South Africa has one of the richest and diverse floras in the world with over 30000 species of higher plants. There are approximately 3000 species of medicinal plants in South Africa. The discovery of active compounds in medicinal plants plays a strategic role in the phytochemical investigation of crude plant extracts. Secondary metabolites of medicinal plants are a major source of drugs for the treatment of various health disorders. Cardiac glycosides are one of the subgroups of steroids modified from terpenoids. The existence of cardiac glycosides in some plant species often indicates toxicity. Among the contagious elements, diseases caused by viruses are one of the major causes of death, disability, and social and economic disruption for millions of people. Viruses also cause many important plant diseases and are responsible for huge losses in crop production and quality in all parts of the world. According to literature, plants containing cardiac glycosides demonstrate potential for the discovery of more effective new drugs in the treatment of infection especially viral contagions. The main objectives of this study were to evaluate the antiviral (HSV-2 and PVYN), antibacterial and cytotoxic activities of South African plants containing cardiac glycosides. Furthermore, isolation and identification of compounds with an emphasis on cardiac glycosides was carried out. Eight plants known to contain cardiac glycoside compounds were selected. Leaves of Gomphocarpus fruticosus, Nerium oleander, Cotyledon orbiculata, the fruits and leaves of Strophanthus speciosus, the bulbs of Bowiea volubilis and Merwilla plumbea were selected to examine their antimicrobial activities, toxicity, antiviral and to isolate potential pure compounds.
The ethanol extracts of all selected plants were screened for antibacterial activity against two Gram-positive (Staphylococcus aureus and Enterococcus faecalis) and two Gram-negative (Escherichia coli and Klebsiella pneumonia) pathogens. Plant extracts showed promising antibacterial action against Gram-positive bacteria. The most active extract against both Gram-positive bacteria was C. orbiculata with the MIC value of 1.25 mg/ml.
Cell toxicity was monitored by determining the effect of the ethanolic crude extracts on human embryonic kidney cell line (HEK 293) using the XTT method. All extracts exhibited high toxic effects with IC50 < 100 μg/ml on the tested cell line. The XTT assay was used to determine of the antiviral activity of crude ethanolic extracts on the kidney epithelial cells of African Green Monkey (Vero). The results revealed that the crude ethanolic extracts of all
selected plants exhibited a cytotoxic effect on Vero cells at concentrations lower than their EC50. Consequently, the determination of antiviral activity of the selected plant extracts was not successful.
Based on the chromatographic and bioassay results from the six plants selected for this study, the fruit extract of S. speciosus, belonging to the Apocynaceae family, was chosen for the isolation of compounds, particularly cardiac glycosides. All plant extracts were tested for antibacterial activity against E. coli (Gram-negative) and E. faecalis (Gram-positive) by using bioautography. The general TLC test showed more variety of compounds in the fruit extract of S. speciosus. The results of the bioassay showed promising activity of the fruit extract of S. speciosus and the bulb extract of B. volubilis against Gram-positive bacteria. The chromatographic investigation of the S. speciosus fruit extract led to the isolation of three pure compounds including a cardiac glycoside. The three compounds were identified based on NMR (1D and 2D) and HRMS. The isolated compounds were identified as: a triterpene (ursolic acid methyl ester), a sugar: myo-inositol methyl ester and an unidentified cardiac glycoside. According to literature, it is the first report of the isolation of ursolic acid methyl ester and myo-inositol-methyl ether from S. speciosus fruit extract.
The isolated cardiac glycoside exhibited no inhibitory activity at 1.25 mg/ml (the highest concentration tested) against all four tested bacteria (S. aureus, E. faecalis, E. coli and K. pneumonia). The cytotoxicity and anti-HSV-2 screening of the isolated cardenolide demonstrated the highly toxic effect of this compound on the HEK 293 cell line with 4.62 μg/ml IC50 value and < 25 μg/ml IC50 of Vero cell line. No evidence could be found in the literature of the cytotoxic activity of cardiac glycoside compounds on the HEK 293 cell line.
The ethanolic extracts of all plant extracts and isolated cardenolide were tested against the PVYN in vivo and in vitro. The results revealed that the high concentration (50 mg/ml) of M. plumbea, N. oleander, B. volubilis (fresh bulb), C. orbiculata and isolated cardenolide reduced the PVYN symptoms on tobacco plants in an in vivo experiment. In in vitro analysis, the high concentration (50 mg/ml) of S. speciosus (leaves & fruits), and especially M. plumbea (dry bulb) showed significant antiphytoviral activity. In vivo and in vitro results demonstrated that M. plumbea has potential antiphytoviral activity.