Dr a1

The pH was determined using the pH meter model MW102 with MA 920 electrodes (Milwaukee, Sat Baciu, Romania). The soluble solids (SS) content, in °Brix, was obtained with an ‘Abbe’ digital refractometer, model DR-A1 (ATAGO Co., Bellevue, WA, USA), at 22 °C.

Densities were measured using a density tube meter, the viscosities were measured with a Brooksfield DV2T viscometer. This viscometer was calibrated with distilled water. Viscosity of the samples (η) was obtained under the following conditions; the flow time of 60 s was used to measure the flow time in the hybrid solvents or solvent, T = 303 K and a speed of 50 rpm. The estimated uncertainty of the experimental viscosity was ± 0.02 cP. The refractive index was measured with a digital refractometer (ATAGO DRA1, Japan) with an uncertainty of ± 0.001.
The analytical balance (AND, GR202, Japan) with the precision of ± 0.0001 g was used for the preparation of mixtures in molar basis. The studied hybrid solvents were prepared in well-sealed glass vials to avoid contamination or mixture evaporation. Measurements were done continually after the mixtures preparation. The standard uncertainty of solubility is 0.0014 and density is 0.001 g/cm³.

The proximate composition was determined using the methods of the Association of Official Analytical Chemists (AOAC 1990) [33 ]. This included moisture content (Method N° 934.06), total crude protein (Method N° 960.52), lipid content (Method N° 960.39), and crude ash (Method N° 923.03). The total carbohydrate content was estimated by the difference. Physicochemical characteristics of the samples were measured, including pH with a pH-meter (Orion Star A320, Thermo Scientific Inc., Fort Collins, CO, USA), titratable acidity expressed as gram tartaric acid per 100 mL of GJ, soluble solid content (°Brix) measured with an Abbé refractometer (DR-A1, Atago, Tokyo, Japan), and the surface color measured with a colorimeter (HunterLab, MiniScanTM XE Plus, Reston, VA, USA). The total color difference (ΔE) was calculated using Equation (1).
$$∆E=\sqrt{[{(L*-L_0*)}^2+{(a*-a_0*)}^2+{(b*-b_0*)}^2]}$$
where L* is the lightness of the sample, L₀* is the lightness of the fresh sample, a* is the redness of the sample, a₀* is the redness of the fresh sample, b* is the yellowness of the sample, and b₀* is the yellowness of the fresh sample.

Optimal concentration of KM and FA for treating HCECs was decided based on the result of the CCK-8 test; finally, KM1 and FA100 were supplemented in the buffer solution (BS). The chemical composition of BS is based on the balance salt solution (BSS PLUS^®) for further animal studies. The pH and osmolarity of BS and herbal combination were determined using a pH meter (pH 510; Eutech Instruments, Singapore) and a micro-osmometer (Model 3320; Advanced Instruments, Norwood, MA, USA). A refractometer (DR-A1 ATAGO, Kyoto, Japan) was used to measure the refractive index (RI) of the solution. The BSS PLUS^® is composed by two aliquots: solution (A) with NaCl (3.571 g), KCl (0.189), Na₂HPO₄ (0.207) and NaHCO₃ (1.051) in 480 mL; and solution (B) with CaCl₂, MgCl₂⋅6H₂O, detrose, and glutathione in 20 mL [42 ]. Solutions A and B were freshly prepared before use; they were preservative-free and aseptic after filtration through a 0.22-µm filter. Afterwards, both solutions were mixed before use and abbreviated as BS for further animal testing in this study.