Qualitative And Quantitative Analysis Of Main Type Materials And Purification Of Abundant Components In The Southwest Vespa Venom

Objective: Using colorimetric method, SDS-PAGE fingerprints and spectrophotometry techniques, identification study and contents determination were conducted for the material compositions of wasp venom from southwestern China to provide references for its purified chemicals’ structure elucidation. Six main components of studied venom were purified with the application of HPLC separation,and their structures were identified using mass spectrometry, nuclear magnetic resonance spectroscopy, as well as protein N terminal and C terminal sequencing technologies.Methods: Ninhydrin test, xanthoprotein reaction test, biuret reaction test were adopted to identify the amino acids and polypeptides of the studied wasp venom,whilealpha-naphthol experiment, Fiji Lin test, polysaccharide test were applied to identify its glycosidic ingredients. Alkaline beta-naphthol reagent was used to detect components such as aromatic amines, whereas bismuth potassium iodide test,potassium mercury iodide experiment, iodine potassium iodide, and silicotungstic acid hydrate test were executed for identifying alkaloids. Preliminary understanding the distribution of molecular weights of ingredients of different batches of studied wasp venom was obtained based on SDS-PAGE feature maps. Moreover, the contents of major components of the wasp venom were measured: BCA method, Folin-phenol method, coomassie brilliant blue staining were for peptides and proteins, ninhydrin method for free amino acids, and phenol-sulfuric acid method for polysaccharides.Preparative HPLC was applied in the purification of wasp venom main ingredients,followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and N-terminal sequencing by Edman degradation to determine the relative molecular mass and the structures of peptide components.Results: In the xanthoprotein reaction test, the reference substance and all of the test samples showed the color orange, suggesting the wasp venom contained proteins,while the reference substance and the test samples were purple in ninhydrin test and biuret reaction test, pointing out the existence of proteins, polypeptides and amino acids. In alpha-naphthol identification experiment, the reference substance and the test samples illustrated purple bands, revealing it may contain sugars, and in both Fiji Lin test and polysaccharide test, the presence of brick red precipitates in reference substance and test samples indicated that the wasp venom may contain polysaccharides but no reducing sugars. Amine material identification experiment supported the existence of secondary aromatic amines in the wasp venom, meanwhile,alkaloid constituents were detected in the experiments of bismuth potassium iodide test, potassium mercury iodide experiment, iodine potassium iodide, and silicotungstic acid hydrate test. The investigation on the identification of the major components in this product provides strong support for further chemical research of its material base.Results derived from the content determination of total polypeptide/protein,free amino acids, and total sugars showed that the wasp venom contained rich proteins,polypeptides, amino acids, and polysaccharides.Chemical identities in the studied Wasp venom were isolated and purified by preparative reverse phase HPLC system. Moreover, the protocols and conditions for massive collection of pure compounds using HPLC were established and optimized,making it fulfilled of successfully isolating six compounds (A-F) with the purity of all above 98%. Outcomes of MALDI-TOF-MS and N-terminal Edman sequencing provided information on their molecular structures, including 2 amines and alkaloids,3 peptides and 1 protein belonging to an active protein subfamily.Conclusion:The study revealed that the tested wasp venom contained amino acids,peptides, amines, alkaloids, polysaccharides, excluding reductive sugar substances,and the content of proteins/peptides was richer than other types of substance. Six compounds, including two alkaloids, three peptides, one protein, from the study wasp venom were isolated, purified, and identified.