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A
BRIEF RESUME OF THE INTENDED WORK
6.1 Need of study:
Diabetes is a global health problem and persistent hyperglycemia in diabetic patients
despite appropriate therapeutic measures leads to several complications including
retinopathy, nephropathy and neuropathy1. The intensity and extent of the functional
and anatomical abnormalities of diabetic neuropathy parallel the degree and duration
of hyperglycemia. Acute hyperglycemia decreases nerve function. Chronic
hyperglycemia is associated with the loss of myelinated and unmyelinated fibers,
wallerian degeneration, and blunted nerve-fiber reproduction. Approaches to
preventing or treating neuropathy include the intensive treatment of hyperglycemia,
aldose reductase inhibition, and various symptomatic treatments 2. There are several
lines of evidence that showed the presence of neurological involvement at the stage
of impaired glucose tolerance (IGT)3. The suggestion of an early presence of
neuropathy in IGT is also supported by the Japanese epidemiological survey of IGT
patients that showed high prevalence of subjects with loss of Achilles tendon reflex,
reduced vibration perception threshold, or even reduced nerve conduction velocity4.
Conventional analgesics such as opiates and non-steroidal anti-inflammatory drugs
are not effective in diabetic neuropathic pain5. Antidepressant agents are used to treat
neuropathic pain in humans6. Diabetic oxidative stress induced over production of
superoxide may be responsible for vascular and neuronal complications of painful
neuropathy7. Impaired blood flow seems to contribute to noxious stimulus
hypersensitivity. Vasodilator treatment has been demonstrated to reduce allodynia in
diabetic rats8. Quercetin produced a marked increase in tail-flick latencies in both
diabetic and non diabetic mice9. Combination of oral hypoglycemic agents as been
reported to produce significant glycemic control than treated alone10.
Tulsi (Ocimum sanctum) is one of the aromatic plants, distributed mainly in the
tropical and subtropical regions of the world including India. It is reported that there
was a significant reduction in fasting blood glucose & improvement in glucose
tolerance effect after oral (200 mg/Kg) administration of Ocimum sanctum (Tulsi)
extract mixed with diet for eight weeks to diabetic (Stroptozotocin induced) rats11.
The present study is undertaken to investigate effect of Ocimum sanctum and
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combined therapy of Ocimum sanctum with anti-diabetic agents in diabetic
neuropathy.
6.2 Review of Literature:
Tight glycaemic control has been shown to be effective in slowing the progression of
diabetic neuropathy12. Oxidative nitrosative stress is important determinant of
degenerative and painful pathological conditions in peripheral nerve fibres13
Lycopene, a carotenoid found in tomatoes is a powerful antioxidant and it has
attenuated diabetic neuropathic pain14. Quercetin, a bioflavonoid has been
demonstrated to possess antinociceptive activity in naïve animals15. Ocimum sanctum
(L.), (syn, Tulsi) is an indigenous plant commonly found in India and is
recommended in the ayurveda for the treatment of bronchitis, bronchial asthma,
malaria, diarrhea, dysentery, skin diseases, arthritis, painful eye diseases, chronic
fever and insect bite16. Ocimum sanctum ameliorates axotomy-induced neuropathy
in rats17. Pharmacological studies carried out with this herb suggest that it possesses
hypoglycemic, hypolipidemic, antioxidant properties11, anti-inflammatory, analgesic
and antipyretic activity18, wound healing activity19, and it is reported that Ocimum
sanctum leaf extracts stimulate insulin secretion from perfused pancreas20.
Ocimum sanctum leaves contain various bioactive constituents such as flavanoids
like luteolin, orientin, vicenin;triterpenoids like ursolic acid; fixed oils like palmitic,
stearic, oleic, linoleic, linolenic; essential oil like eugenol, camphor, carvacrol,
caryophyllene, decylaldehyde, nerol, α-pinene, γ-selinene, cirsilineol, cirsimartin,
isothymusin, isothymosin, tannins, etc21.
6.3 Objective of study:
The objective of the present study is to evaluate the antinociceptive action of Ocimum
sanctum leaves in diabetic neuropathic pain.
SPECIFIC OBJECTIVES:

To study the role of methanolic extract of Ocimum sanctum leaves in diabetic
Neuropathic pain.

To study whether oral hypoglycemics can play a vital role in diabetic
neuropathic pain.
2

To study the combined effect of methanolic extract of Ocimum sanctum
leaves with standard oral hypoglycemic agents in diabetic neuropathic pain.

To study the effect of methanolic extract of Ocimum sanctum leaves alone
and combination with oral hypoglycemics on structural integrity of sciatic
nerve.
B
MATERIALS AND METHODS:
7.1 Source of Data:
Data will be obtained from CD-Rom, Internet facilities, Literatures and related
articles from libraries of Krupanidhi College of Pharmacy, Indian Institute of
Sciences, Government College of Pharmacy etc., and other Research
Publications and Journals.
7.2 Method of Collection of Data:
The data collected will be based on animal experimentation as per the parameters
studied under each animal model, which are mentioned under the objectives of the
study.
Preparation of methanolic extract: The methanolic extract will be prepared using
Soxhlet apparatus.
EXPERIMENTAL MODELS
1.
Oral Glucose tolerance test22.
Effect of methanolic extract of Ocimum sanctum leaves alone & in
combination with oral hypoglycemic agents in Streptozotocin induced
diabetic rats22.
The oral glucose tolerance test will be performed on overnight fasted diabetic
rats. Rats will be divided into different groups as follows:
Group 1: Normal control, rats receive saline/vehicle.
Group 2 Methanolic extract (200 mg/kg p.o)17
Group 3: Glibenclamide (4mg/kg)23
Group 4: Pioglitazone (3mg/kg) 24
Group 5: Repiglinide (1mg/kg i.p)25
Group 6: Glebenclamide plus Pioglitazone
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Group 7: Repaglinide plus Pioglitazone
Group 8 Pioglitazone + Aqueous extract (low dose)
Group 9: Repaglinide + Aqueous extract (low dose)
Group 10. Glibenclamide + Aqueous extract (low dose)
Glucose will be fed 90 min after the administration of extracts or drug. Blood
will be withdrawn from the retro orbital sinus under ether inhalation at 30, 60
and 120 min of glucose administration and glucose levels will be estimated.
Food & water intake will be monitored daily & animals will be weighed every
weekend. Blood glucose level will be estimated every 7 days of the treatment.
OGTT will be performed every 15days & HbA1C will be estimated every month
of the treatment. At the end of the treatment period the rats will be sacrificed by
decaptation and the sciatic nerve will be excised and histopathological studies
will be carried out. Lipid peroxidation, superoxide dismutase (SOD), catalase
and the reduced form of glutathione (GSH) will also be estimated from the
sciatic nerve homogenate.
Treatment protocol
A. Induction and assessment of diabetic induced neuropathy in rats.
B. The following diabetic neuropathic animal models26 will be used to evaluate
the antinociceptive effect of Ocimum sanctum leaves aqueous extract
1. Tail immersion ( warm water) test
2. Hot plate test
3. Mechanical allodynia testing
4. Cold allodynia testing
5. Formaldehyde solution testing
6. Gross activity Monitoring
7.3 Does the study require any investigation or interventions to be conducted on
patients or the human or animals? If so please describe briefly:
YES
Study requires investigation on animals. The effects of the drug will be
studied on various parameters using rats as experimental animal model.
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7.4 Has ethical clearance been obtained from your institute
Ethical Committee approval letter is enclosed.
C.
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