Food Chemistry 120 (2010) 10191024

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Food Chemistry
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Antioxidant compounds and antioxidant capacity of Peruvian camu camu (Myrciaria dubia (H.B.K.) McVaugh) fruit at different maturity stages
Rosana Chirinos, Jorge Galarza, Indira Betalleluz-Pallardel, Romina Pedreschi 1, David Campos *
Instituto de Biotecnologa (IBT), Universidad Nacional Agraria La Molina UNALM, Av. La Molina s/n, Lima, Peru

a r t i c l e

i n f o

a b s t r a c t
The antioxidant capacities of ascorbic acid and phenolic compounds present in camu camu fruit were screened during ripening. Ascorbic acid decreased, and anthocyanin, avonol and avanol contents, and DPPH antioxidant capacity increased during ripening. Antioxidant compounds from camu camu were fractionated in two fractions: an ascorbic acid-rich fraction (F-I) and a phenolics-rich fraction (F-II). F-I was the major contributor to the DPPH antioxidant capacity (67.579.3%) and F-II played a minor role (20.732.5%). A total of 30 different phenolic compounds were detected by HPLC-PAD. The presence of catechin, delphinidin 3-glucoside, cyanidin 3-glucoside, ellagic acid and rutin was elucidated. Other phenolic compounds, such as avan-3-ol, avonol, avanone and ellagic acid derivatives, were also present. For the three ripening stages the avan-3-ols and ellagic acid group were the most representative phenolic compounds in this fruit. Acid hydrolysis of F-II revealed the presence mainly of gallic and ellagic acids, suggesting that camu camu fruit possesses important quantities of hydrolysed tannins (galloand/or ellagitannins). These results conrm that camu camu fruit is a promising source of antioxidant phenolics. 2009 Elsevier Ltd. All rights reserved.

Article history: Received 22 June 2009 Received in revised form 17 November 2009 Accepted 20 November 2009

Keywords: Camu camu Myrciaria dubia HPLC-PAD Phenolic compounds Ascorbic acid Maturity stages Antioxidant capacity

1. Introduction Phenolic compounds are widely distributed in plants and in recent years they have gained much attention, due to their antioxidant activity and free radical-scavenging ability with potential benecial implications in human health (Ross & Kasum, 2002). Plant phenolics comprise a great diversity of compounds which can be classied into different groups, based on the number of phenol rings that they contain and on the structural elements that bind these rings to one another (Manach, Scalbert, Morand, Rmsy, & Jimnez, 2004). Distinctions are thus made between phenolic acids (hydroxybenzoic and hydroxycinnamic acids), stilbenes, lignins and avonoids (anthocyanins, avan-3-ols, avones, and avonols, among others). Flavan-3-ols can be found as oligomers and polymers and are known as condensed tannins (Tsao & Deng, 2004). The hydrolysable tannins are a group of phenolics that possess a sugar esteried with gallic acid (gallotannins) or with ellagic acid (ellagitannins) (Khanbabaee & van Ree, 2001). Camu camu is a low-growing shrub found throughout the Amazon rainforest of Colombia, Venezuela, Peru and Brazil that belongs to the Myrtaceae family (IIAP, 2001). Round, red-coloured berry fruits of 2.5-cm diameter with a strong acid taste are produced

* Corresponding author. Tel./fax: +51 1 3495764. E-mail address: dcampos@lamolina.edu.pe (D. Campos). 1 Present address: Food Safety and Quality Unit, Institute for Reference Materials and Measurements, Joint Research Centre, European Union, Geel, Belgium. 0308-8146/$ - see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2009.11.041

(Zapata & Dufour, 1993). Camu camu fruit is an important source of vitamin C with values within the 950 g/kg range (Rodrigues, Menezes, Cabral, Dornier, & Reynes, 2001). Its high vitamin C content has created a demand for this fruit in the natural product market Thus, today camu camu derivatives such as pulp, extract and juice have as main export markets Japan and the EU. Anthocyanins have also been identied and studied in camu camu. Cyanidin 3-glucoside was identied as the major anthocyanin in this fruit, followed by delphinidin 3-glucoside (Zanatta, Cuevas, Bobbio, Winterhalter & Mercadante, 2005). Fruits of the Myrtaceae family have a signicant use history as edible and as traditional medicines in divergent ethnobotanical practices throughout the tropical and subtropical world. For instance, it has been reported that the fruit of three other species in this group (Eugenia foetida, Eugenia uniora and Myrciaria cauliora) displayed a strong antioxidant capacity, using the 1,1-diphenyl-2-picrylhydrazyl chemical (DPPH) assay, due to the presence of antioxidant compounds (Reynertson, Basile, & Kenelly, 2005), such as ascorbic acid, anthocyanins and other phenolic compounds. A study conducted by Ueda et al. (2004) revealed the presence of ellagic acid and its two derivatives, 4-O-methylellagic acid and 4-(a-rhamnopyranosyl)ellagic acid, from leaves of Myrciaria dubia (H.B.K.) McVaugh. Reynertson, Yang, Jiang, Basile and Kenelly (2008), in addition, investigated the main phenolic compounds present in 14 underutilised Myrtaceae fruits, including M. dubia. The presence of cyanidin 3-glucoside, ellagic acid, quercetin, and rutin was conrmed.

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When immature, camu camu fruit displays a green colour that changes during the ripening process to red and purple, due to the presence of anthocyanins (Zanatta et al., 2005). Changes in contents and proles of other phytochemicals, such as vitamin C, phenolic compounds and carotenoids are also evident. Scientic information regarding the changes in antioxidant compounds at different maturity stages is scarce to our knowledge. Thus, the objectives of this study were (1), to evaluate changes in antioxidant capacity and total phenolic contents of camu camu (M. dubia (H.B.K.) McVaugh) fruit at three maturity stages, and (2), to evaluate the changes in the main phenolic compounds present in camu camu fruit at three maturity stages, using high-performance liquid chromatography (HPLC) coupled with a photodiode array detector (PAD). 2. Materials and methods 2.1. Sample material, standards and reagents Camu camu fruits from Iquitos were purchased from a local market in Lima (Peru). The fruit was sorted into three maturity stages according to external colour: full green (initial stage), greenreddish (semi-ripe stage) and red (ripe stage). The soluble solids acidity ratios for the three ripening stages were 4.28, 3.44 and 2.57, respectively. These ratios were determined according to the AOAC (1998) method. Approximately 3.0 kg of fruit per each maturity stage were obtained. In addition the fruit was washed, dried and then stored at 20 C until analysed. Trolox (6-hydroxy-2,5,7,8-tetramethyl chroman-2-carboxylic acid), FolinCiocalteu reagent (2N), p-dimethylaminocinnamaldehyde (DMACA), potassium dihydrogen phosphate, hexadecyltrimethylammonium bromide and 1,2-phenylenediamine (OPDA) were purchased from Sigma Chemicals Co. (St. Louis, MO). Phenolic acids (p-coumaric, o-coumaric, protocatechuic, ferulic, gallic, caffeic, chlorogenic, p-hydroxybenzoic), avonols (quercetin, rutin, myricetin, kaempherol), avones (chrysin) and avanones (naringenin, eriodictyol) were purchased from Sigma Chemicals Co. (St. Louis, MO). Flavan-3-ols (catechin, epicatechin, epicatechin gallate, epigallocatechin, epigallocatechin gallate) and procyanidins (procyanidin B1 and B2) were purchased from ChromaDex (Santa Clara, CA). Anthocyanin standards: delphinidin 3-glucoside, delphinidin 3-rutinoside, cyanidin 3-glucoside, cyanidin 3-rutinoside and peonidin 3-glucoside were purchased from Extrasynthse (Genay, France). Anthocyanin aglycone standards: delphinidin, cyanidin, peonidin, pelargonidin and malvidin were purchased from ChromaDex. HPLC-grade acetonitrile and other solvents and reagents were purchased from Merck (Darmstadt, Germany). 2.2. Sample preparation Fifty grams of camu camu edible portion (peel and esh) were mixed in a blender with 200 ml of 80% (v/v) methanol in a ask wrapped with aluminium foil. This mixture was homogenised for 90 s and ushed with nitrogen for 3 min. The ask was allowed to stand at 4 C for 24 h under agitation (200 rpm). Then, the extract was centrifuged at 4000 rpm for 20 min at 4 C and the supernatant was collected. The supernatant was evaporated in a rotary evaporator at 39 C and the residue was diluted with 25 ml of acidied water (0.01% HCl, v/v). The resulting extract is referred to as crude extract (CE). Twenty-ve millilitres of CE were passed through a C18 Sep-Pak cartridge (10 g, 35 ml; Waters, Milford, MA) (Chirinos et al., 2008; Zheng & Wang, 2003), previously activated with methanol and acidied water (0.01% HCl). Phenolic compounds were absorbed onto the column. Sugars, acids, and other polar compounds were eluted with 70 ml of acidied water (0.01% HCl). The resulting fraction is referred to as F-I. Phenolic compounds were then eluted with 100 ml of acidied methanol

(0.01% HCl). Methanol was then removed under vacuum (39 C) and the residue was redissolved in 20 ml of Milli-Q water. This resulting extract is referred to as puried extract (F-II). Ten millilitres of F-II were then lyophilised. At a later stage, a part of the lyophilised F-II was resuspended in HPLC grade methanol for HPLC-PAD analysis. Ten millilitres of aqueous F-II were used for the determination of antioxidant capacity and phenolic compounds. Extractions and purication procedures were performed in triplicate at each maturity stage. 2.3. Acid hydrolysis of camu camu phenolic compounds F-II was acid-hydrolysed following the protocol proposed by Hertog, Holman, and Venema (1992). Twenty milligrams of dried F-II were hydrolysed using 5 ml of a 50% acidied methanol solution (1.2 M HCl) for 2 h at 90 C. The solution was then adjusted to a nal volume of 15 ml with 50% methanol and was cooled to room temperature and then analysed by HPLC-PAD. The purpose of this step was to release sugar molecules from the phenolic compounds and to elucidate the main aglycone structures of camu camu phenolic compounds. 2.4. HPLC-PAD analysis of phenolic compounds Phenolics proles were determined according to the procedure proposed by Chirinos et al. (2008). F-II was treated either with or without an acid solution, as described above, and the compounds were separated on a reversed-phase HPLC column on a Waters 2695 Separation Module (Waters) equipped with an autoinjector, a 996 photodiode array detector (PAD) and Empower software. Spectral data were recorded from 200 to 700 nm during the whole run. An X-terra RP18 (5 lm, 250 4.6 mm) column (Waters) and a 4.6 2.0 mm guard column were used for phenolic separation at 30 C. The mobile phase was composed of solvent (A) water: acetic acid (94:6, v/v, pH 2.27) and solvent (B) acetonitrile. The solvent gradient was as follows: 0% to 15% B in 40 min, 15% to 45% B in 45 min, and 45% to 100% B in 10 min. A ow rate of 0.5 ml/min was used and 20 ll of sample were injected. Samples and mobile phases were ltered through a 0.22-lm Millipore lter, Type GV (Millipore, Bedford, MA) prior to HPLC injection. Phenolic compounds were identied and quantied by comparing their retention time and UVvisible spectral data with known previously injected standards. 2.5. Antioxidant capacity assay Antioxidant capacity was determined by the DPPH procedure proposed by Brand-Williams, Cubelier and Berset (1995). Absorbance was measured at 515 nm. The antioxidant capacity was calculated as lmol of Trolox equivalents (TE) per gram of fresh weight (FW) from a Trolox standard curve. 2.6. Total phenolics (TP), total anthocyanins (TA) total avanoids (TFA) and total avonoids (TFO) assays TP contents in camu camu extracts were determined with the FolinCiocalteu reagent, as described by Singleton and Rossi (1965), using gallic acid (GAE) as a standard. Absorbance was measured at 755 nm and the results were expressed as mg GAE per 100 g FW. TA contents were measured using the pH differential method (Giusti & Wrolstad, 2001). Absorbance was measured at 520 and 700 nm in pH 1.0 and 4.5 buffers. A molar extinction coefcient of 26, 900 l/cm/mol and a molecular weight of 449.2 were used for anthocyanin calculation. Results were expressed as mg of cyanidin 3-glucoside equivalents (CGE) per 100 g FW. TFA contents were estimated in the camu camu extracts, using the

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chromogen DMACA reagent, following the method proposed by Delcour and Devarebeke (1985), using catechin as standard. Absorbance was measured at 640 nm and results were expressed in mg of catechin equivalents (CE) per 100 g FW. The quantication of TFO (compounds containing avonol and avone families) was carried out by means of the aluminium chloride colorimetric method (Chang, Yang, wen, & Chern, 2002). Results were expressed as mg of quercetin equivalents (QE) per 100 g FW. 2.7. Ascorbic and dehydroascorbic acid assay The ascorbic (AA) and dehydroascorbic acid (DHA) contents in camu camu were estimated according to the method proposed by Zapata and Dufour (1992). Approximately 20 g of edible portion were homogenised in a vortex for 90 s and then rapidly ltered (Whatman No.1) and centrifuged at 10,000 rpm for 5 min at 4 C. The supernatant was recovered and then passed through a C18 Sep-Pak cartridge (3 g, 6 ml; Waters) previously activated with methanol and Milli-Q water, and eluted with 10 ml of methanol followed by 10 ml of Milli-Q water. The rst 5 ml of the eluted solution were discarded and the following 3 ml were recovered. The recovered ascorbic and dehydroascorbic solution was mixed with 1 ml of OPDA solution (332.7 mg/100 ml) and ltered through a 0.22-lm syringe lter. The mixture was allowed to stand at room temperature for 37 min in darkness and then analysed by HPLCPAD. Twenty microlitres of the solution were injected onto a Terra RP18 (5 lm, 250 4.6 mm) column (Waters) and a 4.6 2.0 mm guard column. The mobile phase was methanol/water (5:95, v/v) containing 50 mM potassium dihydrogen phosphate (pH 4.6) and 5 mM hexadecyltrimethylammonium bromide (cetrimide). The mobile phase was ltered through a 0.22-lm Millipore lter, type GV, prior to HPLC analysis. Ascorbic and dehydroascorbic acid were eluted isocratically in 11 min at a ow rate of 1 ml/min. AA and DHA were identied by their retention times and spectral data, as compared to authentic standards. Quantication was accomplished at maximum absorbance detected in the spectrum of ascorbic acid (261 nm) and dehydroascorbic acid (348 nm) by using a standard curve developed for both compounds. Results were expressed as mg AA or DHA per 100 g FW. 2.8. Statistical analysis In all analyses, three replicates were used. Results were processed by using one-way analysis of variance (ANOVA). Duncans test was performed to account for mean differences among different treatments. Differences at p < 0.05 were considered as signicant. SPSS software for Windows 14.0 (SPSS, Chicago, IL) was used to perform all statistical analyses. 3. Results and discussion 3.1. Total phenolics (TP), ascorbic acid (AA), dehydroascorbic acid (DHA) and antioxidant capacity of camu camu fruit at three maturity stages The TP, AA and DHA contents and total antioxidant capacity (expressed as DPPH value) in camu camu fruit at three maturity stages

are presented in Table 1. Total phenolic contents in camu camu increased from the full green to greenreddish stage and then decreased at the red stage. At full maturity (red stage) camu camu displayed a TP of 1320 mg GAE/100 g FW. This value is very high, in comparison to the contents reported for cherries, plums, strawberry (339, 366 and 368 mg GAE/100 g FW, respectively) (Wu et al., 2004), as well as for other berry types, such as blueberry, cranberry, lingonberry (412, 315, 652 mg GAE/100 g of FW, respectively), but lower than the value reported for chokeberry (2556 mg GAE/100 g FW) (Zheng & Wang, 2003). The AA content decreased with the increase of fruit maturity to a value of 2010 mg AA/ 100 g FW at full maturity. AA content was higher than DHA content during the course of the evaluated maturity (12 to 19-fold difference). At the red stage camu camu displayed AA values very close to the values reported by IIAP (2001) (2106 mg AA/100 g of FW) and Justi, Visentainer, Souza and Matsushita (2000) (2050 mg/ 100 g FW), and was within the range indicated by Rodrigues et al. (2001), from 9 to 50 g/kg. Camu camu fruit is characterised by its high AA content, as previously reported (Justi et al., 2000; Zapata et al., 1992). The DPPH antioxidant capacity displayed the same trend as the TP content during ripening, reaching a value of 167 lmol TE/g FW at full maturity. Reynertson et al. (2005) reported the antioxidant potential of seven fruits of the Myrtaceus family as good antioxidant sources by using the DPPH assay. A good linear correlation was established between TP and DPPH antioxidant capacity (r2 = 0.931) but not between AA and DPPH antioxidant capacity (r2 = 0.190). These results suggest that the antioxidant capacity of the fruit is derived from phenolic compounds. However, it is very important to mention the non-specicity of the FolinCiocalteu assay, due to the presence of sugars, ascorbic acid, aromatic amines, which could interfere in the measurement and lead to an overestimation of the TP content (Scholten & Kacprowski, 1992). Thus, the results of TP presented above derived from a combination of reducing compounds that react with the FolinCiocalteu reagent. In order to evaluate the contribution of phenolic compounds to the overall antioxidant capacity of the fruit, camu camu extracts were fractionated by means of solid-phase extraction, so that a phenolics-rich fraction could be obtained. 3.2. Contribution of phenolic compounds to the antioxidant capacity of camu camu at three different maturity stages Solid-phase extraction with a C18 Sep-Pak cartridge yielded two fractions: an aqueous fraction (F-I) and a methanolic fraction (F-II). AA and DHA were only detected in F-I, whereas phenolic compounds were detected in F-II. AA and DHA in F-I for the three maturity stages evaluated were 222.4 and 13.5 (full green), 170.5 and 15.3 (greenreddish) and 179.4 and 11.9 (red) mg AA/100 g of FW and mg DHA/100 g FW; levels of TP in FII were 129 (full green), 155 (greenreddish) and 219 (red) mg GAE/100 g FW. Losses of up to ten fold of AA were found after the fractionation process. DPPH antioxidant capacity in F-I and F-II were 121.2 (full green), 137.8 (greenreddish) and 112.6 (red) lmol TE/100 g FW, and 19.8 (full green), 26.1 (green-reddish), and 32.7 (red) lmol TE/100 g FW,

Table 1 Total phenolics (TP), ascorbic acid (AA), dehydroascorbic acid (DHA) and DPPH antioxidant capacity (DPPH) in camu camu fruit at three maturity stagesa. Maturity stage TP (mg GAE/100 g FW) Full green Greenreddish Red
a b b

AA (mg/100 g FW) 2280 34y 1910 45x 2010 65x

b

DHA (mg/100 g FW) 120 12x 152 9y 121 18x

b

DPPH (lmol TE/g FW)b 153 8x 185 11y 167 11y

1120 47x 1420 193y 1320 102y

SD of three replicate analyses. Means within a column with different letters are signicantly different at p < 0.05.

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respectively. Furthermore, the participation of F-I and F-II in the antioxidant capacity of the fruit at the different evaluated maturity stages, ranged from 67.5% to 79.3% and from 20.7% to 32.5%, respectively. These results demonstrated that the AA-rich fraction was the major contributor to the total antioxidant capacity of camu camu fruit despite the high losses incurred. The contribution of F-I and F-II to the antioxidant capacity are also related to the phenolic compound losses that could have happened during the fractionation process, as well as the antioxidant capacity method used in this study. In order to elucidate the main phenolic families present in camu camu fruit, TA, TFA and TFO were estimated in F-II (Fig. 1) by spectrophometric assays. TA, TFA and TFO contents during ripening increased from 0.8 to 52.6 mg CGE/100 g FW, from 11.6 to 19.2 mg CE/100 g FW and from 7.7 to 13.3 mg QE/100 g FW, respectively. The highest increases were found for TA contents (65.7-fold). Other phenolic compounds not quantied but present in camu camu fruit

were calculated by means of differences of TP content with respect to the addition of TA, TFA and TFO. These unclassied compounds represented the majority of phenolic compounds ranging from 61.6% to 84.9%. At the ripe stage, the TA content for camu camu (52.6 mg CGE/100 g FW) was similar to the value of 54 mg/100 g FW reported for camu camu fruit by Zanatta et al. (2005). This value is also comparable to the values reported for raspberry (10 60 mg CGE/100 g FW), strawberry (2090 mg CGE/100 g FW) and red cabbage (3090 mg CGE/100 g FW) (Awika, Rooney, & Waniska, 2004). The TFA content (11.6 mg CE/100 g FW) was similar to the values reported for yellow mashua tuber (12.4 mg CE/ 100 g FW) (Chirinos et al., 2008) and aa fruit (12 mg CE/100 g FW) (Silva, Souza, Rogez, Rees, & Larondelle, 2007) but lower than that reported for purple mashua tuber (90.7 mg CE/100 g FW) (Chirinos et al., 2008). To elucidate the nature of camu camu phenolic compounds, identication of the main phenolic compounds present in F-II was performed by means of HPLC-PAD. 3.3. HPLC-PAD phenolics prole of camu camu at three maturity stages

Total flavonols (mg QE/100 g FW), total flavanols (mg CE/100g FW), total anthocyanins (CGE/100 g FW) and other phenolic compounds

200

Others

TA

TFA

TFO

150

100

50

0
Initial Semi ripe Maturity stage Ripe

Fig. 1. Distribution of total avonols (TFO), total avanols (TFA), total anthocyanins (TA) and other phenolics in F-II from camu camu at three different maturity stages.

Phenolic determination in F-II was performed at 280, 320, 360 and 520 nm, to facilitate the identication of the different phenolics families present in camu camu. At 280 nm, HPLC-PAD proling (Fig. 2) revealed approximately 30 major peaks as well as several minor peaks for the three maturity stages studied. HPLC proles showed the same types of phenolic compounds, even though differences in concentration of each individual compound were observed. Fraction II contained a variety of different phenolic compounds, such as ellagic acid derivatives, avan-3-ols, anthocyanins, avonols and avanones (Table 1). Peaks identied corresponded to catechin (peak 6), delphinidin 3-glucoside (peak 9), cyanidin 3-glucoside (peak 10), ellagic acid (peak 15) and rutin (peak 18). Identication was performed by comparison of their UV spectra and retention times with those of authentic standards. The other non-identied peaks showed UVvis spectral characteristics similar to avan-3-ols of catechin type (peaks 15, 7, 8, 11, 12, 20, 23 and 27), avanones of naringenin type (peaks 13 and 29) and eriodictyol type (peaks 19 and 30), avonols of rutin type (peaks 14, 16, 17, 22 and 23); and ellagic acid (peaks 2426 and

10

25 21 17 12 1 2 34 6 7 5 9 11 25 17 1 2 34 5 6 7 8 10 11 12 13 14 15 16
18

30 19 29 20 22 23 24 28 26 27

Red

Absorbance at 280 nm

13

16 14 15
18

21 19 20 22 23 24 28 26 27 30 29

Green-reddish

25 17 21 1 2 34 5 6 13 7 8 11 12 14 15 16
18

28 23 22 24 26 27 29

19 20

30

Full green

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Retention time (min)

Fig. 2. HPLC-PAD phenolic proles for F-II from camu camu fruit at three different maturity stages recorded at 280 nm.

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Gallic acid derivative

Gallic acid

Delphinidin

Ellagic acid

Cyanidin

Myricetin

Red

Absorbance at 280 nm

Gallic acid derivative

Gallic acid

Ellagic acid

Green - reddish

Gallic acid derivative

Gallic acid

Ellagic acid

Full green

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Retention time (min)

Fig. 3. HPLC-PAD chromatograms for the hydrolysed F-II fraction at three different maturity stages recorded at 280 nm. Table 2 Chromatographic and spectral characteristics of the main phenolics detected in camu camu fruit at different maturity stagesa by HPLC-PDA. Peak N 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
a b c d e

Retention time (min) 15.34 20.77 22.52 23.80 25.60 28.88 30.29 31.19 32.22 35.91 40.77 46.89 48.67 52.57 53.49 55.31 56.51 57.67 58.30 60.40 62.10 63.51 64.97 67.63 68.35 70.51 72.62 74.24 79.11 86.28

kmax (nm) 241.9, 241.7, 241.7, 239.8, 242.0, 242.9, 240.9, 239.5, 281.9, 280.8, 242.3, 241.5, 289.1 233.5, 254.7, 243.5, 241.2, 257.1, 287.9 242.0, 254.5, 255.4, 235.8, 264.7, 254.7, 237.0, 242.3, 235.8, 289.1 287.9 279.0 279.1 278.1 279.6 278.3 279.6 274.6 278.9 519.8 521.1 279.9 278.4 358.0 366.3 349.7 359.3 357.0 279.1 364.5 354.3 278.3 366.1 360.9 367.5 279.0 341.4

Phenolic compound assignment Flavan-3-ol derivativeb Flavan-3-ol derivativeb Flavan-3-ol derivativeb Flavan-3-ol derivativeb Flavan-3-ol derivativeb Catechin Flavan-3-ol derivativeb Flavan-3-ol derivativeb Delphinidin-3-glucoside Cyanidin-3-glucoside Flavan-3-ol derivativeb Flavan-3-ol derivativeb Flavanone derivativec Flavonol derivatived Ellagic acid Flavonol derivatived Flavonol derivatived Rutin Flavanone derivativee Flavan-3-ol derivativeb Ellagic acid derivative Flavonol derivatived Flavan-3-ol derivativeb Ellagic acid derivative Ellagic acid derivative Ellagic acid derivative Flavan-3-ol derivativeb Ellagic acid derivative Flavanone derivativec Flavanone derivativee

Full green amount (mg/100 g FW) 5.9 5.4 3.8 9.7 1.4 1.3 5.8 5.0 Tr Tr 1.2 5.1 6.9 1.7 7.0 1.2 4.5 5.9 0.6 4.0 5.0 0.6 4.1 3.9 7.8 1.8 2.4 6.1 4.5 0.3

Greenreddish amount (mg/100 g FW) 7.6 5.9 3.9 6.8 3.8 1.7 5.6 4.9 Tr 0.9 3.0 5.9 7.0 1.2 6.0 1.2 4.1 3.8 0.5 2.7 6.4 0.3 3.4 3.6 8.4 1.6 1.5 5.0 4.7 0.3

Red amount (mg/100 g FW) 7.3 5.4 4.2 7.3 1.7 2.2 3.4 7.4 2.2 22.4 3.1 8.2 4.9 1.0 5.1 1.6 3.5 4.0 0.6 1.8 7.0 0.3 2.8 3.6 9.0 1.1 1.3 3.6 6.5 0.4

Mean of three replicates. Quantied as catechin equivalent at 280 nm. Quantied as naringenin equivalents at 280 nm. Quantied as quercetin equivalents at 360 nm. Quantied as eriodictyol equivalents at 280 nm.

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28). The detection of cyanidin 3-glucoside and delphinidin 3-glucoside in camu camu fruit are in agreement with previous results (Reynertson et al., 2008; Zanatta et al., 2005). Ellagic acid and two derivatives have been detected in leaves of M. dubia (H.B.K.) McVaugh (Ueda et al., 2004). In addition, Reynertson et al. (2005) highlighted the presence of catechin, epicatechin, epigallocatechin 3-O-gallate and epicatechin 3-O-gallate, tannins and avonol glycosides in other fruits of the Myrtaceae family. Some years later, Reynertson et al. (2008) found ellagic acid, quercetin, and rutin in M. dubia fruit. In this last study, detection of phenolic compounds belonging to the avan-3-ols family was not evaluated. The presence of avanones is reported for the rst time in this fruit. In order to obtain additional information on the aglycones present in camu camu, an acid hydrolysis was performed. After hydrolysis, the chromatographic proles at 280 nm (Fig. 3) for the three evaluated maturity stages showed three representative peaks: gallic (new peak), ellagic acid and a gallic acid derivative. Furthermore, at full maturity (red fruit) three additional peaks were detected (cyanidin, delphidin and myricetin (new peak)). Peaks were identied based on their UVvis spectral data and their retention times. Gallic and ellagic acids in the hydrolysed F-II were the most representative peaks for the three maturity stages. The detection of gallic and ellagic acid as major peaks might be indicative that camu camu fruit contains important quantities of hydrolysable tannins of gallo- or ellagitannins type. Hydrolysis with strong acids converts gallotannins to gallic acid and the core polyol (Inoue & Hagerman, 1988); also ellagitannins in plants are hydrolysed and ellagic acids are released (Wilson & Hagerman, 1990). From the total amount of phenolic compounds quantied by HPLC-PAD (Table 2), the ellagic acid group, avan-3-ols, avonols, avanones, and anthocyanins for the full green, greenreddish and red stages were 31.6, 31.0 and 29.4 mg/100 g FW; 55.1, 56.7 and 56.1 mg/100 g FW; 13.9, 10.6 and 6.4 mg/100 g FW; 12.3, 12.5 and 12.4 mg/100 g FW, and traces, 0.9 and 24.6 mg/100 g FW, respectively. In order of importance of phenolic compounds families, for the three ripening stages, we found that they were present as follows: avan-3-ol > ellagic acid group > avonol > avanone > anthocyanin (full green stage); avan-3-ol > ellagic acid group > avanone > avonol > anthocyanin (greenreddish stage); and avan-3-ol > ellagic acid group > anthocyanin > avanone > avonol (red stage). Thus, the ellagic acid and avan-3-ols groups represent the main phenolic compounds in camu camu fruit. Several phenolic compounds present in camu camu remained unidentied. Further studies are necessary to elucidate the overall phenolic potential of this fruit.

Acknowledgements The authors are thankful to Fiorella Ochoa and Adelaida Pardo for their technical assistance. This research was supported by the Consejo Nacional de Ciencia y Tecnologa (CONCYTEC, Peru). References
AOAC (1998). Ofcial methods of analysis (16th ed.). Washington, DC: Association of Ofcial Analytical Chemists. Awika, J. M., Rooney, L. W., & Waniska, R. (2004). Anthocyanin from black sorghum and their antioxidant properties. Food Chemistry, 90, 2933001. Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity using the DPPH free radical method. Lebensmittel Wissenchaft und Technologie Food Science and Technology, 28, 2530. Chang, C. C., Yang, M. H., wen, H. M., & Chern, J. C. (2002). Estimation of total avonoid content in propolis by two complementary colorimetric methods. Journal of Food and Drug Analysis, 10, 178182. Chirinos, R., Campos, D., Costa, N., Arbizu, C., Pedreschi, R., & Larondelle, Y. (2008). Phenolic proles of andean mashua (Tropaeolum tuberosum Ruz & Pavn) tubers: Identication and evaluation of their antioxidant capacity contribution. Food Chemistry, 106, 12851298. Delcour, J. A., & Devarebeke, D. J. (1985). A new colorimetric assay for avonoids in pilsner beers. Journal of the Institute of Brewing, 91, 3740. Giusti, M. M., & Wrolstad, R. E. (2001). Anthocyanins. Characterization and measurement with UVvisible Spectroscopy. In R. E. Wrolstad (Ed.), Current protocols in food analytical chemistry (pp. 113). New York: John Wiley & Sons. Unit F 1.2. Hertog, M. G. L., Holman, P. C. H., & Venema, D. P. (1992). Optimization of a quantitative HPLC determination of potentially anticarcinogenic avonoids in vegetables and fruits. Journal of Agricultural and Food Chemistry, 40, 15911598. Inoue, K. H., & Hagerman, A. E. (1988). Determination of gallotannins with rhodanine. Analytical Biochemistry, 169, 363369. Instituto de Investigaciones de la Amazonia Peruana (IIAP). (2001). Sistema de produccin de Camu Camu en Restinga (text in spanish). Justi, K. C., Visentainer, J. V., Souza, N. E., & Matsushita, M. (2000). Nutritional composition and vitamin C stability in stored camu camu (Myrciaria dubia) pulp. Archivos Latinoamericanos de Nutricin, 50, 405408. Khanbabaee, K., & van Ree, T. (2001). Tannins: Classication and denition. Natural Product Reports, 18, 641649. Manach, C., Scalbert, A., Morand, A., Rmsy, C., & Jimnez, L. (2004). Polyphenols: Food sources and bioavailability. American Journal of Clinical Nutrition, 79, 727747. Reynertson, K. A., Basile, M. J., & Kenelly, E. J. (2005). Antioxidant potential of seven Myrtaceous fruits. Ethnobotany Research and Applications, 3, 2535. Reynertson, K. A., Yang, H., Jiang, B., Basile, M.-J., & Kennelly, E. (2008). Quantitative analysis of antiradical phenolic constituents from fourteen edible Myrtaceae fruits. Food Chemistry, 109, 883890. Rodrigues, R. B., Menezes, H. C., Cabral, L. M. C., Dornier, M., & Reynes, M. (2001). An amazonian fruit with a high potential as a natural source of vitamin C: The Camu camu (Myrciaria dubia). Fruits, 56, 345354. Ross, J. A., & Kasum, C. M. (2002). Dietary avonoids: Bioavailability, metabolic effects, and safety. Annual Reviews of Nutrition, 22, 1934. Scholten, G., & Kacprowski, M. (1992). Zur Analytik von Polyphenolen in Wein. Die Weinwissenschaft, 48, 3338. Singleton, V. L., & Rossi, J. A. (1965). Colorimetry of total phenolics with phosphomolybdicphosphotungstic acid reageants. American Journal of Enology and Viticulture, 16, 144158. Silva, E. M., Souza, J. N. S., Rogez, H., Rees, J.-F., & Larondelle, Y. (2007). Antioxidant activities and polyphenolic contents of fteen selected plant species from the Amazonian region. Food Chemistry, 101, 10121018. Tsao, R., & Deng, Z. (2004). Separation procedures for naturally occurring antioxidant phytochemicals. Journal of Chromatography B, 812, 8599. Ueda, H., Kuroiwa, E., Tachibana, Y., Kawanishi, K., Ayala, F., & Moriyasu, M. (2004). Aldose reductase inhibitors from the leaves of Myrciaria dubia (H.B.K.) McVaugh. Phytomedicine, 11, 652656. Wilson, T. C., & Hagerman, A. E. (1990). Quantitative determination of ellagic acid. Journal of Agricultural and Food Chemistry, 38, 16781683. Wu, X., Beecher, G. R., Holden, J. M., Haytowitz, D. B., Gebhardt, S. E., & Prior, R. L. (2004). Lipophilic and hydrophilic antioxidant capacities of common foods in the United States. Journal of Agricultural and Food Chemistry, 52, 40264037. Zanatta, C. F., Cuevas, E., Bobbio, F. O., Win terhalter, P., & Mercadante, A. Z. (2005). Determination of anthocyanins from Camu camu (Myrciaria dubia) by HPLCPDA, HPLC-MS, and NMR. Journal of Agricultural and Food Chemistry, 53, 95319535. Zapata, S., & Dufour, J.-P. (1993). Camu Camu (Myrciaria dubia) (H.B.K.) McVaugh: Chemical composition of fruit. Journal of the Science of Food and Agriculture, 61, 349351. Zapata, S., & Dufour, J.-P. (1992). Ascorbic, dehydroascorbic and isoascorbic acid simultaneous determinations by reverse phase ion interaction HPLC. Journal of Food Science, 57, 506511. Zheng, W., & Wang, S. (2003). Oxygen radical absorbing capacity of phenolics in blueberries, cranberries, chokeberries, and lingonberries. Journal of Agricultural and Food Chemistry, 51, 502509.

4. Conclusions This study provides information concerning the antioxidant potential of camu camu fruit, not only related to its high ascorbic acid but to its phenolics content. Ascorbic acid displayed a signicantly higher contribution to the overall antioxidant capacity of camu camu at the three evaluated maturity stages. In addition to the already recognized and well known ascorbic acid content, camu camu possesses phenolic families such as avan-3-ols (catechin and its derivatives), ellagic acid derivatives, anthocyanins (delphinidin 3-glucoside and cyanidin 3-glucoside) avonols (rutin and its derivatives) and avanones (naringenin and eriodictyol derivatives). These results provide further evidence to promote this fruit as an important source of health-promoting phytochemicals, in addition to its high ascorbic acid content. Furthermore, the presence of hydrolysed tannins (gallo- and ellagitannins) has been shown.