Anti-inflammatory Activity of 80% Ethanolic Extract of Acorus calamus Linn. Leaves in Albino Rats

 

Deepak Kumar Jain1*, Sonika Gupta1, Ruchi Jain2 and Nilesh Jain2

1Truba Institute of Pharmacy, Bypass Road, Bhopal, M.P – 462 036 (India)2Sagar Institute of Research and Technology-Pharmacy, Bypass Road, Bhopal – 462036

*Corresponding Author E-mail: jaindeepak2081@yahoo.com

 

ABSTRACT:

Acorus calamus Linn, (family: Acoraceae) is used in traditional medicine to treat mental fatigue, memory loss, anxiety, bronchitis, sinusitis, headache, flatulence, joint pains febrifuge, hallucinogen, hypotension. It is also used internally in the treatment of digestive complaints, bronchitis, sinusitis diseases in humans. The anti-inflammatory activity of the 80% ethanolic extract of the leaves was investigated with experimental animal models using the carrageenan-induced paw oedema and cotton pellet granuloma tests in rats.  The extract (100 and 200 mg/kg) at 240 min post-treatment caused a significant (p<0.05) reduction in the paw oedema in rats.  The effect of the extract was most pronounced at the dose of 200 mg/kg and was closer to that of indomethacin (10 mg/kg). The exudate formation inhibited by 200 mg/kg of the extract in the cotton pellet granuloma test was comparatively significant. The findings of the study indicate that the 80% ethanolic extract of Acorus calamus leaves possesses anti-inflammatory activity which is probably related to the significant reduction of various biochemicals, viz. histamine, 5-HT, various kinins which are involved in early phases of inflammation. This is a possible rationale for its folkloric use as an anti-inflammatory agent.

 

KEYWORDS: Acorus calamus, Anti-inflammatory activity, Paws oedema, Exudates, Carrageenan.

 


INTRODUCTION:

Acorus calamus Linn. (Acoraceae) is a perennial, semi-aquatic, marshy plant, native to northern Europe and Asia1. The plant is popularly known as sweet flag, sweet root, sweet rush, sweet cane, gladdon, sweet myrtle, myrtle grass, myrtle sedge, cinnamon sedge and vacha2. It is used for mental fatigue, memory loss, anxiety, bronchitis, sinusitis, tension headache, flatulence, joint pains3. The root is aphrodisiac, aromatic, carminative, diaphoretic, emmenagogue, expectorant, febrifuge, hallucinogenic, hypotensive, sedative, stimulant, stomachic and vermifuge4-7. Sweet flag is also used externally to treat skin eruptions, rheumatic pains and neuralgia. An essential oil from the rhizome is used in perfumery and as food flavoring. It is used internally in the treatment of digestive complaints, bronchitis, sinusitis etc8, 9. It’s essential oil contains acorenone, b-gurjunene, isoshyobunine, b-asarone, calamendiol, a-selinene, a-calacorene, calamusenone, camphone and shyobunone10-12. Roots and rhizomes mainly contain monoterpene hydrocarbons, sequestrine ketones, (trans- or alpha) asarone (2, 4, 5-trimethoxy-1-propenylbenzene) and beta-asarone (cis- isomer) 13, 14.

 

A literature survey reveals that no systematic approach has been made to study the anti-inflammatory activity of leaves of this plant. In the present work, we have investigated the anti-inflammatory activity of the ethanolic extract of Acorus calamus using animal models.

 

MATERIALS AND METHODS:

The leaves of Acorus calamus were collected from the local market of Bhopal, Madhya Pradesh. Identification was confirmed by Dr. A. B. Tiwari, Reader, Department of Herbal Physiology and Medicinal Plant, J.N.K.V.V, Jabalpur, (M.P).

 

The material was air dried under shade, pulverized by a mechanical grinder and passed through a 40 mesh and then stored in airtight containers. The powdered leaves (250 g) were extracted with ethanol (80% w/v) for 24 h using a soxhlet extractor15. This ethanolic extract was concentrated to dryness under reduced pressure and controlled temperature (50-60oC) to yield solid masses that were completely free from solvents (18.8% w/w). The pharmacological screening of the crude extract was carried out using standard protocols. The crude extract was suspended in 1% carboxy methyl cellulose (CMC) for administration to albino rats.

 


Table I.  Acute anti-inflammatory activity of Acorus calamus extracts on carrageenan induced rat paw oedema.

Group

Mean of anti-inflammatory response of the extracts by monitoring rat  paw diameter(mm) at time (min)

 

0

30

60

120

180

240

Control CMC

2.8 ± 0.16

4.8 ± 0.69

4.7 ± 0.42

4.4±0.78

4.1± 0.89

4.0± 0.60

Ethanolic extract-100mg/kg b.w

2.8 ± 0.98

4.8 ± 0.23

4.2 ± 0.21

4.0 ± 0.82

3.5 ± 0.48

3.2 ± 0.13

Ethanolic extract-200mg/kg b.w

2.9 ± 0.07

4.6 ± 0.67*

4.4 ± 0.71*

3.9 ± 0.77*

3.4 ± 0.24*

3.0 ± 0.12*

Indomethacin (10mg/kg)

2.8 ± 0.13

4.5 ± 0.53*

4.2 ± 0.52*

3.6± 0.56*

3.2± 0.49*

2.6± 0.14*

*p< 0.05 as compared to control group, Value are mean ± SEM; n=6 in each group.

 

Table II. Effect of Acorus calamus extracts on cotton pellet induced granuloma in albino rats

Treatment

Weight of granuloma (mg)

Pair wise mean difference*

Control

42.852 ± 4.623

-

Ethanolic extract-100mg/kg b.w

21.426 ± 2.834

9.281 ± 2.564a

Ethanolic extract-200mg/kg b.w

18.620 ± 1.093

14.486 ± 2.310 a

Indomethacin (10mg/kg)

16.623 ± 1.630

17.723 ± 2.439 a

Values are mean ± SEM; n=6 in each group. One way ANOVA revealed F=18.462, aP=0.001 indicating significant difference between groups. Further multiple comparisons using Scheffe’s test showed significant difference among treated and control group. *mean difference (±SEM) is between control and each treatment using Scheffe’s test. *aP < 0.001

 

 


Animals:

Healthy inbred Wister albino rats of either sex (150-180 g) were selected for the present investigation. They were housed in a well-ventilated, temperature-controlled (25±1oC) animal room in 12 h light dark cycle. The animals were provided with standard pellet diet and water ad libitum in polypropylene cages. Animals were periodically weighed before and after experiments. The ethanol extract of Acorus calamus was devoid of any mortality or change in behavior up to 1 g/kg orally in albino rats. Based on this observation maximum dose of 100 mg/kg and 200 mg/kg orally was used for acute and chronic treatment in following experiments.

 

Carrageenan induced rat paw oedema:

Animals were divided into 4 groups of 6 animals each as follows: Group-I served as control and Group-II as reference standard were treated with (Indomethacin 10 mg/kg) injection16, 17. Edema was induced by subcutaneous injection of 1% carrageenan into the planter region of right hind paw18. Group-III and Group-IV rats were treated with 100 mg/kg and 200 mg/kg ethanolic extract respectively. The paw volume was measured initially and at 30, 60, 120, 180, and 240 min. after carrageenan injection using plethysmometer.  Percentage inflammation was calculated for comparison. Animals were closely observed for any infection; those, which showed signs of infection, were separated and excluded from the study. The study was approved by the Institutional Animal Ethics Committee (Reg. No-1196/a/08/CPCSEA).

 

Cotton pellet granuloma:

Cotton pellet granuloma was induced according to the method of D’Arcy19. Sterilized cotton pellets each weighing 10mg were implanted in both axilla and groin of each rat under light ether anesthesia. Twenty four rats were divided into four groups as shown in Table II for various treatments for five days. Subsequently, on 6th day all pellets were dissected out under ether anesthesia and dried at 70oC for 6 hours and weight of each granuloma was determined.

 

 

Statistical analysis:

The data were analyzed using one-way analysis of variance. Post-hoc comparisons using Scheffe’s test were carried out for the analysis to determine significant overall effects (P<0.05).

 

RESULTS:

The present preliminary phytochemical investigation on Acorus calamus (Linn.) leaves reveals the presence of flavanoids and terpenoids in 80% ethanolic extracts. Hence we have taken 80% ethanolic extracts of Acorus calamus (Linn.) leaves for evaluation of the anti-inflammatory activity.

 

Carrageenan induced rat paw oedema: This anti-inflammatory activity was dose-dependent and found to be statistically significant at the higher concentration, 200 mg/kg, Table I. The anti-inflammatory activity of indomethacin, a standard reference drug, was also found to be significant.

 

Cotton pellet granuloma: There was dose dependant reduction in granular tissue formation in extract and indomethacin treated rats as shown in Table II. The activity was found to be statistically significant for the dose ranges used.

 

DISCUSSION:

The crude ethanol extract showed presence of multiple chemical constituents with presence of flavanoids and terpenoids. The ethanol extract of Acorus calamus is devoid of toxicity up to 1 g/kg in albino rats. The extract showed dose dependent anti-inflammatory activity, which was found to be statistically significant at higher concentration in acute carrageenan induced rat paw oedema model. However, this activity was less potent as compared to indomethacin. The anti-inflammatory activity of Acorus calamus appears due to significant reduction of various biochemicals, viz. histamine, 5-HT, various kinins which are involved in early phases of inflammation. In the chronic model of cotton pellet implantation the activity was dose dependant and significant reduction in granular tissue formation was recorded. The results were significant when analyzed statistically. Thus, extract shows anti-inflammatory activity at various acute phases of inflammation and on formation of granular tissue. Thus, anti-inflammatory property of Acorus calamus may be attributed to the phytoconstituents present in it, which may be either due to their individual or additive effect that fastens the anti-inflammatory process. At this stage, it is difficult to say which components of the extracts are responsible for this anti-inflammatory activity. However, further phytochemical studies are needed to isolate the active compounds responsible for these pharmacological activities.

 

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Received on 27.02.2010       Modified on 15.03.2010

Accepted on 07.04.2010      © RJPT All right reserved

Research J. Pharm. and Tech.3 (3): July-Sept. 2010; Page 882-884