Enzymolysis polypeptide and use thereof in preparation of medicine for treating lung adenocarcinoma

A technology of enzymatic hydrolysis and compound protease, which is applied in the field of enzymatic hydrolysis of polypeptides and preparation of drugs for the treatment of lung adenocarcinoma, to achieve the effect of promoting apoptosis and preventing drug resistance

Active Publication Date: 2017-06-13
苏州默迪夫生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on the extraction, isolation, purification and anti-tumor aspects of cicada monkey polypeptides

Method used

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  • Enzymolysis polypeptide and use thereof in preparation of medicine for treating lung adenocarcinoma
  • Enzymolysis polypeptide and use thereof in preparation of medicine for treating lung adenocarcinoma
  • Enzymolysis polypeptide and use thereof in preparation of medicine for treating lung adenocarcinoma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The preparation of embodiment 1 enzymolysis polypeptide

[0031] 1. Experimental materials

[0032] 1. Instrument reagents

[0033] The ultrafiltration cup and ultrafiltration membrane were purchased from Shanghai Mosu Scientific Equipment Co., Ltd.;

[0034] Protamex complex protease is Novozymes complex protease.

[0035] 2. Experimental samples

[0036] Fresh cicada monkeys just unearthed from trees were collected, stored in water, and frozen at -20°C. Thaw naturally at room temperature before use.

[0037] 2. Experimental method

[0038] 1. Enzymatic hydrolysis method

[0039] Wash the cicada monkeys and grind them into minced meat. Take 10 g of the minced meat and put it in 50 mL of ultrapure water. Add Protamex complex protease for enzymolysis. The amount of Protamex complex protease added is 0.45% of the weight of the substrate minced meat. The enzymatic hydrolysis conditions are: pH value, 7.0; enzymatic hydrolysis temperature, 45°C; enzymatic hydrolysis ...

Embodiment 2

[0052] Example 2 Effects of Enzymatically Hydrolyzed Polypeptides on the Proliferation and Apoptosis of Lung Adenocarcinoma A549 Cells

[0053] 1. Cell culture: Human lung cancer cell line A549 was cultured in RPMI-1640 medium containing 10% calf serum at 37°C and 5% CO 2 Under normal conditions, cultured and subcultured.

[0054] 2. MTT method to determine the inhibition of tumor cell growth by enzymatic peptides

[0055] A549 cells in the logarithmic growth phase were digested with trypsin and diluted to 2×10 4 / mL, 100 μL per well was inoculated in a 96-well culture plate; the blank culture medium was used as a control, and cisplatin was used as a positive drug. After static culture for 24 h, the old culture solution in each well was discarded. Add 100 μL of culture solution containing different concentrations of enzymatic polypeptide 3 or enzymatic polypeptide 4 to each well of the experimental group, 6 wells in each group. After 48 hours of enzymolysis of the polypeptid...

Embodiment 3

[0065] Example 3 Inhibitory effect of enzymatically hydrolyzed polypeptide on the growth of lung adenocarcinoma A549 cell transplanted tumor in nude mice

[0066] Establishment and grouping of lung adenocarcinoma nude mouse model: Lung adenocarcinoma A549 cells in logarithmic growth phase were digested with 0.25% trypsin to make a single cell suspension, and the number of living cells counted by trypan blue exclusion method accounted for more than 95%. Adjust the cell density to 1×10 7 / ml, 0.2ml of the cell suspension was inoculated subcutaneously on the dorsal side of the right thigh of each nude mouse, and the body weight was measured before operation. Observe daily after inoculation. After the transplanted tumor is visible to the naked eye, measure the transplanted tumor with a vernier caliper until the volume of the transplanted tumor reaches 100mm 3 Nude mice were randomly divided into blank control group, enzymatic polypeptide 3 administration group (4 mg / kg), enzymati...

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Abstract

The invention discloses enzymolysis polypeptide and use thereof in preparation of a medicine for treating lung adenocarcinoma. The enzymolysis polypeptide is prepared from cicada by the following steps: cleaning the cicada, mincing the cicada into meat paste, adding Protamex compound protease for enzymolysis, wherein the enzymolysis conditions are as follows: the pH value is 6.8-7.2, the enzymolysis temperature is 42-47 DEG C, and the enzymolysis time is 7-9 hours; after the enzymolysis is finished, heating to inactivate the compound protease, and cooling to the room temperature; after the cooling, carrying out centrifugation at a rotation speed of 8000rpm-10000rpm for 20-30 minutes, extracting supernatant, and carrying out freeze drying, so as to obtain freeze-dried powder; dissolving the freeze-dried powder with ultrapure water, separating obtained enzymolysis polypeptide by virtue of ultrafiltration membranes with different molecular weight cut-offs, and carrying out freeze-drying on components obtained through ultrafiltration, so as to obtain enzymolysis polypeptides with different molecular weights. According to the enzymolysis polypeptide, the proliferation of A549 cells of the lung adenocarcinoma can be restrained, the apoptosis of the A549 cells can be promoted, the growth of nude mice transplanted tumors of the A549 cells of the lung adenocarcinoma is remarkably inhibited, and drug resistance of the A549 cells of the lung adenocarcinoma to the enzymolysis polypeptide is unlikely to be produced; and toxicology researches prove that the enzymolysis polypeptide has no toxic and side effects.

Description

technical field [0001] The invention belongs to the field of medicine and relates to the preparation of polypeptide drugs, in particular to an enzymatically hydrolyzed polypeptide and its use in preparing drugs for treating lung adenocarcinoma. Background technique [0002] Lung cancer is the most common malignant tumor in the world, and its mortality rate ranks first among malignant tumors. The incidence of lung cancer in my country has been increasing year by year, with an average annual increase of nearly 2%. Lung cancer is divided into multiple tissue types according to different pathological characteristics, and the treatment measures are also different for different types of lung cancer tissue. According to the 2004 edition of the WHO classification, common histopathological types of lung cancer are divided into non-small cell carcinoma (NSCLC) and small cell carcinoma (SCLC). Non-small cell carcinomas are subdivided into squamous cell carcinoma (SCC), adenocarcinoma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/06C07K1/34A61K38/01A61P35/00
CPCA61K38/012C07K1/34C12P21/06
Inventor 郭守河其他发明人请求不公开姓名
Owner 苏州默迪夫生物科技有限公司
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