1. Cytotoxicity Test of Sesamin and Oseltamivir in Pbmcs

Supplementary Data

Supplementary Experimental

1. Cytotoxicity test of sesamin and oseltamivir in PBMCs

For thecytotoxicity and proliferation assay, PBMCs (2×105 cells/ml) were plated in a 96-well plate and incubated overnight. PBMCs were treated with different concentrations of sesamin or oseltamivir (Tamiflu®, USA) for 24 h. For background control, RPMI 1640 medium without cells wasused. Then, 10% of AlamarBlue solution (Sigma-Aldrich®, USA)(Dutkaet. al.1983; King et. al.1984; Liu et. al.1981; Strotmannet. al.1993)was added to theconditional medium and incubated at 37oC with 5% CO2 for 24 h. The absorbance was measured at 540 nm (test wavelength) and 620 nm (reference wavelength) using a micro-plate reader spectrophotometer.The percentage of difference in cytotoxicity between the treated and control group cells was calculated by the following equation:

The results are thatOD1 is 80,586 molar extinction coefficient of oxidized alamarBlue® at 540 nm, OD2is 117,216 molar extinction coefficient of oxidized alamarBlue® at 620 nm, A1 and A2 are absorbance of treated cells at 540 nm and 620 nmrespectively, R1 is 155,677 molar extinction coefficient of reduced alamarBlue® at 540 nm, R2is 14,562 molar extinction coefficient of reduced alamarBlue® at 620 nm, and N1 and N2 areabsorbance of control group cells(media plus alamarBlue® but no cells) at 540 nm and 620 nmrespectively.

The percentage of viability of the treated cells relative to the control group cells was calculated by the following equation:

Supplementary Table Legend

Table S1.The sequence primers of cytokine markers.

Supplementary Figure Legends

Figure S1.The sequence of theneuraminidase enzyme used in this study (PDB ID: 3TI6).

Figure S2.The standard curve of total sialic acid (unit/ml) released from bovine submaxillary mucin.

Figure S3.The effects of sesamin (A) and oseltamivir (B) on PBMCs’ cell toxicity and proliferation. Embryonated chicken eggs were used in the control group. Data are shown as mean ±S.D. of the three independent experiments.

Figure S4.Optimization of influenza type A H1N1 dilution. Conditional media of various embryonated egg dilutions of H1N1-treated PBMCs for 24 hwere measured for IL-1β levels. The highest IL-1β level was found in dilution 1:7,812,500. It was selected for the study of phytochemical effects.

Table S1. The sequence primers of cytokine markers.

Gene / Sequence 5’3’ / Base pair / GenBank Accession No.
IL-1β / F: CCTGTCCTGCGTGTTGAAAGA
R: GGGAACTGGGCAGACTCAAA / 150 / NM_000576.2
TNF-α / F: CCCCAGGGACCTCTCTCTAATC
R: GGTTTGCTACAACATGGGCTACA / 98 / NM_000594.3
IL-2 / F: ACCAGGATGCTCACATTTAAGTTTT
R: GAGGTTTGAGTTCTTCTTCTAGACACTG / 88 / NM_000586.3
GAPDH / F: GAAGGTGAAGGTCGGAGTC
R: GAAGATGGTGATGGGATTTC / 226 / NM_002046.5

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Figure S1.

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Figure S2.

Figure S3.

Figure S4.

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Supplemental Reference

Dutka BJ, Nyholm N, Petersen J (1983) Comparison of several microbiological toxicityscreening tests. Water Research 17:1363-1367.

King EF(1984)A comparative study of methods for assessing the toxicity to bacteria of single chemicals and mixtures. In: Toxicity Screening Procedures Using Bacterial Systems, D. Liu and B.J. Dutka eds., Decker, New York, pp. 175-194.

Liu D(1981)A rapid biochemical test for measuring chemical toxicity. Bull Environmental Contamination Toxicology26:145-149.

Strotmann UJ, Butz B, Bias WR(1993) Thedehydrogenase assay with resazurin:Practical performance as a monitoring system and pH-dependent toxicity of phenolic compounds. Ecotoxicology Environmental Safety 25:79-89.

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