The nutrient broth (NB) medium was purchased from Difco (USA). Other chemicals and reagents used were of very pure and high analytical grade. Test samples of (+)-lariciresinol isolated from R. philippinensis were prepared in 1% dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Germany). Spectrophotometric measurements were made using an enzyme-linked immunosorbent assay (ELISA) instrument (Tecan, Infinite M200, Männedorf, Switzerland) was used.

Two selected foodborne pathogenic bacteria one Gram-positive (Staphylococcus aureus KCTC1621), and one Gram-negative (Escherichia coli O157:H7, shiga-toxin producing bacterium) were procured from the Korean Collection for Type Cultures (KCTC, Korea) and used in this study. All the bacterial strains were grown in the NB and incubated at 37°C. The bacterial strains were maintained on nutrient agar slants at 4°C.

The roots of Rubia philippinensis were collected from Bidoup-Nui Ba National Park, Lamdong province, Vietnam in July 2013, and identified by Dr. Phuong Thien Thuong at the Department of Pharmaceutical Analysis and Herbal Standardization, National Institute of Medicinal Materials, Vietnam. A voucher specimen was deposited at the herbarium of the National Institute of Medicinal Materials, Hanoi, Vietnam, as well as the Laboratory of Pharmacognosy at the College of Pharmacy, Chungnam National University, Daejeon, Korea.

(+)-Lariciresinol was obtained from the roots of R. philippinensis by chromatographic methods. Briefly, the ethanol extract of R. philippinensis (150 g) was suspended in H₂O (1.5 L) and partitioned with CH₂Cl₂ (2 L × 3) to yield CH₂Cl₂ extract. The CH₂Cl₂-soluble fraction (50 g) was subjected to silica gel VLC and eluted with n-hexane-EtOAc (20:1, 10:1, 5:1, 3:1, 2:1) and CHCl₃-MeOH (8:1) to afford six fractions (D-1 → D-6). Fraction D-6 (10 g) was divided into 11 sub-fractions (D-6-1 → D-6-11) using MPLC with a gradient of MeOH-H₂O (10:90 → 100:0, 7 L). (+)-Lariciresinol (t_R 33.0 min, 31 mg) was obtained from D-6-3 (360 mg) by HPLC model 1200 using a Zorbax SB-C18 analytical column (4.6 mm × 150 mm, 5 μm particle size), Agilent, Germany eluting with MeOH-H₂O (45:55, 4 mL/min, UV 254 nm). (+)-Lariciresinol: brownish amorphous powder, ¹H NMR (300 MHz, methanol-d₄): 2.38 (1H, m, H-8), 2.47 (1H, dd, J = 13.1, 11.7, H_a-7’), 2.72 (1H, m, H-8’), 2.91 (1H, dd, J = 13.1, 4.5, H_b-7’), 3.63 (1H, dd, J = 10.8, 6.6, H_a-9), 3.72 (1H, dd, J = 8.1, 6.1, H_a-9’), 3.82 (1H, overlapped, H_b-9), 3.97 (1H, dd, J = 8.1, 6.1, H_b-9’), 4.75 (1H, d, J = 6.9, H-7), 6.63 (1H, dd, J = 8.0, 1.2, H-6’), 6.73 (1H, d, J = 8.0, H-5’), 6.78 (3H, overlapped, H-6, H-5, H-2’), 6.91 (1H, d, J = 1.2, H-2); ¹³C NMR (75 MHz, methanol-d₄): 135.7 (C-1), 110.6 (C-2), 148.9 (C-3), 146.9 (C-4), 116.0 (C-5), 119.8 (C-6), 83.9 (C-7), 53.9 (C-8), 60.4 (C-9), 133.5 (C-1’), 113.4 (C-2’), 148.9 (C-3’), 145.7 (C-4’), 116.2 (C-5’), 122.1 (C-6’), 33.6 (C-7’), 43.8 (C-8’), 73.4 (C-9’), 56.3, 56.3 (2 × OCH₃-3,3’).

Standard agar diffusion method was used for antibacterial assay (Bajpai and Kang, 2010). Petri plates were prepared by pouring 20 ml of LB medium and allowed to solidify. Plates were dried, and 100 μL of standardized inoculum containing approximately 10⁷ CFU/mL of bacterial suspension was poured and uniformly spread. The inoculum was allowed to dry for 5 min. The compound was dissolved in 5% DMSO, and a Whatman No. 1 sterile filter paper disk (6 mm diameter) was impregnated with 50 μL of the compound (+)-lariciresinol corresponding to 250 μg/disk. Negative controls were prepared using the same solvent that was employed to dissolve the sample. The plates were incubated at 37°C for 24 h in a bacterial incubator chamber (Thermo Scientific Ltd., Korea). Antibacterial activity was evaluated by measuring the diameters of the zones of inhibition against the tested bacteria. Each assay in this experiment was replicated three times.

The MIC of (+)-lariciresinol was tested by the two-fold serial dilution method (Bajpai et al., 2013). The (+)-lariciresinol was first dissolved in 5% DMSO, and incorporated into NB medium for bacterial pathogens to obtain a concentration of 500 μg/mL, and serially diluted in NB broth medium to achieve 250, 125, 62.5, 31.25, 15.62, and 7.81 μg/mL, respectively. A 10 μL standardized suspension (approximately 10⁷ CFU/mL) of each tested organism was transferred to each tube. The control tubes containing only bacterial suspensions were incubated at 37°C for 24 h in a bacterial incubator chamber (Thermo Scientific Ltd., Korea). The lowest concentration of (+)-lariciresinol, which did not show any visible growth of test organisms after macroscopic evaluation, was determined as MIC, which was expressed in μg/mL. Further, the concentrations showing complete inhibition of visual growth of bacterial pathogens were identified, and 50 μL of each culture broth was transferred onto the agar plates and incubated for specified time and temperature as mentioned above. The complete absence of growth of bacterial colony forming unit (CFS) on the agar surface is the lowest concentration of the sample and was defined as MBC. Each assay in this experiment was replicated three times.

Freshly grown bacterial colonies of the selected pathogenic bacteria were inoculated in NB medium at 37°C for 24 h, and then bacterial cultures were serially diluted to 10⁷ CFU/mL (Shin et al., 2007). To determine the effect of (+)-lariciresinol on cell viabilities, each of the tubes containing the bacterial suspension (10 μL; approximately 10⁷ CFU/mL) of S. aureus KCTC1621 and E. coli O157:H7 was inoculated with 100 μL of (+)-lariciresinol at its MIC in 890 μL NB broth at 37°C. Samples for viable cell counts were taken out at 0, 40, 80, 120, 160, and 200 min time intervals. Viable plate counts were monitored on NB agar as we previously described (Bajpai et al., 2013). Colonies were counted after incubation for 24 h at 37°C in a bacterial incubator chamber (Thermo Scientific Ltd., Korea). The controls were inoculated without (+)-lariciresinol for each pathogen using the same experimental condition. Assay were performed in triplicate.

Effect of lariciresinol on the efflux of potassium ion from the tested pathogens was determined according to our previously reported method (Bajpai et al., 2013). The concentrations of free potassium ions from S. aureus KCTC1621 and E. coli O157:H7 cell suspensions were measured by a photometric procedure after the exposure of cells to at MIC in sterile peptone water using the Calcium/Potassium ions kit (Quantofix, GmbH, Wiesbaden, Germany). Similarly control was also tested without adding CFS. Results were expressed as the release of extracellular free K⁺ ion (mmol/L) in the growth media at pre-established incubation intervals for 0, 30, 60, 90, and 120 min.

The measurement of the release of 260-nm-absorbing materials from S. aureus KCTC1621 and E. coli O157:H7 cells was carried out in aliquots of 2 mL of the bacterial inocula in sterile peptone water (0.1 g/100 mL). The reaction solution was added of MIC of (+)-lariciresinol and incubated at 37°C. At 0, 30, and 60 min time interval of treatment, cells were centrifuged at 3,500 × g, and the absorbance of the obtained supernatant was measured at 260 nm using a 96-well plate ELISA reader (Magellan5, TECAN, Korea) (Bajpai et al., 2013). Similarly control was also tested without adding (+)-lariciresinol. Results were expressed in terms of optical density (OD) of 260-nm absorbing materials in each interval with respect to the ultimate time.

Scanning electron microscopic (SEM) study was executed according to Kim et al. (2007) to examine the effects of (+)-lariciresinol on the morphological changes in the cell wall of the selected pathogens, S. aureus KCTC1621 and E. coli O157:H7 at MIC. Control samples were prepared without (+)-lariciresinol. Microscopic examination was performed using a S-4300 SEM Analyzer (Hitachi, Japan).

The data were statically analyzed using software package SPSS v.19.0 statistical software package (SPSS Inc., Chicago, IL, USA). All experiments were performed in triplicate and results were expressed the mean ± SD following one-way ANOVA statistical analysis coupled with Duncan’s multiple test.

The ¹H NMR data of purified compound, [α]_D +17 (c 0.05, MeOH), displayed two pairs of aromatic ring systems at δ_H 6.63 (1H, dd, J = 8.0, 1.2, H-6’), 6.73 (1H, d, J = 8.0, H-5’), 6.78 (3H, overlapped, H-6, H-5, H-2’), and 6.91 (1H, d, J = 1.2, H-2). In addition, an oxymethylene moiety at δ_H 3.72 (1H, dd, J = 8.1, 6.1, Ha-9’), 3.97 (1H, dd, J = 8.1, 6.1, Hb-9’), two methine groups at δ_H 2.38 (1H, m, H-8) and 2.72 (1H, m, H-8’), and an oxymethine at δ_H 4.75 (1H, d, J = 6.9, H-7) indicating a tetrahydrofuran substructure were observed in the ¹H NMR data as well. The ¹³C NMR spectrum showed 18 signals for a typical lignan derivative (Figure 1).

In this assay, presence or absence of inhibition zones determined the antibacterial potential of (+)-lariciresinol against the test foodborne pathogenic bacteria. As presented in Table 1, (+)-lariciresinol exhibited a considerable amount of inhibitory effect against the tested foodborne pathogens. It was observed that (+)-lariciresinol exerted a consistent antibacterial effect as diameters of inhibition zones against both Gram-positive and Gram-negative bacteria, which were found in the range of 12.1–14.9 mm (Table 1). The NB broth, used as a negative control, had no inhibitory effect on the growth of the tested pathogens.

In this assay, test foodborne pathogens displayed different susceptibility rates to (+)-lariciresinol, and (+)-lariciresinol exhibited potent inhibitory effect as confirmed by its different MIC and MBC values against both the tested pathogens. As a result, the MIC and MBC values of (+)-lariciresinol against the tested foodborne pathogens were found in the range of 125–250 and 125–250 μg/mL, respectively (Table 1). In this assay, (+)-lariciresinol exhibited consistent antibacterial effect, however, the inhibitory effect of (+)-lariciresinol was more profound against Gram-positive bacterium than Gram-negative bacterium.

In this assay, (+)-lariciresinol when inoculated at MIC, exhibited significant inhibitory effects against the growth of tested pathogens, S. aureus KCTC1621 and E. coli O157:H7, as confirmed by reducing pattern in the bacterial cell viabilities (Figure 2). Exposure to (+)-lariciresinol for 0 to 80 min did not elicit severe inhibition of cell viability, but remarkable declines in the cell viable counts of S. aureus KCTC1621 and E. coli O157:H7 was observed after exposure to (+)-lariciresinol for 160 min. Interestingly, the exposure to (+)-lariciresinol for 200 min completely inhibited the cell viabilities of both tested pathogens (Figure 2).

Release of extracellular K+ ions from the bacterial cells when treated with a suitable antimicrobial could be an indication of the establishment of the bactericidal effect of the tested compound. Interestingly, in this assay, (+)-lariciresinol was able to confirm its bactericidal effect against both the tested pathogens, S. aureus KCTC1621 and E. coli O157:H7 as confirmed by the marked release of free K⁺ ions from the treated bacterial cells as compared to non-treated cells used as a control (Figure 3). The (+)-lariciresinol (at MIC) allowed the K+ ion release of (150–700 mmol/L) and (100–650 mmol/L) against S. aureus KCTC1621 and E. coli O157:H7, respectively (Figures 3A,B).

In this assay, we determined the release of 260-nm absorbing materials from the bacterial cells when treated with (+)-lariciresinol at MIC. Since the excessive release of 260 nm materials (DNA and RNA) may lead to irreversible injury to bacterial cells, and (+)-lariciresinol caused a significant effect on the bacterial cells (Figure 4). Bacterial cells when treated with (+)-lariciresinol allowed significantly higher release of 260 nm materials from S. aureus KCTC1621 (Figure 4A) and E. coli O157:H7 (Figure 4B) when compared with control cells as confirmed by a significant difference in optical densities observed between treatment (2.72–2.99) and control (1.12–1.26) cells measured at 260 nm. In contrast, no changes in the OD of control cells of tested pathogens were observed during the study.

Since exposure to an antimicrobial agent may lead to disruption of bacterial cell wall, we turned to SEM analysis to investigate further the effect of (+)-lariciresinol on the cell wall physiologies and morphologies of S. aureus KCTC1621 and E. coli O157:H7 cells (Figure 5). As was expected, control bacterial cells not exposed the result of (+)-lariciresinol had regular smooth surfaces (Figures 5A,C), whereas those treated with (+)-lariciresinol at MIC showed cell wall damage and lysis (Figures 5B,D).