Advanced
In vitro antibacterial and synergistic effect of phlorotannins isolated from edible brown seaweed Eisenia bicyclis against acne-related bacteria
In vitro antibacterial and synergistic effect of phlorotannins isolated from edible brown seaweed Eisenia bicyclis against acne-related bacteria
ALGAE. 2014. Mar, 29(1): 47-55
Copyright © 2014, The Korean Society of Phycology
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
  • Received : February 02, 2014
  • Accepted : March 03, 2014
  • Published : March 15, 2014
Download
PDF
e-PUB
PubReader
PPT
Export by style
Share
Article
Author
Metrics
Cited by
TagCloud
About the Authors
Jeong-Ha Lee
Department of Microbiology, Pukyong National University, Busan 608-737, Korea
Sung-Hwan Eom
Department of Food Science and Technology, Pukyong National University, Busan 608-737, Korea
Eun-Hye Lee
Department of Food Science and Technology, Pukyong National University, Busan 608-737, Korea
Yeoun-Joong Jung
Department of Food Science and Technology, Pukyong National University, Busan 608-737, Korea
Hyo-Jung Kim
Department of Food Science and Technology, Pukyong National University, Busan 608-737, Korea
Mi-Ra Jo
Food Safety Research Division, National Fisheries Research & Development Institute, Busan 619-705, Korea
Kwang-Tae Son
Food Safety Research Division, National Fisheries Research & Development Institute, Busan 619-705, Korea
Hee-Jung Lee
Food Safety Research Division, National Fisheries Research & Development Institute, Busan 619-705, Korea
Ji Hoe Kim
Food Safety Research Division, National Fisheries Research & Development Institute, Busan 619-705, Korea
Myung-Suk Lee
Department of Microbiology, Pukyong National University, Busan 608-737, Korea
Young-Mog Kim
Department of Food Science and Technology, Pukyong National University, Busan 608-737, Korea
ymkim@pknu.ac.kr
Abstract
To develop effective and safe acne vulgaris therapies with a continuing demand for new solutions, we investigated unique efficacy of an antibacterial agent from marine brown alga Eisenia bicyclis in treating acne vulgaris. The methanolic extract of E. bicyclis exhibited potential antibacterial activity against acne-related bacteria. The ethyl acetate fraction showed the strongest antibacterial activity against the bacteria among solvent fractions. Six compounds (1-6), previously isolated from the ethyl acetate fraction of E. bicyclis , were evaluated for antibacterial activity against acne-related bacteria. Among them, compound 2 (fucofuroeckol-A [FF]) exhibited the highest antibacterial activity against acne-related bacteria with a minimum inhibitory concentration (MIC) ranging from 32 to 128 μg mL -1 . Furthermore, FF clearly reversed the high-level erythromycin and lincomycin resistance of Propionibacterium acnes . The MIC values of erythromycin against P. acnes were dramatically reduced from 2,048 to 1.0 μg mL -1 in combination with MIC of FF (64 μg mL -1 ). The fractional inhibitory concentration indices of erythromycin and lincomycin were measured from 0.500 to 0.751 in combination with 32 or 64 μg mL -1 of FF against all tested P. acnes strains, suggesting that FF-erythromycin and FF-lincomycin combinations exert a weak synergistic effect against P. acnes . The results of this study suggest that the compounds derived from E. bicyclis can be a potential source of natural antibacterial agents and a pharmaceutical component against acnerelated bacteria.
Keywords
INTRODUCTION
Acne vulgaris is a common skin disease affecting children and adolescents. The pathogenesis of acne is multifactorial and complex. There are four important factors that cause acne in humans, such as an increase in sebum secretion, keratinization of the follicle, bacteria, and inflammation ( Farrar and Ingham 2004 ). Propionibacterium acnes , Staphylococcus epidermidis , S. aureus , and Pseudomonas aeruginosa are often involved in the development of abnormal follicular keratinization and inflammation ( Yamaguchi et al. 2009 ). P. acnes and S. epidermidis have been recognized as pus-forming organisms that trigger inflammation in acne. Especially, P. acnes , one of the commonly isolated skin organisms, induces an inflammation of the sebaceous glands in human face, neck, chest or back ( Park et al. 2004 ). The currently available therapeutic option for acne (antibiotic treatment) is usually used to treat acne vulgaris to inhibit inflammation or kill the bacteria. In the case of antibiotic therapy, triclosan, benzoyl peroxide, azelaic acid, retinoid, tetracycline, erythromycin, macrolide, and clindamycin are the most commonly prescribed ( Gollnick et al. 2003 , Ravenscroft 2005 , Han et al. 2010 ). However, these antibiotics are often associated with several side effects, such as the emergence of resistant bacteria, organ damage, and immune hypersensitivity if these medicines are used for a long period ( Kim et al. 2008 ). Therefore, many researchers have tried to develop targeted therapeutic agents with no side effects and high antibacterial activity.
To overcome the problem of side effects, medicinal plants and marine organisms have been investigated for the treatment of acne. Therefore, we investigated the possibility that they may be effective acne treatments based on the previously known anti-methicillin-resistant S. aureus effects of phlorotannin isolated from brown alga Eisenia bicyclis . E. bicyclis is a common perennial phaeophyceae (brown alga) and generally inhabits the region of Ulleung Island in the East sea of Korea. This seaweed has been added to appetizers, casseroles, muffins, pilafs, and soups ( Maegawa 1990 , Yoon et al. 2011 ). The antioxidant activity of E. bicyclis phlorotannins, such as eckol (a trimer), phlorofucofuroeckol A (a pentamer), dieckol and 8,8′-bieckol (hexamers) have been previously described ( Okada et al. 2004 ). This brown alga has also been reported to exhibit several medicinal functions, such as anti-tumor ( Ermakova et al. 2013 ), anti-Alzheimer’s disease ( Ahn et al. 2012 ), anti-atherosclerosis ( Kang et al. 2006 ), anti-inflammatory ( Jung et al. 2013 ), anti-coagulant activities ( Jeong et al. 2009 ), anti-allergic disease and anti-cancer activities ( Shibata et al. 2003 , Yoon et al. 2013 ). In addition, phlorotannins have been known to show potent antimicrobial activity against several microorganisms ( Eom et al. 2013 ).
However, there is no obvious report on the antibacterial activity of phlorotannins from brown alga against acne-related bacteria. Therefore, we demonstrated that phlorotannins isolated from E. bicyclis have high antibacterial effects against acne-related bacteria and may act as alternative and therapeutic agents for acne in this study.
MATERIALS AND METHODS
- Raw materials and extraction
In late September 2010, E. bicyclis was purchased from Ulleung Trading Co. (Ulleung-gun, Korea). A voucher specimen has been deposited in the author’s laboratory. Dried E. bicyclis was finely ground and powdered with a food mixer (HMF-1000A; Hanil Electronics, Seoul, Korea). The dried powder was vacuum-packed and kept at -20℃ until use. The dried E. bicyclis powder (1.0 kg) was extracted with methanol (MeOH; 10 L × 3) at 70℃ for 3 h (3 times) and the solvent was evaporated in vacuo with a rotary evaporator (N-1001S-W; Eyela, Tokyo, Japan). The crude MeOH extract of E. bicyclis was suspended in 10% MeOH (1.0 L) and then partitioned in turn with n -hexane (Hexane), dichloromethane (DCM), ethyl acetate (EtOAc), and n -butanol (BuOH) in sequence. The concentration of each extract was adjusted to 200 mg mL -1 by dissolving in dimethyl sulfoxide under sterile conditions and stored at -70℃ until used.
- Microorganism and culture
The following bacterial strains obtained from the Korean Collection for Type Cultures (KCTC; Daejeon, Korea) were used as indicator microorganisms in the study: P. acnes (KCTC 3314), S. aureus (KCTC 1927), S. epidermidis (KCTC 1370), P. aeruginosa (KCTC 1637), which were used for evaluation of anti-acne-related bacterial effect. Two strains of P. acnes clinical isolates were provided by the Gyeongsang National University Hospital (Jinju, Korea), a member of the National Biobank of Korea.
P. acnes strains were anaerobically cultivated in brain heart infusion broth (BHI; Difco Inc., Detroit, MI, USA) supplemented with 1.0% glucose, and incubated at 37℃ for 24 h in a CO 2 incubator (NAPCO 5400; General Laboratory Supply, Pasadena, TX, USA), in a 10% CO 2 humidified atmosphere. As for determining the growth curves of bacterial cells under optimal growth conditions, cultures of P. acnes were diluted and plated on BHI-agar. The plates were incubated at 37℃ for 24 h in a 10% CO 2 incubator and the number of colony-forming units (CFU) was determined. S. aureus , S. epidermidis , and P. aeruginosa were grown aerobically at 37℃ in tryptic soy broth (TSB; Difco Inc.). The disk diffusion assay was prepared in Mueller-Hinton agar (MHA; Difco Inc.) and the broth dilution method was carried out in Mueller-Hinton broth (MHB; Difco Inc.) according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (formerly National Committee for Clinical Laboratory Standards [NCCLS]).
- Disk diffusion assay
The antibacterial efficacy was evaluated by disk diffusion assay described by the CLSI (2009) . In brief, bacterial strains were cultured in TSB at 37℃ until cells reached at an OD 600 nm of 0.5. One hundred microliter of bacterial culture containing approximately 10 4 -10 5 CFU mL -1 was spread on MHA agar plates. A paper disc (6 mm in diameter) containing 1 mg and 5 mg of each extract was placed in the above MHA plate. After incubating for 24 h at 37℃, the diameter of the inhibition zone was measured on bacterial culture plates. The experiment was carried out three times and the mean values were presented.
- Determination of minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs)
The concentration of MeOH extract and its solvent fractions of E. bicyclis was 200 mg mL -1 . Each extracts was diluted with MHB to obtain a stock solution of 2,048 μg mL -1 . To determine the MIC values of the MeOH extract and its solvent fractions of E. bicyclis , a stock solution of extracts was prepared in microbial culture medium for each microbial species. The MICs are the lowest concentration of MeOH extracts and its solvent fraction of E. bicyclis to inhibit the visible growth of microorganisms after overnight incubation using MHB, which was modified from the methods described for antimicrobial susceptibility testing by the CLSI (2006) . MIC was defined as the lowest concentration of crude extract that inhibited the visual growth after incubating the aerobic bacteria for 18 h and the anaerobic bacteria for 48 h. MICs of the solvent-soluble extracts were determined by the two-fold serial dilution method in 96-well flat-bottomed microtitration plates at final concentration of 7 × 10 5 CFU mL -1 . The microtitration plates were read visually and the MIC of the extracts that exhibited no turbidity was recorded as the MIC. For MBC testing, an aliquot of inoculum was taken with a MIC test well that did not show turbidity, and was poured onto nutrient agar (Difco Inc.) plates for each bacterial species. The agar plates were incubated for 2 days for aerobic bacteria and 5 days for anaerobic bacteria. The MBC value was read as the lowest concentration of the solvent-soluble extracts at which 99.99% or more of the initial inoculum was killed. The MIC and MBC experiments were repeated in triplicate.
- Synergistic effects of fucofuroeckol-A (FF) with tetracycline, erythromycin, and lincomycin againstPropionibacterium acnes
The interaction between FF and antibiotics including tetracycline, erythromycin, and lincomycin (Sigma Chemical Co., St. Louis, MO, USA) against P. acnes was tested by the checkerboard method ( Weig and Müller 2001 , Perea et al. 2002 ). The synergistic effect was evaluated as a fractional inhibitory concentration (FIC) index. With the checkerboard test, the FIC was calculated as the MIC values of an antibiotic or FF in combination divided by the MIC of the antibiotic or FF alone. The FIC was then summed to derive the FIC index, which indicated synergy when index values were determined using the following formulae:
  • FICA= MICAin combination / MICA,
  • FICB= MICBin combination / MICB,
  • FIC index = FICA+ FICB
The interaction was defined as synergistic if the FIC index was <1, additive if the FIC index was 1.0, subadditive if the FIC index was between 1.0 and 2.0, indifferent if the FIC index was 2, and antagonistic if the FIC index >2. Synergy was further subclassified as marked (FIC index, ≤0.50) and weak (FIC index, between 0.50 and 1.0).
- Statistical analysis
In all cases analyses were performed in triplicate and data were averaged over the three measurements. The standard deviation (SD) was also calculated. Significance of differences between average MICs for each individual microorganism were determined by Student’s t test at the 95% significance level using SPSS version 12.0 (SPSS Inc., Chicago, IL, USA).
RESULTS
- Anti-acne related bacteria activity ofEisenia bicyclisextracts
The antibacterial activity of methanol extract and its solvent fractions are presented in Table 1 . The MeOH extract of E. bicyclis exhibited an antibacterial activity against acne-related bacteria, suggesting that the extract contains antibacterial substances against acne-related bacteria. For P. acnes , the clear zones of treatment with the EtOAc-soluble fraction had a diameter of 9.0 mm with a concentration of 1 mg per disc and 12.0-19.0 mm with concentrations of 5 mg per disc. For S. aureus and S. epidermidis , the antibacterial activity of the EtOAc-soluble fraction were noted as 1.0 mg per disc (clear zone, 10.0 mm) and 5.0 mg per disc (clear zone, 16.0 and 20.0 mm, respectively). However, the antimicrobial effects on gram-negative P. aeruginosa were less effective than those on gram-positive bacteria in all the tested extract and fractions. Regardless of solvent fractionation, the water-soluble fraction of the MeOH extract did not exhibit the antibacterial activity against all of the bacteria tested.
Disk diffusion assay of methanol extract and its solvent-soluble fractions fromEisenia bicyclisagainst skin-pathogenic microorganisms
PPT Slide
Lager Image
MeOH, methanolic extract; Hexane, n-hexane-soluble extract; DCM, dichloromethane-soluble extract; EtOAc, ethyl acetate-soluble extract; BuOH, n-butanol-soluble extract; H2O, water-soluble extract.aMethanol extract and its fraction from E. bicyclis were loaded onto a disk (6 mm in diameter).bData are the averages of duplicate experiments.cNo detected antibacterial activity.
- Measurement of MIC and MBC values ofEisenia bicyclisextract
The MIC values of solvent fractions against acne-related bacteria varied depending on the polarity of the solvent. Among solvent-soluble fractions, the EtOAc-soluble fraction showed the lowest MIC values against acne-related bacteria. The EtOAc-soluble extract could completely inhibit the growth of P. acnes strains at 128 and 256 μg mL -1 concentration. The antibacterial activity of the EtOAc-soluble extract against acne-related bacteria was higher than those of other-soluble fractions. Eom et al. (2011) reported that the EtOAc-soluble extract of E. bicyclis exhibited the highest antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and food-pathogen bacteria. The MBC values of EtOAc-soluble fraction against P. acnes strains were determined from 128 to 512 μg mL -1 ( Table 2 ). However, no antibacterial activities of E. bicyclis extracts against P. aeruginosa were observed. It was also observed that the gram-negative bacteria P. aeruginosa showed higher MIC values for all extracts compared to other gram-positive bacteria.
Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of methanol extract and its solvent-soluble fractions fromEisenia bicyclisagainst skin-pathogenic microorganisms
PPT Slide
Lager Image
MeOH, methanolic extract; Hexane, n-hexane-soluble extract; DCM, dichloromethane-soluble extract; EtOAc, ethyl acetate-soluble extract; BuOH, n-butanol-soluble extract; H2O, water-soluble extract.aMIC and MBC values for methanol extract and its solvent-soluble fractions from E. bicyclis are express as μg mL-1.bNo detected antibacterial activity.
- MIC value of isolated phlorotannins fromEisenia bicyclis
According to above results, the EtOAc-soluble fraction of E. bicyclis showed the strongest antibacterial activity against acne-related bacteria. In order to identify an anti-acne substance from the EtOAc-soluble fractions of E. bicyclis , we screened the antibacterial activity of previously isolated phlorotannins against acne-related bacteria. It is reported that marine-derived polyphenols (phlorotannins) are believed as the active components of E. bicyclis ( Eom et al. 2013 ). Recently, we have reported that successive column chromatographic purification of the EtOAc-soluble extract led to the isolation and characterization of six phloroglucinol derivatives: eckol (1), FF (2), 7-phloroeckol (3), dioxinodehydroeckol (4), phlorofucofuroeckol-A (PFF) (5), and dieckol (6) ( Eom et al. 2013 ). In this report, we demonstrate the antimicrobial activities of these compounds against acne-related bacteria. The MIC values of these phlorotannins were in the range of 32 to 64 μg mL -1 and were at least two-folds less than those of tetracycline, erythromycin and lincomycin against P. acnes ( Table 3 ). Among the isolated compounds, compound 2 (FF) had the highest antibacterial activity against acne-related bacteria (32 to 128 μg mL -1 ) ( Table 3 ). Other compounds had also similar antibacterial activities with a MIC values from 64 to 256 μg mL -1 . P. acnes strains were considered to be highly resistant to antibiotics (tetracycline, erythromycin, and lincomycin) in this study. In particular, P. acnes strains with high-level resistance to erythromycin and lincomycin were detected with the MIC values of 2,048 and 1,024 μg mL -1 , respectively. The MIC values of FF against P. acnes (32 to 64 μg mL -1 ) were equal or lower than that of tetracycline against P. acnes (32 μg mL -1 ). S. epidermidis and S. aureus were found to be sensitive for tetracycline, erythromycin, and lincomycin (0.125 to 8 μg mL -1 ).
Minimum inhibitory concentrations (MIC) of phlorotannins isolated fromEisenia bicyclisand antibiotics (tetracycline, erythromycin, and lincomycin) against skin-pathogenic microorganisms
PPT Slide
Lager Image
EK, eckol; FF, fucofuroeckol-A; 7P, 7-phloroeckol; DD, dioxinodehydroeckol; PFF, phlorofucofuroeckol-A; DE, dieckol.
- Synergic effects between FF and antibiotics againstPropionibacterium acnes
As an alternative way, natural materials such as plantderived or marine-derived compounds in combination with traditional medicines against drug-resistant bacteria may be used as an effective approach for restoration of antibiotic activity ( Eom et al. 2013 ). Taylor et al. (2005) reported that green tea components may display synergy with conventional antibiotics against gram-negative bacteria. In addition, the catechin fraction of green tea acted synergistically with ciprofloxacin in a chronic bacterial prostatitis model in the rat ( Lee et al. 2005 ). Based on these reports, the synergistic effect of marine-derived polyphenol on P. acnes was assessed in combination with commercial antibiotics to treat acne. As shown in Table 4 , the MIC values of tetracycline against P. acnes were reduced from 16 to 8 μg mL -1 when administered in combination with 64 μg mL -1 of FF. The MIC values of erythromycin and lincomycin against P. acnes were also dramatically reduced when administered in combination with FF. The FIC indices of antibiotics were in a range from 0.500 to 0.750 in combination with the concentration of FF (64 μg mL -1 ) against P. acnes strains, thereby indicating the marked or weak synergy effect of FF-antibiotics (tetracycline, erythromycin, and lincomycin) combination, suggesting that FF-tetracycline, FF-erythromycin, and FF-lincomycin synergistically inhibited the growth of P. acnes .
Minimum inhibitory concentrations (MIC) and fractional inhibitory concentration (FIC) indices of fucofuroeckol-A in combination with antibiotics used in the treatment of acne
PPT Slide
Lager Image
A, without fucofuroeckol-A; B to C and b to c, fucofuroeckol-A at 32.0 and 64.0 μg mL-1, respectively.aThe FIC index indicated synergistic effect: <0.5, marked synergy; 0.5 to <1.0, weak synergy; 1.0, additive; >1.0 to <2.0, subadditivie; 2.0, indifferent; >2.0, antagonistic.
DISCUSSION
Tetracycline, erythromycin, and lincomycin have been used for decades to treat acne. However, these antibiotics can cause undesirable side effects, including vomiting diarrhea, sore mouth, and skin redness. In addition, antibiotic-resistant P. acnes is a growing problem in many countries due to the overuse of antibiotics ( Davies and Davies 2010 ). Therefore, there is a need to develop new medicines or alternative therapies for acne.
In an effort to decrease usage of antibiotics and discover an alternative therapeutic agent for treating acne infection, we have screened MeOH extract and its soluble extract from a brown alga E. bicyclis to find out anti-acne agents. The relative susceptibility of acne-related bacteria to the potential antimicrobial agent was measured by a clear zone of growth inhibition around the disc. A previous study reported the antimicrobial activities of MeOH extract and its solvent-soluble extract from E. bicyclis against MRSA and food-pathogen bacteria ( Eom et al. 2011 ). The EtOAc-soluble extract showed the highest antibacterial activity against MRSA and S. aureus in disc diffusion (15-24 mm) with the highest total polyphenolic contents ( Eom et al. 2011 ). Choi et al. (2011) reported that the MeOH extracts of brown algae Ecklonia cava , Ecklonia kurome , and Ishige sinicola exhibited antibacterial activity against P. acnes , with 5.3, 5.7, and 6.3 mm at 5 mg per disc, respectively. Thus, E. bicyclis has a similar antibacterial activity against P. acnes when compared with other brown seaweeds.
The present study showed significant correlations between anti-MRSA activity and anti-acne-inducing bacterial activity. In general, gram-negative bacteria are more resistant than gram-positive bacteria to treatment with natural anti-bacterial extracts ( Afolayan 2003 ). The higher resistance of gram-negative bacteria than gram-positive bacteria against different antibacterial compounds is generally attributed to differences in their cell wall and outer membrane ( Nikaido 1996 , McDonnell and Russell 1999 ). In this study, the EtOAc-soluble extract of E. bicyclis exhibited a similar antibacterial activity against acne-related bacteria in comparison to those from E. cava , E. kurome , and I. sinicola . Therefore, the MIC values indicated that the anti-acne-related bacteria activity of E. bicyclis was almost equal to the anti-MRSA activity of E. bicyclis .
According to Eom et al. (2011) , the antibacterial activities of brown algae are related to their total phenolic contents. Marine-derived polyphenols (phlorotannins) are the predominant EtOAc-soluble compound in brown algae ( Choi et al. 2010 ). Among EtOAc-soluble compound, the polyphenol polymers (eckol, PFF, dieckol, and 8,8′-bieckol) exhibited potent antibacterial activities ( Nagayama et al. 2002 , Isnansetyo and Kamei 2009 ). We also reported the isolation of six phloroglucinol derivatives from EtOAc-soluble extract of E. bicyclis with successive column chromatographic purification: eckol (1), FF (2), 7-phloroeckol (3), dioxinodehydroeckol (4), PFF (5), and dieckol (6) ( Eom et al. 2013 ).
Eckol (1), PFF (5), and dieckol (6) have previously been reported to exhibit potential antibacterial activity against MRSA ( Lee et al. 2008 , Choi et al. 2010 , Eom et al. 2013 ). These results are in accordance with those of our study, which found that eckol-type phlorotannins might possess potential anti-bacterial activities. Although several anti- P. acnes agents such as xanthonoid (α-mangostin, MIC = 1.95 μg mL -1 ), tannins (terchebulin, MIC = 250 μg mL -1 ; ellagic acid, MIC = 125 μg mL -1 ; flavogallonic acid dilactone, MIC = 250 μg mL -1 ), flavonoid (kaempferol and quercetin, MIC = 32-64 μg mL -1 ), and terpenoids (rosthornins, MIC = 3.17-25 μg mL -1 ) ( Kubo et al. 2004 , Lim et al. 2007 , Pothitirat et al. 2010 , Muddathir and Mitsunaga 2013 ) from natural sources have been identified, there has been no scientific report on anti- P. acnes activity of phlorotannins.
From our results, it appears that P. acnes is resistant against antibiotics. Because of its resistance to many commonly used antibiotics, there is a need for searching more effective anti-acne agents. As an alternative method, the FIC test for the combination of isolated phlorotannins and antibiotics, a commonly ineffective antibiotic to P. acnes due to resistance, was assessed using the checkerboard test. It has been previously reported that dieckol from E. stolonifera and PFF from E. bicyclis exhibited the synergistic effect in combination with β-lactam antibiotics against MRSA ( Lee et al. 2008 , Eom et al. 2013 ). The results of the checkerboard assay revealed the restoration of antibacterial activity of antibiotics used in this study against the antibiotic-resistant P. acnes in combination with FF. FF (MIC = 64 μg mL -1 ) can remarkably reduce the MIC values of the antibiotics against P. acnes , suggesting that FF may have potential for use as an adjunct in the treatment of antibiotic-resistant P. acnes . Several studies have previously reported synergistic effects between catechin and β-lactam antibiotics occurred at the cell wall ( Zhao et al. 2001 , Taylor et al. 2005 ). Therefore, further investigations are needed to fully elucidate the underlying antimicrobial mechanisms of FF against P. acnes .
In conclusion, the antibacterial activity of the phlorotannins from edible marine brown algae E. bicyclis against anti-acne-related bacteria was evaluated. Since the ethyl acetate fraction showed the strongest antibacterial activity against acne-related bacteria among solvent fractions, antibacterial activity of E. bicyclis extracts against acne-related bacteria may also correlate with their phlorotannins or marine-derived polyphenolic contents. Therefore, previously isolated phlorotannins such as eckol, FF, 7-phloroeckol, dioxinodehydroeckol, PFF, and dieckol were evaluated for antibacterial activity against acne-related bacteria. Among them, FF exhibited the highest antibacterial activity against acne-related bacteria. In addition, FF in combination with other antibiotics is expected to have a therapeutic effect for relieving symptoms against P. acnes with synergy effects. Collectively, phlorotannins can be used as natural antibiotic agents and food supplement as functional ingredients. The results of the present investigation are expected to contribute to the development of an alternative phytotherapeutic agent against antibiotic-resistant P. acnes .
To our knowledge, this is the first report on the antibacterial activity of phlorotannins against acne-related bacteria. The results of the present investigation are expected to contribute to the development of an alternative phytotherapeutic ingredient without any adverse side effects to human body.
Acknowledgements
This work was financially supported by the National Fisheries Research and Development Institute (RP-2014-FS-003). Also, this research was supported by the special fund of Pukyong National University donated by the SKS Trading Co. in Lynnwood, WA, USA in memory of the late Mr. Young Hwan Kang, who had a deep concern for and inspiration in fishery science. We are grateful to the Gyeongsang National University Hospital (Jinju, Korea), a member of the National Biobank of Korea.
References
Afolayan A. J. 2003 Extracts from the shoots of Arctotis arctotoides inhibit the growth of bacteria and fungi Pharm. Biol. http://dx.doi.org/10.1076/phbi.41.1.22.14692 41 22 - 25
Ahn B. R. , Moon H. E. , Kim H. R. , Jung H. A. , Choi J. S. 2012 Neuroprotective effect of edible brown alga Eisenia bicyclis on amyloid beta peptide-induced toxicity in PC12 cells Arch. Pharm. Res. http://dx.doi.org/10.1007/s12272-012-1116-5 35 1989 - 1998
Choi J. -G. , Kang O. -H. , Brice O. -O. , Lee Y. -S. , Chae H. -S. , Oh Y. -C. , Sohn D. -H. , Park H. , Choi H. -G. , Kim S. -G. , Shin D. -W. , Kwon D. -Y. 2010 Antibacterial activity of Ecklonia cava against methicillin-resistant Staphylococcus aureus and Salmonella spp Foodborne Pathog. Dis. http://dx.doi.org/10.1089/fpd.2009.0434 7 435 - 441
Choi J. -S. , Bae H. -J. , Kim S. -J. , Choi I. S. 2011 In vitro antibacterial and anti-inflammatory properties of seaweed extracts against acne inducing bacteria, Propionibacterium acnes J. Environ. Biol. 32 313 - 318
2006 Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically: Approved standard, CLSI document M07-A8 8th ed. CLSI Wayne, PA 68 pp -
2009 Performance standards for antimicrobial susceptibility testing: 18th informational supplement. CLSI document M100-S19 CLSI Wayne, PA 206 pp -
Davies J. , Davies D. 2010 Origins and evolution of antibiotic resistance Microbiol. Mol. Biol. Rev. http://dx.doi.org/10.1128/MMBR.00016-10 74 417 - 433
Eom S. -H. , Kim D. -H. , Lee S. -H. , Yoon N. -Y. , Kim J. H. , Kim T. H. , Chung Y. -H. , Kim S. -B. , Kim Y. -M. , Kim H. -W. , Lee M. -S. , Kim Y. -M. 2013 In vitro antibacterial activity and synergistic antibiotic effects of phlorotannins isolated from Eisenia bicyclis against methicillin‐resistant Staphylococcus aureus Phytother. Res. http://dx.doi.org/10.1002/ptr.4851 27 1260 - 1264
Eom S. -H. , Park J. -H. , Yu D. -U. , Choi J. -I. , Choi J. -D. , Lee M. -S. , Kim Y. -M. 2011 Antimicrobial activity of brown alga Eisenia bicyclis against methicillin-resistant Staphylococcus aureus Fish. Aquat. Sci. 14 251 - 256
Ermakova S. , Men’shova R. , Vishchuk O. , Kim S. -M. , Um B. -H. , Isakov V. , Zvyagintseva T. 2013 Water-soluble polysaccharides from the brown alga Eisenia bicyclis: structural characteristics and antitumor activity Algal Res. http://dx.doi.org/10.1016/j.algal.2012.10.002 2 51 - 58
Farrar M. D. , Ingham E. 2004 Acne: inflammation Clin. Dermatol. http://dx.doi.org/10.1016/j.clindermatol.2004.03.006 22 380 - 384
Gollnick H. , Cunliffe W. , Berson D. , Dreno B. , Finlay A. , Leyden J. J. , Shalita A. R. , Thiboutot D. 2003 Management of acne: a report from a global alliance to improve outcomes in acne J. Am. Acad. Dermatol. http://dx.doi.org/10.1067/mjd.2003.582 49 S1 - S37
Han S. , Lee K. , Yeo J. , Baek H. , Park K. 2010 Antibacterial and anti-inflammatory effects of honeybee (Apis mellifera) venom against acne-inducing bacteria J. Med. Plants Res. 4 459 - 464
Isnansetyo A. , Kamei Y. 2009 Anti-methicillin-resistant Staphylococcus aureus (MRSA) activity of MC21-B, an antibacterial compound produced by the marine bacterium Pseudoalteromonas phenolica O-BC30T Int. J. Antimicrob. Agents http://dx.doi.org/10.1016/j.ijantimicag.2009.02.009 34 131 - 135
Jeong E. -S. , Yoon Y. -H. , Kim J. -K. 2009 Contrasting correlation in the inhibition response of ADP-induced platelet aggregation and the anti-coagulant activities of algal fucoidans derived from Eisenia bicyclis and Undaria pinnatifida sporophylls (Mekabu) Fish. Aquat. Sci. 12 194 - 202
Jung H. A. , Jin S. E. , Ahn B. R. , Lee C. M. , Choi J. S. 2013 Anti-inflammatory activity of edible brown alga Eisenia bicyclis and its constituents fucosterol and phlorotannins in LPS-stimulated RAW264.7 macrophages Food Chem. Toxicol. http://dx.doi.org/10.1016/j.fct.2013.05.061 59 199 - 206
Kang K. A. , Lee K. H. , Chae S. , Zhang R. , Jung M. S. , Ham Y. M. , Baik J. S. , Lee N. H. , Hyun J. W. 2006 Cytoprotective effect of phloroglucinol on oxidative stress induced cell damage via catalase activation J. Cell Biochem. http://dx.doi.org/10.1002/jcb.20668 97 609 - 620
Kim J. Y. , Oh T. H. , Kim B. J. , Kim S. S. , Lee N. H. , Hyun C. G. 2008 Chemical composition and anti-inflammatory effects of essential oil from Farfugium japonicum flower J. Oleo Sci. http://dx.doi.org/10.5650/jos.57.623 57 623 - 628
Kubo I. , Xu Y. , Shimizu K. 2004 Antibacterial activity of ent‐kaurene diterpenoids from Rabdosia rosthornii Phytother. Res. http://dx.doi.org/10.1002/ptr.1421 18 180 - 183
Lee D. -S. , Kang M. -S. , Hwang H. -J. , Eom S. -H. , Yang J. -Y. , Lee M. -S. , Lee W. -J. , Jeon Y. -J. , Choi J. -S. , Kim Y. -M. 2008 Synergistic effect between dieckol from Ecklonia stolonifera and β-lactams against methicillin-resistant Staphylococcus aureus Biotechnol. Bioprocess Eng. http://dx.doi.org/10.1007/s12257-008-0162-9 13 758 - 764
Lee Y. S. , Han C. H. , Kang S. H. , Lee S. -J. , Kim S. W. , Shin O. R. , Sim Y. -C. , Lee S. -J. , Cho Y. -H. 2005 Synergistic effect between catechin and ciprofloxacin on chronic bacterial prostatitis rat model Int. J. Urol. http://dx.doi.org/10.1111/j.1442-2042.2005.01052.x 12 383 - 389
Lim Y. -H. , Kim I. -H. , Seo J. -J. 2007 In vitro activity of kaempferol isolated from the Impatiens balsamina alone and in combination with erythromycin or clindamycin against Propionibacterium acnes J. Microbiol. 45 473 - 477
Maegawa M. 1990 Ecological studies of Eisenia bicyclis (Kjellma) Setchell and Ecklonia cava Kjellman Bull. Fac. Bioresour. Mie Univ. 4 73 - 145
McDonnell G. , Russell A. D. 1999 Antiseptics and disinfectants: activity, action, and resistance Clin. Microbiol. Rev. 12 147 - 179
Muddathir A. M. , Mitsunaga T. 2013 Evaluation of anti-acne activity of selected Sudanese medicinal plants J. Wood Sci. http://dx.doi.org/10.1007/s10086-012-1303-5 59 73 - 79
Nagayama K. , Iwamura Y. , Shibata T. , Hirayama I. , Nakamura T. 2002 Bactericidal activity of phlorotannins from the brown alga Ecklonia kurome J. Antimicrob. Chemother. http://dx.doi.org/10.1093/jac/dkf222 50 889 - 893
Nikaido H. 1996 Multidrug efflux pumps of gram-negative bacteria J. Bacteriol. 178 5853 - 5859
Okada Y. , Ishimaru A. , Suzuki R. , Okuyama T. 2004 A new phloroglucinol derivative from the brown alga Eisenia bicyclis: potential for the effective treatment of diabetic complications J. Nat. Prod. http://dx.doi.org/10.1021/np030323j 67 103 - 105
Park J. , Lee J. , Jung E. , Park Y. , Kim K. , Park B. , Jung K. , Park E. , Kim J. , Park D. 2004 In vitro antibacterial and anti-inflammatory effects of honokiol and magnolol against Propionibacterium sp Eur. J. Pharmacol. http://dx.doi.org/10.1016/j.ejphar.2004.05.047 496 189 - 195
Perea S. , Gonzalez G. , Fothergill A. W. , Kirkpatrick W. R. , Rinaldi M. G. , Patterson T. F. 2002 In vitro interaction of caspofungin acetate with voriconazole against clinical isolates of Aspergillus spp Antimicrob. Agents Chemother. http://dx.doi.org/10.1128/AAC.46.9.3039-3041.2002 46 3039 - 3041
Pothitirat W. , Chomnawang M. T. , Gritsanapan W. 2010 Anti-acne-inducing bacterial activity of mangosteen fruit rind extracts Med. Princ. Pract. http://dx.doi.org/10.1159/000312714 19 281 - 286
Ravenscroft J. 2005 Evidence based update on the management of acne Arch. Dis. Child. Educ. Pract. Ed. 90 ep98 - ep101
Shibata T. , Nagayama K. , Tanaka R. , Yamaguchi K. , Nakamura T. 2003 Inhibitory effects of brown algal phlorotannins on secretory phospholipase A2s, lipoxygenases and cyclooxygenases J. Appl. Phycol. http://dx.doi.org/10.1023/A:1022972221002 15 61 - 66
Taylor P. W. , Hamilton-Miller J. M. T. , Stapleton P. D. 2005 Antimicrobial properties of green tea catechins Food Sci. Technol. Bull. 2 71 - 81
Weig M. , Müller F. -M. C. 2001 Synergism of voriconazole and terbinafine against Candida albicans isolates from human immunodeficiency virus-infected patients with oropharyngeal candidiasis Antimicrob. Agents Chemother. http://dx.doi.org/10.1128/AAC.45.3.966-968.2001 45 966 - 968
Yamaguchi N. , Satoh-Yamaguchi K. , Ono M. 2009 In vitro evaluation of antibacterial, anticollagenase, and antioxidant activities of hop components (Humulus lupulus) addressing acne vulgaris Phytomedicine http://dx.doi.org/10.1016/j.phymed.2008.12.021 16 369 - 376
Yoon N. Y. , Lee S. -H. , Shim K. B. , Lim C. -W. , Lee M. -H. , Cho H. -A. , Xie C. 2013 Quinone reductase induction activity of phlorotannins derived from Eisenia bicyclis in Hepa1c1c7 cells Fish. Aquat. Sci. 16 1 - 5
Yoon N. Y. , Lee S. -H. , Wijesekara I. , Kim S. -K. 2011 In vitro and intracellular antioxidant activities of brown alga Eisenia bicyclis Fish. Aquat. Sci. 14 179 - 185
Zhao W. -H. , Hu Z. -Q. , Okubo S. , Hara Y. , Shimamura T. 2001 Mechanism of synergy between epigallocatechin gallate and β-lactams against methicillin-resistant Staphylococcus aureus Antimicrob. Agents Chemother. http://dx.doi.org/10.1128/AAC.45.6.1737-1742.2001 45 1737 - 1742