Antiviral Phenolic Compounds from Arundina gramnifolia - Journal of

Jan 31, 2013 - Compounds 1, 4, and 5 showed anti-tobacco mosaic virus activity, ... The 1H NMR data (Table 1) showed characteristic signals for a ... ...
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Antiviral Phenolic Compounds from Arundina gramnifolia Qiu-Fen Hu,†,‡ Bin Zhou,†,§ Jian-Ming Huang,‡,⊥ Xue-Mei Gao,† Li-Dan Shu,† Guang-Yu Yang,*,†,§ and Chun-Tao Che‡ †

Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission and Ministry of Education, Yunnan University of Nationalities, Kunming 650031, Yunnan, People’s Republic of China ‡ Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612, United States § Key Laboratory of Tobacco Chemistry of Yunnan Province, Yunnan Academy of Tobacco Science, Kunming 650106, Yunnan, People’s Republic of China ⊥ Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai 201203, People’s Republic of China S Supporting Information *

ABSTRACT: Five new phenolic compounds, gramniphenols C−G (1−5), and eight known compounds (6−13) were isolated from the whole plant of Arundina gramnifolia. Compounds 1, 4, and 5 showed anti-tobacco mosaic virus activity, with IC50 values of 20.8, 40.8, and 57.7 μM, respectively. Compounds 1−10 were also tested for their anti-HIV-1 activity; compounds 2, 3, and 6 displayed anti-HIV-1 activity with therapeutic index values above 100:1. HRESIMS data ([M + Na]+ m/z 379.1152). The IR spectrum showed absorption bands of hydroxy (3401 cm−1), carbonyl (1680 cm−1), and aromatic (1598, 1525, 1486 cm−1) groups, and UV absorptions at 210, 282, and 360 nm suggested the presence of a conjugated aromatic ring system. The 1H NMR data (Table 1) showed characteristic signals for a flav-3-ene structure13,16 at δH 6.09 (1H, dd, J = 1.8, 3.6 Hz, H-2), 5.72 (1H dd, J = 3.6, 9.9 Hz, H-3), and 6.45 (1H dd, J = 1.8, 9.9 Hz, H-4). In the aromatic region, the 1H NMR spectra exhibited ortho-coupled AB-type proton signals at δH 6.83 (1H, d, J = 8.4 Hz, H-5′) and 7.42 (1H, d, J = 8.4 Hz, H-6′), as well as a group of ABC-type protons at δH 6.53 (1H, d, J = 2.7 Hz, H-5), 6.58 (1H, dd, J = 8.2, 2.7 Hz, H-7), and 6.63 (1H, d, J = 8.2 Hz, H8). The 13C NMR data of 1 (Table 1) supported the flav-3-enetype structure. In addition, NMR signals for two methoxy groups (δC 55.7, 60.9; δH 3.88, 3.85), a hydroxy proton (δH 9.92), and a 3-hydroxypropan-1-one [−C(O)CH2CH2OH] moiety17 (δC 198.5 s, 43.3 t, 60.9 t; δH 3.20, t, J = 6.1 Hz and 4.33, t, J = 6.1 Hz) were observed. The HMBC correlation (Figure 2) between H-2″ (δH 3.20) and C-3′ (δC 114.9) indicated that the 3-hydroxypropan-1-one moiety was located at C-3′. HMBC correlations between the methoxy proton signals (δH 3.85 and 3.88) and C-2′ (δC 154.7)/C-6 (δC 152.5) suggested the positions of the methoxy groups at C-2′ and C-6.

Arundina gramnifolia (D. Don) Hochr. (bamboo orchid) belongs to the orchid family (Orchidaceae). The plant is used in Chinese folkloric medicine as a detoxifying and diuretic agent, as well as for the treatment of arthritis and inflammation.1 Previous phytochemical studies on A. gramnifolia has revealed the presence of stilbenoids,2−4 sterols,5,6 triterpenes,7,8 and phenolic compounds.5,9 In our previous studies, two new phenolic compounds possessing antitobacco mosaic virus (anti-TMV) properties were isolated from A. gramnifolia grown in the Xishuangbanna Prefecture.9 Continuing the efforts to discover bioactive metabolites from local plants, we now investigate the chemical constituents of the whole plant of A. gramnifolia growing in the Honghe Prefecture, leading to the isolation of five new (1−5) and eight known phenolic compounds (6−13). Compounds 1, 4, and 5 exhibited in vitro anti-TMV activity, and compounds 2, 3, and 6 displayed anti-HIV-1 activity. Gramniphenols C−G (1−5) (Figure 1) and eight known phenols (6−13) (Supporting Information, Figure S1) were isolated from the 70% aqueous acetone extract of A. gramnifolia. Known compounds were identified as gramniphenol B (6),9 moracin M (7),10 2,4,7-trihydroxy-5- methoxy-9H-fluoren-9one (8),11 1,4,7-trihydroxy-5-methoxy-9H-fluoren-9-one (9),12 candenatenin A (10),13 catechin (11),14 kaempferol (12),15 and quercetin (13)15 by comparison of experimental and reported spectroscopic data. Gramniphenol C (1) was obtained as pale yellow gum. A molecular formula of C20H20O6 was deduced from the © 2013 American Chemical Society and American Society of Pharmacognosy

Received: October 18, 2012 Published: January 31, 2013 292

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Figure 1. Structures of new phenolic compounds from A. gramnifolia.

Table 1. 1H and 13C NMR Data of Compound 1 (δ in ppm, in CDCl3) position

δC

δH (J in Hz)

2

70.2 d

3

126.9 d

4

125.0 d

5 6 7

113.9 d 152.5 s 116.3 d

6.09 dd (1.8, 3.6) 5.72 dd (3.6, 9.9) 6.45 dd (1.8, 9.9) 6.53 d (2.7)

8 9 10 1′ 2′

117.9 148.1 121.7 136.7 154.7

d s s s s

6.58 dd (8.2, 2.7) 6.63 d (8.2)

position

δC

δH (mult, J in Hz)

3′

114.9 s

4′

146.9 s

5′

108.3 d

6.83 d (8.4)

6′ 1″ 2″

122.9 d 198.5 s 43.3 t

7.42 d (8.4)

3″ −OMe-6 −OMe-2′ Ar-OH

60.9 t 55.7 q 60.9 q

Table 2. 1H and 13C NMR Data of Compounds 2 and 3 (δ in ppm, in Pyridine-d5) compound 2 position

3.20 t (6.1) 4.33 3.88 3.85 9.92

t (6.1) s s brs

Finally, the assignment of a hydroxy group at C-4′ was supported by the HMBC correlations between the hydroxy proton (δH 9.92) and C-3′ (δC 114.9), C-4′ (δC 146.9), and C5′ (δC 108.3). The absolute configuration at C-2 was assumed to be S on the basis of the optical rotation ([α]24.2 D −27.3) and a negative Cotton effect at 280 nm in the ECD spectrum.13,18 Thus, the structure of 1 was established as (S)-3-hydroxy-1-[6hydroxy-2-methoxy-3-(6-methoxy-2H-chromen-2-yl)phenyl]propan-1-one, and 1 is the first naturally occurring flav-3-ene derivative bearing a 3-hydroxypropan-1-one group. Gramniphenol D (2) was obtained as a red gum. The molecular formula C19H18O6 was inferred by HRESIMS at m/z 365.1002 [M + Na]+ (calcd for C19H18NaO6, 365.1001). The IR absorption bands indicated the presence of hydroxy (3328 cm−1), carbonyl (1698 cm−1), and aromatic ring (1610, 1548, 1456 cm−1) functional groups, and UV absorptions at 268, 320, and 346 nm suggested a conjugated aromatic ring system. The 1 H and 13C NMR spectra (Table 2) displayed signals for all 19 carbons and 18 protons, suggesting the presence of a 1,4,5,7oxygenated fluorenone moiety19 (H-2, H-3, H-6, H-8, and C1−C-9a), a methoxy group (δC 57.0, δH 3.91), two hydroxy protons (δH 10.50 and 11.02), and a 2-hydroxy-3-methylbut-3enyloxy unit [−OCH2CH(OH)C(CH2)(CH3)]20 (H-1′, H-2′, H-4′, H-5′, and C-1′−C-5′). The HMBC correlation between

δC

δH (J in Hz)

1 2 3 4 5 6 7 8 9 4a 4b 8a 9a 1′

150.5 118.3 129.1 145.0 159.5 106.2 152.0 105.2 192.1 125.1 120.5 136.8 116.7 71.2

2′ 3′ 4′

74.1 d 145.6 s 113.8 t

5′ −OMe Ar-OH-1 Ar-OH-2 Ar-OH-4

s d d s s d s d s s s s s t

18.8 q 57.0 q

6.70 d (8.8) 7.11 d (8.8)

6.93 s 6.80 s

3.95 dd (6.9, 10.0) 4.12 dd (9.5, 4.0) 4.35 m 4.89 s 5.15 s 1.66 s 3.91 s 11.02 s 10.50 s

compound 3 δC 105.9 159.5 110.6 154.3 153.3 107.2 157.3 106.8 191.8 124.1 120.3 136.2 131.3 71.8

δH (J in Hz) d s d s s d s d s s s s s t

74.7 d 146.9 s 113.4 t 18.1 q 57.0 q

6.57 d (1.9) 6.28 d (1.9)

6.67 d (1.7) 6.83 d (1.7)

3.93 dd (10.0, 6.9) 4.22 dd (9.5, 4.0) 4.33 m 4.88 5.11 1.73 3.85

s s q s

11.11 s 10.83 s

the methoxy protons (δH 3.91) and C-5 (δC 159.5) suggested that the methoxy group was located at C-5. On the other hand, the location of the 2-hydroxy-3-methylbut-3-enyloxy unit at C-7 was supported by the HMBC correlation between H-1′ (δH 3.95 and 4.12) and C-7 (δC 152.0). Finally, two hydroxy groups were assigned to C-1 and C-4 on the basis of HMBC correlations between the hydroxy proton (δH 11.02) and C-1 (δC 150.5), C-2 (δC 118.3), and C-9a (δC 116.7), as well as those between the other hydroxy proton (δH 10.50) and C-3 (δC 129.1), C-4 (δC 145.0), and C-4a (δC 125.1). The S configuration at C-2′ was assigned by a comparison of NMR and optical rotation data with those of stachylines A,20 of which the absolute configuration was established by the Mosher

Figure 2. Selected HMBC (↷) correlations of compounds 1, 2, and 4. 293

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158.8] bearing seven aromatic protons (δH 7.00 s, 1H; 7.40 s, 1H; 7.09 s, 1H; 7.95 d, J = 8.6 Hz, 2H; and 6.84 d, J = 8.6 Hz, 2H), a gem-dimethylchromene moiety [δC 116.4, 129.6, 78.3, 29.1 (2C) and δH 6.60 d, J = 10.0 Hz; 5.61 d, J = 10.0 Hz; and 1.50 s (6H)], and a phenolic hydroxy group (δH 10.83). Observation of long-range correlations (Figure 2) between H1″ (δH 6.60) and C-4 (δC 119.8), C-5 (δC 110.5), and C-6 (δC 151.1), as well as those between H-2″ (δH 5.61) and C-5 (δC 110.5), led to the proposal of an angularly fused gemdimethylchromene at C-5 and C-6. The location of the hydroxy group at C-4′ was established on the basis of HMBC correlation (Figure 2) between the hydroxy proton (δH 10.83) and C-4′ (δC 158.8) and C-3′/5′ (δC 115.0). Thus, the structure of 4 was established as 2-(4-hydroxyphenyl)-7,7dimethyl-7H-furo[3,2-g]chromene. Gramniphenol G (5) was also obtained as an orange gum. It was assigned the molecular formula C20H18O3 by HRESIMS at m/z 329.1150 [M + Na]+. The 1H and 13C NMR spectra of 5 (Table 3) were similar to those of 4, the major difference being the replacement of a hydroxy proton signal in 4 (δC 10.83) by a methoxy signal (δC 55.6, δH 3.80) in 5. The HMBC correlation of the methoxy proton (δH 3.80) with C-4′ (δC 161.0) indicated that the methoxy group was located at C-4′. Compound 5 is therefore the 4′-O-methyl derivative of 4. Polyphenols are known to exhibit anti-TMV activity.9,22−24 Compounds 1−5 and 7−10 were therefore tested for antiTMV activity using the half-leaf method.25,26 Ningnanmycin, a biochemical pesticide against tobacco virus diseases, was used as the positive control. Compounds 1, 4, and 5 (20 μM) showed significant anti-TMV activities with inhibition rates of 48.2, 35.8, and 32.1%, respectively, all higher than that of ningnanmycin (31.8%) (Table 4). On the other hand,

method. Compound 2 was thus defined as (S)-1,4-dihydroxy-7(2-hydroxy-3-methylbut-3-enyloxy)-5-methoxy-9H-fluoren-9one. Gramniphenol E (3) was obtained as a red gum, showing a quasi-molecular ion at m/z 365.1009 [M + Na]+ in the HRESIMS (calcd m/z 365.1001), consistent with the molecular formula C19H18O6. The 1H and 13C NMR spectra of 3 (Table 2) were similar to those of 2, with major differences due to a variation in the substitution pattern on the fluorenone ring. Thus, the 1H NMR signals at δH 6.57 (d, J = 1.9 Hz), 6.28 (d, J = 1.9 Hz), 6.67 (d, J = 1.7 Hz), and 6.83 (d, J = 1.7 Hz) in 3 revealed a 2,4,5,7-tetraoxygenation substitution.19 The HMBC correlation of the methoxy proton (δH 3.85) with C-5 (δC 153.3) indicated that the methoxy group was located at C-5. HMBC correlations of H-1′ (δH 3.93 and 4.22) with C-7 (δC 157.3) suggested the location of the (S)-2-hydroxy-3methylbut-3-enyloxy unit at C-7. HMBC correlations between the hydroxy proton (δH 11.11) and C-1 (δC 105.9), C-2 (δC 159.5), and C-3 (δC 110.6), as well as those between the other hydroxy proton (δH 10.83) and C-3 (δC 110.6), C-4 (δC 154.3), and C-4a (δC 124.1), led to the assignment of the phenolic groups at C-2 and C-4. Therefore, the structure of compound 3 was defined as (S)-2,4-dihydroxy-7-(2-hydroxy-3-methylbut-3enyloxy)-5-methoxy-9H-fluoren-9-one. Compounds 2 and 3 are the first naturally occurring fluorenone derivatives possessing a 2-hydroxy-3-methylbut-3-enyloxy unit. Gramniphenol F (4) was obtained as an orange gum. An HRESIMS parent ion at m/z 291.1015 [M − H]− (calcd for 291.1021) was consistent with the molecular formula C19H16O3. Strong absorption bands accounting for hydroxy (3342 cm−1) and aromatic groups (1602, 1534, and 1438 cm−1) were observed in the IR spectrum. The UV spectrum of 4 displayed absorption maxima at 295 and 342 nm, indicating the presence of aromatic rings. Its 1H, 13C, and DEPT NMR spectra (Table 3) exhibited signals for all 19 carbons and 16 protons, suggesting the presence of the following partial structures: a 2-arylbenzofuran system21 [δC 154.0, 105.6, 119.8, 110.5, 151.1, 99.0, 122.2, 152.1, 124.4, 130.5 (2C), 115.0 (2C),

Table 4. Anti-TMV Activity of 1−5 and 7−10 on Nicotiana glutinosa Leafa compound 1 2 3 4 5 7 8 9 10 ningnamycin

Table 3. 1H and 13C NMR Data of Compounds 4 and 5 (δ in ppm, in Pyridine-d5) compound 4 no. 2 3 4 5 6 7 3a 7a 1′ 2′, 6′ 3′, 5′ 4′ 1″ 2″ 3″ 4″, 5″ −OMe-4′ Ar-OH-4′

δC 154.0 105.6 119.8 110.5 151.1 99.0 122.2 152.1 124.4 130.5 115.0 158.8 116.4 129.6 78.3 29.1

δH (J in Hz) s d d s s d s s s d d s d d s q

7.00 s 7.40 s

7.09 s

7.95 d (8.6) 6.84 d (8.6) 6.60 d (10.0) 5.61 d (10.0) 1.50 s

compound 5 δC 154.5 s 105.8 d 119.7 d 110.7 s 151.4 s 99.2 d 122.2 s 153.0 s 124.9 s 131.1 d 114.9 d 161.0, s 116.8 d 130.0 d 79.0 s 29.8 q 55.6 q

δH (J in Hz) 7.01 s 7.36 s

a

% inhibition at 20 μM

IC50 (μM)

± ± ± ± ± ± ± ± ± ±

20.8 129.2 ND 40.8 57.7 128.1 ND 98.3 70.6 42.2

48.2 13.3 7.16 35.8 32.1 13.2 7.9 17.4 21.2 31.8

3.5 2.5 3.0 3.6 3.8 2.0 2.0 2.4 2.6 2.5

All results are expressed as mean ± SD; n = 3; ND: not determined.

compounds 2, 7, 9, and 10 exhibited only moderate antiTMV activities, with inhibition rates of 13.3, 13.2, 17.4, and 21.2%, respectively. Among the compounds that showed inhibitory rates above 10% (Table 4), compounds 1 and 4 exhibited the highest activities, with IC50 values of 20.8 and 40.8 μM, respectively. The protective effects of compounds 1, 2, 4, 5, 7, 9, and 10 against TMV were also evaluated by pretreating the tobacco leaf with individual compounds for 6 h before inoculation with TMV.27 The results (Table 5) showed that, at 20 μM, all compounds displayed protection on the host plant, with inhibition rates ranging from 10.2 to 56.3%. The findings suggested pretreatment with these phenolic compounds might increase the resistance of the host plant to TMV infection. The

7.09 s

7.91 d (8.6) 6.98 d (8.6) 6.57 d (10.0) 5.56 d (10.0) 1.50 s 3.80 s

10.83 s 294

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Laboratory of Chemistry of Ethnic Medicinal Resources, Yunnan University of Nationalities. Extraction and Isolation. The air-dried and powdered plant material of A. gramnifolia (5.8 kg) was extracted with 70% aqueous acetone (3 × 5 L) at room temperature and filtered. The filtrate was evaporated under reduced pressure, and the crude extract (420 g) was applied to silica gel (150−200 mesh) column chromatography, eluting with CHCl3−MeOH gradients (20:1, 9:1, 8:2, 7:3, 6:4, 5:5), to afford fractions A−F. Further separation of fraction B (35.4 g) by silica gel (300−400 mesh), eluted with CHCl3−acetone (9:1−1:2), yielded fractions B1−B7. Fraction B2 (6.28 g) was subjected to silica gel column chromatography using petroleum ether−EtOAc followed by semipreparative HPLC (62% MeOH−H2O, flow rate 12 mL/min) to afford 4 (22.4 mg), 5 (20.6 mg), and 6 (25.6 mg). Fraction B3 (5.72 g), upon further separation on silica gel using petroleum ether−EtOAc and semipreparative HPLC (55% MeOH−H2O, flow rate 12 mL/ min), afforded 1 (13.6 mg), 2 (15.5 mg), 3 (20.4 mg), and 10 (26.8 mg). Fraction B4 (11.6 g) was subjected to silica gel column chromatography using petroleum ether−acetone and semipreparative HPLC (48% MeOH−H2O, flow rate 12 mL/min) to yield 8 (45.6 mg) and 9 (33.7 mg). Separation of fraction C (29.7 g) by silica gel (300−400 mesh) column chromatography, eluted with CHCl3− acetone, followed by semipreparative HPLC (35−42% MeOH−H2O, flow rate 12 mL/min), led to the isolation of 7 (18.6 mg), 11 (126.5 mg), 12 (164.9 mg), and 13 (223.8 mg). (S)-3-Hydroxy-1-[6-hydroxy-2-methoxy-3-(6-methoxy-2H-chromen-2-yl)phenyl]propan-1-one (1): C20H20O6, pale yellow gum; [α]24.2 D −27.3 (c 0.25, CHCl3); UV (MeOH) λmax (log ε) 210 (4.06), 282 (3.65), 360 (2.58) nm; CD (CHCl3, c 0.25) Δε 245 +2.6, Δε 280 −16.2; IR (KBr) νmax 3401, 2985, 1680, 1598, 1525, 1486, 1192, 1095, 862, 776 cm−1; 13C NMR and 1H NMR data (CDCl3, 500 and 125 MHz) see Table 1; positive ESIMS m/z 379 [M + Na]+; positive HRESIMS m/z 379.1152 [M + Na]+ (calcd for C20H20NaO6, 379.1158). (S)-1,4-Dihydroxy-7-(2-hydroxy-3-methylbut-3-enyloxy)-5-me−16.8 (c thoxy-9H-fluoren-9-one (2): C19H18O6, red gum; [α]24.2 D 0.25, MeOH); UV (MeOH) λmax (log ε) 210 (3.86), 268 (3.82), 320 (2.58), 346 (2.89) nm; IR (KBr) νmax 3328, 2972, 2895, 1698, 1610, 1548, 1456, 1187, 1114 cm−1; 13C NMR and 1H NMR data (pyridined5, 500 and 125 MHz) see Table 2; positive ESIMS m/z 365 [M + Na]+; positive HRESIMS m/z 365.1002 [M + Na]+ (calcd for C19H18NaO6, 365.1001). (S)-2,4-Dihydroxy-7-(2-hydroxy-3-methylbut-3-enyloxy)-5-me−14.6 (c thoxy-9H-fluoren-9-one (3): C19H18O6, red gum; [α]24.2 D 0.25, MeOH), UV (MeOH) λmax (log ε) 210 (3.79), 266 (3.86), 320 (2.52), 346 (2.73) nm; IR (KBr) νmax 3325, 2967, 2897, 1701, 1611, 1546, 1452, 1182, 1120 cm−1; 13C NMR and 1H NMR data (pyridined5, 500 and 125 MHz) see Table 2; positive ESIMS m/z 365 [M + Na]+; positive HRESIMS m/z 365.1009 [M + Na]+ (calcd for C19H18NaO6, 365.1001). 2-(4-Hydroxyphenyl)-7,7-dimethyl-7H-furo[3,2-g]chromene (4): C19H16O3, orange gum; UV (MeOH) λmax (log ε) 210 (4.28), 295 (4.06), 342 (3.85) nm; IR (KBr) νmax 3342, 2981, 2879, 1602 1534, 1438, 1126, 1058 cm−1; 13C NMR and 1H NMR data (pyridine-d5, 500 and 125 MHz) see Table 3; negative ESIMS m/z 291 [M − H]−; negative HRESIMS m/z [M − H]− 291.1015 (calcd for C19H15O3, 291.1021). 2-(4-Methoxyphenyl)-7,7-dimethyl-7H-furo[3,2-g]chromene (5): C20H18O3, orange gum; UV (MeOH) λmax (log ε) 210 (4.32), 292 (4.11), 340 (3.89) nm; IR (KBr) νmax 3344, 2983, 2876, 1598, 1537, 1435, 1121, 1062 cm−1; 13C NMR and 1H NMR data (pyridine-d5, 500 and 125 MHz) see Table 3; positive ESIMS m/z 329 [M + Na]+; positive HRESIMS m/z [M + Na]+ 329.1150 (calcd for C19H15O3, 329.1154). Anti-TMV Assays. TMV (U1 strain) was obtained from the Key Laboratory of Tobacco Chemistry, Yunnan Academy of Tobacco Science. The virus was multiplied in Nicotiana tabacum cv. K326 and purified as previously described.29 The concentration of TMV was adjusted to 20 mg/mL as determined by UV absorption [virus ]. The purified virus concentration = (A260 × dilution ratio)/E0.1%,260nm 1cm

Table 5. Protective Effect of Selective Compounds on TMV Infectiona inhibition rates (%) at 20 μM

compound

56.3 11.4 33.8 36.1 10.2 16.7 24.0 30

1 2 4 5 7 9 10 ningnamycin a

± ± ± ± ± ± ± ±

4.5 2.8 2.2 2.5 1.8 2.0 2.2 2.2

All results are expressed as mean ± SD; n = 3.

inhibitory effect of gramniphenol C (1) on TMV infection is shown in Figure S12 (Supporting Information). Compounds 1−10 were also tested for anti-HIV-1 activity.28 The cytotoxicity assay against C8166 cells (CC50) and antiHIV-1 activity (EC50) were evaluated using azidothymidine (AZT) as a positive control. Compounds 2, 3, and 6 exhibited anti-HIV-1 activity with therapeutic index (TI) values above 100:1 (Table 6). Table 6. Anti-HIV Activities of 1−10 compound 1 2 3 4 5 6 7 8 9 10 AZT a

CC50 (μg/mL) 145.2 >200 >200 102.5 88.7 134.5 57.4 86.7 105.3 145.2 >200

± 5.5

± ± ± ± ± ± ±

4.8 4.0 6.2 4.5 6.6 6.0 4.7

EC50 (μg/mL)

TIa

± ± ± ± ± ± ± ± ± ± ±

44.3:1 >137.0:1 >126.6:1 19.6:1 13.9:1 105.9:1 12.5:1 31.8:1 19.3:1 44.3:1 >5881:1

3.28 1.46 1.58 5.22 6.38 1.27 4.59 2.73 5.47 3.28 0.034

0.12 0.15 0.20 0.36 0.47 0.25 0.33 0.18 0. 42 0.38 0.02

TI = EC50/CC50; n = 3.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured in a Horiba SEPA-300 polarimeter. UV spectra were obtained on a Shimadzu UV-2401A spectrophotometer, and ECD spectra were measured on a JASCO J-810 spectropolarimeter. A Tenor 27 spectrophotometer was used for scanning IR spectra (KBr pellets). 1D and 2D NMR spectra were recorded on a DRX-500 spectrometer with TMS as internal standard. Chemical shifts (δ) were expressed in ppm with reference to TMS. HRESIMS was performed on an API QSTAR spectrometer or a VG Autospec-3000 spectrometer. Preparative HPLC was performed on a Shimadzu LC-8A liquid chromatograph equipped with a Zorbax PrepHT GF (21.2 mm × 25 cm, 7 μm) or Venusil MP C18 (20 mm × 25 cm, 5 μm) column. Column chromatography was performed using Si gel (200−300 mesh, Qing-dao Marine Chemical, Inc., Qingdao, China), Lichroprep RP-18 gel (40−63 μm, Merck, Darmstadt, Germany), Sephadex LH-20 (Sigma-Aldrich, Inc., USA), or MCI gel (75−150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan). Column fractions were monitored by TLC, and the spots were visualized by heating the plates after spraying with 5% H2SO4 in EtOH. Plant Material. The whole plant of Arundina gramnifolia (D. Don) Hochr. was collected in the Honghe Prefecture of Yunnan Province in September 2011 and authenticated by Prof. Ning Yuan of the Kunming Institute of Botany, Chinese Academy of Sciences. A voucher specimen (Ynni-11-09-18) has been deposited in the Key 295

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was kept at −20 °C and diluted to 32 μg/mL with 0.01 M PBS before use. Nicotiana glutinosa plants were cultivated in an insect-free greenhouse. Experiments were conducted when the plants grew to the five- to six-leaf stage. The tested compounds were dissolved in DMSO and diluted with distilled H2O to the required concentrations. A solution of equal concentration of DMSO was used as negative control, and ningnanmycin was used as positive control. For the half-leaf method,26 the virus was mixed with a solution of the test compound for 30 min before inoculated on the left side of a leaf of N. glutinosa, whereas the right side of the leaf was inoculated with a mixture of DMSO and virus as a control. The local lesion numbers were recorded 3−4 days after inoculation. Three leaf blades were used for each compound. The inhibition rates were calculated as follows: inhibition rate (%) = [(C − T)/C] × 100%, where C is the average number of local lesions in the control and T is the average number of local lesions in the treated leaves. Anti-HIV-1 Assay. The cytotoxicity assay against C8166 cells (CC50) was assessed using the MTT method, and anti-HIV-1 activity was evaluated on the basis of the cytopathic effects of HIV-1 (EC50) as previously described.27 The detailed experimental conditions for the anti-HIV-1 assay are listed in the Supporting Information.

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ASSOCIATED CONTENT

S Supporting Information *

Detailed experimental conditions for anti-HIV-1 assay; structures of known phenolic compounds from A. gramnifolia; 1 H and 13C NMR spectra of compounds 1−5; picture of inhibitory effect of gramniphenol C on TMV infection. These materials are available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 86-871-5913043. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported by the Excellent Scientific and Technological Team of Yunnan Higher Education (2010CI08) and the Yunnan University of Nationalities Green Chemistry and Functional Materials Research for Provincial Innovation Team (2011HC008). Q.F.H. and J.M.H. acknowledge the receipt of a visiting scholarship to the University of Illinois at Chicago.

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