Article Cite This: J. Nat. Prod. 2017, 80, 2723-2733
pubs.acs.org/jnp
Structures and Antibacterial Properties of Isorugosins H−J, Oligomeric Ellagitannins from Liquidambar formosana with Characteristic Bridging Groups between Sugar Moieties Yuuki Shimozu,† Teruo Kuroda,‡ Tomofusa Tsuchiya,§ and Tsutomu Hatano*,† †
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan Department of Microbiology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima 734-8553, Japan § College of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Nojihigashi, Shiga 525-8577, Japan ‡
S Supporting Information *
ABSTRACT: Three new ellagitannin oligomers, isorugosins H (1), I (2), and J (3), together with 11 known hydrolyzable tannins were isolated from an aqueous acetone extract of the fresh leaves of Liquidambar formosana. Their chemical structures were elucidated based on spectroscopic data and chemical conversion into known hydrolyzable tannins. The bridging mode of the valoneoyl groups between their sugar moieties has been identified only in this plant species. Additionally, the effects of the isorugosins isolated from this species on drug-resistant bacteria were evaluated and showed that isorugosin A (4) exhibited the most potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). The isorugosins also had a suppressing effect on pigment formation in Pseudomonas aeruginosa. The isorugosin−protein complexes were analyzed using sizeexclusion chromatography and polyacrylamide gel electrophoresis to clarify the relationship of their antibacterial properties with their protein interaction potency as hydrolyzable tannins. The results suggested that the antibacterial properties of hydrolyzable tannins are not simply a result of their binding activity to proteins, but are due to other factors such as the accessibility of polyphenolic acyl groups to bacterial membranes.
Liquidambar formosana Hance, a plant species previously regarded as belonging to the family Hamamelidaceae, has recently been reassigned to the family Altingiaceae.1 This plant is a deciduous tree indigenous to central and southern China and is often planted on roadsides in Japan. Its leaves and fruits are used in traditional Chinese medicine to treat enteritis, dysentery, and rheumatic arthralgia.2 In addition to several constituents including flavonoids3 and terpenes,4 hydrolyzable tannins have been isolated from the leaves.5−8 Hydrolyzable tannins are polyphenolic compounds widely distributed in dicotyledonous plants of Angiospermae.9 Among the numerous hydrolyzable tannins characterized to date, oligomeric ellagitannins have received considerable attention because of their vast structural diversity and varied biological activities depending on their structures.10 One of the largest groups of oligomeric ellagitannins has a valoneoyl group that is produced through oxidative C−O coupling between a galloyl © 2017 American Chemical Society and American Society of Pharmacognosy
group of one monomer and a 4,6-hexahydroxydiphenoyl (HHDP) group of the other.11 The modes by which glucose cores interconnect with valoneoyl groups of oligomers are classified into five types, and representative oligomers in which the valoneoyl groups link to anomeric centers of glucose cores are rugosin and isorugosin types, as illustrated in Figure 1. Ellagitannin oligomers with a rugosin-type valoneoyl group have been isolated from various plant species such as Rosa rugosa,12 Coriaria japonica,13 and Corylus heterophylla.14 However, the oligomers possessing an isorugosin-type valoneoyl group have only been isolated from L. formosana. Infectious diseases caused by methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa have Received: June 9, 2017 Published: October 11, 2017 2723
DOI: 10.1021/acs.jnatprod.7b00496 J. Nat. Prod. 2017, 80, 2723−2733
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Figure 1. Structures of rugosin- and isorugosin-type valoneoyl groups.
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RESULTS AND DISCUSSION In this study, the leaves were collected in July and October because of a previous report of the remarkable seasonal changes in the hydrolyzable tannin composition in the leaves of L. formosana.5 An aqueous acetone homogenate of the fresh leaves of L. formosana was extracted successively with Et2O, EtOAc, and n-BuOH. The EtOAc and n-BuOH extracts were subjected to column chromatography on several supports such as Diaion HP-20, Toyopearl HW-40, Sephadex LH-20, and MCI-gel CHP-20P, followed by HPLC purification to obtain three new isorugosin-type ellagitannins, named isorugosins H (1), I (2), and J (3) (Figure 2). In addition, 11 known hydrolyzable tannins, pedunculagin,23 casuarinin,23 1,4,6-tri-O-galloyl-β-D-glucose,24 2,4,6-tri-O-galloyl-D-glucose,25 1,2,4,6-tetra-O-galloyl-β-D-glucose,25 guavin C,26 pterocarinin A,27 and isorugosins A (4),7 B (5),7 D (6),7 and G (7)8 (Figure 3), were also identified. Among these known hydrolyzable tannins, 2,4,6-tri-O-galloyl-D-glucose, guavin C, and pterocarinin A were isolated for the first time from this plant source. These known tannins were identified using spectroscopic data and/or comparisons of reported spectroscopic data. As a result, the differences in the isolated hydrolyzable tannins from the leaves collected in July and October were rather insignificant. Structures of the New Ellagitannin Oligomers. Isorugosin H (1) was obtained as a light-brown amorphous powder. Its molecular formula was determined to be C89H62O57 from ESIMS ion at m/z 2060 [M + NH4]+, elemental analysis, and NMR spectroscopic data. The aromatic region in the 1H NMR spectrum of 1 indicated the presence of five galloyl groups [δH 7.08, 7.02, 6.97, 6.89, and 6.88 (2H each, s)] and two valoneoyl groups [δH 7.17 (1H, s, valoneoyl-HC), 6.64
become widespread problems, especially as nosocomial pathogens.15 One promising reservoir of new antibacterial molecules is plants.16 In fact, our recent study revealed that ellagitannins from Davidia involucrata showed anti-MRSA activity.17 Synergistic effects of ellagitannins and antibiotics against drug-resistant bacteria have also been identified as notable properties of hydrolyzable tannins.18 Accordingly, isorugosins, characteristic ellagitannins from L. formosana, are also expected to be promising antibacterial materials against MRSA and P. aeruginosa. In addition to the aforementioned antibacterial activity, ellagitannins have a wide variety of biochemical and pharmacological properties, including host-mediated antitumor effects and antiviral effects.11 Since tannins possess the fundamental property of binding with proteins, as shown by the formation of water-soluble and/or -insoluble complexes, proteins may participate in the various biological activities of hydrolyzable tannins.19,20 However, despite the necessity of clarifying the biological activities of hydrolyzable tannins, detailed mechanisms for the interactions of hydrolyzable tannins with proteins remain unclear due to the difficulty of conducting this type of analysis. Although we have established evaluation methods for water-soluble tannin−protein complexes using polyacrylamide electrophoresis (PAGE)21 and sizeexclusion chromatography (SEC),22 these analyses have been performed using only a few tannins or polyphenols at a time. In this study, new isorugosin oligomers and 11 known ones were isolated from the leaves of L. formosana. In addition, their antibacterial effects on antibiotic-resistant MRSA and P. aeruginosa were assessed, while the complex-forming ability and sizes of the water-soluble complexes between the isorugosins and the bovine serum albumin were also evaluated. 2724
DOI: 10.1021/acs.jnatprod.7b00496 J. Nat. Prod. 2017, 80, 2723−2733
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Figure 2. Structures of new isorugosins 1−3 isolated from L. formosana. The arrows (H → C) indicate important HMBC correlations.
(1H, s, valoneoyl-HA), 6.23 (1H, s, valoneoyl-HB) and δH 7.20 (1H, s, valoneoyl-HC), 6.61 (1H, s, valoneoyl-HA), 6.15 (1H, s, valoneoyl-HB)].7 The aliphatic region of the spectrum exhibited two sets of sugar proton signals (δH 6.12−3.78) assignable to the protons of fully O-acylated glucose cores (Table 1).7 The 4 C1 glucopyranose cores with β-oriented anomeric acyloxy groups were assigned by the large coupling constants (J1,2 = 7.8/8.4 Hz, J2,3 = J3,4 = J4,5 = 9.6 Hz).28 Based on the large difference in the 1H NMR chemical shifts between the geminal protons at C-6 and C-6′ of the two glucopyranose cores (ΔδH 1.42 for glucose-I, ΔδH 1.33 for glucose-II) of 1, the HHDP moiety of each of the valoneoyl groups was assigned at O-4/O6 and O-4′/O-6′ of the glucopyranose cores, based on the coupling constants of the glucose protons described above.29 Therefore, the five galloyl groups in 1 are located at O-1, O-2,
O-3, O-2′, and O-3′ within the glucopyranose cores. The signals in the 13C NMR spectrum of 1 also identified five galloyl and two valoneoyl groups and two glucopyranose cores (see Experimental Section). The assignments of the signals corresponding to the structural moieties of 1 were based on the correlations in the HSQC and HMBC spectra. The positions of the galloyl groups were verified by the HMBC correlations between galloyl proton signals (δH 7.08, 7.02, 6.97, 6.89, and 6.88) and glucose protons [δH 6.12 (H-1′), 5.52 (H2′), 5.52 (H-3′), 5.54 (H-2), and 5.54 (H-3)] through the respective carbonyl carbon signals [δC 165.3, 166.0, 166.1 (2C), and 166.3] (Figure 2). Among the valoneoyl carbon signals in the 13C NMR spectrum of 1, the signals at δC 147.0/146.4 and 145.2/145.1 were assignable to C-4 (or C-4′) and C-4′ (or C4). These assignments were based on the couplings of these 2725
DOI: 10.1021/acs.jnatprod.7b00496 J. Nat. Prod. 2017, 80, 2723−2733
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Figure 3. Structures of known isorugosins isolated from L. formosana.
attributed to the anisotropic effect of the galloyl moiety of the valoneoyl group.8 The electronic circular dichroism (ECD) spectrum of 1 exhibited a strong positive Cotton effect in the short-wavelength region ([θ]225 +2.7 × 105), indicating that the absolute configuration of both valoneoyl groups was (aS).31 The structure 1 assigned to isorugosin H was substantiated by the treatment of 1 in boiling water to afford the two monomeric ellagitannins 4 and 5. Isorugosin I (2) was obtained as a light-brown amorphous powder. The molecular formula C116H82O74 was assigned from the ESIMS ion at m/z 2676 [M + NH4]+, elemental analysis, and NMR spectroscopic data. The 1H NMR spectrum showed the presence of two valoneoyl groups by the two following three-proton sets [δH 7.16 (1H, s, valoneoyl-HC), 6.62 (1H, s, valoneoyl-HA), 6.156 (1H, s, valoneoyl-HB) and δH 7.18 (1H, s, valoneoyl-HC), 6.63 (1H, s, valoneoyl-HA), 6.162 (1H, s, valoneoyl-HB)].7 Proton signals due to the six galloyl groups (δH 7.10, 7.08, 7.01, 6.97, 6.89, and 6.88) and an HHDP group (δH 6.70 and 6.61) were also present in the aromatic region.8 The aliphatic region of the 1H NMR spectrum displayed three seven-spin systems assignable to the protons of three glucose
carbons (via two bonds) with valoneoyl protons (HA or HB), as indicated by the correlations δH 6.23−δC 147.0/δH 6.15−δC 146.4, and δH 6.64−δC 145.1/δH 6.61−δC 145.2 in the HMBC spectrum of 1. The deshielded signals (δC 147.0/146.4) were assigned to C-4′ of the valoneoyl groups because the C-4 and C-4′ signals of the HHDP group are observed at around δC 145.30 Therefore, the correlations described above indicated that the protons at δH 6.23/6.15 and δH 6.64/6.61 are those of HB and HA of the valoneoyl groups, respectively. The positions of the valoneoyl groups were also substantiated by the following HMBC correlations: correlations of valoneoyl-HB (δH 6.23 and 6.15) and glucose H-4/H-4′ (δH 5.05 and 5.06) through the carbonyl carbon signals at δC 167.6 (2C) and correlations of valoneoyl-HA (δH 6.64 and 6.61) and glucose H2-6/H2-6′ (δH 5.20/3.78 and 5.16/3.83) through the carbonyl carbon signals at δC 168.3 (2C). The orientations of both of the valoneoyl groups were determined to be of the isorugosin type (Figure 2), and as shown in Table 1, the 1H NMR chemical shifts of glucose-II signals of 1 coincided with the corresponding signals of 6, while the H-3 signal of glucose-I of 1 was shielded (ΔδH 0.23) relative to that of 6. This shift was 2726
DOI: 10.1021/acs.jnatprod.7b00496 J. Nat. Prod. 2017, 80, 2723−2733
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(H-1″), 5.51 (H-2″), and 5.53 (H-3″)] via the common carbonyl carbon signals [δC 165.3, 166.1 (3C), 166.3, and 166.7]. Bridging of the HHDP group between O-4/O-6 of glucose-I was confirmed by the HMBC correlations of the HHDP proton signals (δH 6.70 and 6.61) and signals of H-4 (δH 4.99) and H2-6 (δH 5.15/3.75) of glucose-I via the ester carbonyl carbon signals (δC 168.2 and 168.6). Analogously, the HHDP moieties of the valoneoyl group were assigned at O-4′/ O-6′ and O-4″/O-6″ of glucose-II/III and were confirmed by the HMBC correlations between valoneoyl-HB [δH 6.16 (2H)] and H-4 (δH 5.05 and 5.06) of glucose-II/III via the ester carbonyl carbon signals (δC 167.6 and 167.7) and the correlations between valoneoyl-HA (δH 6.61 and 6.63) and H2-6 (δH 5.14/3.79 and 5.18/3.84) of glucose-II/III via ester carbonyl carbon signals (δC 168.3 and 168.4). In the HMBC spectrum, H-1/H-1′ of glucose-I/II (δH 5.85 and 6.04) also showed correlations with the valoneoyl-HC (δH 7.16 and 7.18) through the ester carbonyl carbon (δC 162.8 and 162.9), indicating that the locations of the galloyl parts of both of the valoneoyl groups were at the corresponding anomeric centers (Figure 2). The (aS)-configuration of each of an HHDP group and the HHDP moieties of the valoneoyl groups had a strong positive Cotton effect at 226 nm in the ECD spectrum.31 The remaining hydroxy groups of the three glucopyranose cores except for that at C-3 of glucose-I were acylated by the galloyl groups. Based on these data, structure 2 was assigned to isorugosin I. Isorugosin J (3) was obtained as a light-brown amorphous powder. Its trimeric ellagitannin structure (molecular formula, C130H90O83) was revealed by the ion peak [M + NH4]+ at m/z 2996 using ESIMS, elemental analysis, and NMR spectroscopic data. The 1H NMR spectrum exhibited proton signals assignable to three valoneoyl groups [δH 7.17 (1H, s,
Table 1. NMR Spectroscopic Data (600 MHz, Acetone-d6/ D2O, 9:1) for Isorugosins H (1) and D (6) position glucose-I 1 2 3 4 5 6 glucose-II 1 2 3 4 5 6
isorugosin H (1)
isorugosin D (6)
δH (J in Hz)
δH (J in Hz)
6.05, 5.54, 5.54, 5.06, 4.37, 5.20, 3.78,
d (7.8) dd (9.6, 7.8) t (9.6) t (9.6) dd (9.6, 6.6) dd (13.2, 6.6) d (13.2)
6.09, 5.59, 5.78, 5.18, 4.48, 5.18, 3.84,
d (8.5) dd (10.0, 8.5) t (10.0) t (10.0) dd (10.0, 6) dd (13.5, 6) d (13.5)
6.12, 5.52, 5.52, 5.06, 4.31, 5.16, 3.83,
d (8.4) dd (9.6, 8.4) t (9.6) t (9.6) dd (9.6, 6.6) dd (13.2, 6.6) d (13.2)
6.13, 5.52, 5.52, 5.06, 4.31, 5.27, 3.82,
d (8.0) m m t (10.0) dd (10.0, 6.5) dd (13.0, 6.5) d (13.0)
cores (Table 2). The 4C1 conformations of the glucopyranose cores were indicated by the large coupling constants of their proton signals. The shielding of the H-3 signal of glucose-I (δH 4.14) indicated that the corresponding C-3 hydroxy group was unacylated (Table 2). The 13C NMR spectrum of 2 showed carbon signals consistent with the presence of these acyl groups (see Experimental Section) and the glucose cores (Table 2). The locations of the galloyl moieties on the glucose cores were determined from the HMBC correlations of the galloyl proton signals (δH 7.10, 7.08, 7.01, 6.97, 6.89, and 6.88) with the glucose signals [δH 5.34 (H-2), 5.52 (H-2′), 5.49 (H-3′), 6.12
Table 2. NMR Spectroscopic Data (600 MHz, Acetone-d6/D2O, 9:1) for Isorugosins G (7), I (2), and J (3) isorugosin G (7)
isorugosin I (2)
isorugosin J (3)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
glucose-I 1 2 3 4 5 6
93.23 73.0 73.4 70.6 72.9 63.0
6.06, d (8.4) 5.58, dd (10.2, 8.4) 5.76, t (10.2) 5.16, t (10.2) 4.44, dd (10.2, 6.6) 5.23, dd (13.2, 6.6) 3.80, d (13.2)
93.5 74.2 73.2 72.5 73.1 63.4
5.85, d (8.4) 5.34, dd (9.6, 8.4) 4.14, t (9.6) 4.99, t (9.6) 4.19, dd (9.6, 7.8) 5.15, dd (13.8, 6.6) 3.75, d (12.6)
93.2 71.7 73.0 70.9 72.6 63.00
6.05, d (7.8) 5.52, m 5.52, m 5.07, t (9.0) 4.36, dd (9.0, 7.8) 5.20, dd (13.2, 7.8) 3.77, d (13.2)
glucose-II 1 2 3 4 5 6
93.20 71.7 71.6 70.8 72.8 63.0
6.03, d (7.8) 5.53, t (9.6) 5.50, t (9.6) 5.05, t (9.6) 4.30, dd (10.8, 6.0) 5.13, dd (13.2, 6.0) 3.80, d (13.2)
93.3 72.9 73.0 70.8 72.8 63.04
6.04, d (8.4) 5.52, dd (9.6, 8.4) 5.49, t (9.6) 5.05, t (9.6) 4.30, dd (9.6, 6.6) 5.14, dd (13.8, 6.6) 3.79, d (13.2)
93.3 71.7 73.1 70.8 72.7 63.04
6.04, d (7.8) 5.52, m 5.52, m 5.05, t (9.6) 4.31, dd (10.2, 6.0) 5.14, dd (13.2, 6.0) 3.79, d (13.2)
glucose-III 1 2 3 4 5 6
93.6 71.6 71.6 70.8 72.6 63.0
6.11, d (7.8) 5.50, t (9.6) 5.52, t (9.0) 5.05, t (9.0) 4.31, dd (10.2, 6.0) 5.17, dd (13.2, 6.0) 3.84, d (13.2)
93.6 71.7 71.7 70.9 72.7 63.00
6.12, d (7.8) 5.51, dd (9.6, 7.8) 5.53, t (9.6) 5.06, t (9.6) 4.32, dd (9.6, 6.6) 5.18, dd (13.8, 6.6) 3.84, d (13.2)
93.6 71.6 73.2 70.9 72.8 62.98
6.12, d (7.8) 5.52, m 5.52, m 5.06, t (9.6) 4.31, dd (10.2, 6.0) 5.17, dd (13.2, 6.0) 3.84, d (13.2)
position
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valoneoyl-HC), 6.62 (1H, s, valoneoyl-HA), 6.15 (1H, s, valoneoyl-HB), 7.19 (1H, s, valoneoyl-HC), 6.63 (1H, s, valoneoyl-HA), 6.16 (1H, s, valoneoyl-HB), and 7.21 (1H, s, valoneoyl-HC), 6.64 (1H, s, valoneoyl-HA), 6.24 (1H, s, valoneoyl-HB)], seven galloyl groups (δH 7.09, 7.01, 7.00, 6.97, 6.891, 6.886, and 6.884), and proton signals from three 4 C1 β-glucopyranose cores (Table 2). The 13C NMR data of 3 were consistent with the presence of these constituent units (Table 2 and Experimental Section). Comparisons of the 1H NMR data of 3 with those of 7, an isorugosin trimer previously isolated from L. formosana,8 revealed that almost all the chemical shifts of the signals of 3 coincided with those of 7, except for H-3 of glucose-I. An upfield shift (ΔδH 0.24) of H-3 of glucose-I was regarded to be an anisotropic effect from the galloyl moiety of the neighboring valoneoyl group as mentioned in the structural discussion of 1. These data suggested structure 3 for this compound, where the HHDP group at O-4/O-6 of glucose-I in 7 was substituted with a valoneoyl group. The assigned structure was substantiated by key HMBC correlations depicted in Figure 2. There were correlations of galloyl proton signals (δH 7.09, 7.013, 7.008, 6.97, 6.891, 6.886, and 6.884) and glucose protons [δH 5.55− 5.49 (H-2, 3, 2′, 3′, 2″, and 3″) and 6.12 (H-1″)] through the carbonyl carbon signals at δC 166.2 (2C), 166.15, 166.08 (2C), and 166.0, 165.3; correlations of valoneoyl-HB (δH 6.24, 6.16, and 6.15) and H-4 of glucose-I−III (δH 5.07, 5.06, and 5.05) via carbonyl carbon signals at δC 167.7, 167.64, and 167.5; and correlations of valoneoyl-HA (δH 6.64, 6.63, and 6.62) and H2-6 of glucose-I−III (δH 5.20/3.77, 5.14/3.79, and 5.17/3.84) via carbonyl carbon signals at δC 168.32 and 168.27 (2C). These HMBC correlations also substantiated the isorugosin-type orientations of the valoneoyl groups. The (aS) configurations of all of the HHDP moieties of the valoneoyl groups in 3 were confirmed with a diagnostic Cotton effect at 224 nm in the ECD spectrum.31 Consequently, structure 3 was assigned for isorugosin J. Antibacterial Effects. Early studies have revealed the antibacterial effects of several hydrolyzable tannins against MRSA.18 To clarify the effectiveness of isorugosins, characteristic hydrolyzable tannins solely found in L. formosana, antibacterial assays for monomeric to trimeric (4, 5, 6, and 7) isorugosins were conducted. The effects of isorugosins on MRSA (S. aureus OM481 and OM584), methicillin-sensitive S. aureus (MSSA) (209P), and P. aeruginosa PAO1 were examined using a liquid dilution method.32 The results are summarized in Table 3. Isorugosin monomers 4 and 5 displayed potent antibacterial effects on MRSA [minimum inhibitory concentration (MIC) 29−67 μM] as well as MSSA (58−67 μM). Notably, 4 and 5 exhibited the most potent antibacterial activity against MRSA. On the other hand, 1,2,3,4,6-penta-O-galloyl-β-D-glucose (PGG), which is a representative hydrolyzable tannin, exerted antibacterial effects on both MRSA and MSSA (MIC 68 μM) in a separate experiment (data not shown). Thus, the substitution of two or three galloyl groups with a valoneoyl group induced comparable anti-MRSA effects in these compounds. The isorugosin dimer 6 exhibited moderate effects (34−68 μM), while trimer 7 displayed only a weak effect (91 μM). The induction of cell membrane instability has been regarded as a mechanism for the antibacterial properties of tannins,33 and our results suggest that hydrolyzable tannins with large molecular sizes are detrimental to the effect on the bacterial membranes.
Table 3. Antibacterial Effects of Isorugosins 4−7 MIC (μM) MSSA sample
209P
oxacillin 926 [29]b >1073 [34]b
34
68
>546 [34]b
91
91
>364 [23]b
b
n.t.: not tested. These values are minimum concentrations for inhibiting the formation of green pigments.
On the other hand, none of the isorugosins tested in this study showed antibacterial effects (>364−1073 μM) on P. aeruginosa PAO1 (Table 3). However, the suppression of the formation of green pigments (23−34 μM) was observed, with the monomers exhibiting more potent effects than the oligomers. The suppressing effects on pigment formation were attributed to the expression of pyocyanin or related compounds characteristically secreted from P. aeruginosa.34 Since other hydrolyzable tannins such as PGG, casuarinin, and pedunculagin also inhibited green pigment secretion (data not shown), this effect on P. aeruginosa is regarded to be an important property of hydrolyzable tannins. Two explanations are possible, although the details remain unclear. First, hydrolyzable tannins directly react with secreted pigments (direct inhibition mechanism). Second, hydrolyzable tannins react with autoinducers such as homoserine lactones35 to express pigments (indirect inhibition mechanism). Protein Complexation. Since protein complexation is a fundamental property of tannins, this property might participate in the antibacterial activities of isorugosins. Thus, the complex-forming properties of isorugosins with proteins using SEC and PAGE and bovine serum albumin (BSA) were assessed to identify the relationship between the antibacterial and complexation properties of these ellagitannins. BSA was exposed to isorugosins 1, 4, 5, 6, and 7 in a phosphate buffer (pH 7.0). After a 24 h incubation at 35 °C, the reaction mixtures were analyzed using SEC. As shown in Figure 4A, BSA showed as a major peak in SEC, along with minor peaks due to dimeric and trimeric forms of BSA. In contrast, the peaks corresponding to isorugosins had an observed retention time (tR) around 16 min (Figure 4B−F). The peaks with the reaction mixtures of isorugosin−BSA differed significantly from those of BSA in SEC [e.g., the peaks attributed to water-soluble isorugosin−BSA complexes (tR: 9−12 min)] (Figure 4B−F). These peaks with shorter retention times were consistent with the formation of molecular species much larger than BSA. No peaks were observed around this region (tR: 9−12 min) for solutions in the absence of BSA after incubation (data not shown). For the evaluation of the molecular size of the complexes, the number-average molecular weight (Mn) and the weight-average molecular weight (Mw) were estimated from the SEC peaks. As shown in Table 4, 4 produced larger complexes [Mn 1.9 × 105 (corresponding to a sum of 2.8 molecules of BSA), Mw 2.2 × 105] than 5 [Mn 1.7 × 105 (corresponding to 2728
DOI: 10.1021/acs.jnatprod.7b00496 J. Nat. Prod. 2017, 80, 2723−2733
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Figure 4. SEC profiles of solutions of (A) BSA alone, (B) 4 and BSA, (C) 5 and BSA, (D) 6 and BSA, (E) 1 and BSA, and (F) 7 and BSA.
galloyl and related acyl groups in isorugosins are required for interaction efficiency with proteins (complexes of the dimers and the trimer > those of the monomers). However, crosslinking five molecules of BSA seems to be a limit for the complexes, probably due to the rigidity and bulky structures of isorugosins. The formation of the supermolecules of the isorugosin−BSA complexes was also substantiated by the native- and SDS-PAGE of the reaction mixture. As shown in Figure 5, the motility of the BSA bands changed with slight expansions of the bands in the presence of the isorugosins in both native- and SDS-PAGE. In keeping with the results from SEC, dimers 1 and 6 and trimer 7 showed larger complexes than those observed for the monomers. Notably, these large complexes were not decomposed during the SDS denaturing process. In contrast, the complex of PGG with BSA was decomposed during the SDS denaturing process.21 On the other hand, the size of the PGG complex was much larger [Mn 9.5 × 105 (corresponding to 13.8 molecules of BSA), Mw 1.4 × 106] than those of isorugosins.22 Although the detailed mechanisms of the complexations of hydrolyzable tannins and protein remain unclear, previous reports have suggested that cysteine and lysine participate in
Table 4. Number Average Molecular Weights (Mn) and the Weight Average Molecular Weights (Mw) of Complexes of Isorugosins 1 and 4−7 with BSA complexes Monomeric tannins 4 + BSA 5 + BSA Dimeric tannins 6 + BSA 1 + BSA Trimeric tannin 7 + BSA
Mn
Mw
1.9 × 105 1.7 × 105
2.2 × 105 1.9 × 105
3.3 × 105 3.4 × 105
4.1 × 105 4.1 × 105
3.2 × 105
4.1 × 105
2.5 molecules of BSA), Mw 1.9 × 105]. Dimers 1 and 6 formed larger complexes [Mn 3.3 × 105 (corresponding to 4.8 molecules of BSA), Mw 4.1 × 105; and Mn 3.4 × 105 (corresponding to 4.9 molecules of BSA), Mw 4.1 × 105, respectively] than monomers 4 and 5, and trimer 7 yielded a comparable complex size [Mn 3.2 × 105 (corresponding to 4.6 molecules of BSA), Mw 4.1 × 105] to that of 6. These results suggest that a large number of phenolic hydroxy groups on 2729
DOI: 10.1021/acs.jnatprod.7b00496 J. Nat. Prod. 2017, 80, 2723−2733
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solvent. Analytical reversed phase HPLC was performed on a YMCPack ODS-A A-302 (YMC Co., Ltd.) column (4.6 mm i.d. × 150 mm) eluted with 10 mM H3PO4/10 mM KH2PO4/CH3CN (42.5/42.5/15) (flow rate: 1.0 mL/min; 280 nm UV detector) at 40 °C. Preparative reversed phase HPLC was conducted at 40 °C on a YMC-Pack ODSA A-324 (YMC Co., Ltd.) column (10 mm i.d. × 300 mm) eluted with 10 mM H3PO4/10 mM KH2PO4/CH3CN [either 42.5/42.5/15 (solvent I), 42/42/16 (solvent II), or 46.5/46.5/7 (solvent III), v/v] at a flow rate of 2.0 mL/min with detection at 280 nm. Materials. Fresh leaves of L. formosana were collected in July 2005 and October 2006 at Okayama University. We used Toyopearl HW-40 (coarse and fine grades; TOSOH, Tokyo, Japan), YMC-gel ODS-A (S, 75 μm; YMC), Sephadex LH-20 (GE Healthcare Japan, Tokyo, Japan), Sep-Pak C18 (Nihon Waters, Osaka, Japan), Diaion HP-20, and MCI-gel CHP-20P (Mitsubishi Chemical, Tokyo, Japan) for column chromatography. Isolation and Purification of Hydrolyzable Tannins. Fresh leaves of L. formosana (2.0 kg) collected in July 2005 were homogenized in 70% aqueous acetone at room temperature. The filtered solution was concentrated to 800 mL and extracted successively with Et2O, EtOAc, and n-BuOH to furnish the Et2O (2.0 g), EtOAc (14.5 g), and n-BuOH (44.5 g) extracts and a watersoluble portion (132.3 g). A portion of the EtOAc extract (6.0 g) was subjected to column chromatography over Toyopearl HW-40C (2.2 cm i.d. × 66 cm) with 70% EtOH to yield pedunculagin (94.8 mg) and casuarinin (113.0 mg). Half of the amount of the n-BuOH extract (23.6 g) was subjected to column chromatography on Diaion HP-20 (6.5 cm i.d. × 33 cm) with H2O → aqueous MeOH (20 → 40 → 60 → 80 → 100% MeOH) → H2O/acetone (3/7, v/v). The 40% MeOH eluate from the column (8.3 g) was further applied to column chromatography on Toyopearl HW-40C [2.2 cm i.d. × 61 cm, 700 drops/fraction (fr.)] with 70% EtOH → 70% EtOH/70% acetone (9/ 1 → 8/2, v/v) → 70% acetone (chromatography T1). The combined frs. 111−130 (426.4 mg) from T1 eluted with 70% EtOH were then chromatographed on Sephadex LH-20 (1.1 cm i.d. × 45 cm) with 70% EtOH and on MCI-gel CHP-20P (1.1 cm i.d. × 39 cm) with aqueous MeOH (0 → 20 → 40 → 60 → 100% MeOH) to give isorugosin B (5) (49.5 mg). The combined frs. 131−150 (351.1 mg) from T1 eluted with 70% EtOH were subjected to column chromatography on MCI-gel CHP-20P (1.1 cm i.d. × 39 cm) with aqueous MeOH (0 → 20 → 40 → 60 → 100% MeOH). The 20% MeOH eluate (18.6 mg) was further purified by preparative RP-HPLC with solvent III to give pterocarinin A (6.4 mg). The combined frs. 151−170 (469.8 mg) from T1 eluted with 70% EtOH were separated using MCI-gel CHP-20P (1.1 cm i.d. × 35 cm) with aqueous MeOH (0 → 20 → 30 → 100% MeOH) to give isorugosin A (4) (17.5 mg). The combined frs. 251− 300 (327.6 mg) from T1 eluted with 70% EtOH were separated using MCI-gel CHP-20P (1.1 cm i.d. × 36 cm) with aqueous MeOH (0 → 20 → 40 → 60 → 100% MeOH). Guavin C (11.9 mg) was obtained from the 20% MeOH. Combined frs. 226−335 (193.9 mg) from T1 eluted with 70% EtOH/70% acetone (9/1, v/v) were subjected to column chromatography on MCI-gel CHP-20P (1.1 cm i.d. × 37 cm, 400 drops/fr.) with aqueous MeOH (0 → 20 → 40 → 60 → 100% MeOH). Isorugosin H (1) (15.4 mg) and isorugosin D (6) (9.5 mg) were obtained from frs. 13−20 and frs. 37−55 of the 40% MeOH eluates, respectively. Combined frs. 261−299 (31.2 mg) from T1 eluted with 70% EtOH/70% acetone (8/2, v/v) were subjected to preparative HPLC with solvent II to give isorugosin G (7) (4.4 mg). Combined frs. 201−260 (85.0 mg) from T1 eluted with 70% EtOH/ 70% acetone (8/2, v/v) were chromatographed on MCI-gel CHP-20P (1.1 cm i.d. × 37 cm) with aqueous MeOH (0 → 20 → 40 → 60 → 100% MeOH). Isorugosin G (7) (0.79 mg) and isorugosin I (2) (3.4 mg) were isolated from the 40% MeOH eluate (17.7 mg) via preparative HPLC with solvent I. The 60% MeOH eluate (2.6 g) from the Diaion chromatography was subjected to column chromatography on Toyopearl HW-40C (2.2 cm i.d. × 42 cm) with 70% EtOH → 70% EtOH/70% acetone (9/1, v/v) → 70% acetone (chromatography T2). The 70% acetone eluates from T1 (169.1 mg) and T2 (380.9 mg) were combined and further subjected to column chromatography on Toyopearl HW-40C (1.1 cm i.d. × 43 cm, 500 drops/fr.) with 70%
Figure 5. Reactions of isorugosins and BSA. (A) Native-PAGE analysis of reaction mixture. Lane 1, BSA alone; 2, 4 and BSA; 3, 5 and BSA; 4, 6 and BSA; 5, 1 and BSA; 6, 7 and BSA. (B) SDS-PAGE analysis of reaction mixture. Lane 1, BSA alone; 2, 4 and BSA; 3, 5 and BSA; 4, 6 and BSA; 5, 1 and BSA; 6, 7 and BSA.
this reaction in addition to hydrophobic interactions.19 Our findings suggest that the antibacterial properties of hydrolyzable tannins are not simply due to their binding activity to proteins, but may be due to some other factors such as accessibility of the polyphenolic acyl groups to bacterial membranes. In summary, in addition to four known isorugosins, A (4), B (5), D (6), and G (7), three new isorugosin oligomers were isolated from L. formosana, namely, isorugosins H (1), I (2), and J (3) (Figure 2). As shown in Figure S19 (Supporting Information), isorugosin monomers, dimers, and trimers in the extract of fresh leaves of L. formosana were detected. Their contents were as follows: monomers > dimers > trimers. All three new isorugosins were characteristic ellagitannins having an isorugosin-type valoneoyl group. However, rugosins, the isomers of isorugosins that impact the orientation of the valoneoyl group, have not been found in the fractions obtained from L. formosana. It has been suggested, consistent with a previous report,7 that the specific enzymes catalyzing the formation of isorugosin-type valoneoyl groups are present in L. formosana. Moreover, we showed that isorugosin monomers exerted more effective suppression of MRSA growth and pigment secretion from P. aeruginosa than did the dimer and trimer (Table 3). However, the reactivities of the isorugosin dimers and trimer linked to BSA that formed large supermolecules were much higher (Figures 4 and 5, Table 4) than those of the monomers. These results suggest that the mechanisms for the antibacterial activities cannot be simply explained by protein reactivity, but may be due to other factors, such as accessibility of the polyphenolic acyl groups to bacterial membranes. Appropriate lipophilicity, rigidity, and bulkiness are also key factors. Therefore, further studies are required to estimate the relationship between antibacterial activities and protein reactivity using diverse types of hydrolyzable tannins.
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EXPERIMENTAL SECTION
General Experimental Procedures. The 1D (1H and 13C) and 2D (1H−1H-COSY, HSQC, and HMBC) NMR spectra were recorded on a Varian INOVA AS 600 instrument (600 MHz for 1H and 151 MHz for 13C, Agilent, Santa Clara, CA, USA). Chemical shifts are given in δ (ppm) values relative to that of the solvent signal acetone-d6 (δH 2.04; δC 29.8) on the tetramethylsilane scale. Optical rotations were recorded on a JASCO DIP-1000 digital polarimeter. Elemental analyses were recorded on a Yanaco CHN recorder MT-5. ECD spectra were measured using a JASCO J-720W spectrophotometer. ESIMS was performed with a Micromass Auto Spec OA-TOF spectrometer using 50% MeOH containing 0.1% NH4OAc as a 2730
DOI: 10.1021/acs.jnatprod.7b00496 J. Nat. Prod. 2017, 80, 2723−2733
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EtOH → 70% EtOH/70% acetone (9/1, v/v) → 70% EtOH/70% acetone (8/2, v/v) → 70% acetone. The combined frs. 171−250 (74.2 mg) eluted with 70% EtOH/70% acetone (9/1, v/v) were fractionated using Sep-Pak C18 followed by preparative HPLC with solvent I to give isorugosin G (7) (2.6 mg), isorugosin H (1) (2.4 mg), and isorugosin J (3) (3.5 mg). Fresh leaves of L. formosana (4.0 kg) collected in October 2006 were homogenized in 70% aqueous acetone at room temperature. The homogenate was filtered, then concentrated to 2.6 L, and extracted with Et2O, EtOAc, and n-BuOH, successively, to furnish the Et2O (3.8 g), EtOAc (22.9 g), and n-BuOH (67.6 g) extracts and a water-soluble portion (244.7 g). A portion of the EtOAc extract (15.8 g) was subjected to column chromatography on Diaion HP-20 (6.5 cm i.d. × 33 cm) with H2O → aqueous MeOH (20 → 40 → 60 → 80% MeOH) → H2O/acetone (3/7, v/v). The 40% MeOH eluate (4.7 g) from the Diaion chromatography was further subjected to a column of Toyopearl HW-40C (2.2 cm i.d. × 58 cm, 500 drops/fr.) with 70% EtOH → 70% EtOH/70% acetone (9/1, v/v) → 70% acetone (chromatography T3). The combined frs. 66−90 (205.5 mg) from T3 eluted with 70% EtOH were chromatographed on MCI-gel CHP-20P (1.1 cm i.d. × 36 cm) with aqueous MeOH (0 → 20 → 40% MeOH) to give 1,4,6-tri-O-galloyl-β-D-glucose (9.9 mg) and 2,4,6-tri-O-galloylD-glucose (28.9 mg). The combined frs. 121−150 (215.0 mg) from T3 eluted with 70% EtOH were subjected to column chromatography on MCI-gel CHP-20P (1.1 cm i.d. × 35 cm) with 40% aqueous MeOH to yield 1,2,4,6-tetra-O-galloyl-β-D-glucose (14.1 mg). The n-BuOH extract (67.6 g) was subjected to column chromatography on Diaion HP-20 (10.0 cm i.d. × 35 cm) with H2O → aqueous MeOH (20 → 40 → 60 → 80 → 100% MeOH) → H2O/acetone (3:7, v/v). The 60% MeOH eluate (7.5 g) from the Diaion column was subjected to column chromatography on Toyopearl HW-40C (2.2 cm i.d. × 61 cm) with 70% EtOH → 70% EtOH/70% acetone (9/1, v/v) → 70% acetone. The 70% acetone eluate (638.0 mg) was further purified by Toyopearl HW-40C (2.2 cm i.d. × 40 cm, 500 drops/fr.) with 70% EtOH → 70% EtOH/70% acetone (9/1, v/v) → 70% EtOH/70% acetone (8/2, v/v) → 70% acetone (chromatography T4). The combined frs. 141−321 (149.0 mg) from T4 were fractionated with Sep-Pak C18 followed by preparative HPLC with solvent I to give isorugosin G (7) (2.2 mg), isorugosin I (2) (5.0 mg), and isorugosin J (3) (2.4 mg). Isorugosin G (7): light-brown amorphous powder; [α]25D +49 (c 0.5, MeOH); ECD (MeOH) [θ] (nm) +2.1 × 105 (225), +1.3 × 105 (242), −6.2 × 104 (262), +5.7 × 104 (284); anal. C 49.58%, H 3.15%, calcd for C128H86O78·9H2O, C 49.67%, H 3.52%; 1H NMR (acetoned6/D2O = 9/1, 600 MHz) δ 7.17, 7.15 (each 1H, s, valoneoyl-I, II-HC), 7.07, 7.012, 7.007, 6.96, 6.94, 6.873, 6.871 (each 2H, s, galloyl-H), 6.64, 6.63 (each 1H, s, valoneoyl-I, II-HA), 6.61, 6.47 (each 1H, s, HHDP-H-3, 3′), 6.17, 6.15 (each 1H, s, valoneoyl-I, II-HB), and glucose protons (Table 2). Isorugosin H (1): light-brown amorphous powder; [α]25D +31 (c 0.5, MeOH); ECD (MeOH) [θ] (nm) +2.7 × 105 (225), +9.5 × 104 (242), −7.7 × 104 (260), +8.5 × 104 (282); ESIMS m/z 2060 [M + NH4]+; anal. C 45.44%, H 3.95%, calcd for C89H62O57·17H2O, C 45.49%, H 4.12%; 1H NMR (acetone-d6/D2O = 9/1, 600 MHz) δ 7.20 (1H, s, valoneoyl-II-HC), 7.17 (1H, s, valoneoyl-I-HC), 7.08, 7.02, 6.97, 6.89, 6.88 (each 2H, s, galloyl-H), 6.64 (1H, s, valoneoyl-I-HA), 6.61 (1H, s, valoneoyl-II-HA), 6.23 (1H, s, valoneoyl-I-HB), 6.15 (1H, s, valoneoyl-II-HB), and glucose protons (Table 1); 13C NMR (acetoned6/D2O = 9/1, 151 MHz) δ 168.33 (valoneoyl-I C-7), 168.30 (valoneoyl-II C-7), 167.65 (valoneoyl-II C-7′), 167.60 (valoneoyl-I C7′), 167.5 (valoneoyl-I C-7″), 166.3, 166.13, 166.09, 166.06, 165.3 (galloyl C-7), 162.7 (valoneoyl-II C-7″), 147.0 (valoneoyl-I C-4′), 146.4 (valoneoyl-II C-4′), 146.1, 145.9, 145.50, 145.46 (10C, galloyl C-3, 5), 145.2 (valoneoyl-II C-4), 145.1 (valoneoyl-I C-4), 144.7, 144.63, 144.59, 144.5 (valoneoyl-I, II C-6, 6′), 143.09 (valoneoyl-I C2″), 143.06 (valoneoyl-II C-2″), 141.1 (valoneoyl-I C-4″), 140.5 (valoneoyl-II C-4″), 140.1 (valoneoyl-II C-5″), 140.0 (valoneoyl-I C5″), 139.9, 139.5, 139.4, 139.1, 139.0 (galloyl C-4), 137.8, 137.1 (valoneoyl-I, II C-3″), 137.3 (valoneoyl-I C-5′), 137.1 (valoneoyl-II C5′), 136.5 (valoneoyl-I C-5), 136.4 (valoneoyl-II C-5), 125.65, 125.62
(valoneoyl-I, II C-1), 125.4, 125.2 (valoneoyl-I, II C-1′), 120.09, 120.06, 119.8, 119.7, 119.3 (galloyl C-1), 117.8 (valoneoyl-II C-2), 117.6 (valoneoyl-I C-2), 115.9 (valoneoyl-I C-2′), 115.8 (valoneoyl-II C-2′), 115.1 (valoneoyl-II C-1″), 112.6 (valoneoyl-I C-1″), 110.23, 110.22, 110.13, 110.11, 110.0 (12C, galloyl C-2, 6, valoneoyl-I, II C6″), 108.1 (valoneoyl-I C-3), 107.7 (valoneoyl-II C-3), 105.4 (valoneoyl-I C-3′), 104.9 (valoneoyl-II C-3′), 93.6 (glucose-I C-1), 93.2 (glucose-II C-1′), 73.2 (glucose-I C-3), 72.9 (glucose-II C-3′), 72.7 (glucose-II C-5′), 72.6 (glucose-I C-5), 71.7 (glucose-II C-2′), 71.5 (glucose-I C-2), 70.84 (glucose-I C-4), 70.82 (glucose-II C-4′), 63.01 (glucose-II C-6′), 62.98 (glucose-I C-6). Isorugosin I (2): light-brown amorphous powder; [α]25D +36 (c 0.5, MeOH); ECD (MeOH) [θ] (nm) +2.9 × 105 (226), +1.6 × 105 (241), −8.3 × 104 (260), +8.2 × 104 (282); ESIMS m/z 2676 [M + NH4]+; anal. C 47.94%, H 3.33%, calcd for C116H82O74·13H2O, C 48.14%, H 3.76%; 1H NMR (acetone-d6/D2O = 9/1, 600 MHz) δ 7.18 (1H, s, valoneoyl-II-HC), 7.16 (1H, s, valoneoyl-I-HC), 7.10, 7.08, 7.01, 6.97, 6.89, 6.88 (each 2H, s, galloyl-H), 6.70 (HHDP-H-3′), 6.63 (1H, s, valoneoyl-II-HA), 6.62 (2H, s, HHDP-H-3, valoneoyl-I-HA), 6.16 (1H, s, valoneoyl-II-HB), 6.15 (1H, s, valoneoyl-I-HB), and glucose protons (Table 2); 13C NMR (acetone-d6/D2O = 9/1, 151 MHz) δ 168.6 (HHDP C-7), 168.4 (valoneoyl-I C-7), 168.3 (valoneoyl-II C7), 168.2 (HHDP C-7′), 167.69 (valoneoyl-I C-7′), 167.66 (valoneoylII C-7′), 166.7, 166.3, 166.1 (3C), 165.3 (galloyl C-7), 162.9 (valoneoyl-I C-7″), 162.8 (valoneoyl-II C-7″), 146.4 (valoneoyl-II C4′), 146.1 (valoneoyl-I C-4′), 145.90, 145.88, 145,85, 145.49, 145.48 (12C, galloyl C-3, 5), 145.24, 145.16, 145.1, 145.0 (valoneoyl-I, II C-4, HHDP C-4, 4′), 144.7, 144.59, 144.57, 144.5, 144.3, 144.2 (valoneoylI, II C-6, 6′, HHDP C-6, 6′), 143.13 (valoneoyl-II C-2″), 143.10 (valoneoyl-I C-2″), 141.3, 141.1 (valoneoyl-I, II C-4″), 140.5 (2C, valoneoyl-I, II C-5″), 139.9, 139.5, 139.4, 139.3, 139.04, 139.03 (galloyl C-4), 137.90, 137.86 (valoneoyl-I, II C-3″), 137.1 (2C, valoneoyl-I, II C-5′), 136.5 (HHDP C-5), 136.4 (2C, valoneoyl-II C-5, HHDP C-5′), 136.1 (valoneoyl-I, C-5), 126.4, 126.0, 125.7, 125.6, 125.31, 125.28 (valoneoyl-I, II, HHDP C-1, 1′), 120.5, 120.1 (2C), 119.85, 119.76, 119.3 (galloyl C-1), 117.74 (valoneoyl-I C-2′), 117.70 (valoneoyl-II C-2′), 116.2, 115.83 (HHDP C-2, 2′), 115.82 (valoneoyl-II C-2), 115.7 (valoneoyl-I C-2), 112.7, 112.4 (valoneoylI, II C-1″), 110.3, 110.2, 110.1, 110.02, 109.98, 109.94 (12C, valoneoyl-I, II C-6″, galloyl C-2, 6), 108.2 (HHDP C-3′), 107.98, 107.95, 107.8 (valoneoyl-I, II C-3′, HHDP C-3), 105.1, 105.0 (valoneoyl-I, II C-3), and glucose carbons (Table 2). Isorugosin J (3): light-brown amorphous powder; [α]25D +42 (c 1.0, MeOH); ECD (MeOH) [θ] (nm) +4.1 × 105 (224), +1.2 × 105 (240), −1.2 × 104 (260), +1.4 × 104 (282); ESIMS m/z 2996 [M + NH4]+; anal. C 50.01%, H 3.08%, calcd for C130H90O83·7H2O, C 50.27%, H 3.37%; 1H NMR (acetone-d6/D2O = 9/1, 600 MHz) δ 7.21 (1H, s, valoneoyl-I-HC), 7.19, 7.17 (each 1H, s, valoneoyl-II, III-HC), 7.09, 7.013, 7.008, 7.002, 6.97, 6.891, 6.886, 6.884 (each 2H, s, galloylH), 6.64, 6.63, 6.62 (each 1H, s, valoneoyl-I, II, III-HA), 6.24, 6.16, 6.15 (each 1H, s, valoneoyl-I, II, III-HB), and glucose protons (Table 2); 13C NMR (acetone-d6/D2O = 9/1, 151 MHz) δ 168.32, 168.27 (2C) (valoneoyl-I, II, III C-7), 167.7, 167.64, 167.5 (valoneoyl-I, II, III C-7′), 167.59 (valoneoyl-I C-7″), 166.2 (2C), 166.15, 166.08 (2C), 166.0, 165.3 (galloyl C-7), 162.74, 162.69 (valoneoyl-II, III C-7″), 147.0 (valoneoyl-I C-4′), 146.40, 146.38 (valoneoyl-II, III C-4′), 146.1 (2C), 145.90 (4C), 145.86 (2C), 145.53 (2C), 145.50 (4C) (galloyl C-3, 5), 145.25, 145.18, 145.16 (valoneoyl-I, II, III C-4), 144.72, 144.67, 144.61, 144.60, 144.57, 144.50 (valoneoyl-I, II C-6, 6′), 143.14, 143.12, 143.10 (valoneoyl-I, II, III C-2″), 141.23, 141.16 140.58, 140.57 140.11, 140.07, 139.9, 139.5 (2C), 139.4, 139.1, 139.0 (2C) (valoneoyl-I, II, III C-4″, 5″, galloyl C-4), 137.9 (2C), 137.4 (valoneoyl-I, II, III C-3″), 137.2 (valoneoyl-I C-5′), 137.1 (2C, valoneoyl-II, III C-5′), 136.5, 136.4 (2C) (valoneoyl-I, II, III C-5), 125.8, 125.7 (2C), 125.5, 125.4, 125.3 (valoneoyl-I, II, III C-1, 1′), 120.2 (3C), 120.0, 119.8 (2C), 119.4 (galloyl C-1), 117.7 (2C), 117.6 (valoneoyl-I, II, III C-2′), 115.93, 115.87, 115.86 (valoneoyl-I, II, III C-2), 115.2 (valoneoyl-I C-1″), 112.6, 112.5 (valoneoyl-II, III C-1″), 110.28, 110.25, 110.17, 110.1, 110.0 (17C, valoneoyl-I, II, III C-6″, 2731
DOI: 10.1021/acs.jnatprod.7b00496 J. Nat. Prod. 2017, 80, 2723−2733
Journal of Natural Products galloyl C-2, 6,), 108.1, 108.0, 107.8 (valoneoyl-I, II, III C-3), and glucose carbons (Table 2). Antibacterial Assay. In this study, two strains of MRSA, OM481 and OM584, clinical isolates from Okayama University Hospital that were stored at the Department of Microbiology, were used. Pseudomonas aeruginosa PAO1 was also used in this study. The bacterial cells were precultured in Mueller−Hinton broth at 37 °C under aerobic conditions. They were incubated in the presence of compounds with the concentrations obtained by 2-fold serial dilution at 37 °C without shaking in the same broth for 24 h on 96-well plates as described in a previous paper.30 The lowest concentration among the tested samples at which the visible growth was completely inhibited was regarded as the MIC. MICs are given based on triplicate experiments. DMSO was used for dissolving compounds, and the final concentrations were set at
