Cycloartane-type triterpene glycosides anopanins A-C with monoacyldigalactosylglycerols from Anodendron paniculatum

Phytochemistry 144 (2017) 113e118

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Cycloartane-type triterpene glycosides anopanins A-C with monoacyldigalactosylglycerols from Anodendron paniculatum Viet Duc Ho a, Thi Nhu Hanh Hoang b, Quoc Hung Vo a, Van Kiem Phan c, Tuan Anh Le d, Viet Ty Pham e, Minh Hien Nguyen f, Takeshi Kodama f, Takuya Ito f, Hiroyuki Morita f, Ain Raal g, *, Thi Hoai Nguyen a, ** a

Faculty of Pharmacy, Hue University of Medicine and Pharmacy, Hue University, 06 Ngo Quyen, Hue City, Viet Nam Faculty of Chemistry, Hue University's College of Sciences, 77 Nguyen Hue, Hue City, Viet Nam Institute of Marine Biochemistry, VAST, 18 Hoang Quoc Viet, Caugiay, Hanoi, Viet Nam d Quang Tri Center of Science and Technology, Mientrung Inst. for Scientific Research, VAST, Dien Bien Phu, Dong Ha, Quang Tri, Viet Nam e Faculty of Chemistry, Hue University of Education, Hue University, 34 Le Loi, Hue City, Viet Nam f Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama, 930-0194, Japan g Institute of Pharmacy, University of Tartu, 1 Nooruse str., 50411, Tartu, Estonia b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 March 2017 Received in revised form 14 August 2017 Accepted 8 September 2017

Three cycloartane-type triterpene glycosides (anopanins AeC), together with three known monoacyldigalactosylglycerols gingerglycolipids AeC and (2S)-1-O-palmitoyl-3-O-[a-D-galactopyranosyl(1/6)-b-D-galactopyranosyl]-sn-glycerol, were isolated from the aerial parts of Anodendron paniculatum collected in Viet Nam. The chemical structures of the present compounds were elucidated by means of 1D and 2D NMR and HRESIMS spectroscopy, and by comparing to the reported data in the literature. These compounds did not show significant growth inhibitory activities (IC50 > 100 mg/mL) against the tested cancer cell lines LU-1 (lung adenocarcinoma), KB (epidermoid carcinoma), Hep-G2 (hepatoma cancer), MKN-7 (stomach cancer), and SW-480 (colon adenocarcinoma). © 2017 Elsevier Ltd. All rights reserved.

Keywords: Anodendron paniculatum Apocynaceae Cycloartane-type triterpene glycosides Anopanins AeC Anticancer activity

1. Introduction Anodendron paniculatum (Roxb.) A. DC. is a climbing species of the Apocynaceae family and broadly distributed in Sri Lanka, India, Bangladesh, Burma, and Southeast Asia (Middleton, 1996). The roots of this plant have been used in traditional folk medicine as remedy for vomiting and cough in India (Chi, 1997). Moreover, its latex is used to cure the poison of snake and centipede bites (Forster, 1993). Several cardenolides have been isolated from A. paniculatum in the previous chemical studies (Polonia et al., 1970; Lichtia et al., 1972). To our knowledge, however, the great majority of the phytochemical components of this plant is still undiscovered. Recently, we discovered a new triterpene ester (anopaniester), and successfully isolated it from the aerial parts of A. paniculatum together with established cycloartenol, ursolic acid,

* Corresponding author. ** Corresponding author. E-mail address: [email protected] (A. Raal). http://dx.doi.org/10.1016/j.phytochem.2017.09.004 0031-9422/© 2017 Elsevier Ltd. All rights reserved.

esculenic acid, bis-(2-ethylhexyl) phthalate, desmosterol, stigmasterol, vaniline, and (E)-phytol (Ho et al., 2017). In the present study, three new cycloartane-type triterpene glycosides, anopanins AeC (1e3), and four known monoacyldigalactosylglycerols (4e7) were isolated from the aerial parts of A. paniculatum collected in Viet Nam. The chemical structure and cytotoxic activity of these isolated compounds were thoroughly investigated. 2. Results and discussion Compound 1 was obtained as a white amorphous powder. The HRESIMS of 1 showed a pseudo-molecular ion peak at m/z 1025.5294 [MþNa]þ. Its molecular formula was thus determined to be C50H82O20 by HRESIMS in conjunction with NMR data analysis. The IR spectrum of 1 revealed strong absorption bands corresponding to an ester (1713 cm1), double bond (1632 cm1), ethers (1010, 1070 cm1), and hydroxyl groups (3418 cm1). The 1H NMR spectrum of 1 in pyridine-d5 showed the typical signals for seven tertiary methyl groups (each, 3H, s) at dH 1.13 (H-

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V.D. Ho et al. / Phytochemistry 144 (2017) 113e118

29), 1.25 (H-30), 1.30 (H-28), 1.57 (H-18), 1.66 (H-27), 1.67 (H-26), and 1.69 (H-21); one acetoxy group at dH 2.06 (3H, s), and three anomeric protons at dH 4.86 (1H, d, J ¼ 7.2 Hz), 5.16 (1H, d, J ¼ 7.8 Hz), and 5.41 (1H, d, J ¼ 7.8 Hz). These data suggested the presence of three sugar units with b-configuration. In addition, two protons of a cyclopropyl methylene group at dH 0.22 and 0.49 (each, 1H, d, J ¼ 4.2 Hz, H-19) were observed. Analysis of the 13C NMR, DEPT and HMQC spectra of 1 revealed 50 signals, of which 30 were assigned to an aglycone, 18 to three sugar moieties, and 2 belonged to acetoxy group (dC 171.2 and 22.0). The aglycone of 1 was deduced to be a cycloartane-type triterpene with two olefinic carbons of tri-substituted double bond (dC 125.1 and 132.5), three oxygenated methine carbons (dC 77.2, 79.0, and 89.4), an oxygenated quaternary carbon (dC 76.7), and seven quaternary methyl carbons (15.8, 18.6, 20.7, 21.2, 21.7, 26.2, and 26.4). All proton and carbon signals of 1 were assigned by means of 2DNMR, including HMQC, HMBC, and 1H-1H COSY (Fig. 2). The HMBC correlations of H3-28 (dH 1.30) and H3-29 (dH 1.13) to C-3 (dC 89.4), C-4 (dC 41.7), and C-5 (dC 48.1) indicated the locations of the oxygenated carbon and gem-dimethyl groups at C-3 and C-4 in the A ring, respectively. Similarly, the HMBC correlations between H321 (dH 1.69) and C-17 (dC 56.5)/C-20 (dC 76.7)/C-22 (dC 79.0), between H3-26 (dH 1.67)/H3-27 (dH 1.66) and C-24 (dC 125.1)/C-25 (dC 132.5) confirmed the position of two hydroxyl groups at C-20 and C-22 as well as the double bond (D-24) in the side chain moiety. In addition, the acetoxy group at C-16 (dC 77.2) was assigned from the HMBC correlation between H-16 (dH 6.20) and carboxyl carbon at dC 171.2. Thus, the aglycone was considered to be 3,16,20,22tetraoxygenated cycloart-24-ene. The stereochemistry of aglycone moiety of 1 was determined on the basis of the ROESY experiment (Fig. 3). The ROESY correlation between H-3 (dH 3.38) and H-1a (dH 1.43)/H-5 (dH 1.26)/H3-28, between H-2b (dH 1.84) and H3-29 suggested the a-orientation for H-3, H-5 and H3-28 as well as the chair conformation for A ring (Fig. 3). In addition, the J values of 11.4 and 4.2 Hz between H-3 and H-2 (dH 1.84, 2.27) are typical for the 3a-axial H of cycloartane triterpenoids (Inada et al., 1997). Furthermore, the ROESY correlations between H-16 and H3-18/H-22 (dH 3.92), between H-17 (dH 3.19) and H-30, and the lack of correlation between H-16 and H3-30 (dH 1.25) revealed the a-orientations of 16-OAc and H-17. The 20R configuration was determined by the ROESY correlations of H3-21 to H-12b (dH 1.91)/H-17, of 20-OH (dH 5.61) to H-16/H3-18 and the lack of cross-peaks of H3-21/H3-18, 20-OH/H-17 (Bedir et al., 2001; Kuo et al., 2005; Yalçın et al., 2012). This was strengthened by the agreement of chemical shift of C-21 (dC 21.7) with those of 20R,22x isomers [dC: 21.1 (20R,22R), 21.9 (20R,22S)], but quite different from those of 20S,22x isomers of cholestane-3b,20,22-triol [dC: 23.8 (20S,22S), 24.2 (20S,22R)] (Riccio et al., 1985). The absolute configuration of C-22 chiral center was deduced by comparing dC values of C-20 and C-22 of 1 with those (in C5D5N) of 20R,22R and 20R,22S forms. In the 13C NMR spectrum of 20R,22R form such as in cholestane-3b,20R,22R-triol (Hikino et al., 1975), vitexirone (Kubo et al., 1990), frondoside A7-3, frondoside A7-4 (Silchenko et al., 2007), and stachysterone C (Ling et al., 2009), the carbons C-20 and C-22 resonated at the close values of chemical shifts (DdC values less than 0.4 ppm). In contrast, these values differed about 2 ppm in the 20R,22S form (Hikino et al., 1975; Silchenko et al., 2007). The signals of C-20, C-22 in 13C NMR spectrum of 1 were found at 76.7 and 79.0 ppm, respectively. Therefore, the 20R,22S configuration was suggested for 1. This suggestion was further confirmed by the ROE correlations of 22-OH (dH 5.64) to H3-21/H23a (dH 2.58), of H-22 to H-16, and the lack of correlations between 22-OH and H-16/20-OH/H-23b (dH 2.93) (Omobuwajo et al., 1996; Tigoufack et al., 2010). The monosaccharide composition of a sugar moiety was D-

glucose, and this was determined by acid hydrolysis of 1 followed by reduction, per-acetylated derivatization and GCMS analysis (see Experimental Section). Furthermore, the 13C NMR signals of the sugar moiety in 1 (Table 2) were closely similar to those of shatavarin IX (Hayes et al., 2008). These findings indicate that the sequence of the sugar linkages is [b-D-glucopyranosyl(1/2)][b-Dglucopyranosyl(1/4)]-b-D-glucopyranosyl which was supported by the HMBC correlations from H-100 (dH 5.41) to C-20 (dC 82.2), from H-1000 (dH 5.16) to C-40 (dC 81.6). The attachment of sugar moiety at C-3 of the aglycone via glycosidic bond was deduced from the HMBC correlations of H-10 (dH 4.86) to C-3 (dC 89.4), and H-3 (dH 3.38) to C-10 (dC 105.1). Consequently, the chemical structure of 1 was elucidated to be (3b,16a,20R,22S)-16-acetoxy-20,22dihydroxycycloart-24-ene 3-O-[b-D-glucopyranosyl-(1/2)][b-Dglucopyranosyl-(1/4)]-b-D-glucopyranoside, named anopanin A. Compound 2 was isolated as a white amorphous powder. The HRESIMS exhibited a quasi-molecular ion peak at m/z 1027.5452 [MþNa]þ, corresponding to the molecular formula C50H84O20. The 1 H and 13C NMR spectroscopic data (Tables 1 and 2) were closely similar to those of 1. The significant differences between 1 and 2 were the absence of 20,22-dihydroxy groups and 24-D in 1 and the presence of 24,25-dihydroxy groups in 2, as confirmed by the HMBC correlations of H3-21 (dH 1.02) to C-17 (dC 58.1), C-20 (dC 34.7), and C-22 (dC 33.7), of H3-26 (dH 1.52) and H3-27 (dH 1.55) to C24 (dC 79.6) and C-25 (dC 73.2) (Fig. 2). Moreover, the relative stereo-structure of the aglycone in 2 had the same pattern as that of 1. This finding was also supported by the ROESY test. The absolute configuration of C-24 (dC 79.6) was determined to be R by comparing the chemical shift values of C-24 in cyclounifolioside C (24R: dC 80.3) and cyclocantogenin (24S: dC 77.0) (Kucherbaev et al., 2002; Zhao et al., 2008). Thus, compound 2 was assigned as (3b,16a,24R)-16-acetoxy-24,25-dihydroxycycloartane 3-O-[b-Dglucopyranosyl-(1/2)][b-D-glucopyranosyl-(1/4)]-b-D-glucopyranoside, and was given the trivial name anopanin B. Compound 3 was isolated as a white amorphous powder, and its molecular formula was determined as C50H82O20 based on the data obtained with NMR and HRESIMS. The 1D-NMR spectroscopic data of 3 were similar to those of 1 and 2, except for the side chain. The 1 H1H COSY correlations of H3-21/H-20/H-22/H2-23/H-24 as well as the HMBC correlations of H3-26 (dH 1.31) and H3-27 (dH 1.53) to C-24 (dC 78.3) and C-25 (dC 82.8) suggested the presence of 5-ethyl3-hydroxy-2,2-dimethyltetrahydrofuran moiety (Fig. 2). The linkage of C-17 and C-20 was confirmed by the HMBC correlations from H-17 (dH 2.51) to C-20 (dC 38.3), from H-20 (dH 1.68) to C-17 (dC 56.2), and from H3-21 (dH 1.21) to C-17/C-20/C-22 (dC 76.3). The absolute configuration of two chiral centers at C-22 and C-24 was elucidated by a ROESY test and by comparing the dC values of the side chain with those of related compounds. The carbon signal of C24 at dC 78.3 was assigned for S configuration by comparing to that of cyclocanthoside B [24S: dC 77.1] and cycloasgenin B [24R: dC 80.5] (Hirotani et al., 1994; Ahmad et al., 1998). The strong ROESY correlations ranging from H-22 (dH 4.19) and H-24 (dH 4.28) to H3-26 (dH 1.31), and from 24-OH (dH 6.41) to H3-27 (dH 1.53) suggested that both H-22 and H-24 located at the same side in the tetrahydrofuran ring. Therefore, the absolute configuration of C-22 was verified to be S form. Consequently, the compound 3 was elucidated to be (3b,16a,22S,24S)-16-acetoxy-22,25-epoxy-24-hydroxy cycloartane 3-O-[b-D-glucopyranosyl-(1/2)][b-D-glucopyranosyl(1/4)]-b-D-glucopyranoside, named anopanin C. The remaining compounds were identified as gingerglycolipids AeC (4e6) (Yoshikawa et al., 1994) and (2S)-1-O-palmitoyl-3-O-[aD-galactopyranosyl-(1/6)-b-D-galactopyranosyl]-sn-glycerol (7) (Kim et al., 2004) (Fig. 1). Their structures were established by means of spectroscopic (HRESIMS, 1H- and 13C-NMR), and the results were in a good agreement with the previous studies. To our

V.D. Ho et al. / Phytochemistry 144 (2017) 113e118 Table 1 1 H (600 MHz) and

13

115

C (150 MHz) NMR data for aglycone moieties of 1e3 in pyridine-d5 [d (ppm), J (Hz)].

Position

1

2

dC 1 2 3 4 5 6

32.5 30.3 89.4 41.7 48.1 21.5

7 8 9 10 11 12 13 14 15

26.9 47.9 19.6 26.8 27.5 34.2 48.2 48.2 46.2

16 16-OAc 17 18 19

77.2 171.2 22.0 56.5 21.2 30.7

20 20-OH 21 22 22-OH 23 24 24-OH 25 26 27 28 29 30

76.7 e 21.7 79.0b e 31.0 125.1 e 132.5 26.4 18.6 26.2 15.8 20.7

dH

3

dC

1.10 m, 1.43 m 1.84 m, 2.27 m 3.38 dd (11.4, 4.2) e 1.26a 0.68 q (12.6) 1.51a 1.07a, 1.23a 1.52a e e 1.12a, 2.06a 1.91a, 1.92a e e 1.62 brd (13.8) 2.17 dd (14.4, 8.4) 6.20 dd (7.8, 7.2) e 2.06 s 3.19 d (6.6) 1.57 s 0.22 d (4.2) 0.49 d (4.2) e 5.61 s 1.69 s 3.92a 5.64a 2.58 m, 2.93 m 5.65a e e 1.67 s 1.66 s 1.30 s 1.13 s 1.25 s

dH a

32.5 30.3 89.4 41.7 48.2 21.5

a

1.11 , 1.43 1.84 m, 2.27 m 3.38 dd (11.4, 4.2) e 1.26a 0.68 q (12.6) 1.51a 1.03a, 1.22a 1.52a e e 1.06a, 1.98a 1.60a, 1.66a e e 1.48a 2.01a 5.27 dd (6.0, 6.0) e 2.15 s 2.08 dd (10.2, 6.6) 1.01 s 0.20 d (4.2) 0.46 d (4.2) 1.78a e 1.02 d (6.0) 1.74a e 1.88a 3.76 m e e 1.52 s 1.55 s 1.30 s 1.14 s 1.13 s

26.8 48.0 19.8 26.8 27.1 33.5 47.0 48.0 46.2 80.8 171.3 22.0 58.1 19.5 30.4 34.7 e 19.1 33.7 e 28.5 79.6 e 73.2 26.6 26.4 26.2 15.8 20.3

dC

dH

32.5 30.3 89.4 41.7 48.2 21.5

1.07a, 1.42a 1.84 m, 2.27a 3.38 dd (11.4, 4.2) e 1.25a 0.68 q (12.0) 1.51a 1.01a, 1.23a 1.52a e e 1.04a, 1.92a 1.62a, 1.66a e e 1.48a 2.01 m 5.33 dd (7.8, 7.2) e 2.21 s 2.51 dd (10.8, 6.0) 1.03 s 0.20 d (3.6) 0.46 d (3.6) 1.68 m e 1.21 d (6.6) 4.19 m e 2.10 m, 2.29 m 4.28a 6.41 d (4.8) e 1.31 s 1.53 s 1.30 s 1.14 s 1.06 s

26.7 48.0 19.8 26.8 27.1 33.5 47.1 48.0 46.2 80.3 171.0 22.1 56.2 19.4 30.4 38.3 e 12.8 76.3 e 39.6 78.3 e 82.8 27.1 24.2 26.2 15.8 20.2

Assignments were done by DEPT, HMQC, HMBC, COSY, and ROESY experiments. a Overlapping signals. b Signal missing in 13C NMR spectrum.

Table 2 1 H (600 MHz) and Position

3-O-glc 10 20 30 40 50 60 2′-glc 100 200 300 400 500 600 4′-glc 1000 2000 3000 4000 5000 6000

13

C (150 MHz) NMR data for sugar moieties of 1e3 in pyridine-d5 [d (ppm), J (Hz)]. 1

2

dC

dH

105.1 82.2 77.1 81.6 76.5 62.6

4.86 4.29 4.30 4.25 3.83 4.49

106.0 77.6 78.7 72.2 78.7 63.3 105.4 75.3 78.4 71.9 78.9 62.7

3

dC

dH

d (7.2) m m m ddd (9.6, 3.6, 3.0) m, 4.53 m

105.1 82.2 77.1 81.6 76.5 62.6

4.86 4.29 4.30 4.26 3.84 4.49

5.41 4.10 4.23 4.32 3.93 4.48

d (7.8) dd (8.4, 7.8) m m ddd (9.6, 3.6, 3.6) m, 4.52 m

106.0 77.6 78.7 72.2 78.7 63.3

5.16 4.09 4.25 4.24 4.00 4.31

d (7.8) dd (8.4, 7.8) m m m m, 4.54 m

105.4 75.3 78.3 71.9 78.9 62.7

Assignments were done by DEPT, HMQC, HMBC, COSY, and ROESY experiments. Glc: b-D-glucopyranosyl.

dC

dH

d (7.2) m m m ddd (9.6, 3.6, 3.0) m, 4.53 m

105.1 82.2 77.1 81.6 76.5 62.6

4.86 4.29 4.30 4.25 3.83 4.48

d (7.8) m m m ddd (9.6, 3.6, 3.0) m, 4.53 m

5.42 4.10 4.23 4.34 3.94 4.48

d (7.8) dd (9.6, 7.8) m dd (9.6, 9.0) ddd (9.6, 3.6, 3.6) m, 4.52 m

105.9 77.6 78.6 72.2 78.7 63.3

5.42 4.10 4.23 4.35 3.94 4.46

d (7.2) m m m ddd (9.6, 3.6, 3.6) m, 4.51 m

5.16 4.09 4.26 4.24 4.00 4.32

d (7.8) dd (8.4, 7.8) m dd (9.0, 9.0) m m, 4.51 m

105.4 75.3 78.3 71.9 78.9 62.7

5.16 4.09 4.25 4.24 4.00 4.32

d (8.4) m m m m m, 4.50 m

116

V.D. Ho et al. / Phytochemistry 144 (2017) 113e118

Fig. 1. Chemical structure of isolated compounds 17.

Fig. 2. Key HMBC (1H/13C, arrows) and COSY (bold lines) correlations of 1e3.

knowledge, these glycolipids were isolated from this genus for the first time. The cytotoxic properties of the isolated compounds 1e7 against LU-1 (lung adenocarcinoma), KB (epidermoid carcinoma), Hep-G2 (hepatoma cancer), MKN-7 (stomach cancer), SW-480 (colon adenocarcinoma) cancer cell lines were tested by a sulforhodamine B assay. For details about the method see Ho et al. (2015). The present compounds, however, did not show any significant growth inhibitory activities (IC50 > 100 mg/mL) against the tested cancer cell lines. 3. Experimental 3.1. General experimental procedures Melting points were determined with a Buchi Melting Point B545 apparatus (Sigma-Aldrich, Missouri, USA). Infrared spectra

were recorded with an IR Prestige-21 spectrometer (Shimadzu, Kyoto, Japan). Optical rotations were measured by using a JASCO P2100 polarimeter (Hachioji, Tokyo, Japan). The 1D and 2D NMR experiments were performed on a Varian Union 600 spectrometer (Varian, California, USA) with TMS as an internal reference. HRESIMS data were measured on a LTQ Orbitrap XL mass spectrometer (Thermo Scientific, Massachusetts, USA). Column chromatography was performed using silica gel (60 N, spherical, neutral, 40e50 mm, Kanto Chemical Co., Inc., Tokyo, Japan), Cosmosil 75C18-OPN (Nacalai Tesque Inc., Kyoto, Japan), YMC RP-18 (Fuji Silysia Chemical Ltd, Kasugai, Aichi, Japan), Sephadex LH-20 (Dowex® 50WX2100, SigmaeAldrich, USA), and Diaion HP-20 (Mitsubishi Chem. Co., Tokyo, Japan). Analytical TLC was performed on pre-coated silica gel 60F254 and RP-18 F254 plates (0.25 or 0.50 mm thickness, Merck KGaA, Darmstadt, Germany). GCMS experiments were carried out, using GCMS-QP2010 Plus (Shimadzu, Kyoto, Japan). The cell lines, LU-1 (lung adenocarcinoma), KB (epidermoid carcinoma), Hep-G2

V.D. Ho et al. / Phytochemistry 144 (2017) 113e118

117

Fig. 3. Key ROESY correlations (dash lines) for aglycone moiety of 1.

(hepatoma cancer), MKN-7 (stomach cancer), SW-480 (colon adenocarcinoma) were used for the cytotoxic activity determinations. Cell culture flasks and 96-well plates (Corning Inc., Corning, NY, USA) were used in the cytotoxic activity tests. The ELISA Plate Reader (Bio-Rad, California, USA) was used to measure the absorbance of the cells in the cytotoxicity assay. 3.2. Plant material The aerial parts of A. paniculatum A. DC. were collected from Quang Tri province, Viet Nam (N17 30 21.400 E107 040 24.400 ) in June, 2014 and were identified by Dr. Nguyen The Cuong, Institute of Ecology and Biological Resources, VAST, Viet Nam. A voucher specimen (AV03) was deposited at the Faculty of Pharmacy, Hue University of Medicine and Pharmacy, Viet Nam.

(150 MHz, pyridine-d5): see Tables 1 and 2; HRESIMS m/z 1025.5294 [MþNa]þ (calcd. for C50H82O20Na, 1025.5297). 3.3.2. Anopanin B (2) White amorphous powder; mp: 222e224  C; ½a22 D þ77.7 (c 0.1, MeOH); IR (KBr) nmax (cm1): 3443, 2930, 1730, 1647, 1460, 1377, 1252, 1074, 1028; 1H NMR (600 MHz, pyridine-d5) and 13C NMR (150 MHz, pyridine-d5): see Tables 1 and 2; HRESIMS m/z 1027.5452 [MþNa]þ (calcd. for C50H84O20Na, 1027.5454). 3.3.3. Anopanin C (3) White amorphous powder; mp: 218e219  C; ½a22 D þ66.7 (c 0.1, MeOH); IR (KBr) nmax (cm1): 3428, 2932, 1709, 1630, 1456, 1381, 1261, 1072, 1028; 1H NMR (600 MHz, pyridine-d5) and 13C NMR (150 MHz, pyridine-d5): see Tables 1 and 2; HRESIMS m/z 1025.5293 [MþNa]þ (calcd. for C50H82O20Na, 1025.5297).

3.3. Extraction and isolation The dried aerial parts of A. paniculatum (2.5 kg) were extracted with MeOH (10.0 L 3 times) at room temperature to yield 105 g of a dark solid extract. This was then suspended in water and successively partitioned with chloroform (CHCl3) and ethyl acetate (EtOAc) (each, 2.0 L 3 times) to obtain the CHCl3 (AC, 50.7 g), the EtOAc (AE, 10.2 g), and the water (AW, 27.5 g) layers after removal of the solvents in vacuo. The water-soluble fraction AW was subjected to a Diaion HP-20 column and eluted with stepwise additions of MeOH in water (0%, 25%, 50%, 75%, and 100%) to obtain four subfractions, AW1AW4. Fraction AW2 (10.5 g) was chromatographed on a silica gel column eluting with CHCl3eMeOHewater (3:1:0.1, v/v) to obtain 7 smaller fractions, AW2.1eAW2.7. Fraction AW2.4 (550 mg) was chromatographed on a Sephadex LH-20 column eluting with MeOHewater (4:1, v/v), followed by an YMC RP18 column eluting with MeOHeMeCNewater (3:2:3, v/v) to yield 1 (32.2 mg), 2 (10.8 mg), and 3 (25.6 mg). Fraction AW4 (4.2 g) was chromatographed on a silica gel column eluting with CHCl3e MeOHewater (5:1:0.1, v/v) to obtain 5 sub-fractions, AW4.1eAW4.5. Fraction AW4.3 (700 mg) was then applied to an YMC RP-18 column eluting with MeOHewater (6:1, v/v) to yield 4 (12.5 mg), 5 (10.7 mg), 6 (13.8 mg), and 7 (9.6 mg). 3.3.1. Anopanin A (1) White amorphous powder; mp: 213e215  C; ½a22 D -19.9 (c 0.1, MeOH); IR (KBr) nmax (cm1): 3418, 2932, 1713, 1632, 145, 1383, 1265, 1070, 1030; 1H NMR (600 MHz, pyridine-d5) and 13C NMR

3.3.4. The monosaccharide composition of Compound 1 Alditol per-acetate derivatives were prepared by using the method reported by Pettolino et al. (2012) with a slight modification. Compound 1 (5 mg) was hydrolyzed with 4 M trifluoroacetic acid (1 mL) in a sealed tube at 105  C for 4 h. After cooling, the reaction mixture was diluted with 4 mL water and extracted with dichloromethane (2 mL x 3 times). The aqueous layer was evaporated with methanol under reduced pressure. The residue was then dissolved in deionized water (1 mL) and incubated with sodium borohydride (33 mg) at 40  C for 90 min. After reduction, three drops of glacial acetic acid was slowly added to the tube. The mixture was evaporated to a dry state in a filtered air flow at 37  C. The dry sample was dissolved in 2 mL of methanoleglacial acetic acid (95:5, v/v) and then evaporated in vacuo. This step was repeated three times to remove boric acid completely. The obtained alditols were acetylated with 1 mL of acetic anhydrideepyridine (1:1, v/v) at 110  C for 2 h. After cooling to room temperature, the acetylation mixture was diluted with 4 mL water and partitioned with dichloromethane (2 mL x 2 times). The combined dichloromethane extract was washed with 5 mL water (3 times) and then placed in heating block (37  C) to yield alditol hexaacetate derivatives as pale yellow oil. The resulting derivatives were analyzed by GCMS with a DB-5 capillary column (30 m  0.25 mm i.d.). Helium was used a carrier gas at a flow rate of 1.20 mL/min. Column, injector, detector were set at 80, 220, and 230  C, respectively. The oven temperature was programed as follows: initially 80  C for 2 min, increased to

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180  C at 10  C/min and held at 180  C for 2 min, increased to 220  C at 2  C/min and held at 220  C for 5 min, further increased to 240  C at 2  C/min and held for 5 min at the final temperature. The results indicated that the single peak of 1 at 26.65 min coincides with the corresponding peak of the standard D-glucose derivative (SigmaAldrich) with a tR value at 26.65 min. 3.3.5. SRB assay for evaluating cytotoxic activity The cytotoxic activity of the isolated compounds from the aerial parts of A. paniculatum was tested by sulforhodamine B assay against the growth of twomonolayer human cancer cell lines (LU-1, KB, Hep-G2, MKN-7 and SW-480). Stock cultures were grown in T75 flasks containing 50 mL of Dulbecco's Modified Eagle Medium (DMEM) with 2 mM L-glutamine, 1.5 g/L sodium bicarbonate and 10% Fetal Bovine Serum (FBS). The medium was changed at 48-h intervals. The cells were dissociated with 0.05% Trypsin-EDTA, sub-cultured every 3e5 days with the ratio of (1:3) and incubated at 37  C under a humidified 5% carbon dioxide atmosphere. Tumor cells were cultivated in a humidified atmosphere of 5% CO2 at 37  C for 48 h. Cell viability was examined by a sulforhodamine B (SRB) method intended for cell density determination and based on the measurement of cellular protein content (Monks et al., 1991). Viable cells were seeded in the growth medium (180 mL) into 96well microplates (4  104 cells per well) and allowed to attach overnight. The tested samples were added carefully into each well of 96-well plates and the cultivation was continued under the same conditions for another 72 h. Thereafter, the medium was removed and the remaining cell monolayers were fixed with cold 20% (w/v) trichloroacetic acid for 1 h at 4  C and stained by 1X SRB staining solution at room temperature for 30 min. The unbound dye was subsequently removed by washing repeatedly with 1% (v/v) acetic acid. The protein-bound dye was dissolved in 10 mM Tris base solution for optical density determination at 515 nm on an ELISA Plate Reader (Bio-Rad). DMSO 10% was used a blank sample and ellipticine was used as positive control. The cytotoxicity activity was determined using four doses (100 mg/mL, 20 mg/mL, 4 mg/mL, and 0.8 mg/mL), and calculating a half maximal inhibitory concentration, IC50 (TableCurve Version 4.0, …). All experiments were prepared in triplicatee. The inhibition rate (IR) of cells was calculated by the following formula: IR% ¼ {100%  [(absorbancet e absorbance0)/(absorbancec  absorbance0)]  100}, where IR ¼ inhibition rate of cell growth, absorbancet ¼ average optical density value at day 3; absorbance0 ¼ average optical density value at time-zero; absorbancec ¼ average optical density value of the blank DMSO control sample. Acknowledgements This work was supported in part by a grant for the 2015 international exchange program from the University of Toyama (H. V. D. and H. M.), a grant from the Ministry of Education and Training, Viet Nam (H. V. D., ID No. B2017-DHH-50), a grant from the Japan Society for the Promotion of Science (ID No. P14412), a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (T.I. and H.M.), and a grant from the Kobayashi International Scholarship Foundation (H.M.). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.phytochem.2017.09.004.

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