Research On Improving The Quality Control Standard Of Whole Scorpion

Objective:Objective to establish the TLC identification method of scorpion medicinal materials,screen the target protein of Scorpion by proteomics technology,and establish the determination method for detecting the content of target protein,so as to provide guarantee for the control and evaluation of the quality of scorpion,improve its quality control standard and ensure the clinical safety of drug use.Method:(1)Through morphological and microscopic identification,30 batches of scorpion from Hebei,Henan,Shanxi,Shandong and Liaoning and 7 batches of scorpion medicinal materials provided by a pharmaceutical factory were confirmed to be accurate.(2)Through the determination of water,ash,extract,heavy metals and harmful elements in 37 batches of scorpion medicinal materials,as well as aflatoxin B and G,the quality of the collected medicinal materials was determined.(3)By optimizing the conditions of TLC,the method of TLC identification of Scorpion was studied.(4)HRMS combined with proteomics technology and protein sequencing method based on bottom up were used to find the target protein and its corresponding characteristic peptide for the quality control of scorpion medicinal materials,and the characteristic peptide and its isotope internal standard were synthesized.(5)HPLC-MS method was used,and the mobile phase of 0.1%formic acid aqueous solution(V/V)and acetonitrile were gradient eluted.Mass spectrometry was carried out by ESI positive ion mode,and MRM was used to collect the signal.The characteristic peptide was taken as the reference substance,the internal standard of the isotope was the internal standard,and the content determination method of protein in Scorpio was studied.Result:(1)Based on character identification and microscopic identification,it was confirmed that the characteristics of the collected samples were in accordance with the relevant provisions of the Pharmacopoeia of the people's Republic of China(Volume I,2020 Edition).(2)The moisture content of 37 batches of scorpion ranged from 3.9%to 23.8%,the total ash content ranged from 5.9%to 38.2%,the acid insoluble ash content ranged from 0.05%to 1.31%,the extract(dilute ethanol)content ranged from 14.5%to 49.4%,and the extract(95%ethanol)content ranged from 8.9%to 33.9%;the detection results of heavy metals and harmful elements showed that the contents of cadmium,arsenic and copper were 1.19?4.49 mg/kg,0.23?1.73 mg/kg and 94.2?306.7 mg/kg,the content of lead was between lower than the limit of quantitation and 0.63 mg/kg,and the content of mercury was between lower than the limit of detection and 0.24 mg/kg;Aflatoxin(B1,B2,G1 and G2)were not detected in the samples.(3)In the TLC identification method,n-butanol-glacial acetic acid-water(3:1:1)and cyclohexane acetone(4:1)were used as the developing agents respectively,and the extracts of scorpion and scorpion reference herbs were developed at the same time.The results showed that the samples had the same color spots at the same position based on the chromatogram of reference herbs.(4)HRMS was used to analyze scorpion protein extracted by PBS.The target protein of ?-actin for quality control of scorpion medicinal materials was determined.The corresponding characteristic peptide sequence of the protein was ?APPER(isoleucine isoleucine alanine proline proline glutamic acid arginine).The characteristic peptide and its isotope were synthesized Internal standard(IIA(13C3,15N pper).(5)The results showed that the linear range of the characteristic peptide of ?-actin was 0.1?40 nmol/L,and the correlation coefficient(r)was above 0.999.The detection limit was 0.04 mg/kg and the limit of quantification was 0.13 mg/kg.The recoveries were 87%?93%,RSD of repeatability and intermediate precision were 5.2%(n=6)and 4.8%(n=5),respectively.The test solution was stable within 12 h.The content of ?-actin in 37 batches of scorpion samples ranged from 4.66 mg/kg to 100.26 mg/kg.Conclusion:The TLC method established in this study can be used for the qualitative identification of scorpion.The determination results of heavy metals and harmful elements in the samples showed that the contents of lead,cadmium,arsenic,mercury and copper in scorpion should be controlled in the quality standard.Based on high-resolution mass spectrometry combined with proteomics technology,it was found that scorpion ?-actin can be used as the target protein for the quality control of scorpion,and its characteristic peptide can be used as the reference material for the determination of specific protein content in scorpion,thus becoming a means to reflect the content of scorpion medicinal materials.The above research results and related methods can play a positive reference significance for improving the quality standard of scorpion medicinal materials.