Note pubs.acs.org/jnp
Tetranorsesquiterpenoids and Santalane-Type Sesquiterpenoids from Illicium lanceolatum and Their Antimicrobial Activity against the Oral Pathogen Porphyromonas gingivalis Miwa Kubo,*,† Yuri Nishikawa,† Kenichi Harada,† Masataka Oda,‡ Jian-Mei Huang,§ Hisanori Domon,‡ Yutaka Terao,‡ and Yoshiyasu Fukuyama*,† †
Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan Division of Microbiology and Infectious Diseases, Niigata University Graduate School of Medical and Dental Sciences, 2-5274, Gakkocho-dori, Chuo-ku, Niigata 951-8514, Japan § School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, People’s Republic of China ‡
S Supporting Information *
ABSTRACT: The methanol extract of the leaves of Illicium lanceolatum, indigenous to Fujian Province, People’s Republic of China, was found to exhibit antimicrobial activity against the periodontal pathogen Porphyromonas gingivalis, and a bioassayguided fractionation led to the isolation of two new compounds, 1 and 2, along with two known santalane-type sesquiterpenoids, 3 and 4. The structures of lanceolactone A (1) and lanceolactone B (2) were elucidated by analyzing their 2D NMR spectroscopic data. Compounds 1 and 2 were assigned as new tetranorsesquiterpenoids with a spiroacetal ring and tricyclic structure, respectively. Compound 3 (α-santal-11en-10-one) showed potent antimicrobial activity against the oral pathogen P. gingivalis.
D
ental caries and periodontal diseases are oral infections that affect a large proportion of the global population.
Table 1. NMR Spectroscopic Data (600 MHz, CDCl3) of Lanceolactones A (1) and B (2) lanceolactone A (1) position
δC
1 2 3 4 5 6 7 8
170.0 119.4 163.9 115.2 34.3 36.1 87.5 142.2
9
112.2
10 11
27.7 12.6
δH (J in Hz) 5.86 q (1.4)
2.08, m; 2.21, m 2.18, m; 2.25, m 5.92, dd, (10.7, 1.1) 5.06, dd (10.7, 1.1) 1.52, s 2.05, d (1.4)
lanceolactone B (2) δC 39.2 84.5 45.7 29.4 28.1 46.0 46.4 38.4
δH (J in Hz) 1.78, m; 1.84, m 4.13, dd (6.7, 1.4)
Figure 1. Key (A) 1H−1H COSY and HMBC and (B) NOESY correlations of lanceolactone A (1).
1.29, m; 1.78, m 1.13, m; 1.77, m 1.96, brdd (4.4, 4.4) 2.36, d (19.7); 2.65, d (19.7)
172.3 17.4 12.4
0.92, s 1.02, s
Figure 2. Key (A) 1H−1H COSY and HMBC and (B) NOESY correlations of lanceolactone B (2).
The cariogenic organism Streptococcus mutans is considered the principal etiological agent in the formation of dental caries due to its aciduric, acidogenic, and adhesion properties.1 The periodontopathogenic bacterium Porphyromonas gingivalis has been identified as a key pathogen contributing to chronic periodontitis,2 as the establishment of P. gingivalis in subgingival sites induces an inflammatory response that leads to gingival © XXXX American Chemical Society and American Society of Pharmacognosy
Received: March 19, 2015
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DOI: 10.1021/acs.jnatprod.5b00237 J. Nat. Prod. XXXX, XXX, XXX−XXX
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tissue destruction and the progressive loss of alveolar bone around the teeth.3 Hence, discovering new growth inhibitors of P. gingivalis is of potential importance. Our continuing studies on Illicium species have resulted in the isolation of sesquiterpenoids with neurotrophic properties,4 such as merrilactone A5 and jiadifenin6 and jiadifenolide7 from I. merrillianum and I. jiadifengpi, respectively. Illicium lanceolatum A. C. Sm. (Illiciaceae) is a medicinal plant of the genus Illicium with the Chinese name “Mangcao” or “Hongduhui”. Its roots and leaves have been shown to have anti-inflammatory and analgesic activities and are used in the treatment of bruises, internal injuries, and back pain.8 In the course of research to discover natural products with antimicrobial activity, a MeOH extract of the leaves of I. lanceolatum was found to exhibit antimicrobial activity against P. gingivalis. Bioassay-guided fractionation resulted in the isolation of two new tetranorsesquiterpenoids, compounds 1 and 2, along with two known compounds, 3 and 4. In this paper, we report the structures of 1 and 2 and antimicrobial activities of all four isolated compounds. The MeOH extract of the dried leaves of I. lanceolatum was fractionated by passage over silica gel and Sephadex LH-20 and finally purified by reversed-phase HPLC, leading to the
Scheme 1. Plausible Biosynthesis of 1 and 2
Figure 3. Effects of compounds 1−4 on the growth of P. gingivalis (panel A), S. mutans (panel B), S. mitis (panel C), S. oralis (panel D), and S. sobrinus (panel E). Data are the means ± standard deviations of triplicate assays (**, p < 0.005; *, p < 0.01 significantly different from the control with no compounds). B
DOI: 10.1021/acs.jnatprod.5b00237 J. Nat. Prod. XXXX, XXX, XXX−XXX
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1) showed signals that were assigned to two tertiary methyl groups (δH 0.92, 1.02), three methylenes [δH 1.13 (m), 1.77 (m); δC 28.1 (C-5); δH 1.29 (m), 1.78 (m); δC 29.4 (C-4); δH 1.78 (m), 1.84 (m); δC 39.2 (C-1)], a methine [δH 1.96 (brdd, J = 4.4, 4.4 Hz); δC 45.9 (C-6)], an oxymethine [δH 4.13 (dd, J = 6.7, 1.4 Hz); δC 84.5 (C-2)], and an isolated methylene [δH 2.36 (d, J = 19.7 Hz), 2.65 (d, J = 19.7 Hz); δC 38.4 (C-8)]. The 13C NMR spectrum and the molecular formula indicated that 2 is also a tetranorsesquiterpenoid. Analysis of 1H−1H COSY, HMQC, and HMBC NMR experiments was carried out (Figure 2A). The HMBC correlations of H3-10 to C-7 and H3-11 to C-3 indicated that the methyl groups are connected to C-7 and C-3, respectively. The HMBC correlations of H-1 and H-2 to C-3, H-6 and H-1 to C-7, and H3-11 to C-4 led to the formation of a bicyclo[2.2.1]heptane framework. The HMBC correlations of H3-10 with C-8 and H-8 with C-3 and C-7 indicated that C-8 is connected to the C-7 quaternary carbon. In addition, the ester carbonyl signal at δC 172.4 (C-9) showed HMBC correlations with H-2 and H-8. On considering its four degrees of unsaturation, 2 was assumed to have a tricyclic carbon skeleton. The data obtained allowed C-8 and C-9 to be connected through an oxygen atom, resulting in the construction of a δlactone ring. Thus, the spectroscopic values described above were used to propose the planar structure of 2. The relative configuration of this compound was elucidated based on the NOESY data, as shown in Figure 2B. The proposed tricyclic framework automatically fixed the stereochemistry of C-2, C-3, C-6, and C-7. In fact, the NOESY correlations suggested the proposed stereochemistry. The absolute configuration was determined according to the sector rule.11 The CD spectrum of this compound showed a negative Cotton effect (Δε −0.77) at 216 nm, and the absolute configuration was assigned as shown. Thus, compound 2 (lanceolactone B) was determined also as a unique tricyclic tetranorsesquiterpenoid. The following two plausible biosynthetic routes were proposed for 1 and 2: nerolidol would be oxidized to a, which would undergo an oxidative cleavage, leading to the tetranor compound b, and then acetal formation between C-4 and C-7 and a sequential lactonization would give rise to 1. On the other hand, isocampherenol would be oxidized to give rise to c, which would undergo a sequential oxidative cleavage to give rise to d, and then would undergo lactonization leading to 2 (Scheme 1). Compounds 1−4 were evaluated for their antibacterial activities against the periodontopathogenic P. gingivalis and the cariogenic S. mutans, Streptococcus mitis, Streptococcus sobrinus, and Streptococcus oralis at concentrations between 5 and 20 μg/mL (Figure 3). While compounds 1−4 had no effects on the growth of S. mitis, S. oralis, or S. sobrinus, compound 3 markedly inhibited the growth of P. gingivalis and S. mutans at 10−20 and 20 μg/mL, respectively. Compound 4 was inactive against P. gingivalis and S. mutans. These results supported the unsaturated carbonyl group as being important for the activity of the santalane-type sesquiterpenoid against P. gingivalis and S. mutans. In conclusion, two new tetranorsesquiterpenoids, 1 and 2, and two previously known santalane-type sesquiterpenoids, 3 and 4, were isolated from the leaves of I. lanceolatum. Among these compounds, compound 3 showed potent antimicrobial activity against the oral pathogen P. gingivalis and S. mutans. Futher studies are warranted to confirm the ability of products containing compound 3, such as mouthwash, toothpaste, and
isolation of new compounds 1 and 2 along with the previously known compounds α -santal-11-en-10-one (3)9 and 11hydroxy-α-santal-9-ene (4).9
Compound 1, [α]D 46.4 (c 0.03, CHCl3), gave the molecular formula C11H14O3, as deduced from the HRCIMS at m/z 195.1016 [M + H]+, which suggested the presence of five degrees of unsaturation. The IR spectrum displayed an absorption due to a γ-lactone moiety at 1765 cm−1. The 1H and 13C NMR data of 1 (Table 1) indicated the presence of a tertiary methyl group (δH 1.52), a vinyl methyl group [δH 2.05 (d, J = 1.4 Hz)], two methylenes [δH 2.08 (m), 2.21 (m); δC 34.3 (C-5); δH 2.18 (m), 2.25 (m); δC 36.1 (C-6)], a trisubstituted olefin [δH 5.86 (q, J = 1.4 Hz); δC 119.4 (C-2)], and a vinyl group [δH 5.06 (dd, J = 10.7, 1.1 Hz), 5.25 (dd, J = 17.4, 1.1 Hz); δC 112.2 (C-9); δH 5.92 (dd, J = 17.4, 10.7 Hz); δC 142.2 (C-8)]. The 13C NMR spectrum of 1 showed 11 carbon signals. Furthermore, its molecular formula indicated that the carbon number of 1 was four less than that of normal sesquiterpenoids, implying that 1 is a tetranorsesquiterpenoid. As summarized in Figure 1, 1H−1H COSY, HMQC, and HMBC NMR experiments were carried out. The 1H−1H COSY and HMQC spectra for 1 indicated the presence of three partial structures, as depicted in Figure 1 by bold lines, and four quaternary carbons at δC 87.5, 115.2, 163.9, and 170.0. Connectivities between these partial structures and the quaternary carbons were established by the HMBC experiment. The HMBC correlations of H3-10 and H-9 to C-7 indicated that the methyl (C-10) and vinyl groups were connected to C7. The ester carbon signal (δC 170.0) showed HMBC correlations with H-2 and the HMBC correlations of H-2/C3 and C-4 and H3-11/C-2 and C-3, thereby allowing a γ-lactone ring to be formed. The HMBC correlations of H-5/C-4 and H6/C-7 revealed the presence of a tetrahydrofuran ring. In addition, the five degrees of unsaturation and the above spectroscopic data disclosed that 1 has a spiroacetal ring fused at the C-4 position. Thus, the planar structure 1 could be proposed accordingly. The relative configuration of 1 was elucidated from the NOESY data, as shown in Figure 1B. Thus, H-9 showed a cross-peak to H3-11, indicating that the carbonyl of the spirolactone occurs in an exo orientation with the methyl group at C-7 in a β-configuration. The absolute configuration of 1 was determined by the CD exciton chirality method,10 and the CD spectrum showed a positive n−π* Cotton effect (Δε +1.38) at 246 nm and a negative π−π* Cotton effect (Δε −1.69) at 219 nm. Thus, compound 1, named lanceolactone A, was determined to have a unique tetranorsesquiterpenoid structure, as shown. Compound 2, [α]D −55.6 (c 0.02, CHCl3), gave the molecular formula C11H16O2, as deduced by HREIMS. Its IR spectrum showed absorption bands at 1736 cm−1, ascribable to a δ-lactone moiety. The 1H and 13C NMR spectra of 2 (Table C
DOI: 10.1021/acs.jnatprod.5b00237 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Tesque). The degree of turbidity in the broth culture was measured at an absorbance of 620 nm using a microplate reader (Thermo Scientific). Statistical Analysis. Data of the bioassay were evaluated using Student’s t test with Graph Pad Prism statistical software (Graph Pad Software, Inc., La Jolla, CA, USA), and p < 0.01 was considered statistically significant.
gum, to alter the composition of oral microflora in humans, thereby improving oral health.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations ([α]D) were obtained on a JASCO P-1030 digital polarimeter. IR and UV spectra were recorded on a JASCO FT-IR 410 infrared and Shimadzu UV-1650PC spectrophotometer, respectively. High-resolution mass spectrometric measurements were acquired on a MStation JMS-700. NMR experiments were performed on a Varian Unity and JEOL 600 or 500 MHz NMR spectrometer. 1H and 13C spectra were referenced to peaks of TMS. Silica gel column chromatography (CC) was carried out on Wako C-300 and Kiselgel 60 (70−230 and 230−400 mesh). HPLC was performed on a JASCO PU-2080 equipped with a JASCO RI-2031 and a UV-2075 detector. Plant Material. The leaves of Illicium lanceolatum were collected in Wuyishan, Fujian Province, People’s Republic of China, in September 2004, identified by one of the authors (J.-M.H.), and a voucher specimen (1798LF) has been deposited at Tokushima Bunri University. Extraction and Isolation. The dried leaves of I. lanceolatum (1.0 kg) were powdered and extracted with MeOH at room temperature to give 500 g of extract. The MeOH extract (371.6 g) of I. lanceolatum, which was mixed with Celite (371.6 g) and the solvent, was completely removed in vacuo to give a solid, which was then pulverized. The resultant powder was packed into a glass column and then eluted in turn with n-hexane, CH2Cl2, EtOAc, and MeOH to give four fractions (1−4). Fraction 2 (48.6 g) initially was first subjected to silica gel chromatography eluted with n-hexane−EtOAc (4:1) to give fractions 5−14. Fraction 11 (1.6 g) was subjected to silica gel chromatography eluted with CH2Cl2−EtOAc (10:0.3 → 4:1) to give fractions 15−23. Fraction 18 (66.3 mg) was subjected to reversed-phase chromatography eluted with CH3CN−H2O (9:11) to give fractions 24−26. Fraction 25 (37.3 mg) was purified by reversed-phase HPLC (Cosmosil 5C18MS-II, ⦶ 10 × 250 mm) using MeOH−H2O (3:2) to give lanceolactone A (1) (5.6 mg) (tR 14.4 min) and lanceolactone B (2) (3.7 mg) (tR 17.2 min). Fraction 6 (1.7 g) was subjected to silica gel chromatography eluted with n-hexane−toluene (7:3) to give fractions 27−35. Fraction 30 (126.8 mg) was purified by silica gel chromatography eluting with n-hexane−toluene (7:3) to give 11hydroxy-α-santal-9-ene (4) (8.5 mg) and α-santal-11-en-10-one (3) (24.1 mg). Lanceolactone A (1): colorless oil; [α]D 46.4 (c 0.03, CHCl3); UV (EtOH) λmax (log ε) 226 (2.13), 203 (2.64) nm; CD (1.9 × 10−3 M, EtOH) λmax (Δε) 219 (−1.69), 246 (+1.38) nm; IR (ATR) νmax 1765 cm−1; 1H and 13C NMR data, see Table 1; HRCIMS m/z 195.1016 [M + H]+ (calcd for C11H15O3, 195.1021). Lanceolactone B (2): colorless oil; [α]D −55.6 (c 0.02, CHCl3); UV (EtOH) λmax (log ε) 205 (2.63) nm; CD (1.7 × 10−3 M, EtOH) λmax (Δε) 216 (−0.77) nm; IR (ATR) νmax 1736 cm−1; 1H and 13C NMR data, see Table 1; HREIMS m/z 180.1163 [M]+ (calcd for C11H16O2, 180.1150). Bioassays. P. gingivalis (ATCC33277) was incubated anaerobically at 37 °C to an optical density of 1.0 at 620 nm. Precultured P. gingivalis (5 μL) was incubated anaerobically with various concentrations of compounds 1−4 and in 200 μL of Gifu anaerobic medium (GAM, Nissui, Tokyo, Japan) at 37 °C for 100 h in a 96-well plate (BD Falcon, Franklin Lakes, NJ, USA). Compounds 1−4 and the MeOH extract of I. lanceolateum were dissolved in ethanol (Nacalai Tesque, Kyoto, Japan). The degree of turbidity in the broth culture was measured at an absorbance of 620 nm using a microplate reader (Thermo Scientific, Waltham, MA, USA). S. mutans (MT8148), S. mitis (ATCC903), S. sobrinus (MT10186), and S. oralis (SK23) were incubated at 37 °C to an optical density of 1.0 at 620 nm. Precultured S. mitis, S. sobrinus, and S. oralis (5 μL) were incubated with various concentrations of compounds 1−4 and in 200 μL of brain heart infusion medium (BHI, Gibco) at 37 °C for 24 h in a 96-well plate (BD Falcon). Compounds were dissolved in ethanol (Nacalai
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ASSOCIATED CONTENT
S Supporting Information *
1
H and 13C NMR, COSY, HMQC, HMBC, and NOESY spectra of 1 and 2. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.jnatprod.5b00237.
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AUTHOR INFORMATION
Corresponding Authors
*Phone: +81 (88) 602-8436. E-mail: [email protected] (M. Kubo). *E-mail: [email protected] (Y. Fukuyama). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Dr. M. Tanaka and Dr. Y. Okamoto (TBU) for measuring the 600 MHz NMR spectroscopic and mass spectrometric data.
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REFERENCES
(1) Takahashi, N.; Nyvad, B. J. Dent. Res. 2011, 90, 294−303. (2) Feng, Z.; Weinberg, A. Periodontology 2000 2006, 40, 50−76. (3) Liu, Y. C.; Lerner, U. H.; Teng, Y. T. Periodontology 2000 2010, 52, 163−206. (4) Fukuyama, Y.; Huang, J.-M. Studies in Natural Products Chemistry; Rahman, A., Ed.; Elsevier: Amsterdam, 2005; Vol. 32, pp 395−429. (5) Huang, J.-M.; Yokoyama, R.; Yang, C.-S.; Fukuyama, Y. Tetrahedron Lett. 2000, 41, 6111−6114. (6) Yokoyama, R.; Huang, J.-M.; Yang, C.-S.; Fukuyama, Y. J. Nat. Prod. 2002, 65, 527−531. (7) Kubo, M.; Okada, C.; Huang, J.-M.; Harada, K.; Hioki, H.; Fukuyama, Y. Org. Lett. 2009, 11, 5190−5193. (8) Liang, J.; Huang, B. K.; Wang, G. W. Nat. Prod. Res. 2012, 26, 1712−1714. (9) Ngo, K.-S.; Brown, G. D. Phytochemistry 1999, 50, 1213−1218. (10) Gawronski, J. K.; Van Oeveren, A.; Van der Deen, H.; Leung, C. W.; Feringa, B. L. J. Org. Chem. 1996, 61, 1513−1515. (11) Jennings, J. P.; Klyne, W.; Scopes, P. M. J. Chem. Soc. 1965, 7211−7227.
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DOI: 10.1021/acs.jnatprod.5b00237 J. Nat. Prod. XXXX, XXX, XXX−XXX
Tetranorsesquiterpenoids and Santalane-Type Sesquiterpenoids from Illicium lanceolatum and Their Antimicrobial Activity against the Oral Pathogen Porphyromonas gingivalis Miwa Kubo,*,† Yuri Nishikawa,† Kenichi Harada,† Masataka Oda,‡ Jian-Mei Huang,§ Hisanori Domon,‡ Yutaka Terao,‡ and Yoshiyasu Fukuyama*,† †
Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan Division of Microbiology and Infectious Diseases, Niigata University Graduate School of Medical and Dental Sciences, 2-5274, Gakkocho-dori, Chuo-ku, Niigata 951-8514, Japan § School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, People’s Republic of China ‡
S Supporting Information *
ABSTRACT: The methanol extract of the leaves of Illicium lanceolatum, indigenous to Fujian Province, People’s Republic of China, was found to exhibit antimicrobial activity against the periodontal pathogen Porphyromonas gingivalis, and a bioassayguided fractionation led to the isolation of two new compounds, 1 and 2, along with two known santalane-type sesquiterpenoids, 3 and 4. The structures of lanceolactone A (1) and lanceolactone B (2) were elucidated by analyzing their 2D NMR spectroscopic data. Compounds 1 and 2 were assigned as new tetranorsesquiterpenoids with a spiroacetal ring and tricyclic structure, respectively. Compound 3 (α-santal-11en-10-one) showed potent antimicrobial activity against the oral pathogen P. gingivalis.
D
ental caries and periodontal diseases are oral infections that affect a large proportion of the global population.
Table 1. NMR Spectroscopic Data (600 MHz, CDCl3) of Lanceolactones A (1) and B (2) lanceolactone A (1) position
δC
1 2 3 4 5 6 7 8
170.0 119.4 163.9 115.2 34.3 36.1 87.5 142.2
9
112.2
10 11
27.7 12.6
δH (J in Hz) 5.86 q (1.4)
2.08, m; 2.21, m 2.18, m; 2.25, m 5.92, dd, (10.7, 1.1) 5.06, dd (10.7, 1.1) 1.52, s 2.05, d (1.4)
lanceolactone B (2) δC 39.2 84.5 45.7 29.4 28.1 46.0 46.4 38.4
δH (J in Hz) 1.78, m; 1.84, m 4.13, dd (6.7, 1.4)
Figure 1. Key (A) 1H−1H COSY and HMBC and (B) NOESY correlations of lanceolactone A (1).
1.29, m; 1.78, m 1.13, m; 1.77, m 1.96, brdd (4.4, 4.4) 2.36, d (19.7); 2.65, d (19.7)
172.3 17.4 12.4
0.92, s 1.02, s
Figure 2. Key (A) 1H−1H COSY and HMBC and (B) NOESY correlations of lanceolactone B (2).
The cariogenic organism Streptococcus mutans is considered the principal etiological agent in the formation of dental caries due to its aciduric, acidogenic, and adhesion properties.1 The periodontopathogenic bacterium Porphyromonas gingivalis has been identified as a key pathogen contributing to chronic periodontitis,2 as the establishment of P. gingivalis in subgingival sites induces an inflammatory response that leads to gingival © XXXX American Chemical Society and American Society of Pharmacognosy
Received: March 19, 2015
A
DOI: 10.1021/acs.jnatprod.5b00237 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
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tissue destruction and the progressive loss of alveolar bone around the teeth.3 Hence, discovering new growth inhibitors of P. gingivalis is of potential importance. Our continuing studies on Illicium species have resulted in the isolation of sesquiterpenoids with neurotrophic properties,4 such as merrilactone A5 and jiadifenin6 and jiadifenolide7 from I. merrillianum and I. jiadifengpi, respectively. Illicium lanceolatum A. C. Sm. (Illiciaceae) is a medicinal plant of the genus Illicium with the Chinese name “Mangcao” or “Hongduhui”. Its roots and leaves have been shown to have anti-inflammatory and analgesic activities and are used in the treatment of bruises, internal injuries, and back pain.8 In the course of research to discover natural products with antimicrobial activity, a MeOH extract of the leaves of I. lanceolatum was found to exhibit antimicrobial activity against P. gingivalis. Bioassay-guided fractionation resulted in the isolation of two new tetranorsesquiterpenoids, compounds 1 and 2, along with two known compounds, 3 and 4. In this paper, we report the structures of 1 and 2 and antimicrobial activities of all four isolated compounds. The MeOH extract of the dried leaves of I. lanceolatum was fractionated by passage over silica gel and Sephadex LH-20 and finally purified by reversed-phase HPLC, leading to the
Scheme 1. Plausible Biosynthesis of 1 and 2
Figure 3. Effects of compounds 1−4 on the growth of P. gingivalis (panel A), S. mutans (panel B), S. mitis (panel C), S. oralis (panel D), and S. sobrinus (panel E). Data are the means ± standard deviations of triplicate assays (**, p < 0.005; *, p < 0.01 significantly different from the control with no compounds). B
DOI: 10.1021/acs.jnatprod.5b00237 J. Nat. Prod. XXXX, XXX, XXX−XXX
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1) showed signals that were assigned to two tertiary methyl groups (δH 0.92, 1.02), three methylenes [δH 1.13 (m), 1.77 (m); δC 28.1 (C-5); δH 1.29 (m), 1.78 (m); δC 29.4 (C-4); δH 1.78 (m), 1.84 (m); δC 39.2 (C-1)], a methine [δH 1.96 (brdd, J = 4.4, 4.4 Hz); δC 45.9 (C-6)], an oxymethine [δH 4.13 (dd, J = 6.7, 1.4 Hz); δC 84.5 (C-2)], and an isolated methylene [δH 2.36 (d, J = 19.7 Hz), 2.65 (d, J = 19.7 Hz); δC 38.4 (C-8)]. The 13C NMR spectrum and the molecular formula indicated that 2 is also a tetranorsesquiterpenoid. Analysis of 1H−1H COSY, HMQC, and HMBC NMR experiments was carried out (Figure 2A). The HMBC correlations of H3-10 to C-7 and H3-11 to C-3 indicated that the methyl groups are connected to C-7 and C-3, respectively. The HMBC correlations of H-1 and H-2 to C-3, H-6 and H-1 to C-7, and H3-11 to C-4 led to the formation of a bicyclo[2.2.1]heptane framework. The HMBC correlations of H3-10 with C-8 and H-8 with C-3 and C-7 indicated that C-8 is connected to the C-7 quaternary carbon. In addition, the ester carbonyl signal at δC 172.4 (C-9) showed HMBC correlations with H-2 and H-8. On considering its four degrees of unsaturation, 2 was assumed to have a tricyclic carbon skeleton. The data obtained allowed C-8 and C-9 to be connected through an oxygen atom, resulting in the construction of a δlactone ring. Thus, the spectroscopic values described above were used to propose the planar structure of 2. The relative configuration of this compound was elucidated based on the NOESY data, as shown in Figure 2B. The proposed tricyclic framework automatically fixed the stereochemistry of C-2, C-3, C-6, and C-7. In fact, the NOESY correlations suggested the proposed stereochemistry. The absolute configuration was determined according to the sector rule.11 The CD spectrum of this compound showed a negative Cotton effect (Δε −0.77) at 216 nm, and the absolute configuration was assigned as shown. Thus, compound 2 (lanceolactone B) was determined also as a unique tricyclic tetranorsesquiterpenoid. The following two plausible biosynthetic routes were proposed for 1 and 2: nerolidol would be oxidized to a, which would undergo an oxidative cleavage, leading to the tetranor compound b, and then acetal formation between C-4 and C-7 and a sequential lactonization would give rise to 1. On the other hand, isocampherenol would be oxidized to give rise to c, which would undergo a sequential oxidative cleavage to give rise to d, and then would undergo lactonization leading to 2 (Scheme 1). Compounds 1−4 were evaluated for their antibacterial activities against the periodontopathogenic P. gingivalis and the cariogenic S. mutans, Streptococcus mitis, Streptococcus sobrinus, and Streptococcus oralis at concentrations between 5 and 20 μg/mL (Figure 3). While compounds 1−4 had no effects on the growth of S. mitis, S. oralis, or S. sobrinus, compound 3 markedly inhibited the growth of P. gingivalis and S. mutans at 10−20 and 20 μg/mL, respectively. Compound 4 was inactive against P. gingivalis and S. mutans. These results supported the unsaturated carbonyl group as being important for the activity of the santalane-type sesquiterpenoid against P. gingivalis and S. mutans. In conclusion, two new tetranorsesquiterpenoids, 1 and 2, and two previously known santalane-type sesquiterpenoids, 3 and 4, were isolated from the leaves of I. lanceolatum. Among these compounds, compound 3 showed potent antimicrobial activity against the oral pathogen P. gingivalis and S. mutans. Futher studies are warranted to confirm the ability of products containing compound 3, such as mouthwash, toothpaste, and
isolation of new compounds 1 and 2 along with the previously known compounds α -santal-11-en-10-one (3)9 and 11hydroxy-α-santal-9-ene (4).9
Compound 1, [α]D 46.4 (c 0.03, CHCl3), gave the molecular formula C11H14O3, as deduced from the HRCIMS at m/z 195.1016 [M + H]+, which suggested the presence of five degrees of unsaturation. The IR spectrum displayed an absorption due to a γ-lactone moiety at 1765 cm−1. The 1H and 13C NMR data of 1 (Table 1) indicated the presence of a tertiary methyl group (δH 1.52), a vinyl methyl group [δH 2.05 (d, J = 1.4 Hz)], two methylenes [δH 2.08 (m), 2.21 (m); δC 34.3 (C-5); δH 2.18 (m), 2.25 (m); δC 36.1 (C-6)], a trisubstituted olefin [δH 5.86 (q, J = 1.4 Hz); δC 119.4 (C-2)], and a vinyl group [δH 5.06 (dd, J = 10.7, 1.1 Hz), 5.25 (dd, J = 17.4, 1.1 Hz); δC 112.2 (C-9); δH 5.92 (dd, J = 17.4, 10.7 Hz); δC 142.2 (C-8)]. The 13C NMR spectrum of 1 showed 11 carbon signals. Furthermore, its molecular formula indicated that the carbon number of 1 was four less than that of normal sesquiterpenoids, implying that 1 is a tetranorsesquiterpenoid. As summarized in Figure 1, 1H−1H COSY, HMQC, and HMBC NMR experiments were carried out. The 1H−1H COSY and HMQC spectra for 1 indicated the presence of three partial structures, as depicted in Figure 1 by bold lines, and four quaternary carbons at δC 87.5, 115.2, 163.9, and 170.0. Connectivities between these partial structures and the quaternary carbons were established by the HMBC experiment. The HMBC correlations of H3-10 and H-9 to C-7 indicated that the methyl (C-10) and vinyl groups were connected to C7. The ester carbon signal (δC 170.0) showed HMBC correlations with H-2 and the HMBC correlations of H-2/C3 and C-4 and H3-11/C-2 and C-3, thereby allowing a γ-lactone ring to be formed. The HMBC correlations of H-5/C-4 and H6/C-7 revealed the presence of a tetrahydrofuran ring. In addition, the five degrees of unsaturation and the above spectroscopic data disclosed that 1 has a spiroacetal ring fused at the C-4 position. Thus, the planar structure 1 could be proposed accordingly. The relative configuration of 1 was elucidated from the NOESY data, as shown in Figure 1B. Thus, H-9 showed a cross-peak to H3-11, indicating that the carbonyl of the spirolactone occurs in an exo orientation with the methyl group at C-7 in a β-configuration. The absolute configuration of 1 was determined by the CD exciton chirality method,10 and the CD spectrum showed a positive n−π* Cotton effect (Δε +1.38) at 246 nm and a negative π−π* Cotton effect (Δε −1.69) at 219 nm. Thus, compound 1, named lanceolactone A, was determined to have a unique tetranorsesquiterpenoid structure, as shown. Compound 2, [α]D −55.6 (c 0.02, CHCl3), gave the molecular formula C11H16O2, as deduced by HREIMS. Its IR spectrum showed absorption bands at 1736 cm−1, ascribable to a δ-lactone moiety. The 1H and 13C NMR spectra of 2 (Table C
DOI: 10.1021/acs.jnatprod.5b00237 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Note
Tesque). The degree of turbidity in the broth culture was measured at an absorbance of 620 nm using a microplate reader (Thermo Scientific). Statistical Analysis. Data of the bioassay were evaluated using Student’s t test with Graph Pad Prism statistical software (Graph Pad Software, Inc., La Jolla, CA, USA), and p < 0.01 was considered statistically significant.
gum, to alter the composition of oral microflora in humans, thereby improving oral health.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations ([α]D) were obtained on a JASCO P-1030 digital polarimeter. IR and UV spectra were recorded on a JASCO FT-IR 410 infrared and Shimadzu UV-1650PC spectrophotometer, respectively. High-resolution mass spectrometric measurements were acquired on a MStation JMS-700. NMR experiments were performed on a Varian Unity and JEOL 600 or 500 MHz NMR spectrometer. 1H and 13C spectra were referenced to peaks of TMS. Silica gel column chromatography (CC) was carried out on Wako C-300 and Kiselgel 60 (70−230 and 230−400 mesh). HPLC was performed on a JASCO PU-2080 equipped with a JASCO RI-2031 and a UV-2075 detector. Plant Material. The leaves of Illicium lanceolatum were collected in Wuyishan, Fujian Province, People’s Republic of China, in September 2004, identified by one of the authors (J.-M.H.), and a voucher specimen (1798LF) has been deposited at Tokushima Bunri University. Extraction and Isolation. The dried leaves of I. lanceolatum (1.0 kg) were powdered and extracted with MeOH at room temperature to give 500 g of extract. The MeOH extract (371.6 g) of I. lanceolatum, which was mixed with Celite (371.6 g) and the solvent, was completely removed in vacuo to give a solid, which was then pulverized. The resultant powder was packed into a glass column and then eluted in turn with n-hexane, CH2Cl2, EtOAc, and MeOH to give four fractions (1−4). Fraction 2 (48.6 g) initially was first subjected to silica gel chromatography eluted with n-hexane−EtOAc (4:1) to give fractions 5−14. Fraction 11 (1.6 g) was subjected to silica gel chromatography eluted with CH2Cl2−EtOAc (10:0.3 → 4:1) to give fractions 15−23. Fraction 18 (66.3 mg) was subjected to reversed-phase chromatography eluted with CH3CN−H2O (9:11) to give fractions 24−26. Fraction 25 (37.3 mg) was purified by reversed-phase HPLC (Cosmosil 5C18MS-II, ⦶ 10 × 250 mm) using MeOH−H2O (3:2) to give lanceolactone A (1) (5.6 mg) (tR 14.4 min) and lanceolactone B (2) (3.7 mg) (tR 17.2 min). Fraction 6 (1.7 g) was subjected to silica gel chromatography eluted with n-hexane−toluene (7:3) to give fractions 27−35. Fraction 30 (126.8 mg) was purified by silica gel chromatography eluting with n-hexane−toluene (7:3) to give 11hydroxy-α-santal-9-ene (4) (8.5 mg) and α-santal-11-en-10-one (3) (24.1 mg). Lanceolactone A (1): colorless oil; [α]D 46.4 (c 0.03, CHCl3); UV (EtOH) λmax (log ε) 226 (2.13), 203 (2.64) nm; CD (1.9 × 10−3 M, EtOH) λmax (Δε) 219 (−1.69), 246 (+1.38) nm; IR (ATR) νmax 1765 cm−1; 1H and 13C NMR data, see Table 1; HRCIMS m/z 195.1016 [M + H]+ (calcd for C11H15O3, 195.1021). Lanceolactone B (2): colorless oil; [α]D −55.6 (c 0.02, CHCl3); UV (EtOH) λmax (log ε) 205 (2.63) nm; CD (1.7 × 10−3 M, EtOH) λmax (Δε) 216 (−0.77) nm; IR (ATR) νmax 1736 cm−1; 1H and 13C NMR data, see Table 1; HREIMS m/z 180.1163 [M]+ (calcd for C11H16O2, 180.1150). Bioassays. P. gingivalis (ATCC33277) was incubated anaerobically at 37 °C to an optical density of 1.0 at 620 nm. Precultured P. gingivalis (5 μL) was incubated anaerobically with various concentrations of compounds 1−4 and in 200 μL of Gifu anaerobic medium (GAM, Nissui, Tokyo, Japan) at 37 °C for 100 h in a 96-well plate (BD Falcon, Franklin Lakes, NJ, USA). Compounds 1−4 and the MeOH extract of I. lanceolateum were dissolved in ethanol (Nacalai Tesque, Kyoto, Japan). The degree of turbidity in the broth culture was measured at an absorbance of 620 nm using a microplate reader (Thermo Scientific, Waltham, MA, USA). S. mutans (MT8148), S. mitis (ATCC903), S. sobrinus (MT10186), and S. oralis (SK23) were incubated at 37 °C to an optical density of 1.0 at 620 nm. Precultured S. mitis, S. sobrinus, and S. oralis (5 μL) were incubated with various concentrations of compounds 1−4 and in 200 μL of brain heart infusion medium (BHI, Gibco) at 37 °C for 24 h in a 96-well plate (BD Falcon). Compounds were dissolved in ethanol (Nacalai
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ASSOCIATED CONTENT
S Supporting Information *
1
H and 13C NMR, COSY, HMQC, HMBC, and NOESY spectra of 1 and 2. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.jnatprod.5b00237.
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AUTHOR INFORMATION
Corresponding Authors
*Phone: +81 (88) 602-8436. E-mail: [email protected] (M. Kubo). *E-mail: [email protected] (Y. Fukuyama). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Dr. M. Tanaka and Dr. Y. Okamoto (TBU) for measuring the 600 MHz NMR spectroscopic and mass spectrometric data.
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REFERENCES
(1) Takahashi, N.; Nyvad, B. J. Dent. Res. 2011, 90, 294−303. (2) Feng, Z.; Weinberg, A. Periodontology 2000 2006, 40, 50−76. (3) Liu, Y. C.; Lerner, U. H.; Teng, Y. T. Periodontology 2000 2010, 52, 163−206. (4) Fukuyama, Y.; Huang, J.-M. Studies in Natural Products Chemistry; Rahman, A., Ed.; Elsevier: Amsterdam, 2005; Vol. 32, pp 395−429. (5) Huang, J.-M.; Yokoyama, R.; Yang, C.-S.; Fukuyama, Y. Tetrahedron Lett. 2000, 41, 6111−6114. (6) Yokoyama, R.; Huang, J.-M.; Yang, C.-S.; Fukuyama, Y. J. Nat. Prod. 2002, 65, 527−531. (7) Kubo, M.; Okada, C.; Huang, J.-M.; Harada, K.; Hioki, H.; Fukuyama, Y. Org. Lett. 2009, 11, 5190−5193. (8) Liang, J.; Huang, B. K.; Wang, G. W. Nat. Prod. Res. 2012, 26, 1712−1714. (9) Ngo, K.-S.; Brown, G. D. Phytochemistry 1999, 50, 1213−1218. (10) Gawronski, J. K.; Van Oeveren, A.; Van der Deen, H.; Leung, C. W.; Feringa, B. L. J. Org. Chem. 1996, 61, 1513−1515. (11) Jennings, J. P.; Klyne, W.; Scopes, P. M. J. Chem. Soc. 1965, 7211−7227.
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DOI: 10.1021/acs.jnatprod.5b00237 J. Nat. Prod. XXXX, XXX, XXX−XXX
