High Performance Liquid Chromatography (HPLC) Partially Purified Lantadene B Bioactive Pentacylic Triterpenoid from Lantana camara var. aculeate Leaves and It’s Trypanicidal Activity

Abstract

Leaves of Lantana camara were freshly harvested, dried in the shade and pounded. Obtained leaf powder (2g) L. camara was cold extracted with 3 x 40-ml MeCN. Purification of the extract went through thin layer chromatography, column chromatography, preparative thin layer chromatography and fractional crystallization. Pooled fractions that contained bio constituent of latandene B as shown on thin layer chromatography were taken for HPLC analysis after being dissolved in HPLC grade methanol. Aliquots (20 µl) were taken for HPLC analysis. The HPLC system used was from Waters (USA) with 510 pump, Rheodyne injector with a 20-µl loop, 490E multichannel detector and Millennium 2010 dataprocessor. The detector was set at 210 nm. Purified and spectroscopic determined lantadene B content of young and matured leaves of L. camara was 347.0 ± 3.0 and 522.3 ± 37.1. Identified Lantadene B was screened against Trypanosoma evansi for trypanocidal activity on Vero cells grown in Dulbecco's Modified Eagle Medium (DMEM) and supplemented with foetal calf serum (FCS) 20-40% at appropriate conditions. In vitro cytotoxicity test of lantadene B at concentrations (1.56-100 µg ml-1) was done on Vero cells but without FCS. In vitro trypanocidal activity varied from immobilization, reduction and to the killing of trypanosomes in corresponding ELISA plate wells. At 250 µg ml-1of purified Lantadene B, there was drastic reduction of average mean trypanosomes count (40.±0.0 to 1.667±0.33) was observed. At 500 µg ml-1 of the test lantadene B, there was complete killing of trypanosomes (40.±0.0 to 0.00±0.00) at 8 h of incubation, which was statistically the same as diminazine aceturate (50 µg ml-1) at 4 h. Trypanosomes counts decreased in concentration and time –dependent manner with significant difference (P \(\le\) 0.05 to 0. 01)). During in vitro cytotoxicity test, Purified lantadene B and diminazine aceturate standard drug, were cytotoxic to Vero cells at all concentrations except at concentrations of 6.25-1.56 µg ml-1. Lantadene B and diminazine aceturate were cytotoxic to Vero cells at the same concentration. Lantadene B was responsible for higher anti-trypanosomal activity, which could be a future candidate compound for a new trypanocide.