Investigation of antimycobacterial and antiviral activities of extracts and compounds isolated from Malawian medicinal plants

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Kamuzu University of Health Sciences
Infectious diseases accounts for approximately one-half of all the deaths that occurs in tropical countries including Malawi. Some of these infectious diseases are caused by microorganism such as mycobacteria and viruses. However, the threat of antimicrobial resistance is growing at an alarming rate and the situation has been aggravated in these developing countries due to so many factors including presence of multidrug resistants’ strains and over prescriptions. Therefore, the purpose of present study was to investigate the phytochemical constitutes and compounds isolated from Aeschynomene nyassana, Euphorbia whyteana, Euphorbia cooperi, Flueggea. virosa, Phyllanthus amarus, Ericae milanjiana and Rhus acuminatissima medicinal plants, determine their antimicrobial activities and cytotoxic properties. Ethnobotanical survey was conducted and several methods were used to identify and confirm the selections of plants understudy. Search engine on several online databases (Google Scholar, PubMed, MEDLINE, Scopus, Cochrane Library, and Science Direct) were used to identify medicinal plants with antiviral and antimycobacterial activities. The plants found were then matched with published and unpublished ethnobotanical survey data and database on the Flora of Malawi, Chewa Medical Botany by Brian Morris, Useful Plants of Nyasaland by Jessie Williamson and other sources. Seven plants, were identified for this research and these were extracted twice with solvents such as methanol, ethyl acetated and dichloromethane. Part of the methanol/water extracts obtained were subjected to alkaloid extraction scheme while the other part was subjected to column chromatograph. 1,3,6 – tri(3,4,5-trihydroxyphenyl) acetyl ester - β-Dglucopyranose, 1,2,3,4,6 – penta(3,4,5-trihydroxyphenyl) acetyl ester - β-D- glucopyranose, 1,2,3,6 – tetra(3,4,5-trihydroxyphenyl) acetyl ester - β-D- glucopyranose, 2,3,6 – tri(3,4,5- trihydroxyphenyl) acetyl ester - β-D- glucopyranose, compounds were obtained from Hexane: Ethyl acetate 50%; 80%; 90% and Ethyl acetate: Methanol 5% and the compounds were identified from the NMR spectra while Betulinic acid was obtained as a white amorphous powder, soluble in methanol, having been eluted with methylene chloride: methanol 9:1 from the silica gel column. The NMR spectral data was compared in reference literature and showed to be very similar to those of Betulinic acid compound. Benzoxylanthaquinone was identified by direct comparison of the spectroscopic data with those published literatures. The compound, Cyclanoline, greyish in colour was obtained from alkaloid extraction scheme and identified by direct comparison of the spectroscopic data with those published literatures. Mass spectra(ESIMS) were obtained with a Thermo-Finningan LCD DECA mass spectrometer and HRESIMS spectra were measured with a FTHRMS-Orbitrap (Thermo-Finnigan) mass spectrometer. The compound E5 m/z 695.33, yellowish in colour was obtained from alkaloid extraction scheme and identified by direct comparison of the spectroscopic data with those published literatures. Mass spectra(ESI-MS) were obtained with a Thermo-Finningan LCD DECA mass spectrometer and HRESIMS spectra were measured with a FTHRMS-Orbitrap (Thermo- Finnigan) mass spectrometer. The resultant crude extracts, fractions and compounds were used in the phytochemical screening and antimicrobial analysis. The phytochemical analysis involved spectrophotometric screening for pro/antioxidant properties of the crude extracts, fractions and compounds using HPTLC, DPPH, FRAP, Reducing power, Crocin bleaching assay, Cupric reducing antioxidant capacity and Nitric oxide radical scavenging activities. Cell viability and cytotoxicity studies were conducted using the Trypan blue dye exclusion, (3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT and Mammalian macrophage cytotoxicity assays while heavy metals analysing was performed using atomic absorption spectrometry. The antimycobacterial activities of crude extracts, fractions and compounds were assessed using microplate alamar blue assay. Compounds obtained were further assessed for drug susceptibility against resistant Mycobacterium tuberculosis using the BACTEC™ MGIT 320 system. The results of the review study identified 50 different families of medicinal plants comprising 90 plants species that are frequently used by traditional healers to treat various diseases in Malawi such as sexually transmitted infections, diarrhoea/dysentery, skin infections/rash, respiratory infection, cold/cough, fever, tuberculosis and herpes zoster. Furthermore, these medicinal plants also demonstrated some anti-viral and antimycobacterial activities. The compounds/compound groups identified in this study were categorized into eight distinct groups of flavonoids, alkaloids, saponins, phenolics, lignins, xanthones, proteins and peptide. In phytochemical analysis, results of crude extracts, fractions and compounds showed moderate DPPH scavenging activity, a dose dependent decrease in NO scavenging activity except for R. acuminatissinia while A. nyassana revealed reducing power ability that was significantly greater than standard Ascorbic acid. In HPTLC analysis, DPPH active spots showed pale-yellow coloured spots for the 4 plants understudy while the phenolic active blue colour spots were observed to be more visible on R. acuminatissinia and E. milanjiana. In PBMC viability analysis, the lymphocytes treated with Rhus acuminatissima and Ericae milanjiana had the lowest cell percent viability. The plants understudy also demonstrated a decrease in percent viability with increased time for all the plants extracts except for E. milanjiana and A. nyassana which showed some stimulative effects. A. nyassana and Euphorbia whyteana demonstrated relatively high cytotoxicity as compared to E. milanjiana, R. acuminatissima and standard drug, Doxorubicin. In heavy metal analysis, all the plants understudy displayed the least levels of toxic metals concentration in the order of Cadmium˂Chromium˂Lead. In the antimycobacterial analysis, F. virosa and E. milanjiana crude extracts displayed Minimum Inhibitory Concentration of 128 ug/ml and 512 ug/ml respectively while A. nyassana ANX fraction showed MIC of 256 ug/ml for M. smegmatis. E. cooperi, E. milanjiana and E. whyteana crude extracts displayed MIC value of 512 ug/ml while E. whyteana W1 fraction had MIC value of 252 ug/ml for M. ulcerans. The selectivity index (SI) values of plants understudy ranged from <0.082 – 2.20 and considerably good SI were observed in F. virosa, E. whyteana crude extracts and E. whyteana W1 fraction at 2.20, 0.625 viii and 0.985 respectively. In synergistic antimycobacterial analysis, the synergistic drug action showed Betulinic acid (EM8), 1,2,3,6 – tetra(3,4,5-trihydroxyphenyl) acetyl ester - β-Dglucopyranose (FV8) and Cyclanoline (C5) exhibited synergistic antimycobacterial activity at a final concentration of 18 ug/ml. The good synergistic results (susceptibility) were observed in combined EM8+INH and FV8+INH while a moderate synergistic result was observed in C5+INH indicating an additive effect of the combination. However, antagonistic activity (resistance) was observed in combined EM9+ETH and E1+ETH. Therefore, it can be concluded from the results that all the plants understudy showed considerable antimycobacterial activities against M. tuberculosis, M. ulcerans and M. smegmatis. The compounds obtained from the plants understudy also demonstrated synergistic properties with the first-line tuberculosis drugs, rifampicin; isoniazid ethambutol and streptomycin. Consequently, this study recommends that further studies to be undertaken in order to determine the mechanism of action of the novel compounds in order to unravel its exact potential to inhibit several pathogenic microbes especially resistant M. tuberculosis and M. ulcerans and also to evaluate whether its true pharmacological activities exist. Currently, an all-oral and less toxic treatment regimen of Buruli ulcer is been sought after and encouraged by WHO.