Omar, Alfaqih Hussain
(2020)
The effect 0f pyrogallol 0n the ph of plasmodium falciparum digestive vacuole.
Masters thesis, Universiti Sains Malaysia.
Abstract
Quercus infectoria, a highly potent medicinal herb is widely used in a
Malay culture in several applications. Previous studies proved the antimalarial activity
of Q. infectoria, however the exact phytochemical constituents leading to the
antimalarial activity of this plant remains searchable despite years of intensive research
efforts. It has been reported that pyrogallol is found among the phytoconstituents of Q.
infectoria. Pyrogallol, an organic compound, has a capability of generating free
radicals like other antimalarial drugs such as artemisinin. This has made artemisinin
an acceptable drug to predict the mechanism of action of pyrogallol against P.
falciparum in this study. It has been reported that reactive oxygen species (ROS)
generated by artemisinin in the parasite’s digestive vacuole through endoperoxide
bridge activation may cause parasite death. Another study reported that artemisinin
displays direct inhibition of V-type H+-ATPase, a proton pump located on the digestive
vacuole’s membrane in which the inactivation might cause pH alteration of this
organelle. Hence, the flow cytometry-based assay was performed to measure the
digestive vacuole pH after treatment with pyrogallol and artemisinin. The malarial
SYBR Green 1 fluorescence-based (MSF) assay was used to determine the
antimalarial activity of pyrogallol against the chloroquine-sensitive strain (3D7) of P.
falciparum by determining the 50% inhibitory concentration (IC50). A pH calibration
curve was constructed by using fluorescein isothiocyanate (FITC)-dextran, a
ratiometric pH indicator incorporated into the digestive vacuole of isolated trophozoite
stage parasites suspended in buffers of various pH values in the presence of an
ionophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP). The CCCP-free
parasite displayed the steady-state digestive vacuole pH of 5.42 ± 0.11, hence
validating the potential application of the generated pH calibration curve.
Subsequently, the concentrations of IC50-4 hours (60 nM) and near IC50-4 hours (15 and 30
nM) of artemisinin were selected from the previous study to ensure the pH change of
the digestive vacuole observed in the subsequent experiment was not resulted from
parasite death. The assay of 4-hour drug pulse with artemisinin (15, 30 and 60 nM)
was carried out by using mid trophozoite stage parasites to determine the pH change
of the digestive vacuole. The selected concentrations of artemisinin increased the pH
of the digestive vacuole by 1 (15 nM, pH = 6.6 ± 0.1), 1.48 (30 nM, pH = 7.1 ± 0.08)
and 1.6 pH unit (60 nM, pH = 7.3 ± 0.1), respectively as compared with the untreated
digestive vacuole (pH = 5.6 ± 0.1). The same result of the pH change of the digestive
vacuole induced by a standard proton pump inhibitor, concanamycin A was observed.
The result indicates that artemisinin might inhibit the V-type H+-ATPase, causing the
pH change of the digestive vacuole. In conclusion, this study contributes to a better
understanding on the mechanism of action of pyrogallol based on the results obtained
by using artemisinin as a drug to predict the compound’s activity.
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