Indian Journal of Clinical Biochemistry, 2006 / 21 (2) 181-188

Halim Eshrat M. and A. K. Mukhopadhyay
Department of Laboratory Medicine, AII India Institute of Medical Sciences, Ansari Nagar, New Delhi - 110029, India ABSTRACT
This study was carried out to see the effect of the aqueous extract of Ocimum sanctum Linn (Tulsi) with
Vitamin E on biochemical parameters and retinopathy in the streptozotocin-induced diabetic albino male rats.
Adult albino male rats weighing 150-200gm were made diabetic by intraperitoneal injection of streptozotocin
in the dose 60 mg/kg in citrate buffer (pH 6.3). The diabetic animals were left for one month to develop
retinopathy. Biochemical parameters like plasma glucose, oral glucose tolerance and glycosylated hemoglobin
HbA , were measured along with lipid profile, and enzymes like glutathione peroxidase (GPX), lipid peroxidase

(LPO), superoxide dismutase (SOD), catalase (CAT) and glutathione -S- transferase (GST) in normal, untreateddiabetic rats and diabetic rats treated with Ocimum sanctum L extracts and vitamin E. Fluorescein angiographytest was done for assessing retinopathy. Results on biochemical parameters were analyzed statistically byusing ANOVA followed by Dunnet’s ‘t’- test. A p-value of < 0.05 was considered as significant. Evaluation ofbiochemical profile in treated groups showed statistically significant reduction in plasma levels of glucose,HbA , lipid profile and LPO, and elevation of GPX, SOD, CAT and GST. Treatment of the diabetic animals with Ocimum sanctum and Vitamin E, alone and in combination for 16 weeks showed reversal of most of theparameters studied including plasma glucose levels. Angiography showed improvement in retinal changesfollowing combined antidiabetic treatment. KEY WORDS
Ocimum sanctum, Vitamin E, Diabetes mellitus, Diabetic retinopathy, Lipid peroxidation, Antioxidants.
vessels. DR is the leading cause of blindness in peoplebetween 20 and 74 years of age(1,2,3). Of all the complications Besides complications like cardiovascular changes, of diabetes mellitus, DR and age related macular degeneration nephropathy and neuropathy, diabetic retinopathy (DR) is a (AMD) are most prevalent in the Western World. In India, it common complication of both Type I and Type II diabetes was the seventeenth cause of blindness twenty years ago.
mellitus (DM). Diabetic retinopathy is the most severe of the Today the diabetes related blindness has rapidly ascended to several ocular complications of diabetes. Advances in the sixth position (4,5). DR is a progressive disease that treatment over the post 40 years have greatly reduced the passes through following stages, (1) no apparent DR, (11) non- risk of blindness from this disease. However, as diabetes is proliferative DR, (111) proliferative (DR) and (18) vitreous so common that retinopathy remains an important problem hemorrhage resulting in poor visual acuity. Non-proliferative (1). Blindness, like other complications of diabetes, is usually DR is characterized by the presence of venous dilatation, caused by persistent high blood glucose level damaging blood microaneurysm formation, retinal hemorrhage, retinal edema,and hard exudates (4). The incidence of DR was found Address for Correspondence:
significantly high in the Hispanic (especially Cuban, Mexican)
diabetic patients surveyed in the third National Health and
control of blood –glucose level, retards progression of diabetic microvascular disease, but the effect on macrovascular complication is unknown (7). The benefits of improved glycemic and lipid control on complications of DM are now E-mail : [email protected]/ [email protected] almost confirmed. Enhanced oxidative stress along with 181
Indian Journal of Clinical Biochemistry, 2006 / 21 (2)
decreased antioxidative status causes progression of the disease. Antioxidant defenses are located both inside and outside the cell. The benefits of antioxidant on delaying development of complication are also reported. Vitamin E Diabetic rat which were fed Vitamin E 544 (α-tocopherol), a chain–breaking lipid soluble antioxidant, has mg / kg orally daily for 16 weeks (n=5).
been used in medicine in the treatment of cataract, arteriosclerosis, cancer, tardive dyskinesia, immunological and extract of Ocimum sanctum 250 mg / kg hematological disorders and peripheral neuropathy (8). Heart and Vitamin E 544 mg / kg daily orally for disease is the leading cause of diabetes related death because chronically elevated blood sugar level is associated witharteriosclerosis, hypertriglyceridema, decreased HDL level, During the course of the experiment, the animals were fed hypertension and heart attack (9). However, there is no study with standard pellet diet ad libitum and had free access to assessing its effect on DR. Currently available therapeutic water (12). All the animals were cared according to the rules options for non-insulin-dependent diabetes mellitus (NIDDM) and regulations of the Institutional Animal Ethics Committee such as dietary modification, oral hypoglycemic drugs and (IAEC) guidelines of Jamia Hamdard University, New Delhi insulin have limitations of their own. A diet with plenty of fruits, where the animals were reared (First author’s earlier place of nuts, grains and vegetables is rich in vitamin-E and has remarkable antioxidant effects (10). These effects seem toprotect the organs from stressful effect of hyperglycemia.
II . Streptozoticin, Vitamin E and Aqueous extract of Tulsi
Plants have always been the source of drugs since ancienttime. Many of the currently available drugs have been derived Streptozotocin (STZ) and capsule of Vitamin E (544 mg) were directly or indirectly from plants. Many herbal medicines have obtained from Sigma, USA. Tulsi leaves were collected from been recommended for the treatment of diabetes. Tulsi Indian Agriculture Research Institute New Delhi. Identification (Ocimum sanctum- family Lamiaceae Linde) is one of such was done from National Bureau of Plant Genetic Research drugs, used in diabetes mellitus. The effect of Ocimum (NBPGR), Pusa Campus, New Delhi. Ocimum santum sanctum (Tulsi) on the plasma glucose, serum lipid, peroxidase collected belongs to family Lamiaceae Lindi and has purple and antioxidant enzymes are documented (11,12). However, leaves (photograph submitted). The leaves were air dried at the effect of Tulsi and vitamin E individually or in combination room temperature and powdered in a grinder. An aqueous on diabetic retinopathy has not been studied earlier. The extract was produced by soaking the powdered leaves in present study is aimed to demonstrate the effect of Tulsi and distilled water at room temperature for 24 hours. After adequate vitamin E on biochemical parameters as well as on well- stirring the mixture was centrifuged and filtered. The developed retinopathy in diabetic male albino rat.
supernatant was collected and processed as described earlier(10). The supernatant was lyophilized in lyophilizer and the MATERIALS AND METHODS
extract was reconstituted in sterile water at room temperature.
I. Animals: Diabetic and Control
The rats were fed with reconstituted extract by means offeeding tube at a dose of 250 mg / kg body weight.
Healthy adult Male Wistar albino rats of 150-200 gram weightwere used for the study. They were fed with a standard diet III. Collection of blood sample
and water ad libitum. Twenty-five rats were made diabetic byintraperitoneal injection of streptozotocin (STZ), 60 mg/kg Blood was collected at the beginning and at the end of the dissolved in citrate buffer at pH 6.3. Five rats were injected experiment retroorbitally from the inner canthus of the eye with same volume of only citrate buffer and were used as using micro hematocrit capillaries (Mucaps) in oxalate–sodium non-diabetic healthy controls. The diabetic rats developed changes in their retina and developed diabetic retinopathy afterone month. The diabetic rats were divided into following four IV. Preparation of tissue extracts for enzyme assay.
groups, each having five rats. Five diabetic rats, which expiredduring the experiment, were excluded from the study.
All animals were sacrificed by cervical dislocation under lightether anesthesia. Immediately after sacrifice, the livers were dissected out, washed in the ice-cold saline, and the homogenates were prepared in Tris HCL buffer (pH 7.4). The 182
Antioxidative Activity of Ocimum sanctum
homogenate was centrifuged at 3000 rpm for 30 minutes at Protein estimation
4°C in a refrigerated centrifuge (Sorvall RC-5B). Thesupernatant containing the cytosolic fraction was used for Protein was estimated according to the method of Lowry et al enzyme assays. In the similar way, brain extract and (20), using bovine serum albumin as the standard.
VI. Retinal examination
V. Estimation of biochemical parameters.
Glucose and glucose tolerance test (GTT)

Fluorescein Angiography is the method used to observe retinalblood vessels. Fluoroscein dye was injected into a vein in the Glucose was estimated at the beginning and end of the leg, as described (23,24). The dye travels through the blood experiment (16 weeks following) by glucose oxidase method vessels including those in eyes. The photograph of the retina using the kit from Ranbaxy Labs, New Delhi India. Standard was taken at Dr Rajendra Prasad Centre for Ophthalmic oral glucose tolerance test was carried out as described by Sciences, AIIMS, New Delhi with Zeiss FF 450 plus IR Camera Eshrat Halim.M (13) at the beginning and at the end of 16 (Carlzeis Meditec AG, 07740 Jena, Germany). A qualified ophthalmologist (acknowledged at the end) carried out thepre - and post- treatment fundus examination.
Glycosylated hemoglobin (HbA )
Statistical Analysis
Glycosylated hemoglobin was estimated in the blood by usinga kit from Stanger Immunodiagnostics.
The data of biochemical parameters were analyzed. Resultsare presented as mean ± SEM for all experiments. The Assay of antioxidant enzymes
significance of difference between data pairs was evaluatedby analysis of variance (ANOVA) followed by Dunnet’s ‘t’ test.
All the enzyme activities were measured in the supernatantfraction of tissue homogenate separately from liver, heart and brain and the numerical average of three measurements wereconsidered. The activity of total Superoxide dismutase (SOD) Blood glucose : The effects of oral administration of
was measured by the method of Beers, (14). The assay of aqueous extract of Tulsi leaves alone and in Catalase (CAT) was performed by method of Aebi (15,16).
combination with vitamin E in diabetic rat are shown in Glutathione peroxides (GPX) and Glutathione-S-transferase Table1. Whereas in diabetic untreated rat plasma (GST) activities were estimated by the method of Lawrence glucose continued to rise from 258.9±4.02 mg/dl to and Gupta (17,18). Lipid peroxidase (LPO) was determined 335.7 ±5.99mg/dl, in diabetic rats having treatment with by the method using the thiobarbituric acid reaction (19). The Tulsi alone, plasma glucose fell from 232.4±7.66mg/dl amounts of malondialdehyde (MDA) in brain, liver, cardiac andmuscle tissue were also estimated by method of Okhawa and Comparative effect of aqueous extract of leaf of Ocimum sanctum
Lowry (20,21). Erythrocyte catalase and GPX were also Linn (Tulsi) and vitamin-E on plasma glucose level in Control, and
measured before treatment and following combined therapy.
streptozotocin - induced diabetic rats at the end of 16 weeks.
Lipid Profile
Plasma glucose mg/dl, mean ± SE
Total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and triglycerides (TG) were estimated by enzymatic methods employing kits from Orthodiagnostics systems. VLDL and LDL-C level was calculated by the standard formula asdescribed. LDL cholesterol was estimated by using Friedewald Ocimum sanctum Linn (Tulsi ) WT (1972) formula as follows: LDL in mg % = total cholesterol – (HDL-C –1/5 triglycerides) (22).
Ocimum sanctum Linn (Tulsi ) Values are mean ± SE of five animals in each group.
aP<0.01; bP<0.05 183
Indian Journal of Clinical Biochemistry, 2006 / 21 (2)
Comparative Effect of aqueous leaf extract of Ocimum sanctum Linn (Tulsi), and Vitamin E on
glucose tolerance after 16 weeks, in Control and STZ induced diabetic rats.
Plasma glucose mg/dl, mean ± SE
Diabetic + Ocimumsanctum Linn ( Tulsi ) Diabetic + Vitamin E + Ocimumsanctum Linn (Tulsi ) Values are mean ± SE of five animals in each group.
* P< 0.05 when compared with untreated diabetic group.
to 85.22±4.59mg/dl. When treated with Vitamin E alone, which received Vitamin E and Tulsi together, there was plasma glucose fell from 231.24 ±4.87mg/dl to reduction in 2 hours post load plasma glucose level, 175.34±2.36mg/dl and, when treated with Tulsi and there was no reduction and instead a mild rise of plasma vitamin E in combination the plasma glucose fell from glucose from 86.3±10.87 mg/dl to 93.6 ±10.60 mg/dl in 220.90±5.37 mg/dl to 71.33±1.03mg/dl. Ocimum diabetic rats treated with Tulsi alone. Vitamin E is seen sanctum extract was found more effective than Vitamin to be more effective in this context.
Glycosylated hemoglobin (HbA ) and Lipid profile
Glucose Tolerance Test (GTT) : Table 2 shows the
(Table 3) :Table 3 shows the effect of aqueous extract
effect of aqueous extract of Tulsi leaves and vitamin E of Tulsi and Vitamin E, alone and in combination, on on glucose tolerance (GTT) in normal and in diabetic value of HbA , total protein and lipid profile measured rats after 16 weeks of treatment. Control group showed at the end of 16 weeks in control, diabetic untreated mild rise of blood glucose from 94.5 ±11.54 mg/dl to and diabetic treated rats. HbA did not show any 105.0 ±5.77 mg/dl in 2 hours following the glucose load.
alteration in diabetic rat treated with both Tulsi and In untreated diabetic group, 2 hours post load level of Vitamin E. Without treatment HbA level rose up to plasma glucose was 274.0 ±37.92 mg/dl. Whereas in more than double while when treated alone with Tulsi the group treated with vitamin E alone and in the group or Vitamin E it showed mild rise. Total cholesterol and Comparative effect of aqueous extract of Ocimum sanctum Linn (Tulsi) and Vitamin E on glycosylated hemoglobin (HbA ), protein and
lipid profile in control, diabetic untreated and diabetic treated rats at the end of 16 weeks.
80.0±1.88 a 172.0 ± 5.55a 93.17± 5.63a Val Values are mean ± SE of five animals in each group.
Comparison of each diabetic treated experimental group are shown with the diabetic untreated.
α P < 0.005 when compared with control group 184
Antioxidative Activity of Ocimum sanctum
Effect of treatment with aqueous extract of Ocimum sanctum Linn (Tulsi) and Vitamin E, alone and in combination, for 16 weeks on
LPO, SOD, CAT, Glutathione peroxidase (GPX) and Glutathione transferase (GST) activities in tissue extract ), in control diabetic
untreated and diabetic treated rats at the end of 16 weeks.
3. Diabetic + Ocimum sanctum * P Values are mean ± SE of five animals in each group.
* P *P< 0.001 , (Values are mean ± SE of five animals in each group) /Values are expressed as Lipid (peroxides (nm) of TBA reactants / mg protein;SOD - Units / mg protein / CAT - nmol of H O / min /mg protein / GPX - nmole of glutathione oxidized / min / mg protein / LPO - nmol of mal malondialdehyde /mg protein, and / GST - μmole of H O utilized / min / mg protein.
triglyceride levels showed maximum reduction with level rose significantly. Treatment with Vitamin E alone LPO and other Antioxidant enzymes : Table 4 shows
Retinal changes : Fundus fluorescien angiography of
the effect of treatment at the end of 16 week on liver the right eye of diabetic rat shows dot & blot enzymes like LPO, SOD, CAT, GPX and GST. While hemorrhages (as indicated by white arrows in Fig.1).
there was remarked reduction of LPO in all treated Hard exudates (as indicated by black arrows in Fig. 1), group as compared to control, there was rise of SOD, few cotton wool spots and microaneurysms are also CAT, GPX and GST level as an effect of treatment.
seen. There was also macular edema. Fundoscopicexamination following 16 weeks of combination Erythrocyte GPX and Catalase : Measurement of
treatment (Vitamin E and Ocimum sanctum) showed erythrocyte GPX and catalase activities shows reduction of erythrocyte catalase level (Table 5) following hemorrhages and exudates (Fig 2). The vision also Malondialdehyde (MDA) in brain, liver and muscle
tissue :
Table 6 shows MDA level in brain, liver and
muscle tissue in normal, diabetic untreated and diabetic
rats treated with vitamin E. Without treatment, MDA
Erythrocyte GPX and Catalase activities in control, diabetics and
diabetic treated rats at the end of 16 weeks.
Diabetic + Vitamin-E +Ocimun sanctum Values are mean ± SE of five animals in each group.
** P < 0.05; when compared with control group.
Units for catalase (nm) of H O decomposed / min/mg protein.
Indian Journal of Clinical Biochemistry, 2006 / 21 (2)
hypoglycemic damage and prevent diabetic retinopathy inexperimental animals (29). Beside hypoglycemic controlfamilial and genetic factors have been stressed to take part inthe pathogenesis of complications like nephropathy andretinopathy (30).
In the present experimental set up of streptozotocin-induceddiabetes in rat, hypoglycemic effect of Ocimum sanctum Linnis found to be remarkable (Table 1). Vitamin-E in this respectis effective but seems to be less potent than Ocimumsanctum Linn. This is consistent with the data alreadypublished by Hussain et al (11), and Sarkar et al (31).
However in 2 hour GTT, Vitamin E is seen to be at least aseffective as Ocimum sanctum Linn in reducing blood glucoselevel (Table 2). Hypolipidemic effect, however, seems to be additive when combined treatment with Ocimum sanctum Amounts of malondialdehyde (MDA) in brain, liver and
Linn and Vitamin E are given together (Table 3). Vitamin E muscle rat tissue (nmol wt).
therapy has remarkable effect in reduction of LPO enzymeand elevation of SOD, CAT, GPX and GST (Table 3). This finding confirms known antioxidant effect of Vitamin E (32).
Similar effect of Vitamin E is observed in reduction ofmalonaldehyde level in liver, brain and myocardial 292.58± 23.91 146.27±14.84 220.12±19.68 Diabetic + Vitamin E 231.32±15.30* 114.82±16.32* 170.0±15.76* * P < 0.05; when compared with diabetic untreated group.
In the last few years extensive work has been done on (Values are mean ± SE of five animals in each group).
antioxidant effects of Ocimum sanctum Linn. Ocimum sanctumpretreatment may also have neuroprotective effect in rat, DISCUSSION
particular in reperfusion injury (33). Long-term oral feeding ofO sanctum, in rats offered significant protection against Several biochemical mechanisms have been proposed as isoproterenol-induced myocardial necrosis through a unique explanations for progression of diabetic retinopathy. Four major property of enhancement of endogenous antioxidants (34).
molecular mechanisms have been implicated in Prior intrapertoneal administration of O sanctum in dose 5 hyperglycemia–mediated vascular damage namely, increased and 10 mg /kg has been shown to have potential anticataract polyol pathway flux, increased hexosamine pathway flux, activity in selenite – induced experimental cataractgenesis.
increased AGE formation, and activation of Protein Kinase C Such effect is also supported by concomitant restoration of (PKC) isoforms through de novo synthesis of the lipid second antioxidant defense system and inhibition of protein messenger DAG. Several clinical trials and studies have clearly insolubilization of rat lenses (35). Aqueous and alcohol shown that improved glycemic control is strongly associated extracts of Ocimum sanctum, have significant in vivo activity with decreased development or regression of diabetic against hypercholesterolemia induced lipid peroxidation activity complications in both type I & II DM. Hyperglycemia leads to in erythrocyte and provides liver and aortic tissue a kind of increased protein glycosylation, which is an important source protection against hypercholesteroemia–induced peroxidative of free radicals. Garg et al (25) focuses on free radical formation in hyperglycemic conditions. Wolf et al (26) stresseson non-enzymic glycosylation and subsequent browning Several clinical trials although have clearly shown that reaction. Hyperglycemia induced synthesis of diacylglyceral improved glycemic control is strongly associated with which activates Protein Kinase-C has been highlighted by decreased development or regression of diabetic complication PKC-DRS study group (27). Cusick et al (28) has clearly shown in both types I and II DM (28), the most remarkable effect of that Vitamin E in dietary formulation (in dose of 500 IU daily) combined therapy with Ocimum sanctum Linn and Vitamin E could reduce age related eye disease (ARED). Benfotiamine is, however, seen in diabetic retinopathy. All the features of has been shown to block three major pathways of retinopathy regressed and the vision improved completely. This 186
Antioxidative Activity of Ocimum sanctum
could be explained by hypoglycemic effect of Ocimum sanctum 8. Jain, S. K. (1999) Should high – dose vitamin E Linn combined with antioxidant effect of Vitamin E. Given supplementation be recommended to patients? Diabetes alone, they are not as effective as to cause regression of retinopathic changes as when those are used in combination.
9. Sivaram, P. (1994) In New Developments in cardiology The uniqueness of the present study, therefore, is the and cardiac surgery. Eds. Tehran, N., Kumar, A. Escort demonstration of the effect of combined therapy with Ocimum Heart Institute and Research Center, (New Delhi), 1.
sanctum Linn and Vitamin E to reverse the changes of diabeticretinopathy.
10. Jain, S.K. (1996) The effect of modest vitamin E supplementation on lipid peroxidation products and other Treatment of the diabetic animals with Ocimum sanctum Linn cardiovascular risk factors in diabetic patients. Lipids, and Vitamin E, alone and in combination, for 16 weeks showed reversal of most of the parameters like plasma glucose and 11. Eshrat, Halim M.A. Hussain, Kaiser Jamil and Mala Rao.
lipid levels. Retinal angiography showed remarkable (2001) Hypoglycemic, Hypolipidemic and antioxidant improvement in the retinal changes and in fact reversal of properties of Tulsi (Ocimum sanctum Linn) on changes following combination treatment.
streptozotocin induced diabetes in rats. Ind J ClinBiochem, 2, 190 -194.
12. Eshrat, H. M. (2002) Reversal of diabetic retinopathy in streptozotocin induced diabetic rats using traditional Indian First author gratefully acknowledges University Grants antidiabetic plant, Azadirachta indica (L.) Ind.J. Clin.
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