Methodology for the Isolation and Detection of Sesquiterpene Lactones in Neurolaena Lobata Kristine Trotta Sesquiterpene lactones (SLs) are a class of terpene-derived organic compounds that are characterized by their polycyclic C 15 structure and five-membered lactone ring. They occur in abundance in plants of the family Asteraceae, and have been shown to be bioactive components in some traditional plant medicines that treat inflammation and intestinal worms, among myriad other conditions. 1 Evidence for the effectiveness of Neurolaena lobata in weakening and killing <strong>Brugia</strong> pahangi nematodes 2 (related to the <strong>Brugia</strong> malayi nematodes that cause lymphatic filariasis) indicate that SLs may be at least partially responsible for the plant’s bioactivity. Identification of the SLs is necessary for attempting to elucidate the molecular mechanism of N. lobata’s lethality against B. pahangi. An extraction of 5 g dried, crushed N. lobata (The Arvigo Institute, LLC) was prepared with 50% aqueous methanol twice by reflux at 50°C for 30 minutes 3 to yield 100 mL of a transparent brown liquid. The extract was washed with 300 mL ethyl acetate, 3 and the bright yellow organic layer was separated and left to evaporate. This yielded a yellow residue that was reconstituted in dichloromethane, gravity filtered 3 , divided into aliquots, and vacuum centrifuged to evaporate the solvent. Aliquots were reconstituted and consolidated in a total of 20 mL 50% aqueous methanol. 7 g aluminum oxide was added 3 . Filtration yielded approximately 15 mL of a transparent yellow liquid. Compound separation and detection was performed by preparative thin layer chromatography using silica gel GF as the stationary phase and toluene/ethyl acetate (3:2) as the mobile phase. 4 Preparative TLC yielded five distinct spots per lane (Figure 1) with the following mean R f values: (1) 0.27, (2) 0.42, (3) 0.52, (4) 0.62, (5) 0.69. This suggests that there may be at least five detectable varieties of SLs in N. lobata. Spots 1, 2, and 3 are consistent with previous findings: 0.29 (Neurolenin D), 0.42 (Neurolenin C), and 0.53/0.54 (Neurolenins B and/or F), respectively 4 (Figure 2). Spots 4 and 5 may be impurities or less polar SLs that may have not been previously described. Sesquiterpene lactones may have an important role in Neurolaena lobata’s ability to kill B. pahangi nematodes. This preliminary work in detecting SLs in N. lobata is an essential step in the way of discovering the molecular mechanism for this plant’s bioactivity. Future studies of this plant will involve large-scale extractions to attempt to purify SLs for structural analysis by NMR and IR, as well as applications of individual SLs in B. pahangi cultures to assess the efficacy of each isolated compound on worm mortality. (Supported by Howard Hughes Medical Institute) Advisor: Steven Williams References: 1 Chaturvedi, D. Sequiterpene lactones: Structural diversity and their biological activities. In Opportunity, Challenge and Scope of Natural Products in Medicinal Chemistry [Online]. Tiwari V.K. and Mishra B.B., Ed; Research Signpost: Kerala India, 2011; Chapter 10, pp 313-334. http://www.trnres.com/ebook/uploads/tiwari/ T_1302158793Tiwari-10.pdf (accessed 10 June 2012). 2 Fujimaki, Y., et al. Macrofilaricidal and microfilaricidal effects of Neurolaena lobata, a Guatemalan medicinal plant, on <strong>Brugia</strong> pahangi. Journal of Helminthology [Online] 2005. 79, 23-28. http://journals.cambridge.org/action/displayAbstract?fromPage=online&aid=712552 (accessed 1 June 2012) 3 Passreiter, C.M. Quantification of Sesquiterpene Lactones in Leaves of Neurolaena lobata. Phytochemical Analysis [Online] 1998. 9, 67-70. http://onlinelibrary.wiley. com/doi/10.1002/(SICI)1099-1565(199803/04)9:2%3C67::AID-PCA389%3E3.0.CO;2-V/abstract (accessed 1 June 2012). 4 Passreiter, C.M., Wendisch D, Gondol D. Sesquiterpene Lactones from Neurolaena lobata. Phytochemistry [Online] 1995. 39, 133-137. http://www.sciencedirect.com/ science/article/pii/003194229400863O (accessed 13 June 2012). 2012 53
Figure 1: Compound separation of the methanol extract of N. lobata under UV detection (254 nm). All replicates of this material under the given conditions yielded five distinct spots. Figure 2: Proposed sesquiterpene lactones detected by preparative TLC in accordance with Passreiter, Wendisch, and Gondol (1995). 4 Though all compounds have the same carbon backbone, they vary by functional groups, as displayed in red. Note that ‘ival’ is an abbreviation for ‘isovaleric acid’, a five-carbon acidic functional group, and that ‘OAc’ is an abbreviation for ‘acetoxy group’, an oxygen-modified acetyl group. 2012 54
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