The use of many of South Africa’s medicinal plants has shown marked increase with over 27 million users in South Africa alone. Most plants are still being unsustainably wild-harvested, a major concern for biodiversity conservation. Commercial interest in certain more commonly-used species has increased, with potential to cultivate medicinal plants on a more sustainable basis. Focus has shifted from conventional use of synthetic fertilisers, pesticides and fungicides to more organic methods of plant propagation. Aqueous extract derived from earthworm composted food waste (vermitea) was used to study the germination and rooting success of selected species. Also survival and growth performance of selected plants grown in a medium amended with commercial NPK fertiliser was compared to those grown in the same medium amended with compost and to those grown in the same medium amended with compost with weekly applications of vermitea. No change in germination success was noted. Vermitea showed promising results on the rooting of cuttings. The application of NPK improved growth performance (biomass) significantly for all species tested. However, they had lower root:shoot ratios as well as lower survival rates compared to plants under the compost and compost/vermitea treatments. The improved survival of these plants highlights the potential of these organic treatments on the propagation of selected medicinal plants.

The incidence of Type 2 Diabetes Mellittus (T2DM) is increasing world wide. In Africa the limited access to health care and the insidious course of the disease lead to more severe illness and diabetic complications. There is a need to find alternative approaches to treatment and prevention that address the problems and needs of Africa. Sutherlandia frutescens (S.frutescens) is a traditional herbal plant with known anti-diabetic properties, the precise mechanism of action of S.frutescens is not known. In order to develop new approaches for treatment and prevention of T2DM the pathophysiology of T2DM must be understood. T2DM is the final outcome of a multi-organ disease characterized by early defects in muscle, adipocytes, hepatocytes and pancreatic β-cells. In this study the role of the liver was investigated because of its central role in glucose and lipid metabolism. It is hard to differentiate between all the influences in an in vivo model, so the aim of this study was to develop an in vitro model of T2DM in Chang liver cells and to determine if S.frutescens can reverse the state of insulin resistance in this model. Different culture media conditions were screened to identify a method that can be used as the T2DM model in Chang liver cells. Serum free medium (MCBD-201) supplemented with human diabetic serum, (2.5%-10%), high insulin concentrations (0.1μM-1μM), high fructose concentrations (1-10mM). and a combination of high insulin and high fructose was used for this screening. Chang liver cells cultured in MCBD-201 medium supplemented with 1mM fructose and 0.1μM insulin showed reduced glucose uptake and increased lipid accumulation. The effect of two S.frutescens extracts, two anti-diabetic drugs, metformin and ciglitazone, and a hypolipidemic drug ciprofibrate were determined and shown to increase glucose uptake and reduce lipid accumulation. It was postulated that exposing the cells to excess nutrients in the form of high fructose would stimulate the cells to become adipogenic and accumulate lipids, which would interfere with the glucose uptake and induce insulin resistance. Gene expression of PPARγ, PPARα, and SREBP-1 transcription factors regulating lipid metabolism was determined in Chang liver cells cultured in insulin resistance inducing medium over a 48 hour time course. The expression of PPARγ, known to stimulate adipogenesis was increased after 6, 24 and 48 hours of exposure (P(H1)<0.0001). The expression of PPARα, known to stimulate β-oxidation expression, was significantly decreased after 24 hours of exposure (P(H1)<0.0001). The presence of the plant extracts in the insulin resistance inducing media protect against this increase in adipogenesis and decrease in β-oxidation after 48 hours of exposure by increasing PPARα expression and decreasing PPARγ expression. A PCR Array was performed which identified 32 more potential molecular targets of S.frutescens. Five of the 32 targets identified with the PCR Array were validated using qRT-PCR. These genes play a role in lipid and glucose metabolism and protection against oxidative stress and inflammation. In summary a cellular model of insulin resistace in hepatocytes has been established and the capacity of S.frutescens to reverse this process has been demonstrated by acting as a dual PPARγ/α agonist. New genes have been identified in the development of insulin resistance and as targets of S.frutescens.

Infectious diseases such as pneumonia still pose a major global health concern. Currently, the world is facing widespread emergence of acquired bacterial resistance to antibiotics which constitute one of the chief causes of infectious diseases. The accumulation of different antibiotic resistance mechanisms within the same strains has induced the appearance of the so called “superbugs”, or “multiple-drug resistant bacteria”. Due to antibiotic resistance, attention is currently being drawn towards biologically active components isolated from plant species commonly used as herbal medicine, as they may offer a new source of antibacterial, antifungal and antiviral activities. This is the basis of this study. In this study four medicinal plants namely, Cassia abbreviata, Geranium incanum, Pelargonium hortorum and Tecoma capensis were investigated for their antimicrobial potential. In vitro antimicrobial activity using agar disc diffusion method, agar dilution method and broth microdilution plate determination of minimum inhibitory concentration (MIC), were carried out against ATCC (American Type Culture Collection) strains and clinical isolates known to cause pneumonia. Aqueous, methanol and acetone extracts from the selected plants were thus tested against strains of Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli and Candida albicans. The plants exhibited pronounced antimicrobial activity and were more active against Gram-positive bacteria than Gram-negative bacteria. During agar disc diffusion method, the highest inhibition zone was demonstrated by the acetone extract of P. hortorum (IZ=22mm and AI=0.73) against the reference strain of S. pneumoniae (ATCC 49619). The range of zones of inhibition in diameter across strains of S. pneumoniae and H. influenzae was 7mm to 22mm with activity index range of 0.23 to 0.74. The lowest MIC produced by medicinal plants in the study during agar disc diffusion method against S. pneumoniae and H. influenzae strains, was 2.5mg/ml. In broth microdilution plate assay, the lowest MIC demonstrated by C. abbreviata, T. capensis and P. hortorum extracts on tested bacteria was 0.031mg/ml and that of G. incanum was 0.063mg/ml. Candida albicans strains were only inhibited at 20mg/ml by the study plants. The highest activity among the individual extracts was shown by P. hortorum methanol extract which inhibited 71% of the studied bacteria. T. capensis methanol extract was the least and inhibited only 17% of the tested bacteria. The strains of Klebsiella pneumoniae showed the highest resistance to medicinal plants employed in this study. Traditional preparation of selected medicinal plants did not show any significant antimicrobial activity. Bioactive analysis of compounds on study plants was carried out using standard methods which revealed the presence of anthraquinones, flavonoids, phytosterol, saponins, tannins and triterpenoids. Comparison of the inhibitory effect of the plant extracts against some broad spectrum antibiotics revealed that the tested medicinal plants showed greater antimicrobial activity than standard antibiotics. However, there was no correlation between the antibiotic susceptibility patterns of the bacteria and the effects of the plants, signifying that plants probably function through different mechanisms. Bioautographic findings on thin-layer chromatography plate, exhibited clear zones of inhibition of bacterial growth with the Rf value range of 0.09 to 0.94. Anti-mutagenic activity was assayed by the Ames mutagenicity test in the plate-incorporation method using histidine mutants of S. typhimurium strains TA 100. The selected plant extracts at 2.5mg/ml and 5mg/ml did not induce mutagenesis in the absence of liver-metabolizing enzymes. The study results indicated that the selected plants are capable of inhibiting the growth of the studied pathogenic microorganisms to a varied degree. The leaves of G. incanum, P. hortorum, T. capensis as well as the stem bark of C. abbreviata could be novel sources of antimicrobial agents that might have broad spectrum activity. The anti-mutagenic properties of the studied medicinal plants may also provide additional health supplemental value to the other claimed therapeutic properties of the plants.

Obesity can lead to Type 2 Diabetes, both conditions increase in association with physical inactivity and high-energy diets, resulting in elevated blood glucose, decreased insulin sensitivity and increased insulin resistance. Sutherlandia frutescens (S.frutescens), an anti-diabetic plant, reverses and prevents insulin resistance in a rat model and human cell culture model. Gene expression analysis in hepatocyte cultures, identified genes down regulated in insulin resistance and up regulated by S.frutescens. These included genes encoding vesicle transporter proteins, hypothesised to be linked to hepatic lipid accumulation and lipid droplet formation during insulin resistance. The aim of this study was to investigate critical genes involved in lipid droplet formation, vesicle assembly and transport in high fat diet (HFD)-induced insulin resistant rat liver tissue during the development of insulin resistance and the reversal of these changes by S.frutescens. Rats were fed a low fat diet (LFD) or HFD supplemented with S.frutescens for 2, 4 and 8 weeks. Rats fed a HFD for 12 weeks developed insulin resistance, confirmed by plasma glucose and insulin levels (compared to normal controls). Groups of these rats were gavaged with S. frutescens (50mg/kg BW), Metformin (13mg/kg BW) or water for a further 4 weeks and starved for 12 hours, anaesthetized and blood removed by heart puncture. Liver was stored in RNA-Later™ for qRT-PCR and snap-frozen in liquid nitrogen for western blotting and confocal microscopy analysis. Changes in expression of vesicle transporter genes VAMP3 and NSF were analysed by qRT-PCR and changes in the protein expression by western blotting analysis. Proteins were localised within the liver by confocal immunohistochemistry using ZEN lite™ software. Statistical analysis was performed using One-Way ANOVA and unpaired t-test. mRNA gene expression of vesicle transport components VAMP3, NSF and SNAP25 showed relatively moderate changes with considerable individual variation within control or experimental groups. Uncorrelated changes in mRNA and protein products were found and may be due to differential regulation by siRNA. Proteins also showed altered staining patterns in high fat diet rats that reverted towards normal on S. frutescens treatment, potentially reflecting functional changes associated with transport of lipid-filled vesicles.

A follow-up of traditional medicinal plants uses is an important tool in highlighting their therapeutic potentials, as they have been found to be a source of a wide range of bioactive compounds that can be used as base compounds for new pharmaceutical drugs. This study therefore focuses on assessing the anti-Listerial properties of the seeds of Garcinia kola (Heckel) plant, which is a traditional medicinal plant of west and central African origin, and was and is still used to traditionally treat several ailments. Four different solvents crude extracts of the seeds were assessed for their anti-Listerial activities in-vitro, against a panel of 42 Listeria bacteria, which included Listeria monocytogenes, Listeria ivanovii and Listeria grayi species. At 10 mg/ml concentration the aqueous extract had activity against 29% of the test isolates while the other three crude extracts namely dichloromethane, n-hexane and the methanol extracts had activity against 45% of the test bacteria. The minimum inhibitory concentration (MIC) ranges of the extracts were 0.079-0.313 mg/ml for the dichloromethane extract; 0.079-0.625 mg/ml for the n-hexane extract; 0.157-0.625 mg/ml for the methanol extract; and 10->10 mg/ml for the aqueous extract. The minimum bactericidal concentration (MBC) ranges of the extracts were 0.625–10 mg/ml for both the n-hexane and the dichloromethane extract; 5-10 mg/ml for the methanol extract; and those for the aqueous extract were above 10 mg/ml against all the susceptible Listeria isolates. The rate of kill analysis was then determined for the three most active crude extracts that is excluding the aqueous extract and it was assessed against four representative Listeria species namely L. monocytogenes (LAL 8), L. grayi (LAL 15), L. ivanovii (LEL 30) and L. ivanovii (LEL 18). All the three extracts showed a general trend of being concentration and time dependent in their rate of kill profiles such that most bacteria cells were killed at the highest test concentration of 4× MIC value after the maximum exposure time of 2 h. The n-hexane, dichloromethane and methanol extracts were bactericidal against 4, 3 and 1 isolates out of the four test Listeria isolates respectively.

The n-Hexane, dichloromethane, methanol and aqueous crude extracts of Garcinia kola (Heckel) seeds were screened for their anti-Vibrio activities against 50 Vibrio bacteria isolated from wastewater final effluents. The 50 isolates consisted of different Vibrio species namely V. fluvialis (14), V. vulnificus (12), V. parahaemolyticus (12), V. metschnikovii (3) and 9 others unidentified to the specie level. The n-Hexane, dichloromethane and methanol extracts had activities against 16 (32 percent) of the Vibrio isolates, while the aqueous extracts had activities against 12 (24 percent) all at a screening concentration of 10 mg/ml. The minimum inhibitory concentrations (MICs) were 0.313-0.625 mg/ml, 0.313-0.625 mg/ml, 0.313-2.5 mg/ml and 10 mg/ml for n-Hexane, dichloromethane, methanol and aqueous extracts respectively. Rate of kill studies were carried out against three different Vibrio species namely V. vulnificus (AL042), V. parahaemolyticus (AL049) and V. fluvialis ( AL040) using the n-Hexane, dichloromethane and methanol extracts at 1× to 4 × MICs and 2 hour exposure. About 96.3 percent, 82.2 percent, and 78.1 percent (V. fluvialis AL040); 92.6 percent, 87.8 percent and 68.9 percent (V. parahaemolyticus AL049); and 91.6 percent, 64.4 percent, 60 percent (V. vulnificus AL042) of the bacteria were killed by the crude n-Hexane, dichloromethane and methanol extracts respectively after 2 hour exposure time at 4× MIC. The patterns of activity were bacteriostatic, with the n-Hexane extracts being most effective in activity. We conclude that the Garcinia kola seeds have promise in the treatment and management of infections caused by Vibrio species.

Variations in the yield, chemical composition, antibacterial, and antioxidant properties of the essential oils of Rosmarinus officinalis L. cultivated in Alice, Eastern Cape of South Africa over a period of 12 months using the solvent-free microwave extraction and traditional hydrodistillation methods were evaluated. The GC-MS analyses of the essential oils revealed the presence of 33 compounds with 1,8-cineole, a-pinene, camphor, verbenone, bornyl acetate and camphene constituting about 80 percent of the oils throughout the period of investigation, with the solvent-free microwave extraction method generally yielding more of the major components than the hydrodistillation method. Each of the major components of the oils varied in quantity and quality of yield at different periods of the year. The method of extraction and time of harvest are of importance to the quantity and quality of essential oil of Rosmarinus officinalis. Higher amounts of oxygenated monoterpenes such as borneol, camphor, terpene- 4-ol, linalool, a-terpeneol were present in the oil of SFME in comparison with HD. However, HD oil contained more monoterpene hydrocarbons such as a-pinene, camphene, β-pinene, myrcene, a-phellanderene, 1,8-cineole, trans- β-ocimene, γ-teprinene, and cis-sabinene hydrate than SFME extracted oil. Accumulation of monoterpene alcohols and ketones was observed during maturation process of Rosmarinus leaves. Quantitative evaluation of antibacterial activity, minimum inhibitory concentration values were determined using a serial microplate dilution method. The essential oils obtained using both methods of extraction were active against all the bacteria tested at a concentration of 10 mg mL-1. The minimum inhibitory concentrations for the SFME extracted oils ranged between 0.23 and 1.88 mg mL-1, while those of the HD extracted oils varied between 0.94 and 7.5 mg mL-1, thus suggesting that the oil obtained by solvent free microwave extraction was more active against bacteria than the oil obtained through hydrodistillation. The antioxidant and free radical scavenging activity of the obtained oils were tested by means of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH+) assay and β- carotene bleaching test. In the DPPH+ assay, while the free radical scavenging activity of the oil obtained by SFME method showed percentage inhibitions of between 48.8 percent and 67 percent, the HD derived oil showed inhibitions of between 52.2 percent and 65.30 percent at concentrations of 0.33, 0.50 and 1.0 mg mL-1, respectively. In the β-carotene bleaching assay, the percentage inhibition increased with increasing concentration of both oils with a higher antioxidant activity of the oil obtained through the SFME than the HD method. Thin layer chromatography (TLC) was used to analyze the chemical composition of the extracts using three eluent solvent systems of varying polarities i. e. CEF, BEA and EMW and sprayed with vanillin-sulfuric acid. The chemical composition of the different extracts was similar with the exception of methanol and water extracts which had only one or two visible compounds after treating with vanillin-spray reagent. To evaluate the number of antibacterial compounds present in the fractions, bioautography was used against two most important nosocomial microorganisms. S. aureus (Gram positive) and E. coli (Gram negative). Nearly all the crude serial extraction fractions contained compounds that inhibited the growth of E. coli. The hexane extract had the most lines of inhibition followed by ethyl acetate. Bioassay-guided fractionation against E. coli was used to isolate antibacterial compounds. The largest number of antibacterial compounds occurred in the hexane fraction. Furthermore we tried to complete the characterization by extracting and studying other biologically important plant metabolites such as phenolic compounds to evaluate the antioxidant capacity of Rosmarinus extracts

Hermannia incana Cav. (Sterculiaceae), known as sweet yellow bells, is a medicinal plant used by the people of the Eastern Cape for the treatment of stomach-ache and diarrhoea. It has purgative and diaphoretic effects. It is a prostrate herb with yellow flowers and sparsely hairy and slightly glandular leaves, occurring in grassland and marshes in the Eastern Cape Province of South Africa. Based on the ethnomedical uses of this plant, the research project was designed to evaluate its antidiarrhoeal and toxicological properties. An ethnobotanical study of plants used for the treatment of diarrhoea in the Eastern Cape Province was carried out, using a questionnaire which was administered to herbalists, traditional healers and rural dwellers. This survey indicated a total of 17 plant species from 14 families. Elephantorrhiza elephantine (Burch.) Skeels, Hermannia incana Cav., Pelargonium reniforme Curt., Alepidea amatymbica Eckl. & Zeyh. and Bulbine latifolia (L.f.) Roem. et Schult. were the most frequently mentioned and highly recommended plants for the treatment of diarrhoea by both the traditional healers and rural dwellers. The root, bark and leaves are the common parts of plants used, while decoctions and infusions are the main methods of preparation. The agar dilution method was used to study the antimicrobial activity. The methanol extracts of the plant showed appreciable activity against Gram-positive and Gram-negative bacteria at concentrations ranging from 0.5 to 7.0 mg/ml. The acetone and water extracts of both the leaves and the roots showed moderate activity against Gram positive bacteria and less activity against Gram negative bacteria. All the extracts inhibited the growth of the fungi Aspergillus flavus, Aspergillus niger, and Mucor hiemalis with growth inhibition ranging from 54.31 percent to 96.67 percent at 0.1-10 mg/ml. None of the extracts suppressed the growth of Candida albicans at the maximum concentration (10 mg/ml) tested. iii In the in vivo antidiarrhoeal evaluation using Wistar rats, the aqueous extract at all the doses tested, significantly prolonged the time of induction of diarrhoea and also reduced the frequency of diarrhoeal episodes and fecal parameters (total number, number of wet, fresh and dry weight and water content of the faeces). The percentage inhibition of defecation and intestinal content (enteropooling) were increased in dose dependent manner. The doses also reduced the intestinal transit time of charcoal, masses and volumes of intestinal fluid (gastrointestinal motility). These results are indications of antidiarrhoeal property of H. incana leaf extract with the 600 mg/kg body weight of the extract being the most effective. In the toxicological evaluation using Wistar rats, the oral administration of the extract did not produce any significant effect on the liver and kidney body weight ratios, RBC, HB, PCV, MCV MCH, MCHC, RCDW, WBC, neutrophils, monocytes and basophils cholesterol, triacylglycerol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol and atherogenic index. The extract also did not affect the levels of sodium, potassium, chloride, inorganic phosphorus, urea, creatinine, total protein, globulin, albumin, total and conjugated bilirubin. The activities of alkaline phosphatase, gamma glutamyl transferase and alanine aminotransaminase in the serum were increased by the extract whereas aspartate aminotransaminase was decreased. The levels of LUC, platelets, lymphocytes and eosinophils were significantly affected at 600 mg/kg body weight. The available evidence in this study suggests that the extract of H. incana leaf is mild, parameter and dose specific. The structure and distribution of foliar appendages on the leaves of this plant were investigated with the JEOL (JSM-6390LV) scanning electron microscope (SEM). Both glandular and non-glandular trichomes were observed. Long stalked glandular trichomes were present on both the abaxial and adaxial surfaces while short stalked glandular trichomes were present only on the adaxial surface. Glandular trichomes were capitate while nonglandular trichomes were stellate with many arms. Energy dispersive X-ray spectroscopyiv SEM showed that Al, Ca, K, Na, Ti and Si were the major constituents of the crystals analyzed from the leaf surfaces. The phytochemical screening of H. incana revealed the presence of bioactive antidiarrhoeal agents such as alkaloids, tannins, saponins, phenolics, triterpenes, cardiac glycosides, flavonoids, cardenolides and dienolides. Two flavonoids, epicatechin and 3, 5, 7, 2’ tetra-hydroxy flavone-3- O--D-glucopyranoside were isolated from the leaves of the plant through bio-active guided fractionation. Both these compounds were screened against diarrhoea causative organisms (Echerichia coli, Shigella flexneri, Bacillus cereus and Staphylococcus aureus) and exhibiting minimum inhibitory concentrations ranging from 12.5 to 100 μg/ml. The findings from this research have generally justified the traditional use of this plant for the treatment of diarrhoea in this province.

The use of medicinal plants for treatment of skin infections and improvement of skin tone was assessed. The study covered Amathole and OR Tambo municipalities of the Eastern Cape Province of South Africa. The study sought to identify and document some medicinal plants which are used by the indigenous people on the skin, evaluate some biological properties which attribute to their use for therapeutic use. This report provides ethnobotanical data on some plant which were identified. Six medicinal plants (Kniphofia drepanophylla, Gnidia capitata, Hypoxis hemerocallidea, Syzgium cordatum, Macaranga capensis and Protorhus longifolia) which were implicated for treatment of skin diseases were screened for their antibacterial and antioxidant properties. Guided questionnaires were used to interview and gather ethnobotanical information from the traditional healers. Members of the communities which were indicated to be knowledgeable on use of medicinal plants in the areas were visited and interviewed. The results revealed that a total of 45 plant species distributed in 41 Genera belonging to 28 Families are used for treatment of skin infections and improvement of skin texture. The plant medicines were reported to be used as remedies against common skin problem such as wounds, pimples, acnes and itches. Some were reported to have anti-inflammatory effect on the skin while other were reported to oil dry skin and prevent skin dryness and dry eczematous conditions. Others like Cassipourea flanaganii, and Spirostachys africana were commonly used to enlighten skin and protect skin from ultra-violet radiation. The most common mode of preparation of these medicinal plants was pastes or decoctions. Sometimes crushed plant materials were put in bathing water or boiled and steamed on affected parts. It was reported that mixing pastes with oil, animal fat and milk improve medicinal properties. Water, methanol and acetone extracts of K. drepanophylla, G. capitata, H. hemerocallidea, S. cordatum, M. capensis and P. longifolia were screened against five strains of Gram negative bacteria. The results revealed that water extracts from five plant species inhibited two or more strains of bacteria with the most common minimum inhibitory concentrations (MICs) ranging between 5,0 to 10,0 mg/ml with the exception of aqueous extracts of K. drepanophylla which failed to inhibit all strains of bacteria. The screening of methanol extract of these plants for antioxidant and free radical activity revealed a significant activity with 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) free radical (DPPH). The extracts of P. longifolia, G. capitata, M. capensis, S. cordutum and H. hemerocallidea showed significantly higher free radical scavenging activity with IC50 of ranging from about 11.0 to 41.2 μg/ ml while the IC50 value for K. drepanophylla could not be determined at 100 μg/ ml. Generally the study showed that medicinal plants still play a very important role in the health care delivery system, especially in the O R Tambo and Amathole municipalities of the Eastern Cape. Botanical medicine remains pivotal in the treatment of skin ailments and improvement of the skin tone.

M.Sc.