Method for preparing clitocine and applications in antineoplastic medicaments

A technology of critocin and drugs, applied in the field of medicine, can solve the problem of high mortality

Inactive Publication Date: 2008-12-31
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Malignant tumor is the most serious disease that threatens the physical and mental health of human beings today, with a high mortality rate. At present, there is no report of a drug with a high cure rate. Therefore, the development of anti-malignant tumor drugs has long been the direction of people's efforts.

Method used

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  • Method for preparing clitocine and applications in antineoplastic medicaments
  • Method for preparing clitocine and applications in antineoplastic medicaments
  • Method for preparing clitocine and applications in antineoplastic medicaments

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Extraction, separation and purification of anti-tumor active compounds from fresh Pleurotus ostreatus fruiting bodies and their structure identification

[0022] experimental method

[0023] 1. Extraction and purification: The raw material fresh Pleurotus ostreatus fruiting bodies (2.7kg) are vacuum freeze-dried and crushed by a grinder. The resulting 340g of bacterial powder is immersed in 1.5L of 95% (v / v) alcohol, and the fresh solvent is replaced once in the meantime. The two extracts were combined and concentrated under reduced pressure to obtain an alcohol extract (25.7g). The extract was extracted with ethyl acetate and concentrated under reduced pressure to obtain an ethyl acetate extract (15.7g). Take 15g ethyl acetate extract on silica gel column, CH 2 Cl 2 / MeOH (10:0-0:10) gradient elution, collect cells to screen the active elution peak (6.5g), and then apply the reverse phase column RP-18 (ODS, 50μm, Φ25×420mm), MeOH / H 2 O(1:9) elution, and the acti...

Embodiment 2

[0030] Example 2: Test of the inhibitory activity of clitocine on the proliferation of human tumor cells

[0031] Experimental material method

[0032] 1. Human tumor cell lines and sources: A total of six tumor lines are breast cancer Bcap37, breast cancer MCF-7, cervical cancer Hela, liver cancer SMMC-7721, liver cancer HepG2, gastric cancer SGC-7901, all from American Type Culture Collection.

[0033] 2. Dissolve clitocine in DMSO to make 1mg / ml mother liquid, place it in the refrigerator at -20℃, dilute it with culture medium to the required concentration for use in the experiment, and make MTT (product of Sigma company) make 1mg / ml mother liquid with phosphate buffer. Place in a refrigerator at 4°C for later use.

[0034] 3. Cell culture: All cells were cultured in DMEM complete medium (Invitrogen), plus 5% newborn calf serum (Invitrogen), 2mmol / L L-glutamine. Choose logarithmic growth phase cells, with 1×10 4 The density was inoculated into 96-well plates, added with differe...

Embodiment 3

[0043] Example 3: Related mechanisms of clitocine inhibiting the proliferation of liver cancer HepG2 cells

[0044] 1. DNA ladder-like strip experiment method: tumor cells HepG2 were treated with different concentrations of clitocine for 48h and collected, and lysed at 45℃ for 1h (5mM Tris-HCl, 100mM EDTA, 1%(w / v)SDS, and Proteinase K), genomic DNA was extracted with phenol / chloroform / isoamyl alcohol (25:24:1), precipitated with 70% alcohol at -20°C, RNA was removed with RNase, and 40μg of the same amount of DNA was added to 1.2% agarose Electrophoresis on the gel and photographed by a gel imager.

[0045] The experimental results are attached Figure 5 .

[0046] One of the most typical characteristics of cell apoptosis is that after the cell receives the drug, the DNA will be cut at the nucleosome junctions by nucleases, resulting in DNA fragments of 180-200bp or an integer multiple of them. During agarose gel electrophoresis Presents regular strips of ladders, which is an impor...

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Abstract

The invention provides a preparation method for cobratoxin; as raw material, fresh giant clitocybe fruitbody is frozen and dried through vacuum and then crushed; the obtained powders are dipped in alcohol; the water extract is decompressed and concentrated into liquid extract which is then extracted with ethyl acetate and concentrated into liquid extract through decompression; the liquid extract is respectively treated with silica gel column, reversed phase RP-RP-18 and RP-RP-C8 preparation columns to get the target material. The cobratoxin provided by the invention has wide broad-spectrum anti-tumor activity and has a strong growth inhibition effect on six kinds of tumors through anti-tumor activity in vitro detection; wherein, the cobratoxin has particularly prominent killing effect on HepG2 liver cancer cells, with the half of the proliferation inhibitory concentration IC50 reaching 0.24 micron; nude mice experiments show that 3mg / kg dose of cobratoxin can significantly inhibit the growth of human hepatocellular carcinoma HepG2 nude mice xenografts, with the xenograft inhibiting rate reaching 40.1% . The invention has great significance on the development of fungal resources in China and can be applied to the preparation of anti-tumor activity drugs.

Description

Technical field [0001] The present invention relates to the technical field of medicine, in particular to the extraction of active ingredients from natural fungi and their uses, in particular to a method for extracting the active ingredient clitocine from the fruiting bodies of Leucopaxillus giganteus, and its application in the preparation of anti-tumor drugs Application and related mechanisms in Background technique [0002] Leucopaxillus giganteus (Leucopaxillus giganteus) belongs to the Tricholomaceae family. It grows solitary or in groups on grasslands in summer and autumn, and sometimes grows on grasslands in forests. Distributed in Hebei, Inner Mongolia, Jilin, Liaoning, Shanxi, Heilongjiang, Qinghai, Xinjiang and other places. There are few research reports on this bacteria in the treatment of tumors. Malignant tumors are the most serious diseases threatening human physical and mental health, with high mortality. There are no reports of drugs with high cure rates. Therefo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H19/067A61K31/7064A61K36/06A61P35/00
Inventor 刘非燕任刚傅承新吴平
Owner ZHEJIANG UNIV
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