ABSTRACT GENETIC VARIABILITY IN HYDRASTIS CANADENSIS L. USING RAPD ANALYSIS FEBRUARY 2009 KERRY J. KELLEY, B.A. MOUNT HOLYOKE COLLEGE M.A. UNIVERSITY OF MASSACHUSETTS AMHERST Directed by: Professor Lyle Craker Hydrastis canadensis L. (goldenseal) is an endangered perennial wildflower species native to eastern North America. In this study, several populations of goldenseal, (both cultivated and wild type) were analyzed for genetic variability. The samples were collected from plant populations in North Carolina, Ohio, Pennsylvania and West Virginia and preserved using silica gel during collection. Random amplified polymorphic DNA (RAPD) analysis technique was used to generate DNA profiles from individual plants and to estimate genetic variability between groups (cultivated and wild type), among populations within groups and within populations using analysis of molecular variance (AMOVA) and a UPGMA clustering phenogram. Our results demonstrate that the bulk of genetic diversity may be within and among populations, but not between groups. This indicates the need for preservation and conservation efforts at the population level. The next step would be to study goldenseal populations more in depth for underlying causes of the genetic variability observed in this study. Further study of genetic variability with different molecular markers may be needed to clarify the level of diversity for the species at the group level. Increased knowledge of genetic variability and the identification of accessions of goldenseal would prove useful for reintroduction and cultivation strategies.

<p>We hypothesize that South African medicinal plants contain compounds that can act in synergism with synthetic antifungal compounds. Four fungicides - Sporekill&trade / , Rovral&trade / , Terminator&trade / and Teldor&trade / at doses 0.1, 0.2, 0.4 and 0.8 mL L-1 and plant species Galenia africana, Elytropappus rhinocerotis and Tulbaghia violacea were tested aloneand in different combinations for their potency (efficacy) on radial growth inhibition of Botrytis cinerea strains on potato dextrose plates. Four doses of plant extract for each of the respective plant species were used. A total of 48 combinations were tested for each strain. Mixtures of plant extracts were far more effective in controlling strains compared to the individual components alone, representing significant levels of in vitro synergistic interactions. Combinations of these components represent an attractive future prospect for the development of new management strategies for controlling B. cinerea. Since the in vitro tests of these mixtures showed inhibitory activity, the mixtures were tested for activity in assays on Granny Smith apples. In vitro tests can be used to screen mixtures to obtain information on their inhibitory activity on a pathogen, however, the environmental conditions of the fruit and the ability of the pathogen to grow into the fruit cannot be simulated in vivo. A series of two-fold doses of medicinal plant extracts were combined with fungicides to conduct decay inhibition studies. The incidence of gray mold was significantly reduced by mixtures of plant extracts and fungicides. Under conditions similar to those in commercial storage, a drench treatment with G. africana and Rovral&trade / significantly (p=0.05) inhibit gray mold on the apples and was more effective than the plant extract and fungicide alone. The treatments exerted synergistic effects and were markedly better than the components applied alone. The wound colonization assay was used for optimal decay control. In a drench, much higher volumes of the treatments are used to ensure that the components of the suspension are deposited evenly over the entire fruit surface. Drenching of fruit to apply other chemicals is an established practise in the pome (fleshy) fruit industry, and simplifies the commercial application of the mixtures, as no additional infrastructure at commercial packing houses will be required. This approach not only makes it possible to reduce fungicide concentrations while maintaining adequate decay control, but also ensures a reduction of the chemical residue on the fruit.</p>

<p>We hypothesize that South African medicinal plants contain compounds that can act in synergism with synthetic antifungal compounds. Four fungicides - Sporekill&trade / , Rovral&trade / , Terminator&trade / and Teldor&trade / at doses 0.1, 0.2, 0.4 and 0.8 mL L-1 and plant species Galenia africana, Elytropappus rhinocerotis and Tulbaghia violacea were tested aloneand in different combinations for their potency (efficacy) on radial growth inhibition of Botrytis cinerea strains on potato dextrose plates. Four doses of plant extract for each of the respective plant species were used. A total of 48 combinations were tested for each strain. Mixtures of plant extracts were far more effective in controlling strains compared to the individual components alone, representing significant levels of in vitro synergistic interactions. Combinations of these components represent an attractive future prospect for the development of new management strategies for controlling B. cinerea. Since the in vitro tests of these mixtures showed inhibitory activity, the mixtures were tested for activity in assays on Granny Smith apples. In vitro tests can be used to screen mixtures to obtain information on their inhibitory activity on a pathogen, however, the environmental conditions of the fruit and the ability of the pathogen to grow into the fruit cannot be simulated in vivo. A series of two-fold doses of medicinal plant extracts were combined with fungicides to conduct decay inhibition studies. The incidence of gray mold was significantly reduced by mixtures of plant extracts and fungicides. Under conditions similar to those in commercial storage, a drench treatment with G. africana and Rovral&trade / significantly (p=0.05) inhibit gray mold on the apples and was more effective than the plant extract and fungicide alone. The treatments exerted synergistic effects and were markedly better than the components applied alone. The wound colonization assay was used for optimal decay control. In a drench, much higher volumes of the treatments are used to ensure that the components of the suspension are deposited evenly over the entire fruit surface. Drenching of fruit to apply other chemicals is an established practise in the pome (fleshy) fruit industry, and simplifies the commercial application of the mixtures, as no additional infrastructure at commercial packing houses will be required. This approach not only makes it possible to reduce fungicide concentrations while maintaining adequate decay control, but also ensures a reduction of the chemical residue on the fruit.</p>

Philosophiae Doctor - PhD / We hypothesize that South African medicinal plants contain compounds that can act in synergism with synthetic antifungal compounds. Four fungicides - Sporekill™, Rovral™, Terminator™ and Teldor™ at doses 0.1, 0.2, 0.4 and 0.8 mL L-1 and plant species Galenia africana, Elytropappus rhinocerotis and Tulbaghia violacea were tested aloneand in different combinations for their potency (efficacy) on radial growth inhibition of Botrytis cinerea strains on potato dextrose plates. Four doses of plant extract for each of the respective plant species were used. A total of 48 combinations were tested for each strain. Mixtures of plant extracts were far more effective in controlling strains compared to the individual components alone, representing significant levels of in vitro synergistic interactions. Combinations of these components represent an attractive future prospect for the development of new management strategies for controlling B. cinerea. Since the in vitro tests of these mixtures showed inhibitory activity, the mixtures were tested for activity in assays on Granny Smith apples. In vitro tests can be used to screen mixtures to obtain information on their inhibitory activity on a pathogen, however, the environmental conditions of the fruit and the ability of the pathogen to grow into the fruit cannot be simulated in vivo. A series of two-fold doses of medicinal plant extracts were combined with fungicides to conduct decay inhibition studies. The incidence of gray mold was significantly reduced by mixtures of plant extracts and fungicides. Under conditions similar to those in commercial storage, a drench treatment with G. africana and Rovral™ significantly (p=0.05) inhibit gray mold on the apples and was more effective than the plant extract and fungicide alone. The treatments exerted synergistic effects and were markedly better than the components applied alone. The wound colonization assay was used for optimal decay control. In a drench, much higher volumes of the treatments are used to ensure that the components of the suspension are deposited evenly over the entire fruit surface. Drenching of fruit to apply other chemicals is an established practise in the pome (fleshy) fruit industry, and simplifies the commercial application of the mixtures, as no additional infrastructure at commercial packing houses will be required. This approach not only makes it possible to reduce fungicide concentrations while maintaining adequate decay control, but also ensures a reduction of the chemical residue on the fruit. / South Africa

Philosophiae Doctor - PhD / We hypothesize that South African medicinal plants contain compounds that can act in synergism with synthetic antifungal compounds. Four fungicides - Sporekill™, Rovral™, Terminator™ and Teldor™ at doses 0.1, 0.2, 0.4 and 0.8 mL L-1 and plant species Galenia africana, Elytropappus rhinocerotis and Tulbaghia violacea were tested alone and in different combinations for their potency (efficacy) on radial growth inhibition of Botrytis cinerea strains on potato dextrose plates. Four doses of plant extract for each of the respective plant species were used. A total of 48 combinations were tested for each strain. Mixtures of plant extracts were far more effective in controlling strains compared to the individual components alone, representing significant levels of in vitro synergistic interactions. Combinations of these components represent an attractive future prospect for the development of new management strategies for controlling B. cinerea. Since the in vitro tests of these mixtures showed inhibitory activity, the mixtures were tested for activity in assays on Granny Smith apples. In vitro tests can be used to screen mixtures to obtain information on their inhibitory activity on a pathogen, however, the environmental conditions of the fruit and the ability of the pathogen to grow into the fruit cannot be simulated in vivo. A series of two-fold doses of medicinal plant extracts were combined with fungicides to conduct decay inhibition studies. The incidence of gray mold was significantly reduced by mixtures of plant extracts and fungicides. Under conditions similar to those in commercial storage, a drench treatment with G. Africana and Rovral™ significantly (p=0.05) inhibit gray mold on the apples and was more effective than the plant extract and fungicide alone. The treatments exerted synergistic effects and were markedly better than the components applied alone. The wound colonization assay was used for optimal decay control. In a drench, much higher volumes of the treatments are used to ensure that the components of the suspension are deposited evenly over the entire fruit surface

The study evaluated the extent and threat of bark harvesting of plant species for medicinal purposes in the Venda region and investigated possibilities of the sustainability of these practices. Approximately 30% of the woody plant species in Venda were found to have medicinal properties in their bark, but only about 12% of these species are commonly traded in muthi shops in the region. Fifty-eight medicinal plant species are commonly harvested for medicinal properties in their bark and found in muthi shops in the region. These 58 species were scored for the possible threat of bark harvesting to the species’ survival using 20 ecologically relevant plant population traits. The most vulnerable species were Adansonia digitata, Adenia spinosa, Albizia adianthifolia, Albizia versicolor, Brackenridgea zanguebarica, Croton megalobotrys, and Warburgia salutaris. Of these species Brackenridgea zanguebarica and Warburgia salutaris are amongst the ten most traded medicinal plant species in Venda region. Elaeodendron transvaalense and Brackenridgea zanguebarica, the two species investigated in detail in this study, were amongst the most commonly traded medicinal plant species in Venda region. Analysis of size class distributions showed that both species had growing and healthy populations, exhibiting J-shaped population curves, centroids left-skewed from the midpoint of the size class distribution, and a fine-grained status. However, size-class distributions in both species revealed certain classes that needed monitoring since they were negatively affected by bark harvesting. Adult individuals of B. zanguebarica showed a high degree of bark regeneration as a response to bark removal from medicine men. The crown health status of E. transvaalense was generally good although some individuals, contributing 9% of the sample, had dead crowns. A linear relationship was noticed between areas harvested and stem circumference, which is understandable considering the large surface area of harvestable bark on bigger individuals. Matrix modeling of E. transvaalense revealed that the vegetative stage should be targeted for management action. An assessment of the adequacy of the Brackenridgea Nature Reserve, an initiative aimed at protecting Brackenridgea zanguebarica, revealed that the reserve size is not enough for conservation of a viable population. The method flagged out potential growth habitat for B. zanguebarica around the current reserve, which could be incorporated to enlarge the conservation area, which could be incorporated to enlarge the conservation area. Four different scenarios were analysed on how best to conserve the species. Assuming a 50% reduction in human-related activities, such as cultivation, harvesting and livestock grazing, it is recommended that the reserve be enlarged from its current 110 ha to 366 ha to maintain a viable population into the future. Finally, the study recommended the adoption of an integrated approach to achieve sustainability of bark harvesting in the Venda region. Only by selecting best practices from both indigenous and conventional conservation techniques will the conservation of natural resources that are of important to local communities, be successful. An action plan that will involve the formation of an association by all stakeholders interested in the sustainable utilization of natural resources must be developed. The association must be governed by a constitution with a clear mission statement and the harvesting of natural resources should be done in line with a collection policy. / Thesis (PhD)--University of Pretoria, 2012. / Plant Science / unrestricted

Thesis (MSc. (Microbiology)) -- University of Limpopo, 2017 / This study was aimed at isolating and evaluating the antibacterial, antioxidant and cytotoxic effects of active compounds from selected medicinal plant species. The biological activities and cytotoxic effects of Commelina africana, Dombeya rotundifolia, Elephantorrhiza elephantina and Schkuhria pinnata were investigated based on their traditional uses. The plants were extracted with different solvents of varying polarities. Methanol was the best extractant with the highest yield. The presence of different phytoconstituents was screened using standard methods; this revealed the presence of terpenoids, flavonoids, tannins, and saponins. Meanwhile, the phytochemicals were screened on TLC, where different colours observed indicated the presence of different compounds. The total phenolic, tannin and flavonoid content were estimated using spectrophotometric methods. D. rotundifolia had the highest amounts of phenol, tannin and flavonoid content, while S. pinnata had the least. The antibacterial and antioxidant screening employed both qualitative and quantitative assays. Although not separated, this study demonstrated the antioxidant potential of D. rotundifolia extracts developed in BEA and CEF. D. rotundifolia also demonstrated the highest free radical scavenging and reducing power antioxidant potential. These activities could be attributed to the high phenolic, tannin and flavonoid content observed. All the selected plants had antibacterial potential against E. coli and P. aeruginosa. Only S. pinnata extracts were active against E. faecalis and S. aureus. The MIC values of the plants and ampicillin ranged from 0.02 to 2.50 mg/mL and 0.02 to 0.08 mg/mL, respectively. The plants were combined to determine any interaction effects in exhibiting antibacterial activity. Among all the possible combinations, combinations with S. pinnata had improved antibacterial activity. The effects of D. rotundifolia and S. pinnata extracts on the inhibition of the production of reactive oxygen species on LPS-induced RAW 264.7 macrophages was determined using the DCFHD-A assay. Both plants demonstrated anti-inflammatory potential by inhibiting the production of ROS in a dose-dependent manner. Cell viability assays revealed that S. pinnata had cytotoxic effects on African green monkey kidney (Vero) cells with the lowest LC50 (25 µg/mL). Bioassay guided fractionation and nuclear magnetic resonance spectroscopy of dichloromethane extracts of S. pinnata on column chromatography afforded isolation and characterisation of three sesquiterpene lactones. These were identified as 6-(2, 3- xxvi dihydroangeloylory)-6α-hydroxyinuviscolide, heliangolide and eucannabiolide. All three compounds had antibacterial, anti-inflammatory and cytotoxic effects. The compounds may serve as leads for synthesis of less toxic therapeutic antibacterial and anti-inflammatory drugs in the pharmaceutical industry. / National Research Foundation (NRF)

The aim of this study was to isolate and characterize antifungal compounds from the most active medicinal plant species that could be used to address secondary infection problems in immunocompromised patients. An ethnobotanical study was conducted and 45 medicinal plant species used traditionally to treat candidiasis and related infections in HIV/AIDS patients were identified and documented. The most popular plant species used included Acacia caffra, Clerodendrum glabrum, Croton gratissimus, Elaeodendron transvaalense, Faurea saligna, Hippocratea longipetiolata, Osyris lanceolata, Richardia brasiliensis, Schkuhria pinnata, Schotia brachypetala, Spilanthes acmella, Strychnos potatorum, Vangueria infausta subsp. infausta and Withania somnifera. The plant parts used in the therapeutic preparations were roots (26.7%), bark (22.2%), and a combination of roots and bark (17.7%). Decoctions (44.4%), infusions (20%) and macerations (17.7%) were used. Most of the herbal remedies were administered orally. Chemical profiles of the plant species were established by using thin layer chromatography. Leaf extracts of these plant species were tested for antimicrobial activity against two common pathogenic fungal species in humans (Candida albicans and Cryptococcus neoformans) and four nosocomial bacteria (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa) using a two-fold serial microdilution method and bioautography. All plant species investigated had some degree of antimicrobial activity against the test microorganisms. The hexane and the acetone extracts of Clerodendrum glabrum, Hippocratea longipetiolata, Schkuhria pinnata and Withania somnifera were the most active with MIC values ranging from 0.06 to 0.08 mg/ml. The most susceptible pathogen to the test samples was C. neoformans while C. albicans was resistant to most of the plant extracts. The water extracts of Withania somnifera and Hippocratea longipetiolata (14%) had MIC < 1 mg/ml against C. albicans. C. neoformans was susceptible to nine water plant extracts (64%) with MIC < 1 mg/ml and the promising activity was observed in Hippocratea longipetiolata and Faurea saligna extracts with MIC values of 0.16 and 0.31 mg/ml respectively. The hexane extract of C. glabrum was the most active against C. albicans with an MIC value of 0.06 mg/ml and total activity of 550 ml/g. In the bioautography, most plant extracts tested had few active compounds, others had no active components at all and this may be attributed to the disruption of synergism by the thin layer chromatography. C. glabrum had eight active antifungal compounds on bioautograms and most of these components were observed in the EMW solvent system. Based on this and its wide distribution in rural areas, C. glabrum was chosen for further study. The antioxidant activity and possible immune boosting potential of the species were determined using 1, 1-diphenyl-2-picrylhydrazyl radical (DPPH), 2, 2’ azinobis-(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and ferric reducing antioxidant power (FRAP) assays. In the DPPH qualitative assay, the aqueous plant extracts had several prominent antioxidant components than the organic plant extracts. The aqueous plant extracts which had the most prominent antioxidant activity were F. saligna with 8 compounds, followed by E. transvaalense, H. longipetiolata O. lanceolata, R. brasiliensis and S.brachypetala, with five compounds each and their Rf values ranged from 0;06 to 0.94. This appears to validate the ethnomedicinal use of the plant species to some extent because decoction is the most common method used in the preparation of the remedy by the traditional healers. With regard to the organic plant extracts, only one plant extract, F. saligna had two prominent antioxidant components at Rf values 0.81 and 0.88. A third of the plant species had a high level of free radical scavenging activities in the DPPH, ABTS and FRAP assays. However, all plant extracts had lower antioxidant activity than the positive control (Trolox) used. The selected plant species were also evaluated for their in vitro toxicity against the Vero monkey kidney cell line using 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. The acetone plant extracts of O. lanceolata, S. acmella, S. pinnata and S. brachypetala had high cytotoxic activity against Vero cells with IC50 values of 13.7±0, 19.9±0.001, 21.6±0.001 and 28.34±0.001 μg/ml respectively. However, their IC50 values were higher than that of the positive control, doxorubicin (IC50 = 9.9±0 μg/ml). The rest of the acetone plant extracts (64%) had moderate cytotoxic activity (30 < IC50<100 μg/ml). The aqueous plant extracts were relatively non-toxic to the Vero cells with IC50 values ranging from 137 to > 500 µg/ml. This supports the use of aqueous extracts in the traditional medicine. However, their low selectivity index values ranging from 0.26 to 1.68 suggest that the plant extracts are probably suitable for external use only. Fractionation of the hexane extract of the leaves of C. glabrum by chromatographic techniques yielded six fractions of which fractions C and D had significant antifungal activity (average MIC value = 0.1 mg/ml) against C. albicans and C. neoformans. From these fractions, one new triterpenoid, 3-(1-oxobutyl)-11α-hydroxytaraxast-20(30)-ene-24,28-dioic acid (clerodendrumic acid) (1) was isolated along with known heptadecanoic acid (2). C. albicans was relatively insensitive to clerodendrumic acid (1) (MIC value = 125 µg/mL) and was resistant to heptadecanoic acid (2) (MIC value = 188 µg/ml). Compounds 1 and 2 were non-toxic against monkey kidney Vero cells in vitro with IC50 values of 202.6 and 108.4 µg/ml respectively. Due to its low antifungal activity, the novel compound clerodendrumic acid (1) is not a viable candidate for drug development which could be used to combat candidiasis and related fungal infections. However, due to its relative safety, it may possibly be used as a lead compound to produce new chemically modified active derivatives or could be used together with known antibiotics to mitigate their undesirable side effects. To the best of our knowledge, the isolation of a novel, clerodendrumic acid (1) and a known heptadecanoic acid (2) compounds from leaf extracts of C. glabrum is reported herein for the first time. The results obtained from this study generally substantiate the rationale behind the use of the selected plant species in the traditional medicine to treat candidiasis and related infections to some extent. This study showed the potential of studying traditional medicine in the search for effective plant extracts or new lead compounds that could be developed into drugs for combating microbial infections among the rural poor people. / Thesis (PhD)--University of Pretoria, 2013. / gm2013 / Paraclinical Sciences / Unrestricted