Original Full Length Article Turbo methanol extract inhibits bone resorption through regulation of T cell function Babita Balakrishnan a , Madhavi M. Indap a , Surya P. Singh b , C. Murali Krishna b , Shubhada V. Chiplunkar c, ⁎ a Department of Zoology, The D. G. Ruparel College, Mahim, Mumbai 40016, India b Chilakapati Laboratory, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai 410210, India c Chiplunkar Laboratory, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai 410210, India abstract article info Article history: Received 2 April 2013 Revised 10 September 2013 Accepted 11 October 2013 Available online 18 October 2013 Edited by: David Burr Keywords: Turbo methanol extract Osteoporosis Bone resorption Osteoclastogenesis Raman spectroscopy TNFα Marine organisms have bioactive potential which has tremendous pharmaceutical promise. Emerging evidence highlights the importance of the interplay between bone and the immune system of which T lymphocytes and their product act as key regulators of bone resorption. In the present investigation we have analyzed the anti- osteoporotic effect of turbo methanol extract (TME) in the reversal of bone resoprtion. Forty-two female Swiss albino mice were used and randomly assigned into sham-operated group (sham) and six ovariectomized (OVX) subgroups, i.e. OVX with vehicle (OVX) that received daily oral administration of water ad libitum; OVX with estradiol (2 mg/kg/day); and OVX with different doses of TME i.e. TME 100 mg/kg, TME 50 mg/kg, TME 25 mg/kg and TME 12.5 mg/kg. Oral administration of TME or estradiol started on the second week after ovariectomy for a period of 4 weeks. We observed that the administration of TME increased the trabeculation in tibia and reduced the atrophy in the uterus. TME significantly decreased the serum alkaline phosphatase (ALP) and acid phosphatase (ACP) activity in OVX mice. Micro CT analysis revealed that the TME administration preserved the bone volume, connectivity density, trabecular number, trabecular thickness and trabecular separation in OVX mice. Bone mineralization was measured in different groups of mice by Raman spectroscopy. Reversal of bone resorption was observed in TME treated group of mice. To further investigate the mechanism of action of TME, we analyzed the T lymphocyte proliferation and profiles of cytokine TNFα and sRANKL in TME treated ovariectomized mice. Decrease in the elevation of T cell subsets was observed after the supplementation with TME. The extract significantly lowered the T cell proliferation responses to mitogens, phorbol 12-myristate 13-acetate (PMA) and ionomycin (Io) and phytohemagglutinin (PHA). A marked reduction in TNFα and sRANKL secretion in serum and TNFα in cell free supernatants of activated T lymphocytes was observed upon TME administration. TME could significantly inhibit the in vitro osteoclastogenesis and the bone resorption observed using artificial calcium coated slides. Collectively, these results indicate that TME has the potential to inhibit bone resorption and may prove to be a potential candidate for the development of an anti-osteoporosis drug. © 2013 Elsevier Inc. All rights reserved. Introduction Osteoporosis characterized by the loss of bone mass and strength that leads to fragility fractures has probably existed throughout human history but only recently became a major clinical dilemma as human life span increased. Postmenopausal osteoporosis has become a foremost problem with significant morbidity and mortality. After the onset of menopause, the cessation of the estrogen leads to increased bone turnover with an increase in bone resorption by osteoclasts, resulting in decreased bone mass [1]. This progressive bone loss caused by the sharp decrease in ovarian estrogen production can be prevented by estrogen replacement therapy (ERT) [2]. However, estrogen therapy recently became a subject of debate because clinical studies revealed an increased risk of breast cancer and coronary artery disease in women who take estrogen supplement [3]. Thus it is necessary to develop naturally occurring compounds with less undesirable side effects that can substitute or reduce the need for the drugs used currently. Estrogen deficiency induced osteoporosis leads to a marked stimulation of bone resorption which is caused primarily by increased osteoclast (OC) formation. Osteoclast formation is regulated by the production of RANKL (receptor activator of nuclear factor kappa-B ligand) and M-CSF (macrophage-colony stimulating factor) under the influence of stimulators of resorption leading to the maturation and activation of the osteoclast so formed to resorb bone. RANKL exists in two forms, soluble and membrane bound. Soluble form is secreted by T lymphocytes and membrane bound RANKL is expressed by the Bone 58 (2014) 114–125 Abbreviations: TME, turbo methanol extract; OVX, ovariectomized mice; ALP, alkaline phosphatase; ACP, acid phosphatase; TNFα, tumor necrosis factor α; PMA, phorbol 12-myristate 13-acetate; Io, ionomycin; PHA, phytohemagglutinin; RANKL, receptor activator of nuclear factor kappa-B ligand; M-CSF, macrophage-colony stimulating factor; sRANKL, soluble RANKL; OC, osteoclast; ERT, estrogen replacement therapy; α-MEM, α-minimum essential medium; FBS, fetal bovine serum. ⁎ Corresponding author. Fax: +91 22 27405085. E-mail address: schiplunkar@actrec.gov.in (S.V. Chiplunkar). 8756-3282/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bone.2013.10.008 Contents lists available at ScienceDirect Bone journal homepage: www.elsevier.com/locate/bone