Brugia Malayi - Clark Science Center - Smith College

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 Brugia pahangi nematodes 2 (related to
the Brugia 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 Brugia 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).
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