Anti-oxidative and anti-inflammatory activities of caffeoyl hemiterpene glycosides from Spiraea prunifolia

Anti-oxidative and anti-inflammatory activities of caffeoyl hemiterpene glycosides from Spiraea prunifolia

Phytochemistry 96 (2013) 430–436 Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem Anti-...

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Phytochemistry 96 (2013) 430–436

Contents lists available at ScienceDirect

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

Anti-oxidative and anti-inflammatory activities of caffeoyl hemiterpene glycosides from Spiraea prunifolia Sang Hee Park, Kwan Hee Park, Myeong Hwan Oh, Han Hyuk Kim, Kang In Choe, So Ra Kim, Kwang Jun Park, Min Won Lee ⇑ Department of Pharmacognosy, College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea

a r t i c l e

i n f o

Article history: Received 6 March 2013 Received in revised form 15 June 2013 Available online 22 October 2013 Keywords: Spiraea prunifolia Rosaceae Caffeoyl hemiterpenes Hemiterpene glycosides DPPH radical scavenging activity NBT superoxide scavenging activity NO production

a b s t r a c t Activity guided isolation of a Spiraea prunifolia extract yielded five caffeoyl hemiterpene glycosides: 40 -(6-O-caffeoyl-b-D-glucopyranosyl)-20 -methyl butyric acid, 1-O-caffeoyl-6-O-(40 -hydroxy-20 -methy1,2-O-dicaffeoyl-6-O-(40 -hydroxy-20 -methylene-butyroyl)-b-Dlene-butyroyl)-b-D-glucopyranoside, 0 and glucopyranoside, 1-O-caffeoyl-6-O-(4 -caffeoyl-20 -methylene-butyroyl)-b-D-glucopyranoside, 1-O-caffeoyl-6-O-(40 -caffeoyl-30 -hydroxy-20 -methylene-butyroyl)-b-D-glucopyranoside, and nine known compounds. Structures were elucidated by analysis of 1D and 2D NMR spectra and FAB-MS. To evaluate the anti-oxidative and anti-inflammatory properties of all fourteen compounds, DPPH radical scavenging, NBT superoxide scavenging, and inhibition of nitric oxide production in LPS-stimulated RAW264.7 cells were examined. Three of the caffeoyl hemiterpene glycosides exhibited potent anti-oxidative and antiinflammatory activities compared with Vitamin C and L-NMMA, which were used as positive controls. Ó 2013 Published by Elsevier Ltd.

1. Introduction Spiraea prunifolia (Rosaceae), commonly called ‘Bridal wreath’, is widespread in northeast Asia. The young leaves, fruits, and roots of S. prunifolia have been used for the treatment of malaria, fever, and emetic conditions in traditional medicine (Choudhary et al., 2009; Oh et al., 2001; So et al., 1999a,b). Spiraea species have also been reported to contain various diterpenes, diterpene alkaloids, terpenoid glycosides, and flavonoids (Hou et al., 2009). In the present study, phytochemical isolation yielded five new caffeoyl hemiterpene glycosides (1–5), along with nine known phenolic compounds. Structures were elucidated by analysis of 1D and 2D NMR spectroscopic data. All of the isolated compounds were evaluated for anti-oxidative and anti-inflammatory properties by measuring their DPPH radical scavenging activity, NBT superoxide scavenging activity, and ability to inhibit nitric oxide production in LPS-stimulated RAW264.7 cells. 2. Results and discussion 2.1. Structure elucidation Compound 1 had a molecular formula of C20H26O11 as indicated by HR-FAB-MS data (m/z 441.1396 [MH], Calcd. C20H25O11, 441.1397). Its 1H NMR spectrum (Table 1) was consistent with a ⇑ Corresponding author. Tel.: +82 2 820 5602; fax: +82 2 822 9778. E-mail addresses: [email protected], [email protected] (M.W. Lee). 0031-9422/$ - see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.phytochem.2013.09.017

caffeoyl moiety in the aromatic region as determined by the presence of an aromatic ABX-spin system with a meta-coupled aromatic signal [d 7.05 (1H, d, J = 2.1 Hz, H-5)], ortho-coupled aromatic resonance [d 6.77 (1H, d, J = 8.4 Hz, H-8)] and ortho– meta-coupled aromatic signal [d 6.95 (1H, dd, J = 2.1, 8.4 Hz, H9)] for a 3,4-dihydroxyphenyl unit, and two olefinic protons [d 6.29 and 7.57 (1H, d, J = 15.9 Hz, H-2,3)] for a trans-configured double bond. In addition, the 1H and 13C NMR spectra of 1 showed the presence of a glucopyranosyl moiety with one methylene [d 4.32 (1H, dd, J = 6.0, 12.0 Hz, glc-6a) and 4.48 (1H, dd, J = 2.1, 12.0 Hz, glc-6b)], five methines [d 4.29 (1H, d, J = 7.8 Hz, glc-1), 3.51 (1H, ddd, J = 9.0, 6.0, 2.4 Hz, glc-5), 3.41 (1H, t, J = 9.0 Hz, glc-3), 3.37 (1H, t, J = 9.0 Hz, glc-4), and 3.21 (1H, dd, J = 9.0, 7.8 Hz, glc-2)], and six carbons (d 63.3, 70.3, 73.6, 74.0, 76.4 and 103.0). The large coupling constant of the anomeric proton at d 4.28 (J = 7.8 Hz, glc1) indicated that the glucopyranoside was in a b-configuration. The 1 H and 13C NMR spectra of 1 also showed the presence of a hemiterpene moiety with a single methyl group [d 1.12 (3H, d, J = 6.6 Hz, H-50 )], two methylenes [d 1.65, 1.97 (each 1H, m, H-30 ) and 3.60, 3.89 (each 1H, m, H-40 )], one methine [d 2.49 (1H, m, H-20 )], and five carbons (d 16.9, 33.7, 37.9, 67.8, 181.7). The 1H–1H COSY spectrum of 1 (Fig. 2) showed a correlation of the methyl group proton signal (H-50 ) with the methine proton (H-20 ). Further, the methine proton was also correlated with a methylene at d 1.65/1.97 (H-30 ). In addition, the two methylenes were correlated with each other. These data led to the elucidation of the hemiterpene structure as 40 -hydroxy-20 -methyl-butyrate. The connectivities of the caffeoyl,

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S.H. Park et al. / Phytochemistry 96 (2013) 430–436 Table 1 Ha and

1

13 b

C NMR spectroscopic data for compounds 1–5.

Position

1 d H (J in Hz)

Glucopyranosyl moiety 1 4.29 d (7.8) 2 3.21 dd (9.0, 7.8) 3 3.41 t (9.0) 4 3.37 t (9.0) 5 3.51 ddd (9.0, 6.0, 2.4) 6a 4.32 dd (12.0, 6.0) 4.28 dd (12.0, 5.4) 6b 4.48 dd (12.0, 2.4) Hemiterpene moiety 10 20 2.49 m 30 1.65/1.97 m 40 3.60/3.89 m 50 1.12 d (6.6) Caffeoyl moiety 100 200 6.29 d (15.9) 7.57 d (15.9) 300 400 500 7.05 d (2.1) 600 700 800 6.77 d (8.4) 900 6.95 dd (8.4, 2.1) 100 0 200 0 300 0 400 0 500 0 600 0 700 0 800 0 900 0 a b

2

3

dC

d H (J in Hz)

103.0 73.6 76.4 70.3 74.0 63.3 63.3

5.56 3.45 3.48 3.41 3.67

181.7 37.9 33.7 67.8 16.9 167.7 113.5 145.8 126.3 113.8 145.4 148.2 115.1 121.5

d (7.8) dd (9.0, 7.8) t (9.0) t (9.0) m

4.33 dd (12.0, 5.4) 4.48 dd (12.0, 1.8)

2.53 t (6.6) 3.66 t (6.6) 5.69/6.24 d (1.2)

6.30 d (15.9) 7.66 d (15.9) 7.06 d (1.8)

6.78 d (8.4) 6.97 dd (8.4, 1.8)

dC

4

d H (J in Hz)

dC

94.3 72.5 76.4 69.9 74.7

5.78 5.06 3.75 3.55 3.76

63.1

4.32 dd (12.0, 6.0) 4.52 dd (12.0, 2.1)

166.7 137.1 34.9 60.2 126.7 166.2 112.8 147.0 126.1 113.8 145.4 148.5 115.1 121.9

d (8.4) dd (9.6, 8.4) t (9.6) t (9.6) ddd (9.6, 5.4, 2.1)

2.56 t (6.6) 3.68 t (6.6) 5.71/6.27 d (1.2)

6.28 d (15.6) 7.58 d (15.6) 7.02 d (1.8)

6.76 d (8.4) 6.96 dd (8.4, 1.8) 6.19 d (15.6) 7.58 d (15.6) 7.01 d (1.8)

6.75 d (8.1) 6.94 dd (8.1, 1.8)

5

d H (J in Hz)

92.4 72.6 74.6 70.1 74.9

5.57 3.45 3.47 3.42 3.69

63.4

4.31 dd (12.0, 6.0) 4.52 dd (12.0, 1.8)

166.8 137.1 34.9 60.2 126.7 166.9 113.1 147.5 126.2 113.9 145.4 148.6 115.1 122.0 165.6 112.2 146.4 126.0 113.7 145.3 148.3 115.1 121.7

d (7.8) dd (9.0, 7.8) t (9.0) t (9.0) ddd (9.0, 6.0, 1.8)

2.70 t (6.6) 4.33 m 5.71/6.26 d (1.2)

6.28 d (15.9) 7.63 d (15.9) 7.05 d (1.8)

6.77 d (8.1) 6.93 dd (8.1, 1.8) 6.21 d (15.9) 7.50 d (15.9) 7.02 d (1.8)

6.74 d (8.1) 6.91 dd (8.1, 1.8)

dC 94.2 72.5 76.5 70.0 74.7

d H (J in Hz) 5.56 3.49 3.58 3.51 3.74

d (7.8) dd (9.0, 7.8) t (9.0) t (9.0) ddd (9.0, 6.0, 2.1)

dC 94.2 72.6 76.4 70.0 74.6

63.2 4.56 dd (12.0, 2.1) 166.5 136.8 31.4 62.4 127.1 167.7 113.6 147.0 126.3 113.9 145.4 148.4 115.1 121.9 166.1 112.9 145.6 126.2 113.8 145.3 148.1 115.1 121.6

4.81 t (4.8) 4.28 d (4.8) 6.06/6.41 br s

6.28 d (15.6) 7.63 d (15.6) 7.05 d (1.8)

6.75 d (7.8) 6.91 dd (7.8, 1.8) 6.25 d (15.6) 7.55 d (15.6) 7.03 d (2.4)

6.77 d (8.4) 6.93 dd (8.4, 1.8)

165.5 140.2 68.0 66.9 126.2 167.7 113.5 147.0 126.4 113.9 145.4 148.4 115.1 121.9 166.1 112.9 145.8 126.1 113.8 145.3 148.1 115.1 121.6

NMR data were measured in CD3OD at 600 MHz. NMR data were measured in CD3OD at 150 MHz.

glucose, and hemiterpene groups were elucidated by HMBC correlation signals. The HMBC spectrum of 1 (Fig. 2) established a correlation between the H-1 of glucose and the C-40 of hemiterpene. The HMBC spectrum of 1 also showed a correlation between H-6 of glucose and carbonyl C-100 of the caffeoyl moiety. Based on these data, 1 was determined to be 40 -(6-O-caffeoyl-b-D-glucopyranosyl)-20 methyl butyric acid (Fig. 1). The naturally occurring caffeoyl hemiterpene glycosides have been rarely reported – aohada-glycosides (A, B and C) from Ilex macropoda (Fuchino et al., 1997) and rotundarpenosides (A and B) from Ilex rotunda (Kim et al., 2012) and hymenosides (N, O, P and V) from Hymenophyllum barbatum (Toyota et al., 2002). Comparing with these, 1 possessed different type of hemiterpene or sugar moiety. Compound 2 had a molecular formula of C20H24O11 as indicated by HR-FAB-MS data (m/z 439.1240 [MH], Calcd. C20H23O11, 439.1240). Its 1H NMR spectrum (Table 1) was consistent with a caffeoyl moiety in the aromatic region as determined by the presence of an aromatic ABX-spin system, meta-coupled aromatic signal [d 7.06 (1H, d, J = 1.8 Hz, H-500 )], ortho-coupled aromatic resonance [d 6.78 (1H, d, J = 8.4 Hz, H-800 )] and ortho–meta-coupled aromatic signal [d 6.97 (1H, dd, J = 1.8, 8.4 Hz, H-900 )] for a 3,4-dihydroxyphenyl unit, and two olefinic protons [d 6.30 and 7.66 (1H, d, J = 15.9 Hz, H200 ,300 )] for a trans-configured double bond. In addition, the 1H and 13 C NMR spectra of 2 indicated the presence of a glucopyranosyl moiety with one methylene [d 4.28 (1H, dd, J = 5.4, 12.0 Hz, glc-6a) and 4.48 (1H, dd, J = 1.8, 12.0 Hz, glc-6b)], five methines [d 5.56 (1H, d, J = 7.8 Hz, glc-1), 3.67 (1H, m, glc-5), 3.48 (1H, t, J = 9.0 Hz, glc-3), 3.45 (1H, dd, J = 9.0, 7.8 Hz, glc-2), and 3.41 (1H, t, J = 9.0 Hz, glc-

4)], and six carbons (d 63.3, 69.9, 72.5, 74.7, 76.4 and 94.3). The large coupling constant of the anomeric proton at d 5.56 (J = 7.8 Hz, glc-1) indicated that the glucopyranoside as in a b-configuration. The 1H and 13C NMR spectra of 2 also established the presence of a hemiterpene moiety, exomethylene protons [d 5.69 and 6.24 (each 1H, d, J = 1.2 Hz, H-50 )], two methylenes [d 2.53 (2H, t, J = 6.6 Hz, H-30 ), d 3.66 (2H, t, J = 6.6 Hz, H-40 )], and five carbons (d 34.9, 60.2, 126.7, 137.1, 166.7). The HMBC spectrum of 2 (Fig. 3) showed correlations between the exomethylene proton signals (H-50 ) with the quaternary carbon at d 166.7 (C-10 ) and the methylene carbon at d 34.9 (C-30 ). These data led to the elucidation of the hemiterpene structure as 40 -hydroxy-20 -methylene-butyrate. The connectivities of the caffeoyl, glucose, and hemiterpene groups were elucidated by HMBC correlation signals. The HMBC spectrum of 2 showed a correlation between the H-1 of glucose and carbonyl C-100 of the caffeoyl moiety. The HMBC spectrum of 2 also showed a correlation between H-6 of glucose and C-10 of the hemiterpene. Therefore, 2 was deduced as 1-O-caffeoyl-6-O-(40 -hydroxy-20 -methylene-butyroyl)-b-D-glucopyranose (Fig. 1). The hemiterpene glycoside portion of compound 2, excluding the caffeoyl moiety, was previously isolated from a Tulipa species and named 6-Tuliposide A (Christensen, 1999, 1995a,b; Christensen and Kristiansen, 1999, 1995; Tschesche et al., 1968). Thus, 2 was named 1-caffeoyl-6-tuliposide A. Compound 3 was isolated as a pale yellow amorphous powder. Its HR-FAB-MS (m/z 601.1556 [MH] Calcd. for C29H29O14, 601.1557) indicated that the molecular formula was C29H30O14. The 1H and 13C NMR spectra of 3 (Table 2) were similar to those of 2 except for the presence of other signals for a caffeoyl moiety,

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O

5"

3"

OH

O

2"

2'

1'

6

6

9"

O

HO HO

R1

O

O

OH

8"

O

1

OH

2'

3'

4'

O 1'

OH

4'

3' 5'

O

HO HO

8" 9"

1

OR3

2 3 4 5

HO

1

2

6

OH

5'

HO

O

1

2

OH

OR2

3

O

OH O

O

O 1"

HO

3'

O

5' 8

HO OH OH

11

O

2 2'

6

OH

5'

R1

R2 = H, R3 = H R2 = H, R3 = H R2 = Caffeoyl, R3 = H R2 = H, R3 = Caffeoyl

6'

OH

OH

2'

OH

2' 6

9'

7 R1 = H, 8 R1 = OH, 9 R1 = OH, 10 R1 = OH,

6' 8

8'

O

8

6

5"

OR3

HO HO

9

HO

3"

O

R2 = H, R3 = H R2 = H, R3 = Caffeoyl R2 = Caffeoyl, R3 = H R2 = Caffeoyl, R3 = H

R1 = H, R1 = H, R1 = H, R1 = OH,

O

3

5

OH

2"

O

5'

1

OR2

3

OH OH

R

OH 12 13 14

OH OH Xylose

Fig. 1. Structures of the compounds isolated from S. prunifolia.

O 5"

O H H 6 HO HO

9"

O 1

OH H

8"

OH OH

O

O 1' OH

4' 5' 1 1

H H-COSY HMBC

Fig. 2. Key correlations of 1H–1H COSY and HMBC spectra for compound 1.

an ABX spin system [dH 6.75 (d, J = 8.1 Hz, H-8000 ), dH 6.94 (dd, J = 8.1, 1.8 Hz, H-900 0 ), dH 7.01 (d, J = 1.8 Hz, H-500 0 )], and two olefinic protons [dH 6.19 and dH 7.58 (J = 15.6 Hz)] of a trans-configured double bond. The HMBC spectrum of 3 (Fig. 3) showed correlations between the anomeric glucose proton and C-100 of the caffeoyl moiety, as well as correlations between glucose C-6 protons and C-10 of the hemiterpene in the same manner as for 2. In addition, the HMBC spectrum of 3 showed a correlation between the C-2 proton of glucose and another caffeoyl carbonyl carbon (C-100 0 ). Thus, 3 was deduced as 1,2-O-dicaffeoyl-6-O-(40 -hydroxy-20 -methylenebutyroyl)-b-D-glucopyranoside (Fig. 1) and tentatively named 1,2-dicaffeoyl-6-tuliposide A.

Compound 4 was isolated as a brown amorphous powder. Its HR-negative FAB-MS (m/z 601.1561 [MH] Calcd. for C29H29O14, 601.1557) indicated that molecular formula was C29H30O14. The 1 H NMR spectra of 4 (Table 2) showed the presence of two caffeoyl groups [dH 7.63 (d, J = 15.9, H-300 ), dH 7.50 (d, J = 15.9, H-3000 ), dH 7.05 (d, J = 1.8, H-500 ), dH 7.02 (d, J = 1.8, H-500 0 ), dH 6.93 (dd, J = 8.1, 1.8, H900 ), dH 6.91 (dd, J = 8.1, 1.8, H-900 0 ), dH 6.77 (d, J = 8.1, H-800 ), dH 6.74 (d, J = 8.1, H-8000 ), dH 6.28 (d, J = 15.9, H-200 ), dH 6.21 (d, J = 15.9, H2000 )], a b-glucosyl moiety [dH 5.57 (d, J = 7.8 Hz, glc-1), dH 4.32 (dd, J = 12.0, 6.0 Hz, glc-6a), dH 4.52 (dd, J = 12.0, 1.8, glc-6b), dH 3.69 (ddd, J = 9.0, 6.0, 1.8 Hz, glc-5), dH 3.47 (t, J = 9.0 Hz, glc-3), dH 3.45 (dd, J = 9.0, 7.8 Hz, glc-2), dH 3.42 (t, J = 9.0 Hz, glc-4) and a hemiterpene moiety [dH 6.26 (d, J = 1.2 Hz, H-50 a), dH 5.71 (d, J = 1.2 Hz, H-50 b), dH 4.33 (m, H-40 ), dH 2.70 (t, J = 6.6 Hz, H-30 )]. The HMBC spectrum of 4 (Fig. 3) showed correlations between the anomeric glucose proton and C-100 of the caffeoyl moiety and also showed correlations between glucose C-6 protons and C-10 of the hemiterpene in the same manner as for 2. In addition, the HMBC spectrum of 4 showed a correlation between the C-40 proton of the hemiterpene moiety and another caffeoyl carbonyl carbon (C-100 0 ). Thus, compound 4 was elucidated as 1-O-caffeoyl-6-O(40 -caffeoyl-20 -methylene-butyroyl)-b-D-glucose (Fig. 1) and named 1,40 -dicaffeoyl-6-tuliposide A; compound 4 is a structural isomer of 3. The molecular formula of compound 5 was determined as C29H30O15 by HR-FAB-MS (m/z 617.1505 [MH] Calcd. for

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O

O H H 6 4

O 1'

2'

3'

OH

4'

H H 6

H 5' H

8"

O

5

OH 1 O 1" OH 3 2 OH H O

9"

7"

2" 3"

4"

OH

5"

6"

HO HO

OH

O 1'

2'

H 5' H HO

OH

4'

3'

O

5"

1"

O 1

9" 8"

H O

OH OH

1"'

O

OH

OH 2

OH

O H H 6

O 1'

2'

3'

H 5' H

HO HO

3

O OH

4'

O

HH

1"'

5"'

O

O

OH

H H 6

OH 1

H

O 1" O

5"

HO HO

OH

O 1'

OH

OH 2'

O

4' 3'

H 5' H

HH

1"'

OH

5"'

O

OH

O 1

OH H

O 1" O

5"

OH

5

4

Fig. 3. Key correlations for HMBC spectra of compounds 2–5.

Table 2 Biological activities of compounds isolated from the leaves of S. prunifolia. Compounds

DPPH radical scavenging activity IC50 ± SD (lM)a

NBT superoxide scavenging activity IC50 ± SD (lM)a

Inhibitory activity on NO production IC50 ± SD (lM)a

1 2 3 4 5 6 7 8 9 10 11 12 13 14

11.55 ± 0.37 20.81 ± 0.39 16.48 ± 0.30 18.90 ± 1.43 14.46 ± 0.33 42.62 ± 1.68 100< 31.69 ± 1.58 19.80 ± 0.54 18.94 ± 0.50 17.92 ± 1.20 21.77 ± 0.12 24.21 ± 0.63 20.95 ± 0.33 24.99 ± 0.35

51.30 ± 2.46 53.47 ± 4.86 13.34 ± 2.49 13.93 ± 2.31 15.41 ± 1.02 27.25 ± 2.79 100< 70.94 ± 0.91 16.89 ± 1.54 29.18 ± 2.56 24.30 ± 3.72 31.67 ± 1.24 25.64 ± 1.41 24.85 ± 1.57 44.48 ± 3.12

100< 15.76 ± 0.37 19.20 ± 0.27 100< 50.88 ± 2.19 100< 100< 100< 100< 100< 100< 100< 100< 100<

L-Ascorbic L-NMMA a b

acidb

b

17.10 ± 0.61

Values are presented as the mean ± SD (n = 3). Positive controls.

C29H29O15, 617.1506). Its 1H NMR spectra (Table 2) showed the presence of two caffeoyl groups [dH 7.63 (d, J = 15.6 Hz, H-300 ), dH 7.55 (d, J = 15.6 Hz, H-300 0 ), dH 7.05 (d, J = 1.8 Hz, H-500 ), dH 7.03 (d, J = 2.4 Hz, H-5000 ), dH 6.91 (dd, J = 7.8, 1.8 Hz, H-900 ), dH 6.93 (dd, J = 8.4, 1.8 Hz, H-900 0 ), dH 6.77 (d, J = 8.4 Hz, H-8000 ), dH 6.75 (d, J = 7.8 Hz, H-800 ), dH 6.28 (d, J = 15.6 Hz, H-200 ), dH 6.25 (d, J = 15.6 Hz, H-200 0 )], a b-glucosyl moiety [dH 5.56 (d, J = 7.8 Hz, H1), dH 4.31 (dd, J = 12.0, 6.0 Hz, H-6a), dH 4.56 (dd, J = 12.0, 2.4 Hz, H-6b), dH 3.74 (ddd, J = 9.0, 6.0, 2.1 Hz, H-5), dH 3.58 (t, J = 9.0 Hz, H-3), dH 3.51 (t, J = 9.0 Hz, H-4), dH 3.49 (dd, J = 9.0, 7.8 Hz, H-2)], and a hemiterpene moiety [dH 6.41 and 6.06 (br s, H-50 ), dH 4.81 (t, J = 4.8 Hz, H-30 ), dH 4.28 (d, J = 4.8 Hz, H-40 )]. The NMR spectroscopic data of 5 were highly similar with those of 4, except that the C-30 signal of the hemiterpene was shifted downfield. These data indicated that the 5 had an additional hydroxyl group located

at C-30 of the hemiterpene moiety. Thus, 5 was elucidated as 1-Ocaffeoyl-6-O-(40 -caffeoyl-30 -hydroxy-20 -methylene-butyroyl)-b-Dglucopyranose (Fig. 1). The hemiterpene glycoside portion of 5, except for the caffeoyl moieties, is known as 6-Tuliposide B, which has been isolated previously from a Tulipa species (Christensen, 1999; Tschesche et al., 1968). Thus, 5 was named 1,40 -dicaffeoyl6-tuliposide B. The nine known compounds were identified as caffeic acid (6) (Andary et al., 1989), 1-O-coumaroyl-b-D-glucopyranose (7) (Hiradate et al., 2004), 1-O-caffeoyl-b-D-glucopyranose (8), 1,2-Odicaffeoyl-b-D-glucopyranose (9) (Jiang et al., 2005, 2001), 1,6-Odicaffeoyl-b-D-glucopyranose (10) (Hamerski et al., 2005; Jiang et al., 2001), quercetin-3-O-glucopyranose (11) (Fossen et al., 1998), (+)-catechin (12) (Lu and Foo, 1999), ()-epicatechin (13) (Lu and Foo, 1999; Nonaka and Nishioka, 1999), and

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(+)-catechin-3-O-b-D-xylopyranose (14) (Liimatainen et al., 2012). The spectroscopic data of 6–14 were compared with values reported in the literature. 2.2. Anti-oxidative activity In order to assess the anti-oxidative activity of the compounds isolated from the leaves of S. prunifolia, the effect of the compounds on DPPH radical scavenging activity (Choudhary et al., 2009; Hatano et al., 1989) was measured. According to the results (Table 2), all of the compounds exhibited potent anti-oxidative activities except for compound 7. In particular, the new caffeoyl hemiterpene glycosides (1–5) and dicaffeoyl glycosides (9, 10) were more potent than L-ascorbic acid (IC50 = 24.99 ± 0.35 lM), which was used as a positive control. Compound 1, which has a 40 -hydroxy-20 -methyl-butyrate substituted hemiterpene moiety, exhibited the most activity in this assay. It is thus hypothesized that the carboxyl group of the butyrate stabilized the free radicals by acting as an electron-donating group. Furthermore, 5 exhibited strong radical scavenging activity and was better than that of the other caffeoyl tuliposides (2–4). Interestingly, the only structural difference between 4 and 5 was a hydroxyl group at the C-30 position of the hemiterpene moiety (Fig. 1). Thus, the hydroxyl group at the C-30 of hemiterpene may have a positive effect towards stabilization of radicals. These results suggested that the hemiterpene moiety was important for the free radical scavenging activities of 1–5. NBT superoxide scavenging was also measured to evaluate the anti-oxidative activities of the isolated compounds. As shown in Table 3, all isolates except 7 showed strong superoxide scavenging activities compared with L-ascorbic acid (IC50 = 44.48 ± 3.12 lM), which was used as a positive control. Especially, compounds bearing two caffeoyl groups (3–5, 9–10) exhibited better anti-oxidative activities than those containing only one caffeoyl moiety. These results suggested that the number of caffeoyl groups in the isolated compounds was important for elimination of superoxide. 2.3. Anti-inflammatory activity To evaluate the anti-inflammatory activities of the isolated compounds, next tested was their ability to inhibit the production of NO production in RAW264.7 cells treated with LPS, which induces over-expression of iNOS and models an inflammatory condition (Hobbs et al., 1999). Among the compounds isolated from S. prunifolia, 2 (IC50 = 15.76 ± 0.37 lM) and 3 (IC50 = 19.20 ± 0.27 lM) showed strong inhibitory activity towards NO production and were similar to that of the positive control L-NMMA (IC50 = 15.07 ± 0.86 lM), while 5 (IC50 = 50.88 ± 2.19 lM) only mildly inhibited NO production. None of the other compounds were active at concentrations less than 100 lM. A common structural feature of the two active compounds (2 and 3) was the hemiterpene moiety, which lacked a caffeoyl group at the 40 -OH position. Consistent with this observation, the presence of a substituted caffeoyl group at the 40 -OH position of the hemiterpene moiety (4) abrogated the inhibition of NO production, even though 4 is a structural isomer of 3. These results suggested that the structure of the hemiterpene moiety in the isolated compounds had a decisive effect on cell permeability and inhibition of NO production.

(2–5) have not been described previously. All of the isolated compounds, except 1-O-coumaroyl-b-D-glucopyranoside (7), exhibited potent DPPH radical scavenging activity and NBT superoxide scavenging activity. In addition, 1-caffeoyl-6-tuliposide A (2), 1,2-dicaffeoyl-6-tuliposide A (3) and 1,40 -dicaffeoyl-6-tuliposide B (5) inhibited NO production in RAW264.7 cells. 4. Experimental 4.1. General experimental procedures Celite 545 (Duksan Pure Chemicals), Sephadex LH-20 (GE Healthcare Bio-Science AB), MCI-gel CHP 20P (Mitsubishi Chemical), and ODS-B gel (Daiso) were used for column chromatography (CC). TLC was performed using a pre-coated silica gel 60 F254 plate (Merck). 1D (1H, 13H) and 2D (1H–1H COSY, HMBC, HSQC) NMR spectra were acquired with a Gemini 2000 (300 MHz for 1H NMR) and VNS (Varian, 600 MHz for 1H NMR and 13C NMR) using CD3OD as the solvent. Optical rotations were measured with a Jasco DIP-370 polarimeter. High-resolution FAB-MS data were obtained with a JEOL JMS-600 W and JMS-700. 4.2. Plant material S. prunifolia leaves were collected from the Korea National Arboretum, Pocheon-City Gyeonggi Province, Korea, during September 2011. 4.3. Extraction and isolation Leaves of S. prunifolia (5.5 kg) were extracted with acetone-H2O (Sol, 4:1, v/v, 280 g) at room temperature and concentrated in vacuo to afford a crude extract. After acetone evaporation, the extract was filtered through Celite 545 and eluted with water and acetone. The water-soluble filtrate (204 g) was subjected to Sephadex LH-20 CC and eluted with a H2O–MeOH gradient (from 100:0 to 0:100), yielding 9 fractions (SP-1 to 9). Repeated CC of fraction SP-5 (11.8 g), using a MCI gel column with a gradient solvent system of H2O: MeOH (from 80:20 to 0:100), yielded 7 (470 mg) and 8 (3.3 g), respectively. Fraction SP-6 (17.5 g) was separated using a MCI gel column with a gradient solvent system of H2O: MeOH (from 80:20 to 0:100) to yield 2 (2.0 g). Sub-fractions of SP-6 were passed through an ODS gel column with a gradient solvent system of H2O: MeOH (from 80:20 to 0:100) to give 1 (27 mg) and 14 (16 mg). Fraction SP-7 (12.2 g) was separated using a MCI gel column and an ODS silica gel column with a H2O: MeOH gradient (from 70:30 to 0:100) yielding 5 (397 mg), 6 (1.0 g), 11 (140 mg), 12 (112 mg), and 13 (400 mg), respectively. Fraction SP-8 (8.1 g) was applied to a MCI gel column using a gradient solvent system of H2O: MeOH (from 80:20 to 0:100) yielding 3 (140 mg) and 4 (112 mg). Sub-fractions of SP-8 were passed through an ODS gel column using a isocratic solvent of H2O: MeOH (60:40) to give 9 (127 mg) and 10 (300 mg).

3. Conclusions

4.3.1. 40 -(6-O-Caffeoyl-b-D-glucopyranosyl)-20 -methyl butyric acid (1) Pale yellow amorphous powder; [a]D25: 21 (c 0.001, MeOH); UV (MeOH): kmax (log e) 285 (3.67) 329 (3.84) nm; for 1H and 13C NMR spectroscopic data, see Table 1; HR-FAB-MS: m/z 441.1396 [MH] (Calcd for C20H25O11, 441.1397).

Five new caffeoyl hemiterpene glycosides (1–5) and nine known phenolic compounds (6–14) were isolated from leaves of S. prunifolia. Although a tuliposide derivative bearing a cinnamoyl group has been reported (Hou et al., 2009), caffeoyl tuliposides

4.3.2. 1-O-caffeoyl-6-O-(40 -hydroxy-20 -methylene-butyroyl)-b-Dglucopyranose (2) Pale yellow amorphous powder; [a]D25: +4.5 (c 0.01, MeOH); UV (MeOH): kmax (log e) 288 (3.76) 335 (4.02) nm; for 1H and 13C

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NMR spectroscopic data, see Table 1; HR-FAB-MS: m/z 439.1240 [MH] (Calcd for C20H23O11, 439.1240). 4.3.3. 1,2-O-dicaffeoyl-6-O-(40 -hydroxy-20 -methylene-butyroyl)-b-Dglucopyranose (3) Pale yellow amorphous powder; [a]D25: +1.4 (c 0.01, MeOH); UV (MeOH): kmax (log e) 288 (3.81) 330 (3.99) nm; for 1H and 13C NMR spectroscopic data, see Table 2; HR-FAB-MS: m/z 601.1556 [MH] (Calcd for C29H29O14, 601.1557). 4.3.4. 1-O-caffeoyl-6-O-(40 -caffeoyl-20 -methylene-butyroyl)-b-Dglucopyranose (4) Brown amorphous powder; [a]D25: +8.9 (c 0.01, MeOH); UV (MeOH): kmax (log e) 288 (3.97) 331 (4.18) nm; for 1H and 13C NMR spectroscopic data, see Table 2; HR-FAB-MS: m/z 601.1561 [MH] (Calcd for C29H29O14, 601.1557). 4.3.5. 1-O-caffeoyl-6-O-(40 -caffeoyl-30 -hydroxy-20 -methylenebutyroyl)-b-D-glucopyranose (5) Brown amorphous powder; [a]D25: +13.8 (c 0.01, MeOH); UV (MeOH): kmax (log e) 288 (3.97) 332 (4.16); for 1H and 13C NMR spectroscopic data, see Table 2; HR-FAB-MS: m/z 617.1505 [MH] (Calcd for C29H29O15, 617.1506). 4.4. Measurement of DPPH free radical scavenging activity DPPH (0.1 mM) was dissolved in absolute EtOH. Various concentrations of test samples (12.5, 25, 50 and 100 lM) in a volume of 20 lL were added to 180 lL of the DPPH solution. After mixing gently and incubating for 30 min, the optical density was measured at 518 nm using an ELISA reader (TECAN). Free radical scavenging activity was calculated as an inhibition rate (%) = [1  (sample O.D./control O.D.)]  100 and IC50 values were defined as the concentration that could scavenge 50% of DPPH free radicals. L-ascorbic acid was used as positive control. 4.5. Measurement of NBT superoxide scavenging activity A reaction mixture with a final volume of 632 lL was prepared with 50 mM phosphate buffer (pH 7.5) containing EDTA (0.05 mM), hypoxanthine (0.2 mM), NBT (63 lL, 1 mM), sample (63 lL), and of xanthine oxidase (63 lL, 1.2 U/lL) in an Eppendorf tube; xanthine oxidase was added last. The subsequent rate of NBT reduction was determined on the basis of sequential spectrophotometric determination of the change in absorbance at 590 nm using an ELISA reader (TECAN). Results were expressed as the percent inhibition of NBT reduction with respect to the reaction mixture without sample (buffer only). Superoxide anion scavenging activity was calculated as an inhibition rate (%) = [(1  (sample O.D.  blank O.D.)/(control O.D.  blank O.D.))  100] and IC50 values that were defined as the concentration at which 50% of NBT/Superoxide anions were scavenged. L-Ascorbic acid was used as positive control. 4.6. Measurement of inhibitory activity on NO production The murine macrophage cell line RAW264.7 was purchased from the Korean Cell Line Bank. Cells were grown at 37 °C in a humidified atmosphere (5% CO2) in DMEM containing 10% fetal bovine serum and 10 IU/mL penicillin G. Cells (3  104 cells/well) were seeded onto 96-well plates and incubated under the same conditions for 2 h. Next, test samples (12.5, 25, 50 and 100 lM) were added along with 1 lg/mL LPS (lipopolysaccharide) and incubated for 24 h. Griess reagent (0.1% naphthylethylene-diamine and 1% sulfanilamide in 5% H3PO4 solution) was added to the supernatant obtained from treated cells. The absorbance of the samples

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was then read at 540 nm using an ELISA reader and the amount of nitrite in each sample was calculated based on a sodium nitrite standard curve (Green et al., 1982). The inhibitory activity against NO production was calculated as an inhibition rate (%) = [1  (sample O.D.  blank O.D.)/(control O.D.  blank O.D.)]  100 and the IC50 values were determined. L-NMMA, an inhibitor of nitric oxide synthase, was used as a positive control. 4.7. MTT assay The cytotoxicity of compounds was assessed by MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Mosmann, 1983). After performing the NO measurement assay of samples, the remaining medium was removed and 100 lL of MTT (0.5 mg/mL) was added to each well. After a 4 h incubation at 37 °C, the produced MTT-formazan was dissolved in 100 lL of DMSO. The absorbance of MTT-formazan was measured at 540 nm using an ELISA reader. Cytotoxicity was calculated as the percent of cell viability (%) = [(sample O.D./blank O.D.)  100]. Acknowledgments This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0022929). References Andary, C., Ravn, H., Wylde, R., Heitz, A., Motteflorac, E., 1989. Crassifolioside, a caffeic acid glycoside ester from Plantago crassifolia. Phytochemistry 28, 288– 290. Choudhary, M.I., Naheed, N., Abbaskhan, A., Ali, S., Atta-ur-Rahman, 2009. Hemiterpene glucosides and other constituents from Spiraea canescens. Phytochemistry 70, 1467–1473. Christensen, L.P., 1995a. A further tuliposide from Alstroemeria revoluta. Phytochemistry 40, 49–51. Christensen, L.P., 1995b. Tuliposides from Alstroemeria revoluta. Phytochemistry 38, 1371–1373. Christensen, L.P., 1999. Tuliposides from Tulipa sylvestris and T. turkestanica. Phytochemistry 51, 969–974. Christensen, L.P., Kristiansen, K., 1995. Isolation and quantification of a new tuliposide (Tuliposide-D) by HPLC in Alstroemeria. Contact Dermatitis 33, 188– 192. Christensen, L.P., Kristiansen, K., 1999. Isolation and quantification of tuliposides and tulipalins in tulips (Tulipa) by high-performance liquid chromatography. Contact Dermatitis 40, 300–309. Fossen, T., Pedersen, A.T., Andersen, Ø.M., 1998. Flavonoids from red onion (Allium cepa). Phytochemistry 47, 281–285. Fuchino, H., Tachibana, H., Tanaka, N., 1997. Three new hemiterpene glycosides from Ilex macropoda. Chem. Pharm. Bull. 45, 1533–1535. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum, S.R., 1982. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal. Biochem. 126, 131–138. Hamerski, L., Bomm, M.D., Silva, D.H.S., Young, M.C.M., Furlan, M., Eberlin, M.N., Castro-Gamboa, I., Cavalheiro, A.J., Bolzani, V.D., 2005. Phenylpropanoid glucosides from leaves of Coussarea hydrangeifolia (Rubiaceae). Phytochemistry 66, 1927–1932. Hatano, T., Edamatsu, R., Hiramatsu, M., Mori, A., Fujita, Y., Yasuhara, T., Yoshida, T., Okuda, T., 1989. Effects of the interaction of tannins with co-exist substances. IV. Effects of tannins and ralated polyphenols on superoxide anion radical, and on 1,1-diphenyl-2-picrylhydrazyl radical. Chem. Pharm. Bull. 37, 2016. Hiradate, S., Morita, S., Sugie, H., Fujii, Y., Harada, J., 2004. Phytotoxic cis-cinnamoyl glucosides from Spiraea thunbergii. Phytochemistry 65, 731–739. Hobbs, A., Higgs, A., Moncada, S., 1999. Inhibition of nitric oxide synthase as a potential therapeutic target. Annu. Rev. Pharmacol. Toxicol. 39, 191–220. Hou, T.P., Teng, Y., Sun, Q., Yu, Z.Y., 2009. A new fungitoxic metabolite from Spiraea alpina Pall. Fitoterapia 80, 237–240. Jiang, Z.H., Hirose, Y., Iwata, H., Sakamoto, S., Tanaka, T., Kouno, I., 2001. Caffeoyl, coumaroyl, galloyl, and hexahydroxydiphenoyl glucoses from Balanophora japonica. Chem. Pharm. Bull. 49, 887–892. Jiang, Z.H., Wang, J.R., Li, M., Liu, Z.Q., Chau, K.Y., Zhao, C., Liu, L., 2005. Hemiterpene glucosides with anti-platelet aggregation activities from Ilex pubescens. J. Nat. Prod. 68, 397–399. Kim, M.H., Park, K.H., Oh, M.H., Kim, H.H., Choe, K.I., Park, S.H., Lee, M.W., 2012. Two new hemiterpene glycosides from the leaves of Ilex rotunda. Thunb. Arch. Pharm. Res. 35, 1779–1784.

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