Variety and ripening impact on phenolic composition and antioxidant activity of mandarin (Citrus reticulate Blanco) and bitter orange (Citrus aurantium L.) seeds extracts

Industrial Crops and Products 39 (2012) 74–80

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Variety and ripening impact on phenolic composition and antioxidant activity of mandarin (Citrus reticulate Blanco) and bitter orange (Citrus aurantium L.) seeds extracts Ikram Moulehi, Soumaya Bourgou ∗ , Ines Ourghemmi, Moufida Saidani Tounsi Laboratoire des Substances Bioactives Centre de Biotechnologie à la Technopole de Borj-Cedria, Tunisia

a r t i c l e

i n f o

Article history: Received 11 October 2011 Received in revised form 8 February 2012 Accepted 10 February 2012 Keywords: Mandarin Bitter orange Seeds Phenolic composition Antioxidant activity Ripening

a b s t r a c t Changes of antioxidants in the seeds of mandarin (Citrus reticulata) and bitter orange (Citrus aurantium) fruits during three stages of ripening (IM, SM and CM) were evaluated. Comparison between the two varieties showed that Mandarin exhibited the highest total polyphenols and flavonoids contents at IM and SM stages, respectively while bitter orange possesses the highest tannins content at SM stage. A total of 22 phenolic compounds were identified in mandarin and bitter orange seeds, including hydroxybenzoic acids (3), hydroxycinnamic acids (6), flavanones (3), flavanols (2), flavonols (3), flavones (3), simple phenol (1) and coumarin (1). The percentages of phenolic classes were found to vary during ripening which extents depended on the variety. During mandarin ripening, IM, SM and CM stages were characterized respectively by the presence of naringin, hesperidin and gallic acid as the most abundant mandarin compounds. In the case of bitter orange, naringin and neohesperidin were the major compounds during fruit maturation. Antioxidant activity was employed by two complementary test systems namely DPPH free radical scavenging and ␤-carotene/linoleic acid systems. The antioxidant capacity varied during ripening with bitter orange activity being higher than that of mandarin. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Nowadays, antioxidants have gained more importance because of their positive involvement as health promoters. In this context, several epidemiological studies have associated the consumption of phenolic compounds with lower risks of different types of disorders such as cancer (Yang et al., 2011) and cardiovascular diseases (Hooper et al., 2008), Besides, when added to foods, antioxidants minimize rancidity, retard the formation of toxic oxidation products, maintain nutritional quality, and increase shelf life of food products (Jadhav et al., 1996). The genus Citrus encompasses several orange types-sweet and sour oranges, tangerines (mandarins), tangors, and tangelos. Citrus fruits are one of the important horticultural crops, with worldwide agricultural production over 100 million metric tons per year. Also providing an ample supply of vitamin C, folic acid, potassium, and pectin, citrus contains a host of antioxidant phytophenolics that can potentially protect health. In fact, extensive studies focused on the edible portion of citrus showed that juices and extracts posses

∗ Corresponding author. Tel.: +216 71430855; fax: +216 79412638. E-mail address: soumaya [email protected] (S. Bourgou). 0926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2012.02.013

important antioxidant potential and that they represent a significant source of phenolic compounds mainly phenolic acids and flavanones (Rapisarda et al., 1999; Peterson et al., 2006; Gattuso et al., 2007; Wang et al., 2007; Jayaprakasha et al., 2008; Kelebek et al., 2008; Barreca et al., 2010; Ramful et al., 2010). About 80% of the citrus harvest is used by the juice industry. However, during the citrus juice extraction process, large quantities of by-products are produced (55% of the weight products). They are constituted by peels, seeds remaining after juice extraction and are a serious environmental problem for disposal. Thus new aspects concerning the use of these by-products for further exploitation on the production of food additives or supplements with high nutritional value and economically attractive have gained increasingly interest. Recent studies showed that citrus by-products are a source of bioactive compounds; peels are rich on secondary metabolites such as essential oil (Sahraoui et al., 2011) and phenolic compounds (Li et al., 2006; Ortuno et al., 1995; Khan et al., 2010; He et al., 2011). Moreover, citrus pomace has been reported to contain natural antioxidants such as phenolic acids and flavonoids (Kim et al., 2008; Hayat et al., 2010). However, little is known about the bioactive potential of the seeds. Yusof et al. (1990) analysing the content of naringin in a variety of Mexican citrus have detected this flavanone in the seeds of Rough lime. Sun et al. (2010) investigated the

I. Moulehi et al. / Industrial Crops and Products 39 (2012) 74–80

flavonoids composition of the different parts of the Chinese mandarin fruit and identified naringin, hesperidin, didymin, tangeretin and nobiletin in the seeds. In addition, the chemical composition and the antioxidant activity of the whole fruit and by-products of citrus can vary depending on cultivar and ripening stage (Castillo et al., 1993; Sun et al., 2005; Huang et al., 2007). However, such changes were not described in the seeds of mandarin and bitter orange. Thus, the aims of the present work were to investigate the phenolic composition and the antioxidant activities of the seeds of two local citrus cultivars; mandarin and bitter orange and to study the changes of these parameters during fruit maturation and ripening. Hopefully, the results will prove useful for the utilization of phenolics in citrus fruit seeds.

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calibration curve with catechin. The calibration curve range was 0–500 ␮g/mL (R2 = 0.987). Triplicates measurements were taken for all samples. 2.5. Total condensed tannins

2. Materials and methods

Total tannin content was measured using the modified vanillin assay described by Sun et al. (1998). Three millilitres of 4% methanol vanillin solution and 1.5 mL of concentrated H2 SO4 were added to 50 ␮l of suitably diluted sample. The mixture was kept for 15 min, and the absorbance was measured at 500 nm against methanol as a blank. The amount of total condensed tannins was expressed as mg (+)-catechin equivalent per gram of dry weight (␮g CE/g DW) through the calibration curve with catechin. The calibration curve range was 0–400 ␮g/mL (R2 = 0.999). Triplicates measurements were taken for all samples.

2.1. Plant material

2.6. Identification of phenolic compounds using RP-HPLC

Citrus fruits used in this study originate from Menzel Bouzelfa in the North East of Tunisia (latitude 36◦ 42 13.17 ; longitude 10◦ 29 46.93 ). Fruits of bitter orange (Citrus aurantium) and mandarin (Citrus reticulata Blanco) were harvested at three stages of ripening according fruit colour changes: immature, IM (green); semimature, SM (yellow); and commercial mature, CM (orange). These Citrus fruits were used for juice extraction and the seeds of every species of Citrus were separated from the waste product. The seeds were washed and dried at ambient temperature in the dark until used.

The methanol extracts were injected to RP-HPLC. The phenolic compound analysis was carried out using an Agilent Technologies 1100 series liquid chromatograph (RP-HPLC) coupled with an UV–Vis multiwavelength detector. The separation was carried out on a 250 × 4.6-mm, 4-␮m Hypersil ODS C18 reversed phase column at ambient temperature. The mobile phase consisted of acetonitrile (solvent A) and water with 0.2% sulphuric acid (solvent B). The flow rate was kept at 0.5 ml/min. The gradient programme was as follows: 15%A/85%B 0–12 min, 40%A/60% B 12–14 min, 60%A/40%B 14–18 min, 80%A/20% B 18–20 min, 90%A/10%B 20–24 min, 100% A 24–28 min (Bourgou et al., 2008). The injection volume was 20 ␮l, and peaks were monitored at 280 nm. Samples were filtered through a 0.45 ␮m membrane filter before injection. Phenolic compounds were identified according to their retention times and spectral characteristics of their peaks against those of standards, as well as by spiking the sample with standards. Analyses were performed in triplicate.

2.2. Polyphenol extraction The seeds were finely ground with a type A10 blade-carbide gringing (Ika-Werk, Staufen, Germany). 3 g of this ground material was extracted by stirring with 30 mL of pure methanol for 30 min. The extracts were then kept for 24 h at 4 ◦ C, filtered through a Whatman No. 4 filter paper to obtain a 25 ml final volume, evaporated under vacuum to dryness and stored at 4 ◦ C until analyzed. 2.3. Total polyphenols content Total phenolics content was assayed using the Folin–Ciocalteu reagent, following Singleton’s method slightly modified by Dewanto et al. (2002). An aliquot (0.125 mL) of a suitable diluted acetone sample was added to 0.5 mL of deionized water and 0.125 mL of the Folin–Ciocalteu reagent. The mixture was shaken and allowed to stand for 6 min, before adding 1.25 mL of 7% sodium carbonate (Na2 CO3 ) solution. The solution was then adjusted with deionized water to a final volume of 3 mL and mixed thoroughly. After incubation for 90 min at 23 ◦ C, the absorbance versus prepared blank was read at 760 nm. Total phenolic contents were expressed as mg gallic acid equivalents per gram of dry weight (␮g GAE/g DW) through the calibration curve with gallic acid. The calibration curve range was 0–400 ␮g/mL (R2 = 0.99). Triplicate measurements were taken for all samples.

2.7. Antioxidant activity assays 2.7.1. DPPH assay The electron donation capacity of the obtained extracts was measured by bleaching of the purple-coloured solution of 1,1diphenyl-2-picrylhydrazyl radical (DPPH) according to the method of Hanato et al. (1998). 1 mL of different concentrations of extracts was added to 0.5 mL of a 0.2 mmol/l DPPH methanolic solution. The mixture was shaken vigorously and kept at room temperature for 30 min. The absorbance of the resulting solution was then measured at 517 nm after 30 min. The antiradical activity was expressed as IC50 (␮g/mL), the concentration required to cause a 50% DPPH inhibition. The ability to scavenge the DPPH radical was calculated using the following equation: DPPH scavenging effect (%) =

A − A  0 1 A0

× 100

2.4. Flavonoids content

where A0 is the absorbance of the control at 30 min, and A1 is the absorbance of the sample at 30 min. BHT was used as a positive control. Samples were analyzed in triplicate.

Total flavonoid content was measured according to Dewanto et al. (2002). 250 ␮l of the sample appropriately diluted was mixed with 75 ␮l NaNO2 (sodium nitrite, 5%). After 6 min, 150 ␮l of 10% aluminium chloride (AlCl3 ) and 500 ␮l of NaOH (1 M) were added to the mixture. Finally, the mixture was adjusted to 2.5 mL with distilled water. The absorbance versus prepared blank was read at 510 nm. Total flavonoid content was expressed as mg catechin equivalents per gram of dry weight (␮g CE/g DW) through the

2.7.2. ˇ-Carotene bleaching test A modified method described by Koleva et al. (2002) was employed. ␤-Carotene (2 mg) was dissolved in 20 mL chloroform. Then, 4 mL of this solution were added to linoleic acid (40 mg) and Tween 40 (400 mg). Chloroform was evaporated under vacuum at 40 ◦ C and 100 mL of oxygenated ultra-pure water was added, then the emulsion was vigorously shaken. The emulsion (3 mL) was added to a tube containing 0.2 mL of different concentrations of

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mandarin

2,5

bitter orange

mandarin 3,0

a

a

2,5

1,5

a b

b

1,0

c

c

mg Eq Cat/ g DW

mg Eq Gallic acid/g DW

2,0

bitter orange

2,0

b

b

a

a

b

1,5 1,0 0,5

0,5

0,0

IM

SM

CM

IM

SM

CM

0,0 IM

SM

CM

IM

SM

CM

Fig. 1. Total polyphenols content of methanol extracts of Citrus seeds during ripening. Means of three replicates (values with different superscripts are significantly different at p < 0.05).

Fig. 2. Total flavonoids content of methanol extracts of Citrus seeds during ripening. Means of three replicates (values with different superscripts are significantly different at p < 0.05).

3.2. Total flavonoids content

3. Results and discussion

The amounts of total flavonoids in the seeds the two citrus varieties are shown in Fig. 2. As for polyphenols content, flavonoids accumulation pattern during fruit ripening was different between the two varieties. Indeed, the highest value was reached in the semimature stage for the two varieties but it was altered after that in mandarin while it remained stable in bitter orange. Independently to repining stage, the flavonoids content ranged from 1.31 to 2.52 mg CE/g DW. We noticed that mandarin was richer than bitter orange in flavonoids. As for polyphenols, data on the seeds total flavonoids content of citrus are absent. Concerning other citrus by-products, Ramful et al. (2010) found that the total flavonoids content of the peel of Mauritian mandarin varied according to the studied variety and was classified as low (<2 mg/g FW) or high (>3.6 mg/g FW). In addition, Guimarães et al. (2010) reported that the peel polar fractions obtained from grapefruit, lemon, lime and sweet orange were rich on total flavonoids varying from 0.32 to 1.69 mg CE/g extract.

3.1. Total polyphenols content

3.3. Total tannins content

It is believed that major components of antioxidant activity in edible plants are polyphenolic compounds. Thus, it is necessary to extract polyphenolic compounds effectively when antioxidant activities are measured. In this context, we chose methanol as solvent to extract the bioactive compounds from citrus varieties since it is well known to be an effective solvent for antioxidants extraction (Siddhuraju and Becker, 2003). The total polyphenols (TP) content of two Tunisian citrus varieties harvested at three ripening stages (immature, IM; semimature, SM; and commercial mature, CM) is presented in Fig. 1 and was found to vary from 0.68 to 2.11 mg EGA/g DW with mandarin exhibiting higher content than bitter orange. The changes of TP content from the two varieties showed different trends during ripening; it was reduced during ripening for mandarin with the highest content reached at first ripening stage (immature) while it increased during ripening for bitter orange to reach 1.35 mg GAE/g DW at CM stage. Data on the total polyphenols extracted from citrus seeds or not available, however, the obtained TP contents in our study were higher than that reported by Garau et al. (2007) for other bitter orange and mandarin by-products. In fact, these authors showed that peels of bitter orange and mandarin contained a total phenolic content of 0.51 and 0.61 mg EGA/100 g DM, respectively. Nevertheless, Ramful et al. (2010) reported high total phenolic content in the peel of four Mauritian mandarin varieties, varying from 2.65 to 6.92 mg/g fresh weight. Such differences with our study could be due to the effect of several factors including origin, climatic conditions and practical conditions as well as the chosen cultivar and organ (Amaral et al., 2010).

As it is shown in Fig. 3, the tannin content in citrus varieties ranged from 0.12 to 0.37 mg CE/g DW with bitter orange being richer than mandarin. The effect of repining stage was not significant on C. reticulate tannin content which remained stable with a value about 0.12 mg CE/g DW. However, the bitter orange tannin content varied significantly during the repining stages, in fact, the highest value was reached in the semimature stage and was slightly reduced in the mature stage while the smallest value was found at the immature stage. Independently to ripening stage, the found tannin contents were higher than that reported in the literature for

% Inhibition =

At − Ct × 100 C0 − Ct

where At and Ct are the absorbance values measured for the test sample and control, respectively, after incubation for 120 min, and C0 is the absorbance values for the control measured at zero time during the incubation. The results are expressed as IC50 values (mg/mL), the concentration required to cause a 50% ␤-carotene bleaching inhibition.

mandarin

0,5

bitter orange a

0,4 mg Eq Cat/g DW

extract. The absorbance was immediately measured at 470 nm and the test emulsion was incubated in a water bath at 50 ◦ C for 120 min, when the absorbance was measured again. In the negative control, the extract was substituted with an equal volume of methanol. The antioxidant activity (%) was evaluated in terms of the bleaching of the ␤-carotene using the following formula:

b

0,3 c 0,2

a

a

IM

SM

a

0,1 0,0 CM

IM

SM

CM

Fig. 3. Total tannins content of methanol extracts of Citrus seeds during ripening. Means of three replicates (values with different superscripts are significantly different at p < 0.05).

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Table 1 Variation of phenolic composition during ripening in mandarin. Compounds

IM

SM

CM

Flavonoids Epigallocatechin Catechin Rutin Naringin Hesperidin Quercetin Amentoflavone Flavone Phenolic acids Gallic acid Cafeic acid Chlorogenic acid Vanillic acid Syringic acid Ferulic acid Rosmarinic acid trans-2-Hydroxicinnamic acid NI

29.26 ± 1.05b 1.27 ± 0.5a 6.07 ± 0.75a 0.88 ± 0.03b 10.87 ± 1.22a 2.83 ± 0.25b 3.62 ± 0.03a 0.68 ± 0.02a 3.04 ± 0.03a 41.39 ± 2.45b 19.01 ± 2.33b 4.76 ± 0.21a 3.71 ± 0.66a 0.26 ± 0.01b 0.42 ± 0.02a 2.35 ± 0.32b 0.68 ± 0.01a 10.20 ± 1.11a 29.35

36.21 ± 1.52a 3.08 ± 0.05a 7.92 ± 0.90a 2.96 ± 0.25a 0.45 ± 0.01b 16.76 ± 2.33a 0.41 ± 0.01b 0.56 ± 0.01a 4.07 ± 0.75a 25.32 ± 0.33c 6.72 ± 0.03c 1.68 ± 0.02b 2.64 ± 0.03a 1.61 ± 0.15a 0.75 ± 0.04a 5.05 ± 0.45a 0.90 ± 0.03a 5.97 ± 0.12b 38.47

6.69 ± 0.05c ND ND 2.50 ± 0.02 a 0.73 ± 0.03 b 0.15 ± 0.01b 0.76 ± 0.02b 0.95 ± 0.03a 1.6 ± 0.05b 71.39 ± 2.73a 60.91 ± 2.77a ND ND ND ND 1.84 ± 0.02c ND 8.64 ± 1.11a 21.92

Values (means of three replicates ± SD) with different superscripts are significantly different at p < 0.05).

other Rutaceae species such as Newbouldia laevis and Zanthoxylum zanthoxyloïdes (1.18 and 3.19 equiv. g tannic acid/100 g DW, Azando et al., 2011). 3.4. Identification of phenolic compounds Polyphenol qualitative determination of the Citrus seeds was performed by RP-HPLC analysis. As it is shown in Table 1, mandarin was rich on both flavonoids and phenolic acids during the two first ripening stages. In fact, the two classes were present at 29.26 and 41.39%, respectively during IS stage and 36.21 and 25.32%, respectively during SM stage. Nevertheless, at the end of the ripening, mandarin phenolic composition became dominated by phenolic acids (71.39% of the phenolic compounds) while flavonoids were present with a low percentage (6.69% of the phenolic compounds). Differently to mandarin, the phenolic composition of bitter orange was dominated by the flavonoids as major classes during ripening; this classes represented 49.82, 56.95 and 55.97% of the total compounds at IS, SM and CM stages, respectively. Phenolic acids were present at a moderate level; about 20% at IS and SM stages and

decreased to 16.80% at CM stage (Table 2). Thus, flavonoids and phenolic acids displayed opposite trend during ripening for both Citrus varieties which is in accordance with their biosynthetic route where all flavonoids derived from the chalcone which results from the condensation of malonyl CoA with p-coumaroyl CoA. The latter p-coumaroyl CoA is supplied from the phenylpropanoid pathway, which is responsible for phenolic acids biosynthesis (Macheix et al., 2005). Analysis of seeds individual compounds leads to the identification of 16 compounds in mandarin including eight flavonoids, three benzoic acids and 5 hydroxycinnamic acids and 18 compounds in bitter orange including 11 flavonoids, 3 benzoic acids, 3 hydroxycinnamic acids and one coumarin (Tables 1 and 2). Moreover, results indicated that the phenolic composition was stage-dependant in both varieties. Indeed, in the case of mandarin, gallic acid was the most represented compound (19.01%) at IS stage followed closely by naringin and trans-2-hydroxicinnamic acid (10.78 and 10.20%, respectively). However, the levels of these compounds were lowered during SM stage while hesperidin increased to become the major compound (16.76%). The mature fruit was

Table 2 Variation of phenolic composition during ripening in bitter orange. Compounds

IM

SM

CM

Flavonoids Epigallocatechin Naringin Hesperidin Neohesperidin Naphtorecinol Apigenin Quercetin Resorcinol Catechin Rutin Kaempherol Phenolic acids Gallic acid Vanillic acid Syringic acid Rosmarinic acid p-Coumaric acid trans-2-Hydroxicinnamic acid Coumarin NI

49.82 ± 1.43a 3.05 ± 0.5a 13.29 ± 0.78c 4.14 ± 0.35b 14.59 ± 1.33a 0.19 ± 0.02a 0.55 ± 0.01b 0.73 ± 0.03a 2.43 ± 0.05a 4.05 ± 0.45a 3.40 ± 0.25c 3.40 ± 0.05b 21.64 ± 0.22a 5.23 ± 0.20a 0.62 ± 0.10a 1.60 ± 0.11a 8.18 ± 0.15a 3.35 ± 0.12a 2.66 ± 0.45a 0.096 ± 0.01a 28.44 ± 2.33

56.95 ± 1.21a 1.62 ± 0.05b 15.03 ± 1.11b 7.03 ± 0.33a 9.40 ± 0.35c 0.19 ± 0.03a 1.3 ± 0.01a 0.10 ± 0.02a 2.70 ± 0.13a 4.91 ± 0.15a 5.52 ± 0.55 b 9.15 ± 0.35a 19.09 ± 0.43a 4.77 ± 0.33a 1.08 ± 0.32a 1.22 ± 0.02a 4.53 ± 0.15b 4.09 ± 0.14a 3.40 ± 0.66a 0.072 ± 0.01a 23.89 ± 3.22

55.97 ± 1.25a 1.81 ± 0.11b 19.29 ± 1.15a 3.48 ± 0.03b 12.37 ± 0.45b ND 0.55 ± 0.01b 0.06 ± 0.01b 1.80 ± 0.03b 0.91 ± 0.05b 7.34 ± 0.75a 8.36 ± 0.55a 16.80 ± 0.25a 4.60 ± 0.20a 1.08 ± 0.02a 1.65 ± 0.03a 4.13 ± 0.33b 3.13 ± 0.05a 2.21 ± 0.02a ND 27.23 ± 2.11

Values (means of three replicates ± SD) with different superscripts are significantly different at p < 0.05).

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mainly marked by an important increase of gallic acid level which reached 60.91% while the rest of the compounds were lowly represented except for trans-2-hydroxicinnamic acid present with a moderate percentage of 8.64% (Table 1). The only results available from other authors on the phenolic composition of mandarin seeds were focused on the analysis of the glycosylated flavanones content only; Bocco et al. (1998) detected hesperidin, neoeriocitrin, narirutin and naringin in the seeds of mandarin from South Africa while recently Sun et al. (2010) detected naringin, hesperidin, didymin, tangeretin and nobiletin, in the seeds of the Chinese mandarin. The two authors reported that naringin and hesperidin were the major flavonoids. On the other hand, richness of the by-product pomaces of two citrus mandarin on phenolic acids was reported by Hayat et al. (2010). These authors identified five phenolic acids including gallic, p-hydroxybenzoic, vanillic, p-coumaric and ferulic acids. Concerning bitter orange, 18 compounds were identified and the extracts were characterized by the predominance of the flavanones glycosides (naringin, hesperidin and neohesperidin) which represented 32.02, 31.46 and 35.14% of the analysed compounds at IM, SM and CM stages, respectively (Table 2). Naringin and neohesperidin were found to be the major compounds during ripening with the highest neohesperidin level found at the immature stage (14.59%) while the highest naringin level was reached at maturity (19.29%, Table 2). Our results concerning the mature stage are in accordance with those of Bocco et al. (1998) how demonstrated that bitter orange seeds were characterized by naringin and neophesperidin as the major free phenolics. Moreover, Castillo et al. (1993) analysed the level of these favanones in different bitter orange organs during the development and showed that neohesperidin and naringin levels were higher in the first stages of fruit development. Such difference with our results could be due to that these authors studied the phenolic composition of the whole fruit, not only the seed fraction. The relative levels of these molecules were reported to be highly characteristic of a given organ and are markedly affected by the age of the developing organ (Castillo et al., 1993). Naringin, hesperidin and neohesperidin are flavanones glycosides and are known to accumulate specifically in Citrus species. They have different sugar moieties which influence taste; naringin and neohesperidin are neohesperidosides with a bitter taste due to the sugar neohesperidose while the sugar rutinose causes the hesperidin to have a neutral taste (Peterson et al., 2006). Our results showed that the bitter orange extracts contained also appreciable levels of flavanols (catechin and epigallocatechin) and flavonols (querectin, rutin and kaempferol) with the highest total amount reached at the IS stage (7.10%) for flavanols and CM stage for flavonols (15.76%, Table 2). Moreover, hydroxybenzoic acids (gallic, vanillic and syringic acids) and hydroxycinnamic acids (p-coumaric, rosmarinic, trans-2-hyroxycinnamic acids) were also present with appreciable total levels varying from 7.07 to 14.19% during ripening (Table 2). Differently to us, Bocco et al. (1998) identified caffeic, p-coumaric, ferulic and sinapinic acids as the main phenolics in the bitter orange seed extract which were not found in our samples except for p-coumaric acid. The difference could be due to the fact that the authors used alkaline hydrolysis to release the phenolic acids (Bocco et al., 1998) since they were present in bound form in the extract. Moreover, hydrolysis conditions were reported to affect significantly the phenolic acids which can be degraded (Kim et al., 2006). This could further explain the absence of the phenolic acids identified in our study in the extracts obtained by Bocco et al. (1998). 3.5. Antioxidant activity Total antioxidant activities of the plant extracts cannot be evaluated by any single method; two or more methods should always

Table 3 Antiradical (DPPH) activity of mandarin and bitter orange extracts during ripening. IC50 (␮g/ml)

Mandarin Bitter orange

IM

SM

CM

577.00 ± 7.92a 303.33 ± 23.56a

279.33 ± 11.89b 246.67 ± 13.07ab

210 ± 37.0c 188.67 ± 58.16b

Values (means of three replicates ± SD) with different superscripts are significantly different at p < 0.05).

be employed in order to evaluate the total antioxidative effects of vegetables (Nuutila et al., 2003). Thus, we applied two complementary test systems namely ␤-carotene–linoleic acid and DPPH for evaluating the antioxidant capacities of the extracts studied here. Variations of antioxidant activities were found during ripening of citrus fruits as Tables 3 and 4 show. DPPH radical scavenging activity showed similar trends for both varieties and was found to increase during ripening. The highest antioxidant capacity was reached at maturity with IC50 values of 210.00 and 188.70 ␮g/ml for mandarin and bitter orange, respectively (Table 3). However, significant difference on antiradical activity was found among the two varieties. In fact, bitter orange exhibited about twice higher activity than bitter orange. In the case of linoleic system, oxidation of linoleic acid was inhibited by both varieties (Table 4) and as for DPPH assay, bitter orange exhibited higher antioxidant capacity than mandarin. Moreover, the antioxidant activity varied during ripening; mandarin showed the highest capacity during the mature stage (IC50 value of 3.65 mg/ml) while bitter orange reached the highest values at semi mature stage (IC50 value of 1.80 mg/ml). On the other hand, our study demonstrated that the antioxidant activities of bitter orange were correlated with the polyphenols (r = −0.76 and −0.82, for DPPH and linoleic acid inhibition, respectively) and flavonoids (r = −0.90 and −0.80 for DPPH and linoleic acid inhibition, respectively) contents, however, such correlations were not found in the case of mandarin. Generally, a positive correlation between the phenolic content and antioxidant capacity is reported (Maisuthisakul et al., 2007) but recently it has been shown that the antioxidant activity of extracts is roughly connected to their phenolic composition and strongly depends upon their phenolic structures (Kwee and Niemeyer, 2011). Our RP-HPLC analysis showed that the phenolic composition of mandarin was characterized by the presence of high level hesperidin at the middle stage (Table 1, Fig. 4). It has been shown that hesperidin is able to inhibit lipid peroxidation since it has been reported to act as a powerful superoxide, singlet oxygen and hydroxyl radicals scavengers and to react with peroxy radicals involving termination of radical chain reactions (Torel et al., 1986; Pradeep et al., 2008). On the other hand, the increase of the antiradical activity at CM is correlated to a significant increase of gallic acid level (Table 1). This phenolic acid has been reported as an efficient DPPH antiradical compound which may be attributed to its proton-donating ability (Zhou et al., 2006). This compound is characterized by the presence of galloyl moiety (three –OH groups) in the B ring (Fig. 4) which contribute to the high scavenging reactive oxygen species (ROS) activity (Villano Table 4 ␤-Carotene bleaching capacity of mandarin and bitter orange extracts during ripening. IC50 (mg/ml)

Mandarin Bitter orange

IM

SM

CM

4.90 ± 0.79a 2.93 ± 0.60a

4.33 ± 0.14ab 1.80 ± 0.22b

3.65 ± 0.73b 1.91 ± 0.02b

Values (means of three replicates ± SD) with different superscripts are significantly different at p < 0.05).

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Fig. 4. Majors compounds of mandarin and bitter orange seeds. Source: Structures from Harborne and Baxter (1999).

et al., 2007). In the case of bitter orange, the increase of antioxidant activities during ripening could be related to the enhancement of naringin and kampferol levels as well as to the high level of neohesperidin (Table 2). Neohesperidin and naringin has been reported to exhibit antioxidant and radical scavenger property and to offer protection against lipid peroxidation which is linked to the presence of effective antioxidant structures (Fig. 4) including hydroxylation and C2–C3 double bond in conjugation with a 4-oxo function (Di Majo et al., 2005; Hamdan et al., 2011). Moreover, kampferol could participate to the antioxidant activities of bitter orange since it is a powerful antioxidant compound (Yang et al., 2009).

4. Conclusion Significant variation in total polyphenols, flavonoids and tannins contents as well as antioxidant activity between citrus seeds varieties and during ripening, indicates that the potential efficacy of antioxidants vary considerably with both variety as well maturity stage. The findings from this study indicated also that the seeds of mandarin and bitter orange are considered valuable, as they provide components with potential for industrial and pharmacological applications as antioxidants. Moreover, the significant changes of the phenolic composition during ripening suggest that Mandarin seeds are a promising source for the extractions of gallic acid at the maturity (CM stage) while bitter orange seeds could be used as a potential source of neohesperidin at immature stage and naringin at maturity.

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