Indian Journal of Experimental Biology Vol. 43. August 2005. pp. 7 1 5-72 1

Effect of standardized extract of Ocimum sanctum Linn. on gastric mucosal offensive and defensive factors

R K Goel. K Sairam. M Dorababu. T Prabha & Ch V Rao Department of Pharmacology. Institute of Medical Sciences. Banaras Hindu University. Varanasi 22 1 005. India

Received 20 October 2004; revised 9 May 2005

The standardized methanolic extract of leaves of O. sanctum (OSE; eugenol content 5%) given in doses of 50-200 mglkg. orally. twice daily for five days showed dose-dependent nicer protective effect against cold restraint stress induced gastric ulcers. Optimal effective dose ( 1 00 mglkg) of OSE showed significant ulcer protection against ethanol and pyloric ligation-induced gastric ulcers. but was ineffective against aspirin-induced ulcers. OSE significantly healed ulcers induced by 50% acetic acid after 5 and 10 days treatment. OSE ( 100mg/kg) significantly inhibited the offensive acid-pepsin secretion and lipid peroxidation and increased the gastric defensive factors like mucin secretion. cellular mucus. and life span of mucosal cells and had antioxidant effect. but did not induce mucosal cell proliferation. The results indicate that the ulcer protective and healing effects of OSE may be due to its effects both on offensive and defensive mucosal factors.

Keywords: Antioxidants. Eugenol. Gastric ulcer protection and healing. Mucosal offensive and defensive factors. Ocimum sanctum.

Ocimum sanctum Linn. (Family- Labiatae) known as holy basil, is reported to · possess several pharmacological I and adaptogenic properties2,.3 . Adaptogens are medicinal substances which cause a state of non-specifically induced resistance4. This definition fits into the Ayurvedic concept of rasayanas. Rasayanas are drugs that promote longevity and prevent diseases by providing strength and immuniti. Many rasayanas have also been reported to possess significant antiulcer activitl-8, as stress is one of the. important factors responsible for ulcerogenesis9• Though antiulcerogenic activity lO, 1 I of O. sanctum (OS) have been reported due to its anti­secretory activity, the role of defensive mucosal factors, whose imbalance with the offensive factors leads to gastric ulcers 12 needs exploration for better understanding of anti ulcerogenic activity of this plant.

Eugenol forms the major active constituent of OS, even though other minor constituents like fixed oils l3 and flavones 1 4 have also been reported to have pharmacological activities. Eugenol is the major constituent responsible for anti-stress activity2 and significant anti-oxidant activity 15 . Fixed oils of OS have been reported to possess antiulcer activity and anti-inflammatory activity. Most of the natural drugs

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• . 1 6 1 7 d " d . . . 1 8 possessmg antI-stress ' an antI-OX} ant actIVItIes have also been reported to have anti-ulcer activity and in accordance, the anti ulcerogenic activity of juice of fresh leaves of OS was reported by Sairam et al. 1 9

.

Recent report on OS also indicated its antiulcerogenic and ulcer healing properties2o.

In view of these information, the present study has been undertaken to evaluate the gastric ulcer protective and healing effects of methanolic extract of fresh leaves of OS, standardized to eugenol and also to elucidate its possible mechanism of action by studying its effect on various offensive factors like acid-pepsin and lipid peroxidation and defensive factors like mucin secretion, cellular mucus, life span of mucosal cells, cell proliferation and anti-oxidants in albino rats.

Materials and Methods Animals-Inbred Charles-Foster (CF) albino rats

( 130- 1 80 g), of either sex, obtained from the central animal house of the Institute used for the study, were kept in the departmental animal house at 26°±2° C, 44-56% RH, 10 : 1 4 hr L:D cycle for 1 week before and during the experiments. The animals were provided with standard rodent pellet diet (Hind Lever) and the food was withdrawn 1 8-24 hr before the experiment though water was allowed ad libitum. 'Principles of laboratory animal care'

7 16 INDIAN J EXP BIOL, AUGUST 2005

(NIH publication no. 82-23, revised 1985) guidelines were followed.

Drug treatment-Fresh leaves of cultivated variety of OS were collected in the month of December from the Ayurvedic garden of the Institute and were identified with the standard sample preserved in the Department of Dravyaguna, Institute of Medical Sciences, Varanasi. The fresh leaves were size reduced and macerated with methanol for 7 days. The extract was filtered. vacuum dried and stored in a refrigerator until further use. The yield was 6.04%. The methanolic extract of OS (OSE) was quantified for the essential oil, eugenol by HPTLC using a CAMAG assembly (evaluation soft ware © 1 990 TLC system; Scanner II. Y, 3 . 14IPC/CTS version), toluene: ethylacetate (93 :7) as developing solvent, eugenol (Sigma, U.S.A.) as standard reference compound and quenching at 260nm 1 5. 2 1 . The percentage of eugenol

was 5 .0%. The doses were fixed based on our earlier studies on the fresh juice of OS 19.

Treatment protocol-OSE, suspended in 1 % carboxy methyl cellulose (CMC) in distilled water in doses of 50, 100 and 200 mglkg were administered orally twice daily at 1000 hrs and 1 600 hrs respectively for five days for dose response study. Further, the optimal effective dose of 100 mglkg of OSE and standard ulcer protective drug sucralfate (SF[) in the dose of 250 mg/kg, twice daily for 5 days was used for ulcer protective studies, gastric secretion and mucosal studies, and up to 10 days for ulcer healing study. Control group of animals received suspension of 1 % CMC in distilled water.

Anti-ulcer study-Antiulcer activity was studied by using cold restraint stress (CRS)-, aspirin (ASP)-, ethanol (EtOH)- and pyloric ligation (PL)- induced gastric ulcer (GU) models while, ulcer healing study was studied against acetic acid (AA)- induced chronic gastric ulcer model as described earlier22. A dose dependant study for the selection of optimal ulcer protective dose was undertaken by using 50, 1 00, 200 mg/kg of OSE against CRS-induced gastric ulcer. Statistical significance was calculated by using unpaired Student's t test.

Gastric secretion stlldy-The gastric juice was collected 4 hr after PL and centrifuged for 5 min at 2000 rpm and the volume of the supernatant was expressed as mlll 00g body weight. Total acid output was determined by titrating with 0.01 N NaOH, using phenolphthalein . as indicator and is expressed as

/lEq/ml concentration or /lEq/4hr as output. Peptic

activity was determined using hemoglobin as

substrate and has been expressed as /lmol of

tyrosine/ml as concentration or J,Lmol of tyrosine/4 hr

as output23. Dissolved mucosubstances were estimated in the 90% alcoholic precipitate of the gastric juice. The precipitate, thus obtained was either dissolved in 1 ml of 0. 1 N NaOH or 1 ml of 0. 1 N H2S04. The former was used for the estimation of protein24, tOlal hexoses, hexosamine and fucose, while the latter w as used for the estimation of sialic acid25. The results are

expressed in J,Lg/ml. The ratio of total carbohydrate

(TC) (sum of total hexoses, hexosamine, fucose and sialic acid) to protein (P) has been taken as the indt!x of mucin activity25. DNA content was estimated and

expressed as �glrnl gastric juice/l 00g weight of rat2/'. Estimation of mucosal glycoproteins-Samples of

gastric mucosal scraping were homogenized in distilled water and treated with 90% ethanol and were subjected for the estimation of carbohydrates and proteins using the methods described above for gastric • . 27 JUIce contents .

Statistical analysis of data were done by using unpaired Student' s t test.

Cell proliferation

Estimation of DNA in gastric mucosa-Muco:-al scraping was homogenized in 2.5 ml of ice cooled 0.6 N perchloric acid (PCA). DNA 28 and protein24 were estimated. The concentration of DNA is expressed as

�g DNA/mg protein. Measurement of glandular weights of

stomach-The weights of the whole stomach (rumen and glandular portion) and rumen were taken and the weight of the glandular portion was calculated. The weights of the glandular portions are expressed in mg/l OO g body weight of animals. Statistical analysis was done by Student' s t test.

Estimation of free radical generation-OSE ( 100 mg/kg) was given orally, daily for 5 days and on day 6 of experiment, 1 hr prior to subjecting the animals to CRS. The animals were then sacrificed and the ulcer index was calculated as described earlier. The gastric fundic mucosal scrap was homogenized by using a Potter-Elvehjem glass homogenizer for 30 sec either in ice cold 0.9% saline (5%) for estimation of lipid peroxidation29 and superoxide dismutase30 or in MI150 phosphate buffer (2%) for estimation of catalase3 l .

(a) Measurement of lipid peroxidation (LPO)

GOEL et. al. : OCIMUM SANCTUM & GASTRIC MUCOSAL OFFENSIVFJDEFENSIVE FACfORS 7 1 7

LPO levels were estimated in terms of malondialdehyde (MDA). To 0.4 ml of the homogenate was added 0.2 ml of 8. 1 % sodium dodecyl sulphate (SDS), 1 .5 ml of 20% acetic acid solution (adjusted to pH 3 .5 with NaOH), and 1 .5 ml of 0.8% aqueous solution of thiobarbituric acid (TBA). The mixture was made up to 4 ml with

distilled water, and then heated in an oil bath at 95° C for 60 min. After cooling with tap water, 1 ml of distilled water and · 5 ml mixture of n-butanol and pyridine ( 15 : 1 , v/v) were added and shaken vigorously. After centrifugation at 4000 rpm for 10 . min the organic layer was taken and its absorbance at 532 nm was measured against blank containing 0.4 ml of distilled water in place of sample. 1 , 1 ,3,3- tetra­methoxypropane was used as external standard and the level of LPO was expressed as nmol

·MDAlg wet

tissue29•

(b) Superoxide dismutase (SOD) SOD was estimated as per Kakkar et al 30. The

inhibition of reduction of nitro blue tetrazolium (NBT) to blue colored formozan in presence of phenazine metha sulphate (PMS) and NADH was measured at 560 nm using n-butanol as blank. Briefly, to 0.4 ml of the homogenate was added 1 .2 ml of sodium pyrophosphate buffer (PH 8.3, 0.052 M), 0. 1 ml of 1 86 JLM of PMS, 0.3 ml of 300 JLM NBT and 0.8ml of distilled water was added to make up the volume up to 3 m!. The reaction was started by the addition of 0.2 ml of NADH (780 JLM). It was

incubated at 30° C for 60 sec. The reaction was then stopped by the addition of 1 ml of glacial acetic acid. The reaction mixture was stirred vigorously and shaken with 4 ml of n-butanol. The mixture was allowed to stand for 10 min, centrifuged and butanol layer was taken out. Colour intensity of the chromogen in the butanol was measured against butanol at 560 nm using Spectronic-20 absorptiometer. A system devoid of enzyme served as control. One unit of enzyme activity is defined as enzyme concentration required to decrease the rate of reaction by 50% in one min under the assay conditions. The results were expressed as units (U) of SOD activity/g wet tissue.

(c) Catalase (CAT) Gastric mucosal scrap was homogenized (2%) in

Ml150 phosphate buffer at 1 °_4°C and centrifuged. Sediment was stirred in cold phosphate buffer and was allowed to stand in the cold with occasiopal

shaking. The extraction was repeated twice and the

combined supernatant was used for the assay. The supernatant was diluted ten times wjth distilled water and 0.04 ml of the diluted supernatant was taken for the assay. Decomposition of H202 in presence of catalase was followed at 240 nm. Results were expressed as units (U) of CAT activity/g wet tissue3 ) .

Results OSE (50-200 mg/kg, po, twice daily for 5 days)

showed dose-dependant ulcer protective effect against 2 hr cold restraint stress induced gastric ulcers. Further, optimal effective dose of OSE showed significant ulcer protective effect against ethanol and 4 hr pyloric l igation induced gastric ulcers but tended to decrease ulcer index against aspirin-induced ulcers (Table 1 ). OSE showed significant ulcer healing against 50% acetic acid induced gastric ulcers after 5 and 10 days treatment (Table 2).

OSE in the dose of 100 mg/kg significantly decreased the gastric juice volume, acid and peptic output (Table 3). OSE in the same dose showed a

Table I -Effect of methanolic extract of O. sanctum (OSE) and sucralfate (SFT) on ethanol (EtOH, 1 00%, I mV200g, po, I hr)-, aspirin (ASP, 200 mg/kg, po, 4 hr)-, 2 hr cold restraint stress (CRS)- and 4 hr pylorus ligation (PL)-induced gastric u leers in rats

[Values are mean ± SE from 8 animals in each group]

Oral treatment Ulcer index Protection (mg/kg, bd for 5 days) (%)

CRS-induced ulcers

Control 1 7.8 ± 3.2 OSE 50 9.8 ± 2.2 44.9

1 00 6.9 ± 2.3· 6 1 .2 200 3.4 ± 1 .9b 80.9

SFT 250 7.2 ± 1 .9" 59.6 EtOH - induced ulcers (mm2/rat)

Control 26.9 ± 4.3 OSE 100 8.9 ± 2.9b 67. 1 SFT 250 7.5 ± 2.5b 72.3 ASP- induced ulcers

Control 1 9.3 ± 3.2

OSE 1 00 1 2.8 ± 2.9 33.7 SFT 250 5.2 ± 1 .2b 73. 1 PL- induced ulcers

Control 13 .3 ± 3.7 OSE 1 00 7.2 ± 1 .9a 7 1 .4 SFT 250 2.3 ± 1 .0c 76.7

P values: a< 0.05; b< 0.0 1 ; c< 0.001 . Statistical analysis was done by unpaired Student's t test.

7 1 8 INDIAN J EXP BIOL, AUGUST 2005

tendency to increase the total carbohydrates (TC, which is the sum of the individual carbohydrates, like total hexoses, hexoseamine, fucose and sialic acid) and significantly decreased the protein (P) content of the gastric juice (Table 4), leading to significant mcrease in the TC: P ratio in the gastric juice, the ;narker for mucin secretion. It also significantly decreased cell shedding as evidenced from the Jecrease in DNA content of the gastric juice, which is indicative of increase in life span of cells (Table 3). Again OSE in the above dose significantly increased the TC:P ratio in the gastric mucosa with significant increase in sialic acid indicating enhancement of mucosal glycoproteins, the source of dissolved mucin in the gastric juice (Table 4). OSE did not show any effect on cell proliferation as shown by no changes in �g DNA/mg protein or weight of glandular portion of the stomach (Table 5). Cold restraint stress significantly caused gastric ulcers with increased lipid peroxidation with concomitant increase in superoxide dismutase (SOD) and decrease in catalase (CAT) levels compared to control unstressed group. OSE treatment (100 mg/kg) decreased the ulcer index significantly with decrease in LPO and SOD and increase in CAT levels near to the control unstressed values (Table 6).

Table 2- Effect of OSE on 50% acetic acid-induced (healing) gastric ulcers in rats

[Values are mean ± SE from 8 animals in each group, Figures in parenthesis are per cent healing]

Oral 5 days 10 days treatment (mg/kg, bd) Ulcer Incidence Ulcer Incidence

index of index of perforations perforations (%)

(%) Control 20.2 ± 3.5 80 1 1 .0 ± 1 .9 40.0

OSE 100 7.0 ± I .3b 0.0 5.0 ± 1 .8' 0.0

(65.3) (54.5) P values: '< 0.05; b< 0.0 1 . Statistical analysis was done by unpaired Student's I test.

Discussion Extract of O. sanctum in the dose of 1 00 mg/kg

showed ulcer protective effect in acute GU induc�d by ethanol, cold restraint stress and pyloric ligation and healed chronic GU induced by acetic acid though its protective effect against aspirin induced GU w as less marked. As ulcers are essentially due to imbalances between offensive and defensive muco�al factors 12, the aCtIVIty of OSE in the above experimental models can be explained based on the'>e factors.

In PL-induced ulcers, OSE in the dose of 100 mg/kg significantly decreased the gastric jui�e volume, acid and peptic output indicating decrease in offensive acid and pepsin secretion. On the defensive factors, OSE significantly increased the TC: P ratio in the gastric juice, which is taken as a reliable marker for mucin secretion32. This was due to the tendency to increase dissolved mucopolysaccharides and decrease in protein content of the gastric juice. Decrease in protein content of the gastric j uice indicates increased mucosal resistance as the increase in proteins in the gastric j uice during ulceration is mostly from shedded mucosal cells and bleeding28• This is further substantiated by decrease in DNA content of the gastric juice, which is a reliable index for cell exfoliation. Decrease in DNA content indicates increase in life span of mucosal cells26. OSE abo significantly i ncreased the TC:P ratio of the gastric mucosa indicating increase in cellular mucus27. This increase was due to increase in individual carbohydrates specifically sialic acid, which was increased significantly. Mucus is an important pre­epithelial defensive factor, which also serves as the first line of defense against ulcerogens33• OSE did not show any effect on cell proliferation as seen from the absence of any significant changes in �g DNA/mg protein in the gastric mucosa and glandular weights of the stomach. Hence protection afforded by OSE should involve the process of restitution rather than cell proliferation34. PL-induced ulcers are caused

Table 3-- Effect of OSE and SFf on gastric juice volume, acid, pepsin and DNA content [Values are mean ± SE from 8 animals in each group]

Oral treatment Volume (mJ /lOOg) Acid output (r..I.Eq/4hr) Pepsin output (I..I.mo1/4 hr) DNA (J.lg/mlllOO g) (mg /kg, bd for 5 days)

Control 2.39 ± 0.35 352.8 ± 47.6 588.5 ± 55.6 17.9 ± 1 .3

OSE 100 1 .41 ± 0. 1 6a 147. 1 ± 23. 1 b 337.4 ± 67. 1a 1 3.0 ± LOb SFf 250 1 .93 ± 0. 1 8 297.8 ± 32.4 229.7 ± 1 1 .4a I Ll ± l . lb

P values: a< 0.05; b< 0.01 . Statistical analysis was done by unpaired Student's t test.

GOEL et. al. : DC/MUM SANCTUM & GASTRIC MUCOSAL OFFENSIVEIDEFENSIVE FACTORS 7 1 9

predominantly due to enhanced acid-pepsin secretion leading to breakdown of mucosal barrierl 2 . Hence the decrease in acid pepsin secretion and increase in the mucosal protective factors may be involved in the protection afforded by OSE in PL-induced ulcers.

OSE also offered significant protection against gastric ulcers induced by ethanol and cold restraint stress. The mechanisms of ulcerations induced by ethanol and cold restraint stress are different from one another. Ulcerations by ethanol are caused due to perturbations of superficial mucosal cells, notably the mucosal mast cell leading to release of vasoactive mediators including histamine, thus causing damage to gastric mucosa35• As ethanol induced ulcers are independent of luminal acid36, the increase in mucosal protective factors should then be the major factors responsible for ulcer protective property of OSE rather than the decrease in acid secretion. CRS­induced ulcers are due to both psychological and physiological factors37• The role of acid is questionable and decrease in mucin secretion has been reported during stress-induced ulcers38. Hence,

Table 5-- Effect of OSE and SFT on cell proliferation and weight of glandular portion of stomach in pylorus-ligated rats

[Values are mean ± SE from 8 animals in each group]

Oral treatment Weight of Cell prol iferation (mglkg, bd for glandular DNA Protein Ilg DNA 5 days) portion of Img protein

Control

OSE 100

stomach (Ilg/loo mg wet tissue) (mg/lOO g body weight)

640.5 ± 5 1 .5 588.2 ± 4 1 .3 5605 ± 470 1 04.9 ± 7.9

777.0 + 97.7 617.3 ± 53.7 5996 + 495 102.5 + 8.3

protection afforded by OSE in stress-induced ulcers may principally involve the mucosal defensive factors, which were significantly increased.

However, OSE ( l Oa mg/kg) did not offer significant protection against aspirin-induced ulcers. This may be due to various reasons as many factors, both topical and systemic are reported to be involved in ulcers induced by aspirin . Aspirin is reported to interfere with PG synthesis, increase acid secretion and back diffusion of H+ ions and thus leading to breaking up of mucosal barrier39. 4o

. OSE significantly decreased acid secretion and increased mucin secretion, which should be capable of inhibiting back diffusion of H + ions. However a tendency to decrease aspirin-induced GU with OSE was observed. This may be due to the difference in the dose and amount of active principle/s present in the test extracts of leaves of O. sanctum used or the amount of aspirin

Table 6- Effect of OSE on LPO, SOD and CAT activities in rat gastric mucosa

[ Values are mean ± SE from 8 animals in each group[

Treatment (mglkg. bd for 5 days)

Control (unstressed)

Ulcer LPO (MDA, index nmoles/mg of

protein)

o ± 0 0.22 ± 0.D2

Stress 20.2 ± 3.4' 0.49 ± 0.05"

Stress+ OSE 6.2 ± 2. 1 ' 0. 1 8 ± 0.0 I b 1 00

SOD CAT

(Units/mg of protein) 80.4 ± 8.4 28.6 ± 2.5

1 75.6 ± 7.2" 1 2.9 ± 1 .2"

7 1 .6 ± 7.2b 3 1 .8 ± 2.2"

P values: '. '< 0.01 . ". b< 0.00 I . ' and " compared to control group, ' and h compared to stress group. Statistical analysis was done by unpaired Student's I test.

Table 4-- Effect of OSE and SFT on gastric juice mucoproteins and mucosal glycoproteins [Values are mean ± SE from 8 animals in each group]

Treatment Protein (P) Total Hexosamine Fucose (iii) Sialic acid TC TC:P (mg/kg , po bd hexoses (i) (ii) (iv) (i+ii+iii+iv) for 5 days )

Mucoprotein (�g/ml)

Control 454.5 ± 1 7.7 39 1 . 1± 20. 1 1 68.3 ± 8.6 67.3 ± 2.68 29.3 ± 2. 1 656.0 ± 30.7 1 .55 ± 0. 1 3 OSE 1 00 348.7 ± 1 7.4 c 497.0 ± 5 1 .5 165. 1 ± 1 8.2 57.2 ± 5.7 34.8 ± 2.6 754. 1 ± 57.4 2. 1 9 ± O. 16 b

SFT 250 326.8 ± 25.2c 525.8 ± 49.2" 1 95.4 ± 20. 1 75.8 ± 8. 1 52.6 ± 5.8 h 849.6 ± 62.3 " 2.60 ± 0.22b

Glycoprotein (ltg/lOO mg wet tissue)

Control 501 6 ± 330 241 5 ± 1 87 1472 ± 1 27 368 ± 53 1 07 ± 20 4362 ± 234 0.90 ± 0.08 OSE 100 4149 ± 329 2822 ± 305 1 729 ± 1 94 302 ± 1 9 1 45 ± 17 4998 ± 435 1 .24 ± 0. 12" SFT 250 431 2 ± 4 1 1 3 1 20 ± 3 1 7 1 852 ± 206 427 ± 48 1 79 ± 1 8" 5578 ± 32 1 b 1 .29 ± 0. 1 1 a

P values: "< 0.05; b< 0.0 1 . Statistical analysis was done by unpaired Student's t test. .

720 INDIAN J EXP BIOL, AUGUST 2005

used to produce GU. Most of the workers have used oral aspirin either in the dose of 100 mg/kg lOA I or 1 50 mg/kg

20 to induce GU or have studied the ulcer protective effect of aqueous extract4 1 , fixed oil 1 0 or ethanolic extract20 of leaves of 0. sanctum against aspirin-induced GU. Recently, a significant ulcer protective effect of dried juice of leaves of OS has been observed against aspirin-induced GU though at a higher dose of 500 mg/kg (unpublished data). This could be due to biological variation shown by test substances of herbal origin due to change in soil , weather, temperature etc that could lead to change in the amount of active principle/s present in theml2.

OSE not only protected gastric ulcers due to different ulcerogens but also significantly healed ulcers induced by acetic acid both after 5 and 10 days of treatment. Acetic acid induced ulcers are thought to be mostly due to increase in gastric secretion and volume of acid output and subsequent pyloric obstruction42. Hence, the healing effect may be predominantly due to decrease in acid secretion supported by augmented Increase in mucosal defensive factors.

Free radicals, a common factor in the aetiopathogenesis of ulcers by different ulcer models43

are thought to play a decisive role in stress-induced ulcers37 and as OSE has been reported to have adaptogenic property, the cold restraint stress model was chosen to study the effect of OSE on free radicals. OSE significantly reduced lipid peroxidation in stressed rat gastric mucosa. OS has been reported to possess significant antiuxidant properties in rat brain regions l 5 . During acute stress LPO and SOD were significantly increased while CAT level was found to be decreased significantly. Induction of SOD may be due to stress-induced increase in free radicals that could lead to increase generation of reactive

. oxygen species (ROS) from free radicals that are finally removed by CAT. During mucosal damage SOD scavenges the super oxide radical Oz-, one of the ROS responsible for lipid peroxidation44 and may lead to increased generation of H202' and its accumulation due to decreased CAT level (which scavenge H202-along with gastric peroximes) and may lead to further mucosal damage45. During stress gastric peroximes have been reported to be inactivated46, which may further aggravate the mucosal damage. This evidently caused increased lipid peroxidation and mucosal damage as seen from the increase in ulcer index during acute stress. OSE effectively alleviated stress-

induced ulcers by reversing the increased level l )f LPO and SOD and decreased level of CAT back neaf to the control values suggesting decrease in oxidative damage. Thus, the anti ulcerogenic activity of OSE may also involve its anti-oxidant effect apart from i ts effects on other defensive factors.

Thus, the ulcer protective and healing effects t)f OSE may be due to its effects on both offensive and defensive factors . The antioxidant property of OSE could contribute towards its activity . Further, work ( ,n other mucosal factors like nitric oxide, prostaglandins, cAMP etc. would provide more insight into the activity of OSE.

Acknowledgement RKG is thankful to Indian Council of Medical

Research for grant-in-aid. The authors are thankful (0 Indian Herbs Ltd, Saharanpuf, India, for technical facilities for chemical standardization of the extract.

References Kritikar K R & Basu B D, in Indian Medicinal Plants, edilCd by B J F Cainus and K S Mhaskar, Vol I I I (Bhishen Singh Mahendra Paul Singh Co . • Dehradan). 1 935. 19.

2 Sen P, Maiti P C, Puri S, Audulov N A & Valdman A Y, Mechanism of anti-stress activity of Ocimum sanctum Linn. eugenol and Tinospora malabarica in experimental animals, Indian J Exp BioI. 30 ( 1 992) 592.

3 Rege N N, Thatte U N & Dahanukar S A, Adaptogenic properties of six rasayana herbs used in Ayurvedic medicil l�, Phytother Res. 13 ( 1 999) 275.

4 Brekhman I I & Dardymov I V, New substances of pl;,I1t origin which increase non-specific resistance, Ann Rev Pharmacol. I X ( 1 969) 4 19.

5 Sharma P V, Citi tsastana to Siddhistana, in Caraka samhilll. Vol 4 (Chakhamba Orientalia, Delhi), 1 994, 3.

6 Rao Ch V, Sairam K & Goel R K, Experimental evaluati<ln of Bacopa monniera on rat gastric ulceration and secreti( 'Il. Indian J Physio/ Pharmacol. 44 (2000) 35.

7 Sairam K. Rao Ch V & Gael R K, Effect of Centella asiatica Linn on physical and chemical factors induced gastric ulceration and secretion, Indian J Exp Bioi. 39 (2001 ) 137 .

8 Sairam K, Rao Ch V & Goel R K, Effect of Convolvulus piuricaulis Chois on gastric ulceration and secretion in ruts. Indian J Exp Bioi. 39, (200 1 ) 350.

9 McGuigan J E, Peptic ulcer and gastritis. in HarriUlls principles of internal medicine, edited by J D Wilson, E Braunwald, K J Isselbacher, R G Peterdorf, J B J B Martin. A S Fauchi and R K Root. 1 2th edition (McGraw-Hili, N,:w York). 199 1 , 1 229.

1 0 Mandai S, Das D N, De K, Ray K, Chaudhuri S B, SahamJ C

C & Chowdhuri M K. Ocimum sanctum Linn a study ,)n gastric ulceration and gastric secretion in rats, Indian J Physiol Pharmacal. 37 ( 1 993) 9 1 .

1 1 Singh S & Majumdar D K, Evaluation of gastric antiulcer activity of fixed oil of Ocimum sanctum (Holy Basil). J Ethnopharmacol. 65 ( 1 999) 1 3 .

GOEL et. al. : OCIMUM SANCTUM & GASTRIC MUCOSAL OFFENSIVEIDEFENSIVE FACTORS 721

12 Goel R K & Bhattacharya S K, Gastroduodenal mucosal defense and mucosal protective agents, Indian J Exp Bioi, 29 ( 1 99 1 ) 70 1 .

13 Singh S, Comparative evaluation of anti-inflammatory potential of fixed oil of different species of Ocimum and its possible mechanism of action, Indian J Exp Bioi, 36 ( 1 998) 1 028.

14 Uma Devi P, Ganasoundari A, Rao B S & Srinivasan K K, In vivo radioprotection by Ocimum flavonoids: Survival of mice, Radiation Res, 1 5 1 ( 1999) 74.

1 5 Bhattacharya S K , Bhattacharya A & Ghosal S , Antioxidant activity of Ocimum sanctum, Indian J Med Biochem, 1 ( 1 997) 1 2.

1 6 Bhattacharya S K & Ghosal S, Concerning the anti­ulcerogenic action of sitoindosides IV, Phytother Res I ( 1 987) 95.

17 Ghosal S, Singh S K, Kumar Y & Srivatsava R, Anti­ulcerogenic activity of fulvic acids and 4-metoxy-6-carbomethyl biphenyl isolated from shilagit, Phytother Res, 2 ( 1 988) 1 87.

18 Goel R K, Sairam K, Rao Ch V & Raman A, Role of gastric antioxidant and anti-Helicobacter pylori activities in the anti ulcerogenic activity of banana (Musa sapientum var. paradisiaca), Indian J Exp Bioi, 39 (2001 ) 7 1 9.

19 Sairam K, Rao Ch V & Goel R K, Effect of Ocimum sanctum Linn on peptic ulcers and gastric mucosal offensive and defensive factors, Biomed, 20 (4) (2000) 260.

20 Dharmani P, Kuchibhotla V K, Maurya R, Srivatava S, Sharma S & Palit G, Evaluation of anti-ulcerogenic and ulcer-healing properties of Ocimum sanctum Linn, J Ethnopharmacol, 93 (2-3) (2004) 1 97.

21 Wagner H, Bladt S & Zgainski E M, Plant drug analysis, translated from German by T A Scoot (Springer-Verlag, Berlin) 1 984, 22.

22 Sairam K, Rao Ch V, Dorababu M, Agrawal V K & Goel R K, Antiulcerogenic effect of ethanolic extract of Emblica officinalis: an experimental study, J Ethnopharmacol, 82 (2002) 1 .

23 Debnath P K, Gode K D, Govinda Das D & Sanyal A K, Effect of propranolol on gastric secretion in albino rats, British J Pharmacol, 5 1 ( 1 974) 2 1 3 .

24 Lowry 0 H, Rosenborough N J, Farr A L & Randal R J, Protein measurement with folin phenol reagent, J Bioi Chem, 193 ( 195 1 ) 265.

25 Sanyal A K, Mitra P K & Goel R K, A modified method to estimate dissolved mucosubstances in gastric juice, Indian J Exp Bioi, 2 1 ( 1 983) 78.

26 Mukhopadhyaya K, Bhattacharya D, Chakrabarti A, Goel R K & Sanyal A K, Effect of banana powder (Musa sapientum var. paradisiaca) on gastric mucosal shedding, J Ethnopharmacol, 2 1 ( 1 987) 1 1 .

27 Goel R K, Maiti R N & Mukhopadhyay K, Effect of Tamrabhasma, an indigenous preparation of copper, on rat gastric mucosal resistance, Indian J Exp Bioi, 32 ( 1 994) 559.

28 Goel R, Gupta S, Shankar R & Sanyal A K, Antiulcerogenic effect of Banana powder (Musa sapientum var. Paradisiaca)

and its effect on mucosal resistance,' J Ethnopharmacol, 1 8 ( 1 986) 33.

29 Okhawa H, Ohish N & Yagi K, Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction, Anal Biochem, 95 ( 1979) 35 1 .

30 Kakkar P, Das B & Viswanathan P N, A modified spectrophotometric assay of superoxide dismutase, Indian J Biochem Biophy, 2 1 ( 1 984) 1 30.

3 1 Luck H, Catalase, in Methods of enzymatic analysis, edited by H U Bergmeyar, (Verlag Chemie, Academic press, New York) 1 963, 885.

32 Goel R K, Govinda Das D & Sanyal A K, Effect of vegetable banana power on changes induced by ulcerogenic agents on dissolved mucosubstances in gastric juice, Indian J Gastroenterol, 4 ( 1985) 249.

\

33 Williams S E &Turnberg L A, Retardation of acid diffusion by pig gastric mucus: A potential role in mucosal protection, Gastroenterol, 79 ( 1 980) 299.

34 Svanes K, Takeachi K, Ito S & Silen W, Effect of luminal pH and nutrient bicarbonate concentration on restitution after gastric surface injury, Surgery, 94 ( 1 983) 494.

35 Miller T A & Henagan J M, Indomethacin decreases resistance of gastric barrier to disruption by alcohol, Dig Dis Sci, 29 ( 1 984) 1 4 1 .

3 6 Tamawski A , Hollander D , Gergely M S & Skachura J , Comparision of antacid, sucralfate, cimitidine and ranitidine in protection of gastric mucosa against ethanol injury, American J Med, 79 (Suppl. 2c) ( 1 985) 1 9 .

37 Miller T A, Mechanisms of stress-related mucosal damage, American J Med, 83 ( 1 987) 8 .

38 Guth P, The role of microcirculation and the mast cell in stress ulcer, in Peptic ulcer edited by C J Pfeiffer, (Munksgaard, Copenhagen) 1 97 1 , 22 1 .

3 9 Vane J R , Inhibition o f prostaglandin synthesis as a mechanism of action for aspirin like drugs, Nat News Bioi, 23 1 ( 1 97 1 ) 232.

40 Schoen R T & Vender R J, Mechanism of non-steroidal anti­inflammatory induced gastric damage, Am J Med, 86 ( 1 989) 449.

4 1 Singh S , Majumdar D K & Rehman H M , Chemical and pharmacological studies on fixed oil of Ocimum sanctum, Indian J Exp Bioi, 34 ( 1 996) 1 2 1 2.

42 Okabe S & Pfeiffer C J, Chronicity of acetic acid ulcer in the rat stomach, Dig Dis, 7 ( 1 972) 6 1 9.

43 Cochran T, Stefanko J, Moore C & Saik R, Dimethylsulfoxide protection against gastric stress ulceration, Curr Surg, 40 ( 1 983) 435.

44 Fridovich I, Biological effects of superoxide radical, Arch Biochem Bi()phy, 247 ( 1 986) 1 .

45 Das D, Bandyopadhyay D, Bhattacharya M & Banarjee R K, Hydroxyl radical is the m�or causative factor in stress induced gastric ulceration, Free radical Bioi Med, 23 ( 1 997) 8.

46 Boyd S C, Sasame H A & Boyd M R, Gastric glutathione depletion and acute ulcerogen(;sis by diethylmalate given subcutaneously to rats, Life Sci, 28 ( 198 1 ) 2987.