Research On Identification Of Sika Deer Antler And F1 Hybridization Antler Of Cervus Nippon And Cervus Elaphus

As a traditional and precious medicinal material,velvet antler has a very high medicinal value.However,its quality evaluation and identification methods are not perfect.It is difficult to distinguish the sika deer and F1 hybridization Antler of Cervus nippon and Cervus elaphus.In this experiment,the sika deer and F1Hybridization Antler of Cervus nippon and Cervus elaphus was used as experimental material.Comparative experiment on F1 Hybridization Antler of Cervus nippon and Cervus elaphus from five identification methods?Content analysis,chromatographic identification,SDS-PAGE electrophoresis identification,spectral identification,DNA molecular identification?,Determining the best method for identification,Provide scientific basis for actual production and application.Content determination analysis:The protein,moisture,ash,crude fat and phospholipid contents of two kinds of antler were determined by Kjeldahl method,high temperature drying method,burning method,Soxhlet extraction method and spectrophotometry;The contents of inorganic elements Ca,P,Na,K,Mg,Fe,Zn,Mn and Cu were determined by atomic absorption spectrometry combined with spectrophotometry;The contents of As,Pb,Cd,Cr and Hg were determined by inductively coupled plasma mass spectrometry;The amino acid contents of the two velvet antlers was determined by amino acid analyzer combined with spectrophotometry;Statistical analysis was performed using spss analysis software.Results:The crude ash content of sika deer antler was significantly higher than that of F1 Hybridization Antler of Cervus nippon and Cervus elaphus?P<0.01?,and the total phospholipid content of sika deer antler was significantly higher than that of F1 Hybridization Antler of Cervus nippon and Cervus elaphus?P<0.05?.The content of Mn?Fe?Cu?Pb?Cd and in sika deer antler in the main element of sika deer velvet was significantly lower than that in hybrid F1 generation?P<0.01?.The sodium content of sika deer antler was significantly lower than that of F1Hybridization Antler of Cervus nippon and Cervus elaphus?P<0.05?,The essential trace elements of sika deer antler were significantly lower than those of hybrid F1generation.?P<0.01?,The content of manganese,iron and copper in Sika deer antler was significantly lower than that in hybrid F1 generation velvet antler?P<0.01?.The total content of heavy metals in F1 Hybridization Antler of Cervus nippon and Cervus elaphus was significantly higher than that of Sika velvet?P<0.01?,and the contents of lead,cadmium and mercury were significantly higher than that of Sika velvet?P<0.05?.The total amino acid contents of sika deer antler was higher than that of F1hybrid velvet antler,but the difference was not significant.The content of methionine in sika deer antler was significantly lower than that in the F1 Hybridization Antler of Cervus nippon and Cervus elaphus?P<0.05?.The content of histidine in sika antler was lower than that in F1 Hybridization Antler of Cervus nippon and Cervus elaphus?P<0.01?.Chromatographic identification:This experiment is in accordance with the relevant provisions of the 2015 edition of the Chinese Pharmacopoeia,and comprehensive thin layer chromatography to identify other various herbs,Optimized from thin-layer chromatographic conditions,color processing conditions,and spotting amount,so choose the most suitable method for identification.Results:There is no significant difference between the two velvet layer maps.Identification by high performance liquid chromatography,Chromatographic conditions AgilentHCC18Column?4.6mm×250mm,5?m?,Mobile phase with 0.1%phosphoric acid aqueous solution-methanol?30:70?and acetonitrile-0.1%phosphoric acid water?30:70?,flow rate is 0.5mL·min^-1,detection wavelength is 280 nm,Column temperature is 25?,The injection volume is 20?L,It is concluded that the sika antler has characteristic peaks at the peak of 6 min.SDS-PAGE electrophoretic identification:Comparison of two velvet antler proteins extracted with water,Analysis of protein differences between two velvet antlers by SDS-PAGE?Results:Separation of two water-soluble proteins from pilose antler by 12.5%,10%and 7.5%gel separation system,and their protein bands were different.The number of protein bands of sika deer antler was higher than that of hybrid F1 deer antler.In 10%and 7.5%gel separation system,the protein bands of two kinds of velvet antler were significantly different.The sika deer antler showed bands at 116-97.2KD,while the F1 Hybridization Antler of Cervus nippon and Cervus elaphus had no bands.Spectral discrimination:This paper uses an UV spectrophotometer to scan the wavelengths of two antler extracts at 190⁴00 nm.The results showed that the ultraviolet peak of the aqueous extract of sika deer antler appeared at 204 nm.And the ultraviolet peak of the F1 Hybridization Antler of Cervus nippon and Cervus elaphus appeared at 210nm.At 200nm,The F1 Hybridization Antler of Cervus nippon and Cervus elaphus appeared in the peaks and valleys,sika deer antler has no such peaks and valleys at 200nm;The spectra of two kinds of velvet antler in the wavelength range of 4000⁴50cm-1 were scanned by infrared spectrometer.Infrared absorption peaks of two kinds of velvet antler were analyzed,The characteristic absorption peaks related to o-h stretching vibration of velvet antler of sika deer were observed at3500³000cm^-1,while the characteristic absorption peaks related to o-h stretching vibration of hybrid velvet antler were not observed at 3500³000cm-1.Two kinds of velvet antler can be distinguished by ultraviolet spectrophotometry and infrared spectrophotometry.Identification of DNA molecular diversity:Random amplified DNA polymorphism?RAPD?technique was used to explore the difference of DNA polymorphism between two kinds of velvet antler.Two kinds of antler DNA were extracted by blood tissue cell genome extraction kit and 20 random primers were selected to amplify the DNA of two kinds of deer antler.The PCR reaction system was 2×Taq PCR Master Mix 12.5?L,random primer 1uL?12.5?mol·L-1?,DNA template 1uL?0.5?g?,sterile double distilled water 10.5?L,total system 25?L.The cycle parameters were set to:94?pre-denaturation 1 min,94?deformation 1 min,45?annealing 1.5 min,72?extension 1.5 min,cycle 5 times;94?denaturation 1min,50?annealing 1.5 min,72?extension 1 min,cycle 35 Times,72?extension for 5 minutes and the amplified product was run for 30 min in 80v agarose electrophoresis.The random primer electropherogram was obtained and 20 primer amplification maps of two antlers were compared.There were no bands in primers 1?4?5?9?11?16?17and 18 of sika deer antler and hybrid F1 deer antler,but there were bands in primers 6?7?10?13?14?15 and 20.Hybrid F1 deer antler had 3 specific bands in primer 3 and 4 specific bands in primer 12.Sika deer antler had 1 specific band in primer 2,3 specific bands in primer 8 and 4 specific bands in primer 19.Among the 7 primers that could amplify the bands,25 bands were found in Sika Deer antler,while 23 were found in F1 Hybridization Antler of Cervus nippon and Cervus elaphus.The F1 Hybridization Antler of Cervus nippon and Cervus elaphus has similar nutritional components with sika deer antler and has high research value.This study provides an important basis for further development and utilization of deer antler.From the perspective of identification,TLC can be used as a simple and convenient identification method to identify the difference between antler and antler fakes,and to facilitate the identification of commercially available authentic antlers.HPLC identification has a wide range of differential analysis,strong separation ability,fast analysis time and reliable qualitative analysis results,which can identify two kinds of near-end antler;Polyacrylamide gel electrophoresis has strong resolution and high recognition,and is suitable for identifying antler species from the difference of protein between two kinds of antler;Spectrometric determination of pilose antler varieties has the characteristics of high accuracy and good precision.RAPD technology is rich in polymorphism,high detection rate and simple technology.It is suitable for rapid identification of antler.In this experiment,the RAPD method can distinguish the two velvet antlers well and provide a good genetic basis for the identification of the two velvet antlers.