Quality Evaluation And Antioxidant Study Of Rana Chensinensis Ovum Based On Unsaturated Fatty Acid

Rana chensinensis ovum（RCO）,the ovum of the female Rana temporaria chensinensis David,is the main by-product in the production of the valuable traditional Chinese medicine Ranae Oviductus,which is rich in nutrients and has great nutritional value and development prospects.At present,there are few studies on RCO quality evaluation methods,and there is a lack of efficient and comprehensive evidence-based methods for quality evaluation and regulatory assessment.However,complex factors such as geographical origin,breeding,harvesting and processing lead to uneven quality of RCO,which in turn directly affects the quality of related products.In order to guarantee the quality of RCO and the products,this study was committed to establish a quality evaluation method based on unsaturated fatty acids（UFAs）,the main active component of RCO,and to investigate the differences in the activity of RCO from different origins and their correlation with quality control indexes in terms of antioxidant activity initially.Firstly,in this study,21 batches of RCO from different origins and different harvesting time were determined by the optimized HPLC method to establish fingerprint,and reference profile was generated by the averaging method and similarity analysis was performed.Cluster analysis（HCA）and principal component analysis（PCA）were used to visualize the similarities and differences among the RCO samples.The results showed that the HPLC fingerprint of RCO matched a total of 20 common peaks,and the average percentage of non-common peak area was less than 5%.The similarity between the 21 batches of RCO and the reference profile R（20）was greater than 0.900,and the reference profile generated by the fingerprint could be used for the identification and consistency evaluation of RCO.Seven unsaturated fatty acid peaks were identified among the 20 common peaks in the fingerprint of RCO,namely eicosapentaenoic acid（EPA）,α-linolenic acid（ALA）,docosahexaenoic acid（DHA）,arachidonic acid（ARA）,docosapentaenoic acid（DPA）,linoleic acid（LA）and oleic acid（OA）,and the seven UFAs play an important role in the quality evaluation of RCO.HCA and PCA divided 21 batches of RCO into three categories based on the semi-quantitative information of peak areas of the common components,and the classification results showed certain regional similarity.Secondly,the quantitative analysis of multi-components by single-marker（QAMS）of EPA,ALA,DHA,ARA,DPA,LA and OA of seven main UFAs identified in RCO was established with ALA as an internal reference.Different column temperatures,flow rates,HPLC systems and columns were used to evaluate the durability of QAMS.The relative retention time of chromatographic peaks under different HPLC system and column conditions were used for localization of the peaks in QAMS.The conventional external standard method（ESM）was used as a reference to verify the feasibility of QAMS.The results showed that the relative correction factors of the seven UFAs with ALA as the internal were F_ALA/EPA=1.7411、FALA/ALA=1.0000、FALA/DHA=2.3179、FALA/ARA=1.6609、FALA/DPA=1.7696、FALA/LA=0.7106 and F_ALA/OA=0.1535.There were no significant fluctuations in the relative retention time of chromatographic peaks under different HPLC systems and columns,which could provide a basis for QAMS to locate the chromatographic peaks.The relative error（RE%）of the quantitative results of seven UFAs in 21 batches of RCO by ESM and QAMS is between–5%and+5%,and cosθwas greater than 0.9999,demonstrating the good feasibility of QAMS with a relatively wide range of RCO sample sources.The QAMS method can achieve the simultaneous quantification of the seven major UFAs in RCO with high efficiency and low cost.Finally,the quality of 17 batches of RCO from different origins was evaluated based on UFAs content,UFAs category and category ratio of UFAs.DPPH and ABTS free radical scavenging effects were used in antioxidant studies on RCO of different origins.Gray correlation and bivariate correlation analysis were used to correlate the in antioxidant activity of RCO with the content of UFAs.The results showed that the contents of the seven UFAs in 17 batches of RCO from different origins showed OA>LA>ALA>ARA>EPA≈DHA>DPA.The RCO with the highest monounsaturated fatty acid content was mainly from Huadian,Jilin Province（S9,S12 and S13）and Yanbian Korean Autonomous Region,Jilin Province（S4 and S5）.The RCO S9 from Huadian（Jilin Province）has the highest total UFAs（7）content.The RCO with the highest content of different types of UFAs was mainly from Huadian,Jilin Province（S9 and S12）.The mean values ofω-3 UFAs,ω-6 UFAs andω-9 UFAs ratios of RCO from different origins were 1:1.08:1.77.The gray correlation degree between antioxidant activity and UFAs content of RCO from different origin was greater than 0.60.Bivariate correlation analysis showed that the antioxidant activity of RCO was positively correlated with its UFAs content,in which DHA,ARA,DPA,LA,OA（ω-9 UFAs）,ω-3 UFAs,ω-6 UFAs and total UFAs（7）content were significantly and positively correlated with DPPH free radical scavenging,and DHA,ARA,LA,OA（ω-9 UFAs）,ω-6 UFAs and total UFAs（7）content were significantly and positively correlated with ABTS free radical scavenging.In conclusion,this study established a quality evaluation method of RCO fingerprint combined with chemometric analysis through HPLC analysis of RCO,which could be used for identification and consistency evaluation of RCO.Based on the seven UFAs identified by the fingerprint of RCO,a QAMS method was established to evaluate the quality of RCO with high efficiency and low cost.The free radical scavenging effect and UFAs content of RCO from different origins were used to investigate the differences of antioxidant activity and correlation with quality control indexes of RCO from different origins,so as to provide a reference basis for the quality evaluation and development and utilization of RCO.