Variation of active constituents in Euclea natalensis based on seedling stages, seasons, and fertilizers

Euclea natalensis A.DC. belongs to the Ebenaceae family, and is extensively distributed along the eastern coast of southern Africa. Many Euclea species are widely gathered by indigenous people because of their medicinal properties. Roots of these plant species are frequently used to treat respiratory complications such as chest pains, bronchitis, pleurisy and asthma. Ground root powder is topically applied in cases of leprosy and is used by some ethnic groups to treat toothache and headache. The bioactivity encountered is attributable to naphthoquinones, which are common phenolic compounds in the Ebenaceae family. Naphthoquinones isolated from E. natalensis (shinanolone, 7-methyljuglone, diospyrin, isodiospyrin and neodiospyrin) have exhibited a broad spectrum of antimicrobial activity. The demand for these products will escalate due the amount of plant material required to further research. We need to explore techniques that can maximize their productivity. The present study was conducted on E. natalensis, in an attempt to establish if there exists any correlation between the accumulation of naphthoquinones and stages of seedling growth, seasonal fluctuations and application of fertilizers. A possible correlation between seedling growth stages and the accumulation of naphthoquinones (shinanolone, 7-methyljuglone and diospyrin) was investigated in seeds and seedlings of Euclea natalensis. In this study, the seeds represented the first stage, whereas the second seedling stage was defined as the stage when the radicles were about 6 cm long. The lengths of the seedlings at the third, fourth and fifth seedling stages were 9 cm, 12 cm and 16 cm respectively. Plant materials collected from the five seedling stages were separately extracted using chloroform and the naphthoquinones were then quantified by means of High Performance Liquid Chromatography (HPLC). Mobile phase of MeCN: H2O: AcOH (62.5: 32.5: 5) was used as an eluent in an isocratic mode and at a flow rate of 0.8 ml/min. Standard curves of each of the four compounds were obtained by making a series of dilutions in the concentration range of 22.5 µg/ml to 2.25 µg/ml. Ten microlitres of each dilution was injected three times into the HPLC, and the run time for each injection was 20 minutes. Calibration curves were then generated and used for the quantification of each compound. Shinanolone, which was the only naphthoquinone detectible in seeds, accumulated at variable rates (P<0.01) and no trend could be established between its synthesis and seedling growth. The content of shinanolone ranged from 87.5 mg/kg dry weight (dw) in seeds to a high mean value of 1047 mg/kg (dw) during the fourth seedling stage. A significant correlation (P<0.01) was found between the mean concentrations of 7-methyljuglone and seedling growth. 7-Methyljuglone was quantified at a high mean level of 5003 mg/kg during the third seedling stage and was not detected in the seed samples. A positive correlation (P<0.01) was established between the concentration of diospyrin and seedling stages. Diospyrin was detected at an elevated mean concentration of 6182 mg/kg during the fifth seedling stage, which was higher than the other quantified naphthoquinones. Seasonal variation of naphthoquinones (shinanolone, 7-methyljuglone, diospyrin, isodiospyrin and neodiospyrin) was investigated from eleven plants of E. natalensis subsp. natalensis growing in natural populations, over a period of four seasons. The roots were harvested, dried, extracted and analysed as in the previous study. The mean levels of shinanolone and 7-methyljuglone were found to be uniform in all the seasons and no statistically significant variation could be found between seasonal changes and their mean concentrations. Accumulation of isodiospyrin and neodiospyrin varied significantly with seasonal changes (P<0.05). These two bioactive naphthoquinones were detected only in summer and autumn respectively, and not in winter. A statistically significant variation (P<0.05) was established between the levels of diospyrin and seasonal fluctuations. Diospyrin was detected at a mean concentration of 3190 mg/kg (dw) during spring, which was higher than the other naphthoquinones quantified in all four seasons. The effect of NPK fertilizers on growth performance and accumulation of naphthoquinones (shinanolone, 7-methyljuglone, diospyrin, isodiospyrin and neodiospyrin) in seedlings of E. natalensis grown in shade and under field conditions was investigated. Each group was subdivided into four subgroups, which were then subjected to four respective treatments of water-soluble foliar feed (2:1:2 (44) NPK) at three different concentrations. Treatments tested were as follows: Treatment 1 at 40 g/l, Treatment 2 and Treatment 3 at 20 g/l and 10 g/l respectively. The control group received only supplemental water. The first harvest was conducted after 6 months of application of fertilizers and the second one was done after 12 months of treatment. Roots and shoots were harvested and analysed separately. The naphthoquinones were quantified as previously described. The bioactivity of root extracts from seedlings was tested against Mycobacterium smegmatis and extracts with lower MIC were further tested on M. tuberculosis. Growth parameters differed between the two groups, with seedlings from the shadehouse showing more plant vigour than the field grown plants. No significant interaction could be established between the measured growth factors and treatment. A significant interaction (P<0.001) was found between Treatment 2 and shadehouse seedlings. Treatment 2 enhanced vegetative performance with the mean values of fresh weight of shoots and roots being twice as much as their respective control mean values. A significantly positive correlation was established between the concentration of shinanolone (P<0.01), isodiospyrin (P<0.05) and neodiospyrin (P<0.05) with fertilization from field-grown seedlings. Application of NPK fertilizers significantly (P<0.05) increased the accumulation of neodiospyrin in seedlings subjected to shadehouse conditions. The most potent naphthoquinone, 7-methyljuglone, was found to be abundant in all the extracts and was quantified at a high mean concentration of 10200 mg/kg from shadehouse seedlings. Root extracts of E. natalensis seedlings grown under field conditions were generally more active against the bacterial strain of M. smegmatis as compared to extracts acquired from roots of seedlings maintained under a shadehouse setting. A lowest minimum inhibitory concentration (MIC) of 0.78mg/ml against M. smegmatis was observed from the second harvest of field-cultivated seedlings of the control and Treatment 1 subgroups. The MIC values for shadehouse seedlings ranged from 1.6 to 6.3 mg/ml. Minimum bactericidal concentration (MBC) values from all the extracts tested were relatively higher than their respective MIC’s. Root extracts of E. natalensis were more active against M. tuberculosis and their MIC values were lower than the tested concentrations. Extracts acquired from field-grown seedlings were more active against M. smegmatis with a lowest MIC value of 0.78 mg/ml. Extracts from the control group and Treatment 1, which had less application of fertilizers were more active against strains of M. tuberculosis with MIC value of 10 µg/ml. This shows the selectivity of E. natalensis against the mycobacterial strain of M. tuberculosis. Based on the findings, synthesis and accumulation of naphthoquinones in E. natalensis is highly variable within individuals of the species investigated. Naphthoquinones accumulate in relatively higher amounts in roots of E. natalensis than in the aboveground structures, which validate their harvest by indigenous people. The concentration of shinanolone varied slightly and its production increased with seedling growth. The synthesis of 7-methyljuglone is independent of fertilisation as its accumulation was enhanced in seedlings subjected to control treatment. Neodiospyrin and isodiospyrin were always present in every sample obtained from the seedlings but they were not detectible in every profile of samples from mature plants. Diospyrin is the only naphthoquinone that was detected in every sample analysed and also quantified in high concentrations from mature plants harvested in spring. The study showed that depending on the requirement of a particular naphthoquinone for research, one could target the seasons and seedling stages recommended from this study. This study also showed that field-cultivated seedlings produced more potent naphthoquinones than the ones subjected to controlled environments.Dissertation (MSc (Plant Science))--University of Pretoria, 2008.Plant Production and Soil Scienceunrestricte

Nmr-based metabolomic study of medicinal plants used against malaria and the isolation of bioactive alkaloids

Despite the significant advances achieved in lessening the burden of malaria and other tropical diseases in recent years, protozoal infections remain a major cause of mortality in many developing countries, with malaria accounting for a large proportion of the recorded mortality. The main aim of this study was to bioprospect indigenous plant species for novel antiplasmodial plant products by means of NMRbased metabolomics. In this study, an ethnobotanical criterion was followed in collecting twenty indigenous plant species used to treat malaria or its symptoms by Vha-Venda people living in Mutale Municipality of Limpopo Province, South Africa. Plant samples were extracted in dichloromethane:50% methanol (1:1), separated into polar and nonpolar fractions, and tested on Trypanosoma brucei rhodesiense, T. cruzi, Leishmania donovani and Plasmodium falciparum. Plant extracts were correspondingly subjected to an antiproliferative bioassay against mammalian skeletal myoblast cells. The current study is the first scientific account on the significant antileishmanial efficacy (IC50 ? 5 ?g/ml) of Bridelia mollis (Phyllanthaceae), Vangueria infausta subsp. infausta (Rubiaceae), Syzygium cordatum (Myrtaceae) and Xylopia parviflora (Annonaceae), as well as high antitrypanosomal activity (IC50 = 3.45 ?g/ml) of Albizia versicolor (Fabaceae). Ten plant extracts exhibited significant in vitro antiplasmodial activity (IC50 ? 5 ?g/ml), with Tabernaemontana elegans (IC50 = 0.331 ?g/m and IC50 = 0.834 ?g/m) and V. infausta subsp. infausta (IC50 = 1.84 ?g/ml) being the best samples. This is the first scientific report to document significant antiplasmodial activity of extracts from T. elegans. The findings of this study substantiate the rationale for adopting an ethnopharmacological approach when bioprospecting medicinal plants for antiplasmodial compounds. Dichloromethane extracts were subjected to 1H NMR-based metabolomic analysis, where each crude extract was reconstituted in CDCl3, analysed on a Varian 600 MHz spectrometer and the acquired 1H NMR spectra were then analysed collectively using multivariate data analysis (MDA). Principal Component Analysis (PCA) could not separate the analysed profiles according to the detected antiplasmodial bioactivity. Application of supervised Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) on the 1H NMR profiles resulted in a discrimination pattern that could be correlated to the observed antimalarial bioactivity. A contribution plot generated from the OPLS-DA scoring plot illustrated the classes of compounds responsible for the observed grouping. Further phytochemical analyses were conducted on lipophilic extracts of T. elegans and V. infausta subsp. infausta. These best candidates were fractionated, purified and identified based on conventional chromatographic and spectroscopic techniques. Two known indole alkaloids isolated from T. elegans, were identified as dregamine and tabernaemontanine. The antiplasmodial activity of these acyl indole alkaloids has previously been established and ranges from moderate to good. Three compounds were isolated from V. infausta subsp. infausta of which two were identified as friedelin (IC50 = 3.01 ?g/ml) and morindolide (IC50 = 18.5 ?g/ml). While these compounds have been previously identified, this is the first account of their occurrence in the genus Vangueria and their antiplasmodial activity. An unidentified compound with significant antiplasmodial activity (IC50 = 0.143 ?g/ml) was also isolated. The study demonstrated the potential of discovering novel antiplasmodial scaffolds from medicinal plants.Thesis (PhD)--University of Pretoria, 2016.Plant SciencePhDUnrestricte

1H NMR-based metabolomics of antimalarial plant species traditionally used by Vha-Venda people in Limpopo Province, South Africa and isolation of antiplasmodial compounds

Please read abstract in the article.The National Research Foundation (South Africa)http://www.elsevier.com/locate/jethpharmhj2020Plant Production and Soil Scienc

Variation in the content of naphthoquinones in seeds and seedlings of Euclea natalensis

A correlation between plant growth and accumulation of naphthoquinones (shinanolone (1), 7-methyljuglone (2) and diospyrin (3)) was investigated in seeds and seedlings of Euclea natalensis A.DC. In this study, the seeds represented the first stage whereas the second seedling stage was defined as the stage, when the radicles were about 6 cm in length. The lengths of the seedlings at the third, fourth and fifth seedling stages were 9 cm, 12 cm and 16 cm respectively. Plant materials collected from the five seedling stages were separately extracted using chloroform and the naphthoquinones were then quantified by means of High Performance Liquid Chromatography (HPLC). Shinanolone (1), which was the only naphthoquinone detectible from seeds, accumulated at variable rates (P < 0.01) and no trend could be established between its synthesis and seedling growth. The content of shinanolone (1) ranged from 87.5 mg/kg in seeds (first stage) to a high mean value of 1047 mg/kg during the fourth seedling stage. A significant correlation (P < 0.01) was found between the mean concentrations of 7-methyljuglone (2) and seedling growth. 7-Methyljuglone (2) was quantified at a high mean level of 5003 mg/kg during the third seedling stage and was not detected from the seed samples. A positive correlation (P < 0.01) was established between the concentration of diospyrin (3) and seedling stages. Diospyrin (3) was detected at an elevated mean concentration of 6182 mg/kg during the fifth seedling stage, which was higher than the other quantified naphthoquinones

Anti-inflammatory potential of South African medicinal plants used for the treatment of sexually transmitted infections

Please read abstract in the article.The National Research Foundation (NRF Grant no: 99022 and 98980) South Africa and the University of Pretoria.http://www.elsevier.com/locate/sajb2020-09-01hj2020Paraclinical SciencesPlant Production and Soil ScienceZoology and Entomolog