Rutin analysis by liquid chromatography and mass spectrometry

Rutin hydrate (Sigma-Aldrich) was dissolved in methanol and diluted with water to give four separate standards containing 0.1, 0.125, 0.25 and 0.5 µg in 10 µL. Each standard (10 µL) was analyzed on a Shimadzu LCMS 2010A (Japan) using a diphenyl column (250 × 4.6 mm, 5 µm pore size, Vydac, (USA), a buffer system of 0.005% formic acid in water and B solution, and acetonitrile (0–50% B) over 30 min at a flow rate of 1 mL/min.

The dried Ip leaves (50 g) were added to 250 mL of distilled water at 70°C, and left to stand for 15 min, simulating the classic preparation. Thereafter, the infusion was sterilized with a fiberglass filter (2.7 µm). Final treatment concentration was 10%. Rutin hydrate (Sigma-Aldrich, USA) was diluted in distilled water, sterilized by filtration and used at the concentration found in the Ip infusion (40 µg/mL) (15). Both products at the indicated concentrations were added separately to liquid YPD cultures 1 h before irradiation (room temperature). Ip infusion was also added 5 min before irradiation in order to elucidate the kinetics of the putative protection. Exposure times corresponded approximately to 0.33 and 0.03 of cell cycle duration.

Irradiation was performed in Compton effect range, with a ⁶⁰Co source (Nordion 220, (Canada), mean photon energy E=1.25 MeV, and dose rate of 13.4 kGy/h. The dosimetry was performed with a polymethyl methacrylate Harwell Amber S 3042 dosimeter (United Kingdom). Absorbed dose was 0 ≤D ≤200 Gy. Samples were irradiated in YPD liquid medium with or without Ip or R. After treatment, controls and treated samples were kept on ice before further processing.

Relative surviving fraction was determined as a function of the absorbed doses. Based on survival curves (Figure 1) an absorbed dose of 200 Gy was selected for combination treatments. Aliquots of cells were plated in solid nutrient medium YPDA: YPD + 2% agar (DIFCO Laboratories, USA) and incubated at 30°C for 72 h. Survival was calculated as surviving fraction: S(x,y) = Ns/No, where Ns is the number of surviving cells capable of generating visible clones/mL; No is the total number of treated cells/mL; x the absorbed dose of radiation; and y the doses of the putative protectors (16,17).

To determine mutation frequency, cell samples of SC7K lys2-3 were plated after treatment on omission media (OM: 2% dextrose, 0.67% nitrogenous base yeast (Sigma-Aldrich, USA), 2% agar) (17,18) and incubated at 30°C for 21 days. Thereafter, the number of revertants lys → LYS were scored (12,13). Mutation frequency M(x,y) and mutation yield Y(x,y) were calculated as: M(x,y) = Nm / Ns; Y(x,y) = Nm / No, where Nm is the number of mutants per mL, No is the number of treated cells per mL, x the absorbed dose, and y the dose of modulators (16,19,20).