Fitoterapia 95 (2014) 16–21
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Five new sesquiterpenoids from Dalbergia odorifera Hao Wang a,b, Wen-Hua Dong a, Wen-Jian Zuo a, Shuai Liu a, Hui-Min Zhong b, Wen-Li Mei a,⁎, Hao-Fu Dai a,⁎⁎ a Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China b College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, China
a r t i c l e
i n f o
Article history: Received 8 January 2014 Accepted in revised form 21 February 2014 Available online 5 March 2014 Keywords: Dalbergia odorifrea T. Chen Leguminosae Sesquiterpenoids Antibacterial activity
a b s t r a c t Five new sesquiterpenes (1–5) along with ten known ones were isolated from the heartwood of Dalbergia odorifrea T. Chen. Their structures were determined by spectroscopic techniques including MS, UV, IR, 1D and 2D NMR. Bioassay results showed that compounds 1 and 9 had inhibitory effect on Candida albicans, and compound 9 exhibited inhibitory effects on Staphylococcus aureus by paper disk diffusion method. © 2014 Published by Elsevier B.V.
1. Introduction The heartwood of Dalbergia odorifera T. Chen. (Leguminosae) is a traditional Chinese medicine and has been used to treat blood disorder, ischemia, swelling, necrosis and rheumatic pain [1]. It is indigenous to Hainan Province, China. Results of chemical studies showed that D. odorifera mainly contained flavonoids and volatile oil. Previous phytochemical studies of D. odorifera were focused on phenolic components, such as flavonoids, biflavones and their interesting biological properties [2–7]. Those studies revealed that the essential oil of D. odorifera was chiefly composed of trans-nerolidol, β-bisabolene and trans-β-farnesene. In total, less than ten sesquiterpenoids
were examined separately from D. odorifera, and this type of compound did not attract considerable attention. In the previous paper [8], we reported the extraction, analysis and antibacterial activity of the volatile oil. The major components of volatile oil were sesquiterpenoids, and the oil showed activity against Staphylococcus aureus and MRSA. Our continuous study on this drug led to the characterization of fifteen sesquiterpenoids. This paper describes the isolation, structure elucidation and antibacterial activity of five new sesquiterpenoids (1–5) and ten known ones from the heartwood of D. odorifera. 2. Experimental 2.1. General
⁎ Correspondence to: W. Mei, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Xueyuan Road 4, Longhua District, Haikou 571101, Hainan, PR China. Tel./fax: +86 898 6698 7529. ⁎⁎ Correspondence to: H. Dai, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Xueyuan Road 4, Longhua District, Haikou 571101, Hainan, PR China. Tel./fax: +86 898 6696 1869. E-mail addresses: [email protected] (W.-L. Mei), [email protected] (H.-F. Dai).
http://dx.doi.org/10.1016/j.fitote.2014.02.013 0367-326X/© 2014 Published by Elsevier B.V.
Optical rotations were recorded using a Rudolph Autopol III polarimeter (Rudolph, Flanders, America). Melting points were determined on a Beijing Taike X-5 stage apparatus (Beijing Taike Instrument Company, China) and are uncorrected. UV spectra were recorded on a Shimadzu UV-2550 spectrometer (Beckman, America). IR absorptions were obtained on a Nicolet 380 FT-IR instrument (Thermo, America) using KBr pellets. 1H-, 13C- and
H. Wang et al. / Fitoterapia 95 (2014) 16–21
2D-NMR spectra were recorded on Bruker Avance 500 NMR spectrometers (Bruker, Germany), using TMS as an internal standard. HRMS were measured with an API QSTAR Pulsar mass spectrometer (Bruker, Germany). Column chromatography was performed with silica gel (60–80, 200–300 mesh, Qingdao Marine Chemical Co. Ltd., China, and Sephadex LH-20, Merck, Germany). TLC was carried out on silica gel G precoated plates (Qingdao Marine Chemical Co. Ltd.), and spots were detected by spraying with 5% H2SO4 in EtOH followed by heating. Kanamycin sulfate was produced by Sangon Biotech Co., Ltd, fluconazole capsules was produced by Shandong Lvyin Pharmaceutical Co., Ltd. 2.2. Plant material The dried heartwood of D. odorifera was purchased from the Haikou Free Market of Agricultural Products, Hainan Province, China, in October 2010. The specimen was identified by Dr. Xi-Long Zheng of the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural sciences, where a voucher specimen (no. 20101009) has been deposited. 2.3. Extraction and isolation The dried and crushed heartwood of D. odorifera (8.4 kg) was extracted three times with 95% (v/v) ethanol (50 L) at room temperature for three weeks totally. The solution was evaporated under reduced pressure to obtain an extract (0.9 kg). This extract was suspended in distilled water and then partitioned with petroleum ether, ethyl acetate and n-butanol, respectively. The EtOAc-soluble residue (477 g) was submitted to column chromatography (CC) over silica gel (silica H, 3.1 kg, 15 × 50 cm) and eluted with CHCl3–MeOH (v/v, 1:0, 100:1, 50:1, 25:1, 15:1, 10:1, 5:1, 2:1, 1:1 and 0:1, each 8.0 L) of increasing polarity resulting in eighteen fractions (Fr.1–Fr.18). Fr.3 (6.2 g) was subjected to silica gel CC (200–300 mesh, 2.5 × 48 cm, 60 g) with a gradient elution of petroleum ether–Me2CO (1:0, 100:1, 50:1, 25:1, 15:1, 10:1, 3:1 and 0:1, each 1.5 L), resulting in twenty-seven fractions (Fr.3.1– Fr.3.27). Fr.3.3 was separated by Sephadex LH-20 (1.4 × 42 cm) using CHCl3–MeOH (1:1), followed by silica gel eluted with petroleum ether–CHCl3 (7:3), to give 7 and 8 (751.6 mg). Fr.3.6 was separated by Sephadex LH-20 (1.4 × 42 cm, 23 g) using CHCl3–MeOH (1:1), followed by silica gel eluted with petroleum ether–CHCl3 (1:1), to give 1 (20.7 mg). Fr.3.7 was separated by Sephadex LH-20 (1.4 × 42 cm, 23 g) using CHCl3–MeOH (1:1), followed by silica gel eluted with petroleum ether–CHCl3 (1:1), to give 9 (120.6 mg). Fr.3.10 was separated by Sephadex LH-20 (1.4 × 42 cm, 23 g) using CHCl3–MeOH (1:1), followed by silica gel eluted with petroleum ether–CHCl3 (1:1), to give 13 (155.3 mg). Fr.4 (30.2 g) was separated by Sephadex LH-20 (3 × 100 cm) using CHCl3–MeOH (1:1) to afford eight fractions (Fr.4.1–Fr.4.8). Fr.4.1 (4.1 g) was subjected to silica gel CC (200–300 mesh, 2.5 × 48 cm, 60 g) with a gradient elution of petroleum ether-Me2CO (1:0, 100:1, 50:1 and 20:1, each 1.5 L), resulting in twenty fractions (Fr.4.1.1–Fr.4.1.20). Fr.4.1.10 was separated by silica gel eluted with petroleum ether–Me2CO (100:1), to give 4 (27.5 mg) and 10 (28.4 mg). Fr.7 (20.8 g) was separated by Sephadex LH-20 (3 × 100 cm) using MeOH to afford six fractions (Fr.7.1–Fr.7.8). Fr.7.1 was subjected
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to silica gel CC (200–300 mesh, 2.5 × 48 cm, 60 g) with a gradient elution of petroleum ether-Me2CO (1:0, 100:1, 50:1, 25:1, 15:1 and 10:1, each 1.5 L), resulting in sixteen fractions (Fr.7.1.1–Fr.7.1.16). Fr.7.1.3 was separated by Sephadex LH-20 (1.4 × 42 cm) using CHCl3–MeOH (1:1), followed by silica gel eluted with CHCl3 to give 14 (6.3 mg). Fr.7.1.4 was separated by Sephadex LH-20 (1.4 × 42 cm) using CHCl3–MeOH (1:1), followed by silica gel eluted with CHCl3–MeOH (200:1) to give 5 (2.3 mg) and 15 (27.6 mg). Fr.7.1.6 was separated by Sephadex LH-20 (1.4 × 42 cm) using CHCl3–MeOH (1:1), followed by silica gel eluted with CHCl3–MeOH (150:1) to give 2 (27.8 mg) and 3 (11.2 mg). Fr.9 (6.2 g) was separated by Sephadex LH-20 (3 × 100 cm) using MeOH to afford seven fractions (Fr.9.1–Fr.9.7). Fr.9.2 was separated by Sephadex LH-20 (3 × 100 cm) using CHCl3–MeOH (1:1) to afford three fractions (Fr.9.2.1–Fr.9.2.3). Fr.9.2.3 was separated by silica gel eluted with CHCl3–MeOH (100:1) to give 6 (28.5 mg), 11 (30.4 mg) and 12 (18.9 mg). 2.3.1. rel-(3R,6R,7S)-3,7,11-trimethyl-3,7-epoxy-1,10-dodecadien6-ol (1) White amorphous solid; m.p. 57.6–58.9 °C; [α]30 D + 40 (c = 3.0, MeOH); UV (MeOH): λmax (log ε) 202 (1.18) and 232 (0.07) nm; IR (KBr): υmax 3436, 2926, 1635, 1539, 1444, 1074 and 740 cm−1; 1H- and 13C-NMR data see Tables 1 and 2; HR-EI-MS m/z 238.1938 [M]+ (calcd. for C15H26O2, 238.1933). 2.3.2. rel-(3S,6R,7S,9E)-3,7,11-trimethyl-3,6-epoxy-1,9,11dodecatrien-7-ol (2) Colorless oil; [α]30 D + 38 (c = 3.4, MeOH); UV (MeOH): λmax (log ε) 204 (1.86), 215 (1.58), 222 (1.59) nm; IR (KBr): υmax 3445, 2971, 2929, 1633, 1452, 1374, 1043 and 922 cm−1; 1Hand 13C-NMR data see Tables 1 and 2; HR-EI-MS m/z 236.1768 [M]+ (calcd. for C15H24O2, 236.1776). 2.3.3. rel-(3S,6R,7S)-3,7,11-trimethyl-3,6-epoxy-1-dodecen-7,11diol (3) Colorless oil; [α]30 D + 44 (c = 1.1, MeOH); UV (MeOH): λmax (log ε) 205 (1.70) nm; IR (KBr): υmax 3447, 2970, 1641, 1459, 1372, 1165, 1047 and 914 cm−1; 1H- and 13C-NMR data see Tables 1 and 2; HR-EI-MS m/z 256.2030 [M]+ (calcd. for C15H28O3, 256.2038). 2.3.4. rel-(3S,6R,7S,10S)-2,6,10-trimethyl-3,6;7,10-diepoxy-2dodecen-11-ol (4) Colorless oil; [α]30 D + 28 (c = 2.5, MeOH); UV (MeOH): λmax (log ε) 202 (0.35) nm; IR (KBr): υmax 3447, 1639, 1457, 1376, 1313, 1243, 1157, 1089, 949 and 919 cm−1; 1H- and 13 C-NMR data see Tables 1 and 2; HR-EI-MS m/z 254.1877 [M]+ (calcd. for C15H26O3, 254.1882). 2.3.5. (3S,5E)-3,11-dimethyl-7-methylenedodaca-1,5,10-trien-3ol (5) Colorless oil; [α]30 D + 13 (c = 0.70, MeOH); UV (MeOH): λmax (log ε) 205 (2.27), 232 (1.01), 285 (0.33) nm; IR (KBr): υmax 3426, 2926, 1640, 1449, 1374, 1049 and 925 cm−1; 1H- and 13 C-NMR data see Tables 1 and 2; HR-EI-MS m/z 220.1830 [M]+ (calcd. for C15H24O, 220.1827).
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Table 1 1 H-NMR data of compounds 1–5.a Pos.
1b
2
3
4
5
1
5.19 dd (17.4, 1.3); 5.00 dd (10.8, 1.3) 5.96 dd (17.4, 10.8)
5.20 d (17.4); 5.03 d (10.8) 5.98 dd (17.4, 10.8)
5.19 d (17.4); 4.96 d (10.8) 5.95 dd (17.4, 10.8)
5.24 dd (17.3, 1.0); 5.09 dd (10.7, 1.0) 5.97 dd (17.3, 10.7)
4
4.96 d (17.6); 4.96 d (11.3) 5.95 ddd (17.6, 11.3, 0.79) 2.10 m
1.91 m; 1.78 m
1.92 m; 1.82 m
1.95 m 1.74 m
5 6 8
1.72 m 3.53 dd (10.7, 5.0) 1.52 m
1.92 m; 1.83 m 3.90 t (7.1) 1.49 m; 1.35 m
1.82 m 3.97 t (6.6) 1.95 m; 1.69 m
dd (13.4, 6.9); dd (13.4, 8.6) m d (15.8) m; 2.19 m
9 10 12 13 14 15
2.09 5.13 1.67 1.61 1.13 1.13
1.93 m; 1.84 m 3.87 t (7.2) 2.36 dd (13.9, 6.5); 2.16 dd (13.9, 8.5) 5.70 ddd (15.5, 8.5, 6.6) 6.18 d (15.5) 4.89 s 1.84 s 1.20 s 1.31 s
2.41 2.33 5.70 6.16 2.22
1.46 1.48 1.22 1.21 1.22 1.31
1.83 3.77 1.10 1.19 1.17 1.28
2.08 5.16 1.72 1.63 4.97 1.32
m t (6.8) s s s; 4.95 s s
2
a b
m t (7.1) s s s s
m m s s s s
m t (7.6) s s s s
All Compounds were measured at 500 MHz in CDCl3. Chemical shifts are given in ppm; J values are in parentheses and reported in Hz.
2.4. Bioassay for antibacterial activity
3. Results and discussion
All the isolated compounds were tested for in vitro antibacterial activity against Candida albicans and S. aureus (obtained from Hainan Medical University) by the filter paper disk agar diffusion method [9]. The C. albicans was cultured using yeast peptone dextrose and the S. aureus was cultured using nutrient agar. A total of 25 μL 20 mg/mL of compounds 1–15 were impregnated on sterile filter paper disks (6 mm diameter), respectively, and then applied aseptically to the surface of the agar plates. Fluconazole (25 μL 26 mg/mL) was used as positive control of C. albicans and kanamycin sulfate (25 μL 0.64 mg/mL) was used as positive control of S. aureus. As an expression of the antibacterial activities, the diameters of inhibition zones including the 6-mm disk diameter were measured after 24 h of incubation at room temperature. Experiments were done in triplicate, and the results were presented as mean values ± SD.
Five new sesquiterpenes (1–5) (Fig. 1), along with ten known sesquiterpenes, were isolated from the heartwood of Dalbergia odorifrea T. Chen. The known compounds were identified as rel-(S,E)-2-[(S)-2,2-dimethyl-1,3-dioxolan-4yl]-6,10-dimethylundeca-5,9-dien-2-ol (6) [10,11], rel(3S,6R,7S)-3,7,11-trimethyl-3,6-epoxy-1,10-dodecadien-7-ol (7) [12,13], rel-(3S,6S,7R)-3,7,11-trimethyl-3,6-epoxy-1,10dodecadien-7-ol (8) [12,13], 6α-hydroxycyclonerolidiol (9) [14–17], rel-(3S,6R,7S,10R)-2,6,10-trimethyl-3,6;7,10-diepoxy11-dodecen-2-ol (10) [18], neroplofurol (11) [19], 3,7,11trimethyldodeca-1,10-diene-3,6,7-triol (12) [20], rel-(2R,2′R,5′ S)-2,5′-dimethyl-5′-vinylhexahydro-2,2′-bifuran-5(2H)-one (13) [21,22], crocinervolide (14) [23] and (E)-7-hydroxy6,10-dimethylundeca-5,9-dien-2-one (15) [24], by interpreting spectral data and making comparisons with literature values.
Table 2 13 C-NMR data for compounds 1–5.a Pos.
1b
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
110.7 146.6 73.3 32.8 25.7 72.5 77.3 41.6 21.7 125.2 131.3 25.8 17.8 19.6 31.8
a b
2 t d s t t d s t t d s q q q q
All Compounds were measured at 125 MHz in CDCl3. Chemical shifts are given in ppm.
111.8 144.4 82.8 38.0 26.2 84.9 73.3 41.5 125.5 136.2 142.1 115.8 18.8 24.6 26.8
3 t d s t t d s t d d s t q q q
111.8 144.5 82.6 38.0 26.2 85.2 73.0 38.0 18.4 44.6 71.2 29.6 29.2 24.4 26.2
4 t d s t t d s t t t s q q q q
111.4 144.6 83.0 37.9 27.4 84.7 84.6 34.8 26.5 87.0 70.7 23.7 27.8 24.1 26.1
5 t d s t t d s t t d s q q q q
112.1 t 144.8 d 72.8 s 45.9 t 124.1 d 136.4 d 145.7 s 32.2 t 26.9 t 124.2 d 131.8 s 25.7 q 17.9 q 114.7 t 27.6 q
H. Wang et al. / Fitoterapia 95 (2014) 16–21
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Fig. 1. Structures of compounds 1–5.
Compound 1 was obtained as a white amorphous solid. The molecular formula was determined to be C15H26O2 from the molecular ion peak [M]+ at m/z 238.1938 (calcd. 238.1933) in the HREIMS. The data of 1H- and 13C-NMR spectra of 1 (Tables 1 and 2) were very similar to those of the known compound 9. The NMR spectra clearly showed that a vinyl and a prenyl end group were present in both compounds. It is proposed that compounds 1 and 9 are two diastereomers of 6-hydroxycyclonerolidiol as their HMBC spectra showed the same correlations. Comparing the data of 1H- and 13C-NMR spectra of THP 4 and THP 15 [14], especially the 13C-NMR data of C-8, 3-OH/Me-14 were regarded to be cis-oriented. The relative configurations were also supported by the key correlations of H-6 with H-8 (Fig. 2). The difference between 1 and 9 is the relative configuration of the chiral center at C-3. Since the relative configurations of 9 are reported, the relative configuration of the chiral center at C-3 is certain. In addition, comparing the data of 1H- and 13C-NMR spectra of tetrahydro2,2,6-trimethyl-6-vinyl-2H-pyran-3-ols [16], especially the 1Hand 13C-NMR data of Me-15, the NMR data of compound 1 are more close to (3S,6S)-4. Thus, 1 was elucidated to be rel(3R,6R,7S)-3,7,11-trimethyl-3,7-epoxy-1,10-dodecadien-6-ol. Compound 2 was obtained as colorless oil. The molecular formula was determined to be C15H26O2 as derived from HREIMS ([M]+ at m/z 236.1776, calcd. 236.1768) and 1H- and 13 C-NMR spectral data (Tables 1 and 2). The data of 1H- and 13 C-NMR spectra of 2 were very similar to those of the known compounds 7 and 8. The difference was the presence of a double bond and the absence of a methyl group. The HMBC
correlations (Fig. 2) from Me-13 to C-10/C-11/C-12 and from H-10 to C-8/C-11/C-12/C-13 proved that the two double bonds were located at C-9/C-10 and C-11/C-12 to form conjugated double bond. Comprehensive analysis of the 1Hand 13C-NMR data and 2D-NMR spectra of 2 established the structure of compound 2. The coupling constant value of 15.5 Hz between H-9 and H-10 suggested the two protons were trans-oriented. The relative configuration of 2 was deduced from the correlations of H-10 with H-12, Me-13 with H-9 and Me-15 with H-6 in the NOESY spectrum. And the result was further confirmed by comparing the data of 1 H- and 13C-NMR spectra of 7 and 8 as the differences of functional groups between 2 and 7 have little impact on the chemical shifts. The 1H-NMR data of H-2 and H-6 are more close to compound 7. Therefore, the structure of 2 was determined to be rel-(3S,6R,7S,9E)-3,7,11-trimethyl-3,6epoxy-1,9,11-dodecatrien-7-ol. Compound 3 was obtained as colorless oil. The molecular formula was determined to be C15H28O3 from the molecular ion peak [M]+ at m/z 256.2030 (calcd. 256.2038) in the HREIMS. The data of 1H- and 13C-NMR spectra of 3 (Tables 1 and 2) were very similar to those of the known compounds 7 and 8. The difference was the presence of a quaternary carbon connected to an oxygen atom and the absence of a double bond. The HMBC correlations (Fig. 2) from Me-12 and Me-13 to C-11 proved that the OH group was located on C-11. The relative configuration of 3 was assigned by comparing the data of 1H- and 13C-NMR spectra of 7 and 8. The NMR data of 3 are more close to 7. Comprehensive analysis of the 1H- and 13C-NMR data and 2D-NMR spectra
Fig. 2. Key HMBC and ROESY correlations for compounds 1–5.
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H. Wang et al. / Fitoterapia 95 (2014) 16–21
Table 3 Antimicrobial activity of compounds 1–15 (mm). Compounds
Candida albicans
Staphylococcus aureus
1 2–8 9 10–15 Fluconazolea Kanamycin sulfatea
10.86 ± 0.06 – 9.21 ± 0.05 – 30.64 ± 0.24
– – 11.02 ± 0.08 – 24.52 ± 0.18
Notes: Each value represents the mean ± SD (n = 3); 20 mg/mL of each compound. ‘–,’ inactive. a Positive control fluconazole (26 mg/mL) and kanamycin sulfate (0.64 mg/mL).
established the structure of compound 3. Thus, the structure of 3 was determined to be rel-(3S,6R,7S)-3,7,11-trimethyl-3,6epoxy-1-dodecen-7,11-diol. Compound 4 was obtained as colorless oil. The molecular formula was determined to be C15H26O3 from the molecular ion peak [M]+ at m/z 254.1877 (calcd. 254.1882) in the HREIMS. The data of 1H- and 13C-NMR spectra of 4 (Tables 1 and 2) were very similar to those of the known compound 10. Compounds 4 and 10 are also two diastereomers as the same correlations in HMBC spectra. The ROESY correlation (Fig. 2) of H-6 with Me-15 suggested H-6 and Me-15 were cis-oriented. And the NMR data of Me-15 are similar to the NMR data of Me-15 in compound 10. As H-10 showed no correlation with Me-14, so H-10 and Me-14 were trans-oriented. And chemical shifts of H-14 and C-11 give the fine agreement with the chemical shifts of neroplofurol [25]. The transconfiguration of H-6 with Me-14 was assigned by comparing the NMR data of 10, 13 and glabrescol [26]. The coupling constant of H-6 is more close to the coupling constants of H-6 in compounds 10 and 13. Thus, the structure of 4 was determined to be rel-(3S,6R,7S,10S)-2,6,10-Trimethyl-3,6;7,10-diepoxy-11dodecen-2-ol.
Compound 5 was obtained as colorless oil. The molecular formula was determined to be C15H24O as derived from HREIMS ([M]+ at m/z 220.1827, calcd. 220.1830) and 1H- and 13 C-NMR spectral data (Tables 1 and 2). Analysis of the 1H, 13 C and HMQC-NMR date of 5 revealed the presence of three methyl groups, one exchange protons and eight olefinic carbons. The major differences observed in the NMR spectra for 5 relative to those of 12 revealed their similar structural features except for the presence of four olefinic carbons and the absence of two hydroxyls in 5. The HMBC correlations (Fig. 2) from H-14 to C-6/C-7/C-8 and the NOESY correlations of H-5 with H-14 suggested a cis-conjugated double bond in 5. Comprehensive analysis of the 1H- and 13C-NMR data and 2D-NMR spectra established the structure of compound 5. The stereochemistry of 5 was proposed to be 3S compared to (3R)-nerolidol [27], based on its specific rotation value ([α]30 D + 13° (c = 0.70, MeOH)). Thus, the structure of 5 was determined to be (3S,5E)-3,11-dimethyl-7-methylenedodaca-1,5,10-trien-3-ol. Compounds 1 and 9 had inhibitory effect on C. albicans with inhibition zone diameters of 10.86 mm and 9.21 mm, while compound 9 exhibited inhibitory effects on S. aureus with inhibition zone diameters of 11.02 mm by paper disk diffusion method (Table 3). We analyzed the phenomenon based on their structures. It is possible that 6-hydroxycyclonerolidiol could eliminate antibacterial activity in vitro by ring-opening isomerization of pyran ring. If there were no pyran ring within these molecules, a change in the number of double bonds and hydroxyls will not cause antibacterial activity. Pyran ring was found to be essential for the antibacterial activity. And the antibacterial activity is influenced by relative configurations as well. The cytotoxic result of the compound 1 is published by Dong-Li Li et al. [28]. However, its structure was misidentified. Additionally, antithrombotic and antiplatelet activities of 1 and 7 were measured by Yi Tao and Yi Wang [7], the results showed that their anti-platelet activity was
Scheme 1. Plausible biosynthetic pathway of compounds 1–15.
H. Wang et al. / Fitoterapia 95 (2014) 16–21
strong at middle and high concentrations. Unfortunately, the chemical structure of 1 was misidentified as well. Additionally, the NMR data used for assignment of the absolute configuration of the chiral centra using MTPA do not adhere to the prerequisites reported by Seco et al. [29], leading to a loss of confidence in the assignment in that article. Compound 15 should be resulted from a reaction with acetone during the isolation procedure. All of the compounds may be degraded at room temperature. Compounds 7 and 8 were degraded under dry conditions in the absence of light at room temperature with one year, since the two methyl signals at δH1.6–1.7 were completely disappeared in the 1H-NMR spectra. These compounds most likely are biotransformed from nerolidol, and a possible biogenetic pathway of these compounds was proposed as shown in Scheme 1. In summary, five new sesquiterpenes along with ten known ones were isolated from the heartwood of D. odorifrea. The structures of the five new compounds were determined using UV, IR, HRMS and NMR. These sesquiterpenes were generated from nerolidol through oxidation, dehydrogenation and degradation. All the isolated compounds were tested for in vitro antibacterial activity against C. albicans and S. aureus by the filter paper disk agar diffusion method. Bioassay results showed that compounds 1 and 9 had inhibitory effect on C. albicans, and compound 9 exhibited inhibitory effects on S. aureus. Many of these compounds are volatile chemicals and may be degraded at room temperature. Taken together, these results provide basis for the utilization of D. odorifera. Acknowledgments This research was financially supported by Special Fund for Agro-scientific Research in the Public Interest (201303117), National Support Science and Technology Subject (2013BAI11B04) and Major Technology Project of Hainan Province (ZDZX2013008-4). Appendix A. Supplementary data 1
H- and 13C-NMR spectra of the compounds 1–5 are available as Supporting Information. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/ j.fitote.2014.02.013. References [1] The state Pharmacopoeia Commission of PR China. Pharmacopoeia of the People's Republic of China, 1. Beijing: Chemical Industry Press; 2000. p. 184. [2] Zhao Q, Guo JX, Zhang YY. Chemical and pharmacological research progress of Chinese drug “JiangXiang” (Lignum Dalbergiae Odoriferae). J Chin Pharm Sci 2000;9:1–5. [3] Hou JP, Wu H, Ho CT, Weng XC. Antioxidant activity of polyphenolic compounds from Dalbergia odorifera T. Chen. Pak J Nutr 2011;10:694–701. [4] Lee SH, Kim JY, Seo GS, Kim YC, Sohn DH. Isoliquiritigenin, from Dalbergia odorifera, up-regulates antiinflammatory heme oxygenase-1 expression in RAW264.7 macrophages. Inflamm Res 2009;58:257–62.
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Five new sesquiterpenoids from Dalbergia odorifera Hao Wang a,b, Wen-Hua Dong a, Wen-Jian Zuo a, Shuai Liu a, Hui-Min Zhong b, Wen-Li Mei a,⁎, Hao-Fu Dai a,⁎⁎ a Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China b College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, China
a r t i c l e
i n f o
Article history: Received 8 January 2014 Accepted in revised form 21 February 2014 Available online 5 March 2014 Keywords: Dalbergia odorifrea T. Chen Leguminosae Sesquiterpenoids Antibacterial activity
a b s t r a c t Five new sesquiterpenes (1–5) along with ten known ones were isolated from the heartwood of Dalbergia odorifrea T. Chen. Their structures were determined by spectroscopic techniques including MS, UV, IR, 1D and 2D NMR. Bioassay results showed that compounds 1 and 9 had inhibitory effect on Candida albicans, and compound 9 exhibited inhibitory effects on Staphylococcus aureus by paper disk diffusion method. © 2014 Published by Elsevier B.V.
1. Introduction The heartwood of Dalbergia odorifera T. Chen. (Leguminosae) is a traditional Chinese medicine and has been used to treat blood disorder, ischemia, swelling, necrosis and rheumatic pain [1]. It is indigenous to Hainan Province, China. Results of chemical studies showed that D. odorifera mainly contained flavonoids and volatile oil. Previous phytochemical studies of D. odorifera were focused on phenolic components, such as flavonoids, biflavones and their interesting biological properties [2–7]. Those studies revealed that the essential oil of D. odorifera was chiefly composed of trans-nerolidol, β-bisabolene and trans-β-farnesene. In total, less than ten sesquiterpenoids
were examined separately from D. odorifera, and this type of compound did not attract considerable attention. In the previous paper [8], we reported the extraction, analysis and antibacterial activity of the volatile oil. The major components of volatile oil were sesquiterpenoids, and the oil showed activity against Staphylococcus aureus and MRSA. Our continuous study on this drug led to the characterization of fifteen sesquiterpenoids. This paper describes the isolation, structure elucidation and antibacterial activity of five new sesquiterpenoids (1–5) and ten known ones from the heartwood of D. odorifera. 2. Experimental 2.1. General
⁎ Correspondence to: W. Mei, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Xueyuan Road 4, Longhua District, Haikou 571101, Hainan, PR China. Tel./fax: +86 898 6698 7529. ⁎⁎ Correspondence to: H. Dai, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Xueyuan Road 4, Longhua District, Haikou 571101, Hainan, PR China. Tel./fax: +86 898 6696 1869. E-mail addresses: [email protected] (W.-L. Mei), [email protected] (H.-F. Dai).
http://dx.doi.org/10.1016/j.fitote.2014.02.013 0367-326X/© 2014 Published by Elsevier B.V.
Optical rotations were recorded using a Rudolph Autopol III polarimeter (Rudolph, Flanders, America). Melting points were determined on a Beijing Taike X-5 stage apparatus (Beijing Taike Instrument Company, China) and are uncorrected. UV spectra were recorded on a Shimadzu UV-2550 spectrometer (Beckman, America). IR absorptions were obtained on a Nicolet 380 FT-IR instrument (Thermo, America) using KBr pellets. 1H-, 13C- and
H. Wang et al. / Fitoterapia 95 (2014) 16–21
2D-NMR spectra were recorded on Bruker Avance 500 NMR spectrometers (Bruker, Germany), using TMS as an internal standard. HRMS were measured with an API QSTAR Pulsar mass spectrometer (Bruker, Germany). Column chromatography was performed with silica gel (60–80, 200–300 mesh, Qingdao Marine Chemical Co. Ltd., China, and Sephadex LH-20, Merck, Germany). TLC was carried out on silica gel G precoated plates (Qingdao Marine Chemical Co. Ltd.), and spots were detected by spraying with 5% H2SO4 in EtOH followed by heating. Kanamycin sulfate was produced by Sangon Biotech Co., Ltd, fluconazole capsules was produced by Shandong Lvyin Pharmaceutical Co., Ltd. 2.2. Plant material The dried heartwood of D. odorifera was purchased from the Haikou Free Market of Agricultural Products, Hainan Province, China, in October 2010. The specimen was identified by Dr. Xi-Long Zheng of the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural sciences, where a voucher specimen (no. 20101009) has been deposited. 2.3. Extraction and isolation The dried and crushed heartwood of D. odorifera (8.4 kg) was extracted three times with 95% (v/v) ethanol (50 L) at room temperature for three weeks totally. The solution was evaporated under reduced pressure to obtain an extract (0.9 kg). This extract was suspended in distilled water and then partitioned with petroleum ether, ethyl acetate and n-butanol, respectively. The EtOAc-soluble residue (477 g) was submitted to column chromatography (CC) over silica gel (silica H, 3.1 kg, 15 × 50 cm) and eluted with CHCl3–MeOH (v/v, 1:0, 100:1, 50:1, 25:1, 15:1, 10:1, 5:1, 2:1, 1:1 and 0:1, each 8.0 L) of increasing polarity resulting in eighteen fractions (Fr.1–Fr.18). Fr.3 (6.2 g) was subjected to silica gel CC (200–300 mesh, 2.5 × 48 cm, 60 g) with a gradient elution of petroleum ether–Me2CO (1:0, 100:1, 50:1, 25:1, 15:1, 10:1, 3:1 and 0:1, each 1.5 L), resulting in twenty-seven fractions (Fr.3.1– Fr.3.27). Fr.3.3 was separated by Sephadex LH-20 (1.4 × 42 cm) using CHCl3–MeOH (1:1), followed by silica gel eluted with petroleum ether–CHCl3 (7:3), to give 7 and 8 (751.6 mg). Fr.3.6 was separated by Sephadex LH-20 (1.4 × 42 cm, 23 g) using CHCl3–MeOH (1:1), followed by silica gel eluted with petroleum ether–CHCl3 (1:1), to give 1 (20.7 mg). Fr.3.7 was separated by Sephadex LH-20 (1.4 × 42 cm, 23 g) using CHCl3–MeOH (1:1), followed by silica gel eluted with petroleum ether–CHCl3 (1:1), to give 9 (120.6 mg). Fr.3.10 was separated by Sephadex LH-20 (1.4 × 42 cm, 23 g) using CHCl3–MeOH (1:1), followed by silica gel eluted with petroleum ether–CHCl3 (1:1), to give 13 (155.3 mg). Fr.4 (30.2 g) was separated by Sephadex LH-20 (3 × 100 cm) using CHCl3–MeOH (1:1) to afford eight fractions (Fr.4.1–Fr.4.8). Fr.4.1 (4.1 g) was subjected to silica gel CC (200–300 mesh, 2.5 × 48 cm, 60 g) with a gradient elution of petroleum ether-Me2CO (1:0, 100:1, 50:1 and 20:1, each 1.5 L), resulting in twenty fractions (Fr.4.1.1–Fr.4.1.20). Fr.4.1.10 was separated by silica gel eluted with petroleum ether–Me2CO (100:1), to give 4 (27.5 mg) and 10 (28.4 mg). Fr.7 (20.8 g) was separated by Sephadex LH-20 (3 × 100 cm) using MeOH to afford six fractions (Fr.7.1–Fr.7.8). Fr.7.1 was subjected
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to silica gel CC (200–300 mesh, 2.5 × 48 cm, 60 g) with a gradient elution of petroleum ether-Me2CO (1:0, 100:1, 50:1, 25:1, 15:1 and 10:1, each 1.5 L), resulting in sixteen fractions (Fr.7.1.1–Fr.7.1.16). Fr.7.1.3 was separated by Sephadex LH-20 (1.4 × 42 cm) using CHCl3–MeOH (1:1), followed by silica gel eluted with CHCl3 to give 14 (6.3 mg). Fr.7.1.4 was separated by Sephadex LH-20 (1.4 × 42 cm) using CHCl3–MeOH (1:1), followed by silica gel eluted with CHCl3–MeOH (200:1) to give 5 (2.3 mg) and 15 (27.6 mg). Fr.7.1.6 was separated by Sephadex LH-20 (1.4 × 42 cm) using CHCl3–MeOH (1:1), followed by silica gel eluted with CHCl3–MeOH (150:1) to give 2 (27.8 mg) and 3 (11.2 mg). Fr.9 (6.2 g) was separated by Sephadex LH-20 (3 × 100 cm) using MeOH to afford seven fractions (Fr.9.1–Fr.9.7). Fr.9.2 was separated by Sephadex LH-20 (3 × 100 cm) using CHCl3–MeOH (1:1) to afford three fractions (Fr.9.2.1–Fr.9.2.3). Fr.9.2.3 was separated by silica gel eluted with CHCl3–MeOH (100:1) to give 6 (28.5 mg), 11 (30.4 mg) and 12 (18.9 mg). 2.3.1. rel-(3R,6R,7S)-3,7,11-trimethyl-3,7-epoxy-1,10-dodecadien6-ol (1) White amorphous solid; m.p. 57.6–58.9 °C; [α]30 D + 40 (c = 3.0, MeOH); UV (MeOH): λmax (log ε) 202 (1.18) and 232 (0.07) nm; IR (KBr): υmax 3436, 2926, 1635, 1539, 1444, 1074 and 740 cm−1; 1H- and 13C-NMR data see Tables 1 and 2; HR-EI-MS m/z 238.1938 [M]+ (calcd. for C15H26O2, 238.1933). 2.3.2. rel-(3S,6R,7S,9E)-3,7,11-trimethyl-3,6-epoxy-1,9,11dodecatrien-7-ol (2) Colorless oil; [α]30 D + 38 (c = 3.4, MeOH); UV (MeOH): λmax (log ε) 204 (1.86), 215 (1.58), 222 (1.59) nm; IR (KBr): υmax 3445, 2971, 2929, 1633, 1452, 1374, 1043 and 922 cm−1; 1Hand 13C-NMR data see Tables 1 and 2; HR-EI-MS m/z 236.1768 [M]+ (calcd. for C15H24O2, 236.1776). 2.3.3. rel-(3S,6R,7S)-3,7,11-trimethyl-3,6-epoxy-1-dodecen-7,11diol (3) Colorless oil; [α]30 D + 44 (c = 1.1, MeOH); UV (MeOH): λmax (log ε) 205 (1.70) nm; IR (KBr): υmax 3447, 2970, 1641, 1459, 1372, 1165, 1047 and 914 cm−1; 1H- and 13C-NMR data see Tables 1 and 2; HR-EI-MS m/z 256.2030 [M]+ (calcd. for C15H28O3, 256.2038). 2.3.4. rel-(3S,6R,7S,10S)-2,6,10-trimethyl-3,6;7,10-diepoxy-2dodecen-11-ol (4) Colorless oil; [α]30 D + 28 (c = 2.5, MeOH); UV (MeOH): λmax (log ε) 202 (0.35) nm; IR (KBr): υmax 3447, 1639, 1457, 1376, 1313, 1243, 1157, 1089, 949 and 919 cm−1; 1H- and 13 C-NMR data see Tables 1 and 2; HR-EI-MS m/z 254.1877 [M]+ (calcd. for C15H26O3, 254.1882). 2.3.5. (3S,5E)-3,11-dimethyl-7-methylenedodaca-1,5,10-trien-3ol (5) Colorless oil; [α]30 D + 13 (c = 0.70, MeOH); UV (MeOH): λmax (log ε) 205 (2.27), 232 (1.01), 285 (0.33) nm; IR (KBr): υmax 3426, 2926, 1640, 1449, 1374, 1049 and 925 cm−1; 1H- and 13 C-NMR data see Tables 1 and 2; HR-EI-MS m/z 220.1830 [M]+ (calcd. for C15H24O, 220.1827).
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H. Wang et al. / Fitoterapia 95 (2014) 16–21
Table 1 1 H-NMR data of compounds 1–5.a Pos.
1b
2
3
4
5
1
5.19 dd (17.4, 1.3); 5.00 dd (10.8, 1.3) 5.96 dd (17.4, 10.8)
5.20 d (17.4); 5.03 d (10.8) 5.98 dd (17.4, 10.8)
5.19 d (17.4); 4.96 d (10.8) 5.95 dd (17.4, 10.8)
5.24 dd (17.3, 1.0); 5.09 dd (10.7, 1.0) 5.97 dd (17.3, 10.7)
4
4.96 d (17.6); 4.96 d (11.3) 5.95 ddd (17.6, 11.3, 0.79) 2.10 m
1.91 m; 1.78 m
1.92 m; 1.82 m
1.95 m 1.74 m
5 6 8
1.72 m 3.53 dd (10.7, 5.0) 1.52 m
1.92 m; 1.83 m 3.90 t (7.1) 1.49 m; 1.35 m
1.82 m 3.97 t (6.6) 1.95 m; 1.69 m
dd (13.4, 6.9); dd (13.4, 8.6) m d (15.8) m; 2.19 m
9 10 12 13 14 15
2.09 5.13 1.67 1.61 1.13 1.13
1.93 m; 1.84 m 3.87 t (7.2) 2.36 dd (13.9, 6.5); 2.16 dd (13.9, 8.5) 5.70 ddd (15.5, 8.5, 6.6) 6.18 d (15.5) 4.89 s 1.84 s 1.20 s 1.31 s
2.41 2.33 5.70 6.16 2.22
1.46 1.48 1.22 1.21 1.22 1.31
1.83 3.77 1.10 1.19 1.17 1.28
2.08 5.16 1.72 1.63 4.97 1.32
m t (6.8) s s s; 4.95 s s
2
a b
m t (7.1) s s s s
m m s s s s
m t (7.6) s s s s
All Compounds were measured at 500 MHz in CDCl3. Chemical shifts are given in ppm; J values are in parentheses and reported in Hz.
2.4. Bioassay for antibacterial activity
3. Results and discussion
All the isolated compounds were tested for in vitro antibacterial activity against Candida albicans and S. aureus (obtained from Hainan Medical University) by the filter paper disk agar diffusion method [9]. The C. albicans was cultured using yeast peptone dextrose and the S. aureus was cultured using nutrient agar. A total of 25 μL 20 mg/mL of compounds 1–15 were impregnated on sterile filter paper disks (6 mm diameter), respectively, and then applied aseptically to the surface of the agar plates. Fluconazole (25 μL 26 mg/mL) was used as positive control of C. albicans and kanamycin sulfate (25 μL 0.64 mg/mL) was used as positive control of S. aureus. As an expression of the antibacterial activities, the diameters of inhibition zones including the 6-mm disk diameter were measured after 24 h of incubation at room temperature. Experiments were done in triplicate, and the results were presented as mean values ± SD.
Five new sesquiterpenes (1–5) (Fig. 1), along with ten known sesquiterpenes, were isolated from the heartwood of Dalbergia odorifrea T. Chen. The known compounds were identified as rel-(S,E)-2-[(S)-2,2-dimethyl-1,3-dioxolan-4yl]-6,10-dimethylundeca-5,9-dien-2-ol (6) [10,11], rel(3S,6R,7S)-3,7,11-trimethyl-3,6-epoxy-1,10-dodecadien-7-ol (7) [12,13], rel-(3S,6S,7R)-3,7,11-trimethyl-3,6-epoxy-1,10dodecadien-7-ol (8) [12,13], 6α-hydroxycyclonerolidiol (9) [14–17], rel-(3S,6R,7S,10R)-2,6,10-trimethyl-3,6;7,10-diepoxy11-dodecen-2-ol (10) [18], neroplofurol (11) [19], 3,7,11trimethyldodeca-1,10-diene-3,6,7-triol (12) [20], rel-(2R,2′R,5′ S)-2,5′-dimethyl-5′-vinylhexahydro-2,2′-bifuran-5(2H)-one (13) [21,22], crocinervolide (14) [23] and (E)-7-hydroxy6,10-dimethylundeca-5,9-dien-2-one (15) [24], by interpreting spectral data and making comparisons with literature values.
Table 2 13 C-NMR data for compounds 1–5.a Pos.
1b
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
110.7 146.6 73.3 32.8 25.7 72.5 77.3 41.6 21.7 125.2 131.3 25.8 17.8 19.6 31.8
a b
2 t d s t t d s t t d s q q q q
All Compounds were measured at 125 MHz in CDCl3. Chemical shifts are given in ppm.
111.8 144.4 82.8 38.0 26.2 84.9 73.3 41.5 125.5 136.2 142.1 115.8 18.8 24.6 26.8
3 t d s t t d s t d d s t q q q
111.8 144.5 82.6 38.0 26.2 85.2 73.0 38.0 18.4 44.6 71.2 29.6 29.2 24.4 26.2
4 t d s t t d s t t t s q q q q
111.4 144.6 83.0 37.9 27.4 84.7 84.6 34.8 26.5 87.0 70.7 23.7 27.8 24.1 26.1
5 t d s t t d s t t d s q q q q
112.1 t 144.8 d 72.8 s 45.9 t 124.1 d 136.4 d 145.7 s 32.2 t 26.9 t 124.2 d 131.8 s 25.7 q 17.9 q 114.7 t 27.6 q
H. Wang et al. / Fitoterapia 95 (2014) 16–21
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Fig. 1. Structures of compounds 1–5.
Compound 1 was obtained as a white amorphous solid. The molecular formula was determined to be C15H26O2 from the molecular ion peak [M]+ at m/z 238.1938 (calcd. 238.1933) in the HREIMS. The data of 1H- and 13C-NMR spectra of 1 (Tables 1 and 2) were very similar to those of the known compound 9. The NMR spectra clearly showed that a vinyl and a prenyl end group were present in both compounds. It is proposed that compounds 1 and 9 are two diastereomers of 6-hydroxycyclonerolidiol as their HMBC spectra showed the same correlations. Comparing the data of 1H- and 13C-NMR spectra of THP 4 and THP 15 [14], especially the 13C-NMR data of C-8, 3-OH/Me-14 were regarded to be cis-oriented. The relative configurations were also supported by the key correlations of H-6 with H-8 (Fig. 2). The difference between 1 and 9 is the relative configuration of the chiral center at C-3. Since the relative configurations of 9 are reported, the relative configuration of the chiral center at C-3 is certain. In addition, comparing the data of 1H- and 13C-NMR spectra of tetrahydro2,2,6-trimethyl-6-vinyl-2H-pyran-3-ols [16], especially the 1Hand 13C-NMR data of Me-15, the NMR data of compound 1 are more close to (3S,6S)-4. Thus, 1 was elucidated to be rel(3R,6R,7S)-3,7,11-trimethyl-3,7-epoxy-1,10-dodecadien-6-ol. Compound 2 was obtained as colorless oil. The molecular formula was determined to be C15H26O2 as derived from HREIMS ([M]+ at m/z 236.1776, calcd. 236.1768) and 1H- and 13 C-NMR spectral data (Tables 1 and 2). The data of 1H- and 13 C-NMR spectra of 2 were very similar to those of the known compounds 7 and 8. The difference was the presence of a double bond and the absence of a methyl group. The HMBC
correlations (Fig. 2) from Me-13 to C-10/C-11/C-12 and from H-10 to C-8/C-11/C-12/C-13 proved that the two double bonds were located at C-9/C-10 and C-11/C-12 to form conjugated double bond. Comprehensive analysis of the 1Hand 13C-NMR data and 2D-NMR spectra of 2 established the structure of compound 2. The coupling constant value of 15.5 Hz between H-9 and H-10 suggested the two protons were trans-oriented. The relative configuration of 2 was deduced from the correlations of H-10 with H-12, Me-13 with H-9 and Me-15 with H-6 in the NOESY spectrum. And the result was further confirmed by comparing the data of 1 H- and 13C-NMR spectra of 7 and 8 as the differences of functional groups between 2 and 7 have little impact on the chemical shifts. The 1H-NMR data of H-2 and H-6 are more close to compound 7. Therefore, the structure of 2 was determined to be rel-(3S,6R,7S,9E)-3,7,11-trimethyl-3,6epoxy-1,9,11-dodecatrien-7-ol. Compound 3 was obtained as colorless oil. The molecular formula was determined to be C15H28O3 from the molecular ion peak [M]+ at m/z 256.2030 (calcd. 256.2038) in the HREIMS. The data of 1H- and 13C-NMR spectra of 3 (Tables 1 and 2) were very similar to those of the known compounds 7 and 8. The difference was the presence of a quaternary carbon connected to an oxygen atom and the absence of a double bond. The HMBC correlations (Fig. 2) from Me-12 and Me-13 to C-11 proved that the OH group was located on C-11. The relative configuration of 3 was assigned by comparing the data of 1H- and 13C-NMR spectra of 7 and 8. The NMR data of 3 are more close to 7. Comprehensive analysis of the 1H- and 13C-NMR data and 2D-NMR spectra
Fig. 2. Key HMBC and ROESY correlations for compounds 1–5.
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H. Wang et al. / Fitoterapia 95 (2014) 16–21
Table 3 Antimicrobial activity of compounds 1–15 (mm). Compounds
Candida albicans
Staphylococcus aureus
1 2–8 9 10–15 Fluconazolea Kanamycin sulfatea
10.86 ± 0.06 – 9.21 ± 0.05 – 30.64 ± 0.24
– – 11.02 ± 0.08 – 24.52 ± 0.18
Notes: Each value represents the mean ± SD (n = 3); 20 mg/mL of each compound. ‘–,’ inactive. a Positive control fluconazole (26 mg/mL) and kanamycin sulfate (0.64 mg/mL).
established the structure of compound 3. Thus, the structure of 3 was determined to be rel-(3S,6R,7S)-3,7,11-trimethyl-3,6epoxy-1-dodecen-7,11-diol. Compound 4 was obtained as colorless oil. The molecular formula was determined to be C15H26O3 from the molecular ion peak [M]+ at m/z 254.1877 (calcd. 254.1882) in the HREIMS. The data of 1H- and 13C-NMR spectra of 4 (Tables 1 and 2) were very similar to those of the known compound 10. Compounds 4 and 10 are also two diastereomers as the same correlations in HMBC spectra. The ROESY correlation (Fig. 2) of H-6 with Me-15 suggested H-6 and Me-15 were cis-oriented. And the NMR data of Me-15 are similar to the NMR data of Me-15 in compound 10. As H-10 showed no correlation with Me-14, so H-10 and Me-14 were trans-oriented. And chemical shifts of H-14 and C-11 give the fine agreement with the chemical shifts of neroplofurol [25]. The transconfiguration of H-6 with Me-14 was assigned by comparing the NMR data of 10, 13 and glabrescol [26]. The coupling constant of H-6 is more close to the coupling constants of H-6 in compounds 10 and 13. Thus, the structure of 4 was determined to be rel-(3S,6R,7S,10S)-2,6,10-Trimethyl-3,6;7,10-diepoxy-11dodecen-2-ol.
Compound 5 was obtained as colorless oil. The molecular formula was determined to be C15H24O as derived from HREIMS ([M]+ at m/z 220.1827, calcd. 220.1830) and 1H- and 13 C-NMR spectral data (Tables 1 and 2). Analysis of the 1H, 13 C and HMQC-NMR date of 5 revealed the presence of three methyl groups, one exchange protons and eight olefinic carbons. The major differences observed in the NMR spectra for 5 relative to those of 12 revealed their similar structural features except for the presence of four olefinic carbons and the absence of two hydroxyls in 5. The HMBC correlations (Fig. 2) from H-14 to C-6/C-7/C-8 and the NOESY correlations of H-5 with H-14 suggested a cis-conjugated double bond in 5. Comprehensive analysis of the 1H- and 13C-NMR data and 2D-NMR spectra established the structure of compound 5. The stereochemistry of 5 was proposed to be 3S compared to (3R)-nerolidol [27], based on its specific rotation value ([α]30 D + 13° (c = 0.70, MeOH)). Thus, the structure of 5 was determined to be (3S,5E)-3,11-dimethyl-7-methylenedodaca-1,5,10-trien-3-ol. Compounds 1 and 9 had inhibitory effect on C. albicans with inhibition zone diameters of 10.86 mm and 9.21 mm, while compound 9 exhibited inhibitory effects on S. aureus with inhibition zone diameters of 11.02 mm by paper disk diffusion method (Table 3). We analyzed the phenomenon based on their structures. It is possible that 6-hydroxycyclonerolidiol could eliminate antibacterial activity in vitro by ring-opening isomerization of pyran ring. If there were no pyran ring within these molecules, a change in the number of double bonds and hydroxyls will not cause antibacterial activity. Pyran ring was found to be essential for the antibacterial activity. And the antibacterial activity is influenced by relative configurations as well. The cytotoxic result of the compound 1 is published by Dong-Li Li et al. [28]. However, its structure was misidentified. Additionally, antithrombotic and antiplatelet activities of 1 and 7 were measured by Yi Tao and Yi Wang [7], the results showed that their anti-platelet activity was
Scheme 1. Plausible biosynthetic pathway of compounds 1–15.
H. Wang et al. / Fitoterapia 95 (2014) 16–21
strong at middle and high concentrations. Unfortunately, the chemical structure of 1 was misidentified as well. Additionally, the NMR data used for assignment of the absolute configuration of the chiral centra using MTPA do not adhere to the prerequisites reported by Seco et al. [29], leading to a loss of confidence in the assignment in that article. Compound 15 should be resulted from a reaction with acetone during the isolation procedure. All of the compounds may be degraded at room temperature. Compounds 7 and 8 were degraded under dry conditions in the absence of light at room temperature with one year, since the two methyl signals at δH1.6–1.7 were completely disappeared in the 1H-NMR spectra. These compounds most likely are biotransformed from nerolidol, and a possible biogenetic pathway of these compounds was proposed as shown in Scheme 1. In summary, five new sesquiterpenes along with ten known ones were isolated from the heartwood of D. odorifrea. The structures of the five new compounds were determined using UV, IR, HRMS and NMR. These sesquiterpenes were generated from nerolidol through oxidation, dehydrogenation and degradation. All the isolated compounds were tested for in vitro antibacterial activity against C. albicans and S. aureus by the filter paper disk agar diffusion method. Bioassay results showed that compounds 1 and 9 had inhibitory effect on C. albicans, and compound 9 exhibited inhibitory effects on S. aureus. Many of these compounds are volatile chemicals and may be degraded at room temperature. Taken together, these results provide basis for the utilization of D. odorifera. Acknowledgments This research was financially supported by Special Fund for Agro-scientific Research in the Public Interest (201303117), National Support Science and Technology Subject (2013BAI11B04) and Major Technology Project of Hainan Province (ZDZX2013008-4). Appendix A. Supplementary data 1
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