| The raw material derived from animal origin is one of the three parts of traditional Chinese medicine(TCM).It is widely used in the clinical practice,possessing a definite therapeutic effect.It's difficult to study its effective substances and improve its quality control because of enriching a lot of macro-molecules.Totality-of-the-evidence(TOE)approach is described in the final revised version of Botanical Drug Development Guidance for Industry issued by FDA for the quality control of botanical raw material with unclear effective ingredients.TOE emphasizes to clarify the chemical compositions and ensure the quality consistency by manufacturing process control,concerning the original varieties,harvesting and processing as well as finished products,etc.,which is especially suitable for the quality control of animal raw material.Toad venom is prepared from the dried secretion of parotid gland and skin gland of Bufo bufo gargarizans Cantoror or B.melanostictus Schneider.It is not only one of the 28 kinds of poisonous TCMs in the toxic drug list issued by the State Council of the People's Republic of China,but also one of the 42 kinds of wild TCMs(grade?)in the key protection list.As a processed product,toad venom lost the appearance information of animal species,which makes its quality control more difficult.Thus,the study on the quality control of animal raw material is carried out under the approach of TOE,taking toad venom as an example.Toad venom contains micro-molecules of bufadienolides and indoles alkaloids as well as macromolecules of proteins.In the previous investigations,the chemical difference among the different bufo species,concerning bufadienolides and indole alkaloids,and the content change of these small molecules during the processing after harvesting were studied.In the present continuous study,the protein content and profiling were investigated by combining the quantitative method,SDS-PAGE and Nano LC-MS/MS analysis.Based on the interesting finding on small molecule and macro-molecule research,the quality standard of toad venom and its processed slices,toad venom power,was drafted.The detailed investigations are summarized as follows:1 protein content and profiling in the samples of toad venomThe extraction method of total protein in toad venom was established by investigating the solvent types,compositions and different p H values.The total protein content in the different batches of toad venom was determined by Bradford method.The total protein content of 47 batches of samples varied from 6.90 to 28.3%.Proteomics technique was used to analyze protein profiling difference bwteen the legal species(B.bufo gargarizans Cantor or B.melanostictus Schneider)and other bufo species,B.andrewsi Schmidt and B.raddei Strauch.1.1 SDS-PAGE analysis and marker proteinsThe SDS-PAGE method was used to analyze the protein bands of different bufo species.Three species,B.bufo gargarizans,B.andrewsi and B.raddei have main protein band at40-55 KD,however,B.melanostictus has no band at the same molecule weight.Moreover,B.melanostictus has marker band in the>180 KD range,compared with other three species.For B.gargarizans and B.andrewsi,a marker band at 130 KD was observed.The former had no protein band.There is a protein band at 25-35 KD in the samples of B.gargarizans and it disappeared in the samples of B.raddei.It is considered as a distinguished band for B.gargarizans and B.raddei.1.2 Nano LC-MS/MS analysis and marker proteinsUsing Nano LC-MS/MS,the differential protein analysis of eight toad venom samples from four bufo species was carried out.Based on Proteome Discoverer 2.2 platform,1357proteins were identified by searching cane toad.v2.2.Proteins.Fasta database.Gene functional annotation by Gene Ontology analysis and KEGG enrichment indicated the identified proteins included REDOX enzyme,hydrolytic enzymes and transporters,etc.,participated in various biological processes such as glycolysis,transport and translation.The statistical analysis showed that there were 49 specific proteins in the legal speies(B.bufo gargarizans and B.melanostictus),which were distinct from the related confusable species,B.andrewsi and B.raddei.26 specific proteins were observed in B.gargarizans and B.andrewsi,distinguishing from other two species.22 specific proteins present in B.gargarizans were different from other species.Similarly,82 proteins in B.melanostictus,5proteins in B.raddei,and 60 proteins in B.andrewsi were characteristic,distinguishing other species,respectively.According to the highly reliable peptides in the identified proteins,the characteristic peptides were obtained.ILKDQGYSTGIIGK for B.melanostictus and QLLAGGMAGAVSR for B.andrewsi were distinguished from other species.SLLTGPSQLK in B.gargarizans could be used to differentiate B.melanostictus.TGAFDWSHVAK in B.gargarizans could be used to differentiate B.andrewsi.FLENLNNFVK in B.melanostictus could be used to differentiate B.andrewsi.TTDMMFGGK,QVVEEPSPQLPAGK,MIGPFFDTLSTK,ITPADSQLQR,DLTAEQAAER and LVDNTEDWHPR were characteristic for B.andrewsi,distingushing from B.melanostictus.YIAVIIHPDQK was characteristic for B.melanostictus,distinguishing from B.raddei.The SIEVE 2.0 software was also used to find the characteristic ion among different bufo species.The mass data including mass/charge ratio,retention time and peak area were obtained and then subjected to the PCA and OPLS-DA analysis to screen the characteristic ions between different bufo species.The following data pairs of m/z to t _R:were obtained:B.gargarizans(530.3290-15.40)and B.andrewsi(964.5004-19.63,352.5574-18.11,1055.6564-18.31 and 527.3066-20.87),B.gargarizans(461.2529-66.23 and 622.8214-65.24)and B.raddei,B.melanostictus(948.0197-65.76,1269.6597-67.10,512.3225-52.24,1269.6581-64.98 and 644.3872-40.02)and B.andrewsi,B.melanostictus(47 characteristic ions)and B.raddei,B.andrewsi(34 characteristic ions)and B.raddei,B.raddei and the other three other species(8 characteristic ions).1.3 Effect of drying methods on proteins in toad venomThe effects of different drying methods,including hot air-drying at 105?,80?and60?,vacuum-drying at 60?,freeze-drying and shade-drying on the protein contents and molecule weight distribution in toad venom was investigated.There was no significant color change for freeze-drying samples before and after processing and other samples took on the increasing deepened yellow or dark yellow color.The total protein content of the different samples was shown in the following change trend:freeze-drying>vacuum-drying>shade drying>hot air-drying at 80?>hot air-drying at 60?>hot air-drying at 105?.SDS-PAGE analysis showed that the sample obtained by hot air-drying at 105?had less protein bands than other drying methods.The sample obtained by hot air-drying at 60?had the same protein band numbers as those of freeze-drying and vacuum-drying,but took on a relative light color.Different analytical methods suggested that the protein components significantly changed with the temperature rise,and some proteins could be degraded at higher temperature.2 Improvement of quality standards for toad venom and its processed slicesBased on comprehensive investigation on bufadienollides,indole alkaloids and proteins,combination with the consideration on gradual improvement strategy of mandatory quality standard and the current manufaturing practice,bufadienolides were still used as marker conponents for quality control of toad venom.The improved overall quality control methods and standards of toad venom were proposed,including using the optimized TLC method for qualitative identification,HPLC characteristic chromatogram and content determination under the same chromatographic conditions.The relative correction factors(RCFs)of QAMS was improved,and it was used to determine the content of bufadienolides in 38 batches of toad venom,and the reasonable content limit was suggsted.With reference to the proposed revision method of toad venom,the different batches of processed toad venom powder were analyzed including appearance description,TLC examination,HPLC characteristic chromatogram,moisture content and the total amount of five bufadienolides,and then the quality standard of toad venom powder was drafted.2.1 TLC examinationThe preparation method of the sample solution for TLC examination was simplified and unified according to the method decribed in quantitative analysis.2.2 HPLC characteristic chromatogramHPLC characteristic chromatogram of five bufadienolides was established,displaying gamabufotalin bufotalin,bufalin,cinobufagin and resibufogenin as characteristic peaks,by using the same chromatographic conditions as those describd in quantitative analysis.2.3 validation of RCFsIn the previous study,QAMS method was established for the five bufadienolides in toad venom.For the better application,RCFs at different detection wavelengths were calculated using different calculation methods(multiple correction method and slope method).RCFs were validated among different operators in the same laboratory and different laboratories.Finally,RCFs of cinobufagin to gamabufotalin,bufotalin,bufalin and resibufogenin were determined as 0.942,1.03,0.923 and 1.04,respectively.There was no significant difference between the total amount of five bufadienolides in 38 batches of toad venom calculated by RCFs mentioned above and by the external standard method.2.4 Suggestion on the content limitAccording to the total amount of five bufadienolides in 38 batches of toad venom samples and the current quality situation of commercial toad venom,it is suggested that the sum of five bufadienolides should be not less than 9.0%caculated as the dry product. |
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