Food Research International 44 (2011) 2088–2099

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Polyphenol composition, vitamin C content and antioxidant capacity of Mauritian citrus fruit pulps Deena Ramful a, Evelyne Tarnus b, Okezie I. Aruoma c, Emmanuel Bourdon b, Theeshan Bahorun d,⁎ a

Department of Agricultural and Food Science, Faculty of Agriculture, University of Mauritius, Réduit, Mauritius 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 Department of Pharmaceutical and Biomedical Sciences, Touro College of Pharmacy, NY, USA d Department of Biosciences, Faculty of Science, University of Mauritius, Réduit, Mauritius b c

a r t i c l e

i n f o

Article history: Received 20 November 2010 Accepted 30 March 2011 Keywords: Citrus fruits Pulp Flavonoids Vitamin C TSS/TA ratio Antioxidants

a b s t r a c t The pulp extracts of twenty-one varieties of citrus fruits (oranges, satsumah, clementine, mandarins, tangor, bergamot, lemon, tangelos, kumquat, calamondin and pamplemousses), commonly grown in Mauritius, were characterised in terms of their total soluble solids (TSS), titratable acidity (TA), polyphenol composition and vitamin C contents. Total phenolics ranged from 406.3 ± 14 to 1694 ± 19 μg g− 1 fresh weight (FW). Total flavonoids varied between 133 ± 6 and 965 ± 7 μg g− 1 FW and vitamin C contents were from 166 ± 19 μg/mL to 677 ± 22 μg/mL. The pulp of a pamplemousse variety had the highest TSS/TA ratio whereas lemon pulps had lowest TSS/TA ratios. The antioxidant activities of the pulp extracts were assessed and total phenolics correlated strongly with the trolox equivalent antioxidant capacity (TEAC), ferric reducing antioxidant capacity (FRAP) and hypochlorous acid (HOCl) scavenging activity assays. Based on their antioxidant activities, nine citrus fruits namely, one orange, tangor, kumquat, calamondin and pamplemousse variety and two mandarin and tangelo varieties were further characterised for their flavanone, flavonol and flavone levels by HPLC. Hesperidin (6.89 ± 0.06 to 26.98 ± 0.07 mg/g FW) and narirutin (0.27 ± 0.01 and 20.91 ± 0.10 mg/g FW) were present at high concentrations compared to the other flavonoid glycosides in the pulp extracts. Naringin was detected only in pulp extracts of pamplemousses. In the light of the data obtained, citrus fruit pulps represent an important source of phytochemicals with potent antioxidant capacity. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Nutritional studies are now laying more emphasis on the protective and health-promoting effects of fresh fruits and vegetables. According to the United States Department of Agriculture (USDA) dietary guidelines (Dietary Guidelines Advisory Committee (DGAC), 2005), a range of 2½ to 6½ cups (5 to 13 servings) of fruits and vegetables is recommended each day for the 1200- to 3200-calorie level diet. Epidemiological studies have demonstrated that there is a significant positive relationship between fruit and vegetable consumption and reduced risk of chronic diseases (Liu, Manson, Lee, Cole, Hennekens, Willet et al., 2000; John, Ziebland, Yudkin, Roe, & Neil, 2002; Dauchet, Amouyel, Hercberg, & Dallongeville, 2006). An optimal mix of essential vitamins, minerals, fibres and bioactive phytochemicals such as alkaloids, carotenoids, nitrogenous compounds and polyphenolics are reported to contribute to these beneficial effects. Antioxidant polyphenols, by virtue of their hydrogen and electron donating abilities and metal chelating effects

⁎ Corresponding author at: Faculty of Science, University of Mauritius, Réduit, Mauritius. Tel.: + 230 4037471; fax: + 230 4656928. E-mail address: [email protected] (T. Bahorun). 0963-9969/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2011.03.056

(Lindsay & Astley, 2002; Valko, Rhodes, Moncol, Izakovic, & Mazur 2006), exhibit a wide range of biological properties including antiallergenicity, anti-atherogenicity, anti-inflammatory, anti-microbial, anti-thrombotic, cardioprotective and vasodilatory actions ( Middleton, Kandaswami, & Theoharides, 2000; Puupponen-Pimiä, Nohynek, Meier, Kähkönen, Heinonen, Hopia et al., 2001; Samman, Lyons Wall, & Cook, 1998). Tropical countries like Mauritius enjoy the right balance of sunshine and rainfall for the growth of a wide range of exotic fruits, delicious in taste and refreshing in flavour and aroma. Citrus genus is one of the most important fruit tree crop in the world and in Mauritius, citrus fruits are the second most consumed fruits, after bananas (Central Statistics Office (CSO), 2008), as fresh produce, juice, marmalades, jams and paste. Consumption of citrus fruit or juice appears to be associated with improved blood lipid profiles (Kurowska, Spence, Jordan, Wetmore, Freeman, Piche et al., 2000), survival in the elderly (Fortes, Forastiere, Farchi, Rapiti, Pastori & Perucci, 2000), lower risk of cancers (Benavente-Garcia & Castillo, 2008), lowering of blood pressure (Adibelli, Dilek, & Akpolat, 2009), reduced risks of stroke (Chen, Ward, Graubard, Heineman, Markin, Potischman et al., 2002), cardiovascular and coronary heart diseases and obesity (González-Molina, Domínguez-Perles, Moreno, & García-Viguera,

D. Ramful et al. / Food Research International 44 (2011) 2088–2099

2010). The health promoting effects of citrus have been mainly associated with its antioxidant vitamin C and flavonoid contents. More than sixty flavonoid compounds have so far been identified in Citrus sp. and a majority of them can be regrouped into flavanones, flavones and flavonols existing as glycoside or aglycone forms (Benavente-Garcia, Castillo, Marin, Ortuno, & Del Rio, 1997). The polyphenolic and vitamin C composition and in vitro antioxidant activities of flavedo extracts of 21 citrus fruit varieties grown in Mauritius have been recently investigated by our group (Ramful, Bahorun, Bourdon, Tarnus, & Aruoma, 2010; Ramful, Tarnus, Rondeau, Robert da Silva, Bahorun, & Bourdon, 2010). In this present study, we report the polyphenolic composition, total soluble solids/ titratable acidity ratio (TSS/TA) and in vitro antioxidant potencies of the pulp extracts of these 21 citrus fruit varieties.

Table 1 Scientific and common names, variety and harvest dates of citrus fruits analysed. Scientific name

Common name

Variety

Harvest month

Variety and harvest code

Citrus sinensis

Orange

Citrus unshiu

Satsumah

Valencia late Washington Navel Owari

Citrus clementina

Clementine

Commune

Citrus reticulata

Mandarin

Fairchild

Aug Mar May Mar May Mar May Apr May May Jun Jun Aug Aug Aug Jun Aug Apr

1 2A 2B A B A B 1A 1B 2A 2B 3A 3B 4 5 A B –

Apr May Jun Aug Aug Jun Aug Apr Jun Jun Aug May Aug May Aug May Aug Aug

A B 1A 1B 2 3A 3B A B A B 1A 1B 2A 2B 3A 3B 4

Dancy Beauty

2. Methods and materials 2.1. Standards and chemicals 2,2′-Azino-bis(3-ethylbenzthiozoline-6)-sulfonic acid (ABTS) and Folin and Ciocalteu's phenol reagent were purchased 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-2carboxylic 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). HPLCgrade acetonitrile and methanol were obtained from Merck (Darmstadt, Germany). All other reagents used were of analytical grade.

C. reticulata × C. Tangor sinensis Citrus aurantium ssp. Bergamot bergamia Citrus meyeri Lemon C. reticulata × C. paradisis

Tangelo

2.3. Extraction The extraction procedure used was adapted from Franke, Custer, Arakaki, and Murphy (2004) and Chun et al. (2003). 500 mg of freezedried citrus pulp powder was weighed in a plastic screw-capped tube and 5 mL of 80% methanol was added. The tube contents were

Suhugan Fizu Elendale – Meyer Mineola Orlando Ugli

Fortunella margarita Citrus mitis

Kumquat

Nagami

Calamondin

–

Citrus maxima

Pamplemousses (Pummelo)

Rainking Kaopan Pink Chandler

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 for sale 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. At least 10 fruits of each variety were carefully washed under running tap water and patted dry. The peel was carefully removed with a manual peeler and the pulp obtained was homogenised in a Waring Commercial blender (Dynamics Corporation, CT, USA). Weighed portions of the pulp 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. It is noteworthy that there is a general tendency to mix the terms ‘pamplemousses’ and ‘grapefruits’ even though they are two distinct citrus varieties. The variety analysed in this study, is the pamplemousses(Citrus grandis or Citrus maxima) which originates from South East Asia and has an extremely thick peel covering its pulp which is either yellow, pale pink or pale green in colour. Graprefruit (citrus × paradise) is a hybrid of the pamplemousses and sweet orange (Citrus maxima × Citrus sinensis). Its peel is thinner than the pamplemousses and its pulp is either yellow or deep pink in colour.

2089

vortexed and left to macerate overnight at 4 °C. Centrifugation was then carried out at 4500 rpm for 15 min and the clear supernatant obtained was decanted into a clean vial and stored at −20 °C. 5 mL of 80% methanol was added to the residue and the same procedure was repeated. 2 mL of 80% methanol was subsequently added to the residue to obtain a final volume of 12 mL. 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 water-bath 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 μg mL− 1). Results are expressed in μg of gallic acid g− 1 FW of plant material. 2.5. Total flavonoid content Total flavonoids were measured using a colorimetric assay adapted from Zhishen, Mengcheng, and Jianming (1999). 150 μL of 5% aqueous

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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 μL of 10% aqueous AlCl3 was added. 1 mL of 1 M NaOH was added 1 min after the addition of aluminium chloride. The solution was mixed 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 μg mL− 1). Results are expressed in μg of quercetin g− 1 FW of plant material. 2.6. Total vitamin C content The 2, 6-dichloroindophenol titrimetric method (AOAC, 1995a) was used to determine the vitamin C content of pulp extracts. Fresh fruit pulp was homogenised in a blender and filtered. 5 mL of the clear juice obtained was diluted to 50 mL with metaphosphoric acid–acetic acid solution and 7 mL was titrated against standard indophenol solution. Extractions and titrations were performed in triplicates. Results were expressed in μg ascorbic acid mL− 1 pulp juice. 2.7. Titratable acidity The AOAC Official Method for titratable acidity of fruit products was used (AOAC, 1995b). Fruit pulp was homogenised in a Waring blender and filtered using muslin cloth. 10 mL of the juice obtained was diluted to 100 mL with distilled water and transferred into a 250 mL beaker which was placed over a magnetic stirrer to provide a continuous stirring of the sample solution. A pH meter probe (calibrated with standard buffer solutions of pH 7 and pH 9 respectively) was then immersed into the solution and 0.1 N NaOH was added, quite rapidly, from a burette until a pH of 6 was reached. The alkali was then slowly added to attain a pH of 7 and the volume of NaOH used was recorded. Titration was completed by adding 4 drops of NaOH at a time and the total volume of NaOH and pH reading after each addition were recorded. The 4 drops of NaOH were added until the pH of the sample exceeded 8.1. Determinations were performed in triplicates. The data for titration corresponding to pH 8.1 was then interpolated from the values obtained. Titratable acidity was expressed in g citric acid 100 mL− 1 pulp juice.

2.10. 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)-striazine (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. A 50 μL aliquot of sample was added to 150 μL of distilled water, followed by 1.5 mL of FRAP reagent. The absorbance was read at 593 nm after a 4 min incubation at 37 °C. A calibration curve of ferrous sulphate (0–1.2 mM) was used and results are expressed as μmol Fe2+ g− 1 fresh weight. 2.11. Hypochlorous acid (HOCl) scavenging assay The HOCl assay was adapted from Weiss, Klein, Slivka, and Wei (1982). Both HOCl and ClO− are potent oxidants and thus harmful in excessive amounts in vivo. In this model, taurine, a β-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 μL taurine (10 mM), 100 μL extract (variable concentrations), 100 μL 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 μL 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− 1). 2.12. High performance liquid chromatography

2.8. Total soluble solids (TSS) Juice was extracted from the citrus fruits manually using a citrus squeezer and then filtered using muslin cloth. 1 drop of pulp juice was placed on a hand refractometer (Bellingham + Stanley, Germany) prism plate of range 0–50%, which was then covered. The reading on the prism scale was noted to one decimal place. The temperature of the sample at the time of measurement was also recorded. The degree (°) Brix of the juice was then calculated and temperature correction applied. 2.9. 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-6sulfonic 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 (1997) 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 min on a Helios-Alpha spectrophotometer (Unicam Ltd, UK) maintained at 20 °C by a peltier thermostat. Calculations were made with respect to a dose–response curve of Trolox (concentration range: 0–100 μM) and the TEAC values are expressed in μmol Trolox g− 1 fresh weight.

2.12.1. Sample preparation Based on the results obtained from the TEAC, FRAP and HOCl assays, nine pulp extracts, with most potent antioxidant capacities, were selected for flavonoid glycoside analyses by HPLC. 1 g of lyophilized fruit powders were extracted with 12 mL of 80% aqueous methanol (HPLC grade) following the same procedure as described in Section 2.3. Samples were filtered on Milipore (0.22 μm) before use. 2.12.2. Chromatographic conditions Chromatographic conditions were adapted from Mouly, Gaydou, and Auffray (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 μm), 30 μL of extract was injected on a Waters Spherisorb ODS-2 column (5 μm particle size, 80 pore size, 4.6 mm id × 150 mm). The solvents used were: A, water–acetonitrile (90:10, v/v; pH adjusted to 2.35 using phosphoric acid) 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− 1. The identification and quantification of the flavonoids investigated were determined from retention time and peak area in comparison with the

D. Ramful et al. / Food Research International 44 (2011) 2088–2099

with their total phenolics exceeding 1100 μg g− 1 FW. The lowest amounts were observed in Lemon A and B (467±7 μg g− 1 FW and 406± 14 μg g− 1 FW respectively). Significant differences (pb 0.05) were observed between total phenolic levels of pulps of similar varieties of citrus harvested at different periods except for Orange 2, Satsumah and Tangor. The pulp extract of Pamplemousse 2A contained the highest level of total flavonoids (p b 0.05) with 965 ± 7 μg g− 1 FW (Fig. 2) followed by the one from Pamplemousse 1A (p b 0.05) with a content of 880 ± 5 μg g− 1 FW. Clémentines A and B, Mandarins 2A, 2B, 3A, 3B, 4 and 5, Lemons A and B, Kumquats A and B and Pamplemousse 1B all had total flavonoid levels below the 400 μg g− 1 FW mark with the lowest value (p b 0.05) measured in Lemon B (133 ± 6 μg g− 1 FW). The vitamin C content of the pulp extracts are shown in Fig. 3. Pulp juice of the different Pamplemousse varieties had high vitamin C levels. Pamplemousse 4 had the maximum amount (p b 0.05) with 677 ± 22 μg/mL while Satsumahs A and B, Mandarins 1B, 3A, 3B and 4, Lemons A and B and Tangelo 1A had low vitamin C levels that were not significantly different from each other. No significant differences were observed between vitamin C content of pulps of similar varieties harvested at different periods except for Tangor, Tangelo 3, Kumquat, Pamplemousse 1 and Pamplemousse 2. Taking into consideration the large distribution of total phenolics, flavonoids and vitamin C in the pulp extracts, we propose 3 groupings of these phytochemicals into 1) high level, 2) medium level and 3) low level (Table 2).

standards used. The standards, poncirin, rhoifolin, didymin, naringin, rutin, diosmin, isorhoifolin, neohesperidin, hesperidin, neoeriocitrin and narirutin, were prepared at a stock concentration of 200 μg mL− 1. Calibration standard samples containing the standards each at 20, 40, 100 and 200 μg mL− 1 were prepared by appropriate dilutions with methanol from the stock solutions and filtered on Milipore (0.22 μm) before use. The linearity of the assay was demonstrated by assaying calibration standards in duplicates 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 analyses 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 one-way ANOVA followed by Tukey's HSD post-test using Prism™ v4.0 software (GraphPad® Software, San-Diego, 2003). The differences were accepted as significant when P b 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™ v4.0 software (GraphPad® Software, San-Diego, 2003).

3.2. Titratable acidity and total soluble solids The TSS, TA and TSS/TA ratio of the citrus pulp juices are shown in Table 3. The TSS of the citrus pulps is expressed in terms of °Brix. Tangor B was characterised by the highest TSS value (13.19%) followed by the kumquats (Kumquat A: 13.12%; Kumquat B: 12.21%). Tangelos 1A, 1B, 2 and 3A, Orange 1, Tangor A, Mandarines 3B and 5, Calamondins A and B and Pamplemousse 2B also had °Brix greater than 10%. The lowest TSS value was recorded for Lemon A with a °Brix of 5.75%.

3. Results 3.1. Phenolic and vitamin C contents The total phenolic composition of the pulp extracts is presented in Fig. 1. The highest amounts (pb 0.05) of total phenolics were assayed in Kumquat B (1694 ± 19 μg g− 1 FW) followed by Kumquat A (1412 ± 16 μg g− 1 FW). Pulp extracts of Calamondin ranked third on the list 1750

a

Gallic acid equivalent (µg/g FW)

1500

b c

1250

1000

2091

d g gh

h k kl

e

ef

f h

i

j l

m mn

750

no

k

m

m

op

pq

r

r

mn

op

q

r

s t

pq s

u

500

v

250

Pamplemousses 4

Pamplemousses 3B

Pamplemousses 3A

Pamplemousses 2B

Pamplemousses 2A

Pamplemousses 1B

Calamondin B

Pamplemousses 1A

Kumquat B

Calamondin A

Kumquat A

Tangelo 3B

Tangelo 2

Tangelo 3A

Tangelo 1B

Tangelo 1A

Lemon A

Lemon B

Tangor B

Bergamot

Tangor A

Mandarine 5

Mandarine 4

Mandarine 3B

Mandarine 3A

Mandarine 2B

Mandarine 2A

Mandarine 1B

Clementine B

Mandarine 1A

Satsumah B

Clementine A

Orange 2B

Satsumah A

Orange 1

Orange 2A

0

Fig. 1. Total phenolic content of citrus pulp extracts. Data represent mean values (bars) with standard errors (n = 2). Different letters between columns represent significant differences between samples (p b 0.05). Letter a denotes sample having highest total phenolics and letter v denotes sample having lowest phenolic content.

2092

D. Ramful et al. / Food Research International 44 (2011) 2088–2099 1000

a b

900

Quercetin equivalent (µg/g FW)

800

c

700

e e

600

k jkl

f

g

h 500

i

ijk

l

cd

de

e

gh

gh ij

jkl

ij

jkl

m

400

opq st

300

pqr

rs

s

mn

nop no

qr st

t

u

200

v 100

Pamplemousses 4

Pamplemousses 3B

Pamplemousses 3A

Pamplemousses 2B

Pamplemousses 2A

Pamplemousses 1B

Calamondin B

Pamplemousses 1A

Kumquat B

Calamondin A

Kumquat A

Tangelo 3B

Tangelo 2

Tangelo 3A

Tangelo 1B

Lemon B

Tangelo 1A

Lemon A

Tangor B

Bergamot

Tangor A

Mandarine 5

Mandarine 4

Mandarine 3B

Mandarine 3A

Mandarine 2B

Mandarine 2A

Mandarine 1B

Clementine B

Mandarine 1A

Satsumah B

Clementine A

Orange 2B

Satsumah A

Orange 1

Orange 2A

0

Fig. 2. Total flavonoid contents of citrus pulp extracts. Data represent mean values (bars) with standard errors (n = 2). Different letters between columns represent significant differences between samples (p b 0.05). Letter a denotes sample having highest total flavonoids and letter v denotes sample having lowest flavonoid content.

highest TSS/TA ratio, followed by Mandarine 4 and 5. Lemon pulps had the lowest TSS/TA ratio (1.3–1.5).

Pulp juice of Calamondin B was the most acidic with a TA value of 6.33% whilst the juice of Pamplemousse B was the least acidic with a TA value of 0.36%. The lemons and calamondins showed high TA values (N4%). Citrus pulps characterised by low titratable acidity (b1%) were Tangelos 1A, 1B, 2 and 3A ,Pamplemousses 1B, 2B and 4, Mandarines 1A, 1B, 2A, 2B, 3A, 3B, 4 and 5, Oranges 2A and 2B, Satsumahs A and B and Clementines A and B. The TSS/TA ratio gives an indication of the sweetness of fruits. The higher the TSS/TA ratio, the sweeter is the fruit. Pamplemousses 4 was characterised by the

3.3. Antioxidant activities The Trolox Equivalent Antioxidant Capacity (TEAC), the Ferric Reducing Antioxidant Power (FRAP) and the Hypochlorous acid (HOCl) scavenging activities of the pulp extracts are given in Table 4.

a

700

bc

b

600

bc c

Ascorbic acid (µg/ml)

500

de

d efg

400

def

def

efg fg

fg

fgh

fgh fgh

fgh

ghi

de

def

ghi

hi ij 300

jk

jkl

klm

ij

ij jk

klm

klm

200

klm lm

klm lm

m

Pamplemousses 4

Pamplemousses 3B

Pamplemousses 3A

Pamplemousses 2B

Pamplemousses 2A

Pamplemousses 1B

Calamondin B

Pamplemousses 1A

Calamondin A

Kumquat B

Kumquat A

Tangelo 3B

Tangelo 3A

Tangelo 2

Tangelo 1B

Tangelo 1A

Lemon B

Lemon A

Bergamot

Tangor B

Tangor A

Mandarine 5

Mandarine 4

Mandarine 3B

Mandarine 3A

Mandarine 2B

Mandarine 2A

Mandarine 1B

Clementine B

Mandarine 1A

Satsumah B

Clementine A

Orange 2B

Satsumah A

Orange 1

0

Orange 2A

100

Fig. 3. Total vitamin C content of citrus pulp extracts. Data represent mean values (bars) with standard errors (n = 2). Different letters between columns represent significant differences between samples (p b 0.05). Letter a denotes sample having highest total vitamin C content and letter m denotes sample having lowest vitamin C content.

D. Ramful et al. / Food Research International 44 (2011) 2088–2099 Table 2 Classification of citrus fruits according to total phenolic, flavonoid and vitamin C levels in pulp extracts. Low

Medium

High

b 750 μg/g FW ▪Mandarins 1A, 2B, 3A, 3B and 4 ▪Tangelo 2 ▪Pamplemousses 1, 3B and 4 ▪Clémentines A and B ▪Lemons A and B ▪Bergamot

750–950 μg/g FW ▪Mandarins 1B, 2A and 5 ▪Tangelos 1B and 2 ▪Pamplemousses 1A, 2B and 3A ▪Oranges 2A and 2B ▪Satsumahs A andB

N 950 μg/g FW ▪Tangelos 1A, 3A and 3B ▪Pamplemousses 2A ▪Orange 1 ▪Tangors A and B ▪Kumquats A and B ▪Calamondins A and B

Total b 400 μg/g FW flavonoids ▪Mandarins 2A, 2B, 3A, 3B, 4 and 5 ▪Pamplemousse 1B ▪Clémentines A and B ▪Lemons A and B ▪Kumquats A and B

400–600 μg/g FW ▪Mandarin 1A ▪Tangelos 1B, 2 and 3B ▪Pamplemousse 3B ▪Oranges 1, 2A and 2B ▪Tangors A and B ▪Satsumahs A and B ▪Bergamot ▪Calamondins A and B

N 600 μg/g FW ▪Mandarin 1B ▪Tangelos 1A and 3A ▪Pamplemousses 1A, 2A, 2B, 3A and 4

b 300 μg/mL ▪Mandarins 1A, 1B, 3A, 3B and 4 ▪Tangelos 1A and 1B ▪Lemons A and B ▪Satsumahs A and B ▪Bergamot

300–500 μg/mL ▪Mandarins 2A, 2B and 5 ▪Tangelos 2 and 3B ▪Pamplemousses 1A, 2B, 3A and 3B ▪Clémentines A and B ▪Oranges 1, 2A and 2B ▪Tangors A and B ▪Kumquat B ▪Calamondins A and B

N 500 μg/mL ▪Tangelo 3A ▪Pamplemousses 1B, 2A and 4 ▪Kumquat A

Total phenolic

Total vitamin C

The free radical scavenging capacity of the pulp extracts, based on their TEAC values did not differ widely and were in the range 2.60 ± 0.02 μmol/g FW for Lemon B to 9.92 ± 0.11 μmol/g FW for Kumquat B. Orange 1, Tangors A and B, Tangelo 1A, Tangelos 3A and 3B, Kumquats A and B, Calamondins A and B, Pamplemousses 1A and 2A had TEAC values greater than 6 μmol/g FW. FRAP values of the pulp extracts ranged from 3.3 to 10.4 μmol/g FW. Analogous to results obtained in the TEAC assay, the pulp extract of Kumquat B had the highest FRAP value (pb 0.05) (10.4 ± 0.32 μmol/g FW). The FRAP values of Oranges 1, 2A and 2B, Mandarins 1B and 5, Tangors A and B, Tangelos 1A, 2, 3A and 3B, Kumquat A and Pamplemousses 2A and 2B pulps were greater than 6 μmol/g FW. Mandarin 4 was characterised by the lowest value (pb 0.05) (3.3± 0.14 μmol/g FW). The HOCl scavenging property of the extracts was expressed in terms of IC50 which represents the concentration of pulp (mg FW/mL) needed to achieve 50% scavenging of hypochlorite. Low IC50 values are reflective of high HOCl scavenging property of the extract. IC50 values for pulp extracts were in the range 52.5 to 175 mg FW/mL. The extract from Kumquat A had the lowest value. IC50 values for Tangelo 1A, Tangelo 3A, Pamplemousses 1A and 2A were not significantly different from Kumquat A (p N 0.05); being less than 60 mg FW/mL.

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Table 3 TSS, TA and TSS/TA ratio of citrus pulp juices. Citrus variety

TSS (%)a,c

TA (%)b,c

TSS/TA ratio

Orange 1 Orange 2A Orange 2B Satsumah A Satsumah B Clementine A Clementine B Mandarine 1A Mandarine 1B Mandarine 2A Mandarine 2B Mandarine 3A Mandarine 3B Mandarine 4 Mandarine 5 Tangor A Tangor B Bergamot Lemon A Lemon B Tangelo 1A Tangelo 1B Tangelo 2 Tangelo 3A Tangelo 3B Kumquat A Kumquat B Calamondin A Calamondin B Pamplemousse Pamplemousse Pamplemousse Pamplemousse Pamplemousse Pamplemousse Pamplemousse

11.55 ± 0.19 6.64 ± 0.13 8.85 ± 0.05 6.58 ± 0.07 8.52 ± 0.03 7.84 ± 0.10 9.17 ± 0.06 8.74 ± 0.07 9.64 ± 0.15 8.84 ± 0.06 9.55 ± 0.06 8.49 ± 0.06 11.11 ± 0.08 9.07 ± 0.04 10.13 ± 0.07 11.25 ± 0.14 13.19 ± 0.12 6.64 ± 0.04 5.76 ± 0.14 7.18 ± 0.02 10.11 ± 0.08 12.07 ± 0.04 10.15 ± 0.08 10.08 ± 0.05 9.50 ± 0.02 12.21 ± 0.21 13.12 ± 0.07 11.11 ± 0.06 11.10 ± 0.05 7.85 ± 0.04 8.58 ± 0.05 8.37 ± 0.05 10.10 ± 0.05 8.92 ± 0.02 9.08 ± 0.05 8.59 ± 0.05

1.15 ± 0.07 0.74 ± 0.06 0.59 ± 0.05 0.73 ± 0.08 0.51 ± 0.05 0.64 ± 0.07 0.65 ± 0.04 0.44 ± 0.06 0.61 ± 0.06 0.75 ± 0.05 0.68 ± 0.04 0.72 ± 0.04 0.77 ± 0.07 0.45 ± 0.07 0.48 ± 0.04 1.25 ± 0.08 1.28 ± 0.06 2.45 ± 0.16 4.51 ± 0.15 4.65 ± 0.14 0.78 ± 0.02 0.96 ± 0.06 0.80 ± 0.02 0.90 ± 0.03 1.32 ± 0.10 1.34 ± 0.06 1.83 ± 0.09 5.16 ± 0.06 6.33 ± 0.07 1.43 ± 0.07 0.75 ± 0.06 1.15 ± 0.08 0.82 ± 0.10 1.26 ± 0.05 1.15 ± 0.07 0.74 ± 0.04

10.07 9.02 15.01 9.02 16.71 12.25 14.11 19.72 15.71 11.79 14.05 11.85 14.49 20.30 21.25 9.02 10.33 2.71 1.28 1.54 13.02 12.53 12.74 11.16 7.18 9.09 7.18 2.15 1.75 5.48 11.49 7.29 12.32 7.06 9.11 23.65

a b c

1A 1B 2A 2B 3A 3B 4

Total soluble solids. Titratable acidity. Data expressed as mean ± SE.

lowest IC50 value in the HOCl assay (Table 4). On the other hand, Lemon A and B, with lowest amount of total phenolics had very low antioxidant capacity in the three assays (Table 4). Total flavonoid levels showed moderate correlation with TEAC values (r = 0.43) and poor correlation with FRAP values (r = 0.10). However, a good correlation was obtained between HOCl assay values and total flavonoid content (r = 0.64). A very low correlation was noted between FRAP values and total vitamin C contents of pulp extracts (r = 0.17, P N 0.05). Nevertheless, both TEAC and HOCl assay values showed moderate correlations with vitamin C levels of pulp extracts (TEAC, r = 0.35; HOCl, r = 0.44). Based on the three in vitro antioxidant assays, the following pulp extracts were found to have highest antioxidant capacities: Orange 2A, Mandarin 1B, Mandarin 5, Tangor A, Tangelo 1A, Tangelo 3A, Kumquat B, Calamondin A and Pamplemousse 2A. Their flavonoid profile was further investigated using high performance liquid chromatography.

3.4. Correlation between phenolic contents and antioxidant capacities 3.5. HPLC analysis of flavonoids With a view to rationalising the antioxidant potential of the citrus pulp extracts in terms of their polyphenol and vitamin contents, linear regression plots were generated and the Pearson correlation coefficients were calculated (Fig. 4). A striking correlation between total phenolics and antioxidant capacity of the extracts was noted (TEAC, r = 0.95; FRAP, r = 0.73; HOCl, r = 0.71). Kumquat A and B with highest levels of total phenolics (1412 ± 16 and 1694 ± 19 μg g− 1 FW, respectively) (Fig. 1) showed highest TEAC and FRAP values and

Representative HPLC profiles of the extracts are given in Figs. 5 and 6. Identification of the flavonoids 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 characterised in the pulp extracts: poncirin, dydimin, hesperidin, neohesperidin, neoeriocitrin and narirutin (Figs. 5a and 6a). One flavonol glycoside

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D. Ramful et al. / Food Research International 44 (2011) 2088–2099

Table 4 Antioxidant activities of pulp extracts of citrus fruits. Data expressed as mean value ± standard error (n = 2). Citrus fruit

Code

TEACI

FRAPII

HOClIII

Orange

1 2A 2B A B A B 1A 1B 2A 2B 3A 3B 4 5 A B – A B 1A 1B 2 3A 3B A B A B 1A 1B 2A 2B 3A 3B 4

6.44 ± 0.18c d e 5.55 ± 0.19g h 5.29 ± 0.10h i j 4.35 ± 0.06k l m n 4.33 ± 0.10k l m n 3.48 ± 0.07o p 3.89 ± 0.11l m n o 4.74 ± 0.16i j k 4.43 ± 0.12k l m n 4.53 ± 0.14k l m 3.84 ± 0.13m n o 3.22 ± 0.06o p q 3.72 ± 0.08n o 2.92 ± 0.06p q 5.63 ± 0.19f g h 6.24 ± 0.20e f g 6.32 ± 0.13d e f 3.97 ± 0.05l m n o 3.84 ± 0.04m n o 2.60 ± 0.02q 7.11 ± 0.19b c 5.33 ± 0.16h i 4.36 ± 0.07k l m n 6.42 ± 0.22c d e 6.73 ± 0.15c d e 7.79 ± 0.20b 9.92 ± 0.11a 7.71 ± 0.21b 7.02 ± 0.11b c d 6.44 ± 0.18c d e 3.76 ± 0.05n o 7.60 ± 0.18b 4.65 ± 0.16i j k l 4.54 ± 0.09j k l m 4.57 ± 0.11j k l m 4.60 ± 0.09i j k l

6.84 ± 0.06g 6.40 ± 0.04h 6.00 ± 0.03i j 4.20 ± 0.03s t 4.61 ± 0.05p q r 4.35 ± 0.01r s 4.68 ± 0.03p q 5.08 ± 0.03n o 6.28 ± 0.04h i 5.67 ± 0.06k l 4.14 ± 0.06s t u 4.42 ± 0.05q r s 4.55 ± 0.06q r 3.27 ± 0.06w 8.22 ± 0.04c d 9.22 ± 0.04b 9.44 ± 0.09b 3.85 ± 0.04u v 3.60 ± 0.06v 4.20 ± 0.05s t 7.83 ± 0.07e 5.63 ± 0.03l 7.11 ± 0.09f 8.40 ± 0.04c 5.96 ± 0.04j k 8.05 ± 0.06d e 10.44 ± 0.13a 5.30 ± 0.07m n 5.32 ± 0.05m n 4.14 ± 0.02s t u 4.39 ± 0.03q r s 6.33 ± 0.04h 6.36 ± 0.04h 5.56 ± 0.08l m 4.88 ± 0.02o p 3.94 ± 0.05t u

65.7 ± 1.1m n o 68.6 ± 0.9l m n 66.0 ± 0.4m n 85.5 ± 1.8i j 75.9 ± 1.7k l 105.8 ± 0.6g h 106.2 ± 0.9f g h 81.1 ± 0.8j k 99.6 ± 1.7h 100.1 ± 0.5h 134.9 ± 0.8b c 137.2 ± 1.1b 175.0 ± 2.7a 174.2 ± 3.0a 114.6 ± 0.7e 99.8 ± 0.7h 91.6 ± 1.5i 111.7 ± 3.4e f g 113.3 ± 1.0e f 124.7 ± 0.7d 53.6 ± 0.3q 71.0 ± 0.7l m 103.4 ± 0.7h 57.7 ± 1.5p q 69.4 ± 1.4l m n 52.5 ± 0.3q 63.3 ± 0.7n o p 69.1 ± 1.3l m n 71.0 ± 0.5l m 55.3 ± 1.1q 86.9 ± 1.0i j 58.3 ± 1.3o p q 63.3 ± 0.6n o p 84.1 ± 1.0i j 127.7 ± 1.2c d 75.7 ± 1.7k l

Satsumah Clémentine Mandarin

Tangor Bergamot Lemon Tangelo

Kumquat Calamondin Pamplemousses

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 b 0.05). I μmol Trolox/g fresh weight. II μmol Fe(II)/g fresh weight. III IC50 mg fresh weight/mL.

(rutin) and three flavone glycosides (rhoifolin, diosmin and isorhoifolin) were also identified in the extracts (Figs. 5b and 6b). Naringin was detected only in Pamplemousse 2A extracts. The levels of flavonoid glycosides in the citrus pulps were quantified and are presented 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 (R2 N 0.99) for all the compounds in the range of concentration tested. Hesperidin and narirutin were found to be present at high concentrations compared with the other flavonoid glycosides in the pulp extracts analysed (Table 5). Levels of hesperidin ranged from 6.89 ± 0.06 mg/g FW (Calamondin A) to 26.98 ± 0.07 mg/g FW (Tangor A) and based on its content the extracts could be ranked in the following order: Tangor A N Mandarin 1B = Orange 2A N Tangelo 1A N Mandarin 5 = Tangelo 3A N Kumquat B N Calamondin A. Hesperidin was not identified in pulp extracts of Pamplemousse 2A. Narirutin contents ranged from 0.27 ± 0.01 mg/g FW (Pamplemousse 2A) to 20.91 ± 0.10 mg/g FW (Tangelo 1A) and based on its level the following rank order of the pulp extracts could be established: Tangelo 1A N Orange 2A = Mandarin 1B N Tangelo 3A N Kumquat B = Mandarin 5 N Calamondin A N Pamplemousses 2A. Narirutin was not detected in pulp extracts of Tangor A. Didymin and isorhoifolin were present in all the pulp extracts. Diosmin was present only in Calamondin A while rhoifolin was measured only in Tangelo 1A and Pamplemousse 2A. Rutin and

neoeriocitrin were absent from pulp extracts of Kumquat B and Calamondin A. Neohesperidin was present in three pulp extracts namely, Tangor A, Calamondin A and Pamplemousse 2A. Highest levels of poncirin (3.93 ± 0.01 mg/g FW), didymin (15.5 ± 0.10 mg/g FW) and naringin (1.83 ± 0.03 mg/g FW) (p b 0.05) were quantified in Kumquat B whilst Pamplemousse 2A showed highest levels of rhoifolin (9.51 ± 0.02 mg/g FW). Topmost levels of rutin, isorhoifolin, neohesperidin and neoeriocitrin were recorded in Orange 2A (0.85 ± 0.02 mg/g FW), Mandarin 1B (1.24 ± 0.01 mg/g FW), Calamondin A (6.46 ± 0.07 mg/g FW) and Mandarin 5 (1.81 ± 0.07 mg/g FW), respectively. 4. Discussion Significant health risks and benefits are associated with dietary food choice. Consequently, knowledge of the bioactive constituents of the diet components remains a relevant guideline in the selection of foods that will maximise health benefits. In this respect, polyphenols from fruits, vegetables and plant-based beverages have been increasingly studied for their antioxidant properties and action mechanisms in diseases (Wang, Melnyk, Tsao, & Marcone, 2011; Fraga, Galleano, Verstraeten, & Oteiza, 2010). In addition to the total phenolic, flavonoid and vitamin C content, that may account for the antioxidant propensities of citrus fruit pulps, this present study also provides meaningful data on titratable acidity and total soluble solids of the pulp extracts. Total phenols were assessed using the Folin Ciocalteau method and results are expressed as gallic acid equivalents (GAE). The indicative total phenolics in the pulp extracts were in the range 406– 1694 μg/g FW and are comparable to values reported by Gorinstein, Martin-Belloso, Park, Haruenkit, Lojek, Ciz et al. (2001) for three citrus pulps which varied between 1350 and 1640 μg/g FW. These levels, however, were lower than those of flavedo extracts of the same varieties which ranged from 1882 to 7667 μg/g FW (Ramful, Bahorun, et al., 2010). Gorinstein et al. (2001) also reported total polyphenols in the peels of lemons, oranges and grapefruits to be significantly higher than in the peeled fruits whilst quince peel extracts had three fold higher amounts than that of the pulp (Fattouch, Caboni, Coroneo, Tuberoso, Angioni, Dessi et al., 2007). Polyphenols exhibit a broad spectrum of biomedical functions including antibacterial, anti-inflammatory, antiallergic, antithrombotic, antiviral, anticarcinogenic, hepatoprotective, and vasodilatory actions (Middleton et al., 2000; Sies, 2010). In addition, they can exert modulatory actions in cells by interacting with a wide spectrum of molecular targets central to the cell signalling machinery (Senthil Murugan, Vidya Priyadarsini, Ramalingan, Hara, Karunagan, &Nagini, 2010). Many of these biological functions stem mostly from their free radical scavenging and antioxidant activity (Soobrattee, Neergheen, Luximon-Ramma, Aruoma, & Bahorun, 2005). In this study the antioxidant activities of the pulp extracts were evaluated using 3 independent methods: the TEAC, FRAP and HOCl assays. While there are many methods for determining antioxidant activity of plant extracts, each has its own limitations. Moreover, it has been argued that the antioxidant activity of extracts cannot be reasonably validated by a single method due to the complex nature of phytochemicals and their interactions, thus the importance of using multi assay systems with different indices (Pérez-Jiménez et al., 2008). The free radical scavenging activities of the citrus pulp extracts as assessed by TEAC assay were in the range 2.6 to 9.9 μmol/g FW with highest values obtained in Kumquat pulp. Comparable results were obtained in the FRAP assay whereby Kumquat pulp showed the highest ferric reducing potential (10.4 μmol/g FW, p b 0.05) and in the HOCl assay where extract of the same variety had the lowest IC50 value (52.5). Thus, data from the TEAC, FRAP and HOCl assays were consistent with each other. Based on these three assays, the following

D. Ramful et al. / Food Research International 44 (2011) 2088–2099

7.5 5.0

r 2=0.90 Pearson r=0.95

2.5

12.5

0.025

10.0

0.020

7.5

0.015

1/IC50

10.0

FRAP (µmol/g FW)

TEAC (µmol/g FW)

12.5

5.0

r 2=0.53 Pearson r=0.73

2.5

0

500

1500

1000

2000

2000

0

7.5 5.0

r 2=0.19 Pearson r=0.43 250

500

750

1000

5.0

0

250

500

750

1000

0.000

1250

0

r 2=0.12 Pearson r=0.35

2.5

0

100 200 300 400 500 600 700 800

Total vitamin C (µg/g FW)

500

750

1000

1250

0.020

10.0

0.015

7.5 5.0

r 2=0.03 Pearson r=0.17*

2.5 0.0

0.0

250

Total flavonoids (µg/g FW)

1/IC50

5.0

r 2=0.41 Pearson r= 0.64

Total flavonoids (µg/g FW)

FRAP (µmol/g FW)

7.5

2000

0.010 0.005

r 2=0.01 Pearson r=0.10

2.5

12.5

10.0

1500

0.015

7.5

Total flavonoids (µg/g FW) 12.5

1000

0.020

10.0

0.0

1250

500

Total phenol (µg/g FW)

1/IC50

10.0

FRAP (µmol/g FW)

TEAC (µmol/g FW)

1500

1000

12.5

0.0

TEAC (µmol/g FW)

500

Total phenol (µg/g FW)

12.5

0

r 2=0.50 Pearson r= 0.71

0.000 0

Total phenol (µg/g FW)

2.5

0.010 0.005

0.0

0.0

2095

0

100 200 300 400 500 600 700 800

Total vitamin C (µg/g FW)

0.010 0.005

r 2=0.19 Pearson r= 0.44

0.000 0

100 200 300 400 500 600 700 800

Total vitamin C (µg/g FW)

Fig. 4. 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 citrus pulp extracts. All correlations were significant at the 0.05 level (2-tailed) except for values marked with an asterix (*).

citrus pulps were earmarked as most potent antioxidants: Kumquat, Tangelos 1 and 3, Tangor, Calamondin, Oranges 1 and 2, Pamplemousse 2. On the other hand, Lemon, Mandarins 3 and 4, Clementine and Bergamot can be classified in the low antioxidant activity range. In a previous study, the antioxidant activities of flavedo extracts of the same citrus fruits were investigated using similar assay sytems (Ramful, Bahorun, et al. 2010). The TEAC values of the flavedo extracts ranged from 11.0 to 46.1 μmol/g FW, FRAP values were between 13.7 and 81.3 μmol/g FW whilst IC50 values varied from 3.7 to 17.8 indicating much higher antioxidant indices for flavedo extracts. These data are consistent with those of Wang, Zhou, and Lin (2011) who report the antioxidant activity of peel extracts of Citrus sulcata to be twice that of the pulp extracts using the TEAC and DPPH assays. It is interesting to compare the data obtained here for Mauritian citrus fruits with those obtained previously for similar or different types of fruits using the same assays. In assessing the antioxidant activities of a variety of 30 fruits consumed in Italy, Pellegrini et al. (2003) report the TEAC values of yellow grapefruit, orange and tangerine to be 3.05, 8.74 and 4.16 μmol Trolox/g FW respectively whilst FRAP values of the same fruits were 10.20, 20.50 and 9.60 μmol Fe2+/g FW respectively. Data for FRAP values of citrus fruits consumed in China were of the order of 22.9, 18.9, 14.3, 5.0 and 3.9 μmol/g for pulp fractions of Lukan tangerine, orange, lemon, kumquat and pomelo respectively (Guo et al., 2003). In the light of these comparisons, Chinese and Italian oranges and Chinese lemon seem to have higher antioxidant capacities than Mauritian cultivars whilst reported FRAP values for Chinese Kumquat were lower compared to this study. Native Australian lemon and limes, on the other hand, had higher FRAP values ranging from 12.6 to 53.9 μmol/g FW (Konczak, Zabaras, Dunstan, & Aguas 2010). The variations measured can be

attributed to several factors including extraction methods and solvents used, geographical origin, cultivar and harvest or storage time (Van der Sluis, Dekker, De Jager, & Jongen, 2001). Halvorsen, Holte, Myhrstad, Barikmo, Hvattum, Remberg et al. (2002) reported that geographic origins influenced the FRAP data in fruit samples collected from different regions in the world. For instance, the antioxidant capacities of orange samples collected from Spain, Holland and Zenta showed this disparity as they ranged from 0.83 mmol/L to 1.50 mmol/L. Hypochlorous acid (HOCl) is produced in vivo by the oxidation of chloride ions by neutrophil-derived myeloperoxidase in the presence of H2O2 at sites of inflammation (Gutteridge, 1995). The non-specific oxidising and chlorinating OCl−radical is involved in a variety of chemical and biochemical processes including its reaction with various biomolecules such as proteins, carbohydrates, phospholipids and DNA (Prutz, 1996). Thus, effective HOCl scavengers are of interest in the management of oxidative stress. No literature data is available on the assessment of the HOCl scavenging activity of citrus fruits. However, there have been investigations on other fruit and plant extracts. Thus, in assessing the OCl− radical scavenging capacities of 17 fruits and 11 vegetables grown in Mauritius, Bahorun, Neergheen, Soobrattee, Luximon-Ramma, and Aruoma (2007) identified Chinese guava and broccoli to be the most potent, with IC50 values of 4.11 and 43.79 mg FW/mL respectively. The observed antioxidant activities of the citrus pulps might be a function of their polyphenolic contents as depicted by the strong correlations obtained between total phenolics and the TEAC, FRAP and HOCl data whereby coefficients (r) ranged from 0.71 to 0.95. Gorinstein et al. (2004) found a high correlation between antioxidant activities of two citrus fruits from Israel, Jaffa sweeties and Jaffa white

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A mAU

1750 1500 1250 1000 750 500

8

9 250

11

4

1

0 15

20

25

30

35

40

50 45

min

35

40

50 45

min

B mAU 600 500 400 300 200

6

100

7 0 15

20

25

30

Fig. 5. (A). HPLC profile of pulp extract of Calamondin A at 280 nm. 1: poncirin; 4: dydimin; 8: neohesperidin; 9: hesperidin; 11: narirutin. (B). HPLC profile of pulp extract of Calamondin A at 330 nm. 6: diosmin; 7: isorhoifolin.

grapefruits, and their total phenol content. A high correlation was also observed between antioxidant effectiveness of fresh orange juices and their total phenol content (Rapisarda et al., 1999). Literature data abounds in reports where the same type of linear correlation between antioxidant activities and phenolic contents has been found in plant extracts (Neergheen, Soobrattee, Bahorun, & Aruoma, 2006), fruits (Luximon-Ramma, Bahorun, & Crozier, 2003), vegetables (Bahorun, Luximon-Ramma, Crozier, & Aruoma, 2004), fruits juices (Gil, TomásBarberán, Hess-Pierce, Holcroft, & Kader, 2000) and wines (Burns, Gardner, McPhail, O'Neil, Crawford, Morecroft et al., 2000). It is thus suggested that the antioxidant activity of the citrus extracts is not likely to be ascribed to the property of an individual compound but rather to the synergistic actions of several phytochemicals. Citrus fruits represent a popular source of vitamin C. Pulp extracts of Pamplemousses 1B, 2A and 4, Kumquat A and Tangelo 3A contained the highest vitamin C levels (N500 μg/mL). Cano, Medina, and Bermejo (2008) reported that orange varieties had higher vitamin C concentrations than mandarin, clementine, satsume and hybrid varieties supporting the argument of wide variation of vitamin C content in literature. Vitamin C levels in fruits and vegetables can be influenced by various factors such as genotypic differences, climatic conditions and cultural practices (Lee & Kader, 2000). Rapisarda, Pannuzzo, Romano, and Russo (2003), using the 2,6-dichloroindophenol method measured 470 μg/mL vitamin C in pigmented (blood) orange juice. Using reversed phase HPLC, Gardner, White, McPhail, and Duthie (2000) reported the vitamin C content of commercial

orange juices to be 217 μg/mL whilst values in the range 250–613 μg/ mL were recorded by Sánchez-Moreno, Plaza, de Ancos, and Cano (2003) for fresh and commercial orange juices. In the present study, the contribution of vitamin C to the antioxidant activities of citrus pulp juices was found to range from very poor (FRAP: r = 0.17, p N 0.05) to moderate (TEAC: r = 0.35; HOCl: r = 0.44). Vitamin C was also reported previously to have no significant contribution to the antioxidant potential of Mauritian citrus flavedos, as evidenced by the negative correlations obtained between TEAC, FRAP and HOCl antioxidant capacity and vitamin C content (Ramful, Bahorun, et al. 2010). Luximon-Ramma et al. (2003) reported negative correlations between TEAC and FRAP assays and vitamin C content of Mauritian exotic fruits. The ascorbate content was shown to play a minor role in the antioxidant effectiveness of fresh orange juices in various in vitro systems (Rapisarda et al., 1999). Miller and Rice-Evans (1997) have underlined the significant contributory role of polyphenols in the total antioxidant activity of long-life orange juice even if vitamin C was present at higher concentration. However, there are contrasting reports whereby vitamin C makes significant contributions to antioxidant activity of citrus fruit juices. Thus, Gardner et al. (2000) reported that although vitamin C accounted for less than 5% of the antioxidant potential of apple and pineapple juices, it contributed to 65–100% of the antioxidant activity of beverages derived from citrus fruits. Sánchez-Moreno et al. (2003) indicated a very high correlation between ascorbic acid and total vitamin C content of commercial orange juices and their free radical scavenging capacities. The total

D. Ramful et al. / Food Research International 44 (2011) 2088–2099

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A mAU

11 9

800

600

400

4

200

10

1

0 15

20

25

30

35

40

50 45

min

30

35

40

50 45

min

B mAU 350 300 250 200 150 100 50

3

72

0 15

20

25

Fig. 6. (A). HPLC profile of pulp extract of Tangelo 1A at 280 nm. 1: poncirin; 4: dydimin; 9: hesperidin; 10: neoeriocitrin; 11: narirutin. (B). HPLC profile of pulp extract of Tangelo 1A at 330 nm. 2: rhoifolin; 3: rutin; 7: isorhoifolin.

antioxidant activity, as measured by the TEAC assay, of fresh orange juices highly correlated (r= 0.73) with the ascorbic acid concentration (Proteggente, Saija, De Pasquale, & Rice-Evans, 2003). Flavonoid derivatives, expressed in quercetin equivalents, in Mauritian citrus pulps were in the range 133 and 965 μg g− 1 FW with highest values reported in Pamplemousse 2A. Total flavonoid content of the pulp extracts was much lower than their flavedo counterparts that contained mostly values N2000 μg/g FW (Ramful, Bahorun, et al. 2010). Citrus fruits contain a wide range of flavonoid constituents which are encompassed in the flavanones, flavones and flavonols sub-classes (Nogata et al., 2006). HPLC analyses of the nine pulp extracts showed that, in line with literature data (Cano et al., 2008), hesperidin was the most abundant flavanone glycoside. The

latter was found at concentrations ranging from 7 to 27 mg/g FW, followed by narirutin (0.3–21 mg/g FW). Hesperidin was present in all the varieties except for Pamplemousse 2A whilst narirutin was absent only in Tangor A. The amount of flavonoid glycosides in the pulp extracts was much lower than the flavedo extracts of Mauritian citrus fruits, a trend that was consistent with the phenolic and vitamin C contents as well as the antioxidant activities (Ramful, Bahorun, et al. 2010; Ramful, Tarnus, et al. 2010). Berhow, Tisserat, Kanes, and Vandercook (1998) reported that the concentration of flavanones was greater in the citrus albedo whilst the levels of flavones and flavonols decreased in the following order flavedo N albedo N juice sacs. Total soluble solids (TSS) and titratable acidity (TA) of the fruits represent important quality parameters. Their flavour and palatability

Table 5 Flavanone, flavone and flavonol glycoside levels in pulp 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 b 0.05). Citrus fruit

Poncirin

Rhoifolin

Orange 2A Mandarine 1B Mandarine 5 Tangor A Tangelo 1A Tangelo 3A Kumquat B Calamondin A Pamplemousse 2A

0.12±0.00g 0.44±0.00c 0.18±0.01f 0.35±0.01d 0.71±0.02b 0.14±0.00fg 3.93±0.01a 0.27±0.02e ND

ND 2.70±0.04e ND 7.38±0.09b ND 0.95±0.01f ND 4.86±0.02c 0.56±0.01b 3.60±0.10d ND 0.77±0.01f ND 15.53±0.05a ND 0.39±0.01g 9.51±0.02a 0.05±0.00h

Didymin

Naringin

Rutin

Diosmin

Isorhoifolin

Neo-hesperidin Hesperidin

ND ND ND ND ND ND ND ND 1.63±0.01

0.85±0.02a 0.75±0.01bc 0.78±0.02b 0.74±0.00bc 0.73±0.01c 0.63±0.01d ND ND 0.68±0.01d

ND ND ND ND ND ND ND 1.21±0.02 ND

0.46±0.01d 1.24±0.01a 0.34±0.01f 0.69±0.01c 0.41±0.01e 1.17±0.01b 0.03±0.00g 0.43±0.01de 0.35±0.00f

ND ND ND 0.73 ± 0.01c ND ND ND 6.46 ± 0.07a 1.40 ± 0.03b

19.93±0.26b 20.35±0.24b 15.10±0.09d 26.98±0.07a 17.04±0.12c 14.95±0.02d 13.97±0.05e 6.89±0.06f ND

Neoeriocitrin Narirutin 1.07 ± 0.02c 1.25 ± 0.01b 1.81 ± 0.07a 1.34 ± 0.04b 0.56 ± 0.02e 0.93 ± 0.01c ND ND 0.74 ± 0.01d

16.77±0.23b 16.36±0.20b 5.02±0.10d ND 20.91±0.10a 13.22±0.02c 5.11 ± 0.04d 1.70 ± 0.01e 0.27 ± 0.01f

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are functions of relative levels of TSS, organic acids, and the presence or absence of various aromatic or bitter juice constituents (Davies & Albrigo, 1994). The TSS/TA ratio provides a clear indication of the sweetness of fruits, i.e. the higher the TSS/TA ratio, the sweeter is the fruit. The TSS/TA ratios of Mauritian citrus pulps varied between 1.3 and 23.7 and are comparable to values reported by Xu, Liu, Chen, Ye, Maa and Shi (2008) for 15 citrus varieties cultivated in China which ranged from 1.79 in lemon to 17.09 in Pommelo var Miyou. In the present study, pulps that were characterised by the low TSS/TA ratio (b3) (Table 3) were bergamot, calamondin and lemon indicating that they are less likely to be consumed as fresh fruits but rather in the form of sweetened beverages, jams or pastes. The citrus fruits having TSS/TA ratio greater than 10 are more likely to be consumed as fresh fruits due to their inherent sweetness. This is an important parameter for consideration in the development of functional juices with prophylactic properties as it is instrumental for the determination of the amount of sugar needed to be added for an acceptable product to the consumer. Major efforts have been launched to find therapeutic means to improve the management of health and diseases. Recent data show that supplemental natural antioxidants represent a potential strategy as adjunct therapy. This study provides additional data that citrus pulps contain a host of antioxidant polyphenolics that can potentially protect health. Based on the present data the following citrus fruit pulps can thus be encouraged for consumption as fresh produce or for processing into juices having functional properties to maintain wellness and to manage chronic diseases of overt inflammation in humans: Kumquat Nagami, Tangelo Mineola, Tangelo Ugli, Calamondin, Pamplemousses Kaopan, Tangor Elendale, Mandarin Fairchild and Orange (Washington Navel). 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 Adibelli, Z., Dilek, M., & Akpolat, T. (2009). Lemon juice as an alternative therapy in hypertension in Turkey. International Journal of Cardiology, 135, e58–e59. AOAC (1995a). AOAC official method 981.12. In P. Cunniff (Ed.), AOAC official methods of analysis of AOAC international. USA: AOAC International. AOAC (1995b). AOAC official method 942.15. In P. Cunniff (Ed.), AOAC official methods of analysis of AOAC international. USA: AOAC International. 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. Journal of the Science of Food and Agriculture, 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 J. N. Losso, F. Shahidi, & D. Bagchi (Eds.), Anti-angiogenic functional and medicinal foods (pp. 149–176). New York: CRC Press, Taylor & Francis Group. Benavente-Garcia, O., & Castillo, J. (2008). Update on uses and properties of Citrus flavonoids: New findings in anticancer, cardiovascular, and anti-inflammatory activity. Journal of Agricultural and Food Chemistry, 56, 6185–6205. Benavente-Garcia, O., Castillo, J., Marin, F. R., Ortuno, A., & Del Rio, J. A. (1997). Use and properties of Citrus flavonoids. Journal of Agricultural and Food Chemistry, 45, 4506–4515. Benzie, I. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of ‘antioxidant power’: The FRAP assay. Analytical Biochemistry, 239, 70–76. Berhow, M., Tisserat, Brent, Kanes, K., & Vandercook, C. (1998). Survey of phenolic compounds produced in citrus. Technical bulletin number 1856. : United States Department of Agriculture, Agricultural Research Service Available on: http:// www.ars.usda.gov/IS/np/phenolics.htm

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