Analgesic Activity of Thuja orientalis Leaves

 

PV Andhale2, RS Jadhav2, RD Bhalke1*, VD Tambe2 and MH Kolhe2

1Dept. of Pharmacognosy, Sanjivani College of Pharmaceutical Education and Research, Kopergaon-423603

2Department of Pharmacognosy, Pravara Rural College of Pharmacy, Pravaranagar, A/P-Loni (413 736), Tal- Rahata, Dist-Ahmednagar. (M.S.)

*Corresponding Author E-mail: rasikabhalke@yahoo.co.in

 

ABSTRACT:

Present study reports analgesic activity of petroleum ether, chloroform, methanolic and aqueous extract of leaves of Thuja orientalis. Hot plate and tail immersion methods were used for evaluation of central analgesic activity and acetic acid induced writhing model was used for evaluation of peripheral analgesic activity. Hot plate acetic acid induced writhing model was used for evaluation of central analgesic activity as well as peripheral analgesic activity. Results indicate that petroleum ether extract of leaves at 50 mg/kg, i.p. dose produced a significant increase in reaction time (P<0.05) in tail immersion method and increased in response latency period (P<0.05) in hot plate method, against standard drug pentazocin. Aqueous extract of leaves of Thuja orientalis significantly (P<0.05) attenuated the number of writhing when compared to standard drug paracetamol in acetic acid induced writhing model.

 

KEYWORDS: Thuja orientalis, hot plate method, tail immersion method, acetic acid induced writhing model.

 


INTRODUCTION:

Thuja orientalis L. Family: Cupressaceae is indigenous to temperate regions of Asia and America and grows wildly in parts of western Himalayas1. It is highly aromatic and resinous shrub that widely cultivated in gardens located in temperate and semi-temperate areas2. Thuja was an old remedy for delayed menstruation also it is a stimulant to smooth muscles such as those of uterus and bronchial passages so it is used for treatment of Bronchitis3 and also as cough suppressant in traditional Chinese medicine4. Externally it is used as a wash for infectious skin disease such as impetigo and scabies5. Previous work on Thuja orientalis L. has focused on the terpenoid and flavonoid composition6-10. The present work evaluates the analgesic activity of various extracts of leaves of Thuja orientalis.

 

MATERIALS AND METHODS:

Plant material:

Leaves of Thuja orientalis were collected in November 2008 from Ahmednagar district of Maharashtra , India and authenticated by Dept. of Botany P.V.P.College Loni.   (Voucher specimen was deposited).

 

Preparation of extracts:

Leaves were shade dried, powdered and subjected to successive solvent extraction in Soxhlet extractor using petroleum ether (60-80oC), chloroform, methanol and water as solvents. The extracts were concentrated by vacuum distillation and then dried in open air4.

 

Animals:

Healthy wistar albino mice of either sex and of approximately the same age, weighing about 20-25 gm were used for study. They were housed in polypropylene cages maintained under standard condition (12hour light/12 hour dark cycle; 30±4°C, 36-60 humidity).

 

The experimental protocol was subjected to the scrutiny of the Institutional Animal Ethical Committee and was cleared by the same before starting.

 

Evaluation of antinociceptive activity:

Hot plate method:

Central analgesic activity of petroleum ether, chloroform, methanolic and aqueous extract of leaves of Thuja orientalis was evaluated using hot plate method. The mice of either sex were divided into six groups of six animals each. The first group served as control and received only vehicle (2% DMF), second group was administered standard drug pentazocine lactate (50mg/kg, i.p.) dissolved in 2% DMF in water for injection. The animals of third to sixth group were treated with petroleum ether, chloroform, methanolic and aqueous extract of leaves of Thuja orientalis (50 mg/kg, i.p.) suspended in 2%DMF in saline water respectively.


Table 1: Effect of various extracts leaves of Thuja orientalis on thermic stimulus-induced pain in mice (Hot plate test).

Treatment

Latency to lick the paws (sec±SEM)

Pre drug reaction time reaction time

30 min

60 min

90 min

120 min

150 min

180min

Pentazocine

7.46± 0.34

11.45±1.07

15.84±0.18

19.09±0.58

6.06±0.78

4.96±0.32

3.84±0.63*

Pet ether

6.57±0.94

12.03±0.5*

19.10±b0.29

16.52±0.35

14.12±0.44

8.95±0.28

2.02±0.16*

Chloroform

3.57±0.94

6.93±0.5*

7.31=b0.29

5.57±0.35

2.65±0.44

2.95±0.28

1.33±0.16

Methanol

4.46±0.5

6.28±0.88

8.98±0.44

3.48±0.37

2.96±0.20*

2.02±0.58

1.92±0.6

Aqueous

4.46±0.5

5.28±0.88

11.98±0.44*

7.48±0.37

3.66±0.20

2.07±0.58*

2.02±0.6

All the values are expressed as mean±SEM; n=6, * P<0.05, significant compared to control. All the extracts and pentazocine were given intraperitoneally at 50 mg/kg .

 

Table2. Effect of various extracts leaves of Thuja orientalis on tail immersion method.

Treatment

(Dose 50 mg/kg i.p.)

Reaction time (Sec)±SEM

Predrug reaction time

30 min

60 min

90 min

120min

150min

180min

Vehicle

(2% DMF)

5.34±0.648

5.65±0.796

5.89±1.238

6.21±0.796

6.13±0.697

6.67±0.593

7.34±0.602

Pentazocine

8.68±0.232*

9.14±0.732*

13.56±0.746*

16.28±0.862*

19.82±0.883*

19.34±0.925*

17.23±0.773*

Pet. ether

6.37±0.641

7.24±0.464

8.87±0.384*

15.56±0.312*

13.42±0.922*

13.76±0.835*

10.45±0.616*

Chloroform

6.29±0.814

9.49±0.377*

10.45±0.174*

11.93±0.892*

10.32±0.353*

816.12±0.645*

4.56±0.404*

Methanol

9.23±0.105*

5.45±0.194*

9.23±0.234*

10.89±0.212*

12.11±0.532*

8.42±0.100*

7.16±0.780*

Aqueous

8.23±0.127*

10.28±0.400*

12.66±0.227*

11.42±0.573*

10.52±0.118*

9.80±0.932*

5.45±0.376*

All the values are expressed as mean ± SEM; n=6, *P<0.01, significant compared to control. All the extracts were given intraperitoneally at 50 mg/kg dose.

 

 


Mice were placed individually on the hot plate maintained at 55±10C. The basal reaction time was noted before and at 30, 60, 90, 120, 150, 180 min after the administration of treatment. The experiment was terminated 20sec after their placement on the hot plate to avoid damage to the paws. Zero minute reading was the pre -drug reaction time5.

 

Tail immersion method:6- 8

Central analgesic activity of petroleum ether, chloroform, methanolic and aqueous extract of leaves of Thuja orientalis was evaluated using tail immersion method. The mice of either sex were divided into six groups of six animals each. The first group served as control and received only vehicle (2% DMF), second group was administered standard drug pentazocine lactate (50mg/kg, i.p.) dissolved in 2% DMF in water for injection. The animals of third to seventh group were treated with petroleum ether, chloroform, methanolic and aqueous extract of leaves of Thuja orientalis (50 mg/kg, i.p.) suspended in 2%DMF in saline water respectively.

 

The lower 5 cm portion of the tail was immersed in water bath maintained at 550C. The time in seconds for mice to withdrawal the tail from the water was recorded as the reaction time, with a cut of period of immersion set as 20 seconds.  The basal reaction time was noted before and at 30, 60, 90, 120, 150, 180 min after the administration of treatment.

 

Acetic acid-induced writhing test:9

Peripheral analgesic activity was evaluated using acetic acid-induced writhing test. Mice of either sex were prescreened 48 hrs before the actual experiment and those sensitive to acetic acid-induced writhing were divided into six groups, of six animals each. . The first group served as control and received only vehicle (2% DMF), second group received the standard drug paracetamol (50mg/kg i.p.) dissolved in 2% DMF in water for injection and animals of third to sixth group were treated with petroleum ether, chloroform, methanolic and aqueous extract of leaves of Thuja orientalis (50 mg/kg, i.p.) suspended in 2%DMF in saline water respectively 30 minutes before intraperitoneal injection of 0.1 ml of 0.6% solution of acetic acid. Mice were placed individually in glass beakers after administration of acetic acid and five minutes were allowed to elapse. The mice were then observed for a further period of 30 minutes and the number of writhes recorded for each animal.

 

Statistical significance:

The results were analyzed for statistical significance using students‘t’ test. P<0.05 was considered as significant.

 

RESULTS AND DISSCUSSION:

Analgesic activity:

The hotplate method and tail immersion test are considered to be selective to examine compounds acting through opioid receptor, the extract increased mean basal latency which indicates that it may act via centrally mediated analgesic mechanism9, 10.

 

Among all the extracts, the petroleum ether extract of leaves at 50 mg/kg, i.p. dose produced a significant increased in response latency period (P<0.05) in hot plate method (Table 1) and increase in reaction time (P<0.05) in tail immersion method (Table 2), against standard drug pentazocin. Thermic painful stimuli are known to be selective to centrally active drugs reference11 . Prostaglandins and bradykinins are suggested to play an important role in analgesia12, 13. Some sterols and triterpenes are responsible for analgesic activity14. As phytochemical tests showed presence of these constituents in petroleum ether extracts, they may be exerting their action by suppressing the formation of prostaglandin and bradykinins or antagonizing their action.

Table 3: Effect of various extracts of Thuja orientalis on acetic acid induced writhing in mice:

Treatment

Number of writhing

Vehicle

62.45±0.341

Paracetamol

35.22±0.729

Pet ether

42.45±0.824

Chloroform

47.07±0.510

Methanol

41.07±0.510

Aqueous

32.00±0.824

All the values expressed as mean±SEM; n=6, P<0.05 significant compared to control. All the extracts were given intraperitoneally at 50 mg/kg dose and paracetamol was given intraperitoneally at 50 mg/kg dose.

 

Aqueous extract of leaves of Thuja orientalis significantly (P<0.05) attenuated the number of writhing when compared to standard drug paracetamol in acetic acid induced writhing model (Table 3). It was observed that onset of writhing was delayed and duration of writhing as shortened. Acetic acid is known to trigger the production of noxious substances within the peritoneum, which induces the writhing response15. The effect of the extracts in reducing writhing may be an indication that they depressed the production of noxious substances. The acetic acid induced writhing response is a sensitive procedure to evaluate peripherally acting analgesics. The response is thought to be mediated by peritoneal mast cells 16, acid sensing ion channels17 and the prostaglandin pathways.

 

CONCLUSION:

Petroleum ether extract of Thuja orientalis showed significant central analgesic activity and it is may be due to presence of sterols, whereas aqueous extract showed peripheral analgesic activity.

 

REFERENCES:

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16.     Ronaldo AR, Mariana LV, Sara MT, Adriana BPP, Steve P, Ferreira SH, Fernando QC Involvement of resident macrophages and mast cells in the writhing nociceptive response induced by zymosan and acetic acid in mice. Eur. J. Pharmacol. 2000; 387: 111-118.

17.     Voilley N Acid-Sensing Ion Channels (ASICs): New targets for the analgesic effects of Non-Steroid Anti-Inflammatory Drugs (NSAIDs). Curr. Drug Targets- Inflam. Aller. 2004; 3: 71-79.

 

 

 

 

Received on 25.09.2009       Modified on 15.11.2009

Accepted on 19.12.2009      © RJPT All right reserved

Research J. Pharm. and Tech. 3(2): April- June 2010; Page 435-437