Abstract
We screened extracts of 342 species of marine algae collected from Japanese coastlines for antibacterial activity against Propionibacterium acnes, and found a novel antibacterial compound, which we named Sargafuran, from the MeOH extract of the marine brown alga, Sargassum macrocarpum. Sargafuran has low cytotoxicity, and the MIC against P. acnes was 15 μg ml−1, showing a broad antibacterial activity against Gram-positive bacteria. A time-kill study showed that Sargafuran was bactericidal and completely killed P. acnes at 4 × MIC by lysing bacterial cells. These results suggest that Sargafuran might be useful as a lead compound to develop new types of anti-P. acnes substances and new skin care cosmetics to prevent or improve acne.
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Introduction
Acne vulgaris is a common skin disease, affecting about 70–80% of adolescents and young adults. It is a multifactorial disease of the pilosebaceous unit.1 Propionibacterium acnes, a common skin organism, is most often recognized in acne vulgaris and produces a number of virulence factors. Clindamycin and erythromycin are most commonly used as topical antibiotics against P. acnes.2
In our laboratory, we screened the biological activities of marine algae collected from the Japanese coastline and found several bioactive compounds,3, 4, 5, 6 including an antibacterial compound.7 Other reports show that some distinct seaweeds contain antimicrobial substances against both Gram-positive and -negative bacteria.8, 9 Thus, marine algae are a promising bioresource to find new antibacterial compounds against P. acnes and to develop new natural cosmetic products to prevent acne. In this study, we screened a total of 342 species of marine algae, collected from the Japanese coastline, for activity against P. acnes, and found a novel anti-P. acnes compound from one of these algae.
Materials and methods
Bacterial strains and media
P. acnes (ATCC 11827) was used for the screening of antibacterial activity of marine algal extracts. This test strain was cultured with Reinforced Clostridial Medium (Sigma-Aldrich, Tokyo, Japan). Bacteria used for the antibacterial spectrum assay were methicillin-resistant Staphylococcus aureus (ATCC 33591), methicillin-sensitive S. aureus (ATCC 25923), Bacillus subtilis (IFO 14419), Escherichia coli (NBRC 12734), Enterococcus faecium (NBRC 3826), Enterococcus faecalis (NBRC 3971), Enterococcus serolicida (NG 8206), Streptococcus mutans (NBRC 13955), Streptococcus pneumoniae (GTC 261), Streptococcus pyogenes (GTC 262), Pseudomonas aeruginosa (IFO 13736) and Vibrio alginolyticus (V7) as well as two strains of P. acnes (ATCC 1187 and ATCC 25746). These strains were cultured with Tryptic Soy Agar medium (Difco Laboratories, Detroit, MI, USA), except for P. acnes and V. alginolyticus which was cultured in ZoBell 2216E agar medium.10
Marine algae collection and preparation of extracts
During the period from April 1994 to August 2003, marine algae samples were collected at 96 points from north to south along the Japanese coastline and were stored at −20 °C until needed. The marine algae extracts were prepared as described in our earlier paper.11
Screening assay for anti-P. acnes activity
Sterilized paper disks (ϕ8 mm; Advantec, Tokyo, Japan) permeated with 50 μl each of phosphate-buffered saline or MeOH algal extract and dried completely were placed on double-layer agar plates inoculated with 5.0 × 106 CFU ml−1 of P. acnes strain. These plates were incubated at 37 °C for 2 days under anaerobic condition. After incubation, the zones of inhibition were measured and recorded.
Isolation and purification of Sargafuran
One liter of the MeOH extract from the marine brown alga Sargassum macrocarpum (250 g wet weight) was partitioned with a chloroform/water (1:1) mixture. The chloroform/MeOH fraction was concentrated to dryness and redissolved in n-hexane/acetone (6:1), and then subjected to a silica gel-60 (Merck, Darmstadt, Germany) column chromatography under continuous elution with n-hexane/acetone (6:1, 5:1 and 4:1) and MeOH. The active fractions were pooled and concentrated, and then further chromatographed on a second silica gel column, eluting with n-hexane/acetyl ethyl ester (10:1, 6:1 and 3:1) and MeOH, and on a reverse-phase Cosmosil (Nacalai Tesque Inc., Kyoto, Japan) column, eluting with 60–80% MeOH containing 0.1% trifluoroacetic acid (Sigma-Aldrich). The active fractions were finally purified with an HPLC on a reversed-phase column (Mightysil RP-8 GP, ∅ 4.6 × 250 mm; Kanto Chemical Co. Inc., Tokyo, Japan), eluting with a gradient of acetonitrile and water containing 0.1% trifluoroacetic acid.
Spectrometric analyses of Sargafuran
Optical rotations were measured with an SEPT-200 polarimeter (Horiba, Tokyo, Japan). UV spectra were recorded on a U-3210 spectrophotometer (Hitachi, Tokyo, Japan) and IR spectra on a model 1720 spectrometer (Perkin-Elmer, Waltham, MA, USA). NMR spectra were recorded in CDCl3 on a JEOL lambda 500 NMR spectrometer (JEOL, Tokyo, Japan). Chemical shifts are shown as δ values from TMS as the internal reference. Peak multiplicities are quoted in Hz. Mass spectra were measured on a JMS-700 spectrometer (JEOL).
Antibacterial test
The MICs of Sargafuran were determined by the standard microdilution method described by the National Committee for Clinical Laboratory Standards,12 using a Tryptic Soy Broth medium (Difco Laboratories), except for P. acnes and V. alginolyticus which was in ZoBell 2216E broth medium. The final volume of Tryptic Soy Broth, Reinforced Clostridial Medium or ZoBell broth medium containing Sargafuran was 100 μl per well to give a starting inoculum density of 5 × 105 CFU ml−1.
Time-kill curve experiment
The time-kill experiment was conducted by the method described by Aeschlimann and Rybak13 and Etenza et al.14 The experiments were conducted in test tubes containing 15 ml of fresh Reinforced Clostridial Medium inoculated with an overnight culture of P. acnes (ATCC 11827) to give an initial bacterial density of 106 cells per ml. The inoculation was carried out immediately after the addition of Sargafuran or Clindamycin (Sigma-Aldrich) at the final concentrations of MIC, 2 × MIC and 4 × MIC and incubated at 37 °C for 2 days under anaerobic condition.
Bacteriolytic assay
The seed culture of P. acnes (ATCC 11827) on an RCA plate was resuspended in Reinforced Clostridial Medium and washed twice with sterile 10 mM Tris-HCl buffer (pH 7.6). The absorbance was adjusted to 0.1 at 660 nm. The bacterial cell suspension (5 ml each) was aliquoted in sterile test tubes and exposed to Sargafuran at various concentrations or to Achromopeptidase (Wako Pure Chemical Industries Ltd, Tokyo, Japan) at 134 μg ml−1 as a positive control. Untreated bacterial suspensions were used as negative controls. The concentration of MeOH (the solvent of Sargafuran) in each tube was less than 0.1% (v/v). The test tubes were incubated at 37 °C under anaerobic conditions. The absorbance at 660 nm was measured at 0, 0.15, 0.5, 0.45, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 h after incubation, and the relative absorbance was calculated by dividing each absorbance by that of the negative control. Each treatment was conducted in duplicate.
Cytotoxicity test
The cytotoxic activity of Sargafuran was evaluated as described earlier.15 Human normal dermal fibroblasts (Morinaga Institute of Biological Science, Yokohama, Japan) were prepared at 5 × 103 cells per 100 μl per well in a 96-well plate. Then Sargafuran, serially diluted twofold, was added to give final concentrations ranging from 3.8 to 480 μg ml−1. The cells were incubated at 37 °C for 3 days. The proliferation of cells was evaluated by an 3-[4,5-dimethylthiazol-2-ly]-2,5-diphenyltetrazolium bromide (MTT) assay16 and the growth rate relative to the control treatment was calculated.
Results
Marine algae with anti-P. acnes activity
We found 13 species of marine algae with anti-P. acnes activity, based on disk diffusion assays, using the extracts from a total of 342 species of marine algae collected from the Japanese coastline. The algae are Laurencia brongniartii, Laurencia okamurae, Osonthalia corymbifera, Rhodomela teres, Dictyopteris divaricata, Dictyopteris undulata, Ishige okamurai, Padina crassa, Sargassum fulvellum, S. macrocarpum, Sargassum siliquastrum, Sargassum yezoense and Zonaria diesingiana, showing approximate MIC ranges of 62.5–1000 μg per disk against P. acnes (Table 1). Most of the positive marine algae were brown algae and showed a relatively high anti-P. acnes activity at 62.5–250 μg per disk.
Purification of the antibacterial compound, Sargafuran, from S. macrocarpum
As described above, several marine algae showed promising anti-P. acnes activity. One of those positive algae, S. macrocarpum, is widely distributed throughout the Japanese coastline and can be collected in large amounts during any season. Thus, we selected this algal species to proceed with the isolation and purification of the antibacterial compound against P. acnes from the MeOH extract. After several purification steps, starting from 250 g wet weight of S. macrocarpum, 3.6 mg of the antibacterial compound, which we named Sargafuran, was obtained.
Structure determination of Sargafuran
Sargafuran was isolated as a colorless oil [UV λmax (CHCl3) nm (log ɛ): 241 (4.27), 263 (3.79); [α]D–1.26°(CHCl3, c 0.95)]. The molecular formula was determined to be C27H36O4 (found 424.2593, calcd. 424.2614) by high resolution electron impact mass spectrometry (HR-EI-MS). The IR spectrum showed absorption bands at 1686 and 3400 cm–1 attributable to a carbonyl and hydroxyl group, respectively. 1H- and 13C-NMR spectroscopic data indicate that Sargafuran has five singlet methyl groups (C-18, C-19, C-21, C-26 and C-27), seven olefinic bonds, six aliphatic methylenes, a carbonyl group and a quaternary carbon attached to oxygen. Taken together with the presence of seven olefinic bonds and a carbonyl group, 10 units of unsaturation in Sargafuran indicate that the compound has two rings in its structure. 1H-1H COSY spectrum revealed the linkages among the methylenes, and olefinic protons (H-6 to H-8, H-10 to H-12 and H-14 to H-16), in addition to two sets of connection between olefinic protons (H-2/H-3 and H-23/H-24). The connections in the triene chain (C-6 to C-21) were established from the HMBC correlations (H-8/C-10, 19, H-19/C-9, 10, H-10/C-9, H-11/C-9, 13, H-12/C-14, 20, H-14/C-13, 20, H-18/C-16, 17 and H-21/C-16, 17) among the olefinic methines (H-8, H-12 and H-16), methyl groups (H-18, -19 and -21) and a carbonyl carbon (C-20). The geometry of olefinic bonds was determined to be E (C-8/C-9) and Z (C-12/C-13), respectively, by NOESY correlations (H-7/H-19, H-8/H-10 and H-12/H-14). The 2-methyl furan moiety (C-22 to C-26) was revealed by the HMBC correlations (H-23/C-22, 25, H-24/C-22, 25 and H-26/C-24, 25) and the small JH−H coupling value between H-23 and H-24 (J=3 Hz). Additional HMBC correlations (H-2/C-1, -4, H-3/C-1, -4 and H-27/C-1, -2, -5) indicated the presence of 1-methyl-cyclopenta-2,4-dienol moiety (C-1 to C5, and C-27). The remaining carbonyl carbon (C-20) was attributed to a carboxylic acid. The cyclopentadiene moiety was connected with C-6 of the triene chain at C-5 and with C-22 of methylfuran at C-4 on the basis of HMBC correlations (H-7/C-5, H-6/C-1, H-3/C-22 and H-23/C-4), and this was further supported by a strong fragment ion at m/z 175 (100%) in the EI-MS spectrum. Thus, the structure of Sargafuran was determined as shown in Figure 1. The assignments of 1H- and 13C-NMR signals are indicated in Table 2.
Minimum inhibitory concentration
Sargafuran showed antibacterial activity against P. acnes at 15 μg ml−1. At the same concentration, Sargafuran also showed antibacterial activity against other Gram-positive bacteria, S. pyogenes and S. pneumoniae, and a Gram-negative bacterium, V. alginolyticus (Table 3). However, this compound did not inhibit the bacterial growth of the Gram-negative bacteria, E. coli and P. aeruginosa, and another Gram-positive bacterium, S. mutans, even at a concentration of 120 μg ml−1.
Bactericidal activity of Sargafuran
The time-kill study showed that Sargafuran decreased the bacterial counts of P. acnes at 1/2 × MIC (7.5 μg ml−1) during a 16-h exposure (Figure 2). At MIC (15 μg ml−1), Sargafuran completely killed the P. acnes strain tested within 4 h. However, Clindamycin did not show bactericidal activity against the P. acnes strain tested even after 72 h of incubation at 4 × MIC (0.1 μg ml−1). This bactericidal property of Sargafuran seems to be very promising for developing new types of antibiotics against the multi-drug-resistant P. acnes strains.
Bacteriolytic activity
The reduction in the absorbance of P. acnes cell suspensions was not observed in the presence of Sargafuran, at up to 4 × MIC (60 μg ml−1), until the end of the incubation period (Figure 3). In contrast, the absorbance of P. acnes cell suspensions treated with Achromopeptidase at 134 μg ml−1 as the positive control was reduced drastically in the early incubation period. These results indicated that Sargafuran did not lyse P. acnes cells.
Cytotoxicity
An MTT assay showed that Sargafuran was not cytotoxic to human normal dermal fibroblast cells (Figure 4). At concentrations up to 2 × the MIC (30 μg ml−1), it did not significantly affect cell viability and did not reduce cell viability by 50% at any concentrations up to 480 μg ml−1.
Discussion
Acne vulgaris is a multifactorial disease with an unclear etiology and pathogenesis. The factors known to cause acne vulgaris include follicular hyperkeratosis, sebum secretion, P. acnes and inflammation.17 Recently, cosmetics and toiletries containing natural products, such as extracts of herbs, Chinese plant medicine and seaweed, have been prevalent in commercial markets because of consumer concerns over synthetic chemical ingredients.
This study was conducted to screen marine algae collected from the Japan coastline for antibacterial activity against P. acnes to develop new types of cosmetics to treat acne. We found a new anti-P. acnes compound, Sargafuran, from the marine brown alga, S. macrocarpum. We also have reported two neurostimulating substances, sargaquinoic acid and sargachromenol, from this alga18, 19 and determined their structures to be 2-methylquinone-type compounds having side chain (C-20 unit). The 2-methylquinone moiety and C-20 side chain of these compounds would be biosynthesized from sikimic acid (C-7 unit) and geranyl geranyl diphospate (C-20 unit). It is likely that the biosynthetic pathway of Sargafuran carbon skeleton would share with the pathway of sargaquinoic acid and sargachromenol skeletons, because Sargafuran has a similar side chain and the same number of carbons. Sargafuran was stable against heating up to 60 °C, pH 4–7 and irradiation for 24 h. These properties are suitable for cosmetics or skin care products to prevent acne. Clindamycin is generally used for curing acne as a clinical treatment. However, Clindamycin was shown to be bacteriastatic even at 4 × MIC, whereas Sargafuran showed bactericidal activity against P. acnes. This is a superior property because bactericidal activity minimizes the chance of development of resistance. Takahashi et al.20 have also reported that eucalyptus leaf extracts and constituent flavonoids showed great antibacterial activity against Gram-positive bacteria, including P. acnes. The marine brown alga, S. macrocarpum, containing Sargafuran might be a good candidate from which to develop and produce the anti-acne cosmetics or skin care products in the near future.
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Acknowledgements
We are grateful to T Takeda (Hokkaido Kushiro Fisheries Experimental Station, Hokkaido, Japan) for kindly donating V. alginolyticus strain tested in this study.
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Kamei, Y., Sueyoshi, M., Hayashi, Ki. et al. The novel anti-Propionibacterium acnes compound, Sargafuran, found in the marine brown alga Sargassum macrocarpum. J Antibiot 62, 259–263 (2009). https://doi.org/10.1038/ja.2009.25
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DOI: https://doi.org/10.1038/ja.2009.25
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