EVALUATION OF ANTITUMOUR PROPERTIES OF SIMAROUBA GLAUCA (‘LAKSHMI TARU’) AND THESPESIA POPULNEA (‘POOVARASU’) IN EXPERIMENTAL MAMMARY TUMOUR MODELS IN RATS
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Date
2017-12-30
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COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR
Abstract
The present study was undertaken to evaluate the antitumour properties of
Simarouba glauca (‘Lakshmi taru’) and Thespesia populnea (‘Poovarasu’) in in
vitro cancer cell line and in DMBA (7, 12 dimethyl benz[a]anthracene) induced
mammary tumours in rats. The crude alcoholic extracts and different fractions of
S. glauca leaves and T. populnea bark were screened for their cytotoxic property
in MCF-7 human breast carcinoma cell line. Based on the IC50 value, chloroform
soluble fractions (CSF) of both S. glauca and T. populnea were found to be
superior in cytotoxic action than crude extracts and other fractions, and hence
CSFs of both the plants were selected for further in vitro and in vivo studies.
In in vitro haemolytic assay, CSF of S. glauca did not produce haemolysis
in any of the tested concentrations whereas CSF of T. populnea showed
haemolytic activity in a dose dependent manner starting from the concentration 40
µg/mL. Chloroform soluble fractions of S. glauca and T. populnea exhibited a
superior in vitro antioxidant effect in a dose dependent manner when compared
with vitamin C standard. On phase contrast microscopy and Acridine orange/
ethidium bromide (AOEB) dual staining, MCF-7 cells treated with CSFs of S.
glauca and T. populnea exhibited marked morphological and nuclear alterations
which were characteristics of apoptosis. DNA ladder assay and TUNEL assay was
conducted to detect the apoptotic DNA fragmentation if any, occurred in MCF-7
cells after treatment with the plant fractions. A typical ladder pattern characteristic
of apoptosis was not observed in DNA ladder assay whereas, there was an
increase in positive reactions noticed in TUNEL assay which indicates an increase
in DNA fragmentation after the plant fraction treatments.
In acute toxicity study (OECD Guidelines no. 423) in rats, there was no
apparent toxic symptoms or mortality observed in both S. glauca and T. populnea
treatment groups up to an oral dose of 2000 mg/kg body weight. For in vivo
antitumour studies, DMBA induced rat mammary tumour model was used.
Experimental study was conducted for 14 days with 12 animals in each group.
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After treatment with S. glauca and T. populnea CSFs at the dose rates 50 mg/kg
and 100 mg/kg, no significant changes were noticed on tumour volume, tumour
weight and ratio of tumour weight to body weight. The elevated mean serum
LDH levels decreased significantly in the treatment groups on days 7 and 14.
When compared with normal control, SOD, CAT and GSH levels were noticed to
increase whereas lipid peroxidation level reduced in the mammary tumour tissues
after the plant fraction treatments. In AO/EB dual staining, an increased apoptotic
and necrotic cell density was observed in both the plant fraction treatment groups
in dose dependent manner. After treatment with CSFs of S. glauca and T.
populnea, the antiapoptotic gene Bcl2 expression was downregulated both in in
vitro and in vivo studies. On histopathological examination of the mammary
tumour masses using H & E staining, a progressive reduction in cellularity and
apoptotic changes were noticed in the plant fraction treated groups.
Thus in the present study, CSF of ethanolic extract of S. glauca leaf and
CSF of methanolic extract of T. populnea bark exhibited a dose dependent
cytotoxic and antitumour activity both in vitro and in vivo. The plant fractions
were effective in inducing apoptotic cell death and may be considered as potent
sources for isolating therapeutic molecules for cancer treatment.
Description
Thesis Submitted in partial fulfilment of the requirement for the degree of Doctor of Philosophy in Veterinary Pharmacology and Toxicology