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Item Enhancement of abiotic stress tolerance of lessertia frutescents using plant growth promoting rhizobacteria.(Tshwane University of Technology, 2024-03-01)Lessertia (L.) frutescens is among the medicinal plants which caught the interest of conservationists as it is over exploited and risks becoming extinct. Moreover, the worsening climate conditions in South Africa may negatively impact the efficacy and yield of L. frutescens. Despite the multipurpose medicinal benefits associated with L. frutescens, little has been done to conserve the plant. To provide an environmentally friendly solution to the challenges surrounding L. frutescens, this study aimed to explore the use of plant growth promoting rhizobacteria to induce drought and salt stress tolerance in L. frutescens. The rhizobacteria was trapped using a homologous host L. frutescens from rhizosphere soils sampled in the Western Cape and Northern Cape, South Africa, under glasshouse conditions. A total of 111 bacterial isolates with varying morphological characteristics were obtained and 67 authenticated with the homologous host. The genetic diversity, phylogenetic relationships, tolerance to extreme conditions (drought, pH and salinity) and plant growth promoting properties (phosphorus solubilising and indole acetic acid capability) of the authenticated microsymbionts were evaluated for accurate selection of three isolates with superior biomass yield, drought and salt tolerance for further studies. Genetic diversity dendogram clustered the 67 test isolates into 7 major clades, with most isolates grouping according to their province of origin. Meanwhile, the phylogenetic study that targeted multiple genes (16s rRNA, glnII, gyrB, atpD, nifH and nodC) identified genus Mesorhizobium (M.) as the species that formed symbiotic relationship with Lessertia frutescens in the Western Cape and Northern Cape provinces. The specific species involved in the nodulation of L. frutescens had high sequence similarity with M. atlanticum, M. australicum and M. shonense. Furthermore, the tested microsymbionts exhibited markedly different indole acetic acid (IAA) content and phosphorus solubilising (PSB) ability. Majority (81%) of the isolates recorded IAA value less than 10%, while only 48% exhibited ability to solubilise phosphorus. Interestingly, the isolates from the Western Cape province recorded outstanding performance in the pH and salt stress tolerance assay, while the Northern Cape isolates performed better in the drought tolerance assay. These observations were attributed to the ability of rhizobacteria isolates to evolve and device mechanism for survival under extreme conditions, demonstrating the significance of exploring the microsymbionts in the Western Cape and Northern Cape provinces to combat drought and salt induced stress. Ultimately, three rhizobial microsymbionts TUTLFNC33 (biomass yield), TUTLFNC37 (drought tolerance) and TUTLFWC74 (salt tolerance) were selected to ascertain their remarkable traits in a glasshouse pot trial experiment. A glasshouse pot trial experiment was set up to evaluate the three selected rhizobacteria isolates for their performance when used to inoculate L. frutescens under drought and salt stress. Isolate TUTLFNC37 was found to be the most effective to enhance drought and salt stress tolerance in Lessertia frutescens. The rhizobacteria isolate applied various mechanisms including root proliferation, stomatal closure and osmolyte proline accumulation, to enhance tolerance of L. frutescens to drought and salt stress. Most importantly, the concentrations of the metabolites that are unique to Lessertia frutescens (sutherlandiosides A – D and sutherlandins A – D) remained stable under various levels of stress when the plant was inoculated with TUTLFNC37. This study is the first to explore microsymbionts in the Western Cape and Northern Cape provinces for enhancement of drought and stress tolerance in L. frutescens. Although further studies are required to verify the performance of TUTLFNC37 under a field setting and abiotic stress conditions, the findings of this study form significant part of the preliminary work towards developing biological inoculants for Lessertia frutescens and possibly other leguminous medicinal plants.Item HPTLC fingerprinting of croton gratissimus leaf extract with preparative HPLC-MS-isolated marker compounds.(Elsevier, 2017-10-14)The leaves, root and bark of the aromatic African indigenous plant, Croton gratissimus Burch. (Euphorbiaceae), are widely used in traditional medicine to treat coughs, chest complaints, rheumatism, abdominal disorders and fever among others. In Afrikaans it is referred to as “Koorsbessie” which alludes to its traditional use as a pyrogenic. The chemical composition of plants is very complex and analysis and quality control can be very challenging due to natural variability. In addition, very few reference standards from plants, especially from Africa, are commercially available. Due to its visual nature and the holistic fingerprint produced, high performance thin layer chromatography (HPTLC) is often recommended for the quality control of plant material. The aim of this study was to develop an HPTLC fingerprint and isolate marker compounds for inclusion on HPTLC plates to enable quality control. Croton gratissimus leaf samples were collected from various parts of South Africa and extraction was optimised. HPTLC fingerprints were developed and optimised and images were captured before and after derivatisation under UV (254 nm, and 366 nm) and white light. Preparative high performance liquid chromatography hyphenated to mass spectrometry (HPLC-MS) was used to isolate marker compounds. Method development and optimisation determined the following: most efficient extraction solvent = methanol:water (8:2 v/v); mobile phase = ethylacetate:acetic acid:formic acid:water (100:11:11:27 v/v/v/v); and derivatising agent = natural product reagent. UPLC-MS analysis and 1D NMR spectroscopy were used to characterise and identify compound 1 as isoorientin and compound 2 as kaempferol-3-β-D-(6″-O-trans-p-coumaroyl) glucopyranoside, which correlated well with published spectral data. The final HPTLC fingerprint with biomarkers included showed good separation for profiling purposes and well-defined bands. The biomarkers at retention factor (Rf) 0.30 and Rf 0.69 for isoorientin and kaempferol-3-β-D-(6″-O-trans-p-coumaroyl) glucopyranoside, respectively, were present in all samples but varied quantitatively. The HPTLC method developed provided a good fingerprint for species authentication. Preparative HPLC-MS played a major role in successfully isolating marker compounds to be used for the quality control of C.gratissimus.Item The role of the South African Journal of Botany as a vehicle to promote medicinal plant research– A bibliometric appraisal.(Elsevier, 2018-07-16)South Africa's unique flora and extensive use of African traditional medicines has created a unique research opportunity on medicinal plants for local and international scientists. In this paper, submitted to a special issue of South African Journal of Botany(SAJB) dedicated to “sub-Saharan Ethnobotany”, we aim to explore the research landscape on medicinal plant research as published in SAJB. A bibliometric assessment (1982–2017) showed that medicinal plant research represents 24% of the published volume in SAJB and that contributions on this topic are increasing exponentially. Although most papers are broad-basedinvitro screening studies, the range of topics covered is impressive. Antimicrobial (41%) and anti-oxidant (21%) studies dominate the research space with fewer studies focusing on diseases highly relevant to the African continent, e.g. malaria (3%) and tuberculosis (2%). Although the SAJB has contributed greatly to disseminating knowledge on important species (e.g. Aspalathus and Cyclopia) several taxa of potential economic relevance remain poorly investigated (e.g. Adansonia, Bulbine etc.). The citation footprint and authorship networks which have been established attest the pivotal role the SAJB has played to build research capacity and create awareness in this important field of research.Item Phytochemical profiling and quality control of terminalia sericea burch. ex dc. using hptlc metabolomics.(MDPI, 2021-01-05)Terminalia sericea is used throughout Africa for the treatment of a variety of conditions and has been identified as a potential commercial plant. The study was aimed at establishing a high-performance thin layer chromatography (HPTLC) chemical fingerprint for T. sericea root bark as a reference for quality control and exploring chemical variation within the species using HPTLC metabo3lomics. Forty-two root bark samples were collected from ten populations in South Africa and extracted with dichloromethane: methanol (1:1). An HPTLC method was optimized to resolve the major compounds from other sample components. Dichloromethane: ethyl acetate: methanol: formic acid (90:10:30:1) was used as the developing solvent and the plates were visualized using 10% sulfuric acid in methanol as derivatizing agent. The concentrations of three major bioactive compounds, sericic acid, sericoside and resveratrol-3-O-β-rutinoside, in the extracts were determined using a validated ultra-performance liquid chromatography-photodiode array (UPLC-PDA) detection method. The rTLC software (written in the R-programming language) was used to select the most informative retardation factor (Rf) ranges from the images of the analysed sample extracts. Further chemometric models, including principal component analysis (PCA) and hierarchical cluster analysis (HCA), were constructed using the web-based high throughput metabolomic software. The rTLC chemometric models were compared with the models previously obtained from ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS). A characteristic fingerprint containing clear bands for the three bioactive compounds was established. All three bioactive compounds were present in all the samples, although their corresponding band intensities varied. The intensities correlated with the UPLC-PDA results, in that samples containing a high concentration of a particular compound, displayed a more intense band. Chemometric analysis using HCA revealed two chemotypes, and the subsequent construction of a loadings plot indicated that sericic acid and sericoside were responsible for the chemotypic variation; with sericoside concentrated in Chemotype 1, while sericic acid was more abundant in Chemotype 2. A characteristic chemical fingerprint with clearly distinguishable features was established for T. sericea root bark that can be used for species authentication, and to select samples with high concentrations of a particular marker compound(s). Different chemotypes, potentially differing in their therapeutic potency towards a particular target, could be distinguished. The models revealed the three analytes as biomarkers, corresponding to results reported for UPLC-MS profiling and thereby indicating that HPTLC is a suitable technique for the quality control of T. sericea root bark.Item A quality control perspective on devil’s claw, harpagophytum procumbens and H. zeyheri: Phytochemical analysis and DNA barcoding.(Elsevier, 2021-09-23)Devil’s claw is a popular natural anti-inflammatory phytomedicine. The preparation of Devil’s claw products is complicated by the similarity between the morphotypes of Harpagophytum procumbens and H. zeyheri. This study investigated the potential of a validated ultra-high performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) method to investigate the harpagoside content of methanol extracts of the secondary tubers of both species. In addition, DNA barcoding was used to determine the identity of the Harpagophytum species included in commercial Devil’s claw products. Herbal reference material of H. procumbens and H. zeyheri, originally harvested in southern Africa, was morphologically authenticated and commercial products containing Harpagophytum were purchased on the internet using the search terms “Harpagophytum” or “Devil’s claw.” High-performance thin-layer chromatography (HPTLC) and UHPLC-MS was used for the qualitative and quantitative determination of harpagoside content in commercial products. The UHPLC-MS method developed had a limit of detection (LOD) of 0.165 mg/mL and a limit of quantification (LOQ) of 0.499 mg/mL. Quantitative UHPLC-MS analysis revealed high variability in the harpagoside content for both species; H. procumbens (0.17-4.37%) and H. zeyheri (0.00-3.07%). Only 41% of H. procumbens and 17% of H. zeyheri raw materials contained harpagoside. Using HPTLC, 90% of the commercial products tested were determined to contain harpagoside. The results were congruent with the UHPLC-MS analysis where the harpagoside content was out of specification for 10% of analyzed commercial products. The harpagoside content in commercial products varied from not detected to 3.50%. PCR amplification followed by comparative sequencing analysis of trnL-F, and ycf1 plastid gene amplicons indicated specific sites in trnL-F at positions 118 and 146 and at position 538 in ycf1 amplicons suitable to distinguish the two Harpagophytum species. These sites were used to barcode the herbal products and Character-based Barcode Recognition Obtained with Nucleotide eXposes (BRONX) analyses verified the taxonomic identity of the Harpagophytum in the samples. The results revealed that 76% (16/21) of the commercial products tested were incorrectly labelled as H. procumbens and, instead, contained H. zeyheri. Our study suggests that DNA barcoding in combination with the UHPLC-MS and HPTLC could significantly support herbal product authentication. This study contributed knowledge and methods that can be implemented to enhance the quality of commercial Devil’s claw products.
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