1.0 INTRODUCTION:
Malaria is one of the most important infectious
diseases in the world that is caused by intracellular parasitic protozoa of the
genus Plasmodium and it is transmitted via the bite of an
infected female Anopheles sp mosquito. During
infection, Plasmodium passes over several stages including an intra-erythrocytic
stage, in which the parasite degrades 60–80% of host hemoglobin that is used as
food support for its development and growth. Hemoglobin is oxidized to
methemoglobin within parasite digestive vacuole and is hydrolyzed by aspartic
proteases into free heme (Fe3+) (ferriprotoporphyrin IX) and denatured globin.
Globin is hydrolyzed by cysteine proteases (i.e. falciparum) and exopeptidases
into small peptides and amino acids used for protein synthesis. Digestion of
hemoglobin releases large quantities of heme (Fe3+) that accumulate and
reaches high concentrations (up to 300–500 mM). Those high concentrations are
thought to be toxic for Plasmodium through membrane disruption, lipid
peroxidation, and protein and DNA oxidation. Free heme (Fe3+) might also
interfere with the hemoglobin degradation pathway. Plasmodium uses a
system to detoxify heme (Fe3+) called biocrystallization based on the formation
of hemozoin pigment which appears as a dark black crystalline spot (a dark
brown pigment) in red blood cells of infected patients.
2.0 METHODOLOGY:
·
Different concentrations (0–2
mg/mL in DMSO) of the extracts will be incubated with 3mM of hematin, 10 mM
oleic acid, and 1M HCl.
·
The final volume will be
adjusted to 1mL using sodium acetate buffer, pH 5.
·
Similarly the positive control
chloroquine diphosphate will be prepared.
·
Finally, the reaction mixtures
will be incubated overnight at 37˚C with constant gentle shaking.
·
After incubation, samples will
be centrifuged at 14,000rpm, 10 min, at 21˚C and the hemozoin pellet will be
repeatedly washed with incubation (15 min at 37˚C with regular shaking) in 2.5%
(w/v) sodium dodecyl sulfate (SDS) in phosphate-buffered saline followed by a
final wash in 0.1 M sodium bicarbonate until the supernatant will clear
(usually 3–8 washes).
· After the final wash, the
supernatant will be removed and the pellets will be dissolved in 1 mL of 0.1 M
NaOH.
·
Absorbance will be measured at
400nm using a spectrophotometer. The results will be recorded as %inhibition of
heme.
· Crystallization compared to the negative control (DMSO) using the following equation:
(%)
percentage inhibition = [(AN−AS)/AN] × 100
Where
AN: absorbance of negative control and AS is the absorbance of test samples.
3.0 ENDPOINT PARAMETER(S):
·
% Percentage inhibition activity
4.0 REFERENCE(S):
4.1 Solmaz
Asnaashari, Fariba
Heshmati Afshar, Sedigheh
Bamdad Moghadam, and Abbas
Delazar. Evaluation of In
Vitro Antimalarial Activity of Different Extracts of Eremostachys
azerbaijanica Rech.f. Iran
J Pharm Res. 2016
Summer; 15(3): 523–529.
4.2 Mahdi Mojarrab, Ali Shiravand, Abbas Delazar,
and Fariba Heshmati Afshar. Evaluation of In Vitro Antimalarial Activity of
Different Extracts of Artemisia aucheri Boiss. and A. armeniaca Lam. and
Fractions of the Most Potent Extracts. The Scientific World Journal Volume
2014, Article ID 825370, 6 pages http://dx.doi.org/10.1155/2014/825370.
4.3 Herraiz, T., Guillén, H., González-Peña,
D. et al. Antimalarial Quinoline Drugs Inhibit β-Hematin and Increase
Free Hemin Catalyzing Peroxidative Reactions and Inhibition of Cysteine
Proteases. Sci Rep 9, 15398 (2019). https://doi.org/10.1038/s41598-019-51604-z.
END OF DOCUMENTS
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