Identification of phenolic compounds in piper sarmentosum roxb. leaves methanolic extract and its cytotoxicity studies against pathogenic acanthamoeba spp

Abstract

Phenolic compounds are plant secondary metabolites that are beneficial to human health. Previous studies reported that phenolic compounds could lower the risk of heart diseases, occurrence of cancers and many microbial infections. Piper sarmentosum or locally known as Kaduk is a tropical herb that has long history in traditional medicines and food. These benefits are significantly dependent on the phenolic content. In this study, the crude methanolic extract of Piper sarmentosum leaves were analyzed for phenolic compounds identification and its anti-amoebic properties against two pathogenic Acanthamoeba strains namely Acanthamoeba castelanii and Acanthamoeba sp. of Hospital Kuala Lumpur (HKL) isolate. The total phenolic content in P. sarmentosum leaves methanolic extract was first determined by using Folin-Ciocalteu assay. The compounds were further characterized by using gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS) and reverse phase-high performance liquid chromatography (RP-HPLC) analyses. The cytotoxicity of the plant extract was examined using four assays: determination of IC50 by eosin dye method, cell morphological analysis using inverted light and scanning electron microscopies, cell membrane integrity assessment by acridine orange/propidium iodide (AO/PI) staining, and determination of cell death mode by DNA fragmentation assay. The total phenolic content was found to be 142.72 mg[GAE]/g of the extract. A total of 36 phenolic compounds were identified from the three high-resolution chromatography analyses. The IC50 values obtained were 74.64 μg/mL for A. castelanii while 22.13 μg/mL for Acanthamoeba sp. (HKL isolate). Microscopy analyses showed that the extract caused cell encystment indicated by distinctive morphological changes on cell shape and organelles, shortening of acanthopodia and disruption on cell membrane integrity. The extract was also discovered to promote apoptosis in both Acanthamoeba species. The result provided the evidence that crude methanolic extract of P. sarmentosum leaves contains active phenolic compounds contributing to its anti-acanthamoebic properties.