Biological activity of Sinularia notanda

Abstract

In 1963 the study of marine natural products was just beginning and there were only a handful of public records with only one of these publications reporting a new compound. There has since been 9220 papers published, reporting on 24 662 new compounds. Annually a variety of cembrane diterpenoids with a range of biological activities are reportedly isolated from marine soft coral, and most notably these compounds possess anticancer properties. The investigation of a marine soft coral, Sinularia notanda, for novel bioactive compounds is presented here. The aim was to identify compounds active against key HIV enzymes as well as against cervical cancer, an opportunistic malignancy affecting many HIV positive women in Sub-Saharan Africa. A methanol extract of S. notanda was prepared and the cytotoxicity of the crude extract tested against a cervical cancer (HeLa) cell line using sodium 2,3-bis-(2-methoxy-4-nitro- 5-sulfophenyl)-2H-tetrazolium- 5-carboxanilide (XTT). The crude extract was also tested for HIV-1 protease inhibition using a direct enzyme assay, and antioxidant activity determined using 1,1-diphenyl-2picrylhydrazyl (DPPH). Bioassay guided fractionation, column chromatography and thin layer chromatography were used to identify active fractions from the total extract as well as to isolate biologically active compounds. The isolated compounds were tested for cytotoxic activity against four different cell lines. Nuclear magnetic resonance spectroscopy and x-ray crystallography were used for structure determination. The crude ethyl acetate fraction of S. notanda had a 50% cytotoxic concentration (CC50) of 33.82 ?g/ml, exhibited moderate inhibition of HIV-1 protease (between 40 and 60% inhibition), was unable to inhibit reverse transcriptase and showed antioxidant activity at a concentration (IC50) of 76.15 ?g/ml. In comparison, the crude methanol fraction had a CC50 of 145.80 ?g/ml, showed significantly lower protease inhibition (p < 0.05), and showed antioxidant activity at an IC50 of 27.16 ?g/ml. Extracts from natural sources including soft coral are routinely screened for free radical scavenging ability and the DPPH assay is one of the fastest methods to do so. Free radicals are known to induce oxidative damage to biomolecules which can eventually lead to diseases such as cancer. Oxidative damage can also be caused by HIV infection and oxidative stress levels may be exacerbated by antiretroviral treatment. Free radical scavenging antioxidants can provide protection against the damage caused by reactive oxygen species and are therefore considered as important nutraceuticals. Structural elucidation of a crystal isolated from S. notanda using NMR and x-ray crystallography confirmed a unique cembrane diterpenoid structure. The identified compound [(1R,3R,5S,12R,13S,E)-12-hydroxy-5,9,13-trimethyl-16-methylene-4,14-dioxatricyclo[11.3.2.03,5]octadec-8-en-15-one] showed moderate HIV-1 protease inhibition. This compound was abbreviated as CPD1. A second isolated compound designated E7 was found to be toxic to human leukemic monocyte lymphoma (U937) cells at 25 ?g/ml resulting in cell viability of less than 10%. Structure elucidation of E7 by 1D and 2D NMR as well as mass spectrometric analysis confirmed this compound to be structurally identical to the one isolated as a crystal and it also exhibited similar cytotoxic behaviour. Although this is not the first time CPD1 has been isolated from a coral of the Sinularia genus, data presented in this dissertation represents the first time that the compound was isolated from S. notanda. In Sinularia flexiblis CPD1 was isolated as a ketone (carbon-oxygen double bond at C12) while in the current study it was isolated from S. notanda as an alcohol (hydroxyl group at C12). Ketones are produced from the oxidation of secondary alcohols and the change in functional groups at C12 of CPD1 could be attributed to different organic solvents being used for initial extraction. A third compound was isolated and showed < 50% inhibition of HeLa cell growth and > 90% inhibition of U937 cell growth at 50 ?g/ml. Structure elucidation data identified the compound as 3-caffeoylquinic acid. This compound is not synthesised by the coral itself but is produced by algae that the coral ingested. The cembrane diterpenoid isolated from S. notanda in this study showed moderate inhibition of HIV-1 protease and selective cytotoxicity towards the U937 lymphoma cell line (selectivity index > 2). These responses are not unusual as cembrane diterpenoids with anti-cancer potential are increasingly being isolated from soft corals.