IMMUNOMODULATORY ACTIVITY OF INDIGOFERA TINCTORIA LEAF EXTRACT ON IN VITRO MACROPHAGE RESPONSES AND LYMPHOCYTE PROLIFERATION
Objective: In this study the effects of ethanolic and aqueous extracts of Indigofera tinctoria were investigated on the functions of peritoneal macrophages and peripheral blood lymphocytes isolated from Wistar rats through in vitro studies.
Methods: Macrophage functions were determined through Nitric oxide (NO) estimation, Arginase production, lysosomal, phagocytic activities and analysis of proinflammatory cytokines (TNF-Î± and IL-1Î²) production. The lymphocytes proliferation was analyzed by MTT assay.
Results: The results indicated that both ethanolic and aqueous extracts of I. tinctoria were enhanced nitric oxide (NO) production at 50 Âµg/ml whereas it suppressed the production of arginase at 100 Âµg/ml by the macrophage. Moreover lysosomal and phagocytic activity of the macrophage was enhanced by the ethanolic and aqueous extracts at a dose of 100 Âµg/ml. The extracts also enhanced the production of pro-inflammatory cytokines by the macrophages. With the lymphocytes, the proliferation was significantly increased by ethanolic and aqueous extracts in the EC50 value of 32.27 Âµg/ml and 49.06 Âµg/ml after 48 h incubation.
Conclusion: Collectively the results demonstrated that both extracts of I. tinctoria enhanced innate as well as adaptive immune response and proved the immunostimulating potential of the plant I. tinctoria.
Keywords: Indigofera tinctoria, Immunomodulation, Macrophage, Lymphocyte proliferation
Stanilove SA, Dobreva ZG, Slavov ES, Mitera LD. C3 binding glycoprotein from Cuscuta europea induced different cytokine profiles from human PBMC compared to other plant and bacterial immunomodulators. Int Immunopharmacol 2005;5:723-34.
Jakhar R, Paul S, Chauhan AK, Kang SC. Morin hydrate augments phagocytosis mechanism and inhibits LPS-induced autophagic signaling in murine macrophages. Int Immunopharmacol 2014;22:356-65.
Harizi H, Chaabane F, Ghedira K, Chekir-Ghedira L. Inhibition of pro-inflammatory macrophage responses and lymphocyte proliferation in vitro by ethyl acetate leaf extract from Daphne gnidium. Cell Immunol 2011;267:94-101.
Littman DR, Rudensky AY. TH17 and regulatory T-cells in mediating and restraining inflammation. Cell 2010;19:845-58.
Mogensen TH. Pathogen recognition and inflammatory signaling in innate immune defenses. Clin Microbiol Rev 2009;22:240-73.
Thomas JK, Barbara AO, Richard AG. Eds. Kuby Immunology. 6th Edition. W. H. Freeman and Company, London; 2006.
Janeway CA, Travers P, Walport M, Copra JD. Eds. Immunobiology: Immune System in Health and Disease. 7th Edition. TX: Garland Publishing, Houston; 2008.
Salem ML. Immunomodulatory and therapeutic properties of Nigella sativa seed. Intl Immunopharmacol 2005;15:1749-70.
Tyagi KP, Rai VK, Pahira RK, Kumar SS, Singh Y, Sharma M, et al. Preliminary phytochemical screening and evaluation of anti-inflammatory activities of ethanolic extract of leaves of Indigofera tinctoria linn. J Curr Pharm Res 2010;3:47-50.
Kameswaran TR, Ramanibai R. The anti-proliferative activity of a flavanoidal fraction of Indigofera tinctoria is through cell cycle arrest and apoptotic pathway in A-549 cell lines. J Biol Sci 2008;8:584-90.
Ramanibai R, Boothapandi M, Madhavarani A. Preliminary phytochemical screening and in vitro antioxidant activities of aqueous extract of Indigofera tinctoria and Indigofera astragalina. Int J Drug Res Technol 2014;4:46-54.
Han R. Highlights on the studies of anti-cancer drugs delivered from plants in china. Stem Cells 1994;12:53-63.
Singh B, Saxena AK, Chandan BK, Bhardwaj V, Gupta VN, Suri OP, et al. Hepatoprotective activity of indigo toneâ€“a bioactive fraction from Indigofera tinctoria linn. Phytother Res 2001;15:294-7.
Moretao MP, Buchi DF, Gorin PA, Iacomini M, Oliveira MB. Effect of an acidic heteropolysaccharide (ARAGAL) from the gum of Anadenanthera colubrina (Angico branco) on peritoneal macrophage function. Immunol Lett 2003;31:175-85.
Sladowski D, Steer S, Clothier R, Balls M. An improved MTT assay. J Immunol Meth 1993;157:203-7.
Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and [N]-nitrate in biological fluids. Anal Biochem 1982;126:131-8.
Munder M, Eichmann K, Modolell M. Alternative metabolic states in murine macrophages reflected by the nitric oxide synthase/arginase balance: Competitive regulation by CD4+T-cells correlates with TH1/TH2 phenotype. J Immunol 1998;160:5347-54.
Plytycz B, Rozanowska M, Seljelid R. Quantification of neutral red pinocytosis by small numbers of adherent cells: comparative studies. Folia Biol 1992;40:3-9.
Suzuki I, Tanaka H, Kinoshita A, Oikawa S, Osawa M, Yadomae T. Effect of orally-administered ï¢-glucan on macrophage function in mice. Int J Immunopharmacol 1990;12:675-84.
Rainard P. A colorimetric microassay for opsonins by reduction of NBT in phagocytizing bovine polymorphs. J Imunol Meth 1986;90:197-201.
Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application for proliferation and cytotoxicity assays. J Imunol Meth 1983;65:55-63.
Boyum A. Isolation of mononuclear cells and granulocytes from human blood: isolation of mononuclear cells by one centrifugation and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl 1968;97:77-89.
Davicino R, Mattar A, Casali Y, Porporatto C, Correa SG, Micalizzi B. In vivo immunomodulatory effects of aqueous extracts of Larrea divaricata Cav. Immunopharmacol Immunotoxicol 2007;29:351-66.
Limem-Ben Amor I, Harizi H, Ghedira K, Chekir-Ghedira L. Leaf extracts from Phlomis crinita Cav. subs. mauritanica Munby affects immune cell functions in vitro, Immunopharmacol Immunotoxicol 2010;29:1-6.
Agarwal SS, Singh VK. Immunomodulators: a review of studies on Indian medicinal plants and synthetic peptides: part I. Medicinal plants. Proc Indian Nat Sci Acad 1999;65:179-204.
Limem-Ben Amor I, Skandrani I, Boubaker J, Ben SghaÃ¯er M, Neffati A, Bhouri W, et al. Investigation of the biological activity of polar extracts isolated from Phlomis crinita Cav ssp. mauritanica Munby. Drug Chem Toxicol 2009;32:38-46.
Chen TL, Chang CC, Lin YL, Ueng YF, Chen R. Signal-transducing mechanisms of ketamine-caused inhibition of IL-1 gene expression in LPS-stimulated murine macrophage-like RAW264.7 cells. Toxicol Appl Pharmacol 2009;240:15-25.
Li X, Xu W. TLR4-mediated activation of macrophages by polysaccharide fraction from Polyporus umbellatus (pers.) Fries. J Ethnopharmacol 2011;135:1-6.
Elmowalid G, Amar AM, Ahmad AA. Nigella sativa seed extract: Enhancement of sheep macrophage immune functions in vitro. Res Vet Sci 2013;95:437-43.
Gobert AP, Cheng Y, Akhtar M, Mersey BD, Blumberg DR, Cross RK, et al. Protective role of arginase in a mouse model of colitis. J Immunol 2004;173:2109-17.
Zhang CX, Dai ZR. Immunomodulatory activities on macrophage of a polysaccharide from Sipunculus nudus L. Food Chem Toxicol 2011;49:2961-7.
Cheng A, Wan F, Wang J, Jin Z, Xu X. Macrophage immunomodulatory activity of polysaccharides isolated from Glycyrrhiza uralensis fish. Int Immunopharmacol 2008;8:43-50.
Bune AJ, Hayman AR, Evans MJ, Cox TM. Mice lacking tartrate-resistant acid phosphatase (Acp 5) have disordered macrophage inflammatory responses and reduced clearance of the pathogen, Staphylococcus aureus. Immunology 2001;102:103-13.
Manosroi A, Saraphanchotiwitthaya A, Manosroi J. Immunomodulatory activities of Clausena excavata Burm. f. wood extracts. J Ethnopharmacol 2003;89:155-60.
Delcenserie V, Martel D, Lamoureux M, Amiot J, Boutin Y, Roy D. Immunomodulatory effects of probiotics in the intestinal tract. Curr Issues Mol Biol 2008;10:37-54.
Manosroi A, Saraphanchotiwitthaya A, Manosroi J. In vitro immunomodulatory effect of Pouteria cambodiana (Pierre ex Dubard) Baehni extract. J Ethnopharmacol 2005;101:90-4.
Li D, Fu Y, Zhang W, Su G, Liu B, Guo M, et al. Salidroside attenuates inflammatory responses by suppressing NF-ï«B and mitogen-activated protein kinase activation in LPS-induced mastitis in mice. Inflamm Res 2013;62:9-15.
Chen ML, Wu S, Tsai TC, Wang LK, Chou WM, Tsai FM. Effect of aqueous extract of Tournefortia sarmentosa on the regulation of macrophage immune response. Intl Immunopharmacol 2013;17:1002-8.
Ho CY, Lau CB, Kim CF, Leung KN, Fung KP, Tse TF, et al. Differential effect of Coriolus versicolor (Yunzhi) extract on cytokine production by murine lymphocytes in vitro. Int Immunopharmacol 2004;4:1549-57.
Kim MJ, Kim HN, Kang KS. The methanol extract of Dioscoreae rhizoma inhibits pro-inflammatory cytokines and mediators in the synoviocytes of rheumatoid arthritis. Intl Immunopharmacol 2004;4:1489-97.