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Carotenoids and pro-vitamin A value of white fleshed Brazilian sweet potatoes (Ipomoea batatas Lam.)
JOURNALOF
FOODCOMPOSITION
Carotenoids
AND
ANALYSIS
1,341-352
(1988)
and Pro-Vitamin A Value of White Fleshed Brazilian Sweet Potatoes (Ipomoea batatas Lam.) LIGIA B. ALMEIDA’
AND MARILENE
V. C. PENTEADO
Department0 de Alimentos e Nutri@o Experimental, Faculdade de Citbcias Farmact?uticas, Universidade de Sio Paula, Caixa Postal 30786, SZo Paula, Brazil Received March 20, 1988, and in revised form September 15, 1988 The main carotenoids of white fleshed sweet potatoes (Ipomoea batatas Lam.) consumed in .%o Paula, Brazil, were identified and their pro-vitamin A values were estimated. Raw and cooked samples obtained from CEAGESP (the main market of Sb Paulo) during the period of June 1984 till June 1986 were analyzed. The carotenoids in the raw samples were identified as luteochrome (19 I &lOO g), P-carotene 5,6,5’6’diepoxide (I 14 &lo0 g), p-carotene (6 1 wg/ 100 g), p-carotene 5,6-monoepoxide (25 pg/lOO g), l-carotene (22 &IO0 g), ol-zeacarotene (19 & 100 g), and neurosporene (13 pg/ 100 g). The pro-vitamin A value for raw sweet potatoes was 11.1 retinol equivalents and was decreased to 7.9 with 10 min of cooking. o 1988 Academic PI-es. Inc.
INTRODUCTION
The carotenoid pigments, belonging to the chemical classification of terpenoids, are widespread in nature and have a technological and nutritional importance as they are natural colorants and some act as pro-vitamin A (Bauernfeind, 198 1). Although it is a country with a high production of vegetables that are sources of carotenoids, Brazil has, in some regions, problems of hypovitaminosis A (Batista, 1969; Roncada, 1983). Researchers in the food field have suggested a reevaluation of the Food Composition Tables data of carotenoid values and vitamin A from vegetable foods (Beecher and Khachik, 1984; Rodriguez-Amaya, 1985). In other countries, the common sweet potatoes have an orange color with a high content of P-carotene. This kind of sweet potato is not marketed in the Slo Paulo region of Brazil. It is used only in the dessert and candy industries. “White” fleshed sweet potatoes, with color varying from white to light yellow, are marketed and consumed in Sao Paula. Their composition has not been previously reported. In 1985, at the CEAGESP (Companhia de Entrepostos e Armazens Gerais de SBo Paulo), the main market of Slo Paulo, 1,126,163 boxes of 25 kg of sweet potatoes were sold. The “Funda$io Instituto Brasileiro de Geografia e Estatistica” (ENDEF) values for the Sao Paul0 state region showed a range of 2446 to 11,439 metric tons for a year’s consumption of sweet potatoes. The purpose of this study was to determine the main carotenoids of the white fleshed sweet potato consumed in Sao Paulo and to estimate the pro-vitamin A activity of these roots and the loss of this activity after cooking. ’ To whom correspondence should be addressed. 341
0889-1575/88 $3.00 Copyright Q 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
342
ALMEIDA
AND PENTEADO TABLE 1
CHARACTERISTICSOFSOME CAROTENOIDSFROMWHITE FLESHEDSWEETPOTATO (Zpomoeu bat&as Lam.) CONSUMED IN SKo PAULO, BRAZIL Fraction
Identification
1 2 3
&carotene 5,6,5’,6’-diepoxide &carotene 5,6-epoxide p-carotene
4 5
Absorption on petroleum ether (nm)
Rf values on TLC
(424)
436 441 447
467 470 474
0.96 0.98 0.99
Luteochrome ol-zeacarotene
396 398
419 421
446 447
0.97 0.98
6
Neurosporene
413
436
466
0.94
7
{-carotene
378
399
424
0.99
413 419
Chemical reactions Epoxide tests Epoxide tests tram test Epoxide test Epoxide tests tram test Epoxide test tram test Epoxide tests -
+ + + + + + -
EXPERIMENTAL
Sample descriptions.Samples of white fleshed sweet potatoes were collected monthly at CEAGESP from June 1984 to June 1986. The exact cultivar is unknown, but the main cultivars for this region are IAC 66-118 (Monalisa) and IAC 27 1 (Monteiro, 1986). Sweet potatoes are available in this market all year round. Each lot of approximately 1 kg of the roots was peeled, cut in small pieces (smaller than 1 cm), and homogenized. Samples of 200 g of sweet potatoes were used for the analysis of raw roots. Separate samples of 200 g of sweet potatoes were cooked and analyzed. The cooking was done in boiling water for 10 min. The analyzes were initiated on the day the samples arrived and the experimental procedure was conducted without artifical light or direct sunlight exposure. Analytical grade solvents and reagents were used. Petroleum ether and acetone were distilled prior to use. Roche’s p-carotene standard was used for comparison of spectrum and Rfvalues of P-carotene. p- and {-carotene from carrots were used to compare the characteristics of these carotenoids. Standards obtained by synthesis in the Organic Chemistry Institut of Zurich University were used for luteochrome and 5,6,5’,6’-diepoxide-P-carotene. No standards were available for the other carotenoids. ANALYTICAL
METHODOLOGY
The method used was similar to that employed by Almeida et al. (1986, 1988) which was based on the procedure of Rodriguez et al. (1976). All the steps took place with protection against light exposure and the material with the pigments was covered with dark cloth and/or aluminum paper. The method consisted of
(a) Extraction Small pieces of roots were blended with cooled acetone (5°C) in a Waring Blendor at moderate speed for 3 min and the extract was filtered under suction. The solid
CAROTENOIDS
IN WHITE
SWEET POTATOES
343
,-
I-
I-
! I
I
I./ i
\\
t 35 WAVELENGTH
FIG. I. Absorption spectrum offl-carotene 5,6,5’,6’-diepoxide of raw white fleshed sweet potato (Ipomoea batatas Lam.), in ethanol, before (-) and after (---) the HCI reaction.
materials were reextracted with cooled acetone four more times. The carotenoids were transferred to 100 ml of light petroleum in a separation funnel and the extract was washed free of acetone by repeated additions of water. (b) Saponification
The pigments dissolved in light petroleum were treated with the same volume (approximately 200 ml) of methanolic potassium hydroxide (10%) at room temperature overnight. The alkali was removed by thorough washing in a separation funnel, and the pigment solution was dried with Na2S04 and concentrated on a rotary evaporator at 30°C. This saponification procedure has been reported to be the mildest of the several common saponification procedures (Kimura and Rodrigues-Amaya, 1987). (c) Column Chromatography
The carotenoids were separated by column chromatography in a 2 X 30 cm glass column using MgO:HyflosuperceJ (1:2) to a height of about 25cm, packed.by press-
344
ALMEIDA
AND PENTEADO
II I ./’ J
1
/
400
450
500
WAVELENGTH FIG.
2. Absorption spectrum of B-carotene 5,6-epoxide of raw white fleshed sweet potato (Ipomoea baza-
tas Lam.), in ethanol, before (-) and after (---) the HCl reaction.
ing down with a cork stopper, and with approximately 0.3 cm of anhydrous Na2S04 on the top. The mobile phase was a solution of 3% acetone in light petroleum. Each band was cut out and eluted with acetone, transferred to light petroleum, and evap orated. (d) Absorption Spectra The absorption spectra were determined with a Perkin-Elmer photometer with a R 1OOA recorder. (e) Thin-Layer
Lambda 3B spectro-
Chromatography
The TLC was developed using 20 X 20-cm plates with 0.25 mm thickness of silica gel 60 G, using a solution of 3% methanol in benzene as mobile phase.
(f) Chemical Reactions (Davies, 1976) Epoxide tests: -The chromatography plate, with the pigments adsorbed, was exposed to HCl vapor. The reaction is positive when the yellow spots become blue or green.
CAROTENOIDS
IN WHITE
I
I
400
345
SWEET POTATOES
450
w
1
500 WAVELENGTH
(nm)
FIG. 3. Absorption spectrum of &carotene of raw white fleshed sweet potato (Ipomoea batatas Lam.), in light petroleum, before (-) and after (---) the iodine isomerization.
-The presence of the 5,6-epoxide group was detected by the decrease of 20 nm in the absorption spectrum, after adding drops of 0.1 N HCl to an alcoholic solution of the pigment. This change occurs by the transformation of 5,6-epoxide group to a 5,8epoxide. c&tram isomerization: -The cis-tram isomers were detected by adding an iodine solution in petroleum ether to the cuvette immediately after recording the spectrum. After 5 min of exposure to light, the spectrum was taken again. In the caseof all-trans isomers there is an hypsochromic shift (to shorter wavelengths) as well as an increase in the cis peak. In the case of cis isomers, there is normally a bathochromic shift (to higher wavelengths) or no shift.
(g) Quantification The quantification of each carotenoid was done as recommended by Davies ( 1976) using the maximal absorbances and applying Beer’s law. The calculation of the carotenoid value was done by the utilization of the following:
pg of carotenoidlg
maximum solution absorbance X volume X lo6 of sample = 100 X E& X weight of sample
Some of the extinction coefficients (Ei&,) used were those cited by Davies ( 1976) as
346
ALMEIDA
AND PENTEADO
-\\
‘\ I-
I
400
450 WAVELENGTH
a00 ( nd
FIG. 4. Absorption spectrum of luteochrome (&carotene 5,6,5’,8’-diepoxide) of raw white fleshed sweet potato (Zpomoeu bututus Lam.), in ethanol, before (-) and after (---) the HCl reaction.
follows: 2592 for &carotene, 2450 for cY-zeacarotene, 29 I8 for neurosporene, and 2555 for l-carotene. For two epoxides the E f“, from Acemoglu and Eugster ( 1984) was used: 5,6,5’,6’diepoxide p-carotene (27 10) and luteochrome (29 16). For @carotene 5,6-epoxide the value of E& = 2549 from Eschenmoser and Eugster ( 1978) was used. The quantification of the vitamin A value was done based on the pro-vitamin A activity of each carotenoid assuming 2 1% of activity for P-carotene 5,6-epoxide, 100% of activity for ,&carotene, and none for the others (Bauernfeind, 1981). It was assumed that 6 pg of P-carotene, which has 100% of pro-vitamin A activity, corresponded to 10 IU of vitamin A or 1 retinol equivalent, according to National Academy of Sciences/National Research Council (1980).
(h) Identification The identification of these compounds was based on the position of the bands in the column, absorption spectra, chemical reactions, and comparison with the literature (Isler, 197 1; Davies, 1976; Zechmeister, 1962).
CAROTENOIDS
IN WHITE
347
SWEET POTATOES
400
450 WAVELENGTH
500 trim)
FIG. 5. Absorption spectrum of cy-zeacarotene of raw white fleshed sweet potato (Ipomoea in light petroleum, before (-) and after (---) the iodine isomerization.
batatas
Lam.),
The qualitative identification of the carotenoids was based on the UV/VIS spectrum and chemical reactions. This qualitative identification was confirmed by other techniques such as HPLC, NMR, and CD spectra (Almeida et al., 1986, 1988). RESULTS Seven carotenoids were identified in extracts of white fleshed sweet potatoes prepared as outlined in this paper. Their characteristics are listed in Table 1 and their structures can be found in Isler (197 1) numbered: 133(&carotene 5,6,5’,6’-diepoxide), 114(&carotene 5,6-epoxide), 3(P-carotene), 136(luteochrome), 12(cY-zeacarotene), 22(neurosporene), and 26({-carotene). Their absorption spectra are shown on Figs. 1 to 7. Data of Rfvalues in Table 1 were obtained with the purpose of checking the presence of certain functional groups. The main pigment was luteochrome (19 1 pg/ 100 g), followed by P-carotene 5,6,5’6’-diepoxide (114 &lo0 g), P-carotene (6 1 &lo0 g), P-carotene 5,6-epoxide (25 pg/lOO g), c-carotene (22 pg/lOO g), cw-zeacarotene ( 19 pg/ 100 g), and neurosporene(l3pg/lOOg). Of the seven carotenoids found, the only ones active as pro-vitamin A were pcarotene 5,6-epoxide (with 2 1% of activity) and P-carotene (with 100% of pro-vitamin A activity). As sweet potato is consumed cooked, we made eight paired analyses of
348
ALMEIDA
AND PENTEADO
._ 1
400
1
430
600 WAVELENGTH
( nm)
FIG.6. Absorption spectrum of neurosporene of raw white fleshed sweet potato (Ipomoea in light petroleum, before (-) and after (---) the iodine isomerization.
batatas
Lam.),
raw and cooked samples for the determination of the loss of pro-vitamin A activity from cookjng. The results are shown in Table 2. For the comparison of the results (pg/lOO g) between raw and cooked sweet potatoes and to verify the significance of statistical differences between them, we used the Student paired t test (Mood, 1963). We found for &carotene a loss of 26% by cooking, significant at 1% level, and for P-carotene 5,6-monoepoxide a loss of 5670, also significant at 1%. The first fraction was identified as ,&carotene 5,6,5’,6’-diepoxide and showed a 40nm shift in the epoxide test, indicating the presence of two epoxide groups in the position 5,6 and 5’,6’. The second fraction was identified as @-carotene 5,6-epoxide and presented a 20-nm shift in the epoxide test. The third fraction was identified as P-carotene and showed a shift to lower wavelengths after the addition of the iodine solution, characteristjc of an all-truns isomer. The fourth fraction was identified as
CAROTENOIDS
IN WHITE
400
SWEET POTATOES
450 WAVELENGTHt
349
500 nm)
FIG. 7. Absorption spectrum of c-carotene of raw white fleshed sweet potato (Zpomoea batotas Lam.), in light petroleum.
luteochrome, also an epoxide, and showed a 20-nm shift in the epoxide test; the shift is characteristic of a 5,6-epoxide. The others fractions were identified as a-zeacarotene, neurosporene, and [-carotene. We verified that the seven identified carotenoids correspond to 43.8% of luteochrome, 25.6% of &carotene 5,6,5’,6’-diepoxide, 13.7% of o-carotene, 5.6% of /3-carotene 5,6 epoxide, 4.9% of <-carotene, 4.3% of a-zeacarotene, and 2.9% of neurosporene. DISCUSSION
Martin (1983) found the following pigments in white fleshed sweet potatoes from Puerto Rico: phytofluene, S-carotene, neurosporene, /3-zeacarotene, and P-carotene. From the 15 cultivars studied, the author found the following as main pigments: p-
350
ALMEIDA
AND PENTEADO TABLE 2
CAROTENOIDS CONTENT (RETINOL EQUIVALENTS
WITH PRO-VITAMIN A ACTIVITY g) OF RAW AND CORKED (Ipomoeabatatas Lam.) CONSUMED IN
&lo0
Raw sweet potato
(&lo0 g) AND VITAMIN A VALUE WHITE FLESHED SWEET POTATOES So PAULO, BRAZIL
Cooked sweet potato
Fraction
Carotenoid
Vitamin A
Carotenoid
Vitamin A
No. of samples
j3-carotene 5,6,-epoxide B-carotene
25+ II” 611-28”
0.9 10.2
II+ 4” 4.5 * 19”
0.4 7.5
8 8
86
11.1
56
7.9
Total ’ Means + SD.
carotene in six varieties, ,&zeacarotene in seven varieties, and neurosporene in two varieties. In the same study the author mentioned the presence of epoxides that give an unpleasant green color to cooked sweet potatoes. However, there were no quantitative data nor the identification of epoxides. Of all the carotenoids mentioned by the author, the only ones found in our study were b-carotene, c-carotene, and neurosporene. Luteochrome has not been reported to be present in other sweet potatoes. Thus, the Brazilian sweet potato, having luteochrome as a primary carotenoid pigments, is in a unique carotenoid composition category. In a previous study, Almeida et al. (1986) made the first identification of luteochrome as a naturally occurring carotenoid and showed by HPLC, NMR, and CD spectra to consist of a mixture of (5R,6S,5'R,8'R)- and (5R,6S,5’R,8’S)-5,6,5’-8’,diepoxi-5,6,5’,8’,tetrahydro-pBcarotene. Many authors have studied the orange colored sweet potato. Goddard and Matthews (1979) mentioned the USDA 1978 study (USDA, 1978) which reported a variation in vitamin A content from 2 175 to 3950 IU/lOO g. Bureau and Bushway (1986) using a HPLC technique, analyzed the carotenoids of 22 fruits and vegetables from the United States; the sweet potato had one of the highest pro-vitamin A contents: 882.97 to 1712.42 retinol equivalents/l00 g. The values obtained from white fleshed sweet potato are quite different: 111 IU of vitamin A/ 100 g for raw roots and 79 IU of vitamin A/ 100 g for cooked roots, corresponding to 11 and 8 retinol equivalents, respectively. Although its occurrence is limited in nature, cY-zeacarotene was reported by Petzold et al. ( 1959) when it was isolated from corn. Buchecker and Eugster ( 1973) reported in their study the configuration of this pigment. This identification of a-zeacarotene in this work is one of the very few cases in which it has been found in nature. More work needs to be done to confirm its structure. We recognize that MgC03 is frequently added during extraction of carotenoids. However, we did not do so because sweet potato is not an acid food. Other roots such as carrots and “mandioquinha” (Arracacia xanthorrhiza Bancr.) have been analyzed using the same extraction (Almeida and Penteado, 1987a,b) and no epoxides were found. Therefore, we do not believe that epoxides found in the white sweet potatoes were artifacts of the extraction procedure.
CAROTENOIDS
IN WHITE
SWEET POTATOES
351
The pro-vitamin A carotenoids in cooked samples were not measured for this study, but will be investigated at a later date. ACKNOWLEDGMENTS Thanks are due to the Funda$o de Amparo i Pesquisa de S&o Paulo and Coordenacb de Aperfeicoamento de Pessoal de Nivel Superior for graduate fellowships granted to the first author. We also thank Financiadora de Estudos e Projetos for financial support and Produtos Roche Quimicos e Farmachticos S. A. for the standards of carotenoids. The authors are grateful to Dr. Delia Rodriguez-Amaya (Universidade Estadual de Campinas-SP, Brazil) for her kind and valuable assistance in this paper.
REFERENCES ACEMOGLU, M., AND EUGSTER,C. H. (1984). Luteochrome: Spektroskopische, chiroptische und chromatographische eingenschaften. Helv. Chim. Acta 67,2226-2230. ALMEIDA, L. B., AND PENTEADO, M. V. C. (1987a). Carotenoides e valor pro-vitaminico A de mandioquinha (Arracacia xanthorrhiza Bancr.) consumida em Sb Paulo. Rev. Farm. Bioquim. Univ. SLio Paul0 23(1),52-57.
ALMEIDA, L. B., AND FENTEAW, M. V. C. (1987b). Carotenoides corn atividade pro-vitaminica A de cenouras (Daucus carota L.) comercializadas em Sio Paulo, Brasil. Rev. Farm. Bioquim. Univ. Srio Paul0 23(2), 133-140. ALMEIDA, L. B., PENTEADO, M. V. C., BRITTON, Cl., UEBELHART, P., ACEMOGLU, M., AND EUGSTER, C. H. (1988). Isolation and absolute configuration of &P-carotene diepoxide. Helv. Chim. Acta 71( 1), 31-32.
ALMEIDA, L. B., PENTEADO, M. V. C., SIMPSON, K. L., BRITTON, G., ACEMOGLU, M., AND EUGSTER. C. H. (1986). Isolation and characterisation of (5R,6S,SR,8’R)-and (5R,6&5’R$‘S)-Luteochrome from Brazilian sweet potatoes (Zpomoea batatas Lam.). Helv. Chim. Acta 69(7), 1554-l 558. BATISTA, M. (1969). Consideracoes sobre o problema da vitamina A no nordeste brasileiro. Hospital (Rio de Janeiro) 75(3), 31-45. BAUERNFEIND, J. C. (Ed.) (198 1). Carotenoids as Colorants and Vitamin A Precursors. Academic Press, New York. BEECHER,G. R., AND KHACHIK, F. ( 1984). Evaluation of vitamin A and carotenoid data in food composition tables. J. Natl. Cancer Inst. (US.) 73(6), 1397-1404. Boletim Anual CEAGESP (1985). Sio Paulo. pp. l-99. BUCHECKER, R., AND EUGSTER, C. H. (1973). Absolute konfiguration von a-zeacarotin, a-apo-8-carotinal und cY-apo-8-carotinol. Helv. Chim. Acta 56(3), 110-I 11. BUREAU, J. L., AND BUSHWAY, R. J. (1986). HPLC determination of carotenoids in fruits and vegetables in the United States. J. Food Sci. 51( 1), 128- 130. DAVIES, B. H. (1976). Carotenoids. In (T. W. GOODWIN Ed.) Chemistry and Biochemistry of Plant Pigments, 2nd ed., Vol. 2, pp. 38-165. Academic Press, London. ESCHENMOSER,W., AND EUGSTER, C. H. (1978). Synthese und chiralitiit von (5S,6R)-5,6-epoxy-5,6dihydro-fi-Scarotin und (SR,6R)-5,6-dihydro-&%carotin,5,6-diol, einem carotinoid mit ungewohnlichen eingenschaften. Helv. Chim. Acta 61(2), 822-83 1. Fundacio Instituto Brasileiro de Geografia e Estahstica (1977). Consumo Alimentar: Antropometria, Pte 3: Regib II, Sb Paulo, Regiao IV, Minas Gerais e Espirito Santo, Rio de Janeiro, p. 35-38. Estudo National da Despesa Familiar, v. 1; dados preliminares, t. 1. (ENDEF) GODDARD, M. S., AND MATTHEWS, R. H. (1979). Current knowledge of nutritive values of vegetables. Food Technol. 33(2), 7 l-73. ISLER, 0. (197 1). Carotenoids, p. 799. Basle, Birkiauser. KIMURA, M., AND RODRIGUEZ-AMAYA, D. B. (1987). Assessment of the saponification step in the quantitative determination of carotenoids and pro-vitamin A. In Proceedings of 8th International Symposium on Carotenoids, (Speech). Boston, Vol. 8. MARTIN, F. W. (1983). The carotenoids pigments of white-fleshed sweet potatoes. J. Agric. Univ. P. R. 67(4),494-500.
352
ALMEIDA
AND PENTEADO
MONTEIRO, D. A. (1986). Batatadoce (Zpomoea batatas (L.) Lam.). Bol. Inst. Agron. Camp&as 200,4041. Moor, A. L., AND GRAYBILL, F. A. (1963). Introduction to the Theory of Statistics, 2nd ed. McGrawHill, New York. National Academy of Sciences/National Research Council (1980). Recommended Dietary Allowance. 9th ed., pp. 55-60. Washington, DC. PETZOLD, E. N., QUACKENBUSH, F. W., AND MCQUISTAN, M. (1959). Zeacarotenes, new provitamins A from corn. Arch. B&hem. Biophys. 82, 117- 124. RODRIGUEZ, D. B., RAYMUNW, L. C., LEE, T. C., SIMPSON, K. L., AND CHICHESTER, C. 0. (1976). Carotenoid pigments changes in ripening Momordica charantia fruits. Ann. Bot. (London)40,6 15-624. RODRIGUEZ-AMAYA, D. B. (1985). OS carotenoides coma precursores de vitamina A. Bol. Sot. Bras. Cit%c. Tecnol. Aliment. Campinas 19(4), 227-242. RONCADA, M. J. (1983). Hypovitaminosis A: A public health problem. Znt.J. Vitam.Nutr. Res.Suppl. 24, 17-22. USDA (1978). Nutritional analysis of sweet potatoes. Report S-101 Regional Tech. Comm., U.S. Dept. of Agriculture, Washington, D.C. Apud: GODDARD, M. S. & MATTHEWS, R. H. (1979)-Current knowledge of nutritive values of vegetables. Food Technol. 33(2), 7 1-73. ZECHMEISTER, L. ( 1962). cis-trans Zsomeric Carotenoids, Vitamin A and AryIpolyenes, p. 119. Academic Press, New York.
FOODCOMPOSITION
Carotenoids
AND
ANALYSIS
1,341-352
(1988)
and Pro-Vitamin A Value of White Fleshed Brazilian Sweet Potatoes (Ipomoea batatas Lam.) LIGIA B. ALMEIDA’
AND MARILENE
V. C. PENTEADO
Department0 de Alimentos e Nutri@o Experimental, Faculdade de Citbcias Farmact?uticas, Universidade de Sio Paula, Caixa Postal 30786, SZo Paula, Brazil Received March 20, 1988, and in revised form September 15, 1988 The main carotenoids of white fleshed sweet potatoes (Ipomoea batatas Lam.) consumed in .%o Paula, Brazil, were identified and their pro-vitamin A values were estimated. Raw and cooked samples obtained from CEAGESP (the main market of Sb Paulo) during the period of June 1984 till June 1986 were analyzed. The carotenoids in the raw samples were identified as luteochrome (19 I &lOO g), P-carotene 5,6,5’6’diepoxide (I 14 &lo0 g), p-carotene (6 1 wg/ 100 g), p-carotene 5,6-monoepoxide (25 pg/lOO g), l-carotene (22 &IO0 g), ol-zeacarotene (19 & 100 g), and neurosporene (13 pg/ 100 g). The pro-vitamin A value for raw sweet potatoes was 11.1 retinol equivalents and was decreased to 7.9 with 10 min of cooking. o 1988 Academic PI-es. Inc.
INTRODUCTION
The carotenoid pigments, belonging to the chemical classification of terpenoids, are widespread in nature and have a technological and nutritional importance as they are natural colorants and some act as pro-vitamin A (Bauernfeind, 198 1). Although it is a country with a high production of vegetables that are sources of carotenoids, Brazil has, in some regions, problems of hypovitaminosis A (Batista, 1969; Roncada, 1983). Researchers in the food field have suggested a reevaluation of the Food Composition Tables data of carotenoid values and vitamin A from vegetable foods (Beecher and Khachik, 1984; Rodriguez-Amaya, 1985). In other countries, the common sweet potatoes have an orange color with a high content of P-carotene. This kind of sweet potato is not marketed in the Slo Paulo region of Brazil. It is used only in the dessert and candy industries. “White” fleshed sweet potatoes, with color varying from white to light yellow, are marketed and consumed in Sao Paula. Their composition has not been previously reported. In 1985, at the CEAGESP (Companhia de Entrepostos e Armazens Gerais de SBo Paulo), the main market of Slo Paulo, 1,126,163 boxes of 25 kg of sweet potatoes were sold. The “Funda$io Instituto Brasileiro de Geografia e Estatistica” (ENDEF) values for the Sao Paul0 state region showed a range of 2446 to 11,439 metric tons for a year’s consumption of sweet potatoes. The purpose of this study was to determine the main carotenoids of the white fleshed sweet potato consumed in Sao Paulo and to estimate the pro-vitamin A activity of these roots and the loss of this activity after cooking. ’ To whom correspondence should be addressed. 341
0889-1575/88 $3.00 Copyright Q 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
342
ALMEIDA
AND PENTEADO TABLE 1
CHARACTERISTICSOFSOME CAROTENOIDSFROMWHITE FLESHEDSWEETPOTATO (Zpomoeu bat&as Lam.) CONSUMED IN SKo PAULO, BRAZIL Fraction
Identification
1 2 3
&carotene 5,6,5’,6’-diepoxide &carotene 5,6-epoxide p-carotene
4 5
Absorption on petroleum ether (nm)
Rf values on TLC
(424)
436 441 447
467 470 474
0.96 0.98 0.99
Luteochrome ol-zeacarotene
396 398
419 421
446 447
0.97 0.98
6
Neurosporene
413
436
466
0.94
7
{-carotene
378
399
424
0.99
413 419
Chemical reactions Epoxide tests Epoxide tests tram test Epoxide test Epoxide tests tram test Epoxide test tram test Epoxide tests -
+ + + + + + -
EXPERIMENTAL
Sample descriptions.Samples of white fleshed sweet potatoes were collected monthly at CEAGESP from June 1984 to June 1986. The exact cultivar is unknown, but the main cultivars for this region are IAC 66-118 (Monalisa) and IAC 27 1 (Monteiro, 1986). Sweet potatoes are available in this market all year round. Each lot of approximately 1 kg of the roots was peeled, cut in small pieces (smaller than 1 cm), and homogenized. Samples of 200 g of sweet potatoes were used for the analysis of raw roots. Separate samples of 200 g of sweet potatoes were cooked and analyzed. The cooking was done in boiling water for 10 min. The analyzes were initiated on the day the samples arrived and the experimental procedure was conducted without artifical light or direct sunlight exposure. Analytical grade solvents and reagents were used. Petroleum ether and acetone were distilled prior to use. Roche’s p-carotene standard was used for comparison of spectrum and Rfvalues of P-carotene. p- and {-carotene from carrots were used to compare the characteristics of these carotenoids. Standards obtained by synthesis in the Organic Chemistry Institut of Zurich University were used for luteochrome and 5,6,5’,6’-diepoxide-P-carotene. No standards were available for the other carotenoids. ANALYTICAL
METHODOLOGY
The method used was similar to that employed by Almeida et al. (1986, 1988) which was based on the procedure of Rodriguez et al. (1976). All the steps took place with protection against light exposure and the material with the pigments was covered with dark cloth and/or aluminum paper. The method consisted of
(a) Extraction Small pieces of roots were blended with cooled acetone (5°C) in a Waring Blendor at moderate speed for 3 min and the extract was filtered under suction. The solid
CAROTENOIDS
IN WHITE
SWEET POTATOES
343
,-
I-
I-
! I
I
I./ i
\\
t 35 WAVELENGTH
FIG. I. Absorption spectrum offl-carotene 5,6,5’,6’-diepoxide of raw white fleshed sweet potato (Ipomoea batatas Lam.), in ethanol, before (-) and after (---) the HCI reaction.
materials were reextracted with cooled acetone four more times. The carotenoids were transferred to 100 ml of light petroleum in a separation funnel and the extract was washed free of acetone by repeated additions of water. (b) Saponification
The pigments dissolved in light petroleum were treated with the same volume (approximately 200 ml) of methanolic potassium hydroxide (10%) at room temperature overnight. The alkali was removed by thorough washing in a separation funnel, and the pigment solution was dried with Na2S04 and concentrated on a rotary evaporator at 30°C. This saponification procedure has been reported to be the mildest of the several common saponification procedures (Kimura and Rodrigues-Amaya, 1987). (c) Column Chromatography
The carotenoids were separated by column chromatography in a 2 X 30 cm glass column using MgO:HyflosuperceJ (1:2) to a height of about 25cm, packed.by press-
344
ALMEIDA
AND PENTEADO
II I ./’ J
1
/
400
450
500
WAVELENGTH FIG.
2. Absorption spectrum of B-carotene 5,6-epoxide of raw white fleshed sweet potato (Ipomoea baza-
tas Lam.), in ethanol, before (-) and after (---) the HCl reaction.
ing down with a cork stopper, and with approximately 0.3 cm of anhydrous Na2S04 on the top. The mobile phase was a solution of 3% acetone in light petroleum. Each band was cut out and eluted with acetone, transferred to light petroleum, and evap orated. (d) Absorption Spectra The absorption spectra were determined with a Perkin-Elmer photometer with a R 1OOA recorder. (e) Thin-Layer
Lambda 3B spectro-
Chromatography
The TLC was developed using 20 X 20-cm plates with 0.25 mm thickness of silica gel 60 G, using a solution of 3% methanol in benzene as mobile phase.
(f) Chemical Reactions (Davies, 1976) Epoxide tests: -The chromatography plate, with the pigments adsorbed, was exposed to HCl vapor. The reaction is positive when the yellow spots become blue or green.
CAROTENOIDS
IN WHITE
I
I
400
345
SWEET POTATOES
450
w
1
500 WAVELENGTH
(nm)
FIG. 3. Absorption spectrum of &carotene of raw white fleshed sweet potato (Ipomoea batatas Lam.), in light petroleum, before (-) and after (---) the iodine isomerization.
-The presence of the 5,6-epoxide group was detected by the decrease of 20 nm in the absorption spectrum, after adding drops of 0.1 N HCl to an alcoholic solution of the pigment. This change occurs by the transformation of 5,6-epoxide group to a 5,8epoxide. c&tram isomerization: -The cis-tram isomers were detected by adding an iodine solution in petroleum ether to the cuvette immediately after recording the spectrum. After 5 min of exposure to light, the spectrum was taken again. In the caseof all-trans isomers there is an hypsochromic shift (to shorter wavelengths) as well as an increase in the cis peak. In the case of cis isomers, there is normally a bathochromic shift (to higher wavelengths) or no shift.
(g) Quantification The quantification of each carotenoid was done as recommended by Davies ( 1976) using the maximal absorbances and applying Beer’s law. The calculation of the carotenoid value was done by the utilization of the following:
pg of carotenoidlg
maximum solution absorbance X volume X lo6 of sample = 100 X E& X weight of sample
Some of the extinction coefficients (Ei&,) used were those cited by Davies ( 1976) as
346
ALMEIDA
AND PENTEADO
-\\
‘\ I-
I
400
450 WAVELENGTH
a00 ( nd
FIG. 4. Absorption spectrum of luteochrome (&carotene 5,6,5’,8’-diepoxide) of raw white fleshed sweet potato (Zpomoeu bututus Lam.), in ethanol, before (-) and after (---) the HCl reaction.
follows: 2592 for &carotene, 2450 for cY-zeacarotene, 29 I8 for neurosporene, and 2555 for l-carotene. For two epoxides the E f“, from Acemoglu and Eugster ( 1984) was used: 5,6,5’,6’diepoxide p-carotene (27 10) and luteochrome (29 16). For @carotene 5,6-epoxide the value of E& = 2549 from Eschenmoser and Eugster ( 1978) was used. The quantification of the vitamin A value was done based on the pro-vitamin A activity of each carotenoid assuming 2 1% of activity for P-carotene 5,6-epoxide, 100% of activity for ,&carotene, and none for the others (Bauernfeind, 1981). It was assumed that 6 pg of P-carotene, which has 100% of pro-vitamin A activity, corresponded to 10 IU of vitamin A or 1 retinol equivalent, according to National Academy of Sciences/National Research Council (1980).
(h) Identification The identification of these compounds was based on the position of the bands in the column, absorption spectra, chemical reactions, and comparison with the literature (Isler, 197 1; Davies, 1976; Zechmeister, 1962).
CAROTENOIDS
IN WHITE
347
SWEET POTATOES
400
450 WAVELENGTH
500 trim)
FIG. 5. Absorption spectrum of cy-zeacarotene of raw white fleshed sweet potato (Ipomoea in light petroleum, before (-) and after (---) the iodine isomerization.
batatas
Lam.),
The qualitative identification of the carotenoids was based on the UV/VIS spectrum and chemical reactions. This qualitative identification was confirmed by other techniques such as HPLC, NMR, and CD spectra (Almeida et al., 1986, 1988). RESULTS Seven carotenoids were identified in extracts of white fleshed sweet potatoes prepared as outlined in this paper. Their characteristics are listed in Table 1 and their structures can be found in Isler (197 1) numbered: 133(&carotene 5,6,5’,6’-diepoxide), 114(&carotene 5,6-epoxide), 3(P-carotene), 136(luteochrome), 12(cY-zeacarotene), 22(neurosporene), and 26({-carotene). Their absorption spectra are shown on Figs. 1 to 7. Data of Rfvalues in Table 1 were obtained with the purpose of checking the presence of certain functional groups. The main pigment was luteochrome (19 1 pg/ 100 g), followed by P-carotene 5,6,5’6’-diepoxide (114 &lo0 g), P-carotene (6 1 &lo0 g), P-carotene 5,6-epoxide (25 pg/lOO g), c-carotene (22 pg/lOO g), cw-zeacarotene ( 19 pg/ 100 g), and neurosporene(l3pg/lOOg). Of the seven carotenoids found, the only ones active as pro-vitamin A were pcarotene 5,6-epoxide (with 2 1% of activity) and P-carotene (with 100% of pro-vitamin A activity). As sweet potato is consumed cooked, we made eight paired analyses of
348
ALMEIDA
AND PENTEADO
._ 1
400
1
430
600 WAVELENGTH
( nm)
FIG.6. Absorption spectrum of neurosporene of raw white fleshed sweet potato (Ipomoea in light petroleum, before (-) and after (---) the iodine isomerization.
batatas
Lam.),
raw and cooked samples for the determination of the loss of pro-vitamin A activity from cookjng. The results are shown in Table 2. For the comparison of the results (pg/lOO g) between raw and cooked sweet potatoes and to verify the significance of statistical differences between them, we used the Student paired t test (Mood, 1963). We found for &carotene a loss of 26% by cooking, significant at 1% level, and for P-carotene 5,6-monoepoxide a loss of 5670, also significant at 1%. The first fraction was identified as ,&carotene 5,6,5’,6’-diepoxide and showed a 40nm shift in the epoxide test, indicating the presence of two epoxide groups in the position 5,6 and 5’,6’. The second fraction was identified as @-carotene 5,6-epoxide and presented a 20-nm shift in the epoxide test. The third fraction was identified as P-carotene and showed a shift to lower wavelengths after the addition of the iodine solution, characteristjc of an all-truns isomer. The fourth fraction was identified as
CAROTENOIDS
IN WHITE
400
SWEET POTATOES
450 WAVELENGTHt
349
500 nm)
FIG. 7. Absorption spectrum of c-carotene of raw white fleshed sweet potato (Zpomoea batotas Lam.), in light petroleum.
luteochrome, also an epoxide, and showed a 20-nm shift in the epoxide test; the shift is characteristic of a 5,6-epoxide. The others fractions were identified as a-zeacarotene, neurosporene, and [-carotene. We verified that the seven identified carotenoids correspond to 43.8% of luteochrome, 25.6% of &carotene 5,6,5’,6’-diepoxide, 13.7% of o-carotene, 5.6% of /3-carotene 5,6 epoxide, 4.9% of <-carotene, 4.3% of a-zeacarotene, and 2.9% of neurosporene. DISCUSSION
Martin (1983) found the following pigments in white fleshed sweet potatoes from Puerto Rico: phytofluene, S-carotene, neurosporene, /3-zeacarotene, and P-carotene. From the 15 cultivars studied, the author found the following as main pigments: p-
350
ALMEIDA
AND PENTEADO TABLE 2
CAROTENOIDS CONTENT (RETINOL EQUIVALENTS
WITH PRO-VITAMIN A ACTIVITY g) OF RAW AND CORKED (Ipomoeabatatas Lam.) CONSUMED IN
&lo0
Raw sweet potato
(&lo0 g) AND VITAMIN A VALUE WHITE FLESHED SWEET POTATOES So PAULO, BRAZIL
Cooked sweet potato
Fraction
Carotenoid
Vitamin A
Carotenoid
Vitamin A
No. of samples
j3-carotene 5,6,-epoxide B-carotene
25+ II” 611-28”
0.9 10.2
II+ 4” 4.5 * 19”
0.4 7.5
8 8
86
11.1
56
7.9
Total ’ Means + SD.
carotene in six varieties, ,&zeacarotene in seven varieties, and neurosporene in two varieties. In the same study the author mentioned the presence of epoxides that give an unpleasant green color to cooked sweet potatoes. However, there were no quantitative data nor the identification of epoxides. Of all the carotenoids mentioned by the author, the only ones found in our study were b-carotene, c-carotene, and neurosporene. Luteochrome has not been reported to be present in other sweet potatoes. Thus, the Brazilian sweet potato, having luteochrome as a primary carotenoid pigments, is in a unique carotenoid composition category. In a previous study, Almeida et al. (1986) made the first identification of luteochrome as a naturally occurring carotenoid and showed by HPLC, NMR, and CD spectra to consist of a mixture of (5R,6S,5'R,8'R)- and (5R,6S,5’R,8’S)-5,6,5’-8’,diepoxi-5,6,5’,8’,tetrahydro-pBcarotene. Many authors have studied the orange colored sweet potato. Goddard and Matthews (1979) mentioned the USDA 1978 study (USDA, 1978) which reported a variation in vitamin A content from 2 175 to 3950 IU/lOO g. Bureau and Bushway (1986) using a HPLC technique, analyzed the carotenoids of 22 fruits and vegetables from the United States; the sweet potato had one of the highest pro-vitamin A contents: 882.97 to 1712.42 retinol equivalents/l00 g. The values obtained from white fleshed sweet potato are quite different: 111 IU of vitamin A/ 100 g for raw roots and 79 IU of vitamin A/ 100 g for cooked roots, corresponding to 11 and 8 retinol equivalents, respectively. Although its occurrence is limited in nature, cY-zeacarotene was reported by Petzold et al. ( 1959) when it was isolated from corn. Buchecker and Eugster ( 1973) reported in their study the configuration of this pigment. This identification of a-zeacarotene in this work is one of the very few cases in which it has been found in nature. More work needs to be done to confirm its structure. We recognize that MgC03 is frequently added during extraction of carotenoids. However, we did not do so because sweet potato is not an acid food. Other roots such as carrots and “mandioquinha” (Arracacia xanthorrhiza Bancr.) have been analyzed using the same extraction (Almeida and Penteado, 1987a,b) and no epoxides were found. Therefore, we do not believe that epoxides found in the white sweet potatoes were artifacts of the extraction procedure.
CAROTENOIDS
IN WHITE
SWEET POTATOES
351
The pro-vitamin A carotenoids in cooked samples were not measured for this study, but will be investigated at a later date. ACKNOWLEDGMENTS Thanks are due to the Funda$o de Amparo i Pesquisa de S&o Paulo and Coordenacb de Aperfeicoamento de Pessoal de Nivel Superior for graduate fellowships granted to the first author. We also thank Financiadora de Estudos e Projetos for financial support and Produtos Roche Quimicos e Farmachticos S. A. for the standards of carotenoids. The authors are grateful to Dr. Delia Rodriguez-Amaya (Universidade Estadual de Campinas-SP, Brazil) for her kind and valuable assistance in this paper.
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ALMEIDA, L. B., AND FENTEAW, M. V. C. (1987b). Carotenoides corn atividade pro-vitaminica A de cenouras (Daucus carota L.) comercializadas em Sio Paulo, Brasil. Rev. Farm. Bioquim. Univ. Srio Paul0 23(2), 133-140. ALMEIDA, L. B., PENTEADO, M. V. C., BRITTON, Cl., UEBELHART, P., ACEMOGLU, M., AND EUGSTER, C. H. (1988). Isolation and absolute configuration of &P-carotene diepoxide. Helv. Chim. Acta 71( 1), 31-32.
ALMEIDA, L. B., PENTEADO, M. V. C., SIMPSON, K. L., BRITTON, G., ACEMOGLU, M., AND EUGSTER. C. H. (1986). Isolation and characterisation of (5R,6S,SR,8’R)-and (5R,6&5’R$‘S)-Luteochrome from Brazilian sweet potatoes (Zpomoea batatas Lam.). Helv. Chim. Acta 69(7), 1554-l 558. BATISTA, M. (1969). Consideracoes sobre o problema da vitamina A no nordeste brasileiro. Hospital (Rio de Janeiro) 75(3), 31-45. BAUERNFEIND, J. C. (Ed.) (198 1). Carotenoids as Colorants and Vitamin A Precursors. Academic Press, New York. BEECHER,G. R., AND KHACHIK, F. ( 1984). Evaluation of vitamin A and carotenoid data in food composition tables. J. Natl. Cancer Inst. (US.) 73(6), 1397-1404. Boletim Anual CEAGESP (1985). Sio Paulo. pp. l-99. BUCHECKER, R., AND EUGSTER, C. H. (1973). Absolute konfiguration von a-zeacarotin, a-apo-8-carotinal und cY-apo-8-carotinol. Helv. Chim. Acta 56(3), 110-I 11. BUREAU, J. L., AND BUSHWAY, R. J. (1986). HPLC determination of carotenoids in fruits and vegetables in the United States. J. Food Sci. 51( 1), 128- 130. DAVIES, B. H. (1976). Carotenoids. In (T. W. GOODWIN Ed.) Chemistry and Biochemistry of Plant Pigments, 2nd ed., Vol. 2, pp. 38-165. Academic Press, London. ESCHENMOSER,W., AND EUGSTER, C. H. (1978). Synthese und chiralitiit von (5S,6R)-5,6-epoxy-5,6dihydro-fi-Scarotin und (SR,6R)-5,6-dihydro-&%carotin,5,6-diol, einem carotinoid mit ungewohnlichen eingenschaften. Helv. Chim. Acta 61(2), 822-83 1. Fundacio Instituto Brasileiro de Geografia e Estahstica (1977). Consumo Alimentar: Antropometria, Pte 3: Regib II, Sb Paulo, Regiao IV, Minas Gerais e Espirito Santo, Rio de Janeiro, p. 35-38. Estudo National da Despesa Familiar, v. 1; dados preliminares, t. 1. (ENDEF) GODDARD, M. S., AND MATTHEWS, R. H. (1979). Current knowledge of nutritive values of vegetables. Food Technol. 33(2), 7 l-73. ISLER, 0. (197 1). Carotenoids, p. 799. Basle, Birkiauser. KIMURA, M., AND RODRIGUEZ-AMAYA, D. B. (1987). Assessment of the saponification step in the quantitative determination of carotenoids and pro-vitamin A. In Proceedings of 8th International Symposium on Carotenoids, (Speech). Boston, Vol. 8. MARTIN, F. W. (1983). The carotenoids pigments of white-fleshed sweet potatoes. J. Agric. Univ. P. R. 67(4),494-500.
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AND PENTEADO
MONTEIRO, D. A. (1986). Batatadoce (Zpomoea batatas (L.) Lam.). Bol. Inst. Agron. Camp&as 200,4041. Moor, A. L., AND GRAYBILL, F. A. (1963). Introduction to the Theory of Statistics, 2nd ed. McGrawHill, New York. National Academy of Sciences/National Research Council (1980). Recommended Dietary Allowance. 9th ed., pp. 55-60. Washington, DC. PETZOLD, E. N., QUACKENBUSH, F. W., AND MCQUISTAN, M. (1959). Zeacarotenes, new provitamins A from corn. Arch. B&hem. Biophys. 82, 117- 124. RODRIGUEZ, D. B., RAYMUNW, L. C., LEE, T. C., SIMPSON, K. L., AND CHICHESTER, C. 0. (1976). Carotenoid pigments changes in ripening Momordica charantia fruits. Ann. Bot. (London)40,6 15-624. RODRIGUEZ-AMAYA, D. B. (1985). OS carotenoides coma precursores de vitamina A. Bol. Sot. Bras. Cit%c. Tecnol. Aliment. Campinas 19(4), 227-242. RONCADA, M. J. (1983). Hypovitaminosis A: A public health problem. Znt.J. Vitam.Nutr. Res.Suppl. 24, 17-22. USDA (1978). Nutritional analysis of sweet potatoes. Report S-101 Regional Tech. Comm., U.S. Dept. of Agriculture, Washington, D.C. Apud: GODDARD, M. S. & MATTHEWS, R. H. (1979)-Current knowledge of nutritive values of vegetables. Food Technol. 33(2), 7 1-73. ZECHMEISTER, L. ( 1962). cis-trans Zsomeric Carotenoids, Vitamin A and AryIpolyenes, p. 119. Academic Press, New York.