Anti-inflammatory activity of Ethanolic Extract of the Roots of the Plant Caesalpinia sappan Linn.

 

Jennifer F1*, Thomas AK2, Fernandes R1 amd Subrhamanyam EVS1

1Department of Pharma. Chemistry, NGSM Institute of Pharmaceutical Sciences, Paneer, Deralakatte P.O, Mangalore -574160 India.

2Nirmala college of Pharmacy,Kerala India.

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

 

ABSTRACT:

In the present study the anti-inflammatory activity of ethanolic extract of the roots of the plant Caesalpinia sappan. (Family: Caesalpiniaceae) was carried out. The preliminary phytochemical investigation revealed the presence of carbohydrates, proteins, triterpenoids, flavonoids, steroids and tannins. The ethanolic extract was subjected to anti-inflammatory study by Carrageenan induced paw edema model and Cotton pellet granuloma method in rats. The results of anti-inflammatory  activities of ethanolic extract of the roots of the plant Caesalpinia sappan were compared with control and found to be statistically significant (P<0.01) at dose levels of 100mg/kg, 200mg/kg and 400mg/kg.

 

KEYWORDS: Anti-inflammatory; cotton pellet granuloma; Caesalpinia sappan; Caesalpiniaceae


 

INTRODUCTION:

Caesalpinia sappan belongs to the family Caesalpiniaceae commonly known as Brazil or sappan wood1.Various pharmacological activities of the plant other than the roots were reported like anti-bacterial, antifungal, antiviral, anti inflammatory properties2 . In view of the fact that there are no systematic studies on analgesic, anti-inflammatory and antipyretic activities of ethanolic extract of the roots of the plant Caesalpinia sappan, and the present study has been conducted.

 

MATERIALS AND METHODS:

Plant material and preparation of extract

In the present study the roots of the plant Caesalpinia sappan were collected from Muvattupuzha, Kerala, India, during the month of November- December 2005. It was identified and authenticated by Prof. Dr. J G Ray, Dept. of Botany S.B College, Changanacherry, Kerala. The roots were then dried in shade and chopped into small pieces and powdered (500g). The extraction was carried out using 95% ethanol by continuous soxhlet extraction method and extract were vacuum dried. (Yield: 10.2 % w/w)Preliminary phytochemical analysis was performed for testing the different chemical groups present in ethanolic extract by standard procedure3.

 

Animals and Housing condition:

Laboratory bred adult albino Wister rats of either sex weighing between 150-200 g were selected for the study. The animals were kept at constant temperature (25+ 20 C), humidity (55%), and light and dark cycle (12/12h light/dark). The animals were deprived of food for 24h before experiment but allowed free access to drinking water ad libitum.

 

The experiment protocol has been approved by the Institutional Animal Ethics Committee of K.S. Hegde Medical Academy, Deralakatte, Mangalore, India (Ref No.IAEC/037/2006). The study was in accordance with the guidelines provided by committee for the purpose of control and supervision of experiments on animal (CPCSEA)

 

Acute toxicity study

The acute oral toxicity study (AOT) of ethanolic roots extract of Caesalpinia  sappan of  was carried out in female albino rats as per staircase method4 and OECD guidelines 4255.The animals were fasted 12hours prior to the dosing. Animals were administered orally with a single dose of extract suspended in 0.6% CMC and observed for 2-3 hours for general, behavioral, neurological, autonomic profiles6  and death for a period of 24hours and for 14 days after administration of the root extract

 

ANTI-INFLAMMATORY SCREENING METHOD:

Carrageenan-induced acute paw edema in rats: 7-10

In this study 1% carrageenan was used in a dose of 0.1 ml as a phlogistic agent (irritant) injected subcutaneous into the plantar aspect of the left hind paw in rats.            The paw volume was measured immediately after the carrageenan injection, which served as the initial volume both in control and drug treated animals. First group was served as vehicle control. The second group was treated with Diclofenac Na 13.5mg/kg bodyweight (p.o) 3rd, 4th and 5th group 100mg/kg, 200mg/kg, 400mg/kg bodyweight (p.o) ethanolic roots extract of Caesalpinia sappan   (suspended in 5% Na CMC).After thirty minutes of the drug administered, the animals, 0.1ml of 1% carrageenin was injected subcutaneous to the right hind paw of all the rats.

 

Immediate after the injection of carrageenin the paw volumes were measured in a mercury plethysmograph. Thereafter the paw volume was measured at 60,90and 120 minutes. The amounts of edema in the drug treated groups were compared in relation to the control group with the corresponding time intervals. The percentage inhibition by the drugs was calculated using the formula,

 

                % edema inhibition =

Where, Vc= volume of paw edema in control group.

Vt = volume of paw edema in drug treated group.

The results were expressed as % inhibition of edema over the untreated control group.

 

Cotton pellet granuloma method:7,9,11

The method has been showed that foreign body granulomas were provoked in rats by subcutaneous implantation of pellets of cotton. After several days, histological giant cells and undifferentiated connective tissue can be observed besides the fluid infiltration. The amount of newly formed connective tissue can be measured by weighing the dried pellets after removal.

 

Male albino rats with an average weight of 200gm were selected for the study. They were divided into 5 different groups, consisting six each in a group.  The first group served as control. The second group served as standard drug treated. 3rd, 4th and 5th groups 100mg/kg, 200mg/kg, 400mg/kg bodyweight (p.o) ethanolic roots extract of Caesalpinia sappan (suspended in 0.6% Na CMC). The rats anaesthetized using pentobarbitone. The dorsal skin shaved and disinfected with 70% ethanol. An incision was made in the lumbar region. By a blunted forceps subcutaneous tunnels were formed and presterillized cotton pellets weighing about 20mg ± 0.05 mg (the cotton pellets were sterilized in an autoclave for 30-45 minutes under 15 lb pressures) was placed on both axillae and groin regions. The animals are treated for 0-9 days with standard and test drugs to the different group respectively.

 

On the 10th day the animals were anaesthetized with ether and pellets were removed, cleaned and dried at 60-70°C for 6 hours.   Granuloma weight was obtained by deducting the weight of the cotton pellets on ‘0’ day (i.e., before start of the experiment) from the weight of the cotton pellet on ‘9th’ day (i.e., at the end of the experiment). The average weight of the pellets of the control group as well as of the test group was calculated. The percent change of granuloma weight relative to vehicle control group was determined.

Statistical Analysis:

The data obtained were expressed as mean ± SEM. The data of analgesic, anti- inflammatory and antipyretic activities of ethanolic extract of the roots of Caesalpinia sappan were compared with control and analyzed by one way analysis of variance (ANOVA) followed by Dunnett's 't’ test. P value less than 0.01 was considered as statistically significant.

 

RESULTS:

Effect on Carrageenan-induced acute paw edema model:

Caesalpinia sappan root extract exhibited the effect on Carrageenan-induced acute paw edema in a dose dependent manner (Table 1).Treatmant with ethanolic root extract at the dose of 100,200 and 400 mg/kg body weight of the experimental animals showed significant (p<0.01)reduce in the paw edema at 60, 90 and 120 minutes when compared to control.

 

Effect on Cotton pellet granuloma method:

Table 2 shows the effect of drug treatment on the mean weights of cotton pellet. The ethanolic root extract of at dose levels (100, 200 and 400 mg/kg) inhibited the granuloma tissue formation. Also showed significant (p<0.01) dose proportionate inhibited effect on the granuloma weight when compared to control

 

DISCUSSION:
The preliminary phytochemical screening of the ethanolic extract of the roots of Caesalpinia sappan revealed the presence of Carbohydrates, Proteins, Triterpenoids, Flavonoids, Steroids and Tannins. The rats treated with oral administration of ethanolic extract of the roots of Caesalpinia sappan reduced acute paw oedema volume as compared to the control. However it exhibited a dose dependent activity. Ethanolic extract of the roots of Caesalpinia sappan also exhibited significant reduction (P<0.01) of the granulation mass formation at a dose of 100,200,400mg/kg body weight. However it exhibited a dose dependent anti-inflammatory activity.The carrageenan test was selected because of its sensitivity in detecting orally active anti-inflammatory agent particularly in the acute phase of inflammation12.  The carrageenan induced paw oedema model in rats is known to be sensitive to cyclooxygenase (COX) inhibitors and has been used to evaluate the effect of non-steroidal anti-inflammatory agents, which primarily inhibit the enzyme COX involved in prostaglandin synthesis. Based on the result, it can be inferred that the inhibitory effect of ethanolic extract of the roots of Caesalpinia sappan on carrageenan induced inflammation in rats may be due to the inhibition of enzyme cyclooxygenase leading to the inhibition of prostaglandin synthesis.  But lipooxygenase inhibitions also possess significant anti-inflammatory activity against carrageenan induced paw oedema13. Hence the inhibitory effect of ethanolic extract of the roots of Caesalpinia sappan on carrageenan induced paw oedema may also due its inhibitory effect on lipooxygenase pathway14.

 

 


Table-1: Effect of ethanolic extract of roots of Caesalpinia sappan on  carrageenan   induced paw oedema in rats.

Treatment

Dose mg/kg

30 min

60 min

90 min

120 min

Control

-

0.0733+0.0056

0.1133+0.0056

0.16+0.0063

0.2217+0.0087

Diclofenac sodium

13.5

0.0250+0.0092**

0.0317+0.0079**

0.033+0.0056**

0.043+0.0056**

Ethanolic extract of Cs

100

0.055+0.005

0.085+0.0076**

0.1183+0.006**

0.1467+0.0084**

Ethanolic extract of Cs

200

0.0500+0.0063

0.0683+0.0065**

0.073+0.0056**

0.0867+0.0056**

Ethanolic extract of Cs

400

0.04+0.0063**

0.0583+0.006**

0.07+0.0052**

0.053+0.0109**

All values are expressed as mean ± SEM (n = 6), *P < 0.05 significant compared to control, ** P < 0.01 significant compared to control

 

 

Table-2: Effect of ethanolic extract of roots of Caesalpinia sappan on cotton pellet  granuloma model in rats.

Treatment

Dose mg/kg

Weight of cotton pellet (mg)

Weight of granuloma(mg) Mean ± SEM

Before

After

Control

-

20.06

100.25

80.19±0.6408

Diclofenac sodium

13.5

20.08

51.35

31.30±0.7509*

Ethanolic extract of Cs

100

19.89

90.5

70.61±0.7414*

Ethanolic extract of Cs

200

20.15

73.55

53.23±0.1475*

Ethanolic extract of  Cs

450

20.14

64.6

44.46±0.1344*

All values are expressed as mean ± SEM (n = 6), * P < 0.01 significant compared to control.

 

 


The ethanolic extract of the roots of Caesalpinia sappan effectively reduce the cotton pellet induced granuloma, suggesting its activity in the proliferative phase of inflammation. Based on the results obtained, it is likely that the mechanism of action of the roots extract is similar to that of non-steroidal anti-inflammatory drugs, namely inhibition of prostaglandin biosynthesis.

 

REFERENCES:

1.         Kirthikar KR, Basu BD. Indian Medicinal Plants vol II, 2nd ed, 1935; 847-8.

2.         Bhattacharyee SK. Hand book of medicinal plants. 1999; 2:1-3

3.        Kokate CK.  Practical  Pharmacognosy. New Delhi, India: Vallabh Prakashan, 1994: 107-110

4.        Ghosh MN. Fundamentals of Pharmacology, 3rd edn, Hilton and Company, Kolkatta, 2005 ,190.

5.        OECD 425 guidelines, OECD guidelines for testing chemicals, 1/20, 2001,1-26

6.        Turner MA. In: Screening methods in Pharmacology, New York, Academic Press: 1965.26

7.        Vogel HG and Vogel WH. (eds), Drug discovery and evaluation of   Pharmacological  assays, Springer Verlag Berlin Heidelberg, New York 1997, 370,372,382,383.

8.        Basu A and Nagchaudhauri AK. Preliminary studies on the anti-inflammatory and analgesic activities of Calotropis procera root extract, Journal of Pharmacology  1991 31 319-324.

9.        Karunakar N, Pillai KK, Husain SZ and Rao M. Investigation of anti-inflammatory activity of jigrine, Indian J. Physiology and Pharmacology, 1997, 41(2): 134-38.

10.     Winter CA, Risely EA, Nuss GW. Carrageenan induced oedema in hind paw of the rat  as assay for anti-inflammatory drugs. Pro Soc Exp Biol Med.1962; 111:544

11.     Thirupathy Kumaresan P, Ananda Vijaya Kumar PR and Rajasekaran A., Anti-inflammatory activity of Hibiscus esculantus, Indian Drugs, 2001, 38(8): 426-427

12.     DiRosa M, Giroud JP, Willoughby DA, Studies of the acute inflammatory response induced in rats in different sites by carrageenan and turpentine. Journal  of Pathology.  1971; 104 :15-29.

13.     Chawla AS, Singh M, Murthy MS, Gupta MP, Singh H. Anti-inflammatory action of ferulic acid and esters in carrageenan induced rat paw oedema model. Ind J Exp Biol.1987; 25:187-9.

14.     Lino CS, Traveira ML, Viana GSB, Motos FJA. Analgesic and anti- inflammatory activity of Justicia pectoralis jacq and its main constituents:coumarin and umbelliferone.Phytother Res.1997;11:211-5


 

 


 

 

 

 

 

Received on 03.07.2009          Modified on 01.09.2009

Accepted on 06.10.2009         © RJPT All right reserved

Research J. Pharm. and Tech. 3(1): Jan.-Mar. 2010; Page 99-101