Mass spectrometry and synchrotron-FTIR microspectroscopy reveal the anti-inflammatory activity of Bua Bok extracts.

Abstract

Bua Bok or Centella asiatica (CA) is an Asian vegetable with anti-inflammatory benefits. Asiaticoside, asiatic acid, madecassoside and madecassic have been characterised as major active ingredients with a wide range of pharmacological advantages. In manufacturing processes, high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LCMS) are used to routinely determine the active compounds in raw materials. This research aims to explore anti-inflammatory properties, characterise metabolites and observe the biochemical changes of the inflammatory induced macrophages after pretreatment with the potential extracted fractions. Bua Bok leaf extracts were prepared. Macrophages were pretreated with non-toxic fractions to determine the anti-inflammatory action. Tentative metabolites of effective fractions were identified by LC-MS. Synchrotron fourier-transform infrared (S-FTIR) microspectroscopy was utilised to observe the biochemical change of the lipopolysaccharide (LPS)-induced cells after pretreatment with potential fractions. Fractions of ethyl acetate, 30% and 100% ethanol highly increased the nitrile scavenging and suppressed the function of phospholipase A2 . Fractions of 70% and 100% ethanol strongly decreased nitric oxide production. The comparison of 39 chemical compounds was presented. The change of proteins was improved after pretreatment of macrophages with fraction 70% ethanol. Fraction of 100% ethanol revealed the lipid accumulation was lower than 70% ethanol and diclofenac. While the anti-inflammatory actions of 70% and 100% ethanol were similar. S-FTIR expressed they inhibited inflammatory response with the distinct features of biomolecules. The S-FTIR, LC-MS and biological assay confidently provided the efficient strategies to inform the advantage of herbal extract on cellular organisation instead of a single compound.