Research Centre for Plant Growth and Development

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Ameliorative effects of botanicals and rhizobacteria on the growth of Pelargonium sidoides and Solanum lycopersicum infested with Meloidogyne incognita.
(2021) Sithole, Nokuthula Thulisile.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.
Abstract available in PDF.
Antidiabetic and phytochemical properties of four selected medicinal plants.
(2019) Ratsoma, Manchela Francinah.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.
Abstract available in pdf.
A comparative evaluation of the biological activities and phytochemical properties in Ehretia obtusifolia and Ehretia rigida.
(2021) Mnikathi, Mzamo Mpendulo Ntethelelo.; Finnie, Jeffrey Franklin.; Van Staden, Johannes.
Ehretia from the Boraginaceae family is predominantly found in parts of Asia and North America, with fewer species found in Africa, Europe, and Australia. The genus consists of more than 150 species, and species such as E. microphylla, E. accuminata, E. laevis have been reported on their medicinal prowess. Distribution of Ehretia in Southern Africa is found among a variety of habitats such as the lush forests of the Eastern Cape and Arid parts of Namibia. In South Africa, two species have been identified, namely E. rigida and E. obtusifolia and are used in traditional medicine. African and Asian countries traditional medicine is highly recommended because of its affordability. The aim of the study was to establish a baseline and compare different biological activities and phytochemical properties exhibited by the two South African species. In the study, phenolics, saponins, flavonoids, and tannins were detected in bark, roots, and leaves, of both species, but no detection of alkaloidsf. E. obtusifolia had a higher quantity of flavonoids than E. rigida. Both species exhibited high phenolic quantities in leaves with E. rigida having the highest quantity. Condensed tannins were found with a higher content in leaves than roots and bark for both species, with E. rigida containing higher quantities. E. rigida had the lower MIC’s compared to E. obtusifolia (0.195 mg/ml against M. luteus from ethyl-acetate root extracts). E. rigida had more samples with a MIC lower than 1 mg/ml than E. obtusifolia. The lowest MIC for leaves was 0.39 from ethyl-acetate extracts against S. aureus while methanol bark extracts also achieved 0.39 mg/ml against M. luteus. E. obtusifolia’s lowest MIC was 0.195 mg/ml from methanol leaf extracts against K. pneumoniae. Activity against C. albican was not as good as against the bacterial strains, as the lowest MIC was 0.78 mg/ml for both species. E. rigida and E. obtusifolia had dose-dependent antioxidant activity, with methanol and ethyl-acetate bark, leaf, and root extracts having the highest activities for both species. This study revealed that in comparison to literature, the activity achieved was similar or better when compared to the likes of E. laevis extracts. The α-glucosidase inhibitory activity reported in this study was dose-dependent. The relationship between antioxidant activity and antidiabetic activity is well documented and this study found that extracts with high antioxidant activity also had similarly high α-glucosidase inhibitory activity. It was E. rigida methanol extracts from bark and roots which exhibited the highest activities compared to E. obtusifolia. However, based on the dosedependent activity, E. obtusifolia is more potent because of the higher activity observed at the lowest concentrations. The study demonstrated that both species have good ethnopharmacological properties and were rich in phytochemicals, particularly phenolics and flavonoids. With E. rigida being the least studied of the two species with only one reported study, it was important to carry out this investigation as it has yielded further evidence that the genus Ehretia has multiple species with medicinal potential.
The effect of organic biostimulants and the mode of application on the growth and biochemical composition of Amaranthus hybridus L.
(2020) Ngoroyemoto, Nelson.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.; Kulkarni, Manoj G.
Abstract available in PDF.
Elicitation, metabolomic analysis, and identification of antidiabetic compounds from selected indigenous plants = Ukuvuselelwa, Uhlaziyo Lokugayeka, nokuhlonzwa Kwezingxube Zesinqindasifo Sikashukela Ethathwe Ezihlahleni Zendabuko Eziqokiwe.
(2022) Ogbe, Abdulazeez Adeola.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.
Diabetes mellitus (DM) is an endocrine disorder associated with high blood glucose levels accompanied by disruptions in the metabolism of fat, proteins and carbohydrates. DM is a chronic, non-communicable and medically incurable disease affecting millions of people globally, resulting in high morbidity and mortality rates, especially with the lingering coronavirus disease of 2019 (Covid-19). The use of western antidiabetic medicine has posed many challenges due to their perceived overall safety, treatment failure and cost. Many African communities rely on medicinal plants and their bioactive compounds as sources of medicine as a consequence of the poor state of health facilities, shortage of medical doctors and unaffordability of treatments. For this reason, this study partly evaluated the phytochemical contents, in vitro antioxidant and hypoglycaemic potentials of eleven indigenous plants using five different solvents. Putative hypoglycaemic agents from one of the most promising and readily available species were also identified using in silico molecular modelling. Secondary metabolites and their pharmacological activity have been reported as the basis for the wide use of plants in traditional medicine. However, due to the indiscriminate harvesting and environmental pressure, many valuable indigenous plant species have gone into extinction or are at least threatened. Moreover, plants' bioactive compounds are often produced in minute quantities, and prevailing environmental conditions further influence their concentrations in plants. Thus, due to indigenous plants' industrial and medicinal value, deliberate cultivation and elicitation strategies have been adopted for the en masse production of uniform indigenous plants and to influence the quality and quantity of their active principles. Thus, this study also assessed the effects of individual and co-inoculation of two isolated drought-resistant and growth promoting endophytes on the growth, drought tolerance, medicinal efficacy and metabolome changes in the leaves of Endostemon obtusifolius. In this research, the eleven plants were selected based on the traditional uses of the plants (or their related available species) for treating various ailments, including DM. The preliminary phytochemical quantification results revealed that the highest concentrations of phenolics, flavonoids and tannins were found in the crude extracts of Combretum krausssii, Lippia javanica, Psidium guajava, Pentanassia prenulloides, E. obtusifolius, Syzgium cordatum, Pachira aquatic and Catha edulis. The inhibitory effects of the crude extracts against the digestive enzymes α-amylase and α-glucosidase also showed that the crude extracts of C. edulis, C. krausssii, L. javanica, P. aquatica, P. guajava, P. prenulloides, E. obtusifolius and S. cordatum displayed excellent in vitro antioxidant and antidiabetic properties. These results validate the extensive use of these plants in the treatment of DM in many African communities. Furthermore, the 80% ethanol (v/v) leaf extract of S. cordatum (one of the most active and readily accessible specie from the previous study) was fractionated into four sub-extracts [petroleum ether (PE), dichloromethane (DCM), ethyl acetate (EtOAc) and water], and their phytochemical content, in vitro antioxidant and antidiabetic capacities were evaluated. Although the EtOAc extract was the richest of the sub-extracts in total phenolics, all four sub-extracts of S. cordatum showed good in vitro free radical scavenging and hypoglycaemic activities. In silico modelling evaluation of some (34) bioactive principles found in the Gas Chromatography-Mass Spectroscopy (GC-MS) analysis of the PE, DCM and EtOAc sub-extracts revealed that 21 compounds including andrographolide, benzylidene-iditol, cubenol and deoxyspergualin and bis[3,3,4,7-tetramethyl-1,3-2H-benzofuran-1-yl]-ether returned binding energy scores ≤ -7.5 kcal/mol against α-amylase and α-glucosidase enzymes indicative of their hypoglycaemic potentials. The physicochemical and toxicological properties of andrographolide, benzylidene-iditol, bis[3,3,4,7-tetramethyl-1,3-2H-benzofuran-1-yl]-ether and cubenol were predicted to be soluble with high gastrointestinal solubility and non-toxic following Lipinski's rule of five and Veber's rule. Thus, these results indicate that these compounds are potential candidates for oral drugs. The drought tolerance and in vitro plant growth-promoting properties of some endophytes isolated from E. obtusifolius (another active antidiabetic plant identified from the previous experiment) was evaluated. A total of 26 culturable endophytes (twelve fungi and fourteen bacteria) were isolated from the organs (leaf and root) of E. obtusifolius. These endophytic species displayed varying in vitro drought stress tolerance and plant-growth-promoting capacities. Two promising drought stress-tolerant and plant-growth-enhancing endophytic species (Fusarium oxysporum and Paenibacillus polymyxa) were subsequently identified using molecular tools. The identified bacterium (P. polymyxa) and fungus (F. oxysporum) exhibited a symbiotic relationship in an in vitro dual culture experiment. Paenibacillus polymyxa and F. oxysporum individual and co-inoculation differential effects on their host under varying water regimes was further evaluated. The plants were raised with or without endophyte infection under three watering regimes for two months, and their therapeutic efficacy, physiological, biochemical and metabolic responses were assessed. In this study, drought stress markedly affected the growth and hypoglycaemic potentials of E. obtusifolius. On the other hand, endophyte inoculation generally enhanced the dry shoot and root biomass, chlorophyll contents and fluorescence, total soluble sugar, relative water content, proline contents and superoxide dismutase activities in the leaves of E. obtusifolius, whereas their electrolyte leakage and malondialdehyde contents were lowered. As for phytochemical accumulation, while the total phenolic contents were slightly enhanced by the inoculation of endophytes in the leaves of E. obtusifolius, the flavonoid contents of the plant increased as the water deficit worsened. The EtOAc crude extracts' free radical scavenging capacity across the treatments remained unchanged; their in vitro α-glucosidase activity was negatively affected under moderate and severe drought stress but improved with endophyte inoculation. The metabolome difference between the twelve treatments was evaluated using GC-MS based metabolomics. The bi-plot PCA result revealed that the metabolome of fungal inoculated moderately stressed E. obtusifolius correlated less with the other E. obtusifolius plants under different treatments. Additionally, a heatmap of eight differential metabolites showed that the most responsive treatment (the co-inoculated severely drought-stressed plants) produced the highest quantities of non-protein amino acids and organic acids known to protect plant cells during abiotic stress. The leaf extracts of S. cordatum and E. obtusifolius showed remarkable antioxidant and antidiabetic potentials in this study. Although the putative active principles of these plants were identified using GC-MS analysis, proper isolation and quantification of these compounds can be explored by future studies. Moreover, some culturable endophytic species were isolated from the E. obtusifolius organs. Paenibacillus polymyxa and F. oxysporum showed their drought stress mitigating capacity in E. obtusifolius under varying water regimes. Although the concentration of some identified antidiabetic compounds in E. obtusifolius were up regulated, the mechanism involved in this observation requires further investigations. IQOQA Isifo sikashukela (Diabetes mellitus - DM) siwukungasebenzi kwezitho zangaphakathi okuhambisana nobuphezulu kwamazinga eglukhosi egazini, kuhambisana nokuphazamiseka kokugayeka kwamafutha, izakhamzimba ezingamaphrotheni nezinikimandla. IDM iyisifo esingelapheki, esingathelelani nesingelapheki ngokwemithi, esiphethe izigidi zabantu emhlabeni jikelele, kuphethe ngamazinga okugula aphezulu nokufa imbala, ikakhulu esimeni sokungakhawuki kwesifo ukhuvethe (coronavirus disease of 2019 - Covid-19). Ukusetshenziswa kwemithi eyizinqindimandla zesifo sikashukela yasentshonalanga sekudale izinselelo eziningi ngenxa yokucabangeka ukuthi kuthinta ukuphepha kwayo jikelele, ukwehluleka ukwelapha nezindleko. Iningi lemiphakathi yase-Afrika yethembele ezihlahleni eziyimithi (yokwelapha) nezingxube zayo ezinokuphilayo njengesizinda semithi ngokomphumela wesimo esingenele sezinsiza zezempilo, ukwesweleka kodokotela abelaphayo nokungameleki kwezindleko zokwelashwa. Ngalesi sizathu-ke, ingxenye yalolu cwaningo yahlola okuqukethwe ngamakhemikhali ezihlahla, ngezivikelizinhlayiya ezifakwe eshubhini lokuhlola nama-ejenti anezinga eliphezulu likashukela egazini avela kolunye uhlobo olwethembisayo nolutholakala kalula olwatholwa kusetshenziswa ukumodela kwemolekhula ngekhompiyutha. Kwabikwa ukuthi ukugayeka kokudla kwezinga lesibili, nomnyakazo wakho wezokwelapha ngemithi kuyisisekelo sokusetshenziswa kabanzi kwezihlahla emithini yendabuko. Nokho-ke, ngenxa yokuvunwa okunga okungakhethi nengcindezi yezokuvikelwa kwezemvelo, iningi lezinhlobo zezihlahla zendabuko zenani eliphezulu seziphelile kungenjalo-ke zisengcupheni. Naphezu-ke, izingxube ezinokuphilayo zivama ukukhiqizwa ngezamba ezincanyana, nezimo zokuvikelwa kwezemvelo ezikhona ziphinda zibe nomthelela wazo ogxile ezihlahleni. Ngakho-ke, ngenxa yenani lezihlahla zendabuko ngokwezimboni nakwezokwelapha, sekufakwe ngokwenhloso amasu okuzitshala nokuzivuselela ukuze zikhiqizele ngobuningi bazo nangokwefana kwezihlahla zendabuko nokuba nomthelela kohlonze nasebuningini kwemithetho esebenzayo. Ngalokho-ke lolu cwaningo lwahlola imithelela yokunye nokugonywa okuhlanganisiwe kokukhethiwe kokuhlala ezinhlayiyeni eziphilayo zokukhula, okumelana nesomiso nokukuthaza ukukhula, ukumelana nesomiso, ukwenza kahle ekwelapheni nezinguquko zokugayeka emaqabungeni e-Endostemon obtusifolius. Naphezu-ke, kwahlolwa i-ethanol (v/v) emuncwe emaqabungeni engama-80%, ye- S. cordatum (engenye yezinhlobo ezitholakala kalula nesebenzayo esukela ocwaningweni lwaphambilini) yacozululwa yaba ngokumunciwe okuncanyana [petroleum ether (PE), dichloromethane (DCM), ethyl acetate (EtOAc) namanzi], nokwamakhemikhali ezihlahla kwawo, nezivikelizinhlayiya ezifakwe eshubhini lokuhlola, nokukwazi ukulwa nesifo sikashukela. Nakuba isimuncwa i-EtOAc kwakuyiyo ecebe kunazo zonke izimuncwa ezincane zamafenolikhi aphelele, zonke izimucwa ezincane zozine ze- S. cordatum zakhombisa ukukwazi ukuthungatha okukhululekile nokunamandla nokulwa nezinga eliphezulu likashukela egazini. Ukuhlolwa kokumodelwa ngokwekhompiyutha, kwalokho okunokuphilayo okungama (34) ngokwemitheshwana yokunokuphilayo okwatholwa ohlaziyweni lwe-Gas Chromatography-Mass Spectroscopy (GC-MS) kwezimuncwa ezincane ze- PE, DCM ne-EtOAc zakhombisa ukuthi izingxube ezingama-21 kubalwa kuzo ne- andrographolide, benzylidene-iditol, cubenol ne-deoxyspergualin nebis[3,3,4,7-tetramethyl-1,3-2H-benzofuran-1-yl]-ether kwabuyisa imiphumela yamandla okubophezela ≤ -7.5 kcal/mol aphikisana nama-enzayimu e-α-amylase ne-a-glucosidase akhombisa ukuthi angakwazi ukulwa nezinga eliphezulu lesifo sikashukela. Okuqukethwe yiphysicochemical netoxicological andrographolide, benzylidene-iditol, bis[3,3,4,7-tetramethyl-1,3-2H-benzofuran-1-yl]-ether necubenol kwabikezelwa ngokukwazi ukugayeka ngokomgudusisu-mathumbu wokugayeka nokungabi nasihlungu kulandela umthetho kaLipinski wokuhlanu nomthetho weVeber. Ngalokho-ke, le miphumela ikhombisa ukuthi lezi zingxube zinganethuba lokungenela ukuba yimithi ephuzwayo. Kwahlolwa ukukwazi ukumela isomiso nokukwazi ukukhuthaza ukukhuliswa kwezinhlaka zezihlahla eshubhini lokho okuhlala kwezinye izinhlayiya zokuphilayo okwakhishwa ku- E. obtusifolius (esinye sezihlahla esilwa nesifo sikashukela esahlonzwa elingeni laphambilini). Isamba salokho okuhlala ezinhlayiyeni zokuphilayo sokukhulisekayo okungama-26 (ishuminambili lesikhunta namagciwane ayishumi nane) kwehluswa ezithweni (amaqabunga nezimpande) ze- E. obtusifolius. Lezi zinhlobo ezinokuhlala ezinhlayiyeni zalokho okuphilayo zakhombisa ukumelana nesomiso okwahlukene uma zifakwa eshubhini lokuhlola nokukwazi ukuba ngokukhuthazwa kokukhula kwezihlahla. Kwagcina sekutholwe izihlahla ezimbili ezikwazi ukumelana nokuhlukunyezwa yisomiso nokukwazi ukukhuthaza ukukhuliswa kwezihlahla kohlobo okuhlala kulo okuphilayo (Fusarium oxysporum nePaenibacillus polymyxa) kusetshenziswa amathuluzi anobumokhula. Ubugciwane obatholakala (P. polymyxa) nobukhunta (F. oxysporum) bakhombisa ubudlelwano obunokwencikana elingeni leshubhu lokuhlola elikhulisa ngakubili. Kwaphinda kwahlolwa iPaenibacillus polymyxa ne-F. oxysporum okungakunye nomgomo ohambisana nemithelela ehlusayo kokungumgcini wakho ezinhlotsheni zamanzi ezahlukene. Kwahlolwa izihlahla ezakhuliswa ngaphandle kokufakwa okuhlala kokuphilayo, ngaphansi kwezinhlobo ezintathu zokuchelela ngezinyanga ezimbili, nokusebenza kahle kokukwazi ukwelapha kwazo, ngokomzimba, ngokwamakhemikhali aphilayo nokwenza kwakho ekugayweni kokudla. Kulolu cwaningo, ukuhlukumeza kwesomiso kwaba nomthelela omkhulu ekukhuleni nokukwazi ukuvimba izinga eliphezulu likashukela kwe- E. obtusifolius. Ngakolunye uhlangothi, ukugonywa kwalokho okuhlala kokuphilayo kwavama ukukhuthaza ibhayomasi yezithombo nezimpande, okuqukethwe yiklorofili nokukhanyayo, ushukela oncibilika ngokuphelele, isikalo samanzi esinokuhambisana, okuqukethwe ngokwenziwa yiproline nesuperoxide okwenzeka ekuhlakazekeni kabili emaqabungeni e-E. obtusifolius, kanti kwehliswa ukuvuza kwe-elekthrolaythi yakho nokuqukethwe yimalondialdehyde. Ngokokunqwabelana kwamakhemikhali ezihlahla, ngenkathi okuqukethwe kwefenolikkhi kwakukhuthazwa ngukugonywa kwalokho okuhlala kokuphilayo emaqabungeni e-E. obtusifolius, okuqukethwe yiflavanoydi kwesihlahla kwakhula ngenkathi ukuncipha kwamanzi kudlanga. Okwe-EtOAc okungagayiwe okumuncwe ngomthamo okhululekile nokokuzingela okumawala kuzo zonke izinhlobo zokwelapha kwahlala kungaguqukile; ukusebenza kwe- α-glucosidase eshubhini lokuhlola kwatheleleka kabi ngaphansi kokuhlukunyezwa yisomiso esiphakathi nendawo nesadlulele, kodwa kwathuthuka ngokugonywa ngokuhlala kokuphilayo. Umehluko ekugayweni phakathi kokwelaphayo okuyishumi nambili wahlolwa kusetshenziswa i- GC-MS egxile kwezokugaya. Umphumela we bi-plot PCA waveza ukuthi ukugayeka kwesikhutha esigonyiwe kwahlukumeza ngokuphakathi kwendawo i- E. obtusifolius ehambisana kancane nezinye izihlahla ze- E. obtusifolius ngaphansi kokwelashwa okwahlukile. Ngokwengeziwe-ke, ibalazwe lokushisa kokugaya okunomehluko okuyisishiyagalombili kwakhombisa ukuthi ukwelapha okuzwela kakhulu (kwezihlahla ezigonywe ngokuhambisana ezihlukunyezwe kakhulu yisomiso) kwakhiqiza izibalo eziphezulu zezakhimaphrotheyni nama-esidi emvelo aziwa ngokuvikela izinhlayiya zezihlahla ngenkathi yokuhlukunyezwa ngokungenampilo. Okumuncwe emaqabungeni e- cordatum ne-E. obtusifolius kwakhombisa ubukhona kwezivikelizihlahla nokulwa nesifo sikashukela kulolu cwaningo. Nakuba imitheshwana esebenzayo ehlawumbiselwayo kwalezi zihlahla yabukwa kusetshenziswa uhlaziyo iGC-MS, ukwahluswa okufanele nokubalwa kwalezi zingxube kungahlolwa ezifundweni zangomuso. Naphezu-ke ezinye izinhlobo ezikhulisekayo zalokho okuhlala kokuphilayo zehluswa ezingxenyeni ze-E. obtusifolius. IPaenibacillus polymyxa ne-F. oxysporum kwakhombisa ukukwazi ukunciphisa ukukhandlwa yisomiso kwe- in E. obtusifolius okwalawulwa ngokuphakama, ukusebenza okwafakwa ekwethameleni kudinga okunye ukuhlolwa.
Enhancing phenolic compound production in medicinal plants.
(2017) Paine, Christine Susan.; Finnie, Jeffrey Franklin.; Van Staden, Johannes.
Abstract available in PDF file.
Evaluation of plants used in African traditional medicine for asthma and related conditions.
(2014) Motlhatlego, Katlego Ellena.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.
Traditional medicine is a form of discipline that has been applied within most South African societies with the objective of enhancing the physical and psychological health system in the country. Asthma is a complex inflammatory disease that involves the narrowing of the airways. The prevalence of asthma is increasing worldwide, and this chronic disease has been identified as a significant cause of morbidity and mortality. Asthma poses a major threat to health across the population of South Africa. The adverse effects of current treatments have encouraged the use of traditional medicine. The primary aim of the research study was to evaluate the efficacy of plants used in African traditional medicine against asthma and chest infections. This was achieved by screening Adansonia digitata, Ballota africana, Catha edulis, Datura stramonium, Pelargonium sidoides, Siphonochilus aethiopicus, Xerophyta retinervis and Zantedeschia aethiopica for their pharmacological properties against key bacteria; Staphylococcus aureus (ATCC 12600), Klebsiella pneumonia (ATCC 13883), Streptococcus pyogenes (ATCC 12344) and Haemophilus parainfluenzae (ATCC 7901) as well as the fungus Candida albicans (ATCC 10231) these microorganisms are known to cause chest infections. In the microdilution antibacterial assay, the crude extracts of the screened medicinal plants showed activity at minimal inhibitory concentrations (MICs) ranging from 0.098 to >12.5 mg/ml. In the disc-diffusion assay, only the ethanol extract of stems from Siphonochilus aethiopicus and water extract of leaves from Zantedeschia aethiopica showed zones of inhibition of 13.24 and 21.10 mm. All the other screened extracts showed no zones of inhibition, which may possibly indicate that plants were ineffective against Haemophilus parainfluenzae. One or more extracts from the tested plants were effective against one or both Gram-positive bacteria investigated in the study. There was no good antifungal activity shown in the study as the MIC and minimal fungicidal concentrations (MFCs) values were higher than 1 mg/ml. Genotoxicity of medicinal plant extracts that showed good antibacterial activity ≤ 0.5 mg/ml was evaluated using the Salmonella microsome assay without S9 metabolic activation. Two strains of Salmonella TA98 and TA102 were used in the Ames test. Most tested extracts were non-mutagenic in the Ames test except for the Siphonochilus aethiopicus roots which showed a dose dependent increase. The ethanolic crude extracts were screened in an immunological assay to determine the level of competitive binding to the receptors for the treatment of asthma and related conditions. Histamine is intimately associated with allergies. Datura stramonium flowers and fruits experienced remarkable histamine binding of approximately 97% at both concentrations (400 and 800 μg/ml). The immunological activity may be attributed to the various phytochemical constituents in the crude extracts. Ballota africana leaves and stems, Datura stramonium flowers and fruits, roots and stems as well as Zantedeschia aethiopica leaves showed excellent affinity with histamine ranging between 88 and 97% and these medicinal plants could potentially serve as a new effective antihistamine when compared to the currently available pharmaceuticals. Most of the medicinal plants tested may potentially provide remedies for asthma and related conditions such as eczema, rhinitis (hayfever), anaphylaxis, sinusitis, chronic obstructive pulmonary disease (COPD), emphysema, bronchiectasis and bronchitis.
Flower abscission in potted Plectranthus.
(2013) Rice, Laura Jane.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.; Ascough, Glendon D.
Transport and post-harvest handling of flowers both cut and potted is one of the greatest challenges in the horticulture industry (REDMAN et al., 2002). Ethylene-induced flower abscission is responsible for the loss of crops (KIM et al., 2007). Flower abscission is greater when plants are transported (ABEBIE et al., 2005). This limits the sale of flowers and potted plants to areas close to the site of production and prevents export opportunities. South Africa is home to many spectacular species with great horticultural potential (RICE et al., 2011). Unfortunately however, development of a number of these species for export is difficult due to transport-induced flower abscission. Transport-induced flower abscission is a problem experienced by Dr Gert Brits, a breeder of Plectranthus in Stellenbosch in South Africa. In this study a number of Dr Brits’s Plectranthus varieties were used as model plants to understand the process of transport-induced flower abscission and develop a protocol for the prevention of such abscission. Flow cytometry was used to determine the ploidy levels of each of the varieties. It was important to be aware of this during the experiments as varieties with different ploidy levels have been reported to behave differently under stressful environmental conditions. Of the eight varieties examined, three were diploid (2n), one was triploid (3n), three were tetraploid (4n) and one was a mixopliod (2n/4n) variety. To determine the effects of packaging plants during transport and the effects of darkness on flower abscission, plants were packaged into perspex chambers and kept either in a 16 h photoperiod or in darkness for 96 h. Every 24 h the number of open and unopened flowers that had abscised was recorded. Both packaging and darkness increased flower abscission of open and unopened flowers in all eight varieties. Four varieties preferentially abscised open flowers; while the remaining four preferentially abscised unopened flowers. All eight varieties were exposed to different concentrations of ethylene (0, 0.1, 0.25 0.5, 1 and 2 μll-1) to determine their level of ethylene sensitivity. All of the Plectranthus varieties were determined to be extremely sensitive to ethylene. With 100% flower abscission occurring within 24 h at 1 and 2 μll-1 in all varieties. In order to determine what internal changes were causing this increase in flower abscission under these conditions, the changes in the expression of key ethylene biosynthetic enzymes, cytokinin content and carbohydrates in the flowers were examined. ACS and ACO are the two key enzymes in the ethylene biosynthetic pathway (JOHNSON & ECKER, 1998). Changes in the levels of mRNAs coding for these two enzymes were examined when plants were packaged and put into the dark. In general there was an upregulation of the ethylene biosynthetic pathway and in turn this may have increased ethylene production by the plants under simulated transport conditions. However, the changes were not large enough to be solely responsible for the increased flower abscission observed under simulated transport conditions. The concentrations of 43 cytokinins were measured in pedicle tissue from plants which had been kept in the dark for 0, 24, 48, 72 and 96 h. Of the 43 cytokinins measured 21 were below the level of detection. Concentrations for the remaining 22 cytokinins at each of the time points were examined and it was found that in general cytokinin concentrations increase when plants are packaged and put into the dark. DHZ-type cytokinins remained stable during the 96 h continuous dark monitoring period, with most of the changes observed in the tZ and iP types. Peaks in cytokinin concentrations are often followed by an increase in flower abscission, indicating that an increase in cytokinin concentrations may be one of the factors causing the increase in transport-induced flower abscission. Only glucose and fructose were detected in peduncle tissue. Changes in glucose and fructose over 24 h in the greenhouse and over 0, 24, 48, 72 and 96 h in simulated transport conditions were measured. During the day, glucose and fructose levels increased towards the afternoon and evening and decreased in the early morning. This is consistent with studies conducted on other species (ALONI et al., 1996). When plants were put into the dark, glucose and fructose levels increased slightly at 24 h and then decreased to levels similar to those measured in control plants. Although there were changes in glucose and fructose level in simulated transport conditions, they were very slight and it is unlikely that these changes are not responsible for the transport-induced flower abscission. These results suggest that the observed transport-induced flower abscission is the result of increased cytokinin concentrations and expression of ACO and ACS genes when plants are packaged and put into the dark. These changes in turn cause an increase in ethylene production by the plants, and the build-up of ethylene in the transport container causes flowers to abscise. Ethylene perception by the plant is the step which could be targeted to prevent flower abscission. A number of ethylene antagonists block the ethylene receptors in the plant and in so doing prevent the receptors from binding ethylene and transducing the abscission signal. 1-MCP isone such ethylene antagonist. To test whether 1-MCP could be used for the prevention of flower abscission in Plectranthus, plants were placed in sealed perspex chambers in the light and in the dark and treated with 100 nll-1 1-MCP for a single 6 h treatment, or for 6 h every day prior to continuous exposure to ethylene. 1-MCP treatment greatly reduced ethylene- and transport-induced flower abscission when plants were treated continuously, but reduced flower abscission for the first 24 h when pre-treated with a single 6 h exposure to 1-MCP.Transport-induced flower abscission in Plectranthus is the result of exposure to ethylene. The increase in ethylene production by the plants in transport conditions is likely due to an upregulation of the ethylene biosynthetic pathway and an increase in cytokinin concentrations or movement in the pedicle tissue. This transport-induced flower abscission can be prevented by continuous treatment with 100 nll-1 1-MCP during the transport period. By using 1-MCP plants can be transported for up to 4 d and the opportunity for export is made possible.
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.
Impacts of climate change on cowpea (Vigna unguiculata L. Walp) treated with biostimulants.
(2020) Voko, Mxolisi Peter.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.
Vigna unguiculata L. Walp, commonly known as cowpea, is a warm-season herbaceous legume considered native in Africa and Asia. The crop is traditionally consumed as both a leafy vegetable and staple pulse. Although the growth behaviour and nutritional composition of V. unguiculata have been explored by the research community, information regarding the plant’s response to biostimulants under abiotic stresses remain limited. Therefore, studies of this nature are pertinent, especially in the presence of climate change which manifests into global warming, drought episodes and dissipating of natural resources. Hence, a better understanding of the effects of temperature and drought stress on V. unguiculata physiology, morphology, nutrition and phytochemistry are important to ensure high yields which is important for meeting the goals of global food security. Firstly, this study investigated the effects of seed priming with biostimulants [vermicompost leachate, VCL (1:20 v/v), commercial seaweed extract Kelpak®, KEL (0.6%) and smoke-water, SW (1:1000 v/v)] and distilled water (dH2O) on V. unguiculata germination and seedling parameters under constant day/night temperatures of 30/30, 35/35 and 40/40 °C in Conviron® plant chambers. In addition, hydroponic experiments were set-up to evaluate biostimulant efficacy on rooting. Secondly, post-germination effects of VCL 1:20 (v/v), KEL (0.6%) and SW 1:1000 (v/v) were evaluated under similar temperature conditions to ascertain the influence on morphological parameters 28 days after sowing (DAS). Thirdly, postgermination effects of VCL 1:20 (v/v), KEL (0.6%) and SW 1:1000 (v/v) were investigated under different watering regimes using greenhouse protocols to ascertain biostimulation influence on growth variables and flowering after 13 weeks. Finally, the effects of VCL 1:20 (v/v), KEL (0.6%) and SW 1:1000 (v/v) were tested on V. unguiculata’s photosynthetic pigments [chlorophyll a, b, (a + b) and carotenoids], carbohydrates, proteins and phytochemicals (total phenolics and flavonoids) grown for 11 weeks under different watering regimes in the greenhouse. Despite the biostimulants not differing significantly with the corresponding controls, KEL and SW induced marked germination at 30, 35 and 40 °C while VCL being potent under 30 and 40 °C. Seed priming (i.e. biostimulant and hydropriming) significantly improved shoot length and root length over non-priming, with biostimulant-priming being more effective under 40 °C by also inducing significantly higher leaf number, fresh weights and seedling areas compared to non-primed controls. At 30 °C, priming with the three biostimulants improved peduncle diameter, fresh weight and established a significant increase on root length and dry biomass over hydropriming. VCL was most effective at promoting shoot length, root elongation and dry weight under 30 °C while KEL was most effective in increasing seedling leaves, shoot length, root number, fresh and dry biomass of plants exposed to 35 °C. Although overall, biostimulant-non-priming was second best after biostimulantpriming, non-priming with biostimulants was able to promote key variables compared to both controls (i.e. non-primed and hydroprimed controls). Hydroponic results revealed that non-priming with VCL and SW increased root number by 3 and 4-fold, respectively, at 40 °C. SW also stands out at enhancing a significant increase in leaf number and seedling area whereas KEL was the most significant solution promoting shoot length and seedling area. At 30 °C, SW-non-priming promoted significant root elongation, improved fresh and dry weights while VCL was best at promoting root number and root length. KEL and SW also exhibited post-germination effects over the control at 40 °C by improving leaf number on a weekly basis. Fresh and dry weight were improved similarly with significant improvements at 30 °C by KEL and SW. Increasing watering regimes from once to thrice a week significantly increased the number of leaves, root length and flower number. The number of nodules, however, did not differ significantly. Restricting watering frequency to once a week significantly increased shoot length, root length and leaf area in SW-treated plants compared to the control. Shoot length and root length of KEL-treated plants were also increased similarly. Remarkably, VCL increased the number of nodules and shoot length by 4 and 3-fold, respectively. Relative leaf weekly growth in SW, VCL and KEL was higher by 1, 4 and 5 leave(s), respectively, after 11 weeks under high water deficits. This foliage increase remained high by more than 4 leaves in KEL and SW-treated plants watered twice and thrice a week. Accompanying increase in number of flowers was only established in SW water-stressed plants. However, raising watering frequency to twice a week increased flower number in SW, VCL and KEL-treated plants by 2, 4 and 7-fold, respectively, compared to the control. This floral increase was still comparatively high by 2, 4 and 2-fold, respectively, in plants watered thrice a week with biostimulants. VCL also induced a marked significant increase on root length, peduncle diameter and dry weights of plants watered thrice a week. Decreasing substrate water availability from thrice to once a week induced a general increase in leaf soluble proteins, total phenolics and flavonoids. This watering transition significantly enhanced root soluble carbohydrates and proteins while root phenolics and flavonoids markedly declined. VCL, KEL and SW promoted leaf carbohydrates coupled with significant increases in those of roots of SW plants compared to the corresponding controls. Remarkably, leaf soluble proteins of biostimulant plants significantly declined to within the ranges of the plants watered twice and thrice a week. Root proteins were significantly greater to those of leaves in high water-stressed plants and statistically the same to those of roots of plants watered twice and thrice a week. Total phenolics and flavonoids of foliage of the biostimulant plants were lowered and relatively the same in the different watering regimes. Root total phenolics were highly inhibited in less watered plants and gradually increased with an increase in watering regimes. Similar trends were established in flavonoids although they were greater than in the corresponding controls. Biostimulant photosynthetic pigments [i.e. chlorophyll a, b, (a + b) and carotenoid contents] did not differ significantly with those of the control in plants watered once a week. However, the three biostimulants were able to improve chlorophyll a and a + b. KEL and SW induced higher increase in chlorophyll b and carotenoid concentrations. The biostimulants increased chlorophyll a in 3-day-watered plants by more than 2-fold. These biostimulants also improved the chlorophyll a + b and carotenoid contents. Both increasing and decreasing trends in compatible solutes (i.e. soluble sugars and proteins), photosynthetic pigments and phytochemicals under water deficits indicated biostimulant-induced capacity in cowpea for osmotic adjustment, drought tolerance or adaptive mechanisms to water stress. These findings demonstrated the biological potential of VCL, KEL and SW to improve germination, seedling/plant growth and yield in legumes even under temperature stress and drought stress. Thus, establishing their stress amelioration properties to offset negative impacts of climate change on plants and yield.
In vitro propagation, phytochemistry and pharmacology of the blood lily, Scadoxus puniceus.
(2016) Naidoo, Devashan.; Finnie, Jeffrey Franklin.; Van Staden, Johannes.
Abstract available in PDF file.
Micropropagation and pharmacological evaluation of Boophone disticha.
(2013) Cheesman, Lee.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.; Light, Marnie Elizabeth.
Boophone disticha (L.f.) Herb is one of the most widely distributed bulbous species in southern Africa. Of Africa’s many bulbous plants, it is widely known for its poisonous and medicinal properties. It is of considerable ethnobotanical interest in traditional medicine because of its hallucinogenic alkaloids and it has great potential as an ornamental due to its fan-shaped foliage and large umbel of bright pink to deep red flowers. In South Africa, many bulbous plants are used in traditional medicine which are collected from wild populations. The high demand for trade and use of such plants, that are destructively harvested, places an enormous pressure on natural populations. According to the Red List of South African Plants, the conservation status of B. disticha has been listed as ‘declining’. It is, therefore, important to develop conservation strategies for these medicinal plants, such as the development of alternative propagation methods. Micropropagation is a useful technique for rapid clonal multiplication of plant material which could alleviate the pressure on the wild plant populations, as well as potentially producing useful secondary metabolites. The in vitro induction of storage organs is especially beneficial as it can limit the loss of plants during acclimatization since bulblets are generally hardier than shoots or plantlets. Thus, the main aim of this research was to establish a micropropagation protocol which could be a valuable tool for conservation of this plant species. In addition, B. disticha plants were assessed in various ethnopharmacological assays to evaluate their medicinal properties, and a preliminary study on the population genetics was also conducted. As part of the development of a suitable micropropagation protocol, the effect of environmental and physiological factors on the initiation and growth of bulblets were investigated. These factors included the effect of various plant growth regulators, carbohydrates, temperature, photoperiod and liquid culture. Different explants (i.e. ovaries, anthers, filaments, pedicels, embryos, seeds and bulb twin-scales) were tested to determine which explants were the most suitable for subsequent experiments. Although success was limited, twin-scales proved to be the most suitable explant and it was demonstrated that activated charcoal, ascorbic acid and N6- benzyladenine were required as media supplements. Antimicrobial activity was tested between different plant parts and seasons. The plant parts (roots, leaves, outer and inner bulb scales) were extracted with a range of differing polarity solvents. These were screened for antibacterial activity against Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae, and for antifungal activity against Candida albicans. Extracts from roots of plants collected in spring and summer showed the best antimicrobial activity against B. subtilis, E. coli and K. pneumoniae, indicating that plant part and collection time do affect activity. In vitro grown bulblets also showed antimicrobial activity, demonstrating that antibacterial properties were maintained in cultured plantlets. Extracts from plants collected in summer were tested for mutagenicity using the Ames test (Salmonella/microsome assay; plate incorporation method, with or without metabolic activation). None of the extracts tested were found to induce mutations and also did not modify the effect of the mutagenic compounds (2AA with S9 and 4NQO without S9). Although the results do not indicate a mutagenic response, this does not necessarily confirm that it is not mutagenic nor carcinogenic to other bacterial strains, however, B. disticha must be used with caution, especially considering the levels of alkaloids in the plant. The two major constituent alkaloids of B. disticha were identified as buphanidrine and distichamine. In the antibacterial assay, both compounds exhibited broad-spectrum micromolarlevel activity against the two Gram-positive and two Gram-negative bacteria tested. The best MIC value, of 0.063 mg/ml, was found for bupanidrine/distichamine against S. aureus, E. coli and K. pneumonia. The isolated compounds were tested and found to be neither mutagenic nor toxic at the concentrations tested. Thus, buphanidrine and distichamine are thought to be the constituents likely responsible for the medicinal properties of the plant. To determine the level of genetic variation between different populations of B. disticha, plants were collected from six wild populations in KwaZulu-Natal, South Africa. DNA was isolated and tested for genetic variation using ten Inter Simple Sequence Repeat (ISSR) primers. The level of inter-population polymorphism ranged between 23% and 39%, showing that the populations had low genetic polymorphism. From the genetic distance results, it was found that the Midmar and Umgeni Valley populations are closely related, and these populations are similar to two sister populations. The Amatikulu and Lions River populations were similar but slightly different to the other populations. Antimicrobial assays showed minor difference in activity from the six wild populations. Although the micropropagation of B. disticha had limited success, this study did develop a successful decontamination protocol as well as determine the most useful explant and supplements. This information provides an important starting point for the development of a successful micropropagation protocol for the conservation of B. disticha. Since, B. disticha is an important medicinal plant in South Africa, this study has also deepened our understanding of the constituents that could be responsible for the medicinal properties of B. disticha and, in so doing, confirmed the value of this plant for use in traditional medicine in South Africa.
Micropropagation of Brunsvigia undulata F.M. Leight.
(2009) Rice, Laura Jane.; Finnie, Jeffrey Franklin.; Van Staden, Johannes.
Many South African medicinal plants face the threat of over-collection for use in traditional medicines. Many bulbous plants suffer as the whole plant is removed from the wild so that the bulb may be used for medicine. Micropropagation is a technique which can be used as an alternative to conventional propagation methods. Micropropagation produces many plantlets in a relatively short period of time. Different plant parts of Brunsvigia undulata F.M. Leight, a rare South African species of medicinal value, were used in an attempt to produce in vitro plantlets using micropropagation techniques. Although leaf and floral explants were successfully formed from seedling explants and twin-scales. Seeds germinated quickly in culture. Seedlings which grew from seeds were cut into sections and used to initiate bulblets. Seedling explants formed bulblets, shoots and callus best when the explants included a meristematic region. Callus from seedling explants formed shoot clusters readily when placed on hormone-free MURASHIGE and SKOOG (1962) (MS) medium. Shoots from shoot clusters formed bulblets and rooted on medium supplemented with IBA. The greatest rooting response was achieved by bulblets on 1 mgl-1 IBA. The callus which was left after shoot clusters were separated was placed back onto hormone-free MS medium. Callus explants continued to form shoot clusters. Twin-scales, cut from large parent bulbs, were cultured on 25 hormone treatments. Bulblets formed on twin-scales even in the absence of plant growth hormones. Bulblets formed by twin-scales were used to determine the effects of both medium constituents and environmental factors on bulblet multiplication. Bulblet multiplication was greatest when bulblets were split in half and cultured as half-bulblets. Optimal multiplication was achieved on hormone-free MS, with 4% sucrose, kept at high temperatures in the dark. Bulblets were successfully initiated and multiplied from both seedlings and twin-scales. Bulblets which were produced via both protocols were acclimatized relatively easily. Both explant types could be used to mass propagate Brunsvigia undulata.
Micropropagation of three Brachystelma species and investigations of their phytochemical content and antioxidant activity.
(2017) Hlophe, Nqobile Prisca.; Finnie, Jeffrey Franklin.; Van Staden, Johannes.
Local communities in most African countries possess a wealth of knowledge on the uses of plants in the environment. Documentation of this knowledge is not a common practice among these communities and therefore the knowledge is in the process of disappearing as the younger generation is more inclined towards the western lifestyle. The knowledge of African communities on the use of medicinal plants has provided and continues to provide leads towards therapeutic concepts which accelerate the pace of drug discovery. This makes the use of medicinal plants an important line of research to be pursued. The use of plants, particularly medicinal plants, as a resource is generally accompanied by the concern of over exploitation. Exponential population growth along with newly emerging and resistant diseases as well as habitat destruction due to human developmental activities are among the reasons for the decline of plant biodiversity. There is therefore an urgent need to develop effective means of conservation specific to Brachystelma species as a medicinal group, and also to document not only the uses but also the constituents and pharmacological activity of these plants. Brachystelma R. Br. ex Sims is a genus of geophytic plants used traditionally in some parts of the world including East Africa, southern Africa, West Africa, northern and western India. Apart from being used as a food source, they are used for the treatment of illnesses such as colds, chest pains, wounds, and as an appetite suppressant and for enhancing fertility. The aim of this study was to establish efficient micropropagation protocols for Brachystelma species, namely B. ngomense (Endangered - EN), B. pulchellum (Vulnerable - VU) and B. pygmaeum (Least Concern - LC), as a means of ensuring their survival, and to explore their phytochemical and pharmacological properties. A number of Brachystelma species have been listed as medicinal herbs. There is currently no documented approach towards the mass propagation of Brachystelma species and there are no available scientific studies validating the ethnomedicinal use of these plants. Traditional uses indicate that Brachystelma is commonly used and this is accompanied by the concern that wild populations, which are the only available ones, are under threat for various reasons. Development of optimal tissue culture protocols such as micropropagation and callus cultures may alleviate conservation concerns. In addition, these protocols would make available the possibility of utilization of the plant as a daily food and towards production of biological compounds with potential health benefits. The development of a micropropagation protocol for the three Brachystelma species made use of nodal explants (~10 mm in length). The effects of different concentrations of N6-benzyladenine (BA), 2-isopentenyladenine (iP) and meta-topolin riboside (mTR) supplemented into Murashige and Skoog (MS) (1962) media were tested over a period of 6 weeks. The specific concentrations used were 1.0, 5.0, 10 and 25 μM for each of the cytokinins without auxins. An increase in concentration of all the cytokinin treatments was found to typically result in significantly higher shoot proliferation. However, each species differed in response to each specific cytokinin, the optimal concentrations were 25 μM mTR, 25 μM iP and 25 μM BA for B. ngomense, B. pulchellum and B. pygmaeum respectively. The conclusion drawn from these results is that the application of BA, iP or mTR is beneficial for in vitro shoot proliferation from nodal explants of the three species. Regenerated shoots were rooted ex vitro after exposure to a 3 min pulse treatment using 100 mgl-1 indole-3-butyric acid (IBA). Shoots regenerated from a plain MS medium were typically found to have rooted prior to IBA treatment, however, these shoots as well as the ones derived from other treatments yielded poor rooting ex vitro. Survival of these shoots in the greenhouse was short-lived. As a result, acclimatization of all three species has been extremely limited thus the micropropagation protocol is not effective on a commercial scale. A number of assays were used to evaluate the phytochemical content of the three species of Brachystelma. Vacuum filtered methanolic extracts were used for the determination of phenolics and flavonoids. Absorbance readings obtained using a spectrophotometer were further converted to concentration of compound per gram of extract. The phenolic and flavonoid contents varied between the three species. Higher phenolic and flavonoid content was found in leaf extracts. The effect of plant growth regulators (PGRs) i.e. BA, iP and mTR on specific phenolic acids was also observed. The cytokinin treatments were found to have a stimulatory effect on some of the phenolic acids. Pharmacological properties against oxidative stress were tested using the 2,2-Diphenyl-1-picryl hydrazyl (DPPH) and Oxygen radical absorbance capacity (ORAC) assays. The potency of the antioxidant activity of the three species was compared to the standard antioxidant, ascorbic acid. The activity of the standard antioxidant was found to not be significantly different to the plant extracts. B. ngomense leaf extracts (13.5 μg/ml) were more potent in comparison to ascorbic acid (14.5 μg/ml). The effect of plant growth regulators (PGRs) i.e. BA, iP and mTR on antioxidant activity was also observed. In some instances, cytokinin-treated plant extracts showed an increased antioxidant capacity compared to those not treated.
Pharmacological evaluation of South African medicinal plants used for treating tuberculosis and related symptoms.
(2014) Madikizela, Balungile.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.
Respiratory ailments are major human killers, especially in developing countries including South Africa. Tuberculosis is one of the most prevalent infectious respiratory tract disease posing a major threat to human healthcare worldwide. This disease is a highly contagious airborne bacterial disease that usually infects the lungs and sometimes other body parts. Tuberculosis spreads easily in overcrowded conditions from one person with an active respiratory disease to another via droplets that are emitted when they sneeze or cough. Approximately two million deaths that occur worldwide per annum are caused by tuberculosis and about 285,000 cases occur in South Africa. This is the seventh highest total number in the world. The emergence of drug-resistant tuberculosis and other pathogenic diseases over the past decades makes this disease a serious threat to human health worldwide. Emerging drug-resistant tuberculosis strains and the long duration of treatment has established an urgent need to search for new effective agents. According to a 2012 report by the World Health Organisation (WHO), South Africa, China, India and Russia are the countries with the highest prevalence of multi drug-resistant (MDR) tuberculosis. Most researchers in South Africa have focused on evaluating the antimycobacterial activity of medicinal plants against bacterial strains that cause tuberculosis, but there has not been sufficient focus on the related ailments. Therefore, one of the aims of the present study was the evaluation of the antimicrobial properties of the selected medicinal plants against Mycobacterium species and other bacterial strains related to respiratory infection. The floral diversity of South Africa has a potential for yielding new bioactive compounds, therefore pharmacological screening of plant extracts from this region is important. The aim of this study was the pharmacological evaluation of plants that are used traditionally in South Africa to treat tuberculosis and related symptoms against microorganisms that cause respiratory ailments, and the identification of compounds from antimicrobial active plant extracts. Ten plants: Abrus precatorius subsp. africanus (leaves and seeds), Asparagus africanus (leaves), Asparagus falcatus (leaves), Brunsvigia grandiflora (bulb), Ficus sur (bark and roots), Indigofera arrecta (leaves and roots), Leonotis intermedia (leaves and stem), Pentanisia prunelloides (leaves and roots), Polygala fruticosa (whole plant), and Terminalia phanerophlebia (leaves, roots and twigs) were selected based on a survey of available literature of medicinal plants used in South Africa for the treatment of tuberculosis and related symptoms. Ground plant material from different plant parts of the 10 plants were extracted sequentially with four solvents: petroleum ether (PE), dichloromethane (DCM), 80% ethanol (EtOH) as well as water, and a total of 68 extracts were produced. The plant extracts of the selected plants were evaluated for antibacterial activity against four microorganisms (Klebsiella pneumoniae, Staphylococcus aureus, Mycobacterium aurum A+ and Mycobacterium tuberculosis H37Ra) associated with respiratory infections using the microdilution assay. Cyclooxygenase-2 (COX-2) enzyme was used to evaluate the anti-inflammatory activity of the extracts. The Ames test and mitochondrial reduction (MTT) assays were used to establish toxicity of the extracts that showed noteworthy antimicrobial activity against the tested bacterial strains (Klebsiella pneumoniae, Staphylococcus aureus, Mycobacterium aurum A+ and Mycobacterium tuberculosis H37Ra). The extracts were tested for genotoxicity against Salmonella typhimurium (TA98 and TA100 strains) and cytotoxicity using monkey kidney Vero cells. Based on good antimicrobial activity observed, compounds were isolated from Terminalia phanerophlebia (leaves). Crude extracts obtained from 80% methanol (MeOH) of Terminalia phanerophlebia were successively extracted with hexane, DCM, ethyl acetate (EtOAc) and n-butanol. The fractions and isolated compounds obtained were tested for their antibacterial activity against Mycobacterium aurum A+, Mycobacterium tuberculosis H37Ra, Staphylococcus aureus and Klebsiella pneumoniae. Structure elucidation was carried out using NMR (1D and 2D) spectroscopic methods. This investigation revealed the pharmacological potential of the 10 plants used in South Africa for traditional treatment of tuberculosis and related symptoms: Abrus precatorius subsp. africanus (leaves and seeds), Asparagus africanus (leaves), Asparagus falcatus (leaves), Brunsvigia grandiflora (bulb), Ficus sur (bark and roots), Indigofera arrecta (leaves and roots), Leonotis intermedia (leaves and stem), Pentanisia prunelloides (leaves and roots), Polygala fruticosa (whole plant), and Terminalia phanerophlebia (leaves, roots and twigs). The minimum inhibitory concentration (MIC) values of the tested plant extracts ranged from 0.098 to 12.5 mg/ml. Out of 68 extracts tested from different plant parts of the 10 plant species, 18 showed good antimicrobial activity against at least one or more of the microbial strains tested with MIC values ranging from 0.098 to 0.78 mg/ml. For anti-inflammatory results, only three extracts showed high inhibition (˃ 70%) of the COX-2 enzyme. In the Ames test using Salmonella typhimurium (TA98 and TA100 tester strains), all the extracts tested were non-genotoxic. However, in the MTT assay nine extracts demonstrated cytotoxicity. Bioguided fractionation of the EtOAc fraction of Terminalia phanerophlebia (leaves) afforded two bioactive compounds: methyl gallate (methyl-3,4,5-trihydroxybenzoate) (1) and a phenylpropanoid glucoside; 1,6-di-O-coumaroyl glucopyranoside (2). These compounds are reported from Terminalia phanerophlebia for the first time. Both compounds showed good antimicrobial activity against all bacterial strains tested with MIC values ranging from 0.063 to 0.25 mg/ml. Inhibition of Mycobacterium tuberculosis by 1,6-di-O-coumaroyl glucopyranoside (2) at a MIC value of 0.063 mg/ml was noteworthy, as this bacterial strain is reported to be the leading cause of tuberculosis worldwide. The good antimicrobial property of Abrus precatorius subsps. africanus, Asparagus africanus, Asparagus falcatus, Terminalia phanerophlebia, Indigofera arrecta, Ficus sur, Leonotis intermedia and Pentanisia prunelloides partially authenticate their traditional use in the treatment of respiratory diseases. However, these plants must be used with caution as some of their extracts showed cytotoxicity against Vero cells. The results observed in this study indicate that Abrus precatorius subsp. africanus, Asparagus africanus, Asparagus falcatus, Ficus sur, Pentanisia prunelloides and Terminalia phanerophlebia could be investigated further against drug-resistant tuberculosis strains. Good antimicrobial activity exhibited by the compounds isolated from Terminalia phanerophlebia (leaves) authenticate the traditional use of this plant in treating tuberculosis and its related symptoms. Compound (2), 1,6-di-O-coumaroyl glucopyranoside showed noteworthy activity against a Mycobacterium tuberculosis strain H37Ra (0.063 mg/ml), therefore this compound could potentially serve as a lead in tuberculosis drug discovery.
A phylogeny-based comparative study of the phytochemical and pharmacological characteristics of Croton species occurring in KwaZulu-Natal.
(2021) Mathe, Tanya.; Bytebier, Benny Leopold Germaine.; Finnie, Jeffrey Franklin.; Van Staden, Johannes.
Ethnobotanical enquiries often lead to the discovery of phytocompounds with pharmacological activities. Against this background a comparative and quantitative evaluation of the phytochemical and antioxidant activity of extracts from six Croton species; C. gratissimus Burch., C. sylvaticus Hochst., C. menyhartii Pax, C. pseudopulchellus Pax, C. steenkampianus Gerstner and C. rivularis Müll.Arg., all collected from KwaZulu-Natal (KZN), South Africa, was conducted. The analysis included a comparison of the different plant organs to explore the possibility of using leaves rather than bark for medicinal purposes. The latter would result in less destructive harvesting and would contribute to sustainable use of these medicinal plant resources. Extraction of the different plants and their organs were done in water and, in different organics solvents, including methanol (MeOH), dichloromethane (DCM), and petroleum ether (PE). The extracts were screened for antibacterial and antifungal activities using the microdilution technique. All of the tested plant samples showed some notable antibacterial activity in one or two of their organs, except for C. rivularis, which was the only species in the list that had no record of medicinal use. The most potent antibacterial activity was exhibited by the dichloromethane (DCM) extracts of C. steenkampianus leaves and the petroleum ether (PE) extracts of C. pseudopulchellus stem bark, both sampled from the Durban Botanic Gardens, which yielded a minimum inhibition concentration (MIC) value of 0.04 mg/ml against Enterococcus faecalis (E. faecalis). The DCM stem bark and leaf extracts, as well as the PE twig extracts of C. pseudopulchellus, (Durban Botanic Gardens) also exhibited noteworthy activities against S. aureus (MIC value of 0.08 mg/ml). A broad spectrum of activity was observed in the DCM and PE twig extracts of C. sylvaticus collected from Umdoni Park, with a MIC ranging from 0.31-0.94 mg/ml. This activity was against E. faecalis, Staphylococcus aureus (S. aureus) and Klebsiella pneumoniae (K. pneunomiae). Noteworthy antifungal activity against Candida albicans was only observed in one extract, the MeOH leaf extract of C steenkampianus collected at Kosi Bay. The MIC of this extract was 0.6 mg/ml. Water extracts did not show any antimicrobial activity. The results of the pharmacological study suggested that the aerial plant organs, such as the leaves and the twigs, could replace bark as they exhibited significant antimicrobial activity when compared to the preferred bark. The study also revealed that the same species collected from different regions may not necessarily exhibit similar biological activities, as pharmacological activities are as a result of the phytochemicals present in the plant, which are triggered by environmental stimuli. The phenolic profiles of aqueous (50%) methanol extracts obtained from the plants were assessed using the Folin and Ciocalteu (Folin C), butanol-hydrochloric acid and aluminium chloride assays. Aqueous (50%) methanol extracts were also run on thin layer chromatography plates and the plates were later stained with the Dragendorff reagent to determine the possible presence of alkaloids. Antioxidant activity was determined with two different assays, β-carotene /Linoleic model system and 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging assay. The overall EC50 values of the different Croton species displayed by the DPPH assay ranged from 1.76 to 5.35 μg/ml. The β-carotene /Linoleic model system displayed antioxidant activity that ranged between 48.66 to 81.97%. In the phytochemical study; the leaf extracts of C. pseudopulchellus from Mkuze exhibiting the highest phenolic content at 23.8±1.1 mg GAE/g Dry Weight. The highest condensed tannin content was from the leaf extracts of C. gratissimus from Southport at a concentration of 31.3± 0.1 mg CCE/g Dry Weight. The highest flavonoid content observed in the leaf extracts of C. gratissimus from Southport at a concentration of 31.2±0.7 mg CE/g Dry Weight. Higher phytochemical contents were also observed in the leaves and twigs. These phytochemicals are believed to be the reason for most of the notable antimicrobial activities exhibited by these plant organs. The mutagenic potential of the most biologically active Croton extracts was tested. An Ames with two Salmonella tester strains (TA98 and TA102) revealed that the species are not toxic as they did not produce His+ revertant colonies in Salmonella tester strains that were more than twice the number of His+ revertant produced by the positive control, Nitroquinoline-N-oxide (4-NQO). Standard DNA barcodes of the Croton species occurring in KZN were generated, since these are useful in plant identification and authentication. These were used to run a phylogenetic analysis in order to assess whether the phytochemical profile is clade-specific. The results showed that the quantity and quality of phytochemicals closely related species may vary. DNA barcoding may be a useful tool in medicinal plant identification, especially where fragments of the plants are traded and morphological identification is not possible. In this study, the biological activity of different parts of six Croton species occurring in KZN, was investigated with the aim of replacing bark as the source of local medicine with other plant parts (leave and twigs) that can be more sustainably harvested, as this will contribute to the conservation of these species.
Propagation of Sceletium tortuosum (L.) N. E. Br.: a South African medicinal plant.
(2019) Sreekissoon, Amrisha.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.
Abstract available in PDF.
The role of biostimulants on the physiology, nutrition, phytochemistry and endogenous phytohormone content in Ceratotheca triloba under abiotic stress conditions.
(2017) Masondo, Nqobile Andile.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.
Abstract available in PDF file.
Strigolactone hormonal actions in plant tolerance to heat and chilling stress, adventitious root development, and seedling de-etiolation.
(2020) Omoarelojie, Luke Odianose.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.; Kulkarni, Manoj.
Abstract available in PDF.
The use of traditional medicinal plants for treating dermatological diseases and wound healing in KwaZulu-Natal, South Africa.
(2018) Shanaz, Ghuman.; Coopoosamy, Roger M.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.
Abstract available in PDF file.

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