As part of an investigation of the biological activities of South African plants, a phytochemical and pharmacological investigation was carried out on five indigenous Vitex species; V. obovata ssp. obovata, V. obovata ssp. wilmsii, V. pooara, V. rehmannii and V. zeyheri. The chemical composition of the essential oils was determined using gas chromatography and mass spectrometry, and 1,8-cineole, α-copaene, caryophyllene oxide and γ-muurolene were found to be the most abundant constituents in the essential oils of both V. obovata ssp. obovata and V. obovata ssp. wilmsii. The essential oil of V. zeyheri showed high levels of 1,8-cineole, globulol, linalool and trans-pinocarveol. Spathulenol, caryophyllene oxide, elema-1,11-dien-15-ol and caryophyl-ladienol II were shown to be the major compounds in the essential oil of V. rehmannii, while limonene, β-selinene, cryptone, humulene epoxide II and caryophyllene oxide predominated in the essential oil of V. pooara. It was also revealed that the essential oil of V. pooara contained more hydrocarbon constituents whereas the other four taxa produce more oxygenated terpenes.
Using the minimum inhibitory concentration assay, the antimicrobial activity of the essential oils and non-volatile compounds were assessed on three Gram-positive bacteria, Staphylococcus aureus, Bacillus cereus and Enterococcus faecalis, two Gram-negative bacteria, Escherichia coli and Salmonella typhimurium, and one yeast, Cryptococcus neoformans. In general, the non-volatile fractions showed good antimicrobial activity, with V. obovata ssp. obovata and V. rehmannii being the most active. The essential oils were found to be poorly active, with the essential oil of V. zeyheri displaying higher activity. Gram-positive bacteria and the yeast were found to be more sensitive to both the essential oils and non-volatile compounds than Gram-negative bacteria. The antimicrobial activity of the essential oils may be ascribed to the oxygenated constituents, such as 1,8-cineole, linalool and carvacrol.
Through bioactivity-guided fractionation, the compound responsible for the antimicrobial activity of the non-volatile components was isolated from V. rehmannii and identified using nuclear magnetic resonance spectroscopy as 12S,16S/R-dihydroxy-ent-labda-7,13-dien-15,16-olide, a labdane-type diterpene. Using high performance liquid chromatography, this compound was detected in all the investigated species and it is most probable that it is responsible for the antimicrobial activity. Cirsimaritin, a flavonoid, was also isolated and identified from V. rehmannii and was shown to be present in the five taxa. For both cirsimaritin and 12S,16S/R-dihydroxy-ent-labda-7,13-dien-15,16-olide, this is the first report on their isolation and identification from the genus Vitex.
The anti-oxidant properties of the non-volatile compounds of the studied species were also investigated using the DPPH assay. All species, apart from V. pooara exhibited DPPH scavenging activity. This activity may be assigned to the flavonoids, especially cirsimaritin which was detected in all the species, and α-tocopherol (vitamin E) which was found in V. obovata ssp. wilmsii, and the diterpenoids. This is the first report on the presence of vitamin E in the genus Vitex.
The anti-inflammatory activity of both the essential oils and non-volatile compounds was assessed using the 5-lipoxygenase assay. Only the essential oils exhibited activity, with the essential oil of V. pooara being the most active. The 5-lipoxygenase inhibitory activity of the essential oils may be due to the terpenes and sesquiterpenes, and the greater activity of the essential oil of V. pooara may be attributed to higher levels of limonene, and to the presence of two esters, (Z)-3-hexenyl benzoate and (E)-2-hexenyl benzoate.
Finally, the antimalarial activity and toxicity profile of the non-volatile compounds, the labdane diterpene isolated from V. rehmannii and the essential oils of V. obovata ssp. obovata, V. obovata ssp. wilmsii and V. pooara, were investigated. The antimalarial activity was assessed on the chloroquine-resistant Gambian FCR-3 strain of Plasmodium falciparum using tritiated hypoxanthine incorporation. The toxicity was determined on Graham cells using microculture tetrazolium cellular viability assay.
All the test compounds revealed antimalarial properties and high toxicity, with V. obovata ssp. obovata and V. rehmannii extracts being the most toxic compared to other extracts. Because of the high toxicity profile of these test compounds, their anti-plasmodial activity was presumably associated with their cytotoxicity. The general cytotoxicity of 12S,16S/R-dihydroxy-ent-labda-7,13-dien-15,16-olide may also account for the antimicrobial activity, antimalarial properties and toxicity exhibited by the investigated species.
Cluster analysis of the essential oil constituents showed that V. pooara is an outlier, while the other four species are united into a single coherent cluster. Chemotaxonomy thus confirmed the results of the previous anatomical and taxonomical study on the indigenous Vitex species in which it was reported that V. pooara belongs to the subgenus Vitex while V. rehmannii, V. obovata ssp. obovata, V. obovata ssp. wilmsii and V. zeyheri belong to the subgenus Holmskiodiopsis (Bredenkamp and Botha, 1996).