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Antioxidant and Anti-Inflammatory Phenolic Glycosides from Clematis tashiroi Li-Jie Zhang,† Hung-Tse Huang,† Shih-Yen Huang,‡ Zhi-Hu Lin,† Chien-Chang Shen,† Wei-Jern Tsai,† and Yao-Haur Kuo*,†,§,⊥ †

National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei 112, Taiwan, Republic of China Endemic Species Research Institute, Nantou County 552, Taiwan, Republic of China § Graduate Institute of Integrated Medicine, China Medical University, Taichung 404, Taiwan, Republic of China ⊥ Ph.D. Program for the Clinical Drug Discovery from Botanical Herbs, College of Pharmacy, Taipei Medical University, Taipei 110, Taiwan, Republic of China ‡

S Supporting Information *

ABSTRACT: From the 95% EtOH extract of dried aerial parts of Clematis tashiroi, eight new and four known phenolic (caffeic acid, coumaric acid, ferrulic acid) glycosides were isolated and characterized. The structures of the new isolates (clematisides A−H) were elucidated by spectroscopic data interpretation as trans-4-O-(6-O-trans-caffeoyl-β-Dglucopyranosyl)-9-O-β- D -glucopyranosyl caffeic acid (1), trans-4-O-(6-O-trans-feruloyl-β- D -glucopyranosyll)-9-Oβ-D-glucopyranosyl caffeic acid (2), trans-4-O-(6-O-trans-p-coumaroyl-β-D-glucopyranosyl)-9-O-β-D-glucopyranosyl caffeic acid (3), trans-4-O-(6-O-trans-caffeoyl-β-D-glucopyranosyl)-9-O-β-D-glucopyranosyl p-coumaric acid (4), trans-3-O-(6-O-transcaffeoyl-β-D-glucopyranosyl)-9-O-β-D-glucopyranosyl caffeic acid (5), trans-3-O-(6-O-trans-p-coumaroyl-β-D-glucopyranosyl)-9O-β-D-glucopyranosyl caffeic acid (6), 6-(3′,4′-dihydroxystyryl)-2-pyrone-4-O-(6-O-trans-caffeoyl)-β-D-glucopyranoside (7), and 6-(3′,4′-dihydroxystyryl)-2-pyrone-4-O-{6-O-[4-O-(6-O-trans-caffeoyl)-β-D-glucopyranosyl]-trans-caffeoyl}-β-D-glucopyranoside (8), respectively. In a DPPH radical-scavenging test, compounds 1, 7, and 8 showed more potent antioxidant activity than that of the positive control, vitamin E. In addition, compound 7 also showed inhibitory activity in an antinitric oxide release assay. hexapetala16,17 and C. armandii18,19 are rich in flavonoids and lignans, and C. mandshurica20−25 is known to contain saponins, phenolic glycosides, and flavonoids. However, phytochemical and bioactive studies on the C. tashiroi have not been reported. Herein are documented the isolation and structural elucidation of eight new phenolic glycosides (clematisides A to H, 1−8) from the aerial parts of C. tashiroi. Evaluations of the DPPH free-radical-scavenging and antinitric oxide release activities of these isolates are also reported.

T

he genus Clematis, one of the largest genera in the family Ranunculaceae, comprises 300 widely distributed species, ranging in habitat from tropical regions to frigid zones and from sea level to high altitudes, and has 18 species and two varieties in Taiwan. Clematis tashiroi Maxim. is only distributed in Taiwan and the Ryukyu Islands in Japan from low to middle altitudes (50−2800 m).1 Some plants of this genus including C. chinensis, C. hexapetala, and C. mandshurica (C. ternif lora var. mandshurica) are used in traditional Chinese medicine for the treatment of rheumatism and collectively are termed “Weilingxian”.2 In addition, the dried rattans of both C. armandii and C. montana, named “Chuanmutong”, are also used in Chinese medicine.3 The constituents of several Clematis species have been investigated. Thus, C. chinensis4−11 and C. montana12−15 were found to contain saponins, both C. © 2015 American Chemical Society and American Society of Pharmacognosy

Received: February 13, 2015 Published: July 6, 2015 1586

DOI: 10.1021/acs.jnatprod.5b00154 J. Nat. Prod. 2015, 78, 1586−1592

Journal of Natural Products

Article

Figure 1. Key HMBC correlations of compound 1.

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trans-4-O-(6-O-trans-caffeoyl-β-D-glucopyranosyl)-9-O-β-D-glucopyranosyl caffeic acid, and this compound was assigned the trivial name, clematiside A. Clematiside B (2) was isolated as a yellow amorphous powder, and its HRESIMS gave a quasimolecular ion at m/z 703.1848 [M + Na]+ (C31H36O17Na). The UV, IR, 1H, and 13C NMR spectra of 2 as well as the substitution pattern were similar to those of 1 (Tables 1 and 2) except for the presence of a methoxy signal in 2. The methoxy group was located at C-3′ based on HMBC correlations (H-5′/C-3′, H3−OMe/C-3′). Thus, the structure of 2 was elucidated as trans-4-O-(6-O-trans-feruloyl-βD-glucopyranosyl)-9-O-β-D-glucopyranosyl caffeic acid. Compounds 3 and 4 gave the same HRESIMS data and similar NMR spectra. Their common molecular formula was established as C30H34O16 from the HRESIMS data {[M + Na]+ m/z 673.1718 (3) and 673.1709 (4)}. Both 3 and 4 were found to possess two glucopyranose units and two phenylpropanoid moieties, whereas one aromatic ring with an AMX coupling pattern in 1 was replaced by a aryl group with an A2B2 pattern in 3 and 4. Furthermore, the molecular formula of 3 and 4 showed one less oxygen than that of 1, indicating a trans-caffeoyl moiety to be replaced by a trans-p-coumaroyl unit in each case. The location of the p-coumaroyl moiety in 3 was determined at Glc G-6′ (HMBC correlation: H-G-6′/C-9′), and the same unit in 4 was found to be linked between two glucose units via Glc G-1O-C-9 and Glc G-1′-O-C-4 (HMBC correlations: H-G-1/C-9, H-G-1′/C-4). Accordingly, the structures of 3 and 4 (clematisides C and D) were determined as trans-4-O-(6-O-trans-pcoumaroyl-β-D-glucopyranosyl)-9-O-β-D-glucopyranosyl caffeic acid and trans-4-O-(6-O-trans-caffeoyl-β-D-glucopyranosyl)-9-Oβ-D-glucopyranosyl p-coumaric acid, respectively. Compound 5 gave the same molecular formula and similar NMR data to 1, indicating the occurrence of the same major structural units. The difference between them was that the linkage position of glucose G-1′ was at C-3 in 5. Thus, the structure of 5 (clematiside E) was determined as trans-3-O-(6-O-trans-caffeoylβ-D-glucopyranosyl)-9-O-β-D-glucopyranosyl caffeic acid. Compound 6 (clematiside F) gave the same HRESIMS data and similar NMR data to 3. Like compound 5, the Glc G-1′ was located at C-3 in 6. Thus, the structure of 6 was determined as trans-3-O-(6-O-trans-p-coumaroyl-β-D-glucopyranosyl)-9-Oβ-D-glucopyranosyl caffeic acid. Compound 7 was obtained as a yellowish powder. The HRESIMS indicated a molecular formula of C28H26O13 (m/z 569.1306 [M − H]−). The IR spectrum displayed bands for hydroxy (3383 cm−1), carbonyl (1683 cm−1), double bond (1634 cm−1), and aromatic (1605, 1536 cm−1) groups, and the UV spectrum showed absorption maxima at 335, 298, 248, and 219 nm. On observing the 1H and 13C NMR spectra, resonances were evident for a 6-(3′,4′-dihydroxystyryl)-4-hydroxy-2-pyrone

RESULTS AND DISCUSSION

The dried aerial parts of C. tashiroi (2.4 kg) were extracted with 95% EtOH at 50 °C three times. The EtOH extract was concentrated and partitioned with n-hexane and H2O. Then, the water layer was chromatographed on Diaion HP-20, Sephadex LH-20, and ODS columns sequentially. The subfractions obtained were subjected to preparative reversed-phase HPLC to yield eight new (1−8) and four known phenolic glycosides. The HRESIMS of clematiside A (1) afforded the molecular formula, C30H34O17, from the quasimolecular ion at m/z 689.1722 [M + Na]+. The IR spectrum displayed bands for hydroxy (3355 cm−1), carbonyl (1699 cm−1), double bond (1631 cm−1), and aromatic (1595, 1514 cm−1) groups, and the UV spectrum exhibited absorption maxima at 324, 294, 243, and 217 nm. The 1H NMR spectrum showed signals for two pairs of trans-olefinic protons [δH 7.87 and 6.56 (each 1H, d, J = 16.0 Hz, H-7 and H-8), 7.93 and 6.61 (each 1H, d, J = 16.0 Hz, H-7′ and H-8′)], and two aryl moieties with an AMX coupling pattern [δH 7.45 (d, J = 2.0 Hz, H-2), 7.56 (d, J = 8.5 Hz, H-5), 7.09 (dd, J = 8.5, 2.0 Hz, H-6) and 7.55 (br s, H-2′), 7.22 (d, J = 8.0 Hz, H-5′), 7.09 (dd, J = 8.5, 2.0 Hz, H-6′)], together with 18 sp2 carbons in the 13C NMR spectrum, suggesting that 1 contains two caffeoyl moieties.26 Moreover, two anomeric signals of two glucose units designated H-G-1 [δH 6.47 (d, J = 7.5 Hz)] and H-G-1′ [5.54 (d, J = 7.5 Hz)], eight oxygenated methine signals [δH 4.30, 4.32, 4.35, and 4.08 (H-G-2 to H-G-5), 4.22, 4.29, 4.14, and 4.20 (H-G-2′ to H-G-5′)], and two oxygenated methylene signals [δH 4.40, 4.49 (each 1H, H-G-6), 5.08, 4.88 (each 1H, H-G-6′)] were found in the 1H NMR spectrum. This evidence, together with the HRESIMS data, suggested that 1 possesses two caffeoyl moieties and two hexose units. The saccharide moieties were confirmed as β-glucopyranose units with a D-configuration based on acid hydrolysis of 1.27 The HMBC spectrum of 1 (Figure 1) showed long-range correlations between the carbonyl carbons of two caffeoyl groups at δC 166.09 and 167.42 and H-G-1 and H-G-6′, respectively, and between C-4 and H-G-1′. Accordingly, the structure of 1 was determined as 1587



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Table 1. 1H NMR Data of Compounds 1 to 6 (J in Hz)a position 2 3 5 6 7 8 G-1 G-2 G-3 G-4 G-5 G-6 2′ 3′ 5′ 6′ 7′ 8′ G-1′ G-2′ G-3′ G-4′ G-5′ G-6′

1

2

3

4

7.45 d (2.0)

7.06 d (1.8)

7.45 d (2.0)

7.56 d (8.5) 7.09 dd (8.5, 2.0) 7.87 d (16.0) 6.56 d (16.0) 6.47 d (7.5) 4.30 t (7.5) 4.32 t (7.5) 4.35 t (7.5) 4.08 m 4.49 br d (10.0) 4.40 dd (10.0, 4.5) 7.55 br s

7.11 d (8.4) 6.86 dd (8.4, 1.8) 7.52 d (15.9) 6.18 d (15.9) 5.56 d (7.8) 3.45 m 3.45 m 3.41 m 3.41 m 3.86 br d (9.6) 3.70 dd (9.6, 3.0) 7.17 d (1.8)

7.22 d (8.0) 7.09 dd (8.5, 2.0) 7.93 d (16.0) 6.61 d (16.0) 5.54 d (7.5) 4.22 t (7.5) 4.29 m 4.14 t (9.0) 4.20 m 5.08 br d (11.0) 4.88 dd (11.0, 5.5)

6.85 d (7.8) 7.07 dd (7.8, 1.8) 7.62 d (15.9) 6.38 d (15.9) 4.80 ob 3.55 t (9.0) 3.51 t (9.0) 3.41 m 3.75 td (8.4, 2.4) 4.56 dd (12.0, 2.4) 4.38 dd (12.0, 7.8) 3.88 s 3H

7.59 d (8.5) 7.10 dd (8.5, 2.0) 7.86 d (16.0) 6.53 d (16.0) 6.46 d (8.0) 4.34 m 4.36 m 4.38 m 4.08 m 4.51 dd (12.5, 2.5) 4.40 dd (12.5, 5.0) 7.54 d (8.5) 7.22 d (8.5) 7.22 d (8.5) 7.54 d (8.5) 7.92 d (16.0) 6.61 d (16.0) 5.59 d (7.5) 4.32 t (7.5) 4.38 m 4.18 t (9.0) 4.28 m 5.15 dd (11.5, 2.0) 4.89 dd (11.5, 6.5)

OCH3

5

6

7.48 d (9.0) 7.30 d (9.0) 7.30 d (9.0) 7.48 d (9.0) 7.83 d (16.0) 6.49 d (16.0) 6.48 d (7.5) 4.32 m 4.33 m 4.37 m 4.09 m 4.50 dd (12.0, 2.0) 4.40 dd (12.0, 4.5) 7.55 br s

7.36 d (1.8)

7.36 d (2.4)

6.86 d (8.4) 7.17 dd (8.4, 1.8) 7.64 d (16.2) 6.34 d (16.2) 5.57 d (7.8) 3.44 m 3.47 t (8.4) 3.38 t (8.4) 3.41 m 3.85 dd (12.0, 1.8) 3.70 dd (12.0, 5.4) 7.00 d (1.8)

7.22 d (8.0) 7.09 dd (8.0, 2.0) 7.92 d (16.0) 6.62 d (16.0) 5.62 d (7.5) 4.32 m 4.33 m 4.20 t (9.0) 4.28 m 5.12 br d (10.0) 4.88 dd (10.0, 6.5)

6.76 d (8.0) 6.88 dd (8.0, 1.8) 7.51 d (16.2) 6.25 d (16.2) 4.85 o 3.55 t (9.0) 3.52 t (9.0) 3.43 t (9.0) 3.76 ddd (9.0, 6.6, 2.4) 4.56 dd (12.0, 2.4) 4.37 dd (12.0, 6.6)

6.85 d (8.4) 7.17 dd (8.4, 2.4) 7.65 d (16.2) 6.36 d (16.2) 5.56 d (7.8) 3.44 m 3.47 t (8.4) 3.38 t (8.4) 3.42 m 3.83 dd (12.0, 1.8) 3.69 dd (12.0, 4.8) 7.39 d (8.4) 6.79 d (8.4) 6.79 d (8.4) 7.39 d (8.4) 7.57 d (16.2) 6.31 d (16.2) 4.85 o 3.54 t (9.0) 3.52 t (9.0) 3.43 t (9.0) 3.77 ddd (9.0, 7.2, 2.4) 4.55 dd (12.0, 2.4) 4.38 dd (12.0, 7.2)

a

Compounds 1, 3, and 4 were measured in pyridine-d5 (500 MHz), and compounds 2, 5, and 6 were measured in methanol-d4 (600 MHz). b “o”: overlapped.

Table 2. 13C NMR Data of Compounds 1 to 6a position

1

2

3

4

5

6

position

1

2

3

4

5

6

1 2 3 4 5 6 7 8 9 G-1 G-2 G-3 G-4 G-5 G-6 OCH3

130.3 s 116.6 d 149.5 s 148.9 s 118.6 d 121.1 d 146.0 d 116.7 d 166.1 s 96.1 d 74.2 d 78.5 d 71.0 d 79.5 d 62.2 t

130.7 s 116.4 d 148.5 s 148.8 s 118.0 d 122.0 d 147.3 d 116.5 d 167.2 s 95.8 d 73.9 d 78.0 d 71.0 d 78.8 d 62.3 t 56.5 q


128.6 s 130.3 d 117.3 d 160.3 s 117.3 d 130.3 d 146.0 d 116.4 d 166.1 s 96.1 d 74.2 d 78.5 d 71.0 d 79.5 d 62.2 t



1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ G-1′ G-2′ G-3′ G-4′ G-5′ G-6′







a

Compounds 1, 3, and 4 were measured in pyridine-d5 (125 MHz), and compounds 2, 5, and 6 were measured in methanol-d4 (150 MHz).

unit28 (a trans double band, an AMX coupled aryl moiety, and a 4,6-disubstituted 2-pyrone unit), a trans-caffeoyl moiety, and a β-glucopyranose unit. The NMR data of compound 7 are shown in Tables 3 and 4. The linkage of the pyrone and the caffeoyl moieties to the glucose unit was determined at Glc C-1 and Glc C-6, respectively, based on the HMBC correlations of H-G-1/ C-4, H-G-6/C-9″ (Figure 2). Accordingly, the structure of 7 (clematiside G) was determined as 6-(3′,4′-dihydroxystyryl)-2pyrone-4-O-(6-O-trans-caffeoyl)-β-D-glucopyranoside.

Compound 8 was obtained as a yellowish powder, and its molecular formula was established as C43H42O21 from the HRESIMS data at m/z 893.2127 [M − H]−. In the 1H and 13C NMR spectra of 8, the resonances for two trans-caffeoyl units, two β-D-glucopyranose moieties, and one 6-(3′,4′-dihydroxystyryl)-4-hydroxy-2-pyrone unit were observed, indicating that the structure of 8 was similar to that of 7 except for an additional (6-O-trans-caffeoyl)-β-D-glucopyranoside unit in 8. From the above evidence, together with HMBC correlations of H-G-1/C-4, 1588



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Table 3. 1H NMR Data of 7 and 8 (J in Hz, in Methanol-d4, 600 MHz) position

7

8

position

7

8

position

8

3 5 2′ 5′ 6′ 7′ 8′ G-1 G-2 G-3 G-4

6.23 d (2.4) 5.78 d (2.4) 7.00 d (1.8) 6.74 d (8.4) 6.91 dd (8.4, 1.8) 7.28 d (15.6) 6.57 d (15.6) 5.09 d (7.2) 3.50 m 3.51 m 3.40 t (9.0)

6.17 br s 5.72 d (1.8) 6.97 br s 6.74 d (7.8) 6.87 br d (7.8) 7.21 d (15.6) 6.49 d (15.6) 5.10 d (6.6) 3.54 m 3.54 m 3.40 m

G-5 G-6

3.79 ddd (9.0, 7.8, 1.8) 4.58 dd (12.0, 1.8) 4.24 dd (12.0, 7.8) 7.06 d (1.8) 6.75 d (8.4) 6.95 dd (8.4, 1.8) 7.54 d (16.2) 6.32 d (16.2)

3.73 dd (9.6, 7.8) 4.61 brd (11.4) 4.26 dd (11.4, 7.8) 7.04 d (1.2) 7.03 d (8.4) 6.93 brd (8.4) 7.46 d (15.6) 6.24 d (15.6) 4.82 d (7.2) 3.52 m

G-3′ G-4′ G-5′ G-6′

3.53 m 3.42 m 3.79 dd (9.6, 7.8) 4.58 br d (12.0) 4.31 dd (12.0, 7.8) 6.99 br s 6.74 d (7.8) 6.88 br d (7.8) 7.50 d (15.6) 6.25 d (15.6)

2″ 5″ 6″ 7″ 8″ G-1′ G-2′

2‴ 5‴ 6‴ 7‴ 8‴

Table 4. 13C NMR Data of Compounds 7 and 8 (150 MHz, Methanol-d4) position

7

8

position

7

8

position

8

2 3 4 5 6 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′

166.7 s 92.6 d 171.5 s 101.1 d 161.8 s 128.7 s 114.9 d 146.8 s 148.8 s 116.5 d 122.1 d 137.6 d 116.7 d

166.6 s 92.8 d 171.3 s 100.9 d 161.7 s 128.8 s 115.0 d 146.8 s 148.7 s 116.6 d 122.0 d 137.6 d 116.7 d

G-1 G-2 G-3 G-4 G-5 G-6 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″

100.8 d 74.4 d 77.7 d 71.6 d 76.0 d 64.6 t 127.7 s 115.2 d 146.7 s 149.7 s 116.6 d 123.3 d 147.5 d 114.6 d 169.1 s


G-1′ G-2′ G-3′ G-4′ G-5′ G-6′ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 7‴ 8‴ 9‴


Table 5. Antioxidant Activity of Compounds 1−12 (DPPH Test)

Figure 2. Key HMBC correlations of compound 7.

H-G-6/C-9″, H-G-1′/C-4″, and H-G-6′/C-9‴, the structure of 8 (clematiside H) was elucidated as 6-(3′,4′-dihydroxystyryl)-2pyrone-4-O-{6-O-[4-O-(6-O-trans-caffeoyl)-β-D-glucopyranosyl]-trans-caffeoyl}-β-D-glucopyranoside. Moreover, four known compounds (9−12) were also isolated from the 95% EtOH extract of C. tashiroi. By comparing their NMR and MS data with those reported in the literature, compounds 9−12 were identified as 6-O-trans-p-coumaroyl29 D-glucopyranose (9), trans-caffeic acid 4-O-β-D-glucopyrano29 side (10), 6-O-(trans-caffeoyl)-D-glucopyranose (11),30 and 1-O-(trans-caffeoyl)-β-D-glucopyranose (12).29 In a DPPH radical-scavenging test, compounds 1, 7, and 8 showed more potent antioxidant activity than that of the positive control, vitamin E (Table 5). In the antinitric oxide release activity assay, compound 7 showed an inhibitory effect (IC50 = 14.9 ± 0.30 μM), whereas the other tested compounds did not show any pronounced inhibition effect at the tested concentration (20 μg/mL), compared with the positive control quercetin (IC50 = 1.8 ± 0.18 μM).

compound

removal effect (%)a

ED50 (μM)

1 2 3 4 5 6 7 8 9 10 11 12 vitamin E

99.8 ± 0.60 74.8 ± 1.51 7.8 ± 3.11 99.7 ± 0.15 99.4 ± 0.30 32.2 ± 3.64 99.0 ± 0.60 98.8 ± 0.15 5.0 ± 1.36 91.2 ± 3.17 9.8 ± 3.51 99.3 ± 0.15 -c

13.3 ± 0.37 87.0 ± 0.28 (−)b 47.0 ± 0.28 39.9 ± 1.06 (−) 13.0 ± 0.38 17.0 ± 0.23 (−) 175.4 ± 0.70 (−) 29.1 ± 0.32 27.8 ± 0.86

Concentration of removal effect: 100 μg/mL. determined. cRemoval effect not tested.

a

b

ED50 value not

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EXPERIMENTAL SECTION General Experimental Procedures. Optical rotations were obtained on a JASCO P-2000 polarimeter. IR spectra were recorded on a Mattson Genesis II IR-FT spectrometer. NMR spectra were recorded on Varian Unity Inova-500 and VNMRS-600 MHz spectrometers. HRESIMS were recorded on a Shimadzu IT-TOF HR mass spectrometer. For column chromatography, Diaion HP 20 (Mitsubishi Chemical),

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column chromatography eluted with 65% aq MeOH to yield 10 subfractions (KCT27-1 to KCT27-10). Fraction KCT27-6 (1.392 g) was subjected to separation over an ODS SPE column eluted with a gradient mobile phase (20 to 100% MeOH in H2O, v/v) to afford five subfractions, KCT276-1 to KCT276-5. Fraction KCT276-4 (60.4 mg) was purified by preparative HPLC (22% MeCN in H2O) to afford 8 (15.4 mg). Fraction KCT27-7 (0.438 g) was purified on an ODS SPE column eluted with a gradient mobile phase (20% to 70% MeOH in H2O) to afford 7 (142 mg). Clematiside A (1). Yellowish powder, [α]25 D −272.6 (c 0.2, MeOH); UV λmax (MeOH) (log ε) 324 (4.83), 294 (4.84), 243 (4.74), 217 (4.86) nm; IR (neat) vmax 3355, 2917, 1699, 1631, 1595, 1514, 1447 cm−1; 1H and 13C NMR data are shown in Tables 1 and 2; HRESIMS m/z 689.1722 [M + Na]+ (calcd for C30H34O17Na, 689.1688). Clematiside B (2). Yellow amorphous powder, [α]25 D −71.9 (c 0.2, MeOH); UV λmax (MeOH) (log ε) 320 (4.49), 293 (4.48), 238 (4.41), 218 (4.50) nm; IR (neat) vmax 3354, 2917, 1700, 1634, 1589, 1511, 1430 cm−1; 1H and 13C NMR data are shown in Tables 1 and 2; HRESIMS m/z 703.1848 [M + Na]+ (calcd for C31H36O17Na, 703.1844). Clematiside C (3). Yellowish powder, [α]25 D −91.3 (c 0.2, MeOH); UV λmax (MeOH) (log ε) 310 (4.47), 297 (4.49), 229 (4.34), 219 (4.38) nm; IR (neat) vmax 3357, 2925, 1701, 1634, 1593, 1511, 1442 cm−1; 1H and 13C NMR data are shown in Tables 1 and 2; HRESIMS m/z 673.1718 [M + Na]+ (calcd for C30H34O16Na, 673.1739). Clematiside D (4). Yellowish powder, [α]25 D −83.1 (c 0.2, MeOH); UV λmax (MeOH) (log ε) 305 (4.34), 296 (4.36), 232 (sh, 4.17), 220 (4.30) nm; IR (neat) vmax 3358, 2924, 1699, 1634, 1596, 1514, 1449 cm−1; 1H and 13C NMR data are shown in Tables 1 and 2; HRESIMS m/z 673.1709 [M + Na]+ (calcd for C30H34O16Na, 673.1739). Clematiside E (5). Yellowish powder, [α]25 D −95.4 (c 0.2, MeOH); UV λmax (MeOH) (log ε) 322 (4.41), 298 (sh,4.31), 234 (sh, 4.31), 217 (4.40) nm; IR (neat) vmax 3338, 2933, 1695, 1634, 1593, 1511, 1442 cm−1; 1H and 13C NMR data are shown in Tables 1 and 2; HRESIMS m/z 665.1743 [M − H]− (calcd for C30H33O17, 665.1771). Clematiside F (6). Yellowish powder, [α]25 D −141.1 (c 0.2, MeOH); UV λmax (MeOH) (log ε) 315 (4.55), 303 (sh, 4.49), 230 (4.45) nm; IR (neat) vmax 3358, 2921, 1703, 1634, 1601, 1516, 1450 cm−1; 1H and 13C NMR data are shown in Tables 1 and 2; HRESIMS m/z 673.1729 [M + Na]+ (calcd for C30H34O16Na, 673.1739). Clematiside G (7). Yellowish powder, [α]25 D −68.4 (c 0.2, MeOH); UV λmax (MeOH) (log ε) 335 (4.41), 298 (4.25), 248 (4.27), 219 (4.57) nm; IR (neat) vmax 3383, 1683, 1634, 1605, 1536, 1438 cm−1; 1H and 13C NMR data are shown in Tables 3 and 4; HRESIMS m/z 569.1306 [M − H]− (calcd for C28H25O13, 569.1299). Clematiside H (8). Yellowish powder, [α]25 D −98.0 (c 0.2, MeOH); UV λmax (MeOH) (log ε) 324 (4.52), 294 (4.51), 242 (sh, 4.46), 218 (4.71) nm; IR (neat) vmax 3387, 1703, 1634, 1605, 1516, 1438 cm−1; 1H and 13C NMR data are shown in Tables 3 and 4; HRESIMS m/z 893.2127 [M − H]− (calcd for C43H41O21, 893.2140). Acid Hydrolysis of Glycosides 1−8. A methanol solution of compound 1 (3.0 mg) was spotted on a TLC plate (20 × 10 cm) to form a narrow band, and this plate was placed in an atmosphere of concentrated HCl for 30 min. The HCl and H2O were then evaporated in a vacuum oven. The plate was developed

Sephadex LH-20 (GE Healthcare), and ODS (Merck, 40−63 μm) were used. Precoated silica gel (Merck 60 F254) plates were used for TLC and the plates were visualized by spraying with 5% H2SO4 followed by heating at 110 °C. HPLC separations were performed on a 250 × 20 mm i.d. preparative Cosmosil 5C18 AR-II column (Nacalai Tesque, Inc.) with a Shimadzu LC-8A pump and a SPD-20 UV detector. Plant Material. The aerial parts of C. tashiroi (2.4 kg) were collected at Heping District, Taichung City, Taiwan, in August 2012 and identified by Dr. Shih-Yen Huang, Endemic Species Research Institute, Nantou County, Taiwan. A voucher specimen (no. NRICM20120807) has been deposited in the National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei, Taiwan. Extraction and Isolation. The dried aerial parts of T. tashiroi (2.4 kg) were extracted with 95% EtOH (20 L) at 50 °C three times. The EtOH extract was concentrated under reduced pressure. The concentrated EtOH extract (2.5 L in H2O) was partitioned with n-hexane to give 30 g of n-hexane extract. Then, the H2O layer was loaded onto a Diaion HP-20 column, and successively eluted with 100% H2O, 50% aq EtOH, 80% aq EtOH, 93% aq EtOH, and 100% EtOAc. Fraction II (50% aq EtOH, KCT2, 90 g) was separated on a Sephadex LH-20 column eluted with MeOH to yield 10 fractions (KCT2-1 to KCT2-10). Fraction KCT2-5 (40 g) was chromatographed on a column containing Sephadex LH-20 and eluted with 65% aq MeOH to yield 12 fractions (KCT25-1 to KCT25-12). Fraction KCT25-4 (4.5 g) was subjected to ODS flash column chromatography eluted with a gradient mobile phase (10 to 100% MeOH in H2O, v/v) to give eight fractions, KCT254-1 to KCT254-8. Fraction KCT254-1 (10% aq MeOH, 2.686 g) was subjected to Sephadex LH-20 column chromatography eluted with 60% MeOH to afford four subfractions (KCT2541-1 to KCT2541-4). Subfraction KCT2541-2 (1.512 g) was purified by ODS flash column chromatography (5% aq MeOH) to afford 10 (122.1 mg). Subfraction KCT2541-3 (172 mg) was further purified by reversed-phase preparative HPLC (250 × 20 mm i.d., Cosmosil 5 C18 AR-II column, 10% MeCN in H2O, 8 mL/min; the same column and flow rate were used in subsequent preparative HPLC experiments) to afford 11 (79.1 mg) and 12 (24.4 mg). Fraction KCT254-2 (20% aq MeOH, 0.640 g) was subjected to separation over Sephadex LH-20, eluted with 60% MeOH, to afford six subfractions (KCT2542-1 to KCT2542-6). Subfraction KCT2542-3 (0.337 g) was purified by reversed-phase preparative HPLC (12% MeCN in H2O) to give 9 (22.4 mg). Fraction KCT254-3 (30% aq MeOH, 0.606 g) was subjected to passage over a Sephadex LH-20 column, eluted with 60% MeOH, to afford five subfractions (KCT2543-1 to KCT2543-5). The precipitate isolated from KCT2543-3 was found to be pure compound 4 (7.6 mg) and the residual solution of KCT2543-3 was purified by preparative HPLC (17.5% MeCN in H2O) to afford 5 (8.2 mg) and 6 (8.0 mg). Fraction KCT2543-5 (55 mg) was purified by reversed-phase preparative HPLC (16% MeCN in H2O) to afford 1 (6.7 mg). Fraction KCT254-4 (40% aq MeOH, 0.759 g) was subjected to purification over Sephadex LH-20 column, eluted with 60% MeOH, to afford four subfractions (KCT2544-1 to KCT2544-4). Fraction KCT2544-2 (252 mg) was separated by preparative HPLC (28% MeCN in H2O) to afford seven subfractions (KCT25442-1 to KCT25442-7). Subfraction KCT25442-2 (48.2 mg) was purified by preparative HPLC (16% MeCN in H2O), to afford 2 (8.5 mg) and 3 (7.8 mg). Fraction KCT2-7 (4.8 g) was dealt with the same manner as KCT2-5 and purified by Sephadex LH-20 1590


Journal of Natural Products in CHCl3-MeOH (3:1, saturated with H2O). To detect the glycoside band, part of the TLC plate was cut off, sprayed with 5% H2SO4 in EtOH, and then heated at 110 °C. The glycoside band was removed from the rest of TLC plate. The glycoside was dissolved in EtOH and subjected to HPLC (Shimadzu HPLC equipped with LC-20AT pump, RID-10A detector, and SCL10AVP system controller; column: Chiralpak AD-H column (Daicel), 4.6 × 250 mm, 5 μm; mobile phase: EtOH-n-hexaneTFA (3:7:0.05, v/v); flow rate: 0.5 mL/min).34 Under these conditions, the sugar units were identified by comparison with authentic samples: tR (min) 14.33 (D-glucose), 14.72 (L-glucose). Compounds 2 to 8 were treated in the same manner. For all tested compounds, glucose was identified in the D-form. Scavenging Activity of 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) Radicals. The DPPH free-radical-scavenging activities of the 12 compounds (1−12) isolated were measured using the method of Rangkadilok et al.31 and Chung et al.,32 with minor modifications. An aliquot of each sample (120 μL, 10−100 μg/mL) or (±)-α-tocopherol (vitamin E 10−40 μg/mL, Sigma, 96.0%, HPLC) was mixed with 30 μL of 0.75 mM DPPH methanol solution in 96-well microplate. The mixture was shaken vigorously with an orbital shaker in the dark at room temperature for 30 min, and then the absorbance was measured at 517 nm with an ELISA reader. Methanol was used as negative control, and the positive control was vitamin E [(±)-α-tocopherol]. The final results were reported as the concentrations of ED50, a concentration of a compound that scavenged 50% DPPH radicals in the reaction solution. Cell Culture and NO Measurement. The macrophage cell line RAW 264.7 was obtained from ATCC (Rockville, MD) and cultured in DMEM containing 5% heat-inactivated fetal calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin, and grown at 37 °C with 5% CO2 in fully humidified air. Cells were plated at a density of 2 × 105 cells/well in 96-well culture plates and stimulated with LPS (1000 ng/mL) in the presence or absence of different concentrations of tested compounds for 24 h simultaneously. All compounds were dissolved in DMSO and further diluted with sterile PBS. Nitrite (NO2−) accumulation in the medium was used as an indicator of NO production, which was measured by adding the Griess reagent (1% sulfanilamide and 0.1% naphthylenediamine in 5% phosphoric acid). NaNO2 was used to generate a standard curve, and nitrite production was determined by measuring optical density at 550 nm. All experiments were performed in triplicate. NO production by LPS stimulation was designated as 100% for each experiment. Quercetin (Sigma, 98.0% HPLC) was employed as a positive control.33

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ACKNOWLEDGMENTS

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REFERENCES

The authors thank the National Ministry of Science and Technology, Republic of China (NSC 98-2320-B-077-005MY3) for a grant to Y.-H.K. We are grateful to Dr. ChienChang Shen and Ms. Fei-Pei Kao, as well as Dr. Ming-Jaw Don and Mr. Tai-Hung Chen, NRICM, for technical assistance for the NMR and LRMS measurements, respectively. The Instrument Center of National Taiwan University for HRMS measurement is also acknowledged.

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ASSOCIATED CONTENT

S Supporting Information *

1

H and 13C NMR spectra of compounds 1−8. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00154.

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*E-mail: [email protected]. Tel: +886-2-28201999 ext. 7051. Fax: +886-2-28236150. Notes

The authors declare no competing financial interest. 1591



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(30) Hussein, S. A. M.; Ayoub, N. A.; Nawwar, M. A. M. Phytochemistry 2003, 63, 905−912. (31) Rangkadilok, N.; Sitthimonchai, S.; Worasuttayangkurn, L.; Mahidol, C.; Ruchirawat, M.; Satayavivad, J. Food Chem. Toxicol. 2007, 45, 328−36. (32) Chung, Y. C.; Chang, C. T.; Chao, W. W.; Lin, C. F.; Chou, S. T. J. Agric. Food Chem. 2002, 50, 2454−2458. (33) Zhang, L.-J.; Liu, H.-K.; Hsiao, P.-C.; Kuo, Y.-L.-M.; Lee, I.-J.; Wu, T.-S.; Chiou, W.-F.; Kuo, Y.-H. J. Agric. Food Chem. 2011, 59, 1131− 1137. (34) Lopes, J. F.; Gaspar, E. M. S. M. J. Chromatogr. A 2008, 1188, 34− 42.

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