Supplementary Data
Supplementary Experimental
1. Cytotoxicity test of sesamin and oseltamivir in PBMCs
For the cytotoxicity 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 was used. Then, 10% of AlamarBlue solution (Sigma-Aldrich®, USA) (Dutka et. al. 1983;
King et. al. 1984; Liu et. al. 1981; Strotmann et. al. 1993) was added to the conditional 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:
%𝑟𝑒𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑜𝑓 𝑎𝑙𝑎𝑚𝑎𝑟𝐵𝑙𝑢𝑒 = [
(𝑂𝐷2 × 𝐴1) − (𝑂𝐷1 × 𝐴2)
] × 100
(𝑅1 × 𝑁2) − (𝑅2 × 𝑁1)
The results are that OD1 is 80,586 molar extinction coefficient of oxidized alamarBlue® at 540
nm, OD2 is 117,216 molar extinction coefficient of oxidized alamarBlue® at 620 nm, A1 and
A2 are absorbance of treated cells at 540 nm and 620 nm respectively, R1 is 155,677 molar
extinction coefficient of reduced alamarBlue® at 540 nm, R2 is 14,562 molar extinction
coefficient of reduced alamarBlue® at 620 nm, and N1 and N2 are absorbance of control group
cells (media plus alamarBlue® but no cells) at 540 nm and 620 nm respectively.
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The percentage of viability of the treated cells relative to the control group cells was calculated
by the following equation:
%𝑐𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 = [
%𝑟𝑒𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑜𝑓 𝑎𝑙𝑎𝑚𝑎𝑟𝐵𝑙𝑢𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
] × 100
%𝑟𝑒𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑜𝑓 𝑎𝑙𝑎𝑚𝑎𝑟𝐵𝑙𝑢𝑒 𝑜𝑓 𝑅𝑃𝑀𝐼1640
2
Supplementary Table Legend
Table S1.
The sequence primers of cytokine markers.
Supplementary Figure Legends
Figure S1. The sequence of the neuraminidase 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 h were 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.
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Table S1. The sequence primers of cytokine markers.
GenBank
Gene
Sequence 5’3’
Base pair
Accession No.
F: CCTGTCCTGCGTGTTGAAAGA
IL-1β
R: GGGAACTGGGCAGACTCAAA
150
NM_000576.2
98
NM_000594.3
88
NM_000586.3
226
NM_002046.5
F: CCCCAGGGACCTCTCTCTAATC
TNF-α
R: GGTTTGCTACAACATGGGCTACA
F: ACCAGGATGCTCACATTTAAGTTTT
IL-2
R: GAGGTTTGAGTTCTTCTTCTAGACACTG
F: GAAGGTGAAGGTCGGAGTC
GAPDH
R: GAAGATGGTGATGGGATTTC
4
Figure S1.
5
Figure S2.
6
Figure S3.
7
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 Toxicology 26: 145-149.
Strotmann UJ, Butz B, Bias WR (1993) The dehydrogenase 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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