[Molecular identification of animal species of Bufonis Venenum from secretions].

The animal species is one of the key factors affecting the quality of Bufonis Venenum. The quality of Bufonis Venenum derived from Bufo bufo gargarizans is significantly higher than that from B. melanostictus. Since Bufonis Venenum is from secretions, the conventional identification methods are difficult to identify the animal species due to the lack of the appearance and morphology of the animals. The rapid development of molecular identification technology has provided new methods for the identification of Bufonis Venenum. However, because of the low content and serve degradation of residual DNA in secretions, the research on the molecular identification of Chinese medicinal materials from secretions remains to be carried out. To understand the animal species of Bufonis Venenum, this study collected 83 samples of Bufonis Venenum, including 7 commercially available samples, 5 reference medicinal materials, and 71 animal samples from which Bufonis Venenum was prepared according to the method in the 2020 edition of the Chinese Pharmacopoeia. Different DNA extraction methods were used and compared, and the mitochondrial 16S rRNA gene fragments were amplified, on the basis of which the phylogenetic trees were built. Finally, molecular identification of the animal species of the samples was performed. The results showed that the DNA extracted from Bufonis Venenum by the reagent kit had good quality, and 16S rRNA sequences were successfully amplified from 80 out of the 83 samples. In addition, 71 16S rRNA sequences of the animal species of Bufonis Venenum were downloaded from GenBank. The phylogenetic trees constructed based on the neighbor-joining(NJ) method and the Bayesian inference(BI) method showed that the samples derived from B. bufo gargarizans and B. melanostictus were clustered into separate monophyletic clades, with the support of 100%(NJ) and 1.00(BI), respectively. The animal species of both commercially available samples and reference medicinal materials were B. bufo gargarizans. In conclusion, DNA can be extracted from Bufonis Venenum derived from secretions, and the 16S rRNA gene sequences can be amplified, which can be used for molecular identification of the animal species of Bufonis Venenum. The findings provide a reference for the quality control of Bufonis Venenum and the identification of animal species of medicinal materials derived from secretions.

[Effect of Total Ravonoids of Herba Epimedium on BMP-2/RunX2/OSX Signaling Pathway during Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells].

OBJECTIVE: To explore the effect of total flavonoids of Herba Epimedium (FHE) on BMP-2/RunX2/OSX signaling pathway in promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). METHODS: Passage 3 BMSCs were randomly divided into the control group, the experimental group, and the inhibitor group. BMSCs in the control group were cultured in 0.2% dimethyl sulfoxide + Osteogenuxic Supplement (OS) fluid + DMEM/F12 culture media. BMSCs in the experimental group were intervened by 20 microg/mL FHE. BMSCs in the inhibitor group were intervened by 20 microg/mL FHE and 1 microg/mL NOGGIN recombinant protein. At day 9 alkaline phosphatase (ALP) activity was measured. Calcium nodules were stained by alizarin red staining and the density was observed. The transcription expression of osteogenic differentiation-related proteins (type I collagen, osteocalcin, and osteopontin) and related factors of BMP-2/RunX2/OSX signaling pathway was assayed by RT-PCR. RESULTS: Compared with the control group, ALP activities were enhanced and the density of calcium nodules significantly increased; type I collagen, osteocalcin, and osteopontin expression levels were increased in the experimental group. The expression of osteogenesis-related transcription factor was also increased in the experimental group. Noggin recombinant protein inhibited FHE promoting BMSCs osteogenesis in the inhibitor group. Compared with the experimental group, ALP activity decreased (P < 0.05), the density of calcium nodules was lowered, expression levels of type I collagen, osteocalcin, osteopontin significantly decreased (P < 0.05) in the inhibitor group. CONCLUSION: 20 microg/mL FHE promoted osteogenic differentiation process of BMSCs by BMP-2/RunX2/OSX signaling pathway.