Fitoterapia 103 (2015) 33–45

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Triterpene saponins with anti-inflammatory activity from the stems of Entada phaseoloides Hui Xiong, Yanan Zheng, Guangzhong Yang, Huixia Wang, Zhinan Mei ⁎ College of Pharmacy, South-Central University for Nationalities, Wuhan 430074, PR China

a r t i c l e

i n f o

Article history: Received 15 January 2015 Accepted in revised form 1 March 2015 Available online 7 March 2015 Keywords: Entada phaseoloides Leguminosae Anti-inflammation Triterpene saponins

a b s t r a c t The phytochemical investigation of the ethanol extract from the stems of Entada phaseoloides (L.) Merr (also called “Guo Gang Long”) led to the isolation of eleven triterpene saponins (1–11). Their structures were elucidated by extensive spectroscopic methods including 1D- (1H and 13C) and 2D-NMR (1H–1H COSY, HSQC, HMBC, HSQC-TOCSY and ROESY) experiments as well as ESIMS analysis and hydrolysis. These saponins comprised entagenic acid as the main aglycon, saccharide moieties at C-3 and C-28, and esterification of C-2 or C-3 hydroxyl group of the terminal β-Dglucopyranose unit with a monoterpenic acid. To further explain the clinical applications of “Guo Gang Long” for its anti-inflammatory effect, the inhibitory activities on the production of NO of the saponins and the related aglycon, entagenic acid (12), were evaluated in vitro. The compounds containing a free hydroxyl at C-3 of aglycon (1 and 4) and entagenic acid showed significant activities against NO production in lipopolysaccharide-stimulated mouse macrophage RAW264.7 cells with IC50 values of 25.08, 20.13 and 23.48 μM, respectively. And the three compounds could also inhibit the release of pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-6 and IL-8. © 2015 Published by Elsevier B.V.

1. Introduction Entada phaseoloides (L.) Merr. (Leguminosae), belonging to the Entada genus, is a traditional Chinese folk medicine and distributed widely in south of China. Both the seed kernels and stems of E. phaseoloides are well-known medicines to treat various diseases for a long time in minority area of China, but their clinical pharmacologies are different. The seed kernels of E. phaseoloides have been commonly used as folk medicine for the treatment of constipation and stomach ulcer. While, the stems of E. phaseoloides also called “Guo Gang Long”, have effects of dispelling wind and dampness, and possess especially powerful in the treatment of inflammatory disorder, such as arthritis and rheumatism [1,2]. Previous phytochemical studies on E. phaseoloides led to the isolation and identification of triterpene saponins [3–6], phenylacetic acid derivatives [7–10],

⁎ Corresponding author at: College of Pharmacy, South-Central University for Nationalities, No. 708 Minyuan Road, Wuhan 430074, PR China. Tel.: +86 27 67843713. E-mail address: [email protected] (Z. Mei).

http://dx.doi.org/10.1016/j.fitote.2015.03.001 0367-326X/© 2015 Published by Elsevier B.V.

flavonoids [11–13] and sulfur-containing amides [6,10,14,15]. Triterpene saponins isolated from the seed kernels of E. phaseoloides have been proved to be the main bioactive principles against the diabetes and inducing apoptosis of cancer cells in our previous research [16,17]. The stems of E. phaseoloides are also rich in triterpene saponins [3,4], however, whether these triterpene saponins responsible for its anti-inflammatory effects are still unknown. To explain the anti-inflammatory effects of “Guo Gang Long”, the isolation and structure elucidation of new triterpene saponins (1–11, Fig. 1), as well as their inhibitory effects on lipopolysaccharide (LPS)induced NO and pro-inflammatory cytokine (TNF-α, IL-1β, IL-6 and IL-8) production in RAW 264.7 cells are described in this study. 2. Experimental 2.1. General experimental procedures Optical rotations were determined on a JASCO P-1020 automatic digital polarimeter. The NMR spectra were obtained

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H. Xiong et al. / Fitoterapia 103 (2015) 33–45

Fig. 1. Chemical structures of compounds 1–11.

on a BRUKER DRX-500 spectrometer with TMSi as internal reference. High resolution mass spectra were acquired on an AutoSpec Premier P776 mass spectrometer or an Agilent G6230 TOF mass spectrometer. ESIMS analyses were performed using a BRUKER HCT Esquire 3000 mass spectrometer or an Agilent G6230 TOF mass spectrometer. IR spectra were recorded on a Thermo Scientific Nicolet 6700 spectrometer with KBr pellets. Semi-preparative HPLC was performed on DIONEX Ultimate 3000 with a Diode array detector, and Phenomenex column

(Luna 5u C18 100A, 10.0 × 250.0 mm, 5 μm). Analytical HPLC was carried out on DIONEX Ultimate 3000 with a Varivable Wavelength detector, and Agilent column (Agilent HC-C18, 4.6 × 250.0 mm, 5 μm). Column chromatographies were carried out on silica gel (200–300, or 300–400 mesh) (Qingdao Haiyang Chemical Co. Ltd.), ODS (50 μm, YMC, Japan), Sephadex LH-20 (20–150 μm, GE Healthcare, USA), and MCI GEL (CHP20P, 120 μm, Mitsubishi Chemical, Japan). Thinlayer chromatography was performed using silica gel (GF254,

H. Xiong et al. / Fitoterapia 103 (2015) 33–45

Qingdao Haiyang Chemical Co. Ltd., GF254). The HPLC pure methanol was purchased from Tedia Co. Ltd. L-cysteine methyl ester, and standard sugars D-glucose, N-acetyl-D-glucosamine, D-xylose, and L-arabinose in the analysis of HPLC experiments were purchased from Aladdin Industrial Co. Ltd., while D-apiose and O-tolyl isothiocyanate were obtained from Sigma-Aldrich and J&K Scientific Co. Ltd., respectively. The other chemical reagents were purchased from Sinopharm Chemical Reagent Co. Ltd. 2.2. Plant material The stems of E. phaseoloides were collected from Xishuangbanna, Yunnan Province, China and identified by Prof. Dingrong Wan, College of Pharmacy, South-Central University for Nationalities. A voucher specimen (No. EP201112) was deposited in the herbarium of College of Pharmacy, South-Central University for Nationalities. 2.3. Extraction and isolation The dried and powdered stems (9 kg) of E. phaseoloides were extracted tree times with 70% ethanol at room temperature (3 × 63 L, 24 h). The combined ethanol extracts were extracted consecutively with petroleum ether (3 × 4 L), EtOAc (3 × 4 L) and n-BuOH (3 × 4 L). The n-BuOH soluble fraction (173.9 g) was subjected to CC over silica gel (200–300 mesh, 13 × 117 cm, 2500 g) and eluted with a range of different CH2Cl2–MeOH gradients [100:0 (20 L), 95:5 (20 L), 9:1 (40 L), 8:2 (37 L), 7:3 (20 L), 6:4 (15 L), 1:1 (15 L), 4:6 (15 L), 0:100 (10 L)] to give 172 fractions with each fraction being 1000 mL in size (fractions 1–172). These fractions were combined into groups based on their TLC patterns to give 13 major subfractions (Fr. 1–13). Fr.5 (13.17 g) was purified by CC over MCI (120 μm, 4.5 × 29 cm, 200 g) using different gradient mixtures of H2O–MeOH [9:1 (3 L), 8:2 (5 L), 7:3 (6 L), 6:4 (7 L), 5:5 (7 L), 4:6 (5 L), 3:7 (4 L), 0:10 (3.5 L)] to afford 180 fractions (each fraction 200 mL). These fractions were combined into groups based on their Rf values by TLC to give 10 subfractions (Fr. 5.1–5.10). Fr. 5.9 (1.22 g) was submitted to Sephadex LH20 (20–150 μm, 1.8 × 127 cm) eluting with MeOH (1.75 L) to give 200 fractions (each fraction 8.0 mL). These fractions were combined using the same method described for Fr. 5 to give 5 subfractions (Fr. 5.9.1–5.9.5). Fr. 5.9.1 (372 mg) was purified by semi-preparative RP-HPLC with a gradient of H2O (containing 1‰ TFA)-CH3CN [60:40 (0 min)–60:40 (5 min)–55:45 (10 min)–55:45 (35 min)–50:50 (43 min); 3.0 mL/min, 212 nm)] to give compounds 1 (13.0 mg, tR 28.78 min), 2 (6.6 mg, tR 30.47 min), 3 (5.7 mg, tR 38.29 min) and 4 (15.9 mg, tR 42.19 min). Fr. 6 (21.1 g) was purified by CC over ODS (50 μm, 4.5 × 26 cm, 250 g) using different gradient mixtures of H2O–MeOH [95:5 (5 L), 9:1 (6 L), 8:2 (6 L), 7:3 (7 L), 6:4 (4 L), 4:6 (4 L), 3:7 (4 L)] to afford 170 fractions (each fraction 200 mL). These fractions were combined into groups based on their Rf values by TLC to give 8 subfractions (Fr. 6.1–6.8). Fr. 6.7 (2.12 g) was purified by CC over silica gel (300–400 mesh, 2.5 × 26 cm, 67.5 g) eluting with different CH2Cl2–MeOH gradients [10:0 (0.5 L), 9:1 (0.5 L), 5:1 (0.8 L), 3:1 (0.8 L), 1:1 (0.7 L), 1:2 (0.9 L), 1:4 (0.5 L), 0:10 (0.5 L)] to give 80 fractions (each fraction 60 mL). These fractions were combined according to

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the same procedure described for Fr. 6, to give nine subfractions (Fr. 6.7.1–6.7.9). Fr. 6.7.4 (387 mg) was purified by semi-preparative RP-HPLC with a gradient of H2O (containing 1‰ TFA)-CH3CN [65:35 (0 min)–65:35 (5 min)–60:40 (15 min)–60:40 (25 min), 3.0 mL/min, 212 nm)] to yield 5 (32.9 mg, tR 18.45 min) and 6 (6.5 mg, tR 21.81 min). Fr. 6.7.6 (168 mg) was purified by the same manner with Fr. 6.7.4 to yield 7 (36.1 mg, tR 21.67 min). Fr. 8 (22.6 g) was submitted to a column of polyamide (60–100 mesh, 6.0 × 45 cm, 314 g) eluting with different H2O– MeOH gradients [10:0 (5.0 L), 9:1 (7.0 L), 8:2 (10.0 L), 7:3 (12.0 L), 1:1 (5.0 L), 3:7 (5.0 L)] to afford 88 fractions (each fraction 500 mL). These fractions were combined using the same method described for Fr. 6, to give eight subfractions (Fr. 8.1–8.8). Fr. 8.5 (1.49 g) was further purified by CC over silica gel (300–400 mesh, 2.5 × 18 cm, 42.7 g) eluting with different CH2Cl2–MeOH gradients [10:0 (0.2 L), 4:1 (0.4 L), 2:1 (0.6 L), 1:1 (0.6 L), 1:2 (0.4 L), 0:10 (0.2 L)] to give 50 fractions (each fraction 50 mL). Fr. 8.5.3 (134 mg) further purified by semi-preparative RP-HPLC with a gradient of H2O (containing 1‰ TFA)-CH3CN [67:23 (0 min)–67:23 (35 min)–50:50 (55 min); 3.0 mL/min, 212 nm)] to give compounds 9 (28.0 mg, tR 30.40 min), 10 (7.0 mg, tR 46.5 min), and 11 (18.3 mg, tR 50.80 min). Fr. 9 (25.39 g) was submitted to a column of polyamide (60–100 mesh, 6.0 × 48 cm, 340 g) eluting with different H2O– MeOH gradients [10:0 (6.0 L), 8:2 (5.0 L), 7:3 (5.0 L), 1:1 (5.0 L), 3:7 (5.0 L)] to afford 88 fractions (each fraction 500 mL). According to the same method with Fr. 8, Fr. 9.1 was collected (460 mg) and further purified by semi-preparative RP-HPLC with a gradient of H2O (containing 1‰ TFA)-CH3CN [67:23 (0 min)–67:23 (30 min)–55:45 (40 min)–55:45 (47 min); 3.0 mL/min, 212 nm)] to give compound 8 (175.3 mg, tR 22.64 min). 2.4. Entagenic acid 28-O-[3-O-(2E,6R)-2,6-dimethyl-6-hydroxy2,7-octadienoyl]-β-D-glucopyranosyl-(1 → 4)-[β-D-xylopyranosyl(1 → 2)]-β-D-glucopyranosyl ester (1) White amorphous powder; [α]20 D − 11.4 (c 0.10, MeOH); IR (KBr) νmax 3422, 2946, 1685, 1654, 1208, 1076, 1046 cm−1; HRESIMS m/z 1109.5868 [M − H]− (calc. for 1109.5896); ESIMS (positive-ion) m/z 1133 [M + Na]+, 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz): see Tables 1 and 2. 2.5. Entagenic acid 28-O-[2-O-(2E,6R)-2,6-dimethyl-6-hydroxy2,7-octadienoyl]-β-D-glucopyranosyl-(1 → 4)-[β-D-xylopyranosyl(1 → 2)]-β-D-glucopyranosyl ester (2) White amorphous powder; [α]20 D − 14.3 (c 0.68, MeOH); IR (KBr) νmax 3420, 2928, 1682, 1205, 1030 cm−1; HRESIMS m/z 1109.5876 [M − H]− (calc. for 1109.5896); ESIMS (positiveion) m/z 1133 [M + Na]+, 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz): see Tables 1 and 2. 2.6. Entagenic acid 28-O-[2-O-(2E,6R)-2,6-dimethyl-6-hydroxy2,7-octadienoyl]-β-D-glucopyranosyl-(1 → 4)-[β-D-xylopyranosyl(1 → 2)]-(6-O-acetyl)-β-D-glucopyranosyl ester (3) White amorphous powder; [α]20 D − 4.2 (c 0.47, MeOH); IR (KBr) νmax 3421, 2970, 1735, 1701, 1239, 1076 cm−1; HRESIMS

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H. Xiong et al. / Fitoterapia 103 (2015) 33–45

Table 1 The 1H NMR data of compounds 1–5a,b (500 MHz, J in Hz, CD3OD). Position

1

2

3

4

5

1 2 3 5 6 7 9 11 12 15 16 18 19 21 22 23 24 25 26 27 29 30 Sugars 1 2 3 4 5 6

1.72 (m) 1.64 (m), 1.58 (m) 3.16 (dd, 11.4,4.4) 0.74 (m) 1.38 (m), 1.54 (m) 0.96 (m), 1.62 (m) 1.54 (m) 1.90 (m) 5.40 (brs) 3.85 (brs) 4.34 (d, 4.0) 2.98 (dd, 14.0, 4.0) 2.32 (t, 13.5), 1.02 (m) 1.98 (m), 1.18 (d, 13.0) 1.88 (m) 0.97 (s) 0.76 (s) 0.96 (s) 0.77 (s) 1.30 (s) 0.89 (s) 1.00 (s) 28-O-glc I 5.46 (d, 8.5) 3.70 (m) 3.80 (t, 9.0) 3.70 (m) 3.45 (m) 3.85 (dd, 12.0, 3.0), 3.75 (dd, 12.0, 5.5)

1.71 (m) 1.61 (m), 1.54 (m) 3.13 (d-like, 11.0) 0.72 (m) 1.37 (m), 1.51 (m) 0.98 (m), 1.64 (m) 1.53 (m) 1.90 (m) 5.38 (brs) 3.87 (d, 4.0) 4.32 (d, 3.0) 2.97 (dd, 14.5, 4.0) 2.29 (t, 14.0), 1.00 (m) 1.98 (m), 1.18 (d, 13.0) 1.86 (m) 0.96 (s) 0.77 (s) 0.95 (s) 0.82 (s) 1.29 (s) 0.88 (s) 0.97 (s) 28-O-glc I 5.44 (d, 8.0) 3.63 (m) 3.80 (t, 8.0) 3.68 (m) 3.38 (m) 3.65 (dd, 13.0, 3.5), 3.55 (m)

xyl 4.73 (d, 8.0) 3.20 (t, 8.0) 3.33 (m) 3.52 (t, 9.5) 3.95 (dd, 11.5, 5.5), 3.19 (m)

xyl 4.72 (d, 7.5) 3.18 (t, 8.0) 3.38 (m) 3.55 (m) 3.92 (d, 12.0), 3.15 (dd, 11.5, 5.5)

1.69 (m) 1.60 (m), 1.56 (m) 3.15 (d-like, 10.5) 0.71 (m) 1.37 (m), 1.51 (m) 0.98 (m), 1.63 (m) 1.53 (m) 1.85 (m) 5.36 (brs) 3.92 (d, 4.0) 4.29 (d, 4.0) 3.00 (dd, 14.0, 4.0) 2.29 (t, 14.0), 1.00 (m) 1.97 (m), 1.16 (d, 10.5) 1.98 (m), 1.78 (d, 11.0) 0.92 (s) 0.77 (s) 0.90 (s) 0.78 (s) 1.28 (s) 0.88 (s) 0.96 (s) 28-O-glc I 5.45 (d, 8.0) 3.66 (t, 9.0) 3.82 (t, 10.5) 3.56 (m) 3.56 (m) 4.10 (d, 11.5), 4.00 (dd, 11.5, 4.5) 2.05 (s) xyl 4.70 (d, 8.0) 3.14 (t, 10.5) 3.30b 3.47 (m) 3.86 (dd, 11.0, 3.5), 3.15 (dd, 11.5, 5.0)

1.70 (m) 1.63 (m), 1.53 (m) 3.13 (dd, 11.5, 4.5) 0.77 (m) 1.38 (t, 13.0), 1.53 (m) 0.97 (m), 1.63 (m) 1.53 (m) 1.85 (m) 5.38 (brs) 3.89 (brs) 4.29 (d, 3.0) 3.00 (dd, 14.0, 4.5) 2.29 (t, 14.0), 1.00 (m) 1.97 (m), 1.17 (m) 1.97 (m), 1.80 (t, 9.0) 0.96 (s) 0.77 (s) 0.93 (s) 0.81 (s) 1.29 (s) 0.89 (s) 1.03 (s) 28-O-glc I 5.45 (d, 7.5) 3.70 (t, 10.5) 3.83 (t, 10.5) 3.62 (t, 10.0) 3.68 (m) 4.36 (dd, 12.0, 3.0), 4.24 (dd, 12.0, 4.5) 2.05 (s) xyl 4.71 (d, 7.5) 3.19 (t, 10.0) 3.38 (t, 10.5) 3.50 (m) 3.92 (dd, 11.0, 3.0), 3.16 (dd, 11.5, 5.0)

1.68 (m) 1.67 (m), 1.91 (m) 3.10 (dd, 12.0, 4.0) 0.73 (m) 1.37 (m), 1.49 (m) 0.95 (m), 1.62 (m) 1.53 (m) 1.83 (m) 5.35 (brs) 3.86 (d, 3.0) 4.27 (d, 3.0) 2.98 (dd, 14.0, 4.0) 2.29 (t, 13.0), 0.98 (m) 1.96 (m), 1.15 (m) 1.97 (m), 1.77 (d, 9.0) 0.95 (s) 0.75 (s) 0.92 (s) 0.77 (s) 1.30 (s) 0.88 (s) 1.02 (s) 3-O-glcNAc 4.43 (d, 7.5) 3.67 (m) 3.44 (m) 3.32b 3.24 (m) 3.84 (dd, 12.5, 3.0), 3.67 (m) 2.13 (s) 28-O-glc I 5.45 (d, 8.0) 3.67 (t, 8.0) 3.81 (t, 10.5) 3.55 (m) 3.55 (m)

Me (−Ac) 1 2 3 4 5 6 Me (−Ac) 1 2 3 4 5 6

1 2 3 4 5 6 MT 3 4 5 7 8 9 10 a b

glc II 4.53 (d, 7.5) 3.40 (d, 8.0) 5.00 (t, 9.5) 3.52 (t, 8.5) 3.40 (m)

glc II 4.65 (d, 8.0) 4.80 (t, 9.5) 3.55 (m) 3.40 (m) 3.52 (t, 9.0)

glc II 4.56 (d, 8.0) 4.80 (t, 9.5) 3.55 (m) 3.39 (m) 3.39 (m)

glc II 4.43 (d, 8.0) 3.40 (d, 8.0) 5.00 (t, 9.0) 3.50 (m) 3.40 (m)

3.86 (dd, 12.0, 3.0), 3.69 (m)

3.90 (dd, 13.0, 2.5), 3.69 (dd, 13.0, 4.5)

3.93 (brd, 12.0), 3.68 (dd, 12.0, 5.0)

3.88 (dd, 11.0, 3.0), 3.68 (dd, 12.0, 5.5)

4.10 (d, 12.0), 4.00 (d, 12.0) 2.04 (s) xyl 4.70 (d, 8.0) 3.14 (t, 9.5) 3.30b 3.47 (m) 3.87 (d, 12.0), 3.13 (dd, 11.0, 3.0)

glc II 4.54 (d, 8.0) 4.80 (t, 9.5) 3.55 (m) 3.38 (m) 3.38 (m) 3.94 (d, 12.0), 3.69 (m) 6.83 (td, 7.5, 1.0) 2.24 (m) 1.66 (m) 5.92 (dd, 17.5, 11.0) 5.20 (dd, 17.5, 1.5), 5.05 (dd, 11.0, 1.5) 1.86 (s) 1.28 (s)

6.86 (td, 7.5, 1.0) 2.27 (m) 1.66 (m) 5.92 (dd, 17.5, 11.0) 5.21 (dd, 17.5, 1.5), 5.00 (dd, 11.0, 1.5) 1.85 (s) 1.28 (s)

6.84 (td, 7.5, 1.5) 2.24 (m) 1.66 (m) 5.92 (dd, 17.5, 11.0) 5.20 (dd, 17.5, 1.5), 5.05 (dd, 11.0, 1.5) 1.82 (s) 1.28 (s)

6.83 (td, 7.5, 1.0) 2.24 (m) 1.66 (m) 5.92 (dd, 17.5, 11.0) 5.20 (dd, 17.5, 1.5), 5.05 (dd, 11.0, 1.5) 1.85 (s) 1.27 (s)

These assignments are based on data from the DEPT, 1H-1H COSY, HSQC, HSQC-TOCSY, HMBC and ROESY experiments. Overlapped with other signals.

6.83 (td, 7.5, 1.0) 2.21 (m) 1.62 (m) 5.92 (dd, 17.5, 11.0) 5.23 (dd, 17.5, 1.5), 5.00 (dd, 11.0, 1.5) 1.85 (s) 1.27 (s)

H. Xiong et al. / Fitoterapia 103 (2015) 33–45

m/z 1151.5974 [M − H]− (calc. for 1151.6002); ESIMS (positive-ion) m/z 1175 [M + Na]+, 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz): see Tables 1 and 2. 2.7. Entagenic acid 28-O-[3-O-(2E,6R)-2,6-dimethyl-6-hydroxy2,7-octadienoyl]-β-D-glucopyranosyl-(1 → 4)-[β-D-xylopyranosyl(1 → 2)]-(6-O-acetyl)-β-D-glucopyranosyl ester (4) [α]20 D

− 11.8 (c 0.11, MeOH); IR White amorphous powder; (KBr) νmax 3419, 2926, 1734, 1248, 1077, 1034 cm−1; HRESIMS m/z 1151.5984 [M − H]− (calc. for 1151.6002); ESIMS (positive-ion) m/z 1175 [M + Na]+, 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz): see Tables 1 and 2. 2.8. 3β-O-2-Acetamido-2-deoxy-β-D-glucopyranosylentagenic acid 28-O-[2-O-(2E,6R)-2,6-dimethyl-6-hydroxy-2,7-octadienoyl]-βD-glucopyranosyl-(1 → 4)-[β-D-xylopyranosyl-(1 → 2)]-(6-Oacetyl)-β-D-glucopyranosyl ester (5) White amorphous powder; [α]20 D − 5.9 (c 0.31, MeOH); IR (KBr) νmax 3411, 2972, 1654, 1072, 1047 cm−1; HRESIMS m/z 1354.6714 [M − H]− (calc. for 1354.6796); ESIMS (positiveion) m/z 1378 [M + Na]+, 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz): see Tables 1 and 2. 2.9. 3β-O-β-D-Glucopyranosylentagenic acid 28-O-β-Dapiofuranosyl-(1 → 3)-β- D -xylopyranosyl-(1 → 2)]-[(2-O(2E,6R)-2,6-dimethyl-6-hydroxy-2,7-octadienoyl)-β-D-glucopyranosyl-(1 → 4)]-(6-O-acetyl)-β-D-glucopyranosyl ester (6) White amorphous powder; [α]20 D − 11.2 (c 0.31, MeOH); IR (KBr) νmax 3421, 2943, 1735, 1654, 1239, 1077, 1044 cm−1; HRESIMS m/z 1445.6865 [M − H]− (calc. for 1445.6953); ESIMS (positive-ion) m/z 1469 [M + Na]+, 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz): see Tables 2 and 3. 2.10. 3β-O-2-Acetamido-2-deoxy-β-D-glucopyranosylentagenic acid 28-O-β-D-apiofuranosyl-(1 → 3)-β-D-xylopyranosyl(1 → 2)-[(2-O-(2E,6R)-2,6-dimethyl-6-hydroxy-2,7-octadienoyl)β-D-glucopyranosyl-(1 → 4)]-(6-O-acetyl)-β-D-glucopyranosyl ester (7) White amorphous powder; [α]20 D − 7.6 (c 0.24, MeOH); IR (KBr) νmax 3424, 2926, 1735, 1654, 1238, 1076, 1027 cm−1; HRESIMS m/z 1486.7210 [M − H]− (calc. for 1486.7218); ESIMS (positive-ion) m/z 1510 [M + Na]+, 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz): see Tables 2 and 3. 2.11. 3β-O-β-D-Xylopyranosyl-(1 → 3)-α-L-arabinopyranosyl(1 → 6)-[β-D-glucopyranosyl-(1 → 4)]-2-acetamido-2-deoxy- βD-glucopyranosylentagenic acid 28-O-[2-O-(2E,6R)-2,6-dimethyl6-hydroxy-2,7-octadienoyl]-β-D-glucopyranosyl-(1 → 4)]-[βD-xylopyranosyl-(1 → 2)]-(6-O-acetyl)-β-D-glucopyranosyl ester (8) White amorphous powder; [α]20 D − 14.5 (c 0.85, MeOH); IR (KBr) νmax 3421, 2928, 1735, 1650, 1375, 1240, 1076, 1046 cm−1; HRESIMS m/z 1780.8113 [M − H]− (calc. for 1780.8169); ESIMS (negative-ion) m/z 1781 [M − H]−, 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz): see Tables 2 and 3.

37

2.12. 3β-O-β-D-Xylopyranosyl-(1 → 3)-α-L-arabinopyranosyl(1 → 6)-[β-D-glucopyranosyl-(1 → 4)]-2-acetamido-2-deoxy-βD-glucopyranosylentagenic acid 28-O-β-D-apiofuranosyl(1 → 3)-β-D -xylopyranosyl-(1 → 2)-[(3-O-(2E,6R)-2,6-dimethyl-6-hydroxy-2,7-octadienoyl)-β-D-glucopyranosyl-(1 → 4)](6-O-acetyl)-β-D-glucopyranosyl ester (9) White amorphous powder; [α]20 D − 13.1 (c 0.82, MeOH); IR (KBr) νmax 3421, 2938, 1735, 1650, 1205, 1076, 1044 cm−1; HRESIMS m/z 1912.8570 [M − H]− (calc. for 1912.8592); ESIMS (negative-ion) m/z 1913 [M − H]−, 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz): see Tables 2 and 3. 2.13. 3β-O-β-D-Xylopyranosyl-(1 → 3)-α-L-arabinopyranosyl(1 → 6)-[β-D-glucopyranosyl-(1 → 4)]-2-acetamido-2-deoxy-βD-glucopyranosylechinocystic acid 28-O-β-D-apiofuranosyl(1 → 3)-β-D-xylopyranosyl-(1 → 2)-[(3-O-(2E,6R)-2,6-dimethyl6-hydroxy-2,7-octadienoyl)-β-D-glucopyranosyl-(1 → 4)]-(6-Oacetyl)-β-D-glucopyranosyl ester (10) White amorphous powder; [α]20 D − 17.5 (c 0.39, MeOH); IR (KBr) νmax 3421, 2926, 1735, 1650, 1206, 1076, 1047 cm−1; HRESIMS m/z 1896.8694 [M − H]− (calc. for 1896.8643); ESIMS (negative-ion) m/z 1897 [M − H]−, 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz): see Tables 2 and 3. 2.14. 3β-O-β-D-Xylopyranosyl-(1 → 3)-α-L-arabinopyranosyl(1 → 6)-[β-D-glucopyranosyl-(1 → 4)]-2-acetamido-2-deoxyβ-D-glucopyranosyloleanolic acid 28-O-[2-O-(2E,6R)-2,6-dimethyl6-hydroxy-2,7-octadienoyl]-β-D-glucopyranosyl-(1 → 4)]-[βD-xylopyranosyl-(1 → 2)]-(6-O-acetyl)-β- D-glucopyranosyl eater (11) White amorphous powder; [α]20 D − 6.9 (c 0.54, MeOH); IR (KBr) νmax 3421, 2942, 1745, 1650, 1375, 1205, 1076 cm−1; HRESIMS m/z 1748.8262 [M − H]− (calc. for 1748.8643); ESIMS (negative-ion) m/z 1748 [M − H]−, 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz): see Tables 2 and 3. 2.15. Acid hydrolysis and derivatization of 1–11 Each compound (2.0 mg) was hydrolyzed with 4 N aqueous TFA (5 mL) for 3 h at 95 °C in a water bath. The mixture was diluted in water (8 mL) and extracted with CH2Cl2 (3 × 5 mL), and then the aq. layer was evaporated under reduced pressure to remove TFA. Furthermore, L-cysteine methyl ester hydrochloride (2.0 mg) was added into the residue of sugars and dissolved in anhydrous pyridine (1.0 mL). The mixture solution was refluxed at 60 °C in a water bath for 1.5 h. Then O-tolyl isothiocyanate (10 μL) was added to the mixture and heated further for 1 h. The reaction mixture was directly analyzed by Analytical HPLC. Analytical HPLC was performed on an Agilent column (Agilent HC-C18, 4.6 × 250.0 mm, 5 μm) at 30 °C with an isocratic elution of 25% CH3CN–H2O (containing 1‰ TFA, 0.8 mL/min, 250 nm) for 50 min [18]. Retention times for authentic sugars after being derivatized were 9.55 min (N-acetyl-D-glucosamine), 14.28 min (D-glucose), 15.70 min (L-arabinose), 16.20 min (D-xylose), and 27.64 min (D-apiose), respectively. By comparison of the retention times with the

38

H. Xiong et al. / Fitoterapia 103 (2015) 33–45

Table 2 The 13C NMR data of compounds 1–11a,b (125 MHz, CD3OD). Position

1

2

3

4

5

6

7

8

9

10

11

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 3-O-Sugars 1 2 3 4 5 6 Me (−Ac) −Ac

37.5 27.8 79.7 40.0 56.7 19.6 39.8 42.4 48.0 38.3 24.5 125.8 144.6 48.5 69.1 79.1 48.5 42.1 47.2 30.9 36.3 32.1 28.7 16.4 16.1 18.3 20.5 176.8 33.4 24.8

37.5 27.9 79.7 40.1 56.6 19.6 39.8 42.4 48.0 38.3 24.6 125.7 144.9 48.5 69.2 79.2 48.5 42.4 47.2 31.3 36.5 32.1 28.7 16.2 16.2 18.8 20.5 176.8 33.4 24.8

37.7 27.8 79.7 39.8 56.5 19.6 40.3 42.1 47.0 38.3 24.7 125.8 144.9 48.5 69 78.8 48.5 42.5 47.0 31.4 36.5 32.1 28.2 16.4 16.4 18.8 20.4 176.9 33.4 24.7

37.5 27.8 79.7 40.1 56.5 19.6 39.8 42.1 48.0 38.2 24.5 125.8 144.7 48.5 69 78.8 48.5 42.1 47.0 31.4 36.6 32.3 28.2 16.4 16.4 18.5 20.5 176.8 33.5 24.6

37.5 26.9 91.0 40.0 56.8 19.6 39.9 42.2 48.0 38.0 24.7 125.9 144.8 48.5 69.1 79.4 48.5 42.1 47.0 31.4 36.5 32.2 28.7 17.1 16.3 18.5 20.4 176.9 33.4 24.9 glcNAc 104.9 57.9 77.7 72.3 76.0 62.9 23.6 170.1

37.5 27.1 90.8 40.1 57.1 19.6 40.1 42.2 48.0 38.0 24.7 125.9 145.0 48.5 69.1 79.4 48.5 42.2 47.1 31.4 36.5 32.3 28.6 17.1 16.4 18.5 20.4 176.9 33.4 24.9 glc I 105.0 75.7 77.6 71.8 79.0 62.6

37.5 26.9 90.8 40.0 57.0 19.5 40.1 42.2 48.7 38.0 24.7 125.9 144.8 48.5 69.1 79.5 48.5 42.2 47.1 31.4 36.5 32.2 28.9 17.4 16.8 18.8 20.4 176.9 33.7 25.2 glcNAc 104.9 57.9 75.9 72.0 77.3 62.6 23.2 173.7

glc I 93.5 79.4 77.1 79.7 77.1 61.3

glc I 93.7 79.5 77.0 79.2 78.2 61.2

xyl 105.5

xyl 105.2

glc I 93.5 79.3 77.0 80.9 74.0 63.9 20.9 171.9 xyl 105.1

glc I 93.5 79.5 77.0 80.5 74.3 64.7 20.6 172.5 xyl 105.2

glc I 93.5 79.0 77.0 80.9 74.0 63.9 20.9 172.0 xyl 105.2

glc II 93.5 79.0 77.0 80.6 73.7 63.6 20.9 172.0 xyl 105.3

glc I 93.5 78.7 76.7 80.5 78.7 63.6 20.9 172.0 xyl I 104.9

37.5 27 91.1 40.0 56.7 19.6 39.9 42.2 48.7 38.0 24.7 125.1 144.5 48.5 69 79.3 48.5 42.5 47.0 31.4 36.5 32.2 28.6 17.1 16.4 18.5 20.5 176.8 33.6 24.9 glcNAc 105.0 57.3 73.9 80.6 75.1 68.3 23.2 173.3 glc I 104.3 74.9 77.7 71.4 76.4 62.4 ara 103.7 73.9 83.0 69.4 66.5 xyl I 107.7 76.4 77.7 71.1 67.4 glc II 93.7 78.8 76.8 80.0 73.9 64.2 20.9 171.9 xyl II 105.2

37.5 27 91.0 39.9 56.7 19.6 39.9 42.2 48.0 37.9 24.5 125.8 145.0 48.5 69.1 78 48.5 42.5 48.3 31.4 36.5 32.3 28.6 17.1 16.3 18.5 20.5 176.9 33.5 24.5 glcNAc 104.6 57.3 73.9 80.6 75.1 68.3 23.2 173.3 glc I 104.3 74.9 77.7 71.1 78 62.4 ara 103.7 73.9 83.1 69.4 66.5 xyl I 107.6 76.4 78.0 71.1 67.4 glc II 93.6 79.0 76.4 80.7 74.4 64.0 20.9 172.4 xyl II 105.0

34.3 27.1 91.0 39.9 57.2 19.4 39.9 42.3 48.0 37.9 24.5 123.6 144.0 48.5 36.8 74.7 48.5 41.8 47.3 31.4 36.3 31.1 28.4 17.2 16.2 18.5 27.8 176.9 33.1 25.1 glcNAc 104.8 57.3 73.9 80.8 74.9 68.3 23.1 173.3 glc I 104.3 74.7 77.7 71.4 78 62.4 ara 103.5 73.9 83.1 69.4 66.5 xyl I 107.5 75.4 78.0 71.2 67.4 glc II 93.6 78.9 77.0 80.8 74.4 63.9 20.9 172.4 xyl II 105.0

39.8 27.0 90.9 40.0. 57.0 19.4 34.1 42.5 48.2 37.9 24.6 124.6 145.0 48.5 27.0 23.7 48.5 42.5 47.1 31.6 34.9 33.3 28.6 17.1 16.1 17.9 26.5 178 33.5 24.2 glcNAc 104.8 57.4 73.9 80.7 75.1 68.4 23.1 173.3 glc I 104.3 74.9 78.0 71.4 75.8 62.4 ara 103.7 73.9 83.2 69.4 66.5 xyl I 107.6 76.5 77.9 71.2 67.4 glc II 93.5 78.7 77.0 80.8 73.6 64 20.9 172.4 xyl II 105.0

1 2 3 4 5 6 1 2 3 4 5 1 2 3 4 5 28-O-Sugars 1 2 3 4 5 6 −Me (−Ac) −Ac 1

H. Xiong et al. / Fitoterapia 103 (2015) 33–45

39

Table 2 (continued) Position

1

2

3

4

5

6

7

8

9

10

11

2 3 4 5

75.5 78.0 71.0 67.2 glc II 104.5 73.3 78.8 69.4 78.0 61.2

75.5 78.0 71.0 67.3 glc II 102.3 75.3 76.2 71.5 79.7 62.4

75.7 77.9 71.1 67.2 glc II 102.6 75.3 76.2 71.6 78.4 62.4

75.7 77.9 71.2 67.2 glc II 104.5 73.2 79.4 71 78.0 62.2

75.7 78.0 71.1 67.2 glc II 102.6 75.3 76.2 71.7 78.4 62.5

75.4 85.3 69.5 66.9 glc III 102.3 75.4 76.2 71.7 78.0 62.1 api 111.3 77.3 80.0 74.7 65.1

75.4 85.0 70.8 66.9 xyl II 102.6 75.0 75.9 71.4 78.1 62.1 api 111.3 77.5 80.2 74.7 65.1

75.7 78.0 71.2 67.2 glc III 102.6 75.3 76.0 71.6 78.2 62.4

75.2 85.3 69.8 66.9 glc III 104.7 73.3 78.9 69.7 78.1 62.3 api 111.2 77.9 80.5 75.0 65.4

75.1 85.3 70.0 66.7 glc III 104.2 73.3 78.9 69.7 78.1 62.2 api 111.2 78.00 80.5 75.1 65.2

75.8 77.9 71.2 67.1 glc III 102.6 75.1 76.2 71.6 78.3 62.4

169.4 128.7 144.0 24.6 41.7 73.6 145.9 112.4 12.5 27.8

168.5 128.4 144.7 24.6 41.5 73.6 146.0 112.4 12.5 27.9

168.4 128.0 145.3 24.7 41.6 73.6 146.2 112.4 12.5 27.9

169.3 128.7 143.7 24.5 42.1 73.6 145.9 112.4 12.5 27.8

168.5 128.3 145.1 24.7 41.6 73.6 146.1 112.5 12.5 27.9

168.5 128.3 144.9 24.7 41.6 73.6 146.2 112.4 12.5 27.9

168.5 128.5 143.9 24.7 41.6 73.6 146.0 112.4 12.5 27.9

168.4 128.0 144.5 24.7 41.6 73.6 146.1 112.4 12.5 27.9

169.6 128.8 143.9 24.5 41.7 73.6 146.0 112.4 12.5 27.8

169.6 128.8 144.5 24.6 41.7 73.6 146.0 112.2 12.4 28.2

169.6 128.8 144.5 24.6 41.7 73.6 146.0 112.5 12.5 27.9

1 2 3 4 5 6 1 2 3 4 5 MT 1 2 3 4 5 6 7 8 9 10 a b

These assignments are based on data from the DEPT, 1H-1H COSY, HSQC, HSQC-TOCSY, HMBC and ROESY experiments. Overlapped with other signals.

standards, the absolute configuration of sugars in each hydrolysis was established. Fr. 9.1 (50 mg) rich in compound 8 was hydrolyzed by the same manner with the other compounds. And the CH2Cl2 extract was chromatographed by RP-HPLC using H2O (containing 1‰ TFA)-CH3CN (45:55, v/v, 4 mL/min, 212 nm) to yield entagenic acid (12).

2.16. Alkaline hydrolysis Fr. 5.9.1 (50 mg) rich in compounds 1–4 was hydrolyzed with 0.2 M NaOH (20 mL) and MeOH (4 mL) for 10 h at room temperature. After adjusting the pH to 5.0 with 0.2 M HCl, the reaction mixture was extracted successively with EtOAc and n-BuOH. The EtOAc extract was further purified [19,20] to afford the MT moiety [(2E,6R)-2,6-dimethyl-6-hydroxy-2,7octadienoic acid (13)]. The other compounds were hydrolyzed as described for Fr. 5.9.1 to give the MT moiety.

2.17. Cell culture and treatment RAW264.7 cells (CCTCC) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum. Cells were grown at 37 °C and in a humidified incubator in an atmosphere of 5% CO2. In all experiments, cells were incubated in the presence or absence of various concentrations of test compounds, which were added LPS (10 μg/mL) treatment.

2.18. MTT assay for measuring cell viability The cytotoxic effects of the compounds were evaluated by a MTT assay. Cells were seeded in 96-well plates (1 × 104 cells/well) and incubated for 24 h. After this incubation period, cells were treated with various concentrations of compounds (15, 30 and 60 μM), DEX (15 μM) and LPS (10 μg/mL) at 37 °C in 5% CO2 for 48 h. After treatment, 10 μL of MTT (5 mg/mL) dissolved in DMEM was added to each well, followed by incubation for 4 h. The medium was aspirated, and the formazan crystals were dissolved in 150 μL of DMSO for 15 min. Optical density of each well at 492 nm was determined with a microplate reader. Cell viability in response to treatment was calculated as percentage of control cells treated with solvent DMSO at the final concentration 0.1%:% viable cells = (100 × OD treated cells) / OD control cells. 2.19. Measurement of nitric oxide production Production of NO was determined by measuring the accumulated level of nitrite, an indicator of NO in the supernatant after 48 h of LPS treatment with or without different concentrations of test compounds. After preincubation of cells for 24 h, DEX (15 μM), the different concentrations of test compounds were added, together with LPS. The cells were further incubated at 37 °C in 5% CO2 for 48 h. The quantity of nitrite in the culture medium was measured as an indicator of NO production. Amounts of nitrite, a stable metabolite of NO, were measured using Griess reagent (Beyotime Institute of Biotechnology, China).

40

H. Xiong et al. / Fitoterapia 103 (2015) 33–45

Table 3 The 1H NMR data of compounds 6–11a,b (500 MHz, J in Hz, CD3OD). Position

6

1 2 3 5 6

1.68 (m) 1.90 (m), 1.67 (m) 3.16 (d, 10.5) 0.73 (m) 1.51 (m), 1.35 (m)

7

0.96 (dd, 11.5, 5.0), 1.62 (m) 1.51 (m) 1.84 (m) 5.35 (brs) 3.90 (brs) 4.28 (brs) 2.98 (d, 14.0) 2.30 (t, 12.0), 1.00 (m)

9 11 12 15 16 18 19 21 22 23 24 25 26 27 29 30 3-O-Sugars 1 2 3 4 5 6

1.98 (d, 10.0), 1.15 (m) 1.95 (m), 1.78 (m) 1.28 (s) 0.78 (s) 0.93 (s) 0.84 (s) 1.00 (s) 0.88 (s) 1.05 (s) glc I 4.32 (d, 7.5) 3.18 (m) 3.24 (m) 3.30b 3.34 (m) 3.83 (dd, 14.0, 2.0), 3.68 (m)

Me (−Ac)

7

1

11 1.58 (m), 0.98b 1.87 (m), 1.67 (m) 3.13 (d, 10.0) 0.75 (m) 1.52 (m), 1.38 (m)

0.96 (m), 1.63 (m)

0.98 (m), 1.62 (m)

1.29 (m)

1.54 (m) 1.87 (m) 5.39 (brs) 3.92 (d, 3.0) 4.30 (d, 3.5) 3.03 (dd, 11.5, 4.0) 2.34 (t, 12.0), 1.02 (m)

1.50 (d, 10.5) 1.89 (m) 5.35 (brs) 3.85 (brs) 4.28 (d, 3.0) 2.97 (d, 11.0) 2.30 (t, 13.0), 1.00 (m)

1.53 (m) 1.86 (m) 5.37 (brs) 3.84 (brs) 4.29 (d, 3.5) 2.99 (dd, 13.0, 4.0) 2.35 (m), 1.02 (m)

1.55 (m) 1.86 (m) 5.20 (brs) 1.67 (m) 1.67 (m) 2.88 (dd, 13.0, 2.5) 1.70 (d, 9.0), 1.11 (m)

2.00 (m), 1.19 (m)

1.96 (m), 1.16 (m)

1.98 (d, 9.0), 1.82 (t, 7.5) 0.95 (s) 0.75 (s) 0.92 (s) 0.77 (s) 1.28 (s) 0.88 (s) 0.96 (s) glcNAc 4.46 (d, 7.0) 3.69 (m) 3.58 (t, 8.0) 3.35b 3.28 (m) 3.90 (dd, 10.0, 2.0), 3.71 (m) 1.99 (s)

1.96 (m), 1.79 (m)

1.96 (t, 10.5), 1.17 (d, 9.0) 1.96 (m), 1.79 (m)

1.62 (m) 1.88 (m) 5.30 (brs) 1.30 (m), 1.80 (m) 4.53 (d, 5.0) 2.99 (d, 10.0) 2.30 (t, 13.0), 1.01 (dd, 14.0, 5.0) 1.93 (m), 1.13 (t, 10.5)

0.95 (s) 0.76 (s) 0.92 (s) 0.77 (s) 1.28 (s) 0.88 (s) 1.05 (s) glcNAc 4.45 (d, 7.0) 3.70 (m) 3.56 (m) 4.01 (t, 10.5) 3.48 (m) 4.10 (d-like, 11.5), 3.99 (d, 12.0) 1.94 (s) glc I 4.63 (d, 8.5) 3.21 (t, 9.0) 3.35 (m) 3.35 (m) 3.55 (m) 3.82 (m), 3.69 (m) ara 4.54 (d, 5.5) 3.70 (m) 3.72 (m) 3.83 (m) 3.84 (d, 12.0), 3.51 (d, 10.5) xyl I 4.47 (d, 7.5) 3.25 (d, 9.0) 3.34 (m) 3.48 (m) 4.06 (dd, 12.5, 5.5), 3.24 (m) glc II 5.45 (d, 7.0) 3.69 (m) 3.82 (m) 3.55 (m) 3.62 (t, 10.5) 4.10 (d, 11.5), 4.05 (d, 12.5) 2.04 (s) xyl II 4.74 (d, 7.5)

0.95 (s) 0.76 (s) 0.93 (s) 0.77 (s) 1.29 (s) 0.88 (s) 1.00 (s) glcNAc 4.43 (d, 8.5) 3.70 (m) 3.62 (t, 8.5) 4.00 (m) 3.49 (d, 9.0) 4.11 (d, 10.5), 3.90 (dd, 11.0, 5.0) 1.94 (s) glc I 4.63 (d, 8.5) 3.21 (t, 9.0) 3.35 (m) 3.50 (m) 3.55 (m) 3.84 (m), 3.68 (m) ara 4.54 (d, 6.0) 3.72 (m) 3.70 (m) 3.83 (m) 3.84 (d, 12.0), 3.50 (d, 12.0) xyl I 4.47 (d, 7.5) 3.26 (m) 3.35 (m) 3.49 (m) 4.00 (dd, 11.5, 6.0), 3.24 (m) glc II 5.47 (d, 8.0) 3.69 (m) 3.84 (m) 3.62 (d, 8.5) 3.69 (m) 4.36 (d, 12.0), 4.28 (dd, 11.5, 3.5) 2.06 (s) xyl II 4.74 (d, 7.5)

1 2 3 4 5

Me (−Ac)

10 1.51 (m),1.38 1.87b, 1.68 (d, 11.0) 3.13 (dd, 11.0, 4.5) 0.77 (m) 1.52 (m), 1.39 (m)

1.68 (m) 1.86 (m), 1.65 (m) 3.13 (dd, 10.0, 3.5) 0.74 (m) 1.48 (m), 1.36 (m)

1 2 3 4 5

glc II 5.45 (d, 8.0) 3.68 (m) 3.81 (t, 10.5) 3.55 (m) 3.55 (m) 4.10 (d, 12.0), 4.01 (dd, 12.0, 5.0) 2.04 (s) xyl 4.73 (d, 7.5)

9 1.68 (m) 1.86 (m), 1.67 (m) 3.12 (dd, 11.0, 4.0) 0.73 (m) 1.51 (d, 9.5), 1.36 (t, 10.0) 0.97 (m), 1.52 (m)

1.71 (d, 12.5) 1.94 (m), 1.68 (m) 3.13 (dd, 10.0, 3.5) 0.77 (m) 1.54 (m), 1.35 (d, 10.5), 0.98 (m), 1.66 (m)

1 2 3 4 5 6

28-O-Sugars 1 2 3 4 5 6

8

glc I 5.50 (d, 7.0) 3.71 (m) 3.85 (t, 10.5) 3.59 (t, 9.5) 3.59 (t, 9.5) 4.14 (d, 11.0), 4.05 (dd, 13.0, 2.5) 2.08 (s) xyl 4.73 (d, 7.5)

b

1.86 (m), 1.76 (m) 0.95 (s) 0.74 (s) 0.93 (s) 0.77 (s) 1.30 (s) 0.87 (s) 0.96 (s) glcNAc 4.42 (d, 7.5) 3.68 (m) 3.61 (m) 4.03 (d, 10.5) 3.49 (m) 4.11 (dd, 11.0, 5.0), 3.92 (m) 1.94 (s) glc I 4.64 (d, 7.5) 3.29 (m) 3.34 (t, 8.5) 3.35 (t, 8.5) 3.38-3.40 (m) 3.82 (d, 11.0) ara 4.54 (d, 5.0) 3.72 (m) 3.70 (m) 3.83 (m) 3.85 (m), 3.51 (m) xyl I 4.45 (d, 8.0) 3.28b 3.33 (m) 3.42 (m) 3.90 (m), 3.24 (d, 10.0) glc II 5.44 (d, 7.5) 3.69 (m) 3.82 (m) 3.62 (d, 8.5) 3.69 (m) 4.40 (d, 12.0), 4.28 (dd, 12.0, 5.0) 2.05 (s) xyl II 4.72 (d, 7.5)

1.38 (t, 11.0), 1.20 (brd, 12.0) 1.46 (brd, 12.5), 1.81 (m) 0.95 (s) 0.73 (s) 0.91 (s) 0.76 (s) 1.13 (s) 0.90 (s) 0.96 (s) glcNAc 4.45 (d, 8.5) 3.68 (m) 3.62 (t, 8.5) 4.01 (m) 3.49 (t, 9.0) 4.12 (d, 11.5), 3.92 (d, 11.5) 1.94 (s) glc I 4.63 (d, 7.5) 3.20 (t, 8.5) 3.35 (m) 3.48 (t, 9.0) 3.49 (t, 9.0) 3.87 (d, 12.0), 3.69 (m) ara 4.54 (d, 6.5) 3.71 (m) 3.70 (m) 3.82 (m) 3.84 (d, 12.0), 3.52 (d, 11.5) xyl I 4.46 (d, 7.5) 3.25 (t, 8.0) 3.30b 3.35 (m) 3.81 (dd, 11.5, 5.0), 3.24 (dd, 12.0, 4.5) glc II 5.45 (d, 8.0) 3.69 (m) 3.83 (m) 3.55 (m) 3.56 (m) 4.12 (d, 11.0), 4.04 (m) 2.04 (s) xyl II 4.69 (d, 8.0)

H. Xiong et al. / Fitoterapia 103 (2015) 33–45

41

Table 3 (continued) Position

6

7

8

9

10

11

2 3 4 5

3.27 (d, 10.5) 3.37 (m) 3.52 (m) 3.92 (d, 12.0), 3.16 (d, 11.0) glc III 4.54 (d, 8.0) 4.82 (t, 7.5) 3.55 (m) 3.30 (m) 3.38 (m) 3.92 (d, 13.5), 3.68 (m) api 5.20 (d, 3.0) 3.97 (d, 2.0) 4.10 (d, 9.5), 3.78 (d, 9.5) 3.60 (brs)

3.20 (t, 10.5) 3.42 (m) 3.50 (d, 10.5) 3.93 (d, 11.0), 3.19 (d-like, 10.5) glc II 4.60 (d, 7.0) 4.85 (t, 7.5) 3.58 (d, 8.0) 3.42 (m) 3.42 (m) 3.95 (dd, 12.5, 3.5), 3.68 (m) api 5.29 (d, 3.0) 4.00 (brs) 4.15 (d, 9.0), 3.82 (dd, 9.0, 3.0) 3.64 (brs)

3.16 (t, 9.5) 3.33b 3.48 (m) 3.86 (d, 13.0), 3.14 (d, 12.0) glc III 4.56 (d, 7.0) 4.80 (t, 9.5) 3.57 (m) 3.39 (m) 3.39 (m) 3.91 (dd, 12.0, 4.0), 3.69 (m)

3.19 (t, 8.0) 3.37 (t, 9.0) 3.44 (m) 3.94 (d, 11.5), 3.18 (brd, 10.0) glc III 4.42 (d, 7.5) 3.39 (m) 5.00 (t, 9.5) 3.50 (m) 3.35 (m) 3.89 (dd, 11.5, 4.5), 3.69 (m) api 5.26 (d, 2.5) 3.97 (d, 2.0) 4.12 (d, 9.5), 3.79 (d, 10.5) 3.60 (brs)

3.19 (t, 8.0) 3.35 (t, 8.5) 3.50 (m) 3.90 (m), 3.17 (dd, 13.0, 4.5) glc III 4.44 (d, 8.0) 3.38 (t, 8.0) 4.97 (t, 9.5) 3.50 (m) 3.30 (m) 3.90 (m), 3.68 (m)

3.18 (t, 9.0) 3.30b 3.35 (m) 3.81 (dd, 11.5, 5.0), 3.15 (dd, 12.5, 5.5) glc III 4.56 (d, 8.0) 4.80 (t, 9.0) 3.56 (m) 3.39 (m) 3.39 (m) 3.92 (d, 11.5), 3.69 (m)

6.83 (td, 7.5, 1.0) 2.24 (m) 1.60 (m) 5.92 (dd, 17.5, 11.0) 5.27 (dd, 17.5, 1.5), 5.05 (dd, 11.0, 1.5) 1.82 (s) 1.28 (s)

6.87 (td, 7.5, 1.0) 2.24 (m) 1.60 (m) 5.96 (dd, 17.5, 11.0) 5.28 (dd, 17.5, 1.5), 5.10 (dd, 11.0, 1.5) 1.82 (s) 1.28 (s)

6.83 (td, 7.5, 1.0) 2.18 (m) 1.60 (m) 5.92 (dd, 16.5, 10.5) 5.22 (dd, 17.0, 1.5), 5.06 (dd, 10.5, 1.5) 1.83 (s) 1.28 (s)

6.83 (td, 7.5, 1.0) 2.20 (m) 1.63 (m) 5.92 (dd, 17.5, 10.5) 5.18 (dd, 17.0, 1.5), 5.04 (dd, 10.5, 1.5) 1.85 (s) 1.27 (s)

6.83 (td, 7.5, 1.0) 2.18 (m) 1.62 (m) 5.92 (dd, 17.5, 11.0) 5.20 (dd, 17.5, 1.5), 5.06 (dd, 11.0, 1.5) 1.85 (s) 1.27 (s)

1 2 3 4 5 6 Sugar 1 2 4 5 MT 3 4 5 7 8 9 10 a b

api 5.26 (d, 2.5) 3.96 (d, 2.0) 4.12 (d, 9.5), 3.80 (d, 10.0) 3.60 (brs) 6.83 (td, 7.5, 1.0) 2.26 (m) 1.62 (m) 5.92 (dd, 17.5, 11.5) 5.22 (dd, 17.5, 1.5), 5.04 (dd, 11.0, 1.5) 1.85 (s) 1.27 (s)

These assignments are based on data from the DEPT, 1H-1H COSY, HSQC, HSQC-TOCSY, HMBC and ROESY experiments. Overlapped with other signals.

2.20. Quantification of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-8) production After pre-incubation of cells for 24 h, the cells were treated LPS with or without different concentrations of test compounds. The effect of test compounds on the cytokine (TNF-α, IL-1β, IL-6 and IL-8) productions from the LPS treated RAW 264.7 cell was determined after stimulation and intervention lasting for 48 h by ELISA kits (BeingLay Biotechnology, China) according to the instructions provided by the manufacturer. 2.21. Statistical analysis Data are expressed as mean ± SEM. Statistical differences were evaluated by analysis of variance for comparison between each group using GraphPad Prism 5 (GraphPad Software, Inc., CA). A value of P b 0.05 was considered statistically significant. 3. Results and discussion 3.1. Characterization of the compounds After extraction by ethanol 70% of the stems of E. phaseoloides, the resulting extract was subjected to multiple chromatographic steps to afford eleven new compounds 1–11, along with two hydrolysis products (entagenic acid (12), and (2E,6R)-2,6-dimethyl-6-hydroxy-2,7-octadienoic acid (13)). The structures of new compounds were identified by extensive spectroscopic methods including 1D- (1H and 13C) and 2D-NMR (DQF-COSY, HSQC, HMBC, HSQC-TOCSY and ROESY) experiments

in combination with mass spectrometry (HRESIMS and ESIMS) and hydrolysis. The NMR spectra of two known compounds (12–13) were in accordance with the corresponding literature [6,19]. Compound 1, an amorphous powder, had a molecular formula of C57H90O21 deduced from the [M − H]− ion at m/z 1109.5868 (calc. for 1109.5896) in the negative HRESIMS, as well as its NMR data. Data from the 1H NMR and 13C NMR spectra (Tables 1 and 2) indicated a triterpene saponin structure. The 1H NMR spectrum of the aglycon portion displayed signals for seven tertiary methyl groups at δH 0.97, 0.76, 0.96, 0.77, 1.30, 0.89 and 1.00, for an olefinic proton at δH 5.40 (brs), a typical signal of H-3ax attached to a hydroxylated carbon at δH 3.16 (dd, 11.4, 4.4), and two vicinal oxygenated methine protons at δH 3.85 (brs) and 4.34 (4.0). These signals along with the carbon resonances in the 13C NMR spectrum (Table 2) suggested the aglycon characteristic for entagenic acid, as the chemotaxonomic marker triterpene saponins isolated from the genus of Entada [6,21]. The 1H NMR of 1 exhibited three anomeric protons at δH 5.46 (d, 8.5), 4.73 (d, 8.0) and 4.53 (d, 7.5), which gave correlations in the HSQC spectrum with the three anomeric carbon signals at δC 93.5, 105.5, and 104.5, respectively, confirming that compound 1 contains three sugar units. After acid hydrolysis, the sugar units were confirmed to be D-glucose and D-xylose in a ratio of 2:1, which were identified by HPLC analysis of their derivatives [18]. The β-configurations of D-glucose and D-xylose units were deduced from the coupling constants of correlation anomeric protons and intraresidue ROE relationships. Complete assignments of the resonances of each sugar unit were achieved by 1D and 2D NMR analyses. By extensive NMR spectra and HPLC analysis,

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H. Xiong et al. / Fitoterapia 103 (2015) 33–45

Fig. 2. Inhibitory effects of compounds 1, 4, and 12 on TNF-a, IL-1β, IL-6, and IL-8 productions stimulated by LPS in RAW 264.7 cells. Cells were treated LPS with or without compounds 1, 4, and 12 (15, 30, and 60 μM). The cytokines (TNF-a, IL-1β, IL-6, and IL-8) were quantified in supernatants using ELISA assays. Dexamethasone (Dex) was used as positive reference compound. Data are means ± SEM from three independent experiments. Statistical analysis was performed by ANOVA followed by t-test (n = 3; ## P b 0.01 vs. normal control; * P b 0.05 vs. LPS; **P b 0.01 vs. LPS).

the monosaccharide moieties were identified as one β-Dxylopyranosyl (xyl) and two glucopyranosyl (glc I and glc II) units. The HMBC cross peaks at δH 5.46 (H-1 of glc I) / δC 176.8 (C-28 of aglycon), δH 4.73 (H-1 of xyl) / δC 79.4 (C-2 of glc I), and δH 4.53 (H-1 of glc II) / δC 79.7 (C-4 of glc I) revealed the sequence glc II-(1 → 4)-[xyl-(1 → 2)]-glc I to be linked at C-28 of aglycon portion. The 13C NMR spectrum exhibited 47 carbons, of which 30 were assigned to the aglycon, 17 to the three sugar units comprising two glucoses and one xylose, and remaining 10 carbons, which were assigned to a monoterpene unit (MT), due to the presence of two tertiary methyl (δH 1.86, 1.28), three coupled olefinic protons [δH 5.92 (dd, 17.5, 11.0), 5.20 (dd, 17.5, 1.5) and 5.05 (dd, 11.0, 1.5)], and one olefinic proton [δH 6.83 (td, 7.5, 1.0)]. To elucidate the structure of MT moiety, the MT moiety was obtained from the alkaline hydrolysis residues of saponin. By comparison the NMR data and optical activity ([α]20D − 14.5 (c 0.35, MeOH)) with the literature [19,20], the MT moiety was identified as (2E,6R)-2,6-dimethyl-6hydroxy-2,7-octadienoic acid. The down-field shift of H-3 of glc II (δH 5.00) suggested the esterification of the hydroxyl group at glc II C-3 by MT unit. This was further confirmed by the HMBC correlation between the glc II H-3 (δH 5.00) and MT C-1 (δC 169.4). Based on the above analyses, the structure of 1 was established as entagenic acid 28-O-[3-O-(2E,6R)-2,6-dimethyl-6-hydroxy-2,7-octadienoyl]-

β-D-glucopyranosyl-(1 → 4)-[β-D-xylopyranosyl-(1 → 2)]-βester. Compound 2 showed the same molecular formula of C57H90O21 as 1, established from the [M − H]− ion at m/z 1109.5876 (calc. for 1109.5896) in the negative HRESIMS, as well as its NMR data. Acid hydrolysis of 2 allowed the identification of the same sugar units as 1, containing two Dglucoses and one D-xylose, as determined by a same manner with 1. Analysis of its NMR data and comparison those of 1 showed that they possess the same aglycon and sugar chain at C-28 of aglycon, except for the linkage site of MT group in the sugar chain. The down-field shift of glc II H-2 (δH 4.80) indicated that MT unit is linked to C-2 of glc II. This hypothesis was also confirmed by the observation of cross-peak correlation between the H-2 of glc II (δH 4.80) and C-1 of MT (δC 168.5) in the HMBC spectrum. Therefore, compound 2 was defined as entagenic acid 28-O-[2-O-(2E,6R)-2,6-dimethyl6-hydroxy-2,7-octadienoyl]-β-D-glucopyranosyl-(1 → 4)-[βD-xylopyranosyl-(1 → 2)]-β-D-glucopyranosyl ester. Compound 3 was assigned the molecular formula C59H92O22 from the [M − H]− peak at m/z 1151.5974 in HRESIMS and NMR spectroscopic data. The 1H and 13C NMR spectra of compound 3 in comparison to those of 2 showed the presence of signals at δH 2.05 (3H, s) / δC 20.9 and δC 171.9 (a carbonyl function), a typical of an acetyl group linked at C-6 of glc I in compound 3 (δH 4.10, 4.00 / δC 63.9 in 3 versus δH 3.65, 3.55 / δC 61.2 in 2). The linkage site of acetyl group was further confirmed by the cross-peak between the H-6 of glc I and the carbonyl function of the acetyl group in the HMBC spectrum, and the cross-peak between the H-1 (δH 5.45) and C-6 (δC 63.9) of D-glucopyranosyl

H. Xiong et al. / Fitoterapia 103 (2015) 33–45

43

Table 4 Inhibitory effect of compounds on NO production in LPS-activated RAW264.7 cellsa,b. Compound

1

4

5

7

8

9

11

12

IC50 (μM) Cell viability (%)

25.08 ± 1.8 112.3 ± 0.3

20.13 ± 1.7 110.3 ± 1.9

37.72 ± 2.9 108.3 ± 4.1

72.50 ± 1.1 15.2 ± 0.9

93.12 ± 6.3 67.6 ± 5.3

N100 13.7 ± 0.05

70.57 ± 0.5 48.4 ± 4.3

23.48 ± 1.9 93.8 ± 0.9

a b

Results are averages of three independent experiments, and data are expressed as mean ± SEM. Cell viability after treatment with 60 μM of each compound is expressed as a percentage (%) of the LPS only treatment group.

glc I in the HSQC-TOCSY spectrum. These observations were used to assign the structure of 3 as entagenic acid 28-O-[2-O-(2E,6R)-2,6-dimethyl-6-hydroxy-2,7→ 4)-[β-Doctadienoyl]-β-D-glucopyranosyl-(1 xylopyranosyl-(1 → 2)]-(6-O-acetyl)-β-D-glucopyranosyl ester. Compound 4 showed the same molecular formula of C59H92O22 as 3, established from the [M − H]− ion at m/z 1151.5984 (calc. for 1151.6002) in the negative HRESIMS and its NMR spectroscopic data. Comparison of the NMR data for 4 and 3 revealed that the compounds are similar except for the linkage site of MT group in the sugar chain, which is connected to C-3 (δC 79.4) of glc II in 4 instead of to C-2 (δC 75.3) of glc II in 3, according to the HMBC correlation observed between the H-3 of glc II (δH 5.00) and C-1 of MT (δC 169.3). Thus, the structure of compound 4 was established as entagenic acid 28-O-[3-O-(2E,6R)-2,6-dimethyl-6-hydroxy-2,7-octadienoyl]β-D-glucopyranosyl-(1 → 4)-[β-D-xylopyranosyl-(1 → 2)]-(6O-acetyl)-β-D-glucopyranosyl ester. Compound 5 had a molecular formula of C67H105NO27, established from the [M − H]− ion at m/z 1354.6714 (calc. for 1354.6796) in the negative HRESIMS and its NMR spectroscopic data. The 1H and 13C NMR spectra indicated that 5 was also a triterpene saponin with the entagenic acid as the aglycon. The chemical shift values of C-3 (δC 91.0) and C-28 (δC 176.9) of aglycon portion indicated that 5 was a bisdesmosidic glycoside. Acid hydrolysis of 5 allowed the sugar components as 2-acetamido-2-deoxy-D-glucopyranose (D-glcNAc), D-glucose and D-xylose identified by using the same method as 1. A comparison of the NMR data of 5 with those of 4 showed that 5 differed from 4 only by the presence of an additional sugar signals in 5 [an anomeric proton at δH 4.43 (d, 7.5), an anomeric carbon at δC 104.9, other carbons at δC 23.6, 57.9, 77.7, 72.3, 76.0, and 170.1], which was determined to D-glcNAc unit. Cross peak between the H-1 of glcNAc (δH 4.43) and C-3 of aglycon (δC 91.0) indicated that the glcNAc was located at C-3 of aglycon. Thus, compound 5 was defined as 3β-O-2-acetamido2-deoxy-β-D-glucopyranosylentagenic acid 28-O-[2-O-(2E,6R)2,6-dimethyl-6-hydroxy-2,7-octadienoyl]-β-D-glucopyranosyl(1 → 4)-[β- D -xylopyranosyl-(1 → 2)]-(6-O-acetyl)-β-Dglucopyranosyl ester. Compound 6 had a molecular formula of C70H110O31, determined from the [M − H]− ion peak at m/z 1445.6865 (calc. for 1445.6953) in the HRESIMS and its NMR spectroscopic data. The NMR data of 6 were similar to those of 3, except for the presence of two additional sugars. The two additional sugar units were determined to be D-glucose and D-apiose, according to the same manner used to determine the sugars in 1. The HMBC cross-peaks at δH 4.32 (H-1 of glc I)/δC 90.8 (C-3 of aglycon) and δH 5.20 (H-1 of api)/δC 85.3 (C-3 of xyl) indicated that the glc I linked at C-3 of aglycon and the api linked at C-3 of xyl. These observations were used to assign

the structure of 6 as 3β-O-β-D-glucopyranosylentagenic acid 28-O-β-D-apiofuranosyl-(1 → 3)-β-D-xylopyranosyl-(1 → 2)][(2-O-(2E,6R)-2,6-dimethyl-6-hydroxy-2,7-octadienoyl)-β-Dglucopyranosyl-(1 → 4)]-(6-O-acetyl)-β-D-glucopyranosyl ester. Compound 7 had a molecular formula of C72H113NO31, determined from the [M − H]− ion peak at m/z 1486.7210 (calc. for 1486.7218) in the HRESIMS and its NMR spectroscopic data. In a comparison of NMR spectra of 7 with those of 6, the data were identical, except for the signals at δC (104.9, 57.9, 75.9, 72.0, 77.3 and 62.6) due to 2-acetamido-2-deoxy-Dglucopyranosyl moiety of 7 instead of the signals at δC (105.0, 75.7, 77.6, 71.8, 79.0 and 62.6) due to D-glucopyranosyl moiety linked at C-3 of aglycon of 6, suggesting that the sugar unit linked to C-3 of aglycon was 2-(acetamido)-2-deoxy-Dglucopyranosyl. Thus, the structure of 7 was determined as 3βacid O-2-acetamido-2-deoxy-β-D-glucopyranosylentagenic 28-O-β-D-apiofuranosyl-(1 → 3)-β-D-xylopyranosyl-(1 → 2)[(2-O-(2E,6R)-2,6-dimethyl-6-hydroxy-2,7-octadienoyl)-β-Dglucopyranosyl-(1 → 4)]-(6-O-acetyl)-β-D-glucopyranosyl ester. Compound 8 had a molecular formula of C83H131NO40, determined from the [M − H]− ion peak at m/z 1780.8113 (calc. for 1780.8169) in the HRESIMS and its NMR spectroscopic data. Its spectral features also suggested 8 to be a glycosylated at C-3 and C-28 positions triterpen glycoside based on entagenic acid as an aglycon (Tables 2 and 3). Acid hydrolysis of 8 yielded D-glcNAc, D-glucose, L-arabinose and Dxylose by the same manner with the other compounds. The assignments of sugar resonances (1D and 2D NMR) revealed that 8 and 5 possessed the same sugar arrangement at aglycon C-28, but differed in the sugar portion at aglycon C-3. The linkages of the sugar units at entagenic acid C-3 were concluded from the cross peaks correlating the resonances of glcNAc H-1 (δH 4.45) and aglycon C-3 (δC 91.1), ara H-1 (δH 4.54) and glcNAc C-6 (δC 68.3), xyl II H-1 (δH 4.47) and ara C-3 (δC 83.0), glc I H-1 (δH 4.63) and glcNAc C-4 (δC 80.6). Consequently, the structure of 8 was established as 3β-O-β-D-xylopyranosyl-(1 → 3)-α-L-arabinopyranosyl-(1 → 6)[β-D-glucopyranosyl-(1 → 4)]-2-acetamido-2-deoxy-β-Dglucopyranosylentagenic acid 28-O-[2-O-(2E,6R)-2,6-dimethyl-6-hydroxy-2,7-octadienoyl]-β-D-glucopyranosyl(1 → 4)]-[β-D-xylopyranosyl-(1 → 2)]-(6-O-acetyl)-β-Dglucopyranosyl ester. Compound 9 had a molecular formula of C88H139NO44, as evidenced from its HRESIMS data ([M − H]− m/z 1912.8570, calc. for 1912.8592) and its NMR spectroscopic data. The 1H and 13 C NMR signals of aglycon moiety of 9 were superimposable on those of 8. Moreover, for the sugar portion of 9 in comparison with that of 8, the occurrence of an additional pentose was observed, which was assigned to an api unit (δC 111.2, 77.9, 80.5, 75.0 and 65.4). The assignments of the signals of sugars were further allowed by extensive 1D and

44

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2D NMR analysis. The linkage site of the api was confirmed by the HMBC correlation between the api H-1 (δH 5.26) and xyl II C-3 (δC 85.3). In addition, the down-field shift of the proton H-3 (δH 5.00) of the glc III unit in 9, suggesting that the MT group linked at C-3 of the glc III unit. This was confirmed by the HMBC cross-peak observed between H-3 (δH 5.00) of the glc III and the carbonyl carbon (δC 169.6) of the MT group. On the basis of all these studies, the structure of compound 9 was established as 3β-O-β-D-xylopyranosyl-(1 → 3)-α-Larabinopyranosyl-(1 → 6)-[β-D-glucopyranosyl-(1 → 4)]-2acetamido-2-deoxy-β-D-glucopyranosylentagenic acid 28-Oβ-D-apiofuranosyl-(1 → 3)-β-D-xylopyranosyl-(1 → 2)-[(3O-(2E,6R)-2,6-dimethyl-6-hydroxy-2,7-octadienoyl)-β-Dglucopyranosyl-(1 → 4)]-(6-O-acetyl)-β-D-glucopyranosyl ester. Compound 10 had a molecular formula of C88H139NO44, as evidenced from its HRESIMS data ([M − H]− m/z 1912.8570, calc. for 1912.8592) and its NMR spectroscopic data. Acid hydrolysis of 10 afforded D-glucose, D-xylose, D-glcNAc, L-ara and D-apiose through HPLC analysis. Analysis of the NMR data and comparison with those of 9 showed that they possess the same saccharide chains at C-3 and C-28, while the two compounds are based on different aglycons. The main difference was the absence of one hydroxyl group linked at C15 positions of the aglycon (δH 1.30, 1.80 / δC 36.8 in 10 versus δH 3.84 / δC 69.1 in 9), implying the aglycon of 10 was echinocystic acid, a common triterpene aglycon of triterpenoid saponins [19]. Thus, saponin 10 was elucidated as 3β-O-β-D-xylopyranosyl-(1 → 3)-α-L-arabinopyranosyl(1 → 6)-[β-D-glucopyranosyl-(1 → 4)]-2-acetamido-2-deoxyβ-D-glucopyranosylechinocystic acid 28-O-β-D-apiofuranosyl(1 → 3)-β-D -xylopyranosyl-(1 → 2)-[(3-O-(2E,6R)-2,6dimethyl-6-hydroxy-2,7-octadienoyl)-β-D-glucopyranosyl(1 → 4)]-(6-O-acetyl)-β-D-glucopyranosyl ester. Compound 11 had the molecular formula C83H139NO38, as deduced from the HRESIMS ([M − H]− , m/z 1748.8271, calc. for 1748.8262). According to the NMR analysis and the basic hydrolysis results, 11 possessed almost the same structure than 8 except a difference at the C-15 and C-16 positions of the aglycon. The NMR data showed signals at δH 1.67 (H-15)/δC 27.0 (C-15) and δH 1.67 (H-16)/δC 23.7 (C-16) instead of δH 3.85 (H-15)/δC 69.0 (C-15) and δH 4.28 (H-16)/δC 79.3 (C-16) of the entagenic acid of compound 8. These data suggested the identification of oleanolic acid as the aglycon [21]. The structure of compound 11 was thus established as 3β-O-βD -xylopyranosyl-(1 → 3)-α-L -arabinopyranosyl-(1 → 6)[β-D-glucopyranosyl-(1 → 4)]-2-acetamido-2-deoxy-β-D glucopyranosyloleanolic acid 28-O-[2-O-(2E,6R)-2,6dimethyl-6-hydroxy-2,7-octadienoyl]-β-D-glucopyranosyl-(1 → 4)][β-D-xylopyranosyl-(1 → 2)]-(6-O-acetyl)-β-D-glucopyranosyl eater. 3.2. Bioactivity To explain the anti-inflammatory effects of “Guo Gang Long”, the activities of the compounds (1, 4–5, 7–9, 11, and entagenic acid (12)) on the inflammatory response were investigated in vitro. The anti-inflammatory effects were evaluated by the inhibitory activity on LPS-stimulated inflammation factor-releases (NO, TNF-α, IL-1β, IL-6 and IL-8) of RAW

264.7 in vitro [22]. Because triterpenoids isolated from the genus Entada have been reported to inhibit cell proliferation and induce apoptosis in cancer cells [16,19], we also examined the cytotoxic effect of compounds on RAW 264.7 cells. Compounds 1, 4–5 and entagenic acid reduced significantly the production of NO in LPS-induced RAW 264.7 cells at the tested concentrations (15, 30, and 60 μM) with IC50 values of 25.08, 20.13, 37.72, and 23.48 μM, respectively, which indicated that the monosaccharides linked to C3-OH of entagenic acid may potently reduce the anti-inflammatory activity, and the monosaccharides located at C-28 of entagenic acid had no obvious influence on the anti-inflammatory activity. Compounds 1, 4 and entagenic acid could also significantly inhibit the levels of proinflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-8) at the tested doses (Fig. 2). Compounds 7–8 and 11 showed the inhibition of NO production at the concentrations of 60 μM, due to their cellular toxicity on RAW 264.7 cells (Table 4). It should be noted that, the cell viabilities of compounds 5, 7–9 and 11 at the concentration of 60 μM (Table 4), demonstrating that the introduction of the sugar chain (β-D-apiofuranosyl-(1 → 3)-β-D-xylopyranosyl(1 → 2)-[β-D-glucopyranosyl-(1 → 4)]-(6-O-acetyl)-β-Dglucopyranosyl) to C-28 of aglycon could favor cytotoxic activity, and the presence of terminal apiose played an important role in cytotoxic activity. Acknowledgments This work was financially supported by the Hubei Province Natural Science Foundation of China (No. 2013CFA013) and the Fundamental Research Funds for the Central Universities (No. CZW14044). Appendix A. Supplementary data The HRESIMS, 1D and 2D NMR spectra of 1–11 are available in the Supporting information. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote. 2015.03.001. References [1] Nanjing University of Traditional Chinese Medicine. Dictionary of Chinese Materia Medica. Shanghai: Shanghai Science and Technology Publishing House; 2006. [2] Chinese Materia Medica Editorial Committee. Chinese Materia Medica. Shanghai: Scientific and Technical Press; 1999. [3] Okada Y, Shibata S, Javellana AMJ, Kamo O. Entada saponins (ES) II and IV from the bark of Entada phaseollides. Chem Pharm Bull 1988;36:1264–9. [4] Okada Y, Shibata S, Ikekawa T, Javellana AMJ, Kamo O. Entada saponin-III, a saponin isolated from the bark of Entada phaseoloides. Phytochemistry 1987;26:2789–96. [5] Xiong H, Mei Z, Yang G, Mo S, Yang X, Zhang P, et al. Triterpene saponins from Entada phaseoloides. Helv Chim Acta 2013;96:1579–89. [6] Iwamoto Y, Sugimoto S, Harinantenaina L, Matsunami K, Otsuka H. Entadosides A–D, triterpene saponins and a glucoside of the sulphurcontaining amide from the kernel nuts of Entada phaseoloides (L.) Merrill. J Nat Med 2012;66:321–8. [7] Chen L, Zhang Y, Ding G, Ba M, Guo Y, Zou Z. Two new derivatives of 2,5dihydroxyphenylacetic acid from the kernel of Entada phaseoloides. Molecules 2013;18:1477–82. [8] Dong Y, Shi H, Yang H, Peng Y, Wang M, Li X. Antioxidant phenolic compounds from the stems of Entada phaseoloides. Chem Biodivers 2012; 9:68–79. [9] Singh O, Ali M, Akhtar N. Phenolic acid glucosides from the seeds of Entada phaseoloides Merill. J Asian Nat Prod Res 2011;13:682–7.

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