U 5100

The scavenging activity of DPPH stable free radicals in analyzed extracts as well as acceptor fluids was assessed as previously described [72 (link)]. Briefly, 150 μL of the tested samples was mixed with 2850 μL of 0.3 mM DPPH solution (in 96% (v/v) ethanol). The DPPH working solution was diluted with 70% (v/v) ethanol until its absorbance at 517 nm was 1.00 ± 0.02. The incubation time of the samples in the dark was 10 min. Analyses at a wavelength of 517 nm were performed with a UV–Vis Spectrophotometer (Hitachi U-5100, Tokyo, Japan). Antioxidant activity determined by the DPPH method was expressed as % of DPPH radical scavenging, which was calculated from the formula: $$\mathrm{\%}\mathrm{D}\mathrm{P}\mathrm{P}\mathrm{H}\mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}=\frac{\mathrm{A}\mathrm{c}-\mathrm{A}\mathrm{s}}{\mathrm{A}\mathrm{c}}\times 100$$
where
The total polyphenol content (TPC) in analyzed extracts as well as acceptor fluids was determined using Folin–Ciocalteu method, according to the methodology described earlier [11 (link)]. Briefly, 150 μL of the tested sample, 150 μL of Folin–Ciocalteu reagent (diluted tenfold with water.), 1350 μL of 0.01 M sodium carbonate solution, and 1350 μL of distilled water were mixed and subjected to a 15 min incubation at room temperature. Absorbance measurements were made at a wavelength of 765 nm (UV–Vis Spectrophotometer Hitachi U-5100, Hitachi, Tokyo, Japan). The results were expressed as gallic acid (GA) equivalents in µg·mL⁻¹. Three independent measurements were made.

The solubility of RES in different types of oils and emulsifiers were determined. An excess of RES powder was added into each vehicle followed by vortex mixing for 30 s. Then, the mixture was shaken at 37 °C in a water bath for 48 h and centrifuged at 10,000 rpm for 15 min (Mikro 22R, Hettich Zentrifugen, Tuttlingen, Germany) to separate the undissolved RES. The clear supernatant was diluted with appropriate 50% (v/v) methanol and measured spectrophotometrically at 310 nm (U-5100, Hitachi, Tokyo, Japan). The calibration cure of RES solution, constructed by UV-visible spectrophotometer (U-5100, Hitachi) at 310 nm, was in the range of 1–25 µg/mL.
The RES-SNEDDS preparation procedures were modified from a previous report [49 (link)]. Briefly, in the dark environment, RES (120 mg) was dissolved in 12 grams of oil phase (Capryol 90) by magnetic stirring to completely dissolve. The mixture of surfactant (Cremophor EL) and co-surfactant (Tween 20) were added in dropwise to prepare of a total weight of 20 grams and the resulting mixture was stirred for 30 min. Different ratios of oil phase, surfactants were prepared and examined to fine an optimal RES-SNEDDS. The formulation components of RES-SNEDDS were listed in Table 1.

Growth parameters were measured for all plantlets, including height, root collar diameter, and above- and belowground biomass. The dry weights of the shoots and roots were recorded after oven-drying at 70 °C until they reached a constant mass. The foliar nutrient concentration was determined on dried material in the tested plantlets. Dried leaves were milled and passed through a 0.25 mm sieve, and a sample of 0.5 g of leaf powder was taken for digestion with H₂SO₄ and H₂O₂ (v/v) 1:4. Nitrogen, P and K contents were determined by the Kjeldahl Method (Foss Kjeltec 8400; Foss Analytics, Hillerød, Hovedstaden, Denmark), ultraviolet–visible spectrophotometry (Hitachi U-5100; Hitachi, Tokyo, Japan) and atomic absorption spectrophotometry (Hitachi Polarized Zeeman Atomic Absorption Spectrophotometer ZA3000; Hitachi, Tokyo, Japan), respectively. In addition, the ratio of root:shoot growth was calculated to reflect the robustness of the plantlets and the efficiency of AMF inoculation (Tobar, Azcón & Barea, 1994 (link)). Six replicates were performed for each treatment and control.