Doctoral Degrees (Research Centre for Plant Growth and Development)

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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.
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.
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 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.
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.
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.
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.
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.
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.
The physiological response of cut carnation flowers to ethanol and acetaldehyde post-harvest treatments.
(2000) Podd, Lindsey Alice.; Van Staden, Johannes.
A replacement for silver thiosulphate as a commercial post-harvest treatment needs to be found. The longevity of cut carnation flowers is extended by all concentrations of ethanol tested. Compared to a water control, the vase-life of ethanol-treated flowers is between 150 and 250% longer. The greatest longevity increases are recorded with 3% ethanol. The use of ethanol as a post-harvest treatment was tested. The longevity increase as a result of ethanol application only occurs if the ethanol is applied as a holding solution. Pulse treatments are not effective at delaying the senescence of the flowers. The sooner the ethanol is applied, the greater the increase in vase life. If ethanol treatment is halted at any point during the experiment, the longevity of the flowers is reduced. It was observed that the longer the stems of ethanol-treated flowers, the greater the longevity increases. The ethanol holding solution does not prevent the action of external ethylene, thereby restricting the potential of ethanol as a commercial post-harvest treatment. Physiologically, flowers treated with ethanol exhibit a different senescence process to control flowers. The typical in-rolling of the petals of carnation flowers is not seen, instead the petals appear burnt. The ovaries are also notably effected by ethanol, being smaller and more yellow in colour. Ethanol treatment results in longevity increases by inhibiting the formation of ethylene, the plant hormone responsible for senescence. The concentration of the direct precursor to ethylene, ACC, as well as the activity of the enzyme that converts ACC to ethylene, ACC oxidase, is reduced to almost zero in the tissues of treated flowers. Another physiological factor affected by ethanol treatment is the carbohydrate status of the flowers. The normal sink activity of the ovary is inhibited by ethanol treatment. Although the carbohydrate content of the petals is found to decrease sharply in ethanol-treated flowers, these carbohydrates are not relocated to the ovary. The ovary does not increase in dry matter or chlorophyll content. The carbohydrate content decreases as a result of ethanol treatment, and when ¹⁴C sucrose was applied to petals, no radioactivity was recovered in the ovary. The petals and ovary are the organs most effect by ethanol activity, as when ¹⁴C ethanol was applied to cut carnation flowers as a pulse, the majority of the radioactivity was discovered here. The protein content of cells of both organs decreases significantly compared to control flowers. This is a total protein loss, rather than the destruction of specific systems. If the activity of alcohol dehydrogenase is prevented in ethanol-treated flowers, inhibiting the conversion of ethanol to acetaldehyde, no longevity increases are seen. The airspace surrounding treated flowers was found to contain ethanol and small amounts of acetaldehyde. The tissues of flowers treated with ethanol show an increase in the acetaldehyde content, as well as the ethanol content, especially in the ovary. The application of acetaldehyde directly to cut carnation flowers as a holding solution resulted in the vase life of the flowers increased by 150%. To determine the effectiveness of acetaldehyde as a post-harvest treatment, various concentrations of acetaldehyde were applied to cut carnation ftowers as a pulse treatment and a holding solution. Pulse treatments did not increase the vase life of flowers, and resulted in a number of negative effects in the flower. A holding solution of acetaldehyde does increase the longevity of cut carnation flowers, provided it is above a certain concentration. Treatments at concentrations below 1% acetaldehyde appear to promote flower senescence. The use of acetaldehyde as a post-harvest treatment has many of the same disadvantages as ethanol treatment. Acetaldehyde must also be applied as a holding solution for as long as possible. If removed from this solution, death of the organ occurred quickly. Acetaldehyde is also ineffective against external ethylene. A negative effect of acetaldehyde not found in ethanol-treated flowers, is that the longer the stem of cut carnation flowers, the shorter the resultant vase life. Physiologically the responses in cut carnation flowers were very similar to those seen in ethanol-treated flowers. Acetaldehyde inhibited the formation of ethylene completely. Almost no ACC can be found in treated tissues, and the action of ACC oxidase is completely reduced. The petals of acetaldehyde-treated flowers suffer from severe petal browning, rather than in rolling. The ovaries are particularly badly effected by treatment. There are large scale losses in fresh weight and chlorophyll content. The latter results in the ovaries appearing yellow in colour. They also show a loss in structure. The sink activity of these ovaries is destroyed. Like ethanol-treated flowers, the carbohydrate content of both the petals and ovaries are dramatically reduced. When ¹⁴C sucrose was applied to one of the. petals, almost no radioactivity was recorded in the ovary. There. is also a major loss in general protein content, slightly more severe than in ethanol-treated flowers. The conversion of ethanol to acetaldehyde is necessary in order to achieve longevity increases in ethanol-treated flowers. If the conversion of this acetaldehyde to ethanol is prevented in acetaldehyde-treated flower there is once again no longevity increase. Both ethanol and acetaldehyde are required within the system to result in increased longevity. Although ethanol and acetaldehyde treatments result in decreases in the total protein content of the flowers, certain enzymes remain active. Alcohol dehydrogenase is a bi-directional enzyme, capable of converting ethanol to acetaldehyde and then back to ethanol again. The activity of this enzyme, in both orientations, is increased in ethanol and acetaldehyde-treated flowers. The activity of pyruvate decarboxylase, which converts pyruvate to acetaldehyde, is also increased as a result of both treatments. The similarities of the physiological response of cut carnation flowers to ethanol and acetaldehyde post-harvest treatments, and the increased activity of these enzymes, indicate that the effect of both compounds on longevity is closely linked.
Stress related responses in soybean.
(2000) Liu, Tao.; Van Staden, Johannes.
Environmental stresses such as drought, salinity and low temperature have been major selective forces throughout plant evolution and are important factors which limit crop plant distribution and agricultural productivity. An understanding of how crops adapt to adverse conditions is not only of theoretical interest, but also has considerable practical value . Low-temperature stress subtraction libraries were constructed in a pBluescript vector with the two-step-PCR amplified cDNAs using subtractive hybridization. One insert cs18 was obtained and the sequence analysis of insert cs18 revealed that the insert cDNA had a 76% homology with the sequences of the corresponding portion of glucose dehydrogenase from Thermoplasma acidophilum and 62.0% homology with a genomic DNA of Arabidopsis. Four clones, cs18-13, cs18-14, cs18-15, and cs18-16 from low-temperature stress soybean root conventional cDNA library have been confirmed to have inserts that could hybridize to the cs18 insert. One cDNA with a Xba I and Xho I fragment of approximately 3,500 bp in length corresponded to the insert cs18 , which probably encodes for glucose dehydrogenase, was obtained. Northern blot analysis indicated that cs18 mRNA was highly expressed in soybean root but moderately expressed in leaves under low temperature. Changes in the nuclei of meristematic root cells in response to severe salinity were studied. Roots are in direct contact with the surrounding solution . Thus, they are the first to encounter the saline medium and are potentially the first site of damage or line of defence under salt stress. Nuclear deformation or degradation in the soybean root meristem with 150 mM or higher NaCI led to sequential cell degradation, cell death and cessation of plant growth . However, this study indicates that an increase in CaCI[2] concentration up to 5 mM could partially prevent salt injury to the cells. Tissue culture is an excellent tool for elucidat ing the correlation between plant organizational levels and salt tolerance because of the possibility it offers for studying the physiology of intact plantlets together with that of organs and single cells using homogenous plant material under uniform environmental conditions. One NaCI-tolerant cell line (R100) was isolated during this study. The R100 callus cell line was significantly more tolerant to salt than the salt-sensitive line (S100) during exposure to salt stress. Salt tolerance in this culture was characterized by an altered growth behaviour, reduced cell volume and relative water content, and accumulation of Na+, Cl ¯, K+, proline and sugars when grown under salt stress and with its subsequent relief. The selection of this salt tolerant cell line has potential for contributing new genetic variability to plant breeders. Sugars are not only important energy sources and structural components in plants , they are also central regulatory molecules controlling physiology, metabolism, cell cycle , development, and gene expression in plants. The concentrations of glucose and fructose increased during salt stress and after relieving salt stress, at a rate closely corresponding to the increase in relative water content. Their accumulation was the earliest response detected during the removing of salt stress indicating that glucose and fructose may play important roles during salt-stress.
Alkaloids from three South African Crinum species.
(2000) Elgorashi, Esameldin Elzein.; Van Staden, Johannes.; Drewes, Siegfried Ernst.
The alkaloid content of three Crinum species namely C. bulbispermum, C. macowanii and C. moorei was investigated. The ethanolic extracts of C. bulbispermum yielded seven compounds. The new alkaloids 8α-ethoxyprecriwelline, N-desmethyl-8α-ethoxypretazettine and N-desmethyl-8β-ethoxypretazettine were isolated for the first time from a natural source. In addition, the known alkaloids bulbispermine, crinamine, 6-hydroxycrinamine and 3-O-acetylhamayne were isolated in this study. The ethanolic extracts of C. moorei were found to contain Iycorine, 1-O-acetyllycorine, crinine, 3-O-acetyllycrinine, epibuphanisine, powelline, crinamidine, undulatine, epivittatine, 1-epideacetylbowdensine, cherylline and the new alkaloids mooreine and 3-[4'-(2'-aminoethyl)phenoxy]bulbispermine. The alkaloids crinine, lycorine, bulbispermine, cherylline and hamayne were obtained from the ethanolic extracts of C. macowanii. In addition, the amine tyramine was identified during the isolation process. Dilute HCl solution extraction followed by GC analysis was used to investigate organ-to-organ and seasonal variation of alkaloids in each Crinum species, as well as the interspecific variation in these alkaloids over two consecutive years. Twelve alkaloids were identified, including crinine, epibuphanisine, powelline, crinamine, crinamidine, 6-hydroxycrinamine, 1-epideacetylbowdensine, 3-O-acetylhamayne, undulatine, Iycorine, 1-O-acetyllycorine and cherylline. Alkaloids were detected in all organs of C. moorei and C. macowanii. However, alkaloids were not detected in the leaves of C. bulbispermum. Organ-to-organ and seasonal variations in the total yield and total ring types of these alkaloids were noticed. Organ-to-organ and seasonal statistical variations were also detected for some of the individual alkaloids detected in each of these species. The results also showed that C. moorei had the highest levels of all individual alkaloids except crinamine when compared to C. bulbispermum and C. macowanii. Quantitatively, the detected alkaloids chemotaxonomically separated C. moorei from C. bulbispermum and C. macowanii. The results also indicated that C. macowanii is more closely related to C. bulbispermum. Qualitatively, Iycorine, 1-O-acetyllycorine, cherylline, crinamidine, 1-epideacetylbowdensine, crinine, crinamine and 3-O-acetylhamayne were detected in both C. moorei and C. macowanii, indicating the close relationship of these species.
Monocarpic senescence in Bidens pilosa L.
(2000) Zobolo, Alpheus Mpilo.; Van Staden, Johannes.
Senescence was examined in the economic weed Bidens pilosa, with the objectives to a) determine the effects of deflowering and defruiting on growth, chlorophyll content, photosynthesis and transpiration; b) to identify the stage of development of the head at which the flowers, seeds/fruit produce senescence signals; and c) to test for senescence activity in plant extracts made from the receptacles and leaves of Bidens pilosa. Total chlorophyll content in the controls, in association with the development of fruit, was lower in the final harvests when compared with earlier harvests in both pot and field-grown plant experiments. Deflowered Bidens pilosa plants had a higher chlorophyll concentration than both defruited and control plants in both pot and field-grown plants. Stem death of the control plants was higher than that of deflowered plants in both field and pot experiments. The present results suggest that deflowering is essential if the leaves are to be harvested commercially because it retards senescence and maintains growth. Fruit and flower heads were responsible for the reduction in leaf and stem growth after flowering in Bidens pilosa. Removing these organs slowed plant decline, suggesting that the flower head and especially the fruit are responsible for senescence. In contrast, the fruit were the main organs responsible for the decline in leaf chlorophyll concentration. In pot-grown plants in full sunlight, photosynthesis and transpiration were low in deflowered plants compared with the control and defruited plants 45 days after treatment, and it coincided with a low stomatal conductance. These results suggest that stomatal conductance played a role in lowering photosynthesis in deflowered plants. In contrast, the control plants had a higher stomatal conductance than deflowered plants 75 days after treatment, yet photosynthesis and transpiration rates were the same in both treatments. Thus stomatal conductance alone does not successfully explain differences in photosynthesis in these treatments. The dry weight of head with mature dry fruit was higher in plants grown at high light intensities than at medium or low light intensities. It coincided with a greater decline in chlorophyll concentration in the leaf nearest to the head and fruit. In contrast, photosynthesis was the same at all light intensities in the leaf nearest to the head and fruit. This suggests that high light accelerated the process of fruit maturation of the fruit which then influenced senescence in the leaf nearest to the flower head. Ethanolic and water extracts of senescent receptacles purified using paper chromatography, induced senescence of leaves in light but not in the dark. In ethanolic extracts, activity was detected in R[f]s 0.1, 0.2 and 0.3. In water extracts, activity was detected in R[f] 0.1. Senescent leaf extracts purified using column chromatography also induced senescence in light under greenhouse conditions. At high concentrations, activity was detected in fraction 10 eluted with ethyl acetate: methanol (55:45); fraction 11 eluted with ethyl acetate: methanol (50:50); fraction 12 eluted with methanol (100%) and in fraction 13 eluted with ethylacetate : isopropanol: water: acetic acid (52:28:28:4). Under growth room conditions, activity was detected in fractions 12, eluted with methanol (100%) and 13, eluted with ethyl acetate: isopropanol: water: acetic acid (52:28:28:4) in the presence of light. Fraction 1 (R[f] 0.00-0.10) from senescent receptacles, non-senescent and senescent leaves, obtained following thin layer chromatography of ethanolic extracts induced senescence under light. Fraction 1 was eluted with methanol. This fraction lacked activity when eluted with ethyl acetate. Fraction 4 (R[f] 0.25 - 0.35) from non-senescent leaf extracts, which co-chromatographed with 4-chloroindole acetic acid, gave activity in bean cuttings kept under continuous low light. Senescent leaf extracts showed no activity. Fraction 7 (R[f] 0.9 - 1.0) from non-senescent leaf extracts, also induced senescence in bean cuttings under light. The same Fraction from senescent leaf extracts lacked activity.
The effect of charcoal on tissue morphogenesis in vitro.
(2000) Pan, Manjing.; Van Staden, Johannes.
The effect of activated charcoal, autoclaving and culture media on sucrose hydrolysis in tissue culture media was investigated. Activated charcoal acidified an aqueous sucrose (5%) solution and culture media by about 1 to 2 units after autoclaving . Sucrose hydrolysis in tissue culture media and/or aqueous sucrose (5%) solutions containing activated charcoal (buffered to pH 5.8) was dependent on both the hydrogen ion concentration (pH) and autoclaving. After autoclaving, 70%, 56% and 53% sucrose hydrolysis were respectively recorded in a 5.0% sucrose solution, Murashige and Skoog (MS) and Gamborg B5 (B5) liquid media in the presence of 1.0% activated charcoal, added before autoclaving . In the absence of activated charcoal, autoclaving resulted in about 20% of the sucrose being hydrolysed The adsorption of 2, 4-dichlorophenoxyacetic acid (2,4-D) by activated charcoal from methanol and aqueous solutions was determinated using HPLC. The amount of the added 2,4-D decreased in both methanol and aqueous solutions in the presence of activated charcoal, compared with those in the absence of activated charcoal. In methanol and aqueous solutions, activated charcoal used at the level of 0.1% significantly reduced 2,4-D. About 68.4% and 60.9% respectively of the added 2,4-D was adsorbed by activated charcoal (1.0%) from these solutions. The changes of inorganic elements in MS-salt solutions, in the presence of activated charcoal, were analysed by SEM-EDX. The concentrations of magnesium (Mg), calcium (Ca), iron (Fe) and zinc (Zn) deceased in the presence of activated charcoal, while the concentrations of potassium (K), copper (Cu), manganese (Mn), phosphorus (P), and sulphur (S) increased in the MS salt solution in the presence of activated charcoal compared with no activated charcoal in the medium. This suggests that activated charcoal adsorbed calcium, magnesium, iron and zinc and released copper, manganese, phosphorus and sulphur. Rooting occurred when 7-day-old seedling hypocotyls of Daucus carota L. Cape Market were placed on MS medium supplemented with 2,4-D, and IAN/NAA in the presence of activated charcoal. Hypocotyls did not produce roots on the 2,4-D containing media in the absence of activated charcoal. The roots were produced polarly on the NAA/IAA-containing media in the presence of activated charcoal. No-polarity of root formation was observed on media supplemented with NAA/IAA without activated charcoal. Different responses of hypocotyls to a series of 2,4-D concentrations (0.5, 1.0, 3.05.0, 8.0, and 10.0 mg l ¯¹) were observed on media supplemented with 0.02, 0.1 and 0.5% activated charcoal. In the NAA/IAA containing media in the presence of activated charcoal, root number per hypocotyl decreased. Root number perhypocotyl, on the media supplemented with NAA and IAA, increased when hypocotyls were pre-cultured on MS medium supplemented with 2,4-D (1.0 mg l ¯¹) for 2-3 days. When hypocotyls were pre-cultured on a 2,4-D containing MS medium for 5 days, embryos emerged from the hypocotyls directly on the medium supplemented with 2,4-D in the presence of activated charcoal. Addition of activated charcoal to MS medium supplemented with 2,4-D resulted in somatic embryogenesis of Daucus carota. Somatic embryos were not formed on the medium in the absence of activated charcoal. In suspension culture, the incorporation of 0.01 to 1.0% concentrations of activated charcoal to the MS medium, irrespective of 2,4-D, increased the number of somatic embryos produced. The maximum number of somatic embryos were produced with 1.0% activated charcoal. Further development of embryos of Daucus carota occurred on the media in the presence of activated charcoal, and the embryos subsequently regenerated normal plantlets. Abnormal somatic embryos followed the addition of 3.0% activated charcoal to the medium.
An evaluation of plants used in eastern Nigeria in the treatment of epilepsy and convulsion.
(2002) Ogbonnia, Steve Okwudili.; Van Staden, Johannes.
Schumanniophyton magnificum and Glypheae brevis are important medicinal plants growing wild in the West African rain forest. They are used in folkloric medicine in the treatment of epilepsy and convulsion as well as for some other diseases. The purpose of this work was to investigate the aspect of folkloric use in order to support folkloric claims and document the findings. The extracts were prepared from ground plant material by a continuous extraction method. Five hundred grams of ground plant material were continuously de-fatted with 2 L petroleum ether (60°- 80°) in a Soxhlet apparatus for about 5 h. The resulting marc was dried and the chemical constituents extracted hot in a Soxhlet apparatus for about 8 to 10 h with 2 L aqueous ethanol (70%). The efficacy of the extraction method was confirmed using standard bioassays and phytochemical analyses. The anti-convulsant activity of the crude extracts was evaluated in vivo against chemically induced convulsions using three different animal models, namely the strychnine, the picrotoxin and the pentylenetetrazole tests. The acute and delayed toxicity test results showed that in all the animal models investigated very high doses, about four times higher than the protective doses of the extracts, were required to kill 50% of the population of animal used. Phytochemical assays of the extracts indicated the presence of alkaloids only in S. magnificum root extract and glycosides in extracts from both species. The glycosides were positive to Baljet, Xanthydrol and Keller-Kiliani tests for cardiac glycosides. S. magnificum and G. brevis chemical constituents were initially isolated with a sequential fractionation method starting with a highly non-polar solvent and gradually increasing to a more polar solvent. The fractions were pooled on the basis of TLC similarity profiles when viewed under the UV light at 254 and 366 nm and were found to have two and four major UV absorbing fractions for S. magnificum and G. brevis respectively. Radio-receptor binding tests were used to assess the anti-convulsant activities of the hydro-alcoholic crude extracts, the organic and aqueous fractions of the crude extracts, partially purified components and pure components in in vitro tests against some standard GABA[A] receptor antagonists, muscimol and isoguvacine respectively. The anti-convulsant activities resided in the aqueous fractions of the hydro-alcoholic crude extracts of both plants. The purely organic fractions of G. brevis demonstrated no activity while all the fractions of the aqueous component demonstrated some degree of activity. The anti-convulsant activity of S. magnificum was found only in one fraction-Fraction 1. This Fraction was further investigated and one of the components appear to be responsible for the activity. The structure of the active constituent was 5,7dihdroxy-2 methylbenzopyran-4-one, a noreugenin. A second bioactive compound, schumanniofoside, was identified from Fraction M[5.2] from S. magnificum.
Crinum moorei : propagation and secondary metabolite production in vitro.
(2002) Fennell, Catherine W.; Van Staden, Johannes.
As an alternative to conventional methods of vegetative propagation, micropropagation attracts much attention, because the levels of multiplication are increased, somaclonal variation is limited and disease-free material can be obtained. The technique is invaluable to the conservation of Crinum species belonging to the Amaryllidaceae which, as a group, possesses several biological features that make them particularly vulnerable. This is in addition to other problems relating to their value as horticultural material, traditional medicines and sources of phytochemicals of interest to medical science. Two in vitro systems are widely used for the propagation of amaryllidaceous species; regeneration from young floral stem explants and from twin-scales excised from bulbs. Although plantlet regeneration could be obtained from peduncle explants of Crinum moorei, a complex of factors including: the age of the floral stem; explant position and; hormonal factors, limited growth. Callus production was poor and indirect organogenesis could not be achieved. Twin-scales were used for the induction of somatic embryos. Morphologically these were different depending on the concentrations of 2,4-D and BA used in the induction medium. Although some of them went on to germinate, the use of somatic embryos for large-scale culture is not an efficient micropropagation route, owing to the low frequency of both embryo production and germination and to the long culture times. Regeneration of shoots and bulblets could, however, be readily induced from twin-scales using a series of modified MS media, and this despite the fact that explants from the bulb were more difficult to decontaminate than the above ground parts. Shoots arose in the axes of the twin-scales close to the basal plate. Initiation was greatest on a basic Murashige and Skoog medium, containing 4 g ℓ ¯¹ sucrose, and in the dark. No hormones were required. At high concentrations, the hormones stimulated abnormal organogenesis. Bulbing of the shoots was further enhanced using higher than normal levels of sucrose i.e. 6% and 5 g ℓ ¯¹ activated charcoal. The response was also influenced by the size of the twin-scale and its position in the parent bulb. Greater numbers of bulblets with larger diameters developed in large twin-scales from an intermediate position between the inner and outer scales. Furthermore, light, and a temperature of 25°C were required for normal bulblet development. Bulblets grown in this manner were used as a source of secondary explants by splitting them vertically in half. The addition of 10 mg ℓ ¯¹ BA resulted in multiple shoot development. In a liquid-shake culture system, this same multiplication medium induced the formation of meristemoid clusters whose rates of proliferation were higher than that achieved for shoot multiplication on either solid or static liquid media. The advantage of using meristematic clusters is that shoot hyperhydricity is avoided. Furthermore, the clusters can be mechanically separated; making the system ideal for automated plant production. Shoot morphogenesis, followed by the formation of bulblets occurred on solid MS media containing activated charcoal or high concentrations (6%) of sucrose. The induction of bulblets by sucrose was, however, slower, which may be beneficial for long-term storage and conservation ex situ. Compared to smaller bulblets, bulblets with a diameter of approximately 9 mm, acclimatized readily and grew rapidly after transferring them to the soil in greenhouse conditions. Biotechnological processes such as cell and tissue culture provide an ideal system for producing secondary metabolites, especially where their production in situ is hampered by poor resource availability or when chemical synthesis is difficult. In vitro produced Crinum moorei bulblets were found to contain nine alkaloids of the Amaryllidaceae type; three of which were released into the culture medium. Light was essential for alkaloid biosynthesis while the inclusion of BA and activated charcoal stimulated the production of specific alkaloids.
Cultivation of Combretum bracteosum (Hochst.) Brandis.
(2001) Koen, Kerry Jacqueline.; Van Staden, Johannes.
In maximizing South Africa's floral diversity, plant propagators have begun exploiting the rich array of indigenous plants, especially those with horticultural potential. Plants previously unavailable to the professional and amateur gardeners alike, are legally becoming common-place in nurseries. However, in promoting the trade of indigenous plants to nursery-owners, rapid, easy and cost effective methods of propagating these plants need to be established. Combretum bracteosum is one such indigenous plant, the aesthetic appeal thereof exhibits great potential for ornamentation, especially when flowering. In facilitating the introduction of Combretum bracteosum into nurseries, small gardens or even pots, investigations carried out aimed to determine and analyse quick and easy methods of propagating this plant. Of the various propagation techniques considered, only one, micropropagation, required specialized skill and training prior to carrying out the relevant procedures and protocols. The two other techniques used, which are accessible to most plant propagators, were seed germination and propagation from cuttings. Propagation by seed germination yielded less than optimal results from a commercial perspective. Although the hard pericarp surrounding the embryo did not impose any dormancy inducing mechanisms, such as the restriction of water uptake or the leaching of an inhibitory compounds, it did act as a mechanical barrier to the emerging radicle and roots. Recommendations for optimal Combretum bracteosum seed germination would be to remove the protective pericarp completely, incubate imbibed embryos in complete darkness at 25°C. After radicle emergence the germinating embryos could be moved into an alternating light: dark cycle. A more viable and simpler alternative to seed germination, was propagation by stem cuttings. Treating the cuttings with 10% and 50% or 75% of the commercially available Kelpak concentrate (using the Soak Method and Quick-dip Methods respectively), provided the most promising results, with the rapid development of roots and subsequent vegetative growth. Synthetic hormones such as IBA and NAA were also applied to the cuttings both alone or in combination however, although callus growth was profuse, root development was slow and unsubstantial, if any at all. Therefore, in recommending a protocol for the successful rooting of Combretum bracteosum cuttings taken during spring, summer or early autumn, the application of Kelpak at either 10% (Soak Method) or 50% (Quick-dip Method) of the full strength solution, is advised. Subsequent to hormone treatment, the cuttings still required attention with regard to nutrient supplementation as well as atmospheric moisture and temperature regulation. Success in generating Combretum bracteosum plantlets was obtained by germinating the seed in vitro as well as stimulating axillary shoot elongation from nodal explants. Placing the sterilized Combretum bracteosum embryo onto a nutrient rich basal medium (containing no hormones) was sufficient to stimulate 100% germination. The frequent poor availability of the seed may hamper the use of in vitro seed germination for commercial propagation purposes. The use of nodal explants from in vitro germinated stock plants, is a rapid and reliable means of generating a large seedling stock. Nodal explants excised from the newly developed shoot were subsequently placed onto 0.5 mg.ℓ ¯¹ BA which encouraged axillary bud elongation. After elongation, the lateral shoots were removed and placed onto a rooting medium (1.0 mg.ℓ ¯¹ IBA). The more mature nodal explants, collected from parent plants growing in vivo, required either a BA: NAA hormone combination or Kelpak to stimulate axillary shoot elongation, with the latter being most successful. Root initiation followed the protocol described above. Once rooted plantlets were hardened off they displayed a strong and vigorous growth, which is desirable from a commercial perspective. Upon maturity, the habit of many indigenous trees and shrubs could become too big for confined spaces such as the urban garden. Therefore, determining a means of modifying the plants' habit in order to maintain its suitability as a smaller garden plant was important. Treating the Combretum bracteosum plants with a 50 mg.ℓ ¯¹ paclobutrazol soil drench proved most successful, with the desired effects being visible within a few weeks of initial application. No negative morphological or developmental effects were noted on plants treated with the dwarfing agent, conversely however, the treated Combretum bracteosum plants were compact and bushy, with considerable visual appeal and aesthetic attractiveness.
Evaluation of anthelmintic, antiamoebic and antibacterial activity in traditional South African medicinal plants.
(2001) McGaw, Lyndy Joy.; Van Staden, Johannes.; Jäger, Anna Katharina.
Traditional medicine in southern Africa draws upon a vast selection of plants to treat gastrointestinal disorders such as diarrhoea and intestinal parasites. The evaluation of these plants for biological activity is necessary, both to substantiate the use of these plants by healers, and also a possible lead for new drugs or herbal preparations. After a survey of the existing ethnobotanical literature, plants used to treat stomach ailments such as diarrhoea, dysentery or intestinal worm infestations were selected and submitted to bioassays according to their traditional uses. Extracts of the chosen plants were made using the solvents hexane, ethanol and water, to ensure the extraction of compounds with a wide range of polarity. In total, 138 extracts were tested for antibacterial activity, 72 for anthelmintic activity, and 42 for antiamoebic activity. Antibacterial activity was evaluated using the disc-diffusion assay, and Minimal Inhibitory Concentration (MIC) values were determined using a microdilution assay. The extracts were tested against the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus, and the Gram-negative bacteria Escherichia coli and Klebsiella pneumoniae. Ethanolic extracts showed the greatest activity and Gram-positive bacteria were the most susceptible microorganisms. The free-living nematode Caenorhabditis elegans, which is morphologically similar to parasitic nematodes, was used in two different assays to evaluate anthelmintic activity. A microdilution technique was employed to investigate antiamoebic activity against the enteropathogenic Entamoeba histolytica, the causal organism of amoebic dysentery. These assays were suitable for the screening of a large number of extracts at one time. Several plants exhibited significant activity against these test organisms. Many species of plants belonging to the family Combretaceae are used in southern African traditional medicine against a variety of ailments, including abdominal complaints, bilharzia and diarrhoea. Extracts of powdered leaf material of 24 species belonging to the Combretaceae were prepared using the solvents ethyl acetate, acetone, methanol and water. These extracts were screened for anthelmintic activity. Significant activity was exhibited by C. apiculatum, C. hereroense and C. mossambicense. The most anthelmintic activity was shown by acetone extracts, followed by ethyl acetate, water and then methanol extracts. The aromatic rhizomes of Acarus calamus L. are used extensively in traditional medicine worldwide. They reportedly relieve stomach cramps and dysentery, and are used as anthelmintics. Rhizome extracts of A. calamus growing in KwaZulu-Natal, South Africa, exhibited anthelmintic and antibacterial activity in the initial general screening. Using bioassay-guided fractionation, the phenylpropanoid β-asarone was isolated from the rhizome. This compound possessed both anthelmintic and antibacterial activity. It has previously been isolated from A. calamus, and a related species, A. gramineus. Different varieties of A. calamus exhibit different levels of β-asarone, with the diploid variety containing none of the compound. Mammalian toxicity and carcinogenicity of asarones has been demonstrated by other researchers, supporting the discouragement of the medicinal use of Acarus calamus by traditional healers in South Africa. Schotia brachypetala was another plant to show good antibacterial activity in the initial screening. The roots and bark of S. brachypetala are used in South African traditional medicine as a remedy for dysentery and diarrhoea. The lack of pharmacological and chemical data on this plant prompted a further investigation into its antibacterial activity. The differences in activity of ethanol and water extracts with respect to plant part, season and geographical position were analysed. No extreme fluctuations in activity were noted. Two other Schotia species, S. afra and S. capitata, were included in the study, and both displayed good antibacterial activity. The storage of the plant, either as dried, ground plant material at room temperature, or as an extract residue at -15°C, had little effect on the antibacterial activity. Preparing the extracts from fresh or dry material also did not notably affect the activity. In general, the ethanolic extracts were more active than the aqueous extracts. The chemical profiles on TLC chromatograms were compared and found to be very similar in the case of ethanol extracts prepared in different months of the year, and from different trees. The extracts of the three species, and of the leaves stored under various conditions, as well as extracts prepared from fresh or dry material, also showed similar TLC fingerprints. However, various plant parts of S. brachypetala showed distinctly different chemical compositions. The leaves of S. brachypetala showed slightly higher antibacterial activity than the roots. Fractionation of the ethanol extract of the dried leaves using liquid-liquid partitioning and chromatographic techniques yielded 9,12,15-octadecatrienoic (linolenic) acid and methyl-5, 11,14,17-eicosatetraenoate. These fatty acids displayed antibacterial activity against the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus, and activity to a lesser extent against the Gram-negative Escherichia coli and Klebsiella pneumoniae. Linolenic acid is known to have antibacterial activity. The screening of plants for biological activity yielded valuable preliminary information about the plants used by traditional healers to treat gastrointestinal illnesses. The isolation of biologically active compounds from two highly active plants was achieved.
A pharmacological study of some Nigerian medicinal plants.
(2005) Chukwujekwu, Jude Chinedu.; Van Staden, Johannes.
Petroleum ether, dichloromethane, and 80% ethanol extracts of 15 plant species collected in Nigeria were screened for in vitro antibacterial, anti-inflammatory and antimalarial activities. Antibacterial activity was tested using the agar diffusion method, while the minimum inhibitory concentrations (MIC) of the active extracts were determined using the microtitre serial dilution method. Most antibacterial activity detected was against Gram-positive bacteria with Staphylococcus aureus being the most susceptible. The highest activity was found in petroleum ether and dichloromethane leaf extracts of Mallotus oppositifolius; petroleum ether, dichloromethane and ethanolic root extracts of Newbouldia laevis; and ethanolic root extracts of Morinda lucida and Canthium subcordatum. Against the Gram-negative bacterium Escherichia coli, the highest activity was found in dichloromethane leaf extracts of Newbouldia laevis, ethanolic root extracts of Phyllanthus amarus, Mallotus oppositifolius, and Canthium subcordatum. A total of 60 plant extracts were screened for antiplasmodial activity. A chloroquine sensitive strain of Plasmodium falciparum (D10) was used. In the assay, the parasite lactate dehydrogenase (pLDH) activity was used to measure parasite viability. About 11 extracts showed promising activity with an IC₅₀ ranging from 2.5 to 13.4 µg/ml. The petroleum ether leaf extract of Hyptis suaveolens had the highest activity (IC₅₀ = 2.5 µg/ml). The cyclooxygenase (COX-1 and COX-2) assays were used to test for anti-inflammatory activity. All the plant species, with the exception of Hedranthera barteri and Picralima nitida showed anti-inflammatory activity. Apart for a few ethanolic extracts, all the activities were recorded with petroleum ether and dichloromethane extracts. Employing bioassay-guided activity fractionation, an antibacterial anthraquinone identified as emodin was isolated from ethanolic root extract of Senna occidentalis. Although this compound had been isolated from other sources, this was the first report of isolation from Senna occidentalis. Using a similar approach a novel antimalarial diterpenoid was isolated from the petroleum ether leaves extract of Hyptis suaveolens. It had IC₅₀ of 0.1 µg/ml. This new compound is worthy of further investigation and may act as an important lead compound for future antimalarial drugs.
Anti-bacterial and anti-inflammatory activity of medicinal plants used traditionally in Lesotho.
(2003) Shale, Thato Lucy.; Van Staden, Johannes.; Stirk, Wendy Ann.
A significant potion of the population in Lesotho relies on traditional medicine to meet its health care requirements. Traditional healers and herbalists were interviewed from Qacha's Nek (Highlands) and Mohale's Hoek (Lowlands) districts in Lesotho on plants used by the Basotho in traditional remedies. Fifteen plants were reported to be used for bacterial infections while thirteen plants were used for diseases associated with inflammation . Plant roots were most often used to make water extracts. Mainly high altitude plants are used with lowland healers obtaining most of their plant material from the highlands, either by collecting them or buying them from highland gatherers. Leaves and roots of plants used to treat bacterial infections were extracted with hexane, methanol and water and the respective extracts screened at 100 mg ml¯¹ for anti-bacterial activity using the disc diffusion bioassay. Seven species displayed very high anti-bacterial activity against both Gram-positive and Gram-negative bacteria. A number of plant extracts had medium inhibitory activity, mostly against Gram-positive bacteria. This activity was mainly found in the root extracts. Six of the thirteen plants screened for anti-inflammatory activity using the cyclooxygenase-1 (COX-1) bioassay had activity above 90%. Hexane and methanol extracts were the most active while water extracts usually had lower activity. Malva parviflora, Eriocephalus punctulatus and Asparagus microraphis exhibited high anti-inflammatory activity from hexane, methanol and water extracts made from leaf and root material. High anti-bacterial activity was also recorded from M. parviflora and E. punctulatus hexane, methanol and water extracts. An investigation on seasonal variation and plant part substitution in medicinal activities for these plants was carried out. Extracts of M. parviflora collected between June 1999 and July 2001 showed variation in anti-bacterial activity. Extracts made from leaves and roots inhibited the growth of both Gram-positive and Gram-negative bacteria. More bacterial strains were inhibited by extracts made from roots collected in cooler months. However, a trend in seasonal activity was not evident for either the roots or leaves because there was no detection of activity in some of the extracts made within the same months or seasons of the adjacent years. Variation in anti-inflammatory was detected for M. parviflora extracts. E. punctulatus leaf extracts did not exhibit any seasonal variation in anti-bacterial activity. Anti-inflammatory activity of E. punctulatus showed seasonal variation with the highest activity noted when material was collected during the cooler months and a decline in activity when collections were made during the warmer months. Hexane, methanol and water extracts made from leaves and roots of A. microraphis did not show any seasonal variation in anti-inflammatory activity. Thus, M. parviflora and E. punctulatus should be collected during the cooler months while A. microraphis can be collected throughout the year. Traditional healers, herbalists and vendors need to be encouraged to use aerial parts in substitution of ground parts which are reported to be highly utilized. Effect of storage on anti-bacterial and anti-inflammatory activities of M. parviflora, E. punctulatus and A. microraphis were monitored. Dried, ground leaf and root material of the three plants was stored in a cold room, at room temperature and in the Botanical Garden where the material was exposed to high and large changes in temperature. Dried hexane and methanol extracts made from leaves and roots of these plants were stored in a cold room and at room temperature. Initially, storage of the plant material under the three storage conditions caused an increase in antibacterial activity of the hexane, methanol and water extracts made from leaf and root material of M. parviflora and E. punctulatus. Storage for a longer period resulted in a decrease in inhibitory activity. TLC fingerprints developed from hexane and methanol extracts made from M. parviflora and E. punctulatus stored in a cold room and at room temperature showed a consistent number and colour of spots during the initial storage period. Prolonged storage resulted in a decline in the number and colour of detected spots. The stored hexane and methanol extracts made from leaves and roots showed a similar trend of increases and decreases in anti-bacterial activity as well as changes in spots with the storage of the extracts. Testing of the effect on anti-inflammatory activity of hexane, methanol and water extracts made from leaves and roots of M. parviflora, E. punctulatus and A. microraphis showed no change in inhibitory activity of hexane extracts obtained from the material and the extracts stored at the three storage conditions. Methanol and water extracts made from leaves exhibited an increase in activity with prolonged storage. Generally, the stability of the inhibitory activity was longer for the stored dried material than the plant extracts. Isolation of biological active compounds from M. parviflora was not successful due to loss in anti-bacterial activity as a result of collection of plant material from a different locality. Anti-inflammatory compounds could not be isolated due to insufficient amount and the synergistic effect of the active compounds . The purified compounds exhibited loss of activity following HPLC purification which then re-appeared upon recombining the fractions. A number of compounds were detected from essential oils of E. punctulatus using GC. Fractions containing these compounds gave positive anti-bacterial activity in the disc-diffusion , bioautographic and MIC bioassays as well as high anti-inflammatory activity with COX-1 and COX-2 anti-inflammatory bioassays. No anti-inflammatory compounds were isolated from A. microraphis.

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