J Nat Med (2015) 69:135–141 DOI 10.1007/s11418-014-0869-1
NOTE
Chemical structures of constituents from the flowers of Osmanthus fragrans var. aurantiacus Jiang Liu • Seikou Nakamura • Bin Xu • Takahiro Matsumoto • Tomoe Ohta Katsuyoshi Fujimoto • Keiko Ogawa • Masashi Fukaya • Shiori Miyake • Masayuki Yoshikawa • Hisashi Matsuda
•
Received: 17 July 2014 / Accepted: 23 August 2014 / Published online: 10 October 2014 Ó The Japanese Society of Pharmacognosy and Springer Japan 2014
Keywords Osmanthus fragrans var. aurantiacus Floraosmanoside Floraosmanolactone Megastigmane c-Decalactone Medicinal flower
and a secoiridoid glucoside (ligstroside) were isolated from the flowers [1–4]. In particular, several lignans was reported to show inhibitory effects on nitric oxide (NO) production [2]. In the course of our studies on bioactive constituents from medicinal flowers [5–14], we examined the constituents from the flowers of O. fragrans var. aurantiacus cultivated in Guangxi Zhuang Autonomous Region of China. We also examined the inhibitory effects of the isolated principal constituents on NO production in lipopolysaccharide (LPS)-activated RAW264.7 macrophages. Here, we describe the isolation and structure elucidation of three new megastigmane glycosides named floraosmanosides I (1), II (2), and III (3) and a new cdecalactone named floraosmanolactone I (4) and the inhibitory effects of the constituents on NO production.
Introduction
Results and discussion
The Oleaceae plant, Osmanthus fragrans var. aurantiacus, is an evergreen tree that is widely cultivated in many countries including China and Japan. The flower has been used in food products and perfume. In previous studies, lignans, sterols, a pentacyclic triterpene (pomolic acid),
The MeOH extract (43.8 % from the dried flowers of O. fragrans var. aurantiacus cultivated in Guangxi Zhuang Autonomous Region, China) was partitioned into an nhexane-H2O (1:1, v/v) mixture to furnish an n-hexanesoluble fraction (4.6 %) and an aqueous layer. The aqueous layer was further extracted with CHCl3 to give CHCl3(2.8 %) and H2O- (36.4 %) soluble fractions. The CHCl3soluble fraction was subjected to normal phase and reversed-phase silica gel column chromatography and repeated HPLC to give floraosmanoside I (1, 0.00077 %), II (2, 0.0063 %), and III (3, 0.00011 %) and floraosmanolactone I (4, 0.0011 %) together with 16 known compounds, geranyl-b-primeveroside (5, 0.00036 %) [15], jasminoside O (6, 0.0019 %) [16], foliamenthic acid (7, 0.00026 %) [17], 10-hydroxygeraniol (8, 0.00088 %) [18], 10-acetoxyligustroside (9, 0.0035 %) [19], ligustroside (10,
Abstract Three new megastigmane glycosides named floraosmanosides I–III and a new c-decalactone named floraosmanolactone I together with 16 known constituents were isolated from the flowers of Osmanthus fragrans var. aurantiacus cultivated in Guangxi Zhuang Autonomous Region, China. The chemical structures of the new compounds were elucidated on the basis of chemical and physicochemical evidence. Among them, ligustroside and (?)-pinoresinol significantly inhibited nitric oxide production in lipopolysaccharide-activated RAW264.7 macrophages.
J. Liu Key Laboratory of Crop Ecophysiology and Farming System in Southwest China, Ministry of Agriculture, Sichuan Agricultural University, Chengdu 611130, People’s Republic of China S. Nakamura B. Xu T. Matsumoto T. Ohta K. Fujimoto K. Ogawa M. Fukaya S. Miyake M. Yoshikawa H. Matsuda (&) Department of Pharmacognosy, Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan e-mail: [email protected]
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0.011 %) [20], 30 -O-D-glucopyranosyl ligustroside (11, 0.00050 %) [21], oleoacteoside (12, 0.00091 %) [22], isoacteoside (13, 0.00036 %) [23], (?)-epipinoresinol (14, 0.00040 %) [24], (?)-phillygenin (15, 0.0042 %) [25], (?)-pinoresinol (16, 0.0020 %) [24], trans-p-coumaric acid (17, 0.00012 %), trans-ferulic acid (18, 0.0018 %), p-tyrosol (19, 0.0027 %), and vanillic acid (20, 0.0027 %) (Fig. 1). Floraosmanosides I (1) and II (2) were obtained as a white amorphous powder with negative specific rotations 26 (1: ½a24 D - 8.4; 2: ½aD - 48.9, both in MeOH), respectively. The IR spectra of 1 and 2 showed absorption bands assignable to hydroxy and ether functions [1: 3400 and 1,076 cm-1, 2: 3,400 and 1,078 cm-1]. FABMS in the positive-ion mode of 1 and 2 revealed the common quasimolecular ion [M ? Na]? at m/z 513 from which the molecular formula C24H42O10 was deduced via HRMS and 13 C-NMR data. Acid hydrolysis of 1 and 2 with 1 M HCl liberated D-glucose and D-xylose, which were identified by HPLC of their tolylthiocarbamoyl thiazolidine derivatives [26]. The 1H-NMR (CD3OD) and 13C-NMR (Table 1) spectra of 1 and 2, which were assigned by various NMR experiments, showed signals assignable to aglycone parts {1: d 0.99 (6H, s, H3-11, 12), 1.18 (3H, d, J = 6.2 Hz, H310), 1.42 (2H, m, H2-2), 1.53, 1.64 (1H each, both m, H28), 1.58 (2H, m, H2-3), 1.60 (3H, s, H3-13), 1.89 (2H, m, H2-4), 1.99, 2.19 (1H each, both m, H2-7), 3.84 (1H, m, H-9), 2: d 1.00 (6H, s, H3-11, 12), 1.25 (3H, d, J = 6.2 Hz,
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H3-10), 1.42 (2H, m, H2-2), 1.53, 1.65 (1H each, both m, H2-8), 1.57 (2H, m, H2-3), 1.60 (3H, s, H3-13), 1.90 (2H, m, H2-4), 2.00, 2.19 (1H each, both m, H2-7), 3.82 (1H, m, H-9)}, a b-D-glucopyranosyl moiety [1: d 4.32 (1H, d, J = ca. 7 Hz, H-10 ), 2: d 4.33 (1H, d, J = 7.6 Hz, H-10 )], and a b-D-xylopyranosyl moiety [1: d 4.33 (1H, d, J = ca. 7 Hz, H-100 ), 2: d 4.33 (1H, d, J = 7.6 Hz, H-100 )]. The proton and carbon signals due to the diglycosyl moiety in the 1H- and 13C-NMR spectra of 1 and 2 were superimposable on those of geranyl-b-primeveroside (5), while the proton and carbon signals assignable to the aglycone parts of 1 and 2 resembled those of 4-(2,6,6-trimethylcyclohex1-enyl)butan-2-ol [27], except for the signals around the 9-position. The structures of 1 and 2 were characterized by means of DQF COSY and HMBC experiments (Fig. 2), i.e., the DQF COSY data of 1 and 2 indicated the presence of the partial structures written in bold lines and long-range correlations in the HMBC experiment of 1 and 2 were observed between the following proton and carbon—H2-2 and C-1, 3, 4, 6; H2-3 and C-1, 2, 4; H2-4 and C-2, 3, 5, 6, 13; H2-7 and C-5, 6, 9; H-8 and C-9; H-9 and C-7; H3-10 and C-8, 9; H3-11,12 and C-1, 2, 6; H3-13 and C-4, 5, 6; H-10 and C-9; H-10 and C-60 . The absolute stereochemistries at the 9-position of 1 and 2 were determined by application of the empirical rules of 13C-NMR chemical shift reported by Matsunami et al. [28]. Namely, the 13CNMR signals [dc 76.1–76.4 (C-9); 19.8–19.9 (C-10); 102.2–102.4 (C-10 )] of megastigmanes with 4-(2,6,6-
Fig. 1 Structures of the compounds isolated from O. fragrans var. auranticus
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J Nat Med (2015) 69:135–141 Table 1 13C-NMR (125 MHz) data of compounds 1–4 (1–3: CD3OD, 4: CDCl3)
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Position
Interchangeable
2
3
Position
1
2
3
Position
4
1
36.0
35.9
33.0
10
102.3
103.9
100.8
2
177.4
2
41.1
41.1
32.7
20
74.9
74.8
74.9
3
28.8
3
20.6
20.6
24.0
30
78.1
78.0
78.2
4
28.0
4
33.8
33.7
122.1
40
71.5
71.4
71.6
5
81.0
5
127.9
127.9
135.2
50
76.9
76.8
76.9
10
35.4
6
138.5
138.3
55.5
60
69.8
69.7
69.9
20
25.2a
136.1
1
00
105.6
0
3
29.0a
00
0
7
a
1
25.7
25.3
105.5
105.4
8
39.1
38.1
134.1
2
75.1
75.1
74.9
4
25.5
9
76.3
78.0
75.4
300
77.6
77.6
77.8
50
32.5
10
19.8
21.9
22.4
400
71.2
71.1
71.2
60
62.8
00
66.8
66.8
66.9
a
11
29.2
29.1
27.4
12
29.2
29.1
28.0a
13
20.2
20.2
23.5
5
Fig. 2 Important 2D NMR correlations of 1–4
trimethylcyclohex-1-enyl)butan-2-ol moiety and R-configuration at the 9-position were shifted upfield relative to those [dc 77.9 (C-9); 21.8 (C-10); 103.9 (C-10 )] of megastigmanes with S-configuration. The 9-, 10-, and 10 -carbon signals of 1 were observed at dc 76.3 (C-9), 19.8 (C-10), and 102.3 (C-10 ), so that the C-9 configuration of 1 was determined to be R. On the other hand, the 9-, 10-, and 10 carbon signals of 2 were observed at dc 78.0 (C-9), 21.9 (C10), and 103.9 (C-10 ), so that the C-9 configuration of 2 was determined to be S. This evidence led us to formulate the structures of floraosmanosides I (1) and II (2) as shown. Floraosmanoside III (3), obtained as a white amorphous powder with positive specific rotation (½a27 D ? 58.1 in MeOH), showed absorption bands due to hydroxy and ether functions in the IR spectrum. Positive-ion FABMS revealed a quasimolecular ion [M?Na]? at m/z 511 from which the molecular formula C24H40O10 was deduced via HRMS and 13C-NMR data. The 1H-NMR and 13C-NMR (Table 1) spectra of 3 showed signals assignable to a megastigmane part and a sugar moiety. The proton and carbon signals due to the diglycosyl moiety in the 1H- and 13 C-NMR spectra of 3 were superimposable on those of 1
and 2, while the proton and carbon signals assignable to the aglycone parts of 3 resembled those of 4-(2,6,6-trimethylcyclohex-2-enyl)but-3-en-2-ol [29], except for the signals around the 9-position. The structure of 3 was characterized by means of DQF COSY and HMBC experiments (Fig. 2). The linkage positions of the glycosides were determined by a HMBC experiment, which showed a long-range correlation between H-10 and C-9; H-100 and C-60 . Furthermore, the configuration of the 9-position of 3 was deduced by comparison of the 13CNMR data [28]. Namely, the 13C-NMR signals [dc 77.3–79.1 (C-9); 21.2–21.8 (C-10); 102.2–103.0 (C-10 )] of megastigmanes with but-3-en-2-ol moiety and R-configuration at the 9-position were shifted upfield relative to those [dc 74.7–76.3 (C-9); 22.3–22.6 (C-10); 100.5–101.7 (C-10 )] of megastigmanes with S-configuration. The 9-, 10-, and 10 carbon signals of 3 were observed at dc 75.4 (C-9), 22.4 (C10), and 100.8 (C-10 ), so that the C-9 configuration of 3 was deduced to be S. This evidence led us to formulate the structure of floraosmanoside III (3) as shown. Floraosmanolactone I (4), obtained as a colorless oil, showed absorption bands at 3,400 and 1,771 cm-1
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assignable to hydroxy and c-lactone functions in the IR spectrum. The molecular formula C10H18O3 of 4 was determined from the positive-ion FABMS (m/z 209 [M ? Na]?) and by HRFABMS measurement. The 1H-NMR (CDCl3) and 13C-NMR (Table 1) spectra of 4 showed signals assignable to a methylene and a methine bearing an oxygen function {[d 3.65 (2H, t, J = 6.8 Hz, H2-60 ), 4.49 (1H, m, H-5)]} and a c-lactone carbon [dC 177.4 (C-2)]. The relative structure of 3 was characterized by DQF COSY and HMBC experiments as shown in Fig. 2. The absolute stereostructure of the 4-position of 4 was characterized by comparison of the optical rotation of 4 with those of known c-butylolactones with a side chain at the 5position. Namely, the specific rotation of a known compound, (R)-dihydro-5-(30 -hydroxypropyl)-2(3H)-furanone, with 5R orientation was reported to show positive {½a21 D ? 11.5 (EtOH)}, while the optical rotation of a known compound, (S)-dihydro-5-(30 -hydroxypropyl)2(3H)-furanone, with 5S orientation was reported to show negative {[½a21 D - 11.4 (EtOH)} [30]. Since 4 showed positive specific rotation {½a17 D ? 15.0 (EtOH)}, the absolute stereostructure of the 5-position of 4 was determined to be R orientation. On the basis of this evidence, floraosmanolactone I (4) was determined to be (R)-dihydro5-(60 -hydroxyhexyl)-2(3H)-furanone. Next, in the course of our studies on NO production inhibitors from natural medicine [31, 32], the inhibitory effects of the principal constituents [4–7, 9–12, 15, 16, and 19] on NO production in LPS-activated RAW264.7 macrophages were examined. Among them, ligustroside (10) [inhibition (%): 17.8 ± 2.3 (P \ 0.01) at 30 lM, 40.7 ± 3.3 (P \ 0.01) at 100 lM] [33], (?)-phillygenin (15) [inhibition (%): 21.3 ± 2.6 (P \ 0.05) at 30 lM, 66.5 ± 1.0 (P \ 0.01) at 100 lM] [2], and (?)-pinoresinol (16) [inhibition (%): 20.5 ± 3.4 (P \ 0.01) at 30 lM, 53.1 ± 1.4 (P \ 0.01) at 100 lM] [34, 35], which were already reported to show the inhibitory effect on NO production, also displayed the inhibitory effect without cytotoxic effects using the MTT assay. However, the NO inhibitory effects of 10, 15, and 16 were weaker than those of a previous reported reference compound, caffeic acid phenethyl ester (CAPE) [IC50: 3.1 lM] [31]. On the other hand, other compounds did not show inhibitory effects on NO production (\21.0 % at 100 lM).
Experimental General The following instruments were used to obtain physical data—specific rotations, a Horiba SEPA-300 digital
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polarimeter (l = 5 cm); IR spectra, a Thermo Electron Nexus 470; EIMS and HREIMS, JEOL JMS-GCMATE mass spectrometer; FABMS and HRFABMS, a JEOL JMS-SX 102A mass spectrometer; 1H-NMR spectra, JEOL JNM-ECA 600 (600 MHz) spectrometer; 13C-NMR spectra, JEOL JNM-ECA 600 (150 MHz) spectrometers; HPLC, a Shimadzu RID-6A refractive index and SPD10Avp UV-VIS detectors. COSMOSIL 5C18-MS-II and COSMOSIL 5C18-PAQ (250 9 4.6 mm i.d. and 250 9 20 mm i.d.) columns were used for analytical and preparative purposes. The following materials were used for chromatography—normal phase silica gel column chromatography, Silica gel BW-200 (Fuji Silysia Chemical, Ltd., 150–350 mesh); reversed-phase silica gel column chromatography, Chromatorex ODS DM1020T (Fuji Silysia Chemical, Ltd., 100–200 mesh); TLC, precoated TLC plates with Silica gel 60F254 (Merck, 0.25 mm) (ordinary phase) and Silica gel RP-18 F254S (Merck, 0.25 mm) (reversed phase); reversed-phase HPTLC, precoated TLC plates with Silica gel RP-18 WF254S (Merck, 0.25 mm). Detection was achieved by spraying with 1 % Ce(SO4)2– 10 % aqueous H2SO4 followed by heating. Plant material The dried flowers of O. fragrans var. aurantiacus cultivated in Guangxi Zhuang Autonomous Region, China were commercial products purchased from Koshiro Co. Ltd (Osaka, Japan) in 2011 (KPU Medicinal Flower-2011-OF). Extraction and isolation The flowers (2.0 kg) of Osmanthus fragrans var. aurantiacus were extracted three times with methanol under reflux for 3 h. Evaporation of the solvent under reduced pressure provided a methanol extract (874.9 g, 43.8 %). A part of the MeOH extract (824.7 g) was partitioned into an n-hexaneH2O (1:1, v/v) mixture to furnish an n-hexane-soluble fraction (85.7 g, 4.6 %) and an aqueous phase. The aqueous phase was further extracted with CHCl3 to give a CHCl3soluble fraction (53.3 g, 2.8 %) and an H2O-soluble fraction (685.4 g, 37.6 %). The CHCl3-soluble fraction (48.25 g) was subjected to normal phase silica gel column chromatography [700 g, n-hexane ? n-hexane:EtOAc (2:1, v/v) ? CHCl3 ? CHCl3:MeOH (10:1, v/v) ? CHCl3:MeOH:H2O (10:3:1 ? 7:3:1 ? 6:4:1, v/v/v, lower layer)] to give 6 fractions [Fr. 1 (47 mg), Fr. 2 (649 mg), Fr. 3 (3.96 g), Fr. 4 (26.04 g), Fr. 5 (5.77 g), Fr. 6 (8.90 g)]. A part of fraction 4 (20.15 g) was subjected to reversed-phase silica gel column chromatography [450 g, MeOH:H2O (10:90 ? 20:80 ? 40:60 ? 50:50 ? 60:40 ? 80:20, v/v) ? MeOH] to give 24 fractions [Fr. 4–1 (170 mg), Fr. 4–2 (280 mg), Fr. 4–3 (570 mg), Fr. 4–4 (690 mg), Fr. 4–5
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(380 mg), Fr. 4–6 (380 mg), Fr. 4–7 (350 mg), Fr. 4–8 (640 mg), Fr. 4–9 (400 mg), Fr. 4–10 (1.25 g), Fr. 4–11 (280 mg), Fr. 4–12 (630 mg), Fr. 4–13 (340 mg), Fr. 4–14 (220 mg), Fr. 4–15 (340 mg), Fr. 4–16 (350 mg), Fr. 4–17 (260 mg), Fr. 4–18 (400 mg), Fr. 4–19 (1000 mg), Fr. 4–20 (2.42 g), Fr. 4–21 (5.88 g), Fr. 4–22 (3.67 g), Fr. 4–23 (930 mg), and Fr. 4–24 (1.21 g)]. Fr. 4–4 (690 mg) was purified by HPLC [H2O, COSMOSIL 5C18-PAQ] to give 19 (36 mg). Fr. 4–5 (380 mg) was purified by HPLC [MeOH: H2O (30:70), COSMOSIL 5C18-MS-II] to give 20 (36 mg). Fr. 4–8 (640 mg) was purified by HPLC [MeOH: H2O (30: 70), COSMOSIL 5C18-MS-II] to give 4 (15 mg) and 18 (24 mg). Fr. 4–9 (400 mg) was purified by HPLC [MeOH:H2O (40:60), COSMOSIL 5C18-MS-II] to give 7 (3.5 mg), 8 (11.7 mg), and 17 (1.6 mg). Fr. 4–10 (1.25 g) was subjected to Sephadex LH-20 column chromatography (MeOH) and HPLC [MeOH:H2O (50:50), COSMOSIL 5C18-MS-II] to give 14 (5.3 mg), and 16 (26 mg). Fr. 4–12 (630 mg) was purified by HPLC [MeOH:H2O (55:45), COSMOSIL 5C18-MS-II] to give 15 (55 mg). Fraction 5 (5.77 g) was subjected to reversed-phase silica gel column chromatography [700 g, MeOH:H2O (40:60 ? 60:40 ?70: 30 ? 80:20 ? 90:10, v/v) ? MeOH] to give 9 fractions [Fr. 5–1 (100 mg), Fr. 5–2 (402 mg), Fr. 5–3 (551 mg), Fr. 5–4 (302 mg), Fr. 5–5 (485 mg), Fr. 5–6 (426 mg), Fr. 5–7 (143 mg), Fr. 5–8 (313 mg), Fr. 5–9 (1.56 g)]. Fr. 5–3 (551 mg) was purified by HPLC [MeOH: H2O (45:55), COSMOSIL 5C18-MS-II] to give 9 (59 mg), 10 (181 mg), and 11 (8.5 mg). Fr. 5–5 (485 mg) was subjected to Sephadex LH-20 column chromatography (MeOH) and HPLC [MeOH:H2O (60:40), COSMOSIL 5C18-MS-II] to give 5 (6.0 mg). Fraction 5–6 (426 mg) was purified by HPLC [MeOH:H2O (70:30), COSMOSIL 5C18-MS-II] to give 1 (13 mg), 2 (108 mg), and 3 (1.8 mg). Fraction 6 (8.90 g) was subjected to reversed-phase silica gel column chromatography [700 g, MeOH:H2O (20:80 ? 80:20, v/v) ? MeOH] to give 11 fractions [Fr. 6–1 (100 mg), Fr. 6–2 (721 mg), Fr. 6–3 (90 mg), Fr. 6–4 (100 mg), Fr. 6–5 (309 mg), Fr. 6–6 (1.72 g), Fr. 6–7 (125 mg), Fr. 6–8 (69 mg), Fr. 6–9 (134 mg), Fr. 6–10 (189 mg), Fr. 6–11 (166 mg)]. Fraction 6–9 (134 mg) was purified by HPLC [MeOH:H2O (40:60), COSMOSIL 5C18-MS-II] to give 13 (6.3 mg). Fraction 6–10 (189 mg) was purified by HPLC [MeOH:H2O (50:50), COSMOSIL 5C18-MS-II] to give 12 (16 mg). Fraction 6–11 (166 mg) was purified by HPLC [MeOH:H2O (60:40), COSMOSIL 5C18-MS-II] to give 6 (32 mg). Floraosmanoside I (1): a white amorphous powder; 24 ½aD - 8.4 (c = 0.66, MeOH); IR (KBr): mmax 3400, 1076 cm-1; 1H-NMR (CD3OD, 600 MHz) d 0.99 (6H, s, H3-11, 12), 1.18 (3H, d, J = 6.2 Hz, H3-10), 1.42 (2H, m, H2-2), 1.53, 1.64 (1H each, both m, H2-8), 1.58 (2H, m, H2-
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3), 1.60 (3H, s, H3-13), 1.89 (2H, m, H2-4), 1.99, 2.19 (1H each, both m, H2-7), 3.84 (1H, m, H-9), 4.32 (1H, d, J = ca. 7 Hz, H-10 ), 4.33 (1H, d, J = ca. 7 Hz, 100 -H); 13CNMR: given in Table 1; positive-ion FAB-MS: m/z 513 [M ? Na]?; HR-FAB-MS: m/z 513.2682 (calculated for C24H42O10Na [M?Na]?: m/z 513.2676). Floraosmanoside II (2): a white amorphous powder; ½a26 D –48.9 (c = 0.64, MeOH); IR (KBr): mmax 3400, 1078 cm-1; 1H-NMR (CD3OD, 600 MHz) d 1.00 (6H, s, H3-11, 12), 1.25 (3H, d, J = 6.2 Hz, H3-10), 1.42 (2H, m, H2-2), 1.53, 1.65 (1H each, both m, H2-8), 1.57 (2H, m, H23), 1.60 (3H, s, H3-13), 1.90 (2H, m, H2-4), 2.00, 2.19 (1H each, both m, H2-7), 3.82 (1H, m, H-9), 4.33 (2H, d, J = 7.6 Hz, H-10 , 100 ); 13C-NMR: given in Table 1; positive-ion FAB-MS: m/z 513 [M ? Na]?; HR-FAB-MS: m/z 513.2681 (calculated for C24H42O10Na [M?Na]?: m/z 513.2676). Floraosmanoside III (3): a white amorphous powder; ½a27 D ? 58.1 (c = 0.09, MeOH); IR (KBr): mmax 3400, 1075 cm-1; 1H-NMR (CD3OD, 600 MHz) d 0.83, 0.89 (3H each, both s, H3-12, 11, interchangeable), 1.18, 1.49 (2H, m, H2-2), 1.26 (3H, d, J = 6.9 Hz, H3-10), 1.63 (3H, br s, H3-13), 2.02 (2H, m, H2-3), 2.14 (2H, d, J = 8.9 Hz, H-6), 4.30 (1H, d, J = 7.6 Hz, H-10 ’), 4.33 (1H, d, J = 8.3 Hz, H-10 ), 4.40 (1H, m, H-9), 5.34 (1H, dd, J = 8.2, 15.2 Hz, H-8), 5.41 (1H, br s like, H-4), 5.52 (1H, dd, J = 8.9, 15.2 Hz, H-7); 13C-NMR: given in Table 1; positive-ion FAB-MS: m/z 511 [M ? Na]?; HR-FAB-MS: m/z 511.2514 (calculated for C24H40O10Na [M?Na]?: m/z 511.2519). Floraosmanolactone I (4): a colorless oil; ½a25 D ? 13.8 17 (c = 0.75, MeOH), ½aD ? 15.0° (c = 0.42, EtOH); IR (KBr): mmax 3400, 1771, 1184 cm-1; 1H-NMR (CDCl3, 600 MHz) d 1.38 (6H, m, H2-20 , 30 , 40 ), 1.57 (2H, m, H250 ), 1.59, 1.75 (1H each, both m, H2-10 ), 1.86, 2.33 (1H each, both m, H2-4), 2.53 (2H, m, H2-3), 3.65 (2H, t, J = 6.8 Hz, H2-60 ), 4.49 (1H, m, H-5)]; 13C-NMR: given in Table 1; positive-ion FAB-MS: m/z 209 [M ? Na]?; HR-FAB-MS: m/z 209.1161 (calculated for C10H18O3Na [M?Na]?: m/z 209.1154). Acid hydrolyses Compounds 1 and 2 (each 1.0 mg) were dissolved in 5 % aqueous H2SO4–1,4-dioxane (1:1, v/v, 1.0 ml), and each solution was heated at 90 °C for 3 h. After extraction three times with EtOAc, the aqueous layer was neutralized with Amberlite IRA-400 (OH- form). After drying in vacuo, the residue was dissolved in pyridine (0.5 mL) containing Lcysteine methyl ester hydrochloride (0.5 mg) and heated at 60 °C for 1 h. A solution of o-tolylisothiocyanate (0.5 mg)
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in pyridine (0.1 mL) was added to the mixture and heated at 60 °C for 1 h. The reaction mixture was analyzed by reversed-phase HPLC [column: COSMOSIL 5C18-AR-II (Nacalai Tesque), 250 9 4.6 mm i.d. (5 lm); mobile phase: MeCN–H2O in 1 % AcOH (18:82, v/v); detection: UV (250 nm); flow rate: 0.8 ml/min; column temperature: 35 °C] to identify the derivatives of constituent monosaccharides (D-glucose and D-xylose) in 1 and 2 by comparison of their retention times with those of authentic samples (tR: D-glucose; 48.9 min, L-glucose; 41.2 min, D-xylose; 56.9 min, L-xylose; 53.0 min).
Statistical analysis
Cell culture
References
The murine macrophage cells (RAW264.7, ATCC No. TIB-71) were obtained from Dainippon Pharmaceutical, Osaka, Japan and cultured in Dulbecco’s modified Eagle’s medium (DMEM, high glucose) supplemented with 10 % fetal calf serum, penicillin (100 U/mL), and streptomycin (100 lg/mL) (Sigma Chemical Co., St. Louis, MO, USA). The cells were incubated at 37 °C in 5 % CO2/air.
1. Yoo KH, Park J-H, Lee DK, Fu YY, Baek NI, Chung IS (2013) Pomolic acid induces apoptosis in SK-OV-3 human ovarian adenocarcinoma cells through the mitochondrial-mediated intrinsic and death receptor-induced extrinsic pathways. Oncol Lett 5:386–390 2. Lee DG, Lee SM, Bang MH, Park H-J, Lee TH, Kim YH, Kim JY, Baek NI (2011) Lignans from the flowers of Osmanthus fragrans var. aurantiacus and their inhibition effect on NO production. Arch Pharm Res 34:2029–2035 3. Lee DG, Park JH, Yoo K-H, Chung I-S, Lee Y-H, Lee J-K, Han D-S, Cho S-M, Baek N-I (2011) 24-ethylcholesta-4,24(28)-dien3,6-dione from Osmanthus fragrans var. aurantiacus flowers inhibits the growth of human colon cancer cell line, HCT-116. J Korean Soc Appl Bi 54:206–210 4. Lee D-G, Choi J-S, Yeon S-W, Cui E-J, Park H-J, Yoo J-S, Chung I-S, Baek N-I (2010) Secoiridoid glycoside from the flowers of Osmanthus fragrans var. aurantiacus makino inhibited the activity of b-secretase. J Korean Soc Appl Bi 53:371–374 5. Matsumoto T, Nakamura S, Fujimoto K, Ohta T, Ogawa K, Yoshikawa M, Matsuda H (2014) Structure of constituents isolated from the flower buds of Cananga odorata and their inhibitory effects on aldose reductase. J Nat Med 68:1–8. doi:10.1007/ s11418-014-0843-y 6. Fujimoto K, Nakamura S, Matsumoto T, Ohta T, Yoshikawa M, Ogawa K, Kashiwazaki E, Matsuda H (2014) Structures of acylated sucroses from the flower buds of Prunus mume. J Nat Med 68:481–487 7. Matsumoto T, Nakamura S, Ohta T, Fujimoto K, Yoshikawa M, Ogawa K, Matsuda H (2014) A rare glutamine derivative from the flower buds of daylily. Org Lett 16:3076–3078 8. Matsumoto T, Nakamura S, Nakashima S, Fujimoto K, Yoshikawa M, Ohta T, Ogawa K, Matsuda H (2014) Lignan dicarboxylates and terpenoids from the flower buds of Cananga odorata and their inhibitory effects on melanogenesis. J Nat Prod 77:990–999 9. Nakamura S, Fujimoto K, Matsumoto T, Nakashima S, Ohta T, Ogawa K, Matsuda H, Yoshikawa M (2013) Acylated sucroses and acylated quinic acids analogs from the flower buds of Prunus mume and their inhibitory effects on melanogenesis. Phytochemistry 92:128–136 10. Liu J, Nakamura S, Matsuda H, Yoshikawa M (2013) Hydrangeamines A and B, novel polyketide-type pseudoalkaloidcoupled secoiridoid glycosides from the flowers of Hydrangea macrophylla var. thunbergii. Tetrahedron Lett 54:32–34 11. Nakamura S, Fujimoto K, Matsumoto T, Ohta T, Ogawa K, Tamura H, Matsuda H, Yoshikawa M (2013) Structures of acylated sucroses and an acylated flavonol glycoside and
Effects on NO production in LPS-activated RAW264.7 macrophages The total amount of nitrite in a medium is used as an indicator of NO synthesis. The screening test for NO production using RAW264.7 macrophages was described previously [31, 32]. Briefly, RAW264.7 cells were cultured in DMEM, and the suspension seeded into a 96-well microplate at 2.5 9 105 cells/100 lL/well. After 6 h, nonadherent cells were removed by washing with PBS, the adherent cells were cultured in 100 lL of fresh medium containing the test compounds for 10 min, and then 100 lL of the medium containing LPS (from E. coli, 055: B5, Sigma) was added to stimulate the cells for 18 h (final concentration of LPS, 10 lg/mL). The nitrite concentration was measured from the supernatant by a Griess reaction. NO production in each well was assessed by measuring the accumulation of nitrite in the culture medium using Griess reagent. Inhibition (%) was calculated using the following formula and the IC50 was determined graphically (n = 4). Inhibition ð% Þ ¼ ½ðABÞ=ðAC Þ 100 A–C: Nitrite concentration (lg/mL) A: LPS (?), Sample (-); B: LPS (?), Sample (?); C: LPS (-), Sample (-) Cytotoxicity was evaluated by the 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetric assay according to the previous report [31].
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All data are expressed as the mean ± S.E.M. The data analysis was performed with a one-way analysis of variance (1-ANOVA), followed by Dunnett’s test. A p value of \0.05 was considered to be significant. Acknowledgments This work was supported in part by a Ministry of Education, Culture, Sports, Science and Technology (MEXT)Supported Program for the Strategic Research Foundation at Private Universities and by JSPS KAKENHI Grant Number 26460135.
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NOTE
Chemical structures of constituents from the flowers of Osmanthus fragrans var. aurantiacus Jiang Liu • Seikou Nakamura • Bin Xu • Takahiro Matsumoto • Tomoe Ohta Katsuyoshi Fujimoto • Keiko Ogawa • Masashi Fukaya • Shiori Miyake • Masayuki Yoshikawa • Hisashi Matsuda
•
Received: 17 July 2014 / Accepted: 23 August 2014 / Published online: 10 October 2014 Ó The Japanese Society of Pharmacognosy and Springer Japan 2014
Keywords Osmanthus fragrans var. aurantiacus Floraosmanoside Floraosmanolactone Megastigmane c-Decalactone Medicinal flower
and a secoiridoid glucoside (ligstroside) were isolated from the flowers [1–4]. In particular, several lignans was reported to show inhibitory effects on nitric oxide (NO) production [2]. In the course of our studies on bioactive constituents from medicinal flowers [5–14], we examined the constituents from the flowers of O. fragrans var. aurantiacus cultivated in Guangxi Zhuang Autonomous Region of China. We also examined the inhibitory effects of the isolated principal constituents on NO production in lipopolysaccharide (LPS)-activated RAW264.7 macrophages. Here, we describe the isolation and structure elucidation of three new megastigmane glycosides named floraosmanosides I (1), II (2), and III (3) and a new cdecalactone named floraosmanolactone I (4) and the inhibitory effects of the constituents on NO production.
Introduction
Results and discussion
The Oleaceae plant, Osmanthus fragrans var. aurantiacus, is an evergreen tree that is widely cultivated in many countries including China and Japan. The flower has been used in food products and perfume. In previous studies, lignans, sterols, a pentacyclic triterpene (pomolic acid),
The MeOH extract (43.8 % from the dried flowers of O. fragrans var. aurantiacus cultivated in Guangxi Zhuang Autonomous Region, China) was partitioned into an nhexane-H2O (1:1, v/v) mixture to furnish an n-hexanesoluble fraction (4.6 %) and an aqueous layer. The aqueous layer was further extracted with CHCl3 to give CHCl3(2.8 %) and H2O- (36.4 %) soluble fractions. The CHCl3soluble fraction was subjected to normal phase and reversed-phase silica gel column chromatography and repeated HPLC to give floraosmanoside I (1, 0.00077 %), II (2, 0.0063 %), and III (3, 0.00011 %) and floraosmanolactone I (4, 0.0011 %) together with 16 known compounds, geranyl-b-primeveroside (5, 0.00036 %) [15], jasminoside O (6, 0.0019 %) [16], foliamenthic acid (7, 0.00026 %) [17], 10-hydroxygeraniol (8, 0.00088 %) [18], 10-acetoxyligustroside (9, 0.0035 %) [19], ligustroside (10,
Abstract Three new megastigmane glycosides named floraosmanosides I–III and a new c-decalactone named floraosmanolactone I together with 16 known constituents were isolated from the flowers of Osmanthus fragrans var. aurantiacus cultivated in Guangxi Zhuang Autonomous Region, China. The chemical structures of the new compounds were elucidated on the basis of chemical and physicochemical evidence. Among them, ligustroside and (?)-pinoresinol significantly inhibited nitric oxide production in lipopolysaccharide-activated RAW264.7 macrophages.
J. Liu Key Laboratory of Crop Ecophysiology and Farming System in Southwest China, Ministry of Agriculture, Sichuan Agricultural University, Chengdu 611130, People’s Republic of China S. Nakamura B. Xu T. Matsumoto T. Ohta K. Fujimoto K. Ogawa M. Fukaya S. Miyake M. Yoshikawa H. Matsuda (&) Department of Pharmacognosy, Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan e-mail: [email protected]
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0.011 %) [20], 30 -O-D-glucopyranosyl ligustroside (11, 0.00050 %) [21], oleoacteoside (12, 0.00091 %) [22], isoacteoside (13, 0.00036 %) [23], (?)-epipinoresinol (14, 0.00040 %) [24], (?)-phillygenin (15, 0.0042 %) [25], (?)-pinoresinol (16, 0.0020 %) [24], trans-p-coumaric acid (17, 0.00012 %), trans-ferulic acid (18, 0.0018 %), p-tyrosol (19, 0.0027 %), and vanillic acid (20, 0.0027 %) (Fig. 1). Floraosmanosides I (1) and II (2) were obtained as a white amorphous powder with negative specific rotations 26 (1: ½a24 D - 8.4; 2: ½aD - 48.9, both in MeOH), respectively. The IR spectra of 1 and 2 showed absorption bands assignable to hydroxy and ether functions [1: 3400 and 1,076 cm-1, 2: 3,400 and 1,078 cm-1]. FABMS in the positive-ion mode of 1 and 2 revealed the common quasimolecular ion [M ? Na]? at m/z 513 from which the molecular formula C24H42O10 was deduced via HRMS and 13 C-NMR data. Acid hydrolysis of 1 and 2 with 1 M HCl liberated D-glucose and D-xylose, which were identified by HPLC of their tolylthiocarbamoyl thiazolidine derivatives [26]. The 1H-NMR (CD3OD) and 13C-NMR (Table 1) spectra of 1 and 2, which were assigned by various NMR experiments, showed signals assignable to aglycone parts {1: d 0.99 (6H, s, H3-11, 12), 1.18 (3H, d, J = 6.2 Hz, H310), 1.42 (2H, m, H2-2), 1.53, 1.64 (1H each, both m, H28), 1.58 (2H, m, H2-3), 1.60 (3H, s, H3-13), 1.89 (2H, m, H2-4), 1.99, 2.19 (1H each, both m, H2-7), 3.84 (1H, m, H-9), 2: d 1.00 (6H, s, H3-11, 12), 1.25 (3H, d, J = 6.2 Hz,
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H3-10), 1.42 (2H, m, H2-2), 1.53, 1.65 (1H each, both m, H2-8), 1.57 (2H, m, H2-3), 1.60 (3H, s, H3-13), 1.90 (2H, m, H2-4), 2.00, 2.19 (1H each, both m, H2-7), 3.82 (1H, m, H-9)}, a b-D-glucopyranosyl moiety [1: d 4.32 (1H, d, J = ca. 7 Hz, H-10 ), 2: d 4.33 (1H, d, J = 7.6 Hz, H-10 )], and a b-D-xylopyranosyl moiety [1: d 4.33 (1H, d, J = ca. 7 Hz, H-100 ), 2: d 4.33 (1H, d, J = 7.6 Hz, H-100 )]. The proton and carbon signals due to the diglycosyl moiety in the 1H- and 13C-NMR spectra of 1 and 2 were superimposable on those of geranyl-b-primeveroside (5), while the proton and carbon signals assignable to the aglycone parts of 1 and 2 resembled those of 4-(2,6,6-trimethylcyclohex1-enyl)butan-2-ol [27], except for the signals around the 9-position. The structures of 1 and 2 were characterized by means of DQF COSY and HMBC experiments (Fig. 2), i.e., the DQF COSY data of 1 and 2 indicated the presence of the partial structures written in bold lines and long-range correlations in the HMBC experiment of 1 and 2 were observed between the following proton and carbon—H2-2 and C-1, 3, 4, 6; H2-3 and C-1, 2, 4; H2-4 and C-2, 3, 5, 6, 13; H2-7 and C-5, 6, 9; H-8 and C-9; H-9 and C-7; H3-10 and C-8, 9; H3-11,12 and C-1, 2, 6; H3-13 and C-4, 5, 6; H-10 and C-9; H-10 and C-60 . The absolute stereochemistries at the 9-position of 1 and 2 were determined by application of the empirical rules of 13C-NMR chemical shift reported by Matsunami et al. [28]. Namely, the 13CNMR signals [dc 76.1–76.4 (C-9); 19.8–19.9 (C-10); 102.2–102.4 (C-10 )] of megastigmanes with 4-(2,6,6-
Fig. 1 Structures of the compounds isolated from O. fragrans var. auranticus
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J Nat Med (2015) 69:135–141 Table 1 13C-NMR (125 MHz) data of compounds 1–4 (1–3: CD3OD, 4: CDCl3)
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Position
Interchangeable
2
3
Position
1
2
3
Position
4
1
36.0
35.9
33.0
10
102.3
103.9
100.8
2
177.4
2
41.1
41.1
32.7
20
74.9
74.8
74.9
3
28.8
3
20.6
20.6
24.0
30
78.1
78.0
78.2
4
28.0
4
33.8
33.7
122.1
40
71.5
71.4
71.6
5
81.0
5
127.9
127.9
135.2
50
76.9
76.8
76.9
10
35.4
6
138.5
138.3
55.5
60
69.8
69.7
69.9
20
25.2a
136.1
1
00
105.6
0
3
29.0a
00
0
7
a
1
25.7
25.3
105.5
105.4
8
39.1
38.1
134.1
2
75.1
75.1
74.9
4
25.5
9
76.3
78.0
75.4
300
77.6
77.6
77.8
50
32.5
10
19.8
21.9
22.4
400
71.2
71.1
71.2
60
62.8
00
66.8
66.8
66.9
a
11
29.2
29.1
27.4
12
29.2
29.1
28.0a
13
20.2
20.2
23.5
5
Fig. 2 Important 2D NMR correlations of 1–4
trimethylcyclohex-1-enyl)butan-2-ol moiety and R-configuration at the 9-position were shifted upfield relative to those [dc 77.9 (C-9); 21.8 (C-10); 103.9 (C-10 )] of megastigmanes with S-configuration. The 9-, 10-, and 10 -carbon signals of 1 were observed at dc 76.3 (C-9), 19.8 (C-10), and 102.3 (C-10 ), so that the C-9 configuration of 1 was determined to be R. On the other hand, the 9-, 10-, and 10 carbon signals of 2 were observed at dc 78.0 (C-9), 21.9 (C10), and 103.9 (C-10 ), so that the C-9 configuration of 2 was determined to be S. This evidence led us to formulate the structures of floraosmanosides I (1) and II (2) as shown. Floraosmanoside III (3), obtained as a white amorphous powder with positive specific rotation (½a27 D ? 58.1 in MeOH), showed absorption bands due to hydroxy and ether functions in the IR spectrum. Positive-ion FABMS revealed a quasimolecular ion [M?Na]? at m/z 511 from which the molecular formula C24H40O10 was deduced via HRMS and 13C-NMR data. The 1H-NMR and 13C-NMR (Table 1) spectra of 3 showed signals assignable to a megastigmane part and a sugar moiety. The proton and carbon signals due to the diglycosyl moiety in the 1H- and 13 C-NMR spectra of 3 were superimposable on those of 1
and 2, while the proton and carbon signals assignable to the aglycone parts of 3 resembled those of 4-(2,6,6-trimethylcyclohex-2-enyl)but-3-en-2-ol [29], except for the signals around the 9-position. The structure of 3 was characterized by means of DQF COSY and HMBC experiments (Fig. 2). The linkage positions of the glycosides were determined by a HMBC experiment, which showed a long-range correlation between H-10 and C-9; H-100 and C-60 . Furthermore, the configuration of the 9-position of 3 was deduced by comparison of the 13CNMR data [28]. Namely, the 13C-NMR signals [dc 77.3–79.1 (C-9); 21.2–21.8 (C-10); 102.2–103.0 (C-10 )] of megastigmanes with but-3-en-2-ol moiety and R-configuration at the 9-position were shifted upfield relative to those [dc 74.7–76.3 (C-9); 22.3–22.6 (C-10); 100.5–101.7 (C-10 )] of megastigmanes with S-configuration. The 9-, 10-, and 10 carbon signals of 3 were observed at dc 75.4 (C-9), 22.4 (C10), and 100.8 (C-10 ), so that the C-9 configuration of 3 was deduced to be S. This evidence led us to formulate the structure of floraosmanoside III (3) as shown. Floraosmanolactone I (4), obtained as a colorless oil, showed absorption bands at 3,400 and 1,771 cm-1
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138
assignable to hydroxy and c-lactone functions in the IR spectrum. The molecular formula C10H18O3 of 4 was determined from the positive-ion FABMS (m/z 209 [M ? Na]?) and by HRFABMS measurement. The 1H-NMR (CDCl3) and 13C-NMR (Table 1) spectra of 4 showed signals assignable to a methylene and a methine bearing an oxygen function {[d 3.65 (2H, t, J = 6.8 Hz, H2-60 ), 4.49 (1H, m, H-5)]} and a c-lactone carbon [dC 177.4 (C-2)]. The relative structure of 3 was characterized by DQF COSY and HMBC experiments as shown in Fig. 2. The absolute stereostructure of the 4-position of 4 was characterized by comparison of the optical rotation of 4 with those of known c-butylolactones with a side chain at the 5position. Namely, the specific rotation of a known compound, (R)-dihydro-5-(30 -hydroxypropyl)-2(3H)-furanone, with 5R orientation was reported to show positive {½a21 D ? 11.5 (EtOH)}, while the optical rotation of a known compound, (S)-dihydro-5-(30 -hydroxypropyl)2(3H)-furanone, with 5S orientation was reported to show negative {[½a21 D - 11.4 (EtOH)} [30]. Since 4 showed positive specific rotation {½a17 D ? 15.0 (EtOH)}, the absolute stereostructure of the 5-position of 4 was determined to be R orientation. On the basis of this evidence, floraosmanolactone I (4) was determined to be (R)-dihydro5-(60 -hydroxyhexyl)-2(3H)-furanone. Next, in the course of our studies on NO production inhibitors from natural medicine [31, 32], the inhibitory effects of the principal constituents [4–7, 9–12, 15, 16, and 19] on NO production in LPS-activated RAW264.7 macrophages were examined. Among them, ligustroside (10) [inhibition (%): 17.8 ± 2.3 (P \ 0.01) at 30 lM, 40.7 ± 3.3 (P \ 0.01) at 100 lM] [33], (?)-phillygenin (15) [inhibition (%): 21.3 ± 2.6 (P \ 0.05) at 30 lM, 66.5 ± 1.0 (P \ 0.01) at 100 lM] [2], and (?)-pinoresinol (16) [inhibition (%): 20.5 ± 3.4 (P \ 0.01) at 30 lM, 53.1 ± 1.4 (P \ 0.01) at 100 lM] [34, 35], which were already reported to show the inhibitory effect on NO production, also displayed the inhibitory effect without cytotoxic effects using the MTT assay. However, the NO inhibitory effects of 10, 15, and 16 were weaker than those of a previous reported reference compound, caffeic acid phenethyl ester (CAPE) [IC50: 3.1 lM] [31]. On the other hand, other compounds did not show inhibitory effects on NO production (\21.0 % at 100 lM).
Experimental General The following instruments were used to obtain physical data—specific rotations, a Horiba SEPA-300 digital
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polarimeter (l = 5 cm); IR spectra, a Thermo Electron Nexus 470; EIMS and HREIMS, JEOL JMS-GCMATE mass spectrometer; FABMS and HRFABMS, a JEOL JMS-SX 102A mass spectrometer; 1H-NMR spectra, JEOL JNM-ECA 600 (600 MHz) spectrometer; 13C-NMR spectra, JEOL JNM-ECA 600 (150 MHz) spectrometers; HPLC, a Shimadzu RID-6A refractive index and SPD10Avp UV-VIS detectors. COSMOSIL 5C18-MS-II and COSMOSIL 5C18-PAQ (250 9 4.6 mm i.d. and 250 9 20 mm i.d.) columns were used for analytical and preparative purposes. The following materials were used for chromatography—normal phase silica gel column chromatography, Silica gel BW-200 (Fuji Silysia Chemical, Ltd., 150–350 mesh); reversed-phase silica gel column chromatography, Chromatorex ODS DM1020T (Fuji Silysia Chemical, Ltd., 100–200 mesh); TLC, precoated TLC plates with Silica gel 60F254 (Merck, 0.25 mm) (ordinary phase) and Silica gel RP-18 F254S (Merck, 0.25 mm) (reversed phase); reversed-phase HPTLC, precoated TLC plates with Silica gel RP-18 WF254S (Merck, 0.25 mm). Detection was achieved by spraying with 1 % Ce(SO4)2– 10 % aqueous H2SO4 followed by heating. Plant material The dried flowers of O. fragrans var. aurantiacus cultivated in Guangxi Zhuang Autonomous Region, China were commercial products purchased from Koshiro Co. Ltd (Osaka, Japan) in 2011 (KPU Medicinal Flower-2011-OF). Extraction and isolation The flowers (2.0 kg) of Osmanthus fragrans var. aurantiacus were extracted three times with methanol under reflux for 3 h. Evaporation of the solvent under reduced pressure provided a methanol extract (874.9 g, 43.8 %). A part of the MeOH extract (824.7 g) was partitioned into an n-hexaneH2O (1:1, v/v) mixture to furnish an n-hexane-soluble fraction (85.7 g, 4.6 %) and an aqueous phase. The aqueous phase was further extracted with CHCl3 to give a CHCl3soluble fraction (53.3 g, 2.8 %) and an H2O-soluble fraction (685.4 g, 37.6 %). The CHCl3-soluble fraction (48.25 g) was subjected to normal phase silica gel column chromatography [700 g, n-hexane ? n-hexane:EtOAc (2:1, v/v) ? CHCl3 ? CHCl3:MeOH (10:1, v/v) ? CHCl3:MeOH:H2O (10:3:1 ? 7:3:1 ? 6:4:1, v/v/v, lower layer)] to give 6 fractions [Fr. 1 (47 mg), Fr. 2 (649 mg), Fr. 3 (3.96 g), Fr. 4 (26.04 g), Fr. 5 (5.77 g), Fr. 6 (8.90 g)]. A part of fraction 4 (20.15 g) was subjected to reversed-phase silica gel column chromatography [450 g, MeOH:H2O (10:90 ? 20:80 ? 40:60 ? 50:50 ? 60:40 ? 80:20, v/v) ? MeOH] to give 24 fractions [Fr. 4–1 (170 mg), Fr. 4–2 (280 mg), Fr. 4–3 (570 mg), Fr. 4–4 (690 mg), Fr. 4–5
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(380 mg), Fr. 4–6 (380 mg), Fr. 4–7 (350 mg), Fr. 4–8 (640 mg), Fr. 4–9 (400 mg), Fr. 4–10 (1.25 g), Fr. 4–11 (280 mg), Fr. 4–12 (630 mg), Fr. 4–13 (340 mg), Fr. 4–14 (220 mg), Fr. 4–15 (340 mg), Fr. 4–16 (350 mg), Fr. 4–17 (260 mg), Fr. 4–18 (400 mg), Fr. 4–19 (1000 mg), Fr. 4–20 (2.42 g), Fr. 4–21 (5.88 g), Fr. 4–22 (3.67 g), Fr. 4–23 (930 mg), and Fr. 4–24 (1.21 g)]. Fr. 4–4 (690 mg) was purified by HPLC [H2O, COSMOSIL 5C18-PAQ] to give 19 (36 mg). Fr. 4–5 (380 mg) was purified by HPLC [MeOH: H2O (30:70), COSMOSIL 5C18-MS-II] to give 20 (36 mg). Fr. 4–8 (640 mg) was purified by HPLC [MeOH: H2O (30: 70), COSMOSIL 5C18-MS-II] to give 4 (15 mg) and 18 (24 mg). Fr. 4–9 (400 mg) was purified by HPLC [MeOH:H2O (40:60), COSMOSIL 5C18-MS-II] to give 7 (3.5 mg), 8 (11.7 mg), and 17 (1.6 mg). Fr. 4–10 (1.25 g) was subjected to Sephadex LH-20 column chromatography (MeOH) and HPLC [MeOH:H2O (50:50), COSMOSIL 5C18-MS-II] to give 14 (5.3 mg), and 16 (26 mg). Fr. 4–12 (630 mg) was purified by HPLC [MeOH:H2O (55:45), COSMOSIL 5C18-MS-II] to give 15 (55 mg). Fraction 5 (5.77 g) was subjected to reversed-phase silica gel column chromatography [700 g, MeOH:H2O (40:60 ? 60:40 ?70: 30 ? 80:20 ? 90:10, v/v) ? MeOH] to give 9 fractions [Fr. 5–1 (100 mg), Fr. 5–2 (402 mg), Fr. 5–3 (551 mg), Fr. 5–4 (302 mg), Fr. 5–5 (485 mg), Fr. 5–6 (426 mg), Fr. 5–7 (143 mg), Fr. 5–8 (313 mg), Fr. 5–9 (1.56 g)]. Fr. 5–3 (551 mg) was purified by HPLC [MeOH: H2O (45:55), COSMOSIL 5C18-MS-II] to give 9 (59 mg), 10 (181 mg), and 11 (8.5 mg). Fr. 5–5 (485 mg) was subjected to Sephadex LH-20 column chromatography (MeOH) and HPLC [MeOH:H2O (60:40), COSMOSIL 5C18-MS-II] to give 5 (6.0 mg). Fraction 5–6 (426 mg) was purified by HPLC [MeOH:H2O (70:30), COSMOSIL 5C18-MS-II] to give 1 (13 mg), 2 (108 mg), and 3 (1.8 mg). Fraction 6 (8.90 g) was subjected to reversed-phase silica gel column chromatography [700 g, MeOH:H2O (20:80 ? 80:20, v/v) ? MeOH] to give 11 fractions [Fr. 6–1 (100 mg), Fr. 6–2 (721 mg), Fr. 6–3 (90 mg), Fr. 6–4 (100 mg), Fr. 6–5 (309 mg), Fr. 6–6 (1.72 g), Fr. 6–7 (125 mg), Fr. 6–8 (69 mg), Fr. 6–9 (134 mg), Fr. 6–10 (189 mg), Fr. 6–11 (166 mg)]. Fraction 6–9 (134 mg) was purified by HPLC [MeOH:H2O (40:60), COSMOSIL 5C18-MS-II] to give 13 (6.3 mg). Fraction 6–10 (189 mg) was purified by HPLC [MeOH:H2O (50:50), COSMOSIL 5C18-MS-II] to give 12 (16 mg). Fraction 6–11 (166 mg) was purified by HPLC [MeOH:H2O (60:40), COSMOSIL 5C18-MS-II] to give 6 (32 mg). Floraosmanoside I (1): a white amorphous powder; 24 ½aD - 8.4 (c = 0.66, MeOH); IR (KBr): mmax 3400, 1076 cm-1; 1H-NMR (CD3OD, 600 MHz) d 0.99 (6H, s, H3-11, 12), 1.18 (3H, d, J = 6.2 Hz, H3-10), 1.42 (2H, m, H2-2), 1.53, 1.64 (1H each, both m, H2-8), 1.58 (2H, m, H2-
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3), 1.60 (3H, s, H3-13), 1.89 (2H, m, H2-4), 1.99, 2.19 (1H each, both m, H2-7), 3.84 (1H, m, H-9), 4.32 (1H, d, J = ca. 7 Hz, H-10 ), 4.33 (1H, d, J = ca. 7 Hz, 100 -H); 13CNMR: given in Table 1; positive-ion FAB-MS: m/z 513 [M ? Na]?; HR-FAB-MS: m/z 513.2682 (calculated for C24H42O10Na [M?Na]?: m/z 513.2676). Floraosmanoside II (2): a white amorphous powder; ½a26 D –48.9 (c = 0.64, MeOH); IR (KBr): mmax 3400, 1078 cm-1; 1H-NMR (CD3OD, 600 MHz) d 1.00 (6H, s, H3-11, 12), 1.25 (3H, d, J = 6.2 Hz, H3-10), 1.42 (2H, m, H2-2), 1.53, 1.65 (1H each, both m, H2-8), 1.57 (2H, m, H23), 1.60 (3H, s, H3-13), 1.90 (2H, m, H2-4), 2.00, 2.19 (1H each, both m, H2-7), 3.82 (1H, m, H-9), 4.33 (2H, d, J = 7.6 Hz, H-10 , 100 ); 13C-NMR: given in Table 1; positive-ion FAB-MS: m/z 513 [M ? Na]?; HR-FAB-MS: m/z 513.2681 (calculated for C24H42O10Na [M?Na]?: m/z 513.2676). Floraosmanoside III (3): a white amorphous powder; ½a27 D ? 58.1 (c = 0.09, MeOH); IR (KBr): mmax 3400, 1075 cm-1; 1H-NMR (CD3OD, 600 MHz) d 0.83, 0.89 (3H each, both s, H3-12, 11, interchangeable), 1.18, 1.49 (2H, m, H2-2), 1.26 (3H, d, J = 6.9 Hz, H3-10), 1.63 (3H, br s, H3-13), 2.02 (2H, m, H2-3), 2.14 (2H, d, J = 8.9 Hz, H-6), 4.30 (1H, d, J = 7.6 Hz, H-10 ’), 4.33 (1H, d, J = 8.3 Hz, H-10 ), 4.40 (1H, m, H-9), 5.34 (1H, dd, J = 8.2, 15.2 Hz, H-8), 5.41 (1H, br s like, H-4), 5.52 (1H, dd, J = 8.9, 15.2 Hz, H-7); 13C-NMR: given in Table 1; positive-ion FAB-MS: m/z 511 [M ? Na]?; HR-FAB-MS: m/z 511.2514 (calculated for C24H40O10Na [M?Na]?: m/z 511.2519). Floraosmanolactone I (4): a colorless oil; ½a25 D ? 13.8 17 (c = 0.75, MeOH), ½aD ? 15.0° (c = 0.42, EtOH); IR (KBr): mmax 3400, 1771, 1184 cm-1; 1H-NMR (CDCl3, 600 MHz) d 1.38 (6H, m, H2-20 , 30 , 40 ), 1.57 (2H, m, H250 ), 1.59, 1.75 (1H each, both m, H2-10 ), 1.86, 2.33 (1H each, both m, H2-4), 2.53 (2H, m, H2-3), 3.65 (2H, t, J = 6.8 Hz, H2-60 ), 4.49 (1H, m, H-5)]; 13C-NMR: given in Table 1; positive-ion FAB-MS: m/z 209 [M ? Na]?; HR-FAB-MS: m/z 209.1161 (calculated for C10H18O3Na [M?Na]?: m/z 209.1154). Acid hydrolyses Compounds 1 and 2 (each 1.0 mg) were dissolved in 5 % aqueous H2SO4–1,4-dioxane (1:1, v/v, 1.0 ml), and each solution was heated at 90 °C for 3 h. After extraction three times with EtOAc, the aqueous layer was neutralized with Amberlite IRA-400 (OH- form). After drying in vacuo, the residue was dissolved in pyridine (0.5 mL) containing Lcysteine methyl ester hydrochloride (0.5 mg) and heated at 60 °C for 1 h. A solution of o-tolylisothiocyanate (0.5 mg)
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in pyridine (0.1 mL) was added to the mixture and heated at 60 °C for 1 h. The reaction mixture was analyzed by reversed-phase HPLC [column: COSMOSIL 5C18-AR-II (Nacalai Tesque), 250 9 4.6 mm i.d. (5 lm); mobile phase: MeCN–H2O in 1 % AcOH (18:82, v/v); detection: UV (250 nm); flow rate: 0.8 ml/min; column temperature: 35 °C] to identify the derivatives of constituent monosaccharides (D-glucose and D-xylose) in 1 and 2 by comparison of their retention times with those of authentic samples (tR: D-glucose; 48.9 min, L-glucose; 41.2 min, D-xylose; 56.9 min, L-xylose; 53.0 min).
Statistical analysis
Cell culture
References
The murine macrophage cells (RAW264.7, ATCC No. TIB-71) were obtained from Dainippon Pharmaceutical, Osaka, Japan and cultured in Dulbecco’s modified Eagle’s medium (DMEM, high glucose) supplemented with 10 % fetal calf serum, penicillin (100 U/mL), and streptomycin (100 lg/mL) (Sigma Chemical Co., St. Louis, MO, USA). The cells were incubated at 37 °C in 5 % CO2/air.
1. Yoo KH, Park J-H, Lee DK, Fu YY, Baek NI, Chung IS (2013) Pomolic acid induces apoptosis in SK-OV-3 human ovarian adenocarcinoma cells through the mitochondrial-mediated intrinsic and death receptor-induced extrinsic pathways. Oncol Lett 5:386–390 2. Lee DG, Lee SM, Bang MH, Park H-J, Lee TH, Kim YH, Kim JY, Baek NI (2011) Lignans from the flowers of Osmanthus fragrans var. aurantiacus and their inhibition effect on NO production. Arch Pharm Res 34:2029–2035 3. Lee DG, Park JH, Yoo K-H, Chung I-S, Lee Y-H, Lee J-K, Han D-S, Cho S-M, Baek N-I (2011) 24-ethylcholesta-4,24(28)-dien3,6-dione from Osmanthus fragrans var. aurantiacus flowers inhibits the growth of human colon cancer cell line, HCT-116. J Korean Soc Appl Bi 54:206–210 4. Lee D-G, Choi J-S, Yeon S-W, Cui E-J, Park H-J, Yoo J-S, Chung I-S, Baek N-I (2010) Secoiridoid glycoside from the flowers of Osmanthus fragrans var. aurantiacus makino inhibited the activity of b-secretase. J Korean Soc Appl Bi 53:371–374 5. Matsumoto T, Nakamura S, Fujimoto K, Ohta T, Ogawa K, Yoshikawa M, Matsuda H (2014) Structure of constituents isolated from the flower buds of Cananga odorata and their inhibitory effects on aldose reductase. J Nat Med 68:1–8. doi:10.1007/ s11418-014-0843-y 6. Fujimoto K, Nakamura S, Matsumoto T, Ohta T, Yoshikawa M, Ogawa K, Kashiwazaki E, Matsuda H (2014) Structures of acylated sucroses from the flower buds of Prunus mume. J Nat Med 68:481–487 7. Matsumoto T, Nakamura S, Ohta T, Fujimoto K, Yoshikawa M, Ogawa K, Matsuda H (2014) A rare glutamine derivative from the flower buds of daylily. Org Lett 16:3076–3078 8. Matsumoto T, Nakamura S, Nakashima S, Fujimoto K, Yoshikawa M, Ohta T, Ogawa K, Matsuda H (2014) Lignan dicarboxylates and terpenoids from the flower buds of Cananga odorata and their inhibitory effects on melanogenesis. J Nat Prod 77:990–999 9. Nakamura S, Fujimoto K, Matsumoto T, Nakashima S, Ohta T, Ogawa K, Matsuda H, Yoshikawa M (2013) Acylated sucroses and acylated quinic acids analogs from the flower buds of Prunus mume and their inhibitory effects on melanogenesis. Phytochemistry 92:128–136 10. Liu J, Nakamura S, Matsuda H, Yoshikawa M (2013) Hydrangeamines A and B, novel polyketide-type pseudoalkaloidcoupled secoiridoid glycosides from the flowers of Hydrangea macrophylla var. thunbergii. Tetrahedron Lett 54:32–34 11. Nakamura S, Fujimoto K, Matsumoto T, Ohta T, Ogawa K, Tamura H, Matsuda H, Yoshikawa M (2013) Structures of acylated sucroses and an acylated flavonol glycoside and
Effects on NO production in LPS-activated RAW264.7 macrophages The total amount of nitrite in a medium is used as an indicator of NO synthesis. The screening test for NO production using RAW264.7 macrophages was described previously [31, 32]. Briefly, RAW264.7 cells were cultured in DMEM, and the suspension seeded into a 96-well microplate at 2.5 9 105 cells/100 lL/well. After 6 h, nonadherent cells were removed by washing with PBS, the adherent cells were cultured in 100 lL of fresh medium containing the test compounds for 10 min, and then 100 lL of the medium containing LPS (from E. coli, 055: B5, Sigma) was added to stimulate the cells for 18 h (final concentration of LPS, 10 lg/mL). The nitrite concentration was measured from the supernatant by a Griess reaction. NO production in each well was assessed by measuring the accumulation of nitrite in the culture medium using Griess reagent. Inhibition (%) was calculated using the following formula and the IC50 was determined graphically (n = 4). Inhibition ð% Þ ¼ ½ðABÞ=ðAC Þ 100 A–C: Nitrite concentration (lg/mL) A: LPS (?), Sample (-); B: LPS (?), Sample (?); C: LPS (-), Sample (-) Cytotoxicity was evaluated by the 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetric assay according to the previous report [31].
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All data are expressed as the mean ± S.E.M. The data analysis was performed with a one-way analysis of variance (1-ANOVA), followed by Dunnett’s test. A p value of \0.05 was considered to be significant. Acknowledgments This work was supported in part by a Ministry of Education, Culture, Sports, Science and Technology (MEXT)Supported Program for the Strategic Research Foundation at Private Universities and by JSPS KAKENHI Grant Number 26460135.
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