Anti-arthritic and anti-inflammatory effect of Cetylmyristeolate against Freund’s adjuvant induced arthritis model

Kayalvizhi M.K.¹*, Vanitha Samuel¹, P. Nirmala¹and K. Balamurugan²

¹Division of Pharmacology, RMMC and H, Annamalai University, Annamalai Nagar, Chidambaram

²Department of Pharmacy, Annamalai University, Annamalai Nagar, Chidambaram.

ABSTRACT:

To evaluate the effect of Cetylmyristeolate (CMO) in Freund’s complete adjuvant induced arthritis in rats. The experimental study was evaluated on male and female Wister rats weighing (150 – 200gms). 0.5 ml of Freund’s complete adjuvant (FCA) in the left tibiotarsal joint (IA) was injected to induce arthritis, CMO-500mg; 1g and CMO100mg + MSM200mg + glucosamine 500mg /day for 21 days were given. Paw edema by plesthysmometer and arthritic index were studied, the results showed marked reduction of arthritic and inflammatory effect by CMO500mg/day and 1gm/day as well as the combination of 100mg/day + glucosamine 500mg and MSM 200mg /day. Percentage inhibition of paw volume edema was statistically significant and reductions in the arthritic index scores were comparable with reference standard drug dexamethasone. From the results of this study, it was concluded that CMO as well as the combinations produced a statistically significant effect in reducing carrageenan induced arthritis in rats.

KEYWORDS: Cetyl myristoleate (CMO), Glucosamine sulphate, Methyl sulfonyl methane (MSM), Dexamethasone, Arthritis.

INTRODUCTION:

Rheumatoid arthritis (RA) is an autoimmune disorder affecting 2% of the adult population. Although the peak incidence of arthritis occurs between the ages of 35±45, the prevalence of the disorder increases with age, due to the aggregate of early and late onset cases⁽¹⁾. RA is characterized by intermittent are ups of aching/burning joint pain. If untreated, the disease can ultimately result in irreversible joint damage, disability and premature death. The causes of RA are unknown, and there is no known cure for or prevention of the disease^(2,
3). RA affects a disproportionate number of young- to middle aged adults, but its impact on older adults may be greater, given an increased incidence of concomitant disease for that population⁽⁴⁾. Arthritis is thought to affect more than 50 million Americans, and is generally accepted to be the leading cause of movement limitation and disability. It deserves and receives a great deal of research and medical attention. There are hundreds of drugs, procedures, medical aids and devices directed at coping with the many manifestations of the disease. Given this degree of complexity, certainly no one agent alone could ever be expected to manage or cure "arthritis" in its entirety ^{(5, 6)}. Non steroidal antiinflammatory drugs have been the main stay of treatment in both these conditions. But their wide spread use is associated with significant toxic effects especially on the gastrointestinal tract, liver and kidneys. The discovery of Cetyl Myristoleate an ester of fatty acid natural substances shown to be a less toxic and more effective means of achieving relief.

Freund’s complete adjuvant induced arthritis in rats is an excellent experimental model and is well documented^{(7, 8)}.

TABLE 1: CHRONIC STUDY FOR PERCENTAGE INHIBITION OF PAW VOLUME

Groups	Day 1		Day 7		Day 14		Day 21		Two way ANOVA with repeated measures	‘p’ value
Groups	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Two way ANOVA with repeated measures	‘p’ value
Group I	4.367	0.0516	4.367	0.0516	4.367	0.0516	4.367	0.0516
GroupII	4.650	0.0548	4.833	0.0516	5.000	0.0894	5.183	0.0753	Group ‘F’ 90.926	0.000
GroupIII	4.417	0.0408	4.567	0.0516	4.467	0.516	4.350	0.0548	Day = 95.140	0.000
GroupIV	4.417	0.0408	4.533	0.0516	4.433	0.0516	4.350	0.0548	Day 79.288 * Group	0.000
Group V	4.433	0.0516	4.600	0.0894	4.500	0.0894	4.400	0.894	-	-
GroupVI	4.417	0.0408	4.517	0.0408	4.417	0.408	4.317	0.0408	-	-

N = 6 in each group, two way ANOVA, test Mauchly’s test, p < 0.01 = s, P < 0.001 = HS

FIGURE 1: CHRONIC STUDY FOR PERCENTAGE INHIBITION OF PAW VOLUME

TABLE 2: Results of arthritic index

Sl.No	Treatment (P.O)	Primary lesions	Secondary lesions
1	Control	-	-
2	Freund’s adjuvantinduced	++++	++++
3	Dexamethasone treated	+++	+
4	Cetylmyristoleate 500mg	+++	+
5	Cetylmyristoleate 1g	++	-
6	Cetylmyristoleate Methyl Sulphonyl methane + Glucosamine	++	+

++++ Maximum / Severe, +++ Moderate, ++ Mild, + Nil / Very mild, N = 6 groups

There are innumerable drugs for manifestations of the disease yet; there remains an urgent need for finding pharmacological therapies for arthritis which are effective, relatively safe and least toxic. By keeping the above factors it was aimed to treat Freund’s complete adjuvant induced arthritis in rats through a natural substance called Cetylmyristeolate and compare its efficacy alone and in combination with other conventionally used preparations⁽⁹⁾.

MATERIALS AND METHODS:

Animals

The institutional Animal ethical committee, RMMC and H (Registration No. 160/1999 CPCSEA) Annamalai University, Annamalai Nagar, India approved the experimental design (proposal No. 549, dated 20.02.2008). A total of 36 albino Wistar rats of 180-200g were used for the study. Animals were housed in well ventilated room (temperature 23 ± 2°C, humidity 65 - 70% and 12h light / dark cycle, provided standard pellet feeds and water ad libitum at central animal house, Rajah Muthaih Medical College and Hospital, Annamalai University. All studies were conducted in accordance with committee for the purpose of control and supervision on experiments and animals (CPCSEA) norms and the national institute of Health guidelines “Guide for the care and use of Laboratory Animals”⁽¹⁰⁾.

MEASUREMENT OF CHRONIC INFLAMMATION

(Freund’s complete adjuvant induced arthritis model)

Rats were divided into six groups. Experimental Arthritis was induced by injecting 0.5 ml of Freund’s complete adjuvant into the tibiotarsal joint of left hind paw intrarticularly groups II to VI. On the 1^st day of the study, the paw volume was measured by using volume displacing plethysmometer from day 1 to day 21 of the study. Test drugs were administered to group-IV (CMO-500mg; group-V CMO-1g; group-VI CMO-100mg + MSM-200mg+ glucosamine 500mg /day for 21 days; while group III received the reference standard dexamethasone at dose of 0.5mg/body weight/p.o for a period of 21 days, the edema formation and percentage inhibition of paw volume were calculated on days 1,7,14 and 21 for all the groups. Rats Paw edema by plesthysmometer and arthritic index were studied⁽¹¹⁾.

Discussion:

The mono articular arthritis induced by Freund’s adjuvant showed an increased paw volume, which remained consistent throughout the study period in group II, which was the non drug treatment. But in group III to VI there was as statistically significant reduction in the percentage inhibition of the paw volume edema (Table. 1 Figure. 1).The anti inflammatory activity (chronic study) in freund’s complete adjuvant induced arthritis at 2 doses of CMO500mg/day and 1gm/day as well as the combination of 100mg/day + glucosamine 500mg and MSM 200mg /day was evaluated^(12). The percentage inhibition of paw volume edema was statistically significant in groups IV and V with maximal lowering being observed in group VI. The reduction in the arthritic index (Table. 2). scores in groups VI were comparable with the effects observed with dexamethasone in group III. CMO at both doses showed a statistically significant reduction lower the arthritic index (Table. 2). It was presumed that CMO a medium chain fatty acid is not an analgesic parse but reduces inflammation by normalizing hyper immune responses.

Prostaglandins play a significant role in different phases of inflammatory response. In addition inflammatory mediators such as 5HT and Kinins also play a vital role. Like EFA’s CMO presumably turns off the fires of chronic inflammation by serving as a mediator of prostaglandin formation and metabolism. It was speculated that CMO stimulates the production of prostaglandin series 1 and 3 which were known to suppress inflammation (anti-inflammatory) unlike the series 6 prostaglandins were known to enhance inflammation (pro-inflammatory). This probably accounts for its anti- inflammatory action. As the exudative phase of inflammation subsides, the initial stages of the reparative or third phase are set in motion. The fibroblast, which is the dominant cell in the wounded zone first proliferates then synthesizes extra cellular materials including new collagen fibers and acidic mucopolysaccride which are laid down to form new connective tissue matrix^(13).CMO facilitates cartilage building andalso serves as a surfactant/ lubricant for the damaged joints. Several studies have documented the actions of Glucosamine, a mucopolysaccride which prevents inflammation by decreasing the generation of superoxide radical⁽¹⁴⁾. MSM– a naturally occurring nutrient also restores flexibility andpermeability of the cell wall thus aiding in the repair of worn out ligaments andtendons^(15).

Given alone these drugs produce mediocre results and higher dose of these compounds such as glucosamine, given in 1 – 2gm / day produces adverse effects. But this combination, selected on basis of varying mechanism of actions, caused a profound, statistically significant lowering of most serological parameters. This presumably potentiating effect of CMO was observed even at the minimal dose ranges of 100 mg. From the results of this study it was concluded that CMO, a acetylated fatty acid ester produced a statistically significant effect in reducing adjuvant induced arthritis⁽¹⁵⁾.

Though the actual mechanism of action of CMO as an anti-inflammatory agent is still hypothetical, its effect in reducing the signs of inflammation and improving joint mobility with the least possible adverse effects makes it a remarkable drug in the treatment of degenerative conditions like OA and auto immune diseases like RA.

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Received on 09.01.2012

Modified on 18.01.2012

Accepted on 24.01.2012

Research J. Pharmacology and Pharmacodynamics. 4(2): March - April, 2012, 91-93