Herbalogical studies of Ranae oviductus (Hamayou) and its species identification using DNA barcoding method

Abstract

<p indent=0mm>Ranae oviductus is a rare and precious Chinese medicine; however, there are many challenges connected with identifying this medicine, such as presence of many adulterants, the controversy over its origin, and the complexity of its classification history. First, we performed textual criticism of herbal literature and modern research data, constructed a phylogenetic relationship using whole mitochondrial genomes, and created a spatial overlay for the traditional production areas of Ranae oviductus and animal geographical distribution. The results obtained confirm that <italic>Rana chensinensis</italic>, <italic>R. dybowskii</italic>, and <italic>R. amurensis</italic> can be considered as a valid species for Ranae oviductus and that the <italic>Chinese Pharmacopoeia’s </italic>use of <italic>Rana temporaria chensinensis</italic> David. as the scientific name for Chinese brown frog is inappropriate. We further believe that the original species for Ranae oviductus could be <italic>R. dybowskii</italic>. Second, a debonding solution was prepared and used to pretreat medicinal materials to overcome the harmful effects of high expansion properties of Ranae oviductus during its DNA extraction. A new polymerase chain reaction primer pair was designed to resolve nonspecific amplification of cytochrome c oxidase subunit I (<italic>COI</italic>) sequences of Ranae oviductus using universal primers. A DNA barcoding reference library was established that contained 259 <italic>COI</italic> barcodes for 43 species covering Ranae oviductus and most of its adulterants. Finally, the DNA barcoding procedure for the identification of Ranae oviductus was verified based on simulation of real identification practices using commercially available medicinal materials. The results indicate that there are several adulterants, such as frog oil (<italic>Pelophylax nigromaculatus</italic>) and bullfrog oil (<italic>R. catesbeiana</italic>), in the herbal market. This study shows that DNA barcoding can successfully distinguish Ranae oviductus and its adulterants. In addition, the procedure established in this study can provide strong support for the safety of Ranae oviductus and protect the interests of consumers.