Food Sci. Biotechnol. 20(2): 555-560 (2011) DOI 10.1007/s10068-011-0078-6

RESEARCH NOTE

Isolation and Identification of Cinnamic Acid Amides as Antioxidants from Allium fistulosum L. and Their Free Radical Scavenging Activity Gee-Woo Seo, Jeong-Yong Cho, Jae-Hak Moon, and Keun-Hyung Park

Received: 18 October 2010 / Revised: 22 December 2010 / Accepted: 23 December 2010 / Published Online: 30 April 2011 © KoSFoST and Springer 2011

Gee-Woo Seo, Jeong-Yong Cho, Jae-Hak Moon, Keun-Hyung Park ( ) Department of Food Science & Technology, and Functional Food Research Center, Chonnam National University, Gwangju 500-757, Korea Tel: +82-62-530-2143; Fax: +82-62-530-2149 E-mail: [email protected]

onion is a popular flavoring vegetable in Asian countries including China, Japan, and Korea (1). The plant is widely cultivated in southern areas of Korea, especially Jindo. Welsh onion has traditionally been used for salads and cooking. In Korea, the roots and trunks have long been used as a traditional folk medicine for the treatment of febrile disease, headache, abdominal pain, diarrhea, and habitual abortion (2). In addition, several studies have recently reported that Welsh onion showed various biological effects such as anti-aggregation (3,4), anti-hypertensive (57), antioxidant (6-8), hepatoprotective (9), and antifungal (10) activities. The Welsh onion contains abundant sulfur-based compounds that are responsible for its flavor, especially propenyl propyl disulfides (11). Fistulosides (steroidal saponins) and fistulosin (octadecyl 3-hydroxyindole) isolated from the edible parts and the roots of the onion have been identified as antifungal compounds (2,10). However, the various beneficial compounds of the Welsh onion have not yet been thoroughly investigated. Previously, we isolated and purified antioxidants from the EtOAc-soluble acidic fraction of Welsh onion MeOH extract using a guided DPPH radical scavenging assay (12). The results of that study revealed the presence of 4 phenolic compounds (4hydroxybenzoic acid, 4-hydroxy-3-methoxy-benzoic acid, 4-hydroxycinnamic acid, and 4-hydroxy-3-methoxycinnamic acid), which were also found to have antioxidant activities (12). In the present study, 4 additional antioxidative compounds were isolated from the EtOAcsoluble neutral fraction of Welsh onion MeOH extract.

Gee-Woo Seo Food Safety Management Division, Gwangju Regional Korea Food & Drug Administration, Gwangju 500-757, Korea

Materials and Methods

Jeong-Yong Cho Department of Food Engineering and Solar Salt Biotechnology Research Center, Mokpo National University, Muan, Jeonnam 534-729, Korea

Plant material Welsh onion (var. Keumjang) was cultivated in Jindo, Korea and harvested in December.

Abstract Four compounds obtained from the ethyl acetate-soluble neutral fraction of methanol extracts of the Welsh onion (Allium fistulosum L.) were purified by column chromatography using silica gel, octadecyl silane (ODS), and Sephadex LH-20 with a guided DPPH radical scavenging assay. After purification, the compounds were isolated by ODS-HPLC. The isolated compounds were identified as N-trans-feruloyl-3'-methoxytyramine (1), Ncis-feruloyl-3'-methoxytyramine (2), N-trans-p-coumaroyltyramine (3), and 3,5,7-trihydroxyflavone (kaempferol, 4) based on the spectroscopic data of NMR and MS. To the best of our knowledge, this is the first study to identify compound 1-3 in the Welsh onion. Compound 1 and 2 showed significantly (p<0.05) higher DPPH radical scavenging activities than compound 3. Keywords: Allium fistulosum, Welsh onion, cinnamic acid amide, DPPH radical, antioxidant

Introduction Welsh onion (green onion) is a perennial herb that is classified as an Allium species, which includes onion (Allium cepa L.) and garlic (Allium sativum L.). Welsh

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After the roots were removed, the edible parts were washed with water and the samples were stored at −70oC until use. Chemicals DPPH and α-tocopherol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Methanol-d4 (CD3OD) was obtained from Merck (Darmstadt, Germany). All other chemicals used in this study were of reagent grade and were obtained from commercial sources. Isolation of antioxidative compounds from MeOH extract The fresh edible parts (9.0 kg) of the plant were homogenized with methanol (MeOH, 36 L) using a homogenizer (BM-2 Nissei bio-mixer; Nihonseiki Kaiseiki Ltd., Tokyo, Japan). After extraction for 24 h at room temperature, the mixture was filtered with a glass filter (G3). The filtrate was then concentrated in vacuum using a rotary evaporator (N-2N; Eyela, Tokyo, Japan). The MeOH extracts were subsequently suspended in phosphate buffer (0.2 M NaH2PO4-0.2 M Na2HPO4, pH 8.0, 2 L) and partitioned with ethyl acetate (EtOAc, 2 L, 3 times) to give an EtOAc-soluble neutral fraction. The aqueous layer was adjusted to pH 3.0 with 1.0 N HCl and then partitioned with EtOAc (5 L, 3 times) to yield the EtOAc-soluble acidic fraction. The EtOAc-soluble neutral fraction was subjected to column (3.0×65 cm) chromatography on silica gel (Kieselgel 60, 70-230 mesh; Merck). The elution was conducted using n-hexane/EtOAc/MeOH =10:4:1, 8:6:1, 6:8:1, 4:10:1, and 2:12:1 (v/v, each 1.5 L). Fraction A was chromatographed on a Sephadex LH-20 column (25100 mesh; Pharmacia Fine Chemicals, Uppsala, Sweden, 1.9×90 cm, total volume 250 mL) using MeOH/CHCl3= 4:1 (v/v) as the solvent. The active fraction was further fractionated on an octadecyl silane (ODS) column (1.0×23 cm, 25-100 mesh; Pharmacia Fine Chemicals) and eluted with H2O/MeOH (7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:10, v/v, step-wise system, each 140 mL). Fraction B was subjected to column (2.0×25 cm) chromatography on a silica gel (Merck) and eluted with n-hexane/EtOAc/ MeOH=12:3:1 and 11:4:1 (v/v, each 1.5 L). The active fraction was chromatographed on a Sephadex LH-20 as described above. The active fractions were then subjected to HPLC on an ODS column (µBondapak, 7.8×300 mm, 10 µm; Waters, Milford, MA, USA) at a flow rate of 8.0 mL/min and a wavelength of 254 nm using 60 or 40% MeOH [0.1% trifluoroacetic acid (TFA), pH 2.6] as the mobile phases. Structural analysis NMR spectra were obtained using an unitINOVA 500 spectrometer (Varian, Walnut Creek, CA, USA) with solvents as the internal standards. All of the isolated compounds were dissolved with CD3OD. Chemical shifts were referenced to residual CHD2OD at δ=3.31 ppm in the 1H-NMR spectrum and δ=49.15 ppm

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in the 13C-NMR spectrum. Mass spectral data were analyzed by fast atom bombardment-mass spectrometry (FAB-MS, JMS-HX100; Jeol, Tokyo, Japan) with a matrix ingredient (3-nitrobenzyl alcohol). Compound 1: white powder; 1H-NMR (CD3OD, 500 MHz) δ 7.11 (1H, d, J=2.0 Hz, H-2), 6.79 (1H, d, J=8.3 Hz, H-5), 7.03 (1H, dd, J=8.3, 2.0 Hz, H-6), 7.44 (1H, d, J=16.0 Hz, H-7), 6.41 (1H, d, J=16.0 Hz, H-8), 3.88 (3H, s, H-3-OCH3), 6.82 (1H, d, J=2.0 Hz, H-2'), 6.73 (1H, d, J=8.3 Hz, H-5'), 6.67 (1H, dd, J=8.3, 2.0 Hz, H-6'), 2.77 (2H, t, J=7.5 Hz, H-7'), 3.49 (2H, t, J=7.5 Hz, H-8'), 3.83 (3H, s, H-3'-OCH3); 13C-NMR (CD3OD, 125 MHz) δ 169.4 (C-9), 150.0 (C-4), 149.5 (C-3), 149.1 (C-3'), 146.2 (C-4'), 142.2 (C-7), 132.2 (C-1'), 128.4 (C-1), 123.3 (C-6), 122.4 (C-6'), 118.9 (C-8), 116.6 (C-5), 116.4 (C-5'), 113.6 (C-2'), 111.7 (C-2), 56.5 (-OCH3, C-3, 3'), 42.6 (C-8'), 36.4 (C-7'); FABMS (positive) m/z 344 [M+H]+ and 366 [M+Na]+. Compound 2: white powder; 1H-NMR (CD3OD, 500 MHz) δ 7.36 (1H, d, J=2.0 Hz, H-2), 6.73 (1H, d, J=8.0 Hz, H-5), 6.93 (1H, dd, J=8.3, 2.0 Hz, H-6), 6.61 (1H, d, J=12.5 Hz, H-7), 5.82 (1H, d, J=12.5 Hz, H-8), 3.83 (3H, s, H-3-OCH3), 6.77 (1H, d, J=2.0 Hz, H-2'), 6.69 (1H, d, J=8.0 Hz, H-5'), 6.61 (1H, dd, J=8.0, 2.0 Hz, H-6'), 3.42 (2H, t, J=7.5 Hz, H-7'), 2.71 (2H, t, J=7.5 Hz, H-8'), 3.78 (3H, s, H-3'-OCH3); FABMS (positive) m/z 344 [M+H]+ and 366 [M+Na]+. Compound 3: white powder; 1H-NMR (CD3OD, 500 MHz) δ 7.40 (1H, br. d, J=8.0 Hz, H-2), 6.79 (1H, br. d, J=8.0 Hz, H-3), 6.79 (1H, br. d, J=8.0 Hz, H-5), 7.40 (1H, br. d, J=8.0 Hz, H-6), 7.44 (1H, d, J=15.5 Hz, H-7), 6.38 (1H, d, J=15.5 Hz, H-8), 7.05 (1H, br. d, J=8.0 Hz, H-2'), 6.71 (1H, br. d, J=8.0 Hz, H-3'), 6.71 (1H, br. d, J=8.0 Hz, H-5'), 7.05 (1H, br. d, J=8.0 Hz, H-6'), 2.76 (2H, t, J=7.5 Hz, H-7'), 3.46 (2H, t, J=7.5 Hz, H-8'); FABMS (positive) m/z 284 [M+H]+ and 307 [ M+Na]+. Compound 4: yellow powder; 1H-NMR (CD3OD, 500 MHz) δ 8.56 (2H, br. d, J=7.5 Hz, H-2', 6'), 7.33 (2H, br. d, J=7.5 Hz, H-3', 5'), 6.77 (1H, d. s, H-6), 6.86 (1H, d. s, H-8). Assay of DPPH radical scavenging The free radical scavenging activities of the isolated compounds were evaluated by a DPPH radical assay according to the method described by Abe et al. (13), with slight modification. Briefly, a methanol solution (100 µL) of each compound (50 µM) was added to DPPH radical ethanol solution (900 µL, final concentration, 100 µM). The solution was then mixed and allowed to stand for 30 min in the dark, after which the free radical scavenging activity of each compound was quantified by observing the decolorization of DPPH at 517 nm. The DPPH radical scavenging activities of extract, fractions, and the isolated compounds (final concentration, 50 µM) were also determined as the percentage decrease

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Fig. 1. Isolation and purification of compound 1-4 from MeOH extracts of Welsh onion.

when compared to the absorbance of a blank. An assay for purification of the antioxidative compounds was conducted by spraying the DPPH solution on a TLC (Silica gel 60 F254, 0.25 mm thickness, Merck) using the method described by Takao et al. (14), with slight modification. Briefly, each fraction was purified by various types of column chromatography and ODS-HPLC, after which they were spotted onto the TLC plate and developed with suitable solvents. After spraying the plates with 200 µM DPPH free radical EtOH solution, the fractions that showed decolorization of the spot were considered to have antioxidative activity. Statistical analysis The antioxidative activities of the isolated compounds were expressed as the mean±standard deviation (SD) using the SPSS (Statistical Package for Social Sciences Inc., Chicago, IL, USA) 17.0 package programs. Specifically, groups were compared by one-way analysis of variance (ANOVA), followed by Scheffe’s test. A p<0.05 was considered to indicate statistical significance.

Results and Discussion Antioxidative activity of EtOAc-soluble neutral fractions of MeOH extract The edible parts (fresh 9.0 kg) of the Welsh onion were partitioned with EtOAc according to the

difference in the dissociation degree to give EtOAc-soluble acidic (6.6 g) and EtOAc-soluble neutral (18.8 g) fractions (12). These fractions showed DPPH radical scavenging activity. The identification of 4 phenolic compounds from the EtOAc-soluble acidic fraction of MeOH extracts has already been reported (12). To isolate additional antioxidative compounds in the Welsh onion, the EtOAc-soluble neutral fraction (18.8 g) was subjected to silica gel column chromatography using n-hexane/EtOAc/MeOH to give 6 fractions (A-F). Each fraction was then developed on TLC and sprayed with DPPH free radical EtOH solution. All of the fractions showed DPPH free radical scavenging activity (data not shown). In particular, fractions A (n-hexane/ EtOAc/MeOH=8:6:1, v/v, 702.7 mg) and B (n-hexane/ EtOAc/MeOH=10:4:1, v/v, 1.2 g) showed higher DPPH radical scavenging activity than other fractions. Fraction A (n-hexane/EtOAc/MeOH=8:6:1, v/v, 702.7 mg) was further fractionated on a Sephadex LH-20 column chromatograph to yield active fraction A2 [elution volume/bed volume (Ve/Vt) 0.78-1.02, 69.3 mg] and other fractions. Fraction A2-a (60% MeOH, 15 mg) obtained following ODS column chromatography of fraction A2 (69.3 mg) was subjected to ODS-HPLC (60% MeOH) and 4 peaks were detected on the chromatogram. Of these 4 compounds, 3 active compounds [1 (tR 23.92 min, white powder, 1.3 mg), 2 (tR 14.39 min, white powder, 0.5 mg), 3 (tR 19.05 min, white

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Fig. 2. Chemical structures of compound 1-4 isolated and identified from Welsh onion extract.

powder, 0.7 mg] were isolated by repeated ODS-HPLC. Fraction B (n-hexane/EtOAc/MeOH=10:4:1, v/v, 1.2 g), which was obtained another active fraction after silica gel column chromatography, was further purified using a silica gel column, after which active fraction B1 (n-hexane/ EtOAc/MeOH=11:3:1, v/v, 160 mg) was re-fractionated by Sephadex LH-20 column chromatography to yield active fraction B1-a [Ve/Vt=1.57-1.72, 3.0 mg]. Compound 4 (tR 17.5 min, white powder, 2.6 mg) was isolated from fraction B1-a (3.0 mg) by ODS-HPLC using 40% MeOH as a mobile phase. The isolation and purification procedures of the 4 active compounds (1-4) are indicated in Fig. 1. The structure of the isolated compounds was elucidated by NMR and MS spectroscopic data. Structural determination of compound 1-4 The presence of pseudomocular ion peaks at m/z 344 [M+H]+ and 366 [M+Na]+ in the electron spray ionization (ESI)MS negative spectrum of 1 indicated that the molecular weight is 343. The 1H-NMR spectrum of 1 showed the presence of 2 tri-substituted benzene rings of 6 sp2 carbon proton signals at δ 7.11 (1H, d, J=2.0 Hz, H-2), 6.79 (1H, d, J=8.3 Hz, H-5), 7.03 (1H, dd, J=8.3, 2.0 Hz, H-6), 6.82 (1H, d, J=2.0 Hz, H-2'), 6.73 (1H, d, J=8.3 Hz, H-5'), and 6.67 (1H, dd, J=8.3, 2.0 Hz, H-6'). In addition, 2 methylene proton signals [δ 3.49 (2H, t, J=7.5 Hz, H-8'), 2.77 (2H, t, J=7.5 Hz, H-7')], 2 double bond proton signals [δ 6.41 (1H, d, J=16.0 Hz, H-8), 7.44 (1H, d, J=16.0 Hz, H-7)], and 2 methoxyl proton signals [δ 3.88 (3H, s, OCH3 of C-3), 3.83 (3H, s, -OCH3 of C-3')] were detected. The trans configuration of a double bond could be derived from the coupling constant value (J=16.0 Hz) of proton signals at [δ 7.44 (H-8) and 6.41 (H-7)] in the 1H-NMR spectrum. The 1H-NMR spectrum of 1 was supported by the 13C-NMR spectrum, which revealed the presence of 19

carbon signals including a carbonyl carbon (δ 169.4, C-9), 14 sp2 carbons (δ 150-111.7), and 4 sp3 carbons (δ 56.536.4). The FAB-MS and 1D-NMR spectroscopic analyses suggested that 1 was a tyramine derivative coupled with a methyl group and ferulic acid (3-methoxy-4-hydroxycinnamic acid). In the heteronuclear multiple bond correction (HMBC) spectrum (data not shown), the partial structures of 1 were assigned as tyramine and ferulic acid. Moreover, the HMBC correlation of the methoxyl proton signal of δ 3.83 (3H, s) and the carbonyl carbon signal of δ 149.1 (C-3') indicated that the methoxyl group is coupled with the C-3 position of tyramine. Furthermore, the cross peak from the methylene proton signal (δ 3.49, H-8') to the carbonyl carbon signal (δ 169.4, C-9) indicated that the amine group of 3-methoxytyramine was harbored within the carbonyl group of ferulic acid. Moreover, the 1H- and 13 C-NMR spectra of 1 agreed with those of N-trans-3-Omethylcaffeoyl 3'-O-methyldopamine isolated from Chenopodium album reported in the relevant literature (15). Therefore, 1 was unambiguously identified as Ntrans-feruloyl-3'-methoxy-tyramine (Fig. 2). The 1H-NMR spectrum of 2 was closely related to that of 1 except for 2 sp2 methine proton signals [δ 6.61 (1H, d, J=12.5 Hz, H-7) and 5.82 (1H, d, J=12.5 Hz, H-8)]. The coupling constant value (J=12.5 Hz) of the proton signals of δ 6.61 (H-7) and 5.82 (H-8) indicated that a double bond differs from that in 1 in that it is in the cis form. In addition, the molecular weight of this compound was found to be 343 based on the detection of pseudomolecular ion peaks (m/z 344 [M+H]+ and 366 [M+Na]+) in the ESI-MS (positive) spectrum. Therefore, 2 was identified as N-cisferuloyl-3'-methoxytyramine based on comparison with other available NMR spectra isolated from Annona cherimola (16) (Fig. 2). The 1H-NMR spectrum of 3 revealed the presence of

Cinnamic Acid Amides in Allium fistulosum

para-substituted aromatic ring proton signals of the AA'BB' system at δ 7.40 (2H, br. d, J=8.0 Hz, H-2, 6), 6.79 (2H, br. d, J=8.0 Hz, H-3, 5), 7.05 (2H, br. d, J=8.0 Hz, H-2', 6'), and 6.71 (2H, br. d, J=8.0 Hz, H-3', 5'). When compared to the 1H-NMR spectrum of 1, it was suggested that the structure of 3 was N-trans-p-coumaroyltyramine without 2 methoxyl groups in 1. The 1H-NMR spectrum of 3 was consistent with that of N-trans-p-coumaroyltyramine that was isolated and identified from the twigs of Celtis chinensis (17). In addition, the molecular weight (283) of 3 was confirmed by the presence of pseudomolecular ion peaks of m/z 284 [M+H]+ and 307 [M+Na]+ in the ESI-MS negative spectrum. Therefore, 3 was identified as N-trans-p-coumaroyltyramine, which differs from the absence of 2 methoxy groups of 1 (Fig. 2). The 1H-NMR spectrum of 4 exhibited the presence of a typical flavonoid from A ring proton signals [δ 6.77 (1H, d, 7.5 Hz, H-6), δ 6.86 (1H, d, 7.5 Hz, H-8)] and parasubstituted B ring proton signals [δ 8.56 (2H, br. d, 7.5 Hz, H-2', 6'), 7.33 (2H, br. d, 7.5 Hz, H-3', 5')]. Specifically, the AA'BB' system of sp2 carbon proton signals [δ 8.56 (2H, br. d, J=7.5 Hz, H-2', 6'), 7.33 (2H, br. d, J=7.5 Hz, H-3', 5')] on the B ring of flavonol suggested that 4 is a kaempferol. The 1H-NMR spectrum of 4 was consistent with that of kaempferol isolated from the aerial parts of Lespedeza cuneata (18). Therefore, the structure of 4 was unambiguously identified as 3,5,7-trihydroxyflavone (kaempferol) (Fig. 2). DPPH radical scavenging activity of the isolated compounds The antioxidative activities of the isolated compounds at the same concentration (50 µM) were determined by the DPPH radical scavenging method (Fig. 3). Compound 1 and 2 showed relatively higher DPPH radical scavenging activities than 3, which has a parasubstituted hydroxyl group. It is well known that the radical scavenging activities of phenolic derivatives are critically dependent on the number of phenolic hydroxyl groups as well as the presence of methoxyl groups, thiols, and amides (19-21). The radical scavenging activity of 1 and 2, which have different double bonds, did not differ significantly. The DPPH radical scavenging activity of kaempferol (4) was also similar to 1 and 2. Four antioxidative compounds were isolated from the MeOH extracts of A. fistulosum and identified as N-transferuloyl-3'-methoxydopamine (1), N-cis-feruloyl-3'-methoxytyramine (2), N-trans-p-coumaroyltyramine (3), and 3,5,7trihydroxyflavone (kaempferol) (4) based on NMR and MS analyses. In previous studies, compound 1 and 2 have been identified from Allium species, while 1 has been isolated from A. sativum L. (22), and 1 and 2 have been found in A. tuberosum (23). Identification of 3 in Canabis sativa (24), Solanum indicum (25), and Celtis chinensis

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Fig. 3. DPPH radical scavenging activities of compounds isolated from Welsh onion extract. Final concentration of the DPPH radical was 100 µM, while those of compound 1-4 were 50 µM. Data shown are the mean±SD (n=3); a,bDifferent letters indicate a significant difference (p<0.05).

(17) has been also been reported. However, to the best of our knowledge, this is the first time that 3 was identified in an Allium species as well as the Welsh onion. Some studies have reported that cinnamic acid amides have nitric oxide inhibition (26) and anti-inflammatory (27) effects. Therefore, we were interested in whether 3 also exerts such biological activities. In addition, the occurrence of 4 together with quercetin in this plant has been reported with its antioxidative, lipid-lowering, and antihypertensive effects (7). Compound 1-3 isolated in the present study are newly identified as antioxidative compounds from Welsh onion. The crude extracts of Welsh onion have previously been reported to have strong antioxidative and antihypertensive activities in vitro and in vivo (6,7). The 4 compounds identified in this study containing compounds isolated from our previous study (12) may contribute to the antioxidative effect of the Welsh onion.

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