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The human immunodeficiency virus (HIV) is one of the most common and dreaded diseases of the 21th century. Today, the disease is still spreading with increasing incidence. “Since the beginning of the epidemic, almost 75 million people have been infected with the HIV virus and about 36 million people have died of HIV. Globally, 35.3 million [32.2–38.8 million] people were living with HIV at the end of 2012” (http://www.who.int/gho/hiv/en/). Several studies have been conducted on herbs under a multitude of ethnobotanical grounds. The use of medicinal plants for the management of HIV has become a common practice especially, in the Eastern Cape Province of South Africa (Wilfred Otang Mbeng, 2013 PhD thesis, UFH). At the beginning of this programme, an ethnomedicinal survey of plants used for the management of HIV infection was carried out in targeted areas of the Province and information on the names of plants, the parts and the methods of preparation were collected. The survey revealed that 18 species representing 12 families were found to be commonly used for the management of HIV, as well as other opportunistic diseases such as tuberculosis, diabetes mellitus, sores, high blood pressure, etc. Carpobrotus edulis was selected for this research because it was the most frequently used in the Province. The foliar micro morphological contents of the plant, its phytochemical and antioxidant activity, in vitro antimicrobial activity, inhibitory effect against HIV-1 protease and reverse transcriptase, mechanisms of action and cytotoxicity were investigated. In terms of the foliar micro morphological contents in plants, an electron microscopy scanning (SEM) was completed. Investigation revealed that both glandular tricomes and calcium oxalate crystals (CaOX) were observed. Consequently, it is hypothesized that the bioactive therapeutic compounds secreted by C. edulis may be produced in the glandular trichomes. An investigation of phytochemical content of the plant extracts (C. edulis) was completed using four solvent extracts (hexane, acetone, ethanol and water). Results of the phytochemical analysis showed that proanthocyanidins (86.9 ± 0.005%) where highest in the aqueous extract with phenolics at 55.7 ± 0.404% in acetone extract, tannin at 48.9 ± 0.28% in ethanol extract, while the hexane extract had the highest levels of flavonoids (0.12 ± 0.05%) and flavonols (0.12 ± 0.05%). Antioxidant studies of the various extracts revealed that aqueous and ethanol extracts were found to be the best solvents for antioxidant activity in C. edulis leaves. GC-MS analysis of the essential oil from C. edulis leaves revealed that the essential oil contained at least 28 compounds. These included, in order of abundance: Oxygenated monoterpenes (36.61%); fatty acids esters (19.25%); oxygenated diterpenes (19.24%); monoterpenes (10.6%); sesquiterpenes (3.58%); and diterpenes (1.43%). Similarly, a GC-MS analysis of the crude hexane, acetone and ethanol extracts from C. edulis leaves identified a total of 59 compounds. Of the 59 compounds, 12 major phyto-metabolites that are active against infectious diseases were identified. To comfirm the potential use of C. edulis to treat infectious disease, antifungal activity of the crude essential oil extract and the four solvent extracts were tested against Candida albicans, Candida krusei, Candida glabrata, Candida rugosa and Cryptococcus neoformans strains. The essential oil extract was found to be the most active against all the fungal strains tested and performed better than the four extracts used various solvents used to extract (hexane, acetone, ethanol and water) the C. edulis leaves were tested for antibacterial and anti HIV-1 reverse transcriptase (RT) activity. The results indicated that both gram-positive and gram-negative isolates were inhibited by the extracts (hexane, acetone and ethanol) but antimicrobial activity was observed for the water extract. The lowest minimum inhibitory concentration values were obtained for the ethanol extract, followed by acetone and hexane extracts. No inhibition of HIV-1 reverse transcriptase was observed for any of the leaf extracts, even up to concentrations of 16 mg/ml. The potential inhibitory activities of the various solvent extracts against HIV-1 protease were evaluated at four different concentrations (16, 1.6, 0.16 and 0.016 mg/ml). Results indicated that the water extract showed almost 100% inhibition of HIV-1 protease activity, with an IC50 of 0.86 mg/ml leaf extract. Other solvent extracts (hexane, acetone and ethanol) however, did not show any inhibition activity above that observed for the DMSO control. The metabolic components in the water extract were subjected to LC-MS/MS analysis, which identified at least 91 compounds present in the water extract. Further studies involving the molecular modelling need to be carried out to confirm the inhibitory potential of these compounds. The cytotoxicity of the water extract of C. edulis leaves was also screened using human Chang liver cells at concentrations ranging bewteen 0.005 mg/ml and 1 mg/ml. Results indicated that the water extracts were not toxic. In conclusion the results from this study support the use of water extracts of C. edulis leaves by traditional healers to treat HIV infections and have identified possible mechanisms of action of the water extracts of C. edulis.

The chemical profiling, characterization of Aloe products and phytochemical properties of Aloe vera were studied. The adulteration of commercial products derived from medicinal plants has been a major muddle for both the society and the pharmaceuticalindustry. Economically motivated adulteration includes the potential for contaminated, sub‐potent or counterfeit medication to enter the supply chain at several levels, from the production of raw ingredients through to the point of retail sale. Darwin’s theory of evolution states that, species undergo genetic variation with time to adapt to environmental changes. Therefore, the same species growing in widely different habitats may drift from the original genetic makeup as a mechanism of adaptation and that may result in them having different chemical profiles. Therefore this study aimed at investigating the phytochemical properties of Aloe vera growing in South Africa. Also, this study aims to utilize Thin Layer Chromatography to profile this plant, as well as use Infra Red spectroscopy to characterize commercial Aloe vera products. A large quantity of Aloe vera plant was collected from AloeWay, Iphofolo Game Farm, Polokwane in the Limpopo province of South Africa. The identity of the plant was confirmedrom literature and authenticated by Professor DS Grierson of Botany Department, University of Fort Hare, Alice. The plant leaves were divided into two portions. One portion was extracted fresh while the other was cut into pieces and oven dried at 400C then and milled to a homogenous powder once dried completely. The phytochemical composition of the gel and leaf extracts revealed the presence of alkaloids, flavonoids, saponins, tannins and phenols at different concentrations. Results showed that the dry plant material yielded more phytochemicals than the fresh plant material. In particular, it was found that the acetone extract showed much more amounts ofphychemicals than the dichloromethane and aqueous extracts. The percentage compositions of phenols (71.86), flavonols (36.61), proanthocyanidins (82.71), saponins (37.73) and alkaloids (13.29) were significantly high in the acetone extract, followed by the dichloromthane extract with values of 46.85, 37.73, 49.51, 89.0 and 11.11 respectively, while the least composition was found in the aqueous extract. Furthermore, flavonoids were somewhat high in composition in both the aqueous extract of the dried and of the fresh plant material while others were very low. Tannins levels were significantly very low in all the solvent extracts. It was found that the acetone extract showed great amounts of phytochemicals than dichloromethane and aqueous extracts. Since A. vera is used in the treatment of different ailments such as skin wounds and abrasions, eczema, constipation, rheumatoid arthritis etc, the medicinal uses of this plant could be associated to such analysed bioactive compounds. Acetone, hexane, ethanol, water and dichloromethane were used to extract the Aloe vera leaf and the best solvent extract was determined. Thin layer chromatography was used to profile the leaf extracts with the aim of documenting the main phytochemicals present in the Aloe vera growing in South Africa. The best spraying reagent was determined. Fourier transform infrared spectrophotometer was used to validate the presence of Aloe vera ingredients in commercial products. The yield extraction ability of the solvent was the order: water>ethanol> hexane >dichloromethane and acetone for the dry portion. However, for the plant extracted fresh, the order of yield produced was ethanol-acetone-dichloromethane > and water. The different solvent systems separated the compounds differently. Hexane: acetone: ethanol (20 : 5: 2) and Benzene: ethanol: ammonium (80): ethanol (10): ammonium solvent systems were noted to be the best mobile phase as they gave the best separation compared to other systems.EMW [ethyl acetate (81): methanol (11): water (8)] showed better separation than the other two separating solvent systems. Vanillin- sulphuric acid spray was seen to be the best spraying reagent as compared to vanillin- phosphoric acid. Fourier transform infrared spectrophotometer validated the presence aloe ingredients in aloe vera commercial products.

The contamination of heavy metals in the environment is a looming concern worldwide. Egeria densa (Planch) (Submerged aquatic plant) from two ponds: Site A with co-ordinates (32º 48’22.04”S; 26°48’58.79” E) and Site B with co-ordinates (32°48’33.25”S; 26°48’33.25”S) in Alice (Eastern Cape) was evaluated for its ability to absorb heavy metals, phytochemical constituents, antimicrobial activity and ultra-structure using standard analytic procedures. Cadmium (Cd), copper (Cu), iron (Fe), lead (Pb), manganese (Mn), and zinc (Zn) were measured in water, sediments and plant. The concentrations of these metal elements were determined with use of Inductively Coupled Plasma- Optical Emission Spectrometry (ICP-OES). In sediments, the heavy metals (mg/kg) decreased in the order of their average concentration as follows: Fe (40.320) > Zn (1.259) > Pb (0.564) > Mn (0.186) > Cu (0.037) in Pond 1 whereas in Pond 2 Fe (61.527) > Cd (0.999) > Mn (0.648) > Pb (0.586) > Zn (0.156) > Cu (0.045). The highest concentration of Fe was detected in both sites and Cu being the least. The concentrations of the metals in the plants sample (from Pond 1) were found in order of Mn > Pb > Cu > Fe whereas cadmium and zinc were not detected, while the concentration in Pond 2 decreases in order of Zn > Mn > Pb > Cd > Fe > Cu. In the water samples, concentrations of heavy metals (mg/L) decreased in the order of their average concentrations as follows: Pb (35.36) > Fe (3.07) > Mn (0.238) > Cu (0.104), both cadmium and zinc were below the limit of detection in Pond 1, whereas in Pond 2 the concentrations decreased as follows: Pb (13.033) >Fe (1.69) > Cu (0.270) > Mn (0.248) > Cd (0.004) and Zinc was not detected. Phytochemical analyses of the plant extracts revealed the presence of phenols, flavonoids, proanthocyanidin, flavonols, saponins, alkaloid and tannins in all the extracts (water, acetone and n-hexane). Both acetone and water extracts, showed high concentration of proanthocyanidin, while tannin was the lowest in acetone extract. Antimicrobial evaluation using, Gram positive (Staphylococcus aureus, Bacillus pumilus, Bacillus cereus, Streptococcus pyogenes, Enterococcus faecalis) and Gram negative (Klebsiella pneumonia, Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris and Serratia marcescens) bacteria showed negative results for all the strain, except Streptococcus pyogenes which was inhibited at MIC of 0.1 mg/ml. Scanning electron microscopy (SEM) of ultra-structure of Egeria densa, showed that certain bacteria attached to the leaf, However more work has to be done on E. densa to verify the mechanism by which it accumulates heavy metals. The study shows that E. densa has a potential of accumulating heavy metals especial Manganese in plant.

MSc (Microbiology) / Department of Microbiology / Background: The medicinal value of plants lies in some chemical substances that produce a definite physiological action in the human body. The secondary metabolites help the plants to survive hash conditions and could be used by humans as supplements of their health, as foods additives or for medicinal purposes. This bioactive compounds are not always beneficial to human beings, and some of this plants bioactive compounds can be toxic or genotoxic to human cells. This study used several methods to evaluate of phytochemical constituents and mutagenic properties of Coccinia rehmanni and Jatropha zeyheri plant extracts. Methodology: Methanol was used for extraction of the bioactive compounds from the two selected plants, filtered with Whatman filter paper and evaporated with rotary evaporator. The extracts were fractionated using open column chromatography. Chemical and TLC methods were used to determine phytochemicals of the study plants extracts and fractions. The plants extracts and fractions were tested against Vero cell lines in order to evaluate cytotoxicity and genotoxicity of the plants. NucRed and LTR Hoechst 33342 dyes were used for cytotoxicity and genotoxicity respectively. For the evaluation of cytotoxicity and genotoxicity Quantification of live and dead cells for the screening assay was performed using the ImageXpress Micro XLS Widefield Microscope and acquired images analyses using the MetaXpress software and Multi-Wavelength Cell Scoring Application Module. Antimutagenicity of plants extracts was observed using PARP universal colorimetric assay kit. Acquired data was transferred to an EXCEL spreadsheet and data was analyzed. Results and discussion: C. rehmanni (12.03%) yielded more extract than J. Zeyheri (8.20%). the two plants had different compound composition and were in different stages of maturity. The study revealed the domination of Terpenoids, Cardiac glycosides, Phenolic and tannis. With an exception of two fraction fractions all the fractions was found to be toxic to an extent were genotoxicity of such fraction could not be concluded. The reason for such extreme toxicity could be due to the influence of the retained alcohol during rotary evaporation. xvi | P a g e Conclusion: this study provides and add to existing knowledge on the phytochemicals mutagenicity and anti-mutagenicity of C. rehmanni and J. Zeyheri medicinal plants. The study serves as scientific proof that extensive use of this plant in traditional medicine for treatment of various ailments may lead to some irreversible damages. / NRF

MSc (Microbiology) / Department of Microbiology / BACKGROUND: Antibiotic resistance amongst microbial pathogens has become a challenge over past decades, bringing about genuine and frequently deadly contaminations that can't be dealt with by ordinary means. This has led to a search on developing solutions to this problem by searching for new source of antimicrobial agents or chemically altering the existing ones. Traditional medicinal plants and nanoparticles are highly targeted as promising agents to address the challenge. Pyrenacantha grandiflora Baill from Icacenaceae family possess pharmaceutical activities and is used by Vhavenda people to cure gastrointestinal related infections, diarrhea and tooth pain. OBJECTIVES: The present study aimed to synthesize, characterize and evaluate the efficacy of Pyranacantha grandiflora extracts alone and when conjugated with selected nanoparticles against pathogenic microorganisms. Furthermore, this study investigated the efficacy of selected antibiotics when conjugated with nanoparticles against selected pathogenic microbes. METHODS: Pyrenacantha grandiflora Baill (tubers) were collected from Masisi area. Bioactive compounds were extracted using different solvents such as methanol, acetone, hot water, dichloromethane and chloroform. Preliminary phytochemical screening was done to identify different phytochemicals in the extracts and their functional groups were identified by Fourier Transform Infrared spectroscopy (FTIR). Extracts were further assessed for their total phenolic and flavonoids content. Thin layer chromatography was used to separate the compounds from the plant extracts and active compounds/group of compounds were identified by bioautography. The antioxidant ability of the extracts to scavenge free radical DPPH was also determined. Silver and gold nanoparticles were synthesized using chemical and biological methods, characterized by Ultraviolet-Visible Spectrophotometry (UV-VIS), Transmission Electron Microscopy (TEM) and Energy dispersive X-ray analysis (EDX). Plant extracts, nanoparticles and antibiotics were xix conjugated differently, and conjugates were analyzed by FTIR and their antimicrobial activities were evaluated against different bacteria and fungi. The conjugates were tested for antimicrobial activity against extended beta-lactamase producing Escherichia coli (ATCC 35218), Escherichia coli (ATCC 25922), methicillin-resistant Staphylococcus aureus (ATCC 25923), methicillin-susceptible Staphylococcus aureus (ATCC 33591) and beta-lactamase producing Klebsiella pneumonia (ATCC 700603) using agar diffusion assay and the minimum inhibitory concentrations (MIC) were determined using the microdilution method. The minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) were determined by sub-culturing from the MIC plates on Mueller-Hinton Agar. RESULTS: Pyrenacantha grandiflora was found to contain phenolics, saponins, alkaloids, tannin, steroids, terpenoids and flavonoids. FTIR spectroscopic studies revealed different characteristic peak values with various functional compounds similar in most extracts but differed with transmittance values. The total phenolic contents in the examined extracts ranged from 14.167 to 19.02 mg GA/g. The total flavonoid content in the examined extracts ranged from 26.603 to 34.621 mg QE/g. Thin-layer chromatography revealed various Rf values and when analyzed with bioautography, well-defined inhibition zones within the Rf value of 0.236 was identified against E. coli and K. pneumonia. The MICs of the extracts were determined, and all the extracts showed some antimicrobial activity against all tested strains ranging from 0.06-7.5 mg ml/g. Some extracts appeared to be fungicidal and hot water extracts were more active against Cryptococcus neoformans with the MFC value of 0.06 mg/ml. The methanol extract was also active against most tested strains including Candida tropicalis with the minimum fungicidal concentration value of 3.75 mg/ml. Pyrenacantha grandiflora tuber extracts conjugated with silver or gold nanoparticles exhibited a good antibacterial activity against all bacterial strain used and very few were able to exhibit bactericidal activity. Penicillin showed improvement of antibacterial activity xx when conjugated with compounds from the acetone extracts and vancomycin was found to be more effective when conjugated with silver nanoparticles and water extracts. CONCLUSION: The present study validated the efficacy of conjugated P. grandiflora tuber extracts which is used in traditional medicine. The results revealed that water extracts which are generally used by the traditional healers are active against most microorganisms tested as well as methanol and acetone extracts and the synergistic effect was observed when they were conjugated to gold and silver nanoparticles. The results of the present investigation clearly indicate that antimicrobial activity of Pyrenacantha grandiflora Baill tuber when conjugated with selected nanoparticles and antibiotics vary with test strain and the type of solvent used during extraction, thus giving hope for future development of drug leads. / NRF