WO2011032360A1 - Peptide mc-jj2 issu de momordica charantia et son procédé de synthèse sur phase solide assistée par micro-ondes - Google Patents

Peptide mc-jj2 issu de momordica charantia et son procédé de synthèse sur phase solide assistée par micro-ondes Download PDF

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WO2011032360A1
WO2011032360A1 PCT/CN2010/001422 CN2010001422W WO2011032360A1 WO 2011032360 A1 WO2011032360 A1 WO 2011032360A1 CN 2010001422 W CN2010001422 W CN 2010001422W WO 2011032360 A1 WO2011032360 A1 WO 2011032360A1
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ala
lys
gly
ser
tyr
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PCT/CN2010/001422
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黄文龙
金晶
钱海
张惠斌
陈巍
王敬杰
杜阔
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中国药科大学
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Priority claimed from CN200910183320A external-priority patent/CN101665529A/zh
Priority claimed from CN200910183315A external-priority patent/CN101665528A/zh
Priority claimed from CN200910183316A external-priority patent/CN101781361A/zh
Priority claimed from CN200910183317A external-priority patent/CN101691398A/zh
Application filed by 中国药科大学 filed Critical 中国药科大学
Publication of WO2011032360A1 publication Critical patent/WO2011032360A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/045—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers using devices to improve synthesis, e.g. reactors, special vessels
    • 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
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to Momordica charantia MC-JJ2 polypeptide and a method thereof for synthesizing bitter melon MC-JJ2 polypeptide by microwave-assisted solid phase. Background technique
  • Diabetes is an important disease that threatens human health today. About 5% to 7% of people worldwide suffer from diabetes. Therefore, diabetes is considered to be the third leading cause of human disability and death after cardiovascular and cerebrovascular diseases and malignant tumors.
  • Type I diabetes is an autoimmune disease. It refers to insulin deficiency caused by autoimmunity and other causes. It is prone to ketosis and must be treated with insulin. It is more common in adolescents. This type of diabetes is rare, accounting for only about 5%.
  • Type II diabetes accounts for about 95% of all diabetic patients. Type II diabetes is caused by a disorder of islet cell metabolism, insufficient insulin secretion, or insulin resistance. It is a metabolic disease caused by insulin resistance mainly accompanied by insufficient insulin secretion, or insulin resistance caused by relatively insufficient insulin secretion. Insufficient insulin secretion, or insulin resistance, can cause excessive glucose in the blood. When the blood sugar level exceeds the renal threshold, glucose is transferred to the urine to produce glucoseuria.
  • Momordica charantia is a vegetable that has a long history of safe eating.
  • many fruits with hypoglycemic activity have been found in the fruit of bitter gourd.
  • Some extracts of bitter gourd fruit, or tea drinks made directly from bitter gourd fruit, have been recommended for use by diabetics.
  • Bitter gourd ingredients have been patented as a drug for the treatment of diabetes.
  • the patents or products introduced from the scientific literature and the existing patents or products are mainly based on the knowledge of a known component in many hypoglycemic active ingredients of bitter gourd, and it is not realized that the components with hypoglycemic activity in bitter gourd are known.
  • some active ingredients with blood sugar lowering in bitter gourd are unknown. Therefore, the limitations of existing literature, patents or products are also obvious.
  • U.S. Patent No. 6,391,854 B1 and Chinese Patent CN 98805001 provide a water-soluble extract of bitter gourd - Called MC6, MC6 is characterized by three peptides. Its preparation method is characterized by centrifugation of clarified bitter gourd juice through 30kDal, lOkDal and 3kDal ultrafiltration membranes. MC6 has a molecular weight of less than 10 kDal and can be retained by the lOkDal ultrafiltration membrane for the 3kDal membrane.
  • the active MC6 is characterized by migration in a single band on SDS-PAGE gel electrophoresis with a molecular weight of less than 10 kDal and contains three peptides. Also provided are the peptide components of MC6, referred to as MC6. 1, and derivatives and mimetics of MC6.1. Active MC6, MC6. 1, MC6. 2, MC6. 3 have hypoglycemic effects, especially for the treatment of diabetes, wherein these active drugs are preferably administered orally. In this patent, the bitter melon used is also active after hypoglycemia orally.
  • MC6 does not increase the activity of insulin levels in plasma of model animals.
  • MC6 and peptide-k are proteins with hypoglycemic activity in bitter gourd.
  • MC6 is soluble in water and polypeptide-k is insoluble in water. According to the preparation method of MC6, the polypeptide-k in bitter gourd is lost. According to the preparation method of polypeptide-k, MC6 in bitter gourd was also lost.
  • the bitter melon protein is prepared; the filtrate is concentrated in vacuo at 60 ° C, dissolved in 2 times water, degreased with 2 times diethyl ether, concentrated in vacuo at 50 ° C to recover ether, 60 ° C vacuum recovery of ethanol to make total saponins of crude bitter gourd, and then The anhydrous acetone is precipitated and washed several times at 4 ° C, and concentrated under vacuum at 30 ° C to prepare total saponins of bitter gourd. Finally, the total saponins, bitter gourd protein powder and bitter gourd residue are mixed at a weight ratio of 10:2:88. , filled into capsules to make bitter goose hypoglycemic capsules. It has the advantages of wide source of raw materials, simple process, convenient taking, low price, no toxic and side effects, and can be used as an auxiliary preparation for treating diabetes.
  • the invention discloses a method for preparing natural momordicin and hypoglycemic agent
  • the method comprises the following steps: a. cutting the mature bitter gourd into pieces and drying; b. soaking the dried bitter gourd pieces in water and extracting the infusion; c. concentrating the bitter melon extract into a concentrated liquid; d. Bioconcentration of the concentrated solution to obtain a highly active degradation liquid; e. Performing constant temperature drying of the degradation liquid to spray granulation; f. Loading the granule into a capsule into a medicine; the method has simple process equipment and meets large-scale production requirements. , the product yield is high, the treatment effect is good, no toxic side effects.
  • the invention discloses a traditional Chinese medicine for treating diabetes, the main components of the traditional Chinese medicine are animal pancreas, bitter melon, pumpkin, scutellaria, yam, production
  • the method comprises the following steps: pulverizing the animal's pancreas, mixing with the extract of bitter gourd, pumpkin and astragalus obtained by water extraction or alcohol precipitation, and adding yam powder, the medicine has the benefits of qi, spleen and moistening, nourishing yin and reducing fire power. It has the effect of improving the body's immune function, promoting insulin secretion and lowering blood sugar.
  • the invention relates to the field of biopharmaceuticals.
  • the hypoglycemic active protein new component PA is extracted from the plant material bitter gourd, and the material purification treatment in the preparation process - low temperature freezing - crushing - acid alcohol impregnation - high speed homogenization - acid alcohol extraction - suction filtration - centrifugal purification - Precipitation-redissolution-Sephadex gel column-ultraviolet detection fraction collection-concentration-drying bitter melon hypoglycemic active protein new component PA preparation method, which is characterized by isoelectric precipitation and gel column, Method of collection by the UV detection division. The inventor believes that the substance has the advantages of simple separation and extraction, low consumption of organic solvents, high yield, and the like, and the effect of blood sugar lowering is obvious by the animal test.
  • bitter gourd seeds contain rich plant insulin and are an effective component for treating diabetes.
  • the technical feature of the method of the invention is that the bitter gourd seed is shelled, the kernel is crushed by 60 or 100 mesh sieve, and then extracted with acidic ethanol, and the supernatant is centrifuged, and the volume of NaCl is added to the volume of 3% to 5%. 5 Mol / L NaOH to adjust the solution to P H value of 7. 0 ⁇ 7.5 solution became cloudy, centrifuged, to remove the liquid, washed with acetone three times, and dried under vacuum to obtain crude plant insulin.
  • the gel affinity chromatography was carried out by dissolving 0.1 L Tris. cl solution (pH 7.5), and the eluted peak solution was collected, and concentrated under vacuum with acetone or ethanol to obtain a plant-based insulin product.
  • the method can extract and prepare high-purity plant insulin components, and can be used for the treatment of diabetes.
  • a bitter melon lozenge and a preparation method thereof the invention is a bitter melon lozenge and a preparation method thereof.
  • the bitter melon lozenge mainly comprises bitter gourd powder, dextrin, citric acid and mannitol.
  • fresh bitter gourd is firstly juiced, filtered, freeze-dried to obtain bitter gourd lyophilized powder, and then mixed with other ingredients and pressed. Into pieces.
  • the preparation method of the invention is scientific and reasonable, and fully retains various nutrients in the bitter gourd, and the prepared tablet contains no activity of bitter gourd at normal temperature, and has the functions of clearing away heat and detoxifying, widening thirst, stimulating thirst, lowering blood sugar, and improving immunity.
  • the ability to suppress cancer cells and other functions is a convenient necessity for daily life, especially in disaster relief, military operations, tourism, and expeditions, to quickly replenish physical strength and improve immunity.
  • the Sovereignty Item (A61K35/78) requires: a bitter melon lozenge that is characterized by bitter melon powder, dextrin, mannitol, spices, pigments, and magnesium stearate.
  • bitter gourd saponin is a tetracyclic three mushroom compound, which has many different components and is hardly soluble in water. It can be extracted from dried bitter gourd fruit by ethanol (Chang Fenggang: Chemical constituents of bitter gourd, Chinese herbal medicine, 1995) , 26 (10) 507-510).
  • Momordica saponin does not affect the plasma insulin content in the animal model, mainly to promote the synthesis of muscle glycogen and hepatic glycogen (Wang Xianyuan, Jin Hong, Xu Zhiqin, etc.: hypoglycemic effect and mechanism of bitter melon saponin, amino acid and biological resources, 2001 , 23 (3) : 42-45), and thus has significant hypoglycemic effects (Sun Shuqing, pharmacological and clinical research overview of bitter gourd, Chinese herbal medicine, 1997, (8): 428-429), animal test proved that its blood sugar is lowered The amplitude is even better than "Yi Jiang Tang” (Sun Baoying et al., Experimental study on pharmacodynamics of Momordica saponin, Henan Journal of Traditional Chinese Medicine, 1994, 9 (6): 19-21).
  • the water-soluble polypeptide MC6 the water-insoluble polypeptide-k and the bitter melon saponin belong to each other due to differences in their chemical properties. Moreover, the bitter melon hypoglycemic active ingredient is not only known saponins and proteins.
  • hypoglycemic functional actives of bitter gourd are known, and there is also an unknown hypoglycemic function of bitter gourd, which is actually the result of synergistic action of various components having hypoglycemic activity contained therein.
  • active ingredients it is difficult to extract them simultaneously with a solvent because of their different chemical properties.
  • hypoglycemic active of bitter gourd a component which is not heat-resistant is contained, and the extraction process should not be heated.
  • the fresh bitter gourd pulp tissue cells are fully disrupted by refining, and all the contents of the cells, including the hypoglycemic active ingredients dissolved in water and poorly soluble in water, are released into the pulp.
  • the crude fiber in the pulp (from the cell wall of the pulp) is filtered off and then freeze-dried.
  • This lyophilized product of bitter gourd juice undoubtedly contains a relatively comprehensive hypoglycemic active ingredient.
  • the content of hypoglycemic active substances in bitter gourd juice is actually very low, and the direct freeze-drying is expensive.
  • the sugar substances present in bitter gourd juice are easily deliquescent, so that the freeze-dried products cannot be divided under normal conditions at all. Packing, storage and use. Although a large amount of drying aid or moisture-proofing agent can be added to solve the problem of deliquescence of the freeze-dried product of bitter gourd juice, it is impossible to obtain a high content of bitter melon hypoglycemic product.
  • the preparation of the active ingredients of bitter gourd disclosed in the literature and patents is basically the extraction of one or several specific chemical components by physical and chemical methods, or a certain solvent of bitter gourd under heating conditions (
  • the total extracts, such as water, ethanol or mixtures thereof, do not mention how to use the hypoglycemic active ingredients of the chemically unknown in bitter gourd, or the thermal damage of certain thermosensitive hypoglycemic actives in bitter gourd.
  • the polypeptide can be obtained by genetic engineering or chemical synthesis.
  • the method of genetic engineering is superior to chemical methods in obtaining long peptides (amino acid residue lengths greater than 50) or proteins, but the chemical synthesis methods of peptides, especially after the appearance of solid phase synthesis strategies, are prepared to have amino acid residues less than 40 in length.
  • Polypeptides or small peptides have incomparable flexibility, diversity and efficiency in genetic engineering methods.
  • Merrifield founded and developed a method for solid phase synthesis of peptides. There are two strategies for solid phase peptide synthesis: Boc/Bzl orthogonal protection solid phase synthesis strategy and Fmoc/tBu orthogonal protection solid phase synthesis strategy.
  • the solid phase synthesis method established by Merrifield is Boc/Bzl orthogonal protection solid phase. Synthesis strategy.
  • There are some disadvantages in the Boc/Bzl orthogonal protection solid phase synthesis strategy such as many side reactions, harsh conditions, and loss of the polypeptide chain from the solid phase during the process of extending the peptide chain.
  • the subsequent Fmoc/tBu orthogonal protection solid phase synthesis strategy has a milder reaction conditions.
  • microwave technology has been applied to the solid phase synthesis of peptides in recent years, the synthesis technology of peptides has made a leap. Microwaves promote chemical reactions because they rapidly rotate polar molecules in the microwave field, making some reaction rates 10 to 1000 times faster than conventional heating methods, and the yield is greatly improved.
  • a first object of the present invention is to obtain a polypeptide having a hypoglycemic activity from a polypeptide having a hypoglycemic activity, a homologous plant bitter gourd, to obtain a MC-JJ2 polypeptide having a higher hypoglycemic activity.
  • the present invention provides a bitter melon MC-JJ2 polypeptide having hypoglycemic activity, wherein the bitter melon MC-JJ2 polypeptide comprises the amino acid sequence shown in SEQ. ID NO: 1.
  • Xaa 2 is Ala, Gly, Tyr, Ser or D-Ala
  • Xaa 3 is Ala, Gly, Tyr or Ser
  • Xaa4 3 ⁇ 4 Lys, Ser or D-Ala Xaa 5 3 ⁇ 4 Ala, Gly, Cys, Arg, Lys, Met, Ser or D-Ala.
  • the present invention also provides a bitter melon MC-JJ2163 polypeptide having hypoglycemic activity, wherein the bitter melon MC-JJ2163 polypeptide comprises the amino acid sequence shown in SEQ. ID NO:108.
  • ⁇ 1 is eight 13, 1 «5, 0 ⁇ or 0-eight 1 &;
  • Xaa 2 is Ala, Tyr, Gly or D-Ala;
  • Xaa 3 is Ala, Tyr, Gly or D-Ala
  • Xa ⁇ is Ala, Lys, Gly or D-Ala
  • Xaa 5 is Ala, Lys, Gly or D-Ala
  • Xaae is Ala, Asn, Gly or D-Ala
  • Xaa 7 is Ala, His, Gly or D-Ala.
  • the inventors of the present application found that the use of microwaves to promote solid phase synthesis of polypeptides can overcome difficult peptide sequences, synthesize peptides that cannot be obtained by conventional solid phase methods, and the synthesis time is greatly shortened, and the purity and yield of the crude peptides are significantly improved, so that the products are Purification is very convenient, as long as a preparative HPLC can obtain a higher purity product; in addition, it is also very convenient to use D-type amino acids and unnatural amino acids to modify the polypeptide to find higher activity and longer biological half-life.
  • the polypeptide which is even more impossible by genetic engineering technology.
  • a second object of the present invention is to provide a solid phase preparation method for the above polypeptide, which utilizes a microwave-assisted Fmoc/tBu orthogonal protection solid phase synthesis strategy to efficiently and rapidly synthesize the above polypeptide.
  • a microwave-promoted Fraoc/tBu orthogonal protection solid phase synthesis strategy is adopted, and a resin carrying the first Fmoc-protected amino acid is synthesized on a solid phase carrier, and the ninhydrin method is negative, and the Finoc is removed.
  • the protecting group obtains the resin carrying the first amino acid residue; then proceeds to the next coupling cycle, repeats the coupling and deprotection steps with different protected amino acids according to the corresponding peptide sequence, and sequentially prolongs the desired amino acid sequence, and synthesizes
  • the resin carrying the corresponding polypeptide is obtained, and finally the polypeptide is cleaved from the resin with a lysing agent to obtain a crude polypeptide.
  • the crude polypeptide is purified by preparative high performance liquid chromatography and freeze-dried to obtain a pure polypeptide.
  • the synthesis of the resin carrying the first Fnioc protected amino acid is carried out by coupling the Fmoc-protected amino acid with an activator under microwave irradiation and then coupling with a solid phase carrier, and 1-hydroxy-benzotriazine is added to the reaction.
  • the azole (H0BT) or its derivative inhibits racemization and neutralizes the acidity of the reaction using an organic base.
  • the solid phase carrier used is Rink resin, Wang resin or 2-chlorotrityl chloride resin;
  • the activator is dicyclohexyl carbon dioxide Amine (DCC), hydrazine, ⁇ '-diisopropylcarbodiimide (DIC), hydrazine, hydrazine "-carbonyldiimidazole (CDI), 1-ethyl-(3-dimethylaminopropyl) carbon Diimine hydrochloride (EDC.
  • HC1 2-(7-azobenzotriazole) - N, N, N', N' - tetramethylurea hexafluorophosphate (HATU), benzo Triazole-N, N, N', N'-tetramethylurea hexafluorophosphate (HBTU) or the 1-hydroxy-benzotriazole (H0BT) derivative used is N-hydroxysuccinimide (H0SU), 1-hydroxy-7-azobenzotriazole (H0AT) or 3-hydroxy-1, 2,3-benzotriazin-4 (3H)-ketone (H00BT); organic base is three Ethylamine (TEA), N-methylmorpholine (NMM) or diisopropylethylamine (DIEA); microwave promotion conditions are: microwave frequency 2450MHz, reaction temperature: 20 ⁇ 100 ° C, reaction time is 5 ⁇ 15min .
  • TAA Ethylamine
  • NMM N-methylmorpholine
  • DIEA diisopropylethy
  • the removal of the Fmoc protecting group is carried out by a microwave-assisted reaction using a solution of 1-light-benzotriazole (H0BT) containing hexahydropyridine containing 0.1 mol. L- ', using dimethyl Formamide (DM F), dimethyl sulfoxide (DMS0), or N-methylpyrrolidone (NMP) is the reaction solvent.
  • the microwave promotion conditions are: microwave frequency 2450 MHz, reaction temperature: 20 to 100 ° C, reaction time is l 10 10 min.
  • the proposed MC-JJ2 polypeptide or MC-JJ2163 polypeptide can improve the stability and prolong the action time based on the retention of hypoglycemic activity.
  • Microwave-assisted solid phase synthesis of the polypeptide greatly increases the rate of coupling reaction.
  • Conventional solid phase synthesis methods fully couple an amino acid to the resin, often ranging from 2 hours to 20 hours, or even longer.
  • Microwave promotion only takes about 10 minutes on average; conventional solid phase synthesis methods take Fnioc protecting groups, which usually take 30 minutes to 1 hour, while microwave promotion only takes about 5 minutes on average, which greatly improves peptide synthesis. The efficiency of the synthesis cycle is shortened.
  • Microwave-promoted solid phase synthesis The purity of the crude product obtained by the synthesis of the polypeptide is greater than 85%, which is greatly improved compared with the conventional solid phase synthesis method, which facilitates the subsequent purification work, and only needs to be purified once in a liquid phase, and lyophilized. Get the target pure product.
  • the microwave-assisted solid phase method for synthesizing the polypeptide has low cost. Due to the high coupling efficiency, the average amino acid required to be protected needs only a 2-fold excess, which is much lower than the conventional solid phase synthesis method requires 4 to 5 times excess.
  • the method of microwave-assisted solid phase synthesis of the polypeptide is easy to automate and large-scale, which makes it more suitable for industrial production.
  • the polypeptide is prepared by the microwave-assisted solid phase synthesis technology provided by the invention, and the yield is high, the synthesis cycle is short, the crude product is easy to be purified, the production cost is low, and the industrial automation is easy to produce.
  • the prepared MC-JJ2 polypeptide has high stability and long action time and is suitable as an active ingredient for treating diabetes drugs. detailed description
  • Et 3 N triethylamine; Draw: N-methylmorpholine; DIEA: ⁇ , ⁇ '-diisopropylethylamine; DMF: dimethylformamide; DM SO: dimethyl sulfoxide; DCM: Chloroform; Fmoc: N-9 methoxycarbonyl; DIC: N, N, -diisopropylcarbodiimide; CDI : ⁇ , ⁇ '-carbonyldiimidazole; DMAP: 4-dimethylaminopyridine; H0SU : ⁇ -hydroxysuccinimide; EDC.
  • HC1 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; HATU: 2-(7-azobenzotriazine) Azole) ⁇ , ⁇ , 1 ⁇ '-tetramethylurea hexafluorophosphate; HBTU: benzotriazole-oxime, oxime, ⁇ ', ⁇ '-tetramethylurea hexafluorophosphate; HCTU: 6- Chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate; H0AT: 1-hydroxy-7-azobenzotriazole; H0BT: 1-hydroxy-benzotriazine PyBOP: benzotriazol-1-yl-oxytripyrrylphosphonium hexafluorophosphate; HPLC: high performance liquid chromatography; ESI-MS: electrospray ionization mass spectrometry; Gly: glycine; Ser: se
  • Fmoc- Ser- OH (0. 04 ol), HBTU (0. 04 ol), H0BT (0. 04 ol) and DIPEA (0. 08 mmol) were dissolved in 10 mL of NMP, and this solution was added to the above.
  • the reaction was carried out in a microwave reactor for 7 min, the microwave power was 25 W, the reaction temperature was controlled at 50 ° C, and air cooling was performed using an air compressor. After the reaction was completed, the reaction solution was filtered off, and 7 mL of DCM and NMP were used to wash the resin 3 Times.
  • the coupling efficiency of the resin is legally determined by the ninhydrin method or bromophenol blue, and the color reaction is negative to enter the next coupling cycle.
  • the ninhydrin method a small amount of resin particles were washed with ethanol, and placed in a transparent vial, 5% ninhydrin ethanol, KCN pyridine solution (2 ml 0.001 M KCN diluted in 98 ml pyridine), and 2 drops of 80% phenol ethanol solution. Heating at 10 CTC for 5 minutes, if the resin is blue, it is positive.
  • Bromophenol blue method Take a small amount of resin particles and wash with diacetyl acetamide. Add 3 drops of 1% bromophenol blue dimethylacetamide solution to a transparent vial and shake for 3 minutes at room temperature. That is positive.
  • the crude product of Ala 2 - MC-JJ2-(1 10)-NH 2 obtained above was dissolved in a small amount of water, and the crude product was purified by the reversed phase HPLC.
  • C18 reverse phase preparative column (340 mm ⁇ 28 5 Wn) was used for purification; mobile phase A: 0.1% TFA/water (V/V), mobile phase B: 0, 1% TFA/acetonitrile (V/V); mobile phase gradient : Mobile phase B13% 15%, 30 min; flow rate 6 mL/min ; detection wavelength 214 nm.
  • the collected solution was lyophilized to a pure product to finally obtain a pure product of 29.7 mg.
  • Example 2 106 the polypeptide of Example 2 106 was synthesized according to the corresponding sequence, and the respective molecular weights were confirmed by electrospray ionization mass spectrometry (ESI-MS).
  • ESI-MS electrospray ionization mass spectrometry
  • mice Male Kunming mice (body weight 18 ⁇ 22 g) of 10 weeks old were randomly divided into groups of 6 rats. The tail vein was injected with alloxan to be administered at a dose of 60 mg/kg. After 72 hours, the mice were fasted for 5 to 6 hours to measure blood glucose. The mice with blood glucose values ranging from 13 to 23 were randomly assigned to each group. only. The modeled mice were intraperitoneally injected with MC-JJ2 polypeptide for 10 consecutive days. The blood glucose level was measured with a blood glucose meter at 0 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes.
  • hypoglycemic activity of the MC-JJ2 polypeptide was stronger than that of metformin.
  • the coupling efficiency of the resin is legally determined by the ninhydrin method or bromophenol blue, and the color reaction is negative to enter the next coupling cycle.
  • the ninhydrin method a small amount of resin particles were washed with ethanol, and placed in a transparent vial, 5% ninhydrin ethanol, KCN pyridine solution (2 ml 0.001 M KCN diluted in 98 ml pyridine), and 2 drops of 80% phenol ethanol solution. Heating at 10 CTC for 5 minutes, if the resin is blue, it is positive.
  • Bromophenol blue method Take a small amount of resin particles and wash with diacetyl acetamide. Add 3 drops of 1% bromophenol blue dimethylacetamide solution to a transparent vial and shake for 3 minutes at room temperature. That is positive.
  • the crude product of Ala 2 - MC-JJ2163-(1 21)-NH2 obtained above was dissolved in a small amount of water, and the crude product was purified by the reversed phase HPLC.
  • Purified medium was prepared by C18 reverse phase preparative column (340 X 28 5 Mm); mobile phase A: 0.1% TFA/water (V/V), mobile phase B: 0.1% TFA/ Acetonitrile (V/V); mobile phase gradient: mobile phase B 13% ⁇ 15%, 30 min; flow rate 6 mL/min ; detection wavelength 214 nm.
  • Example 108 the polypeptides of Examples 109 to 222 were synthesized according to the corresponding sequences, and the respective molecular weights were confirmed by electrospray ionization mass spectrometry (ESI-MS).
  • ESI-MS electrospray ionization mass spectrometry
  • Gly-His-Pro-Tyr-Tyr-Ser- lie- Gly-Lys-Ser- Lys- Thr-Asn- Met-Lys ⁇ Gly- Met-Ala- Gly_Ala- Ala
  • mice Male Kunming mice (body weight 18 ⁇ 22 g) of 10 weeks old were randomly divided into groups of 6 rats. The tail vein was injected with alloxan to be administered at a dose of 60 mg/kg. After 72 hours, the mice were fasted for 5 to 6 hours to measure blood glucose. The mice with blood glucose values ranging from 13 to 23 were randomly assigned to each group. only. The modeled mice were intraperitoneally injected with MC-JJ2 polypeptide for 10 days. The blood glucose level was measured by a blood glucose meter at 0 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes.
  • the MC-JJ2163 polypeptide has a lower blood glucose lowering activity than metformin.

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Abstract

Cette invention concerne les peptides MC-JJ2 et MC-JJ2163 issus de Momordica charantia ayant une activité hypoglycémique et leur procédé de synthèse sur phase solide assistée par micro-ondes. Les peptides MC-JJ2 et MC-JJ2163 comprennent les séquences d'acides aminés de SEQ ID N°: 1 et SEQ ID N°: 108, respectivement.
PCT/CN2010/001422 2009-09-17 2010-09-17 Peptide mc-jj2 issu de momordica charantia et son procédé de synthèse sur phase solide assistée par micro-ondes WO2011032360A1 (fr)

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CN200910183320A CN101665529A (zh) 2009-09-17 2009-09-17 微波促进固相合成苦瓜mc-jj22多肽类似物及其应用
CN200910183315A CN101665528A (zh) 2009-09-17 2009-09-17 微波促进固相合成苦瓜mc-jj2多肽类似物及其应用
CN200910183316A CN101781361A (zh) 2009-09-17 2009-09-17 微波促进固相合成苦瓜mc-jj2163多肽类似物及其应用
CN200910183316.8 2009-09-17
CN200910183320.4 2009-09-17
CN200910183317.2 2009-09-17
CN200910183317A CN101691398A (zh) 2009-09-17 2009-09-17 微波促进固相合成苦瓜mc-jj2162多肽类似物及其应用
CN200910183315.3 2009-09-17

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CN101691398A (zh) * 2009-09-17 2010-04-07 中国药科大学 微波促进固相合成苦瓜mc-jj2162多肽类似物及其应用
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