Filter paper number 42

Barley (Hordeum vulgare L.) was cultivated in 2015 in the experimental field at the National Institute of Crop Science, Rural Development Administration, Jeonbuk, Korea. BS was prepared using the procedure reported in the literature [23 (link)]. Barley seeds were washed twice using deionized distilled water and imbibed in water at 18°C for 24 hr. The imbibed seeds were germinated in 65% humidity at 16°C in a normal light cycle (16/8 hr day/night). The germinating seeds and seedlings were harvested in liquid nitrogen 7 days after germination. The collected leaves were freeze-dried immediately after sampling. Prior to obtaining the BSE, the leaves were pulverised to 100 mesh. The masses of all the samples were based on dry weight. To determine the antiosteoporotic activities, the pulverised seeds (10 g) were extracted with 20 ml of the prethanol for 2 days at 4°C in the dark. The crude extracts were filtered through Whatman Number 42 filter paper to remove the sediment. The solvent was evaporated, and the prethanol extracts were obtained from the BS.

Determination of the samples’ antioxidant activity was done with the DPPH assay according to Subianto et al. (2013 (link)). In preparation for the assay, distilled water was added to the samples (1:1), centrifuged for 30 min at 5000 rpm, and filtered with Whatman number 42 filter paper. The supernatants were diluted fivefold to obtain the samples for DPPH assay. The samples and the DPPH solution reacted for 30 min and the absorbance (A) was measured at 517 nm in a UV–Vis spectrophotometer (Shimadzu, Japan). The Gallic acid standard curve was conducted with methanol as the solvent. The samples’ antioxidant activity was expressed in milligrams of Gallic acid equivalent per gram of yogurt (mg GAE·g⁻¹) and calculated according to Eq. (4): $$\%\text{Inhibition}=\left\{\left(\text{A control}-\text{A sample}\right)/\left(\text{A control}\right)\right\}\times 100\%.$$