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Toxicology 278 (2010) 75–87

Contents lists available at ScienceDirect

Toxicology

journa l homepage: www.e lsev ier .com/ locate / tox ico l

ioactive phenolics and antioxidant propensity of flavedo extracts of Mauritianitrus fruits: Potential prophylactic ingredients for functional foods application

eena Ramfula, Theeshan Bahorunb,∗, Emmanuel Bourdonc, Evelyne Tarnusc, Okezie I. Aruomad

Department of Agricultural and Food Science, Faculty of Agriculture, University of Mauritius, Réduit, MauritiusDepartment of Biosciences, Faculty of Science, University of Mauritius, Réduit, MauritiusLaboratoire de Biochimie et Génétique Moléculaire (LBGM), Groupe d’Etude sur l’Inflammation Chronique et l’Obésité (GEICO), Université de Saint Denis de La Réunion, FranceDepartment of Pharmaceutical and Biomedical Sciences, Touro College of Pharmacy, New York, USA

r t i c l e i n f o

rticle history:eceived 26 November 2009eceived in revised form 11 January 2010ccepted 18 January 2010vailable online 25 January 2010

eywords:itrus fruitslavedolavonoidsitamin C

a b s t r a c t

The flavedo extracts of twenty-one varieties of citrus fruits (oranges, satsumah, clementine, mandarins,tangor, bergamot, lemon, tangelos, kumquat, calamondin and pamplemousses) grown in Mauritius wereexamined for their total phenolic, flavonoid and vitamin C contents and antioxidant activities. Totalphenolics correlated strongly with the trolox equivalent antioxidant capacity (TEAC), ferric reducingantioxidant capacity (FRAP) and hypochlorous acid (HOCl) scavenging activity assays (r > 0.85). Basedon their antioxidant activities in these three assays nine citrus fruits namely, one orange, clementine,tangor and pamplemousse variety, two tangelo varieties and three mandarin varieties, were furthercharacterized for their flavanone, flavonol and flavone levels by HPLC and their antioxidant activitieswere assessed by the copper-phenanthroline and iron chelation assays. The flavanone, hesperidin, waspresent at the highest concentrations in all flavedo extracts except for pamplemousses where it was

ntioxidantsood ingredientsunctional foods

not detected. Contents in hesperidin ranged from 83 ± 0.06 to 234 ± 1.73 mg/g FW. Poncirin, didymin,diosmin, isorhoifolin and narirutin were also present in all extracts whereas naringin was present onlyin one mandarin variety. The nine flavedo extracts exhibited good DNA protecting ability in the cuphenassay with IC50 values ranging from 6.3 ± 0.46 to 23.0 ± 0.48 mg FW/mL. Essentially the flavedos wereable to chelate metal ions however, tangor was most effective with an IC50 value of 9.1 ± 0.08 mg FW/mL.The flavedo extracts of citrus fruits represent a significant source of phenolic antioxidants with potential

or the

prophylactic properties f

. Introduction

The role played by dietary factors on health status has long beenecognised but it has been only recently that epidemiological andlinical studies have provided a clearer insight on the chemicalnd physiological mechanisms of the effects of bioactive foods onuman health (Shahidi, 2009). Phytophenolics play a crucial role inealth promotion and disease prevention by mechanisms related toell differentiation, deactivation of pro-carcinogenes, maintenancef DNA repair, inhibition of N-nitrosamine formation and changef estrogen metabolism, amongst others (Shahidi, 2004). Majorechanisms for the antioxidant effect of phenolics in functional

oods include free radical scavenging and metal chelation activi-ies. Reactive oxygen species (ROS), such as the superoxide radicalO2

•−), hydrogen peroxide (H2O2), hypochlorous acid (HOCl) andhe hydroxyl radical (HO•) have been recognised to play a determin-

∗ Corresponding author.E-mail address: tbahorun@uom.ac.mu (T. Bahorun).

300-483X/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.tox.2010.01.012

development of functional foods.© 2010 Elsevier Ireland Ltd. All rights reserved.

ing role in the pathogenesis of several human diseases (Halliwell,1996; Halliwell et al., 1992; Aruoma, 1994, 2003). ROS-inducedoxidation can result in cell membrane disintegration, membraneprotein damage and DNA mutation, which can further initiate orpropagate the development of diseases including cancer (Huanget al., 2001), diabetes (Boynes, 1991), neurodegenerative diseases(Perry et al., 2000), the process of aging (Hensley and Floyd, 2002)and cardiovascular dysfunctions (Hool, 2006). Phenolic compoundssuch as phenolic acids, flavonoids, stilbenes, tannins and lignanscan scavenge free radicals and quench ROS and therefore provideeffective means for preventing and treating free radical-mediateddiseases.

Mauritius is a tropical island in the Indian Ocean with a relativelyhigh prevalence of cardiovascular diseases, cancers and diabetes(Central Statistic Office, 2007). This has triggered interest in the

study of the phytochemistry and antioxidant capacity of the Mauri-tian diet, which comprises a wide variety of exotic fruits, vegetablesand beverages (Luximon-Ramma et al., 2003; Bahorun et al., 2004,2007, 2010). Citrus (Citrus L. from Rutaceae) is one of the mostpopular world fruit crops that, besides providing an ample sup-

7 icology 278 (2010) 75–87

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6 D. Ramful et al. / Tox

ly of vitamin C, folic acid, potassium and pectin, contains a host ofctive phytochemicals that can protect health. Citrus species of var-ous origins have been assessed for their phenolic constituents andntioxidant activities (Proteggente et al., 2003; Gorinstein et al.,004; Anagnostopoulou et al., 2006; Guimarães et al., 2009). Cit-us fruits, citrus fruit extracts and citrus flavonoids exhibit a wideange of promising biological properties including antiatherogenic,nti-inflammatory and antitumor activity, inhibition of blood clotsnd strong antioxidant activity (Middleton and Kandaswami, 1994;ontanari et al., 1998; Samman et al., 1996). Citrus is consumedostly as fresh produce and juice and most often the peel is dis-

arded. This represents a huge waste as citrus peels are reportedo possess highest amounts of flavonoids compared to other partsf the fruit (Manthey and Grohmann, 2001). Citrus peels are sub-ivided into the epicarp or flavedo and mesocarp or albedo. Theavedo is the colored peripheral surface of the peel while the albedo

s the white soft middle layer of the peel (Fig. 1).The phytophenolic composition and in vitro antioxidant activ-

ties of the flavedo extracts of 21 citrus fruit varieties (Table 1)rown in Mauritius were determined. From the initial results, ninef the flavedo extracts (Orange 2B, Clementine A, Mandarin 1A,A and 5, Tangor A, Tangelo 1A and 2 and Pamplemousses 2B)

ere further characterized for their flavanone, flavonol and flavone

evels, their ability to protect DNA damage and their iron chelat-ng activity. There has been so far no report on the nutritionalnd health-promoting values of Mauritian citrus varieties. Thusauritian citrus varieties could be important sources of dietary

able 1cientific and common names, variety and harvest dates of citrus fruits analysed.

Scientific name Common name Variety

Citrus sinensis Orange Valencia lWashingt

Citrus unshiu Satsumah Owari

Citrus clementina Clementine Commun

Citrus reticulata Mandarin Fairchild

Dancy

Beauty

SuhuganFizu

C. reticulata × C. sinensis Tangor Elendale

Citrus aurantium ssp. bergamia Bergamot –

Citrus meyeri Lemon Meyer

C. reticulata × C. paradisis Tangelo Mineola

OrlandoUgli

Fortunella margarita Kumquat Nagami

Citrus mitis Calamondin –

Citrus grandis Pamplemousses Rainking

Kaopan

Pink

Chandler

Fig. 1. Anatomy of citrus fruit showing the flavedo (the orange peripheral surface ofthe peel or epicarp), albedo (the white soft fibre middle layer of the peel or mesocarp)and the pulp (the inside layer of the fruit with juicy vesicles).

polyphenolic antioxidant compounds that may have potential ben-efits in health and disease management.

2. Materials and methods

2.1. Chemicals

2,2′-Azino-bis(3-ethylbenzthiozoline-6)-sulfonic acid (ABTS), Folin & Ciocal-teu’s phenol reagent and 2-Aminoethanesulfonic acid (Taurine) were purchased

Harvest month Variety and harvest code

ate August 1on Navel March 2A

May 2B

March AMay B

e March AMay B

April 1AMay 1BMay 2AJune 2BJune 3AAugust 3BAugust 4August 5

June AAugust B

April –

April AMay B

June 1AAugust 1BAugust 2June 3AAugust 3B

April AJune B

June AAugust B

May 1AAugust 1BMay 2AAugust 2BMay 3AAugust 3BAugust 4

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D. Ramful et al. / Tox

rom Sigma (St. Louis, MO, USA). 2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ) was fromnalytical Rasayan, s.d. fiNe-CHEM Limited (Mumbai, India). Metaphosphoric acidas from Sigma Chemical Co. (St. Louis, MO). 2,6-Dichloroindophenol indophenol

odium salt was from Alpha Chemika (Mumbai, India). l-Ascorbic acid was fromHD Laboratory Supplies (Poole, England). 6-Hydroxy-2,5,7,8-tetramethylchroman--carboxylic acid, 97% (Trolox) was from Sigma–Aldrich Chemie (Steinheim,ermany). Gallic acid, quercetin, rutin, diosmin, rhoifolin, isorhoifolin, neoeriocitrin,oncirin, narirutin, neohesperidin, didymin, hesperidin and naringin (HPLC grade)ere from Extrasynthèse (Genay, France). HPLC-grade acetonitrile and methanolere obtained from Merck (Darmstadt, Germany). All other reagents used were of

nalytical grade.

.2. Plant material

Citrus fruits (Table 1) were obtained from La Compagnie Agricole de Labour-onnais situated at Mapou, in the north of Mauritius. Fruits were harvested at theature stage when they were ready to be placed on the market or ready for process-

ng. Some varieties were sampled twice at different periods of the harvest season toetermine the effect of harvest time on tested parameters. After harvest, the fruitsere rapidly processed on the same day. They were carefully washed under running

ap water and patted dry. The flavedo of at least 10 fruits of each variety was care-ully removed with a manual peeler and cut into small pieces. Weighed portions ofhe peripheral peel of pooled samples of each variety were lyophilised for 48 h andhe freeze-dried weight was determined. Samples were ground into a fine powdern a coffee grinder and stored in airtight containers at −4 ◦C until analysed.

.3. Extraction

The extraction procedure used was adapted from Franke et al. (2004) and Chunt al. (2003). A known amount of powdered freeze-dried citrus tissues was exhaus-ively extracted in 80% aqueous methanol at 4 ◦C for three consecutive days. Afterentrifugation at 4500 rpm for 15 min, supernatants of all three extractions wereooled and stored at −20 ◦C until used for the determination of total phenol andotal flavonoids and for the antioxidant assays.

.4. Total phenolic content

The Folin–Ciocalteu assay, adapted from Singleton and Rossi (1965), was usedor the determination of total phenolics present in the citrus fruit extracts. To.25 mL of diluted extract, 3.5 mL of distilled water was added followed by 0.25 mLf Folin–Ciocalteu reagent (Merck). A blank was prepared using 0.25 mL of 80%ethanol instead of plant extract. After 3 min, 1 mL of 20% sodium carbonate was

dded. Tube contents were vortexed before being incubated for 40 min in a water-ath set at 40 ◦C. The absorbance of the blue coloration formed was read at 685 nmgainst the blank standard. Total phenolics were calculated with respect to galliccid standard curve (concentration range: 0–12 �g/mL). Results are expressed ing of gallic acid/g fresh weight of plant material.

.5. Total flavonoid content

Total flavonoids were measured using a colorimetric assay adapted from Zhishent al. (1999). 150 �L of 5% aqueous NaNO2 was added to an aliquot (2.5 mL) of eachxtract and the mixture was vortexed. A reagent blank using 80% aqueous methanolnstead of sample was prepared. After 5 min, 150 �L of 10% aqueous AlCl3 was added.mL of 1 M NaOH was added 1 min after the addition of aluminium chloride. Solutionas mixed well and the absorbance was measured against the blank at 510 nm. Totalavonoids were calculated with respect to quercetin standard curve (concentrationange: 50–200 �g/mL). Results are expressed in �g of quercetin g/fresh weight oflant material.

.6. Total vitamin C content

The 2,6-dichloroindophenol titrimetric method (AOAC, 1995) was used to deter-ine the vitamin C content of flavedo extracts. 100 g of the plant material was cut

nto small pieces and blended in a Waring blander with 150 mL of metaphosphoriccid–acetic acid solution. After filtration and appropriate dilutions with metaphos-horic acid–acetic acid solution as determined by the extract colour intensity, 7 mLf the diluted solution was titrated against standard indophenol solution. Resultsre expressed in �g ascorbic acid/g fresh weight.

.7. Trolox equivalent antioxidant capacity (TEAC) assay

The TEAC assay measures the relative ability of antioxidant substances tocavenge the 2,2′-azino-bis(3,ethyl benz-thiazoline-6-sulfonic acid) radical cation

ABTS•+), compared with standard amounts of the synthetic antioxidant Trolox, theater-soluble vitamin E analogue. The method of Campos and Lissi (1996) was used.

o 3 mL of the ABTS•+ solution generated by a reaction between ABTS (0.5 mM)nd activated MnO2 (1 mM) in phosphate buffer (0.1 M, pH 7), 0.5 mL of dilutedlant extract was added. Decay in absorbance was monitored at 734 nm for 15 minsn a Helios-Alpha spectrophotometer (Unicam Ltd., UK) maintained at 20 ◦C by a

y 278 (2010) 75–87 77

peltier thermostat. Calculations were made with respect to a dose–response curveof Trolox (concentration range: 0–100 �M) and the TEAC values are expressed in�mol Trolox/g fresh weight.

2.8. Ferric reducing antioxidant power (FRAP) assay

The FRAP assay was carried according to the procedure described by Benzie andStrain (1996). The principle of this method is based on the ability of substancesto reduce Fe(III)-2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ) complex to Fe(II)-TPTZ, theresulting intense blue colour being linearly related to the amount of reductant(antioxidant) present. The FRAP reagent consisting of 20 mL of 10 mM TPTZ solu-tion in 40 mM HCl and 20 mL of 20 mM ferric chloride in 200 mL of 0.25 M sodiumacetate buffer (pH 3.6) was freshly prepared and warmed at 37 ◦C. A 50 �L aliquot ofsample was added to 150 �L of distilled water, followed by 1.5 mL of FRAP reagent.The absorbance was read at 593 nm after 4 min incubation at 37 ◦C. A calibrationcurve of ferrous sulphate (0–1.2 mM) was used and results are expressed as �molFe2+/g fresh weight.

2.9. Hypochlorous acid (HOCl) scavenging assay

The HOCl assay was adapted from Weiss et al. (1982). Both HOCl and ClO− arepotent oxidants and thus harmful in excessive amounts in vivo. In this model, tau-rine, a �-amino acid, is used as a representation compound capable of reacting withHOCl/ClO− at diffusion controlled rates to form a stable and quantitable taurine chlo-ramine derivative. The antioxidant capacity was based on the ability of the extractto scavenge hypochlorous acid. HOCl was prepared by adjusting the pH of a 1% (v/v)solution of NaOCl to 6.2 with dilute sulphuric acid. The working concentration of thestock solution was determined spectrophotometrically by measuring its absorbanceat 235 nm and applying a molar extinction coefficient of 100. The reaction mixturecontained 100 �L taurine (10 mM), 100 �L extract (variable concentrations), 100 �LHOCl (1 mM) and phosphate saline buffer (pH 7.4) in a final volume of 1 mL. Afterincubation at room temperature for 10 min, the sample was then assayed for taurinechloramine by adding 10 �L of potassium iodide (2 M) to the reaction mixture. Thiscomplex has the ability to oxidise I− ions into I2 producing a yellow coloration. Theabsorbance of the reaction mixture was read at 350 nm. Results are expressed asIC50 values (mg fresh weight/mL).

2.10. Copper-phenanthroline (Cuphen) assay

The ability of the copper-phenanthroline complex to degrade DNA in the pres-ence of a reducing agent has been adopted as a method for assessing the antioxidantpropensities of dietary biofactors with antioxidant potentials (Aruoma, 1993, 1994;Gutteridge and Halliwell, 1982). Reaction mixture contained in a final volume of1.2 mL the following reagents in order of addition indicated: 100 �L of 1.8 mM 1,10-phenanthroline hydrate (stock solution made up in water having initially dissolvedthe crystals in 50 �L ethanol), 100 �L of 1.2 mM copper(II) chloride, 100 �L DNA(2.75 mg/mL), 100 �L of 120 mM KH2PO4–KOH buffer at pH 7.4, 100 �L distilledwater, 100 �L ascorbic acid (stock solution: 2.88 mM) and 600 �L of methanolic cit-rus extracts, serially diluted. After incubation at 37 ◦C for 1 h, 100 �L of 0.1 M EDTAwas added to stop the reaction. DNA damage was assessed by adding 1 mL of 1%(w/v) TBA and 1 mL of 25% (v/v) HCl followed by 15 min incubation at 80 ◦C. Thepink chromogen formed was extracted into butan-1-ol and the absorbance mea-sured at 532 nm. Results are expressed in terms of IC50 (mg FW/mL able to inhibit50% of DNA damage).

2.11. Iron(II) chelating activity

The method of Dorman et al. (2003) was adapted to assess the chelating activ-ity of the citrus extracts on iron(II) ions. The reaction mixture containing 950 �Lof extract serially diluted with 80% methanol, and 50 �L of 0.5 mM FeCl2·4H2O wasincubated for 5 min at room temperature. 50 �L of 2.5 mM ferrozine was then addedand was allowed to equilibrate for 10 min at room temperature. The purple col-oration formed was read at 562 nm. The control contained 80% methanol instead ofthe extract. The chelating activity was calculated according to the equation givenbelow and results are expressed as mean IC50 (mg FW/mL).

Metal chelating activity (%) =[

Abscontrol − Abssample

Abscontrol

]× 100

2.12. High performance liquid chromatography

2.12.1. Sample preparation

extracts (Orange 2B, Clementine A, Mandarin 1A, 2A and 5, Tangor A, Tangelo 1A and2 and Pamplemousses 2B) with most potent antioxidant capacities were selectedfor flavonoid glycosides quantification by HPLC. Known weights of lyophilized fruitpowders were extracted with 80% of aqueous methanol (HPLC grade) followingthe same procedure as described in Section 2.3. Samples were filtered on Milipore(0.22 �m) before use.

7 icolog

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8 D. Ramful et al. / Tox

.12.2. Chromatographic conditionsChromatographic conditions were adapted from Mouly et al. (1998). A HP1100

eries HPLC equipped with a vacuum degasser, quaternary pump, autosampler, ther-ostatted column compartment, diode array detector and HP Chemstation for data

ollection and analysis was used. After filtration on Millipore (0.22 �m), 30 �L ofxtract was injected on a Waters Spherisorb ODS-2 column (5 �m particle size,0 Å pore size, 4.6 mm id × 150 mm). The solvents used were: A, water–acetonitrile90:10, v/v; pH 2.35) and B, acetonitrile. The gradient profile was as follows:–12 min 0–8% B, 12–43 min 8–34% B, 43–44 min 34–70% B, 44–59 min 70% B,9–60 min back to 0% B. The diode array detector was set at 280 nm for the quanti-ative determination of flavanone glycosides and at 330 nm for flavone and flavonollycosides. The column temperature was 25 ◦C and the flow rate was fixed at.7 mL/min. The identification and quantification of the flavonoids investigatedere determined from retention time and peak area in comparison with the stan-ards used. The standards, poncirin, rhoifolin, didymin, naringin, rutin, diosmin,

sorhoifolin, neohesperidin, hesperidin, neoeriocitrin and narirutin, were preparedt a stock concentration of 200 �g/mL. Calibration standard samples containing thetandards each at 20, 40, 100 and 200 �g/mL were prepared by appropriate dilu-ions with methanol from the stock solutions and filtered on Milipore (0.22 �m)efore use. The linearity of the assay was demonstrated by assaying calibrationtandards in duplicate at four separate concentrations on two separate occasions.alibration curves were obtained by plotting the peak area of the standards ver-us their concentrations. Concentrations of each of the eleven flavonoid glycosidesn citrus fruit samples were determined by application of the obtained standardurve.

.13. Statistical analysis

Simple regression analysis was performed to calculate the dose–responseelationship of the standard solutions used for calibration as well as testamples. Unicam Vision 32 software (Unicam, Ltd., UK) was used to eval-ate initial and final antioxidant rate values for the TEAC assay. Data arexpressed as the means ± standard error of mean (SE) from two independentxperiments performed in triplicates. Mean differences were determined by one-ay ANOVA followed by Tukey’s HSD post-test using PrismTM v4.0 software

®

GraphPad Software, San-Diego, 2003). The differences were accepted as sig-ificant when P < 0.05 and are denoted by different letters. Linear regressionlots were generated and correlations between antioxidant activities and totalhenol, flavonoids and vitamin C contents were computed as Pearson’s correla-ion coefficient (r) using PrismTM v4.0 software (GraphPad® Software, San-Diego,003).

ig. 2. Total phenolic content of flavedo extracts of citrus fruits. Data represent mean valuignificant differences between samples (p < 0.05). Letter a denotes sample having highes

y 278 (2010) 75–87

3. Results

3.1. Phenolic and vitamin C contents

The total phenolic composition of the flavedo extracts is pre-sented in Fig. 2. The amount of total phenolics varied widely andranged from 1882 ± 65 �g/g FW in Lemon B to 7667 ± 57 �g/g FWin Tangor A. The total phenolics content in flavedo of Tangor Awas significantly higher (p < 0.05) than in the other extracts. Nosignificant differences (p > 0.05) were observed in the phenolic lev-els of Mandarin 1A, 2B, 5 and Tangor B, all ranking second on thelist. Orange 2B, Pamplemousses 2B, Mandarin 2A, Clementine Aand Tangelo 1A ranked third in terms of total phenolic content(p > 0.05). Significant differences (p < 0.05) were observed betweentotal phenolic levels of flavedo of similar varieties of citrus har-vested at different periods except for Pamplemousses 3. Highestlevels of total flavonoids (p < 0.05) were obtained in extracts of Tan-gelo 2 (5615 ± 93 �g/g FW) followed by Mandarin 1B (p < 0.05) and1A (p < 0.05) (5237 ± 68 �g/g FW and 5027 ± 89 �g/g FW respec-tively) (Fig. 3). Flavedo extracts of the Tangor variety, which hadthe highest total phenols, contained relatively low levels of totalflavonoids. Extracts of Lemon B and Calamondin A, whose flavonoidcontents were not significantly different (p > 0.05), ranked last onthe list.

The vitamin C composition of the flavedo extracts are shownin Fig. 4. Vitamin C content ranged between 344 ± 8 �g/g FW and1475 ± 15 �g/g FW. The majority of the pamplemousses varietiescontained the highest levels and flavedo of Pamplemousses 1B wasthe richest with 1475 ± 37 �g/g FW (p < 0.05). Tangelo 3B flavedo

ranked after the pamplemousses with a value of 1002 ± 46 �g/gFW. No significant difference was found between vitamin C con-tent in flavedo extracts of Lemon A and Kumquat B (p > 0.05) bothhaving the lowest amounts (388 ± 44 �g/g FW and 345 ± 21 �g/gFW respectively).

es (bars) with standard errors (n = 2). Different letters between columns representt total phenolics and letter r denotes sample having lowest phenolics content.

D. Ramful et al. / Toxicology 278 (2010) 75–87 79

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ig. 3. Total flavonoid contents of flavedo extracts of citrus fruits. Data represent meignificant differences between samples (p < 0.05). Letter a denotes sample having h

In the light of the large distribution of total phenolics, flavonoidsnd vitamin C in the flavedo extracts, we propose 3 groupings ofhese phytochemicals into (1) high level, (2) medium level and (3)ow level (Table 2).

ig. 4. Total vitamin C content of flavedo extracts of citrus fruits. *Flavedo and albedo aifferent letters between columns represent significant differences between samples (p <enotes sample having lowest vitamin C content.

ues (bars) with standard errors (n = 2). Different letters between columns representt total flavonoids and letter w denotes sample having lowest flavonoids content.

3.2. Antioxidant capacities

The Trolox Equivalent Antioxidant Capacity (TEAC), the FerricReducing Antioxidant Power (FRAP) and the Hypochlorous acid

nalysed together. Data represent mean values (bars) with standard errors (n = 2).0.05). Letter a denotes sample having highest total vitamin C content and letter n

80 D. Ramful et al. / Toxicology 278 (2010) 75–87

Table 2Classification of citrus fruits according to total phenolic, flavonoid and vitamin C levels in flavedo extracts.

Low Medium High

Total phenolic <3000 �g/g FW 3000–5500 �g/g FW >5500 �g/g FW• Mandarin 4 • Clémentine B • Clémentine A• Lemon A and B • Orange 1 • Orange 2A and 2B• Kumquat B • Mandarin 3A and B • Mandarin 1A, 1B, 2A, 2B and 5• Calamondin B • Satsumah A andB • Tangelo 1A and 2• Pamplemousses 1A • Tangelo 3 • Tangor A and B• Bergamot • Kumquat A • Pamplemousses 2B

• Pamplemousses 1B, 2A, 3A, 3B and 4• Calamondin A

Total flavonoids <2000 �g/g FW 2000–3600 �g/g FW >3600 �g/g FW• Mandarin 3A, 3B and 4 • Clémentine B • Clémentine A• Lemon A and B • Orange 1, 2A and 2B • Mandarin 1A, 1B, 2A and 2B• Kumquat A and B • Satsumah A and B • Tangelo 2• Calamondin A and B • Tangelo 1A, 1B, 3A and 3B • Pamplemousses 2B, 3A, and 4• Pamplemousses 1B • Pamplemousses 1A, 2A and 3B

• Tangor A and B• Bergamot

Total vitamin Ca <600 �g/g FW 600–1000 �g/g FW >1000 �g/g FW• Mandarin 1B • Clémentine B • Tangelo 3B• Mandarin 3B • Orange 1 and 2B • Pamplemousses 1A, 1B, 2B, 3A, 3B and 4• Lemon A • Mandarine 2A, 2B, 3A, 4 and 5• Kumquat B • Satsumah B

TA

I

f

• Tangelo 1A and 2 • Tangelo 1B• Pamplemouss• Tangor A and• Calamondin A

able 3ntioxidant activities of flavedo extracts of citrus fruits. Data expressed as mean value ±

Citrus fruit Code TEACI

Orange 1 21.6 ± 0.2hij

2A 26.1 ± 1.5fg

2B 31.2 ± 0.4d

Satsumah A 25.5 ± 0.1fg

B 20.0 ± 0.1ijk

Clémentine A 36.2 ± 0.1c

B 18.4 ± 0.6jklm

Mandarin 1A 43.4 ± 1.5ab

1B 31.5 ± 1.4d

2A 42.9 ± 0.7ab

2B 44.0 ± 0.5ab

3A 19.2 ± 0.2jkl

3B 15.1 ± 0.1mno

4 16.4 ± 0.2lmn

5 30.7 ± 0.1de

Tangor A 46.1 ± 0.7a

B 37.0 ± 0.6c

Bergamot – 12.8 ± 0.2op

Lemon A 13.3 ± 0.1nop

B 11.5 ± 0.2p

Tangelo 1A 43.1 ± 0.2ab

1B 23.3 ± 0. ghi

2 43.85 ± 1.2ab

3A 24.0 ± 0. gh

3B 31.2 ± 0.4d

Kumquat A 12.1 ± 0.2op

B 18.0 ± 0.4klm

Calamondin A 24.3 ± 0.1 gh

B 11.0 ± 0.2p

Pamplemousses 1A 11.7 ± 0.1p

1B 14.0 ± 0.3nop

2A 18.0 ± 0.9klm

2B 40.8 ± 0.3b

3A 21.6 ± 0.3hij

3B 23.1 ± 0. ghi

4 27.7 ± 0.7ef

�mol Trolox/g fresh weight; II�mol Fe(II)/g fresh weight; IIIIC50 mg fresh weight/mL. Sigollowed by Tukey’s multiple comparison test. Different superscripts between rows repre

es 2ABand B

standard error (n = 2).

FRAPII HOClIII

37.6 ± 0.1i 6.57 ± 0.23jk

48.2 ± 0. g 4.95 ± 0.05mno

56.7 ± 0.9cde 3.98 ± 0.10pq

32.8 ± 0.8k 7.68 ± 0.09i

41.4 ± 0.3h 6.66 ± 0.13j

67.6 ± 0.6b 3.80 ± 0.10pq

39.0 ± 0.2i 7.68 ± 0.04i

81.3 ± 0.3a 4.53 ± 0.04mnop

58.4 ± 1.0c 3.70 ± 0.06q

55.9 ± 0.4de 4.23 ± 0.06opq

56.7 ± 0.1cde 5.23 ± 0.03lm

29.2 ± 0.2l 13.62 ± 0.23c

20.9 ± 0.1p 14.24 ± 0.67bc

21.1 ± 0.1p 11.00 ± 0.09fg

53.2 ± 0.3f 4.98 ± 0.03mno

57.72 ± 0.4cd 4.41 ± 0.04nopq

57.8 ± 0.3cd 5.13 ± 0.06lmn

21.6 ± 0.5op 14.62 ± 0.06b

26.7 ± 0.3m 10.70 ± 0.0 g

21.2 ± 0.1p 14.36 ± 0.29bc

55.03 ± 0.4ef 5.17 ± 0.06lmn

38.0 ± 0.2i 9.15 ± 0.09h

55.2 ± 0.6ef 4.54 ± 0.10mnop

21.0 ± 0.4p 7.18 ± 0.14ij

35.0 ± 0.1j 9.11 ± 0.16h

13.9 ± 0.2r 10.49 ± 0.1 g

13.7 ± 0.1r 10.84 ± 0.19fg

24.3 ± 0.3n 12.78 ± 0.06d

14.2 ± 0.1r 17.78 ± 0.30a

14.7 ± 0.1r 12.14 ± 0.33de

22.8 ± 0.2nop 11.46 ± 0.04ef

32.6 ± 0.4k 5.86 ± 0.07kl

47.5 ± 0. g 4.53 ± 0.12mnop

23.7 ± 0.3no 5.25 ± 0.07lm

17.3 ± 0.1q 6.44 ± 0.18jk

21.1 ± 0.2p 6.98 ± 0.09ij

nificance testing among the different samples was performed by one-way ANOVAsent significant differences between samples (p < 0.05).

D. Ramful et al. / Toxicology 278 (2010) 75–87 81

Table 4Classification of citrus fruits according to the antioxidant activities of their flavedo extracts as measured by the TEAC, FRAP and HOCl scavenging assays.

Low Medium High

TEAC <20 �mol/g FW 20–35 �mol/g FW >35 �mol/g FW• Clémentine B • Orange 1, 2A and 2B • Clémentine A• Mandarin 3A, 3B and 4 • Mandarin 1B and 5 • Mandarine 1A, 2A and 2B• Lemon A and B • Satsumah A and B • Tangelo 1A and 2• Kumquat A and B • Tangelo 1B, 3A and 3B • Pamplemousses 2B• Calamondin B • Pamplemousses 3A, 3B and 4 • Tangor A and B• Pamplemousses 1A, 1B and 2A • Calamondin A• Bergamot

FRAP <30 �mol/g FW 30–50 �mol/g FW >50 �mol/g FW• Mandarin 3A, 3B and 4 • Clémentine B • Clémentine A• Lemon A and B • Orange 1 and 2A • Orange 2B• Kumquat A and B • Satsumah A and B • Mandarine 1A, 1B, 2A, 2B and 5• Calamondin A and B • Tangelo 1B and 3B • Tangelo 1A and 2• Tangelo 3A • Pamplemousses 2A and 2B • Tangor A and B• Pamplemousses 1A, 1B, 3A, 3B and 4• Bergamot

HOCl >10 mg FW/mL 5–10 mg FW/mL <5 mg FW/mL• Mandarin 3A, 3B and 4 • Clémentine B • Clémentine A• Lemon A and B • Orange 1 • Orange 2A and 2B• Kumquat A and B • Mandarin 2B • Mandarin 1A, 1B, 2A and 5• Calamondin A and B • Satsumah A and B • Tangelo 2

TangePampTango

(Tc(

Aa(AMTRflP

(C1tbF(

i((bhCnvF

gsspI62

tial in all assayed systems whilst extracts characterised by low totalphenolic levels exhibited poor antioxidant capacities. For instance,Tangor A which had highest phenolic content (7667 ± 57 �g/gFW) (Fig. 2) showed high TEAC (46.1 ± 0.7 �mol/g FW) and FRAP

• Pamplemousses 1A and 1B •• Bergamot •

•

HOCl) scavenging activities of the flavedo extracts are given inable 3. Based on these values the antioxidant extracts could beategorised in (1) high, (2) moderate and (3) low activity groupsTable 4).

TEAC values ranged from 11.0 ± 0.21 �mol/g FW (Calamondin) to 46.1 ± 0.72 �mol/g FW (Tangor A). In the high antioxidantctivity group (>35 �mol/g FW), there was no significant differencep > 0.05) among Tangor A, Mandarin 1A, 2A, 2B, Tangelo 1A and 2.mong the extracts with moderate activities (20–35 �mol/g FW),andarin 1B and 5, Tangelo 3B and Orange 2B topped the list with

EAC values not significantly different from each other (p > 0.05).elatively low TEAC values (<15 �mol/g FW) were obtained inavedo extracts of Lemon A and B, Kumquat A, Calamondin B andamplemousses 1A and 1B.

Mandarine 1A flavedo had the highest (p < 0.05) FRAP value81.3 ± 0.33 �mol/g FW) (Tables 4 and 5). Extracts of Orange 2B,lementine A, Mandarin 1B, 2A, 2B and 5, Tangor A and B, TangeloA and Tangelo 2 were also characterised by FRAP values greaterhan 50 �mol/g FW. The lowest ferric reducing capacity was showny Kumquat A and B, Calamondin B and Pamplemousses 1A withRAP values not significantly different from each other (p > 0.05)14 �mol/g FW).

The HOCl scavenging property of the extracts was expressedn terms of IC50 which represents the concentration of flavedomg FW/mL) needed to achieve 50% scavenging of hypochloriteTables 3 and 4). Mandarin 1B flavedo extract was characterisedy the lowest IC50 value (3.70 ± 0.06 mg FW/mL) indicating theighest efficacy to scavenge hypochlorite. Extracts of Orange 2B,lémentine A, Mandarin 2A and Tangor A had IC50 values not sig-ificantly different from Mandarin 1B (p > 0.05). The highest IC50alue (p < 0.05) was measured for Calamondin B (17.78 ± 0.30 mgW/mL).

Nine flavedo extracts (Mandarine 1A, 2A and 5, Tangor A, Tan-elo 1A and 2, Orange 2B, Clementine A and Pamplemousses 2B),elected for their high antioxidant potential in TEAC, FRAP and HOCl

cavenging systems, were further characterised for their ability torotect DNA damage and for their iron chelating activity (Fig. 5).

n the Cuphen assay, the IC50 values of the extracts ranged from.3 ± 0.5 mg FW/mL (Tangelo 1A) to 23.0 ± 0.5 mg FW/mL (Tangelo). Tangelo 1A, Tangor A, Clementine A and Pamplemousses 2B

lo 1A, 1B, 3A and 3B • Pamplemousses 2Blemousses 2A, 3A, 3B and 4 • Tangor Ar B

offered greatest protection against DNA damage while Mandarine1A, 2A, Tangelo 2 and Orange 2B were relatively weak protectants.The iron chelating activities of the flavedos were between 7.7 ± 0.1and 27.5 ± 0.2 mg FW/mL. All the citrus flavedos were relativelygood Fe2+ ion chelator except Mandarine 5, Tangelo 2 and Pample-mousses 2B, the latter being least effective. Clementine A was themost potent with an IC50 value of 7.7 ± 0.1 mg FW/mL (p < 0.05).

3.3. Correlation between phenolic contents and antioxidantcapacities

With a view to rationalizing the antioxidant potential of theflavedo extracts in terms of their phytophenolic constituents, lin-ear regression plots were generated and the Pearson correlationcoefficients were calculated (Fig. 6). A striking correlation betweentotal phenolics and antioxidant capacity of flavedo extracts wasnoted (TEAC, r = 0.92; FRAP, r = 0.88; HOCl, r = 0.87). Extracts withthe highest phenolic contents had the highest antioxidant poten-

Fig. 5. DNA protecting and iron(II) chelating activities of citrus flavedo extracts. Datarepresent mean values (bars) with standard errors (n = 2). Different letters betweencolumns of same colour represent significant differences between samples (p < 0.05).

82 D. Ramful et al. / Toxicolog

Tab

le5

Flav

anon

e,fl

avon

ean

dfl

avon

olgl

ycos

ides

leve

lsin

flav

edo

extr

acts

ofci

tru

sfr

uit

s.D

ata

exp

ress

edin

mg/

gFW

asm

ean

valu

e±

stan

dar

der

ror

ofm

ean

(n=

2).N

D:

Not

det

ecte

d.S

ign

ifica

nce

test

ing

amon

gth

ed

iffe

ren

tsa

mp

les

was

per

form

edby

one-

way

AN

OV

Afo

llow

edby

Tuke

y’s

mu

ltip

leco

mp

aris

onte

st.D

iffe

ren

tsu

per

scri

pts

betw

een

row

sre

pre

sen

tsi

gnifi

can

td

iffe

ren

ces

betw

een

sam

ple

s(p

<0.

05).

Cit

rus

fru

itPo

nci

rin

Rh

oifo

lin

Did

ymin

Nar

ingi

nR

uti

nD

iosm

inIs

orh

oifo

lin

Neo

hes

per

idin

Hes

per

idin

Neo

erio

citr

inN

arir

uti

n

Ora

nge

2B2.

49±

0.02

gN

D8.

69±

0.03

cN

D8.

16±

0.06

h4.

55±

0.04

f3.

23±

0.02

g3.

20±

0.08

f22

5.4

±2.

0b8.

80±

0.12

e16

.52

±0.

15c

Clé

men

tin

eA

6.97

±0.

12d

ND

4.45

±0.

08d

efN

D33

.13

±0.

34b

5.20

±0.

10e

6.35

±0.

05d

ND

130.

1±

1.2f

31.0

9±

0.47

b5.

05±

0.14

g

Man

dar

ine

1A18

.85

±0.

20a

10.3

9±

0.05

a11

.34

±0.

08b

19.4

9±

0.18

42.1

3±

0.27

a18

.06

±0.

08a

14.1

4±

0.07

aN

D17

0.5

±0.

3d34

.65

±0.

17a

6.22

±0.

08f

Man

dar

ine

2A8.

69±

0.13

bN

D4.

27±

0.05

fN

D21

.00

±0.

15c

6.29

±c 0

.07b

5.43

±0.

07e

7.16

±0.

08c

194.

3±

1.0c

17.6

1±

0.04

c13

.54

±0.

12d

Man

dar

ine

55.

40±

0.18

e4.

54±

0.06

d3.

22±

0.12

gN

D18

.10

±0.

29d

4.01

±0.

07g

4.10

±0.

09f

7.09

±0.

05c

90.2

±0.

7g18

.30

±0.

25c

11.5

5±

0.05

e

Tan

gor

A4.

73±

0.15

f7.

86±

0.03

c13

.94

±0.

21a

ND

10.6

2±

0.09

g5.

91±

0.11

d8.

90±

0.16

b5.

66±

0.10

e23

4.1

±1.

7a8.

43±

0.09

e20

.03

±0.

11b

Tan

gelo

1A5.

61±

0.18

eN

D5.

26±

0.10

dN

D11

.71

±0.

14f

5.66

±0.

06d

e5.

13±

0.03

e10

.33

±0.

15b

163.

2±

0.9e

15.7

5±

0.20

d13

.99

±0.

16d

Tan

gelo

27.

68±

0.03

c9.

06±

0.01

b4.

80±

0.05

de

ND

13.0

9±

0.38

ee6.

58±

0.14

b8.

20±

0.02

c11

.67

±0.

02a

83.4

±0.

1h15

.71

±0.

06d

21.2

3±

0.17

a

Pam

ple

mou

sses

2B1.

73±

0.02

h1.

93±

0.01

e3.

33±

0.03

gN

DN

D5.

98±

0.20

cd1.

72±

0.04

h6.

68±

0.13

dN

DN

D13

.53

±0.

09d

y 278 (2010) 75–87

(57.7 ± 0.4 �mol/g FW) values and low IC50 value (4.41 ± 0.04 mgFW/mL) in the HOCl assay (Table 3). On the other hand, Lemon B,with lowest amount of total phenolics (1882 ± 65 �g/g FW) (Fig. 2)had very low antioxidant capacity in the TEAC (11.5 ± 0.2 �mol/gFW), FRAP (21.2 ± 0.1 �mol/g FW) and HOCl (14.36 ± 0.29 mgFW/mL) assays (Table 3). The flavonoid levels also showed goodinfluence on the antioxidant capacities of the extracts in all antiox-idative systems as evidenced by the correlation coefficient values(TEAC, r = 0.75; FRAP, r = 0.66; HOCl, r = 0.80). Thus, fruits with high-est levels of total flavonoids namely, Tangelo 2 (5615 ± 93 �g/gFW), Mandarin 1B (5237 ± 68 �g/g FW) and 1A (5027 ± 89 �g/gFW) (Fig. 3) exhibited highest antioxidant capacities (Table 3). Verylow negative correlations were obtained between total vitamin Clevels and antioxidant capacity of the extracts (TEAC, r = −0.07;FRAP, r = −0.27; HOCl, r = −0.08). The coefficient values were notsignificant (p > 0.05). Flavedo extracts rich in vitamin C namely, thevarieties of pamplemousses, had low TEAC and FRAP values andhigh IC50 values in the HOCl assay in most cases.

3.4. Analysis of the flavonoid profile by high performance liquidchromatography

The flavonoid profile of the nine selected flavedo extracts wereanalysed by HPLC. Representative HPLC profiles of the extracts aregiven in Figs. 7 and 8. Identification of the compounds was basedon the retention times in comparison with authentic standards attwo wavelengths: 280 nm for the determination of flavanone glyco-sides and 330 nm for flavone and flavonol glycosides. The followingflavanone glycosides were detected in the flavedo extracts: pon-cirin, dydimin, naringin, hesperidin, neohesperidin, neoeriocitrinand narirutin (Figs. 7a and 8a). One flavonol glycoside (rutin) andthree flavone glycosides (rhoifolin, diosmin and isorhoifolin) werealso identified in the flavedo extracts (Figs. 7b and 8b).

Flavonoid glycoside levels in the flavedo extracts are shownin Table 5. The values were calculated using calibration plots ofpeak-area vs. concentration of the pure compounds. The results ofcalibration showed good linearity (R2 > 0.99) for all the compoundsin the range of concentration tested. Hesperidin was present athighest concentrations in all citrus flavedos except in Pample-mousses 2B where it was not detected. Hesperidin contents rangedfrom 83 ± 0.06 mg/g FW (Tangelo 2) to 234 ± 1.73 mg/g FW (Tan-gor A). Poncirin, didymin, diosmin, isorhoifolin and narirutin wereidentified in all flavedo extracts while naringin was present onlyin Mandarin 1A. Highest levels of poncirin (12.9 ± 0.20 mg/g FW),rhoifolin (10.4 ± 0.05 mg/g FW), rutin (42.1 ± 0.27 mg/g FW), dios-min (18.1 ± 0.08 mg/g FW), isorhoifolin (14.1 ± 0.07 mg/g FW) andneoeriocitrin (34.6 ± 0.17 mg/g FW) were quantified in Mandarin1A (p < 0.05). Topmost levels of didymin (13.9 ± 0.21 mg/g FW)were quantified in Tangor A (p < 0.05) whilst Tangelo 2 showedhighest concentrations of neohesperidin (11.7 ± 0.02 mg/g FW) andnarirutin (21.2 ± 0.17 mg/g FW) (p < 0.05).

4. Discussion

Interest has considerably increased in finding natural antioxi-dants which can impact on the management of a variety of clinicalconditions and maintenance of health. The present study deter-mined the antioxidant capacities of Mauritian citrus flavedos andthe composition of total phenols, flavonoids and vitamin C that maycontribute to the antioxidant activities of the citrus fruits. Total phe-

nols were evaluated using the Folin–Ciocalteu method. Literaturereports have argued that the method overestimates the contentof phenolic compounds, primarily because other agents presentin food, such as carotenoids, amino acids, sugars and vitamin C,can interfere (Singleton and Rossi, 1965; Vinson et al., 2001). It has

D. Ramful et al. / Toxicology 278 (2010) 75–87 83

F AP vaa e signi

bmo(f

TC

ig. 6. Linear regression plots and Pearson’s correlation coefficients of TEAC and FRnd total vitamin C contents of flavedo extracts of citrus fruits. All correlations wer

een documented that using the 6-dichloroindophenol titrimetric

ethod, phenolic extracts give a response corresponding to 20%

f vitamin C content measured in fresh homogenised fruit extractsLuximon-Ramma et al., 2003). Total phenolic contents are there-ore only indicative of the amount of polyphenols in the flavedo

able 6omparative literature data on total phenol content of peel (flavedo + albedo) extracts of

Citrus fruit Total polyphenols(�g/g FW)

Method of extraction

Grapefruits 1550a Homogenisation of 10 g of fresh peel95% ethanol followed by boiling in waSweet Oranges 1790a

Lemons 1900a

White grapefruits 282b Vortexing of 50 mg of lyophilised sam5 mL 80% methanol for 1 min. Heatingfor 3 h with vortexing every 30 minJaffa sweetie grapefruits 376b

Lemons (cv. Meyer) 598a Extraction of 2 g of frozen citrus peelwith 16 mL of 72% ethanol for 3 h.Centrifugation, filtration and evaporasolvent under pressure. Dissolving ofwater

Lemons (cv. Yenben) 1190a

Grapefruit 1616a

Mandarin (cv. Ellendale) 1211a

Sweet orange (cv. Navel) 736a

Lemons 1882–2828 Extraction of 100 mg of lyophilised sawith 12 mL of 80% methanol over 3 dCentrifugation, decantation and use oas is

Mandarins 2649–6923Sweet orange 4509–6470

a Values were converted from original values expressed in mg/100 g FW.b Values were converted from original values expressed in �moL/g FW.

lues and 1/IC50values for HOCl assay with respect to total phenols, total flavonoidsficant at the 0.05 level (two-tailed) except for values marked with an asterisk (*).

extracts and on this basis some orange, clementine, mandarin, tan-

gor, tangelo and pamplemousses extracts were found to contain thehighest levels (>5500 �g/g FW) (Fig. 1 and Table 2). These results arein accordance with others who indicated that peels are an impor-tant source of phenolics (Bocco et al., 1998). The levels in this study

citrus fruits measured by the Folin–Ciocalteu assay.

Expression of results Origin Reference

in 125 mLterbath

Chlorogenic acidequivalent

Grown in Israel Gorinsteinet al. (2001)

ple inat 90 ◦C

Gallic acid equivalent Grown in Israel Gorinsteinet al. (2004)

powder

tion ofextract in

Gallic acid equivalent Bought in NewZealand

Li et al. (2006)

mpleays.f extract

Gallic acid equivalent Grown in Mauritius Present study

84 D. Ramful et al. / Toxicology 278 (2010) 75–87

F Dydimfl rhoifo

afosco

2tilicweF(bs(i(eafIff(fll

ig. 7. (a) HPLC profile of flavedo extract of Mandarin 1A at 280 nm. 1: Poncirin; 4:avedo extract of Mandarin 1A at 330 nm. 2: Rhoifolin; 3: Rutin; 6: Diosmin; 7: Iso

re much higher than those measured in peels of similar varietiesrom Israel and New Zealand (Table 6) using the same methodol-gy indicating that the contents can be influenced by various factorsuch as genotypic differences, geographical and climatic conditions,ultural practices, harvest time and extraction methods amongstthers (Van der Sluis et al., 2001).

The free radical scavenging capacities/reducing powers of the1 flavedo extracts were evaluated by three independent methods,he TEAC, FRAP and HOCl assays. Flavedo extracts had wide antiox-dant potential ranges, thereby supporting their classification asow, moderate and high (Table 4). The antioxidant potential exhib-ted by Clementine A, Mandarin 1A, 2A, Tangor A and Tangelo 2an be associated with high levels of phenolics, of which flavonoidsere major components. This is clearly demonstrated by the lin-

ar regression plots and Pearson’s correlation coefficients of TEAC,RAP and HOCl IC50 values against total phenols and flavonoidsFig. 6). Gorinstein et al. (2004) found a similar high correlationetween antioxidant activities of two citrus fruits from Israel, Jaffaweeties and Jaffa white grapefruits, and their total phenols contentR2 = 0.94). Similar linear correlation between antioxidant activ-ty and phenolic content have been reported for plant extractsNeergheen et al., 2006; Soobrattee et al., 2008), beverages (Richellet al., 2001; Luximon-Ramma et al., 2005), vegetables (Bahorun etl., 2004), juices, (Gil et al., 2000), wines (Burns et al., 2000) andresh and processed edible seaweeds (Jimenez-Escrig et al., 2000).t is also interesting to compare the antioxidant data obtained here

or Mauritian citrus flavedo extracts with those obtained previouslyor similar types of fruit parts using the same assays. Guo et al.2003) reported FRAP values for the peel fractions (comprising theavedo and albedo) of the Chinese fruits Lukan tangerine, orange,

emon, kumquat and pomelo as 69.4, 56.9, 23.0, 2.5 and 18.4 �mol/g

in; 5: Naringin; 9: Hesperidin; 10: Neoeriocitrin; 11: Narirutin. (b) HPLC profile oflin.

respectively. FRAP values of orange and lemon extracts were com-parable to those of this study whilst the values for kumquat andpomelo were lower.

Some flavedo extracts, however, were found to have higheror lower antioxidant activities in one assay system comparedto the others. This confirms that there is no universal methodthat can measure the antioxidant capacity of all samples accu-rately and consistently. Clearly, matching radical source and systemcharacteristics to antioxidant reaction mechanisms is critical inthe selection of appropriate antioxidant capacity assay assessingmethods (Prior et al., 2005). Along this line, nine flavedo extractsexhibiting most potent antioxidant activities in the TEAC, FRAPand HOCl assays were further assessed for their ability to modu-late metal ion dependent free radical reaction. The metal complexcopper 1,10-phenanthroline is known to promote hydroxyl rad-ical formation from molecular oxygen by redox-cycling and istherefore a suitable agent for the stimulation of oxidative DNA dam-age (Aruoma, 1993; Halliwell, 1997). Indeed DNA fragmentationdetected in different cells treated with copper phenanthroline isconsidered to result from direct attack upon DNA by the hydroxylradical (Tsang et al., 1996). DNA damage, such as single and doublestrand breakage, base modification, cross-linking of DNA with otherbiomolecules particularly proteins, are reported to be early eventsof cancer, cardiovascular diseases, diabetes, cataract and neurologi-cal disorders (Cadet et al., 1997) and phytochemicals have profoundchemopreventive effects through modulation of molecular events

that damage DNA (Bisht et al., 2008). The level of protectionagainst copper-phenanthroline-mediated oxidative DNA damagewere in the following order for the flavedo extracts: Tangelo1A > Clementine A > Tangor A > Pamplemousses 2B > Mandarine5 > Mandarine 1A ≈ Orange 2B > Mandarine 2A > Tangelo 2.

D. Ramful et al. / Toxicology 278 (2010) 75–87 85

F imin;o

tolahr(baapmAhtwHTc

mvbai2epov

ig. 8. (a) HPLC profile of flavedo extract of Tangor A at 280 nm. 1: Poncirin; 4: Dydf flavedo extract of Tangor A at 330 nm. 3: Rutin; 6: Diosmin; 7: Isorhoifolin.

Among the transition metals, iron is known as the most impor-ant lipid prooxidant due to its high reactivity. The ferrous statef iron accelerates lipid oxidation by breaking down hydrogen andipid peroxides to reactive free radicals via the Fenton reaction. Thegents that can attenuate the action of these bivalent metal ionsave been classified as secondary antioxidants which retard theate of radical initiation reaction by the elimination of initiatorsVaya and Aviram, 2001). Ferrozine forms a complex with free Fe2+

ut the extent of the complexation is reduced when the Fe2+ is lessvailable by being bound onto the plant extracts (or a chelatinggent) for example. In the presence of chelating agents, the com-lex formation of ferrous ion and ferrozine is altered and this can beonitored by decrease in the absorbance at 562 nm. Benherlal andrumughan (2008) reported that phytochemicals/extracts withigh antioxidant activity but without iron chelation capacity failedo protect DNA in Fenton’s system, suggesting that iron chelationas an essential requirement for extracts studied here to retardO• generation by Fenton’s reaction. In this study Clementine A,angor A and Mandarin 1A and 5 were the most potent Fe2+ ionhelator.

Mauritian citrus flavedos are moderately rich sources of vita-in C with the highest values measured in the pamplemousses

arieties (maximum: 1475 ± 37 �g/g FW (p < 0.05)). No compara-le data are available on the same extract type but Abeysinghe etl. (2007) reported values ranging between 2540 and 4430 �g/g FWn juice sacs, 1420 and 3260 �g/g FW in segment membranes and

610 and 3890 �g/g FW in segments of mandarin and orange vari-ties. Vitamin C does not contribute significantly to the antioxidantotential of the flavedos, as evidenced by the negative correlationsbtained between TEAC, FRAP and HOCl antioxidant capacity anditamin C content (Fig. 7). This is very much consistent with the

8: Neohesperidin; 9: Hesperidin; 10: Neoeriocitrin; 11: Narirutin. (b) HPLC profile

literature report indicating that vitamin C makes little contribu-tion or does not contribute at all to the total antioxidant capacityof fruit and vegetable extracts (Prior et al., 1998; Kalt et al., 1999;Bahorun et al., 2007). This can be argued to be reflected by the resultfrom the Cu-phenanthroline studies where the ability of the fruitphenolics to chelate copper ions and modulate their redox poten-tials was demonstrated suggesting that metal chelation could bemore important. However, in other reported citrus research, Vita-min C was found to be a main contributor to the Total AntioxidantCapacity (TAC) (Gardner et al., 2000; Yoo et al., 2004; Abeysinghe etal., 2007). This suggests a wide variation in vitamin C contributionin different fruit species and even different cultivars within citrusspecies.

Flavonoid derivatives, expressed in quercetin equivalents, inMauritian citrus flavedos were generally high (>2000 �g/g FW forthe majority of samples analysed) (Table 2). Factors, including dif-ferences in variety and high sunlight conditions (a characteristicfeature of tropical Mauritius), which can induce the accumula-tion of flavonoids (Li et al., 1993) are probably responsible for therelatively high yield. Using the same assay system but with cat-echin as standard, Gorinstein et al. (2004) reported that peeledJaffa sweeties (a grapefruit hybrid) and white grapefruits con-tained 471 and 377 �g/g FW while 925 and 744 �g/g FW weremeasured in their respective peels. Three types of flavonoids occurin citrus fruits: flavanones, flavones and flavonols. HPLC analysesof nine flavedo extracts showed that, consistent with literature

data (Londono-Londono et al., 2010), the flavanone glycoside hes-peridin was present at highest concentrations (83–234 mg/g FW)in all the extracts except for Pamplemousses 2B. The flavanoneglycosides poncirin, didymin, narirutin and flavone glycosidesdiosmin and isorhoifolin were present in all flavedo extracts

8 icolog

wMd(tcOpayaiuasoppaiww

C

A

asd

R

A

A

A

A

A

A

B

B

B

B

B

B

B

6 D. Ramful et al. / Tox

hereas the flavanone glycoside naringin was present only inandarin 1A. The presence of naringin was observed in Man-

arin 1A despite its reported absence from Mandarin varietiesTomás-Barberán and Clifford, 2000). Several reports highlighthe structure–antioxidant activity relationships of flavonoid sub-lasses in citrus extracts. Data evidence suggests that glycosylation,-methylation, O-glycosylation influence greatly the antioxidantotency of citrus flavonoids (Di Majo et al., 2005). Antioxidantctivity decreases with glycosylation and is enhanced with hydrox-lation and the presence of C2–C3 double bond in conjugation with4-oxo function (Rice-Evans et al., 1996). Whilst the flavonoids

n the flavedo extracts may contribute significantly, other yetncharacterized phytochemicals, may also contribute to the overallntioxidant effect of the flavedos. Overall citrus flavedos repre-ent a major source of polyphenolic antioxidants. Large amountsf citrus peels are generated as by-product wastes of the juicerocessing industry and represent an untapped resource whichotentially can be judiciously used as functional food ingredientsnd prophylactic agents. Further studies on the effective antiox-dants contained in these fruit fractions and the mechanisms by

hich they could protect against disease development are highlyarranted.

onflict of interest

The authors declared no conflict of interest.

cknowledgements

This work was supported by the University of Mauritius. Theuthors wish to thank the University of Mauritius for a postgraduatecholarship awarded to Deena Ramful and the Compagnie Agricolee Labourdonnais for providing citrus samples.

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