Fitoterapia 92 (2014) 127–132
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New ursane-type triterpenoid saponins from the stem bark of Schefflera heptaphylla Chun Wu a,1, Ying-Hui Duan b,1, Wei Tang a, Man-Mei Li a, Xia Wu a, Guo-Cai Wang a, Wen-Cai Ye a, Guang-Xiong Zhou a,⁎, Yao-Lan Li a,⁎ a b
Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, People's Republic of China Xiamen Institute for Drug Control, Xiamen 361012, People's Republic of China
a r t i c l e
i n f o
Article history: Received 10 August 2013 Accepted in revised form 10 October 2013 Available online 19 October 2013 Keywords: Araliaceae Schefflera heptaphylla Triterpenoid saponin Anti-inflammatory activities
a b s t r a c t Phytochemical investigation on the stem bark of Schefflera heptaphylla led to the isolation of five new ursane-type triterpenoid saponins (1–5). Their structures were determined on the basis of spectroscopic and chemical methods. It is noteworthy in this study that the genins of all compounds are reported for the first time. All compounds isolated from this plant were evaluated for their inhibitory activities on lipopolysaccharide-induced nitric oxide production in RAW264.7 cells, and compounds 2 and 5 showed weak anti-inflammatory activities under their non-cytotoxic concentrations. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Schefflera heptaphylla (L.) Frodin (Araliaceae) is a mediumsized, evergreen tree up to 25 m tall, bole up to 80 cm in diameter. It is used as a folk remedy for the treatment of pain, inflammation, and common cold. It is also a principal ingredient of an herbal tea formulation widely used to treat common cold in southern China [1–3]. Previous phytochemical studies on S. heptaphylla showed that the plant is rich in triterpenoids and triterpenoid glycosides [1,4–10]. In the previous research, we had isolated and identified some triterpenoid saponins, including scheffursoside D, scheffursoside F, scheffoleoside A, scheffoleoside D, and acankoreoside A, from the stem bark of S. heptaphylla [5]. As part of our continuing search for bioactive constituents from S. heptaphylla, a 95% EtOH extract of the stem bark of S. heptaphylla had been investigated, and five ⁎ Corresponding authors at: Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, 601 West Huangpu Avenue, Guangzhou 510632, PR China. Tel.: +86 20 85221469; fax: +86 20 85221559. E-mail addresses: [email protected] (G.-X. Zhou), [email protected] (Y.-L. Li). 1 These authors contributed equally to this work. 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.10.006
new ursane-type triterpenoid saponins (1–5) were obtained (Fig. 1). In addition, all the new compounds were evaluated for their anti-inflammatory activities on lipopolysaccharide (LPS)induced nitric oxide (NO) production in RAW264.7 cells. In this paper, we described the isolation, structural elucidation, and anti-inflammatory activities of these triterpenoid saponins. 2. Experimental 2.1. General methods Optical rotations were carried out using a JASCO P-1030 automatic digital polarimeter. IR spectra were measured on a JASCO FT/IR-480 plus infrared spectrometer with KBr pellets. 1D and 2D NMR spectra were recorded on a Bruker AV-400 spectrometer with TMS as the internal standard, and chemical shifts were expressed in δ values (ppm). HRESIMS data were detected on an Agilent 6210 LC/MSD TOF mass spectrometer. Silica gel (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), ODS silica gel (50 μm, YMC, Kyoto, Japan), and Sephadex LH-20 (Pharmacia, Uppsala, Sweden) were used for column chromatography (CC). Analytical high-performance liquid chromatography (HPLC) was carried out on a Waters
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Fig. 1. Chemical structures of compounds 1–5.
chromatograph equipped with an evaporative light-scattering detector, a P680 pump, and a reversed phase (RP) C18 column (5 μm, 4.6 mm × 250 mm, Cosmosil, Kyoto, Japan). Semipreparative HPLC was performed on an Agilent 1200 unit with DAD detector and a RP C18 column (5 μm, 10 mm × 250 mm; Cosmosil, Kyoto, Japan). Preparative HPLC was carried out on a Varian chromatograph equipped with a Prostar 215 pump and a Prostar 325 UV–Vis detector with a RP C18 column (5 μm, 20 mm × 250 mm; Cosmosil, Kyoto, Japan). Thin-layer chromatography (TLC) was performed using pre-coated silica-gel plates (GF254, Yantai Chemical Industry Research Institute, Yantai, China). All the reagents were purchased from Tianjin Damao Chemical Company (Tianjin, China). L-cysteine methyl ester and standard sugars D-glucose (D-Glc), L-glucose (L-Glc), and L-rhamnose (L-Rha) in the analysis of HPLC experiments were purchased from Adamasbeta Company (Basel, Switzerland). O-Tolyl isothiocyanate and dexamethasone were purchased from Sigma Company (Sigma, St. Louis, MO, USA).
2.3. Extraction and isolation
2.2. Plant material
The dried and powdered stem bark of S. heptaphylla (10 kg) was soaked in 95% EtOH at room temperature for five times. The solution was evaporated under reduced pressure to obtain an extract (1.3 kg). This extract was suspended in distilled water, and then partitioned with petroleum ether, EtOAc, and n-BuOH, respectively. The n-BuOH-soluble residue (100 g) was subjected to silica gel column and eluted with CHCl3–MeOH (100:0; 90:10; 80:20; 70:30; 60:40; 50:50; 30:70; 0:100) in gradient to yield 40 fractions (Fr 1–40) based on their TLC patterns. Fr 12 (5.3 g) was separated on an ODS gel column (180 g, 3.5 cm × 40 cm) eluted with H2O– MeOH (80:20, 70:30, 60:40, 40:60, 20:80, 0:100, each 3 L), to give 15 subfractions (Sfr 1–15). Sfr 12 (250 mg) was subjected to preparative HPLC using 70% MeOH–H2O (7 mL/min) to give compounds 1 (7.4 mg) and 2 (5.1 mg). Sfr 10 (210 mg) was subjected to semi-preparative HPLC using 63% MeOH–H2O (3 mL/min) to give compound 3 (11.3 mg). Sfr 6 (318 mg) was subjected to preparative HPLC using 62% MeOH–H2O (7 mL/min) to give compounds 4 (13.4 mg) and 5 (21.9 mg).
The dried stem bark of S. heptaphylla was collected from Yulin, Guangxi, China, in August 2008, and was authenticated by Mr Zhen-Qiu Mai, a senior herbalist at the Chinese Medicinal Material Company, Guangdong, China. A voucher specimen with accession (No. SH20090301) has been deposited in the herbarium of College of Pharmacy, Jinan University.
2.3.1. 3-oxo-urs-20-en-23,28-dioic acid 28-O-α-L-rhamnopyranosyl(1 → 4)-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranoside (1) Amorphous powder; C48H76O19; [α]25D −2.35° (c 1.03, MeOH); IR (KBr) νmax: 3420, 2935, 1724, 1070 cm−1; HRESIMS (positive-ion mode) m/z 979.4880 [M + Na]+ (calcd. for C48H76O19Na: 979.4878); 1H NMR (C5D5N, 400 MHz) and 13C NMR (C5D5N, 100 MHz) data: see Tables 1 and 2.
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Table 1 1 H NMR data of compounds 1–5 (C5D5N, J in Hz).a Position
1
1 2 3 4 5 6 7 9 11 12 13 15 16 18 19 20 21 22 23 24 25 26 27 29 30 Glc-I 1′ 2′ 3′ 4′ 5′ 6′
1.82 2.32 – – 2.18 1.23 1.28 1.31 1.21 1.71 2.60 1.18 2.68 1.33 2.42 – 5.46 2.05 – 1.06 1.18 0.99 0.93 0.92 1.68 6.28 4.13 4.15 4.33 4.09 4.35 4.61 4.93 4.06 4.16 4.42 3.65 4.14 5.87 4.58 4.55
Glc-II 1″ 2″ 3″ 4″ 5″ 6″ Rha 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ Glc-III 1′′′′ 2′′′′ 3′′′′ 4′′′′ 5′′′′ 6′′′′ a
2 m, 1.95 m m, 2.42 m
br d (11.0) m, 1.69 m m m m, 1.39 m m m m, 1.94 m m m m br d (7.2) m, 2.58 dd (15.2, 6.8) s s s s d (6.4) s d (8.1) m m m m m, br s d (7.8) t (8.5) m t (9.1) d (9.1) m, 4.21 m br s br s m
1.74 1.83 4.31 – 2.54 1.17 1.24 1.67 1.15 1.77 2.61 1.20 2.61 1.60 2.34 – 5.45 2.09 – 1.47 0.87 1.25 0.97 1.02 1.70 6.29 4.41 4.53 4.65 4.41 4.38 4.50 5.00 4.32 4.50 4.61 4.25 4.32
3 m, 1.92 m m, 2.09 m br s br d (11.3) m, 1.70 m m m m, 1.45 m m m m, 1.92 m m m m d (6.5) m, 2.58 dd (14.7, 7.1) s s s s d (6.5) s d (8.0) m m m m m, br s d (7.8) m m m m m, 4.42 m
4.26 m 4.96 m 1.72 d (6.0)
1.74 1.87 4.56 – 2.53 1.17 1.25 1.64 1.21 1.82 2.62 1.21 2.62 1.62 2.35 – 5.42 2.01 – 1.43 0.78 1.17 0.90 1.01 1.67 6.26 4.06 4.13 4.31 4.19 4.35 4.70 4.92 3.93 4.03 4.32 3.66 4.10 5.89 4.59 4.55
m, 1.92 m m, 2.01 m br s
4.26 5.00 1.72 6.43 4.24 4.03 4.29 4.31 4.41
m m d (6.1) d (7.7) m m m m m, 4.58 m
br d (11.4) m, 1.71 m m m m, 1.44 m m m m, 1.93 m m m m d (6.9) m, 2.56 m s s s s d (6.5) s d (8.0) m m m m m, br s d (7.8) m m m d (9.0) m, 4.24 m br s br s m
4
5
1.57 m, 2.45 m 2.42 m, 2.47 m – 3.70 m 1.01 m 1.10 m, 1.57 m 1.28 m, 1.55 m 1.56 m 1.86 m 5.47 br s – 1.16 m, 2.39 m 1.94 m 2.54 d (11.4) 1.43 m 0.93 m 1.38 m 1.83 m, 1.94 m 1.08 d (6.3) – 1.06 s 1.21 s 1.15 s 0.95 d (6.5) 0.93 d (6.5) 6.21 d (8.0) 4.11 m 4.18 m 4.31 m 4.10 m 4.33 m, 4.68 br s 5.00 d (7.8) 3.96 m 4.21 m 4.43 t (9.3) 3.70 d (8.6) 4.11 m, 4.29 m 5.87 br s 4.70 br s 4.55 dd (3.2, 9.4) 4.34 m 4.94 m 1.71 d (6.2)
1.70 1.87 4.22 – 2.51 1.19 1.24 1.67 1.14 1.73 1.52 1.19 1.74 1.00 1.57 – 1.53 1.50 – 1.44 0.87 0.90 0.67 0.83 1.26 6.46 4.25 4.04 4.33 4.32 4.36
m, 1.94 m m, 2.01 m br s br d (11.0) m, 1.67 m m m m, 1.50 m m m m, 1.90 m m m m m, 1.82 m m s s s s d (6.9) s d (7.7) m m m m m, 4.42 m
Signals are designated as follows: s, singlet; br s, broad singlet; d, doublet; dd, doublet of doublets; m, multiplet.
2.3.2. 3α-hydroxy-urs-20-en-23,28-dioic acid 28-O-β-Dglucopyranosyl-(1 → 6)-β-D-glucopyranoside (2) Amorphous powder; C42H66O15; [α]25D −0.82° (c 1.10, MeOH); IR (KBr) νmax: 3423, 2930, 1721, 1079 cm-1; HRESIMS (positive-ion mode) m/z 833.4306 [M + Na]+ (calcd. for C42H66O15Na: 833.4299); 1H NMR (C5D5N, 400 MHz) and 13C NMR (C5D5N, 100 MHz) data: see Tables 1 and 2. 2.3.3. 3α-hydroxy-urs-20-en-23,28-dioic acid 23-O-β-Dglucopyranosyl, 28-O-α-L-rhamnopyranosyl-(1 → 4)-β-Dglucopyranosyl-(1 → 6)-β-D-glucopyranoside (3) Amorphous powder; C54H86O24; [α]25D +0.53° (c 0.94, MeOH); IR (KBr) νmax: 3427, 2928, 1728, 1075 cm−1; HRESIMS
(positive-ion mode) m/z 1141.5404 [M + Na]+ (calcd. for C54H86O24Na: 1141.5401); 1H NMR (C5D5N, 400 MHz) and 13C NMR (C5D5N, 100 MHz) data: see Tables 1 and 2.
2.3.4. 3-oxo-urs-12-en-24-nor-oic acid 28-O-α-L-rhamnopyranosyl(1 → 4)-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranoside (4) Amorphous powder; C47H74O17; [α]25D +1.32° (c 1.14, MeOH); IR (KBr) νmax: 3437, 2920, 1730, 1669, 1076 cm−1; HRESIMS (positive-ion mode) m/z 933.4816 [M + Na]+ (calcd. for C47H74O17Na: 933.4818); 1H NMR (C5D5N, 400 MHz) and 13C NMR (C5D5N, 100 MHz) data: see Tables 1 and 2.
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2.4. Determination of NO production and the cell viability assay
Table 2 13 C NMR data of compounds 1–5 (C5D5N). Position
1
2
3
4
5
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Glc-I 1′ 2′ 3′ 4′ 5′ 6′ Glc-II 1″ 2″ 3″ 4″ 5″ 6″ Rha 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ Glc-III 1′′′′ 2′′′′ 3′′′′ 4′′′ 5′′′′ 6′′′′
37.0 37.9 212.7 49.9 45.0 23.0 33.9 40.9 50.3 37.3 22.8 28.2 39.9 42.8 29.8 33.6 49.9 49.7 37.3 143.6 118.0 38.0 180.3 12.4 16.7 15.4 14.1 174.9 17.8 22.6 95.7 74.4 77.6 70.8 76.9 70.8 105.4 75.8 77.6 78.7 78.4 61.9 103.1 71.6 73.1 73.2 70.8 19.0
33.6 26.8 72.1 51.6 45.5 22.3 33.8 40.0 51.6 37.9 22.4 26.8 40.0 42.5 30.0 34.8 49.7 49.9 38.1 143.7 118.0 38.1 180.2 18.5 17.5 17.3 15.6 175.0 24.1 22.8 95.7 74.8 78.4 71.4 75.7 69.8 105.6 75.6 76.9 79.0 78.9 63.2
33.4 26.6 73.1 50.2 45.1 22.2 33.7 42.4 51.5 38.1 22.2 26.6 39.9 42.8 29.8 34.8 50.2 49.8 37.7 143.5 117.9 38.0 176.5 17.5 17.9 17.1 15.4 174.8 24.0 22.7 95.7 74.5 77.6 70.8 76.8 69.7 105.5 75.8 76.9 78.8 78.4 61.8 103.2 73.1 73.2 73.5 70.8 19.0 97.1 75.1 80.1 71.7 79.0 62.7
40.6 37.9 212.3 44.9 53.9 22.4 32.6 40.1 45.7 36.9 24.4 126.2 138.9 42.8 28.9 24.8 48.7 53.5 39.6 39.3 31.0 37.0 12.2 – 13.6 18.0 23.9 176.5 17.9 21.5 95.9 74.1 77.5 70.6 76.8 69.8 105.2 75.6 76.9 78.6 78.2 61.7 102.9 72.8 73.0 73.1 70.6 19.0
33.4 26.6 73.5 53.3 45.1 22.1 34.5 42.8 51.3 37.7 21.5 26.6 42.6 41.8 28.4 32.4 53.3 48.6 42.6 84.3 27.7 43.7 176.4 17.3 17.8 17.2 14.3 177.1 16.6 24.6 97.0 75.1 80.1 71.6 78.9 62.7
2.3.5. 3α-hydroxy-20β-hydroxyursan-23,28-dioic acid δ-lactone 23-O-β-D-glucopyranoside (5) Amorphous powder; C36H56O10; [α]25D − 1.00° (c 1.00, MeOH); IR (KBr) νmax: 3423, 2937, 1760, 1740, 1074 cm− 1; HRESIMS (positive-ion mode) m/z 671.3765 [M + Na]+ (calcd. for C36H56O10Na: 671.3766); 1H NMR (C5D5N, 400 MHz) and 13C NMR (C5D5N, 100 MHz) data: see Tables 1 and 2.
The anti-inflammatory activities of the compounds were evaluated by determining the amount of nitrite, a stable oxidized product in cell culture supernatant as described previously [15]. Briefly, cells (6 × 104 cells/well) were cultured in 48-well plates overnight and replaced by the media containing 1 μg/mL of LPS (Sigma, St. Louis, MO, USA) and different concentrations of the compounds. After culturing for 48 h, 50 μL of the supernatant was piped out and mixed with an equal volume of Griess reagent (Sigma, St. Louis, MO, USA) for 15 min. The absorbance at 540 nm was measured with a microplate reader (Molecular Devices, Emax, Sunnyvale, CA, USA). Nitrite concentrations in the supernatant were determined by comparison with a sodium nitrite standard curve. The inhibitory rate was calculated according to the formula: Inhibition (%) = 100% − [amount of nitrite (LPS + compound) / amount of nitrite (LPS)] × 100%. Cell viability was measured with the MTT-based colorimetric assay. 2.5. Determination of absolute configurations of sugars Compound 1 (3.0 mg) was dissolved in 1 M HCl (2 mL) and heated at 85 °C for 2 h. The mixture was evaporated to dryness under vacuum. The residue was dissolved in pyridine (2 mL) containing L-cysteine methyl ester (3.0 mg) and heated at 60 °C for 2 h. Then, O-tolyl isothiocyanate (10 μL) was added to the mixture, which was heated at 60 °C for 2 h. The reaction mixture was directly analyzed by reversed-phase HPLC. Analytical HPLC was performed on a RP C18 column (5 μm, 4.6 mm × 250 mm) at 30 °C with isocratic elution of 25% CH3CN–H2O containing 0.08% formic acid for 50 min and subsequent washing of the column with 90% CH3CN–H2O at a flow rate of 0.8 mL/min. Peaks were detected by a UV detector at 250 nm. Identification of D-glucose and L-rhamnose was carried out for 1, giving peaks at tR 23.2 and 35.3 min. The standard sugars such as D-, L-glucose, and L-rhamnose were subjected to the same method. The peaks of standard sugar derivatives were recorded at tR 23.30 (D-Glc), 21.28 (L-Glc), and 35.25 (L-Rha) min. Sugars from compounds 2–5 were also identified by the same procedure. 3. Results and discussion Compound 1, obtained as an amorphous powder, has a molecular formula of C48H76O19 established on the basis of its HRESIMS data ([M + Na]+ m/z 979.4880). Its 1H NMR spectrum suggested the presence of three sugar anomeric protons at δH 4.93 (1H, d, J = 7.8 Hz, H-1″of Glc-II), 5.87 (1H, br s, H-1″ of Rha) and 6.28 (1H, d, J = 8.1 Hz, H-1′ of Glc-I), giving the HSQC correlations with three anomeric carbons at δC 105.4, 103.1 and 95.7, respectively (Table 2), confirming that compound 1 contains three sugar units. Acid hydrolysis of 1 afforded D-glucose and L-rhamnose through HPLC analysis according to the method of Tanaka et al. [11]. The structure of the saccharide chain was established as α-L-rhamnopyranosyl-(1 → 4)-β-Dglucopyranosyl-(1 → 6)-β-D-glucopyranosyl based on the HMBC correlations of H-1‴/C-4″ (δC 78.7), and H-1″/C-6′ (δC 70.8). In addition, the NMR spectra of 1 also revealed 30 carbon signals including five tertiary methyl groups at δH/δC 0.93 (s, H3-27)/14.1, 0.99 (s, H3-26)/15.4, 1.06 (s, H3-24)/12.4, 1.18 (s, H3-25)/16.7, and 1.68 (s, H3-30)/22.6, one secondary methyl groups at δH/δC 0.92
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(d, J = 6.4 Hz, H3-29)/17.8, a tri-substituted double bond at δH/δC 5.46 (1H, br d, J = 7.2 Hz, H-21)/118.0 (C-21), and 143.6 (C-20), suggesting the carbon skeleton of 1 to be ursane [12–14]. In addition, the 13C NMR spectrum showed an ester carbonyl carbon at δC 174.9 (C-28), a carboxylic group at δC 180.3 (C-23), and a ketocarbonyl carbon at δC 212.7 (C-3). The ketocarbonyl was assigned for C-3 due to the HMBC correlations of H3-24, H-5/C-3, H3-24/C-3, C-5 and C-23. The double bond was placed at Δ20,21 on the basis of the HMBC correlations of H3-29/C-20, H3-30/C-20, and C-21, H-21/C-30. The sugar chain was linked to C-28, according to the upfield shift of C-28 (δC 174.9). This was further confirmed by the HMBC correlation of H-1′/C-28. Thus, the structure of 1 was elucidated as 3-oxo-urs-20-en-23,28-dioic acid 28-O-αL -rhamnopyranosyl-(1 → 4)-β-D-glucopyranosyl-(1 → 6)-β-Dglucopyranoside. Compound 2 was obtained as an amorphous powder. Its molecular formula was determined to be C42H66O15 from the quasi-molecular ion peak [M + Na]+ at m/z 833.4306 in the HRESIMS. The NMR signals of 2 were analogous to those of 1 except that a hydroxyl group [δH/δC 4.31 (1H, br s, H-3)/72.1] replaced the ketocarbonyl group of 1, and the α-rhamnose was absent in 2. ROESY correlation of H-3/H3-24 (δH 1.47), as well as the absence of the ROESY correlation between H-3 and H-5 (δH 2.54), indicated the β-orientation of H-3. Thus, compound 2 was assigned as 3α-hydroxy-urs-20-en-23,28-dioic acid 28-O-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranoside. Compound 3, an amorphous powder, was established to be C54H86O24 due to the HRESIMS data ([M + Na]+ m/z 1141.5404). In contrast with compound 1, the NMR spectra of 3 showed the presence of an extra anomeric proton and carbon signals at δH/δC 6.43 (1H, d, J = 7.7 Hz, H-1′′′′ of Glc-III)/97.1, an additional methylene group at δH/δC 4.41 (m, H-6′′′′ of Glc-III) and 4.58 (m, H-6′′′′ of Glc-III)/62.7, indicating the presence of a third glucosyl unit in the molecule. This glucosyl unit was located at C-23, according to the upfield shift of C-23 (δC 176.5), and further confirmed by the HMBC correlation of H-1′′′′/C-23. Moreover, a methine [δH/δC 4.56 (1H, br s, H-3)/73.1] with hydroxyl group replaced the ketocarbonyl group of 1. The obvious ROESY correlation of H-3/H3-24 (δH 1.43) and the absence of the ROESY correlation between H-3 and H-5 (δH 2.53) indicated the β-orientation of H-3. Thus, compound 3 was concluded to be 3α-hydroxy-urs-20-en-23,28-dioic acid 23-O-β-Dglucopyranosyl, 28-O-α-L-rhamnopyranosyl-(1 → 4)-β-Dglucopyranosyl-(1 → 6)-β-D-glucopyranoside. Compound 4 was obtained as an amorphous powder. Its HRESIMS spectrum showed a quasi-molecular ion peak at m/z 933.4816 [M + Na]+, ascribable to the molecular formula C47H74O17. The 1D NMR data of 4 (Tables 1 and 2) exhibited three tertiary methyl groups at δH/δC 1.06 (s, H3-25)/13.6, 1.15 (s, H3-27)/23.9 and 1.21 (s, H3-26)/18.0, three secondary methyl groups at δH/δC 0.93 (d, J = 6.5 Hz, H3-30)/21.5, 0.95 (d, J = 6.5 Hz, H3-29)/17.9, 1.08 (d, J = 6.5 Hz, H3-23)/12.2, one methine group at δH/δC 2.54 (1H, d, J = 11.4 Hz, H-18)/ 53.5, a tri-substituted double bond at δH/δC 5.47 (1H, br s, H-12)/126.2 (C-12) and 138.9 (C-13), and an ester carbonyl carbon at δC 176.5 (C-28), indicating an ursan-12-en-oic acid as an aglycon [12]. The comparison of the NMR spectra of its saccharide moiety with those of 1, helped establish the chain as α-L-rhamnopyranosyl-(1 → 4)-β-D-glucopyranosyl-(1 → 6)-βD-glucopyranosyl, and the chain was also positioned at C-28. The
131
13
C NMR spectrum of 4 (Table 2) showed 47 carbon signals, of which 18 signals were assigned to the saccharide moiety. The remaining 29 carbon signals were attributed to a nor-triterpene moiety including six methyl carbons (δC 12.2, 13.6, 17.9, 18.0, 23.9 and 21.5), two olefinic carbons [δC 126.2 (C-12) and 138.9 (C-13)], and a ketone carbon [δC 212.3 (C-3)]. The ketone carbon was assigned for C-3 due to the HMBC correlations of H-1 (δH 2.45)/C-2 (δC 37.9), C-3, C-5 (δC 53.9), C-10 (δC 36.9) and C-25 (δC 13.6). There was only one methyl group at δH/δC 1.08 (3H, d, J = 6.3 Hz)/12.2 connecting to C-4, instead of two methyl groups as found at C-4 in common usual ursane triterpenes, which was proved by the HMBC correlations of H-4 (δH 3.70)/C-3, C-5 and C-23, H-5 (δH 1.01)/C-4 (δC 44.9), C-10 and C-25, and H-23/C-4 and C-5 (δC 53.9). The α-configuration of the methyl group at C-4 was confirmed by the ROESY correlation between H-4/H-25. Thus, compound 4 was determined as 3-oxo-urs-12-en-24-nor-oic acid 28-O-α-L-rhamnopyranosyl(1 → 4)-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranoside. Compound 5 also was an amorphous powder. Its molecular formula, C36H56O10, was determined from the HRESIMS positiveion peak at m/z 671.3765 ([M + Na]+). The NMR spectra of 5 were analogous to those of 3 except for the different signals attributed to ring E and the absence of the sugar chain at C-28. The presence of a δ-lactone ring was suggested by the appearance of an absorption band at 1740 cm−1 in its IR spectrum and a carbon signal at δC 177.1 (C-28) in the 13C NMR spectrum. The oxygen-bearing quaternary carbon signal observed at δC 84.3 (C-20) as well as the HMBC correlations from H-18 (δH 1.00), H-16 (δH 1.19) and H-22 (δH 1.50) to C-28, and from H-29 (δH 0.83), H-30 (δH 1.26) and H-19 (δH 1.56) to C-20, substantially provided information for a δ-lactone ring formed between C-20 and C-28. Thus, the structure of 5 was elucidated as 3α-hydroxy-20β-hydroxyursan-23,28-dioic acid δ-lactone 23-O-β-D-glucopyranoside. It is worth noting that the genins of compounds 1–5 are reported for the first time. Although the oleanane skeleton occurs frequently in Schefflera plants, the ursane type is very rarely in the genus. From our knowledge, this is the first time that α-amyrane-type compounds with a Δ20,21 double bond have been found in the Araliaceae. All isolates were evaluated for their inhibitory effects on the release of NO from macrophages using LPS-induced RAW264.7 cells as a model system. First of all, the non-cytotoxic concentrations of the compounds toward macrophage RAW264.7 cells were determined with 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay. Compounds 2 and 5 exhibited weak inhibitory effects against NO production in Table 3 The inhibitory effects of compounds on the NO secretion of mouse macrophage RAW 264.7 cells stimulated by LPS for 48 h (means ± SD, n = 4). Inhibition (%)
Concentration (μM)
Compounds
80
2 3 4 5 Dexamethasone
38.3 17.0 26.8 42.7 –
40 ± ± ± ±
3.2 2.7 3.0 3.2
18.7 16.5 16.2 45.7 –
20 ± ± ± ±
1.3 2.5 2.2 4.3
12.7 11.8 10.1 20.7 62.8
10 ± ± ± ± ±
2.4 1.7 1.4 3.2 2.5
NE NE NE 19.6 ± 3.8 61.7 ± 2.1
Note: NE indicated no effect; – indicated not detected due to cytotoxic concentration.
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their non-cytotoxic concentrations, as shown in Table 3. The positive control, dexamethasone, gave a 61.7 ± 2.1% (n = 4) inhibition at 10 μM. Conflict of interest The authors have no conflict of interest to report. Acknowledgments This research program was supported financially by the National Natural Science Foundation of China (No. 81202429, 81273390), Postdoctoral Science Foundation (2012M521656), and the grant (2009B090600117) from Guangdong province. Appendix A. Supplementary data All the HRESIMS and NMR spectra are available as Supporting Information. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2013.10.006. References [1] Sung TV, Steglich W, Adam G. Triterpene glycosides from Schefflera octophylla. Phytochemistry 1991;30:2349–56. [2] Li YL, Paul PHB, Vincent ECO. Antiviral activity and mode of action of caffeoylquinic acids from Schefflera heptaphylla (L.) Frodin. Antiviral Res 2005;68:1–9.
[3] Kong YC. Herbal medicines and teas of Hong Kong. Hong Kong: Commercial Press; 2000 90–1. [4] Li YL, Jiang RW, Linda SMO, Paul PHB, Vincent ECO. Antiviral triterpenoids from the medicinal plant Schefflera heptaphylla. Phytother Res 2007;21:466–70. [5] Wu C, Wang L, Yang XX, Duan YH, Dai Y, Jiang RW, et al. A new ursane-type triterpenoid from Schefflera heptaphylla (L.) Frodin. J Asian Nat Prod Res 2011;13:434–9. [6] Kitajima J, Shindo M, Tanaka Y. Two new triterpenoids sulfates from the leaves of Schefflera octophylla. Chem Pharm Bull 1990;38:714–6. [7] Kitajima J, Tanaka Y. Two new triterpenoid glycosides from the leaves of Schefflera octophylla. Chem Pharm Bull 1989;37:2727–30. [8] Lischewski M, Ty PD, Schmidt J, Preiss A, Phiet HV, Adam G. 3α,11αdihydroxy-lup-20(29)-ene-23,28-dioic acid from Schefflera octophylla. Phytochemistry 1984;23:1695–7. [9] Schmidt J, Nam VV, Lischewski M, Phiet HV, Kuhnt C, Adam G. Longchain fatty acid esters of 3α-hydroxy-lup-20(29)-ene-23,28-dioic acid and other triterpenoid constituents from the bark of Schefflera octophylla. Phytochemistry 1984;23:2081–2. [10] Maeda C, Ohtani K, Kasai R, Yamasaki K, Nguyen MD, Nguyen TN, et al. Oleanane and ursane glycosides from Schefflera octophylla. Phytochemistry 1994;37:1131–7. [11] Tanaka T, Nakashima T, Ueda T, Tomii K, Kouno I. Facile discrimination of aldose enantiomers by reversed-phase HPLC. Chem Pharm Bull 2007;55:899–901. [12] Mahato SB, Kundu AP. 13C-NMR spectra of pentacyclic triterpenoids-a compilation and some salient features. Phytochemistry 1994;37:1517–75. [13] Tatiana LE, Rivero-Cruz JF, Su BN, Chai HB, Cordell GA, Pezzuto JM, et al. Constituents of the leaves and twigs of Calyptranthes pallens collected from an experimental plot in Southern Florida. J Nat Prod 2005;68:577–80. [14] Yu QL, Duan HQ, Gao WY, Takaishi Y. A new triterpene and a saponin from Centella asiatica. Chin Chem Lett 2007;18:62–4. [15] Islam MN, Ishita IJ, Jin SE, Choi RJ, Lee CM, Kim YS, et al. Anti-inflammatory activity of edible brown alga Saccharina japonica and its constituents pheophorbide a and pheophytin a in LPS-stimulated RAW 264.7 macrophage cells. Food Chem Toxicol 2013;55:541–8.
Contents lists available at ScienceDirect
Fitoterapia journal homepage: www.elsevier.com/locate/fitote
New ursane-type triterpenoid saponins from the stem bark of Schefflera heptaphylla Chun Wu a,1, Ying-Hui Duan b,1, Wei Tang a, Man-Mei Li a, Xia Wu a, Guo-Cai Wang a, Wen-Cai Ye a, Guang-Xiong Zhou a,⁎, Yao-Lan Li a,⁎ a b
Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, People's Republic of China Xiamen Institute for Drug Control, Xiamen 361012, People's Republic of China
a r t i c l e
i n f o
Article history: Received 10 August 2013 Accepted in revised form 10 October 2013 Available online 19 October 2013 Keywords: Araliaceae Schefflera heptaphylla Triterpenoid saponin Anti-inflammatory activities
a b s t r a c t Phytochemical investigation on the stem bark of Schefflera heptaphylla led to the isolation of five new ursane-type triterpenoid saponins (1–5). Their structures were determined on the basis of spectroscopic and chemical methods. It is noteworthy in this study that the genins of all compounds are reported for the first time. All compounds isolated from this plant were evaluated for their inhibitory activities on lipopolysaccharide-induced nitric oxide production in RAW264.7 cells, and compounds 2 and 5 showed weak anti-inflammatory activities under their non-cytotoxic concentrations. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Schefflera heptaphylla (L.) Frodin (Araliaceae) is a mediumsized, evergreen tree up to 25 m tall, bole up to 80 cm in diameter. It is used as a folk remedy for the treatment of pain, inflammation, and common cold. It is also a principal ingredient of an herbal tea formulation widely used to treat common cold in southern China [1–3]. Previous phytochemical studies on S. heptaphylla showed that the plant is rich in triterpenoids and triterpenoid glycosides [1,4–10]. In the previous research, we had isolated and identified some triterpenoid saponins, including scheffursoside D, scheffursoside F, scheffoleoside A, scheffoleoside D, and acankoreoside A, from the stem bark of S. heptaphylla [5]. As part of our continuing search for bioactive constituents from S. heptaphylla, a 95% EtOH extract of the stem bark of S. heptaphylla had been investigated, and five ⁎ Corresponding authors at: Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, 601 West Huangpu Avenue, Guangzhou 510632, PR China. Tel.: +86 20 85221469; fax: +86 20 85221559. E-mail addresses: [email protected] (G.-X. Zhou), [email protected] (Y.-L. Li). 1 These authors contributed equally to this work. 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.10.006
new ursane-type triterpenoid saponins (1–5) were obtained (Fig. 1). In addition, all the new compounds were evaluated for their anti-inflammatory activities on lipopolysaccharide (LPS)induced nitric oxide (NO) production in RAW264.7 cells. In this paper, we described the isolation, structural elucidation, and anti-inflammatory activities of these triterpenoid saponins. 2. Experimental 2.1. General methods Optical rotations were carried out using a JASCO P-1030 automatic digital polarimeter. IR spectra were measured on a JASCO FT/IR-480 plus infrared spectrometer with KBr pellets. 1D and 2D NMR spectra were recorded on a Bruker AV-400 spectrometer with TMS as the internal standard, and chemical shifts were expressed in δ values (ppm). HRESIMS data were detected on an Agilent 6210 LC/MSD TOF mass spectrometer. Silica gel (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), ODS silica gel (50 μm, YMC, Kyoto, Japan), and Sephadex LH-20 (Pharmacia, Uppsala, Sweden) were used for column chromatography (CC). Analytical high-performance liquid chromatography (HPLC) was carried out on a Waters
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Fig. 1. Chemical structures of compounds 1–5.
chromatograph equipped with an evaporative light-scattering detector, a P680 pump, and a reversed phase (RP) C18 column (5 μm, 4.6 mm × 250 mm, Cosmosil, Kyoto, Japan). Semipreparative HPLC was performed on an Agilent 1200 unit with DAD detector and a RP C18 column (5 μm, 10 mm × 250 mm; Cosmosil, Kyoto, Japan). Preparative HPLC was carried out on a Varian chromatograph equipped with a Prostar 215 pump and a Prostar 325 UV–Vis detector with a RP C18 column (5 μm, 20 mm × 250 mm; Cosmosil, Kyoto, Japan). Thin-layer chromatography (TLC) was performed using pre-coated silica-gel plates (GF254, Yantai Chemical Industry Research Institute, Yantai, China). All the reagents were purchased from Tianjin Damao Chemical Company (Tianjin, China). L-cysteine methyl ester and standard sugars D-glucose (D-Glc), L-glucose (L-Glc), and L-rhamnose (L-Rha) in the analysis of HPLC experiments were purchased from Adamasbeta Company (Basel, Switzerland). O-Tolyl isothiocyanate and dexamethasone were purchased from Sigma Company (Sigma, St. Louis, MO, USA).
2.3. Extraction and isolation
2.2. Plant material
The dried and powdered stem bark of S. heptaphylla (10 kg) was soaked in 95% EtOH at room temperature for five times. The solution was evaporated under reduced pressure to obtain an extract (1.3 kg). This extract was suspended in distilled water, and then partitioned with petroleum ether, EtOAc, and n-BuOH, respectively. The n-BuOH-soluble residue (100 g) was subjected to silica gel column and eluted with CHCl3–MeOH (100:0; 90:10; 80:20; 70:30; 60:40; 50:50; 30:70; 0:100) in gradient to yield 40 fractions (Fr 1–40) based on their TLC patterns. Fr 12 (5.3 g) was separated on an ODS gel column (180 g, 3.5 cm × 40 cm) eluted with H2O– MeOH (80:20, 70:30, 60:40, 40:60, 20:80, 0:100, each 3 L), to give 15 subfractions (Sfr 1–15). Sfr 12 (250 mg) was subjected to preparative HPLC using 70% MeOH–H2O (7 mL/min) to give compounds 1 (7.4 mg) and 2 (5.1 mg). Sfr 10 (210 mg) was subjected to semi-preparative HPLC using 63% MeOH–H2O (3 mL/min) to give compound 3 (11.3 mg). Sfr 6 (318 mg) was subjected to preparative HPLC using 62% MeOH–H2O (7 mL/min) to give compounds 4 (13.4 mg) and 5 (21.9 mg).
The dried stem bark of S. heptaphylla was collected from Yulin, Guangxi, China, in August 2008, and was authenticated by Mr Zhen-Qiu Mai, a senior herbalist at the Chinese Medicinal Material Company, Guangdong, China. A voucher specimen with accession (No. SH20090301) has been deposited in the herbarium of College of Pharmacy, Jinan University.
2.3.1. 3-oxo-urs-20-en-23,28-dioic acid 28-O-α-L-rhamnopyranosyl(1 → 4)-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranoside (1) Amorphous powder; C48H76O19; [α]25D −2.35° (c 1.03, MeOH); IR (KBr) νmax: 3420, 2935, 1724, 1070 cm−1; HRESIMS (positive-ion mode) m/z 979.4880 [M + Na]+ (calcd. for C48H76O19Na: 979.4878); 1H NMR (C5D5N, 400 MHz) and 13C NMR (C5D5N, 100 MHz) data: see Tables 1 and 2.
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129
Table 1 1 H NMR data of compounds 1–5 (C5D5N, J in Hz).a Position
1
1 2 3 4 5 6 7 9 11 12 13 15 16 18 19 20 21 22 23 24 25 26 27 29 30 Glc-I 1′ 2′ 3′ 4′ 5′ 6′
1.82 2.32 – – 2.18 1.23 1.28 1.31 1.21 1.71 2.60 1.18 2.68 1.33 2.42 – 5.46 2.05 – 1.06 1.18 0.99 0.93 0.92 1.68 6.28 4.13 4.15 4.33 4.09 4.35 4.61 4.93 4.06 4.16 4.42 3.65 4.14 5.87 4.58 4.55
Glc-II 1″ 2″ 3″ 4″ 5″ 6″ Rha 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ Glc-III 1′′′′ 2′′′′ 3′′′′ 4′′′′ 5′′′′ 6′′′′ a
2 m, 1.95 m m, 2.42 m
br d (11.0) m, 1.69 m m m m, 1.39 m m m m, 1.94 m m m m br d (7.2) m, 2.58 dd (15.2, 6.8) s s s s d (6.4) s d (8.1) m m m m m, br s d (7.8) t (8.5) m t (9.1) d (9.1) m, 4.21 m br s br s m
1.74 1.83 4.31 – 2.54 1.17 1.24 1.67 1.15 1.77 2.61 1.20 2.61 1.60 2.34 – 5.45 2.09 – 1.47 0.87 1.25 0.97 1.02 1.70 6.29 4.41 4.53 4.65 4.41 4.38 4.50 5.00 4.32 4.50 4.61 4.25 4.32
3 m, 1.92 m m, 2.09 m br s br d (11.3) m, 1.70 m m m m, 1.45 m m m m, 1.92 m m m m d (6.5) m, 2.58 dd (14.7, 7.1) s s s s d (6.5) s d (8.0) m m m m m, br s d (7.8) m m m m m, 4.42 m
4.26 m 4.96 m 1.72 d (6.0)
1.74 1.87 4.56 – 2.53 1.17 1.25 1.64 1.21 1.82 2.62 1.21 2.62 1.62 2.35 – 5.42 2.01 – 1.43 0.78 1.17 0.90 1.01 1.67 6.26 4.06 4.13 4.31 4.19 4.35 4.70 4.92 3.93 4.03 4.32 3.66 4.10 5.89 4.59 4.55
m, 1.92 m m, 2.01 m br s
4.26 5.00 1.72 6.43 4.24 4.03 4.29 4.31 4.41
m m d (6.1) d (7.7) m m m m m, 4.58 m
br d (11.4) m, 1.71 m m m m, 1.44 m m m m, 1.93 m m m m d (6.9) m, 2.56 m s s s s d (6.5) s d (8.0) m m m m m, br s d (7.8) m m m d (9.0) m, 4.24 m br s br s m
4
5
1.57 m, 2.45 m 2.42 m, 2.47 m – 3.70 m 1.01 m 1.10 m, 1.57 m 1.28 m, 1.55 m 1.56 m 1.86 m 5.47 br s – 1.16 m, 2.39 m 1.94 m 2.54 d (11.4) 1.43 m 0.93 m 1.38 m 1.83 m, 1.94 m 1.08 d (6.3) – 1.06 s 1.21 s 1.15 s 0.95 d (6.5) 0.93 d (6.5) 6.21 d (8.0) 4.11 m 4.18 m 4.31 m 4.10 m 4.33 m, 4.68 br s 5.00 d (7.8) 3.96 m 4.21 m 4.43 t (9.3) 3.70 d (8.6) 4.11 m, 4.29 m 5.87 br s 4.70 br s 4.55 dd (3.2, 9.4) 4.34 m 4.94 m 1.71 d (6.2)
1.70 1.87 4.22 – 2.51 1.19 1.24 1.67 1.14 1.73 1.52 1.19 1.74 1.00 1.57 – 1.53 1.50 – 1.44 0.87 0.90 0.67 0.83 1.26 6.46 4.25 4.04 4.33 4.32 4.36
m, 1.94 m m, 2.01 m br s br d (11.0) m, 1.67 m m m m, 1.50 m m m m, 1.90 m m m m m, 1.82 m m s s s s d (6.9) s d (7.7) m m m m m, 4.42 m
Signals are designated as follows: s, singlet; br s, broad singlet; d, doublet; dd, doublet of doublets; m, multiplet.
2.3.2. 3α-hydroxy-urs-20-en-23,28-dioic acid 28-O-β-Dglucopyranosyl-(1 → 6)-β-D-glucopyranoside (2) Amorphous powder; C42H66O15; [α]25D −0.82° (c 1.10, MeOH); IR (KBr) νmax: 3423, 2930, 1721, 1079 cm-1; HRESIMS (positive-ion mode) m/z 833.4306 [M + Na]+ (calcd. for C42H66O15Na: 833.4299); 1H NMR (C5D5N, 400 MHz) and 13C NMR (C5D5N, 100 MHz) data: see Tables 1 and 2. 2.3.3. 3α-hydroxy-urs-20-en-23,28-dioic acid 23-O-β-Dglucopyranosyl, 28-O-α-L-rhamnopyranosyl-(1 → 4)-β-Dglucopyranosyl-(1 → 6)-β-D-glucopyranoside (3) Amorphous powder; C54H86O24; [α]25D +0.53° (c 0.94, MeOH); IR (KBr) νmax: 3427, 2928, 1728, 1075 cm−1; HRESIMS
(positive-ion mode) m/z 1141.5404 [M + Na]+ (calcd. for C54H86O24Na: 1141.5401); 1H NMR (C5D5N, 400 MHz) and 13C NMR (C5D5N, 100 MHz) data: see Tables 1 and 2.
2.3.4. 3-oxo-urs-12-en-24-nor-oic acid 28-O-α-L-rhamnopyranosyl(1 → 4)-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranoside (4) Amorphous powder; C47H74O17; [α]25D +1.32° (c 1.14, MeOH); IR (KBr) νmax: 3437, 2920, 1730, 1669, 1076 cm−1; HRESIMS (positive-ion mode) m/z 933.4816 [M + Na]+ (calcd. for C47H74O17Na: 933.4818); 1H NMR (C5D5N, 400 MHz) and 13C NMR (C5D5N, 100 MHz) data: see Tables 1 and 2.
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2.4. Determination of NO production and the cell viability assay
Table 2 13 C NMR data of compounds 1–5 (C5D5N). Position
1
2
3
4
5
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Glc-I 1′ 2′ 3′ 4′ 5′ 6′ Glc-II 1″ 2″ 3″ 4″ 5″ 6″ Rha 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ Glc-III 1′′′′ 2′′′′ 3′′′′ 4′′′ 5′′′′ 6′′′′
37.0 37.9 212.7 49.9 45.0 23.0 33.9 40.9 50.3 37.3 22.8 28.2 39.9 42.8 29.8 33.6 49.9 49.7 37.3 143.6 118.0 38.0 180.3 12.4 16.7 15.4 14.1 174.9 17.8 22.6 95.7 74.4 77.6 70.8 76.9 70.8 105.4 75.8 77.6 78.7 78.4 61.9 103.1 71.6 73.1 73.2 70.8 19.0
33.6 26.8 72.1 51.6 45.5 22.3 33.8 40.0 51.6 37.9 22.4 26.8 40.0 42.5 30.0 34.8 49.7 49.9 38.1 143.7 118.0 38.1 180.2 18.5 17.5 17.3 15.6 175.0 24.1 22.8 95.7 74.8 78.4 71.4 75.7 69.8 105.6 75.6 76.9 79.0 78.9 63.2
33.4 26.6 73.1 50.2 45.1 22.2 33.7 42.4 51.5 38.1 22.2 26.6 39.9 42.8 29.8 34.8 50.2 49.8 37.7 143.5 117.9 38.0 176.5 17.5 17.9 17.1 15.4 174.8 24.0 22.7 95.7 74.5 77.6 70.8 76.8 69.7 105.5 75.8 76.9 78.8 78.4 61.8 103.2 73.1 73.2 73.5 70.8 19.0 97.1 75.1 80.1 71.7 79.0 62.7
40.6 37.9 212.3 44.9 53.9 22.4 32.6 40.1 45.7 36.9 24.4 126.2 138.9 42.8 28.9 24.8 48.7 53.5 39.6 39.3 31.0 37.0 12.2 – 13.6 18.0 23.9 176.5 17.9 21.5 95.9 74.1 77.5 70.6 76.8 69.8 105.2 75.6 76.9 78.6 78.2 61.7 102.9 72.8 73.0 73.1 70.6 19.0
33.4 26.6 73.5 53.3 45.1 22.1 34.5 42.8 51.3 37.7 21.5 26.6 42.6 41.8 28.4 32.4 53.3 48.6 42.6 84.3 27.7 43.7 176.4 17.3 17.8 17.2 14.3 177.1 16.6 24.6 97.0 75.1 80.1 71.6 78.9 62.7
2.3.5. 3α-hydroxy-20β-hydroxyursan-23,28-dioic acid δ-lactone 23-O-β-D-glucopyranoside (5) Amorphous powder; C36H56O10; [α]25D − 1.00° (c 1.00, MeOH); IR (KBr) νmax: 3423, 2937, 1760, 1740, 1074 cm− 1; HRESIMS (positive-ion mode) m/z 671.3765 [M + Na]+ (calcd. for C36H56O10Na: 671.3766); 1H NMR (C5D5N, 400 MHz) and 13C NMR (C5D5N, 100 MHz) data: see Tables 1 and 2.
The anti-inflammatory activities of the compounds were evaluated by determining the amount of nitrite, a stable oxidized product in cell culture supernatant as described previously [15]. Briefly, cells (6 × 104 cells/well) were cultured in 48-well plates overnight and replaced by the media containing 1 μg/mL of LPS (Sigma, St. Louis, MO, USA) and different concentrations of the compounds. After culturing for 48 h, 50 μL of the supernatant was piped out and mixed with an equal volume of Griess reagent (Sigma, St. Louis, MO, USA) for 15 min. The absorbance at 540 nm was measured with a microplate reader (Molecular Devices, Emax, Sunnyvale, CA, USA). Nitrite concentrations in the supernatant were determined by comparison with a sodium nitrite standard curve. The inhibitory rate was calculated according to the formula: Inhibition (%) = 100% − [amount of nitrite (LPS + compound) / amount of nitrite (LPS)] × 100%. Cell viability was measured with the MTT-based colorimetric assay. 2.5. Determination of absolute configurations of sugars Compound 1 (3.0 mg) was dissolved in 1 M HCl (2 mL) and heated at 85 °C for 2 h. The mixture was evaporated to dryness under vacuum. The residue was dissolved in pyridine (2 mL) containing L-cysteine methyl ester (3.0 mg) and heated at 60 °C for 2 h. Then, O-tolyl isothiocyanate (10 μL) was added to the mixture, which was heated at 60 °C for 2 h. The reaction mixture was directly analyzed by reversed-phase HPLC. Analytical HPLC was performed on a RP C18 column (5 μm, 4.6 mm × 250 mm) at 30 °C with isocratic elution of 25% CH3CN–H2O containing 0.08% formic acid for 50 min and subsequent washing of the column with 90% CH3CN–H2O at a flow rate of 0.8 mL/min. Peaks were detected by a UV detector at 250 nm. Identification of D-glucose and L-rhamnose was carried out for 1, giving peaks at tR 23.2 and 35.3 min. The standard sugars such as D-, L-glucose, and L-rhamnose were subjected to the same method. The peaks of standard sugar derivatives were recorded at tR 23.30 (D-Glc), 21.28 (L-Glc), and 35.25 (L-Rha) min. Sugars from compounds 2–5 were also identified by the same procedure. 3. Results and discussion Compound 1, obtained as an amorphous powder, has a molecular formula of C48H76O19 established on the basis of its HRESIMS data ([M + Na]+ m/z 979.4880). Its 1H NMR spectrum suggested the presence of three sugar anomeric protons at δH 4.93 (1H, d, J = 7.8 Hz, H-1″of Glc-II), 5.87 (1H, br s, H-1″ of Rha) and 6.28 (1H, d, J = 8.1 Hz, H-1′ of Glc-I), giving the HSQC correlations with three anomeric carbons at δC 105.4, 103.1 and 95.7, respectively (Table 2), confirming that compound 1 contains three sugar units. Acid hydrolysis of 1 afforded D-glucose and L-rhamnose through HPLC analysis according to the method of Tanaka et al. [11]. The structure of the saccharide chain was established as α-L-rhamnopyranosyl-(1 → 4)-β-Dglucopyranosyl-(1 → 6)-β-D-glucopyranosyl based on the HMBC correlations of H-1‴/C-4″ (δC 78.7), and H-1″/C-6′ (δC 70.8). In addition, the NMR spectra of 1 also revealed 30 carbon signals including five tertiary methyl groups at δH/δC 0.93 (s, H3-27)/14.1, 0.99 (s, H3-26)/15.4, 1.06 (s, H3-24)/12.4, 1.18 (s, H3-25)/16.7, and 1.68 (s, H3-30)/22.6, one secondary methyl groups at δH/δC 0.92
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(d, J = 6.4 Hz, H3-29)/17.8, a tri-substituted double bond at δH/δC 5.46 (1H, br d, J = 7.2 Hz, H-21)/118.0 (C-21), and 143.6 (C-20), suggesting the carbon skeleton of 1 to be ursane [12–14]. In addition, the 13C NMR spectrum showed an ester carbonyl carbon at δC 174.9 (C-28), a carboxylic group at δC 180.3 (C-23), and a ketocarbonyl carbon at δC 212.7 (C-3). The ketocarbonyl was assigned for C-3 due to the HMBC correlations of H3-24, H-5/C-3, H3-24/C-3, C-5 and C-23. The double bond was placed at Δ20,21 on the basis of the HMBC correlations of H3-29/C-20, H3-30/C-20, and C-21, H-21/C-30. The sugar chain was linked to C-28, according to the upfield shift of C-28 (δC 174.9). This was further confirmed by the HMBC correlation of H-1′/C-28. Thus, the structure of 1 was elucidated as 3-oxo-urs-20-en-23,28-dioic acid 28-O-αL -rhamnopyranosyl-(1 → 4)-β-D-glucopyranosyl-(1 → 6)-β-Dglucopyranoside. Compound 2 was obtained as an amorphous powder. Its molecular formula was determined to be C42H66O15 from the quasi-molecular ion peak [M + Na]+ at m/z 833.4306 in the HRESIMS. The NMR signals of 2 were analogous to those of 1 except that a hydroxyl group [δH/δC 4.31 (1H, br s, H-3)/72.1] replaced the ketocarbonyl group of 1, and the α-rhamnose was absent in 2. ROESY correlation of H-3/H3-24 (δH 1.47), as well as the absence of the ROESY correlation between H-3 and H-5 (δH 2.54), indicated the β-orientation of H-3. Thus, compound 2 was assigned as 3α-hydroxy-urs-20-en-23,28-dioic acid 28-O-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranoside. Compound 3, an amorphous powder, was established to be C54H86O24 due to the HRESIMS data ([M + Na]+ m/z 1141.5404). In contrast with compound 1, the NMR spectra of 3 showed the presence of an extra anomeric proton and carbon signals at δH/δC 6.43 (1H, d, J = 7.7 Hz, H-1′′′′ of Glc-III)/97.1, an additional methylene group at δH/δC 4.41 (m, H-6′′′′ of Glc-III) and 4.58 (m, H-6′′′′ of Glc-III)/62.7, indicating the presence of a third glucosyl unit in the molecule. This glucosyl unit was located at C-23, according to the upfield shift of C-23 (δC 176.5), and further confirmed by the HMBC correlation of H-1′′′′/C-23. Moreover, a methine [δH/δC 4.56 (1H, br s, H-3)/73.1] with hydroxyl group replaced the ketocarbonyl group of 1. The obvious ROESY correlation of H-3/H3-24 (δH 1.43) and the absence of the ROESY correlation between H-3 and H-5 (δH 2.53) indicated the β-orientation of H-3. Thus, compound 3 was concluded to be 3α-hydroxy-urs-20-en-23,28-dioic acid 23-O-β-Dglucopyranosyl, 28-O-α-L-rhamnopyranosyl-(1 → 4)-β-Dglucopyranosyl-(1 → 6)-β-D-glucopyranoside. Compound 4 was obtained as an amorphous powder. Its HRESIMS spectrum showed a quasi-molecular ion peak at m/z 933.4816 [M + Na]+, ascribable to the molecular formula C47H74O17. The 1D NMR data of 4 (Tables 1 and 2) exhibited three tertiary methyl groups at δH/δC 1.06 (s, H3-25)/13.6, 1.15 (s, H3-27)/23.9 and 1.21 (s, H3-26)/18.0, three secondary methyl groups at δH/δC 0.93 (d, J = 6.5 Hz, H3-30)/21.5, 0.95 (d, J = 6.5 Hz, H3-29)/17.9, 1.08 (d, J = 6.5 Hz, H3-23)/12.2, one methine group at δH/δC 2.54 (1H, d, J = 11.4 Hz, H-18)/ 53.5, a tri-substituted double bond at δH/δC 5.47 (1H, br s, H-12)/126.2 (C-12) and 138.9 (C-13), and an ester carbonyl carbon at δC 176.5 (C-28), indicating an ursan-12-en-oic acid as an aglycon [12]. The comparison of the NMR spectra of its saccharide moiety with those of 1, helped establish the chain as α-L-rhamnopyranosyl-(1 → 4)-β-D-glucopyranosyl-(1 → 6)-βD-glucopyranosyl, and the chain was also positioned at C-28. The
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13
C NMR spectrum of 4 (Table 2) showed 47 carbon signals, of which 18 signals were assigned to the saccharide moiety. The remaining 29 carbon signals were attributed to a nor-triterpene moiety including six methyl carbons (δC 12.2, 13.6, 17.9, 18.0, 23.9 and 21.5), two olefinic carbons [δC 126.2 (C-12) and 138.9 (C-13)], and a ketone carbon [δC 212.3 (C-3)]. The ketone carbon was assigned for C-3 due to the HMBC correlations of H-1 (δH 2.45)/C-2 (δC 37.9), C-3, C-5 (δC 53.9), C-10 (δC 36.9) and C-25 (δC 13.6). There was only one methyl group at δH/δC 1.08 (3H, d, J = 6.3 Hz)/12.2 connecting to C-4, instead of two methyl groups as found at C-4 in common usual ursane triterpenes, which was proved by the HMBC correlations of H-4 (δH 3.70)/C-3, C-5 and C-23, H-5 (δH 1.01)/C-4 (δC 44.9), C-10 and C-25, and H-23/C-4 and C-5 (δC 53.9). The α-configuration of the methyl group at C-4 was confirmed by the ROESY correlation between H-4/H-25. Thus, compound 4 was determined as 3-oxo-urs-12-en-24-nor-oic acid 28-O-α-L-rhamnopyranosyl(1 → 4)-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranoside. Compound 5 also was an amorphous powder. Its molecular formula, C36H56O10, was determined from the HRESIMS positiveion peak at m/z 671.3765 ([M + Na]+). The NMR spectra of 5 were analogous to those of 3 except for the different signals attributed to ring E and the absence of the sugar chain at C-28. The presence of a δ-lactone ring was suggested by the appearance of an absorption band at 1740 cm−1 in its IR spectrum and a carbon signal at δC 177.1 (C-28) in the 13C NMR spectrum. The oxygen-bearing quaternary carbon signal observed at δC 84.3 (C-20) as well as the HMBC correlations from H-18 (δH 1.00), H-16 (δH 1.19) and H-22 (δH 1.50) to C-28, and from H-29 (δH 0.83), H-30 (δH 1.26) and H-19 (δH 1.56) to C-20, substantially provided information for a δ-lactone ring formed between C-20 and C-28. Thus, the structure of 5 was elucidated as 3α-hydroxy-20β-hydroxyursan-23,28-dioic acid δ-lactone 23-O-β-D-glucopyranoside. It is worth noting that the genins of compounds 1–5 are reported for the first time. Although the oleanane skeleton occurs frequently in Schefflera plants, the ursane type is very rarely in the genus. From our knowledge, this is the first time that α-amyrane-type compounds with a Δ20,21 double bond have been found in the Araliaceae. All isolates were evaluated for their inhibitory effects on the release of NO from macrophages using LPS-induced RAW264.7 cells as a model system. First of all, the non-cytotoxic concentrations of the compounds toward macrophage RAW264.7 cells were determined with 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay. Compounds 2 and 5 exhibited weak inhibitory effects against NO production in Table 3 The inhibitory effects of compounds on the NO secretion of mouse macrophage RAW 264.7 cells stimulated by LPS for 48 h (means ± SD, n = 4). Inhibition (%)
Concentration (μM)
Compounds
80
2 3 4 5 Dexamethasone
38.3 17.0 26.8 42.7 –
40 ± ± ± ±
3.2 2.7 3.0 3.2
18.7 16.5 16.2 45.7 –
20 ± ± ± ±
1.3 2.5 2.2 4.3
12.7 11.8 10.1 20.7 62.8
10 ± ± ± ± ±
2.4 1.7 1.4 3.2 2.5
NE NE NE 19.6 ± 3.8 61.7 ± 2.1
Note: NE indicated no effect; – indicated not detected due to cytotoxic concentration.
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their non-cytotoxic concentrations, as shown in Table 3. The positive control, dexamethasone, gave a 61.7 ± 2.1% (n = 4) inhibition at 10 μM. Conflict of interest The authors have no conflict of interest to report. Acknowledgments This research program was supported financially by the National Natural Science Foundation of China (No. 81202429, 81273390), Postdoctoral Science Foundation (2012M521656), and the grant (2009B090600117) from Guangdong province. Appendix A. Supplementary data All the HRESIMS and NMR spectra are available as Supporting Information. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2013.10.006. References [1] Sung TV, Steglich W, Adam G. Triterpene glycosides from Schefflera octophylla. Phytochemistry 1991;30:2349–56. [2] Li YL, Paul PHB, Vincent ECO. Antiviral activity and mode of action of caffeoylquinic acids from Schefflera heptaphylla (L.) Frodin. Antiviral Res 2005;68:1–9.
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