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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2017
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Massey University
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Abstract
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.
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Keywords
Osteoporosis, Nutritional aspects, Tea, Selenium in human nutrition, Research Subject Categories::INTERDISCIPLINARY RESEARCH AREAS::Domestic science and nutrition