Oleanolic acid and hederagenin glycosides from Weigela stelzneri

Phytochemistry xxx (2016) xxx–xxx

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Oleanolic acid and hederagenin glycosides from Weigela stelzneri Abdelmalek Rezgui a, Anne-Claire Mitaine-Offer a, Tomofumi Miyamoto b, Chiaki Tanaka b, Stéphanie Delemasure c, Patrick Dutartre c, Marie-Aleth Lacaille-Dubois a,⇑ a

Laboratoire de Pharmacognosie, EA 4267, FDE, Université de Bourgogne Franche-Comté, UFR Sciences de Santé, 7, Bd. Jeanne d’Arc, BP 87900, 21079 Dijon Cedex, France Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan c Cohiro, UFR Sciences de Santé, 7, Bd. Jeanne d’Arc, BP 87900, 21079 Dijon Cedex, France b

a r t i c l e

i n f o

Article history: Received 20 October 2015 Received in revised form 9 December 2015 Accepted 22 December 2015 Available online xxxx Keywords: Weigela stelzneri (Caprifoliaceae) Oleanolic acid Hederagenin Glycoside Cytotoxicity Anti-inflammatory NMR

a b s t r a c t Four previously undescribed and one known oleanolic acid glycosides were isolated from the roots of Weigela stelzneri, and one previously undescribed and three known hederagenin glycosides were isolated from the leaves. Their structures were elucidated mainly by 2D NMR spectroscopic analysis and mass spectrometry as 3-O-b-D-glucopyranosyl-(1 ? 2)-[b-D-xylopyranosyl-(1 ? 4)]-b-D-xylopyranosyl-(1 ? 4)-b-D-xylopyranosyl-(1 ? 3)-a-L-rhamnopyranosyl-(1 ? 2)-a-L-arabinopyranosyloleanolic acid, 3-O-bD-glucopyranosyl-(1 ? 2)-[b-D-xylopyranosyl-(1 ? 4)]-b-D-xylopyranosyl-(1 ? 4)-b-D-xylopyranosyl(1 ? 3)-a-L-rhamnopyranosyl-(1 ? 2)-b-D-xylopyranosyloleanolic acid, 3-O-b-D-glucopyranosyl-(1 ? 2)-[b-D-glucopyranosyl-(1 ? 4)]-b-D-xylopyranosyl-(1 ? 4)-b-D-xylopyranosyl-(1 ? 3)-a-L-rhamnopyranosyl-(1 ? 2)-b-D-xylopyranosyloleanolic acid, 3-O-b-D-glucopyranosyl-(1 ? 2)-[b-D-xylopyranosyl(1 ? 4)]-b-D-xylopyranosyl-(1 ? 4)-b-D-xylopyranosyl-(1 ? 3)-a-L-rhamnopyranosyl-(1 ? 2)-a-L-arabinopyranosyloleanolic acid 28-O-b-D-glucopyranosyl-(1 ? 6)-b-D-glucopyranosyl ester, and 3-O-b-D28-O-b-D-xylopyranosyl-(1 ? 6)-[a-Lglucopyranosyl-(1 ? 2)-a-L-arabinopyranosylhederagenin rhamnopyranosyl-(1 ? 2)]-b-D-glucopyranosyl ester. The majority of the isolated compounds were evaluated for their cytotoxicity against two tumor cell lines (SW480 and EMT-6), and for their anti-inflammatory activity. The compounds 3-O-b-D-glucopyranosyl-(1 ? 2)-[b-D-xylopyranosyl-(1 ? 4)]-b-D-xylopyranosyl-(1 ? 4)-b-D-xylopyranosyl-(1 ? 3)-a-Lrhamnopyranosyl-(1 ? 2)-a-L-arabinopyranosyloleanolic acid and 3-O-b-D-glucopyranosyl-(1 ? 2)-[b-Dxylopyranosyl-(1 ? 4)]-b-D-xylopyranosyl-(1 ? 4)-b-D-xylopyranosyl-(1 ? 3)-a-L-rhamnopyranosyl(1 ? 2)-b-D-xylopyranosyloleanolic acid exhibited the strongest cytotoxicity on both cancer cell lines. They revealed a 50% significant inhibitory effect of the IL-1b production by PBMCs stimulated with LPS at a concentration inducing a very low toxicity of 23% and 28%, respectively. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction Historically, the genus Weigela or Weigelia contains ten species from Japan, China, Korea and Manchuria but many cultivars were produced, over 200 (FOBS, 2015). Among the few cultivar widely cultivated, Weigela stelzneri is a hybrid shrub of medium size with tender pink flowers and deciduous green leaves. This genus used to belong to the Caprifoliaceae family, subfamily Diervilloideae according to the APGIII classification (The Angiosperm phylogeny group, 2009). As the species Weigela hortensis (Murayama et al., 2003), and Weigela subsessilis (Won et al., 2015) were already described as a source of oleanane-type glycosides, we decided to complete the chemotaxonomic data about saponins from the

⇑ Corresponding author. E-mail address: [email protected] (M.-A. Lacaille-Dubois).

genus Weigela and thus the Caprifoliaceae family, by the phytochemical study of W. stelzneri. In this paper, we report the isolation and the characterization of nine oleanane-type saponins, five of them being previously undescribed. Furthermore, the antiproliferative activity of the isolated compounds against two cancer cell lines (SW480 and EMT-6) was assessed using the XTT method compared to positive controls, methotrexate, and etoposide. In addition, the study of the modulation of the production of the pro-inflammatory cytokine, interleukin IL-1b by PBMCs stimulated with LPS, was performed. 2. Results and discussion An aqueous ethanolic (3:7) root extract of W. stelzneri was subjected to multiple chromatographic steps over silica gel yielding compounds 1–4 (Fig. 1) and the known 3-O-b-D-xylopyranosyl(1 ? 3)-a-L-rhamnopyranosyl-(1 ? 2)-a-L-arabinopyranosylolean-

http://dx.doi.org/10.1016/j.phytochem.2015.12.016 0031-9422/Ó 2016 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Rezgui, A., et al. Oleanolic acid and hederagenin glycosides from Weigela stelzneri. Phytochemistry (2016), http://dx.doi. org/10.1016/j.phytochem.2015.12.016

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A. Rezgui et al. / Phytochemistry xxx (2016) xxx–xxx

Fig. 1. Structures of compounds 1–5.

olic acid (6) (Wang et al., 2011). From the aqueous ethanolic (3:7) extract of the leaves, compound 5 was isolated (Fig. 1) with the known 3-O-a-L-arabinopyranosylhederagenin 28-O-b-D-xylopyranosyl-(1 ? 6)-[a-L-rhamnopyranosyl-(1 ? 2)]-b-D-glucopyranosyl

ester (loniceroside A) (7) (Won et al., 2015), 3-O-a-L-arabinopyranosylhederagenin 28-O-b-D-glucopyranosyl-(1 ? 6)-[a-Lrhamnopyranosyl-(1 ? 2)]-b-D-glucopyranosyl ester (loniceroside K) (8) (Won et al., 2015), and 3-O-a-L-arabinopyranosylhedera-

Please cite this article in press as: Rezgui, A., et al. Oleanolic acid and hederagenin glycosides from Weigela stelzneri. Phytochemistry (2016), http://dx.doi. org/10.1016/j.phytochem.2015.12.016

A. Rezgui et al. / Phytochemistry xxx (2016) xxx–xxx

genin 28-O-b-D-glucopyranosyl-(1 ? 6)-b-D-glucopyranosyl ester (akebia saponin D) (9) (Ren et al., 2008) (Fig. 2). The elucidation of their structures was performed mainly by 600 MHz 2D NMR analysis (1H–1H COSY, TOCSY, NOESY, HSQC, HMBC), in combination with mass spectrometry by ESIMS (positive-ion mode) and FABMS (negative-ion mode). Compounds 1–9 were isolated as white amorphous powders. The monosaccharides obtained by acid hydrolysis of 1–5 were identified by comparison on TLC with authentic samples as glucose (Glc), xylose (Xyl), arabinose (Ara) and rhamnose (Rha) for 1, 4, 5 and Glc, Xyl, Rha for 2, 3. The absolute configurations of the sugars were determined to be D for Glc and Xyl, and L for Ara and Rha by GC analysis according to a method previously described (Hara et al., 1987). The HRESIMS of compound 1 established its molecular formula as C62H100O28. Its ESIMS (positive-ion mode) displayed a quasimolecular ion peak [M+Na]+ at m/z 1315 indicating a molecular weight of 1292. The 1H NMR spectrum of the aglycon part of 1 displayed signals assignable to seven angular methyl groups at dH 0.72, 0.86, 0.88, 0.91, 0.99, 1.17, and 1.21 (3H s, each), one olefinic proton at dH 5.39 (t-like) (H-12), and one oxygen-bearing methine protons at dH 3.18 (dd, J = 12.1, 3.1 Hz) (H-3). The HMBC spectrum showed 2 JH-C and 3JH-C couplings from the methyl protons (CH3 23, 24, 25, 26, 27, 29 and 30), which allowed the assignments of most carbons and protons of this aglycon. The deshielded chemical shift of C-28

3

observed at dC 179.6 suggested the presence of a carbonyl group of a carboxylic acid function. By comparison of these data with those of the literature, this genin was identified as oleanolic acid (Zheng et al., 2004). The 1H NMR spectrum of 1 in the sugar region displayed signals of six anomeric protons at dH 4.75 (d, J = 6.2 Hz), 4.77 (d, J = 8.1 Hz), 5.09 (d, J = 7.1 Hz), 5.12 (d, J = 7.4 Hz), 5.66 (d, J = 5.2 Hz), 5.98 (br s), which gave correlation, in the HSQC spectrum, with six anomeric carbon signals at dC 104.8, 102.1, 103.0, 105.8, 102.8 and 100.8, respectively. The ring protons of the monosaccharide residues were assigned starting from the readily identifiable anomeric protons by means of the 1H–1H COSY, TOCSY, HSQC, HMBC experiments. The relatively large 3JH-1,H-2 value of the Glc, Xyl and Ara (5.2–8.1 Hz) in their pyranose form indicated a b anomeric orientation for Glc and Xyl, and an a anomeric orientation for Ara. The large 1JH-1,C-1 value of the Rha (166 Hz) confirmed that the anomeric proton was equatorial (a-pyranoid anomeric form). Units of one b-D-glucopyranosyl, three b-D-xylopyranosyl, one a-L-arabinopyranosyl, and one a-L-rhamnopyranosyl were thus identified (Table 2). The linkages between these six sugars were established using mainly HMBC and NOESY spectra: the HMBC correlation at dH 4.75 (d, J = 6.2 Hz) (Ara-1)/dC 88.2 (C-3) and the NOESY correlation at dH 4.75 (d, J = 6.2 Hz) (Ara-1)/dH 3.18 (dd, J = 12.1, 3.1 Hz) (C-3) confirmed the glycosidic linkage of the Ara at the C-3 position of the aglycon. The HMBC cross-peak at dH 4.40 (dd, J = 6.9, 6.2 Hz) (Ara-2)/dC 100.8 (Rha-1) and the NOESY cross-peak at dH 4.40 (dd, J = 6.9, 6.2 Hz) (Ara-2)/dH 5.98 (br s) (Rha-1), proved the (1 ? 2) linkage between Rha and Ara. The correlation at dH 5.12 (d, J = 7.4 Hz) (Xyl I-1)/dC 81.5 (Rha-3) in the HMBC spectrum and at dH 5.12 (d, J = 7.4 Hz) (Xyl I-1)/dH 4.55 (dd, J = 9.0, 2.5 Hz) (Rha-3) in the NOESY spectrum, confirmed the (1 ? 3) linkage between Xyl I and Rha. The HMBC cross-peak at dH 4.77 (d, J = 8.1 Hz) (Xyl II-1)/dC 77.0 (Xyl I-4) and NOESY crosspeak at dH 4.77 (d, J = 8.1 Hz) (Xyl II-1)/dH 4.10 (Xyl I-4) proved the (1 ? 4) linkage between Xyl II and Xyl I. Finally, the structure analysis of the terminal sequence b-D-glucopyranosyl-(1 ? 2)-[b? 4)]-b-D-xylopyranosyl was based upon the HMBC cross-peak at dH 4.30 (Xyl II-4)/dC 103.0 (Xyl III-1) and the NOESY correlation at dH 5.66 (d, J = 5.2 Hz) (Glc I-1)/dH 3.89 (dd, J = 8.6, 8.1 Hz) (Xyl II-2). On the basis of the above results, the structure of 1 was elucidated as 3-O-b-D-glucopyranosyl-(1 ? 2)-[b-D-xylopyranosyl(1 ? 4)]-b-D-xylopyranosyl-(1 ? 4)-b-D-xylopyranosyl-(1 ? 3)-aD-xylopyranosyl-(1

? 2)-a-L-arabinopyranosyloleanolic acid. The HRESIMS of compound 2 established its molecular formula as C62H100O28, as compound 1. Its ESIMS (positive-ion mode) showed the same quasimolecular ion peak [M+Na]+ at m/z 1315 indicating a molecular weight of 1292. All the NMR signals observed for 2 was comparable to those of 1, excepted the sugar linked at the C-3 position of the aglycon. Its ring protons were assigned starting from anomeric protons at dH 4.68 (d, J = 6.4 Hz) by means of the 1H–1H COSY, TOCSY, HSQC, HMBC experiments and it was thus identified as a b-D-xylopyranosyl moiety. The NOESY correlation at dH 4.68 (d, J = 6.4 Hz) (Xyl I-1)/dH 3.23 (dd, J = 11.9, 3.8 Hz) (C-3) and at dH 4.08 (Xyl I-2)/dH 6.31 (br s) (Rha-1), and the HMBC correlation at dH 4.08 (Xyl I-2)/dC 101.0 (Rha-1), revealed the a-L-rhamnopyranosyl-(1 ? 2)-b-D-xylopyranosyloleanolic acid sequence. The structure of compound 2 was thus established as 3-O-b-Dglucopyranosyl-(1 ? 2)-[b-D-xylopyranosyl-(1 ? 4)]-b-D-xylopyranosyl-(1 ? 4)-b-D-xylopyranosyl-(1 ? 3)-a-L-rhamnopyranosyl(1 ? 2)- b-D-xylopyranosyloleanolic acid. The HRESIMS of compound 3 established its molecular formula as C63H102O29. Its FABMS (negative-ion mode) displayed a quasimolecular ion peak [M
H]
at m/z 1321 indicating a molecular weight of 1322, and another fragment ion peak at m/z 1159 [(M
H)
162]
corresponded to the loss of one hexosyl moiety. L-rhamnopyranosyl-(1

Fig. 2. Structures of compounds 7–9.

Please cite this article in press as: Rezgui, A., et al. Oleanolic acid and hederagenin glycosides from Weigela stelzneri. Phytochemistry (2016), http://dx.doi. org/10.1016/j.phytochem.2015.12.016

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A. Rezgui et al. / Phytochemistry xxx (2016) xxx–xxx

six anomeric carbon signals at dC 104.5, 102.4, 104.3, 102.7, 100.8, 95.2, respectively. Two more anomeric protons were overlapped at dH 5.13 (d, J = 6.9 Hz), which correlated in the HSQC spectrum with two anomeric carbons at dC 103.1 and 106.0. Complete assignments of the resonances of each sugar were achieved by extensive 2D NMR analysis (COSY, TOCSY, HSQC, and HMBC). Units of three b-D-xylopyranosyl, three b-D-glucopyranosyl, one a-L-arabinopyranosyl, and one a-L-rhamnopyranosyl were thus identified. The 3-Oheterosidic linkage and the structure of the oligosaccharidic chain were established using mainly the NOESY spectrum: a cross-peak at dH 4.76 (d, J = 6.4 Hz) (Ara-1)/dH 3.21 (dd, J = 11.9, 3.7 Hz) (C-3) confirmed the linkage of Ara at the C-3 position of the aglycon. The NOESY cross-peaks at dH 4.43 (dd, J = 6.7, 6.4 Hz) (Ara-2)/dH 6.02 (br s) (Rha-1), dH 5.13 (d, J = 6.9 Hz) (Xyl I-1)/dH 4.57 (Rha3), dH 4.78 (d, J = 7.9 Hz) (Xyl II-1)/dH 4.12 (Xyl I-4), and dH 5.13 (d, J = 6.9 Hz) (Xyl III-1)/dH 4.32 (Xyl II-4) and the HMBC correlation at dH 3.90 (dd, J = 8.8, 7.9 Hz) (Xyl II-2) dC 102.7 (Glc I-1), allowed to establish the structure of the prosapogenin as 3-O-b-D-glucopyranosyl-(1 ? 2)-[b- D -xylopyranosyl-(1 ? 4)]-b- D -xylopyranosyl(1 ? 4)-b-D-xylopyranosyl-(1 ? 3)-a-L-rhamnopyranosyl-(1 ? 2)a-L-arabinopyranosyloleanolic acid. Moreover, the C-28 position of the aglycon at dC 176.5 was esterified by an oligosaccharidic chain which was analyzed on the basis of NOESY and HMBC spectra: the HMBC cross-peak at dH 6.09 (d, J = 8.1 Hz) (Glc II-1)/dC 176.5 (C-28), proved the esterification of C-28 position by the Glc II residue. The HMBC correlation at dH 4.88 (d, J = 7.9 Hz) (Glc III-1)/dC 68.6 (Glc II-6) and the NOESY cross-peak at dH 4.88 d, J = 7.9 Hz) (Glc III-1)/dH 4.59 (Glc II-6) confirmed the linkage (1 ? 6) between Glc III and Glc II. According to the above results, the structure of 4 was elucidated as 3-O-b-D-glucopyranosyl-(1 ? 2)-[b-D-xylopyranosyl-(1 ? 4)]-b-

The NMR analysis of 3 led to the identification of the same partial glycoside as 2, b-D-xylopyranosyl-(1 ? 4)-b-D-xylopyranosyl(1 ? 3)-a-L-rhamnopyranosyl-(1 ? 2)-b-D-xylopyranosyloleanolic acid. The difference between the two compounds was located in the terminal part of the oligosaccharidic chain. In the HMBC spectrum, a correlation between dH 5.22 (d, J = 7.1 Hz) (Glc II-1) and dC 74.0 (Xyl III-2) showed a substitution of the position Xyl III-2 by a b-D-glucopyranosyl moiety (Glc II). Another HMBC correlation between dH 5.42 (d, J = 5.0 Hz) (Glc I-1) and dC 81.1 (Xyl III-4) showed a substitution of the position Xyl III-4 by a b-D-glucopyranosyl moiety (Glc I) instead of a b-D-xylopyranosyl moiety (Xyl IV) in compound 2. The structure of compound 3 was thus established as 3-O-b-Dglucopyranosyl-(1 ? 2)-[b-D-glucopyranosyl-(1 ? 4)]-b-D-xylopyranosyl-(1 ? 4)-b-D-xylopyranosyl-(1 ? 3)-a-L-rhamnopyranosyl(1 ? 2)-b-D-xylopyranosyloleanolic acid. The HRESIMS of compound 4 established its molecular formula as C74H120O38. Its ESIMS (positive-ion mode) displayed a quasimolecular ion peak [M+Na]+ at m/z 1639 indicating a molecular weight of 1616. Its FABMS (negative-ion mode) showed fragment ion peaks at m/z 1291 [(M
H)
162
162]
, 1159 [(M
H)
162
162
132]
, 997 [(M
H)
162
162
132
162]
, 865 [(M
H)
162
162
132
162
132]
, 733 [(M
H)
162
162
132
162
132
132]
, 587 [(M
H)
162
162
132
162
132
132
146]
, 455 [(M
H)
162
162
132
162
132
132
146
132]
, due to the successive loss of three hexosyl, four pentosyl and one deoxyhexosyl moieties. As in compounds 1–3, the aglycon of 4 was identified by 2D NMR analysis as oleanolic acid. A signal at dC 176.5 suggested an esterification with an osidic part at the C-28 position instead of a free carboxylic group in 1–3. The 1H NMR spectrum of 4 displayed signals of six anomeric protons at dH 4.76 (d, J = 6.4 Hz), 4.78 (d, J = 7.9 Hz), 4.88 (d, J = 7.9 Hz), 5.72 (d, J = 5.2 Hz), 6.02 (br s), 6.09 (d, J = 8.1 Hz), which gave correlations, in the HSQC spectrum, with

? 4)-b-D-xylopyranosyl-(1 ? 3)-a-L-rhamnopyranosyl-(1 ? 2)-a-L-arabinopyranosyloleanolic acid 28-O-b-Dglucopyranosyl-(1 ? 6)-b-D-glucopyranosyl ester.

D-xylopyranosyl-(1

Table 1 C NMR and 1H NMR Data of the aglycons of compounds 1–5 in Pyridine-d5 (d ppm).

13

1

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

2

3

4

5

dC

dH

dC

dH

dC

dH

dC

dH

dC

dH

38.2 26.1 88.2 39.1 55.5 18.0 32.8 39.3 47.7 36.6 23.3 121.9 144.6 41.5 27.8 23.2 46.5 41.7 46.2 30.6 33.6 32.5 27.7 16.6 15.1 16.6 25.7 179.6 32.9 23.4

0.84, 1.40 1.73, 1.96 3.18 dd (12.1, 3.1) – 0.72 1.18, 1.42 1.18, 1.36 – 1.54 – 1.80, 1.88 5.39 t-like – – 1.12, 2.06 1.90, 2.01 – 3.18 dd (12.1, 3.1) 1.18, 1.70 – nd 1.68, 1.88 1.17 s 0.99 s 0.72 s 0.88 s 1.21 s – 0.86 s 0.91 s

38.4 26.0 88.3 39.3 55.7 18.1 32.5 39.2 47.7 36.6 23.4 122.0 144.6 41.6 27.9 23.2 46.5 41.8 46.2 30.6 33.6 32.5 27.8 16.8 15.2 17.1 25.8 179.8 33.0 23.5

0.88, 1.42 1.72, 1.94 3.23 dd (11.9, 3.8) – 0.75 1.18, 1.44 1.36, nd – 1.54 – 1.82, 1.89 5.40 t-like – – 1.11, 2.09 1.91, 2.02 – 3.19 dd (14.0, 3.0) 1.19, 1.71 – nd 1.70, 1.90 1.25 s 1.08 s 0.74 s 0.88 s 1.21 s – 0.86 s 0.92 s

38.7 26.0 88.3 39.3 55.7 18.2 32.8 39.2 47.8 36.8 23.4 122.1 144.6 41.7 27.8 23.3 46.6 41.7 46.3 30.5 34.0 32.8 27.8 16.9 15.2 17.1 25.9 179.8 33.0 23.5

0.86, 1.43 1.74, 1.94 3.23 dd (11.9, 3.8) – 0.75 1.18, 1.48 1.17, 1.37 – 1.57 – 1.82, 1.90 5.40 t-like – – 1.12, 2.08 1.90, 2.01 – 3.20 dd (14.0, 3.0) 1.19, 1.71 – nd 1.71, 1.92 1.26 s 1.09 s 0.74 s 0.89 s 1.22 s – 0.86 s 0.92 s

38.6 26.1 88.5 39.5 55.7 18.0 32.6 39.2 47.7 36.6 23.4 122.4 143.9 41.8 28.0 23.0 46.7 41.3 45.9 30.3 33.6 32.0 27.8 16.7 15.1 17.1 25.7 176.5 32.7 23.3

0.85, 1.42 1.67, 1.97 3.21 dd (11.9, 3.7) – 0.71 br d (12.6) 1.17, 1.37 1.22, 1.35 – 1.54 – 1.82, 1.90 5.35 t-like – – 1.10, 2.18 1.90, 2.02 – 3.08 dd (12.5, 3.1) 1.15, 1.66 – nd 1.64, 1.81 1.24 s 1.01 s 0.77 s 0.97 s 1.18 s – 0.82 s 0.80 s

38.2 25.9 81.5 42.8 47.6 18.0 32.5 39.7 48.0 36.4 23.1 122.0 143.2 41.9 28.3 23.2 46.6 41.2 46.3 30.3 33.0 32.0 63.5 13.1 15.8 17.2 25.8 176.8 33.0 23.8

0.82, 1.48 1.90, 2.05 4.06 m – 1.51 d (11.4) 1.30, 1.62 nd – 1.65 – 1.83, 1.92 5.37 t-like – – 1.42, 1.99 1.92, 2.03 – 3.05 dd (12.1, 3.0) 1.11, 1.66 – 1.54, 1.60 1.69, 1.76 3.55 d (10.9), 4.08 0.88 s 0.92 s 1.02 s 1.12 s – 0.77 s 0.84 s

Overlapped proton signals are reported without designated multiplicity. Nd: not determined.

Please cite this article in press as: Rezgui, A., et al. Oleanolic acid and hederagenin glycosides from Weigela stelzneri. Phytochemistry (2016), http://dx.doi. org/10.1016/j.phytochem.2015.12.016

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A. Rezgui et al. / Phytochemistry xxx (2016) xxx–xxx

The HRESIMS of compound 5 established its molecular formula as C58H94O26. Its ESIMS (positive-ion mode) displayed a quasimolecular ion peak [M+Na]+ at m/z 1229 indicating a molecular weight of 1206. The HSQC spectrum allowed the assignments of 1H and 13C signals of a classical oleanane-type aglycon with six methyl groups as singlets at dH 0.77/dC 33.0, dH 0.84/dC 23.8, dH 0.88/dC 13.1, dH 0.92/ dC 15.8, dH 1.02/dC 17.2, dH 1.12/dC 25.8, an olefinic proton at dH 5.37 (t-like)/dC 122.0 (CH-12), one oxygen-bearing methine proton

at dH 4.06 (m)/dC 81.5 (CH-3), and one primary alcoholic function at dH 3.55 (d, J = 10.9 Hz), 4.08/dC 63.5 (CH2-23). A HMBC cross-peak at dH 0.88 (s) (H3-24)/dC 63.5 (C-23) and a NOESY cross-peak at dH 4.06 (m) (H-3a)/dH 3.55 (d, J = 10.9 Hz) (H-23), suggested the location of the primary alcoholic function at C-23. These data allowed the identification of the genin as (3b)-3,23-dihydroxy-olean-12en-28-oic acid, known as hederagenin (Table 1) (Wang et al., 2011). The 1H NMR spectrum of 5 showed signals of five anomeric protons at dH 4.76 (d, J = 7.4 Hz), 5.09 (d, J = 5.5 Hz), 5.13 (d, J = 5.5 Hz),

Table 2 C NMR and 1H NMR Data of the sugar moieties of compounds 1–5 in Pyridine-d5 (d ppm).

13

No.

1

2

dC

dH

Ara-1 2

104.8 75.1

3 4 5

dC

3 dC

4 dH

5

dC

dH

dC

dH

4.75 d (6.2) 4.40 dd (6.9, 6.2)

104.5 75.0

103.0 79.2

5.09 d (5.5) 4.51

73.3 68.3 64.5

4.18 dd (6.9, 1.2) 4.24 3.75, 4.24

73.7 68.8 64.6

4.76 d (6.4) 4.43 dd (6.7, 6.4) 4.19 4.22 3.76, 4.26

72.1 68.0 64.2

Rha-1 2 3 4 5

100.8 71.1 81.5 72.0 69.3

5.98 4.75 4.55 4.34 4.46

100.8 71.0 71.4 72.9 69.2

4.06 4.26 3.66 dd (11.7, 1.9), 4.18 6.29 br s 4.70 br s 4.44 4.24 4.38

6 Xyl I-1 2 3 4 5

17.9 105.8 74.5 75.0 77.0 63.9

17.9 104.5 73.8 76.9 70.3 66.1

1.65 4.76 3.86 4.06 4.06 3.53

Xyl II-1 2

104.5 75.1 77.0 70.5 77.4 61.4 94.1 75.1 78.0 70.0 76.8 68.1

5.13 d (5.5) 3.98 4.10 4.12 3.75 m 4.20, 4.37 5.98 d (7.9) 4.24 4.15 4.23 3.99 4.18, 4.51

101.0 71.2 81.8 72.4 69.3

6.31 br s 4.85 br s 4.62 4.39 t (9.5) 4.61

101.8 71.1 81.9 72.1 69.3

6.32 br s 4.86 br s 4.63 4.40 4.62

100.8 71.3 81.7 72.1 69.2

1.46 d (6.0) 5.12 d (7.4) 3.96 4.02 4.10 3.49 t (11.1), 4.27

18.1 105.6 78.7 76.8 nd 64.6

18.2 105.6 78.7 77.0 68.7 65.9

4.77 d (8.1) 3.89 dd (8.6, 8.1)

106.1 74.7

106.1 74.8

1.54 4.69 4.10 4.07 4.24 3.64 4.40 5.18 4.00

17.8 106.0 74.4 75.0 77.0 64.0

102.1 74.2

1.54 d (6.0) 4.68 d (6.4) 4.08 4.07 nd 3.63 t (12.0), 4.54 dd (12.0, 3.6) 5.17 d (7.4) 3.98

3 4 5

73.3 79.6 64.4

75.3 77.1 64.4

4.03 4.13 3.54 t (11.2), 4.30

75.2 77.4 64.1

Xyl III1 2

103.0

4.23 4.30 3.62 t (11.7), 4.52 dd (11.7, 3.4) 5.09 d (7.1)

102.4

4.78 d (7.6)

102.3

74.0

3.94

73.6

3.90 dd (8.6, 8.1)

74.0

3 4 5

76.4 70.2 66.1

4.08 4.06 3.60 t (9.3), 4.31

73.8 79.7 66.6

4.27 4.31 3.76, 4.26

nd 81.1 66.1

102.9

5.13 d (7.2)

73.9 77.0 70.4 66.5 102.8 72.7 76.5 70.3 77.0 60.9

3.96 4.07 4.08 3.61, 4.33 5.70 d (5.0) 4.05 4.08 4.02 3.75 4.30, 4.32

Xyl IV1 2 3 4 5 Glc I-1 2 3 4 5 6 Glc II-1 2 3 4 5 6 Glc III1 2

102.8 72.4 76.4 69.9 76.4 60.4

br s br s dd (9.0, 2.5)

dH

dq (9.3, 6.0)

5.66 d (5.2) 4.05 4.06 4.02 3.75 4.27, 4.28

103.7 75.0 77.3 70.2 77.7 61.8 102.7 73.9 76.2 70.0 76.7 60.5

3 4 5 6

4.05 4.12 3.55 t (10.6), 4.30 4.83 d (6.7)

73.5 79.4 64.3 103.1

6.02 4.76 4.57 4.36 4.48 5.7) 1.47 5.13 3.96 4.04 4.12 3.52 4.30 4.78 3.90 7.9) 4.26 4.32 3.64 4.55 5.13

3.93 dd (8.3, 8.1) nd 4.43 3.63 t (11.4), 4.26

74.6

3.97

76.7 70.6 66.2

4.08 4.09 3.61 t (9.8), 4.33

5.42 d (5.0) 4.01 4.09 4.10 3.84 4.14, 4.35 5.22 d (7.1) 4.01 4.08 4.10 3.84 4.28, 4.31

102.7 72.6 76.7 70.0 76.8 60.6 95.2 72.6 77.6 70.2 77.2 68.6 104.3

5.72 d (5.2) 4.07 4.08 4.03 3.74 4.29, 4.31 6.09 d (8.1) 4.07 4.18 4.23 4.00 4.23, 4.59 4.88 d (7.9)

74.3

3.88 dd (8.9, 7.9) 4.10 4.02 3.77 4.16, 4.34

d (6.0) d (6.4)

t (11.4), d (7.8)

102.4 74.3

77.5 71.0 77.6 61.8

br s br s

dq (8.6, d (5.7) d (6.9)

t (11.4),

d (6.0) d (7.4) dd (8.1,7.4)

t (9.8), 4.20

d (7.9) dd (8.8,

t (11.5), d (6.9)

Overlapped proton signals are reported without designated multiplicity. Nd: not determined.

Please cite this article in press as: Rezgui, A., et al. Oleanolic acid and hederagenin glycosides from Weigela stelzneri. Phytochemistry (2016), http://dx.doi. org/10.1016/j.phytochem.2015.12.016

6

A. Rezgui et al. / Phytochemistry xxx (2016) xxx–xxx

5.98 (d, J = 7.9 Hz), and 6.29 (br s), which gave correlations in the HSQC spectrum with five anomeric carbon signals at dC 104.5, 103.0, 104.5, 94.1, and 100.8, respectively. Units of two b-D-glucopyranosyl, one b-D-xylopyranosyl, one a-L-arabinopyranosyl, and one a-L-rhamnopyranosyl moieties were identified (Table 2). A HMBC correlation at dH 5.13 (d, J = 5.5 Hz) (Glc I-1)/dC 79.2 (Ara-2) and a reverse one at dH 4.51 (Ara-2)/dC 104.5 (Glc I-1) Table 3 Concentration of compounds 1–3, 5, 7–9 that inhibits the proliferation of two tumor cell lines by half (IC50).a,b IC50 (lM) Tissue Cell line Compounds 1 2 3, 5, 7–9 a

Colon SW480 (human)

Breast EMT-6 (rat)

3.84 ± 0.43 0.97 ± 0.03 >22.69

3.22 ± 0.02 1.26 ± 0.01 >22.69

The values are means ± standard errors of experiments carried out in triplicates. Positive controls: etoposide 20 lM is IC50 in EMT6 and SW480, and methotrexate 20 lM is IC50 in SW480 and IC80 in EMT6. b

revealed the b-D-glucopyranosyl-(1 ? 2)-a-L-arabinopyranosyl sequence linked to the aglycon. Another correlations in the HMBC spectrum between dH 4.24 (Glc II-2) and dC 100.8 (Rha-1), and between dH 4.76 (d, J = 7.4 Hz) (Xyl-1) and dC 68.1 (Glc II-6), allowed to establish the structure of the sequence which esterified the C-28 position of the hederagenin as b-D-xylopyranosyl-(1 ? 6)[a-L-rhamnopyranosyl-(1 ? 2)]-b-D-glucopyranosyl. This was proved by an HMBC correlation at dH 5.98 (d, J = 7.9 Hz) (Glc II1)/dC 176.8 (C-28) and by the upfield value at dC 94.1 (Glc II-1) in accordance with a classical chemical shift of an anomeric carbon in an ester linkage with the aglycon (Zheng et al., 2004). The structure of 5 was elucidated as 3-O-b-D-glucopyranosyl(1 ? 2)-a-L-arabinopyranosylhederagenin 28-O-b-D-xylopyranosyl(1 ? 6)-[a-L-rhamnopyranosyl-(1 ? 2)]-b-D-glucopyranosyl ester. In the literature, close structures of oleanolic glycosides 1–4, scabiosaponins and hookerosides, were isolated from Scabiosa tschiliensis, Dipsacaceae (Zheng et al., 2004). But now, the species of the Dipsacaceae family are placed in the Caprifoliaceae family as Weigela. In the same way, close hederagenin glycosides, named lonicerosides, were already isolated from Lonicera japonica and

Fig. 3. Effets of compounds 1 (A) and 2 (B) on IL-1b production by LPS-stimulated PBMCs (bar chart) and PBMC viability (line plot). ZVAD was used as an anti-inflammatory reference. ***P < 0.001, as compared to the cells stimulated with LPS.

Please cite this article in press as: Rezgui, A., et al. Oleanolic acid and hederagenin glycosides from Weigela stelzneri. Phytochemistry (2016), http://dx.doi. org/10.1016/j.phytochem.2015.12.016

A. Rezgui et al. / Phytochemistry xxx (2016) xxx–xxx

Weigela subsessilis also from the Caprifoliaceae (Kuroda et al., 2014; Won et al., 2015). The phytochemistry is thus in accordance with the chemotaxonomy. These isolated compounds 1–3, 5, 7–9, monodesmoside and bidesmoside saponins, were tested to evaluate the biological interest. Their cytotoxic activity was determined for 48 h against a human colorectal cancer cell line (SW480), and a mouse mammary cancer cell line (EMT6) by a XTT assay (Jost et al., 1992), in concentrations ranging from 0.8 to 32.3 lM (Table 3). The positive controls were etoposide and methotrexate tested at 20 lM. Etoposide decreased SW480 and EMT-6 cell proliferation by 50%. EMT-6 cancer cells exhibit greater sensitivity to methotrexate than SW480 cancer cells, with a potent reduction in cell viability of 80% and 50%, respectively. All bidemosidic hederagenin derivatives 5, 7–9 were considered inactive on both cell lines (Table 3). The negative effect on the cytotoxicity of the primary alcoholic function at C-23 and/or the ester group at C-28, was already described in the literature (Bang et al., 2005). On the other hand, two monodesmosidic oleanolic acid glycosides 1,2 exerted the strongest cytotoxicity on both cancer cell lines (Table 3). These two saponins had very similar structures but the only difference was the structure of the monosaccharide linked at the C-3 position of the oleanolic acid: a-L-arabinopyranosyl for 1 and b-D-xylopyranosyl for 2. The presence of the sequence a-L-rhamnopyranosyl-(1 ? 2)-a-L-arabinopyranosyloleanolic acid or a-L-rhamnopyranosyl-(1 ? 2)-b-Dxylopyranosyloleanolic, might have a role in the cytotoxicity (Bang et al., 2005). But compound 3, which possessed almost the same structure than 2 except a terminal glucose at C-4 of Xyl III instead of a terminal xylose, was inactive; more glycosides should be tested to propose clear structure/activity relationships. The seven compounds 1–3, 5, 7–9 were then tested for their anti-inflammatory activity by determination of the modulation of the IL-1b production (Mariathasan et al., 2006). Chronic inflammation has been identified as a key determinant in the progression of more than 25% of cancers (Mantovani et al., 2008). IL-1b is a major pro-inflammatory cytokine involved in tumor pathogenesis (Dinarello, 2010). In all experiments conducted in triplicate, LPS induced a strong increase of IL-1b production by human peripheral blood mononuclear cells (PBMCs). An anti-inflammatory reference (Z-VAD, a caspase-1 inhibitor) prevents this production (p < 0.001). Once again, only compounds 1 and 2 modulated the production of IL1-b by the PBMCs stimulated by LPS (Fig. 3). 1 and 2 decreased significantly the IL-1b production by PBMCs stimulated by LPS of approximately 50% at the 2.3 lM for 1 and 0.8 lM for 2 (p < 0.001). At these concentrations, compounds 1 and 2 induce a very low toxicity of 23% and 28%, respectively, on PBMCs treated for 24 h. 3. Experimental 3.1. General experimental procedures Optical rotations values were recorded on a AA-OR automatic polarimeter. The 1D and 2D NMR spectra (1H and 13C NMR, 1H–1H COSY, TOCSY, NOESY, HSQC and HMBC) were performed using a UNITY-600 spectrometer at the operating frequency of 600 MHz on a Varian INOVA 600 instrument equipped with a SUN 4 L-X computer system (600 MHz for 1H and 150 MHz for 13C spectra). Conventional pulse sequences were used for COSY, HSQC, and HMBC spectra. TOCSY spectra were acquired using the standard MLEV17 spin-locking sequence and a 90 ms mixing time. The mixing time in the NOESY experiment was set to 500 ms. The carbon type (CH3, CH2, CH) was determined by DEPT experiments. Chemical shifts are reported as d values (ppm), referenced with respect to the residual solvent signal of C5D5N, and coupling constants (J) were measured in Hz. The samples were solubilized in pyridine-d5. HRE-

7

SIMS (positive-ion mode) and ESIMS (positive-ion mode) were carried out on a Q-TOF 1-micromass spectrometer. FABMS were conducted in the negative-ion mode on a Jeol SX-102 instrument. Isolations of compounds were carried out using column chromatography (CC) on Sephadex LH-20 (550 mm  20 mm, GE Healthcare Bio-Sciences AB), and vacuum liquid chromatography (VLC) on reversed-phase RP-18 silica gel (75–200 lm, Silicycle). Mediumpressure liquid chromatography (MPLC) was performed on silica gel 60 (Merck, 15–40 lm) with a Gilson M 305 pump (25 SC head pump, M 805 manometric module), a Büchi glass column (460 mm  25 mm and 460 mm  15 mm), and a Büchi precolumn (110 mm  15 mm). Thin-layer chromatography (TLC, Silicycle) and high-performance thin-layer chromatography (HPTLC, Merck) were carried out on precoated silica gel plates 60F254, solvent system CHCl3/MeOH/H2O/AcOH 60:32:7:1. The spray reagent for saponins was vanillin reagent (1% vanillin in EtOH/H2SO4, 50:1). 3.2. Plant material Weigela stelzneri was provided in 2011 from JardilandÒ (Dijon, France). A voucher specimen (N° 20111001) was deposited in the herbarium of the Laboratory of Pharmacognosy, Université de Bourgogne Franche-Comté, Dijon, France. 3.3. Extraction and isolation The dried, powdered roots of W. stelzneri (18 g) were refluxed three times with EtOH–H2O (7/3, 3  1 L) for 1 h. After evaporation of the solvent under vacuum, the resulting extract (6 g) was submitted to VLC (RP-18 silica gel, H2O, MeOH/H2O 50:50 and MeOH). The fraction eluted with MeOH (192.2 mg) was fractionated by CC (Sephadex LH-20, MeOH) to give a fraction rich in saponin (148.0 mg). The latter was submitted to a MPLC on silica gel 60 (15–40 lm, CHCl3/MeOH/H2O 70:30:5; 60:32:7) yielding 8 fractions (F1–F8) and a pure compound, 1 (10.3 mg) in the fraction F4. The remaining fractions were combined and fractionated again by successive MPLC on silica gel 60 (15–40 lm, CHCl3/MeOH/H2O 70:30:5; 60:32:7) to give compounds 2 (4.4 mg), 3 (4.5 mg), 4 (6.2 mg) and 6 (5.3 mg). The same protocol was used for the extraction of the leaves of W. stelzneri (57 g). The resulting extract (16 g) was submitted to VLC on silica gel 60 (15–40 lm, CHCl3/MeOH/H2O 80:20:2; 70:30:5; 60:32:7) yielding 5 fractions (F1–F5) and a pure compound, 7 (14.0 mg) in the fraction F5. The remaining fractions were combined and fractionated again by successive MPLC on silica gel 60 (15–40 lm, CHCl3/MeOH/H2O 80:20:2; 70:30:5; 60:32:7) affording compounds 5 (20.6 mg), 8 (12.3 mg), and 9 (21.0 mg). 3.4. Compound characterization 3.4.1. 3-O-b-D-glucopyranosyl-(1 ? 2)-[b-D-xylopyranosyl-(1 ? 4)]b-D-xylopyranosyl-(1 ? 4)-b-D-xylopyranosyl-(1 ? 3)-a-L-rhamnopyranosyl-(1 ? 2)-a-L-arabinopyranosyloleanolic acid (1) 1 White, amorphous powder; ½a25 D =
28 (c 0.75, MeOH); H NMR 13 (pyridine-d5, 600 MHz) and C NMR (pyridine-d5, 150 MHz), see Tables 1 and 2; ESIMS (positive-ion mode) m/z 1315 [M+Na]+, HRESIMS (positive-ion mode) m/z 1315.6293 [M+Na]+ (calcd. for C62H100O28Na, 1315,6299).

3.4.2. 3-O-b-D-glucopyranosyl-(1 ? 2)-[b-D-xylopyranosyl-(1 ? 4)]b-D-xylopyranosyl-(1 ? 4)-b-D-xylopyranosyl-(1 ? 3)-a-L-rhamnopyranosyl-(1 ? 2)-b-D-xylopyranosyloleanolic acid (2) 1 White, amorphous powder; ½a25 D =
28 (c 0.75, MeOH); H NMR (pyridine-d5, 600 MHz) and 13C NMR (pyridine-d5, 150 MHz), see Tables 1 and 2; ESIMS (positive-ion mode) m/z 1315 [M+Na]+,

Please cite this article in press as: Rezgui, A., et al. Oleanolic acid and hederagenin glycosides from Weigela stelzneri. Phytochemistry (2016), http://dx.doi. org/10.1016/j.phytochem.2015.12.016

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A. Rezgui et al. / Phytochemistry xxx (2016) xxx–xxx

HRESIMS (positive-ion mode) m/z 1315.6293 [M+Na]+ (calcd. for C62H100O28Na, 1315,6299). 3.4.3. 3-O-b-D-glucopyranosyl-(1 ? 2)-[b-D-glucopyranosyl-(1 ? 4)]b-D-xylopyranosyl-(1 ? 4)-b-D-xylopyranosyl-(1 ? 3)-a-L-rhamnopyranosyl-(1 ? 2)-b-D-xylopyranosyloleanolic acid (3) 1 White, amorphous powder; ½a25 D =
19 (c 0.75, MeOH); H NMR 13 (pyridine-d5, 600 MHz) and C NMR (pyridine-d5, 150 MHz), see Tables 1 and 2; FABMS (negative-ion mode) m/z 1321 [M
H]
, 1159 [(M
H)
162]
, HRESIMS (positive-ion mode) m/z 1345.6411 [M+Na]+ (calcd. for C63H102O29Na, 1345.6404).

3.4.4. 3-O-b-D-glucopyranosyl-(1 ? 2)-[b-D-xylopyranosyl-(1 ? 4)]b-D-xylopyranosyl-(1 ? 4)-b-D-xylopyranosyl-(1 ? 3)-a-L-rhamnopyranosyl-(1 ? 2)-a-L-arabinopyranosyloleanolic acid 28-O-b-D-glucopyranosyl-(1 ? 6)-b-D-glucopyranosyl ester (4) 1 White, amorphous powder; ½a25 D =
12 (c 0.10, MeOH); H NMR (pyridine-d5, 600 MHz) and 13C NMR (pyridine-d5, 150 MHz), see Tables 1 and 2; ESIMS (positive-ion mode) m/z 1639 [M+Na]+, FABMS (negative-ion mode) m/z 1291 [(M
H)
162
162]
, 1159 [(M
H)
162
162
132]
, 997 [(M
H)
162
162
132
162]
, 865 [(M
H)
162
162
132
162
132]
, 733 [(M
H)
162
162
132
162
132
132]
, 587 [(M
H)
162
162
132
162
132
132
146]
, 455 [(M
H)
162
162
132
162
132
132
146
132]
, HRESIMS (positive-ion mode) m/z 1639.7350 [M+Na]+ (calcd. for C74H120O38Na, 1639.7355).

3.4.5. 3-O-b-D-glucopyranosyl-(1 ? 2)-a-Larabinopyranosylhederagenin 28-O-b-D-xylopyranosyl-(1 ? 6)-[a-Lrhamnopyranosyl-(1 ? 2)]-b-D-glucopyranosyl ester (5) 25 D

1

White, amorphous powder; ½a =
21 (c 0.75, MeOH); H NMR (pyridine-d5, 600 MHz) and 13C NMR (pyridine-d5, 150 MHz), see Tables 1 and 2; ESIMS (positive-ion mode) m/z 1229 [M+Na]+, HRESIMS (positive-ion mode) m/z 1229.5940 [M+Na]+ (calcd. for C58H94O26Na, 1229.5931). 3.5. Acid hydrolysis and GC analysis Each compound (3 mg) was hydrolyzed with 2 N aq. CF3COOH (5 mL) for 3 h at 95 °C. After extraction with CH2Cl2 (3  5 mL), the aq. layer was repeatedly evaporated to dryness with MeOH until neutral, and then analyzed by TLC over silica gel (CHCl3/ MeOH/H2O 8:5:1) by comparison with authentic samples. Furthermore, the residue of sugars was dissolved in anhydrous pyridine (100 lL), and L-cysteine methyl ester hydrochloride (0.06 mol/L) was added. The mixture was stirred at 60 °C for 1 h, then 150 lL of HMDS-TMCS (hexamethyldisilazane/trimethylchlorosilane 3:1) was added, and the mixture was stirred at 60 °C for another 30 min. The precipitate was centrifuged off, and the supernatant was concentrated under a N2 stream. The residue was partitioned between n-hexane and H2O (0.1 mL each), and the hexane layer (1 lL) was analyzed by GC (Hara et al., 1987). The absolute configurations were determined by comparing the retention times with thiazolidine derivatives prepared in a similar way from standard sugars (Sigma–Aldrich). 3.6. Biological assays 3.6.1. XTT cytotoxicity assay Compounds 1–3, 5, 7–9 were tested for cytotoxicity during 48 h against human colorectal (SW480) and mouse mammary (EMT6) cancer cell lines, provided by the Cohiro society, Dijon, France. The bioassay was carried out according to the XTT method (Jost et al., 1992). The positive controls were etoposide and methotrex-

ate tested at 20 lM. Etoposide decreased SW480 and EMT-6 cell proliferation by 50%. EMT-6 cancer cells exhibit greater sensitivity to methotrexate than SW480 cancer cells, with a potent reduction in cell viability of 80% and 50%, respectively. 3.6.2. Anti-inflammatory activity Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coat of healthy donor by centrifugation on Ficoll of density 1.077 g/mL and were seeded at 6.67  105 cells/mL in a 96-well microplate in Roswell Park Memorial Institute medium (RPMI-1640) containing fetal bovine serum (10%), penicillin (100 U/mL), streptomycin (100 lg/mL), amphotericin B (250 ng/mL), and incubated at 5% CO2 and 37 °C. PBMCs were pre-treated with compounds 1–3, 5, 7–9 at different concentrations (0.8–32.3 lM). After 1 h, PBMCs were stimulated for 24 h with LPS (Escherichia coli, 0128:B12, Sigma) (10 ng/mL). Z-VAD-FMK (5 lM), a Caspase-1 inhibitor, was used as anti-inflammatory reference. All experiments were done in triplicate. Production of the inflammatory cytokine IL-1b was determined in the supernatant using ELISA assay according to the protocol provided with the Kit (eBioscience, San Diego, USA). Statistical analysis: Data are expressed as mean ± SEM. Statistical differences were evaluated by analysis of variance (One-way ANOVA followed by Bonferroni t-test) for comparison between each group using SigmaPlot 11.0 (Systat software; San Jose, CA). A value of P < 0.05 was considered statistically significant. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2015. 12.016. References Bang, S.-C., Lee, J.-H., Song, G.-Y., Kim, D.-H., Yoon, M.-Y., Ahn, B.-Z., 2005. Antitumor activity of Pulsatilla koreana saponins and their structure–activity relationship. Chem. Pharm. Bull. 53, 1451–1454. Dinarello, C.A., 2010. Why not treat human cancer with interleukin-1 blockade? Cancer Metastasis Rev. 29, 317–329. FOBS, Friends of the Botanical Gardens, Sheffield, 2015, http://www.fobssheffield. co.uk/weigelas.html. Hara, S., Okabe, H., Mihashi, K., 1987. Gas-liquid chromatographic separation of aldose enantiomers as trimethylsilyl ethers of methyl 2-(polyhydroxyalkyl) thiazolidine-4(R)-carboxylates. Chem. Pharm. Bull. 35, 501–506. Jost, L.M., Kikwood, J.M., Whiteside, T.L., 1992. Improved short- and long-term XTTbased colorimetric cellular cytotoxicity assay for melanoma and other tumor cells. J. Immunol. Methods 147, 153–165. Kuroda, M., Shizume, T., Mimaki, Y., 2014. Triterpene glycosides from the stems and leaves of Lonicera japonica. Chem. Pharm. Bull. 62, 92–96. Mantovani, A., Allavena, P., Sica, A., Balkwill, F., 2008. Cancer-related inflammation. Nature 454, 436–444. Mariathasan, S., Weiss, D.S., Newton, K., McBride, J., O’Rourke, K., Roose-Girma, M., Lee, W.P., Weinrauch, Y., Monack, D.M., Dixit, V.M., 2006. Cryopyrin activates the inflammasome in response to toxins and ATP. Nature 440, 228–232. Murayama, T., Kasahara, A., Shiono, Y., Ikeda, M., 2003. Structure elucidation of a triterpene glycoside isolated from Weigela hortensis. Nat. Med. 57, 181–184. Ren, M.-T., Chen, J., Song, Y., Sheng, L.-S., Li, P., Qi, L.-W., 2008. Identification and quantification of 32 bioactive compounds in Lonicera species by high performance liquid chromatography coupled with time-of-flight mass spectrometry. J. Pharm. Biomed. Anal. 48, 1351–1360. The Angiosperm phylogeny group, 2009. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG III. Bot. J. Linn. Soc. 161, 105–121. Wang, X.-Y., Chen, X.-L., Tang, H.-F., Gao, H., Tian, X.-R., Zhang, P.-H., 2011. Cytotoxic triterpenoid saponins from the rhizomes of Anemone taipaiensis. Planta Med. 77, 1550–1554. Won, Y.-M., Seong, Z.-K., Kim, J.-L., Kim, H.-S., Song, H.-H., Kim, D.-Y., Kim, J.-H., Oh, S.-R., Cho, H.-W., Cho, J.-H., Lee, H.-K., 2015. Triterpene glycosides with stimulatory activity on melanogenesis from the aerial parts of Weigela subsessilis. Arch. Pharmacal Res. 38, 1541–1551. Zheng, Q., Koike, K., Han, L.-K., Okuda, H., Nikaido, T., 2004. New biologically active triterpenoid saponins from Scabiosa tschiliensis. J. Nat. Prod. 67, 604–613.

Please cite this article in press as: Rezgui, A., et al. Oleanolic acid and hederagenin glycosides from Weigela stelzneri. Phytochemistry (2016), http://dx.doi. org/10.1016/j.phytochem.2015.12.016