Cytotoxic Resin Glycosides from Ipomoea aquatica and Their Effects

Article pubs.acs.org/jnp

Cytotoxic Resin Glycosides from Ipomoea aquatica and Their Effects on Intracellular Ca2+ Concentrations Bo-Yi Fan,† Yu-Cheng Gu,‡ Ye He,† Zhong-Rui Li,† Jian-Guang Luo,*,† and Ling-Yi Kong*,† †

State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China ‡ Syngenta, Jealott’s Hill International Research Centre, Bracknell, Berkshire RG42 6EY, United Kingdom S Supporting Information *

ABSTRACT: Eleven new resin glycosides, aquaterins I−XI (1−11), were isolated from the whole plants of Ipomoea aquatica. The structures of 1−11 were elucidated by a combination of spectroscopic and chemical methods. They were found to be partially acylated tetra- or pentasaccharides derived from simonic acid B and operculinic acids A and C. The site of the aglycone macrolactonization was placed at C-2 or C-3 of the second saccharide moiety, while the two acylating residues could be located at C-2 (or C-3) of the second rhamnose unit and at C4 (or C-3) on the third rhamnose moiety. All compounds were evaluated for cytotoxicity against a small panel of human cancer cell lines. Compound 4 exhibited the most potent activity against HepG2 cells with an IC50 value of 2.4 μM. Cell cycle analysis revealed 4 to inhibit the proliferation of HepG2 cells via G0/G1 arrest and apoptosis induction. In addition, compounds 1−4, 7, 9, and 10 were found to elevate Ca2+ in HepG2 cells, which might be involved in the regulation of the cytotoxic activities observed.

R

Convolvulaceae,11−13 11 new resin glycosides, aquaterins I− XI (1−11), were isolated from the whole plants of I. aquatica. The cytotoxic properties of the isolates were screened, and their effects on intracellular Ca2+ levels in HepG2 cells were also evaluated.

esin glycosides are amphipathic glycolipids that are composed of monohydroxy and dihydroxy C14 and C16 fatty acids glycosidically linked to oligosaccharide chains with an ester linkage between the fatty acid and the oligosaccharide chain to form a macrolactone ring.1 They have been found mainly in plants of the Convolvulaceae and are responsible for the drastic purgative action of some important species in this family used in traditional medicine throughout the world since ancient times.1 Chemical investigations on these resin glycosides were initiated in the middle of the 19th century. To date, hundreds of resin glycosides have been reported, some of which exhibit cytotoxic, neuroprotective, sedative, and vasorelaxant activities, and reversal effects on multidrug resistance in both microbial pathogens and mammalian cancer cells.2−5 Notably, several resin glycosides have shown ion-transport activities on Na+, K+, and Ca2+ ions,6,7 and Ca2+ influx was proved to be the basis of the contractile effect of tricolorin A, a typical resin glycoside, on the guinea pig ileum.8 Thus, the effects of resin glycosides on cellular Ca2+ homeostasis deserve further investigation. Ipomoea aquatica Forsk. (Convolvulaceae) is an annual or perennial herbaceous plant used both for food and in folk medicine in mainland China.9 Previous phytochemical investigations of this plant have led to the isolation of several alkaloids, carotenoids, and flavonoids.10 However, there is no previous report on the resin glycosides from I. aquatica. Hence, as part of an effort to identify additional structurally and biologically diverse resin glycosides from plants in the © 2014 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION A 95% EtOH extract from the whole plants of I. aquatica was suspended in H2O, then partitioned successively with petroleum ether, dichloromethane (CH2Cl2), and ethyl acetate (EtOAc). The CH2Cl2-soluble fraction was subjected to column chromatography over silica gel and ODS and then further purified by preparative HPLC to afford 11 resin glycosides (1−11). Aquaterin I (1) was obtained as a white powder. Its molecular formula was established as C57H98O24 by the HRESIMS at m/z 1189.6343 [M + Na] + (calcd for C57H98O24Na, 1189.6340). The 1H NMR spectrum (Table 1) of 1 exhibited five anomeric proton signals at δH 4.75 (d, J = 7.0 Hz), 5.49 (br s), 6.14 (br s), 5.92 (br s), 5.66 (br s) and three primary methyl signals at δH 0.89 (t, J = 7.0 Hz), 0.85 (t, J = 7.5 Hz), and 0.79 (t, J = 7.0 Hz). The 13C NMR spectrum (Table 2) showed signals that could be ascribed to three carboxyl carbons (δC 175.9, 173.9, 173.6) and five anomeric carbons (δC 104.8, 99.2, 99.7, 104.2, 105.3). These data, together with the Received: June 27, 2014 Published: October 14, 2014 2264

dx.doi.org/10.1021/np5005246 | J. Nat. Prod. 2014, 77, 2264−2272

Journal of Natural Products

Article

pair of isomers. The 1H and 13C NMR spectra (Tables 1 and 2) of 2 were similar to those of compound 1. The major difference between the 13C NMR spectra of 1 and 2 was evident in the high-field region (20 to 45 ppm), representative of the methylene signals of the fatty acyl groups. Therefore, it was deduced that 2 is a homologue of 1, with a C8 rather than a C6 fatty acid side chain present, which was further supported by the key HMBC correlation from Rha″ H-4 (δH 5.87) to C-1 (δC 174.0) of Octa (n-octanoyl). Consequently, the structure of 2 was determined as (11S)-jalapinolic acid 11-O-α-L-rhamnopyranosyl-(1→3)-O-[4-O-octan oyl-α-L-rhamopyranosyl-(1→ 4)]-O-[2-O-(2S-methylbutanoyl)]-α-L-rhamnopyranosyl-(1→ 4)-O-α- L -rhamnopyranosyl-(1→2)-O-β- D -fucopyranoside(1,2″-lactone). The 1H and 13C NMR spectra (Tables 1 and 2) of 3 were similar to those of 2, with the only difference being at the site of the lactonization. In the HMBC spectrum of 3, the H-3 proton of Rha resonated at δH 5.64 and showed an HMBC correlation to the carbonyl group that resonated at δC 175.1 (C-1 of Jal), which suggested the lactone bond was linked at C-3 of Rha in 3 rather than at C-2 of Rha as in 2. Therefore, the structure of 3 was determined as shown. Aquaterin IV (4) was purified as a white powder. The molecular formula was established as C60H104O24 by the HRESIMS at m/z 1231.6814 [M + Na]+, or 14 mass units more than that of 3. Basic hydrolysis of this compound afforded simonic acid B, n-octanoic acid, and n-hexanoic acid, suggesting compound 4 to be a derivative of 3 with an n-hexanoyl unit rather than a 2-methylbutanoyl moiety. 1D NMR analysis (Tables 1 and 2) and the HMBC correlation between H-2 (δH 5.84) of Rha′ and C-1 (δC 173.4) of Hexa, together with the key fragment at m/z 813 in the MS spectrum (Supporting Information), confirmed 4 as the derivative of 3 with the substitution of an n-hexanoyl unit for a 2-methylbutanoyl group. Accordingly, the structure of 4 was determined as (11S)jalapinolic acid 11-O-α-L-rhamnopyranosyl-(1→3)-O-[4-O-noctanoyl-α-L-rhamnopyranosyl-(1→4)]-O-[2-O-n-hexanoyl]-αL-rhamnopyranosyl-(1→4)-O-α-L-rhamnopyranosyl-(1→2)-Oβ-D-fucopyranoside-(1,3″-lactone). Aquaterins V−IX (5−9), white powders, gave quasimolecular ions of [M + Na]+ at m/z 1259.7121 (C62H108O24Na), 1259.7132 (C62H108O24Na), 1259.7148 (C62H108O24Na), 1287.7437 (C64H112O24Na), and 1287.7423 (C64H112O24Na), respectively. Each compound exhibited three methyl triplets in the range δH 0.80−1.00 due to three fatty acyl groups, five anomeric protons, and three paramagnetically shifted nonanomeric ring protons in the 1H NMR spectra (Tables 1 and 3) and three ester carbonyl carbon signals and five anomeric carbon signals in the 13C NMR spectra (Table 2). Furthermore, reflux of 5−9 in 5% KOH generated simonic acid B and noctanoic acid from 5−9 and n-decanoic acid from 8 and 9. It was therefore deduced that compounds 5−7 are isomeric, and 8 and 9 are another pair of isomers. On the basis of the 1H NMR spectra, it could be seen that all of these compounds have three acylation sites on the oligosaccharide cores. Through the detailed analysis of HMBC spectra, the lactone bond was determined to be linked at C-2 of Rha in 5, 6, and 8 and at C-3 of Rha in 7 and 9, while the other two acylation sites were determined to be at C-2 of Rha′ and C-4 of Rha″ in 5 and 7−9 and at C-2 of Rha′ and C-3 of Rha″ in 6. Thus, the structures of 5−7 were established as shown, which were also confirmed by their MS fragmentation patterns (Supporting Information).14 However, the structures of 8 and 9 were hard to determine

overlapped aliphatic NMR signals, indicated that 1 is a resin glycoside composed of five sugar units and three fatty acyl groups.11,12 Moreover, in the positive ESIMS spectrum, apart from the fragment ions produced by glycosidic cleavage of the sugar moieties, i.e., m/z 1043 [M + Na − C 6 H 10 O 4 (methylpentose)]+, 799 [945 − C6H10O4 (methylpentose)]+, the presence of fragment ions at m/z 945 [1043 − 98]+ and 569 [799 − 146 − 84]+ suggested that compound 1 may contain hexanoyl (C6H10O, 98) and methylbutanoyl (C5H8O, 84) residues (Supporting Information).14 This result was confirmed by the alkaline hydrolysis of 1, which afforded a water-soluble glycoside acid in the aqueous phase and 2methylbutyric acid and n-hexanoic acid in the CHCl3 layer.11,12 The configuration of 2-methylbutyric acid was determined as S by comparison of its optical rotation value ([α]27D +17.6) with that of an authentic sample.11,12 The glycoside acid was identified as simonic acid B based on the MS and NMR spectroscopic data.15 The locations of the acyl groups on the oligosaccharide core were determined by the HMBC crosspeaks from δH 5.87 (H-4, Rha′′) to δC 173.9 (n-hexanoyl, Hexa), from δH 5.99 (H-2, Rha′) to δC 175.9 (2Smethylbutanoyl, Mba), and from δH 5.97 (H-2, Rha) to δC 173.6 (11S-hydroxyhexadecanoyl, Jal) (Supporting Information). Therefore, compound 1 was established as (11S)jalapinolic acid 11-O-α-L-rhamnopyranosyl-(1 → 3)-O-[4-Ohexanoyl-α-L-rhamnopyranosyl-(1→4)]-O-[2-O-(2S-methylbutanoyl)]-α-L-rhamnopyranosyl-(1→4)-O-α-L-rhamnopyranosyl(1→2)-O-β-D-fucopyranoside-(1,2″-lactone). Aquaterins II (2) and III (3) showed the same molecular formula, C59H102O24, according to their HRESIMS data at m/z 1217.6656 and 1217.6654 [M + Na]+ (calcd 1217.6653), respectively. Alkaline hydrolysis of 2 and 3 afforded simonic acid B in the aqueous phase and 2-methylbutyric acid and noctanoic acid in the CHCl3 layer, suggesting that 2 and 3 are a 2265



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Table 1. 1H NMR Spectroscopic Data for 1−6 (500 MHz, Pyridine-d5)a 1

2

3

4

5

6

position

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

Fuc-1 2 3 4 5 6 Rha-1 2 3 4 5 6 Rha′-1 2 3 4 5 6 Rha″-1 2 3 4 5 6 Rha‴-1 2 3 4 5 6 Jal -2 Jal-11 Jal-16 Mba-4 Mba-2-Me Hexa-6 Octa-8 Octa′-8

4.75 d (7.5) 4.18 dd (9.5, 7.5) 4.09 dd (9.5, 3.0) 4.00 d (3.0) 3.79 q (6.5) 1.52 d (6.5) 5.49 br s 5.97 br s 5.04 br d (9.5) 4.27 dd (9.5, 9.5) 4.42* 1.63 d (6.0) 6.14 br s 5.99 br s 4.63 dd (9.0, 3.0) 4.24* 4.44* 1.67 d (6.0) 5.92 br s 4.74 br s 4.53 dd (9.5, 3.0) 5.87 dd (10.0, 10.0) 4.38* 1.43 d (6.0) 5.66 br s 4.87 br s 4.43* 4.26 * 4.28* 1.58 d (6.0) 2.45 m; 2.25 m 3.87 m 0.89 t (7.0) 0.85 t (7.5) 1.08 d (7.0) 0.79 t (7.0)

4.75 d (7.5) 4.17 dd (9.5, 7.5) 4.09 dd (9.5, 3.5) 4.01 d (3.5) 3.79 q (6.5) 1.53 d (6.5) 5.49 br s 5.97 br s 5.04 dd (10.0, 3.0) 4.28 dd (10.0, 10.0) 4.42* 1.63 d (6.0) 6.14 br s 5.99 br s 4.63 dd (9.5, 3.0) 4.24* 4.44* 1.67 d (6.0) 5.92 br s 4.74 br s 4.53 dd (9.5, 3.0) 5.87 dd (9.5, 9.5) 4.38* 1.45 d (6.0) 5.66 br s 4.87 br s 4.43* 4.26 dd (9.5, 9.5) 4.28* 1.58 d (6.0) 2.45 m; 2.24 m 3.87 m 0.89 t (6.5) 0.86 t (7.5) 1.08 d (7.0)

4.83 d (8.0) 4.54 dd (8.0, 9.0) 4.21 dd (9.0, 2.5) 3.94 d (2.5) 3.84 q (6.5) 1.53 d (6.5) 6.36 br s 5.33 br s 5.64* 4.69 dd (10.0, 10.0) 5.03 dq (10.0, 6.5) 1.58 d (6.5) 5.65 br s 5.80 br s 4.56* 4.24* 4.34* 1.61 d (6.5) 5.89 br s 4.70 br s 4.47 dd (10.0, 3.0) 5.85 dd (10.0, 10.0) 4.36* 1.43 d (6.0) 5.62 br s 4.83 br s 4.45 dd (9.5, 3.0) 4.25 dd (9.5, 9.5) 4.29* 1.74 d (6.0) 2.78 m; 2.25 m 3.89 m 0.95 t (7.0) 0.90 t (7.5) 1.15 d (7.0)

4.84 d (8.0) 4.55* 4.22 dd (10.0, 3.5) 3.94 d (3.5) 3.84 q (6.5) 1.54 d (6.5) 6.37 br s 5.34 br s 5.63 dd (10.0, 3.0) 4.66 dd (10.0, 10.0) 5.04 dq (10.0, 6.0) 1.60 d (6.0) 5.68 br s 5.84 br s 4.56* 4.30 dd (9.5, 9.5) 4.36 dq (9.5, 6.0) 1.62 d (6.5) 5.94 br s 4.67 br s 4.46 dd (10.0, 3.5) 5.83 dd (10.0, 10.0) 4.36 dq (9.5, 6.0) 1.43 d (6.0) 5.60 br s 4.82 br s 4.55* 4.25 dd (10.0, 10.0) 4.31* 1.73 d (6.0) 2.95 m; 2.28 m 3.89 m 0.96 t (7.0)

4.75 d (7.5) 4.18 dd (9.5, 7.5) 4.09 dd (9.5, 3.5) 4.01 d (3.5) 3.79 q (6.5) 1.53 d (6.5) 5.50 br s 5.97 br s 5.03 dd (9.5, 3.0) 4.23 dd (9.5, 9.5) 4.37 dq (9.5, 6.0) 1.63 d (6.0) 6.16 br s 6.02 br s 4.61 dd (9.0, 3.0) 4.30 dd (9.5, 9.0) 4.46 dq (9.5, 6.0) 1.66 d (6.0) 5.93 br s 4.72 br s 4.52 dd (10.0, 3.0) 5.84 dd (10.0, 10.0) 4.38 dq (10.0, 6.0) 1.44 d (6.0) 5.62 br s 4.83 br s 4.50 dd (9.5, 3.5) 4.25 dd (9.5, 9.5) 4.29* 1.61 d (6.0)

4.79 d (7.5) 4.20 dd (9.5, 7.5) 4.13 dd (9.5, 3.0) 4.02 d (3.0) 3.80 q (6.5) 1.54 d (6.5) 5.52 br s 5.99 br s 5.03* 4.26 dd (10.0, 10.0) 4.48* 1.60 d (6.0) 6.10 br s 6.04 br s 4.66 dd (9.5, 3.5) 4.35 dd (9.5, 9.5) 4.31* 1.60 d (6.5) 5.99 br s 4.91 br s 5.87 dd (9.0, 3.0) 4.46* 4.50* 1.69 d (6.0) 5.66 br s 4.83 br s 4.49 dd (9.5, 3.5) 4.20 dd (9.5, 9.5) 4.30* 1.58 d (6.0)

2.45 m; 2.27 m 3.87 m 0.90 t (7.0)

2.43 m; 2.24 m 3.90 m 0.89 t (7.0)

0.83 t (7.0)

0.83 t (7.0)

0.84 t (7.0) 0.83 t (7.0)

0.77 t (7.0) 0.83 t (7.0)

0.80 t (7.0) 0.85 t (7.0)

a

Chemical shifts marked with an asterisk (*) indicate overlapped signals. Abbreviations: Fuc = fucose; Rha = rhamnose; Jal = 11hydroxyhexadecanoyl; Mba = 2S-methylbutanoyl; Hexa = n-hexanoyl; Octa = n-octanoyl; Me = methyl.

oligosaccharide skeletons. The aglycone (Jal) was located at C3 of Rha in 10 and at C-2 of Rha in 11, while the two Octa groups were placed at C-2 of Rha′ and C-4 of Rha″ in 10 and at C-3 of Rha′ and C-4 of Rha″ in 11. Accordingly, the structures of compounds 10 and 11 were elucidated as shown. Although the structures of compounds 1−11 were firmly established, the assignments of some carbonyl carbon signals in compounds 7, 9, and 11 still remained ambiguous because of the seriously overlapped HMBC cross-peaks between the carbonyl carbon signals and the proton signals of sugars. However, on the basis of the observation of the carbonyl carbon signals in other compounds, which have been assigned unambiguously through the HMBC correlations, it was noted that all the carbonyl carbons of the Octa groups linked with the C-4 of Rha″ shared a similar chemical shift at δC 173.9−174.0 (2−5, 8, and 10) and that the carbonyl carbons linked with the C-2 of Rha′, whether Hexa, Octa, or Deca, shared the similar chemical shift δC 173.3−173.4 when such compounds are

because the long-chain fatty acyl residue at each site was difficult to determine by NMR spectroscopy due to the seriously overlapped methylene signals. Nevertheless, the positive ESIMS data could be applied to solve this problem.14 Both 8 and 9 showed a diagnostic fragment ion peak at m/z 869 (Supporting Information), corresponding to [M + Na − 146 (C6H10O4, methylpentose) − 126 (C8H14O, octanoyl) − 146 (C6H10O4, methylpentose)]+, which suggested that the Deca (n-decanoyl) group is located at the Rha′ C-2 and that the residue on Rha″ C-4 must be an Octa group. Thus, the structures of 8 and 9 were defined as shown. Aquaterins X (10) and XI (11) were also obtained as white powders. Their molecular formulas were established as C62H108O25 and C56H98O20, respectively, according to their HRESIMS data. Basic hydrolysis afforded operculinic acid A16 for 10 and operculinic acid C16 for 11, except for n-octanoic acid. A combination of HMBC and ESIMS experiments led to assignments of the exact locations of the ester linkages on the 2266



Article

Table 2. 13C NMR Spectroscopic Data for 1−11 (125 MHz, Pyridine-d5)a 1

2

3

4

5

6

7

8

9

10

11

position

δC

δC

δC

δC

δC

δC

δC

δC

δC

δC

δC

Fuc-1 2 3 4 5 6 Rha-1 2 3 4 5 6 Rha′-1 2 3 4 5 6 Rha″-1 2 3 4 5 6 Rha‴-1 2 3 4 5 6 Glc-1 2 3 4 5 6 Jal-1 Jal -2 Jal -11 Jal -16 Mba-1 Mba-2 Mba-4 Mba-2-Me Hexa-1 Hexa-6 Octa-1 Octa-8 Octa′-1 Octa′-8 Deca-1 Deca-10

104.8 80.8 73.8 73.4 71.3 17.8 99.2 74.4 70.4 80.6 69.1 19.2 99.7 73.6 80.0 80.6 69.0 19.9 104.2 73.1 70.7 75.6 68.7 18.4 105.3 72.9 73.0 74.0 71.0 19.0

104.8 80.8 73.8 73.4 71.3 17.8 99.2 74.4 70.4 80.6 69.1 19.3 99.7 73.6 80.0 80.6 69.1 19.9 104.2 73.1 70.7 75.6 68.7 18.4 105.3 72.9 73.0 74.0 71.0 19.0

102.1 73.9 77.1 74.0 71.7 17.7 100.7 70.3 78.5 77.2 68.4 19.7 99.4 73.2 80.1 80.3 69.0 19.2 104.3 73.0 70.7 75.6 68.6 18.3 105.0 73.2 73.3 74.1 71.0 19.3

102.1 73.9 77.1 74.0 71.7 17.7 100.7 70.3 78.3 78.4 68.4 19.6 99.6 73.5 80.7 79.7 68.8 19.2 104.1 73.0 70.7 75.6 68.6 18.3 104.8 73.1 73.1 74.2 71.2 19.3

104.8 80.7 73.8 73.4 71.3 17.8 99.2 74.4 70.3 80.6 69.1 19.3 99.7 73.7 80.3 80.0 69.0 19.9 104.0 73.1 70.8 75.6 68.7 18.4 105.1 72.9 73.1 74.1 71.2 19.0

104.8 80.7 73.8 73.4 70.8 17.8 99.2 74.3 70.2 81.1 69.1 19.9 99.9 73.7 80.1 79.5 69.0 19.2 104.0 70.8 76.5 71.3 71.5 18.8 104.8 73.1 73.1 74.2 71.1 19.0

102.1 73.9 77.1 74.0 71.7 17.7 100.7 70.3 78.3 78.5 68.4 19.6 99.7 73.5 80.7 79.7 68.8 19.3 104.1 73.0 70.7 75.7 68.6 18.4 104.8 73.1 73.1 74.2 71.2 19.3

104.8 80.7 73.8 73.4 71.3 17.8 99.2 74.4 70.3 80.6 69.1 19.3 99.7 73.7 80.3 80.0 69.0 19.9 104.0 73.1 70.8 75.6 68.7 18.4 105.1 72.9 73.1 74.1 71.2 19.0

102.1 73.9 77.1 74.0 71.7 17.7 100.7 70.3 78.3 78.5 68.4 19.6 99.7 73.5 80.7 79.7 68.8 19.2 104.1 73.0 70.7 75.6 68.6 18.4 104.8 73.1 73.1 74.2 71.2 19.2

102.0 74.0 77.0 74.1 71.7 17.6 100.6 70.5 78.4 77.2 68.6 19.6 99.8 72.8 80.7 79.0 68.5 19.2 103.8 72.8 70.7 75.7 68.5 18.5

104.8 80.6 73.9 73.4 71.3 17.8 99.1 74.4 70.7 81.5 69.2 19.8 103.8 70.6 76.2 79.1 69.2 19.2 103.9 73.2 70.7 75.6 68.7 18.4

105.3 76.0 78.9 71.3 78.5 63.1 175.0 34.7 80.0 14.9

173.6 34.8 82.9 14.7

174.0 14.6 173.9 14.6

174.0 14.6 173.8 14.7

173.6 35.1 82.8 14.7 175.9 42.0 12.2 17.3 173.9 14.4

173.6 35.2 82.8 14.7 175.9

175.1 34.4 80.0 14.9 175.8

12.2 17.3

12.3 17.4

174.0 14.6

173.9 14.6

175.3 34.2 79.9 14.9

173.4 14.4 173.9 14.7

173.6 35.2 82.8 14.7

173.5 35.7 82.8 14.7

175.3 35.2 79.9 14.9

173.6 35.2 82.8 14.7

175.3 34.2 79.9 14.9

174.0 14.6 173.4 14.7

174.3 14.6 173.3 14.7

173.9 14.6 173.4 14.7

174.0 14.6

173.9 14.6

173.4 14.7

173.4 14.7

a

Abbreviations: Fuc = fucose; Rha = rhamnose; Glc = glucose; Jal = 11-hydroxyhexadecanoyl; Mba = 2S-methylbutanoyl; Hexa = n-hexanoyl; Octa = n-octanoyl; Deca = n-decanoyl; Me = methyl.

derived from simonic acid B (4−6 and 8). Also, it was found that the chemical shift of the acyl carbon of Jal was near δC 173.6 when an 18-membered ring is formed (1, 2, 5, 6, 8) and was near δC 175.3 when a 19-membered ring is formed (3, 4,

10). Therefore, on combining the analysis of HMBC correlations and the comparison of the signals with the analogous units, the carbonyl carbon signals in compounds 7, 9, and 11 were assigned successfully (Table 2). 2267



Article

Table 3. 1H NMR Spectroscopic Data for 7−11 (500 MHz, Pyridine-d5)a 7

8

9

10

11

position

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

Fuc-1 2 3 4 5 6 Rha-1 2 3 4 5 6 Rha′-1 2 3 4 5 6 Rha″-1 2 3 4 5 6 Rha‴-1 2 3 4 5 6 Glc-1 2 3 4 5 6 Jal-2 Jal-11 Jal-16 Octa-8 Octa′-8 Deca-10

4.82 d (8.0) 4.54* 4.21 dd (9.0, 3.5) 3.94 d (3.5) 3.83 q (6.5) 1.54 d (6.5) 6.35 br s 5.32 br s 5.63 dd (10.0, 2.5) 4.65 dd (10.0, 10.0) 5.03 dq (10.0, 6.0) 1.60 d (6.0) 5.67 br s 5.84 br s 4.55* 4.28 dd (9.5, 9.5) 4.36* 1.62 d (6.0) 5.92 br s 4.66 br s 4.46 dd (10.0, 3.5) 5.83 dd (10.0, 10.0) 4.36* 1.44 d (6.5) 5.58 br s 4.80 br s 4.45* 4.25 dd (10.0, 10.0) 4.30* 1.72 d (6.0)

4.84 d (7.5) 4.54* 4.21 dd (9.5, 3.0) 3.94 d (3.0) 3.84 q (6.5) 1.53 d (6.5) 6.37 br s 5.34 br s 5.63 dd (10.0, 2.5) 4.66 dd (10.0, 10.0) 5.03 dq (10.0, 6.0) 1.60 d (6.0) 5.68 br s 5.85 br s 4.55* 4.27 dd (9.5, 9.5) 4.36* 1.62 d (6.5) 5.95 br s 4.66 br s 4.47 dd (10.0, 3.0) 5.84 dd (10.0, 10.0) 4.36* 1.44 d (6.0) 5.60 br s 4.82 br s 4.54* 4.25 dd (9.5, 9.5) 4.31* 1.73 d (6.0)

4.85 d (8.0) 4.55 dd (9.0, 8.0) 4.23 dd (9.0, 3.5) 3.94 d (3.5) 3.84 q (6.0) 1.54 d (6.0) 6.37 br s 5.27 br s 5.68 dd (10.0, 3.0) 4.70 dd (10.0, 10.0) 5.01 dq (10.0, 6.0) 1.61 d (6.0) 5.65 br s 6.02 br s 4.65 dd (9.0, 3.0) 4.34* 4.40* 1.64 d (6.5) 6.25 br s 4.94 br s 4.50 dd (9.5, 3.0) 5.80 dd (9.5, 9.5) 4.40* 1.45d (6.0)

4.75 d (7.5) 4.18 dd (9.5, 7.5) 4.10 dd (9.5, 3.5) 4.00 d (3.5) 3.80 q (6.5) 1.53 d (6.5) 5.51 br s 5.94 br s 5.04 br d (9.5) 4.26 dd (9.5, 9.5) 4.49 dq* 1.67 d (6.5) 6.23 br s 4.93 br s 5.86 dd (9.5, 3.5) 4.62 dd (9.5, 9.5) 4.46* 1.68 d (6.5) 5.79 br s 4.60 br s 4.55 dd (9.5, 3.0) 5.86 dd (9.5, 9.5) 4.37 dq (9.5, 6.0) 1.42 d (6.0)

5.12 d (6.0) 3.98 br d (8.5) 4.15 dd (9.0, 9.0) 4.21 dd (9.0, 9.0) 3.91* 4.51*, 4.36* 2.70 m; 2.29 m 3.94m 0.95 t (7.0) 0.83 t (7.0) 0.83 t (7.0)

2.44m; 2.23 m 3.87 m 0.80 t (7.0) 0.80 t (7.0) 0.82 t (7.0)

2.95 3.89 0.97 0.85 0.86

m; 2.29 m m t (7.0) t (7.0) t (7.0)

4.75 d (7.5) 4.17 dd (9.5, 4.08 dd (9.5, 4.01 d (3.5) 3.79 q (6.5) 1.52 d (6.5) 5.50 br s 5.95 br s 5.02 dd (9.5, 4.22 dd (9.5, 4.36 dq (9.5, 1.64 d (6.0) 6.14 br s 6.01 br s 4.59 dd (9.0, 4.29 dd (9.0, 4.45 dq (9.0, 1.66 d (6.0) 5.92 br s 4.71 br s 4.51 dd (9.5, 5.83 dd (9.5, 4.37 dq (9.5, 1.44 d (6.0) 5.60 br s 4.82 br s 4.49 dd (9.0, 4.24 dd (9.0, 4.29* 1.61 d (6.0)

2.43 3.87 0.90 0.84

7.5) 3.5)

3.0) 9.5) 6.0)

3.0) 9.0) 6.0)

3.0) 9.5) 6.0)

3.5) 9.0)

m; 2.27 m m t (7.0) t (7.0)

2.95 3.89 0.97 0.84

0.88 t (7.0)

m; 2.28 m m t (7.0) t (7.0)

0.87 t (7.0)

a

Chemical shifts marked with an asterisk (*) indicate overlapped signals. Abbreviations: Fuc = fucose; Rha = rhamnose; Glc = glucose; Jal = 11hydroxyhexadecanoyl; Octa = n-octanoyl; Deca = n-decanoyl.

Table 4. Cytotoxicity of Compounds 1−4, 7, 9, and 10 (IC50, μM)a compound 1 2 3 4 7 9 10 cis-platinum doxorubicin a

SMMC-7721 >10 7.8 9.5 6.3 9.0 >10 >10 10.4 1.1

± ± ± ±

0.9 1.2 0.9 0.9

± 0.4 ± 0.1

HepG2 8.6 4.5 3.7 2.4 5.2 4.9 8.5 6.4 0.36

± ± ± ± ± ± ± ± ±

1.1 1.0 0.4 0.3 0.5 0.8 0.8 0.2 0.05

MG-63 >10 5.4 7.7 5.3 3.7 6.0 5.4 9.3 0.74

± ± ± ± ± ± ± ±

0.9 0.9 0.4 0.6 0.7 0.8 0.1 0.11

U2-OS 8.2 5.6 9.4 4.5 9.6 >10 >10 8.1 0.65

± ± ± ± ±

0.4 0.8 0.9 0.6 1.0

± 0.3 ± 0.08

MCF-7 9.8 5.5 4.1 4.2 5.0 9.8 7.4 8.2 0.46

± ± ± ± ± ± ± ± ±

0.6 0.9 0.4 0.7 0.8 1.6 1.0 0.5 0.05

MCF-7/ADR 5.4 5.6 >10 >10 >10 >10 >10 101.2 1574.2

± 0.9 ± 0.7

± 4.1 ± 30.1

Compounds 5, 6, 8, and 11 were inactive at 10 μM. Each value represents the mean ± SEM from three independent experiments.

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Article

Figure 1. G0/G1 cell cycle arrest and apoptosis in HepG2 cells by compound 4. (A) Histograms showing the number of cell channels (vertical axis) vs DNA content (horizontal axis). (B) Columns showing the percentage of HepG2 cells in each phase of the cell cycle. (C) Columns showing the percentage of sub-G1 cells. Each value represents the mean ± SEM of three independent experiments; *p < 0.05, **p < 0.01, as compared with the control.

Figure 2. Effects of compounds 1−11 on intracellular Ca2+ concentrations in HepG2 cells. (A) Alteration of fluorescence intensities when the cells were treated with compounds 1−11. (B) Scatter plots representing inhibitory rate on HepG2 cells vs intracellular Ca2+ fluorescence intensities for compounds 1−11. Each value represents the mean ± SEM of three independent experiments; *p < 0.05, **p < 0.01, as compared with the control.

that they might possess MDR-selective toxicity.17 Since compounds 1 and 2 both share an 18-membered ring, and their 19-membered ring analogues 3, 4, 7, 9, and 10 did not exhibit such effects, it can be assumed that the 18-membered macrocyclic structure is crucial for their cytotoxicity on multidrug-resistant cells, which may serve as a valuable starting point for the design and synthesis of new agents for treating the multidrug-resistant cancer types. To clarify the cytotoxic mechanism of these resin glycosides, the effect of compound 4 on the cell-cycle progress of HepG2 cells was analyzed by flow cytometry.18 The results showed a significant increase in the numbers of G0/G1 cells in a dosedependent manner (Figure 1A and B). Meanwhile, a sub-G1 peak was also observed at a high concentration (8 μM), indicating the activation of apoptosis (Figure 1C). Thus, it is deduced that 4 inhibits the proliferation of HepG2 cells via G0/ G1 cell cycle arrest and apoptosis induction. Since intracellular Ca2+ is a key regulator of many cellular processes,19,20 and some resin glycosides have been shown to transport Ca2+ across the plasma membrance,7,8 efforts were made to explore whether compounds 1−11 could affect cellular Ca2+ homeostasis in HepG2 cells.21 As shown in Figure 2A, compounds 1−4, 7, 9, and 10 at 30 μM induced significant elevations of Ca2+ in HepG2 cells. In particular, the Ca2+

As many reports on the cytotoxicity of resin glycosides are now available,2 the cytotoxic activities of compounds 1−11 were tested toward the human breast cancer (MCF-7, MCF-7/ ADR), human hepatoma (SMMC-7721, HepG2), and human osteosarcoma (MG-63, U2-OS) cell lines. The screening results are expressed as IC50 values and are summarized in Table 4. Compounds 1−4, 7, 9, and 10 exhibited weakly cytotoxic activities against all or part of the selected drug-sensitive cancer cells MCF-7, SMMC-7721, HepG2, MG-63, and U2-OS, while 1 and 2 displayed inhibitory effects against the multidrugresistant cell line MCF/ADR.3 It was found that, when compared to an 18-membered ring, some resin glycosides with a 19-membered ring showed increased activity against drug-sensitive cancer cells (7 vs 5, 9 vs 8). However, those having an opposite effect on multidrug-resistant MCF/ADR cells (3 vs 2) were evident, suggesting the influence of the macrocyclic structure on the resultant cytotoxic activity is not straightforward. The acyl groups also seem to affect the cytotoxic activities, as evidenced by the comparison of the activities of 5, 6, and 8 with those of 1 and 2. Moreover, it is noteworthy that compounds 1 and 2 exhibited comparable cytotoxic effects against both MCF-7 and its resistant counterpart, MCF-7/ADR, which suggests that they cannot be recognized by multidrug resistance (MDR) transporters or 2269



Article

Aquaterin II (2): white powder; [α]28D −34.8 (c 0.31, MeOH); IR (KBr) νmax 3424, 2929, 2857, 1736, 1639, 1461, 1384, 1136, 1063 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 1217.9 [M + Na]+ and 1230.0 [M + Cl]−; HRESIMS m/z 1217.6654 [M + Na]+ (calcd for C59H102O24Na, 1217.6653). Aquaterin III (3): white powder; [α]27D −42.9 (c 0.19, MeOH); IR (KBr) νmax 3449, 2932, 2858, 1740, 1461, 1383, 1137, 1063 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 1218.1 [M + Na]+ and 1230.0 [M + Cl]−; HRESIMS m/z 1217.6656 [M + Na]+ (calcd for C59H102O24Na, 1217.6653). Aquaterin IV (4): white powder; [α]27D −45.6 (c 0.14, MeOH); IR (KBr) νmax 3451, 2930, 2857, 1727, 1384, 1136, 1059 cm−1; 1H and 13 C NMR data, see Tables 1 and 2; ESIMS m/z 1232.0 [M + Na]+ and 1244.0 [M + Cl]−; HRESIMS m/z 1231.6814 [M + Na]+ (calcd for C60H104O24Na, 1231.6810). Aquaterin V (5): white powder; [α]26D −33.8 (c 0.43, MeOH); IR (KBr) νmax 3418, 2929, 2858, 1736, 1637, 1458, 1384, 1136, 1063 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 1260.1 [M + Na]+ and 1272.0 [M + Cl]−; HRESIMS m/z 1259.7132 [M + Na]+ (calcd for C62H108O24Na, 1259.7123). Aquaterin VI (6): white powder; [α]27D −13.3 (c 0.17, MeOH); IR (KBr) νmax 3426, 2932, 2860, 1739, 1461, 1386, 1138, 1065 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 1260.1 [M + Na]+ and 1272.0 [M + Cl]−; HRESIMS m/z 1259.7148 [M + Na]+ (calcd for C62H108O24Na, 1259.7123). Aquaterin VII (7): white powder; [α]27D −63.9 (c 0.43, MeOH); IR (KBr) νmax 3442, 2932, 2858, 1732, 1640, 1461, 1384, 1136, 1062 cm−1; 1H and 13C NMR data, see Tables 2 and 3; ESIMS m/z 1259.9 [M + Na]+ and 1272.0 [M + Cl]−; HRESIMS m/z 1259.7121 [M + Na]+ (calcd for C62H108O24Na, 1259.7123). Aquaterin VIII (8): white powder; [α]27D −24.0 (c 0.21, MeOH); IR (KBr) νmax 3425, 2929, 2857, 1736, 1641, 1459, 1384, 1136, 1063 cm−1; 1H and 13C NMR data, see Tables 2 and 3; ESIMS m/z 1288.2 [M + Na]+ and 1300.1 [M + Cl]−; HRESIMS m/z 1287.7423 [M + Na]+ (calcd for C64H112O24Na, 1287.7436). Aquaterin IX (9): white powder; [α]28D −47.8 (c 0.21, MeOH); IR (KBr) νmax 3442, 2931, 2858, 1738, 1635, 1460, 1384, 1136, 1062 cm−1; 1H and 13C NMR data, see Tables 2 and 3; ESIMS m/z 1288.1 [M + Na]+ and 1300.1 [M + Cl]−; HRESIMS m/z 1287.7437 [M + Na]+ (calcd for C64H112O24Na, 1287.7436). Aquaterin X (10): white powder; [α]28D −31.6 (c 0.15, MeOH); IR (KBr) νmax 3420, 2932, 2860, 1739, 1461, 1386, 1138, 1060 cm−1; 1H and 13C NMR data, see Tables 2 and 3; ESIMS m/z 1276.2 [M + Na]+ and 1287.9 [M + Cl]−; HRESIMS m/z 1275.7066 [M + Na]+ (calcd for C62H108O25Na, 1275.7072). Aquaterin XI (11): white powder; [α]27D −39.4 (c 0.26, MeOH); IR (KBr) νmax 3419, 2929, 2857, 1737, 1644, 1458, 1384, 1136, 1064 cm−1; 1H and 13C NMR data, see Tables 2 and 3; ESIMS m/z 1114.1 [M + Na]+ and 1126.1 [M + Cl]−; HRESIMS m/z 1113.6545 [M + Na]+ (calcd for C56H98O20Na, 1113.6544). Alkaline Hydrolysis of Compounds 1−11. Compounds 1−11 (3.0 mg each) in 5% KOH (3 mL) were separately refluxed at 80 °C for 2 h. The reaction mixtures were acidified to pH 4 and extracted with CHCl3 (3 mL × 3). The CHCl3 layer was then washed with water, dried over anhydrous Na2SO4, concentrated in vacuo, and then analyzed by GC-MS on a Shimadzu GCMS-QP2010 Ultra in EI at 70 eV under the following conditions (30 m × 0.25 mm × 0.25 μm, RTX5MS column; He, 0.8 mL/min; 35 °C, 3 min; 35−300 °C, Δ 10 °C/ min). The peaks of organic acids in each CHCl3 layer were identified by comparison with authentic samples as 2-methylbutanoic acid (tR 8.970 min; m/z 87 (19), 74 (100), 57 (54), 41 (50), 29 (40)) in 1−3; n-hexanoic acid (tR 10.925 min; m/z 99 (1), 87 (14), 73 (58), 60 (100), 41 (28)) in 1 and 4; n-octanoic acid (tR 14.700 min; m/z 144 [M]+ (1), 115 (11), 101 (27), 85(32), 73 (67), 60 (100), 43 (54)) in 1−11; and n-decanoic acid (tR 16.000 min; m/z 172 [M]+ (4), 143 (10), 129 (51), 115 (15), 87 (19), 73 (100), 60 (96), 57 (54), 55 (41), 43 (62)) in 8 and 9. The CHCl3 extract (0.7 mg) from the alkaline hydrolysis of 1 was purified by preparative HPLC eluted with MeOH− H2O (25:75) to give 2-methylbutyric acid (0.3 mg), which was assigned with the S-configuration by comparing the optical rotation

fluorescence intensity increased by over 200% when cells were treated with 1 or 10. Moreover, it is interesting to note that the inhibitory effect on HepG2 cells of these compounds (30 μM) is correlated positively with elevated intracellular Ca 2+ concentrations (Figure 2B), suggesting that the increase in Ca2+ might be involved in the regulation of their cytotoxic activities.22 This supports the hypothesis proposed by Pereda− Miranda and colleagues that some biological potential of the resin glycosides may rely on their pore-forming properties to provoke an imbalance in cellular homeostasis.6,23

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were obtained on a JASCO P-1020 polarimeter. IR spectra were recorded on KBr disks on a Bruker Tensor 27 IR spectrometer. 1D and 2D NMR spectra were recorded in pyridine-d5 on a Bruker AV-500 NMR instrument at 500 MHz (1H) and 125 MHz (13C). Mass spectra were acquired using an MS Agilent 1100 series LC/MSD ion-trap mass spectrometer (ESIMS) and an Agilent 6520B Q-TOF spectrometer (HRESIMS), respectively. The GC-MS was performed on a Shimadzu GCMS-QP2010 Ultra. Column chromatography (CC) was carried out using silica gel (Qingdao Marine Chemical Co., Ltd., Qingdao, People’s Republic of China) and ODS (40−63 μm, Fuji, Tokyo, Japan). Preparative HPLC was run on an Agilent 1100 series system equipped with a Shim-pack RP-C18 column (20 × 200 mm). An Alltech 2000 evaporative light scattering detector (ELSD) was combined with HPLC for detection of compounds. All solvents were of analytical grade. Plant Material. The whole plants of I. aquatica were collected in Dongshan, Nanjing, Jiangsu Province, People’s Republic of China, in November 2012. The plant material was authenticated by Prof. MinJian Qin, Department of Medicinal Plants, China Pharmaceutical University. A voucher specimen (no. 121108) was deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University. The whole plants were air-dried and were then powered using a grinder. Extraction and Isolation. The dried powder of I. aquatica (5 kg) was exhaustively extracted with 95% EtOH (3 × 3 h). The extract was then combined and concentrated under reduced pressure, using a 50 °C water bath. The crude extract (400 g) was suspended in 2.0 L of water and then successively extracted with petroleum ether, dichloromethane (CH2Cl2), and ethyl acetate (EtOAc). The CH2Cl2-soluble fraction (30g) was chromatographed on a silica gel column eluted with CH2Cl2−MeOH (14:1, 5:1, 1:1, 0:1) to afford three pooled fractions (Fr. 1−3). Fr. 2 was applied to an ODS column using a continuous gradient of MeOH−H2O (30:70−100:0, v/v) to afford 11 fractions (Fr. 2.1−2.11). Fr. 2.2 was further separated by silica gel column chromatography using CH2Cl2−MeOH (12:1), to give four subfractions (Fr. 2.2.1−2.2.4). Purification of Fr. 2.2.2 was then performed on preparative HPLC with MeOH−H2O (83:17), yielding 3 (9 mg) and 4 (4 mg). Fr. 2.3 and 2.6 were separately subjected to preparative HPLC using MeOH−H2O (89:11 and 92:8, respectively) to give 7 (31 mg) and 10 (4 mg), respectively. Fr. 2.4 was chromatographed over a silica gel column eluted with CH2Cl2− MeOH (10:1) to give three subfractions (Fr. 2.4.1−2.4.3). Fr. 2.4.2 and 2.4.3 were separately purified by preparative HPLC with MeOH− H2O (90:10 and 89:11, respectively) to give 9 (9 mg) and 6 (6 mg). Separation of Fr. 2.7 was carried out by preparative HPLC with MeOH−H2O (93:7) to give 1 (11 mg), 2 (52 mg), and 11 (9 mg). Compounds 5 (110 mg) and 8 (80 mg) were obtained from Fr. 2.10 and 2.11 by preparative HPLC purification with MeOH−H2O (95:5 and 94:6, respectively), respectively. Aquaterin I (1): white powder; [α]27D −45.4 (c 0.23, MeOH); IR (KBr) νmax 3423, 2929, 2857, 1737, 1637, 1460, 1383, 1135, 1063 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 1190.0 [M + Na]+ and 1201.9 [M + Cl]−; HRESIMS m/z 1189.6343 [M + Na]+ (calcd for C57H98O24Na, 1189.6340). 2270



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([α]27D +17.6) with that of authentic 2S-methylbutyric acid.11,12 The aqueous phase obtained from compounds 1−11 was partitioned separately with n-BuOH (3 mL × 3) and purified on an open column containing ODS to afford simonic acid B15 for 1−9, operculinic acid A16 for 10, and operculinic acid C16 for 11, by comparing the mass spectrometric and NMR spectroscopic data with authentic samples or with literature data. Cytotoxicity Assays. Human hepatoma cell lines HepG2 and SMMC-7721, human breast carcinoma cell lines MCF-7 and MCF-7/ ADR, and human osteosarcoma cell lines MG-63 and U2-OS were purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, People’s Republic of China). HepG2, MCF-7, and MG-63 cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco Invitrogen Corp., Carlsbad, CA, USA). SMMC-7721, U2-OS, and MCF-7/ADR cell lines were cultured in modified RPMI-1640 medium (Gibco Invitrogen Corp.). The cells were all supplemented with 10% fetal bovine serum (Sijiqing, Hangzhou, People’s Republic of China), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C with 5% CO2. The cytotoxic activity was investigated using the MTT colorimetric method.24 Briefly, cells were seeded in a 96-well plate at a density of 5 × 103 cells per well in 190 μL of medium for 12 h. Then, the cells were exposed to the test compounds or positive controls. After 48 h exposure, 20 μL of MTT (5 mg/mL) was added, and the cells were incubated at 37 °C for an additional 4 h. The supernatants were then aspirated, and 150 μL of DMSO was added to each well. The absorbance was measured at 570 nm using a microplate reader (Spectramax Plus 384, Molecular Devices, Sunnyvale, CA, USA). The cytotoxic activity was expressed as IC50 values (the concentration of a compound that inhibited the proliferation rate of tumor cells by 50% as compared to the untreated control cells). All experiments were carried out in triplicate and repeated twice. cis-Platinum and doxorubicin were used as positive controls. In the experiments to test the inhibitory effects on HepG2, cells were incubated with 30 μM compounds 1−11 for 12 h followed by MTT incubation, as described above. Cell Cycle Analysis via Propidium Iodide Staining Assay. HepG2 cells were seeded in six-well culture plates (2.5 × 105 cells per well). After incubation overnight, various concentrations (2, 4, 8 μM) of compound 4 or 0.1% DMSO were added, and the cells were incubated for 48 h. Then, the cells were harvested, washed with PBS twice, fixed in ice-chilled 70% EtOH for 12 h at 4 °C, and again incubated for 30 min at 37 °C in a PBS solution containing 1 mg/mL RNase A. Propidium iodide (Beyotime, Nantong, People’s Republic of China) was added for DNA content analysis. The analysis was carried out using a BD FACS Calibur flowcytometer (Becton & Dickinson Co., Miami, FL, USA), and the data were sorted with the CellQuest program from Becton−Dickinson.18 Intracellular Free Ca2+ Measurement. The concentration of intracellular Ca2+ concentration was measured using the fluorescence Ca2+ indicator Fluo-3 AM according to a previous report.21 HepG2 cells were harvested after exposure to the test compounds (30 μM) for 6 h, washed with PBS twice, and loaded with 2 μM Fluo-3 AM (Beyotime) for 45 min in the dark. Then the cells were rinsed twice and incubated for another 20 min at 37 °C to ensure that Fluo-3 AM had completely transformed into Fluo-3 in the cells. Detection of intracellular Ca2+ was carried out by a BD Accuri C6 flow cytometer (Becton & Dickinson Co.). The Ca2+ concentration was expressed as the mean fluorescence intensity of each treated group compared with that of the vehicle control group. Each value represented the mean ± SEM of three independent experiments. Statistical Analysis. All results were recorded as the means ± SEM for at least three independent experiments. Data were analyzed by one-way analysis of ANOVA using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA). A p value less than 0.05 was considered statistically significant.

Article

ASSOCIATED CONTENT

S Supporting Information *

1D NMR, 2D NMR, ESIMS, and HRESIMS spectra of 1−11 are available free of charge via the Internet at http://pubs.acs. org.

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AUTHOR INFORMATION

Corresponding Authors

*Tel/Fax: +86-25-8327-1405. E-mail: [email protected] (J.-G. Luo). *Tel/Fax: +86-25-8327-1405. E-mail: [email protected] (L.-Y. Kong). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported financially by the Program for Changjiang Scholars and Innovative Research Team in University (IRT1193), Program for New Century Excellent Talents in University (NCET-12-0977), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). B.Y.F. was supported by the 111 Project (No. 111-2-07).

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REFERENCES

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