ISOLATION, CHARACTERISATION AND BIOLOGICAL ACTIVITY OF SOME COMPOUNDS FROM RAPANEA MELANOPHLOEOS (L.) MEZ. by THABILE LUKHELE Dissertation in fulfilment of the requirement for the degree MASTER in CHEMISTRY in the FACULTY OF SCIENCE of the UNIVERSITY OF JOHANNESBURG Supervisor : Prof. R.W.M. Krause Co-supervisor : Mrs D.K. Olivier DECLARATION I hereby declare that this dissertation, which I herewith submit for the research qualification MASTERS DEGREE IN CHEMISTRY to the University of Johannesburg, Department of Chemical Technology, is, apart from the recognised assistance of my supervisors, my own work and has not previously been submitted by me to another institution to obtain a research diploma or degree. _______________________________ on this ____ day of _______________ (Candidate) _______________________________ on this ____ day of _______________ (Supervisor) _______________________________ on this ____ day of _______________ (Co-supervisor) i DEDICATION This work is a true reflection of God‟s greatness, immeasurable love and faithfulness. It is dedicated to my family without whose support I would never have been able to complete it. Bo Mhlantiwendlunkhulu, thank you for your support, you mean the world to me. To my dearest dad; QomazithawaNgwane, Nabonyoni yasindvwa sisila kusuka esihlahleni, thank you for believing in me and always supporting me. You are my pillar of strength and my inspiration. To my lovely mom; Dlamini wekunene, Langeni lekutsenga eSwatini, a big thank you for your love, prayers and always reminding me that there is an end to every process. To my late big sister, thank you for your love. I know you would have been very proud of this work. Wherever you are, you will always be a bigger part of my life, I miss you dearly. ii ACKNOWLEDGEMENTS The following individuals and institutions are sincerely acknowledged for their invaluable contributions towards the success of this work: My supervisors, Professor R.W.M Krause and Mrs D.K. Olivier for their supervision, advice and guidance throughout this work. Professor Sandy van Vuuren for her assistance with the biological tests. Dr Anna Moteete for helping with the deposition of herbarium specimens. Mr Gugu Mavuso from the University of Swaziland and Ms I Johnson for their assistance with plant collection. My friends, Bheki, Dumie, Innocent, Mfanaleni, Nikiwe, Njabu, Unathi, and Vinny for their support, love and for always offering a shoulder to lean on. From the bottom of my heart, thank you for being a part of my life, it‟s an honour and a blessing. My colleagues, Dr Derek Tantoh Ndinteh, Mr Smart Mpofu and Xavier Siwe Noundou for being the best lab mates. I am grateful for your advice; help with some experiments and of course the crazy moments we shared. May God richly bless you and your families. The University of Johannesburg through the Postgraduate Bursaries for financial support. The Almighty God, for providing me with the opportunity and forever engulfing me with his love. Even when I pass through fire I know you are there and in the darkest hour you shine as bright as day because you alone are God. iii ABSTRACT The continued use and popularity of plant based traditional medicine necessitates scientific validation of the therapeutic potential of medicinal plants through phytochemical and pharmacological screening as well as the isolation and identification of bioactive compounds. Since the pharmacological effectiveness of medicinal plants is affected by several intrinsic and extrinsic factors, studies on the variations on chemical composition and biological activity are important as well. These provide a scientific rationale for using plants from different regions for the same medicinal purposes and allow traditional healers and consumers to make informed decisions with regard to the collection and use. Rapanea melanophloeos is a popular medicinal plant from the Myrsinaceae family widely distributed in southern Africa. It‟s bark, fruits and rarely the leaves are used traditionally for ailments ranging from stomach disorders, respiratory problems to disorders of the nervous system. Available chemical information reports on the accumulation of benzoquinones as major compounds, as well as some triterpenoid saponins and tannins. In view of the plant‟s wide distribution and medicinal use of different plant parts, this study comparatively evaluated the chemical composition of various crude extracts of the leaves, fruits and bark of plants collected from six localities. This was coupled with antibacterial tests to evaluate the therapeutic potential of different solvent extracts of the leaves, fruits and bark as well as the isolation of bioactive compounds from the fruits. Plants were collected from six different localities between Swaziland and South Africa and sequentially extracted with petroleum ether, ethyl acetate, methanol and water as a series of increasingly polar solvents. Thin layer chromatography (TLC), Gas-Chromatography/Mass spectrometry (GC-MS) and High Performance Liquid Chromatography (HPLC) were used to obtain a semi-quantitative chemical composition profile of different extracts. The TLC fingerprints of petroleum ether, ethyl acetate and methanol extracts showed the accumulation of non-polar terpenes, benzoquinones, saponins, tannins and flavonoids in the three plant parts. Slight variations in the chemical composition of the leaves, bark, and fruits were noted. Some components occurred in specific plant parts and others iv occurred in all three plants. The chemical profile of the leaves and bark were quite comparable in most instances with the fruits showing a generally different profile. This confirms previous literature reports on the comparability of leaves and the bark accounting for interchangeable use in traditional medicine. From GC-MS analysis five compounds were observed to accumulate in the three plant parts with considerable quantitative variations and random plant to plant variations. Three of these were tentatively identified as (-)-spathulenol, caryophyllene oxide and α-cardinol. Caryophyllene oxide was identified as the major compound in the leaves and the fruits and as expected the concentrations of the five compounds were the highest in the fruits and lowest in the bark. Screening for amino acids using GC-MS provided some insight into the traditional uses of the plant for neuroactive disorders. About 29 free amino acids (standard and non- standard) were identified and quantified in all plant parts. These showed minor qualitative variations and remarkable quantitative variations between the leaves, fruits and the bark. The fruits accumulated higher amino acid concentrations as compared to the leaves and the bark. HPLC analysis as well showed minor differences in the chemical profile of the leaves, fruits and bark. Three major compounds were observed to occur in the leaves, fruits and bark with minor variations in their quantities. No distinct geographical trend was noted except for random plant to plant variations within and between populations. Broad spectrum antibacterial activity of crude extracts of the bark, fruits, and leaves against B. cereus, E. faecalis, S. epidermidis, E. coli, K. pneumoniae and P. aeruginosa was noted. This provides an important basis for the justification of traditional medicinal uses of the plant against skin, stomach and respiratory infections. The highest activity was noted against gram positive strains with the fruit exhibiting superior activity than the bark and leaves. Medium polar compounds extractable with ethyl acetate were the most active, while the polar compounds (extractable in methanol) were somewhat surprisingly the least active. In all extracts compounds are likely to work synergistically to effect the observed activity, since no individual compound was active from the bio-autography assays. v Although no single bioactive compound was localized from an auto-biography assay, an attempt was made to isolate some compounds from the fruits. About six compounds were isolated; two from the polar fraction and four from the non-polar fraction. Interestingly, preliminary spectroscopic analysis showed the lack of a benzoquinoid skeleton in five of the isolated compounds although benzoquinones, which are characteristic of Myrsinaceae plants were identified by TLC. From spectral analysis, one compound was successfully characterised as pentacos-4- ene-6,7-dione. Re-isolation of the remaining four compounds so as to fully resolve their structures is continuing in our lab. Overall the study provided some justification into the traditional medicinal uses of R. melanophloeos. Although the leaves, fruits and bark showed some variations in their chemical profile, they don‟t have a pronounced effect on the antibacterial activity as all plant parts showed good antibacterial activity. Plants from different localities as well had similar chemical profiles only with minor quantitative variations and comparable antibacterial activity. vi CONFERENCE PRESENTATIONS AND PUBLICATIONS Some of the work in this dissertation has been presented in conferences and prepared for publication. Conference presentations: Lukhele T., Krause R.W.M., Olivier D.K., Isolation, Characterization and Biological Activity of Some Compounds from R. melanophloeos (L.) Mez. Oral Presentation. University of Johannesburg, Department of Botany and Plant Biotechnology, Annual Postgraduate Symposium. 22 October 2008. Lukhele T., Krause R.W.M., Olivier D.K., Phytochemical and Biological Screening of Some Compounds from