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From Asparagus Microraphis ANTIMBACTERIAL AND ANTIMINFLAMMATORY ACTIVITY OF MEO'CINAL PLANTS USED TRADITIONALLY IN LESOTHO by THATO LUCY SHALE Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in the School of Botany and Zoology University of Natal, Pietermaritzburg March 2003 DECLARATION The experimental work described in this research was carried out in the Research Centre for Plant Growth and Development, School of Botany and Zoology, University of Natal Pietermaritzburg. The work was supervised by Professor J. van Staden and Doctor W. Stirk from January 2000 to February 2003. The studies presented are the results of my own investigations , except where the work of others is acknowledged, and have not been submitted in any form to another Institute. Thato Lucy Shale I declare the above statement to be true. Professo J. van Staden (SUPERVISOR) Doctor W. Stirk (CO-SUPERVISOR) March 2003 11 DEDICATION In loving memory of my mother, Mathato Beauty Shale III ACKNOWLEDGEMENTS I would like to thank Professor J. van Staden for his great assistance in supervising this research throughout the time taken . His advices, and encouragement are greatly appreciated . The co-supervision from Dr. W. Stirk has contributed a lot to the success of this research. Her availability, encouragement and suggestions helped me greatly. The assistance and support from Research Centre members, academic and technical staff from the School of Botany and Zoology are greatly appreciated. I would like to thank traditional healers and herbalists from Lesotho for valuable information and assistance on medicinal plants . Or. Boshoff (Cape Technikon) is thanked for the analysis of essential oils. The National Manpower Developmental Secretariat and National Research Foundation are acknowledged for financial assistance given for this research. The support and encouragement from my family and friends gave me courage to overcome difficult times and I am very grateful. My special thanks to Molisana for his love and support. Most of all, I would like to thank God for everything. iv ABSTRACT A significant potion of the population in Lesotho relies on traditional medicine to meet its health care requirements. Traditional healers and herbalists were interviewed from Qacha's Nek (Highlands) and Mohale's Hoek (Lowlands) districts in Lesotho on plants used by the Basotho in traditional remedies. Fifteen plants were reported to be used for bacterial infections while thirteen plants were used for diseases associated with inflammation . Plant roots were most often used to make water extracts. Mainly high altitude plants are used with lowland healers obtaining most of their plant material from the highlands, either by collecting them or buying them from highland gatherers. Leaves and roots of plants used to treat bacterial infections were extracted with hexane, methanol and water and the respective extracts screened at 100 mg rnl" for anti-bacterial activity using the disc diffusion bioassay. Seven species displayed very high anti-bacterial activity against both Gram-positive and Gram-negative bacteria. A number of plant extracts had medium inhibitory activity, mostly against Gram-positive bacteria. This activity was mainly found in the root extracts. Six of the thirteen plants screened for anti-inflammatory activity using the cyclooxygenase-1 (COX-1) bioassay had activity above 90%. Hexane and methanol extracts were the most active while water extracts usually had lower activity. Ma/va parviflora, Eriocepha/us punctulatus and Asparagus microraphis exhibited high anti-inflammatory activity from hexane, methanol and water extracts made from leaf and root material. High anti-bacterial activity was also recorded from M. v parviflora and .E. punctulatus hexane, methanol and water extracts. An investigation on seasonal variation and plant part substitution in medicinal activities for these plants was carried out. Extracts of M. parviflora collected between June 1999 and July 2001 showed variation in anti-bacterial activity. Extracts made from leaves and roots inhibited the growth of both Gram-positive and Gram-negative bacteria. More bacterial strains were inhibited by extracts made from roots collected in cooler months. However, a trend in seasonal activity was not evident for either the roots or leaves because there was no detection of activity in some of the extracts made within the same months or seasons of the adjacent years. Variation in anti-inflammatory was detected for M. parviflora extracts. E. punctulatus leaf extracts did not exhibit any seasonal variation in anti-bacterial activity. Anti-inflammatory activity of E. punctulatus showed seasonal variation with the highest activity noted when material was collected during the cooler months and a decline in activity when collections were made during the warmer months. Hexane, methanol and water extracts made from leaves and roots of A. microraphis did not show any seasonal variation in anti-inflammatory activity. Thus, M. parviflora and E. punctulatus should be collected during the cooler months while A. microraphis can be collected throughout the year. Traditional healers, herbalists and vendors need to be encouraged to use aerial parts in substitution of ground parts which are reported to be highly utilized. Effect of storage on anti-bacterial and anti-inflammatory activities of M. parviflora, E. punctulatus and A. microraphis were monitored. Dried, ground leaf and root material of the three plants was stored in a cold room, at room temperature and in the VI Botanical Garden where the material was exposed to high and large changes in temperature. Dried hexane and methanol extracts made from leaves and roots of these plants were stored in a cold room and at room temperature. Initially, storage of the plant material under the three storage conditions caused an increase in anti­ bacterial activity of the hexane, methanol and water extracts made from leaf and root material of M. parviflora and E. punctulatus. Storage for a longer period resulted in a decrease in inhibitory activity. TLC fingerprints developed from hexane and methanol extracts made from M. parviflora and E. punctulatus stored in a cold room and at room temperature showed a consistent number and colour of spots during the initial storage period. Prolonged storage resulted in a decline in the number and colour of detected spots. The stored hexane and methanol extracts made from leaves and roots showed a similar trend of increases and decreases in anti-bacterial activity as well as changes in spots with the storage of the extracts. Isolation of biological active compounds from M. parviflora was not successful due to loss in anti-bacterial activity as a result of collection of plant material from a different locality. Anti-inflammatory compounds could not be isolated due to insufficient vu amount and the synergistic effect of the active compounds . The purified compounds exhibited loss of activity following HPLC purification which then re-appeared upon re­ combining the fractions. A number of compounds were detected from essential oils of E. punctulatus using GC. Fractions containing these compounds gave positive anti-bacterial activity in the disc-diffusion , bioautographic and MIC bioassays as well as high anti-inflammatory activity with COX-1 and COX-2 anti-inflammatory bioassays. No anti-inflammatory compounds were isolated from A. microraphis. V111 PUBLICATIONS AND CONFERENCE PROCEEDINGS A) Oral papers T.L Shale, W .A Stirk and J. van Staden (1998). Screening of medicinal plants used in Lesotho for anti-bacterial and anti-inflammatory activity. 4th Annual Symposium on Indigenous Plant Use Research in Kwazulu-Natal (SIPUR). Department of Botany, University of Natal Pietermaritzburg. T.L Shale, W.A Stirk and J. van Staden (1999). Screening of medicinal plants used in Lesotho for anti-bacterial and anti-inflammatory activity. South African Association of Botanists (SMB) zs" Annual Congress. University of Transkei, Umtata. Shale, T.L, W.A Stirk and J. van Staden (2000). Anti-bacterial and anti-inflammatory activity of Malva parviflora. South African Association of Botanists (SMB) zs" Annual Congress . University of Potschefstroom . Shale, T.L, W.A Stirk and J . van Staden (2001). Collection and storage of Ma/va parviflora and Asparagus microraphis for medicinal uses. South African Association of Botanists (SMB) 2ih Annual Congress. Rand Afrikaans University B) Posters Shale, T.L , W.A Stirk and J. van Staden (1999). Anti-bacterial and anti-inflammatory activities of medicinal plants used traditionally in Lesotho. 2000 Years of Natural IX Products Research - Past, Present and Future . 5th Joint Meeting of the American Society of Pharmacognosy, Association Francaise pou l' Enseignement et la Recherche en Pharmacognosie, Gesellschaft fur Arzneipflanzenforschung and the Phytochemical Society of Europe. Amsterdam, The Netherlands. c) Published papers T.L Shale, W .A Stirk and J. van Staden (1999) . Screening of medicinal plants used in Lesotho for anti-bacterial and anti-inflammatory activity. Journal of Ethnopharmacology 67: 347-354 d) Papers in preparation T.L Shale, W.A Stirk and J. van Staden. Seasonal variation in medicinal activities and plant part substitution for Malva parviflora, Eriocephalus punctulatus and Asparagus microraphis. (In prep) T.L Shale, W.A Stirk and J. van Staden. Effects of storage on anti-bacterial and anti­ inflammatory activity of M. parviflora, E. punctulatus and A. microraphis. (In prep) T.L Shale, W.A Stirk and J. van Staden.
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