Protective Effect of Tulsi (Ocimum Sanctum) on Lipid Peroxidation in Stress Induced by Anemic Hypoxia in Rabbits
(Received on April 8, 2002)

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.







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