Indian J Physiol Pharmacol 2002;46 (4);

Inhibitory Role of Syzygium Cumini on Autacoid-Induced Inflammation in Rats
S. MURUGANANDAN, S. PANT, K. SRINIVASAN, S. CHANDRA,
S. K. TANDAN, J. LAL* AND V. RAVI PRAKASH
Division of Pharmacology and Toxicology,
Indian Veterinary Research Institute,
Izatnagar – 243 122
(Received on May 25, 2002)

Abstract: The ethanolic extract of Syzygium cumini bark has been reported to possess anti-inflammatory activity in our previous studies. The present study is an attempt to elucidate the anti-inflammatory activity of S. Cumini bark against inflammation induced by individual autacoid insult. Histamine (1 mg/ml), 5-HT (1 mg/ml), bradykinin (0.02 mg/ml) and PGE₂ (0.001 mg/ml) were used as inflammogens. One of these agents (0.1 ml) was injected s.c. into the right hind paw of each rat. The ethanolic extract of S. cumini bark was tested at the doses of 100, 300 and 1000 mg/kg, p.o. The results indicated the anti-inflammatory activity of S. cumini bark in histamine, 5-HT and PGE2-induced rat paw oedema. However, there was no such significant inhibition of oedema volume observed in bradykinin-induced rat paw oedema at any dose level. Thus, it is concluded that S. cumini exhibits inhibitory role on inflammatory response to histamine, 5-HT and PGE₂.

Key words:     Syzygium cumini           histamine                      5-HT   

bradykinin                    PGE₂                anti-inflammatory activity

Introduction
Methods
Results
Discussion
Reference

INTRODUCTION

Syzygium cumini Syn. Eugenia jambolana L. (Myrtaceae), commonly known as Black berry, Black plum, Jambul or Java Plum, is large evergreen, glabrous tree, which grows up to 30 m in height. It is distributed throughout India, Sri Lanka, Malaya, and Australia. Inflammatory diseases constitute a complex biological response to an injurious agent, involving several important mediators. Amongst the chemical mediators, the two amines, histamine and serotonin are especially important due to their ready release from performed stores, as well as being the first mediators released during inflammation. The kinin system generates vasoactive peptides from plasma proteins called kininogens by specific proteases, viz., kallikreins, which result in the ultimate release of bradykinin that causes increased vascular permeability in the later phase (1). Biologically active lipid mediators released as a result of membrane lipid remodeling to diverse stimuli are the arachidonic acid metabolites as prostaglandins, leukotrienes and lipoxins, which are also involved in the pathogenesis of pain, fever and inflammation (2). Chemical mediators like, histamine, 5-HT, bradykinin an prostaglandins are involved in carrageenin-induced acute inflammation (3, 4). In our previous study, we have reported the anti-inflammatory activity of ethanolic extract of Syzygium cumini bark in carrageenin, kaolin-carrageenin and formaldehyde-induced inflammation along with cotton pellet granuloma formation (5). The present study was aimed to evaluate the anti-inflammatory effect of S. cumini following individual autacoid insult.
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METHODS

Animals:
Wistar albino rats (150-200 g) of either sex were employed in the present study. The animals were obtained from Laboratory Animal Resource Section of Indian Veterinary Research Institute, Izatnagar, India. They were fed on a balanced diet procured from the Feed Technology Unit of the Institute. The experimental protocol was approved by Institute Animal Ethics Committee.

Plant Material:
S. cumini bark was collected from fully maturated (~ 50 years old) tree in October 1998, from the premises of the Indian Veterinary Research Institute, Izatnagar, India. The plant material was botanically authenticated by the Department of Botany, Bareilly College, Bareilly, India and the herbarium specimen (ID 2000:22) was deposited at the Indigenous Drug Laboratory of the Division of Pharmacology and Toxicology, Indian Veterinary Research Institute, Izatnagar, India.

Extract preparation:
The stem bark of S. cumini was shade-dried for a week and then powdered finely, sieved through a muslim cloth and refluxed in 70% ethanol for 8 h. This was concentrated to a semi-solid mass under reduced pressure and made free from alcohol (yield: 2%). Appropriate dilutions of this extract were made in normal saline.

Preliminary phytochemical screening of the extract:
In the preliminary phytochemical screening (6), this 70% ethanolic extract of S. cumini bark gave positive test for tannins and was negative for terpenoids, flavonoids, alkaloids and glycosides.

Histamine-induced hind paw oedema:
The anti-inflammatory activity of S. cumini bark extract was studied in albino rats, which were randomly allotted to five groups of six animals each, using histamine (1 mg/kg) as phlogistic agent (7). Histamine solution (0.1 ml) was injected into the right hind foot of each rat. Thirty minutes prior to this injection, S. cumini bark extract (100, 300 and 1000 mg/kg, p.o.) was administered to the first three groups. The fourth group was administered standard anti-inflammatory agent, phenylbutazone (100 mg/kg, p.o.). The last group, which received a proportionate volume of normal saline, served as control. The normal paw volume (0 h) and the volume of injected paw were measured plethysmometrically. The per cent inhibition of oedema volume in the treated groups was calculated by comparison with control groups.

5-HT-induced hind paw oedema:
The same procedure as was used in histamine-induced hind paw oedema was adopted, the only difference being phlogistic agent. In this, 0.1 ml of 5-HT (1 mg/ml) was injected into the sub plantar tissue of right-hand paw (7).

Bradykinin-induced hind paw oedema:
Bradykinin at a concentration of 0.02 mg/ml was used as phlogistic agent and per cent inhibition was calculated as described earlier (7).

PGE₂-induced hind paw oedema:
PGE₂ solution (0.001 mg/ml) was employed for inducing hind paw oedema in rats. Other procedures remained same as above (7).

Statistical analysis:
Statistical analysis of data was done using Student’s ‘t’-test as per the method described by Snedecor and Cochran (8).
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RESULTS

Effect on histamine-induced inflammation:
As shown in Table I the sub-plantar injection of histamine (0.1 ml of 1 mg/ml solution) induced inflammation with a mean oedema volume of 0.31 ± 0.02 ml in control group. S. cumini bark extract (100, 300 and 1000 mg/kg, p.o.) significantly (P<0.001) decreased the oedema volume with the inhibition being 48.38, 51.61 and 70.96%, respectively. The reference drug, phenylbutazone (100mg/kg), showed significant (P<0.001) inhibition of oedema volume to the extent of 51.61%.

TABLE I: Effect of S. cumini bark extract on autocoid-induced hind paw oedema in rats

*Oedema volume values are expressed in ml (Mean ± S.EM.)
  Number of animals n = 6
*P<0.05 as compared to control
**P<0.001 vs. control
The figure in parentheses indicate the per cent inhibitation of oedema volume
^bVehicle : normal saline solution

Effect on 5-HT-induced inflammation:
The mean paw oedema volume produced by the injection of 5-HT (1 mg/ml) was 0.45 ± 0.01 ml. S. cumini bark extract at 100 mg/kg (P<0.001) significantly inhibited the oedema volume to the extent of 15.56, 28.89 and 46.67%, respectively, as compared to control group. The reference drug phenylbutazone (100 mg/kg) showed significant anti-inflammatory effect (P<0.001) with the inhibition of oedema volume being 46.67%.

Effect on bradykinin-induced inflammation:
The mean paw oedema volume induced by bradykinin (0.02 mg/ml) was 0.31 ± 0.02 ml in control group. In this experiment, S. cumini bark extract showed no significant inhibition of oedema volume at any dose level, as compared to the controls. However, phenylbutazone (100 mg/kg) had significant (P<0.001) anti-inflammatory activity.

Effect on PGE₂-induced inflammation:
The mean paw oedema volume induced by PGE₂ (0.001 mg/ml) was 0.35 ± 0.01 ml. S. cumini bark extract (300 and 1000 mg/kg) significantly (P<0.001) inhibited the oedema volume to the extent of 28.57 and 45.71 per cent, respectively. But at the dose of 100 mg/kg, the extract failed to produced any significant inhibition of oedema. Phenylbutazone (100 mg/kg) significantly (P<0.001) inhibited the oedema volume to the extent of 51.43%, as compared to control group.
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DISCUSSION

In order to elucidate the principal autacoids that may be involved in the anti inflammatory activity of S. cumini bark, trials were conducted with histamine (1 mg/ml), 5-HT (1 mg/ml), bradykinin (0.02 mg/ml) and PGE₂ (0.001 mg/ml) induced oedema in rats. S. cumini bark extract exhibited maximum anti-inflammatory activity against histamine and 5-HT induced inflammation and the anti-inflamatory effect was in a dose-dependent manner. Significant decrease in oedema volume was also produced against PGE₂-induced inflammation at 300 and 1000 mg/kg dose levels. However, S. cumini bark extract did not affect the mean oedema volume produced by bradykinin at any of the dose level (100, 300 or 1000 mg/kg) that was employed. Tannin was found to be an important constituent of S. cumini bark by phytochemical screening. Tannins are known to inhibit the histamine responses (9) and also to inhibit PG synthesis (10). This further substantiates that histaminergic, serotonergic and PGE₂-mediated, but not bradykinin-mediated, mechanism might be involved in the anti-inflammatory action of S. cumini bark. The non-steroidal anti-inflammatory drug (phenylbutazone) produced similar anti-inflammatory action against all the autacoid-induced inflammations. Further, the absence of ulcerogenic side effects (a major NSAID side effect) adds to the therapeutic value of S. cumini bark (5).

In conclusion, the present study had demonstrated the promising anti-inflammatory action of S. cumini bark involving inhibitory mechanisms on histamine, 5-HT and PGE₂-mediated inflammatory actions that deserves further detailed investigations prior to clinical application.

ACKNOWLEDGEMENTS

The authors thank Dr. B.N. Pandey, Department of Botany, Bareilly College, Barilly (India) for botanically identifying the plant specimen.
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REFERENCES

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10.              Nakazato K, Takeo T. Anti-inflammatory effect of oolong tea polyphenols. Nippon-Nogeikagaku-Kaishi 1998; 72: 51-54.
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