| dc.description.abstract | Quest for bioactives that confer protection against chemotherapy induced cardio
toxicity is a front- line area of cardio oncology research. Attempts have been made in
recent years to find out the effects of medicinal mushroom bioactives for this purpose.
Morel mushrooms have been used in traditional medicine in Asian countries to treat
asthma, wound healing, cough, cold, indigestion, excessive phlegm and breathlessness.
Morchella esculenta, commonly known as Guchhi in India is highly prized culinary
morel mushroom. Investigation carried out in our laboratory have demonstrated that
mycelium of M esculenta possessed significant antioxidant, anti-tumour and
hepatoprotective activities. Experimental studies were carried out on the protective
effect of bioactive extract of M. esculenta fruiting bodies against chemotherapy drug
induced cardiotoxicity using Swiss albino mice model. The findings are reported in this
thesis.
Several bioactive compounds are reported to be present in the fruiting bodies of
M. esculenta. This might be attributed for its therapeutic properties. Methanolic extract
of the fruiting bodies of M.esculenta was used in the current study. Chemical finger
print of methanolic extract (ME) was accomplished by HPTLC analysis. Bioactive
constituents were identified by LCMS analysis. The results showed a large number of
bioactive compounds were present in the methanol extract of M. esculenta. Some of the
major
compounds
present
were
5-eicosapentaenoicacid(C20H30O2),
8-
hydroxyoctadecadienoic acid(C18H32O3), 4,4-dipo-zetacarotene(C30H44), CynarosideA
( C21H32O10). These compounds might be responsible for the observed cardio protective
effect of the extract.
Despite the use of M. esculenta as a widely used edible mushroom worldwide
including India, little is known about its safety. So, in order to find the safety, toxicity
studies were conducted using Swiss Albino mice. Acute oral toxicity study was
conducted following the OECD-423 guidelines. The results revealed that the mushroom
does not cause any toxic effects in animals at dosages of 5000 and 2000 mg/kg b.wt.
The animals were active and no changes in behavioural pattern was observed. Body
weight, feed and water intake were also found normal. Thus, methanolic extract of M.
esculenta (ME) was found safe for oral consumption. Further sub-chronic toxicity
studies were carried out using ME at doses of 100, 250 and 500 mg/kg. b.wt . No
toxicity was observed and all animals remained healthy throughout the study period. Doxorubicin and Cyclophosphamide are extensively used chemotherapy drugs.
These drugs show severe cardiotoxicity especially in
higher doses. Several
mechanisms have been proposed for the cardiotoxic effects of
doxorubicin and
cyclophosphamide. Oxidative stress is one of the primary mechanisms by which both
of these chemotherapeutic drugs induce harmful effects. Hence, the current study was
designed to find whether the antioxidant capability of ME would be capable to mitigate
these negative consequences.
The cardioprotective study of ME was carried out in three stages i.e., effect on
doxorubicin-
induced
cardiotoxicity;
effect
on
cyclophosphamide-
induced
cardiotoxicity and effect on combination of cyclophosphamide and doxorubicin-
induced cardiotoxicity. Doxorubicin (DOX), Cyclophosphamide(CP) and their
combination (CD) are extensively used treatment options for cancer. Cardiotoxicity
induced by these drugs is an impediment in their clinical use. Hence levels of cardiac
injury makers
were studied. The activities of Creatine kinase-MB, Lactate
dehydrogenase and Troponin I levels consequent to the administration of CP, DOX and
CP+DOX were studied using diagnostic kits. Depletion of endogenous antioxidant
levels in myocardium was also determined by spectrophotometric assays. Nrf-2, iNOS,
NF-
-PCR
analysis. To assess the mitochondrial dysfunction, TCA cycle and electron transport
chain complex enzymes activities were evaluated. Cardiac tissue damage caused by CP,
DOX and CP+DOX was assessed by histopathological examination.
Cardiac injury marker levels elevated by CP, DOX and CP+DOX treatments
were significantly downregulated by ME. Endogenous antioxidants such as SOD, GPx,
and GSH depleted by CP, DOX and CP+DOX administration was restored to almost
normal level by ME. This indicated the effect of ME to ameliorate oxidative stress
caused by chemotherapy drugs DOX, CP and DOX+CP administration leading to
myocardial injury. Histopathological observation supported these findings. CP, DOX
and CP+DOX-induced decline of antioxidant status and expression of nuclear factor
erythroid 2-related factor 2 was restored by ME. CP, DOX and CP+DOX-induced
expression of NF-
-6, IL1-
-
(500mg/kg). Depleted levels of mitochondrial enzyme activities consequent to
chemotherapy were also restored by ME. Since bioactive extracts of morel mushrooms were found to possess profound
antioxidant activity, the possible interference of these extracts with antineoplastic
activity of chemotherapy drugs is often surmised. Hence another study was undertaken
to evaluate the effect of doxorubicin and cyclophosphamide on solid tumour bearing
mice treated with bioactive extract of M.esculenta. Solid tumour was induced by
limbs of Swiss albino mice. Animals were administered with various concentrations of
methanol extract of M. esculenta following tumour induction. Tumour growth (volume
and mass) was measured for four weeks after tumour induction. Cardioprotective effect
of methanolic extract was assessed by determining cardiac injury markers levels in
serum, antioxidant status in myocardium and histopathology of heart tissue. The results
showed significant cardioprotective effect of methanol extract of M. esculenta on
tumour bearing mice The findings also suggested that methanol extract of M. esculenta
did not delimit the therapeutic effect of doxorubicin and cyclophosphamide despite its
profound antioxidant capabilities.
In silico studies using Auto dock tools were performed to determine the
interaction of major bioactives of ME with KEAP-1 protein which plays a regulatory
role in NRF-2 induced antioxidant defence mechanism. The results showed that the
major bioactives interacted with Cys-151 residue of KEAP-1 which is found to have a
pivotal role in KEAP1-NRF2 interaction.
This thesis is a compilation of the comprehensive investigation and
experimental findings on the cardioprotective effect of bioactive extract of morel
mushroom Morchella esculenta against chemotherapy induced cardiotoxicity
doxorubicin and cyclophosphamide and their combination. The studies are compiled
in 10 chapters: Introduction (Chapter I), Review of literature (Chapter II), Materials
and Methods (Chapter III), Extraction and identification of major bioactive compounds
(Chapter IV), Acute oral toxicity study of methanolic extract of M. esculenta fruiting
body (Chapter V), Evaluation of protective effect of methanolic extract of M. esculenta
fruiting body against doxorubicin induced cardiotoxicity(Chapter VI), Evaluation of
protective effect of methanolic extract of M. esculenta fruiting body against
cyclophosphamide induced cardiotoxicity(Chapter VII), Evaluation of protective effect
of methanolic extract of M. esculenta fruiting body against combination treatment ofcyclophosphamide and doxorubicin induced cardiotoxicity(Chapter VIII), Evaluation
of anti-tumour activity of doxorubicin, cyclophosphamide and combination of
cyclophosphamide + doxorubicin vis-à-vis cardioprotective effect of M.esculenta
(Chapter IX). In silico studies for evaluating interaction of bioactives with KEAP-1
(Chapter X).
Keywords: Medicinal mushrooms, M. esculenta, Oxidative stress, Antioxidants, Anti-
tumour, Chemotherapy, Cardio protection, Mitochondrial dysfunction, Doxorubicin,
Cyclophosphamide, Gene expression, Combination chemotherapy. | en_US |
