Membrane stabilizing activity a possible mechanism

Fitoterapia 70 Ž1999. 251]257

Membrane stabilizing activity } a possible mechanism of action for the anti-inflammatory activity of Cedrus deodara wood oil U.A. ShindeU , A.S. Phadke, A.M. Nair, A.A. Mungantiwar, V.J. Dikshit, M.N. Saraf Department of Pharmacology, Bombay College of Pharmacy, Kalina, Mumbai 400 098, India Received 24 March 1998; accepted 4 December 1998

Abstract The volatile oil of the wood of Cedrus deodara Ž50 and 100 mgrkg, p.o.. produced a significant inhibition of compound 48r80 and nystatin-induced rat paw edema. It also inhibited heat- as well as hypotonic solution-induced haemolysis of erythrocytes in vitro. The anti-inflammatory activity of the oil could be due to its membrane stabilizing action. Q 1999 Elsevier Science B.V. All rights reserved. Keywords: Cedrus deodara; Anti-inflammatory activity; Membrane stabilizing action

1. Introduction Cedrus deodara ŽRoxb.. Loud. ŽPinaceae . ŽSanskrit } Devadaru; HindirMarathi } Deodar; English } Cedar. is a graceful, ornamental, evergreen tree growing U

Corresponding author. Department of Pharmacology, L.M. College of Pharmacy, Navrangpura, Ahmedabad } 380 009, Gujarat, India. Tel.: q91-79-6440-746; fax: q91-79-6444-865; e-mail: [email protected] 0367-326Xr99r$ - see front matter Q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 6 7 - 3 2 6 X Ž 9 9 . 0 0 0 3 0 - 1

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U.A. Shinde et al. r Fitoterapia 70 (1999) 251]257

extensively on the slopes of the Himalayas. Deodar forests are common from Kashmir Žaltitude of 1500]3000 m., especially Krishnaganga, Kishtwar and Jhelum, to Garhwal w1x. Cedrus deodara has been used since ancient times in Ayurvedic Medical Practice for the treatment of rheumatoid arthritis w2x. Lack of scientific data to support the ancient literature claims prompted us to study the anti-inflammatory activity of C. deodara wood oil. In a previous work we have evaluated the effect of C. deodara wood oil in both acute and chronic inflammatory models, such as carrageenan-induced paw edema and Freund’s complete adjuvant-induced polyarthritis in rats. Results of these studies indicated the possibility that the oil interfered in the release andror action of mediators like histamine, serotonin, prostaglandins, leukotrienes, etc. w3x. Therefore, with the aim to investigate the possible mechanism of its anti-inflammatory activity, in the present study C. deodara wood oil was further evaluated using two in vivo models, i.e. compound 48r80 and nystatin-induced paw edemas in rats and an in vitro model, i.e. the effect on erythrocyte membrane. The principal constituents of the oil are sesquiterpenes, i.e. a-himachalene Ž12.5%. and b-himachalene Ž43%. w1x. Associated with them are sesquiterpene alcohols Žhimachalol, allohimachalol, himadarol, isocentdarol, and centdarol. w4x. Isolation and characterization of atlantone Ž trans-atlantone 12%, cis-atlantone 0.4%. has been reported w5x.

2. Experimental 2.1. Plant material Cedrus deodara wood, obtained from a commercial supplier, was identified and authenticated by Dr K.S. Laddha, Department of Pharmacognosy, University Department of Chemical Technology, Mumbai, India. A voucher specimen was deposited at the Bombay College of Pharmacy, Kalina, Mumbai. 2.2. Preparation of the extract The plant material, cut into small pieces, was subjected to steam distillation. On cooling, the essential oil was separated from the aqueous layer in 0.5% Žwrw. yield, dried over anhydrous sodium sulphate, and stored in an amber coloured glass bottle in a cool place. The oil was emulsified by using acacia and tragacanth mixture, and administered orally at doses of 50 and 100 mgrkg for in vivo studies. Solutions prepared in alcohol Ž25]200 m grml. were used for the in vitro study. 2.3. Drugs Ketotifen fumarate and acetyl salicylic acid ŽASA. were generous gift samples from FDC Ltd., Mumbai and German Remedies Ltd., Mumbai, respectively.


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2.4. Animals Male Wistar rats Ž150]200 g. were used for the study. Animals were housed under standard conditions of temperature Ž23 " 18C., relative humidity Ž55 " 10%., 12r12 h lightrdark cycle and fed with standard pellet diet ŽGold Mohur brand, Lipton India Ltd.. with water ad libitum. 2.5. Effect of compound 48r80-induced paw edema in rats The method of West was used w6x. Rats were injected with 0.1-ml solution of compound 48r80 Ž10 m grpaw. in saline into the subplantar region of the right hind paw. The paw volume was measured before the experiment and 10, 20, 30, 60, 120, 180 min after the injection of compound 48r80 by the volume displacement method. The oil Ž50 and 100 mgrkg. and ketotifen Ž1 mgrkg., as a reference standard, were administered orally 1 h before the induction of inflammation. The effect of the treatment was expressed as % inhibition of inflammation in comparison with the control group which received 10 mlrkg of vehicle wmixture of acacia Ž3%. and tragacanth Ž0.1% of acacia. in distilled waterx. 2.6. Effect on nystatin-induced paw edema in rats The edema was induced according to Arrigoni-Martelli et al. w7x. Rats were injected with 0.1 ml Ž30 000 U. nystatin into the subcutaneous tissue of the right hind paw as an 8.5% suspension in sterile saline. The hind paw volume was measured at 0 h Žbefore injection of nystatin ., 1, 4, 8, 15, 24, 72, 96, 120, 144 and 168 h by the volume displacement method. The oil Ž50 and 100 mgrkg. and dexamethasone Ž0.09 mgrkg., as a reference standard, were administered orally 1 h before the induction of inflammation. The effect of the treatment was expressed as % inhibition of inflammation in comparison with control group which received 10 mlrkg of vehicle Žmixture of acacia and tragacanth in distilled water.. 2.7. Effect on haemolysis The method of Abe et al. was used w8x. 2.7.1. Erythrocyte suspension Whole blood was collected from rats under ether anaesthesia. Heparin was used to prevent clotting. The blood was washed three times with 0.9% saline. The volume of saline was measured and reconstituted as a 40% Žvrv. suspension with isotonic buffer solution ŽpH 7.4. which contained in 1 l of distilled water: NaH 2 PO4 ? 2H 2 O, 0.26 g; Na 2 HPO4 , 1.15 g; NaCl, 9 g Ž10 mM sodium phosphate buffer.. 2.7.2. Heat-induced haemolysis Portions Ž5 ml. of the isotonic buffer containing 50, 100 and 200 m grml of

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alcoholic solution of C. deodara wood oil were put into two duplicate sets of centrifuge tubes. The vehicle, in the same amount, was added to another tube as control. Erythrocyte suspension Ž30 m l. was added to each tube and mixed gently by inversion. One pair of the tubes was incubated at 548C for 20 min in a water bath. The other pair was maintained at 0]58C in an ice bath. The reaction mixture was centrifuged for 3 min at 1300 g and the absorbance ŽO.D.. of the supernatant was measured at 540 nm using Shimadzu UV-160A spectrophotometer. Acetyl salicylic acid ŽASA. 200 m grml, was used as a reference standard. 2.7.3. Hypotonic solution-induced haemolysis The isotonic buffer solution was composed of 154 mM NaCl in 10 mM sodium phosphate buffer ŽpH 7.4.. The experiments were carried out in duplicate pairs. Stock erythrocyte suspension Ž30 m l. was mixed with 5 ml of the hypotonic solution containing the C. deodara wood oil at concentrations of 50, 100 and 200 m grml, while the control sample was mixed with drug free solution. The mixtures were incubated for 10 min at room temperature, and centrifuged for 3 min at 1300 g and the absorbance ŽO.D.. of the supernatant was measured at 540 nm. ASA Ž200 m grml., was used as a reference standard. 2.7.4. Calculation The percentage inhibition or acceleration of haemolysis in tests (b) and (c) was calculated according to the equation: % acceleration or inhibition of haemolysis s 100x 1 y

OD 2 y OD1 OD 3 y OD1

Where OD1 s test sample unheated or in isotonic solution; OD 2 s test sample heated or in hypotonic solution; and OD 3 s control sample heated or in hypotonic solution. 2.8. Statistical analysis Data were statistically analyzed by Student’s t-test.

3. Results and discussion Cedrus deodara wood oil Ž50 and 100 mgrkg, p.o.. significantly inhibited the edema formation induced by compound 48r80. The effect was found inferior to that of ketotifen at 1 mgrkg, p.o. ŽFig. 1.. Compound 48r80 is one of the more potent releasers of histamine and other mediators of inflammation from mast cell granules, which on release lead to immediate inflammatory response w9x. The


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Fig. 1. Effect of C. deodara wood oil on the time of course of compound 48r80-induced paw edema in U rats. Values are mean " S.D., n s 6; P - 0.05 vs n control, Student’s t-test.

inhibition of compound 48r80-induced edema is suggestive of its probable mast cell stabilization activity. Cedrus deodara wood oil Ž50 and 100 mgrkg, p.o.. significantly inhibited the edema due to nystatin in a dose-dependent manner ŽFig. 2.. Nystatin, a polyene antibiotic induces edema by its membrane labilizing action, thereby releasing hydrolytic enzymes which play an important role in promoting the inflammation. It

Fig. 2. Effect of C. deodara wood oil on the time course of nystatin-induced paw edema in rats. Values U are mean " S.D., n s 6; P - 0.05 vs n control, Student’s t-test.


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Table 1 Effect of C. deodara wood oil on heat-induced and hypotonic solution-induced haemolysis of erythrocyte membrane Treatment

Concentration Ž m grml.

Haemolysis inhibition Ž%. Heat-induced

Hypotonic solution-induced

Control ŽVehicle 10 mlrkg, p.o..

]

]

]

C. deodara

50 100 200

27.18 " 1.15U 48.25 " 0.84U 63.77 " 0.82U

73.15 " 0.53U 77.17 " 0.08U 79.18 " 0.03U

ASA

200

25.12 " 1.72U

76.23 " 1.35U

Values are mean " S.D., n s 6; U P - 0.001 vs. control, Student’s t-test.

has also been demonstrated that prostaglandins are also involved in this edema. Inhibition of nystatin-induced edema suggests a possible stabilizing action on lysosomal membranes and inhibition of prostaglandin synthesis. These results confirm the already reported anti-inflammatory activity of C. deodara wood oil on carrageenan-induced paw edema in rats w3x. To confirm the membrane stabilizing action of C. deodara wood oil observed in the above two models, experiments were performed on the erythrocyte membrane. Cedrus deodara was found to inhibit the haemolysis of erythrocytes induced by both hypotonic solution and heat ŽTable 1.. A possible explanation for the stabilizing activity of C. deodara oil could be an increase in the surface arearvolume ratio of the cells which could be brought about by an expansion of membrane or shrinkage of the cell, and an interaction with membrane proteins w8x. Moreover, it has also been shown that the deformability and cell volume of erythrocytes is closely related to the intracellular content of calcium. Hence, it may be speculated that the cytoprotective effect on erythrocyte membrane may be due to the ability of the test oil to alter the influx of calcium into the erythrocytes. The present investigation suggests that the membrane stabilizing activity of C. deodara wood oil may be playing a significant role in its anti-inflammatory activity.

Acknowledgements Thanks are due to Mrs Vaishali Dikshit, Dr Mandar Kodgule and Dr Shridhar Narayan for their help and advice.

References w1x Gulati BC. Cultivation and utilization of aromatic plants. In: Atal CK, Kapur BM, editors. Regional Research Laboratory, Jammu-Tawi, India, 1977:640]652.


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w2x Kirtikar KR, Basu BD. Indian medicinal plants, vol. III. 2nd ed. Lalit Mohan Basu, Allahabad, India, 1933:2390]2392. w3x Shinde UA, Phadke AS, Nair AM, Mungantiwar AA, Dikshit VJ, Saraf MN. J Ethnopharmacol, in press. w4x Kar K, Puri VN, Patnaik GK et al. J Pharm Sci 1975;64:258. w5x Puri VN, Kar K, Patnaik GK, Dhawan BN, Kulshreshtha BK, Rastogi RP. Ind J Exp Biol 1975;13:369. w6x West GB. Int Archs Allergy Appl Immunol 1983;72:284. w7x Arrigoni-Martelli E, Schiatti P, Selva D. Pharmacology 1971;5:215. w8x Abe H, Katada K, Orita M, Nishikibe M. J Pharm Pharmacol 1991;43:22. w9x Ghooi RB, Rath S, Bhinde MB. Asp Allergy Appl Immunol 1980;13:41.