Study of anti oxidant and anti-inflammatory activity from ethanol fraction of Thuja occidentalis Linn.

Dubey SK¹* and Batra A²

¹Sanjeevan College of Pharmacy, Dausa (Raj.) India.

²Dept. of Botany, University of Rajasthan, Jaipur (Raj.) India.

ABSTRACT

The present study was carried out to evaluate the antioxidant (in vitro) and anti-inflammatory (in vivo) activities of ethanol fraction of Thuja occidentalis Linn. (Cupressaceae). 1, 1-Diphenyl-2-picryl-hydrazyl (DPPH) radical, superoxide anion radical, hydroxyl radical scavenging, nitric oxide scavenging and lipid peroxidation were carried out to evaluate the antioxidant potential of the extract. Anti-inflammatory activity was performed by carrageenan induced paw oedema and cotton pellet granuloma models. Both the in vitro and in vivo were found significant.

Keywords: Thuja occidentalis, Anti-inflammatory, Antioxidant activity.

INTRODUCTION

Thuja occidentalis, commonly known as Arbor vitae or white cedar, is indigenous to North America and is grown in Europe as an ornamental tree. In folk medicine, Thuja occidentalis has been used to treat bronchial catarrh, enuresis, cystitis, psoriasis, uterine carcinomas, amenorrhea and rheumatism (1). Extract of this plant has shown anti viral, anti diarrhoeal activity (2, 3). It has been reported to increase the proliferation of spleen cells as well as increase in TNF-α, IL-6 and IL-1 activity in serum and also have protective effect against radiation-induced toxicity (4). Today it is mainly used in homeopathy as mother tincture or dilution. The aim of the present investigation was to evaluate the possible anti-inflammatory activity of Thuja occidentalis aerial part based on antioxidant activity.

MATERIALS AND METHODS

Plant material

Fresh aerial part (twigs) of Thuja occidentalis were collected from Jaipur, Rajasthan, India, in October-2007 and were authenticated by experts of Deptt. Of Botany University of Rajasthan, Jaipur. The voucher specimen is preserved for further research in our laboratory.

Preparation of extract

Shade dried and powdered twigs (40-mesh size, 1kg) were soxhlet extracted with 90% EtOH (Dept. of Botany), the solvent was removed and the residue was triturated with hot (65⁰C) petroleum ether (60-80⁰C). Solvent was evaporated from the petroleum ether soluble portion and the residue dissolved in ethanol. On removal of the ethanol by evaporation, a semi solid reddish brown mass (12.76g) was obtained. This fraction is abbreviated as EFTO (Ethanolic Fraction of Thuja occidentalis ).

Phytochemical investigations showed the presence of flavonoids (quercetin, kaempherol), tannic acids, polysaccharides and proteins.

Chemicals

Naphthylethylenediamine dihydrochloride,Thiobarbituric acid was obtained from Loba Chemie, India. Carrageenan, 1,1-Diphenyl-2-picryl hydrazyl (DPPH), NADH and Nitroblue tetrazolium (NBT) were obtained from Sigma Chemicals, St. Louis, USA. Deoxy ribose was obtained from Merck India.

Diclofenac sodium (Novartis), Dimethyl sulphoxide, ethylene diamine tetra acetic acid (EDTA), ferrous sulphate, trichloro acetic acid, Hydrogen peroxide (H₂O₂), Ascorbic acid, Mannitol, Potassium dihydrogen phosphate, potassium hydroxide, phenazine methosulfate were of analytical grade and obtained from Ranbaxy fine chemicals.

Animals

Wistar albino rats of either sex, weighing 175-220 g, provided by the Sanjeevan college of Pharmacy, Dausa, Rajasthan, India, were used. Animals were maintained under standard environmental conditions and had free access to standard pellet food (Hindustan lever, India) and water. The animals were maintained as per the norms of IAEC and the experiments were cleared by IAEC and the local institutional ethical committee.

Determination of Anti oxidant activity

Assay of lipid per oxidation

The extent of lipid peroxidation in goat liver homogenate was measured in vitro in terms of formation of thiobarbituric acid reactive substances (TBARS) by using standard method⁵with minor modifications⁶ with the help of spectrophotometer (Shimadzu model 1601).

Goat liver was purchased from local slutter house. Its lobes were dried between blotting papers (to remove excess blood) and were cut into small pieces with a heavy-duty blade. They were then homogenized in glass-Teflon homogenizing tubes in cold phosphate buffer saline (pH 7.4). It was centrifuged at 2000rpm for 10 min, and supernatant was diluted with phosphate buffer saline up to final concentration of protein 0.8-1.5 mg/0.1ml. protein concentration was measured by using standard method of Lowery et.al.⁷ to study the comparative response, the experiments was divided into seven groups. Liver homogenates (5%, 3ml) was aliquoted to seven different 35mm glass Petri dishes. The first two groups were treated as control and standard where buffer and Vit. E were added. In the third to seventh group, different concentrations of EFTO were added. Lipid peroxidation was initiated by adding 100µl of 15mM ferrous sulphate solution to 3 ml of liver homogenate. After 30 min, 100µl of this reaction mixture was taken in a tube containing 1.5ml of 10% trichloro acetic acid. After 10 min, tubes were centrifuged and supernatant was separated and mixed with 1.5ml of 0.67% thio barbituric acid. The mixture was heated in a water bath at 85⁰C for 30 min, and in boiling water bath to complete the reaction. The intensity of pink colored complex formed was measured at 535 nm .

The percentage of inhibition of lipid peroxidation was calculated by compring the results of the test with those of controls as per the following formula i.e. Eq. (1);

Inhibition(%) = (Control Absorbance- Test Absorbance) X 100/Control absorbance. ______Eq. (1)

DPPH radical scavenging activity

DPPH scavenging activity was measured by spectrophotometric method at 517 nm ⁸. To a methanolic solution of DPPH (100µM, 2.95 ml), 0.05 ml of EFTO and standard compound Vit. E were added in different concentrations. Equal amount of Methanol (0.05ml) was added to a control. After 30 min. absorbance was measured in triplicate. The percentage of scavenging was calculated by comparing the control and test samples with the Eq. (1).

Hydroxyl radical scavenging activity

Hydroxyl radical scavenging activity is a competition between deoxyribose and EFTO for hydroxyl radical generated by the Fe³⁺- ascorbate-EDTA-H₂O₂ system (Fenton reaction) according to the method of Kunchandy and Rao ⁹. The reaction mixture containing final volume of 1.0 ml, 100µl 2-deoxy-ribose, 500µl of the various concentrations of EFTO and standard compound (Mannitol 50mM) in KH₂PO₄-KOH buffer (20mM, pH 7.4), 200µl 1.04mm H₂O₂ and 100µL 1.0mM ascorbic acid was incubated at 37⁰C for 1 hour. One milliliter 1% trichloro acetic acid was added to each test tube and incubated at 100⁰C for 20 min. after cooling at room temperature; absorbance was measured at 532nm against a control preparation containing deoxyribose and buffer. Percent inhibition was determined by comparing the results of tests and control samples with the above mentioned Eq. (1)

Super oxide anion scavenging activity

The superoxide scavenging activity of EFTO was determined by the method described by Nishimik et.al¹⁰. with slight modification. About 1.0ml NBT solution containing 156µM NBT dissolved in 1.0 ml 100mM phosphate buffer , pH 7.4, 1.0 ml NADH solution containing 468µM NADH dissolved in 1.0 ml 100mM phosphate buffer, pH 74., and 0.1 ml of various concentrations of EFTO and standard compound Vit. E was mixed and the reaction was started by adding 100µl of phenazine methosulfate solution containing 60µM phenazine methosulfate in 100mM phosphate buffer, pH 7.4. The reaction mixture was incubated at 25⁰C for 5 min, and absorbance at 560nm was measured against control sample. Percent inhibition was determined by comparing the results of the test and control samples with above mentioned Eq. (1)

Nitric oxide scavenging activity

Sodium nitropruside (10mM) in phosphate buffered saline was mixed with different concentrations of EFTO (100, 150, 200, 250 and 300µg) dissolved in methanol and incubated at room temperature for 150 min. The same reaction mixture without the sample but with equivalent amount of solvent served as control. After, the incubation period , 0.5ml of Griess reagent (1% sulphanilamide, 2% H₃PO₄ and 0.1% naphthylethylenediamine dihydrochloride) was added. The absorbance of the chromophore formed during diazotization of nitrite with sulphanilamide and subsequent coupling with naphthylethylenediamine , was read at 546nm. Curcumin was used as positive control ¹¹ .

Anti inflammatory activity

Carrageenan induced paw oedema

Inflammation was induced by injecting 0.05ml of 1% carrageenan sodium salt (Sigma) subcutaneously in the sub-plantar region of the rat right hind paw

Table -1; Effect of ethanol fraction of Thuja occidentalis (EFTO) on different antioxidant models.

Concentration (µg/ml)	% Inhibition
Concentration (µg/ml)	Lipid peroxidation	DPPH	Hydroxyl radical	Superoxide radical	Nitric oxide scavenging
300	61.52±0.13*	73.35±1.04*	80.38±2.28*	89.79±0.29*	71.55±0.95*
250	58.17±0.33*	62.66±1.34*	79.54±1.24*	86.05±.24*	61.95±0.85*
200	57.39±0.65*	50.40±1.73*	65.37±2.27*	83.52±0.14*	58.54±0.23*
150	42.14±0.37	43.51±2.41	46.28±1.89	50.06±0.62*	52.98±0.66*
100	33.95±1.80	16.82±2.75	32.14±2.24	41.38±0.56	38.34±1.45
Vitamin E(5mM)	68.32±0.42*	67.29±0.71*	--NT--	70.39±0.13*	--NT--
Mannitol(50mM)	--NT--	--NT--	89.64±1.10*	--NT--	--NT--
Curcumin	--NT--	--NT--	--NT--	--NT--	85.38±0.22*
IC₅₀ (µg/ml)	195.60	202.46	158.59	124.11	155.84

Values are mean ±SEM of three replicates. (NT- Not Tested) *P<0.05 VS control; student’s t-test.

Table 2: Anti-inflammatory effects of EFTO (ethanolic fraction of Thuja occidentalis Linn.) on Carrageenan-induced rat paw oedema.

Treatment	Dose (mg/kg; i.p)	Percentage of hind paw oedema at time (h) after carrageenan
Treatment	Dose (mg/kg; i.p)	0h	1h	2h	3h	4h	6h	24h
Control	Water 10 ml/kg. i.p	100.00±2.80	170.10 ±5.54	210.50 ±7.25	212.80 ±8.88	215.42 ±6.50	218.00 ±8.12	208.16 ±6.66
EFTO	200	100.00±3.82	118.70 ±4.35*	129.69 ±5.55*	143.53 ±2.50*	152.65 ±2.69*	157.65 ±2.47*	130.40 ±2.50*
EFTO	400	100.00±4.23	109.89 ±2.30*	115.65 ±1.60*	116.16 ±2.30*	113.56 ±2.65*	112.46 ±2.79*	111.35 ±5.29*

Values are mean ±S.E (n=6); *P<0.05 vs control: student‘s t-test.

Table 3: Effect of EFTO (ethanolic fraction of Thuja occidentalis) on Cotton Pellet granuloma in rats.

Treatment	Dose (mg/kg. p.o. for 14 days)	Percentage increase of cotton pellet weight
Control	Water (10 ml/kg)	119.5±14.70
EFTO	200	71.6±9.29*
EFTO	400	41.3±3.90*
Diclofenac sodium	4.0 (i.p) once	40.2±3.32*

Values (100 x final weight/initial weight) are mean ± S.E (n=6); *P<0.05 vs control; student’s t-test.

Table- 4: Effect of Ethanolic Fraction of Thuja occidentalis (EFTO) on Peritoneal Permeability Test in rat

Group	Treatment	Dose Mg/kg/day	Absorbance (at 625 nm)	% inhibition
I	Control	--	0.447±0.017	--
II	Aspirin	300	0.355±0.02***	19.29
III	EFTO	200	0.235±0.01***	51.15
IV	EFTO	400	0.093±0.016***	85.91

according to Winter et.al ¹². The EFTO (ethanolic fraction of Thuja occidentalis) (200 and 400 mg/kg) was administered intra peritoneally, 30 min after carrageenan injection while control group received saline (10ml/kg, i.p). The hind paw volume was measured plethysmometrically before and after carrageenan injection, at hourly interval for 6h and then at 24h.

Cotton pellet granuloma

A 20-mg sterilized cotton pellet was implanted subcutaneously on the back of the neck in rats under ether anaesthesia. Animals in treated group received the EFTO (200and 400 mg/kg, p.o), once daily for 14 consecutive days. Animals in the control group received only the vehicle (10ml/kg, p.o). Diclofenac sodium (4mg/kg, i.p) was given as reference drug in a fourth group. On the 14^th day, the animals were sacrificed with ether, the pellet’s granuloma were removed, freed from extraneous tissue, dried overnight at 55±0.5⁰ C, and weighed¹³.

Acetic acid-induced vascular permeability

Four groups of adult rats were designed with six rats in each group weighing 190-225g, Group-I to Group-IV received Vehicle (0.1ml s.c.), Aspirin (300 mg/kg bw s.c.), EFTO (200 mg/kg bw s.c.) and EFTO (400 mg/kg bw s.c.), respectively.

Three hours later, each mice received 4ml of 0.05N acetic acid in 0.9% saline intra peritoneally, followed by 0.1ml of 4% Pontamine sky blue dye intravenously. One hour after the administration of the dye the animals were sacrificed. The peritoneal cavity was opened and drained. The exudates were centrifuged for 10 min at 1500 rpm and 0.5ml of the supernatant was diluted with 4.5ml of physiological saline. Dye concentration was measured in a spectrophotometer at 625nm saline (0.9% NaCl) was used as blank. The mean percentage of light absorption was then calculated for both control and drug treated groups ¹⁴.

STATISTICAL ANALYSIS

Values for anti-inflammatory activity were expressed as "mean increase in paw volume ±SEM". The significance of difference between means was determined by student's t-test values of p<0.05 were considered significant.

RESULTS AND DISCUSSION

Antioxidant activity

Assay of lipid peroxidation

The results presented in Table-1 showed that the ethanol extract of the Thuja occidentalis inhibited FeSO₄ induced lipid peroxidation in a dose dependent manner. The extract at 300µg/ml exhibited maximum inhibition (61.516±0.131 %) of lipid peroxidation nearly equal to the inhibition produced by Vit. C. The IC₅₀ value was found to be 195.60µg/ml.The inhibition could be caused by the absence of ferryl-perferryl complex or by changing the ratio of Fe³⁺/Fe²⁺ or by reducing the rate of conversion of ferrous to ferric or by changing the iron itself or combination thereof ¹⁵.

DPPH scavenging activity

DPPH is a stable free radical that can accept an electron or hydrogen radical to become a diamagnetic molecule. Due to its odd electron, the methanolic solution of DPPH shows a strong absorption at 517nm. DPPH radical reacts with suitable reducing agent, then electrons become paired off, and the solution loses color stoichometrically with the number of electrons taken up ¹⁶. Such reactivity has been widely used to test the ability of compound/plant extracts to act as free radical scavengers. Reduction of the DPPH radicals can be observed by the decrease in absorbance at 517nm. The DPPH scavenging activity of the extract was found to be 73.346±1.040 % at 300µg/ml compared with standard drug Vit. C at 5mM 78.296 ±0.708. The IC₅₀ value was found to be 202.457µg/ml.

Hydroxyl radical scavenging activity

Hydroxyl radicals are the major active oxygen species causing lipid peroxidation and enormous biological damage ¹⁷. Ferric-EDTA was incubated with H₂O₂ and ascorbic acid at pH 7.4. Hydroxyl radicals were formed in free solution and were detected by their ability to degrade 2-deoxy-2-ribose into fragments that formed a pink chromogen upon heating with TBA at low pH ¹⁸. When the test compound were added to the reaction mixture they removed hydroxyl radicals from the sugar and prevented their degradation. The ethanol extract of Thuja occidentalis significantly inhibited (80.38±2.28%) degradation of deoxy-ribose mediated by hydroxyl radicals at the dose of 300µg/ml (Table-1), compared to that of known scavenger Mannitol (50mM). the concentration of ethanol extract of Thuja occidentalis needed for 50% inhibition was 158.59µg/ml.

Superoxide scavenging activity

Superoxide radical O₂ is a highly toxic species and is generated by numerous biological and photochemical reactions. Both aerobic and anaerobic organisms possess superoxide dismutase enzymes that catalyze the breakdown of superoxide radical ¹⁹. Reduced phenazine methosulfate assay was used to measure the superoxide dismutase activity of EFTO. The results presented in Table -1 showed that the scavenging activity of the extract was 89.79±0.296% at 300µg/ml and IC₅₀ was found to be 124.11µg/ml.

Nitric oxide scavenging activity

EFTO (ethanolic fraction of Thuja occidentalis) showed moderate NO scavenging activity, with IC₅₀ of 155.85µg/ml and has its activity was comparable with that of curcumin (Table-1). The role of free radical oxidative stress in various disease conditions has been well established²⁰. Herbal drug containing radical scavenger activity are well known for their therapeutic activity²¹ .The role of free radical in inflammation particularly of NO scavenging activity is known well-documented²². Overproduction of NO is known to be an important mediator of inflammation state²³. Therefore, the extract was studied for anti-inflammatory activity.

The EFTO (ethanolic fraction of Thuja occidentalis) in the dose of 200 and 400 (mg/kg, i.p), significantly reduced the increase in hind paw oedema induced by carrageenan, with effects starting from 1h after carrageenan administration and lasting for 24h (Table-2).

The sub-acute oral treatment with EFTO (ethanolic fraction of Thuja occidentalis), at the same doses used (200and400 mg/kg, p.o for 14 days), significantly reduced the increase in weights of cotton pellets, compared with the control group (Table-3).

The results of the present study show that the EFTO (ethanolic fraction of Thuja occidentalis) possess anti-inflammatory properties, demonstrated following single intra peritoneally administration (carrageenan induced hind paw oedema) and repeated oral administration (cotton pellet granuloma). Further, studies are needed to elucidate the mode of action and to isolate and characterize the active constituent responsible for this property. This study also confirmed that the EFTO (Ethanolic Fraction of Thuja occidentalis) decreased the permeability of small vessels as evident from the inhibitory effect observed on dye leakage (Table-4) presumably by causing a reduction in peritoneal capillary permeability.

REFERENCES:

1) L.C.Chang , L.L Song , E.J.Park , L. Luyengi , K.J. Lee and R. Norman, Journal of Natural Product., 63, 1235 (2000).

2) S.H. Nam and M.Y. Kang, Pharmaceutical Biotechnology., 42, 409 (2005).

3) L. Deb , S.K. Dubey , A.K. Jain , A. Jain , G.S. Pandian and S.P. Rout, Indian Drugs., 44,319 (2007).

4) N. Belal , C. Bodinet , M. Tegtmeier and U. Lindequist, Advance Access Publication eCAM., 69 (2005).

5) H. Ohkawa , N. Oshishi and K. Yagi, Anal Biochem., 95, 351 (1979).

6) S. Pandey, M. Sharma, P. Chaturvedi and Y.B. Tripathi, Experimental journal of Biology., 193,265 (1995).

7) O.H. Lowery , N.J. Rosenbrough , A.L. Farr and R.J. Randall, Biol. Chem., 193, 265 (1951).

8) N. Sreejayan and M.N.A. Rao, J. Pharm. Pharmacology.,58,237(1990).

9) E. Kunchandy andM.N.A. Rao, (Int. J. Pharmco.,. 58, 237 (1990).

10) M. Nishimiki , N.A. Rao , N. Appaji and K. Yagi, Biochem. Biophy. Res. Comm., 46, 849 (1972).

11) N. Sreejayan and M. N. A. Rao, Journ. Of Pharmacy and Pharmacology., 49, 105(1997).

12) C. A. Winter, E.A. Risley and G.W. Nuss, Proceedings of the Society for Experimental Biology and Medicine., 111, 544 (1962).

13) P.F. D’Arcy, E.M. Haward, R.W. Muggleton and S.B. Townsend, J. Pharm. And Pharmacology., 12,659 (1960).

14) NagChaudhari, A.K., Pal, S., Gomes,A. and Bhattacharya, S.1990.Antiinflammatory and related action of Syzygium cumini seed extracts. Phytotherapy Res. 4:5-10.

15) J.M. Braugghler , C.A. Duncan and I.R.Chase, J. Biol. Chem., 261, 10282(1986).

16) M.S. Blois, Nature., 29,1199 (1958).

17) L.W. Aurand , N.H. Boonnmen and G.G. Gidding, J Diar Sci ., 60, 363 (1977).

18) O.I. Aruoma , M.J. Laughton and B. Halliwell , Biochem J., 264,863 (1989).

19) R. Govindarajan , M. Vijaykumar , A.K.S. Rawat and S. Mehrotra, Ind. J. Exp. Bio. , 41, 875 (2003).

20) T.P.A. Devasagayam, J.P. Kamat, H. Mohan and P.C. Kesavan, Biochem Biophys acta.,63, 1282 (1996).

21) R. Chander, V. Srivastava, J.S. Tendon and N.K. Kapoor, Int. J. Pharmacog., 33, 135 (1995).

22) A. Moneada, R.M.J. Palmer and E.A Higgs, Pharmacol Rev., 43, 109 (1991).

23) E.J.Moody, B.A.Simon and R.A. Johns, Goodman and Gilman’s ‘The Pharmacological basis of Therapeutics’ , McGraw - Hill Medical Publication Division, New York, p394. (2001).

Received on 04.06.2009

Accepted on 22.07.2009

Research J. Science and Tech. 1(1): July-Aug. 2009: 39-42