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Method for separation of antitumor polypeptide compound from Arca subcrenata Lischke and application

A peptide compound, anti-tumor activity technology, applied in the field of medicine, can solve the problems of too simple method, inability to quantify, and inability to develop and utilize drugs

Inactive Publication Date: 2013-08-21
于荣敏
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] After searching the literature, it is found that there is no report on the study of extracting active substances from cockles in foreign countries
In China, the research on cockles mainly focuses on the separation of polysaccharide components of cockles, its antioxidant or immune activity, and the activity of hydrolyzate of cockles, but there are few studies on the polypeptides of cockles proteins, such as: Chinese Patent Application No. 98110478.9 discloses the technology of "an anti-cancer marine biological extract", the method used is too simple, the obtained substance is complex and cannot be quantified, and it cannot be developed and utilized as a drug; while Chinese patent application number 2005100043797.4 discloses "An anti-cancer peptide extract of cockles and its preparation method" and 200610043222.7 disclosed a "method for extracting small molecule active polypeptides from cockles". components have been studied; in addition, Chinese patent application number 200510043846.4 discloses "a cockle glycopeptide extract and its preparation method"
The above patent documents only describe the process of extracting active crude protein and polypeptide substances from cockles, and the extracted products are crude composite components with low purity and unclear basis of active substances
So far, there have been no research reports on the extraction and isolation of anti-tumor polypeptide compounds with high purity and clear material basis from clams and their physical and chemical properties and structural analysis

Method used

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  • Method for separation of antitumor polypeptide compound from Arca subcrenata Lischke and application
  • Method for separation of antitumor polypeptide compound from Arca subcrenata Lischke and application
  • Method for separation of antitumor polypeptide compound from Arca subcrenata Lischke and application

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Embodiment 1

[0012] Shell the cockles, take its contents, wash three times with low-temperature distilled water, weigh after drying, add extraction buffer (pH=8.0, 0.03mol / L, sodium phosphate buffer) according to the weight ratio of 1:3, use High-speed tissue masher, homogenate at intervals of 8000rpm for 3min until fine and lump-free, place the homogenate in a low-frequency ultrasonic instrument for ultrasonic extraction for 40min, use a low-temperature high-speed centrifuge, 10000rpm, 4°C, centrifuge for 30min, remove slag, measure After measuring the volume of the supernatant, put it in an ice bath environment, add ammonium sulfate with 70% saturation, continue stirring and salting out for 1 hour, remove the precipitate by high-speed centrifugation, take the supernatant, measure the volume, and add ammonium sulfate with 100% saturation , continue stirring and salting out for 1 hour, centrifuge for 30 minutes (10,000 rpm, 4° C.), and collect the precipitate. Dissolve the precipitate with...

Embodiment 2

[0014] MTT method was used to track and screen the anti-tumor activity during the whole extraction and separation process.

[0015] Tumor cells in the logarithmic growth phase (adherent cells need to be digested with trypsin) were made into cells containing 3-4×10 3 cells / ml single cell suspension (cell density), respectively inoculated in sterile 96-well culture plates, 100 μl / well, in 5% CO 2 , cultured in a 37°C incubator. After the cells were cultured for 12 hours, a blank control group (adding an equal volume of RPMI1640), a positive control (cisplatin) and an administration group were set up. Three replicate wells were set up for each concentration group, and drugs were added. The total reaction volume of each well was 200 μl. Cultivate in the incubator for 48h. 4 hours before the end of the experiment, add 20 μl of human MTT (5 mg / ml) and continue to culture for 4 hours, discard the supernatant, add DMSO 200 μl / well, place on a shaker and shake for 10 minutes, and mea...

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Abstract

The invention relates to separation of an active polypeptide compound from Arca subcrenata Lischke and pharmaceutical use thereof. The preparation method comprises: shelling the Arca subcrenata Lischke, cleaning the content, adding a buffer solution, conducting homogenizing, extraction, centrifugation and slag removal, taking the supernatant, carrying out fractional precipitation by ammonium sulfate and dialysis desalting so as to obtain an antitumor active component; subjecting the antitumor active component to dialysis desalting and freeze drying, then performing ion exchange column chromatography, respectively collecting active peaks, then conducting hydrophobic column chromatography to realize separation and purification, thus obtaining an antitumor polypeptide compound with purity of 99%. The method provided in the invention creatively uses a modern extraction separation technology to separate the active polypeptide compound with a clear material basis and a molecular weight of 20491.0Da from Arca subcrenata Lischke. The compound has significant anti-tumor activity. In vitro anti-tumor experiments show that the compound has a tumor inhibition rate up to 87%, and small toxic and side effects, thus being applicable to development and application of novel antitumor drugs.

Description

technical field [0001] The invention belongs to the technical field of medicine, and relates to a polypeptide compound with anti-tumor activity obtained from a marine organism hairy clam (Arca subcrenata Lischke) through a series of steps of extraction, separation, chromatographic purification, etc. The compound is used for antitumor medicine. Background technique [0002] Hair clam (Arca subcrenata Lischke) belongs to the phylum Mollusca, belongs to the clam family of the clambranchia and order dentate order, and is one of the marine organisms with rich resources in our country. The nutritional and medicinal value of cockles are very high, and its shell and meat have medicinal value, and its medicinal use has a long history. Its shell is the corrugated seed of traditional Chinese medicine, which is recorded in "Shen Nong's Materia Medica" and successive dynasties of herbal medicines. " and "Yi Lin Zhuan Yao" and other traditional Chinese medicines are recorded in modern a...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K1/20C07K1/18A61K38/17A61P35/00
Inventor 于荣敏陈莉莉朱建华宋丽艳
Owner 于荣敏