Bioactivity Guided Separation and Purification of Secondary Metabolites of Nymphoides indica

Abstract

As a continuing work of Professor Chowdhury Faiz Hossain’s research group crude MeOH Extract of Nymphoides indica showed potent antibacterial effect. The activity was Reconfirmed from the 6 gm MeOH extract of Nymphoides indica. Highest effect was found in Salmonella typhi which was 14 mm diameter of zone of inhibition in dose of 1000 μg/disc. On continuation of the following work large scale of Nymphoides indica collected from Panchagarh. The leaves were crashed and 250 gm of dried crashed leaves were obtained. Extraction of Nymphoides indica leaves (250 gm) at room temperature by maceration with EtOH yielded 42 gm (16.8 % dry weight) extract. The EtOH extract of Nymphoides indica was subjected to vacuum liquid chromatography (VLC) and five different fractions were collected using five different solvents; Fractions were- Fraction-1 (0.28 gm) eluted with 5000 ml n-hexane, Fraction-2 (0.63 gm) eluted 2000 ml CH2Cl2, Fraction-3 (3.97 gm) eluted with 5000 ml EtOAc, Fraction-4 (20.2 gm) eluted with 5000 ml acetone and Fraction-5 (0.74 gm) eluted with 5000 ml MeOH. The antimicrobial effect was further tested by using the VLC fractions. The effect was found in the fractions of n-hexane, CH2Cl2 and EtOAc. Highest effect was found with EtOAc fraction that was 18 mm diameter of zone of inhibition in Staphylococcus aureus in dose of 100 μg/disc. Fraction-2 of VLC fraction was separated by open column chromatography with silica gel which gave and the fractions were collected by monitoring the thin layer chromatography (TLC) which gave 18 fractions. Among them Fr-2-3 & Fr-2-4 yielded a colorless crystal, NIH-3 (17 mg). This compound was UV inactive and charring with MeOH and H2SO4 (9:1) gave dark black color. Based on 1H-NMR analysis, structure of NIH-3 was proposed as shown below (Figure-i). To the best of our knowledge the proposed structure of NIH-3 is novel. The compound may be a triterpenoid and have a structural simillarity with a triterpenoid that was lupeol. Rather than the lupeol, there was an xii extra double bond between C12 and C13 in NIH-3. 13CNMR, Mass spectroscopy, 1H-1H COSY, HMQC, HMBC and NOSY are required to confirm the proposed structure. Then Fraction-3 was subjected to open column chromatography and 35 fractions were separated according to different color bands in the column and by monitoring the TLC. Further chemical separation and testing biological activities of these fractions are yet to be done.