Identification Of Horn-derived Chinese Medicines Based On DNA Sequencing And Proteomic Analysis

Deer,also known as Cervids,is one of the Artiodactyl families.Cervus nippon,Cervus elephaus,Rangifer tarandus,and Rusa unicolor Kerr are four legitimate Cervid species that live and are naturally distributed in Asia.Cervus antler development is regulated by a variety of circumstances,thus each deer species has its own distinct shape and size.Antlers have economic and therapeutic worth,and are used to enhance kidney function,strengthen bones,boost bone marrow,and replenish the blood in afflicted persons.It’s also utilized to treat vacuity issues such limb discomfort and cold limbs,which is good for individuals recovering from surgery.The size and shape of entire antlers that are attached to the skull were traditionally used to identify antler species.It was difficult to identify them,and based on that,one of the most common difficulties in China’s deer antler industry is fraudulent substitution or mislabeling of expensive deer antlers with cheaper ones which causes health problems to consumers.Therefore,there is a continuous need for the development of genetic and protein marker to discriminate between genuine and fraudulent antlers.In this study,a molecular methodology was developed for identifying adulterant Cervus antler utilizing various methodologies such as DNA sequences and PCR-based technology,which may be used for particular rapid identification of some counterfeit antler products,identification of different protein and protein content was determined using the SDS-PAGE electrophoresis,BCA method which was employed and the results revealed that the cell SDS-PAGE profile of the processed cervus antler and its molecular weight has several visible protein bands.Furthermore,analyzing the amount of amino acid,red deer antlers are rich in amino acids.At present,about 19 kinds of amino acids and 8 kinds of essential amino acids have been identified,result showed that cervus antler,Saiga Tataricae Cornu have 5 types of amino acids,of which proline is the highest,the content of glycine was the second,and the content of Serine was the lowest.Furthermore,average content of total amino acids in processed product,the percentage was 60.1%and 40%respectively.The main parts of the study are as follow:1.PCR amplification for identification of Saiga Tataricae Cornu14 pairs of partial primer were firstly designed by Oligo and DNAMAN software,after PCR amplification of DNA template of Saiga Tataricae Cornu and its adulterants,three pairs primer were selected according to the presence and brightness of the target band.The PCR amplification condition and reaction system were optimized and finally the identification of commercial antelope horn.Under the optimal condition of PCR,the concentration of ST-1,OA-3,and CH-1primers were observed.The results showed that the amplification was good when the concentration degrees were 0.2μM,0.3μM,and 0.02μM,the cycle number was 34,and annealing temperature was 60~oC,the specificity of PCR reaction was good and there was no cross reaction between primers.In addition,the two-way sequencing results of the amplified products were highly matched with the gene database,indicating that the amplification condition were reliable.Furthermore,when the annealing temperature was low,the three set of primers showed different degrees of nonspecific amplification.As the annealing temperature increased the primer showed different level of non-specific increase.In addition,the established specific primer amplification technology was used to identify the fake antler pieces and antler powder on the market.The results showed that among the 8 batches of processed antler products on the market,the target DNA fragment of 2 batches of antler was amplified by primer PST and identified as genuine antlers.The other 6batches of antelope were fake Ovis aries and Capra hircus antlers which were amplified by primer POA and PCH.It was seen that the proportion of common fake products made of horn is higher than35%.2.DNA molecular identification of Cervi Cornu PantotrichumBased on the species-specific primer amplification,we establish the identification technology of antler which has the advantages of strong specificity.DNAMAN and Oligo software were used to design the primers for amplification of DNA of Cervus antlers,and their counterfeits.Three pairs of primer namely,PC,PRT,and PRU,were further selected according to the lightness and darkness of the target strip.Then the PCR amplification condition were optimized,the specificity and the sensitivity were investigated,finally the established detection method was used to identify the authenticity of the commercial antler.Secondly,under the optimized PCR conditions,the number of cycles was 33,the primer concentration of PC,PRT,and PRU were 0.2μM,0.4μM,and 0.2μM respectively and the annealing temperature was 64~oC,62~oC,and 60~oC,the specificity of PCR reaction was good and there was no cross reaction between primers.In addition,the two-way sequencing results of the amplified products were highly matched with the gene database,indicating that the amplification condition were reliable.Furthermore,when the annealing temperature was low,the three set of primers showed different degrees of nonspecific amplification.As the annealing temperature increased the DNA template respondent with the primer.In addition,this technique can be used to identify the adulterants in antlers,PCR amplification condition was optimized,when the annealing temperature was increased to 64~oC,62~oC,and 60~oC respectively.The corresponding DNA template reacted with primers,PC,PRT,and PRU,no nonspecific amplification bands were observed by electrophoresis.The established specific primer amplification technology was used to identify the fake Cervus antler pieces and antler powder on the market.The results showed that among the 14 batches of processed antler products on the market,the target DNA fragment of 8 batches antler were amplified by primer PC and identified as genuine antlers,the 6 other batches were identified as fake Reindeer antlers.At the same time,these commodities were not observed after PU with primer,indicating that there were no counterfeits made from Rusa unicolor Kerr.It was seen that the proportion of common fake products of horn is about40%.3.SDS-PAGE analysis of protein in Cervus and Pilose antlersThis chapter explores the dynamic changes of protein in the process of processing the medicinal parts of basal animals.The experimental results showed that the protein in the medicinal parts of antler were more abundant than those of antlers and the protein of Cervus antler was distributed between 14.4k D and 116 k D and the protein of antler were more abundant than those of antler.Among them,there are four other protein bands I:70.2 k D,II:66.5 k D,III:45 k D,IV:18.4 k D in antler in the SDS-PAGE protein profile,while there are two clear and one unstable band A:762 k D,B:662 k D,C:18.4 k D in antler.These protein band can be used as to identify them.In addition,SDS-PAGE analysis was carried out on the protein of 3 batches of pilose antler and 5 batches of pilose antler decoction pieces.It was found that 2 batches of 3 batches of pilose antler decoction pieces had the characteristics of antler protein band distributed and one batch in accordance with the characteristics of SDS-PAGE protein profile of pilose antler.Therefore,it was identified that the 2 above batches of commercial pilose antler all accordance with the characteristics of antler protein profile.It provides a scientific basis for quality control of professionally treated and evaluated highly treated products,and the new strategy can ensure its safety and effectiveness of natural medicines.4.Protein content determination of Cervus and Pilose antlers by BCAThe protein content of Cervus and Pilose antler was determined by BCA method.The results showed that there is some difference in the protein content of various batches of commercial Cervus and pilose antlers.The protein percentage was from 30.26%to 49.29%,while most of the protein content of Cervus antler is higher than pilose antlers.The protein content of three batches of Pilose antler are different from five batches of Cervus antlers,which may be related to factors such as inconsistent and production process of medicinal parts.In addition,this part of the research work lay a foundation for the analysis of protein by SDS-PAGE,2-DE,etc.5.Determination of amino acid in Cervus antlersThe amino acid content in five batches of Cervus antler used in this experiment were Glycine,Alanine,Aspartic acid and Proline respectively.Glycine,Proline and Alanine are the characteristic amino acid of other commodities on the market and the common characteristic amino acid of traditional Chinese medicine commodities,indicating that the chemical component of femoral protein.4 kinds of amino acid in commodities on the market were determined.It was showed that the types and content of amino acid were rich in Cervus antler then in other antler produced by different manufacture.Finally,after comparing,it is found that several amino acids with high content Gly,Asp are hydro amino acid while the content of hydrophobic amino acid low except Ala and Pro,the content of hydrophobic amino acid is higher.There are differences in the content of amino acid in antler from different manufactures,refer to the preparation method of antler in the 2020 edition of Chinese Pharmacopoeia in which raw material decorating time and the proportion of auxiliary materials may affect the content of the amino acid in the product.