Faculty of Applied Sciences
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Item Isolation, characterization and biological activities of terpenoids from Gunnera perpensa(2018) Mammo, Fitsum K.; Mohanlall, Viresh; Shode, Francis O.; Odhav, BhartiFive pentacyclic triterpenes were identified from the leaves and stalks of Gunnera perpensa. These were ursolic acid (I), 3-β-hydroxyolean-12-en-27-oic acid (II) (β-peltoboykinolic acid), 3- β-hydroxyurs-12-en-27-oic acid (III) (α-peltoboykinolic acid), 3-α-hydroxyolean-12-en-27-oic acid (IV), and 3α-3, 19-dihydroxyurs-12-en-28-oic acid (V) (pomolic acid). These compounds were extracted using ethyl acetate and separated by silica gel column chromatography by the aid of TLC. Their structures were elucidated by 1H and 13C NMR spectroscopy and identified by comparing their spectral data with those reported in the literature. High-Performance Liquid Chromatography (HPLC) was used to identify those compounds that were difficult to separate using CC. Disc diffusion assay was used to evaluate the antimicrobial potentials of the crude extracts and the isolated compounds against bacterial and fungal species. The sickle-cell normalisation and free radical scavenging potentials of the crude extracts and the isolated compounds were done using Emmel’s test and DPPH (2, 2 dyphenyl-2-picryl hydrate) photometric assay, respectively. The anticancer potential of the isolated compounds was performed using the 3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The antimicrobial results showed that the crude extracts GP/A3 and GP/C4 were highly active against S. aureus, B. cereus, E. coli and P. aeruginosa with MIC values ranging from 0.75 to 1.5 µg/ml. Significant antibacterial activity was observed with compound X against S. aureus, S. epidermidis and E. faecalis. Neither of the crude extracts nor the isolates showed antifungal activity.The partial fraction (compound Y) showed the highest normalisation rate to the sickle- cells with 82.135 % standardisation at 144.93 µg/ml. The crude extracts GP/A2, GP/B3, GP/A3, and GP/B2 exhibited the highest radical scavenging capacity against the free radical 2,2- diphenyl-1-picrylhydrazyl with 96, 95, 94.8, and 94.3 percentage values, respectively.While the free radical scavenging potentials of the isolates were found to be less. The highest cytotoxicity activity was recorded by the compounds X and Y followed by the moderate activity of compounds I, II, III and IV with equal percentage death values of, 54.58. The tested compounds were found to be more toxic to the MCF-7 than HEK293 cell line. In conclusion, this is the first study to discover pentacyclic triterpenes from the leaves and stalks of G. perpensa. The crude extracts and their isolates together with the two partial fractions (compounds X and Y) were found to be responsible and add value to the existing known potential of G. perpensa. These compounds have shown antimicrobial, anti-sickling, anti-oxidant and anticancer properties. The probability of getting a cure for Sickle Cell Anemia patients from G. perpensa is possible through direct use or after chemical modification of the compounds. The high anti-oxidant potential of the crude extracts will serve as a starting point for further studies to identify and characterise undiscovered secondary metabolites. The anticancer properties of the isolated compounds encourage future studies against different cancer cell lines. Moreover, none of the crude extracts and isolated compounds were found to be toxic to brine shrimp larvae which indicate its safety in any human health related applications. It is highly recommended to further purify the partially purified compounds (X and Y) as they showed high biological activities that can add value to new drug development.Item Bioactive compounds from South African plants against Mycobacterium tuberculosis(2016) Singh, Alveera; Odhav, Bharti; Coovadia, YacoobMycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB) has infected approximately one-third of the world population, with 9.6 million TB cases in 2014. The emergence of multi-drug resistant (MDR) and extensively-drug resistant (XDR) strains of MTB has further complicated the problem of TB control. It is now imperative that novel antimycobacterial compounds are discovered in order to treat infections and reduce the duration of current TB therapy courses. For centuries, medicinal plants have been used globally worldwide for the treatment and prevention of various ailments. This occurs particularly in developing countries where infectious diseases are endemic and modern health facilities and services are inadequate. In recent years, the use and search for plant drug derivatives have been fast-tracked. Ethnopharmacologists, botanists, microbiologists, and natural product chemists are trying to discover phytochemicals which could be developed for the treatment of infectious diseases, especially TB. Plants are rich in a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids, and flavonoids, which have been found in vitro to have antimycobacterial activity. In the search for new lead compounds, nine medicinal plant species, Buddleja saligna, Capparis tomentosa, Carpobrotus dimidiatus, Dichrostachys cinerea, Ekerbergia capensis, Ficus Sur, Gunnera perpensa, Leonotis leonurus and Tetradenia riparia were collected in Kwa-Zulu Natal (KZN) following report of their therapeutic use in traditional medicine to treat symptoms and infections related to TB. They were tested in vitro for their activity against Mycobacterium smegmatis, Mycobacterium tuberculosis H37Rv (ATCC 25177) and three well-characterized clinical isolates of MDR-TB and XDR-TB using the agar incorporation method. The minimum inhibitory concentration of the active plant extracts was determined using the broth microdilution method. Our findings show that five of the nine plants screened have antimycobacterial activity with concentrations ranging from 125 µg/ml to 1000 µg/ml. The aqueous extracts of G. perpensa and T. riparia; and the methanolic extracts of B. saligna, C. tomentosa, and C. dimidiatus possessed significant activity against M. smegmatis, M. tuberculosis H37Rv (ATCC 25177) and the three well-characterized clinical isolates of MDR-TB and XDR-TB. The cytotoxic effect of the active plant extracts was evaluated against the mouse BALB/C monocyte-macrophage (J774.2) and peripheral blood mononuclear cells (PBMCs). The toxic effects of the active plant extracts were evaluated using the brine shrimp lethality assay. Except for a high concentration of G. perpensa none of the other plants which possessed antimycobacterial activity showed any toxic or cytotoxic activity. The active plant extracts were thereafter assessed to determine if they had any effect on the survival or death of mycobacterial species, M. smegmatis, bound within the macrophage (J774.2) cell line at a concentration of 100 µg/ml. B. saligna had inactivated most of the phagocytosed bacilli after 24 hours of treatment therefore, it has a bactericidal effect on the mycobacteria located within the mouse macrophage. A phytochemical investigation of the leaves of B. saligna led to the isolation of two isomeric pentacyclic triterpene compounds namely Oleanolic Acid (OA) and Ursolic Acid (UA) using thin layer chromatography followed by silica gel column chromatography. The structures of these compounds were fully characterized by detailed NMR investigations, which included 1H and 13C NMR. Ursolic acid was isolated from this plant for the first time. Two-dimensional (2D) and three-dimensional (3D) quantitative structure-activity relationship (QSAR) studies were carried out to provide insight on the interaction of the compounds with the enzyme. Molecular docking studies predicted the free binding energy of the triterpenes inside the steroid binding pocket of Mycobacterium tuberculosis fadA5 thiolase compared to a reported inhibitor. Thus, their ability to inhibit the growth of Mycobacterium tuberculosis was predicted and was confirmed to possess significant antimycobacterial activity when tested against M. smegmatis, M. tuberculosis H37Rv (ATCC 25177), clinical isolates of MDR-TB and XDR-TB using the Microplate Alamar Blue Plate (MABA) assay. The present study has scientifically validated the traditional use of medicinal plant B. saligna.Item Bioactivity of famine food plants from the family: Amaranthaceae(2009) Singh, Alveera; Odhav, Bharti; Reddy, LaliniInformation regarding the nutritional value of wild food plants in Africa and current information varies from source to source. Prior to commercialization of wild foods the nutritional, ethnobotanical, medical, chemical, anthropological and toxicity requires investigation. Plants from the Amaranthaceae family were chosen because the family is characterized by several species which are used by indigenous communities as a source of nutrition in different plants of the world. The focus of this study was to investigate the nutritional and biological activities of three plants from the Amaranthaceae family viz. Achyranthes aspera, Alternanthera sessilis and Guilleminea densa that are considered famine plants. This study aimed to determine the nutritional value (proximate, minerals and vitamins), biological activity, toxicity and potential of a tissue culture system for three species from the family Amaranthaceae. Nutritional analysis comprised of determining moisture, ash, protein, fat, carbohydrate, dietary fibre and energy. Mineral analysis of calcium, copper, iron, magnesium, manganese, phosphorus, sodium and zinc was performed by microwave digestion and then analyzed by ICP Spectrophotometry. Vitamin A, Vitamin B1, Vitamin B2, Vitamin B3 and Vitamin C were also analyzed. For biological and safety analyses aqueous and methanolic extracts were prepared. Anti-oxidative and anti-inflammatory properties of the extracts were tested; antimicrobial activity was tested by evaluating the bactericidal, fungal effect and minimum inhibitory concentration on selected bacteria and fungi using the agar disk diffusion method. Anti mosquito potential was determined by setting up repellency, larvacidal assay and insecticidal assay. The safety and toxicity analysis was carried out by measuring cytotoxicity, toxicity and mutagenicity. The potential of an in vitro tissue culture system of A. aspera, A. sessilis and G. densa was determined using micropropagation. A. aspera indicated significant amounts moisture, ash, dietary fibre, protein, vitamin B1, vitamin B2, magnesium and manganese. Plant extracts of A. aspera had antibacterial activity against the Gram negative bacteria Esherichia coli, Pseudomas aeroginosa and Salmonella typhi; Gram positive bacteria Staphylococcus epidermis and Staphylococcus aureus. The methanolic extract had antifungal activity against Sacchromyces cerevisiae and exhibited significant free radical scavenging activity as well as 85% repellency against Anopheles arabiensis. The aqueous extract stimulated the growth of the K562 (Chronic Myclogenous Leukaemia) cell line and the plant extracts showed no mutagenicity or toxicity. A. sessilis indicated significant levels of ash, dietary fibre, protein, energy, vitamin A, vitamin B1, vitamin B2, vitamin B3, iron, magnesium and manganese present. Plant extracts of A. sessilis had antibacterial activity against Gram negative bacteria P. aeroginosa and Gram positive bacteria S. epidermis. The plant also showed antifungal activity against the yeasts S. cerevisiae and Candida albicans. The methanolic plant extract showed excellent antioxidant activity. The aqueous plant extract stimulated the growth of the K562 cell line and the plant extracts possessed no mutagenicity or toxicity. This plant grew well in a tissue culture system where it was propagated from callus to a fully grown plant able to survive in environmental conditions. G. densa has ash and dietary fibre, vitamin B2, vitamin B3 and iron. The plant extracts had antibacterial activity against Gram negative bacteria E. coli, P. aeroginosa and Klebsiella. oxytoca; Gram positive bacteria Baccilus stereathermophilus and S. aureus. The plant also has antifungal activity against C. albicans and significant repellency activity against A. arabiensis where it showed 100% repellency. This plant was not found to be mutagenic or toxic. The results obtained from this study show promising potential for the plants to be exploited as famine food plants. The nutritional value, biological activity and ability to micropropagate A. aspera, A. sessilis and G. densa indicates a good potential for purposes of harnessing biotechnological products.Item Evaluation of traditional South African leafy plants for their safety in human consumption(2007) Mudzwiri, Mashudu; Reddy, Lalini; Odhav, BhartiEighteen traditionally leafy vegetables consumed as food or medicinal compounds by a majority of people in the KwaZulu Natal province of South Africa were analysed for the presence of potentially harmful chemicals (antinutrients) and for their toxicity and mutagenicity. The purpose of the study was to determine whether leafy vegetables were safe for human consumption. Chemical analysis showed that none of the vegetables contained cyanogenic glycosides, however all the vegetables contained oxalic acid ranging from 24.1 mg/ml to 798.2 mg/ml with Solanum nigrum, Portulaca oleracea and Mormodica balsamina showing the highest concentrations. Most of the vegetables contained negligible amounts of phytic acid and saponins, except for Momordica balsamina (3.01 mg/ml and 1.83 mg/ml, respectively). Fourteen of the plants contained alkaloids with Portulaca oleracea having the highest content (1.53 g total alkaloids/5 g leaf material). Eight of the plants were found to inhibit trypsin activity. These chemical analyses were carried out in duplicate and the mean and standard deviation were used. The Ames test revealed that none of the leafy vegetables produced a mutagenic frequency above 1, except 10 000 µg/ml organic extract of Senna occidentalis (mutagenecity considered at mutagenic frequency above 2), thus none were considered mutagenic. All 18 organic extracts did not kill off more than 50% brine shrimp and were thus considered non-toxic. On the other hand the aqueous extracts of seven vegetables, namely, Physalis viscosa, Amaranthus dubius, Justicia flava, Bidens pilosa, Senna occidentalis, Chenopodium album and Ceratotheca triloba, killed more than 50% of the shrimp and are thus considered toxic above 100 µg/ml. The MTT assay carried out on the organic extracts indicated that 17 vegetables did not kill off more than 50% of HepG2 cells and were thus considered non-cytotoxic. The aqueous extracts of four vegetables, namely, Justicia flava, Asystasia gangetica, Momordica balsamin and Senna occidentalis, however killed more than 50% of the shrimp and were thus considered cytotoxic above 1 000 µg/ml. It may be concluded from the antinutrient analyses and the bioassays on the 18 vegetables that caution needs to be maintained with the consumption of certain leafy vegetables included in this study, especially Senna occidentalis.