Bezoar Bovis is derived from cattle gallstones and is an expensive crude drug with antipyretic, cardiotonic, and sedative effects. Australia and South America are known origins of Bezoar Bovis, but Australian-produced Bezoar Bovis is considered high quality and commands high prices. Therefore, there is a risk of counterfeit origin sales. To ensure proper origin labeling in distribution, a testing method capable of determining the origin of Bezoar Bovis is necessary. We attempted to determine the origin of Bezoar Bovis through genetic analysis and metagenomic analysis of its bacterial flora.

We obtained 42 Bezoar Bovis samples (25 Australian, 12 Brazilian, 5 Argentine) from crude drug manufacturers and traditional Chinese medicine pharmacies. After extracting DNA from 200 mg of powdered samples, we performed single nucleotide polymorphism (SNP) array analysis, mitochondrial DNA D-loop region analysis, and metagenomic analysis of the bacterial 16S rRNA region.

SNP analysis revealed no SNPs with statistically significant differences in gene frequency. Principal component analysis and STRUCTURE analysis also failed to yield results characterizing origin. For SNP genotyping, likely due to DNA degradation, only 645 SNPs could be typed in over 80% of the 53,128 SNPs across all samples. This limited information was considered one reason origin determination was unsuccessful. Mitochondrial DNA analysis yielded sequences from all samples, but no origin-specific clusters were observed. Metagenomic analysis produced polymerase chain reaction (PCR) amplification products from 28 samples. Principal component analysis using unweighted UniFrac distance separated samples into two major clusters: Australian and South American. Although some South American samples were found within the Australian cluster, no Australian samples were present in the South American cluster. Since unweighted UniFrac distance is qualitative and unaffected by the abundance of individual species, our investigation of indicator bacteria suggested that genera such as Brevundimonas, Sphingobacterium, Ochrobactrum, and Achromobacter were influential. Given that the South American cluster contained no Australian samples, we believe that bacterial community analysis could be used to determine origin as South American. Since metagenomic analysis proved more useful than genetic analysis, this suggests that the land on which cattle are raised is more relevant than their genetic background in determining the origin of Bezoar Bovis.

Authors

• Hiroaki Nakanishi (Juntendo University School of Medicine, Japan)
• Aya Takada (Saitama Medical University, Japan)
• Katsumi Yoneyama (Saitama Medical University, Japan)
• Kentaro Sakai (Tokyo Medical Examiner’s Office, Japan)
• Kazuyuki Saito (Juntendo University School of Medicine, Japan)