Chinese Journal of Natural Medicines 2015, 13(4): 02990302

Chinese Journal of Natural Medicines

Two new conjugated ketonic fatty acids from the stem bark of Juglans mandshurica YAO Da-Lei1, ZHANG Chang-Hao1Δ, LI Ren2, LUO Jie1, JIN Mei1, 2,PIAO Jin-Hua1, ZHENG Ming-Shan1, CUI Jiong-Mo1, SON Jong Keun3, LI Gao1* 1

Key Laboratory of Natural Resources of Changbai Mountain and Functional Molecules, Ministry of Education, Yanbian Univer-

sity College of Pharmacy, Yanji 133002, China; 2

Department of Pharmacy, Yanbian University Hospital, Yanji 133000, China;

3

College of Pharmacy, Yeungnam University, Gyongsan 712-749, Korea Available online 20 Apr. 2015

[ABSTRACT] The present study was designed to isolate and characterize novel chemical constituents of the stem bark of Juglans mandshurica Maxim. (Juglandaceae).The chemical constituents were isolated and purified by various chromatographic techniques. The structures of the compounds were elucidated on the basis of spectral data (1D and 2D NMR, HR-ESI-MS, CD, UV, and IR) and by the comparisons of spectroscopic data with the reported values in the literatures. Two long chain polyunsaturated fatty acids (1 and 2) were obtained and identified as (S)-(8E,10E)-12-hydroxy-7-oxo-8,10-octadecadienoic acid (1) and (S)-(8E, 10E)-12-hydroxy7-oxo-8,10-octadecadienoic acid methyl ester (2). To the best of our knowledge, this is the first report on the isolation and structural elucidation of the two new conjugated ketonic fatty acids from this genus. [KEY WORDS] Juglans mandshurica; Juglandaceae; Conjugated ketonic fatty acid

[CLC Number] R284.1

[Document code] A

[Article ID] 2095-6975(2015)04-0299-04

Introduction The stem bark of Juglans mandshurica Maxim. (Juglandaceae) has long been used as a folk medicine for the treatment of cancer in China [1]. A number of compounds, such as naphthoquinones, naphthalenyl glucosides, tetralones, flavonoids, diarylheptanoids and galloyl glycosides, have previously been isolated from Juglans species [2-19], and some of these compounds have been shown to have cytotoxicity, anti-oxidant activity, and inhibitory effects on topoisomerases I and II, DNA polymerase, and RNase H activity of HIV-1 reverse transcriptase [3,7,9,12-19]. The long chain polyunsaturated fatty acids (LCPUFAs) are imperative to plant survival and have diverse [Received on] 28-Mar.-2014 [Research funding] This work was supported by the National Natural Science Foundation of China (Nos. 81160386, 81260474, 30911140276). [*Corresponding author] Tel: 86-433-2436022. E-mail: gli@ybu. edu.cn. Δ Co-first author. All the authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved

roles throughout the course of plant development; they often have 18 or more carbons and two or more double bonds [20]. Conjugated ketonic fatty acids as LCPUFAs have been found in mushrooms[21-24] and other natural plants [25-26], showing cytotoxic activity, aldehyde dehydrogenase inhibitory activity and toxic effects on brine shrimp larvae [21,24-26]. In the continuation of our studies on this plant [7-11], we recently isolated two new conjugated ketonic fatty acids (Fig. 1) from the chloroform solvent fraction of the methanolic extract of stem bark of J. mandshurica. Herein, we describe the structural determination of the two compounds on the basis of spectroscopic studies. It is hoped these results will provide a basis for future investigation towards the best use of these compounds and the plant in the treatment of human diseases.

Results and Discussion Two conjugated ketonic fatty acids were isolated from a CHCl3 solvent fraction of the MeOH extract of the stem bark of J. mandshurica by repetitive column chromatography and preparative HPLC using a RP-C18 column.

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Fig. 1 Structures of compounds 1 and 2

Compound 1 had the molecular formula C18H30O4 as determined by the HR-ESI-MS, 13C NMR, and DEPT spectral data. 1H NMR spectrum showed signals due to two conjugated E,E-form enone systems [δ 6.14 (1H, d, J = 16.0 Hz, H-8), 6.33 (1H, dd, J = 3.8, 16.0 Hz, H-11), 6.33 (1H, dd, J = 10.4, 16.0 Hz, H-10), 7.12 (1H, dd, J = 10.4, 16.0 Hz, H-9)], and one methine [4.20 (1H, m, H-12)], 10 methylene [1.33 (10H, m, H-4, 14, 15, 16, 17 ), 1.58 (6H, m, H-3, 5, 13), 2.34 (2H, t, J = 7.2 Hz, H-2), and 2.55 (2H, t, J = 7.2 Hz, H-6)], and one methyl [0.89 (3H, t, J = 7.2 Hz, H-18)], which could be attributed to the terminal methyl to methylene groups of aliphatic chain. The 13C NMR and DEPT spectra exhibited a total of 18 car-

Fig. 2

1

bon signals, including characteristic signals due to a carboxylic acid at δ 177.9 (C-1), a ketone at δ 201.5 (C-7), and four olefinic carbons [δ 127.9 (C-10), 129.7 (C-8), 142.2 (C-11), and 146.2 (C-9)], along with a hydroxymethine carbon at δ 72.0 (C-12). The 1H-1H COSY (Fig. 2) spectrum showed connectivity among H-2, H-3, H-4, H-5 and H-6, and among H-8, H-9, H-10, H-11, H-12, H-13 and H-14, and between H-17 and H-18. In the HMBC spectrum, the locations of a carbonyl carbon (C-7) and a carboxylic acid (C-1) were indicated by the cross peaks between C-7 and H-5, H-6, H-8 and H-9, and those between C-1 and H-2 and H-3. Based on the above data, Compound 1 was determined to be (8E, 10E)-12-hydroxy-7-oxo-8,10-octadecadienoic acid. A racemic mixture of Compound 1 was isolated from the fungus Pleurocybella porrigens by Amakrura et al.[23] When the optical rotation [] 25 –8.83° (c = 0.50, MeOH) was measured, it was D not due to a racemic mixture compound. While the CD spectrum of Compound 1 exhibited the negative Cotton effect at 217.7 nm, indicating that the absolute stereochemistry of C-12 was S configuration [24]. Thus Compound 1 was determined to be (S)-(8E,10E)-12-hydroxy-7-oxo-8,10-octadecadienoic acid.

H-1H COSY and HMBC correlations of compounds 1 and 2

Compound 2 had the molecular formula C19H32O4 as determined by the HR-ESI-MS, 13C NMR, and DEPT spectral data. The 1H and 13C NMR data were very similar to those of Compound 1, indicating that this compound was C18 fatty acid having an α, β, γ, δ-dienone group and the methyl ester of Compound 1. The location of the methoxyl group was identified by a cross-peak between the C-1 and C-1-OCH3 in the HMBC spectrum (Fig. 2). Also, Compound 2 was optically active and the optical rotation [α] 25 –7.20° (c D = 0.11, MeOH) was detected. The CD spectrum of Compound 2 exhibited the negative Cotton effect at 218.1 nm, indicating that the absolute stereochemistry of C-12 was S configuration [24] . The structure of Compound 2 was therefore determined to be (S)-(8E,10E)-12-hydroxy-7-oxo-8,10-octadecadienoic acid methyl ester.

Experimental General experimental procedures Optical rotations were measured using a Rudolph Auto-

Autopol I (Rudolph Research Analytical, Hackettstown, NJ, USA) automatic polarimeter. CD spectra were recorded on a JASCO J-810 spectropolarimeter (JASCO Analytical Instruments, Easton, MD, USA). UV spectra were measured on a Dynamica Halo DB-20 UV/Visible double beam spectrophotometer (Dynamica Pty Ltd., Prahran, Victoria, Australia). NMR spectra were recorded on a Bruker 400 MHz instrument (AV-400, Berne, Switzerland) for Compound 1, and on a Bruker 500 MHz instrument (AV-500, Berne, Switzerland) for Compound 2. CDCl3 was used as solvent. Chemical shifts were reported in ppm downfield from TMS. The HR-ESI-MS spectra were measured with a Bruke micrOTOF QII mass spectrometer (Bruker Daltonics, Fremont, CA, USA) equipped with an electrospray ionization (ESI) source. Flash chromatography was performed on a Bona-Agela CHEETAHTM MP200 system (Bonna-AgelaTechnologies, Tianjin, China). For preparative HPLC, LC-10AD pump (Shimadzu, Kyoto, Japan), SPD-10A detector (Shimadzu), and Shim-Pack Prep-ODS (20 mm  250 mm) column (Shimadzu) were used. Column chromatography was performed using silica gel (70–230,

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200–300 and 270–400 mesh, Branch of Qingdao Marine Chemical Co., Ltd., Qingdao, China). Sephadex LH-20 was purchased from GE Healthcare Bio-Sciences AB (GE Healthcare, Uppsala, Sweden). TLC was performed with precoated Silica gel GF254 glass plates (GF254, Branch of Qingdao Marine Chemical Co., Ltd., Qingdao, China). All other chemicals and solvents were of analytical grade and used without further purification. Plant materials The stem bark of J. mandshurica was collected in August 2006 in a mountainous area of Yanji City, Jilin Province, P. R. China, and were identified and authenticated by Prof. LV Hui-Zi (College of Pharmacy, Yanbian University, Yanji, China). A voucher specimen (YBH-HT-0112) had been deposited at the College of Pharmacy, Yanbian University. Extraction and isolation The stem bark of J. mandshurica (10 kg) was extracted thrice with refluxing MeOH (70%), and concentrated under reduced pressure. The crude extract was mixed with H2O (1 L) to form a suspension and then partitioned successively with CHCl3, EtOAc, and BuOH (1 L each  3). The CHCl3 solvent fraction (50 g) was fractionated with a flash chromatography system using a Bonna-Agela G46049-1 cartridge (CHCl3–MeOH, 1 : 99 to 100 : 0, V/V) to obtain 10 subfractions, A–J. The fraction Table 1 1H NMR and 13C NMR data for Compounds 1 (1H, 400 MHz; 13C, 100 MHz; CDCl3) and 2 (1H, 500 MHz; 13C, 125 MHz; CDCl3) Position 1

1

2

δH (J Hz)

δC

δH (J Hz)

δC



177.9



174.4

1-OCH3





3.66 s

51.6

2

2.34 t (7.3)

34.0

2.28 t (6.0)

34.2

3

1.58 m

24.8

1.57 m

25.0

4

1.33m

29.1

1.29 m

29.2

5

1.58 m

24.4

1.57 m

24.4

6

2.55 t (7.2)

40.6

2.53 t (5.9)

40.8

7



201.5



201.0

8

6.15 d (16.0)

129.7

6.15 d (16.0)

129.9

9

7.12 dd (10.4, 16.0) 146.2

7.12 dd (10.3, 16.0)

141.7

10

6.33 dd (10.4, 16.0) 127.9

6.33 dd (10.3, 16.0)

128.0

11

6.14 dd (3.8, 16.0)

142.2

6.14 dd (3.6, 16.0)

145.9

12

4.2 m

72.0

4.23 dd (5.5, 11.0)

72.2

13

1.58 m

36.8

1.57 m

36.9

14

1.33 m

27.5

1.29 m

27.6

15

1.33 m

29.1

1.25 m

29.9

16

1.33 m

28.9

1.25 m

29.9

17

1.33 m

22.7

1.29 m

22.7

18

0.89 m

14.1

0.89 t (5.6)

14.2

G (1.5 g) was further separated using a flash chromatography system (MeOH–H2O, 2 : 8 to 9 : 1, V/V) to obtain 15 subfractions (G1–G15). Fraction G5 (180 mg) was applied to a Sephadex LH-20 column eluted with MeOH to give a major fraction, which was purified by preparative HPLC (MeOH–H2O, 5 : 5 to 9 : 1, V/V) to yield Compound 1 (9 mg). Fraction G6 (160 mg) was purified on a flash chromatography system with MeOH–H2O (3 : 7 to 9 : 1, V/V), followed by preparative HPLC with MeOH–H2O gradients (5 : 5 to 9 : 1, V/V) to afford Compound 2 (13.5 mg).

Identification (S)-(8E, 10E)-12-hydroxy-7-oxo-8,10-octadecadienoic –8.83° (c = 0.50, MeOH); acid (1) Brown solid (9 mg), [] 25 D CD (MeOH) Δε217.7 –4.83; UV (MeOH) λmax (logε) 268.0 (2.382), 206.0 (1.300); IR vmax (KBr) cm–1 3 429, 2 930, 2 858, 2 364, 1 716, 1 466, 1 202, 1 009; 1H and 13C NMR data (Table 1); and HR-ESI-MS m/z 309.206 6 [M – H]– (Calcd. for C18H29O4, 309.206 6). (S)-(8E, 10E)-12-hydroxy-7-oxo-8,10-octadecadienoic acid methyl ester (2) Yellow solid (13.5 mg), [] 25 –7.20° (c D = 0.11, MeOH); CD (MeOH) Δε218.1 –5.57; UV (MeOH) λmax (logε) 275.0 (1.668), 226.0 (2.776); IR vmax (KBr) cm–1 3 439, 2 930, 2 858, 2 365, 1 736, 1 466, 1 380, 1 178; 1H and 13 C NMR data (Table 1); and ESI-MS m/z 325.231 2 [M + H]+ (Calcd. for C19H33O4, 325.237 9).

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Cite this article as: YAO Da-Lei, ZHANG Chang-Hao, LI Ren, LUO Jie, JIN Mei, PIAO Jin-Hua, ZHENG Ming-Shan, CUI Jiong-Mo, SON Jong Keun, LI Gao. Two new conjugated ketonic fatty acids from the stem bark of Juglans mandshurica[J]. Chinese Journal of Natural Medicines, 2015, 13(4): 299-302

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Two new conjugated ketonic fatty acids from the stem bark of Juglans mandshurica.

The present study was designed to isolate and characterize novel chemical constituents of the stem bark of Juglans mandshurica Maxim. (Juglandaceae). ...
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