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Cytotoxic cardenolides and sesquiterpenoids from the fruits of Reevesia formosana
Phytochemistry xxx (2016) 1e9
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Cytotoxic cardenolides and sesquiterpenoids from the fruits of Reevesia formosana Pei-Yu Hsiao a, Shiow-Ju Lee b, Ih-Sheng Chen a, c, d, Hsing-Yu Hsu b, Hsun-Shuo Chang a, c, d, e, * a
Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, 807, Taiwan, ROC Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, 350, Taiwan, ROC School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, 807, Taiwan, ROC d Research Center for Natural Products and Drug Development, Kaohsiung Medical University, Kaohsiung, 807, Taiwan, ROC e Center for Infectious Disease and Cancer Research (CICAR), Kaohsiung Medical University, Kaohsiung, 807, Taiwan, ROC b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 March 2016 Received in revised form 9 June 2016 Accepted 23 June 2016 Available online xxx
Bioassay-guided fractionation of the fruits of Reevesia formosana led to isolation of three cardenolides (reevesioside J, reevesioside K, and epi-reevesioside K), three sesquiterpenoids (reevesiterpenol C, reevesiterpenol D, and reevesiterpenol E), and two glycosides (reevesianin A and reevesianin B), along with 46 known compounds. Their structures were determined using spectroscopic techniques. In addition to the reported cytotoxic cardenolides, reevesioside J and strophanthidin exhibited moderate cytotoxicity against the cell lines MCF-7, NCI-H460, and HepG2, with IC50 values of 0.39 ± 0.06 mM and 1.06 ± 0.12 mM for MCF-7, 0.12 ± 0.01 mM and 0.29 ± 0.01 mM for NCI-H460, and 1.09 ± 0.02 mM and 1.72 ± 0.02 mM for HepG2, respectively. Reevesiterpenol E also exhibited the best selective cytotoxicity to the NCI-H460 cell line, with an IC50 value of 3.15 ± 0.22 mM. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Reevesia formosana Sterculiaceae Cardenolides Sesquiterpenoids Cytotoxicity
1. Introduction The Sterculiaceae family, with its rich fruits and oily seeds, has long been used in traditional treatments and as supplements (Al Muqarrabun and Ahmat, 2015). Among these genera, Reevesia, with its unique fruit forms and glamorous inflorescence, has not been well studied. This inspired an investigation of the endemic species Reevesia formosana Sprague (Sterculiaceae), which is found in the low forests of the Hengchun Peninsula, Pingtung in southern Taiwan (Li and Lo, 1993). A previous investigation of the chemical constituents of the root of R. formosana identified new cardenolides that exhibit potent cytotoxicity against the cancer cell lines MCF-7, NCI-H460, and HepG2 in vitro (Chang et al., 2013). The methanolic extract of the fruits of this species also exhibited potent cytotoxicity against the above three cancer cells. A bioassay-guided fractionation of the active EtOAc-soluble layer of the fruits led to isolation of 54 compounds, including three new cardenolides (reevesioside J
* Corresponding author. Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, 807, Taiwan, ROC. E-mail address: [email protected] (H.-S. Chang).
(1), reevesioside K (2), and epi-reevesioside K (3)), three new sesquiterpenoids (reevesiterpenol C (4), reevesiterpenol D (5), and reevesiterpenol E (6)), and two new glycosides (reevesianin A (7) and reevesianin B (8) (Fig. 1)) and 46 known compounds. The structures of these compounds were elucidated using spectroscopic analyses, and an evaluation of their cytotoxicities is also described.
2. Results and discussion Compound 1 was identified as an optically active colorless syrup with ½a22 D e13.0 (c 0.05, MeOH). Its molecular formula was determined to be C30H44O8 by ESIMS and HRESIMS analyses. The IR spectrum indicated the presence of a hydroxy group (3394 cm1) and an a,b-unsaturated g-lactone ring (1775, 1738, and 1629 cm1). According to a previous investigation of R. formosana (Chang et al., 2013), the MS, IR, 1H, and 13C NMR spectra suggested that the substance was a characteristic cardenolide glycoside of this species. The 1H and 13C NMR spectra had signals for an a,b-unsaturated glactone at d 4.81 (1H, dd, J ¼ 18.0, 1.8 Hz, H-21b), 4.98 (1H, dd, J ¼ 18.0, 1.2 Hz, H-21a), and 5.87 (1H, br s, H-22); two methyl groups at d 0.87 (3H, s, H-18) and 0.94 (3H, s, H-19), as well as oxygen-
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bearing quaternary carbon (d 85.6, C-14), indicating the aglycone to be a 3-O-substituted digitoxigenin (Beale et al., 1988). HMBC (Fig. 2) correlations were observed between: H-18 and C-12 (d 40.1), C-13 (d 49.6), C-14, and C-17 (d 50.9); H-19 and C-1 (d 30.1), C-5 (d 36.1), C-9 (d 35.8), and C-10 (d 35.1); and H-17 [(d 2.78) (1H, dd, J ¼ 9.6, 5.4 Hz)] and C-13, C-21 (d 73.4), and C-22 (d 117.7). These further confirmed the aglycone moiety of the structure. From the NOESY (Fig. 3) correlations between H-19 and H-5 (d 1.77) and H-8 (d 1.55), and between H-18 and H-8, H-21, and H-22, the relative configuration of digitoxigenin was deduced. An anomeric proton at d 4.69 (1H, d, J ¼ 7.8 Hz, H-10 ) indicated the presence of a sugar moiety with the following signals: a methyl group at d 1.37 (3H, d, J ¼ 6.0 Hz, H-60 ); four oxymethines at d 3.24 (1H, dd, J ¼ 9.6, 7.8 Hz, H-20 ), 3.34 (1H, dq, J ¼ 9.0, 6.0 Hz, H-50 ), 3.36 (1H, t, J ¼ 9.6 Hz, H-30 ), and 3.58 (1H, dd, J ¼ 9.6, 9.0 Hz, H-40 ); and a methylenedioxy group at d 5.14 (1H, d, J ¼ 0.6 Hz, H-70 b), and d 5.17 (1H, d, J ¼ 0.6 Hz, H70 a). From the NOESY correlations between H-10 and H-30 , and H-50 and between H-20 and H-40 , the relative configuration of the sugar moiety was deduced. Combining the HMBC and COSY analyses with the J values (>7.8 Hz) of H-10 ~ H-50 , the methylenedioxy group was determined to adopt an equatorial orientation at C-20 and C-30 . From the above signals, this sugar moiety was identified as a 6deoxy-2,3-methylenedioxy-b-glucopyranosyl unit. The anomeric proton H-10 exhibited a 3J-correlation with C-3 (d 74.6), and the H-3 signal was present as a broad singlet. Thus the 6-deoxy-2,3methylenedioxy-b-glucopyranosyl unit was determined to be connected to the C-3 of the digitoxigenin moiety in a b-orientation (Shi et al., 2010). Therefore, the structure of 1 was determined to be digitoxigenin-6-deoxy-2,3-methylenedioxy-b-glucopyranoside and named reevesioside J. Compound 2 was identified as an optically active colorless syrup with ½a23 D þ36.0 (c 0.1, MeOH). Its molecular formula was determined to be C30H38O10 by ESIMS and HRESIMS analyses. The IR spectrum of the compound suggested the presence of a strophanthidin-like aglycone with an absorption of an aldehyde group at 1714 cm1, an absence of the a,b-unsaturated g-lactone ring, and presence of a saturated g-lactone ring at 1780 cm1. The
substance had three methylene groups, indicated by d 3.70 (1H, d, J ¼ 10.2 Hz, H-18b) and 4.12 (1H, dd, J ¼ 10.2, 3.0 Hz, H-18a), 2.27 (1H, dd, J ¼ 18.0, 1.8 Hz, H-22b) and 3.30 (1H, d, J ¼ 18.0 Hz, H-22a), and 4.02 (1H, d, J ¼ 10.2 Hz, H-21b) and 4.43 (1H, dd, J ¼ 10.2, 1.2 Hz, H-21a), respectively. The IR spectrum and the presence of the methylene groups suggested that the substance may be an example of an 18,20-epoxy derivative, such as reevesioside G (14). This type of derivative has been previously isolated from the root of R. formosana (Chang et al., 2013). Comparison of the 1H NMR spectra of 2 and reevesioside G (14) (Chang et al., 2013), which bears an 18,20-epoxide in its structure, showed that the two compounds were similar, but 2 exhibited an olefinic proton at d 5.82 (1H, d, J ¼ 1.8 Hz, H-15). Compound 2 also had double bond signals in its 13C NMR spectrum at d 186.4 (C-14) and 125.6 (C-15) and a carbonyl carbon at d 204.4 (C-16). HMBC (Fig. 2) correlations between H-15 and C-13 (d 57.0), C-14, C-16, and C-17 (d 59.7), together with the 12 degrees of unsaturation, suggested a cyclopentenone moiety in 2, which suggested the presence of an aglycone different from that of reevesioside G. Additional absorptions at 1698 cm1 for the carbonyl group and 1614 cm1 for the double bond in the IR spectrum also supported the presence of a cyclopentenone in 2. The connections of a saturated lactone ring and an aldehyde group were confirmed through the correlations between: H-21 and C-20 (d 85.8) and C-23 (d 174.5); H-22 and C-20, C-21 (d 74.6), and C-23; and H-19 (d 9.92) and C-10 (d 54.2) of HMBC. For the absolute configuration of the spiro moiety at C-20, the NOESY (Fig. 3) correlations were observed between H-21a and H-18b, and H-21b and H-17 (d 2.28). Lack of correlations between H-22 and H17 or H-18, however, indicated a S-configuration at C-20 of 2 (Chang et al., 2013). An anomeric proton at d 4.48 (1H, d, J ¼ 6.6 Hz, H-10 ) also suggested the presence of a sugar moiety including one methyl group [d 1.24 (3H, d, J ¼ 6.6 Hz, H-60 )], one methylene [d 1.74 (1H, m, H-40 b) and 2.15 (1H, m, H-40 a)], three oxymethines [d 3.83 (1H, dd, J ¼ 6.6, 5.4 Hz, H-20 ), 3.83 (1H, m, H-50 ), 4.15 (1H, td, J ¼ 5.4, 1.8 Hz, H-30 )], and one methylenedioxy group [d 4.91 (1H, s, H-70 b), 5.23 (1H, s, H-70 a)]. The sugar moiety was identified as a unique reevesiosyl group that has only been previously isolated from this
Fig. 1. Structures of compounds 1e8 from the fruits of R. formosana.
Please cite this article in press as: Hsiao, P.-Y., et al., Cytotoxic cardenolides and sesquiterpenoids from the fruits of Reevesia formosana, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.06.009
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Fig. 2. Key HMBC (/), and COSY (━) correlations of 18.
species (Chang et al., 2013). As H-10 exhibited a 3J-correlation toward C-3 (d 73.1) and a broad triplet with a small J value of H-3 [d 4.21 (1H, br t, J ¼ 2.7 Hz)], the reevesiosyl group connected to the C3 of the aglycone was determined to exist in a b-orientation (Shi et al., 2010). On the basis of the above evidence, structure 2 was elucidated and named reevesioside K. Compound 3 was similar to 2 in that it was also an optically active colorless syrup with ½a23 D þ25.9 (c 0.06, MeOH). The same molecular formula, C30H38O10, was proposed with a [MþNa]þ m/z of 581.2360, as shown by the ESIMS and HRESIMS analyses. From the similarities between their 1H NMR spectra, a slight difference in the structures of compounds 3 and 2 was found through the chemical shifts of the methylene groups of C-18 [d 3.63 (1H, d, J ¼ 10.2 Hz, H-18b), 4.12 (1H, d, J ¼ 10.2 Hz, H-18a)], C-21 [d 4.13 (1H, d, J ¼ 10.5 Hz, H-21b), 4.94 (1H, d, J ¼ 10.5 Hz, H-21a)], and C-22 [d 2.45 (1H, d, J ¼ 17.4 Hz, H-22b), 2.79 (1H, d, J ¼ 17.4 Hz, H-22a)]. The aglycone of 3 resembled that of 2, as an 18,20-epoxide, whereas
NOESY (Fig. 3) correlations were found between H-22a and H-18b and H-22b and H-17 (d 2.42) instead of H-21. Moreover, there was no correlation between H-21 and H-18 or H-17, which indicated an absolute configuration of 20R for the spiro of 3 (Chang et al., 2013). The sugar moiety of 3 was also identified as the reevesiosyl group from the 1H NMR, 13C NMR, HMBC, COSY, and NOESY spectroscopic analyses. With the HMBC (Fig. 2) correlation of H-10 (d 4.48) and C-3 (d 73.1) and the presence of a broad triplet of H-3, the connection of the sugar moiety to the aglycone of 3 was found to be the same as that of 2. The two substances were different only in the configuration at C-20, which was consistent with reevesioside G (14) and epi-reevesioside G (15) (Chang et al., 2013) for both the NOESY correlations and chemical shifts of C-17 and C-22. Thus, 3 was determined to be the epimer of 2 and was named epi-reevesioside K. Compound 4 was obtained as an optically active yellowish oil with ½a23 D þ30.2 (c 0.04, CHCl3), and its molecular formula was
Please cite this article in press as: Hsiao, P.-Y., et al., Cytotoxic cardenolides and sesquiterpenoids from the fruits of Reevesia formosana, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.06.009
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Fig. 3. Key NOESY(4) correlations of 18.
found to be C15H20O4, as determined by ESIMS and HRESIMS analyses, suggesting that the structure was a sesquiterpenoid with six degrees of unsaturation. The IR spectrum indicated the presence of a hydroxy group (3421 cm1) and a carbonyl group (1655 cm1), with UV absorption maxima at 206 and 276 nm. The above physical data and the 1H and 13C NMR spectra (Table 2) of 4 were similar to those of furanosesquiterpenoid-hibiscone B (Ferreira et al., 1980), except for the presence of a hydroxy group at d 1.53 (1H, br s, OH-7, D2O exchangeable) and an oxymethine at d 4.28 (1H, dd, J ¼ 3.6, 3.0 Hz, H-7) in 4 rather than the methylene group of hibiscone B. The HRESIMS of 4 also indicated it contained one more oxygen atom than hibiscone B. According to the HMBC (Fig. 2) correlations, the OH-7 group exhibited a 2,3J-correlation between C-6 (d 44.2), C7 (d 72.2) and C-8 (d 66.6), which confirmed the substitution of a
hydroxy group at C-7. The relative configuration of 4 was determined to be identical to hibiscone B based on the NOESY (Fig. 3) correlations between H-7 and H-6 (d 1.61) and H-8 (d 4.70) and between H-5 (d 3.32) and H-12 (d 1.36) and H-13 (d 2.11). Thus the structure of 4 was elucidated, and the compound was named reevesiterpenol C. The physical data of compound 5 were similar to those of 4, whereas the molecular formula was determined to be C15H20O5, as established by HRESIMS analyses, suggesting that this compound contains one more oxygen atom than 4. Compound 5 exhibited similar 1H and 13C NMR spectra to 4, except for a hydroxyisopropyl group [dH 1.40 (3H, s, H-14), 1.43 (3H, s, H-15); dC 29.0 (C-15), 32.0 (C-14), 73.8 (C-13)] of 5 at C-6 was in the same place as the isopropyl group of 4 at C-6. The HMBC (Fig. 2) correlations between H-
Please cite this article in press as: Hsiao, P.-Y., et al., Cytotoxic cardenolides and sesquiterpenoids from the fruits of Reevesia formosana, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.06.009
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14 and H-15 toward C-6 (d 49.9) and C-13, respectively, also confirmed that the hydroxyisopropyl group was connected at C-6. The structure of 5 was elucidated, and the compound was named reevesiterpenol D. Compound 6 was obtained as a brownish oil. The ESIMS and HRESIMS analyses indicated that the molecular formula was C16H16O5 and that it had nine degrees of unsaturation. The UV absorption maxima at 225, 254, and 373 nm underwent a bathochromic shift after addition of aqueous KOH, suggesting the presence of a phenolic moiety. Through analyses of the 13C NMR and DEPT spectra, the substance was determined to be a sesquiterpenoid. As the physical data were similar to those of hibiscolactone A (Ferreira et al., 1980), the 1H NMR spectrum of 6 indicated the presence of one more methoxy group (d 3.91) and one less olefinic proton than in hibiscolactone A. From the HMBC (Fig. 2) correlation between the methoxy group (d 3.91) and C-7 (d 145.7) and between H-13 [d 3.85 (1H, sept, J ¼ 7.2 Hz)] and C-7, along with the HRESIMS data, the attachment of one methoxy group to C-7 was established. The structure of 6 was suggested as shown, and the compound was assigned the trivial name reevesiterpenol E. Compound 7 was obtained as an optically active brownish syrup with ½a23 D e40.9 (c 0.06, MeOH), and the molecular formula of C27H34O15 was determined on the basis of ESIMS and HRESIMS data (m/z 621.1792 [MþNa]þ). The IR spectrum indicated the presence of a hydroxy group (3410 cm1), a carbonyl group (1699 cm1), and an aromatic ring (1602, 1514, 1457 cm1). The above moieties corresponded to the data found in the UV spectrum and the bathochromic shift of the compound. The 1H NMR spectrum showed two aromatic ring signals, three meta-coupled protons [d 6.02 (1H, t, J ¼ 2.1 Hz, H-4), 6.15 (1H, t, J ¼ 2.1 Hz, H-6), 6.19 (1H, t, J ¼ 2.1 Hz, H-2)], and one ABX system of a benzene ring [d 6.89 (1H, d, J ¼ 8.4 Hz, H-5000 ), 7.48 (1H, d, J ¼ 1.8 Hz, H-2000 ), 7.55 (1H, dd, J ¼ 8.4, 1.8 Hz, H-6000 )]. The three methoxy groups [d 3.65, 3.84, and 3.87] connected to aromatic rings were determined to be OCH3-5, OCH33000 , and OCH3-4000 , respectively, by the 3J-correlations in the HMBC. Furthermore, two anomeric protons [d 4.94 (1H, d, J ¼ 7.8 Hz, H-10 ), 5.54 (1H, d, J ¼ 1.2 Hz, H-100 )] suggested the existence of two glycosyl residues. Considering the J values and the 13C NMR data, and in accordance with previous investigations (Tian et al., 2013; Wang et al., 2006), the two residues were identified as b-glucopyranosyl and b-apiofuranosyl, respectively. The 100 /20 O-linkage was supported by the fact that H-100 had correlations with C-20 (d 79.2) in the HMBC spectrum (Fig. 2). Furthermore, the correlations of H-2000 and H-6000 with the ester carbonyl group (d 167.0, C¼O) suggested the presence of a veratroyl group connected to an ester carbonyl group. Because the 3J-correlations of H-10 with C-1 (d 163.2) and of H-500 (d 4.32, 500 b/d 4.34, 500 a) with d 167.0 (C¼O) were observed, the two phenolic groups and the glycosyl residues were determined to be connected. Compound 7, as a phenolic glycoside, was elucidated to be 3-hydroxy-5-methoxyphenyl-1-O-b-(500 -Overatroyl)-apiofuranosyl-(100 /20 )-b-glucopyranoside and was named reevesianin A. All of the physical data for compound 8 were similar to those for 7, a phenolic glycoside, and compound 8 exhibited a molecular formula of C26H32O15. This was established by HRESIMS (m/z 607.1635 [MþNa]þ). When comparing the 13C NMR spectra of 8 and 7, their sugar moieties were confirmed to be identical. As for the 1H NMR spectra, only two methoxy groups [d 3.60 (3H, s, OCH3-5) and 3.87 (3H, s, OCH3-3000 )] in the two separated aromatic rings were observed. Thus, the ABX spin system was regarded as a vanilloyl group in 8. Based on the 3J-correlations between H-10 (d 4.89) and C-1 (d 160.5); between H-2000 (d 7.46), H-6000 (d 7.46), H-500 (d 4.27, 500 b/d 4.33, 500 a) and d 167.8 (C¼O); and between H-10 ’ (d 5.47) and C-20 (d 78.8), the structure of 8 was suggested to be 3-hydroxy-5methoxyphenyl-1-O-b-(500 -O-vanilloyl)-apiofuranosyl-(100 /20 )-b-
5
glucopyranoside, and the compound was named reevesianin B. All of the known compounds were identified by comparing their NMR and physical data with published literature on reevesioside A (9) (Chang et al., 2013), reevesioside B (10) (Chang et al., 2013), reevesioside F (11) (Chang et al., 2013), epi-reevesioside F (12) (Chang et al., 2013), strophanthidin (13) (Kopp et al., 1992), a mixture of reevesioside G (14) and epi-reevesioside G (15) (Chang et al., 2013), a mixture of reevesioside I (16) and epi-reevesioside I (17) (Chang et al., 2013), ()-(2S,3R)-dehydrodiconiferyl alcoholg0 -methyl ether (18) (Ohta et al., 1979), (þ)-trans-dehydrodiconiferyl alcohol (19) (Yoshizawa et al., 1990), (þ)-erythrobuddlenol B (20) (Cutillo et al., 2003), (þ)-threo-buddlenol B (21) (Houghton, 1985), (±)-erythro-guaiacylglycerol-b-O-40 -sinapyl ether (22) (Machida et al., 2008), ()-erythro-guaiacylglycerol-b-O40 -coniferyl ether (23) (Gan et al., 2008), ()-threo-guaiacylglycerol-b-O-40 -coniferyl ether (24) (Gan et al., 2008), cleomiscosin B (25) (Ranjan and Sahai, 2009), cleomiscosin D (26) (Kumar et al., 1988), (þ)-lariciresinol (27) (Wang et al., 2010), ()-5,50 - dimethoxylariciresinol (28) (Ida et al., 1994), ()-syringaresinol (29) (Vermes et al., 1991), ()-medioresinol (30) (Marti et al., 2013), ()-pinoresinol (31) (Cutillo et al., 2003), hibiscone C (32) (Ferreira et al., 1980), hibiscone D (33) (Ferreira et al., 1980), oleanolic acid (34) (Martinez et al., 2013), betulinic acid (35) (Pohjala et al., 2009), a mixture of b-sitosterol (36) and stigmasterol (37) (Xu et al., 2013), a mixture of stigmasta-5-en-3b-ol-7-one (38) and stigmasta-5,22en-3b-ol-7-one (39) (Choi and Doyle, 2007), ergosterol peroxide (40) (Takaishi et al., 1991), 4-methoxybenzoic acid (41) (Villano et al., 2014), 4-hydroxybenzoic acid (42) (Azizuddin et al., 2010), protocatechuic acid (43) (Zhu et al., 2010), 3,4-dimethoxybenzoic acid (44) (Alagiri and Prabhu, 2011), vanillic acid (45) (Kang et al., 2014), methyl vanillate (46) (Kuo and Li, 1997), syringic acid (47) (Zhao et al., 2012), cinnamic acid (48) (Du et al., 2011), (E)-4hydroxycinnamic acid (49) (Lavoie et al., 2013), caffeic acid (50) (Chung et al., 2011), 3,4,5-trimethoxyphenyl-b-D-glucopyranoside (51) (Achenbach and Benirschke, 1997), ()-epicatechin (52) (Yokozawa et al., 2002), proanthocyanidin A2 (53) (Lou et al., 1999), and (þ)-abscisic acid (54) (Todoroki et al., 2000). All isolates were evaluated for their cytotoxicity levels, excluding reevesiosides A (9), B (10), F (11), G (14), and I (16) and epi-reevesiosides F (12), G (15), and I (17), which were also isolated from the root of this species. These compounds had been already evaluated, along with three other known compounds, (þ)-transdehydrodiconiferyl alcohol (19), proanthocyanidin A2 (53), and (þ)-abscisic acid (54). The results for the active compounds are listed in Table 4, and the most bioactive compounds were among the new compounds. Reevesioside J (1), bearing the aglycone of digitoxigenin, was the most potent of all, with moderate IC50 values against the cancer cell lines MCF-7, NCI-H460, and HepG2 of 0.39 ± 0.06 mM, 0.12 ± 0.01 mM, and 1.09 ± 0.02 mM, respectively. Reevesioside K (2) and epi-reevesioside K (3), with an 18,20epoxide group exhibited weaker potency than reevesioside J (1), with a digitoxigenin group as the aglycone. This result was consistent with a previous bioactivity investigation of the root of R. formosana (Chang et al., 2013). Reevesioside K (2) and epireevesioside K (3), which contain a cyclopentenone group, exhibited much weaker cytotoxic activities compared with reevesioside G (14) and epi-reevesioside G (15). However, epi-reevesioside K (3) presented selective cytotoxicity against NCI-H460 cells, with an IC50 value of 4.08 ± 0.07 mM. Observing the activities of the epimers reevesiosides G (14), H (Chang et al., 2013), and K (Chang et al., 2013) and epi-reevesiosides G (15), H (Chang et al., 2013), and K (Chang et al., 2013), the 20R-form exhibited more favorable activities than the 20S-form (Chang et al., 2013). For the two furanosesquiterpenoids reevesiterpenols C (4) and D (5), the connection of an isopropyl group to C-6 instead of a
Please cite this article in press as: Hsiao, P.-Y., et al., Cytotoxic cardenolides and sesquiterpenoids from the fruits of Reevesia formosana, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.06.009
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hydroxyisopropyl group made 4 more cytotoxic than 5 (IC50 > 50 mM) toward NCI-H460 cells. Reevesiterpenol E (6), bearing a naphtholactone group, exhibited a more potent cytotoxicity than reevesiterpenols C (4) and D (5) and exhibited selective cytotoxicity to NCI-H460 cells, with an IC50 value of 3.15 ± 0.22 mM. As for the known compounds strophanthidin (13) and oleanolic acid (34), though several studies have discussed their cytotoxicity, their respective activities against NCI-H460 cells and HepG2 cells were presented for the first time in this study. The new compounds reevesiterpenols CeE (4e6) shared similar furanosesquiterpenoid structures with a series of hibiscones from Hibiscus elatus (Malvaceae) (Ferreira et al., 1980). Furanosesquiterpenoids were obtained mostly from Malvaceae in previous studies; thus, their occurrence in Sterculiaceae enhances the relationship of these two families of Malvales in chemotaxonomy.
3. Conclusions In this study, eight new compounds, including cardenolides (reevesioside J (1), reevesioside K (2), and epi-reevesioside K (3)), sesquiterpenoids (reevesiterpenol C (4), reevesiterpenol D (5), and reevesiterpenol E (6)), and glycosides (reevesianin A (7) and reevesianin B (8)), together with 46 known compounds were isolated from the fruits of R. formosana. Among the isolates, cardenolides, reevesioside J (1) and strophanthidin (13) exhibited moderate cytotoxicity against the MCF-7, NCI-H460, and HepG2 cell lines, with IC50 values of 0.39 ± 0.06 mM and 1.06 ± 0.12 mM, 0.12 ± 0.01 mM and 0.29 ± 0.01 mM, and 1.09 ± 0.02 mM and 1.72 ± 0.02 mM, respectively, and reevesiterpenol E (6) exhibited selective cytotoxicity to NCI-H460 cells, with an IC50 value of 3.15 ± 0.22 mM. The bioassay results in this study suggested that R. formosana is a prospective species for the discovery of anticancer compounds and that the new cardenolides and furanosesquiterpenoids found in its fruits could potentially support development of anticancer therapies.
4. Experimental 4.1. General experimental procedures Optical rotations were obtained using a Jasco P-2000 polarimeter. The UV spectra were measured with a JASCO V-530 UV/VIS spectrophotometer, and the IR spectra (ATR) were recorded using a JASCO FT/IR-4600 spectrometer. For the NMR spectra, the 1D (1H, 13 C, DEPT) and 2D (COSY, NOESY, HSQC, HMBC) spectra using CDCl3 (1H, d 7.26; 13C, d 77.0), acetone-d6 (1H, d 2.05; 13C, d 30.5), and CD3OD (1H, d 3.31; 13C, d 49.0) as the solvents, were obtained with a Varian Unity Plus-400 spectrometer (400 MHz for 1H NMR and 100 MHz for 13C NMR) and Varian VNMRS-600 spectrometer (600 MHz for 1H NMR and 150 MHz for 13C NMR). The chemical shifts were displayed as d (ppm). The low-resolution mass spectra were measured on a Waters ZQ 4000 and a VG Biotech Quattro 5022; high-resolution mass spectra were measured with a JOELJMS-HX 100 mass spectrometer. The gels used for column chromatography (CC) were Sephadex LH-20, silica gel (60e200 mm and 15e35 mm, Silicycle), and SilicaBond C18 (17%) 60A (40e63 mm, Silicycle). Silica gels 60 F254 (Merck) and RP-18 F254 (Merck) were used for preparative TLC. HPLC (pump L-7110, Hitachi; UV/VIS detector UV-975 JASCO; integrator LC-NetII/ADC, JASCO; 5m C18 100 Å New Column 250 10.0 mm, Phenomenex Kinetex) was used for further purification.
4.2. Plant material Fruits of R. formosana were collected from the Mudan Village, Pingtung County, Taiwan, in December 2012. They were identified by Prof. Ih-Sheng Chen of Kaohsiung Medical University, Kaohsiung, Taiwan. A voucher specimen (Chen 6117) is deposited in the Herbarium of the School of Pharmacy, College of Pharmacy, Kaohsiung Medical University. 4.3. Extraction and isolation Dried fruits of R. formosana (4.4 kg) were extracted with cold MeOH (15 L 4) at room temperature, with each extraction lasting for three days. The MeOH extract (100 g) was partitioned with EtOAc and H2O into EtOAc-soluble (13 g), H2O-soluble (74 g), and insoluble parts (11 g). The MeOH extract, along with the three other parts, all exhibited cytotoxicity against cancer cell lines MCF-7, NCIH460, and HepG2, with survival rates <5% (at 30 mg/ml). The EtOAcsoluble fraction (13 g) was subjected to silica gel CC (60e200 mesh, 1.4 kg), eluted with hexane-acetone in a gradient, to provide 15 fractions (Fr. A-1eFr. A-15). Among these fractions, Fr. A-12eFr. A15 showed cytotoxicity towards the above cell lines. Fr. A-6 (1.3 g) was crystallized from MeOH to obtain a mixture of 36 and 37 (1.0 g). Fr. A-8 (516 mg) was eluted with hexane-EtOAc (5:1) by MPLC and purified by prep. TLC (CH2Cl2-MeOH, 15:1) to obtain 32 (2.2 mg), 33 (0.5 mg), 34 (1.8 mg), 35 (3.1 mg), 41 (0.7 mg), and 46 (0.5 mg), respectively. Fr. A-9 (476 mg) was subjected to MPLC, eluted with hexane-acetone (5:1), to afford a mixture of 38 and 39 (9.7 mg), and 40 (5.7 mg). Fr. A-12 (207 mg), applied to MPLC (CH2Cl2-MeOH, 30:1) afforded 11 fractions (Fr. A-12-1eFr. A-12-11). Compound 31 (2.4 mg) was obtained from Fr. A-12-2 (26.5 mg). Fr. A-12-4 (70.4 mg) was subjected to MPLC (RP-18, acetone-H2O, 1:2) to afford eight fractions (Fr. A-12-4-1eFr. A-12-4-8), and Fr. A-12-4-1 (11.2 mg) yielded 44 (1.7 mg) and 48 (0.8 mg). Fr. A-12-4-5 (13.3 mg) underwent preparative HPLC (RP-18, 250 10.0 mm, 5 mm, Phenomenex) to afford 6 (1.1 mg, tR 16.8 min, 2 ml/min), and the following HPLC experiment was conducted with the same instrument and flow rate conditions. Fr. A-12-7 was subjected to MPLC (RP-18, acetone-H2O, 1:2) to obtain 42 (0.9 mg) and 54 (1.2 mg). Fr. A-13 (695 mg) was placed on a Sephadex LH-20 column, and the column was eluted with MeOH to give over 17 fractions (Fr. A-13-1eFr. A-13-17). Fr. A-13-9 (248 mg) was further purified with MPLC (RP-18, acetone-H2O, 2:3) to yield 11 fractions (Fr. A-13-9-1eFr. A-13-9-11). Fr. A-13-9-3 (82.3 mg) was purified by preparative HPLC. A mixture of 16 and 17 (0.5 mg, tR 7.7 min), 4 (1.6 mg, tR 8.1 min), and 13 (2.7 mg, tR 11.1 min) were eluted with CH3CN-H2O (1:1). Fr. A-13-9-8 was purified by RP-18 TLC (MeOHH2O, 2:1) to afford 1 (0.9 mg), 9 (12.0 mg), 10 (4.3 mg), 11 (1.7 mg), and 12 (2.4 mg), respectively. Fr. A-13-11 (166 mg) was treated with RP-18 TLC (acetone-H2O 1:3) to obtain 27 (9.8 mg), 29 (12.6 mg), 30 (0.8 mg), and 45 (2.4 mg). This fraction went through preparative HPLC (RP-18, CH3CN-H2O, 1:1) to obtain a mixture of 14 and 15 (2.0 mg, tR 9.8 min), 2 (0.5 mg, tR 11.9 min) and 3 (1.3 mg, tR 12.1 min). Fr. A-13-13 (54.0 mg) was subjected to MPLC (RP-18, acetone-H2O, 2:3) to produce 25 (1.0 mg) and 49 (5.7 mg). Fr. A-14 (3.2 g) was passed over a Sephadex LH-20 column (MeOH) and eluted into 20 fractions (Fr. A-14-1eFr. A-14-20). Fr. A-14-9 (885 mg) was further purified with MeOH-H2O (1:1) by MPLC (RP18) and divided into 13 fractions (Fr. A-14-9-1eFr. A-14-9-13). Fr. A14-9-3 (86.2 mg) was 51 (3.1 mg). Fr. A-14-9-4 (243.5 mg) was subjected to MPLC (silica gel, CH2Cl2-MeOH, 20:1) and divided into 14 fractions (Fr. A-14-9-4-1eFr. A-14-9-4-14). Fr. A-14-9-4-1 (50.0 mg) was treated with preparative HPLC (RP-18, CH3CN-H2O, 1:2) to afford 23 (2.3 mg, tR 13.3 min) and 24 (3.0 mg, tR 14.0 min). Fr. A-14-9-4-5 (82.6 mg) was applied to RP-18 TLC (MeOH-H2O, 2:3)
Please cite this article in press as: Hsiao, P.-Y., et al., Cytotoxic cardenolides and sesquiterpenoids from the fruits of Reevesia formosana, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.06.009
P.-Y. Hsiao et al. / Phytochemistry xxx (2016) 1e9
7
Table 1 1 H and 13C NMR spectroscopic data for compounds 1e3.a Position
1
2
dH 1a/b 2a/b 3 4a/b 5 6a/b 7a/b 8 9 10 11a/b 12a/b 13 14 15a/b 16a/b 17 18a/b
1.49, 1.74, 4.07, 1.77, 1.77, 1.87, 1.68, 1.55, 1.61, e 1.42, 1.51, e e 2.12, 2.15, 2.78, 0.87,
m m/1.52, br s m/1.51, m m/1.29, m/1.22, m m
19 20 21a/b
0.94, e 4.98, 4.81, 5.87,
s
22a/b
e 4.69, 3.24, 3.36, 3.58, 3.34, 1.37, 5.17, 5.14,
23 10 20 30 40 a/b 50 60 7’a/b a 1
H and
13
m m m m
m/1.22, m m/1.39, m
m/1.69, m m/1.86, m dd (9.6, 5.4) s
dd (18.0, 1.2)/ dd (18.0, 1.8) br s
d (7.8) dd (9.6, 7.8) t (9.6) dd (9.6, 9.0) dq (9.0, 6.0) d (6.0) d (0.6)/ d (0.6)
dC
dH
30.1 26.5 74.6 30.4 36.1 26.5 21.4 41.9 35.8 35.1 21.1 40.1 49.6 85.6 33.2 26.9 50.9 15.8
2.46, 2.02, 4.21, 1.97, e 1.75, 2.05, 1.49, 3.02, e 1.77, 2.12, e e 5.82, e 2.28, 4.12, 3.70, 9.92, e 4.43, 4.02, 3.30, 2.27, e 4.48, 3.83, 4.15, 2.15, 3.83, 1.24, 5.23,
23.5 174.4 73.4 117.7 174.4 100.0 77.0 81.6 74.2 73.8 17.1 96.9
3
m/1.68, m m/1.47, m br t (2.7) m/1.74, m m m/1.58, m m m m m/2.04, m
d (1.8) s dd (10.2, 3.0)/ d (10.2) d (1.8) dd (10.2, 1.2)/ d (10.2) d (18.0)/ dd (18.0, 1.8) d (6.6) dd (6.6, 5.4) td (5.4, 1.8) m/1.74, m m d (6.6) s/4.91, s
dC
dH
dC
18.5 25.1 73.1 34.2 73.3 36.1 26.1 43.1 37.9 54.2 22.9 36.6 57.0 186.4 125.6 204.4 59.7 72.9
2.47, m/1.68, m 2.00, m/1.47, m 4.22, br t (2.7) 1.96, m/1.72, m e 2.03, m/1.75, m 2.04, m/1.54, m 1.45, m 3.02, m e 1.77, m/1.44, m 2.17, m/1.60, m e e 5.81, d (1.8) e 2.42, s 4.12, d (10.2)/ 3.63, d (10.2) 9.92 d (1.8) e 4.94, d (10.5)/ 4.13, d (10.5) 2.79, d (17.4)/ 2.45, d (17.4) e 4.48, d (6.6) 3.83, dd (6.6, 5.4) 4.15 m 2.14, m/1.73, m 3.83, m 1.24, d (6.0) 5.23, s/4.91, s
18.5 25.1 73.1 34.2 73.3 36.1 26.1 42.9 37.9 54.2 22.8 36.4 56.6 186.4 125.6 204.4 61.5 73.18
208.3 85.8 74.6 35.4 174.5 99.5 73.8 74.6 34.4 67.2 20.9 95.5
208.3 85.3 73.16 39.1 173.9 99.5 73.8 74.6 34.4 67.2 20.9 95.5
C NMR data (d) were measured in CDCl3 at 600 and 150 MHz for 1,2 and 3.
Table 2 1 H and 13C NMR spectroscopic data for compounds 4e6.a Position
1 2 3 4a/b 5 6 7 8 9 10 11 12 13 14 15 OH-7b OH-8b OH-13b OCH3-7
4
5
6
dH
dC
dH
dC
dH
dC
e e 2.77, 2.12, 1.90, 3.32, 1.61, 4.28, 4.70, e e 7.72, 1.36, 2.11, 1.12, 1.13, 1.53, 1.75, e e
143.9 188.9 42.9 36.8
e e 2.58, qdd (7.8, 4.2, 2.4) 2.55, ddd (13.5, 4.5, 2.4)/ 2.05, ddd (13.5,12.0, 4.2) 3.52, ddd (12.0, 11.7, 4.5) 1.85, dd (11.7, 1.2) 4.35, dd (1.8, 1.2) 4.64, br d (1.8) e e 7.79, s 1.28, d (7.8) e 1.40, s 1.43, s 3.35, 4.09, 4.29 (OH-7 or OH-8 or OH-13)
145.1 189.3 44.8 40.9
e e e 7.62, d (0.6)
132.0 135.0 128.1 121.0
26.3 49.9 74.5 67.3 126.3 144.6 148.2 16.8 73.8 32.0 29.0 e
e e e e e e e 2.47, 3.85, 1.55, 1.55, e e e 3.91,
117.9 146.2 145.7 150.4 100.4 126.5 165.7 17.3 28.1 22.6 22.6 e e e 62.1
qdd (7.2, 4.7, 2.1) ddd (12.0, 4.8, 2.1)/ ddd (12.0, 12.0, 4.7) ddd (12.0, 12.0, 4.8) br dd (12.0, 3.6) dd (3.6, 3.0) dd (5.7, 3.0)
s d (7.2) m d (7.2) d (7.2) br s d (5.7)
24.4 44.2 72.2 66.6 123.5 142.5 146.9 16.0 26.9 19.0 21.5 e e e e
e
e
d (1.2) sept (7.2) d (7.2) d (7.2)
s
a 1 b
H and 13C NMR data (d) were measured in CDCl3 at 600 and 150 MHz for 4 and 6, and acetone-d6 for 5. D2O exchangeable.
to obtain 5 (1.3 mg) and 47 (3.5 mg). Fr. A-14-9-4-9 (36.1 mg) was purified by MPLC (silica gel, CH2Cl2-MeOH, 9:1) and then treated with RP-18 TLC (acetone-H2O, 1:2). This was repeated five times to produce 8 (3.3 mg). With further application of preparative HPLC
(RP-18, CH3CN-H2O, 1:3), 7 (0.9 mg, tR 12.5 min) was obtained. Fr. A14-9-5 (144.6 mg) was purified with RP-18 TLC (acetone-H2O, 1:1) to yield 19 (4.7 mg), 22 (2.9 mg), and 28 (4.7 mg). Fr. A-14-9-8 (146 mg) was subjected to MPLC (CH2Cl2-MeOH, 25:1) and then
Please cite this article in press as: Hsiao, P.-Y., et al., Cytotoxic cardenolides and sesquiterpenoids from the fruits of Reevesia formosana, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.06.009
8
P.-Y. Hsiao et al. / Phytochemistry xxx (2016) 1e9 13 C NMR spectroscopic data, see Table 1; ESIMS m/z 533 [MþH]þ; HRESIMS m/z 533.3107 [MþH]þ (calcd. for C30H45O8, 533.3109).
Table 3 1 H and 13C NMR spectroscopic data for compounds 7 and 8.a Position
1 2 3 4 5 6 10 20 30 40 50 6’a/b 100 200 300 400 a/b 00
5 a/b 1000 2000 3000 4000 5000 6000 OCH3-5 OCH3-3000 OCH3-4000 C¼O
7
8
dH
dC
dH
dC
e 6.19, e 6.02, e 6.15, 4.94, 3.67, 3.66, 3.47, 3.46, 3.84, 5.54, 4.11, e 4.30, 3.91, 4.34, 4.32, e 7.48, e e 6.89, 7.55, 3.65, 3.84, 3.87, e
163.2 98.0 161.2 97.1 163.2 95.5 100.8 79.2 78.3 72.4 78.2 63.3 110.8 79.3 79.4 75.7
e 6.07, t (2.2) e 5.92, t (2.2) e 6.05, t (2.2) 4.89 d (8.0) 3.63, m 3.66, m 3.35, m 3.38, m 3.87, m/3.69, m 5.47, d (1.2) 4.03, d (1.2) e 4.28, d (9.6)/ 3.90, d (9.6) 4.33, d (11.4)/ 4.27, d (11.4) e 7.46, d (2.0) e e 6.76, d (8.8) 7.46, dd (8.8, 2.0) 3.60, s 3.87, s e e
160.5 97.2 160.2 96.5 162.8 94.9 100.3 78.8 78.4 71.4 78.1 62.5 110.5 78.8 79.3 75.3
t (2.1) t (2.1) t (2.1) d (7.8) m m m m m/3.66, m d (1.2) br d (1.2) d d d d
(9.9)/ (9.9) (12.6)/ (12.6)
d (1.8)
d (8.4) dd (8.4, 1.8) s s s
68.0 123.9 114.0 150.6 155.2 112.4 125.1 56.1 56.9 56.9 167.0
67.8 122.1 113.7 148.6 152.9 115.9 125.3 55.5 56.4 e 167.8
a 1 H and 13C NMR data (d) were measured in acetone-d6 at 600 and 150 MHz for 7, and in CD3OD at 400 and 100 MHz for 8.
4.3.2. Reevesioside K (2) Colorless syrup; ½a23 D þ36.0 (c 0.1, MeOH), IR (ATR) ymax 3445 (OH), 1780 (lactone ring), 1714 (CHO), 1698 (C¼O), 1614 (C¼C) cm1; for 1H and 13C NMR spectroscopic data, see Table 1; ESIMS m/ z 581 [MþNa]þ; HRESIMS m/z 581.2354 [MþNa]þ (calcd. for C30H38O10Na, 581.2357). 4.3.3. epi-Reevesioside K (3) Colorless syrup; ½a23 D þ25.9 (c 0.06, MeOH); IR (ATR) ymax 3435 (OH), 1780 (lactone ring), 1711 (CHO), 1693 (C¼O), 1613 (C¼C) cm1; for 1H and 13C NMR spectroscopic data, see Table 1; ESIMS m/z 581 [MþNa]þ; HRESIMS m/z 581.2360 [MþNa]þ (calcd. for C30H38O10Na, 581.2357). 4.3.4. Reevesiterpenol C (4) Yellowish oil; ½a23 D þ30.2 (c 0.04, CHCl3); UV (MeOH) lmax (log ε) 206 (3.52), 276 (3.86) nm; IR (ATR) ymax 3421 (OH), 1655 (C¼O) cm1; for 1H and 13C NMR spectroscopic data, see Table 2; ESIMS m/ z 287 [MþNa]þ; HRESIMS m/z 287.1252 [MþNa]þ (calcd. for C15H20O4Na, 287.1254). 4.3.5. Reevesiterpenol D (5) Yellowish oil; ½a24 D þ39.8 (c 0.08, CHCl3); UV (MeOH) lmax (log ε) 205 (3.80), 276 (3.94) nm; IR (ATR) ymax 3368 (OH), 1655 (C¼O) cm1; for 1H and 13C NMR spectroscopic data, see Table 2; ESIMS m/ z 303 [MþNa]þ; HRESIMS m/z 303.1205 [MþNa]þ (calcd. for C15H20O5Na, 303.1203).
Table 4 Cytotoxicity (IC50 values) of isolates from the fruits of R. formosana. Compounds
Reevesioside J (1) Reevesioside K (2) epi-Reevesioside K (3) Reevesiterpenol C (4) Reevesiterpenol E (6) Strophanthidin (13) Hibiscone C (32) ()-(2S,3R)-Dehydrodiconiferyl alcohol-g0 -methyl ether (18) Oleanolic acid (34) Betulinic acid (35) Actinomycin D a a
IC50 (mM) MCF-7
NCI-H460
HepG2
0.39 ± 0.06 >50 25.73 ± 7.34 40.14 ± 3.17 27.53 ± 4.34 1.06 ± 0.12 38.63 ± 0.50 >50 4.57 ± 0.55 >50 0.01 ± 0.001
0.12 ± 0.01 34.38 ± 0.66 4.08 ± 0.07 7.06 ± 1.31 3.15 ± 0.22 0.29 ± 0.01 32.21 ± 4.78 28.88 ± 8.01 1.54 ± 0.18 33.67 ± 1.68 0.02 ± 0.005
1.09 ± 0.02 >50 >50 >50 46.12 ± 2.59 1.72 ± 0.02 34.71 ± 3.79 >50 7.60 ± 1.42 >50 0.10 ± 0.015
Positive control.
divided into nine fractions (Fr. A-14-9-8-1eFr. A-14-9-8-9), where Fr. A-14-9-8-1 yielded 26 (1.4 mg). By way of preparative HPLC (RP18, CH3CN-H2O, 1:2), Fr. A-14-9-8-6 (18.2 mg) afforded 20 (1.7 mg, tR 14.0 min) and 21 (1.5 mg, tR 15.9 min). Fr. A-14-9-12 was purified by RP-18 TLC (acetone-H2O 1:1) to produce 18 (2.0 mg). Fr. A-14-12 (141.9 mg) was further treated with RP-18 TLC (CH3CN-H2O, 1:3) and thus provided 43 (4.9 mg) and 50 (4.2 mg). Fr. A-14-14 (294.3 mg) and Fr. A-14-17 (158 mg) were passed through a Sephadex LH-20 column (MeOH) to yield 52 (29.1 mg) and 53 (2.8 mg), respectively.
4.3.1. Reevesioside J (1) Colorless syrup; ½a22 D e13.0 (c 0.05, MeOH), IR (ATR) ymax 3394 (OH), 1775, 1738, 1629 (a,b-unsaturated g-lactone) cm1; for 1H and
4.3.6. Reevesiterpenol E (6) Brownish oil; UV (MeOH) lmax (log ε) 225 (4.33), 254 (4.19), 373 (3.77) nm; UV(MeOHþKOH) lmax (log ε) 210 (4.56), 238 (2.43), 272 sh (1.98), 399 (3.94) nm; IR (ATR) ymax 3237 (OH), 1727, 1650 (lactone ring), 1482, 1451 (aromatic ring) cm1; for 1H and 13C NMR spectroscopic data, see Table 2; ESIMS m/z 289 [MþH]þ; HRESIMS m/z 311.0888 [MþNa]þ (calcd. for C16H16O5Na, 311.0890). 4.3.7. Reevesianin A (7) Brownish syrup; ½a23 D e40.9 (c 0.06, MeOH); UV (MeOH) lmax (log ε) 209 (4.31), 225 sh (4.16), 262 (3.77), 288 (3.64) nm; UV (MeOHþKOH) lmax (log ε) 213 (4.72), 245 sh (3.91), 266 sh (3.80), 289 (3.71) nm; IR (ATR) ymax 3410 (OH), 1699 (C¼O), 1602, 1514, 1457 (aromatic ring) cm1; for 1H and 13C NMR spectroscopic data,
Please cite this article in press as: Hsiao, P.-Y., et al., Cytotoxic cardenolides and sesquiterpenoids from the fruits of Reevesia formosana, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.06.009
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see Table 3; ESIMS m/z 621 [MþNa]þ; HRESIMS m/z 621.1792 [MþNa]þ (calcd. for C27H34O15Na, 621.1790). 4.3.8. Reevesianin B (8) Brownish syrup; ½a23 D e46.1 (c 0.16, MeOH); UV (MeOH) lmax (log ε) 209 (3.68), 224 sh (4.28), 264 (3.95), 298 sh (3.68) nm; UV (MeOHþKOH) lmax (log ε) 212 (4.71), 239 sh (4.14), 283 sh (3.89), 315 (4.17) nm; IR (ATR) ymax 3386 (OH), 1697 (C¼O), 1600, 1515, 1457 (aromatic ring) cm1; for 1H and 13C NMR spectroscopic data, see Table 3; ESIMS m/z 607 [MþNa]þ; HRESIMS m/z 607.1635 [MþNa]þ (calcd. for C26H32O15Na, 607.1633). 4.4. Cytotoxicity assay Human cancer cells, MCF-7 (human breast adenocarcinoma), NCI-H460 (non-small-cell lung cancer), and HepG2 (liver hepatocellular cells), were cultured in DMEM containing 10% fetal calf serum and nonessential amino acids and seeded in 96-well microtiter plates at 6500, 2500, and 10,000 cells/well, respectively. The sequential treatments of the cell cultures and the cytotoxicity assays were performed as previously described (Chang et al., 2009). Acknowledgment This work was supported by the National Science Council of Taiwan (NSC102-2320-B037-007) and was supported partially by the Kaohsiung Medical University “Aim for the Top Universities Grant,” Grant No. KMU-TP103H01, KMU-TP103H05, and KMUTP104E43. We also thank Dr. Ming-Jen Cheng and Dr. Chu-Hung Lin for their valuable suggestions on structural elucidation. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.phytochem.2016.06.009. References Achenbach, H., Benirschke, G., 1997. Joannesialactone and other compounds from Joannesia princeps. Phytochemistry 45, 149e157. Al Muqarrabun, L.M.R., Ahmat, N., 2015. Medicinal uses, phytochemistry and pharmacology of family Sterculiaceae: A review. Eur. J. Med. Chem. 92, 514e530. Alagiri, K., Prabhu, K.R., 2011. Efficient synthesis of carbonyl compounds: oxidation of azides and alcohols catalyzed by vanadium pentoxide in water using tertbutylhydroperoxide. Tetrahedron 67, 8544e8551. Azizuddin, Makhmoor, T., Choudhary, M.I., 2010. Radical scavenging potential of compounds isolated from Vitex agnus-castus. Turk. J. Chem. 34, 119e126. Beale, J.M., Floss, H.G., Lehmann, T., Luckner, M., 1988. Digitoxigenin-3b-O-[b-Dfucopyranosyl-4'-b-D-glucopyranoside], the main cardenolide of somatic embryos of Digitalis lanata. Phytochemistry 27, 3143e3146. Chang, H.S., Chiang, M.Y., Hsu, H.Y., Yang, C.W., Lin, C.H., Lee, S.J., Chen, I.S., 2013. Cytotoxic cardenolide glycosides from the root of Reevesia formosana. Phytochemistry 87, 86e95. Chang, H.S., Lin, Y.J., Lee, S.J., Yang, C.W., Lin, W.Y., Tsai, I.L., Chen, I.S., 2009. Cytotoxic alkyl benzoquinones and alkyl phenols from Ardisia virens. Phytochemistry 70, 2064e2071. Choi, H., Doyle, M.P., 2007. Optimal TBHP allylic oxidation of D5-steroids catalyzed by dirhodium caprolactamate. Org. Lett. 9, 5349e5352. Chung, C.P., Hsia, S.M., Lee, M.Y., Chen, H.J., Cheng, F., Chan, L.C., Kuo, Y.H., Lin, Y.L., Chiang, W.C., 2011. Gastroprotective activities of Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf) on the growth of the stomach cancer ags cell line and indomethacin-induced gastric ulcers. J. Agric. Food Chem. 59, 6025e6033. Cutillo, F., D'Abrosca, B., DellaGreca, M., Fiorentino, A., Zarrelli, A., 2003. Lignans and neolignans from Brassica fruticulosa: Effects on seed germination and plant growth. J. Agric. Food Chem. 51, 6165e6172. Du, Z., Zhou, W., Bai, L., Wang, F., Wang, J.X., 2011. In situ generation of palladium nanoparticles: Reusable, ligand-free Heck reaction in PEG-400 assisted by focused microwave irradiation. Synlett 369e372.
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Please cite this article in press as: Hsiao, P.-Y., et al., Cytotoxic cardenolides and sesquiterpenoids from the fruits of Reevesia formosana, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.06.009
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Cytotoxic cardenolides and sesquiterpenoids from the fruits of Reevesia formosana Pei-Yu Hsiao a, Shiow-Ju Lee b, Ih-Sheng Chen a, c, d, Hsing-Yu Hsu b, Hsun-Shuo Chang a, c, d, e, * a
Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, 807, Taiwan, ROC Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, 350, Taiwan, ROC School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, 807, Taiwan, ROC d Research Center for Natural Products and Drug Development, Kaohsiung Medical University, Kaohsiung, 807, Taiwan, ROC e Center for Infectious Disease and Cancer Research (CICAR), Kaohsiung Medical University, Kaohsiung, 807, Taiwan, ROC b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 March 2016 Received in revised form 9 June 2016 Accepted 23 June 2016 Available online xxx
Bioassay-guided fractionation of the fruits of Reevesia formosana led to isolation of three cardenolides (reevesioside J, reevesioside K, and epi-reevesioside K), three sesquiterpenoids (reevesiterpenol C, reevesiterpenol D, and reevesiterpenol E), and two glycosides (reevesianin A and reevesianin B), along with 46 known compounds. Their structures were determined using spectroscopic techniques. In addition to the reported cytotoxic cardenolides, reevesioside J and strophanthidin exhibited moderate cytotoxicity against the cell lines MCF-7, NCI-H460, and HepG2, with IC50 values of 0.39 ± 0.06 mM and 1.06 ± 0.12 mM for MCF-7, 0.12 ± 0.01 mM and 0.29 ± 0.01 mM for NCI-H460, and 1.09 ± 0.02 mM and 1.72 ± 0.02 mM for HepG2, respectively. Reevesiterpenol E also exhibited the best selective cytotoxicity to the NCI-H460 cell line, with an IC50 value of 3.15 ± 0.22 mM. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Reevesia formosana Sterculiaceae Cardenolides Sesquiterpenoids Cytotoxicity
1. Introduction The Sterculiaceae family, with its rich fruits and oily seeds, has long been used in traditional treatments and as supplements (Al Muqarrabun and Ahmat, 2015). Among these genera, Reevesia, with its unique fruit forms and glamorous inflorescence, has not been well studied. This inspired an investigation of the endemic species Reevesia formosana Sprague (Sterculiaceae), which is found in the low forests of the Hengchun Peninsula, Pingtung in southern Taiwan (Li and Lo, 1993). A previous investigation of the chemical constituents of the root of R. formosana identified new cardenolides that exhibit potent cytotoxicity against the cancer cell lines MCF-7, NCI-H460, and HepG2 in vitro (Chang et al., 2013). The methanolic extract of the fruits of this species also exhibited potent cytotoxicity against the above three cancer cells. A bioassay-guided fractionation of the active EtOAc-soluble layer of the fruits led to isolation of 54 compounds, including three new cardenolides (reevesioside J
* Corresponding author. Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, 807, Taiwan, ROC. E-mail address: [email protected] (H.-S. Chang).
(1), reevesioside K (2), and epi-reevesioside K (3)), three new sesquiterpenoids (reevesiterpenol C (4), reevesiterpenol D (5), and reevesiterpenol E (6)), and two new glycosides (reevesianin A (7) and reevesianin B (8) (Fig. 1)) and 46 known compounds. The structures of these compounds were elucidated using spectroscopic analyses, and an evaluation of their cytotoxicities is also described.
2. Results and discussion Compound 1 was identified as an optically active colorless syrup with ½a22 D e13.0 (c 0.05, MeOH). Its molecular formula was determined to be C30H44O8 by ESIMS and HRESIMS analyses. The IR spectrum indicated the presence of a hydroxy group (3394 cm1) and an a,b-unsaturated g-lactone ring (1775, 1738, and 1629 cm1). According to a previous investigation of R. formosana (Chang et al., 2013), the MS, IR, 1H, and 13C NMR spectra suggested that the substance was a characteristic cardenolide glycoside of this species. The 1H and 13C NMR spectra had signals for an a,b-unsaturated glactone at d 4.81 (1H, dd, J ¼ 18.0, 1.8 Hz, H-21b), 4.98 (1H, dd, J ¼ 18.0, 1.2 Hz, H-21a), and 5.87 (1H, br s, H-22); two methyl groups at d 0.87 (3H, s, H-18) and 0.94 (3H, s, H-19), as well as oxygen-
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bearing quaternary carbon (d 85.6, C-14), indicating the aglycone to be a 3-O-substituted digitoxigenin (Beale et al., 1988). HMBC (Fig. 2) correlations were observed between: H-18 and C-12 (d 40.1), C-13 (d 49.6), C-14, and C-17 (d 50.9); H-19 and C-1 (d 30.1), C-5 (d 36.1), C-9 (d 35.8), and C-10 (d 35.1); and H-17 [(d 2.78) (1H, dd, J ¼ 9.6, 5.4 Hz)] and C-13, C-21 (d 73.4), and C-22 (d 117.7). These further confirmed the aglycone moiety of the structure. From the NOESY (Fig. 3) correlations between H-19 and H-5 (d 1.77) and H-8 (d 1.55), and between H-18 and H-8, H-21, and H-22, the relative configuration of digitoxigenin was deduced. An anomeric proton at d 4.69 (1H, d, J ¼ 7.8 Hz, H-10 ) indicated the presence of a sugar moiety with the following signals: a methyl group at d 1.37 (3H, d, J ¼ 6.0 Hz, H-60 ); four oxymethines at d 3.24 (1H, dd, J ¼ 9.6, 7.8 Hz, H-20 ), 3.34 (1H, dq, J ¼ 9.0, 6.0 Hz, H-50 ), 3.36 (1H, t, J ¼ 9.6 Hz, H-30 ), and 3.58 (1H, dd, J ¼ 9.6, 9.0 Hz, H-40 ); and a methylenedioxy group at d 5.14 (1H, d, J ¼ 0.6 Hz, H-70 b), and d 5.17 (1H, d, J ¼ 0.6 Hz, H70 a). From the NOESY correlations between H-10 and H-30 , and H-50 and between H-20 and H-40 , the relative configuration of the sugar moiety was deduced. Combining the HMBC and COSY analyses with the J values (>7.8 Hz) of H-10 ~ H-50 , the methylenedioxy group was determined to adopt an equatorial orientation at C-20 and C-30 . From the above signals, this sugar moiety was identified as a 6deoxy-2,3-methylenedioxy-b-glucopyranosyl unit. The anomeric proton H-10 exhibited a 3J-correlation with C-3 (d 74.6), and the H-3 signal was present as a broad singlet. Thus the 6-deoxy-2,3methylenedioxy-b-glucopyranosyl unit was determined to be connected to the C-3 of the digitoxigenin moiety in a b-orientation (Shi et al., 2010). Therefore, the structure of 1 was determined to be digitoxigenin-6-deoxy-2,3-methylenedioxy-b-glucopyranoside and named reevesioside J. Compound 2 was identified as an optically active colorless syrup with ½a23 D þ36.0 (c 0.1, MeOH). Its molecular formula was determined to be C30H38O10 by ESIMS and HRESIMS analyses. The IR spectrum of the compound suggested the presence of a strophanthidin-like aglycone with an absorption of an aldehyde group at 1714 cm1, an absence of the a,b-unsaturated g-lactone ring, and presence of a saturated g-lactone ring at 1780 cm1. The
substance had three methylene groups, indicated by d 3.70 (1H, d, J ¼ 10.2 Hz, H-18b) and 4.12 (1H, dd, J ¼ 10.2, 3.0 Hz, H-18a), 2.27 (1H, dd, J ¼ 18.0, 1.8 Hz, H-22b) and 3.30 (1H, d, J ¼ 18.0 Hz, H-22a), and 4.02 (1H, d, J ¼ 10.2 Hz, H-21b) and 4.43 (1H, dd, J ¼ 10.2, 1.2 Hz, H-21a), respectively. The IR spectrum and the presence of the methylene groups suggested that the substance may be an example of an 18,20-epoxy derivative, such as reevesioside G (14). This type of derivative has been previously isolated from the root of R. formosana (Chang et al., 2013). Comparison of the 1H NMR spectra of 2 and reevesioside G (14) (Chang et al., 2013), which bears an 18,20-epoxide in its structure, showed that the two compounds were similar, but 2 exhibited an olefinic proton at d 5.82 (1H, d, J ¼ 1.8 Hz, H-15). Compound 2 also had double bond signals in its 13C NMR spectrum at d 186.4 (C-14) and 125.6 (C-15) and a carbonyl carbon at d 204.4 (C-16). HMBC (Fig. 2) correlations between H-15 and C-13 (d 57.0), C-14, C-16, and C-17 (d 59.7), together with the 12 degrees of unsaturation, suggested a cyclopentenone moiety in 2, which suggested the presence of an aglycone different from that of reevesioside G. Additional absorptions at 1698 cm1 for the carbonyl group and 1614 cm1 for the double bond in the IR spectrum also supported the presence of a cyclopentenone in 2. The connections of a saturated lactone ring and an aldehyde group were confirmed through the correlations between: H-21 and C-20 (d 85.8) and C-23 (d 174.5); H-22 and C-20, C-21 (d 74.6), and C-23; and H-19 (d 9.92) and C-10 (d 54.2) of HMBC. For the absolute configuration of the spiro moiety at C-20, the NOESY (Fig. 3) correlations were observed between H-21a and H-18b, and H-21b and H-17 (d 2.28). Lack of correlations between H-22 and H17 or H-18, however, indicated a S-configuration at C-20 of 2 (Chang et al., 2013). An anomeric proton at d 4.48 (1H, d, J ¼ 6.6 Hz, H-10 ) also suggested the presence of a sugar moiety including one methyl group [d 1.24 (3H, d, J ¼ 6.6 Hz, H-60 )], one methylene [d 1.74 (1H, m, H-40 b) and 2.15 (1H, m, H-40 a)], three oxymethines [d 3.83 (1H, dd, J ¼ 6.6, 5.4 Hz, H-20 ), 3.83 (1H, m, H-50 ), 4.15 (1H, td, J ¼ 5.4, 1.8 Hz, H-30 )], and one methylenedioxy group [d 4.91 (1H, s, H-70 b), 5.23 (1H, s, H-70 a)]. The sugar moiety was identified as a unique reevesiosyl group that has only been previously isolated from this
Fig. 1. Structures of compounds 1e8 from the fruits of R. formosana.
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3
Fig. 2. Key HMBC (/), and COSY (━) correlations of 18.
species (Chang et al., 2013). As H-10 exhibited a 3J-correlation toward C-3 (d 73.1) and a broad triplet with a small J value of H-3 [d 4.21 (1H, br t, J ¼ 2.7 Hz)], the reevesiosyl group connected to the C3 of the aglycone was determined to exist in a b-orientation (Shi et al., 2010). On the basis of the above evidence, structure 2 was elucidated and named reevesioside K. Compound 3 was similar to 2 in that it was also an optically active colorless syrup with ½a23 D þ25.9 (c 0.06, MeOH). The same molecular formula, C30H38O10, was proposed with a [MþNa]þ m/z of 581.2360, as shown by the ESIMS and HRESIMS analyses. From the similarities between their 1H NMR spectra, a slight difference in the structures of compounds 3 and 2 was found through the chemical shifts of the methylene groups of C-18 [d 3.63 (1H, d, J ¼ 10.2 Hz, H-18b), 4.12 (1H, d, J ¼ 10.2 Hz, H-18a)], C-21 [d 4.13 (1H, d, J ¼ 10.5 Hz, H-21b), 4.94 (1H, d, J ¼ 10.5 Hz, H-21a)], and C-22 [d 2.45 (1H, d, J ¼ 17.4 Hz, H-22b), 2.79 (1H, d, J ¼ 17.4 Hz, H-22a)]. The aglycone of 3 resembled that of 2, as an 18,20-epoxide, whereas
NOESY (Fig. 3) correlations were found between H-22a and H-18b and H-22b and H-17 (d 2.42) instead of H-21. Moreover, there was no correlation between H-21 and H-18 or H-17, which indicated an absolute configuration of 20R for the spiro of 3 (Chang et al., 2013). The sugar moiety of 3 was also identified as the reevesiosyl group from the 1H NMR, 13C NMR, HMBC, COSY, and NOESY spectroscopic analyses. With the HMBC (Fig. 2) correlation of H-10 (d 4.48) and C-3 (d 73.1) and the presence of a broad triplet of H-3, the connection of the sugar moiety to the aglycone of 3 was found to be the same as that of 2. The two substances were different only in the configuration at C-20, which was consistent with reevesioside G (14) and epi-reevesioside G (15) (Chang et al., 2013) for both the NOESY correlations and chemical shifts of C-17 and C-22. Thus, 3 was determined to be the epimer of 2 and was named epi-reevesioside K. Compound 4 was obtained as an optically active yellowish oil with ½a23 D þ30.2 (c 0.04, CHCl3), and its molecular formula was
Please cite this article in press as: Hsiao, P.-Y., et al., Cytotoxic cardenolides and sesquiterpenoids from the fruits of Reevesia formosana, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.06.009
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Fig. 3. Key NOESY(4) correlations of 18.
found to be C15H20O4, as determined by ESIMS and HRESIMS analyses, suggesting that the structure was a sesquiterpenoid with six degrees of unsaturation. The IR spectrum indicated the presence of a hydroxy group (3421 cm1) and a carbonyl group (1655 cm1), with UV absorption maxima at 206 and 276 nm. The above physical data and the 1H and 13C NMR spectra (Table 2) of 4 were similar to those of furanosesquiterpenoid-hibiscone B (Ferreira et al., 1980), except for the presence of a hydroxy group at d 1.53 (1H, br s, OH-7, D2O exchangeable) and an oxymethine at d 4.28 (1H, dd, J ¼ 3.6, 3.0 Hz, H-7) in 4 rather than the methylene group of hibiscone B. The HRESIMS of 4 also indicated it contained one more oxygen atom than hibiscone B. According to the HMBC (Fig. 2) correlations, the OH-7 group exhibited a 2,3J-correlation between C-6 (d 44.2), C7 (d 72.2) and C-8 (d 66.6), which confirmed the substitution of a
hydroxy group at C-7. The relative configuration of 4 was determined to be identical to hibiscone B based on the NOESY (Fig. 3) correlations between H-7 and H-6 (d 1.61) and H-8 (d 4.70) and between H-5 (d 3.32) and H-12 (d 1.36) and H-13 (d 2.11). Thus the structure of 4 was elucidated, and the compound was named reevesiterpenol C. The physical data of compound 5 were similar to those of 4, whereas the molecular formula was determined to be C15H20O5, as established by HRESIMS analyses, suggesting that this compound contains one more oxygen atom than 4. Compound 5 exhibited similar 1H and 13C NMR spectra to 4, except for a hydroxyisopropyl group [dH 1.40 (3H, s, H-14), 1.43 (3H, s, H-15); dC 29.0 (C-15), 32.0 (C-14), 73.8 (C-13)] of 5 at C-6 was in the same place as the isopropyl group of 4 at C-6. The HMBC (Fig. 2) correlations between H-
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14 and H-15 toward C-6 (d 49.9) and C-13, respectively, also confirmed that the hydroxyisopropyl group was connected at C-6. The structure of 5 was elucidated, and the compound was named reevesiterpenol D. Compound 6 was obtained as a brownish oil. The ESIMS and HRESIMS analyses indicated that the molecular formula was C16H16O5 and that it had nine degrees of unsaturation. The UV absorption maxima at 225, 254, and 373 nm underwent a bathochromic shift after addition of aqueous KOH, suggesting the presence of a phenolic moiety. Through analyses of the 13C NMR and DEPT spectra, the substance was determined to be a sesquiterpenoid. As the physical data were similar to those of hibiscolactone A (Ferreira et al., 1980), the 1H NMR spectrum of 6 indicated the presence of one more methoxy group (d 3.91) and one less olefinic proton than in hibiscolactone A. From the HMBC (Fig. 2) correlation between the methoxy group (d 3.91) and C-7 (d 145.7) and between H-13 [d 3.85 (1H, sept, J ¼ 7.2 Hz)] and C-7, along with the HRESIMS data, the attachment of one methoxy group to C-7 was established. The structure of 6 was suggested as shown, and the compound was assigned the trivial name reevesiterpenol E. Compound 7 was obtained as an optically active brownish syrup with ½a23 D e40.9 (c 0.06, MeOH), and the molecular formula of C27H34O15 was determined on the basis of ESIMS and HRESIMS data (m/z 621.1792 [MþNa]þ). The IR spectrum indicated the presence of a hydroxy group (3410 cm1), a carbonyl group (1699 cm1), and an aromatic ring (1602, 1514, 1457 cm1). The above moieties corresponded to the data found in the UV spectrum and the bathochromic shift of the compound. The 1H NMR spectrum showed two aromatic ring signals, three meta-coupled protons [d 6.02 (1H, t, J ¼ 2.1 Hz, H-4), 6.15 (1H, t, J ¼ 2.1 Hz, H-6), 6.19 (1H, t, J ¼ 2.1 Hz, H-2)], and one ABX system of a benzene ring [d 6.89 (1H, d, J ¼ 8.4 Hz, H-5000 ), 7.48 (1H, d, J ¼ 1.8 Hz, H-2000 ), 7.55 (1H, dd, J ¼ 8.4, 1.8 Hz, H-6000 )]. The three methoxy groups [d 3.65, 3.84, and 3.87] connected to aromatic rings were determined to be OCH3-5, OCH33000 , and OCH3-4000 , respectively, by the 3J-correlations in the HMBC. Furthermore, two anomeric protons [d 4.94 (1H, d, J ¼ 7.8 Hz, H-10 ), 5.54 (1H, d, J ¼ 1.2 Hz, H-100 )] suggested the existence of two glycosyl residues. Considering the J values and the 13C NMR data, and in accordance with previous investigations (Tian et al., 2013; Wang et al., 2006), the two residues were identified as b-glucopyranosyl and b-apiofuranosyl, respectively. The 100 /20 O-linkage was supported by the fact that H-100 had correlations with C-20 (d 79.2) in the HMBC spectrum (Fig. 2). Furthermore, the correlations of H-2000 and H-6000 with the ester carbonyl group (d 167.0, C¼O) suggested the presence of a veratroyl group connected to an ester carbonyl group. Because the 3J-correlations of H-10 with C-1 (d 163.2) and of H-500 (d 4.32, 500 b/d 4.34, 500 a) with d 167.0 (C¼O) were observed, the two phenolic groups and the glycosyl residues were determined to be connected. Compound 7, as a phenolic glycoside, was elucidated to be 3-hydroxy-5-methoxyphenyl-1-O-b-(500 -Overatroyl)-apiofuranosyl-(100 /20 )-b-glucopyranoside and was named reevesianin A. All of the physical data for compound 8 were similar to those for 7, a phenolic glycoside, and compound 8 exhibited a molecular formula of C26H32O15. This was established by HRESIMS (m/z 607.1635 [MþNa]þ). When comparing the 13C NMR spectra of 8 and 7, their sugar moieties were confirmed to be identical. As for the 1H NMR spectra, only two methoxy groups [d 3.60 (3H, s, OCH3-5) and 3.87 (3H, s, OCH3-3000 )] in the two separated aromatic rings were observed. Thus, the ABX spin system was regarded as a vanilloyl group in 8. Based on the 3J-correlations between H-10 (d 4.89) and C-1 (d 160.5); between H-2000 (d 7.46), H-6000 (d 7.46), H-500 (d 4.27, 500 b/d 4.33, 500 a) and d 167.8 (C¼O); and between H-10 ’ (d 5.47) and C-20 (d 78.8), the structure of 8 was suggested to be 3-hydroxy-5methoxyphenyl-1-O-b-(500 -O-vanilloyl)-apiofuranosyl-(100 /20 )-b-
5
glucopyranoside, and the compound was named reevesianin B. All of the known compounds were identified by comparing their NMR and physical data with published literature on reevesioside A (9) (Chang et al., 2013), reevesioside B (10) (Chang et al., 2013), reevesioside F (11) (Chang et al., 2013), epi-reevesioside F (12) (Chang et al., 2013), strophanthidin (13) (Kopp et al., 1992), a mixture of reevesioside G (14) and epi-reevesioside G (15) (Chang et al., 2013), a mixture of reevesioside I (16) and epi-reevesioside I (17) (Chang et al., 2013), ()-(2S,3R)-dehydrodiconiferyl alcoholg0 -methyl ether (18) (Ohta et al., 1979), (þ)-trans-dehydrodiconiferyl alcohol (19) (Yoshizawa et al., 1990), (þ)-erythrobuddlenol B (20) (Cutillo et al., 2003), (þ)-threo-buddlenol B (21) (Houghton, 1985), (±)-erythro-guaiacylglycerol-b-O-40 -sinapyl ether (22) (Machida et al., 2008), ()-erythro-guaiacylglycerol-b-O40 -coniferyl ether (23) (Gan et al., 2008), ()-threo-guaiacylglycerol-b-O-40 -coniferyl ether (24) (Gan et al., 2008), cleomiscosin B (25) (Ranjan and Sahai, 2009), cleomiscosin D (26) (Kumar et al., 1988), (þ)-lariciresinol (27) (Wang et al., 2010), ()-5,50 - dimethoxylariciresinol (28) (Ida et al., 1994), ()-syringaresinol (29) (Vermes et al., 1991), ()-medioresinol (30) (Marti et al., 2013), ()-pinoresinol (31) (Cutillo et al., 2003), hibiscone C (32) (Ferreira et al., 1980), hibiscone D (33) (Ferreira et al., 1980), oleanolic acid (34) (Martinez et al., 2013), betulinic acid (35) (Pohjala et al., 2009), a mixture of b-sitosterol (36) and stigmasterol (37) (Xu et al., 2013), a mixture of stigmasta-5-en-3b-ol-7-one (38) and stigmasta-5,22en-3b-ol-7-one (39) (Choi and Doyle, 2007), ergosterol peroxide (40) (Takaishi et al., 1991), 4-methoxybenzoic acid (41) (Villano et al., 2014), 4-hydroxybenzoic acid (42) (Azizuddin et al., 2010), protocatechuic acid (43) (Zhu et al., 2010), 3,4-dimethoxybenzoic acid (44) (Alagiri and Prabhu, 2011), vanillic acid (45) (Kang et al., 2014), methyl vanillate (46) (Kuo and Li, 1997), syringic acid (47) (Zhao et al., 2012), cinnamic acid (48) (Du et al., 2011), (E)-4hydroxycinnamic acid (49) (Lavoie et al., 2013), caffeic acid (50) (Chung et al., 2011), 3,4,5-trimethoxyphenyl-b-D-glucopyranoside (51) (Achenbach and Benirschke, 1997), ()-epicatechin (52) (Yokozawa et al., 2002), proanthocyanidin A2 (53) (Lou et al., 1999), and (þ)-abscisic acid (54) (Todoroki et al., 2000). All isolates were evaluated for their cytotoxicity levels, excluding reevesiosides A (9), B (10), F (11), G (14), and I (16) and epi-reevesiosides F (12), G (15), and I (17), which were also isolated from the root of this species. These compounds had been already evaluated, along with three other known compounds, (þ)-transdehydrodiconiferyl alcohol (19), proanthocyanidin A2 (53), and (þ)-abscisic acid (54). The results for the active compounds are listed in Table 4, and the most bioactive compounds were among the new compounds. Reevesioside J (1), bearing the aglycone of digitoxigenin, was the most potent of all, with moderate IC50 values against the cancer cell lines MCF-7, NCI-H460, and HepG2 of 0.39 ± 0.06 mM, 0.12 ± 0.01 mM, and 1.09 ± 0.02 mM, respectively. Reevesioside K (2) and epi-reevesioside K (3), with an 18,20epoxide group exhibited weaker potency than reevesioside J (1), with a digitoxigenin group as the aglycone. This result was consistent with a previous bioactivity investigation of the root of R. formosana (Chang et al., 2013). Reevesioside K (2) and epireevesioside K (3), which contain a cyclopentenone group, exhibited much weaker cytotoxic activities compared with reevesioside G (14) and epi-reevesioside G (15). However, epi-reevesioside K (3) presented selective cytotoxicity against NCI-H460 cells, with an IC50 value of 4.08 ± 0.07 mM. Observing the activities of the epimers reevesiosides G (14), H (Chang et al., 2013), and K (Chang et al., 2013) and epi-reevesiosides G (15), H (Chang et al., 2013), and K (Chang et al., 2013), the 20R-form exhibited more favorable activities than the 20S-form (Chang et al., 2013). For the two furanosesquiterpenoids reevesiterpenols C (4) and D (5), the connection of an isopropyl group to C-6 instead of a
Please cite this article in press as: Hsiao, P.-Y., et al., Cytotoxic cardenolides and sesquiterpenoids from the fruits of Reevesia formosana, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.06.009
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P.-Y. Hsiao et al. / Phytochemistry xxx (2016) 1e9
hydroxyisopropyl group made 4 more cytotoxic than 5 (IC50 > 50 mM) toward NCI-H460 cells. Reevesiterpenol E (6), bearing a naphtholactone group, exhibited a more potent cytotoxicity than reevesiterpenols C (4) and D (5) and exhibited selective cytotoxicity to NCI-H460 cells, with an IC50 value of 3.15 ± 0.22 mM. As for the known compounds strophanthidin (13) and oleanolic acid (34), though several studies have discussed their cytotoxicity, their respective activities against NCI-H460 cells and HepG2 cells were presented for the first time in this study. The new compounds reevesiterpenols CeE (4e6) shared similar furanosesquiterpenoid structures with a series of hibiscones from Hibiscus elatus (Malvaceae) (Ferreira et al., 1980). Furanosesquiterpenoids were obtained mostly from Malvaceae in previous studies; thus, their occurrence in Sterculiaceae enhances the relationship of these two families of Malvales in chemotaxonomy.
3. Conclusions In this study, eight new compounds, including cardenolides (reevesioside J (1), reevesioside K (2), and epi-reevesioside K (3)), sesquiterpenoids (reevesiterpenol C (4), reevesiterpenol D (5), and reevesiterpenol E (6)), and glycosides (reevesianin A (7) and reevesianin B (8)), together with 46 known compounds were isolated from the fruits of R. formosana. Among the isolates, cardenolides, reevesioside J (1) and strophanthidin (13) exhibited moderate cytotoxicity against the MCF-7, NCI-H460, and HepG2 cell lines, with IC50 values of 0.39 ± 0.06 mM and 1.06 ± 0.12 mM, 0.12 ± 0.01 mM and 0.29 ± 0.01 mM, and 1.09 ± 0.02 mM and 1.72 ± 0.02 mM, respectively, and reevesiterpenol E (6) exhibited selective cytotoxicity to NCI-H460 cells, with an IC50 value of 3.15 ± 0.22 mM. The bioassay results in this study suggested that R. formosana is a prospective species for the discovery of anticancer compounds and that the new cardenolides and furanosesquiterpenoids found in its fruits could potentially support development of anticancer therapies.
4. Experimental 4.1. General experimental procedures Optical rotations were obtained using a Jasco P-2000 polarimeter. The UV spectra were measured with a JASCO V-530 UV/VIS spectrophotometer, and the IR spectra (ATR) were recorded using a JASCO FT/IR-4600 spectrometer. For the NMR spectra, the 1D (1H, 13 C, DEPT) and 2D (COSY, NOESY, HSQC, HMBC) spectra using CDCl3 (1H, d 7.26; 13C, d 77.0), acetone-d6 (1H, d 2.05; 13C, d 30.5), and CD3OD (1H, d 3.31; 13C, d 49.0) as the solvents, were obtained with a Varian Unity Plus-400 spectrometer (400 MHz for 1H NMR and 100 MHz for 13C NMR) and Varian VNMRS-600 spectrometer (600 MHz for 1H NMR and 150 MHz for 13C NMR). The chemical shifts were displayed as d (ppm). The low-resolution mass spectra were measured on a Waters ZQ 4000 and a VG Biotech Quattro 5022; high-resolution mass spectra were measured with a JOELJMS-HX 100 mass spectrometer. The gels used for column chromatography (CC) were Sephadex LH-20, silica gel (60e200 mm and 15e35 mm, Silicycle), and SilicaBond C18 (17%) 60A (40e63 mm, Silicycle). Silica gels 60 F254 (Merck) and RP-18 F254 (Merck) were used for preparative TLC. HPLC (pump L-7110, Hitachi; UV/VIS detector UV-975 JASCO; integrator LC-NetII/ADC, JASCO; 5m C18 100 Å New Column 250 10.0 mm, Phenomenex Kinetex) was used for further purification.
4.2. Plant material Fruits of R. formosana were collected from the Mudan Village, Pingtung County, Taiwan, in December 2012. They were identified by Prof. Ih-Sheng Chen of Kaohsiung Medical University, Kaohsiung, Taiwan. A voucher specimen (Chen 6117) is deposited in the Herbarium of the School of Pharmacy, College of Pharmacy, Kaohsiung Medical University. 4.3. Extraction and isolation Dried fruits of R. formosana (4.4 kg) were extracted with cold MeOH (15 L 4) at room temperature, with each extraction lasting for three days. The MeOH extract (100 g) was partitioned with EtOAc and H2O into EtOAc-soluble (13 g), H2O-soluble (74 g), and insoluble parts (11 g). The MeOH extract, along with the three other parts, all exhibited cytotoxicity against cancer cell lines MCF-7, NCIH460, and HepG2, with survival rates <5% (at 30 mg/ml). The EtOAcsoluble fraction (13 g) was subjected to silica gel CC (60e200 mesh, 1.4 kg), eluted with hexane-acetone in a gradient, to provide 15 fractions (Fr. A-1eFr. A-15). Among these fractions, Fr. A-12eFr. A15 showed cytotoxicity towards the above cell lines. Fr. A-6 (1.3 g) was crystallized from MeOH to obtain a mixture of 36 and 37 (1.0 g). Fr. A-8 (516 mg) was eluted with hexane-EtOAc (5:1) by MPLC and purified by prep. TLC (CH2Cl2-MeOH, 15:1) to obtain 32 (2.2 mg), 33 (0.5 mg), 34 (1.8 mg), 35 (3.1 mg), 41 (0.7 mg), and 46 (0.5 mg), respectively. Fr. A-9 (476 mg) was subjected to MPLC, eluted with hexane-acetone (5:1), to afford a mixture of 38 and 39 (9.7 mg), and 40 (5.7 mg). Fr. A-12 (207 mg), applied to MPLC (CH2Cl2-MeOH, 30:1) afforded 11 fractions (Fr. A-12-1eFr. A-12-11). Compound 31 (2.4 mg) was obtained from Fr. A-12-2 (26.5 mg). Fr. A-12-4 (70.4 mg) was subjected to MPLC (RP-18, acetone-H2O, 1:2) to afford eight fractions (Fr. A-12-4-1eFr. A-12-4-8), and Fr. A-12-4-1 (11.2 mg) yielded 44 (1.7 mg) and 48 (0.8 mg). Fr. A-12-4-5 (13.3 mg) underwent preparative HPLC (RP-18, 250 10.0 mm, 5 mm, Phenomenex) to afford 6 (1.1 mg, tR 16.8 min, 2 ml/min), and the following HPLC experiment was conducted with the same instrument and flow rate conditions. Fr. A-12-7 was subjected to MPLC (RP-18, acetone-H2O, 1:2) to obtain 42 (0.9 mg) and 54 (1.2 mg). Fr. A-13 (695 mg) was placed on a Sephadex LH-20 column, and the column was eluted with MeOH to give over 17 fractions (Fr. A-13-1eFr. A-13-17). Fr. A-13-9 (248 mg) was further purified with MPLC (RP-18, acetone-H2O, 2:3) to yield 11 fractions (Fr. A-13-9-1eFr. A-13-9-11). Fr. A-13-9-3 (82.3 mg) was purified by preparative HPLC. A mixture of 16 and 17 (0.5 mg, tR 7.7 min), 4 (1.6 mg, tR 8.1 min), and 13 (2.7 mg, tR 11.1 min) were eluted with CH3CN-H2O (1:1). Fr. A-13-9-8 was purified by RP-18 TLC (MeOHH2O, 2:1) to afford 1 (0.9 mg), 9 (12.0 mg), 10 (4.3 mg), 11 (1.7 mg), and 12 (2.4 mg), respectively. Fr. A-13-11 (166 mg) was treated with RP-18 TLC (acetone-H2O 1:3) to obtain 27 (9.8 mg), 29 (12.6 mg), 30 (0.8 mg), and 45 (2.4 mg). This fraction went through preparative HPLC (RP-18, CH3CN-H2O, 1:1) to obtain a mixture of 14 and 15 (2.0 mg, tR 9.8 min), 2 (0.5 mg, tR 11.9 min) and 3 (1.3 mg, tR 12.1 min). Fr. A-13-13 (54.0 mg) was subjected to MPLC (RP-18, acetone-H2O, 2:3) to produce 25 (1.0 mg) and 49 (5.7 mg). Fr. A-14 (3.2 g) was passed over a Sephadex LH-20 column (MeOH) and eluted into 20 fractions (Fr. A-14-1eFr. A-14-20). Fr. A-14-9 (885 mg) was further purified with MeOH-H2O (1:1) by MPLC (RP18) and divided into 13 fractions (Fr. A-14-9-1eFr. A-14-9-13). Fr. A14-9-3 (86.2 mg) was 51 (3.1 mg). Fr. A-14-9-4 (243.5 mg) was subjected to MPLC (silica gel, CH2Cl2-MeOH, 20:1) and divided into 14 fractions (Fr. A-14-9-4-1eFr. A-14-9-4-14). Fr. A-14-9-4-1 (50.0 mg) was treated with preparative HPLC (RP-18, CH3CN-H2O, 1:2) to afford 23 (2.3 mg, tR 13.3 min) and 24 (3.0 mg, tR 14.0 min). Fr. A-14-9-4-5 (82.6 mg) was applied to RP-18 TLC (MeOH-H2O, 2:3)
Please cite this article in press as: Hsiao, P.-Y., et al., Cytotoxic cardenolides and sesquiterpenoids from the fruits of Reevesia formosana, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.06.009
P.-Y. Hsiao et al. / Phytochemistry xxx (2016) 1e9
7
Table 1 1 H and 13C NMR spectroscopic data for compounds 1e3.a Position
1
2
dH 1a/b 2a/b 3 4a/b 5 6a/b 7a/b 8 9 10 11a/b 12a/b 13 14 15a/b 16a/b 17 18a/b
1.49, 1.74, 4.07, 1.77, 1.77, 1.87, 1.68, 1.55, 1.61, e 1.42, 1.51, e e 2.12, 2.15, 2.78, 0.87,
m m/1.52, br s m/1.51, m m/1.29, m/1.22, m m
19 20 21a/b
0.94, e 4.98, 4.81, 5.87,
s
22a/b
e 4.69, 3.24, 3.36, 3.58, 3.34, 1.37, 5.17, 5.14,
23 10 20 30 40 a/b 50 60 7’a/b a 1
H and
13
m m m m
m/1.22, m m/1.39, m
m/1.69, m m/1.86, m dd (9.6, 5.4) s
dd (18.0, 1.2)/ dd (18.0, 1.8) br s
d (7.8) dd (9.6, 7.8) t (9.6) dd (9.6, 9.0) dq (9.0, 6.0) d (6.0) d (0.6)/ d (0.6)
dC
dH
30.1 26.5 74.6 30.4 36.1 26.5 21.4 41.9 35.8 35.1 21.1 40.1 49.6 85.6 33.2 26.9 50.9 15.8
2.46, 2.02, 4.21, 1.97, e 1.75, 2.05, 1.49, 3.02, e 1.77, 2.12, e e 5.82, e 2.28, 4.12, 3.70, 9.92, e 4.43, 4.02, 3.30, 2.27, e 4.48, 3.83, 4.15, 2.15, 3.83, 1.24, 5.23,
23.5 174.4 73.4 117.7 174.4 100.0 77.0 81.6 74.2 73.8 17.1 96.9
3
m/1.68, m m/1.47, m br t (2.7) m/1.74, m m m/1.58, m m m m m/2.04, m
d (1.8) s dd (10.2, 3.0)/ d (10.2) d (1.8) dd (10.2, 1.2)/ d (10.2) d (18.0)/ dd (18.0, 1.8) d (6.6) dd (6.6, 5.4) td (5.4, 1.8) m/1.74, m m d (6.6) s/4.91, s
dC
dH
dC
18.5 25.1 73.1 34.2 73.3 36.1 26.1 43.1 37.9 54.2 22.9 36.6 57.0 186.4 125.6 204.4 59.7 72.9
2.47, m/1.68, m 2.00, m/1.47, m 4.22, br t (2.7) 1.96, m/1.72, m e 2.03, m/1.75, m 2.04, m/1.54, m 1.45, m 3.02, m e 1.77, m/1.44, m 2.17, m/1.60, m e e 5.81, d (1.8) e 2.42, s 4.12, d (10.2)/ 3.63, d (10.2) 9.92 d (1.8) e 4.94, d (10.5)/ 4.13, d (10.5) 2.79, d (17.4)/ 2.45, d (17.4) e 4.48, d (6.6) 3.83, dd (6.6, 5.4) 4.15 m 2.14, m/1.73, m 3.83, m 1.24, d (6.0) 5.23, s/4.91, s
18.5 25.1 73.1 34.2 73.3 36.1 26.1 42.9 37.9 54.2 22.8 36.4 56.6 186.4 125.6 204.4 61.5 73.18
208.3 85.8 74.6 35.4 174.5 99.5 73.8 74.6 34.4 67.2 20.9 95.5
208.3 85.3 73.16 39.1 173.9 99.5 73.8 74.6 34.4 67.2 20.9 95.5
C NMR data (d) were measured in CDCl3 at 600 and 150 MHz for 1,2 and 3.
Table 2 1 H and 13C NMR spectroscopic data for compounds 4e6.a Position
1 2 3 4a/b 5 6 7 8 9 10 11 12 13 14 15 OH-7b OH-8b OH-13b OCH3-7
4
5
6
dH
dC
dH
dC
dH
dC
e e 2.77, 2.12, 1.90, 3.32, 1.61, 4.28, 4.70, e e 7.72, 1.36, 2.11, 1.12, 1.13, 1.53, 1.75, e e
143.9 188.9 42.9 36.8
e e 2.58, qdd (7.8, 4.2, 2.4) 2.55, ddd (13.5, 4.5, 2.4)/ 2.05, ddd (13.5,12.0, 4.2) 3.52, ddd (12.0, 11.7, 4.5) 1.85, dd (11.7, 1.2) 4.35, dd (1.8, 1.2) 4.64, br d (1.8) e e 7.79, s 1.28, d (7.8) e 1.40, s 1.43, s 3.35, 4.09, 4.29 (OH-7 or OH-8 or OH-13)
145.1 189.3 44.8 40.9
e e e 7.62, d (0.6)
132.0 135.0 128.1 121.0
26.3 49.9 74.5 67.3 126.3 144.6 148.2 16.8 73.8 32.0 29.0 e
e e e e e e e 2.47, 3.85, 1.55, 1.55, e e e 3.91,
117.9 146.2 145.7 150.4 100.4 126.5 165.7 17.3 28.1 22.6 22.6 e e e 62.1
qdd (7.2, 4.7, 2.1) ddd (12.0, 4.8, 2.1)/ ddd (12.0, 12.0, 4.7) ddd (12.0, 12.0, 4.8) br dd (12.0, 3.6) dd (3.6, 3.0) dd (5.7, 3.0)
s d (7.2) m d (7.2) d (7.2) br s d (5.7)
24.4 44.2 72.2 66.6 123.5 142.5 146.9 16.0 26.9 19.0 21.5 e e e e
e
e
d (1.2) sept (7.2) d (7.2) d (7.2)
s
a 1 b
H and 13C NMR data (d) were measured in CDCl3 at 600 and 150 MHz for 4 and 6, and acetone-d6 for 5. D2O exchangeable.
to obtain 5 (1.3 mg) and 47 (3.5 mg). Fr. A-14-9-4-9 (36.1 mg) was purified by MPLC (silica gel, CH2Cl2-MeOH, 9:1) and then treated with RP-18 TLC (acetone-H2O, 1:2). This was repeated five times to produce 8 (3.3 mg). With further application of preparative HPLC
(RP-18, CH3CN-H2O, 1:3), 7 (0.9 mg, tR 12.5 min) was obtained. Fr. A14-9-5 (144.6 mg) was purified with RP-18 TLC (acetone-H2O, 1:1) to yield 19 (4.7 mg), 22 (2.9 mg), and 28 (4.7 mg). Fr. A-14-9-8 (146 mg) was subjected to MPLC (CH2Cl2-MeOH, 25:1) and then
Please cite this article in press as: Hsiao, P.-Y., et al., Cytotoxic cardenolides and sesquiterpenoids from the fruits of Reevesia formosana, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.06.009
8
P.-Y. Hsiao et al. / Phytochemistry xxx (2016) 1e9 13 C NMR spectroscopic data, see Table 1; ESIMS m/z 533 [MþH]þ; HRESIMS m/z 533.3107 [MþH]þ (calcd. for C30H45O8, 533.3109).
Table 3 1 H and 13C NMR spectroscopic data for compounds 7 and 8.a Position
1 2 3 4 5 6 10 20 30 40 50 6’a/b 100 200 300 400 a/b 00
5 a/b 1000 2000 3000 4000 5000 6000 OCH3-5 OCH3-3000 OCH3-4000 C¼O
7
8
dH
dC
dH
dC
e 6.19, e 6.02, e 6.15, 4.94, 3.67, 3.66, 3.47, 3.46, 3.84, 5.54, 4.11, e 4.30, 3.91, 4.34, 4.32, e 7.48, e e 6.89, 7.55, 3.65, 3.84, 3.87, e
163.2 98.0 161.2 97.1 163.2 95.5 100.8 79.2 78.3 72.4 78.2 63.3 110.8 79.3 79.4 75.7
e 6.07, t (2.2) e 5.92, t (2.2) e 6.05, t (2.2) 4.89 d (8.0) 3.63, m 3.66, m 3.35, m 3.38, m 3.87, m/3.69, m 5.47, d (1.2) 4.03, d (1.2) e 4.28, d (9.6)/ 3.90, d (9.6) 4.33, d (11.4)/ 4.27, d (11.4) e 7.46, d (2.0) e e 6.76, d (8.8) 7.46, dd (8.8, 2.0) 3.60, s 3.87, s e e
160.5 97.2 160.2 96.5 162.8 94.9 100.3 78.8 78.4 71.4 78.1 62.5 110.5 78.8 79.3 75.3
t (2.1) t (2.1) t (2.1) d (7.8) m m m m m/3.66, m d (1.2) br d (1.2) d d d d
(9.9)/ (9.9) (12.6)/ (12.6)
d (1.8)
d (8.4) dd (8.4, 1.8) s s s
68.0 123.9 114.0 150.6 155.2 112.4 125.1 56.1 56.9 56.9 167.0
67.8 122.1 113.7 148.6 152.9 115.9 125.3 55.5 56.4 e 167.8
a 1 H and 13C NMR data (d) were measured in acetone-d6 at 600 and 150 MHz for 7, and in CD3OD at 400 and 100 MHz for 8.
4.3.2. Reevesioside K (2) Colorless syrup; ½a23 D þ36.0 (c 0.1, MeOH), IR (ATR) ymax 3445 (OH), 1780 (lactone ring), 1714 (CHO), 1698 (C¼O), 1614 (C¼C) cm1; for 1H and 13C NMR spectroscopic data, see Table 1; ESIMS m/ z 581 [MþNa]þ; HRESIMS m/z 581.2354 [MþNa]þ (calcd. for C30H38O10Na, 581.2357). 4.3.3. epi-Reevesioside K (3) Colorless syrup; ½a23 D þ25.9 (c 0.06, MeOH); IR (ATR) ymax 3435 (OH), 1780 (lactone ring), 1711 (CHO), 1693 (C¼O), 1613 (C¼C) cm1; for 1H and 13C NMR spectroscopic data, see Table 1; ESIMS m/z 581 [MþNa]þ; HRESIMS m/z 581.2360 [MþNa]þ (calcd. for C30H38O10Na, 581.2357). 4.3.4. Reevesiterpenol C (4) Yellowish oil; ½a23 D þ30.2 (c 0.04, CHCl3); UV (MeOH) lmax (log ε) 206 (3.52), 276 (3.86) nm; IR (ATR) ymax 3421 (OH), 1655 (C¼O) cm1; for 1H and 13C NMR spectroscopic data, see Table 2; ESIMS m/ z 287 [MþNa]þ; HRESIMS m/z 287.1252 [MþNa]þ (calcd. for C15H20O4Na, 287.1254). 4.3.5. Reevesiterpenol D (5) Yellowish oil; ½a24 D þ39.8 (c 0.08, CHCl3); UV (MeOH) lmax (log ε) 205 (3.80), 276 (3.94) nm; IR (ATR) ymax 3368 (OH), 1655 (C¼O) cm1; for 1H and 13C NMR spectroscopic data, see Table 2; ESIMS m/ z 303 [MþNa]þ; HRESIMS m/z 303.1205 [MþNa]þ (calcd. for C15H20O5Na, 303.1203).
Table 4 Cytotoxicity (IC50 values) of isolates from the fruits of R. formosana. Compounds
Reevesioside J (1) Reevesioside K (2) epi-Reevesioside K (3) Reevesiterpenol C (4) Reevesiterpenol E (6) Strophanthidin (13) Hibiscone C (32) ()-(2S,3R)-Dehydrodiconiferyl alcohol-g0 -methyl ether (18) Oleanolic acid (34) Betulinic acid (35) Actinomycin D a a
IC50 (mM) MCF-7
NCI-H460
HepG2
0.39 ± 0.06 >50 25.73 ± 7.34 40.14 ± 3.17 27.53 ± 4.34 1.06 ± 0.12 38.63 ± 0.50 >50 4.57 ± 0.55 >50 0.01 ± 0.001
0.12 ± 0.01 34.38 ± 0.66 4.08 ± 0.07 7.06 ± 1.31 3.15 ± 0.22 0.29 ± 0.01 32.21 ± 4.78 28.88 ± 8.01 1.54 ± 0.18 33.67 ± 1.68 0.02 ± 0.005
1.09 ± 0.02 >50 >50 >50 46.12 ± 2.59 1.72 ± 0.02 34.71 ± 3.79 >50 7.60 ± 1.42 >50 0.10 ± 0.015
Positive control.
divided into nine fractions (Fr. A-14-9-8-1eFr. A-14-9-8-9), where Fr. A-14-9-8-1 yielded 26 (1.4 mg). By way of preparative HPLC (RP18, CH3CN-H2O, 1:2), Fr. A-14-9-8-6 (18.2 mg) afforded 20 (1.7 mg, tR 14.0 min) and 21 (1.5 mg, tR 15.9 min). Fr. A-14-9-12 was purified by RP-18 TLC (acetone-H2O 1:1) to produce 18 (2.0 mg). Fr. A-14-12 (141.9 mg) was further treated with RP-18 TLC (CH3CN-H2O, 1:3) and thus provided 43 (4.9 mg) and 50 (4.2 mg). Fr. A-14-14 (294.3 mg) and Fr. A-14-17 (158 mg) were passed through a Sephadex LH-20 column (MeOH) to yield 52 (29.1 mg) and 53 (2.8 mg), respectively.
4.3.1. Reevesioside J (1) Colorless syrup; ½a22 D e13.0 (c 0.05, MeOH), IR (ATR) ymax 3394 (OH), 1775, 1738, 1629 (a,b-unsaturated g-lactone) cm1; for 1H and
4.3.6. Reevesiterpenol E (6) Brownish oil; UV (MeOH) lmax (log ε) 225 (4.33), 254 (4.19), 373 (3.77) nm; UV(MeOHþKOH) lmax (log ε) 210 (4.56), 238 (2.43), 272 sh (1.98), 399 (3.94) nm; IR (ATR) ymax 3237 (OH), 1727, 1650 (lactone ring), 1482, 1451 (aromatic ring) cm1; for 1H and 13C NMR spectroscopic data, see Table 2; ESIMS m/z 289 [MþH]þ; HRESIMS m/z 311.0888 [MþNa]þ (calcd. for C16H16O5Na, 311.0890). 4.3.7. Reevesianin A (7) Brownish syrup; ½a23 D e40.9 (c 0.06, MeOH); UV (MeOH) lmax (log ε) 209 (4.31), 225 sh (4.16), 262 (3.77), 288 (3.64) nm; UV (MeOHþKOH) lmax (log ε) 213 (4.72), 245 sh (3.91), 266 sh (3.80), 289 (3.71) nm; IR (ATR) ymax 3410 (OH), 1699 (C¼O), 1602, 1514, 1457 (aromatic ring) cm1; for 1H and 13C NMR spectroscopic data,
Please cite this article in press as: Hsiao, P.-Y., et al., Cytotoxic cardenolides and sesquiterpenoids from the fruits of Reevesia formosana, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.06.009
P.-Y. Hsiao et al. / Phytochemistry xxx (2016) 1e9
see Table 3; ESIMS m/z 621 [MþNa]þ; HRESIMS m/z 621.1792 [MþNa]þ (calcd. for C27H34O15Na, 621.1790). 4.3.8. Reevesianin B (8) Brownish syrup; ½a23 D e46.1 (c 0.16, MeOH); UV (MeOH) lmax (log ε) 209 (3.68), 224 sh (4.28), 264 (3.95), 298 sh (3.68) nm; UV (MeOHþKOH) lmax (log ε) 212 (4.71), 239 sh (4.14), 283 sh (3.89), 315 (4.17) nm; IR (ATR) ymax 3386 (OH), 1697 (C¼O), 1600, 1515, 1457 (aromatic ring) cm1; for 1H and 13C NMR spectroscopic data, see Table 3; ESIMS m/z 607 [MþNa]þ; HRESIMS m/z 607.1635 [MþNa]þ (calcd. for C26H32O15Na, 607.1633). 4.4. Cytotoxicity assay Human cancer cells, MCF-7 (human breast adenocarcinoma), NCI-H460 (non-small-cell lung cancer), and HepG2 (liver hepatocellular cells), were cultured in DMEM containing 10% fetal calf serum and nonessential amino acids and seeded in 96-well microtiter plates at 6500, 2500, and 10,000 cells/well, respectively. The sequential treatments of the cell cultures and the cytotoxicity assays were performed as previously described (Chang et al., 2009). Acknowledgment This work was supported by the National Science Council of Taiwan (NSC102-2320-B037-007) and was supported partially by the Kaohsiung Medical University “Aim for the Top Universities Grant,” Grant No. KMU-TP103H01, KMU-TP103H05, and KMUTP104E43. We also thank Dr. Ming-Jen Cheng and Dr. Chu-Hung Lin for their valuable suggestions on structural elucidation. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.phytochem.2016.06.009. References Achenbach, H., Benirschke, G., 1997. Joannesialactone and other compounds from Joannesia princeps. Phytochemistry 45, 149e157. Al Muqarrabun, L.M.R., Ahmat, N., 2015. Medicinal uses, phytochemistry and pharmacology of family Sterculiaceae: A review. Eur. J. Med. Chem. 92, 514e530. Alagiri, K., Prabhu, K.R., 2011. Efficient synthesis of carbonyl compounds: oxidation of azides and alcohols catalyzed by vanadium pentoxide in water using tertbutylhydroperoxide. Tetrahedron 67, 8544e8551. Azizuddin, Makhmoor, T., Choudhary, M.I., 2010. Radical scavenging potential of compounds isolated from Vitex agnus-castus. Turk. J. Chem. 34, 119e126. Beale, J.M., Floss, H.G., Lehmann, T., Luckner, M., 1988. Digitoxigenin-3b-O-[b-Dfucopyranosyl-4'-b-D-glucopyranoside], the main cardenolide of somatic embryos of Digitalis lanata. Phytochemistry 27, 3143e3146. Chang, H.S., Chiang, M.Y., Hsu, H.Y., Yang, C.W., Lin, C.H., Lee, S.J., Chen, I.S., 2013. Cytotoxic cardenolide glycosides from the root of Reevesia formosana. Phytochemistry 87, 86e95. Chang, H.S., Lin, Y.J., Lee, S.J., Yang, C.W., Lin, W.Y., Tsai, I.L., Chen, I.S., 2009. Cytotoxic alkyl benzoquinones and alkyl phenols from Ardisia virens. Phytochemistry 70, 2064e2071. Choi, H., Doyle, M.P., 2007. Optimal TBHP allylic oxidation of D5-steroids catalyzed by dirhodium caprolactamate. Org. Lett. 9, 5349e5352. Chung, C.P., Hsia, S.M., Lee, M.Y., Chen, H.J., Cheng, F., Chan, L.C., Kuo, Y.H., Lin, Y.L., Chiang, W.C., 2011. Gastroprotective activities of Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf) on the growth of the stomach cancer ags cell line and indomethacin-induced gastric ulcers. J. Agric. Food Chem. 59, 6025e6033. Cutillo, F., D'Abrosca, B., DellaGreca, M., Fiorentino, A., Zarrelli, A., 2003. Lignans and neolignans from Brassica fruticulosa: Effects on seed germination and plant growth. J. Agric. Food Chem. 51, 6165e6172. Du, Z., Zhou, W., Bai, L., Wang, F., Wang, J.X., 2011. In situ generation of palladium nanoparticles: Reusable, ligand-free Heck reaction in PEG-400 assisted by focused microwave irradiation. Synlett 369e372.
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Please cite this article in press as: Hsiao, P.-Y., et al., Cytotoxic cardenolides and sesquiterpenoids from the fruits of Reevesia formosana, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.06.009