BLACK TEA PREVENTS MONONUCLEAR CELL OXIDATIVE STRESS-INDUCED BONE LOSS 1287 Copyright © 2009 John Wiley & Sons, Ltd. Phytother. Res. 23, 1287–1294 (2009) DOI: 10.1002/ptr Copyright © 2009 John Wiley & Sons, Ltd. PHYTOTHERAPY RESEARCH Phytother. Res. 23, 1287–1294 (2009) Published online 10 March 2009 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/ptr.2719 Protective Action of Aqueous Black Tea (Camellia sinensis) Extract (BTE) against Ovariectomy-induced Oxidative Stress of Mononuclear Cells and its Associated Progression of Bone Loss Asankur Sekhar Das, Maitrayee Mukherjee, Dolan Das and Chandan Mitra* Department of Physiology, Presidency College, Kolkata 700 073, India The protective action of aqueous black tea extract (BTE) against ovariectomy-induced oxidative stress of mononuclear cells and its associated progression of bone loss was demonstrated in this study. Eighteen female adult 6-month-old Wistar albino rats were divided into three groups: sham-control (A), bilaterally ovariectomized (B) and bilaterally ovariectomized + BTE supplemented (C). Studies included the measurement of oxidative (nitric oxide, lipid peroxidation) and antioxidative (superoxide dismutase, catalase) markers, inflammatory cytokines (IL-6, TNF- α α α), osteoclast differentiation factor (RANKL) and bone resorption markers (tartrate- resistant acid phosphatase and hydroxyproline). Also quantitative histomorphometry and histological studies were undertaken. The bone breaking force was measured. The results indicate that BTE was effective in preserving and restoring skeletal health by reducing the number of active osteoclasts. Such changes with BTE supplementation were steadily linked with the reduced oxidative stress of mononuclear cells, serum levels of bone resorbing cytokines, osteoclast differentiation factor and resorption markers. The results of the bone breaking force, histological and histomorphometric analyses further supported the hypothesis. This study suggests that BTE has both protective and restorative actions against ovariectomy-induced mononuclear cell oxidative stress and associated bone loss. Copyright © 2009 John Wiley & Sons, Ltd. Keywords: ovariectomy; mononuclear cell oxidative stress; cytokines; bone loss; aqueous black tea extract. Received 28 June 2008 Revised 10 September 2008 Accepted 11 September 2008 * Correspondence to: Professor Chandan Mitra, 14/17A, Golf Club Road, Kolkata 700 033, India. E-mail: chandan_mitrapresi@yahoo.com Contract/grant sponsor: National Tea Research Foundation (NTRF), Government of India; contract/grant number: RL:17(20)/2001/809. INTRODUCTION Evidence suggests that the decline in ovarian function with menopause is associated with spontaneous increases in many proinflammatory cytokines. Among those cyto- kines that have drawn the most attention are IL-1, IL-6 and TNF-α. Spontaneous increases in the expres- sion and secretion of these proinflammatory cytokines with estrogen deficiency were first noted several years ago in ex vivo cultures of circulating monocytes (Pacifici et al., 1989, 1991), bone marrow macrophages (Kitazawa et al., 1994; Bismar et al., 1995) and osteoblasts (Passeri et al., 1993). It is now well known that these cytokines are stimulatory to bone resorption by increas- ing the activity and differentiation of osteoclasts, the primary bone-resorbing cells. Interestingly, these osteo- clasts are hematopoietic in origin and derived from the monocyte/macrophage lineage (Yamada et al., 2003; Reddy, 2004). This understanding that the osteoclast has a relation with the macrophage, a cell that secretes and is regulated by inflammatory cytokines itself, raised the likelihood that the same molecules also regulate the bone resorptive cells. In fact, TNF-α and IL-1 proved agonistic in this regard (Teitelbaum, 2004). Not only that, macrophages are also widely recognized to play a central role in the immune and inflammatory re- sponse (Newsholme et al., 1996) and have been ascribed as a major source of TNF-α in vivo (Yang et al., 1998). Another mononuclear cell that plays a role in the pathogenesis of postmenopausal bone loss is the T lymphocyte (Teitelbaum, 2004). The observation that T-cell deficient nu/nu (nude) mice are protected from ovariectomy-induced bone loss strongly suggests the role of these cells in estrogen deficiency induced bone loss and indicates a relationship between skeletal regu- lation and immune system (Cenci et al., 2000). Reports further indicate that the mechanism by which estrogen deprivation enhances osteoclast recruitment appears to involve T-cell production of TNF-α and IL-1 (Teitelbaum, 2004). It was reported earlier that TNF-α is known to augment the production and activity of the osteoclasto- genic molecule RANKL (Lam et al., 2000; Pfeilschifter et al., 2002) and potently stimulate estrogen regulated cytokines to mediate ovariectomy-induced bone loss, namely, IL-1, IL-6 and macrophage colony-stimulating factor (Pfeilschifter et al., 2002). In addition, an in vivo study by Cenci et al. (2003) revealed that estrogen deficiency is able to expand the number of TNF- producing T-cells by increasing T-cell proliferation and the lifespan of active T-cells in ovariectomized mice. Reports also suggest that ovariectomy selectively stimulates B