Article pubs.acs.org/jnp
Bistinospinosides A and B, Dimeric Clerodane Diterpene Glycosides from Tinospora sagittata Wei Li,* Chao Huang, Qingbo Liu, and Kazuo Koike Faculty of Pharmaceutical Sciences, Toho University, Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan S Supporting Information *
ABSTRACT: Two dimeric clerodane diterpene glycosides, namely, bistinospinosides A (1) and B (2), were isolated from the roots of Tinospora sagittata. Their structures were elucidated by extensive spectroscopic data interpretation. The compounds feature an unusual 1,4-epoxycyclohexane ring in their structures and may be biosynthetically constructed via an intermolecular Diels−Alder [4+2] cycloaddition from the corresponding clerodane diterpene. The compounds were evaluated in a nitric oxide inhibitory assay using J774.1 macrophage-like cells.
C
Bistinospinoside A (1) was isolated as a colorless solid, [α]16 D +19.7 (c 0.23, MeOH). The molecular formula of 1 was established as C52H64O22 by HRESIMS (m/z 1063.3782 [M + Na]+; calcd for C52H64O22Na, 1063.3787). The IR spectrum of 1 showed absorptions at 3423 (hydroxy group) and 1751 (lactone) cm−1. The 13C NMR spectrum showed 51 wellresolved resonances and one signal overlapped by the solvent resonance of pyridine-d5 at δC 150.0 (Table 1). These carbons were further classified into four methyls, eight methylenes, 28 methines, and 12 quaternary carbons by analysis of the 1H, DEPT, and HMQC NMR data. Characteristic resonances with close chemical shifts mostly appeared in pairs (Table 1), suggesting the dimeric structure of 1. Detailed analysis of the 1D and 2D NMR data disclosed that the 1H and 13C NMR resonances could be assigned to two monomeric units, designated as parts A and B (Figure 1). Both of these contained a β-glucopyranosyl moiety with the resonance for the anomeric resonances at δH 5.77 (1H, d, J = 7.5 Hz) and 5.32 (1H, d, J = 7.4 Hz), respectively. The βglucopyranosyl moieties were both determined to be in the Dform by GLC analysis after acid hydrolysis. Furthermore, the 1 H and 13C NMR resonances assignable to part A included a set of characteristic downfield resonances at δH 6.73 (1H, brd, J = 1.7 Hz, H-14), 7.67 (1H, brt, J = 1.7 Hz, H-15), and 7.82 (1H, brd, J = 0.4 Hz, H-16) and carbon resonances at δC 126.3 (C13), 109.4 (C-14), 144.4 (C-15), and 140.6 (C-16), which
lerodane diterpenoids constitute one of the largest and, yet, still rapidly growing groups of naturally occurring diterpenoids.1 Depending on the relationship of rings A and B in the skeleton, clerodane diterpenoids can be divided into two classes, namely, cis- and trans-clerodanes. Extensive modification of the clerodane skeleton during their biosynthesis results in a large variation in their chemical structures, with a wide range of biological effects having been observed, including antifeedant, antiulcer, cytotoxic, anti-inflammatory, and antibacterial activities.2 Tinospora sagittata (Oliv.) Gagnep (Menispermaceae) is widely distributed in southern mainland China. Its tuberous roots have been used in traditional Chinese medicine to relieve sore throats, treat superficial infection, and prevent diarrhea.3 Previous chemical investigations have demonstrated the presence of sterols,4 alkaloids,5 and diterpenoids.6 Among them, clerodane furanoditerpenoids are characteristic constituents, which have shown cytotoxic,6 antifeedant,7 and antiinflammatory activities.8
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RESULTS AND DISCUSSION As a part of an ongoing chemical investigation of the diterpenoids from the roots of T. sagittata, two novel dimeric clerodane diterpene glycosides, bistinospinosides A (1) and B (2), were isolated. Herein are reported the isolation and structural elucidation of compounds 1 and 2. Notably, they possess an unprecedented polycyclic skeleton with a 1,4epoxycyclohexane ring system. A plausible biosynthetic pathway is proposed for these compounds. © 2017 American Chemical Society and American Society of Pharmacognosy
Received: April 14, 2017 Published: August 24, 2017 2478
DOI: 10.1021/acs.jnatprod.7b00324 J. Nat. Prod. 2017, 80, 2478−2483
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indicated the presence of a furan ring. The NMR spectra also showed resonances assignable to two oxygenated methine protons at δH 5.10 (1H, brs, H-1) and 5.90 (1H, dd, J = 12.1, 4.0 Hz, H-12) and two carbonyl moieties at δC 174.1 (C-17) and 173.1 (C-18). When the NMR data of part A were compared with columbin glucoside (3, Scheme 1),9 which was previously isolated from the same plant, they revealed superimposable resonances assignable to the B and C rings, but differences in the A ring. The marked differences observed were the replacement of Δ2,3 olefin carbon resonances at δC 130.5 (C-2) and 131.2 (C-3) in the A ring of columbin glucoside by two methines at δC 44.2 (C-2) and 43.4 (C-3) in part A. The 1H and 13C NMR resonances assignable to part B also showed great similarities to those of 3, except that a pair of conjugated double bonds of the furan ring was replaced by a double bond at δC 150.0 (C-13′) and 133.5 (C-14′) and two oxygenated methines at δC 82.5 (C-15′) and 78.8 (C-16′). The connection positions between parts A and B were found to be C-15′ to C-2 and C-16′ to C-3, constructing an unusual 1,4epoxycyclohexane ring, as deduced from the key 1H−1H COSY correlations between δH 2.37 (H-2) and 5.08 (H-15′), and 3.15 (H-3) and 6.37 (H-16′), as well as the HMBC correlations of δH 5.08 (H-15′) to δC 75.4 (C-1), 43.4 (C-3), and 78.8 (C-16′) and δH 6.37 (H-16′) to δC 44.2 (C-2), 85.1 (C-4), and 82.5 (C15′). The relative configuration of 1 was elucidated by analysis of the NOESY correlations (Figure 2) and coupling constants and Table 1. 1H and 13C NMR Spectroscopic Data of Compounds 1 and 2 (Pyridine-d5) 1 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Glc-1 Glc-2 Glc-3 Glc-4 Glc-5 Glc-6
δH (J in Hz) 5.10, brs 2.37, dd (8.0, 1.1) 3.15, d (8.0)
2.40, dd (14.0, 5.5) 1.85, overlap 2.90, m 2.01 overlap 2.53, brdd (8.1, 3.7) 2.10, 2.46, 2.01, 5.90,
s dd (14.9, 4.0) overlap dd (12.1, 4.0)
6.73, brd (1.7) 7.67, brt (1.7) 7.82, brd (0.4)
1.53, 1.32, 5.77, 4.11, 4.26, 4.13, 4.02, 4.58, 4.28,
s s d (7.5) t (7.8) overlap t (8.0) m dd (11.7, 2.0) overlap
1
2 δC
δH (J in Hz)
δC
75.4 44.2 43.4 85.1 42.7 26.0
5.21, brs 3.14, dd (10.0, 4.6) 3.87, dd (10.0, 4.3)
72.7 45.0 40.4 85.5 44.4 26.1
1′ 2′ 3′ 4′ 5′ 6′
17.5
7′
44.2 35.1 49.2 41.7
8′ 9′ 10′ 11′
17.6 44.0 35.0 48.2 41.7 71.2 126.3 109.4 144.4 140.6 174.1 173.1 22.3 28.2 99.9 76.2 79.0 71.8 78.6 62.9
2.25, 1.77, 2.88, 1.96, 2.51,
overlap overlap overlap overlap brd (11.3)
2.29, 2.44, 1.96, 6.01,
s dd (14.6, 3.8) overlap dd (12.2, 3.8)
6.65, brd (1.3) 7.62, brt (1.3) 7.72, brs
1.82, 1.24, 5.50, 4.16, 4.28, 4.16, 4.06, 4.65, 4.32,
s s d (7.3) t (7.5) overlap overlap m dd (11.5, 1.7) overlap
position
71.3 126.2 109.4 144.3 140.5 174.2 172.9 22.1 27.9 100.0 75.7 78.9 71.6 78.6 62.8
12′ 13′ 14′ 15′ 16′ 17′ 18′ 19′ 20′ Glc-1′ Glc-2′ Glc-3′ Glc-4′ Glc-5′ Glc-6′
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δH (J in Hz) 5.39, d (5.3) 6.49, dd (8.4, 5.3) 7.29, dd (8.4, 1.3)
2.20, 1.80, 2.73, 1.89, 2.64,
overlap overlap m m dd (10.7, 1.6)
2.04, 2.52, 2.20, 5.84,
s overlap overlap dd (11.9, 3.5)
6.51, brd (1.3) 5.08, brs 6.37, brs
1.38, 1.27, 5.32, 4.18, 4.23, 4.24, 3.96, 4.51, 4.31,
s s d (7.4) t (7.8) overlap overlap m dd (12.0, 2.3) dd (12.0, 5.5)
2 δC
δH (J in Hz)
δC
73.4 130.7 133.3 86.9 39.4 26.8
4.97, d (5.1) 6.42, dd (8.3, 5.1) 7.26, dd (8.3, 0.9)
73.2 130.7 133.5 87.1 40.7 26.0
17.8 44.8 35.7 46.9 39.7 72.9 150.0 133.5 82.5 78.8 174.0 172.2 24.5 27.9 101.9 75.4 78.8 71.5 78.7 62.5
2.25, 1.77, 2.25, 1.77, 2.84,
overlap overlap overlap overlap d (9.2)
1.73, 2.30, 1.96, 5.51,
s overlap overlap dd (8.7, 3.2)
7.02, s 5.88, d (4.3) 5.38, d (4.6)
1.31, 1.02, 5.34, 4.22, 4.26, 4.29, 3.99, 4.52, 4.32,
s s d (7.6) t (8.9) overlap overlap m dd (12.1, 2.3) overlap
17.0 41.9 36.3 56.8 44.7 71.9 147.4 131.9 82.9 79.8 174.0 172.1 25.9 24.7 101.9 75.5 78.8 71.5 78.6 62.4
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Figure 1. Key 1H−1H COSY and HMBC correlations of compounds 1 and 2.
Scheme 1. Hypothetical Biosynthetic Pathway for Compounds 1 and 2
14), 7.62 (1H, brt, J = 1.3 Hz, H-15), and 7.72 (1H, brs, H-16) and δC 126.2 (C-13), 109.4 (C-14), 144.3 (C-15), and 140.5 (C-16), and an α,β-unsaturated carbonyl moiety at δH 6.42 (1H, dd, J = 8.3, 5.1 Hz, H-2′) and 7.26 (1H, dd, J = 8.3, 0.9 Hz, H-2′) and δC 130.7 (C-2′), 133.5 (C-3′), and 172.1 (C18′). In addition, the 1H NMR spectra also showed the presence of two β-glucopyranosyl moieties with the resonances for the anomeric protons at δH 5.50 (1H, d, J = 7.3 Hz) and 5.34 (1H, d, J = 7.6 Hz), and the sugar suits were determined to be in the D-form by GLC analysis after acid hydrolysis. Compound 2 was also presumed to be a dimeric clerodane furanoditerpenoid, since the characteristics of the 13C NMR resonances appearing in pairs were similar to those of compound 1. Detailed analysis of the 1H and 13C NMR data suggested that the part A structure in 2 is the same as 1, with the part B structure being different. The chemical shift of C-8′ was shifted upfield from δC 44.8 in 1 to δC 41.9 in 2, suggesting the configuration at C-8′ in part B of 2 to be different from that in part B of 1. Part B was assigned as a derivative of isocolumbin glucoside (Scheme 1, 4) (the C-8 epimer of 3),10 which was confirmed by the NOESY correlations between δH 1.02 (H3-19′) and 2.84 (H-8′), and 2.84 (H-8′) and 5.51 (H12′) (Figure 2). The linkage pattern of parts A and B was
by molecular simulation. The NOESY correlations between H2/H-3, H-2/H-10, H-3/H-10, H-3/H3-19, and H-10/H3-19 indicated the α-orientation of H-2 and H-3. Furthermore, the significant NOESY correlations of H-2/H-16′ and H-2/H-14′ suggested that the 1,4-epoxy moiety should be β-oriented. The small coupling constant values between H-3 and H-16′ (J = ca. 0 Hz) and H-2 and H-15′ (J = 1.1 Hz) suggested the dihedral angles between these protons are near 90°. Computer-modeled 3D structure analysis of 1 using Discovery Studio version 2.1 supported the dihedral angles between H-3 and H-16′, H-2, and H-15′ being almost 90°, when the 1,4-epoxy moiety is βoriented (Figure 3). In contrast, if the 1,4-epoxy moiety is αoriented, the dihedral angles between these protons were calculated to be almost 40°. Bistinospinoside B (2) was isolated as a colorless solid, [α]16 D +4.8 (c 0.64, MeOH−H2O 1:1). The molecular formula of 2 was established as C52H64O22 from the HRESIMS peak at m/z 1063.3817 [M + Na]+ (calcd for C52H64O22Na, 1063.3787). The 1H and 13C NMR spectra (Table 1) displayed typical resonances for four tertiary methyls at δH 1.82 (H3-19), 1.24 (H3-20), 1.31 (H3-19′), and 1.02 (H3-20′) and δC 22.1 (C-19), 27.9 (C-20), 25.9 (C-19′), and 24.7 (C-20′), a furan ring having downfield resonances at δH 6.65 (1H, brd, J = 1.3 Hz, H2480
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glucoside (4), through a [4+2] cycloaddition reaction in an endo formation. Generally, the Diels−Alder reaction proceeds chemically under high temperatures and/or is catalyzed with a Lewis acid.12 Since the isolation procedure was carried out without high temperature (below 40 °C) or a Lewis acid catalyst, and the presence of 1 and 2 as natural products was confirmed in the plant MeOH extract, compounds 1 and 2 were considered as natural products rather than extraction artifacts. Nitric oxide (NO) plays key roles in immune and inflammatory responses, and the inhibitor of NO release may be considered as a therapeutic agent for treatment of inflammatory diseases.11 Taking into account the traditional usage of the roots of T. sagittata, and some clerodane diterpenoids isolated from T. sagittata having been reported with NO inhibition activities,8 bistinospinosides A (1) and B (2) were evaluated for NO inhibition activity using exposure to lipopolysaccharide (LPS) and TNFγ-induced NO production in the J774.1 macrophage-like cell line. However, compounds 1 and 2 did not show any discernible NO inhibitory effects (IC50 > 50 μM) in this assay. Likewise their biosynthesis precursor columbin glucoside (3) and columbin were also inactive in this assay.9
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Figure 2. Key NOESY correlations of compounds 1 and 2.
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured on a JASCO P-2200 polarimeter in a 0.5 dm cell. The UV spectra were obtained with a Shimadzu UV-160 spectrophotometer. Electronic circular dichroism (ECD) spectra were recorded on a JASCO J-720W spectropolarimeter. The IR spectra were measured on a JASCO FT/IR-4100 Fourier transform infrared spectrometer using the KBr disk method. The NMR spectra were measured on a JEOL ECA-500 spectrometer with the deuterated solvent used as the internal reference, and the chemical shifts are expressed in δ (ppm) units. HRTOFESIMS was conducted using a JEOL JMST100LP AccuTOF LC-Plus mass spectrometer. Diaion HP20 (Mitsubishi Chemical Corporation, Tokyo, Japan), silica gel (silica gel 60N, Kanto Chemical Co., Inc., Tokyo, Japan), and ODS (100− 200 mesh, Chromatorex DM1020T ODS, Fuji Silysia Chemical Co., Ltd., Aichi, Japan) were used for column chromatography. For preparative HPLC, a Waters 515 HPLC pump, equipped with a Shodex RI-101 differential refractometer detector and a JASCO UV970 intelligent UV/vis detector, was used. For reversed-phase HPLC separation, an RP-C18 silica gel column (YMC-Pack Pro C18, 150 × 20 mm) was used at a flow rate of 5.0 mL/min. Plant Material. The roots of Tinospora sagittata (one year old), which originated from Guizhou Province, People’s Republic of China, were purchased in December 2003 and identified by Professor Qishi Sun (Shenyang Pharmaceutical University). A voucher specimen (TH307) has been deposited at the Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Toho University, Japan. Extraction and Isolation. The roots (1.4 kg) were extracted with MeOH, then partitioned between EtOAc and H2O as in a previous study.12 The EtOAc fraction (37.8 g) was subjected to silica gel column chromatography and eluted with a gradient of CHCl3− MeOH−H2O to produce five fractions (1−5). Fraction 4 (10.4 g) was further subjected to passage over an ODS column and eluted with MeOH−H2O (2:3) to afford subfraction 4-1 (6.1 g). This subfraction was separated over a silica gel column and eluted with CHCl3−MeOH (9:1) to afford seven additional subfractions. Subfraction 4-1-5 (0.7 g) was purified by preparative HPLC with MeOH−H2O (9:11) to afford compounds 1 (6 mg) and 2 (15 mg). Bistinospinoside A (1): colorless solid; [α]16 D +19.7 (c 0.23, MeOH); UV (MeOH) λmax (log ε) 205 (4.03) nm; ECD (MeOH) [θ]22 (nm) −53 136 (205), −42 683 (208), 13 808 (238); IR (KBr) νmax 3423, 2925, 2849, 1751, 1708, 1637, 1458, 1385, 1210, 1163, 1075, 1027 cm−1; 1H and 13C NMR spectroscopic data, see Table 1; ESIMS m/z
determined in 2 as being from C-2 to C-16′ and C-3 to C-15′ by the HMBC correlations of H-1 with C-16′, H-2 with C-13′, and H-3 with C-14′ (Figure 1). More importantly, key 1H−1H COSY correlations between H-2 and H-16′, and H-3 and H15′, supported the above connection between parts A and B. In the NOESY spectrum, the correlations of H-2/H-3, H-2/H-10, H-3/H3-19, and H-10/H3-19 were observed, while no correlations of H-3/H-14′ and H-2/H-15′ were evident, suggesting that the 1,4-epoxy moieties in 2 and 1 are oppositely oriented. The dihedral angles of H-2/H-16′ and H-3/H-15′ were calculated using Discovery Studio version 2.1 as being about 40°, which are in good agreement with their corresponding coupling constants of JH‑2,H‑16′ = 4.6 Hz and JH‑3,H‑15′ = 4.3 Hz, and therefore the 1,4-expoxy moiety must be α-oriented (Figure 3). Since the related clerodane diterpenoid columbin glucoside (3), which may be a precursor of compounds 1 and 2, has also been isolated from the same plant,9 these compounds were assumed to have the same absolute configurations. Thus, the structures of bistinospinosides A (1) and B (2) were elucidated as shown. The Diels−Alder reaction has been reported to be involved in the construction of the ring structure during the biosynthesis of various types of natural products, including polyketides, terpenoids, and alkaloids.11 Bistinospinosides A (1) and B (2) feature an unusual 1,4-epoxycyclohexane ring in their structures, which may be constructed biosynthetically via an intermolecular Diels−Alder [4+2] cycloaddition from the corresponding monomeric clerodane diterpenes 3 and/or 4 (Scheme 1). However, the fusion pattern of the two compounds is different, since compound 1 may be biosynthesized via Diels−Alder addition of the two hypothetical columbin glucoside units depicted. In this cycloaddition reaction, the furan ring of part B (conjugated diene) approaching the double bond of part A (dienophile) resulted in an exo formation of the dimeric adduct. Compound 2 may be formed from columbin glucoside (3) and isocolumbin 2481
DOI: 10.1021/acs.jnatprod.7b00324 J. Nat. Prod. 2017, 80, 2478−2483
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Figure 3. Molecular simulation of compounds 1 and 2. 1063.3 [M + Na]+; HRESIMS m/z 1063.3782 [M + Na]+ (calcd for C52H64O22Na, 1063.3787). Bistinospinoside B (2): colorless solid; [α]16 D +4.8 (c 0.64, MeOH− H2O, 1:1); UV (MeOH) λmax (log ε) 208 (4.17) nm; ECD (MeOH) [θ]22 (nm) −62 594 (207), 15 826 (237); IR (KBr) νmax 3436, 2924, 2855, 1743, 1637, 1458, 1384, 1208, 1234, 1192, 1164, 1105, 1075, 1041 cm−1; 1H and 13C NMR spectroscopic data, see Table 1; ESIMS m/z 1063.3 [M + Na]+; HRESIMS m/z 1063.3817 [M + Na]+ (calcd for C52H64O22Na, 1063.3787). Acid Hydrolysis and Determination of the Absolute Configuration of Sugars. A solution of each of 1 and 2 (0.5 mg) in 1 M HCl (dioxane−H2O, 1:1, 200 μL) was heated at 100 °C for 1 h under an Ar atmosphere. After the dioxane was removed, the solution was extracted with EtOAc (1 mL × 3) to remove the aglycone. The aqueous layer was neutralized by passing it through an ion-exchange resin (Amberlite MB-3, Organo, Tokyo, Japan) column, then concentrated under reduced pressure to dryness, to provide a residue of the sugar fraction. The residue was dissolved in pyridine (0.1 mL),
to which 0.08 M L-cysteine methyl ester hydrochloride in pyridine (1.5 mL) was added. The mixture was kept at 60 °C for 1.5 h. After drying in vacuo, the residue was silylated with 1-trimethylsilylimidazole (0.1 mL) for 2 h. The mixture was partitioned between n-hexane and H2O (0.3 mL each), and the n-hexane extract was analyzed by GC-MS under the following conditions: capillary column, EQUITY-1 (30 m × 0.25 mm × 0.25 μm, Supelco); column temperature, 230 °C; injection temperature, 250 °C; carrier gas, N2. On the basis of the acid hydrolysate of 1 and 2, D-glucose was confirmed by comparison of the retention times of their derivatives with those of the D-glucose and Lglucose derivatives prepared in a similar way, which showed retention times of 11.02 and 11.43 min, respectively. Nitric Oxide Inhibitory Assay. The J774.1 cell line was purchased from Riken Cell Bank (Tsukuba, Japan) and cultured in RPMI 1640 medium supplemented with penicillin G (100 units/mL), streptomycin (100 mg/mL), and 10% fetal bovine serum. The cells were seeded in 96-well plastic plates with 1 × 106 cells/well and allowed to adhere for 2 h at 37 °C in a humidified atmosphere containing 5% CO2. Then, 2482
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(9) Huang, C.; Li, W.; Ma, F.; Li, Q.; Asada, Y.; Koike, K. Chem. Pharm. Bull. 2012, 60, 1324−1328. (10) Achenbach, H.; Hemrich, H. Phytochemistry 1991, 30, 1957− 1962. (11) Strowig, T.; Henao-Mejia, J.; Elinav, E.; Flavell, R. Nature 2012, 481, 278−286. (12) Wei, K.; Li, W.; Koike, K.; Nikaido, T. Org. Lett. 2005, 7, 2833− 2835.
the medium was replaced with fresh medium, containing LPS (10 ng/ mL) and TNFγ (40 U/mL) together with test compound at various concentration and then incubated for 48 h. NO production was determined by measuring the accumulation of nitrite in the culture supernatant using Griess reagent. Briefly, 100 mL of the supernatant from incubates was mixed with an equal volume of Griess reagent (1% sulfanilamide and 0.1% naphthalene-diamide dihydrochloride in 2.5% H3PO4) and was allowed to stand for 10 min at 37 °C in a humidified atmosphere containing 5% CO2. Cell viability was determined using the mitochondrial respiration-dependent MTT reduction method. Absorbance at 550 nm was measured using an ImmunoMini NJ-2300 microplate reader. Polymyxin B (IC50 8.5 ± 0.9 μM) was used as positive control. Cell Viability. Cell viability was determined using the mitochondrial respiration-dependent MTT reduction method. After transferring the required supernatant to another plate for the Griess assay, the remaining supernatant was aspirated from the 96-well plates, and 100 μL of fresh medium and 10 μL of MTT (5 mg/mL PBS) were added to each well. The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2. After incubating for 4 h, the medium was removed and the violet crystals of formazan in viable cells were dissolved in DMSO. Absorbance at 550 nm was measured using an ImmunoMini NJ-2300 reader.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00324. NMR spectra of compounds 1 and 2 and chemical calculation method (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Tel: +81-47-4721161. Fax: +81-47-4721404. E-mail: liwei@ phar.toho-u.ac.jp. ORCID
Wei Li: 0000-0003-4143-8597 Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Li, R.; Morris-Natschke, S. L.; Lee, K. H. Nat. Prod. Rep. 2016, 33, 1166−1226. (2) (a) Castro, A.; Coll, J.; Arfan, M. J. Nat. Prod. 2011, 74, 1036− 1041. (b) Hiruma-Lima, C. A.; Spadari-Bratfisch, R. C.; GrassiKassisse, D. M.; Brito, A. R. S. Planta Med. 1999, 65, 325−330. (c) Dai, S. J.; Zhang, L.; Xiao, K.; Han, Q. T. Bioorg. Med. Chem. Lett. 2016, 26, 1750−1753. (d) Lv, H. W.; Luo, J. G.; Zhu, M. D.; Shan, S. M.; Kong, L. Y. Chem. Pharm. Bull. 2014, 62, 472−476. (e) Murthy, M. M.; Subramanyam, M.; Bindu, M. H.; Annapurna, J. Fitoterapia 2005, 76, 336−339. (3) State of Administration of Traditional Chinese Medicine of People’s Republic of China. Zhong Huang Ben Cao; Shanghai Scientific and Technical Publishers: Shanghai, 1999; pp 1988−1990. (4) Shi, Q. R.; Shen, Y. H.; Zhang, C.; Liu, R. H.; Zhang, W. D. Zhongguo Tianran Yaowu 2008, 6, 186−190. (5) Liu, X.; Hu, Z.; Shi, Q.; Zeng, H.; Shen, Y.; Jin, H.; Zhang, W. Arch. Pharmacal Res. 2010, 33, 981−987. (6) Shi, L. M.; Li, R. Q.; Liu, W. H. Helv. Chim. Acta 2008, 91, 978− 982. (7) Zhang, Y.; Shi, Q.; Shi, P.; Zhang, W.; Cheng, Y. Rapid Commun. Mass Spectrom. 2006, 20, 2328−2342. (8) Li, W.; Huang, C.; Li, S.; Ma, F.; Li, Q.; Asada, Y.; Koike, K. Planta Med. 2012, 78, 82−85. 2483
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