Flavonoid-Rich Fraction from Sageretia theezans

JOURNAL OF MEDICINAL FOOD J Med Food 12 (6) 2009, 1310–1315 # Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition DOI: 10.1089=jmf.2008.1309

Flavonoid-Rich Fraction from Sageretia theezans Leaves Scavenges Reactive Oxygen Radical Species and Increases the Resistance of Low-Density Lipoprotein to Oxidation Shin-Kyo Chung,1 C.-Y. Oliver Chen,2 and Jeffrey B. Blumberg 2 1

School of Life and Food Sciences, College of Agriculture and Life Sciences, Kyungpook National University, Daegu, Republic of Korea; and 2Antioxidants Research Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts, USA

ABSTRACT To explore their bioactive fractions, Sageretia theezans leaves were extracted with 60% acetone and then fractionated sequentially with hexane, ethyl acetate, and water. Reactive oxygen radical species (ROS) (HOCl, ONOO, and O2) scavenging activity, oxygen radical absorbance capacity (ORAC) value, and total phenolic content of each fraction were investigated. The ethyl acetate fraction had the largest total phenolic content and ORAC value and displayed the strongest ROS scavenging activity. The ethyl acetate fraction at 2 mmol of gallic acid equivalents=L prolonged the lag time of low-density lipoprotein oxidation by 260% compared to the control. In the ethyl acetate fraction, 7-O-methylmearnsetin 3-O-rhamnoside and the other nine flavonols were characterized using high-performance liquid chromatography with electrochemical detection. The total flavonoid content in the ethyl acetate fraction was 460 mg=g. 7-O-Methylmyricetin 3-Orhamnoside and 7-O-methylmearnsetin 3-O-rhamnoside were the predominant flavonoids, making up 36% and 17% of the total flavonoid content, respectively. KEY WORDS: high-performance liquid chromatography with electrochemical detection oxidation 7-O-methylmearnsetin 3-O-rhamnoside 7-O-methylmyricetin 3-O-rhamnoside species scavenger Sageretia theezans

low-density lipoprotein reactive oxygen radical

Sageretia theezans is a semi-evergreen spinescent, 1–3 m tall shrub of the Rhamnaceae family that grows along in the southern seashores of East Asia. It has been used as a tisane for treating cold and fever in Korea and a folk medicine for hepatitis in China.7 However, there have been few reports on its chemical composition8 and medicinal effects, with the exception of the inhibitory effect on human immunodeficiency virus type 1 protease activity.9 Recently, we have identified a few flavonoids in S. theezans leaves, including a novel flavonol glycoside, 7-O-methylmearnsetin 3-O-rhamnoside (7,40 -Odimethylmyricetin 3-O-a-l-rhamnopyranoside).10 In an effort to further identify the biological activity of the leaves, the crude acetone extract was sequentially fractionated with n-hexane, ethyl acetate, and water, and then ROS scavenging activity and the resistance of LDL to oxidation in the presence of the fractions were examined. The flavonoid composition of the ethyl acetate fraction was also characterized because we found it possessed the greatest bioactivity.

INTRODUCTION

R

eactive oxygen radical species (ROS) are generated endogenously by NADP(H) oxidase, xanthine oxidase (XOD), and nitric oxide (NO) synthase, as well as by the mitochondrial electron transport chain. Superoxide radical anion (O2) has a long half-life and reacts with NO to generate peroxynitrite (ONOO). Peroxynitrite, as a strong oxidant and nitrating agent, oxidizes low-density lipoprotein (LDL) and causes injury to endothelial and smooth muscle cells.1 Myeloperoxidase, secreted by stimulated phagocytes in human atherosclerotic lesions, produces hypochlorite (HOCl).2,3 Highly reactive HOCl modifies proteins to yield secondary products like chloramines and induces lipid peroxidation.4 Overproduction of these ROS contributes to the etiology of numerous chronic diseases,1,5 particularly when antioxidant defenses are compromised. Plant polyphenols that act as ROS scavengers have been widely studied for their role in the prevention of chronic diseases. For example, green tea and wine polyphenols have been found to reduce the risk of atherosclerosis and cardiovascular disease.6

MATERIALS AND METHODS Manuscript received 6 October 2008. Revision accepted 1 September 2009.

Plant material

Address correspondence to: Dr. Shin-Kyo Chung, Professor, School of Life and Food Sciences, College of Agriculture and Life Sciences, Kyungpook National University, Daegu, 702-701, Republic of Korea, E-mail: [email protected]

S. theezans leaves were collected at the seashore of Kohung, Republic of Korea, and identified by Min-Sup Chung,

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FLAVONOID-RICH FRACTION FROM S. THEEZANS

Ph.D. in the Department of Agricultural Biology at Kyungpook National University. Thereafter the leaves were dried in the shade, pulverized, and kept in the freezer (308C). A specimen voucher has been preserved in the laboratory of Shin-Kyo Chung, Ph.D. Chemicals and instruments All organic solvents were analytical grade from Merck (Darmstadt, Germany), except for high-performance liquid chromatography (HPLC) reagents ( J.T. Baker, Phillipsburg, NJ, USA). Dihydrorhodamine-123 (DHR-123) and peroxynitrite were obtained from Molecular Probes (Eugene, OR, USA) and Cayman (Ann Arbor, MI, USA), respectively. Quercetin dihydrate, gallic acid, hypochlorite, ferrocyanide, 1,1-diphenyl-2-picrylhydrazyl (DPPH), XOD, xanthine, nitro blue tetrazolium (NBT), FolinCiocalteu reagent, and other chemicals for ROS scavenging assays and total phenolic content were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Chemicals used for the LDL isolation and conjugated dienes (CD) determination assays were also obtained from Sigma. Myricetin 3-O-rhamnoside, 7-O-methylmyricetin 3-Orhamnoside, 7-O-methylmearnsetin 3-O-rhamnoside, kaempferol 3-O-rhamnoside, and 7-O-methylquercetin 3-O-rhamnoside were isolated and characterized as previously described.10 Myricetin, quercetin, isorhamnetin, and kaempferol were purchased from Sigma. In addition, 7-Omethylmearnsetin was prepared by the acid hydrolysis of 7-O-methylmearnsetin 3-O-rhamnoside as a reference flavonoid compound. A Shimadzu (Kyoto, Japan) ultraviolet spectrophotometer (model UV1601) and a FLUOstar Optima microplate reader (BMG Labtechnologies, Inc., Durham, NC, USA) were used for ROS scavenging assays, total phenolic content, and LDL oxidation assay. An HPLC system with electrochemical detection (ECD) (Coularray 5600A, ESA Inc., Chelmsford, MA, USA) was used for the flavonoid analysis. A Beckman (Palo Alto, CA, USA) centrifuge (model L8-M) was used for the isolation of human LDL. Extraction and fractionation The extraction was executed in triplicate. In brief, 10 g of S. theezans leaf powder was defatted twice with 100 mL of CHCl3 and extracted twice with 100 mL of 60% acetone for 12 hours at room temperature. The solvent in the combined filtrates was removed at room temperature using a rotary evaporator, leaving the crude acetone extract. After preparation of a 10% (vol=wt) methanol slurry, the crude acetone extract was fractionated sequentially with 300 mL of each n-hexane, ethyl acetate, and water. The n-hexane, ethyl acetate, and water fractions and crude acetone extract were used for the determinations of bioactivity. The yields of crude extract and hexane, ethyl acetate, and water fractions obtained from 10 g of S. theezans leaves were 2.1639 0.0902 g, 0.0012 0.0004 g, 0.1357 0.0102 g, and 1.6872 0.0462 g, respectively.

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ROS scavenging activities The HOCl scavenging activity was tested using the modification of the ferrocyanide [Fe(II)CN)6] oxidation method.11 The reaction mixture solution containing 100 mL of samples and 450 mL of HOCl (0.4 mM) was incubated for 5 minutes at room temperature. After addition of 450 mL of K4Fe(II)(CN)6 (20 mM), the resulting mixture was incubated for 5 minutes at room temperature, and the absorbance was measured at 420 nm using a spectrophotometer. Intraand inter-day coefficients of variation (CV) values were 0.9% and 2.9%, respectively. The ONOO scavenging activity was measured by a modified method12 of utilizing DHR-123 oxidation. Intraand inter-day CV values were 4.7% and 3.6%, respectively. The O2 scavenging activity was assessed by the spectrophotometric determination of the reduction product of NBT in a xanthine=XOD system.13 In brief, following 10 minutes of incubation of the antioxidant at room temperature with a reaction mixture of 50 mmol=L NBT, 50 mmol=L xanthine, and 0.05 U=mL XOD (final concentrations), the absorbance was measured at 560 nm. Intra- and inter-day CV values were 1.9% and 7.7%, respectively. The DPPH scavenging assay was determined using the method described by Blois.14 In brief, the reaction mixtures containing 100 mmol=L DPPH in methanol and samples or standard solutions were incubated at 378C for 30 minutes in the dark, and the absorbance was measured at 517 nm using a microplate reader. Intra- and inter-day CV values were 1.4% and 7.6%, respectively. The ROS activities by all above assays were expressed as percentage inhibition of the control without antioxidant. Test fraction concentrations of 125, 100, 50, and 25 mg=mL were used for HOCl, O2, DPPH, and ONOO scavenging assays, respectively. Quercetin (50 mmol=L) was used as a reference antioxidant. Total phenolic content Total phenolic content was measured colorimetrically using the Folin-Ciocalteu assay.15 The total phenolic content was expressed as mmol of gallic acid equivalent (GAE)=g. Oxygen radical absorbance capacity (ORAC) assay The ORAC assay was performed in a microplate reader (378C) equipped with fluorescence filters (excitation 485 nm, emission 520 nm).16 ORAC values were calculated from a standard curve established using Trolox at 5–50 mmol=L and expressed as mmol of Trolox equivalents (TE)=g. The test fraction concentrations were 10 mg=mL. Intra- and inter-day CV values were 3.0% and 7.3%, respectively. Resistance of LDL to Cu2þ-induced oxidation Plasma was prepared from blood collected into EDTAtreated tubes from 10 nonfasted healthy volunteers and pooled. The study protocol was approved by the Institutional Review Board of the Tufts Medical Center=Tufts University

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(Boston, MA). LDL (1.019–1.063 g=mL) was isolated from the EDTA-treated plasma using an ultracentrifugation protocol,17 stored at 48C in the dark under N2, and used within 2 days. Prior to the assay, NaCl and EDTA were removed using a PD-10 column (Amersham Pharmacia Biotech, Uppsala, Sweden). The LDL concentration was calculated from the protein content determined using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). The resistance of LDL to oxidation was assessed by the method of Esterbauer et al.18 In brief, a total of 1 mL of reaction mixture containing LDL (100 mg=mL), diluted ethyl acetate fraction (1 and 2 mmol of GAE=L), and CuSO4 (10 mmol=L) solution was incubated at 378C, and the formation of CD was monitored at 234 nm over a 3-hour period using a spectrophotometer equipped with a six-position automatic sample changer. The lag time was calculated as the intercept of the lag and propagate phase at the abscissa in the absorbance–time plot. Flavonoid determination Flavonoids in the ethyl acetate fraction from S. theezans leaves were determined by an HPLC=ECD method.19 The stock solution of 1 mg=mL ethyl acetate fraction in methanol was diluted to 10 mg=mL with methanol and then analyzed on a Zorbax SB-C18 column (4.6150 mm) (Agilent Technologies, Santa Clara, CA, USA) with a gradient elution of mobile phase A (75 mM citric acid and 25 mM ammonium acetate in 10% acetonitrile) and mobile phase B (75 mM citric acid and 25 mM ammonium acetate in 50% acetonitrile) at the flow rate of 0.6 mL=minute. Detection was achieved with potentials applied from 60 to 720 mV with 60 mV increments. Myricetin 3-O-rhamnoside, 7-O-methylmyricetin 3-O-rhamnoside, kaempferol 3-O-rhamnoside, 7-O-methylmearnsetin 3-O-rhamnoside, 7-O-methylquercetin 3-O-rhamnoside, myricetin, quercetin, isorhamnetin, kaempferol, and 7-O-methylmearnsetin were used as reference compounds for flavonoid identification and quantification. The content of each flavonoid compound was determined based on the standard curve of the reference flavonoids. Statistical treatment All of the experiments were executed in triplicate, and the values are reported as mean SD values. The significance between the fractions in ROS scavenging activities, total phenolic content, and ORAC value was determined using analysis of variance and Tukey’s HSD test. SAS version 9.1 (2003) (SAS Institute Inc., Cary, NC, USA) was used for statistical analyses, with P < .05 considered statistically significant. RESULTS AND DISCUSSION

reference antioxidant are shown in Figure 1. The ethyl acetate fraction showed the highest radical scavenging activities against HOCl, ONOO, O2, and DPPH, followed by the crude acetone extract. The strong activities of the ethyl acetate fraction were consistent with those reported by others.20 In contrast to the significant activity differences among the fractions shown in the other assays, the O2 scavenging activities showed similar higher activities among all the fractions. This result suggests that antioxidant in the hexane and water fraction were more reactive toward O2 than toward the other three ROS. ORAC assay has been commonly used to evaluate the total antioxidant capacity of plant foods.21 We found the ethyl acetate fraction had the highest ORAC value (7.7-fold higher than the hexane and water fractions) (Table 1). Similarly, the ethyl acetate fraction had the highest total phenolic content, which was 5.13-fold higher than the hexane and water fraction. In agreement with the findings of Kappel et al.,22 there was a good correlation between total phenolic content and ORAC value (r ¼ 0.9917). The descending order of the ratio of ORAC value to total phenolic content was ethyl acetate fraction > acetone crude extract > water fraction > hexane fraction. This observation is consistent with other reports that ethyl acetate is very efficient for extracting phenolic antioxidants from fruits and herbs.23,24 In summary, our results show that antioxidants in the ethyl acetate fraction of S. theezans leaves could serve as a potent natural antioxidant ingredient because of their strong ROS scavenging activities and high phenolic content. Effect of the ethyl acetate fraction on resistance of LDL to oxidation As the ethyl acetate fraction displayed the strongest ROS scavenging activity, we further examined the antioxidant effect of this fraction on the LDL to Cu2þ-induced oxidation in vitro. This assay has been widely used to assess the potency of antioxidants to protect LDL against oxidation because of the contribution of oxidized LDL to the development of atherosclerosis and cardiovascular disease.25 We found that antioxidants in the ethyl acetate fraction enhanced the resistance of LDL to oxidation in a dosedependent manner, with a lag time extension from the control by the ethyl acetate fraction at 1 and 2 mmol=L being 47 and 91 minutes, respectively (Fig. 2). Further, the lag time of LDL oxidation with an addition of the ethyl acetate fraction at 2 mmol=L was comparable to that with 0.5 mmol=L quercetin. The mechanism by which antioxidants from S. theezans leaves contribute to protect LDL against oxidation in S. theezans leaves remains to be determined. We suggested that almond skin polyphenols might enhance the resistance of LDL to oxidation by stabilizing LDL conformation and protecting apoprotein-B tryptophan from oxidation.26

ROS scavenging activities and total antioxidant capacity of fractions

Flavonoid characterization in the ethyl acetate fraction

The ROS scavenging activities of the three fractions and crude acetone extract of S. theezans leaves and quercetin as

Because the ethyl acetate fraction from S. theezans leaves displayed the highest antioxidant activity, its flavonoid

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FIG. 1. ROS scavenging activities of the solvent fractions from S. theezans leaves. The test concentrations were as follows: quercetin, 50 mmol=L; fractions, 125, 25, 100, and 50 mg=mL for HOCl, ONOO, O2, and DPPH assays, respectively. Values with different letters on the same assay significantly different, tested by a Turkey’s HSD test, P .05. EtOAc, ethyl acetate.

Table 1. The ORAC Value and Total Phenolic Content of the Solvent Fractions from S. theezans Leaves Fraction

ORAC (mmol=g TE)

Total phenolic content (mmol=g GAE)

a

a

3.4 0.5a 0.0 0.0b 3.5 0.2a 2.5 0.3c

740 20 0.0 0.0b 2,270 50c 370 10d

2,540 250 700 80c 7,930 300b 910 90c

Acetone Hexane Ethyl acetate Water

ORAC=total phenolic

Means with different letters in the same column are significantly different by Tukey’s HSD test.

2 CON STE 1 µM STE 2 µM Q

0.5 µM

Absorbance at 234 nm

1.5

FIG. 2. Effect of the ethyl acetate fraction from S. theezans leaves on CD formation during Cu2þ-induced LDL oxidation. CON, control treatment; STE, treatment with ethyl acetate fraction; Q, quercetin.

1

0.5

0 0

30

60

90

Incubation time (min)

120

150

180

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CHUNG ET AL.

FIG. 3. The flavonoid profile of the ethyl acetate fraction from S. theezans leaves analyzed by HPLC=ECD with peaks identified as follows: 1, myricetin 3-Orhamnoside; 2, myricetin; 3, 7-O-methylmyricitrin; 4, kaempferol 3-O-rhamnoside; 5, quercetin; 6, 7-O-methylmearnsetin 3-O-rhamnoside; 7, 7-O-methylquercetin 3-O-rhamnoside; 8, kaempferol; 9, isorhamnetin; 10, 7-O-methylmearnsetin. Color images available online at www. liebertonline.com=jmf.

profile was further characterized using HPLC-ECD. After retention times and redox traces were substantiated with authentic standards, 10 flavonols were identified, including kaempferol, isorhamnetin, mearnsetin, and myricetin and their glycosides (Fig. 3 and Table 2). Interestingly, all of the identified flavonol glycosides are rhamnose conjugates, whereas glucose conjugates are found commonly in the plant leaves. Among these flavonols, 7-O-methylmyricetin 3-O-rhamnoside was the major flavonoid, accounting for 36% of the total flavonoid content. Furthermore, myricetin and its rhamnoside, including 7-O-methyl myricitrin, made up 57.6% of the total flavonoid content, while mearnsetin (40 -methylmyricetin) and its rhamnoside made up 21.2%. Although more compounds remain to be characterized, the total content of 10 identified flavonoids made up 46.6% of the total amount of the ethyl acetate fraction. Among the identified flavonols, 7-O-methylmearnsetin 3-O-rhamnoside is a novel compound isolated from S. theezans for the first time.10 To date, mearnsetin and its conjugates have been rarely reported in the literature.27 Flavonoids may slow the pathogenesis of atherosclerosis and cardiovascular disease by protecting LDL from oxidation through their

Table 2. The Flavonoid Composition and Contents of the Ethyl Acetate Fraction from S. theezans Leaves Peak number 1 2 3 4 5 6 7 8 9 10

Flavonoid

Contents (mg=g)

Myricetin 3-O-rhamnoside Myricetin 7-O-Methylmyricetin 3-O-rhamnoside Kaempferol 3-O-rhamnoside Quercetin 7-O-Methylmearnsetin 3-O-rhamnoside 7-O-Methylquercetin 3-O-rhamnoside Kaempferol Isorhamnetin 7-O-Methylmearnsetin Total

42.4 1.3 56.5 0.3 167 4.2 45.9 1.7 36.8 3.2 80.6 8.0 11.6 0.1 3.3 0.3 1.3 0.1 17.7 0.4 463.1 4.5

ROS scavenging effects by both antioxidant recycling and transient metal chelating activity.28–32 Epidemiological evidence also suggests an inverse relationship between intake of dietary flavonoids and risk of cardiovascular disease.33,34 Therefore, we suggest the flavonoid-rich ethyl acetate fraction of S. theezans leaves may be a candidate material for use in functional foods and dietary supplements.

ACKNOWLEDGMENTS This work was supported by Korea Research Foundation grant KRF-2008-313-F00169 funded by the Korean Government and by U.S. Department of Agriculture= Agricultural Research Service Cooperative Agreement number 58-1950-7-707. AUTHOR DISCLOSURE STATEMENT No competing financial interests exist. REFERENCES 1. Yokoyama M: Oxidant stress and atherosclerosis. Curr Opin Pharmacol 2004;4:110–115. 2. Fu X, Kassim SY, Parks WC, Heinecke JW: Hypochlorous acid oxygenates the cysteine switch domain of pro-matrilysin (MMP-7): a mechanism for matrix metalloproteinase activation and atherosclerotic plaque rupture by myeloperoxidase. J Biol Chem 2001;276:41279–41287. 3. Sugiyama S, Okada Y, Sukhova GK, Virmani R, Heinecke JW, Libby P: Macrophage myeloperoxidase regulation by granulocyte macrophage colony-stimulating factor in human atherosclerosis and implications in acute coronary syndromes. Am J Pathol 2001;158:879–891. 4. Hazell LJ, Davies MJ, Stocker R: Secondary radicals derived from chloramines of apolipoprotein B-100 contribute to HOClinduced lipid peroxidation of low-density lipoproteins. Biochem J 1999;339:489–495. 5. Heitzer T, Schlinzig T, Krohn K, Meiertz T, Munzel T: Endothelial dysfunction, oxidative stress, and risk of cardiovascular


6. 7.

8.

9.

10.

11.

12.

13.

14. 15.

16.

17.

18.

19.

20.

events in patients with coronary artery disease. Circulation 2001; 104:2673–2678. Manach C, Mazur A, Scalbert A: Polyphenols and prevention of cardiovascular diseases, Curr Opin Lipidol 2005;16:77–84. Wu F, Wang M, Fan H, Zhao Z, Zhao B: Chemical constituents of the roots of Sageretia theezans and effect on experimental liver injury. Chin Trad Herb Drugs 1987;18:389–390. Xu LZ, Yang XJ, Li B: Chemical constituents of Sageretia theezans Brongn. [in Chinese]. Zhongguo Zhong Yao Za Zhi 1994;19:675–676, 702. Park JC, Hur JM, Park JG, Hatano T, Yoshida T, Miyashiro H, Min BS, Hattori M: Inhibitory effects of Korean medicinal plants and camellia tannin H from Camellia japonica on human immunodeficiency virus type 1 protease. Phytother Res 2002;16: 422–426. Chung SK, Kim YC, Takaya Y, Terashima K, Niwa M: Novel flavonol glycoside, 7-O-methyl mearnsitrin, from S. theezans and its antioxidant effects. J Agric Food Chem 2004;52:4664–4668. Zhu BZ, Carr AC, Frei B: Pyrrrolidine dithiocarbanmate is a potent antioxidant against hypochlorous acid-induced protein damage. FEBS Lett 2002;532:80–84. Choi RH, Choi JS, Han YN, Bae S, Chung HY: Peroxynitrite scavenging activity of herb extracts. Phytother Res 2002;16:364– 367. Kim DO, Chun OK, Kim YJ, Moon HY, Lee CY: Quantification of polyphenolics and their antioxidant capacity in fresh plums. J Agric Food Chem 2003;51:6509–6515. Blois MS: Antioxidant determinations by the use of a stable free radical. Nature 1958:181;1199–1200. Singleton VL, Orthofer R, Lamuela-Raventos RM: Analysis of total phenolic and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods Enzymol 1999;299: 152–178. Ou B, Hampsch-Woodill M, Prior RL: Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe. J Agric Food Chem 2001;49:4619–4926. Chung BH, Wilkinson T, Geer JC, Segrest JP: Preparative and quantitative isolation of plasma lipoproteins: rapid, single discontinuous density gradient ultracentrifugation in a vertical rotor. J Lipid Res 1980;21:284–291. Esterbauer H, Striegl G, Puhl H, Rotheneder M: Continuous monitoring of in vitro oxidation of human low-density lipoprotein. Free Radic Res Commun 1989;6:67–75. Chen CY, Milbury PE, Lapsley K, Blumberg JB: Flavonoids from almond skins are bioavailable and act synergistically with vitamins C and E to enhance hamster and human LDL resistance to oxidation. J Nutr 2005;135:1366–1373. Kim JY, Kim HS, Kang HS, Choi JS, Yokozawa T, Chung HY: Antioxidant potential of dimethyl lithospermate isolated from Salvia miltiorrhiza (red sage) against peroxynitrite. J Med Food 2008;11:21–28.

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21. Prior RL, Wu X, Schaich K: Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J Agric Food Chem 2005;53:4290– 4302. 22. Kappel VD, Costa GM, Scola G, Silva FA, Landell MF, Valente P, Souza DG, Vanz DC, Reqinatto FH, Moreira JC: Phenolic content and antioxidant and antimicrobial properties of fruits of Capsicum baccatum L. var. pendulum at different maturity stages. J Med Food 2008;11:267–274. 23. Termentzi A, Kefalas P, Kokkalou E: Antioxidant activities of various extracts and fractions of Sorbus domestica fruits at different maturity stages. Food Chem 2006;98:599–608. 24. Parejo I, Viladomat F, Bastida J, Rosas-Romero A, Flerlage N, Burillo J, Codina C: Comparison between the radical scavenging activity and antioxidant activity of six distilled and nondistilled Mediterranean herbs and aromatic plants. J Agric Food Chem 2002;50:6882–6890. 25. Kondo K, Matsumoto A, Kurata H, Tanahashi H, Koda H, Amachi T, Itakura H: Inhibition of oxidation of low-density lipoprotein with red wine. Lancet 1994;344:193–194. 26. Chen CY, Milbury PE, Chung SK, Blumberg J: Effect of almond skin polyphenolics and quercetin on human LDL and apolipoprotein B-100 oxidation and conformation. J Nutr Biochem 2007; 18:7857–7894. 27. Rao CP, Hanumaiah T, Vemuri VSS, Rao KVJ: Flavonol 3-glycosides from the leaves of Flemingia stricta. Phytochemistry 1983;22:621–622. 28. McPhail DB, Hartley RC, Gardner PT, Duthie GG: Kinetic and stoichiometric assessment of the antioxidant activity of flavonoids by electron spin resonance spectroscopy. J Agric Food Chem 2003;51:1684–1690. 29. Xiao J, Suzuki M, Jlang X, Chen X, Yamamoto K, Ren F, Xu M: Influence of B-ring hydroxylation on interactions of flavonols with bovine serum albumin. J Agric Food Chem 2008:56;2350– 2356. 30. Miura S, Watanabe J, Sano M, Tomita T, Osawa T, Hara Y, Tomita I: Effects of various natural antioxidants on the Cu2þmediated oxidative modification of low-density lipoprotein. Biol Pharm Bull 1995;18:1–4. 31. Salvayre AN, Salvayre R: Quercetin prevents the cytotoxicity of oxidized LDL on lymphoid cell lines. Free Radic Biol Med 1992;12:101–106. 32. Moon JH, Nakata R, Oshima S, Inakuma T, Terao J: Accumulation of quercetin conjugates in blood plasma after the short term ingestion of onion by women. Am J Physiol Regul Integr Comp Physiol 2000;279:R461–467. 33. Hertog MGL, Feskens EJM, Kromhout D: Antioxidant flavonols and coronary heart disease risk: ten year follow-up of the Zutphen Elderly Study. Lancet 1997;349:699. 34. Yochum L, Kushi LH, Meyer K, Folsom AR: Dietary flavonoid intake and risk of cardiovascular disease in postmenopausal women. Am J Epidemiol 1999;149:943–949.