Diastereomeric Ellagitannin Isomers from

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Diastereomeric Ellagitannin Isomers from Penthorum chinense Manami Era,† Yosuke Matsuo,† Takuya Shii,† Yoshinori Saito,† Takashi Tanaka,*,† and Zhi-Hong Jiang‡ †

Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-Machi, Nagasaki 852-8521, Japan Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau 999078, PR China

‡

S Supporting Information *

ABSTRACT: From the dried stem of Penthorum chinense (Penthoraceae), 1-O-galloyl-4,6-(R)-hexahydroxydiphenoyl (HHDP)-β-D-glucose and 2′,4′,6′-trihydroxyacetophenone 4′O-[4,6-(R)-HHDP]-β-D-glucoside were isolated together with their (S)-HHDP isomers. Ellagitannins with a 4,6-(S)-HHDPglucose moiety are widely distributed in the plant kingdom; however, 4,6-(R)-HHDP glucoses are extremely rare. Lowest-energy conformers of 1-O-galloyl-(S)- and (R)-HHDPglucopyranoses were derived by density functional theory calculations, and the calculated 1H and 13C NMR chemical shifts and the 1H−1H coupling constants were in agreement with the experimental values. The results revealed a conformational difference of the diastereomeric macrocyclic ester rings. In addition, a new compound, 1′,3′,5′-trihydroxybenzene 1′-O-[4,6-(S)-HHDP]-βD-glucoside, was also isolated.

T

he medicinal herb Penthorum chinense PURSH (Penthoraceae) is used mainly in China. Experimental evidence suggests that this herb has hepatoprotective effects and antioxidant activity.1−8 Previous chemical studies of P. chinense revealed the presence of hexahydroxydiphenoyl (HHDP) esters of flavonoid glucosides and related compounds,9 and the flavonoids are postulated to be responsible for the various activities. In addition to biological activities, the chemical constituents of this plant are also intriguing from a chemotaxonomical viewpoint because P. chinense was originally classified into Saxifragaceae in Cronquist’s classification system and recently became independent as Penthoraceae in the Angiosperm Phylogeny Group III classification system.10 Penthoraceae is a small family including only a few species and belongs to an order of Saxifragales. Families in this order, such as Altingiaceae,11 Cercidiphylaceae,12 Paeoniaceae,13 and Saxifragaceae,14 include many ellagitannin-rich plants. Preliminary HPLC analysis of the aqueous acetone extract of the dried stem of P. chinense revealed the presence of many uncharacterized phenolic compounds besides the major known flavonoids, and separation of the aqueous acetone extract resulted in the isolation of three new HHDP esters. This paper deals with the isolation and structure elucidation of the new compounds and discusses the conformation of the (S)and (R)-HHDP-glucopyranoses.

chromatography, and the resulting fractions were further separated by column chromatography using Sephadex LH-20, Diaion HP20SS, MCI gel CHP20P, Toyopearl Butyl-650C, Cosmosil 75C18-OPN, and Chromatorex ODS to afford three new compounds 1−3 together with 2′,4′,6′-trihydroxyacetophenone 4′-O-β-glucoside (8),15 strictinin (4),16 and 2′,4′,6′trihydroxyacetophenone 4′-O-[4,6-(S)-HHDP]-β-glucoside (5) (Figure 1).6 The known compounds were identified by comparison of their spectroscopic data with data acquired from authentic samples and reported data. HPLC analysis of the extract showed prominent peaks of 6 and 7, indicating that these compounds are the main constituents of this plant. These compounds are rare in nature and only found in Phyllanthus tenellus (Phyllanthaceae)17 and Stylogyne cauliflora (Primulaceae).18 Neostrictinin (1) was obtained as a tan amorphous powder showing a dark-blue coloration with ethanolic FeCl3 reagent on TLC, and the Rf value was the same as that of 4. 13C NMR data and HRFABMS, showing an [M + H]+ ion at m/z 635.0876 (calcd for C27H23O18: 635.0884), indicated that the molecular formula C27H22O18 was also the same as that of 4. Comparison of 1H NMR data of 1 with those of 4 (Table 1) suggested the presence of galloyl (δ 7.14, 2H, s) and HHDP (δ 7.06, 6.81, each 1H, s) ester groups attached to a hexose moiety. The large coupling constants of the hexose protons indicated that the sugar is a 4C1-glycopyranose. The D configuration of the glucosyl moiety was determined via acid hydrolysis followed by HPLC analysis of the thiazolidine derivatives prepared by reaction with L-cysteine and o-tolylisothiocyanate.19 The downfield shifts of H1 (δ 5.72), H4 (δ 4.83), and H6 (δ 4.56) of the glucosyl unit indicated that C1, C4, and C6 were

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RESULTS AND DISCUSSION The dried stem of P. chinense was extracted with 60% aqueous acetone, and the extract was concentrated under reduced pressure to remove the acetone. The precipitate formed in the aqueous solution was mainly composed of pinocembrin 7-O[4″,6″-(S)-HHDP]-β-D-glucoside (6) and pinocembrin 7-O[3″-O-galloyl-4″,6″-(S)-HHDP]-β-D-glucoside (7).6 The watersoluble part was fractionated by Sephadex LH-20 column © XXXX American Chemical Society and American Society of Pharmacognosy

Received: May 17, 2015

A

DOI: 10.1021/acs.jnatprod.5b00439 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 1. Structures of compounds 1−8 and selected HMBC and NOESY correlations of 1.

conformational search of 1 and 4 with the MMFF94 force field, the obtained conformers within an energy window of 6 kcal/mol were optimized at the B3LYP/6-31G(d,p) level to afford each lowest-energy conformer of 1 and 4 (Figure 2). The 1 H and 13C NMR chemical shifts and 1H−1H coupling constants of the lowest-energy conformers of 1 and 4 were estimated by density functional theory (DFT) calculations using the GIAO method at the mPW1PW91-SCRF/6-311+G(2d,p) level (for NMR chemical shifts)22 or the B3LYP/631G(d,p)u+1s level (for 1H−1H coupling constants).23 The calculated values were in agreement with the experimental data of 1 and 4 (Supporting Information), supporting the validity of the structures depicted in Figure 2. The NOE association between glucose H4 and HHDP H3″ (δ 7.06) was also consistent with a calculated distance of 2.3 Å between these protons. In contrast, the distance in the structure of 4 was estimated to be 4.7 Å. The glucose H4 signals of the (R)- and (S)-HHDP isomers showed similar chemical shifts (δ 4.83 and 4.88, respectively), and these signals resonated further upfield compared to that of 1,4,6-trigalloyl glucose (δ 5.25). This observation suggested that the chemical shift of the glucose proton was affected by complex anisotropic effects from the ester carbonyl groups and aromatic rings.21 The molecular formula of compound 2 is the same as that of 5, on the basis of 13C NMR and HRFABMS data giving an [M + H]+ ion at m/z 633.1085 (calcd for C36H31O17: 633.1114). The 1H and 13C NMR data of 2 (Table 1) are also similar to those of 5 and showed signals attributable to a symmetric 2,4,6trihydroxyacetophenone unit, an HHDP group, and a 4,6acylated glycosidic glucopyranosyl moiety, indicating that 2 and 5 possess the same molecular structures. A significant difference between the two compounds was the large deshielding of C4 (δC 82.6), shielding of HHDP C2″ and C2‴, and the appearance of the H6a relatively further upfield in the 13C and 1H NMR spectra of 2 (δ 4.64; 5: δ 5.24). The differences were similar to those observed in comparison of the spectra of 1 and 4. The ECD Cotton effects of 2 (226 nm (Δε −20.91) and 260 nm (Δε +7.16)) were the same as those of 1, indicating that the HHDP ester of 2 is R-configured. Thus, the

acylated with galloyl and HHDP esters. The large differences in the chemical shifts of the H6 methylene protons were similar to those of 4, suggesting that the HHDP ester moiety was attached to C4 and C6 of the glucosyl unit. This was confirmed by HMBC correlations of H4 and H6 of the glucosyl unit with the HHDP ester carbons and of the anomeric proton with the galloyl ester carbon (Figure 1). These observations indicated that the molecular structure of 1 is the same as that of 4. The electronic circular dichroism (ECD) spectrum of 1 showed a positive Cotton effect at 261 nm (Δε +7.01) and a negative Cotton effect at 228 nm (Δε −12.84), which had signs opposite those observed for 4. Thus, the HHDP group of 1 was R-configured. Accordingly, the structure of 1 was determined to be 1-O-galloyl-4,6-(R)-HHDP-β-D-glucose and named neostrictinin. A diagnostic feature of ellagitannins with (S)-HHDP esters at C4 and C6 of the glucosyl moiety, such as 4 (Table 1), is the unusual deshielding (δ 5.18) of one of the H6 methylene protons (H6a) in the 1H NMR spectra compared to that of the H6 methylene protons of 1,4,6-trigalloyl glucose (δ 4.48 and 4.15, Figure S7 in the Supporting Information).20 This deshielding is due to the anisotropic effect of the HHDP ester moiety attached to the glucose C6, which is based on a posteriori considerations.21 In contrast, the glucose H6a signal of 1 appeared further upfield (δ 4.56), suggesting conformational differences of the macrocyclic ester rings of these isomers. This was also supported by the following three spectroscopic differences. First, the coupling constant between glucose H5 and H6b of 1 (J5,6b = 9.8 Hz) was much larger than that in 4 (J5,6b < 2 Hz). Second, the carbon signals attributable to the glucose C4 (ΔδC 8.4) and C6 (ΔδC 2.0) of 1 were deshielded compared to those of 4 (Table 1). Third, significant shielding of HHDP C2″ and C2‴ (δC 123.1 and 123.6) of 1 compared to those of 4 (δC 126.5 and 126.9) was observed, which suggested lower electron density at these carbons in 1 than in 4. This reflected the difference in electron delocalization caused by different dihedral angles between conjugated carboxylic groups and aromatic rings of the HHDP moieties. Further investigation of the conformational differences was accomplished by computational methods. Following the B

DOI: 10.1021/acs.jnatprod.5b00439 J. Nat. Prod. XXXX, XXX, XXX−XXX

C

1 2 3 4 5 6a 6b 1 2, 6 3, 5 4 CO CH3 1″, 1‴ 2″, 2‴ 3″, 3‴ 4″, 4‴ 5″, 5‴ 6″, 6‴ 7″, 7‴

d (8.2) dd (8.2, 9.5) dd (8.9, 9.5) t (8.9) ddd (5.6, 8.9, 9.8) dd (5.6, 10.8) dd (9.8, 10.8)

7.06, 6.81, each s

7.14, s

5.72, 3.68, 3.96, 4.83, 4.04, 4.56, 3.52,

H

1

115.9, 123.1, 108.5, 144.1, 135.6, 145.3, 168.7,

120.4 110.1 146.0 139.4 165.5

94.5 73.0 76.0 81.2 71.0 65.7

13

116.0 123.6 109.2 145.1 137.0 145.8 168.1

C d (8.1) dd (8.1, 9.6) t (9.6) t (9.6) br dd (6.3, 9.6) dd (6.3, 13.2) br d (13.2)

6.70, 6.58, each s

7.18, s

5.71, 3.68, 3.80, 4.88, 4.09, 5.18, 3.75,

H

1

4a,d C

115.7, 116.0 126.5, 126.9 107.9, 108.2 144.5, 144.4e 136.2, 136.5 145.1e 168.1, 168.3

120.8 110.3 146.1 139.4 165.4

95.9 74.7 75.6 72.8 73.2 63.7

13

1

H

2b,d d (7.6) dd (7.6, 8.9) t (8.9) t (8.9) br dt (5.6, 8.9) dd (5.6, 10.7) br t (10.7) 107.3 165.5 96.2 164.7 205.3 33.0 116.8, 122.9, 109.0, 144.8, 137.1, 145.9, 169.3,

100.1 74.3 76.1 82.6 70.9 66.3

13

117.3 123.0 109.7 145.8 138.3 146.5 171.5

C

H

1

br dt (5.6, 8.9) dd (5.6, 10.7) br t (10.7)

dd (7.6, 8.9) t (8.9)

6.71, 6.58, each s

2.64, s

6.10, s

− 3.58, 3.74, −g 4.09, 5.24, 3.87, g

5b,d

107.3 165.5 96.2 164.9 205.4 33.0 116.8, 126.5, 108.6, 144.9, 137.6, 145.9, 169.9,

116.6 126.3 108.3 144.8 137.4 145.8 169.6

C

101.4 73.1e 75.7f 73.0e 75.3f 64.3

13

Assignments may be interchanged in each column. gOverlapped with

e,f

7.00, 6.81, each s

2.62, s

6.03, s

5.01, 3.53, 3.87, 4.81, 4.05, 4.64, 3.52,

In acetone-d6. bIn methanol-d4. cUsing 500 MHz for 1H and 125 MHz for 13C. dUsing 400 MHz for 1H and 100 MHz for 13C. HOD signal.

a

4,6-HHDP

galloyl or aglycone

glucose

position

1a,c

Table 1. 1H and 13C NMR Data for New Compounds 1 and 2 and Known Compounds 4 and 5

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observed in the spectra of the only previously isolated 4,6-(R)HHDP-glucose from Alangium chinense (Cornaceae, Cornales, Asterids),26 which taxonomically has a distant relationship with Penthoraceae. Further studies on the ellagitannins of this plant are currently in progress.

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EXPERIMENTAL SECTION

General Experimental Procedures. Ultraviolet (UV) spectra were obtained on a Jasco V-560 UV/vis spectrophotometer (Jasco, Tokyo, Japan). Optical rotations were measured with a JASCO DIP370 digital polarimeter. The ECD spectra were measured with a Jasco J-725N spectrophotometer (Jasco). 1H and 13C NMR spectra were recorded on a Varian Unity Plus 500 spectrometer (Agilent Technologies, Santa Clara, CA, USA) operating at 500 and 125 MHz for the 1H and 13C nuclei, respectively. NMR spectra were also recorded on a JEOL JNM-AL 400 spectrometer (JEOL Ltd., Tokyo, Japan) operating at 400 and 100 MHz for the 1H and 13C nuclei, respectively. ESI-MS were obtained using a JEOL JMS-T100TD spectrometer (JEOL Ltd.). FAB-MS were recorded on a JMS700N spectrometer (JEOL Ltd.) using m-nitrobenzyl alcohol or glycerol as the matrix. Column chromatography was carried out using Sephadex LH-20 (25−100 mm, GE Healthcare U.K. Ltd., Little Chalfont, United Kingdom), MCI-gel CHP20P (75−150 mm, Mitsubishi Chemical Co., Tokyo, Japan), Diaion HP20SS (Mitsubishi Chemical Co.), Toyopearl Butyl-650C (Tosoh Bioscience Japan, Tokyo, Japan), Cosmosil 75C18OPN (Nacalai Tesque Inc., Kyoto, Japan), and Chromatorex ODS (Fuji Silysia Chemical Ltd., Kasugai, Japan) columns. TLC was carried out on precoated Kieselgel 60 F254 plates (0.2 mm thickness, Merck, Darmstadt, Germany) with toluene−ethyl formate−formic acid (1:7:1, v/v) and CHCl3−MeOH−H2O (7:3:0.5, v/v) mixtures being used as the eluents. The spots were detected using UV illumination and by spraying with 2% ethanolic FeCl3 (for phenolic compounds), a 5% anisaldehyde in ethanolic 5% H2SO4 (for proanthocyanidins), or 5% H2SO4 solution followed by heating. Analytical HPLC was carried out on a Cosmosil 5C18-AR-II (Nacalai Tesque Inc., Kyoto, Japan) column (250 mm × 4.6 mm I.D.) with a gradient elution of 4−30% (39 min) and 30−75% (15 min) CH3CN in 50 mM H3PO4 at 35 °C (flow rate, 0.8 mL/min; detection, Jasco photodiode array detector MD-2010). Plant Material. Dried stem material of P. chinese produced in December 2010 in Sichuan, China, was purchased at a local market by Dr. Zhi-Hong Jiang. The voucher specimen was deposited in the Graduate School of Biomedical Sciences, Nagasaki University. Extraction and Separation. Dried stem material (1.0 kg) of P. chinese was crushed by a Waring blender and extracted with 60% acetone (3 × 3 L). After evaporation of the solvent using a rotary evaporator, the resulting precipitates (Fr. 13, 42.3 g) were collected by filtration. The filtrate was fractionated by Diaion HP20SS column chromatography (7 cm I.D. × 34 cm) with H2O containing increasing proportions of MeOH (0−100%, 10% stepwise elution, each 1 L) and finally with 60% acetone (3 L) to give 12 fractions: Fr. 1 (105 g), Fr. 2 (2.5 g), Fr. 3 (1.6 g), Fr. 4 (3.2 g), Fr. 5 (4.5 g), Fr. 6 (5.6 g), Fr. 7 (5.0 g), Fr. 8 (2.2 g), Fr. 9 (1.2 g), Fr. 10 (1.1 g), Fr. 11 (1.7 g), and Fr. 12 (3.8 g). Fr. 4 (3.2 g) was further fractionated by Sephadex LH-20 (4 cm I.D. × 25 cm, 0−100% MeOH) into 10 fractions: Fr. 4a−4r. Fr. 4k (184 mg) was separated by MCI gel CHP20P (0−50% MeOH in H2O) and Toyopearl Butyl-650C column chromatography (0−70% MeOH in H2O) to yield 3 (14 mg). Successive chromatography of Fr. 4m (576 mg) using Sephadex LH-20 (0−40% H2O in EtOH), MCIgel CHP20P (0−70% MeOH in H2O), and Toyopearl Butyl-650C (0−70% MeOH in H2O) afforded 4 (203 mg) and 1 (20 mg). 2′,4′,6′Trihydroxyacetophenone 4′-O-β-glucoside (8) (268 mg) was obtained from Fr. 5 (4.5 g) by Sephadex LH-20 (0−100% MeOH in H2O) and MCI-gel CHP20P (0−70% MeOH in H2O) column chromatography procedures. Similar chromatographic separation of Fr. 9 (1.2 g) with Sephadex LH-20, MCI-gel CHP20P, and Cosmosil 75C18-OPN (0− 100% MeOH) gave 2 (16 mg). A part (10.2 g) of Fr. 13, the waterinsoluble precipitates, was dissolved in 40% MeOH and subjected to Sephadex LH-20 column chromatography (5 cm I.D. × 25 cm) with 40−100% MeOH (20% stepwise, each 300 mL) to give 10 fractions:

Figure 2. Most stable conformations of 1 and 4.

structure of compound 2 was defined as 2′,4′,6′-trihydroxyacetophenone 4′-O-[4,6-(R)-HHDP]-β-D-glucoside. The 1H and 13C NMR spectra of 3 resembled those of 5, except for the appearance of aromatic signals attributable to a phloroglucinol ring rather than the trihydroxyacetophenone moiety of 5. Lack of the acetyl unit was also shown by the [M + H]+ peak at m/z 591 in the FAB-MS analysis. The location of the HHDP ester at C4 and C6 of the glucose moiety was apparent from the deshielding of glucose H4 and H6a, and the (S)-configuration was defined by the Cotton effects in the ECD spectrum (237 nm (Δε +26.15) and 260 nm (Δε −11.51)). Therefore, the structure of 3 was defined as 1,3,5-trihydroxybenzene 1-O-[4,6-(S)-HHDP]-β-D-glucoside. Thus, the major polyphenols of Penthorum chinense are 4,6(S)-HHDP esters of pinocembrin 7-O-glycoside (6 and 7). Minor constituents identified in this study also have HHDP groups at C4 and C6 of a glucosyl moiety, except for compound 8. In the biogenesis of ellagitannins, HHDP esters are postulated to be generated by oxidative coupling between two galloyl groups on the glucopyranose core, and the atropisomerism of the biphenyl bond depends on the locations of the two galloyl groups on the glucose core.20 The HHDP groups attached to C4 and C6, and C2 and C3 of the 4C1− glucose core usually have the S-biphenyl bond. Many successful biomimetic chemical syntheses of ellagitannins support this hypothesis, in which metal-catalyzed oxidative coupling between two galloyl groups attached to glucose 4 and 6 positions exclusively yield (S)-HHDP esters.24,25 Therefore, production of (R)-HHDP esters at glucose 4 and 6 positions is an exception in ellagitannin biosynthesis. In this study, the spectroscopic characteristics of the 4,6-(R)-HHDP glucosyl moiety were demonstrated to involve a much larger 1H NMR J5,6b coupling constant (ca. 9 Hz; S diastereomer: J5,6b < 2 Hz), a large deshielding of the glucosyl C4 carbon (ΔδC ≈ 9), and shielding of the HHDP C2 carbons bearing carboxylic groups (Δδ ≈ −3) in the 13C NMR spectra. These features were also D

DOI: 10.1021/acs.jnatprod.5b00439 J. Nat. Prod. XXXX, XXX, XXX−XXX

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the lowest-energy conformers were calculated at the B3LYP-SCRF/631G(d,p)u+1s (using only the Fermi contact term)//B3LYP-SCRF/631G(d,p) level in acetone (PCM).23 Calculated NMR chemical shifts and 1H−1H coupling constants were linearly corrected for the experimental data. All DFT calculations were carried out using Gaussian 09.27 GaussView was used to draw the molecular structures.28

Fr. 13a−13j. Fr. 13f (633 mg) was separated by MCI-gel CHP20P column chromatography (3 cm I.D. × 15 cm, 60−90% MeOH) to give 5 (44 mg). Diaion HP20S (3 cm I.D. × 20 cm with 30−100% MeOH) column chromatography of Fr. 13g (1.45 g) afforded 6 (523 mg). Fr. 13i (910 mg) was separated by Diaion HP20S (3 cm I.D. × 20 cm, with 80−100% MeOH) and Chromatorex ODS (2 cm I.D. × 15 cm with 10−100% MeOH) to yield 7 (850 mg). Neostrictinin (1). Pale brown amorphous powder; [α]30D +11 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 233 (4.65), 277 (4.55); ECD (MeOH) Δε (nm) +12.84 (205), −12.84 (228), +7.01 (261), −1.06 (289), +0.69 (311); IR νmax cm−1 3400, 1705, 1611; FABMS (positive, matrix: glycerol) m/z [M + H]+ 635; HRFABMS m/z [M + H]+ 635.0876 (calcd for C27H23O18, 635.0884); 1H NMR (acetone-d6, 500 MHz) and 13C NMR (acetone-d6, 125 MHz) are included in Table 1. Acid Hydrolysis of Compound 1. The absolute configuration of the sugar moiety was determined according to the method previously developed by our group with modifications.19 Compound 1 (1 mg) was hydrolyzed with 10% trifluoroacetic acid (0.1 mL) in a screwcapped vial (1 mL) at 90 °C for 7 h. The resulting mixture was lyophilized. The residue was dissolved in 0.1 mL of pyridine containing L-cysteine HCl (1.0 mg) and heated at 60 °C for 1 h. To the mixture was added o-tolylisothiocyanate (2 μL), and this mixture heated at 60 °C for 1 h. The final mixture was cooled to ambient temperature and directly analyzed by HPLC. The retention time of the peak at 34.83 min coincided with that of the thiazolidine derivatives of D-glucose (L-glucose: 35.48 min.). 2′,4′,6′-Trihydroxyacetophenone 4′-O-[4,6-(R)-HHDP]-β-Glucoside (2). Brown amorphous powder; [α]24D −38 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 222 (4.63), 237 (4.36), 277 (4.41), 322 (3.65); ECD (MeOH) Δε (nm) +19.16 (201), −20.91 (226), +7.16 (260), −5.24 (285); IR νmax cm−1 3386, 1722, 1626; FABMS (positive, matrix: thioglycerol) m/z [M + H]+ 633; HRFABMS m/z 633.1085 (calcd for C36H31O17, 633.1114); 1H NMR (methanol-d4, 400 MHz) and 13C NMR (methanol-d4, 100 MHz) are included in Table 1. 1′,3′,5′-Trihydroxybenzene 1′-O-[4,6-(S)-HHDP]-β-Glucoside (3). Brown amorphous powder; [α]30D −31 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 227 (3.88), 275 (3.40); ECD (MeOH) Δε (nm) +26.15 (237), −11.51 (260), +3.44 (282), −2.30 (313); IR νmax cm−1 3372, 1714, 1604; FAB-MS (positive, matrix: glycerol) m/z [M + H]+ 591; HR-FAB-MS m/z 591.0970 (calcd for C26H23O16, 591.0986); 1H NMR (methanol-d4, 400 MHz, δ) 6.64, 6.52 (each 1H, each s, H3″, 3‴), 6.02 (2H, d, J = 2.2 Hz, H2, 6), 5.93 (1H, t, J = 2.2 Hz, H4), 5.21 (1H, dd, J = 5.9, 13.0 Hz, glc-6), 4.79 (1H, d, J = 9.8 Hz, glc-4), 3.97 (1H, dd, J = 5.9, 9.8 Hz, glc-5), 3.81 (1H, d, J = 13.0 Hz, glc-6), 3.64 (1H, t, J = 9.8 Hz, glc-3), 3.49 (1H, dd, J = 7.8, 9.8 Hz, glc-2); 13C NMR (methanol-d4, 100 MHz, δ) 169.6, 170.0 (C7″, 7‴), 160.2 (C3, 5), 160.7 (C1), 145.8, 145.9 (C4″, 4‴), 144.8, 144.9 (C6″, 6‴), 137.4, 137.6 (C5″, 5‴), 126.4, 126.5 (C2″, 2‴), 116.6, 116.8 (C1″, 1‴), 108.3, 108.6 (C3″, 3‴), 102.7 (glc-1), 98.2 (C4), 96.9, 98.2 (C2, 6), 75.5, 75.8 (glc-3, 5), 72.9, 73.3 (glc-2, 4), 64.4 (glc-6). Strictinin (4). ECD (MeOH) Δε (nm) −9.06 (206), +59.26 (236), −19.57 (262), +8.90 (282), −4.19 (315). 2′,4′,6′-Trihydroxyacetophenone 4′-O-[4,6-(S)-HHDP]-β-Glucoside (5). Brown amorphous powder; [α]23D −80 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 223 (4.69), 234 (4.54), 276 (4.52), 335 (3.80); ECD (MeOH) Δε (nm) +6.02 (237), 0 (252), −2.29 (264), 0 (386); IR νmax cm−1 3376, 1724, 1623; FAB-MS (positive, matrix: glycerol + NaCl) m/z [M + H]+ 633; HR-FAB-MS m/z 633.1103 (calcd for C36H31O17, 633.1114); 1H NMR (methanol-d4, 400 MHz) and 13C NMR (methanol-d4, 100 MHz) are included in Table 1. Computational Methods. The conformational search was carried out using a Monte Carlo method at the MMFF94 force field with Spartan’14 (Wave function, Inc., Irvine, CA, USA). Obtained lowenergy conformers within 6 kcal/mol were optimized at the B3LYPSCRF/6-31G(d,p) level in acetone (PCM). The vibrational frequencies were also calculated at the same level to confirm their stability, and no imaginary frequencies were found. NMR chemical shifts of the lowest-energy conformers were calculated using the GIAO method at the mPW1PW91-SCRF/6-311+G(2d,p)//B3LYP-SCRF/ 6-31G(d,p) level in acetone (PCM).22 1H−1H coupling constants of

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ASSOCIATED CONTENT

S Supporting Information *

1

H NMR and 13C NMR spectra of 1−3, 2D NMR spectra of 1, and computational methods and results. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00439.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +81-95-819-2432. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Japan Society for the Promotion of Science KAKENHI (grant nos. 26460125 and 25870532). We are grateful to Mr. K. Inada, Mr. N. Yamaguchi, and Mr. N. Tsuda (Nagasaki University Joint Research Center) for NMR and MS measurements.

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REFERENCES

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