Bioactive Phenolic Components from the Twigs of Atalantia buxifolia

Jul 5, 2018 - In the 13C NMR (Table 2) and DEPT spectra of 1, seven carbon signals were ..... 1–30 against human liver cancer (HepG2), human breast ...
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Bioactive Phenolic Components from the Twigs of Atalantia buxifolia Fang-Rong Chang,†,§ Pei-Shian Li,† Rosa Huang Liu,⊥ Hao-Chun Hu,† Tsong-Long Hwang,∥ Jin-Ching Lee,† Shu-Li Chen,† Yang-Chang Wu,†,¶ and Yuan-Bin Cheng*,†,‡,¶

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†

Graduate Institute of Natural Products, College of Pharmacy, and ‡Center for Infectious Disease and Cancer Research, Kaohsiung Medical University, Kaohsiung 807, Taiwan § National Research Institute of Chinese Medicine, Taipei 112, Taiwan ⊥ School of Nutrition, College of Health Care and Management, Chung Shan Medical University, Taichung 402, Taiwan ∥ Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan ¶ Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan S Supporting Information *

ABSTRACT: Five new compounds named buxifoximes A−C (1−3), buxifobenzoate (4), and 7-O-(7′-peroxygeranyl) coumarin (5), together with 25 known compounds, were identified from the twigs of Atalantia buxifolia. Compounds 1−3 are unique secondary metabolites with the aldoxime functionality. The structures of the isolates were determined on the basis of spectroscopic data analyses, and the structure of 1 was confirmed by an X-ray single-crystallographic analysis. With respect to bioactivity, antidengue virus, anti-inflammatory, and cytotoxic activities of all purified compounds were tested and evaluated. Compound 1 showed a significant antiinflammatory effect by inhibiting superoxide anion generation with an IC50 value of 4.8 ± 0.7 μM. Among the acridone alkaloids, 5-hydroxy-N-methylseverifoline (23) exhibited antidengue activity (IC50 = 5.3 ± 0.4 μM), and atalaphyllinine (20) demonstrated cytotoxicity (IC50 = 6.5 ± 0.0 μM) against the human liver cancer cell line, HepG2.

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Atalantia buxifolia (Poir.) Oliv. ex Benth. (family Rutaceae) is an evergreen citrus distributed across the coastal regions of Southeast Asia. This plant is generally regarded as a folk medicine for the treatment of coughs, malaria, and rheumatism in China. In previous pharmacological studies, the extract or pure compounds isolated from Atalantia plants were proven to have antiallergic,1 antibacterial,2 antifeedant,3 cytotoxic,4 expectorant,5 and antimalarial6 activities. In addition, early phytochemical research revealed that acridone alkaloids,7−9 coumarins,10 limonoids,11 sesquiterpenoids,12 tyramines,13 and oximes14 were the major constituents of Atalantia plants. In this bioactivity study, the MeOH extract of the twigs of A. buxifolia showed anti-inflammatory effects at a concentration of 10 μg/mL by inhibiting superoxide anion generation (100.80 ± 0.44%) and elastase release (115.99 ± 3.06%) from human neutrophils in response to N-formylmethionylleucylphenylalanine (fMLF). In addition, the extract also exhibited antihepatitis C virus activity in a Western blot assay. Thus, this plant material was investigated for bioactive secondary metabolites. Herein, the separation, structural elucidation, cytotoxicity, and anti-inflammatory and antiviral activities of the compounds are reported. © XXXX American Chemical Society and American Society of Pharmacognosy

RESULTS AND DISCUSSION

The MeOH extract of the twigs of A. buxifolia was partitioned into EtOAc/H2O (1:1). The EtOAc soluble portion was isolated by a series of liquid column chromatography processes. Five new compounds (1−5) and 25 known compounds [citaldoxime (6),15 methyl 4-hydroxybenzoate (7),16 methyl 3′-hydroxy-2′-methoxybenzoate (8),17 auraptene (9), 18 7-[7′-hydroxy-3′,7′-dimethylocta-2′,5′-dienyloxy]coumarin (10), 19 7-(6′-hydroxy-3′,7′-dimethyl-2′E,7′octadienyloxy)coumarin (11),20 peroxyschinilenol (12),21 imperatorin (13),22 5-methoxypsoralen (14),22 isoimperatorin (15),23 2′,2′-dimethylpyranocoumarin (16),23 methyl transferulate (17),24 2-methoxy-6-methylnaphthoquinone (18),25 atalaphyllidine (19),8 atalaphyllinine (20),8 N-methylseverifoline (21),8 N-methylatalaphylline (22),8 5-hydroxy-Nmethylseverifoline (23),8 glycocitrine-I (24),8 severifoline (25),8 5-methoxynoracronycine (26),26 N-methylacronicine (27),11 3-taraxeranone (28),27 atalantoflavone (29),9 and angustifolin (30)28] were identified via their spectroscopic data (Figure S35, Supporting Information). Received: November 8, 2017

A

DOI: 10.1021/acs.jnatprod.7b00938 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Chart 1

Table 1. 1H NMR Spectroscopic Data of Compounds 1−5a no.

1

2 3 4 5 6 8 1′ 2′ 3′ 4′ 5′ 6′ 8′ 9′ 10′ 7-OMe 5′-OMe N-OMe

8.07, d (8.9) 6.90, d (8.9)

8.12, d (9.0) 7.03, d (9.0)

8.12, d (9.0) 6.94, d (9.0)

8.00, d (9.0) 6.90, d (9.0)

6.90, d (8.9) 8.07, d (8.9) 7.95, s

7.03, 8.12, 7.91, 6.62, 5.58, 3.22, 1.36,

6.94, 8.12, 7.92, 4.79, 6.91,

6.90, d (9.0) 8.00, d (9.0)

4.09, s

2

d (9.0) d (9.0) s d (15.0) dd (15.0, 7.0) quint (7.0) d (7.0)

3.72, s 4.09, s

3

d (9.0) d (9.0) s dd (5.8, 1.2) m

1.95, s

3.77, s 4.09, s

4

4.77, dd (5.8, 1.2) 6.90, m 1.95, s

5 6.25, 7.63, 7.36, 6.84, 6.81, 4.61, 5.49,

d (9.5) d (9.5) d (8.5) dd (8.5, 2.4) d (2.4) d (6.7) d (6.7)

2.82, 5.66, 5.63, 1.34, 1.34, 1.75,

d (6.0) dt (15.8, 6.0) d (15.8) s s s

3.89, s 3.77, s

a

Data were measured in CDCl3 at 400 MHz. Chemical shifts are in ppm; J values in Hz are in parentheses.

glyoxal O-methylaldoxime (Figure S33, Supporting Information).30 The difference between 1 and the phenylglyoxal Omethylaldoxime is that 1 possesses a para-hydroxy group. According to the above 2D NMR spectroscopic data, the structure of 1 was defined. Compound 1 was crystallized from MeOH/H2O, and its X-ray single-crystallographic analysis (Figure 2) confirmed the structure of buxifoxime A (1) as (E)2-(4-hydroxyphenyl)-2-oxoacetaldehyde O-methyl oxime. Buxifoxime B (2) was obtained as a yellow amorphous powder and exhibited the molecular formula C15H17NO5 by a sodium adduct ion at m/z 314.1001. The IR spectrum of 2 indicated the presence of CO (1730 cm−1), CN (1684 cm−1), CC (1597 cm−1), and N−O (930 cm−1) groups. The IR and 1D NMR spectroscopic data of 2 (Tables 1 and 2) resembled part of 1, implying these two compounds were congeners. The HMBC correlations (Figure 1) from H-3/H-5 to C-1′ (δC 141.7) suggested that the hydroxy functionality of 1 was replaced by an ether-linked moiety in 2. In the COSY spectrum, correlations of H-1′ (δH 6.62)/H-2′ (δH 5.58)/H-3′ (δH 3.22)/Me-4′ (δH 1.36) indicated these protons are contiguous. The HMBC correlations from both Me-4′ and 5′-OMe (δH 3.72) to C-5′ (δC 175.8) showed that an ester carbonyl (C-5′) is adjacent to C-3′. The E geometry of the Δ1′(2′) double bond was confirmed by their coupling constant (JH‑1′/H‑2′ = 15.0 Hz). The absolute configuration of C-3′ was assigned to be S on the basis of the similar trend between the ECD spectrum of 2 and the calculated spectrum of the 3′S

Buxifoxime A (1) was obtained as yellowish cubic crystals after crystallization from MeOH and H2O. The HRESIMS data showed a sodium adduct ion at m/z 202.0476 [M + Na]+, consistent with a molecular formula of C9H9NO3Na (six indices of hydrogen deficiency). The IR spectrum exhibited absorption bands at 927, 1593, 1684, and 3290 cm−1, which are characteristic of N−O, CC, CN, and OH functionalities, respectively.29 The 1H NMR data of 1 (Table 1) indicated the presence of a methoxy group (δH 4.09), an oxime methine (δH 7.95), and a para-disubstituted benzene ring [δH 8.07, d, J = 8.9 Hz (2H) and 6.90, d, J = 8.9 Hz (2H)]. In the 13 C NMR (Table 2) and DEPT spectra of 1, seven carbon signals were detected. These signals resulted from a methoxy (δC 63.3), four aromatic methines [δC 115.4 (2C) and 132.8 (2C)], two aromatic carbons (δC 128.8 and 160.7), an olefinic methine (δC 147.0), and a carbonyl carbon (δC 186.4). In the COSY spectrum of 1 (Figure 1), correlations between H-2/H6 (δH 8.07) and H-3/H-5 (δH 6.90) were found. This proton sequence and the HMBC correlations (Figure 1) from H-3/H5 to C-4 (δC 160.7) indicated a hydroxy group attached at C-4. In addition, the HMBC cross-peaks from H-2/H-6 and H-8 (δH 7.95) to C-7 (δC 186.4) were used to connect C-1/C-7/C8. In the HMBC spectrum, the methoxy protons did not correlate to any carbon, which implied this methoxy group was attached to a nitrogen atom. These NMR data suggested that 1 had a 7-oxo-7-(4-hydroxyphenyl)acetaldehyde oxime framework, which is similar to the synthetic compound, phenylB

DOI: 10.1021/acs.jnatprod.7b00938 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products Table 2.

13

Article

C NMR Spectroscopic Data of Compounds 1−5a

no. 1 2 3 4 4a 5 6 7 8 8a 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 7-OMe 5′-OMe N-OMe

1

2

3

4

5

128.8, 132.8, 115.4, 160.7,

C CH CH C

130.5, 132.5, 115.8, 162.5,

C CH CH C

130.4, 132.6, 114.3, 162.5,

C CH CH C

130.2, 131.7, 114.2, 161.9,

115.4, 132.8, 186.4, 147.0,

CH CH C CH

115.8, 132.5, 186.4, 147.2,

CH CH C CH

114.3, 132.6, 186.3, 147.2,

CH CH C CH

114.2, CH 131.7, CH 166.7, C

141.7, CH 114.7, CH 38.5, CH 17.9, CH3 175.8, C

64.9, CH2 135.7, CH 129.2, C 13.1, CH3 167.5, C

64.9, CH2 135.8, CH 123.1, C 13.1, CH3 167.6, C

52.1, CH3 63.4, CH3

52.1, CH3 63.3, CH3

63.3, CH3

C CH CH C

162.0, C 113.0, CH 143.5, CH 112.5, C 128.7, CH 113.3, CH 161.3, C 101.5, CH 155.8, C 65.4, CH2 119.6, CH 140.7, C 42.3, CH2 128.4, CH 135.8, CH 82.1, C 24.3, CH3 24.3, CH3 16.8, CH3

51.9, CH3 52.1, CH3

a

Data were measured in CDCl3 at 100 MHz.

Figure 1. COSY (bold bond) and selected HMBC (arrow) correlations of 1−3.

Figure 2. Perspective drawing of the X-ray structure of 1.

spectrum. The IR and MS data of 3 were similar to those of 2, suggesting that they were structural congeners. The 1H and 13 C NMR spectra of 3 were similar to those of 2, apart from the absence of two methines (δH 6.62; δC 141.7 and δH 3.22; δC 38.5) in 2 and the presence of an oxymethylene (δH 4.79; δC 64.9) in 3. These NMR differences suggested that the Δ1′(2′) double bond in 2 became a Δ2′(3′) double bond in 3. This was supported by the COSY correlation of H2-1′ (δH 4.79)/H-2′ (δH 6.91) and the HMBC correlation of Me-4′ (δH 1.95)/C-2′ (δC 135.7), C-3′ (δC 129.2), and C-5′ (δC 167.5) (Figure 1). The presence of the NOESY correlation of Me-4′/

enantiomers (Figure 3). The specific rotation of the (3′S) and (3′R) enantiomers was also calculated with Gaussian 9.0 software. The calculations were done by the gauge-invariant atomic orbitals (GIAOs) method using the 6311++G(2df, 2pd) basis set. The calculated specific rotations of (3′S)-2 and (3′R)-2 were −183 and +183, respectively. The experimental specific rotation of 2 was −467, consistent with the negative value of (3′S)-2, and thus confirmed the (3′S) configuration of 2. Buxifoxime C (3) was obtained as a yellow amorphous powder. The molecular formula, C15H17NO5, was deduced by the sodium adduct ion at m/z 314.1000 in the HRESIMS C

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correlations from both Me-8′ (δH 1.34) and Me-9′ (δH 1.34) to C-6′ (δC 135.8) and C-7′ (δC 82.1) were used to establish the connection between C-6′ and C-7′. Finally, the HMBC correlation from H-1′ to C-7 revealed a geranyl side chain attached to the coumarin moiety through an ether bond. The hydroperoxy group attaching at C-7′ was inferred from the deshielded carbon chemical shift (δC 82.1) and the molecular formula of 5. This assignment was supported by comparison of the NMR data with those of peroxyschinilenol (Figure S34, Supporting Information) isolated from Zanthoxylum schinifolium.21 Therefore, structure 5 was assigned as 7-O-(7′peroxygeranyl)coumarin. The cytotoxicities of compounds 1−30 against human liver cancer (HepG2), human breast cancer (MDA-MB-231), and human lung cancer (A549) cells were screened and are collated in Table 3. The results showed that all new

Figure 3. Calculated and experimental CD spectra of 2.

H2-1′ and the absence of the NOESY correlation of Me-4′/H2′ proved the E geometry of the Δ2′(3′) double bond. Consequently, the structure of buxifoxime C (3) was determined as shown. The HRESIMS data provided a molecular formula of C14H16O5, for buxifobenzoate (4). Inspection of the IR revealed the presence of C−H (2923 cm−1), CO (1709 cm−1), and CC (1613 cm−1) functionalities. The 1H NMR data of 4 showed the presence of a methyl at δH 1.95 (Me-4′), a methoxy at δH 3.77 (5′-OMe), an olefinic methine at δH 6.90 (H-2′), an oxymethylene at δH 4.77 (H2-1′), and a paradisubstituted benzene moiety at δH 8.12 (H-2/H-6) and δH 6.94 (H-3/H-5). The data were highly similar to those of 3 (Table 1), suggesting compounds 4 and 3 had similar structures. The HMBC cross-peaks from H-2/H-6 and 7OMe (δH 3.89) to C-7 (δC 166.7) indicated the presence of a methoxycarbonyl group at C-1. Hence, the two substituents of the para-disubstituted benzene moiety were defined, and the structure of buxifobenzoate C (4) was assigned as shown. 7-O-(7′-Peroxygeranyl)coumarin (5) was isolated as a pale yellow oil. The molecular formula of 5, C19H22O5, was deduced by the sodium adduct ion at m/z 353.1359 [M + Na]+. The IR absorptions at 1613, 1724, and 3385 cm−1 revealed the presence of CC, CO, and OH functional groups, respectively. The UV absorption bands at 322 nm31 and the aromatic proton signals at δH 6.25 (d, J = 9.5 Hz; H-3), 7.63 (d, J = 9.5 Hz; H-4), 7.36 (d, J = 8.5 Hz; H-5), 6.84 (dd, J = 8.5 and 2.4 Hz; H-6), and 6.81 (d, J = 2.4 Hz; H-8)32 suggested that compound 5 was a 7-O-substituted coumarin. The coumarin skeleton was confirmed by the HMBC crosspeaks (Figure 4) of H-3/C-2 (δC 162.0); H-4/C-4a (δC

Table 3. Cytotoxicity (IC50, μM) against Human Liver Cancer (HepG2), Human Breast Cancer (MDA-MB-231), and Human Lung Cancer (A549) Cellsa

a

compound

HepG2

MDA-MB-231

A549

19 20 doxorubicinb

7.3 ± 0.0 6.5 ± 0.0 0.3 ± 0.0

NA NA 1.2 ± 0.0

7.8 ± 0.0 7.8 ± 0.1 0.6 ± 0.1

Inactive compounds are omitted. bPositive control.

compounds were not cytotoxic (IC50 > 20 μg/mL), and most of the acridone alkaloids were cytotoxic. Atalaphyllinine (20) was found to be the most active compound, exhibiting IC50 values less than 10 μM toward HepG2 and A549 cell lines. The compounds were also assayed for their antiinflammatory effects by inhibiting superoxide anion generation and elastase release from human neutrophils in response to fMLF (Table 4). Imperatorin (13) showed an inhibiting effect Table 4. Effects of Active Compounds on Superoxide Anion Generation and Elastase Release in fMLF/CB-Induced Human Neutrophilsa superoxide anion

elastase release

compound

IC50 (μM)

IC50 (μM)

1 13 15 16 23 LY294002b

4.8 ± 0.2 ± 2.6 ± 4.7 ± >10 2.1 ±

>10 >10 >10 >10 3.7 ± 1.6 5.1 ± 1.1

0.7 0.0 0.9 1.1 0.4

Results are expressed as means ± SEM (n = 3 or 4). No replication. b Positive control. a

Figure 4. COSY (bold bond) and selected HMBC (arrow) correlations of 5.

(IC50 = 0.2 ± 0.0 μM) against superoxide anion generation. 5Hydroxy-N-methylseverifoline (23) showed an anti-inflammatory effect (IC50 = 3.7 ± 1.6 μM) by downregulating elastase release. Notably, the two compounds were more effective than the positive control LY294002. In the preliminary screening, the extract of A. buxifolia showed antiviral activity against hepatitis C virus (HCV). The isolated compounds were therefore evaluated for antidengue virus (DENV), which belongs to the same family as HCV. In the antiviral assay against DENV-2, compound 23 showed activity with an EC50 value of 5.3 ± 0.4 μM, and the selectivity index (CC50/EC50) of compound 23 was 6.3.

112.5), C-8a (δC 155.8); H-5/C-4a, C-8a; H-6/C-7 (δC 161.3); H-8/C-7, C-8a. Three methyl groups (δH 1.34, 1.34, and 1.75), an aliphatic methylene (δH 2.86), an oxymethylene (δH 4.61), and three olefinic methines (δH 5.49, 5.66, and 5.63) were also observed in the 1H NMR spectrum, which implied the presence of a geranyl side chain. Two proton coupling systems, H-1′ (δH 4.61)/H-2′ (δH 5.49) and H-4′ (δH 2.86)/H-5′ (δH 5.66)/H-6′ (δH 5.63), were evidenced by the COSY spectrum. These two portions were linked by the HMBC correlations from Me-8′ (δH 1.75) to C-2′ (δC 119.6), C-3′ (δC 140.7), and C-4′ (δC 42.3). In addition, HMBC D

DOI: 10.1021/acs.jnatprod.7b00938 J. Nat. Prod. XXXX, XXX, XXX−XXX

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In conclusion, five new and 25 known compounds were identified. Among the isolates, oximes 1−3 and 6 were relatively minor constituents. The formation of these rare compounds may be involved in plant defense or serve as intermediates in nitrate reduction.33 Immersion of citaldoxime (6) in MeOH for 10 days showed that its hydroxy group was retained, confirming that the new compounds 1−3 with Omethyloxime functionality are naturally occurring metabolites. On the other hand, acridone alkaloids not only represented the major components of A. buxifolia but also showed activity in bioassays. The findings suggested that acridone alkaloids are potential agents for anti-inflammatory and anti-DENV drug development.

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10−4A−10−4C. Fraction 10−4A (65.6 mg) was separated by NPHPLC (Si, n-hexane/EtOAc = 8:1, isocratic) to produce 29 (13.8 mg). Fraction 10−4B (1.2 g) was separated by MeOH in a Sephadex LH-20 column to afford 24 (85.6 mg) and 18 (0.7 mg). Fraction 10− 4C (3.7 g) was separated by a Sephadex LH-20 column eluted with MeOH to give subfractions 10−4C1 to 10−4C6. Subfraction 10− 4C3 (35.5 mg) was subjected to NP-HPLC (Si, n-hexane/EtOAc = 12:1, isocratic) to yield 13 (2.2 mg), 14 (14.3 mg), 17 (2.4 mg), and 30 (7.0 mg). Buxifoxime A (1): Yellow cubic crystals; mp 119−120 °C; UV (MeOH) λmax (log ε) 229 (3.1), 309 (3.3) nm; IR (neat) vmax 3290,1684, 1593, 1513, 1445, 1335, 1249, 1073, 1027, 927, 845, 613 cm−1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS m/z 202.0476 (calcd for C9H9NO3Na, 202.0475). Buxifoxime B (2): Yellow amorphous powder; [α]D20 −467 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 209 (3.3), 304 (3.0) nm; CD (MeOH) λmax (Δ ε) 220 (2.79), 239 (1.80), 307 (−2.82), 308 (−2.85) nm; IR (neat) vmax 2924, 2855, 2358, 1855, 1730, 1684, 1597, 1245, 1028, 930, 693 cm−1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS m/z 314.1001 (calcd for C15H17NO5Na, 314.0999). Buxifoxime C (3): Yellow amorphous powder; UV (MeOH) λmax (log ε) 216 (3.5), 303 (3.6) nm; IR (neat) vmax 2924, 1722, 1684, 1600, 1442, 1260, 927, 836, 722 cm−1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS m/z 314.1000 (calcd for C15H17NO5Na, 314.0999). Buxifobenzoate (4): White amorphous powder; UV (MeOH) λmax (log ε) 208 (3.2), 256 (3.1) nm; IR (neat) vmax 2923, 1709, 1613, 1111, 965, 771 cm−1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS m/z 287.0891 (calcd for C14H16O5Na, 287.0890). 7-O-(7′-Peroxygeranyl)coumarin (5): White amorphous powder; UV (MeOH) λmax (log ε) 211 (3.1), 322 (3.0) nm; IR (neat) vmax 3385, 2925, 1724, 1614, 1395, 1281, 1232, 1131, 995, 839 cm−1; for 1 H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS m/z 353.1358 (calcd for C19H22O5Na, 353.1359). X-ray Crystallographic Analysis. Colorless crystals of 1 were obtained in a mixture of MeOH and H2O. Crystal data were collected at 200 K on a Bruker APEX DUO single-crystal diffractometer with a montel-mirror microfocus Cu Kα radiation (wavelength = 1.54178 Å). The structures were solved by direct methods and refined using SHELXS-97. Crystal data of 1: orthorhombic crystal (0.20 × 0.15 × 0.10 mm); space group Pbca; unit cell dimensions a = 8.7826(2) Å, b = 13.1341(4) Å, c = 16.1714(3) Å, V = 1865.40(8) Å3; Z = 8; d = 1.276 Mg/m3; 6934 independent reflections were measured and 1703 independent reflections were observed [R(int) = 0.0312]. Completeness to θ = 67.99°: 100.0%. Absorption correction: semiempirical from equivalents. Max and min transmission: 1.00000 and 0.86706. The structure was solved by direct methods and refined by a fullmatrix least-squares on F2. Final R indices [I > 2σ(I)]: R1 = 0.0447, wR2 = 0.1409. The crystallographic data of compound 1 were deposited at the Cambridge Crystallographic Data Centre and the deposition number, CCDC 1584461, was given. Cytotoxic Assay. All tested cell lines were obtained from the Food Industry Research and Development Institute (FIRDI, Hsinchu, Taiwan). Cytotoxicities of the components were evaluated using the MTT assay. Doxorubicin was selected as the positive control. Absorbance at 550 nm was obtained by an ELISA reader (Thermo Fisher Corporation, Waltham, MA, USA). Anti-inflammatory Assay. In the superoxide anion generation assay, neutrophils were equilibrated in ferricytochrome c at the concentration of 0.6 mg/mL. The other methods for superoxide anion generation and elastase release were the same as those published by Hwang.33 Anti-DENV Assay. The preparation details of DENV infected Huh-7 cells, evaluation of anti-DENV RdRp and protease activity, and statistical analysis protocols were the same as that in a previously published paper.34

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotation data were recorded on a Jasco P-2000 polarimeter. Ultraviolet spectra were recorded on a JASCO V-530 UV/vis spectrophotometer. IR spectra were measured by a PerkinElmer system 2000 FT-IR infrared spectrophotometer. NMR spectroscopic data were acquired with a JEOL ECS 400 MHz spectrometer and performed at room temperature, using CDCl3 as solvent. Chemical shifts were calibrated by the solvent peaks for CDCl3 (δH 7.26 and δC 77.0 ppm). HRESIMS data were recorded on a Bruker Daltonics APEX II α30e mass spectrometer. Single-crystal X-ray diffraction data were analyzed by a Bruker D8 VENTURE single-crystal XRD equipped with Oxford Cryostream 800+. Sephadex LH-20 (GE, Uppsala, Sweden) and Silica 60 (Merck, Darmstadt, Germany) gels were used for column chromatography. Luna CN, phenylhexyl, and silica semipreparative columns (Phenomenex, Torrance, CA, USA) were equipped for HPLC. The HPLC equipment comprised a Shimazu LC-20AT pump, a CBM-20A communication bus module, and an SPD-M20A diode array detector. Plant Material. The plant material of A. buxifolia was collected in the mountain area of Kaohsiung city, Taiwan, in August 2014. The specimen was identified by the corresponding author Dr. Y.-B. Cheng. A voucher specimen (code no. KMU-AB) was deposited in Dr. Celica Koo Botanic Conservation Center. Extraction and Isolation. The shade-dried twigs of A. buxifolia (17.0 kg) were powdered and extracted with MeOH (20 L) to give a crude extract. This crude extract was partitioned between water and EtOAc to obtain two portions. The EtOAc portion was further partitioned with hexanes and 75% MeOH(aq). The 75% MeOH layer (143.3 g) was separated by a Si gel open column stepwise eluting with hexanes/EtOAc/MeOH to furnish 15 fractions and 28 (113.3 mg). Fraction 6 (1.6 g) was subjected to a Sephadex LH-20 column eluting with MeOH to yield subfractions 6−1−6−3. Subfraction 6−3 (366.1 mg) was separated by a Si gel column to give 9 (169.1 mg), fractions 6−3A (46.1 mg), and 6−3B (30.0 mg). Fraction 6−3A was separated by RP-HPLC [phenyl-hexyl, 77% MeOH(aq), isocratic] to afford 3 (1.6 mg) and 5 (2.0 mg). Fraction 6−3B (2.1 g) was also separated by RP-HPLC [CN, 28% MeCN(aq), isocratic] to yield 1 (4.4 mg), 10 (8.0 mg), 11 (1.3 mg), and 12 (1.1 mg). Fraction 7 (3.2 g) was subjected to a Sephadex LH-20 column eluted with MeOH to produce subfractions 7−1−7−7. Subfraction 7−3 (658.4 mg) was further subjected to a Si gel open column to give 22 (244.7 mg) and fractions 7−3A to 7−3G. Fraction 7−3B (53.2 mg) was separated by NP-HPLC (Si, n-hexane/EtOAc = 18:1, isocratic) to afford 2 (7.3 mg), 15 (11.8 mg), 16 (20.4 mg), 21 (12.3 mg), 26 (1.0 mg), and 27 (1.1 mg). Fraction 7−3D (52.6 mg) was chromatographed using HPLC (Si, n-hexane/EtOAc = 3:1, isocratic) to give 6 (6.8 mg), 23 (17.7 mg), and 25 (12.1 mg). Fraction 7−3E (137.5 mg) was separated by NP-HPLC (Si, n-hexane/EtOAc = 8:1, isocratic) to produce 4 (27.5 mg), 7 (10.1 mg), and 8 (4.0 mg). Fraction 10 (38.1 g) was subjected to a Si gel open column stepwise eluting with CH2Cl2/acetone to yield subfractions 10−1−10−6. Subfraction 10−4 (7.5 g) was purified by another Si gel column stepwise eluting with nhexane/EtOAc to give 19 (89.9 mg), 20 (189.6 mg), and fractions E

DOI: 10.1021/acs.jnatprod.7b00938 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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(23) Znati, M.; Ben Jannet, H.; Cazaux, S. Molecules 2014, 19, 16959−16975. (24) Schmidt, B.; Wolf, F. J. Org. Chem. 2017, 82, 4386−4395. (25) Boisvert, L.; Brassard, P. J. Org. Chem. 1988, 53, 4052−4059. (26) Wu, T.-S. Phytochemistry 1987, 26, 871−872. (27) Trinh, T. T.; Tran, V. S.; Katrin, F. J. Chem. 2008, 46, 515− 520. Kumar, V.; Vallipuram, K.; Adebajo, A. C. Phytochemistry 1995, 40, 1563−1565. (28) Nakanishi, K. Infrared Absorption Spectroscopy; Holden-Day Inc.: San Francisco, CA, 1962. (29) Kreutz, O. C.; Segura, C. M.; Rodrigues, J. A. R.; Moran, P. J. S. Tetrahedron: Asymmetry 2000, 11, 2107−2115. (30) Vitório, F.; Pereira, T. M.; Castro, R. N.; Guedes, G. P.; Graebin, C. S.; Kümmerle, A. E. New J. Chem. 2015, 39, 2323−2332. (31) Gadakh, S. K.; Dey, S.; Sudalai, A. J. Org. Chem. 2015, 80, 11544−11550. (32) Ashani, Y.; Silman, I. Hydroxylamines and Oximes: Biological Properties and Potential Uses as Therapeutic Agents, PATAI’s Chemistry of Functional Groups; John Wiley & Sons, 2010. (33) Yu, H.-P.; Hsieh, P.-W.; Chang, Y.-J.; Chung, P.-J.; Kuo, L.-M.; Hwang, T.-L. Free Radical Biol. Med. 2011, 50, 1737−1748. (34) Lee, J.-C.; Chang, F.-R.; Chen, S.-R.; Wu, Y.-H.; Hu, H.-C.; Wu, Y.-C.; Backlund, A.; Cheng, Y.-B. Mar. Drugs 2016, 14, 151.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00938. HRESIMS, 1 H, 13 C, and 2D NMR spectra of compounds 1−5 (PDF) X-ray data (CIF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +886-7-3121101, ext. 2197. Fax: +886-7-3114773. Email: [email protected]. ORCID

Yuan-Bin Cheng: 0000-0001-6581-1320 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This phytochemical investigation was sponsored by Ministry of Science and Technology, Taiwan (MOST106-2320-B-037-016 and MOST 106-2320-B-037-008-MY2). This research was also supported by a grant from Kaohsiung Medical University Research Foundation (105 KMUOR02).

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REFERENCES

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DOI: 10.1021/acs.jnatprod.7b00938 J. Nat. Prod. XXXX, XXX, XXX−XXX