Determination of the phytochemical, antioxidant and anti-inflammatory properties of selected Maltese medicinal plants

Abstract

An inflammatory response is characterised by an increase in the generation of free radicals. Overproduction of free radicals may cause excessive oxidative stress and indiscriminate host-tissue damage. For many inflammatory conditions there is constant demand for better therapeutic alternatives, in part due to the unfavourable adverse effect profile of many existing drugs. A number of scientific reports document that plant-derived phenolic compounds, triterpenoids and alkaloids act as key players in the modulation of inflammatory pathways. The overall scope of the study was to delineate the phytochemical profile and determine the antioxidant properties of the local varieties of 4 selected medicinal plants chosen on the basis of ethno-pharmacological records. The findings provided the basis for bioassays designed to elucidate the molecular mechanisms underlying the anti-inflammatory activity of the extracts in human cell models of inflammation. Extracts were preparcd from the rind fraction of Aloe vera leaves and the whole aerial parts of Chiliadenus bocconei, Calendula suffruticosa and Ruta chalepensis using 5 separate solvents. Each of the extracts and the crude gel and exudate from Aloe vera leaf were studied for the total content of phenols, alkaloids and triterpenoids using spectrophotometric assays. In addition antioxidant properties were studied using free radical-scavenging activity. Based on their phytochemical content and antioxidant activity, 6 from a total of 22 extracts that were investigated were selected for further study. Specifically optimized assays were used to investigate the effect of the extracts on the release of tissue necrosis factor-alpha (TNF-a) and interleukinl- beta (IL-β~). In addition assays were carried out to determine the effect of the extracts on the transcriptional activity of nuclear factor-kappa B (NF-KB) in human cell models of inflammation. Those extracts that emerged with the best profile in the phytochemical and antioxidant studies were the following: Aloe vera exudate; the ethanolic and acetone extracts of Ruta chalepensis; and the aqueous, ethanolic and acetone extracts of Chiliadenus bocconei. These extracts significantly suppressed the LPS (bacterial lipopolysaccharide)-induced release of TNF-a and IL-l~ from phorbol ester- . differentiated U937 cells. The acetone extract of Chiliadenus bocconei and the ethanolic extract of Ruta chalepensis demonstrated the best profiles in terms of both potency and efficacy in the inhibition of the release of TNFa and IL-l respectively Another significant finding was the increased activity of NF-KB that was induced by the extracts from Ruta chalepensis in human embryonic kidney cells (HEK293) that had been stably transfected with a reporter gene that expressed luciferase under the control NF-KB response elements. In addition, significant positive statistical correlations were found between alkaloid content, transcriptional activity of NF-KB and increased EC)o valucs for DPPH frce radical scavenging activity. This is the first scientific study that documents the suppressive activity of extracts from Chiliadenus bocconei and Ruta chalepensis on the release of the pro-inflammatory cytokines TNFa and IL-l ~ from human cell lines. Another significant finding was the increased transcriptional activity of NF-KB that was induced by extracts from Ruta chalepensis. In addition, the outcomes of the study point to the alkaloids in these extracts as potential inducers of the transcriptional activity of NF -KB through a mechanism that could involve the generation of increased intracellular oxidative stress. Increased transcriptional activity of NF -KB has been linked to cytotoxic activity in certain tumour cells. Fractionation of the extracts and delineation of the activity of the fractions on pro-inflammatory mediators is thus warranted. In addition further studies on Ruta chalepensis could lead to the identification of sources of compounds with potential anti-tumour activity in the chemotherapeutic field.