Protective
Effect of Tulsi (Ocimum Sanctum) on Lipid Peroxidation in
Stress Induced by Anemic Hypoxia in Rabbits
(Received on April 8, 2002)
by
JYOTI SETHI*, SUSHMA SOOD*-, SHASHI
SETH**
AND ANJANA TALWAR*
Departments of 'Physiology and **Biochemistry,
Pt BD Sharma PGIMS,
Rohtak - 124 001
Sir,
Ocimum sanctum Linn (commonly known
as holy basil in India) is considered as a sacred plant in
India, to which several medicinal properties
are attributed in Ayurveda, the Indian system of medicine. A chemical analytical study by gas liquid chromatography
revealed that essential oil from OS contained 70% eugenol
(1). The leaves of this plant contain apigenin, apigenin-7-0
glucuronide, molludistin, orientin and urosolic acid (2).
The leaves of the plant have been used
as an expectorant, diaphoretic, anti-emetic, anti-cancer,
anti-helminthic, antiseptic, analgesic and also as a tonic
rejuvinator or vitaliser to induce longevity and disease free
healthy life (3, 4). Many
reports are available regarding the anti-stressor activity
of this plant material on the changes induced by different
stress agents (5, 6).
The present study was conducted to assess
the oxidative damage due to stress imposed by anemic hypoxia
induced by sodium nitrite administration and to assess the
effect of ingestion of fresh Ocimum Sanctum leaves on this
oxidative damage by measuring Plasma Malondialdehyde (MDA).
Forty albino rabbits of either sex weighing
1.5-2.5 kg were maintained under standard conditions and received
food and water ad libitum.
The animals were divided into two equal groups
Control group
(n=20) - Maintained
on normal diet for 30 days.
Test group (n=20) - Received supplementation
of 2 gm fresh leaves of Ocimum Sanctum for 30 days.
Blood samples were taken for estimation
of haemoglobin, blood glucose and plasma malondialdehyde in
both the groups of rabbits at the beginning of the study (day
1) and after one month (day 30) of maintenance on respective
diets. Rabbits of both
the groups were subjected to the following operative procedure
and oxidative stress after 30 days.
After overnight fasting, rabbits were
anaesthetized with intravenous injection of urethane (1.5
gm/kg body weight). Tracheostomy
was performed and femoral vein was cannulated for taking blood
samples for assessment of haemoglobin, glucose and plasma
MDA levels. One hour
after anaesthesia, anemic hypoxia was induced chemically by
injecting the rabbits intraperitoneally with 15 mg NANO2/100 gm body weight (7). Sodium nitrite used for induction of anemic
hypoxia led to formation of methaemoglobin and free radicals. Blood samples were taken 40 minutes after oxidative
stress for estimation of haemoglobin and glucose for assessment
of stress and plasma MDA was done to assess the oxidative
damage induced by the stress.
Haemoglobin was estimated by sahli's method and blood
glucose was estimated by enzymatic method. Malondialdehyde (MDA) estimation: The lipid
peroxidation products react with thiobarbituric acid forming
a pink coloured adduct on boiling which was measured at 548
nm. Results were expressed
in nmol/ml (8).
There
was no difference in body weight gain, and food consumption
pattern in both control and test group were comparable group
during the period of study. No acute or chronic adverse symptoms were observed
in the test group with the dose employed.
Levels of haemoglobin on day I and day
30 (before and after stress) are shown in Table I for the
control and test group. There
was 27.45% decrease in haemoglobin levels after stress in
control and 20.86% in test group.
Dietary supplementation of tulsi leaves
for 30 days led to a decrease in blood sugar levels on day
30 (before stress) in test group (26%) which was highly significant
statistically (P<0.001). After stress there was a significant
increase in the blood sugar levels in both, control (127.74%)
and test (46.72%) group; although the increase was less in
test group which had received tulsi leaves supplementation
in diet as compared to control group (P<0.01).
Dietary supplementation of tulsi leaves
for 30 days led to statistically significant decrease in the
plasma MDA levels in test group (P<0.001) as shown in Table
1. There was a 42.40% decrease in plasma MDA levels after
30 days in test group. Oxidative stress led to increase in the plasma
MDA levels after stress in both control (154%) and test (112%)
group, although the increase was less in test group which
had received dietary supplementation of tulsi leaves.
In the present study, fresh leaves of
Ocimum sanctum were used.
Satyavati et al have recommended the evaluation of
plant substances in the forms, in which they are usually used
in practice (9). Dose
of tulsi leaves supplemented in diet of test group was 2 gm
per day. This dose
was in accordance with the dose used by Sarkar et al (10).
Stress is known to produce immunosupression
and studies have revealed that Ocimum sanctum possesses adaptogenic
properties when tested against a battery of tests in mice
and rats (11). The
alterations in brain adrenegric neurotransmitters caused by
stress were inhibited by Ocimum sanctum (12).
Sembulingam et al reported the normalizing activity
of Ocimum sanctum extract on increased corticosterone level,
total and differential count in rats induced by acute noise
stress (13). Lately it has been found that acute noise stress
exposure increased the level of' free radicals in the blood
and brain of albino rats (14).
Acute exposure to anemic hypoxia (oxidative
stress) produces a wide range of physiological and biochemical
changes in an organism. Oxygen free radicals and other reactive
oxygen species were postulated by many investigators to be
among the causal factors for those biochemical changes. Hypothetically,
the mechanisms by which the free radicals are generated during
hypoxia are complex and depend on multiple interacting factors.
Lack of oxygen at cytochrome oxidase step might give
rise to leakage of partially reduced 0 2- species, coupled
with a rapid fall in cellular ATP due to diminished aerobic
oxidation may result in the alteration of ionic transport
with cytosolic calcium overload.
This elevated AMP concentration and an increase in
the catabolic processes appear to be the main mechanism underlying
the generation of free radicals in anemic hypoxia. One of the most common effects of an exacerbated
free radical formation in living tissues is the peroxidation
of polyunsaturated fatty acids (7).
Increased lipid peroxide formation has been demonstrated
in several tissues in stressed rats, suggesting that free
radical generation is increased under stress (15).
Table I
click to see full view
|
Table I: Levels of hemoglobin (Hb)
(gm/100ml), blood sugar (mgm/100ml) and plasma MDA (nmol/ml)
in control and test rabbits (n = 20) on day 1 and day
30 before and after oxidative stress.
|
Haemoglobin level as an index of oxidative
stress showed lesser decline in test group as compared to
control group. The
chemical method used for the induction of anemic hypoxia in
the present study led to formation of methaemoglobin and free
radicals (7). Prior
treatment with Ocimum sanctum might be responsible for this
lesser decline in haemoglobin after anemic hypoxia in the
test group. Perhaps formation of methaemoglobin and free
radicals was less in tulsi treated rabbits.
Hypoglycemic effect of OS leaves is
well documented (16, 17).
Oxidative stress led to marked increase in the levels
of blood sugar from base level, more so in control group (127.7%)
than in test group (46.7%). Since blood glucose is strongly
influenced by glucocorticoids, hence elevation of blood glucose
levels in response to hypoxia could be explained by the stimulation
of the hypothalamopituitary adrenal axis, that will trigger
the release of corticotropin releasing factor from hypothalamus
with a subsequent increase in ACTH secretion.
These results are in accordance with Shaheen et al
who had reported that hypoxia was associated with enhancement
of metabolic processes as reflected by elevation of catabolic
hormones and blood sugar levels as well as increased brain
Na+K+ ATPase activity.
They had also reported that elevation in plasma corticosterone
level in response to anemic hypoxia was greater than those
reported after starvation and immobilization (7).
In our study, lipid peroxidation as
indicated by MDA level significantly decreased in test group
which had received dietary supplementation of tulsi.
However, oxidative stress led to marked increase in
MDA levels in both the groups.
Increased MDA levels in both groups due to oxidative
stress induced in the present study suggest the role of free
radicals in the pathogenesis of anemic hypoxia. The increase in MDA levels after oxidative stress
in test group was less as compared to the increase seen in
control group and was significant statistically.
This could be due to conversion of lipid peroxides
to alcohol derivatives and not MDA due to protective action
of Ocimum sanctum against oxidative stress induced by anemic
hypoxia. Urosolic acid
isolated from the Ocimum sanctum extract is found to be very
effective in reducing the free radical level (18).
Contrary to this, Kelm et al demonstrated that eugenol
is responsible for its free radical scavenging action (1).
Hence the stress alleviating effect of Ocimum sanctum
may be due to the free radical scavenging effect of Ocimum
Sanctum.
Further studies are required to substantiate
the protective role of Ocimum sanctum against oxidative stress
induced by anemic hypoxia and to examine the role of inherent
antioxidant defense system in this protection offered.
REFERENCES
1.
Kelm
MA, Nair MG, Strasburg GM, De Witt DI.
Antioxidant and cyclooxygenase inhibitory phenolic
compounds from Ocimum Sanctum Linn. Phytomedicine
2000; 7(l): 7-13.
2.
Nair
AGR, Gunasegaran R. Chemical Investigation of certain south
Indian plants. Indian
J Chem 1982; 21:979-980.
3.
Kirtikar
KR, Basu BD. In: Bratter
E, Carris JF, Bhaskar KS, editors. Indian Medicinal plants. Vol. 3.
Delhi: Periodical Experts Book Agency; 1991;
p. 1955.
4.
Nadkarni AK. In: Nadkirni's
Indian Materia Medica. Bombay: Popular Book Depot; 1954; p.868.
5.
Seetalakshmi
B, Nirasappa AP, Kenchaveerappa S. Protective effect of Ocimuin
Sanctum in experimental liver injury in albino rats. Indian
J Pharmacol 1982; 4: 63-70.
6.
Sakina MR, Dandiya
PC, Hamdard ME,
Hameed A. Preliminary psychopharmacological evaluation of
Ocimum Sanctum leaf extract. J
Ethnopharmacol 1990; 28(2): 143-150.
7.
Shaheen AA, EI-Fattah
AA, Gad MZ.
Effect of various stressors on the level of lipid peroxide,
antioxidants and Na K ATPase
activity in rat brain. Experentia
1996; 52: 336-339.
8.
Kumar R, Seth
RK, Sekhon MS,
Bhargava JS. Serum lipid peroxide and other enzyme
levels of patients suffering from thermal injury. Burn 1995; 21:96-97.
9.
Satyavati GV, Raina MK,
Sharma M, Medicinal plants of India. Vol. I, New Delhi: ICMR, 1976.
10.
Sarkar
A;Pandey DN.Pant MC. A report on the effects of Ocimum Sanctum
(Tulsi) leaves and seeds on blood uric acid, urea and urine
volume in normal albino rabbits. Indian J Physiol Pharmacol 1990; 34(1):
61-62.
11.
Maity
TK, Mandal SC, Saha BP, Pal M. Effect of Ocimum Sanctum
roots extract on swimming performance in mice. Phytother Res
2000; 14(2): 120-121.
12.
Singh
N, Misra N, Srivastava AK, Dixit KS, Gupta GP. Effect of anti-stress plants on biochemical changes
during stress reaction. Indian
J Pharmacol 1991; 23:137-142.
13.
Sembulingam
K, Sembulingam P, Namasivayam A. Effect of Ocimum Sanctum
Linn on changes in leucocytes of albino rats induced by acute
noise stress. Indian J Physiol Pharmacol 1999; 43(1): 137-140.
14.
Kumar
A, Mathangi N, Namasivayam DC. Noise induced changes in free radical scavenging enzymes
in the blood and brain of albino rats. Med
Sci Res 1998; 26:811-812.
15.
Ader
R. In: Ballieu RE, Fielding JF, Affate
AL, editors. Breakdown in human adaptation to stress
towards a multidisciplinary approach. Boston: Martumus-Ninjhoff; 1984; p.653.
16.
Sarkar
A, Pant MC. A comparative study of the hypoglycemic action
of the seeds and fresh leaves of Ocimum Sanctum (Tulsi).
Indian J Physiol Pharmacol 1989; 33:197-198.
17.
Mani
UV, Rai V, lyer U. Effect of Tulsi (Ocimum Sanctum) leaf
powder supplementation on blood sugar levels, serum lipids
and tissue lipids in diabetic rats. Plant Foods Hum Nutr 1997;
50: 9-16.
18.
Balanehru
S, Nagarajan B. Protective effect of oleanolic acid and urosolic
acid against lipid peroxidation. Biochem Int 1991; 24(5):
981-990.