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Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20

In vitro and in vivo antimicrobial efficacy of natural plant-derived compounds against Vibrio cholerae of O1 El Tor Inaba serotype a

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Hyung-Ip Kim , Ji-Ae Kim , Eun-Jin Choi , Jason B. Harris , Seong-Yeop Jeong , Seok-Jun f

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Son , Younghoon Kim & Ok Sarah Shin

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Department of Biomedical Sciences, College of Medicine, Korea University, Seoul, Republic of Korea b

Asian Pacific Influenza Institute, College of Medicine, Korea University, Seoul, Republic of Korea

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Division of Infectious Diseases, Massachusetts General Hospital, Boston, MA, USA

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Department of Medicine, Harvard Medical School, Boston, MA, USA

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Microbial Institute for Fermentation Industry (MIFI), Sunchang-gun, Korea

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BK21 Plus Graduate Program, Department of Animal Science and Institute Agricultural Science & Technology, Chonbuk National University, Jeonju, Korea g

Department of Microbiology, College of Medicine, Korea University, Seoul, Republic of Korea Published online: 17 Dec 2014.

To cite this article: Hyung-Ip Kim, Ji-Ae Kim, Eun-Jin Choi, Jason B. Harris, Seong-Yeop Jeong, Seok-Jun Son, Younghoon Kim & Ok Sarah Shin (2014): In vitro and in vivo antimicrobial efficacy of natural plant-derived compounds against Vibrio cholerae of O1 El Tor Inaba serotype, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2014.991685 To link to this article: http://dx.doi.org/10.1080/09168451.2014.991685

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Bioscience, Biotechnology, and Biochemistry, 2014

In vitro and in vivo antimicrobial efficacy of natural plant-derived compounds against Vibrio cholerae of O1 El Tor Inaba serotype Hyung-Ip Kim1, Ji-Ae Kim1, Eun-Jin Choi2, Jason B. Harris3,4, Seong-Yeop Jeong5, Seok-Jun Son6, Younghoon Kim6,*,a and Ok Sarah Shin1,7,a Department of Biomedical Sciences, College of Medicine, Korea University, Seoul, Republic of Korea; 2Asian Pacific Influenza Institute, College of Medicine, Korea University, Seoul, Republic of Korea; 3Division of Infectious Diseases, Massachusetts General Hospital, Boston, MA, USA; 4Department of Medicine, Harvard Medical School, Boston, MA, USA; 5Microbial Institute for Fermentation Industry (MIFI), Sunchang-gun, Korea; 6BK21 Plus Graduate Program, Department of Animal Science and Institute Agricultural Science & Technology, Chonbuk National University, Jeonju, Korea; 7Department of Microbiology, College of Medicine, Korea University, Seoul, Republic of Korea

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Received September 3, 2014; accepted October 6, 2014 http://dx.doi.org/10.1080/09168451.2014.991685

In this study, we investigated antibacterial activities of 20 plant-derived natural compounds against Gram-negative enteric pathogens. We found that both flavonoids and non-flavonoids, including honokiol and magnolol, possess specific antibacterial activities against V. cholerae, but not against other species of Gram-negative bacterium which we tested. Using various antibacterial assays, we determined that there was a dose-dependent bactericidal and biofilm inhibitory activity of honokiol and magnolol against Vibrio cholerae. In addition to antibacterial activities, these molecules also induced an attenuating effect on reactive oxygen species (ROS) production and pro-inflammatory responses generated by macrophages in response to lipopolysaccharides (LPS). Additionally, Caenorhabditis elegans lethality assay revealed that honokiol and magnolol have an ability to extend a lifespan of V. choleraeinfected worms, contributing to prolonged survival of worms after lethal infection. Altogether, our data show for the first time that honokiol and magnolol may be considered as attractive protective or preventive food adjuncts for cholera. Key words:

V. cholerae; antibacterial effect; honokiol; magnolol; C. elegans

Introduction Antimicrobial resistance is a global concern, resulting in prolonged hospitalizations, greater risk of death, and higher health care costs. In developing countries, multidrug-resistant enteric pathogens, such as Vibrio cholerae, Escherichia coli, and Shigella species, cause significant mortality and morbidity. Cholera, caused by *Corresponding author. Email: [email protected] a These authors contributed equally to this study. © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry

bacterium V. cholerae, is a severe diarrheal disease.1) Approximately 3 to 5 million cases of cholera occur annually, resulting in more than 100,000 deaths. During the current cholera pandemic, circulating V. cholerae O1 El Tor strains have developed antimicrobial resistance to major classes of antibiotics used to treat cholera, including tetracyclines and more recently fluoroquinolones.2,3) Furthermore, other enteric pathogens are also rapidly developing resistance to currently available antibiotics. Thus, it is important and necessary to make efforts to discover and develop novel and more effective antimicrobial agents against enteric pathogens. Food-grade plant extracts have proved to be effective antibacterial agents, and there is a synergy for using combination of antibiotics and plant-derived extracts. In this study, we tested several flavonoid and non-flavonoid compounds for antimicrobial activity against Gram-negative bacterium, including V. cholerae. Among them, baicalein is a flavonoid compound isolated from Scutellaria baicalensis Georgi (Huang Qin), a traditional Chinese medicinal herb, which has been proven to possess antiviral, antioxidative, antithrombotic, and anticancer activities.4) Honokiol and magnolol are polyphenols isolated from Magnolia species and have been shown to have anti-angiogenic, anti-tumor, and anti-inflammatory effects.5–7) In addition, honokiol and magnolol have been known to show antimicrobial activity against several microorganisms, including Propionibacterium species, Acinetobacter baumannii, Acinetobacter lwoffii, and periodontopathic microorganisms, such as Porphyromonas gingivalis, Prevotella gingivalis, Actinobacillus actinomycetemcomitans, Capnocytophaga gingivalis, and Veillonella disper.8−10) Antifungal activity of honokiol and magnolol against fungal pathogens (Candida albicans, Candida tropicalis, Trichosporon belgeii, Trichophyton mentagrophytes,

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Microsporium gypseum, Epidermophyton floccosum, Aspergillus niger, and Cryptococcus neoformans) were also demonstrated.11) Although an antimicrobial role of these plant-derived compounds against V. cholerae has not been previously described, the anti-inflammatory properties of quercetin and resveratrol during V. cholerae infection have been suggested by previous studies. Quercetin, a flavonoid present in plants such as onions, ginkgo biloba, and tea, causes a significant reduction of pro-inflammatory chemokine, interleukin-8 (IL-8), production in human intestinal epithelial cells infected with V. cholerae in a dose and time-dependent manner. Furthermore, quercetin treatment of V. cholerae-infected suckling mice led to a significant reduction of neutrophil infiltration in the intestinal epithelial layer of the animals.12) Resveratrol found in grapes and red wine is also another polyphenolic compound, which has shown to block cholera toxin (CT)-induced cyclic AMP (cAMP) accumulation in Vero cells.13) In this report, we examined the ability of 20 food-grade natural compounds derived from plant on antibacterial effect against Gram-negative enteric pathogen, V. cholerae. Our findings suggest that honokiol and magnolol possess specific antimicrobial activities against V. cholerae, but not against other enteric pathogens. Furthermore, these molecules induced both bacteriostatic and bactericidal effects, as well as anti-biofilm activities, and anti-inflammatory effects in V. cholerae LPS-stimulated mouse macrophage cells. We also measured the effect of treatment with honokiol or magnolol in a C. elegans/ V. cholerae infection model and found that these compounds delayed worms from V. cholerae-mediated killing. Taken together, our findings suggest that natural plantderived compounds may have protective or preventive potential for the treatment for cholera and other enteric foodborne pathogenic diseases and that a C. elegans as a host organism model may be useful for antimicrobial screening against V. cholerae.

Paper disk diffusion assay. Sterile paper disks (Advantec paper disk) of 8 mm diameter were used. 20 µL of 100 mg/mL sample solutions was applied on the sterile filter paper disks with a micropipette in an aseptic condition. Similarly, blank disks and other disks were prepared to serve as negative control and test sample, respectively. Plates were incubated in 37 °C incubator for 24 h before diameter of zones of was measured. Bacterial growth assay and MIC. Growth curves of V. cholerae were conducted to test for antimicrobial effects of natural polyphenolic compounds. Stationary cultures of the bacteria were diluted 100-fold into Luria-Bertani (LB) (Fisher Scientific, Pittsburgh, PA, USA) broth in a 96-well plate and exposed to concentrations of 10 μg/mL of natural compounds (baicalein, honokiol, magnolol, quercetin, and resveratrol) along with a DMSO only control and gentamicin as a positive control. The bacterial growth was measured after 1, 2, 4, and 8 h by spectrophotometer. For determination of MIC, stationary phase cultures of the bacteria were diluted 100-fold into LB broth in a 96-well plate and exposed to a twofold dilution series (100, 20, 10, 5, 2.5, 1.25, and 0.625 µg/mL) of natural compounds along with DMSO only control. Growth was assayed in duplicate wells over a 24 h period by monitoring the absorbance at OD 600 nm. Bactericidal assay. Bacteria were cultured overnight in LB broth with the appropriate antibiotics at 37 °C. For assays of cells from log-phase cultures, overnight cultures were diluted 1:100 in LB medium and grown to an optical density at 600 nm (OD600) of 0.5. Cells were washed twice in phosphate buffered saline (PBS). Bacterial suspension was added to 96-wellplates, and 100 µg/mL solutions of the natural compounds were incubated with cells for 24 h at 37 °C. Cells were spread onto LB plates to enumerate colony forming units (CFUs).

Materials and methods Reagents and bacteria. The following natural compounds were obtained from Korea Aging Tissue Bank (Busan, Korea); baicalein, curcumin, (+)-catechin, (−)-catechin, epicatechin, quercetin, saponin, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rg1, oligonol (phenolic product containing catechin-type monomers and oligomers of proanthocyanidin), reseveratrol, and ursodeoxycholic acid (UDCA). Emodin, honokiol, magnolol, wogonin, and rutin were generously given by Korea National Research Resource Bank (KNRRB) (Iksan, Korea). All the molecules were suspended in dimethyl sulfoxide (DMSO) at a concentration of 10 mg/mL. V. cholerae O1 El Tor serotype Inaba strain N16961 was a kind gift of Dr. Sang-Sun Yoon at Yonsei University School of Medicine (Seoul, Korea). Three subtypes of Escherichia coli O157:H7 (American Type Culture Collection (ATCC, USA) 43889, 43890, 35150), Pseudomonas aeroginosa (ATCC 15692), Salmonella typhimurim (ATCC 14028), and Shigella flexneri (ATCC 12022) were obtained from ATCC.

Biofilm assay. Overnight cultures of V. cholerae were inoculated at a 1:100 dilution into LB broth and incubated in tubes and in 96-well plates for 48 h at 25 °C. Subsequently, the tubes were rinsed with distilled water and then stained with crystal violet. After 10 min, the tubes were rinsed. The biofilm-associated crystal violet was resuspended with 95% EtOH, and the OD570 of the resulting suspension was measured. Quantitation of biofilms by crystal violet stain was described previously.14) Mouse macrophages. RAW 264.7 cells were obtained from ATCC and maintained in Dulbecco odified Eagle Medium (DMEM) (Lonza, USA) containing 10% fetal bovine serum (FBS) (Hyclone, USA) and penicillin–streptomycin (Lonza, USA) at 37 °C. Bone marrow derive macrophages (BMDM) cells from 2–3 months old C57BL/6 mice (Dbl animal science, Korea) were differentiated in RPMI (Lonza, USA) supplemented with 20% heat-inactivated FBS (Hyclone,

Natural antibacterial compounds against Vibrio cholerae

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USA) and 30% L929 cell (ATCC, USA) supernatants containing macrophage-stimulating factor (M-CSF). Bone marrow cells were cultured for 6–7 days, and fresh medium was added at day 3. Cells were harvested with RPMI and plated at a density of 1 × 106 per mL in RPMI supplemented with 10% FBS. Macrophages were cultured for at least 24 h before stimulation.

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Nitric oxide (NO) assay. For NO detection, the quantity of nitrite in the culture medium was measured as an indicator of NO production by NO detection kit (InTron, Korea). Briefly, 100 μL of cell culture medium was mixed with 50 μL of N1 buffer (substrate solution) followed by 50 μL of N2 buffer (coloring solution). Subsequently, the mixture was incubated at room temperature for 10 min, and the absorbance at 540 nm was measured in a microplate reader. Reactive oxygen species (ROS) assay. Raw cells (1.0 × 105 cells/mL) were cultured in 96 well plates for overnight. Next day, honokiol and magnolol were added to cells at concentrations of 10 μg/mL for 2 h. Cells were then treated with E. coli LPS, V. cholerae O1 LPS (derived from either the Ogawa or Inaba serotypes) at 1 μg/mL. After 24 h, cells were washed with PBS twice and stained with 10 μM dichlorofluorescin diacetate (DCF-DA) (Sigma, USA) in PBS for 30 min in the dark. Cells were then washed with PBS twice and extracted with PBS for 10 min at 37 °C. Fluorescence was recorded with an excitation wavelength of 490 nm and an emission wavelength of 525 nm. For mitochondrial ROS measurement, RAW 264.7 cells were plated in 6-well dishes and stimulated with LPS as described above. Culture medium was removed, washed with PBS, then incubated with MitoSOX (Invitrogen, USA) at 2.5 μM final concentration in serum-free DMEM media for 30 min at 37 °C. Cells were washed with warmed PBS, removed from plates with cold PBS containing 1.0 mM EDTA by pipetting, pelleted at 1,500 rpm for 3 min, immediately resuspended in cold PBS containing 1% FBS, and subjected to flow cytometry analysis (FACS). Unstained controls were treated similarly, except that treatments and dyes were omitted. Tumor necrosis factor-α (TNF-α) ELISA. Culture supernatants were collected after 24 h. Murine TNF-α in culture supernatants were measured by TNF-α ELISA kit (Biolegend, USA) according to the manufacturer’s protocol. Survival assays using C. elegans in vivo host. The C. elegans fer-15(b26)II;fem-1(hc17) nematode was selected for liquid assay experiments because it is unable to produce progeny at 25 °C,15) and were infected as described previously,16) with minor modifications. Worms were cultured and maintained on nematode growth medium (NGM) agar containing lawns of E. coli OP50. To assess the susceptibility of C. elegans to infection by V. cholerae, the bacteria were cultured in brain heart infusion media (BHI) at 37 °C.

Fig. 1. Structure of flavonoids and non-flavonoids, which induce antibacterial activities against V. cholerae. Notes: (A) Baicalein, (B) Honokiol, (C) Magnolol, (D) Quercetin, and (E) Resveratrol.

Table 1. Antibacterial activity of natural compounds against V. cholerae by disk diffusion assay. Compound Baicalein (+)-Catechin (−)-Catechin Curcumin Emodin Epicatechin Ginsenoside Rb2 Ginsenoside Rc Ginsenoside Rd Ginsenoside Re Ginsenoside Rg1 Honokiol Magnolol Oligonol Quercetin Resveratrol Rutin Saponin Ursodeoxy cholic acid Wogonin Gentamicin

Zone diameter (mm) 11.667 ± 0.577 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 15.333 ± 0.577 13.000 ± 1.000 0.000 ± 0.000 10.050 ± 0.707 13.333 ± 0.577 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 25.667 ± 1.155

Note: “0” indicates no sensitivity or zone of inhibition lower than 8 mm. The data shown are presented as average ± SD for three independent experiments.

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H. Kim et al. Table 2.

Antibacterial activity of natural compounds against Gram-negative bacterium by disk diffusion assay.

Diameter of clear zone (mm)

Baicalein

Honokiol

Magnolol

Quercetin

Resveratrol

Gentamicin

E.coli ATCC 43889 E. coli ATCC 43890 E. coli ATCC 35150 P. aeroginosa ATCC 15692 S. typhimurium ATCC 14028 S. flexeneri ATCC 12022 V. cholerae ATCC 39315

0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 11.667 ± 0.577

0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 15.333 ± 0.577

0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 13.000 ± 1.000

0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 10.050 ± 0.707

0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 13.333 ± 0.577

25.667 ± 1.155 25.667 ± 1.155 25.667 ± 1.155 25.667 ± 1.155 25.667 ± 1.155 25.667 ± 1.155 25.667 ± 1.155

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Note: “0” indicates no sensitivity or zone of inhibition lower than 8 mm. The data shown are presented as average + SD for three independent experiments.

Fig. 2. Effect of natural compounds on V. cholerae growth kinetics and killing. Notes: (A) V. cholerae growth was measured at different timepoints (1, 2, 4, 8 h) in the presence of baicalein, honokiol, magnolol, quercetin, or resveratrol at a concentration of 10 μg/mL. Growth is expressed as the optical density measured at 600 nm. (B) Baicalein, honokiol, magnolol, quercetin, and resveratrol at a concentration of 100 μg/mL were incubated with V. cholerae. The viability of V. cholerae cells was evaluated by counting colony forming units (CFU) after 24 h of incubation in LB medium at 37 °C in the absence or presence of increasing concentrations of natural compounds. The data shown are presented as mean ± SD for three independent experiments. *p < 0.05 for comparison with the DMSO-treated control.

Synchronized young adult/L4 worms were infected on lawns of V. cholerae strains for 24 h at 25 °C. After washing three times with M9 buffer,16) worms were

transferred into the wells of 6-well microtiter plates (30 worms per well). Each well contained 2 mL of assay medium (20% BHI:80% M9, v/v) supplemented with

Natural antibacterial compounds against Vibrio cholerae

various concentrations of honokiol or magnolol (1 and 10 µg/mL). DMSO only was employed as control. The plates were incubated at 25 °C and examined for viability at 24 h intervals for 12 days using a Nikon SMZ645 dissecting microscope. Biological replicates of each experiment were conducted.

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Statistical analysis. Data are reported as mean ± SD. Statistical analysis was performed in Prism 5 (Graph Pad Software, Inc. San Diego, USA) using Student’s t-test. Differences in C. elegans survival were tested for significance by Kaplan–Meier and logrank tests (STATA6; STATA, College Station, TX, USA). A two tailed p value of < 0.05 was considered significant.

Results Antimicrobial activity of flavonoids and nonflavonoids compounds against V. cholerae Antimicrobial activities of natural plant-derived compounds were examined by disk diffusion using 20 μL of 100 mg/mL compounds/disk against Gram-negative enteric pathogens, including V. cholerae. Gentamicin was employed as a positive control. We tested 20 different compounds: baicalein, (+) -catechin, (−)-catechin, curcumin, emodin, epicatechin, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rg1, honokiol, magnolol, oligonol, quercetin, reseveratrol, rutin, saponin, ursodeoxycholic acid (UDCA), and wogonin. The chemical structures of baicalein, honokiol, magnolol, quercetin, and Resveratroi are shown in Fig. 1. Among these 20 compounds, several flavonoids (baicalein and quercetin) and non-flavonoids (honokiol, magnolol, and resveratrol) were found to have significant antibacterial activities against V. cholerae, although their activities were less potent than those of gentamicin (11.667 ± 0.577 mm for baicalein, 15.333 ± 0.577 mm for honokiol, 13.000 ± 1.000 mm for magnolol, 10.050 ± 0.707 mm for quercetin, and 13.333 ± 0.577 mm for resveratrol, compared with gentamicin 25.667 ± 1.155 mm) as shown in Table 1. We also tested whether these compounds have similar antimicrobial activities against other enteric pathogens, such as E. coli, P. aeroginosa, S. typhimurium, and Shigella species. Interestingly, there was no significant antibacterial effect of the compounds on these pathogens, emphasizing V. cholerae-specific antibacterial efficacy of these plant-derived natural compounds (Table 2). For compounds that demonstrated activity in the disk diffusion assay, we performed a bacterial growth assay to test if these compounds inhibit bacterial growth. There were small yet statistically significant decreases in V. cholerae growth in baicalein, honokiol, magnolol, quercetin, or resveratrol-treated samples beginning at 1, 2, 4, and 8 h post stimulation (Fig. 2(A)). Antimicrobial activities of honokiol and magnolol were further evaluated by determining the minimum inhibitory concentration (MIC). As shown in Table 3, honokiol inhibited the growth of V. cholerae at 0.625 µg/mL, whereas magnolol inhibited the growth of bacterial cells at 5 µg/mL. In addition to bacteriostatic effect, we wanted

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Table 3. Minimum inhibitory concentration (MIC) of natural compounds against V. cholerae. Compounds

MIC (µg/mL)

Baicalein Honokiol Magnolol Quercetin Resveratrol

1.25 0.625 5 0.625 0.625

Note: Cells were grown to mid-log phase in the absence or presence of baicalein, honokiol, magnolol, quercetin, or resveratrol, serial-diluted in LB medium at 2-fold increments with concentrations in two staggered dilution series.

to test if these molecules have a bactericidal effect as well. As shown in Fig. 2(B), honokiol and magnolol treatment significantly decreased V. cholerae recovery, resulting in approximately 4 logs lower bacterial cell counting, compared with DMSO control alone. Biofilm formation is important for survival of bacteria, and biofilms are thought to play an important role in the survival of aquatic V. cholerae and in the pathogenesis of cholera.17–22) To examine, if honokiol and magnolol treatment interfere with biofilm formation, V. cholerae cultures were treated with various concentrations of baicalein, honokiol, magnolol, quercetin, or resveratrol. Honokiol- or magnolol-treated V. cholerae cultures showed significantly reduced biofilm formation, while baicalein, quercetin, or resveratrol did not inhibit biofilm formation (Fig. 3). This suggests that honokiol and magnolol may specifically target pathways required for V. cholerae biofilm formation in vitro.

Fig. 3. Honokiol and magnolol inhibit biofilm formation. Notes: Crystal violet staining of V. cholerae biofilms shows that honokiol and magnolol (10 μg/mL) reduced the biofilm biomass accumulating on the sides of culture tubes after 48 h of growth. The amount of crystal violet retained by biofilms was quantitated using optical density measurements (OD 570 nm). The data shown are presented as mean ± SD for three independent experiments. *p < 0.05 for comparison with the DMSO-treated control.

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Antioxidant and anti-inflammatory effect of honokiol and magnolol in LPS-stimulated mouse macrophages Given that V. cholerae LPS can induce increased production of pro-inflammatory mediators in various cell types,23) we wanted to determine if honokiol and magnolol would modulate cellular immune responses after LPS stimulation. First, we performed MTT assay on E. coli or V. cholerae LPS Ogawa and Inaba-stimulated mouse macrophages RAW 264.7 cells. There was no significant decrease in % cell viability in cells treated with honokiol or magnolol at a concentration of 10 μg/mL in response to E. coli or V. cholerae LPS (data not shown). We next tested whether ROS production in response to LPS stimulation is modulated by honokiol or magnolol treatment. The addition of honokiol and magnolol in E. coli or V. cholerae LPS Ogawa and Inaba -stimulated mouse macrophage cells resulted in a significant inhibition of ROS generation from these cells (honokiol or magnolol-treated cultures [70–75% inhibition]), shown in Fig. 4(A). Mitochondria, as a major site of ROS production in cells, have been shown to contribute to bactericidal activities in macrophages. 24) Thus,

we also tested whether mitochondrial superoxide production is altered upon treatment with honokiol or magnolol. Activation of Toll-like receptor 4 (TLR4) signaling pathways in response to LPS has been shown to up-regulate mitochondrial ROS (mROS) production.24) Consistent with previous findings, we observed that the exposure of cells to LPS augmented mROS production by FACS. However, addition of honokiol or magnolol resulted in the reduction of mROS, compared with LPS, shown by flow cytometry (Fig. 4(B)). This data potentially suggests that honokiol and magnolol may induce antioxidant activities in response to LPS stimulation. Pro-inflammatory cytokines (including TNF-α, interleukin-6 (IL-6) and interleukin-1β (IL-1β) and mediators (NO) play important roles in inflammatory process.25) Since NO and TNF-α are two of the most important inflammatory mediators in macrophages, we next measured the effects of honokiol and magnolol on LPS-induced releases of NO from RAW264.7 cells and the production of TNF-α from bone marrow-derived macrophages (BMDMs). Cells were pretreated with honokiol/magnolol for 2 h followed by stimulation with

Fig. 4. Effect of honokiol and magnolol on ROS production in V. cholerae LPS-stimulated mouse macrophages. Notes: Raw 264.7 cells were grown in 96-well plates. Next day, honokiol and magnolol (10 µg/mL) were added to cells for 2 h before stimulation with E. coli LPS, V. cholerae Ogawa and Inaba LPS (1.0 µg/mL). Total cellular ROS production (A) was measured. Data shown are expressed as mean ± SD for three independent experiments.*p < 0.05 for comparison with the DMSO-treated control. Mitochondrial superoxide formation levels (B) were determined by measuring MitoSOX staining of the cells, and analyzed by flow cytometry analysis (FACS). The graph shown is representatives of two independent experiments.

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Fig. 5. Effect of honokiol and magnolol on cellular inflammatory response in V. cholerae LPS-stimulated mouse macrophages. Notes: RAW 264.7 or BMDM cells were grown in 96-well plates. Next day, honokiol and magnolol (10 µg/mL each) were added to cells for 2 h before stimulation with E. coli LPS, V. cholerae Ogawa and Inaba LPS (1.0 µg/mL). Cell supernatant was collected after 24 h and NO from Raw 264.7 cells (A), and TNF-α from BMDMs (B) production were measured. Mean concentration ± SD from three independent experiments is shown.

1 μg/mL of E. coli or V. cholerae LPS Ogawa and Inaba LPS for overnight. V. cholerae LPS stimulation of RAW264.7 cells resulted in up-regulation of NO release, and both honokiol and magnolol suppressed LPS-induced production of NO (Fig. 5(A)). Furthermore, as shown in Fig. 5(B), TNF-α levels were downregulated upon the addition of honokiol and magnolol followed by V. cholerae LPS stimulation in BMDMs, suggesting that honokiol/magnolol induce anti-inflammatory response upon V. cholerae LPS stimulation in primary macrophage cells. Honokiol and magnolol treatment results in decreased lethality rate in C. elegans during V. cholerae infection C. elegans has been used as an invertebrate host model to identify and assess virulence factors of several human pathogens including V. cholera.26,27) We tested an ability of honokiol and magnolol to induce anti-microbial activities in vivo using C. elegans liquidkilling assays.16) Consistent with previously results, V. cholerae infection in C. elegans led to death of worms within ~8 days. The treatment with honokiol or magnolol increased a lifespan of V. cholerae-infected worms significantly (honokiol (p = 0.3350 for 1 µg/mL and p = 0.0098 for 10 µg/mL compared with worms adding on DMSO control, respectively) and magnolol (p = 0.0020 for 1 µg/mL and p = 0.0001 for 10 µg/mL compared with worms adding on DMSO control, respectively)) (Fig. 6). This data suggests that these compounds contribute to host defense in vivo in C. elegans against enteric pathogens.

Discussion Plant-derived natural compounds offer a promising strategy to overcome bacterial resistance mechanisms and restore the effectiveness of antibiotics. In our study, we report antibacterial, antioxidant, and anti-inflammatory activities of honokiol and magnolol against V. cholerae. Using various methods, such as disk diffusion, bacterial growth, and killing assays, we determined that there was a dose-dependent bactericidal activity of magnolol and honokiol against V. cholerae, and a significant biofilm formation inhibition caused by honokiol and magnolol. In addition to antibacterial activities, these molecules also induced an attenuating effect on ROS production and pro-inflammatory responses generated by macrophages in response to E. coli and V. cholerae LPS. Additionally, C. elegans lethality assay revealed that honokiol and magnolol have an ability to extend a lifespan of infected worms, contributing to prolonged survival of worms after lethal infection. Our data suggest that the growth of honokiol and magnolol-treated biofilms was significantly inhibited, whereas biofilm inhibition by baicalein, quercetin, and resveratrol was not significant (Fig. 2). Biofilms are formed by surface-associated communities of bacteria, which produce extracellular polymeric matrix (EPM) that decreases their susceptibility to host immunity and antimicrobial agents.17,28) The formation of biofilms during the proliferation of V. cholerae is linked to its pathogenesis.22,29) Therefore, our findings suggesting honokiol and magnolol as biofilm

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Fig. 6. Honokiol/magnolol delays killing during V. cholerae infection in C. elegans. Notes: Lethality analysis was performed in C. elegans fer-15(b26) II;fem-1(hc17) worms that were fed with V. cholerae. Worms were transferred into the wells of 6-well microtiter plates (30 worms per well), containing medium supplemented with various concentrations of honokiol or magnolol (1 and 10 g/mL). DMSO was employed as control. Agar plates were kept at 25 °C and scored for survivors at 24 h intervals. Data were plotted according to a Kaplan–Meier method, and survival curves were compared using the log-rank test.

inhibiting agents may suggest a promising source for the development of a potential biofilm inhibition against V. cholerae, similar to arctic actinomycetes that was suggested as potential inhibitors of V. cholerae biofilm.30) In general, the outer membrane of V. cholerae has been reported to have a smooth-type lipopolysaccharide (LPS) that constitutes an effective permeability barrier for extracellular molecules. Thus, these cells are highly sensitive to a wide range of chemicals.31) Indeed, V. cholerae possess the highest mean values of susceptibility to ten different plant polyphenols.32) Taken together, our results indicate that these cell surface features of V. cholerae may be causative of the critical susceptibility to honokiol and magnolol compared to other pathogenic bacteria. In addition, in vivo antimicrobial role of plant-derived natural extracts against V. cholerae has been recently described by Faruque group. They show that methanol extract of V. negundo leaves, commonly found in Bangladesh and India, exhibited strong vibriocidal activity, inducing protection from V. cholerae infection-mediated killing in infant mouse model.33) Given that honokiol and

magnolol have shown antimicrobial activities in vitro, we attempted an in vivo study (liquid-based lethality assay) to determine the potential anti-infectivity of these compounds in C. elegans model. C. elegans has been used as an excellent model to identify virulence factors and study the pathogenesis of bacterial pathogens, such as Salmonella Typhimurium, Pseudomonas aeruginosa, and Vibrio cholerae.26,34–36) Our data indicate that honokiol and magnolol increase the survival rate of infected worms and even low concentration at a 1 µg/mL can provide a prolonged survival of C. elegans. These data suggest that C. elegans model can be utilized as an in vivo antimicrobial screening system and natural and synthetic compounds can be further investigated as novel therapeutic agents against V. cholerae infection. In addition to honokiol and magnolol, flavonoids (baicalein and quercetin) and polyphenolic compound (resveratrol) also showed similar in vitro antibacterial effect against V. cholerae. Baicalein has previously been shown to significantly restore the effectiveness of β-lactam antibiotics and tetracycline against methicillinresistant Staphylococcus aureus (MRSA),37) but the role of baicalein for cholera has not been demonstrated. Quercetin was implicated to play an immunomodulatory role to suppress nuclear factor-κB (NF-κB) activation in response to V. cholerae stimulation in intestinal epithelial cells. In vivo, quercetin administration produced a significant reduction of neutrophil infiltration in the intestinal epithelial layer of suckling mouse12) and was proven to be effective anti-diarrheal agent.38) Lastly, the role of resveratrol in cholera infection was also studied by Morinaga et al. indicating that resveratrol inhibited CT-induced cAMP accumulation in Vero cells by suppressing the internalization of CT.13) Combined with our results, it is interesting to note that flavonoids and polyphenolic compounds found in food may have some protective activity against cholera infection. Although mechanisms for the antimicrobial actions of honokiol and magnolol have yet to be uncovered, our data for the first time provide promising evidence for specific antimicrobial function of honokiol and magnolol against V. cholerae infection. The spread of drug-resistant V. cholerae strains is a major clinical problem, and the ineffectiveness in antibiotic treatment necessitates finding new modes of prevention and containment of the disease, cholera. Given potential bacteriostatic and bactericidal activities of honokiol and magnolol against V. cholerae, we can conclude that food-grade honokiol and magnolol may be introduced as possible protective or preventive agents for cholera in food industry.

Acknowledgement This work was supported by the Korean Health Technology R&D Projects (R1306922), Ministry for Health & Welfare, Republic of Korea (O.S.S), and by the Cooperative Research Program for Agriculture Science & Technology Development (PJ009990032014), Rural Development Administration, Republic of Korea (S.Y.J.).

Natural antibacterial compounds against Vibrio cholerae

Funding Korean Health Technology R&D Projects (R1306922), Ministry for Health & Welfare, Republic of Korea (O.S.S), and by the Cooperative Research Program for Agriculture Science & Technology Development (PJ009990032014), Rural Development Administration, Republic of Korea (S.Y.J.).

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