INTRODUCTION:

Inflammation is the reaction of vascular connective tissue to local injury. Acute inflammation is immediate and early response to an injurious agent and its major components are vascular events.Chronic inflammation is a prolonged duration (weeks andmonths) in which active inflammation of tissue destruction and attempts at repair are proceeding simultaneously.

Immune and inflammatory response are involved in many diseases and development of drugs to control these process is a major concern of pharma industry.^1-6

Inflammatory Mediators:

Inflammatory Mediators like histamine, cytokines, arachidonic acid metabolites, eicosanoids prostaglandins, leukotrienes, omega-3 polyunsaturated fatty acids, platelet-activating factor, Plasma proteins. NO (Nitric Oxide) plays a major role in causation of events. Currently NSAIDS andsteroids control inflammation by targeting of cellular events they prevents inflammation. iNOS (Inducible nitric oxide synthatase) is involved in the synthesis of NO is induced whenever inflammatory cells are activated by cytokines.^6-10

E.g.: TNF-α [tumor nechrosis factor], inter feron –gamma NO inactivates by anti proteases such as antitrypsin, decreases destruction of extra vascular matrix, inhibitsiNOS and posses anti-inflammatory by Diazepam.Diazepam binds with peripheral benzodiazepapine receptors in macrophases and endothelial cells to inhibit iNOS and reduce the inflammation. It acting on neutrophills, monocytes and other inflammatory cells.It also inhibits diapedesis and chromo toxins of the cells towards inflammatory stimulus.It also inhibits cytokine secretion by the cells in adrenal cortex it increases the synthesis and release of endogenous glucocortocoid cortisol.^10-15

Inflammation is mainly two types:

1. ACUTE INFLAMMATION:

Inflammation, usually of sudden onset, marked by the classical signs, in which vascular and oxidative processes predominate.^15-20

2. CHRONIC INFLAMMATION:

Prolonged and persistent inflammation marked chiefly by new connective tissue formation; it may be a continuation of an acute form or a prolonged low-grade form.

Inflammatory mediators are Histamine, Cytokine, Arachidonic acid metabolites, eicosanoids prostaglandins, leukotrienes, omega-3 polyunsaturated fatty acids, platelet-activating factor, Plasma proteins.^20-22

MATERIALS AND METHODS:

Materials:

DIAZEPAM: Diazepam first marketed as Valium by Hoffmann-La Roche, is a benzodiazepine drug. It is commonly used for treating anxiety, panic attacks, insomnia, seizures including status epilepticus, muscle spasms (such as in cases of tetanus), restless legs syndrome, alcohol withdrawal, benzodiazepine withdrawal, Opiate withdrawal syndrome and Ménière's disease. It may also be used before certain medical procedures (such as endoscopies) to reduce tension and anxiety, and in some surgical procedures to induce amnesia.

ACECLOFENAC:

Aceclofenac Sodium Anti Inflammatory Drug It has higher anti-inflammatory action than conventional NSAIDs. It is a cytokine inhibitor. Aceclofenac works by blocking the action of a substance in the body called cyclo-oxygenase. Cyclo-oxygenase is involved in the production of prostaglandins (chemicals in the body) which cause pain, swelling and inflammation. Aceclofenac is the glycolic acid ester of diclofenac. Aceclofenac is an orally administered phenyl acetic acid derivatives with effects on a variety of inflammatory mediators.

CARRAGEENAN:

It is a family of linear sulfated polysaccharides that are extracted from red edible seaweeds. They are widely used in the food industry, for their gelling, thickening, and stabilizing properties. Their main application is in dairy and meat products, due to their strong binding to food proteins. It is used for gived inflammation to mice.

SWISS ALBINO MICE:

Animals used :	specific pathogen-free SWISS ALBINO Mice
Weight Range :	25-30gms
Sex :	Female
Age of Animal :	8-9 weeks old
Method :	Animals were divided into 5 groups, of each group containing 6 animals .Swiss albino 30 mice's are divided in 5 groups.

INDUCING AGENTS ARE :

Group-1 : Carrageenan injection à 0.2 ml in subplantar injection

Group-2: Aceclofenac sodium à 25 mg/kg

Group-3: Diazepam à 4 mg/kg

Group-4: Combination (diazepam andaceclofenac sodium)

Group-5 : Called as negative control

Animal was taken from each group and anatomical marking made at the level of malleolus of the right hind paw of animal in order to fix a constant level up to which a animal paw dipped in the plethysmograph every time.All the animals were administered corresponding dose of the respective drug intrapertonially prior to subplantar injection of carrageenan. Paw volume measured by dipping into the mercury in the plethysmometer. Similar readings were taken at 30, 60,120 mins After carrageenan injection the time for onset of paw licking (in sec number of licks) in early phase and late phase.

RESULTS AND DISCUSSION:

Results:

Table 1: Onset of paw licking of various groups of animals

S. NO	GROUP	ANIMAL NO.	TIME (SEC)	MEAN
1	CARRAGEENAN	1 2 3 4 5 6	41 40 39 42 38 39	40
2	ACECLOFENAC SODIIUM	1 2 3 4 5 6	104 105 106 104 103 105	104.5
3	DIAZEPAM	1 2 3 4 5 6	220 221 223 235 220 219	223
4	COMBINATION(ACECLOFENAC SODIUM+DIAZEPAM)	1 2 3 4 5 6	95 96 94 93 92 98	94.6

Table 2: Paw licking (early phase) of various groups of animals

S. NO	GROUP	ANIMAL NO.	TIME (SEC)	MEAN
1	CARRAGEENAN	1 2 3 4 5 6	39 34 42 35 36 35	36.8
2	ACECLOFENAC SODIIUM	1 2 3 4 5 6	10 11 9 8 12 14	10.66
3	DIAZEPAM	1 2 3 4 5 6	3 4 2 3 5 4	3.5
4	COMBINATION(ACECLOFENAC SODIUM+DIAZEPAM)	1 2 3 4 5 6	10 11 8 9 11 12	8.6

Table 3:Paw score of various groups of animals

S. NO	GROUP	ANIMAL NO.	PAW SCORE	MEAN
1	CARRAGEENAN	1 2 3 4 5 6	3 2 3 3 3 3	2.8
2	ACECLOFENAC SODIIUM	1 2 3 4 5 6	2 2 2 3 2 2	2.1
3	DIAZEPAM	1 2 3 4 5 6	1 1 1 1 1 1	1
4	COMBINATION (ACECLOFENAC SODIUM+DIAZEPAM)	1 2 3 4 5 6	2 3 2 2 3 3	2.5
5	NORMAL	1 2 3 4 5 6	0	0

Table 4: Paw licking (late phase)of various groups of animals

S. NO	GROUP	ANIMAL NO.	TIME (SEC)	MEAN
1	CARRAGEENAN	1 2 3 4 5 6	10 11 12 13 14 11	11.8
2	ACECLOFENAC SODIIUM	1 2 3 4 5 6	5 6 7 4 5 4	5.1
3	DIAZEPAM	1 2 3 4 5 6	2 3 4 3 2 2	2.6
4	COMBINATION(ACECLOFENAC SODIUM+DIAZEPAM)	1 2 3 4 5 6	4 5 6 7 4 3	4.8

Table 5: Changes in paw volume of various groups of animals

S. NO	GROUP	N	30(MIN) MEAN	60(MIN) MEAN	120(MIN) MEAN
1	CARRAGEENAN	1	0.31	0.38	0.57
2	ACECLOFENAC SODIIUM	1	0.21	0.25	0.32
3	DIAZEPAM	1	0.13	0.11	0.15
4	COMBINATION (ACECLOFENAC SODIUM+ DIAZEPAM)	1	0.1	0.23	0.32

DISCUSSION:

— Diazepam binds to peripheral benzodiazepine receptor(PBR) in macrophages and endothelial cells to inhibit I NOS and reduce the formation of NO. Nitric oxide plays a major role in causation of vascular events of inflammation.

— iNOS, Enzyme involved in the synthesis of NO,is induced whenever inflammatory cells are activated by cytokines(ex:TNF,IFN-gamma) or other agents.

— It is used in stress and anxiety, generalized anxiety disorder, muscle spasms, insomnia, sleep disorder, depression.

— Diclofenac sodium acts by inhibiting cyclo-oxygenase and produces anti-inflammatory action. It is used in arthritis, pain, back pain - low, inflammation, osteoarthritis, rheumatoid arthritis.

— In most clinical situations aceclofeac sodium and diazepam are used in combination.we compared diazepam and aceclofenac sodium on carrageenan induced paw oedema .we found that there is no additive or synergistic effect of these two drugs in inflammation.

Analysis of paw edema in mice: Unpaired ‘t’ test was used for comparison between each group and to each time interval. For mice (Table no: 1): There was significant reduction in the paw volume for all the groups compared to control group. There was significant reduction in the paw volume with Diazepam in the dose of 4mg/kg body weight compared to aceclofenac sodium. Reduction of paw volume with diazepam 4mg/kg body weight was comparable to aceclofenac sodium, combination with control group of animlas. A significant reduction of paw volume with diazepam 4mg/kg body weight was more compared to aceclofenac Sodium and combination

Analysis of onset of paw score in and mice:

We observed a significant reduction of score of diagepam(4mg/kg), aceclofenac sodium,combination compared with control. By using unpaired t-test p<0.05 compared to control group of animals.

CONCLUSION:

Carrageenan induces inflammation is a good animal model to induce acute inflammation in mice.

The present study data support that the Carrageenan may act collaboratively to elicit inflammation leads to rise in paw volume. We compared the change in paw volume, pawlicking, paw score of aceclofenac sodium, diazepam, combination groups with respect to positive control group.

Diazepam(4mg/kg) had better anti inflammatory activity compared to aceclofenac sodium. However combination of diazepam and Aceclofenac sodium did not produce any additive/synergistic effect.

REFERENCES:

1. D.E.TRENTHAM, AS. TOWNES and A.H. KANG, J Exp Med 146 857-868 (1977).

2. Gabriel S. E., The epidemiology of rheumatoid arthritis. Rheum Dis Clin North Am. 27 (2001); 269–81.

3. Saegusa M., Murakami M., Nakatani Y. et al. Contribution of membrane-associated prostaglandin E2 synthase to bone resorption. J. Cell. Physiol. 197 (2003); 348–356.

4. T. Tanioka, Y. Nakatani, N. Semmyo, M. Murakami, I. Kudo, Molecular identification of cytosolic prostaglandin E2 synthase that is functionally coupledwith cyclooxygenase-1 in immediate prostaglandin E2 biosynthesis, J. Biol. Chem. 275 (2000); 32775 – 32782.

5. Winter CA, Risley EA, Nauss GW. Carrageenan induced edema in hind paw of rats as an assay for anti-inflammatory drugs. ProcSocexpBiol Med. 111: 544-547, (1962).

6. 7.F. S. Laroux, K. P. Pavlick, I. N. Hines, S. Kawachi, H. Harada, S. Bharwani, J. M. Hoffman and M. B. Grisham., Acta Physiologica Scandinavia., 2001, 173 (1), 113.

7. Bertram G Katzung., Basic and Clinical pharmacology., McGraw-Hill Medical., 2006, 10th edition, 308.

8. Kumar, Abbas and Fausto., Robbins and Cotran Pathologic Basis of Disease., Saunders., 2004, 7thedition, 48.

9. Ricardo Lazzarini, Paulo César Maiorka, Jun Liu, Vassilios Papadopoulos and Joao Palermo-Neto., Science Direct – Life sciences., 2006, 78 (26), 3027.

10. R.C. Farges., Current medicinal chemistry – Anti inflammatory and anti allergy agents., 2006, 2 (4), 409.

11. R. Sandra,Torres, Tania S. Frode, M. Geisson, G. Nardi, Natalio Vita, Rennee Reeb, Pascual, Ferrara, M. Rosa, Ribeiro-do-Valle and Roseli C. Farges., Science Direct: European journal of

12. Magdalena Bujalska, Jan Tatarkiewicz, Anna de Corde and Stanisław Witold Gumułka., Pharmacology., 2008, 81(2), 151.

13. Christopher J. Morris., Methods in Molecular Biology., 2003, 225(2), 115.

14. Edward F. Webb and Don E. Griswold., Journal of Pharmacological Methods., 1984,12 (2), 149

15. Ferrero-Miliani L, Nielsen OH, Andersen PS, Girardin SE (February 2007). "Chronic inflammation: importance of NOD2 and NALP3 in interleukin-1beta generation". Clin. Exp.Immunol. 147 (2): 061127015327006––.

16. Abbas A.B.; Lichtman A.H. (2009). "Ch.2 Innate Immunity". In Saunders (Elsevier). Basic Immunology. Functions and disorders of the immune system (3rd ed.). ISBN 978-1-4160-4688-2.

17. Cotran; Kumar, Collins (1998). Robbins Pathologic Basis of Disease. Philadelphia: W.B Saunders Company. ISBN 0-7216-7335-X.

18. Parakrama Chandrasoma, Clive R. Taylor (ca. 2005). "Part A. General Pathology, Section II. The Host Response to Injury, Chapter 3. The Acute Inflammatory Response, sub-section Cardinal Clinical Signs". Concise Pathology (3rd edition (Computer file) ed.). New York, N.Y.: McGraw-Hill. ISBN 0-8385-1499-5. OCLC 150148447. Retrieved 2008-11-05.

19. A Massage Therapist Guide to Pathology Ruth Werner (2009). A massage Therapist Guide to Pathology (4th ed.). Philadelphia, PA and Baltimore, MD: Wolters Kluwer.

20. Vogel, Wolfgang H.; Berke, Andreas (2009). Brief History of Vision and Ocular Medicine. Kugler Publications. p. 97. ISBN 90-6299-220-X.

21. Porth, Carol (2007). Essentials of pahtophysiology: concepts of altered health states. Hagerstown, MD: Lippincott Williams and Wilkins. p. 270. ISBN 0-7817-7087-4.

22. Dormandy, Thomas (2006). The worst of evils: man's fight against pain. New Haven, Conn: Yale University Press. p. 22. ISBN 0-300-11322-6.

Received on 01.12.2017 Modified on 11.03.2018

Res. J. Pharmacology and Pharmacodynamics.2018; 10(2): 61-65.

DOI: 10.5958/2321-5836.2018.00010.1

GROUP	ONSET OF PAW LICKING	NO.OF LICKS		PAW VOLUME				PAW SCORE
GROUP	ONSET OF PAW LICKING	EARLY	LATE	0min	30min	60min	120min	PAW SCORE
CARRAGEENAN (0.1%)	39.8	36.67	11.83	0	0.3	0.38	0.57	3
ACECLOFENAC SODIUM(25mg/kg)	104.5	10.67	5.17	0	0.2	0.27	0.32	2.5
DIAZEPAM(4mg/kg)	22.3	3.5	2.67	0	0.1	0.12	0.15	2
COMBINATION	94.6	10.17	4.83	0	0.1	0.23	0.32	2.1