Kamal, Laina Zarisa Mohd
(2019)
Antifungal Activities Of Three Alkaloids From Ruta Angustifolia (L.) Pers. Against ATCC Susceptible Strains Of Candida Albicans And Candida Glabrata.
PhD thesis, Universiti Sains Malaysia.
Abstract
The vital ability of medicinal plants to resist pathogenic microorganisms has
led many scientists to isolate the active compounds and further investigate their
mechanism of action. This is important as to fight the growing incidence of drugresistant
pathogens. Overall, this study is focusing towards searching for new
antifungal agent from alkaloids belong to Rutaceae family. The bioassay guided
isolation was performed to isolate the antifungal active alkaloids. The impacts of
these alkaloids on selected targeted genes have been experimented through
quantitative real-time polymerase chain reaction (qRT-PCR). Leaves of the plant
Ruta angustifolia (L.) Pers. was extracted and fractionated by using standard
chromatographic techniques. Through bioassay-guided isolation, combined fraction
of H3-3 and H4-8, fraction H10-9 and DA-10 yielded a total of three antifungal
active alkaloids. These isolated alkaloids were then characterized and compared to
the standard alkaloids by means of Thin Layer Chromatography (TLC) profile,
melting point and maximum absorption of UV spectra in methanol (UVλ max-
MeOH). The identification was further confirmed by HPLC spectroscopic data and
was characterized as acridone, furoquinoline and 4-quinolone so named arborinine,
skimmianine and graveoline respectively. The antifungal activites of the three
alkaloids were tested against Candida albicans and Candida glabrata of ATCC
strains with that of the standard antibiotics fluconazole. Broth microdilution assay of the alkaloids gave Minimum Inhibitory Concentration (MIC) values ranging from
250 μg/ml to 1000 μg/ml. Minimum Fungicidal Concentration (MFC) values were
recorded at 500 μg/ml and 1000 μg/ml. The results from qRT-PCR analysis in
C. albicans revealed that arborinine, skimmianine and graveoline down regulate all
genes related to the central metabolism of Candida which are PCK, ICL and MLS.
However, in the regulation of resistant genes, there is a decreased in ERG11, CDR2
and MDR after treated with arborinine, skimmianine and graveoline respectively. As
in C. glabrata gene expression analysis, arborinine treated cells showed a significant
decrease against three targeted genes namely ICL1, PCK1 and MLS1. Skimmianine
and graveoline impact on the gene expression of the cell revealed the same impact
when both of the compounds were able to down regulate ICL1 and PCK1 while MLS
and ERG11 increased. The three isolated compounds were also able to reduce protein
expression of Caicl1 in C. albicans through Western Blot. Overall, this study has
proven that arborinine, skimmianine and graveoline can be remarkable component as
antifungal agents with potential capacity to inhibit the growth and suppress the
expression of the targeted genes and protein of C. albicans and C. glabrata.
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