Sravan Kumar A. V. G.*
Department
of Pharmacology, Krupanidhi College of Pharmacy, Bangalore
ABSTRACT:
Diabetic
complications involve cardiovascular system, kidneys and nerves. GIT is a prime
target of diabetic autonomic neuropathy. Delayed small intestinal transit and megacolon
have been demonstrated in streptozotocin treated diabetic rats. Increased lipid
peroxidation and accelerated advanced lipoxidation end product formation,
possibly catalyzed by hyperglycemia and oxidative stress, may play a critical
role in the development of neurovascular complications in diabetes. The health-promoting
activity of Ocimum sanctum seems to be related to the
antioxidant (free radical scavenging) activity. Ocimum sanctum leaves
extract treatment (200mg/kg/day) for 10 weeks to high fat diet-fed plus low
dose streptozotocin diabetic rats significantly reversed both reduced
contractile response of distal colon to acetylcholine and delayed transmit of
charcoal meal in small intestine compared to diabetic control. The significant
effect of Ocimum in reversing the
increased plasma lipid peroxidation level in diabetic rats may be due to its
antioxidant property. In conclusion, the present study suggest that Ocimum sanctum may be useful in preventing type II diabetes induced delay
in intestinal motility and since, Ocimum
sanctum is already in clinical use it
may be evaluated for preventive diabetis induced delay in intestinal motility
in patients at risk of developing autonomic neuropathy.
KEYWORDS: Ocimum sanctum,
colonic contractility, Intestinal transit, Diabetic gastroparesis, Rat.
INTRODUCTION:
Diabetic
complication involves cardiovascular system, kidneys and nerves [1]. Gastrointestinal
tract is the prime target of diabetic autonomic neuropathy. Diabetic animal
models exhibit changes in gastrointestinal function that resemble the
abnormalities manifested in human disease. Delayed small intestinal transit and
mega colon have been demonstrated in streptozotocin treated diabetic rats[2-3].
There are several
pathways which contributes to the development of diabetic neuropathy includes
increased activation of polyol pathway, oxidative stress, advance glycation end
product formation, nerve hypoxia/ischemia and reduction of nerve growth factor
support [4-7].
Oxidative stress
plays a main role in contributing to neural and vascular complications [8-9].
The reactive
oxygen species once formed tend to deplete anti-oxidant defences (superoxide
dismutase, catalase and glutathione peroxidase), rendering the affected cells
and tissues more susceptible to oxidative damage. Increased lipid peroxidation
and accelerated advanced lipoxidation end products formation possibly catalysed
by hyperglycaemia and oxidative stress which may play a critical role in the development
of neurovascular complications in diabetes[10].
Dietary
supplements of antioxidants are required to achieve an increase in antioxidant
status which may diminish oxidative stress associated with diabetes mellitus[11].
Ocimum sanctum leaves contains rich
amount of flavonoids i.e., luteolin, vicenin and orientin, it also contains
essential oil Eugenol as a major part which play a main role in protecting
human body against reactive oxygen species[12]. Ocimum
sanctum reported to have many
beneficial effects on human health, including anti-inflammatory,
anti-nociceptive, anti-cancer, anti-ulcer, anti-hypercholesteramic[13].
The health promoting activity of Ocimum
sanctum related to the antioxidant
(free radical scavenging) activity [14]. The involvement of
oxidative stress in the development of functional changes in gastrointestinal
tract and the effect of Ocimum sanctum on such changes are less
documented.
The aim of
present study is to examine the effect of Ocimum
sanctum leaves extract treatment on
altered response of distal colon to exogenous acetylcholine and small
intestinal transit of charcoal meal in type 2 diabetes.
MATERIAL
AND METHODS:
Chemicals:
Streptozotocin
was purchased from Sigma Aldrich. The feed ingredients such as casein (Himedia
laboratories, Mumbai), dl-methionine (Loba chemie, Mumbai), vitamin and mineral
mix (sarabhai chemicals) were procured from commercial sources. Ocimum sanctum leaves extract(ethanolic) from green chem industries,
Bangalore. Glipizide was obtained from Ranbaxy research laboratories. The
compoundswere administered orally as suspension by mixing with vehicle 1%
Na-CMC at a dose volume of 2ml/kg body weight of rats.
Preparation of fructose diet:
Fructose diet was
prepared by the method mentioned elsewhere[15] and consists of 660 g
of fructose, 100g protein, 80g fat, 0.04 g zinc carbonate, 5g vitamin mixture,
5g mineral mixture and cellulose 150g, all commercial grades.
Experimental animals:
Male
Sprague-dawley rats (SD) rats (160-180 g) were housed in standard polypropylene
cages (three rats/cage) and maintained under control room temperature (22 ± 2̊C) and humidity (55 ± 5%) with 12:12 h light and dark
cycle . All the rats were provided with normal pellet diet (Amrut diet, New
delhi) and water ad libitum, prior to diet manipulation. Institutional animal
ethics committee approved the experimental protocol; Animals were maintained
under standard conditions in animal house approved by the committee for the
purpose of control and supervision of experiments on animals(CPCSEA).
Development of high Fat diet fed/STZ treated type 2 diabetic rats :
The model of type
2 diabetic like rats was established according to the method reported earlier[16]
with modification. Animals were fed with high fat diet (HFD), once a day
for two weeks. After two weeks, animals were fasted overnight and were injected
intraperitonially with streptozotocin(35mg/kg) dissolved in 0.1mol/l citrate
buffer (PH 4.4). The rats with non-fasting BGL of ≥300 mg /dl were
considered diabetic and selected for further pharmacological studies.
Experimental protocol:
The control rats
were divided into two groups of 6 rats each (I-II). Group I was treated with
NPD (non-pellet diet) and group II was treated with Ocimum sanctum leaves
extract(200mg/kg,p.o). The fat-fed /STZ diabetic rats(BGL of ≥ 200 mg/dl)
were randomly divided into 3 groups Group III to Group V animal groups
consisting of ten rats each such that their biochemical parameters similar to
each other. Group III animals served as diabetic control which receives 1%
Na-CMC (2ml/kg, P.o). Group IV animals received Ocimum sanctum leaves
extract (200 mg/kg) and group V was treated with the Glipizide(5 mg/kg) an
insulin secretagouge[17]. All the substance above was administered
intragastrically for 10 weeks and treatment schedule was started one day before
the administration of STZ.
Experimental procedure:
Charcoal meal administration
At the end of the treatment period, overnight fasted animals of
different groups were administered; p.o, 2ml/rat with charcoal meal (10%
charcoal in 5% gum acacia) and 20 min later the rats were killed by cervical dislocation.
The abdomen was opened and the intestine was removed from pyloric junction to
caecal end. Then colon was separated and kept in continuously aerated Tyrode’s
solution. The farthest distance travelled by the charcoal meal through the
small intestine and total length of the intestine were measured.
Gastrointestinal transit was expressed as the percentage of the distance
travelled by the charcoal meal relative to the total length of small intestine[18].
Contractile response of colonic
smooth muscle
Immediately after cleansing the colon, 1 cm of distal colon was
mounted under a resting tension of 0.5g in an organ bath (40 ml) containing
continuously aerated Tyrode’s solution. The temperature was maintained at 37̊ ± 1̊C
throughout the experiment and the tissue was allowed to equilibrate for 30 min
before exposing to acetylcholine. A primary dose of 100 ng of acetylcholine was
tested before starting the actual concentration response curve. The contractile
responses were recorded isotonically using a student’s physiograph. At
the end of the initial equilibration period dose response curves were obtained
for ascending dose of acetylcholine.ED50 values of acetylcholine were
calculated from the graph plotted using percent response against log dose.
Lipid peroxidation in plasma
Lipid peroxidation in plasma was estimated by measuring
malondialdehyde (MDA) level in plasma. Amount of malondialdehyde formed was
quantified by reaction with thiobarbituric acid as reported previously[19].
Statistical analysis
Data
are presented as the mean ± SE from 8 rats per group. Comparison of mean values
among the various groups was performed by one way ANNOVA. For the single
comparison between the groups unpaired Student’s t-test was used. P values less
than 0.05 were considered significant.
Table 1: Effect of Ocimum sanctum leaves extract and glipizide on blood glucose level and
lipid peroxidation (Values are mean ±SE).
|
Groups |
Blood glucose level(mg/dl) |
MDA(nmol/100ml of plasma) |
|
Normal Control |
92.34 ± 0.8 |
410.2±18.32 |
|
Normal Control + Ocimum sanctum extract(200mg/kg) |
89.2 ± 0.6 |
|
|
Diabetic control |
302.4 ± 0.4 |
832.5± 26.50 |
|
Diabetic +
Glipizide |
152± 0.4b |
470.2± 11.27c |
|
Diabetic + Ocimum sanctum leaves extract(200mg/kg) |
139 ± 0.9a |
422.75± 12.27a |
P value: Pa<0.001, Pb<0.01 and Pc<0.05
when compared with diabetic control.
RESULTS:
Baseline body weights and blood glucose level were similar in
all the groups. Table1 shows the mean blood sugar level at the end of 6 weeks
treatment. Six weeks after injection of streptozotocin, the diabetic rats had
significantly higher body weights (data not shown) and there was an increase in
blood sugar level when compared with their age matched non-diabetic controls.
Treatment with glipizide and Ocimum sanctum leaves extract significantly
decreased blood glucose towards normal levels.
ED50 of
acetylcholine (Fig 1 A), per cent transit of charcoal meal in small intestine
(Fig 1B) and plasma MDA values(table 1) showed a significant difference among
the groups. The untreated diabetic rats showed a significant increase in ED50
of acetylcholine (p< 0.001), plasma MDA values (p<0.001) and a
significant reduction of transit of charcoal meal(p<0.001) compared to
normal controls. OSLE treated diabetic rats significantly reduced ED50 of
acetylcholine (p<0.001) and increased the per cent distance travelled by
charcoal meal (p<0.001) compared to HFD fed /low dose streptozotocin
diabetic control. The effect of Glipizide treated diabetic rats showed less significant
effect (p<0.01) when compared to diabetic control.
Contractile response of distal colon to exogenous acetylcholine
(Fig 1 A) and Small intestinal transit of charcoal meal (Fig 1 B)l in
non-diabetic and HFD fed/low dose STZ –diabetic rats. P value: ^ P < 0.001
when compared with control. # P < 0.05 when compared with diabetic control.
*P < 0.01when compared with diabetic control.
FIG 1A
FIG 1 B
DISCUSSION:
Metabolic
syndrome is characterised by a group of pathological changes including obesity,
hypertriglyceridemia,
impaired glucose tolerance and insulin resistance. A modified diet (fructose
diet) was adopted to induce insulin resistant because the role of fructose in
the development of diabetic complications was well documented [15]and
injection of a single dose of STZ induced a diabetic state similar to
prediabetic, insulin resistant state in humans[16]. Streptozotocin
targets pancreatic β cells, leading to serum insulin reduction. The rodent
model induced by high fat diet feeding followed by a low dose streptozotocin
injection, stimulates the natural history and the metabolic characteristics of
patients with type 2 diabetes[16]. Distal colons from untreated
diabetic rats were found to be less sensitive to ACH and thereby delay in
transit of intestinal content. These observationsare in agreement with previous
report [2].There is diminished release or production of
neurotransmitter, i.e; acetylcholine, to enhanced degradation of the neurotransmitter,
or to diminished end organ sensitivities to the neurotransmitter itself[20].
There was a parallel increase in lipid peroxidation level in diabetic rats[21].
Increased lipid peroxidation and accelerated advanced lipoxidation end product
formation, possibly catalyzed by hyperglycemia and oxidative stress, may play a
critical role in the development of neurovascular complications in diabetes[10].
Treatment with glipizide, OSLE (Ocimum
sanctum leaves extract) produced a
significant reversal of all parameters measured, suggesting the role of
hyperglycemia and oxidative stress involvement in diabetic complications. OSLE
had more profound effect than glipizide. Ocimum
sanctum leaves contains different flavonoids, namely vicenin,
luteolin and orientin are reported to be present in Ocimum sanctum leaves. Ocimum sanctum leaves extract has proven to protect against the
development of diabetic gastropathy by inhibition of lipid peroxidation and
restoration of antioxidant enzymes in diabetic rats. Earlier, Eugenol has also
been reported to produce significant improvement in small intestine transit
percentage and this activity has been attributed to the antioxidant activity of
Eugenol [22]. The reduced contractile response of
colonic smooth muscle to exogenous acetylcholine may be the result of excessive
degradation of acetylcholine by tissue acetylcholine esterase, diminished
muscarinic receptor sensitivity or density or defective interaction between
muscarinic receptor and intracellular contractile process. The myogenic
phenomenon in distal small intestine of diabetic rats is not affected[20]. Studies of the responsiveness of
diabetes colonic smooth muscle to acetylcholine are limited, whereas vascular
in experimental diabetic show altered sensitivity to acetylcholine [23].
Therefore oxidative stress may induce changes in muscarinic receptor density
and binding affinity leading to reduced cholinergic response and thus OSLE may
play vital role in abolishing these changes. Impaired cholinergic response of
distal small intestinal smooth muscle has been reported and thus treatment with
OSLE improved intestinal motility and enhanced intestinal transit of charcoal
meal in diabetic rats.
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Received on 15.09.2012
Modified on 28.09.2012
Accepted on 25.10.2012
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Research J. Pharmacology and
Pharmacodynamics. 4(6): November
–December 2012, 341-345