Nuclear Factor Erythroid 2-Related Factor 2 Activating Triterpenoid

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Nuclear Factor Erythroid 2‑Related Factor 2 Activating Triterpenoid Saponins from Camellia japonica Roots Kiwon Ko,† Lilik D. Wahyudi,‡,§ Yong-Soo Kwon,† Jung-Hwan Kim,*,§ and Heejung Yang*,† †

College of Pharmacy, Kangwon National University, Chuncheon 24341, Korea Department of Convergence Medical Science (BK21 Plus), Gyeongsang National University, Jinju, 52727, Korea § Department of Pharmacology, School of Medicine, Institute of Health Sciences, Gyeongsang National University, Jinju 52727, Korea Downloaded via UNIV OF TEXAS AT EL PASO on November 6, 2018 at 02:35:40 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: Oxidative stress due to the presence of excess reactive oxygen species may cause cancers, aging, and many other conditions. Nuclear factor erythroid 2-related factor 2 (Nrf2) may control abnormal oxidative stress as a transcription factor by inducing antioxidant-related genes via antioxidant response elements (AREs) in the gene promoters. The 11 triterpenoid saponins (1−11) isolated from Camellia japonica roots were tested for ARE-luciferase activity and Nrf2 accumulation in human keratinocytes (HaCaT cells). The ARE-luciferase activity was significantly increased by compounds 1−11 (25 μM) as a result of nuclear Nrf2 accumulation in the cells. Thus, these compounds may contribute to the induction of Nrf2 activity against oxidative damage in cells.

N

new compounds (1−11) were isolated from the MeOH extract of C. japonica roots, and their biological activities on the activation of Nrf2 signaling were investigated.

uclear factor erythroid 2-related factor 2 (Nrf2) controls the redox system to prevent cellular damage against insults of oxidative stress. To activate the system, Nrf2 binds to the antioxidant response element (ARE, 5′-TGACNNNGC-3) in the promoter region of its target genes.1−3 The activation of Nrf2 can be stimulated by phosphorylation of some kinases such as cAMP-activated protein kinase (AMPK), mitogen-activated protein kinases (MAPKs), protein kinase C (PKC), and protein kinase B (Akt).4−7 After Nrf2 translocation into the nucleus by stimuli, cytoprotective proteins such as glutathione peroxidase (GPx), heme oxygenase-1, catalase, and γ-glutamylcysteine ligase catalytic subunit (GCLC), superoxide dismutase (SOD), and phase-2 detoxification enzymes such as NAD(P)H-quinone oxidoreductase 1 (NQO1), glutathione S-transferase (GST), and UDP-glucuronosyltransferase (UGT) can be induced.1,8 Camellia japonica L. (Theaceae), a perennial tree native to East Asian countries such as Korea, Japan, and China, has been used as a medicinal herb and in cosmetics. The various parts of the tree have been used for different purposes; for example, the flowers are used for melanogenesis inhibition and inhibition of porcine epidemic diarrhea virus (PEDV) replication,9−12 the stems for their anti-inflammatory and cytotoxic activities,13,14 the seeds for their ethanol absorption inhibitory effects,15,16 the fruits as anti-inflammatory agents and for treating gastric ulcers and breast cancer,17,18 and the leaves as antioxidants.19 Although the chemical and biological properties of the roots of other members of the Camellia family, such as C. sinensis and C. oliferia, have been previously studied,20−25 the secondary metabolites of C. japonica roots are reported herein for the first time. Eleven © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

The n-BuOH fraction (45 g), the subfraction of an 80% MeOH extract (476 g) of C. japonica roots, was subjected to repeated column chromatography and HPLC to give 11 new compounds (1−11), the structures of which were defined by NMR spectroscopic techniques and MS analyses. Compound 1 was obtained as an amorphous powder and exhibited an [M − H]− ion at m/z 1167.5956 (calcd for 1167.5951), indicating a molecular formula of C59H92O23. The 1 H NMR spectroscopic data exhibited characteristic signals of a triterpenoid at δH 0.81, 0.89, 0.95, 1.06, 1.28, and 1.42 (all s, 3H, H-26, 25, 24, 29, 30, and 27) for six methyl groups and δH 5.41 (br s, 1H, H-12) for an olefinic group (Table 1). The significant chemical shifts of C-12 and C-13 at δC 125.6 and 141.5, respectively, in the 13C NMR spectrum demonstrated that 1 is an oleanane-type triterpenoid.26 The HSQC experiment showed deshielded chemical shifts corresponding to the carbons of two oxymethylene groups at δH/δC 3.73 and 4.38 (both m, 2H, H-23)/64.4 (C-23) and 3.51 and 3.67 (both m, 2H, H-28)/ 63.9 (C-28) and four oxymethine groups at δH/δC 4.34 (m, 1H, H-3)/81.1 (C-3), 5.62 (br s, 1H, H-16)/72.0 (C-16), 5.92 (d, J Received: May 11, 2018

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DOI: 10.1021/acs.jnatprod.8b00374 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

The anomeric proton chemical shifts of the glucuronic acid and arabinose moieties are similar and are difficult to differentiate without the HSQC data (Figure S3, Supporting Information). Accordingly, the structure of compound 1 was assigned as 16αO-acetyl-21β-O-angeloyl-23,28-dihydroxy-22α-O-(2methylbutanoyl)olean-12-ene 3β-O-α-L-rhamnopyranosyl-(1→ 3)-α-L-arabinopyranosyl-(1→3)-β-D-glucuronopyranoside. Compound 2 was obtained as an amorphous powder. The HRESIMS data (m/z 1125.5859 [M − H]−, calcd for 1125.5845) indicated that the molecular formula of 2 was C57H90O22, suggesting that it is an analogue of 1. The significant changes in the chemical shifts of C-12 (δC 124.4, −1.2 ppm), C13 (δC 143.3, +1.8 ppm), C-15 (δC 35.3, +3.8 ppm), and C-16 (δC 68.7, −3.3 ppm) in the 13C NMR data and H-16 (δH 4.48, −1.16 ppm) and H-21 (δH 6.67, +0.75 ppm) in the 1H NMR data indicated the absence of the acetyl group at C-16, which was confirmed by the correlations between H-15 (δH 1.60) and C-14 (δC 42.1), C-16 (δC 68.7), C-17 (δC 48.6), and C-27 (δC 28.0) in the HMBC experiment (Figure 1). Accordingly, the structure of compound 2 was assigned as 21β-O-angeloyl-16α,23,28trihydroxy-22α-O-(2-methylbutanoyl)olean-12-ene 3β-O-α-Lrhamnopyranosyl-(1→3)-α-L-arabinopyranosyl-(1→3)-β-Dglucuronopyranoside. Compound 3 was isolated as an amorphous powder. The molecular formula of compound 3, C57H90O22, was established from its HRESIMS data (m/z 1125.5856 [M − H]−, calcd for 1125.5845). Compound 3 was deduced to be a C-22 derivative of 2 based on the NMR spectra. The significant changes in the chemical shifts of C-4′′′′′ (δC 23.0, +10.6 ppm) and C-5′′′′′ (δC 22.9, +5.7 ppm) showed the 3-MB group was located at C-22 by 2D NMR data (Figure 1). Accordingly, the structure of compound 3 was assigned as 21β-O-angeloyl-16α,23,28trihydroxy-22α-O-(3-methylbutanoyl)olean-12-ene 3β-O-α-Lrhamnopyranosyl-(1→3)-α-L-arabinopyranosyl-(1→3)-β-Dglucuronopyranoside. Compound 4, an amorphous powder with an HRESIMS [M − H]− ion at m/z 1141.5800 (calcd for 1141.5795), was assigned a molecular formula of C57H90O23. The structure of 4 was similar to that of 2 except for the substituent at C-15. The significant change in the chemical shift of C-15 (δC 68.0, +32.8 ppm) indicated the presence of a hydroxy group at C-15. Correlations between H-15 (δH 4.19) and C-8 (δC 42.0), C-14 (δC 48.2), and C-27 (δC 21.7) were observed in the HMBC experiment (Figure 1). The NOESY correlations between H-15 (δH 4.19), H-16 (δH 4.36), and H-18 (δH 3.05) suggested that both the 15- and 16-OH groups are α-oriented (Figure 2). Accordingly, the structure of compound 4 was assigned as 21βO-angeloyl-15α,16α,23,28-tetrahydroxy-22α-O-(2methylbutanoyl)olean-12-ene 3β-O-α-L-rhamnopyranosyl-(1→ 3)-α-L-arabinopyranosyl-(1→3)-β-D-glucuronopyranoside. The molecular formula of compound 5, an amorphous powder, was assigned as C57H88O23 by HRESIMS analysis (m/z 1139.5658 [M − H]−, calcd for 1139.5638). Compound 5 was found to be a C-22 derivative of 4 based on the 2D NMR spectroscopic data. The significant changes in the chemical shifts of C-2′′′′′ (δC 129.4, +87.5 ppm) and C-3′′′′′ (δC 137.6, +110.2 ppm) showed that the functional group at C-22 was a tigloyl (Tig) moiety based on the HMBC experiments (Figure 1).23 Accordingly, the structure of compound 5 was defined as 21β-Oangeloyl-15α,16α,23,28-tetrahydroxy-22α-O-tigloylolean-12ene 3β-O-α-L-rhamnopyranosyl-(1→3)-α-L-arabinopyranosyl(1→3)-β-D-glucuronopyranoside.

Scheme 1. Chemical Structures of Compounds 1−11

= 10.4 Hz, 1H, H-21)/78.5 (C-21), and 6.24 (d, J = 10.4 Hz, 1H, H-22)/72.9 (C-22); these data suggested that 1 is a highly oxygenated triterpenoid-type compound. In addition, the presence of an acetyl (Ac), an angeloyl (Ang), and a 2methylbutanoyl (2-MB) substituent was evident from the signals at δH 2.54 (s, 3H, H-2′′′′′′) for the Ac moiety; δH 2.02 (s, 3H, H-5⁗), 2.13 (dd, J = 7.2, 1.3 Hz, 3H, H-4⁗), and 6.09 (m, 1H, H-3⁗) for the Ang moiety; and δH 0.91 (m, 3H, H4′′′′′), 1.19 (d, J = 7.0 Hz, 3H, H-5′′′′′), 1.48 (m, 1H, H-3′′′′′), 1.80 (m, 1H, H-3′′′′′), and 2.49 (m, 1H, H-2′′′′′) for the 2-MB moiety. The Ac, Ang, and 2-MB moieties were located at C-16, C-21, and C-22, respectively, based on the HMBC correlations between H-16 (δH 5.62) and C-1′′′′′′ (δC 170.3), H-21 (δH 5.92) and C-1⁗ (δC 167.9), and H-22 (δH 6.24) and C-1′′′′′ (δC 176.4) (Figure 1). The NOESY spectra showed correlations between H-21 (δH 5.92) and H-29 (δH 1.06) as well as those between H-22 (δH 6.24) and H-30 (δH 1.28), suggesting that H21 and H-22 are α- and β-oriented, respectively. Consequently, the Ang and 2-MB moieties were attached at C-21 and C-22 as β and α substituents, respectively. Additionally, the NOESY correlations between H-16 (δH 5.62) and H-18 (δH 3.06) suggested that the 16-OAc group is α-oriented (Figure 2). The signals at δH 5.18 (d, J = 8.3 Hz, 1H, H-1′), 5.19 (m, 1H, H-1″), and 6.06 (br s, 1H, H-1‴) were correlated to C-1′ (δC 105.6), C1″ (δC 106.6), and C-1‴ (δC 104.3) and indicated the presence of three sugar units based on the HSQC spectrum. The HMBC correlations between H-1′ (δH5.18) and C-3 (δC 81.1), H-1″ (δH 5.19) and C-3′ (δC 86.5), and H-1‴ (δH 6.06) and C-3″ (δC 80.8) showed that a trisaccharide moiety was connected to the hydroxyl group at C-3 of the aglycone. Acid hydrolysis of 1 yielded D-glucuronic acid (22.49 min), L-arabinose (24.58 min), and L-rhamnose (38.09 min) (Figure S59, Supporting Information).27 The anomeric protons of the glucuronopyranosyl, arabinopyranosyl, and rhamnopyranosyl moieties were β (δH 5.18 (d, J = 8.3 Hz, 1H, H-1′), α (δH 5.19, m, 1H, H-1″), and α (δH 6.06, br s, 1H, H-1‴) oriented based on the coupling constants and anomeric proton chemical shifts, respectively.28 B



Article

Table 1. NMR Spectroscopic Data for Compounds 1−6 (Pyridine-d5, 150 MHz for 1 position

δC, type

1

39.2, CH2 26.5, CH2 81.1, CH 43.9, C 47.5, CH 18.4, CH2 33.2, CH2 41.5, C 47.5, CH 37.0, C 24.3, CH2 125.6, CH 141.5, C 40.4, C 31.5, CH2 72.0, CH 47.4, C 39.9, CH 47.7, CH2 36.5, C 78.5, CH 72.9, CH 64.4, CH2

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

24 25 26 27 28

29 30 21-OAng 1⁗ 2⁗ 3⁗

4⁗ 5⁗

14.1, CH3 16.6, CH3 17.2, CH3 27.4, CH3 63.9, CH2 29.8, CH3 20.2, CH3

167.9, C 128.6, C 139.9, CH 16.6, CH3 21.5, CH3

2 δH (J in Hz)

0.92 m, 1.44 m 1.99 m, 2.28 m 4.34 m

1.77 m 1.31 m, 1.73 m 1.13 m, 1.52 m 1.67 m

1.77 m, 1.87 m 5.41 br s

1.55 m, 1.87 m 5.62 br s

3.06 m 1.45 m, 2.64 m 5.92 d (10.4) 6.24 d (10.4) 3.73 m, 4.38 m 0.95 s 0.89 s 0.81 s 1.42 s 3.51 d (10.5), 3.67 m 1.06 s 1.28 s

6.09 m

2.13 dd (7.2, 1.3) 2.02 s

3

δC, type

δH (J in Hz)

δC, type


0.99 m, 1.48 m


2.01 m, 2.30 m 4.38 m

1.74 m 1.38 m, 1.79 m 1.32 m, 1.79 m

1.86 m

1.84 m, 1.91 m 5.42 br s

1.60 d (4.3), 1.89 m 4.48 br s

3.11 m 1.40 m, 3.10 m

6.67 d (10.2) 6.29 d (10.2) 3.75 d (10.7), 4.38 d (10.1)

14.2, CH3 16.7, CH3 17.4, CH3 28.0, CH3 64.0, CH2

0.98 s

29.9, CH3 20.8, CH3

1.07 s

168.0, C 129.2, C 138.9, CH 16.5, CH3 21.6, CH3

0.94 s 0.92 s 1.83 s 3.43 d (10.6), 3.70 d (10.6)

1.32 s

6.08 m

2.17 dd (7.2, 1.2) 2.06 s

14.2, CH3 16.8, CH3 17.4, CH3 28.0, CH3 64.1, CH2 29.9, CH3 20.8, CH3

168.1, C 129.3, C 138.4, CH 16.5, CH3 21.6, CH3

13

C and 600 MHz for 1H)

4 δH (J in Hz)

0.98 m, 1.49 m 2.00 m, 2.30 m 4.36 m

1.74 m 1.39 m, 1.80 m 1.33 m, 1.80 m 1.87 m

1.86 m, 1.94 m 5.42 br s

1.62 m, 1.90 m 4.47 br s


δC, type

δH (J in Hz)

δC, type

39.4, CH2 26.6, CH2 81.5, CH 43.9, C 47.7, CH 19.0, CH2 36.9, CH2 42.0, C 47.7, CH 37.4, C 24.5, CH2 125.9, CH 144.2, C 48.2, C 68.0, CH 73.5, CH 48.9, C 41.3, CH 47.2, CH2 36.8, C 79.1, CH 73.6, CH 64.7, CH2

0.95 m, 1.46 m 1.97 m, 2.25 m 4.34 m

39.5, CH2 26.5, CH2 79.4, CH 43.9 47.7, CH 19.0, CH2 36.8, CH2 42.0, C 47.7, CH 37.3, C 24.5, CH2 125.9, CH 144.2, C 48.2, C 68.0, CH 73.7, CH 49.1, C 41.3, CH 47.3, CH2 36.7, C 79.0, CH 74.1, CH 64.7, CH2

14.2, CH3 16.9, CH3 18.1, CH3 21.7, CH3 63.4, CH2 29.8, CH3 20.7, CH3

168.1, C 129.2, C 139.0, CH

6.06 m

2.17 d (7.1) 2.06 s

5

16.5, CH3 21.5, CH3

1.76 m 1.40 m, 1.82 m 2.14 m, 2.23 m 1.82 m

1.81 m, 1.90 m 5.48 br s

4.19 br s 4.36 br s

3.05 m 1.37 m, 3.03 m 6.63 d (10.2) 6.25 d (10.2) 3.71 d (10.4), 4.31 m 0.94 s 0.92 s 1.04 s 1.80 s 3.46 d (10.5), 3.76 m 1.04 s 1.28 s

6.04 ddd (14.3, 7.0, 0.9) 2.14 d (7.2) 2.02 s

14.2, CH3 16.9, CH3 18.1, CH3 21.8, CH3 63.3, CH2 29.9, CH3 20.6, CH3

168.4, C 129.6, C 137.1, CH 16.3, CH3 21.4, CH3

6 δH (J in Hz)

0.98 m, 1.49 m 2.01 m, 2.31 m 4.38 m

1.80 m 1.44 m, 1.86 m 2.16 m, 2.28 m 1.87 m

1.86 m, 1.94 m 5.54 br s

4.21 m 4.45 m


5.92 dd (7.1, 1.4) 2.06 d (7.1) 2.00 s

δC, type

δH (J in Hz)

39.4, CH2 26.5, CH2 80.5, CH 43.9, C 47.7, CH 19.0, CH2 36.8, CH2 42.0, C 47.7, CH 37.3, C 24.5, CH2 125.9, CH 144.2, C 48.2, C 68.0, CH 74.0, CH 48.8, C 41.4, CH 47.3, CH2 36.9, C 79.0, CH 73.8, CH 64.6, CH2

0.99 m, 1.50 m 2.01 m, 2.30 m 4.37 m

1.80 m 1.44 m, 1.86 m 2.16 m, 2.28 m

1.86 m

1.86 m, 1.94 m 5.54 br s

4.25 m 4.45 m

3.10 m 1.42 m, 3.10 m

6.73 d (10.2) 6.36 d (10.2) 3.72 d (10.6), 4.33 m

14.2, CH3 16.9, CH3 18.1, CH3 21.8, CH3 63.5, CH2

0.97 s

29.9, CH3 20.7, CH3

1.09 s

168.2 129.4 137.9, CH 16.4, CH3 21.5, CH3

0.97 s 1.08 s 1.86 s 3.53 d (10.5), 3.80 d (10.5)

1.34 s

6.00 ddd (14.3, 7.1, 0.9) 2.11 d (7.1) 2.03 s

22-O-2MB C



Article

Table 1. continued 1 position

δC, type

1′′′′′ 2′′′′′

176.4, C 42.2, CH 27.2, CH2 12.4, CH3 17.3, CH3

3′′′′′ 4′′′′′ 5′′′′′

2 δH (J in Hz)

2.49 m 1.48 m, 1.80 m 0.91 m 1.19 d (7.0)

δC, type 177.1, C 42.1, CH 27.4, CH2 12.4, CH3 17.2, CH3

δH (J in Hz)

3 δC, type

4 δH (J in Hz)

2.28 m 1.36 m, 1.72 m 0.80 t (7.3) 1.11 d (7.0)

22-O-3MB 1′′′′′ 2′′′′′

173.5, C 44.2, CH2 26.1, CH 23.0, CH3 22.9, CH3

3′′′′′ 4′′′′′ 5′′′′′

δC, type 177.0, C 41.9, CH 27.4, CH2 12.3, CH3 17.1, CH3

δH (J in Hz)

5 δC, type

6 δH (J in Hz)

δC, type

2.03 m 1.20 m, 1.57 m 0.64 t (7.3) 0.99 d (6.9)

2.11 m 2.09 m 0.81 d (6) 0.85 d (5.9)

22-OAng 1′′′′′ 2′′′′′ 3′′′′′

168.6, C 129.6, C 136.3, CH 16.2, CH3 21.1, CH3

4′′′′′ 5′′′′′ 22-OTig 1′′′′′ 2′′′′′ 3′′′′′

168.7, C 129.4, C 137.6, CH 14.4, CH3 12.7, CH3

4′′′′′ 5′′′′′ 16-Ac 1′′′′′′ 2′′′′′′ 3-O-DGlcA 1′ 2′ 3′ 4′ 5′ 6′ 3′-O-LAra 1″ 2″ 3″ 4″

170.3, C 22.5, CH3

105.6, CH 74.6, CH 86.5, CH 72.1, CH 77.2, CH 174.1, C

106.6, CH 72.4, CH 80.8, CH 69.8, CH

δH (J in Hz)

5.81 dt(7.0, 6.3) 1.99 d (7.0) 1.77 s

6.81 d (7.1) 1.33 br s 1.76 s

2.54 s

5.18 d (8.3) 4.09 m 4.24 m 4.40 m 4.42 m

5.19 m 4.69 m 4.29 m 4.45 m

105.8, CH 74.6, CH 86.7, CH 72.0, CH 77.0, CH 174.0, C

106.7, CH 72.5, CH 80.9, CH 69.8, CH

5.19 m 4.11 m 4.22 m 4.42 m 4.45 m

5.19 m 4.67 m 4.28 m 4.44 m

105.6, CH 74.5, CH 86.7, CH 72.1, CH 77.2, CH 174.0, C

106.4, CH 72.5, CH 80.8, CH 69.8, CH

5.15 m

105.6, CH 74.7, CH 86.8, CH 71.8, CH 77.3, CH 173.7, C

4.07 m 4.20 m 4.42 m 4.45 m

5.15 m

106.6, CH 72.5, CH 80.9, CH 69.8, CH

4.66 m 4.24 m 4.41 m

D

5.15 m 4.05 m 4.17 m 4.38 m 4.41 m

5.14 m 4.62 t (8.2) 4.23 m 4.40 m

105.6, CH 74.1, CH 85.9, CH 72.0, CH 77.2, CH 174.0, C

106.0, CH 72.0, CH 80.7, CH 69.8, CH

5.15 m 4.07 m 4.22 m 4.38 m 4.40 m

5.14 m 4.69 m 4.25 m 4.41 m

105.1, CH 74.5, CH 86.4, CH 72.1, CH 76.7, CH 174.1, C

106.4, CH 72.2, CH 80.8, CH 69.8, CH

5.14 m 4.08 m 4.21 m 4.31 m 4.32 m

5.16 m 4.68 m 4.26 m 4.43 m



Article

Table 1. continued 1

2 δH (J in Hz)

5

6 δH (J in Hz)

position

δC, type

δC, type

δH (J in Hz)

δC, type

68.7, CH2

3.81 m, 4.30 m

68.6, CH

3.80 d (12.0), 4.30 m

68.6, CH2

3.78 m, 4.31 m

68.6, CH2

3.76 m, 4.26 m

68.7, CH2

3.78 m, 4.29 m

68.6, CH2

3.79 m, 4.31 m

104.3, CH 72.8, CH 73.1, CH 74.6, CH 70.6, CH 19.1, CH3

6.06 br s

104.3, CH 72.8, CH 73.1, CH 74.6, CH 70.6, CH 19.1, CH3

6.06 br s

104.3, CH 72.7, CH 73.0, CH 74.6, CH 70.6, CH 19.1, CH3

6.03 br s

104.3, CH 72.7, CH 73.0, CH 74.6, CH 70.6, CH 19.1, CH3

6.01 br s

104.3, CH 72.7, CH 73.1, CH 74.6, CH 70.6, CH 19.1, CH3

6.03 br s

104.3, CH 72.7, CH 73.0, CH 74.6, CH 70.6, CH 19.1, CH3

6.04 br s

3″-O-LRha 1‴ 2‴ 3‴ 4‴ 5‴ 6‴

4.76 m 4.58 m 4.33 m 4.62 m 1.69 d (6.2)

4.75 m 4.58 m 4.33 m 4.62 m 1.69 d (6.2)

δC, type

4 δH (J in Hz)

5″

δC, type

δH (J in Hz)

3

4.73 m 4.57 m 4.32 m 4.59 m 1.68 d (5.7)

4.71 br s 4.54 m 4.28 m 4.57 m 1.64 d (6.0)

δC, type

4.75 m 4.58 m 4.32 m 4.60 m 1.67 d (6.0)

δH (J in Hz)

4.76 m 4.59 m 4.33 m 4.61 m 1.68 d (5.8)

Figure 1. Key HMBC and 1H−1H COSY correlations of compounds 1−11.

anosyl-(1→3)-α-L-arabinopyranosyl-(1→3)-β-D-glucuronopyranoside. Compound 7, an amorphous powder, was deduced to have the molecular formula C61H100O23 by HRESIMS analysis (m/z 1199.6528 [M − H]−, calcd for 1199.6577). The significant changes in the chemical shifts of C-15 (δC 69.8, +1.8 ppm), C-16 (δC 71.6, −1.9 ppm), C-4′′′′′ (δC 22.8, +10.5 ppm), and C-5′′′′′ (δC 22.8, +5.7 ppm) showed that the functional groups connected to C-15, C-16, and C-22 are Ac, Ac, and 3-MB, respectively, by 2D NMR data (Figure 1). The NMR

Compound 6 was an amorphous powder with a molecular formula of C57H88O23, as determined from its HRESIMS data (m/z 1139.5660 [M − H]−, calcd for 1139.5638). The 1H and 13 C NMR spectroscopic data of 6 indicated that it is also a derivative of 4. The significant changes in the chemical shifts of C-4′′′′′ (δC 16.2, +1.8 ppm) and C-5′′′′′ (δC 21.1, +8.4 ppm) and 2D NMR experiments showed the presence of an Ang moiety at C-22 (Figure 1). Accordingly, the structure of compound 6 was assigned as 21β,22α-di-O-angeloyl15α,16α,23,28-tetrahydroxyolean-12-ene 3β-O-α-L-rhamnopyrE



Article

Compound 9, an amorphous powder, had a molecular formula of C57H92O23 based on its HRESIMS data (m/z 1143.5934 [M − H]−, calcd for 1143.5951). The NMR spectroscopic data revealed that its structure was similar to 8 except for the substituent at C-22. The significant changes in the chemical shifts of C-2′′′′′ (δC 42.4, −87.6 ppm) and C-3′′′′′ (δC 27.7, −108.9 ppm) showed that the functional group at C-22 was a 2-MB moiety by 2D NMR data (Figure 1). Accordingly, the structure of compound 9 was assigned as 16α,21β,23,28tetrahydroxy-22α-O-(2-methylbutanoyl)-15α-O-(3methylbutanoyl)olean-12-ene 3β-O-α-L-rhamnopyranosyl-(1→ 3)-α-L-arabinopyranosyl-(1→3)-β-D-glucuronopyranoside. Compound 10 was purified as an amorphous powder with a molecular formula of C57H88O23 (m/z 1139.5679 [M − H]−, calcd for 1139.5638). The structure of 10 was similar to 4, and the main differences were the deshielded shifts of C-4 (δC 56.0, +12.1 ppm) and C-23 (δC 207.0, +142.3 ppm) and the upfield shift of C-24 (δC 10.9, −3.3 ppm). The NMR data showed signals for a formyl group at δH/δC 9.73 (s, 1H, H-23)/207.0 (C23), which was attached to C-23 based on the correlations between C-24 (δC 10.9), C-5 (δC 56.0), and H-23 (δH 9.73) in the HMBC experiment (Figure 1). Accordingly, the structure of compound 10 was assigned as 21β-O-angeloyl-15α,16α,28trihydroxy-22α-O-(2-methylbutanoyl)olean-12-en-23-al 3β-Oα-L-rhamnopyranosyl-(1→3)-α-L-arabinopyranosyl-(1→3)-βD-glucuronopyranoside. The molecular formula of compound 11, an amorphous powder, was assigned as C57H88O23 based on its HRESIMS data (m/z 1139.5687 [M − H]−, calcd for 1139.5638). The NMR spectra showed similar signals to those of 10 except for the resonances for the substituent at C-22. The chemical shifts of C4′′′′′ (δC 22.8, +10.5 ppm) and C-5′′′′′ (δC 22.9, +5.8 ppm) showed that the functional group attached to C-22 is a 3-MB moiety by 2D NMR data (Figure 1). Accordingly, the structure of compound 11 was assigned as 21β-O-angeloyl-15α,16α,28trihydroxy-22α-O-(3-methylbutanoyl)olean-12-en-23-al 3β-Oα-L-rhamnopyranosyl-(1→3)-α-L-arabinopyranosyl-(1→3)-βD-glucuronopyranoside. Compounds 1 and 4−11 did not show any significant cytotoxicity against HaCaT cells at concentrations of 3.125 to 25 μM for 24 h, while compounds 2 and 3 slightly affected cell viability (72.7% and 69.7%) at 25 μM (Figure S58, Supporting Information). Since the activation of ARE-luciferase in HaCaT cells is mainly regulated by Nrf2 through binding to the ARE sequences in the promoter region of target genes, the changes in the expression of Nrf2 after treatment with compounds 1−11 were investigated (Figure 3A). Compounds 1−11 increased the ARE-luciferase activity compared with the vehicle-treated cells. Treatment with compounds 4, 5, and 6, having hydroxy moieties at C-15 and C-23, increased ARE-luciferase activities more than 2-fold at concentrations of 25 μM. In addition, compounds 1− 11 increased Nrf2 accumulation in the nucleus compared to the vehicle after 6 h of treatment (Figure 3B). In conclusion, 11 new triterpenoid saponins containing a triglycosidic moiety of α-L-rhamnopyranosyl-(1→3)-α-L-arabinopyranosyl-(1→3)-β-D-glucuronopyranosyl at C-3 and different ester moieties, such as acetyl, angeloyl, 2-methylbutanoyl, and 3-methylbutanoyl, were isolated from C. japonica roots. Compounds 4, 5, 6, 8, and 11 significantly increased Nrf2 activity. This suggests that triterpenoid saponins from C. japonica roots are potential therapeutic candidates for protection against oxidative stress via Nrf2 activation.

Figure 2. Key NOESY correlations of compounds 1 and 4.

spectroscopic data showed signals for two acetyl groups at δH/δC 5.68 (d, J = 3.6 Hz, 1H, H-15)/69.8 (C-15) and 5.89 (d, J = 4.3 Hz, 1H, H-16)/71.6 (C-16). The locations of these two acetyl moieties were determined from the correlations from H-15 (δH 5.68) to C-1′′′′′′ (δC 170.9) and C-2′′′′′′ (δC 21.9) and from H16 (δH 5.89) to C-1′′′′′′′ (δC 170.4) and C-2′′′′′′′ (δC 22.2). Accordingly, the structure of compound 7 was assigned as 15α,16α-di-O-acetyl-21β-O-angeloyl-23,28-dihydroxy-22α-O(3-methylbutanoyl)olean-12-ene 3β-O-α-L-rhamnopyranosyl(1→3)-α-L-arabinopyranosyl-(1→3)-β-D-glucuronopyranoside. Compound 8, an amorphous powder, was deduced to have the molecular formula C57H90O23 by HRESIMS analysis (m/z 1141.5800 [M − H]−, calcd 1141.5795). Its structure was similar to 4 except for the substituents at C-15, C-21, and C-22. The significant changes in the chemical shifts of C-15 (δC72.7, +4.7 ppm), C-21 (δC 77.0, −2.1 ppm), C-22 (δC 76.9, +3.3 ppm), C-2⁗ (δC130.0, +4.7 ppm), and C-3⁗ (δC 136.6, +3.3 ppm) showed that the functional groups at C-15, C-21, and C22 were 3-MB, OH, and Ang, respectively (Figure 1). The NMR data showed signals at δH 0.73 (d, J = 6.6 Hz, 3H, H-4⁗), 0.85 (d, J = 6.5 Hz, 3H, H-5⁗), 1.88 and 2.00 (both m, 2H, H-2⁗), and 2.06 (m, 1H, H-3⁗) for 3-MB, and the location of this group was defined from the correlations between H-15 (δH 5.60) and C-1⁗ (δC 173.0) (Figure 1). Accordingly, the structure of compound 8 was assigned as 22α-O-angeloyl-16α,21β,23,28tetrahydroxy-15α-O-(3-methylbutanoyl)olean-12-ene 3β-O-αL-rhamnopyranosyl-(1→3)-α-L-arabinopyranosyl-(1→3)-β-Dglucuronopyranoside. F



Article

analytical separations] (YMC Co. Ltd., Japan). The purities of the sugar standards, D-glucuronic acid, L-rhamnose, L-arabinose, and D-arabinose, purchased from TCI (Tokyo, Japan), were above 98%. Anhydrous pyridine (Sigma-Aldrich), L-cysteine methyl ester hydrochloride, and otolylisothiocyanate (TCI, >98%) were used for the sugar derivatization experiments prior to HPLC analysis. Primary antibodies against Nrf2 and Histon H3 and secondary antibodies were purchased from Abcam (Cambridge, UK). Plant Material. Dried roots of C. japonica (11.5 kg) were collected by Mr. Sunho Choi in Haenam, Jeollanam Province, Korea, in August 2016. The material was identified by Prof. Yong Soo Kwon in College of Pharmacy, Kangwon National University, and stored in the Herbarium of Kangwon National University (KNUP-CJ-1). Extraction and Isolation. The dried roots of C. japonica (11.5 kg) were extracted twice with 80% MeOH in an ultrasonic bath for 3 h. The 80% MeOH extract (476 g) was partitioned using an n-BuOH (1 L × 4) and H2O (1 L) mixture to obtain an n-BuOH-soluble (45.3 g) fraction and a H2O-soluble (380.7 g) fraction. The n-BuOH extract was separated on a Diaion HP-20 resin column with a gradient solvent system consisting of a mixture of H2O and MeOH (each 1 L; 100:0, 80:20, 60:40, 40:60, 20:80, 0:100, and 0:100) to obtain seven fractions (B1−7). Fraction B6 (7 g) was subjected to RP silica gel CC [H2O/ MeOH = 50:50 → 40:60 → 30:70 → 20:80 → 10:90 → MeOH], which yielded 14 fractions (B6-1−14). B6-7 (290 mg) was separated by preparative HPLC [MeOH/H2O = 60:40 → MeOH] to give fraction B6-7-7 (35 mg), which was purified by preparative HPLC [MeCN/ H2O in 0.1% FA = 50:50] to give compound 8 (5.1 mg). B6-8 (200 mg) was separated by preparative HPLC [MeOH/H2O = 60:40 → MeOH] to give B6-8-3 (156 mg), which was purified by preparative HPLC [MeOH/H2O = 75:25] to give compound 9. B6-10 (300 mg) was subjected to preparative HPLC [MeOH/H2O = 72:28 → MeOH] to give B6-10-3 (150 mg), which was purified by preparative HPLC [MeCN/H2O in 0.1% FA = 50:50] to give compounds 4 (9.8 mg), 5 (3.1 mg), 6 (4.6 mg), 7 (5.9 mg), 10 (6.4 mg), and 11 (6.1 mg). The separation of B7 (11 g) was performed by reversed-phase silica gel CC [H2O/MeOH = 50:50 → 40:60 → 30:70 → 20:80 → 10:90 → MeOH], which yielded 25 fractions (B7-1−25). B7-18 (200 mg) was partitioned by preparative HPLC [MeCN/H2O = 70:30] to give four fractions (B7-18-1−7-18-4). Further separation of B7-18-2 (28 mg) was performed by preparative HPLC [MeCN/H2O in 0.1% FA = 50:50] to give compounds 1 (3.2 mg), 2 (4.5 mg), and 3 (5.2 mg). 16α-O-Acetyl-21β-O-angeloyl-23,28-dihydroxy-22α-O-(2methylbutanoyl)olean-12-ene 3β-O-α-L-rhamnopyranosyl-(1→3)α-L-arabinopyranosyl-(1→3)-β-D-glucuronopyranoside (1): white, amorphous powder; [α]25D −15.9 (c 0.01 MeOH); 1H and 13C NMR, see Table 1; HRESIMS m/z 1167.5956 [M − H]− (calcd for C59H92O23 1167.5951). 21β-O-Angeloyl-16α,23,28-trihydroxy-22α-O-(2methylbutanoyl)olean-12-ene 3β-O-α-L-rhamnopyranosyl-(1→3)α-L-arabinopyranosyl-(1→3)-β-D-glucuronopyranoside (2): white, amorphous powder; [α]25D −3.9 (c 0.01 MeOH); 1H and 13C NMR, see Table 1; HRESIMS m/z 1125.5859 [M − H]− (calcd for C57H90O22 1125.5845). 21β-O-Angeloyl-16α,23,28-trihydroxy-22α-O-(3methylbutanoyl)olean-12-ene 3β-O-α-L-rhamnopyranosyl-(1→3)α-L-arabinopyranosyl-(1→3)-β-D-glucuronopyranoside (3): white, amorphous powder; [α]25D −1.8 (c 0.16 MeOH); 1H and 13C NMR, see Table 1; HRESIMS m/z 1125.5856 [M − H]− (calcd for C57H90O22 1125.5845). 21β-O-Angeloyl-15α,16α,23,28-tetrahydroxy-22α-O-(2methylbutanoyl)olean-12-ene 3β-O-α-L-rhamnopyranosyl-(1→3)α-L-arabinopyranosyl-(1→3)-β-D-glucuronopyranoside (4): white, amorphous powder; [α]25D −4.0 (c 0.11 MeOH); 1H and 13C NMR, see Table 1; HRESIMS m/z 1141.5800 [M − H]− (calcd for C57H90O23 1141.5795). 21β-O-Angeloyl-15α,16α,23,28-tetrahydroxy-22α-O-tigloylolean-12-ene 3β-O-α-L-rhamnopyranosyl-(1→3)-α-L-arabinopyranosyl-(1→3)-β-D-glucuronopyranoside (5): white, amorphous powder; [α]25D −9.8 (c 0.02 MeOH); 1H and 13C NMR, see Table 1; HRESIMS m/z 1139.5658 [M − H] − (calcd for C 57 H 88 O 23 1139.5638).

Figure 3. Compounds 1−11 induced ARE luciferase activity and promoted Nrf2 nuclear accumulation in HaCaT ARE cells. (A) HaCaT-ARE cells were treated with 25 μM of compounds 1−11 for 6 h followed by ARE-luciferase activity was measured. (B) Cells were treated with 25 μM of compound 1−11 for 6 h and horseradish peroxide-conjugated secondary antibodies, and the expression of Nrf2 in the nucleus was analyzed by Western blot. Histon H3 was used as nuclear protein loading control. *p < 0.05, **p < 0.01, ***p < 0.001.

■

EXPERIMENTAL SECTION

General Experimental Procedures. The optical rotations were measured using a JASCO P-2000 polarimeter. High-resolution mass (HRMS) spectra were recorded on a Waters Xevo-G2 mass spectrometer coupled with a Waters UPLC system. The 1D and 2D NMR spectra were recorded on a Bruker Avance II 600 (Bruker, Germany) spectrometer using pyridine-d5 as the solvent in the Central Laboratory of Kangwon National University (Chuncheon, Korea) and the National Center for Inter-University Research Facilities of Seoul National University. Column chromatography (CC) was performed with normal-phase silica gel 60 (Merck, Germany), reversed-phase YMC ODS-A C18 (75 μm, YMC Co. Ltd., Japan), and Diaion HP-20 (Mitsubishi Chemical Industries Ltd., Japan). TLC experiments were performed on precoated silica gel 60 RP-18 F254s plates (Merck, Germany). Preparative HPLC separations were conducted on a 1525 binary HPLC pump (Waters, USA), while analytical HPLC experiments were done on an Agilent 1260 Infinity quaternary LC instrument (Agilent, Santa Clara, CA, USA). The columns used were a HECTORM C18 [250 × 21.2 mm i.d. (5 μm) for preparative separations] (RSTech, Korea) and a YMC-Triart C18 [250 × 4.6 mm i.d. (5 μm) for G



Article

Table 2. NMR Spectroscopic Data for Compounds 7−11 (Pyridine-d5, 150 MHz for 7 position

δC, type

δH (J in Hz)

8 δC, type

δH (J in Hz)

39.3, CH2

0.94 m, 1.47 m

39.4, CH2

1.00 m, 1.48 m

39.4, CH2

2

26.5, CH2

1.99 m, 2.30 m

26.6, CH2

1.99 m, 2.31 m

26.4, CH2

3 4 5 6

80.7, 43.9, 47.0, 18.9,

CH C CH CH2

4.36 m

4.40 m

7

35.6, CH2

8 9 10 11

42.2, 47.6, 37.2, 24.3,

12 13 14 15 16 17 18 19

128.1, CH 141.1, C 47.0, C 69.8, CH 71.6, CH 48.5, C 40.6, CH 47.3, CH2

20 21 22 23

36.5, 78.6, 72.0, 64.3,

C CH CH CH2

24 25 26 27 28

14.2, 16.7, 18.0, 21.4, 63.0,

CH3 CH3 CH3 CH3 CH2

29 30 15-O-3MB 1⁗ 2⁗

29.7, CH3 20.0, CH3

3⁗ 4⁗ 5⁗ 15-Ac 1′′′′′′ 2′′′′′′ 16-Ac 1′′′′′′′ 2′′′′′′′ 21-OAng 1⁗ 2⁗ 3⁗

1.77 m 1.31 m, 1.72 m

81.0, 43.9, 47.3, 19.0,

CH C CH CH2

1.81 m 1.40 m, 1.81 m

81.3, 43.9, 47.2, 19.0,

1.32 m, 2.02 m

35.8, CH2

1.42 m, 2.15 m

35.9, CH2

1.78 m 1.79 m, 1.93 m 5.61 br s

5.68 d (3.6) 5.89 d (4.3) 3.20 m 1.51 m, 2.74 m

5.78 d (10.3) 6.27 d (10.3) 3.75 d (10.2), 4.35 m 0.94 s 0.90 s 1.03 s 1.29 s 3.75 d (10.2), 3.89 d (10.5) 1.06 s 1.29 s

170.9, C 21.9, CH3

2.03 s

170.4, C 22.2, CH3

2.55 s

168.0, C 128.6, C 139.7, CH

4⁗

16.6, CH3

5⁗

21.5, CH3

42.2, 47.6, 37.3, 24.4,

C CH C CH2

126.7, CH 143.2, C 47.6, C 72.7, CH 72.0, CH 49.3, C 41.7, CH 47.8, CH2 37.5, 77.0, 76.9, 64.5,

C CH CH CH2

14.1, 16.8, 18.0, 22.0, 63.6,

CH3 CH3 CH3 CH3 CH2

1.87 m 1.86 m, 1.96 m 5.62 br s

5.60 br s 4.69 br s 3.24 m 1.48 m, 3.09 m

5.03 d (9.9) 6.32 d (9.8) 3.73 d (10.6), 4.35 m 0.96 s 0.93 s 1.08 s 2.05 s 3.75 d (10.6), 3.95 d (10.4)

C and 600 MHz for 1H)

9 δC, type

1

C CH C CH2

13

42.2, 47.6, 37.5, 24.4,

CH C CH CH2

C CH C CH2

126.7, CH 143.2, C 47.6, C 72.6, CH 71.7, CH 49.4, C 41.6, CH 47.8, CH2 37.3, 77.0, 76.5, 64.5,

C CH CH CH2

14.1, 16.8, 18.0, 22.0, 63.4,

CH3 CH3 CH3 CH3 CH2

30.6, CH3 19.7, CH3

1.27 s 1.43 s

30.6, CH3 19.7, CH3

173.0, C 44.0, CH2

1.88 m, 2.00 m

173.0, C 44.0, CH2

25.6, CH 22.7, CH3 22.9, CH3

2.06 m 0.73 d (6.6) 0.85 d (6.5)

25.5, CH 22.7, CH3 23.0, CH3

δH (J in Hz) 1.00 m, 1.50 m 2.00 m, 2.30 m 4.39 m 1.81 m 1.40 m, 1.81 m 1.42 m, 2.15 m 1.87 m 1.87 m, 1.96 m 5.60 br s

5.56 br s 4.66 br s 3.24 m 1.46 m, 3.06 m 5.00 d (9.7) 6.20 d (9.8) 3.74 m, 4.35 m 0.96 s 0.94 s 1.09 s 2.06 s 3.71 m, 3.93 d (10.5) 1.28 s 1.41 s

10 δC, type

2.10 dd (7.1, 1.1) 1.99 s

0.99 m, 1.48 m 38.8, CH2

25.6, CH2

2.26 m

25.6, CH2

82.3, 56.0, 47.5, 21.2,

4.21 m

CH C CH CH2

CH C CH CH2

82.2, 56.0, 1.81 m 47.5, 1.13 m, 1.49 m 21.2,

36.5, CH2

2.08 m

36.5, CH2

42.2, 47.8, 36.5, 24.4,

1.86 m

C CH C CH2

C CH C CH2

125.6, CH 144.2, C 48.3, C 68.0, CH 73.5, CH 49.0, C 41.3, CH 47.2, CH2 36.8, C 79.1, CH 73.5, CH 207.0, CH 10.9, CH3 16.4, CH3 18.0, CH3 21.7, CH3 63.4, CH2 29.9, CH3 20.7, CH3

42.1, 47.8, 36.5, 1.82 m, 1.92 m 24.4, 5.53 br s

125.6, CH 144.2, C 48.3, C 4.19 br s 68.0, CH 4.40 br s 73.3, CH 49.0, C 3.11 m 41.4, CH 1.44 m, 3.07 m 47.3, CH2

6.68 d (10.2) 6.29 d (10.2) 9.73 s 1.33 s 0.87 s 1.01 s 1.87 s 3.50 d (10.5), 3.78 m 1.12 s 1.33 s

36.8, C 79.2, CH 73.9, CH 207.0, CH 10.9, CH3 16.4, CH3 18.0, CH3 21.6, CH3 63.6, CH2 29.9, CH3 20.7, CH3

δH (J in Hz) 0.98 m, 1.48 m 2.28 m 4.22 m 1.82 m 1.14 m, 1.50 m 2.09 m

1.74 m 1.82 m, 1.91 m 5.53 br s

4.19 m 4.40 m 3.10 m 1.44 m, 3.09 m 6.66 d (10.3) 6.26 d (10.2) 9.74 s 1.33 s 0.87 s 1.02 s 1.87 s 3.50 d (10.4), 3.76 m 1.12 s 1.33 s

1.95 m, 2.04 m 2.07 m 0.75 d (6.3) 0.87 br s

16.5, CH3 21.5, CH3 H

11 δC, type

38.8, CH2

168.1, C 129.1, C 139.1, CH

6.06 m

δH (J in Hz)

6.09 ddd (14.2, 7.1, 1.1) 2.18 dd (7.2, 1.3) 2.06 s

168.2 129.3 138.6, CH 16.5, CH3 21.5, CH3

6.06 m 2.17 dd (7.2, 1.3) 2.05 s



Article

Table 2. continued 7 position

δC, type

8

δH (J in Hz)

δC, type

9

δH (J in Hz)

22-O-2MB 1′′′′′ 2′′′′′ 3′′′′′ 4′′′′′ 5′′′′′ 22-O-3MB 1′′′′′ 2′′′′′ 3′′′′′ 4′′′′′ 5′′′′′ 22-OAng 1′′′′′ 2′′′′′ 3′′′′′ 4′′′′′ 5′′′′′ 3-O-DGlcA 1′ 2′ 3′ 4′ 5′ 6′ 3′-O-LAra 1″ 2″ 3″ 4″ 5″ 3″-O-LRha 1‴ 2‴ 3‴ 4‴ 5‴ 6‴

δC, type

178.2, C 42.4, CH 27.7, CH2 12.4, CH3 17.4, CH3

172.9, C 43.9, CH2 26.0, CH 22.8, CH3 22.8, CH3

2.23 2.14 0.82 0.84

2.44 m 1.48 m, 1.81 m 0.93 m 1.16 d (7.0)

11

δC, type

δH (J in Hz)

177.1, C 41.9, CH 27.4, CH2

2.08 m 1.23 m, 1.60 m

12.3, CH3 17.1, CH3

0.68 t (7.4) 1.03 d (7.0)

5.14 m 4.07 m 4.20 m

δC, type

173.3, C 44.0, CH2 26.1, CH 22.8, CH3 22.9, CH3

m m d (6.6) d (6.6)

168.9, C 130.0, C 136.6, CH 16.3, CH3 21.4, CH3

105.3, CH 74.5, CH 86.5, CH

10

δH (J in Hz)

105.5, CH 74.6, CH 86.5, CH

δH (J in Hz)

1.94 1.97 0.78 0.72

m m d (6.3) d (6.2)

5.81 dt (7.1, 6.1) 2.02 d (7.1) 1.95 s

5.16 m 4.08 m 4.19 m

105.4, CH 74.6, CH 86.4, CH

5.17 m 4.09 m 4.20 m

105.3, CH 74.5, CH 86.2, CH

5.17 m 4.09 m 4.29 m

105.4, CH 74.6, CH 86.2, CH5 72.1, CH 77.2, CH 174.1, C

5.16 m 4.08 m 4.21 m

71.9, CH 76.7, CH 174.1, C

4.31 m 4.32 m

72.0, CH 77.1, CH 174.1, C

4.36 m 4.37 m

72.1, CH 77.2, CH 174.1, C

4.40 m 4.45 m

71.9, CH 77.2, CH 174.0, C

4.39 m 4.42 m

106.4, CH 72.4, CH 80.8, CH 69.8, CH 68.6, CH2

5.17 m

106.6, CH 72.4, CH 80.8, CH 69.8, CH 68.6, CH2

5.17 m

106.6, CH 72.4, CH 80.8, CH 69.8, CH 68.6, CH2

5.17 m

106.5, CH 72.5, CH 80.8, CH 69.8, CH 68.6, CH2

5.20 m

106.5, CH 4.66 m 72.5, CH 4.26 m 80.9, CH 4.43 m 69.8, CH 3.78 m, 4.29 m 68.5, CH2

5.20 m

104.3, CH 72.7, CH 73.0, CH 74.6, CH 70.6, CH 19.1, CH3

6.04 br s

6.04 br s

104.3, CH 72.7, CH 73.0, CH 74.6, CH 70.6, CH 19.1, CH3

4.67 m 4.27 m 4.43 m 3.80 d (12.2), 4.30 m

6.04 br s 4.75 4.58 4.32 4.61 1.68

br s m m m d (5.7)

104.3, CH 72.7, CH 73.1, CH 74.6, CH 70.6, CH 19.1, CH3

4.66 m 4.27 m 4.43 m 3.78 d (12.1), 4.28 m

6.05 br s 4.74 4.57 4.32 4.61 1.67

m m m m d (6.1)

104.3, CH 72.7, CH 73.1, CH 74.6, CH 70.6, CH 19.1, CH3

4.66 m 4.26 m 4.43 m 3.79 m, 4.29 m

6.05 br s 4.75 4.58 4.33 4.61 1.64

m m m m d(6.0)

4.74 4.57 4.32 4.61 1.67

m m m m d (5.7)

104.3, CH 72.7, CH 73.1, CH 74.6, CH 70.6, CH 19.1, CH3

4.36 m 4.37 m

4.66 m 4.26 m 4.42 m 3.79 m, 4.29 m

4.74 4.58 4.32 4.61 1.67

m m m m d (4.6)

α-L-arabinopyranosyl-(1→3)-β-D-glucuronopyranoside (8): [α]25D −6.6 (c 0.14 MeOH); 1H and 13C NMR, see Table 2; HRESIMS m/ z 1141.5800 [M − H]− (calcd for C57H90O23 1141.5795). 16α,21β,23,28-Tetrahydroxy-22α-O-(2-methylbutanoyl)-15α-O(3-methylbutanoyl)olean-12-ene 3β-O-α-L-rhamnopyranosyl-(1→ 3)-α- L -arabinopyranosyl-(1→3)-β- D -glucuronopyranoside (9): [α]25D −6.4 (c 0.16 MeOH); 1H and 13C NMR, see Table 2; HRESIMS m/z 1143.5934 [M − H] − (calcd for C 57 H 92 O 23 1143.5951). 21β-O-Angeloyl-15α,16α,28-trihydroxy-22α-O-(2methylbutanoyl)olean-12-en-23-al 3β-O-α-L-rhamnopyranosyl(1→3)-α-L-arabinopyranosyl-(1→3)-β-D-glucuronopyranoside (10):

21β,22α-Di-O-angeloyl-15α,16α,23,28-tetrahydroxyolean-12ene 3β-O-α-L-rhamnopyranosyl-(1→3)-α-L-arabinopyranosyl-(1→ 3)-β-D-glucuronopyranoside (6): white, amorphous powder; [α]25D −4.5 (c 0.13 MeOH); 1H and 13C NMR, see Table 1; HRESIMS m/z 1139.5660 [M − H]− (calcd for C57H88O23 1139.5638). 15α,16α-Di-O-acetyl-21β-O-angeloyl-23,28-dihydroxy-22α-O-(3methylbutanoyl)olean-12-ene 3β-O-α-L-rhamnopyranosyl-(1→3)α-L-arabinopyranosyl-(1→3)-β-D-glucuronopyranoside (7): [α]25D −7.2 (c 0.19 MeOH); 1H and 13C NMR, see Table 2; HRESIMS m/ z 1199.6528 [M − H]− (calcd for C61H100O23 1199.6577). 22α-O-Angeloyl-16α,21β,23,28-tetrahydroxy-15α-O-(3methylbutanoyl)olean-12-ene 3β-O-α-L-rhamnopyranosyl-(1→3)I



Article

[α]25D −4.1 (c 0.17 MeOH); 1H and 13C NMR, see Table 2; HRESIMS m/z 1139.5679 [M − H]− (calcd for C57H88O23 1139.5638). 21β-O-Angeloyl-15α,16α,28-trihydroxy-22α-O-(3methylbutanoyl)olean-12-en-23-al 3β-O-α-L-rhamnopyranosyl(1→3)-α-L-arabinopyranosyl-(1→3)-β-D-glucuronopyranoside (11): [α]25D −4.1 (c 0.17 MeOH); 1H and 13C NMR, see Table 2; HRESIMS m/z 1139.5687 [M − H]− (calcd for C57H88O23 1139.5638). Acid Hydrolysis and Determination of the Absolute Configurations of the Sugars. The absolute configurations of the constituent monosaccharide moieties of compounds 1−11 were determined with a slight modification of a literature precedure.27 Compounds 1−11 (each 1.0 mg) were dissolved in 1 mL of 2 M HCl (H2O/1,4-dioxane, 1:1, v/v), and each solution was heated at 90 °C for 4 h. After cooling, the solvent was evaporated under N2, and the residue was redissolved in water and extracted with EtOAc (3 × 3 mL). The water layer was concentrated under N2, and the remaining portion was dissolved in 300 μL of pyridine containing 1 mg of L-cysteine methyl ester hydrochloride. After heating at 60 °C for 1 h, 10 μL of otolylisothiocyanate was reacted with the mixture at 60 °C for 1 h. The reactant was directly measured by an HPLC system equipped with a YMC-Triart C18 column (YMC Co. Ltd., Japan, 250 × 4.6 mm, 5 μm) with isocratic elution with MeCN/0.1% H3PO4 for 50 min. The injection volume was 10 μL, and the flow rate was 0.8 mL/min. The UV data were recorded at 250 nm. The derivatives of D-glucuronic acid, Larabinose, and L-rhamnose in compounds 1−11 were determined by comparison to the retention times of the standards (tR: D-glucuronic acid 22.5 min, L-arabinose 24.6 min, D-arabinose 26.0 min, and Lrhamnose 38.1 min). Cell Culture. HaCaT cells were purchased from American Type Culture Collection (ATCC), and HaCaT-ARE cells, stably transfected with 3xARE elements, were kindly gifted by Prof Young-Sam Keum of Dongguk University.29 The cells were cultured in RPMI 1640 media with 10% fetal bovine serum supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B and stored at 37 °C in a humidified incubator supplied with 5% CO2 and 95% air. Cell Viability Assay. The cytotoxic effects of compounds 1−11 on HaCaT cells were measured by an MTT assay. Briefly, after incubating in 48-well plates overnight, the cells were treated with different concentrations of compounds 1−11 for 24 h. Each well was treated with 20 μL of MTT solution (5 mg/mL) and incubated for 2 h. After removing the media, DMSO was added and the soluble formazan was measured at 570 nm by an ELISA plate reader (Tecan Instruments, Switzerland). ARE-Luciferase Assay. To determine whether compounds 1−11 induce Nrf2 activation after treating in HaCaT-ARE cells with compounds 1−11, the ARE-luciferase activity was measured by a luciferase assay kit (Promega, USA). Briefly, after incubating the cells in 48-well plates overnight, the cells were treated with different concentrations of compounds 1−11 for 6 h. After lysing with 100 μL of passive lysis buffer for 15 min at room temperature with shaking, the ARE-luciferase activity was measured according to the manufacturer′s directions and was calculated after normalization with total protein concentration. Western Blot Analysis. HaCaT cells were incubated in 60 mm dishes until 60−70% confluency prior to drug treatment. The cells were treated with compounds 1−11 or DMSO (0.1%) at the appropriate concentration and time, and the nuclear protein was extracted using MPER (Thermo Scientific) in the presence of a protease inhibitor cocktail for 5 min on ice. The samples were centrifuged at 15 000 rpm for 15 min. BCA reagent (Thermo Scientific) was used to measure the protein concentration at 570 nm. The proteins (25 μg) were electrophoresed on 10% SDS-polyacrylamide gel and transferred to nitrocellulose membranes in Tris-glycine buffer containing 20% MeOH. A solution of 5% nonfat dry milk in PBS-T (phosphate-buffered saline, 0.1% Tween20) was applied to block the membranes for 1 h, and the samples were incubated with the primary antibodies. After washing the membrane with PBS-T, the samples were incubated with HRP secondary antibodies for 1 h, and the proteins were analyzed using an ECL kit (Bio-Rad) under a ChemiDoc image analyzer (Bio-Rad).

Preparation of Nuclear and Cytosolic Extracts. Proteins were extracted by M-PER buffer (Thermo Scientific). After treatment, the cells were lysed using M-PER for 10 min on ice to release the cytosolic proteins. The cell lysates were separated into cytosolic and nuclear proteins by centrifugation at 3000 rpm for 4 min. The pellet was resuspended to collect the nuclear proteins in M-PER. After sonication of both groups of proteins and centrifugation at 12 000 rpm for 10 min, the cytosolic and nuclear proteins were measured using the BCA reagent. Statistical Analysis. The results of the biological assays are expressed as the mean ± standard deviation (SD) from three independent experiments, and the statistical significance was analyzed using a t test with p-values of *p < 0.05, **p < 0.01, and ***p < 0.001 by comparison with the control.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00374.

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1D (1H and 13C) and 2D (HSQC and HMBC) NMR spectra for compounds 1−11; the HaCaT cell viability of 1−11 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Tel (J. H. Kim): (+82)-55-772-8072. E-mail: junghwan.kim@ gnu.ac.kr. *Tel (H. Yang): (+82)-33-250-6919. E-mail: heejyang@ kangwon.ac.kr. ORCID

Heejung Yang: 0000-0001-5986-9024 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2018R1C1B6002574).

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REFERENCES

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