Neuroprotective Xanthones from the Root Bark of Cudrania

Jul 22, 2014 - Natural Products Research Institute, College of Pharmacy, Seoul ... Jaeyoung Kwon , Young Hye Seo , Jae-Eun Lee , Eun-Kyoung Seo , Shen...
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Neuroprotective Xanthones from the Root Bark of Cudrania tricuspidata Jaeyoung Kwon,† Nguyen Tuan Hiep,† Dong-Woo Kim,‡ Bang Yeon Hwang,§ Hak Ju Lee,⊥ Woongchon Mar,*,‡ and Dongho Lee*,† †

Department of Biosystems and Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Korea Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul 151-742, Korea § College of Pharmacy, Chungbuk National University, Cheongju 361-763, Korea ⊥ Korea Forest Research Institute, Seoul 130-712, Korea ‡

S Supporting Information *

ABSTRACT: Seventeen new prenylated xanthones (1−17) were isolated from an ethyl acetate-soluble extract of root bark of Cudrania tricuspidata together with 17 previously identified xanthones. The structures of the new compounds were elucidated by spectroscopic methods. Six new compounds (3, 7, 8, 9, 15, and 16) and six known compounds (18−23) showed neuroprotective effects against 6-hydroxydopamine-induced cell death in human neuroblastoma SH-SY5Y cells, with EC50 values of 0.7− 16.6 μM.

P

Herein, we describe the isolation, structural elucidation, and biological evaluation of these isolates.

arkinson’s disease is one of the most common neurodegenerative disorders. It is characterized by the selective degeneration of nigrostriatal dopaminergic neurons and the presence of Lewy bodies, which are abnormal aggregates of protein in nerve cells.1−3 Although recent studies have suggested that oxidative stress and mitochondrial dysfunction can be major contributing factors in Parkinson’s disease,4 its exact pathogenesis has not been fully characterized, and a cure for this disease is still unknown.5 Cudrania tricuspidata (Carr.) Bureau, a small thorny tree belonging to the family Moraceae, is mainly distributed in East Asia, and its roots have long been used to treat contusion, hemoptysis, hematemesis, lumbago, and spermatorrhea.6 Phytochemical studies of C. tricuspidata have revealed that the major constituents of this plant are various types of xanthones and flavonoids, and these constituents have shown cytotoxic,7−9 antioxidant,9,10 antiatherosclerotic,11 anti-inflammatory,11 and hepatoprotective activities.12,13 In a search to discover neuroprotective metabolites from natural products, a MeOH extract of C. tricuspidata showed significant protective effects against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in human neuroblastoma SH-SY5Y cells. 6-OHDA, as a neurotoxin, elicits dopaminergic neuronal cell death in modeling Parkinson’s disease.14 This investigation of C. tricuspidata led to the isolation of six new compounds (3, 7, 8, 9, 15, and 16) and six known compounds (18−23) that were found to be active (0.7−16.6 μM). In addition, 11 new and 11 known compounds were isolated and found to be inactive. © 2014 American Chemical Society and American Society of Pharmacognosy

■

RESULTS AND DISCUSSION

Compound 1 was isolated as a yellow, amorphous solid, and its molecular formula of C23H22O6 was determined by HRESIMS at m/z 395.1500 [M + H]+, suggesting 13 degrees of unsaturation. The 1H NMR spectrum (Table 1) showed the presence of an intramolecular hydrogen-bonded hydroxy group at δH 13.32 (1H, s, OH-1) and three aromatic protons at δH 7.45 (1H, d, J = 9.0 Hz, H-5), 7.40 (1H, d, J = 9.0 Hz, H-6), and 6.14 (1H, s, H-2). Additionally, a 2,2-dimethylpyran group at δH 6.86 (1H, d, J = 10.0 Hz, H-11), 5.74 (1H, d, J = 10.0 Hz, H-12), and 1.48 (6H, s, Me-14, 15) and a 2-hydroxy-3methylbut-3-enyl group at δH 5.12 (1H, s, H-19a), 4.86 (1H, s, H-19b), 4.53 (1H, d, J = 10.0 Hz, H-17), 4.11 (1H, dd, J = 13.0, 2.5 Hz, H-16a), 3.12 (1H, dd, J = 13.0, 10.0 Hz, H-16b), and 1.96 (3H, s, Me-20) were observed. The 13C NMR spectrum (Table 2) showed 23 carbon signals, including one carbonyl carbon, two aromatic rings, and two C5 groups. The 1D-NMR data suggested that 1 has a 1-hydroxyxanthone skeleton with two C5 groups, and the HMBC cross-peak of H-2/C-1 (δC 164.0) confirmed that the chelated hydroxy group is located at C-1. Moreover, the HMBC cross-peaks of H-11/C-4a (δC 152.2) and H-16/C-8a (δC 119.8) suggested that the 2,2dimethylpyran group is fused at C-3 and C-4, and that the 2Received: April 28, 2014 Published: July 22, 2014 1893

dx.doi.org/10.1021/np500364x | J. Nat. Prod. 2014, 77, 1893−1901

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Chart 1

a 1,1-dimethylallyl group at δH 6.44 (1H, dd, J = 18.0, 10.5 Hz, H-14), 5.43 (1H, d, J = 18.0 Hz, H-15a), 5.36 (1H, d, J = 10.5 Hz, H-15b), and 1.68 (6H, s, Me-12, 13), a 2-oxo-3-methylbut3-enyl group at δH 6.18 (1H, s, H-19a), 5.86 (1H, s, H-19b), 4.70 (2H, s, H-16), and 1.99 (3H, s, Me-20), and a methoxy group at δH 3.76 (3H, s, OMe-7). The positions of all substituents were determined by detailed analysis of the HMBC cross-peaks observed. The HMBC cross-peaks of Me-12, 13/C4 (δC 109.1), H-16/C-8a (δC 111.7), and OMe-7/C-7 (δC 143.9) suggested that the 1,1-dimethylallyl, 2-oxo-3-methylbut3-enyl, and methoxy groups are attached at C-4, C-8, and C-7, respectively. Accordingly, the structure of new compound 3 (cudratrixanthone C) was assigned as shown. However, in contrast to the other compounds, the NMR spectroscopic data for 3 were obtained using CDCl3 because it was found to be unstable in acetone-d6. The molecular formula of compound 4 (cudratrixanthone D) was established as C24H24O8 by HRESIMS at m/z 441.1563 [M + H]+. The 1H and 13C NMR spectra of 4 (Tables 1 and 2) were similar to those of 3, which showed a 1,3,6,7tetraoxygenated xanthone skeleton with methoxy and 2-oxo3-methylbut-3-enyl groups. The only difference was that a 1,1dimethylallyl group of 3 was replaced by a 2-hydroxymethyl3,3-dimethyldihydrofuran group in 4, which appeared at δH 4.48 (1H, t, J = 6.0, H-14), 3.91 (2H, d, J = 6.0 Hz, H-15), 1.65 (3H, s, Me-12), and 1.43 (3H, s, Me-13). The position of this group was determined by an HMBC correlation of Me-12, 13/ C-4 (δC 112.1), and the positions of the remaining substituents

hydroxy-3-methylbut-3-enyl group is attached at C-8, respectively. Therefore, the new compound 1 was assigned as shown and has been given the trivial name cudratrixanthone A. The molecular formula of compound 2 was determined as C23H22O6 by HRESIMS at m/z 395.1496 [M + H]+, corresponding to 13 degrees of unsaturation. The 1H NMR spectrum (Table 1) showed a sharp signal at δH 13.42 (1H, s, OH-1), characteristic of a 1-hydroxyxanthone skeleton, and two aromatic protons at δH 7.58 (1H, s, H-8) and 6.35 (1H, s, H-4). Also, resonances for a 2,3,3-trimethyldihydrofuran group at δH 4.53 (1H, q, J = 6.5 Hz, H-14), 1.49 (3H, s, Me-12), 1.40 (3H, d, J = 6.5 Hz, Me-15), and 1.24 (3H, s, Me-13) and a 2,2dimethylpyran group at δH 6.91 (1H, d, J = 10.0 Hz, H-16), 5.91 (1H, d, J = 10.0 Hz, H-17), and 1.53 (6H, s, Me-19, 20) were observed. The HMBC cross-peak of OH-1/C-1 (δC 159.2) suggested a chelated hydroxy group to be located at C-1. Also, the HMBC cross-peaks of Me-12, 13/C-2 (δC 117.3) and H-16/C-4b (δ C 147.9) indicated that the 2,3,3trimethyldihydrofuran group is fused at C-2 and C-3 and the 2,2-dimethylpyran group is located at C-5 and C-6, respectively. Thus, the structure of the new compound 2 (cudratrixanthone B) was assigned as shown. Compound 3 gave a molecular formula of C23H22O6, deduced from the HRESIMS peak at m/z 425.1587 [M + H]+. The 1H and 13C NMR spectra of 3 (Tables 1 and 2) showed also that it has a 1,3,6,7-tetraoxygenated xanthone skeleton with two C5 groups. Its 1H NMR spectrum revealed a chelated hydroxy group at δH 13.21 (1H, s, OH-1), two aromatic protons at δH 6.92 (1H, s, H-5) and 6.19 (1H, s, H-2), 1894

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Table 1. 1H NMR Spectroscopic Data for Compounds 1−9 1a

position

2a

3b 6.19, s

4a 6.10, s

5a 6.14, s

6a

7a

2 3 4 4a 4b 5 6 7 8 8a 9 9a 11

6.14, s

6.27, s

12

5.74, d (10.0)

1.49, s

1.68, s

1.65, s

1.64, s

13 14

1.48, s

1.24, s 4.53, q (6.5)

1.68, s 6.44, dd (18.0, 10.5)

1.43, s 4.48, t (6.0)

1.42, s 4.48, td (6.0, 2.0)

15

1.48, s

1.40, d (6.5)

5.43, d (18.0)

3.91, d (6.0)

3.92, dd (6.5, 2.0)

4.78, s

3.65, dd (12.0, 4.0) 3.56, dd (12.5, 9.0) 4.40, d (8.5)

4.14, dd (13.5, 2.5) 3.10, dd (13.0, 10.0) 4.53, d (10.0)

4.95, s 4.76, s 1.91, s 3.87, s 13.71, s

5.13, s 4.87, s 1.96, s

5.14, s 4.88, s 1.96, s

13.20, s

13.61, s

6.92, s

7.05, s

7.05, s

7.41, d (9.0) 7.38, d (9.0)

17 18 19 20 OMe OH-1 a

6.38, s

7.49, s

7.40, s

1.68, s

1.47, s

1.49, s

1,68, s 6.38, dd (17.5, 10.5)

1.21, s 4.55, q (6.5)

1.25, s 4.50, q (6.5)

5.03, dd (17.5, 1.0) 4.92, dd (10.5, 1.0) 4.22, d (13.5)

1.41, d (6.5)

1.39, d (6.5)

3.59, d (7.0)

5.31, t (7.0)

3.22, dd (17.0, 5.0) 2.92, dd (17.0, 6.5) 3.98, dd (6.5, 5.0)

1.88, s

1.44, s

1.65, s

1.39, s

6.85, s

3.20, dd (14.5, 4.5) 3.07, dd (14.5, 7.5) 4.48, dd (7.0, 4.0) 4.89, s 4.73, s 1.88, s

5.36, d (10.5) 16

6.51, s

7.58, s

6.86, d (10.0)

4.11, dd (13.0, 2.5) 3.12, dd (13.0, 10.0) 4.53, d (10.0)

6.91, d (10.0)

5.12, s 4.86, s 1.96, s

1.53, s

13.32, s

13.42, s

4.70, s

5.91, d (10.0)

1.53, s

6.18,s 5.86, s 1.99, s 3.76, s 13.21, s

6.24, s 5.88, s 1.93, s 3.73, s 13.44, s

9a

6.26, s

6.35, s

7.45, d (9.0) 7.40, d (9.0)

8a

3.08, dd (13.0, 10.0) 4.52, d (10.0)

13.55, s

Acetone-d6. bCDCl3.

H]+. Its 1H and 13C NMR spectra (Tables 1 and 2) were similar to those of 3, but lacked a methoxy signal and indicated the presence of a 2-hydroxy-3-methylbut-3-enyl group, instead of a 2-oxo-3-methylbut-3-enyl group. The structure of 7, including the positions of all the functional groups, was confirmed by 2DNMR studies. Therefore, the new compound 7 (cudratrixanthone G) was deduced as shown. The molecular formula of compound 8 was determined as C23H24O6 by HRESIMS at m/z 397.1643 [M + H]+. The 1DNMR data of this substance (Tables 1 and 2) were comparable to those of cudratricusxanthone J,15 with the exception of the substitution pattern of the aromatic ring. The HMBC correlation of Me-12, 13/C-2 (δC 118.8) and the unusual absence of any hydrogen-bonded hydroxy group suggested that a 2,3,3-trimethyldihydrofuran group is attached at C-1 and C-2. In addition, a 3,3-dimethylallyl (prenyl) group at δH 5.31 (1H, t, J = 7.0 Hz, H-17), 3.59 (1H, d, J = 7.0 Hz, H-16), 1.88 (3H, s, Me-19), and 1.66 (3H, s, Me-20) was observed, and its position was determined on the basis of the HMBC correlation of H16/C-4b (δC 150.2). Consequently, the structure of the new compound 8 (cudratrixanthone H) was determined as shown.

were deduced by additional HMBC correlations. Consequently, the new compound 4 was determined as shown. The molecular formula of compound 5 was determined to be C24H26O8 by HRESIMS at m/z 443.1707 [M + H]+. The 1H and 13C NMR spectroscopic data for 5 (Tables 1 and 2) were superimposable with those of 4, except that a 2-oxo-3methylbut-3-enyl group of 4 was replaced by a 2-hydroxy-3methylbut-3-enyl group in 5. From an analysis of the 1H−1H COSY, HSQC, and HMBC data, the new compound 5 (cudratrixanthone E) was determined to be as shown. Compound 6 (cudratrixanthone F) was obtained as a yellow, amorphous solid, and its elemental formula was determined to be C23H24O7 from the HRESIMS molecular ion peak at m/z 413.1580 [M + H]+. According to the 1H and 13C NMR spectra (Tables 1 and 2), compound 6 has the same skeleton as that of 1, with the only difference between these two compounds being that the 2,2-dimethylpyran group present in 1 was changed to the 2-hydroxy-3-methylbut-3-enyl group in 6, as confirmed by 1 H−1H COSY, HSQC, and HMBC spectra. Thus, the new compound 6 was assigned as shown. The molecular formula of compound 7 was found to be C23H24O7 by positive-ion HRESIMS at m/z 413.1594 [M + 1895

dx.doi.org/10.1021/np500364x | J. Nat. Prod. 2014, 77, 1893−1901

a

1896

164.0 99.2 161.3 101.2 152.2 152.5 118.0 125.8 154.4 127.4 119.8 184.5 104.7 115.5 128.1 79.1 28.4 28.4 34.4 78.0 149.3 110.2 18.6

1 2 3 4 4a 4b 5 6 7 8 8a 9 9a 11 12 13 14 15 16 17 18 19 20 OMe

159.2 117.3 166.4 89.8 158.5 147.9 109.3 146.8 144.1 110.3 114.3 180.9 104.1 43.9 25.4 20.8 91.6 14.5 115.7 131.3 79.2 28.0 28.0

2a

161.7 100.2 162.1 109.1 154.9 154.7 102.1 155.1 143.9 129.9 111.7 181.8 104.2 40.8 28.0 28.0 149.3 113.2 37.4 199.0 144.8 123.8 17.8 62.4

3b,d 164.0 92.9 165.6 112.1 152.1 154.7 102.5 156.5 144.6 131.3 111.4 181.5 103.2 43.0 26.5 20.4 94.5 60.8 37.0 197.9 144.8 122.8 17.1 60.3

4a 165.0 93.9 166.5 112.8 152.9 155.9 103.3 157.8 145.9 136.2 112.3 183.3 104.4 43.9 27.4 21.4 95.5 61.3 34.3 76.3 150.1 109.7 18.4 61.1

5a,d 162.7 99.1 165.0 104.2 155.8 152.5 117.8 125.5 154.1 127.3 119.6 184.6 104.3 30.0 76.3 148.2 110.4 18.4 34.4 78.0 149.4 110.2 18.6

6a,d 162.1 99.6 162.4 111.2 154.3 154.0 101.8 156.2 143.0 127.4 111.7 183.7 104.5 41.7 28.2 28.2 151.5 108.1 34.3 78.4 149.1 110.2 18.7

7a,d 160.7 118.8 159.6 96.4 158.8 150.2 116.1 149.9 142.5 107.6 115.8 174.5 103.7 43.9 25.9 21.1 91.5 14.6 23.2 122.8 132.1 18.1 26.0

8a 159.0 117.3 166.3 89.9 158.7 148.9 109.5 150.0 144.5 106.4 113.8 181.1 104.2 44.1 25.5 20.9 91.7 14.6 26.6 68.5 80.2 25.5 21.3

9a,d

Acetone-d6. bCDCl3. cPyridine-d5. dSignals derived from HSQC and HMBC experiments. eNo signal detected.

1a

position

Table 2. 13C NMR Spectroscopic Data for Compounds 1−17 158.7 117.4 166.5 90.0 158.8 148.0 110.4 143.7 147.0 109.3 114.4 181.0 104.2 43.6 26.8 20.6 95.6 61.3 115.8 131.5 79.4 28.1 28.1

10a,d

153.1 103.6 153.8 139.0 121.0 108.4 183.2 104.6 43.9 27.4 21.4 95.5 61.4 121.6 133.7 76.8 27.1 27.1

e

165.3 93.9 166.4 112.8

11a 164.6 93.9 166.3 113.4 153.8 150.5 100.1 156.4 142.0 127.3 107.0 181.2 104.3 44.5 26.1 21.7 91.6 14.5 109.6 149.1

12a,d 159.5 117.3 166.4 89.8 158.6 156.4 115.9 148.4 140.5 110.5 115.0 181.0 104.1 44.1 25.5 20.9 91.7 14.6 28.5 93.3 71.5 25.6 25.9

13a

153.8 103.6 153.3 139.0 121.0 108.5 183.1 104.5 44.4 26.1 21.6 91.6 14.5 121.6 133.6 76.8 27.1 27.1

e

164.9 93.8 166.4 112.9

14a 161.9 99.8 163.9 111.9 156.7 149.6 99.9 156.1 141.3 128.3 106.7 181.6 104.3 41.9 29.7 29.7 151.5 108.0 105.8 133.9 160.3 114.8 19.4

15a,d 162.9 95.7 164.8 112.1 155.8 153.8 102.1 154.7 144.1 127.4 111.1 183.6 104.6 41.3 29.6 29.6 151.4 107.0 34.6 77.7 149.7 110.1 18.8 55.7

16c

162.0 107.4 165.0 113.2 151.5 151.8 135.7 157.2 114.3 122.3 115.1 180.9 103.6 22.3 122.8 132.1 17.8 25.8 44.9 26.1 21.8 91.4 14.6 62.0

17a

Journal of Natural Products Article

dx.doi.org/10.1021/np500364x | J. Nat. Prod. 2014, 77, 1893−1901

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Table 3. 1H NMR Spectroscopic Data for Compounds 10−17 10a

position 2 3 4 4a 4b 5 6 7 8 8a 9 9a 11 12 13 14 15

a

11a 6.13, s

12a

15a

16b

6.11, s

6.33, s

6.58, s

6.93, s

6.91, s

7.12, s

17a

6.32, s

6.94, s

7.05, s

7.46, s

s s t (6.0) d (6.0)

14a

6.17, s

6.42, s

1.57, 1.37, 4.47, 3.90,

13a

7.02, d (8.5) 7.85, d (9.0)

7.47, s

1.65, 1.43, 4.49, 3.91,

s s t (6.0) d (6.0)

1.60, 1.32, 4.57, 1.41,

s s q (6.5) d (6.5)

16

6.95, d (10.0)

8.04, d (10.5)

7.74, d (2.0)

17 18 19

5.94, d (10.5)

5.94, d (10.0)

8.08, d (2.0)

1.53, s

20 OMe OH-1

1.49, 1.24, 4.53, 1.39,

s s q (7.0) d (7.0)

1.57, 1.30, 4.55, 1.40,

s s q (6.5) d (6.5)

1.70, 1.70, 6.41, 5.05, 4.93, 7.62,

s s dd (17.5, 11.0) d (17.5) d (11.0) s

3.49, dd (16.0, 8.5) 3.42, dd (16.0, 10.0) 4.96, dd (10.0, 8.5)

8.04, d (10.0)

1.47, s

1.34, s

1.47, s

1.53, s

1.47, s

1.28, s

1.47, s

5.89, s 5.32, s 2.23, s

13.46, s

13.66, s

13.59, s

13.67, s

13.50, s

13.58, s

5.93, d (10.0)

1.74, 1.74, 6.43, 5.05, 4.93, 4.22, 3.75, 5.08,

s s dd (17.5) dd (17.5, 1.0) dd (10.5, 1.5) dd (13.5, 2.5) dd (13.0, 10.0) d (9.5)

5.49, s 5.05, s 2.19, s 3.72, s 14.48, s

3.28, d (7.5) 5.27, t (7.5) 1.77, s 1.65, s

1.64, s 1.36, s 4.53, q (6.5) 1.44, d (6.5) 4.02, s 13.63, s

Acetone-d6. bPyridine-d5.

and K) were found to be regioisomers and were assigned as shown. Compound 12, isolated as a yellow and amorphous solid, was shown to have a molecular formula of C20H16O6 by HRESIMS at m/z 353.1030 [M + H]+. The 1H and 13C NMR spectra of 12 (Tables 2 and 3) indicated that it also possesses the 1,3,6,7tetraoxygenated xanthone skeleton with a 2,3,3-trimethyldihydrofuran group. Furthermore, an additional furan ring at δH 8.08 (1H, d, J = 2.0 Hz, H-17) and 7.74 (1H, d, J = 2.0 Hz, H16) was observed. The position of this furan ring was determined from the HMBC cross-peak of H-17/C-8 (δC 127.3), and the positions of the remaining substituents were deduced by other HMBC correlations. Consequently, the new compound 12 (cudratrixanthone L) was determined as shown. The molecular formula of compound 13 was found to be C23H24O7, as deduced by analysis of the molecular ion peak at m/z 413.1596 [M + H]+ in the HRESIMS. Its 1H and 13C NMR spectra (Tables 2 and 3) were similar to those of 9, except that the 2-hydroxy-3,3-dimethyldihydropyran group present was changed to a 2-(1-hydroxy-1-methylethyl)dihydrofuran group in 13, which appeared at δH 4.96 (1H, dd, J = 10.0, 8.5 Hz, H-17), 3.49 (1H, dd, J = 16.0, 8.5 Hz, H16a), 3.42 (1H, dd, J = 16.0, 10.0 Hz, H-16b), 1.34 (3H, s, Me19), and 1.28 (3H, s, Me-20). The locations of all substituents were confirmed by the HMBC correlations, such as Me-12, 13/ C-2 (δC 117.3) and H-16/C-4b (δC 156.4). Thus, the structure of the new compound 13 (cudratrixanthone M) was deduced as shown. Compound 14 was isolated as a yellow, amorphous solid, and the molecular formula of C23H22O6 was determined by HRESIMS at m/z 395.1498 [M + H]+, suggesting 13 degrees

Compound 9 (cudratrixanthone I) was isolated as a light yellow, amorphous solid with the molecular formula of C23H24O7 deduced from its HRESIMS at m/z 413.1596 [M + H]+. The 1H and 13C NMR spectroscopic data (Tables 1 and 2) exhibited a characteristic 1,3,6,7-tetraoxygenated xanthone skeleton similar to that of 2, except for the presence of a 3hydroxy-2,2-dimethyldihydropyran group at δH 3.98 (1H, dd, J = 6.5, 5.0 Hz, H-17), 3.22 (1H, dd, J = 17.0, 5.0 Hz, H-16a), 2.92 (1H, dd, J = 17.0, 6.5 Hz, H-16b), 1.44 (3H, s, Me-19), and 1.39 (3H, s, Me-20), instead of a 2,2-dimethylpyran group. This group was located at C-5 and C-6, as confirmed by the HMBC correlation of H-16/C-5 (δC 109.5). Accordingly, the new compound 9 was assigned as shown. HRESIMS showed that compounds 10 and 11 gave the same molecular formula, C23H22O7. Careful comparison of the 1H and 13C NMR spectra of 10 and 11 (Tables 2 and 3) revealed that they share the same 1,3,6,7-tetraoxygenated xanthone skeleton with a 2-hydroxymethyl-3,3-dimethyldihydrofuran and a 2,2-dimethylpyran ring. They were found to differ in terms of the substitution pattern of the aromatic ring, as confirmed by 2D-NMR experiments including 1H−1H COSY, HSQC, and HMBC correlations. In compound 10, HMBC cross-peaks of Me-12, 13/C-2 (δC 117.4) and H-16/C-4b (δC 148.0) indicated that the 2-hydroxymethyl-3,3-dimethyldihydrofuran ring is fused at C-2 and C-3 and the 2,2-dimethylpyran group is located at C-5 and C-6, respectively. In contrast, the furan ring of 11 is fused at C-3 and C-4, and the pyran group is located at C-7 and C-8, respectively, as indicated by HMBC correlations of Me-12, 13/C-4 (δC 112.9) and H-17/C-8 (δC 121.0). Therefore, the new compounds 10 and 11 (cudratrixanthones J 1897

dx.doi.org/10.1021/np500364x | J. Nat. Prod. 2014, 77, 1893−1901

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of unsaturation. The 1H and 13C NMR spectra (Tables 2 and 3) were closely similar to those of 11, except that a 2,3,3trimethyldihydrofuran ring was present in 14 instead of a 2hydroxymethyl-3,3-dimethyldihydrofuran ring. The HMBC cross-peaks of Me-12, 13/C-4 (δC 112.9) and H-16/C-8a (δC 108.5) indicated a 2,3,3-trimethyldihydrofuran group to be fused at C-3 and C-4 and a 2,2-dimethylpyran group to be attached at C-7 and C-8, respectively. Accordingly, the structure of 14 (cudratrixanthone N) was elucidated as shown. Compound 15 gave a molecular formula of C23H20O6, as deduced by HRESIMS at m/z 393.1338 [M + H]+. The 1DNMR data (Tables 2 and 3) showed the characteristic signals of a 1,3,6,7-tetraoxygenated xanthone skeleton with the presence of a 1,1-dimethylallyl group. Moreover, an additional 2-(1methylethenyl)furan group at δH 7.62 (1H, s, H-16), 5.89 (1H, s, H-19a), 5.32 (1H, s, H-19b), and 2.23 (3H, s, Me-20) was observed. According to the HMBC cross-peaks of Me-12, 13/ C-4 (δC 111.9) and H-16/C-8 (δC 128.3), the 1,1-dimethylallyl group could be located at C-4 and the 2-(1-methylethenyl)furan group is fused at C-7 and C-8, respectively. Consequently, the new compound 15 (cudratrixanthone O) was determined as shown. Compound 16 showed a molecular formula of C24H26O7 by HRESIMS at m/z 427.1745 [M + H]+. The 1H and 13C NMR spectra (Tables 2 and 3) were comparable to those of compound 7, except for the presence of a methoxy group. The methoxy group was assigned at C-3 from the HMBC cross-peak of OMe-3/C-3 (δC 164.8), and the structure of the new compound 16 (3-O-methylcudratrixanthone G) was assigned as shown. Compound 17 was obtained as a yellow, amorphous solid with the elemental formula C24H26O6, showing a molecular ion peak at m/z 411.1796 [M + H]+ in the HRESIMS. The 1H and 13 C NMR spectra (Tables 2 and 3) were similar to those of formoxanthone C, 16 indicating that 17 has a 1,3,5,6tetraoxygenated xanthone skeleton with 2,3,3-trimethyldihydrofuran and prenyl groups. In contrast to formoxanthone C, 17 was found to have a methoxy group, and the HMBC correlation of OMe-5/C-5 (δC 135.7) indicated the methoxy group to be attached at C-5. Consequently, the structure of the new compound 17 (5-O-methylformoxanthone C) was assigned as shown. Additionally, 17 previously reported compounds were assigned as cudraxanthone A (18),17 6-deoxyisojacareubin (19),19 nigrolineaxanthone F (20),20 neriifolone A (21),21 cudraxanthone L (22),22 gerontoxanthone C (23),17 8-(1,1dimethyl-2-propenyl)-5,11-dihydroxy-9-methoxy-3,3dimethylpyrano[3,2-a]xanthen-12(3H)-one,18 cudraxanthone B,18 cudraxanthone C,18 cudraxanthone N,22 cudratricusxanthone A,8 1,3,5-trihydroxy-2-(3-methylbut-2-enyl)xanthone,19 isocudraxanthone K,13 cudraxanthone D,18 cudratricusxanthone J,15 6-deoxy-γ-mangostin, 23 and cudraxanthone M22 by comparing their spectroscopic data with published literature values. All isolated compounds were evaluated for their neuroprotective effects against 6-OHDA-induced neurotoxicity in human neuroblastoma SH-SY5Y cells, using a 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The EC50 values of 12 compounds are summarized in Table 4. Compounds 15 and 21 were the most active xanthones, with EC50 values of 0.8 and 0.7 μM, respectively, which were approximately seven times more potent than curcumin, the positive control used for this assay. Compounds

Table 4. Protective Effects of Compounds against 6-OHDAInduced Cell Death in Human Neuroblastoma SH-SY5Y Cells compound

EC50 (μM)a

3 7 8 9 15 16 18 19 20 21 22 23 curcuminb

7.2 16.6 2.4 2.2 0.8 2.3 4.5 5.1 15.5 0.7 8.2 3.0 6.0

± ± ± ± ± ± ± ± ± ± ± ± ±

0.33 0.34 0.12 0.07 0.04 0.13 0.21 0.25 0.52 0.09 0.12 0.20 0.14

a EC50 was defined as the concentration giving half maximal protection against 6-OHDA-induced cell death and expressed as the mean ± SD of triplicate determinations. bPositive control.

8, 9, 16, and 23 also showed prominent neuroprotective activities, with EC50 values of 2.2−3.0 μM. In addition, 18 and 19 had moderate activities, with the EC50 values of 4.5−5.1 μM, and 3, 7, 20, 22, and 23 showed marginal activities with EC50 values of 7.2−16.6 μM. Through the analysis of the bioassay results and the structural characteristics of the isolated compounds, clear structure−activity relationships could not be determined. It has been previously reported that some natural phenolic compounds such as curcumin, naringenin, and mangiferin prevent 6-OHDA-induced cell death, and further research including mechanism studies has been carried out.24,25 The present study describes the isolation and characterization of xanthones from the root bark of C. tricuspidata, with 17 new compounds elucidated, and some of these xanthones showed neuroprotective activities against 6-OHDA-induced cell death. These findings indicate that further research into C. tricuspidata and its constituents would be valuable, as these neuroprotective compounds might provide useful bioactive molecules for the treatment of neurodegenerative disorders.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a JASCO P-2000 polarimeter using MeOH as the solvent. The UV and IR spectra were recorded on Mecasys Optizen POP and Varian 640 FT-IR spectrometers, respectively. NMR experiments were conducted on a Varian 500 MHz NMR spectrometer with chemical shifts given in ppm (δ), and MS and HRMS were recorded on a Waters Q-TOF Micromass spectrometer. Column chromatography was performed using silica gel (70−230 mesh, Merck, Darmstadt, Germany), C18 reversed-phase (RP) silica gel (12 μm, YMC, Kyoto, Japan), and Sephadex LH-20 (18−111 μm, GE Healthcare AB, Stockholm, Sweden). Thin-layer chromatography was performed using precoated silica gel 60 F254 plates (0.25 mm, Merck). HPLC was carried out using a Varian system comprising a 210 pump and a 335 UV detector with a YMC J’sphere ODS-H80 column (4 μm, 250 × 10 mm i.d., YMC, Kyoto, Japan) and a YMC Pack ODS-A column (5 μm, 250 × 20 mm i.d., YMC, Kyoto, Japan). MTT and 6-OHDA (≥97.0%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Curcumin (≥98.5%) was purchased from Enzo Life Science (Farmingdale, NY, USA). Plant Material. The root bark of C. tricuspidata was collected by the Korea Forest Research Institute, Southern Forest Research Center, 1898

dx.doi.org/10.1021/np500364x | J. Nat. Prod. 2014, 77, 1893−1901

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(MeOH/H2O, 7:3 to 1:0) to afford cudratrixanthone H (8, 3.6 mg), cudratrixanthone I (9, 3.4 mg), cudraxanthone L (22, 3.9 mg), and isocudraxanthone K (5.5 mg). Fr 4.5.2.7 (185.0 mg) was separated using a silica gel column (CHCl3/acetone, 50:1 to 1:1) to give seven fractions and purified by RP HPLC (MeOH/H2O, 7:3 to 1:0) to give cudratrixanthone L (12, 2.7 mg) and cudratrixanthone M (13, 7.6 mg). Fr 4.5.2.8 (490.0 mg) was chromatographed on a silica gel column (n-hexane/CHCl3/acetone, 1:5:0 to 0:5:1) followed by RP HPLC (MeOH/H2O, 7:3 to 1:0), affording cudratrixanthone N (14, 2.7 mg), cudratrixanthone O (15, 2.7 mg), 3-O-methylcudratrixanthone G (16, 6.5 mg), gerontoxanthone C (23, 12.6 mg), cudraxanthone D (40.0 mg), cudratricusxanthone J (4.3 mg), and 6deoxy-γ-mangostin (8.1 mg). Fr 4.5.2.9 (670.0 mg) was separated on a silica gel column (CHCl3/acetone, 70:1 to 5:1) followed by RP HPLC (MeOH/H2O, 7:3 to 1:0) to give 5-O-methylformoxanthone C (17, 3.3 mg) and cudraxanthone M (24.6 mg). Cudratrixanthone A (1): amorphous, yellow powder; [α]23D +6.6 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 220 (4.1) 275 (4.3), 337 (3.7) nm; IR νmax (ATR) 3266, 2923, 1646, 1581, 1461, 1265, 1207, 1153, 1114 cm−1; 1H and 13C NMR data (500 MHz, acetone-d6), see Tables 1 and 2; HMBC correlations OH-1/C-1, C-2, C-9a; H-2/C-1, C-3, C-4, C-9a; H-5/C-7, C-8a; H-6/C-4b, C-8; H-11/C-3, C-4a, C13; H-12/C-4, C-13; Me-14, 15/C-12, C-13; H-16/C-7, C-8, C-8a, C17; H-19/C-18, C-20; Me-20/C-17, C-18, C-19; ESIMS (negative) m/ z 393 [M − H]−; ESIMS (positive) m/z 395 [M + H]+; HRESIMS m/ z 395.1500 [M + H]+ (calcd for C23H23O6, 395.1495). Cudratrixanthone B (2): amorphous, yellow powder; [α]23D −6.2 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 247 (4.6), 259 (4.7), 295 (4.1), 335 (4.3), 372 (4.1) nm; IR νmax (ATR) 3371, 2974, 2924, 1658, 1612, 1454, 1381, 1304, 1161, 1126, 1062 cm−1; 1H and 13C NMR data (500 MHz, acetone-d6), see Tables 1 and 2; HMBC correlations OH-1/C-1, C-2, C-9a; H-4/C-2, C-3, C-4a, C-9a; H-8/C-6, C-7, C-9; Me-12, 13/C-11, C-14; H-14/C-12, C-13; Me-15/C-11, C-14; H-16/ C-4b, C-18; H-17/C-5, C-18; Me-19, 20/C-16, C-18; ESIMS (negative) m/z 393 [M − H]−; ESIMS (positive) m/z 395 [M + H]+; HRESIMS m/z 395.1496 [M + H]+ (calcd for C23H23O6, 395.1495). Cudratrixanthone C (3): amorphous, yellow powder; UV (MeOH) λmax (log ε) 241 (4.3), 258 (4.2), 275 (4.0), 317 (4.1), 357 (3.8) nm; IR νmax (ATR) 3382, 2921, 1647, 1599, 1489, 1450, 1276, 1158, 1082, 1076, 1026 cm−1; 1H and 13C NMR data (500 MHz, CDCl3), see Tables 1 and 2; HMBC correlations OH-1/C-1, C-9a; H-2/C-1, C-4, C-9a; H-5/C-6, C-7, C-8a; Me-12, 13/C-4, C-11, C-14; H-15/C-11; H-16/C-7, C-8, C-8a, C-17; H-19/C-17, C-18; Me-20/C-17, C-18; 7OMe/C-7; ESIMS (negative) m/z 423 [M − H]−; ESIMS (positive) m/z 425 [M + H]+; HRESIMS m/z 425.1587 [M + H]+ (calcd for C24H25O7, 425.1600). Cudratrixanthone D (4): amorphous, yellow powder; [α]23D +4.6 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 240 (3.9), 257 (3.9), 276 (3.6), 316 (3.7), 356 (3.4) nm; IR νmax (ATR) 3352, 2923, 1646, 1585, 1466, 1278, 1157, 1021 cm−1; 1H and 13C NMR data (500 MHz, acetone-d6), see Tables 1 and 2; HMBC correlations OH-1/C-1, C-2, C-9a; H-2/C-3, C-4, C-9a; H-5/C-4b, C-6, C-7, C-8a; Me-12, 13/C-4, C-11, C-14; H-14/C-12, C-13; H-15/C-11, C-14; H-16/C-8, C-8a, C7, C-17; H-19/C-17, C-20; Me-20/C-17, C-18, C-19; 7-OMe/C-7; ESIMS (negative) m/z 439 [M − H]−; ESIMS (positive) m/z 441 [M + H]+; HRESIMS m/z 441.1563 [M + H]+ (calcd for C24H25O8, 441.1549). Cudratrixanthone E (5): amorphous, yellow powder; [α]23D +2.9 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 240 (3.9), 258 (3.8), 317 (3.6), 363 (3.5) nm; IR νmax (ATR) 3344, 2920, 1647, 1597, 1454, 1375, 1273, 1161, 1080, 1030 cm−1; 1H and 13C NMR data (500 MHz, acetone-d6), see Tables 1 and 2; HMBC correlations OH-1/C-1, C-2, C-9a; H-2/C-4, C-9a; H-5/C-4b, C-6, C-7, C-8a; Me-12, 13/C-4, C11, C-14; H-15/C-14; H-16/C-7, C-8, C-17; H-19/C-17, C-20; Me20/C-17, C-18, C-19; 7-OMe/C-7; ESIMS (negative) m/z 441 [M − H]−; ESIMS (positive) m/z 443 [M + H]+; HRESIMS m/z 443.1707 [M + H]+ (calcd for C24H27O8, 443.1706). Cudratrixanthone F (6): amorphous, yellow powder; [α]23D +3.9 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 239 (3.9), 265 (4.0), 318

Jinju, Korea, in September 2008. A voucher specimen (accession number KH1-4-090814) was deposited at the Department of Biosystems and Biotechnology, Korea University, Seoul, Korea. Extraction and Isolation. The dried root bark of C. tricuspidata (13.0 kg) was ground and extracted with MeOH (48 L, 20 L, 18 L) at room temperature, and the combined extracts were evaporated in vacuo at 35 °C. The MeOH extract showed protective effects against 6-OHDA-induced cell death in human neuroblastoma SH-SY5Y cells, with an EC50 value of 13.4 μg/mL. The dark brown residue (702.1 g) was suspended in H2O and then partitioned with n-hexane and EtOAc, sequentially, to give an EtOAc-soluble extract (213.0 g) and a hexanesoluble residue (69.2 g). The EtOAc extract was subjected to silica gel column chromatography (CHCl3/MeOH, 1:0 to 1:1) to yield seven fractions (Fr 1 to Fr 7). Fr 4 (36.0 g) was fractionated on a silica gel column (n-hexane/EtOAc, 30:1 to 0:1) to afford eight fractions (Fr 4.1 to Fr 4.8). Fr 4.4 (4.8 g) was applied to a silica gel column (n-hexane/ CHCl3/MeOH, 1:10:0 to 0:5:1) to give 10 fractions (Fr 4.4.1 to Fr 4.4.10). Fr 4.4.4 (233.5 mg) was repeatedly passed over a silica gel column (hexane/EtOAc, 20:1 to 5:1) to yield eight fractions (Fr 4.4.4.1 to Fr 4.4.4.8). Fr 4.4.4.2 (10.5 mg) was then purified using RP HPLC (MeOH/H2O, 9:1 to 1:0) to afford cudraxanthone A (18, 2.2 mg). Fr 4.4.4.4 (7.2 mg) was separated by RP HPLC (MeOH/H2O, 9:1) to give 8-(1,1-dimethyl-2-propenyl)-5,11-dihydroxy-9-methoxy3,3-dimethylpyrano[3,2-a]xanthen-12(3H)-one (2.1 mg). Fr 4.4.7 (2.5 g) was fractionated on a silica gel MPLC column (CHCl3/acetone, 1:0 to 0:1) to yield eight fractions (Fr 4.4.7.1 to Fr 4.4.7.8). Fr 4.4.7.2 (1.1 g) was repeatedly separated on a silica gel MPLC column (CHCl3/ MeOH, 1:0 to 1:1) to afford four fractions (Fr 4.4.7.2.1 to Fr 4.4.7.2.4). Fr 4.4.7.2.1 (522.8 mg) was precipitated with MeOH, and the MeOH-soluble layer was further chromatographed on a C18 RP silica gel column (MeOH/H2O, 9:1 to 1:0) followed by RP HPLC (MeOH/H2O, 3:1) to give cudraxanthone B (10.0 mg) and cudraxanthone C (3.5 mg). Fr 4.4.7.2.2 (120.0 mg) was fractionated using a C18 RP silica gel column (MeOH/H2O, 8:2 to 1:0) followed by RP HPLC (MeOH/H2O, 7:3 to 1:0) to afford cudratrixanthone A (1, 4.1 mg), cudratrixanthone B (2, 10.0 mg), and 6-deoxyisojacareubin (19, 1.3 mg). Fr 4.4.7.4 (350.0 mg) was separated using a C18 RP silica gel column (MeOH/H2O, 8:2 to 1:0) and purified by RP HPLC (MeOH/H2O, 7:3 to 9:1) to give cudratrixanthone C (3, 3.4 mg). Fr 4.4.7.6 (280.0 mg) was chromatographed on a C18 RP silica gel column (MeOH/H2O, 9:1 to 1:0) followed by RP HPLC (MeOH/H2O, 7:3) to afford nigrolineaxanthone F (20, 2.8 mg). Fr 4.4.8 (418.8 mg) was separated on a silica gel column (CHCl3/MeOH, 100:1 to 10:1) to yield 13 fractions (Fr 4.4.8.1 to Fr 4.4.8.13). Fr 4.4.8.9 (25.9 mg) and Fr 4.4.8.10 (16.0 mg) were separated by RP HPLC (MeOH/H2O, 3:2 to 1:0) to give cudratrixanthone D (4, 1.8 mg) and cudraxanthone N (1.8 mg), respectively. Fr 4.4.8.11 (50.0 mg) was separated by RP HPLC (MeOH/H2O, 1:1 to 1:0) to give cudratrixanthone E (5, 2.6 mg). Fr 4.4.9 (667.0 mg) was separated using Sephadex LH-20 (CHCl3/MeOH, 1:1) and purified by RP HPLC (MeOH/H2O, 13:7 to 1:0) to afford cudratrixanthone F (6, 2.5 mg). Fr 4.5 (7.2 g) was fractionated on a silica gel column (CHCl3/ MeOH, 1:0 to 0:1) to yield seven fractions (Fr 4.5.1 to Fr 4.5.7). Fr 4.5.2 (4.3 g) was chromatographed on a C18 RP silica gel column (MeOH/H2O, 8:2 to 1:0) to yield 15 fractions (Fr 4.5.2.1 to Fr 4.5.2.15). Fr 4.5.2.3 (270.0 mg) was separated on a silica gel MPLC column (n-hexane/EtOAc, 5:1 to 1:1) to afford eight fractions (Fr 4.5.2.3.1 to Fr 4.5.2.3.8). Fr 4.5.2.3.2 (39.0 mg) was separated by RP HPLC (MeOH/H2O, 3:2 to 1:4) to give 1,3,5-trihydroxy-2-(3methylbut-2-enyl)xanthone (2.0 mg). Fr 4.5.2.3.8 (21.7 mg) was purified using RP HPLC (MeOH/H2O, 7:3 to 9:1) to give cudratrixanthone G (7, 9.8 mg). Fr 4.5.2.5 (69.4 mg) was separated by RP HPLC (MeOH/H2O, 7:3 to 1:0) to give neriifolone A (21, 1.6 mg) and cudratricusxanthone A (37.0 mg). Fr 4.5.2.6 (420 mg) was separated on a silica gel MPLC column (CHCl3/MeOH, 100:1 to 1:1) to yield six fractions (Fr 4.5.2.6.1 to Fr 4.5.2.6.6). Fr 4.5.2.6.1 (33.1 mg) was purified using RP HPLC (MeOH/H2O, 3:1 to 17:3) to afford cudratrixanthone J (10, 2.2 mg) and cudratrixanthone K (11, 2.4 mg). Fr 4.5.2.6.2 (230.0 mg) was fractionated on a silica gel column (CHCl3/acetone, 10:1) to give seven fractions followed by RP HPLC 1899

dx.doi.org/10.1021/np500364x | J. Nat. Prod. 2014, 77, 1893−1901

Journal of Natural Products

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(3.6), 369 (3.3) nm; IR νmax (ATR) 3334, 2970, 2924, 1678, 1643, 1493, 1450, 1382, 1295, 1203, 1194, 1142 cm−1; 1H and 13C NMR data (500 MHz, acetone-d6), see Tables 1 and 2; HMBC correlations OH-1/C-1, C-2, C-9a; H-2/C-1, C-3, C-9a; H-5/C-7, C-8a; H-6/C4b, C-8; H-11/C-3, C-4, C-4a, C-12; H-14/C-12, C-15; Me-15/C-12, C-13, C-14; H-16/C-7, C-8, C-8a; H-19/C-17, C-20; Me-20/C-17, C18, C-19; ESIMS (negative) m/z 411 [M − H]−; ESIMS (positive) m/ z 413 [M + H]+; HRESIMS m/z 413.1580 [M + H]+ (calcd for C23H25O7, 413.1600). Cudratrixanthone G (7): amorphous, yellow powder; [α]23D −2.7 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 243 (3.7), 262 (3.8), 321 (3.6), 369 (3.3) nm; IR νmax (ATR) 3422, 3024, 2919, 1685, 1601, 1493, 1450, 1194, 1026 cm−1; 1H and 13C NMR data (500 MHz, acetone-d6), see Tables 1 and 2; HMBC correlations OH-1/C-1, C-2, C-9a; H-2/C-3, C-4, C-9a; H-5/C-4b, C-7, C-8a; Me-12, 13/C-4, C11, C-14; H-14/C-11, C-12, C-13; H-15/C-11, C-14; H-16/C-7, C-8, C-8a, C-17; H-17/C-8; H-19/C-17, C-18, C-20; Me-20/C-17, C-18, C-19; ESIMS (negative) m/z 411 [M − H]−; ESIMS (positive) m/z 413 [M + H]+; HRESIMS m/z 413.1594 [M + H]+ (calcd for C23H25O7, 413.1600). Cudratrixanthone H (8): amorphous, yellow powder; [α]23D +7.4 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 244 (3.8), 256 (3.8), 295 (3.4), 313 (3.4), 362 (3.4) nm; IR νmax (ATR) 3358, 2974, 2920, 1683, 1608, 1450, 1382, 1295, 1203, 1146, 1076 cm−1; 1H and 13C NMR data (500 MHz, acetone-d6), see Tables 1 and 2; HMBC correlations H-4/C-2, C-3, C-9a; H-8/C-6, C-7, C-9; Me-12, 13/C-2, C-11, C-14; H-14/C-12, C-13; Me-15/C-11, C-14; H-16/C-4b, C-5, C-17, C-18; H-17/C-19, C-20; Me-19, 20/C-17, C-18; ESIMS (negative) m/z 395 [M − H]−; ESIMS (positive) m/z 397 [M + H]+; HRESIMS m/z 397.1643 [M + H]+ (calcd for C23H25O6, 397.1651). Cudratrixanthone I (9): amorphous, yellow powder; [α]23D +4.0 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 246 (4.0), 259 (4.0), 280 (3.5), 321 (3.8), 363 (3.5) nm; IR νmax (ATR) 3429, 2975, 2921, 1667, 1623, 1467, 1379, 1293, 1188, 1140, 1069 cm−1; 1H and 13C NMR data (500 MHz, acetone-d6), see Tables 1 and 2; HMBC correlations OH-1/C-1, C-2, C-9a; H-4/C-2, C-3, C-4a, C-9a; H-8/C-4b, C-6, C-7, C-9; Me-12, 13/C-2, C-11, C-14; H-14/C-12, C-13; Me-15/C-11, C14; H-16/C-5, C-6, C-16, C-17; Me-19, 20/C-16, C-17; ESIMS (negative) m/z 411 [M − H]−; ESIMS (positive) m/z 413 [M + H]+; HRESIMS m/z 413.1596 [M + H]+ (calcd for C23H25O7, 413.1600). Cudratrixanthone J (10): amorphous, yellow powder; [α]23D −2.6 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 248 (3.9), 260 (4.0), 335 (3.6), 375 (3.4) nm; IR νmax (ATR) 3387, 3024, 2920, 1677, 1608, 1489, 1450, 1378, 1304, 1189, 1027 cm−1; 1H and 13C NMR data (500 MHz, acetone-d6), see Tables 2 and 3; HMBC correlations OH-1/C-1, C-2, C-9a; H-4/C-2, C-3, C-4a, C-9a; H-8/C-4b, C-6, C-7, C-9; Me12, 13/C-2, C-11, C-14; H-14/C-13; H-15/C-11, C-14; H-16/C-4b, C-18; H-17/C-5, C-18; Me-19, 20/C-17, C-18; ESIMS (negative) m/z 409 [M − H]−; ESIMS (positive) m/z 411 [M + H]+; HRESIMS m/z 411.1442 [M + H]+ (calcd for C23H23O7, 411.1444). Cudratrixanthone K (11): amorphous, yellow powder; [α]23D +2.8 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 246 (4.4), 266 (4.4), 287 (3.9), 334 (4.3), 383 (3.8) nm; IR νmax (ATR) 3353, 2970, 2920, 1644, 1577, 1489, 1450, 1370, 1292, 1161, 1026, cm−1; 1H and 13C NMR data (500 MHz, acetone-d6), see Tables 2 and 3; HMBC correlations OH-1/C-1, C-2, C-9a; H-2/C-4, C-9a; H-5/C-6, C-7, C-8a; Me-12, 13/C-4, C-11, C-14; H-14/C-12, C-13; H-15/C-11; H-16/C-7, C-18; H-17/C-8, C-18; Me-19, 20/C-17, C-18; ESIMS (negative) m/z 409 [M − H]−; ESIMS (positive) m/z 411 [M + H]+; HRESIMS m/z 411.1438 [M + H]+ (calcd for C23H23O7, 411.1444). Cudratrixanthone L (12): amorphous, yellow powder; [α]23D −1.7 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 237 (3.9), 259 (3.8), 279 (3.4), 289 (3.5), 328 (3.8), 359 (3.7) nm; IR νmax (ATR) 3356, 2921, 1647, 1587, 1489, 1422, 1272, 1160, 1092 cm−1; 1H and 13C NMR data (500 MHz, acetone-d6), see Tables 2 and 3; HMBC correlations OH-1/C-1, C-2, C-9a; H-2/C-3, C-4, C-9a; H-5/C-4b, C-6, C-7, C-8a; Me-12, 13/C-4, C-11, C-14; H-14/C-12, C-13; Me-15/C-11, C-14; H16/C-7, C-8, C-17; H-17/C-7, C-8, C-16; ESIMS (negative) m/z 351 [M − H]−; ESIMS (positive) m/z 353 [M + H]+; HRESIMS m/z 353.1030 [M + H]+ (calcd for C20H17O6, 353.1025).

Cudratrixanthone M (13): amorphous, yellow powder; [α]23D −16.1 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 246 (4.7), 257 (4.7), 284 (4.1), 326 (4.5), 359 (4.3) nm; IR νmax (ATR) 3367, 2974, 2921, 1657, 1603, 1473, 1452, 1347, 1159, 1060, 1092 cm−1; 1H and 13 C NMR data (500 MHz, acetone-d6), see Tables 2 and 3; HMBC correlations OH-1/C-1, C-2, C-9a; H-4/C-2, C-3, C-4a, C-9a; H-8/C4b, C-6, C-7, C-9; Me-12, 13/C-2, C-11, C-14; H-14/C-12, C-13; Me15/C-11, C-14; H-16/C-4b, C-5, C-17, C-18; Me-19, 20/C-17, C-18; ESIMS (negative) m/z 411 [M − H]−; ESIMS (positive) m/z 413 [M + H]+; HRESIMS m/z 413.1596 [M + H]+ (calcd for C23H23O6, 413.1600). Cudratrixanthone N (14): amorphous, yellow powder; [α]22D −3.5 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 246 (4.2), 266 (4.2), 287 (3.7), 335 (4.1), 383 (3.6) nm; IR νmax (ATR) 3357, 2923, 1640, 1572, 1446, 1290, 1158, 1058 cm−1; 1H and 13C NMR data (500 MHz, acetone-d6), see Tables 2 and 3; HMBC correlations OH-1/C-1, C-2, C-9a; H-2/C-1, C-3, C-4, C-9a; H-5/C-4b, C-7, C-8a; Me-12, 13/C-4, C-11, C-14; H-14/C-12, C-13; Me-15/C-11, C-14; H-16/C-7, C-8a, C-18; H-17/C-16, C-18; Me-19, 20/C-17, C-18; ESIMS (negative) m/ z 393 [M − H]−; ESIMS (positive) m/z 395 [M + H]+; HRESIMS m/ z 395.1498 [M + H]+ (calcd for C23H25O7, 395.1495). Cudratrixanthone O (15): amorphous, yellow powder; UV (MeOH) λmax (log ε) 246 (4.2), 257 (4.1), 350 (4.0) nm; IR νmax (ATR) 3399, 2924, 1603, 1409, 1279, 1189 cm−1; 1H and 13C NMR data (500 MHz, acetone-d6), see Tables 2 and 3; HMBC correlations OH-1/C-1, C-2, C-9a; H-2/C-1, C-3, C-4, C-9a; H-5/C-6, C-7, C-8a; Me-12, 13/C-4, C-11, C-14; H-14/C-11, C-12, C-13; H-15/C-11, C14; H-16/C-7, C-8, C-18; H-19/C-17, C-18, C-20; Me-20/C-17, C-18, C-19; ESIMS (negative) m/z 391 [M − H]−; ESIMS (positive) m/z 393 [M + H]+; HRESIMS m/z 393.1338 [M + H]+ (calcd for C23H21O6, 393.1338). 3-O-Methylcudratrixanthone G (16): amorphous, yellow powder; [α]23D +3.1 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 241 (4.3), 260 (4.4), 277 (4.0), 317 (4.1), 374 (3.9) nm; IR νmax (ATR) 3479, 3310, 2973, 2924, 1575, 1451, 1675, 1275, 1203, 1163, 1038 cm−1; 1H and 13 C NMR data (500 MHz, pyridine-d5), see Tables 2 and 3; HMBC correlations OH-1/C-1, C-2, C-9a; H-2/C-1, C-3, C-4, C-9a; H-5/C6, C-7, C-8a; Me-12, 13/C-4, C-11, C-14; H-14/C-12, C-13; H-15/C11, C-14; H-16/C-7, C-8, C-8a, C-17; H-19/C-17, C-20; Me-20/C-17, C-18, C-19; OMe-3/C-6; ESIMS (negative) m/z 425 [M − H]−; ESIMS (positive) m/z 427 [M + H]+; HRESIMS m/z 427.1745 [M + H]+ (calcd for C24H27O7, 427.1757). 5-O-Methylformoxanthone C (17): amorphous, yellow powder; [α]23D −2.9 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 247 (4.4), 259 (4.2), 285 (3.8), 323 (4.1), 360 (3.8) nm; IR νmax (ATR) 3353, 2972, 2921, 1590, 1443, 1380, 1304, 1199, 1129 cm−1; 1H and 13C NMR data (500 MHz, acetone-d6), see Tables 2 and 3; HMBC correlations OH-1/C-1, C-2, C-9a; H-2/C-1, C-3, C-4, C-9a; H-7/C-5, C-8a; H-8/ C-4b, C-6, C-8; H-11/C-1, C-2, C-3, C-12, C-13; H-12/C-11, C-14, C-15; Me-14, 15/C-12, C-13; Me-17, 18/C-4, C-16, C-19; H-19/C17, C-18; Me-20/C-16, C-19; 5-OMe/C-5; ESIMS (negative) m/z 409 [M − H]−; ESIMS (positive) m/z 411 [M + H]+; HRESIMS m/z 411.1796 [M + H]+ (calcd for C24H27O6, 411.1808). Assay for Neuroprotective Activity. Human neuroblastoma SHSY5Y cells were cultured in 96-well plates at a density of 2 × 104 cells/ well in 200 μL for 24 h. The cells were treated with 100 μM 6hydroxydopamine and various concentrations of extracts and compounds for an additional 24 h. Cell viability was determined by treatment with MTT dissolved in 0.5 mg/mL of phosphate-buffered saline (PBS) at 37 °C for 4 h. The PBS was then carefully removed, and formazan crystals were dissolved using dimethyl sulfoxide. The absorbance of this solution was then measured at 540 nm using a microplate reader. Neuroprotection against 6-OHDA-induced cell death was calculated using a semilogarithmic graph depicting the relationship between at least four different concentrations of compounds and their percentage effects. Samples were tested in triplicate, and the mean values with standard deviation were used. All results were typically expressed as EC50, and curcumin was used as a positive control. 1900

dx.doi.org/10.1021/np500364x | J. Nat. Prod. 2014, 77, 1893−1901

Journal of Natural Products

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Article

(24) Zbarsky, V.; Datla, K. P.; Parkar, S.; Rai, D. K.; Aruoma, O. I.; Dexter, D. T. Free Radical Res. 2005, 39, 1119−1125. (25) Rao, V. S.; Carvalho, A. C.; Trevisan, M. T. S.; Andrade, G. M.; Júnior, H. V. N.; Moraes, M. O.; Iury, H. I. M. H.; Morais, T. C.; Santos, F. A. Pharmacol. Rep. 2012, 64, 848−856.

ASSOCIATED CONTENT

S Supporting Information *

Mass and 1H and 13C NMR spectra of 17 new compounds (1− 17) and 1H NMR data of 17 known compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +82-2-880-2473. Fax: +82-2-888-9122. E-mail: mars@ snu.ac.kr (W. Mar). *Tel: +82-2-3290-3017. Fax: +82-2-953-0737. E-mail: [email protected] (D. Lee). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant from the Korea University, the Korea Forest Research Institute, and the National Research Foundation of Korea (NRF) (grant NRF2013R1A1A2008111).

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REFERENCES

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dx.doi.org/10.1021/np500364x | J. Nat. Prod. 2014, 77, 1893−1901