New cycloartane triterpene glycosides from Thalictrum ramosum

Phytochemistry Letters 15 (2016) 108–112

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New cycloartane triterpene glycosides from Thalictrum ramosum Fan-Cheng Menga , Cen Yuana , Xiao-Jun Huanga,b , Wen-Jing Wanga,b , Li-Gen Lina , Xian-Tao Zhangc, Hao-Yan Jiaoc , Qing-Wen Zhanga,* a

State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao SAR, China Guangdong Research Institute of Traditional Chinese Medicine, Guangzhou 510520, China c Institute of Traditional Chinese Medicine, Guangdong Food and Drug Vocational College, Guangzhou, Guangdong 510520, China b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 21 September 2015 Received in revised form 27 November 2015 Accepted 11 December 2015 Available online xxx

Three new cycloartane triterpene glycosides, cycloramosides A–C (1–3), were isolated from Thalictrum ramosum B. Boivin. Those compounds were determined as 3-O-a-L-arabinopyranosyl-3b,16b,24S,25,30pentaolcycloartane-16-O-b-D-glucopyranoside (1), 3-O-a-L-arabinopyranosyl-3b,16b,24S,25,30-pentaolcycloartane-24-O-b-D-glucopyranoside (2) and 3-O-a-L-arabinopyranosyl-3b,16b,24S,25-tetrahydoxylcycloartane-16-O-b-D-glucopyranoside (3), respectively. Their structures were elucidated by hydrolysis and extensive spectroscopic methods including 1D and 2D NMR experiments (1H, 13C, DEPT, COSY, ROESY, HSQC, HMBC) along with HR-ESI–MS analyses. ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

Keywords: Thalictrum ramosum Cycloartane triterpeniod Saponin Cycloramosides A–C

1. Introduction The genus Thalictrum (Ranunculaceae) is commonly known as ‘meadow rue’. Some Thalictrum spp. have been used in Traditional Chinese Medicine for a couple of thousand years for its antiinflammation and antibacterial effects. So far approximately 150 species of Thalictrum have been discovered globally, 67 of which have been found in China. Among those species found in China, 29 have been utilized in medicinal practices and 14 of them are frequently used as substitutes for Coptidis Rhizoma. Because their appearance is similar to that of horsetail (‘mawei’ in Chinese) and their effects resemble that of Coptidis Rhizoma, these herbal Thalictrum spp. are often referred to as ‘mawei huanglian’ or ‘mawei lian’, being important ingredients of Tibetan medicine. (Delectis Florae Reipublicae Popularis Sinicae Agendae Academiae Sinicae Edita, 1979; Dimaer, 2012). Numerous natural products, such as alkaloids, triterpenes, flavonoids, steroids and organic acids, have previously been isolated from Thalictrum spp. Recent studies have revealed the important associations of Thalictrum spp. with their antitumor, anti-inflammation, and antivirus activities, as well as effects on the cardio-vascular, autoimmune, and central nervous system. (Lutskii et al., 2005; Liu, 2010; Chen and Xu, 2000; Lin et al., 1994, 1999; Varadinova et al., 1996) As an advancement from our previous investigation of isolation tcylcoartane triterpene glycosides from

* Corresponding author. Fax: +86 853 2884 1358. E-mail addresses: [email protected], [email protected] (Q.-W. Zhang).

the plants in Ranunculaceae family (Zhang et al., 2012, 2001a,b; Ye et al., 1999), we report in this study the isolation of three new cycloartane saponins (Fig. 1) from Thalictrum ramosum B. Boivin. Their structures were elucidated by hydrolysis followed by extensive spectroscopic methods including 1D and 2D NMR experiments (1H, 13C, DEPT, COSY, ROESY, HSQC and HMBC) and HR-ESI–MS. 2. Results and discussion Compound 1 was obtained as a white amorphous powder. The molecular formula of 1 was determined to be C41H70O14, based upon its HR-ESI–MS data (m/z 809.4653 [M + Na]+, calcd for C41H70O14Na 809.4658). The 1H NMR spectrum of 1 showed the cycloartane-type triterpene characteristic signals due to a cyclopropane methylene group (Zhang et al., 2013, 1999, 2001a,b.) at dH 0.35 (1H, d, J = 3.7 Hz) and 0.23 (1H, d, J = 3.7 Hz), six methyl groups at dH 1.55 (3H, s), 1.52 (6H, s), 1.24 (3H, s), 1.03 (3H, d, J = 6.6 Hz), and 0.86 (3H, s), three methine proton signals at dH 4.41 (1H, m), 3.98 (1H, dd, J = 8.6, 1.0 Hz) and 3.70 (1H, dd, J = 11.6, 4.5 Hz), as well as an oxygenated methylene proton signals at dH 4.51 (1H, d, J = 11.4 Hz) and 3.79 (1H, d, J = 11.4 Hz). In the 13C and DEPT NMR spectra of 1, 41 carbon signals were observed including six methyl carbons (dC 26.2, 25.8, 21.3, 20.5, 19.8 and 17.5), three oxygenated methine carbons (dC 89.5, 82.8 and 78.2), as well as an oxygenated methylene carbons (dC 63.3). Comparison of the 13C NMR spectrum of 1 with those of the known compounds (3b,16b,24S)-cycloartane-3,16,24,25,30-pentol

http://dx.doi.org/10.1016/j.phytol.2015.12.001 1874-3900/ ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

F.-C. Meng et al. / Phytochemistry Letters 15 (2016) 108–112

109

Fig. 2. Key NOE correlations of aglycone of 1.

Fig. 1. Structures of compounds 1–4.

3,25-di-b-D-glucopyranoside (Sun et al., 2008) and 3-O-b-Dglucopyranosyl (24S)-cycloartane-3b,16b,24,25,30-pentaol 25-Ob-D-glucopyranosyl-(1!6)-b-D-glucopyranoside (Zhang et al., 2013) indicated that the aglycone of 1 was 24S-cycloartane3b,16b,24,25,30-pentaol (cyclofoetigenin B, a known compound). This was further confirmed by the 1H–1H COSY, HSQC, HMBC and ROESY data and acid hydrolysis. In the ROESY spectrum of 1 (Fig. 2), NOE correlations between H-5 (dH 1.39) and H-3 (dH 3.70)/H3-28 (dH 0.86), between H-3 (dH 3.70) and H-29 (dH 1.55), as well as between H-16 (dH 4.41) and H3-28 (dH 0.86)/H-17 (dH 1.86) were observed. Thus, the relative configurations of the oxygenated carbons were determined to be b-OH for C-3 and C-16. The absolute configuration of C-24 could be determined to be S by the 13C NMR chemical shift of C-24 (dC 78.2).

According to the literature data, whereas the chemical shift of C-24 is in the range of 77.1 - 78.6 ppm for the 24 S configuration (Özipek et al., 2005; Naubeev et al., 2007; Kim et al., 2008; Horo et al., 2012; Savran et al., 2012; Tigoufack et al., 2010), while the chemical shift of C-24 is in the range of 79.6–81.0 ppm for the 24R configuration (Tigoufack et al., 2010; Lu et al., 2012; Karabey et al., 2012; Naubeev et al., 2014; Zhao et al., 2008, 2011; Naubeev et al., 2011). The above deduction was also supported by acid hydrolysis. As expected, acid hydrolysis of compound 1 gave a secondary sapogenin, compound 4 (24S-lanost-9(11)-ene-3b, 16b, 24, 25, 30-pentanol) (Ganenko et al., 1986), presumably due to the easy opened 9,19 three-membered ring in acidic condition. Furthermore, two anomeric protons at dH 4.94 (d, J = 6.3 Hz) and 4.74 (d, J = 7.8 Hz) were observed in the 1H NMR spectrum. In the 13 C NMR spectrum, 11 carbons signals at dC 106.8, 106.4, 78.5, 78.1, 75.6, 74.3, 72.7, 71.8, 69.0, 66.1, 62.9 were additionally observed. These data showed the presence of one arabinopyranosyl unit and one glycopyranosyl unit. The 1H and 13C chemical shifts of the two sugar units were assigned unambiguously through a combination of HSQC and HMBC spectroscopic analyses. The linkage sites were determined by the HMBC spectroscopic analysis, which showed the key correlations between H-1 (dH 4.94) of arabinose and C-3 (dC 89.5) of aglycone, and between H-1 (dH 4.74) of the glucose and C-16 (dC 82.8) of aglycone. Acid hydrolysis of 1 also yielded L-arabinose and D-glucose, which were determined by HPLC analysis. The relative anomeric configuration of L-arabinose and D-glucose moieties were elucidated as a and b, respectively, based on the 3JH1–H2 coupling constants (J = 6.3 Hz and J = 7.8 Hz, respectively). Thus, the structure of compound 1 was elucidated as 3-O-a-L-arabinopyranosyl-3b,16b,24S,25,30-pentaolcycloartane-16-O-b-D-glucopyranoside, named Cycloramoside A. Compound 2 was obtained as a white amorphous powder. The HR-ESI–MS of 2 (m/z 809.4655 [M + Na]+, calcd for C41H70O14Na 809.4658) supported a molecular formula of C41H70O14, indicating that compound 2 was the isomeric compound of compound 1. Comparison of the 13C NMR spectrum of 2 with compound 1 showed a good agreement between the two, except for C-16 and C-24. The chemical shifts of C-16 and C-24 for compound 2 were 71.1 and 90.5, respectively, while the chemical shifts of C-16 and C-24 for compound 1 were 82.8 and 78.2, respectively. Therefore, we speculated that the aglycone of 2 was the same with that of compound 1 and the linkage sites of the sugar units differed between compounds 2 and 1. This deduction was further confirmed by assigning all the 1H and 13C NMR signals of 2 with the assistance of 1H-1H COSY, HSQC, HMBC and NOESY experiments. In the HMBC spectrum, key correlations between H-1 (dH 4.96) of arabinose and C-3 (dC 89.4) of aglycone, and between H-1 (dH 5.09) of the glucose and C-24 (dC 90.5) of aglycone were observed. The absolute configuration of C-24 could be determined to be S by the 13C NMR chemical shift of C-24 (90.5) (Zhang et al., 1999; Çalis et al., 1999; Bedir et al., 1999, 1998; Polat et al., 2009; Yalçın et al., 2012) Acid hydrolysis of 2 yielded L-arabinose and D-glucose, which were determined by HPLC analysis. The relative anomeric configuration of L-arabinose and D-glucose moieties were also elucidated as a and b, respectively, based on the 3J H1-H2 coupling

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constants (J = 6.3 Hz and J = 7.9 Hz). On the basis of the aforementioned information, the structure of compound 2 was established as 3-O-a-L-arabinopyranosyl-3b,16b,24S,25,30-pentaolcycloartane-24-O-b-D-glucopyranoside, denoted as Cycloramoside B. Compound 3 was obtained as a white amorphous powder. The molecular formula of 3 was determined to be C41H70O13 from its HR-ESI–MS data (m/z 793.4704 [M + Na]+, calcd for C41H70O13Na 793.4714). Compared with the 1H NMR specturm of 1, the 1H NMR data of 3 was in agreement with compound 1 but a methyl signal at dH 1.04 (3H, s) for 3 instead of an oxygenated methylene protons at dH 4.51 (1H, d, J = 11.4 Hz) and 3.79 (1H, d, J = 11.4 Hz) for 1. The same result could be found in the 13C NMR spectra of 1 and 3. One more

methyl carbon was found in the 13C NMR spectrums of 3 than was observed in that of compound 1, while the oxygenated methylene carbon (dC 63.3) was not observed. With the assistance of 1H-1H COSY, HSQC, HMBC and NOESY experiments, all of the 1H and 13C NMR signals of 3 were assigned. The aglycone of 3 was identified as cyclofoetigenin A. The absolute configuration of C-24 was determined to be S by the 13C NMR chemical shift of C-24 (78.1). Acid hydrolysis of 3 yielded L-arabinose and D-glucose. The linkage sites were determined by the HMBC spectral analysis, which showed the key correlations between H-1 (dH 4.82) of arabinose and C-3 (dC 88.5) of aglycone, and between H-1 (dH 4.77) of the glucose and C-16 (dC 82.8) of aglycone. The 3JH1-H2 coupling constants (J = 7.1 Hz and J = 7.8 Hz) of L-arabinose and D-glucose indicated the a and b

Table 1 NMR data of compounds 1,2, 3 and 4 (d in ppm and J in Hz). 1a

2a

3a

4a

dC

dH (J)

dC

dH (J)

dC

dH (J)

dC

dH (J)

1

32.0

32.0

3 4 5 6

89.5 45.0 47.8 21.9

7

26.3

8 9 10 11

48.5 21.1 25.6 26.6

1.88 (1H, m) 1.53 (1H, m) 2.14 (1H, m) 2.07 (1H, m) 3.67 (1H, dd, 11.6, 4.6) – 1.12 (1H, overlap) 1.82 (1H, m) 1.45 (1H, m) 1.65 (1H,m) 1.34 (1H, m) 2.32 (1H, overlap) – – 5.34 (1H, br d, 5.6)

12

32.9

13 14 15

45.7 46.9 48.2

16 17 18 19

82.8 57.8 19.8 30.0

20 21 22

29.6 17.5 33.5

23

28.9

24 25 26 27 28 29 30

78.2 72.8 26.2 25.8 20.5 21.3 63.3

1.53 (1H, m) 1.22 (1H, m) 2.37 (1H, m) 1.97 (1H, m) 3.50 (1H, dd, 11.6, 4.3) – 1.29 (1H, dd, 12.3, 4.3) 1.58 (1H, m) 0.56 (1H, m) 1.95 (1H,m) 1.02 (1H, m) 1.41 (1H, dd, 12.7, 4.8) – – 1.17(1H, m) 0.96 (1H, m) 2.34 (1H, m) 1.63 (1H, m) – – 2.22 (1H, dd, 13.9, 8.0) 2.05 (1H, dd, 14.2, 4.1) 4.44 (1H, m) 1.88 (1H, m) 1.27 (3H, s) 0.41 (1H, d, 3.5) 0.23 (1H, d, 3.9) 2.41 (1H, m) 1.05 (3H, d, 6.6) 1.67 (1H, m) 1.62 (1H, m) 2.14 (1H, m) 1.87 (1H, m) 4.00 (1H, dd, 9.7, 1.2) – 1.54 (3H, s) 1.54 (3H, s) 0.88 (3H, s) 1.32 (3H, s) 1.04 (3H,s)

36.4

30.0

Ara-1 Ara-2 Ara-3 Ara-4 Ara-5

106.4 72.7 74.3 69.0 66.1

Glu-1 Glu-2 Glu-3 Glu-4 Glu-5 Glu-6

106.8 75.6 78.1 71.8 78.5 62.9

1.54 (1H, m) 1.19 (1H, m) 2.42 (1H, m) 2.15 (1H, m) 3.73 (1H, dd, 11.6, 4.8) – 1.58 (1H, overlap) 1.66 (1H, m) 0.97 (1H, m) 1.97 (1H,m) 1.03 (1H, m) 1.45 (1H,dd, 13.0, 6.1) – – 1.27 (1H, m) 0.97 (1H, m) 2.47 (1H, m) 1.69 (1H, d, 12.3) – – 2.08 (1H, dd, 13.0, 8.3) 1.69 (1H, overlap) 4.74 (1H, m) 1.78 (1H, dd, 10.3, 7.6) 1.41 (3H, s) 0.48 (1H, d, 3.7) 0.28 (1H, d, 3.7) 2.35 (1H, m) 1.08 (3H, d, 6.5) 1.71 (1H, m) 1.60 (1H, m) 2.15(1H, m) 1.86 (1H, m) 3.95 (1H, dd, 8.8, 2.2) – 1.36 (3H, s) 1.43 (3H, s) 0.88 (3H, s) 1.58 (3H, s) 4.55 (1H, d, 11.1) 3.84 (1H, d, 10.0) 4.96 (1H, d, 6.3) 4.48 (1H, dd, 7.9, 6.6) 4.27 (1H, dd, 8.7, 3.1) 4.39 (1H, m) 4.39 (1H, d, 11.0) 3.85 (1H, d, 10.0) 5.09 (1H, d, 7.9) 4.09 (1H, t, 8.4) 3.95 (1H, dd, 8.8, 2.0) 4.22 (1H, d, 6.0) 4.23 (1H, m) 4.54 (1H, dd, 11.1, 2.3) 4.32 (1H, dd, 11.6, 5.6)

32.2

2

1.57 (1H, m) 1.21 (1H, m) 2.36 (1H, m) 2.09 (1H, m) 3.70 (1H, dd, 11.6, 4.5) – 1.39 (1H, dd, 12.3, 4.3) 1.62 (1H, m) 0.80 (1H, m) 1.95 (1H,m) 0.98 (1H, m) 1.34 (1H, dd, 12.5, 4.1) – – 1.15 (1H, m) 0.90 (1H, m) 2.33 (1H, m) 1.59 (1H, d, 12.3) – – 2.19 (1H, dd, 14.0, 8.0) 2.01 (1H, dd, 14.0, 4.2) 4.41 (1H, m) 1.86 (1H, m) 1.24 (3H, s) 0.35 (1H, d, 3.7) 0.23 (1H, d, 3.7) 2.39 (1H, m) 1.03 (3H, d, 6.6) 1.66 (1H, m) 1.60 (1H, m) 2.12 (1H, m) 1.85 (1H, m) 3.98 (1H, dd, 8.6, 1.0) – 1.52 (3H, s) 1.52 (3H, s) 0.86 (3H, s) 1.55 (3H, s) 4.51 (1H, d, 11.4) 3.79 (1H, d, 11.4) 4.94 (1H, d, 6.3) 4.45 (1H, dd, 8.0, 6.3) 4.25 (1H, dd, 8.0, 3.1) 4.38 (1H, m) 4.37 (1H, d, 9.9) 3.83 (1H, d, 9.9) 4.74 (1H, d, 7.8) 4.01 (1H, t, 7.8) 3.89 (1H, m) 4.20 (1H, d, 7.8) 4.18 (1H, m) 4.52 (1H, d, 11.5) 4.35 (1H, dd, 11.5, 5.1)

a

Measured in pyridine-d5.

30.1 89.4 46.7 48.8 21.4 26.4 47.9 22.0 25.6 26.8 32.3 45.0 45.7 49.4 71.1 57.6 19.6 30.0 29.8 17.7 33.3 29.3 90.5 72.0 27.1 24.9 20.4 21.2 63.3 106.4 72.8 74.4 69.0 66.2 105.6 75.5 78.4 71.6 78.4 62.5

30.0 88.5 41.3 47.5 21.0 26.4 48.0 19.9 26.3 26.1 32.9 45.8 47.0 48.1 82.8 57.8 19.6 30.0 29.6 17.5 33.5 28.9 78.1 72.8 26.3 25.8 20.4 25.7 15.4 107.5 72.9 74.7 69.6 66.8 106.9 75.7 78.3 71.8 78.6 63.0

4.82 (1H, d, 7.1) 4.48 (1H, dd, 8.6, 7.1) 4.19 (1H, dd, 8.9, 3.5) 4.34 (1H, m) 4.33 (1H, dd, 12.0, 2.6) 3.82 (1H, d, 10.7) 4.77 (1H, d, 7.8) 4.03 (1H, t, 8.3) 4.21 (1H, dd, 7.3, 4.4) 4.22 (1H, d, 6.6) 3.92 (1H, m) 4.55 (1H, dd, 11.6, 2.4) 4.37 (1H, dd, 11.6, 5.4)

28.9 80.1 43.3 53.6 21.9 28.8 42.0 148.8 39.5 115.2 37.3 44.6 45.1 47.0 71.8 55.9 15.7 23.6 28.6 18.3 33.0 27.8 77.1 72.5 26.4 25.8 19.4 23.7 64.5

2.16 (1H, m) 2.01 (1H, m) – – 2.17 (1H, dd, 12.9, 8.3) 1.80 (1H, dd, 13.0, 4.7) 4.76 (1H, m) 1.85 (1H, m) 1.14 (3H, s) 1.11 (3H, s) 2.40 (1H, m) 1.12 (3H, d, 6.6) 2.32 (1H, m) 1.54 (1H, m) 2.01 (1H, m) 1.86 (1H, m) 3.90 (1H, dd, 10.9, 2.1) – 1.50 (3H, s) 1.47 (3H, s) 0.81 (3H, s) 1.55 (3H, s) 4.58 (1H, d, 10.9) 3.73 (1H, d, 10.9)

F.-C. Meng et al. / Phytochemistry Letters 15 (2016) 108–112

anomeric configuration of these two sugar moieties, respectively. Therefore, the structure of 3 was elucidated as 3-O-a-L-arabinopyranosyl-3b,16b,24S,25-tetrahydoxylcycloartane-16-O-b-D-glucopyranoside, which is denoted as Cycloramoside C. 3. Experimental 3.1. General experimental procedures HR-ESI–MS measurements were carried out on a Thermo LTQ Orbitrap XL mass spectrometer (Thermo Electron, Bremen, Germany). IR spectra were recorded on a PerkinElmer Spectrum 100 FT-IR Spectrometer with KBr pellets (PerkinElmer, Waltham, USA). Optical rotations were measured on a PerkinElmer Model 341 polarimeter at the sodium D line at 20.0  C (PerkinElmer, Waltham, USA). 1D and 2D NMR spectra were recorded on a Bruker AV-400 spectrometer and a Bruker Ascend 600 spectrometer (C5D5N used as solvent and TMS as an internal standard, Fallanden, Switzerland). HPLC was performed on a Waters 2695 Chromatograph equipped with a 250 mm  4.6 mm i.d. Cosmosil 5C18-MS-II column (Nacalai Tesque Inc., Kyoto, Japan) at 25  C. Column chromatography was performed on ODS (Alltech, USA), silica gel (200–300 mesh, Qingdao Marine Chemical Group Co., Qingdao, China), Macroporous resin (D101, Haiguang Chemical Group Co., Tianjin, China), Sephadex LH-20 (Pharmacia, USA); The solvents of analytical grade, used for extraction, gel chromatography and macroporous resin column chromatography, were supplied by Kaitong Chemical Co., Ltd. (Tianjin, China). Solvents applied for preparative column and HPLC analysis were of HPLC grade and purchased from Merck (Darmstadt, Germany). Distilled water was purified with a Millipore Milli Q-Plus system (Millipore, Milford, MA, USA). L-arabinose, D-Glucose and L-cysteine methyl ester were purchased from Aladdin (Aladdin, CA, USA). O-tolyl isothiocyanate was purchased from Sigma (Signma–Aldrich, Missouri, USA).

3.2. Plant material The rhizomes of T. ramosum B. Bovin were collected in the Emei, Sichuan Province, China in 2012, and authenticated by Dr. ChunFeng Qiao (Institute of Chinese Medical Sciences, University of Macau). A voucher specimen was deposited in Institute of Traditional Chinese Medicine, Guangdong Food and Drug Vocational College.

3.3. Extraction and isolation Dried plants of T. ramosum were powdered and extracted with 95% ethanol (3  20 L) under reflux. The ethanol extract was suspended in water and then successively extracted with petroleum ether, EtOAc and n-BuOH. The n-BuOH solution was concentrated and give a residue (344 g), which was separated by a silica gel column using CHCl3-MeOH (1:0!1:1) as the eluent, affording 5 fractions (Fr1-5). Fr-4 (25 g) was purified by a silica gel column using CDCl3-MeOH (10:1 ! 6:1 ! 3:1) as the eluent to afford 5 subfractions 1–5. Subfraction 3 (6.8 g) was further purified by silica gel column (CDCl3-MeOH 20:1) to afford compound 1 (3.3 g). Subfraction 2 (3.1 g) was subjected to silica gel (60 g, 30 g, 20 g) column chromatography with the solvent system CHCl3– MeOH (20:1), repeatedly. TLC monitored during eluting. Finally, compound 1 (830 mg) and 2 (13 mg) was obtained. Subfraction 4 was subjected to silica gel (30 g, 20 g) column chromatography with the solvent system CHCl3–MeOH (20:1 !10:1), repeatedly. The eluent was monitored via TLC. Finally, compound 3 (9 mg) was purified.

111

3.3.1. 3-O-a-L-arabinopyranosyl-3b,16b,24S,25,30pentaolcycloartane-16-O-b-D-glucopyranoside (1)

Amorphous white powder, C41H70O14, ½a20 D = + 45.7 (c 0.20 MeOH), IR (KBr) nmax: 3398, 2963, 2936, 1641, 1451, 1381, 1164, 1076, 1033 cm1; HR-ESI–MS m/z 809.4653 [M + Na]+ (calcd for C41H70O14Na, 809.4658); 1H NMR (C5D5N, 500 MHz) and 13C (C5D5N, 125 MHz) data see Table 1. 3.3.2. 3-O-a-L-arabinopyranosyl-3b,16b,24S,25,30pentaolcycloartane-24-O-b-D-glucopyranoside (2) Amorphous white powder, C41H70O14, ½a20 D = + 24.4 (c 0.16 MeOH); IR (KBr) nmax: 3411, 2936, 1639, 1452, 1382, 1306, 1260, 1172, 1076 cm1; HR-ESI–MS m/z 809.4655 [M + Na]+ (calcd for C41H70O14Na, 809.4658); 1H NMR (C5D5N, 600 MHz) and 13C (C5D5N, 150 MHz) data see Table 1. 3.3.3. 3-O-a-L-arabinopyranosyl-3b,16b,24S,25tetrahydoxylcycloartane-16-O-b-D-glucopyranoside (3) Amorphous white powder, C41H70O13, ½a20 D = + 39.3 (c 0.12 MeOH), IR (KBr) nmax: 3411, 2934, 1647, 1452, 1383, 1254, 1147, 1074 cm1; HR-ESI–MS m/z 793.4704 [M + Na]+ (calcd for C41H70O13Na, 793.4714); 1H NMR (C5D5N, 600 MHz) and 13C (C5D5N, 150 MHz) data see Table 1. 3.3.4. 24S-lanost-9(11)-ene-3b,16b,24,25,30-pentanol (4)

Amorphous white powder, C30H52O5, ½a20 D = + 52.1 (c 0.28 MeOH), IR (KBr) nmax: 3401, 2939, 2871, 1630, 1449, 1377, 1155, 1107, 1086, 1040; HR-ESI–MS m/z 515.3715 [M + Na]+ (calcd for C30H52O5Na, 515.3712); 1H NMR (C5D5N, 600 MHz) and 13C (C5D5N, 150 MHz) data see Table 1. 3.4. Acid hydrolysis Compound 1 (200 mg) was added into 100 mL of 1 M HCl and refluxed for 1 h at 80  C. After filtration, the solution was extracted with 100 mL of chloroform for 3 times. The chloroform layer was combined. After removal of the chloroform, the residue was subjected to silica gel chromatography, using chloroform-methanol (30:1) as eluent, thus yielding compound 4 (24S -lanost-9(11)ene-3b, 16b, 24, 25, 30-pentanol, 19.6 mg). The solution of each compound (1–3, each 2 mg) in 2 mL of 2 M HCl was kept at 80  C water-bath for 2 h. After removal of the solvent, the residue was re-dissolved in pyridine (0.5 mL). L-cysteine methyl ester (5 mg) was added and the mixture was heated at 60  C for 1 h, and then O-tolyl isothiocyanate (5 mg) was added to the mixture and heated further for 30 min (Tanaka et al., 1997). Finally the solution was passed through a 0.45 mm syringe filter for HPLC analysis (Waters 2695, 250 mm  4.6 mm i.d. Cosmosil 5C18-MS-II, acetonitrile: 0.5% aqueous trifluoroactic acid solution: 25: 75, flow rate 0.8 mL/ min, l = 250 nm). The standard solutions of arabinose and glucose (each 5 mg) were treated as described above. The peaks of each monosaccharide derivative were observed at tR (min): 1: D-glucose 17.17, L-arabinose 19.24; 2: D-glucose 17.17, L-arabinose 19.24; 3: D-glucose 17.18, L-arabinose 19.26 (reference L-glucose 15.79, D-glucose 17.13, L-arabinose 19.33 and D-ara 20.79). Acknowledgements This project was supported by grants from Macao Science and Technology Development Fund (FDCT/042/2014/A1), University of Macau (MYRG2014-00162-ICMS-QRCM), the National Natural Science Foundation of China (81273407) and the Medical Scientific Research Foundation of Guangdong Province (B2013074).

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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. phytol.2015.12.001.

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