Polygonumosides A–D, Stilbene Derivatives from Processed Roots of

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Polygonumosides A−D, Stilbene Derivatives from Processed Roots of Polygonum multif lorum Shi-Lun Yan,† Yan-Fang Su,*,†,§ Lei Chen,‡ Meng Que,† Xiu-Mei Gao,§ and Jun-Biao Chang⊥ †

Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, School of Pharmaceutical Science & Technology, Tianjin University, Tianjin 300072, People’s Republic of China ‡ Tianjin Tasly Modern TCM Resources Co., Ltd, Tianjin 300410, People’s Republic of China § Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, People’s Republic of China ⊥ College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450052, People’s Republic of China S Supporting Information *

ABSTRACT: Four new stilbene derivatives, polygonumosides A−D (1−4), were isolated from the processed roots of Polygonum multif lorum. Their structures were elucidated by spectroscopic analysis, including 1D and 2D NMR and ECD. Polygonumosides A (1) and B (2), possessing an unprecedented tetracyclic skeleton, were assigned as 2S- and 2R-2-(4hydroxyphenyl)-9,10,11-trihydroxy-2H-benzo[c]furo[2,3-f ]chromen-7(3H)-one-4-O-β-D-glucopyranosides, respectively, while polygonumosides C (3) and D (4) were assigned as a pair of diastereomeric stilbene glucoside dimers.

NMR spectra of 1 showed resonances at δH 7.36 (2H, d, J = 8.5 Hz) and 6.74 (2H, d, J = 8.5 Hz) for a 4-substituted phenyl group, resonances at δH 5.29 (1H, d, J = 9.0 Hz), 4.09 (1H, br d, J = 18.0 Hz), and 3.53 (1H, dd, J = 18.0, 9.0 Hz) for an oxygenated 1,2-ethylidene group, a resonance at δC 160.2 indicating a carbonyl group, and resonances at δH 4.65 (1H, d, J = 7.5 Hz) and δC 105.5, 74.0, 76.1, 69.7, 77.6, and 61.0 suggesting a β-glucopyranosyl (Glc) residue (Table 1).3 The Dconfiguration of the glucosyl moiety was determined by GC analysis after acidic hydrolysis and chiral derivatization.12 The other resonances at δH 6.78 (1H, s) and 7.46 (1H, s) and 12 carbon resonances between δC 109.0 and 149.3 (Table 1) were assigned to two pentasubstituted phenyl groups. The connectivity of the partial structures was established by combined analysis of 1D and 2D (1H, 13C, HSQC, and HMBC) NMR data. The NMR data of 1 suggested a substituted dibenzo[b,d]pyrone group,4 which was confirmed by HMBC correlations of H-6/C-4, C-5, C-7, and C-15 and H11/C-9, C-12, C-13, C-15, and C-16. The HMBC correlations from H-2 to C-17 and from H-3 to C-4 and C-5 confirmed a dihydrobenzofuran substructure.5 The C−H long-range correlations between H-2 and C-2′ (6′) and between H-3 and C-1′ and the relative deshielding of C-2 (δ 79.8) suggested that the 4-hydroxyphenyl group was connected to C-2. The long-range correlation between Glc-1 and C-5 indicated that

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n China, the dried roots of Polygonum multif lorum Thunb. (Polygonaceae) have long been used as detoxifying and purgative agents, while the processed roots have been used in the preparation of tonics and as nutritional supplements.1 Compounds isolated from the roots of P. multif lorum include anthraquinones, stilbenoids, and flavonoids to name but a few.2 We conducted a chemical study on the processed roots of P. multif lorum, and herein we report the isolation and structure elucidation of four new stilbene derivatives, polygonumosides A−D (1−4).

Polygonumoside A (1) was purified as a cream-colored powder and exhibited a pseudomolecular ion [M − H]− peak in the negative HRESIMS at m/z 555.1139, consistent with the molecular formula C27H24O13, which was supported by the 13C NMR data. The IR spectrum of 1 showed strong absorptions at 3346, 1703, and 1611 cm−1 assignable to hydroxy, carbonyl, and aromatic functionalities, respectively. The 1H and 13C © XXXX American Chemical Society and American Society of Pharmacognosy

Received: September 23, 2013

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The absolute configurations of 1 and 2 were determined by quantum chemical calculation of their ECD spectra using Gaussian 09 software.6 Considering the rigid structure of the aglycone,7 the 2R diastereoisomer was geometrically optimized using DFT at the B3LYP/6-31+G(d) level in DMSO to afford a preferred conformer. The ECD spectrum was calculated using the TDDFT method at the B3LYP-SCRF/6-31+G(d) level in DMSO. The calculated ECD curve showed good agreement with the experimental ECD curve of 2 and exhibited a Cotton effect opposite to that of 1 (Figure 2). Thus the absolute configurations of C-2 in 1 and 2 were assigned as S and R, respectively.

Table 1. NMR Data for Polygonumosides A (1) and B (2) in DMSO-d6 (δ in ppm, J in Hz) 1a no. 2 3

δC, type 79.8, CH 38.7, CH2

2b δH

5.29, d (9.0) 4.09, d (18.0)

δC, type 79.9, CH 40.0, CH2

3.53, dd (18.0, 9.0) 4 5 6 7 9 10 11 12 13 14 15 16 17 1′ 2′/6′ 3′/5′ 4′ Glc-1 2 3 4 5 6

109.0, 140.4, 103.4, 147.6, 160.2, 131.4, 110.4, 145.6, 143.5, 149.3, 118.7, 110.7, 143.2, 131.3, 127.4, 114.8, 156.6, 105.5, 74.0, 76.1, 69.7, 77.6, 61.0,

C C CH C C C C C C C C C C C CH CH C CH CH CH CH CH CH2

6.78, s

7.46, s

7.36, d (8.5) 6.74, d (8.5) 4.65, d (7.5) 3.33c 3.27c 3.15c 3.19c 3.66, br d (10.5) 3.45, dd (11.5, 5.0)

108.7, 140.7, 103.2, 147.7, 160.1, 132.0, 110.4, 145.5, 143.5, 149.3, 118.3, 110.5, 143.3, 131.2, 128.1, 114.9, 156.9, 106.2, 73.9, 76.1, 69.7, 77.5, 61.0,

C C CH C C C CH C C C C C C C CH CH C CH CH CH CH CH CH2

δH 5.14, d (9.6) 3.98, dd (18.6, 9.6) 3.74, d (18.6)

6.80, s

7.46, s

7.35, d (8.4) 6.74, d (8.4)

Figure 2. Comparison of the experimental and calculated ECD spectra of 1 and 2.

4.61, d (7.8) 3.33c 3.26c 3.16c 3.24c 3.70, br d (11.2) 3.47, dd (15.4, 6.0)

Polygonumoside C (3) was isolated as a brown, amorphous solid. The molecular formula of C40H44O19 was assigned by the pseudomolecular [M − H]− peak at m/z 827.2408, in combination with 13C NMR spectroscopic analysis. The IR spectrum showed absorption bands for hydroxy groups and aromatic rings. The 1H NMR spectrum displayed resonances assignable to two 4-substituted phenyl groups via AA′XX′ spin systems [δH 7.22 (2H, d, J = 8.5 Hz), 6.75 (2H, d, J = 8.5 Hz); 7.24 (2H, d, J = 8.5 Hz), 6.69 (2H, d, J = 8.5 Hz)], a 1,2,3,5tetrasubstituted phenyl group [δH 6.20 (1H, d, J = 3.0 Hz), 6.15 (1H, d, J = 3.0 Hz)], an E double bond [δH 6.58 (1H, d, J = 16.5 Hz), 6.00 (1H, d, J = 16.5 Hz)], and two aliphatic protons [δH 6.44 (1H, d, J = 3.5 Hz), 4.96 (1H, d, J = 3.5 Hz)]. An aromatic proton singlet at δH 6.32 indicated the presence of a pentasubstituted phenyl group. The 1H and 13C NMR spectra also showed resonances at δH 4.44 (1H, d, J = 7.5 Hz), 4.36 (1H, d, J = 7.5 Hz) and δC 105.5, 75.7, 78.1, 71.5, 79.0, 63.0, 108.2, 75.6, 78.1, 71.0, 78.3, and 62.4 indicative of two βglucopyranosyl residues (Table 2). The D-configuration of the sugar moieties was determined using the same method as for 1. Following the complete assignment of 1H and 13C NMR resonances of the partial structures by NMR analysis (DEPT, HSQC, 1H−1H COSY, and HMBC), their connectivities were completed by further HMBC analysis (Figure 3). The correlations of H-7a/C-2a(6a) and C-9a, H-8a/C-10a and C14a, H-7b/C-2b(6b), and H-8b/C-10b and C-14b indicated that 3 contained two stilbene units, and the correlations between H-8b and C-14a and between H-7b and C-13a and C9a suggested that the two stilbene units were connected through a C-7b−C-14a bond. The two β-glucopyranosyl residues were located by HMBC correlations and placed at H-Glca-1 to C-10a and H-Glcb-1 to C-10b. Thus, polygonumoside C (3) was assigned as 5-[2-hydroxy-1-(4-hydroxyphenyl)2-(3,5-dihydroxy-2-β- D -glucopyranosyloxyphenyl)ethyl]-6-

a

Recorded at 500 MHz (1H) and 125 MHz (13C). bRecorded at 600 MHz (1H) and 150 MHz (13C). cOverlapped. All assignments were based on HSQC and HMBC experiments.

the sugar moiety was linked to C-5. Thus the structure of polygonumoside A (1) was established as 2-(4-hydroxy)phenyl-9,10,11-trihydroxy-2H-benzo[c]furo[2,3-f ]chromen7(3H)-one-4-O-β-D-glucopyranoside, leaving only the absolute configuration of C-2 to be determined.

Figure 1. Structure and selected HMBC correlations of 1.

Polygonumoside B (2) was purified as a cream-colored powder and exhibited a pseudomolecular [M − H]− at m/z 555.1139, combined with 13C NMR data indicating the same molecular formula as 1. Comparison of the NMR data of 2 with those of 1 indicated that compounds 1 and 2 were a pair of C-2 diastereoisomers. B

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Table 2. NMR Data for Polygonumosides C (3) and D (4) in Methanol-d4 (δ in ppm, J in Hz)a 3 no. 1a 2a/6a 3a/5a 4a 7a 8a 9a 10a 11a 12a 13a 14a Glca-1 2 3 4 5 6 a

δC, type 131.1, 129.2, 116.7, 158.2, 135.3, 123.6, 137.5, 138.3, 150.1, 105.4, 155.3, 118.9, 108.3, 75.7, 78.1, 71.5, 79.0, 63.0,

C CH CH C CH CH C C C CH C C CH CH CH CH CH CH2

4 δH

7.22, d (8.5) 6.75, d (8.5) 6.00, d (16.5) 6.58, d (16.5)

6.32, s

4.36, d (7.5) 3.34b 3.32b 3.27b 3.20b 3.95, dd (11.0, 1.0) 3.73, dd (12.0, 6.0)

δC, type 130.2, 129.2, 116.6, 158.2, 135.0, 123.2, 137.6, 137.5, 150.4, 105.7, 154.9, 119.7, 107.3, 75.2, 77.6, 70.9, 78.1, 62.2,

C CH CH C CH CH C C C CH C C CH CH CH CH CH CH2

3 δH

no.

7.09, d (8.5) 6.71, d (8.5) 5.60, d (16.5) 6.40, d (16.5)

6.31, s

4.44, d (7.5) 3.37b 3.31b 3.39b 3.26b 3.77, dd (11.5, 1.5) 3.68, dd (11.5, 4.5)

1b 2b/6b 3b/5b 4b 7b 8b 9b 10b 11b 12b 13b 14b Glcb-1 2 3 4 5 6

δC, type 135.0, 130.6, 116.0, 156.3, 50.5, 72.3, 140.1, 137.2, 151.2, 103.4, 155.7, 106.3, 108.2, 75.6, 78.3, 71.0, 79.0, 62.4,

C CH CH C CH CH C C C CH C CH CH CH CH CH CH CH2

4 δH 7.24, d (8.5) 6.69, d (8.5) 4.96, d (3.5) 6.44, d (3.5)

6.20, d (3.0) 6.15, d (3.0) 4.44, d (7.5) 3.35b 3.32b 3.43b 3.18, m 3.68, d (3.0)

δC, type 134.8, 129.6, 115.9, 156.3, 49.9, 69.2, 139.2, 136.9, 151.3, 103.5, 155.5, 107.1, 106.8, 75.4, 77.6, 70.2, 76.9, 61.6,

C CH CH C CH CH C C C CH C CH CH CH CH CH CH CH2

δH 7.19, d (8.5) 6.70, d (8.5) 4.70, br s 6.29, d (2.0)

6.32, d (3.0) 5.98, d (3.0) 4.14, d (7.5) 3.25b 2.82, dd (9.0, 9.0) 3.23b 2.32, br d (9.5) 3.19, dd (12.0, 2.5) 2.90, dd (12.0, 2.5)

Recorded at 500 MHz (1H) and 125 MHz (13C). bOverlapped. All assignments were based on DEPT, HSQC, COSY, and HMBC experiments.

Figure 4. Newman projection of 4 showing selected NOE correlations. Figure 3. Selected COSY and HMBC correlations of 3.

2b/H-Glcb-6b was also observed. This NOE association was possible only if the configurations of 7b and 8b were S and R, respectively. Thus, the absolute configurations of 4 and 3 were assigned as (7bS, 8bR) and (7bR, 8bS), respectively. Polygonumosides A (1) and B (2) are the first stilbenes possessing 2H-benzo[c]furo[2,3-f ]chromen-7(3H)-one skeletons. Polygonumosides C (3) and D (4), a pair of diastereomeric dimeric stilbene glucosides, are the first dimeric stilbenes to be reported from P. multif lorum. A comparison of extracted ion chromatograms of processed and unprocessed roots indicated that the contents of polygonumosides A−D (1−4) increased significantly during the processing. Since the processed roots of P. multif lorum are used as tonics and nutritional supplements, especially antiaging in Traditional Chinese Medicine,1,11 and antioxidants are regarded as promising agents against aging,10 antioxidant activity was measured for the new compounds. Compounds 1 and 2 displayed significant scavenging of DPPH free radicals (IC50 298 and 569 μM respectively), while compounds 3 and 4 showed weak activity (IC50 > 2000 μM). The positive control was ascorbic acid (IC50 573 μM).

[(E)-4-hydroxystyryl]benzene-2,4-diol-1-O-β-D-glucopyranoside. The coupling constant (3.0 Hz) between H-7b and H-8b suggested an erythro arrangement.8 Polygonumoside D (4) was isolated as brown, amorphous solid. The ESIMS spectrum of 4 showed a pseudomolecular [M − H]− at m/z 827.2403, calculated for a formula of C40H44O19, the same as that of 3, which is co-established by 13C NMR data. Analysis of 1D and 2D NMR data indicated that 4 had the same gross structure as 3. The small coupling constant (3.0 Hz) between H-7b and H-8b suggested their erythro arrangement. The absolute configuration of 4 was determined by analysis of its NOESY spectrum, together with consideration of anisotropy (Figure 4).9 The NOE interactions of H-7b with H-8b and H-8a, H-8b with H-Glcb-1, and H-2a with H-Glcb-1 suggested ring A1, H-7b, H-8b, and Glcb were cofacial. The NOE interactions of H-2a with H-Glcb-1 and H-Glcb-5 and the relative shielding of protons of Glcb-1 (δH 4.14), Glcb-3 (2.82), and Glcb-5 (2.32) indicated that Glcb was close to ring A1 and that the cofacial protons Glcb-1, Glcb-3, and Glcb-5 were located in the anisotropic region of ring A1. An NOE association for HC

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Polygonumoside D (4): brown, amorphous solid; [α]25D −0.5 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 280 (4.72); IR (KBr) νmax 3422, 1611; 1H and 13C NMR, see Table 2; HRESIMS m/z 827.2403 (calcd for [M − H]− C40H43O19, 827.2399). Determination of the Absolute Configuration of Glucosyl Moieties (ref 12). A solution of each compound (3.0 mg) in 1 M HCl (H2O/ethylene oxide, 1:1, 2 mL) was refluxed at 90 °C for 2 h. The solution was evaporated, and the residue was partitioned between H2O and EtOAc. The H2O layer was concentrated to dryness and mixed with L-cysteine methyl ester hydrochloride (2 mg) in pyridine (1 mL), followed by refluxing at 60 °C for 2 h. Then trimethylsilylimidazole (0.2 mL) was added, and the mixture was refluxed at 60 °C for another 2 h. After drying the solution, the residue was partitioned between H2O (2 mL) and cyclohexane (2 mL) three times. The cyclohexane layer was concentrated to 1 mL and subjected to GC analysis (column: HP-5, 30 m × 0.32 mm × 0.25 μm; detector: FID; detector temperature: 280 °C; injected temperature: 250 °C; column temperature: 160 °C, 4 °C/min, 200 °C, 10 °C/min, 240 °C, held for 15 min; carrier gas: N2). The hydrolysates of 1−4 gave peaks at 27.6 min. The derivatives of authentic D- and L-glucose gave peaks at 27.6 and 28.5 min, respectively. DPPH Scavenging Assay. Compounds 1 and 2 were tested for their antioxidant activity using the DPPH free radical scavenging method.13 A solution of DPPH in EtOH (120 μM, 2.9 mL) was mixed with different concentrations of each test compound (200−1000 μM, 0.1 mL). After 30 min of incubation at 37 °C, the absorbance of the reaction mixture at 517 nm was measured. The reaction solution without DPPH was used as a blank. Each sample was tested in triplicate, and the concentration required for 50% reduction (IC50) of DPPH was determined graphically using ascorbic acid as a positive control (IC50: 574 μM). HPLC-MS Analysis. Powdered sample (0.5 g) was refluxed with 50 mL of EtOH (75%) for 2 h. The supernatant was filtered and concentrated to 5 mL and filtered through a 0.45 μm membrane, and 10 μL samples were analyzed with HPLC and LC-MS. Peaks 1, 2, 3, and 4 in the EICs were identified as compounds 4, 3, 1, and 2 by comparing retention times and MS data with authentic samples. Both the processed and unprocessed roots were tested in triplicate.

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were acquired on a Rudolph Research Analytical Autopol II automatic polarimeter. UV spectra were obtained on an Agilent Cary 60 UV− visible spectrophotometer. ECD spectra were obtained on a Jasco J810 spectropolarimeter. IR spectra were measured on a Bruker Tensor 27 spectrometer with KBr pellets. 1D and 2D NMR spectra were recorded on Bruker AV-500 and AV-600 spectrometers. HRESIMS spectra were recorded on a Varian QFT-ESI mass spectrometer. HPLC-MS was performed on an Agilent 1200 Series equipped with a 6310 Ion Trap LC-MS eluted with MeOH/H2O at a flow rate of 1 mL/min by a continuous gradient in which MeOH was adjusted to 10, 60, and 100% at 0, 35, and 40 min, respectively, and the MS scan range was set as 800−860, 380−430, and 530−580 amu at 0, 17, and 26 min, respectively. GC analysis was performed on an Agilent 6820 GC. Agilent SB C18 and SB C18 PreHT were used for HPLC analysis and separation. Silica gel (100−200 and 200−300 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), polyamide (60−100 mesh, Taizhou Luqiao Sijia Biochemical Plastic Factory, Zhejiang, China), and LiChroprep RP-18 (40−63 μm; Merck, Germany) were used for column chromatography. Ascorbic acid was purchased from Tianjin Jiangtian Chemical Technology Co. Ltd. (Tianjin, China). L-Cysteine methyl ester hydrochloride was purchased from Heowns (Tianjin, China). Trimethylsilylimidazole and D- and L-glucose were purchased from Alfa Aesar (Tianjin) Chemical Co., Ltd. (Tianjin, China). DPPH (2,2-diphenyl-1-picrylhydrazyl) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Plant Material. The roots of P. multif lorum were purchased from Xinyang Medicine Company in Henan and identified by Dr. Tianxiang Li from Tianjin University of TCM, Tianjin, China. A voucher specimen (No. S200812001) was deposited at the Tianjin University of TCM, Tianjin, China. The roots were steamed with a water decoction of black soybean under high pressure (0.11 MPa) at 121 °C for 6 h according to the processing methods documented in the Chinese Pharmacopoeia (2005).11 Extraction and Isolation. The processed roots (38 kg) of P. multif lorum were refluxed twice with 90% aqueous EtOH (70 L) and then twice with 60% aqueous EtOH (70 L). The extracts were combined and concentrated to give a residue (4 kg), which was suspended in H2O to a final volume of 10 L and then sequentially partitioned with petroleum ether (60−90 °C), CHCl3, EtOAc, and nBuOH. The n-BuOH extract (900 g) was subjected to D101 macroporous resin CC and eluted with H2O, followed by increasing concentrations of EtOH (30, 50, 95%) in H2O to yield five fractions. The 30% EtOH eluate (400 g) was separated on silica gel CC (EtOAc/MeOH/H2O) to afford 122 fractions (F1−F122). Compound 1 (100 mg) was precipitated from F43 to F49. F31−F53 (18 g) were combined and purified further by silica gel CC (CH2Cl2/MeOH/H2O) to yield 30 subfractions (SF1−SF30). SF19−SF23 (2.5 g) were combined and subjected to polyamide CC to yield compound 2 (10 mg). F67−F103 (24 g) were chromatographed over silica gel (CH2Cl2/MeOH/H2O) followed by ODS (MeOH/H2O) and finally purified by HPLC to give compounds 3 (40 mg) and 4 (50 mg). Polygonumoside A (1): cream-colored powder; [α]25D +3.9 (c 0.09, DMSO); UV (DMSO) λmax (log ε) 268 (4.54), 295 (sh) (4.04), 341 (3.83); IR (KBr) νmax 3346, 1704, 1611, 1515, 1447,1348; 1H and 13C NMR, see Table 1; HRESIMS m/z 555.1139 (calcd for [M − H]− C27H23O13, 555.1139). Polygonumoside B (2): cream-colored powder; [α]25D −2.7 (c 0.09, DMSO); UV (DMSO) λmax (log ε) 268 (4.29), 295 (sh) (3.82), 341 (3.63); IR (KBr) νmax 3354, 1704, 1614, 1517, 1445,1348; 1H and 13C NMR, see Table 1; HRESIMS m/z 555.1139 (calcd for [M − H]− C27H23O13, 555.1139). Polygonumoside C (3): brown, amorphous solid; [α]25D +0.3 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 280 (4.55); IR (KBr) νmax 3430, 1618, 1517, 1449, 1348; 1H and 13C NMR, see Table 2; HRESIMS m/ z 827.2408 (calcd for [M − H]− C40H43O19, 827.2399).

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ASSOCIATED CONTENT

S Supporting Information *

HRESIMS, UV, CD, IR, and 1D and 2D NMR spectra of compounds 1−4 and HPLC-MS data for processed and unprocessed roots of P. multif lorum. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 86 22 27402885. Fax: 86 22 27892025. E-mail: suyfphd@ sina.com. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Dr. X. Zhang (Lab of Computational Chemistry and Drug Design, Laboratory of Chemical Genomics, Peking University Shenzhen Graduate School, Shenzhen, China) for the ECD calculation. Financial support from Program for New Century Excellent Talents in University (NCET-09-0589) and Tianjin Municipal Science and Technology Commission (No. 11ZCGHHZ00800) is gratefully acknowledged.

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REFERENCES

(1) Li, J. B.; Lin, M. Trad. Herb. Drug 1993, 24, 115−118.

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