Comparative LC-ESIMS-Based Metabolite Profiling of Senna italica with Senna alexandrina and Evaluating Their Hepatotoxicity.

Senna Mill. (Fabaceae) is an important medicinal plant distributed worldwide. Senna alexandrina (S. alexandrina), the officinal species of the genus, is one of the most well-known herbal medicines traditionally used to treat constipation and digestive diseases. Senna italica (S. italica), another species of the genus, is native to an area ranging from Africa to the Indian subcontinent, including Iran. In Iran, this plant has been used traditionally as a laxative. However, very little phytochemical information and pharmacological reports investigating its safety of use are available. In the current study, we compared LC-ESIMS metabolite profiles of the methanol extract of S. italica with that of S. alexandrina and measured the content of sennosides A and B as the biomarkers in this genus. By this, we were able to examine the feasibility of using S. italica as a laxative agent like S. alexandrina. In addition, the hepatotoxicity of both species was evaluated against HepG2 cancer cell lines using HPLC-based activity profiling to localize the hepatotoxic components and evaluate their safety of use. Interestingly, the results showed that the phytochemical profiles of the plants were similar but with some differences, particularly in their relative contents. Glycosylated flavonoids, anthraquinones, dianthrones, benzochromenones, and benzophenones constituted the main components in both species. Nevertheless, some differences, particularly in the relative amount of some compounds, were observed. According to the LC-MS results, the amounts of sennoside A in S. alexandrina and S. italica were 1.85 ± 0.095% and 1.00 ± 0.38%, respectively. Moreover, the amounts of sennoside B in S. alexandrina and S. italica were 0.41 ± 0.12 % and 0.32 ± 0.17%, respectively. Furthermore, although both extracts showed significant hepatotoxicity at concentrations of 50 and 100 µg/mL, they were almost non-toxic at lower concentrations. Taken together, according to the results, the metabolite profiles of S. italica and S. alexandrina showed many compounds in common. However, further phytochemical, pharmacological, and clinical studies are necessary to examine the efficacy and safety of S. italica as a laxative agent.

The Biological Effects of Bilirubin Photoisomers.

Although phototherapy was introduced as early as 1950's, the potential biological effects of bilirubin photoisomers (PI) generated during phototherapy remain unclear. The aim of our study was to isolate bilirubin PI in their pure forms and to assess their biological effects in vitro. The three major bilirubin PI (ZE- and EZ-bilirubin and Z-lumirubin) were prepared by photo-irradiation of unconjugated bilirubin. The individual photoproducts were chromatographically separated (TLC, HPLC), and their identities verified by mass spectrometry. The role of Z-lumirubin (the principle bilirubin PI) on the dissociation of bilirubin from albumin was tested by several methods: peroxidase, fluorescence quenching, and circular dichroism. The biological effects of major bilirubin PI (cell viability, expression of selected genes, cell cycle progression) were tested on the SH-SY5Y human neuroblastoma cell line. Lumirubin was found to have a binding site on human serum albumin, in the subdomain IB (or at a close distance to it); and thus, different from that of bilirubin. Its binding constant to albumin was much lower when compared with bilirubin, and lumirubin did not affect the level of unbound bilirubin (Bf). Compared to unconjugated bilirubin, bilirubin PI did not have any effect on either SH-SY5Y cell viability, the expression of genes involved in bilirubin metabolism or cell cycle progression, nor in modulation of the cell cycle phase. The principle bilirubin PI do not interfere with bilirubin albumin binding, and do not exert any toxic effect on human neuroblastoma cells.

Mitochondria play a central role in apoptosis induced by alpha-tocopheryl succinate, an agent with antineoplastic activity: comparison with receptor-mediated pro-apoptotic signaling.

alpha-Tocopheryl succinate (alpha-TOS) is a semisynthetic vitamin E analogue with high pro-apoptotic and anti-neoplastic activity [Weber, T et al. (2002) Clin. Cancer Res. 8, 863-869]. Previous studies suggested that it acts through destabilization of subcellular organelles, including mitochondria, but compelling evidence is missing. Cells treated with alpha-TOS showed altered mitochondrial structure, generation of free radicals, activation of the sphingomyelin cycle, relocalization of cytochrome c and Smac/Diablo, and activation of multiple caspases. A pan-caspase inhibitor suppressed caspase-3 and -6 activation and phosphatidyl serine externalization, but not decrease of mitochondrial membrane potential or generation of radicals. For alpha-TOS, but not Fas or TRAIL, apoptosis was suppressed by caspase-9 inhibition, while TRAIL- and Fas-resistant cells overexpressing cFLIP or CrmA were susceptible to alpha-TOS. The central role of mitochondria was confirmed by resistance of mtDNA-deficient cells to alpha-TOS, by regulation of alpha-TOS apoptosis by Bcl-2 family members, and by anti-apoptotic activity of mitochondrially targeted radical scavengers. Co-treatment with alpha-TOS and anti-Fas IgM showed their cooperative effect, probably by signaling via different, convergent pathways. These data provide an insight into the molecular mechanism, by which alpha-TOS kills malignant cells, and advocate its testing as a potential anticancer agent or adjuvant.