Evaluation of Anti-inflammatory Potential of Cyathocline lyrata Cass Plant Extract by using Carrageenan Induced and Formalin Induced Rat Paw Edema.

 

K.G. Malviya1*, Dr. U.D. Shivhare2, Dr. Preeti Srivastav1, S.C. Shivhare1

1MJRP College of Heath Care and Allied Sciences, MJRP University, Jaipur India.

2Sharad Pawar College of Pharmacy, Nagpur India

*Corresponding Author E-mail: kmalviya81@gmail.com

 

 

ABSTRACT

The present study was aimed at evaluating the chloroform extracts of whole part of Cyathocline lyrata cass anti-inflammatory activity. Anti-inflammatory activity of chloroform extracts of whole part of Cyathocline lyrata cass was evaluated by  carrageenan  induced  hind paw edema and Formalin induced paw edema method. Chloroform extracts of whole part of Cyathocline  lyrata cass produced significant Anti-inflammatory activity when evaluated by carrageenan  induced  hind paw edema and Formalin induced paw edema method respectively. The  interpretation  of  the  results was  done  after  subjecting  the  data  obtained from various studies to statistical analysis which included one-way ANOVA followed by post-hoc with Dunnets t-test. The results suggest that the chloroform extracts of whole part of Cyathocline  lyrata cass posses  anti-inflammatory.

 

KEYWORDS: Cyathocline lyrata cass, Anti-inflammatory, carrageenan induced  hind paw edema , formalin paw edema.

 

 


INTRODUCTION:

Cyathocline  lyrata  is annual herb, growing  to 20-25  cm  hight,  branched  grooved  stem  has soft hair covering  it. Whole Plant is strongly aromatic. Alternatively arrange stalkless leaves are toothed covered with soft hair and flowers occurs in corymbs at the end  of  branched  in purple color. Cyathocline  lyrata widely spread  in Himalyas range,  Assam,  India  and also available in Local  area  of Betul M.P. Cyathocline  lyrata  is  well  known  drug  in Indigeneous system of medicine for its various used as a bitter  tonic. It acts as germicide and appetizer.  The essential oil of aerial part of Cyathocline lyrata had show fairly pharmacological activity.  It also shows anthelmatic, insect repellant and anti-microbial activity.. Inflammation is the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells or irritants.  Inflammation is a protective attempt by the organism to remove the injurious stimuli as well as initiate the healing process for the tissue.

 

Non  steroidal  anti-inflammatory  drugs  (NSAIDs)  are  widely  used  in  the treatment  of  pain,  fever  and  inflammation. However these drugs have no side effects especially on the gastro intestinal tract.

 

Therefore there is a need to search for novel anti-inflammatory  agents  from  natural  source,  which  could  be  used  in  medicine  and  as additives  to  Nutraceuticals. Many natural products have been reported to have anti-inflammatory action. An  attempt  is made  during  this  research work  to  evaluate  the  chloroform extracts of whole part of Cyathocline  lyrata  for anti-inflammatory activities.

 

MATERIAL AND METHOD:

Plant material collection and authentication:

The entire plant of Cyathocline lyrata Cass were collected from local area of Betual (M.P.) in the month of June, 2012. The plant specimen was confirmed by Dr. Madhuri Modak, Professor, Dept. of Botany, Motilal Vigyan Mahavidyalaya, Bhopal (M.P). A voucher specimen (Her/Bot/1212.90-374) is deposited at the herbarium of MJPR University, Jaipur (Rajasthan).

 

Evaluation of In vivo anti-inflammatory activity:

Procurement of Experimental Animals:

Male albino rats (100-150 g) of approximate same age were used in the present studies was procured from VNS College of pharmacy, Bhopal India.  The animals were fed with standard pellet diet and water ad libitum. All the animals were housed in polypropylene cages. The animals were kept under alternate cycle of 12 hours of darkness and light. The animals were acclimatized to the laboratory condition for 1 week before starting the experiment. The animals were fasted for at least 12 hours before the onset of each activity. The animals received the drug treatments by oral gavage tube. The preparation of chloroform extract the next step was to formulate a suspension of extracts of Cyathocline lyrata Cass which was subjected to animal studies. Suspension of the extract was made by suspending in 0.5% CMC.

 

Acute toxicity studies:

This study is needful before pharmacological screening on animals. The acute oral toxicity study of Cyathocline lyrata chloroform extracts was carried out according to OECD 423 guideline (Organization for Economic Cooperation and Development) which is based on a stepwise procedure with the use of a minimum number of animals per step. All animals were received respective dose for seven days and observed mortality on 7th day. The Animal Male albino rats (100-150 g) were treated with the drug of chloroform extract Cyathocline lyrata.

 

Screening of Anti-inflammatory Activity:

Carrageenan induced paw oedema[9]

Both in- vivo and in- vitro methods are available for the evaluation of anti-inflammatory agent but among the in-vivo method the carrageenan induced rat paw oedema assay is belived to be one of the most reliable and also the most widely used.

        

Male Swiss albino rats weighing 100- 150 gm were used. The animals were put on standard diet and water was provided ad libitum. The animals were fasted over night before the experimentation. The rats were divided into six groups (n=6).  The anti-inflammatory activity of extract was assessed by the method described by Winter et al. Rats were divided into four groups

 

Group I  Normal control, without treatment,

 

Group II: Negative control, intraplantar injection of 0.1 ml of 1 %( w/v) carrageenan on 7th Day.

 

Group III: Positive control, received Diclofenac potassium 10mg/kg BW p.o. on 7th day before 1hr prior to intraplantar injection of 0.1 ml of carrageenan

 

Group IV: Rats received chloroform extract of Cyathocline lyrata (100-200 mg/kg) for 7 days p.o. and then after 1hr of last dose, intraplantar injection of 0.1 ml of carrageenan.

Group V: Rats chloroform extract of Cyathocline lyrata (200-400 mg/kg) for 7 days p.o. and then after 1hr of last dose, intraplantar  injection of 0.1 ml of carrageenan

 

Since the LD50 has not been determined during the acute toxicity study, the doses for this study where selected by trial and error method. The standard and extract were given orally to the animal. The perimeter of paw was measured by using vernier caliper mesurments were taken at 0,1,2,3 hours after the administration of the carrageenan.

 

The percentage inhibition of edema was calculated by the following equation:

% Inhibition of edema= 100 ()

Where Vc is the edema volume in the control group and Vt is the edema volume in tested groups [Il-Ok Lee, You-Seong Jeong, 2002].

 

Formalin induced paw edema [10]

In the experiment, a total of 24 rats were used. The rats were divided into 5 groups

 

Comprising of 6 animals in each group as follows:

Group I: Normal control, without treatment,

 

Group II: Negative control, intraplantar injection of 0.1 ml of formalin on 7th day.

 

Group III Positive control, received Diclofenac potassium 10mg/kg BW p.o. on 7th day before 1hr of intraplantar injection of 0.1 ml of formalin

 

Group IV Rats received chloroform extract of Cyathocline lyrata (100-200 mg/kg) for 7 days p.o. and then after 1hr of last dose, intraplantar injection of 0.1 ml of formalin.

 

Group V Rats chloroform extract of Cyathocline lyrata (200-400 mg/kg) for 7 days p.o. and then after 1hr of last dose, intraplantar injection of 0.1 ml of formalin.

 

Acute inflammation was caused by injecting 0.1 ml of formalin (40%) into the sub-plantar region of the right hind paw of each rat. The paw volume was measured plethysmometrically at 0 h, 1 h, 2 h, 3 h, 4h, 5h and 6h after the formalin injection. Edema was expressed as mean increase in paw volume relative to control animals. The percentage inhibition of edema was calculated by the following equation:

% Inhibition of edema= 100 (),

Where Vc is the edema volume in the control group and Vt is the edema volume in tested groups

 

 


RESULT AND DISCUSSION:

Acute toxicity study:

Acute toxicity studies for chloroform extract of Cyathocline lyrata was conducted as per OECD guidelines 423 using albino rats. Each animal was administered extracts by oral route. There was no change in normal behavioral pattern of  animals  and  no  sign  and  symptoms  of  toxicity  were  observed  during  the observations which was done continuously for  the first  two hours and  then observed up  to  twenty four hours and then for seven days for mortality.

 

Screening of Anti-inflammatory activity:

Carrageenan induced paw edema:


 

 


Table 1: Effect of Chloroform Extract of Cyathocline lyrata on Carrageenan Induced Paw Edema in Rats

Treatment

Right hind paw volume in ml

% Inhibition

30 min

1 h

2 h

3 h

4 h

30 min

1 h

2 h

3 h

4 h

Control

4.25± 0.35

4.15±

0.3

4.12±

0.29

4.19±

0.29

4.2±

0.33

_

_

_

_

_

DP (10 mg/kg)

4.05± 0.36

3.44±

0.3*

3.18±

0.29*

3.02±

0.11**

2.88±

0.10**

4.70

17.10

22.43

27.92

31.26

CECL  (100 mg/kg)

4.19±

0.36

3.49±

0.27

3.39±

0.23

3.29±

0.16

3.16±

0.27*

1.41

15.90

17.42

21.47

24.58

CECL  (200 mg/kg)

4.09±

0.36

3.4±

0.23

3.11±

0.21*

2.92±

0.21**

2.79±

0.23**

3.76

18.07

24.10

30.31

33.41

CECL (400 mg/kg)

4.01±

0.4

3.2±

0.3*

2.99±

0.29**

2.82±

0.21**

2.77±

0.24**

5.64

22.89

26.96

32.69

33.89

All values are expressed as a Mean ± S.E.M, n=6. Results were analyzed using one way ANOVA followed by Dennett’s comparison multiple test. DP- Diclofenac Potassium, CECL- chloroform extract of Cyathocline lyrata. *p<0.05 was used to indicate statistical significance when compare to control.


 


 

Figure 1:  Carrageenan induced paw edema of chloroform extract of Cyathocline lyrata cass

 

Figure 2:  Carrageenan induced Paw edema Percentage inhibition of chloroform extract Cyathocline lyrata cass   Formalin induced paw edema

Table 2: Effect of Chloroform Extract of Cyathocline lyrata on Formalin Induced Paw Edema in Rats

Treatment

Right hind paw volume in ml

% inhibition

30 min

1 h

2 h

3 h

4 h

30 min

1 h

2 h

3 h

4 h

Control

7.39±

0.55

7.15±

0.3

6.92±

0.29

7.02±

0.29

7.02±

0.32

_

_

-

-

_

DP

(10 mg/kg)

7.25±

0.57

5.14±

0.39*

4.11±

0.29**

4.01±

0.31**

4.88±

0.29**

1.89

28.11

43.49

42.87

32.22

CECL 

(100 mg/kg)

7.29±

0.57

6.09±

0.45

5.56±

0.29

5.17±

0.26*

5.39±

0.47*

1.35

3.49

26.73

26.35

25.13

CECL 

(200 mg/kg)

7.19±

0.55

6.01±

0.09

5.11±

0.09*

4.92±

0.09**

4.79±

0.39**

2.70

15.94

30.34

29.91

33.47

CECL

(400 mg/kg)

6.45±

0.49

6.83±

0.03

4.65±

0.29*

4.12±

0.29**

4.29±

0.24**

12.71

18.46

41.90

41.31

40.41

All values are expressed as a Mean ± S.E.M, n=6. Results were analyzed using one way ANOVA followed by Dennett’s comparison multiple test. DP- Diclofenac Potassium, CECL- chloroform extract of Cyathocline lyrata. *p<0.05 was used to indicate statistical significance when compare to control.


 

 


Figure 3: Formalin induced paw edema of Cyathocline lyrata cass extact

 

Figure 4: Formalin induced Paw edema Percentage inhibition of Cyathocline lyrata cass extract

 


 

CONCLUSION:

The results obtained in different pharmacological evaluation of cyathocline lyrata cass has  anti-inflammatory effect. The results have been obtained in carefully controlled experiments with laboratory animals, where psychological factors can presumably be ruled out. In all the tests the responses have been assessed by actual measurement and not by subjective comparisons which may be influenced by the observer. Therefore the statistical validity of the findings has been proved and they provide a scientific foundation for the use of the biologically active ingredients of Cyathocline lyrata in inflammatory conditions and explain the clinical effectiveness of the plant. In our study we have made an attempt to prove its efficacy in experimental animals. Further study can be done in human subjects.

 

ACKNOWLEDGEMENTS:

Researchers are very much thankful to the Dept. of Botany, Motilal Vigyan Mahavidyalaya, Bhopal (M.P). MJRP College of Heath Care and Allied Sciences, MJRP University Jaipur, Sharad Pawar College of Pharmacy, Nagpur for providing necessary facilities.

 

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Received on 14.04.2013       Accepted on 08.05.2013     

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Asian J. Res. Pharm. Sci.  2013; Vol. 3: Issue 2, Pg 90-94