Cytotoxic triterpenoid saponins from Lysimachia foenum-graecum

Phytochemistry xxx (2017) 1e10

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Cytotoxic triterpenoid saponins from Lysimachia foenum-graecum Lu-Mei Dai a, 1, Ri-Zhen Huang a, b, 1, Bin Zhang a, Jing Hua a, Heng-Shan Wang a, **, Dong Liang a, * a State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmaceutical Sciences, Guangxi Normal University, Guilin 541004, People's Republic of China b Department of Pharmaceutical Engineering, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, People's Republic of China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 September 2016 Received in revised form 22 January 2017 Accepted 28 January 2017 Available online xxx

Eleven oleanane-type triterpenoid saponins, foegraecumosides AeK, and eight known ones, were isolated from the aerial parts of Lysimachia foenum-graecum. Their structures were elucidated by spectroscopic data analyses and chemical methods. All isolated saponins were evaluated for their cytotoxicity against four human cancer cell lines (NCI-H460, MGC-803, HepG2, and T24). Seven saponins containing the aglycone cyclamiretin A exhibited moderate cytotoxicity against all tested human cancer cell lines, with IC50 values of 9.3e24.5 mM. Simultaneously, the cytotoxic activities of foegraecumosides A and B, lysichriside A, ardisiacrispins A and B, cyclaminorin, and 3-O-a-L-rhamnopyranosyl-(1 / 2)-b-D-glucopyranosyl-(1 / 4)-a-L-arabinopyranosyl-cyclamiretin A were tested on drug-resistant lung cancer cell lines (A549 and A549/CDDP, respectively). Ardisiacrispin B displayed moderate cytotoxicity against A549/ CDDP, with an IC50 value of 8.7 mM and a resistant factor (RF) of 0.9. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Lysimachia foenum-graecum Myrsinaceae Triterpenoid saponins Cytotoxic activities

1. Introduction The genus Lysimachia of the family Primulaceae, comprising about 180 species, is widely distributed throughout the world. However, data from recent phylogenetic analyses suggested its relocation to the family Myrsinaceae (Podolak et al., 2013). L. foenumgraecum is one of the best-known plants from this genus, distributed mainly in the Guangxi and Yunnan Provinces of China. The aerial parts of this plant have been used not only as a perfumery plant and an insect repellent, but also for the treatment of colds and headaches in traditional Chinese medicine (The Health Administration of Beijing, 1998). Previous phytochemical investigation of this species led to isolation of several triterpenoid

* Corresponding author. State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmaceutical Science, Guangxi Normal University, 15 Yucai Road, Guilin, 541004, People's Republic of China. ** Corresponding author. State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmaceutical Science, Guangxi Normal University, 15 Yucai Road, Guilin, 541004, People's Republic of China. E-mail addresses: [email protected] (H.-S. Wang), [email protected] (D. Liang). 1 These authors contributed equally to this work.

saponins and flavonoids (Shen et al., 2005; Li et al., 2007, 2009a,b; 2010). In a continuing search for bioactive constituents from medicinal plants in the Guangxi Zhuang Autonomous Region, the aerial parts of L. foenum-graecum were investigated and eleven new oleanane-type triterpenoid saponins, named foegraecumosides AeK (1e11) (Fig. 1) were isolated, together with eight known ones (12e19) (Fig. 1). The isolated saponins were evaluated for their cytotoxic activities against a panel of drug-sensitive and drugresistant human cancer cell lines. Herein, described are the isolation, structural elucidation, and biological assays of these compounds. 2. Results and discussion The aerial parts of L. foenum-graecum were collected from the Jinxiu County of Guangxi Province and extracted with 95% aqueous EtOH. The EtOH extract was suspended in H2O and partitioned successively with EtOAc and n-BuOH. The n-BuOH-soluble extract was subjected to macroporous resin column chromatography to give a crude saponin fraction, which showed cytotoxicity against three human cancer cell lines (HepG2, MGC-803, and T24) with IC50 values of 2.6e7.1 mg/mL. The crude saponin fraction was subjected to further column chromatography and purified by preparative HPLC, to afford eleven new (1e11) and eight known (12e19)

http://dx.doi.org/10.1016/j.phytochem.2017.01.021 0031-9422/© 2017 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Dai, L.-M., et al., Cytotoxic triterpenoid saponins from Lysimachia foenum-graecum, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.021

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L.-M. Dai et al. / Phytochemistry xxx (2017) 1e10

Fig. 1. Structures of compounds 1e19.

oleanane-type triterpenoid saponins. All of the new compounds (1e11) were obtained as amorphous powders. Acid hydrolysis of each compound (1e11) and subsequent HPLC analysis of the sugars using an optical detector (Yoshikawa et al., 2003, 2007) allowed the characterisation of D-glucose, L-arabinose, and L-rhamnose for compounds 1, 3, 4, 6, 7, and 10, D-glucose and L-arabinose for compounds 2 and 9, and D-glucose, L-arabinose, and D-xylose for compounds 5, 8, and 11, respectively. Foegraecumoside A (1) possessed the molecular formula of C55H88O23 on the basis of its positive-ion HRESIMS (m/z 1139.5608 [M þ Na]þ, calcd 1139.5609) and 13C NMR data (Table 2). Its 1H NMR spectrum (Table 1) showed six methyl singlets at dH 0.83, 0.99, 1.02, 1.21, 1.27, and 1.51, one oxygenated methylene at dH 3.14 and 3.52 (each 1H, d, J ¼ 7.5 Hz), and one aldehyde proton at d 9.60 (1H, s), which were unambiguously designated by HSQC experiment. Its 13 C NMR (Table 2) and DEPT spectra displayed six sp3 carbon resonances at dC 16.38, 16.42, 18.5, 19.7, 24.1, and 28.0, an oxygenated methylene at dC 77.6, a quaternary carbon resonance at dC 86.4, and an aldehyde carbon at dC 207.5. The NOESY correlation of H-30 (dH 9.60)/H-18 (dH 1.35) confirmed the b-orientation of the aldehyde group. These data indicated that 1 was based on a 13,28-epoxy-

oleanane skeleton. On the basis of its 2D NMR (1He1H COSY, HSQC, and HMBC) analyses and comparison with literature data, the aglycone of 1 was identified as 3b,16a-dihydroxy-13b,28-epoxyoleanan-30-al (cyclamiretin A) (Dong et al., 2011). The 1H NMR spectrum of 1 exhibited signals of four sugar anomeric protons at dH 4.87 (1H, d, J ¼ 5.0 Hz), 5.12 (1H, d, J ¼ 8.0 Hz), 5.38 (1H, d, J ¼ 7.5 Hz), and 6.35 (1H, br s), which correlated to four anomeric carbons at dC 104.4, 103.5, 105.0, and 101.6, respectively, according to the HSQC spectrum. The extensive 2D NMR spectrocopic analyses together with results of acid hydrolysis of 1 allowed the characterisation of two b-D-glucopyranosyls (Glc), one a-L-rhamnopyranosyl (Rha), and one a-Larabinopyranosyl (Ara), respectively. In the same way, these sugars were identified in compounds 3, 4, 6, 7, and 10. The coupling constants also confirmed the b-glycosidic linkages for two glucopyranosyl units. The arabinopyranosyl unit was determined to be the a-anomer on the basis of: the 3JH1,H2 value (5.0 Hz) and the correlations of Ara-H-1/Ara-H-3 and Ara-H-1/Ara-H-5 in the NOESY spectrum (Chang et al., 2007; Liang et al., 2011), as well as the 1H non-splitting pattern (br s) and the large JC1-H1 coupling constants (171 Hz) which confirmed the a-anomeric orientation of the

Please cite this article in press as: Dai, L.-M., et al., Cytotoxic triterpenoid saponins from Lysimachia foenum-graecum, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.021

L.-M. Dai et al. / Phytochemistry xxx (2017) 1e10

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Table 1 1 H NMR spectroscopic data of compounds 1e8.a position 1

2

3

4

5

6

7

8

1a

1.61, br d (13.0)

1.67, m

1.61, m

1.61, m

1.50, m

1b 2a

0.83 (overlapped) 2.00 (overlapped)

0.87, m 2.05 (overlapped)

0.82, m 1.99, m

0.93, m 1.89, m

1.64 (overlapped) 0.84, m 2.02, m

0.87, m 1.99 (overlapped)

1.49 (overlapped) 0.90, m 1.98, m

2b

1.81, m

1.83, m

1.82, m

1.79, m

1.79, m

1.82, m

1.48 (overlapped) 0.85, m 2.00 (overlapped) 1.76, m

3

3.17, dd (11.5, 4.0)

3.20, dd (11.5, 4.0)

3.06, dd (11.5, 4.0)

0.68, d (11.5) 1.44 (overlapped)

0.72, d (11.5) 1.46 (overlapped)

6b

1.37 (overlapped)

1.39 (overlapped)

1.37 (overlapped)

7a

1.50 (overlapped)

1.53 (overlapped)

1.51, m

1.69, dd (13.0, 4.0)

7b

1.16, m

1.19, m

1.16, m

1.25, br d (13.0)

3.12, dd (11.5, 4.0) 0.62, d (10.0) 1.40 (overlapped) 1.31 (overlapped) 1.31 (overlapped) 1.04, m

3.15, dd (11.5, 4.0)

5 6a

3.13, dd (11.5, 4.0) 0.64, d (11.5) 1.38 (overlapped) 1.32, m

9

1.23, m

1.27, m

1.25, m

2.00, m

1.20, m

1.69, dd (10.0, 7.0)

11a

1.71, dd (12.5, 4.5)

1.73, dd (12.5, 4.0)

2.03 (overlapped)

1.69, m

1.87, m

11b

1.43 (overlapped)

1.44 (overlapped)

1.63, m

12a

2.07 (overlapped)

2.11 (overlapped)

1.39 (overlapped) 1.99, m

12b

1.41 (overlapped)

1.43 (overlapped)

1.72, dd (13.5, 4.5) 1.43 (overlapped) 2.11 (overlapped) 1.46, m

15a

2.16, dd (14.5, 5.0)

2.18, dd (14.5, 4.5)

15b

1.43 (overlapped)

1.47, m

16

4.18 (overlapped)

4.19 (overlapped)

18

1.36, m

1.37, dd (14.5, 2.0)

19a

2.82, dd (13.5, 13.0)

2.83, dd (14.5, 13.0)

19b

2.10 (overlapped)

2.10 (overlapped)

21a

2.52, ddd (13.5, 13.5, 2.52, ddd (13.5, 13.5, 2.94, dd (12.0, 5.0) 5.0) 12.0) 2.04 (overlapped) 2.06 (overlapped) 2.53, dd (12.0, 5.5) 1.92, m 1.94, m 4.13 (overlapped) 1.55, m 1.56, dd (13.5, 5.0) 1.21, s 1.22, s 1.14, s 1.02, s 1.05, s 1.00, s 0.83, s 0.88, s 0.80, s 1.27, s 1.29, s 1.26, s 1.51, s 1.52, s 1.59, s 3.52, d (7.5) 3.53, d (7.5) 3.93, d (7.5) 3.14, d (7.5) 3.15, d (7.5) 3.52, d (7.5) 0.99, s 0.99, s 1.06, s 9.60, s 9.61, s 9.64, s

2.55, ddd (13.5, 13.0, 1.62 5.0) (overlapped) 2.09, dd (13.5, 4.0) 1.39 (overlapped) 1.96, m 1.65 (overlapped) 1.66, m 1.48, m 1.14, s 1.14, s 1.00, s 0.97, s 0.84, s 0.81, s 1.33, s 1.22, s 2.03, s 1.23, s 3.57, d (7.5) 3.58, d (8.0) 3.21, d (7.5) 3.25, d (8.0) 0.96, s 1.08, s 9.68, s 4.33, d (11.0) 4.00, d (11.0) 2.14, s

Ara at C-3

Ara at C-3

Ara at C-3

Ara at C-3

Ara at C-3

Ara at C-3

Ara at C-3

1 2

4.87, d (5.0) 4.53, dd (7.0, 5.0)

4.86, d (5.5) 4.48, m

4.94, br s 4.56, br d (7.0)

4.90, d (4.0) 4.53, m

4.92, d (5.0) 4.54 (overlapped)

3

4.43 (overlapped)

4.37, m

4.49, m

4.48 (overlapped)

4 5a

4.48, m 4.45, m

4.46 (overlapped) 4.39, dd (12.0, 5.0)

4.87, d (5.0) 4.53, dd (7.5, 5.0) 4.43 (overlapped) 4.48, m 4.45, m

5b

3.76 (overlapped)

3.77, dd (12.0, 2.5)

4.58, m 4.56, m 4.40, dd (12.0, 4.37, dd (12.0, 5.5) 5.5) 3.80, br d (12.0) 3.77 (overlapped)

5.04, d (3.5) 4.49 (overlapped) 4.50 (overlapped) 4.47, m 4.28, dd (11.5, 6.0) 3.83, dd (11.5, 2.5)

21b 22a 22b 23 24 25 26 27 28a 28b 29 30a 30b 16-OAc 28-OAc 30-OAc

0.75, d (11.0) 1.42 (overlapped)

1.24, m 1.55, m

1.24, m 1.31, m 1.15, m

5.40, br s

5.59, br s

5.34, br s

1.42 (overlapped) 2.16, m

2.17, dd (14.5, 3.5)

1.33, m

1.59, br d (14.5)

4.23 (overlapped)

5.25, br d (4.0)

4.65 (overlapped)

2.28, br d (14.0) 2.00 (overlapped) 1.66, d (14.0) 1.55 (overlapped) 4.81, br s 5.44, br s

1.77 (overlapped)

1.64 2.31, dd (13.5, 4.5) (overlapped) 2.26, dd (14.0, 2.77, dd (13.5, 13.5) 13.5) 1.73, br d (13.0) 1.98 (overlapped)

4.14, m

3.29, m 3.27, m

1.27, m

3.11, dd (11.5, 4.5) 0.66, d (11.0) 1.44, m

1.30 (overlapped) 1.73, dd (10.5, 7.0) 1.85 (overlapped)

2.21, dd (14.5, 2.33, dd (14.5, 5.5) 5.5) 1.54, br d (14.5) 1.50, d (14.5) 5.02 (overlapped) 1.40 (overlapped) 2.98, dd (13.5, 13.5) 2.08, m

0.70, d (11.5) 1.42, m

1.80 (overlapped) 3.19, dd (11.5, 4.5) 0.76, d (11.0) 1.48 (overlapped) 1.29 (overlapped) 1.60, m

2.71, dd (12.5, 2.0) 2.85, dd (13.5, 12.5) 2.44, br d (12.5)

2.44, ddd (13.0, 13.0, 2.58, m 5.0) 2.15, br d (13.0) 2.36, br d (13.0)

2.54, m

2.09, 1.15, 1.02, 0.83, 0.89, 1.79, 3.69, 3.44, 1.00, 9.76,

1.22, 1.03, 0.84, 0.94, 1.85, 3.82, 3.54, 1.48,

dd (13.0, 5.0) s s s s s d (10.5) d (10.5) s s

s s s s s d (10.5) d (10.5) s

2.07, s

4.46 (overlapped) 4.56 (overlapped) 4.38, dd (12.0, 5.0) 3.78 (overlapped)

1.60, m 1.79, m

2.32, dd (14.5, 3.0) 2.23, dd (14.0, 13.0) 1.54 (overlapped) 1.54 (overlapped) 1.48 (overlapped) 1.74, m 1.69, 1.16, 0.98, 0.83, 0.89, 1.41, 4.14, 3.90, 1.05, 4.36, 4.09, 2.17, 2.10, 2.07,

m s s s s s d (11.5) d (11.5) s d (10.5) d (10.5) s s s

Ara at C-3

5.03, d (4.0) 4.50 (overlapped) 4.51 (overlapped) 4.47, m 4.29, dd (11.5, 6.0) 3.75, br d (12.0) 3.84, dd (11.5, 3.0) (continued on next page)

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L.-M. Dai et al. / Phytochemistry xxx (2017) 1e10

Table 1 (continued ) position 1

2

3

4

5

6

7

8

Glc-I

Glc-I

Glc-I

Glc-I

Glc

Glc-I

Glc-I

Glc

1 2

5.38, d (7.5) 4.05, dd (8.0, 7.5)

5.05, d (7.5) 4.01 (overlapped)

5.32, d (7.5) 4.04, m

4.31, dd (9.0, 9.0)

4.09, dd (9.0, 8.5)

4.27, m

5.20, d (8.0) 4.09 (overlapped) 4.22, m

5.35, d (7.5) 4.06, m

3 4

4.09, dd (9.5, 9.0)

4.04, m

5.35, d (7.5) 4.06 (overlapped) 4.30 (overlapped) 4.23, m

4.22, m

4.20, m

4.20, m

5

4.18 (overlapped)

3.79, m

4.91, br d (11.5) 4.75, dd (11.5, 5.0)

4.80, dd (12.0, 2.0) 4.69, dd (12.0, 5.5)

3.72 (overlapped) 4.35, m 4.31 (overlapped)

4.04, m

6a 6b

4.05 4.02, m (overlapped) 4.49, br d (12.0) 4.46 (overlapped) 4.40, dd (12.0, 4.36 (overlapped) 5.5)

5.39, 4.05, 7.5) 4.31, 9.0) 4.08, 8.5) 4.19,

OAc

2.01, s

2.00, s

Glc-II

Glc-II

Glc-II

Glc-II

Xyl

Glc-II

Glc-II

Xyl

1 2

5.12, d (8.0) 4.25 (overlapped)

5.14, d (7.5) 4.02 (overlapped)

5.20, d (7.5) 4.24 (overlapped)

5.25, d (7.0) 4.08, m

5.23, d (7.5) 4.24 (overlapped)

3

4.17 (overlapped)

4.17 (overlapped)

5.22, d (8.0) 4.27 (overlapped) 4.19, m

4.17, m

4.11, m

4.18, m

4

4.12, dd (9.5, 9.5)

4.20 (overlapped)

4.08, dd (9.0, 8.5)

4.16, m

4.10, dd (9.0, 9.0)

5.27, d (7.0) 4.09 (overlapped) 4.11 (overlapped) 4.18, m

5a

3.76 (overlapped)

3.85, m

4.12, dd (9.0, 9.0) 3.78, m

3.79 (overlapped)

4.43, dd (11.0, 5.0) 3.72 (overlapped)

3.78 (overlapped)

5.14, d (8.0) 4.25 (overlapped) 4.17, dd (9.0, 8.5) 4.11, dd (9.5, 9.0) 3.78, m

6a

4.42, m

4.45 (overlapped)

4.45, br d (12.0) 4.43, m

4.44, br d (12.0)

6b

4.26 (overlapped)

4.32, dd (12.0, 5.0)

4.28 (overlapped)

4.25 (overlapped)

4.26 (overlapped)

5b

4.28, m

4.48, dd (11.0, 1.5) 4.35, dd (11.0, 5.0)

d (7.5) dd (9.0,

5.22, d (7.5) 4.11 (overlapped) dd (9.0, 4.23 (overlapped) dd (9.0, 4.22 (overlapped) m 3.72 (overlapped) 4.91, br d (12.0) 4.36, br d (10.5) 4.76, dd (12.0, 4.32, m 5.0) 2.02, s

4.43, dd (11.5, 5.0) 3.73 (overlapped)

4.43 (overlapped) 4.27 (overlapped)

Rha

Rha

Rha

Rha

Rha

1 2 3

6.35, br s 4.70, br s 4.67, dd (9.5, 3.0)

6.35, br s 4.69, br s 4.64, br d (9.0)

6.38, br s 4.70, br s 4.66 (overlapped)

4

4.25 (overlapped)

4.24 (overlapped)

4.24 (overlapped)

5 6

5.00, m 1.78, d (6.0)

6.39, br s 4.72, br s 4.68, dd (9.5, 3.0) 4.27 (overlapped) 5.03, m 1.79, d (6.0)

4.99, m 1.76, d (6.0)

5.00, m 1.79 (overlapped)

6.36, br s 4.70, br s 4.67, dd (9.5, 3.0) 4.26 (overlapped) 5.01, m 1.79, d (6.5)

a 1 H NMR data (d) were measured in pyridine-d5 at 500 MHz. Coupling constants (J) in Hz are given in parentheses. The assignments were based on 1He1H COSY, HSQC, HMBC, and NOESY experiments.

rhamnopyranosyl unit (Tommasi et al., 1993; Sahu et al., 1995). The sequence and linkage positions of the sugar chain were subsequently deduced from an HMBC experiment. In the HMBC spectrum, correlations from GlcI-H-1 (dH 5.38) to Ara-C-2 (dC 80.7), GlcII-H-1 (dH 5.12) to Ara-C-4 (dC 75.4), Rha-H-1 (dH 6.35) to GlcII-C2 (dC 77.5), as well as Ara-H-1 (dH 4.87) to C-3 (dC 89.1) were observed. Besides, an acetoxy group (dC 20.8 and 170.9, dH 2.01) was assigned at GlcI-C-6 by the HMBC from GlcІ-H2-6 (dH 4.91, 4.75) to the carbon at dC 170.9. Hence, foegraecumoside A (1) was 3b-{a-Lrhamnopyranosyl-(1 / 2)-b-D-glucopyranosyl-(1 / 4)-[b-D-6-Oacetylglucopyranosyl-(1 / 2)]-a-L-arabinopyranosyloxy}-cyclamiretin A. Foegraecumoside B (2) gave the molecular formula of C49H78O19 from HRESIMS (m/z 993.5020 [MþNa]þ, calcd 993.5030) and 13C NMR data. Its NMR data assignable to the aglycone moiety were identical to those of 1 (Tables 1 and 2). Comparison of its 1H and 13C NMR data attributable to the sugar portion with those of 1, together with results of acid hydrolysis, suggested that the terminal a-Lrhamnopyranosyl moiety present in 1 was absent in 2. This conclusion was confirmed by the HMBC: Ara-H-1 (dH 4.86) to C-3 (dC 89.2); GlcI-H-1 (dH 5.05) to Ara-C-2 (dC 81.3); GlcII-H-1 (dH 5.14) to Ara-C-4 (dC 77.1); and GlcI-H2-6 (dH 4.80, 4.69) to the carbonyl carbon (dC 170.9) of the acetyl group. Thus, foegraecumoside B (2) was 3b-{b-D-6-O-acetylglucopyranosyl-(1 / 2)-[b-D-glucopyranosyl-

(1 / 4)]-a-L-arabinopyranosyloxy}-cyclamiretin A. Foegraecumoside C (3) had a molecular formula of C53H86O23 by HRESIMS (m/z 1113.5433 [MþNa]þ, calcd 1113.5452) and 13C NMR data. Comparison of its 1H and 13C NMR data (Tables 1 and 2) with those of 1 established that the aglycone of 3 contained an additional oxymethine signal resonating at dC 74.2 (dH 4.13). The HMBC from H2-21 (dH 2.94, 2.53) and H-28b (dH 3.52) to dC 74.2 located the hydroxy group at C-22 in 3, while the NOESY cross-peaks of H-22 (dH 4.13)/H-18 (dH 1.40) and H-22/H-28b confirmed the b-orientation of H-22. Thus, the aglycone of 3 was defined as 3b,16a,22atrihydroxy-13b,28-epoxy-oleanan-30-al (Mu et al., 2015). The extensive 2D NMR spectrocopic analyses, together with results of acid hydrolysis of 3, suggested that the NMR signals attributable to the sugar moieties of 3 were similar to those of 1 except for the absence of the acetyl group at GlcI-C-6 in 3, which was reported previously in ardisiacrispin B (13) (Jansakul et al., 1987). This consequence was established by analysis of the NOESY correlations of: Ara-H-1 (dH 4.94)/H-3 (dH 3.13); GlcI-H-1 (dH 5.35)/Ara-H2 (dH 4.56); GlcII-H-1 (dH 5.22)/Ara-H-4 (dH 4.58); and Rha-H-1 (dH 6.39)/GlcII-H-2 (dH 4.27). Accordingly, foegraecumoside C (3) was elucidated as 16a,22a-dihydroxy-3b-{a-L-rhamnopyranosyl-(1/2) -b-D-glucopyranosyl-(1 / 4)-[b-D-glucopyranosyl-(1 / 2)]-a-Larabinopyranosyloxy}-13b,28-epoxy-oleanan-30-al. Foegraecumoside D (4) was assigned the molecular formula of

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5

Table 2 13 C NMR spectroscopic data of compounds 1e8a. position

1

2

3

4

5

6

7

8

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 16-OAc

39.2, CH2 26.5, CH2 89.1, CH 39.6, C 55.7, CH 18.0, CH2 34.4, CH2 42.6, C 50.5, CH 36.9, C 19.1, CH2 32.7, CH2 86.4, C 44.6, C 36.8, CH2 76.9, CH 44.0, C 53.3, CH 33.4, CH2 48.3, C 30.5, CH2 32.3, CH2 28.0, CH3 16.42, CH3 16.38, CH3 18.5, CH3 19.7, CH3 77.6, CH2 24.1, CH3 207.5, CH

39.3, CH2 26.7, CH2 89.2, CH 39.7, C 55.7, CH 18.0, CH2 34.4, CH2 42.6, C 50.5, CH 36.9, C 19.2, CH2 32.7, CH2 86.4, C 44.6, C 36.8, CH2 76.9, CH 44.1, C 53.3, CH 33.4, CH2 48.3, C 30.5, CH2 32.3, CH2 28.0, CH3 16.5, CH3 16.4, CH3 18.6, CH3 19.8, CH3 77.7, CH2 24.1, CH3 207.5, CH

39.2, CH2 26.5, CH2 89.2, CH 39.6, C 55.6, CH 17.9, CH2 34.3, CH2 42.7, C 50.4, CH 36.9, C 19.2, CH2 32.9, CH2 86.6, C 45.5, C 36.7, CH2 70.3, CH 49.4, C 52.9, CH 32.7, CH2 49.5, C 39.8, CH2 74.2, CH 28.0, CH3 16.5, CH3 16.3, CH3 18.5, CH3 20.0, CH3 77.7, CH2 23.9, CH3 207.3, CH

39.0, CH2 26.4, CH2 89.1, CH 39.6, C 56.0, CH 18.0, CH2 35.2, CH2 42.8, C 44.6, CH 36.6, C 29.7, CH2 77.8, CH 87.3, C 45.0, C 38.5, CH2 77.2, CH 44.8, C 54.6, CH 35.4, CH2 48.6, C 30.1, CH2 32.3, CH2 28.0, CH3 16.4, CH3 16.9, CH3 19.0, CH3 19.2, CH3 77.99, CH2 24.1, CH3 208.0, CH

39.2, CH2 26.5, CH2 88.8, CH 39.6, C 55.6, CH 17.9, CH2 34.3, CH2 42.6, C 50.5, CH 36.9, C 19.0, CH2 32.4, CH2 86.1, C 44.2, C 33.6, CH2 78.4, CH 43.4, C 50.2, CH 33.4, CH2 35.1, C 32.1, CH2 30.0, CH2 28.1, CH3 16.6, CH3 16.4, CH3 18.4, CH3 19.6, CH3 77.0, CH2 28.7, CH3 67.7, CH2 169.9, C 21.9, CH3

38.9, CH2 26.4, CH2 89.1, CH 39.5, C 55.8, CH 18.5, CH2 33.1, CH2 40.1, C 47.0, CH 36.8, C 23.8, CH2 122.9, CH 144.5, C 41.9, C 34.8, CH2 73.9, CH 40.5, C 43.5, CH 42.0, CH2 47.4, C 30.7, CH2 31.3, CH2 28.1, CH3 16.7, CH3 15.7, CH3 16.9, CH3 27.6, CH3 70.0, CH2 24.3, CH3 207.7, CH

38.9, CH2 26.4, CH2 89.1, CH 39.5, C 55.8, CH 18.5, CH2 33.2, CH2 40.5, C 47.1, CH 36.9, C 23.8, CH2 122.7, CH 145.1, C 41.9, C 34.9, CH2 73.8, CH 40.1, C 44.2, CH 44.8, CH2 44.5, C 33.9, CH2 32.5, CH2 28.1, CH3 16.6, CH3 15.7, CH3 17.0, CH3 27.4, CH3 71.0, CH2 29.3, CH3 180.6, C

38.9, CH2 26.3, CH2 88.7, CH 39.5, C 55.7, CH 18.3, CH2 33.0, CH2 40.0, C 47.0, CH 36.8, C 23.7, CH2 124.8, CH 141.7, C 41.3, C 31.1, CH2 75.9, CH 38.3, C 41.6, CH 42.4, CH2 34.3, C 31.4, CH2 28.6, CH2 28.2, CH3 16.8, CH3 15.7, CH3 16.9, CH3 26.9, CH3 70.6, CH2 27.9, CH3 68.4, CH2 170.1, C 21.8, CH3 171.0, C 20.8, CH3 170.8, C 20.6, CH3

28-OAc 30-OAc

1 2 3 4 5

1 2 3 4 5 6 OAc

1 2 3 4 5 6

1 2 3 4 5 6

170.9, C 20.7, CH3 Ara at C-3

Ara at C-3

Ara at C-3

Ara at C-3

Ara at C-3

Ara at C-3

Ara at C-3

Ara at C-3

104.4, CH 80.7, CH 72.4, CH 75.4, CH 63.6, CH2

104.4, CH 81.3, CH 72.7, CH 77.1, CH 63.9, CH2

104.4, CH 80.7, CH 72.4, CH 74.7, CH 63.5, CH2

104.4, CH 80.6, CH 72.3, CH 74.66, CH 63.5, CH2

104.1, CH 80.4, CH 72.7, CH 67.5, CH 63.9, CH2

104.4, CH 80.7, CH 72.4, CH 74.7, CH 63.6, CH2

104.4, CH 80.8, CH 72.2, CH 75.5, CH 63.7, CH2

104.1, CH 80.4, CH 72.7, CH 67.6, CH 64.0, CH2

Glc-I

Glc-I

Glc-I

Glc-I

Glc

Glc-I

Glc-I

Glc

105.0, CH 76.2, CH 77.8, CH 71.2, CH 75.0, CH 64.7, CH2 170.9, C 20.8, CH3

105.7, CH 76.0, CH 78.0, CH 71.0, CH 75.2, CH 64.6, CH2 170.9, C 20.8, CH3

105.4, CH 76.3, CH 78.1, CH 71.8, CH 78.1, CH 62.9, CH2

105.3, CH 76.3, CH 77.99, CH 71.7, CH 78.03, CH 62.8, CH2

103.8, CH 84.6, CH 77.9, CH 71.5, CH 77.7, CH 62.6, CH2

105.4, CH 76.3, CH 78.1, CH 71.8, CH 78.1, CH 62.9, CH2

105.1, CH 76.3, CH 77.8, CH 71.2, CH 75.0, CH 64.7, CH2 170.9, C 20.9, CH3

103.8, CH 84.7, CH 77.9, CH 71.5, CH 77.76, CH 62.5, CH2

Glc-II

Glc-II

Glc-II

Glc-II

Xyl

Glc-II

Glc-II

Xyl

103.5, CH 77.5, CH 79.5, CH 71.8, CH 78.3, CH 62.6, CH2

105.7, CH 75.7, CH 78.3, CH 71.5, CH 78.6, CH 62.6, CH2

103.1, CH 77.4, CH 79.5, CH 71.9, CH 78.3, CH 62.6, CH2

103.1, CH 77.4, CH 79.5, CH 71.9, CH 78.3, CH 62.6, CH2

106.9, CH 76.1, CH 77.8, CH 70.8, CH 67.4, CH2

103.1, CH 77.3, CH 79.5, CH 71.9, CH 78.3, CH 62.6, CH2

103.5, CH 77.5, CH 79.5, CH 71.8, CH 78.3, CH 62.6, CH2

107.0, CH 76.1, CH 77.81, CH 70.9, CH 67.5, CH2

Rha

Rha

Rha

Rha

Rha

101.6, CH 72.4, CH 72.6, CH 74.5, CH 69.6, CH 18.8, CH3

101.6, CH 72.4, CH 72.7, CH 74.8, CH 69.4, CH 18.9, CH3

101.5, CH 72.3, CH 72.6, CH 74.74, CH 69.4, CH 18.8, CH3

101.5, CH 72.4, CH 72.7, CH 74.8, CH 69.4, CH 18.9, CH3

101.7, CH 72.4, CH 72.7, CH 74.6, CH 69.6, CH 18.8, CH3

a 13

C NMR data (d) were measured in pyridine-d5 at 125 MHz. The assignments were based on 1He1H COSY, HSQC, HMBC, and NOESY experiments.

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L.-M. Dai et al. / Phytochemistry xxx (2017) 1e10

C53H86O23 in accordance with its HRESIMS (m/z 1113.5432 [M þ Na]þ, calcd 1113.5452). The sugar chain was equal to that of 3 by comparing their 1H and 13C NMR data (Tables 1 and 2). Moreover, the NMR signals of the aglycone of 4 were parallel to those of 3, apart from the presence of an additional hydroxy group at C-12 (dC 77.8) in 4 rather than at C-22 in 3. This was confirmed by the HMBC from H-12 (dH 4.14) to C-9 (dC 44.6), C-13 (dC 87.3), and C-14 (dC 45.0). Further, the NOESY correlation of H-12/H-18 (dH 1.77) indicated that H-12 was b-oriented. Thus, the structure of foegraecumoside D (4) was 12a,16a-dihydroxy-3b-{a-Lrhamnopyranosyl-(1 / 2)-b-D-glucopyranosyl-(1 / 4)-[b-D-glucopyranosyl-(1 / 2)]-a-L-arabinopyranosyloxy}-13b,28-epoxyoleanan-30-al. Foegraecumoside E (5) gave the molecular formula of C50H80O19 on the basis of its HRESIMS (m/z 1007.5173 [MþNa]þ, calcd 1007.5186) and 13C NMR data (Table 2). Its 13C NMR spectrum displayed 50 carbon resonances, of which 30 belonged to the aglycone, 16 to the sugar moiety, and the remaining four to two acetyl groups. The HMBC from H-16 (dH 5.25) to the carbonyl carbon (dC 169.9) of one acetyl group, C-14 (dC 44.2), and C-18 (dC 50.2) and from H2-30 (dH 4.00, 4.33) to the carbonyl carbon (dC 170.9) of the other acetyl group, C-19 (dC 33.4), C-20 (dC 35.1), and C-29 (dC 28.7), together with the NOESY correlation of H2-30/H-18 (dH 1.64), located the two acetyl groups at C-16 and C-30. Additionally, NOESY cross-peaks of H-3 (dH 3.12)/H-5 (dH 0.62) and H-16/H-18 were also observed. Thus, the aglycone of 5 was defined as 16a,30-diacetoxy-13b,28epoxy-oleanan-3b-ol. The extensive 2D NMR spectrocopic analyses, together with results of acid hydrolysis of 5, allowed for characterisation of one b-D-glucopyranosyl (Glc), one a-L-arabinopyranosyl (Ara), and one b-D-xylopyranosyl (Xyl). The sugar sequence of 5 was in agreement with that of heterogenoside E (Huang et al., 2011) by comparison of their 1H and 13C NMR data (Tables 1 and 2). This conclusion was confirmed by HMBC correlations from Ara-H-1 (dH 5.04) to C-3 (dC 88.8), Glc-H-1 (dH 5.20) to Ara-C-2 (dC 80.4), and Xyl-H-1 (dH 5.25) to Glc-C-2 (dC 84.6). Consequently, foegraecumoside E (5) was assigned as 16a,30diacetoxy-3b-{b-D-xylopyranosyl-(1 / 2)-b-D-glucopyranosyl(1 / 2)-a-L-arabinopyranosyloxy}-13b,28-epoxy-oleanane. The molecular formula of foegraecumoside F (6) was determined as C53H86O22 on the basis of its HRESIMS (m/z 1097.5505 [M þ Na]þ, calcd 1097.5503). Its 1D NMR data (Tables 1 and 2) displayed six methyl singlets at dH 0.83, 0.89, 1.00, 1.02, 1.15, 1.79, an oxygenated methylene group at dH 3.44 and 3.69 (each 1H, d, J ¼ 10.5 Hz), an olefinic proton at dH 5.40 (br s) coupled with two typical olefinic carbon resonances at dC 122.9 and 144.5, and an aldehyde group resonating at dH 9.76 (dC 207.7), which were confirmed by HSQC analysis. The HMBC from H-19a (dH 2.77) and H3-29 (dH 1.00) to dC 207.7, as well as the NOESY correlation of dH 9.76/H-18 (dH 2.31) confirmed that the aldehyde group was located at C-30 in 6. NOESY correlations of H-3 (dH 3.15)/H-5 (dH 0.70) and H-16 (dH 4.65)/H-28a (dH 3.69) indicated an a-orientation of H-3 and a b-orientation of H-16. Thus, the aglycone of 6 was identified as 3b,16a,28-trihydroxy-olean-12-en-30-al (cyclamiretin D) (Lavaud et al., 1994). Comparison of the 1H and 13C NMR data of 6 with those of 3 indicated that the signals of their sugar moieties were superimposable, suggesting that the sugar sequence at C-3 was the same as that in 3. Therefore, foegraecumoside F (6) was determined as 3b-{a-L-rhamnopyranosyl-(1 / 2)-b-D-glucopyranosyl-(1 / 4)-[b-D-glucopyranosyl-(1 / 2)]-a-L-arabinopyranosyloxy}-cyclamiretin D. Foegraecumoside G (7) possessed the molecular formula of C55H88O24 on the basis of its HRESIMS at m/z 1155.5536 [MþNa]þ (calcd 1155.5558). Its NMR data of the aglycone moiety (Tables 1 and 2) was similar to those of 6, except that the aldehyde group at C-30 in 6 was replaced by a carboxy group (dC 180.6) in 7. This

conclusion was confirmed by the HMBC from H3-29 (dH 1.48) to C19 (dC 44.8), C-20 (dC 44.5), C-21 (dC 33.9), and C-30 (dC 180.6), as well as from the NOESY correlations of H3-29/H-19a (dH 2.85) and H-19a/H3-27 (dH 1.85). Then the aglycone of 7 was determined to be 3b,16a,28-trihydroxy-olean-12-en-30-oic acid (Koike et al., 1999). The sugar sequence of 7 was identical with that of 1 by comparison of their 1H and 13C NMR data (Tables 1 and 2) together with results of acid hydrolysis. On the basis of the above analysis, foegraecumoside G (7) was elucidated as 16a,28-dihydroxy-3b-{a-Lrhamnopyranosyl-(1 / 2)-b-D-glucopyranosyl-(1 / 4)-[b-D-glucopyranosyl-(1 / 2)]-a-L-arabinopyranosyloxy}-olean-12-en-30oic acid. Foegraecumoside H (8) had a molecular formula of C52H82O20 on the basis of its HRESIMS (m/z 1049.5276 [M þ Na]þ, calcd 1049.5292) and 13C NMR data (Table 2). NMR analysis indicated that its aglycone contained three acetyl groups compared to 6, and where the aldehyde group at C-30 in 6 was replaced by an oxygenated methylenic functionality in 8. The HMBC from H2-30 (dH 4.09, 4.36) to the carbonyl carbon (dC 170.8) of one acetyl group, C-19 (dC 42.4), C-21 (dC 31.4), and C-29 (dC 27.9), together with the NOESY correlation of H2-30/H-18 (dH 2.32), confirmed that one acetyl group was linked to the C-30 oxygenated methylene. In the HMBC spectrum, the correlations from H-16 (dH 5.44) to dC 170.1, C14 (dC 41.3), and C-18 (dC 41.6), H2-28 (dH 3.90, 4.14) to dC 171.0, C-16 (dC 75.9), C-18, and C-22 (dC 28.6) located the other two acetyl groups at C-16 and C-28. The sugar chain of 8 was identified the same as that of 5 by comparing their 1H and 13C NMR data (Tables 1 and 2) together with results of acid hydrolysis. Thus, foegraecumoside H (8) was elucidated as 16a,28,30-triacetoxy-3b-{bD-xylopyranosyl-(1 / 2)-b-D-glucopyranosyl-(1 / 2)-a-L-arabinopyranosyloxy}-olean-12-ene. Foegraecumoside I (9) was assigned the molecular formula of C49H76O19 from its HRESIMS (m/z 991.4868 [M þ Na]þ, calcd 991.4873) and 13C NMR data (Table 3). Its 1D NMR data (Table 3) indicated the presence of six tertiary methyl groups at dH 0.78, 0.85, 1.05, 1.24, 1.30, 1.33, a pair of geminal protons at dH 4.30 and 4.72 (each 1H, d, J ¼ 11.5 Hz), an olefinic proton at dH 5.24 coupled with sp2-hybridized carbons at dC 124.3 and 140.6, and a carboxylic carbon resonance at dC 177.5, which were confirmed by the HSQC spectrum. The NMR data assignable to the aglycone moiety of 9 showed high similarity with those of 7 (Tables 1 and 2), while the downfield shift of H2-28 (dH 4.30, 4.72) and key HMBC from H-28b (dH 4.30) to C-30 (dC 177.5) indicated that 9 was based on a lactone ring from C-28 to C-30. Thus, the aglycone was identified as 3b,16adihydroxy-olean-12-en-30,28-lactone (Yayli et al., 1998). The sugar chain at C-3 was identical with that of 2 by comparison of their 1H and 13C NMR data (Table 1e3) together with results of acid hydrolysis. Hence, foegraecumoside I (9) was assigned as 3b-{b-D-6O-acetylglucopyranosyl-(1 / 2)-[b-D-glucopyranosyl-(1 / 4)]-aL-arabinopyranosyloxy}-16a-hydroxy-olean-12-en-30,28-lactone. Foegraecumoside J (10) was assigned the molecular formula of C53H84O22 from its HRESIMS (m/z 1095.5363 [M þ Na]þ, calcd 1095.5346) and 13C NMR data (Table 3). Comparing the 1H and 13C NMR data of 10 with those of 9 showed that it had the same aglycone as 9, but differed in its saccharide units. Then the sugar chain at C-3 of 10 was determined to be the same as that of 3 by careful comparison of their 1H and 13C NMR data (Table 1e3). Accordingly, foegraecumoside J (10) was established as 16a-hydroxy-3b-{a-L-rhamnopyranosyl-(1 / 2)-b-D-glucopyranosyl(1 / 4)-[b-D-glucopyranosyl-(1 / 2)]-a-L-arabinopyranosyloxy}olean-12-en-30,28-lactone. Foegraecumoside K (11) gave the molecular formula of C46H72O17 on the basis of its HRESIMS (m/z 919.4655 [M þ Na]þ, calcd 919.4662) and 13C NMR data (Table 3). Its NMR analysis indicated it possessed the same aglycone as 9 and had an identical

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7

Table 3 1 H and 13C NMR spectroscopic data of compounds 9e11.a position

1a 1b 2a 2b 3 4 5 6a 6b 7a 7b 8 9 10 11 12 13 14 15a 15b 16 17 18 19a 19b 20 21a 21b 22a 22b 23 24 25 26 27 28a 28b 29 30

9

10

dC, type

dH

dC, type

dH

dC, type

1.48, m 0.88, m 2.04, m 1.81 (overlapped) 3.20, dd (11.5, 4.5)

38.7, CH2

1.43 (overlapped) 0.80, m 1.97, m 1.77 (overlapped) 3.12, dd (12.0, 4.5)

38.6, CH2

1.45, dd (9.5, 3.0) 0.83, m 2.02 (overlapped) 1.78 (overlapped) 3.14, dd (11.5, 4.5)

38.6, CH2

0.74, d (11.5) 1.51 (overlapped) 1.31 (overlapped) 1.41, m 1.37, m 1.51 (overlapped) 1.83 (overlapped) 5.24, br s

2.11, dd (14.5, 5.0) 1.54 (overlapped) 3.66, m 2.74, dd (13.0, 9.0) 1.83 (overlapped) 1.69, dd (13.5, 13.0) 2.02 (overlapped) 1.58, m 2.16, m 2.03 (overlapped) 1.24, s 1.05, s 0.85, s 0.78, s 1.30, s 4.72, d (11.5) 4.30, d (11.5) 1.33, s

26.5, CH2 89.1, 39.5, 56.0, 18.4,

CH C CH CH2

32.6, CH2 40.3, C 47.2, CH 37.0, C 23.7, CH2 124.3, CH 140.6, C 43.0, C 37.3, CH2 74.4, 40.4, 41.7, 42.1,

CH C CH CH2

42.2, C 28.7, CH2 22.2, CH2 28.1, 16.6, 15.6, 16.0, 28.5, 81.6,

CH3 CH3 CH3 CH3 CH3 CH2

27.4, CH3 177.5, C

Ara at C-3 1 2 3 4 5a 5b

4.84, d (5.5) 4.49, m 4.37, m 4.46 (overlapped) 4.38, dd (12.0, 4.5) 3.76, dd (12.0, 2.0)

1 2 3 4 5 6a 6b OAc

5.07, 4.03, 4.10, 4.06, 3.80, 4.81, 4.71, 2.01,

1.45, dd (10.5, 7.5) 1.80 (overlapped) 5.22, br s

2.09, dd (14.0, 5.0) 1.52, dd (14.0, 12.5) 3.65, dd (12.5, 5.0) 2.74, dd (13.0, 8.5) 1.82, m 1.68, dd (13.0, 13.0) 2.02 (overlapped) 1.59, m 2.15, m 2.02 (overlapped) 1.15, s 1.00, s 0.77, s 0.74, s 1.29, s 4.72, d (11.5) 4.30, d (11.5) 1.34, s

104.4, CH 81.3, CH 72.7, CH 77.2, CH 64.0, CH2

4.93, br s 4.58, br d (7.0) 4.49 (overlapped) 4.57, m 4.39, dd (11.5, 5.0) 3.78, br d (11.5)

88.9, 39.4, 55.8, 18.3,

CH C CH CH2

32.5, CH2 40.28, C 47.1, CH 36.8, C 23.6, CH2 124.2, CH 140.4, C 42.9, C 37.2, CH2 74.4, CH 40.25, C 41.6, CH 42.0, CH2 42.1, C 28.6, CH2 22.2, CH2 28.0, 16.6, 15.5, 15.8, 28.4, 81.5,

CH3 CH3 CH3 CH3 CH3 CH2

27.4, CH3 177.5, C

105.7, CH 76.0, CH 78.0, CH 71.0, CH 75.2, CH 64.6, CH2

5.38, d (8.0) 4.08, dd (9.0, 8.0) 4.28 (overlapped) 4.24, dd (9.0, 9.0) 4.07 (overlapped) 4.51, dd (11.5, 2.0) 4.38, dd (11.5, 5.0)

4.47, d (12.0) 4.34, dd (12.0, 5.0)

1.48, dd (11.0, 6.5) 1.81 (overlapped) 5.23, br s

2.11, dd (14.5, 4.5) 1.53, dd (14.5, 12.0) 3.66, dd (12.0, 4.5) 2.73, dd (13.0, 8.5) 1.82 (overlapped) 1.69, dd (13.5, 13.0) 2.03 (overlapped) 1.59, m 2.16, m 2.02 (overlapped) 1.15, s 0.97, s 0.79, s 0.76, s 1.30, s 4.72, d (11.5) 4.30, d (11.5) 1.33, s

104.4, CH 80.8, CH 72.4, CH 74.7, CH 63.7, CH2

5.06b 4.52 (overlapped) 4.53 (overlapped) 4.49, m 4.31 (overlapped) 3.86, dd (11.5, 2.5)

105.4, CH 76.4, CH 78.0, CH 71.7, CH 78.1, CH 62.8, CH2

5.24, d (8.0) 4.12 (overlapped) 4.25 (overlapped) 4.24 (overlapped) 3.72 (overlapped) 4.38, dd (12.0, 2.0) 4.35, dd (12.0, 4.5

88.6, 39.4, 55.8, 18.3,

CH C CH CH2

32.5, CH2 40.32, C 47.1, CH 36.8, C 23.6, CH2 124.2, CH 140.5, C 42.9, C 37.2, CH2 74.4, CH 40.28, C 41.6, CH 42.0, CH2 42.1, C 28.6, CH2 22.1, CH2 28.1, 16.8, 15.6, 15.9, 28.4, 81.6,

CH3 CH3 CH3 CH3 CH3 CH2

27.4, CH3 177.6, C

104.1, CH 80.4, CH 72.7, CH 67.55, CH 64.0, CH2

Glc 103.8, CH 84.7, CH 77.9, CH 71.3, CH 77.8, CH 62.4, CH2

170.9, C 20.8, CH3 Glc-II

d (8.0) dd (8.5, 8.0) dd (9.0, 9.0) dd (9.0, 9.0) ddd (9.0, 5.5, 2.5)

0.66, d (11.0) 1.43 (overlapped) 1.28, m 1.40, m 1.37, m

26.3, CH2

Ara at C-3

Glc-I d (8.0) dd (8.5, 8.0) dd (9.0, 8.5) m ddd (9.0, 5.0, 2.0) dd (11.5, 2.0) dd (11.5, 4.5) s

Glc-II 5.16, 4.03, 4.18, 4.23, 3.87,

0.64, d (11.5) 1.42 (overlapped) 1.23, m 1.36, m 1.30, m

26.3, CH2

Ara at C-3

Glc-I

1 2 3 4 5a 5b 6a 6b

11

dH

Xyl

105.7, CH 75.7, CH 78.3, CH 71.5, CH 78.6, CH

5.24, d (8.0) 4.28 (overlapped) 4.20, dd (9.0, 9.0) 4.13, dd (9.0, 9.0) 3.80, m

103.1, CH 77.2, CH 79.5, CH 71.8, CH 78.4, CH

62.6, CH2

4.46, dd (12.0, 2.0) 4.29, dd (12.0, 5.0)

62.6, CH2

5.28, d (7.0) 4.10, dd (8.5, 7.0) 4.13, m 4.18, m 4.44, dd (11.5, 5.0) 3.73 (overlapped)

107.0, CH 76.1, CH 77.8, CH 70.8, CH 67.46, CH2

Rha 1 2

6.42, br s 4.73, br s

101.6, CH 72.4, CH (continued on next page)

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L.-M. Dai et al. / Phytochemistry xxx (2017) 1e10

Table 3 (continued ) position

9

10

dH

dC, type

3 4 5 6

11

dH

dC, type

4.69, dd (9.5, 3.5) 4.28 (overlapped) 5.04, m 1.80, d (6.0)

72.7, 74.8, 69.4, 18.9,

dH

dC, type

CH CH CH CH3

a 1 H NMR data (d) were measured in pyridine-d5 at 500 MHz. Coupling constants (J) in Hz are given in parentheses.13C NMR data (d) were measured in pyridine-d5 at 125 MHz. The assignments were based on 1He1H COSY, HSQC, HMBC, and NOESY experiments. b Signal overlapped by solvent peaks.

sugar structure at C-3 with 5, by comparison of their 1H and 13C NMR data. Thus, foegraecumoside K (11) was identified as 16ahydroxy-3b-{b-D-xylopyranosyl-(1 / 2)-b-D-glucopyranosyl(1 / 2)-a-L-arabinopyranosyloxy}-olean-12-en-30,28-lactone. The known triterpenoid saponins isolated from the aerial parts of L. foenum-graecum were identified as lysichriside A (12) (Tian et al., 2008), ardisiacrispin B (13) (Jansakul et al., 1987), ardisiacrispin A (14) (Jansakul et al., 1987), cyclaminorin (15) (Çalis¸ et al., 1997), 3-O-a-L-rhamnopyranosyl-(1 / 2)-b-D-glucopyranosyl(1 / 4)-a-L-arabinopyranosyl-cyclamiretin A (16) (Lavaud et al., 1994), ardisimamilloside A (17) (Huang et al., 2000), ardisimamilloside B (18) (Huang et al., 2000), and ardisimamilloside H (19) (Huang et al., 2003), respectively, by comparison of their spectroscopic data with literature data. All isolated saponins were evaluated for their cytotoxic activities against four human cancer cell lines (NCI-H460, MGC-803, HepG2, and T24) with doxorubicin as a positive control (Table 4). Saponins with the aglycone cyclamiretin A (1, 2, and 12e16) exhibited moderate cytotoxicity against all tested human cancer cell lines, with IC50 values of 9.3e24.5 mM, while the other saponins (3e11 and 17e19) were inactive (IC50 > 50 mM). Among the 13b,28-epoxyoleanan-30-al-type triterpenoid saponins and by comparing the cytotoxicity of 1 and 2 with 18 and 19, the results suggested that the hydroxy group at C-16 might have an effect on the activity. Simultaneously, the cytotoxic activities of compounds 1, 2, and 12e16 were tested on drug-sensitive and drug-resistant lung cancer cell lines (A549 and A549/CDDP, respectively). As shown in Table 5, compound 13 displayed moderate cytotoxicity against A549/CDDP, with an IC50 value of 8.7 mM and a resistant factor (RF) of 0.9. Furthermore, all of the tested compounds showed no cytotoxicity against the human normal liver cell line HL-7702 (IC50 > 50 mM), which suggested that the triterpenoid saponins from the n-BuOH-soluble extract of L. foenum-graecum might have potential as a starting point for the development of new anticancer drugs.

Table 4 Cytotoxic Activity of compounds 1e19 By the MTT method. compd.

1 2 12 13 14 15 16 Doxorubicinb

IC50 (mM)a NCI-H460

MGC-803

HepG2

T24

HL-7702

20.6 ± 1.7 19.6 ± 1.5 19.4 ± 1.2 11.5 ± 1.4 10.4 ± 1.0 10.5 ± 1.1 17.3 ± 1.0 1.3 ± 1.5

24.5 ± 0.9 16.6 ± 1.5 20.4 ± 1.2 9.3 ± 0.6 10.1 ± 0.8 13.3 ± 1.2 13.0 ± 1.2 4.9 ± 1.7

24.1 ± 1.2 13.1 ± 1.9 17.5 ± 1.3 9.4 ± 1.5 11.5 ± 0.9 17.3 ± 1.3 14.7 ± 1.7 2.5 ± 1.4

18.8 ± 1.1 16.2 ± 1.5 14.5 ± 1.3 9.8 ± 1.3 11.0 ± 1.2 10.4 ± 1.4 13.9 ± 1.1 1.6 ± 0.8

>50 >50 >50 >50 >50 >50 >50 13.7 ± 1.3

a Results are expressed as means ± SD (n ¼ 3). Compounds 3e11 and 17e19 were inactive against all cell lines tested (IC50 > 50 mM). b Positive control.

Table 5 Cytotoxic Activities of Compounds 1, 2, and 12e16 on Drug-resistant Lung Cancer Cell Lines (A549 and A549/CDDP cells, respectively). compd.

1 2 12 13 14 15 16 Doxorubicinb a b

IC50 (mM)a A549

A549/CDDP

RF (resistant factor)

17.5 ± 1.3 17.7 ± 1.8 14.3 ± 1.2 9.6 ± 1.7 12.5 ± 1.8 13.5 ± 1.8 16.8 ± 1.0 0.5 ± 1.2

17.4 ± 1.6 14.1 ± 2.1 14.2 ± 1.5 8.7 ± 1.6 10.9 ± 1.1 15.7 ± 1.4 16.1 ± 2.0 >50

1.0 0.8 1.0 0.9 0.9 1.2 1.0 e

Results are expressed as means ± SD (n ¼ 3). Positive control.

3. Conclusions Phytochemical study of the aerial parts of L. foenum-graecum generated eleven new oleanane-type triterpenoid saponins (1e11) and eight known ones (12e19). Saponins with the aglycone cyclamiretin A (1, 2, and 12e16) exhibited moderate cytotoxicity against all tested human cancer cell lines (NCI-H460, MGC-803, HepG2, and T24), with IC50 values of 9.3e24.5 mM. Simultaneously, compound 13 displayed moderate cytotoxicity against A549/CDDP, with an IC50 value of 8.7 mM and a resistant factor (RF) of 0.9. This work enriched the diversity of triterpenoid saponins of the genus Lysimachia, and the bioassay results suggested that L. foenum-graecum might be a prospective species for the discovery of anticancer compounds. 4. Experimental 4.1. General experimental procedures Optical rotations were obtained using a PerkinElmer model 341 polarimeter, whereas IR spectra were acquired on a PerkinElmer Spectrum Two FT-IR spectrometer. NMR data were measured using Bruker AVANCE 500 MHz spectrometers, with compounds dissolved in pyridine-d5. Chemical shifts are expressed in d (ppm) and referenced to residual solvent signals for pyridine-d5 (dH 7.19 ppm, dC 123.5 ppm). HRESIMS measurements were performed on a Thermo-Scientific Exactive spectrometer. HPLC was carried out on a Shimadzu LC-6AD apparatus with a RID-10A detector, using a YMCPack ODS-A column (250 mm  20 mm, 5 mm) for preparative HPLC and a YMC-Pack ODS-A column (250 mm  10 mm, 5 mm) for semipreparative HPLC. Analytical HPLC was conducted on a Jasco LC-4000 instrument. Column chromatography (CC) was conducted using silica gel (200e300 mesh, Qingdao Marine Chemical Factory, China), ODS (50 mm, YMC, Japan), and MCI gel (CHP20, 75e150 mm, Mitsubishi Chemical Corporation, Japan), respectively. Precoated silica gel GF254 plates (Qingdao Marine Chemical Factory) were

Please cite this article in press as: Dai, L.-M., et al., Cytotoxic triterpenoid saponins from Lysimachia foenum-graecum, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.021

L.-M. Dai et al. / Phytochemistry xxx (2017) 1e10

used for TLC. Spots were visualized by spraying with 10% H2SO4 in EtOH followed by heating. 4.2. Plant material Aerial parts of L. foenum-graecum were collected in Jinxiu, Guangxi Zhuang Autonomous Region, People's Republic of China, in July 2014. The plant material was identified by Professor Shao-Qing Tang (Guangxi Normal University). A voucher specimen (No. LF2014022) is deposited in the State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmaceutical Sciences, Guangxi Normal University, Guilin. 4.3. Extraction and isolation Aerial parts of L. foenum-graecum (14.7 kg) were extracted three times (each for 3 h) under conditions of reflux using EtOH:H2O (95:5, v/v; 75 L  3). After removing the solvent of combined extracts under reduced pressure, the dried residue (1.6 kg) was suspended in H2O and partitioned with EtOAc and n-BuOH, successively. The n-BuOH-soluble extract was subjected to macroporous resin CC, eluted with H2O and H2O:EtOH (80:20; 50:50; 30:70; and 5:95; v/v). Subsequently, the EtOH-H2O (70:30, v/v) fraction (228 g) was subjected to silica gel CC using a gradient of CH2Cl2-MeOH (10:1 / 3:1, v/v, then MeOH) to yield fractions AeF. Fraction B (1 g) was further separated by RP-C18 silica gel CC, eluted with MeOH:H2O (73:27 / 79:21, v/v, then MeOH) to afford four subfractions (B1eB4). Subfraction B2 (222.4 mg) was purified by preparative HPLC, eluted with CH3CN:H2O (48:52, v/v, at 8 mL/min) to yield compound 19 (24.9 mg, tR 15.9 min). Fraction C (7 g) was subjected to RP-C18 CC, eluted with MeOH:H2O (44:56 / 70:30, v/ v, then MeOH) to give 15 subfractions (C1eC15). Subfractions C3 (105.5 mg), C4 (84.1 mg), C5 (170.1 mg), and C6 (78.0 mg) were further purified by preparative HPLC, eluted with CH3CN:H2O (34:66, v/v) at a flow rate of 8 mL/min to afford compounds 11 (9.8 mg, tR 22.8 min), 9 (16.1 mg, tR 28.8 min), and 15 (15.7 mg, tR 43.7 min), and compound 12 (13.1 mg, tR 32.7 min) was eluted with CH3CN:H2O (38:62, v/v), respectively. Subfraction C7 (148.9 mg) was also subjected to preparative HPLC, eluted with CH3CN:H2O (36:64, v/v, at 8 mL/min) to afford compounds 1 (31.2 mg, tR 54.1 min) and 2 (12.6 mg, tR 60.1 min). Subfractions C9 (148.9 mg), C10 (74.8 mg), C11 (110.3 mg), and C12 (84.0 mg) were subjected to preparative HPLC at a flow rate of 8 mL/min with a gradient elution of CH3CN:H2O (38:62 / 43:57, v/v) to yield compounds 16 (24.1 mg, tR 42.3 min), 18 (12.7 mg, tR 53.5 min), 8 (12.4 mg, tR 47.1 min), and 5 (8.0 mg, tR 37.6 min), respectively. Fraction E (92.8 g) was fractionated using MCI gel CC with MeOH:H2O (50:50 / 100:0, v/v) to obtain 10 subfractions (E1eE10). E3 (5.9 g) was further purified by RP-C18 CC using MeOH:H2O (50:50 / 70:30, v/v, then MeOH) to yield 18 subfractions (E3-1 to E3-18). Subfractions E3-7 (159.0 mg), E3-10 (90.0 mg), and E3-12 (104.6 mg) were purified by semi-preparative HPLC (3 mL/min) using a MeOH:H2O gradient (60:40 / 65:35, v/v) to afford compounds 10 (10.2 mg, tR 23.2 min, 14 (13.6 mg, tR 29.8 min), and 13 (30.4 mg, tR 23.6 min), respectively. E6 (3.5 g) was subjected to RPC18 CC and eluted with MeOH:H2O (50:50 / 65:35, v/v, then MeOH) to yield 11 subfractions (E6-1 to E6-11). E6-2 (171.6 mg) was purified by preparative HPLC, eluted with CH3CN:H2O (28:72, v/v, at 8 mL/min) to afford compounds 3 (11 mg, tR 16.6 min) and 17 (17.7 mg, tR 35.5 min). E6-4 (92.6 mg), E6-6 (94.0 mg), and E6-9 (76.6 mg) were also subjected to preparative HPLC, eluted with a CH3CN:H2O gradient (23:77 / 30:70, v/v, at 8 mL/min) to yield compounds 7 (13.0 mg, tR 32.7 min), 6 (14.1 mg, tR 29.7 min), and 4 (16.2 mg, tR 32.0 min), respectively.

9

4.3.1. Foegraecumoside A (1) Amorphous powder, [a]20D 16 (c 0.1, MeOH); IR (KBr) nmax 3425, 2930, 1731, 1637, 1384, 1253, 1075, 615 cm1; For 1H NMR (pyridine-d5, 500 MHz) and 13C NMR (pyridine-d5, 125 MHz) spectroscopic data, see Tables 1 and 2; positive-ion HRESIMS m/z 1139.5608 [M þ Na]þ (calcd for C55H88O23Na, 1139.5609). 4.3.2. Foegraecumoside B (2) Amorphous powder, [a]20D 4 (c 0.1, MeOH); IR (KBr) nmax 3426, 2930, 1730, 1384, 1253, 1075, 606 cm1; For 1H NMR (pyridine-d5, 500 MHz) and 13C NMR (pyridine-d5, 125 MHz) spectroscopic data, see Tables 1 and 2; positive-ion HRESIMS m/z 993.5020 [M þ Na]þ (calcd for C49H78O19Na, 993.5030). 4.3.3. Foegraecumoside C (3) Amorphous powder, [a]20D 18 (c 0.1, MeOH); IR (KBr) nmax 3436, 2940, 1730, 1637, 1384, 1084, 587 cm1; For 1H NMR (pyridine-d5, 500 MHz) and 13C NMR (pyridine-d5, 125 MHz) spectroscopic data, see Tables 1 and 2; positive-ion HRESIMS m/z 1113.5433 [M þ Na]þ (calcd for C53H86O23Na, 1113.5452). 4.3.4. Foegraecumoside D (4) Amorphous powder, [a]20D 10 (c 0.1, MeOH); IR (KBr) nmax 3426, 2930, 1721, 1637, 1384, 1075, 615 cm1; For 1H NMR (pyridine-d5, 500 MHz) and 13C NMR (pyridine-d5, 125 MHz) spectroscopic data, see Tables 1 and 2; positive-ion HRESIMS m/z 1113.5432 [M þ Na]þ (calcd for C53H86O23Na, 1113.5452). 4.3.5. Foegraecumoside E (5) Amorphous powder, [a]20D 17 (c 0.1, MeOH); IR (KBr) nmax 3436, 2940, 1739, 1637, 1384, 1243, 1047, 615 cm1; For 1H NMR (pyridine-d5, 500 MHz) and 13C NMR (pyridine-d5, 125 MHz) spectroscopic data, see Tables 1 and 2; positive-ion HRESIMS m/z 1007.5173 [M þ Na]þ (calcd for C50H80O19Na, 1007.5186). 4.3.6. Foegraecumoside F (6) Amorphous powder, [a]20D 6 (c 0.1, MeOH); IR (KBr) nmax 3436, 2940, 1637, 1384, 1075, 541 cm1; For 1H NMR (pyridine-d5, 500 MHz) and 13C NMR (pyridine-d5, 125 MHz) spectroscopic data, see Tables 1 and 2; positive-ion HRESIMS m/z 1097.5505 [M þ Na]þ (calcd for C53H86O22Na, 1097.5503). 4.3.7. Foegraecumoside G (7) Amorphous powder, [a]20D 6 (c 0.1, MeOH); IR (KBr) nmax 3436, 2940, 1730, 1637, 1384, 1253, 1075, 541 cm1; For 1H NMR (pyridine-d5, 500 MHz) and 13C NMR (pyridine-d5, 125 MHz) spectroscopic data, see Tables 1 and 2; positive-ion HRESIMS m/z 1155.5536 [M þ Na]þ (calcd for C55H88O24Na, 1155.5558). 4.3.8. Foegraecumoside H (8) Amorphous powder, [a]20D 8 (c 0.1, MeOH); IR (KBr) nmax 3436, 2940, 1740, 1637, 1384, 1243, 1047, 615 cm1; For 1H NMR (pyridined5, 500 MHz) and 13C NMR (pyridine-d5, 125 MHz) spectroscopic data, see Tables 1 and 2; positive-ion HRESIMS m/z 1049.5276 [M þ Na]þ (calcd for C52H82O20Na, 1049.5292). 4.3.9. Foegraecumoside I (9) Amorphous powder, [a]20D 41 (c 0.1, MeOH); IR (KBr) nmax 3436, 2940, 1721, 1637, 1384, 1253, 1084, 606 cm1; For 1H NMR (pyridine-d5, 500 MHz) and 13C NMR (pyridine-d5, 125 MHz) spectroscopic data, see Table 3; positive-ion HRESIMS m/z 991.4868 [M þ Na]þ (calcd for C49H76O19Na, 991.4873). 4.3.10. Foegraecumoside J (10) Amorphous powder, [a]20D 49 (c 0.1, MeOH); IR (KBr) nmax

Please cite this article in press as: Dai, L.-M., et al., Cytotoxic triterpenoid saponins from Lysimachia foenum-graecum, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.021

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L.-M. Dai et al. / Phytochemistry xxx (2017) 1e10

3436, 2940, 1712, 1384, 1074, 624 cm1; For 1H NMR (pyridine-d5, 500 MHz) and 13C NMR (pyridine-d5, 125 MHz) spectroscopic data, see Table 3; positive-ion HRESIMS m/z 1095.5363 [M þ Na]þ (calcd for C53H84O22Na, 1095.5346). 4.3.11. Foegraecumoside K (11) Amorphous powder, [a]20D 41 (c 0.1, MeOH); IR (KBr) nmax 3436, 2940, 1712, 1636, 1393, 1075, 615 cm1; For 1H NMR (pyridine-d5, 500 MHz) and 13C NMR (pyridine-d5, 125 MHz) spectroscopic data, see Table 3; positive-ion HRESIMS m/z 919.4655 [M þ Na]þ (calcd for C46H72O17Na, 919.4662). 4.4. Acid hydrolysis of the saponins and determination of the absolute configuration of the monosaccharides A solution of each compound (2 mg) in 1 M HCl (1,4-dioxane-H2O, 1:1, 5 mL) was stirred at 80  C for 8 h. After cooling, each reaction mixture was extracted with CH2Cl2. Each aqueous layer was evaporated under vacuum, diluted with H2O repeatedly to furnish a neutral residue. Then each residue was subjected to HPLC (Jasco LC-4000) under the following conditions: column, Shodex Asahipak NH2P50 4E (250 mm  4.6 mm, 5 mm); detection, Jasco OR-4090 optical rotation detector. For the sugar units composed of glucose, arabinose, and rhamnose (1, 3, 4, 6, 7, and 10), or glucose and arabinose (2 and 9), or D-glucose, L-arabinose, and L-rhamnose were confirmed by comparison of their retention times and optical rotations with those of authentic samples (Yoshikawa et al., 2003, 2007), tR (CH3CN:H2O, 78:22, v/v, 1 mL/min): 6.5 min (L-rhamnose, negative optical rotation), 7.7 min (L-arabinose, positive optical rotation), and 11.3 min (Dglucose, positive optical rotation). The absolute configurations of the sugar units in 5, 8, and 11 were determined in a similar way, tR (CH3CN:H2O, 84:16, v/v, 1 mL/min): 14.8 min (L-arabinose, positive optical rotation), 16.0 min (D-xylose, positive optical rotation), and 28.0 min (D-glucose, positive optical rotation). 4.5. Cytotoxicity assay Compounds 1e19 (purity of each compound > 95%, analyzed by HPLC with a RID-10A detector) were tested for cytotoxicity against NCI-H460 (human lung cancer cell line), BGC-803 (human gastric cancer cell line), HePG2 (human liver cancer cell line), T24 (human bladder cancer cell line), and HL-7702 (human normal liver cell line), and compounds 1, 2, and 12e16 against A549 (human lung cancer cell line) and A549/CDDP (human cisplatin-resistant lung cancer cell line) by means of the MTT method as described (Huang et al., 2015; Hua et al., 2015). Cell lines were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences. All were individually cultured in 96-well microtitre plates at a cell density of 1  105 cells/well in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum. Plates were incubated at 37  C in a humidified atmosphere with 5% CO2 overnight. Cells were treated in triplicate with five concentrations (2.5, 5, 10, 20, and 50 mM) of the tested compounds for 48 h, with doxorubicin as positive control. Cells were stained with 10 mL (10 mg/mL) of MTT in the incubator at 37  C for about 4 h. After removal of the supernatant, DMSO (100 mL) was added to dissolve the formazan crystals. The absorbance was read by a microplate reader at 570/630 nm, with the data processed by Student's t-test with a significance level of P < 0.05 using SPSS software (17.0; SPSS, Inc., Chicago, IL, USA). All the tests were repeated in three independent experiments. Acknowledgments We gratefully acknowledge financial support from the National Natural Science Foundation of China (21462006 and 21431001),

the gs2:Ministry of Education of China (IRT_16R15), the Natural Science Foundation of Guangxi (2014GXNSFBA118042 and 2016GXNSFGA380005), the Department of Education of Guangxi (ZD2014024), the project of State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, Ministry of Science and Technology of China (CMEMR2016-A02), and Guangxi Normal University (2013ZD001). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.phytochem.2017.01.021. References Chang, X.L., Li, W., Jia, Z.H., Satou, T., Fushiya, S., Koike, K., 2007. Biologically active triterpenoid saponins from Ardisia japonica. J. Nat. Prod. 70, 179e187. _ Yürüker, A., Tanker, N., Wright, A.D., Sticher, O., 1997. Triterpene saponins Çalis¸, I., from Cyclamen coum var. coum. Planta Med. 63, 166e170. Dong, W.W., Liu, X., Li, X.B., Yang, D.J., Ding, L.H., 2011. A new triterpene saponin from Androsace integra. Fitoterapia 82, 782e785. 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Please cite this article in press as: Dai, L.-M., et al., Cytotoxic triterpenoid saponins from Lysimachia foenum-graecum, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.021