Snake venom cytotoxin, preparation method and application thereof

Abstract

The invention discloses a snake poison cytotoxin, the preparation method and the application. The method of preparing the snake poison cytotoxin in the invention includes the following steps: firstly, the snake poison is conducted an ion-exchange chromatography and then the eluent between a first absorption peak and a second absorption peak on the absorbance curve with a detection wavelength of 280nm in the eluent is gathered; secondly, the eluent obtained in the first step is conducted an exclusion chromatography, water is used as a moving phase, and the second absorption peak on the absorbance curve with a detection wavelength of 280 nm in the eluent is gathered to obtain the snake poison cytotoxin. The saw-scale viper poison cytotoxin in the invention can induce the human laryngeal cancer cell Hep-2 to an apoptosis and the capability of inducing Cell Hep-2 to an apoptosis is stronger than Pingyangmycin with a viscosity of 100 Mug / ml when the snake poison cytotoxin has a concentration of 0.01 mg per milliliter. In addition, experiments of undue toxicity prove that the cytotoxin has slight side effects. The saw-scale viper poison cytotoxin in the invention can be used as an anti-cancer drug to turn biological anti-cancer drugs into a reality.

Description

technical field

[0001] The invention relates to a snake venom cytotoxin and its preparation method and application. Background technique

[0002] At present, the anticancer drugs used clinically are mainly chemical drugs, which are often called chemotherapy drugs. The common disadvantage of this class of drugs is serious side effects, which can generally cause severe vomiting and hair loss in patients. Therefore, biological anticancer drugs with less side effects have become a hot topic in research in recent years, mainly focusing on the research of snake venom and centipede toxin.

[0003] The venom of some snake species contains cytotoxins that can destroy the structure of tumor cells. There have been related reports before. For example, cells with anti-cancer effects have been found in the venom of the American copperhead and the venom of the akistrodon viper in southern Anhui, my country. Toxins, these discoveries are still in the stage of academic research and have not...

Examples

Embodiment 1

[0020] Embodiment 1, the preparation of snake venom cytotoxin

[0021] In this example, the lyophilized powder of viper venom (purchased from Jingshi Kaitai Pharmaceutical Co., Ltd.) was used as the raw material.

[0022] The extracted experimental steps are as follows:

[0023] 1. Snake venom dissolving: Dissolve 8 g of Viper saw scales lyophilized powder in 100 ml of Tris-HCl buffer solution (pH 8.2, 0.05 mol / L).

[0024] 2. Remove impurities: Add 6ml of saturated ammonium sulfate solution to the snake venom solution obtained in step 1, mix and place at room temperature for 1 hour, centrifuge at 3000rpm for 10 minutes, discard the precipitate, and keep the supernatant solution.

[0025] 3. Desalination by dialysis: seal all the supernatant obtained in step 2 in a dialysis bag with a molecular weight cut-off of 14000D, then put the dialysis bag into distilled water for dialysis for 15 hours, change the water every 3 hours, and the volume of distilled water is 1200ml. The di...

Embodiment 2

[0032] Example 2, Functional verification of viper venom cytotoxin

[0033] 1. Validation of anti-cancer function of viper venom cytotoxin

[0034] Taking the human laryngeal cancer cell line Hep-2 (provided by the Institute of Head and Neck Surgery, Liaoning Medical College) as the target cell, the efficiency comparison of the snake venom cytotoxin and pingyangmycin inducing the apoptosis of cancer cells was observed. The experimental method refers to the literature (Tai Jun, Wang Xuefeng, Pingyangmycin-induced apoptosis of human laryngeal carcinoma He ep-2 cells experimental research "Journal of Liaoning Medical College" 2004Aug.25 (4) 5-7 pages), the details are as follows:

[0035] Cultivation of the human laryngeal carcinoma cell line Hep-2 cell line: the cells were inoculated in the complete culture medium of RPMI-1640 (fetal bovine serum 10%, penicillin 100u / ml, streptomycin 100ug / ml), placed at 37°C, containing 5%CO 2 Cultured in an incubator.

[0036] Take 20ml of ...

technical field

[0001] The invention relates to a snake venom cytotoxin and its preparation method and application. Background technique

[0002] At present, the anticancer drugs used clinically are mainly chemical drugs, which are often called chemotherapy drugs. The common disadvantage of this class of drugs is serious side effects, which can generally cause severe vomiting and hair loss in patients. Therefore, biological anticancer drugs with less side effects have become a hot topic in research in recent years, mainly focusing on the research of snake venom and centipede toxin.

[0003] The venom of some snake species contains cytotoxins that can destroy the structure of tumor cells. There have been related reports before. For example, cells with anti-cancer effects have been found in the venom of the American copperhead and the venom of the akistrodon viper in southern Anhui, my country. Toxins, these discoveries are still in the stage of academic research and have not...

Examples

Embodiment 1

[0020] Embodiment 1, the preparation of snake venom cytotoxin

[0021] In this example, the lyophilized powder of viper venom (purchased from Jingshi Kaitai Pharmaceutical Co., Ltd.) was used as the raw material.

[0022] The extracted experimental steps are as follows:

[0023] 1. Snake venom dissolving: Dissolve 8 g of Viper saw scales lyophilized powder in 100 ml of Tris-HCl buffer solution (pH 8.2, 0.05 mol / L).

[0024] 2. Remove impurities: Add 6ml of saturated ammonium sulfate solution to the snake venom solution obtained in step 1, mix and place at room temperature for 1 hour, centrifuge at 3000rpm for 10 minutes, discard the precipitate, and keep the supernatant solution.

[0025] 3. Desalination by dialysis: seal all the supernatant obtained in step 2 in a dialysis bag with a molecular weight cut-off of 14000D, then put the dialysis bag into distilled water for dialysis for 15 hours, change the water every 3 hours, and the volume of distilled water is 1200ml. The di...

Embodiment 2

[0032] Example 2, Functional verification of viper venom cytotoxin

[0033] 1. Validation of anti-cancer function of viper venom cytotoxin

[0034] Taking the human laryngeal cancer cell line Hep-2 (provided by the Institute of Head and Neck Surgery, Liaoning Medical College) as the target cell, the efficiency comparison of the snake venom cytotoxin and pingyangmycin inducing the apoptosis of cancer cells was observed. The experimental method refers to the literature (Tai Jun, Wang Xuefeng, Pingyangmycin-induced apoptosis of human laryngeal carcinoma He ep-2 cells experimental research "Journal of Liaoning Medical College" 2004Aug.25 (4) 5-7 pages), the details are as follows:

[0035] Cultivation of the human laryngeal carcinoma cell line Hep-2 cell line: the cells were inoculated in the complete culture medium of RPMI-1640 (fetal bovine serum 10%, penicillin 100u / ml, streptomycin 100ug / ml), placed at 37°C, containing 5%CO 2 Cultured in an incubator.

[0036] Take 20ml of ...

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