Antimicrobial effect of phlorotannins from marine brown algae

Antimicrobial effect of phlorotannins from marine brown algae Sung-Hwan Eom , Young-Mog Kim Se-Kwon Kim a Marine Bioprocess Research Center, Pukyong National University, Busan 608-737, Republic of Koreab Department of Food Science and Technology, Pukyong National University, Busan 608-737, Republic of Koreac Marine Biochemistry Laboratory, Department of Chemistry, Pukyong National University, Busan 608-737, Republic of Korea Marine organisms exhibit a rich chemical content that possess unique structural features as compared to terrestrial metabolites. Among marine resources, marine algae are a rich source of chemically diverse compounds with the possibility of their potential use as a novel class of artificial food ingredients and antimicrobial agents. The objective of this brief review is to identify new candidate drugs for antimicro-bial activity against food-borne pathogenic bacteria. Bioactive compounds derived from brown algae are discussed, namely phlorotannins, that have anti-microbial effects and therefore may be useful to explore as potential antimicrobial agents for the food and pharmaceutical industries.
Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3251 Phlorotannins from marine brown algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3253 Antibacterial effect of phlorotannins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3253 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3254 Conflict of Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3254Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3254References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3254 the marine biodiversity of the ocean can be expected to yieldnew therapeutic agents ( Since the 1970s, more than 21,855 structurally diverse, bioac- Increasing resistance of clinically important bacteria to existing tive natural products with an astounding array of biological activ- antibiotics is a major problem throughout the world ities have been discovered from marine microbes, algae and ). Over the past 20 years, investigators from virtually invertebrates Even though, the ocean covers every corner of the world have documented that increasing pro- more than 70% of the earth’s surface, we only use less than 10% portions of Staphylococcus aureus are resistant to penicillin and of the total ocean area (In particular, many marine other antibiotics. As a result, the majority of S. aureus are swamped organisms live in complex habitats exposed to extreme conditions with methicillin-resistant S. aureus (MRSA). In spite of the available and in adapting to new environment surroundings, they produce a effective treatments against serious infections due to MRSA, high wide variety of secondary metabolites which cannot be found in mortality rates are still a major concern. There are a few new other organisms. Moreover, considering its great taxonomic diver- agents in development that can be expected to benefit the situa- sity, investigations related to the search for new bioactive com- tion in the next decade ). Over the past 50 years, pounds from the marine environment can be seen as an almost S. aureus has become resistant to most antibiotics except vancomy- unlimited field. In addition, since the biological productivity of ter- cin and other glycopeptides. Recently, these antibiotics have been restrial ecosystems has also perhaps reached what it can achieve; the mainstay of treatment for the multi drug-resistant S. aureusand therefore the possibility that vancomycin resistance mighttransfer from vancomycin-resistant enterococci to multi drug- ⇑ Corresponding author. Tel.: +82 516297094; fax: +82 516297099.
resistant S. aureus has been extremely worrying 0278-6915/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
S.-H. Eom et al. / Food and Chemical Toxicology 50 (2012) 3251–3255 ). Moreover, the emergence of several newly discovered MRSA terrestrial metabolites (). In the marine environ- showed antibiotic resistance to vancomycin and teicoplanin ment, where all surfaces are constantly exposed to the threat of (As an alternative to vancomycin for treatment of surface colonisation, sessile organisms remain relatively free from S. aureus infection, the new antimicrobial agents such as linezolid, biofouling Furthermore, the chemical com- quinupristin/dalfopristin, daptomycin, tigecycline, and fidaxomicin pounds produced by marine organisms are less well known than are being used for the most severe infections ).
those of their terrestrial counterparts. Among marine organisms, One of the ways of preventing antibiotic resistance is by using edible seaweeds have been identified as an under-exploited plant new compounds which are not based on the existing synthetic resource and a source of functional foods. It is believed that the antimicrobial agents. Thus, the search for novel natural sources physiological and genetic characteristics of seaweeds differ com- from marine ecosystems could lead to the isolation of new antibi- pared to those of terrestrial plants. They are extensively used in otics ). Many organisms produce marine natural food and medicine The ability of seaweeds to pro- products that possess unique structural features as compared to duce secondary metabolites of antimicrobial value, such as volatile Fig. 1. Structures phlorotannins derived from marine algae [ phloroglucinol (1), eckol (2), fucofuroeckol-A (3), phlorofucofuroeckol-A (4), dioxinodehydroeckol (5), 8,80-bieckol(6), 7-phloroeckol (7), & dieckol (8)].
S.-H. Eom et al. / Food and Chemical Toxicology 50 (2012) 3251–3255 phlorotannin compounds such as eckol, phlorofucofuroeckol A and dieckol, and 8,80-bieckol have been isolated from E. kurome and E. bicyclis Phlorotannins in E. Arborea possess a strong anti-allergic effect and their structures were elu- cidated as eckol, 6,60-bieckol, 6,80-bieckol, 8,80-bieckol, phlorofu- phlorotannins as polyphenolic secondary metabolites are found cofuroeckol-A, and phlorofucofuroeckol-B ().
Moreover, 6,60-Bieckol diphlorethohydroxycarmalol, and phloro- Thus, the screen for antimicrobial agents as safe alternatives glucinol have been isolated from brown algae I. okamurae ( and secondary metabolites from marine algae is attracting atten- Collectively, phlorotannins can be used functional tion in the food industry. This review focuses on phlorotannins de- ingredients in the food and pharmaceutical industries.
rived from marine algae and presents their potential application asantimicrobial agents.
Some synthetic preservatives and additives used in the food industry have been evaluated as toxic to various cells and organs, Marine algae have become an important source of pharmaco- mutagens and tumor promoters over long-term use ( logically active metabolites. Also, they are widely distributed and Therefore, recently there has been a great abundant throughout the coastal areas of many countries. In addi- deal of interest in searching for novel natural antibiotics and these tion, they are a source of useful secondary metabolites such as studies have shown that phlorotannins in brown algae can act as agar, carragenean and alginate with interesting pharmaceutical potential antimicrobial agents that may be useful in the food properties Among marine algae, brown algae have been reported to contain higher phlorotannin contents as marine phenolic compounds (). Phlorotannins The isolated and characterized phlorotannins (1–8) from brown consist of polymers of phloroglucinol (1,3,5-tryhydroxybenzene) algae with antimicrobial activity are presented in such as units and are formed in the acetate–malonate pathway in marine phloroglucinol (1), eckol (2), fucofuroeckol-A (3), phlorofucofuroec- algae. Furthermore these phlorotannins are highly hydrophilic kol-A (4), dioxinodehydroeckol (5), 8,80-bieckol (6), 7-phloroeckol components with a wide range of molecular sizes (126–650 kDa) (7), and dieckol (8). In addition, triphloroethol A, 6,60-bieckol and 8,4000-dieckol have been reported. These isolated phlorotannins have Several phlorotannins purified from brown seaweeds such as been shown to have antimicrobial effect against food-borne Ecklonia cava, E. kurome, E. stolonifera, Eisenia aborea, Eisenia pathogenic bacteria, antibiotic resistance bacteria, and human tinea bicyclis, Ishige okamurae, Pelvetia siliquosa have medicinal and phar- maceutical benefits and have shown strong anti-oxidant, anti- Dieckol purified from E. cava has fungicidal activity ( inflammatory, anti-viral, anti-tumor, anti-diabetes and anti-cancer It has shown a potent antifungal activity against Trichophy- ton rubrum associated with dermatophytic nail infections in hu- mans. In addition, it has shown a potent inhibition of cell Eckol, dieckol, and phloroglucinol from E. cava have shown po- membrane integrity as well as cell metabolism against T. rubrum.
tential for skin whitening effect () and anti-hyper- The MIC (minimum inhibitory concentration) values for eckol from tensive effect E. cava also contains other E. cava indicates potent antimicrobial activity against methicillin- phlorotannins including 6,60-bieckol, 8,80-bieckol, 8,4000-dieckol, resistant S. aureus (MRSA) in the range of 125–250 lg/mL dioxinodehydroeckol, fucodiphlorethol G, phlorofucofuroeckol-A, Dieckol isolated from E. stolonifera may possess stron- ger anti-MRSA activity than eckol and the MICs of dieckol were in Table 1Phlorotannin compounds with antibacterial effect.
Inhibition of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) dieckol (8)dioxinodehydroeckol(5)fucofuroeckol-A (3)7-phloroeckol (7)Phlorofucofuroeckol-A (4) Inhibition of S. aureus, MRSA, Salmonella sp.
. Inhibition of Campylobacter jejuni, Escherichia coli, Salmonella enteritidis, Salmonella Inhibition of S. aureus and MRSA, bacillus subtilis.
Inhibition of Acinetobacter sp. Klebsiella pneumonia, Legionella birminghamensis, Salmonella typhimurium, Shigella flexneri a IC50: concentration of a compound required for 50% inhibition in vitro.
  MIC: minimum inhibitory concentration.
à MBC: minimum bactericidal concentration.
S.-H. Eom et al. / Food and Chemical Toxicology 50 (2012) 3251–3255 the ranges of 32–64 lg/mL ). Although the current methicillin-resistant Staphylococcus aureus and Salmonella spp. Food borne knowledge on the relationship between the structure and activity Cox, S., Abu-Ghannam, N., Gupta, S., 2010. An assessment of the antioxidant and of the active phlorotannins is limited, the physiological activity antimicrobial activity of six species of edible Irish seaweeds. Food Res. Int. J. 17, may depend on the degree of polymerization of phlorotannin Demirel, Z., Yilmaz-Koz, F.F., Karabay-Yavasoglu, U.N., Ozdemir, G., Sukatar, A., 2009. Antimicrobial and antioxidant activity of brown algae from the Aegean addition, in a comparison of other phlorotannins, using catechin Sea. J. Serb. Chem. Soc. 74, 619–628.
derivatives as positive control, it has been reported that the MICs Eom, S.H., 2012. Anti-MRSA (methicillin-resistant Staphylococcus aureus) substance of (À)-epigallocatechin, (À)-EGCg, (+)-gallocatechin and (À)-gallo- isolated from Eisenia bicyclis and its action mechanism. Ph.D. Dissertation.
Pukyong National University, Busan, pp. 45.
catechin from green tea (Camellia sinensis) against MRSA were Eom, S.H., Kang, Y.M., Park, J.H., Yu, D.U., Jeong, E.T., Lee, M.S., Kim, Y.M., 2011.
64 lg/mL (Thus, the anti-MRSA activity of Enhancement of polyphenol content and antioxidant activity of brown alga phlorotannins isolated E. bicyclis was superior to or equal to those Eisenia bicyclis extract by microbial fermentation. J. Fish. Aquat. Sci. 14, 192–197.
of catechins derived from green tea ().
Eom, S.H., Lee, S.H., Yoon, N.Y., Jung, W.K., Jeon, Y.J., Kim, S.K., Lee, M.K., Kim, Y.M., Phlorotannins from E. kurome have been reported to show bac- 2012. a-Glucosidase-and a-amylase-inhibitory activities of phlorotannins from tericidal activity against food-borne pathogenic bacteria ( Eisenia bicyclis. J. Sci. Food Agric. 92, 2084–2090.
Moreover, the oral administration of the phlorotan- Gould, F.K., Brindle, R., Chadwick, P.R., Fraise, A.P., Hill, S., Nathwani, D., Ridgway, G.L., Spry, M.J., Warren, R.E., 2009. Guidelines (2008) for the prophylaxis and nins at a dosage rate of 170–1500 mg/kg bw/day for 14 days in treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections in mice ‘‘did not report any cytotoxic effect’’. The interactions be- the United Kingdom. J. Antimicrob. Chemother. 63, 849–861.
tween bacterial proteins and phlorotannins were considered to Gressler, V., Stein, É.M., Dörr, F., Fujii, M.T., Colepicolo, P., Pinto, E., 2011.
Sesquiterpenes from the essential oil of Laurencia dendroidea (Ceramiales, play an important role in the bactericidal action of phlorotannins Rhodophyta): isolation, biological activities and distribution among seaweeds.
(Therefore it is thought that phlorotannins from Rev. Bras. Farmacogn. 21, 248–254.
brown algae could be very useful in the food and pharmaceutical Gupta, S., Abu-Ghannam, N., 2011. Recent developments in the application of seaweeds or seaweed extracts as a means for enhancing the safety and quality industries as antibiotic agents. In addition to phlorotannins, brown attributes of foods. Innovat. Food Sci. Emerg. Tech. 12, 600–609.
algae include various health enhancing compounds such as fuco- Heo, S.J., Jeon, Y.J., 2005. Antioxidant effect and protecting effect against cell damage xanthin, sulphated polysaccharides, sterols, polyunsaturated fatty by enzymatic hydrolysates from marine algae. J. Korean Soc. Food Sci. Nut. 10,31–41.
Heo, S.J., Ko, S.C., Cha, S.H., Kang, D.H., Park, H.S., Choi, Y.U., Kim, D., Jung, W.K., Jeon, Y.J., 2009. Effect of phlorotannins isolated from Ecklonia cava on melanogenesisand their protective effect against photo-oxidative stress induced by UV-B radiation. Toxicol. In Vitro 23, 1123–1130.
Kahl, R., Kahl, G., 1983. Effect of dietary antioxidants on benzo(a)pyrene metabolism Marine natural products provide a rich source of chemically di- in rat liver microsomes. Toxicology 28, 229–233.
Kaplan, S.L., Mason Jr., E.O., 1998. Management of infections due to antibiotic- verse compounds that can be used to develop novel, potential, and resistant Streptococcus pneumoniae. Clin. Microbiol. Rev. 11, 628–644.
useful therapeutic agents. Certain marine products have been re- Kim, A.R., Shin, T.S., Lee, M.S., Park, J.Y., Park, K.E., Yoon, N.Y., Kim, J.S., Choi, J.S., Jang, ported to exhibit antimicrobial effects against several pathogens.
B.C., Byun, D.S., Park, N.K., Kim, H.R., 2009. Isolation and Identification ofPhlorotannins from Ecklonia stolonifera with antioxidant and anti-inflammatory Hence, in an effort to discover an alternative antibiotic, marine properties. J. Agric. Food Chem. 57, 3483–3489.
organisms have attracted much attention since more pathogens Kim, J.H., Lee, D.S., Lim, C.W., Park, H.Y., Park, J.H., 2002. Antibacterial activity of sea- are becoming resistant to antibiotics due to over-prescription. In mustard, Laminaria japonica extracts on the cariogenic bacteria Streptococcusmutans. J. Korean Fish. Soc. 35, 191–195.
this brief review, phlorotannins derived from brown algae have Kotnala, S., Garg, A., Chatterji, A., 2009. Screening for the presence of antimicrobial been considered from the perspective of their potential antimicro- activity in few Indian seaweeds. Pertanika. J. Trop. Agric. Sci. 32, 69– bial activity. Further work will be necessary to show whether they Larsen, T.O., Smedsgaard, J., Nielsen, K.F., Hansen, M.E., Frisvad, J.C., 2005.
can be effective as broad spectrum antibiotics against food-borne Phenotypic taxonomy and metabolite profiling in microbial drug discovery.
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 b-lactams against methicillin-resistant Staphylococcus aureus.
Biotechnol. Bioprocess Eng. 13, 758–764.
Lee, M.H., Lee, K.B., Oh, S.M., Lee, B.H., Chee, H.Y. Antifungal activities of dieckol The authors declare that there are no conflicts of interest.
isolated from the marine brown alga Ecklonia cava against. Trichophyton rubrum.
Food Sci. Biotechnol. 53 (2010) 504–507.
Li, Y., Qian, Z.J., Ryu, B., Lee, S.H., Kim, M.M., Kim, S.K., 2009. Chemical components and its antioxidant properties in vitro: an edible marine brown alga, Eckloniacava. Bioorg. Med. Chem. 17, 1963–1973.
This study was supported by a grant from The Marine Bioproc- Nagayama, K., Iwamura, Y., Shibata, T., Hirayama, I., Nakamura, T., 2002. Bactericidal activity of phlorotannins from the brown alga Ecklonia kurome. Antimicrob.
ess Research Center of the Marine Bio 21 Project funded by The Ministry of Land, Transport and Maritime, Republic of Korea.
Patra, J.K., Rath, S.K., Jena, K.B., Rathod, V.K., Thatoi, H., 2008. Evaluation of antioxidant and antimicrobial activity of seaweed (Sargassum sp.) extract: astudy on inhibition of glutathione-S-transferase activity. Turk. J. Biol. 32, 119– Ragan, M.A., Glombitza, K.W., 1986. Phlorotannins, brown algal polyphenols. In: Round, F.E., Chapman, D.J. (Eds.), Progress in Psychological Research, Vol. 4.
Ahn, M.J., Yoon, K.D., Min, S.Y., Lee, J.S., Kim, J.H., Kim, T.G., Kim, S.H., Kim, N.G., Huh, Biopress Ltd., Bristol, pp. 129–241.
H., Kim, J., 2004. Inhibition of HIV-1 reverse transcriptase and protease by Rhimou, B., Hassane, R., José, M., Nathalie, B., 2010. The antibacterial potential of the phlorotannins from the brown alga Ecklonia cava. Biol. Pharm. Bull. 27, 544– seaweeds (Rhodophyceae) of the Strait of Gibraltar and the Mediterranean Coast of Morocco. Afr. J. Biotechnol. 9, 6365–6372.
Blunt, J.W., Copp, B.R., Munro, M.H.G., Northcote, P.T., Prinsep, M.R., 2012. Marine Sasaki, M., Maki, J.I., Oshiman, K.I., Matsumura, Y., Tsuchido, T., 2005.
natural products. Nat. Prod. Rep. 29, 144–222.
Biodegradation of bisphenol A by cells and cell lysate from Sphingomonas sp.
Bugni, T.S., Ireland, C.M., 2004. Marine-derived fungi: a chemically and biologically strain AO1. Biodegradation 16, 449–459.
diverse group of microorganisms. Nat. Product Rep. 21, 143–163.
Schito, G.C., 2006. The importance of the development of antibiotic resistance in Bush, L.M., 2011. Best Alternative to vancomycin for serious methicillin-resistant Staphylococcus aureus. Clin. Microbiol. Infect. 12, 3–8.
Staphylococcus aureus Infections: Let’s just say it. Clin. Infect. Dis. 53, 965–966.
Schultes, R.E., 1978. The kingdom of plants. In: Thomson, W.A.R. (Ed.), Medicines Cha, J.Y., Lee, B.J., Je, J.Y., Kang, Y.M., Kim, Y.M., Cho, Y.S., 2011. GABA-enriched From The Earth. Mc Graw-Hill Book Co., New York, p. 208.
fermented Laminaria japonica protects against alcoholic hepatotoxicity in Shanmughapriya, S., Manilal, A., Sujith, S., Selvin, J., Kiran, G.S., Natarajaseenivasan, Sprague-Dawley rats. J. Fish. Aquat. Sci. 14, 79–88.
K., 2008. Antimicrobial activity of seaweeds extracts against multiresistant Choi, J.G., Kang, O.H., Brice, O.O., Lee, Y.S., Chae, H.S., Oh, Y.C., Sohn, D.H., Park, H., pathogens. Ann. Microbiol. 58, 535–541.
Choi, H.G., Kim, S.G., 2010. Antibacterial activity of Ecklonia cava against S.-H. Eom et al. / Food and Chemical Toxicology 50 (2012) 3251–3255 Stapleton, P.D., Shah, S., Anderson, J.C., Hara, Y., Hamilton-Miller, J.M.T., Taylor, P.W., Weigel, L.M., Clewell, D.B., Gill, S.R., Clark, N.C., McDougal, L.K., Flannagan, S.E., 2004. Modulation of b-lactam resistance in Staphylococcus aureus by catechins Shetty, J.F.J., Killgore, G.E., Tenover, F.C., 2003. Genetic analysis of a high-level and gallates. Int. J. Antimicrob. Agents 23, 462–467.
vancomycin-resistant isolate of Staphylococcus aureus. Science 302, 1569–1571.
Sugiura, Y., Matsuda, K., Yamada, Y., Nishikawa, M., Shioya, K., Katsuzaki, H., Imai, K., Wijesekara, I., Kim, S.K., 2010. Angiotensin-I-converting enzyme (ACE) inhibitors from marine resources: prospects in the pharmaceutical industry. Mar. Drugs 8, phlorofucofuroeckol-B, from an edible brown alga, Eisenia arborea. Biosci.
Biotechnol. Biochem. 70, 2807–2811.
Wijesinghe, W.A.J.P., Ko, S.C., Jeon, Y.J., 2011. Effect of phlorotannins isolated from Tan, R.X., Zou, W.X., 2001. Endophytes: a rich source of functional metabolites. Nat.
Ecklonia cava on angiotensin I-converting enzyme (ACE) inhibitory activity.
Taskin, E., Ozturk, M., Kurt, O., 2007. Antibacterial activities of some marine algae Zou, Y., Qian, Z.J., Li, Y., Kim, M.M., Lee, S.H., Kim, S.K., 2008. Antioxidant effects of from the Aegean Sea (Turkey). Afr. J. Biotechnol. 6, 2746–2751.
phlorotannins isolated from Ishige okamurae in free radical mediated oxidative Wang, Y., Xu, Z., Bach, S.J., McAllister, T.A., 2009. Sensitivity of Escherichia coli systems. J. Agricult. Food Chem. 56, 7001–7009.
O157:H7 to seaweed (Ascophyllum nodosum) phlorotannins and terrestrialtannins. Asian–Austral. J. Anim. Sci. 22, 238–245.

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