Phytochemical and Biological Investigation of two Bangladeshi Medicinal plants, Leucas aspera and Curcuma zedoria

Abstract

Extraction of Leucas aspera leaves (50.00 g) and Curcuma zedoria rhizome (1.00 kg) at room temperature by maceration with MeOH yielded 10.20 g (20.40% of dry weight) and 136.15 g (13.60% of dry weight) extracts, respectively. The MeOH extract of Leucas aspera was separated by open column chromatography with silica gel using step gradient of Hex-EtOAc- MeOH. The fractions were collected by monitoring with thin layer chromatography (TLC). Analysis of TLC of the MeOH extract and further column fractions did not show any characteristic spot of lignan derivatives. Elution with Hex-EtOAc (9:1) yielded Fr-3′َ (0.85 g) and Fr-4 (0.55 g), respectively. On concentration of the solvent of Fr-3′َ and Fr-4 yielded two colorless crystals LA-1 (12.00 mg) and LA-2 (7.00 mg), respectively. TLC analysis revealed that these compounds were UV inactive and charring with MeOH and H2SO4 (9:1) on TLC plate gave characteristic bright red color. This characteristic charring indicated the compounds were steroidal derivatives. Fr-16 (3.00 g), eluted with MeOH showed strong antioxidant activity. But further purification was not carried out due to absence of lignan derivatives as well as the small quantity. The MeOH extract of Leucas aspera did not show any significant antimicrobial activity up to a concentration of 1 mg/disc against Escherichia coli, Staphylococcus aureus, Shigella dysentery, Salmonella typhi and Candida albicans. The MeOH extract of Curcuma zedoria showed significant antimicrobial activity. The zone of inhibitions were 12, 9.5, 8.5, 12 and 8 (mm) against Escherichia coli, Bacillus subcutis, Shigella boydii, Salmonella paratyphi and Saccharomyces cereviceae, respectively at a dose of 1000 μg/disc. The MeOH extract of Curcuma zedoria was subjected to vacuum liquid chromatography (VLC) and five different fractions were collected using five different solvents. The fractions were – Fr-1 (14.37 g, eluting solvent: n hexane), Fr-2 (20.28 g, eluting solvent dichloromethane or DCM), Fr-3 (16.50 g, eluting solvent EtOAc), Fr-4 (18.30 g, eluting solvent Acetone) and Fr- 5 (9.87 g, eluting solvent MeOH). Among the five fractions Fr-1 and Fr-2 were subjected to open column chromatography. Fr-1 of Curcuma zedoria was separated by open column chromatography with silica gel using step gradient of Hex-EtOAc-MeOH. The fractions were collected by monitoring with TLC. Fr-1-42 yielded a colorless crystal, CZH-42 (60.00 mg). This compound was UV inactive and charring with methanol and H2SO4 (9:1) gave characteristic 2 | P a g e bright red color which indicated the compound was a steroidal derivative. Structure of CZH-42 was elucidated by the analysis of 1H-NMR spectral data which revealed that the compound could be campesterol, a plant steroid. Further analysis such as 13C-NMR and Mass Spectroscopy (MS) are required for confirmation. As per our knowledge, based on the literature review, this is the first time report of champesterol isolation from Curcuma zedoria. CZH-42 showed no antioxidant activity. Fr-2 was then subjected to open column chromatography with silica gel using step gradient of Hex-EtOAc-MeOH and 11 fractions were obtained monitoring TLC. All of these fractions were mixtures of compounds which are needed to be separated by further column chromatography to isolate pure bio-active compounds. Bioactivities of these fractions are also needed to be evaluated.