目的 对基胡蜂蜂毒的组成进行分析及对其体外生物活性进行评价。方法 采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和超高效液相色谱-质谱联用技术对基胡蜂蜂毒中的蛋白质和多肽成分的相对分子质量组成进行分析。采用MTT比色法评价蜂毒的体外抗炎及抗肿瘤活性,牛津杯法评价蜂抗菌活性,并将结果与基胡蜂醇提物进行比较。结果 基胡蜂蜂毒中蛋白质主要分布在11~48×103内,且在26、34和43×103附近有3条明显的蛋白条带,为蜂毒的主要过敏原成分。多肽类分子的相对分子质量呈无规律的多峰式分布,其中85%的多肽分子集中在300~2 500内。就生物活性而言,基胡蜂蜂毒可抑制脂多糖(LPS)诱导的RAW264.7细胞增殖,25 μg·mL-1浓度下的蜂毒对细胞的抑制率高达67.67%,且100 μg·mL-1的抗炎效果是同浓度地塞米松的10倍;对HT-29,HepG2,B16肿瘤细胞的生长繁殖具有显著的杀伤作用;对金黄色葡萄球菌,大肠埃希氏菌和白色念珠菌具有生长抑制作用;而基胡蜂醇提物仅表现出对LPS诱导的RAW264.7炎症细胞增殖抑制活性,且抑制效果仅为蜂毒的1/60。结论 本实验利用现代分离及分析技术对基胡蜂蜂毒的组成及生物活性进行了相对系统的研究,由结果可知,基胡蜂蜂毒含有丰富的肽类物质,为后续蜂毒成分研究方法的选择提供方向;此外,基胡蜂蜂毒具有抗炎、抗肿瘤、抗菌活性,具有潜在的药用开发价值,实验为后续该毒素的现代化深入研究提供了参考。
Abstract
OBJECTIVE To analyze the composition of Vespa basalis (Smith) and evaluate its biological activity in vitro. METHODS Using SDS-PAGE and UPLC-ESI-Q-TOF-MS to analyze the relative molecular mass composition of proteins and peptides in the venom. The MTT method was used to evaluate the anti-inflammatory and anti-tumor activities and the Oxford Cup method was used to study the antibacterial effect of the venom. In addition, the results were compared with the ethanol extract of the Vespa basalis adult. RESULTS The proteins in the venom was mainly distributed in the range of 11-48×103, and there were 3 more obvious protein bands around 26, 34 and 43×103 respectively,which were the main allergen components of wasp venom. The relative molecular masses of peptides presented an irregular multimodal distribution and 85% were concentrated in the 300-2 500 range. In terms of biological activity, Vespa basalis venom had an inhibitory effect on the proliferation of RAW264.7 cells induced by lipopolysaccharide LPS. The inhibitory rate of 25 μg·mL-1 venom was 67.67%, and the anti-inflammatory activity of 100 μg·mL-1 was 10 times that of dexamethasone with the same concentration. It had significant killing effect on the growth breeding of HT-29, HepG2, and B16 tumor cells. In addition, it exhibited a growth inhibitory effect on Staphylococcus aureus, Escherichia coli and Candida albicans. While the ethanol extract only showed an inhibitory activity on the proliferation of induced RAW264.7 cells, and the inhibitory effect was only about 1/60 of venom. CONCLUSION This experiment used modern separation and analysis techniques to conduct a relatively systematic study on the composition and biological activity of Vespa basalis venom. From the results, it can be seen that venom is rich in peptides substances, which provided a direction for the selection of follow-up research methods of the venom; In addition to the anti-inflammatory activity, Vespa basalis venom also had anti-tumor and antibacterial effects, indicating that it has potential medicinal purposes development value, and which provided a reference for subsequent in-depth research on the modernization of this venom.
关键词
基胡蜂 /
蜂毒 /
醇提物 /
炎症 /
抗肿瘤活性 /
抗菌活性
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Key words
Vespa basalis /
wasp venom /
ethanol extract /
inflammation /
antitumor activity /
an-tibacterial activity
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中图分类号：
R96
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基金
国家自然科学基金项目资助(81903924, 81703742, 82060765); 云南省科技厅重点项目资助(2017FA050); 云南省自然科学基金项目资助(2019FB121);大理州重点科技支撑专项计划资助(D2019NA01)
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