Method for preparing melittin

A technology of melittin and bee venom, applied in the biological field, can solve the problems of long extraction time, cumbersome preparation process, and long production cycle, and achieve the effects of shortening the preparation time, simplifying the extraction process, and reducing the preparation cost

Inactive Publication Date: 2017-08-18
UNIV OF JINAN
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Preparation of melittin by means of solvent extraction, three-column chromatography, combination of solvent extraction and three-column chromatography, etc., the preparation process of the product is cumbersome, the production cycle is long, and the amount of organic solvent used is large
Kreil (Kreil, G. Structure of melittin isolated from two species of honey bees [J]. Febs Letters, 1973,33(2): 241-244.) reported the method for preparing melittin by solvent extraction, and the yield of melittin About 20% (mass fraction), the purity is 60.3% (mass fraction), the preparation method is simple, extracting 1g melittin requires 200mL n-butanol, the demand for n-butanol is large, causing serious environmental pollution
Liu Ling and Yang Wenchao (1. Liu Ling, Li Changquan, Huang Xueqiang. Purification method of melittin and its anti-tumor effect in vitro[J]. China Biochemical Drug Impurities. 2003,24(4):16-17 2. Yang Wenchao, melittin Peptide separation and purification and its anti-radiation mechanism [D], Fujian Agriculture and Forestry University, 2007.) reported the preparation of melittin by three-column chromatography, the yield of melittin was about 48.67% (mass fraction), and the purity was 97.32% (mass fraction), high purity, which solves the problem of large amount of organic solvent, but the extraction process is complicated and the extraction time is long
Zhang Wenli and Xu Peng (1. Zhang Wenli, Sun Jingyuan. Separation and purification method of melittin [P], China, 101089017A.2007-12-19. 2. Xu Peng, Ouyang Yongwei, Huang Jingyao, Zhang Guiying, Xiao Cheng, Liu Bo . Experimental study on the separation and purification of melittin from bee venom. Chinese herbal medicine [J]. 2000, 31(12): 892-894.) reported the preparation of melittin by combining solvent extraction and chromatography, and the yield of melittin About 47% (mass fraction), the purity is electrophoretic pure, which shortens the time required for three-column chromatography, and Kreil (Kreil, G. Structure of melittin isolated from two species of honey bees [J]. Febs Letters, 1973, 33(2): 241-244.) Compared with the preparation method, the use of n-butanol is reduced, but a large amount of n-butanol is still needed in the extraction process, which is not conducive to environmental protection
[0005] The above separation and purification method has complex production process, long production cycle, low yield, low purity and serious pollution

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] ①Removal of bee venom

[0031] Add crude bee venom to a buffer solution with a pH of 4.75 to make the concentration 1.0 mg / mL, and centrifuge at 3000 r / min for 5 min to remove insoluble impurities to obtain a supernatant;

[0032] The crude bee venom is obtained by dissolving the venom discharged from bee stings with distilled water and freeze-drying;

[0033] The buffer is acetic acid-sodium acetate buffer solution;

[0034] The impurity removal is the removal of insoluble substances in the buffer solution.

[0035] ② Separation of melittin

[0036] Pass the supernatant obtained in ① through a Sephadex G-25 column, elute with distilled water, control the flow rate at 28mL / h, measure the molecular weight of each separated component, the molecular weight collected under the Sephadex column is 2840Da The chromatographic components were freeze-dried to obtain solid samples;

[0037] The Sephadex G-25 is represented by the English letter G for dextran, and G reflects th...

Embodiment 2

[0047] ①Removal of bee venom

[0048]Add the crude bee venom to a buffer solution with a pH of 5.25, 1.5mg / mL, and centrifuge at 3200r / min for 6min to remove insoluble impurities to obtain a supernatant;

[0049] The crude bee venom is obtained by dissolving the venom discharged from bee stings with distilled water and freeze-drying;

[0050] The buffer is acetic acid-sodium acetate buffer solution;

[0051] The impurity removal is the removal of insoluble substances in the buffer solution.

[0052] ② Separation of melittin

[0053] Pass the supernatant obtained in ① through a Sephadex G-25 column, elute with distilled water, the flow rate is controlled at 30mL / h, measure the molecular weight of each separated component, and the molecular weight collected under the Sephadex column is 2840Da The chromatographic components were freeze-dried to obtain solid samples;

[0054] The Sephadex G-25 is represented by the English letter G for dextran, and G reflects the crosslinking ...

Embodiment 3

[0064] ①Removal of bee venom

[0065] Add the crude bee venom to a buffer solution with a pH of 4.5, 0.5mg / mL, and centrifuge at 2800r / min for 4min to remove insoluble impurities to obtain a supernatant;

[0066] The crude bee venom is obtained by dissolving the venom discharged from bee stings with distilled water and freeze-drying;

[0067] The buffer is acetic acid-sodium acetate buffer solution;

[0068] The impurity removal is the removal of insoluble substances in the buffer solution.

[0069] ② Separation of melittin

[0070] Pass the supernatant obtained in ① through a Sephadex G-25 column, elute with distilled water, and control the flow rate at 26mL / h, measure the molecular weight of each separated component, and the molecular weight collected under the Sephadex column is 2840Da The chromatographic components were freeze-dried to obtain solid samples;

[0071] The Sephadex G-25 is represented by the English letter G for dextran, and G reflects the crosslinking de...

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PUM

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Abstract

The invention discloses a method for separating and purifying melittin, and belongs to the technical field of biology. The melittin is a polypeptide extracted from bee venom; the melittin is obtained by adopting sephadex chromatography, freeze drying and combining a separation method including centrifuging and ethanol precipitation. The melittin disclosed by the invention has relatively high yield, the production time is short, and a process is simple; the purity is electrophoretically pure; an original structure and the highest biological activity of the melittin are kept, and the melittin can be applied to the field of medicines and the like.

Description

technical field [0001] The invention relates to a method for preparing melittin, which belongs to the field of biotechnology. Background technique [0002] Bee venom is a transparent liquid with aromatic odor secreted by worker bee venom glands. It is mainly composed of polypeptides, active enzymes and non-peptide substances. Protein polypeptide substances are the main components of bee venom, accounting for about 70% of bee venom -80%. Melittin is a polypeptide composed of 26 amino acid residues, with a molecular weight of 2840Da, accounting for about 50% of the dry weight of bee venom, and its primary structure amino acid residue sequence is NH 2 -Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Glu-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Glu -Gln-COOH. Melittin has anti-inflammatory, antibacterial, anti-cancer, anti-radiation, and inhibition of platelet aggregation effects, and can effectively treat many diseases such as frozen shoulder, rheumatoid arthritis, and rh...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C07K1/16
CPCC07K14/43572
Inventor 王元秀孙敏丁良
Owner UNIV OF JINAN
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