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Silkworm chrysalis polypeptide and its preparation method and application

A technology of silkworm chrysalis, diabetes, applied in the field of medicine

Active Publication Date: 2016-11-30
肽能九号生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no well-developed compounds extracted from silkworm chrysalis for the prevention or treatment of diabetes

Method used

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  • Silkworm chrysalis polypeptide and its preparation method and application
  • Silkworm chrysalis polypeptide and its preparation method and application
  • Silkworm chrysalis polypeptide and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0011] Preparation method of polypeptide

[0012] Boil silkworm chrysalis in water for 1.5 hours, then cool to room temperature; add 0.5% neutral protease and 1% compound protease to hydrolyze for 2 hours; use macroporous resin to separate protein according to molecular weight, and then use semi-preparative RP-HPLC to separate and purify the obtained product After freeze-drying, it is silkworm chrysalis polypeptide 2. The silkworm chrysalis polypeptide 2 extracted by the above method is identified by mass spectrometry, and its sequence is PYERQADEPETKVTKKI, which is a new sequence. It can also be synthesized by Shanghai Sangong.

Embodiment 2

[0014] In Vitro Cell Viability Determination of Silkworm Chrysalis Polypeptide 2

[0015] The MTT colorimetric method was used. Insulinoma cells (INS-1 cells) with logarithmic growth were added to 96-well culture plate at a volume of 1.0×105 and cultured for 24 hours. Different concentrations of the experimental drug silkworm chrysalis polypeptide 2 were added to the experimental wells and positive drug control wells; the blank group was added same volume of solvent. Set up five duplicate wells in every well, culture 48h, add MTT in every hole, after 4h of action, add DMSO, hatch 30min, measure the absorbance A value at 570nm place of microplate reader, according to the formula cell growth proliferation rate=(experimental group absorbance value / Absorbance value of control group -1)×100%. The calculated EC50 of the experimental drug was 25.76nmol / L.

Embodiment 3

[0017] In vivo hypoglycemic experiment of silkworm chrysalis polypeptide 2

[0018] Kunming mice were fed with high-fat and high-sugar to a body weight of 18-22 g, half male and half female, fasted for 24 hours before the experiment, and intraperitoneally injected with streptozotocin (STZ) 50 mg / kg (1% citrate buffer). liquid solution preparation). One week later, blood glucose (BS) was measured by blood collection from the tail vein of the mice. If the BS was higher than 16mmol / L, the modeling was successful. After modeling, the mice were divided into 3 groups, 10 in each group. Diabetes model group (DM group): subcutaneous injection of the same amount of PBS buffer; control group (Bai Sugar): 20 μg / kg subcutaneous injection dissolved in 0.1mol / L PBS buffer), twice a day for 15 consecutive days; sample group (silkworm chrysalis polypeptide 2): the same as the control group; another 10 normal mice were taken as the blank group. On the 15th day after administration, blood was...

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Abstract

The invention relates to the field of medicines and in particular relates to a silkworm pupa polypeptide 2 which can be used for preventing or treating diabetes mellitus. The sequence of the polypeptide 2 is PYERQADEPETKVTKKI. A preparation method of the polypeptide 2 comprises the following steps: (1) decoction; (2) enzymolysis; (3) protein separation; (4) separation and purification through semipreparative RP-HPLC (reverse phase high-performance liquid chromatography); (5) freeze-drying, wherein neutral protease and compound protease are adopted for enzymolysis; macroporous resin and sephadex are adopted for protein separation. The invention provides the application of the polypeptide 2 to treatment of diabetes mellitus and complications of diabetes mellitus, wherein the complications of diabetes mellitus include diabetic nephropathy, diabetic hypertension, diabetic eye diseases and diabetic neuropathy. The silkworm pupa polypeptide 2 is extracted from silkworm pupae, has good safety and can be used for effectively preventing or treating diabetes mellitus.

Description

Technical field: [0001] The invention relates to the field of medicine, in particular to a polypeptide extract for treating diabetes. Background technique [0002] Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia. It is a syndrome of glucose, protein and lipid metabolism disorders caused by insufficient insulin or defects in insulin cell metabolism. In recent years, the number of diabetic patients has gradually increased, and is divided into type I diabetes and type II diabetes. Among them, the number of patients with type II diabetes accounts for 85-90% of the total number of diabetes, which seriously threatens the health and life of patients. If diabetes is not well controlled, it may cause many complications, such as diabetic nephropathy, retinopathy, high blood pressure, etc. [0003] At present, most patients treat diabetes by injecting insulin or oral hypoglycemic drugs to help control blood sugar in the body, or regulating insulin se...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C07K1/20C12P21/06A61K38/10A61P3/10A61P13/12A61P9/12A61P27/02A61P25/00
Inventor 罗瑞雪
Owner 肽能九号生物科技有限公司
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