Investigation of antimycobacterial and antiviral activities of extracts and compounds isolated from Malawian medicinal plants
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 r...
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| Format: | Thesis |
| Language: | English |
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Kamuzu University of Health Sciences
2022
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| Online Access: | http://nkhokwe.kuhes.ac.mw/handle/20.500.12845/647 |
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Add Tag | Summary: | 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and unpublished ethnobotanical survey data and database on the Flora of Malawi, Chewa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
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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. |
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