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Vascular [calcium ions (Ca2+)] was analysed using a colorimetric calcium assay.
[Ca2+] from the vessels was determined using a colorimetric Calcium Detection Kit (Abcam; Cambridge, UK) following the manufacturer's instructions.
For osteoclast-resorptive function analyses, osteoclast differentiation was conducted in OsteoAssay bone plates (Lonza, Basel, Switzerland), and osteoclast activity was quantified as calcium release from bone into culture medium using a colorimetric calcium detection kit (Abcam, Cambridge, Massachusetts).
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Plasma total calcium concentration (mM) was measured using a colorimetric assay (o-Cresolphtalein, calcium kit, Spinreact, Girona, Spain) in duplicates, and differences between groups were analyzed by two-way ANOVA followed by Tukey' test at a statistical significance level of p < 0.05.
Synovial fluid calcium concentrations were determined using a colorimetric assay based on formation of a chromogenic complex between free calcium and o-cresolphthalein (Abcam, Cambridge, UK).
The concentration of total calcium was measured in the culture supernatants after resorption using a colorimetric assay and a Hitachi 912 Automatic Analyzer (Roche Diagnostics).
Mg2+ was measured using a colorimetric technique, and phosphate was measured using an ultraviolet technique.
Total flavonoids content was determined using a colorimetric method as described by Heimler et al. [26].
DPPH was determined using a colorimetric method as described by Chen and Ho [29].
Condensed tannin content was determined using a colorimetric method as described by Broadhurst and Jones [27].
Urinary Crt was measured using a colorimetric activity assay (R&D Systems, UK).
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