Journal of Phytology 2011, 3(1): 33-38 ISSN: 2075-6240

Phytopharmacology Available Online: www.journal-phytology.com

REGULAR ARTICLE

EVALUATION OF ANALGESIC AND ANTI-INFLAMMATORY

ACTIVITIES OF TERMINALIA ARJUNA LEAF
Moulisha Biswas1, Kaushik Biswas2, Tarun K Karan3, Sanjib Bhattacharya4, Ashoke K
Ghosh5 , Pallab K Haldar3*
1Bengal
Institute of Pharmaceutical Sciences, Kalyani, Nadia 741235, India
2BCDA
College of Pharmacy & Technology, Hridaypur, Kolkata 700127, India
3Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032, India
4Bengal School of Technology, Sugandha, Hooghly 712102, India
5College of Pharmacy, IFTM, Moradabad, Uttar Pradesh 244001, India

SUMMARY
Terminalia arjuna Roxb. (Combretaceae), commonly known as Arjuna is traditionally
used for several medicinal purposes in India. The present study assessed analgesic and
anti-inflammatory activities of its leaf. Dried and crushed leaves of Terminalia arjuna were
defatted with petroleum ether and then extracted with methanol. The methanol extract
(META) at the doses of 100 mg/kg and 200 mg/kg body weight was subjected for
evaluation of analgesic and anti-inflammatory activities in experimental animal models.
Analgesic activity was evaluated by acetic acid-induced writhing, hot plate and formalin
tests in Swiss albino mice; and acute anti-inflammatory activity was evaluated by
carrageenan, histamine and dextran-induced paw oedema in Wister albino rats. Aspirin
and indomethacin were employed as reference drugs for analgesic and anti-
inflammatory studies respectively. In the present study, the methanol extract of the
leaves of Terminalia arjuna demonstrated significant analgesic and acute anti-
inflammatory activities in the tested models.
Key words: Terminalia arjuna Roxb., Analgesic, Anti-inflammatory, Pain
Moulisha Biswas et al. Evaluation of analgesic and anti-inflammatory activities of Terminalia arjuna leaf. J Phytol 3/1 (2011) 33-38.

*Corresponding Author, Email: [email protected], Tel.: + 91 9433230566; Fax: + 91 3324146046

1. Introduction
Terminalia arjuna Roxb. (Combretaceae), calcium, 13% tannin and aluminum,
commonly known as Arjuna is traditionally magnesium, organic acids, colouring matter
used for several medicinal purposes in India. and other substances [1]. In Indian
It is a large tree, often with buttressed trunk, traditional medicine, the fruits of the plant
smooth grey bark and about 20 - 25 m in are used as a tonic [2]. Externally, its leaves
height. Its leaves are usually sub-opposite, are used as a cover on sores and ulcer. The
oblong or elliptic-long, pale dark green bark is anti-dysenteric, antipyretic, astringent,
above and pale-brown beneath, 10-20 cm cardiotonic, lithotriptic and tonic while the
long and hard. The flowers are yellowish- powder of the bark acts as a diuretic in
white while the fruits are 2.5-5.0 cm ovoid or cirrhosis of liver and gives relief in
ovoid-oblong, fibrous-woody, and glabrous. symptomatic hypertension [3]. A decoction
It is common on the banks of rivers, streams of the thick bark made with milk is given
and dry watercourses in sub-Himalayan tract, every morning on an empty stomach or its
West Bengal as well as in central and South powder with milk and gurh as a cardiotonic
India. The bark of the plant is known to [4]. The bark powder is also given with
contain a crystalline compound, arjunine, a honey in fractures and contusions with
lactone, arjunetin, essential oil and reducing echymosis. Furthermore, the extract of the
sugar. Besides these, it also contains 34 % bark, as an astringent, is used for cleaning
calcium carbonate, 9% of other salts of sores, ulcers and cancers, etc. An ointment
 Moulisha Biswas et al./J Phytol 3/1 (2011) 33-38

made from the bark by mixing with honey is water ad libitum. The mice were
used to cure acne while the ashes of the bark acclimatized to laboratory conditions for 7
are prescribed in scorpion stings [5]. The days before commencement of the
present work was undertaken to evaluate the experiment. All experimental procedures
methanol extract of the leaves of T. arjuna for were reviewed and approved by University
its analgesic and anti-inflammatory activity. Animal Ethics Committee, Jadavpur
University.
2. Materials and Methods
Plant material Evaluation of analgesic activity
The leaves of Terminalia arjuna were Acetic acid induced writhing test
collected in January 2008 from Nadia, West Swiss albino mice were divided into four
Bengal, India. The plant material was groups (n = 6). Group I received acetic acid
taxonomically identified by Dr. Lakhmi (1% v/v, 10 ml/kg b.w., i.p.) and writhing
Narashimhan, Scientist, Botanical Survey of reflex was noted for the period of 15 minutes.
India, Central National Herbarium, Howrah, Group II received aspirin (100 mg/kg b.w.
West Bengal, India. The voucher specimen p.o.) Group III and IV received META at the
[CNH/I-I/(216)/2008/Tech.II/216] and the doses of 100 mg/kg and 200 mg/kg b.w., p.o.
herbarium were preserved in the laboratory respectively. 30 min after aspirin and META
of Bengal Institute of Pharmaceutical administration, group II and III received
Sciences, Kalyani 741235, India for future acetic acid (1% v/v, 10 ml/kg b.w., i.p.) and
reference. writhing reflex was noted for the period of 15
min [7, 8].
Extraction
The powdered plant material (400 g) was Hot plate assay
macerated at room temperature (24-26ºC) The hot plate test was carried out as
with methanol (850 ml) for 4 days with described by previous workers [7]. Four
occasional shaking, followed by re- groups of mice (n = 6) were treated orally
maceration with the same solvent for 3 more with META (100 and 200 mg/kg b.w. p.o.),
days. The macerates were combined, filtered morphine sulphate (10 mg/kg b.w. p.o.) and
and distilled off in reduced pressure. The normal saline (5 ml/kg b.w.). Mice were
resulting concentrate was vacuum dried at placed on a hot plate (Bibby Sterilin, UK)
40°C to yield the dry extract (META, yield: maintained at 55 ± 1°C and the reaction
21.45% w/w). The dry extract was kept in a latency (in seconds) for licking of hind paw
vacuum desiccator until use. Preliminary or jumping noted. The mice which reacted
phytochemical studies of META revealed the within 15 sec and which did not show large
presence of alkaloids, triterpenoids, tannins variation when tested on four separated
and flavonoids [6]. occasions were selected for studies.
Recordings were taken before treatment with
Animals the different drugs and 1, 2, 3, 4, 5 h post
Adult male Swiss albino mice of about 2 treatment. Results were expressed as the
months of age weighing 20 ± 2 g were difference between the baseline reaction
obtained from the Laboratory Animal Centre, latency and the reaction latency at recorded
Department of Pharmaceutical Technology, times.
Jadavpur University, Kolkata, India. The
mice were grouped and housed in Formalin assay
polyacrylic cages (38×23×10 cm) with not The formalin test was carried out as
more than four animals per cage and described by previous workers [9]. Four
maintained under standard laboratory groups of mice (n = 6) were treated orally
conditions (temperature 25 ± 2ºC with with META (100 and 200 mg/kg), aspirin
dark/light cycle 12/12 h). They were (100 mg/kg) and normal saline (5 ml/kg
allowed free access to standard dry pellet b.w.). Formalin solution (1% v/v) was
diet (Hindustan Lever, Kolkata, India) and injected into the sub-plantar region of the
right hind paw of the animals 30 min post-
 Moulisha Biswas et al./J Phytol 3/1 (2011) 33-38

treatment. The number of times paw was Histamine induced rat paw oedema
licked/bitten within the time frames of 0-5 The rats were divided into four groups
min (neurogenic phase) and 15-30 min containing six rats in each group. 0.1 ml of
(inflammatory phase) after formalin 1.0% histamine sulphate in normal saline
administration was counted. (0.9% w/v NaCl) was injected to the sub
plantar region of right hind paw. The META
Evaluation of anti-inflammatory activity was administered to the rats 1 h before
Carrageenan induced rat paw oedema histamine injection. Different groups were
The rats were divided into four groups treated as follows:
containing six rats in each group. 0.1 ml of Group I: Histamine (0.1 ml of 1.0%
1.0% carrageenan in normal saline (0.9% w/v histamine/rat to the sub plantar region).
NaCl) was injected to the sub plantar region Group II: Histamine + Indomethacin (10
of right hind paw. The META was mg/kg b. w., p. o.)
administered to the rats 1 h before Group III and IV: Histamine + META
carrageenan injection. Different groups were (100 mg/kg and 200 mg/kg b.w., p.o.
treated as follows: respectively). The paw volume was
Group I: Carrageenan (0.1 ml of 1.0% measured initially and at 1, 2, 3 and 4 h after
carrageenan/rat to the sub plantar region). histamine injection, using Plethysmograph,
Group II: Carrageenan + Indomethacin inflammation was calculated for comparison
(10 mg/kg b. w., p. o.) [10, 11].
Group II and IV: Carrageenan + META
(100 mg/kg and 200 mg/kg b. w., p. o. Statistical analysis
respectively). The paw volume was The values are represented as mean ±
measured initially and at 1, 2, 3 and 4 h after standard error of mean (SEM). Statistical
carrageenan injection, using Plethysmograph, significance was analyzed by One way
inflammation was calculated for comparison ANOVA with Tukey-Kramer multiple
[10, 11]. comparison post test. P values of < 0.001
were considered as statistically significant.
Dextran induced rat paw oedema
The rats were divided into four groups 3. Results and Discussion
containing six rats in each group. 0.1 ml of The present study evaluated the
1.0% dextran in normal saline (0.9% w/v analgesic and anti-inflammatory activities of
NaCl) was injected to the sub plantar region methanol extract from T. arjuna leaf in
of right hind paw. The META was experimental rodent models. The results are
administered to the rats 1 h before dextran presented in Tables 1-5. The acetic acid
injection. Different groups were treated as induced writhing test is normally used to
follows: evaluate the peripheral analgesic effect of
Group I: Dextran (0.1 ml of 1.0% drugs and chemicals. The response is
dextran/rat to the sub plantar region). thought to be mediated by peritoneal mast
Group II: Dextran + Indomethacin (10 cells, acid sensing ion channels and the
mg/kg b. w., p. o.) prostaglandin pathway [14, 15]. Therefore, it
Group III and IV: Dextran + META (100 may be inferred that the inhibitory effect of
mg/kg and 200 mg/kg b.w., p.o. the compound could be due to the inhibition
respectively). The paw volume was of prostaglandin pathway. Effectiveness in
measured initially and at 1, 2, 3 and 4 h after the hot plate test, on the other hand,
dextran injection, using Plethysmograph, indicated central analgesic action of META.
inflammation was calculated for comparison
[10, 11].
 Moulisha Biswas et al./J Phytol 3/1 (2011) 33-38

Table 1. Analgesic effect of META on acetic acid induced writhing in mice

Treatment Dose Mean no of % Inhibition
writhing ±SEM
Acetic acid (1% v/v) 10 ml/kg 52.83 ±1.400 -
Acetic acid + Aspirin 100 mg/kg 17.66 ±1.606* 66.57
Acetic acid + META 100 mg/kg 26.00 ±1.261* 50.79
Acetic acid + META 200 mg/kg 21.05±1.371* 60.16
Values are mean ± SEM (n = 6). * p < 0.001 when compared to control

Table 2. Analgesic effect of META on hot plate test in mice

Treatment Dose % Inhibition
Normal saline 5 ml/kg -
Morphine sulphate 10 mg/kg 67.56*
META 100 mg/kg 51.79*
META 200 mg/kg 66.16*
Values are mean ± SEM (n=6). *p < 0.001 when compared to control

Table 3. Analgesic effect of META on formalin induced pain in mice

Treatment Dose % Inhibition
Normal saline 5 ml/kg -----
Aspirin 100 mg/kg 70.55*
META 100 mg/kg 53.74*
META 200 mg/kg 69.18*
Values are mean ± SEM (n = 6). *p < 0.001 when compared to control

Table 4. Anti-inflammatory effect of META on carrageenan induced rat paw oedema

Treatment 1h 2h 3h 4h %Inhibiti
on
Carrageenan (1% 0.73±0.0 1.40±0.57 1.80±0.57 1.66±0.08 -
w/v) 8
Carrageenan + 0.20±0.0 0.50±0.05* 0.36±0.03* 0.23±0.03* 86.12
Indomethacin (10 5*
mg/kg)
Carrageenan + 0.33±0.0 0.58±0.03* 0.45±0.05* 0.36±0.03* 78.34
META (100 mg/kg) 5*
Carrageenan + 0.31±0.0 0.51±0.07* 0.41±0.04* 0.32±0.06* 80.70
META (100 mg/kg) 6*
Values are mean ± SEM (n = 6). *p < 0.001 when compared to control

Table 5. Anti-inflammatory effect of META on dextran induced rat paw oedema

Treatment 1h 2h 3h 4h %
Inhibition
Dextran (1% w/v) 1.13±0.08 1.90±0.57 1.76±0.57 1.67±0.08 -
Dextran + 0.29±0.05* 0.58±0.05* 0.46±0.03* 0.38±0.03* 77.25
Indomethacin (10 *
mg/kg)
Dextran + META (100 0.33±0.05* 0.69±0.03* 0.58±0.05* 0.46±0.03* 72.50
mg/kg)
Dextran + META (200 0.31±0.06* 0.61±0.07* 0.52±0.04* 0.43±0.06* 74.25
mg/kg)
Values are mean ± SEM (n = 6). *p < 0.001 when compared to control
 Moulisha Biswas et al./J Phytol 3/1 (2011) 33-38

Table 6. Anti-inflammatory effect of META on Histamine induced rat paw edema

Treatment 1h 2h 3h 4h %
Inhibition
Histamine (1% w/v) 1.33±0.08 1.96±0.57 1.86±0.57 1.78±0.08 -
Histamine + 0.32±0.05 0.59±0.05* 0.51±0.03* 0.47±0.03* 73.60
Indomethacin (10 *
mg/kg)
Histamine + META 0.41±0.05 0.79±0.03* 0.68±0.05* 0.58±0.03* 67.42
(100 mg/kg) *
Histamine + META 0.38±0.06 0.69±0.07* 0.62±0.04* 0.49±0.06* 72.47
(200 mg/kg) *
Values are mean ± SEM (n = 6). *p < 0.001 when compared to control

The carrageenan-induced hind paw India for the necessary facilities and also to
oedema model in rats is known to be the the authority of Bengal Institute of
acute inflammatory model sensitive to Pharmaceutical Sciences, Kalyani 741235,
cyclooxygenase (COX) inhibitors and has India for great support.
been used to evaluate the effect of non-
steroidal anti-inflammatory agents (NSAID), References
which primarily inhibit the cyclooxygenase 1. Anonymous. 1976. Indian Raw Materials.
involved in prostaglandin (PG) synthesis. In Vol.VI New Delhi. Publication and
case of the time course of oedema Information Directorate. Council of
development in carrageenan induced paw Scientific and Industrial Research, New
edema model in rats is generally two phases Delhi.
are found. The first phase, which occurs 2. Anonymous. 1976. Useful Medicinal
between 0 to 2.5 h of injection of the Plants. Vol. VI. Publications &
phlogistic agent, has been attributed to the Information Directorate. Council of
release of histamine or serotonin. The edema Scientific and Industrial Research, New
volume reaches to its maximum Delhi.
approximately 3 h post treatment and then 3. Chatterjee, A. S.C. Pakrashi. 1994. The
begin to decline. The second phase of Treatise on Indian Medicinal Plants. Vol.
inflammatory reaction which is measured at III. Publication and Information
3h is caused by the release of bradykinin, Directorate. Council of Scientific and
protease, prostaglandin and lysosome [14, Industrial Research. New Delhi.
15]. Therefore, it can be inferred that the 4. Chopra, R.N., S.L. Nayar, I.C. Chopra.
inhibitory effect of the extract on the 1956. Glossary of Indian Medicinal
carrageenan induced inflammation could be Plants.. Council of Scientific and
due to the inhibition of enzyme Industrial Research. New Delhi.
cyclooxygenase leading to inhibition of 5. Chopra, R.N., I.C. Chopra, K.L. Handa,
prostaglandin synthesis. L.D. Kapur. 1958. Indigenous Drugs of
Thus, the results of the present study India. U.N. Dhur & Sons Pvt. Ltd.,
demonstrates that the methanol extract Calcutta.
obtained from leaves of Terminalia arjuna 6. Mukherjee, P.K. 2007. Quality Control of
exhibited analgesic activity and acute anti- Herbal Drugs. Business Horizons
inflammatory activity in the tested models Pharmaceutical Publishers, New Delhi.
which was found to be the most effective at 7. Parkes, M.W., J.T. Pickens. 1965.
higher concentrations employed. However, a Conditions influencing the inhibition of
more extensive study is necessary to analgesic drugs of the response to intra
determine the exact mechanism(s) of action peritoneal injections of
of the extracts and its active compound(s). phenylbenzoquine in mice. Brit. J.
Acknowledgements Pharmacol., 25: 81-87.
The authors are thankful to the authority 8. Biswas, M., K. Biswas, A.K. Ghosh, P.K.
of Jadavpur University, Kolkata 700032, Haldar. 2009. A Pentacyclic triterpenoid
 Moulisha Biswas et al./J Phytol 3/1 (2011) 33-38

possessing analgesic activity from the Roxb. in albino rats. Indian J. Pharmacol.,
fruits of Dregea volubilis. Phcog. Mag., 5: 32: 375-377.
90-92. 12. Biswas, M., K. Biswas, A.K. Ghosh, P.K.
9. Hunskaar S., O.B. Fasmar, K. Hole. 1985. Haldar. 2009. A Pentacyclic triterpenoid
Formalin test in mice: a useful technique possessing analgesic and anti-
for evaluating mild analgesics. J. inflammatory activities from the fruits of
Neurosci. Meth. 14: 69-76 Dregea volubilis: Oriental Pharm. Exp.
10. Biswas, M., K. Biswas, A.K. Ghosh, P.K. Med., 9: 315-319.
Haldar. 2009. A pentacyclic triterpenoid 13. Sutradhar RK, A.M. Rahman, M. Ahmad,
possessing anti-inflammatory activity S.C. Bachar, A. Saha, S.K. Guha. 2006.
from the fruits of Dregea volubilis. Phcog. Bioactive alkaloid from Sida cordifolia
Mag., 5: 64-68. Linn. with analgesic and anti-
11. Majumder, A.M., D.G. Naik, C.N. inflammatory activities. Iranian J.
Dandgee, H.M. Puntambekar. 2000. Anti- Pharmacol. Ther. 5, 175-178.
inflammatory Activity of Curcuma amada