New hydroxy fatty acid from the root bark of Morus alba L

New hydroxy fatty acid from the root bark of Morus alba L. Jae-Woo Jung, Ji-Hae Park, Kyeong-Hwa Seo, Eun-Ji Oh, Dae-Young Lee, DongWook Lim, Daeseok Han, MyoungChong Song & Nam-In Baek Journal of the Korean Society for Applied Biological Chemistry ISSN 1738-2203 J Korean Soc Appl Biol Chem DOI 10.1007/s13765-015-0071-5 1 23 Your article is protected by copyright and all rights are held exclusively by The Korean Society for Applied Biological Chemistry. This e-offprint is for personal use only and shall not be self-archived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com”. 1 23 Author's personal copy J Korean Soc Appl Biol Chem DOI 10.1007/s13765-015-0071-5 Online ISSN 2234-344X Print ISSN 1738-2203 NOTE New hydroxy fatty acid from the root bark of Morus alba L. Jae-Woo Jung . Ji-Hae Park . Kyeong-Hwa Seo . Eun-Ji Oh . Dae-Young Lee . Dong-Wook Lim . Daeseok Han . Myoung-Chong Song . Nam-In Baek Received: 17 February 2015 / Accepted: 10 April 2015 Ó The Korean Society for Applied Biological Chemistry 2015 Abstract A new hydroxyl fatty acid, named sangbaimoric acid (1), was isolated from the root bark of Morus alba L. The chemical structure of compound 1 was established as 2-methoxyoctadeca-3Z,5Z-dienoic acid on the basis of spectroscopic analyses including nuclear magnetic resonance, high-resolution electronic ionization mass spectroscopy, and infrared spectroscopy experiments. Keywords 2-Methoxyoctadeca-3Z,5Z-dienoic acid
Morus alba L.
Nuclear magnetic resonance
Sangbaimoric acid Bai-Pi’’ and are used for a variety of medicinal purposes, primarily in South Asia. Previously, many phytochemical compounds such as flavonoids, Diels–Alder type adducts, coumarins, stilbenes, and triterpenoids (Hano et al. 1988; Piao et al. 2009; Jung et al. 2014) have been isolated from the root bark of M. alba. These compounds have been reported to show anti-oxidant, anti-inflammatory, anticancer, and anti-microbial activities (Dat et al. 2010; Yang et al. 2011; Yang and Lee 2012; Naik et al. 2015). In order to identify new biological constituents of the root bark of M. alba, we conducted a phytochemical study. Introduction Materials and Methods The mulberry tree, Morus alba L. (Moraceae), a deciduous broad-leaved tree, is native to Thailand and also widely distributed in Europe, America, and Asia (Sohn et al. 2009). The root barks of mulberry trees are named ‘‘Sang- The root barks of M. alba were extracted with 80 % MeOH and the concentrated extract was successively partitioned by polarity gradient using ethyl acetate (EtOAc), normalbutylalcohol (n-BuOH), and H2O. From the EtOAc fraction, repeated open column chromatography (c.c.) through the silica gel (SiO2), octadecyl SiO2 (ODS), and sephadex LH-20 afforded a new fatty acid, which was subsequently identified by spectroscopic data analyses including nuclear magnetic resonance (NMR), high-resolution electronic ionization mass spectroscopy (HR/EI/MS), and infrared spectroscopy (IR) experiments. In our study, dried and powdered root barks of M. alba (10 kg) were extracted with 80 % MeOH (68 L 9 3) at room temperature for 24 h. Next, the concentrated MeOH extract (1.7 kg) was suspended in 2 L water and successively extracted with EtOAc (2 L 9 2) and n-BuOH (1.8 L 9 3). The organic and aqueous layers were concentrated to produce residues of the EtOAc fraction (MRE, 580 g), the nBuOH fraction (MRB, 114 g), and the H2O fraction (MRW, 1006 g), respectively. The EtOAc fraction (122 g) was J.-W. Jung
J.-H. Park
K.-H. Seo
E.-J. Oh
N.-I. Baek (&) Department of Oriental Medicine Biotechnology, Graduate School of Biotechnology, Kyung Hee University, Yongin 446-701, Republic of Korea e-mail: nibaek@khu.ac.kr D.-Y. Lee Department of Herbal Crop Research, National Institute of Horticultural and Herbal Science, RDA, Eumseong 369-873, Republic of Korea D.-W. Lim
D. Han Division of Metabolism and Functionality Research, Korea Food Research Institute, Sungnam 463-746, Republic of Korea M.-C. Song Intelligent Synthetic Biology Center, KAIST, Daejeon 305-701, Republic of Korea 123 Author's personal copy J Korean Soc Appl Biol Chem fractionated by SiO2 c.c. (12.5 9 17 cm) eluting with nhexane–EtOAc (4:1 ? 2:1 ? 1:1, 27 L of each) and CHCl3–MeOH (10:1, 27 L) to yield 41 fractions (MRE-1 to MRE-41). Fraction MRE-23 [848 mg, elution volume/total volume (Ve/Vt) 0.422–0.528] was applied to ODS c.c. (4.5 9 6 cm) and eluted with MeOH-H2O (3:1 ? 8:1, 3 L of each), yielding 12 fractions (MRE-23-1 to MRE-23-12). Subfraction MRE-23-9 (120 mg, Ve/Vt 0.507–0.651) was subjected to sephadex LH-20 c.c. (1.5 9 60 cm) and eluted with MeOH–H2O (4:1, 0.7 L) to yield five fractions (MRE23-9-1 to MRE-23-9-5) including compound 1, MRE-23-9-1 [12 mg (yield 0.0006 %), Ve/Vt 0.00–0.12, TLC (ODS) Rf 0.51, MeOH–H2O = 12:1]. Compound 1 (sangbaimoric acid): Yellow oil; [a]25 D ?19.1° (c 0.82, MeOH); IR (KBr, m): 3450, 1732, 1681 cm-1; HR/EI/MS m/z 310.2513 [M]? (calcd. for C19H34O3 310.2509); 1H-NMR (400 MHz, CD3OD, dH): 6.48 (1H, dd, J = 10.8, 10.8 Hz, H-4), 5.99 (1H, dd, J = 10.8, 10.8 Hz, H-5), 5.45 (1H, dt, J = 10.8, 8.0 Hz, H-6), 5.41 (1H, dd, J = 10.8, 8.0 Hz, H-3), 3.61 (1H, d, J = 8.0 Hz, H-2), 3.22 (3H, s, –OCH3), 2.19 (2H, m, H-7), 1.39 (2H, m, H-8), 1.31-1.28 (14H, m, H-9 * H-15), 0.89 (3H, t, J = 6.4 Hz, H-18); 13C-NMR (100 MHz, CD3OD, dC): 172.88 (C-1), 134.39 (C-3), 133.64 (C-6), 129.44 (C-4), 128.97 (C-5), 83.75 (C-2), 56.32 (–OCH3), 36.62 (C-8), 32.49 (C-16 or 17), 30.53 * 30.36 (C-10, 11, 12, 13, 14, 15), 28.57 (C-7), 26.39 (C-9), 23.56 (C-16 or 17), 14.39 (C-18). Results and Discussion The fatty acid is a carboxylic acid with a long aliphatic chain that was either saturated or unsaturated. C16 and C18 unsaturated fatty acids are more common in the plant kingdom. In addition, hydroxyl fatty acids with C16 and C18 chains are observed often, and have been reported to possess several biological activities such as anti-bacterial (Kurata et al. 2010), anti-inflammatory (Masayuki et al. 1998), and anti-melanogenesis effects (Fujita et al. 2010). In this study, we isolated and identified a new hydroxyl fatty acid from the root barks of M. alba. Interestingly, this compound is a hydroxyl fatty acid with a C-2 hydroxylated moiety, which has been rarely reported in the literature. Fig. 1 Chemical structure of compound 1 isolated from the root bark of Morus alba L. 123 Compound 1 was isolated as a yellow oil, was UV absorption active, and appeared as a brown spot color on TLC plates upon spraying with 10 % sulfuric acid followed by heating. The molecular weight was determined to be 310 from the molecular ion peak m/z 310 [M]? in the EI/MS spectrum, and a molecular formula of C19H34O3 was elucidated on the basis of the high-resolution molecular ion peak m/z 310.2509 [M]? (calcd for 310.2513, C19H34O3) in the HR/EI/MS. The IR absorbance bands of hydroxyl (3450 cm-1), carbonyl (1732 cm-1), and double bond (1681 cm-1) groups were observed. In the 1H-NMR spectrum, four olefinic methine signals at dH 6.48 (1H, dd, J = 10.8, 10.8 Hz, H-4), 5.99 (1H, dd, J = 10.8, 10.8 Hz, H-5), 5.45 (1H, dt, J = 10.8, 8.0 Hz, H-6), and 5.41 (1H, dd, J = 8.0, 10.8 Hz, H-3) were observed. In the oxygenated proton region, one oxygenated methine signal at dH 3.61 (1H, d, J = 8.0 Hz, H-2) and one oxygenated methyl signal at dH 3.22 (3H, s, –OCH3) were detected. In the high magnetic fields, nine methylene signals including overlapping signals at dH 2.19 (2H, m, H-7), 1.39 (2H, m, H-8), and 1.31–1.28 (14H, m, H-9 * 15) were detected, along with a terminal methyl signal at 0.89 (3H, t, J = 6.4 Hz, H-18). According to the above-described proton signals, compound 1 was assumed to be an unsaturated fatty acid that included one hydroxyl group. The 13 C-NMR spectrum showed 19 carbon signals including one methoxy carbon signal. Specifically, the observed carbon signals were as follows: one carbonyl at dC 172.88 (C-1); four olefine methines at dC 134.39 (C-3), 133.64 (C-6), 129.44 (C-4), and 128.97 (C-5); one oxygenated methine at dC 83.75 (C-2); and one methoxy at dC 56.32 (–OCH3). In addition, at high magnetic fields, 11 methylene carbon signals at dC 23.56–36.62 (C-7 * 17) and terminal methyl carbon signal at dC 14.39 (C-18) were observed, indicating the presence of a C18 fatty acid with two double bonds, a hydroxyl group, and a methoxy group. The locations of the functional groups in compound 1 were determined by 2D NMR (gCOSY, gHSQC, gHMBC) experiments. In the gCOSY experiments, the connectivity of C-2 to C-8 and C-16 to C-18 was successfully determined on the basis of the correlations among neighboring proton signals. In the gHMBC experiment, the oxygenated methine proton signal at dH 3.61 (H-2) showed the cross Author's personal copy J Korean Soc Appl Biol Chem peaks with the carbonyl carbon signal at dC 172.88 (C-1) and the methoxy signal at dC 56.32 (–OCH3), indicating the position of the carbonyl, methoxy, and hydroxy groups. Likewise, the stereostructures of both double bonds were, respectively, identified as 3Z and 5Z from the coupling constants (J) between the olefine methine proton signals (H-3/H-4: J = 10.8 Hz, H-5/H-6: J = 10.8 Hz). The stereochemistry of the chiral carbon, C-2, was not specified in this study. Taken together, the structure of compound 1 was determined to be 2-methoxyoctadeca-3Z,5Z-dienoic acid, a new hydroxy fatty acid, which we named sangbaimoric acid (Fig. 1). Acknowledgments This study was financially supported from the Korea Food Research Institute (Grant Number 20140398U0054101S 00100) and the Kyung Hee University (sabbatical year project: 20150142), Republic of Korea. References Dat NT, Binh PTX, Quynh LTP, Minh CV, Huong HT, Lee JJ (2010) Cytotoxic prenylated flavonoids from Morus alba. Fitoterapia 81:1224–1227 Fujita H, Hongo M, Mochizuki M, Yokoyama K, Tanaka Y (2010) Inhibitory effects of 16-hydroxy-9-oxo-10E,12E,14E-octadecatrienoic acid (corchorifatty acid B) isolated from Melissa officinalis linne on melanogenesis. Exp Dermatol 20:420–424 Hano Y, Suzuki S, Nomura T, Litakam Y (1988) Absolute configuration of natural Diel–Alder type adducts from the Morus root bark. Heterocycles 27:2315–2325 Jung JW, Park JH, Jung YJ, Lee CH, Han DS, Baek NI (2014) Isolation and identification of triterpenoids from the Mulberry (Morus alba) root bark. J Appl Biol Chem 57:295–299 Kurata I, Umekita M, Sawa T, Hattori S, Hayashi C, Kinoshita N, Homma Y, Igarashi M, Hamada M, Watanabe T, Sawa R, Naganawa H, Takahashi Y, Akamatsu Y (2010) Paleic acid, a fatty acid from Paenibacillus sp.: taxonomy, fermentation, isolation, structure determination, and anti-Mannheimia and -Pasteurella activity. J Antibiot 63:519–523 Masayuki Y, Yoshiyuki M, Hiromi S, Satoshi Y, Masami S, Johji Y, Hisashi M (1998) Medicinal foodstuffs. XIV. On the bioactive constituents of moroheiya. (2): new fatty acids, corchorifatty acids A, B, C, D, E, and F, from the leaves of Corchorus olitorius L. (Tiliaceae): structures and inhibitory effect on NO production in mouse peritoneal macrophages. Chem Pharm Bull 46:1008–1014 Naik R, Harmalkar DS, Xuezhen X, Jang K, Lee K (2015) Bioactive benzofuran derivatives: Moracins A–Z in medicinal chemistry. Eur J Med Chem 90:379–393 Piao SJ, Qiu F, Chem LX, Pan Y, Dou DQ (2009) New stilbene, benzofuran, and coumarin glycosides from Morus alba. Helv Chim Acta 92:579–587 Sohn BH, Park JH, Lee DY, Cho JG, Kim YS, Jung IS, Kang PD, Baek NI (2009) Isolation and identification of lipids from the silkworm (Bombyx mori) droppings. J Korean Soc Appl Biol Chem 52:336–341 Yang JY, Lee HS (2012) Evaluation of antioxidant and antibacterial activities of morin isolated from mulberry fruits (Morus alba L.). J Korean Soc Appl Biol Chem 55:485–489 Yang ZG, Matsuzaki K, Takamatsu S, Kitanaka S (2011) Inhibitory effects of constituents from Morus alba var. multicaulis on differentiation of 3T3-L1 cells and nitric oxide production in RAW264.7 cells. Molecules 16:6010–6022 123 View publication stats