CHEMICAL COMPOSITION AND BIOLOGICAL POTENTIAL OF THE VOLATILE AND NON VOLATILE CONSTITUENTS OF TARCHONANTHUS CAMPHORATUS AND TARCHONANTHUS TRILOBUS VAR GALPINNI OF KWAZULU – NATAL PROVINCE. BY NANYONGA SARAH KIWANUKA (200901240) B.Ed. (Hons) Chemistry/Physics (MUK) Dip. Ed. (MUK) M.Sc. Chemistry (MUK) A thesis submitted to the Department of Chemistry, Faculty of Science and Agriculture in fulfilment of the Requirements for the Degree of Doctor of Philosophy, University of Zululand, Kwa-Dlangezwa, South Africa. Promoter: Prof. O.A. Oyedeji Co-promoters: Prof. A.R. Opoku Dr. F.B Lewu i ABSTRACT The chemical composition and the biological activities of the volatile extracts (essential oils) and non-volatile (solvent extracts) of the aerial parts Tarchonanthus camphoratus and Tarchonanthus trilobus var galpinni collected from Sangoyana and Ubombo respectively, in the Kwa-Zulu Natal province were investigated in this study. The essential oils were separately extracted from the fresh leaves, dry leaves and dry stem by hydro-distillation and their chemical constituents determined by Gas chromatography/mass spectrometry (GC/MS). The essential oils investigated were of the dry leaf of T. camphoratus harvested in 2009 and of the fresh leaf, dry leaf and dry stem of both T. camphoratus leaf and T. trilobus var galpinni harvested in 2010 and 2011. The compounds present in all the essential oils were categorised as monoterpene hydrocarbons, sesquiterpene hydrocarbons, oxygenated monoterpenes, oxygenated sesquiterpenes and others but they differed in the type and percentage composition. Sesquiterpene hydrocarbons dominated all the essential oils with β–caryophyllene and allo-aromadendrene present in all the essential oils either as minor or major compounds. The oxygenated monoterpene 1,8-cineole, which is suspected to be responsible for the camphor smell of T. camphoratus, was present in all the essential oils of the leaves and stem of T. camphoratus. δ-Cadinene, a sesquiterpene hydrocarbon, featured in all the essential oils except of the fresh and dry leaf of T. camphoratus harvested in 2011. The chemical constituents in the non volatile extracts were determined by Pyrolysis-Gas chromatography/mass spectrometry (Py-GC/MS). A variety of compounds categorised as hydrocarbons, tetraterpenes, triterpenoids, phenols, fatty acids, fatty alcohols, steroids and sterols among others were identified in the extracts. Phenols were identified in the aqueous, methanol, dichloromethane leaf extracts and the methanol bark extract of T. camphoratus and in the dichloromethane leaf and ethyl acetate leaf extracts of T. trilobus ii var galpinni. Steroids were identified in the bark extracts of T. camphoratus and in the methanol leaf extract of T. trilobus var galpinni. Hydrocarbons were identified in all the solvent/non volatile extracts but were more dominant in the dichloromethane bark extracts of both T. camphoratus and T. trilobus var galpinni. Triterpenoids were identified in the dichloromethane leaf extracts, ethyl acetate bark extracts of both species, in the methanol leaf of T. trilobus var galpinni and in the methanol bark extract of T. camphoratus. The preliminary phytochemical screening of the extracts also revealed the presence of phenols, flavonoids, saponins and tannins in some of the extracts. The volatile and non volatile extracts were screened for their antibacterial activity, antioxidant activity and cytotoxic activities and the volatile extracts were further screened for their insecticidal activities. The extracts were screened for antibacterial activity against 4 Gram positive and 5 Gram negative bacteria. The zones of inhibition of the fresh leaves, dry leaves and dry stem essential oils of T. camphoratus ranged from 7.3 – 14.0, 7.3 – 16.7 and 13.5 – 20.4 mm respectively and the zones of inhibition of the fresh leaf oil of T. trilobus var galpinni ranged from 8.2 – 21.8 mm. The volatile extracts exhibited higher percent inhibition than the non volatile extracts and the dry stem essential oil of T. camphoratus showed the highest antibacterial activity of all the extracts. The non volatile extracts exhibited significant antioxidant potential when tested by DPPH and ABTS•+ radical scavenging assays plus reducing power assay but showed poor nitric oxide inhibition and iron chelating ability. The volatile extracts generally showed poor antioxidant activity in all the antioxidant assays. The essential oils of both the dry leaf of T. camphoratus and fresh leaf of T. trilobus var galpinni showed the highest larval mortality of 100% which was observed at a concentration of 300 ppm after 24h whereas the lowest mortality after 24h was 20% at 25 ppm for T. camphoratus and 20% at 50 ppm for T. trilobus var galpinni. Insecticidal activity of the iii volatile extracts against stored product pests, S. zeamais and S. oryzae revealed a lack of contact and fumigation toxicity by essential oils of both plant species. The essential oil of the dry leaf of T. camphoratus, however, showed repellent activity of over 50% after 24h at all the concentrations used on both S. zeamais and S. oryzae. Cytotoxic investigation of the volatile and non volatile extracts revealed that the extracts generally had low toxicity. This study is the first to provide the chemical profiles and biological activities of the volatile and non volatile extracts of T. camphoratus and T. trilobus var galpinni from Kwa-Zulu Natal. The volatile extracts could be considered as potential alternatives, whether alone or in combination, to synthetic antibiotics, larvicides and repellents. The non volatile extracts on the other hand, could be potential sources of antioxidants that could have great importance as therapeutic agents. iv CONTRIBUTIONS TO KNOWLEDGE 1. PAPERS PUBLISHED (PEER REVIEWED) i) Nanyonga SK., Opoku A., Lewu FB., Oyedeji OA. 2012. Chemical Composition and Larvicidal Activity of the Essential Oil of Tarchonanthus camphoratus Against Anopheles arabiensis Mosquito Larvae. Journal of Essential Oil Bearing Plants. 15 (2): 288-295. ii) Nanyonga SK., Opoku A., Lewu FB., Oyedeji OA. 2013. Variation in chemical composition and antibacterial activity of the essential oil of fresh and dry leaves and dry stem of Tarchonanthus camphoratus. Journal of Medicinal Plants Research. 7 (8): 442-447. iii) Nanyonga SK., Opoku A., Lewu FB., Oyedeji OA., Moganavelli S. 2013. Chemical composition, antioxidant activity and cytotoxicity of the essential oils of the leaves and stem of Tarchonanthus camphoratus. African Journal of Pharmacy and Pharmacology. 7(7): 360- 367. iv) Nanyonga SK., Opoku A., Lewu FB., Oyedeji OA., Singh M., Opeoluwa O. 2013. Antioxidant activity and cytotoxocity of the leaf and bark extracts of Tarchonanthus camphoratus. Tropical Journal of Pharmaceutical Research. 12(3): 377-383. 2. PAPERS IN PRESS i) Nanyonga SK., Opoku A., Lewu FB., Oyedeji OA. 2013. Insecticidal activities and chemical composition of the essential oil from Tarchonanthus camphoratus (L.), leaves against Sitophilus zeamais Motschulsky, and Sitophilus oryzae (L.). African Journal of Agricultural Research. v 3. PAPERS SUBMITTED FOR EDITORIAL CONSIDERATION i) Nanyonga SK., Opoku A., Lewu FB., Oyedeji OA. 2013. The chemical composition, larvicidal and antibacterial activities of the essential oil of Tarchonanthus trilobus var galpinii. Journal of Essential Oil Bearing Plants. 4. PAPERS PRESENTED AT CONFERENCES / SYMPOSIUM i) Nanyonga SK., Opoku A., Lewu FB., Oyedeji OA. 2010. Chemical composition and some biological properties of the essential oil of Tarchonanthus camphoratus. A paper presented at the 13th Indigenous Plant Use Forum (IPUF) Annual conference. Keimoes City Hall, Nothern Cape, South Africa. 28th June – 1st July. ii) Nanyonga SK., Opoku A., Lewu FB., Oyedeji OA. 2010. Chemical composition and larvicidal activity of the essential oil of Tarchonanthus camphoratus. A paper presented at the Annual Faculty of Science and Agriculture Research Symposium, University of Zululand, KwaDlangezwa, South Africa. iii) Nanyonga SK., Opoku A., Lewu FB., Oyedeji OA. 2011. Chemical composition of the essential oil and insecticidal activities of the essential oil and solvent extracts of Tarchonanthus camphoratus. A paper presented at the Walter Sisulu University 2011 Joint International Research Conference. East London, South Africa. iv) Nanyonga SK., Opoku A., Lewu FB., Oyedeji OA. 2011. Antioxidant Activity of the Essential Oil and Various Extracts of Tarchonanthus camphoratus from Kwa-Zulu Natal.A paper presented at the Annual Faculty of Science and Agriculture Research Symposium, University of Zululand, KwaDlangezwa, South Africa. 27th October. vi v) Nanyonga SK., Opoku A., Lewu FB., Oyedeji OA. 2011. Antioxidant Activity and Cytotoxicity of the Essential Oil and Various Extracts of Tarchonanthus camphoratus from Kwa-Zulu Natal. A poster presented at the International Conference of Natural Products 2011. (ICNP2011). Universiti Putra Malaysia. 14-16 November. vi) Nanyonga SK., Opoku A., Lewu FB., Oyedeji OA. 2012. The chemical composition, larvicidal and anti bacterial activities of the essential oil of Tarchonanthus trilobus var galpinii. A poster presented at the III International Symposium for Medicinal and Nutraceutical Plants. Aracaju Convention Center. Brazil. 14th -19th October. AWARD Best poster presenter. International Conference of Natural Products 2011. (ICNP2011). Universiti Putra Malaysia. vii EPILOGUE TO GOD BE THE GLORY viii DEDICATION To my father Mr. Jenkins Kiwanuka and my mother Ms Catherine Nakalema