Pharmacological, toxicological and phytochemical evaluation of helichrysum petiolare hilliard & b.l. burtt - an indigenous plant traditionally used in the treatment of diabetes in the eastern cape province of South Africa
- Authors: Aladejana, Adebowale Emmanuel https://orcid.org/0000-0003-1871-926X
- Date: 2022-04
- Subjects: Diabetes -- Alternative treatment , Traditional medicine , Medicinal plants
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/22787 , vital:52755
- Description: Diabetes mellitus is one of the leading causes of death in South Africa, and it has already placed significant stress on the country’s health sector and economy. The orthodox hypoglycaemic drugs are not only ineffective in the management of the disease and its complications, but they also possess unwanted side effects. The need for alternative non-toxic drugs is therefore imperative. Various studies have listed several medicinal plants that can be successfully used in the herbal treatment of diabetes and have investigated them for their anti-diabetic potentials in vivo and/or in vitro. Out of the different potential herbal species, plants belonging to the Asteraceae family possess highly potent hypoglycaemic properties with negligible toxicities. Five Asteraceae plants widely used in different parts of South Africa for the treatment of diabetes were reviewed. The review provided an update of scientific evidence on the hypoglycaemic properties of the plants. However, Helichrysum petiolare was studied extensively in this study for its antidiabetic activity H. petiolare has been listed in many ethnobotanical surveys as a plant with potent hypoglycaemic potential, this, however, has not been properly verified in scientific literature and there has hardly been any study on the essential oil and nutritional composition, and antioxidant, antidiabetic, and cytotoxicity potentials of the plant. The effects of hydro-distillation (HD) and solvent-free microwave extraction (SFME) methods on the chemical constituents of H. petiolare-derived essential oils were evaluated. The SFME method had a higher yield of essential oil than the HD. There were substantial amounts of monoterpenes, monoterpene alcohols, sesquiterpenes, and sesquiterpene alcohols in both essential oils obtained, but these compounds were more profound in the SFME derived essential oil which has 62 compounds compared to the 52 derived through HD. The SFME derived essential oil can therefore be said to be of better quality than the HD method. The compounds obtained in the essential oils have high pharmaceutical and cosmetic value, and as observed in this study, their quantity is dependent on the method of extraction (Ibáñez and Blázquez, 2021; Kaur et al., 2021). The proximate analysis of the whole plant of H. petiolare showed high levels of Acid Detergent Fibre (ADF), vitamins (A, C and E), Neutral Detergent Fibre (NDF), and minerals. The high ADF level is believed to be responsible for the low energy, fat and carbohydrate levels observed in the study. The result showed a high level of oxalate and therefore suggests cooking of the plant before human consumption. Overall nutrition and mineral compositions of the plant showed that H. petiolare is immensely rich in vital nutrients that are of great importance to health and metabolism; these nutrients are suggested to be partly responsible for the plant’s useful medicinal properties. The phytochemical contents of the acetone (ACQ), ethanol (ETQ), and boiled (BAQ) and cold (CAQ) aqueous whole-plant extracts of Helichrysum petiolare were determined using standard phytochemical reaction methods. ABTS, DPPH, NO and TAC assays were used to evaluate their antioxidant properties. The highest total phenolic content (212,963 mg/g) was reported in the BAQ extract, while the ETQ had the highest flavonoid (172.393 mg/g) and proanthocyanidin contents (65.855 mg/g). Alkaloids, flavonols, and saponin were highest in the ACQ extract, while the CAQ had the lowest phytochemical content. Among the extracts, the BAQ had the highest DPPH•+ (IC50 0.02 mg/mL) and ABTS•+ (IC50 0.07) inhibition capacities, while the ETQ exhibited the highest NO• Inhibition (IC50 0.41 mg/mL) and TAC (IC50 0.19 mg/mL). These findings justify the use of H. petiolare in traditional medicine and further recommend the ETQ and BAQ extracts of the plant as more effective extracts for medicinal treatment. The hepatotoxicity (cytotoxicity, mitotoxicity and lipotoxicity) potential of the BAQ, CAQ and ETQ extracts of Helichrysum petiolare was evaluated using standard procedures. The results showed negligible BAQ and CAQ cytotoxicities, which were further, corroborated by stability in the mitochondrial membrane potentials and were congruent with the CAQ and BAQ results for steatosis and phospholipidosis. The data suggested favourable CAQ and BAQ toxicity profiles with limited risks for hepatotoxicity. The ETQ extract, however, showed significantly high levels of cytotoxicity and lipotoxicity, and a low level of mitotoxicity. Our result suggested a potential risk of the ETQ extract for hepatotoxicity but appears partly independent of direct mitochondrial involvement. Glucose uptake assay showed significantly increased glucose uptake in the BAQ and CAQ treated L6 and C3A cell lines. The CAQ extract enhanced glucose uptake more in the L6 myocytes than in the C3A cell-lines hepatocytes. The BAQ extract showed higher levels of inhibition on α–amylase and α-glucosidase activities as compared to CAQ. The BAQ and CAQ extracts of H. petiolare may, therefore, contain pharmacologically active and relatively non-toxic hypoglycaemic chemicals, which may be effective substitutes in the treatment of diabetes mellitus. This study provides up to date scientific information on the use of H. petiolare in the treatment of diabetes mellitus in the Eastern Cape of South Africa. It justifies the use of this plant in herbal medicine and sheds more light on its previously vaguely understood nutritional and medicinal potentials. More studies, however, need to be done to isolate, identify and purify the constituent bioactive compound(s). Their dosage of application and mode of action also needs to be understood. , Thesis (PhD) -- Faculty of Science and Agriculture, 2022
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- Date Issued: 2022-04
Anti-oxidant and cytotoxicity activity against the HCC70 Breast cancer cell line of aqueous and methanolic extracts of two red algae species from the Eastern Cape Coast, South Africa
- Authors: Basera, Afra Tsitsi
- Date: 2021
- Subjects: Drug resistance in cancer cells , Oxidative stress
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/22589 , vital:52586
- Description: Cancer, despite extensive research, remains one of the leading causes of mortality. Research into plant-derived compounds has gained traction, as some are not cytotoxic towards healthy cells and pose fewer side effects. Marine algae produce secondary metabolites, protecting them from oxidative stress, which causes cardiovascular diseases, cancer, and diabetes. Therefore, this study aimed to evaluate the anticancer potential of selected red algae species, collected from Humewood Beach, Gqeberha in South Africa, against HCC70 (human breast adenocarcinoma) cancer cells. Identification of the species was made through DNA barcoding of the rbcL gene. Methanolic and aqueous extracts of Gelidium pristoides and Laurencia natalensis were evaluated for antioxidant activity, phytochemical content, and anticancer potential. The phytochemical composition was determined using the phenol, flavonoid, flavonol, tannin, and proanthocyanidin content assays. Antioxidant potential was evaluated using the Ferric reducing antioxidant potential, Diphenyl-1- Picrylhydrazyl (DPPH), nitric oxide, 2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging activity, and total antioxidant capacity (TAC) assays. The resazurin assay was used to determine the antiproliferative and cytotoxic activity of the methanolic extracts and solid-phase extraction fractions against the HCC70 breast cancer cells. The red algae species were identified as G. pristoides and L. natalensis. The aqueous extract of G. pristoides exhibited significantly higher phenols (24.0 mg/g GAE), tannins (31.6 mg/g TAE), and flavonols content (20.5 mg/g RE) than its methanolic extract, or the aqueous and methanolic extracts of L. natalensis. Methanolic extracts of G. pristoides and L. natalensis exhibited significantly higher proanthocyanidin contents (19.4 mg/g and 15.7 mg/g CE) than their respective aqueous extracts (13.0 mg/g and 11.8 mg/g CE). Flavonoids, however, were absent in both species. The aqueous and methanol extracts of Laurencia natalensis and Gelidium pristoides exhibited low inhibitory activities (less than 50%) against DPPH, ABTS, and nitric oxide radicals. Both species also showed low ferric reducing power. All the extracts were antiproliferative and cytotoxic against the HCC70 breast cancer cells, with L. natalensis exhibiting more significant inhibitory activity. However, its Solid Phase Extraction fractions did not display antiproliferative potential, unlike fraction 4 of G. pristoides. The study results suggest that both G. pristoides and L. natalensis possess bioactive compounds with potential anticancer activity. , Thesis (MSc) -- Faculty of Science and Agriculture, 2021
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- Date Issued: 2021
Spatial and seasonal variations of water quality determinants and pollutants as fitness-for-use and compliance assessments of the Mzimvubu catchment water resources for the proposed Mzimvubu Water Project, South Africa
- Authors: Mutingwende, Nhamo
- Date: 2018
- Subjects: Water quality -- South Africa Water quality management
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10353/9308 , vital:34320
- Description: The Department of Water Affairs as of late reported plans to build two substantial stockpiling dams in the Mzimvubu Catchment. The Mzimvubu stream basin is probably one of most prominent and undeveloped basin in South Africa. This is notwithstanding high yearly rainfall, high ecological status, high tourism potential, and appropriateness for afforestation, dryland/rainfed and water system agribusiness. Hence, the Department of Water Affairs researched the capability of building a multipurpose dam in the Mzimvubu catchment to catalyse financial and social improvement. The proposed dam will be based on the Tsitsa River. Often, scientific studies related to dam construction concentrate more on discovering the most, in fact, accessible place to construct it, than on the long haul socio-natural issues that come in its preparation. The water quality of the Tsitsa River, its tributaries and the underground drinking water sources within the Mzimvubu catchment are most likely to change once the dam wall is completed. Surface water resources are susceptible to chemical, physical, microbiological contamination and the so-called emerging pollutants either, through human or natural activities. A comprehensive baseline study on the water quality of the Mzimvubu water resources regarding traces of emerging pollutants and water quality determinants (physical, chemical and microbial) pre-dam construction is therefore essential. The objective of the water quality section of this study was to perform an in-depth analysis of water quality in the study area to form a baseline for future studies on how the built dam may affect these. The approach was to assess the spatial and seasonal variations of the pollutants (pharmaceuticals and pesticides) and water quality determinants for all water sources most likely to be affected by the development of the dam. The fitness-for-use and compliance assessments were conducted to assess if the current water resources are fit-for-use for various categories of use and if they comply with various water quality standards and guidelines as determined by the Department of Water Affairs and Forestry. Department of Water Affairs and Forestry is the overseer of South Africa's water assets, and its central goal is to keep up the fitness-for-use of water on a sustained basis. Water samples (500ml) were collected from sixteen (16) sample points, ranging from the proposed mouth of the dam to the N2 bridge point of the Tsitsa River. Points were selected where the Tsitsa River was accessible using the dam project development roads or where tributaries to the Tsitsa River were accessible using dam development roads. Taps/groundwater sources were sampled from the five selected villages. Monthly samples were collected upstream and downstream of the proposed dam wall, from June 2015 to April 2017.Seventeen (17) water quality indices were therefore analysed at sixteen sampling sites, over a two year period. The AB SCIEX TripleTOF™5600 LC/MS/MS was used to screen for pharmaceutical and pesticide pollutants. All the water quality indices were analysed using the AL400 Aqua lytic photometer, and the microbial analysis was done using the Rand Water Method Number 1.2.2.09.1 for enumerating the amount of E. coli and coliforms in the water samples (Rand Water, 2010f). The South African Water Quality Guidelines, Volumes 1 to 7 (DWS, 1996a-g) were used to assess the fitness-for-use of the water sources. To confirm the compliance of the water resources to various standards and guidelines, the water quality data were assessed against international and national guidelines and standards i.e. the WHO guideline, South African water quality guidelines (domestic, irrigation, livestock and watering, aquaculture, and aquatic ecosystems), and the SANS: 241 (2015) standard for drinking water. Non-parametric statistics were utilised to ascertain the changeability, which is a measure of how water quality may vary after some time. With non-parametric insights, the interquartile extent, which lies between the 25th and the 75th percentile, was utilised to depict inconstancy. The median value (50th percentile) was used as an indication of the central tendency or average. The 90th percentile was included as it can be used to assess the frequency of excursions into higher and possibly unacceptable water quality conditions. 3D Sigma plot was used to graphical present the spatial and seasonal variations of water quality indices and emerging pollutants against their concentrations. Fundamental statistical properties and correlations of water quality variables from the Tsitsa River, Tsitsa River tributaries and the drinking water sources were examined using SAS descriptive statistics. The water quality was determined to be of relatively sound quality, based on the comparison with guidelines and standards for the various intended uses, even though some of the water quality determinants were non-compliant and were “unacceptable” regarding fitness for purpose. The water quality of the Ntabalenga dam would most probably be affected by natural influences (for example rainfall, weathering and geological composition) and anthropogenic factors through non-point source pollution from agriculture activities, human settlements (pit latrines and open defecation) as well as industrial activities in the Maclear and Tsolo towns (wastewater treatments plants effluent, hospital effluent). The Tsitsa River had the highest number of non-compliances, especially to the World Health Organisation and Department of Water and Sanitation aquaculture guidelines. Therefore, the Tsitsa River’s water quality would be a significant factor that could compromise the water quality of the water collected in the dam. The human settlement conditions and agricultural inputs seem to be the factors contributing most to contamination of the surface water of the catchment area. The lack of sanitation systems and facilities means that community members have to use the bush and rivers for ablutions, thus contributing to microbial contamination of the environment. The direct application of manure and fertilisers on the fields by farmers further exacerbates microbial contamination and high nutrient inputs into the environment as observed in elevated microbial and phosphate contaminants during the study period. The data obtained from the analysis of pesticides and pharmaceuticals confirmed the contamination of the drinking water sources, the Tsitsa River and its tributaries with pesticides and pharmaceuticals through non-point source pollution. The origins of these pharmaceutical contaminants were identified as the pit latrines, open defecation and wastewater treatment plant effluent, while agricultural application of pesticides was identified as the source of pesticides in surface waters. If not monitored closely, the presence of these emerging pollutants will negatively affect the quality of the dam water both at spatial and temporal scales once the dam wall is completed. Pit latrines and wastewater treatment plants are a significant source of non-point source pollution. The results of this study will add to the ongoing efforts on water quality remediation by recording the spatial and seasonal variations in water quality across various water sources within the study area. The study also provides a baseline for future water quality fitness-for-use and compliance assessments. By these findings and conclusions, it is recommended that a long-term continuous monitoring programme be implemented, especially in areas where increased agricultural activities have been observed. Monitoring should be implemented for the Tsitsa River, its tributaries, and selected drinking water sources which showed the highest number of non-compliances and microbial contamination. All anthropogenic activities in the catchment areas of these sources, both upstream and downstream of the proposed dam wall, must be monitored and strictly managed to prevent and mitigate their possible impacts. Specific emphasis should be placed on agricultural development, which should be controlled to ensure sustainable livestock and cropping practises. Sanitation facilities, systems and community programmes should be put in place to minimise microbial contamination. It would be beneficial for the Department of Water and Sanitation office responsible for the Mzimvubu water resources to establish a central database for all information concerning the water quality of their water resources including the findings in this report. The database must be freely accessible to the residents of the Mzimvubu catchment.
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- Date Issued: 2018
Antidiabetic and toxicological properties of Dianthus thunbergii (Caryophyllaceae) roots and Hypoxis argentea (Hypoxidaceae) corms
- Authors: Akinrinde, Akinleye Stephen
- Date: 2017
- Subjects: Medicinal plants--South Africa--Eastern Cape Traditional medicine--South Africa--Eastern Cape Diabetics--Alternative treatment--South Africa
- Language: English
- Type: Thesis , Doctoral , Biochemistry
- Identifier: http://hdl.handle.net/10353/16036 , vital:40606
- Description: Diabetes mellitus is a chronic metabolic disorder primarily characterized by elevated blood glucose levels. Its rapidly increasing prevalence as a major non-communicable disease is now a growing concern for both developed and developing countries. The development of safe, cost-effective and pharmacologically-effective medications continues to a major challenge of antidiabetic management. Since most conventional antidiabetic drugs are costly and present with numerous adverse effects, alternatives are increasingly being sought from natural sources, most notably medicinal plants, as viable approaches to tackling the scourge of this disease. In the Eastern Cape Province of South Africa, the roots of Dianthus thunbergii and corms of Hypoxis argentea are frequently used traditionally for the treatment of diabetes mellitus, although no scientific evidence exists to validate their effectiveness for diabetes management. The studies conducted in the resent research were, therefore, aimed at investigating the antidiabetic and toxicological properties of these plants, in an attempt to providing data towards validating their use in traditional management of diabetes mellitus. Aqueous and ethanol extracts of the underground parts of D. thunbergii and H. argentea were initially subjected to analysis of their phytochemical composition, relative to standard compounds, and the nature of their in-vitro antioxidant activities using standard spectrophotometric methods. The potentials of these extracts for cytotoxicity and/or cell proliferation were evaluated using MTT assay in HepG2 cells and Crystal violet assay in INS-1 cells. These activities were further examined in INS-1 cells using live cell fluorescence imaging techniques. To evaluate the antidiabetic properties of the extracts, they were screened for their inhibitory effects on the activities of different enzymes including α-amylase, α-glucosidase, porcine pancreatic lipase, Dipeptidyl peptidase IV (DPP-IV), collagenase and the drug metabolizing enzyme, CYP3A4, while also assessing their effects on protein glycation using in- vitro visible and fluorescence spectrophotometric approaches. Cell culture procedures were carried out to evaluate the effects of the extracts on glucose utilization in HepG2 cells and L6 myotubes; nitric oxide production in RAW 264.7 macrophages; glucose metabolism in INS-1 cells, as well as triglyceride accumulation in 3T3-L1 pre-adipocytes. Furthermore, identification of compounds present in the aqueous and ethanol extracts was carried out by Liquid chromatography- Mass spectrometry (LC-MS), while volatile oils extracted from fresh and dried parts of the two plants by hydrodistillation were also analyzed by Gas chromatography-Mass spectrometry (GC-MS). The ethanol extracts of both D. thunbergii and H. argentea contained higher amounts of total phenols, flavonoids, tannins, proanthocyanidins and alkaloids, when compared with the aqueous extracts. This finding was in direct correlation with the antioxidant activities of the extracts, with the ethanol extracts of both plants demonstrating stronger scavenging activities against hydrogen peroxide, nitric oxide, ABTS and DPPH radicals, while also exhibiting higher ferric reducing antioxidant potentials, when compared with the aqueous extracts, and in some cases, the standard antioxidants, Vitamin C, butylated hydroxytoluene and rutin. The aqueous extracts of D. thunbergii exhibited the highest toxicity in HepG2 cells with IC50 < 50 μg/ml, while also producing a concentration-dependent reduction in the viability of INS-1 cells up to 41.81percent at 50 μg/ml. Both extracts of H. argentea, however, did not produce any significant toxicity in these cells. Fluorescence imaging of live INS-1 cells using Hoechst and propidium iodide staining revealed stimulation of cell proliferation by H. argentea, while the cytotoxicity of D. thunbergii was further confirmed. H. argentea caused stimulation of glucose uptake in HepG2 cells up to 119.58 percent at 100 μg/ml and as much as 116.96 percent in L6 myotubes at 50 μg/ml, without showing toxicity to these cells. D. thunbergii produced 18.39 percent increase in L6 glucose uptake above untreated control; although its effect on HepG2 glucose uptake was irrelevant as significant toxicity was produced in these cells. H. argentea produced a concentration-dependent reduction in nitric oxide production in RAW macrophages, although not as effectively as the positive control, aminoguanidine. Again, the toxicity of D. thunbergii to this cell line precludes the relevance of nitric oxide inhibition as an antidiabetic mechanism for this plant. D. thunbergii produced a concentration-dependent increase in 3T3-L1 triglyceride accumulation, as measured by Oil red O staining, compared to untreated cells, while H. argentea exerted no significant alterations in pre- adipocyte differentiation. Generally, the two plants produced weak inhibition of the activities of the various enzymes measured, suggesting that this mechanism may not play a major role in the activities of these plants as possible antidiabetic agents. GC-MS analysis revealed major differences in the volatile oil composition between fresh and dried plant parts for both plants. Most notably, total terpenoid content of D. thunbergii oils reduced significantly from 77.17 percent in the fresh root oil to 47.58 percent in the dried root oil. Total terpenoid content was much lower in H. argentea oils, but similarly reduced from 10.58 percent in the fresh corm oil to 4.00 percent in the dried corm oil. LC-MS analysis enabled the tentative identification of compounds including phenolic glycosides, flavonoids, alkaloids, terpenoids saponins and sapogenins, many of which have been reported in literature to exert bioactivities relevant to the ones elucidated in the present study. Overall, H. argentea exhibited antidiabetic properties that may be mediated by its stimulation of glucose uptake in HepG2 and L6 cells; stimulation of proliferation in INS-1 cells; lack of stimulation of 3T3-L1 triglyceride accumulation and a tendency to reduce nitric oxide production in RAW macrophages. These activities suggest that H. argentea has promise for further investigations as an antidiabetic agent. On the contrary, D. thunbergii exhibited significant toxicity to HepG2 cells, INS-1 cells and RAW macrophages. Its cytotoxicity at the concentrations investigated in the present studies raises significant concerns about any potential antidiabetic applications for this plant.
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- Date Issued: 2017
Differential regulation of wheat (Triticum aestivum) callose synthases during Russian wheat aphid (diuraphis noxia)infestation
- Authors: Ngwenya, Nompilo
- Date: 2017
- Subjects: Russian wheat aphid Wheat -- Diseases and pests
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10353/8100 , vital:31522
- Description: Plant production serves as the primary food source for all living beings and is continuously being affected by many biotic and abiotic factors. Wheat, one of the most consumed cereal crops in the world and the second most cultivated crop after maize in South Africa, is continuously being affected by Russian wheat aphid (RWA) since 1978. To counteract the effect of this aphid, the first resistant wheat cultivar was developed in 1984 in South Africa containing the Dn1 resistance gene. However, the mechanism of action of the resistance is still not fully understood to date. The feeding of the RWA on the wheat induces callose deposition especially in susceptible wheat cultivars, linking the β-1.3-polysaccharide (callose) to the resistance mechanism. Callose is synthesized by callose synthases, also known as glucan synthase-like genes (GSL). Not much is known about the mechanism of action of callose synthases or how they are regulated in response to the RWA. Bioinformatics tools, such as those available at NCBI, were used to identify the wheat callose synthases genes. The proteins encoded by the identified wheat callose synthases genes, were then analyzed for conserved domain motifs using the MEME suite and InterproScan, sequence similarities using ClustaL Omega and transmembrane domains using HMMTOP and ProtCamp. These were then compared with those of the already characterized Arabidopsis callose synthase proteins in an attempt to identify the wheat callose synthase(s) that responds to aphid feeding. Further bioinformatics studies were carried out to identify the presence of biotic stress associated cis-acting regulatory elements found in the 1.5 kbp upstream region of the start codon of the Arabidopsis callose synthase genes. Eight partial wheat callose synthase sequences were identified and two of these (TaGSL2 and TaGSL22) showed high similarities to the AtGSL5, which is up-regulated in response to aphid feeding in Arabidopsis. Six of the wheat callose synthase genes were mediated to code for the functional callose synthases proteins: TaGSL3, 8, 12, 19, 22 and 23, and analyzed for conserved protein motifs. Based on the sequence similarities and conserved protein domains, TaGSL2 and TaGSL22 were found to be the most similar to AtGSL5 and most likely to respond to RWA infestation. Cis-acting regulatory element analyses confirmed the possibility of TaGSL22 being responsible for callose deposition in wheat as biotic stress associated cis-acting regulatory elements, Box W1, TC- rich element and W- box were all found in the 1.5 kbp upstream of the TaGSL22 coding region. Callose was quantified in both susceptible and resistant wheat cultivars using the aniline blue fluorescence method. When infested with RWA-SA1, the susceptible wheat cultivar (Tugela) deposited significantly higher amounts of callose, compared to the resistant wheat cultivar (Tugela DN), which deposited little, to no callose with respect to the control samples. Transcription analysis, of the TaGSL2 and TaGSL22 in RWA-SA2 infested Tugela and Tugela DN wheat cultivars, was performed using real time polymerase chain reaction (qPCR). Both TaGSL2 and TaGSL22 genes were up-regulated in Tugela and Tugela DN wheat cultivars in response to RWA-SA2 infestation, with TaGSL22 being more expressed than TaGSL2 in both cultivars, indicating that RWA-SA2 is able to overcome the resistance of Tugela DN and cause callose deposition. However, significantly higher expression of both genes was still observed in the susceptible, Tugela wheat cultivar. This study therefore confirms that callose deposition is associated with RWA-SA1 feeding, only in the susceptible wheat cultivar, Tugela and not the resistant cultivar, Tugela DN. However, during RWA-SA2 feeding, two of the eight identified callose synthases in wheat, are up-regulated in response to RWA-SA2 feeding, in both the resistant and susceptible wheat cultivars with higher levels observed in the susceptible wheat cultivar when compared to the resistant wheat cultivar. TaGSL2 and TaGSL22 are therefore implicated in the callose deposition observed in the susceptible and resistant wheat cultivars, after RWA-SA2 infestation. Further studies are required to confirm the differential regulation of the two wheat callose synthases proteins during RWA infestation and their possible role in the resistance mechanism of the resistant wheat cultivar, Tugela DN.
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- Date Issued: 2017
Mechanism and synchronicity of wheat (Triticum aestivum) resistance to leaf rust (Puccinia triticina) and Russian wheat aphid (Duiraphis noxia) SA1
- Authors: Njom, Henry Akum
- Date: 2016
- Subjects: Wheat -- Disease and pest resistance Bacterial diseases of plants Russian wheat aphid
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10353/2700 , vital:28056
- Description: Wheat (Triticum aestivum and T. Durum) is an extremely important agronomic crop produced worldwide. Wheat consumption has doubled in the last 30 years with approximately 600 million tons consumed per annum. According to the International Maize and Wheat Improvement Center, worldwide wheat demand will increase over 40 percent by 2020, while land as well as resources available for the production will decrease significantly if the current trend prevails. The wheat industry is challenged with abiotic and biotic stressors that lead to reduction in crop yields. Increase knowledge of wheat’s biochemical constitution and functional biology is of paramount importance to improve wheat so as to meet with this demand. Pesticides and fungicides are being used to control biotic stress imposed by insect pest and fungi pathogens but these chemicals pose a risk to the environment and human health. To this effect, there is re-evaluation of pesticides currently in use by the Environmental Protection Agency, via mandates of the 1996 Food Quality Protection Act and those with higher perceived risks are banned. Genetic resistance is now a more environmental friendly and effective method of controlling insect pest and rust diseases of wheat than the costly spraying with pesticides and fungicides. Although, resistant cultivars effectively prevent current prevailing pathotypes of leaf rust and biotypes of Russian wheat aphid from attacking wheat, new pathotypes and biotypes of the pathogen/pest may develop and infect resistant cultivars. Therefore, breeders are continually searching for new sources of resistance. Proteomic approaches can be utilised to ascertain target enzymes and proteins from resistant lines that could be utilised to augment the natural tolerance of agronomically favourable varieties of wheat. With this ultimate goal in mind, the aim of this study was to elucidate the mechanism and synchronicity of wheat resistance to leaf rust (Puccinia triticina) and Russian wheat aphid (Duiraphis noxia) SA1. To determine the resistance mechanism of the wheat cultivars to leaf rust infection and Russian wheat aphid infestation, a proteomics approach using two-dimensional gel electrophoresis was used in order to determine the effect of RWA SA1 on the wheat cultivars proteome. Differentially expressed proteins that were up or down regulated (appearing or disappearing) were identified using PDQuestTM Basic 2-DE Gel analysis software. Proteins bands of interest were in-gel trypsin digested as per the protocol described in Schevchenko et al. (2007) and analysed using a Dionex Ultimate 3000 RSLC system coupled to an AB Sciex 6600 TripleTOF mass spectrometer. Protein pilot v5 using Paragon search engine (AB Sciex) was used for comparison of the obtained MS/MS spectra with a custom database containing sequences of Puccinia triticina (Uniprot Swissprot), Triticum aestivum (Uniprot TrEMBL) and Russian wheat aphid (Uniprot TrEMBL) as well as a list of sequences from common contaminating proteins. Proteins with a threshold of ≥99.9 percent confidence were reported. A total of 72 proteins were putatively identified from the 37 protein spots excised originating from either leaf rust or Russian wheat aphid experiments. Sixty-three of these proteins were associated with wheat response to stress imposed by RWA SA1 feeding while 39 were associated with infection by Puccinia triticina. Several enzymes involved in the Calvin cycle, electron transport and ATP synthesis were observed to be differentially regulated suggesting greater metabolic requirements in the wheat plants following aphid infestation and leaf rust infection. Proteins directly associated with photosynthesis were also differentially regulated following RWA SA1 infestation and P.
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- Date Issued: 2016
A comparative study of the in vitro antidiabetic properties, cytotoxicity and mechanism of action of Albuca bracteata and Albuca setosa bulb extracts
- Authors: Odeyemi, Samuel Wale
- Date: 2015
- Subjects: Medicinal plants Herbs -- Therapeutic use Diabetics -- Alternative treatment
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10353/3154 , vital:28327
- Description: The search for cheap, non toxic and readily available antidiabetic drugs has been a challenge for researchers and the pharmaceutical industries. Diabetes mellitus is a metabolic disease characterized by defects in the synthesis of insulin and/or insensitivity to the action of insulin at the target cells. The disease has been on the increase mostly in developing countries where large proportions of the population have little access to good medical care due to either accessibility or non availability of synthetic drugs. This has led to the use of medicinal plants to treat diabetes because it is safe, cheap and with few side effects. There is little scientific evidence on the dosages, active compounds, mechanisms of action and toxicity of these traditionally used plants. Two of the most frequently used plants; Albuca setosa and Albuca bracteata were investigated in this study. The qualitative analysis of different extractions of these plants revealed the presence of phenolics, alkaloids, tannins and saponins. The antioxidant properties of aqueous, acetone and methanollic extracts of Albuca setosa and Albuca bracteata were investigated using models such as Diphenyl-1-Picrylhydrazyl (DPPH), 2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), Ferric ion reducing antioxidant potential (FRAP), Nitric Oxide and Hydrogen Peroxide (H2O2). Both plants revealed inhibitions against DPPH in a concentration - dependent manner with Albuca setosa (0.330 mg/ml) showing higher activity than Albuca bracteata (0.647 mg/ml) determined from the IC50. The aqueous extract of Albuca setosa showed a higher inhibition against DPPH radical compared to the Albuca bracteata aqueous extract at all concentrations investigated. The isolated saponins from Albuca bracteata had a higher DPPH scavenging activity than the crude methanolic extract of the plant in a concentration - dependent manner but are significantly different from each other at 0.4, 0.6 and 1.0 mg/ml only. The IC50 of the saponins was also observed to be higher than the crude extracts and standards.The Albuca setosa aqueous extract showed a higher percentage inhibition of ABTS radicals than Albuca bracteata at all the concentrations investigated. Overall, the Albuca setosa aqueous extract (0.0809 mg/ml) showed maximum activity against ABTS radicals. The iron reducing power was significantly higher (P < 0.05) in the methanolic extract of both plants compared to the aqueous counterpart. Overall, the Albuca bracteata aqueous extract (0.344 mg/ml) showed maximum activity as indicated by the IC50. The aqueous extracts of both plants also revealed percentage inhibitions in a concentration - dependent manner against NO2. The aqueous extract of Albuca bracteata bulb was more active against nitric oxide and hydrogen peroxide inhibition. In this study, the cytotoxicity of the extracts was evaluated at a high dose of 100 μg/ml on Chang liver cells and determined using MTT, crystal violet, glucose consumption, lactate production and lactate dehydrogenase release and FRAP. The aqueous extracts of both Albuca setosa and Albuca bracteata were non-toxic on Chang liver cells at the concentrations investigated. The MTT revealed that the aqueous extract of Albuca setosa bulb had the optimum cell viability of 108.09 percent while the acetonic extract of Albuca bracteata showed the least cell viability (37.72 percent) compared with the control. The crystal violet test also revealed the acetone extract of Albuca bracteata to have the least percentage of cell viability at 31.47 percent, while the aqueous extract of Albuca setosa showed the maximum cell viability at 112.5 percent. The aqueous extracts of both plants showed higher percentage cell density on the second day of incubation from the proliferation assay. All the tested samples were observed to consume more glucose than the blank except for the methanollic and acetone extracts of Albuca bracteata bulb. The aqueous and methanolic extracts of Albuca setosa bulbs produced the highest lactate with 120.2 μg/ml and 113.7 μg/ml respectively. The acetone extracts of both Albuca setosa and Albuca bracteata revealed toxicity with a higher lactate dehydrogenase release compared to the control.
- Full Text:
- Date Issued: 2015
Optimisation of expression of a rice (Oryza sativa L.cv Nipponbare) plant natriuretic peptide (OsPNP-B) and its functional characterisation
- Authors: Affun, Ogheneochuko Janet
- Date: 2012
- Subjects: Rice , Plant hormones , Water-electrolyte imbalances
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/24354 , vital:62644
- Description: Maintenance of water and solute homeostasis is a key requirement for living systems, and in vertebrates, homeostasis is in part achieved by natriuretic peptides (NP), a family of peptide hormones. A related family of peptide hormones have also been found in the plant kingdom. Plant natriuretic peptides (PNP) are a novel class of plant proteins with two closely related homologous genes (AtPNP-A and AtPNP-B) identified in the model plant Arabidopsis thaliana. AtPNP-A has been extensively studied and evidence obtained points to a role in plant water homeostasis. No research has been conducted on the function of PNP-B proteins. In this study, we focus on the bioinformatic analysis of the PNP-B gene in various plants, as well investigating whether PNP-B plays a role in water homeostasis in rice plants exposed to drought stress. Basic local alignment search tool (BLAST) queries of the ―The Gene Index‖, EST and available plant genome databases revealed the presence of the PNP-B mRNA in rice, oil seed rape, oak, leafy spurge, poplar, sugarcane, pepper, cotton, apple and maize. All the identified genome sequences contained a predicted intron/insert, which was not present in the related mRNA sequences. However, RT-PCR analysis revealed the presence of the 101 bp insert in the 976 bp amplified rice PNP-B (OsPNP-B) cDNA and therefore expression was optimized for the protein encoded by only the exon2 sequence as this contained the predicted active site region of PNP-B. OsPNP-B was shown to be translated to a protein of 14 kDa with a sequence similarity to AtPNP-B (54percent), AtPNP-A (37percent) and CjBAp12 (55percent), suggesting two possible functions for PNP-B viz water homeostasis and/or pathogenesis defence. To determine whether PNP-B is involved in water homeostasis, total protein extracted from 4 weeks old (4 leaves stage) rice plants subjected to drought treatment for a period of 24, 48, 72, 120, 168 and 240hrs were resolved by 17percent SDS-PAGE and analysed by western blot analysis. The PNP-B protein was found to be down-regulated during drought stress, implying that PNP-B may play a role in water homeostasis through the release of water from cells rather than the up-take of water as seen At-PNP-A. PNP-B could therefore also be involved in plant defence mechanisms to pathogens where plants induce desiccation of infected leaves, thereby ridding the plant of the relevant pathogen. , Thesis (MSc) -- Faculty of Science and Agriculture, 2012
- Full Text:
- Date Issued: 2012
Identification of cis-elements and transacting factors involved in the abiotic stress responses of plants
- Authors: Maclear, Athlee
- Date: 2005 , 2013-06-10
- Subjects: Plants -- Effect of stress on , Proteins -- Analysis , Bioinformatics , DNA , Plant genetics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4074 , http://hdl.handle.net/10962/d1007236 , Plants -- Effect of stress on , Proteins -- Analysis , Bioinformatics , DNA , Plant genetics
- Description: Many stress situations limit plant growth, resulting in crop production difficulties. Population growth, limited availability and over-utilization of arable land, and intolerant crop species have resulted in tremendous strain being placed on agriculturalists to produce enough to sustain the world's population. An understanding of the principles involved in plant resistance to environmental stress will enable scientists to harness these mechanisms to create stress-tolerant crop species, thus increasing crop production, and enabling the farming of previously unproductive land. This research project uses computational and bioinformatics techniques to explore the promoter regions of genes, encoding proteins that are up- or down-regulated in response to specific abiotic stresses, with the aim of identifying common patterns in the cis-elements governing the regulation of these abiotic stress responsive genes. An initial dataset of fifty known genes encoding for proteins reported to be up- or down-regulated in response to plant stresses that result in water-deficit at the cellular level viz. drought, low temperature, and salinity, were identified, and a postgreSQL database created to store relevant information pertaining to these genes and the proteins encoded by them. The genomic DNA was obtained where possible, and the promoter and intron regions identified. The Neural Network Promoter Prediction (NNPP) software package was used to predict the transcription start signal (TSS) and the promoter searching software tool, TESS (Transcription Element Search Software) used to identify known and user-defined cis-elements within the promoter regions of these genes. Currently available promoter prediction software analysis tools are reported to predict one promoter per kilobase of DNA, whilst functional promoters are thought to only occur one in 30-40 kilobases, which indicates that a large perccntage of predictions are likely to be false positives (pedersen et. al., 1999). NNPP was chosen as it was rated as the highest performing promoter prediction software tool by Fickett and Hatzigeorgiou (1997) in a thorough review of eukaryotic promoter prediction algorithms, however results were less than promising as very few predicted TSS were identified in the area 50 bps up- and downstream of the gene start site, where biologically functional TSSs are known to occur (Reese, 2000; Fickett and Hatzigeorgiou, 1997). TESS results seemed to support the hypothesis that drought, low-temperature and high salinity plant stress response proteins have similar as-elements in their promoter regions, and suggested links to various other gene regulation mechanisms viz. gibberellin-, light-, auxin- and development-regulated gene expression, highlighting the vast complexity of plant stress response processes. Although far from conclusive, results provide a valuable basis for future comparative promoter studies that will attempt to deduce possible common transcriptional initiation of abiotic stress response genes. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2005