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Abstract:
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Diabetes mellitus is a heterogeneous metabolic disorder characterized by hyperglycemia
with disturbances in carbohydrate, protein and lipid metabolism resulting from defects in
insulin secretion, insulin action or both. Currently there are 387 million people with
diabetes worldwide and is expected to affect 592 million people by 2035. Insulin
resistance in peripheral tissues and pancreatic beta cell dysfunction are the major
challenges in the pathophysiology of diabetes. Diabetic secondary complications (like
liver cirrhosis, retinopathy, microvascular and macrovascular complications) arise from
persistent hyperglycemia and dyslipidemia can be disabling or even life threatening.
Current medications are effective for control and management of hyperglycemia but
undesirable effects, inefficiency against secondary complications and high cost are still
serious issues in the present prognosis of this disorder. Hence the search for more
effective and safer therapeutic agents of natural origin has been found to be highly
demanding and attract attention in the present drug discovery research. The data
available from Ayurveda on various medicinal plants for treatment of diabetes can
efficiently yield potential new lead as antidiabetic agents. For wider acceptability and
popularity of herbal remedies available in Ayurveda scientific validation by the
elucidation of mechanism of action is very much essential. Modern biological techniques
are available now to elucidate the biochemical basis of the effectiveness of these
medicinal plants. Keeping this idea the research programme under this thesis has been
planned to evaluate the molecular mechanism responsible for the antidiabetic property of
Symplocos cochinchinensis, the main ingredient of Nishakathakadi Kashayam, a wellknown
Ayurvedic antidiabetic preparation. A general introduction of diabetes, its
pathophysiology, secondary complications and current treatment options, innovative
solutions based on phytomedicine etc has been described in Chapter 1.
The effect of Symplocos cochinchinensis (SC), on various in vitro biochemical targets
relevant to diabetes is depicted in Chapter 2 including the preparation of plant extract.
Since diabetes is a multifactorial disease, ethanolic extract of the bark of SC (SCE) and its
fractions (hexane, dichloromethane, ethyl acetate and 90 % ethanol) were evaluated by in
vitro methods against multiple targets such as control of postprandial hyperglycemia,
insulin resistance, oxidative stress, pancreatic beta cell proliferation, inhibition of protein
glycation, protein tyrosine phosphatase-1B (PTP-1B) and dipeptidyl peptidase-IV (DPPxxi
IV). Among the extracts, SCE exhibited comparatively better activity like alpha
glucosidase inhibition, insulin dependent glucose uptake (3 fold increase) in L6
myotubes, pancreatic beta cell regeneration in RIN-m5F and reduced triglyceride
accumulation in 3T3-L1 cells, protection from hyperglycemia induced generation of
reactive oxygen species in HepG2 cells with moderate antiglycation and PTP-1B
inhibition. Chemical characterization by HPLC revealed the superiority of SCE over other
extracts due to presence of bioactives (beta-sitosterol, phloretin 2’glucoside, oleanolic
acid) in addition to minerals like magnesium, calcium, potassium, sodium, zinc and
manganese. So SCE has been subjected to oral sucrose tolerance test (OGTT) to evaluate
its antihyperglycemic property in mild diabetic and diabetic animal models. SCE showed
significant antihyperglycemic activity in in vivo diabetic models.
Chapter 3 highlights the beneficial effects of hydroethanol extract of Symplocos
cochinchinensis (SCE) against hyperglycemia associated secondary complications in
streptozotocin (60 mg/kg body weight) induced diabetic rat model. Proper sanction had
been obtained for all the animal experiments from CSIR-CDRI institutional animal ethics
committee. The experimental groups consist of normal control (NC), N + SCE 500 mg/kg
bwd, diabetic control (DC), D + metformin 100 mg/kg bwd, D + SCE 250 and D + SCE
500. SCEs and metformin were administered daily for 21 days and sacrificed on day 22.
Oral glucose tolerance test, plasma insulin, % HbA1c, urea, creatinine, aspartate
aminotransferase (AST), alanine aminotransferase (ALT), albumin, total protein etc. were
analysed. Aldose reductase (AR) activity in the eye lens was also checked. On day 21,
DC rats showed significantly abnormal glucose response, HOMA-IR, % HbA1c,
decreased activity of antioxidant enzymes and GSH, elevated AR activity, hepatic and
renal oxidative stress markers compared to NC. DC rats also exhibited increased level of
plasma urea and creatinine. Treatment with SCE protected from the deleterious alterations
of biochemical parameters in a dose dependent manner including histopathological
alterations in pancreas. SCE 500 exhibited significant glucose lowering effect and
decreased HOMA-IR, % HbA1c, lens AR activity, and hepatic, renal oxidative stress and
function markers compared to DC group. Considerable amount of liver and muscle
glycogen was replenished by SCE treatment in diabetic animals. Although metformin
showed better effect, the activity of SCE was very much comparable with this drug.
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The possible molecular mechanism behind the protective property of S. cochinchinensis
against the insulin resistance in peripheral tissue as well as dyslipidemia in in vivo high
fructose saturated fat diet model is described in Chapter 4. Initially animal were fed a
high fructose saturated fat (HFS) diet for a period of 8 weeks to develop insulin resistance
and dyslipidemia. The normal diet control (ND), ND + SCE 500 mg/kg bwd, high
fructose saturated fat diet control (HFS), HFS + metformin 100 mg/kg bwd, HFS + SCE
250 and HFS + SCE 500 were the experimental groups. SCEs and metformin were
administered daily for the next 3 weeks and sacrificed at the end of 11th week. At the end
of week 11, HFS rats showed significantly abnormal glucose and insulin tolerance,
HOMA-IR, % HbA1c, adiponectin, lipid profile, liver glycolytic and gluconeogenic
enzyme activities, liver and muscle triglyceride accumulation compared to ND. HFS rats
also exhibited increased level of plasma inflammatory cytokines, upregulated mRNA
level of gluconeogenic and lipogenic genes in liver. HFS exhibited the increased
expression of GLUT-2 in liver and decreased expression of GLUT-4 in muscle and
adipose. SCE treatment also preserved the architecture of pancreas, liver, and kidney
tissues. Treatment with SCE reversed the alterations of biochemical parameters, improved
insulin sensitivity by modifying gene expression in liver, muscle and adipose tissues.
Overall results suggest that SC mediates the antidiabetic activity mainly via alpha
glucosidase inhibition, improved insulin sensitivity, with antiglycation and antioxidant
activities. |