Browsing by Author "Stirk, Wendy Ann."

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Anti-bacterial and anti-inflammatory activity of medicinal plants used traditionally in Lesotho.
(2003) Shale, Thato Lucy.; Van Staden, Johannes.; Stirk, Wendy Ann.
A significant potion of the population in Lesotho relies on traditional medicine to meet its health care requirements. Traditional healers and herbalists were interviewed from Qacha's Nek (Highlands) and Mohale's Hoek (Lowlands) districts in Lesotho on plants used by the Basotho in traditional remedies. Fifteen plants were reported to be used for bacterial infections while thirteen plants were used for diseases associated with inflammation . Plant roots were most often used to make water extracts. Mainly high altitude plants are used with lowland healers obtaining most of their plant material from the highlands, either by collecting them or buying them from highland gatherers. Leaves and roots of plants used to treat bacterial infections were extracted with hexane, methanol and water and the respective extracts screened at 100 mg ml¯¹ for anti-bacterial activity using the disc diffusion bioassay. Seven species displayed very high anti-bacterial activity against both Gram-positive and Gram-negative bacteria. A number of plant extracts had medium inhibitory activity, mostly against Gram-positive bacteria. This activity was mainly found in the root extracts. Six of the thirteen plants screened for anti-inflammatory activity using the cyclooxygenase-1 (COX-1) bioassay had activity above 90%. Hexane and methanol extracts were the most active while water extracts usually had lower activity. Malva parviflora, Eriocephalus punctulatus and Asparagus microraphis exhibited high anti-inflammatory activity from hexane, methanol and water extracts made from leaf and root material. High anti-bacterial activity was also recorded from M. parviflora and E. punctulatus hexane, methanol and water extracts. An investigation on seasonal variation and plant part substitution in medicinal activities for these plants was carried out. Extracts of M. parviflora collected between June 1999 and July 2001 showed variation in anti-bacterial activity. Extracts made from leaves and roots inhibited the growth of both Gram-positive and Gram-negative bacteria. More bacterial strains were inhibited by extracts made from roots collected in cooler months. However, a trend in seasonal activity was not evident for either the roots or leaves because there was no detection of activity in some of the extracts made within the same months or seasons of the adjacent years. Variation in anti-inflammatory was detected for M. parviflora extracts. E. punctulatus leaf extracts did not exhibit any seasonal variation in anti-bacterial activity. Anti-inflammatory activity of E. punctulatus showed seasonal variation with the highest activity noted when material was collected during the cooler months and a decline in activity when collections were made during the warmer months. Hexane, methanol and water extracts made from leaves and roots of A. microraphis did not show any seasonal variation in anti-inflammatory activity. Thus, M. parviflora and E. punctulatus should be collected during the cooler months while A. microraphis can be collected throughout the year. Traditional healers, herbalists and vendors need to be encouraged to use aerial parts in substitution of ground parts which are reported to be highly utilized. Effect of storage on anti-bacterial and anti-inflammatory activities of M. parviflora, E. punctulatus and A. microraphis were monitored. Dried, ground leaf and root material of the three plants was stored in a cold room, at room temperature and in the Botanical Garden where the material was exposed to high and large changes in temperature. Dried hexane and methanol extracts made from leaves and roots of these plants were stored in a cold room and at room temperature. Initially, storage of the plant material under the three storage conditions caused an increase in antibacterial activity of the hexane, methanol and water extracts made from leaf and root material of M. parviflora and E. punctulatus. Storage for a longer period resulted in a decrease in inhibitory activity. TLC fingerprints developed from hexane and methanol extracts made from M. parviflora and E. punctulatus stored in a cold room and at room temperature showed a consistent number and colour of spots during the initial storage period. Prolonged storage resulted in a decline in the number and colour of detected spots. The stored hexane and methanol extracts made from leaves and roots showed a similar trend of increases and decreases in anti-bacterial activity as well as changes in spots with the storage of the extracts. Testing of the effect on anti-inflammatory activity of hexane, methanol and water extracts made from leaves and roots of M. parviflora, E. punctulatus and A. microraphis showed no change in inhibitory activity of hexane extracts obtained from the material and the extracts stored at the three storage conditions. Methanol and water extracts made from leaves exhibited an increase in activity with prolonged storage. Generally, the stability of the inhibitory activity was longer for the stored dried material than the plant extracts. Isolation of biological active compounds from M. parviflora was not successful due to loss in anti-bacterial activity as a result of collection of plant material from a different locality. Anti-inflammatory compounds could not be isolated due to insufficient amount and the synergistic effect of the active compounds . The purified compounds exhibited loss of activity following HPLC purification which then re-appeared upon recombining the fractions. A number of compounds were detected from essential oils of E. punctulatus using GC. Fractions containing these compounds gave positive anti-bacterial activity in the disc-diffusion , bioautographic and MIC bioassays as well as high anti-inflammatory activity with COX-1 and COX-2 anti-inflammatory bioassays. No anti-inflammatory compounds were isolated from A. microraphis.
Aspects of structure, growth and morphogenesis in a new filamentous red alga (Ceramiaceae, Rhodophyta)
(1993) Stirk, Wendy Ann.; Van Staden, Johannes.
Pteroceramium, a descriptive name given to an undescribed winged species closely related to Ceramium, has uniaxial filamentous thallus construction with pseudodichotomous branching. Alternate branches become dominant. This pattern of growth is referred to as cellulosympodial growth. All growth is from an apical cell which cuts off subapical cells. The subapical cells develop into axial cells. Each axial cell cuts off six pericentral cells in a ring around its apical pole. The pericentral cells divide further to form the cortical band. Pc1 always forms on the outer face of the thallus as determined by the preceding pseudodichotomy and gives rise to the larger outer wing which is a lateral expansion of the cortical band. The smaller inner wing forms from Pc6 on the inner face. The other pericentral cells give rise apically to uniseriate spines. The pericentral cells also give rise to rhizoids and adventitious lateral branches. Each axial cell has a large central vacuole with a few peripheral chloroplasts, mitochondria and floridean starch granules. The smaller wing cells have a much denser cytoplasm with fewer small vacuoles, many chloroplasts which are more closely packed together and more floridean starch granules than axial cells. Chloroplasts have a typical Rhodophyta ultrastructure with single, evenly spaced thylakoids with phycobilisomes. Pit connections have a plug core but no plug cap. Pteroceramium has a typical Polysiphonia-type triphasic life history. The carposporophyte is naked and tetraspores are produced in a characteristic decussate cruciate arrangement. The effect of a number of physical and chemical factors on growth and morphogenesis was investigated. Pteroceramium grew best at irradiance levels between 79 μmol m⁻² S¯¹ and 129 μmol m⁻² S¯¹ with growth being limited at 30 μmol m⁻² S-I. The largest axial cells and wings were obtained from the material grown at 79 μmol m⁻² S¯¹ and the smallest measurements for material grown at 129 μmol m⁻² S¯¹. Monochromatic light fields of red, green and blue caused reduced growth rates compared to the control replicates grown in a white light from both incandescent and fluorescent lights. Light quality had no effect on morphogenesis. The critical daylength for maximum rates of cell elongation was 10 hours or longer, although 16 hours light caused a decrease in final axial cell volume. Optimum temperatures for growth of Pteroceramium were between 20°C and 25°C with growth decreasing at 15°C and 30°C. Axial cell volume was reduced and wing size was stunted at these two extreme temperatures tested. Scouring by sand caused axial cells to decrease in volume although the wings were unaffected. Smothering by sand did not prevent growth although axial cells and wings were greatly decreased in size, with the wings consisting of only one or two other cells. Tumbling to disrupt gravity did not affect the angle of each pseudodichotomy. Decreased levels of nitrogen and phosphorus limited growth but had little effect on axial cell volume and wing development. Pteroceramium was stenohaline with maximum growth at 34°/[00] and reduced growth at 300/[00] and 40°/[00]. Pteroceramium grew best at pH 7.5 and pH 8.5 with decreased growth at pH 6.5 and pH 5.5. The various pHs tested had little effect on morphogenesis. The best photosynthetic responses were obtained from material preconditioned at 80 μmol m⁻² S¯¹ compared with that at 30 μmol m⁻² S¯¹ and 150 μmol m⁻² S¯¹. There was a decrease in pigment content with increasing irradiance at which the alga was grown. Phycoerythrin was the dominant pigment. Exposure to a high irradiance (3000 μmol m⁻² S¯¹) for 30 minutes or longer inhibited photosynthesis. Plants did not fully recover even 24 hours later, indicating that this damage was permanent. Pteroceramium was able to acclimatize slowly over a week to temperature changes within the range of 15°C to 25°C. Rapid increases of 5°C within this temperature range increased photosynthetic performance and a rapid drop of 5°C decreased photosynthetic performance. However, a 10°C increase or drop reduced Pteroceramium's photosynthetic performance. Photosynthetic rates were decreased in alkaline conditions and increased in acidic conditions. Pteroceramium has well defined developmental patterns with basal band growth of axial cells and tip growth in the rhizoids. The pericentral cells are formed in a set sequence similar to Ceramium species with Pcl forming on the outer face, Pc2 and Pc3 forming on the lower and upper surface nearest to Pel respectively, Pc4 and PcS forming on the lower and upper surface respectively farthest from Pel, and Pc6 forming on the inner face. This sequence is unaffected by the direction of illumination or gravity. Exogenous application of plant hormones (IAA, GA3 and kinetin) in the concentration range of 10[-9] M to 10[-5] M had no effect on growth and morphogenesis in Pteroceramium. Application of polyamines and their precursors caused a decrease in growth and a reduction in cell size at concentrations higher than 10[-4] M. Polyamine inhibitors caused a reduction in growth and cell size at concentrations higher than 10[-5] M. Arginine increased growth at concentrations 10[-5] M and 10[-6] M. High power liquid chromatography (HPLC) separation of Pteroceramium extracts indicated that spermidine was present in Pteroceramium at approximately 38 μg spermidine g¯¹ fresh weight. The apical tip exerts an apical dominance effect on the subordinate branches, suppressing their elongation. Removal of the dominant apical tip increased adventitious branch formation. This effect was not reversed by application of exogenous IAA at concentrations of 10[-9] M to 10[-4] M.
Cadmium induces hypodermal periderm formation in the roots of the monocotyledonous medicinal plant Merwilla plumbea.
(Annals of Botany Company., 2010) Lux, Alexander.; Vaculık, Marek.; Martinka, Michal.; Liskova, Desana.; Kulkarni, Manoj G.; Stirk, Wendy Ann.; Van Staden, Johannes.
Background and Aims. Merwilla plumbea is an important African medicinal plant. As the plants grow in soils contaminated with metals from mining activities, the danger of human intoxication exists. An experiment with plants exposed to cadmium (Cd) was performed to investigate the response of M. plumbea to this heavy metal, its uptake and translocation to plant organs and reaction of root tissues. Methods. Plants grown from seeds were cultivated in controlled conditions. Hydroponic cultivation is not suitable for this species as roots do not tolerate aquatic conditions, and additional stress by Cd treatment results in total root growth inhibition and death. After cultivation in perlite the plants exposed to 1 and 5 mg Cd L-1 in half-strength Hoagland’s solution were compared with control plants. Growth parameters were evaluated, Cd content was determined by inductively coupled plasma mass spectroscopy (ICP-MS) and root structure was investigated using various staining procedures, including the fluorescent stain Fluorol yellow 088 to detect suberin deposition in cell walls. Key Results. The plants exposed to Cd were significantly reduced in growth. Most of the Cd taken up by plants after 4 weeks cultivation was retained in roots, and only a small amount was translocated to bulbs and leaves. In reaction to higher Cd concentrations, roots developed a hypodermal periderm close to the root tip. Cells produced by cork cambium impregnate their cell walls by suberin. Conclusions. It is suggested that the hypodermal periderm is developed in young root parts in reaction to Cd toxicity to protect the root from radial uptake of Cd ions. Secondary meristems are usually not present in monocotyledonous species. Another interpretation explaining formation of protective suberized layers as a result of periclinal divisions of the hypodermis is discussed. This process may represent an as yet unknown defence reaction of roots when exposed to elemental stress.
Characterization and control of micropropagation problems in aloe, devil's claw and banana.
(2008) Bairu, Michael Wolday.; Van Staden, Johannes.; Stirk, Wendy Ann.
The development of the science of micropropagation from the very initial concept of totipotency to the modern day advancement and sophistication has been affected by a wide range of problems such as hyperhydricity, shoot-tip necrosis and somaclonal variation. These problems are largely the result of the obvious fact of trying to grow plants in an environment that is different from the one plants are used to naturally. The extent of these problems ranges from minor technical inconvenience to significant economic loss. Characterization and control of micropropagation problems has been one of the priorities of plant tissue culture research due to the enormous contribution of this discipline for plant production, improvement and conservation. The prevalence and severity of these tissue culture problems varies widely among plant species. The rationale of this research project was therefore, to identify plant species most affected by the problems studied, characterize the problem and find mechanism(s) to control or minimize the damage caused by the problem. The literatures reviewed provide sufficient background information for the experimental chapters. Due to the different nature of the problems and variation in the plant species they affect, the model plant, the methodologies used and parameters analysed were also different. The findings of these investigations, in their own different way, addressed certain problems that individually and collectively pose difficulties to the micropropagation industry. The difference in the content of the experimental chapters is therefore the result of the broader objective of the research project to tackle such difficulties. The success and failure of tissue culture system greatly depends on the choice of PGR’s. This choice can be made based on comparative study of their biological activity. Some promising reports on the role of topolins in micropropagation led to the idea of testing these cytokinins for their potential in tissue culture. As a prerequisite to subsequent investigations, the biological activity of some selected topolins and BA derivatives was tested using the soybean callus bioassay. The activity of the cytokinins tested varied significantly. The results demonstrated that the structure of a cytokinin dictates its activity. Modifications of side-chain improved the activity of oT but had no effect on pT. The presence of the methyl group had an enhancing effect on cytokinin activity of topolins or at least it did not reduce it. BA derivatives BA9THP (conjugated at N9 position), 3FBA and 2Cl6(3OHBA)R (halogenated derivatives) also showed good cytokinin activity and hold good promise for future research. In an attempt to alleviate hyperhydricity in Aloe polyphylla and optimize the micropropagation protocol, meta-topolin and its derivatives were tested at various concentrations together with BA and zeatin. Of all the cytokinins tested mT produced the best results in terms of shoot and root growth. Five μM was found to be the optimum concentration at which complete control of hyperhydricity was achieved without compromising shoot and root growth. Plantlets rooted in a multiplication media. BA generally had a negative effect on growth and development both in vitro and ex vitro. Acclimatization of plantlets was achieved easily by initially transferring plantlets to a mist house (for three weeks) followed by transfer to the greenhouse. The type of cytokinin also had an effect on ex vitro growth with BA-treated plants producing the lowest shoot and root biomass. Various experiments were conducted to characterize and control factors affecting STN in Harpagophytum procumbens. Media type and strength, PGR, carbon sources, sub-culturing, calcium and boron were tested. Results indicated that all of the tissue culture components tested affected STN. From the different media types tested, half strength was MS found to be the preferred medium. Increasing cytokinin concentration increased the incidence of STN and the problem was aggravated by the addition of auxin to the multiplication medium. Optimum shoot multiplication was achieved by omitting auxin and using the cytokinin mTR. Plantlets produced basal callus which interfered with rooting. The quantity of this basal callus was minimum when mTR was used. Sub-culturing plantlets onto fresh medium every two weeks helped minimize STN. Off all the sugars tested 3% sucrose was optimum. Other sugars either aggravated STN or inhibited growth when compared at equi-molar concentration. Increasing the concentration of either Ca or B prevented the development of necrotic shoots. When the concentration of both elements is increased simultaneously negative effects on both growth and STN were observed. Using 6 mM Ca in half strength MS medium was optimum. B was toxic at higher concentrations. Plantlets rooted readily in half strength cytokinin-free MS media supplemented with 2.5 μM IAA. Rooted plantlets produced using the optimized protocol were acclimatized successfully by transferring directly to a greenhouse in a 1:1 ratio of sand and soil mixture. The effect of meta-toplins on micropropagation and somaclonal variation of banana was investigated. Tissue cultured explants of cultivars ‘Williams’ and ‘Grand Naine’ were cultured in MS media containing the cytokinins BA, mT, MemT, MemTR and mTR at various concentrations. Results of the investigation revealed that superior multiplication and lower abnormality index was recorded from the mTR and mT treatments at 22.2 μM concentration. These treatments, however, had an inhibitory effect on rooting. The effect of these treatments (22.2 μM mT and mTR) in comparison with equi-molar concentration of BA on somaclonal variation of ‘Williams’ banana was tested using RAPD-PCR at the 7th multiplication cycle. No significant difference was found between the treatments. It should however be highlighted that cultures were initially maintained for three multiplication cycles in media containing BA. The inherent stability and initial effect of BA could have influenced the results.
Cytokinins and the germination of Tagetes minuta L.
(2003) Gold, John David.; Van Staden, Johannes.; Stirk, Wendy Ann.
Tagetes minuta L. is a weedy herb that has been a rich source of fragrant oils, used as in the perfume and flavour industry. T. minuta achenes germinate erratically under field conditions. However, at the optimal germination temperature of 25 °C, 100 % germination is attained within 48 h of imbibition. The achenes are thermoinhibited at 35 °C. The aims of this project were to assess the role of cytokinins (CKs) in normal germination at 25 °C, and to investigate the factors that regulate thermoinhibition at 35 °C. CKs were extracted from achenes germinating at 25 °C at 0, 24; 48; 96 and 144 h after imbibition. Two different purification techniques were used, namely Dowex cation exchange resin followed by paper chromatography, or high performance liquid chromatography (HPLC). CK-like activity was tested with the soybean callus bioassay. With both techniques, a peak in CK-like activity appeared 24 h after imbibition, which coincides with the period during which most of the achenes germinated. For quantitative analysis, HPLC\mass spectrometry (MS) techniques were used. The isoprenoid CKs were far more abundant in T. minuta achenes than the aromatic CKs. cis-Zeatin (cZ) and its derivatives were the most abundant CKs. In total, 19 CK compounds were detected, including 4 free bases and a number of corresponding conjugates. Benzyladenine (BA) was the only aromatic CK detected. There was no common time at which active free base maximal concentrations were detected, suggesting that different CKs may have specific roles in the germination process, and thus peak at different times. This in turn suggests that germination is not a single process, but rather a correlative process involving a number of events, with specific CKs having specific roles relating to these correlative events. There is sufficient evidence obtained from both the soybean callus bioassay and HPLC/MS analysis to suggest that CKs have an active role in T. minuta germination. A decline in free BA during germination without corresponding conjugation, suggests that BA is actively used in early germination processes, possibly in the stimulation of DNA synthesis. Secondly, there was a distinct dihydrozeatin (DHZ) peak obtained at 24 h. Roughly 75 % of the achenes germinate between 16 and 26 h, thus it is likely that DHZ has an active role during the germination of T. minuta. Although CKs are probably not involved in the breaking of dormancy per se, the distinct peak in CK-like activity obtained in the bioassays, 24 h after imbibition, suggests that CKs have an active role in the germination of T. minuta. With respect to the regulation of thermoinhibition, a number of exogenous treatments were applied, including hormones [gibberellins (GA₄₊₇), abscisic acid (ABA), ethylene and a number of CKs], adenosine triphosphate (ATP) and incubation in 100 % oxygen. ABA was extracted from thermoinhibited and germinating achenes to assess the role of ABA in thermoinhibition and germination. While exogenous 0.1 mg L¯¹ GA₄₊₇ application slightly improved normal germination at 25°C, no treatments were effective in alleviating thermoinhbibition in T. minuta achenes. Thermoinhibition in T. minuta achenes may be under hormonal regulation, as there is strong evidence for the role of ABA in the maintenance of dormancy and thermoinhbition. High ABA levels were found in dry control samples. Additionally, exogenous ABA application inhibited normal germination, and the commencement of germination was accompanied by a decrease in endogenous ABA levels. A number of experiments relating to the imposition of thermoinhibition were carried out. Thermoinhibition appears to be very rapidly imposed. Germination is rapidly inhibited following shifting to higher thermoinhibitory temperatures, even after prolonged exposure to optimal germination temperatures. Results suggest active de novo biosynthesis of ABA in thermoinhibited achenes. Active biosynthesis of ABA during thermoinhibition suggests that this phytohormone is essential in the maintenance of thermoinhibition of T. minuta achenes. It thus appears that ABA is synthesized in the achenes in response to elevated temperatures that are unfavourable for germination to proceed. Unfavourable environmental conditions result in an achene-mediated inhibition of germination, which appears to be initiated and maintained by elevated levels of endogenous ABA.
Heavy metals in South African medicinal plants with refence to safety, efficacy and quality.
(2014) Okem, Ambrose.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.; Stirk, Wendy Ann.; Southway, Colin.; Street, Renée Anne.
The trend in commercialization of medicinal plant products reflects the excessive exploitation of medicinal plants from the wild populations. Due to widespread soil pollution, there is a likelihood that medicinal plants could be harvested from heavy metal-contaminated soils and thus pose a potential health threat to consumers. Unregulated procurement coupled with the unhygienic trading environment, poor post-harvest handling and processing, represent major routes of heavy metal contamination in medicinal plant products. A comparative screening was carried out to assess the levels of heavy metal contamination in some frequently used South African medicinal plants obtained from out-door traditional medicinal markets and muthi shops. Plant samples were digested using a microwave-assisted acid digestion system and the elemental content determined using inductively coupled plasma optical emission spectrophotometry (ICP-OES). There was multi-elemental contamination in the investigated medicinal plants with elevated levels of Fe, Al and Mn detected in most of the samples and levels of As and Hg were above the World Health Organization limits of 1 mg kg-1 and 2 μg kg-1 respectively. The high levels of metal contaminations in some of the investigated medicinal plants is a health concern and urgent measures are needed to protect the health of consumers. Samples were quantified for their total phenolic and flavonoid contents as well as screened for antibacterial activity. Variable phenolic and flavonoid composition and antibacterial activity showed that the quality and efficacy of medicinal plants sold at traditional medicine markets is compromised. Data obtained from elemental analysis was subjected to hierarchical cluster analysis which categorized samples into four main groups with samples within a group having relatively similar metal analyte compositions. Hierarchical cluster analysis proved to be a valuable tool in this preliminary screening of heavy metal contamination in medicinal plants and can potentially be used to develop a large database for easy monitoring of plant species with hyperaccumulative potentials. Information such as site of collection, plant species and plant part could be a valuable approach to ensure safety, efficacy and quality of medicinal plants sold at traditional medicine markets. Exposure to Cd and Al for six weeks in a pot trial induced responses in Bulbine natalensis, Drimia elata and Hypoxis hemerocallidea and these included variations in heavy metal uptake, growth parameters and physiological changes. Generally, application of Cd and Al at low concentrations (2 and 500 mg L-1 respectively) enhanced growth parameters in the three plant species compared to the control plants. However, at the highest concentrations of Cd 10 and Al 1500 mg L-1 respectively, there was significant growth inhibition. Hypoxis hemerocallidea exhibited good tolerance to Al exposure up to 1000 mg L-1 compared to the other plant species. Some of the physiological changes such as accumulation of free-proline increased progressively with increasing heavy metal treatments in all the investigated plant species. The combined treatment of Cd 5:Al 1000 mg L-1 exhibited synergistic effects on the uptake and accumulation of Cd and Al with values of about 83 and 918 mg kg-1 respectively in the bulbs of D. elata. In B. natalensis, the combined treatment of Cd 10:Al 1500 mg L-1 resulted in the highest amount of Cd (67 mg kg-1) in the bulb samples while the highest amount of Al (1607 mg kg-1) was recorded after treatment with Cd 5:Al 1000 mg L-1. There was an antagonistic effect on the uptake and accumulation of Cd in H. hemerocallidea in the combined treatments. Energy dispersive X-ray analysis of the abaxial leaf surface indicated that more Al was translocated to the shoot in H. hemerocallidea compared to Cd. The bulbs and corms of the investigated medicinal plants are the most extensively utilized plant parts in traditional medicine. High levels of Cd and Al in the bulbs and corms raise public health concerns. Analysis of photosynthetic pigments showed total chlorophyll progressively decrease with increasing heavy metal stress in all three plant species. The effect of Cd and Al on chlorophyll fluorescence in H. hemerocallidea was investigated. Non-photochemical quenching (NPQ) was adversely affected in most of the heavy metal-treated plants indicating a photoinactivation of photosystem II (PSII) reaction centres. In the present study, increasing heavy metal treatment resulted in the inability of H. hemerocallidea to utilize the absorbed light energy leading to oxidative stress. Exposure to Cd and Al treatments for six weeks induced several ultrastructural changes in H. hemerocallidea including damage to the cortical cells and an increase in xylem size. Transmission electron microscopy revealed a complete breakdown of the thylakoids at the highest Cd treatment and the application of Al at moderate and the highest treatment significantly reduced the size of the chloroplasts. These ultrastructural changes could possibly explain the reduced chlorophyll fluorescence and the amounts of total chlorophyll recorded at the higher levels of heavy metal treatments. Biosynthesis and accumulation of secondary metabolites under heavy metal stress were variable in the investigated plants. The moderate Cd treatment at Cd 5 mg L-1 up-regulated the synthesis of total phenolics slightly compared to the controls in B. natalensis. All the other heavy metal treatments down-regulated the synthesis of total phenolics and flavonoids compared to the control plants in B. natalensis. Application of Cd and Al at the lowest concentrations, 2 and 500 mg L-1 respectively up-regulated the synthesis and accumulation of both phenolics and flavonoids in D. elata compared to the control plants. In H. hemerocallidea, the highest amounts of total phenolics and flavonoids were recorded at the moderate Cd treatment (5 mg L-1). High performance liquid chromatography showed a significant decrease in the levels of hypoxoside, a bioactive compound in H. hemerocallidea after heavy metal exposure. The lowest amount of hypoxoside was recorded at the highest concentration of the combined treatment (Cd 10:Al 1500 mg L-1). These variable responses to heavy metal stress indicated the need for in-depth research on changes of secondary metabolites in medicinal plants exposed to heavy metals in order to ensure ultimate quality and efficacy of medicinal plant products. There was a progressive decrease in antioxidant activity as measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging in the bulb extracts of B. natalensis and D. elata. The lowest treatment of Al (500 mg L-1) had slightly higher DPPH activity compared to the positive control (ascorbic acid). Extracts of H. hemerocallidea exhibited a progressive increase in DPPH activity with increasing heavy metal treatments. There was a significant decrease in the DPPH activity at the highest Cd application (10 mg L-1) compared to the control plants indicating a loss in the biosynthesis of important bioactive compounds at high levels of heavy metal exposure. Cadmium applied at low and moderate concentrations enhanced antibacterial activity (0.78 mg mL-1) against Staphylococcus aureus in B. natalensis compared to the control plant extracts. However, there was poor antibacterial activity against Escherichia coli in all the heavy metal-treated plants in B. natalensis. Application of Cd and AL at low concentration in D. elata enhanced good antibacterial activity (0.78 mg mL-1) against E. coli which is less susceptible to antibiotics than S. aureus. Extracts from all Cd-treated plants as well as low and moderate Al-treated H. hemerocallidea plants exhibited the good antibacterial activity against S. aureus compared to the control plants. Plants treated with the combined Cd 2:Al 500 mg L-1 treatment also had good activity against S. aureus. However, all the extracts of H. hemerocallidea exhibited poor activity against E. coli. The responses of plants to Cd and Al varied depending on the species. Their ability to accumulate elevated levels of heavy metals raises concerns not only on the safety of these products but also the issues regarding the quality and efficacy of plants grown on heavy metal contaminated soils. The findings presented in this thesis highlight the need for stringent monitoring of heavy metal contamination in medicinal plant material sold at traditional medicine markets and the need for safe and sustainable cultivation of important medicinal plants. This will ensure that medicinal plant products are of a standard quality, safe from toxic contaminants and consistent in terms of phytochemical compositions.
Heavy metals in South African medicinal plants.
(2008) Street, Renée Anne.; Van Staden, Johannes.; Stirk, Wendy Ann.; Southway, Colin.
Plants are able to take up and accumulate certain environmental contaminants such as heavy metals. When the plants are ingested by man, these contaminants are transferred along the food chain. Due to the poorly regulated medicinal plant trade in South Africa, many opportunities exist for heavy metal contamination of medicinal plants namely contaminated harvest sites as well as poor drying, processing, storage, transport and manufacturing conditions. The concentrations of five heavy metals (As, Cd, Co, Ni, Pb) and six microelements (B, Cu, Fe, Mn, Mo, Zn) were determined in some commonly used South African medicinal plants obtained from street markets. Elemental content was determined using inductively coupled plasma optical emission spectrophotometry (ICP-OES). Some of the medicinal plant samples investigated contained As and Cd at levels exceeding the World Health Organization limits of 1 and 0.3 mg kg-1 respectively. Lead and Ni were detected in all the samples. Elevated Fe and Mn levels were recorded in certain plant species. The results revealed multiple metal contamination in some medicinal plant parts sold in local markets and is thus grounds for concern. The effects of Cd application on growth parameters of some medicinal plant species belonging to the Hyacinthaceae (Albuca setosa, Eucomis autumnalis, Eucomis humilis, Merwilla plumbea) gave insight into heavy metal accumulation and distribution in these species. Application of Cd at 5 mg l-1 over a 12 week period reduced growth in A. setosa. The medicinally used A. setosa bulbs accumulated 37 mg kg-1 Cd after 12 weeks. Cadmium application at 2 mg l-1 over a six week period had no effect on growth parameters of E. autumnalis or E. humilis. However, a substantial difference in total Cd accumulation was detected in the plants (40.2 and 15.3 mg kg-1 respectively). Cadmium application at 2 mg l-1 significantly reduced the fresh weight of leaves, bulbs and roots of M. plumbea. Although most of the Cd was stored in the roots, the medicinally used bulbs accumulated up to 11.6 mg kg-1 when applied at 10 mg l-1. The antagonistic effect between Cd and Zn treatments and their effect on micronutrient distribution in M. plumbea were investigated. Five treatments were evaluated: (1) Hoagland’s nutrient solution (HS) (control) (2) HS + Cd 2 mg l-1 (single) (3) HS + Cd 2 mg l-1 + Zn 50 mg l-1 (combination) (4) HS + Cd 2 mg l-1 + Zn 100 mg l-1 (combination) (5) HS + Cd 2 mg l-1 + Zn 150 mg l-1 (combination). Cadmium readily accumulated in leaves, bulbs and roots of M. plumbea when supplied at 2 mg l-1. Zinc at 50 mg l-1 led to increased Cd accumulation. However, further increases in Zn concentration showed an antagonistic effect of Zn on Cd uptake and accumulation. Thus, increasing Zn levels in soils may be favourable for reducing toxic Cd accumulation in M. plumbea plants. Boron was not significantly affected by the addition of Cd to the media. However, with an increase in Zn, leaf B content increased while the B content in the bulbs and roots decreased. Copper and Mo levels were not significantly affected by treatments with Cd or Cd/Zn combinations. Compared to the control, Cd and Cd/Zn applications caused an increase in Mn content in leaves, bulbs and roots. Iron levels of M. plumbea were not significantly affected by Cd in the media. However, with an increase of Zn in the Cd-containing media, Fe content in the leaves, bulbs and roots increased. Tulbaghia violacea is one of the few medicinal plants that is also frequently used as a leafy vegetable. Application of Cd at 2 and 5 mg l-1 to T. violacea of varying sizes (small 8 - 10 g, medium 16 - 20 g, large 80 – 95 g) elicited a difference in growth response, Cd accumulation and micronutrient distribution. Leaf length and fresh weight of leaves of the medium-size plants decreased with application of Cd at 2 mg l-1 whilst 5 mg l-1 Cd significantly decreased the number of leaves in small-sized plants. Small plants accumulated more Cd in the leaves than medium- or large-sized plants. Application of Cd at 2 mg l-1 and 5 mg l-1 lowered the leaf Cu, Fe, Mo and Zn contents in small- and medium-size plants. This study indicated that T. violacea has the ability to accumulate Cd. In addition, plant size plays an important role with regards to Cd accumulation and elemental distribution. The effect of various nutrient applications (10%, 50% and 100% Hoagland’s nutrient solutions (HS); and HS deficient in N, P or K) on growth parameters and micronutrient distribution in Dioscorea dregeana were investigated. Irrigating plants with 50% HS resulted in better growth performance, whereas a deficiency of either N, P or K negatively affected seedling growth. Plants grown in 10% HS contained higher total B, Fe and Mo levels compared to seedlings grown in 50% and 100% HS. Compared to the control, P deficiency resulted in a Fe increase in the leaves, tuber and roots while a lack of P and K significantly increased total Mn content in D. dregeana. The effect of excess Zn (100, 200 and 300 mg l-1) on growth performance, chlorophyll content and microelemental distribution on Dioscorea sylvatica was investigated. Growth parameters showed a significant decrease when supplied with Zn at 100 mg l-1. Zinc phytotoxicity was evident by the reduction in chlorophyll content. Highest Zn concentrations were detected in the roots. Certain micronutrients appear to be redistributed due to Zn toxicity. The effect of microelements (Cu, Zn) and heavy metals (Cd, Pb, Hg) on germination and seedling development of Bowiea volubilis, Eucomis autumnalis and Merwilla plumbea was investigated. Copper and Zn applied at 1 mg l.1 significantly reduced the percentage germination of E. autumnalis. Low concentrations (. 1 mg l.1) of Cu and Zn negatively affected the root growth of all three species. Mercury concentrations of 0.5 and 1 mg l.1 significantly decreased the percentage germination of B. volubilis and E. autumnalis respectively. Cadmium and Hg at 2 mg l.1 showed a negative effect on the root growth of B. volubilis. Concentrations of 0.5 mg l.1 of all heavy metals tested significantly decreased shoot length of M. plumbea. The effect of Cd on biological activity (anti-inflammatory, antibacterial and antifungal) of medicinal plants with previously confirmed activity was evaluated. When supplied with Cd at 2 mg l-1, Eucomis humilis bulbous extracts showed lower anti-inflammatory activity than the control for both COX-1 and COX-2 activity. Eucomis autumnalis bulbous extracts had greater COX-1 activity compared to the control. However, Cd suppressed the activity of COX-2. Compared with non-Cd-treated Merwilla plumbea plants (control), those supplied with Cd at 10 mg l-1 showed increased antibacterial activity against Bacillus subtilis, Klebsiella pneumoniae and Staphylococcus aureus. However, no change in activity against Escherichia coli was observed. Cadmium accumulation in the bulbs had no effect on antifungal activity of Tulbaghia violacea. Thus, optimized agricultural practices are essential for quality control of cultivated medicinal plants. The studies presented in this thesis collectively answer several questions related to heavy metal involvement in South African medicinal plants. The findings substantiate the need to regulate and monitor the South African medicinal plant trade against heavy metal contamination which will in turn provide a product of safety and quality to the consumer.
Polyamines in Ecklonia maxima and their effects on plant growth.
(2012) Papenfus, Heino Benoni.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.; Stirk, Wendy Ann.
Kelpak®, a seaweed concentrate (SWC) prepared from the brown seaweed Ecklonia maxima (Osbeck) Papenfuss, improves overall plant mass and fruit yield in a variety of crops. The main active principals isolated from Kelpak® are cytokinins and auxins. Although these compounds are partly responsible for the growth promoting effect observed with Kelpak® application, they do not fully account for the complete effect of Kelpak® treatment. For this reason the focus has turned to polyamines (PAs) which are found in all cells of plants, animals and microorganisms, including eukaryotic algae. Polyamines also have growth promoting effects in plants. A study was carried out to investigate the PA levels in E. maxima and Kelpak® through a biennial cycle and to investigate if the PAs present in Kelpak® may have an effect on root growth, alleviating nutrient deficiency and the transport and accumulation of PAs in plants. To determine the amount of PA in the stipes, fronds and SWC prepared from E. maxima, samples were collected monthly over a two-year period (June 2009-June 2011). Extracts were benzoylated and quantified using a Varian HPLC. Putrescine concentrations ranged from 15.98-54.46 μg.g⁻¹, 6.01-40.46 μg.g⁻¹ and 50.66-220.49 μg.g⁻¹ DW in the stipe, fronds and SWC, respectively. Spermine concentrations ranged from 1.02-35.44 μg.g⁻¹, 1.05-26.92 μg.g⁻¹ and 7.28-118.52 μg.g⁻¹ DW in the stipe, fronds and SWC, respectively. Spermidine concentrations fell below the detection threshold. This is the first report of PAs being detected in a SWC. The seasonal pattern established for the stipe, frond and SWC followed the same trend over a biennial cycle. Polyamines accumulated in the seaweed tissue during periods of active growth and as a stress response elicited by rough wave action. This PA trend was similar to the cytokinin trend reported by MOONEY and VAN STADEN (1984b) for Sargassum heterophyllum which suggests that PAs play an important role in the hormone cascade during active growth. Routine monthly screening of Kelpak® carried out in the Research Centre for Plant Growth and Development indicated that Kelpak® consistently resulted in more rooting in the mung bean bioassay than the IBA control. The potential root promoting effect of PAs were investigated. Individually applied PAs did not increase rooting in the mung bean bioassay, but a synergistic relationship was observed between Put (10⁻³ M) and IBA (10⁻⁴ M). When applied together, rooting increased significantly above Put (10⁻³ M) and IBA (10⁻⁴ M) applied separately. The Put-auxin combination produced a similar number of roots to those treated with Kelpak®. It is possible that the PAs present in Kelpak® have a synergistic effect with auxins present in Kelpak® to promote root development and growth. Several physiological effects of Kelpak® and PAs on plant growth were investigated in a series of pot trials. Kelpak® significantly improved the growth of P- and K-deficient okra seedlings and masked the detrimental effects exerted by P- and K-deficiency. The application of PAs (10⁻⁴ M) significantly improved the seedling vigour index (SVI) of okra seedlings subjected to N-deficiency. The statistical difference was attributed to the N-containing growth regulators and polyamines being degraded and metabolized by the okra seedlings. Polyamine application did not alleviate P- and K-deficiency but increased root growth significantly in seedlings receiving an adequate supply of nutrients. It is likely that the additional PAs supported auxin-mediated root growth. A pot trial with okra plants was conducted to establish if the PAs in Kelpak®, applied as a soil drench or foliar application, are absorbed and translocated in a plant. Plants were also treated with Put, Spm, Spd to establish if PAs can be absorbed and translocated. Once the fruit had matured, plants were harvested and the endogenous PA content quantified by HPLC in the roots, stems and fruits. Applying PAs as a soil drench was not as effective as a foliar spray at increasing the PA content in the different plant parts. Kelpak® treatment (0.4%) did not contribute more PAs in any plant part. Spermidine concentrations were higher, in the various plant parts, than Put or Spm, irrespective of the mode of application. The application of Put, Spd and Spm increased Spd concentrations in the roots. Considering that Spd is the main PA produced in the roots and that exogenously applied PAs are readily converted to Spd, it seems evident that Spd is the preferred PA for long-distance transport in plants. The cytokinins and auxins in Kelpak® play an important role in stimulating growth in plants. It is, however, the totality of different compounds in Kelpak® that gives it its unique growth stimulating ability. Polyamines, occurring within the seaweed contribute to this activity, having an active role in root production and thus increased plant growth.
Root-stimulating activity from various gelling agents used in tissue culture.
(2003) Arthur, Georgina Dede.; Van Staden, Johannes.; Stirk, Wendy Ann.
Extracts of gelling agents have been shown to stimulate rooting and this study was initiated to investigate the presence of root stimulating substances in gelling agents. After screening a number of gelling agents, four were selected, namely; Agar Bacteriological, Agar Commercial Gel, Difco Bacto Agar and Gelrite were selected and examined for the presence of root-stimulating substances using mungbean bioassay. Water extracts of Agar Bacteriological, Agar Commercial Gel and Difco Bactol Agar stimulated rooting of mungbean cuttings. Addition of Charcoal neither reduced nor increased rooting produced by the water extract of the first two agars but when added in conjunction with Difco Bacto Agar rooting was reduced. Autoclaving, however reduced rooting in extracts of the gelling agents. The possibility that root-stimulating substances may not be the same in all the gelling agents can not be excluded. Extraction of Gelrite with water was problematical and was therefore excluded. IBA solution and water extracts of the gelling agents separately promoted good rooting in mungbeans cuttings. Rooting in extracts of autoclaved frozen-thawed gelling agents was poor, however, IBA + gelling agents gave high rooting at the 100% concentration and this could possibly be due to an additive effect of the IBA. Addition of charcoal reduced rooting significantly in extracts of IBA + gelling agents. Using 80% acidic methanol, reasonable levels of rooting substances were obtained from the residue extract of this complex (IBA + gelling agent+charcoal) of all the gelling agents except Gelrite indicating that root-promoting substances were adsorbed by charcoal. The low rooting in the presence of the Gelrite extract was attributed to the matrix of the polymer of the Gelrite. Ethyl acetate fractionated extracts (EA-pH 8.0; EA-pH 3.0; and Aqueous) obtained from the four gelling agents stimulated rooting indicating the presence of numerous root promoting substances. Gelrite gave good rooting with both the 50 and 100% concentrations of all the fractions. Purified water and ethanol extracts of the gelling agents exhibited auxin-like activity when separated by paper chromatography and compared with IBA and IAA standards. Using HPLC, IAA was quantified in all the gelling agents with Difco Bacto Agar and Agar Commercial Gel having the highest IAA concentration and Gelrite the lowest IAA concentration. IAA concentration in Agar Bacteriological was a third of the level detected in Difco Bacto Agar. The information from this work may enable researchers to consider gelling agents as sources of auxin-like compounds and other plant hormones as well as support media for use in tissue culture procedures and also increase the enthuse for further research into the nutrient types and levels in gelling agents.