Cytoprotective dihydronaphthalenones from the wood of Catalpa ovata

Phytochemistry 147 (2018) 14e20

Contents lists available at ScienceDirect

Phytochemistry journal homepage: www.elsevier.com/locate/phytochem

Cytoprotective dihydronaphthalenones from the wood of Catalpa ovata Yun-Seo Kil a, Yang Kang So b, Min Jung Choi a, Ah-Reum Han b, Chang Hyun Jin b, Eun Kyoung Seo a, * a b

Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul, 03760, South Korea Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup-si, Jeollabuk-do, 56212, South Korea

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 July 2017 Received in revised form 24 October 2017 Accepted 14 December 2017

Three previously undescribed dihydronaphthalenones, 7-hydroxycatalponol, (4S)-3,4-dihydro-4hydroxy-2-[(2R)-2,3-dihydroxy-3-methylbutylidene]naphthalen-1(2H)-one, and (6S)-5,6-dihydro-6hydroxy-2,2-dimethyl-2H-benzo[h]chromen-4(3H)-one and one phthalide, (±)-3-(5-hydroxy-5-methyl2-oxohex-3-en-1-yl)isobenzofuran-1(3H)-one, were isolated from the wood of Catalpa ovata G. Don (Bignoniaceae), together with six known compounds. The structures of the previously undescribed compounds were elucidated by interpretation of 1D and 2D NMR data. The absolute configurations of the dihydronaphthalenones were deduced by analysis of the ECD data and application of Mosher ester methodology. All isolates were investigated for their cytoprotective effects against hydrogen peroxide (H2O2)-induced oxidative damage in HepG2 cells. Moreover, the mRNA expression levels of antioxidant enzymes such as heme oxygenase-1 (HO-1) and NAD(P)H:quinine oxidoreductase 1 (NQO1) in HepG2 cells were examined by RT-PCR analysis. As a result, catalponol and epi-catalponol showed antioxidant activities via directly scavenging of intracellular ROS and inducing the antioxidant enzymes in vitro. © 2017 Published by Elsevier Ltd.

Chemical compounds: (2R,3R,4R)-3,4-Dihydro-3,4-dihydroxy-2-(3methyl-2-butenyl)naphthalen-1(2H)-one (PubChem CID: 642916) (2S,3R,4R)-3,4-Dihydro-3,4-dihydroxy-2-(3methyl-2-butenyl)naphthalen-1(2H)-one (PubChem CID: 10562371) Catalponol (PubChem CID: 169570) epi-Catalponol (PubChem CID: 155494) 9-Methoxy-a-lapachone (PubChem CID: 442754) Catalpalactone (PubChem CID: 3014018) Keywords: Catalpa ovata Bignoniaceae Dihydronaphthalenone Phthalide Cytoprotective effects Reactive oxygen species Heme oxygenase-1 NAD(P)H:quinine oxidoreductase 1

1. Introduction Catalpa ovata G. Don is a tall tree, which belongs to the family Bignoniaceae (Inouye et al., 1967). C9-type iridoids are * Corresponding author. E-mail address: [email protected] (E.K. Seo). https://doi.org/10.1016/j.phytochem.2017.12.009 0031-9422/© 2017 Published by Elsevier Ltd.

predominantly found in the stem bark (Young et al., 1992); otherwise, phthalide (Inouye et al., 1967) and naphthoquinone derivatives (Inouye et al., 1971, 1975a, 1975b; Inoue et al., 1979; Fujiwara et al., 1998; Park et al., 2010) have been identified as the major components of the wood. The phthalide and naphthoquinones were shown to suppress the activation of Epstein-Barr virus early antigen (EBV-EA) induced by 12-O-tetradecan-

Y.-S. Kil et al. / Phytochemistry 147 (2018) 14e20

oylphorbol 13-acetate (TPA) in Raji cells (Fujiwara et al., 1998), as well as to inhibit the production of nitric oxide (NO) induced by lipopolysaccharide in RAW 264.7 cells (Park et al., 2010). Hydrogen peroxide (H2O2) is one of the reactive oxygen species (ROS), and the accumulation of metabolically generated ROS in cells can induce oxidative stress which causes damage to proteins, lipids, and DNA (Giorgio et al., 2007). However, H2O2 has been shown to be involved in intracellular signaling at low concentration as well as in host defense and oxidative biosynthesis. For these purposes, H2O2 is generated in a tightly regulated manner (Stone and Yang, 2006). Moreover, antioxidants such as peroxidases and catalases are balanced by H2O2 as its effects obviously differ depending on the concentration in cells (Stone and Yang, 2006; Giorgio et al., 2007). Inflammatory conditions cause overproduction of ROS including H2O2, which can lead to dysregulation of the H2O2 level with loss of molecular functions in cells. In our ongoing study, natural products have been evaluated to discover active compounds that can regulate high-level H2O2 in an unbalanced status. A previous study on C. ovata suggested phthalide and naphthoquinones as promising anti-inflammatory agents based on their inhibitory effects on NO production (Park et al., 2010). The traditional use of C. ovata for treating inflammatory diseases has raised the possibility of finding new active natural products obtained from the medicinal plant. In the present study, three previously undescribed dihydronaphthalenones (1, 6, and 7) and one phthalide (9) were isolated from the wood of C. ovata together with six known compounds (2e5, 8, and 10). All isolates were tested for their cytoprotective effects against H2O2-induced oxidative damage in HepG2 cells. 2. Results and discussion 2.1. Phytochemical investigation Three previously undescribed dihydronaphthalenones, 7-hydroxycatalponol (1), (4S)-3,4-dihydro-4-hydroxy-2-[(2R)-2,3-dihydroxy-3-methylbutylidene]naphthalen-1(2H)-one (6), and (6S)5,6-dihydro-6-hydroxy-2,2-dimethyl-2H-benzo[h]chromen-4(3H)one (7) and one phthalide, (±)-3-(5-hydroxy-5-methyl-2-oxohex3-en-1-yl)isobenzofuran-1(3H)-one (9), were isolated from the wood of C. ovata, together with 6 known compounds: (2R,3R,4R)3,4-dihydro-3,4-dihydroxy-2-(3-methyl-2-butenyl)naphthalen1(2H)-one (2) (Peraza-Sanchez et al., 2000), (2S,3R,4R)-3,4dihydro-3,4-dihydroxy-2-(3-methyl-2-butenyl)naphthalen-1(2H)one (3) (Peraza-Sanchez et al., 2000), catalponol (4) (Inoue et al., 1980), epi-catalponol (5) (Inoue et al., 1980), 9-methoxy-a-lapachone (8) (Inouye et al., 1971), and catalpalactone (10) (Inouye et al., 1965) (Fig. 1). Compounds 2 and 3 were identified from the genus Catalpa for the first time. Compound 1 was isolated as a colorless amorphous solid with a positive specific rotation ([a]25D þ16, c 0.1, MeOH). The molecular formula of 1 was established as C15H18O3 by the HRESIMS ([M þ Na]þ, m/z 269.1148). The 1H and 13C NMR spectra showed resonances for an olefinic methine [dH 5.15 (1H, br t, J ¼ 7.7 Hz)/dC 121.2 (C-12)], an oxygenated methine [dH 4.95 (1H, m)/dC 68.3 (C-4)], an extra methine [dH 2.52 (1H, m)/dC 46.6 (C-2)], two methylenes [dH 2.50 and 1.77 (each 1H, m)/dC 38.9 (C-3), and 2.71 (1H, m, H-11a) and 2.28 (1H, dt, J ¼ 14.6, 7.7 Hz, H-11b)/28.0 (C-11)], and two methyl groups [dH 1.72 (3H, s)/dC 25.9 (C-14) and 1.65 (3H, s)/17.9 (C-15)], along with signals for a ABX system appearing at dH 7.62 (1H, br d, J ¼ 8.4 Hz)/dC 127.7 (C-5), 7.12 (1H, dd, J ¼ 8.4, 2.8 Hz)/ 121.5 (C-6), 7.47 (1H, d, J ¼ 2.8 Hz)/112.5 (C-8), dC 155.3 (C-7), 132.5 (C-9), and 139.1 (C-10) (Table 1). A carbonyl carbon signal was observed at dC 198.6 (C-1). These observations were comparable to those reported for catpalponol (Garcia et al., 2010) except for the

15

Fig. 1. Chemical structures of isolates 1e10 from C. ovata.

presence of the hydroxyl group at C-7. The ambiguous assignment of the 1H and 13C NMR data was confirmed by the important COSY and HMBC correlations including the HMBC correlation from H-5 to C-7 (Fig. 2). The (2R,4S)-configuration was supported by the ECD data showing negative Cotton effects at 260 and 302 nm, and a positive Cotton effects at 341 nm (Anh et al., 1997; Liu et al., 2004; Evidente et al., 2011). Accordingly, the structure of 1 was elucidated as a previously undescribed compound (2R,4S)-3,4-dihydro-4,7dihydroxy-2-(3-methylbut-2-en-1-yl)naphthalen-1(2H)-one, namely 7-hydroxycatalponol. Compound 6 was obtained as a colorless amorphous solid with a molecular formula of C15H18O4 as determined by the HRESIMS ([M þ Na]þ, m/z 285.1100). The 1H and 13C NMR data of 6 exhibited signals for the dihydronaphthalenone skeleton, similar to those of 1 (Table 1). The ABX system in 1 was replaced by a AA0 BB0 system at dH 7.53 (1H, br d, J ¼ 7.6 Hz)/dC 127.2 (C-5), 7.62 (1H, td, J ¼ 7.6, 1.5 Hz)/134.2 (C-6), 7.47 (1H, br td, J ¼ 7.6, 1.2 Hz)/129.0 (C-7), 8.13 (1H, dd, J ¼ 7.6, 1.5 Hz)/128.4 (C-8), dC 131.7 (C-9), and 144.0 (C-10). Instead of the methine proton signal of C-2 in the 1H NMR data of 1, a quaternary carbon signal of an exocyclic double bond was observed at dC 134.3 (C-2) in the 13C NMR of 6. In addition, the methylene protons in 6 appeared downfield at dH 3.10 (ddd, J ¼ 14.7, 5.8, 1.6 Hz, H-3a) and 3.05 (ddd, J ¼ 14.7, 4.1, 1.6 Hz, H-3b) compared to those in 1. The 1H NMR spectrum showed an exocyclic olefinic proton at dH 7.01 (dt, J ¼ 10.8, 1.6 Hz, H-11), an oxygenated methine proton at dH 4.67 (d, J ¼ 10.8 Hz, H-12), and two methyl groups at dH 1.35 (s, H3-14) and 1.33 (s, H3-15). One oxygenated quaternary carbon resonated at dC 73.5 (C-13). The above 1H and 13C NMR signals were assignable to the presence of the 2,3-dihydroxy-3methylbutylidene group, and the location of the substitute group was determined as C-2 of the dihydronaphthalenone skeleton by the HMBC correlations of H2-3/C-2, C-11 and H-11/C-1 (Fig. 2). The preliminary assignment of the 1H and 13C NMR data was confirmed by detailed analysis of 1H-1H COSY, 1H-13C HSQC, and 1H-13C HMBC spectra as well as by comparison of data with those reported for catalpalenone, with a methoxy group at C-12 (Park et al., 2010). The absolute configurations at C-4 and C-12 were suggested as “S” and “R”, respectively, by the Mosher ester method (Fig. 3). Cotton effects observed in the ECD data [ECD (MeOH) Dε (nm) 3.25 (253), 3.86 (289)] also provided evidence for the (S)-configuration at C-4 (Liu et al., 2004). Therefore, the structure of 6 was elucidated as a previously undescribed compound (4S)-3,4-dihydro-4-hydroxy-2[(2R)-2,3-dihydroxy-3-methylbutylidene]naphthalen-1(2H)-one.

16

Y.-S. Kil et al. / Phytochemistry 147 (2018) 14e20

Table 1 1 H (400 MHz) and Position

13

C (100 MHz) NMR spectroscopic data for 1, 6, 7, and 9.a

1

6

dC

dH mult (J in Hz)

dC

1 2 3

198.6 46.6 38.9

2.52 m 2.50, 1.77 m

186.1 134.3 34.9

4 5 6 7 8 9 10

68.3 127.7 121.5 155.3 112.5 132.5 139.1

4.95 m 7.62 br d (8.4) 7.12 dd (8.4, 2.8)

11 12 13 14 15 16 OH-4

28.0 121.2 134.1 25.9 17.9

2.71 m, 2.28 dt (14.6, 7.7) 5.15 br t (7.7)

a

7.47 d (2.8)

1.72 s 1.65 s

67.3 127.2 134.2 129.0 128.4 131.7 144.0 135.9 64.6 73.5 26.0 25.3

1.90 br d (7.9)

7

dH mult (J in Hz)

3.10 3.05 5.02 7.53 7.62 7.47 8.13

ddd (14.7, 5.8, 1.6), ddd (14.7, 4.1, 1.6) br s br d (7.6) td (7.6, 1.5) br td (7.6, 1.2) dd (7.6, 1.5)

7.01 dt (10.8, 1.6) 4.67 d (10.8) 1.35 s 1.33 s 2.65 br s

9

dC

dH mult (J in Hz)

160.9 105.9 28.0 67.3 126.5 131.5 128.3 124.6 128.7 140.9 191.8 47.5 80.5 26.6 26.3

dC

dH mult (J in Hz)

170.1 2.87 2.82 4.87 7.52 7.48 7.40 7.79

dd (16.2, 6.0), dd (16.2, 6.8) br q (6.3) br d (7.6) td (7.6, 1.3) td (7.6, 1.6) dd (7.6, 1.3)

2.68, 2.63 d (16.6) 1.53 s 1.52 s

77.2

6.02 br t (6.6)

122.6 134.2 129.4 125.8 125.9 149.5 45.5

7.51 7.67 7.54 7.91

196.1 125.5 154.3 70.9 29.3 29.4

br dd (7.8, 0.9) td (7.8, 1.1) br t (7.8) br d (7.8)

3.33 dd (17.2, 6.0), 3.03 dd (17.2, 6.8) 6.37 d (15.8) 6.91 d (15.8) 1.392 s 1.389 s

1.75 d (6.0)

Data were measured in CDCl3. TMS was used as internal standard.

Compound 7 was isolated as a colorless amorphous solid and its molecular formula was established as C15H16O3 by the HRESIMS ([M þ H]þ, m/z 245.1174). The 1H and 13C NMR data were comparable to those of 6, showing the AA'BB0 system [dH 7.52 (1H, br d, J ¼ 7.6 Hz)/dC 126.5 (C-5), 7.48 (1H, td, J ¼ 7.6, 1.3 Hz)/131.5 (C-6), 7.40 (1H, td, J ¼ 7.6, 1.6 Hz)/128.3 (C-7), 7.79 (1H, dd, J ¼ 7.6, 1.3 Hz)/ 124.6 (C-8)], the oxygenated methine [dH 4.87 (1H, br q, J ¼ 6.3 Hz)/ dC 67.3 (C-4)], and the methylene [dH 2.87 (1H, dd, J ¼ 16.2, 6.0 Hz, H-3a) and 2.82 (1H, dd, J ¼ 16.2, 6.8 Hz, H-3b/dC 28.0 (C-3)] (Table 1). Additionally, one methylene group appeared at dH 2.68 and 2.63 (each 1H, d, J ¼ 16.6 Hz, H-12a and H-12b), and two methyl groups were observed at dH 1.53 (s, H3-14) and 1.52 (s, H3-15). The HMBC cross peaks of H2-3/C-1, C-2, H-4/C-2, and H-8/C-1 correlated with two olefinic quaternary carbons [dC 160.9 (C-1) and 105.9 (C2)], which indicated that 7 has a dihydronaphthalenol skeleton, and not the dihydronaphthalenone skeleton of 6 (Fig. 2). Moreover, the presence of an O-heterocyclic ring fused with the

dihydronaphthalenol skeleton at the C-1/C-2 bond was confirmed by the HMBC correlations of H2-3/C-11, H2-12/C-11, C-13, H3-14/C12, C-13, and H3-15/C-12, C-13 as well as by the chemical shift value of C-13 (dC 80.5). The (S)-configuration at C-4 was established by analyzing the result of the Mosher ester reaction (Fig. 3) and the ECD data [ECD (MeOH) Dε (nm) 1.26 (252), 2.05 (295)] (Liu et al., 2004). Thus, the structure of 7 was elucidated as a previously undescribed compound (6S)-5,6-dihydro-6-hydroxy-2,2-dimethyl2H-benzo[h]chromen-4(3H)-one. Compound 9 was isolated as a colorless amorphous solid and its molecular formula was determined as C15H16O4, based on the HRESIMS (m/z 261.1122 [M þ H]þ). The 1H and 13C NMR data exhibited resonances at dH 6.02 (br t, J ¼ 6.6 Hz)/dC 77.2 (C-3), 7.51 (br dd, J ¼ 7.8, 0.9 Hz)/122.6 (C-4), 7.67 (td, J ¼ 7.8, 1.1 Hz)/134.2 (C5), 7.54 (br t, J ¼ 7.8 Hz)/129.4 (C-6), 7.91 (br d, J ¼ 7.8 Hz)/125.8 (C7), dC 170.1 (C-1), 125.9 (C-8), and 149.5 (C-9) (Table 1), which were attributable to the presence of a 3-substituted phthalide skeleton, as reported for catalpalactone (Inouye et al., 1967). In the 1H NMR spectrum, one methylene and two methyl groups were observed at dH 3.33 (dd, J ¼ 17.2, 6.0 Hz, H-10a) and 3.03 (dd, J ¼ 17.2, 6.8 Hz, H10b), 1.392 (s, H3-15), and 1.389 (s, H3-16), respectively. Two olefinic proton signals appeared as doublets at dH 6.37 and 6.91 (d, J ¼ 15.8 Hz, H-12 and H-13, respectively). The 13C NMR spectrum showed an additional carbonyl carbon signal at dC 196.1 (C-11), together with an oxygenated quaternary carbon signal at dC 70.9 (C-

Fig. 2. Key 1H-1H COSY (▬) and HMBC (d) correlations of 1, 6, 7, and 9.

Fig. 3. Values of (dS-dR) of the MTPA esters of 6 and 7.

Y.-S. Kil et al. / Phytochemistry 147 (2018) 14e20

14). The remaining 1H and 13C NMR signals were ascribed to a 5hydroxy-5-methyl-2-oxohex-3-en-1-yl group, which was supported by the HMBC correlations of H-10/C-11, H-12/C-11, C-14, H13/C-11, C-14, C-15, C-16, H3-15/C-14, and H3-16/C-14 with the COSY correlation between H-12 and H-13 (Fig. 2). The position of the 5-hydroxy-5-methyl-2-oxohex-3-en-1-yl group at C-3 of the phthalide skeleton was determined by the important HMBC cross peaks of H-3/C-11 and H2-10/C-9 and the COSY correlation between H-3 and H2-10. The specific rotation of 9 was measured as [a]25D ±0 (c 0.1, MeOH), indicating its racemic characteristic, as found in catalpalactone (Inouye et al., 1967). Thus, the structure of 9 was elucidated as a previously undescribed compound (±)-3-(5hydroxy-5-methyl-2-oxohex-3-en-1-yl)isobenzofuran-1(3H)-one. 2.2. Cytoprotective effects against H2O2-induced oxidative damage in HepG2 cells To identify the effect of 1e10 on intracellular ROS generation, HepG2 cells were treated with H2O2, and ROS generation was significantly increased in H2O2-treated HepG2 cells compared to untreated control cells. Pretreatment with 2e5 and 7 attenuated the increment (p < .05) of H2O2-induced ROS content. The result suggests that 2e5 and 7 protect HepG2 cells against oxidative stress by their direct radical scavenging activities (Fig. 4A). Cytotoxicity of compounds was determined by using the EZ-Cytox cell viability assay kit, and cytotoxicity was not observed following treatment with any of the compounds (data none shown). The effect of compounds on H2O2-induced cell death in HepG2 cells was also investigated to explore whether 1e10 protect cells against H2O2-induced cell injury. Cells were pre-treated with 50 mM of 1e10 for 1 h prior to exposure of 2 mM H2O2, and the degree of cell death was assessed after 24 h. Compounds 1, 4, and 5 significantly reduced H2O2-induced cell death in HepG2 cells (Fig. 4B). The result led to the question of whether these compounds induce antioxidant enzymes, heme oxygenase-1 (HO-1) and NAD(P)

17

H:quinine oxidoreductase 1 (NQO1). The mRNA expression levels of HO-1 and NQO1 in HepG2 cells were examined by RT-PCR analysis. As a result, 1, 4, 5, and 8e10 up-regulated the mRNA expression levels of HO-1, and 4 and 5 also induced the expression of NQO-1 in HepG2 cells at 4 h (Fig. 4C and D). Therefore, 4 and 5 showed antioxidant activities via directly scavenging of intracellular ROS and inducing antioxidant enzymes such as HO-1 and NQO1 in vitro. 3. Conclusion Three previously undescribed dihydronaphthalenones (1, 6, and 7) and one phthalide (9) were identified from the wood of C. ovata together with six known compounds (2e5, 8, and 10). The cytoprotective effects of the isolates against H2O2-induced oxidative damage in HepG2 cells were investigated. As a result, 2e5 and 7 directly attenuated H2O2-induced intracellular ROS generation. Moreover, 4 and 5 induced antioxidant enzymes such as HO-1 and NQO-1 in HepG2 cell. The cytoprotective activity of those compounds in H2O2-treated HepG2 cells may be due to synergistic effect of direct and indirect antioxidant activities. The present study implies that dihydronaphthalenones of C. ovata can be developed as cytoprotective natural products that act against high-level H2O2. 4. Experimental 4.1. General Optical rotations were obtained on a P-1010 polarimeter. UV spectra were recorded on a U-3000 spectrophotometer. ECD spectra were measured with a Jasco J-810 spectropolarimeter. NMR spectra were run on a Varian Unity Inova 400 MHz FT-NMR instrument with tetramethylsilane (TMS) as internal standard, and the data were processed by using MestReNova 6.0 software (Mestrelab Research SL, Santiago de Compostela, Spain). HRESIMS for previously undescribed compounds were performed on Waters

Fig. 4. Cytoprotective effects of 1e10 in H2O2-treated HepG2 cells. (A) H2O2-stimulate ROS generation in HepG2 cells by flow cytometry (FACS). (B) Cell viability, (C) HO-1 mRNA level and (D) NQO1 mRNA level were measured by real time-PCR. mRNA expression level of genes were normalized with b-actin. The results are presented as the means ±SDs of three replicates of on represent experiment. #p < .05 vs. the non-treated cells, *p < .05 vs. the cells treated with only H2O2.

18

Y.-S. Kil et al. / Phytochemistry 147 (2018) 14e20

Acquity UPLC system coupled to a Micromass Q-Tof Micro mass spectrometer and Agilent 6220 Accurate-Mass TOF LC/MS system. HRESIMS for MTPA esters were obtained using Agilent 6550A iFunnel Q-TOF LC/MS system at the Korea Basic Science Institute (Seoul, Korea). Silica gel (230e400 mesh, Merck, Germany), RP-18 (YMC gel ODS-A, 120 Å, S-150 mm, YMC Co., Japan), and Sephadex LH-20 (GE Healthcare Bio-Science AB, Uppsala, Sweden) were used for column chromatography (CC). Thin-layer chromatographic (TLC) analysis was performed on Kieselgel 60 F254 (silica gel, 0.25 mm layer thickness, Merck, Germany) and RP-18 F254s (Merck, Germany) plates, with visualization under UV light (254 and 365 nm) and 10% (v/v) sulfuric acid spray followed by heating (120  C, 5 min). MPLC was performed using CombiFlash (Teledyne Isco Inc., USA), equipped with RediSep Rf C18 column (130 g, Teledyne Isco Inc., USA). Preparative HPLC was conducted using an Acme 9000 system (Young Lin, Korea), equipped with YMC-Pack Pro C18 column (5 mm, 250 mm  20 mm i.d.). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin were purchased from Hyclone (Logan, UT, USA). The RNeasy kit was purchased from QIAGEN (Valencia, CA, USA), the EZ-Cytox Cell Viability assay kit from DAEIL Lab (Seoul, Korea), and the 1st Strand cDNA Synthesis kit and SYBR premix from Takara Bio Inc. (Kyoto, Japan). Hydrogen peroxide (H2O2) was obtained from Sigma Chemical Co., (St. Louis, MO, USA). The 5(and-6)-carboxy-20 70 -dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) was purchased from Invitrogen (Carlsbad, CA, USA).

and 5.6.11.5 (25, 10, and 3 mg, respectively) were purified by preparative HPLC using MeOH-H2O (4:1, 2 mL/min) to give 7 (7.2 mg; tR 33.6 min), 6 (3.6 mg; tR 32.7 min), and 1 (1.2 mg; tR 33.9 min), respectively. 4.3.1. 7-Hydroxycatalponol (1, (2R,4S)-3,4-dihydro-4,7-dihydroxy2-(3-methylbut-2-en-1-yl)naphthalen-1(2H)-one) Colorless amorphous solid; [a]25D þ16 (c 0.1, MeOH); UV (MeOH) lmax (log ε) 222 (4.21), 253 (3.82), 321 (3.32) nm; ECD (MeOH) Dε (nm) 0.84 (260), 3.78 (302), þ2.84 (341); 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see Table 1; HRESIMS m/z 269.1148 [M þ Na]þ (calcd for C15H18O3Na, 269.1148). 4.3.2. (4S)-3,4-Dihydro-4-hydroxy-2-[(2R)-2,3-dihydroxy-3methylbutylidene]naphthalen-1(2H)-one (6) Colorless amorphous solid; [a]25D 118 (c 0.1, MeOH); UV (MeOH) lmax (log ε) 270 (4.15) nm; ECD (MeOH) Dε (nm) 3.25 (253), 3.86 (289); 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see Table 1; HRESIMS m/z 285.1100 [M þ Na]þ (calcd for C15H18O4Na, 285.1097).

4.2. Plant materials

4.3.3. (6S)-5,6-Dihydro-6-hydroxy-2,2-dimethyl-2H-benzo[h] chromen-4(3H)-one (7) Colorless amorphous solid; [a]25D 83 (c 0.1, MeOH); UV (MeOH) lmax (log ε) 226 (3.84), 234 (3.96), 241 (3.95), 256 (3.62), 302 (3.91), 325 (4.07); ECD (MeOH) Dε (nm) 1.26 (252), 2.05 (295); 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see Table 1; HRESIMS m/z 245.1174 [M þ H]þ (calcd for C15H17O3, 245.1172).

The wood of Catalpa ovata G. Don (Bignoniaceae) was collected at Medicinal Plant Garden, College of Pharmacy, Ewha Womans University (coordinates 37 340 07.600 N, 126 560 58.700 E), in March 2014, and identified by Professor Je-hyun Lee (College of Oriental Medicine, Dongguk University). A voucher specimen (no. EAB343) has been deposited at the Natural Product Chemistry Laboratory, College of Pharmacy, Ewha Womans University.

4.3.4. (±)-3-(5-Hydroxy-5-methyl-2-oxohex-3-en-1-yl) isobenzofuran-1(3H)-one (9) Colorless amorphous solid; [a]25D ±0 (c 0.1, MeOH); UV (MeOH) lmax (log ε) 226 (4.34), 274 (3.49), 281 (3.52), 302 (3.41) nm; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see Table 1; HRESIMS m/z 261.1122 [M þ H]þ (calcd for C15H17O4, 261.1121).

4.3. Extraction and isolation

4.4. Preparation of Mosher ester derivatives of 6 and 7

The dried and chopped wood of C. ovata (20 kg) was extracted with MeOH (4  54 L). The concentrated MeOH extract (1.5 kg) was suspended in distilled water (1.5 L) and then partitioned sequentially with hexanes (10  2 L), EtOAc (10  2 L), and BuOH (8  2 L). The EtOAc extract (700 g) was subjected to silica gel CC using gradient mixtures of hexanes-EtOAc (99:1 / 1:4) and EtOAcMeOH (1:0 / 0:1) to afford seven fractions (Fr. 1-Fr. 7). A part (1.3 g) of Fr. 3 (230 g) was subjected to silica gel CC using CH2Cl2acetone (19:1) as a solvent to afford three subfractions with 4 (290 mg) and 5 (470 mg). Fr. 5 (140 g) was chromatographed over silica gel eluted by a gradient solvent system of CH2Cl2-MeOH (1:0 / 4:1) to give nine subfractions (Fr. 5.1-Fr. 5.9) and 10 (60 g). Compound 8 (2.8 g) was obtained from Fr. 5.3 (16 g) by MPLC (MeOH-H2O 2:3 / 1:1, 30 mL/min). Fr. 5.5 (1.1 g) was subjected to MPLC by elution with MeOH-H2O (7:13 / 4:1, 20 mL/min) to yield 28 subfractions (Fr. 5.5.01-Fr. 5.5.28). Fr. 5.5.13 (46 mg) was purified by preparative HPLC using MeOH-H2O (3:2, 2 mL/min) for elution to afford 9 (1.7 mg; tR 42.8 min). Fr. 5.5.20 (110 mg) was subjected to RP-18 CC by elution with MeOH-H2O (7:3) to give 3 (63 mg). Compound 2 (85 mg) was prepared from Fr. 5.5.22 (95 mg) by RP-18 CC using MeOH-H2O (3:2) as a solvent. Fr. 5.6 (4 g) was fractionated by MPLC using MeOH-H2O gradient mixtures (7:13 / 4:1, 20 mL/ min) to obtain 21 subfractions (Fr. 5.6.01-Fr. 5.6.21). Fr. 5.6.11 (50 mg) was separated by Sephadex LH-20 with 100% MeOH to yield six subfractions (Fr. 5.6.11.1-Fr. 5.6.11.6). Frs. 5.6.11.1, 5.6.11.2,

Each compound (1 mg) was transferred to a vial, and completely dried under the vacuum. Pyridine-d5 (1 mL) was added to the vial, and the solution was divided to two clean NMR tubes (0.5 mL each). (S)-(þ)-a-Methoxy-a-(trifluoromethyl)phenylacetyl chloride [(S)MTPA-Cl] and (R)-MTPA-Cl were immediately added into each NMR tube, respectively, together with 4-dimethylaminopyridine (DMAP) under an N2 gas stream, and then the NMR tubes were shaken to make sure of even mixing. The sealed NMR tubes were incubated in a water bath (40  C) for 4 h. 1H NMR data were obtained directly from the reaction NMR tubes (Su et al., 2002). 1D-NOESY experiments were used for ambiguous assignment of 1H NMR data. The molecular formulas of the MTPA esters were confirmed by HRESIMS. (R)-MTPA ester of 6 (6r) 1H NMR (pyridine-d5, 400 MHz): dH 7.756 (br d, J ¼ 10.7 Hz, H-11), 7.556 (d, J ¼ 8.0 Hz, H-5), 6.625 (br t, J ¼ 4.6 Hz, H-4), 5.283 (d, J ¼ 10.7 Hz, H-12), 3.641 (m, H2-3), 1.576 (s, H3-14), 1.512 (s, H3-15); HRESIMS m/z 461.1573 [M þ H]þ (calcd for C25H24F3O5, 461.1576). (S)-MTPA ester of 6 (6s) 1H NMR (pyridine-d5, 400 MHz): dH 7.799 (br d, J ¼ 10.3 Hz, H-11), 7.759 (d, J ¼ 7.2 Hz, H-5), 6.637 (br t, J ¼ 3.8 Hz, H-4), 5.117 (d, J ¼ 10.3 Hz, H12), 3.547 (m, H2-3), 1.500 (s, H3-14), 1.373 (s, H3-15). (R)-MTPA ester of 7 (7r) 1H NMR (pyridine-d5, 400 MHz): dH 7.602 (m, H-5), 6.446 (t, J ¼ 4.5 Hz, H-4), 3.533 (dd, J ¼ 17.5, 4.5 Hz, H-3a), 2.903 (dd, J ¼ 17.5, 4.5 Hz, H-3b), 2.715 (d, J ¼ 16.6 Hz, H-12a), 2.641 (d, J ¼ 16.6 Hz, H-12b), 1.419 (s, H3-14), 1.333 (s, H3-15); HRESIMS m/z 717.1894 [M þ Na]þ (calcd for C35H32F6O8Na, 717.1899). (S)-MTPA

Y.-S. Kil et al. / Phytochemistry 147 (2018) 14e20

ester of 7 (7s) 1H NMR (pyridine-d5, 400 MHz): dH 7.681 (m, H-5), 6.431 (br t, J ¼ 3.9 Hz, H-4), 3.557 (dd, J ¼ 17.5, 3.9 Hz, H-3a), 2.813 (dd, J ¼ 17.5, 3.9 Hz, H-3b), 2.682 (d, J ¼ 16.6 Hz, H-12a), 2.564 (d, J ¼ 16.6 Hz, H-12b), 1.411 (s, H3-14), 1.260 (s, H3-15). 4.5. Cell culture and cytotoxicity Human hepatocyte carcinoma HepG2 cells, obtained from the American Type Culture Collection, were cultured in DMEM supplemented with 10% FBS, 100 units/mL of penicillin, and 100 mg/ mL of streptomycin. Cells were maintained in a humidified incubator at 37  C with 5% carbon dioxide (CO2) atmosphere. Cell cytotoxicity of compounds was measured using an EZ-Cytox cell viability assay kit (Daeil Lab, Seoul, Korea). Briefly, cells were cultured in a 96-well plate at a density of 1  105 cells/mL for 24 h. After treatment with each compounds (50 mM) for 24 h, 10 mL of the kit solution was added to each well, followed by incubation for 4 h at 37  C under 5% CO2. Their cytotoxicities were determined using an ELISA reader (Benchmark Plus, BioRad) at an absorbance of 480 nm. The relative cell viability was calculated and compared with the absorbance of the untreated control group. 4.6. Measurement of intracellular ROS accumulation HepG2 cells were seeded into 6-well plates at a density of 1  105 cells/mL and incubated for 24 h. After treatment with compounds at 50 mM for 1 h, H2O2 (2 mM) was added to plates for 30 min. Cells were then incubated with 10 mM carboxyH2DCFDA for 20 min, then cells were washed and harvested. Cells were immediately examined using a flow cytometer (Cytomics FC500; Beckman, Miami, FL, USA). The fluorescence intensity measuring the oxidation of DCF-DA by ROS represents the relative steady state of ROS generation in the cells (Rushworth et al., 2008). 4.7. Cell viability assay The EZ-Cytox cell viability assay kit was used to measure cell viability. The cells were cultured in a 96-well plate at a density of 1  105 cells/mL for 24 h. Compounds were dissolved in DMSO and incubated with the cells at 50 mM for 1 h and then treated H2O2 (2 mM) for an additional 24 h. After the incubation period, 10 mL solution of cell viability assay kit was added to each well and incubated for 4 h at 37  C and 5% CO2. The index of cell viability was determined by measuring formazan production using a spectrophotometer (Benchmark Plus, Bio-Rad, Hercules, CA, USA) at an absorbance of 480 nm with a reference wavelength of 650 nm. The results are presented as means ± standard deviation (SD) of five replicates for one representative experiment. 4.8. Quantitative real-time polymerase chain reaction (RT-PCR) The cells were cultured in a 6-well plate at a density of 1  105 cells/mL for 24 h. They were treated with compounds at 50 mM for 4 h. Total RNA was isolated using the RNeasy kit according to the manufacturer's protocol. The 1st Strand cDNA Synthesis kit was used for reverse transcription according to the manufacturer's protocol. A Chromm4 real-time PCR detection system (Bio-Rad) and iTaq™ SYBRR Green Supermix (Bio-Rad) were used for the RT-PCR amplification of HO-1, NQO-1, and b-actin using the following conditions: 50 cycles at 94  C for 20 s, 60  C for 20 s and 72  C for 30 s. All the reactions were repeated independently at least three times to ensure the reproducibility of the

19

results. Primers for HO-1, NQO-1 and b-actin were purchased from Bioneer Corp. (Daejeon, Korea) and the following sense and antisense primers were used: HO-1, forward primer 50 - CAGGCAGAGAATGCTGAGTT-30 , reverse primer 50 - GGCTTTC-TGGGCAATCTTT30 ; NQO-1, forward primer 50 - ATGTATGACAAAGGACCCTT-30 , reverse primer 50 - TCCCTTGCAGAGAGTACATG-30 ; b-actin, forward primer 50 -TGAGAGGGAAATCGTGCGTGAC-30 , reverse primer 50 GCTCGTTGCCAATAGTGATGACC-30 . 4.9. Statistical methods Data were expressed as mean ± standard deviation (SD), and a statistical analysis for a single comparison was performed using a Student's t-test. A p < .05 was considered to be significant for all analyses. Acknowledgments This work was supported by Ewha Womans University. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.phytochem.2017.12.009. References Anh, N.H., Ripperger, H., Porzel, A., Sung, T.V., Adam, G., 1997. Tetralones from Ancistrocladus cochinchinensis. Phytochemistry 44, 549e551. Evidente, A., Superchi, S., Cimmino, A., Mazzeo, G., Mugnai, L., Rubiales, D., Andolfi, A., Villegas-Fernandez, A.M., 2011. Regiolone and isosclerone, two enantiomeric phytotoxic naphthalenone pentaketides: computational assignment of absolute configuration and its relationship with phytotoxic activity. Eur. J. Org. Chem. 28, 5564e5570. Fujiwara, A., Mori, T., Iida, A., Ueda, S., Hano, Y., Nomura, T., Tokuda, H., Nishino, H., 1998. Antitumor-promoting naphthoquinones from catalpa ovata. J. Nat. Prod. 61, 629e632. Garcia, A.E., Ouizem, S., Cheng, X., Romanens, P., Kuendig, E.P., 2010. Efficient enantioselective syntheses of sertraline, 2-epicatalponol and catalponol from tetralin-1,4-dione. Adv. Synth. Catal. 352, 2306e2314. Giorgio, M., Trinei, M., Migliaccio, E., Pelicci, P.G., 2007. Hydrogen peroxide: a metabolic by-product or a common mediator of ageing signals? Nat. Rev. Mol. Cell Biol. 8, 722e728. Inoue, K., Shiobara, Y., Chen, C.-C., Sakuyama, S., Inouye, H., 1979. Quinones and related compounds in higher plants. VII. Supplementary studies on the constituents of the wood of Catalpa ovata. Yakugaku Zasshi 99, 500e504. Inoue, K., Inouye, H., Taga, T., Fujita, R., Osaki, K., Kuriyama, K., 1980. Quinones and related compounds in higher plants. IX. Absolute structures of catalponol and its congeners. Chem. Pharm. Bull. 28, 1224e1229. Inouye, H., Okuda, T., Hirata, Y., Nagakura, N., Yoshizaki, M., 1965. Structure of catalpalactone. Tetrahedron Lett. 1261e1264. Inouye, H., Okuda, T., Hirata, Y., Nagakura, N., Yoshizaki, M., 1967. Structure of catalpalactone, a new phthalide from catalpa wood. Chem. Pharm. Bull. 15, 782e792. Inouye, H., Okuda, T., Hayashi, T., 1971. Naphthoquinone derivatives form Catalpa ovata. Tetrahedron Lett. 3615e3618. Inouye, H., Okuda, T., Hayashi, T., 1975a. Quinones and related compounds in higher plants. II. Naphthoquinones and related compounds from Catalpa wood. Chem. Pharm. Bull. 23, 384e391. Inouye, H., Hayashi, T., Shingu, T., 1975b. Quinones and related compounds in higher plants. III. Absolute structure of catalponol, a naphthoquinone congener of Catalpa ovata. Chem. Pharm. Bull. 23, 392e399. Liu, L., Li, W., Koike, K., Zhang, S., Nikaido, T., 2004. New a-tetralonyl glucosides from the fruit of Juglans mandshurica. Chem. Pharm. Bull. 52, 566e569. Park, B.M., Hong, S.S., Lee, C., Lee, M.S., Kang, S.J., Shin, Y.S., Jung, J.-K., Hong, J.T., Kim, Y., Lee, M.K., Hwang, B.Y., 2010. Naphthoquinones from Catalpa ovata and their inhibitory effects on the production of nitric oxide. Arch. Pharmacal Res. 33, 381e385. Peraza-Sanchez, S.R., Chavez, D., Chai, H.-B., Shin, Y.G., Garcia, R., Mejia, M., Fairchild, C.R., Lane, K.E., Menendez, A.T., Farnsworth, N.R., Cordell, G.A., Pezzuto, J.M., Kinghorn, A.D., 2000. Cytotoxic constituents of the roots of Ekmanianthe longiflora. J. Nat. Prod. 63, 492e495. Rushworth, S.A., MacEwan, D.J., O'Connell, M.A., 2008. Lipopolysaccharide-induced expression of NAD(P)H: quinone oxidoreductase 1 and heme Oxygenase-1 protects against excessive inflammatory responses in human monocytes. J. Immunol. 181, 6730e6737.

20

Y.-S. Kil et al. / Phytochemistry 147 (2018) 14e20

Stone, J.R., Yang, S., 2006. Hydrogen peroxide: a signaling messenger. Antioxid. Redox Signal. 8, 243e270. Su, B.-N., Park, E.J., Mbwambo, Z.H., Santarsiero, B.D., Mesecar, A.D., Fong, H.H.S., Pezzuto, J.M., Kinghorn, A.D., 2002. New chemical constituents of Euphorbia

quinquecostata and absolute configuration assignment by a convenient mosher ester procedure carried out in NMR tubes. J. Nat. Prod. 65, 1278e1282. Young, H.S., Kim, M.S., Park, H.J., Chung, H.Y., Choi, J.S., 1992. Phytochemical study on Catalpa ovata. Arch. Pharmacal Res. 15, 322e327.