Chemical Constituents of the Rhizomes of Bletilla formosana and

Article pubs.acs.org/jnp

Chemical Constituents of the Rhizomes of Bletilla formosana and Their Potential Anti-inflammatory Activity Che-Wei Lin,† Tsong-Long Hwang,‡,§,⊥ Fu-An Chen,∥ Chia-Hsin Huang,# Hsin-Yi Hung,*,¶ and Tian-Shung Wu*,∥,¶ †

Department of Chemistry National Cheng Kung University, Tainan 70101, Taiwan Graduate Institute of Natural Products, Chang Gung University, Taoyuan 33302, Taiwan § Research Center for Industry of Human Ecology and Graduate Institute of Health Industry Technology, Chang Gung University of Science and Technology, Taoyuan 333, Taiwan ⊥ Department of Anesthesiology, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan ∥ Department of Pharmacy and Graduate Institute of Pharmaceutical Technology, Tajen University, Pingtung 90741, Taiwan # Department of Biological Science and Technology, Chung Hwa University of Medical Technology, Tainan 71703, Taiwan ¶ School of Pharmacy, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan 70101, Taiwan ‡

S Supporting Information *

ABSTRACT: Nine new phenanthrenes (1−9) and a new benzyl glycoside (10) together with 45 known compounds were isolated from the rhizomes of Bletilla formosana. The structures of 1−10 were elucidated primarily on the basis of their 1D and 2D NMR spectroscopic data. Most of the isolated compounds were evaluated for their anti-inflammatory activities. The results showed that IC50 values for the inhibition of superoxide anion generation and elastase release ranged from 0.2 to 6.5 μM and 0.3 to 5.7 μM, respectively. Structure−activity relationships of the isolated compounds were also investigated. The inhibitory potencies were determined as phenanthrenes > bibenzyls > biphenanthrenes.

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T

RESULTS AND DISCUSSION The rhizomes of B. formosana were extracted with EtOH, and the extracts were concentrated under reduced pressure to give a dark brown syrup. The EtOH crude extract was subjected to column chromatography to afford five major fractions (BF 1− 5) and yielded 10 new compounds (1−10) and 45 known compounds. The known compounds were isolated and identified from their physical and spectroscopic data. The structures of the new substances were characterized as described below. Bleformin A (1) was obtained as a colorless powder with a molecular formula of C23H20O5 by HRESIMS (m/z 399.1205 [M + Na]+, calcd for 399.1203). The UV absorption maxima at λmax 310, 289, and 264 nm were characteristic of a phenanthrene skeleton.13 The IR spectrum showed absorptions at 3302 (OH) and 1547 (benzenoid) cm−1. The 1H NMR spectrum of 1 (Table 1) showed signals for two methoxy groups at δH 3.94 (3H, s) and 3.99 (3H, s), a methylene group of a p-hydroxybenzyl unit at δH 4.41 (2H, s), two coupled aromatic protons at δH 7.31 and 9.33 (2H, d, J = 9.2 Hz), and a p-hydroxybenzyl group at δH 6.66 (2H, d, J = 8.4 Hz) and 7.06

he genus Bletilla (Orchidaceae) is distributed mainly in mainland China and Japan and is used in folk and traditional medicine for the treatment of bleeding, colds, esophagitis, erosive gastritis, and burns.1,2 In previous studies, Bletilla species have been shown to exhibit shortening of the time for blood clotting, to afford mucosal protection, and to have antibactericidal, antifungal, antioxidant, antityrosinase, anti-inflammatory, and cytotoxic effects.3−7 Bletilla formosana (Hayata) Schltr. is the only member of the genus found in Taiwan8 and is a perennial herb, found on mountain slopes at up to ca. 2200 m altitude. 8 Previous phytochemical investigations on Bletilla species have led to the isolation of phenanthrene derivatives, stilbenes, bibenzyls, flavonoids, and phenolic compounds.9−12 However, only two prior phytochemical studies have been performed on B. formosana.9,12 In the present work, three new phenanthrenes (1−3), six new biphenanthrenes (4−9), a new benzyl ester glycoside (10), and 45 known compounds were isolated from extracts of B. formosana. Described herein are the isolation and structural elucidation of these new compounds and the determination of their absolute configurations through 2D-NMR spectroscopic analysis. The isolated compounds were evaluated for their in vitro antineutrophilic inflammatory effects. © XXXX American Chemical Society and American Society of Pharmacognosy

Received: February 6, 2016

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DOI: 10.1021/acs.jnatprod.6b00118 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

which five were attached to aromatic oxygen carbons at δC 156.1, 153.3, 152.3, 149.9, and 142.9, two methoxy group carbons occurred at δC 61.2 and 60.0, and the resonance at δC 30.5 was assigned to a methylene unit of a p-hydroxybenzyl group. The 1H and 13C NMR spectroscopic data of 1 were found to be similar to those of nudol (11)14 (Table 1) and differed only at the C-8 linkage to a p-hydroxybenzyl group in 1. Analysis of the 2J and 3J correlations in the HMBC spectrum (Figure 1) from H-1′ to C-2′, C-7, and C-8 supported the connection of a p-hydroxybenzyl group at C-8. The signals at δH 3.99 (3H, s, OCH3-3) and 3.94 (3H, s, OCH3-4) correlated with C-3 and C-4 in the HMBC spectrum, respectively. On the basis of the above results, the structure of compound 1 was assigned as shown. Bleformin B (2) was obtained as a colorless powder. Its molecular formula was established as C23H20O5 by HRESIMS (m/z 399.1206 [M + Na]+, calcd for 399.1208), showing the same molecular formula as compound 1. In addition, the UV and IR spectra of these two compounds were also similar. The 1 H NMR spectroscopic data of 2 indicated significant differences from those of 1 in the signals at δH 9.38 (1H, d, J = 9.2 Hz), 7.20 (1H, d, J = 2.8 Hz), and 7.17 (1H, dd, J = 9.2,

(2H, d, J = 8.4 Hz). The signals at δH 7.50 (1H, d, J = 9.2 Hz) and 7.80 (1H, d, J = 9.2 Hz) were assigned to a CH(9)− CH(10) fragment in a phenanthrene moiety from the COSY spectrum. The 13C NMR spectrum of 1 revealed 23 signals, of

Table 1. 1H and 13C NMR Spectroscopic Data of Compounds 1−3, 11, and 23 1a position

δC

1 2 3 4 4a 4b 5 6 7 8 8a 9 10 10a OCH3-2 OCH3-3 OCH3-4 OCH3-4′ OCH3-6′ 1′ 1″ 2′ 3′ 4′ 5′ 6′ 7′ 2″ 3″ 4″ 5″ 6″ 7″

109.4 149.9 142.9 152.3 119.5 125.0 126.9 117.1 153.3 121.6 132.9 123.6 127.5 129.7

7.10 s

61.2 60.0

3.99 s 3.94 s

30.5 133.1 130.0 115.8 156.1 115.8 130.0

δH (J in Hz)

9.33 d (9.2) 7.31 d (9.2)

7.80 d (9.2) 7.50 d (9.2)

4.41 s

7.06 d (8.4) 6.66 d (8.4) 6.66 d (8.4) 7.06 d (8.4)

2a δC 118.6 147.6 142.1 150.3 119.5 124.6 129.2 117.6 155.8 112.0 134.2 126.9 124.2 128.8 61.4 59.9

30.7 132.9 130.0 115.8 156.2 115.8 130.0

3b

δH (J in Hz)

δC

9.38 d (9.2) 7.17 dd (9.2, 2.8)

112.3 155.3 98.8 157.9 116.6 126.1 130.0 113.2 155.9 114.6 140.2 30.5 28.0 141.3

7.20 d (2.8) 7.48 d (9.2) 7.81d (9.2)

4.05 s 3.94 s

4.38 s

7.06 d (8.4) 6.67 d (8.4) 6.67 d (8.4) 7.06 d (8.4)

δH (J in Hz)

6.53 s

8.05 d (8.4) 6.67 dd (8.4, 2.4) 6.65 d (2.4) 2.50 m 2.33 m

55.5

3.85 s

55.8 38.9 38.3 144.1 109.4 156.9 110.1 159.6 103.8 144.5 116.2 158.3 113.6 130.1 120.3

3.65 s 2.85 m 2.87 m 6.51 d (1.4)

6.49 d (1.4) 6.76 m 6.66 d (7.7) 7.10 t (7.7) 6.74 m

11a δC

δH (J in Hz)

108.3 147.4 140.8 150.6 118.7 123.9 128.3 116.5 153.4 111.8 133.4 126.3 127.0 129.4

7.18 s

61.2 59.7

4.09 s 3.96 s

9.34 d (10.0) 7.22 m 7.21 m 7.44 d (8.8) 7.48 d (8.8)

23a δC

δH (J in Hz)

116.1 157.7 98.8 154.7 116.6 126.2 130.1 113.3 155.9 114.6 140.1 30.6 28.0 140.7 55.6

3.86 s

55.6

3.61 s

36.9 37.5 144.1 115.6 160.2 98.0 158.9 108.9 144.7 116.0 158.1 113.4 129.9 120.2

2.58 m 2.57 m

6.58 s

8.08 d (8.8) 6.67 dd (8.8, 2.8) 6.64 d (2.8) 2.50 m 2.30 m

6.42 s 6.48 d (2.0) 6.48 d (2.0) 6.54 dd (8.0, 2.4) 6.95 t (7.6) 6.43 m

a1

H and 13C NMR data (δ) were measured in acetone-d6 at 400 and 100 MHz. b1H and 13C NMR data (δ) were measured in acetone-d6 at 700 and 175 MHz. B



Article

Bleformin C (3) was obtained as a colorless powder. The molecular formula of 3 was established as C30H28O6 by HRESIMS (m/z 507.1778 [M + Na]+, calcd for 507.1778). UV absorption maxima were observed at λmax 274, 267, and 260 nm. The IR spectrum of 3 revealed strong absorption bands due to an aromatic ring (1458 cm−1) and hydroxy groups (3435 cm−1). Comparison of the 1H and 13C NMR and UV spectroscopic data of 3 with phochinenin K (23)15 indicated that they are closely related phenanthrene analogues. A detailed comparison of the 2D NMR data of 3 revealed a number of differences from those of compound 23. In the 1H NMR spectrum, three aromatic proton signals at δH 8.05 (1H, d, J = 8.4 Hz), 6.67 (1H, dd, J = 8.4, 2.4 Hz), and 6.65 (1H, d, J = 2.4 Hz) were assigned as belonging to an aromatic ring ABX system in a 9,10-dihydrophenanthrene structure. In addition, four aromatic proton signals at δH 7.10 (1H, t, J = 7.7 Hz), 6.76 (1H, m), 6.74 (1H, m), and 6.66 (1H, d, J = 7.7 Hz) were assigned to H-5″, H-2″, H-6″, and H-4″ in a bibenzyl aromatic ring from the 1H−1H COSY cross-peaks and coupling patterns. Two protons at δH 6.51 (1H, d, J = 1.4 Hz) and 6.49 (1H, d, J = 1.4 Hz) in the aromatic bibenzyl ring were assigned as belonging to an aromatic ring AB system. Two methoxy signals resonated at δH 3.85 (3H, s) and 3.65 (3H, s), two methylene groups of a bibenzyl unit appeared at δH 2.87 (2H, m) and 2.85 (2H, m), and two methylene group protons at δH 2.50 (2H, m) and 2.33 (2H, m) were also observed and could be assigned to the H-9, H-10 fragment in a 9,10-dihydrophenanthrene structure. The 13C NMR spectrum of 3 revealed 30 signals, with six of these attached to aromatic oxygen carbons (δC 159.6, 158.3, 157.9, 156.9, 155.9, and 155.3), along with two methoxy carbons (δC 55.8 and 55.5), two methylene carbons of a bibenzyl unit (δC 38.9 and 38.3), and two methylene carbons (δC 30.5 and 28.0). The HMBC spectrum (Figure 1) showed correlations of H-3 and H-10 to C-1 and C-4a. Moreover, H-3′ and H-7′ showed correlations with C-5′, C-3′, C-7′, and C-1′, while the OCH3-4 and OCH3-6′ signals showed correlations with C-4 and C-6′, respectively. Thus, the methoxy groups were assigned as being substituted at C-4 and C-6′. According to the above observations, a 1,5′-linkage between the two 9,10dihydrophenanthrene and bibenzyl monomers was determined. Therefore, the structure of 3 was established as shown. Bleformin D (4) was obtained as a colorless powder. The molecular formula of 4 was established as C37H32O7 by HRESIMS (m/z 611.2041 [M + Na]+, calcd for 611.2040). The UV spectrum revealed absorption maxima at λmax 297 and 282 nm, typical of phenanthrene absorption peaks.13 The presence of a hydroxy group and an aromatic ring was verified by IR absorption peaks at 3383 and 1510, 1454 cm−1, respectively. Interpretation of the 1H and 13C NMR spectra (Tables 2 and 3) indicated 4 to be related structurally to blestrianol A (22),16 with the signals of a p-hydroxybenzyl group observed for 4 in place of the resonances of H-1′. The 1H NMR spectrum showed resonances for 10 aromatic protons, constituting an AA′XX′ system (δH 7.08, d, J = 8.8 Hz and 7.00, d, J = 8.8 Hz), with an aromatic ring of a p-hydroxybenzyl group and two ABX systems (δH 8.10, d, J = 6.8 Hz; 6.69, m; 6.67, m and δH 8.08, d, J = 8.0 Hz; 6.68, m; 6.66, m). A COSY experiment was used to corroborate these couplings. The 1H NMR spectrum of 4 also showed resonances for two methoxy groups (δH 3.87, s and 3.24, s), with a methylene proton of a p-hydroxybenzyl group (δH 4.10, d, J = 15.6 Hz and 4.02, d, J = 15.6 Hz) verified from the HSQC spectrum. Four methylene protons at δH 2.67 (2H, m), 2.59 (2H, m), 2.58 (2H, m), and 2.48 (2H, m) could be

Figure 1. Selected HMBC (→) and COSY (bold) correlations for compounds 1−10.

2.8 Hz), due to an aromatic ring ABX system. The HMBC correlations (Figure 1) from H-1′ to C-2′, C-1, C-2, and C-10a supported the connection of a p-hydroxybenzyl group at C-1. The 1H and 13C NMR spectra of 2 (Table 1) were similar to those of 1, differing only in the C-1 linkage of a benzyl group at C-1 in 2. Thus, the structure of 2 was established as shown.

C



Article

Table 2. 1H NMR Spectroscopic Data of Compounds 4−10, 22, 27, and 44 position

4a

5a

6a

7b

8a

9a

2 3

7.53 d (8.8) 6.99 d (8.8)

6.63 s

6.99 s

7.00 s

7.01 s

8.08 d (8.0) 6.66 m

9.25 s

9.51 d (10.4) 7.18 m

9.51 d (7.0) 7.19 dd (7.0, 2.8)

8.11 d (8.4) 6.73 dd (8.4, 2.8)

9.41 d (10.0) 7.20 m

8

6.68 m

7.19 s

7.17 m

2.58 m

10

2.48 m

7.36 d (8.8) 6.93 d (8.8)

7.37 d (8.0) 7.03 d (8.0)

6.67 d (2.8) 2.54 m

7.19 m

9

7.18 d (2.8) 7.38 d (7.0) 7.02 d (7.0)

4 5 6 7

10A: 2.37 m 10B: 2.28 m

6.97 s

7.01 s

7.03 s

8.10 d (6.8) 6.67 m

9.15 s

9.25 s

9.32 d (7.0) 7.25 d (7.0)

8′

6.69 m

7.29 s

7.19 m

9′

2.59 m

10′

2.67 m

7.41 d (8.8) 6.90 d (8.8)

7.37 d (8.0) 6.93 d (8.0)

7.77 d (7.0) 7.09 d (7.0)

4.23 s 4.19 s 4.07 s

4.20 s 4.23 s

4.20 s 4.19 s

2″ 3″ 4″ 5″ 6″

7″ 1‴

27a

44c

6.61 s

7.00 s

8.08 d (8.4) 6.69 m

9.25 s

6.65 m

7.19 s

2.56 m

7.36 d (9.2) 6.93 d (9.2)

2.47 m

7.27 d (8.8) 7.08 d (8.8) 8.11 d (8.4) 6.73 dd (8.4, 2.8)

8.16 d (8.8) 6.76 dd (8.8, 2.8)

6.67 d (2.8) 2.54 m

6.65 d (2.8) 2.49 m

10A: 10B: 3.88 3.88 3.75 3.75

10A: 10B: 4.04 3.92 4.01 3.81

7′

OCH3-3 OCH3-3′ OCH3-4 OCH3-4′ OCH3-6 OCH3-6′ OCH3-7′ OCH3-8′ 1″

22a

7.55 d (8.8) 7.10 d (8.8) 7.10 d (8.8) 7.55 d (8.8) 7.65 d (16.0) 6.42 d (16.0)

6.66 s

3′

6′

6.99 d (8.8) 7.53 d (8.8) 6.91 d (12.0) 5.87 d (12.0)

7.40 d (9.2) 7.21 d (9.2)

1′ 2′

4′ 5′

10c

3.87 s 3.24 s

2.37 m 2.28 m s s s s

7.08 d (8.8) 7.27 d (8.8) 5.09 d (10.4)

2.30 m 2.15 m s s s s

7.00 s

8.11 d (8.4) 6.71 m

9.25 s

6.72 m

7.19 s

2.71 m

7.36 d (9.2) 6.93 d (9.2)

2.71 m

3.88 s 3.27 s

4.07 s

4.23 4.23 4.07 4.07

7.35 d (8.8) 7.08 d (8.8) 7.08 d (8.8) 7.35 d (8.8) 5.16 s

s s s s

3.96 s 3.82 s 1″A: 4.10 d (15.6) 1″B: 4.02 d (15.6) 7.08 d (8.8) 7.00 m 7.00 m

4.93 m

4.95 d (7.6)

3.46 m 3.50−3.41 m

3.45 m 3.48−3.40 m

3.39 m 3.50−3.41 m 3.89 dd (12.0, 2.0) 3.69 ddd (12.0, 5.6, 2.0)

3.39 m 3.48−3.40 m 3.88 d (12.0) 3.69 dd (12.0, 5.6)

7.08 d (8.8) 4.91 m

2‴ 3‴

3.46 m 3.50−3.41 m D

4.91 d (7.6) 3.45 m 3.48−3.40 m DOI: 10.1021/acs.jnatprod.6b00118 J. Nat. Prod. XXXX, XXX, XXX−XXX


Article

Table 2. continued position

4a

5a

6a

7b

8a

9a

4‴ 5‴ 6‴

10c 3.39 m 3.50−3.41 m 3.89 dd (12.0, 2.0) 3.69 ddd (12.0, 5.6, 2.0)

22a

27a

44c 3.39 m 3.48−3.40 m 3.88 d (12.0) 3.69 dd (12.0, 5.6)

a1

H NMR data (δ) were measured in acetone-d6 at 400 MHz. b1H NMR data (δ) were measured in acetone-d6 at 700 MHz. c1H NMR data (δ) were measured in methanol-d4 at 400 MHz.

assigned to H-10′, H-9′, H-9, and H-10 in a dimeric dihydrophenanthrene structure. The 13C NMR spectrum of 4 revealed 37 signals, of which seven were attached to aromatic oxygen carbons (δC 158.4, 156.4, 156.2, 156.1, 156.0, 155.8, and 153.2), including two methyl carbons (δC 59.7 and 55.5) and a methylene of a benzyl group (δC 31.5). In the HMBC experiment (Figure 1), C-1″ was inferred from the correlation of HA-1″ and HB-1″ with C-2′, C-10a′, C-1′, C-2″, C-3″, and C7″. Correlations observed for H-10, HA-1″, and HB-1″ with C1′ showed C-1′ to be substituted with a p-hydroxybenzyl group. The signals of OCH3-4 and OCH3-4′ showed correlations with C-4 and C-4′, respectively. Therefore, C-4 and C-4′ were both assigned with a methoxy group substituent. Thus, the structure of 4 was established as shown. Bleformin E (5) was obtained as a colorless powder. The molecular formula of this compound was established as C14H23O2 by HRESIMS (m/z 561.1522 [M + Na]+, calcd for 561.1525). Bleformin E (5) was found to be similar to agrostonin (27)17 according to its 1H and 13C NMR and HRESIMS data. The main difference between these compounds was evident in the methoxy group arrangement. In the 1 H NMR spectrum, the signals of two protons at δH 9.25 (1H, s) and 9.15 (1H, s) were assigned at H-5 and H-5′ of a biphenanthrene unit. In addition, the 1H NMR data showed four aromatic proton signals at δH 7.41 (1H, d, J = 8.8 Hz) coupled with a signal at δH 6.90 (1H, d, J = 8.8 Hz), and δH 7.36 (1H, d, J = 8.8 Hz) coupled with 6.93 (1H, d, J = 8.8 Hz, H-10), and hence could be assigned to typical H-9,10 and H9′,10′ protons of a biphenanthrene derivative.18 The 1H NMR spectrum revealed the presence of four methoxy group signals (δH 4.23, 4.19, 4.07, and 3.96). The 13C NMR spectrum of 5 revealed 32 signals attributable to a biphenanthrene structure, with eight of these attached to aromatic oxygen carbons (δC 160.5, 160.2, 155.3,155.1, 148.5, 147.4, 147.1, and 146.0), and four methoxy group carbons (δC 56.2, 56.1, 56.0, and 55.9) were evident. The 1H−13C correlations determined via the HMBC spectrum (Figure 1) showed correlations from H-3 to C-1 and C-4; H-5 to C-6 and C-7; H-9 to C-8 and C-4b; H-10 to C-1; OCH3-4 to C-4; OCH3-6 to C-6; H-3′ to C-1′ and C4′; H-5′ to C-6′ and C-7′; H-8′ to C-7′, C-8a′, and C-4a′; H-9′ to C-8′ and C-4b′; H-10 to C-1′; OCH3-4′ to C-4′; and OCH37′ to C-7′, respectively. According to the above correlation data, a 1,1′-linkage between the two phenanthrene moieties was determined. Thus, the structure of 5 was established as shown. Bleformin F (6) was obtained as a colorless powder. The molecular formula of this compound was established as C31H24O7 by HRESIMS (m/z 531.1417 [M + Na]+, calcd for 531.1414). Compound 6 showed signals in its NMR and UV spectra very similar to those of 5. However, the C-6 proton signal in 5 was replaced by a methoxy group signal in 6, and the OH-6′ and OCH3-7′ resonances in 5 were transposed in 6. In

the 1H NMR spectrum, a proton appeared at δH 9.51 (1H, d, J = 10.4 Hz), reminiscent of H-5 and H-5′ of a biphenanthrene. In the HMBC experiment (Figure 1), correlations between H5′, H-8′, and OCH3-6′ with C-6′ showed C-6′ to be substituted with a methoxy group. Additionally, H-3′ and OCH3-4′ correlated with C-4′, indicating that C-4′ is substituted with a methoxy group. Thus, the structure of 6 was established as shown. Bleformin G (7) was obtained as a colorless powder. The molecular formula of 7 was established as C31H24O7 by HRESIMS (m/z 531.1415 [M + Na]+, calcd for 531.1414). Comparison of the 1H and 13C NMR, UV, and IR spectroscopic E



Article

Table 3. 13C NMR Spectroscopic Data of Compounds 4−10, 22, 27, and 44 position

4a

5a

6a

7b

8a

9a

10c

22a

27a

44c

1 2 3 4 4a 4b 5 6 7 8 8a 9 10 10a 1′ 2′ 3′ 4′ 4a′ 4b′ 5′ 6′ 7′ 8′ 8a′ 9′ 10′ 10a′ OCH3-3 OCH3-3′ OCH3-4 OCH3-4′ OCH3-6 OCH3-6′ OCH3-7′ OCH3-8′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴

112.1 156.2 99.1 158.4 116.7 126.0 130.0 113.3 156.0 114.6 140.2 30.4 27.0 141.5 121.7 153.2 116.8 155.8 120.8 125.9 129.4 113.9 156.4 114.6 139.9 30.5 28.2 139.8

109.8 155.1 100.0 160.2 116.3 125.8 109.6 148.5 146.0 112.2 128.1 127.9 123.3 135.4 108.9 155.3 99.8 160.5 115.9 126.8 109.9 147.4 147.1 113.4 127.2 128.3 122.6 135.7

110.0 155.1 100.0 160.2 116.5 125.3 130.2 117.4 155.3 112.0 134.1 128.0 125.6 135.1 109.8 155.2 100.3 160.3 116.3 125.8 109.7 148.5 146.0 112.2 128.1 128.3 123.3 135.4

110.0 155.1 100.4 160.3 116.4 125.3 130.2 117.4 155.3 111.9 134.1 128.3 125.5 135.0 110.1 155.4 100.3 160.1 116.5 126.1 125.4 117.9 146.6 141.9 127.5 121.5 125.5 135.1

112.9 155.4 98.9 158.1 116.7 126.1 130.1 113.3 156.0 114.6 140.2 30.5 28.3 140.8 111.9 155.3 117.2 157.8 120.3 125.6 129.1 114.1 156.4 115.1 140.1 30.7 31.1 141.1

109.8 155.1 100.0 160.2 116.3 125.8 109.7 148.5 146.0 112.2 128.1 127.9 123.3 135.4 109.8 155.1 100.0 160.2 116.3 125.8 109.7 148.5 146.0 112.2 128.1 127.9 123.3 135.4

129.9 130.9 118.0 160.9

56.2 55.9 56.1

55.9 56.1

56.0 55.9

116.4 148.4 142.5 151.3 119.3 124.5 129.1 117.6 155.8 112.1 134.4 126.8 125.3 129.2 118.5 147.8 140.6 151.4 120.7 125.5 129.3 114.0 156.6 114.9 140.4 30.5 27.4 135.1 61.3 61.1 60.1 60.4

130.4 132.7 117.0 159.7

55.5 59.7

119.2 147.9 140.5 151.0 120.5 125.5 129.3 113.9 156.6 114.9 140.4 30.5 27.4 134.6 119.2 147.9 140.5 151.0 120.5 125.5 129.3 113.9 156.6 114.9 140.4 30.5 27.4 134.6 61.1 61.1 60.3 60.3

55.6 59.6

56.1 56.1 56.0 56.0

117.0 132.7 143.8 118.7 168.0

131.3 131.2 117.7 159.1

117.7 131.2 66.8

56.0

118.0 130.9 146.0 116.9 168.7

131.6 131.0 117.8 159.1

117.8 131.0 67.0

56.0 61.3 31.5 132.9 129.9 115.7 156.1 115.7 129.9

102.0 74.9 78.2 71.4 77.9 62.5

101.8 74.8 78.2 71.3 77.9 62.5

102.3 75.0 78.3 71.4 78.0 62.5

102.2 74.9 78.3 71.4 78.0 62.6

a13 C NMR data (δ) were measured in acetone-d6 at 100 MHz. b13C NMR data (δ) were measured in acetone-d6 at 175 MHz. c13C NMR data (δ) were measured in methanol-d4 at 100 MHz.

8′ indicated that C-8′ is substituted with a methoxy group. Thus, the structure of 7 was established as shown. Bleformin H (8) was obtained as a colorless powder. The molecular formula of this compound was established as C32H30O8 by HRESIMS (m/z 565.1830 [M + Na]+, calcd for 561.1833). Comparison of the 1H and 13C NMR and UV spectroscopic data of 8 with analogous values for erianthridin (16)19 indicated them to be very similar. The presence of two

data with those of 6 showed many similarities in the signals. The only difference was the methoxy group substituted at C-6′ instead of C-8′ (Tables 2 and 3). The 1H NMR data revealed a downfield aromatic proton at δH 9.32 (1H, d, J = 7.0 Hz), shown as a sharp signal, was H-5′, which coupled with H-6′ (δH 7.25, 1H, d, J = 7.0 Hz). In the HMBC experiment (Figure 1), a key correlation observed for H-6′, H-9′, and OCH3-8′ with CF



Article

4-methoxy-9,10-dihydrophenanthrene (20),22 ephemeranthoquinone (21),23 blestrianol A (22),16 phochinenin K (23),15 4,4′-dimethoxy-9,10-dihydro-[6,10-biphenanthrene]-2,2′,7,7′tetraol (24),24 gymconopin C (25),25 cirrhopetalanthrin (26),25 agrostonin (27),17 blesttriarene B (28),24 biestriarene A (29),26 3-O-methyldihydropinosylvin (30),27 batatasin III (31),19 3′-O-methylbatatasin III (32),28 gigantol (33),29 3′,5dihydroxy-2-(4-hydroxybenzyl)-3-methoxybibenzyl (34),30 3,3′-dihydroxy-4-(4-hydroxybenzyl)-5-methoxybibenzyl (35),31 bulbocodin D (36),32 3,3′-dihydroxy-2-(4-hydroxybenzyl)-5methoxybibenzyl (37),22 2′,6′-bis(p-hydroxybenzyl)-3′,5-dimethoxy-3-hydroxybibenzyl (38),33 3,3′-dihydroxy-2′,6′-bis(p-hydroxybenzyl)-5-methoxybibenzyl (39),31 β-sitosteryl-3-O-β-Dglucoside (40),34 4-hydroxybenzyl ethyl ether (41),35 4hydroxybenzaldehyde (42),36 militarin (43),37 shancigusin I (44),20 1-(4-β-D-glucopyranosyloxybenzyl) (2R)-4-methyl-2isobutyl malate, 2 4 gastrodin, 3 8 dactylorhin A, 3 7 5hydroxymethylfuraldehyde,39 densiflorol B,40 (2S)-5,2′-dihydroxy-7-methoxyflavanone,41 3,5-dimethoxy-3′-hydroxybibenzyl,42 β-sitosterol and stigmasterol,43 ethyl (E)-4-hydroxycinnamate,44 and ethyl (Z)-4-hydroxycinnamate45 and by comparison of their spectroscopic data with those reported in the literature. The above results indicated that the representative chemotypes of B. formosana are biphenanthrenes, phenanthrenes, and bibenzyls. Several of the new and known compounds obtained were subjected to testing for their inhibitory activity of superoxide anion generation and elastase release by human neutrophils in response to N-formyl-L-methionyl-L-leucyl-Lphenylalanine/cytochalasin B (FMLP/CB) (Table 4). Idelalisib (CAL-101) was used as a positive control and inhibited the generation of superoxide anion and the release of elastase in FMLP-activated human neutrophils with IC50 values of 0.1 ± 0.1 and 0.3 ± 0.1 μM, respectively. As shown in Table 4, the bibenzyl 39 with a 2,6-di-p-hydroxybenzyl group exhibited the most potent inhibitory activities against both superoxide anion generation and elastase release with IC50 values of 0.7 and 0.7 μM, respectively. For this compound subtype, a 4′-hydroxy group and compounds with a greater number of phydroxybenzyl groups showed the most potent activities. For the phenanthene chemotype, as shown in Table 4, compound 18 with a 3-p-hydroxybenzyl group and a 9,10-dihydrophenanthene unit exhibited the most potent inhibitory activity against superoxide anion generation with an IC50 value of 0.2 μM. Compound 23 with a 1,6′-linkage between the batatasin III and a 9,10-dihydrophenanthene unit exhibited the most potent inhibitory activity against elastase release with an IC50 value of 0.6 μM. Among the biphenanthene derivatives, compound 4, with a 1′-p-hydroxybenzyl group and a 1,3′-linkage between the 9,10-dihydrophenanthene and 9′,10′-dihydrophenanthene units, exhibited the most potent inhibitory activity against superoxide anion generation, with an IC50 value of 0.2 μM. Compound 27, with 6,6′-dimethoxy groups and a 1,1′-linkage between the 9,10-phenanthene and 9′,10′-phenanthene units, exhibited the most potent inhibitory activity against elastase release, with an IC50 value of 0.3 μM. In conclusion, the inhibitory potency for the three chemotypes obtained from B. formosana rhizomes can be summarized in the order of potency: phenanthrenes > bibenzyls > biphenanthrenes.

monomeric phenanthrene units of 8 was evident from the molecular formula, which indicated this compound to be a dimer of erianthridin (16). The 1H NMR spectrum appeared as two sharp aromatic proton signals for H-5 and H-5′ at δH 8.11 (2H, d, J = 8.4 Hz), and the H-9, -10, -9′, and -10′ signals at δH 2.54 (4H, m), 2.37 (2H, m), and 2.28 (2H, m) in a symmetrical biphenanthrene were also observed. Two aromatic methoxy protons at δH 3.88 (6H, s) and 3.75 (6H, s) were also found. In the HMBC experiment (Figure 1), correlations were observed from H-5 and H-5′ to C-7, C-7′, C-8a, C-8a′, C-4a, and C-4a′, while the HAB-10 and HAB-10′ signals correlated with C-1 and C-1′. Therefore, two phenanthrene moieties were linked between C-l and C-1′ in the molecule of 8. Finally, the OCH3-3, -3′ and OCH3-4, -4′ signals correlated with C-3, -3′ and C-4, -4′ in the HMBC spectrum. Thus, C-3, -3′, -4, and -4′ were assigned as substituted with methoxy groups from their HMBC correlations (Figure 1). The structure of 8 was established as shown. Bleformin I (9) was obtained as a colorless powder. The molecular formula of 9 was established as C32H28O8 by HRESIMS (m/z 563.1677 [M + Na]+, calcd for 563.1676). The 1 H and 13C NMR spectroscopic data of 9 (Tables 2 and 3) indicated them to be very similar to 8, differing only in that one 9,10-dihydrophenanthrene unit was replaced by a 9,10phenanthrene moiety. In the 1H NMR spectrum of 9, the presence of two sets of downfield aromatic proton signals at δH 9.41 and 7.20 (each 1H, J = 10.0 Hz) was observed for H-5′ and H-6′ and at δH 8.16 and 6.76 (each 1H, J = 8.8 Hz) for H-5 and H-6 in a biphenanthrene unit. Moreover, proton signals were observed at δH 2.49 (2H, m), 2.30 (1H, m), and 2.15 (1H, m) for two methylene groups. Furthermore, the HMBC correlations (Figure 1) of H-9 with C-10a, C-8, and C-4b and of HAB-10 with C-8a, C-4a, and C-1 were determined. Thus, the structure of 9 was established as shown. Bleformin J (10) was obtained as a colorless powder. The HRESIMS (m/z 617.1839 [M + Na]+, calcd for 617.1840) revealed that the molecular formula of 10 is C28H34O14. The 1H and 13C NMR spectra of 10 (Tables 2 and 3) were found to be similar to those of shancigusin I (44),20 except that a cis configuration for the olefinic protons of H-7 and H-8 at δH 6.91 and 5.87 with a coupling constant of 12.0 Hz was determined. The 1H and 13C NMR spectra of 10 showed the presence of a cis-configured olefinic group, two aromatic ring AA′XX′ systems (δH 7.53, 7.27, 7.08, 6.99; δC 132.7, 131.2, 117.7, 117.0), two methylene protons (δH 5.09; δC 66.8), two anomeric protons (δH 4.93, 4.91; δC 102.0, 102.3), and eight oxymethine signals (δH 3.50−3.41, 3.46, 3.39; δC 78.3, 78.2, 78.0, 77.9, 75.0, 74.9, 71.4). Analysis of the HMBC spectrum (Figure 1) of 10 further clarified the presence of two hexose units (based on the correlations between H-1″/C-3″, C-5″; H2″/C-1″, C-3″, C-4″; H-1‴/C-3‴, C-5‴; and H-2‴/C-1‴, C3‴, C-4‴). Furthermore, the HMBC spectrum showed correlations between H-7′/C-2′, C-6′, C-9; H-1″/C-4; and H-1‴/C-4′. Thus, the structure of 10 was established as shown. The structures of the known compounds were identified as nudol (11),14 2,7-dihydroxy-3,4,6-trimethoxyphenanthrene (12),19 3,7-dihydroxy-2,4-dimethoxyphenanthrene (13),19 1(4′-hydroxybenzyl)-4-methoxyphenanthrene-2,7-diol (14),21 coeionin (15),19 erianthridin (16),19 2,7-dihydroxy-3,4,6trimethoxy-9,10-dihydrophenanthrene (17),21 3-(4-hydroxybenzyl)-4-methoxy-9,10-dihydrophenanthrene-2,7-diol (18),21 2,7-dihydroxy-1-(p-hydroxybenzyl)-4-methoxy-9,10-dihydrophenanthrene (19),21 2,7-dihydroxy-1,3-bis(p-hydroxybenzyl)-

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a JASCO P-2000 digital polarimeter using a 0.5 dm

G



Article

mm × 250 mm, i.d.; 5 μm) was used. Silica gel (70−230 and 230−400 mesh; Merck), Diaion HP-20 resin (Mitsubishi, Chemical, Tokyo, Japan), and Spherical C18 100 Å reversed-phase silica gel (RP-18; particle size 20−40 μm; Silicycle) were used for column chromatography, and silica gel 60 F254 (Merck) and RP-18 F254S (Merck) were used for TLC. Plant Material. The rhizomes of Bletilla formosana were collected at Baoshan, Hsin-Chu Country, Taiwan, in July 2013. The plant material was identified and authenticated by Assoc. Prof. Dr. ChangSheng Kuoh, Department of Life Sciences, National Cheng Kung University, Tainan, Taiwan. A voucher specimen (TSWu-20130711) was deposited in the Department of Chemistry, National Cheng Kung University, Taiwan. Extraction and Isolation. The ground air-dried whole plants of B. formosana (10 kg) were extracted with EtOH (50 L × 7) at 60 °C and refluxed for 8 h. The EtOH extracts were combined and evaporated under reduced pressure to give 818 g of a residue. The crude extract was subjected to column chromatography over Diaion HP-20 and eluted with H2O and ethanol mixtures in a step gradient manner to afford five fractions, BF1−BF5, on the basis of TLC analysis. Fraction BF3 (205.7 g) was subjected to column chromatography over RP-18 gel and eluted with H2O and MeOH step gradient mixtures to afford six fractions, BF3-1−6. Repeated column chromatography of fraction BF3-2 over RP-18 gel with H2O− MeOH (1:1) yielded seven fractions, BF3-2-1−7. Fraction BF3-2-1 was chromatographed over RP-18 silica gel eluted with H2O−MeOH (7:3) to give gastrodin (21.5 mg). In turn, fraction BF3-2-4 was chromatographed over RP-18 silica gel eluted with H2O−MeOH (1:1) mixtures to give dactylorhin A (37.5 mg), shancigusin I (44) (4.8 mg), 5-hydroxymethylfuraldehyde (0.8 mg), and bleformin J (10) (2.6 mg). Fraction BF3-2-5 was purified by recrystallization to give militarin (43) (35 g). Separation of fraction BF3-3 was performed by silica gel chromatography with n-hexane−diisopropyl ether (1:3) to afford seven fractions, BF3-3-1−7. Fraction BF3-3-3 was chromatographed over silica gel eluted with CHCl2−MeOH (10:1) to afford (2R)-1-(4β-D-glucopyranosyloxybenzyl)-4-methyl-2-isobutyl malate (29.3 mg). Fraction BF4 (52.4 g) was subjected to column chromatography over silica gel and eluted with CHCl3 and MeOH mixtures in a step gradient manner to afford 11 fractions, BF4-1−11. Separation of fraction BF4-4 was performed by further silica gel chromatography with n-hexane−diisopropyl ether (2.5:1) to afford 11 fractions, BF4-41−11. Fraction BF4-4-3 was chromatographed over silica gel eluted with n-hexane−CHCl3 gradient mixtures (1:4) to afford fraction BF44-3-1-2. BF4-4-3-1 was purified by HPLC (MeOH−H2O, 70:30, flow rate, 2.0 mL/min) and afforded 3′-O-methylbatatasin III (32) (7.33 mg, tR = 32.2 min), 5-dimethoxy-3′-hydroxybibenzyl (1.71 mg, tR = 33.7 min), and (2S)-5,2′-dihydroxy-7-methoxyflavanone (2.7 mg, tR = 43.1 min). Purification of fraction BF4-4-3-2 by HPLC (MeOH−H2O, 65:35, flow rate 2.0 mL/min) afforded ethyl (Z)-4-hydroxycinnamate (1.0 mg, tR = 19.9 min) and ethyl (E)-4-hydroxycinnamate (1.6 mg, tR = 21.5 min). Fraction BF4-4-6 was chromatographed over silica gel eluted with n-hexane−CHCl3 (1:3) to give 2,7-dihydroxy-3,4,6trimethoxy-9,10-dihydrophenanthrene (17) (6.2 mg). Fraction BF44-7 was chromatographed on silica gel with n-hexane−CHCl3 (1:4) to afford 2,7-dihydroxy-3,4,6-trimethoxyphenanthrene (12) (4.9 mg). Repeated column chromatography of fraction BF4-5 over silica gel with n-hexane−diisopropyl ether (1:1) yielded 11 fractions, BF4-5-1− 11. Fraction BF4-5-2 was purified by recrystallization to obtain 4hydroxybenzyl ethyl ether (41) (67.3 mg). Fraction BF4-5-3 was chromatographed over silica gel eluted with n-hexane−diisopropyl ether gradient mixtures (2:1) to give 4-hydroxybenzaldehyde (42) (18.3 mg). Fraction BF4-5-4 was chromatographed over silica gel eluted with n-hexane−diisopropyl ether (2:1) to give erianthridin (16) (11.0 mg). Fraction BF4-5-5 was chromatographed over RP-18 HPLC (MeOH−H2O, 60:40, flow rate 2.0 mL/min) to give nudol (11) (2.6 mg, tR = 22.5 min). BF4-5-5-4 purification by HPLC (MeOH−H2O, 50:50, flow rate 2.0 mL/min) afforded bleformin H (8) (3.6 mg, tR = 150.2 min) and bleformin I (9) (1.8 mg, tR = 159.5 min). Fractions BF4-5-6 and BF2-5-7 were purified by recrystallization to obtain gigantol (33) (15.1 mg) and 3,7-dihydroxy-2,4-dimethoxyphenan-

Table 4. Inhibitory Effects of Purified Compounds from B. formosana on Superoxide Anion Generation and Elastase Release by Human Neutrophils in Response to N-Formyl-Lmethionyl-L-leucyl-L-phenylalanine/Cytochalasin B (FMLP/ CB) superoxide anion

elastase release

IC50 (μM)a

IC50 (μM)a

compound 1 2 3 4 5 6 8 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 CAL-101b

1.4 1.6 4.9 0.2 2.8 1.9 2.5 1.7 2.0 2.6 0.5 0.4 0.3 0.2 0.2 0.2 1.1 >10 1.8 0.4 4.2 1.5 1.4 1.5 0.9 2.4 6.5 0.9 6.5 2.1 1.7 2.2 1.1 3.6 6.3 0.7 >10 >10 >10 >10 0.1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.3 0.2 1.1 0.1 0.3 0.3 0.3 0.1 0.2 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.3

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.2 0.1 0.5 0.3 0.1 0.2 0.1 1.1 1.4 0.1 1.6 0.3 0.2 0.4 0.1 0.3 0.7 0.2

*** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** ***

± 0.1

*** *** ***

1.3 ± 0.2 1.9 ± 0.3 >10 0.4 ± 0.1 1.6 ± 0.1 1.7 ± 0.1 >10 >10 2.1 ± 0.4 4.9 ± 0.4 0.9 ± 0.3 >10 2.0 ± 0.3 5.7 ± 0.4 2.0 ± 0.3 2.0 ± 0.7 1.8 ± 0.3 >10 1.4 ± 0.4 0.6 ± 0.1 1.2 ± 0.1 0.5 ± 0.2 0.6 ± 0.2 0.3 ± 0.1 0.6 ± 0.1 0.7 ± 0.1 >10 4.7 ± 0.9 >10 3.2 ± 0.7 2.8 ± 1.0 0.9 ± 0.4 1.1 ± 0.2 2.2 ± 0.9 4.2 ± 0.9 0.7 ± 0.1 >10 >10 3.3 ± 0.2 >10 0.3 ± 0.1

*** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** ** *** ** *** *** *** *** *** *** *** * *** *** * ***

a

Concentration necessary for 50% inhibition (IC50). Results are presented as means ± SEM (n = 3 or 4); *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control value. bThis phosphatidylinositol3-kinase inhibitor was used as a positive control for inhibition of superoxide anion generation and elastase release (n = 3). cell. UV spectra were obtained with a Hitachi UV-3210 spectrophotometer, and IR spectra were measured with a Shimadzu FTIR Prestige-21 spectrometer. The 1H and 13C NMR spectra were recorded using Bruker AVIII-400 and AVIII-700 NMR spectrometers with TMS as the internal reference, and chemical shifts are expressed in δ units (ppm). The ESIMS and HRESIMS were taken on a Bruker Daltonics APEX II 30e spectrometer. HPLC separations were performed on a Shimadzu LC20AT pump with a Shimadzu SPDM20A detector. For HPLC, a Merck Hibar Purospher Star RP-18e (10 H



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1456, 1273 cm−1; 1H NMR (acetone-d6, 400 MHz) and 13C NMR (acetone-d6, 100 MHz), see Table 1; HRESIMS m/z 399.1206 [M + Na]+ (calcd for C23H20O5Na, 399.1208). Bleformin C (3): colorless powder, UV (MeOH) λmax (log ε) 274 (4.01), 267 (4.02), 260 (4.00) nm; IR νmax 3435, 2924, 2855, 1707, 1611, 1458 cm−1; 1H NMR (acetone-d6, 700 MHz) and 13C NMR (acetone-d6, 175 MHz), see Table 1; HRESIMS m/z 507.1778 [M + Na]+ (calcd for C30H28O6Na, 507.1778). Bleformin D (4): colorless powder, UV (MeOH) λmax (log ε) 297 (4.36), 282 (4.51) nm; IR νmax 3383, 2930, 2853, 1701, 1607, 1589, 1510, 1454 cm−1; 1H NMR (acetone-d6, 400 MHz) and 13C NMR (acetone-d6, 100 MHz), see Tables 2 and 3; HRESIMS m/z 611.2041 [M + Na]+ (calcd for C37H32O7Na, 611.2040). Bleformin E (5): colorless powder, UV (MeOH) λmax (log ε) 301 (3.91), 266 (4.61) nm; IR νmax 3404, 2926, 2855, 1589, 1472, 1454, 1265 cm−1; 1H NMR (acetone-d6, 400 MHz) and 13C NMR (acetoned6, 100 MHz), see Tables 2 and 3; HRESIMS m/z 561.1522 [M + Na]+ (calcd for C32H26O8Na, 561.1525). Bleformin F (6): colorless powder, UV (MeOH) λmax (log ε) 309 (3.91), 286 (4.17), 265 (4.69), 258 (4.61) nm; IR νmax 3410, 3393, 2930, 2864, 1699, 1603, 1460 cm−1; 1H NMR (acetone-d6, 400 MHz) and 13C NMR (acetone-d6, 100 MHz), see Tables 2 and 3; HRESIMS m/z 531.1417 [M + Na]+ (calcd for C31H24O7Na, 531.1414). Bleformin G (7): colorless powder, UV (MeOH) λmax (log ε) 312 (4.19), 288 (4.40), 264 (4.77), 254 (4.70), 246 (4.62), 211 (4.54) nm; IR νmax 3395, 2924, 2855, 1695, 1599, 1458 cm−1; 1H NMR (acetoned6, 700 MHz) and 13C NMR (acetone-d6, 175 MHz), see Tables 2 and 3; HRESIMS m/z 531.1415 [M + Na]+ (calcd for C31H24O7Na, 531.1414). Bleformin H (8): colorless powder, UV (MeOH) λmax (log ε) 282 (4.35), 205 (4.74) nm; IR νmax 3443, 2961, 2855, 1707, 1643, 1454 cm−1; 1H NMR (acetone-d6, 400 MHz) and 13C NMR (acetone-d6, 100 MHz), see Tables 2 and 3; HRESIMS m/z 565.1830 [M + Na]+ (calcd for C32H30O8Na, 565.1833). Bleformin I (9): colorless powder, UV (MeOH) λmax (log ε) 308 (4.11), 264 (4.67), 205 (4.69) nm; IR νmax 3389, 2926, 2857, 1701, 1614, 1572, 1505, 1456 cm−1; 1H NMR (acetone-d6, 400 MHz) and 13 C NMR (acetone-d6, 100 MHz), see Tables 2 and 3; HRESIMS m/z 563.1677 [M + Na]+ (calcd for C32H28O8Na, 563.1676). Bleformin J (10): colorless powder, UV (MeOH) λmax (log ε) 300 (3.56), 285 (3.55), 227 (3.56), 218 (3.58) nm; IR νmax 3378, 2926, 2863, 1705, 1606, 1511, 1452, 1401, 1236, 1171, 1074 cm−1; 1H NMR (methanol-d4, 400 MHz) and 13C NMR (methanol-d4, 100 MHz), see Tables 2 and 3; HRESIMS m/z 617.1839 [M + Na]+ (calcd for C28H34O14Na, 617.1841). Determination of Sugar Configuration of Compound 10. Authentic samples of D-glucose and L-glucose (1 mg) were separately dissolved in pyridine (0.5 mL) containing L-cysteine methyl ester (5.0 mg) and heated at 60 °C for 1 h, and then o-isothiocyanate (5 mg) was added to the mixture and reacted at room temperature for an additional 1 h. The reaction mixture (2 mL) was analyzed by HPLC (Purospher STAR RP-8e column; 5 m, 250 × 4.6 mm) and detected at 250 nm.46 The peaks at 29.6 and 32.2 min were from derivatives of Lglucose and D -glucose (Figures S99 and S100, Supporting Information). Glycoside 10 (0.5 mg) was hydrolyzed by heating in 0.5 M HCl (0.1 mL) and neutralized with Amberlite IRA400. After drying in vacuo, the residue was dissolved in pyridine (0.1 mL) containing Lcysteine methyl ester hydrochloride (0.5 mg) and heated at 60 °C for 1 h. A 0.1 mL solution of o-toryl isothiocyanate (0.5 mg) in pyridine was added to the mixture, which reacted at room temperature for an additional 1 h. HPLC analysis of the methyl 2-(polyhydroxyhexyl)-3(o-tolylthiocarbamoyl)-thiazolidine-4(R)-carboxylate obtained from the hydrolysate of glycoside 10 showed a peak at 32.1 min, corresponding to a D-glucose derivative (Figure S101, Supporting Information) Preparation of Human Neutrophils. Neutrophils were isolated with a standard method of dextran sedimentation prior to centrifugation in a Ficoll Hypaque apparatus and hypotonic lysis of erythrocytes. The whole blood was obtained from healthy donors

threne (13) (36.4 mg), respectively. Fraction BF4-5-8 was chromatographed over silica gel eluted with CHCl3−diisopropyl ether (19:1) to give bleformin B (2) (5.5 mg). Separation of fraction BF4-5-8-1 by HPLC (MeOH−H2O, 80:20, flow rate 2.0 mL/min) afforded phemeranthoquinone (21) (3.6 mg, tR = 9.9 min) and densiflorol B (48) (1.4 mg, tR = 8.5 min). Fraction BF4-5-10 was chromatographed over silica gel eluted with n-hexane−acetone (2:1) to give agrostonin (27) (7.3 mg) and bleformin E (5) (4.0 mg). Repeated column chromatography of fraction BF4-6 over silica gel with n-hexane− EtOAc (1:1) yielded 14 fractions, BF4-6-1−14. Of these, fractions BF4-6-2 and BF4-6-5 were purified by recrystallization to give batatasin III (31) (499 mg) and coeionin (15) (160 mg), respectively. Fraction BF2-6-10 was chromatographed over silica gel eluted with CHCl2−diisopropyl ether (20:1) to give bleformin F (6) (7.4 mg), and purification of fraction BF4-6-10-6 by HPLC (MeOH−H2O, 60:40, flow rate 2.0 mL/min) gave bleformin G (7) (1.0 mg, tR = 15.1 min). Repeated column chromatography of fraction BF4-7 over silica gel with n-hexane−diisopropyl ether (1:4) yielded 18 fractions, BF4-71−18. Fraction BF4-7-5 was chromatographed over silica gel eluted with CHCl3−diisopropyl ether (10:1) to give 3-(4-hydroxybenzyl)-4methoxy-9,10-dihydrophenanthrene-2,7-diol (18) (11.6 mg). Fraction BF4-7-6 was purified by recrystallization to give 3′-dihydroxy-4-(4hydroxybenzyl)-5-methoxybibenzyl (35) (10.1 mg). Fraction BF4-7-8 was chromatographed over silica gel eluted with CHCl2−acetone (29:1) to give 7-dihydroxy-1-(p-hydroxybenzyl)-4-methoxy-9,10-dihydrophenanthrene (19) (152 mg), 5-dihydroxy-2-(4-hydroxybenzyl)-3methoxybibenzyl (34) (56.1 mg), bleformin A (1) (5.7 mg), 3′dihydroxy-2-(4-hydroxybenzyl)-5-methoxybibenzyl (37) (18.9 mg), 1(4′-hydroxybenzyl)-4-methoxyphenanthrene-2,7-diol (14) (20.4 mg), and 2,7-dihydroxy-1,3-bis(p-hydroxybenzyl)-4-methoxy-9,10-dihydrophenanthrene (20) (2.1 mg). Fraction BF4-7-9 was chromatographed over silica gel eluted with CHCl3−diisopropyl ether (6:1) to give 6′bis(p-hydroxybenzyl)-3′,5-dimethoxy-3-hydroxybibenzyl (38) (12.2 mg) and bulbocodin D (36) (15.5 mg). Fraction BF4-7-11 was chromatographed over silica gel eluted with CHCl3−diisopropyl ether (6:1) to give 3′-dihydroxy-2′,6′-bis(p-hydroxybenzyl)-5-methoxybibenzyl (39) (190 mg). Fraction BF4-7-12 was chromatographed over silica gel eluted with CHCl3−MeOH (30:1) to give biestriarene A (29) (29.5 mg), blestrianol A (22) (6.7 mg), gymconopin C (25) (9.3 mg), and 4,4′-dimethoxy-9,10-dyhydro-[6,10-biphenanthrene]2,7,2′,7′-tetraol (24) (3.6 mg). Fraction BF4-7-13 was chromatographed over silica gel eluted with CHCl3−MeOH (30:1) to give fractions BF4-7-13-1−7. Purification of fraction BF4-7-13-2 by HPLC (MeOH−H2O, 50:50, flow rate 2.0 mL/min) gave blesttriarene B (28) (12.4 mg, tR = 96.0 min) and cirrhopetalanthrin (26) (8.9 mg, tR = 105.0 min). Purification of BF4-7-13-5 by HPLC (MeOH−H2O, 60:40, flow rate 2.0 mL/min) gave phochinenin K (23) (11.5 mg, tR = 18.3 min) and bleformin D (4) (18.6 mg, tR = 46.1 min). Finally, purification of BF4-7-13-3 by HPLC (MeOH−H2O, 55:45, flow rate 2.0 mL/min) gave bleformin C (3) (12.2 mg, tR = 18.3 min). Fraction BF5 (35.1 g) was subjected to column chromatography over silica gel and eluted with CHCl3 and MeOH step gradient mixtures to afford 11 fractions, BF5−1−9. Repeated column chromatography of fraction BF5-3 over silica gel with n-hexane− EtOAc (7:1) yielded 17 fractions, BF5-3-1−17. Fraction BF5-3-10 was purified by recrystallization to give β-sitosteryl-3-O-β-D-glucoside (40) (97.1 mg). BF1-4 was chromatographed over silica gel eluted with nhexane−diisopropyl ether (2:1) to afford 10 fractions, BF5-4-1−10. Fraction BF5-4-3 was chromatographed over silica gel eluted with nhexane−CHCl2 (1:2) to give 3-O-methyldihydropinosylvin (30) (15.7 mg). Finally, fraction BF5-4-4 was chromatographed over silica gel eluted with n-hexane−CHCl2 (1:2) to give a β-sitosterol and stigmasterol mixture. Bleformin A (1): colorless powder, UV (MeOH) λmax (log ε) 310 (3.77), 297 (3.80), 289 (3.95), 264 (4.51) nm; IR νmax 3331, 3302, 3287, 2926, 2857, 1614, 1547 cm−1; 1H NMR (acetone-d6, 400 MHz) and 13C NMR (acetone-d6, 100 MHz), see Table 1; HRESIMS m/z 399.1205 [M + Na]+ (calcd for C23H20O5Na, 399.1203). Bleformin B (2): colorless powder, UV (MeOH) λmax (log ε) 310 (3.35), 264 (4.05), 208 (3.90) nm; IR νmax 3375, 2926, 2857, 1611, I



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(20−30 years old) by venipuncture,47 using a protocol approved by the Institutional Review Board at Chang Gung Memorial Hospital (IRB protocol number: 102-1595A3). The purified neutrophils were resuspended in a Ca2+-free Hank’s balanced salt solution (HBSS) buffer at pH 7.4 and were maintained at 4 °C before use. Measurement of Superoxide Anion Generation. A superoxide anion generation assay used was based on the reduction of ferricytochrome c inhibited by superoxide dismutase (SOD).47 In brief, the neutrophils (6 × 105 cells/mL) were equilibrated with 0.5 mg/mL ferricytochrome c and 1 mM Ca2+ at 37 °C for 2 min and then incubated with each test compound or an equal volume of vehicle (0.1% DMSO, negative control) for 5 min. Cells were incubated with cytochalasin B (CB, 1 μg/mL) for 3 min, before activation by formylL-methionyl-L-leucyl-L-phenylalanine (FMLP, 100 nM). The changes in absorbance with reduction of ferricytochrome c at 550 nm were monitored continuously in a double-beam, six-cell positioned spectrophotometer (Hitachi U-3010, Tokyo, Japan) with constant stirring. Calculations were based on differences in the reactions with and without SOD (100 U/mL) divided by the extinction coefficient for the reduction of ferricytochrome c (ε = 21.1/mM/10 mm). CAL101 was used as a positive control. Elastase Release Assays. Degranulation of azurophilic granules was determined by an elastase release assay, as described previously.47 Experiments were performed using MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide as the elastase substrate. Briefly, neutrophils (6 × 105 cells/mL) were equilibrated in MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (100 μM) at 37 °C for 2 min and then incubated with a test compound or an equal volume of vehicle (0.1% DMSO, negative control) for 5 min. Cells were activated by 100 nM FMLP and 0.5 μg/mL CB, and changes in absorbance at 405 nm were monitored continuously to assay elastase release. The results were expressed as the percentage of elastase release in the FMLP/CB-activated, drug-free control system. CAL-101 was used as a positive control.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00118. Copies of the spectra of compounds 1−10 and the HPLC profile of compound 10 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: 886-6-2353535, ext 6803. Fax: 886-6-2373149. E-mail: [email protected] (H.-Y. Hung). *Tel: 886-6-2757575, ext 65333. Fax: 886-6-2740552. E-mail: [email protected] (T.-S. Wu.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are thankful for the financial support from the Ministry of Science and Technology of Republic of China granted to T.-S. Wu.

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REFERENCES

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