A PHYTOCHEMICAL AND PHARMACOLOGICAL INVESTIGATION OF INDIGENOUS AGATHOSMA SPECIES Aneesa Moolla A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Pharmacy Johannesburg, South Africa, 2005. DECLARATION I, Aneesa Moolla, declare that this dissertation is my own work. It is being submitted for the Degree of Master of Pharmacy at the University of the Witwatersrand, Johannesburg. It has not been submitted before for any degree or examination at this or any other University. Signature: …………………… Date: …………………... ii DEDICATION To my parents Hassim Ahmed Moolla and Sara Bibi Moolla. iii ACKNOWLEDGEMENTS Firstly I would like to sincerely thank my supervisor, Prof. Alvaro M. Viljoen, for the knowledge impacted on me, his guidance and full support throughout the research period and for his motivation towards the completion of this research. His enthusiasm and interest towards the research project encouraged me a great deal. There are not enough words to express my gratitude. I am also indebted to Mrs Sandy F. van Vuuren and Dr Robyn L. van Zyl who tirelessly supervised the antimicrobial and toxicity components of this study. My sincere thanks to Prof. K. Hüsnü C. Başer, Dr Betül Demirci and Dr Temel Özek for access to their database to document the chemical composition of the essential oils; and for their assistance and expertise in generating the gas chromatography-mass spectroscopy (GC-MS) results. Their hospitality during my visit to Anadolu University is highly appreciated. A special thanks to Dr P. Steenkamp and Mr N.M. Harding for their technical guidance on the high performance liquid chromatography analyses. I am indebted to Mr Terry Trinder-Smith for identifying the plant material and assisting with the collection thereof. I would like to extend my sincere thanks to the National Research Foundation (Indigenous Knowledge Systems) and the Medical Faculty Research Endowment Fund, University of the Witwatersrand for financial support. Finally, I would like to acknowledge the technical and administrative staff of the Department of Pharmacy and Pharmacology, all my friends and colleagues at the iv University of the Witwatersrand, and finally all the people who helped me in ways I cannot detail here. v ABSTRACT As part of an investigation of the biological activities of South African plants and due to their extensive traditional use and lack of scientific evidence, a phytochemical and pharmacological investigation was performed on 17 indigenous Agathosma species (19 samples). The chemical composition of the essential oils was determined using gas chromatography coupled to mass spectroscopy (GC-MS). Analysis resulted in the identification of 333 compounds. To evaluate the chemical similarities and differences, cluster analysis was used to assess the essential oil composition of the samples. The results showed qualitative and quantitative differences amongst the taxa. The essential oils of Agathosma hirsuta and A. zwartbergense are particularly rich in citronellal, hence they are tightly clustered in the dendrogram obtained from the cluster analysis. Linalool, myrcene and limonene are the major constituents of both A. capensis (Gamka) and A. capensis (Besemfontein). Qualitative and quantitative differences are noted in the chemical compositions of the leaf oils of Agathosma capensis (Gamka) and A. capensis (Besemfontein). Agathosma arida and A. lanata are united in a single cluster due to the compounds β-pinene, linalool and spathulenol being major components in both species. The presence of 1,8-cineole in large quantities in both Agathosma namaquensis (23.5%) and A. ovalifolia (9.7%), unites them in a single cluster. A wide chemical variability for the essential oils of indigenous Agathosma species has been demonstrated. There was considerable variation in the percentage oil yield of the essential oils. Agathosma hirsuta produced the highest yield (1.15%) whilst A. ovalifolia produced the lowest yield (0.16%). vi Previous studies have revealed that the coumarin and flavonoid components of Agathosma species are responsible for their biological activities. High performance liquid chromatography (HPLC) was used to document the non-volatile composition of Agathosma species and to establish if phenolic patterns were present amongst the species. All species were found to be rich in flavonoids (i.e. flavones and flavonols). Many of the compounds detected were common to most of the species. A pure coumarin, puberulin, was identified in the diethyl ether extract of Agathosma ovata (round-leaf) and detected in the dichloromethane and methanol (1:1) extract of A. namaquensis. Agathosma species have been used traditionally to treat a wide variety of infections. They has been used as a cough remedy, for the treatment of colds and flu, kidney and urinary tract infections, for the treatment of cholera and other stomach ailments. Based on the extensive use and lack of scientific evidence, a study was embarked upon to determine its bioactivity. Using the disc diffusion assay as a preliminary screening and thereafter the minimum inhibitory concentration (MIC) assay, the antimicrobial activity of the essential oils and non-volatile compounds was assessed on two Gram-positive bacteria, Staphylococcus aureus and Bacillus cereus, one Gram-negative bacterium, Klebsiella pneumoniae, and one yeast, Candida albicans. All of the extracts proved to be active against the four pathogens tested with the exception of Agathosma bathii which showed poor activity against Klebsiella pneumoniae (MIC value of 32mg/ml). The extracts exhibited stronger activity against the pathogens as compared to the essential oils. Both the essential oils and extracts exhibited higher activity towards the Gram-positive bacteria than the Gram-negative bacterium, with the extract of Agathosma ovata (round-leaf) displaying the greatest vii activity against Staphylococcus aureus (MIC value of 0.156mg/ml) and Bacillus cereus (MIC value of 0.125mg/ml). The extract of Agathosma parva displayed the greatest activity against Candida albicans and Klebsiella pneumoniae (MIC value of 1.5mg/ml). Amongst the essential oils, Agathosma pungens proved to be the most active against the Gram-positive pathogen, Bacillus cereus (MIC value of 3mg/ml). Agathosma collina was the most active against Candida albicans (MIC value of 3mg/ml) whilst A. zwartbergense proved to be the least active against most of the tested pathogens. The antimicrobial activity of the essential oils may be ascribed to oxygenated constituents, such as 1,8-cineole, linalool and carvacrol. The activity of the extracts may be ascribed to constituents such as flavonoids, coumarins and alkaloids. Due to the availability and accessibility of Agathosma ovata, a seasonal variation study was performed on the chemical composition of the essential oils and how this may impact on the antimicrobial activity. Furthermore, this species has recently been earmarked for commercial development by the flavour and fragrance industry and information on variability is required to establish the harvesting protocol. Ten samples were harvested in total. There was a substantial variation in the oil yield throughout the year, ranging from 0.23% in early Spring to 0.85% in late Autumn. A higher yield was observed during the flowering season as compared to the non-flowering season. Oil yields were low during Summer (0.44%-0.48%) which may have been due to the low oil content in stems and higher proportion of stems after flowering. The proportion of oil-rich green leaves also decreased markedly, hence affecting the yield. Overall the yields were dependant on the season harvested and proportion of plant parts distilled. viii The chemical composition of the essential oils was determined using GC-MS and resulted in the identification of 145 compounds in 10 of the samples. All samples contained a large number of common monoterpenes and had very similar compositions, with minor quantitative variation. Some components common to all samples include: sabinene, p-cymene, β-pinene, α-pinene, α-thujene, myrcene, limonene, linalool and terpinen-4-ol. Sabinene was found to be the most dominant component in all samples, ranging between 25.6% and 44.4%. Myrcene levels dropped sharply between the beginning of Spring and end of Summer, from 14.9% to 1.0%. β-pinene followed a similar trend, peaking during Spring and decreasing during the Summer months. The lowest levels of linalool (4.3%), myrcene (1.0%), β-pinene (3.9%), limonene (1.9%) and sabinene (25.6%), occurred during the Summer months when the temperatures were high. There was a Springtime increase in the levels of β- pinene, terpinen-4-ol, linalool, sabinene, limonene and p-cymene in the non-flowering Agathosma ovata. These changes may have been due to the higher proportion of young leaves during Spring, which may have oil compositions slightly different to those of mature leaves. A rare thiol derivative (tr) that could not be identified was detected in the March sample. Many of the changes were associated with flowering and the results obtained reveal that the chemical composition of the essential oil of Agathosma ovata is subject to seasonal variation. Using the MIC assay, the antimicrobial activity of the essential oils was assessed on two Gram-positive bacteria, Staphylococcus aureus and Bacillus cereus, one Gram- negative bacterium, Klebsiella pneumoniae, and one yeast, Candida albicans. The study