Bioactive phenolics and antioxidant propensity of flavedo extracts of ...

Toxicology 278 (2010) 75–87 

Contents lists available at ScienceDirect 

Toxicology 

journal homepage: www.elsevier.com/locate/toxicol 

Bioactive phenolics and antioxidant propensity of flavedo extracts of Mauritian 

citrus fruits: Potential prophylactic ingredients for functional foods application 

Deena Ramful a , Theeshan Bahorun b,∗ , Emmanuel Bourdon c , Evelyne Tarnus c , Okezie I. Aruoma d 

a Department of Agricultural and Food Science, Faculty of Agriculture, University of Mauritius, Réduit, Mauritius 

b Department of Biosciences, Faculty of Science, University of Mauritius, Réduit, Mauritius 

c Laboratoire de Biochimie et Génétique Moléculaire (LBGM), Groupe d’Etude sur l’Inflammation Chronique et l’Obésité (GEICO), Université de Saint Denis de La Réunion, France 

d Department of Pharmaceutical and Biomedical Sciences, Touro College of Pharmacy, New York, USA 

article info 

Article history: 

Received 26 November 2009 

Received in revised form 11 January 2010 

Accepted 18 January 2010 

Available online 25 January 2010 

Keywords: 

Citrus fruits 

Flavedo 

Flavonoids 

Vitamin C 

Antioxidants 

Food ingredients 

Functional foods 

1. Introduction 

abstract 

The role played by dietary factors on health status has long been 

recognised but it has been only recently that epidemiological and 

clinical studies have provided a clearer insight on the chemical 

and physiological mechanisms of the effects of bioactive foods on 

human health (Shahidi, 2009). Phytophenolics play a crucial role in 

health promotion and disease prevention by mechanisms related to 

cell differentiation, deactivation of pro-carcinogenes, maintenance 

of DNA repair, inhibition of N-nitrosamine formation and change 

of estrogen metabolism, amongst others (Shahidi, 2004). Major 

mechanisms for the antioxidant effect of phenolics in functional 

foods include free radical scavenging and metal chelation activities. 

Reactive oxygen species (ROS), such as the superoxide radical 

(O 2 •− ), hydrogen peroxide (H2O 2), hypochlorous acid (HOCl) and 

the hydroxyl radical (HO • ) have been recognised to play a determin- 

∗ Corresponding author. 

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

0300-483X/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. 

doi:10.1016/j.tox.2010.01.012 

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 were 

examined for their total phenolic, flavonoid and vitamin C contents and antioxidant activities. Total 

phenolics correlated strongly with the trolox equivalent antioxidant capacity (TEAC), ferric reducing 

antioxidant capacity (FRAP) and hypochlorous acid (HOCl) scavenging activity assays (r > 0.85). Based 

on 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 further 

characterized for their flavanone, flavonol and flavone levels by HPLC and their antioxidant activities 

were assessed by the copper-phenanthroline and iron chelation assays. The flavanone, hesperidin, was 

present at the highest concentrations in all flavedo extracts except for pamplemousses where it was 

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 only 

in one mandarin variety. The nine flavedo extracts exhibited good DNA protecting ability in the cuphen 

assay with IC50 values ranging from 6.3 ± 0.46 to 23.0 ± 0.48 mg FW/mL. Essentially the flavedos were 

able 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 

prophylactic properties for the 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-induced 

oxidation can result in cell membrane disintegration, membrane 

protein damage and DNA mutation, which can further initiate or 

propagate the development of diseases including cancer (Huang 

et 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 compounds 

such as phenolic acids, flavonoids, stilbenes, tannins and lignans 

can scavenge free radicals and quench ROS and therefore provide 

effective means for preventing and treating free radical-mediated 

diseases. 

Mauritius is a tropical island in the Indian Ocean with a relatively 

high 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 Mauritian 

diet, which comprises a wide variety of exotic fruits, vegetables 

and beverages (Luximon-Ramma et al., 2003; Bahorun et al., 2004, 

2007, 2010). Citrus (Citrus L. from Rutaceae) is one of the most 

popular world fruit crops that, besides providing an ample sup-

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

ply of vitamin C, folic acid, potassium and pectin, contains a host of 

active phytochemicals that can protect health. Citrus species of various 

origins have been assessed for their phenolic constituents and 

antioxidant activities (Proteggente et al., 2003; Gorinstein et al., 

2004; Anagnostopoulou et al., 2006; Guimarães et al., 2009). Citrus 

fruits, citrus fruit extracts and citrus flavonoids exhibit a wide 

range of promising biological properties including antiatherogenic, 

anti-inflammatory and antitumor activity, inhibition of blood clots 

and strong antioxidant activity (Middleton and Kandaswami, 1994; 

Montanari et al., 1998; Samman et al., 1996). Citrus is consumed 

mostly as fresh produce and juice and most often the peel is discarded. 

This represents a huge waste as citrus peels are reported 

to possess highest amounts of flavonoids compared to other parts 

of the fruit (Manthey and Grohmann, 2001). Citrus peels are subdivided 

into the epicarp or flavedo and mesocarp or albedo. The 

flavedo is the colored peripheral surface of the peel while the albedo 

is the white soft middle layer of the peel (Fig. 1). 

The phytophenolic composition and in vitro antioxidant activities 

of the flavedo extracts of 21 citrus fruit varieties (Table 1) 

grown in Mauritius were determined. From the initial results, nine 

of the flavedo extracts (Orange 2B, Clementine A, Mandarin 1A, 

2A and 5, Tangor A, Tangelo 1A and 2 and Pamplemousses 2B) 

were further characterized for their flavanone, flavonol and flavone 

levels, their ability to protect DNA damage and their iron chelating 

activity. There has been so far no report on the nutritional 

and health-promoting values of Mauritian citrus varieties. Thus 

Mauritian citrus varieties could be important sources of dietary 

Table 1 

Scientific and common names, variety and harvest dates of citrus fruits analysed. 

Fig. 1. Anatomy of citrus fruit showing the flavedo (the orange peripheral surface of 

the 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 benefits 

in health and disease management. 

2. Materials and methods 

2.1. Chemicals 

2,2 ′ -Azino-bis(3-ethylbenzthiozoline-6)-sulfonic acid (ABTS), Folin & Ciocalteu’s 

phenol reagent and 2-Aminoethanesulfonic acid (Taurine) were purchased 

Scientific name Common name Variety Harvest month Variety and harvest code 

Citrus sinensis Orange Valencia late August 1 

Washington Navel March 2A 

May 2B 

Citrus unshiu Satsumah Owari March A 

May B 

Citrus clementina Clementine Commune March A 

May B 

Citrus reticulata Mandarin Fairchild April 1A 

May 1B 

Dancy May 2A 

June 2B 

Beauty June 3A 

August 3B 

Suhugan August 4 

Fizu August 5 

C. reticulata × C. sinensis Tangor Elendale June A 

August B 

Citrus aurantium ssp. bergamia Bergamot – April – 

Citrus meyeri Lemon Meyer April A 

May B 

C. reticulata × C. paradisis Tangelo Mineola June 1A 

August 1B 

Orlando August 2 

Ugli June 3A 

August 3B 

Fortunella margarita Kumquat Nagami April A 

June B 

Citrus mitis Calamondin 

– 

June A 

August B 

Citrus grandis Pamplemousses Rainking May 1A 

August 1B 

Kaopan May 2A 

August 2B 

Pink May 3A 

August 3B 

Chandler August 4

from Sigma (St. Louis, MO, USA). 2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ) was from 

Analytical Rasayan, s.d. fiNe-CHEM Limited (Mumbai, India). Metaphosphoric acid 

was from Sigma Chemical Co. (St. Louis, MO). 2,6-Dichloroindophenol indophenol 

sodium salt was from Alpha Chemika (Mumbai, India). l-Ascorbic acid was from 

BHD Laboratory Supplies (Poole, England). 6-Hydroxy-2,5,7,8-tetramethylchroman- 

2-carboxylic acid, 97% (Trolox) was from Sigma–Aldrich Chemie (Steinheim, 

Germany). Gallic acid, quercetin, rutin, diosmin, rhoifolin, isorhoifolin, neoeriocitrin, 

poncirin, narirutin, neohesperidin, didymin, hesperidin and naringin (HPLC grade) 

were from Extrasynthèse (Genay, France). HPLC-grade acetonitrile and methanol 

were obtained from Merck (Darmstadt, Germany). All other reagents used were of 

analytical grade. 

2.2. Plant material 

Citrus fruits (Table 1) were obtained from La Compagnie Agricole de Labourdonnais 

situated at Mapou, in the north of Mauritius. Fruits were harvested at the 

mature stage when they were ready to be placed on the market or ready for processing. 

Some varieties were sampled twice at different periods of the harvest season to 

determine the effect of harvest time on tested parameters. After harvest, the fruits 

were rapidly processed on the same day. They were carefully washed under running 

tap water and patted dry. The flavedo of at least 10 fruits of each variety was carefully 

removed with a manual peeler and cut into small pieces. Weighed portions of 

the peripheral peel of pooled samples of each variety were lyophilised for 48 h and 

the freeze-dried weight was determined. Samples were ground into a fine powder 

in a coffee grinder and stored in airtight containers at −4 ◦ C until analysed. 

2.3. Extraction 

The extraction procedure used was adapted from Franke et al. (2004) and Chun 

et al. (2003). A known amount of powdered freeze-dried citrus tissues was exhaustively 

extracted in 80% aqueous methanol at 4 ◦ C for three consecutive days. After 

centrifugation at 4500 rpm for 15 min, supernatants of all three extractions were 

pooled and stored at −20 ◦ C until used for the determination of total phenol and 

total flavonoids and for the antioxidant assays. 

2.4. Total phenolic content 

The Folin–Ciocalteu assay, adapted from Singleton and Rossi (1965), was used 

for the determination of total phenolics present in the citrus fruit extracts. To 

0.25 mL of diluted extract, 3.5 mL of distilled water was added followed by 0.25 mL 

of Folin–Ciocalteu reagent (Merck). A blank was prepared using 0.25 mL of 80% 

methanol instead of plant extract. After 3 min, 1 mL of 20% sodium carbonate was 

added. Tube contents were vortexed before being incubated for 40 min in a waterbath 

set at 40 ◦ C. The absorbance of the blue coloration formed was read at 685 nm 

against the blank standard. Total phenolics were calculated with respect to gallic 

acid standard curve (concentration range: 0–12 g/mL). Results are expressed in 

g of gallic acid/g fresh weight of plant material. 

2.5. Total flavonoid content 

Total flavonoids were measured using a colorimetric assay adapted from Zhishen 

et al. (1999). 150 L of 5% aqueous NaNO2 was added to an aliquot (2.5 mL) of each 

extract and the mixture was vortexed. A reagent blank using 80% aqueous methanol 

instead of sample was prepared. After 5 min, 150 L of 10% aqueous AlCl3 was added. 

1 mL of 1 M NaOH was added 1 min after the addition of aluminium chloride. Solution 

was mixed well and the absorbance was measured against the blank at 510 nm. Total 

flavonoids were calculated with respect to quercetin standard curve (concentration 

range: 50–200 g/mL). Results are expressed in g of quercetin g/fresh weight of 

plant material. 

2.6. Total vitamin C content 

The 2,6-dichloroindophenol titrimetric method (AOAC, 1995) was used to determine 

the vitamin C content of flavedo extracts. 100 g of the plant material was cut 

into small pieces and blended in a Waring blander with 150 mL of metaphosphoric 

acid–acetic acid solution. After filtration and appropriate dilutions with metaphosphoric 

acid–acetic acid solution as determined by the extract colour intensity, 7 mL 

of the diluted solution was titrated against standard indophenol solution. Results 

are expressed in g ascorbic acid/g fresh weight. 

2.7. Trolox equivalent antioxidant capacity (TEAC) assay 

The TEAC assay measures the relative ability of antioxidant substances to 

scavenge the 2,2 ′ -azino-bis(3,ethyl benz-thiazoline-6-sulfonic acid) radical cation 

(ABTS •+ ), compared with standard amounts of the synthetic antioxidant Trolox, the 

water-soluble vitamin E analogue. The method of Campos and Lissi (1996) was used. 

To 3 mL of the ABTS •+ solution generated by a reaction between ABTS (0.5 mM) 

and activated MnO2 (1 mM) in phosphate buffer (0.1 M, pH 7), 0.5 mL of diluted 

plant extract was added. Decay in absorbance was monitored at 734 nm for 15 mins 

on a Helios-Alpha spectrophotometer (Unicam Ltd., UK) maintained at 20 ◦ Cbya 

D. Ramful et al. / Toxicology 278 (2010) 75–87 77 

peltier thermostat. Calculations were made with respect to a dose–response curve 

of 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 and 

Strain (1996). The principle of this method is based on the ability of substances 

to reduce Fe(III)-2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ) complex to Fe(II)-TPTZ, the 

resulting intense blue colour being linearly related to the amount of reductant 

(antioxidant) present. The FRAP reagent consisting of 20 mL of 10 mM TPTZ solution 

in 40 mM HCl and 20 mL of 20 mM ferric chloride in 200 mL of 0.25 M sodium 

acetate buffer (pH 3.6) was freshly prepared and warmed at 37 ◦ C.A50L aliquot of 

sample 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 calibration 

curve of ferrous sulphate (0–1.2 mM) was used and results are expressed as mol 

Fe 2+ /g fresh weight. 

2.9. Hypochlorous acid (HOCl) scavenging assay 

The HOCl assay was adapted from Weiss et al. (1982). Both HOCl and ClO − are 

potent oxidants and thus harmful in excessive amounts in vivo. In this model, taurine, 

a -amino acid, is used as a representation compound capable of reacting with 

HOCl/ClO − at diffusion controlled rates to form a stable and quantitable taurine chloramine 

derivative. The antioxidant capacity was based on the ability of the extract 

to 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 the 

stock solution was determined spectrophotometrically by measuring its absorbance 

at 235 nm and applying a molar extinction coefficient of 100. The reaction mixture 

contained 100 L taurine (10 mM), 100 L extract (variable concentrations), 100 L 

HOCl (1 mM) and phosphate saline buffer (pH 7.4) in a final volume of 1 mL. After 

incubation at room temperature for 10 min, the sample was then assayed for taurine 

chloramine by adding 10 L of potassium iodide (2 M) to the reaction mixture. This 

complex has the ability to oxidise I − ions into I2 producing a yellow coloration. The 

absorbance of the reaction mixture was read at 350 nm. Results are expressed as 

IC50 values (mg fresh weight/mL). 

2.10. Copper-phenanthroline (Cuphen) assay 

The ability of the copper-phenanthroline complex to degrade DNA in the presence 

of a reducing agent has been adopted as a method for assessing the antioxidant 

propensities of dietary biofactors with antioxidant potentials (Aruoma, 1993, 1994; 

Gutteridge and Halliwell, 1982). Reaction mixture contained in a final volume of 

1.2 mL the following reagents in order of addition indicated: 100 L of 1.8 mM 1,10phenanthroline 

hydrate (stock solution made up in water having initially dissolved 

the 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 distilled 

water, 100 L ascorbic acid (stock solution: 2.88 mM) and 600 L of methanolic citrus 

extracts, serially diluted. After incubation at 37 ◦ C for 1 h, 100 L of 0.1 M EDTA 

was 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. The 

pink chromogen formed was extracted into butan-1-ol and the absorbance measured 

at 532 nm. Results are expressed in terms of IC50 (mg FW/mL able to inhibit 

50% of DNA damage). 

2.11. Iron(II) chelating activity 

The method of Dorman et al. (2003) was adapted to assess the chelating activity 

of the citrus extracts on iron(II) ions. The reaction mixture containing 950 L 

of extract serially diluted with 80% methanol, and 50 L of 0.5 mM FeCl2·4H2O was 

incubated for 5 min at room temperature. 50 L of 2.5 mM ferrozine was then added 

and was allowed to equilibrate for 10 min at room temperature. The purple coloration 

formed was read at 562 nm. The control contained 80% methanol instead of 

the extract. The chelating activity was calculated according to the equation given 

below and results are expressed as mean IC50 (mg FW/mL). 

 

Abscontrol − Abssample Metal chelating activity (%) = 

× 100 

Abscontrol 2.12. High performance liquid chromatography 

2.12.1. Sample preparation 

Based on the results obtained from the TEAC, FRAP and HOCl assays, nine epicarp 

extracts (Orange 2B, Clementine A, Mandarin 1A, 2A and 5, Tangor A, Tangelo 1A and 

2 and Pamplemousses 2B) with most potent antioxidant capacities were selected 

for flavonoid glycosides quantification by HPLC. Known weights of lyophilized fruit 

powders were extracted with 80% of aqueous methanol (HPLC grade) following 

the same procedure as described in Section 2.3. Samples were filtered on Milipore 

(0.22 m) before use.


2.12.2. Chromatographic conditions 

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

series HPLC equipped with a vacuum degasser, quaternary pump, autosampler, thermostatted 

column compartment, diode array detector and HP Chemstation for data 

collection and analysis was used. After filtration on Millipore (0.22 m), 30 L of 

extract was injected on a Waters Spherisorb ODS-2 column (5 m particle size, 

80 Å pore size, 4.6 mm id × 150 mm). The solvents used were: A, water–acetonitrile 

(90:10, v/v; pH 2.35) and B, acetonitrile. The gradient profile was as follows: 

0–12 min 0–8% B, 12–43 min 8–34% B, 43–44 min 34–70% B, 44–59 min 70% B, 

59–60 min back to 0% B. The diode array detector was set at 280 nm for the quantitative 

determination of flavanone glycosides and at 330 nm for flavone and flavonol 

glycosides. The column temperature was 25 ◦ C and the flow rate was fixed at 

0.7 mL/min. The identification and quantification of the flavonoids investigated 

were determined from retention time and peak area in comparison with the standards 

used. The standards, poncirin, rhoifolin, didymin, naringin, rutin, diosmin, 

isorhoifolin, neohesperidin, hesperidin, neoeriocitrin and narirutin, were prepared 

at a stock concentration of 200 g/mL. Calibration standard samples containing the 

standards each at 20, 40, 100 and 200 g/mL were prepared by appropriate dilutions 

with methanol from the stock solutions and filtered on Milipore (0.22 m) 

before use. The linearity of the assay was demonstrated by assaying calibration 

standards in duplicate at four separate concentrations on two separate occasions. 

Calibration curves were obtained by plotting the peak area of the standards versus 

their concentrations. Concentrations of each of the eleven flavonoid glycosides 

in citrus fruit samples were determined by application of the obtained standard 

curve. 

2.13. Statistical analysis 

Simple regression analysis was performed to calculate the dose–response 

relationship of the standard solutions used for calibration as well as test 

samples. Unicam Vision 32 software (Unicam, Ltd., UK) was used to evaluate 

initial and final antioxidant rate values for the TEAC assay. Data are 

expressed as the means ± standard error of mean (SE) from two independent 

experiments performed in triplicates. Mean differences were determined by oneway 

ANOVA followed by Tukey’s HSD post-test using Prism TM v4.0 software 

(GraphPad ® Software, San-Diego, 2003). The differences were accepted as significant 

when P < 0.05 and are denoted by different letters. Linear regression 

plots were generated and correlations between antioxidant activities and total 

phenol, flavonoids and vitamin C contents were computed as Pearson’s correlation 

coefficient (r) using Prism TM v4.0 software (GraphPad ® Software, San-Diego, 

2003). 

3. Results 

3.1. Phenolic and vitamin C contents 

The total phenolic composition of the flavedo extracts is presented 

in Fig. 2. The amount of total phenolics varied widely and 

ranged from 1882 ± 65 g/g FW in Lemon B to 7667 ± 57 g/g FW 

in Tangor A. The total phenolics content in flavedo of Tangor A 

was significantly higher (p < 0.05) than in the other extracts. No 

significant differences (p > 0.05) were observed in the phenolic levels 

of Mandarin 1A, 2B, 5 and Tangor B, all ranking second on the 

list. Orange 2B, Pamplemousses 2B, Mandarin 2A, Clementine A 

and Tangelo 1A ranked third in terms of total phenolic content 

(p > 0.05). Significant differences (p < 0.05) were observed between 

total phenolic levels of flavedo of similar varieties of citrus harvested 

at different periods except for Pamplemousses 3. Highest 

levels of total flavonoids (p < 0.05) were obtained in extracts of Tangelo 

2 (5615 ± 93 g/g FW) followed by Mandarin 1B (p < 0.05) and 

1A (p < 0.05) (5237 ± 68 g/g FW and 5027 ± 89 g/g FW respectively) 

(Fig. 3). Flavedo extracts of the Tangor variety, which had 

the highest total phenols, contained relatively low levels of total 

flavonoids. Extracts of Lemon B and Calamondin A, whose flavonoid 

contents were not significantly different (p > 0.05), ranked last on 

the list. 

The vitamin C composition of the flavedo extracts are shown 

in Fig. 4. Vitamin C content ranged between 344 ± 8 g/g FW and 

1475 ± 15 g/g FW. The majority of the pamplemousses varieties 

contained the highest levels and flavedo of Pamplemousses 1B was 

the richest with 1475 ± 37 g/g FW (p < 0.05). Tangelo 3B flavedo 

ranked after the pamplemousses with a value of 1002 ± 46 g/g 

FW. No significant difference was found between vitamin C content 

in flavedo extracts of Lemon A and Kumquat B (p > 0.05) both 

having the lowest amounts (388 ± 44 g/g FW and 345 ± 21 g/g 

FW respectively). 

Fig. 2. Total phenolic content of flavedo extracts of citrus fruits. Data represent mean values (bars) with standard errors (n = 2). Different letters between columns represent 

significant differences between samples (p < 0.05). Letter a denotes sample having highest total phenolics and letter r denotes sample having lowest phenolics content.


Fig. 3. Total flavonoid contents of flavedo extracts of citrus fruits. Data represent mean values (bars) with standard errors (n = 2). Different letters between columns represent 

significant differences between samples (p < 0.05). Letter a denotes sample having highest total flavonoids and letter w denotes sample having lowest flavonoids content. 

In the light of the large distribution of total phenolics, flavonoids 

and vitamin C in the flavedo extracts, we propose 3 groupings of 

these phytochemicals into (1) high level, (2) medium level and (3) 

low level (Table 2). 

3.2. Antioxidant capacities 

The Trolox Equivalent Antioxidant Capacity (TEAC), the Ferric 

Reducing Antioxidant Power (FRAP) and the Hypochlorous acid 

Fig. 4. Total vitamin C content of flavedo extracts of citrus fruits. * Flavedo and albedo analysed together. Data represent mean values (bars) with standard errors (n = 2). 

Different letters between columns represent significant differences between samples (p < 0.05). Letter a denotes sample having highest total vitamin C content and letter n 

denotes sample having lowest vitamin C content.


Table 2 

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

Low Medium High 

Total phenolic 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 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 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 

• Tangelo 1A and 2 • Tangelo 1B 

• Pamplemousses 2A 

• Tangor A and B 

• Calamondin A and B 

Table 3 

Antioxidant activities of flavedo extracts of citrus fruits. Data expressed as mean value ± standard error (n = 2). 

Citrus fruit Code TEAC I FRAP II HOCl III 

Orange 1 21.6 ± 0.2 hij 37.6 ± 0.1 i 6.57 ± 0.23 jk 

2A 26.1 ± 1.5 fg 48.2 ± 0. g 4.95 ± 0.05 mno 

2B 31.2 ± 0.4 d 56.7 ± 0.9 cde 3.98 ± 0.10 pq 

Satsumah A 25.5 ± 0.1 fg 32.8 ± 0.8 k 7.68 ± 0.09 i 

B 20.0 ± 0.1 ijk 41.4 ± 0.3 h 6.66 ± 0.13 j 

Clémentine A 36.2 ± 0.1 c 67.6 ± 0.6 b 3.80 ± 0.10 pq 

B 18.4 ± 0.6 jklm 39.0 ± 0.2 i 7.68 ± 0.04 i 

Mandarin 1A 43.4 ± 1.5 ab 81.3 ± 0.3 a 4.53 ± 0.04 mnop 

1B 31.5 ± 1.4 d 58.4 ± 1.0 c 3.70 ± 0.06 q 

2A 42.9 ± 0.7 ab 55.9 ± 0.4 de 4.23 ± 0.06 opq 

2B 44.0 ± 0.5 ab 56.7 ± 0.1 cde 5.23 ± 0.03 lm 

3A 19.2 ± 0.2 jkl 29.2 ± 0.2 l 13.62 ± 0.23 c 

3B 15.1 ± 0.1 mno 20.9 ± 0.1 p 14.24 ± 0.67 bc 

4 16.4 ± 0.2 lmn 21.1 ± 0.1 p 11.00 ± 0.09 fg 

5 30.7 ± 0.1 de 53.2 ± 0.3 f 4.98 ± 0.03 mno 

Tangor A 46.1 ± 0.7 a 57.72 ± 0.4 cd 4.41 ± 0.04 nopq 

B 37.0 ± 0.6 c 57.8 ± 0.3 cd 5.13 ± 0.06 lmn 

Bergamot – 12.8 ± 0.2 op 21.6 ± 0.5 op 14.62 ± 0.06 b 

Lemon A 13.3 ± 0.1 nop 26.7 ± 0.3 m 10.70 ± 0.0 g 

B 11.5 ± 0.2 p 21.2 ± 0.1 p 14.36 ± 0.29 bc 

Tangelo 1A 43.1 ± 0.2 ab 55.03 ± 0.4 ef 5.17 ± 0.06 lmn 

1B 23.3 ± 0. ghi 38.0 ± 0.2 i 9.15 ± 0.09 h 

2 43.85 ± 1.2 ab 55.2 ± 0.6 ef 4.54 ± 0.10 mnop 

3A 24.0 ± 0. gh 21.0 ± 0.4 p 7.18 ± 0.14 ij 

3B 31.2 ± 0.4 d 35.0 ± 0.1 j 9.11 ± 0.16 h 

Kumquat A 12.1 ± 0.2 op 13.9 ± 0.2 r 10.49 ± 0.1 g 

B 18.0 ± 0.4 klm 13.7 ± 0.1 r 10.84 ± 0.19 fg 

Calamondin A 24.3 ± 0.1 gh 24.3 ± 0.3 n 12.78 ± 0.06 d 

B 11.0 ± 0.2 p 14.2 ± 0.1 r 17.78 ± 0.30 a 

Pamplemousses 1A 11.7 ± 0.1 p 14.7 ± 0.1 r 12.14 ± 0.33 de 

1B 14.0 ± 0.3 nop 22.8 ± 0.2 nop 11.46 ± 0.04 ef 

2A 18.0 ± 0.9 klm 32.6 ± 0.4 k 5.86 ± 0.07 kl 

2B 40.8 ± 0.3 b 47.5 ± 0. g 4.53 ± 0.12 mnop 

3A 21.6 ± 0.3 hij 23.7 ± 0.3 no 5.25 ± 0.07 lm 

3B 23.1 ± 0. ghi 17.3 ± 0.1 q 6.44 ± 0.18 jk 

4 27.7 ± 0.7 ef 21.1 ± 0.2 p 6.98 ± 0.09 ij 

I mol Trolox/g fresh weight; II mol Fe(II)/g fresh weight; III IC50 mg fresh weight/mL. Significance testing among the different samples was performed by one-way ANOVA 

followed by Tukey’s multiple comparison test. Different superscripts between rows represent significant differences between samples (p < 0.05).


Table 4 

Classification 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 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 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 35 mol/g FW), there was no significant difference 

(p > 0.05) among Tangor A, Mandarin 1A, 2A, 2B, Tangelo 1A and 2. 

Among the extracts with moderate activities (20–35 mol/g FW), 

Mandarin 1B and 5, Tangelo 3B and Orange 2B topped the list with 

TEAC values not significantly different from each other (p > 0.05). 

Relatively low TEAC values ( 0.05) 

(14 mol/g FW). 

The HOCl scavenging property of the extracts was expressed 

in terms of IC 50 which represents the concentration of flavedo 

(mg FW/mL) needed to achieve 50% scavenging of hypochlorite 

(Tables 3 and 4). Mandarin 1B flavedo extract was characterised 

by the lowest IC 50 value (3.70 ± 0.06 mg FW/mL) indicating the 

highest efficacy to scavenge hypochlorite. Extracts of Orange 2B, 

Clémentine A, Mandarin 2A and Tangor A had IC 50 values not significantly 

different from Mandarin 1B (p > 0.05). The highest IC 50 

value (p < 0.05) was measured for Calamondin B (17.78 ± 0.30 mg 

FW/mL). 

Nine flavedo extracts (Mandarine 1A, 2A and 5, Tangor A, Tangelo 

1A and 2, Orange 2B, Clementine A and Pamplemousses 2B), 

selected for their high antioxidant potential in TEAC, FRAP and HOCl 

scavenging systems, were further characterised for their ability to 

protect DNA damage and for their iron chelating activity (Fig. 5). 

In the Cuphen assay, the IC 50 values of the extracts ranged from 

6.3 ± 0.5 mg FW/mL (Tangelo 1A) to 23.0 ± 0.5 mg FW/mL (Tangelo 

2). Tangelo 1A, Tangor A, Clementine A and Pamplemousses 2B 

offered greatest protection against DNA damage while Mandarine 

1A, 2A, Tangelo 2 and Orange 2B were relatively weak protectants. 

The iron chelating activities of the flavedos were between 7.7 ± 0.1 

and 27.5 ± 0.2 mg FW/mL. All the citrus flavedos were relatively 

good Fe 2+ ion chelator except Mandarine 5, Tangelo 2 and Pamplemousses 

2B, the latter being least effective. Clementine A was the 

most potent with an IC 50 value of 7.7 ± 0.1 mg FW/mL (p < 0.05). 

3.3. Correlation between phenolic contents and antioxidant 

capacities 

With a view to rationalizing the antioxidant potential of the 

flavedo extracts in terms of their phytophenolic constituents, linear 

regression plots were generated and the Pearson correlation 

coefficients were calculated (Fig. 6). A striking correlation between 

total phenolics and antioxidant capacity of flavedo extracts was 

noted (TEAC, r = 0.92; FRAP, r = 0.88; HOCl, r = 0.87). Extracts with 

the highest phenolic contents had the highest antioxidant potential 

in all assayed systems whilst extracts characterised by low total 

phenolic levels exhibited poor antioxidant capacities. For instance, 

Tangor A which had highest phenolic content (7667 ± 57 g/g 

FW) (Fig. 2) showed high TEAC (46.1 ± 0.7 mol/g FW) and FRAP 

Fig. 5. DNA protecting and iron(II) chelating activities of citrus flavedo extracts. Data 

represent mean values (bars) with standard errors (n = 2). Different letters between 

columns of same colour represent significant differences between samples (p < 0.05).


Table 5 

Flavanone, flavone and flavonol glycosides levels in flavedo extracts of citrus fruits. Data expressed in mg/g FW as mean value ± standard error of mean (n = 2). ND: Not detected. Significance testing among the different samples 

was performed by one-way ANOVA followed by Tukey’s multiple comparison test. Different superscripts between rows represent significant differences between samples (p < 0.05). 

Citrus fruit Poncirin Rhoifolin Didymin Naringin Rutin Diosmin Isorhoifolin Neohesperidin Hesperidin Neoeriocitrin Narirutin 

Orange 2B 2.49 ± 0.02g ND 8.69 ± 0.03c ND 8.16 ± 0.06h 4.55 ± 0.04f 3.23 ± 0.02g 3.20 ± 0.08f 225.4 ± 2.0b 8.80 ± 0.12e 16.52 ± 0.15c Clémentine A 6.97 ± 0.12d ND 4.45 ± 0.08def ND 33.13 ± 0.34b 5.20 ± 0.10e 6.35 ± 0.05d ND 130.1 ± 1.2f 31.09 ± 0.47b 5.05 ± 0.14g Mandarine 1A 18.85 ± 0.20a 10.39 ± 0.05a 11.34 ± 0.08b 19.49 ± 0.18 42.13 ± 0.27a 18.06 ± 0.08a 14.14 ± 0.07a ND 170.5 ± 0.3d 34.65 ± 0.17a 6.22 ± 0.08f Mandarine 2A 8.69 ± 0.13b ND 4.27 ± 0.05f ND 21.00 ± 0.15c 6.29 ± c0.07b 5.43 ± 0.07e 7.16 ± 0.08c 194.3 ± 1.0c 17.61 ± 0.04c 13.54 ± 0.12d Mandarine 5 5.40 ± 0.18e 4.54 ± 0.06d 3.22 ± 0.12g ND 18.10 ± 0.29d 4.01 ± 0.07g 4.10 ± 0.09f 7.09 ± 0.05c 90.2 ± 0.7g 18.30 ± 0.25c 11.55 ± 0.05e Tangor A 4.73 ± 0.15f 7.86 ± 0.03c 13.94 ± 0.21a ND 10.62 ± 0.09g 5.91 ± 0.11d 8.90 ± 0.16b 5.66 ± 0.10e 234.1 ± 1.7a 8.43 ± 0.09e 20.03 ± 0.11b Tangelo 1A 5.61 ± 0.18e ND 5.26 ± 0.10d ND 11.71 ± 0.14f 5.66 ± 0.06de 5.13 ± 0.03e 10.33 ± 0.15b 163.2 ± 0.9e 15.75 ± 0.20d 13.99 ± 0.16d Tangelo 2 7.68 ± 0.03c 9.06 ± 0.01b 4.80 ± 0.05de ND 13.09 ± 0.38ee 6.58 ± 0.14b 8.20 ± 0.02c 11.67 ± 0.02a 83.4 ± 0.1h 15.71 ± 0.06d 21.23 ± 0.17a Pamplemousses 2B 1.73 ± 0.02h 1.93 ± 0.01e 3.33 ± 0.03g ND ND 5.98 ± 0.20cd 1.72 ± 0.04h 6.68 ± 0.13d ND ND 13.53 ± 0.09d (57.7 ± 0.4 mol/g FW) values and low IC 50 value (4.41 ± 0.04 mg 

FW/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/g 

FW), FRAP (21.2 ± 0.1 mol/g FW) and HOCl (14.36 ± 0.29 mg 

FW/mL) assays (Table 3). The flavonoid levels also showed good 

influence on the antioxidant capacities of the extracts in all antioxidative 

systems as evidenced by the correlation coefficient values 

(TEAC, r = 0.75; FRAP, r = 0.66; HOCl, r = 0.80). Thus, fruits with highest 

levels of total flavonoids namely, Tangelo 2 (5615 ± 93 g/g 

FW), Mandarin 1B (5237 ± 68 g/g FW) and 1A (5027 ± 89 g/g 

FW) (Fig. 3) exhibited highest antioxidant capacities (Table 3). Very 

low negative correlations were obtained between total vitamin C 

levels and antioxidant capacity of the extracts (TEAC, r = −0.07; 

FRAP, r = −0.27; HOCl, r = −0.08). The coefficient values were not 

significant (p > 0.05). Flavedo extracts rich in vitamin C namely, the 

varieties of pamplemousses, had low TEAC and FRAP values and 

high IC 50 values in the HOCl assay in most cases. 

3.4. Analysis of the flavonoid profile by high performance liquid 

chromatography 

The flavonoid profile of the nine selected flavedo extracts were 

analysed by HPLC. Representative HPLC profiles of the extracts are 

given in Figs. 7 and 8. Identification of the compounds was based 

on the retention times in comparison with authentic standards at 

two wavelengths: 280 nm for the determination of flavanone glycosides 

and 330 nm for flavone and flavonol glycosides. The following 

flavanone glycosides were detected in the flavedo extracts: poncirin, 

dydimin, naringin, hesperidin, neohesperidin, neoeriocitrin 

and narirutin (Figs. 7a and 8a). One flavonol glycoside (rutin) and 

three flavone glycosides (rhoifolin, diosmin and isorhoifolin) were 

also identified in the flavedo extracts (Figs. 7b and 8b). 

Flavonoid glycoside levels in the flavedo extracts are shown 

in Table 5. The values were calculated using calibration plots of 

peak-area vs. concentration of the pure compounds. The results of 

calibration showed good linearity (R 2 > 0.99) for all the compounds 

in the range of concentration tested. Hesperidin was present at 

highest concentrations in all citrus flavedos except in Pamplemousses 

2B where it was not detected. Hesperidin contents ranged 

from 83 ± 0.06 mg/g FW (Tangelo 2) to 234 ± 1.73 mg/g FW (Tangor 

A). Poncirin, didymin, diosmin, isorhoifolin and narirutin were 

identified in all flavedo extracts while naringin was present only 

in 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), diosmin 

(18.1 ± 0.08 mg/g FW), isorhoifolin (14.1 ± 0.07 mg/g FW) and 

neoeriocitrin (34.6 ± 0.17 mg/g FW) were quantified in Mandarin 

1A (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 showed 

highest concentrations of neohesperidin (11.7 ± 0.02 mg/g FW) and 

narirutin (21.2 ± 0.17 mg/g FW) (p < 0.05). 

4. Discussion 

Interest has considerably increased in finding natural antioxidants 

which can impact on the management of a variety of clinical 

conditions and maintenance of health. The present study determined 

the antioxidant capacities of Mauritian citrus flavedos and 

the composition of total phenols, flavonoids and vitamin C that may 

contribute to the antioxidant activities of the citrus fruits. Total phenols 

were evaluated using the Folin–Ciocalteu method. Literature 

reports have argued that the method overestimates the content 

of phenolic compounds, primarily because other agents present 

in food, such as carotenoids, amino acids, sugars and vitamin C, 

can interfere (Singleton and Rossi, 1965; Vinson et al., 2001). It has


Fig. 6. Linear regression plots and Pearson’s correlation coefficients of TEAC and FRAP values and 1/IC50values for HOCl assay with respect to total phenols, total flavonoids 

and total vitamin C contents of flavedo extracts of citrus fruits. All correlations were significant at the 0.05 level (two-tailed) except for values marked with an asterisk (*). 

been documented that using the 6-dichloroindophenol titrimetric 

method, phenolic extracts give a response corresponding to 20% 

of vitamin C content measured in fresh homogenised fruit extracts 

(Luximon-Ramma et al., 2003). Total phenolic contents are therefore 

only indicative of the amount of polyphenols in the flavedo 

extracts and on this basis some orange, clementine, mandarin, tangor, 

tangelo and pamplemousses extracts were found to contain the 

highest levels (>5500 g/g FW) (Fig. 1 and Table 2). These results are 

in accordance with others who indicated that peels are an important 

source of phenolics (Bocco et al., 1998). The levels in this study 

Table 6 

Comparative literature data on total phenol content of peel (flavedo + albedo) extracts of citrus fruits measured by the Folin–Ciocalteu assay. 

Citrus fruit Total polyphenols 

(g/g FW) 

Grapefruits 1550a Sweet Oranges 1790a Lemons 1900a White grapefruits 282 b 

Jaffa sweetie grapefruits 376 b 

Lemons (cv. Meyer) 598a Lemons (cv. Yenben) 1190a Grapefruit 1616a Mandarin (cv. Ellendale) 1211a Sweet orange (cv. Navel) 736a Method of extraction Expression of results Origin Reference 

Homogenisation of 10 g of fresh peel in 125 mL 

95% ethanol followed by boiling in waterbath 

Vortexing of 50 mg of lyophilised sample in 

5 mL 80% methanol for 1 min. Heating at 90 ◦ C 

for 3 h with vortexing every 30 min 

Extraction of 2 g of frozen citrus peel powder 

with 16 mL of 72% ethanol for 3 h. 

Centrifugation, filtration and evaporation of 

solvent under pressure. Dissolving of extract in 

water 

Lemons 1882–2828 Extraction of 100 mg of lyophilised sample 

Mandarins 2649–6923 

Sweet orange 4509–6470 

with 12 mL of 80% methanol over 3 days. 

Centrifugation, decantation and use of extract 

as is 

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. 

Chlorogenic acid 

equivalent 

Grown in Israel Gorinstein 

et al. (2001) 

Gallic acid equivalent Grown in Israel Gorinstein 

et al. (2004) 

Gallic acid equivalent Bought in New 

Zealand 

Li et al. (2006) 

Gallic acid equivalent Grown in Mauritius Present study


Fig. 7. (a) HPLC profile of flavedo extract of Mandarin 1A at 280 nm. 1: Poncirin; 4: Dydimin; 5: Naringin; 9: Hesperidin; 10: Neoeriocitrin; 11: Narirutin. (b) HPLC profile of 

flavedo extract of Mandarin 1A at 330 nm. 2: Rhoifolin; 3: Rutin; 6: Diosmin; 7: Isorhoifolin. 

are much higher than those measured in peels of similar varieties 

from Israel and New Zealand (Table 6) using the same methodology 

indicating that the contents can be influenced by various factors 

such as genotypic differences, geographical and climatic conditions, 

cultural practices, harvest time and extraction methods amongst 

others (Van der Sluis et al., 2001). 

The free radical scavenging capacities/reducing powers of the 

21 flavedo extracts were evaluated by three independent methods, 

the TEAC, FRAP and HOCl assays. Flavedo extracts had wide antioxidant 

potential ranges, thereby supporting their classification as 

low, moderate and high (Table 4). The antioxidant potential exhibited 

by Clementine A, Mandarin 1A, 2A, Tangor A and Tangelo 2 

can be associated with high levels of phenolics, of which flavonoids 

were major components. This is clearly demonstrated by the linear 

regression plots and Pearson’s correlation coefficients of TEAC, 

FRAP and HOCl IC 50 values against total phenols and flavonoids 

(Fig. 6). Gorinstein et al. (2004) found a similar high correlation 

between antioxidant activities of two citrus fruits from Israel, Jaffa 

sweeties and Jaffa white grapefruits, and their total phenols content 

(R 2 = 0.94). Similar linear correlation between antioxidant activity 

and phenolic content have been reported for plant extracts 

(Neergheen et al., 2006; Soobrattee et al., 2008), beverages (Richelle 

et al., 2001; Luximon-Ramma et al., 2005), vegetables (Bahorun et 

al., 2004), juices, (Gil et al., 2000), wines (Burns et al., 2000) and 

fresh and processed edible seaweeds (Jimenez-Escrig et al., 2000). 

It is also interesting to compare the antioxidant data obtained here 

for Mauritian citrus flavedo extracts with those obtained previously 

for similar types of fruit parts using the same assays. Guo et al. 

(2003) reported FRAP values for the peel fractions (comprising the 

flavedo and albedo) of the Chinese fruits Lukan tangerine, orange, 

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

respectively. FRAP values of orange and lemon extracts were comparable 

to those of this study whilst the values for kumquat and 

pomelo were lower. 

Some flavedo extracts, however, were found to have higher 

or lower antioxidant activities in one assay system compared 

to the others. This confirms that there is no universal method 

that can measure the antioxidant capacity of all samples accurately 

and consistently. Clearly, matching radical source and system 

characteristics to antioxidant reaction mechanisms is critical in 

the selection of appropriate antioxidant capacity assay assessing 

methods (Prior et al., 2005). Along this line, nine flavedo extracts 

exhibiting most potent antioxidant activities in the TEAC, FRAP 

and HOCl assays were further assessed for their ability to modulate 

metal ion dependent free radical reaction. The metal complex 

copper 1,10-phenanthroline is known to promote hydroxyl radical 

formation from molecular oxygen by redox-cycling and is 

therefore a suitable agent for the stimulation of oxidative DNA damage 

(Aruoma, 1993; Halliwell, 1997). Indeed DNA fragmentation 

detected in different cells treated with copper phenanthroline is 

considered to result from direct attack upon DNA by the hydroxyl 

radical (Tsang et al., 1996). DNA damage, such as single and double 

strand breakage, base modification, cross-linking of DNA with other 

biomolecules particularly proteins, are reported to be early events 

of cancer, cardiovascular diseases, diabetes, cataract and neurological 

disorders (Cadet et al., 1997) and phytochemicals have profound 

chemopreventive effects through modulation of molecular events 

that damage DNA (Bisht et al., 2008). The level of protection 

against copper-phenanthroline-mediated oxidative DNA damage 

were in the following order for the flavedo extracts: Tangelo 

1A > Clementine A > Tangor A > Pamplemousses 2B > Mandarine 

5 > Mandarine 1A ≈ Orange 2B > Mandarine 2A > Tangelo 2.


Fig. 8. (a) HPLC profile of flavedo extract of Tangor A at 280 nm. 1: Poncirin; 4: Dydimin; 8: Neohesperidin; 9: Hesperidin; 10: Neoeriocitrin; 11: Narirutin. (b) HPLC profile 

of flavedo extract of Tangor A at 330 nm. 3: Rutin; 6: Diosmin; 7: Isorhoifolin. 

Among the transition metals, iron is known as the most important 

lipid prooxidant due to its high reactivity. The ferrous state 

of iron accelerates lipid oxidation by breaking down hydrogen and 

lipid peroxides to reactive free radicals via the Fenton reaction. The 

agents that can attenuate the action of these bivalent metal ions 

have been classified as secondary antioxidants which retard the 

rate of radical initiation reaction by the elimination of initiators 

(Vaya and Aviram, 2001). Ferrozine forms a complex with free Fe 2+ 

but the extent of the complexation is reduced when the Fe 2+ is less 

available by being bound onto the plant extracts (or a chelating 

agent) for example. In the presence of chelating agents, the complex 

formation of ferrous ion and ferrozine is altered and this can be 

monitored by decrease in the absorbance at 562 nm. Benherlal and 

Arumughan (2008) reported that phytochemicals/extracts with 

high antioxidant activity but without iron chelation capacity failed 

to protect DNA in Fenton’s system, suggesting that iron chelation 

was an essential requirement for extracts studied here to retard 

HO • generation by Fenton’s reaction. In this study Clementine A, 

Tangor A and Mandarin 1A and 5 were the most potent Fe 2+ ion 

chelator. 

Mauritian citrus flavedos are moderately rich sources of vitamin 

C with the highest values measured in the pamplemousses 

varieties (maximum: 1475 ± 37 g/g FW (p < 0.05)). No comparable 

data are available on the same extract type but Abeysinghe et 

al. (2007) reported values ranging between 2540 and 4430 g/g FW 

in juice sacs, 1420 and 3260 g/g FW in segment membranes and 

2610 and 3890 g/g FW in segments of mandarin and orange varieties. 

Vitamin C does not contribute significantly to the antioxidant 

potential of the flavedos, as evidenced by the negative correlations 

obtained between TEAC, FRAP and HOCl antioxidant capacity and 

vitamin C content (Fig. 7). This is very much consistent with the 

literature report indicating that vitamin C makes little contribution 

or does not contribute at all to the total antioxidant capacity 

of 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 result 

from the Cu-phenanthroline studies where the ability of the fruit 

phenolics to chelate copper ions and modulate their redox potentials 

was demonstrated suggesting that metal chelation could be 

more important. However, in other reported citrus research, Vitamin 

C was found to be a main contributor to the Total Antioxidant 

Capacity (TAC) (Gardner et al., 2000; Yoo et al., 2004; Abeysinghe et 

al., 2007). This suggests a wide variation in vitamin C contribution 

in different fruit species and even different cultivars within citrus 

species. 

Flavonoid derivatives, expressed in quercetin equivalents, in 

Mauritian citrus flavedos were generally high (>2000 g/g FW for 

the majority of samples analysed) (Table 2). Factors, including differences 

in variety and high sunlight conditions (a characteristic 

feature of tropical Mauritius), which can induce the accumulation 

of flavonoids (Li et al., 1993) are probably responsible for the 

relatively high yield. Using the same assay system but with catechin 

as standard, Gorinstein et al. (2004) reported that peeled 

Jaffa sweeties (a grapefruit hybrid) and white grapefruits contained 

471 and 377 g/g FW while 925 and 744 g/g FW were 

measured in their respective peels. Three types of flavonoids occur 

in citrus fruits: flavanones, flavones and flavonols. HPLC analyses 

of nine flavedo extracts showed that, consistent with literature 

data (Londoño-Londoño et al., 2010), the flavanone glycoside hesperidin 

was present at highest concentrations (83–234 mg/g FW) 

in all the extracts except for Pamplemousses 2B. The flavanone 

glycosides poncirin, didymin, narirutin and flavone glycosides 

diosmin and isorhoifolin were present in all flavedo extracts


whereas the flavanone glycoside naringin was present only in 

Mandarin 1A. The presence of naringin was observed in Mandarin 

1A despite its reported absence from Mandarin varieties 

(Tomás-Barberán and Clifford, 2000). Several reports highlight 

the structure–antioxidant activity relationships of flavonoid subclasses 

in citrus extracts. Data evidence suggests that glycosylation, 

O-methylation, O-glycosylation influence greatly the antioxidant 

potency of citrus flavonoids (Di Majo et al., 2005). Antioxidant 

activity decreases with glycosylation and is enhanced with hydroxylation 

and the presence of C2–C3 double bond in conjugation with 

a 4-oxo function (Rice-Evans et al., 1996). Whilst the flavonoids 

in the flavedo extracts may contribute significantly, other yet 

uncharacterized phytochemicals, may also contribute to the overall 

antioxidant effect of the flavedos. Overall citrus flavedos represent 

a major source of polyphenolic antioxidants. Large amounts 

of citrus peels are generated as by-product wastes of the juice 

processing industry and represent an untapped resource which 

potentially can be judiciously used as functional food ingredients 

and prophylactic agents. Further studies on the effective antioxidants 

contained in these fruit fractions and the mechanisms by 

which they could protect against disease development are highly 

warranted. 

Conflict of interest 

The authors declared no conflict of interest. 

Acknowledgements 

This work was supported by the University of Mauritius. The 

authors wish to thank the University of Mauritius for a postgraduate 

scholarship awarded to Deena Ramful and the Compagnie Agricole 

de Labourdonnais for providing citrus samples. 

References 

Abeysinghe, D.C., Li, X., Sun, C.D., Zhang, W.S., Zhou, C.H., Chen, K.S., 2007. Bioactive 

compounds and antioxidant capacities in different edible tissues of citrus fruit 

of four species. Food Chem. 104, 1338–1344. 

Anagnostopoulou, M.A., Kefalas, P., Papageorgiou, V.P., Assimopoulou, A.N., Boskou, 

D., 2006. Radical scavenging activity of various extracts and fractions of sweet 

orange flavedo (Citrus sinensis). Food Chem. 94, 19–25. 

AOAC, 1995. AOAC Official Method 981.12, in: Cunniff, P. (Ed.), AOAC Official Methods 

of Analysis of AOAC International. AOAC International, USA, pp. 16–17. 

Aruoma, O.I., 1993. Use of DNA damage as a measure of pro-oxidant actions of antioxidant 

food additives and nutrient components. In: Halliwell, B., Aruoma, O.I. 

(Eds.), DNA and Free Radicals. Eliis Horwood, London, pp. 315–317. 

Aruoma, O.I., 1994. Nutrition and health aspects of free radicals and antioxidants. 

Food Chem. Toxicol. 32, 671–683. 

Aruoma, O.I., 2003. Methodological considerations for characterizing potential 

antioxidant actions of bioactive components in plant foods. Mutat. Res. 523-524, 

9–20. 

Bahorun, T., Luximon-Ramma, A., Crozier, A., Aruoma, O.I., 2004. Total phenol, 

flavonoid, proanthocyanidin and vitamin C levels and antioxidant activities of 

Mauritian vegetables. J. Sci. Food Agric. 84, 1553–1561. 

Bahorun, T., Neergheen, V.S., Soobrattee, M.A., Luximon-Ramma, V.A., Aruoma, O.I., 

2007. Prophylactic phenolic antioxidants in functional foods of tropical island 

states of the Mascarene Archipelago (Indian Ocean). In: Losso, J.N., Shahidi, F., 

Bagchi, D. (Eds.), Anti-angiogenic Functional and Medicinal Foods. CRC Press, 

Taylor and Francis Group, New York. 

Bahorun, T., Luximon-Ramma, A., Gunness, T.K., Sookar, D., Bhoyroo, S., Jugessur, 

R., Reebye, D., Googoolye, K., Crozier, A., Aruoma, O.I., 2010. The effect of black 

tea on the levels of uric acid and C-reactive protein in humans susceptible to 

cardiovascular diseases. Toxicology 278, 68–74. 

Benherlal, P.S., Arumughan, C., 2008. Studies on modulation of DNA integrity in 

Fenton’s system by phytochemicals. Mutat. Res. 648, 1–8. 

Benzie, I.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a measure 

of ‘antioxidant power’: the FRAP assay. Anal. Biochem. 239, 70–76. 

Bisht, K., Wagner, K.H., Bulmer, A.C., 1996. Curcumin, resveratrol and flavanoids 

as anti-inflammatory, cyto-and DNA protective dietary compounds. Toxicology 

278, 88–100. 

Bocco, A., Cuvelier, M.E., Richard, H., Berset, C., 1998. Antioxidant activity and 

phenolic composition of citrus peel and seed extracts. J. Agric. Food Chem. 4, 

2123–2129. 

Boynes, J.W., 1991. Role of oxidative stress in the development of complication in 

diabetes. Diabetes 40, 405–411. 

Burns, J., Gardner, P.T., O’Neil, J., Crawford, S., Morecroft, I., McPhail, D.B., Lister, C., 

Matthews, D., MacLean, M.R., Lean, M.E., Duthie, G.G., Crozier, A., 2000. Relationship 

among antioxidant activity, vasodilation capacity, and phenolic content of 

red wines. J. Agric. Food Chem. 48, 220–230. 

Cadet, J., Berger, M., Douki, T., Ravanat, J.L., 1997. Oxidative damage to DNA: 

formation, measurement and biological significance. Rev. Physiol. Biochem. 

Pharmacol. 131, 1–87. 

Campos, A., Lissi, E., 1996. Kinetics of the reaction between 2,2 ′ -azinobis(3ethyl)benzthiazolinesulfonis 

acid (ABTS) derived radical cations and phenols. 

Int. J. Chem. Kinet. 29, 219–223. 

Central Statistic Office, 2007. http://www.gov.mu/portal/goc/iso/mif06/health 

(Accessed on 7 August 2009). 

Chun, O.K., Kim, D.-O., Moon, H.Y., Kang, H.G., Lee, C.Y., 2003. Contribution of individual 

polyphenolics to total antioxidant capacity of plums. J. Agric. Food Chem. 

51, 7240–7245. 

Di Majo, D., Giammanco, M., La Guardia, M., Tripoli, E., Giammanco, S., Finotti, E., 

2005. Flavanones in citrus fruit: structure–antioxidant activity relationships. 

Food Res. Int. 38, 1161–1166. 

Dorman, H.T.D., Kosar, M., Kahlos, K., Holm, Y., Hittunen, R., 2003. Antioxidant 

properties and composition of aqueous extracts from Mentha species, hybrids, 

varieties and cultivars. J. Agric. Food Chem. 51 (16), 4563–4569. 

Franke, A.A., Custer, L.J., Arakaki, C., Murphy, S.P., 2004. Vitamin C and flavonoid 

levels of fruits and vegetables consumed in Hawaii. J. Food Comp. Anal. 17, 

1–35. 

Gardner, P.T., White, T.A.C., McPhail, D.B., Duthie, G.G., 2000. The relative contributions 

of vitamin C, carotenoid and phenolics to the antioxidant potential of fruit 

juices. Food Chem. 68, 471–474. 

Gil, M.I., Tomas-Barberan, F.A., Hess-Pierce, B., Holcroft, D.M., Kader, A.A., 2000. 

Antioxidant activity of pomegranate juice and its relationship with phenolic 

composition and processing. J. Agric. Food Chem. 48, 4581–4589. 

Gorinstein, S., Martin-Belloso, O., Park, Y.-S., Haruenkit, R., Lojek, A., Ciz, M., Caspi, 

A., Libman, I., Trakhtenberg, S., 2001. Comparison of some biochemical characteristics 

of different citrus fruits. Food Chem. 74, 309–315. 

Gorinstein, S., Cvikrova, M., Machackova, I., Haruenkit, R., Park, Y.S., Jung, S.T., 

Yamamoto, K., Ayala, A.L.M., Katrich, E., Trakhtenberg, S., 2004. Characterization 

of antioxidant compounds in Jaffa sweeties and white grapefruits. Food 

Chem. 84, 503–510. 

Guimarães, R., Barros, L., Barreira, J.C.M., Sousa, M.J., Carvalho, A.M., Ferreira, 

I.C.F.R., 2009. Targeting excessive free radicals with peels and juices 

of citrus fruits: grapefruit, lemon, lime and orange. Food Chem. Toxicol., 

doi:10.1016/j.fct.2009.09.022. 

Guo, C., Yang, J., Wei, J., Li, Y., Xu, J., Jiang, Y., 2003. Antioxidant activities of peel, pulp 

and seed fractions of common fruits as determined by FRAP assay. Nutr. Res. 23, 

1719–1726. 

Gutteridge, J.M.C., Halliwell, B., 1982. The role of superoxide and hydroxyl radicals in 

the degradation of DNA and deoxyribose induced by a copper-phenanthroline 

complex. Biochem. Pharmacol. 31 (17), 2801–2805. 

Halliwell, B., 1996. Antioxidants in human health and disease. Ann. Re. Nutr. 16, 

33–50. 

Halliwell, B., 1997. Antioxidants: the basics—what they are and how to evaluate 

them. Adv. Pharmacol. 38, 3–20. 

Halliwell, B., Gutteridge, J.M.C., Cross, C.E., 1992. Free radicals, antioxidants and 

human disease: where are we now? J. Lab. Clin. Med. 119, 598–620. 

Hensley, K., Floyd, R.A., 2002. Reactive oxygen species and protein oxidation in aging: 

a look back, a look ahead. Arch. Biochem. Biophys. 397, 377–383. 

Hool, L.C., 2006. Reactive oxygen species in cardiac signaling: from mitochondria to 

plasma membrane ion channels. Clin. Exp. Pharmacol. Physiol. 33, 146–151. 

Huang, R.P., Golard, A., Hossain, M.Z., Huang, R., Liu, Y.G., Boynton, A.L., 2001. Hydrogen 

peroxide promotes transformation of rat liver non-neoplastic epithelial 

cells through activation of epidermal growth factor receptor. Mol. Carninog. 30, 

209–217. 

Jimenez-Escrig, A., Jimenez-Jimenez, I., Pulido, R., Saura-Calixto, F., 2000. Antioxidant 

activity of fresh and processed edible seaweeds. J. Agric. Food Chem. 81, 

530–534. 

Kalt, W., Forney, C.F., Martin, A., Prior, R.L., 1999. Antioxidant capacity, vitamin C, 

phenolics, and anthocyanins after fresh storage of small fruits. J. Agric. Food 

Chem. 47, 4638–4644. 

Li, Y., Ou-Lee, T.-M., Raba, R., Amundson, R.G., Last, R.L., 1993. Arabidopsis flavonoid 

mutants are hypersensitive to UV-B irradiation. Plant Cell 5, 171–175. 

Li, S., Lo, C.Y., Ho, C.T., 2006. Hydroxylated polymethoxyflavones and methyl 

flavonoids in sweet orange (Citrus sinensis) flavedo. J. Agric. Food Chem. 54, 

4176–4185. 

Londoño-Londoño, J., de Lima, V.R., Lara, O., Gil, A., Pasa, T.B.C., Arango, G.J., Pineda, 

J.R.R., 2010. Clean recovery of antioxidant flavonoids from citrus peel: optimizing 

an aqueous ultrasound-assisted extraction method. Food Chem. 119, 81–87. 

Luximon-Ramma, A., Bahorun, T., Crozier, A., 2003. Antioxidant actions and phenolic 

and vitamin C contents of common Mauritian exotic fruits. J. Sci. Food Agric. 83, 

496–502. 

Luximon-Ramma, A., Bahorun, T., Crozier, A., Zbarsky, V., Datla, K.K., Dexter, D.T., 

Aruoma, O.I., 2005. Characterization of the antioxidant functions of flavonoids 

and proanthocyanidins in Mauritian black teas. Food Res. Int. 38, 357–367. 

Manthey, J.A., Grohmann, K., 2001. Phenols in citrus peel byproducts. Concentrations 

of hydroxycinnamates and polymethoxylated flavones in citrus peel molasses. 

J. Agric. Food Chem. 49, 3268–3273.

Middleton, E., Kandaswami, C., 1994. Potential health-promoting properties of Citrus 

flavonoids. Food Technol. 11, 115–119. 

Montanari, A., Chen, J., Widmer, W., 1998. Citrus flavonoids: a review of past biological 

activity against disease. In: Manthey, J.A., Buslig, B.S. (Eds.), Flavonoids 

in the Living System. Plenum Press, New York, pp. 103–113. 

Mouly, P., Gaydou, E.M., Auffray, A., 1998. Simultaneous separation of flavanone 

glycosides and polymethoxylated flavones in citrus juices using liquid chromatography. 

J. Chromatogr. A 800, 171–179. 

Neergheen, V.S., Soobrattee, M.A., Bahorun, T., Aruoma, O.I., 2006. Characterization 

of the phenolic constituents of Mauritian endemic plants as determinants of 

their antioxidant activities in vitro. J. Plant Physiol. 163, 787–799. 

Perry, G., Raine, K.A., Nunomura, A., Watayc, T., Sayre, L.M., Smith, M.A., 2000. How 

important is oxidative damage? Lessons form Alzheimer’s disease. Free Radic. 

Biol. Med. 28, 831–834. 

Prior, R.L., Cao, G., Martin, A., Sofic, E., Mcewen, J., O’Brien, B., Lischner, N., Ehlenfeldt, 

M., Kalt, W., Krewer, G., Mainland, C.M., 1998. Antioxidant capacity as influenced 

by total phenolic and anthocyanin content, maturity and variety of Vaccinium 

species. J. Agric. Food Chem. 46, 2686–2693. 

Prior, R.I., Wu, X.L., Schaich, K., 2005. Standardized methods for the determination 

of antioxidant capacity and phenolics in foods and dietary supplements. J. Agric. 

Food Chem. 53, 4290–4302. 

Proteggente, A.R., Saija, A., De Pasquale, A., Rice-Evans, C.A., 2003. The compositional 

characterisation and antioxidant activity of fresh juices from Sicilian 

sweet orange (Citrus sinensis L. Osbeck) varieties. Free Radic. Res. 37, 681–687. 

Rice-Evans, C.A., Miller, N.J., Paganga, G., 1996. Structure–antioxidant activity relationships 

of flavonoids and phenolic acids. Free Radic. Biol. Med. 20, 933–956. 

Richelle, M., Tavazzi, I., Offord, E., 2001. Comparison of the antioxidant activity of 

commonly consumed polyphenolic beverages (coffee, cocoa, and tea) prepared 

per cup serving. J. Agric. Food Chem. 49, 3438–3442. 

Samman, S., Wall, P.M.L., Cook, N.C., 1996. Flavonoids and coronary heart disease: 

dietary perspectives. In: Manthey, J.A., Buslig, B.S. (Eds.), Flavonoids in the Living 

System. Plenum Press, New York, pp. 469–481. 


Shahidi, F., 2004. Functional foods: their role in health promotion and disease prevention. 

J. Food Sci. 69, R146–R149. 

Shahidi, F., 2009. Nutraceuticals and functional foods: whole versus processed foods. 

Trends Food Sci. Technol. 20, 376–387. 

Singleton, V.L., Rossi, J.A., 1965. Colorimetry of total phenolics with 

phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic. 16, 

144–153. 

Soobrattee, M.A., Bahorun, T., Neergheen, V.S., Googoolye, K., Aruoma, O.I., 2008. 

Assessment of the content of phenolics and antioxidant actions of the Rubiaceae, 

Ebenaceae, Celastraceae, Erythroxylaceae and Sterculaceae families of Mauritian 

endemic plants. Toxicol. in Vitro 22, 45–56. 

Tomás-Barberán, F.A., Clifford, M.N., 2000. Flavanones, chalcones and dihydrochalcones 

– nature, occurrence and dietary burden. J. Sci. Food Agric. 80, 1073–1080. 

Tsang, S.Y., Tam, S.C., Bremner, L., Burkitt, M.J., 1996. Copper-1,10-phenanthrolin 

induces internucleosomal DNA fragmentation in HepG2 cells, resulting from 

direct oxidation by the hydroxyl radical. Biochem. J. 317, 13–16. 

Van der Sluis, A.A., Dekker, M., De Jager, A., Jongen, W.M.F., 2001. Activity and concentration 

of polyphenolic antioxidants in apple: effect of cultivar, harvest year, 

and storage conditions. J. Agric. Food Chem. 49, 3606–3613. 

Vaya, J., Aviram, M., 2001. Nutritional antioxidants mechanisms of action, analyses of 

activities and medical applications. Curr. Med. Chem. Immunol. Endocr. Metab. 

Agents 1, 99–117. 

Vinson, J.A., Su, X., Zubik, L., Bose, P., 2001. Phenol antioxidant quantity and quality 

in foods: fruits. J. Agric. Food Chem. 49, 5315–5321. 

Weiss, S.J., Klein, R., Slivka, A., Wei, M., 1982. Chlorination of taurine by the human 

neutrophils. J. Clin. Invest. 10, 598–607. 

Yoo, K.M., Lee, K.W., Park, J.B., Lee, H.J., Hwang, I.K., 2004. Variation in major antioxidants 

and total antioxidant activity of Yuzu (Citrus Junos Sieb ex Tanaka) during 

maturation and between cultivars. J. Agric. Food Chem. 52, 5907–5913. 

Zhishen, J., Mengcheng, T., Jianming, W., 1999. The determination of flavonoid contents 

of mulberry and their scavenging effects on superoxide radicals. Food 

Chem. 64, 555–559.

Bioactive phenolics and antioxidant propensity of flavedo extracts of ...

Create successful ePaper yourself

Delete template?

Save as template?