The nutrient broth (NB) medium was purchased from Difco Ltd., USA. Highly pure quality reagents and chemicals were employed for the test assays. Test samples of jineol were prepared in 1% dimethyl sulfoxide (DMSO) (Sigma–Aldrich, Germany). For absorbance reading, an enzyme-linked immunosorbent assay (ELISA) (Tecan, Infinite M200, Männedorf, Switzerland) was used.

Staphylococcus aureus KCTC1621 (Gram+), and E. coli O157:H7 (Gram-) foodborne pathogens procured from the Korean Collection for Type Cultures (KCTC, Korea) were used in this study. For the growth and culture of strains, NB was used and cultures were incubated at 37°C, followed by maintenance of strains on nutrient agar (NA) slants at 4°C. To reactivate the cultures, cultures were taken out and a loop-full of colonies were inoculated in the fresh NB medium, and incubated for 24 h and 37^oC. Further, sub-culturing was maintained in NB medium.

Dried Scolopendra subspinipes mutilans specimens were purchased from the herbal market at Geumsan, South Korea, and identified by in-charge of the department. A voucher specimen (CNU-INS 1408) was deposited at the Pharmacognosy Laboratory of the College of Pharmacy, Chungnam National University (Daejeon, South Korea).

Jineol was isolated from specimens using a chromatographic approach (Lee et al., 2016). Briefly, the dried ethanol (EtOH) extract (110.0 g) was suspended in water and fractionated successively with ethyl acetate (EtOAc) and then n-butanol (BuOH) to yield EtOAc-soluble (60.0 g) and n-BuOH-soluble (8.0 g) fractions, and residue (40.0 g). The EtOAc-soluble fraction was subjected to vacuum-liquid chromatography (VLC) using hexane-EtOAc 40:1, 20:1, 10:1, and 4:1; hexane-EtOAc-MeOH 2:1:0.2; CHCl₃-MeOH 6:1; CHCl₃-MeOH-H₂O 3:1:0.1 and then washed with MeOH to yield eight fractions (E1–E8). Fraction E4 (8.0 g), which was obtained by eluting with hexane-EtOAc 4:1 was partitioned with hexane and MeOH to afford jineol (60.0 mg).

Jineol: yellowish amorphous powder; ¹H NMR (300 MHz, CD₃OD) δ_H 8.46 (1H, d, J = 2.4 Hz, H-2), 7.42 (1H, d, J = 2.4 Hz, H-4), 7.29 (1H, t, J = 8.0 Hz, H-6), 7.14 (1H, d, J = 8.0 Hz, H-5), 6.87 (1H, d, J = 8.0 Hz, H-7), ¹³C NMR (75 MHz, CD₃OD) δc 154.2 (C-8), 153.0 (C-3), 142.3 (C-2), 134.8 (C-8a), 131.9 (C-4a), 129.0 (C-6), 117.9 (C-5), 117.2 (C-4), 109.0 (C-7) (Moon et al., 1996).

A method of agar diffusion was employed to determine the antibacterial activity of jineol using Luria-Bertani (LB) agar plates (Bajpai and Kang, 2010). To make the desired concentrations (0, 10¹, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, and 10⁹ cells/mL) of the tested strains, pathogens were cultured in NB medium following incubation at 37°C for 24 h and serially diluted. Further, a method of the microbial plate-count was employed to determine the viable cell numbers. A 100 μL inoculum containing 10⁷ CFU/mL was poured on dried agar plates and spread uniformly using a bacterial plate spreader followed by drying at room temperature. The compound was dissolved in 5% DMSO, and finally 50 μL jineol solution, corresponding to 250 μg/disk was impregnated on a sterilized filter paper (Whatman No. 1) disk. The same solvent used for dissolving the sample was also tested as a negative control. Further, plates followed the incubation of 37°C for 24 h, and zones of inhibition around the disks were measured to confirm the antibacterial activity of test compound in triplicate measurements.

A method of twofold serial dilution was employed to determine the MIC of jineol (Bajpai et al., 2013). At first, jineol was dissolved in the DMSO (5%), followed by incorporation into the NB medium to make a 500 μg/mL solution of jineol. Further, serial dilutions of jineol solution were made in NB to obtain 250, 125, 62.5, 31.25, 15.62, and 7.81 μg/mL concentrations of jineol. A 10 μL standardized suspension (˜10⁷ CFU/mL) of each tested organism was inoculated into each tube. Controls were devoid of sample and contained only bacterial inoculum. No bacterial growth in the lowest concentration (μg/mL) of jineol following macroscopic analysis confirmed the MIC of jineol. The cultures (50 μL each) in which jineol concentrations did not show any visual bacterial growth were spread on NA plates in triplicates following incubation of 37°C for 24 h. Finally, the lowest concentration which completely inhibited the formation of CFU on NA plate was referred as MBC of jineol.

Freshly grown bacterial colonies of the selected pathogenic bacteria were inoculated in NB and incubated 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 jineol on cell viabilities, each of the tubes containing the bacterial suspension (10 μL; ∼10⁷ CFU/mL) of S. aureus KCTC1621 and E. coli O157:H7 was inoculated with 100 μL of jineol at its MIC in 890 μL NB broth at 37°C. A time interval of 0, 40, 80, 120, 160, and 200 min was followed to take the sample for counting cell viabilities on NA plates (Bajpai et al., 2013). Counting of CFU was performed after 24 h of incubation at 37°C. Controls were prepared in a similar manner except the treatment of jineol in triplicate.

A previously developed method was adopted for the determination of the effect of jineol on K⁺ ion efflux from the cells of tested bacteria (Bajpai et al., 2013). The concentration of free K⁺ ions from the cell suspensions of S. aureus KCTC1621 and E. coli O157:H7 was determined for 0, 30, 60, 90, and 120 min following the jineol exposure to bacterial cells at MIC employing sterilized peptone-water. Photometeric measurement of extracellular K⁺ ion was performed at each above-mentioned time interval, using a commercial kit for Calcium/Potassium detection. Controls were also tested in a similar way without the addition of jineol. Data were presented as the release of extracellular K⁺ ion concentration (mmol/L) in a triplicate set.

The measurements of the release of 260-nm-absorbing components (DNA/RNA) from S. aureus KCTC1621 and E. coli O157:H7 cells were performed in a 2 mL aliquot of the bacterial inoculum in a sterilized peptone-water. The loss of 260 nm cellular materials is considered a good indication of the antimicrobial efficacy of any test compound. The reaction solution was added of MIC of jineol following incubation at 37°C. Subsequently, cultures treated at 0, 30, and 60 min were collected separately following centrifugation (3,500 × g, 10 min) to obtain the cell free supernatants and read for absorbance at 260-nm using an ELISA (Bajpai et al., 2013). Controls tested devoid of jineol. Data were collected at each time point and presented as optical densities (ODs) of the samples.

Scanning electron microscopic (SEM) study was executed according to Kim et al. (2007) to examine the effects of jineol 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 jineol. Microscopic examination was performed using a S-4300 SEM Analyzer (Hitachi, Japan).

Experiments were carried out in a set of triplicate, and the data obtained were presented as mean ± SD following one-way ANOVA statistical analysis coupled with Duncan’s multiple test.

¹H NMR data showed signals for five aromatic protons at δ_H 8.46 (1H, d, J = 2.4 Hz, H-2), 7.42 (1H, d, J = 2.4 Hz, H-4), 7.29 (1H, t, J = 8.0 Hz, H-6), 7.14 (1H, d, J = 8.0 Hz, H-5), and 6.87 (1H, d, J = 8.0 Hz, H-7). Typical proton signals for 3-hydroxy quinoline alkaloid were observed at 8.46 (1H, d, J = 2.4 Hz, H-2) and 7.42 (1H, d, J = 2.4 Hz, H-4). Inspection of the ¹³C NMR spectra revealed nine aromatic carbon signals. Based on its NMR spectroscopic data analyses, the compound was identified as 3,8-dihydroxyquinoline (jineol) (Moon et al., 1996) (Figure 1).

This study showed antibacterial effects of the test compound jineol as confirmed by the presence of inhibitory zones in agar plates against the tested foodborne pathogenic bacteria, S. aureus KCTC1621 and E. coli O157:H7. In this assay, jineol exhibited a significant inhibitory effect against both the employed foodborne pathogenic bacteria. The inhibitory effect of jineol in agar plates was confirmed through the diameters of zones of inhibition, which were found to be 11.6–13.6 mm (Table 1). It was observed that jineol exhibited antibacterial effects against both the tested bacterial isolates.

This assay revealed different susceptibilities of test compound jineol against the tested foodborne pathogens as confirmed by the low and variable MIC and MBC values. As a result, the MIC and MBC values of jineol against the tested pathogens were ranged 62.5–125, and 125–250 μg/mL, respectively (Table 1). In this assay, it was observed that jineol had inhibitory effects against both Gram-positive and Gram-negative bacteria.

This assay confirmed the antibacterial potential of quinoline alkaloid compound jineol, as confirmed by the reduction in the cell viabilities of the tested pathogens, S. aureus KCTC-1621 and E. coli O157:H7 when inoculated at MIC (Figure 2). Bacterial pathogens when exposed to test compound jineol for 80 min showed no remarkable decrease in the cell viabilities. However, exposure of jineol to pathogen for 160 and 200 min completely inhibited the growth of both the tested pathogens, S. aureus KCTC-1621 and E. coli O157:H7, respectively (Figure 2).

Loss of extracellular K⁺ ions from the bacterial cells upon the treatment of specific antimicrobial indicates loss of cell integrity, thus establishing its antimicrobial effect againt the tested bacteria. It was found in this study that jineol at the used concentration exhibited significant antibacterial effect as confirmed by the significant release of K+ ions from the treated bacterial cells of S. aureus KCTC-1621 (Figure 3A) and E. coli O157:H7 (Figure 3B). Jineol exhibited time dependent inhibitory effect in this assay. However, no significant release of K+ ions from the control sets were observed.

Since loss of 260 nm cellular materials is considered a good indication of the antimicrobial efficacy of any test compound, this assay confirmed that bacterial cells of tested foodborne pathogens, S. aureus KCTC-1621 and E. coli O157:H7 upon treatment with jineol at MIC had a severe inhibitory effect in terms of release of 260 nm absorbing materials (DNA and RNA) from them (Figure 4). As a result, it was observed that bacterial cells of tested foodborne pathogens, S. aureus KCTC-1621 (1.62-3.12) and E. coli O157:H7 (1.15-2.98) showed significant differences in their respective ODs as compared to control groups (1.62–1.65) and (1.15–1.18) measured at 260 nm. No significant differences in ODs of control groups were observed in this assay.

It is a vital phenomenon that exposure of the antimicrobial agent to bacterial cells results in the disruption of cell wall, therefore, we performed SEM analysis to further confirm the deteriorating effects of jineol on the cell wall physiologies and morphologies of S. aureus KCTC-1621 and E. coli O157:H7 cells (Figure 5). As a result, jineol significantly altered the cell wall morphology of both the tested pathogens S. aureus KCTC-1621 and E. coli O157:H7 used at MIC with clear visualization of cell wall damage and cell lysis (Figures 5B,D). However, as expected, cells without treatment as a control had intact shape and no morphological changes were observed (Figures 5A,C).