IN VITRO CYTOTOXICITY ACTIVITY AND IN VIVO ORAL TOXICITY OF Euphorbia hirta
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Date
2014-08
Authors
PING, KWAN YUET
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Abstract
Euphorbia hirta is an annual plant that has been widely used in traditional medicine
to treat various diseases. In vitro cytotoxicity, genotoxicity properties and in vivo
toxicological evaluation of E. hirta methanol extract were investigated in this study.
Various pharmacognostical parameters evaluation was carried out as per World
Health Organization (WHO) guidelines procedure for the standardization of E. hirta
extract. The chemical constituent aspect of standardization involves quantification
of the main chemical components in E. hirta. The GC-MS analysis was used for
quantification of 1,3,4,5-tetrahydroxycyclohexanecarboxylic acid in the extract was
rapid, accurate and linear. Six major peaks in the range of 600 - 1500 and 2800 -
3400 cm-1 were observed in the FTIR spectra. The concentrations of heavy metals
determined in E. hirta extract were well below the permissible limit. In 2,2-
Diphenyl-1-picylhydrazyl radical (DPPH) assay, the extract showed a general
consistency on antioxidant activity above 50%, independent of the extraction time.
The potential cytotoxicity effect of E. hirta was investigated using brine shrimp
lethality assay and the cytotoxicity assay against cancer cells. The extract of E. hirta
showed significant toxicity against brine shrimp with (Lethal Concentration) LC50
value of 620.38μg/mL (24 h). Comparison with positive control potassium
dichromate signifies that cytotoxicity exhibited by the methanol extract have
moderate activity. The 3-(4, 5-dimethyl thiazol-2yl)-2, 5-diphenyltetrazolium
bromide (MTT) cytotoxicity assay showed that E. hirta inhibited MCF-7 cell
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viability with the (Inhibitory Concentration) IC50 values 25.26 μg/ml in a dose and
time-dependent manner. Microscopic studies showed that E. hirta treated cells
exhibited marked morphological features characteristic of apoptosis. E. hirta extract
also had an ignorable influence on the lactate dehydrogenase (LDH) leakage and
generating intracellular reactive oxygen species (ROS). Therefore, E. hirta might
induce the cytotoxicity via a ROS-independent mechanism in MCF-7 cells. The
Annexin V/Propidium Iodide flow cytometry study confirmed that E. hirta extract
induced apoptosis in MCF-7 cells. E. hirta extract treatment also resulted in DNA
fragmentation in MCF-7 cells. Moreover, E. hirta treatment resulted in the
accumulation of cells at the S and G2/M phases as well as apoptosis. The caspase
activity study revealed that E. hirta extract induced apoptosis through the caspase-3
independent pathway by the activation of caspase-2, 6, 8 and 9. To identify the
cytotoxic compound, E. hirta extract was subjected to bioassay-guided fractionation.
E. hirta hexane subfraction, namely EH Hex 4 demonstrated highest activity among
all the fractions tested. Further Liquid Chromatography–Mass Spectrometry (LC-MS)
analysis of EH Hex 4 led to identification of 3,3’,4’,5,7-Pentahydroxy-8-(5-oxo-2-
pyrrolidinyl) flavone as the likely cytotoxic agent in E. hirta extract. The data
obtained from this study revealed that E. hirta induced apoptotic cell death and
suggests that E. hirta could be used as an apoptosis-inducing anti-cancer agent for
breast cancer treatment with further detailed studies. The potential genotoxic effect
of E. hirta was investigated using Allium cepa and comet assay. In A. cepa assay, the
result showed that the methanol extract of E. hirta exerted significant genotoxic and
mitodepressive effects at 1000 μg/mL. A dose-dependent increase of chromosome
aberrations was also observed such as stickiness, c-mitosis, bridges and vagrant
chromosomes. Micronucleated cells were also observed at interphase. In the comet
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assay, the treatment of 25 μg/mL of E. hirta extract for 72 h on MCF-7 cells caused
an increase in DNA damage by approximately 48.16% compared to the unchallenged
control which confirmed the genotoxic effect of E. hirta extract. The acute and subchronic
oral toxicity of E. hirta was evaluated in Sprague Dawley rats. The extract at
a single dose of 5000 mg/kg did not produce treatment related signs of toxicity or
mortality in the animals tested during the 14-day observation period. Therefore, the
(Lethal Dose) LD50 of this plant was estimated to be more than 5000 mg/kg. In the
subchronic toxicity study, the administration of 50 mg/kg, 250 mg/kg, 1000
mg/kg/day of E. hirta extract per body weight revealed no significant difference in
the body weight change, haematological and biochemical parameters, relative organ
weights and gross findings compared to the control group. Macropathology and
histopathology examination of all organs did not reveal morphological alteration.
Analyses of these results with the information of signs, behaviour and health
monitoring could lead to the conclusion that the long-term oral administration of
E. hirta extract for 90 days does not cause toxicity. In conclusion, in vitro and in vivo
toxicity results obtained in this study proved the importance of both methods to
obtain comprehensive toxicological evaluation of natural product agents.
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Keywords
IN VITRO CYTOTOXICITY ACTIVITY