Isolation and characterisation of medicinal compounds from Phyllanthus Niruri L

Abstract

In many countries, Phyllanthus niruri L is one of the most popular alternatives of natural herbal remedy to overcomemany symptoms due to its wide range of therapeutic uses.Even though a considerable number of research projectshave been conducted in order to reveal the pharmacological activities of Phyllanthus niruri L, and that a number of reports have been produced mentioning its pharmacological effect, the rich constituents of this plant are yet to be comprehensively studied, particularly with regards to the nature of the biological activities that the compounds have.Accordingly, the main focus of this study is the exploration of Phyllanthus niruri L as a new candidate of natural compound. This was done through a series of bioassay- guided plant extraction and isolation protocols.Phyllanthus niruri L crude extracts were tested againstplasmodium, cancer cell lines, and platelet aggregation. Guided by the bioassay results, the isolation procedureswere performed using advance chromatography techniques, in order to purify the most - active substances thatrepresent the final candidate natural product.This study also employed flow cytometry and proteomics studies, as an attempt to identify the fundamental principlesof the mechanism of action of the isolated compounds.The results demonstrated that Phyllanthus niruri Lextracts showed a potency as antiplasmodial, anti-cancer, as well as anti-platelet agent. In inhibiting plasmodiumfalciparum growth, the potency of the extracts from the most to the least potent activity was methanol > water > ethanol > chloroform > hexane (IC 50values were 1.6 μg/ml, 9.6 μg/ml, 25 μg/ml, and 141 μg/ml, respectively).With regards to its anti-cancer effect, Phyllanthus niruri Lextracts showed a significant cytotoxic effect on human Caucasian lung large cell carcinoma (COR-L23), human acute T lymphoblastic leukaemia (MOLT-4), andhuman caucasian chronic myelogenous leukaemia (K562).Among all extracts, methanol extract demonstrated the strongest cytotoxicity effect with a low IC 50 values for all cell lines tested (IC 50 values for COR-L23, MOLT- 4, and K562 was 48.92 ± 0.52 μg/ml,42.21 ± 4.98 μg/ml, and 139.28 ± 19.02 μg/ml, respectively). Methanol, water, ethanol, and hexane extracts did not show any toxicity towards normal fibroblast cell line (3T3). However,chloroform extract demonstrated a toxic effect to the normal cells line (IC 50 value 164.3 ± 8.4 μg/ml).x The antiplatelet activity of Phyllanthus niruri Lwas further explored in this study due to a remarkable inhibitory effect of methanolic extract observed on ADP- induced platelet aggregation. With the aid of bioassay- guided isolation protocol, the study has isolated fourcompounds from the methanolic extract, which demonstrated a potency in preventing in-vitro platelet aggregation induced by ADP (compound 1, 2 , 3, and 6). The IC 50of each compound was 179.9 ± 2.67 μg/ml (compound1), 31.91 ± 1.86 μg/ml (compound 2),77.68±6.44 μg/ml (compound 3), and 43.35 ± 6.44 μg/ml (compound 6).Compound 2 was identified as corilagin or [3,5-dihydroxy-2-(3,4,5-trihydroxybenzoyl)oxy -6-[(3,4,5-trihydroxybenzoyl)oxymethyl]oxan-4-yl]3,4,5-trihydroxybenzoate. The finding of this study demonstrated that corilagin altered the G-protein signalling pathway in a selective manner by impeding Gq-protein cascade. Corilagin might act through G13-mediated signalling;however it showed no significant effect on Gi-mediated signalling pathway. Consequently, corilagin inhibited platelet shape changes, granule secretion, and platelet aggregate formation, which was suggested to take place by the inhibition of the elevation of intracellular Ca2+ level due to the inactivation of PLCβ. Although corilaginshowed no observable inhibitory effect on the initial activation of the major platelet glycoprotein, GPIIb/IIIa, the findings suggested that the interaction between the activated integrin and the related ligands is affected, which results in the inhibition of further amplification of platelet aggregation. Overall,this study confirmed the antiplatelet activity of corilagin and explainedits coherent mode of actions, which support the future development of corilagin, isolated from Phyllanthus niruriL as a natural-sourced antiplatelet compound