3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item A phytochemical and pharmacological study of ten Commiphora species indigenous to South Africa(2008-09-29T09:54:28Z) Paraskeva, Maria PenelopeCommiphora species (from which myrrh is obtained) has been a source of several novel and bio-active natural compounds. Traditionally, Commiphora (Burseraceae) is used in southern Africa for the treatment of ulcers, fevers, and as a remedy for snake and scorpion bites. In western Africa, the macerated stem is used in the treatment of rheumatic conditions. The resin of some Commiphora species is applied topically to aid in wound healing. Documented uses include antibacterial and antifungal properties, as well as cytotoxic, cytostatic and anti-oxidant activity. The botanical diversity of this genus in South Africa warrants a study of this plant group, to provide scientific evidence for the traditional use of Commiphora species in African healing rites. Ten Commiphora species were investigated. Fresh plant material of the selected species were identified and collected from natural populations in the Limpopo Province. Active compounds, viz. kaempferol and dihydrokaempferol, in C. glandulosa (stem) were isolated using bioassay-guided fractionation and identified using nuclear magnetic resonance spectroscopy. The stem and leaf extracts of each species were analysed for in vitro anti-oxidant, antimicrobial, anti-inflammatory, anticancer activity, as well as cytotoxicity. The anti-oxidant activity of the extracts was investigated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and the 2,2’-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS) assays. Extracts generally exhibited poor anti-oxidant activity in the DPPH assay, with the exception of C. schimperi (stem), C. neglecta (stem), C. tenuipetiolata (stem and leaf), and C. edulis (stem), which possessed IC50 values ranging between 7.31 μg/ml and 10.81 μg/ml. Isolated compounds were subjected to the DPPH assay to determine the anti-oxidant potential of each compound, separately and in combination to establish possible synergistic, antagonistic or additive effects. The flavonol, kaempferol (IC50 = 3.32 μg/ml) showed exceptional radical scavenging activity, in contrast to the low activity displayed by dihydrokaempferol (IC50 = 301.57 μg/ml), their combination being antagonistic. Greater anti-oxidant activity was observed for most species in the ABTS assay when compared to the results obtained in the DPPH assay. The best activity was observed for the stem extracts of C. neglecta (IC50 = 7.28 μg/ml) and C. mollis (IC50 = 8.82 μg/ml). In vitro antimicrobial efficacy was determined against Gram-positive and Gram-negative bacteria as well as yeasts using the MIC microtiter plate assay. A greater selectivity was exhibited by the extracts against the Gram-positive bacteria and yeast than against the Gram-negative bacteria. Using death kinetics studies (time-kill studies), the rate at which the antimicrobial agent kills pathogens over a 24-hour period was determined. The antibacterial activity of Commiphora marlothii (stem) was observed to begin at ca. 30 min of the exposure of S. aureus to the different concentrations of plant extract. All concentrations exhibited antibacterial activity, with a complete bactericidal effect achieved by all test concentrations by the 24th hour. Commiphora pyracanthoides (stem) displayed anti-inflammatory activity through good inhibition of the 5-LOX enzyme (IC50 = 27.86 μg/ml). The ability of extracts and kaempferol to inhibit the in vitro growth of three human cancer cell lines, namely the colon adenocarcinoma (HT-29), breast adenocarcinoma (MCF-7), and the neuronal glioblastoma (SF-268), was evaluated using the sulforhodamine (SRB) antiproliferative assay. The most active Commiphora species against the HT-29 cells were C. glandulosa (leaf and stem) and C. marlothii (leaf). The MCF-7 cell line was the most sensitive to indigenous Commiphora species, with C. edulis (leaf and stem), C. glandulosa (leaf and stem), C. marlothii (leaf), C. pyracanthoides (leaf and stem), C. schimperi (stem), and C. viminea (stem) all possessing an inhibition greater than 80% at 100 μg/ml. Commiphora glandulosa (leaf and stem) and C. pyracanthoides (leaf and stem) were the two most active species against the SF-268 cells, with IC50 values ranging between 68.50 μg/ml and 71.45 μg/ml. The inhibition of the cancer cell proliferation by kaempferol in all three-cancer cell lines was determined, with IC50 values of 9.78 μg/ml in HT-29 cells, 20.21 μg/ml in MCF-7 cells and 43.83 μg/ml in SF-268 cells. The microculture tetrazolium cellular viability (MTT) assay was used to determine the cellular toxicity of the extracts against transformed human kidney epithelium (Graham) cells. Commiphora glandulosa (stem) proved to be most toxic (IC50 = 30.5 μg/ml). The IC50 values for all other extracts were in excess of 95 μg/ml suggesting low in vitro toxicity for the majority of the species. A phytochemical investigation of the non-volatile constituents of the leaf and stems was conducted using high performance liquid chromatography (HPLC). The HPLC profiles and UV spectra of the stem extracts, and the representative flavonoid patterns in the leaf extracts of the species indicate that a similarity exists in their chemical fingerprint.Item The antimicrobial properties and chemical composition of leaf extracts and essential oils of indigenous Pteronia species(2008-06-30T12:07:12Z) Coovadia, Zubair HoosenAbstract The genus Pteronia consists of approximately 80 species which are widely distributed in southern Africa. For hundreds of years the indigenous people of southern Africa have turned to the earth in order to provide healing for their people. The genus Pteronia has been amongst the first species to be used by the San and Khoi-San people for treating infections and stomach ailments. Ten species were selected for the purpose of this report. The essential oils were isolated by using a Clevenger-type apparatus while the non-volatiles were extracted with acetone and methanol. The essential oils and extracts were assessed for antimicrobial activity. The disc diffusion assays included three Gram-negative bacteria; Escherichia coli, Yersinia enterocolitica and Klebsiella pneumoniae, three Gram-positive bacteria; Staphylococcus aureus, Bacillus subtilis and Bacillus cereus as well as one yeast; Candida albicans. Results indicated that the species were primarily active against Gram-positive organisms. The minimum inhibitory concentration of the ten most active species (essential oils and extracts) were determined using the microdilution method. The most promising activity was noted for P. fasiculata which had a MIC of 0.22 mg/ml against S. aureus, 0.39 mg/ml against B. cereus and 2.08 mg/ml against B. subtilis. The essential oils analysis by GC/MS revealed two chemotypes. In Pteronia pallens, P. empetrifolia and P. flexicaulis rare compounds, such as presilphiperfolol-7-ene, 7-α-(H)-silphiperfol-5-ene, 7-β-(H)-silphiperfol-5-ene, α-campholene aldehyde, silphiperfol-5-ene, camaroonan-7-α-ol, silphiperfol-7- β -ol, presilphiperfolan-9- α -ol and presilphiperfolan-8-ol (a major compound in Pteronia pallens) were recorded. A cluster analysis of the essential oil data indicated that individual collections of P. camphorata within a population were tightly clustered. Similarly, P. pallens sampled from three different localities were also united in the cluster analysis. These results suggest minimal within and between population variations for some of the species studied.Item The phytochemistry and microbial activity of selected indigenous Helichrysum species(2008-06-10T07:01:25Z) Reddy, DakshinaABSTRACT Helichrysum (Asteraceae) is a large genus consisting of approximately 500 species of which 245 taxa are indigenous to southern Africa. Many Helichrysum species are widely used by the indigenous population to treat various ailments, including coughs, colds, fever, infections, headache, menstrual pain and as a treatment for wounds. Medicinal uses are often not species-specific but often depend on the local availability. Guided by the traditional use and the lack of scientific information, nine species of Helichrysum were selected for this study. The essential oils were obtained through hydrodistillation and methanol and acetone extracts of the plant material were prepared. The essential oil composition was determined using GC-MS. The oil profiles were mostly dominated by the presence of monoterpenes such as a-pinene, 1,8-cineole and p-cymene. Monoterpenes were largely absent in the essential oil of H. felinum, but this oil was rich in sesquiterpenes with high yields of b-caryophyllene. The antimicrobial activity of the essential oils and plant extracts were of interest due to their traditional use as an antiseptic. The antimicrobial activity of the essential oils and extracts was determined by disc diffusion assays and, following this, the most active species were further investigated using the minimum inhibition concentration (MIC) assay. Helichrysum dasyanthum displayed the best activity against B. cereus (MIC = 16 mg/ml) and was the only extract that exhibited activity against all three fungal strains tested (C. neoformans, 1 mm; C. albicans, 3 mm; and A. alternata, 2 mm). The essential oil of H. petiolare and H. felinum exhibited the most pronounced activity against the fungal strains in the disc diffusion assay (C. albicans, 2mm).Item A laboratory model for studying inhalation therapy in traditional healing rites(2008-06-04T11:58:18Z) Braithwaite, Miles CharlesABSTRACT The burning of selected indigenous plants and the inhalation of the smoke liberated from them has been a widely accepted and practised form of administration in traditional healing therapy dating back to as far as the Koi and San, and is a method still widely practised in South Africa today. Inhalation has various advantages as a method of administration in both allopathic and traditional practices. Not only is inhalation a highly effective mode of administration because of its direct and local effect on the lungs for the treatment of respiratory ailments, but also because of its ability to deliver drugs effectively systemically. This study elucidated the rationale behind this widely practised treatment by examining chromatographic and antimicrobial data. Five plants that are commonly administered traditionally through inhalation were chosen: Heteropyxis natalensis, Myrothamnus flabellifolius, Artemisia afra, Pellaea calomelanos, and Tarchonanthus camphoratus. An apparatus was designed and constructed and the burning process that occurs in the traditional setting was simulated with the selected plants. The induced volatile fraction (smoke) was captured for analysis. Control solvent extracts were made for each plant using conventional extraction solvents, methanol, acetone, water, and the essential oil of the aromatic plants was also investigated. Antimicrobial assays revealed that the extracts (smoke) obtained after burning had lower minimum inhibitory concentration (MIC) values than the corresponding solvent extracts in most cases. For Klebsiella pneumoniae all five inhalation samples were far more active than the conventional extracts. When tested against the pathogen B. cereus, M. flabellifolius and P. calomelanos inhalation samples proved to exhibit superior antimicrobial activity compared to the respective solvent extracts. Pellaea calomelanos inhalation extract had the lowest MIC values compared to the solvent extracts for all pathogens (P. calomelanos inhalation extract MIC values: 0.53; 1.00; 0.53; 0.53 mg/ml for S. aureus, B. cereus, K. pneumoniae and C. neoformans respectively). Inhalation extracts exhibited different chemical profiles from the solvent extracts of the same plant. For example, A. afra inhalation extract had an abundance of peaks at various retention times from 3.2 to 5.4 minutes, which were not present in the chromatograms of the acetone and methanol extracts of the same plant. These results, albeit preliminary, suggest that the chemistry and antimicrobial activity of plants are influenced by the combustion process which is often used in traditional healing rites.Item The biological activity of specific essential oil constituents(2008-03-13T10:05:44Z) Seatlholo, Tsietsi SamuelABSTRACT Twenty essential oil constituents (EOC′s) from seven structural groups were tested for their antimalarial, antimicrobial (both bacterial and fungal), anti-oxidant, anticholinesterase and toxicity properties. To test for their antimalarial property, the tritiated hypoxanthine incorporation assay was used, while the disc diffusion and minimum inhibitory concentration (MIC) microplate assays were employed for the antimicrobial properties. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) method was used to test the anti-oxidant property and their toxicity profile was assessed with the 3-(4,5-dimethyl-2-thiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cellular viability assay. The anticholinesterase activity was determined using the thin layer chromatography (TLC) bioautographic method. The EOC´s were found to inhibit the growth of Plasmodium falciparum with IC50 values ranging between 0.9 to 1528.8μM with E- and Z-(±)-nerolidol, (-)-pulegone, (+)-α-pinene and linalyl acetate being the most active. In combination p-cymene (the least active) and E- and Z-(±)-nerolidol (the most active) displayed the most synergistic interaction (ΣFIC = 0.09), with their antimalarial activity comparable to that of the interaction between E- and Z-(±)-nerolidol and quinine (ΣFIC = 0.01). Eugenol had the most favourable safety index and was the only EOC with anti-oxidant activity comparable to vitamin C. Combination studies showed that E- and Z-(±)-nerolidol and (-)-pulegone or quinine, p-cymene and γ-terpinene or (-)-pulegone potentiated each other′s toxicity. The EOC´s inhibited the growth of Gram-positive, Gram-negative bacteria and yeast with MIC values ranging from 1.66 to >238.4mM. When combined, synergism was observed between (+)-β-pinene and carvacrol or γ-terpinene; γ-terpinene and geranyl acetate when tested against Staphylococcus aureus, while (+)-β-pinene and (-)-menthone showed antagonism against C. albicans. The combinations of EOC′s and a standard antimicrobial resulted in synergistic interactions between carvacrol and ciprofloxacin against Bacillus cereus, eugenol and ciprofloxacin against Eschericia coli, carvacrol and amphotericin B against C. albicans. The trans-geraniol and E- and Z-(±)-nerolidol combination demonstrated an additive interaction against B. cereus, while for eugenol and E- and Z-(±)-nerolidol an indifferent interaction against E. coli was noted. These results show that the biological activities of EOC′s can vary when used alone and in combination. They do have the potential to be used as templates for novel drugs and as adjuncts to modern medicines in the combat against drug resistance.Item The biological activity and phytochemistry of selected Hermannia species(2006-10-31T11:57:46Z) Essop, Ayesha BibiTraditional medicines form a significant part of the lives of many people around the world and in South Africa almost 60 % of people consult traditional healers in addition to the modern medical services available. Plants form a significant part of traditional healing and hence, selected species of a traditionally used plant genus, Hermannia, were chosen for biological and chemical investigation to determine a scientific basis for the traditional use of these plants. A phytochemical investigation was carried out, firstly using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) and then isolation and identification of compounds from various Hermannia species. TLC analysis indicated significant similarities between the various species with only H. saccifera displaying chemical anomalies. This was further corroborated by the HPLC analysis although very conservative profiles were produced. Isolation of compounds from H. saccifera yielded a novel labdane compound, E-17, 19-diacetoxy - 15 - hydroxylabda - 7,13 - diene, as well as two flavones, 5,8- dihydroxy-6,7,4’- trimethoxyflavone and cirsimaritin which have previously been isolated. In addition, two commonly found compounds, lupeol and β- sitosterol were isolated from H. cuneifolia and H. salviifolia respectively. This is the first report on the isolation and identification of all five compounds from Hermannia species. Antimicrobial activity was assessed using two methods i.e. minimum inhibitory concentrations as well as the death kinetics assay. Minimum inhibitory concentrations were determined using four Gram-positive and two Gram-negative bacteria as well as two yeasts. All species investigated indicated antimicrobial activity with H. saccifera showing good activity against S. aureus and B. cereus. E-17, 19-diacetoxy - 15 - hydroxylabda - 7,13 - diene isolated from H. saccifera indicated activity (MIC = 23.6 μg/ml against S.aureus) although the activity was less than that of the crude extract (MIC = 19.5 μg/ml), thus, demonstrating that there are a number of compound contributing to the promising activity of the crude extract. This was further corroborated by the bioautograms developed of the H. saccifera extract. Time-kill studies on H. saccifera against S. aureus indicated that at concentrations of 0.1, 0.25 and 0.5 % bacteriostatic activity was observed while at 0.75% the extract achieved complete bactericidal activity after 240min. Free radical scavenging activity was assessed using the 2,2-diphenyl-1-picrylhydrazy (DPPH) and 2,2′-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS) assays. Ten of the twelve species indicated good activity with H. cuneifolia demonstrating the most promising activity (IC50 = 10.26 μg/ml for DPPH and 10.32 μg/ml for ABTS). Two of the isolated compound, 5,8- dihydroxy-6,7,4’- trimethoxyflavone and cirsimaritin displayed insignificant activity. The 5-lipoxygenase assay was used to assess the anti-inflammatory activity of Hermannia species. All species exhibited intermediate activity with the exception of H. cuneifolia (IC50 = 15.32 μg/ml). In addition, four isolated compounds, 5,8- dihydroxy-6,7,4’- trimethoxyflavone, cirsimaritin, lupeol and β-sitosterol showed moderate inhibition of the enzyme indicating that while these compounds do contribute to the activity of the extracts they are not individually responsible for any significant activity. Antimalarial activity was assessed using the titrated hypoxanthine incorporation assay while toxicity was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cell proliferation assay. Only three species indicated any good antimalarial activity i.e. H. saccifera, H. muricata and mostly H. trifurca (IC50 = 25.30, 28.17 and 18.80 μg/ml respectively). However, the activity of H. saccifera and H. trifurca are probably due to a general cytotoxicity as these species exhibited a low safety index. All other species appear safe for use. Several Hermannia species have indicated in vitro biological activity in a number of assays which is related to their use in traditional medicines to treat a number of disease states. Hence, a scientific basis, albeit in vitro, has been established for the use Hermannia species in traditional healing.