jfbc_267
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DOI: 10.1111/j.1745-4514.2009.00267.x
PATTERNS OF CAROTENOID PIGMENTS EXTRACTED FROM TWO ORANGE PEEL WASTES (VALENCIA AND NAVEL VAR.) VERONICA S. CHEDEA1,2, PANAGIOTIS KEFALAS2 and CARMEN SOCACIU1 1 Department of Chemistry and Biochemistry University of Agricultural Sciences and Veterinary Medicine 3-5 Manastur Street, 400372 Cluj-Napoca, Romania 2
Department of Food Quality and Chemistry of Natural Products Mediterranean Agronomic Institute of Chania Centre International de Hautes Etudes Agronomique Méditerraneénnes Crete, Greece Accepted for Publication April 3, 2008
ABSTRACT The peel waste from two orange varieties (Valencia and Navel) was analyzed. The saponified extract was analyzed for the carotenoid content by thin-layer chromatography and high-performance liquid chromatography. Identification was made by ultraviolet-Vis spectrometry. Our studies revealed that the waste of Valencia orange peels is rich in apocarotenoids like 10⬘-apo-b-caroten-10⬘-ol, 10⬘-apo-b-caroten-10⬘-al, 8⬘-apo-b-caroten-8⬘-ol, 8⬘-apo-b-caroten-8⬘-al and 6⬘-apo-b-caroten-6⬘-ol representing more than 60% of the total carotenoids. Also identified were b-chryptoxanthin and, in minor quantities, dihydroxycarotenoids (zeaxanthin and its epoxides). b-Cryptoxanthin, which exhibits provitamin A activity, was the main compound in Navel variety extract. z-Carotene as well as some dihydroxy derivatives were also present. The amount of apocarotenoids in this variety is very low. The data reveal important differences of carotenoid composition depending on orange variety: Valencia variety was richer in short-chain apocarotenoids while Navel variety in nonpolar carotenoids. PRACTICAL APPLICATIONS The citrus fruit residues, remaining after juice extraction, represent approximately half of the wet mass of the whole fruit, including the peel 3
Corresponding author. TEL: 0040-264-595825, 596384(5,6,7) ext. 213; FAX: 0040-264-593792; EMAIL:
[email protected]
Journal of Food Biochemistry 34 (2010) 101–110. © 2010, The Author(s) Journal compilation © 2010, Wiley Periodicals, Inc.
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(flavedo and albedo) and almost one-fourth of the whole fruit mass. Such a waste can be used as a good source for extracting bioactive molecules like carotenoid pigments, pectins and terpenes (essential oils). The technological applications given by the carotenoid composition of the orange peels may help the industrial processors to find new ways of increasing the profit by recuperating important bioactive molecules and also reducing the considerable problem of waste disposal. Such extracts can be very useful as functional additives for food matrices. The presence of z-carotene proves the fact that there is a great amount of phytoene in Navel variety, which is a compound commercially desirable, so the orange peel waste from Navel variety oranges can be taken as a commercial source of phytoene. b-Cryptoxanthin, which exhibits provitamin A activity, was also present. Its capacity to quench single oxygen and to be used as dairy product additive (e.g., butter colorant) is very useful in practice (medicine and food industry).
INTRODUCTION Oranges contain around 20 carotenoids, but only red grapefruit has a high beta-carotene content (Gross 1997; Rincon et al. 2005). Other carotenoids, such as lutein, zeaxanthin and beta-cryptoxanthin, can be found in significant quantities in tangerines and oranges. Like beta-carotene, these other carotenoids also possess significant antioxidant activity and lutein; zeaxanthin also protects against age-related macular degeneration. Red grapefruit contains a high level of lycopene. Among dietary carotenoids, lycopene has the highest antioxidant activity (Curl and Bailey 1956, 1961; Britton et al. 1995). With its numerous cultivated varieties, the sweet orange (Citrus sinensis Osbeck) constitutes one of the world’s most popular fruit crops. Sweet oranges are citrus fruits (Citrus spp.), which are regarded as rich sources of vitamin C (ascorbic acid) and other organic acids (Hui 1999). Today, oranges are primarily used fresh or prepared as frozen juice concentrate. The by-products of juice making (the pulp, rind and seed) are utilized as cattle feed and molasses (for alcohol or feed) as well as food flavors and aromas, perfumes, pharmaceuticals and soaps. The extracts of rinds and seeds include pectins and oils. When fermented, orange juices produce vinegar and liqueurs. Oranges are permitted to ripen on trees before harvesting; most of them receive treatments with ethylene to enhance their orange color and to ship well to the markets (Janick et al. 1981). Before, Europeans viewed oranges as food, other population used their trees, flowers and fruits as ornaments, seasonings and for aroma. Originally, Citrus uses included beautification, embalming, mothballs, aphrodisiacs, protection from poisons and cure fever and colic (Gross 1997).
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The yellow-to-orange color of all citrus fruits is due to the presence of carotenoids located in the plastids, both of the flavedo and of the internal juice vesicles. In the early stages of fruit ripeness, the external color is masked by chlorophyll. With advancing maturity, the yellow color appears in various tints, from light yellow to deep orange, due to the variation in type and amount of different carotenoids. The color distribution depends on species and cultivar differences (Curl and Bailey 1956, 1961). One of the most complex carotenoid patterns can be found in citrus, compared with other fruits. As characterized by a high content of epoxides and furanoxides, this fraction accounts for about 70% of the total carotenoids. In peels, which have a higher carotenogenic capacity than the endocarp, the carotenoid level may be from two to six times higher. About 70% of the total fruit carotenoids are found in the peel (Curl and Bailey 1956, 1961). In the technological conditions when the juice is separated from the rest of the fruit, important amounts of orange peel waste were monitored. This waste can represent a very important source of carotenoids of different types: provitamin A, apocarotenoids and epoxidated forms. The purpose of our study was the characterization of carotenoid pattern in two varieties of orange waste (Valencia and Navel) using different analytical methods like thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) and UV-Vis spectrometry for detection and identification.
MATERIAL AND METHODS The orange peel waste was obtained in February 2005 from Biochim, a juice factory in Chania, Crete, Greece, which valorizes Citrus fruit. Two varieties of orange, Valencia and Navel, were used, being harvested from the nearby Platanias and Apokoronas. After the juice extraction, the waste was kept in a freezer at -18C, and then submitted to analysis. Extraction Procedures Two different extraction procedures (solvents) were made for Valencia variety and one for Navel variety. Procedure 1 (Valencia and Navel Variety). Aliquots of homogenized waste (200 g) were weighed and mixed first with 400 mL acetone for extraction. The extraction was carried out at room temperature, at darkness, for 12 h with magnetic stirring. After extraction, the mixture was filtered and the total extract in acetone was evaporated under vacuum at 40°.
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The residue was extracted with petroleum ether (EP), and a third extraction was carried out with diethyl ether (EE). The extracts with EP and EE were mixed and the solvent mixture was evaporated under vacuum at 40° in a Rotavapor. The total dry extract (collected from acetone, EP, EE extractions) was dissolved in 50 mL EP and mixed with 100 mL solution of 10% KOH in methanol. The extract was left to saponify for 12 h at room temperature and then washed with 100 mL EE and 200 mL water added in a separation funnel. The two phases (the upper lipophilic phase containing the carotenoids, and the lower one, aqueous solution containing flavonoids and residues) were successively partitioned with water and re-extracted in EE. The extract was washed with a concentrated solution of NaCl several times and then with water several times until the water washings were neutral to phenophthalein. The ether extract was then concentrated in a Rotavapor almost to dryness, then transferred to a small flask and concentrated to dryness, removing the last traces of solvent by flushing the flask with nitrogen. Procedure 2 (Valencia Variety). Aliquots of 500 g homogenized orange peel waste were extracted with 1,000 mL of a mixture of acetone and hexane, 6:4. After 12 h of extraction, by magnetic stirring, the extract was filtered, transferred to EP and EE, and saponification, washings and all other procedures were carried out as in procedure 1. Chromatographic Analysis The stored extracts resulted by procedure 1 were dissolved in acetone and submitted to TLC on silica using successively three solvents: (1) petroleum; (2) petroleum : EE = 1:1; and (3) EE. For preparative separation, a silica plate was also used and large strips were applied using the dried extracts dissolved in acetone. The elution system used a mixture of petroleum : EE = 1:1. After the separation, the relevant zones were scraped off, eluted with acetone and their spectra were determined with a JASCO V-500 spectrophotometer, Cluj-Napoca, Romania. A Hewlett Packard (series II) 1090 liquid chromatograph equipped with a Diode Array Detector, using a LiChrospher 100 RP-18 (250 ¥ 4 mm, 5 mm particle size, Merck, Darmstadt, Germany) column which, protected by a guard column containing the same stationary phase, was used for highpressure column analysis. HPLC-grade acetonitrile and ethyl acetate from Sigma (St. Louis, MO) were used, and the other reagents and solvents were of guaranteed or analytical grade. For the HPLC analysis, two kinds of saponified extracts were used, one obtained by procedure 2 from the Valencia variety, and the other from
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the Navel variety by procedure 1. The carotenoid standards used were b-carotene, lutein and lycopene and the analysis was monitored at 450, 447 and 471 nm. The column was equilibrated with solvent A (acetonitrile/water, 9:1, containing 0.1% triethylamine) for 15 min. The extract was dissolved in a small volume of solvent (ethyl acetate) and 20 mL sample was injected. A linear gradient, from a mixture of solvent A (acetonitrile : water = 9:1 and triethylamine 0.1%) decreased from 100 to 0%, while solvent B (ethyl acetate) increased from 0 to 100% in 25 min, was applied.
UV-Vis Spectrometry The strips corresponding to the Valencia variety extract in saponified or nonsaponified form were scrapped from the TLC plate and submitted to UV-Vis spectrometric analysis (360–460 nm), giving us a general idea about the major groups of carotenoids found in this extract. The identification of individual carotenoids separated by HPLC was made by co-chromatography with standards, e.g., lutein, b-carotene and lycopene, or by UV-Vis spectrometry of each peak, comparatively with literature data.
RESULTS AND DISCUSION Fingerprint of Orange Extracts by Vis Spectrometry Figure 1 represent the Vis spectra of the carotenoid mixture scratched from the major TLC band separated from the total extract of Valencia orange waste. Four large peaks are noticed, absorbing at 380, 400, 425, 450 nm, corresponding to a mixture of apocarotenoids (l < 450 nm). The highest intensity was noticed for peaks at 400 and 425 nm. After saponification, for the TLC separation, the shape of the same major band’s spectra is changed (Fig. 2). Four peaks can be identified here, but their relative intensity and lmax were slightly modified. The major peak appears at 428 followed by peaks at 403 nm, 453 nm and at 383 nm as a shoulder. Looking at the relative absorption of these peaks comparing with Fig. 1’s peak, one can observe that saponification favored the carotenoids absorbing at 428 nm and 450–453 nm with a bathocromic shift of 3 nm. The two figures give a general view of the extracts main compounds, and even if they are not accurate in quantitative appreciation, they give a rapid and suggestive view of extract composition.
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FIG. 1. THE UV-VIS SPECTRUM OF THE MOST INTENSE STRIP OF THE TOTAL EXTRACT OF VALENCIA ORANGE WASTE SEPARATED BY THIN-LAYER CHROMATOGRAPHY (RETENTION FACTOR [RF] = 0.35)
FIG. 2. THE ULTRAVIOLET-VIS SPECTRUM OF THE MOST INTENSE STRIP, SAME STRIP AS IN FIG. 1, BUT FROM THE SAPONIFIED EXTRACT OF VALENCIA ORANGE WASTE SEPARATED BY THIN-LAYER CHROMATOGRAPHY (RETENTION FACTOR = 0.3)
HPLC-Vis Analysis Figure 3 represents the HPLC of the saponified extract of the Valencia variety. Lutein and b-carotene were also added to each sample as internal standards. Table 1 includes the retention times and the peak identification.
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FIG. 3. THE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY CHROMATOGRAM OF THE SAPONIFIED EXTRACT OF ORANGE PEEL WASTE, VALENCIA VARIETY
TABLE 1. THE CAROTENOID IDENTIFICATION FROM THE VALENCIA VARIETY EXTRACT (SVAL); CO-CROMATOGRAPHY WITH STANDARDS: LUTEIN AND 〉-CAROTENE (TR = 18.5 MIN), (TR = 10.12 MIN) AND VIS SPECTRUM OF EACH COMPOUND (PEAK) WERE USED FOR IDENTIFICATION Peak no. c d 1 2 3 4 + 4a 5 a 7 8 b
tR (min) 2.41 2.609 4.305 5.105 7.22 7.618–7.903 9.242 9.456 10.425 10.725 14.986
% Area
Tentative identification (lmax.)
2.71 1.58 8.34 17 5.73 29.14
1,1 dimethyl, 2-methyl, 2-cyclohexenal 1,1 dimethyl, 2-methyl, 3-propenal, 2-cyclohexene 10′-apo-b-carotene-10′-al, 443 10′-apo-b-carotene-10′-ol, 422,450 8′-apo-b-carotene-8′-al, 465 mixture of 8′-apo-b-carotene-8′-ol (max. at 421,448 nm) 6′-apo-b-carotene-6′-ol (max. at 424,445 nm) 430,455 Dihydroxymonoepoxides 429,456 450,476 zeaxanthin 460,482 dyhydroxycarotenoid b-cryptoxanthin, 455,483
4.63 9.83 1.91 5.09 16.13
The major peaks (nr. 1, 2, 3, 4, 4a) corresponded to short-chain apocarotenoids, groups of 10′-apo-b-caroten-10′-ols, 10′-apo-b-caroten-10′-als, 8′apo-b-carotene-8′-ols, 6′-apo-b-carotene-6′-ols and 8′-apo-b-carotene-8′-al. The apocarotenoids in our orange waste-saponified extract derive by specific
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c
d
FIG. 4. CHEMICAL FORMULAS OF COMPOUNDS (c), 1,1 DIMETHYL, 2-METHYL, 2-CYCLOHEXENAL AND (d), 1,1 DIMETHYL, 2-METHYL, 3-PROPENAL, 2-CYCLOHEXENE IDENTIFIED BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY AT TR OF 2.41 MIN AND, RESPECTIVELY, 2.609 MIN.
FIG. 5. THE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY CHROMATOGRAM OF THE SAPONIFIED EXTRACT OF ORANGE PEEL WASTE, NAVEL VARIETY
cleavage of b-carotene. This cleavage also gives rise to secondary products like compounds c and d identified by HPLC (Fig. 4) at tR of 2.41 min and, respectively, 2.609 min. The compounds c and d were tentatively identified as 1,1 dimethyl, 2-methyl, 2-cyclohexenal (c) and 1,1 dimethyl, 2-methyl, 3-propenal, 2-cyclohexene (d) as presented in Fig. 4. Compound c results when b-carotene is cleaved to 10′-apo-b-carotene-10′-(ol and al). Compound d is formed when b-carotene is cleaved to 8′-apo-b-carotene-8′-(ol and al). b-cryptoxanthin was found also as an important fraction (16%). There were identified also dihydroxyepoxides (tR = 9.24 and tR = 9.46) and dihydroxycarotenoids (tR = 10.42 min and tR = 10.72). No b-carotene was present in this extract. Figure 5 represents the HPLC of the saponified extract of the Navel variety. Table 2 includes the retention times and the peak identification of carotenoid composition of the Navel variety. The major fractions were represented by b-cryptoxanthin and z-carotene. This extract was less rich in apocarotenoids and two unidentified compounds
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TABLE 2. THE CAROTENOID IDENTIFICATION FROM THE NAVEL VARIETY EXTRACT (SNAV); CO-CROMATOGRAPHY WITH STANDARDS: LUTEIN (TR = 10.08 MIN), LYCOPENE (TR = 16.2 MIN) AND 〉-CAROTENE (TR = 18.25 MIN) AND VIS SPECTRUM OF EACH COMPOUND (PEAK) WERE USED FOR IDENTIFICATION Peak no.
tR (min)
% Area
Tentative identification, (lmax.)
c 3 a 6 10 11 b 12 12a
2.41 7.22 9.796 10.085 11.733 12.164 14.678 17.908 18.030
2.71 5.73 15.85 3.42 12 10.23 30.32 8.12 10.41
1,1 dimethyl, 2-methyl, 2-cyclohexenal 8′-apo-b-caroten-8′-al, 465 dihydroxymonoepoxide, 427,457 Zea 5,6-epoxide 316,332 IN 318,336 IN b-cryptoxanthin, 450,478 z-carotene, (15-cis, probably) 402,425 z-carotene, trans isomer, 403,428
were separated at tR = 11.7 and 12.1 min, having lmax at 316,322 and 318,336 nm respectively. The major peak in this case corresponded for b-cryptoxanthin, followed by dihydroxymonoepoxides and x-carotene derived from phytoene. CONCLUSIONS Obtained from one of the most popular fruits, citrus fruits, citrus juices or citrus extracts have distinctive flavors and are used as functional food or natural additives for a wide variety of food products. The by-products may provide also a supplemental revenue source for citrus processors when faced with price fluctuations. Many useful ingredients currently come from citrus and researchers are pressing forward with efforts to squeeze new functionality from this fruit. Recovering phytochemicals from nonjuice citrus components typically involves extraction under specific conditions (Rosenberg et al. 1983). Although researchers have developed methods to extract limonoid glucosides and other substances, methods for many of the more potent phytochemicals remain to be discovered. Product developers always are seeking ways to improve a product’s performance, flavor and health contributions. The various materials obtained from citrus offer benefits in all of those areas (Molnár et al. 2005). The presence of z-carotene proves the fact that there is a great amount of phytoene in Navel variety, which is a compound commercially desirable, so the orange peel waste from Navel variety oranges can be taken as a commercial source of phytoene.
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b-Cryptoxanthin, which exhibits provitamin A activity, being converted to vitamin A in the intestine, was also present. Its capacity to quench single oxygen and to be used as dairy product additive (e.g., butter colorant) is very useful in practice (medicine and food industry). Other apocarotenal and apocarotenol molecules in some other orange extracts were identified. The biological activity of such carotenoid derivatives is very interesting, with recent data suggesting that they have a better bioavailability at cellular level (Molnár et al. 2005).
REFERENCES BRITTON, G., LIAAEN-JENSEN, S. and PFANDER, H. 1995. Carotenoids, Vol. 1A: Isolation and Analysis, Birkhauser Verlag, Basel, Switzerland. CURL, A.L. and BAILEY, G.F. 1956. Orange carotenoids. I. Comparison of Valencia orange peel and pulp. J. Agric. Food Chem. 4, 156–159. CURL, A.L. and BAILEY, G.F. 1961. The carotenoids of navel oranges. J. Food Sci. 26, 442–447. GROSS, J. 1997. Carotenoid pigments in citrus. In Citrus Science and Technology, Vol 1 (S. Nagy, P.E. Shaw and M.K. Veldhuis, eds.) pp. 302–355, AVI Publishing, Westport, CT. HUI, S. Sweet oranges: The biogeography of Citrus sinensis. http:// stephen.cjb.net/resources/orange.html (accessed August 25, 1999). JANICK, J., SCHER, Y., ROBERT, W., WOODS, F.W. and RUTTAN, V.W. 1981. Plant Science: An Introduction to World Crops, 3rd Ed., W.H. Freeman and Company, San Francisco, CA. MOLNÁR, P., KAWASE, M., SATOH, K., SOHARA, Y., TANAK, T., TAN, S., SAKAGAM, H., NAKASHIMA, H., MOTOHASHI, N., GYÉMÁNT, N. ET AL. 2005. Biological activity of carotenoids in red paprika, Valencia orange and Golden delicious apple. Phytother. Res. 19(8), 700–707. RINCON, A.M., VASQUEZ, A.M. and PADILLA, F.C. 2005. Chemical composition and bioactive compounds of flour of orange (Citrus sinensis), tangerine (Citrus reticulata) and grapefruit (Citrus paradisi) peels cultivated in Venezuela. Arch. Latinoam. Nutr. 55(3), 305–310. ROSENBERG, M., MANNHEIM, C.H. and KOPELMAN, I.J. 1983. Carotenoid base food colorant extracted from orange peel by d-limonene -extraction- process and Use. Lebensm.-Wiss. Technol. 17, 270–275.
