Bombyx batryticatus is the dried dead larva of silkworms after infection by a fatal fungi Beauveria bassiana. It's a challenging task to monitor intrinsic biological macromolecules in this natural medicine. In this study, a novel strategy based on comparative proteomic analysis was developed to disclose inherent differences between processed and highly processed Bombyx batryticatus. First, protein and amino acid contents were determined, and the changes in proteins after highly processing the medicine were profiled through electrophoresis-based technologies. Then, the peptides that originated from the differentiated proteins were eluted under reversed-phase NanoLC and sequenced by orbitrap MSD. It was found that both contents had been significantly decreased after processed Bombyx batryticatus was stir-baked with wheat bran. Additionally, protein bands and spots visualized in SDS-PAGE and 2-DE profiles of the processed product were much brighter than those of the highly processed product, allowing them to be distinguished by electrophoresis profiling readily. Moreover, thirty-two signature proteins were identified as potential biomarkers by NanoLC-orbitrap MSD after in-gel tryptic digestion of specific bands and spots. This work provides a solid scientific basis for quality assessment and control of commercial products of this natural medicine, and the developed novel strategy could ensure its safety and efficacy.

A novel non-sequencing approach was developed to detect short DNA fragments ( ca 100 bp) for rapid authentication of two natural products, namely Testudinis Carapax et Plastrum and Trionycis Carapax, based on the difference in mitochondrial genome. Five specifically designed primer reactions were established to target species for reliable identification of their commercial products. They were confirmed to have a high level of inter-species-specificity and good intra-species stability. The limit of detection was estimated to be 1 ng of genomes for all of five assays. Also, the validation results demonstrated that the raw materials and processed products in addition to some of the highly processed products can be conveniently authenticated with good sensitivity and precision by this newly proposed approach. Especially, when reference sample mixtures were assayed, these primer sets have still performed well but not the prevailing COI barcoding technology. These could assist in the discrimination and identification of other animal-derived medicines for their form of raw material, the pulverized and the complex.

A novel PCR technology was developed to detect short DNA fragments using species-specific primers for rapid and non-sequencing authentication of Bombyx batryticatus based on differences in the mitochondrial genome. Three specifically designed primer reactions were established to target species for the reliable identification of their commercial products. They were confirmed to have a high inter-species specificity and intra-species stability. The limit of detection was estimated as 1 ng of genomes for Beauveria bassiana and 100 pg for Bombyx mori and Metarhizium anisopliae. Furthermore, validation results demonstrated that raw materials and their processed products can be conveniently authenticated with good sensitivity and precision using this newly proposed approach. In particular, when counterfeits were assayed, these primer sets performed well, whereas COI barcoding technology did not. These could also assist in the discrimination and identification of adulterates of other animal-derived medicines in their pulverized and processed forms and even in complexes.