U 5100 spectrophotometer

In order to evaluate the encapsulation efficiency of therapeutic compounds in the optimized bilosomal formulation, UV-Vis spectroscopy was applied. Measurements were performed using a Hitachi U-5100 spectrophotometer (Hitachi, Mannheim, Germany) with a 1 cm path length quartz cell. The active cargo content was quantified at λ_CUR = 426 nm and λ_MB = 666 nm after the disruption of bilosomes with THF:water (1:1). The substance concentration was calculated using the calibration plot. The percentage of encapsulation efficiency was quantified using the Equation (1): given below.
$\mathrm{Percentage} \mathrm{encapsulation} \mathrm{efficiency} \left(\mathrm{EE}\%\right)= \frac{drug content in bilosomes}{total drug added}\times 100\%$

The antioxidant activity of infusions was evaluated by several in vitro methods. The ability to scavenge free radicals (RSA [%]) was assessed by the DPPH and ABTS methods. Moreover, the ability of samples to reduce ferric and cupric ions was evaluated using FRAP (ferric reducing antioxidant power), PFRAP (potassium ferricyanide reducing power), and CUPRAC (cupric ion reducing antioxidant capacity) methods. The evaluation of antioxidant activity by DPPH, ABTS, and FRAP methods was performed as described by Muzykiewicz et al. [47 (link)]. To evaluate ferric reducing capacity of infusions, the FRAP method, as described by Apak et al. [48 (link)], and the PFRAP technique (with slight modifications), according to Jayaprakasha et al. [49 (link)], were used. The incubation time was reduced to 10 min and absorbance was measured at 734 nm. The spectrophotometric measurements were performed in 1 cm cuvettes using Hitachi U-5100 spectrophotometer (Japan). In DPPH and ABTS methods the activity was expressed as RSA [%], whereas in CUPRAC technique as Trolox equivalents (TEAC)—mg Trolox/g RM (raw material). The reducing power evaluated using FRAP and PFRAP method was presented as FeSO₄ equivalents—mg FeSO₄/g RM. Three samples were prepared from each extract and the results are presented as an arithmetic mean ± standard deviation (SD).

Hitachi U 5100 spectrophotometer (Japan, Tokyo) connected with computer for controlling of measurement and data analyzing, temperature stabilizer, and glass cuvette with 1 cm optical path was used.

The yeast medium containing the secreted DCcathB protein was purified through an affinity chromatography in a Ni-NTA superflow nickel column (Qiagen). The column was previously equilibrated with buffer containing 10 mM Tris-HCl, 100 mM NaCl and 50 mM NaH₂PO₄, pH 8.0. All purification steps were performed at 4°C. The protein was eluted with the same buffer with increasing imidazole concentrations (10, 25, 50, 75, 100 and 250 mM). The purified protein was analyzed in 12% SDS-PAGE. Fractions containing the purified protein were dialyzed using membranes of 3500 MW (Pierce) for 2 hours at 4°C in buffer containing 10 mM Tris-HCl, 100 mM NaCl and 50 mM NaH₂PO₄, pH 8.0. The protein was then concentrated in the SPD1010 SpeedVac^® System (ThermoSavant) for 3 hours and in Vivaspin™ 3000 MWCO (GE Healthcare) for 1.5 h. The protein concentration was determined using Bradford’s method [47 (link)] in Hitachi U-5100 spectrophotometer.

Glycogen contents in the liver and soleus muscle were measured using the Phenol-sulfuric acid method. First, 50 or 100 mg of liver or soleus muscle, respectively, were homogenized in 0.8 mL of 10% trichloroacetic acid (Wako Pure Chemical Industries Ltd.), followed by centrifugation at 1,900 × g for 10 min for deproteinization. The supernatant (0.4 mL) was mixed with 0.8% ethanol (Wako Pure Chemical Industries Ltd.), followed by centrifugation at 1,900 × g for 10 min to precipitate glycogen. Then, the supernatant was decanted and the precipitated glycogen was r-suspended in 0.5 mL distilled water. Phenol (0.5 mL of 5% stock; Wako Pure Chemical Industries Ltd.) and 2.5 mL concentrated sulfuric acid (Kanto Chemical Co., Inc., Tokyo, Japan) were added to the resuspended glycogen solution, and the mixture was incubated for 20 min at room temperature (20–22 °C). Then, the absorbance was measured at 490 nm using a U-5100 spectrophotometer (Hitachi High-Tech Science Co., Tokyo, Japan). A standard curve was generated using a 40 mg/dL glucose (Wako Pure Chemical Industries Ltd.) solution.

Washed erythrocytes were haemolysed by mixing with 1 mM phenylmethylsulfonyl fluoride and 5P1E buffer (5 mM phosphate buffer, 1 mM EDTA) on ice and centrifuged at 20,400 g, 4°C, for 15 min. The pellets were washed three times with 5P1E buffer to remove the haemoglobin and obtain the erythrocyte membrane fraction. After ensuring equal protein concentrations by measuring the absorbance using the Bradford reagent (Thermo Fisher Scientific, Waltham, MA, USA) and the U‐5100 spectrophotometer (HITACHI, Tokyo, Japan), the erythrocyte membranes were mixed with the sample buffer, electrophoresed on 8% SDS‐polyacrylamide gel and transferred to a PVDF membrane. The PVDF membrane was blocked with 5% skim milk in TBS and incubated with the following primary monoclonal antibodies separately in T‐TBS (TBS and 0.1% Tween 20): rat anti‐ATP11C antibody (11C4; 1:1000¹²), mouse anti‐PLSCR1 antibody (1:5000; Abnova, Taipei, Taiwan) and mouse anti‐actin (1:50 000; Sigma‐Aldrich Inc). Thereafter, the membrane was washed three times and incubated with the following secondary antibodies in T‐TBS: rat IRDye 680RD for ATP11C and mouse IRDye 800 CW for PLSCR1 and actin. Fluorescence was detected and quantitated using a fluorescent scanner (Odyssey; LI‐COR). The levels of ATP11C and PLSCR1 were evaluated relative to the intensity of actin.