The impact of selenium-rich green and black tea water extracts on bone health in vitro, and in an animal model of osteoporosis : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Health Sciences at Massey University, Palmerston North, New Zealand

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The consumption of tea, as a source of dietary antioxidants, is a natural nonpharmacotherapy approach that could provide beneficial effects on bone health and represent an alternative strategy for the prevention and management of osteoporosis throughout one’s life. While the benefits of tea and its bioactive chemical compounds on bone health have been increasingly investigated and reviewed, studies concerning the effects of tea with high selenium content have not yet been conducted. The purpose of the series of studies presented in this thesis was to test the hypotheses that green and black teas with high selenium content would be more effective in preventing postmenopausal bone loss than regular green and black teas, and that the positive effect of these teas on bone (if any), could be due their antioxidant and/or prebiotic-like properties. These hypotheses were investigated through a series of studies involving a variety of cellular assays, a young growing rat model, and an ovariectomy-induced bone loss rat model of postmenopausal osteoporosis. Four different teas derived from Camellia sinensis were assessed for their total phenolic content (TPC), antioxidant properties and prebiotic-like potential, which included a selenium-rich green tea (Se- GTE), a selenium-rich black tea (Se-BTE), a regular green tea (R-GTE) and a regular black tea (R-BTE). Aqueous tea extracts were prepared using different extraction temperatures and times to quantify the extraction efficiencies for TPC and antioxidant properties. TPC was measured using the Folin-Ciocalteu method, antioxidant activity was measured using the ferric-reducing antioxidant power (FRAP) and 2,2-diphenyl-1- picrylhydrazyl (DPPH) assays, and the prebiotic-like effect on two beneficial bacteria (Lactobacillus acidophilus and L. rhamnosus) was determined using the plate agar dilution method. Irrespective of tea selenium content, the results obtained for TPC, antioxidant properties and prebiotic-like potential of the investigated teas were highly variable dependent on the different types of tea. In addition, the optimal time and temperature of tea infusion for maximising TPC was determined to be 90 °C for 5 min (Chapter 4), which was then used as the standard method of preparation for aqueous tea extracts for the subsequent in vitro and in vivo work. Further, the freeze-dried aqueous tea extracts (0.001 to 10 μg/mL) were investigated for their osteogenic effects on murine pre-osteoblastic MC3T3-E1 (Subclone 4) cells, as assessed by the 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), alkaline phosphatase (ALP) activity, and Alizarin Red S (ARS) staining assays. The osteoprotective effect of the freeze-dried aqueous tea extracts against H2O2-induced oxidative stress during osteoblast differentiation was also evaluated. At low concentrations, all tea extracts showed an anabolic effect by enhancing matrix mineralisation in MC3T3-E1 cells. Moreover, the teas were capable of protecting and restoring the differentiated osteoblasts against the dysfunctional effects of H2O2-induced oxidative stress. These in vitro activities were irrespective of the selenium content, and were in a time- and concentration-dependent manner (Chapter 5). Next, their anti-osteoclastogenic effects were assessed by measuring tartrate-resistant acid phosphatase (TRAP) activity in receptor activator of nuclear factor kappa beta ligand (RANKL)-treated RAW 264.7 cells, while the numbers of TRAP-positive osteoclasts (TRAP+ OCLs) with five or more nuclei were quantified. All tea extracts (0.001 to 10 μg/mL), independent of selenium content, suppressed RANKL-induced osteoclastogenesis in a concentration-dependent manner, i.e. mostly significant at the higher concentrations (Chapter 6). In the first animal trial (Chapter 7), the effect consuming tea (1%, w/v) for four weeks on bone mass and strength were examined in young growing male Sprague-Dawley rats. No osteo-stimulative effects on bone parameters (i.e. serum bone resorption biomarker, bone mineral density and bone biomechanics) were observed in the rats during the rapid growth phase following tea consumption. Only Se-GTE showed prebiotic-like potential evaluated by changes in caecal parameters (i.e. decrease in caecal pH, decrease in numbers of Clostridium spp. (perfringens/histolyticum subgroup) and enhanced bacterial β-glucosidase enzyme activity). In the next animal trial (Chapter 8), the effects of eight-week consumption of tea (1%, w/v) on bone loss were assessed in ovariectomised mature adult female Sprague-Dawley rats. Only R-BTE significantly suppressed the serum bone resorption biomarker. Moreover, only Se-GTE and R-BTE demonstrated prebiotic-like potential in modulating intestinal microbiota composition, as seen by a marked decrease in caecal pH and enhanced activity of the bacterial β- glucosidase enzyme. Additionally, serum antioxidant capacity levels of the teas evaluated by FRAP assay in both animal trials showed mixed results. Based on the study findings, it is suggested that tea may exert stimulating effects on bone metabolism part-mediated through its prebiotic influence on gut microbiota, and not via a direct antioxidant mechanism. However, the exact mechanism underlying this effect remains unclear and needs to be investigated further. Taken together, these studies provide new insights into the potential antioxidant and prebiotic roles of teas with different levels of selenium, and their possible impact on bone health.
Osteoporosis, Nutritional aspects, Tea, Selenium in human nutrition, Research Subject Categories::INTERDISCIPLINARY RESEARCH AREAS::Domestic science and nutrition