Cardiotoxicity evaluation and comparison of diterpene alkaloids on zebrafish.

Diterpene alkaloids (DAs) have a broad spectrum of pharmacological activities, but exhibiting extremely serious cardiotoxicity to induce arrhythmia, heart arrest, even death. This study aimed to evaluate the cardiotoxicity of three diester diterpene alkaloids (DDAs) including aconitine (AC), mesaconitine (MAC), hypaconitine (HAC) and three monoester diterpene alkaloids (MDAs) including 14-α-benzoylaconine (BAC), 14-α-benzoylmesaconine (BMAC), 14-α-benzoylhypaconine (BHAC) on zebrafish. Firstly, the zebrafish embryos after a 72-hour post fertilization were treated with different doses of AC, MAC, HAC, and BAC, BMAC and BHAC for 2, 10 and 24 h, respectively. The heart rates of the treated embryos were calculated and the morphological images of body, together with heart fluorescence were obtained. Results demonstrated that AC, MAC, and HAC at low doses (15.6 and 31.3 µM) decreased the heart rates and increased them at high doses (62.5, 125, and 250 µM), while BAC, BMAC, and BHAC decreased the heart rates in the dose range of 31.3-250 µM, but the highest dose (500 µM) of BAC and BMAC increased the heart rates. In addition, AC, MAC, and HAC exhibited serious organic and functional toxicities, while BAC, BMAC, and BHAC did not. It could be induced that DDAs expressed stronger cardiotoxicities than MDAs, which might be due to that they were known as the Na+ channel activators and K+ channel inhibitors, respectively. The ß-acetate at C-8 position, along with the protonated nitrogen on ring A of their chemical structures contributed more for their different cardiotoxicities. This is the first study on cardiotoxicity comparison of DAs, providing references for the rational and safe application of these compounds and some plant species containing them to reduce side effects while retaining therapeutic efficacy.

Molecular authentication and differentiation of Dendrobium species by rDNA ITS region sequence analysis.

Owing to their significant medicinal and edible values, the natural Dendrobium species have underdone over-collection and habitat destruction, and cultivated species emerged for candidates. However, these Dendrobium plants are similar in shape to be easily confused, leading to extreme difficulties for identification based on their morphological and chemical features. In this study, the rDNA ITS region sequence analysis was developed for rapid and accurate identification of thirteen wild and cultivated Dendrobium species belonging to two sections Formosae and Chrysotoxae. By cloning and sequencing the rDNA ITS region genes from 13 Dendrobium species, the phylogenetic relationships among them were analyzed. Results showed that the variation of the ITS region, together with the lengths and Guanine and Cytosine contents of ITS, 5.8s rDNA, ITS1 and ITS2 sequences occurred in the tested Dendrobium species, and which from section Chrysotoxae was higher than that from section Formsae. Phylogenetic analysis based on neighbor-joining and maximum p-arsimony trees indicated that the Dendrobium species of sections Formosae and Chrysotoxae could be well divided into two groups. A majority of Dendrobium species exhibited distinctive ITS2 secondary structures, while for those with close genetic relationships were similar. Therefore, the ITS2 region sequence analysis is simple, quick, and highly reliable that can be used as an effective tool for molecular identification and classification, as well as the reconstruction of the phylogeny of wild and cultivated Dendrobium species belonging to different sections.