Fitoterapia 92 (2014) 127–132

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Fitoterapia

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New ursane-type triterpenoid saponins from the stem bark ofSchefflera heptaphylla

ChunWu 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 Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, People's Republic of Chinab Xiamen Institute for Drug Control, Xiamen 361012, People's Republic of China

a r t i c l e i n f o

⁎ Corresponding authors at: Institute of TraditionalNatural Products, College of Pharmacy, Jinan UniversitAvenue, Guangzhou 510632, PR China. Tel.: +86 20 8585221559.

E-mail addresses: guangxzh@sina.com (G.-X. Zhou(Y.-L. Li).

1 These authors contributed equally to this work.

0367-326X/$ – see front matter © 2013 Elsevier B.V. Ahttp://dx.doi.org/10.1016/j.fitote.2013.10.006

a b s t r a c t

Article history:Received 10 August 2013Accepted in revised form 10 October 2013Available online 19 October 2013

Phytochemical investigation on the stem bark of Schefflera heptaphylla led to the isolation offive new ursane-type triterpenoid saponins (1–5). Their structures were determined on thebasis of spectroscopic and chemical methods. It is noteworthy in this study that the genins ofall compounds are reported for the first time. All compounds isolated from this plant wereevaluated for their inhibitory activities on lipopolysaccharide-induced nitric oxide productionin RAW264.7 cells, and compounds 2 and 5 showed weak anti-inflammatory activities undertheir non-cytotoxic concentrations.

© 2013 Elsevier B.V. All rights reserved.

Keywords:AraliaceaeSchefflera heptaphyllaTriterpenoid saponinAnti-inflammatory activities

1. Introduction

Schefflera heptaphylla (L.) Frodin (Araliaceae) is a medium-sized, evergreen tree up to 25 m tall, bole up to 80 cm indiameter. It is used as a folk remedy for the treatment of pain,inflammation, and common cold. It is also a principal ingredientof an herbal tea formulation widely used to treat common coldin southern China [1–3]. Previous phytochemical studies onS. heptaphylla showed that the plant is rich in triterpenoidsand 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 ofS. heptaphylla [5]. As part of our continuing search for bioactiveconstituents from S. heptaphylla, a 95% EtOH extract of thestem bark of S. heptaphylla had been investigated, and five

Chinese Medicine andy, 601 West Huangpu221469; fax: +86 20

), tliyl@jnu.edu.cn

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new ursane-type triterpenoid saponins (1–5) were obtained(Fig. 1). In addition, all the new compounds were evaluated fortheir anti-inflammatory activities on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 cells. In thispaper, we described the isolation, structural elucidation, andanti-inflammatory activities of these triterpenoid saponins.

2. Experimental

2.1. General methods

Optical rotations were carried out using a JASCO P-1030automatic digital polarimeter. IR spectra were measured on aJASCO FT/IR-480 plus infrared spectrometer with KBr pellets.1D and 2D NMR spectra were recorded on a Bruker AV-400spectrometer with TMS as the internal standard, and chemicalshifts were expressed in δ values (ppm). HRESIMS data weredetected 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 usedfor column chromatography (CC). Analytical high-performanceliquid chromatography (HPLC) was carried out on a Waters

Fig. 1. Chemical structures of compounds 1–5.

128 C. Wu et al. / Fitoterapia 92 (2014) 127–132

chromatograph equipped with an evaporative light-scatteringdetector, a P680 pump, and a reversed phase (RP) C18 column(5 μm, 4.6 mm × 250 mm, Cosmosil, Kyoto, Japan). Semi-preparative HPLC was performed on an Agilent 1200 unitwith DAD detector and a RP C18 column (5 μm, 10 mm ×250 mm; Cosmosil, Kyoto, Japan). Preparative HPLC wascarried out on a Varian chromatograph equippedwith a Prostar215 pump and a Prostar 325 UV–Vis detector with a RP C18column (5 μm, 20 mm × 250 mm; Cosmosil, Kyoto, Japan).Thin-layer chromatography (TLC) was performed usingpre-coated silica-gel plates (GF254, Yantai Chemical IndustryResearch Institute, Yantai, China). All the reagents werepurchased 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 theanalysis of HPLC experiments were purchased from Adamas-beta Company (Basel, Switzerland).O-Tolyl isothiocyanate anddexamethasonewere purchased from Sigma Company (Sigma,St. Louis, MO, USA).

2.2. Plant material

The dried stem bark of S. heptaphylla was collected fromYulin, Guangxi, China, in August 2008, and was authenticatedby Mr Zhen-Qiu Mai, a senior herbalist at the ChineseMedicinal Material Company, Guangdong, China. A voucherspecimen with accession (No. SH20090301) has beendeposited in the herbarium of College of Pharmacy, JinanUniversity.

2.3. Extraction and isolation

The dried and powdered stem bark of S. heptaphylla(10 kg) was soaked in 95% EtOH at room temperature for fivetimes. The solution was evaporated under reduced pressure toobtain an extract (1.3 kg). This extract was suspended indistilled 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 withCHCl3–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 anODS 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), togive 15 subfractions (Sfr 1–15). Sfr 12 (250 mg) was subjectedto preparative HPLC using 70% MeOH–H2O (7 mL/min) to givecompounds 1 (7.4 mg) and 2 (5.1 mg). Sfr 10 (210 mg) wassubjected 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).

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. forC48H76O19Na: 979.4878); 1H NMR (C5D5N, 400 MHz) and 13CNMR (C5D5N, 100 MHz) data: see Tables 1 and 2.

Table 11H NMR data of compounds 1–5 (C5D5N, J in Hz).a

Position 1 2 3 4 5

1 1.82 m, 1.95 m 1.74 m, 1.92 m 1.74 m, 1.92 m 1.57 m, 2.45 m 1.70 m, 1.94 m2 2.32 m, 2.42 m 1.83 m, 2.09 m 1.87 m, 2.01 m 2.42 m, 2.47 m 1.87 m, 2.01 m3 – 4.31 br s 4.56 br s – 4.22 br s4 – – – 3.70 m –

5 2.18 br d (11.0) 2.54 br d (11.3) 2.53 br d (11.4) 1.01 m 2.51 br d (11.0)6 1.23 m, 1.69 m 1.17 m, 1.70 m 1.17 m, 1.71 m 1.10 m, 1.57 m 1.19 m, 1.67 m7 1.28 m 1.24 m 1.25 m 1.28 m, 1.55 m 1.24 m9 1.31 m 1.67 m 1.64 m 1.56 m 1.67 m11 1.21 m, 1.39 m 1.15 m, 1.45 m 1.21 m, 1.44 m 1.86 m 1.14 m, 1.50 m12 1.71 m 1.77 m 1.82 m 5.47 br s 1.73 m13 2.60 m 2.61 m 2.62 m – 1.52 m15 1.18 m, 1.94 m 1.20 m, 1.92 m 1.21 m, 1.93 m 1.16 m, 2.39 m 1.19 m, 1.90 m16 2.68 m 2.61 m 2.62 m 1.94 m 1.74 m18 1.33 m 1.60 m 1.62 m 2.54 d (11.4) 1.00 m19 2.42 m 2.34 m 2.35 m 1.43 m 1.57 m20 – – – 0.93 m –

21 5.46 br d (7.2) 5.45 d (6.5) 5.42 d (6.9) 1.38 m 1.53 m, 1.82 m22 2.05 m, 2.58 dd (15.2, 6.8) 2.09 m, 2.58 dd (14.7, 7.1) 2.01 m, 2.56 m 1.83 m, 1.94 m 1.50 m23 – – – 1.08 d (6.3) –

24 1.06 s 1.47 s 1.43 s – 1.44 s25 1.18 s 0.87 s 0.78 s 1.06 s 0.87 s26 0.99 s 1.25 s 1.17 s 1.21 s 0.90 s27 0.93 s 0.97 s 0.90 s 1.15 s 0.67 s29 0.92 d (6.4) 1.02 d (6.5) 1.01 d (6.5) 0.95 d (6.5) 0.83 d (6.9)30 1.68 s 1.70 s 1.67 s 0.93 d (6.5) 1.26 sGlc-I 1′ 6.28 d (8.1) 6.29 d (8.0) 6.26 d (8.0) 6.21 d (8.0) 6.46 d (7.7)2′ 4.13 m 4.41 m 4.06 m 4.11 m 4.25 m3′ 4.15 m 4.53 m 4.13 m 4.18 m 4.04 m4′ 4.33 m 4.65 m 4.31 m 4.31 m 4.33 m5′ 4.09 m 4.41 m 4.19 m 4.10 m 4.32 m6′ 4.35 m,

4.61 br s4.38 m,4.50 br s

4.35 m,4.70 br s

4.33 m,4.68 br s

4.36 m, 4.42 m

Glc-II 1″ 4.93 d (7.8) 5.00 d (7.8) 4.92 d (7.8) 5.00 d (7.8)2″ 4.06 t (8.5) 4.32 m 3.93 m 3.96 m3″ 4.16 m 4.50 m 4.03 m 4.21 m4″ 4.42 t (9.1) 4.61 m 4.32 m 4.43 t (9.3)5″ 3.65 d (9.1) 4.25 m 3.66 d (9.0) 3.70 d (8.6)6″ 4.14 m, 4.21 m 4.32 m, 4.42 m 4.10 m, 4.24 m 4.11 m, 4.29 mRha 1‴ 5.87 br s 5.89 br s 5.87 br s2‴ 4.58 br s 4.59 br s 4.70 br s3‴ 4.55 m 4.55 m 4.55 dd

(3.2, 9.4)4‴ 4.26 m 4.26 m 4.34 m5‴ 4.96 m 5.00 m 4.94 m6‴ 1.72 d (6.0) 1.72 d (6.1) 1.71 d (6.2)Glc-III 1′′′′ 6.43 d (7.7)2′′′′ 4.24 m3′′′′ 4.03 m4′′′′ 4.29 m5′′′′ 4.31 m6′′′′ 4.41 m, 4.58 m

a Signals are designated as follows: s, singlet; br s, broad singlet; d, doublet; dd, doublet of doublets; m, multiplet.

129C. Wu et al. / Fitoterapia 92 (2014) 127–132

2.3.2. 3α-hydroxy-urs-20-en-23,28-dioic acid 28-O-β-D-glucopyranosyl-(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. forC42H66O15Na: 833.4299); 1H NMR (C5D5N, 400 MHz) and 13CNMR (C5D5N, 100 MHz) data: see Tables 1 and 2.

2.3.3. 3α-hydroxy-urs-20-en-23,28-dioic acid 23-O-β-D-glucopyranosyl, 28-O-α-L-rhamnopyranosyl-(1 → 4)-β-D-glucopyranosyl-(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. forC54H86O24Na: 1141.5401); 1H NMR (C5D5N, 400 MHz) and 13CNMR (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: seeTables 1 and 2.

Table 213C NMR data of compounds 1–5 (C5D5N).

Position 1 2 3 4 5

1 37.0 33.6 33.4 40.6 33.42 37.9 26.8 26.6 37.9 26.63 212.7 72.1 73.1 212.3 73.54 49.9 51.6 50.2 44.9 53.35 45.0 45.5 45.1 53.9 45.16 23.0 22.3 22.2 22.4 22.17 33.9 33.8 33.7 32.6 34.58 40.9 40.0 42.4 40.1 42.89 50.3 51.6 51.5 45.7 51.310 37.3 37.9 38.1 36.9 37.711 22.8 22.4 22.2 24.4 21.512 28.2 26.8 26.6 126.2 26.613 39.9 40.0 39.9 138.9 42.614 42.8 42.5 42.8 42.8 41.815 29.8 30.0 29.8 28.9 28.416 33.6 34.8 34.8 24.8 32.417 49.9 49.7 50.2 48.7 53.318 49.7 49.9 49.8 53.5 48.619 37.3 38.1 37.7 39.6 42.620 143.6 143.7 143.5 39.3 84.321 118.0 118.0 117.9 31.0 27.722 38.0 38.1 38.0 37.0 43.723 180.3 180.2 176.5 12.2 176.424 12.4 18.5 17.5 – 17.325 16.7 17.5 17.9 13.6 17.826 15.4 17.3 17.1 18.0 17.227 14.1 15.6 15.4 23.9 14.328 174.9 175.0 174.8 176.5 177.129 17.8 24.1 24.0 17.9 16.630 22.6 22.8 22.7 21.5 24.6Glc-I 1′ 95.7 95.7 95.7 95.9 97.02′ 74.4 74.8 74.5 74.1 75.13′ 77.6 78.4 77.6 77.5 80.14′ 70.8 71.4 70.8 70.6 71.65′ 76.9 75.7 76.8 76.8 78.96′ 70.8 69.8 69.7 69.8 62.7Glc-II 1″ 105.4 105.6 105.5 105.22″ 75.8 75.6 75.8 75.63″ 77.6 76.9 76.9 76.94″ 78.7 79.0 78.8 78.65″ 78.4 78.9 78.4 78.26″ 61.9 63.2 61.8 61.7Rha 1‴ 103.1 103.2 102.92‴ 71.6 73.1 72.83‴ 73.1 73.2 73.04‴ 73.2 73.5 73.15‴ 70.8 70.8 70.66‴ 19.0 19.0 19.0Glc-III 1′′′′ 97.12′′′′ 75.13′′′′ 80.14′′′ 71.75′′′′ 79.06′′′′ 62.7

130 C. Wu et al. / Fitoterapia 92 (2014) 127–132

2.3.5. 3α-hydroxy-20β-hydroxyursan-23,28-dioic acid δ-lactone23-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: seeTables 1 and 2.

2.4. Determination of NO production and the cell viability assay

The anti-inflammatory activities of the compounds wereevaluated by determining the amount of nitrite, a stable oxidizedproduct in cell culture supernatant as described previously [15].Briefly, cells (6 × 104 cells/well) were cultured in 48-well platesovernight and replaced by the media containing 1 μg/mL of LPS(Sigma, St. Louis, MO, USA) and different concentrations of thecompounds. After culturing for 48 h, 50 μL of the supernatantwas piped out andmixedwith an equal volume of Griess reagent(Sigma, St. Louis, MO, USA) for 15 min. The absorbance at540 nm was measured with a microplate reader (MolecularDevices, Emax, Sunnyvale, CA, USA). Nitrite concentrations in thesupernatant were determined by comparison with a sodiumnitrite standard curve. The inhibitory rate was calculatedaccording to the formula: Inhibition (%) = 100% − [amount ofnitrite (LPS + compound) / amount of nitrite (LPS)] × 100%.Cell viability was measured with the MTT-based colorimetricassay.

2.5. Determination of absolute configurations of sugars

Compound 1 (3.0 mg) was dissolved in 1 M HCl (2 mL) andheated at 85 °C for 2 h. The mixture was evaporated to drynessunder vacuum. The residue was dissolved in pyridine (2 mL)containing L-cysteine methyl ester (3.0 mg) and heated at 60 °Cfor 2 h. Then, O-tolyl isothiocyanate (10 μL) was added to themixture, whichwas heated at 60 °C for 2 h. The reactionmixturewas directly analyzed by reversed-phase HPLC. Analytical HPLCwas performed on a RP C18 column (5 μm, 4.6 mm × 250 mm)at 30 °C with isocratic elution of 25% CH3CN–H2O containing0.08% formic acid for 50 min and subsequent washing of thecolumnwith 90%CH3CN–H2O at a flow rate of 0.8 mL/min. Peakswere detected by a UV detector at 250 nm. Identification ofD-glucose and L-rhamnose was carried out for 1, giving peaks attR 23.2 and 35.3 min. The standard sugars such as D-, L-glucose,and L-rhamnose were subjected to the same method. The peaksof standard sugar derivatives were recorded at tR 23.30 (D-Glc),21.28 (L-Glc), and 35.25 (L-Rha) min. Sugars from compounds2–5 were also identified by the same procedure.

3. Results and discussion

Compound 1, obtained as an amorphous powder, has amolecular formula of C48H76O19 established on the basis of itsHRESIMS data ([M + Na]+m/z 979.4880). Its 1HNMR spectrumsuggested the presence of three sugar anomeric protons at δH4.93 (1H, d, J = 7.8 Hz,H-1″ofGlc-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 theHSQC correlations with three anomeric carbons at δC 105.4,103.1 and 95.7, respectively (Table 2), confirming that compound1 contains three sugar units. Acid hydrolysis of 1 affordedD-glucose and L-rhamnose through HPLC analysis according tothe method of Tanaka et al. [11]. The structure of the saccharidechain was established as α-L-rhamnopyranosyl-(1 → 4)-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranosyl based on the HMBCcorrelations of H-1‴/C-4″ (δC 78.7), and H-1″/C-6′ (δC 70.8). Inaddition, the NMR spectra of 1 also revealed 30 carbon signalsincluding 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, and1.68 (s, H3-30)/22.6, one secondary methyl groups at δH/δC 0.92

Table 3The inhibitory effects of compounds on the NO secretion of mousemacrophage RAW 264.7 cells stimulated by LPS for 48 h (means ± SD,n = 4).

Inhibition (%) Concentration (μM)

Compounds 80 40 20 10

2 38.3 ± 3.2 18.7 ± 1.3 12.7 ± 2.4 NE3 17.0 ± 2.7 16.5 ± 2.5 11.8 ± 1.7 NE4 26.8 ± 3.0 16.2 ± 2.2 10.1 ± 1.4 NE5 42.7 ± 3.2 45.7 ± 4.3 20.7 ± 3.2 19.6 ± 3.8Dexamethasone – – 62.8 ± 2.5 61.7 ± 2.1

Note: NE indicated no effect; – indicated not detected due to cytotoxicconcentration.

131C. Wu et al. / Fitoterapia 92 (2014) 127–132

(d, J = 6.4 Hz, H3-29)/17.8, a tri-substituted double bond at δH/δC5.46 (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]. Inaddition, the 13C NMR spectrum showed an ester carbonyl carbonat δC 174.9 (C-28), a carboxylic group at δC 180.3 (C-23), and aketocarbonyl carbon at δC 212.7 (C-3). The ketocarbonyl wasassigned 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 bondwas placed atΔ20,21 onthe basis of theHMBC correlations of H3-29/C-20, H3-30/C-20, andC-21, H-21/C-30. The sugar chain was linked to C-28, according tothe upfield shift of C-28 (δC 174.9). This was further confirmedby the HMBC correlation of H-1′/C-28. Thus, the structure of1 was elucidated as 3-oxo-urs-20-en-23,28-dioic acid 28-O-α-L-rhamnopyranosyl-(1 → 4)-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranoside.

Compound 2 was obtained as an amorphous powder. Itsmolecular formula was determined to be C42H66O15 from thequasi-molecular ion peak [M + Na]+ at m/z 833.4306 in theHRESIMS. The NMR signals of 2 were analogous to those of 1except that a hydroxyl group [δH/δC 4.31 (1H, br s, H-3)/72.1]replaced the ketocarbonyl group of 1, and the α-rhamnosewas absent in 2. ROESY correlation of H-3/H3-24 (δH 1.47), aswell as the absence of the ROESY correlation between H-3and H-5 (δH 2.54), indicated the β-orientation of H-3. Thus,compound 2was assigned as 3α-hydroxy-urs-20-en-23,28-dioicacid 28-O-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranoside.

Compound 3, an amorphous powder, was established tobe C54H86O24 due to the HRESIMS data ([M + Na]+ m/z1141.5404). In contrast with compound 1, the NMR spectraof 3 showed the presence of an extra anomeric proton andcarbon signals at δH/δC 6.43 (1H, d, J = 7.7 Hz, H-1′′′′ ofGlc-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 themolecule. This glucosyl unit was located at C-23, accordingto the upfield shift of C-23 (δC 176.5), and further confirmedby the HMBC correlation of H-1′′′′/C-23. Moreover, a methine[δH/δC 4.56 (1H, br s, H-3)/73.1] with hydroxyl groupreplaced the ketocarbonyl group of 1. The obvious ROESYcorrelation of H-3/H3-24 (δH 1.43) and the absence of theROESY correlation between H-3 and H-5 (δH 2.53) indicatedthe β-orientation of H-3. Thus, compound 3 was concludedto be 3α-hydroxy-urs-20-en-23,28-dioic acid 23-O-β-D-glucopyranosyl, 28-O-α-L-rhamnopyranosyl-(1 → 4)-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranoside.

Compound 4 was obtained as an amorphous powder. ItsHRESIMS spectrum showed a quasi-molecular ion peak at m/z933.4816 [M + Na]+, ascribable to the molecular formulaC47H74O17. The 1D NMR data of 4 (Tables 1 and 2) exhibitedthree 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 secondarymethyl 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 carbonylcarbon at δC 176.5 (C-28), indicating an ursan-12-en-oic acid asan aglycon [12]. The comparison of the NMR spectra of itssaccharidemoietywith those of 1, helped establish the chain asα-L-rhamnopyranosyl-(1 → 4)-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranosyl, and the chainwas also positioned at C-28. The

13C NMR spectrum of 4 (Table 2) showed 47 carbon signals, ofwhich 18 signals were assigned to the saccharide moiety. Theremaining 29 carbon signals were attributed to a nor-triterpenemoiety 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 carbonwas 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/δC1.08 (3H, d, J = 6.3 Hz)/12.2 connecting to C-4, instead oftwo methyl groups as found at C-4 in common usual ursanetriterpenes, 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-10and C-25, andH-23/C-4 and C-5 (δC 53.9). Theα-configuration ofthemethyl group at C-4was confirmed by the ROESY correlationbetween H-4/H-25. Thus, compound 4 was determined as3-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 molecularformula, C36H56O10, was determined from theHRESIMSpositive-ion peak at m/z 671.3765 ([M + Na]+). The NMR spectra of5 were analogous to those of 3 except for the different signalsattributed to ring E and the absence of the sugar chain atC-28. The presence of a δ-lactone ring was suggested by theappearance of an absorption band at 1740 cm−1 in its IRspectrum and a carbon signal at δC 177.1 (C-28) in the 13C NMRspectrum. The oxygen-bearing quaternary carbon signalobserved at δC 84.3 (C-20) as well as the HMBC correlationsfromH-18 (δH 1.00), H-16 (δH 1.19) andH-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) toC-20, substantially provided information for a δ-lactone ringformed between C-20 and C-28. Thus, the structure of 5 waselucidated as 3α-hydroxy-20β-hydroxyursan-23,28-dioic acidδ-lactone 23-O-β-D-glucopyranoside.

It is worth noting that the genins of compounds 1–5 arereported for the first time. Although the oleanane skeletonoccurs frequently in Schefflera plants, the ursane type is veryrarely in the genus. From our knowledge, this is the first timethat α-amyrane-type compounds with a Δ20,21 double bondhave been found in the Araliaceae.

All isolateswere evaluated for their inhibitory effects on therelease of NO frommacrophages using LPS-induced RAW264.7cells as a model system. First of all, the non-cytotoxic con-centrations of the compounds toward macrophage RAW264.7cells were determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Compounds 2 and5 exhibited weak inhibitory effects against NO production in

132 C. Wu et al. / Fitoterapia 92 (2014) 127–132

their non-cytotoxic concentrations, as shown in Table 3. Thepositive 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 theNational Natural Science Foundation of China (No. 81202429,81273390), Postdoctoral Science Foundation (2012M521656),and the grant (2009B090600117) from Guangdong province.

Appendix A. Supplementary data

All theHRESIMS andNMR spectra are available as SupportingInformation. Supplementary data to this article can be foundonline at http://dx.doi.org/10.1016/j.fitote.2013.10.006.

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