Phenolic compounds from Canna edulis Ker residue and their antioxidant activity

LWT - Food Science and Technology 44 (2011) 2091e2096

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Phenolic compounds from Canna edulis Ker residue and their antioxidant activity Juan Zhang a, b, Zheng-Wu Wang b, *, Qin Mi b a b

School of Life Sciences, Shanghai University, Shanghai 200444, China Department of Food Science and Technology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 November 2010 Received in revised form 16 May 2011 Accepted 25 May 2011

Using the bleaching of the free radical scavenger 1,1-diphenyl-2-picrylhydrazyl (DPPH) as the guiding assay, a novel compound, 4-(3-(3,4-dihydroxyphenyl)acryloyl)-6-hydroxy-1-methoxy-1,2,3,4-tetrahydronaphthalene-2-carboxylic acid, was isolated from water-soluble extract of Canna edulis Ker with ten known compounds: rosmarinic acid, salvianolic acid B, ferulic acid, caffeic acid, 1-caffeoylquinic acid, 3-caffeoylquinic acid, 4-caffeoylquinic acid, 5-caffeoylquinic acid, salicylic acid and gallic acid. The structures were characterized by spectroscopic methods including extensive 2D-NMR techniques. Moreover, antioxidant activities of total extract and the new compound were determined through six different kinds of modes. In view of antioxidant activity confirmed, C. edulis extract and the new compound can be developed as natural food additives. In addition, the correlations between concentration and antioxidant activity and among the modes were analyzed. The results of DPPH and ABTS radical scavenging and FRAP assay exhibited high consistency. However, those for other modes showed difference to a certain extent. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Canna edulis Ker residue Phenolic compounds Structural characterization Antioxidant activity

1. Introduction As reported, phenolic compounds have been reported to possess beneficial bioactivities, such as antioxidant, anticarcinogenic, antibacterial, antimutagenic, anti-inflammatory, antiallergic activities and so on (Meng, Lozano, Bombarda, Gaydou, & Li, 2008). In view of favorable bioactivities, phenolic extract is not only used as functional food ingredients but also provided for preparation of other materials. Furthermore, the phenolic compound contributes to organoleptic properties of final product, such as color, bitterness, flavor and browning. In the process of food production and preservation, quality are always reduced due to oxidation. Many synthetic antioxidants have been used, which has raised problems related to food safety and toxicity (Chang, Ostric-Matijasevic, Hsieh, & Chang, 1977). Therefore, natural antioxidants have emerged as a hot topic to develop food additive in the sight of their safety. Generally, phenolic compounds inhibit oxidation through different mechanisms of action, including scavenging of free radicals (Antolovich, Prenzler, Patsalides, McDonald, & Robards, 2002), quenching of reactive oxygen species, inhibition of oxidative enzymes (Edenharder & Grunhage, 2003), chelation of transition metals or interaction with biomembranes (Liao & Yin, 2000).

* Corresponding author. Tel./fax: þ86 021 34205748. E-mail address: [email protected] (Z.-W. Wang). 0023-6438/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2011.05.021

Canna edulis Ker, belonging to the genus Canna (Cannceae), is largely cultivated in South America, Taiwan, Vietnam, Thailand and China. The dry rhizome of C. edulis contains 70e80 g/100 g starches. Some studies have been conducted on physiochemical properties and modification of C. edulis starch (Zhang & Wang, 2009; Zhang, Chen, Liu, & Wang, 2010). Moreover, several phenolic compounds, composed of phenylpropanoid derivatives, have been characterized from its rhizome, which may be a good source for phenolic extraction (Yun, Satake, Katsuki, & Kunugi, 2004). C. edulis residue, as waste discarded after starch extraction, is a potentially environmental problem because it is highly susceptible to putrefaction as a result of high moisture content. According to our knowledge, the by-product may contain plenty bioactive compounds and been used as value-added materials. Consequently, it is significant to exploit C. edulis residue as a good resource of phenolic extract. In the present study, using the bleaching of the free radical scavenger 1,1-diphenyl-2 -picrylhydrazyl (DPPH) as the guiding assay, phenolic compounds were separated and purified from C. edulis and their structures were determined through spectroscopy including distortionless enhancement by polarization transfer (DEPT), heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple bond connectivity (HMBC), correlation spectroscopy (COSY) and nuclear Overhauser effect spectroscopy (NOESY). Moreover, other five different kinds of modes were used to assay the antioxidant activities of phenolic compounds. The relationships including between concentration and activities and among different modes were discussed.

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2.3. Determination of soluble phenolics, hydrolyzable tannins and insoluble condensed tannins

2 Kg Residue of Canna edulis Ker extracted (3 times) concentrated filtrate precipitate

Soluble phenolics were extracted using sulfite sodium through continuous shaking and its content was determined spectrophotometrically using the FolineCiocalteau’s reagent (Lecumberri et al., 2007). Gallic acid was used as reference. The content of hydrolyzable tannins in the extract was determined according to the method of Reed, McDowell, Van Soest, and Horvath (1982) and tannic acid was used as reference. In the residue obtained after the extraction of phenolic compounds, the content of insoluble condensed tannins was quantified spectrophotometrically at 550 nm, using tannic acid as standard.

concd. aqueous extract extracted with petroleum ether aqueous solution

petroleum ether layer

concd. in vacuo aqueous solution A cc

H2O eluate (sugar)

Fraction A repeated B cc C cc D cc E cc aqueous methanol

A: Diaion HP20 B: TSK gel Toyopearl HW40F C: MCI gel CHP20P D: Cosmosil ODS E: Polyamide cc: column chromatography 4 23 mg 5 32 mg

Fraction F Fraction C Fraction B repeated repeated repeated B cc B cc B cc C cc C cc C cc D cc D cc D cc E cc E cc E cc aqueous aqueous aqueous methanol methanol methanol 6 7 8 9

1 182 mg 2 17 mg 3 59 mg

14 mg 45 mg 47 mg 12 mg

2.4. Extraction and isolation of single compound The dried residues (2 kg) of starch extracted C. edulis rhizome were ground to fine powder passing through 100 meshes. The powder was extracted with 70% ethanol (v/v) under refluxing three times. The suspension was filtered and the filtrates were concentrated under in vacuum to give crude extract. The crude extract was subjected to column chromatography on Diaion HP20 macropore polymeric adsorbent (200e600 mm, Japan Mitshubishi Chemical Co. Ltd, Japan), after removing sugar with water elution, using a stepwise gradient of ethanol (10e90% v/v) to give different fractions. Then these fractions were purified by column chromatography on MCI gel CHP20P (75e150 mm, Japan Mitsubishi Chemical Corporation, Tokyo, Japan), TSK gel Toyopearl HW40F (30e60 mm, Tosoh Co. Ltd., Tokyo, Japan) and Cosmosil ODS (40e80 mm, Nacalai Tesque Inc., Tokyo, Japan) repeatedly, and eluted with aqueous methanol to afford single phenolic compounds (Fig. 1).

10 34 mg 11 51 mg 12 23 mg

Fig. 1. Extraction scheme of phenolic compounds from Canna edulis Ker residue.

2. Materials and methods 2.1. Samples and reagents C. edulis rhizomes were purchased from Guizhou Ziyun Jiahe Chemical Co. Ltd. (Gui Zhou, China). C. edulis residues were obtained after starch extraction according our previous method (Zhang & Wang, 2009). All the chemicals used in the experiment were of analytical grade and water was doubly distilled.

2.5. Structural characterization of single compounds 2.2. General experimental procedures The structure of single compound (Fig. 2) was characterized through various spectral and chromatographic techniques including UV, FT-IR, 1H NMR, 13C NMR, 1He1H COSY, HMQC, HMBC, NOESY, and ESIeTOFeMS.

The UV spectra were obtained on a UVevisible spectrophotometry (DU800, Beckman Coulter Co. Ltd, L.A., USA). The IR spectra were recorded on an Equinox 55 Fourier Transform InfraredeRaman spectrometer (Bruker Co. Ltd., Germany). ESIeTOFeMS was obtained on an LCQ Deca XP Max Liquid Chromatography-Mass (Thermo Scientific Co. Ltd., New York, USA). The 1H, 13C NMR and 2D-NMR data (including HMQC, HMBC, NOESY and 1He1H COSY) were measured on an Avance III 400 spectrometer (Bruker Co. Ltd., Switzerland) operating at 400 MHz for 1H and 101 MHz for 13C.

2.6. Determination of antioxidant activity 2.6.1. DPPH$ radical scavenging The DPPH$ radical scavenging was carried out according to the method of Sánchez-Moreno, Plaza, de Ancos, and Cano (2003). In

HO H3CO 8

O 1 4 10

7 6

5

HO

O O

2

9

9' 8'

O

2'

4

O

OH

4'

O HO

HO 2

OH O HO

HO

OH

R1 5 6 7 8 9

OH

O

OH OH

OH

OH O OH

3

R4

OR O

HO

OH

O

3'

6' 5'

OCH3

O O

OH

OH OH

7' 1'

OH

HO OH

3

1 O

O

R3 R2

R=H R = R1, R2 = OH, R3 = OH, R4 = OH R = R2, R1 = OH, R3 = OH, R4 = OH R = R3, R1 = OH, R2 = OH, R4 = OH R = R4, R1 = OH, R2 = OH, R3 = OH

O O

OH

OH OH

HO

OH OH

10

Fig. 2. Chemical structures of Compounds 1e12.

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brief, sample was mixed with DPPH$ reagent, and the absorbance was measured at 517 nm. 2.6.2. ABTS$þ radical scavenging ABTS assay was based on the method of Re et al. (1999) with slight modifications. Sample or ascorbic acid was added into diluted ABTS_þ solution and absorbance at 734 nm was measured. 2.6.3. The ferric reducing/antioxidant power The ferric reducing/antioxidant power (FRAP) was determined according to the method described by Pulido, Bravo, and SauraCalixto (2000). In brief, FRAP reagent was mixed with the sample, water or tocopherol. Absorbance at 595 nm was measured spectrophotometrically. 2.6.4. Oxidation system of lipid by ferrous sulfate The oxidation of linoleic acid was conducted according to the method of Arabshahi-Delouee, Devi and Urooj (2007). An aqueous solution containing linoleic acid was diluted with Trizma-buffer solution. Lipid peroxidation was initiated by adding ferrous sulfate. The reaction was stopped by adding butylated hydroxytoluene. The solution obtained was mixed with sample. Then, the reacted solution was used for thiobarbituric acid assay (Ohkawa, Ohishi & Yagi, 1979). 2.6.5. b-Carotene-linoleic acid emulsion The method of Reddy, Urooj and Kumar (2005) was used with slight modification. Briefly, b-carotene-linoleic acid emulsion was mixed with the sample and incubated. The absorbance of the oxidized emulsion was measured using a spectrophotometer at 470 nm. 2.6.6. Metal chelating capacity The metal chelating capacity was determined by the method of Du, Li, Ma, and Liang (2009). Sample was mixed with FeCl2$4H2O and ferrozine and the mixture were shaken. After 10 min, the Fe2þ was monitored by measuring the formation of ferrous ioneferrozine complex at 562 nm. 2.7. Statistical analysis All determinations were triplicates, and mean values and standard deviations were calculated. Analysis of variance (ANOVA) was performed and the mean separation was done by LSD (P  0.05) using SPSS 13.0 program for windows (SPSS Inc., IL, USA). 3. Result and discussion 3.1. Preliminary composition Table 1 shows essential composition of polyphenols from C. edulis rhizome and residue. Tannins are defined as any phenolic compound of sufficiently high molecular weight containing sufficient hydroxyls and other suitable groups (i.e. carboxyls) to form effectively strong complexes with protein and other macromolecules under the particular environmental conditions being studied, and classified as two main categories including hydrolyzable tannins and condensed tannins identified more correctly as proanthocyanidins. Hydrolyzable tannins are molecules with a polyol (generally D-glucose) as a central core and the hydroxyl groups of these carbohydrates are partially or totally esterified with phenolic groups like gallic acid (gallotannins) or ellagic acid (ellagitannins). Condensed tannins are oligomers or polymers of flavonoid units (i.e. flavan-3-ol) linked by carbonecarbon bonds not susceptible to cleavage by hydrolysis (Haslam, 1989). C. edulis

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Table 1 The content of soluble polyphenols and tannins and the insoluble condensed tannins in the Canna edulis Ker rhizome and residue. Constituents

Contenta (g GAE/100 g)

Recoveryb (g/100 g)

Soluble phenolics Soluble tannins Insoluble condensed tannins

17.47  0.36a 23.03  0.11a 78.83  3.48a

89.09  1.89b 65.23  2.74b 131.46  3.61a

Rows with the same letters are not significantly different (P  0.05). a in the rhizome of Canna edulis Ker. b in the residue of Canna edulis Ker.

rhizome contains a certain amount of soluble phenols (17.47 g GAE/ 100 g). C. edulis residue owns higher amounts of soluble phenolics, hydrolyzable tannins and insoluble condensed tannins, in comparison with other agricultural and industrial by-products (Balasundram, Sundram, & Samman, 2006). In the residue, the recovery decreases for soluble phenolics and hydrolyzable tannins, and conversely increases for insoluble condensed tannins. The former could be explained for the loss of partial soluble substance in the process of starch extraction. For the latter, it can be attributed to disruption of insoluble condensed tannins and starch complex after starch extraction, leading to abundant extracted insoluble condensed tannins in the residue. Insoluble condensed tannins constitute main component in the total phenolic extract in view of their high content (78.83 g GAE/100 g). As reported, tannins have antioxidant, antimicrobial, antiviral, antimutagenic and antinutrient activities. Moreover, tannins can reduce the risk in the treatment of cardiovascular diseases and diabetes (Serranol, Puupponen-Pimiǎ, Dauer, Aura & Saura-Calixto, 2009). 3.2. Extraction and separation The crude extract was subjected to column chromatography on Diaion HP20 macropore polymeric adsorbent, after removing sugar with water elution, using a stepwise gradient of ethanol (10e90% v/v) to give four fractions labeled A (86 g), B (158 g), C (62 g) and D (58 g) according to their TLC profiles respectively. Fraction A was separated by column chromatography on MCI gel CHP20P, eluted with methanol (10e90% v/v) gradiently to yield two sub-fractions (sub-fractions A-1wA-2). Sub-fraction A-1 was again subject to column chromatography on TSK gel Toyopearl HW40F (30e60 mm, Tosoh) and eluted with water to afford compound 5 (32 mg). Subfraction A-2 was purified by column chromatography on TSK gel Toyopearl HW40F and Cosmosil ODS repeatedly, and eluted with water to afford compounds 4 (23 mg). Fraction B was separated by column chromatography on MCI gel using a gradient of ethanol, to yield three sub-fractions (sub-fractions B-1wB-3). Further repeated purification of sub-fractions B-1, B-2 and B-3 by column chromatography on TSK gel Toyopearl HW40F, using water as eluant, resulted in the isolation of compounds 6 (14 mg), 7 (45 mg), 8 (47 mg) and 9 (12 mg). Fraction C was separated by column chromatography on TSK gel Toyopearl HW40F using a stepwise gradient to yield five sub-fractions (sub-fractions C-1wC-5). Then subfractions C-1, C-2 and C-3 were purified by repeated column chromatography on TSK gel Toyopearl HW40F and Cosmosil ODS, using water as eluant, to afford compounds 1 (182 mg), 2 (17 mg) and 3 (59 mg). Fraction D was separated by MCI gel CHP20P and TSK gel Toyopearl HW40F, to give compounds 10 (34 mg) and 11 (51 mg). 3.3. Structure determination 3.3.1. Structure determination of compound 1 Compound 1, m.p. 152e155  C, was obtained as light yellow powder from a 10% ethanol solution with molecular mass of 384

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determined through ESIeMS spectrum in negative ion mode which exhibited the quasi-molecular ion [MeH] peak at m/z 383.50. UV spectrum exhibits a maximized absorption at 324 nm, which implied the presence of a highly conjugated double bond system. The IR spectrum of compound 1 displays the presence of hydroxyl group (nmax ¼ 3421 cm1), carbonyl group (nmax ¼ 1691 cm1) and aromatic ring (nmax ¼ 1384, 1521 and 1596 cm1). In the 1H NMR spectrum (DMSO-d6) (Table 2) of 1, two ABC spin systems, resonating at dH 6.47 (1H, dd, J ¼ 7.6, 2.0 Hz), 6.59 (1H, d, J ¼ 7.6 Hz), 6.67 (1H, d, J ¼ 2.0 Hz), dH 6.72 (1H, d, J ¼ 8.4 Hz), 6.90 (1H, dd, J ¼ 8.4, 2.0 Hz) and 7.03 (1H, d, J ¼ 2.0 Hz) could be attributed to two 1,3,4trisubstituted benzene rings. The 1H NMR spectrum also exhibits a set of trans olefinic protons at dH 7.40 and 6.20 with coupling constants of 15.6 Hz and one methoxyl group [dH 3.10 (3H, s)], with the assistance from the analysis of HMBC (Fig. 3 (A)) and HMQC experiments. The 13C-NMR spectrum (Table 2) of compound 1 reveals 21 carbon signals as one methyl, one methylenes, eleven methines and eight quaternary carbons assignable to two benzene ring, one olefinic bond, two carbonyl groups (d 166.7 and 173.0), three substituted sp carbons and one sp2 carbon. Full analysis of the 1 He1H COSY, HMBC and HMQC spectra led to the identification of a 1,2,3,4-tetrahydronaphthalene skeleton as shown in Fig. 2. In the HMBC of compound 1, the significant correlations between H-60 (d 6.90) and C-70 (d 144.8), H-70 (d 7.40) and C-10 (d 125.8), C-60 (d 121.1) and C-90 (d 166.7), H-80 (d 6.20) and C-10 (d 125.8) indicate the presence of (3,4-dihydroxyphenyl)acryloyl unit, which is bonded to 4-C of tetrahydronaphthalene in view of the correlations between H-4 (d 4.82) and C-90 (d 166.7), H-80 (d 6.20) and C-3 (d 37.7), and H-80 (d 6.20) and C-10 (d 130.4). The carboxyl moiety is shown to be bonded to C-2, due to the important correlation between H-4 (d 4.82) and carboxyl carbon (d 173.0). The methoxyl moiety is revealed to be bonded to C-1, since an HMBC spectral correlation is observed between methoxyl hydrogen (d 3.10) and C-1(d 67.5). A combination analysis of the HMQC, HMBC and 1He1H COSY experiments allows the unambiguous assignment of all the signals (Table 2). Furthermore, the coupling constants of proton signals on the tetrahydronaphthalene ring demonstrate that the (3,4-dihydroxyphenyl)acryloyl group of compound 1 is equatorial-oriented at C-4 in the conformation. In the NOESY spectrum of compound 1, strong correlations between H-2 (d 3.83), H-3a (d 2.61) and H-4 (d

H

H3C H

O

O

H

OH

HH H

H O

H

OH H H

HO

HH H

H O

O H3C H O

H

H H

HO

H

H

H

H

H

OH H

A

OH

H

B

OH H

OH

Fig. 3. Significant HMBC (A) and NOESY (B) correlations of compound 1.

4.82), and correlation between H-1 (d 3.39) and H-3b (d 2.91) shows that H-2, H-3a and H-4 locates at the same side of the planar, while H-1 and H-3b locates at the opposite side (Fig. 3 (B)). On the basis of the above results, the structure of compound 1 was determined to be 4-(3-(3,4-dihydroxyphenyl)acryloyl)-6-hydroxy-1-methoxy1,2,3,4-tetrahydronaphthalene-2-carboxylic acid. 3.3.2. Structure determination of other compounds Compounds 2, 3, 4 and 5 were obtained as light brown powders and identified as rosmarinic acid, salvianolic acid B, ferulic acid and caffeic acid, respectively, by comparison of their spectral profiles with those in the literature (Khallouki, Hull, & Owen, 2009; Yin et al., 2008). Compounds 6e9 were obtained as oil at low temperature and characterized as 1-caffeoylquinic acid, 3-caffeoylquinic acid, 4-caffeoylquinic acid and 5-caffeoylquinic acid, respectively, in comparison with reference data (Liu et al., 2009; Zhou et al., 2009). Compound 10 was colorless crystal and indentified as salicylic acid, compared by authentic sample. Compound 11 was light brown amorphous powder and characterized as gallic acid (Khallouki et al., 2009) (Fig. 2). 3.4. Antioxidant activity Both phenolic extract and Compound 1 show high DPPH and ABTS radical scavenging activities (Table 3). As reported, the high

Table 2 1 H NMR (400 MHz, DMSO-d6), 13C NMR (101 MHz, DMSO-d6), 1He1H COSY, HMBC and NOESY correlations of compound 1 (DMSO-d6, TMS, dppm). No.

dH

1 2 3a 3b 4 5 6 7 8 9 10 10 20 30 40 50 60 70 80 90 eOCH3 eCOOH

3.39 3.83 2.61 2.91 4.82 6.67 e 6.47 6.59 e e e 7.03 e e 6.72 6.90 7.40 6.20 e 3.10 e

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

overlapped) overlapped) dd, J ¼ 14.8, 10.8) dd, J ¼ 14.8, 2.8) dd, J ¼ 10.8, 2.8) d, J ¼ 2.0)

(1H, dd, J ¼ 7.6, 2.0) (1H, d, J ¼ 7.6)

(1H, d, J ¼ 2.0)

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

d, J dd, d, J d, J

(3H, s)

¼ 8.4) J ¼ 8.4, 2.0) ¼ 15.6) ¼ 15.6)

67.5 (d) 55.6 (d) 37.7 (t) 76.6 117.1 144.0 120.1 115.9 145.4 130.4 125.8 115.6 146.5 149.1 116.4 121.1 144.8 115.2 166.7 53.6 173.0

(d) (d) (s) (d) (d) (s) (s) (s) (d) (s) (s) (d) (d) (d) (d) (s) (q) (s)

1

He1H COSY

HMBC

NOESY

H-2 H-1 H-3b, H-4 H-3a, H-4 H-3a, H-3b H-7 e H-5, H-8 H-7 e e e H-60 e e H-60 H-20 , H-50 H-80 H-70 e e e

e C-3, C-5 e C-2 C-10, C-90 , eCOOH C-3, C-7, C-6 e C-6 C-6, C-10 e e e C-30 , C-40 , C-60 e e C-10 , C-30 C-40 , C-70 C-10 , C-60 , C-90 C-3, C-10, C-10 , C-90 e C-1, eOCH3 e

H-3b H-3a H-3b, H-4, H-5 H-4, H-5 H-5, H-7 H-3a, H-3b, H-4 e H-4 e e e e H-70 e e H-60 H-50 , H-80 H-20 , H-60 , H-80 H-20 , H-60 , H-70 e e e

J. Zhang et al. / LWT - Food Science and Technology 44 (2011) 2091e2096 Table 3 Antioxidant activity and correlation between the content and antioxidant activity of phenolic extract and Compound 1 measured by different modes.

DPPH$ assaya DPPH$ assayb ABTS$þ assay FRAP assay Metal chelating capacity Oxidation system of lipid by ferrous sulfate b-caroteneelinoleic acid emulsion a b

Phenolic extract

Compound 1

6.62  0.06 g/100 g 0.95 6.01  0.83 g/100 g 0.95 6.16  0.013 g/100 g 0.99 33.69  103 mol/g 0.99 48.39  1.34 g/100 g 0.70 3.53  0.36 g/100 g 0.97 38.07  2.19 g/100 g 0.58

7.20  0.35 g/100 g 0.95 6.72  0.37 g/100 g 0.95 7.36  0.04 g/100 g 0.99 102.73  103 mol/g 0.95 48.72  0.24 g/100 g 0.19 49.45  3.12 g/100 g 0.80 76.35  2.37 g/100 g 0.41

as tocopherol equivalent. as ascorbic acid equivalent.

molecular weight phenolics such as tannic acid have more ability to quench the radical and the effectiveness depends on the molecular weight, the number of aromatic rings and nature of hydroxyl group substitution than the specific functional groups (Hagerman et al., 1998; Leong & Shui, 2002). Phenolic compounds capable of donating a hydrogen atom were more effective in scavenging radical, and the effect of antioxidants on radical scavenging is generally due to the hydrogendonating ability (Siddhuraju & Becker, 2007). Moreover, the high radical scavenging activity of Compound 1 might be ascribed to the higher degree of hydroxylation and ortho-dihydroxy groups in the molecular structure, which has been demonstrated to be the most important structural feature of high activity for all tested phenolic compounds (Cai, Sun, Xing, Luo, & Corke, 2006). In the FRAP assay, two samples show strong reducing capacity, especially for Compound 1 (102.73  103 mol/g), attributing to their good ability of donating electrons. The two samples exhibit moderate metal chelating activity and antioxidant activity measured by oxidation system of lipid by ferrous sulfate compared with other compounds in the reference (Maqsood & Benjakul, 2010) and it has been reported that the efficiency in metal chelation varies with the type of phenolic compounds and does not relate with radical scavenging activity and reducing power. The monomeric and polymeric phenolics can chelate Fe (III) and prevented radical formation in the test of metal chelating capacity (Hagerman et al., 1998). The activities of the extract and Compound 1 were evaluated using b caroteneelinoleic acid system and it was found that antioxidant activity was 38.07 and 76.35 g/100 g, respectively, which were lower than those of the extracts from drumstick and carrot tuber (80e83 g/100g) as well as Morus Alba (78.2 g/100 g) (Arabshahi-D et al., 2007).

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Except for the mode of metal chelating capacity, when tested by other five different methods, the antioxidant activity of Compound 1 is much higher than that of the phenolic extract. Regarding to DPPH and ABTS radical scavenging, tannins have stronger activity than quiones, isoflavones and lignans. As shown in Table 1, the phenolic extract is composed of soluble tannins (23.03 g GAE/100 g) and other soluble phenolic compounds, which could be well explained for weaker activity compared to Compound 1. Similar to radical scavenging assay, for FRAP assay, tannic acid could easily donate the electron to Fe (III), thus reducing it to Fe (II). Furthermore, the higher number of hydroxyl groups in the phenolic acids do not increase their reducing power (Medina, Gallardo, Gonzaalez, Lois, & Hedges, 2007). These could explain for low reducing capacity of phenolic extract in comparison with Compound 1. In the measurement of metal chelating capacity, the two samples display almost activities, which could be ascribed for complicated factors influencing on metal chelating of phenolic compounds. Compared with the extract, Compound 1 exhibits much higher inhibition effect on lipid oxidation and this is due to the higher radical scavenging activity of compound 1, thereby lowering the subsequent generation of reactive lipid radicals, which can undergo further chain reactions. In the mode of b-carotene-linoleic acid emulsion, stronger inhibition effect of Compound 1 on b-carotene emulsion oxidation could be also attributed to high radical scavenging ability. For two samples, expect for modes of metal chelating capacity and b-carotene-linoleic acid emulsion, good linear relationships between the content and activity are observed for other four modes, indicating that the assay results of these four methods are consistent (Table 4). Positive correlations are observed when tested using DPPH and ABTS.þ methods and this results accord well with pervious studies which reported that the total antioxidant activities (tested by DPPH and ABTS.þ methods) and phenolic contents of the 112 medicinal plant extracts showed positive and highly significant linear correlations (R2  0.95) (Cai et al., 2006). Good linear correlations are also obtained for both samples in the FRAP assay (R2  0.95), in agreement with previous reports for tannic acid, caffeic acid, ferulic acid and catechin (Maqsood et al., 2009). However, bad correlation between concentration and metal chelating capacity differs from previous studies (Maqsood et al., 2009). It is interesting that negative correlations are detected measured by the mode of the oxidation system of lipid by ferrous sulfate for two samples, which could be ascribed to hydroperoxide degradation producing secondary lipid peroxidation products. In addition, for the mode of b-carotene-linoleic acid emulsion, nonlinear relationships observed approve complexity of inhibition on b-carotene oxidation and releasing from the emulsion. As shown in Table 4, for two samples, good linear relationships among the former three modes including DPPH, ABTS and FRAP assay are observed. However, for the later three modes, the

Table 4 Correlation between different modes for phenolic extract and Compound 1. DPPH$ radical scavenging

FRAP assay

ABTS.þ assay

Metal chelating capacity

Oxidation system of lipid by ferrous sulfate

b-carotene–linoleic acid emulsion mode

1 1 0.98 0.94 0.68 0.82 0.99 0.75 0.99 0.62

1 1 0.68 0.86 0.95 0.81 0.95 0.40

1 1 0.67 0.40 0.72 0.60

1 1 0.48 0.13

1 1

DPPH$ radical scavenging

1 1 FRAP assay 0.94 0.90 0.97 ABTS.þ assay 0.99 Metal chelating capacity 0.79 0.81 Oxidation system of lipid by 0.91 ferrous sulfate 0.81 b-caroteneelinoleic acid emulsion 0.79 mode 0.27

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correlations display difference to a certain extent. Among of them, DPPH and ABTS.þ are popular free radicals for use in assessing radical scavenging activity or antioxidant activity. FRAP assay is based on ability of compounds to reduce TPTZeFe (III) complex to TPTZeFe (II) complex. The three modes above mentioned are related to the capacity of donating electrons. Different from the former modes, metal chelating capacity does not relate with radical scavenging activity and reducing power. With regard to oxidation system of lipid by ferrous sulfate, oxidation is propagated via hydrogen subtraction in the vicinity of double bonds after the initiation stage and influenced by intricate factors. For the mode of b-carotene-linoleic acid emulsion, it is comprised of b-carotene releasing from the emulsion and oxidation. 4. Conclusion Preliminary composition of C. edulis was analyzed and abundant phenols were observed. Using the bleaching of the free radical scavenger 1,1-diphenyl-2-picrylhydrazyl (DPPH) as the guiding assay, the extract was separated and purified through different column chromatography, leading to the isolation of a new compound, 4-(3-(3,4-dihydroxyphenyl)acryloyl)-6-hydroxy-1methoxy-1,2,3,4-tetrahydronaphthalene-2-carboxylic acid, along with ten known compounds. Tested by six kinds of modes, C. edulis extract and Compound 1 exhibit antioxidant activity, suggesting that they can be developed as natural food additives used in the course of food process and storage in the industry. Moreover, Compound 1 could be developed as precursor in the therapy of many diseases like coronary heart disease, cancer, neurodegenerative disorders, etc., in light of its antioxidant and free radical scavenging capacities. Additionally, the relationships between concentration and activity and among the modes were discussed. The results evaluated through DPPH and ABTS radical scavenging as well as FRAP assay exhibit high consistency. However, those from another three modes show difference in varying degrees. Acknowledgements This work is supported by the Shanghai University Young Teachers Training Program. References Antolovich, M., Prenzler, P. D., Patsalides, E., McDonald, S., & Robards, K. (2002). Methods for testing antioxidant activity. The Analyst, 127, 183e198. Arabshahi-Delouee, S., Devi, D. V., & Urooj, A. (2007). Evaluation of antioxidant activity of some plant extracts and their heat, pH and storage stability. Food Chemistry, 100, 1100e1105. Balasundram, N., Sundram, K., & Samman, S. (2006). Phenolic compounds in plants and agri-industrial by-products: antioxidant activity, occurrence, and potential uses. Food Chemistry, 99, 191e203. Cai, Y. Z., Sun, M., Xing, J., Luo, Q., & Corke, H. (2006). Structureeradical scavenging activity relationships of phenolic compounds from traditional Chinese medicinal plants. Life Sciences, 78, 2872e2888. Chang, S. S., Ostric-Matijasevic, B., Hsieh, O. A., & Chang, C. L. (1977). Natural antioxidants from rosemary and sage. Journal of Food Science, 42, 1102e1106. Du, G., Li, M., Ma, F., & Liang, D. (2009). Antioxidant capacity and the relationship with polyphenol and Vitamin C in actinidia fruits. Food Chemistry, 113, 557e562. Edenharder, R., & Grunhage, D. (2003). Free radical scavenging abilities of flavonoidsas mechanism of protection against mutagenicity induced by ter-

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