Lippia javanica is an erect, small woody shrub that grows up to two metres in height, popularly known as “fever tea” or “koorsbossie”. It is distributed throughout southern Africa covering almost the entire country of Swaziland and large parts of South Africa. Its uses range from that of caffeine free tea with fever and pain-relieving activities to treatment of microbial infections such as coughs, colds and other bronchial ailments as well as the basic symptoms of HIV and AIDS. Topical uses also include disinfection and treatment of skin disorders such as dermatitis and dry skin, and even for the treatment of lice and scabies. Furthermore, it is used in combination with Artemisia afra as a remedy against malaria and as a prophylactic against dysentery and diarrhoea. In view of the traditional medicinal importance of L. javanica, it was surprising that not much is known on the polar fraction of this plant. This work was done to explore the presence of the polar compounds of L. javanica which may contribute to the plants’ medicinal properties. The chemical screening was conducted using both the non-polar and polar extracts of the aerial parts of L. javanica collected from various localities in South Africa and Swaziland. The screening involved extraction of the aerial parts of the plant in solvents of different polarities after which TLC showed the presence of essential oils, phenolic glycosides, amino acids, diterpenoids, triterpenoids and other phenolic compounds which may include flavonoids. The chemical variation was further investigated by means of chromatographic techniques such as HPLC and GC/MS. The essential oils of L. javanica demonstrated a dramatic variation both in quality and in quantity within and between natural plant populations. The variation was found to be random and it was not correlated to the geographical distribution of this plant. The polar extracts also showed variation in the chemical compounds, v with clear differences observed between Swaziland and South African populations. Even among the amino acids a considerable variation was observed on a geographical level. Although only the aerial parts of the plants were used in each case, the observed variation could be due to the different developmental stages of the plants and the different harvesting times. We have also established that there are different compounds present in L. rehmannii (toxic Lippia species) and L. javanica which are similar to each other in appearance and they grow in the same regions and similar environments. The results demonstrated the importance of performing chemical variation studies before using these plants and for quality control purposes for marketed products. The extracts of L. javanica were subjected to the isolation of compounds and structural elucidation. Two phenylethanoid glycosides, verbascoside β-[(3,4- dihydroxyphenyl)-ethyl]-(3′-O-α-L-rhamnopyranosyl)-(4′-O-caffeoyl)-β-Dglucopyranoside and isoverbascoside β-[(3,4-dihydroxyphenyl)-ethyl]-(3′-O-α-Lrhamnopyranosyl)-( 6′-O-caffeoyl)-β-D-glucopyranoside, previously unknown in L. javanica were isolated. The structures were established by extensive spectroscopic investigation (IR, UV, FAB-MS, 1D- and 2D-NMR). The discovery of these compounds gave the most important new insight in this study and they are known to possess antioxidant activities1. This activity is still to be investigated in L. javanica. / Mrs. D. K. Olivier Dr. R. W. M. Krause

An experiment was conducted to determine the effects of shade netting on growth, oil yield and compound composition of fever tea (Lippia javaica). The treatments were nine shade nets of different light intensities (100% bird net, 40% green, 10, 12 and 18% white and 30, 40, 55 and 70% black). The layout used was a completely randomized design. The parameters measured were shoot width, fresh shoot mass, the number of plants flowered, plant height and photosynthetically active radiation. Shade netting did not have any effect on the shoot width of L. javanica. Eighteen percent white net was best suited to produce fresh shoot mass, whereas 55% black net, 40% black net and 10% white net produced lower fresh shoot mass. More plants that flowered were obtained under 70% black net than those grown under 30% black net. The number of plants that flowered increased from 22 to 35 with an increase in shading density. The height of plants was not affected by shade netting at 49 DAT. However, at 59 and 67 DAT, the height of the plants was greater under low light intensities (40, 55, and 70% black nets) than that of high light intensity (30% black). Twelve percent white net had the greatest plant height at 102 DAT. Shade netting significantly affected the adsorption and utilization of photosynthetically active radiation (PAR) above, across and below the plants at 102 DAT. Hundred percent bird net had the greatest amount of light intercepted above, across and below the plants. The amount of light intercepted above, below and across the plants was lower for lower shading intensity (30% black) than that of higher shading intensities (40%, 55% and 70% black). To determine shade-netting effect on oil yield and compound composition, fresh leaves (± 200g) of L. javanica were sampled from each shade net for essential oil extraction by hydrodistillation. The oil yield ranged from 0.29% to 0.41%. The oil had a clear to yellowish brown colour. No significant differences were detected for oil yield amongst the shade nets. The chemical compound analysis was carried out by GC/MS. The chemical compounds identified in the essential oil were <font face="symbol">a</font>-pinene, sebinen, myrcene, 1.8 myrcene, ipsenone, ipsedienone, <font face="symbol">b</font>-caryophyllen and germacrene-D. L. javanica plants grown under 10% white net, resulted in significantly higher myrcene content (15.7%) as compared to plants grown in other shade nets. Shade netting did not have any effect on the composition of á-pinene, sebinene, 1.8 myrcene, ipsenone, ipsedienone, <font face="symbol">b</font>-caryophyllen and germacrene-D. For commercial usage of myrcene, white net (10%) would be best suited for L. javanica production. To obtain information about indigenous knowledge of L. javanica, a survey was carried out among street traders, traditional healers, hawkers and farmers in Gauteng. The age group was between 20 and 73. The majority of respondents, who were self employed, were traditional healers followed by farmers. Most (98%) of the respondents indicated that they collected L. javanica from the veld by cutting the plant, whereas other respondents uprooted the whole plant when harvesting. Amongst the different uses of L. javanica mentioned, treatment of colds and coughs through steaming was stated by most (47%) of the respondents. Most (88%) of the respondents felt that it was important to conserve the plant for future generations and also to prevent the plant from becoming extinct. Traditional healers knew the uses and other important aspects of L. javanica than the farmers. / Dissertation (M Inst Agrar (Agronomy))--University of Pretoria, 2007. / Plant Production and Soil Science / unrestricted

Fever tea is one of the important medicinal plants belonging to the family Verbenaceae. The leaves of the plant are used as a remedy to treat malaria, stomach pains, colds and fever. There are different clones of Lippia species available with different medicinal value. Therefore, the purpose of this study was to determine protocols for propagation of fever tea so as to multiply an ideal clone. Vegetative propagation of this plant species by stem cuttings, requirements for seed germination and response of fertigation frequencies and growing medium to growth, yield and quality has not been studied. In this study, factors influencing effective propagation of fever tea were studied. Those factors were: cutting position (apical vs. basal), media (pine bark vs. sand), hormone (seradix No. 2 vs. no hormone) light and temperature requirement for fever tea seed germination and effect of fertigation frequencies (0.4 L/day, 1L/day, 2L/day, 2L/2nd day and 2L/week) and growing media (pine bark vs. sand) on growth, oil yield and oil quality. In vegetative propagation by stem cuttings, measurements made were number of roots per rooted cutting, fresh mass, stem circumference and number of leaves and the rate of rooting during four consecutive sampling dates (5, 10, 15 and 20 days after planting). Apical cuttings rooted earlier by 5 days than basal cuttings at 10 days but at 15-20 days after establishment, both cuttings had good rooting. Fresh mass was not affected by cutting position and rooting media, but cuttings performed slightly better when propagated in pine bark medium than sand medium. Basal cuttings resulted in thicker stems and more leaves as compared to apical cuttings. Seradix No.2 (0.3% IBA) hormone increased the fresh mass, stem circumferences, root number and leaf numbers on both apical and basal cuttings. For the establishment of fever tea stem cuttings, both apical and basal cuttings can be used but pine bark is the ideal medium. The cuttings can be ready for transplanting in 15-20 days after establishment and Seradix No. 2 (0.3% IBA) promotes rooting of fever tea cuttings. The ideal combination of light and temperature for seed germination of fever tea was investigated. Germination was tested at constant temperature regimes (15, 20, 25 and 30¨¬C with continuous light or dark period and alternating temperatures of 20:30 and 16L: 8D (light: dark) combinations respectively. Seeds started to germinate after 8 days from incubation and the last germination was observed at 30 days from incubation. Germination percentages increased at 20-30 constant temperatures and 20/30 alternating temperatures but the difference amongst them was not significant. Higher germination of 86% were achieved when seeds were exposed to continuous light than alternating light and dark. Seeds failed to germinate in continuous darkness. Fever tea seeds were positively photoblastic. The effect of fertigation frequency and growing medium on the growth, yield and quality of fever tea were investigated in a tunnel. Treatments used were five fertigation frequencies (0.5L/day, 1L/day, 2L/day, 2L/2nd day, and 2L/week) and two growing media (pine bark and sand). Measurements made were plant height, stem circumference and number of branches at 8, 16 and 32 weeks after planting. At 8 weeks after planting all fertigation frequencies improved fever tea growth except fertigation frequency of 2L/week. All the fertigation frequencies were ideal to sustain the growth and development of fever tea plants except 2L/week. At 16 weeks after planting there were interactive effects between fertigation frequencies and the growing medium for the plant height of fever tea. Plants fertigated with 2L/day grown in sand media grew taller than all the other fertigation frequencies. At 32 weeks after planting there was a significant effect on the plant height from the main effects of fertigation frequency and growing medium. Plants fertigated with 2L/day were significantly the tallest followed by plants fertigated with 0.5L/day, 1L/day, 2L/2nd day and 2L/week. When plants were younger better plant growth was obtained in pine bark media. Stem circumference and number of branches of fever tea were significantly affected by fertigation frequency and growth medium. At 16 and 32 weeks after planting, plants grown in sand media had thicker stems and more branches as compared to plants grown in pine bark media. The essential oil of fever tea was extracted using hydro-distillation. Fertigation frequency did not affect oil yield. Plants grown in pine bark media yielded more oil than plants grown in sand medium. Microscopical studies using scanning electron microscope were investigated to determine the development of oil glands and trichomes on the abaxial (upper) and adaxial (lower) surfaces of the leaves as affected by fertigation frequency and the growing medium. There were no significant effects on the number of oil glands and trichomes developed on both surfaces of the leaves. Pine bark medium resulted in larger oil glands than sand medium regardless of the treatments, and pine bark also yielded more oil percentages than sand medium. Based on this investigations for commercial production of fever tea essential oil pine bark media is recommended. Chemical compounds of fever tea oil were also not affected by fertigation frequency or growth medium. In this study the chemical compounds detected from essential oils of fever tea were monoterpenes (i.e. ¥á-pinene, sebinen, 1.8 cinede, myrcene, ipsenone and ipsedienone) and the sesquiterpenes (i.e. ¥â-caryophyllene and germacrene-D). Compounds that gave the smallest chemical percentages and the shortest time to be detected were ¥á-pinene, sebinen, 1.8 cinede, myrcene, ipsenone and ipsedienone. Compounds found with the highest chemical percentages with highest peaks were ¥â-caryophyllene and germacrene-D. / Dissertation (MSc (Horticulture))--University of Pretoria, 2007. / Plant Production and Soil Science / unrestricted

MSc (Chemistry) / Department of Chemistry / Malaria, an infectious disease affecting both human beings and other animals, is transmitted by parasitic protozoans belonging to the Plasmodium genus. Malaria is commonly treated with drugs such as quinine, chloroquine, and artesunate. However, the incidence of treatment failure due to drug-drug interactions and parasite resistance is increasing. Therefore, the rich medicinal potential of plants found in nature in Africa is increasingly being explored. The traditional use of Lippia javanica, Sclerocarya birrea, Melia azedarach and Capparis tomentosa for the treatment of malaria is well-known, but the phytochemistry of these four plants is not fully known. Parts of these plants were extracted and column chromatography was used to fractionate the extracts. The antioxidant activities of the fractions were determined using free radical scavenging and reducing power assays, while the cytotoxic, antiplasmodial and antitrypanosomal activities were determined using cell toxicity assay, parasite lactate dehydrogenase (pLDH) and trypanosome assay. The methanol stem bark extract of Melia azedarach (Fraction 2) had the highest phenolic content (59.39 mg GAE/g), while the methanol leaf extract of Melia azedarach had the highest flavonoid content of 188.65 mg QE/g. In the reducing power tests and DPPH free radical scavenging activity, the methanol stem bark extract of Melia azedarach had the lowest IC50 value of 0.1074 μg/mL and an IC0.5 value of 0.5296 μg/mL, respectively. Furthermore, the methanol stem bark extract of Melia azedarach at a concentration of 50 μg/mL showed significant cytotoxicity against HeLa cells (-1.22±0.07 %). The methanol stem bark extract of Melia azedarach at the tested concentration (250 μg/mL) decreased the viability of Plasmodium falciparum to 36.38±11.96 % with an IC50 value of 6.5 μg/mL. Concerning the antitrypanosomal activity, the methanol stem bark extract of Melia azedarach affected the viability of the trypanosomes at the tested concentration (250 μg/mL), giving a viability of 14.05 ± 0.59 %, with an IC50 value of 0.4 μg/mL. The presence of epicatechin (29) and catechin (31) in this extract was confirmed using several spectroscopic techniques (IR, NMR, UPLC-MS and HRMS). / NRF

The evolution of aerobic metabolic processes unavoidably led to the production of reactive oxygen species (ROS). ROS have the ability to cause harmful oxidative damage to biomolecules. Increased ROS generation and subsequent oxidative stress have been associated with aging and neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases as a result of the extreme sensitivity of the central nervous system to damage from ROS. Antioxidant defence systems have co–evolved with aerobic metabolic processes to counteract oxidative damage inflicted by ROS. The impact of neurodegenerative disorders on society is increasing rapidly as the life expectancy of the global population increases. In this day and age, a much younger group of the population is also experiencing neurodegenerative symptoms as a result of the harmful effect of the human immunodeficiency virus (HIV) on the central nervous system. Plants are an invaluable source of medicinal compounds. The use of plants for their healing properties is rooted in ancient times. The aim of this study was to select from twenty one plants, the plant with the most promising antioxidant activity and to determine whether extracts of this plant could act as free radical scavengers, comparing the results to Trolox, a known free radical scavenger. The next step was to isolate and characterize a compound from an extract exhibiting promising antioxidant activity. Bioassay–guided fractionation was followed to achieve this. During screening trials, twenty one plants, namely Berula erecta, Heteromorpha arborescens, Tarchonanthus camphoratus, Vernonia oligocephala, Gymnosporia buxifolia, Acacia karroo, Elephantorrhiza elephantina, Erythrina zeyheri, Leonotis leonurus, Plectranthus ecklonii, P. rehmanii, P. venteri, Salvia auretia, S. runcinata, Solenostemon latifolius, S. rotundifolius, Plumbago auriculata, Clematis brachiata, Vangueria infausta, Physalis peruviana and Lippia javanica were selected from literature, based on reported antioxidant activity within the plant families, for screening of their antioxidant activity. One hundred and ten extracts were prepared from the leaves, using Soxhlet extraction and the solvents petroleum ether (PE), dichloromethane (DCM), ethyl acetate (EtOAc) and ethanol (EtOH), consecutively. The focus during initial screening trials was on chemistry–based assays. The oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) assays were employed for the primary screening of the one hundred and ten leaf extracts. The ORAC assay was used to determine whether the plant extracts were able to scavenge peroxyl radicals and the FRAP assay was used to determine the reducing abilities of the extracts. Quantification of the peroxyl radical scavenging activity by the ORAC assay revealed that activity was observed for most of the extracts, with the ethyl acetate and ethanol extracts of L. javanica exhibiting the most promising activity. This pattern of activity was also found with the reducing capacity evaluated by the FRAP assay in which the EtOAc and EtOH extracts of L. javanica also exhibited the most promising activity. L. javanica was selected for further study by screening for biological activity, employing the nitro–blue tetrazolium (NBT) assay and thiobarbituric acid reactive substances (TBARS) assay. Using a cyanide model to induce neurotoxic effects in rat brain homogenate, the neuroprotective properties of the extracts of L. javanica leaves were examined using the NBT assay and compared to that of Trolox. The NBT assay determines the level of superoxide anions. All the extracts of L. javanica significantly reduced superoxide anion generation at all concentrations used. The petroleum ether and ethyl acetate extracts, at all concentrations, reduced superoxide anion generation to values lower than that of the control, suggesting that these extracts may be able to attenuate normal free radical processes in the brain. The petroleum ether extract exhibited the most promising activity at a concentration of 1.25 and 2.5 mg/ml and also exhibited similar results as the ethyl acetate extract at a lower concentration than the ethyl acetate extract (2.5 mg/ml compared to 5 mg/ml). A toxin–solution consisting of hydrogen peroxide (H2O2), iron(III)chloride (FeCl3) and ascorbic acid was used to induce lipid peroxidation and the ability of the extracts of the leaves of L. javanica to attenuate lipid peroxidation was investigated in rat brain homogenate and compared to that of Trolox. All of the extracts of L. javanica significantly attenuated toxininduced lipid peroxidation at all concentrations used. All of the extracts were also able to significantly attenuate toxin–induced lipid peroxidation to values lower than that of the control. These results suggest that all of the extracts of L. javanica possess the ability to attenuate not only toxin–induced lipid peroxidation, but also lipid peroxidation that occurs during normal processes in the brain. The petroleum ether extract was subjected to bioassay–guided fractionation using column and thin–layer chromatography and the NBT and TBARS assays. Fraction DD1 was investigated by means of nuclear magnetic resonance, infrared and mass spectrometry. The exact structure of fraction DD1 was not elucidated. Considering all the results, it is clear that L. javanica shows great potential as a medicinal plant with antioxidant activity and may therefore be beneficial in diminishing the destructive oxidative effects inflicted by free radicals. There are however still many compounds to be isolated from L. javanica. Key words: Verbenaceae, Lippia javanica, antioxidant, neurodegeneration, oxygen radical absorbance capacity (ORAC), ferric reducing antioxidant power (FRAP), nitro–blue tetrazolium assay (NBT), thiobarbituric acid reactive substances assay (TBARS). / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2011.

The evolution of aerobic metabolic processes unavoidably led to the production of reactive oxygen species (ROS). ROS have the ability to cause harmful oxidative damage to biomolecules. Increased ROS generation and subsequent oxidative stress have been associated with aging and neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases as a result of the extreme sensitivity of the central nervous system to damage from ROS. Antioxidant defence systems have co–evolved with aerobic metabolic processes to counteract oxidative damage inflicted by ROS. The impact of neurodegenerative disorders on society is increasing rapidly as the life expectancy of the global population increases. In this day and age, a much younger group of the population is also experiencing neurodegenerative symptoms as a result of the harmful effect of the human immunodeficiency virus (HIV) on the central nervous system. Plants are an invaluable source of medicinal compounds. The use of plants for their healing properties is rooted in ancient times. The aim of this study was to select from twenty one plants, the plant with the most promising antioxidant activity and to determine whether extracts of this plant could act as free radical scavengers, comparing the results to Trolox, a known free radical scavenger. The next step was to isolate and characterize a compound from an extract exhibiting promising antioxidant activity. Bioassay–guided fractionation was followed to achieve this. During screening trials, twenty one plants, namely Berula erecta, Heteromorpha arborescens, Tarchonanthus camphoratus, Vernonia oligocephala, Gymnosporia buxifolia, Acacia karroo, Elephantorrhiza elephantina, Erythrina zeyheri, Leonotis leonurus, Plectranthus ecklonii, P. rehmanii, P. venteri, Salvia auretia, S. runcinata, Solenostemon latifolius, S. rotundifolius, Plumbago auriculata, Clematis brachiata, Vangueria infausta, Physalis peruviana and Lippia javanica were selected from literature, based on reported antioxidant activity within the plant families, for screening of their antioxidant activity. One hundred and ten extracts were prepared from the leaves, using Soxhlet extraction and the solvents petroleum ether (PE), dichloromethane (DCM), ethyl acetate (EtOAc) and ethanol (EtOH), consecutively. The focus during initial screening trials was on chemistry–based assays. The oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) assays were employed for the primary screening of the one hundred and ten leaf extracts. The ORAC assay was used to determine whether the plant extracts were able to scavenge peroxyl radicals and the FRAP assay was used to determine the reducing abilities of the extracts. Quantification of the peroxyl radical scavenging activity by the ORAC assay revealed that activity was observed for most of the extracts, with the ethyl acetate and ethanol extracts of L. javanica exhibiting the most promising activity. This pattern of activity was also found with the reducing capacity evaluated by the FRAP assay in which the EtOAc and EtOH extracts of L. javanica also exhibited the most promising activity. L. javanica was selected for further study by screening for biological activity, employing the nitro–blue tetrazolium (NBT) assay and thiobarbituric acid reactive substances (TBARS) assay. Using a cyanide model to induce neurotoxic effects in rat brain homogenate, the neuroprotective properties of the extracts of L. javanica leaves were examined using the NBT assay and compared to that of Trolox. The NBT assay determines the level of superoxide anions. All the extracts of L. javanica significantly reduced superoxide anion generation at all concentrations used. The petroleum ether and ethyl acetate extracts, at all concentrations, reduced superoxide anion generation to values lower than that of the control, suggesting that these extracts may be able to attenuate normal free radical processes in the brain. The petroleum ether extract exhibited the most promising activity at a concentration of 1.25 and 2.5 mg/ml and also exhibited similar results as the ethyl acetate extract at a lower concentration than the ethyl acetate extract (2.5 mg/ml compared to 5 mg/ml). A toxin–solution consisting of hydrogen peroxide (H2O2), iron(III)chloride (FeCl3) and ascorbic acid was used to induce lipid peroxidation and the ability of the extracts of the leaves of L. javanica to attenuate lipid peroxidation was investigated in rat brain homogenate and compared to that of Trolox. All of the extracts of L. javanica significantly attenuated toxininduced lipid peroxidation at all concentrations used. All of the extracts were also able to significantly attenuate toxin–induced lipid peroxidation to values lower than that of the control. These results suggest that all of the extracts of L. javanica possess the ability to attenuate not only toxin–induced lipid peroxidation, but also lipid peroxidation that occurs during normal processes in the brain. The petroleum ether extract was subjected to bioassay–guided fractionation using column and thin–layer chromatography and the NBT and TBARS assays. Fraction DD1 was investigated by means of nuclear magnetic resonance, infrared and mass spectrometry. The exact structure of fraction DD1 was not elucidated. Considering all the results, it is clear that L. javanica shows great potential as a medicinal plant with antioxidant activity and may therefore be beneficial in diminishing the destructive oxidative effects inflicted by free radicals. There are however still many compounds to be isolated from L. javanica. Key words: Verbenaceae, Lippia javanica, antioxidant, neurodegeneration, oxygen radical absorbance capacity (ORAC), ferric reducing antioxidant power (FRAP), nitro–blue tetrazolium assay (NBT), thiobarbituric acid reactive substances assay (TBARS). / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2011.