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Indian Journal of Pharmacology, Vol. 37, No. 6, November-December, 2005, pp. 371-375 Research Paper Effect of Aloe vera on nitric oxide production by macrophages during inflammation #*D. Sarkar, **A. Dutta, *M. Das, *K. Sarkar, **C. Mandal, *M. Chatterjee *Department of Pharmacology, Dr. BC Roy Postgraduate Institute of Basic Medical Sciences, I.P.G.M.E.R., 244B Acharya JC Bose Road, Kolkata- 700 020 and **Department of Immunobiology, Indian Institute of Chemical Biology, Kolkata. #Present address: Depatment of Zoology, Dinabandhu Mahavidyalaya, Bongaon, North 24 Parganas, West Bengal. Code Number: ph05098 Abstract OBJECTIVE : To demonstrate the mechanism of action mediating the acute and chronic antiinflammatory activity of leafy exudate of Aloe vera (AVL) in animal models of inflammation.MATERIALS AND METHODS : The acute antiinflammatory activity of AVL was evaluated using carrageenan and dextran as phlogistic agents while its chronic antiinflammatory effect was investigated in a complete Freund's adjuvant-induced model of arthritis. The degree of inflammation in all models was measured plethysmographically. The effect of AVL on nitric oxide production in mouse peritoneal macrophages was measured by the Griess reagent. RESULTS : AVL (25 mg/kg) significantly reduced carrageenan and dextran-induced pedal edema in rats by 61.9% and 61.7%, respectively. In the Freund's adjuvant-induced model of chronic inflammation, AVL showed chronic antiinflammatory activity but failed to decrease the arthritic index indicating the absence of antiarthritic activity. AVL (10 µg/ml) caused a decrease in NO production in macrophages without causing toxicity. CONCLUSION : AVL possesses acute and chronic antiinflammatory activity, which is partly mediated by reduced production of NO, which in turn prevents the release of inflammatory mediators. Keywords: Antiinflammatory activity, acute inflammation, chronic inflammation, arthritis, inflammatory mediators. Introduction Inflammation is generally considered as an essentially protective response to tissue injury caused by noxious physical, chemical or microbiological stimulus. It is a complex process involving various mediators, such as prostaglandins, leukotrienes and platelet activating factor.[1] The major macrophage derived inflammatory mediators such as pro-inflammatory cytokines, tumour necrosis factor-α (TNF-α) and the reactive free radical nitric oxide (NO) synthesized by inducible NO synthase (iNOS), contribute to the development of inflammatory diseases.[2] Thus, inhibition of the excessive production of TNF-α and/or NO could be employed as criteria to evaluate potential antiinflammatory compounds. The current management of inflammatory diseases is limited to the use of antiinflammatory drugs whose chronic administration is associated with several adverse effects. Plant-derived products are slowly emerging as a viable alternative because they are cheap, abundantly available and relatively less toxic. The genus Aloe belongs to the Asphodelaceae family, Alooideae subfamily, and comprises about 420 species of succulent plants. They are indigenous to southern and eastern Africa and Madagascar but have been introduced in other tropical countries, the Mediterranean area and the West Indies.[3] Aloe has been used as a folk medicine for 3000 years and in the last decade Aloe vera is being extensively used in health drinks, topical creams, toiletries and cosmetics.[4] From the leaves of Aloe plants, three types of commercial products are obtained namely, the dried exudate, excreted from the aloin cells present in the zone of the vascular bundle, the gel, a mucilaginous juice present in the centre of the leaf and the oil, extracted by organic solvents.[3] The dried exudate is used as a bitter in alcoholic beverages while the oil, comprising the fatty fraction of the leaf is used primarily in the cosmetic industry as a pigment carrier and soothing agent. Studies have shown that the gel component has antiinflammatory activity[5] that is mediated through antibradykinin[6] activity and inhibition of prostaglandin production.[7] However, no studies have been undertaken with regard to the antiinflammatory activity of the leaf exudate. Accordingly, in this study, we have evaluated the antiinflammatory potential of Aloe vera leaf exudate as also demonstrated that this antiinflammatory activity is mediated partly via reduction of nitric oxide production in macrophages. Materials and Methods Animals Chemicals and drugs Preparation of plant extract Tests for acute inflammation Carrageenan or dextran-induced edema where Ec is the edema volume of the control group and Et is the edema volume of the treated group. Test of chronic inflammation Freund′s complete adjuvant induced arthritic model Primary lesions refer to the edema formation in the injected hind paw that peaks 3-5 days after injection of the phlogistic agent and is measured on day 5 by calculating the percent inhibition of the edema volume of the injected paw using the formula described above. Secondary lesions are immunologically mediated changes characterized by inflammation of the non-injected sites (hindleg, forepaw, ears nose and tail) decrease in weight and occur after a delay of 11-12 days. Accordingly, secondary lesions were evaluated by calculating the percent inhibition of paw volume of the non-injected right paw over control on day 21 and using an arthritic index as the sum of scores according to the method of Schorlemmer.[12] Lesion site Nature of lesion Score Preparation of mouse peritoneal macrophages To elicit activated peritoneal macrophages, mice were injected i.p. with starch [2% in phosphate buffered saline (PBS), 0.02 M, pH 7.2, 2 ml/mice]. After three days, the peritoneal cells comprising principally macrophages were lavaged with sterile PBS (10 ml). The exudate was centrifuged at 4000 rpm for 10 min and the resultant cell pellet washed in PBS and finally resuspended in complete RPMI-1640 phenol red free medium supplemented with 10% fetal calf serum (FCS).[13] Macrophage viability (>95%) was confirmed by the Trypan blue dye exclusion technique.Measurement of nitric oxide (NO) production in macrophages Macrophages obtained by peritoneal lavage as described above were seeded (5 x 106sub/ml) in RPMI 1640 phenol red free medium supplemented with 10% FCS in Petri dishes (35 mm diameter) and incubated at 37°C, 5% CO2 for 4 hrs. AVL (0 - 10.0 µg/ml) was added and incubated for an additional 48 hrs. At the end of 48 h, 0.5 ml of the supernatant was withdrawn and nitrite accumulation as an indicator of NO production was measured using Griess reagent.[14] Briefly, 0.5 ml of Griess reagent [mixture of 1:1 of napthylethylenediamine dihydrochloride (0.1% in water) and sulphanilamide (1% in 5% phosphoric acid)] was added to 0.5 ml of supernatant and incubated in the dark at room temperature (25-30°C) for 10 min. Finally, absorbance at 546 nm was measured spectrophotometrically and a standard curve using sodium nitrite was used to calculate concentrations of nitrite. To demonstrate specificity, macrophages were incubated in the presence of N-monomethyl arginine [(L-NMMA), 0.1mM] an established inhibitor of NO production.Cytotoxicity assay To determine the viability of macrophages in the presence of AVL or DMSO, they were seeded in 96-well tissue culture plates (1 x 106sub cells/ml of RPMI 1640, supplemented with 10% FCS, 0.2 ml per well) and incubated with either AVL (0 - 10 mg/ml) or DMSO (0-1%) alone for 48 hrs. At the end of drug exposure, the MTS assay was performed.[15] Briefly, MTS [{3-(4, 5 dimethylthiazol-2-yl)-5-(carboxymethoxy-phenyl}-2-(4- sulphophenyl)-2H tetrazolium] was prepared (2 mg/ml in PBS) and stored in dark at -20°C. Another stock solution of phenazine methosulphate (PMS) was prepared (0.92 mg/ml in PBS) and stored -20°C. Just before use, MTS and PMS were mixed in the ratio of 20:1 and 20 µl of the solution was added to each well. The plates were then incubated at 37°C for 3 hrs and absorbances measured at 490 nm using a Multiskan ELISA reader (BioRad, USA). Statistical analysis Results AVL possesses strong acute antiinflammatory activity in carrageenan
and dextran-induced animal models of inflammation Chronic
antiinflammatory activity of AVL in a complete Freund′s adjuvant-induced
model of arthritis. With regard to the mean arthritic index, the score of AVL-treated animals was not different from that of the control group being 2.0 vs. 1.8, respectively, indicating that AVL does not possess antiarthritic activity. As expected, the dexamethasone-treated group showed profound antiarthritic activity since it decreased the arthritic index almost 10-fold being 0.17. AVL-treated macrophages showed decreased nitric oxide (NO) production Toxicity of AVL in macrophages Discussion The inhibition of carrageenan-induced inflammation in rats is an established model to screen compounds for potential antiinflammatory activity. According to Vinegar et al . (1987),[16] the development of carrageenan-induced edema is biphasic; the first phase occurs within one hour of carrageenan administration and is attributed to the release of cytoplasmic enzymes, histamine and serotonin, from the mast cells. The second phase (>1.0 h) is mediated by an increased release of prostaglandins in the inflammatory area and continuity between the two phases is provided by kinins. With regard to dextran-induced edema, it has been reported to be mediated mainly by histamine and 5-HT released by the mast cells.[17] Taken together, it suggests that as AVL possesses potent acute antiinflammatory activity [Table - 1][Table - 2], it is mediated possibly due to inhibition of the synthesis and/or release of mediators of inflammation, principally the prostaglandins. This can only be defined after developing models of inflammation using specific mediators. The immunologically mediated complete Freund′s adjuvant arthritic model of chronic inflammation is considered as the best available experimental model of rheumatoid arthritis.[18] This method in rats was originally developed by Pearson et al . (1963),[19] wherein an injection of complete Freund′s adjuvant in the rat hind paw induces inflammation. It has been proposed that bacterial peptido-glycan and muramyl dipeptide are important nonspecific immunogenic contributory components. In this model of immunologically mediated chronic synovial inflammation and arthritis, macrophages play a central role. After activation they are capable of synthesizing mediators such as PGE2 and cytokines such as TNF-α and IL-1. In turn, these synthetic products induce the production of a variety of enzymes which initiate cartilage and bone destruction.[20], [21] NO is an important physiological messenger and effector molecule in many biological systems, including immunological, neuronal and cardiovascular tissues.[22] Nitric oxide, in macrophages, is produced as a free radical by iNOS by catalyzing the oxidation of guanidino nitrogen of L-arginine, thereby converting L- arginine to L-citrulline.[22] NO is an important signalling and effector molecule in inflammation and immunity as it is known to couple with superoxides to form peroxynitrite. These, in turn, induce the production of prostaglandin endoperoxide synthase from monocytes/macrophages resulting in enhanced synthesis of prostaglandins, established mediators of inflammation.[23] The reduced NO production by macrophages in the presence of AVL suggests that AVL by decreasing the release of NO, inhibits the release of inflammatory inhibitors such as prostaglandins resulting in suppression of inflammation. This can be confirmed by measuring the expression of iNOS in macrophages; such studies are ongoing. Acknowledgements The work received financial support from the Council of Scientific and Industrial Research, Govt. of India and the University Grants Commission, New Delhi. DS was a recipient of a Junior Scholarship from the Lady Tata Trust. We express our sincere thanks to Sri Ramphal Mullick for his excellent technical assistance.References
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