Thesis (Ph. D.)(FABI Plant Science))--University of Pretoria, 2009. / Includes bibliographical references.

The methanol extract of Helichrisum pedunculatum was screened for antimicrobial activity up to a concentration of 5 mg/ml using the agar dilution technique. A number of test bacterial isolates, comprising both Gram negative and Gram positive organisms were susceptible to the crude extract of the plant. The minimum inhibitory concentrations (MICs) of the extract ranged between 1 and 5 mg/ml for the susceptible organisms. The MICs of the selected antibiotics, chloramphenicol and penicillin, ranged between 2 and 4 mg/L, and 2 and 32 mg/L respectively against Bacillus cereus, Proteus vulgaris and Staphylococcus aureus OKOH1. Bactericidal activity was determined by the time kill assay. The methanol extract of the plant was not bactericidal at 1 × MIC for B. cereus, P. vulgaris and Staph. aureus OKOH1. At 2 × MIC the extract was bacteriostatic against B. cereus but bactericidal against P. vulgaris and Staph. aureus OKOH1. Combination studies were done at 1/2 × MIC, 1 × MIC and 2 × MIC of the plant extract with 1 × MIC of the antibiotics. Combinations of the plant extract and chloramphenicol resulted in mostly indifferent interactions against P. vulgaris and Staph. aureus OKOH1 but synergistic interactions at higher concentration of the plant extract for B. cereus. Penicillin combinations gave synergistic interactions at lower concentrations of the plant for P.vulgaris and Staph. aureus OKOH1 but was mostly indifferent for B. cereus.

The plant kingdom contributes significantly to the natural products that are used for the treatment of a large number of ailments and disease across the globe. Included in these species is the Helichrysum genus (Asteraceae), which comprises of more then 600 species across Africa of which 244 species are found in South Africa. Helichrysum species are used in many cases for the treatment of coughs, colds, fever, infection, headaches, menstrual pain and are also very popular for wound dressing due to their potential antibacterial properties. The most common Helichrysum species used in traditional medicine and for several medicinal purposes are H. cymosum, H. odoratissimum, H. petiolare and H. nudifolium. Previously published research has shown that several of the Helichrysum species do have antimicrobial activity with the most relevant to this study being the discovery of antiviral activity of H. aureonitens against herpes simplex virus type 1 (HSV-1) as well as the reports of anti-HIV (human immunodeficiency virus) activity of several Helichrysum species. With this knowledge, a more in-depth study was initiated to identify the possible active constituents in South African Helichrysum species against HIV. Due to the need to speed up drug discovery especially against epidemic diseases like HIV, this study investigated a new tool (nuclear magnetic resonance (NMR) – based metabolomics) to speed up drug discovery form natural products especially when anti-viral constituents are investigated. odoratissimum, H. petiolare and H. nudifolium. Previously published research has shown that several of the Helichrysum species do have antimicrobial activity with the most relevant to this study being the discovery of antiviral activity of H. aureonitens against herpes simplex virus type 1 (HSV-1) as well as the reports of anti-HIV (human immunodeficiency virus) activity of several Helichrysum species. With this knowledge, a more in-depth study was initiated to identify the possible active constituents in South African Helichrysum species against HIV. Due to the need to speed up drug discovery especially against epidemic diseases like HIV, this study investigated a new tool (nuclear magnetic resonance (NMR) – based metabolomics) to speed up drug discovery form natural products especially when anti-viral constituents are investigated. In this study very promising anti-HIV results were obtained from several aqueous extracts (1:1 methanol/water) using a full virus model i.e. Helichrysum populifolium (IC50 12 μg/ml), H. appendiculatum (IC50 17 μg/ml), H. cymosum ssp. clavum (IC50 19 μg/ml), H. oxyphyllum (IC50 19 μg/ml) and H. cymosum ssp. cymosum (IC50 21 μg/ml). With the use of NMR-based metabolomics and multivariate data analysis (MVA) the specific characteristic that differentiated the active extracts from the non-active extracts was identified by making use of Orthogonal Projections to Latent Structures – Discriminant Analysis (OPLS-DA). This characteristic was then used as a “blue print” or “fingerprint” to guide the process of fractionation and purification. H. populifolium showed the highest anti-HIV activity and thus was selected as the candidate extract for further analysis. After a very quick and simple chromatographic fractionation process, seven fractions were compared against the activity profile by making use of their NMR profiles, which then visually indicated which of the fractions had the highest similarity. Fraction 6 had the most similar “fingerprint”. The compounds of this active fraction were then identified with the use of liquid chromatography – ion trap – time of flight (LC-IT-TOF) for quick identification. The analysis revealed the presence of five chlorogenic type compounds, 3,4-dicaffeoyl quinic acid (DCQA), 3,5-DCQA, 4,5-DCQA, 1,3,5- tricaffeoyl quinic acid (TCQA) and 5-malonyl-1,3,4-TCQA of which several are well known to have anti-HIV activity ranging from 0.85μM to 12μM. We were thus able to show with this study the possibility of using NMR-based metabolomics guided fractionation to guide the process of fractionation and identification from an active characteristic profile to the active constituents within the active H. populifolium extract. / Thesis (PhD)--University of Pretoria, 2013. / gm2014 / Plant Science / unrestricted

Thesis (Ph. D.)(Botany)--University of Pretoria, 2001. / Acrobat Adobe Reder needed to open files.

For centuries people have used plants as medicine or food additives with varying success to cure and prevent diseases. Written records about medicinal plants date back at least 5 000 years to the Sumerians. According to World Health Organization (WHO) around 80 % of the population in developing countries is dependent on herbal medicine for basic healthcare needs. Even at the dawn of the twenty-first century, 11 % of the 252 drugs considered as basic and essential by WHO were exclusively of flowering plant origin. The genus Helichrysum, belongs to the family Asteraceae and is represented by approximately 600 species in Africa, of which 244 species are indigenous in South Africa. In Helichrysum aureonitens, galangin is one of the flavonol compounds with good medicinal properties. H. aureonitens was targeted to be enhanced via cell suspension culture to potentially produce valuable natural products. In ethanol extracts of cell suspension cultures, galangin was not detected even though it was present in the leaves of the intact plants. Some other compounds were induced in higher amounts in the cells of H. aureonitens suspension cultures when compared to that produced in the intact plants. To find out the reasons for the absence of galangin in the cells of H. aureonitens suspension cultures, some of the intermediates of the 4’-OH biosynthetic pathway for production of flavonols were analyzed by GC-MS, including cinnamic acid, p-coumaric acid, naringenin and kaempferol. None of these were detected in the H. aureonitens cell suspension cultures. The major compound from H. aureonitens cell suspension cultures was isolated and identified as a new chlorophenol compound named 4-chloro-2-(hepta-1,3,5-triyne-1-yl)-phenol (a triyne). This triyne has previously been proposed as being an intermediate in the acetylene biosynthetic pathway in Helichrysum spp., however only the methyl ether form had previously been isolated from the roots of H. coriaceum. The triyne isolated from the H. aureonitens cell suspension cultures in the present study was detected in intact plant extracts, but at very low concentrations. Results of the anti-tuberculosis assay of the cell suspension culture extracts and the triyne showed that the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the ethanol extract of cell suspension cultures against Mycobacterium tuberculosis H37Rv were found to be 1.0 mg/ml and 2.0 mg/ml respectively and the triyne was not active at 200 µg/ml. The ethanol extract of the cell suspension cultures and the triyne were also evaluated for their cytotoxicity against monkey kidney Vero cells and human prostate epithelial carcinoma (DU145) cell lines. The inhibitory concentrations (IC50) of the crude extract and the triyne was found to be 12.11 and 1.51 µg/ml against the Vero cells respectively. The crude extract and the triyne showed similar activity in the prostate cancer cell lines by exhibiting IC50 values of 3.52 and 2.14 µg/ml respectively. The triyne therefore warrants further investigation for its potential as an anticancer drug. Flavonoids represent the major phenolic compounds which are responsible for the medicinal properties in the Helichrysum genus. Some of flavonols, including kaempferol, quercetin, and galangin are also present in H. aureonitens. In this study both galangin and kaempferol (containing a 4’-OH group) were detected in leaf samples of H. aureonitens. But GC-MS analysis of the leaf samples of H. aureonitens did not show the existence of biosynthetic intermediates such as p-coumaric acid and naringenin (compounds having a 4’-OH) while cinnamic acid and some other compounds with no OH at the 4’ position on the B ring, were detected. The chemical structure analysis of the isolated compounds showed that they are pinocembrin chalcone, pinocembrin, pinobanksin and galangin, all containing no OH group at the 4’ position. This indicates that some part of the 4’-OH biosynthetic pathway for 4’-OH flavonoids is not functional in H. aureonitens. Since the only (yet identified) enzyme responsible for hydroxylation at the 4’ position on the B ring is cinnamate 4-hydroxylase (C4H), it can be postulated that C4H might be able to hydroxylate other substrates in H. aureonitens plants. One copy of C4H was isolated and cloned from H. aureonitens. It has 1518-base pairs (including stop codon, TAA) and an open reading frame encoding a 506-amino-acid polypeptide. It showed the highest homologies to Echinacea angustifolia (Asteraceae) C4H with 83.6 % identity on the nucleotide level but 93 % identity on the amino acid level. The genomic DNA sequence of the isolated C4H from H. aureonitens indicates the presence of three introns with a longer size compared to the Arabidopsis thaliana C4H gene structure. The presence of the first intron has not been reported before in the C4H gene from other plants and it is therefore a new finding from the isolated C4H in H. aureonitens. To check the putative isolated C4H, the full length cDNA of C4H was isolated from H. aureonitens and for the first time integrated in a secreted expression vector, pPICZáC, and transformed into Pichia pastoris. After the 48 hrs induction protein was collected, precipitated by ammonium sulphate and finally column purified. The results of SDS-PAGE electrophoresis and Western blot showed the expression of a protein with a size of 50-60 kDa. The calculated mass of C4H with regarding to a polyhistidine tag is about 60.5 kDa. The secreted expression was found as an effective system for the production of a soluble C4H protein with easy purification. / Thesis (PhD)--University of Pretoria, 2009. / Plant Science / unrestricted

There are 500 Helichrysum (Asteraceae) species world wide of which 245 occur in South Africa.The South African species display great morphological diversity and are, therefore classified into 30 groups (Hilliard, 1983). Helichrysum species have been reported for their antimicrobial activities (Rios et al., 1988; Tomas-Barberan et al., 1990; Tomas-Lorente et al., 1989; Mathekga, 1998, Mathekga et al., 2000). Not much information on the bioactivity of compounds isolated from these species is available. In vitro antimicrobial screening methods provide the required preliminary observations to select among crude plant extracts those with potentially useful properties for further chemical and pharmaceutical investigations. In this study we investigated the antimicrobial activities of crude acetone extracts (shaken and homogenized) of twenty-eight Helichrysum species on ten bacteria species and six fungal species. A new phloroglucinol with significant antimicrobial properties was isolated by bioactivity guided fractionation from Helichrysum caespititium. The structure elucidation, conformation and stereochemistry of the new phloroglucinol, 2-methyl-4-[2',4',6'-trihydroxy-3'-(2-methylpropanoyl) phenyl] but-2-enyl acetate (caespitate), was established by high field NMR spectroscopic, crystallographic and MS data. The compound inhibited growth of Bacillus cereus, B. pumilus and Micrococcus kristinae at the very low concentration of 0.5 µg /ml and Staphylococcus aureus at 5.0 µg/ml. Six fungi tested were similarly inhibited at low MICs: Aspergillus flavus and A. niger (1.0 µg /ml), Cladosporium cladosporioides (5 µg/ml), C. cucumerium and C. sphaerospermum (0.5 µg /ml) and Phytophthora capsici at 1.0 µg/ml. The cytotoxicity of most currently used drugs has become a serious problem and efforts are being directed to obtaining new drugs with different structural features. One option favoured is the search for new plant derived non-toxic drugs, as was investigated in this study. Caespitate proved to be non-toxic at biologically active concentrations. Development of resistance to synthetic chemotherapeutic agents is known to occur in modern medicine; for example, resistance to some antibiotics of certain strains of microorganisms. A synergistic antibacterial bioassay demonstrated that the combination of caespitate and caespitin enhanced activity from a concentration range of 5 µg /ml to 0.5 µg /ml down to 0.1 µg /ml to 0.05 µg /ml on Gram-positive bacteria. The synergistic effect was in addition displayed against Gram-negative bacteria. The study of the morphology and ultrastructure of the epicuticular trichomes revealed that trichomes in H. caespititium originate from papillate cell outgrowths which elongate, develop and later polarise into apical, stem and basal parts and that repeated secretions of compounds probably occur from the young three-celled stage, enable us to characterise and relate our observations to their possible functional role in the production of the antimicrobial and other compounds on the leaf surface. South African Helichrysum species are a potentially good source of antimicrobial agents worthy of further investigation as efficient therapeutic compounds and in assisting the primary health care in this part of the world. / Dissertation (PhD (Plant Physiology))--University of Pretoria, 2004. / Plant Science / unrestricted

Please read the abstract in the section 00front of this document / Dissertation (MSc (Plant Physiology))--University of Pretoria, 2005. / Plant Science / unrestricted

From the Helichrysum genus 600 species occur in Africa of which 244 species are found in South Africa. The most commonly used Helichrysum species for medicinal purposes are H. cymosum, H. odoratissimum, H. petiolare and H. nudifolium. The medicinal uses include the treatment of coughs, colds, fever, infection, headaches, menstrual pain and are very popular for wound dressing. Previous published research has shown that H. aureonitens has antiviral properties against Herpes simplex virus type 1 (HSV-1). In this study, further investigation into the Helichrysum species was undertaken, to establish the active constituents responsible for anti-HSV activity using a metabolomics approach. The cytotoxicity of 12 Helichrysum species was investigated and ranged from <3.125 μg/ml to 277.8 μg/ml on the vero cell line. The 12 Helichrysum species also showed various levels of antiviral activity against HSV, with both the water-methanol and chloroform extracts of H. adenocarpum subsp. adenocarpum being the most active extract at 25 μg/ml. In this study the activity of Helichrysum species against HIV-1 RT was also investigated. Helichrysum populifolium was the most active extract, inhibiting the HIV-1 RT enzyme by 63.78 % at 200 μg/ml. The bioactivity data and the spectral nuclear magnetic resonance (NMR) data of al the Helichrysum species from this study was analysed using the SIMCA-P software to discriminate between the different species on the basis of their bioactivity and chemical composition. The samples did not group well on Principal Component Analysis (PCA) but did separate well using the Orthogonal Projection to Latent Structure – Discriminate Analysis (OPLS-DA) on the basis of their activity and NMR spectra data. From the OPLS scoring plots analysis, contribution plots were created which indicated regions responsible for the difference between the species, with these regions being investigated to identify the bioactive constituents. It was thus possible to use metabolomics to discriminate between samples on the basis of their activity and show that it could probably be used in future as a tool to identify active ingredients in medicinal plants and accelerate drug discovery. Copyright / Dissertation (MSc)--University of Pretoria, 2009. / Plant Science / unrestricted

Thesis submitted in fulfilment of the requirements for the degree Master of Technology: Horticulture in the Faculty of Applied Sciences at the Cape Peninsula University of Technology / The high demand for medicinal plants has favoured over-exploitation of wild plants. The search for alternative and sustainable methods of medicinal plant cultivation is imperative and desirable. Biotechnological approaches particularly hydroponic technology has the potential for large scale plant cultivation and production of secondary metabolites. The current study aims at optimizing the production of antimicrobial secondary metabolites by an indigenous South African medicinal plant species (Helichrysum cymosum) through hydroponics N and K fertilization. In Chapter 1, the conceptual framework and justifications of the study are presented. In Chapter 2 the research objective was to discern the optimal potassium (K) supplement level for H. cymosum by evaluating the effects of different hydroponic K levels on growth, K-leaf content, and anti-Fusarium oxysporum f.sp.glycines (Ascomycota: Hypocreales) and total activities. Six weeks old seedlings of H. cymosum were treated with varied concentrations of K in the form of potassium chloride, potassium nitrate and monopotassium phosphate (58.75, 117.5, 235 and 470 ppm). These concentrations were based on a modification of Hoagland’s hydroponic nutrient formula. Plants were maintained under greenhouse conditions and growth parameters (plant height and number of leaves) were recorded weekly. At 8 weeks post treatment, plants were harvested and fresh weights were recorded and tissue nutrient content analysed. Sub-samples of the aerial parts of plants grown in the different treatments were air dried, extracted with acetone and tested against F. oxysporum. Plants exposed to 235 ppm K showed a marked increase in leaf number, plant height and fresh weight. Overall there was no significant difference (P > 0.05) among the treatments with respect to tissue nutrient content; K ranged from 3.56 ± 0.198 to 4.67 ± 0.29 %. The acetone extraction yield increased with increasing K fertilization: 58.75 ppm (16.67 ± 2.35 mg), 117.5 ppm (22.5 ± 4.79 mg), 235 ppm (210 ± 38.5 mg) but dropped to 40 ± 4.08 mg at 470 ppm K. Results from the anti-F. oxysporum bioassay showed that 58.75 and 235 ppm K treatments produced the most bioactive acetone extracts; MIC values of 0.49 and 0.645 mg/l, respectively. Acetone extracts obtained from plants exposed to 235 ppm K yielded the highest total activity, comparatively (P < 0.05). In conclusion, the optimum nutrient K level for growing H. cymosum hydroponically was 235 ppm. Chapter 3 focused on another important macro nutrient N and the objective was to determine the optimum nutrient requirements for growing the medicinal plant, Helichrysum cymosum (L.) (Asteraceae), hydroponically. Experiments were conducted to assess the effects of varied nitrogen (N) concentrations supplied as nitrate and ammonium on growth, tissue nutrient content, antimicrobial and total activities of acetone extracts of aerial parts. Treatments were based on a modified Hoagland’s nutrient formula. Six week old rooted cuttings were treated with 52.5 ppm, 105 ppm, 210 ppm and 420 ppm of N. Leaf number and stem height (cm) were recorded at weekly intervals and leaf analysis conducted. The effects of N treatments on plant growth parameters varied significantly among treatments; 52.5 ppm of N yielded the tallest plants (height) [19.4 ± 0.7 cm], while 105 ppm N yielded the maximum leaf number (68.1 ± 6.2) as well as maximum fresh weight of aerial parts was obtained with 105 ppm (15.12 ± 1.68 g). Nitrogen content of plant tissue ranged between 0.53 ± 0.03 and 4.74 ± 0.29% (d, f, 3, 12; f=14; P ≤ 0.002) depending on treatments. Powdered aerial parts (5 g) of H. cymosum obtained from the different N treatments were extracted with 100 ml of acetone. N treatment significantly affected the yield of crude extracts, which ranged from 87.5 ± 15.5 (52.5 ppm) to 230 ± 23.5 mg (105 ppm). Acetone extracts of plants that were exposed to varied N treatments were screened for anti-Fusarium oxysporum activity using minimum inhibitory concentration (MIC) method. The MIC value (0.073 ± 0.014 mg/ml) obtained with acetone extracts of plants exposed to 52.5 ppm N was significantly lower compared to the MICs of the other N treatments (105 [0.47 ± 0 and 0.705 ± 0.135 mg/ml], 210 [0.234 and 0.47 mg/ml] and 420 ppm [0.29 ± 0.101 mg/ml]) at 24 and 48 hours respectively. However, the total activities of extracts obtained among the four N treatments, which ranged from 0.062 ± 0.02 to 0.26 ± 0.06 ml/g was not statistically different at 24 or 48 hours (P > 0.05). LC-MS analysis of acetone extracts of H. cymosum plants obtained from the four treatments hinted that known anti-microbial agents such as apigenin, quercetin, kaempferol, helihumulone and quinic acids were present in the extracts and the quantity of helihumulone increased with increased nutrient N level. These results suggest that H. cymosum may be cultivated hydroponically and that the antimicrobial activity and/or the phytochemical profile of the crude acetone extracts is affected by nutrient nitrogen levels. Hydroponic cultivation of plants may be able to alleviate to an extent the pressure on wild medicinal plants.

Helichrysum longifolium and H. pedunculatum belong to the Astereceae family and are used extensively in folkloric medicine in South Africa to manage stress-related ailments and as dressings for wounds normally encountered in circumcision rites, bruises, cuts and sores. The in vitro antibacterial time-kill studies, the synergistic potentials, the phytochemical screenings and antioxidant potentials as well as the isolation of the bioactive compounds from the extracts of these two plants were carried out in this study. The in vitro antibacterial activities and time kill regimes of crude extracts of H. pedunculatum was assessed. The extracts was active against both Gram positive and Gram negative bacteria tested at a concentration of 10 mg/ml. Minimum Inhibitory Concentration (MIC) values for all the susceptible bacteria ranged between 0.1 – 35 mg/ml. The average log reduction in viable cell count in time kill assay ranged between 0.17 Log10 to 6.37 Log10 cfu/ml after 6 h of interaction, and between 0.14 Log10 and 6.99 Log10 cfu/ml after 12 h interaction in 1 × MIC and 2 × MIC of the extract. The effect of the aqueous extract was only bacteriostatic on both reference and environmental strains and the clinical isolates were outrightly resistant to aqueous extract. This is worrisome and this could be one reason why, there is an incidence of high death rate resulting from circumcision wounds infection even after treating such wounds with H. pedunculatum leaf. In vitro antibacterial time kill studies of extracts of H. longifolium was assessed. All test bacteria were susceptible to the methanol extract, while none was susceptible to the aqueous extract. Two of the test bacteria were susceptible to the ethyl acetate extract, while ten and seven were susceptible to the acetone and chloroform extracts respectively at the test concentration of 5 mg/ml. The minimum inhibitory concentrations (MICs) ranged between 0.1 and 5.0 mg/ml, while minimum bactericidal concentrations (MBCs) ranged between 1.0 and >5 mg/ml for all the extracts. Average log reductions in viable cell counts for all the extracts ranged between 0.1 Log10 and 7.5 Log10 cfu/ml after 12 h interaction at 1 × MIC and 2 × MIC. Most of the extracts were rapidly bactericidal at 2 × MIC achieving a complete elimination of most of the test organisms within 12 h exposure time. The effect of combinations of the crude extracts of H. pedunculatum leaves and eight antibiotics was investigated by means of checkerboard and time-kill methods. In the checkerboard method, synergies of between 45.83-56.81 percent were observed and this is independent of Gram reaction, with combinations in the aqueous extract yielding largely antagonistic interactions (18.75 percent). The time kill assay also detected synergy that is independent of Gram reaction with a ≥ 3Log10 potentiation of the bactericidal activity of the test antibiotics. We conclude that the crude leaf extracts of H. pedunculatum could be potential source of broad spectrum antibiotics resistance modulating compounds. The interactions between crude extracts of H. longifolium in combination with six first-line antibiotics using both the time-kill and the checkerboard methods were carried out. The time-kill method revealed the highest bactericidal activity exemplified by a 6.7 Log10 reduction in cell density against Salmonella sp. when the extract and Penicillin G are combined at ½ × MIC. Synergistic response constituted about 65 percent, while indifference and antagonism constituted about 28.33 percent and 6.67 percent in the time kill assay, respectively. The checkerboard method also revealed that the extracts improved bactericidal effects of the antibiotics. About 61.67 percent of all the interactions were synergistic, while indifference interactions constituted about 26.67 percent and antagonistic interactions was observed in approximately 11.66 percent. The in vitro antioxidant property and phytochemical constituents of the aqueous crude leaf extracts of H. longifolium and H. pedunculatum was investigated. The scavenging activity on superoxide anions, DPPH, H2O2, NO and ABTS; and the reducing power were determined, as well as the flavonoid, proanthocyanidin and phenolic contents of the extracts. The extracts exhibited scavenging activity in all radicals tested due to the presence of relatively high total phenol and flavonoids contents in the extracts. Our findings suggest that H. longifolium and H. pedunculatum are endowed with antioxidant phytochemicals and could serve as a base for future drugs. Bioactivity-guided fractionation of the leaves of H. longifolium and H. pedunculatum yielded two known compounds. From the n-hexane fraction of H. longifolium a compound was isolated (Stigmasterol) and from the ethyl acetate fraction of H. pedunculatum another compound (β-sitosterol) was isolated. The compounds were isolated and identified using various techniques. The antimicrobial, anti-inflammatory, antioxidant, analgesic and anti-pyretic activities of these compounds have been reported in literatures. In general, the experiments and tests conducted in this study appear to have justified the folkloric medicinal uses of H. longifolium and H. pedunculatum for the treatment of stress related ailments and wound infections and make a substantial contribution to the knowledge base of the use of herbal medicine for the treatment of the microbial infections.