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Bufadienolides from the whole plants of Helleborus foetidus and their cytotoxicity
Phytochemistry 172 (2020) 112277
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Bufadienolides from the whole plants of Helleborus foetidus and their cytotoxicity
T
Tomoki Iguchi, Akihito Yokosuka∗, Riko Kawahata, Madoka Andou, Yoshihiro Mimaki Department of Medicinal Pharmacognosy, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo, 192-0392, Japan
ARTICLE INFO
ABSTRACT
Keywords: Helleborus foetidus Ranunculaceae Bufadienolides Bufadienolide glucosides Cytotoxicity Apoptosis HL-60 cells A549 cells
Two undescribed bufadienolide glucosides and four undescribed bufadienolides were isolated from the whole plants of Helleborus foetidus (Ranunculaceae). Their structures were determined by extensive spectroscopic analysis and the results of hydrolytic cleavage. The isolated compounds exhibited cytotoxic activities against HL60 and A549 cells with IC50 values ranging from 0.019 to 3.0 μM. The isolated compounds also showed the Na+/ K+ ATPase inhibitory activity. The undescribed compound 16β-formyloxy-10,14-dihydroxy-5β-[(β-D-glucopyranosyl)oxy]-19-norbufa-3,20,22-trienolide induced apoptosis in HL-60 cells through a mitochondria-dependent apoptotic pathway.
1. Introduction
2. Results and discussion
Helleborus foetidus L., native to Europe, is an evergreen perennial plant belonging to the family of Ranunculaceae (Tsukamoto, 1989). Anemonin, a quercetin glycoside, a furostanol glycoside, and a few phenolic glucosides have been reported as chemical components of H. foetidus (Prieto et al., 2006). Previously, a cytotoxicity-guided fractionation of a MeOH extract of H. foetidus resulted in the isolation of five bufadienolide derivatives with potent cytotoxicities against HL-60 human leukemia cells (Yokosuka et al., 2018). Bufadienolides represent a type of steroid with a 2-pyrone ring at the C-17 position of the skeleton and show various activities, such as cardiotonic, blood pressure stimulating, local anaesthetic, and cytotoxic activity against cultured tumor cells (Kamano et al., 1998; Nogawa et al., 2001). Although the isolation of many bufadienolides were from animal sources such as a toad venom, some are also distributed in plants such as Helleborus, Drimia, Kalanchoe, and Urginea species (Gao et al., 2011). Ongoing phytochemical studies on the cytotoxic steroidal constituents of H. foetidus led to the isolation of two undescribed bufadienolide glucosides (1 and 2) and four undescribed bufadienolides (3–6). This paper reports the isolation and structure determination of 1–6 and their cytotoxic activities against HL-60 human leukemia cells, A549 human lung adenocarcinoma cells, and TIG-3 human normal diploid lung cells. The Na+/K+ ATPase inhibitory activity of 1–5 was also evaluated. Furthermore, the ability of undescribed bufadienolide glucoside 2 to induce apoptosis in HL-60 cells was investigated.
The fresh whole plants of H. foetidus (3.3 kg) were extracted twice with MeOH (10 L). The fractions eluted with 50% MeOH (5.0 g) and pure MeOH (115 g) were subjected to column chromatography (CC) over Diaion HP-20, silica gel, and ODS silica gel as well as preparative HPLC to give six compounds (1–6) (see Fig. 1). Compound 1 was obtained as an amorphous solid with a molecular formula of C30H40O12, which was elucidated based on its HRESITOFMS (m/z 615.2418 [M + Na]+, calcd for C30H40O12Na: 615.2417) and 13C NMR data (30 carbon signals). The IR spectrum of 1 suggested the presence of hydroxy groups (3383 cm−1) and carbonyl groups (1713 and 1644 cm−1). The UV spectrum showed the absorption maxima indicative of a conjugated system (296 and 206 nm). The 1H NMR spectrum of 1 contained signals for a 2-pyrone ring [δH 8.11 (1H, dd, J = 9.7, 2.5 Hz, H-22), 7.43 (1H, dd, J = 2.5, 0.8 Hz, H-21), and 6.18 (1H, dd, J = 9.7, 0.8 Hz, H-23)], which are characteristic of bufadienolide derivatives, an (Z)-olefinic group [δH 5.91 (1H, m, H-3) and 5.43 (1H, d, J = 9.9 Hz, H-4)], an oxymethine proton [δH 4.46 (1H, ddd, J = 7.7, 7.7, 0.8 Hz, H-16)], an angular methyl group [δH 0.79 (3H, s, Me-18)], and an anomeric proton [5.59 (1H, d, J = 8.0 Hz, H-1’)] (Table 1). The 13C NMR spectrum of 1 displayed signals for a 2-pyrone ring [δC 120.4, 151.7, 152.9, 112.9, and 165.1 (C-20–C-24)], an ester carbonyl carbon [δC 174.3 (C-19)], an olefin [δC 131.1 (C-3) and 134.1 (C-4)], an oxymethine carbon [δC 73.5 (C-16)], two quaternary carbons bearing oxygen atoms [δC 86.8 (C-14) and 71.7 (C-5)], two quaternary
∗
Corresponding author. E-mail address: [email protected] (A. Yokosuka).
https://doi.org/10.1016/j.phytochem.2020.112277 Received 7 October 2019; Received in revised form 14 January 2020; Accepted 14 January 2020 0031-9422/ © 2020 Elsevier Ltd. All rights reserved.
Phytochemistry 172 (2020) 112277
T. Iguchi, et al.
Table 1 1 H NMR Data for 1–6 (CD3OD, 600 MHz). No.
1
1α β 2α β 3 4α β 5 6α β 7α β 8 9 10 11 α β 12 α β 13 14 15 α β 16 α β 17 18 19 20 21 22 23 24 COH Ac Glc1′ 2′ 3′ 4′ 5′ 6′ a b
2.09 2.04 2.08 2.03 5.91 5.43 – 2.50 1.62 2.08 1.02 2.18 1.39 – 1.52 1.21 1.56 1.34 – – 2.33 1.67 4.46
2 m m m m m d (9.9) m m m m ddd (12.0, 12.0, 3.1) m m m ddd (13.3, 13.3, 2.8) ddd (13.3, 2.8, 2.8) dd (14.7, 7.7) dd (14.7, 0.8) ddd (7.7, 7.7, 0.8)
2.73 0.79 – – 7.43 8.11 6.18 –
d (7.7) s
5.59 3.41 3.37 3.36 3.42 3.79 3.67
d (8.0) m m m m dd (11.8, 1.4) m
dd (2.5, 0.8) dd (9.7, 2.5) dd (9.7, 0.8)
2.17 1.71 2.21 2.12 5.89 5.63 – 2.03 1.90 2.07 1.04 1.77 1.47 – 1.65 1.55 1.64 1.45 – – 2.52 1.80 5.62
3 m m m m br d (9.9) d (9.9) m m m m ddd (11.7, 11.7, 2.6) m m m m m dd (12.4, 7.0) br d (12.4) m
3.02 0.86 – – 7.49 8.27 6.24 – 7.91
d (7.1) s
4.68 3.23 3.39 3.32 3.25 3.84 3.68
d (7.8) dd (8.9, 7.8) dd (8.9, 8.9) dd (8.9, 8.9) m dd (11.9, 2.4) dd (11.9, 5.6)
br d (2.3) dd (9.8, 2.3) br d (9.8) s
2.93 1.65 1.91 1.31 4.11 5.71 – 2.25 2.25 2.25 1.12 1.90 1.34 – 3.94
4 m m m m br dd (9.3, 5.7) br s
3.00 1.65 1.92 1.35 4.10 5.70
m m m m ddd (12.1, 12.1, 2.6) dd (12.1, 10.1) ddd (10.1, 10.1, 4.1)
1.72 1.43 – – 2.43 1.71 4.48
dd (13.5, 4.1) dd (13.5,10.1)
2.85 0.84 9.81 – 7.48 8.08 6.19 –
d (72) s s
dd (14.8, 7.3) dd (14.8, 1.2) ddd (7.2, 7.2, 1.2)
dd (2.5, 0.8) dd (9.7, 2.5) dd (9.7, 0.8)
– 2.26 2.26 2.20 1.12 1.92 1.40 – 3.94 1.70 1.48 – – 2.03 1.34 2.19 1.76 2.59 0.76 9.83 – 7.44 7.94 6.28 –
5 m m m m br dd (9.3, 5.7) br s m m m m ddd (12.0, 12.0, 2.8) m m m m ddd (11.0,11.0, 3.0) m m m dd (9.7, 7.0) s s dd (2.5, 0.8) dd (9.7, 2.5) dd (9.7, 0.8)
1.96 1.40 1.83 1.57 4.04 5.44 – 2.43 2.12 2.18 1.09 1.83 1.09 – 1.55 1.55 1.60 1.40 – – 2.38 1.71 4.46 2.75 0.83 – – 7.44 8.12 6.19 –
6 m m m m m d (1.4) m m m m ddd (12.0, 12.0, 3.0) m m m ddd (13.6, 6.1, 3.1) ddd (13.6, 13.6, 3.1) dd (14.8, 7.6) dd (14.8, 0.8) ddd (7.6, 7.6, 0.8) d (7.6) s dd (2.5, 0.8) dd (9.7, 2.5) dd (9.7, 0.8)
3.93 m 2.10 2.10 5.25 2.49 1.76 – 1.79 1.39 2.08 1.29 1.73 1.49 – 1.38 1.38 1.59 1.38 – – 2.57 1.81 4.54
m m br s dd (15.8, 4.6) m
2.80 0.83 1.24 – 7.48 8.16 6.23 –
d (7.7) s s
m m m m ddd (12.1, 12.1, 3.9) m m m m m dd (14.8, 7.7) dd (14.8, 1.0) ddd (7.7, 7.7, 1.0)
dd (2.4, 0.8) dd (9.8, 2.4) dd (9.8, 0.8)
2.05 s
carbons [δC 55.5 (C-10) and 50.5 (C-13)], three methine carbons [δC 59.2 (C-17), 41.4 (C-8), and 39.5 (C-9)], seven methylene carbons [δC 42.3 (C-15), 42.1 (C-12), 37.6 (C-6), 24.7 (C-7), 24.6 (C-11), 23.7 (C-1), and 22.8 (C-2)], a methyl carbon [δC 17.4 (C-18)], and a β-D-glucopyranosyl group [δC 95.9, 74.1, 78.9, 71.0, 78.0, and 62.3] (Table 2). Acid hydrolysis of 1 resulted in the production of D-glucose, whereas the aglycone decomposed under acidic conditions and could not be obtained. Analysis of the 1H–1H COSY and HSQC spectra of 1 indicated that the aglycone moiety of 1 was composed of four spin-coupling systems, corresponding to the following structural fragments: A: H2-1/ H2-2/H-3/H-4, B: H2-6/H2-7/H-8/H-9/H2-11/H2-12, C: H2-15/H-16/H17, and D: H-22/H-23 (Fig. 2). The long-range HMBC correlations from H-1α at δH 2.09, H-1β at δH 2.04, and H-9 at δH 1.39 to C-19 at δC 174.3; from H-1α, H-1β, H-2α at δH 2.08, H-2β at δH 2.03, H-4 at δH 5.43, H-6α at δH 2.50, H-6β at δH 1.62, and H-9 to C-10 at δC 55.5; and from H-3 at δH 5.91, H-4, H-6α, H-6β, H-7α at δH 2.08, and H-7β at δH 1.02 to C-5 at δC 71.7 indicated structural fragments A and B were linked through the C-5 oxygenated quaternary carbon and the C-10 quaternary carbon as well as the presence of a double-bond between C3 and C-4 and an ester carbonyl (C-19) connected to C-10. The threeproton singlet at δH 0.79 assignable to Me-18 showed HMBC cross peaks with δC 42.1 (C-12), 50.5 (C-13), 86.8 (C-14), and 59.2 (C-17), which implied that structural fragment B was linked to C through the C-13 quaternary carbon and the C-14 oxygenated quaternary carbon. The 2pyrone ring was attached to C-17 based on the long-range HMBC correlations from δH 2.73 (H-17) to δC 120.4 (C-20), 151.7 (C-21), and
152.9 (C-22) (Fig. 2). Thus, the aglycone of 1 was revealed to a bufadienolide derivative with an ester carbonyl group at C-19, an (Z)-olefinic group at C-3/C-4, and three hydroxy groups at C-5, C-14, and C-16 (Robien et al., 1987; Shi et al., 2010; Yokosuka et al., 2018). The linkage of the β-D-glucopyranosyl moiety to C-19 through an oxygen atom was confirmed by the 3JC,H HMBC correlation between the signals of the α-anomeric proton at δH 5.59 (J = 8.0 Hz) and the C-19 ester carbonyl carbon at δC 174.3. The NOE correlations between H-4 and H6α, H-2α and H-9, H-12α and H-16/H-17, and Me-18 and H-8/H-11β observed in the phase-sensitive NOESY spectrum (Fig. 3) and the proton spin-coupling constants between H-8 and H-9 (J = 12.0 Hz) (Table 1) indicated that 1 had A/B cis, B/C trans, and C/D cis ring fusions and the 5β, 14β, 16β, and 17β configurations. Therefore, the structure of 1 was determined to be 5β,14β,16β,21-tetrahydroxychola-3,20,22-triene19,24-dioic acid 24,21-lactone 19-β-D-glucopyranosyl ester. Compound 1 is the first bufadienolide derivative in which the C-19 methyl group is oxidized to the carboxylic acid and forms an ester glucosidic linkage. Compound 2 was obtained as an amorphous solid, and its molecular formula was determined to be C30H40O12 based on its HRESITOFMS (m/z 615.2420 [M +Na]+) and 13C NMR data. The NMR spectral features of 2 were closely related to those of 10,14,16β-trihydroxy-5β[(β-D-glucopyranosyl)oxy]-19-norbufa-3,20,22-trienolide (hellebortin A), which was isolated from H. foetidus (Meng et al., 2001; Yokosuka et al., 2018). However, the molecular formula of 2 was larger than that of hellebortin A by CO. The IR (1698 cm−1), 1H NMR [δ 7.91 (1H, s)], and 13C NMR [δ 162.0 (C]O)] data suggested the presence of a 2
Phytochemistry 172 (2020) 112277
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δC 75.2 (Fig. 2). Furthermore, an HMBC cross peak was observed between H-1’ of the β-D-glucopyranosyl group at δH 4.68 (d, J = 7.8 Hz) and C-5 of the aglycone at δC 79.2. Accordingly, the structure of 2 was established as 16β-formyloxy-10,14-dihydroxy-5β-[(β-D-glucopyranosyl)oxy]-19-norbufa-3,20,22-trienolide. The bufadienolide derivative with a formyloxy group at C-3 was isolated from toads native to China (Nogawa et al., 2001). Compound 2 is the first bufadienolide glucoside with a formyloxy group isolated from plant materials. The 1H and 13C NMR spectroscopic data of 3 (C24H30O7) were similar to those of 5β,11α,14β,16β-tetrahydroxy-19-oxobufa-3,20,22trienolide (C24H30O7), which has been isolated from H. foetidus (Yokosuka et al., 2018); however, they differed in the 1H and 13C NMR signals attributable to the A ring (C-1–C-5 and C-10). The signal for the hydroxy-substituted quaternary carbon at δC 73.5 (C-5) was not observed in the data of 3, and instead, signals assignable to an oxymethine unit [δH 4.11 (br dd, J = 9.3, 5.7 Hz, H-3); δC 67.1] were observed. The oxymethine proton at δH 4.11 showed spin-coupling correlations with the olefinic proton at δH 5.71 (br s, H-4) and with the pair of methylene protons at δH 1.91 and 1.31 (each m, H2-2) in the 1H–1H COSY spectrum of 3. The methylene protons exhibited correlations with the terminal methylene protons at δH 2.93 and 1.65 (each m, H2-1). These correlations led us to propose the A ring of 3 to be –C(1)H2–C(2)H2–C(3)(OH)–C(4)H=. Long-range HMBC correlations from the olefinic proton at δH 5.71 (H-4) to the carbons at δC 55.2 (C-10) and 34.8 (C-6) and from the aldehyde proton at δH 9.81 (H-19) to the carbons at δC 32.2 (C-1), 55.6 (C-9), and 55.2 (C-10) were detected (Fig. 2). The B/C trans and C/D cis ring fusions of the bufadienolide skeleton and the C-3β, C-11α, C-16β, and C-17β configurations were elucidated from the NOE correlations in the NOESY spectrum of 3 (Fig. 3) and the proton spin-coupling constants (Table 1). Accordingly, the structure of 3 was formulated as 3β,11α,14β,16β-tetrahydroxy-19oxobufa-4,20,22-trienolide. Compound 4 (C24H30O6) was obtained as an amorphous solid. The 1 H and 13C NMR spectra of 4 were essentially analogous to those of 3. The molecular formula of 4 includes one fewer oxygen atom than that of 3. Comparing the 1H and 13C NMR spectra of 4 with those of 3
Table 2 13 C NMR data for 1–6 (CD3OD, 125 MHz).
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 1' 2' 3' 4' 5' 6' 3-OAc 16-COH
1
2
3
4
5
6
23.7 22.8 131.1 134.1 71.7 37.6 24.7 41.4 39.5 55.5 24.6 42.1 50.5 86.8 42.3 73.5 59.2 17.4 174.3 120.4 151.7 152.9 112.9 165.1 95.9 74.1 78.9 71.0 78.0 62.3
27.8 25.9 132.4 132.9 79.2 32.2 24.4 41.4 41.3 74.8 22.4 41.2 50.7 84.5 41.0 75.2 57.8 17.0 – 119.3 152.7 152.0 113.3 164.5 98.4 75.4 78.3 71.7 77.6 62.8
32.2 30.3 67.1 131.4 139.2 34.8 29.5 44.0 55.6 55.2 68.9 51.1 50.5 84.7 42.7 73.5 58.4 18.9 205.3 120.0 151.9 152.4 113.1 164.9
32.5 30.4 67.1 131.4 139.2 35.0 29.4 44.1 55.7 55.3 69.2 51.7 50.1 84.9 32.7 29.7 51.8 18.2 205.4 124.4 150.7 149.1 115.5 164.7
34.9 29.2 68.4 128.3 142.8 32.7 29.3 42.6 51.1 70.9 21.2 41.7 50.2 85.5 43.0 73.5 59.4 17.2 – 120.5 151.8 152.9 112.9 165.1
74.3 32.2 71.3 36.8 75.6 36.3 25.1 42.2 42.0 44.1 22.8 41.9 50.5 85.9 43.4 73.5 59.7 17.5 13.5 120.7 152.1 153.2 113.2 165.3
162.0
21.9 172.9
formyloxy group in 2. The HMBC spectrum of 2 showed cross peaks between H-16 at δH 5.62 (1H, m) and the carbonyl carbon of the formyl group at δC 162.0 and between the formyl proton at δH 7.91 and C-16 at
Fig. 1. Chemical structures of 1–6. 3
Phytochemistry 172 (2020) 112277
T. Iguchi, et al.
Fig. 2. Key HMBC correlations of 1–3, and 6. The bold lines indicate the 1H–1H coupling, and arrows indicate 1H/13C long-range correlations.
Fig. 3. Key NOE correlations of 1–3, and 6.
showed that the C-16 hydroxymethine proton and carbon signals (δH 4.48/δC 73.5) in 3 were replaced by the methylene proton and carbon signals [δH 2.19 (1H, m) and 1.76 (1H, m)/δC 29.7]. All other signals were observed at almost the same positions in the spectra of these two compounds. Thus, the structure of 4 was elucidated as 3β,11α,14βtrihydroxy-19-oxobufa-4,20,22-trienolide. The 1H and 13C NMR spectral data of 5 (C23H30O6) were also similar to those of 3. However, the signals corresponding to the aldehyde group at C-19 were not observed in the 1H and 13C NMR spectra of 5, and the signals of quaternary carbon C-10 (δC 55.2) and the hydroxymethine proton and carbon signals for C-11 [δH 3.94 (ddd, J = 10.1, 10.1, 4.1 Hz)/δC 68.9] in 3 were replaced by a signals for quaternary carbon
bearing an oxygen atom (δC 70.9) and methylene proton and carbon signals [δH 1.55 (2H, m)/δC 21.2], respectively, in 5. Thus, the structure of 5 was assigned as 3β,10β,14β,16β-tetrahydroxy-19-norbufa-4,20,22trienolide. Compound 6 (C26H36O8) showed spectral features characteristic of a polyhydroxylated bufadienolide derivative. The 1H and 13C NMR spectra of 6 suggested the presence of two angular methyl groups [δH 1.24 (3H, s)/δC 13.5 and δH 0.83 (3H, s)/δC 17.5], three oxymethine units [δH 5.25 (br s)/δC 71.3, δH 4.54 (ddd, J = 7.7, 7.7, 1.0 Hz)/δC 73.5, and δH 3.93 (m)/δC 74.3], two quaternary carbons bearing oxygen atoms [δC 85.9 and 75.6], and an acetyl group [δH 2.05 (3H, s)/δC 172.9 (C]O) and 21.9 (Me)]. The methyl signal at δH 0.83 exhibited 4
Phytochemistry 172 (2020) 112277
T. Iguchi, et al.
long-range HMBC correlations with the carbon signals at δC 42.0 (C12), 50.5 (C-13), 85.9 (C-14), and 59.7 (C-17) and was attributed to Me18. Another methyl signal at δH 1.24, attributable to Me-19, displayed long-range HMBC correlations with the carbon signals at δC 74.3 (C-1), 75.6 (C-5), 41.9 (C-9), and 44.1 (C-10). In the 1H–1H COSY spectrum, the oxymethine proton at δH 5.25 showed spin-coupling correlations with the adjacent methylene groups at δH 2.10 (2H, m, H2-2) and δH 2.49 (1H, dd, J = 15.8, 4.6 Hz, H-4a) and 1.76 (1H, m, H-4b), and assigned as H-3. The other oxymethine proton at δH 4.54 had spincouplings with the methylene group at δH 2.57 (dd, J = 14.8, 7.7 Hz, H15α) and 1.81 (dd, J = 14.8, 1.0 Hz, H-15β), and the methine group at δH 2.80 (d, J = 7.7 Hz, H-17) and was therefore attributed to H-16. A 3 JC,H HMBC correlation was observed between H-3 and the acetyl carbonyl carbon. The above spectroscopic data were indicative of the presence of hydroxy groups at C-1, C-3, C-5, C-14, and C-16, and the C-3 hydroxy group was acetylated. The A/B cis, B/C trans, and C/D cis ring fusions and the 1β, 3β, 5β, 14β, 16β, and 17β configurations were elucidated from NOESY spectrum of 6 (Fig. 3) and the proton spincoupling constants (Table 1). Thus, the structure of 6 was determined to be 3β-acetoxy-1β,5β,14β,16β-tetrahydroxybufa-20,22-dienolide. Compounds 1–6 were evaluated for their in vitro cytotoxic activities against HL-60 and A549 tumor cells and TIG-3 normal cells using a modified MTT assay (Sargent and Taylor, 1989). Although 1–6 showed cytotoxicities against mot only HL-60 and A549 tumor cells but also TIG normal cells, 2 and 4 were much more cytotoxic than the positive controls, etoposide and cisplatin, to both HL-60 and A549 cells, and they showed IC50 values ranging from 0.037 to 0.098 μM (Table 3). On the other hand, 1, 3, 5, and 6 showed moderate cytotoxicity with IC50 values ranging from 0.12 to 3.0 μM. Cardiotonic steroids are categorized as cardenolides or bufadienolides, and have been clinically used for the treatment of arrhythmia and heart failure. They have also been reported to exhibit cytotoxicity through Na+/K+ ATPase inhibition resulting in increased intracellular Ca2+ concentrations, which induces apoptotic cell death in tumor cells (Zeino et al., 2015). Then, the Na+/K+ ATPase inhibitory activities of 1–5 were examined. Compounds 1–5 inhibited Na+/K+ ATPase with IC50 values of 0.11, 1.3, 1.7, 0.061, and 0.30 μM, respectively, which were similar to their average cytotoxicities (IC50 values) against HL-60 and A549 cells (Table 4). These data suggested that their cytotoxicities were partially mediated via the inhibition of Na+/K+ ATPase. Compound 2 was an undescribed bufadienolide derivative and showed potent cytotoxicity against HL-60 and A549 cells. The ability of 2 to induce apoptotic cell death in HL-60 cells was evaluated. Morphologically, fragmented and condensed nuclear chromatins, the accumulation of the sub-G1 cells, the induction of DNA fragmentation, and caspase-3 activation were observed in HL-60 cells treated with 2 (Fig. 4). Furthermore, disruption of the mitochondrial membrane potential and release of cytochrome c into the cytoplasm were observed in 2-treated apoptotic HL-60 cells (Fig. 5). The above data implied that 2 induced apoptosis in HL-60 cells through a mitochondria-dependent pathway.
Table 4 Na+/K+ ATPase inhibitory activities of 1–5 and ouabain.
1 2 3 4 5 6 etoposide cisplatin a
A549
TIG-3
0.27 ± 0.011 0.087 ± 0.0018 3.0 ± 0.31 0.044 ± 0.0021 0.30 ± 0.045 1.0 ± 0.016 0.18 ± 0.00084 1.1 ± 0.019
0.20 ± 0.0075 0.098 ± 0.0075 0.96 ± 0.10 0.019 ± 0.0049 0.12 ± 0.027 0.69 ± 0.023 -a 2.3 ± 0.39
0.035 ± 0.00031 0.15 ± 0.0035 1.50 ± 077 0.020 ± 0.0017 0.16 ± 0.035 0.36 ± 0.0058 _ 1.7 ± 0.025
1 2 3 4 5 Ouabain
1.3 ± 0.16 0.11 ± 0.0058 1.7 ± 0.11 0.061 ± 0.0017 0.30 ± 0.0040 0.035 ± 0.0024
In summary, two undescribed cytotoxic bufadienolide glucosides (1 and 2) and four undescribed bufadienolides (3–6) were obtained from H. foetidus. Their structures were determined by extensive spectroscopic analysis and the results of hydrolytic cleavage. Compound 1 is the first bufadienolide derivative in which the C-19 methyl group is oxidized to the carboxylic acid and forms an ester glucosidic linkage. Compound 2 is the first bufadienolide glucoside with a formyloxy group isolated from plant materials. All the isolated compounds were cytotoxic to cancer cells in vitro, which was suggested to be mediated via the inhibition of Na+/K+ ATPase resulting in increased intracellular Ca2+ concentrations. Compound 2, a bufadienolide glucoside with a formyloxy group at C-16 of the aglycone, induced apoptosis in HL-60 cells through a mitochondria-dependent pathway. 4. Experimental section 4.1. General experimental procedures Optical rotations were measured using a JASCO P-1030 (Tokyo, Japan) automatic digital polarimeter. IR spectra were recorded on a JASCO FT-IR 620 spectrophotometer. UV spectra were measured with a JASCO V-630 UV–Vis spectrophotometer (Tokyo, Japan). NMR spectra were recorded on a Bruker DRX-500 (500 MHz for 1H NMR, Karlsruhe, Germany) and a Bruker DRX-600 (600 MHz for 1H NMR) spectrophotometer using standard Bruker pulse programs. Chemical shifts are given as δ values and are referenced to tetramethylsilane (TMS) as an internal standard. HRESI TOF-MS data were recorded on a Micromass LCT mass spectrometer (Manchester, UK). Diaion HP-20 (Mitsubishi Chemical, Tokyo, Japan), silica gel (Fuji-Silysia Chemical, Aichi, Japan), and ODS silica gel (Nacalai Tesque, Kyoto, Japan) were used for column chromatography. TLC was carried out on precoated silica gel 60 F254 (0.25 mm thick, Merck, Darmstadt, Germany) and RP18 F254s plates (0.25 mm thick, Merck), and spots were visualized by spraying the plates with 10% H2SO4 aqueous solution followed by heating. HPLC separations were performed with a system consisting of an LC-20AD pump (Shimadzu, Kyoto, Japan), a RID-10A refractive index (RI) detector (Shimadzu), and a Rheodyne injection port (Rheodyne LLC, Rohnert Park, CA, USA). A TSK gel ODS-100Z column (10 mm i.d. × 250 mm, 5 μm; Tosoh, Tokyo, Japan) was used for preparative HPLC. HL-60 cells (JCRB 0085), A549 cells (JCRB 0076), and TIG3 cells (JCRB 0510) were obtained from the Human Science Research Resources Bank (Osaka, Japan). The following reagents were obtained from the indicated companies: RPMI 1640 medium, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA); penicillin G sodium salt and streptomycin sulfate (Gibco, Grand Island, NY, USA). All other chemicals used were of biochemical reagent grade.
IC50(μM) HL-60
IC50 (μM)
3. Conclusion
Table 3 Cytotoxic activity of the isolated compounds 1–6, etoposide, and cisplatin. Compounds
Compounds
4.2. Plant material Helleborus foetidus L. (Ranunculaceae) were purchased from the garden center of Fujiengei Ltd. (Okayama, Japan) in October 2014. It
Not determined 5
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Fig. 4. Compound 2 induced apoptosis in HL60 cells. A, Morphology of HL-60 cells treated with 2. HL-60 cells were stained with DAPI after treatment with either 0.5 μM of 2 or 15 μM of etoposide for 20 h and then observed under a fluorescence microscope. B, Cell cycle progression of the HL-60 cells treated with 2. HL-60 cells were treated with either 0.5 μM of 2 or 15 μM of etoposide for 10 h, and the cell cycle distribution was analyzed by a flow cytometer. C, Electrophoretic profile of the DNA of HL60 cells treated with 2. HL-60 cells were incubated with either 0.5 μM of 2 or 15 μM of etoposide for 14 h. DNA was then extracted and analyzed by agarose gel electrophoresis. D, Caspase-3 activity in the lysates of HL-60 cells treated with 2. HL-60 cells were incubated with either 0.5 μM of 2 or 15 μM of etoposide for 12 h, and the caspase-3 activity in the lysates was measured using a caspase-3 colorimetric kit. The data are presented as the mean ± S.E.M. of three experiments. Results significantly different from that of the control group are indicated by * (p < 0.001).
was identified by one of the authors, A. Yokosuka. A voucher specimen was deposited in our laboratory (voucher no. KS-2014-0011, Department of Medicinal Pharmacognosy).
HP-20 column and successively eluted with 30% MeOH, 50% MeOH, MeOH, EtOH, and EtOAc (each 6.0 L). The fractions eluted with 50% MeOH and MeOH demonstrated cytotoxicity against HL-60 cells with IC50 values of 0.15 and 0.23 μg/mL, respectively. The 50% MeOH eluate (5.0 g) was subjected to silica gel CC and eluted with stepwise gradient mixtures of EtOAc-MeOH-H2O (90:10:1; 60:10:1; 40:10:1; 20:10:1, and 0:100:0) to produce 11 fractions (A-K). Fraction C was purified by ODS silica gel CC eluted with CH3CN–H2O (1:4; 1:3; 1:2; and 2:3) and MeOH–H2O (2:3), and silica gel CC eluted with hexane-
4.3. Extraction and isolation Fresh whole plants of H. foetidus (3.3 kg) were extracted twice with MeOH (10 L). After concentrating the MeOH extract under reduced pressure, the viscous concentrate (115 g) was passed through a Diaion
Fig. 5. Compound 2 induced apoptosis through a mitochondria-dependent pathway. A, Mitochondrial membrane morphology of HL-60 cells treated with 2. HL-60 cells were stained with MitoCapture™ reagent after treatment with either 0.5 μM of 2 or 33 μM of cisplatin for 10 h and observed under a fluorescence microscope. B, Release of cytochrome c from mitochondria of HL-60 cells treated with 2. HL-60 cells were treated with either 0.5 μM of 2 or 33 μM of cisplatin for 6 h, and the release of cytochrome c into the cytoplasm was evaluated by Western blot analysis.
6
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Me2CO (1:1; 1:2; and 1:3) to give 3 (3.0 mg). Fraction E was purified by silica gel CC eluted with EtOAc-MeOH-H2O (90:10:1; 60:10:1; 40:10:1; and 20:10:1) and hexane-EtOAc (1:2; 1:4; and 1:9) and ODS silica gel CC eluted with CH3CN–H2O (1:4) to give 4 (3.3 mg). Fraction F was purified by ODS silica gel CC eluted with CH3CN–H2O (1:4; 2:7; 1:3; 1:2; and 2:3), silica gel CC eluted with EtOAc-MeOH-H2O (190:10:1; 90:10:1; and 60:10:1), and preparative HPLC using MeOH–H2O (1:1) and CH3CN–H2O (1:4) as the mobile phase to give 1 (5.3 mg) and 5 (10.0 mg). The MeOH eluate (10 g) was subjected to silica gel CC eluted with a stepwise gradient of CHCl3–MeOH–H2O (90:10:1; 40:10:1; 20:10:1, and 0:100:0) to produce 17 fractions (a–q). Fraction c was purified by ODS silica gel CC eluted with MeOH–H2O (1:1; 3:2; and 4:1) and MeCN–H2O (1:4) and silica gel CC eluted with CHCl3–MeOH–H2O (190:10:1) to give 6 (3.9 mg). Fraction i was purified by ODS silica gel CC eluted with MeCN–H2O (1:4) and MeOH–H2O (2:3) to give 2 (18.2 mg).
column eluted with MeOH–H2O (3:2) followed by MeOH to yield the sugar fractions (0.26 mg from 1 to 0.47 mg from 2). The aglycones of both compounds decomposed under acidic conditions and could not be obtained. All the sugar fractions of 1 and 2 were analyzed using HPLC under following conditions: column: Capcell Pak NH2 UG80 (4.6 mm i.d. x 250 mm, 5 μm, Shiseido, Tokyo, Japan); solvent: MeCN–H2O (17:3); detection: refractive index (RI) and optical rotation (OR); flow rate: 1.0 mL/min. HPLC analysis of the sugar fraction showed the presence of D-glucose (tR = 14.9 min, positive optical rotation). 4.5. Cell culture assay HL-60, A549, and TIG-3 cells were maintained in RPMI 1640 medium (HL-60 cells) or MEM (A549 and TIG-3 cells) containing 10% (v/v) heat-inactivated FBS supplemented with L-glutamine, 100 unit/ mL penicillin G sodium salt, and 100 μg/mL streptomycin sulfate. The cells were stored in a humidified incubator at 37 °C with 5% CO2.
4.3.1. 5β,14β,16β,21-tetrahydroxychola-3,20,22-triene-19,24-dioic acid 24,21-lactone 19-β-D-glucopyranosyl ester (1) Amorphous solid; [α]25 D +74.1 (c 0.10, MeOH); UV (MeOH) λmax 296 (log ε 3.61), 206 (log ε 3.75); IR (film) νmax 3383 (OH), 2925 (CH), 1713 and 1644 (C]O) cm−1; 1H and 13C NMR data see Tables 1 and 2; HRESITOFMS m/z 615.2418 [M+Na]+ (calcd for C30H40O12Na, 615.2417).
4.6. Assay for cytotoxic activity The in vitro cytotoxic activities of the test compounds against HL-60 and A549 tumor cells and TIG-3 normal cells were established using an MTT reduction assay, as previously described (Yokosuka et al., 2014). 4.7. Assay for Na+/K+ ATPase inhibitory activity
4.3.2. 16β-formyloxy-10β,14β-dihydroxy-5β-[(β-D-glucopyranosyl)oxy]19-norbufa-3,20,22- trienolide (2) Amorphous solid; [α]25 D +25.1 (c 0.10, MeOH); UV (MeOH) λmax 298 (log ε 3.35), 204 (log ε 3.68); IR (film) νmax 3403 (OH), 2924 (CH), 1698 and 1630 (C]O) cm−1; 1H and 13C NMR data see Tables 1 and 2; HRESITOFMS m/z 615.2420 [M + Na]+ (calcd for C30H40O12Na, 615.2417).
The Na+/K+ ATPase activities were determined by measuring the amount of inorganic phosphate (Pi) liberated from ATP. Briefly, commercially available Na+/K+ ATPase from the porcine cerebral cortex (EC 3.6.1.7) (Sigma-Aldrich) was incorporated into a reaction mixture containing 24 mM Tris-HCl (pH 7.3, 37 °C), 0.68 mM EDTA, 6.0 mM MgCl2, 2.0 M NaCl, and 45 mM KCl in a final volume of 145 μL. After 10 min of preincubation at 37 °C, the reaction was initiated by the addition of 5 μL of 80 mM ATP and the mixture was incubated at 37 °C for 30 min. Controls were carried out under the same conditions with the addition of 1.0 mM ouabain. After incubation, the reaction solution was diluted with deionized water and the working color solution provided in the Phosphor C Test Kit (Wako Pure Chemical Industries, Osaka, Japan) was added. Then, the color intensity of the mixed solution was measured at 750 nm on SH-1300 microplate reader (Corona Electric, Ibaraki, Japan).
4.3.3. 3β,11α,14β,16β-tetrahydroxy-19-oxobufa-4,20,22-trienolide (3) Amorphous solid; [α]25 D +80.9 (c 0.025, MeOH); UV (MeOH) λmax 298 (log ε 3.41), 205 (log ε 3.93); IR (film) νmax 3384 (OH), 2925 (CH), 1705 and 1654 (C]O) cm−1; 1H and 13C NMR data see Tables 1 and 2; HRESITOFMS m/z 453.1898 [M + Na]+ (calcd for C24H30O7Na, 453.1889). 4.3.4. 3β,11α,14β-trihydroxy-19-oxobufa-4,20,22-trienolide (4) Amorphous solid; [α]25 D +62.1 (c 0.025, MeOH); UV (MeOH) λmax 298 (log ε 3.65), 205 (log ε 4.14); IR (film) νmax 3392 (OH), 2924 (CH), 1711 and 1648 (C]O) cm−1; 1H and 13C NMR data see Tables 1 and 2; HRESITOFMS m/z 437.1949 [M + Na]+ (calcd for C24H30O6Na, 437.1940).
4.8. Assay for apoptosis inducing activity The morphological observations with DAPI staining and cell cycle analysis as well as the detection of DNA fragmentation, cell cycle analysis, activation of caspase-3, disruption of mitochondrial membrane potential (ΔΨm), and release of cytochrome c into the cytosol were performed as previously described (Yokosuka et al., 2018).
4.3.5. 3β,10β,14β,16β-tetrahydroxy-19-norbufa-4,20,22-trienolide (5) Amorphous solid; [α]25 D +30.1 (c 0.10, MeOH); UV (MeOH) λmax 296 (log ε 3.57), 205 (log ε 3.89); IR (film) νmax 3395 (OH), 2936 (CH), 1704 (C]O) cm−1; 11H and 13C NMR data see Tables 1 and 2; HRESITOFMS m/z 425.1940 [M + Na]+ (calcd for C23H30O6Na, 425.1940).
4.9. Statistical analysis For statistical analysis, one-way analysis of variance (ANOVA) followed by Dunnett's test was performed. A probability (p) value of less than 0.001 was considered to represent a statistically significant difference.
4.3.6. 3β-acetoxy-1β,5β,14β,16β-tetrahydroxybufa-20,22-dienolide (6) Amorphous solid; [α]25 D +34.2 (c 0.05, MeOH); UV (MeOH) λmax 294 (log ε 3.57), 204 (log ε 3.86); IR (film) νmax 3388 (OH), 2923 (CH), 1709 and 1629 (C]O) cm−1; 1H and 13C NMR data see Tables 1 and 2; HRESITOFMS m/z 499.2304 [M + Na]+ (calcd for C26H36O8Na, 499.2308).
Declaration of competing interest The authors declare no conflict of interest associated with this manuscript.
4.4. Acid hydrolysis of 1 and 2
Acknowledgements
Compounds 1 (1.2 mg) and 2 (2.2 mg) were independently treated with 0.5 M HCl in dioxane-H2O (1:1, 2.0 mL) at 95 °C for 1 h. The crude hydrolysate was neutralized by passage through an Amberlite IRA-96SB (Organo, Tokyo, Japan) column and subjected to a Diaion HP-20
This work was financially supported in part by the Japan Society for the Promotion of Sciences (JSPS) KAKENHI Grant Numbers JP26860069 and JP18K06735. 7
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Appendix A. Supplementary data
Prieto, J.M., Siciliano, T., Braca, A., 2006. A new acylated quercetin glycoside and other secondary metabolites from Helleborus foetidus. Fitoterapia 77, 203–207. Robien, W., Kopp, B., Schabl, D., Schwarz, H., 1987. Carbon-13 NMR spectroscopy of cardenolides and bufadienolides. Prog. Nucl. Magn. Reson. Spectrosc. 19, 131–181. Sargent, J.M., Taylor, C.G., 1989. Appraisal of the MTT assay as a rapid test of chemosensitivity in acute myeloid leukaemia. Br. J. Canc. 60, 206–210. Shi, L.S., Liao, Y.R., Su, M.J., Lee, A.S., Kuo, P.C., Damu, A.G., Kuo, S.C., Sun, H.D., Lee, K.H., Wu, T.S., 2010. Cardiac glycosides from Antiaris toxicaria with potent cardiotonic activity. J. Nat. Prod. 73, 1214–1222. Tsukamoto, Y. (Ed.), 1989. The Grand Dictionary of Horticulture, vol. 4. Shogakukan, Tokyo, pp. 387–389. Yokosuka, A., Suzuki, T., Tatsuno, S., Mimaki, Y., 2014. Steroidal glycosides from the underground parts of Yucca glauca and their cytotoxic activities. Phytochemistry 101, 109–115. Yokosuka, A., Iguchi, T., Kawahata, R., Mimaki, Y., 2018. Cytotoxic bufadienolides from the whole plants of Helleborus foetidus. Phytochem. Lett. 23, 94–99. Zeino, M., Brenk, R., Gruber, L., Zehl, M., Urban, E., Kopp, B., Efferth, T.J., 2015. Cytotoxicity of cardiotonic steroids in sensitive and multidrug-resistant leukemia cells and the link with Na+/K+-ATPase. Steroid Biochem. Mol. Biol. 150, 97–111.
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.phytochem.2020.112277. References Gao, H., Popescu, R., Kopp, B., Wang, Z., 2011. Bufadienolides and their antitumor activity. Nat. Prod. Rep. 28, 953–969. Kamano, Y., Kotake, A., Hashima, H., Inoue, M., Morita, H., Takeya, K., Itokawa, H., Nandachi, N., Segawa, T., Yukita, A., Saitou, K., Katsuyama, M., Pettit, G., 1998. Structure-cytotoxic activity relationship for the toad poison bufadienolides. Bioorg. Med. Chem. 6, 1103–1115. Meng, Y., Whiting, P., Sik, V., Rees, H.H., Dinan, L., 2001. Ecdysteroids and bufadienolides from Helleborus torquatus (Ranunculaceae). Phytochemistry 57, 401–407. Nogawa, T., Kamano, Y., Yamashita, A., Petit, R.G., 2001. Isolation and structure of five new cancer cell growth inhibitory bufadienolides from the Chinese traditional drug Ch'an Su. J. Nat. Prod. 64, 1148–1152.
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Bufadienolides from the whole plants of Helleborus foetidus and their cytotoxicity
T
Tomoki Iguchi, Akihito Yokosuka∗, Riko Kawahata, Madoka Andou, Yoshihiro Mimaki Department of Medicinal Pharmacognosy, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo, 192-0392, Japan
ARTICLE INFO
ABSTRACT
Keywords: Helleborus foetidus Ranunculaceae Bufadienolides Bufadienolide glucosides Cytotoxicity Apoptosis HL-60 cells A549 cells
Two undescribed bufadienolide glucosides and four undescribed bufadienolides were isolated from the whole plants of Helleborus foetidus (Ranunculaceae). Their structures were determined by extensive spectroscopic analysis and the results of hydrolytic cleavage. The isolated compounds exhibited cytotoxic activities against HL60 and A549 cells with IC50 values ranging from 0.019 to 3.0 μM. The isolated compounds also showed the Na+/ K+ ATPase inhibitory activity. The undescribed compound 16β-formyloxy-10,14-dihydroxy-5β-[(β-D-glucopyranosyl)oxy]-19-norbufa-3,20,22-trienolide induced apoptosis in HL-60 cells through a mitochondria-dependent apoptotic pathway.
1. Introduction
2. Results and discussion
Helleborus foetidus L., native to Europe, is an evergreen perennial plant belonging to the family of Ranunculaceae (Tsukamoto, 1989). Anemonin, a quercetin glycoside, a furostanol glycoside, and a few phenolic glucosides have been reported as chemical components of H. foetidus (Prieto et al., 2006). Previously, a cytotoxicity-guided fractionation of a MeOH extract of H. foetidus resulted in the isolation of five bufadienolide derivatives with potent cytotoxicities against HL-60 human leukemia cells (Yokosuka et al., 2018). Bufadienolides represent a type of steroid with a 2-pyrone ring at the C-17 position of the skeleton and show various activities, such as cardiotonic, blood pressure stimulating, local anaesthetic, and cytotoxic activity against cultured tumor cells (Kamano et al., 1998; Nogawa et al., 2001). Although the isolation of many bufadienolides were from animal sources such as a toad venom, some are also distributed in plants such as Helleborus, Drimia, Kalanchoe, and Urginea species (Gao et al., 2011). Ongoing phytochemical studies on the cytotoxic steroidal constituents of H. foetidus led to the isolation of two undescribed bufadienolide glucosides (1 and 2) and four undescribed bufadienolides (3–6). This paper reports the isolation and structure determination of 1–6 and their cytotoxic activities against HL-60 human leukemia cells, A549 human lung adenocarcinoma cells, and TIG-3 human normal diploid lung cells. The Na+/K+ ATPase inhibitory activity of 1–5 was also evaluated. Furthermore, the ability of undescribed bufadienolide glucoside 2 to induce apoptosis in HL-60 cells was investigated.
The fresh whole plants of H. foetidus (3.3 kg) were extracted twice with MeOH (10 L). The fractions eluted with 50% MeOH (5.0 g) and pure MeOH (115 g) were subjected to column chromatography (CC) over Diaion HP-20, silica gel, and ODS silica gel as well as preparative HPLC to give six compounds (1–6) (see Fig. 1). Compound 1 was obtained as an amorphous solid with a molecular formula of C30H40O12, which was elucidated based on its HRESITOFMS (m/z 615.2418 [M + Na]+, calcd for C30H40O12Na: 615.2417) and 13C NMR data (30 carbon signals). The IR spectrum of 1 suggested the presence of hydroxy groups (3383 cm−1) and carbonyl groups (1713 and 1644 cm−1). The UV spectrum showed the absorption maxima indicative of a conjugated system (296 and 206 nm). The 1H NMR spectrum of 1 contained signals for a 2-pyrone ring [δH 8.11 (1H, dd, J = 9.7, 2.5 Hz, H-22), 7.43 (1H, dd, J = 2.5, 0.8 Hz, H-21), and 6.18 (1H, dd, J = 9.7, 0.8 Hz, H-23)], which are characteristic of bufadienolide derivatives, an (Z)-olefinic group [δH 5.91 (1H, m, H-3) and 5.43 (1H, d, J = 9.9 Hz, H-4)], an oxymethine proton [δH 4.46 (1H, ddd, J = 7.7, 7.7, 0.8 Hz, H-16)], an angular methyl group [δH 0.79 (3H, s, Me-18)], and an anomeric proton [5.59 (1H, d, J = 8.0 Hz, H-1’)] (Table 1). The 13C NMR spectrum of 1 displayed signals for a 2-pyrone ring [δC 120.4, 151.7, 152.9, 112.9, and 165.1 (C-20–C-24)], an ester carbonyl carbon [δC 174.3 (C-19)], an olefin [δC 131.1 (C-3) and 134.1 (C-4)], an oxymethine carbon [δC 73.5 (C-16)], two quaternary carbons bearing oxygen atoms [δC 86.8 (C-14) and 71.7 (C-5)], two quaternary
∗
Corresponding author. E-mail address: [email protected] (A. Yokosuka).
https://doi.org/10.1016/j.phytochem.2020.112277 Received 7 October 2019; Received in revised form 14 January 2020; Accepted 14 January 2020 0031-9422/ © 2020 Elsevier Ltd. All rights reserved.
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Table 1 1 H NMR Data for 1–6 (CD3OD, 600 MHz). No.
1
1α β 2α β 3 4α β 5 6α β 7α β 8 9 10 11 α β 12 α β 13 14 15 α β 16 α β 17 18 19 20 21 22 23 24 COH Ac Glc1′ 2′ 3′ 4′ 5′ 6′ a b
2.09 2.04 2.08 2.03 5.91 5.43 – 2.50 1.62 2.08 1.02 2.18 1.39 – 1.52 1.21 1.56 1.34 – – 2.33 1.67 4.46
2 m m m m m d (9.9) m m m m ddd (12.0, 12.0, 3.1) m m m ddd (13.3, 13.3, 2.8) ddd (13.3, 2.8, 2.8) dd (14.7, 7.7) dd (14.7, 0.8) ddd (7.7, 7.7, 0.8)
2.73 0.79 – – 7.43 8.11 6.18 –
d (7.7) s
5.59 3.41 3.37 3.36 3.42 3.79 3.67
d (8.0) m m m m dd (11.8, 1.4) m
dd (2.5, 0.8) dd (9.7, 2.5) dd (9.7, 0.8)
2.17 1.71 2.21 2.12 5.89 5.63 – 2.03 1.90 2.07 1.04 1.77 1.47 – 1.65 1.55 1.64 1.45 – – 2.52 1.80 5.62
3 m m m m br d (9.9) d (9.9) m m m m ddd (11.7, 11.7, 2.6) m m m m m dd (12.4, 7.0) br d (12.4) m
3.02 0.86 – – 7.49 8.27 6.24 – 7.91
d (7.1) s
4.68 3.23 3.39 3.32 3.25 3.84 3.68
d (7.8) dd (8.9, 7.8) dd (8.9, 8.9) dd (8.9, 8.9) m dd (11.9, 2.4) dd (11.9, 5.6)
br d (2.3) dd (9.8, 2.3) br d (9.8) s
2.93 1.65 1.91 1.31 4.11 5.71 – 2.25 2.25 2.25 1.12 1.90 1.34 – 3.94
4 m m m m br dd (9.3, 5.7) br s
3.00 1.65 1.92 1.35 4.10 5.70
m m m m ddd (12.1, 12.1, 2.6) dd (12.1, 10.1) ddd (10.1, 10.1, 4.1)
1.72 1.43 – – 2.43 1.71 4.48
dd (13.5, 4.1) dd (13.5,10.1)
2.85 0.84 9.81 – 7.48 8.08 6.19 –
d (72) s s
dd (14.8, 7.3) dd (14.8, 1.2) ddd (7.2, 7.2, 1.2)
dd (2.5, 0.8) dd (9.7, 2.5) dd (9.7, 0.8)
– 2.26 2.26 2.20 1.12 1.92 1.40 – 3.94 1.70 1.48 – – 2.03 1.34 2.19 1.76 2.59 0.76 9.83 – 7.44 7.94 6.28 –
5 m m m m br dd (9.3, 5.7) br s m m m m ddd (12.0, 12.0, 2.8) m m m m ddd (11.0,11.0, 3.0) m m m dd (9.7, 7.0) s s dd (2.5, 0.8) dd (9.7, 2.5) dd (9.7, 0.8)
1.96 1.40 1.83 1.57 4.04 5.44 – 2.43 2.12 2.18 1.09 1.83 1.09 – 1.55 1.55 1.60 1.40 – – 2.38 1.71 4.46 2.75 0.83 – – 7.44 8.12 6.19 –
6 m m m m m d (1.4) m m m m ddd (12.0, 12.0, 3.0) m m m ddd (13.6, 6.1, 3.1) ddd (13.6, 13.6, 3.1) dd (14.8, 7.6) dd (14.8, 0.8) ddd (7.6, 7.6, 0.8) d (7.6) s dd (2.5, 0.8) dd (9.7, 2.5) dd (9.7, 0.8)
3.93 m 2.10 2.10 5.25 2.49 1.76 – 1.79 1.39 2.08 1.29 1.73 1.49 – 1.38 1.38 1.59 1.38 – – 2.57 1.81 4.54
m m br s dd (15.8, 4.6) m
2.80 0.83 1.24 – 7.48 8.16 6.23 –
d (7.7) s s
m m m m ddd (12.1, 12.1, 3.9) m m m m m dd (14.8, 7.7) dd (14.8, 1.0) ddd (7.7, 7.7, 1.0)
dd (2.4, 0.8) dd (9.8, 2.4) dd (9.8, 0.8)
2.05 s
carbons [δC 55.5 (C-10) and 50.5 (C-13)], three methine carbons [δC 59.2 (C-17), 41.4 (C-8), and 39.5 (C-9)], seven methylene carbons [δC 42.3 (C-15), 42.1 (C-12), 37.6 (C-6), 24.7 (C-7), 24.6 (C-11), 23.7 (C-1), and 22.8 (C-2)], a methyl carbon [δC 17.4 (C-18)], and a β-D-glucopyranosyl group [δC 95.9, 74.1, 78.9, 71.0, 78.0, and 62.3] (Table 2). Acid hydrolysis of 1 resulted in the production of D-glucose, whereas the aglycone decomposed under acidic conditions and could not be obtained. Analysis of the 1H–1H COSY and HSQC spectra of 1 indicated that the aglycone moiety of 1 was composed of four spin-coupling systems, corresponding to the following structural fragments: A: H2-1/ H2-2/H-3/H-4, B: H2-6/H2-7/H-8/H-9/H2-11/H2-12, C: H2-15/H-16/H17, and D: H-22/H-23 (Fig. 2). The long-range HMBC correlations from H-1α at δH 2.09, H-1β at δH 2.04, and H-9 at δH 1.39 to C-19 at δC 174.3; from H-1α, H-1β, H-2α at δH 2.08, H-2β at δH 2.03, H-4 at δH 5.43, H-6α at δH 2.50, H-6β at δH 1.62, and H-9 to C-10 at δC 55.5; and from H-3 at δH 5.91, H-4, H-6α, H-6β, H-7α at δH 2.08, and H-7β at δH 1.02 to C-5 at δC 71.7 indicated structural fragments A and B were linked through the C-5 oxygenated quaternary carbon and the C-10 quaternary carbon as well as the presence of a double-bond between C3 and C-4 and an ester carbonyl (C-19) connected to C-10. The threeproton singlet at δH 0.79 assignable to Me-18 showed HMBC cross peaks with δC 42.1 (C-12), 50.5 (C-13), 86.8 (C-14), and 59.2 (C-17), which implied that structural fragment B was linked to C through the C-13 quaternary carbon and the C-14 oxygenated quaternary carbon. The 2pyrone ring was attached to C-17 based on the long-range HMBC correlations from δH 2.73 (H-17) to δC 120.4 (C-20), 151.7 (C-21), and
152.9 (C-22) (Fig. 2). Thus, the aglycone of 1 was revealed to a bufadienolide derivative with an ester carbonyl group at C-19, an (Z)-olefinic group at C-3/C-4, and three hydroxy groups at C-5, C-14, and C-16 (Robien et al., 1987; Shi et al., 2010; Yokosuka et al., 2018). The linkage of the β-D-glucopyranosyl moiety to C-19 through an oxygen atom was confirmed by the 3JC,H HMBC correlation between the signals of the α-anomeric proton at δH 5.59 (J = 8.0 Hz) and the C-19 ester carbonyl carbon at δC 174.3. The NOE correlations between H-4 and H6α, H-2α and H-9, H-12α and H-16/H-17, and Me-18 and H-8/H-11β observed in the phase-sensitive NOESY spectrum (Fig. 3) and the proton spin-coupling constants between H-8 and H-9 (J = 12.0 Hz) (Table 1) indicated that 1 had A/B cis, B/C trans, and C/D cis ring fusions and the 5β, 14β, 16β, and 17β configurations. Therefore, the structure of 1 was determined to be 5β,14β,16β,21-tetrahydroxychola-3,20,22-triene19,24-dioic acid 24,21-lactone 19-β-D-glucopyranosyl ester. Compound 1 is the first bufadienolide derivative in which the C-19 methyl group is oxidized to the carboxylic acid and forms an ester glucosidic linkage. Compound 2 was obtained as an amorphous solid, and its molecular formula was determined to be C30H40O12 based on its HRESITOFMS (m/z 615.2420 [M +Na]+) and 13C NMR data. The NMR spectral features of 2 were closely related to those of 10,14,16β-trihydroxy-5β[(β-D-glucopyranosyl)oxy]-19-norbufa-3,20,22-trienolide (hellebortin A), which was isolated from H. foetidus (Meng et al., 2001; Yokosuka et al., 2018). However, the molecular formula of 2 was larger than that of hellebortin A by CO. The IR (1698 cm−1), 1H NMR [δ 7.91 (1H, s)], and 13C NMR [δ 162.0 (C]O)] data suggested the presence of a 2
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δC 75.2 (Fig. 2). Furthermore, an HMBC cross peak was observed between H-1’ of the β-D-glucopyranosyl group at δH 4.68 (d, J = 7.8 Hz) and C-5 of the aglycone at δC 79.2. Accordingly, the structure of 2 was established as 16β-formyloxy-10,14-dihydroxy-5β-[(β-D-glucopyranosyl)oxy]-19-norbufa-3,20,22-trienolide. The bufadienolide derivative with a formyloxy group at C-3 was isolated from toads native to China (Nogawa et al., 2001). Compound 2 is the first bufadienolide glucoside with a formyloxy group isolated from plant materials. The 1H and 13C NMR spectroscopic data of 3 (C24H30O7) were similar to those of 5β,11α,14β,16β-tetrahydroxy-19-oxobufa-3,20,22trienolide (C24H30O7), which has been isolated from H. foetidus (Yokosuka et al., 2018); however, they differed in the 1H and 13C NMR signals attributable to the A ring (C-1–C-5 and C-10). The signal for the hydroxy-substituted quaternary carbon at δC 73.5 (C-5) was not observed in the data of 3, and instead, signals assignable to an oxymethine unit [δH 4.11 (br dd, J = 9.3, 5.7 Hz, H-3); δC 67.1] were observed. The oxymethine proton at δH 4.11 showed spin-coupling correlations with the olefinic proton at δH 5.71 (br s, H-4) and with the pair of methylene protons at δH 1.91 and 1.31 (each m, H2-2) in the 1H–1H COSY spectrum of 3. The methylene protons exhibited correlations with the terminal methylene protons at δH 2.93 and 1.65 (each m, H2-1). These correlations led us to propose the A ring of 3 to be –C(1)H2–C(2)H2–C(3)(OH)–C(4)H=. Long-range HMBC correlations from the olefinic proton at δH 5.71 (H-4) to the carbons at δC 55.2 (C-10) and 34.8 (C-6) and from the aldehyde proton at δH 9.81 (H-19) to the carbons at δC 32.2 (C-1), 55.6 (C-9), and 55.2 (C-10) were detected (Fig. 2). The B/C trans and C/D cis ring fusions of the bufadienolide skeleton and the C-3β, C-11α, C-16β, and C-17β configurations were elucidated from the NOE correlations in the NOESY spectrum of 3 (Fig. 3) and the proton spin-coupling constants (Table 1). Accordingly, the structure of 3 was formulated as 3β,11α,14β,16β-tetrahydroxy-19oxobufa-4,20,22-trienolide. Compound 4 (C24H30O6) was obtained as an amorphous solid. The 1 H and 13C NMR spectra of 4 were essentially analogous to those of 3. The molecular formula of 4 includes one fewer oxygen atom than that of 3. Comparing the 1H and 13C NMR spectra of 4 with those of 3
Table 2 13 C NMR data for 1–6 (CD3OD, 125 MHz).
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 1' 2' 3' 4' 5' 6' 3-OAc 16-COH
1
2
3
4
5
6
23.7 22.8 131.1 134.1 71.7 37.6 24.7 41.4 39.5 55.5 24.6 42.1 50.5 86.8 42.3 73.5 59.2 17.4 174.3 120.4 151.7 152.9 112.9 165.1 95.9 74.1 78.9 71.0 78.0 62.3
27.8 25.9 132.4 132.9 79.2 32.2 24.4 41.4 41.3 74.8 22.4 41.2 50.7 84.5 41.0 75.2 57.8 17.0 – 119.3 152.7 152.0 113.3 164.5 98.4 75.4 78.3 71.7 77.6 62.8
32.2 30.3 67.1 131.4 139.2 34.8 29.5 44.0 55.6 55.2 68.9 51.1 50.5 84.7 42.7 73.5 58.4 18.9 205.3 120.0 151.9 152.4 113.1 164.9
32.5 30.4 67.1 131.4 139.2 35.0 29.4 44.1 55.7 55.3 69.2 51.7 50.1 84.9 32.7 29.7 51.8 18.2 205.4 124.4 150.7 149.1 115.5 164.7
34.9 29.2 68.4 128.3 142.8 32.7 29.3 42.6 51.1 70.9 21.2 41.7 50.2 85.5 43.0 73.5 59.4 17.2 – 120.5 151.8 152.9 112.9 165.1
74.3 32.2 71.3 36.8 75.6 36.3 25.1 42.2 42.0 44.1 22.8 41.9 50.5 85.9 43.4 73.5 59.7 17.5 13.5 120.7 152.1 153.2 113.2 165.3
162.0
21.9 172.9
formyloxy group in 2. The HMBC spectrum of 2 showed cross peaks between H-16 at δH 5.62 (1H, m) and the carbonyl carbon of the formyl group at δC 162.0 and between the formyl proton at δH 7.91 and C-16 at
Fig. 1. Chemical structures of 1–6. 3
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Fig. 2. Key HMBC correlations of 1–3, and 6. The bold lines indicate the 1H–1H coupling, and arrows indicate 1H/13C long-range correlations.
Fig. 3. Key NOE correlations of 1–3, and 6.
showed that the C-16 hydroxymethine proton and carbon signals (δH 4.48/δC 73.5) in 3 were replaced by the methylene proton and carbon signals [δH 2.19 (1H, m) and 1.76 (1H, m)/δC 29.7]. All other signals were observed at almost the same positions in the spectra of these two compounds. Thus, the structure of 4 was elucidated as 3β,11α,14βtrihydroxy-19-oxobufa-4,20,22-trienolide. The 1H and 13C NMR spectral data of 5 (C23H30O6) were also similar to those of 3. However, the signals corresponding to the aldehyde group at C-19 were not observed in the 1H and 13C NMR spectra of 5, and the signals of quaternary carbon C-10 (δC 55.2) and the hydroxymethine proton and carbon signals for C-11 [δH 3.94 (ddd, J = 10.1, 10.1, 4.1 Hz)/δC 68.9] in 3 were replaced by a signals for quaternary carbon
bearing an oxygen atom (δC 70.9) and methylene proton and carbon signals [δH 1.55 (2H, m)/δC 21.2], respectively, in 5. Thus, the structure of 5 was assigned as 3β,10β,14β,16β-tetrahydroxy-19-norbufa-4,20,22trienolide. Compound 6 (C26H36O8) showed spectral features characteristic of a polyhydroxylated bufadienolide derivative. The 1H and 13C NMR spectra of 6 suggested the presence of two angular methyl groups [δH 1.24 (3H, s)/δC 13.5 and δH 0.83 (3H, s)/δC 17.5], three oxymethine units [δH 5.25 (br s)/δC 71.3, δH 4.54 (ddd, J = 7.7, 7.7, 1.0 Hz)/δC 73.5, and δH 3.93 (m)/δC 74.3], two quaternary carbons bearing oxygen atoms [δC 85.9 and 75.6], and an acetyl group [δH 2.05 (3H, s)/δC 172.9 (C]O) and 21.9 (Me)]. The methyl signal at δH 0.83 exhibited 4
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long-range HMBC correlations with the carbon signals at δC 42.0 (C12), 50.5 (C-13), 85.9 (C-14), and 59.7 (C-17) and was attributed to Me18. Another methyl signal at δH 1.24, attributable to Me-19, displayed long-range HMBC correlations with the carbon signals at δC 74.3 (C-1), 75.6 (C-5), 41.9 (C-9), and 44.1 (C-10). In the 1H–1H COSY spectrum, the oxymethine proton at δH 5.25 showed spin-coupling correlations with the adjacent methylene groups at δH 2.10 (2H, m, H2-2) and δH 2.49 (1H, dd, J = 15.8, 4.6 Hz, H-4a) and 1.76 (1H, m, H-4b), and assigned as H-3. The other oxymethine proton at δH 4.54 had spincouplings with the methylene group at δH 2.57 (dd, J = 14.8, 7.7 Hz, H15α) and 1.81 (dd, J = 14.8, 1.0 Hz, H-15β), and the methine group at δH 2.80 (d, J = 7.7 Hz, H-17) and was therefore attributed to H-16. A 3 JC,H HMBC correlation was observed between H-3 and the acetyl carbonyl carbon. The above spectroscopic data were indicative of the presence of hydroxy groups at C-1, C-3, C-5, C-14, and C-16, and the C-3 hydroxy group was acetylated. The A/B cis, B/C trans, and C/D cis ring fusions and the 1β, 3β, 5β, 14β, 16β, and 17β configurations were elucidated from NOESY spectrum of 6 (Fig. 3) and the proton spincoupling constants (Table 1). Thus, the structure of 6 was determined to be 3β-acetoxy-1β,5β,14β,16β-tetrahydroxybufa-20,22-dienolide. Compounds 1–6 were evaluated for their in vitro cytotoxic activities against HL-60 and A549 tumor cells and TIG-3 normal cells using a modified MTT assay (Sargent and Taylor, 1989). Although 1–6 showed cytotoxicities against mot only HL-60 and A549 tumor cells but also TIG normal cells, 2 and 4 were much more cytotoxic than the positive controls, etoposide and cisplatin, to both HL-60 and A549 cells, and they showed IC50 values ranging from 0.037 to 0.098 μM (Table 3). On the other hand, 1, 3, 5, and 6 showed moderate cytotoxicity with IC50 values ranging from 0.12 to 3.0 μM. Cardiotonic steroids are categorized as cardenolides or bufadienolides, and have been clinically used for the treatment of arrhythmia and heart failure. They have also been reported to exhibit cytotoxicity through Na+/K+ ATPase inhibition resulting in increased intracellular Ca2+ concentrations, which induces apoptotic cell death in tumor cells (Zeino et al., 2015). Then, the Na+/K+ ATPase inhibitory activities of 1–5 were examined. Compounds 1–5 inhibited Na+/K+ ATPase with IC50 values of 0.11, 1.3, 1.7, 0.061, and 0.30 μM, respectively, which were similar to their average cytotoxicities (IC50 values) against HL-60 and A549 cells (Table 4). These data suggested that their cytotoxicities were partially mediated via the inhibition of Na+/K+ ATPase. Compound 2 was an undescribed bufadienolide derivative and showed potent cytotoxicity against HL-60 and A549 cells. The ability of 2 to induce apoptotic cell death in HL-60 cells was evaluated. Morphologically, fragmented and condensed nuclear chromatins, the accumulation of the sub-G1 cells, the induction of DNA fragmentation, and caspase-3 activation were observed in HL-60 cells treated with 2 (Fig. 4). Furthermore, disruption of the mitochondrial membrane potential and release of cytochrome c into the cytoplasm were observed in 2-treated apoptotic HL-60 cells (Fig. 5). The above data implied that 2 induced apoptosis in HL-60 cells through a mitochondria-dependent pathway.
Table 4 Na+/K+ ATPase inhibitory activities of 1–5 and ouabain.
1 2 3 4 5 6 etoposide cisplatin a
A549
TIG-3
0.27 ± 0.011 0.087 ± 0.0018 3.0 ± 0.31 0.044 ± 0.0021 0.30 ± 0.045 1.0 ± 0.016 0.18 ± 0.00084 1.1 ± 0.019
0.20 ± 0.0075 0.098 ± 0.0075 0.96 ± 0.10 0.019 ± 0.0049 0.12 ± 0.027 0.69 ± 0.023 -a 2.3 ± 0.39
0.035 ± 0.00031 0.15 ± 0.0035 1.50 ± 077 0.020 ± 0.0017 0.16 ± 0.035 0.36 ± 0.0058 _ 1.7 ± 0.025
1 2 3 4 5 Ouabain
1.3 ± 0.16 0.11 ± 0.0058 1.7 ± 0.11 0.061 ± 0.0017 0.30 ± 0.0040 0.035 ± 0.0024
In summary, two undescribed cytotoxic bufadienolide glucosides (1 and 2) and four undescribed bufadienolides (3–6) were obtained from H. foetidus. Their structures were determined by extensive spectroscopic analysis and the results of hydrolytic cleavage. Compound 1 is the first bufadienolide derivative in which the C-19 methyl group is oxidized to the carboxylic acid and forms an ester glucosidic linkage. Compound 2 is the first bufadienolide glucoside with a formyloxy group isolated from plant materials. All the isolated compounds were cytotoxic to cancer cells in vitro, which was suggested to be mediated via the inhibition of Na+/K+ ATPase resulting in increased intracellular Ca2+ concentrations. Compound 2, a bufadienolide glucoside with a formyloxy group at C-16 of the aglycone, induced apoptosis in HL-60 cells through a mitochondria-dependent pathway. 4. Experimental section 4.1. General experimental procedures Optical rotations were measured using a JASCO P-1030 (Tokyo, Japan) automatic digital polarimeter. IR spectra were recorded on a JASCO FT-IR 620 spectrophotometer. UV spectra were measured with a JASCO V-630 UV–Vis spectrophotometer (Tokyo, Japan). NMR spectra were recorded on a Bruker DRX-500 (500 MHz for 1H NMR, Karlsruhe, Germany) and a Bruker DRX-600 (600 MHz for 1H NMR) spectrophotometer using standard Bruker pulse programs. Chemical shifts are given as δ values and are referenced to tetramethylsilane (TMS) as an internal standard. HRESI TOF-MS data were recorded on a Micromass LCT mass spectrometer (Manchester, UK). Diaion HP-20 (Mitsubishi Chemical, Tokyo, Japan), silica gel (Fuji-Silysia Chemical, Aichi, Japan), and ODS silica gel (Nacalai Tesque, Kyoto, Japan) were used for column chromatography. TLC was carried out on precoated silica gel 60 F254 (0.25 mm thick, Merck, Darmstadt, Germany) and RP18 F254s plates (0.25 mm thick, Merck), and spots were visualized by spraying the plates with 10% H2SO4 aqueous solution followed by heating. HPLC separations were performed with a system consisting of an LC-20AD pump (Shimadzu, Kyoto, Japan), a RID-10A refractive index (RI) detector (Shimadzu), and a Rheodyne injection port (Rheodyne LLC, Rohnert Park, CA, USA). A TSK gel ODS-100Z column (10 mm i.d. × 250 mm, 5 μm; Tosoh, Tokyo, Japan) was used for preparative HPLC. HL-60 cells (JCRB 0085), A549 cells (JCRB 0076), and TIG3 cells (JCRB 0510) were obtained from the Human Science Research Resources Bank (Osaka, Japan). The following reagents were obtained from the indicated companies: RPMI 1640 medium, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA); penicillin G sodium salt and streptomycin sulfate (Gibco, Grand Island, NY, USA). All other chemicals used were of biochemical reagent grade.
IC50(μM) HL-60
IC50 (μM)
3. Conclusion
Table 3 Cytotoxic activity of the isolated compounds 1–6, etoposide, and cisplatin. Compounds
Compounds
4.2. Plant material Helleborus foetidus L. (Ranunculaceae) were purchased from the garden center of Fujiengei Ltd. (Okayama, Japan) in October 2014. It
Not determined 5
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Fig. 4. Compound 2 induced apoptosis in HL60 cells. A, Morphology of HL-60 cells treated with 2. HL-60 cells were stained with DAPI after treatment with either 0.5 μM of 2 or 15 μM of etoposide for 20 h and then observed under a fluorescence microscope. B, Cell cycle progression of the HL-60 cells treated with 2. HL-60 cells were treated with either 0.5 μM of 2 or 15 μM of etoposide for 10 h, and the cell cycle distribution was analyzed by a flow cytometer. C, Electrophoretic profile of the DNA of HL60 cells treated with 2. HL-60 cells were incubated with either 0.5 μM of 2 or 15 μM of etoposide for 14 h. DNA was then extracted and analyzed by agarose gel electrophoresis. D, Caspase-3 activity in the lysates of HL-60 cells treated with 2. HL-60 cells were incubated with either 0.5 μM of 2 or 15 μM of etoposide for 12 h, and the caspase-3 activity in the lysates was measured using a caspase-3 colorimetric kit. The data are presented as the mean ± S.E.M. of three experiments. Results significantly different from that of the control group are indicated by * (p < 0.001).
was identified by one of the authors, A. Yokosuka. A voucher specimen was deposited in our laboratory (voucher no. KS-2014-0011, Department of Medicinal Pharmacognosy).
HP-20 column and successively eluted with 30% MeOH, 50% MeOH, MeOH, EtOH, and EtOAc (each 6.0 L). The fractions eluted with 50% MeOH and MeOH demonstrated cytotoxicity against HL-60 cells with IC50 values of 0.15 and 0.23 μg/mL, respectively. The 50% MeOH eluate (5.0 g) was subjected to silica gel CC and eluted with stepwise gradient mixtures of EtOAc-MeOH-H2O (90:10:1; 60:10:1; 40:10:1; 20:10:1, and 0:100:0) to produce 11 fractions (A-K). Fraction C was purified by ODS silica gel CC eluted with CH3CN–H2O (1:4; 1:3; 1:2; and 2:3) and MeOH–H2O (2:3), and silica gel CC eluted with hexane-
4.3. Extraction and isolation Fresh whole plants of H. foetidus (3.3 kg) were extracted twice with MeOH (10 L). After concentrating the MeOH extract under reduced pressure, the viscous concentrate (115 g) was passed through a Diaion
Fig. 5. Compound 2 induced apoptosis through a mitochondria-dependent pathway. A, Mitochondrial membrane morphology of HL-60 cells treated with 2. HL-60 cells were stained with MitoCapture™ reagent after treatment with either 0.5 μM of 2 or 33 μM of cisplatin for 10 h and observed under a fluorescence microscope. B, Release of cytochrome c from mitochondria of HL-60 cells treated with 2. HL-60 cells were treated with either 0.5 μM of 2 or 33 μM of cisplatin for 6 h, and the release of cytochrome c into the cytoplasm was evaluated by Western blot analysis.
6
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Me2CO (1:1; 1:2; and 1:3) to give 3 (3.0 mg). Fraction E was purified by silica gel CC eluted with EtOAc-MeOH-H2O (90:10:1; 60:10:1; 40:10:1; and 20:10:1) and hexane-EtOAc (1:2; 1:4; and 1:9) and ODS silica gel CC eluted with CH3CN–H2O (1:4) to give 4 (3.3 mg). Fraction F was purified by ODS silica gel CC eluted with CH3CN–H2O (1:4; 2:7; 1:3; 1:2; and 2:3), silica gel CC eluted with EtOAc-MeOH-H2O (190:10:1; 90:10:1; and 60:10:1), and preparative HPLC using MeOH–H2O (1:1) and CH3CN–H2O (1:4) as the mobile phase to give 1 (5.3 mg) and 5 (10.0 mg). The MeOH eluate (10 g) was subjected to silica gel CC eluted with a stepwise gradient of CHCl3–MeOH–H2O (90:10:1; 40:10:1; 20:10:1, and 0:100:0) to produce 17 fractions (a–q). Fraction c was purified by ODS silica gel CC eluted with MeOH–H2O (1:1; 3:2; and 4:1) and MeCN–H2O (1:4) and silica gel CC eluted with CHCl3–MeOH–H2O (190:10:1) to give 6 (3.9 mg). Fraction i was purified by ODS silica gel CC eluted with MeCN–H2O (1:4) and MeOH–H2O (2:3) to give 2 (18.2 mg).
column eluted with MeOH–H2O (3:2) followed by MeOH to yield the sugar fractions (0.26 mg from 1 to 0.47 mg from 2). The aglycones of both compounds decomposed under acidic conditions and could not be obtained. All the sugar fractions of 1 and 2 were analyzed using HPLC under following conditions: column: Capcell Pak NH2 UG80 (4.6 mm i.d. x 250 mm, 5 μm, Shiseido, Tokyo, Japan); solvent: MeCN–H2O (17:3); detection: refractive index (RI) and optical rotation (OR); flow rate: 1.0 mL/min. HPLC analysis of the sugar fraction showed the presence of D-glucose (tR = 14.9 min, positive optical rotation). 4.5. Cell culture assay HL-60, A549, and TIG-3 cells were maintained in RPMI 1640 medium (HL-60 cells) or MEM (A549 and TIG-3 cells) containing 10% (v/v) heat-inactivated FBS supplemented with L-glutamine, 100 unit/ mL penicillin G sodium salt, and 100 μg/mL streptomycin sulfate. The cells were stored in a humidified incubator at 37 °C with 5% CO2.
4.3.1. 5β,14β,16β,21-tetrahydroxychola-3,20,22-triene-19,24-dioic acid 24,21-lactone 19-β-D-glucopyranosyl ester (1) Amorphous solid; [α]25 D +74.1 (c 0.10, MeOH); UV (MeOH) λmax 296 (log ε 3.61), 206 (log ε 3.75); IR (film) νmax 3383 (OH), 2925 (CH), 1713 and 1644 (C]O) cm−1; 1H and 13C NMR data see Tables 1 and 2; HRESITOFMS m/z 615.2418 [M+Na]+ (calcd for C30H40O12Na, 615.2417).
4.6. Assay for cytotoxic activity The in vitro cytotoxic activities of the test compounds against HL-60 and A549 tumor cells and TIG-3 normal cells were established using an MTT reduction assay, as previously described (Yokosuka et al., 2014). 4.7. Assay for Na+/K+ ATPase inhibitory activity
4.3.2. 16β-formyloxy-10β,14β-dihydroxy-5β-[(β-D-glucopyranosyl)oxy]19-norbufa-3,20,22- trienolide (2) Amorphous solid; [α]25 D +25.1 (c 0.10, MeOH); UV (MeOH) λmax 298 (log ε 3.35), 204 (log ε 3.68); IR (film) νmax 3403 (OH), 2924 (CH), 1698 and 1630 (C]O) cm−1; 1H and 13C NMR data see Tables 1 and 2; HRESITOFMS m/z 615.2420 [M + Na]+ (calcd for C30H40O12Na, 615.2417).
The Na+/K+ ATPase activities were determined by measuring the amount of inorganic phosphate (Pi) liberated from ATP. Briefly, commercially available Na+/K+ ATPase from the porcine cerebral cortex (EC 3.6.1.7) (Sigma-Aldrich) was incorporated into a reaction mixture containing 24 mM Tris-HCl (pH 7.3, 37 °C), 0.68 mM EDTA, 6.0 mM MgCl2, 2.0 M NaCl, and 45 mM KCl in a final volume of 145 μL. After 10 min of preincubation at 37 °C, the reaction was initiated by the addition of 5 μL of 80 mM ATP and the mixture was incubated at 37 °C for 30 min. Controls were carried out under the same conditions with the addition of 1.0 mM ouabain. After incubation, the reaction solution was diluted with deionized water and the working color solution provided in the Phosphor C Test Kit (Wako Pure Chemical Industries, Osaka, Japan) was added. Then, the color intensity of the mixed solution was measured at 750 nm on SH-1300 microplate reader (Corona Electric, Ibaraki, Japan).
4.3.3. 3β,11α,14β,16β-tetrahydroxy-19-oxobufa-4,20,22-trienolide (3) Amorphous solid; [α]25 D +80.9 (c 0.025, MeOH); UV (MeOH) λmax 298 (log ε 3.41), 205 (log ε 3.93); IR (film) νmax 3384 (OH), 2925 (CH), 1705 and 1654 (C]O) cm−1; 1H and 13C NMR data see Tables 1 and 2; HRESITOFMS m/z 453.1898 [M + Na]+ (calcd for C24H30O7Na, 453.1889). 4.3.4. 3β,11α,14β-trihydroxy-19-oxobufa-4,20,22-trienolide (4) Amorphous solid; [α]25 D +62.1 (c 0.025, MeOH); UV (MeOH) λmax 298 (log ε 3.65), 205 (log ε 4.14); IR (film) νmax 3392 (OH), 2924 (CH), 1711 and 1648 (C]O) cm−1; 1H and 13C NMR data see Tables 1 and 2; HRESITOFMS m/z 437.1949 [M + Na]+ (calcd for C24H30O6Na, 437.1940).
4.8. Assay for apoptosis inducing activity The morphological observations with DAPI staining and cell cycle analysis as well as the detection of DNA fragmentation, cell cycle analysis, activation of caspase-3, disruption of mitochondrial membrane potential (ΔΨm), and release of cytochrome c into the cytosol were performed as previously described (Yokosuka et al., 2018).
4.3.5. 3β,10β,14β,16β-tetrahydroxy-19-norbufa-4,20,22-trienolide (5) Amorphous solid; [α]25 D +30.1 (c 0.10, MeOH); UV (MeOH) λmax 296 (log ε 3.57), 205 (log ε 3.89); IR (film) νmax 3395 (OH), 2936 (CH), 1704 (C]O) cm−1; 11H and 13C NMR data see Tables 1 and 2; HRESITOFMS m/z 425.1940 [M + Na]+ (calcd for C23H30O6Na, 425.1940).
4.9. Statistical analysis For statistical analysis, one-way analysis of variance (ANOVA) followed by Dunnett's test was performed. A probability (p) value of less than 0.001 was considered to represent a statistically significant difference.
4.3.6. 3β-acetoxy-1β,5β,14β,16β-tetrahydroxybufa-20,22-dienolide (6) Amorphous solid; [α]25 D +34.2 (c 0.05, MeOH); UV (MeOH) λmax 294 (log ε 3.57), 204 (log ε 3.86); IR (film) νmax 3388 (OH), 2923 (CH), 1709 and 1629 (C]O) cm−1; 1H and 13C NMR data see Tables 1 and 2; HRESITOFMS m/z 499.2304 [M + Na]+ (calcd for C26H36O8Na, 499.2308).
Declaration of competing interest The authors declare no conflict of interest associated with this manuscript.
4.4. Acid hydrolysis of 1 and 2
Acknowledgements
Compounds 1 (1.2 mg) and 2 (2.2 mg) were independently treated with 0.5 M HCl in dioxane-H2O (1:1, 2.0 mL) at 95 °C for 1 h. The crude hydrolysate was neutralized by passage through an Amberlite IRA-96SB (Organo, Tokyo, Japan) column and subjected to a Diaion HP-20
This work was financially supported in part by the Japan Society for the Promotion of Sciences (JSPS) KAKENHI Grant Numbers JP26860069 and JP18K06735. 7
Phytochemistry 172 (2020) 112277
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Appendix A. Supplementary data
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