A New Method For Quantitative Evaluation Of Protein Based On IP4 Of Cordyceps Sinensis

Cordyceps sinensis Beck. is a parasitic complex of genus Hepialus larvae parasitized by Cordyceps sinensis (Berk.)Sacc., is a traditional precious Chinese medicine, has the effect of Bushenyifei,bleeding phlegm and so on.The host insect of Cordyceps sinensis is several species belongs to Hepialidae, mainly distributed in Sichuan, Tibet, Qinghai, Gansu, Yunnan, altitude 2800m above an alpine meadow area, located very fragile ecological environment, snow line shift in recent years due to global warming, so distribution areas dwindling. And because of the Cordyceps sinensis special pharmacological effects, the demand of its medicinal and health care is increasing as well domestic as international market,The prices has turned up in recent years,appear a serious shortage of supply and demand, and in the current the artificial propagation is not in mature, resulting in the adulteration is serious. It is reported that counterfeit of Cordyceps sinensis have caused dizziness, nausea and other side effects, seriously affect drug safety. The appearance, powder characteristics and authentic part of counterfeit is very similar to genuine,and the index components according to pharmacopoeia are not unique, it is difficult to form a new operational standards. Identification of Cordyceps sinensis become a hot topic.Proteins as the primary metabolite with stable species-specific and have stable content in vivo as well as between different parts. Our previous studies have demonstrated genuine Cordyceps sinensis has a specific protein, can be used as an index component identification, Then using two-dimensional electrophoresis (2-DE) protein isolated Cordyceps sinensis and its counterfeit, obtained 26 differentially expressed proteins, can be used as qualitative identification of Cordyceps sinensis. In this study,the optical density used as the quantitative method which detect adulterants protein to compensate for the lack of Bradford. We have finished as following based on previous studies carried out1. Verification indicator proteins using 2-DE.This study continue to optimize 2-DE conditions for the establishment of standard operating procedures,based on the previous studies has selected 26ifferentially expressed proteins using two-dimensional electrophoresis (2-DE) analyze Cordyceps sinensis and counterfeit, and by comparing 2-DE protein map of Cordyceps sinensis from different origin and its counterfeits, we verified 21 indicator proteins from 26 differentially expressed proteins by analyse of PDQUEST software.2. Identification analysis of differentially expressed proteins.Mass spectrometry was used analysis the biological function of 21 indicator proteins. The results showed that the biological function include participating in DN A damage repair (4%), participating in mRNA translation, transcription (9%), participating in the immune response (4%), participating in cell division and proliferation processes (4%), participate in transport, a carrier protein function (13%), participating in protein folding, refolding (8%), participating in recovery of unfolded or recovery activity of misfolded protein; and membrane protein, cytoskeleton-associated proteins (38%), participating in endocytosis, cytoskeletal rearrangement, transportation and recycling of membrane proteins, and cell motility related. Functional analysis of differentially expressed proteins was used for filting the bioactive difference protein in latter study.IP4 (indicative protein 4) is a cyanate synthase, an enzyme which catalyzes a cyanate salt solution, Cordyceps sinensis can overcome the toxicity synthase hydrolysis or break down, as a source of nitrogen, combined with previous results of 2-DE, IP4 have a high abundance, good resolution and stability, therefore we determine choose this protein as the protein for laboratory parameters subsequently.3. Preparation and identification of protein IP4.Based on basic information of IP4,established and optimized PAGE, HPLC to search differentially expressed proteins in Cordyceps sinensis,and determine the best method for separation and preparation of protein IP4 is gradient PAGE, the experimental separating conditions is T=5%to 15%, the constant voltage 140v. cutting IP4 and identification by mass spectrometry, recovery IP4 by electric dialysis, recovery rate is 2.51%-3.27%,fanally using HPLC combined PAGE, IEF and other methods identified its purity,4. IP4 polyclonal antibody preparation to establish quantitative analysis and establish the ELISA method for quantitative evaluation.The purified protein IP4 immunized mice to obtain polyclonal antibody, determinate the titer and specificity. Further investigated optimal conditions of detecting antibody IP4 by ELISA:selecting the packet solution, selecting antigen working time,selecting the blocking solution and the closure time, the working concentration of HRP-tapping secondary antibody and the chromogenic substrate time. Finally we test the specificity of ELISA for polyclonal antibody from Cordyceps sinensis. Result showed that the best coating solution is NBS, coating condition is 37℃ overnight, blocking buffer is 1% PBSB, closing time is 2h, HRP-tapping secondary antibody working concentration is 1: 3000, chromogenic substrate time is 20min.Specificity of ELISA for quantitative analysis of polyclonal antibody showed a positive reaction of Cordyceps sinensis, while the adulterants showed a negative reaction, This is indicating that the method can be qualitative identification of Cordyceps sinensis; Then establish a quantitative standard curve, the protein IP4 content in Cordyceps sinensis (SiChuan yajiageng) is 0.492mg/mL accounted for 0.075%of dry Cordyceps sinensis’s quality, accounted for 3.71% of soluble protein, the protein IP4 content in Cordyceps sinensis (Xizanglinzhi) is 0.396mg/mL accounted for 0.063%of dry Cordyceps sinensis’s quality, accounted for 3.22% of soluble protein. The protein IP4 content in Cordyceps sinensis (Qinghaixining) is 0.421mg/mL accounted for 0.052% of dry Cordyceps sinensis’s quality, accounted for 3.37%of soluble protein.IP4 content is different between difference origin, suggesting that ELISA can be used as the method for quantitative evaluation of Cordyceps sinensis’s quality with the IP4 as the indicator.