Indian Journal of Experimental Biology Vol. 37, March 1999, pp. 248-252

Mechanism of action of antiinflammatory effect of fixed oil of Ocimum basilicum Linn.

Surender Singh

College of Pharmacy (University of Delhi), Pushp Vihar, Sector-III New Delhi-II 0 017, India

Received 10 June 1998; revised 26 August 1998

Fixed oil of 0. basilicum was found to possess significant antiinflammatory activity against c{lrrageenan and different other mediator-induced paw edema in rat~. Significant inhibitory effect was also observed in castor oil-induced diarrhoea in rats. It also inhibited arachidonic acid-and leukotriene-induced paw edema. The results of antiinflammatory activity of 0. bflSlizcum support the dual inhibition of arachidonate metabolism as indicated by its activity in inflammation models that are insensitive to selective cyclooxygenase inhibitors. On the basis of these findings, it possible to conclude that 0. basilicum may be a useful antiinflammatory agent which block both cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism.

Ocimum basilicum L.(Labiatae), popularly known as "Kali Tulsi" in Hindi, is a widely grown plant of Hindus. Different parts of the plant have been claimed to be valuable in wide spectrum of diseases I. In earlier reported studtes-;-&xed oil of 0. basilicum was found to possesses significant antiinflammatorl and analgesic3 activity without any noticeable toxicity. Since experimental and clinical studies to determine the antiinflammatory profile of fixed oil of 0. basilicull1 are rare and that preliminary studies show encouragmg results, it was considered worthwhile to take up such an investigation in detail.

I~ the present study, antiinflammatory activity of the fixed oil of 0. basilicum has been investigated in detail usmg different inflammatory mediatorsl phlogistic agents. Attempts have also been made to explore the possible mechanism of antiinflammatory action using castor oil-induced diarrhoea, leukotriene-and arachidonic acid induced paw edema in rats.

Materials and Methods

Dried seeds of 0. basilicum collected from Maidan Garhi, New Delhi , India and authenticated by a resident botanist, Department of Genetics, ICAR, New Delhi , were crushed and macerated in cold petroleum ether (40-60) (S.D. Fine Chemicals Ltd., Mumbai , India) for three days. The extract was separated and petroleum ether was evaporated and oil was filtered . The fixed oil thus obtained was

subjected to the following studies in Wi star strain of albino rats weighing between 120-170g. supplied by Mis. Lucky Zoological Store, New Delhi which were kept under standard conditions and fed on a standard rat diet from Lipton (India) Ltd., New Delhi.

Inflammatory mediators-induced paw edema- In a set of experiment, different infJammatory mediatorslphlogistic agents were used to induce oedema4

. The respective strength of the inflammatory mediator, the volume injected and the time for determination of edema volumes are indicated in parentheses; histamine (10.3 glml, O.lml, 60 min.) (Sigma); serotonin (10.3 glml, O.lml, 30 min) (Sigma);Prostaglandin E2 (10.6 glml O.1ml, 20 min) (S,igma) Bradykinin (2x I 0.5 glml, 0.1 ml, 60 min) (Sigma) and hyaluronidase (2400I.UI ml, O.lml, 20 min) (Rallis India Ltd.) were injected into the hind paw of the rat after thirty minutes administration of 0. basilicum fixed oil or control vehicle (dist.water) intraperitoneally to groups of rats. The edema volume was determined as described by Winter et also

Castor oil Induced diarrhoea-1.n order to investigate the mechanisms that might account for the antiinflammatory and related action of the fixed oil of 0. basilicum, castor oil-induced diarrhoea in rats was used as described by Awounters et al.6

• Fixed oil (1.0, 2.0 and 3.0 ml/kg) or aspirin (100 mg/kg) (Reckitf &: Colman India Ltd.) or dexamethasone (1 mg/kg) (Cadilla Chemicals Ltd., India) or vehicle was administered intraperitoneally one h~ur after the

SURENDER SINGH: MECHANISM OF ACTION OF ANTIINFLAMMATORY EFFECT OF FIXED OIL 249

administration of 20 mg/kg of castor oil (Arora Pharmaceuticals Ltd., New Delhi) orally. Animals were examined for presence or absence of characteristic diarrhoeal dropping on a white paper on the floor of their cages every hour for 4 h. Absence of diarrhoeal dropping was recorded as a positive result indicating possible inhibition of the biosynthesis of prostaglandins.

Leukotriene-induced Injlammation--In order to ascertain whether fixed oil of 0. basilicum had any inhibitory effect on lipoxygenase (the enzyme involved in the lipoxygenase pathway of inflammation) inflammation was induced by a leukotriene, (leukotriene B4) a selective lipoxygenase mediator for inflammation. In this experimental setup, groups of albino rats received either the fixed oil of 0. basilicum, 3.0 .ml/kg or a lip oxygenase inhibitor (caffeic acid) (100 mg/kg) (Sigma) or aspirin (100 mg/kg) or the vehicle (dist. water, 3.0 ml/kg) intraperitoneally. After half an hour, edema was produced by injecting leukotriene (L TB4 methyl ester, Sigma) into subplanter aponeurosis in the right hind paw of rats an? edema were measured after 30 min plethysmogr<;lphically as described earlier.

Arachidonic acid-induced Injlammation-To further explore the mechanism of antiinflammatory action, the effect of the fixed oil on arachidonic acid­induced inflammation in rats was studied as described by Di Martino etaf. Paw edema was induced by a single subplanter injection of 0.1 ml of arachidonic acid in 0.2 M. carbonate buffer (PH 8.43-8.56) into the right hind paw of rats thirty minutes after treatment with either the fixed oil of 0. basilicum. (1.0, 2.0 and 3.0 ml/kg), or aspirin (cyclooxygenase inhibitor) (100 mg/kg) or indomethacin (cyclo­oxygenase inhibitor) (Indian Drugs and Pharma­ceuticals Ltd.) or chlorpheniramine maleate (antihistamine) (25 mg/kg) (Glaxo India Ltd.) or cyproheptadine (antihistamine and anti serotonin) (25 m'g/kg) (Cipla Ltd.) or control vehicle intraperi­to~eally . The edema volume was measured after 30

I

minutes plethysmographically as described earlier.

In order to evaluate the relative contribution of cyclooxygenase inhibition, lipoxygenase inhibition and inhibitory effects of antihistamines on arachi'­donic acid-induced inflammation, in another set of experiment, a cyclooxygenase . inhibitor (indo­methacin) (100 mg/kg) alone or in combination with

either of lipoxygenase inhibitor (caffeic acid-lOO mg/kg) or an antihistamine (chlorpheniramine maleate) (25 mg/kg) was administered intra­peritoneally into groups of rats, thirty minutes before arachidonic acid administration. Similarly, the lipoxygenase inhibitor (caffeic acid) (100 mglkg) was administered either alone or in combination with an antihistamine (chlorpheniramine maleate) (25 mglkg) or the antihistamine (chlorpheniramine maleate) (25 mg/kg) and cyclooxygenase inhibitor (indomethacin) (100 mg/kg). One group of rats received 0. basilicum fixed oil (3.0 ml/kg) and the control received distilled water as usual. The edema volume was measured in the same way as described above.

Results and Discussion At the dose of 3.0 nil/kg intraperitoneally, the

fixed oil of 0. basilicum significantly reduced the paw edema formation induced by various phlogistic agents such as. histamine, serotonin, prostaglandin E2, . bradykinin, and hyaluronidase (Table 1), which suggests that the fixed oil also possesses potential antihistaminic, anti serotonin and anit-prostaglandin actIVIty. Hyaluronidase is known to act by polymerising the hyaluronic acid in capillary endothelium and thereby producing an increase in the vascular permeability8.

Castor oil produced characteristic semisolid diarrhoeal dropping in all the animals of the controls group at all the time intervals (Table 2). At the 1 hr interval, the fixed oil of 0. basilicum at doses of 1.0-3.0 ml/kg delayed and prevented diarrhoea in 50 to 83.33% of the test animals, as against 100% protection afforded by aspirin. At the 2 hr interval, fixed oil in doses 1.0-3.0 ml/kg afforded protection up to 83.33%, fixed oil and aspirin also delayed diarrhoea and afforded significant protection at the 3 and 4 hr intervals. Dexamethasone did not protect the animals against castor oil-induced diarrhoea.

\ . Castor oil-induced diarrhoea in rats has been

reported to be prevented by prostaglandIn inhibitors6•

Accordingly, aspmn a known inhibitor of prostaglandin biosynthesis, significantly prevented diarrhoea at all time intervals of the study, while dexamethasone, a steroidal antiinflammatory drug failed to prevent the diarrhoea, probably because corticosteroids are ineffective inhibitor of prostaglandin synthetase9

• The delay of castor oil­induced diarrhoea has been demonstrated to

250 INDIAN J EXP BIOL, MARCH 1999

characterize aspltm like drugs. Awounters et al.6

tested 44 aspirin like drugs and found that the selective potencies of drugs · in castor oil-induced diarrhoea and in the carrageenan induced test corralated well. The delay of cast9r oil-induced diarrhoes and inhibition of carrageenan-induced inflammation by 0. basilicum fixed oil may be related to inhibitory effect of the oil on prostaglan~in synthesis. In our earlier studies we have reported that the fixed oil of 0. basilicum also possessed significant antiinflammatory' and analgesic activity when administered by intrap.eritoneal route2

•3

•

Therefore it is quite possible that all these effects

could be attributed at least due to inhibition of prostaglandin synthesis.

Fixed oil of 0. basilicum significantly inhibited carrageenan-induced inflammation which involves three distinct phases of mediator relese including histamine and serotonin in first phase, . kin ins in the second phase and prostaglandin in the third phase lO

•

But lipoxygenase inhibitors also possess significant anti inflammatory effect against carrageenan induced paw edema 11 . Therefore, inhibition of carrageenan­induced paw edema by fixed oil of 0. basilicum could also be due to inhibition of lipoxygenase. To explore this possibility, inflammation was induced by

Table I-Effect of O. basilicum fixed oil on histamine-, serotonin-, prostaglandin E2-, br.adykinin-, and hyaluronidase-induced paw edema

[Values, expressed as ml, are mean ±SE from 6 animals in each group]

Group Dose Mean Edema Volume (ml +SE) ( i.p.) Histamine Serotonin Prostaglandin Bradykinin

Control (Dis!. water)

0. basiliclIlIl Fixed oil

3.0 mllkg

3.0 ml/kg

Percent Inhibition

·P< 0.01

0.57± 0.05

O.18± 0.03

68.43 •

0.48± 0.06 0.54± 0.06 0.54± 0.06

0.24± 0.05 0.30± 0.06 0.25±0.04

50.00· 44.45· 53 .71·

Table 2-Effect of O. basilicum fixed oil of on castor oil-induced diarrhoea

[Values, are mean ±SE from 6 animals in each group]

Group Dose % protection from diarrhoea (ip) +1 hr +2 hr +3 hr

Control Castor oil (orally) 0.0 0.0 0.0

o.B. fixed oil + Castor oil (Orally) 1.0 ml!kg 50.00 33.33 0.0

G.B. fixed oil + Castor oil (Orally) 2.0 ml!kg 50.00 66.66 16.66

o.B. fixed oil + Castor oil (Orally) 3.0 ml/kg 83.33 83 .33 66.66

Aspirin + Castor oil (Orall y) 100 mg!kg 100.00 83.33 83.33

Dexamathasone + Castor oil (Orally) \.0 mg !kg 0.0 0.0 0.0

Hyaluronidase

0.54± 0.08

O.24± 0.06

'55.56·

+4 hr

0.0

0.0

0.0

0.0

50.00

0.0

Oral dose of castor oil was 20 mllkg. Fixed oil and reference drugs were given (ip) I hr after the administration of castor oiL

Table 3-Effect of 0. basiliclIlIl fixed oil on L TB. methyl ester-induced edema in rats

[Values are mean ±SE from 6 animals in each group]

Group

Control (Dis!. water)

CafTcl c acid (I ipoxygenase-Inhibitor)

() hClSr/ICIIIII fixed oil

AspiTin

*/) . . OJ) I

Dose

( ip)

3.0 ml/kg

100 mg/kg

3.0 mllkg

100 mg/kg

Edema volume

(m l)

0.S7± 0.06

0.24 ± 0.04

0.26 ± 0.06

0.46 ± 0.0'.)

% Inhibition

57.90 %.

54.39 % ..

19.30%

SURENDER SINGH: MECHANISM OF ACTION OF ANTIINFLAMMATORY EFFECT OF FIXED OIL 251

leukotriene B4' a product of lipoxygenase and the inhibitory effects of fixed oil of 0. basilicum, caffeic acid 12 and aspirin on inflammation were studied, The results show that the fixed oil of 0. basilicum and caffeic acid significantly inhibited edema formation, while aspirin failed to do so, which indicates that 0. basilicum fixed oil may also act by, inhibition of lipoxygenase pathway of arachidonate metabolism.

The metabolites of arachidonic acid formed through cyclooxygenase and lipoxygenase pathways represent two important classes of inflammatory mediators. Prostaglandins, PGE2 in particular, is known to cause or enhance the cardinal signs of inflammation 12. Similarly leukotriene (LTB4) is a mediator of leukocytes activation in inflammation and inhibitors of lipoxygenase may have potential

therapeutic value. The recent description of the phlogistic activities of leukotrienes have stimulated search for agents that inhibit both the cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism13

,14. Arachidonic acid-induced paw edema in rats is an in vivo model to distinguish between cyclooxygenase and lipoxygenase inhibitors7

• The present findings show that neither indomethacin nor aspirin blocked the arachidonic acid-induced edema formation while 0. basilicum fixed oil inhibited the edema formation in a dose-dependent manner (Table 4). Chlorpheniramine maleate' and cypro­heptadine also had inhibitory effect on edema formation which appears to be due to inhibition of mast cell mediator release, suggesting that mast cell mediator release may contribute at least in part to

Table 4- Effect of 0. basilicum fi xed oil and various standard drugs on arachidonic acid induced paw edema in rats

[Values, expressed as ml, are mean ±SE from 6 animals in each group]

Group

Control (Dist. Water)

O. B. fi xed oil

O. B. fi xed oil

O. B. fi xed oil

Aspirin

Indomethacin

Chlorpheniramine Maleate

Cyproheptadine

*P< O.OI

Dose

(ip)

3.0 mllkg

\.0 mllkg

2 .0 mllkg

3.0 mllkg

100 mglkg

10.0 mglkg

25.0 mglkg

25 .0 mglkg

Edema volume

( ml)

0,64 ± 0.04

0.42 ± 0.07

0.32 ± 0.07

0.22 ± 0.04

0.57 ± 0.16

0.60 ± 0.12

0.36 ± 0.04

0.38 ± 0.06

% Inhibition

34.38

50.00 *

65 .63 *

10.94

6.30

43.75*

40.63*

Table 5- Effect of O. basilicum fixed oil and various drug combinations on arachidonic acid induced paw edema in rats

[Values are mean +SE from 6 animals in eac~ group]

Group Dose (ip)

Control (Dist. water) 3.0 mllkg

O. B. fi xed oil 3.0 mllkg

Indomethacin 10.0 mglkg

Caffeic acid 100 mglkg

Indomethac in + Caffeic acid 10.0 mglkg

+ 100 mglkg

Indomethacin +Chlorpheniramine 10.0 mglkg

maleate +25 mglkg

Caffeic acid + Chlorpheniramine 100 mglkg

maleate + 25 mglkg

Indomethacin + Chlorpheniramine 10.0 mglkg

Maleate + Caffe ic acid +25 mglkg

+100 mglkg

*['< 0.01

Edema Volume ( ml)

0.60 ± 0.06

0.24 ± 0.03

0.54 ± 0.10

0.30 ± 0.04

0.27 ± 0.03

0.34 ± 0.03

0.32 ± 0.03

0.22± 0.02

% Inhibition

60.00*

10.00

50.00*

55 .00·

43 .34*

46.67*

63.34*

252 INDIAN J EXP BIOL, MARCH 1999

arachidonic acid-induced paw edema. It has been observed earlier that 0. basilicum fixed

oil inhibited histamine-and serotonin-induced edema and also carrageenan-induced edema which involves release of histamine and serotonin. Hence, the inhibitory effect of 0. basilicum fixed oil on arachidonic acid-induced edema could be partly due to inhibition of mast cell mediator release. Since the percent inhibition of edema with fixed oil of 0. basilicum at 3.0 ml/kg dose (65 .63%) was more than that produced by either aspirin (10.94%) or indomethacin (6.30%) or chlorpheniramine maleat~ (43.75%) or cyproheptadine (40.63%), it appears likely that lipoxygenase inhibitory effect of the oil may be involved in the inhibition of edema formation (Table 4).

In order to evaluate the relative contribution of cyc100xygenase inhibition, lipoxygenase inhibition and inhibitory effect of histamine towards inhibition of arachidonic acid-induced paw edema ' in rats, a series of experiments were carried out. The result (Table 5) sh6ws that the percent inhibition of edema with fixed oil of 0. basilicum at 3.0 ml/kg dose (63.34%) was more than that produced by either indomethacin (10.00%) or caffeic acid (50.00%) and is more or less similar to that produced by the combination of indomethacin and caffeic acid i.e. 55.00%. We know that arachidonic acid-induced paw edema in rats is highly sensitive to inhibition by dual inhibitors of arachidonic acid metabolism, corti­costeroids and antihistamine/antiserotonin agent but is insensitive to cyclooxygenase inhibitors7

• The results of other combination (Table 5) I.e. indomethacin+chlorpheniramine maleate and caffeic acid+chlorpheniramine maleate show that antihistamine agent when combined with a lipoxygenase inhibitor produced higher inhibition (46.67%) compared to its combination with a cyc100xygenase inhibitor, (43.34%). However, when these three were given in combination (indomethacin +caffeic acid+chlorpheniramine maleate), the effect was slightly higher (63.34%) than the effect produced by fixed oil alone (60.00%).

In conclusion, it is possible to speculate that the 0. basilicum fixed oil may have the potential to inhibit

both cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism (dual inhibitor property) which may be supplemented by antihistaminic property of fixed oil and the contribution of lipoxygenase inhibition appears to be more than the rest. Such drugs which affect one or both pathways of arachidonic acid metabolism would be <>f great value in elucidating the respective importance of cyclooxygenase and lipoxygenase products in different inflammatory conditions, which would also help in improving the understanding of the etiology and prognosis of the disease and thereby lead to an improved therapy. However, further studies are required before a conclusion on the exact mechanism of action is reached.

Acknowledgement Thanks are due to different pharmaceutical

companies (cited in text) for providing the drug samples used in the present investigation.

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