Antimicrobial activity of Helichrysum species and the isolation of a new phloroglucinol from Helichrysum caespititium

Abstract

There are 500 Helichrysum (Asteraceae) species world wide of which 245 occur in South Africa.The South African species display great morphological diversity and are, therefore classified into 30 groups (Hilliard, 1983). Helichrysum species have been reported for their antimicrobial activities (Rios et al., 1988; Tomas-Barberan et al., 1990; Tomas-Lorente et al., 1989; Mathekga, 1998, Mathekga et al., 2000). Not much information on the bioactivity of compounds isolated from these species is available. In vitro antimicrobial screening methods provide the required preliminary observations to select among crude plant extracts those with potentially useful properties for further chemical and pharmaceutical investigations. In this study we investigated the antimicrobial activities of crude acetone extracts (shaken and homogenized) of twenty-eight Helichrysum species on ten bacteria species and six fungal species. A new phloroglucinol with significant antimicrobial properties was isolated by bioactivity guided fractionation from Helichrysum caespititium. The structure elucidation, conformation and stereochemistry of the new phloroglucinol, 2-methyl-4-[2',4',6'-trihydroxy-3'-(2-methylpropanoyl) phenyl] but-2-enyl acetate (caespitate), was established by high field NMR spectroscopic, crystallographic and MS data. The compound inhibited growth of Bacillus cereus, B. pumilus and Micrococcus kristinae at the very low concentration of 0.5 µg /ml and Staphylococcus aureus at 5.0 µg/ml. Six fungi tested were similarly inhibited at low MICs: Aspergillus flavus and A. niger (1.0 µg /ml), Cladosporium cladosporioides (5 µg/ml), C. cucumerium and C. sphaerospermum (0.5 µg /ml) and Phytophthora capsici at 1.0 µg/ml. The cytotoxicity of most currently used drugs has become a serious problem and efforts are being directed to obtaining new drugs with different structural features. One option favoured is the search for new plant derived non-toxic drugs, as was investigated in this study. Caespitate proved to be non-toxic at biologically active concentrations. Development of resistance to synthetic chemotherapeutic agents is known to occur in modern medicine; for example, resistance to some antibiotics of certain strains of microorganisms. A synergistic antibacterial bioassay demonstrated that the combination of caespitate and caespitin enhanced activity from a concentration range of 5 µg /ml to 0.5 µg /ml down to 0.1 µg /ml to 0.05 µg /ml on Gram-positive bacteria. The synergistic effect was in addition displayed against Gram-negative bacteria. The study of the morphology and ultrastructure of the epicuticular trichomes revealed that trichomes in H. caespititium originate from papillate cell outgrowths which elongate, develop and later polarise into apical, stem and basal parts and that repeated secretions of compounds probably occur from the young three-celled stage, enable us to characterise and relate our observations to their possible functional role in the production of the antimicrobial and other compounds on the leaf surface. South African Helichrysum species are a potentially good source of antimicrobial agents worthy of further investigation as efficient therapeutic compounds and in assisting the primary health care in this part of the world.