AKADÉMIAI DOKTORI ÉRTEKEZÉS
PAPRIKAKAROTINOIDOK VIZSGÁLATA:
ANALÍZIS, IZOLÁLÁS, SZERKEZETAZONOSÍTÁS
Melléklet
Deli József
Készült
a Pécsi Tudományegyetem Általános Orvostudományi Karának
Orvosi Kémiai Intézetében
Pécs, 2001
2
9*. Deli, J., Molnár P., Matus, Z., Tóth, G. Steck, A: Reisolation of Carotenoid 3,6- Epoxides from Red Paprika (Capsicum annuum)
Helvetica ChimicaActa 79, 1435-1443 (1996)
10*. Deli, J., Matus, Z., Molnár, P., Tóth, G., Décsy, Z., Eugster, C. H.: Epoxidierung von Cucurbitaxanthin A: Herstellung von Cucurbitaxanthin B und seines 5',6'-Epimeren Helvetica ChimicaActa 76,952-956 (1993)
11*. Deli, J., Molnár, P., Matus, Z., Tóth G., Steck, A., Pfander, H.: Isolation of Carotenoids with 3,5,6-Trihydroxy-5,6-dihydro-/?-end Groups from Red Paprika {Capsicum annuum)
Helvetica ChimicaActa 81, 1233-1241 (1998)
12*. Deli, J., Molnár, P., Matus, Z., Tóth G., Steck, A., Pfander, H.: Partial Synthesis and Characterization of Capsokarpoxanthins and 3,6-Epoxycapsanthins
Helvetica ChimicaActa 81,1242-1253 (1998)
13*. Molnár, P., Deli, J., Matus, Z., Tóth G., Steck, A., Pfander, H.: Partial Synthesis and Characterization of Karpoxanthins and Cucurbitaxanthin A Epimers
Helvetica ChimicaActa 82,1994-2002 (1999)
14*. Molnár, P., Deli, J., Matus, Z., Tóth, G., Steck A., Pfander H.: Isolation and Characterization of Mutatoxanthin-epimers from Red Paprika {Capsicum annuum) European Food Research and Technology 211,396-399 (2000)
15*. Deli, J., Matus, Z., Molnár, P., Tóth, G., Szalontai, G., Steck, A., Pfander, H.:
Nigroxanthin (3',4'-Didehydro-yS
>^-carotene-3,6'-diol), a New Carotenoid Isolated from Paprika {Capsicum annuum var. longum nigrum)
Chimin 48,102-104 (1994)
16*. Deli, J., Molnár, P., Matus, Z., Tóth, G., Traber, B., Pfander, H.: „Prenigroxanthin"
[(all-£,3/?,3'5,6'S)-ß,Y-Carotene-3,3'6'-triol], a Novel Carotenoid from Red Paprika {Capsicum annuum)
Tetrahedron Letters 42,1395-1397 (2001)
17*. Deli, J., Matus, Z., Molnár, P., Tóth, G., Steck, A., Pfander, H.: Isolation of Capsanthone ((a//-E,3Ä,5'/2)-3-Hydroxy-/9,x'-carotene-3
,,6
,-dione) from Paprika {Capsicum annuum)
Chimin 49,69-71 (1995)
3
18*. Deli, J., Molnár, P., Matus, Z., Tóth G., Steck, A., Pfander, H.: Isolation and Characterization of 3,5,6-Trihydroxy-Carotenoids from Petals of Lilium tigrinum Chromatographic! 48,27-31 (1998)
19*. Deli, J., Molnár, P., Pfander, H., Tóth, G.: Isolation of Capsanthin 5,6-Epoxide from Lilium tigrinum
Acta Botanica Hungarica 42,105-110 (1999/2000)
20*. Deli, J., Molnár, P., Matus, Z., Tóth G., Steck, A., Niggli, U. A., Pfander, H.:
Aesculaxanthin, a New Carotenoid Isolated from Pollens of Aesculus hippocastanum Helvetica ChimicaActa 81,1815-1820 (1998)
21*. Deli, J., Matus, Z., Tóth, G.: Comparative Study on the Carotenoid Composition in the Buds and Flowers of Different Aesculus species
Chromatographia 51, S179-182 (2000)
22*. Deli, J., Matus, Z., Tóth, G.: Carotenoid Composition in the Fruits of Asparagus officinalis
Journal of Agricultural and Food Chemistry 48,2793-2796 (2000)
23*. Deli, J., Molnár, P., Ősz, E., Tóth, G.: Analysis of Carotenoids in the Fruits of Asparagus falcatus: Isolation of 5,6-Diepikarpoxanthin
Chromatographia 51, S183-187 (2000)
24*. Deli, J., Molnár P., Ősz E., Tóth, G.: Capsoneoxanthin, a New Carotenoid Isolated from the Fruits of Asparagus falcatus
Tetrahedron Letters 41, 8153-8155 (2000) 25 *. Deli, J. : Paprikakarotinoidok bioszintézise
Biokémia 24, 84-87 (2000)
26*. Deli, J.: Thin-Layer Chromatography of Carotenoids
Journal of Planar Chromatography - Modern TLC11,311-312 (1998)
Reprinted from JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 1991, 39. 1907 Copyright © 1991 by the American Chemical Society and reprinted by permission of the copyright owner.
Carotenoid Composition of Yellow Pepper during Ripening: Isolation of /9-Cryptoxanthin 5,6-Epoxide
Zoltán Matus, József Deli, and József Szabolcs*
Institute of Chemistry, Pécs University Medical School, Szigeti út 12, H-7643 Pécs, Hungary
Using a HPLC technique, 54 peaks have been detected in the fruits of yellow pepper; 32 carotenoids (90-94 % of the total carotenoid content) have been completely or tentatively identified. The study was carried out quantitatively with fruits in six different stages of maturation. Violaxanthin, anther- axanthin, zeaxanthin, lutein, /3-cryptoxanthin, /S-carotene, a-cryptoxanthin, a-carotene, and probably
"post-mortem artifacts" (furanoid oxides and cis isomers) were found. /3-Cryptoxanthin 5,6-epoxide, a precursor of cryptocapsin present in red pepper, was also identified. But carotenoids with *-, 5,6- dihydro-3,5,6,-trihydroxy-/J, and 5,6-dihydro-3,6-epoxy-5-hydroxy-/3 (oxabicyclo[2.2.1]) end groups usually found in red pepper were not detected. In the massive biosynthesis of violaxanthin during maturation, the rates of hydroxylation exceeded those of epoxidation.
INTRODUCTION
Although paprika (red pepper), as one of the most important and oldest natural food colors, has been investigated for a long time, its yellow variety, whose color never turns red during ripening, has received little attention (Klaui and Bauernfeind, 1981). The importance of yellow pepper, however, is reflected in its flavoring property and certain vitamin A and C activities. Research on the distribution of carotenoids present in the yellow fruits is also of great importance, as it might provide some clue to the understanding of the biosynthetic pathways of formation not only of yellow carotenoids in yellow paprika but also of both yellow and red carotenoids present in red paprika.
A detailed pioneer work (Cholnoky et al., 1958) on the qualitative and quantitative distribution of carotenoids in yellow paprika (Capsicum annuum lycopersiciforme flavum) revealed that the yellow fruits, although containing a large amount of 5,6-epoxy carotenoids (antheraxanthin, violaxanthin) from which the red carotenoids are formed via pinacolone rearrangement (Weedon, 1971) in red pepper (C. annuum lycopersiciforme rubrum), were lacking the red carotenoids (capsanthin, capsorubin, cryp- tocapsin).
The objectives of this work were to study the quantitative changes of carotenoids in a particular variety of yellow paprika (Szentesi sarga paradicsom paprika) during rip- ening and to correlate the carotenoid biosynthesis of C.
annuum lycopersiciforme flavum to that of C. annuum lycopersiciforme rubrum, first of all, with respect to new minor carotenoids with 5,6-dihydro-3,5,6-trihydroxy-jS and 5,6-dihydro-3,6-epoxy-5-hydroxy-j9 (oxabicyclo[2.2.lj) end groups present in red pepper (Parkes et al., 1986). It was also considered whether cis isomers and furanoid oxides present in pepper were natural or artificial products.
MATERIALS AND METHODS
The yellow peppers used (C. annuum lycopersiciforme fla- vum, cv. Szentesi sarga paradicsom paprika) were obtained commercially in P6cs (southern Hungary) in August 1988.
General methods of handling including routine tests (partition coefficients, epoxide-furanoid oxide rearrangement, reduction of carbonyl functions with metal hydrides, etc.) and column chro- matography have been described elsewhere (Molnar and Szabolcs, 1979). The quantitative determination of the total carotenoid content of fruits was performed in the absence of chlorophylls
a b e d
Or
eJ X r
IOr Or
9 hjOf i r -tT"
0 O H
! 1 "
Antheraxanthin, R = c, Q = b; auroxanthin, R = Q = d; canthaxanthin, R = Q = j; a-carotene, R = a, Q = e; p-carotene, R = Q = a; p-carotene monoepoxide, R = g, Q = a; cryptocapsin, R = a, Q = k; a-cryptoxanthin, R = b, Q = e; p-cryploxanthin, R = b, Q = a; p-cryptoxanthin 5,6-epoxide, R = c, Q = a; lutein, R = b, Q = t; lutein epoxide, R = c, O = f; luteoxanthin, R = c, Q = d; mutatochrome, R = h, Q = a; mutatoxanthin, R = d, Q = b;
neochrome, R = d, Q = i; neoxanthin X, R = c, Q = i; violaxanthin, R = Q = c; zeaxanthin, R = Q = b.
(Davies, 1976). The quantitative determination of chlorophylls was carried out in an ethereal extract (Comar and Zscheile, 1942).
Analytical grade chemicals were used, and authentic samples were taken from our collection. The sample solutions were stored under nitrogen, away from light at -20 °C.
Instruments. Melting points were determined with a Boe- tius hot-stage apparatus and were not corrected. UV-vis spectra were recorded with a Perkin-Elmer 402 instrument. CD spectra were taken in MeOH solutions at room temperature on a Roussel- Juan Dichrographe HI (Jobin Ivon), in quartz cells.
High-Performance Liquid Chromatography. Selection of a Chromatographic Approach. Considering the complexity of carotenoid extracts, the diversities in polarity of carotenoids, and the need for a practical use of monitoring the process of ripening, a reverse phase and gradient elution were used. This technique enabled chromatographic separation of the polar and nonpolar carotenoids.
Apparatus and Chromatographic Conditions. The chro- matographic system consisted of Models 250B and 300B HPLC pumps (Gynkotek), a Glenco injector and a Beckman UV-vis variable-wavelength detector (Ohmacht, 1979). Columns were 250 X 4.6 mm i.d. (Labor MIM) packed with Chromsil C! 86 fim end- 0021-8561/91/1439-1907$02.50/0 © 1991 American Chemical Society
1908 J. Agrte. Food Chem., Vol. 39, No. 11. 1991 Malus et al.
Table I. Miscellaneous properties
stage of maturation
properties 1 2 3 4 5 6
fresh weight, g 596.36 508.19 350.65 311.08 202.50 239.17
dry weight (dw), g 12.92 10.42 6.60 5.86 4.14 4.83
dry weight/fresh weight, % 2.17 2.05 1.88 1.88 2.02 2.02
chlorophyll content, rng/lOO g of dw 63.14 39.21 28.87 2.95 0 0
total carotenoid," mg/100 g of dw 13.19 31.40 105.30 186.81 317.03 488.64
total carotenoid,6 mg/100 g of dw 16.04 22.60 88.67 200.92 263.72 448.78
total carotenoid," mg/100 g of dw 13.00 25.34 98.36 212.10 286.64 476.32
partition coefficient
before saponification 2.03 0.28
after saponification 1.95 2.29
phytoxanthin, % 76.22 89.75
esterified phytoxanthin, % 12.03 75.63
• Calculated by HPLC. 6 Calculated by £{£, = 2300 (Davies, 1976).e Calculated by individual £{;
capped (Labor MIM) and Chromsil CM 6 jim not endcapped (Labor MIM). The eluent was 12% (v/v) H20 in methanol (A), methanol (B), and 50% (v/v) acetone in methanol (C). The gradient program was 100% A, 8 min, to 80% A/20% B in 8 min, to 50% A/50% B in 8 min, to 100% B in 7 min, 100% B 2 min, to 100% C in 6 min, 100% C 5 min (linear steps). The flow rate was 1.5 mL/min, and detection was at 430,450,480,400, and 340 nm.
Preparation of Samples. After evaporation of the saponified carotenoid extracts (see Carotenoid Extraction), the residues were dissolved in a mixture (3:7) of acetone and methanol. The concentration of the samples was approximately 2-8 X 10* M (0.1-0.4 mg/mL); the injection volume was 10-50 IIL. For standard solutions authentic samples were taken directly from our collection or derivatives were freshly prepared from the authentic samples by means of well-known reactions, i.e., epoxide- furanoid oxide rearrangement (Karrer and Jucker, 1950) and trans-cis stereomutation.(Zechmeister, 1962). The derivatives were separated on CaC03 (Biogal) columns with benzene or a mixture of benzene and petroleum ether (40-60 °C). The fura- noid oxide epimers were designated in decreasing order of their adsorption on the CaCOa columns. The standard solutions were prepared as above, and the concentrations were about 2-4 x 10*
M (0.01-0.02 mg/mL); the injection volume was 5-20 IIL. As an internal standard, a 2-4 X 10* M benzene solution of cantha- xanthin [Hoffmann-La Roche, mp 205 °C, recrystallized from benzene-petroleum ether (30-40 °C)] was used. At all times except that of the experiments, the solutions were stored at -20
°C under nitrogen away from light. The total sample handling time was 20 min or less.
Identification of Peaks. To increase the differences in polarity between carotenoids, the carotenoid esters were saponified (see Carotenoid Extraction) before analysis. The peaks in a chro- matogram were identified by means of authentic carotenoid samples, different chemical tests, and appropriate variation of the wavelength of detection (Baranyai et al., 1982). For example, the 5,6-epoxides were converted into furanoid oxides by acid treatment and oxocarotenoids into alcohols by NaBIL reduction;
all derivatives showed different retention times and absorption maxima. Similarly, comparison of the corresponding peak heights at different wavelengths of detection allows differentiation between chromophore systems. To achieve complete conversion and avoid trans-cis isomerization, the chemical tests were performed under strictly controlled conditions.
Quantification. The chromatograms were evaluated quan- titatively by relating the heights of the individual carotenoids to that of canthaxanthin used as an internal standard (Baranyai et al., 1982). The ratios of the 430-nm mole extinctions of the authentic samples in a chromatogram to that of canthaxanthin were used as detector signals to the amount of identified caro- tenoids in the sample introduced. For the quantitation of unidentified pigments a mean value of mole extinction was employed. The minor peaks were evaluated in repeated chro- matograms by using optimized detection limits for them. The coefficient of variation (CV) varied from major (30-10 %) to minor (9-1%) and trace carotenoids (0.9-0.05%), amounting to CV values 0.47-0.79,1.29-4.84, and 7-30, respectively.
Carotenoid Extraction. The fruits in different stages of
ripening were divided into six batches according to their colors from green to orange. Ripe fruits were collected from an open field in August and September 1988. To obtain reliable samples, 200-600 g (fresh weight) of pods, freed from their shells and seeds, were used for extraction. Each of the batches was blended with MeOH and about 1% calcium carbonate. The blendate was allowed to stand in MeOH (100 g of pods requires 300 mL of MeOH) for dehydration. After 18 h, the mixture was filtered and the filter cake extracted with 200 mL of MeOH for 24 h. The extraction was repeated twice with 200 mL of MeOH and finally with Et^O. After suction, the two MeOH and ethereal extracts were combined, transferred to a separatory funnel, diluted with EtsO, washed free from methanol with water, dried over anhydrous NajSO^ evaporated in vacuo to about half-volume, and saponified with 30% KOH-MeOH at room temperature for 18 h. After saponification, monitored by liquid-liquid partition tests, the ethereal solution was washed free from alkali and evaporated to dryness in vacuo, and the residue was dissolved in 100 mL of benzene. Since the first methanolic solution obtained in the process of dehydration also contains polar carotenoids, it was worked up separately. AHquots of the two solutions containing the total carotenoid content were mixed immediately before HPLC analysis.
Preparation of Violaxanthin, Antheraxanthin, Lutein, and Zeaxanthin in Crystalline Form. Ripe fruits, freed from their shells and seeds (3.4 kg of fresh weight), were used for extraction. Further operations were similar to those used for HPLC analysis; however, only the ethereal extract was worked up. After saponification, etc., the residue was dissolved in benzene and the hypophasic pigments were precipitated with hexane (197 mg). The mother liquor was evaporated in vacuo to dryness and dissolved in benzene, and the epiphasic pigments were precip- itated with MeOH (63 mg). Separation of the hypophasic pigments was achieved by column chromatography on calcium carbonate (Biogal) with a 3:2 mixture of benzene and hexane.
The following zones were obtained: band 1 (unidentified), band 2 (violaxanthin), band 3 (antheraxanthin), band 4 [a mixture of lutein (upper part of the zone) and zeaxanthin], and band 5 (unidentified). Rechromatography of the individual pigments on calcium carbonate (Biogal) with benzene gave violaxanthin [crystallized from MeOH; 26 mg; mp 176 °C; Xnu (in benzene) 483, 452, and 426 nm] with 15% hexane in benzene gave an- theraxanthin [crystallized from MeOH; 9.8 mg; mp 181 °C; X ^ nm (in benzene) 487,458 and 434 nm], and with 30% benzene in hexane gave lutein [crystallized from benzene-hexane; 4.0 mg;
mp 165 °C; k m nm (in benzene) 488 and 457 nm] and zeaxan- thin [crystallized from MeOH; 6.8 mg; mp 176 °C; K** (in benzene) 493 and 463 nm].
RESULTS AND DISCUSSION
To avoid trans-cis isomerism and epoxide-furanoid oxide rearrangement, great care was taken during the isolation procedures. The individual carotenoid content and total carotenoid and chlorophyll contents were ex- pressed on the basis of the weightand dry weight of fruits.
After separation of the carotbnoid pigments by HPLC, an
Carotenoid Composition of Yellow Pepper J. Agrlc. Food Chem., Vol. 39, No. 11, 1991 1909 Table II. Relative Carotenoid Content (%) of C. annum Fruit at Six Stages of Maturation
stage of maturation
peak no. pigment I 2 3 4 5 6
0.76 0.44 0.18 0.22 0.16 0.05
0.45 0.39 0.25 0.21 0.19 0.21
0.14 0.18 0.07 0.13 0.07 0.06
0.32 0.33 0.18 0.15 0.10 0.13
0.13 0.15 0.12 0.18 0.08 0.06
0.45 0.79 0.63 0.69 0.49 0.50
0.11 0.14 0.10 0.09 0.08 0.07
0.24 0.14 0.13 0.12 0.07 0.08
0.16 0.22 0.19 0.15 0.07 0.08
0.76 ' 0.71 0.63 0.78 0.36 0.53
0.85 0.79 0.62 0.53 0.35 0.20
0.89 1.11 0.83 1.06 0.46 0.49
1.47 1.55 0.74 0.87 0.36 0.34
0.32 0.23 0.23 0.19 0.14 0.07
5.53 6.06 2.70 2.46 1.04 0.81
3.03 8.87 15.97 20.55 26.53 34.07
3.33 7.33 8.21 8.29 7.44 5.84
1.35 2.41 2.30 1.98 1.86 0.73
0.53 0.96 0.83 1.68 0.64 0.20
1.89 2.60 2.15 1.94 1.60 0.53
1.71 2.41 1.36 0.92 0.59 0.00
0.38 0.27 0.46 0.43 0.77 0.53
0.38 1.64 4.58 6.51 7.79 10.55
0.45 1.09 1.52 1.50 1.28 1.55
0.38 0.77 1.36 1.40 1.85 1.17
0.38 0.90 1.73 1.58 2.18 1.28
0.09 0.09 0.08 0.12 0.07 0.09
0.25 0.20 0.14 0.16 0.14 0.13
37.83 34.17 19.79 13.43 11.52 9.26
2.55 3.81 8.04 7.46 7.73 8.50
0.07 0.00 0.37 0.42 0.51 0.50
0.07 0.22 0.27 0.28 0.38 0.30
0.11 0.15 0.08 0.07 0.11 0.40
1.58 1.25 0.81 0.29 0.52 0.43
3.74 1.98 1.66 1.50 1.20 0.94
0.32 0.28 0.69 0.66 0.63 0.65
0.07 0.07 0.12 0.09 0.09 0.08
0.35 0.16 0.09 0.11 0.12 0.08
0.12 0.14 0.10 0.13 0.12 0.08
0.00 0.02 0.06 0.10 0.09 0.11
0.12 0.12 0.09 0.13 0.13 0.05
0.76 1.41 3.57 3.82 5.39 4.39
0.38 0.60 2.01 2.72 2.50 2.20
0.10 0.16 0.31 0.43 0.28 0.18
0.04 0.09 0.06 0.06 0.05 0.05
0.25 0.23 0.10 0.21 0.15 0.11
0.12 0.13 0.15 0.34 0.23 0.15
0.45 0.20 0.26 0.40 0.31 0.31
0.21 0.16 0.15 0.16 0.15 0.12
0.10 0.07 0.13 0.13 0.12 0.05
1.17 2.11 5.77 6.50 6.22 5.88
19.86 8.32 6.00 4.63 3.59 4.24
2.75 1.01 0.95 0.97 0.71 0.63
13.19 31.40 105.03 186.81 317.03 488.64 2° 1«
4» 3«
5° 6»
7« 8«
9°
11« 10»
12 13«
14 15 16 17 18 19 20 21 22 23 24 25 26 27 28°
29° 30 32« 31 34" 33°
35 36 37 38«
40 39«
41"
42 43°
44 45 46° 47"
48° 49°
51° 50 52« 53 54 55
neoxanthin X + neochrome epimer 3'*
neochrome epimer l6* 4- neochrome epimer 4'*
neoxanthin + neochrome epimer 26* violaxanthin + cis-neochromea6 luteoxanthin epimer 2e 1 luteoxanthin epimer Ie I auroxanthin epimer 2e auroxanthin epimer 3»
auroxanthin epimer Ie 9-cis-violaxanthm lutein epoxide antheraxanthin 13-cù-violaxanthin mutatoxanthin epimer 2e mutatoxanthin epimer Ie lutein
zeaxanthin
9(9')-cis-lutein
13(13')-cis-lutein + 9-cis-zeaxanthin 13-cis-zeaxanthin
canthaxanthin
/3-ciyptoxanthin 5,6-epoxide a-cryptoxanthin
0-cryptoxanthin
mutatochrome a-carotene /S-carotene cis-ß-carotene
total carotenoid, mg/100 g of dw
° Unidentified. 6 Tentatively identified.' The numbers indicate the adsorption affinities, in decreasing order, on a calcium carbonate column.
additional calculation of total carotenoid content was carried put by using the individual molar extinction values for the identified and the average values (E}^ = 2300) for the unidentified components (Davies, 1976). The partition coefficients measured before and after saponification made it possible to determine the percentages of hydrocarbons, phytoxanthins (carotenoid alcohols), and esterified phy- toxanthins present in the fruits (Table I).
The changes of the carotenoids present in the fruits of yellow pepper at six different stages of maturation are shown in Table II. We assume that the different stages of maturation can be characterized more exactly by the total carotenoid content of the fruit as a function of tem- perature, radiation of the sun, etc., than by the duration
of maturation in days, chlorophyll content, or color. (Chlo- rophylls disappear in an early stage of maturation of C.
annuum lycopersiciforme flavurn.) In all the different stages of maturation, the same 54 peaks were found (Figure 1), of which 27 were identified by means of cochromatog- raphy using authentic samples, UV-vis spectra, different chemical tests, etc. (Matus et al., 1981). As a result of mixed peaks, 32 carotenoids were identified in the 27 peaks (Figures 1-3). In the course of ripening the amount of identified carotenoids increased from 90 to 94%. In the ripened fruits, violaxanthin (3S,5E,6S,3'S,5'E,6'S) and an- theraxanthin (3S,5R,6S,3'R) accounted for about 34 and 11% of the total, respectively, zeaxanthin and lutein for about 9 % each, /3-cryptoxanthin and /3-carotene for about
1910 J. Agrlc. Food Chem., Vol. 39, No. 11, 1991 Matus et al.
I J L ï k x )
min. to 35 X 15 20 B » 5 0
Figure 1. HPLC separation of carotenoids in yellow pepper: Chromsil Cis 6 Mm endcapped; detection at 430 nm; other conditions as in text. For peak number, see Table II.
J\ju_
Figure 2. HPLC separation of carotenoids in yellow pepper:
Chromsil Ci8 6 Mm not endcapped; detection at 430 nm; other conditions as in text. For peak number, see Table II.
4% each, a-cryptoxanthin for about 5%, and o-carotene for about 6 %. Numerous furanoid oxides (epimers of au- roxanthin, of luteoxanthin, of mutatoxanthin, and of neochrom) and cis isomers, probably as "post-mortem artifacts", were present in an amount of 10% or less.
The changes of total carotenoid content, total epoxide content including furanoid oxides, and the amount of chlo- rophylls during maturation are presented in Figure 4. By the stage of ripening, characterized by complete disap- pearance of the chlorophylls from the fruits, their total carotenoid content had already reached two-fifths of the total carotenoid and one-third of the total epoxide contents of the ripest fruits. The total carotenoid and total ep- oxide contents of the fruits are increased 40-fold and 105- fold, respectively. It should be noted that a 13-fold increase during maturation had been reported earlier (Cholnoky et al., 1958), which was certainly due to the difficulty of defining maturity and, first of all, to the different varieties of C. annuum lycopersiciforme flavum investigated. At the beginning of maturation phytoxanthins make up 76 % and at its end 90 % of the total carotenoid content. During maturation the percentage of esterified phytoxanthins increases from 12 to 76% of the total carotenoid content (Table I).
Figure 5 shows the changes in milligrams per 100 g of dry weight as a function of ripening for various groups of carotenoids. We obtained practically straight lines with
- I —
( m i n ) ¿0 35 30 25 20 15 TO S 0
Figure 3. HPLC chromatograms after acid treatment: Chrom- sil Cia 6 Mm endcapped; other conditions as in text. For peak number, see Table II. (À) Detection at 430 nm; (B) detection at 450 nm.
different slopes, indicating the rates of formation in decreasing order as follows: epoxides, alcohols, and hydrocarbons. This finding is in agreement with the sequence of carotenoids in the biosynthetic pathway (Scheme I); i.e., the principal epoxide, violaxanthin, is the end product. The same representation for individual car- otenoids is shown in Figure 6. During maturation, the concentration of violaxanthin increases 400-fold and even that of /8-carotene 8-9-fold in spite of the fact that /3-carotene shows a decrease in percentage concentration (Figure 7). The ratio of lutein to zeaxanthin drops from 14.8 to 1.1.
C a r o t e n o l d C o m p o s i t i o n of Y e l l o w P e p p e r J. Agric. Food Chem., Vol. 3 9 , No. 11, 1 9 9 1 1 9 1 1
m g / 1 0 0 g d w
- 4 0 0
- 3 0 0
2 0 0
100
P77Z1 5 , 6 - ond 5 , 8 - * p o x l d « s I I other corotenoid»
m chlorophyll
A 5 6
s t a g e s of m a t u r a t i o n
Figure 4. Changes in total carotenoid and chlorophyll content of C. annuum fruit.
m g t 100g dw
Scheme I. Pathway from 0-Carotene to Violaxanthin in the Fruits of Yellow Pepper
Mutatochrome
( 5 , 8 - E p o x y - 5 , 8 - d i h y d r o - p>, ft -carotene)
/-•-Carotene —
( /b , p - c a r o t e n e )
(b-Cryptoxanthin
Z e a x a n t h i n
/b-Carotene m o n o e p o x t d e (5,6-Epoxy-5,6-dlhydro-
(i -carotene)
t-CrypIo
A n t h e r a x a n t h i n
V i o l a x a n t h i n
/i>-Cryptoxanthin 5,6-ep- oxide ( 5 , 6 - e p o x y - 5 , 6 - d i - hydro- (5, ^j-carotene-3-ol)
Mutatoxanthin e p i m e r s ( 5 , B - e p o x y - 5 , 8 - d i h y d r o -
P>i p> - c a r o t e n e - 3 , 3 ' - d i o l )
Auroxanthin epimers (5,8,5',8'-diepoxy-5,8, 5 ' ,8' - t e t r a h y d r o - f>, (1 - carotene-3,3'-diol)
mgMOOg dw
100 200 300 4 0 0 5 0 0
l o t o l c o r o t e n o i d s ( m g / 1 0 0 g d w )
Figure 5. Pigment changes during ripening. (•) Epoxides; (O) monools + diols; (•) carotenes.
In Figure 7, the percentage distribution of carotenoids is plotted against the total carotenoid content, i.e., as a function of maturity. Antheraxanthin, zeaxanthin, /5-cryp- toxanthin, o-cryptoxanthin, and a-carotene increase and /S-carotene decreases up to the stage disappearance of chlorophyll when they reach a constant equilibrium concentration. A sharp increase of violaxanthin (end product of the biosynthesis of carotenoids with /S end groups) and a sharp decrease of lutein (end product of the biosynthesis of carotenoids with Em e end group) are observed during the whole process of ripening.
In Table II it is demonstrated that furanoid oxides (au- roxanthin, luteoxanthin, mutatoxanthin, mutatochrome) are always present during the process of ripening. The percentage concentration of each of them increases in the early stages of maturation, but after the disappearEmce of chlorophyll there is a steady decrease. The decresising ratios of the different furanoid oxides and the correspond- ing 5,6-epoxides are shown in detail in Figure 8. These decreasing ratios point to a situation in which the smoothly
Figure 6. Carotenoid changes during ripening. (•) Violaxan- thin; (•) antheraxanthin; (A) zeaxanthin; (O) /S-cryptoxanthin;
(C) d-carotene; (•) lutein; (A) a-cryptoxanthin; (N) a-carotene.
rising curves of luteoxanthin plus auroxanthin and those of luteoxanthin plus auroxanthin plus neoxanthin (formed indirectly from zeaxanthin) are converted into nearly horizontal straight lines by plotting milligrams of pigment per 100 g of dry weight vs total carotenoid content, while traas-violaxanthin gives a steeply rising curve (Figure 9).
It is also illustrated how the curve of violaxanthin vs maturation is converted to a linear line by adding vio- laxanthin, the parent compound, to its derivatives step by step.
The isolation of furanoid oxides (=5,8-epoxides), how- ever, always raises the question of whether they represent true natural products or are merely artifacts formed via 5,6-epoxide-furanoid oxide rearrangement during isola- tion. According to data in the literature, the amount of furanoid oxides and their ratio to 5,6-epoxides in plants vary very widely (Szabolcs, 1990; Tóth and Szabolcs, 1970).
Since we detected furanoid oxides in decreasing proportion to the corresponding 5,6-epoxides at every stage of ripening
100 200 300 400 500
t o t o l carotenoids < m g / 1 O 0 g d w )
1912 J. Agric. Food Chem., Vol. 39, No. 11, 1991
lolal corolenotds ( m g / I O O g d w )
Figure 7. Changes in relative carotenoid content during ripening.
( • ) Violaxanthin; (O) antheraxanthin; (A) zeaxanthin; (O) 0-cryp- toxanthin; (€>) /S-carotene; (•) lutein; (A) a-cryptoxanthin; (C) a-carotene.
total corotenoids i m g / 1 0 0 g d w )
Figure 8. Changes in the ratios of different furanoid oxides and the corresponding 5,6-epoxides during ripening. ( • ) Luteo- xanthins/violaxanthin; (O) auroxanthins/violaxanthin; (A) lu- teoxanthins + auroxanthins/violaxanthin; (•) mutatoxan thins/
auroxanthin; (•) total furanoid oxide/total 5,6-epoxide.
and the furanoid oxides with their different epimers were present, we conclude, according to Liaaen-Jensen's def- inition (Liaaen-Jensen, 1990), that the furanoid oxides are post-mortem artifacts. In our experiment not a single furanoid oxide epimer was found without its epimer(s), which was indicative of the lack of stereospecific enzymatic action.
As we have seen so far, cis isomers were always remotely present in the fruits of yellow pepper in the course of maturation, but their percentage concentration (cis form/
cis form plus trans form) decreased with the time of ripening (Figure 10). This tendency is very specific to neoxanthin (the 9-cis form) because the percentage concentration decreases with ripening, as is to be expected, but at the end of ripening the concentration of the 9-cis form reaches even double that of the all-trans form. The curve of cis-violaxanthins also starts from a high value of 40% but falls to 5%. cts-Zeaxan thins, cis-luteins, and cis-f)-carotenes increase slightly with time until the decomposition of chlorophyll, after which they remain at a more or less constant value. Since the 9-cis and 13-cis isomers occur together and their average total concen- tration is only about 5-6 %, we conclude that the mono-cis isomers are likely to be post-mortem artifacts. However,
Matus et al.
total c o r o l t n o i d t ( m g / 1 0 0 g d w )
Figure 9. Changes in the content of violaxanthin and its derivatives during ripening. ( • ) trans-Violaxanthin; (O) trans- + cis-violaxanthin; ( • ) trans- + cis-violaxanthin + luteoxan- thins; (A) trans- + cis-violaxanthin + luteoxanthins + auro- xanthins; (•) trans- + cis-violaxanthin + luteoxanthins + auroxanthins + neoxanthins; (A) luteoxanthins + auroxanthins;
(E) luteoxanthins + auroxanthins + neoxanthins.
totol corottnoidft I mg / tOOg d w )
Figure 10. Changes in percentage concentration of cis isomers during ripening. ( • ) Violaxanthin; (A) zeaxanthin; (C) ^-car- otene; (•) lutein; (o) neoxanthin.
special attention should be paid to the high ratio of 9-cis- neoxanthin to aZi-frans-neoxanthin.
For assessment of the rate of pigment biosynthesis we used what we describe as "integrated carotenoid content", in which the concentration of a carotenoid and those of the others synthesized from it via the biosynthetic pathway are added together. This expression of concentration includes both the actual carotenoid content and the transformed carotenoid content. Thus, the following conversions were considered: /S-carotene —• /S-crypto- xanthin —• zeaxanthin — antheraxanthin —• violaxanthin, a-carotene -*• a-cryptoxanthin —• lutein —• 5,6-lutein ep- oxide and 5,6-epoxides 5,8-epoxides (=furanoid oxides).
For example, the integrated carotenoid content of zea- xanthin is calculated as follows: actual zeaxanthin content (present in the sample) + actual antheraxanthin content (present in the sample) + actual violaxanthin content (present in the sample).
Figure 11 shows the changes of the integrated caro- tenoid contents of 5,6- and 5,8-epoxides, mono- and diols, and carotenes as a function of maturation. The values for the slopes of the straight lines were compared (tga
Carotenoid Composition of Yellow Pepper
300 400 S00
totol carolrnoids {mg/100g dw ) Figure 11. Changes of integrated carotenoid content during ripening. ( • ) Epoxides; (O) monools + diols; ( • ) carotenes.
r i m i loo g dw
» 40 50 60 /S-corotwwIlO5! "Id 'K»g dw)
Figure 12. Changes in integrated carotenoid content during ripening (carotenoids with /3,0-end groups). ( • ) Violaxanthin;
( • ) antheraxanthin; (A) zeaxanthin; (O) /3-cryptoxanthin.
(carotenes) > tga(mono- and diols) > tga(5,6- and 5,8- epoxides), which led to the conclusion that
p(hydroxylation) _ tga2
u(epoxidation) tgat
= 1.44 (±0.04)
Thus, disregarding the distinction between /S and e end groups, between first and second hydroxylation, and between first and second epoxidation, the overall rate of hydroxylation is 1.5 times as great as that of epoxidation.
Furthermore, for comparison of the rates of the first and second steps of hydroxylation of the /S,/S structures and those of the first and second steps of epoxidation of the /3,/S structures (Scheme I), the integrated carotenoid contents were plotted as a function of total carotenoid content (Figure 12). Similar reasoning applied to the evaluation of Figure 11 led to the following proportions:
l>( 1st hydroxylation)
u(2nd hydroxylation) = 1.04 (±0.01) o(lst epoxidation) = 1 3 1 ( ± 0 ( ) 1 )
p(2nd epoxidation)
u(2nd hydroxylation) _ 1 ^ p(lst epoxidation)
It should be apparent that the relationship between the
J. Agric. Food Chem.. Vol. 3 9 , No. 11, 1991 s l o p e s is inevitably as follows:
1913
tga (/3-carotene) > tga(/3-cryptoxanthin) > ...
tga(violaxanthin) Similar graphing of the data for carotenoids with /3,/3 and /3,«-structures led to the conclusion that while the rate of the first hydroxylation step slightly exceeds that of the second for /3,/3 structures, the effects is marked for /3,e structures.
p(hydroxylation at the /3 ring) _ 1 g 2 Q 8 ) i>(hydroxylation at the t ring)
Furthermore, the rate of epoxidation of the /3 end group is much greater for /3,/3 structures than for /S,< structures.
We succeeded in isolating /3-cryptoxanthin 5,6-epoxide [ (3S,5fí,6S)-5,6-epoxy-5,6-dihydro-/3,/3-caroten-3-ol] from nature for the first time: peak 42, 0.1%, XmlI nm (in methanol) 472, 442 nm; XmM (in methanol) after acid treatment 450, 426 nm. The 5,6-epoxy structure is in complete agreement with the structure of cryptocapsin [(3'S,57?)-3'-hydroxy-/S,*-caroten-6'-one), which is formed from /3-cryptoxanthin 5,6-epoxide via a pinacolone rear- rangement in red pepper. Full details of the determina- tion of the structure of /3-cryptoxanthin 5,6-epoxide will be published elsewhere.
We also found /3-carotene 5,8-epoxide (originating from the corresponding 5,6-epoxide) and lutein 5,6-epoxide in traces. This finding agrees with our conclusion that the rate of hydroxylation is greater than that of epoxidation.
So it is asumed that as soon as a little /3-carotene 5,6- epoxide has been formed it begins to transform rapidly into /3-cryptoxanthin 5,6-epoxide and antheraxanthin. In contrast, lutein with its /S,e structure undergoes a very slow process of epoxidation. It should be mentioned that as post-mortem artifacts of lutein 5,6-epoxide neither fla- voxanthin nor chrysanthemaxanthin was found.
Identification of the most important 27 peaks (Table II), totaling 90-94% during ripening, was performed in the HPLC chromatogram, but 27 peaks remained un- identified. Half of the unidentified peaks exceeded the polarity of violaxanthin in the HPLC chromatogram, and they amounted to about 50% of the total of unidentified carotenoids.
Using authentic samples an attempt was made to identify in yellow pepper karpoxanthin and carotenoids with a 5,6-dihydro-3,6-epoxy-/3 (oxabicyclo [2.2.1]) end group present in red pepper (Parkes et al., 1986), but it was not successful. Therefore, we believe that carotenoids with the oxabicyclo[2.2.1] end group are only formed in red pepper, which finding might be utilized in taxonomic studies in the future.
It should be noted that according to our CD measure- ments the antheraxanthin and violaxanthin occurring in red pepper do not differ in absolute configuration from the antheraxanthin and violaxanthin occurring in yellow pepper; i.e., the 3-hydroxy-5,6-epoxy-5,6-dihydro-/3 end groups have the same 3S,5.R,6S absolute configuration.
Thus, the assumption that the lack of formation of cap- santhin and capsorubin in yellow pepper might be due to a difference in absolute configuration between the pre- cursors (antheraxanthin, violaxanthin) must be ruled out.
ACKNOWLEDGMENT
The gifts of yellow paprika from Dr. G. Horváth (Research Institute of Seed Production and Trading Co., Szentes, Hungary) are gratefully acknowledged. We thank Mrs. E. Nyers and Mrs. M. Steiler for skillful assistance.
1914 J. Agrte. Food Chem., Vol. 39, No. 11. 1991 Malus et al.
LITERATURE CITED
Baranyai, M.; Matus, Z.; Szabolcs, J. Determination, by HPLC, of carotenoids in paprika products. Acta Aliment. 1982,11, 309.
Cholnoky, L.; Györgyfy, K.; Nagy, E.; Páncél, M. Acta Chim.
Acad. Sci. Hung. 1958,16, 227.
Comar, C. L.; Zscheile, F. P. Analysis of plant extracts for chlo- rophylls a and b by a photoelectric spectrophotometry method.
Plant Physiol. (Lancaster) 1942,17,198.
Davies, B. H. Carotenoids. In Chemistry and Biochemistry of Plant Pigments, 2nd ed.; Goodwin, T. W., Ed.; Academic Press:
London, 1976; Vol. 2, Chapter 19, p 149.
Karrer, P.; Jucker, E. Carotenoids; Elsevier: Amsterdam, 1950 (English translation by E. A. Braude).
Klaüi, H.; Bauernfeind, J. C. Carotenoids as Food Color. In Car- otenoids as Colorants and Vitamin A Precursors; Bauern- feind, J. C., Ed.; Academic Press: New York, 1981; Chapter 2, pp 66-70,141.
Liaaen-Jensen, S. Artifacts of natural carotenoids—Unintended carotenoid synthesis. In Carotenoids: Chemistry and Biology;
Krinsky, N. I., Matthews-Roth, H. M., Taylor, R. F., Eds.;
Plenum Press: New York, 1990; p 149.
Matus, Z.; Baranyai, M.; Tóth, Gy.; Szabolcs, J. Identification of oxo, epoxy and some cis-carotenoids in High Performance Liquid Chromatography. Chromatographia 1981,14, 337.
Molnár, P.; Szabolcs, J. Alkaline permanganate oxidation of car- otenoid epoxides and furanoids. Acta Chim. Acad. Sci. Hung.
1979, 99, 155.
Ohmahct, R. Chromatographia 1979,12, 565.
Parkes, K. E. B.; Pattenden, G.; Baranyai, M.; Molnár, P.;
Szabolcs, J.; Tóth, Gy. Novel carotenoid 3,6-epoxides from red paprika, Capsicum annuum. Tetrahedron Lett. 1986, 27, 2535.
Szabolcs, J. Plant Carotenoids. In Carotenoids: Chemistry and Biology; Krinsky, N. I., Matthews-Roth, H. M., Taylor, R. F., Eds.; Plenum Press: New York, 1990; p 39.
Tóth, Gy.; Szabolcs, J. Distribution of carotenoids in flowers of Helianthus Annuus, Impatiens Noli Tangere, Ranunculus Acer, Taraxacum Officinale, and in ripe hips of Rosa Canina and Rosa Rubiginosa. Acta Chim. Acad. Sci. Hung. 1970,64,393.
Weedon, B. C. L. Occurrence. In Carotenoids; Isler, 0., Ed.;
Birkhauser Verlag: Basel, 1971; Chapter 2, p 42.
Zechmeister, L. cis-trans Isomeric Carotenoids, Vitamin A and Arylpolyenes; Academic Press: New York, 1962.
Received for review January 2,1991. Revised manuscriptreceived June 26,1991. Accepted July 22,1991.
Registry No. Neoxanthin X, 30743-41-0; neoxanthin, 14660- 91-4; violaxanthin, 126-29-4; 9-cis-violaxanthin, 26927-07-1; an- theraxanthin, 640-03-9; 13-cis-violaxanthin, 75715-58-1; lutein, 127-40-2; zeaxanthin, 144-68-3; 9'-cis-lutein, 79516-56-6; 9-cts- lutein, 29414-89-9; 13'-cis-lutein, 79464-33-8; 13-cis-lutein,32499- 88-0; 9-cts-zeaxanthin, 60497-64-5; 13-cis-zeaxanthin, 60497-65- 6; canthaxanthin, 514-78-3; /3-cryptoxanthin 5,6-epoxide, 17430- 14-7; a-cryptoxanthin, 24480-38-4; /3-cryptoxanthin, 472-70-8;
mutatochrome, 515-06-0; a-carotene, 432-70-2; /3-carotene, 7235- 40-7; cis-/3-carotene, 30430-49-0; /3-carotene 5,8-epoxide, 15678- 54-3; lutein 5,6-epoxide, 28368-08-3.
2 * 2072 Reprinted from JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 1992,40.
Copyright © 1992 by the American Chemical Society and reprinted by permission of the copyright owner.
Carotenoid Composition in the Fruits of Black Paprika
(C a p s i c u mannuum
Variety
longum nigrum)during Ripening
József Deli,* Zoltán Matus, and József Szabolcs
Department of Medical Chemistry, Medical University of Pécs, Szigeti út 12, H-7643 Pécs, Hungary
The changes in the carotenoid pigments of black paprika (Capsicum annuum var. longum nigrum) during maturation have been investigated quantitatively by means of a HPLC technique. In the chromatograms 58 peaks were detected, 34 carotenoids (92-95% of the total carotenoid content) were completely or tentatively identified. The total carotenoid content of the ripe fruits was about 3.2 g/100 g of dry weight, of which capsanthin constituted 42 %, zeaxanthin 8 %, cucurbitaxanthin A (3,6-epoxy- 5,6-dihydro-/3,/3-carotene-5,3'-diol) 6.6%, capsorubin 3.2%, and 0-carotene 7%. The remainder was composed of capsanthin 5,6-epoxide, capsanthin 3,6-epoxide (3,6-epoxy-5,3'-dihydroxy-5,6-dihydro- 0,x-caroten-6'-one), karpoxanthin, violaxanthin, antheraxanthin, zeaxanthin, /3-cryptoxanthin, lutein, and several cis isomers and furanoid oxides. During ripening, an increase in capsanthin and, to a lesser extent, an increase in carotenoids with « and oxabicyclo[2.2.1] end groups were observed.
INTRODUCTION
Although red paprika is one of the most extensively investigated natural carotenoid food colors (Klaüi and Bauernfend, 1981; Camera, 1980, 1985; Camara and Bardat, 1983), the black variety, Capsicum annuum var.
longum nigrum, has not yet been analyzed in detail. As a continuation of our work on paprika carotenoids (Baranyai etal., 1982; Baranyai and Szabolcs, 1976; Parkes et aL, 1986; Matus et al., 1991), the present paper deals with the distribution of yellow and red carotenoids in ripening black paprika, paying special attention to the formation of minor carotenoids with oxabicyclo-/? and 3,5,6- trihydroxy-j3 end groups.
MATERIALS AND METHODS
The black paprika (C. annuum var. longum nigrum cv. Szentesi fekete fűszer paprika) was collected from a research plantation at Szentes (central Hungary) in September 1988and immediately transported to our laboratory. The fruits, which were at different stages of ripening, were divided into six batches according to their black to red colors.
General methods, including sample taking, extraction, workup, instrumentation, and quantitative determination of carotenoids and chlorophyll, were described in a parallel study of yellow paprika (Matus et al., 1991). Mass spectra were recorded on a JEOL MS-01SG-2 spectrometer.
The chromatographic system consisted of Models 250B and 300B HPLC pumps (Gynkotek), a Glenco injector, and a Beckman UV-vis variable-wavelength detector (Ohmacht, 1979). Columns were 250 X 4.6 mm i.d. (Labor MIM) packed with Chromsil Cl8 6 pm endcapped (Labor MIM) and Chromsil CM 6 pm not endcapped (Labor MIM). The eluent was 12% (v/v) H20 in
methanol (A), methanol (B), 50% (v/v) acetone in methanol (C).
The gradient program was 100% A 8 min to 80% A/20% B in 8 min, to 50% A/50% B in 8 min, to 100% B in 7 min, 100% B 2 min, to 100% C in 6 min, 100% C 5 min (linear steps). The flow rate was 1.5 mL/min, and detection was at 430, 450,480, 400, and 340 nm.
Analytical chemicals were used, and authentic samples were taken from our collection. Characteristic data of the authentic minor carotenoids [karpoxanthin, capsanthin 5,6-epoxide, cu- curbitaxanthin A (3,6-epoxy-5,6-dihydro-0,/3-carotene-5,3'-dioI), capsanthin 3,6-epoxide (3,6-epoxy-5,3'-dihydroxy-5,6-dihydro- 0,x-caroten-6'-one)] were published earlier (Parkes et al., 1986).
RESULTS AND DISCUSSION
To avoid pigmentdecompoeition, and epoxide-furanoid oxide and trans-cis rearrangements, the isolation of carotenoids was carried out under nitrogen in darkness using methanol for dehydration at low temperatures (4- 23 °C). The differentstages of ripening were characterized by the total carotenoid content of fruits (Matus et aL, 1991).
During ripening, the changes in total carotenoid content, red carotenoid content, and chlorophyll content are shown in Table L The total and the red carotenoid contents were increased 66- and 214-fold, respectively, while the chlorophyll content was reduced to zero. The ratio of the red and yellow pigments increased from 0.22 to 1.34. Thus, the color of black paprika can range from greenish black to deep red, depending on the concentration of chlorophyll relative to those of red and yellow carotenoids. The ripe fruits of black paprika had a very high carotenoid content, the red variety (Capsicum annuum var. lycopersiciforme Table I. Miscellaneous Properties
stege of maturation
property 1 2 3 4 5 6
fresh wt, g 466.46 279.52 228.48 234.43 226.62 161.67
dry wl, g 27.62 16.01 12.45 11.56 11.50 8.86
dry wt/fresh wt, % 6.93 6.37 6.45 4.93 6.10 6.49
chlorophyll content, mg/100 g of dw 109.36 74.20 44.85 10.02 0 0
total carotenoid, mg/100 g of dw 48.60 162.00 378.90 1753.90 1960.60 3211.00
red component, % 17.71 34.30 43.89 53.28 54.04 57.29
partition coefficient
before saponification 1.12 0.07
after saponification 1.84 3.69
phytoxanthin, % 84.13 91.94
esterified phytoxanthin, % 37.30 93.30
0021-8561/92/1440-2072$03.00/0 © 1992 American Chemical Society
Carotenoids In Black Paprika Fruit Chart I *
ö c " ö r J5c
o b c d
0 Antheraxanthin, R = e, Q = c; canthaxanthin, R = Q = j;
capsanthin, R = c, Q = i; capsanthin 5,6-epoxide, R = e, Q = i;
capsanthin 3,6-epoxide (oxabicyclo,* pigment), R = g, Q = i;
capsanthol, R = c, Q = k; capsochrome, R = f, Q = i; capsorubin, R = Q = i; capsorubol, R = Q = k; a-carotene, R = a, Q = b;
/S-carotene, R = Q = a; cryptocapsin, R = a, Q = i; a-cryptoxanthin, R = c, Q = b; /S-cryptoxanthin, R = c, Q = a; cucurbitaxanthin A (oxabicyclo,/9 pigment), R = g, Q = c; cucurbitaxanthin B (oxabicyclo,¿¡-epoxide pigment), R = g, Q = e; cycloviolaxanthin (oxabicyclo.oxabicyclo pigment), R = Q = g; karpoxanthin, R = h, Q = c; latoxanthin (trihydroxy-/3,¿¡-epoxide pigment), R = h, Q = e; lutein, R = c, Q = d; luteoxanthin, R = e, Q = mactraxanthin (trihydroxy-/S,trihydroxy-/3 pigment), R = Q = h;
mutatoxanthin, R = f, Q = c; violaxanthin, R = Q = e; zeaxanthin, R = Q = c; 3,5,6,3'-tetrahydroxy-5,6-dihydro-/3,*-caroten-6'-one (trihydroxy-d,* pigment), R = h, Q = i; 3,6-epoxy-5,6,5',6'- tetrahydro-/3,d-carotene-5,3',5',6'-tetrol(oxabicyclo,trihydroxy-d pigment), R = g, Q = h.
rubrum) possessing (Cholnoky et al., 1955) only 13% of that of the black variety.
The partition coefficients of the extracts measured before and after saponification and the HPLC analysis revealed that the percentages of the esterified phyto- xanthins had increased from 37 to 93% (Table I), which is in agreement with the literature (Cholnoky et al., 1955).
The changes in the individual carotenoid contents of fruits are given in Table II; 58 peaks were detected by HPLC at all stages of ripening. The pigments were identified by using authentic carotenoids, various chemical
J. Agric. Food Cbem., Vol. 40, No. 11, 1992 2 0 7 3
tests, and different wavelengths of detection (Matus et al., 1981) (Figures 1 and 2).
During maturation, the changes (in milligrams per 100 g of dry weight (dw)] of hydrocarbons, xanthophylls, and epoxy- and ketoxanthophylls are demonstrated in Figure 3. The different slopes of the curves show that the rates of accumulation (rate of formation minus rate of trans- formation) in fruits, in decreasing order, are as follows:
ketoxanthophylls, xanthophylls, hydrocarbons, and ep- oxyxanthophylls. In the case of black paprika, the situation is more complex than that in yellow paprika (Matus et al., 1991); therefore, we limit ourselves to a single conclusion: the rate of pinacol rearrangement exceeds the rate of epoxydation.
The percentage distribution of carotenoids is plotted against total carotenoid content in Figures 4 and 5. From the relative position of the principal capsanthin curve and the antheraxanthin (precursor of capsanthin) curve, it is seen that the sharp increase of capsanthin is accompanied by a moderate decrease of antheraxanthin. Similarly, a moderate increase of the capsorubin curve is associated with a moderate decrease of the violaxanthin (precursor of capsorubin) curve. It is important to recognize that the curves of antheraxanthin and violaxanthin are parallel to each other within measuring errors. ¿¡-Carotene increases until the stage of disappearance of chlorophyll, when it reaches a more or less constant value. In contrast, lutein, which makes up the highest percentage (29%) of caro- tenoids at the very beginning of ripening, plummets to below 0.1%. However, the most important finding is brought out by the curve of cucurbitaxanthin A and capsanthin 3,6-epoxide; the trend of the rate of accumu- lation of the carotenoids with an oxabicyclo end group resembles that of the carotenoids with a * end group. The carotenoids with both oxabicyclo and * end groups are end products in the biosynthetic pathways, so their rates of accumulation are equal to their rates of formation.
During ripening, the changes of pigment concentrations reveal a general increase which is extremely high (470- fold) for capsanthin. The rates of accumulation, in decreasing order, are as follows: capsanthin, zeaxanthin,
¿¡-carotene, cucurbitaxanthin A, ¿¡-cryptoxanthin, cap- sorubin, capsanthin 3,6-epoxide, karpoxanthin, anther- axanthin, violaxanthin, capsanthin 5,6-epoxide, and cryp- tocapsin.
6 5 4 3 2 1
F i g u r e 1. HPLC separation of carotenoids in ripe black pepper. Conditions: Chromsil-Cis 6 iim endcapped, detection at 450 nm, other conditions as in the text. For peak numbers see Table II.
2 0 7 4 J. Agrie. Food Chem., Vol. 40, No. 11, 1 9 9 2 Dell et al.
U W ü k í
mg/IOOg dw
4-
F i g u r e 2. HPLC chromatogram after N a B H4 reduction.
Conditions: Chromsil-Cig 6 pm endcapped, other conditions as in the text. For peak numbers see Table II. Peaks: 7'a and 7'b (from7),capsorubolepimers; 14'a and 141) (from 14),capsanthol 3,6-epoxide epimers; 19'a and 19'b (from 19), capsanthol epimers.
A number of previous investigations have demonstrated that some of the natural carotenoids are isolation artifacts.
In this experiment, the question of isolation artifacts emerges in connection with cis isomers and furanoid oxides (5,8-epoxides), which are always present in the fruits of black paprika (Table II).
The changes of cis isomers in black paprika are similar to those in yellow paprika during the process of ripening (Matus et al., 1991); i.e., the related percentage values (cis form/cis form + trans form) decrease with ripening until about the decomposition of chlorophyll, after which they remain at a more or less constant value. Since coexistence
2000-
1500
1000-
500-
1000 2000 3000
total carotenoids (mg/100g dw ' Figure 3. Pigment changes during ripening. ( • ) Ketocaro- tenoids; (A) epoxides; (O) monools plus diols; ( • ) carotenes.
of the all-trans, 9-cis, and 13-cis forms was consistently observed and the average total amount of the cis isomers was only 5%, we believe that the cis isomers are likely to be "post-mortem artifacts".
Table II. Relative Carotenoid Content (Percent) of C. annuum Fruit at Six Stages of Maturation
stage of maturation stage of maturation
peak pigment 1 2 3 4 5 6 peak pigment 1 2 3 4 5 6
1° 0.16 0.14 0.12 0.08 0.06 0.06 31° 0.07 0.08 0.05 0.04 0.04 0.05
2« 0.07 0.04 0.04 0.05 0.05 0.05 32° 0.07 0.14 0.02 0.04 0.03 0.03
3 trihydroxy-/3,< 0.11 0.09 0.16 0.21 0.17 0.21 33 9(9')-cia-lutein 0.86 0.15 0.04 0.04 0.04 0.04 pigment
4" 0.32 0.25 0.22 0.17 0.18 0.17 34 9-cis-zeaxanthin 1.77 0.38 0.20 0.12 0.11 0.12
5° 0.22 0.16 0.11 0.09 0.08 0.08 35 13(13')-cis-lutein 0.47 0.00 0.00 0.00 0.00 0.00
6° 0.95 0.31 0.26 0.09 0.11 0.08 36 13-cis-zeaxanthin 0.87 1.16 0.72 0.33 0.28 0.25
7 capsorubin 0.57 1.25 1.75 2.35 2.51 3.16 37° 0.18 0.28 0.19 0.11 0.11 0.09
8° 0.00 0.29 0.22 0.27 0.22 0.18 38« 0.09 0.36 0.06 0.15 0.15 0.17
9 epikarpoxanthin 0.38 0.32 0.23 0.19 0.16 0.17 39 cryptocapsin 0.06 0.08 0.13 0.44 0.69 0.68 10 capsanthin 4.82 2.70 2.17 1.15 1.06 0.96 40° cryptocapsin
0.03 0.07 0.04 0.05 0.11 0.07
5,6-epoxide 0.07 0.04 0.05 0.11 0.07
11 karpoxanthin 0.35 1.07 1.61 1.79 1.70 1.67 41°.» 0.28 0.23 0.23 0.06 0.14 0.09
12 capsochrome 0.00 0.36 0.41 0.54 0.62 0.66 42° 0.10 0.12 0.03 0.07 0.11 0.08
13 violaxanthin 3.07 3.87 3.75 1.69 1.46 1.13 43« 0.35 0.43 0.28 0.10 0.09 0.06
14 capsanthin 0.50 1.24 1.75 2.31 2.70 2.85 44 a-cryptoxanthin 0.41 0.57 0.46 0.30 0.25 0.21
3,6-epoxide a-cryptoxanthin
15 9-cis-capsorubin 1.74 0.74 0.57 0.26 0.27 0.26 45 /3-cryptoxanthin 1.41 4.76 5.68 4.83 4.91 3.39 16 13-cis-capsorubin 3.08 1.19 0.86 0.56 0.51 0.55 461 /3-cryptoxanthin
0.38 0.80 0.53
17 cucurbitaxanthin B 0.42 0.74 0.81 0.78 0.87 0.74 47) cis-cryptoxanthins 0.38 0.80 0.80 0.53 0.53 0.43
18 luteoxanthin 0.10 0.18 0.25 0.30 0.27 0.32 48« 0.05 0.08 0.08 0.10 0.09 0.07
19 capsanthin 5.37 22.05 33.46 39.28 40.37 41.58 49° 0.12 0.09 0.05 0.09 0.10 0.08
20 cycloviolaxanthin 0.52 0.68 0.86 0.45 0.46 0.81 50° 0.16 0.07 0.05 0.11 0.11 0.10
21« 0.46 0.34 0.32 0.38 0.51 0.59 51° 0.15 0.10 0.09 0.16 0.16 0.16
22 antheraxanthin 3.42 3.94 3.43 1.67 1.57 1.32 52° 0.29 0.20 0.13 0.19 0.21 0.18
23 mutatoxanthin 1.61 2.23 2.40 3.30 3.46 3.84 53° 0.18 0.14 0.06 0.06 0.09 0.07
epimer 2
24 mutatoxanthin 1.74 0.80 0.57 0.61 0.64 0.75 54° 0.27 0.18 0.09 0.11 0.16 0.18
epimer 1
25 cucurbitaxanthin A 1.08 3.79 4.73 6.32 6.88 6.59 55° 0.06 0.16 0.07 0.06 0.09 0.10
26 9(9')-cis-capsanthin 0.40 1.23 0.40 1.48 1.23 1.61 56 ar-carotene 1.90 1.17 0.57 0.30 0.36 0.31 27 13(13')-c«-
capsanthin 1.17 3.82 2.80 5.45 4.70 5.64 57 /3-carotene 13.97 9.73 6.83 8.38 8.36 7.16 28 lutein 28.49 3.38 0.81 0.09 0.07 0.04 58 cis-0-carotene 1.95 1.11 0.61 0.73 0.60 0.59
29 zeaxanthin 11.96 19.96 16.66 9.59 8.35 8.12 0.60
30 0.27 0.50 0.54 0.85 0.88 0.91 total carotenoids 48.5 162.0 378.9 1753.9 1960.6 3211.0 (mg/100 g of dw)
0 Unidentified. b No reaction with sodium borohydride and dilute acids. c Reaction with sodium borohydride and no reaction with dilute acids.
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