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Sesquiterpenoids from the seeds of Sarcandra glabra and the potential anti-inflammatory effects
Fitoterapia 111 (2016) 7–11
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Sesquiterpenoids from the seeds of Sarcandra glabra and the potential anti-inflammatory effects Saimijiang Yaermaimaiti a,1, Peng Wang a,b,1, Jun Luo a, Rui-Jun Li a, Ling-Yi Kong a,⁎ a b
State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People's Republic of China School of Pharmacy, Yancheng Teachers University, Xiwang Road, Yancheng 224051, People's Republic of China
a r t i c l e
i n f o
Article history: Received 6 February 2016 Received in revised form 29 March 2016 Accepted 30 March 2016 Available online 02 April 2016 Keywords: Sarcandra glabra Sesquiterpenoids Linderanes Eudesmanes Nitric oxide
a b s t r a c t Five new sesquiterpenoids, including two linderanes (1–2) and three eudesmanes (3–5) were isolated from the seeds of Sarcandra glabra. Their structures and relative configurations were established by spectroscopic data analysis. 1 was a rare linderane derivative having an 18-membered triester ring which is a common characteristic in linderane dimers. Compounds 1–5 were investigated for their inhibitory effects on NO production in LPS-induced macrophages and 1 showed moderate bioactivity. © 2016 Elsevier B.V. All rights reserved.
1. Introduction The whole plant of Sarcandra glabra (Thunb.) Nakai (Chloranthaceae) has been used as a Traditional Chinese Medicine (TCM) for the treatment of inflammation and traumatic injuries in China for thousands of years [1]. Modern pharmacological research has also confirmed the traditional applications and curative effects of S. glabra recorded in the ancient books on TCMs [2]. However, there are only a few chemical investigations on the bioactive compounds responsible for the pharmacological effects in S. glabra. Sesquiterpenoids, linderanes and eudesmanes mostly, are reported to be the most important metabolites in S. glabra [2–3]. Particularly, linderane dimers isolated from S. glabra and other plants of the Chloranthaceae family, have attracted a lot more attention of medicinal chemists due to their complex structures and significant bioactivities [4–7]. For instance, some linderane dimers with novel structures and anti-inflammatory activities were isolated from S. glabra in our previous research [8]. In the subsequent investigation of sesquiterpenoids in S. glabra, five new sesquiterpenoid monomers, including two linderanes (1–2) and three eudesmanes (3–5) were isolated from the seeds of S. glabra. Compound 1, named as sarglabolide L, was a rare linderane derivative having an 18-membered triester ring
⁎ Corresponding author. E-mail address: [email protected] (L.-Y. Kong). 1 These authors contributed equally.
http://dx.doi.org/10.1016/j.fitote.2016.03.020 0367-326X/© 2016 Elsevier B.V. All rights reserved.
which is common in linderane dimers [2–3], and also exhibited moderate inhibitory effect on NO production in LPS-induced macrophages. Herein, we report the isolation, structural elucidation and bioassay of the new compounds. 2. Experimental 2.1. General Optical rotations were measured on a JASCO P-1020 polarimeter. HRESIMS experiments were performed using an Agilent UPLC-Q-TOFMS (6520B) spectrometer. UV and IR spectra were recorded on a Shimadzu UV-2450 spectrometer and a Bruker Tensor 27 spectrometer, respectively. NMR spectra were recorded in CDCl3 or CD3OD on a Bruker AV-500 NMR instrument at 500 MHz (1H) and 125 MHz (13C). Silica gel (Qingdao Marine Chemical Co., Ltd., China), ODS (FuJi, Japan), MCI gel (Mitsubishi Chemical, Japan), and Sephadex LH-20 (Pharmacia, Sweden) were employed for separation by column chromatography. MPLC was carried out on a Quiksep system (H&E Co., Ltd., China). Preparative HPLC was performed on a Shimadzu LC-6A instrument with a SPD-10A detector and a shim-pack RP-C18 column (20 × 200 mm, 10 μm). Analytical HPLC was performed on an Agilent 1200 series instrument using a DAD detector and a shim-pack VP–ODS column (150 × 4.6 mm, 5 μm). Authentic L- and D-malic acid samples were purchased from J&K Chemical Ltd., (China). All solvents and reagents were of analytical grade.
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Table 1 1 H (500 MHz) and 13C NMR (125 MHz) data of compounds 1–3. 1a δH (J in Hz) 1 2α 2β 3 4 5 6 7 8 9
1.74, m 0.85, ddd (9.1, 8.5, 6.0) 1.46, m 1.51, m 2.40, dd (13.7, 3.4) 2.63, ddd (17.2, 13.7, 1.6) 2.96, dd (17.2, 3.4)
6.37, s
10 11 12 13 14 15 1’ 2’ 3’ 4’ 5’ 6’ 1” 2” 3”
5.02, br.d (13.3) 4.73, d (13.3) 1.19, s 4.14, d (11.8) 4.71, d (11.8)
6.66, td (5.8, 1.1) 4.60, dd (15.0, 5.7) 5.31, dd (15.0, 5.5) 1.92, d (1.1)
4.48, dd (5.8, 3.4) 2.86, dd (16.7, 5.8) 3.02, dd (16.7, 3.4)
4” a b
2b δC 27.9 12.4 28.4 78.6 64.2 22.1 154.5 148.8 124.5 43.3 120.1 169.0 56.4 22.8 72.3 166.9 130.5 135.1 62.2 13.2
3a
δH (J in Hz)
δC
1.35, td (7.7, 3.4) 0.81, m 0.96, m 1.93, m
δH (J in Hz)
28.6 16.8
2.48, m 2.45, m 2.67, d (13.1)
1.87, d (13.1) 2.67, d (13.1)
1.83, s 0.93, s 4.80, s 4.97, s 4.04, d (7.8) 3.28, br.d (8.9) 3.23, m 3.21, m
3.82, dd (12.4, 5.7) 2.71, dd (17.1, 5.7) 2.64, dd (17.1, 12.4)
δC 73.5 42.6
24.1 152.0 66.8 25.3
6.70, br. d (1.6)
197.7 128.7 154.6 116.3
163.9 107.6 49.6
2.35, m 2.17, m
156.3 22.3 32.7
39.1 124.0 173.7 8.2
1.42, s 1.42, s
38.5 73.5 29.3 29.1
17.9 106.2
1.04, s 1.85, s
15.1 10.5
97.6 78.2 74.4 71.6
2.97, ddd (9.5, 6.0, 2.2) 3.55, dd (11.9, 6.0) 3.75, dd (11.9, 2.2)
78.4 62.6
173.1 67.2 38.3 170.5
Recorded in CDCl3. Recorded in CD3OD.
2.2. Plant material The fresh seeds of S. glabra were collected in Ganzhou, Jiangxi province, PR China in November 2013. The plant material was authenticated by Prof. Mian Zhang, Department of Medicinal Plants, China Pharmaceutical University. A voucher specimen, (No. CSH201311) was deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University.
HPLC to afford 3 (15.2 mg) and 4 (6.6 mg). Fr. 3 (12 g) was also subjected to an MCI gel column and eluted with 40%, 60%, 80% and 100% methanol to afford fractions Fr. 3.1–4. Fr. 3.2 was further chromatographed on an ODS column and purified by preparative HPLC to afford 2 (10.5 mg).
Table 2 1 H (500 MHz) and 13C NMR (125 MHz) data of compounds 4–5 in CDCl3. 4
2.3. Extraction and isolation
δH (J in Hz)
The fresh seeds of S. glabra (10 kg) were air dried and roughly ground. The ground seeds were then extracted with 95% EtOH (3 L) under reflux (4 × 2 h) and the solvent was removed under reduced pressure to afford a brown and odorous crude extract (460 g). This extract was suspended in 2.0 L water and successively extracted with petroleum ether (4 × 2 L) and ethyl acetate (3 × 2 L). The ethyl acetate extract (70 g) was subjected to a silica gel column and eluted with CH2Cl2/MeOH (50:1, 25:1, 10:1, 0:1, v/v), affording four fractions (Fr. 1–4). Fr. 2 (27 g) was further applied to a silica gel column eluted with a continuous gradient of petroleum ether/acetone (3:1 to 1:1, v/v) to afford 30 subfractions (Fr. 2.1–30). Frs. 2.1–15 were combined and chromatographed on an MCI gel column eluted with 60%, 80% and 100% methanol. The 60% methanol eluate was further separated on a reversed-phase MPLC and Sephadex LH-20 gel columns, and purified by preparative HPLC to obtain 1 (2.1 mg) and 5 (3.0 mg). Frs. 2.19–26 were combined and subjected to an MCI gel column eluted with 60%, 80% and 100% methanol. The 60% methanol eluate was further applied to a Sephadex LH-20 gel column, and purified by preparative
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
1.14, m 1.57, m 1.53, m 1.89, dt (13.5, 3.5) 1.33, m 1.75, m 1.21, m 2.38, t (14.0) 2.86, dd (14.5, 3.5) 4.86, dd (11.5, 6.3) 1.00, t (11.8) 2.14, dd (12.0, 6.3)
1.80, s 1.22, s 3.43, d (10.4) 3.60, d (10.4)
5 δC 40.7 17.0 36.2 73.2 49.3 23.4 163.2 78.2 49.9 35.2 120.4 175.1 8.4 19.2 70.0
δH (J in Hz) 1.26, t (11.9) 1.87, br. d (13.3) 3.92, m 1.99, t (11.8) 2.72, dd (12.4, 4.9) 1.93, dd (12.8, 3.7) 2.84, dd (13.9, 12.8) 2.77, dd (13.9, 3.7) 4.84, dd (10.8, 6.8) 1.16, t (11.8) 2.35, dd (12.2, 6.3)
1.83, s 0.92, s 4.72, s 4.99, s
δC 49.5 67.0 46.0 145.4 49.9 25.4 161.9 77.7 47.1 36.8 120.9 174.7 8.4 17.6 109.7
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2.3.1. Compound 1 White powder; [α]23 D −4.6 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 283 (4.06), 205 (4.12) nm; CD (MeOH) 215 (Δε − 0.80), 228 (Δε − 0.40), 258 (Δε + 0.33) nm; IR (KBr) νmax 3121, 2956, 1747, 1715, 1385, 1254, 1209, 959 cm−1; 1H and 13C NMR data: see Table 1; HRESIMS m/z 497.1421 [M + Na]+ (calcd. for C24H26O10Na 497.1418).
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(50 μL) was mixed with an equal volume of Griess reagent in a 96-well plate for 15 min at room temperature before measuring the optical density at 570 nm using a microplate reader. L-NMMA was used as the reference compound. The screening results were expressed as IC50 values. 3. Results and discussion
2.3.2. Compound 2 White powder; [α]23 D −7.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 268 (3.23), 206 (4.16) nm; CD (MeOH) 205 (Δε + 18.4), 229 (Δε − 5.97), 260 (Δε + 3.67) nm; IR (KBr) νmax 3420, 2925, 1752, 1699, 1459, 1387, 1256, 1078, 946 cm−1; 1H and 13C NMR spectral data: see Table 1; HRESIMS m/z 431.1680 [M + Na]+ (calcd. for C21H28O8Na 431.1676). 2.3.3. Compound 3 Colorless oil; [α]23 D +4.6 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 297 (4.23) nm; IR (KBr) νmax 3420, 2974, 2936, 1735, 1701, 1641, 1613, 1459, 1384, 1178, 958 cm−1; 1H and 13C NMR spectral data: see Table 1; HRESIMS m/z 273.1460 [M + Na]+ (calcd. for C15H22O3Na 273.1461). 2.3.4. Compound 4 Colorless oil; [α]23 D +17.6 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 221 (4.30) nm; CD (MeOH) 226 (Δε + 11.2) nm; IR (KBr) νmax 3420, 2932, 2873, 1735, 1641, 1459, 1386, 1254, 1040, 993 cm−1; 1H and 13C NMR spectral data: see Table 2; HRESIMS m/z 289.1408 [M + Na]+ (calcd. for C15H22O4Na 289.1410). 2.3.5. Compound 5 Colorless oil; [α]23 D +65.2 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 219 (4.26) nm; CD (MeOH) 219 (Δε + 35.5) nm; IR (KBr) νmax 3363, 2929, 2858, 1745, 1644, 1434, 1385, 1257, 1039, 894 cm− 1; 1H and 13C NMR spectral data: see Table 2; HRESIMS m/z 249.1484 [M + H]+ (calcd. for C15H21O3, 249.1485). 2.4. Determination of the configuration of C-2″ in compound 1 2.4.1. Preparation of (R)-MTPA ester of dimethyl malate Compound 1 (1.0 mg) was dissolved in 100 μL methanol and added to 100 μL 10% K2CO3 (methanol/water 2:1, v/v). After hydrolysis at 60 °C for 2 h, the reaction mixture was blow-dried, then acidified by adding 0.1 M HCl (500 μL) and extracted with EtOAc (500 μL × 3). The aqueous layer was dried and dissolved in methanol (200 μL). A drop of SOCl2 was added to the solution to catalyze the esterification reaction at room temperature for 12 h. After evaporation, the mixed methyl esters of 4-hydroxytiglic acid and malic acid were dissolved in dry pyridine (100 μL) and reacted with (S)-MTPA chloride (2 μL) overnight to obtain the final products. The corresponding authentic (R)-MTPA esters were also prepared from the commercial D and L-malic acid, respectively. 2.4.2. HPLC analysis of (R)-MTPA esters of dimethyl malate The authentic (R)-MTPA ester of dimethyl D-malate and the authentic (R)-MTPA ester of dimethyl L-malate were analyzed on an Agilent 1200 series instrument using a DAD detector and a shim-pack VP–ODS column (150 × 4.6 mm, 5 μm). The running conditions were a flow rate of 1 mL/min, a column temperature of 25 °C and a detection wavelength at 220 nm. The retention times (tR) of the authentic (R)-MTPA esters were 6.52 and 7.72 min, respectively, with an isocratic elution of MeOH/H2O (65:35, v/v). The (R)-MTPA ester prepared from 1 was also analyzed under the above conditions and had a retention time (tR) of 7.72 min (see supplementary data). 2.5. NO production inhibition assay Raw264.7 cells were seeded into a 96-well plate (105 cells per well) and pretreated with a range of concentrations of 1–5 for 1 h, followed by incubation with or without LPS (100 ng/mL) for 24 h. The supernatant
Compound 1 was obtained as a white powder. Its HRESIMS spectrum showed a quasimolecular ion peak at m/z 497.1421 ([M + Na]+, calcd. for C24H26O10Na, 497.1418), indicating a molecular formula of C24H26O10 for 1. The maximum UV absorption peak at 283 nm suggested the possibility of a large conjugated system in the molecule. The IR spectrum of 1 showed the characteristic absorption peaks of hydroxyl (3121 cm−1) and ester carbonyl (1747, 1715 cm− 1) groups. In the 1H NMR and HSQC spectra of 1, two characteristic upfield signals ascribed to the methylene protons of the cyclopropane ring in a linderane were observed at δH 0.85 (H-2α, ddd, J = 9.1, 8.5, 6.0 Hz) and 1.46 (H-2β, m), suggesting a linderane skeleton for 1 [8]. A tiglyl moiety, common in linderane dimers [8], could be recognized in the 1H NMR spectrum at δH 6.66 (H-3′, td, J = 5.8, 1.1 Hz), 4.60 (H-4′, dd, J = 15.0, 5.7 Hz), 5.31 (H-4′, dd, J = 15.0, 5.5 Hz), and 1.92 (CH3–5′, d, J = 1.1 Hz). The 13C NMR spectrum of 1 exhibited 24 carbon signals that were in agreement with its molecular formula. With the exception of the carbons belonging to the linderane and the tiglyl units, four carbons were remaining, which could be assigned to a malic acid residue according to the NMR signals at δH 4.48 (H-2″, dd, J = 5.8, 3.4 Hz), 2.86 (H-3″, dd, J = 16.7, 5.8 Hz) and 3.02 (H-3″, dd, J = 16.7, 5.8 Hz), and at δC 173.1 (C-1″), 67.2 (C-2″), 38.3 (C-3″) and 170.5 (C-4″). The linderane moiety in the molecule could be easily constructed based on the HMBC correlations (Fig. 2). In addition, the observed HMBC correlations from H-15 to C-1′, H-13 to C-4″, and H-4′ to C-1″ suggested a pendulous triester ring below the linderane framework that was very common in the linderane dimers from the plants of Chloranthaceae [2–3]. The relative configuration of the linderane moiety of 1 was established to be the same as those of the linderane analogues by its ROESY spectrum, which displayed the key ROESY correlations of H-2α/H-1, H-2α/H, H-3/H-15, H-5/H-15 and H-2β/CH3–14. The configuration of the malic residue in the molecule was assigned as L-form by combined methodologies including chemical derivatization and HPLC analysis [8]. Thus, compound 1 was determined to have the structure as shown in Fig.1. Compound 2 was obtained as a white powder. Its molecular formula was established as C21H28O8 by HRESIMS (m/z 431.1680 [M + Na]+, calcd. for C21H28O8Na, 431.1676). The 1H NMR and HSQC spectra of 2 also suggested a linderane unit in the molecule based on the characteristic upfield methylene at δH 0.81 (H-2α, m) and 0.96 (H-2β, m). The fragment ion peak at m/z 247.2 [M + H-162]+ in the ESIMS spectrum indicated that a glucosyl was possibly attached to the linderane. The 1 H and 13C NMR spectra of 2 also clearly exhibited the signals ascribed to the glucose unit. An acid hydrolysis of 2 afforded the free D-glucose that was characterized via TLC with the authentic compound. The anomeric configuration of the glucosyl was assigned as β based on the coupling constant (J = 7.8 Hz) of the anomeric proton. The key HMBC correlation from the anomeric proton (δH 4.04, H-1′) to C-8 (δC 107.6) revealed the location of the glucosyl. The configuration of C-8 was assigned as shown in Fig. 1 due to the key cross peak between H-1′ and CH3–14 in the ROESY spectrum. The previously reported compound chlorajaposide was wrongly determined at the configuration of C-8 and should be the epimer of compound 2 at C-8 [9]. Therefore, compound 2 was finally characterized as shizukanolide 8-O-β-D-glucopyranoside. Compound 3 was obtained as a colorless oil and was assigned a molecular formula C15H22O3 on the basis of an HRESIMS experiment (m/z 273.1460 [M + Na]+, calcd. for C15H22O3Na, 273.1461). The 1H NMR spectrum of 3 showed an olefinic proton at δH 6.70 (H-6, br. d,
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Fig. 1. Structures of compounds 1–5.
J = 1.6 Hz) and an oxygenated methine at δH 3.82 (H-1, dd, J = 12.4, 5.7 Hz) at the low field, and also showed four methyl singlets including two totally overlapped methyl signals at the high field. The 13C NMR spectrum of 3 exhibited 15 carbon signals including a conjugated carbonyl (δC 197.7), two pairs of double bonds (δC 156.2, 154.6, 128.6, 117.3), two oxygenated carbons (δC 73.5, 73.5), and eight aliphatic carbons. Analysis of the 2D NMR (HSQC and HMBC) spectra easily established that the framework of 3 was a eudesmane derivative (Fig. 1). The α-configuration of OH-1 was determined by the key ROESY correlation between H-1 and CH3-14. The OR value of 3 was close to zero and its CD spectrum did not show obvious Cotton effect in the corresponding experiments. A subsequent analysis using a chiral HPLC column was carried out and revealed that 3 was composed of a pair of racemic mixture (see S20 in Supplementary Material). Therefore, compound 3 was identified as shown in Fig. 1. Compound 4 was obtained as a colorless oil and was assigned a molecular formula C15H22O4 by HRESIMS (m/z 289.1408 [M + Na]+, calcd. for C15H22O4Na, 289.1410). According to the 1H and 13C NMR and HSQC spectra, compound 4 contained an oxygenated methine (δH 4.86, δC 78.2, CH-8), an oxygenated methylene (δH 3.43 and 3.60, δC 70.0, CH2–15), an oxygenated quaternary carbon (δC 73.2, C-4), a conjugated ester carbonyl (δC 175.1, C-12), a double bond (δC 163.2, C-7; δC 120.4, C-11) and two methyl groups (δH 1.80, CH3–13; δH 1.22, CH3–14). Detailed analysis of the HMBC spectrum determined its planar structure to be identical to that of a known eudesmane [10]. No obvious correlation between H-8 and CH3–14 was observed in the ROESY spectrum of 4, thus differentiating it from the known one, suggesting the trans-orientation in 4. In addition, the orientation of CH2–15 was assigned as β in 4 by the ROESY correlation between CH2–15 and CH3–14. Thus, compound 4 was determined to be an epimer of the known one at C-8.
Compound 5 was obtained as a colorless oil. Its molecular formula was defined as C15H20O3 by HRESIMS (m/z 249.1484 [M + H]+, calcd. for C15H21O3, 249.1485). The 1H NMR spectrum of 5 revealed two terminal olefinic protons at δH 4.99 (H-15, s) and 4.72 (H-15, s), two oxygenated methine at δH 4.84 (H-8, dd, J = 10.8, 6.8 Hz) and 3.92 (H-2, m), and two methyl groups at δH 1.83 (CH3–13, s) and 0.92 (CH3–14, s). The 13C NMR spectrum of 5 showed 15 carbons that was in accord with its molecular formula, including a conjugated ester carbonyl (δC 174.7), two double bonds (δC 161.9, 145.4, 120.9 and 109.7), two oxygenated carbons (δC 77.7 and 67.0) and eight aliphatic carbons. Analysis of the HMBC spectrum of 5 established its planar structure as that of another eudesmane lactone. The relative configuration of 5 was defined by the observed ROESY correlations of H-2/CH3-14 and H-8/ CH3-14, and the invisible ROESY correlation between H-5 and the above protons. Finally, 5 was characterized as shown in Fig. 1. On the bases of the traditional application of S. glabra as an antiinflammatory herb, the new compounds 1–5, were evaluated for their inhibitory effects on LPS-induced NO production in RAW264.7 cells, and as a result, compound 1 exhibited moderate bioactivity with an IC50 value at 30.65 ± 3.94 μmol/L (L-NMMA was used as positive control, IC50 = 37.46 ± 2.75 μmol/L). The other new compounds did not show any marked bioactivities up to 50 μmol/L. The exceptional bioactivity of 1 might be ascribed to the large ester ring in its molecule, since many linderane dimers from the Chloranthaceae family featuring the large ester ring also showed potent anti-inflammatory effects [8,11].
Conflict of interest statement The authors declare no conflicts of interest.
Fig. 2. Key HMBC and ROESY correlations of compound 1.
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Acknowledgements This research was supported in part by the National Natural Science Foundation of China (81430092, 31470416), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). This research was also supported by the Program for Changjiang Scholars and Innovative Research Team in University (IRT_15R63). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.fitote.2016.03.020. References [1] National pharmacopeia committee, China Pharmacopeia, Part I, China Medical Science Press, Beijing 2015, pp. 223–224.
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Sesquiterpenoids from the seeds of Sarcandra glabra and the potential anti-inflammatory effects Saimijiang Yaermaimaiti a,1, Peng Wang a,b,1, Jun Luo a, Rui-Jun Li a, Ling-Yi Kong a,⁎ a b
State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People's Republic of China School of Pharmacy, Yancheng Teachers University, Xiwang Road, Yancheng 224051, People's Republic of China
a r t i c l e
i n f o
Article history: Received 6 February 2016 Received in revised form 29 March 2016 Accepted 30 March 2016 Available online 02 April 2016 Keywords: Sarcandra glabra Sesquiterpenoids Linderanes Eudesmanes Nitric oxide
a b s t r a c t Five new sesquiterpenoids, including two linderanes (1–2) and three eudesmanes (3–5) were isolated from the seeds of Sarcandra glabra. Their structures and relative configurations were established by spectroscopic data analysis. 1 was a rare linderane derivative having an 18-membered triester ring which is a common characteristic in linderane dimers. Compounds 1–5 were investigated for their inhibitory effects on NO production in LPS-induced macrophages and 1 showed moderate bioactivity. © 2016 Elsevier B.V. All rights reserved.
1. Introduction The whole plant of Sarcandra glabra (Thunb.) Nakai (Chloranthaceae) has been used as a Traditional Chinese Medicine (TCM) for the treatment of inflammation and traumatic injuries in China for thousands of years [1]. Modern pharmacological research has also confirmed the traditional applications and curative effects of S. glabra recorded in the ancient books on TCMs [2]. However, there are only a few chemical investigations on the bioactive compounds responsible for the pharmacological effects in S. glabra. Sesquiterpenoids, linderanes and eudesmanes mostly, are reported to be the most important metabolites in S. glabra [2–3]. Particularly, linderane dimers isolated from S. glabra and other plants of the Chloranthaceae family, have attracted a lot more attention of medicinal chemists due to their complex structures and significant bioactivities [4–7]. For instance, some linderane dimers with novel structures and anti-inflammatory activities were isolated from S. glabra in our previous research [8]. In the subsequent investigation of sesquiterpenoids in S. glabra, five new sesquiterpenoid monomers, including two linderanes (1–2) and three eudesmanes (3–5) were isolated from the seeds of S. glabra. Compound 1, named as sarglabolide L, was a rare linderane derivative having an 18-membered triester ring
⁎ Corresponding author. E-mail address: [email protected] (L.-Y. Kong). 1 These authors contributed equally.
http://dx.doi.org/10.1016/j.fitote.2016.03.020 0367-326X/© 2016 Elsevier B.V. All rights reserved.
which is common in linderane dimers [2–3], and also exhibited moderate inhibitory effect on NO production in LPS-induced macrophages. Herein, we report the isolation, structural elucidation and bioassay of the new compounds. 2. Experimental 2.1. General Optical rotations were measured on a JASCO P-1020 polarimeter. HRESIMS experiments were performed using an Agilent UPLC-Q-TOFMS (6520B) spectrometer. UV and IR spectra were recorded on a Shimadzu UV-2450 spectrometer and a Bruker Tensor 27 spectrometer, respectively. NMR spectra were recorded in CDCl3 or CD3OD on a Bruker AV-500 NMR instrument at 500 MHz (1H) and 125 MHz (13C). Silica gel (Qingdao Marine Chemical Co., Ltd., China), ODS (FuJi, Japan), MCI gel (Mitsubishi Chemical, Japan), and Sephadex LH-20 (Pharmacia, Sweden) were employed for separation by column chromatography. MPLC was carried out on a Quiksep system (H&E Co., Ltd., China). Preparative HPLC was performed on a Shimadzu LC-6A instrument with a SPD-10A detector and a shim-pack RP-C18 column (20 × 200 mm, 10 μm). Analytical HPLC was performed on an Agilent 1200 series instrument using a DAD detector and a shim-pack VP–ODS column (150 × 4.6 mm, 5 μm). Authentic L- and D-malic acid samples were purchased from J&K Chemical Ltd., (China). All solvents and reagents were of analytical grade.
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Table 1 1 H (500 MHz) and 13C NMR (125 MHz) data of compounds 1–3. 1a δH (J in Hz) 1 2α 2β 3 4 5 6 7 8 9
1.74, m 0.85, ddd (9.1, 8.5, 6.0) 1.46, m 1.51, m 2.40, dd (13.7, 3.4) 2.63, ddd (17.2, 13.7, 1.6) 2.96, dd (17.2, 3.4)
6.37, s
10 11 12 13 14 15 1’ 2’ 3’ 4’ 5’ 6’ 1” 2” 3”
5.02, br.d (13.3) 4.73, d (13.3) 1.19, s 4.14, d (11.8) 4.71, d (11.8)
6.66, td (5.8, 1.1) 4.60, dd (15.0, 5.7) 5.31, dd (15.0, 5.5) 1.92, d (1.1)
4.48, dd (5.8, 3.4) 2.86, dd (16.7, 5.8) 3.02, dd (16.7, 3.4)
4” a b
2b δC 27.9 12.4 28.4 78.6 64.2 22.1 154.5 148.8 124.5 43.3 120.1 169.0 56.4 22.8 72.3 166.9 130.5 135.1 62.2 13.2
3a
δH (J in Hz)
δC
1.35, td (7.7, 3.4) 0.81, m 0.96, m 1.93, m
δH (J in Hz)
28.6 16.8
2.48, m 2.45, m 2.67, d (13.1)
1.87, d (13.1) 2.67, d (13.1)
1.83, s 0.93, s 4.80, s 4.97, s 4.04, d (7.8) 3.28, br.d (8.9) 3.23, m 3.21, m
3.82, dd (12.4, 5.7) 2.71, dd (17.1, 5.7) 2.64, dd (17.1, 12.4)
δC 73.5 42.6
24.1 152.0 66.8 25.3
6.70, br. d (1.6)
197.7 128.7 154.6 116.3
163.9 107.6 49.6
2.35, m 2.17, m
156.3 22.3 32.7
39.1 124.0 173.7 8.2
1.42, s 1.42, s
38.5 73.5 29.3 29.1
17.9 106.2
1.04, s 1.85, s
15.1 10.5
97.6 78.2 74.4 71.6
2.97, ddd (9.5, 6.0, 2.2) 3.55, dd (11.9, 6.0) 3.75, dd (11.9, 2.2)
78.4 62.6
173.1 67.2 38.3 170.5
Recorded in CDCl3. Recorded in CD3OD.
2.2. Plant material The fresh seeds of S. glabra were collected in Ganzhou, Jiangxi province, PR China in November 2013. The plant material was authenticated by Prof. Mian Zhang, Department of Medicinal Plants, China Pharmaceutical University. A voucher specimen, (No. CSH201311) was deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University.
HPLC to afford 3 (15.2 mg) and 4 (6.6 mg). Fr. 3 (12 g) was also subjected to an MCI gel column and eluted with 40%, 60%, 80% and 100% methanol to afford fractions Fr. 3.1–4. Fr. 3.2 was further chromatographed on an ODS column and purified by preparative HPLC to afford 2 (10.5 mg).
Table 2 1 H (500 MHz) and 13C NMR (125 MHz) data of compounds 4–5 in CDCl3. 4
2.3. Extraction and isolation
δH (J in Hz)
The fresh seeds of S. glabra (10 kg) were air dried and roughly ground. The ground seeds were then extracted with 95% EtOH (3 L) under reflux (4 × 2 h) and the solvent was removed under reduced pressure to afford a brown and odorous crude extract (460 g). This extract was suspended in 2.0 L water and successively extracted with petroleum ether (4 × 2 L) and ethyl acetate (3 × 2 L). The ethyl acetate extract (70 g) was subjected to a silica gel column and eluted with CH2Cl2/MeOH (50:1, 25:1, 10:1, 0:1, v/v), affording four fractions (Fr. 1–4). Fr. 2 (27 g) was further applied to a silica gel column eluted with a continuous gradient of petroleum ether/acetone (3:1 to 1:1, v/v) to afford 30 subfractions (Fr. 2.1–30). Frs. 2.1–15 were combined and chromatographed on an MCI gel column eluted with 60%, 80% and 100% methanol. The 60% methanol eluate was further separated on a reversed-phase MPLC and Sephadex LH-20 gel columns, and purified by preparative HPLC to obtain 1 (2.1 mg) and 5 (3.0 mg). Frs. 2.19–26 were combined and subjected to an MCI gel column eluted with 60%, 80% and 100% methanol. The 60% methanol eluate was further applied to a Sephadex LH-20 gel column, and purified by preparative
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
1.14, m 1.57, m 1.53, m 1.89, dt (13.5, 3.5) 1.33, m 1.75, m 1.21, m 2.38, t (14.0) 2.86, dd (14.5, 3.5) 4.86, dd (11.5, 6.3) 1.00, t (11.8) 2.14, dd (12.0, 6.3)
1.80, s 1.22, s 3.43, d (10.4) 3.60, d (10.4)
5 δC 40.7 17.0 36.2 73.2 49.3 23.4 163.2 78.2 49.9 35.2 120.4 175.1 8.4 19.2 70.0
δH (J in Hz) 1.26, t (11.9) 1.87, br. d (13.3) 3.92, m 1.99, t (11.8) 2.72, dd (12.4, 4.9) 1.93, dd (12.8, 3.7) 2.84, dd (13.9, 12.8) 2.77, dd (13.9, 3.7) 4.84, dd (10.8, 6.8) 1.16, t (11.8) 2.35, dd (12.2, 6.3)
1.83, s 0.92, s 4.72, s 4.99, s
δC 49.5 67.0 46.0 145.4 49.9 25.4 161.9 77.7 47.1 36.8 120.9 174.7 8.4 17.6 109.7
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2.3.1. Compound 1 White powder; [α]23 D −4.6 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 283 (4.06), 205 (4.12) nm; CD (MeOH) 215 (Δε − 0.80), 228 (Δε − 0.40), 258 (Δε + 0.33) nm; IR (KBr) νmax 3121, 2956, 1747, 1715, 1385, 1254, 1209, 959 cm−1; 1H and 13C NMR data: see Table 1; HRESIMS m/z 497.1421 [M + Na]+ (calcd. for C24H26O10Na 497.1418).
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(50 μL) was mixed with an equal volume of Griess reagent in a 96-well plate for 15 min at room temperature before measuring the optical density at 570 nm using a microplate reader. L-NMMA was used as the reference compound. The screening results were expressed as IC50 values. 3. Results and discussion
2.3.2. Compound 2 White powder; [α]23 D −7.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 268 (3.23), 206 (4.16) nm; CD (MeOH) 205 (Δε + 18.4), 229 (Δε − 5.97), 260 (Δε + 3.67) nm; IR (KBr) νmax 3420, 2925, 1752, 1699, 1459, 1387, 1256, 1078, 946 cm−1; 1H and 13C NMR spectral data: see Table 1; HRESIMS m/z 431.1680 [M + Na]+ (calcd. for C21H28O8Na 431.1676). 2.3.3. Compound 3 Colorless oil; [α]23 D +4.6 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 297 (4.23) nm; IR (KBr) νmax 3420, 2974, 2936, 1735, 1701, 1641, 1613, 1459, 1384, 1178, 958 cm−1; 1H and 13C NMR spectral data: see Table 1; HRESIMS m/z 273.1460 [M + Na]+ (calcd. for C15H22O3Na 273.1461). 2.3.4. Compound 4 Colorless oil; [α]23 D +17.6 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 221 (4.30) nm; CD (MeOH) 226 (Δε + 11.2) nm; IR (KBr) νmax 3420, 2932, 2873, 1735, 1641, 1459, 1386, 1254, 1040, 993 cm−1; 1H and 13C NMR spectral data: see Table 2; HRESIMS m/z 289.1408 [M + Na]+ (calcd. for C15H22O4Na 289.1410). 2.3.5. Compound 5 Colorless oil; [α]23 D +65.2 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 219 (4.26) nm; CD (MeOH) 219 (Δε + 35.5) nm; IR (KBr) νmax 3363, 2929, 2858, 1745, 1644, 1434, 1385, 1257, 1039, 894 cm− 1; 1H and 13C NMR spectral data: see Table 2; HRESIMS m/z 249.1484 [M + H]+ (calcd. for C15H21O3, 249.1485). 2.4. Determination of the configuration of C-2″ in compound 1 2.4.1. Preparation of (R)-MTPA ester of dimethyl malate Compound 1 (1.0 mg) was dissolved in 100 μL methanol and added to 100 μL 10% K2CO3 (methanol/water 2:1, v/v). After hydrolysis at 60 °C for 2 h, the reaction mixture was blow-dried, then acidified by adding 0.1 M HCl (500 μL) and extracted with EtOAc (500 μL × 3). The aqueous layer was dried and dissolved in methanol (200 μL). A drop of SOCl2 was added to the solution to catalyze the esterification reaction at room temperature for 12 h. After evaporation, the mixed methyl esters of 4-hydroxytiglic acid and malic acid were dissolved in dry pyridine (100 μL) and reacted with (S)-MTPA chloride (2 μL) overnight to obtain the final products. The corresponding authentic (R)-MTPA esters were also prepared from the commercial D and L-malic acid, respectively. 2.4.2. HPLC analysis of (R)-MTPA esters of dimethyl malate The authentic (R)-MTPA ester of dimethyl D-malate and the authentic (R)-MTPA ester of dimethyl L-malate were analyzed on an Agilent 1200 series instrument using a DAD detector and a shim-pack VP–ODS column (150 × 4.6 mm, 5 μm). The running conditions were a flow rate of 1 mL/min, a column temperature of 25 °C and a detection wavelength at 220 nm. The retention times (tR) of the authentic (R)-MTPA esters were 6.52 and 7.72 min, respectively, with an isocratic elution of MeOH/H2O (65:35, v/v). The (R)-MTPA ester prepared from 1 was also analyzed under the above conditions and had a retention time (tR) of 7.72 min (see supplementary data). 2.5. NO production inhibition assay Raw264.7 cells were seeded into a 96-well plate (105 cells per well) and pretreated with a range of concentrations of 1–5 for 1 h, followed by incubation with or without LPS (100 ng/mL) for 24 h. The supernatant
Compound 1 was obtained as a white powder. Its HRESIMS spectrum showed a quasimolecular ion peak at m/z 497.1421 ([M + Na]+, calcd. for C24H26O10Na, 497.1418), indicating a molecular formula of C24H26O10 for 1. The maximum UV absorption peak at 283 nm suggested the possibility of a large conjugated system in the molecule. The IR spectrum of 1 showed the characteristic absorption peaks of hydroxyl (3121 cm−1) and ester carbonyl (1747, 1715 cm− 1) groups. In the 1H NMR and HSQC spectra of 1, two characteristic upfield signals ascribed to the methylene protons of the cyclopropane ring in a linderane were observed at δH 0.85 (H-2α, ddd, J = 9.1, 8.5, 6.0 Hz) and 1.46 (H-2β, m), suggesting a linderane skeleton for 1 [8]. A tiglyl moiety, common in linderane dimers [8], could be recognized in the 1H NMR spectrum at δH 6.66 (H-3′, td, J = 5.8, 1.1 Hz), 4.60 (H-4′, dd, J = 15.0, 5.7 Hz), 5.31 (H-4′, dd, J = 15.0, 5.5 Hz), and 1.92 (CH3–5′, d, J = 1.1 Hz). The 13C NMR spectrum of 1 exhibited 24 carbon signals that were in agreement with its molecular formula. With the exception of the carbons belonging to the linderane and the tiglyl units, four carbons were remaining, which could be assigned to a malic acid residue according to the NMR signals at δH 4.48 (H-2″, dd, J = 5.8, 3.4 Hz), 2.86 (H-3″, dd, J = 16.7, 5.8 Hz) and 3.02 (H-3″, dd, J = 16.7, 5.8 Hz), and at δC 173.1 (C-1″), 67.2 (C-2″), 38.3 (C-3″) and 170.5 (C-4″). The linderane moiety in the molecule could be easily constructed based on the HMBC correlations (Fig. 2). In addition, the observed HMBC correlations from H-15 to C-1′, H-13 to C-4″, and H-4′ to C-1″ suggested a pendulous triester ring below the linderane framework that was very common in the linderane dimers from the plants of Chloranthaceae [2–3]. The relative configuration of the linderane moiety of 1 was established to be the same as those of the linderane analogues by its ROESY spectrum, which displayed the key ROESY correlations of H-2α/H-1, H-2α/H, H-3/H-15, H-5/H-15 and H-2β/CH3–14. The configuration of the malic residue in the molecule was assigned as L-form by combined methodologies including chemical derivatization and HPLC analysis [8]. Thus, compound 1 was determined to have the structure as shown in Fig.1. Compound 2 was obtained as a white powder. Its molecular formula was established as C21H28O8 by HRESIMS (m/z 431.1680 [M + Na]+, calcd. for C21H28O8Na, 431.1676). The 1H NMR and HSQC spectra of 2 also suggested a linderane unit in the molecule based on the characteristic upfield methylene at δH 0.81 (H-2α, m) and 0.96 (H-2β, m). The fragment ion peak at m/z 247.2 [M + H-162]+ in the ESIMS spectrum indicated that a glucosyl was possibly attached to the linderane. The 1 H and 13C NMR spectra of 2 also clearly exhibited the signals ascribed to the glucose unit. An acid hydrolysis of 2 afforded the free D-glucose that was characterized via TLC with the authentic compound. The anomeric configuration of the glucosyl was assigned as β based on the coupling constant (J = 7.8 Hz) of the anomeric proton. The key HMBC correlation from the anomeric proton (δH 4.04, H-1′) to C-8 (δC 107.6) revealed the location of the glucosyl. The configuration of C-8 was assigned as shown in Fig. 1 due to the key cross peak between H-1′ and CH3–14 in the ROESY spectrum. The previously reported compound chlorajaposide was wrongly determined at the configuration of C-8 and should be the epimer of compound 2 at C-8 [9]. Therefore, compound 2 was finally characterized as shizukanolide 8-O-β-D-glucopyranoside. Compound 3 was obtained as a colorless oil and was assigned a molecular formula C15H22O3 on the basis of an HRESIMS experiment (m/z 273.1460 [M + Na]+, calcd. for C15H22O3Na, 273.1461). The 1H NMR spectrum of 3 showed an olefinic proton at δH 6.70 (H-6, br. d,
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Fig. 1. Structures of compounds 1–5.
J = 1.6 Hz) and an oxygenated methine at δH 3.82 (H-1, dd, J = 12.4, 5.7 Hz) at the low field, and also showed four methyl singlets including two totally overlapped methyl signals at the high field. The 13C NMR spectrum of 3 exhibited 15 carbon signals including a conjugated carbonyl (δC 197.7), two pairs of double bonds (δC 156.2, 154.6, 128.6, 117.3), two oxygenated carbons (δC 73.5, 73.5), and eight aliphatic carbons. Analysis of the 2D NMR (HSQC and HMBC) spectra easily established that the framework of 3 was a eudesmane derivative (Fig. 1). The α-configuration of OH-1 was determined by the key ROESY correlation between H-1 and CH3-14. The OR value of 3 was close to zero and its CD spectrum did not show obvious Cotton effect in the corresponding experiments. A subsequent analysis using a chiral HPLC column was carried out and revealed that 3 was composed of a pair of racemic mixture (see S20 in Supplementary Material). Therefore, compound 3 was identified as shown in Fig. 1. Compound 4 was obtained as a colorless oil and was assigned a molecular formula C15H22O4 by HRESIMS (m/z 289.1408 [M + Na]+, calcd. for C15H22O4Na, 289.1410). According to the 1H and 13C NMR and HSQC spectra, compound 4 contained an oxygenated methine (δH 4.86, δC 78.2, CH-8), an oxygenated methylene (δH 3.43 and 3.60, δC 70.0, CH2–15), an oxygenated quaternary carbon (δC 73.2, C-4), a conjugated ester carbonyl (δC 175.1, C-12), a double bond (δC 163.2, C-7; δC 120.4, C-11) and two methyl groups (δH 1.80, CH3–13; δH 1.22, CH3–14). Detailed analysis of the HMBC spectrum determined its planar structure to be identical to that of a known eudesmane [10]. No obvious correlation between H-8 and CH3–14 was observed in the ROESY spectrum of 4, thus differentiating it from the known one, suggesting the trans-orientation in 4. In addition, the orientation of CH2–15 was assigned as β in 4 by the ROESY correlation between CH2–15 and CH3–14. Thus, compound 4 was determined to be an epimer of the known one at C-8.
Compound 5 was obtained as a colorless oil. Its molecular formula was defined as C15H20O3 by HRESIMS (m/z 249.1484 [M + H]+, calcd. for C15H21O3, 249.1485). The 1H NMR spectrum of 5 revealed two terminal olefinic protons at δH 4.99 (H-15, s) and 4.72 (H-15, s), two oxygenated methine at δH 4.84 (H-8, dd, J = 10.8, 6.8 Hz) and 3.92 (H-2, m), and two methyl groups at δH 1.83 (CH3–13, s) and 0.92 (CH3–14, s). The 13C NMR spectrum of 5 showed 15 carbons that was in accord with its molecular formula, including a conjugated ester carbonyl (δC 174.7), two double bonds (δC 161.9, 145.4, 120.9 and 109.7), two oxygenated carbons (δC 77.7 and 67.0) and eight aliphatic carbons. Analysis of the HMBC spectrum of 5 established its planar structure as that of another eudesmane lactone. The relative configuration of 5 was defined by the observed ROESY correlations of H-2/CH3-14 and H-8/ CH3-14, and the invisible ROESY correlation between H-5 and the above protons. Finally, 5 was characterized as shown in Fig. 1. On the bases of the traditional application of S. glabra as an antiinflammatory herb, the new compounds 1–5, were evaluated for their inhibitory effects on LPS-induced NO production in RAW264.7 cells, and as a result, compound 1 exhibited moderate bioactivity with an IC50 value at 30.65 ± 3.94 μmol/L (L-NMMA was used as positive control, IC50 = 37.46 ± 2.75 μmol/L). The other new compounds did not show any marked bioactivities up to 50 μmol/L. The exceptional bioactivity of 1 might be ascribed to the large ester ring in its molecule, since many linderane dimers from the Chloranthaceae family featuring the large ester ring also showed potent anti-inflammatory effects [8,11].
Conflict of interest statement The authors declare no conflicts of interest.
Fig. 2. Key HMBC and ROESY correlations of compound 1.
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Acknowledgements This research was supported in part by the National Natural Science Foundation of China (81430092, 31470416), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). This research was also supported by the Program for Changjiang Scholars and Innovative Research Team in University (IRT_15R63). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.fitote.2016.03.020. References [1] National pharmacopeia committee, China Pharmacopeia, Part I, China Medical Science Press, Beijing 2015, pp. 223–224.
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