processed without further purification. It is used in hypoglycemic drugs, which greatly increases the yield and reduces the production cost.
  • Example 12 Type II Diabetes Mice Model Mice Intravenous Injection of Polypeptide Mixtures and Insulin Hypoglycemic Effect Comparison Experiment
  • Peptide The peak between 17.5 and 25 min in Example 1 was collected, and lyophilized, and the obtained polypeptide mixture was prepared into a 2 mg/ml solution with physiological saline for use;
  • Type II diabetes model mice KK rats, Beijing Huakangkang Biotechnology Co., Ltd., 30, weighing 25 ⁇ 2.7g, male, high-fat and high-sugar feed for 1 month;
  • the above-mentioned type 2 diabetes model mice were randomly divided into 3 groups after fasting for 4 hours (not allowed for water), and each group was 10, each of which was labeled with picric acid, and blood glucose was measured by tail vein blood.
  • the model control group was given normal saline 0.05ml/10g tail vein injection once
  • group A peptide hypoglycemic group
  • group B insulin hypoglycemic control group
  • group B 0.05 Mol/10g was injected intravenously with 0.5 U/ml insulin once.
  • Blood glucose changes were measured by blood glucose meter at 15 minutes, 30 minutes, 45 minutes, and 60 minutes after injection. The blood glucose level was mmol/L.
  • intravenous injection of the polypeptide mixture can lower the blood glucose of the type 2 diabetes model mouse, has significant hypoglycemic activity, and the effect is comparable to that of insulin, but does not cause hypoglycemia in the type II diabetes model mouse.
  • Example 13 Type II diabetes model mice subcutaneous injection of the polypeptide mixture of the present invention and insulin hypoglycemic effect control experiment
  • Peptide The peak between 17.5 and 25 min in Example 1 was collected, and lyophilized, and the obtained polypeptide mixture was prepared into a 2 mg/ml solution with physiological saline for use;
  • Type II diabetes model mice KK rats, Beijing Huakangkang Biotechnology Co., Ltd., 30, weighing 25 ⁇ 2.7g, male, high-fat and high-sugar feed for 1 month;
  • the above-mentioned type 2 diabetes model mice were randomly divided into 3 groups after fasting for 4 hours (not allowed for water), and each group was 10, each of which was labeled with picric acid, and blood glucose was measured by tail vein blood.
  • the model control group was given subcutaneous injection of 0.05ml/10g of normal saline in the back, group A (polypeptide hypoglycemic group) was injected subcutaneously with 2mg/ml peptide once in 0.05ml/10g, and group B (insulin hypoglycemic control group) was 0.05.
  • Mol/10g was injected subcutaneously with 0.5 U/ml insulin once a day. Blood glucose changes were measured by blood glucose meter at 15 minutes, 30 minutes, 45 minutes, and 60 minutes after injection. The blood glucose level was mmol/L.
  • subcutaneous injection of the polypeptide mixture can reduce the blood glucose of the type 2 diabetes model mouse, has significant hypoglycemic activity, and the effect is equivalent to insulin, but does not cause hypoglycemia in the type II diabetes model mouse.
  • Example 14 Intramuscular injection of a polypeptide mixture of the invention and a hypoglycemic effect of insulin in a type II diabetes model mouse
  • Peptide The peak between 17.5 and 25 min in Example 1 was collected, and lyophilized, and the obtained polypeptide mixture was prepared into a 2 mg/ml solution with physiological saline for use;
  • Type II diabetes model mice KK rats, Beijing Huakangkang Biotechnology Co., Ltd., 30, weighing 25 ⁇ 2.7g, male, high-fat and high-sugar feed for 1 month;
  • mice were randomly divided into 3 groups, 10 in each group, each of which was labeled with picric acid, and blood glucose was measured by tail vein blood.
  • the model control group was given a saline injection of 0.05 ml/10 g of the lateral thigh muscle once, and a group A (peptide hypoglycemic group) was injected with 2 mg/ml polypeptide once per 0.05 ml/10 g of the lateral thigh muscle.
  • Group B insulin hypoglycemic control group
  • Blood glucose changes were measured by blood glucose meter at 15 minutes, 30 minutes, 45 minutes, and 60 minutes after injection. The blood glucose level was mmol/L.
  • the intramuscular injection of the polypeptide mixture of the present invention can reduce the blood glucose of the type 2 diabetes model mouse, has significant hypoglycemic activity, and has the same effect as insulin, but does not cause hypoglycemia in the type II diabetes model mouse.
  • Polypeptide a peak between 15 and 25 minutes in Example 1 was collected and lyophilized to obtain a composition of the polypeptide;
  • Albumin serum albumin (bovine) standard
  • High Performance Liquid Chromatograph Agilent 1260 High Performance Liquid Chromatograph, Wateres C18 Reversed Phase Column (4.6 x 250 mm, 300 SB, 5 ⁇ m);
  • the polypeptide was subjected to enzymatic hydrolysis using artificial gastric juice and artificial intestinal juice to verify whether the polypeptide was resistant to enzymatic hydrolysis of the digestive enzyme, and the control experiment was performed using albumin.
  • Preparation of artificial gastric juice Dilute hydrochloric acid at a concentration of 1 mol/L, diluted with water, and adjusted to pH 1.5. 1 g of pepsin was added to each 100 ml of the solution, mixed, and filtered for use with a 0.22 um sterile filter.
  • Preparation of artificial intestinal juice Dissolve 6.8 g of potassium dihydrogen phosphate and 500 ml of water, and adjust the pH to 6.8 with 0.4% (w/w) NaOH to a volume of 1 L. 1 g of trypsin was added to each 100 ml of the solution, mixed, and filtered for use with a 0.22 um sterile filter.
  • each polypeptide solution (10 mg/ml) was added to artificial gastric juice (1 ml) and intestinal fluid (1 ml), and the mixture was incubated at 37 ° C for 2 hours in a water bath.
  • albumin dry powder 1 mg was added to artificial gastric juice (1 ml) and intestinal fluid (1 ml), respectively, and incubated for 2 hours at 37 ° C in a water bath.
  • Chromatographic conditions mobile phase, water (containing 0.1% TFA) + acetonitrile (containing 0.1% TFA), acetonitrile eluted from 15% to 50% gradient for 25 min, flow rate 1 ml / min, detection wavelength 214 nm, injection volume 20 ul.
  • the HPLC diagram of the enzymatic hydrolysis of the peptide is the HPLC detection result of the peptide reference substance; the middle is the HPLC detection result of the polypeptide by artificial gastric juice for 2 hours; the bottom is the HPLC detection result of the polypeptide by artificial intestinal juice for 2 hours. .
  • HPLC analysis of white proteolysis (see Figure 7) at the top is the HPLC detection of albumin reference; in the middle is albumin by artificial gastric juice for 2 hours HPLC detection; the bottom is albumin by artificial intestinal hydrolysis 2 hours HPLC Test results.
  • neither the artificial gastric juice nor the intestinal juice can digest the polypeptide of the present invention, but for albumin, gastric juice and intestinal fluid, it can be completely digested, indicating that the polypeptide of the present invention is resistant to digestion by gastrointestinal digestive enzymes. Can be taken orally.
  • Example 16 Type II diabetes model mice were administered with the polypeptide mixture of the present invention and the hypoglycemic effect of metformin
  • Peptide The peak between 17.5 and 25 min in Example 1 was collected, and lyophilized, and the obtained polypeptide mixture was prepared into a 2 mg/ml solution with physiological saline for use;
  • Metformin Hydrochloride Schoua Stop Metformin Hydrochloride Tablets, 0.5g/tablet, Sino-US Shanghai Squibb Pharmaceutical Co., Ltd., prepared with physiological saline to prepare a solution with an effective content of 20mg/ml;
  • Type II diabetes model mice KK rats, Beijing Huakangkang Biotechnology Co., Ltd., 30, weighing 25 ⁇ 2.7g, male, high-fat and high-sugar feed for 1 month;
  • mice were randomly divided into 3 groups, 10 in each group, each of which was labeled with picric acid, and blood glucose was measured by tail vein blood.
  • the model control group was given normal saline 0.05ml/10g once a day
  • group A polypeptide hypoglycemic group
  • group B metalformin control group
  • blood glucose changes were measured by blood glucose meter at 15 minutes, 30 minutes, 45 minutes, and 60 minutes after intragastric administration.
  • the blood glucose level was mmol/L.
  • the peptide mixture of the present invention can reduce the blood sugar of the type II diabetes model mouse, has significant hypoglycemic activity, does not cause hypoglycemia in the type II diabetes model mouse, and the effect is better than metformin.
  • Polypeptide a peak between 15 and 25 minutes in Example 1 was collected and lyophilized to obtain a composition of the polypeptide;
  • High Performance Liquid Chromatograph Agilent 1260 High Performance Liquid Chromatograph, Wateres C18 Reversed Phase Column (4.6 x 250 mm, 300 SB, 5 ⁇ m)
  • the HPLC diagram of the stability of the peptide is the result of three HPLC tests of the stability test of the polypeptide.
  • the top is the initial control.
  • the integral area of the peptide mixture is about 3036.94.
  • the middle is the HPLC detection result after one week of acceleration, and the retention time of the main peak. Without any change, the integral area of the polypeptide mixture was about 2992.66; the bottom was the HPLC detection result after two weeks of acceleration, and the retention time of the main peak did not change, and the integrated area of the polypeptide mixture was about 3011.84. It indicates that the polypeptide is stable during the accelerated test period and can be stored at room temperature, and is suitable for development as a hypoglycemic drug.
  • Example 18 Type II diabetes model mice were injected with the polypeptide mixture of the present invention for therapeutic effect test
  • Peptide The peak between 17.5 and 25 min in Example 1 was collected, lyophilized to obtain a polypeptide mixture, and a solution of 1 mg/ml was prepared by using physiological saline to obtain a peptide mixture injection;
  • Type II diabetes model mice KK rats, Beijing Huakangkang Biotechnology Co., Ltd., 40, weighing 25 ⁇ 2.7g, male, high-fat and high-sugar feed for 1 month;
  • mice were randomly divided into 4 groups, 10 in each group, each of which was labeled with picric acid, and blood glucose was measured by tail vein blood.
  • 0.05 ml/10 g of normal saline was given to the tail vein.
  • Group A intravenous group
  • group B subcutaneous injection group
  • /10 g of the subcutaneous injection of the polypeptide content of 1 mg / ml was injected subcutaneously
  • the injection group of the polypeptide content of 1 mg / ml was injected into the lateral muscle of the thigh in group C (intramuscular injection group).
  • intravenous, subcutaneous injection, intramuscular injection of the polypeptide mixture of the present invention has a clear therapeutic effect on type II diabetes model mice, and there is no significant difference in the hypoglycemic effect of the three administration modes.
  • Example 19 Type II Diabetic Model Mice Therapy of the polypeptide mixture of the present invention and metformin treatment effect, insulin tolerance, oral glucose tolerance comparison and repair of pancreatic ⁇ -cell experiment
  • Peptide The peak between 17.5 and 25 min in Example 1 was collected and lyophilized to obtain a polypeptide mixture
  • Metformin Hydrochloride Schoua Hydrochloride Metformin Tablets, 0.5g/tablet, Sino-US Shanghai Squibb Pharmaceutical Co., Ltd.;
  • Type II diabetes model mice KK rats, Beijing Huakangkang Biotechnology Co., Ltd., 60, weighing 25 ⁇ 2.7g, male, high-fat and high-sugar feed for 1 month;
  • Normal control mice Kunming mice, 20 health and epidemic prevention stations in Hubei province, weighing 20 ⁇ 2g, male, normal feed;
  • Metformin hydrochloride is formulated with physiological saline to prepare a solution having an effective content of 20 mg/ml;
  • the above-mentioned diabetic model rats were randomly divided into 3 groups, 20 in each group, and the normal control mice were a group and 20 in a group. Each of them was labeled with picric acid, and the tail vein blood was taken to determine the initial blood glucose.
  • the model control group and the normal control group were given normal saline 0.05ml/10g once a day for 4 weeks, and group A (metformin treatment group) was given 0.05ml/10g per day for metformin once a day.
  • the drug was administered for 4 weeks;
  • Group B polypeptide treatment group
  • the tail vein blood was collected weekly and the blood glucose level was measured by a blood glucose meter.
  • the blood glucose unit was mmol/L.
  • Insulin tolerance test One hour after the last administration, the diabetic model control group, the metformin treatment group, the peptide treatment group and the normal control group were randomly selected from each group to inject 0.5 U/ml insulin once per 0.05 ml/10 g.
  • the tail vein blood was collected at 0 hours, 0.5 hours, 1 hour, 1.5 hours, and 2 hours, and the blood glucose level was measured by a blood glucose meter, and the blood glucose unit was mmol/L.
  • mice After 1 hour of the last administration, the remaining diabetic model control group, metformin treatment group, peptide treatment group mice and normal control group mice (10 mice in each group) were selected as 2.5g/Kg. Glucose was administered to the mice, and the tail vein blood was collected at 0 hours, 0.5 hours, 1 hour, 1.5 hours, and 2 hours, and the blood glucose level was measured by a blood glucose meter, and the blood glucose unit was mmol/L.
  • mice After 2.5 weeks of treatment, all mice were sacrificed, pancreatic tissue was weighed, and pancreas HE staining and immunohistochemistry were performed.
  • Oral administration of a hypoglycemic agent formulated with a polypeptide mixture can lower the blood glucose level of a type 2 diabetes model mouse and restore it to a normal blood sugar level, so that the hypoglycemic agent can be used for the treatment of type 2 diabetes, and the mixture of the intragastric polypeptide can increase the type II. Insulin tolerance and glucose tolerance in diabetic model mice. At the same time, the weight of the pancreas of the type II diabetes model mice taking the hypoglycemic agent increased, and FIG. 9 showed that the pancreatic ⁇ cells returned to normal. Therefore, the hypoglycemic drug also has the effect of repairing pancreatic ⁇ cells of the type II diabetes model mouse.
  • Example 1 The peak between 17.5 and 25 min in Example 1 was collected and lyophilized to obtain a polypeptide mixture in which the total content of 12 polypeptides of SEQ ID No. 1 - SEQ ID No. 12 was ⁇ 98%, and the polypeptide mixture was abbreviated as polypeptide O.
  • a liquid composition containing polypeptide O was formulated according to Formulation 1 shown in Table 19 below:
  • a liquid composition comprising polypeptide O and an amino acid is formulated according to Formulation 2 shown in Table 20 below:
  • Example 21 The liquid composition of the polypeptide and amino acid of the present invention has hypoglycemic activity
  • Polypeptide Composition A The liquid composition prepared according to Formulation 1 of Example 20 was stored at 40 ⁇ 2 ° C / 75 ⁇ 5% RH for 6 months;
  • Polypeptide Composition B a liquid composition prepared according to Formulation 2 of Example 20, stored at 40 ⁇ 2 ° C / 75 ⁇ 5% RH for 6 months;
  • Polypeptide Composition C a liquid composition formulated on-site according to Formulation 1 of Example 20;
  • Test animals Kunming mice, male, weighing 20 ⁇ 2g, 40, purchased from Hubei Provincial Health and Epidemic Prevention Station;
  • Blood glucose tester and test strip Roche vitality Blood glucose meter and test strips.
  • mice were randomly divided into 4 groups, 10 in each group, each of which was labeled with picric acid, and blood glucose was measured by tail vein blood.
  • 0.1 ml/10 g of normal saline was administered intravenously once, and the other three groups were injected with the compositions A, B, and C once at a dose of 0.1 ml/10 g.
  • Blood glucose changes were measured by blood glucose meter at 15 minutes, 30 minutes, 45 minutes, and 60 minutes after injection. The blood glucose level was mmol/L.
  • the solution containing the polypeptide O was prepared according to the formula 3 shown in the following Table 22, dispensed in a vial, vacuum freeze-dried, and stored at 40 ⁇ 2° C./75 ⁇ 5% RH after packaging:
  • the solution containing the polypeptide O and the amino acid was prepared according to the formula 4 shown in the following Table 23, dispensed in a vial, vacuum freeze-dried, and stored at 40 ⁇ 2° C./75 ⁇ 5% RH after packaging:
  • Peptide Composition D The solid composition prepared according to Formulation 3 in Example 22 was stored at 40 ⁇ 2° C./75 ⁇ 5% RH for 6 months, and was formulated into a solution having a polypeptide O content of 2 mg/ml with physiological saline. spare;
  • Polypeptide Composition E The solid composition prepared according to Formulation 4 in Example 22 was stored at 40 ⁇ 2° C./75 ⁇ 5% RH for 6 months, and was formulated into a solution having a polypeptide O content of 2 mg/ml with physiological saline. spare;
  • Peptide composition F a solid composition prepared in the same manner as in Formulation 2 of Example 22, and a solution having a polypeptide O content of 2 mg/ml was prepared by using physiological saline;
  • Metformin Chaua stopped metformin hydrochloride tablets, 0.5g/tablet, Sino-US Shanghai Squibb Pharmaceutical Co., Ltd., prepared with physiological saline to prepare a suspension with an effective content of 20mg/ml;
  • High fat diet 30% fat, 20% protein, 50% carbohydrate
  • Test animals C57BL/6, male, weighing 20 ⁇ 2g, 100, purchased from Hubei Provincial Health and Epidemic Prevention Station;
  • Blood glucose tester and test strip Roche vitality Blood glucose meter and test strips.
  • the normal control group was fed with normal feed.
  • the mice to be modeled were fed with high-fat diet, and intraperitoneal injection of streptozotocin (STZ) citrate buffer at a dose of 45 mg/kg. After the injection, fasting for 1.5 h, continuous A mouse model of diabetes was established by injection for 5 days. After 4 weeks of intraperitoneal injection of streptozotocin, fasting was performed at 8 o'clock in the previous night, and fasting blood glucose was measured at 8 o'clock in the morning.
  • STZ streptozotocin
  • the fasting blood glucose was selected at 10.0-20.0 mmol/L, accompanied by Diabetic mice with polydipsia and polyuria were randomly divided into 5 groups: model control group, metformin group, composition group D, composition group E, and composition group F, each group of 10, respectively, given by gavage Drugs: normal saline (100 ⁇ L each), metformin (0.05 ml/10 g), composition D (0.05 ml/10 g), composition E (0.05 ml/10 g), composition F (0.05 ml/10 g), given daily The drug was administered once and the dosing period was 4 weeks. Normal control mice were given normal saline (100 ⁇ L each). Diabetic mice were continuously fed with high-fat diet, free to drink water, normal normal mice fed normal feed, free access to water.
  • mice were collected weekly for tail vein blood.
  • the fasting blood glucose level was measured with a blood glucose meter, and the blood glucose unit was mmol/L.
  • composition D the hypoglycemic activity of the solid composition containing the polypeptide O after 6 months of storage
  • composition F the conventional dose of metformin (100 mg/kg).
  • composition E the solid composition of the invention comprising the polypeptide O and the amino acid
  • composition E can effectively control the blood sugar of the STZ combined with the high-fat and high-sugar diet of the type 2 diabetes model mouse, and the blood glucose of the mouse is not significantly different from the normal control group after four weeks of treatment.
  • the curative effect is remarkable, and the effect is better than composition F without added amino acid, which is more obviously superior to metformin. Therefore, DL-methionine not only enhances the stability of the polypeptide O, but also has a synergistic and synergistic effect on the hypoglycemic effect.
  • Example 24 Polypeptide of the Invention and Composition with Amino Acid Improve Glucose and Lipid Metabolism in Diabetic Mice
  • Peptide composition F a solid composition prepared in the same manner as in Formulation 2 of Example 22, and a solution having a polypeptide O content of 2 mg/ml was prepared by using physiological saline;
  • Polypeptide Composition E The solid composition prepared according to Formulation 4 in Example 22 was stored at 40 ⁇ 2° C./75 ⁇ 5% RH for 6 months, and was formulated into a solution having a polypeptide O content of 2 mg/ml with physiological saline. spare;
  • High fat diet 30% fat, 20% protein, 50% carbohydrate
  • Test animals BALB/c, male, weighing 20 ⁇ 2g, 80, purchased from Hubei Provincial Health and Epidemic Prevention Station;
  • Metformin Chaua stopped metformin hydrochloride tablets, 0.5g/tablet, Sino-US Shanghai Squibb Pharmaceutical Co., Ltd., prepared with physiological saline to prepare a suspension with an effective content of 200mg/ml;
  • Blood glucose tester and test strip Roche vitality Blood glucose meter and test paper;
  • Biochemical analyzer and kit Cholesterol (TC) kit, triglyceride (TG) kit, high-density lipoprotein cholesterol (HDL-C) kit purchased from Mindray Biomedical Electronics Co., Ltd., using Mindray Mindray automatic biochemistry Analyzer BS-220 detects.
  • TC Cholesterol
  • TG triglyceride
  • HDL-C high-density lipoprotein cholesterol
  • mice Seven-week-old male BALB/c mice were randomly divided into normal control group (10 rats) and to be modeled (70 rats) after 2 days of adaptive feeding.
  • the normal control group was fed with normal feed.
  • the mice to be modeled were fed with high-fat diet, and a streptozotocin (STZ) citrate buffer was intraperitoneally injected at a dose of 100 mg/kg to establish a diabetic mouse model.
  • STZ streptozotocin
  • mice After 3 days, the mice were fasted for 4 hours (water can not be forbidden), blood was taken from the tip of the tail to measure blood sugar, and diabetic mice with blood glucose greater than 11.0 mmol/L, accompanied by polydipsia and polyuria, were randomly divided into 4 groups: model The control group, the metformin group, the composition group E, and the composition group F, each group of 10, were administered with the corresponding drugs by intragastric administration: physiological saline (100 ⁇ L each), metformin (0.05 ml/10 g), and composition E ( 0.05 ml/10 g), composition F (0.05 ml/10 g), administered once a day for a period of 4 weeks.
  • Normal control mice were given normal saline (100 ⁇ L each). Diabetic mice were continuously fed with high-fat diet, free to drink water, normal normal mice fed normal feed, free access to water.
  • mice were collected weekly for tail vein blood.
  • the fasting blood glucose level was measured with a blood glucose meter, and the blood glucose unit was mmol/L.
  • * represents a significant difference (p ⁇ 0.05) and very significant (p ⁇ 0.01) compared with the model control group; ⁇ , ⁇ ⁇ represents that the composition E group compared with the composition F group, respectively, the difference Significant (p ⁇ 0.05) and very significant (p ⁇ 0.01).
  • the polypeptide of the present invention can significantly reduce blood lipids in a diabetic mouse model of pancreatic damage, and the polypeptide of the present invention and the composition of the amino acid are more effective than the polypeptide alone in reducing blood lipids in a diabetic mouse model of impaired pancreas, It exhibits a synergistic and synergistic effect of the combination of polypeptide and amino acid.
  • Example 25 Composition of Polypeptides and Amino Acids of the Invention for Hypolipidemic Activity
  • Polypeptide Composition E The solid composition prepared according to Formulation 4 in Example 22 was stored at 40 ⁇ 2° C./75 ⁇ 5% RH for 6 months, and was formulated into a solution having a polypeptide O content of 2 mg/ml with physiological saline. spare;
  • High fat diet 78.8% basic feed, 1% cholesterol, 10% egg yolk powder, 10% lard, 0.2% bile salt;
  • Test animals male SD rats, 50, weighing 200 ⁇ 20 g, purchased from Hubei Provincial Health and Epidemic Prevention Station;
  • Biochemical analyzer and kit Cholesterol (TC) kit, triglyceride (TG) kit, high-density lipoprotein cholesterol (HDL-C) kit purchased from Mindray Biomedical Electronics Co., Ltd., using Mindray Mindray automatic biochemistry Analyzer BS-220 detects.
  • TC Cholesterol
  • TG triglyceride
  • HDL-C high-density lipoprotein cholesterol
  • Rats were acclimated to the environment for 3 days, fed normal feed for 5 days, tailed blood was taken 16 h after fasting, centrifuged (3000 rpm), serum was taken, and serum TC, TG, HDL-C were measured.
  • the TC level they were randomly divided into 5 groups, 10 in each group, namely, three dose groups (10, 20, and 30 mg/kg), one high-fat diet control group, and one normal diet control group.
  • three dose groups were given high fat diet and given different doses of the solid composition of the polypeptide and amino acid of the present invention by intragastric administration, the high fat diet control group was given high fat diet and water, and the normal feed control group was given normal feed. And water.
  • the tail blood was taken after fasting for 16 hours, centrifuged (3000 rpm), serum was taken, and serum TC, TG, and HDL-C were measured.
  • Example 26 Oral administration of a composition of the polypeptide and amino acid of the present invention alone to improve blood glucose and glycated hemoglobin (HbA1c) in type 2 diabetic patients
  • composition of the polypeptide and amino acid of the present invention can be orally administered to effectively control blood sugar and normalize glycated hemoglobin.
  • Example 27 The combination of the polypeptide and amino acid of the present invention and metformin improves glycosylated hemoglobin (HbA1c) in type 2 diabetic patients
  • Example 28 The composition of the polypeptide and amino acid of the present invention is combined with metformin and insulin to improve blood sugar and glycated hemoglobin (HbA1c) in type 2 diabetic patients.
  • HbA1c glycated hemoglobin
  • This example shows that when the polypeptide and amino acid composition of the present invention is combined with metformin and insulin, it can effectively improve the blood sugar of patients who cannot effectively control blood sugar after using metformin and insulin, and normalize glycated hemoglobin, and can Reducing the amount of insulin in patients is superior to patients using only metformin and insulin.
  • the patient taking the polypeptide of the present invention and the amino acid composition is stopped after a period of time, and the amount of insulin is halved.
  • the fasting blood glucose and glycated hemoglobin can still be controlled in the normal range, indicating that the polypeptide and amino acid composition of the present invention has the function of repairing diabetes.
  • the function of the patient's impaired pancreas promotes its ability to restore insulin secretion, thus reversing type 2 diabetes.
  • Example 29 Composition of Polypeptides and Amino Acids of the Invention Improves Glucose and Lipid Metabolism in Type 2 Diabetic Patients
  • the solid composition prepared according to Formulation 4 in Example 22 was orally administered on January 5, 2018, orally, 3 times a day, 100 mg/time, without taking other hypoglycemic agents.
  • the fasting blood glucose was reduced to 8.32 mmol/L, the glycated hemoglobin was reduced to 7.7%, and the triglyceride (TG) was reduced to 2.6 mmol/L.
  • fasting blood glucose, glycated hemoglobin and triglyceride decreased by 43%, 27% and 42%, respectively, with significant changes.
  • polypeptides and amino acid compositions of the present invention significantly improve glycolipid metabolism in type 2 diabetic patients.

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Abstract

本发明提供了一种具有降血糖和降血脂活性的多肽及其在制备用于降低哺乳动物血糖和/或血脂，或者预防和/或治疗哺乳动物糖尿病和/或高脂血症的药物中的用途。本发明还提供了包含所述多肽的组合物。

Description

用于治疗代谢系统疾病的多肽及其组合物 技术领域
本发明属于生物医药领域，涉及一种多肽及其组合物，具体涉及一种用于治疗代谢系统疾病的多肽及其组合物。
背景技术
糖尿病是由于体内胰岛素分泌相对或绝对不足、胰岛素在靶细胞不能发挥正常生理作用和/或胰高血糖素分泌过多而引起的糖、蛋白及脂肪代谢紊乱的一种综合性代谢系统疾病，其基本特征是患者长期高血糖。
糖尿病的主要危害在于糖尿病并发症，长期患糖尿病会损害全身多个系统，特别是心血管系统，导致眼、肾、神经、心脏、血管等组织的慢性进行性病变，引起功能缺陷及衰竭，如心脑血管动脉硬化、视网膜及肾脏微血管病变、神经病变和下肢坏疽等。糖尿病性视网膜病变(DR)及糖尿病性肾病(DN)是糖尿病常见的微血管并发症。
世界卫生组织统计，糖尿病是失明、肾衰竭、心脏病发作、中风和下肢截肢的主要病因。目前，糖尿病在老年人中已成为除心脏病、癌症之外的第三大死亡原因。
治疗糖尿病的药物现在主要有胰岛素、口服降糖药物、肠促胰岛素等。现有的这些药物都存在比较明显的缺点，如二甲双胍类易导致乳酸血症，噻唑烷二酮类存在肝毒性以及骨质丢失等副作用，磺脲类药物长期服用易引起低血糖，而长期服用DD-Ⅳ抑制剂容易产生咳嗽，GLP-1易引起患者恶心及呕吐，胰岛素则存在不能口服、贮存及使用不方便、患者有低血糖反应、长期使用导致胰岛素抵抗及皮下脂肪萎缩等系列问题。
目前，临床上使用的降糖药物都存在各种缺陷，且无法治愈糖尿病，患者需要终身用药，因此，急需开发安全有效的降糖新药，尤其是用药方便的口服 药，以满足全球日益增多的糖尿病患者的需求。
同时，糖尿病是由于胰岛素分泌绝对不足或相对不足引起糖、脂肪、蛋白质三大代谢紊乱的一种代谢性疾病，糖尿病患者也常伴有高脂血症，表现为胆固醇、甘油三酯及低密度脂蛋白含量偏高，或高密度脂蛋白含量偏低。
糖尿病与高脂血症密切相关。一方面，胰岛素不足时，糖尿病患者体内脂酶活性也降低，血脂升高，而且，糖尿病本身除糖代谢紊乱外还伴随脂肪、蛋白质和水、电介质的紊乱，常有游离脂肪酸从脂肪库中释放出来，使血中甘油三酯及游离脂肪酸浓度增加，而且，Ⅱ型糖尿病人进食偏多，促使体内脂类合成增多，也导致血脂增加。另一方面，肥胖伴高血脂者，由于胰岛素受体数相对减少，从而产生胰岛素抵抗，反过来易诱发糖尿病。
高血脂是糖尿病的继发症，进而还易引起心、脑血管并发症。据统计，约40％的糖尿病患者存在高脂血症，糖尿病合并高血脂更容易导致脑中风、冠心病、肢体坏死、眼底病变、肾脏病变、神经病变等。因此，需要开发能够在降低糖尿病患者血糖的同时还降低血脂的降糖降脂新药。
发明内容
本发明提供一种多肽及其组合物，同时具有优良的降血糖及降血脂活性，耐胃蛋白酶及胰蛋白酶的酶解，从而既可注射给药，也可口服给药，同时还具有保护并修复胰腺β细胞功能的活性，且不会导致患者产生低血糖反应以及胰岛素抵抗，无任何毒副作用，用药方便安全，可长期稳定贮存，是一种新型降糖、降脂药物。
本发明的发明者研究发现，从豆科植物中提取出来的由多个氨基酸构成的天然多肽和与其结构类似的多肽具有优良的降糖活性，进一步研究发现此类多肽同时还具有优良的降血脂活性，最近研究还发现这些多肽在与某些氨基酸联合使用时能够取得更好的降糖和降血脂效果，从而完成了本发明。
根据本发明的一个方面，本发明涉及一种降低哺乳动物血糖和/或血脂的方 法，其特征在于，对其施用治疗有效量的如下述通式(I)所示序列的多肽中的一种、两种或多种的任意比例的混合物：
X 1SCNGVCSPFX 2X 3PPCGX 4X 5X 6CRCIPX 7GLX 8X 9GX 10CRNPX 11G(I)
其中，X 1-X 11彼此独立地选自A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、W、Y、M ox。
根据本发明的另一个方面，本发明涉及一种预防和/或治疗哺乳动物糖尿病和/或高脂血症的方法，其特征在于，对其施用治疗有效量的如通式(I)所示序列的多肽中的一种、两种或多种的任意比例的混合物。
根据本发明的另一个方面，本发明涉及一种降低哺乳动物血糖和/或血脂的方法，其特征在于，对其施用治疗有效量的如通式(I)所示序列的多肽中的一种、两种或多种的任意比例的混合物，和其他降糖药和/或降脂药。
根据本发明的另一个方面，本发明涉及一种预防和/或治疗哺乳动物糖尿病和/或高脂血症的方法，其特征在于，对其施用治疗有效量的如通式(I)所示序列的多肽中的一种、两种或多种的任意比例的混合物，和其他降糖药和/或降脂药。
根据本发明的另一个方面，本发明涉及如通式(I)所示序列的多肽中的一种、两种或多种的任意比例的混合物在制备用于降低哺乳动物血糖和/或血脂的药物、食品、保健食品或特殊医学用途配方食品中的用途。
根据本发明的另一个方面，本发明涉及如通式(I)所示序列的多肽中的一种、两种或多种的任意比例的混合物在制备用于预防和/或治疗哺乳动物糖尿病和/或高脂血症的药物中的用途。
根据本发明的另一个方面，本发明涉及如通式(I)所示序列的多肽中的一种、两种或多种的任意比例的混合物在制备用于和其他降糖药和/或降脂药联合降低哺乳动物血糖和/或血脂的药物、食品、保健食品或特殊医学用途配方食品中的用途。
根据本发明的另一个方面，本发明涉及如通式(I)所示序列的多肽中的一 种、两种或多种的任意比例的混合物在制备用于和其他降糖药和/或降脂药联合预防和/或治疗哺乳动物糖尿病和/或高脂血症的药物中的用途。
根据本发明的另一个方面，本发明涉及一种用于口服的药物组合物或制剂、食品、保健食品或特殊医学用途配方食品，其含有如通式(I)所示序列的多肽中的一种、两种或多种的任意比例的混合物，以及可药用载体。
根据本发明的另一个方面，本发明涉及一种用于静脉注射、肌肉注射或皮下注射的药物组合物或制剂，其含有如通式(I)所示序列的多肽中的一种、两种或多种的任意比例的混合物，以及可药用载体。
根据本发明的另一个方面，本发明涉及一种组合物，其含有如通式(I)所示序列的多肽中的一种、两种或多种的任意比例的混合物，和至少一种氨基酸。
根据本发明的另一个方面，本发明涉及一种降低哺乳动物血糖和/或血脂的的方法，其特征在于，对其施用治疗有效量的上述组合物。
根据本发明的另一个方面，本发明涉及一种预防和/或治疗哺乳动物糖尿病和/或高脂血症的方法，其特征在于，对其施用治疗有效量的上述组合物。
根据本发明的另一个方面，本发明涉及一种降低哺乳动物血糖和/或血脂的的方法，其特征在于，对其施用治疗有效量的上述组合物，和其他降糖药和/或降脂药。
根据本发明的另一个方面，本发明涉及一种预防和/或治疗哺乳动物糖尿病和/或高脂血症的方法，其特征在于，对其施用治疗有效量的上述组合物，和其他降糖药和/或降脂药。
根据本发明的另一个方面，本发明涉及上述组合物在制备用于降低哺乳动物血糖和/或血脂的药物、食品、保健食品或特殊医学用途配方食品中的用途。
根据本发明的另一个方面，本发明涉及上述组合物在制备用于预防和/或治疗哺乳动物糖尿病和/或高脂血症的药物中的用途。
根据本发明的另一个方面，本发明涉及上述组合物在制备用于和其他降糖药和/或降脂药联合降低哺乳动物血糖和/或血脂的药物、食品、保健食品或特殊 医学用途配方食品中的用途。
根据本发明的另一个方面，本发明涉及上述组合物在制备用于和其他降糖药和/或降脂药联合预防和/或治疗哺乳动物糖尿病和/或高脂血症的药物中的用途。
优选地，哺乳动物例如糖尿病患者在施用本发明的多肽或其组合物前糖化血红蛋白＞7.0％，优选＞7.5％，在施用本发明的多肽或其组合物后糖化血红蛋白＜7.5％，优选＜7.0％。
根据本发明的另一个方面，本发明涉及一种用于口服的药物制剂、食品、保健食品或特殊医学用途配方食品，其含有上述组合物以及可药用载体。
根据本发明的另一个方面，本发明涉及一种用于静脉注射、肌肉注射或皮下注射的药物组合物或制剂，其含有上述组合物以及可药用载体。
在优选的实施方案中，本发明的多肽具有通式(I)所示序列。优选地，X 1为疏水性氨基酸，选自A、L、V、I、P、F、M、W、M ox；X 2为酸性氨基酸，选自E、D；X 3为疏水性氨基酸，选自A、L、V、I、P、F、M、W、M ox；X 4为除半胱氨酸外的极性中性氨基酸，选自G、S、T、Y、N、Q；X 5为除半胱氨酸外的中性氨基酸，选自G、S、T、Y、N、Q、A、L、V、I、P、F、M、W；X 6为疏水性氨基酸，选自A、L、V、I、P、F、M、W、M ox；X 7为除半胱氨酸外的中性氨基酸，选自G、S、T、Y、N、Q、A、L、V、I、P、F、M、W；X 8为疏水性氨基酸，选自A、L、V、I、P、F、M、W、M ox；X 9为疏水性氨基酸，选自A、L、V、I、P、F、M、W、M ox；X 10选自Y、K、N；X 11为除半胱氨酸外的极性中性氨基酸，选自G、S、T、Y、N、Q。更优选地，X 1为A、I或V；X 2为E或D；X 3为M、M ox或I；X 4为S或T；X 5为S或P；X 6为A或L；X 7为V、A或Y；X 8为V、F或L；X 9为V或I；X 10为Y、K或N；X 11为S、T或Y。更优选地，X 2X 3选自DI、EI、DM、EM、DM ox、EM ox中的一种；X 4X 5X 6选自SPL、SPA、SSL、SSA、TPL、TPA、TSL、TSA中的一种；X 8X 9选自VI、VV、FI、FV、LI、LV中的一种。更优选地，本发明的多肽选自具有如下序列多肽中的一种、两种或多种的任意比 例的混合物：
SEQ ID No.1:ASCNGVCSPFEMPPCGTSACRCIPVGLVVGYCRNPSG、
SEQ ID No.2:ASCNGVCSPFEM oxPPCGTSACRCIPVGLVVGYCRNPSG、
SEQ ID No.3:ASCNGVCSPFEMPPCGTSACRCIPVGLVIGYCRNPSG、
SEQ ID No.4:ASCNGVCSPFEM oxPPCGTSACRCIPVGLVIGYCRNPSG、
SEQ ID No.5:ASCNGVCSPFEMPPCGSSACRCIPVGLLIGYCRNPSG、
SEQ ID No.6:ASCNGVCSPFEM oxPPCGSSACRCIPVGLLIGYCRNPSG、
SEQ ID No.7:ASCNGVCSPFEMPPCGTSACRCIPVGLFIGYCRNPSG、
SEQ ID No.8:ASCNGVCSPFEM oxPPCGTSACRCIPVGLFIGYCRNPSG、
SEQ ID No.9:ASCNGVCSPFEMPPCGTSACRCIPYGLFIGYCRNPSG、
SEQ ID No.10:ASCNGVCSPFDIPPCGSPLCRCIPVGLVIGKCRNPYG、
SEQ ID No.11:ISCNGVCSPFDIPPCGSPLCRCIPAGLVIGNCRNPYG、
SEQ ID No.12:VSCNGVCSPFDIPPCGSPLCRCIPAGLVIGKCRNPYG、
SEQ ID No.13:ASCNGVCSPFDIPPCGTPLCRCIPVGLVIGNCRNPYG、
SEQ ID No.14:ASCNGVCSPFDMPPCGSSACRCIPVGLFIGNCRNPYG、
SEQ ID No.15:ASCNGVCSPFDMPPCGSSACRCIPVGLVIGYCRNPYG、
SEQ ID No.16:ASCNGVCSPFDMPPCGSSLCRCIPAGLVVGYCRNPSG、
SEQ ID No.17:ASCNGVCSPFDMPPCGTSACRCIPAGLFIGKCRNPYG、
SEQ ID No.18:ASCNGVCSPFDMPPCGTSACRCIPVGLVVGYCRNPSG、
SEQ ID No.19:ASCNGVCSPFDMPPCGTSLCRCIPAGLFIGYCRNPSG、
SEQ ID No.20:ASCNGVCSPFDMPPCGTSLCRCIPAGLVIGYCRNPYG、
SEQ ID No.21:ASCNGVCSPFEIPPCGSPLCRCIPVGLVIGNCRNPYG、
SEQ ID No.22:ASCNGVCSPFEIPPCGTPLCRCIPAGLVIGKCRNPYG、
SEQ ID No.23:ASCNGVCSPFEIPPCGTPLCRCIPVGLFIGKCRNPSG、
SEQ ID No.24:ASCNGVCSPFEMPPCGSSACRCIPAGLFIGKCRNPYG、
SEQ ID No.25:ASCNGVCSPFEMPPCGSSACRCIPVGLFVGYCRNPYG、
SEQ ID No.26:ASCNGVCSPFEMPPCGSSACRCIPVGLVVGYCRNPSG、
SEQ ID No.27:ASCNGVCSPFEMPPCGSSLCRCIPAGLFIGYCRNPSG、
SEQ ID No.28:ASCNGVCSPFEMPPCGSSLCRCIPAGLVIGYCRNPSG、
SEQ ID No.29:ASCNGVCSPFEMPPCGTSACRCIPAGLFIGNCRNPYG、
SEQ ID No.30:ASCNGVCSPFEMPPCGTSACRCIPAGLVIGYCRNPYG、
SEQ ID No.31:ISCNGVCSPFDIPPCGSPACRCIPVGLVIGKCRNPYG、
SEQ ID No.32:ISCNGVCSPFDIPPCGSPLCRCIPAGLVIGNCRNPSG、
SEQ ID No.33:ISCNGVCSPFDIPPCGSPLCRCIPVGLFIGNCRNPSG、
SEQ ID No.34:ISCNGVCSPFDIPPCGSPLCRCIPVGLVIGYCRNPSG、
SEQ ID No.35:ISCNGVCSPFDIPPCGTPACRCIPVGLVIGNCRNPYG、
SEQ ID No.36:ISCNGVCSPFDIPPCGTPLCRCIPAGLFVGKCRNPYG、
SEQ ID No.37:ISCNGVCSPFDIPPCGTPLCRCIPAGLFIGKCRNPSG、
SEQ ID No.38:ISCNGVCSPFDMPPCGSPACRCIPAGLLIGYCRNPSG、
SEQ ID No.39:ISCNGVCSPFDMPPCGTSACRCIPAGLVIGYCRNPSG、
SEQ ID No.40:ISCNGVCSPFEIPPCGSPLCRCIPAGLFIGKCRNPSG、
SEQ ID No.41:ISCNGVCSPFEIPPCGTPACRCIPVGLFIGKCRNPSG、
SEQ ID No.42:ISCNGVCSPFEIPPCGTPACRCIPAGLVIGKCRNPYG、
SEQ ID No.43:ISCNGVCSPFEIPPCGSPACRCIPVGLVIGNCRNPYG、
SEQ ID No.44:ISCNGVCSPFEIPPCGSSLCRCIPAGLLVGKCRNPSG、
SEQ ID No.45:ISCNGVCSPFEIPPCGTPLCRCIPAGLFIGNCRNPSG、
SEQ ID No.46:ISCNGVCSPFEIPPCGTPLCRCIPAGLVIGYCRNPSG、
SEQ ID No.47:ISCNGVCSPFEIPPCGTSACRCIPAGLVIGYCRNPSG、
SEQ ID No.48:ISCNGVCSPFEMPPCGSSACRCIPAGLVIGYCRNPSG、
SEQ ID No.49:ISCNGVCSPFEMPPCGSSACRCIPAGLFIGYCRNPSG、
SEQ ID No.50:ISCNGVCSPFEMPPCGTSLCRCIPAGLVVGYCRNPSG、
SEQ ID No.51：VSCNGVCSPFDIPPCGTPLCRCIPYGLFVGNCRNPYG、
SEQ ID No.52:VSCNGVCSPFDIPPCGTPACRCIPYGLFVGNCRNPYG、
SEQ ID No.53:VSCNGVCSPFDIPPCGTPLCRCIPAGLVIGNCRNPYG、
SEQ ID No.54:VSCNGVCSPFDIPPCGTPLCRCIPVGLFIGNCRNPSG、
SEQ ID No.55:VSCNGVCSPFDIPPCGTPLCRCIPVGLVIGYCRNPSG、
SEQ ID No.56:VSCNGVCSPFDIPPCGSPLCRCIPVGLFIGKCRNPSG、
SEQ ID No.57:VSCNGVCSPFDMPPCGSSACRCIPAGLFIGNCRNPYG、
SEQ ID No.58:VSCNGVCSPFDMPPCGSSACRCIPAGLVIGYCRNPYG、
SEQ ID No.59:VSCNGVCSPFDMPPCGSSACRCIPVGLVIGYCRNPSG、
SEQ ID No.60:VSCNGVCSPFDMPPCGSSACRCIPVGLFIGYCRNPSG、
SEQ ID No.61:VSCNGVCSPFDMPPCGTSACRCIPAGLFIGYCRNPSG、
SEQ ID No.62:VSCNGVCSPFEIPPCGSPACRCIPVGLVIGKCRNPYG、
SEQ ID No.63:VSCNGVCSPFEIPPCGSPLCRCIPAGLVIGNCRNPYG、
SEQ ID No.64:VSCNGVCSPFEIPPCGSPLCRCIPVGLVIGYCRNPSG、
SEQ ID No.65:VSCNGVCSPFEIPPCGTPACRCIPVGLVIGNCRNPYG、
SEQ ID No.66:VSCNGVCSPFEIPPCGSPLCRCIPVGLFIGNCRNPSG、
SEQ ID No.67:VSCNGVCSPFEIPPCGTPLCRCIPAGLFIGKCRNPSG、
SEQ ID No.68:VSCNGVCSPFEMPPCGSSACRCIPYGLVVGNCRNPSG、
SEQ ID No.69:VSCNGVCSPFEMPPCGTPLCRCIPYGLLIGKCRNPYG、
SEQ ID No.70:VSCNGVCSPFEMPPCGTSACRCIPAGLVIGYCRNPSG、
SEQ ID No.71:VSCNGVCSPFEMPPCGTSACRCIPAGLFIGYCRNPSG。
更优选地，本发明的多肽选自具有如前所述SEQ ID No.1～SEQ ID No.12的多肽中的一种、两种或多种的任意比例的混合物。
本发明的多肽均含有六个半胱氨酸，分别位于第3、7、15、20、22和32位，在肽链内形成三对二硫键，分别是Cys3-Cys20、Cys7-Cys22和Cys15-Cys32，成为“胱氨酸结构模体”(cystine knot motif)，其中，Cys3-Cys20、Cys7-Cys22之间的二硫键形成骨架，Cys15-Cys32之间的二硫键穿透于骨架之中，属于抑制 胱氨酸结构(inhibitor cystine knot，简称ICK)。本发明的多肽还含有14～16个疏水性氨基酸，其中，第1、6、9、10、12、13、14、19、23、24、27、28、29及35位上的氨基酸均为疏水性氨基酸，这些疏水性氨基酸在氨基酸序列中的排列位置高度一致。上述结构特征使得本发明的多肽能够耐胃蛋白酶及胰蛋白酶的酶解，并具有优异的降糖和降脂活性，且无论是口服还是皮下注射、肌肉注射或者是静脉注射均具有降糖和降脂活性。
在一个优选的实施方案中，本发明的多肽及其混合物来自豆科植物的种子或其加工副产物-豆粕的提取物。豆科植物包括但不限于大豆、蚕豆、豌豆、赤豆、绿豆、豇豆、四季豆、扁豆等及其中一种、两种或多种的任意比例的混合物；优选豌豆、大豆、绿豆或其中一种、两种或多种的任意比例的混合物。优选地，所述提取物中含有如前所述SEQ ID No.1～SEQ ID No.12的多肽。优选，所述12个多肽的总含量为至少5％，优选至少8％，进一步优选至少10％，进一步优选至少20％，进一步优选至少30％，进一步优选至少50％，进一步优选至少80％，更进一步优选至少90％，更进一步优选至少95％，更进一步优选至少97％，最优选至少98％。本发明人经研究发现，所述提取物即便在纯度较低的情况下，例如但不限于12个多肽的总含量为10％，就具有良好的降糖和降脂活性。
根据本发明，所述提取物为有机溶剂提取物，所述有机溶剂选自正丁醇、异丙醇、乙醇、甲醇、甲酸、乙酸中的一种、两种或多种有机溶剂的任意比例混合物；优选正丁醇。优选地，提取方法包括如下步骤：
(1)将豆科植物的种子或其加工副产物-豆粕粉碎，加入有机溶剂，低温或常温萃取，离心过滤，收集上清液得多肽粗品；
(2)用反相HPLC对多肽粗品进行纯化，得多肽提取物。
优选，在步骤(1)中用有机溶剂提取之前，对粉碎的豆科植物的种子或其加工副产物豆粕脱色脱脂。进一步优选，用选自氯仿、环己烷、石油醚中的一种、两种或多种有机溶剂的任意比例混合物进行脱色脱脂；最优选用氯仿进行脱色脱脂。
优选，提取温度为0～30℃，最优选为10℃。
优选，提取时间为1～40小时，最优选为20～30小时。
优选地，在本发明中，所述氨基酸选自甲硫氨酸、组氨酸、酪氨酸中的一种、两种或多种的任意比例的混合物。本领域技术人员可以理解，甲硫氨酸、组氨酸、酪氨酸可以为D型、L型或DL型，优选地为L型。
优选地，在本发明中，多肽和氨基酸的重量比为100:1～1:100，更优选地，重量比为50:1～1:50，更优选地，重量比为50:1～1:2，更优选地，重量比为20:1～10:1。
本发明中，含有本发明多肽的组合物和含有本发明多肽和氨基酸的组合物可以是液体组合物或固体组合物。优选地，在本发明中，含有本发明多肽的组合物和含有本发明多肽和氨基酸的组合物中可进一步包含重组人血清白蛋白和/或牛血清白蛋白，用于提高本发明多肽的稳定性，其含量范围为0.5～50mg/ml(在液体组合物中)，更优选1～20mg/ml，更优选5～10mg/ml，或者0.5～50mg/g(在固体组合物中)，更优选1～20mg/g，更优选5～10mg/g。
优选地，在本发明中，含有本发明多肽的组合物和含有本发明多肽和氨基酸的组合物中可进一步包含氯化钠，用于提高离子强度，防止多肽聚集，其含量范围为1～20mg/ml(在液体组合物中)，更优选5～15mg/ml，更优选8～12mg/ml，或者1～20mg/g(在固体组合物中)，更优选5～15mg/g，更优选8～12mg/g。
优选地，在本发明中，含有本发明多肽的组合物和含有本发明多肽和氨基酸的组合物中可进一步包含乙二胺四乙酸二钠，用于络合金属离子，防止多肽氧化，其含量范围为0.05～0.1mg/ml(在液体组合物中)或0.05～0.2mg/g(在固体组合物中)。
优选地，在本发明中，含有本发明多肽的组合物和含有本发明多肽和氨基酸的组合物中可进一步包含乙酸钠、乙酸，用于调节pH及离子强度，增加多肽在水中的溶解性。乙酸钠的含量范围为0.5～3mg/ml(在液体组合物中)，更优 选1～2.5mg/ml，更优选1.5～2mg/ml。
优选地，在本发明中，含有本发明多肽的组合物和含有本发明多肽和氨基酸的组合物中可进一步包含甘油、甘露醇、山梨醇中的一种、两种或多种，用作赋形剂并提高本发明多肽的稳定性，其含量范围为0.5～100mg/ml(在液体组合物中)，更优选1～50mg/ml，更优选8～15mg/ml，或者0.5～100mg/g(在固体组合物中)，更优选1～50mg/g，更优选8～15mg/g。
优选地，当含有本发明多肽的组合物和含有本发明多肽和氨基酸的组合物为液体组合物时，其pH为3.5～6.5，更优选4～5，更优选4.5；优选地，当含有本发明多肽的组合物和含有本发明多肽和氨基酸组合物为固体组合物时，其5％溶液的pH为3.5～6.5，更优选4～5，更优选4.5。
在一个优选的实施方案中，含有本发明多肽和氨基酸的组合物为液体组合物，包含：
(a)如通式(I)所示序列的多肽中的一种、两种或多种的任意比例的混合物；
(b)至少一种氨基酸，选自甲硫氨酸、组氨酸、酪氨酸中的一种、两种或多种的任意比例的混合物；
(c)乙酸钠；
(d)乙酸；
(e)甘油；
(f)注射用水。
在一个优选的实施方案中，含有本发明多肽和氨基酸的组合物为固体组合物，包含：
(a)如通式(I)所示序列的多肽中的一种、两种或多种的任意比例的混合物；
(b)至少一种氨基酸，选自甲硫氨酸、组氨酸、酪氨酸中的一种、两种或多种的任意比例的混合物；
(c)D-甘露醇；
(d)乙二胺四乙酸二钠。优选地，进一步包含氯化钠。
在本发明中，优选地，哺乳动物为人。
本发明的多肽及其组合物能够降低各种原因引起的血糖和/或血脂升高，血糖升高包括但不限于I型和/或II型糖尿病，优选II型糖尿病，血脂升高包括但不限于高脂血症。
本领域技术人员可以理解，本发明的多肽或其组合物可以制成药物组合物或制剂、食品、保健食品或特殊医学用途配方食品。
本领域技术人员可以理解，本领域技术人员通过有限次的常规实验能够确定本发明多肽或其组合物的治疗有效量。本发明多肽或其混合物的治疗有效量例如1～20mg/(kg.d)；含有本发明多肽和氨基酸的组合物的治疗有效量例如1～30mg/(kg.d)。
本领域技术人员可以理解，本发明的多肽或其组合物可以与其他降糖药和/或降脂药联合使用。例如，其他降糖药包括但不限于胰岛素及其衍生物例如长效胰岛素、口服降糖药、肠促胰岛素等及其组合。本领域技术人员可以理解，各种口服降糖药都可以用于本发明，包括但不限于双胍类例如二甲双胍、磺酰脲类促泌剂、噻唑烷二酮类例如格列酮、α-糖苷酶抑制剂类例如阿卡波糖等及其组合。例如，其他降脂药包括但不限于洛伐他丁、辛伐他丁、普伐他丁、非诺贝特、吉非罗齐、苯扎贝特、藻酸双酯钠等。本领域技术人员可以理解，本发明的多肽或其组合物与其他降糖药和/或降脂药可以作为单一组合物同时施用，也可以在分开的组合物中同时施用，还可以在分开的组合物中顺序施用，本领域技术人员可以理解，顺序施用的施用顺序并不影响本发明的多肽或其组合物和其他降糖药和/或降脂药的治疗效果。本发明的多肽或其组合物可以制成适于口服、静脉注射、肌肉注射或皮下注射的各种剂型，包括但不限于，适于口服的片剂(包括但不限于各种包衣片剂、缓释或控释片剂)、锭剂、胶囊剂(包括软胶囊和硬胶囊)、颗粒剂、可分散粉末、水性或油性混悬剂、乳剂、酏剂或 糖浆剂等等；适于静脉注射、肌肉注射或皮下注射的注射液、注射用粉针剂等等。
在将本发明的多肽或其组合物制成药物制剂时，可以进一步含有可药用载体，包括但不限于各种有机或无机药物载体，例如赋形剂、润滑剂、粘合剂、崩解剂、水溶性聚合物、无机盐、溶剂、溶解助剂、悬浮剂、等渗剂、缓冲液、防腐剂、抗氧剂、着色剂、甜味剂、酸味剂、起泡剂和调味剂等等。
本领域技术人员可以理解，本发明的多肽可以商购或者根据本领域已知的各种制备方法获得，包括但不限于从豆科植物中提取、化学合成(例如固相合成)、采用基因重组技术获得重组蛋白的方法等等，优选从豆科植物中提取。
本领域技术人员可以理解，本发明的组合物、药物制剂、食品、保健食品或特殊医学用途配方食品可以通过本领域已知的各种方法制备得到。在一个优选的实施方案中，本发明的多肽或其组合物、药物制剂、食品、保健食品或特殊医学用途配方食品在惰性气体保护下贮存，优选在氮气保护下贮存。
在本发明中，根据国际上通用的氨基酸缩写惯例，氨基酸序列中A代表丙氨酸，C代表半胱氨酸，D代表天冬氨酸，E代表谷氨酸，F代表苯丙氨酸，G代表甘氨酸，H代表组氨酸，I代表异亮氨酸，K代表赖氨酸，L代表亮氨酸，M代表甲硫氨酸，N代表天冬酰胺，P代表脯氨酸，Q代表谷氨酰胺，R代表精氨酸，S代表丝氨酸，T代表苏氨酸，V代表缬氨酸，W代表色氨酸，Y代表酪氨酸。本发明中，M ox代表甲硫氨酸亚砜。
与现有技术相比，本发明具有以下优点和有益效果：
本发明的多肽具有优良的降血糖和降血脂活性，与氨基酸配合后不仅提高了稳定性，而且提高了降血糖和降血脂活性。本发明的多肽及其与氨基酸的组合物耐胃蛋白酶及胰蛋白酶酶解，从而既可以口服给药，又可以静脉注射、肌肉注射或皮下注射，是罕见的四种给药方式均能降血糖和降血脂的药物，用药方式灵活，同时还具有保护并修复胰腺β细胞功能的活性，且不会导致患者产生低血糖反应以及胰岛素抵抗，无药物毒副作用，用药方便安全，可长期稳定 贮存，并能有效预防、治疗Ⅱ型糖尿病及高脂血症，比现有降糖药物能更有效地降低糖尿病患者的糖化血红蛋白，附加心血管获益，减少糖尿病并发症，因此具有明显优势。特别是能够在那些通过二甲双胍、磺酰脲类促泌剂、噻唑烷二酮类、α-糖苷酶抑制剂、胰岛素或其衍生物或其组合无法有效控制血糖的患者中实现血糖及糖化血红蛋白的有效控制。
附图说明
图1.从豆科植物中提取的本发明多肽的HPLC图；
图2.序号为SEQ ID No.7的多肽的氨基酸序列；
图3.序号为SEQ ID No.8的多肽的氨基酸序列；
图4.序号为SEQ ID No.3的多肽的氨基酸序列；
图5.序号为SEQ ID No.10的多肽的氨基酸序列；
图6.本发明多肽的酶解HPLC图；
图7.白蛋白酶解HPLC图；
图8.本发明多肽稳定性考察HPLC图；
图9.胰腺HE染色图和免疫组织化学图。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外，应理解，在阅读了本发明所记载的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本发明所限定的范围。
实施例1从豆科植物中提取本发明的多肽
本发明的多肽可以从豆科植物的种子或加工副产物豆粕中提取。具体提取工艺如下：
取10公斤商品豌豆豆粕，粉碎机粉碎、过筛得到平均粒径100目的颗粒，与50升氯仿混合，搅拌1小时，离心过滤，滤饼用氯仿洗涤，干燥后得到脱色脱脂的豆粕。
上述脱色脱脂的豆粕与100升正丁醇混合，均质机均质30分钟，接冷冻机控制工作温度在10℃，然后转移至夹套式提取罐，10℃下搅拌24小时，离心过滤得上清液，滤饼用正丁醇洗涤，收集、合并上清液。
上清液50℃下减压蒸馏浓缩10倍，浓缩液冷冻干燥，得多肽粗品95.6克。
多肽的纯化及鉴定：
上述多肽粗品用反相液相色谱分离纯化。取适量多肽粗品溶于色谱A液(水+0.1％三氟乙酸)，0.22微米滤膜过滤，滤液上样。安捷伦1260高效液相色谱仪，C18反相色谱柱(Wateres公司，4.6×250mm，300SB，5微米)，流动相：水(含0.1％三氟乙酸)+乙腈(含0.1％三氟乙酸)，乙腈15％到50％梯度淋洗，25min，1ml/min流速，214nm检测，16～25min共得到13个峰(见图1)，分别收集保留时间为16.9min、17.5min、18.1min、18.9min、19.1min、19.9min、20.4min、21.0min、21.2min、22.1min、22.4min、23.3min、23.7min的峰，经MALDI-TOF及Edman降解确认，共含13个肽，氨基酸序列分别如下：
SEQ ID No.1:ASCNGVCSPFEMPPCGTSACRCIPVGLVVGYCRNPSG、
SEQ ID No.2:ASCNGVCSPFEM oxPPCGTSACRCIPVGLVVGYCRNPSG、
SEQ ID No.3:ASCNGVCSPFEMPPCGTSACRCIPVGLVIGYCRNPSG、
SEQ ID No.4:ASCNGVCSPFEM oxPPCGTSACRCIPVGLVIGYCRNPSG、
SEQ ID No.5:ASCNGVCSPFEMPPCGSSACRCIPVGLLIGYCRNPSG、
SEQ ID No.6:ASCNGVCSPFEM oxPPCGSSACRCIPVGLLIGYCRNPSG、
SEQ ID No.7:ASCNGVCSPFEMPPCGTSACRCIPVGLFIGYCRNPSG、
SEQ ID No.8:ASCNGVCSPFEM oxPPCGTSACRCIPVGLFIGYCRNPSG、
SEQ ID No.9:ASCNGVCSPFEMPPCGTSACRCIPYGLFIGYCRNPSG、
SEQ ID No.10:ASCNGVCSPFDIPPCGSPLCRCIPVGLVIGKCRNPYG、
SEQ ID No.11:ISCNGVCSPFDIPPCGSPLCRCIPAGLVIGNCRNPYG、
SEQ ID No.12:VSCNGVCSPFDIPPCGSPLCRCIPAGLVIGKCRNPYG、
SEQ ID No.72:ASCNGVCSPFEMPPCGSSACRCIPVGLVVGYCRHPSG。
实施例2利用固相多肽合成法合成本发明的多肽
本发明的多肽也可用固相多肽合成方法合成，具体工艺如下：
使用Applied Biosystems公司的ABI 433A型多肽合成仪，采用Fmoc/tBu法，NMP溶剂体系，HBTU/HOBt作缩合剂，保护氨基酸侧链所用基团分别是：Ser、Thr及Tyr用叔丁基(tBu)，Asp、Glu用叔丁基酯基，Asn、Gln及Cys用三苯甲基(Trt)，Arg用2,2,4,6,7-五甲基二氢苯并呋喃-5-磺酰基(Pbf)，Lys用2-氯苯甲氧羰基。合成规模0.5mmol，带保护基的氨基酸及缩合剂过量10倍，程序设定后自动完成。反应完成后，树脂用DCM洗涤三次，真空抽干，向多肽固相反应器中加入TFA/TIS/H 2O/Phenol(加入酚可避免半胱氨酸氧化形成二硫键)的混合液充分反应2小时，用氮气除去大部分溶剂，加入冰冻的甲基叔丁基醚沉淀，离心去除上清液，并用冰冻的甲基叔丁基醚洗涤沉淀，沉淀物冷冻干燥，得到还原态的多肽粗品。
100ml 50％异丙醇溶液中加入还原态的多肽粗品10mg，EDTA、Tris-HCl、GSH及GSSG浓度分别为2mM、200mM、1mM、100μM，pH8.6，4℃下反应48小时，加TFA至pH4时反应终止。真空浓缩除去异丙醇，浓缩液冷冻干燥，得到多肽粗品。粗品溶于60％甲醇中，半制备HPLC分离纯化，30-45％乙腈梯度洗脱，冷冻干燥，得产物，产率约为60％，HPLC纯度≥98％。
采用上述固相多肽合成法分别合成氨基酸序列如下的多肽：
SEQ ID No.1:ASCNGVCSPFEMPPCGTSACRCIPVGLVVGYCRNPSG
SEQ ID No.13:ASCNGVCSPFDIPPCGTPLCRCIPVGLVIGNCRNPYG、
SEQ ID No.17:ASCNGVCSPFDMPPCGTSACRCIPAGLFIGKCRNPYG、
SEQ ID No.21:ASCNGVCSPFEIPPCGSPLCRCIPVGLVIGNCRNPYG、
SEQ ID No.26:ASCNGVCSPFEMPPCGSSACRCIPVGLVVGYCRNPSG、
SEQ ID No.36:ISCNGVCSPFDIPPCGTPLCRCIPAGLFVGKCRNPYG、
SEQ ID No.38:ISCNGVCSPFDMPPCGSPACRCIPAGLLIGYCRNPSG、
SEQ ID No.44:ISCNGVCSPFEIPPCGSSLCRCIPAGLLVGKCRNPSG、
SEQ ID No.50:ISCNGVCSPFEMPPCGTSLCRCIPAGLVVGYCRNPSG、
SEQ ID No.52:VSCNGVCSPFDIPPCGTPACRCIPYGLFVGNCRNPYG、
SEQ ID No.61:VSCNGVCSPFDMPPCGTSACRCIPAGLFIGYCRNPSG、
SEQ ID No.63:VSCNGVCSPFEIPPCGSPLCRCIPAGLVIGNCRNPYG、
SEQ ID No.68:VSCNGVCSPFEMPPCGSSACRCIPYGLVVGNCRNPSG。
实施例3正常小鼠尾静脉注射本发明多肽降糖效果实验
1.材料与仪器
多肽：实施例1中SEQ ID No.7的多肽，平均分子量3789，氨基酸序列为ASCNGVCSPFEMPPCGTSACRCIPVGLFIGYCRNPSG(见图2)，HPLC纯度≥98％，用生理盐水配制成1mg/ml的溶液备用；
昆明种小白鼠：湖北省卫生防疫站，40只，体重20±2g，雄性；
血糖测试仪及试纸：北京怡成医疗有限公司；
2.实验方法
上述小白鼠禁食4小时(不禁水)后随机分为4组，每组10只，每只分别染苦味酸作标记，取尾静脉血测定血糖。空白对照组，给予生理盐水0.05ml/10g鼠尾静脉注射1次，A组(多肽高剂量降糖组)按0.10ml/10g鼠尾静脉注射1mg/ml多肽1次，B组(多肽中剂量降糖组)按0.05ml/10g鼠尾静脉注射1mg/ml多肽1次，C组(多肽低剂量降糖组)按0.025ml/10g鼠尾静脉注射1mg/ml多肽1次。于注射后15分钟、30分钟、45分钟、60分钟分别采集尾尖静脉血用血糖仪测定血糖变化，血糖单位mmol/L。
实验结果数据用
Figure PCTCN2018082401-appb-000001
表示，组间差异用t检验，以p＜0.05表示差异具有显 著意义。
3.实验结果见下表1：
表1.正常小鼠尾静脉注射多肽降糖效果
Figure PCTCN2018082401-appb-000002
注：与空白对照组相比，*p<0.05，**p<0.01。
4.结论
从上述实验结果可以看出，正常小鼠尾静脉注射本发明序号为SEQ ID No.7的多肽15分钟后，血糖开始呈下降趋势，降糖作用高剂量组A＞中剂量组B＞低剂量组C，存在明显的量效关系。这表明，静脉注射本发明序号为SEQ ID No.7的多肽可以降低正常小鼠的血糖，因此，该肽具有明确的降糖活性。
实施例4正常小鼠皮下注射本发明多肽降糖效果实验
1.材料与仪器
多肽：实施例1中SEQ ID No.8的多肽，平均分子量3805，氨基酸序列为ASCNGVCSPFEM oxPPCGTSACRCIPVGLFIGYCRNPSG(见图3)，HPLC纯度≥98％，用生理盐水配制成1mg/ml的溶液备用；
昆明种小白鼠：湖北省卫生防疫站，40只，体重20±2g，雄性；
血糖测试仪及试纸：北京怡成医疗有限公司；
2.实验方法
上述小白鼠禁食4小时(不禁水)后随机分为4组，每组10只，每只分别染 苦味酸作标记，取尾静脉血测定血糖。空白对照组，给予生理盐水0.05ml/10g背部皮下注射1次，A组(多肽高剂量降糖组)按0.10ml/10g背部皮下注射1mg/ml多肽1次，B组(多肽中剂量降糖组)按0.05ml/10g背部皮下注射1mg/ml 37肽1次，C组(多肽低剂量降糖组)按0.025ml/10g背部皮下注射1mg/ml多肽1次。于注射后15分钟、30分钟、45分钟、60分钟分别采集尾尖静脉血用血糖仪测定血糖变化，血糖单位mmol/L。
实验结果数据用
Figure PCTCN2018082401-appb-000003
表示，组间差异用t检验，以p＜0.05表示差异具有显著意义。
3.实验结果见下表2：
表2.正常小鼠背部皮下注射多肽降糖效果
Figure PCTCN2018082401-appb-000004
注：与空白对照组相比，*p<0.05，**p<0.01。
4.结论
从上述实验结果可以看出，正常小鼠背部皮下注射本发明序号为SEQ ID No.8的多肽15分钟后，血糖开始呈下降趋势，降糖作用高剂量组A＞中剂量组B＞低剂量组C，存在明显的量效关系。这表明，皮下注射本发明序号为SEQ ID No.8的多肽也可以降低正常小鼠的血糖，因此，该肽具有明确的降糖活性，而且，与序号为SEQ ID No.7的多肽相比，甲硫氨酸氧化为甲硫氨酸亚砜并不影响其降糖活性。
实施例5正常小鼠皮下注射本发明多肽降糖效果对比
1.材料与仪器
多肽：实施例1中SEQ ID No.72的多肽，平均分子量3736，氨基酸序列为ASCNGVCSPFEMPPCGSSACRCIPVGLVVGYCRHPSG，HPLC纯度≥98％，用生理盐水配制成1mg/ml的溶液备用；和
实施例2中SEQ ID No.26的多肽，平均分子量3716，氨基酸序列为ASCNGVCSPFEMPPCGSSACRCIPVGLVVGYCRNPSG，HPLC纯度≥98％，用生理盐水配制成1mg/ml的溶液备用；
昆明种小白鼠：湖北省卫生防疫站，30只，体重20±2g，雄性；
血糖测试仪及试纸：北京怡成医疗有限公司；
2.实验方法
上述小白鼠禁食4小时(不禁水)后随机分为3组，每组10只，每只分别染苦味酸作标记，取尾静脉血测定血糖。空白对照组，给予生理盐水0.05ml/10g背部皮下注射1次，A组(SEQ ID No.72的多肽)按0.05ml/10g背部皮下注射1mg/ml多肽1次，B组(SEQ ID No.26的多肽)按0.05ml/10g背部皮下注射1mg/ml多肽1次。于注射后15分钟、30分钟、45分钟、60分钟分别采集尾尖静脉血用血糖仪测定血糖变化，血糖单位mmol/L。
实验结果数据用
Figure PCTCN2018082401-appb-000005
表示，组间差异用t检验，以p＜0.05表示差异具有显著意义。
3.实验结果见下表3：
表3.正常小鼠背部皮下注射多肽降糖效果
Figure PCTCN2018082401-appb-000006
Figure PCTCN2018082401-appb-000007
注：与空白对照组相比，*p<0.05，**p<0.01。
4.结论
氨基酸序列为ASCNGVCSPFEMPPCGSSACRCIPVGLVVGYCRNPSG的多肽皮下注射可以降低小鼠血糖，有降糖活性。而氨基酸序列为ASCNGVCSPFEMPPCGSSACRCIPVGLVVGYCRHPSG的多肽皮下注射升高小鼠血糖，与前者具有相反作用。二者在结构上的差别仅体现在第34位的氨基酸上，前者为中性氨基酸-天冬酰胺(Asn)，后者为碱性氨基酸-组氨酸(His)。造成二者生物活性不同的可能原因在于后者亲水性强，从C18反相色谱保留时间最短可证实这一点，SEQ ID No.72的多肽为图1中1号峰，保留时间为16.9min，在生理环境下组氨酸质子化，变得更亲水，进而改变多肽的空间构象及其与蛋白质结合的方式，从而不具有降糖活性。
从本实施例还可以看出，多肽第34位的氨基酸为影响降糖活性的关键位点，若为H(组氨酸)，则相应的多肽无降糖活性。
实施例6正常小鼠皮下注射本发明天然提取多肽及固相合成多肽降糖效果对比实验
1.材料与仪器
多肽A：实施例1中SEQ ID No.1的多肽，平均分子量3727，氨基酸序列为ASCNGVCSPFEMPPCGTSACRCIPVGLVVGYCRNPSG，HPLC纯度≥98％，用生理盐水配制成1mg/ml的溶液备用；
多肽B：实施例2中SEQ ID No.1的多肽，平均分子量3727，氨基酸序列为ASCNGVCSPFEMPPCGTSACRCIPVGLVVGYCRNPSG，HPLC纯度≥98％，用生理盐水配制成1mg/ml的溶液备用；
昆明种小白鼠：湖北省卫生防疫站，30只，体重20±2g，雄性；
血糖测试仪及试纸：北京怡成医疗有限公司；
2.实验方法
上述小白鼠禁食4小时(不禁水)后随机分为3组，每组10只，每只分别染苦味酸作标记，取尾静脉血测定血糖。空白对照组，给予生理盐水0.05ml/10g背部皮下注射1次，A组(多肽A)按0.05ml/10g背部皮下注射1mg/ml多肽1次，B组(多肽B)按0.05ml/10g背部皮下注射1mg/ml多肽1次。于注射后15分钟、30分钟、45分钟、60分钟分别采集尾尖静脉血用血糖仪测定血糖变化，血糖单位mmol/L。
实验结果数据用
Figure PCTCN2018082401-appb-000008
表示，组间差异用t检验，以p＜0.05表示差异具有显著意义。
3.实验结果见下表4：
表4.正常小鼠背部皮下注射多肽降糖效果
Figure PCTCN2018082401-appb-000009
注：与空白对照组相比，*p<0.05，**p<0.01。
4.结论
从上述实验结果可以看出，二者降糖活性无显著差异，可见，不同来源但结构相同的本发明多肽降糖活性一样。
实施例7正常小鼠尾静脉注射、皮下注射、肌肉注射本发明多肽降糖效果对比实验
1.材料与仪器
多肽：实施例1中SEQ ID No.3的多肽，平均分子量3741，氨基酸序列为 ASCNGVCSPFEMPPCGTSACRCIPVGLVIGYCRNPSG(见图4)，HPLC纯度≥98％，用生理盐水配制成1mg/ml的溶液备用；
昆明种小白鼠：湖北省卫生防疫站，40只，体重20±2g，雄性；
血糖测试仪及试纸：北京怡成医疗有限公司；
2.实验方法
上述小白鼠禁食4小时(不禁水)后随机分为4组，每组10只，每只分别染苦味酸作标记，取尾静脉血测定血糖。空白对照组，给予生理盐水0.05ml/10g背部皮下注射1次，A组按0.05ml/10g鼠尾静脉注射1mg/ml多肽1次，B组按0.05ml/10g背部皮下注射1mg/ml多肽1次，C组按0.05ml/10g大腿外侧肌肉注射1mg/ml多肽1次。于注射后15分钟、30分钟、45分钟、60分钟分别采集尾尖静脉血用血糖仪测定血糖变化，血糖单位mmol/L。
实验结果数据用
Figure PCTCN2018082401-appb-000010
表示，组间差异用t检验，以p＜0.05表示差异具有显著意义。
3.实验结果见下表5：
表5.正常小鼠尾静脉注射、皮下注射、肌肉注射多肽降糖效果
Figure PCTCN2018082401-appb-000011
注：与空白对照组相比，*p<0.05，**p<0.01。
4.结论
从上述实验结果可以看出，多肽在三种给药方式(静脉注射、皮下注射及肌肉注射)下均具有显著降糖作用，降糖效果间无明显差异。由于皮下注射操作比较方便，可用皮下注射作为本发明多肽及其组合物的降糖活性筛选实验方法。
实施例8正常小鼠皮下注射本发明多肽降糖效果实验
1.材料与仪器
多肽：实施例2中SEQ ID No.13、17、21、26、36、38、44、50、52、61、63、68的多肽，共12个，HPLC纯度≥98％，将上述12个多肽分别用生理盐水配制成1mg/ml的溶液备用；
昆明种小白鼠：湖北省卫生防疫站，130只，体重20±2g，雄性；
血糖测试仪及试纸：北京怡成医疗有限公司；
2.实验方法
上述小白鼠禁食4小时(不禁水)后随机分为13组，每组10只，每只分别染苦味酸作标记，取尾静脉血测定血糖。空白对照组，给予生理盐水0.05ml/10g背部皮下注射1次，其他组按0.05ml/10g分别背部皮下注射1mg/ml 12个多肽1次。于注射后15分钟、30分钟、45分钟、60分钟分别采集尾尖静脉血用血糖仪测定血糖变化，血糖单位mmol/L。
实验结果数据用
Figure PCTCN2018082401-appb-000012
表示，组间差异用t检验，以p＜0.05表示差异具有显著意义。
3.实验结果见下表6：
表6.正常小鼠背部皮下注射多肽降糖效果
Figure PCTCN2018082401-appb-000013
Figure PCTCN2018082401-appb-000014
注：与空白对照组相比，*p<0.05，**p<0.01。
4.结论
序列为X 1SCNGVCSPFX 2X 3PPCGX 4X 5X 6CRCIPX 7GLX 8X 9GX 10CRNPX 11G的多肽中，X 1为A、V、I，X 2X 3为EI、EM、DI、DM，X 4X 5X 6为SPA、SPL、SSA、 SSL、TPA、TPL、TSA、TSL，X 7为A、V、Y，X 8X 9为FI、LI、VI、FV、LV、VV，X 10为K、N、Y，X 11为S、Y，由此排列组合构成的多肽全部具有明确的降糖活性。因此，具有上述通式结构的多肽均具有降糖活性。
实施例9正常小鼠尾静脉注射本发明多肽及胰岛素降糖效果对比实验
1.材料与仪器
胰岛素：江苏万邦生化医药股份有限公司，400U/10ml；
多肽：实施例1中SEQ ID No.11的多肽，平均分子量3788，氨基酸序列为ISCNGVCSPFDIPPCGSPLCRCIPAGLVIGNCRNPYG，HPLC纯度≥98％，用生理盐水配制成2mg/ml的溶液备用；
昆明种小白鼠：湖北省卫生防疫站，30只，体重20±2g，雄性；
血糖测试仪及试纸：北京怡成医疗有限公司；
2.实验方法
上述小白鼠禁食4小时(不禁水)后随机分为3组，每组10只，每只分别染苦味酸作标记，取尾静脉血测定血糖。空白对照组，给予生理盐水0.05ml/10g尾静脉注射1次，A组(多肽降糖组)按0.05ml/10g尾静脉注射2mg/ml多肽1次，B组(胰岛素降糖对照组)按0.05ml/10g尾静脉注射0.5U/ml胰岛素1次。于注射后15分钟、30分钟、45分钟、60分钟分别采集尾尖静脉血用血糖仪测定血糖变化，血糖单位mmol/L。
实验结果数据用
Figure PCTCN2018082401-appb-000015
表示，组间差异用t检验，以p＜0.05表示差异具有显著意义。
3.实验结果见下表7：
表7.正常小鼠尾静脉注射多肽和胰岛素降糖效果
Figure PCTCN2018082401-appb-000016
Figure PCTCN2018082401-appb-000017
注：与空白对照组相比，*p<0.05，**p<0.01。
4.结论
从上述实验结果可以看出，正常小鼠尾静脉注射本发明序号为SEQ ID No.11的多肽与注射胰岛素一样均可以起降糖作用，对小鼠而言，注射10mg/kg体重的多肽与注射2.5U/kg体重的胰岛素，二者降糖效果相当。因此，该肽具有明确的降糖活性。
实施例10正常小鼠灌胃本发明多肽降糖效果实验
1.材料与仪器
多肽：实施例1中SEQ ID No.10的多肽，平均分子量3788，氨基酸序列为ASCNGVCSPFDIPPCGSPLCRCIPVGLVIGKCRNPYG(见图5)，HPLC纯度≥98％，分别用生理盐水及10％葡萄糖溶液配制成1mg/ml的溶液备用；
昆明种小白鼠：湖北省卫生防疫站，30只，体重20±2g，雄性；
血糖测试仪及试纸：北京怡成医疗有限公司；
2.实验方法
上述小白鼠禁食4小时(不禁水)后随机分为3组，每组10只，每只分别染苦味酸作标记，取尾静脉血测定血糖。A组给予10％葡萄糖溶液0.10ml/10g灌胃，B组(多肽组)按0.10ml/10g灌胃1mg/ml多肽生理盐水溶液，C组(多肽+葡萄糖组)按0.10ml/10g灌胃1mg/ml多肽葡萄糖(10％)溶液。于灌胃后15分钟、30分钟、45分钟、60分钟分别采集尾尖静脉血用血糖仪测定血糖变化，血糖单位mmol/L。
实验结果数据用
Figure PCTCN2018082401-appb-000018
表示，组间差异用t检验，以p＜0.05表示差异具有显著意义。
3.实验结果见下表8：
表8.正常小鼠多肽灌胃降糖效果
Figure PCTCN2018082401-appb-000019
注：与A组相比，*p<0.05，**p<0.01。
4.结论：
从上述实验结果可以看出，给正常小鼠灌胃本发明的多肽不会导致正常小鼠低血糖反应，给正常小鼠同时灌胃葡萄糖和本发明的多肽，小鼠血糖不升高，这说明本发明的多肽可以抑制小鼠因摄入葡萄糖而导致的血糖升高，因此，口服本发明的多肽可以稳定餐后血糖在正常范围，间接证明其具有降糖活性。
实施例11正常小鼠皮下注射本发明多肽混合物降糖活性实验
1.材料与仪器
多肽混合物A：实施例1中SEQ ID No.3的多肽与实施例1中SEQ ID No.12的多肽按质量比1:1的比例混合均匀得到多肽混合物A；
多肽混合物B：实施例1中SEQ ID No.1的多肽与实施例1中SEQ ID No.9的多肽按质量比8:1的比例混合均匀得到多肽混合物B；
多肽混合物C：实施例2中SEQ ID No.36的多肽与实施例2中SEQ ID No.68的多肽按质量比1:6的比例混合均匀得到多肽混合物C；
多肽混合物D：收集实施例1中17.5～25min间的峰，冷冻干燥，得到多肽混合物D，其中SEQ ID No.1-SEQ ID No.12共12个多肽的总含量≥98％；
上述各组混合物分别用生理盐水配制成1mg/ml的溶液备用；
昆明种小白鼠：湖北省卫生防疫站，50只，体重20±2g，雄性；
血糖测试仪及试纸：北京怡成医疗有限公司；
2.实验方法
上述小白鼠禁食4小时(不禁水)后随机分为5组，每组10只，每只分别染苦味酸作标记，取尾静脉血测定血糖。空白对照组，给予生理盐水0.05ml/10g背部皮下注射1次，A组(多肽混合物A)按0.05ml/10g背部皮下注射1mg/ml多肽混合物A 1次，B组(多肽混合物B)按0.05ml/10g背部皮下注射1mg/ml多肽混合物B 1次，C组(多肽混合物C)按0.05ml/10g背部皮下注射1mg/ml多肽混合物C 1次，D组(多肽混合物D)按0.05ml/10g背部皮下注射1mg/ml多肽混合物D 1次。于注射后15分钟、30分钟、45分钟、60分钟分别采集尾尖静脉血用血糖仪测定血糖变化，血糖单位mmol/L。
实验结果数据用
Figure PCTCN2018082401-appb-000020
表示，组间差异用t检验，以p＜0.05表示差异具有显著意义。
3.实验结果见下表9：
表9.正常小鼠皮下注射多肽混合物降糖活性
Figure PCTCN2018082401-appb-000021
Figure PCTCN2018082401-appb-000022
注：与空白对照组相比，*p<0.05，**p<0.01。
4.结论
从上述实验结果可以看出，本发明的多肽，其任意比例的组合物也具有优良的降糖活性，复配不会影响多肽的降糖活性。特别是天然提取的多肽混合物(多肽混合物D)也具有显著的降糖活性，这意味从豆科植物中提取的多肽混合物(HPLC中17.5～25min的收集物)不用进一步纯化为单体即可直接用于降糖药物，如此大大提高得率并降低生产成本。
实施例12Ⅱ型糖尿病模型小鼠尾静脉注射本发明多肽混合物及胰岛素降糖效果对比实验
1.材料与仪器
胰岛素：江苏万邦生化医药股份有限公司，400U/10ml；
多肽：收集实施例1中17.5～25min间的峰，冷冻干燥，得到的多肽混合物用生理盐水配制成2mg/ml的溶液备用；
Ⅱ型糖尿病模型小鼠：KK鼠，北京华阜康生物科技股份有限公司，30只，体重25±2.7g，雄性，高脂高糖饲料喂养1个月；
血糖测试仪及试纸：北京怡成医疗有限公司；
2.实验方法
上述Ⅱ型糖尿病模型小鼠禁食4小时(不禁水)后随机分为3组，每组10只，每只分别染苦味酸作标记，取尾静脉血测定血糖。模型对照组给予生理盐水0.05ml/10g尾静脉注射1次，A组(多肽降糖组)按0.05ml/10g尾静脉注射2mg/ml多肽1次，B组(胰岛素降糖对照组)按0.05ml/10g尾静脉注射0.5U/ml胰岛素1次。于 注射后15分钟、30分钟、45分钟、60分钟分别采集尾尖静脉血用血糖仪测定血糖变化，血糖单位mmol/L。
实验结果数据用
Figure PCTCN2018082401-appb-000023
表示，组间差异用t检验，以p＜0.05表示差异具有显著意义。
3.实验结果见下表10：
表10.Ⅱ型糖尿病模型小鼠尾静脉注射多肽和胰岛素降糖效果
Figure PCTCN2018082401-appb-000024
注：与模型对照组相比，*p<0.05，**p<0.01。
4.结论
从上述实验结果可以看出，多肽混合物静脉注射能降低Ⅱ型糖尿病模型小鼠的血糖，具有显著降糖活性，效果与胰岛素相当，但不会导致Ⅱ型糖尿病模型小鼠低血糖。
实施例13Ⅱ型糖尿病模型小鼠皮下注射本发明多肽混合物及胰岛素降糖效果对照实验
1.材料与仪器
胰岛素：江苏万邦生化医药股份有限公司，400U/10ml；
多肽：收集实施例1中17.5～25min间的峰，冷冻干燥，得到的多肽混合物用生理盐水配制成2mg/ml的溶液备用；
Ⅱ型糖尿病模型小鼠：KK鼠，北京华阜康生物科技股份有限公司，30只， 体重25±2.7g，雄性，高脂高糖饲料喂养1个月；
血糖测试仪及试纸：北京怡成医疗有限公司；
2.实验方法
上述Ⅱ型糖尿病模型小鼠禁食4小时(不禁水)后随机分为3组，每组10只，每只分别染苦味酸作标记，取尾静脉血测定血糖。模型对照组给予生理盐水0.05ml/10g背部皮下注射1次，A组(多肽降糖组)按0.05ml/10g背部皮下注射2mg/ml多肽1次，B组(胰岛素降糖对照组)按0.05ml/10g背部皮下注射0.5U/ml胰岛素1次。于注射后15分钟、30分钟、45分钟、60分钟分别采集尾尖静脉血用血糖仪测定血糖变化，血糖单位mmol/L。
实验结果数据用
Figure PCTCN2018082401-appb-000025
表示，组间差异用t检验，以p＜0.05表示差异具有显著意义。
3.实验结果见下表11：
表11.Ⅱ型糖尿病模型小鼠皮下注射多肽和胰岛素降糖效果
Figure PCTCN2018082401-appb-000026
注：与模型对照组相比，*p<0.05，**p<0.01。
4.结论
从上述实验结果可以看出，多肽混合物皮下注射能降低Ⅱ型糖尿病模型小鼠的血糖，具有显著降糖活性，效果与胰岛素相当，但不会导致Ⅱ型糖尿病模型小鼠低血糖。
实施例14Ⅱ型糖尿病模型小鼠肌肉注射本发明多肽混合物及胰岛素降糖效果对照实验
1.材料与仪器
胰岛素：江苏万邦生化医药股份有限公司，400U/10ml；
多肽：收集实施例1中17.5～25min间的峰，冷冻干燥，得到的多肽混合物用生理盐水配制成2mg/ml的溶液备用；
Ⅱ型糖尿病模型小鼠：KK鼠，北京华阜康生物科技股份有限公司，30只，体重25±2.7g，雄性，高脂高糖饲料喂养1个月；
血糖测试仪及试纸：北京怡成医疗有限公司；
2.实验方法
上述小白鼠随机分为3组，每组10只，每只分别染苦味酸作标记，取尾静脉血测定血糖。模型对照组，给予生理盐水0.05ml/10g大腿外侧肌肉注射1次，A组(多肽降糖组)按0.05ml/10g大腿外侧肌肉注射2mg/ml多肽1次，B组(胰岛素降糖对照组)按0.05ml/10g大腿外侧肌肉注射0.5U/ml胰岛素1次。于注射后15分钟、30分钟、45分钟、60分钟分别采集尾尖静脉血用血糖仪测定血糖变化，血糖单位mmol/L。
实验结果数据用
Figure PCTCN2018082401-appb-000027
表示，组间差异用t检验，以p＜0.05表示差异具有显著意义。
3.实验结果见下表12：
表12.Ⅱ型糖尿病模型小鼠肌肉注射多肽和胰岛素降糖效果
Figure PCTCN2018082401-appb-000028
Figure PCTCN2018082401-appb-000029
注：与模型对照组相比，*p<0.05，**p<0.01。
4.结论
从上述实验结果可以看出，本发明的多肽混合物肌肉注射能降低Ⅱ型糖尿病模型小鼠的血糖，具有显著降糖活性，效果与胰岛素相当，但不会导致Ⅱ型糖尿病模型小鼠低血糖。
实施例15本发明多肽混合物对胃蛋白酶及胰蛋白酶的稳定性试验
1.材料与仪器
多肽：收集实施例1中15～25min间的峰，冷冻干燥，得到多肽的组合物；
胃蛋白酶：Sigma公司；
胰蛋白酶：Sigma公司；
白蛋白：血清白蛋白(牛)标准品；
高效液相色谱仪：安捷伦1260高效液相色谱仪，Wateres公司C18反相色谱柱(4.6×250mm，300SB，5μm)；
2.实验方案
使用人工胃液和人工肠液分别对多肽进行酶解，以验证多肽是否耐受消化道酶的酶解，对照实验使用白蛋白进行。
人工胃液配制：取浓度为1mol/L的稀盐酸，加水稀释，将pH调至1.5。每100ml溶液中加入1g胃蛋白酶，混匀，用0.22um的无菌滤头过滤待用。
人工肠液配制：取磷酸二氢钾6.8g加水500ml溶解，用0.4％(w/w)的NaOH回调pH至6.8，定容至1L。每100ml溶液中加入1g胰蛋白酶，混匀，用0.22um的无菌滤头过滤待用。
各取多肽溶液(10mg/ml)10微升分别加入人工胃液(1ml)和肠液中(1ml)混匀，37℃水浴孵育2小时。
各取白蛋白干粉1毫克分别加入人工胃液(1ml)和肠液中(1ml)混匀，37℃水浴孵育2小时。
取上述酶解混合物进行HPLC检测。
色谱条件：流动相，水(含0.1％TFA)+乙腈(含0.1％TFA)，乙腈从15％至50％梯度洗脱25min，流速1ml/min，检测波长214nm，进样量20ul。
3.实验结果
见附图多肽的酶解HPLC图及白蛋白的酶解HPLC图。
多肽的酶解HPLC图(见附图6)最上面为多肽对照品HPLC检测结果；中间为多肽经人工胃液酶解2小时HPLC检测结果；最下面为多肽经人工肠液酶解2小时HPLC检测结果。
白蛋白酶解HPLC图(见附图7)最上面为白蛋白对照品HPLC检测结果；中间为白蛋白经人工胃液酶解2小时HPLC检测结果；最下面为白蛋白经人工肠液酶解2小时HPLC检测结果。
4.结论：
如图所示，无论人工胃液和肠液都不能酶解本发明的多肽，而对于白蛋白，胃液和肠液都可以将其完全酶解，说明本发明的多肽耐受胃肠道消化酶的酶解，可以口服。
实施例16Ⅱ型糖尿病模型小鼠灌胃本发明多肽混合物及二甲双胍降糖效果对照实验
1.材料与仪器
多肽：收集实施例1中17.5～25min间的峰，冷冻干燥，得到的多肽混合物用生理盐水配制成2mg/ml的溶液备用；
盐酸二甲双胍：格华止盐酸二甲双胍片，0.5g/片，中美上海施贵宝制药有限公司，用生理盐水配制成有效含量20mg/ml的溶液备用；
Ⅱ型糖尿病模型小鼠：KK鼠，北京华阜康生物科技股份有限公司，30只， 体重25±2.7g，雄性，高脂高糖饲料喂养1个月；
血糖测试仪及试纸：北京怡成医疗有限公司；
2.实验方法
上述小白鼠随机分为3组，每组10只，每只分别染苦味酸作标记，取尾静脉血测定血糖。模型对照组，给予生理盐水0.05ml/10g灌胃1次，A组(多肽降糖组)给予0.05ml/10g灌胃多肽1次，B组(二甲双胍对照组)给予0.05ml/10g灌胃二甲双胍1次。于灌胃后15分钟、30分钟、45分钟、60分钟分别采集尾尖静脉血用血糖仪测定血糖变化，血糖单位mmol/L。
实验结果数据用
Figure PCTCN2018082401-appb-000030
表示，组间差异用t检验，以p＜0.05表示差异具有显著意义。
3.实验结果见下表13：
表13.Ⅱ型糖尿病模型小鼠灌胃多肽和二甲双胍降糖效果
Figure PCTCN2018082401-appb-000031
注：与模型对照组相比，*p<0.05，**p<0.01。
4.结论
从上述实验结果可以看出，本发明的多肽混合物灌胃能降低Ⅱ型糖尿病模型小鼠的血糖，具有显著降糖活性，不会导致Ⅱ型糖尿病模型小鼠低血糖，且效果较二甲双胍好。
实施例17本发明多肽混合物加速稳定性实验
1.材料与仪器
多肽：收集实施例1中15～25min间的峰，冷冻干燥，得到多肽的组合物；
高效液相色谱仪：安捷伦1260高效液相色谱仪，Wateres公司C18反相色谱柱(4.6×250mm，300SB，5μm)
2.实验方案：将多肽用西林瓶分装置于60℃、湿度70％恒温恒湿箱中进行加速试验，每周取样使用HPLC检测，以确定多肽的稳定性。
3.实验结果
见多肽稳定性考察HPLC图。
4.结论
多肽稳定性考察HPLC图(见图8)是多肽稳定性实验的三次HPLC检测结果，最上面为初始对照品，多肽混合物积分面积约为3036.94；中间为加速一周后HPLC检测结果，主峰的保留时间没有任何变化，多肽混合物积分面积约为2992.66；最下面为加速二周后HPLC检测结果，主峰的保留时间没有任何变化，多肽混合物积分面积约为3011.84。说明多肽在加速试验阶段是稳定的，可常温保存，适合开发成降糖药物。
实施例18Ⅱ型糖尿病模型小鼠注射本发明多肽混合物治疗效果实验
1.材料与仪器
多肽：收集实施例1中17.5～25min间的峰，冷冻干燥，得到多肽混合物，用生理盐水配制成1mg/ml的溶液，得多肽混合物注射液；
Ⅱ型糖尿病模型小鼠：KK鼠，北京华阜康生物科技股份有限公司，40只，体重25±2.7g，雄性，高脂高糖饲料喂养1个月；
血糖测试仪及试纸：北京怡成医疗有限公司；
2.实验方法
上述小白鼠随机分为4组，每组10只，每只分别染苦味酸作标记，取尾静脉血测定血糖。模型对照组，给予生理盐水0.05ml/10g尾静脉注射，A组(静 脉注射组)按0.05ml/10g尾静脉注射多肽含量为1mg/ml的注射液，B组(皮下注射组)按0.05ml/10g背部皮下注射多肽含量为1mg/ml的注射液，C组(肌肉注射组)按0.05ml/10g大腿外侧肌肉注射多肽含量为1mg/ml的注射液。隔日给药，连续4周，每周定时分别采集尾尖静脉血用血糖仪测定血糖变化，血糖单位mmol/L。
实验结果数据用
Figure PCTCN2018082401-appb-000032
表示，组间差异用t检验，以p＜0.05表示差异具有显著意义。
3.实验结果见下表14：
表14.Ⅱ型糖尿病模型小鼠静脉注射、皮下注射、肌肉注射多肽治疗效果
小鼠分组 一周后血糖 二周后血糖 三周后血糖 四周后血糖
模型对照组 9.8±0.7 10.5±1.1 11.6±1.3 13.7±1.2
A 6.3±0.7* 6.5±0.5** 6.4±0.4** 6.2±0.3**
B 6.7±0.5* 6.4±0.4** 6.5±0.3** 6.3±0.3**
C 6.4±0.7* 6.1±0.8** 6.3±0.4** 6.2±0.5**
注：与模型对照组相比，*p<0.05，**p<0.01。
4.结论
从上述实验结果可以看出，静脉注射、皮下注射、肌肉注射本发明的多肽混合物对Ⅱ型糖尿病模型小鼠具有明确治疗效果，三种给药方式的降糖效果无明显差异。
实施例19Ⅱ型糖尿病模型小鼠灌胃本发明多肽混合物及二甲双胍治疗效果、胰岛素耐量、口服糖耐量比较和修复胰腺β细胞实验
1.材料与仪器
多肽：收集实施例1中17.5～25min间的峰，冷冻干燥，得到多肽混合物；
盐酸二甲双胍：格华止盐酸二甲双胍片，0.5g/片，中美上海施贵宝制药有限公司；
胰岛素：江苏万邦生化医药股份有限公司，400U/10ml；
Ⅱ型糖尿病模型小鼠：KK鼠，北京华阜康生物科技股份有限公司，60只，体重25±2.7g，雄性，高脂高糖饲料喂养1个月；
正常对照小鼠：昆明种小白鼠，湖北省卫生防疫站，20只，体重20±2g，雄性，正常饲料喂养；
血糖测试仪及试纸：北京怡成医疗有限公司；
2.实验方法
2.1口服多肽降糖药物制备：将上述多肽混合物与羟丙甲基纤维素按8:2的比例混合均匀，压片，制成药片，每片重量0.5克，多肽总有效含量392mg。灌胃前将药片用生理盐水配制成多肽含量为2mg/ml的混悬液备用；
将盐酸二甲双胍用生理盐水配制成有效含量20mg/ml的溶液备用；
2.2上述糖尿病模型鼠随机分为3组，每组20只，正常对照小鼠为一组，一组20只。每只分别染苦味酸作标记，取尾静脉血测定初始血糖。模型对照组及正常对照组每日分别给予生理盐水0.05ml/10g灌胃1次，连续给药4周；A组(二甲双胍治疗组)每日给予0.05ml/10g灌胃二甲双胍1次，连续给药4周；B组(多肽治疗组)每日给予0.05ml/10g灌胃多肽1次，连续给药4周。每周分别采集尾尖静脉血用血糖仪测定血糖变化，血糖单位mmol/L。
2.3胰岛素耐量实验：末次给药1小时后，糖尿病模型对照组、二甲双胍治疗组、多肽治疗组及正常对照组每组随机选10只分别按0.05ml/10g皮下注射0.5U/ml胰岛素1次。于0小时、0.5小时、1小时、1.5小时及2小时分别采集尾尖静脉血用血糖仪测定血糖变化，血糖单位mmol/L。
2.4口服糖耐量实验：末次给药1小时后，试验2.3选剩下的糖尿病模型对照 组、二甲双胍治疗组、多肽治疗组小鼠及正常对照组小鼠(每组10只)按2.5g/Kg给予小鼠灌胃葡萄糖，于0小时、0.5小时、1小时、1.5小时及2小时分别采集尾尖静脉血用血糖仪测定血糖变化，血糖单位mmol/L。
2.5四周治疗结束后处死所有小鼠，取胰腺组织称重，并进行胰腺HE染色和免疫组织化学检测。
实验结果数据用
Figure PCTCN2018082401-appb-000033
表示，组间差异用t检验，以p＜0.05表示差异具有显著意义。
3.实验结果见下表：
表15.治疗期间各组小鼠血糖
Figure PCTCN2018082401-appb-000034
注：与模型对照组相比，*p<0.05，**p<0.01。
表16.胰岛素耐量实验
Figure PCTCN2018082401-appb-000035
注：与模型对照组相比，*p<0.05，**p<0.01。
表17.口服糖耐量实验
Figure PCTCN2018082401-appb-000036
Figure PCTCN2018082401-appb-000037
注：与模型对照组相比，*p<0.05，**p<0.01。
表18.各组小鼠胰腺重量
小鼠分组 胰腺/体重(％)
模型对照组 1.21±0.04
A 1.32±0.06
B 1.86±0.02**
正常对照组 2.08±0.09
注：与模型对照组相比，*p<0.05，**p<0.01。
4.结论
用多肽混合物配制的降糖药物口服可以降低Ⅱ型糖尿病模型小鼠的血糖值，使其恢复正常血糖值，因此该降糖药物口服可以治疗Ⅱ型糖尿病，而且，灌胃多肽混合物可以提高Ⅱ型糖尿病模型小鼠的胰岛素耐量和糖耐量。同时，服用该降糖药物的Ⅱ型糖尿病模型小鼠胰腺重量增加，图9显示胰腺β细胞恢复正常，因此，该降糖药物还具有修复Ⅱ型糖尿病模型小鼠胰腺β细胞的作用。
实施例20本发明的多肽混合物和氨基酸的液体组合物
收集实施例1中17.5～25min间的峰，冷冻干燥，得到多肽混合物，其中SEQ ID No.1-SEQ ID No.12共12个多肽的总含量≥98％，该多肽混合物简称多肽O。
按如下表19所示配方①配制含有多肽O的液体组合物：
表19.配方①
物质 用量
多肽O 100mg
三水乙酸钠 200mg
乙酸 调pH至4.5
   
85％甘油 1500mg
注射用水 加水至100ml
按如下表20所示配方②配制含有多肽O及氨基酸的液体组合物：
表20.配方②
物质 用量
多肽O 100mg
L-甲硫氨酸 10mg
三水乙酸钠 200mg
乙酸 调pH至4.5
85％甘油 1500mg
注射用水 加水至100ml
将上述按配方①和②配制的液体组合物分别封装于西林瓶中，于40±2℃/75±5％RH条件下贮存。
实施例21本发明的多肽及氨基酸的液体组合物降糖活性
1.材料与仪器
多肽组合物A：按实施例20中配方①配制的液体组合物，于40±2℃/75±5％RH条件下贮存6个月；
多肽组合物B：按实施例20中配方②配制的液体组合物，于40±2℃/75±5％RH条件下贮存6个月；
多肽组合物C：按实施例20中配方①现场配制的液体组合物；
试验动物：昆明种小白鼠，雄性，体重20±2g，40只，购自湖北省卫生防疫站；
血糖测试仪及试纸：罗氏活力
Figure PCTCN2018082401-appb-000038
血糖仪及试纸。
2.实验方法
上述小白鼠进食后随机分为4组，每组10只，每只分别染苦味酸作标记，取尾静脉血测定血糖。空白对照组，给予生理盐水0.1ml/10g尾静脉注射1次，其他三组分别按0.1ml/10g尾静脉注射组合物A、B、C一次。于注射后15分钟、30分钟、45分钟、60分钟分别采集尾尖静脉血用血糖仪测定血糖变化，血糖单位mmol/L。
实验结果数据用
Figure PCTCN2018082401-appb-000039
表示，组间差异用t检验，p＜0.05、p＜0.01分别表示差异具有显著性和非常显著性。
3.实验结果见下表21：
表21.正常小鼠尾静脉注射含有多肽及氨基酸的液体组合物降糖活性
Figure PCTCN2018082401-appb-000040
注：#、##分别代表与空白对照组相比，差异有显著性(p＜0.05)和非常显著性(p＜0.01)；*、**分别代表与组合物A组相比，差异有显著性(p＜0.05)和非常显著性(p＜0.01)；△、△△分别代表组合物B组与组合物C组相比，差异有显著性(p＜0.05)和非常显著性(p＜0.01)。
4.结论
从上述实验结果可以看出，正常小鼠尾静脉分别注射组合物A、B、C 15分 钟后，血糖均开始呈下降趋势，组合物B、C组血糖下降趋势尤为明显，并在45min后均出现低血糖，而组合物A未出现低血糖，但在60min血糖出现反弹。这表明，未加L-甲硫氨酸的组合物A随着贮存时间的增加，降糖活性衰减，而添加了L-甲硫氨酸的组合物B，其降糖活性并未随贮存时间的增加而衰减，且降糖活性优于新鲜制备的组合物C，表现出协同及增效效应。
上述实验证明，添加L-甲硫氨酸可以提高多肽O的稳定性及降糖活性。
实施例22本发明的多肽及氨基酸的固体组合物
按如下表22所示配方③配制含有多肽O的溶液，分装于西林瓶中，真空冷冻干燥，封装后于40±2℃/75±5％RH条件下贮存：
表22.配方③
物质 用量
多肽O 10g
氯化钠 100mg
D-甘露醇 500mg
EDTA二钠盐 2mg
去离子水 加水至100ml
按如下表23所示配方④配制含有多肽O及氨基酸的溶液，分装于西林瓶中，真空冷冻干燥，封装后于40±2℃/75±5％RH条件下贮存：
表23.配方④
物质 用量
多肽O 10g
DL-甲硫氨酸 500mg
D-甘露醇 100mg
   
EDTA二钠盐 2mg
去离子水 加水至100ml
实施例23本发明的多肽及氨基酸的固体组合物的降糖活性
1.材料与仪器
多肽组合物D：按实施例22中配方③制备的固体组合物，于40±2℃/75±5％RH条件下贮存6个月，用生理盐水配制成多肽O含量为2mg/ml的溶液备用；
多肽组合物E：按实施例22中配方④制备的固体组合物，于40±2℃/75±5％RH条件下贮存6个月，用生理盐水配制成多肽O含量为2mg/ml的溶液备用；
多肽组合物F：按实施例22中配方③现场制备的固体组合物，用生理盐水配制成多肽O含量为2mg/ml的溶液备用；
二甲双胍：格华止盐酸二甲双胍片，0.5g/片，中美上海施贵宝制药有限公司，用生理盐水配制成有效含量20mg/ml的混悬液备用；
高脂饲料：脂肪30％，蛋白质20％，碳水化合物50％；
试验动物：C57BL/6，雄性，体重20±2g，100只，购自湖北省卫生防疫站；
血糖测试仪及试纸：罗氏活力
Figure PCTCN2018082401-appb-000041
血糖仪及试纸。
2.实验方法
4周龄雄性C57BL/6小鼠，适应性喂养5天后，随机分为正常对照组(10只)和待造模组(90只)。正常对照组饲以正常饲料，待造模组小鼠饲以高脂饲料，并按45mg/kg剂量腹腔注射链脲佐菌素(STZ)柠檬酸缓冲液，注射完成后禁食1.5h，连续注射5天，建立糖尿病小鼠模型。腹腔注射链脲佐菌素4周后，于前一晚8点禁食，次日早8点取尾尖静脉血测定空腹血糖，挑选出空腹血糖值在10.0～20.0mmol/L，同时伴有多饮、多尿症状的糖尿病小鼠，随机分为5组:模型对照组、二甲双胍组、组合物D组、组合物E组、组合物F组，每组10只,分别通过灌胃给予相应药物:生理盐水(每只100μL)、二甲双胍(0.05ml/10g)、组合物 D(0.05ml/10g)、组合物E(0.05ml/10g)、组合物F(0.05ml/10g)，每天给药1次，给药周期为4周。正常对照组小鼠给予生理盐水(每只100μL)。糖尿病小鼠持续采用高脂饲料喂养,自由饮水,正常对照组小鼠正常饲料喂养,自由饮水。
各组小鼠每周定时采集尾尖静脉血用血糖仪测定一次空腹血糖值，血糖单位mmol/L。
实验结果数据用
Figure PCTCN2018082401-appb-000042
表示，组间差异用t检验，p＜0.05、p＜0.01分别表示差异具有显著性和非常显著性。
3.实验结果见下表24：
表24.II型糖尿病模型小鼠灌胃含有多肽及氨基酸的固体组合物降糖活性
Figure PCTCN2018082401-appb-000043
注：#、##分别代表与模型对照组相比，差异有显著性(p＜0.05)和非常显著性(p＜0.01)；*、**分别代表与二甲双胍组相比，差异有显著性(p＜0.05)和非常显著性(p＜0.01)，△、△△分别代表组合物F组与组合物D组相比，差异有显著性(p＜0.05)和非常显著性(p＜0.01)；★、★★分别代表组合物E 组与组合物F组相比，差异有显著性(p＜0.05)和非常显著性(p＜0.01)。
4.结论
上述实验结果表明，包含多肽O的固体组合物存放6个月后(组合物D)降糖活性与新鲜制备的组合物F相比下降，但好于常规剂量的二甲双胍(100mg/Kg)的降糖效果。本发明包含多肽O与氨基酸的固体组合物(组合物E)可以有效控制STZ联合高脂高糖饲料的二型糖尿病模型小鼠的血糖，治疗四周后小鼠血糖与正常对照组无明显差别，疗效显著，效果优于未添加氨基酸的组合物F，更明显优于二甲双胍。因此，DL-甲硫氨酸不仅提高多肽O的稳定性，对降糖效果具有协同及增效效应。
实施例24本发明的多肽及其与氨基酸的组合物改善糖尿病小鼠的糖脂代谢
1.材料与仪器
多肽组合物F：按实施例22中配方③现场制备的固体组合物，用生理盐水配制成多肽O含量为2mg/ml的溶液备用；
多肽组合物E：按实施例22中配方④制备的固体组合物，于40±2℃/75±5％RH条件下贮存6个月，用生理盐水配制成多肽O含量为2mg/ml的溶液备用；
高脂饲料：脂肪30％，蛋白质20％，碳水化合物50％；
试验动物：BALB/c，雄性，体重20±2g，80只，购自湖北省卫生防疫站；
二甲双胍：格华止盐酸二甲双胍片，0.5g/片，中美上海施贵宝制药有限公司，用生理盐水配制成有效含量200mg/ml的混悬液备用；
血糖测试仪及试纸：罗氏活力
Figure PCTCN2018082401-appb-000044
血糖仪及试纸；
生化分析仪及试剂盒：胆固醇(TC)试剂盒、甘油三酯(TG)试剂盒、高密度脂蛋白胆固醇(HDL-C)试剂盒购自迈瑞生物医疗电子股份公司，用迈瑞Mindray全自动生化分析仪BS-220检测。
2.实验方法
7周龄雄性BALB/c小鼠，适应性喂养2天后，随机分为正常对照组(10只) 和待造模组(70只)。正常对照组饲以正常饲料，待造模组小鼠饲以高脂饲料，并按100mg/kg剂量腹腔注射链脲佐菌素(STZ)柠檬酸缓冲液一次，建立糖尿病小鼠模型。3天后小鼠禁食4h(不禁水),尾尖取血测定血糖，挑选出血糖值大于11.0mmol/L，同时伴有多饮、多尿症状的糖尿病小鼠，随机分为4组:模型对照组、二甲双胍组、组合物E组、本组合物F组，每组10只,分别通过灌胃给予相应药物:生理盐水(每只100μL)、二甲双胍(0.05ml/10g)、组合物E(0.05ml/10g)、组合物F(0.05ml/10g)，每天给药1次，给药周期为4周。正常对照组小鼠给予生理盐水(每只100μL)。糖尿病小鼠持续采用高脂饲料喂养,自由饮水,正常对照组小鼠正常饲料喂养,自由饮水。
各组小鼠每周定时采集尾尖静脉血用血糖仪测定一次空腹血糖值，血糖单位mmol/L。
实验结束后,摘小鼠眼球取血,分离血清，检测血清TC、TG水平。
实验结果数据用
Figure PCTCN2018082401-appb-000045
表示，组间差异用t检验，p＜0.05、p＜0.01分别表示差异具有显著性和非常显著性。
3.实验结果见下表25和表26：
表25.多肽及氨基酸的组合物的降糖活性
Figure PCTCN2018082401-appb-000046
Figure PCTCN2018082401-appb-000047
注：#、##分别代表与模型对照组相比，差异有显著性(p＜0.05)和非常显著性(p＜0.01)；*、**分别代表与二甲双胍组相比，差异有显著性(p＜0.05)和非常显著性(p＜0.01)；△、△△分别代表组合物E组与组合物F组相比，差异有显著性(p＜0.05)和非常显著性(p＜0.01)。
表26.多肽及氨基酸的组合物的降血脂活性
Figure PCTCN2018082401-appb-000048
注：*代表与模型对照组相比，差异有显著性(p＜0.05)和非常显著性(p＜0.01)；△、△△分别代表组合物E组与组合物F组相比，差异有显著性(p＜0.05)和非常显著性(p＜0.01)。
4.结论
上述结果表明，对于STZ造模的胰腺受损模型糖尿病小鼠，超高剂量的二甲双胍(1000mg/Kg，是常用剂量的10倍)依然不能有效控制血糖，单独使用本发明的多肽效果好于二甲双胍，而使用本发明的多肽及氨基酸组合物，四周治疗后小鼠血糖即达到正常水平，本发明的多肽与氨基酸的组合表现出协同及增效效应，比单独使用本发明的多肽时更能有效控制胰腺受损糖尿病小鼠的血糖。
同时，本发明的多肽能够明显降低胰腺受损模型糖尿病小鼠的血脂，且本发明的多肽及氨基酸的组合物较单独的多肽在降低胰腺受损模型糖尿病小鼠的血脂方面效果更好，同样表现出多肽与氨基酸组合的协同及增效效应。
实施例25本发明的多肽及氨基酸的组合物降血脂活性
1.材料与仪器
多肽组合物E：按实施例22中配方④制备的固体组合物，于40±2℃/75±5％RH条件下贮存6个月，用生理盐水配制成多肽O含量为2mg/ml的溶液备用；
高脂饲料：78.8％基础饲料、1％胆固醇、10％蛋黄粉、10％猪油、0.2％胆盐；
试验动物：雄性SD大鼠，50只，体重200±20g，购自湖北省卫生防疫站；
生化分析仪及试剂盒：胆固醇(TC)试剂盒、甘油三酯(TG)试剂盒、高密度脂蛋白胆固醇(HDL-C)试剂盒购自迈瑞生物医疗电子股份公司，用迈瑞Mindray全自动生化分析仪BS-220检测。
2.实验方法
大鼠适应环境3天后，喂饲正常饲料5天，禁食16h后采尾血，离心(3000rpm)，取血清，测血清TC、TG、HDL-C。根据TC水平随机分为5组，每组10只，即三个剂量组(10、20和30mg/kg)、一个高脂饲料对照组和一个正常饲料对照组。试验期内，三个剂量组给予高脂饲料并通过灌胃给予不同剂量的本发明的多肽及氨基酸的固体组合物，高脂饲料对照组给予高脂饲料和水，正常饲料对照组给予正常饲料和水。试验第30天禁食16h后取尾血，离心(3000rpm)，取血清，测血清TC、TG、HDL-C。
实验结果数据用
Figure PCTCN2018082401-appb-000049
表示，组间差异用t检验，p＜0.05、p＜0.01分别表示差异具有显著性和非常显著性。
3.实验结果见下表27：
表27.多肽及氨基酸的组合物的降血脂活性
Figure PCTCN2018082401-appb-000050
Figure PCTCN2018082401-appb-000051
注：#、##分别代表与正常对照组相比，差异有显著性(p＜0.05)和非常显著性(p＜0.01)；*、**分别代表与高脂对照组相比，差异有显著性(p＜0.05)和非常显著性(p＜0.01)。
4.结论
从上述结果可以看出，三个试验组在喂饲高脂饲料的同时给予不同剂量的本发明的多肽及氨基酸的组合物，高、中、低剂量组的SD大鼠血清总胆固醇(TC)及甘油三酯(TG)明显低于高脂对照组，而高密度脂蛋白胆固醇(HDL-C)则明显高于高脂对照组，表明上述本发明的多肽及氨基酸的组合物明确具有改善高脂血症大鼠血脂的作用。
实施例26单独口服本发明的多肽及氨基酸的组合物改善Ⅱ型糖尿病患者的血糖和糖化血红蛋白(HbA1c)
患者江某花，女，58岁，2018年1月19日在医院诊断为糖尿病，空腹血糖高达15.32mmol/L，糖化血红蛋白9.7％。2018年1月25日开始单独口服实施例22中按配方④制备的固体组合物，餐前口服，1次/天，100mg/次，未服用其他降糖药。2018年2月25日去医院复查，空腹血糖降为7.3mmol/L，糖化血红蛋白降为6.7％，仅一个月时间，血糖就得到有效控制，糖化血红蛋白基本正常。
本实施例表明，对于糖尿病初期的患者，仅口服本发明的多肽及氨基酸的组合物即可有效控制血糖，并使其糖化血红蛋白正常化。
实施例27本发明的多肽及氨基酸的组合物与二甲双胍联合用药改善Ⅱ型糖尿病患者的糖化血红蛋白(HbA1c)
患者王某，男，40岁，有家族糖尿病史，2年前诊断出糖尿病，每天口服0.5克二甲双胍一片，空腹血糖长期保持在8.0～10.0间，血糖未能得到有效控制。2018年1月7日医院检查糖化血红蛋白为7.5％，2018年1月8日开始在不改变二甲双胍用药的同时服用实施例22中按配方④制备的固体组合物，餐前口服，3次/天，100mg/次。2018年3月1日医院检查糖化糖化血红蛋白为6.9％，糖化血红蛋白基本正常。
本实施例表明，本发明的多肽及氨基酸的组合物与二甲双胍联合用药时，患者糖化血红蛋白明显改善，趋于正常，效果优于单独使用二甲双胍。
实施例28本发明的多肽及氨基酸的组合物与二甲双胍、胰岛素联合用药改善Ⅱ型糖尿病患者的血糖和糖化血红蛋白(HbA1c)
患者袁某方，男，66岁，糖尿病史20年，常年注射胰岛素，每日50单位短效胰岛素，每晚口服0.5克二甲双胍一片，仍无法有效控制血糖，空腹血糖长期维持在12.0～15.0，2017年11月21日医院检查糖化血红蛋白为12.9％，2017年11月22日开始在不改变二甲双胍及胰岛素用药的同时服用实施例22中按配方④制备的固体组合物，餐前口服，3次/天，100mg/次。持续服用二个月后，空腹血糖维持在5.0～7.0间波动，后停服上述多肽及氨基酸的组合物并将胰岛素注剂量改为25单位，空腹血糖依然稳定在5.0～7.0间。2018年3月3日医院检查糖化糖化血红蛋白为7.0％，糖化血红蛋白基本正常。
本实施例表明，本发明的多肽及氨基酸的组合物与二甲双胍、胰岛素联合用药时，能有效改善使用二甲双胍及胰岛素后仍不能有效控制血糖的患者的血糖，并使其糖化血红蛋白正常化，且可以减少患者胰岛素的用量，效果优于患者只使用二甲双胍及胰岛素。
患者服用本发明的多肽及氨基酸的组合物一段时间后停用，而且胰岛素用量减半，此时空腹血糖及糖化血红蛋白仍能控制在正常范围，说明本发明的多肽及氨基酸的组合物具有修复糖尿病患者受损胰腺的功能，促使其恢复分泌胰 岛素的功能，因此可以逆转Ⅱ型糖尿病。
实施例29本发明的多肽及氨基酸的组合物改善Ⅱ型糖尿病患者的糖脂代谢
患者许某贵，男，49岁，2018年1月4日在医院诊断出糖尿病，空腹血糖高达14.61mmol/L，糖化血红蛋白10.5％，甘油三脂(TG)4.5mmol/L。2018年1月5日开始单独口服实施例22中按配方④制备的固体组合物，餐前口服，3次/天，100mg/次，未服用其他降糖药。2018年3月17日去医院复查，空腹血糖降为8.32mmol/L，糖化血红蛋白降为7.7％，甘油三脂(TG)降为2.6mmol/L。二个多月时间里，空腹血糖、糖化血红蛋白及甘油三脂分别下降了43％、27％及42％，变化显著。
本实施例表明，本发明的多肽及氨基酸的组合物能显著改善Ⅱ型糖尿病患者的糖脂代谢。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (24)

  1. 如下述通式(I)所示序列的多肽中的一种、两种或多种的任意比例的混合物在制备用于降低哺乳动物血糖和/或血脂的药物、食品、保健食品或特殊医学用途配方食品中的用途：
    X 1SCNGVCSPFX 2X 3PPCGX 4X 5X 6CRCIPX 7GLX 8X 9GX 10CRNPX 11G   (I)
    其中，X 1-X 11彼此独立地选自A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、W、Y、M ox。
    优选地，所述哺乳动物为人。
  2. 如下述通式(I)所示序列的多肽中的一种、两种或多种的任意比例的混合物在制备用于预防和/或治疗哺乳动物糖尿病和/或高脂血症的药物中的用途：
    X 1SCNGVCSPFX 2X 3PPCGX 4X 5X 6CRCIPX 7GLX 8X 9GX 10CRNPX 11G   (I)
    其中，X 1-X 11彼此独立地选自A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、W、Y、M ox。
    优选地，所述哺乳动物为人。
    优选地，所述糖尿病为II型糖尿病。
  3. 如下述通式(I)所示序列的多肽中的一种、两种或多种的任意比例的混合物在制备用于和其他降糖药和/或降脂药联合降低哺乳动物血糖和/或血脂的药物、食品、保健食品或特殊医学用途配方食品中的用途：
    X 1SCNGVCSPFX 2X 3PPCGX 4X 5X 6CRCIPX 7GLX 8X 9GX 10CRNPX 11G   (I)
    其中，X 1-X 11彼此独立地选自A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、W、Y、M ox。
    优选地，所述哺乳动物为人。
  4. 如下述通式(I)所示序列的多肽中的一种、两种或多种的任意比例的混 合物在制备用于和其他降糖药和/或降脂药联合预防和/或治疗哺乳动物糖尿病和/或高脂血症的药物中的用途：
    X 1SCNGVCSPFX 2X 3PPCGX 4X 5X 6CRCIPX 7GLX 8X 9GX 10CRNPX 11G   (I)
    其中，X 1-X 11彼此独立地选自A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、W、Y、M ox。
    优选地，所述哺乳动物为人。
    优选地，所述糖尿病为II型糖尿病。
  5. 一种用于口服的药物组合物或制剂、食品、保健食品或特殊医学用途配方食品，其特征在于，含有如下述通式(I)所示序列的多肽中的一种、两种或多种的任意比例的混合物，以及可药用载体：
    X 1SCNGVCSPFX 2X 3PPCGX 4X 5X 6CRCIPX 7GLX 8X 9GX 10CRNPX 11G   (I)
    其中，X 1-X 11彼此独立地选自A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、W、Y、M ox。
    优选地，进一步含有其他降糖药和/或降脂药。
    优选地，所述其他降糖药和/或降脂药选自可口服的降糖药和/或降脂药。
  6. 一种用于静脉注射、肌肉注射或皮下注射的药物组合物或制剂，其特征在于，含有如下述通式(I)所示序列的多肽中的一种、两种或多种的任意比例的混合物，以及可药用载体：
    X 1SCNGVCSPFX 2X 3PPCGX 4X 5X 6CRCIPX 7GLX 8X 9GX 10CRNPX 11G   (I)
    其中，X 1-X 11彼此独立地选自A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、W、Y、M ox。
    优选地，进一步含有其他降糖药和/或降脂药。
    优选地，所述其他降糖药选自胰岛素和/或肠促胰岛素。
  7. 一种降低哺乳动物血糖和/或血脂的方法，其特征在于，对其施用治疗有效量的如下述通式(I)所示序列的多肽中的一种、两种或多种的任意比例的混合物：
    X 1SCNGVCSPFX 2X 3PPCGX 4X 5X 6CRCIPX 7GLX 8X 9GX 10CRNPX 11G   (I)
    其中，X 1-X 11彼此独立地选自A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、W、Y、M ox。
    优选地，所述哺乳动物为人。
  8. 一种预防和/或治疗哺乳动物糖尿病和/或高脂血症的方法，其特征在于，对其施用治疗有效量的如下述通式(I)所示序列的多肽中的一种、两种或多种的任意比例的混合物：
    X 1SCNGVCSPFX 2X 3PPCGX 4X 5X 6CRCIPX 7GLX 8X 9GX 10CRNPX 11G   (I)
    其中，X 1-X 11彼此独立地选自A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、W、Y、M ox。
    优选地，所述哺乳动物为人。
    优选地，所述糖尿病为II型糖尿病。
  9. 一种降低哺乳动物血糖和/或血脂的方法，其特征在于，对其施用治疗有效量的如下述通式(I)所示序列的多肽中的一种、两种或多种的任意比例的混合物，和其他降糖药和/或降脂药：
    X 1SCNGVCSPFX 2X 3PPCGX 4X 5X 6CRCIPX 7GLX 8X 9GX 10CRNPX 11G   (I)
    其中，X 1-X 11彼此独立地选自A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、W、Y、M ox。
    优选地，所述哺乳动物为人。
  10. 一种预防和/或治疗哺乳动物糖尿病和/或高脂血症的方法，其特征在于， 对其施用治疗有效量的如下述通式(I)所示序列的多肽中的一种、两种或多种的任意比例的混合物，和其他降糖药和/或降脂药：
    X 1SCNGVCSPFX 2X 3PPCGX 4X 5X 6CRCIPX 7GLX 8X 9GX 10CRNPX 11G   (I)
    其中，X 1-X 11彼此独立地选自A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、W、Y、M ox。
    优选地，所述哺乳动物为人。
    优选地，所述糖尿病为II型糖尿病。
  11. 一种组合物，其含有如通式(I)所示序列的多肽中的一种、两种或多种的任意比例的混合物，和至少一种氨基酸：
    X 1SCNGVCSPFX 2X 3PPCGX 4X 5X 6CRCIPX 7GLX 8X 9GX 10CRNPX 11G   (I)
    其中，X 1-X 11彼此独立地选自A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、W、Y、M ox。
    优选地，所述氨基酸选自甲硫氨酸、组氨酸、酪氨酸中的一种、两种或多种的任意比例的混合物。
    优选地，甲硫氨酸、组氨酸、酪氨酸为D型、L型或DL型，更优选地为L型。
    优选地，多肽和氨基酸的重量比为100:1～1:100，更优选地，重量比为50:1～1:50，更优选地，重量比为50:1～1:2，更优选地，重量比为20:1～10:1。
    优选地，进一步包含重组人血清白蛋白和/或牛血清白蛋白，其含量范围在液体组合物中为0.5～50mg/ml，更优选1～20mg/ml，更优选5～10mg/ml；或在固体组合物中为0.5～50mg/g，更优选1～20mg/g，更优选5～10mg/g。
    优选地，进一步包含氯化钠，其含量范围在液体组合物中为1～20mg/ml，更优选5～15mg/ml，更优选8～12mg/ml；或在固体组合物中为1～20mg/g，更优选5～15mg/g，更优选8～12mg/g。
    优选地，进一步包含乙二胺四乙酸二钠，其含量范围在液体组合物中为0.05～0.1mg/ml；或在固体组合物中为0.05～0.2mg/g。
    优选地，进一步包含乙酸钠、乙酸，乙酸钠的含量范围在液体组合物中为0.5～3mg/ml，更优选1～2.5mg/ml，更优选1.5～2mg/ml。
    优选地，进一步包含甘油、甘露醇、山梨醇中的一种、两种或多种，其含量范围在液体组合物中为0.5～100mg/ml，更优选1～50mg/ml，更优选8～15mg/ml，或在固体组合物中为0.5～100mg/g，更优选1～50mg/g，更优选8～15mg/g。
    优选地，所述组合物为液体组合物，其pH为3.5～6.5，更优选4～5，更优选4.5。
    优选地，所述组合物为固体组合物，其5％溶液的pH为3.5～6.5，更优选4～5，更优选4.5。
    优选地，所述组合物为液体组合物，包含：
    (a)如通式(I)所示序列的多肽中的一种、两种或多种的任意比例的混合物；
    (b)至少一种氨基酸，选自甲硫氨酸、组氨酸、酪氨酸中的一种、两种或多种的任意比例的混合物；
    (c)乙酸钠；
    (d)乙酸；
    (e)甘油；
    (f)注射用水。
    优选地，所述组合物为固体组合物，包含：
    (a)如通式(I)所示序列的多肽中的一种、两种或多种的任意比例的混合物；
    (b)至少一种氨基酸，选自甲硫氨酸、组氨酸、酪氨酸中的一种、两种或多种的任意比例的混合物；
    (c)D-甘露醇；
    (d)乙二胺四乙酸二钠。优选地，进一步包含氯化钠。
  12. 如权利要求11所述的组合物在制备用于降低哺乳动物血糖和/或血脂的药物、食品、保健食品或特殊医学用途配方食品中的用途。
    优选地，所述哺乳动物为人。
  13. 如权利要求11所述的组合物在制备用于预防和/或治疗哺乳动物糖尿病和/或高脂血症的药物中的用途。
    优选地，所述哺乳动物为人。
    优选地，所述糖尿病为II型糖尿病。
  14. 如权利要求11所述的组合物在制备用于和其他降糖药和/或降脂药联合降低哺乳动物血糖和/或血脂的药物、食品、保健食品或特殊医学用途配方食品中的用途。
    优选地，所述哺乳动物为人。
  15. 如权利要求11所述的组合物在制备用于和其他降糖药和/或降脂药联合预防和/或治疗哺乳动物糖尿病和/或高脂血症的药物中的用途。
    优选地，所述哺乳动物为人。
    优选地，所述糖尿病为II型糖尿病。
  16. 一种用于口服的药物制剂、食品、保健食品或特殊医学用途配方食品，其含有如权利要求11所述的组合物以及可药用载体。
    优选地，进一步含有其他降糖药和/或降脂药。
    优选地，所述其他降糖药和/或降脂药选自可口服的降糖药和/或降脂药。
  17. 一种用于静脉注射、肌肉注射或皮下注射的药物组合物或制剂，其含有如权利要求11所述的组合物以及可药用载体。
    优选地，进一步含有其他降糖药和/或降脂药。
    优选地，所述其他降糖药选自胰岛素和/或肠促胰岛素。
  18. 一种降低哺乳动物血糖和/或血脂的的方法，其特征在于，对其施用治疗有效量的如权利要求11所述的组合物。
    优选地，所述哺乳动物为人。
  19. 一种预防和/或治疗哺乳动物糖尿病和/或高脂血症的方法，其特征在于，对其施用治疗有效量的如权利要求11所述的组合物。
    优选地，所述哺乳动物为人。
    优选地，所述糖尿病为II型糖尿病。
  20. 一种降低哺乳动物血糖和/或血脂的的方法，其特征在于，对其施用治疗有效量的如权利要求11所述的组合物，和其他降糖药和/或降脂药。
    优选地，所述哺乳动物为人。
  21. 一种预防和/或治疗哺乳动物糖尿病和/或高脂血症的方法，其特征在于，对其施用治疗有效量的如权利要求11所述的组合物，和其他降糖药和/或降脂药。
    优选地，所述哺乳动物为人。
    优选地，所述糖尿病为II型糖尿病。
  22. 如前述任一项权利要求所述的用途，所述哺乳动物通过二甲双胍、磺酰脲类促泌剂、噻唑烷二酮类、α-糖苷酶抑制剂、胰岛素或其衍生物、胰岛素或其衍生物或其组合无法有效控制血糖。
  23. 如前述任一项权利要求所述的用途，所述哺乳动物在施用前糖化血红蛋白＞7.0％，优选＞7.5％，在施用后，糖化血红蛋白＜7.5％，优选＜7.0％。
  24. 如权利要求1～23中任一项所述的用途、药物组合物或制剂、食品、保健食品或特殊医学用途配方食品，其特征在于，所述多肽具有通式(I)所示序列。
    优选地，X 1为疏水性氨基酸，选自A、L、V、I、P、F、M、W、M ox；
    X 2为酸性氨基酸，选自E、D；
    X 3为疏水性氨基酸，选自A、L、V、I、P、F、M、W、M ox；
    X 4为除半胱氨酸外的极性中性氨基酸，选自G、S、T、Y、N、Q；
    X 5为除半胱氨酸外的中性氨基酸，选自G、S、T、Y、N、Q、A、L、V、I、P、F、M、W；
    X 6为疏水性氨基酸，选自A、L、V、I、P、F、M、W、M ox；
    X 7为除半胱氨酸外的中性氨基酸，选自G、S、T、Y、N、Q、A、L、V、I、P、F、M、W；
    X 8为疏水性氨基酸，选自A、L、V、I、P、F、M、W、M ox；
    X 9为疏水性氨基酸，选自A、L、V、I、P、F、M、W、M ox；
    X 10选自Y、K、N；
    X 11为除半胱氨酸外的极性中性氨基酸，选自G、S、T、Y、N、Q。
    更优选地，X 1为A、I或V；X 2为E或D；X 3为M、M ox或I；X 4为S或T；X 5为S或P；X 6为A或L；X 7为V、A或Y；X 8为V、F或L；X 9为V或I；X 10为Y、K或N；X 11为S、T或Y。
    更优选地，X 2X 3选自DI、EI、DM、EM、DM ox、EM ox中的一种；X 4X 5X 6选自SPL、SPA、SSL、SSA、TPL、TPA、TSL、TSA中的一种；X 8X 9选自VI、VV、FI、FV、LI、LV中的一种。
    更优选地，所述多肽选自具有如下序列多肽中的一种、两种或多种的任意比例的组合物：
    ASCNGVCSPFEMPPCGTSACRCIPVGLVVGYCRNPSG、
    ASCNGVCSPFEM oxPPCGTSACRCIPVGLVVGYCRNPSG、
    ASCNGVCSPFEMPPCGTSACRCIPVGLVIGYCRNPSG、
    ASCNGVCSPFEM oxPPCGTSACRCIPVGLVIGYCRNPSG、
    ASCNGVCSPFEMPPCGSSACRCIPVGLLIGYCRNPSG、
    ASCNGVCSPFEM oxPPCGSSACRCIPVGLLIGYCRNPSG、
    ASCNGVCSPFEMPPCGTSACRCIPVGLFIGYCRNPSG、
    ASCNGVCSPFEM oxPPCGTSACRCIPVGLFIGYCRNPSG、
    ASCNGVCSPFEMPPCGTSACRCIPYGLFIGYCRNPSG、
    ASCNGVCSPFDIPPCGSPLCRCIPVGLVIGKCRNPYG、
    ISCNGVCSPFDIPPCGSPLCRCIPAGLVIGNCRNPYG、
    VSCNGVCSPFDIPPCGSPLCRCIPAGLVIGKCRNPYG、
    ASCNGVCSPFDIPPCGTPLCRCIPVGLVIGNCRNPYG、
    ASCNGVCSPFDMPPCGSSACRCIPVGLFIGNCRNPYG、
    ASCNGVCSPFDMPPCGSSACRCIPVGLVIGYCRNPYG、
    ASCNGVCSPFDMPPCGSSLCRCIPAGLVVGYCRNPSG、
    ASCNGVCSPFDMPPCGTSACRCIPAGLFIGKCRNPYG、
    ASCNGVCSPFDMPPCGTSACRCIPVGLVVGYCRNPSG、
    ASCNGVCSPFDMPPCGTSLCRCIPAGLFIGYCRNPSG、
    ASCNGVCSPFDMPPCGTSLCRCIPAGLVIGYCRNPYG、
    ASCNGVCSPFEIPPCGSPLCRCIPVGLVIGNCRNPYG、
    ASCNGVCSPFEIPPCGTPLCRCIPAGLVIGKCRNPYG、
    ASCNGVCSPFEIPPCGTPLCRCIPVGLFIGKCRNPSG、
    ASCNGVCSPFEMPPCGSSACRCIPAGLFIGKCRNPYG、
    ASCNGVCSPFEMPPCGSSACRCIPVGLFVGYCRNPYG、
    ASCNGVCSPFEMPPCGSSACRCIPVGLVVGYCRNPSG、
    ASCNGVCSPFEMPPCGSSLCRCIPAGLFIGYCRNPSG、
    ASCNGVCSPFEMPPCGSSLCRCIPAGLVIGYCRNPSG、
    ASCNGVCSPFEMPPCGTSACRCIPAGLFIGNCRNPYG、
    ASCNGVCSPFEMPPCGTSACRCIPAGLVIGYCRNPYG、
    ISCNGVCSPFDIPPCGSPACRCIPVGLVIGKCRNPYG、
    ISCNGVCSPFDIPPCGSPLCRCIPAGLVIGNCRNPSG、
    ISCNGVCSPFDIPPCGSPLCRCIPVGLFIGNCRNPSG、
    ISCNGVCSPFDIPPCGSPLCRCIPVGLVIGYCRNPSG、
    ISCNGVCSPFDIPPCGTPACRCIPVGLVIGNCRNPYG、
    ISCNGVCSPFDIPPCGTPLCRCIPAGLFVGKCRNPYG、
    ISCNGVCSPFDIPPCGTPLCRCIPAGLFIGKCRNPSG、
    ISCNGVCSPFDMPPCGSPACRCIPAGLLIGYCRNPSG、
    ISCNGVCSPFDMPPCGTSACRCIPAGLVIGYCRNPSG、
    ISCNGVCSPFEIPPCGSPLCRCIPAGLFIGKCRNPSG、
    ISCNGVCSPFEIPPCGTPACRCIPVGLFIGKCRNPSG、
    ISCNGVCSPFEIPPCGTPACRCIPAGLVIGKCRNPYG、
    ISCNGVCSPFEIPPCGSPACRCIPVGLVIGNCRNPYG、
    ISCNGVCSPFEIPPCGSSLCRCIPAGLLVGKCRNPSG、
    ISCNGVCSPFEIPPCGTPLCRCIPAGLFIGNCRNPSG、
    ISCNGVCSPFEIPPCGTPLCRCIPAGLVIGYCRNPSG、
    ISCNGVCSPFEIPPCGTSACRCIPAGLVIGYCRNPSG、
    ISCNGVCSPFEMPPCGSSACRCIPAGLVIGYCRNPSG、
    ISCNGVCSPFEMPPCGSSACRCIPAGLFIGYCRNPSG、
    ISCNGVCSPFEMPPCGTSLCRCIPAGLVVGYCRNPSG、
    VSCNGVCSPFDIPPCGTPLCRCIPYGLFVGNCRNPYG、
    VSCNGVCSPFDIPPCGTPACRCIPYGLFVGNCRNPYG、
    VSCNGVCSPFDIPPCGTPLCRCIPAGLVIGNCRNPYG、
    VSCNGVCSPFDIPPCGTPLCRCIPVGLFIGNCRNPSG、
    VSCNGVCSPFDIPPCGTPLCRCIPVGLVIGYCRNPSG、
    VSCNGVCSPFDIPPCGSPLCRCIPVGLFIGKCRNPSG、
    VSCNGVCSPFDMPPCGSSACRCIPAGLFIGNCRNPYG、
    VSCNGVCSPFDMPPCGSSACRCIPAGLVIGYCRNPYG、
    VSCNGVCSPFDMPPCGSSACRCIPVGLVIGYCRNPSG、
    VSCNGVCSPFDMPPCGSSACRCIPVGLFIGYCRNPSG、
    VSCNGVCSPFDMPPCGTSACRCIPAGLFIGYCRNPSG、
    VSCNGVCSPFEIPPCGSPACRCIPVGLVIGKCRNPYG、
    VSCNGVCSPFEIPPCGSPLCRCIPAGLVIGNCRNPYG、
    VSCNGVCSPFEIPPCGSPLCRCIPVGLVIGYCRNPSG、
    VSCNGVCSPFEIPPCGTPACRCIPVGLVIGNCRNPYG、
    VSCNGVCSPFEIPPCGSPLCRCIPVGLFIGNCRNPSG、
    VSCNGVCSPFEIPPCGTPLCRCIPAGLFIGKCRNPSG、
    VSCNGVCSPFEMPPCGSSACRCIPYGLVVGNCRNPSG、
    VSCNGVCSPFEMPPCGTPLCRCIPYGLLIGKCRNPYG、
    VSCNGVCSPFEMPPCGTSACRCIPAGLVIGYCRNPSG、
    VSCNGVCSPFEMPPCGTSACRCIPAGLFIGYCRNPSG。
    更优选地，所述多肽的肽链内含三对二硫键，氨基酸序列中第3位和第20位的两个半胱氨酸之间形成二硫键、第7位和第22位的两个半胱氨酸之间形成二硫键、第15位和第32位的两个半胱氨酸之间形成二硫键。
    更优选地，所述多肽混合物为豆科植物的种子或其加工副产物-豆粕的提取物。
    优选，豆科植物选自大豆、绿豆、豌豆、蚕豆、赤豆、豇豆、四季豆、扁豆及其中一种、两种或多种的任意比例的混合物；最优选，豆科植物为大豆、绿豆、豌豆或其中一种、两种或多种的任意比例的混合物。进一步优选，所述 提取物中含有如下序列的多肽：
    ASCNGVCSPFEMPPCGTSACRCIPVGLVVGYCRNPSG、
    ASCNGVCSPFEM oxPPCGTSACRCIPVGLVVGYCRNPSG、
    ASCNGVCSPFEMPPCGTSACRCIPVGLVIGYCRNPSG、
    ASCNGVCSPFEM oxPPCGTSACRCIPVGLVIGYCRNPSG、
    ASCNGVCSPFEMPPCGSSACRCIPVGLLIGYCRNPSG、
    ASCNGVCSPFEM oxPPCGSSACRCIPVGLLIGYCRNPSG、
    ASCNGVCSPFEMPPCGTSACRCIPVGLFIGYCRNPSG、
    ASCNGVCSPFEM oxPPCGTSACRCIPVGLFIGYCRNPSG、
    ASCNGVCSPFEMPPCGTSACRCIPYGLFIGYCRNPSG、
    ASCNGVCSPFDIPPCGSPLCRCIPVGLVIGKCRNPYG、
    ISCNGVCSPFDIPPCGSPLCRCIPAGLVIGNCRNPYG、
    VSCNGVCSPFDIPPCGSPLCRCIPAGLVIGKCRNPYG。
    进一步优选，所述序列的多肽总含量为至少5％，优选至少8％，进一步优选至少10％，进一步优选至少20％，进一步优选至少30％，进一步优选至少50％，进一步优选至少80％，更进一步优选至少90％，更进一步优选至少95％，更进一步优选至少97％，最优选至少98％。
PCT/CN2018/082401 2017-04-10 2018-04-09 用于治疗代谢系统疾病的多肽及其组合物 WO2018188565A1 (zh)

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