Theses and Dissertations (Microbiology)
http://hdl.handle.net/10386/72
2024-03-28T21:17:59ZIdentification and characterisation of compounds with antimycobacterial activity from the leaves of Dombeya rotundifolia Hochs
http://hdl.handle.net/10386/4304
Identification and characterisation of compounds with antimycobacterial activity from the leaves of Dombeya rotundifolia Hochs
Mashilo, Matsilane Lethabo
Plants are not only an important source of medicines, but also play a significant role
in drug development for the treatment of diseases such as Tuberculosis (TB). TB is a
pulmonary disease that is caused by Mycobacterium tuberculosis complex. The aim
of the study is to identify and characterise antimycobacterial compounds from
Dombeya rotundifolia. The plant was collected from the University of Limpopo, dried
and ground into fine powder. Extraction was done using different solvents that differ
in polarity. The plant was screened and analysed for phytochemicals. Three major
phytochemicals were quantified using reagent assays and analysed using standard
curves. The antioxidant activity of the plant was determined using 2,2-Diphenyl-1-
picrylhydrazyl (DPPH) and ferric reducing power assay. The antibacterial activity of
the plant extracts was tested against Mycobacterium smegmatis using bioautography
and serial microplate broth dilution assay. The antibiofilm activity of the plant extracts
were evaluated using crystal violet assay. The anti-inflammatory activity of the plant
was determined using egg albumin protein denaturation assay. The cytotoxic effects
of the extracts were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) assay on THP-1 cell line. The antimycobacterial
compounds were isolated and purified using bioassay-guided assay incorporated with
column chromatography and preparative TLC. The isolated compounds were
analysed and identified using nuclear magnetic resonance spectroscopy (NMR). The
results obtained in this study showed that water was the best extractant, extracting
57.6 mg of the plant material, followed by methanol with 38.1 mg and hexane as the
least extractant with 12.4 mg. In the phytochemical analysis of TLC plates, there was
a separation of compounds in all the mobile systems, and a clear separation of
compounds was observed in the BEA mobile system, followed by the EMW system.
The plant has all major phytochemicals that are needed for drug development. The
plant extracts had antioxidant activity, which was confirmed by the quantitative assays.
The high inhibitory activity of the extracts was indicated by low MIC values that
inhibited the bacterial growth. The butanol extract had the lowest MIC value (0.13
mg/mL). The plant extracts were able to prevent the formation of biofilm at different
concentrations. The plant was proven to have anti-inflammatory properties by having
a high inhibition capacity to prevent protein denaturation. The cytotoxicity results
showed that the plant was toxic to the cells. The isolated antimycobacterial
compounds were identified as Eicosanoic acid and Docosanoic. They have moderate
activity with the MIC value of 0.25 mg/mL. The study indicated that the isolated
compounds have antimycobacterial activity, which showed that the plant has a
potential to treat TB or symptoms related to TB. However, further studies are needed
to evaluate their toxic effects before use.
Thesis (M.Sc. (Microbiology)) -- University of Limpopo, 2023
2023-01-01T00:00:00ZImprovement of the potability of surface water by using the filtration method
http://hdl.handle.net/10386/4253
Improvement of the potability of surface water by using the filtration method
Malema, Mokaba Shirley
Access to safe drinking water is a major problem globally and it mostly affects people living in low-income countries. The lack of potable water leads to the use of raw water from surface or ground water sources for drinking and other household purposes. A water filtration unit was designed and constructed using fabric, gravel and sand, which were wet-packaged into a 20 L bucket. The efficiency of the filter unit to improve the bio-physicochemical properties of contaminated water was tested using surface waters from rivers in the Sekhukhune area. Physico-chemical parameters tested included turbidity, colour, total suspended solids (TSS), total dissolved solids (TDS), total hardness and pH. Turbidity and colour were the most improved characteristics, where turbidity improved by 69% and colour by 80%. Other parameters such as total hardness, TSS and TDS were non-significantly reduced following treatment with the filtration unit. The amount of soluble solids in raw water was well within allowable limits by WHO standards. Microbiological tests included heterotrophic bacteria, total coliform and faecal coliform counts. The bacterial load was too numerous to count for the untreated water, however, after treatment with the filtration unit, heterotrophic bacterial load decreased to 15 x 10103 CFU/ml, total coliforms to 14 x 10
2
CFU/100ml and faecal coliforms to 11 x 10
2
CFU/100ml. Further treatment with UV sterilization unit resulted in undetectable amount of bacteria. The unit designed in this study can be beneficial in those rural communities were clean water is not available, it is easy to construct and simple to operate and most importantly it reduced contaminants in surface water. The UV unit incorporated in this study is not cost effective, therefore, other household treatment options such as chlorination and boiling which are easily accessible to most communities can be used to further eliminate remaining microorganisms after filtration. The effective shortest boiling time and minimum dosage of Na(OCl2) have been previously tested in our laboratory and total elimination of bacteria was achieved within 2 minutes of rolling boil and after 30 minutes following addition of 5ml/20L of liquid chlorine.
Thesis (M.Sc. (Microbiology)) -- University of Limpopo, 2016
2016-01-01T00:00:00ZAntimycobacterial evaluation, preliminary phytochemical and cytotoxicity studies of cassia petersiana
http://hdl.handle.net/10386/4224
Antimycobacterial evaluation, preliminary phytochemical and cytotoxicity studies of cassia petersiana
Mothupi, Ramokone Florah
This study aimed to investigate antimycobacterial and cytotoxic compounds from
Cassia petersiana. Cassia petersiana was selected for the current study based on its
traditional use for treating tuberculosis (TB) symptoms. Extraction is an important step
in the use of medicinal plants; hence, solvents of varying polarity were employed to
extract a wide range of compounds where chloroform was the best extractant (67 mg).
As there is no relation between the amount of plant material extracted and the
bioactivity of the extracts, standard tests were used to determine the presence of
different phytochemical constituents from Cassia petersiana and the total phenolic,
flavonoid, and tannin contents were quantified using colorimetric assays. It was
revealed that all the tested phytochemical constituents were present, and it was
proven that phenolic compounds were the most abundant, followed by the tannins,
while the flavonoids were the least among the common phytochemical constituents
quantified. The phytochemical compounds were further profiled on thin-layer
chromatography (TLC) and developed in BEA, CEF, and EMW solvent systems.
Colourful compounds which indicated diverse phytochemicals were visualised with
both vanillin-sulphuric acid and ultraviolet light on the phytochemical chromatograms
and good separation of the compounds was from the BEA solvent system. The
qualitative and quantitative antioxidant activity and antimycobacterial activity assays
were used to evaluate the extracts from Cassia petersiana. Minimal antioxidant activity
was observed on the qualitative antioxidant activity profile. These findings correlated
with the minimal quantity of antioxidants from extracts of Cassia petersiana from the
quantitative antioxidant assays; ferric reducing power and DPPH scavenging activity
assays. Cassia petersiana extracts had bioactivity against Mycobacterium smegmatis
as indicated by the lowest MIC value. The cell viability effects of the acetone crude
extract from Cassia petersiana were evaluated against the tryptophan hydroxylase-1
(TPH-1) macrophage cells. Large scale extraction procedure was employed to extract
a sufficient amount of plant material in preparation for the isolation of the bioactive
compound. Bioassay-guided fractionation combined with column chromatography and
TLC were used to isolate and purify the bioactive compound from the n-hexane extract
of Cassia petersiana. The purified isolated compound was elucidated as β-sitosterol,
which showed remarkable bioactivity against Mycobacterium smegmatis only on the
TLC-bioautographic assay, while the quantitative antimycobacterial activity was higher
xx
with the MIC value of 2.5 mg/mL. Although β-sitosterol is known as a good antioxidant,
it showed no antioxidant activity on the qualitative antioxidant activity assay.
Therefore, further studies, including in vivo assay, are recommended on the isolated
compound to evaluate its biological activities before consideration of its use in the
development of alternative drugs.
Thesis (M.Sc.(Microbiology)) -- University of Limpopo, 2022
2022-01-01T00:00:00ZImprovement of the quality and shelf life of traditionally produced sorghum juice by addition of ashes, dried powered leaves and stem obtained from combretum spp
http://hdl.handle.net/10386/4158
Improvement of the quality and shelf life of traditionally produced sorghum juice by addition of ashes, dried powered leaves and stem obtained from combretum spp
Mathipa, Morongwa Mary
Traditional sorghum juice is produced in many African countries for human
consumption. The juice is very rich in calories, B-group vitamins including thiamine,
folic acid, riboflavin, nicotinic acid, and essential amino acids such as lysine. Low
earning income women at village level produce sorghum juice for home consumption
and sale. The short shelf life (2 to 3 days) of sorghum juice is a major problem for both
the brewers and consumers of this drink. The aim of the study was to use 12
Combretum plants to improve the microbiological quality and shelf life of sorghum
juice.
Fresh stems and leaves of C. caffrum, C. vendae, C. erythrophyllum, C. elaegnoides,
C. apiculatum, C. imberbe, C. adenogdium, C. padoides, C. bracteosum, C. kraussii,
C. mkuzense and C. zeyherii were collected at Nelspruit, National Botanical Gardens,
Mpumalanga, South Africa. Voucher specimens and tree labels were used to verify
the identity of the plants. The stems and bark collected were cut into pieces and air dried for 30 days. When dried, the plant material was ground to a fine powder and
stored in paper bags at room temperature. The wood was burnt in an open fire; fuel
was not used to minimise contamination.
The qualitative phytochemical composition of both the leaves and stems of
Combretum plants analysed in this study revealed the presence of saponins, tannins,
terpenoids, steroids, cardiac glycosides and flavonoids. The following
phytoconstituents were lost in the ashes; tannins with the exception of C. mkuzense
and C. padoides; cardiac glycosides and flavonoids. The quantitative phytochemical
analyses revealed that both the leaves, stems and some ashes such as C. apiculatum
and C. vendae contained appreciable levels of phenolic compounds, tannins and
flavonoids.
Quantitative analysis of antioxidant activity, the 2, 2, diphenyl-1-picrylhydrazyl (DPPH)
assay was used as a screen test for the radical scavenging ability of the compounds
present in the different 36 70% acetone extracts. DPPH screening method indicated
great scavenging activity with the 70% acetone leaf extracts of C. kraussii, C. zeyherii
and C. mkuzense. The leaf and stem extracts showed substantial great antioxidant
activity in a concentration-dependent manner. There was a significant decrease in the
antioxidant activity in the ashes (p=001), when compared to both the leaves and the
stems.
The proximate and nutritional analysis of the 70% acetone extracts were performed
by AOAC and ICPE protocols, respectively. The results indicated that all the extracts
had substantial amounts of ash, moisture, protein and energy. Mineral content of the
plant parts was analysed as well, calcium had the highest concentration, while zinc
was lowest in concentration. The mineral content decreased significantly in the stems
(p ≤ 0.05) when compared to the leaves. There was a further decrease in mineral
content with regard to the ashes with the exception of calcium. Based on these
findings, the leaves and ashes of C. adenogonium and C. apiculatum could provide a
good source of calcium in the diet, while C. adenogonium, C. bracteosum and C.
apiculatum had high levels of sodium.
A serial micro-dilution assay was used to determine the minimum inhibitory
concentration (MIC) values for 70% plant extracts using tetrazolium violet reduction as
an indicator of growth. Two Gram-positive (Stapylococcus aureus ATCC 29213 and
Enterobacter faecalis ATCC 29212) and two Gram-negative (Escherichia coli ATCC
25922 and Pseudomonas aeruginosa ATCC 27853) bacterial strains were used in this
study. The leaves had good antibacterial properties with the lowest MIC value being
0.04 mg/ml against E. coli and S. aureus. E. faecalis was found to be resistant against
all the leaves with the exception of C. imberbe. The stem extracts of Combretum spp.
tested in the study showed antimicrobial properties with the lowest MIC value being
0.04 mg/ml against E. coli shown by C. bracteosum. However, E. faecalis was
resistant against all the 12 plants tested. All the test microorganisms showed
resistance to the ashes, with the exception of S. aureus, which was found to be
susceptible to 75% of the test ash extracts with the lowest MIC value of 0.16 mg/ml.
Cytotoxicity and anticancer activity of the acetone extracts of the 12 Combretum plants
were evaluated using tetrazolium-based colorimetric assay (MTT assay) on A549 lung
carcinoma cells. The assays revealed that 50% of the leaf extracts of tested plants
showed cytotoxicity and cell proliferation inhibition in A549 lung carcinoma cells in a
concentration-dependent manner. The A549 cells were more sensitive to the following
plants: C. elaegnoides, C. erythrophyllum C. imberbe, C. kraussii and C. mkuzense.
The following stems extract, C. adenogdium and C. caffrum did not have any anticancer activity, whereas C. apiculatum and C. bracteosum were only able to
reduce cell viability to less than 60%. C. mkuzense, C. padoides, C. vendae and C.
zeyherii acted in a concentration-depended manner with the greatest activity seen at
the highest concentration (1000 µg/ml). The plants had activity at concentrations
between 31.25 and 1000 µg/ml allowing only 20% and 50%, respectively, of the cells
to remain viable. Ashes from C. mkuzense showed good anti-cancer activity at the
highest concentration (1000 µg/ml) reducing cell viability to around 10%.
Enterobacteriaceae, total coliform, S. aureus, B. cereus, E. coli and lactic acid bacteria
viability were studied during the four weeks storage period of prepared sorghum juices.
Juice samples were collected after preparation; the samples were serially diluted using
peptone water. Tempo instrument (Biomereiux) was used to enumerate total coliform,
total aerobic count, E. coli, S. aureus, lactic acid bacteria, enteric bacteria, yeast and
mould using the most probable number following the manufacturers instruction. C.
mkuzense and C. padoides plants were able to inhibit the growth of B. cereus, lactic
acid bacteria and S. aureus during the first three weeks of storage. E. coli was not
present throughout the four weeks storage time. Vitek 2 Compact (Biomereiux) was
used for the characterisation and identification of the dominant bacterial isolates using
biochemical reactions. The isolates were characterised by morphological differences.
Sixty five percent of the isolates were the Enterobacter genus that are commonly found
in soil, water, and sewage.
The nutritional composition and sensory properties of the prepared sorghum juices
treated with Combretum plants were investigated. All the juices had appreciable
amounts of protein, ash and energy. Sorghum juice treated with ashes of C. caffrum,
C. erythrophyllum and C. kraussii had the highest levels of proteins when compared
with other treated sorghum juices. The sorghum juices prepared in the study had
varying levels of trace element or minerals with potassium -(3, 55 – 104 mg/l) and
calcium (3.2-148 mg/l). Similarly, cobalt (-1.22), coppr (-0.99), iron(-0.962),
magnesium (0.004), sodium (-0.145), nickel (-2.7)) and zinc (-1.2)are present in very
low amounts. The juices treated with ashes had relatively higher levels of calcium,
potassium, magnesium and sodium. Sorghum juices treated with the ashes had better
sensory and organoleptic properties when compared with those treated with the
leaves. The juices treated with the ashes of C. caffrum and C. bracteseum were more
accepted by most of the panellist when compared with other treated juices.
Overall, this study presents valuable information on the phytochemical composition,
nutritional composition and antioxidant properties of some Combretum species in
South Africa. It recommended its use as food and in pharmaceutical preparations for
the local industries. In addition, Combretum plants showing the effects tested in this
study may be explored further for development into drugs. functional food as food
preservatives and nutraceutical applications, beside their traditional use
Thesis (Ph.D.(Microbiology)) -- University of Limpopo, 2022
2022-01-01T00:00:00Z