| Objective: Chinese yam,a family member of perennial herbaceous plant tuber,is rich in starch,polysaccharide,protein and other ingredients.Chinese yam is a common form of food and medicine in China.Chinese yam can induce intense itch when contacting with the skin.Pruritus is an unpleasant sensation that causes scratching.It is a common clinical symptom associated with primary skin diseases or systemic diseases.There are endogenous and exogenous substances causing itch.A variety of endogenous and exogenous itch-evoking substances have been found so far.What ingredients in yam causing itch are unclear.In this study through the extraction and purification of fresh yam,we analyzed the active ingredients causing itch,in order to further explore the mechanism of itch by yam.Methods: The effects of physical and chemical factors on the activity of the itch-inducing ingredients in Chinese yam were explored to provide a reference for the analysis of the properties of the active itch-inducing ingredients in Chinese yam and,for the control of subsequent extraction,separation and purification conditions.Q-HPR was used to separate and purify the crude extract of yam protein,and different proteins components were obtained.The itching evaluation and electrophysiological examination of the separated components were carried out.Qualitative analysis of the proteins of Chinese yam and the components though separation and purification were performed by liquid chromatography-mass spectrometry(LC-MS).Gene Ontology(GO)annotation analysis was carried out using Uniprot-GOA database.Inter Pro Scan software and Interpro domain database were used for protein domain annotations and enrichment analyses.STRING and Cytoscape software were used to construct protein-protein interaction networks and conduct modular analysis.The protein domains were compared using the Pfam database.SPSS 21 and Graph Pad 8.0 were used for data analysis and plotting,and P < 0.05 was considered to be statistically significant,which were bilateral tests.Results: Fresh Chinese yam could induce itch.Low temperatures had no effect on the activity of the itch-inducing ingredients,while high temperatures,PH 3 or 10 and80% ethanol could deactivate the itch-inducing ingredients.Oxidation showed no effect on the activity of the itch-inducing ingredients.Four components of yam proteins were isolated and purified,of which one component had itch-inducing activity.Each of the four components activated TRPA1 to some extent.1529 proteins of Chinese yam were identified by mass spectrometry,and protein annotations showed that there were three proteins CATHB2,Os04g0650000 and CCP2 belonging to papain family cysteine protease.GO enrichment analysis showed that these 1529 proteins were mainly involved in the biological processes such as citrate metabolic process,tricarboxylic acid metabolic process,cytoplasmic translation,nucleoside diphosphate phosphorylation,and purine ribonucleotide metabolic process.The cellular components were concentrated in the polysomal ribosome,cytosolic small ribosomal subunit,cell-cell junction,and respiratory chain complex.Molecular functions included glutathione transferase activity,drug binding,copper ion binding,and intramolecular oxidoreductase activity.Protein domain enrichment analysis showed that phosphoglucose isomerase,fructose-bisphosphate aldolase class-l,dimerisation domain,universal stress protein family,aminotransferase class-Ⅲ,papain family cysteine protease,and elongation factor Tu GTP binding domain were involved.Among the four components obtained through the separation and purification,one component had itch-causing activity,which included 235 proteins identified by mass spectrometry.Through the construction of PPI network,it was found that CCP2 was the hub gene connecting two independent networks.In the Pfam database,CCP2 annotated as "papain family cysteine protease",which included 198 amino acids in length.1-195 amino acids of CCP2 aligned with the sequence 23-218 of "Papain family Cysteine protease" model(full length included 218 amino acids).Conclusions: 1.The component of yam proteins containing CCP2 can cause itching.2.CCP2 belongs to papain family cysteine protease.CCP2 may be the active ingredient in Chinese yam that causes pruritus. |
