Carotenoid composition from strawberry tree (Arbutus

unedo L.) fruits.

Raul Delgado-Pelayo and Dámaso Hornero-Méndez*Instituto de la Grasa – CSIC, Chemistry and Biochemistry of Pigments Group, Department of Food Biotechnology, Av. Padre García Tejero, 4, E-41012, Seville (Spain).*hornero@cica.es

The strawberry tree (Arbutus unedo L.) is an evergreen shrub or small tree usually smaller than 5 m, belonging to the Ericaceae family, and typical of the Mediterranean basin and climate, although it can be also found in other regions such as the Near East and Transcaucasia. The fruits are spherical berries, about 1.5-2 cm in diameter, dark red and tasty when fully ripen in autumn and winter. The berries can be eaten as fresh fruits, but they are usually prepared as preserves, jams, jellies, and marmalades. The name 'unedo' is explained by Pliny the Elder as being derived from unum edo "I eat one", which maybe due to the alcoholic content of overrippen fruits. In some countries the fruits are used to produce alcoholic beverages such as wines, liquors, and brandies (e.g. Portuguese medronho, and Koumaro in Greece). Strawberry tree fruits are also well-known in folk medicine as antiseptics, diuretics, and laxatives, while the leaves of the plant are usedas diuretic, urinary antiseptic, antidiarrheic, astringent, depurative, and antihypertensive.

The chemical composition of A. unedo fruits has been study by several authors (1-3), but almost any exhaustive description of the different phytochemicals have been carried out up to date. Recent studies have focused on the description of phenolic compounds, such as flavonoids, phenolic acids and anthocyanins, as a part of the antioxidant compositions of these fruits (4-6). However, very little attention has been paid to the carotenoid composition of these fruits, a part from the early study carried out by Schön in 1935 (7). Some of the studies analyzing the chemical and antioxidant composition of A. unedo fruits, concluded that β-carotene is the major and single carotenoid found in these fruits (2). Therefore, the aim of the present study was to carry out the isolation, identificationand quantification of the carotenoid pigments responsible for the yellow-orange colour of the flesh of the strawberry tree fruits.

Raw materialArbutus unedo fruits were collected during the autumn and winter (October 2009-February 2010) from a tree located at the Instituto de la Grasa in Sevilla, and from trees at the Sierra Norte de Sevilla Natural Park (Sevilla, Spain; 37º37’44.3424”: -6º24’22.4316”). Samples were stored at -30ºC until analysis.Carotenoid extractionTwo grams of fruit were extracted with acetone, until complete exhaustion of colour, according to the procedure of Mínguez-Mosquera and Hornero-Méndez (8). All extracts were pooled and transferred to diethyl ether. The ether phase, containing the carotenes and xanthophylls (free andesterified), was taken to dryness and the residue dissolved in 1 mL of acetone. Saponified extracts were prepared by treating the direct extract with 10% KOH-MeOH. Extracts were kept at -30ºC until HPLC analysis.HPLC analysisCarotenoids were separated by using the HPLC protocol described by Mínguez-Mosquera and Hornero-Méndez (8) with slight modifications. Detection was carried out at 450 nm, and pigment were quantified in the direct extract by using calibration curves prepared with standard stock solutions in the concentration range 5-100 µg/mL.IdentificationRoutine procedures for the identification of carotenoids have been used, which consisted of separation of pigments by TLC and co-chromatography with purified pigments, analysis of the UV-visible and mass spectra, and chemical test for 5,6-epoxide groups. Authentic pigment samples of β-carotene, antheraxanthin, violaxanthin, neoxanthin, β-cryptoxanthin, zeaxanthin and lutein were isolated and purified from natural sources (Capsicum annuum and Menta arvensis). Lutein 5,6-epoxide was isolated from petals of Taraxacum officinale. Furanoid containig pigments (neochrome, luteoxanthin, auroxanthin and mutatoxanthin) were prepared by treating stock solutions of the corresponding 5,6-epoxide xanthophylls with diluted HCl acid. HPLC-MS(APcI) was used for determining molecular mass of pigments by using the conditions Breithaupt and Schwack (9) with some modifications.

Introduction

The chromatograms corresponding to the direct and saponifiedextracts obtained from ripe fruits of strawberry tree are shown in Figure 2 , indicating that more than 90% of the xanthophylls were esterified with fatty acids. Due to the fact that the xanthophyll esters were not well resolved, the identification was carried out using the saponified extracts. At the present we are still conducting the study aimed to identify the nature of the fatty acids involved on xanthophyll esterification by using HPLC-DAD-MS(APCI). Table 1 summarizes the identification of the main carotenoid pigments, including the quantitative composition. The total carotenoid content was about to 43 µg/gof fresh weight. Seventeen carotenoids (including the cisisomers) were identified, being β-carotene the only carotene (up to 7%), and the rest were represented by a whole family of mono- and dihydroxylated xanthophylls and their 5,6-epoxide relatives. Due to the acidity of the fruit, and the softness flesh, a high proportion of the 5,6-epoxide carotenoids were transformed to the corresponding furanoid xanthophylls (this was not avoided when using sodium carbonate during the extraction, indicating their presence in the fruits before the extraction). In fact, although violaxanthin was one of the major pigments (19.7%), luteoxanthin (a derivative of violaxanthincontaining one 5,8-epoxide group) was at higher concentrations (22.4%). Other outstanding pigments were neoxanthin (11.0%), neochrome (5.1%), antheraxanthin(4.2%), lutein 5,6-epoxide (7.4%), zeaxanthin (6.4%) and lutein(12.2%). For the first time, the carotenoid composition of strawberry tree fruits has been studied in detail (10).

1. Ayaz, F.A., Kucukislamoglu, M. Sugar, non-volatile and phenolic acids composition of strawberrytree (Arbutus unedo L. var. ellipsoides) fruits. Journal of Food Composition and Analysis, 2000, 13(2), 171–177.

2. Alarcao-Silva, M.L.C.M.M., Leitao, A.E.B., Azinheira, H.G., Leitao, M.C.A., The arbutus berry: studies on its color and chemical characteristics at two mature stages. Journal of FoodComposition and Analysis, 2001, 14, 27–35.

3. Özcan, M.M., Hacseferogullar, H. The strawberry (Arbutus unedo L.) fruits: Chemical composition, physical properties and mineral contents. Journal of Food Engineer, 2007, 78, 1022–1028.

4. Pawlowska, A.M., Leo, M., Braca, A. Phenolics of Arbutus unedo L. (Ericaceae) Fruits: Identification of anthocyanins and gallic acid derivatives. Journal of Agricultural and FoodChemistry, 2006, 54, 10234-10238.

5. Pallauf, K., Rivas-Gonzalo, J.C., del Castillo, M.D., Cano, M.P., de Pascual-Teresa, S.C. Characterization of the antioxidant composition of strawberry tree (Arbutus unedo L.) fruits. Journal of Food Composition and Analysis, 2008, 21, 273-281.

6. Barros, L., Carvalho, A.M., Morais, J.S., Ferreira, I. Strawberry-tree, blackthorn and rose fruits: Detailed characterisation in nutrients and phytochemicals with antioxidant properties. FoodChemistry, 2010, 1, 247–254.

7. Schön, K. Studies on carotenoids. I. The carotenoids of Diospyros fruits. II. The carotenoids ofarbutus fruits (Arbutus unedo). Biochemistry Journal, 1935, 29(7), 1779–1785.

8. Mínguez-Mosquera, M.I., Hornero-Méndez, D. Separation and quantification of the carotenoidpigments in red peppers (Capsicum annuum L.), paprika and oleoresin by reversed-phase HPLC. J. Agric. Food Chem., 1993, 41, 1616-1620.

9. Breithaupt, D.E., Schwack, W. Determination of free and bound carotenoids in paprika (Capsicum annuum L.) by LC/MS. Eur. Food Res. Technol. 2000, 211, 52-55.

10. Hornero-Méndez, D., Delgado-Pelayo, R. Identification and quantification of carotenoids from strawberry tree (Arbutus unedo L.) fruits. J. Agric. Food Chemistry 2010, submitted for publication.

This work was supported by the Project AGL2007-60092/ALI, Ministeriode Ciencia e Innovación (Spanish Government) and by the Project P08-AGR3477, Consejería de Economía, Innovación y Ciencia (Junta de Andalucía).

Acknowledgements

β-Carotene

OH

OH

Lutein

OH

OH

Zeaxanthin

OHβ-Cryptoxanthin

OH

OH

O

Antheraxanthin

Figure 3 . Chemical structures of the maincarotenoids identifified in strawberry tree (A. unedo L.) fruits.

OH

O

OH

O

OH

O

OHOH

●

Violaxanthin

Neoxanthin

OHO

OHOH

●

Neochrome

OHO

OHO

Auroxanthin

OH

OH

O

Lutein 5,6-epoxide

OH

OH

O

Mutatoxanthin

OH

OHO

O

Luteoxanthin

Figure 2 . HPLC chromatograms corresponding to a direct and a saponified carotenoid extracts obtainedfrom strawberry tree (A. unedo L.) fruits. (See Table1 for peak identities)

10 20 300

4

56

11

101

2

3

12

Retention time (min)

Direct extract

Saponified extract Abs @450nm

Table 1 . Chromatographic and spectroscopic characteristics of carotenoids from strawberrytree (A. unedo L.) fruits. Quantitative analysis.

a. Tentative identification; b. UV-visible maxima in acetone; c. mean ± standard deviation of a triplicate analysis

0.56 ± 0.03569-63330, 422, 444, 474330, 424, 443, 47010.129-cis-Lutein14

0.81 ± 0.08569-54332, 422, 442, 472334, 422, 444, 47210.2213-cis-Lutein15

0.36 ± 0.07553-18428, 450, 478428, 454, 48014.13β-Cryptoxanthin16

537

569

569

585

585

601

601

585

601

601

601

601

601

601

[M+H]+m/z

3.02 ± 1.20-20429, 452, 478430, 454, 48020.68all-trans-β-Carotene17

3.82 ± 0.56-65422, 445, 474424, 449, 4769.56all-trans-Lutein13

2.73 ± 0.86-18430, 452, 479425, 452, 4839.35all-trans-Zeaxanthin12

3.16 ± 0.76+87418, 441, 471418, 441, 4699.12Lutein 5,6-epoxidea11

0.55 ± 0.22-87409, 428, 452411, 430, 4579.00Mutatoxanthin10

1.05 ± 0.30-120377, 398, 423384, 403, 4278.80Auroxanthin9

6.47 ± 2.76+111400, 423, 448397, 420, 4468.57Luteoxanthin isomera8

1.77 ± 0.37+80422, 444, 472425, 449, 4788.33Antheraxanthin7

3.07 ± 0.75+102400, 423, 448400, 424, 4518.17Luteoxanthin6

4.93 ± 0.10+92326, 410, 434, 464327, 414, 438, 4678.039-cis-Violaxanthin5

3.45 ± 1.02+91421, 442, 473419, 443, 4727.28all-trans-Violaxanthin4

2.16 ± 1.08-98398, 421, 448402, 424, 4526.43Neochrome3

2.32 ± 0.22+91327, 416, 438, 468328, 415, 439, 4686.109-cis-Neoxanthin2

2.36 ± 0.06+100416, 440, 470418, 444, 4735.81all-trans-Neoxanthin1

Concentration(µg/g fw) c

Epoxide test%III/IIλmax (nm) according to

bibliography bλmax (nm)Rt (min)CarotenoidPeak

Totally esterifiedxanthophylls

Partially esterifiedxanthophylls

7

8

9

13

1415 16

17

5 611

10

127

8

9

13

12 131415

17

Figure 1 . Botanical characteristics (A), fruits (B) andgeographical distribution (C) of strawberry tree (A. unedo L.).

A C

B

Results and DiscussionMaterials and Methods Literature cited