Centrifugal partition chromatography for antibacterial bio-guided fractionation of Clidemia hirta roots

• Bio-guided fractionation of Clidemia hirta roots by centrifugal partition chromatography.
• Antibacterial activity of ethyl acetate crude extract and fractions.
• Cytotoxicity evaluation on human keratinocytes and fibroblastes.
• Isolation and identification by mass spectrometry and NMR of bio-markers.

Clidemia hirta is an invasive shrub used in traditional medicine to treat some bacterial infections. In order to identify the molecules involved in this antimicrobial activity and to consider C. hirta as a potential source of preservative molecule usable in cosmetic applications, an antibacterial bio-guided screening of C. hirta root ethyl acetate extract was developed. Centrifugal partition chromatography performed using an Arizona N (heptane/ethyl acetate/methanol/water 1:1:1:1 v/v/v/v) system in descending mode, allowed to recover three fractions. For three samples of C. hirta root, independently collected, each crude extract and each fraction were tested against strains recommended in the current regulatory method for cosmetic preservative to evaluate the antimicrobial activity and on keratinocyte and fibroblast cells to verify the absence of skin cell toxicity. The first fraction showed an interesting antibacterial activity but also sometimes skin cell toxicity. Hydrolysable tannins, derivatives of ellagic acid were identified using mass spectrometry in this fraction. The second fraction was both antibacterial and not cytotoxic. Mass spectrometry and NMR experiments confirmed the isolation of only one compound in this fraction which was identified as arjunolic acid.

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