Prenylated Flavonoids from the Stems and Leaves of - American

Jun 23, 2014 - flavonoids. Twenty-eight flavonoids, including 17 flavonoids ... EtOH (4L) to give eight fractions (frs.1−8). ..... hydroxy-8-C-preny...
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Prenylated Flavonoids from the Stems and Leaves of Desmodium caudatum and Evaluation of Their Inhibitory Activity against the Film-Forming Growth of Zygosaccharomyces rouxii F51 Hisako Sasaki,† Hirofumi Shibata,† Kiyoshi Imabayashi,‡ Yoshihisa Takaishi,† and Yoshiki Kashiwada*,† †

Graduate School of Pharmaceutical Sciences and ‡Faculty of Pharmaceutical Sciences, The University of Tokushima, 1-78 Shomachi, Tokushima 770-8505, Japan ABSTRACT: In order to provide scientific evidence for the relationship between the traditional usage, stems and leaves of Desmodium caudatum being used for protecting miso from spoilage, and its Japanese name (miso-naoshi), phytochemical study on the stems and leaves of this plant was carried out. Seven new prenylated flavonoids (1−3, 15−18), together with 19 known compounds (4−14, 19−26), were isolated, and the structures of new compounds were elucidated by extensive spectroscopic analyses. The minimum inhibitory concentrations (MICs) of 28 flavonoids, including 17 compounds (1, 2, 4, 5, 7−14, 20−22, 24, 25) isolated in this study and 11 flavonoids (27−37) previously isolated from the roots of this plant, against the film-forming yeast of Zygosaccharomyces rouxii F51 were determined. Fifteen compounds (2, 4, 5, 11, 12, 14, 21, 22, 25, 27, 28, 32−35) inhibited the film-forming growth of Z. rouxii F51 (MIC values, 7.8−62.5 μg/mL), among which 2″,2″-dimethylpyran(5″,6″:7,8)-5,2′-dihydroxy-4′-methoxy-(2R,3R)-dihydroflavonol (11) demonstrated potent inhibitory activity with an MIC value of 7.8 μg/mL. KEYWORDS: Desmodium caudatum, Fabaceae, prenylated flavonoid, Zygosaccharomyces rouxii

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and thereby the film-forming yeasts might be changed to the non-film-forming form as in the presence of Tween. As a preliminary evaluation, the MeOH extract of the stems and leaves of D. caudatum and its fractions were evaluated for their inhibitory activity against the film forming growth of Z. rouxii F51 in 24-well microtiter plates. The MeOH extract (50 mg/ mL) of the stems and leaves of D. caudatum, as well as the EtOAc-soluble fraction (20 mg/mL) from the MeOH extract of the stems of D. caudatum, exhibited inhibitory effect on the film-forming growth of Z. rouxii F51, which was similar to that of Tween 80 (0.005%) used as a positive control. In contrast, film formation was observed in the well containing the H2Osoluble fraction (20 mg/mL) from the MeOH extract of the stems of D. caudatum, as seen in the well containing DMSO used as a negative control (data not shown). Based on this result, phytochemical examination of the stems and leaves of this plant has been carried out and has resulted in the isolation of seven new prenylated flavonoids, together with 19 known flavonoids. Twenty-eight flavonoids, including 17 flavonoids obtained in this study and 11 flavonoids previously isolated from the roots of this plant, were evaluated for their effect on the film-forming growth of a film-forming strain (F51) of Z. rouxii. In this paper, we describe the structure elucidation of new compounds as well as inhibition of the prenylated flavonoids of D. caudatum against the film-forming growth of Z. rouxii F51.

INTRODUCTION Desmodium caudatum (Thunb.) H. Ohashi (Fabaceae; Japanese name: Miso-naoshi) is a small tree distributed in the west region of Japan. The roots of this plant have been used medicinally to treat rheumatic backache, diarrhea, icteric hepatitis and abscess, and as an anthelmintic.1 As part of our search for bioactive plant metabolites, we previously studied the roots of D. caudatum and reported the isolation and characterization of prenylated flavonoids, as well as their antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and antifungal activities against Aspergillus niger, Penicillium sp., Rhizopus sp., and Trichophyton sp.2,3 The Japanese name of D. caudatum came from the traditional usage that stems and leaves of this plant have been used for protecting miso, one of the traditional Japanese seasonings, from spoilage in Japan.4 Miso is made by the fermentation of soybeans with yeast. Among several yeast species used in miso fermentation, Zygosaccharomyces rouxii is the predominant species.5 This yeast has been used for making fermented foods containing a high concentration of NaCl, including a miso paste as well as soy sauce. In contrast, there is an another osmophilic yeast strain, which is also classified as Z. rouxii, possessing the property of forming a pellicle only on strongly saline media.6 This film-forming yeast often develops a white dry pellicle on the surface of the fermented products during storage, which lead to the spoilage of their taste and flavors.7 Z. rouxii F51, a film-forming strain, forms a heavy film on the surface of the liquid medium. However, it grows in a sedimentary form without forming a film, similarly to non-film-forming strain K122, when it is cultivated in a hypertonic medium containing Tween.7 Considering the traditional usage of D. caudatum, we are interested to determine whether some constituents of this plant have activity for inhibiting film formation of Z. rouxii F51, © 2014 American Chemical Society

Received: Revised: Accepted: Published: 6345

April 30, 2014 June 20, 2014 June 23, 2014 June 23, 2014 dx.doi.org/10.1021/jf5020439 | J. Agric. Food Chem. 2014, 62, 6345−6353

Journal of Agricultural and Food Chemistry

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Article

0:1), each solvent volume 100 mL] to furnish 7 (2.2 mg) [Rf 0.45 (CHCl3/MeOH = 10:1)] and a fraction that was further purified by HPLC on 5C18-AR-II [MeOH−H2O (45:55), flow rate 3.5 mL/min, UV at 280 nm, eluted at 122 min] to give 25 (0.7 mg) [Rf 0.35 (CHCl3/MeOH = 10:1)]. Fr. 3.12 (45.4 mg) was subjected to chromatography over Sephadex LH-20 (2.5 × 27 cm2) [eluted with H2O−MeOH gradient (3:2, 1:1, 2:3, 3:7, 1:4, 1:9, 0:1), each solvent volume 100 mL] and then purified by HPLC on 5C18-AR-II [MeOH− H2O (3:7), flow rate 3.0 mL/min, UV at 280 nm, eluted at 164 min] to yield 3 (0.7 mg) [R f 0.28 (CHCl 3 /MeOH = 10:1)]. Chromatography of Fr. 3.13 (187.3 mg) over Sephadex LH-20 (2.1 × 13 cm2) [eluted with H2O−MeOH gradient (1:1, 2:3, 3:7, 1:4, 1:9, 0:1), each solvent volume 100 mL] furnished 14 (83.8 mg) [Rf 0.40 (CHCl3/MeOH = 10:1)] and a fraction that was further purified by HPLC on 5C18-AR-II [MeOH−H2O (3:7), flow rate 3.0 mL/min, UV at 280 nm, eluted at 84 min] giving 9 (0.4 mg) [Rf 0.48 (CHCl3/ MeOH = 10:1)]. Sephadex LH-20 chromatography (2.5 × 27 cm2) [eluted with H2O−MeOH gradient (1:1, 2:3, 3:7, 1:4, 1:9, 0:1), each solvent volume 100 mL] of fr. 3.14 (47.6 mg) gave 11 (14.0 mg) [Rf 0.55 (CHCl3/MeOH = 10:1)] and 12 (4.3 mg) [Rf 0.60 (CHCl3/ MeOH = 10:1)]. Fraction 4 (781.2 mg) was applied to a MCI gel CHP20P column (3.0 × 24 cm2) with a gradient of H2O−MeOH (7:3, 3:2, 1:1, 2:3, 3:7, 1:4, 1:9, 0:1, each solvent volume 200 mL) to give 22 further fractions (frs. 4.1−4.22). Fr. 4.7 (123.7 mg) was chromatographed over Sephadex LH-20 (3.0 × 30 cm2) [eluted with H2O−MeOH gradient (7:3, 3:2, 1:1, 2:3, 3:7, 1:4, 1:9, 0:1), each solvent volume 100 mL] to afford 20 (3.5 mg) [Rf 0.58 (CHCl3/ MeOH/H2O = 8:2:0.2)]. Sephadex LH-20 chromatography (2.1 × 15 cm2) [eluted with H2O−MeOH gradient (7:3, 3:2, 1:1, 2:3, 3:7, 1:4, 1:9, 0:1), each solvent volume 100 mL] of fr. 4.12 gave 23 (1.2 mg) [Rf 0.65 (CHCl3/MeOH/H2O = 8:2:0.2)]. Fr. 4.15 was subjected to Sephadex LH-20 chromatography (2.6 × 28 cm2) [eluted with H2O− MeOH gradient (7:3, 3:2, 1:1, 2:3, 3:7, 1:4, 1:9, 0:1), each solvent volume 100 mL] to furnish 21 (9.8 mg) [Rf 0.63 (CHCl3/MeOH/ H2O = 8:2:0.2)], 22 (3.6 mg) [Rf 0.65 (CHCl3/MeOH/H2O = 8:2:0.2)], and 11 fractions (frs. 4.15.1−4.15.11). Fr. 4.15.9 (5.5 mg) was purified by HPLC on 5C18-AR-II [MeOH−H2O (3:2), flow rate 3.5 mL/min, UV at 280 nm, eluted at 51 min] to furnish 26 (0.9 mg) [Rf 0.71 (CHCl 3/MeOH/H2O = 8:2:0.2)]. Sephadex LH-20 chromatography (2.6 × 26 cm2) [eluted with H2O−MeOH gradient (7:3, 3:2, 1:1, 2:3, 3:7, 1:4, 1:9, 0:1), each solvent volume 100 mL] of fr. 4.13 (26.0 mg) gave 4 (3.7 mg) [Rf 0.60 (CHCl3/MeOH/H2O = 8:2:0.2)]. The leaves (0.5 kg) of D. caudatum were extracted three times with MeOH (3 L × 3) at room temperature for 3 days. The MeOH extracts were concentrated in vacuo to give a residue (116 g), which was partitioned between EtOAc and H2O. The EtOAc layer was concentrated to give a residue (25 g). The EtOAc-soluble fraction was dissolved in EtOH, loaded onto a Sephadex LH-20 column (6.0 × 20 cm2), and eluted with EtOH (4 L) to give ten fractions (frs. 1−10). The spots of fraction 2 on the TLC plate (TLC solvent; CHCl3/ MeOH = 10:1) were yellow and orange, detected by UV illumination and by spraying with FeCl3 reagent. Fraction 2 (1.7 g) was subjected to chromatography over MCI gel CHP20P (3.6 × 24 cm2) [eluted with H2O−MeOH gradient (7:3, 3:2, 1:1, 2:3, 3:7, 1:4, 1:9, 0:1), each solvent volume 200 mL] to give 20 further fractions (frs. 2.1−2.20). Fr. 2.9 was subjected to chromatography over Sephadex LH-20 (2.6 × 27 cm2) [eluted with H2O−MeOH gradient (7:3, 3:2, 1:1, 2:3, 3:7, 1:4, 1:9, 0:1), each solvent volume 100 mL] to give 13 (1.6 mg) [Rf 0.65 (CHCl3/MeOH/H2O = 8:2:0.2)] and 5 (4.0 mg) [Rf 0.58 (CHCl 3 /MeOH/H 2 O = 8:2:0.2)]. Fr. 2.10 (69.1 mg) was rechromatographed over Sephadex LH-20 (2.0 × 16 cm2) [eluted with H2O−MeOH gradient (7:3, 3:2, 1:1, 2:3, 3:7, 1:4, 1:9, 0:1), each solvent volume 100 mL] to furnish 1 (6.0 mg) [Rf 0.59 (CHCl3/ MeOH/H2O = 8:2:0.2)]. Sephadex LH-20 chromatography (2.6 × 27 cm2) [eluted with H2O−MeOH gradient (7:3, 3:2, 1:1, 2:3, 3:7, 1:4, 1:9, 0:1), each solvent volume 100 mL] of fr. 2.11 (78.9 mg) gave 8 (10.6 mg) [Rf 0.40 (CHCl3/MeOH = 10:1)]. Fr. 2.12 (59.7 mg) was subjected to chromatography over Sephadex LH-20 (2.0 × 16 cm2) [eluted with H2O−MeOH gradient (7:3, 3:2, 1:1, 2:3, 3:7, 1:4, 1:9,

MATERIALS AND METHODS

General Experimental Procedures. Optical rotations were measured on a JASCO P-2200 polarimeter. NMR spectra were recorded on a Bruker AVANCE-500 instrument (1H NMR, 500 MHz; 13 C NMR, 125 MHz) using TMS as an internal standard. High resolution electrospray ionization mass spectra (HRESIMS) were obtained on a Waters LCT Premier. CD spectra were run on a CDJ600 spectropolarimeter (JASCO). Column chromatography was performed with silica gel 60N (63-210 μm, Kanto Kagaku), Sephadex LH-20 (25−100 μm, GE Healthcare Bioscience), and MCI gel CHP20P (75−150 μm, Mitsubishi Chemical Corporation). HPLC was performed on a JASCO apparatus consisting of a PU-980 prep pump, UV-970 UV/VIS (at the wavelength of 280 nm), and Cosmosil 5C18AR-II (5 μm, φ20 × 250 mm2, nacalai tesque), or Cosmosil π-NAP (5 μm, φ20 × 250 mm2, nacalai tesque). TLC was conducted on precoated silica gel 60 F254 (0.20 mm, Merck), and spots were detected by UV illumination and by spraying FeCl3 reagent or cerium sulfate reagent followed by heating. Plant Material. The stems and leaves of Desmodium caudatum (Thunb.) H. Ohashi were collected in Tokushima, Japan, in December 2011. The plant was identified by one of the authors (K.I.), and a voucher specimen (MSO08001) was deposited at the herbarium of Faculty of Pharmaceutical Sciences, The University of Tokushima. Extraction and Isolation of Compounds. The stems (1.9 kg) of D. caudatum were extracted three times with MeOH (5 L × 3) at room temperature for 3 days. The MeOH extracts were concentrated in vacuo to give a residue (132 g), which was partitioned between EtOAc and H2O. The EtOAc layer was concentrated to give a residue (15 g). The EtOAc-soluble fraction was dissolved in EtOH, loaded onto a Sephadex LH-20 column (4.2 × 27 cm2), and eluted with EtOH (4L) to give eight fractions (frs.1−8). The spots of fraction 3 and 4 on the TLC plate (TLC solvent; CHCl3/MeOH = 10:1) were yellow and orange, detected by UV illumination and by spraying with FeCl3 reagent. Fraction 3 (1.3 g) was subjected to chromatography over MCI gel CHP20P (3.0 × 30 cm2) [eluted with H2O−MeOH gradient (1:1, 2:3, 3:7, 1:4, 1:9, 0:1), each solvent volume 200 mL] to give 15 further fractions (frs. 3.1−3.15). Sephadex LH-20 chromatography (3.0 × 30 cm2) [eluted with H2O−MeOH gradient (4:1, 7:3, 1:1, 2:3, 3:7, 1:4, 1:9, 0:1), each solvent volume 100 mL] of fr. 3.6 (136.6 mg) gave 10 (13.9 mg) [Rf 0.35 (CHCl3/MeOH =10:1)]. Fr. 3.8 was subjected to chromatography over Sephadex LH-20 (3.0 × 30 cm2) [eluted with H2O−MeOH gradient (7:3, 3:2, 1:1, 2:3, 3:7, 1:4, 1:9, 0:1), each solvent volume 100 mL] to give 2 (8.4 mg) [Rf 0.35 (CHCl3/MeOH = 10:1)] and 5 (97.3 mg) [Rf 0.38 (CHCl3/MeOH = 10:1)], together with 16 fractions (frs. 3.8.1−3.8.16). Fraction 3.8.9 (2.1 mg) was purified by HPLC on 5C18-AR-II [MeOH−H2O (7:3), flow rate 3.5 mL/min, UV at 280 nm, eluted at 70 min] to furnish 15 (1.0 mg) [Rf 0.18 (CHCl3/MeOH = 10:1)]. Fr. 3.8.10 was separated by HPLC on 5C18-AR-II [MeOH−H2O (7:3), flow rate 3.5 mL/min, UV at 280 nm, eluted at 100 min] and then purified by HPLC on πNAP [MeOH−H2O (7:3), flow rate 3.5 mL/min, UV at 280 nm, eluted at 88 min] to give 13 (2.3 mg) [Rf 0.35 (CHCl3/MeOH = 10:1)]. Fraction 3.9 (61.7 mg) was fractionated by Sephadex LH-20 (2.5 × 27 cm2) [eluted with H2O−MeOH gradient (3:2, 1:1, 2:3, 3:7, 1:4, 1:9, 0:1), each solvent volume 100 mL] into 12 fractions (frs. 3.9.1−3.9.12). Fr. 3.9.7 (6.8 mg) was purified by HPLC on 5C18-AR-II [MeOH−H2O (1:1), flow rate 3.5 mL/min, UV at 280 nm, eluted at 78 min] to furnish 1 (3.2 mg) [Rf 0.38 (CHCl3/MeOH = 10:1)]. Fr. 3.9.11 was separated by HPLC on 5C18-AR-II [MeOH−H2O (1:1), flow rate 3.5 mL/min, UV at 280 nm, eluted at 128 min] to give 6 (1.7 mg) [Rf 0.25 (CHCl3/MeOH = 10:1)]. Fr. 3.10 was subjected to Sephadex LH-20 chromatography (3.0 × 30 cm2) [eluted with H2O− MeOH gradient (7:3, 3:2, 1:1, 2:3, 3:7, 1:4, 1:9, 0:1), each solvent volume 100 mL] to afford 8 (41.0 mg) [Rf 0.48 (CHCl3/MeOH = 10:1)] and a fraction that was further purified by HPLC on 5C18-AR-II [MeOH−H2O (45:55), flow rate 3.5 mL/min, UV at 280 nm, eluted at 98 min] to give 19 (0.5 mg) [Rf 0.33 (CHCl3/MeOH = 10:1)]. Fr. 3.11 (93.2 mg) was chromatographed over Sephadex LH-20 (3.0 × 30 cm2) [eluted with H2O−MeOH gradient (3:2, 1:1, 2:3, 3:7, 1:4, 1:9, 6346

dx.doi.org/10.1021/jf5020439 | J. Agric. Food Chem. 2014, 62, 6345−6353

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Table 1. 1H and 13C-NMR Data for 1, 16, and 18 in Acetone-d6 1 2 3 4 5 6 7 8 9 10 1′ 2′,6′ 3′,5′ 4′ 1″ 2″ 3″ 4″ 5″ 6-Me 5-OH 4′-OMe a

16

δ Ha

position

5.13 (1H, d, 11.5) 4.66 (1H, d, 11.5)

5.90 (1H, s)

7.43 (2H, d, 8.5) 6.90 (2H, d, 8.5) 6.52 (1H, d, 10.5) 5.54 (1H, d, 10.5) 3.60 (1H, d, 12.0) 3.53 (1H, d, 12.0) 1.38 (3H, s)

18

δ Cb

δ Ha

δ Cb

δ Ha

δ Cb

84.5 73.2 198.7 164.3 97.9 163.6 102.8 157.8 102.0 129.0 130.4 116.1 159.0 117.7 124.6 82.4 68.7 23.9

5.10 (1H, d, 11.5) 4.65 (1H, d, 11.5)

84.7 73.6 199.2 158.0 106.2 161.1 102.6 156.1 101.8 129.4 130.6 116.3 159.2 116.5 127.6 79.3 29.0 28.8 7.2

5.43 (1H, d, 11.5) 4.97 (1H, d, 11.5)

1.40 (3H, s) 1.40 (3H, s)

83.6 72.5 198.4 163.5 97.3 161.9 101.7 156.7 101.6 129.6 129.3 113.8 160.0 115.2 126.2 77.8 27.7 27.5

12.6 (1H, s) 3.70 (3H, s)

54.7

7.43 (2H, d, 8.5) 6.89 (2H, d, 8.5) 6.45 (1H, d, 10.0) 5.59 (1H, d, 10.0) 1.42 (3H, s) 1.45 (3H, s) 1.95 (3H, s)

6.31 (1H, s)

7.75 (2H, d, 8.5) 7.09 (2H, d, 8.5) 6.58 (1H, d, 10.5) 5.49 (1H, d, 10.5)

δH, ppm (mult., J in Hz); 500 MHz. bδC, ppm; 125 MHz.

Table 2. 1H and 13C-NMR Data for 2, 3, 15, and 17 in Acetone-d6 2 δHa

position 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 5-OH 7-OMe 3′-OMe a

5.63 (1H, d, 2.0) 4.24 (1H, d, 2.0)

5.06 (1H, s)

6.44 (1H, d, 2.0) 6.40 7.40 3.28 5.21

(1H, (1H, (2H, (1H,

dd, 8.5, 2.0) d, 8.5) d, 7.0) t, 7.0)

1.60 (3H, s) 1.64 (3H, s) 11.9 (1H, s)

3 δC

δH

b

79.1 71.5 196.6 163.4 96.4 165.1 108.4 161.2 101.6 114.8 156.2 103.3 159.3 107.5 131.0 22.3 123.8 131.2 26.0 17.9

a

5.47 (1H, d, 11.5) 4.82 (1H, d, 11.5)

6.17 (1H, s)

6.46 (1H, d, 2.5) 6.42 7.31 3.15 5.11

(1H, (1H, (2H, (1H,

dd, 8.5, 2.5) d, 8.5) d, 7.5) t, 7.5)

1.55 1.56 11.8 3.90

(3H, (3H, (1H, (3H,

s) s) s) s)

15 δC

δH

b

79.4 72.5 199.6 163.3 93.3 165.0 109.7 158.3 101.6 115.0 159.0 103.8 160.7 107.9 130.6 22.2 123.5 131.7 26.1 17.9

5.46 (1H, d, 11.5) 4.77 (1H, d, 11.5)

6.03 (1H, s)

6.46 (1H, d, 2.5) 6.41 7.34 2.57 1.61

(1H, (1H, (2H, (2H,

dd, 8.5, 2.5) d, 8.5) t, 8.0) t, 8.0)

1.13 (3H, s) 1.28 (3H, s) 11.6 (1H, s)

17 δC

a

δH

b

79.3 72.5 199.0 162.7 97.0 165.9 110.0 161.6 101.8 115.9 158.2 104.0 159.9 107.9 130.4 18.2 43.8 70.9 29.5 29.6

a

5.14 (1H, d, 11.5) 4.72 (1H, d, 11.5)

δ Cb

1.42 (3H, s) 1.40 (3H, s)

85.0 73.4 199.0 163.2 98.1 164.4 102.2 158.2 100.2 129.6 112.6 148.5 148.4 115.8 122.4 116.1 127.9 79.3 28.9 28.7

3.87 (3H, s)

56.6

5.92 (1H, s)

7.23 (1H, d, 2.0)

6.88 7.05 6.44 5.59

(1H, (1H, (1H, (1H,

d, 8.0) dd, 2.0, 8.0) d, 10.0) d, 10.0)

56.8

δH, ppm (mult., J in Hz); 500 MHz. bδC, ppm; 125 MHz.

0:1), each solvent volume 100 mL] to yield 19 fractions (frs. 4.8.1− 4.8.19) and then purified by HPLC on π-NAP [MeOH−H2O (7:3), flow rate 3.5 mL/min, UV at 280 nm, eluted at 58 min] to afford 7 (0.7 mg) [Rf 0.75 (CHCl3/MeOH/H2O = 8:2:0.2)]. Fr. 2.14 (50.3 mg) was chromatographed over Sephadex LH-20 (2.0 × 16 cm2) [eluted with H2O−MeOH gradient (7:3, 3:2, 1:1, 3:2, 1:1, 2:3, 3:7, 1:4, 1:9, 0:1), each solvent volume 100 mL] to furnish 9 (3.6 mg) [Rf

0.48 (CHCl3/MeOH = 10:1)] and a fraction that was further purified by HPLC on π-NAP [MeOH−H2O (7:3), flow rate 3.5 mL/min, UV at 280 nm, eluted at 100 min] to yield 14 (12.6 mg) [Rf 0.35 (CHCl3/ MeOH = 10:1)]. Fraction 2.15 (100.7 mg) was fractionated by Sephadex LH-20 (2.6 × 27 cm2) [eluted with H2O−MeOH gradient (7:3, 3:2, 1:1, 2:3, 3:7, 1:4, 1:9, 0:1), each solvent volume 100 mL] into eight fractions (frs. 2.15.1−2.15.8). Fr. 2.15.6 was purified by 6347

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Figure 1. Structures of flavonoids (1−26) isolated from the stems and leaves of D. caudatum and previously isolated flavonoids (27−37) evaluated for anti-film-forming activities. HPLC on π-NAP [MeOH−H2O (7:3), flow rate 3.5 mL/min, UV at 280 nm, eluted at 128 and 160 min, respectively] to furnish 11 (7.9 mg) [Rf 0.45 (CHCl3/MeOH = 10:1)] and 16 (1.3 mg) [Rf 0.31 (CHCl3/MeOH = 10:1)]. Fr. 2.15.2 (9.8 mg) was purified by HPLC on π-NAP [MeOH−H2O (7:3), flow rate 3.5 mL/min, UV at 280 nm, eluted at 138 and 145 min, respectively] to afford 17 (0.5 mg) [Rf 0.65 (CHCl3/MeOH = 10:1)] and 12 (2.7 mg) [Rf 0.68 (CHCl3/MeOH = 10:1)]. Fr. 2.16 (32.3 mg) was chromatographed over Sephadex LH20 (2.0 × 16 cm2) [eluted with H2O−MeOH gradient (1:1, 2:3, 3:7, 1:4, 1:9, 0:1), each solvent volume 100 mL] to furnish 24 (6.2 mg) [Rf 0.50 (CHCl3/MeOH = 10:1)]. Fr. 2.17 (78.9 mg) was rechromatographed over Sephadex LH-20 (2.6 × 27 cm2) [eluted with H2O− MeOH gradient (7:3, 3:2, 1:1, 2:3, 3:7, 1:4, 1:9, 0:1), each solvent volume 100 mL] and by crystallization (aqueous acetone) to give 18 (5.6 mg) [Rf 0.80 (CHCl3/MeOH = 10:1)].

Compound 1. Yellow amorphous powder; [α]20D +10.0 (c 0.11, MeOH). CD (MeOH; c 2.7 × 10−4 M; Δε λmax): 321 (+2.19), 295 (−4.83), 278 (−3.57), 252 (+4.55), 238 (+2.28), 221 (+6.91). HRESIMS: m/z 393.0939, [M + Na]+ (calcd for C20H18O7Na, 393.0950). 1H NMR (acetone-d6) and 13C NMR (acetone-d6) spectroscopic data, see Table 1. Acetylation of 1. Compound 1 (1.0 mg) was treated with dry pyridine (500 μL) and Ac2O (500 μL) overnight at room temperature. The reaction mixture was worked up as usual, and the product was purified by silica chromatography [benzene/acetone (30:1)] to furnish 1a (1.0 mg) as a yellow amorphous powder; [α]20D +2.7 (c 0.10, MeOH). HRESIMS: m/z 561.1384, [M + Na] + (calcd for C20H18O7Na, 561.1373). 1H NMR (CDCl3, 500 MHz): δ 7.49 (2H, d, J = 8.5 Hz, H-2′, 6′), 7.17 (2H, d, J = 8.5 Hz, H-3′, 5′), 6.68 (1H, d, J = 10.5 Hz, H-1″), 6.26 (1H, s, H-6), 5.66 (1H, d, J = 12.0 Hz, H-3), 6348

dx.doi.org/10.1021/jf5020439 | J. Agric. Food Chem. 2014, 62, 6345−6353

Journal of Agricultural and Food Chemistry

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5.49 (1H, d, J = 10.5 Hz, H-2″), 5.40 (1H, d, J = 12.0 Hz, H-2), 4.23 (1H, d, J = 12.0 Hz, H-4″a), 4.12 (1H, d, J = 12.0 Hz, H-4″b), 2.38 (3H, s, 5-OAc), 2.33 (3H, s, 4′-OAc), 2.05 (3H, s, 4″-OAc), 2.02 (3H, s, 3-OAc), 1.47 (3H, s, H-5″). Compound 2. Yellow amorphous powder; [α]20D −75.4 (c 0.23, MeOH). CD (MeOH; c 2.7 × 10−4 M; Δε λmax): 340 (+3.23), 295 (−6.95), 238 (+0.75), 211 (+6.98). HRESIMS: m/z 395.1109, [M + Na]+ (calcd for C20H20O7Na, 395.1109). 1H NMR (acetone-d6) and 13 C NMR (acetone-d6) spectroscopic data, see Table 2. Compound 3. Yellow amorphous powder; [α]20D +2.5 (c 0.10, MeOH). CD (MeOH; c 2.6 × 10−4 M; Δε λmax): 317(−0.33), 295 (−1.24), 248 (−0.47), 202 (+4.20). HRESIMS: m/z 409.1253, [M + Na]+ (calcd for C21H22O7Na, 409.1263). 1H NMR (acetone-d6) and 13 C NMR (acetone-d6) spectroscopic data, see Table 2. Compound 6. Yellow amorphous powder; [α]20D +5.0 (c 0.20, MeOH). CD (MeOH; c 2.7 × 10−4 M; Δε λmax): 326 (+1.76), 295 (−4.40), 247 (+1.92), 223 (+6.30), 210 (+2.55). HRESIMS: m/z 371.1136, [M − H]−(calcd for C20H19O7, 371.1131). 1H NMR (acetone-d6, 500 MHz): δ 7.08 (1H, d, J = 2.0 Hz, H-2′), 6.93 (1H, dd, J = 8.5, 2.0 Hz, H-6′), 6.86 (1H, d, J = 2.0 Hz, H-5′), 6.06 (1H, s, H6), 5.16 (1H, t, J = 5.5 Hz, H-2″), 4.98 (1H, d, J = 11.5 Hz, H-2), 4.55 (1H, d, J = 11.5 Hz, H-3), 3.17 (2H, d, J = 5.5 Hz, H-1″), 1.59 (3H, s, H-4″), 1.56 (3H, s, H-5″). Compound 15. Yellow amorphous powder; [α]20D +34.9 (c 0.12, MeOH). HRESIMS: m/z 371.1113, [M − H]− (calcd for C20H19O7 371.1131). CD (MeOH; c 2.7 × 10−4 M; Δε λmax): 317(+1.13), 295 (−1.78), 224 (+3.99). 1H NMR (acetone-d6) and 13C NMR (acetoned6) spectroscopic data, see Table 2. Compound 16. Yellow amorphous powder; [α]20D −7.1 (c 0.11, MeOH). CD (MeOH; c 2.7 × 10−4 M; Δε λmax): 325 (+0.27), 300 (−3.79), 286 (−2.53), 258(+1.93), 221(+2.96). HRESIMS: m/z 391.1117, [M + Na]+ (calcd for C21H20O6Na, 391.1158). 1H NMR (acetone-d6) and 13C NMR (acetone-d6) spectroscopic data, see Table 1. Compound 17. Yellow amorphous powder; [α]20D +3.9 (c 0.10, MeOH). CD (MeOH; c 2.6 × 10−4 M; Δε λmax): 330 (+0.31), 293 (−0.94), 254 (+0.70). HRESIMS: m/z 407.1091, [M + Na]+ (calcd for C21H20O7Na 407.1107). 1H NMR (acetone-d6) and 13C NMR (acetone-d6) spectroscopic data, see Table 2. Compound 18. Yellow granules, mp 132−138 °C; [α]20D −10.6 (c 0.54, MeOH). HRESIMS: m/z 391.1150, [M + Na]+ (calcd for C21H20O6Na 391.1158). CD (MeOH; c 2.7 × 10−4 M; Δε λmax): 320 (+2.33), 298 (−6.22), 248 (+5.50), 232 (+2.23). 1H NMR (acetoned6) and 13C NMR (acetone-d6) spectroscopic data, see Table 1. Biological Evaluation. Test Microorganisms and Media. Two osmophilic yeasts of Z. rouxii K122 and F51 were kindly provided from Tokushima Prefectural Industrial Technology Center, Tokushima, Japan. Z. rouxii K122, which has been industrially used for misomaking, was used as a typical non-film-forming strain and F51 as a film-forming strain. Both strains of Z. rouxii were cultured in slant agar media of Sabouraud dextrose agar (SDA; Difco) supplemented with 10% NaCl [NaCl 8.4 g] and a soy sauce [10 mL (salt conversion 1.6 g) per 100 mL SDA] and 50 μg/mL chloramphenicol at 25 °C for 2 weeks. MIC Measurement. The MICs of Tween 80 and the isolated compounds (purity >95%) against the film-forming growth of Z. rouxii F51 were determined by the broth microdilution method in 96-well microtiter plates (Well Plate; Becton Dickinson Labware) using Sabouraud dextrose broth (SDB; Difco) supplemented with 10% NaCl [NaCl 8.4 g] and a soy sauce [10 mL (salt conversion 1.6 g) per 100 mL SDB]. The compounds were serially diluted with DMSO to prepare the solutions, which contained from 5000 to 4.8 μg/mL of the samples. Aliquots (5 μL) of the test compound solution were added to wells of the plate, which contained 50 μL of double-strength SDB and 35 μL of water. The 2-week cultures of the test strains were harvested and suspended into saline and finally adjusted to obtain the concentration of 106 CFU/mL. Aliquots (10 μL) of the suspensions were then added into wells of the plate. The plate was incubated at 25 °C for 2 weeks. Optical density (OD) at 540 nm was measured with a microplate reader (Sunrise; TECAN Group Ltd.) on the start day and

after 2-week cultivation. The effects of compounds were evaluated by the difference of OD values on the start day and the 2-week cultivation. When the OD value was increased more than 0.1, compound was considered to have no inhibitory activity against the film-forming growth of Z. rouxii F51. Five microliters of DMSO did not inhibit the film-forming growth of Z. rouxii F51 at all. In contrast, the tested compounds, Tween 80, and DMSO have no effects on the growth of non-film-forming strain K122 on the same evaluation procedure as for Z. rouxii F51.

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RESULTS AND DISCUSSION Structure Elucidation and Identification. The stems of D. caudatum were extracted with MeOH at room temperature, and the MeOH extract was partitioned between EtOAc and H2O. Repeated chromatography of the EtOAc-soluble fraction over Sephadex LH-20 and MCI gel CHP20P and purification by HPLC afforded four new flavonoids (1−3, 15), together with 18 known flavonoids (4 −14, 19−23, 25, 26) (Figure 1). The MeOH extract of leaves of D. caudatum was partitioned between EtOAc and H2O. Repeated chromatography of the EtOAc-soluble fraction over Sephadex LH-20, MCI gel CHP20P, and SiO2 and purification by HPLC afforded four new flavonoids (1, 16−18), together with nine known compounds (5, 7−9, 11−14, 24) (Figure 1). The known compounds were identified as 6-C-prenyl-5,7,2′,4′-tetrahydroxydihydroflavanonol (4),2 8-C-prenyl-5,7,2′,4′-tetrahydroxydihydroflavanonol (5),2 8-C-prenyldihydroisohamnetin (7),8 3hydroxy-8-C-prenylnaringenin (8),9 lespedezaflavanone C (9), 10 dihydrokaempferol (10),11 7,8-(2,2-dimethyl-2Hpyran)-5,2′-dihydroxy-4′-methoxyflavanonol (11),2 glysapinol (12),12 8-C-prenyl-chromanochroman (13),2 yukovanol (14),13 lespecyrtin A1 (19),14 garbanzol (20),15 6-C-prenyl-5,7,2′,4′tetrahydroxydihydroflavonol (21),3 kaempferol (22),16 luteoline (23),17 citrusinol (24),18 leachianone G (25),19 and apigenin (26)20 by comparison of their physical and spectral data with those reported in the literature. The structure of compound 6 was assigned as 8-C-prenyldihydroquercetin by analysis of the spectral data. Since the spectroscopic data of this compound has not been reported previously, the data are shown in the experimental section. Compounds 6, 9, 12, 19, 20, 22, 23, and 26 were isolated from D. caudatum for the first time. The molecular formula of compound 1 was assigned as C20H18O7 by HRESIMS (m/z 393.0950 [M + Na]+). The 1H and 13C NMR spectra showed signals due to a 1,4-disubstituted benzene ring, a 1,2,3,4,5-pentasubstituted benzene ring, two oxygenated methines, a tertiary-methyl, an oxymethylene, a pair of cis-coupled olefinic doublets, and an oxygen-bearing quaternary sp3 carbon (Table 1). These spectroscopic data were similar to those of the flavanonol derivatives with a 2,2dimethyl-2H-pyran ring either at C-6,7 or C-7,8, except for the absence of the signal due to one of the tertiary methyl groups of a 2,2-dimethyl-2H-pyran moiety. The HMBC correlations of the tertiary methyl signal with the oxygen-bearing quaternary sp3 carbon, one of the sp2 methines (δC 124.6), and the oxymethylene suggested that one of methyl groups of 2,2dimethyl-2H-pyran moiety was replaced by a hydroxymethyl group. This was further supported by the fact that acetylation of 1 with Ac2O/pyridine yielded a tetraacetate of 1. The positions of the 2-hydroxymethyl-2-methyl-2H-pyran ring were concluded to be at C-7 and C-8 from the HMBC correlations of H1″ with C-7, C-8, and C-9 and of H-2″ with C-8. The 2,3-trans configuration was assigned from the coupling constant value of H-2/H-3 (11.5 Hz). In addition, the absolute configuration at 6349

dx.doi.org/10.1021/jf5020439 | J. Agric. Food Chem. 2014, 62, 6345−6353

Journal of Agricultural and Food Chemistry

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Figure 2. Selected COSY and HMBC correlations for compounds 1, 2, and 3.

C-2 was assigned as 2R by the CD spectral analysis; the positive Cotton effect at 321 nm and the negative one at 295 nm were similar to those of the related compounds.21−23 However, the configuration of C-3″ could not to be determined by spectral analyses. On the basis of this observation, the structure of 1 was established as shown (Figure 1). Compound 2 gave a [M + Na]+ ion peak at m/z 395.1107 in the HRESIMS, indicating the molecular formula of C20H20O7. The 1H NMR spectrum of 2 quite resembled those of 8-Cprenyl-(+)-dihydromorin (5), except for the signals due to H-2 and H-3 with small coupling constant values. The 1H−1H COSY and HMBC correlations shown in Figure 2 were in good agreement with the positions of the prenyl group at C-8 and the hydroxyl groups at C-5, C-2′, and C-4′. The 2,3-cis configuration was judged from the small coupling constant value of H-2/H-3 (2.0 Hz), and the absolute configuration at the C-2 was assigned as 2R by the CD spectral analysis, in which the positive Cotton effect at 340 nm and the negative one at 295 nm were similar to those of the related compounds.21−23 On the basis of this observation, the structure of 2 was characterized as shown (Figure 1). Compound 3 gave a quasi-molecular ion peak at m/z 409 [M + Na]+ in the ESIMS, and its molecular formula was confirmed as C21H20O7 by HRESIMS. The 1H and 13C NMR spectra of compound 3 were quite similar to those of 5 except for the observation of a methoxy signal [δH 3.90 (3H, s), δC 56.8]. Taking its molecular formula into account, compound 3 was considered to be a monomethyl ether of 5. The location of the methoxy group in 3 was assigned to be at C-7 by the analysis of HMBC and NOESY spectra. The OMe resonance only showed a NOESY correlation with H-6. A planar structure for 3 was assigned by the 1H−1H COSY and HMBC analyses (Figure 2). The 2R configuration in 3 was assigned by the analysis of CD spectra, which showed similar Cotton effects to those found in 1. From this examination, the structure of 3 was assigned as shown (Figure 1). The molecular formula of compound 15 was assigned as C20H20O7 by HRESIMS (m/z 371.1113 [M − H]−). The 1H and 13C NMR spectra were correlated to those of 27 except for the absence of the cis-coupled olefinic signals, but instead signals due to two methylenes [δH 2.57, 1.61 (each 2H, t, J = 8.0 Hz), δC 18.2, 43.8] were observed. This spectral data, coupled with the molecular formula of 3, implied the presence of a 2,2-dimethyl-dihydro-2H-pyran ring. The HMBC correlations of tertiary methyl signals with C-3″ and C-2″, H2″ with C-8, and H-1″ with C-7, C-8, C-9, and C-3″ indicate that the 2,2-dimethyl-dihydro-2H-pyran ring was attached to C7 and C-8 (Figure 3). The 2,3-trans configuration was assigned from the J value of H-2/H-3 (11.5 Hz), and the 2R configuration was elucidated by analysis of the CD spectrum.

Figure 3. Selected COSY and HMBC correlations for compounds 15−18.

Based on this examination, the structure of 15 was elucidated as shown (Figure 1). The molecular formula of 16 was determined as C21H20O6 by HRESIMS (m/z 391.1117 [M + Na]+). The 1H and 13C NMR spectra were well correlated with those of 14. However, an aromatic signal due to the A-ring of flavonoid was absent, but instead a C-methyl proton signal [δH 1.95 (3H, s), δC 7.2] was observed, suggesting compound 16 to be a C-methyl-flavanonol derivative with a 2,2-dimethyl-2H-pyran ring. The HMBC correlations of the H-1″ with C-7, C-8, and C-9 indicated that the 2,2-dimethyl-2H-pyran ring was attached to C-7 and C-8, while the location of the methyl group was concluded to be at C-6 from the HMBC correlations of the methyl signal with C-5, C-6, and C-7 (Figure 3). The trans-configuration of H-2 and H3 was assigned from the J-value of H-2 and Hax-3 (11.5 Hz), 6350

dx.doi.org/10.1021/jf5020439 | J. Agric. Food Chem. 2014, 62, 6345−6353

Journal of Agricultural and Food Chemistry

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Table 3. MIC Values of Compounds against Film-Forming Growth of Zygosaccharomyces rouxii F51, CLog P Values, and Their Isolated Yields (%) From D. caudatum yields (%) isolated from D. caudatum compd

systematic name

MIC (μg/mL)

1 2 4 5 7 8 9 10 11 12 13 14 20 21 22 24 25 27 28 29 30 31 32 33 34 35 36 37

2″-hydroxymethyl-2″-methylpyran-(5″,6″:7,8)-5,4′-(2R,3R)-dihydroxydihydroflavonol 8-(γ,γ-dimethylallyl)-5,7,2′,4′-tetrahydroxy-(2R,3S)-dihydroflavonol 6-(γ,γ-dimethylallyl)-5,7,2′,4′-tetrahydroxy-(2R,3R)-dihydroflavonol 8-(γ,γ-dimethylallyl)-5,7,2′,4′-tetrahydroxy-(2R,3R)-dihydroflavonol 8-(γ,γ-dimethylallyl)-5,7,4′-trihydroxy-3′-methoxy-(2R,3R)-dihydroflavonol 8-(γ,γ-dimethylallyl)-5,7,4′-trihydroxy-(2R,3R)-dihydroflavonol 8,3′-di(γ,γ-dimethylallyl)-5,7,4′-trihydroxy-(2R,3R)-dihydroflavonol 5,7,4′-trihydroxy-(2R,3R)-dihydroflavonol 2″,2″-dimethylpyran-(5″,6″:7,8)-5,2′-dihydroxy-4′-methoxy-(2R,3R)-dihydroflavonol 2″,2″-dimethylpyran-(5″,6″:7,8)-5,3′-dihydroxy-4′-methoxy-(2R,3R)-dihydroflavonol 10-(γ,γ-dimethylallyl)-3,9,13-trihydroxy-6,12-metano-6H,12H-dibenzo[b,f ][1,5]dioxocin 2″,2″-dimethylpyran-(5″,6″:7,8)-5,4′-dihydroxy-(2R,3R)-dihydroflavonol 7,4′-dihydroxy-(2R,3R)-dihydroflavonol 6-(γ,γ-dimethylallyl)-5,7,2′,4′-tetrahydroxyflavonol 5,7,4′-trihydroxyflavonol 2″,2″-dimethylpyran-(5″,6″:7,8)-5,4′-dihydroxyflavonol 8-(γ,γ-dimethylallyl)-5,7,2′,4′-tetrahydroxy- (2R)-flavanone 2″,2″-dimethylpyran-(5″,6″:7,8)-5,2′,4′-trihydroxy-(2R,3R)-dihydroflavonol 2″,2″-dimethylpyran-(5″,6″:6,7)-5,2′,4′-trihydroxy-(2R,3R)-dihydroflavonol 8-(γ,γ-dimethylallyl)-5,7,2′-trihydroxy-4′-methoxy-(2R)-flavanone 8-(γ,γ-dimethylallyl)-5,7,4′-trihydroxy-(2R)-flavanone 5,7,4′-trihydroxy-(2R)-flavanone 2″,2″-dimethylpyran-(5″,6″:7,8)-5,3′,4′-trihydroxy-6-methyl-(2R)-flavanone 6-(γ,γ-dimethylallyl)-5,7,4′-trihydroxyflavonol 2″,2″-dimethylpyran-(5″,6″:7,8)-5,2′,4′-trihydroxyflavonol 2″,2″-dimethylpyran-(5″,6″:6,7)-5,4′-dihydroxy-4′-methoxy-flavonol 2″,2″-dimethylpyran-(5″,6″:7,8)-5,2′,4′-trihydroxy-6-methyl-flavone 2″,2″-dimethylpyran-(5″,6″:7,8)-5,3′,4′-trihydroxy-6-methyl-flavone Tween 80

125.0 62.5 62.5 31.3 125.0 125.0 >125.0 125.0 7.8 31.3 >125.0 15.6 125.0 62.5 15.6 125.0 62.5 62.5 31.3 125.0 >125.0 125.0 31.3 62.5 31.3 15.6 125.0 >125.0