Mikrochimica Acta [Wien] 1976I, 469--476 9 by Springer-Verlag 1976
Ceylon Institute of Scientific and Industrial Research, Colombo 7, Sri Lanka
A Qualitative Study of the Carotenoid Pigments of Sri Lanka Chillies (Capsicum annum) By C. E. C. Lord and A. S. L. Tirimanna
With 2 Figures (Received October 21, 1974. Revised January 6, 1975)
The colour of ripe chillies is an important characteristic in the evaluation of quality. Deterioration of the extractable colour pigments of chillies have been studied by Lease and Lease 1 who showed that the colour was affected by variables such as temperature and rate of drying. It was also shown that during storage, moisture content, atmospheric pressure and temperature were critical factors. The bright red colour of the ripe fruits of chillies is mainly due to the occurrence of carotenoid pigments. These pigments have already been studied using column chromatographic techniques 2-4 and by spectrophotometry 5-v. The use of two dimensional thin layer chromatographic techniques in the study of the carotenoids of chillies is reported in the present communication. Experimental Reagents
All reagents were of analytical grade or comparable purity. Silica gel G (E. Merck) was used for the preparation of the thin layer plates. Hydrochloric acid, ethyl acetate, n-Hexane, ethanol, acetone, light petroleum (b. p. 60--80 ~ and diethyl ether were obtained from British Drug House. Calcium carbonate, magnesium oxide, celite, anhydrous sodium sulphate and potassium hydroxide were also obtained from British Drug House. Chloroform was obtained from Sandberg and Schneidewind, Hamburg 76, West Germany.
470
C.E.C. Lord and A. S. L. Tirimanna:
The following samples of carotenoids were used as authentic samples in co-chromatography and ultraviolet spectrophotometry: sintaxanthin, citranaxanthin, semi-fl-carotenone, neoxanthin, lutein, violaxanthin, zeaxanthin (from United States Department of Agriculture, California), fl-carotene (from Mann Research Laboratories, Inc, New York), capsanthin, canthaxanthin, rhodoxanthin (from Hoffman La Roche Ltd, Switzerland). Three commercial brands of powdered dry ripe chillies (Capsicum annum L; variety Longum) were obtained from the local market. A sample of dry ripe chillies (M. I. hybrid, Capsicum annum L; variety Longum) was obtained from The Agricultural Department, Sri Lanka.
Apparatus Desaga thin layer chromatographic apparatus was used. Glass plates (20 x20 cm) were coated with silica gel G (E. Merck) (0.35 mm thick). The plates were dried for 1 hour and activated for 30 minutes at 1000 just before use. 350 ~1 of the material was spotted on the chromatoplate.
Procedure 5 g of powdered dry chillies were ground for 5 minutes in a mortar with a pinch of calcium carbonate. The material was then extracted in a Waring Blender with 50-ml portions of ethanol, acetone and light petroleum (b. p. 60--80 ~ - - diethyl ether (1 : 1 v/v) successively. The filtered extracts were combined and washed with water until free of ethanol and acetone. The remaining organic layer which contained the carotenoids was dried with anhydrous sodium sulphate, filtered and concentrated under reduced pressure. The oily material obtained was saponified according to the method of Goodwin 8. The unsaponifiable material was extracted with peroxide free diethyl ether and washed thoroughly with distilled water until it was free of alkali, then dried with anhydrous sodium sulphate and concentrated to a volume of approximately 20 ml under reduced pressure. This was chromatographed on a MgO : celite (1 : 1) column using increasing concentrations of acetone in petroleum ether as the developing solvent. One way thin layer chromatograms of the bands which were eluted were developed in hexane : diethyl ether (30 : 70 v/v). An etheraI extract of the material, before and after saponification was subjected to two-dimensional thin layer chromatography. The solvents used were hexane-diethyl ether (30:70 v/v) in the first direction and chloroform-ethyl acetate (80:20 v/v) in the second direction. The principal pigments were identified by spectrophotometry after careful scraping off the pigments from the chromatoplate, by
A Qualitative Study of the Carotenoid Pigments
471
co-chromatography with authentic "markers" and by the relative positions of the compounds on the chromatoplate. The carotenoid extract was subjected to phase-partition between light petroleum and aqueous 90% (v/v) methanol after saponification when it was necessary to elute the spots from the chromatoplate for spectrophotometry in order to separate the carotene hydrocarbons, monohydroxy-xanthophylls, mono and diepoxides of carotene hydrocarbons and monoketo xanthophylls from the di- and polyhydroxy xanthophylls, and their epoxy derivatives. The epoxy carotenoids were identified by the colour reactions of Fioriti and Sims9. Results and Discussion
Several bands were observed when the substance after saponification was passed through the column. The lowest band was light orange, folloWed by a pale yellow, orange, reddish orange, pink, orange-pink and red band. Fraction 1 had an absorption spectrum in various solvents which proved that it was fl-carotene. One-way chromatograms of the other bands showed that they were not pure compounds. Two dimensional thin layer chromatographic separation of an ethereal extract of a commercial brand of powdered chillie (before saponification) is shown in Fig. 1. It was observed that the carotenoids found in appreciable concentration were the esters of capsanthin and capsorubin, fl-carotene, cryptoxanthin ester and zeaxanthin. The fact that capsanthin forms a series of esters has been reported in the literature 8,1~ These workers observed that the red pigment from the skin of ripe paprika yielded a mixture of fatty acids, including oleic, carnabic, myristic and palmitic acids. Egger 1~ showed that in the pericarp of red sweet pepper all the xanthopylls were combined with palmitic, myristic and laurinic acids. It was observed that in some unsaponified commercial brands of chillie powder, more than four identified red pigments which resolved only in the solvent hexane-ether (30:70v/v) were present. These pigments disappeared after saponification with a corresponding increase in the concentrations of capsanthin and capsorubin, indicating that these compounds were probably esters of these two carotenoids. No attempt was made in the identification of the fatty acid moieties of the esters. It was observed that an unidentified compound having higher Rf values than cryptoxanthin and giving a blue colour with concentrated hydrochloric acid disappeared on saponification with a corresponding increase in the cryptoxanthin content. This compound gave a deep blue colour on spraying with concentrated
--
--
--
--
65
86
9
20
21
23
32
36
42
42
49
55
61
68
68
80
91
++
+
+
--
--
+
+
~
-r
+++
++++
++++
T
~
~ ++
+++
T
+ -~+
+
7'
T
+++
+++++
+++++
T
++ -i + + +
+
" "
T
T
+
T
T
+++++
++
--
- -
--
--
T
T
--
--
--
--
+++
+
T
+
++~ ~ +
+++++
+++++
+++
++++
++
+++
--
+++
++
--
T
+++++
--
--
--
--
T
--
--
+
+++§
+++++
+++++
--
++
++ ++§
T
- -
--
--
--
--
+++++
MI Hybrid
of the Carotenoids
Commercial Sample III
Identifiaction
concentration Commercial Sample II
the Tentative
Approximate Commercial Sample I
I. S h o w i n g
Bluish green
--
--
Green
Blue
Blue
--
--
Green
Bluish green
Bluish green
Green
--
--
Bluish green
Bluish green
Blue
--
--
--
--
Epoxy
compound
80 : 20 (v/v).
Unidentified
Unidentified
Unidentified
5--8
Cryptoxanthin
y-carotene
Unidentified
Neoxanthin
Capsorubin
Capsanthin
Violaxanthin
Unidentified
An theraxanthin
Zeaxanthin
Cryptocapsin
Unidentified
Unidentified
Cryptoflavin
Unidentified
Unidentified
/J-carotene
Tentative Identification
acetate
Chillies
Colours after spraying Conc. HC1
of Sri Lanka
following key applies to the approximate concentrations as indicated by visual observation: T a t r a c e , + l o w , +-~ f a i r , + + + m o d e r a t e , ++++ high, +~++* very high. T h e s o l v e n t s y s t e m I is H e x a n e - d i e t h y l e t h e r 3 0 : 7 0 ( v / v ) ; t h e s o l v e n t s y s t e m 11 i s C h l o r o f o r m - e t h y l
--
22
The
--
21
93
17
--
5
16
20
18
15
79
24
14
--
31
13
19
44
12
18
65
70
8
11
72
7
65
84
6
59
85
5
9
95
4
10
90
93
3
Rf values x 100 In solvent In solvent I II
2
No. of Spot
Table
=
~'
>
~,
~"~ B
g"*
"~ U)
.('3
I',-)
--aa~
A Qualitative Study of the Carotenoid Pigments
473
hydrochloric acid s and also answered the picric acid test for epoxides 9. Therefore it was probably a 5,8-epoxy derivative of cryptoxanthin. Its identity could not be confirmed by spectrophotometric measurements because it did not occur in appreciable concentrations.
Before
Sapons~6"ahon
(~
a
0 (?).. "'~ ........ C) ',.. j .
-:??( )
'::;::|
-zT~) r I
:2Z':'
......i'T..,.."75"~ z.'........
...-s~.-.'..., .;.-:. Or/ff/~7"U"~:~
~
Chloroform-E/k S Ace/are 80.ZO v/~
Second Ot?'ec/ion
Two
dimensional T L C
of an ethereal extract of Sri Lanka Chillies
(beforesaponification) Fig. 1. A diagrammatic representation of the chromatoplate. Key for the identification of compounds is in Table I. The solvent system used in the first direction was Hexane-diethyl ether 30 : 70 (v/v) and the solvent system used in the second direction was chloroform-ethyl acetate 80:20 (v/v) Another identified orange pigment occurred in fair concentration, and its spectral properties seem to indicate that it was probably cryptocapsin 3. The occurrence of zeaxanthin was confirmed by cochromatography and spectral properties. A second yellow pigment occupying a position close to zeaxanthin and which disappeared on saponification was probably an ester of zeaxanthin. Violaxanthin occurred in an esterified form as its free occurrence before saponification was not detected. The pigments capsanthin and capsorubin occured only in trace amounts before saponification. Fig. 2 shows the two dimensional separation on a chromatoplate of a saponified extract. It was observed that after saponification the two main pigments, were capsanthin and capsorubin while violaxanthin, capsolutein 5--6 epoxide, cryptoxanthin, zeaxanthin
474
C.E.C. Lord and A. S. L. Tirimanna:
and fl-carotene and neoxanthin were also found in appreciable concentrations. These pigments were identified by their spectral properties, colour reactions and in some cases by co-chromatography. Two unidentified yellow carotenoids present in trace amounts were probably hydroxy-carotenes. In the case of some commercial brands it was observed that two additional red carotenoids occupied adjacent positions to capsanthin and capsorubin on the chromatogram.
Q 0 q~
;-~.~ .,%
(7}'-': ,....
':.,.)
:; zs '..~..... .
Ol,i~ine
,:'...,,
i, Chloroform-Elhyl Ace/ate 80 ZO % Second Di)'ection
Two dimensional TLC of an ethereal extract of Sri Lanka Chillies (after saponification) Fig. 2. A diagrammatic representation of the chromatoplate. Key for the identification of compounds is in Table I. The solvent system used in the first direction was Hexane-diethyl ether 30 : 70 (v/v) and the solvent system used in the second direction was chloroform-ethyl acetate 80:20 (v/v) These two compounds are probably hydroxy capsolutein and hydroxy capsolutein 5--6 epoxide as detected by column chromatographic techniques. It has been shown that in the ripening fruit, after the disappearance of chlorophyll only zeaxanthin and cryptoxanthin take up oxygen and are transformed by it to epoxides of violaxanthin, antheraxanthin and cryptoxanthin mono-epoxide a. At this stage of ripening, the epoxides ceased to deliver their oxygen and were transformed into polyepoxy ketones (capsorubin, capsanthin and cryptocapsin). It was also observed that there was no lutein but a high concentration
A Qualitative Study of the Carotenoid Pigments
475
of zeaxanthin. The distribution of the carotenoid pigments after saponification was in keeping with this observation. Nevertheless, the interesting situation of having high concentrations of capsanthin and capsorubin in capsicum was noteworthy. In other fruits when Table II No. of s p o t 2 9 10 12 13 14 15 17 18 19
Solvent
Absorption Maxima
Identity
Petroleum ether Hexane Chloroform Chloroform Chloroform Ethanol Chloroform Ethanol Hexane Hexane Ethanol Ethanol Petroleum ether Petroleum ether Chloroform Ethanol
425, 450, 475 421, 451, 480 460, 492 430, 460, 490 458, 488 420, 442, 470 427, 450, 484 418, 442, 470 470, 497 444, 469, 504 482, 510 417, 437, 465 432, 460, 490 425, 450, 478 423, 448, 480 380, 402, 428
fl-Carotene Zeaxanthin Antheraxanthin Violaxanthin Capsanthin Capsorubin Neoxanthin 7-Carotene Cryptoxanthin Unidentified
keto carotenoids have been detected, the concentrations of these compounds were low as in the case of canthaxanthin, and the presence of a cyclopentane ring in this class of compounds placed these compounds in a unique position n - l a . It has been shown that the orange yellow colour of one variety of capsicum fruit was controlled by a single gene, the yellow allele of which was recessive to the normal red allele 14. The distribution of the carotenoid pigments of capsicum may be on a genotypic basis. The synthesis of fruit carotenoids probably involves a different series of enzymes. The wide range of carotenoids occurring in fruits may be explained on the basis of a very few genetic mutations which control the formation of the various pigment types. Summary
A Qualitative Study of the Carotenoid Pigments o[ Sri Lant~a Chillies (Capsicum annum) Using two-dimensional thin layer chromatography more than 20 constituents have been separated in the extracts of Sri Lanka chillies before and after saponification. Many of these compounds
476
C.E.C. Lord et al.: A Qualitative Study of the Carotenoid Pigments
were tentatively identified by co-chromatography with authentic samples and by ultraviolet spectrophotometry.
Zusammenfassung Mit Hilfe zweidimensionaler Diinnschicht-Chromatographie wurden aus dem Extrakt von Sri Lanka-Paprika vor und nach der Verseifung mehr als zwanzig Komponenten abgetrennt. Viele davon wurden UV-spektrophotometrisch und durch Vergleich ihrer Chromatogramme mit solchen von Testsubstanzen zu identifizieren versucht.
References I
J. G. Lease and E. T. Lease, Food Tech. 10, 368 (1956). L. V. Cholnoky. Z. Untersuch. Lebensm. 78, 157 (1939). 3 L. V. Cholnoky, K. Gyorgyfy, E. Nagy, and M. Panczel, Acta Chim. Acad. Sci. (Hung) 6, 143 (1955). 4 A. L. Curl. J. Agric. Fd. Chem. 10, 504 (1962). 5 H. V. Schuster and E. E. Lockhart, Food Research 19, 472 (1954). 6 J. B. Moster and N. A. Prater. Food Tech. 6, 459 (1952). 7 J. B. Moster and N. A. Prater, Food Tech. 11, 146 (1957). s T. W. Goodwin, in K. Peach and M. V. Tracey, eds. Modern Methods of Plant Analysis, Vol III, Heidelberg: Springer-Verlag. 1955. S. 272. 9 j. Fioriti and R. Sims, J. Chromatography 32 (4), 761 (1968). 10 K. Egger, Ber. dtsch, botan. Ges. 81, 153 (1968). i1 M. S. Barber, L. M. Jackman, C. K. Warren, and B. C. L. Weedon, J. Chem. Soc. 1961, 4019. 12 H. Faigel and P. Karrer, Helv. Chim. Acta 44, 1257, 1904 (1961). la R. D. G. Cooper, L.M. Jackman, and B. C. L. Weedon. Proc. Chem. Soc. 1962, 215. 14 p. G. Smith. J. Hered 41, 138 (1950). 2
Correspondence and reprints: Dr. A. S. L. Tirimanna, Head, Agro Industries Section, Ceylon Institute of Scientific and Industrial Research, Colombo 7, Sri Lanka.
Ceylon Institute of Scientific and Industrial Research, Colombo 7, Sri Lanka
A Qualitative Study of the Carotenoid Pigments of Sri Lanka Chillies (Capsicum annum) By C. E. C. Lord and A. S. L. Tirimanna
With 2 Figures (Received October 21, 1974. Revised January 6, 1975)
The colour of ripe chillies is an important characteristic in the evaluation of quality. Deterioration of the extractable colour pigments of chillies have been studied by Lease and Lease 1 who showed that the colour was affected by variables such as temperature and rate of drying. It was also shown that during storage, moisture content, atmospheric pressure and temperature were critical factors. The bright red colour of the ripe fruits of chillies is mainly due to the occurrence of carotenoid pigments. These pigments have already been studied using column chromatographic techniques 2-4 and by spectrophotometry 5-v. The use of two dimensional thin layer chromatographic techniques in the study of the carotenoids of chillies is reported in the present communication. Experimental Reagents
All reagents were of analytical grade or comparable purity. Silica gel G (E. Merck) was used for the preparation of the thin layer plates. Hydrochloric acid, ethyl acetate, n-Hexane, ethanol, acetone, light petroleum (b. p. 60--80 ~ and diethyl ether were obtained from British Drug House. Calcium carbonate, magnesium oxide, celite, anhydrous sodium sulphate and potassium hydroxide were also obtained from British Drug House. Chloroform was obtained from Sandberg and Schneidewind, Hamburg 76, West Germany.
470
C.E.C. Lord and A. S. L. Tirimanna:
The following samples of carotenoids were used as authentic samples in co-chromatography and ultraviolet spectrophotometry: sintaxanthin, citranaxanthin, semi-fl-carotenone, neoxanthin, lutein, violaxanthin, zeaxanthin (from United States Department of Agriculture, California), fl-carotene (from Mann Research Laboratories, Inc, New York), capsanthin, canthaxanthin, rhodoxanthin (from Hoffman La Roche Ltd, Switzerland). Three commercial brands of powdered dry ripe chillies (Capsicum annum L; variety Longum) were obtained from the local market. A sample of dry ripe chillies (M. I. hybrid, Capsicum annum L; variety Longum) was obtained from The Agricultural Department, Sri Lanka.
Apparatus Desaga thin layer chromatographic apparatus was used. Glass plates (20 x20 cm) were coated with silica gel G (E. Merck) (0.35 mm thick). The plates were dried for 1 hour and activated for 30 minutes at 1000 just before use. 350 ~1 of the material was spotted on the chromatoplate.
Procedure 5 g of powdered dry chillies were ground for 5 minutes in a mortar with a pinch of calcium carbonate. The material was then extracted in a Waring Blender with 50-ml portions of ethanol, acetone and light petroleum (b. p. 60--80 ~ - - diethyl ether (1 : 1 v/v) successively. The filtered extracts were combined and washed with water until free of ethanol and acetone. The remaining organic layer which contained the carotenoids was dried with anhydrous sodium sulphate, filtered and concentrated under reduced pressure. The oily material obtained was saponified according to the method of Goodwin 8. The unsaponifiable material was extracted with peroxide free diethyl ether and washed thoroughly with distilled water until it was free of alkali, then dried with anhydrous sodium sulphate and concentrated to a volume of approximately 20 ml under reduced pressure. This was chromatographed on a MgO : celite (1 : 1) column using increasing concentrations of acetone in petroleum ether as the developing solvent. One way thin layer chromatograms of the bands which were eluted were developed in hexane : diethyl ether (30 : 70 v/v). An etheraI extract of the material, before and after saponification was subjected to two-dimensional thin layer chromatography. The solvents used were hexane-diethyl ether (30:70 v/v) in the first direction and chloroform-ethyl acetate (80:20 v/v) in the second direction. The principal pigments were identified by spectrophotometry after careful scraping off the pigments from the chromatoplate, by
A Qualitative Study of the Carotenoid Pigments
471
co-chromatography with authentic "markers" and by the relative positions of the compounds on the chromatoplate. The carotenoid extract was subjected to phase-partition between light petroleum and aqueous 90% (v/v) methanol after saponification when it was necessary to elute the spots from the chromatoplate for spectrophotometry in order to separate the carotene hydrocarbons, monohydroxy-xanthophylls, mono and diepoxides of carotene hydrocarbons and monoketo xanthophylls from the di- and polyhydroxy xanthophylls, and their epoxy derivatives. The epoxy carotenoids were identified by the colour reactions of Fioriti and Sims9. Results and Discussion
Several bands were observed when the substance after saponification was passed through the column. The lowest band was light orange, folloWed by a pale yellow, orange, reddish orange, pink, orange-pink and red band. Fraction 1 had an absorption spectrum in various solvents which proved that it was fl-carotene. One-way chromatograms of the other bands showed that they were not pure compounds. Two dimensional thin layer chromatographic separation of an ethereal extract of a commercial brand of powdered chillie (before saponification) is shown in Fig. 1. It was observed that the carotenoids found in appreciable concentration were the esters of capsanthin and capsorubin, fl-carotene, cryptoxanthin ester and zeaxanthin. The fact that capsanthin forms a series of esters has been reported in the literature 8,1~ These workers observed that the red pigment from the skin of ripe paprika yielded a mixture of fatty acids, including oleic, carnabic, myristic and palmitic acids. Egger 1~ showed that in the pericarp of red sweet pepper all the xanthopylls were combined with palmitic, myristic and laurinic acids. It was observed that in some unsaponified commercial brands of chillie powder, more than four identified red pigments which resolved only in the solvent hexane-ether (30:70v/v) were present. These pigments disappeared after saponification with a corresponding increase in the concentrations of capsanthin and capsorubin, indicating that these compounds were probably esters of these two carotenoids. No attempt was made in the identification of the fatty acid moieties of the esters. It was observed that an unidentified compound having higher Rf values than cryptoxanthin and giving a blue colour with concentrated hydrochloric acid disappeared on saponification with a corresponding increase in the cryptoxanthin content. This compound gave a deep blue colour on spraying with concentrated
--
--
--
--
65
86
9
20
21
23
32
36
42
42
49
55
61
68
68
80
91
++
+
+
--
--
+
+
~
-r
+++
++++
++++
T
~
~ ++
+++
T
+ -~+
+
7'
T
+++
+++++
+++++
T
++ -i + + +
+
" "
T
T
+
T
T
+++++
++
--
- -
--
--
T
T
--
--
--
--
+++
+
T
+
++~ ~ +
+++++
+++++
+++
++++
++
+++
--
+++
++
--
T
+++++
--
--
--
--
T
--
--
+
+++§
+++++
+++++
--
++
++ ++§
T
- -
--
--
--
--
+++++
MI Hybrid
of the Carotenoids
Commercial Sample III
Identifiaction
concentration Commercial Sample II
the Tentative
Approximate Commercial Sample I
I. S h o w i n g
Bluish green
--
--
Green
Blue
Blue
--
--
Green
Bluish green
Bluish green
Green
--
--
Bluish green
Bluish green
Blue
--
--
--
--
Epoxy
compound
80 : 20 (v/v).
Unidentified
Unidentified
Unidentified
5--8
Cryptoxanthin
y-carotene
Unidentified
Neoxanthin
Capsorubin
Capsanthin
Violaxanthin
Unidentified
An theraxanthin
Zeaxanthin
Cryptocapsin
Unidentified
Unidentified
Cryptoflavin
Unidentified
Unidentified
/J-carotene
Tentative Identification
acetate
Chillies
Colours after spraying Conc. HC1
of Sri Lanka
following key applies to the approximate concentrations as indicated by visual observation: T a t r a c e , + l o w , +-~ f a i r , + + + m o d e r a t e , ++++ high, +~++* very high. T h e s o l v e n t s y s t e m I is H e x a n e - d i e t h y l e t h e r 3 0 : 7 0 ( v / v ) ; t h e s o l v e n t s y s t e m 11 i s C h l o r o f o r m - e t h y l
--
22
The
--
21
93
17
--
5
16
20
18
15
79
24
14
--
31
13
19
44
12
18
65
70
8
11
72
7
65
84
6
59
85
5
9
95
4
10
90
93
3
Rf values x 100 In solvent In solvent I II
2
No. of Spot
Table
=
~'
>
~,
~"~ B
g"*
"~ U)
.('3
I',-)
--aa~
A Qualitative Study of the Carotenoid Pigments
473
hydrochloric acid s and also answered the picric acid test for epoxides 9. Therefore it was probably a 5,8-epoxy derivative of cryptoxanthin. Its identity could not be confirmed by spectrophotometric measurements because it did not occur in appreciable concentrations.
Before
Sapons~6"ahon
(~
a
0 (?).. "'~ ........ C) ',.. j .
-:??( )
'::;::|
-zT~) r I
:2Z':'
......i'T..,.."75"~ z.'........
...-s~.-.'..., .;.-:. Or/ff/~7"U"~:~
~
Chloroform-E/k S Ace/are 80.ZO v/~
Second Ot?'ec/ion
Two
dimensional T L C
of an ethereal extract of Sri Lanka Chillies
(beforesaponification) Fig. 1. A diagrammatic representation of the chromatoplate. Key for the identification of compounds is in Table I. The solvent system used in the first direction was Hexane-diethyl ether 30 : 70 (v/v) and the solvent system used in the second direction was chloroform-ethyl acetate 80:20 (v/v) Another identified orange pigment occurred in fair concentration, and its spectral properties seem to indicate that it was probably cryptocapsin 3. The occurrence of zeaxanthin was confirmed by cochromatography and spectral properties. A second yellow pigment occupying a position close to zeaxanthin and which disappeared on saponification was probably an ester of zeaxanthin. Violaxanthin occurred in an esterified form as its free occurrence before saponification was not detected. The pigments capsanthin and capsorubin occured only in trace amounts before saponification. Fig. 2 shows the two dimensional separation on a chromatoplate of a saponified extract. It was observed that after saponification the two main pigments, were capsanthin and capsorubin while violaxanthin, capsolutein 5--6 epoxide, cryptoxanthin, zeaxanthin
474
C.E.C. Lord and A. S. L. Tirimanna:
and fl-carotene and neoxanthin were also found in appreciable concentrations. These pigments were identified by their spectral properties, colour reactions and in some cases by co-chromatography. Two unidentified yellow carotenoids present in trace amounts were probably hydroxy-carotenes. In the case of some commercial brands it was observed that two additional red carotenoids occupied adjacent positions to capsanthin and capsorubin on the chromatogram.
Q 0 q~
;-~.~ .,%
(7}'-': ,....
':.,.)
:; zs '..~..... .
Ol,i~ine
,:'...,,
i, Chloroform-Elhyl Ace/ate 80 ZO % Second Di)'ection
Two dimensional TLC of an ethereal extract of Sri Lanka Chillies (after saponification) Fig. 2. A diagrammatic representation of the chromatoplate. Key for the identification of compounds is in Table I. The solvent system used in the first direction was Hexane-diethyl ether 30 : 70 (v/v) and the solvent system used in the second direction was chloroform-ethyl acetate 80:20 (v/v) These two compounds are probably hydroxy capsolutein and hydroxy capsolutein 5--6 epoxide as detected by column chromatographic techniques. It has been shown that in the ripening fruit, after the disappearance of chlorophyll only zeaxanthin and cryptoxanthin take up oxygen and are transformed by it to epoxides of violaxanthin, antheraxanthin and cryptoxanthin mono-epoxide a. At this stage of ripening, the epoxides ceased to deliver their oxygen and were transformed into polyepoxy ketones (capsorubin, capsanthin and cryptocapsin). It was also observed that there was no lutein but a high concentration
A Qualitative Study of the Carotenoid Pigments
475
of zeaxanthin. The distribution of the carotenoid pigments after saponification was in keeping with this observation. Nevertheless, the interesting situation of having high concentrations of capsanthin and capsorubin in capsicum was noteworthy. In other fruits when Table II No. of s p o t 2 9 10 12 13 14 15 17 18 19
Solvent
Absorption Maxima
Identity
Petroleum ether Hexane Chloroform Chloroform Chloroform Ethanol Chloroform Ethanol Hexane Hexane Ethanol Ethanol Petroleum ether Petroleum ether Chloroform Ethanol
425, 450, 475 421, 451, 480 460, 492 430, 460, 490 458, 488 420, 442, 470 427, 450, 484 418, 442, 470 470, 497 444, 469, 504 482, 510 417, 437, 465 432, 460, 490 425, 450, 478 423, 448, 480 380, 402, 428
fl-Carotene Zeaxanthin Antheraxanthin Violaxanthin Capsanthin Capsorubin Neoxanthin 7-Carotene Cryptoxanthin Unidentified
keto carotenoids have been detected, the concentrations of these compounds were low as in the case of canthaxanthin, and the presence of a cyclopentane ring in this class of compounds placed these compounds in a unique position n - l a . It has been shown that the orange yellow colour of one variety of capsicum fruit was controlled by a single gene, the yellow allele of which was recessive to the normal red allele 14. The distribution of the carotenoid pigments of capsicum may be on a genotypic basis. The synthesis of fruit carotenoids probably involves a different series of enzymes. The wide range of carotenoids occurring in fruits may be explained on the basis of a very few genetic mutations which control the formation of the various pigment types. Summary
A Qualitative Study of the Carotenoid Pigments o[ Sri Lant~a Chillies (Capsicum annum) Using two-dimensional thin layer chromatography more than 20 constituents have been separated in the extracts of Sri Lanka chillies before and after saponification. Many of these compounds
476
C.E.C. Lord et al.: A Qualitative Study of the Carotenoid Pigments
were tentatively identified by co-chromatography with authentic samples and by ultraviolet spectrophotometry.
Zusammenfassung Mit Hilfe zweidimensionaler Diinnschicht-Chromatographie wurden aus dem Extrakt von Sri Lanka-Paprika vor und nach der Verseifung mehr als zwanzig Komponenten abgetrennt. Viele davon wurden UV-spektrophotometrisch und durch Vergleich ihrer Chromatogramme mit solchen von Testsubstanzen zu identifizieren versucht.
References I
J. G. Lease and E. T. Lease, Food Tech. 10, 368 (1956). L. V. Cholnoky. Z. Untersuch. Lebensm. 78, 157 (1939). 3 L. V. Cholnoky, K. Gyorgyfy, E. Nagy, and M. Panczel, Acta Chim. Acad. Sci. (Hung) 6, 143 (1955). 4 A. L. Curl. J. Agric. Fd. Chem. 10, 504 (1962). 5 H. V. Schuster and E. E. Lockhart, Food Research 19, 472 (1954). 6 J. B. Moster and N. A. Prater. Food Tech. 6, 459 (1952). 7 J. B. Moster and N. A. Prater, Food Tech. 11, 146 (1957). s T. W. Goodwin, in K. Peach and M. V. Tracey, eds. Modern Methods of Plant Analysis, Vol III, Heidelberg: Springer-Verlag. 1955. S. 272. 9 j. Fioriti and R. Sims, J. Chromatography 32 (4), 761 (1968). 10 K. Egger, Ber. dtsch, botan. Ges. 81, 153 (1968). i1 M. S. Barber, L. M. Jackman, C. K. Warren, and B. C. L. Weedon, J. Chem. Soc. 1961, 4019. 12 H. Faigel and P. Karrer, Helv. Chim. Acta 44, 1257, 1904 (1961). la R. D. G. Cooper, L.M. Jackman, and B. C. L. Weedon. Proc. Chem. Soc. 1962, 215. 14 p. G. Smith. J. Hered 41, 138 (1950). 2
Correspondence and reprints: Dr. A. S. L. Tirimanna, Head, Agro Industries Section, Ceylon Institute of Scientific and Industrial Research, Colombo 7, Sri Lanka.
