| Gelatinous Chinese medicines(GCMs),is a kind of solid glue,which is made of animal skin,horn,nail or bone,decocted with water to get colloid,condensed into thick glue and dried.It is a traditional tonic with the functions of nourishing blood,moistening dryness and hemostasis.It has been used for thousands of years in China.In prevailing Chinese Pharmacopoeia,Asini Corii Colla(ACC),Testudinis Carapacis ET Plastri Colla,and Cervi Cornus Colla are included,while in the Drug standard of the Ministry of Health of the People’s Republic of China,Taurus Corii Colla(TCC),and Scrofa Corii Colla(SCC)are included.In recent years,the consumption of GCMs is increasing rapidly,and the resources of medicinal animals are becoming increasingly scarce.The adulteration of ACC with low-cost bovine and swine skin as raw materials has not only harmed the public health,but also caused unfair competition in the market.Therefore,based on the DNA and specific peptide of animal origin in GCMs,this paper established the key technology to identify the authenticity of GCMs,so as to reliably to reliably identify common counterfeit and adulterated products.In addition,the mixed peptides produced by trypsin hydrolysis of ACC and TCC were separated and their antioxidant activity was studied.The active components were analyzed by MALDI-TOF/TOF-MS to discover potential antioxidant peptides,which helps to clarify the material basis of the nourishing effect of GCMs and has reference value for the development and application of health food containing ACC.The research content is divided into the following parts:(1)Trypsin digestion followed by UPLC-ESI-MS identification of animal skin origin of ACC and TCCTrypsin was used to hydrolyze the protein of ACC,TCC,SCC and Caballus Corii Colla(CCC),then the peptide mixture was obtained.The hydrolysate was analyzed by UPLC-ESI-MS,and the origin of animal skin of ACC and TCC was identified.The characteristic ion of ACC is m/z 539.80,m/z 539.80(double charge)→ m/z 612.40 and m/z 539.80(double charge)→ m/z 923.80 as the specific detection ion pair of ACC;the characteristic ion of TCC is m/z 641.30,m/z 641.30(double charge)→ m/z 726.20 and m/z 641.30(double charge)→ m/z 783.30 as the specific detection ion pair of TCC;the characteristic ion of SCC is m/z 774.00,m/z 774.00(double charge)→ m/z 977.50 and m/z 774.00(double charge)→ m/z 752.30 are used as the special property detection ion pair of SCC;the characteristic ions of CCC are m/z 386.25,m/z 386.25(double charge)→ m/z 556.50 and m/z 386.25(double charge)→ m/z 499.25 are used as the special property detection ion pair of CCC.Both the GCM references and the self-made samples had characteristic ion peaks of the corresponding species.The absence of false positives or false negatives also indicated that the self-made samples meet the standards and could be used in subsequent experiments.The commercially available ACC and TCC were tested,and the results showed that three of the six batches of ACC were only found to contain peptide fragments derived from donkey skin,and were identified as genuine,while the other three batches were found to contain peptide fragments derived from bovine,swine and horse skin,were identified as adulterated;in addition,the three batches of TCC were only found to contain peptide fragments derived from bovine skin,and were identified as genuine.(2)MALDI-TOF/TOF-MS analysis of peptide fragments digested by enzyme in GCMsTrypsin and Collagenase A were used to hydrolyze the protein of ACC,TCC,SCC and CCC.The digested products were analyzed by MALDI-TOF/TOF-MS,and the characteristic peptide fragments were screened out.According to the selected peptide fragments,the animal skin sources of commercial ACC and TCC were identified.The results showed that after trypsin digestion,3,17 and 14 different peptides were screened from ACC,TCC and SCC respectively;however,the characteristic peptide fragments could not be found from CCC.In the detection of commercial samples,among the six batches of ACC,four batches were only found to contain peptide fragments derived from donkey skin and were identified as genuineproducts,while the other two batches were found to contain peptide fragments derived from swine skin and were identified as adulterated;among the three batches of TCC,two batches were only found to contain peptide fragments derived from bovine skin and were detected as authentic products,and the other batch was found to contain peptide fragments derived from swine skin and was identified as adulterated product.After digestion with collagenase A,GCMs mainly form tripeptides with lower molecular weight,and failed to find the characteristic peptides that can be used as the basis for the specific identification based on the MALDI-TOF/TOF-MS analysis results.(3)Identification of animal skin origin of ACC and TCC by specific primer amplificationAccording to the difference of mitochondrial gene sequence among donkey,bovine,swine and horse,four pairs of species-specific primers were designed and screened by using the software of Oligo.Animal-derived DNA was extracted and purified from ACC,TCC,SCC and CCC by SDS cracking-phenol chloroform extraction,the single PCR amplification conditions were optimized.After that,specific verification and sensitivity inspection were conducted,and self-made adulterated samples were detected.At last,the established technology was applied to identify animal sources of commercially products of ACC and TCC.The results showed that the four pairs of primers had good specificity,no non-specific amplification to other species under the optimized amplification conditions,the minimum detection limit is 0.1 ng/μL,which has high sensitivity,and could detect10% adulteration of bovine,swine or horse DNA in ACC and swine or horse DNA in TCC.In addition,the detection results of commercial ACC and TCC products showed that three of the six batches of ACC were only found to contain DNA derived from donkey skin and were identified as authentic products,and the other three batches were found to contain DNA derived from bovine and swine skin and were identified as adulterated products;among the three batches of TCC,one batch was only found to contain DNA derived from bovine skin and was identified as authentic product,onebatch was only found to contain DNA derived from swine skin and was identified as fake product,one batch was not found to contain DNA derived from the four animal origin.(4)Study on the antioxidant activity of peptides of ACC and TCCUsing trypsin to enzymatically digest ACC and TCC,the digested products were separated by RP-HPLC and Sephadex G-50;the resulting fractions were collected and the scavenging rate of OH radicals and DPPH radicals were determined,and the ones with higher antioxidant activity were selected to be analyzed by MALDI-TOF/TOF-MS to discover potential antioxidant peptides.The results showed that the fractions with higher antioxidant activity were mainly composed of peptides with lower molecular weight.The fractions of ACC antioxidant activity,separated by RP-HPLC,Sephadex G-50 and MALDI-TOF/TOF-MS analysis,were found the potential high activity peptides with m/z of 586,904,1048,1077 and 1466.The fractions of ACC antioxidant activity separated by RP-HPLC,Sephadex G-50 and MALDI-TOF/TOF-MS analysis,were found the potential high activity peptides with m/z of 919,1048,1077,1105,1466 and 2382. |
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