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RESEARCH ARTICLE

Comparative Study on Anti-inflammatory and Analgesic Activities of Pure Aceclofenac with the Aceclofenac Loaded Mucoadhesive Microparticles in Rats

Rajesh. M^{1, 2}* and Narayanan. N³

¹Sankaralingam Bhuvaneswari College of Pharmacy, Anaikuttam, Sivakasi- 626130 Tamilnadu, India.

²Periyar Maniammai University, Vallam, Thanjavur- 613403 Tamil Nadu, India.

³Director, Jaya College of Pharmacy, Thirunintravur, Chennai- 602024 Tamilnadu, India.

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

ABSTRACT:

Aceclofenac is a non steroidal Anti-inflammatory drug (NSAID) used for relief of pain and inflammation in osteoarthritis, rheumatoid arthritis and ankylosing spondylitis. Aceclofenac possess remarkable Anti-inflammatory and analgesic properties and the analgesic and Anti-inflammatory efficacy is generally equivalent to that of comparator nonsteroidal Anti-inflammatory drugs (NSAIDs) with similar onset of action. The objective of the present study was to evaluate and compare the Anti-inflammatory and analgesic effect of Aceclofenac loaded mucoadhesive microparticles developed by employing sodium alginate in combination with Carbopol as mucoadhesive polymer (F8) with that of pure Aceclofenac in wistar rats. The Anti-inflammatory and sustaining action of the formulation F8 was evaluated by the carrageenan-induced hind paw edema method and the analgesic activity was determined by tail flick method in wistar rats. The results of Anti-inflammatory activity showed a maximum of 86.99% inhibition of edema with Group III (Pure Aceclofenac) and 82.11% inhibition of edema with Group V (F8) at 4 h after carrageenan treatment. In analgesic study, Group II (pure Aceclofenac) had showed increase in Basal reaction time. Moreover, it showed highly significance with p<0.01 and Group IV (F8) showed significance at p< 0.05 compared to control group.

KEYWORDS: Aceclofenac, Anti-inflammatory activity, Analgesic activity, Carbopol, Mucoadhesive microparticles.

INTRODUCTION:

The treatment of inflammation and pain is an important area of clinical science. In the last decade, non steroidal Anti-inflammatory drugs (NSAIDs) have played a central role in these indications and they are currently considered as the first choice, being one of the most widely prescribed drugs^1,2. Aceclofenac is a non-steroidal Anti-inflammatory (NSAID) drug used extensively in the treatment of rheumatoid arthritis, osteoarthritis and ankylosing spondylitis. Aceclofenac is newer derivative of Diclofenac and having less GIT complication, with short biological half-life of 4 hrs and dosing frequency more than one time make it an ideal candidate for sustain release dosage form³.

Received on 27.12.2014 Modified on 11.01.2015

Research J. Pharm. and Tech. 8(2): Feb. 2015; Page 185-188

DOI: 10.5958/0974-360X.2015.00033.5

The mode of action of Aceclofenac is largely based on the inhibition of prostaglandin synthesis. Aceclofenac is a potent inhibitor of the enzyme cyclooxygenase (Cox), which is involved in the production of prostaglandins⁴. Aceclofenac loaded mucoadhesive microparticles developed by employing sodium alginate in combination with Carbopol as mucoadhesive polymer has been formulated to reduce gastrointestinal tract irritation and to release the drug for prolong period. The present study has been designed to know whether the formulation shows better Anti-inflammatory and analgesic activity.

MATERIALS AND METHODS:

Materials

Aceclofenac was obtained from Micro Labs, Hosur, India. Calcium chloride dihydrate was procured from Leo Chem, Bangalore, India. Carbopol 934P was obtained from Loba Chemie Pvt. Ltd, Mumbai, India. Sodium alginate was procured from Reachem Laboratory Chemicals Pvt. Ltd, Chennai, India. Carrageenan was procured from Spectrochem Pvt. Ltd. Mumbai, India. All other reagents used were of analytical grade.

Methods

Preparation of mucoadhesive microparticles of Aceclofenac by Orifice-Ionic Gelation Method

Sodium alginate (1.0 g) and Carbopol (1.0 g) were dissolved in purified water (32 ml) to form a homogeneous polymer solution. The active core material Aceclofenac (1.0 g) was added to the polymer solution and mixed thoroughly with a stirrer to form a smooth viscous dispersion. The resulting dispersion was then added dropwise into calcium chloride (10%w/v) solution (40 ml) through a syringe with a needle of size No: 18. The added droplets were retained in the calcium chloride solution for 30 min to complete the curing reaction and to produce spherical rigid microcapsules⁵. The microcapsules were collected by decantation and the product thus separated was washed repeatedly with water and dried at 45°C for 12 h.

Experimental Animals

Adult wistar albino rats, weighing (180-220g) were used for the study. The animals were fed with standard pellet diet and water ad libitum. They were housed in colony cages under standard laboratory conditions (12:12h light and day cycle, temperature at 25±2°C and relative humidity at 55±10%). Animals were allowed to acclimate for 7 days to the laboratory conditions before the commencement of experiments. The ethical clearance was obtained from Institutional Animal Ethical Committee (IAEC) of Sankaralingam Bhuvaneswari College of Pharmacy for using animals in the present study.

Experimental Design

The pure drug (Aceclofenac), Marketed Aceclofenac S.R tablet, Aceclofenac loaded mucoadhesive microparticles were suspended in 1% Carboxy methylcellulose (CMC).A required volume was administered orally using oral cannula. Fasted rats that were deprived of food but not water for 24 h prior to experiment were used to assess the effect of pure Aceclofenac, Aceclofenac S.R tablet, Aceclofenac microparticles and CMC on gastric mucosa.

In vivo Anti-inflammatory study ^6-8

The Anti-inflammatory and sustaining action of Aceclofenac microparticles was evaluated by the carrageenan-induced hind paw edema method in Wistar rats. Adult male wistar rats, weighing 180-220 g, were randomly divided into 5 groups each containing 6 rats. Group I as Normal control, Group II as negative control, Group III received Pure Aceclofenac, Group IV received marketed Aceclofenac S.R tablet and Group V received Aceclofenac microparticles (F8). Doses for the rats were calculated based on the mass of the animals (10mg/kg). The suspension of drugs was given orally to Groups III, IV and V using oral cannula. After 30 min of drug administration, rats of all groups (except Group I) were challenged by subcutaneous injection of 1% w/v solution of carrageenan into the plantar site of the hind paw. The paw volumes were measured with a plethysmometer, prior to administration of carrageenan and after 1, 2, 3 and 4 hours of carrageenan administration. The increase in paw volume was calculated as percentage compared with the basal volume. The percentage of inhibition was calculated using the following formula:

% Inhibition = Vc – Vt/ Vc x 100

Where Vc and Vt represent average paw volume of control and treated animals respectively.

In vivo Analgesic study^{7, 9}

The analgesic activity was determined by tail flick method using Analgesiometer. Take basal reaction time to radiant heat by placing the tip (last 1.2 cm) of the tail on the radiant heat source. The tail withdrawal from the heat (Flicking response) is taken as the end point. Normally a rat withdraws its tail within 3-5 sec. A cut off period of 10-12 sec is observed to prevent damage to the tail. Any animal failing to withdraw its tail in 3-5 sec is rejected from the study. Take at least 3-5 basal reaction times for each rat at a gap of 5 minutes to confirm normal behaviour of the animal. The animals were divided into 4 groups (n=6). Group I as Control, Group II received Pure Aceclofenac, Group III received marketed Aceclofenac S.R tablet and Group IV received Aceclofenac microparticles (F8). Doses for the rats were calculated based on the mass of the animals (10mg/kg). The suspension of drugs were given orally to each group except Group I and the reaction time at 1,2,3,4 and 5h were noted and the tail flick latency was measured after the drug administration. The data were then compared with control group.

RESULTS AND DISCUSSION:

Evaluation of Anti-inflammatory Study

The Aceclofenac loaded mucoadhesive microparticles was prepared and subjected for in vivo anti inflammatory study by carrageenan-induced paw edema method in rats. In order to have a better comparison between the anti inflammatory activity of pure aceclofenac, marketed Aceclofenac S.R Tablet and the Aceclofenac loaded mucoadhesive microparticles evaluation was made on the basis of their ability to inhibit the edema produced in hind paw of rats after challenging with the carrageenan. The results are shown in Table 1.

A maximum of 86.99% inhibition of edema was observed with Pure Aceclofenac (Group III) and maximum 82.36% and 82.11% inhibitions of edema was observed with Marketed Aceclofenac S.R tablet (Group IV) and Aceclofenac microparticles F8 (Group V) respectively at 4 h after carrageenan treatment. Pure Aceclofenac (Group-III) was found to be highly significant in reducing the paw edema compared to marketed S.R tablet (Group-IV) and Aceclofenac microparticles F8 (Group-V). Marketed S.R tablet (Group-IV) and Aceclofenac microparticles F8 (Group-V) was found to be equally significant in reducing the paw edema.

Maximum inhibition of edema was observed with Pure Aceclofenac (Group-III) compared to marketed Aceclofenac S.R tablet (Group IV) and Aceclofenac microparticles F8 (Group V).This may be due to slower release of drug in gastrointestinal tract from the mucoadhesive microparticles and marketed S.R tablet because of increased viscosity contributed by swellable hydrophilic polymers.

Evaluation of Analgesic study

The analgesic activity of pure Aceclofenac, marketed Aceclofenac S.R tablet and the Aceclofenac loaded mucoadhesive microparticles were evaluated based on its ability to inhibit the pain produced after challenging with heat reactions.

Group I (Control) have no analgesic effects since there is no significant difference in basal reaction time till 5^th hour. Group II (Pure Aceclofenac) had showed increase in basal reaction time. Moreover, it showed significant (p<0.01) analgesic activity when compared with Group III (marketed Aceclofenac S.R tablet) and Group IV (mucoadhesive microparticles F8). This may be due to rapid absorption of drug from the suspension of pure drug; whereas the drug may be released slowly from the marketed S.R tablet and mucoadhesive microparticles (F8).

Group III and Group IV had showed similar response towards the basal reaction time. Both had showed significance at p< 0.05 compared to control group. The results are shown in Table 2.

Table 1- Anti- Inflammatory effect of Aceclofenac formulations on carrageenan induced hind paw edema in rats

GROUP	Mean increase in paw volume(mL)
GROUP	₁^sthour	₂^nd hour	₃^rd hour	₄^th hour
I (Normal control)	0.080±0.020	0.181±0.004	0.160±0.004	0.14±0.004
II (Negative control)	0.27±0.078^###	0.74±0.004^###	1.17±0.019^###	1.23±0.0232^###
III ( Pure Aceclofenac )	0.090±.001***	0.210±0.031***	0.19±0.004***	0.16±0.004***
IV ( Aceclofenac marketed S.R tablet)	0.105±0.002**	0.220±0.004**	0.220±0.004**	0.217±0.250**
V( Aceclofenac microparticles F8)	0.14±0.006**	0.270±0.009**	0.267±0.004**	0.220±0.004**

The results are expressed as mean ± SEM, (n=6). The data was analyzed by using One way ANOVA followed by Dunnett’s test.

^###p<.0.001, where Group I were compared with Group II. (Significant with Inflammation).

***p<.0.001, **p<0.01, where Group III, IV& V were compared with Group II. (Significant with Anti-inflammatory activity)

Table 2-Analgesic activity of Aceclofenac formulations by tail flick method

Group	Basal reaction time before drug administration (sec)	Basal reaction time after drug administration (sec)
Group		1 h	2 h	3 h	4 h	5 h
I (Control)	2.25±0.25	1.75±0.25	2.25±0.25	3.00±0408	3.25±0.25	3.50±0.28
II ( Pure Aceclofenac )	1.75±0.25	9.50±0.28**	11.00±0.408**	12.25±0.25**	12.75±0.25**	13.25±0.25**
III ( Aceclofenac marketed S.R tablet)	2.00±0.01	7.25±0.25*	9.50±0.288*	11.00±0.408*	12.00±0.408*	12.50±0.408*
IV ( Aceclofenac microparticles F8)	1.50±0.28	6.00±0.408*	8.25±0.25*	9.50±0.28*	10.75±0.478*	11.50±0.288*

The results are expressed as mean ± SEM, (n=6). The data was analyzed by using One way ANOVA followed by Dunnett’s test. **p<0.01, *p<0.05, where Group II, III & IV were compared with Group I.

CONCLUSION:

In Anti-inflammatory study, Group-III (Pure Aceclofenac) was found to be highly significant in reducing the paw edema compared to Group-IV (marketed S.R tablet) and Group-V (Aceclofenac microparticles F8). Group-IV (Marketed S.R tablet) and Group-V (Aceclofenac microparticles F8) were found to be equally significant in reducing the paw edema. In Analgesic study Group II (Pure Aceclofenac) showed significant (p<0.01) analgesic activity when compared with Group III (marketed Aceclofenac S.R tablet) and Group IV (mucoadhesive microparticles). Group III and Group IV had showed similar response towards the basal reaction time (p< 0.05) compared to control group.

ACKNOWLEDGEMENT:

The authors are thankful to Mr. S. Sriram Ashok B.E, Correspondent and Department of Pharmacology, Sankaralingam Bhuvaneswari College of Pharmacy, Anaikuttam, Sivakasi for providing necessary facilities to carry out the work.

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