Fitoterapia 100 (2015) 11–18
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Five new phenolic compounds from Dendrobium aphyllum Dan Yang a,b,d, Liang-Yan Liu c, Zhong-Quan Cheng d, Feng-Qing Xu b, Wei-Wei Fan b, Cheng-Ting Zi a,b, Fa-Wu Dong b, Jun Zhou b, Zhong-Tao Ding a,⁎, Jiang-Miao Hu b,⁎ a
School of Chemical Sciences and Technology of Yunnan University, Kunming 650091, PR China State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China College of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming 650201, PR China d Guilin Normal College, Guilin 541001, PR China b c
a r t i c l e
i n f o
Article history: Received 20 August 2014 Accepted in revised form 28 October 2014 Available online 11 November 2014 Keywords: Dendrobium aphyllum Phenanthrene Bibenzyl derivative NO production
a b s t r a c t One new phenanthrene, aphyllone A (1) and four new bibenzyl derivatives, aphyllone B (2) and aphyllals C–D (3–5), together with nine known compounds (6–14), were isolated from the stems of Dendrobium aphyllum (Roxb.) C. E. Fischer. The structures of these new compounds were elucidated by means of extensive spectroscopic analyses, and the absolute configuration of compound 1 was determined by single crystal X-ray diffraction and quantum calculations. Compounds 6, 8 and 14 inhibited NO production at the concentration of 25 μM in LPS-stimulated RAW264.7 cells with the inhibition (%) of 32.48, 35.68, and 38.50. Compound 2 possessed significant DPPH radical scavenging activity with scavenging percentage of 87.97% at the concentration of 100 μg/mL. © 2014 Published by Elsevier B.V.
Chemical compounds studied in this article: Moscatin (PubChem CID: 194774) Hircinol (PubChem CID: 442705) Moscatilin (PubChem CID: 176096) Gigantol (PubChem CID: 10221179) Batatasin III (PubChem CID: 10466989) Tristin (PubChem CID: 15736297) Dihydroresveratrol (PubChem CID: 185914)
1. Introduction Dendrobium, one of the largest genera in Orchidaceae, containing more than 1100 species identified, is widely distributed in throughout Asia, Europe and Australia. There are about 80 Dendrobium species in China, and the fresh or dried stems of many species have been used as both traditional Chinese and folk remedies for the treatment of various diseases, such as chronic atrophic gastritis, skin aging, fever, cancer and cardiovascular disease for thousands of years, as well as a high-quality
⁎ Corresponding authors. Tel.: +86 871 6522 3264; fax: +86 871 6522 3261. E-mail addresses: [email protected] (Z.-T. Ding), [email protected] (J.-M. Hu).
http://dx.doi.org/10.1016/j.fitote.2014.11.004 0367-326X/© 2014 Published by Elsevier B.V.
health food now [1–3]. The main chemical components of Dendrobium are phenolic compounds, polysaccharides, alkaloids and sesquiterpenoids with multiple biological activities, including immunomodulatory, antioxidant and anti-tumor effects, etc. [4,5]. Chinese Pharmacopoeia (2010 edition) has two monographs of medicinal Dendrobium plants: One is Dendrobii Caulis (Shihu in Chinese), derived from Dendrobium nobile, D. chrysotoxum, D. fimbriatum and other related Dendrobium species [2]. The other is Dendrobii Officinalis Caulis (Tiepi shihu in Chinese), derived from D. officinale [3]. D. aphyllum is grown in large quantities in the Southwest of China and used as one of the most common source for Shihu. Previous investigation on D. aphyllum yielded three polysaccharides with evident immunostimulating activities [6], together with secondary metabolites including bibenzyls, phenanthrenes and
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D. Yang et al. / Fitoterapia 100 (2015) 11–18
flavones, whose activities await elucidation [4,7–9]. In order to supply more evidence to the efficacy and safety of D. aphyllum in clinical applications, we investigated chemical constituents of the stems of D. aphyllum and led to the isolation of fourteen phenolic compounds, including one new phenanthrone, aphyllone A (1) and four new bibenzyl derivatives, aphyllone B (2) and aphyllals C–E (3–5) (Fig. 1), together with nine known compounds moscatin (6) [10], hircinol (7) [11], moscatilin (8) [12], gigantol (9) [13], batatasin III (10) [14], tristin (11) [15], dihydroresveratrol (12) [16], trigonopol B (13) [17] and tricetin 3′,4′,5′-trimethyl ether 7-Oβ-glucopyranoside (14) [18]. The absolute configuration of compound 1 was determined by single crystal X-ray diffraction and quantum calculations. The absolute configurations of compounds 4 and 5 were determined by optical rotations and circular dichroism (CD) spectra. This paper described the isolation and structural elucidation, inhibitory effects on NO production in LPS-stimulated RAW264.7 macrophages, the cytotoxic properties and DPPH radical scavenging activity of the selected isolates.
NMR spectra were recorded on Bruker AM-400, DRX-500 or Av III-600 instruments with TMS as the internal standard (Bruker, Bremerhaven, Germany). The chemical shifts were given in δ (ppm) scale with reference to the solvent signal. Mass spectra were recorded on an API QSTAR time-of-flight spectrometer (MDS Sciqaszex, Concord, Ontario, Canada) or LC-MS-IT-TOF (Shimadzu, Kyoto, Japan) spectrometer. Semi-preparative HPLC was performed on Agilent 1100 liquid chromatography with a ZORBAX SB-C18 (5 μm, 9.4 × 250 mm) column (Agilent, USA) at a flow rate of 3.0 mL/min. Column chromatography was performed on silica gel (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, China). Lichroprep RP-18 gel (40– 63 μm, Merck, Darmstadt, Germany), MCI gel CHP-20P (75– 150 μm, Mitsubishi Chemical Corp., Tokyo, Japan), Sephadex LH-20 (20–150 μm, Amersham Biosciences, Uppsala, Sweden). Fractions were monitored by TLC, and spots were visualized by UV light (254 nm) and sprayed with 5% H2SO4 in ethanol, followed by heating.
2. Experimental
The stems of D. aphyllum (cultivated) were collected in October 2010 from Menglian county, Yunnan Province, PR China, and identified by Prof. Hong Yu. A voucher specimen (No. Zsh12) was deposited at the State Key Laboratory of Phytochemistry and Plant Resource in West China, Kunming Institute of Botany, Chinese Academy of Sciences.
2.1. General X-ray data were collected using a Bruker APEX DUO instrument. Melting points were measured on a XRC-1 micromelting point apparatus (Beijing, PR China) and were uncorrected. Optical rotations were obtained on a Jasco P-1020 digital polarimeter (Horiba, Tokyo, Japan). UV spectra were taken on a Shimadzu UV-2401 PC spectrophotometer (Shimadzu, Kyoto, Japan). CD spectra were recorded with an Applied Photophysics Chirascan spectrometer (Agilent, America). IR spectra were obtained on a Bruker Tensor 27 infrared spectrophotometer (Bruker Optics GmbH, Ettlingen, Germany) with KBr pellets.
2.2. Plant material
2.3. Extraction and isolation The sun-dried stems of D. aphyllum (5 kg) were powdered and extracted with 95% ethanol (20 L × 4, 2 days each time) at room temperature, then filtered. The filtrate was evaporated under reduced pressure to give a crude extract. The extract was suspended in H2O and partitioned successively with ethyl
Fig. 1. Chemical structures of compounds 1–5.
D. Yang et al. / Fitoterapia 100 (2015) 11–18
acetate (EtOAc) and n-butanol. The EtOAc part (150 g) was chromatographed on silica gel column chromatography (Si CC, 1500 g), eluted with CHCl3-MeOH (100:0, 60:1, 20:1, 5:1, v/v), to give four fractions A–D. Fraction B (40 g) was divided into three sub-fractions (B1–B3), by Si CC (400 g) using CHCl3-MeOH (90:1, 60:1, 30:1, 10:1) as the eluent. Fraction B2 (10 g) was subjected to repeated Si CC eluted with CHCl3-MeOH (60:1, 30:1, 20:1) and followed by Sephadex-LH-20 column (CHCl3-MeOH, 1:1) to give compounds 1 (300 mg), 3 (10 mg), 6 (940 mg) and 7 (29 mg). Fraction C (30 g) was loaded on a RP-18 column (MeOHH2O, 30:70, 50:50, 70:30, 90:10) to give four sub-fractions C1–C4. Fraction C2 (10 g) was subjected to Si CC (100 g) eluted with CHCl3-MeOH (60:1, 40:1, 20:1, 10:1) and further purified by Sephadex LH-20 column (CHCl3-MeOH, 1:1) to yield compounds 8 (2147 mg), 9 (23 mg) and 10 (3 mg). Compounds 2 (20 mg), 5 (2 mg) and 11 (40 mg) were obtained from Fraction D (20 g) by Si CC (200 g) using CHCl3-MeOH (40:1, 20:1, 10:1, 5:1) as the eluent and further separated by a RP-18 column (MeOH-H2O, 20:80, 40:60, 60:40, 80:20). The n-BuOH partition (100 g) was chromatographed on Si CC (1000 g), eluted with CHCl3-MeOH (95:5, 90:10, 80:20, 60:40), to afford four sub-fractions (E–H). Fraction E (20 g) was separated by repeated Si CC (CHCl3-MeOH, 40:1, 20:1, 10:1) and Sephadex LH-20 column (MeOH) to yield compounds 12 (28 mg), 13 (4 mg) and 14 (4 mg). Fraction F (10 g) was performed on a MCI CHP-20P gel CC (200 g) and eluted with MeOH-H2O (10:90, 30:70, 60:40, 80:20) to give four fractions (F1–F4). Fraction F1 (3 g) was performed on Si CC (30 g), eluted with CHCl3-MeOH (15:1), and further purified by preparative HPLC MeOH-H2O (25:75) to afford compound 4 (3 mg, tR = 22 min). 2.4. Spectroscopic data Aphyllone A (1): Colorless prismatic crystals (petroleum ether–Me2CO, 5:2); mp 207–208 °C; [α]20 D : +383.0 (c 0.23, MeOH); UV (MeOH) λmax (log ε) 247 (2.76), 213 (2.31), 193 (1.85) nm; CD (c 0.08, MeOH) λ (Δε) 222 (−20.15), 231 (+27.65), 295 (−7.34); IR (KBr) νmax 3426, 1653, 1594, 1458, 1352, 1263, 1222, 1169, 1011, 816, 744 cm−1; 1H and 13C NMR data, see Table 1; positive-ion ESIMS: m/z 281 [M + Na]+; positive-ion HRESIMS [M + H]+ m/z 259.0961 (calcd for C15H15O4, 259.0965). Aphyllone B (2): White amorphous powder; UV (MeOH) λmax (log ε) 202 (4.69), 266 (1.61) nm; IR (KBr) νmax 3441, 1656, 1625, 1516, 1453, 1269, 1228, 1107, 1030 cm−1, 1H and 13C NMR data, see Tables 2 and 3; positive-ion ESIMS: m/z 343 [M + Na]+; positive-ion HREIMS [M]+ m/z 320.1256 (calcd for C17H20O6, 320.1260). Aphyllal C (3): White powder; mp 156–157 °C; UV (MeOH) λmax (log ε) 204 (5.30), 283 (1.19) nm; IR (KBr) νmax 3396, 2925, 1603, 1502, 1442, 1245, 1152, 1039, 977, 927, 837, 810 cm− 1; 1H and 13C NMR data, see Tables 2 and 3; positive-ion ESIMS: m/z 259 [M + H]+; positive-ion HRESIMS [M + H]+ m/z 259.0965 (calcd for C15H15O4, 259.0965). Aphyllal D (4): White amorphous powder; [α]22 D : − 20.3 (c 0.18, MeOH); UV (MeOH) λmax (log ε) 203 (4.31), 281
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Table 1 1 H (500 MHz) and 13C (100 MHz) NMR data of compound 1 in acetone-d6, δ in ppm, J in Hz. δC
Position
δH
1
3.01 (dd, 16.5, 5.2) 2.35 (dd, 16.5, 2.0) – 5.34 (s) – – – – 6.70 (d, 7.5) 7.10 (dd, 7.5, 7.5) 6.60 (d, 7.5) – 6.41 (dd, 9.5, 3.0) 5.64 (dd, 9.5, 2.0) 3.29 (dd, 5.2, 2.0) 3.57 (s)
2 3 4 4a 4b 5 6 7 8 8a 9 10 10a OMe-4
38.2 (t) 196.1 (s) 103.8 (d) 173.2 (s) 77.6 (s) 119.3 (s) 159.1 (s) 117.8 (d) 130.4 (d) 119.1 (d) 135.4 (s) 129.7 (d) 130.1 (d) 42.8 (d) 56.7 (q)
(1.13) nm; CD (c 0.16, MeOH) λ (Δε) 211 (− 4.43), 233 (− 1.87); IR (KBr) νmax 3428, 2925, 1610, 1516, 1384, 1275, 1153, 1033, 843 cm− 1; 1H and 13C NMR data, see Tables 2 and 3; negative-ion ESIMS: m/z 275 [M–H]−; negative-ion HRESIMS [M–H]− m/z 275.0924 (calcd for C15H15O5, 275.0925). Aphyllal E (5): White amorphous powder; [α]22 D : − 30.5 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 203 (4.96), 280 (1.15) nm; CD (c 0.07, MeOH) λ (Δε) 211 (− 7.82), 231 (− 3.24); IR (KBr) νmax 3430, 2925, 1603, 1517, 1457, 1196,
Table 2 1 H NMR data of compounds 2–5 (2 and 5 in acetone-d6, 3 in CDCl3, 4 in CD3OD, δ in ppm, J in Hz). Position 2a 1 2
– 5.87 (s)
3 4
– –
5 6
– 5.87 (s)
7 8
2.03 (m) 2.55 (m)
9 10
– 6.76 (d, 1.9)
11 12 13
– – 6.69 (d, 8.0)
14
6.60 (dd, 8.0, 1.9) – 3.56 (s) 3.56 (s) 3.78 (s)
15 OMe-3 OMe-5 OMe11
3a
4b
5b
– 6.23 (br s) – 6.20 (br s) – 6.23 (br s) 2.73 (m) 2.78 (m)
– 6.22 (d, 2.4)
– 6.40 (br s)
– 6.13 (t, 2.4)
– 6.24 (t, 2.4)
– 6.22 (d, 2.4)
– 6.43 (br s)
4.57 (t, 6.6) 2.88 (dd, 13.8, 7.2) 2.79 (dd, 13.2, 6.6) – 6.55 (d, 1.8)
4.70 (br s) 2.89 (m)
– – 6.64 (d, 7.8)
– – 6.67 (d, 7.8)
6.56 (dd, 7.8, 1.8) – – – 3.72 (s)
6.62 (dd, 7.8, 1.8) – 3.69 (s) – 3.75 (s)
– 6.66 (br s) – – 6.71 (d, 7.9) 6.59 (d, 7.9) 5.92 (s) – – –
– 6.73 (d, 1.8)
Assignments were made based on 1H, 13C NMR, DEPT, HSQC, HMBC, COSY, and ROESY experiments. a NMR data for compounds 2 and 3 were recorded at 500 MHz. b NMR data of compounds 4 and 5 were recorded at 600 MHz.
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D. Yang et al. / Fitoterapia 100 (2015) 11–18
Table 3 13 C NMR data of compounds 2–5 (2 and 5 in acetone-d6, 3 in CDCl3, 4 in CD3OD, δ in ppm). Position
2a
3a
4b
5b
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 OMe-3 OMe-5 OMe-11
70.4 (s) 120.2 (d) 150.3 (s) 176.5 (s) 150.3 (s) 120.2 (d) 45.1 (t) 30.8 (t) 134.2 (s) 112.7 (d) 148.1 (s) 145.5 (s) 115.6 (d) 121.3 (d)
144.7 (s) 108.0 (d) 156.6 (s) 100.5 (d) 156.6 (s) 108.0 (d) 37.9 (t) 37.1 (t) 135.4 (s) 108.9 (d) 147.4 (s) 145.6 (s) 108.1 (d) 121.1 (d) 100.7 (t)
148.3 (s) 106.0 (d) 159.3 (s) 102.3 (d) 159.3 (s) 106.0 (d) 76.9 (d) 46.3 (t) 131.3 (s) 114.4 (d) 148.5 (s) 145.8 (s) 115.8 (d) 123.1 (d)
149.1 (s) 103.7 (d) 161.6 (s) 100.5 (d) 159.0 (s) 106.4 (d) 75.8 (d) 46.6 (t) 131.2 (s) 114.0 (d) 147.7 (s) 145.7 (s) 115.2 (d) 122.8 (d)
55.2 (q) 55.2 (q) 56.1 (q)
55.3 (q) 56.2 (q)
conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2(4- sulfophenyl)-2H-tetrazolium (MTS) (Sigma, St. Louis, MO, USA) [20]. Briefly, 100 μL of adherent cells was seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition, both with an initial density of 1 × 105 cells/mL in 100 μL medium. Each tumor cell line was exposed to the test compound at a concentration of 25 μM in triplicate for 48 h, with cisplatin and paclitaxel (Sigma) as positive controls. After the incubation, MTS (100 μg) was added to each well, and the incubation continued for 4 h at 37 °C. The cells were lysed with 100 μL of 20% SDS–50% DMF after removal of 100 μL medium. The optical density of the lysate was measured at 490 nm in a 96-well microtiter plate reader (Bio-Rad 680). 2.7. DPPH radical scavenging assay
56.0 (q)
Assignments were made based on 1H, 13C NMR, DEPT, HSQC, HMBC,COSY, and ROESY experiments. a NMR data of compounds 2 and 3 were recorded at 100 MHz. b NMR data for compounds 4 and 5 were recorded at 150 MHz.
1154, 1034 cm− 1; 1H and 13C NMR data, see Tables 2 and 3; positive-ion ESIMS: m/z 313 [M + Na]+; positive-ion HRESIMS [M + Na]+ m/z 313.1045 (calcd for C16H18O5Na, 313.1052). 2.5. Nitric oxide production in RAW264.7 macrophages Murine monocytic RAW264.7 macrophages were dispensed into 96-well plates (2 × 105 cells/well) containing RPMI-1640 medium (Hyclone) with 10% FBS under a humidified atmosphere of 5% CO2 at 37 °C. After 24 h preincubation, cells were treated with the compounds (25 μM) dissolved in DMSO, in the presence of 1 μg/mL LPS for 18 h. NO production in each well was assessed by adding 100 μL of Griess reagent (Reagent A & Reagent B, respectively, Sigma) to 100 μL of each supernatant from LPS (Sigma)-treated or LPS- and compound-treated cells in triplicate. After 5 min incubation, the absorbance was measured at 570 nm with a 2104 Envision Multilabel Plate Reader (PerkinElmer Life Sciences, Inc., Boston, MA, USA). MG-132 was used as a positive control [19]. Nitrite concentrations were calculated by regression analysis of a standard curve using sodium nitrite as a standard. The NO inhibition (IH) of each assay was calculated using the following equation: IH (%) = {1 − [(test sample data) − (negative group data)] / [(positive group data) − (negative group data)]} × 100, where positive group data refer to nitrite concentrations of LPS alonetreated cells, negative group data refer to nitrite concentrations of media alone-treated cells and test sample data refer to nitrite concentrations of sample plus LPS-treated cells. 2.6. The cytotoxicity assay The human tumor cell lines HL-60, SMMC-7721, A-549, MCF-7, and SW-480 were used, which were obtained from ATCC (Manassas, VA, USA). All the cells were cultured in RPMI-1640 or DMEM (Hyclone, Logan, UT, USA), supplemented with 10% fetal bovine serum (Hyclone) at 37 °C in a humidified atmosphere with 5% CO2. Cell viability was assessed by
Reaction mixture contained 100 μL ethanol solution of DPPH (25 μg/mL) and 100 μL test compounds (100 μg/mL) in ethanol, respectively. The experiment was performed in triplicate for each of the individual sample. The reaction mixture was incubated for 1 h at 30 °C and the absorbance was measured at 515 nm. The change in absorbance with respect to the control (containing DPPH radical only without sample, expressed as 100% free radicals) was calculated as percentage scavenging. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was used as positive controls. The ability to scavenge DPPH radicals was calculated as follows: DPPH radical scavenging percentage (%) = (OD blank − OD sample) / OD blank × 100 OD blank is the absorbance of blank; OD sample is the absorbance of test compound. 2.8. X-ray crystal structure analysis Colorless crystals of compound 1 were obtained from petroleum ether–Me2CO (5:2). Intensity data were collected at 100 K on a Bruker APEX DUO diffractometer equipped with an APEX II CCD, using Mo Kα radiation. Cell refinement and data reduction were performed with Bruker SAINT. The structures were solved by direct methods using SHELXS-97 [21]. Refinements were performed with SHELXL-97 using full-matrix least-squares, with anisotropic displacement parameters for all the non-hydrogen atoms [21]. The Hatoms were placed in calculated positions and refined using a riding model. Molecular graphics were computed with PLATON. Crystallographic data (excluding structure factor tables) for the structures reported have been deposited with the Cambridge Crystallographic Data Center as supplementary publication no. CCDC 1003248 for compound 1. Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB 1EZ, UK [fax: Int. +44 (0) (1223) (336 033); e-mail: deposit@ccdc. cam. ac. uk]. Crystal data for aphyllone A (1): C15H14O4, M = 258.26, orthorhombic, a = 7.4270(10) Å, b = 7.6615(11) Å, c = 22.110(3) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V = 1258.1(3) Å3, T = 100(2) K, space group P212121, Z = 4, μ (Mo Kα) = 0.099 mm−1, 13045 reflections measured, 3503
D. Yang et al. / Fitoterapia 100 (2015) 11–18
independent reflections (Rint = 0.0268). The final R1 values were 0.0336 (I N 2σ(I)). The final wR(F2) values were 0.0887 (I N 2σ(I)). The final R1 values were 0.0362 (all data). The final wR(F2) values were 0.0901 (all data). The goodness of fit on F2 was 1.062. Flack parameter = 0.0(7). 3. Results and discussion Compound 1 had molecular formula C15H14O4, as deduced from HRESIMS [M + H]+ m/z 259.0961 (calcd for 259.0965) and NMR spectra (Table 1), indicating nine degrees of unsaturation. Its IR spectrum displayed the presence of hydroxyl (3426 cm−1), conjugated carbonyl (1653 cm−1) and aromatic ring (1593, 1458 cm−1) groups. In the 1H NMR spectrum of compound 1, three aromatic protons [H-6 (δH 6.70, d, 7.5 Hz), H-7 (δH 7.10, dd, 7.5, 7.5 Hz) and H-8 (δH 6.60, d, 7.5 Hz)] belong to ring A. The 13C NMR and DEPT spectra exhibited eleven sp2 carbons (one carbonyl, four quaternary carbons, and six methines), four sp3 carbons [one quaternary carbon (oxygenated), one methine, one methylene, and one methoxy, respectively]. The 1H–1H COSY correlations of H-1/H-10a and H-9/H-10, along with the HMBC correlations from H-1 to C-10 (δC 130.1, d), H-9 (δH 6.41, dd, J = 9.5, 3.0 Hz) to C-10a (δC 42.8, d), and from H-3 (δH 5.34, s) to C-2 (δC 196.1, s) and C-4 (δC 173.2, s), established the spin systems for the molecular fragments of C-1–C-10a–C-10–C-9, and an α, β-unsaturated ketone group (C-2–C-3–C-4). In addition, the HMBC correlations from H-9 to C-8 (δC 119.1, d) and C-4b (δC 119.3, s), along with correlations of H-10 to C-4a (δC 77.6, s) and C-8a (δC 135.4, s), clearly indicated that ring B is connected to C-4b and C-8a. Similarly, the HMBC correlations of H-1 to C-10, C-4a and C-3 (δC 103.8, d), along with
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correlations of H-3 to C-4a showed that ring C is connected to C-4a and C-10a (Fig. 2). Thus, compound 1 was suggested to be the skeleton of 1,1,4a,10a-tetrahydrophenanthrene. The location of the methoxy group at C-4 was determined by HMBC correlations from H-OMe (δH 3.57, s) to C-4. One hydroxy group should be attached to the deshielded aromatic carbon C-5 (δC 159.1, s), and the other hydroxy group should be connected to C-4a, which was confirmed by HMBC correlations from H-1, H-3, and H-10 to C-4a. The 1H–1H COSY correlations of H-6/H-7 and H-7/H-8, and the observed NOESY correlations between H-3 and H-MeO, and between H-8 and H-9 further supported the structure of compound 1. Based on the above evidences, the planar structure of compound 1 was elucidated. To confirm the structure and determine the absolute configuration of compound 1, a proper crystal was obtained using an applied single crystal X-ray diffraction with Mo Kα radiation. As shown in Fig. 3, the α-orientations of OH-4a and H-10a were determined by X-ray crystallographic data. The final assignment of compound 1 was achieved using a quantum method by comparing the calculated electronic circular dichroism (ECD) and [α]D value with experimented data [22,23]. The ECD spectra derived from quantum chemical calculations have been used successfully for the stereochemical assignment of small- and medium-sized molecules [24]. Starting from the relative stereochemistry evidenced from the single-crystal X-ray diffraction, the quantum chemical calculation on the ECD spectra was utilized to elucidate its absolute stereochemistry. To determine the absolute configuration of compound 1, the comparison between the experimental and calculated ECD spectra of 1 (Fig. 4) was performed using the time-dependent density functional theory (DFT) method at the B3LYP/6-311++G(2d, p) level. The calculated ECD spectrum was comparable with the
Fig. 2. Selected COSY, HMBC and ROESY correlations of compounds 1, 2, 4, and 5.
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Fig. 3. X-ray crystal structure of compound 1.
measured for compound 1. Similarly, the [α]D calculation was performed on the Gaussian09 program using TD-DFTb3lyp/6–311 g(d,p) level of theory on b3lyp/6–311 g(d,p) optimized geometry (in methanol). The [α]D value calculated (+ 378.9) was similar to the measured (+ 383.0). From the above evidence, the structure of compound 1 was elucidated as (4aR,10aS)-4a,5-dihydroxy-4-methoxy-1,1,10atrihydrophenanthr-2-enone (Fig. 1), named aphyllone A. Compound 2 was obtained as white amorphous powder. A molecular formula of C17H20O6 was established by the HREIMS [M]+ m/z 320.1256 (calcd for 320.1260). The presence of hydroxyl (3441 cm−1) and conjugated carbonyl (1656 cm−1) was deduced from the IR spectrum. The 1H NMR spectrum of 2 showed three 1, 3, 4-trisubstituted aromatic protons [H-10 (δH 6.76, d, J = 1.9 Hz), H-13 (δH 6.69, d, J = 8.0 Hz) and H-14 (δH dd, J = 8.0, 1.9 Hz)], one pair of symmetric olefinic protons H-2, 6 (δH 5.87, s) and methoxys [OMe-3, 5 (δH 3.56, s)], another methoxy signal [OMe-11 (δH 3.78, s)] and two methylene groups [H-8 (δH 2.55, m) and H-7 (δH 2.03, m)]. Its 1H and 13C NMR data (Tables 2 and 3) were similar to those of dumhirone A [25]. The main differences were that two methoxys attached to C-3, 5 (δC 150.3, s) in compound 2 replaced two H-atoms in dumhirone A [25], and the 12-hydroxy-11-methoxyphenyl in compound 2
was present instead of 11-hydroxyphenyl in dumhirone A [25]. The above analyses were supported by two equivalent olefinic protons (H-2 and H-6), the HMBC cross-peaks from H-OMe to C-11 (δC 148.1, s), and from H-OMe to C-3, 5, respectively, and the ROESY correlations of H-OMe with H-10 and H-OMe with H-2, 6, respectively (Fig. 2). Consequently, compound 2 was deduced as 3,5-dimethoxy-1-(12-hydroxy-11methoxyphenethyl)-1-hydroxycyclohexa-2,5-dienone, named aphyllone B (Fig. 1). Compound 3 was isolated as white powder. The molecular formula, C15H14O4, was established by HRESIMS [M + H]+ m/z 259.0965 (calcd for 259.0965). The absorption band at 3396 cm−1 in IR spectrum indicated the presence of hydroxyl groups. The 1H NMR spectrum exhibited the signals of three 1, 3, 4-trisubstituted aromatic protons [H-10 (δH 6.66, br s), H-13 (δH 6.71, d, J = 7.9 Hz) and H-14 (δH 6.59, d, J = 7.9 Hz)], three 1, 3, 5-trisubstituted aromatic protons [H-2, 6 (2H, δH 6.23, br s) and H-4 (δH 6.20, br s)], two methylene groups [H-8 (δH 2.78, m) and H-7 (δH 2.73, m)], and one methylenedioxy group [H-15 (δH 5.92, s)]. Its 1H and 13C NMR data (Tables 2 and 3) resembled those of densiflorol A [26] except that the hydroxyl group attached to C-3 (δC 156.6, s) in compound 3 was present instead of the methoxy group in densiflorol A [26],
Fig. 4. Calculated and experimental ECD spectra of compound 1.
D. Yang et al. / Fitoterapia 100 (2015) 11–18
which was proven by two equivalent aromatic protons (H-2 and H-6) and the absent methoxy in compound 3 (Tables 2 and 3). Thus, compound 3 was defined as 9-[7-(3,5-dihydroxyphenyl)-ethyl]-11a,12a-benzodioxole, named aphyllal C (Fig. 1). Compound 4 was isolated as white amorphous powder. The molecular formula, C15H16O5, was established by HRESIMS [M–H]− m/z 275.0924 (calcd 275.0925). The presence of hydroxyl (3428 cm−1) was deduced from the IR spectrum. The 1H NMR spectrum displayed three 1, 3, 4-trisubstituted aromatic protons [H-10 (δH 6.55, d, J = 1.8 Hz), H-13 (δH 6.64, d, J = 7.8 Hz) and H-14 (δH 6.56, dd, J = 7.8, 1.8 Hz)], three 1, 3, 5-trisubstituted aromatic protons [H-2, 6 (2H, δH 6.22, d, J = 2.4 Hz) and H-4 (δH 6.13, t, J = 2.4 Hz)], one methylene group [H-8 (δH 2.88, dd, J = 13.8, 7.2 Hz) and (δH 2.79, dd, J = 13.2, 6.6 Hz)], one oxygenated methine [H-7 (δH 4.57, t, J =6.6 Hz)], and one methoxy group (δH 3.72, s). Its 1H and 13C NMR data (Tables 2 and 3) were similar to those of nobilin D [27]. The difference was the 3,5-dihydroxyphenyl in compound 4 present instead of the 3,5-dimethoxy-4-hydroxyphenyl in nobilin D [27], which was evidenced by two symmetric aromatic protons (H-2 and H-6), along with the HMBC crosspeak from the only one methoxy to C-11 (δC 148.5, s). The configuration of compound 4 was established to be identical to dendrocandin A [28] based on the similar optical rotation and CD data. Thus, compound 4 was established as (R)-11methoxy-3,5,7,12-tetrahydroxybibenzyl, named aphyllal D. Compound 5 was obtained as white amorphous powder. The molecular formula, C16H18O5, was established by HRESIMS [M + Na]+ m/z 313.1045 (calcd. 313.1052). Its IR bands at 3430 cm− 1 revealed the presence of hydroxyl groups. Comparing the NMR data with those of compound 4 (Tables 2 and 3) indicated that the two compounds were quite similar. The difference was the methoxy attached to C-3 in compound 5 replaced the hydroxy in compound 4, which was confirmed by the HMBC cross-peak from H-OMe (δH 3.69, s) to C-3 (δC 161.6, s) in compound 5. The configuration of compound 5 was established to be identical to compound 4 based on the similar optical rotation and CD data. Therefore, compound 5 was determined as (R)-3,11-dimethoxy-5,7,12-trihydroxybibenzyl, named aphyllal E. Compounds 6–14 were identified by comparison of their NMR data with those in the literatures [10–18]. D. aphyllum is used as one of the most common source for Shihu as both a medicinal herb and a high-quality health food. Due to some phenolic compounds from different Dendrobium species showing significant inhibitory effects on NO production [27,29], and since NO is an essential component of the host innate immune and inflammatory response to a variety of pathogens [30], the anti-inflammatory assay in LPS-stimulated RAW264.7 cells was carried out on compounds (1–4 and 6–14) by MTT assay, at a concentration of 25 μM. As a result, compounds 6, 8 and 14 exhibited inhibitory activities against NO production with the inhibition (%) of 32.48, 35.68, and 38.50, respectively, and compounds 1–4, 7 and 9–13 show inhibitory activities with the inhibition (%) in the range of 12.21 to 27.23 (Table 4). Compound 6 showed higher activity against NO production than compound 7, suggesting that the phenanthrene is a stronger inhibitor of NO production than dihydrophenanthrene. Compound 8 possessed stronger inhibitory effect against NO production than other bibenzyl derivatives
17
(2–4 and 9–13), and these results indicated that more methoxys were favorable for inhibiting NO production. Compounds (1–4 and 6–14) were also evaluated for their in vitro cytotoxicity against five human tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW-480) according to a previously described procedure [20]. However no obviously cytotoxic activity was observed at the concentration of 25 μM. Oxidative stress is believed to be implicated in aging, neurodegeneration, diabetes mellitus, cardiovascular disease and cancer. Antioxidants might be useful in combating these processes and diseases. Considering the use of D. aphyllum for Shihu as both a medicinal herb and a high-quality health food, and some phenolic compounds from different Dendrobium species exhibiting significant antioxidant activities [31–33], compounds 1–3 were also evaluated for their DPPH radical scavenging activities at the concentration of 100 μg/mL. Compound 2 possessed significant DPPH radical scavenging activity with scavenging percentage (87.97%), and compounds 1 and 3 exhibited moderate activities with scavenging percentages (5.25% and 35.28%), respectively. Taken together, these results provide basis for the immunomodulatory and antioxidant effects of the phenolic compounds from D. aphyllum and the utilization of D. aphyllum. Acknowledgments This research was supported by Yunnan province (No. 2012CG001 and 2013IB021) and Pu'er Tea Research Institute commissioned project. We thank the analytical group of the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences for all spectra tests. Appendix A. Supplementary data 1D and 2D NMR, HREIMS, HRESIMS, IR, UV and [a]D spectra of compounds 1–5, CD spectra of compounds 1, 4 and 5, and X-ray crystallographic data for compound 1 are available as Supporting Information. Supplementary data associated with this article can be found, in the online version, at http://dx.doi. org/10.1016/j.fitote.2014.11.004. Table 4 Effects of compounds (1–4 and 6–14) at the concentration of 25 μM on LPS-induced NO production are represented as inhibition %. Compound
NO inhibition (%)
1 2 3 4 6 7 8 9 10 11 12 13 14 MG132a
12.21 19.72 22.07 14.96 32.48 24.88 35.68 24.41 21.60 27.23 25.82 18.31 38.50 50
a
MG132 (0.16 μM) was used as positive control.
18
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[18] Marin PD, Grayer RG, Slavica GJ, Kite GC, Veitch NC. Glycosides of tricetin methyl ethers as chemosystematic markers in Stachys subgenus Betonica. Phytochemistry 2004;65:1247–53. [19] Fan JT, Su J, Peng YM, Li Y, Yan H, Tan NH, et al. Rubiyunnanins C–H, cytotoxiccyclic hexapeptides from Rubia yunnanensis inhibiting nitric oxide production and NF-κB activation. Bioorg Med Chem 2010;18:8226–34. [20] Monks A, Scudiero D, Skehan P, Shoemaker R, Paull K, Vistica D, et al. Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines. J Natl Cancer Inst 1991;83:757–66. [21] Sheldrick GM, Schneider TR. SHELXL: high-resolution refinement. Methods Enzymol 1997;277:319–43. [22] Geng CA, Chen XL, Zhou NJ, Chen H, Ma YB, Huang XY, et al. LC–MS guided isolation of (±)-sweriledugenin A, a pair of enantiomeric lactones, from Swertia leducii. Org Lett 2014;16:370–3. [23] Mazzeo G, Santoro E, Andolfi A, Cimmino A, Troselj P, Petrovic AG, et al. Absolute configurations of fungal and plant metabolites by chiroptical methods. ORD, ECD, and VCD studies on phyllostin, scytolide, and oxysporone. J Nat Prod 2013;76:588–99. [24] Ishida K, Maksimenka K, Fritzsche K, Scherlach K, Bringmann G, Hertweck C. The boat-shaped polyketide resistoflavin results from re-facial central hydroxylation of the discoid metabolite resistomycin. Am Chem Soc 2006;128: 14619–24. [25] Xie CF, Qu JB, Sun B, Guo HF, Lou HX. Dumhirone A, an unusual phenylethyl cyclohexadienone from the Chinese liverwort Dumortiera hirsuta. Biochem Syst Ecol 2007;35:162–5. [26] Fan CQ, Zhao WM, Qin GW. New bibenzyl and phenanthrenedione from Dendrobium densiflorum. Chin Chem Lett 2000;11:705–6. [27] Zhang X, Xu JK, Wang J, Wang NL, Kurihara H, Kitanaka S, et al. Bioactive bibenzyl derivatives and fluorenones from Dendrobium nobile. J Nat Prod 2007;70:24–8. [28] Li Y, Wang CL, Guo SX, Yang JS, Xiao PG. Two new compounds from Dendrobium candidum. Chem Pharm Bull 2008;56:1477–9. [29] Ito M, Matsuzaki K, Wang J, Daikonya A, Wang NL, Yao XS, et al. New phenanthrenes and stilbenes from Dendrobium loddigesii. Chem Pharm Bull 2010;58:628–33. [30] McCartney-Francis NL, Song X, Mizel DE, Wahl SM. Selective inhibition of inducible nitric oxide synthase exacerbates erosive joint disease. J Immunol 2001;166:2734–40. [31] Li Y, Wang CL, Wang YJ, Wang FF, Guo SX, Yang JS, et al. Four new bibenzyl derivatives from Dendrobium candidum. Chem Pharm Bull 2009;57:997–9. [32] Li Y, Wang CL, Wang YJ, Guo SX, Yang JS, Chen XM, et al. Three new bibenzyl derivatives from Dendrobium candidum. Chem Pharm Bull 2009; 57:218–9. [33] Sritularak B, Anuwat M, Likhitwitayawuid K. A new phenanthrenequinone from Dendrobium draconis. J Asian Nat Prod Res 2011;13:251–5.
Contents lists available at ScienceDirect
Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Five new phenolic compounds from Dendrobium aphyllum Dan Yang a,b,d, Liang-Yan Liu c, Zhong-Quan Cheng d, Feng-Qing Xu b, Wei-Wei Fan b, Cheng-Ting Zi a,b, Fa-Wu Dong b, Jun Zhou b, Zhong-Tao Ding a,⁎, Jiang-Miao Hu b,⁎ a
School of Chemical Sciences and Technology of Yunnan University, Kunming 650091, PR China State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China College of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming 650201, PR China d Guilin Normal College, Guilin 541001, PR China b c
a r t i c l e
i n f o
Article history: Received 20 August 2014 Accepted in revised form 28 October 2014 Available online 11 November 2014 Keywords: Dendrobium aphyllum Phenanthrene Bibenzyl derivative NO production
a b s t r a c t One new phenanthrene, aphyllone A (1) and four new bibenzyl derivatives, aphyllone B (2) and aphyllals C–D (3–5), together with nine known compounds (6–14), were isolated from the stems of Dendrobium aphyllum (Roxb.) C. E. Fischer. The structures of these new compounds were elucidated by means of extensive spectroscopic analyses, and the absolute configuration of compound 1 was determined by single crystal X-ray diffraction and quantum calculations. Compounds 6, 8 and 14 inhibited NO production at the concentration of 25 μM in LPS-stimulated RAW264.7 cells with the inhibition (%) of 32.48, 35.68, and 38.50. Compound 2 possessed significant DPPH radical scavenging activity with scavenging percentage of 87.97% at the concentration of 100 μg/mL. © 2014 Published by Elsevier B.V.
Chemical compounds studied in this article: Moscatin (PubChem CID: 194774) Hircinol (PubChem CID: 442705) Moscatilin (PubChem CID: 176096) Gigantol (PubChem CID: 10221179) Batatasin III (PubChem CID: 10466989) Tristin (PubChem CID: 15736297) Dihydroresveratrol (PubChem CID: 185914)
1. Introduction Dendrobium, one of the largest genera in Orchidaceae, containing more than 1100 species identified, is widely distributed in throughout Asia, Europe and Australia. There are about 80 Dendrobium species in China, and the fresh or dried stems of many species have been used as both traditional Chinese and folk remedies for the treatment of various diseases, such as chronic atrophic gastritis, skin aging, fever, cancer and cardiovascular disease for thousands of years, as well as a high-quality
⁎ Corresponding authors. Tel.: +86 871 6522 3264; fax: +86 871 6522 3261. E-mail addresses: [email protected] (Z.-T. Ding), [email protected] (J.-M. Hu).
http://dx.doi.org/10.1016/j.fitote.2014.11.004 0367-326X/© 2014 Published by Elsevier B.V.
health food now [1–3]. The main chemical components of Dendrobium are phenolic compounds, polysaccharides, alkaloids and sesquiterpenoids with multiple biological activities, including immunomodulatory, antioxidant and anti-tumor effects, etc. [4,5]. Chinese Pharmacopoeia (2010 edition) has two monographs of medicinal Dendrobium plants: One is Dendrobii Caulis (Shihu in Chinese), derived from Dendrobium nobile, D. chrysotoxum, D. fimbriatum and other related Dendrobium species [2]. The other is Dendrobii Officinalis Caulis (Tiepi shihu in Chinese), derived from D. officinale [3]. D. aphyllum is grown in large quantities in the Southwest of China and used as one of the most common source for Shihu. Previous investigation on D. aphyllum yielded three polysaccharides with evident immunostimulating activities [6], together with secondary metabolites including bibenzyls, phenanthrenes and
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D. Yang et al. / Fitoterapia 100 (2015) 11–18
flavones, whose activities await elucidation [4,7–9]. In order to supply more evidence to the efficacy and safety of D. aphyllum in clinical applications, we investigated chemical constituents of the stems of D. aphyllum and led to the isolation of fourteen phenolic compounds, including one new phenanthrone, aphyllone A (1) and four new bibenzyl derivatives, aphyllone B (2) and aphyllals C–E (3–5) (Fig. 1), together with nine known compounds moscatin (6) [10], hircinol (7) [11], moscatilin (8) [12], gigantol (9) [13], batatasin III (10) [14], tristin (11) [15], dihydroresveratrol (12) [16], trigonopol B (13) [17] and tricetin 3′,4′,5′-trimethyl ether 7-Oβ-glucopyranoside (14) [18]. The absolute configuration of compound 1 was determined by single crystal X-ray diffraction and quantum calculations. The absolute configurations of compounds 4 and 5 were determined by optical rotations and circular dichroism (CD) spectra. This paper described the isolation and structural elucidation, inhibitory effects on NO production in LPS-stimulated RAW264.7 macrophages, the cytotoxic properties and DPPH radical scavenging activity of the selected isolates.
NMR spectra were recorded on Bruker AM-400, DRX-500 or Av III-600 instruments with TMS as the internal standard (Bruker, Bremerhaven, Germany). The chemical shifts were given in δ (ppm) scale with reference to the solvent signal. Mass spectra were recorded on an API QSTAR time-of-flight spectrometer (MDS Sciqaszex, Concord, Ontario, Canada) or LC-MS-IT-TOF (Shimadzu, Kyoto, Japan) spectrometer. Semi-preparative HPLC was performed on Agilent 1100 liquid chromatography with a ZORBAX SB-C18 (5 μm, 9.4 × 250 mm) column (Agilent, USA) at a flow rate of 3.0 mL/min. Column chromatography was performed on silica gel (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, China). Lichroprep RP-18 gel (40– 63 μm, Merck, Darmstadt, Germany), MCI gel CHP-20P (75– 150 μm, Mitsubishi Chemical Corp., Tokyo, Japan), Sephadex LH-20 (20–150 μm, Amersham Biosciences, Uppsala, Sweden). Fractions were monitored by TLC, and spots were visualized by UV light (254 nm) and sprayed with 5% H2SO4 in ethanol, followed by heating.
2. Experimental
The stems of D. aphyllum (cultivated) were collected in October 2010 from Menglian county, Yunnan Province, PR China, and identified by Prof. Hong Yu. A voucher specimen (No. Zsh12) was deposited at the State Key Laboratory of Phytochemistry and Plant Resource in West China, Kunming Institute of Botany, Chinese Academy of Sciences.
2.1. General X-ray data were collected using a Bruker APEX DUO instrument. Melting points were measured on a XRC-1 micromelting point apparatus (Beijing, PR China) and were uncorrected. Optical rotations were obtained on a Jasco P-1020 digital polarimeter (Horiba, Tokyo, Japan). UV spectra were taken on a Shimadzu UV-2401 PC spectrophotometer (Shimadzu, Kyoto, Japan). CD spectra were recorded with an Applied Photophysics Chirascan spectrometer (Agilent, America). IR spectra were obtained on a Bruker Tensor 27 infrared spectrophotometer (Bruker Optics GmbH, Ettlingen, Germany) with KBr pellets.
2.2. Plant material
2.3. Extraction and isolation The sun-dried stems of D. aphyllum (5 kg) were powdered and extracted with 95% ethanol (20 L × 4, 2 days each time) at room temperature, then filtered. The filtrate was evaporated under reduced pressure to give a crude extract. The extract was suspended in H2O and partitioned successively with ethyl
Fig. 1. Chemical structures of compounds 1–5.
D. Yang et al. / Fitoterapia 100 (2015) 11–18
acetate (EtOAc) and n-butanol. The EtOAc part (150 g) was chromatographed on silica gel column chromatography (Si CC, 1500 g), eluted with CHCl3-MeOH (100:0, 60:1, 20:1, 5:1, v/v), to give four fractions A–D. Fraction B (40 g) was divided into three sub-fractions (B1–B3), by Si CC (400 g) using CHCl3-MeOH (90:1, 60:1, 30:1, 10:1) as the eluent. Fraction B2 (10 g) was subjected to repeated Si CC eluted with CHCl3-MeOH (60:1, 30:1, 20:1) and followed by Sephadex-LH-20 column (CHCl3-MeOH, 1:1) to give compounds 1 (300 mg), 3 (10 mg), 6 (940 mg) and 7 (29 mg). Fraction C (30 g) was loaded on a RP-18 column (MeOHH2O, 30:70, 50:50, 70:30, 90:10) to give four sub-fractions C1–C4. Fraction C2 (10 g) was subjected to Si CC (100 g) eluted with CHCl3-MeOH (60:1, 40:1, 20:1, 10:1) and further purified by Sephadex LH-20 column (CHCl3-MeOH, 1:1) to yield compounds 8 (2147 mg), 9 (23 mg) and 10 (3 mg). Compounds 2 (20 mg), 5 (2 mg) and 11 (40 mg) were obtained from Fraction D (20 g) by Si CC (200 g) using CHCl3-MeOH (40:1, 20:1, 10:1, 5:1) as the eluent and further separated by a RP-18 column (MeOH-H2O, 20:80, 40:60, 60:40, 80:20). The n-BuOH partition (100 g) was chromatographed on Si CC (1000 g), eluted with CHCl3-MeOH (95:5, 90:10, 80:20, 60:40), to afford four sub-fractions (E–H). Fraction E (20 g) was separated by repeated Si CC (CHCl3-MeOH, 40:1, 20:1, 10:1) and Sephadex LH-20 column (MeOH) to yield compounds 12 (28 mg), 13 (4 mg) and 14 (4 mg). Fraction F (10 g) was performed on a MCI CHP-20P gel CC (200 g) and eluted with MeOH-H2O (10:90, 30:70, 60:40, 80:20) to give four fractions (F1–F4). Fraction F1 (3 g) was performed on Si CC (30 g), eluted with CHCl3-MeOH (15:1), and further purified by preparative HPLC MeOH-H2O (25:75) to afford compound 4 (3 mg, tR = 22 min). 2.4. Spectroscopic data Aphyllone A (1): Colorless prismatic crystals (petroleum ether–Me2CO, 5:2); mp 207–208 °C; [α]20 D : +383.0 (c 0.23, MeOH); UV (MeOH) λmax (log ε) 247 (2.76), 213 (2.31), 193 (1.85) nm; CD (c 0.08, MeOH) λ (Δε) 222 (−20.15), 231 (+27.65), 295 (−7.34); IR (KBr) νmax 3426, 1653, 1594, 1458, 1352, 1263, 1222, 1169, 1011, 816, 744 cm−1; 1H and 13C NMR data, see Table 1; positive-ion ESIMS: m/z 281 [M + Na]+; positive-ion HRESIMS [M + H]+ m/z 259.0961 (calcd for C15H15O4, 259.0965). Aphyllone B (2): White amorphous powder; UV (MeOH) λmax (log ε) 202 (4.69), 266 (1.61) nm; IR (KBr) νmax 3441, 1656, 1625, 1516, 1453, 1269, 1228, 1107, 1030 cm−1, 1H and 13C NMR data, see Tables 2 and 3; positive-ion ESIMS: m/z 343 [M + Na]+; positive-ion HREIMS [M]+ m/z 320.1256 (calcd for C17H20O6, 320.1260). Aphyllal C (3): White powder; mp 156–157 °C; UV (MeOH) λmax (log ε) 204 (5.30), 283 (1.19) nm; IR (KBr) νmax 3396, 2925, 1603, 1502, 1442, 1245, 1152, 1039, 977, 927, 837, 810 cm− 1; 1H and 13C NMR data, see Tables 2 and 3; positive-ion ESIMS: m/z 259 [M + H]+; positive-ion HRESIMS [M + H]+ m/z 259.0965 (calcd for C15H15O4, 259.0965). Aphyllal D (4): White amorphous powder; [α]22 D : − 20.3 (c 0.18, MeOH); UV (MeOH) λmax (log ε) 203 (4.31), 281
13
Table 1 1 H (500 MHz) and 13C (100 MHz) NMR data of compound 1 in acetone-d6, δ in ppm, J in Hz. δC
Position
δH
1
3.01 (dd, 16.5, 5.2) 2.35 (dd, 16.5, 2.0) – 5.34 (s) – – – – 6.70 (d, 7.5) 7.10 (dd, 7.5, 7.5) 6.60 (d, 7.5) – 6.41 (dd, 9.5, 3.0) 5.64 (dd, 9.5, 2.0) 3.29 (dd, 5.2, 2.0) 3.57 (s)
2 3 4 4a 4b 5 6 7 8 8a 9 10 10a OMe-4
38.2 (t) 196.1 (s) 103.8 (d) 173.2 (s) 77.6 (s) 119.3 (s) 159.1 (s) 117.8 (d) 130.4 (d) 119.1 (d) 135.4 (s) 129.7 (d) 130.1 (d) 42.8 (d) 56.7 (q)
(1.13) nm; CD (c 0.16, MeOH) λ (Δε) 211 (− 4.43), 233 (− 1.87); IR (KBr) νmax 3428, 2925, 1610, 1516, 1384, 1275, 1153, 1033, 843 cm− 1; 1H and 13C NMR data, see Tables 2 and 3; negative-ion ESIMS: m/z 275 [M–H]−; negative-ion HRESIMS [M–H]− m/z 275.0924 (calcd for C15H15O5, 275.0925). Aphyllal E (5): White amorphous powder; [α]22 D : − 30.5 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 203 (4.96), 280 (1.15) nm; CD (c 0.07, MeOH) λ (Δε) 211 (− 7.82), 231 (− 3.24); IR (KBr) νmax 3430, 2925, 1603, 1517, 1457, 1196,
Table 2 1 H NMR data of compounds 2–5 (2 and 5 in acetone-d6, 3 in CDCl3, 4 in CD3OD, δ in ppm, J in Hz). Position 2a 1 2
– 5.87 (s)
3 4
– –
5 6
– 5.87 (s)
7 8
2.03 (m) 2.55 (m)
9 10
– 6.76 (d, 1.9)
11 12 13
– – 6.69 (d, 8.0)
14
6.60 (dd, 8.0, 1.9) – 3.56 (s) 3.56 (s) 3.78 (s)
15 OMe-3 OMe-5 OMe11
3a
4b
5b
– 6.23 (br s) – 6.20 (br s) – 6.23 (br s) 2.73 (m) 2.78 (m)
– 6.22 (d, 2.4)
– 6.40 (br s)
– 6.13 (t, 2.4)
– 6.24 (t, 2.4)
– 6.22 (d, 2.4)
– 6.43 (br s)
4.57 (t, 6.6) 2.88 (dd, 13.8, 7.2) 2.79 (dd, 13.2, 6.6) – 6.55 (d, 1.8)
4.70 (br s) 2.89 (m)
– – 6.64 (d, 7.8)
– – 6.67 (d, 7.8)
6.56 (dd, 7.8, 1.8) – – – 3.72 (s)
6.62 (dd, 7.8, 1.8) – 3.69 (s) – 3.75 (s)
– 6.66 (br s) – – 6.71 (d, 7.9) 6.59 (d, 7.9) 5.92 (s) – – –
– 6.73 (d, 1.8)
Assignments were made based on 1H, 13C NMR, DEPT, HSQC, HMBC, COSY, and ROESY experiments. a NMR data for compounds 2 and 3 were recorded at 500 MHz. b NMR data of compounds 4 and 5 were recorded at 600 MHz.
14
D. Yang et al. / Fitoterapia 100 (2015) 11–18
Table 3 13 C NMR data of compounds 2–5 (2 and 5 in acetone-d6, 3 in CDCl3, 4 in CD3OD, δ in ppm). Position
2a
3a
4b
5b
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 OMe-3 OMe-5 OMe-11
70.4 (s) 120.2 (d) 150.3 (s) 176.5 (s) 150.3 (s) 120.2 (d) 45.1 (t) 30.8 (t) 134.2 (s) 112.7 (d) 148.1 (s) 145.5 (s) 115.6 (d) 121.3 (d)
144.7 (s) 108.0 (d) 156.6 (s) 100.5 (d) 156.6 (s) 108.0 (d) 37.9 (t) 37.1 (t) 135.4 (s) 108.9 (d) 147.4 (s) 145.6 (s) 108.1 (d) 121.1 (d) 100.7 (t)
148.3 (s) 106.0 (d) 159.3 (s) 102.3 (d) 159.3 (s) 106.0 (d) 76.9 (d) 46.3 (t) 131.3 (s) 114.4 (d) 148.5 (s) 145.8 (s) 115.8 (d) 123.1 (d)
149.1 (s) 103.7 (d) 161.6 (s) 100.5 (d) 159.0 (s) 106.4 (d) 75.8 (d) 46.6 (t) 131.2 (s) 114.0 (d) 147.7 (s) 145.7 (s) 115.2 (d) 122.8 (d)
55.2 (q) 55.2 (q) 56.1 (q)
55.3 (q) 56.2 (q)
conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2(4- sulfophenyl)-2H-tetrazolium (MTS) (Sigma, St. Louis, MO, USA) [20]. Briefly, 100 μL of adherent cells was seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition, both with an initial density of 1 × 105 cells/mL in 100 μL medium. Each tumor cell line was exposed to the test compound at a concentration of 25 μM in triplicate for 48 h, with cisplatin and paclitaxel (Sigma) as positive controls. After the incubation, MTS (100 μg) was added to each well, and the incubation continued for 4 h at 37 °C. The cells were lysed with 100 μL of 20% SDS–50% DMF after removal of 100 μL medium. The optical density of the lysate was measured at 490 nm in a 96-well microtiter plate reader (Bio-Rad 680). 2.7. DPPH radical scavenging assay
56.0 (q)
Assignments were made based on 1H, 13C NMR, DEPT, HSQC, HMBC,COSY, and ROESY experiments. a NMR data of compounds 2 and 3 were recorded at 100 MHz. b NMR data for compounds 4 and 5 were recorded at 150 MHz.
1154, 1034 cm− 1; 1H and 13C NMR data, see Tables 2 and 3; positive-ion ESIMS: m/z 313 [M + Na]+; positive-ion HRESIMS [M + Na]+ m/z 313.1045 (calcd for C16H18O5Na, 313.1052). 2.5. Nitric oxide production in RAW264.7 macrophages Murine monocytic RAW264.7 macrophages were dispensed into 96-well plates (2 × 105 cells/well) containing RPMI-1640 medium (Hyclone) with 10% FBS under a humidified atmosphere of 5% CO2 at 37 °C. After 24 h preincubation, cells were treated with the compounds (25 μM) dissolved in DMSO, in the presence of 1 μg/mL LPS for 18 h. NO production in each well was assessed by adding 100 μL of Griess reagent (Reagent A & Reagent B, respectively, Sigma) to 100 μL of each supernatant from LPS (Sigma)-treated or LPS- and compound-treated cells in triplicate. After 5 min incubation, the absorbance was measured at 570 nm with a 2104 Envision Multilabel Plate Reader (PerkinElmer Life Sciences, Inc., Boston, MA, USA). MG-132 was used as a positive control [19]. Nitrite concentrations were calculated by regression analysis of a standard curve using sodium nitrite as a standard. The NO inhibition (IH) of each assay was calculated using the following equation: IH (%) = {1 − [(test sample data) − (negative group data)] / [(positive group data) − (negative group data)]} × 100, where positive group data refer to nitrite concentrations of LPS alonetreated cells, negative group data refer to nitrite concentrations of media alone-treated cells and test sample data refer to nitrite concentrations of sample plus LPS-treated cells. 2.6. The cytotoxicity assay The human tumor cell lines HL-60, SMMC-7721, A-549, MCF-7, and SW-480 were used, which were obtained from ATCC (Manassas, VA, USA). All the cells were cultured in RPMI-1640 or DMEM (Hyclone, Logan, UT, USA), supplemented with 10% fetal bovine serum (Hyclone) at 37 °C in a humidified atmosphere with 5% CO2. Cell viability was assessed by
Reaction mixture contained 100 μL ethanol solution of DPPH (25 μg/mL) and 100 μL test compounds (100 μg/mL) in ethanol, respectively. The experiment was performed in triplicate for each of the individual sample. The reaction mixture was incubated for 1 h at 30 °C and the absorbance was measured at 515 nm. The change in absorbance with respect to the control (containing DPPH radical only without sample, expressed as 100% free radicals) was calculated as percentage scavenging. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was used as positive controls. The ability to scavenge DPPH radicals was calculated as follows: DPPH radical scavenging percentage (%) = (OD blank − OD sample) / OD blank × 100 OD blank is the absorbance of blank; OD sample is the absorbance of test compound. 2.8. X-ray crystal structure analysis Colorless crystals of compound 1 were obtained from petroleum ether–Me2CO (5:2). Intensity data were collected at 100 K on a Bruker APEX DUO diffractometer equipped with an APEX II CCD, using Mo Kα radiation. Cell refinement and data reduction were performed with Bruker SAINT. The structures were solved by direct methods using SHELXS-97 [21]. Refinements were performed with SHELXL-97 using full-matrix least-squares, with anisotropic displacement parameters for all the non-hydrogen atoms [21]. The Hatoms were placed in calculated positions and refined using a riding model. Molecular graphics were computed with PLATON. Crystallographic data (excluding structure factor tables) for the structures reported have been deposited with the Cambridge Crystallographic Data Center as supplementary publication no. CCDC 1003248 for compound 1. Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB 1EZ, UK [fax: Int. +44 (0) (1223) (336 033); e-mail: deposit@ccdc. cam. ac. uk]. Crystal data for aphyllone A (1): C15H14O4, M = 258.26, orthorhombic, a = 7.4270(10) Å, b = 7.6615(11) Å, c = 22.110(3) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V = 1258.1(3) Å3, T = 100(2) K, space group P212121, Z = 4, μ (Mo Kα) = 0.099 mm−1, 13045 reflections measured, 3503
D. Yang et al. / Fitoterapia 100 (2015) 11–18
independent reflections (Rint = 0.0268). The final R1 values were 0.0336 (I N 2σ(I)). The final wR(F2) values were 0.0887 (I N 2σ(I)). The final R1 values were 0.0362 (all data). The final wR(F2) values were 0.0901 (all data). The goodness of fit on F2 was 1.062. Flack parameter = 0.0(7). 3. Results and discussion Compound 1 had molecular formula C15H14O4, as deduced from HRESIMS [M + H]+ m/z 259.0961 (calcd for 259.0965) and NMR spectra (Table 1), indicating nine degrees of unsaturation. Its IR spectrum displayed the presence of hydroxyl (3426 cm−1), conjugated carbonyl (1653 cm−1) and aromatic ring (1593, 1458 cm−1) groups. In the 1H NMR spectrum of compound 1, three aromatic protons [H-6 (δH 6.70, d, 7.5 Hz), H-7 (δH 7.10, dd, 7.5, 7.5 Hz) and H-8 (δH 6.60, d, 7.5 Hz)] belong to ring A. The 13C NMR and DEPT spectra exhibited eleven sp2 carbons (one carbonyl, four quaternary carbons, and six methines), four sp3 carbons [one quaternary carbon (oxygenated), one methine, one methylene, and one methoxy, respectively]. The 1H–1H COSY correlations of H-1/H-10a and H-9/H-10, along with the HMBC correlations from H-1 to C-10 (δC 130.1, d), H-9 (δH 6.41, dd, J = 9.5, 3.0 Hz) to C-10a (δC 42.8, d), and from H-3 (δH 5.34, s) to C-2 (δC 196.1, s) and C-4 (δC 173.2, s), established the spin systems for the molecular fragments of C-1–C-10a–C-10–C-9, and an α, β-unsaturated ketone group (C-2–C-3–C-4). In addition, the HMBC correlations from H-9 to C-8 (δC 119.1, d) and C-4b (δC 119.3, s), along with correlations of H-10 to C-4a (δC 77.6, s) and C-8a (δC 135.4, s), clearly indicated that ring B is connected to C-4b and C-8a. Similarly, the HMBC correlations of H-1 to C-10, C-4a and C-3 (δC 103.8, d), along with
15
correlations of H-3 to C-4a showed that ring C is connected to C-4a and C-10a (Fig. 2). Thus, compound 1 was suggested to be the skeleton of 1,1,4a,10a-tetrahydrophenanthrene. The location of the methoxy group at C-4 was determined by HMBC correlations from H-OMe (δH 3.57, s) to C-4. One hydroxy group should be attached to the deshielded aromatic carbon C-5 (δC 159.1, s), and the other hydroxy group should be connected to C-4a, which was confirmed by HMBC correlations from H-1, H-3, and H-10 to C-4a. The 1H–1H COSY correlations of H-6/H-7 and H-7/H-8, and the observed NOESY correlations between H-3 and H-MeO, and between H-8 and H-9 further supported the structure of compound 1. Based on the above evidences, the planar structure of compound 1 was elucidated. To confirm the structure and determine the absolute configuration of compound 1, a proper crystal was obtained using an applied single crystal X-ray diffraction with Mo Kα radiation. As shown in Fig. 3, the α-orientations of OH-4a and H-10a were determined by X-ray crystallographic data. The final assignment of compound 1 was achieved using a quantum method by comparing the calculated electronic circular dichroism (ECD) and [α]D value with experimented data [22,23]. The ECD spectra derived from quantum chemical calculations have been used successfully for the stereochemical assignment of small- and medium-sized molecules [24]. Starting from the relative stereochemistry evidenced from the single-crystal X-ray diffraction, the quantum chemical calculation on the ECD spectra was utilized to elucidate its absolute stereochemistry. To determine the absolute configuration of compound 1, the comparison between the experimental and calculated ECD spectra of 1 (Fig. 4) was performed using the time-dependent density functional theory (DFT) method at the B3LYP/6-311++G(2d, p) level. The calculated ECD spectrum was comparable with the
Fig. 2. Selected COSY, HMBC and ROESY correlations of compounds 1, 2, 4, and 5.
16
D. Yang et al. / Fitoterapia 100 (2015) 11–18
Fig. 3. X-ray crystal structure of compound 1.
measured for compound 1. Similarly, the [α]D calculation was performed on the Gaussian09 program using TD-DFTb3lyp/6–311 g(d,p) level of theory on b3lyp/6–311 g(d,p) optimized geometry (in methanol). The [α]D value calculated (+ 378.9) was similar to the measured (+ 383.0). From the above evidence, the structure of compound 1 was elucidated as (4aR,10aS)-4a,5-dihydroxy-4-methoxy-1,1,10atrihydrophenanthr-2-enone (Fig. 1), named aphyllone A. Compound 2 was obtained as white amorphous powder. A molecular formula of C17H20O6 was established by the HREIMS [M]+ m/z 320.1256 (calcd for 320.1260). The presence of hydroxyl (3441 cm−1) and conjugated carbonyl (1656 cm−1) was deduced from the IR spectrum. The 1H NMR spectrum of 2 showed three 1, 3, 4-trisubstituted aromatic protons [H-10 (δH 6.76, d, J = 1.9 Hz), H-13 (δH 6.69, d, J = 8.0 Hz) and H-14 (δH dd, J = 8.0, 1.9 Hz)], one pair of symmetric olefinic protons H-2, 6 (δH 5.87, s) and methoxys [OMe-3, 5 (δH 3.56, s)], another methoxy signal [OMe-11 (δH 3.78, s)] and two methylene groups [H-8 (δH 2.55, m) and H-7 (δH 2.03, m)]. Its 1H and 13C NMR data (Tables 2 and 3) were similar to those of dumhirone A [25]. The main differences were that two methoxys attached to C-3, 5 (δC 150.3, s) in compound 2 replaced two H-atoms in dumhirone A [25], and the 12-hydroxy-11-methoxyphenyl in compound 2
was present instead of 11-hydroxyphenyl in dumhirone A [25]. The above analyses were supported by two equivalent olefinic protons (H-2 and H-6), the HMBC cross-peaks from H-OMe to C-11 (δC 148.1, s), and from H-OMe to C-3, 5, respectively, and the ROESY correlations of H-OMe with H-10 and H-OMe with H-2, 6, respectively (Fig. 2). Consequently, compound 2 was deduced as 3,5-dimethoxy-1-(12-hydroxy-11methoxyphenethyl)-1-hydroxycyclohexa-2,5-dienone, named aphyllone B (Fig. 1). Compound 3 was isolated as white powder. The molecular formula, C15H14O4, was established by HRESIMS [M + H]+ m/z 259.0965 (calcd for 259.0965). The absorption band at 3396 cm−1 in IR spectrum indicated the presence of hydroxyl groups. The 1H NMR spectrum exhibited the signals of three 1, 3, 4-trisubstituted aromatic protons [H-10 (δH 6.66, br s), H-13 (δH 6.71, d, J = 7.9 Hz) and H-14 (δH 6.59, d, J = 7.9 Hz)], three 1, 3, 5-trisubstituted aromatic protons [H-2, 6 (2H, δH 6.23, br s) and H-4 (δH 6.20, br s)], two methylene groups [H-8 (δH 2.78, m) and H-7 (δH 2.73, m)], and one methylenedioxy group [H-15 (δH 5.92, s)]. Its 1H and 13C NMR data (Tables 2 and 3) resembled those of densiflorol A [26] except that the hydroxyl group attached to C-3 (δC 156.6, s) in compound 3 was present instead of the methoxy group in densiflorol A [26],
Fig. 4. Calculated and experimental ECD spectra of compound 1.
D. Yang et al. / Fitoterapia 100 (2015) 11–18
which was proven by two equivalent aromatic protons (H-2 and H-6) and the absent methoxy in compound 3 (Tables 2 and 3). Thus, compound 3 was defined as 9-[7-(3,5-dihydroxyphenyl)-ethyl]-11a,12a-benzodioxole, named aphyllal C (Fig. 1). Compound 4 was isolated as white amorphous powder. The molecular formula, C15H16O5, was established by HRESIMS [M–H]− m/z 275.0924 (calcd 275.0925). The presence of hydroxyl (3428 cm−1) was deduced from the IR spectrum. The 1H NMR spectrum displayed three 1, 3, 4-trisubstituted aromatic protons [H-10 (δH 6.55, d, J = 1.8 Hz), H-13 (δH 6.64, d, J = 7.8 Hz) and H-14 (δH 6.56, dd, J = 7.8, 1.8 Hz)], three 1, 3, 5-trisubstituted aromatic protons [H-2, 6 (2H, δH 6.22, d, J = 2.4 Hz) and H-4 (δH 6.13, t, J = 2.4 Hz)], one methylene group [H-8 (δH 2.88, dd, J = 13.8, 7.2 Hz) and (δH 2.79, dd, J = 13.2, 6.6 Hz)], one oxygenated methine [H-7 (δH 4.57, t, J =6.6 Hz)], and one methoxy group (δH 3.72, s). Its 1H and 13C NMR data (Tables 2 and 3) were similar to those of nobilin D [27]. The difference was the 3,5-dihydroxyphenyl in compound 4 present instead of the 3,5-dimethoxy-4-hydroxyphenyl in nobilin D [27], which was evidenced by two symmetric aromatic protons (H-2 and H-6), along with the HMBC crosspeak from the only one methoxy to C-11 (δC 148.5, s). The configuration of compound 4 was established to be identical to dendrocandin A [28] based on the similar optical rotation and CD data. Thus, compound 4 was established as (R)-11methoxy-3,5,7,12-tetrahydroxybibenzyl, named aphyllal D. Compound 5 was obtained as white amorphous powder. The molecular formula, C16H18O5, was established by HRESIMS [M + Na]+ m/z 313.1045 (calcd. 313.1052). Its IR bands at 3430 cm− 1 revealed the presence of hydroxyl groups. Comparing the NMR data with those of compound 4 (Tables 2 and 3) indicated that the two compounds were quite similar. The difference was the methoxy attached to C-3 in compound 5 replaced the hydroxy in compound 4, which was confirmed by the HMBC cross-peak from H-OMe (δH 3.69, s) to C-3 (δC 161.6, s) in compound 5. The configuration of compound 5 was established to be identical to compound 4 based on the similar optical rotation and CD data. Therefore, compound 5 was determined as (R)-3,11-dimethoxy-5,7,12-trihydroxybibenzyl, named aphyllal E. Compounds 6–14 were identified by comparison of their NMR data with those in the literatures [10–18]. D. aphyllum is used as one of the most common source for Shihu as both a medicinal herb and a high-quality health food. Due to some phenolic compounds from different Dendrobium species showing significant inhibitory effects on NO production [27,29], and since NO is an essential component of the host innate immune and inflammatory response to a variety of pathogens [30], the anti-inflammatory assay in LPS-stimulated RAW264.7 cells was carried out on compounds (1–4 and 6–14) by MTT assay, at a concentration of 25 μM. As a result, compounds 6, 8 and 14 exhibited inhibitory activities against NO production with the inhibition (%) of 32.48, 35.68, and 38.50, respectively, and compounds 1–4, 7 and 9–13 show inhibitory activities with the inhibition (%) in the range of 12.21 to 27.23 (Table 4). Compound 6 showed higher activity against NO production than compound 7, suggesting that the phenanthrene is a stronger inhibitor of NO production than dihydrophenanthrene. Compound 8 possessed stronger inhibitory effect against NO production than other bibenzyl derivatives
17
(2–4 and 9–13), and these results indicated that more methoxys were favorable for inhibiting NO production. Compounds (1–4 and 6–14) were also evaluated for their in vitro cytotoxicity against five human tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW-480) according to a previously described procedure [20]. However no obviously cytotoxic activity was observed at the concentration of 25 μM. Oxidative stress is believed to be implicated in aging, neurodegeneration, diabetes mellitus, cardiovascular disease and cancer. Antioxidants might be useful in combating these processes and diseases. Considering the use of D. aphyllum for Shihu as both a medicinal herb and a high-quality health food, and some phenolic compounds from different Dendrobium species exhibiting significant antioxidant activities [31–33], compounds 1–3 were also evaluated for their DPPH radical scavenging activities at the concentration of 100 μg/mL. Compound 2 possessed significant DPPH radical scavenging activity with scavenging percentage (87.97%), and compounds 1 and 3 exhibited moderate activities with scavenging percentages (5.25% and 35.28%), respectively. Taken together, these results provide basis for the immunomodulatory and antioxidant effects of the phenolic compounds from D. aphyllum and the utilization of D. aphyllum. Acknowledgments This research was supported by Yunnan province (No. 2012CG001 and 2013IB021) and Pu'er Tea Research Institute commissioned project. We thank the analytical group of the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences for all spectra tests. Appendix A. Supplementary data 1D and 2D NMR, HREIMS, HRESIMS, IR, UV and [a]D spectra of compounds 1–5, CD spectra of compounds 1, 4 and 5, and X-ray crystallographic data for compound 1 are available as Supporting Information. Supplementary data associated with this article can be found, in the online version, at http://dx.doi. org/10.1016/j.fitote.2014.11.004. Table 4 Effects of compounds (1–4 and 6–14) at the concentration of 25 μM on LPS-induced NO production are represented as inhibition %. Compound
NO inhibition (%)
1 2 3 4 6 7 8 9 10 11 12 13 14 MG132a
12.21 19.72 22.07 14.96 32.48 24.88 35.68 24.41 21.60 27.23 25.82 18.31 38.50 50
a
MG132 (0.16 μM) was used as positive control.
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