molecules Article
Sesquiterpenoids from Chinese Agarwood Induced by Artificial Holing Wei Li 1,2,† , Ge Liao 1,† , Wen-Hua Dong 1,2 , Fan-Dong Kong 1 , Pei Wang 1 , Hao Wang 1 , Wen-Li Mei 1,2, * and Hao-Fu Dai 1,2, * 1
2
* †
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, Hainan, China;
[email protected] (W.L.);
[email protected] (G.L.);
[email protected] (W.-H.D.);
[email protected] (F.-D.K.);
[email protected] (P.W.);
[email protected] (H.W.) Hainan Engineering Research Center of Agarwood, Haikou 571101, Hainan, China Correspondence:
[email protected] (W.-L.M.);
[email protected] (H.-F.D.); Tel.: +86-898-6698-7529 (W.-L.M.); +86-898-6696-1869 (H.-F.D.) These authors contributed equally to this work.
Academic Editor: Isabel C. F. R. Ferreira Received: 22 January 2016 ; Accepted: 23 February 2016 ; Published: 26 February 2016
Abstract: Two new sesquiterpenoids, 3-oxo-7-hydroxylholosericin A (1) and 1,5;8,12-diepoxy-guaia-12one (2), together with seven known sesquiterpenoids 3–9, were isolated from Chinese agarwood induced by artificial holing originating from Aquilaria sinensis (Lour.) Gilg. Their structures were identified by spectroscopic techniques (UV, IR, 1D and 2D NMR) and MS analyses. The absolute configuration of compound 1 was determined by comparison of its measured CD curve with that of calculated data for 1 and ent-1. The NMR data of 3 were reported in this study for the first time. Compounds 1, 2, 4–6, together with the EtOAc extract showed moderate inhibitory activities against acetylcholinesterase, and compounds 4–6, 8 exhibited antibacterial activities against Staphylococcus aureus or Ralstonia solanacearum. Keywords: sesquiterpenoid; Chinese agarwood induced by artificial holing; Aquilaria sinensis; AChE inhibition activity; antibacterial activity
1. Introduction Chinese agarwood is the resinous wood from the tree of Aquilaria sinensis (Lour.) Gilg (Thymelaeaceae), which is known as a famous traditional medicine, and has been reported as a folk medicine to possess various functions as sedative, analgesic, and digestive, etc. [1,2]. However, Chinese agarwood cannot be generated in healthy wood tissues of A. sinensis but may be produced when an A. sinensis tree is injured by lightning strikes, physical cutting, eaten by moths, burnt, bacterial infections or chemical stimulation, etc. [3,4]. The previous phytochemical research on wild Chinese agarwood from A. sinensis revealed the main constituents of Chinese agarwood were sesquiterpenoids and 2-(2-phenylethyl)chromones [4–6]. It is reported that some sesquiterpenoids and 2-(2-phenylethyl)chromones showed acetylcholinesterase inhibition activities, together with some antibacterial activities [5,7,8]. For better understanding of the chemical components of artificially induced Chinese agarwood, a phytochemical investigation was carried out on Chinese agarwood induced by artificial holing from A. sinensis, which led to the isolation of two new sesquiterpenoids 1–2 with unusual guaiane skeletons, together with seven known sesquiterpenoids 3–9 (Figure 1). The 1D and 2D NMR data of the known compound 3 are reported for the first time. The acetylcholinesterase
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inhibition activities, together with the antibacterial activities against S. aureus and R.against solanacearum of acetylcholinesterase inhibition activities, together with the antibacterial activities S. aureus these isolates were investigated in vitro. and R. solanacearum of these isolates were investigated in vitro.
Figure 1. 1. Chemical structures structures of of compounds compounds 1–9. 1–9. Figure
2. Resultsand andDiscussion Discussion 2. Results Compound Compound 1 was was obtained obtained as as aa colorless colorless oil, oil, and and its its molecular molecular formula formula was was established established as as ´ − C H20 O55by bythe the HR-ESI-MS HR-ESI-MSmolecular molecularpeak peakatatm/z m/z[M [M+ +CF CF 393.1166 (calcd. 280.1311 C15 15H 3COO] (calcd. 280.1311 for 20O 3 COO]393.1166 for C2015OH5)20(Supplementary O5 ) (Supplementary Materials), six degrees of unsaturation. IR spectrum C15H Materials), with with six degrees of unsaturation. The IRThe spectrum clearly ´ 1 ´ 1 −1 −1 clearly demonstrated the presence of hydroxy cm ), carbonyl ) and olefinic demonstrated the presence of hydroxy (3434 cm(3434 ), carbonyl (1630 cm (1630 ) and cm olefinic bond (1597bond cm−1) ´ 1 1 1H-NMR (1597 cm The ) groups. The H-NMR spectroscopic 1) of 1two showed twoat methyls δH 1.35 (3H, groups. spectroscopic data (Tabledata 1) of(Table 1 showed methyls δH 1.35at(3H, s, H-14) s, H-14) and 1.06 (3H, d, J = 7.3 Hz, H-15), one oxygenated methylene [δ 4.62 (1H, dt, J = 12.8, 2.4 Hz, dt, J = 12.8, 2.4 Hz, H-12a) and 1.06 (3H, d, J = 7.3 Hz, H-15), one oxygenated methylene [δH 4.62 (1H, H H-12a) and 4.42dt, (1H, J = 12.8, 2.4 Hz, H-12b)], terminal olefinic methylene[δ[δHH5.28 5.28(1H, (1H, br br t, and 4.42 (1H, J =dt, 12.8, 2.4 Hz, H-12b)], andand oneone terminal olefinic methylene 13 C-NMR 13C-NMR JJ == 2.4, The spectrum (Table 1) of 15 2.4, H-13a) H-13a)and and5.07 5.07(1H, (1H,brbrt,t,J = J =2.4 2.4Hz, Hz,H-13b)]. H-13b)]. The spectrum (Table 1) 1ofexhibited 1 exhibited carbon signals, composed of two methyls, five methylenes, two methines and six quaternary carbons, 15 carbon signals, composed of two methyls, five methylenes, two methines and six quaternary indicative of a possible skeleton. Detailed comparison the NMR data with those of carbons, indicative of asesquiterpenoid possible sesquiterpenoid skeleton. Detailed of comparison of the NMR data holosericin A [9] revealed A that shared thethey same guaiane with an ether bridge linking with those of holosericin [9]they revealed that shared the skeleton same guaiane skeleton with an ether C-1 andlinking C-8, and only by a carbonyl (δCa 219.0) and(δan oxygenated bridge C-1the andtwo C-8,compounds and the twodiffered compounds differed only by carbonyl C 219.0) and an quarternary carbon (δC 75.1) in 1(δreplacing corresponding aliphatic methylene methineand in oxygenated quarternary carbon C 75.1) in the 1 replacing the corresponding aliphaticand methylene holosericin A. This assignment was further proved by key HMBC correlations (Figure 2) from H -15 methine in holosericin A. This assignment was further proved by key HMBC correlations (Figure 3 2) (δ to (δ C-3 (δC 219.0), (δC 48.6) from and H2 -13 (δHH5.28, 5.07) to C-7 H 1.06) to C-3 C-4 (δC 219.0), C-4and (δC C-5 48.6)(δand C-5 and (δC 44.7), from 2-13 (δ H 5.28, 5.07)(δto from H3-15 H 1.06) C 44.7), C 75.1), (δC 151.1) and C-12 (δ 71.3). Thus, the planar structure of compound 1 was established as C-7 (δC-11 C 75.1), C-11 (δ C 151.1) and C-12 (δ C 71.3). Thus, the planar structure of compound 1 was C 3-oxo-7-hydroxylholosericin A. Detailed analysis of ROESY andof NOE difference spectra revealed that established as 3-oxo-7-hydroxylholosericin A. Detailed analysis ROESY and NOE difference spectra the relative configuration the stereogenic C-1, C-4, centers C-5, C-8,C-1, andC-4, C-10C-5, of compound 1 were revealed that the relativeofconfiguration of centers the stereogenic C-8, and C-10 of the same as 1those holosericin A; theofadditional hydroxyl located at C-7 was onlocated the same sidewas of the compound wereofthe same as those holosericin A; the additional hydroxyl at C-7 on 7-membered ring as the epoxy group based on the observation of theon hydroxy proton (δHof5.16) the same side of system the 7-membered ring system as the epoxy group based the observation the enhancement when H-4 (δH 2.16) were irradiated NOEwere difference experiment. The difference measured hydroxy proton (δHthe 5.16) enhancement when the H-4in(δthe H 2.16) irradiated in the NOE CD spectrumThe of 1measured exhibitedCD a characteristic effecta around 300 nm due to nÑπ* transition of experiment. spectrum of 1cotton exhibited characteristic cotton effect around 300 nm the group. In order to determine the absolute of 1,absolute ECD calculations of 1 of and dueketone to n→π* transition of the ketone group. In order configuration to determine the configuration 1, ent-1 the time-dependent density theorydensity (TD-DFT) methodtheory at the(TD-DFT) B3LYP/6-31G(d) ECD using calculations of 1 and ent-1 using thefunctional time-dependent functional method level was performed. conformational distribution search was performed by at the[10] B3LYP/6-31G(d) levelThe [10]preliminary was performed. The preliminary conformational distribution search HyperChem 7.5 by software. The corresponding minimum geometries were further fully optimized by was performed HyperChem 7.5 software. The corresponding minimum geometries were further using DFT at theby B3LYP/6-31G(d) level as implemented in the Gaussian 03 program package. ECD fully optimized using DFT at the B3LYP/6-31G(d) level as implemented in the Gaussian 03The program calculations were performed afterwere optimization of the selected conformers at the B3LYP/6-31G(d) package. The ECD calculations performed after optimization of the selected conformers levels The results showed that the CD curve matched well with thecurve calculated ECDwell for at the[11]. B3LYP/6-31G(d) levels [11]. Themeasured results showed that the measured CD matched 1with and was to that ent-1 3), indicating thethat 1R,4S,5S,7R,8R,10R-configuration. the opposite calculated ECDoffor 1 (Figure and was opposite to of ent-1 (Figure 3), indicating the 1R,4S,5S,7R,8R,10R-configuration.
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Table 1. 1 H (500 MHz) and 13 C (125 MHz) NMR spectral data of compounds 1–3 (δ in ppm, J in Hz). 1c
No. δC
a
δH
2 b,c
a
δH (in DMSO)
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δC
3 b,c δH
73.8
δC 135.4
δH 3 of 8 3 of 8
2.42 d (18.5), 2.40 d (18.5), 45.4 1. 1H (500 MHz) and 13C (125 MHz) NMR spectral 29.6 2.04 m, 1.74 1–3 m (δ in 125.2 5.12 t (6.6) Table data of compounds ppm, J in Hz). d (18.5) 2.05 dNMR (18.5)spectral data Table 1. 1H (500 2.17 MHz) and 13C (125 MHz) of compounds 1–3 (δ in ppm, J in Hz). b,c 2 b,c m 3 2.04 1c 219.0 26.4 1.65 m, 1.14 26.6 m
2 3
No. No.48.6 1 1 44.7 2 2 28.2 3 3 4 4 75.1 5 5 6 112.9 6 7 7 43.3 8 8
2 b,c 3 b,c 1c δC a δH a δH (in DMSO) δC δH δC δH 2.36 dq (2.0, 2.16 dqδ(1.6, 7.0) 39.6 δC 2.21 δdH (1.7) 32.4 δH 2.04 m a δC a89.5 δH7.3) H (in DMSO) δ C 73.8 135.4 89.5 135.4 5 2.12 m 2.00 m2.40 d (18.5), 72.473.8 135.4 2.42 d (18.5), 45.4 29.6 2.04 m, 1.74 m 125.2 5.12 t (6.6) 2.422.17 d (18.5), 2.40 d (18.5), d (18.5) 2.17 br d (14.1), 2.05 d (18.5) 45.4 2.33 br d (14.8), 29.6 2.04m, m, 1.87 1.74 m 2.26 dd 125.2 5.12 t (6.6) 2.17 d (18.5) 2.05 d (18.5) 125.2 5.12mt (6.6) 28.0 6 219.0 26.4 (8.5, 1.6512.3) m, 1.14 m 26.6 2.04 2.26 dd (7.3, 14.8 ) 2.09 dd (7.4, 14.1) 219.048.6 26.4 1.65 m, 1.14 m 26.6 2.04 m m 2.36 dq (2.0, 7.3) 2.16 dq (1.6, 7.0) 39.6 2.21 d (1.7) 32.4 2.04 7 47.239.672.4 2.21 2.01 m 26.6 2.04 m2.04 m 48.644.7 2.36 dq (2.0, 2.16 dq (1.6, d (1.7) 32.4135.4 2.12 7.3) m 2.00 7.0) m 44.7 2.12br md (14.8), 2.00br md (14.1), 72.4 2.33 2.17 2.26 ddd m, 1.87 dd135.4 3.89 28.2 125.2 t (6.6)m 32.4 5.12 2.04 8 80.7 28.0 2.26 m, 1.87 dd 2.33 brdd d (14.8), 2.17 br dd d (14.1), 2.26 (7.3, 14.8 ) 2.09 (7.4, 14.1) (8.5, 12.3) (11.3, 10.2, 3.0) 125.2 5.12 t (6.6) 28.2 28.0 2.26 dd (7.3, 14.8 ) 2.09 dd (7.4, 14.1) (8.5, 12.3) 75.1 47.2 2.01 m 26.6 2.04 m 1.98 2.01 m, 1.93 dd 26.6 2.53 d (13.8), 2.39 d (13.4), 75.1 m ddd 135.4 2.04 m 35.247.2 9 3.89 3.9) 1.77 d (13.4) 112.91.97 d (13.8) 80.7 (12.9, 32.4 2.04 m 3.89 ddd (11.3, 10.2, 3.0) 32.4 112.9 80.7 2.04 m (11.3, 10.2, 3.0) 10 77.1 2.59m, m1.93 dd 125.2 5.12 t (6.6) 2.53 d (13.8), 2.39 d (13.4), 30.3 1.98 9 43.3 35.2 1.98 m, 1.93 dd 135.4 2.531.97 d (13.8), 2.391.77 d (13.4), d (13.8) d (13.4) 42.135.2 (12.9, 9152.5 43.3 11 2.29 m 3.9) 135.426.6 2.04 m 1.97 d (13.8) 1.77 d (13.4) (12.9,2.59 3.9)m 10 77.1 30.3 125.2 5.12 t (6.6) 4.62 dt (12.8, 2.4), 4.49 dt (12.6, 2.2), 10 11 77.1152.5 30.3 2.592.29 m m 125.226.6 5.12 t 2.04 (6.6)m 12 71.3 178.9 42.1 32.4 2.04 m 4.26 dt (12.6, 2.2) 11 152.5 4.42 dt (12.8, 2.29 m 26.6 2.04 m 4.62 dt2.4) (12.8, 2.4), 4.49 dt (12.6, 2.2), 42.1 12 71.3 4.62 dt (12.8, 2.4), 178.9 32.4 2.04 m dt (12.6, 2.2),2.2) dt (12.8, 2.4)5.184.49 dt (12.6, 5.28 br4.42 t (2.4), br t4.26 (2.2), 12 32.4 13 105.8 71.3 12.8178.9 1.23 d (7.0) 23.6 2.04 m1.68 s 4.42 dt (12.8, 2.4) 4.26 dt (12.6, 2.2) 5.18 br t (2.2), 5.07 br t5.28 (2.4)br t (2.4), 4.97 br t (2.2) 13 105.8 12.8 1.23 d (7.0) 23.6 1.68 s 5.285.07 br t br (2.4), 5.184.97 br t br (2.2), t (2.4) t (2.2) 13 105.8 1.23 d 23.623.6 1.68 s 1.68 s 14 28.6 1.35 sbr t (2.4) 1.23 s br t (2.2) 17.012.8 1.21 d (7.0) (7.3) 5.07 4.97 14 28.6 1.35 s 1.23 s 17.0 1.21 d (7.3) 23.6 1.68 s 14 12.4 1.35 1.23 1.210.95 d s 1.68 15 0.96 d (7.0) d (7.3) (7.2) 15 28.612.4 1.06 d (7.3) 1.06sd (7.3) 0.96sd (7.0) 16.817.016.8 0.95 d (7.2) 23.623.6 23.6 1.681.68 s s 157-OH 12.4 1.06 d (7.3) 0.96 d 5.16 (7.0)s 16.8 0.95 d (7.2) 23.6 1.68 s 7-OH 5.16 s 7-OH 5.165.00 s s 10-OH 10-OH 10-OH 5.00 s5.00 s c a b
4
Measured in CD3OD, Measured in CDCl3, Chemical shifts are given in ppm; J values are in a Measured in CD3OD, b Measured in CDCl b Measured 3, c Chemical are in given ppm; Jare values are in Measured in CD3 OD, in CDCl3 , c Chemical shifts shifts are given ppm;inJ values in parentheses parentheses and reported in Hz. andparentheses reported in Hz. and reported in Hz. a
Figure 2. Key HMBC and 1H-1H COSY correlations of compound 1 and 2.
1H11H Figure 2. Key HMBC and1 HH COSY COSY correlations ofof compound 1 and 2. 2. Figure 2. Key HMBC and correlations compound 1 and
15 15
Measured for 1 Measured for11 Cald. for Cald. for 1for ent-1 Cald. Cald. for ent-1
10 10 5 5 CD [mdeg] CD [mdeg] 0 0 -5 -5 -10 -10 -15 200 -15 200
250 250
300 350 Wavelength [nm] 300 350 Wavelength [nm]
400 400
3. Measured CD curves 1 and calculated CDcurves curvesof of 11 and and ent-1. FigureFigure 3. Measured CD curves of 1ofand calculated CD ent-1. Figure 3. Measured CD curves of 1 and calculated CD curves of 1 and ent-1.
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Compound 2 was isolated as a colorless oil. The molecular formula was determined to 21, the 274 basis of HR-ESI-MS (m/z 250.1563 [M]+ , calcd. 250.1569 for C415ofH822 O3 ) be CMolecules 15 H22 O2016, 3 on (Supplementary Materials), indicating five degrees of unsaturation. The 13 C, DEPT NMR spectra Compound 2 was isolated as a colorless oil. The molecular formula was determined to be along with the HSQC experiment indicated the presence of three methyls, four methylenes, five C15H22O3 on the basis of HR-ESI-MS (m/z 250.1563 [M]+, calcd. 250.1569 for C15H22O3) (Supplementary methines and three quaternary carbons, a carbonyl included. The structural units of C-2–C-3–C-4–C-15, Materials), indicating five degrees of unsaturation. The 13C, DEPT NMR spectra along with the 1 H-1 H COSY spectrum. C-11–C-13 C-7–C-8–C-9–C-10–C-14 were undoubtedly determined by the HSQCand experiment indicated the presence of three methyls, four methylenes, five methines and three The fragment C-5–C-1(C-2)–C-10(C-14)–C-9–C-8 was determined by the HMBCC-11–C-13 correlations quaternaryofcarbons, a carbonyl included. The structural units of C-2–C-3–C-4–C-15, and from 1 1 H-10C-7–C-8–C-9–C-10–C-14 (δH 2.59) to C-1(δC 73.8)/C-2(δ were undoubtedly determined by the H-CH80.7)/C-9(δ COSY spectrum. The fragment C 29.6)/C-5(δ C 72.4)/C-8(δ C 35.2)/C-14(δ C 17.0). The HMBC correlation from H-15 (δHwas 0.95) to C-3(δby from (δH 2.26) of C-5–C-1(C-2)–C-10(C-14)–C-9–C-8 determined the HMBC correlations from H-10H-6 (δH 2.59) C 26.4)/C-4(δ C 39.6)/C-5, to C-1(δ 29.6)/C-5(δ C 72.4)/C-8(δ C 80.7)/C-9(δ C 35.2)/C-14(δ C 17.0). 178.9), to C-5/C-7 (δCC 73.8)/C-2(δ 47.2)/C-8C and from H-13 (δH 1.23) to C-7/C-11(δ indicated C 42.1)/C-12(δ C The HMBC correlation from H-15 (δ H 0.95) to C-3(δ C 26.4)/C-4(δ C 39.6)/C-5, from H-6 (δ H 2.26) to C-5/C-7 the fragment of C-3–C-4(C-15)–C-5–C-6–C-7–C-11(C-13)–C-12. By comprehensive analysis of the (δCCOSY 47.2)/C-8 and from H-13 (δH 1.23) to C-7/C-11(δ C 42.1)/C-12(δC 178.9), indicated the fragment of 1 H-1 H spectrum and HMBC correlations (Figure 2), the guaiane skeleton of 2 was deduced. C-3–C-4(C-15)–C-5–C-6–C-7–C-11(C-13)–C-12. By comprehensive analysis of the 1H-1H COSY The 13 C-NMR signals at δC 72.4 and 73.8, both of which were quaternary carbons, indicated that the spectrum and HMBC correlations (Figure 2), the guaiane skeleton of 2 was deduced. The 13C-NMR epoxide ring exists between C-1 (δC 73.8) and C-5 (δC 72.4). The guaiane skeleton, carbonyl group signals at δC 72.4 and 73.8, both of which were quaternary carbons, indicated that the epoxide ring and the epoxide ring 4 (δ degrees of unsaturation, which suggested the presence of one exists between C-1accounted (δC 73.8) andfor C-5 C 72.4). The guaiane skeleton, carbonyl group and the epoxide another The methine signal at δC 80.7 (C-8) andsuggested the carbonyl at δC 178.9 (C-12), combined ringring. accounted for 4 degrees of unsaturation, which the presence of one another ring. The with the HMBC possible to deduce of a combined lactone ring. the planar methinecorrelations, signal at δC were 80.7 (C-8) and the carbonylthe at occurrence δC 178.9 (C-12), withThus the HMBC structure of compound 2 wastoestablished as shown of (Figure 2). ring. The Thus ROESY experiment (Figure 4) correlations, were possible deduce the occurrence a lactone the planar structure of compound 2 was established as shown (Figure 2). The ROESY experiment (Figure 4) showed showed correlations of H-8 (δH 3.89) to Me-14 (δH 1.21, β-oriented), and from H-7 (δH 2.01) to H-8 correlations of H-8suggested (δH 3.89) to that Me-14 (δH H-8, 1.21, β-oriented), fromwere H-7 (δ H 2.01) to H-8 andcorrelations Me-13 and Me-13 (δH 1.23), H-7, Me-13 and and Me-14 β-oriented. The (δH (δ 1.23),2.29) suggested that(δH-7,1.87) H-8, and Me-13 and to Me-14 were The correlations of H-11 (δH 2.29) of H-11 to H-6a H-6a Me-15 (δβ-oriented. H H H 0.95) indicated that H-6a, H-11 and Me-15 to H-6a (δH 1.87) and H-6a to Me-15 (δH 0.95) indicated that H-6a, H-11 and Me-15 were α-oriented. were α-oriented. The stereochemistry of the epoxide ring between C-1 and C-5 of 2, was assigned The stereochemistry of the epoxide ring between C-1 and C-5 of 2, was assigned to be β-oriented to be β-oriented based on the absence of ROESY correlations between H-4 or Me-15 and H-8, which based on the absence of ROESY correlations between H-4 or Me-15 and H-8, which method has been method has used for the similar known compound (+)-1,5-epoxy-nor-ketoguaiene isolated used forbeen the similar known compound (+)-1,5-epoxy-nor-ketoguaiene isolated from agarwood of from agarwood of Aquilaria genus [12]. Based on biosynthetic considerations, the stereogenic center C-4 Aquilaria genus [12]. Based on biosynthetic considerations, the stereogenic center C-4 was proposed was proposed and consequently, the absolute configuration of compound 2 wastoassumed to be to S, be andS,consequently, the absolute configuration of compound 2 was assumed be 1S, 4S, to 5S,be 1S, 7R,7R, 8R,8R, 10S,10S, andand 11R.11R. 4S, 5S,
Figure KeyROESY ROESYcorrelations correlations of 1 and 2. 2. Figure 4. 4. Key ofcompound compound 1 and
The structure of compound1,5,9-trimethyl-1,5,9-cyclododecatriene 1,5,9-trimethyl-1,5,9-cyclododecatriene (3), which waswas isolated from from The structure of compound (3), which isolated agarwood for the first time, was deduced by analyzing the MS, 1D and 2D NMR spectra. The 1D and agarwood for the first time, was deduced by analyzing the MS, 1D and 2D NMR spectra. The 1D 2D NMR data (Table 1) of 3 were also firstly reported in this study. The structures of compounds 4–9, and 2D NMR data (Table 1) of 3 were also firstly reported in this study. The structures of which have been isolated from agarwood, were identified by comparison of their spectroscopic data compounds 4–9, whichinhave been isolated from agarwood, were by comparison of with those reported the literatures as 4-epi-15-hydroxyacorenone (4)identified [13], 7αH-9(10)-ene-11,12their epoxy-8-oxoeremophilane spectroscopic data with(5)those reported in the literatures as 4-epi-15-hydroxyacorenone (4) [13], [7], neopetasane (6) [14], (1β,4αβ,7β,8αβ)-octahydro-7-[1-(hydroxymethyl) 7αH-9(10)-ene-11,12-epoxy-8-oxoeremophilane (5) [7], neopetasane (6) [14], (1β,4αβ,7β,8αβ)-octahydro-7ethenyl]-1,8α-dimethylnaphthalen-4α(2H)-ol (7) [14], valerianol (8) [15], 11-hydroxy-valenc-1(l0)-en[1-(hydroxymethyl)ethenyl]-1,8α-dimethylnaphthalen-4α(2H)-ol (7) [14], from valerianol (8)agarwood [15], 11-hydroxy2-one (9) [16], respectively. Of them, compounds 7 and 9 were isolated Chinese of A. sinensis for the (9) first[16], time.respectively. Among theseOf nine isolated sesquiterpenoids, 5 possesses a valenc-1(l0)-en-2-one them, compounds 7 and 9 compound were isolated from Chinese delicate sweet smell. agarwood of A. sinensis for the first time. Among these nine isolated sesquiterpenoids, compound 5 sesquiterpenoids possesses These a delicate sweet smell. above were evaluated for the inhibitory activity against AChE, and compounds 2–9 were assessed for antibacterial activities against S. aureus and R. solanacearum. The These sesquiterpenoids above were evaluated for the inhibitory activity against AChE, and results showed that compounds 1, 2, 4–6 and the EtOAc extract exhibited different levels of inhibitory compounds 2–9 were assessed for antibacterial activities against S. aureus and R. solanacearum. The results
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showed that compounds 1, 2, 4–6 and the EtOAc extract exhibited different levels of inhibitory activity with inhibition rate arranged from 13.3% to 70.7% (tacrine as the positive control; inhibition rate: 73.3%). Compounds 4, 5 and 8 showed antibacterial activities against both of the two strains, and compound 6 was inhibitory towards R. solanacearum (kanamycin sulfate as the positive control). From this paper, sesquiterpenes 1 and 2 belong to the guaiane type, and compounds 5–9 were all sesquiterpenes with eremophilane skeleton, while 3 and 4 were also sesquiterpenes but not the main type in agarwood. In addition, eleven eudesmane sesquiterpenes were also reported from our previous phytochemical study on this material [17]. Compared with the wild agarwood, the main types of sesquiterpenoids of Chinese agarwood induced by artificial holing are quite similar [6]. 3. Materials and Methods 3.1. General Imformation The IR spectra (KBr pellets) were run on a 380 FT-IR instrument from Nicolet (Thermo, Pittsburgh, PA, USA). The HRMS were recorded with an API QSTAR Pulsar mass spectrometer (Bruker, Bremen, Germany). The UV spectra were obtained from a DU-800 spectrometer (Beckman, Brea, CA, USA). Optical rotations were measured on an Autopol III polarimeter (Rudolph, Hackettstown, NJ, USA). CD spectra were recorded with a J-815 spectrometer (JASCO, Tokyo, Japan). The NMR spectra were recorded on an AV-500 spectrometer (500 MHz for 1 H-NMR and 125 MHz for 13 C-NMR; Bruker), using the solvent residue signal as the internal standard. Column chromatography was performed with ODS gel (20–45 mm, Fuji Silysia Chemical Co. Ltd., Durham, NC, USA), Sephadex LH-20 (Merck, Darmstadt, Germany) and silica gel (60–80, 200–300 mesh, Qingdao Haiyang Chemical Co. Ltd., Qingdao, China). TLC was carried out on silica gel G precoated plates (Qingdao Haiyang Chemical Co. Ltd.), and spots were detected by spraying with 5% H2 SO4 in EtOH followed by heating. 3.2. Plant Material Chinese agarwood induced by artificial holing from A. sinensis were collected from Xishuangbanna, Yunnan province, P.R. China, in November 2012. The botanical identification was made by Associate Prof. Jun Wang, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, where a voucher specimen (No. 20121108) was deposited. 3.3. Extraction and Isolation Dried powdered Chinese agarwood (4.7 Kg) induced by artificial holing was refluxed with 95% EtOH (5 L ˆ 6). The EtOH extract (510.0 g) was suspended in H2 O (2.5 L) and partitioned with EtOAc (2.5 L ˆ 3), and then n-BuOH (2.5 L ˆ 3). The EtOAc extract (310.0 g) was applied to silica gel vacuum liquid chromatography with a step gradient elution of CHCl3 –MeOH (v/v 1:0 to 0:1) to provide nine fractions (Fr.1–Fr.9). Fr.1 (143.2 g) was subjected to silica gel CC and eluted with PE–Me2 CO step gradient (v/v 1:0 to 0:1) to get nine fractions (Fr.1-1–Fr.1-9). Fr.1-1 (3.8 g) was applied to ODS gel eluting with MeOH´H2 O step gradient (v/v 3:7 to 1:0), then submitted to repeated CC on silica gel eluting with PE–CHCl3 (v/v 6:4 to 10:9) and Sephadex LH-20 (CHCl3 –MeOH 1:1) to obtained compounds 2 (3.0 mg), 3 (2.0 mg), 5 (16.0 mg), 6 (18.0 mg), and 8 (62.0 mg). Fr.1-4 (16.8 g) was purified using ODS gel with a step gradient elution of MeOH´H2 O (v/v 3:7 to 1:0), followed by silica gel CC eluting with CHCl3 ´MeOH (v/v 200:1) to obtain compound 4 (4.5 mg). Fr.1-5 (15.8 g) was submitted to column chromatography over silica gel eluted with CHCl3 –MeOH (v/v 200:1 to 100:1), and further purification with Sephadex LH-20 (CHCl3 –MeOH 1:1) to yield compounds 7 (102.0 mg) and 9 (32.0 mg). Compound 1 (4.0 mg) from Fr.1-8 was chromatographed on ODS gel eluting with MeOH´H2 O (v/v 3:7 to 1:0) and then applied to silica gel and eluted with CHCl3 –MeOH (v/v 40:1). 3-Oxo-7-hydroxylholosericin A (1). Colorless oil; C15 H20 O5 ; [αs24 D + 104.0 (c 0.50, MeOH); UV (MeOH) λmax (log ε): 272 (4.78), 254 (4.68); CD (c 0.1, MeOH) λmax (∆ε): 204 (´10.18), 298 (+1.68) nm; IR (KBr) υmax 3434, 2921, 1630, 1597, 1384, 1115, 709 cm´1 ; 1 H NMR (CD3 OD, 500 MHz), 1 H-NMR (DMSO,
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500 MHz) and 13 C-NMR (CD3 OD, 125 MHz) data, see Table 1; HREIMS m/z: [M + CF3 COO]´ 393.1166 (calcd. 280.1311 for C15 H20 O5 ). 1,5;8,12-Diepoxyguaia-12-one (2). Colorless oil; C15 H22 O3 ; [αs24 D ´ 113.0 (c 0.75, MeOH); UV (MeOH) λmax (log ε): 199 (4.12) nm; IR (KBr) υmax 3448, 2929, 1771, 1712, 1456, 1172, 988 cm´1 ; 1 H-NMR (CDCl3 , 500 MHz) and 13 C-NMR (CDCl3 , 125 MHz) data, see Table 1; HREIMS m/z: 250.1563 [M]+ (calcd for C15 H22 O3 , 250.1569). 3.4. AChE Inhibition Activity These above isolated compounds were tested for their acetylcholinesterase inhibitory activities with the spectrophotometric method developed by Ellman [18] with slightly modification. Acetylcholinesterase, 5,51 -dithio-bis-(2-nitrobenzoic) acid (DTNB, Ellman’s reagent), and S-acetylthiocholine iodide were purchased from Sigma Chemical (Saint Louis, MO, USA). The detail experimental procedures were the same as those published previously [8]. The inhibition rates were calculated as follows: % inhibition = (E ´ S)/E ˆ 100 (E is the activity of the enzyme without test compound, and S is the activity of enzyme with test compounds). The values are expressed as the mean ˘ SD of triplicate experiments. The AChE inhibitory activity experiment results are shown in Table 2. Table 2. AChE inhibition activity of the EtOAc extract and compounds 1–9 at 50 µg/mL. Compound
Percentage of Inhibition
Compound
Percentage of Inhibition
1 2 3 4 5
21.1 ˘ 0.8 13.3 ˘ 0.9
