Authentication Of Bombyx Batryticatus And Pupa Bombycis Batryticatus And Chinese Patent Medicine Containing Bombyx Batryticatus And Pupa Bombycis Batryticatus

Bombyx Batryticatus was the dried larva of the insect which was naturally or artificially infected by Beauveria bassiana（Bals.）Vuillant on the 4^thto 5^thinstar larvae of Bombyx mori（BM）.It can expel wind to relieve convulsion,reduce phlegm and resolve masses.Pupa Bombycis Batryticatus is the product which is the pupae of Bombyx mori fermented by Beauveria bassiana（BB）.In recent years,there is a large demand for Bombyx Batryticatus every year in China.Due to the similar morphological characteristics of related species,counterfeit Bombyx Batryticatus appeared in the market,which affected the safety and effectiveness of its clinical medication.The identification of Bombyx Batryticatus mainly depends on morphology with highly subjective.On the other hand,it is very difficult to identify Chinese Patent Medicines（CPM）because of the complicated prescriptions of prepared formulations,including a variety of Traditional Chinese Medicine（TCM）decoction pieces and some excipients,and the various forms of each component.In this study,specific primers were designed and screened according to the mitochondrial genome sequences of Bombyx mori,Beauveria bassiana and their counterfeits,and DNA identification techniques of BM,BB were established.Conventional PCR assay was developed for the identification of Pupa Bombycis Batryticatus in Tiancan Slice and Tiancan Capsule and the authentication of Bombyx Batryticatus in Jinsangqinyin Pills and Zhongfenghuichun Pills.Fluorescence quantitative PCR（qPCR）amplification was used to detect the samples of accelerated test and the quantitative analysis of BM and BB in commercial Jinsangqingyin Pills and Zhongfenghuichun Pills.Then SDS-PAGE and electronic nose technology were employed to explore the difference in proteins and specific smell of Bombyx batryticatus and Pupa Bombycis Batryticatus and CPM containing them.The main research contents are as follows:（1）Three pairs of specific primers were designed based on the whole mitochondrial genomes of Bombyx mori,Beauveria bassiana and Metarhizium anisopliae（MA）.The amplification conditions were established by optimization of cycle number and annealing temperature,and then specificity and sensitivity of PCR assay were investigated.DNA of BM,BB,and MA were amplified to show their bands at 61 bp,75 bp and 79 bp,respectively,when the annealing temperature was62℃and the cycle number was 30;and non-specific amplification bands were not observed.The detection limitations of DNA from BM,BB and MA were 0.10 ng/μL,0.10 ng/μL and 0.010 ng/μL,demonstrating good specificity and high sensitivity.Clear bands could be observed at the position of amplified products of DNA of each species,suggesting that the established conventional PCR technology could be employd to detect Pupa Bombycis Batryticatus.Eventually,all seven batches of commercial Tiancan Tablet and Tiancan Capsule were authenticated as genuine products.（2）Conventional PCR and qPCR were established to identify Bombycis Batryticatus in Jinsangqingyin Pills.After optimization of amplification conditions by conventional PCR,the specificity and sensitivity were investigated,and the commercial Jinsangqingyin Pills were analyzed by using this technique.In details,annealing temperature and primer concentration of qPCR were optimized to establish amplification conditions,which were used to disclose the change of DNA content from Bombycis Batryticatus during processing and storage.The amplification conditions were established by optimization of cycle number and annealing temperature,and then specificity and sensitivity of PCR assay were investigated.DNA of BM,BB,and MA were amplified to show their bands at 82 bp,94 bp and 144bp,respectively,when the annealing temperature was 62℃and the cycle number was35,40 and 40;and non-specific amplification bands were not observed.The detection limitations of DNA from BM,BB and MA were 0.10 ng/μL,0.10 ng/μL and 0.010ng/μL,which indicated that the method had good and high sensitivity.Clear bands could be observed at the position of amplified products of DNA of each species,suggesting that the established conventional PCR technology could be employed to detect Bombycis Batryticatus.Finally,DNA of MB but not MA in all eight batches was detected by conventional PCR assay,and DNA of BB was detected in 6 batches except for batch 2 and batch 5.In addition,when the annealing temperature was 64℃and the primer concentration was 0.60μM,specificity of qPCR amplification reaction was strong,and the dissolution curve was unimodal.The linearity ranges of standard curves of DNA of BM and BB by PM2 and PB2 primers were 10³～10⁷copies/μL and10²～10⁶copies/μL,respectively,and the correlation coefficients were r²>0.990.The DNA content of BB in batches 2 and 5 was 1.15×10³copies/μL and 7.07×10²copies/μL,respectively,which was lower than that of other batches.In addition,during the storage process,the content of DNA from Bombycis Batryticatus of Jinsangqingyin Pills showed an obvious descending trend,which could be the reason that Bombycis Batryticatus contained in two batches of Jinsangqingyin Pills was identified as counterfeit by conventional PCR assay.（3）Conventional PCR and qPCR were established to identify Bombycis Batryticatus inn Zhongfenghuichun Pills.After optimization of amplification conditions by conventional PCR,the specificity and sensitivity were investigated,and the commercial Zhongfenghuichun Pills were analyzed by using this technique.In details,annealing temperature and primer concentration of qPCR were optimized to establish amplification conditions,which were used to disclose the change of DNA content from Bombycis Batryticatus during processing and storage.The amplification conditions were established by optimization of cycle number and annealing temperature,and then specificity and sensitivity of PCR assay were investigated.DNA of BM,BB and MA were amplified to show their bands at 82 bp,94 bp and 144bp,respectively,when the annealing temperature was 60℃and the cycle number was35;and DNA of ES was amplified by 62℃with 34 cycles and non-specific amplification bands were not observed.The detection limitation of DNA in MB,BB,MA and ES were 0.10 ng/μL,1.0 ng/μL,0.10 ng/μL and 0.010 ng/μL,which indicated that the method had specificity and high sensitivity.Finally,DNA of MB but not MA in all six batches was detected by conventional PCR assay,and DNA of BB was detected in 5 batches except for batch 2.In addition,when the annealing temperature was 64℃and the primer pair concentration was 0.40μM,the qPCR amplification reaction specificity was strong,and the dissolution curve was unimodal.The linearity ranges of standard curves of DNA of BM and BB by PM2 and PB2 primers were10⁴～10⁸copies/μL and 10²～10⁶copies/μL,respectively,and the correlation coefficients were r²>0.990.The DNA content of BB in batches 2 was 702 copies/μL,respectively,which was lower than that of other batches.In addition,during the storage process,the content of DNA from Bombycis Batryticatus of Zhongfenghuichun Pills showed an obvious descending trend,which could be the reason that Bombycis Batryticatus contained in two batches of Zhongfenghuichun Pills was identified as counterfeit by conventional PCR.（4）Protein of Bombyx batryticatus and Pupa Bombycis Batryticatus and CPM containing them were extracted by ultrasonic with water and analyzed by SDS-PAGE.The results shown that the protein of processed products were abundant and distributed in molecular weight range from 10 k D to 80 k D,while high-processed Bombycis Batryticatus had not shown clear protein bands.Ten batches of pupa Bombycis Batryticatus and another ten batches of self-made fake product have protein bands at 10 k D or 40 k D,respectively,showing obvious difference for their differentiation.Meanwhile,there was a band at 10 k D in protein analysis of Tiancan Tablet and Tiancan Capsule,which could be originated from pupa Bombycis Batryticatus.This result was the consistent with the previous identification by specific primer PCR assay.In addition,there was no significant difference in proteins between Jinsangqingyin Pills and Zhongfenghuichun Pills.（5）Smell analysis of Bombyx batryticatus and Pupa Bombycis Batryticatus and CPM containing them were conducted by electronic nose technique.In this session,identification models were established for pupa Bombycis Batryticatus and its fake products;processed and high-processed Bombycis Batryticatus;Bombycis Batryticatus and pupa Bombycis Batryticatus;and Jinsangqingyin pills and Zhongfenghuichun pills by clustering analysis（CA）,principal component analysis（PCA）,linear discriminant analysis（LDA）,discriminant factor analysis（DFA）.The results shown that the samples could not clustered well by CA.When PCA or DFA was used,the cumulative variance contribution rates were all greater than 85%,and the samples were divided into two clusters except for pupa Bombycis Batryticatus,processed Bombycis Batryticatus and Jinsangqingyin Pills.While LDA being used,most of the samples could be accurately distinguished except for pupa Bombycis Batryticatus and processed Bombycis Batryticatus.DFA can also be used to identify pupa Bombycis Batryticatus in Tiancan Tablets and Tiancan Capsules,and Bombycis Batryticatus in Zhongfenghuichun Pills.Therefore,electronic nose technology is promising to assist the identification of Bombyx batryticatus and Pupa Bombycis Batryticatus and CPM containing them.