Antimicrobial Activities of Stearidonic and Gamma-Linolenic Acids
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Park et al. Botanical Studies 2013, 54:39 http://www.as-botanicalstudies.com/content/54/1/39 RESEARCH Open Access Antimicrobial activities of stearidonic and gamma-linolenic acids from the green seaweed Enteromorpha linza against several oral pathogenic bacteria Nam-Hee Park1†, Jae-Suk Choi2†, Seon-Yeong Hwang1, Yang-Chun Kim1, Yong-Ki Hong3, Kwang Keun Cho4 and In Soon Choi2,5* Abstract Background: We found that the edible green seaweed Enteromorpha linza displayed potent antimicrobial activity against Prevotella intermedia and Porphyromonas gingivalis. To elucidate the active component of E. linza, isolation procedures were performed. Results: The main active compound was isolated by polarity fractionation, Sephadex LH-20 gel chromatography, and reverse-phase high-performance liquid chromatography (RP-HPLC). The active compounds were eluted at isocratic 95% acetonitrile by RP-HPLC and identified as unsaturated fatty acids, stearidonic acid (SA, C18:4 n-3) and gamma-linolenic acid (GLA, C18:3 n-6) by gas chromatography–mass spectrometry, 1H nuclear magnetic resonance (NMR) spectroscopy, and 13C NMR spectroscopy. The yields of SA and GLA from dried seaweed tissue were 6.33 × 10-3% and 6.47 × 10-3%, respectively. The minimal inhibitory concentration values of SA and GLA were 39.06 μg/mL against P. intermedia and 9.76 μg/mL against P. gingivalis, respectively. SA and GLA were also active against several other oral pathogens, including Aggregatibacter actinomycetemcomitans, Candida albicans, Fusobacterium nucleatum subsp. vincenti, and Streptococcus mutans, at micromolar concentrations. Conclusions: These data suggest that the E. linza extracts SA and GLA are useful antimicrobial agents for the prevention and/or treatment of periodontitis. Keywords: Stearidonic acid; Gamma-linolenic acid; Antimicrobial activity; Enteromorpha linza; Prevotella intermedia; Porphyromonas gingivalis; Oral pathogen Background years, numerous attempts have been made to develop Periodontitis is a chronic inflammatory disease initiated new agents capable of preventing and/or treating peri- by a group of gram-negative periodontal pathogens, odontitis (Park et al. 2005). including Prevotella intermedia and Porphyromonas gin- Interest in marine organisms as natural sources of givalis. Systemic or topical antibiotic therapies have been pharmaceutical agents has increased recently (Newman et al. found to be useful in the treatment of periodontal dis- 2003). Seaweeds produce many secondary metabolites with ease (Slots and Ting 2002). However, antibiotics are a variety of activities; compounds with antiviral, antifungal, commonly known to induce side effects (Falagas and and antibacterial activity have been detected from various Siakavellas 2000) and the development of bacterial re- marine algae (del Val et al. 2001; Newman et al. 2003). sistance (Lopez and Gerwick 1988). Thus, for many In our previous work, we found that the edible green seaweed Enteromorpha linza displays potent antimicro- * Correspondence: [email protected] †Equal contributors bial activity against P. intermedia and P. gingivalis with- 2RIS Center, IACF, Silla University, Sasang-gu, Busan 617-736, Republic of Korea out side effects at a moderate dose (Choi et al. 2012). 5 Depertment of Biological Science, Silla University, Sasang-gu, Busan 617-736, Additionally, a mouth rinse containing E. linza extract Republic of Korea Full list of author information is available at the end of the article has shown clinical effects against gingivitis, as measured © 2013 Park et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Park et al. Botanical Studies 2013, 54:39 Page 2 of 9 http://www.as-botanicalstudies.com/content/54/1/39 by the plaque index (PI), gingival index (GI), bleeding on gradient liquid chromatograph and a Waters 2707 probing (BOP), and activity against two bacterial strains autosampler (Waters Co., Milford, MA, USA) monitored (P. intermedia and P. gingivalis)(Choetal.2011).This at 197 nm. The mobile phase consisted of two solvent sys- mouth rinse produced effects similar to those of Listerine®. tems: acetonitrile with 0.1% trifluoroacetic acid (TFA) and To discover therapeutic agents against periodontitis from distilled water with 0.1% TFA. Elution was performed with the seaweed with few or no side effects and potent anti- an isocratic of 95% acetonitrile with 0.1% TFA (5% dis- microbial activity, we isolated and identified active anti- tilled water added) for 15 min at a flow rate of 4 mL/min. microbial compounds from E. linza extract and present Purification was performed using the same system to yield data regarding its antimicrobial activity against several the pure compound (S14-1: 8.8 mg; S14–2: 9.5 mg; S14–3: oral pathogens. 9.7 mg). Each eluted compound was dried under a stream of nitrogen gas. Methods Algae Analytical methods The green seaweed E. linza was collected from the sea- The purified compound was analyzed on a nuclear mag- shore near Ildo-dong (33°31′15.06″N, 126°32′54.70″E), netic resonance (NMR) spectrometer (JNM-ECP 400; Cheju Island, Korea, in April 2009. After rinsing with JEOL, Tokyo, Japan), operating at 500 and 100 MHz for 1 13 tap water to remove salt and miscellanea, the samples Hand C, respectively, using methanol-d (CD3OD). were dried for 1 day at room temperature using an elec- Gas chromatography–mass spectrometry (GC–MS) was tric fan. Samples were then ground to a powder using a performed on a 5975C gas chromatograph (Agillent, Santa coffee grinder for 5 min and stored at −20°C until use. Clara, CA, USA) equipped with an HP-5MS capillary co- lumn (30 m × 250 μm×0.25 μm). The column tempe- Reagents rature was programmed from an initial temperature of High-performance liquid chromatography (HPLC)-grade 80°C for 2 min and raised to a final temperature of 300°C reagents (or the highest grade available) and culture at a rate of 5°C/min. The samples were converted to me- media were purchased from Sigma Aldrich, Ltd. (Poole, thyl ester derivatives by the protocol reported by Lepage Dorset, UK). All solutions were made with ultrapure and Roy (1986). The injection volume was 1 μL, the car- deionized water (Milli-Q advantage A10 ultrapure water rier gas was helium (flow rate, 1 mL/min) and the split ra- purification system; Millipore, Billerica, MA, USA). tio was 20:1. A computerized search was performed to identify a standard sample for comparison (Sigma 56463: Isolation of antimicrobial compounds methyl stearidonate solution; Sigma L6503: methyl γ- Approximately 150 g of dried E. linza powder was linolenate solution for GC) (Sukatar et al. 2006). The extracted with a 600-mL solution of methanol–water structure of the purified compound was inferred by com- (4:1) at room temperature overnight. The extract was parison with C18H28O2 and C18H30O2 structures from a then filtered through Toyo No. 2 filter paper under re- NMR spectra database (Aires-de-Sousa et al. 2002). duced pressure. This extraction procedure was repeated three times and the extracts were combined. The metha- Test microorganisms and culture media nol–water extract was concentrated to a dark-green resi- In vitro antimicrobial activity assays were performed due (30.2 g) under reduced pressure in an evaporator. with the anaerobic bacteria of P. intermedia KCTC25611 The extract was fractionated according to polarity, as and P. gingivalis KCTC381 obtained from the Korean Col- described by Harborne (1998). The fraction was acidified lection for Type Culture (Deajeon, Korea). These strains to pH 3.0 using 2 M sulfuric acid and extracted three were cultivated on tryptic soy agar (Difco, Sparks, MD, times with chloroform, resulting in a moderately polar USA) media containing 10% sheep blood, 0.1% hemin extract that contained the main antimicrobial activity. (Sigma, St. Louis, MO, USA) and vitamin K1 (Sigma, This fraction III (4.4 g) was loaded on a Sephadex LH-20 USA) in a Bactron IV chamber (SHELLAB, Cornelius, (Sigma LH20100; Sigma Aldrich, Ltd.) gel column (2.2 × OR, USA) at 37°C, 5% CO2,10%H2,and85%N2. 100 cm) using 100% MeOH as the eluent. Each 10-mL fraction was collected at a flow rate of 1 mL/min. The Antimicrobial activity against oral pathogens active fraction, designated as S14 (173 mg), was dried Following identification of the active compounds, their and dissolved in 17.3 mL MeOH for reverse-phase antimicrobial activity was measured against several oral high-performance liquid chromatography (RP-HPLC). pathogens, including anaerobic Aggregatibacter actino- Separation of the three major peaks (S14–1, S14–2, mycetemcomitans KCTC 3698, Candida albicans KCTC and S14-3) was achieved using an Alltima C18 column 17485, Fusobacterium nucleatum subsp. vincenti KCTC (10 mm ID × 25 cm) (W. R. Grace & Co., Columbia, 5105, and Streptococcus mutans KCTC 3065. A. actino- MD, USA). Analysis was performed on a Waters 600 mycetemcomitans was cultivated on brucella medium (Sigma, Park et al. Botanical Studies 2013, 54:39 Page 3 of 9 http://www.as-botanicalstudies.com/content/54/1/39 USA) containing 3% horse serum (Sigma, USA) in a Determination of minimal inhibitory concentration (MIC) Bactron IV chamber. C. albicans was cultivated on RPMI values 1640 medium (Sigma, USA), F. nucleatum subsp. vincenti The Determination of MIC values were performed as was cultivated on Schaedler medium (Sigma, USA), and S. described previously (Choi et al. 2012). Inocula were mutans was cultivated on BHI medium (Sigma, USA) with prepared from 24-h broth cultures and then diluted to 3% horse serum (Sigma, USA). All microorganism incuba- ten volume of the original volume and adjusted to the tions were performed at 37°C. 0.5 McFarland standard solution turbidity containing Figure 1 Purification of antimicrobial compounds isolated from Enteromorpha linza.