Biochem. J. (1962) 85, 1

Carotenoids in Delonix regia (Gul Mohr) Flower

BY F. B. JUNGALWALA AND H. R. CAMADepartment of Biochemistry, Indian Institute of Science, Bangalore 12, India

(Received 6 February 1962)

Delonix regia (Gul Mohr) (family Leguminosae)is a striking ornamental medium-sized tree grownin most tropical countries. It produces clusters oforange-red flowers in April-May. The carotenoidspresent in a variety of different flowers have beenstudied by several workers (e.g. Goodwin, 1952).A comparative study of the carotenoids present invarious floral parts of D. regia has been made togain information about the biogenesis and role ofcarotenoids in the flower.

EXPERIMENTAL

Material&. Fully opened fresh flowers were obtainedfrom trees and the petals, sepals, anthers and filamentswere collected separately.

Light petroleum (b.p. 40460°) was left over KMnO4,washed, distilled, dried over CaCl2 and redistilled beforeuse. Diethyl ether was freshly distilled over reduced ironto remove any peroxides. Commercial acetone, methanoland ethanol were twice distilled before use. Hexane (b.p.60 80°) and acetone-free methanol were used for the phase-partition and iodine-isomerization tests. Ethanol, forspectroscopic use, was refluxed with zinc dust and KOHand distilled.Alumina (E. Merck) for chromatography was used either

as such or after partial deactivation with water or methanolas required (Goodwin & Srisukh, 1949; Taha, 1954).Magnesium oxide (for chromatographic analysis, BritishDrug Houses Ltd.) was mixed with Celite (no. 545, Johns-Manville, U.S.A.) in suitable mixtures.

Extraction of pigments. The flower parts (1 kg. of petalsand sepals, 0-5 kg. of filaments and 50 g. of anthers) weresoaked in ethanol for 2 days in the dark. The plant residueswere filtered off and blended in a Waring Blendor succes-sively with ethanol, acetone and light petroleum-ndiethylether (1:1, v/v). The filtered extracts were combined andwashed with water until free of acetone and ethanol. Theremaining organic layer containing the carotenoids wasdried over anhydrous Na2SO4, filtered and concentrated atreduced pressure.

Saponification of extracts and removal of sterol8. The oilobtained was dissolved in ethanol and saponified under N2with ethanolic 10% (w/v) KOH at 60° for 0.5 hr., and thenleft overnight at room temperature. Unsaponifiablematerial was extracted with diethyl ether, washed free ofalkali, dried over anhydrous Na2SO4, filtered and evapor-ated. Chlorophylls present in sepal extracts were removedby this treatment.The unsaponifiable material was redissolved in light

petroleum and left overnight under N2 at -30°; the pre-cipitated sterols were removed by filtration.

1

Phase-partition of carotenoids between solvents. Thecarotenoid extracts were subjected to phase-partitionbetween light petroleum and aq. 90% (v/v) methanol.Carotene hydrocarbons, monohydroxy xanthophylls, andmono- and di-epoxides of carotene hydrocarbons and ofmonohydroxy xanthophylls are found in the upper layer,whereas the di- and poly-hydroxy xanthophylls and theirepoxy derivatives remain in the lower layer.Column chromatography. The upper- and lower-phase

carotenoids were dissolved in light petroleum before chro-matography. The former group of carotenoids were chro-matographed on an alumina column (3 cm. x 35 cm.) whichhad been partially deactivated by treatment with 5% (v/w)of water. Carotene hydrocarbons did not separate well onthis column and were eluted with light petroleum. Theremaining carotenoids were well resolved when the columnwas eluted (gradient elution) with light petroleum contain-ing increasing amounts of diethyl ether. Fractions (15-20 ml.) were collected and their u.v. and visible spectrawere determined. Fractions showing similar spectra werecombined and concentrated. Individual pigments werefurther purified by rechromatography on more activealumina columns (2 cm. x 30 cm.). The order ofband elutionof monohydroxy xanthophylls and epoxides of carotenehydrocarbons and of monohydroxy xanthophylls obtainedfrom petals of D. regia is given in Table 2.The carotene hydrocarbons eluted from these columns

were rechromatographed as above on an alumina column(3 cm. x 40 cm.) which had been partially deactivated bytreatment with 1% (v/w) of water. Colourless substanceslike phytoene and phytofluene were located by their u.v.fluorescence. The fractions collected were chromatographedagain on fully active alumina. The identification of carotenehydrocarbons from petals of D. regia separated on aluminapartially deactivated with 1% of water is given in Table 1.The separation of ,B-carotene, pigment X and c-carotenewas difficult and longer columns (2 cm. x 45 cm.) wereemployed for satisfactory resolution.The lower-phase carotenoids were adsorbed on a methanol-

deactivated alumina column (3 cm. x 35 cm.) and wereresolved by elution with light petroleum containing increas-ing amounts of acetone. The chromatographic procedurewas repeated three or four times before pure pigments wereobtained. The order of elution of these carotenoids obtainedfrom petals of D. regia is listed in Table 3.

Better resolution of the pigments was obtained by using amagnesium oxide-Celite (1:1, w/w) column (3 cm. x 35 cm.)eluted with light petroleum containing increasing amountsof acetone. Partially resolved pigments were purifiedfurther by rechromatography on magnesium oxide-Celite(3:2 and 2: 1, w/w) columns (2-5 cm. x 35 cm.). Theirelution from a magnesium oxide-Celite (3:2, w/w) columnis illustrated in Table 4. The cis-trans isomers, chrysanthe-

Bioch. 1962, 85

1

F. B. JUNGALWALA AND H. R. CAMA

maxaanthin and flavoxanthin, were eluted as a single bandfrom these columns.The alteration in the order of elution of these pigments

from magnesium oxide-Celite columns oompared with thatfrom methanol-deactivated alumina was verified bychromatography of authentic carotenoid samples.

Separation of carotenoids from sepals, filaments andanthers of D. regia was performed similarly.

Iden*ification of carotenoids. Individual carotenoids wereidentified by comparing their chromatographic propertiesand u.v. and visible spectra with those of authentic caro-tenoid samples (Tables 1, 2 and 4).

Phase-partition of the carotenoids between hexane andaq. methanol (95, 85 and 75%, v/v) acoording to themethod of Petracek & Zechmeister (1956) also helped intheir identification.The cis-trans configuration of the carotenoids was ascer-

tained by the iodine-isomerization test (Zechmeister, 1960).The test was performed by the addition of0 5 ml. of0 001%iodine in hexane to 10 ml. of a solution of carotenoid inhexane in a test tube and exposure of this mixture to lightfrom an incandescent lamp for periods of 5, 15 and 30 mi.The u.v. and visible spectra of the carotenoid in hexanewere recorded before and after exposure to light. Iso-merization of cia8carotenoids leads to slightly higher A.,,values; with trans-carotenoids the shift is to lower A,|values.

Carotenoids having epoxy groups were characterized by amodified conc. HCl-ether test (Curl & Bailey, 1954; Karrer& Jucker, 1950). The isomerization of 5,6-epoxide into5,8-epoxide was studied by observing the change in thespectrum of the carotenoid in light petroleum or ethanolbefore and after addition of a drop of ethanolic 0-O0N-HCI.Hypsochromic (lower-wavelength) shifts of 15-20 and35-45 miz were shown by 5,6-monoepoxy and 5,6-diepoxycarotenoids respectively, with an associated decrease in the

extinction due to the formation of 5,8-epoxy (furanoid)carotenoids (Table 4).

Melting points and mixed melting points were deter-mined for crystalline pigments.

Quantitative determination. Weights of flower componentswere determined after drying at 800.

Total carotenoids of different flower parts were deter-mined by dissolving the unsaponiflable materials in aknown volume of light petroleum and measuring E valuesat 445 mu (Table 5), assuming E1% for the crude mixtureto be that of f-carotene [2500 (Goodwin, 1955)].The concentrations of individual carotenoids (Table 6)

were determined similarly by measuring Em.. and com-paring it with known E1% values at A.. for pure pig-ments (Goodwin, 1955). For unknown pigments E1l%. at.. was assumed to be 2500 as suggested by Goodwin(1954).

Speetrophotometric mea8urement8. The u.v. and visiblespectra of the carotenoids were measured with a Beckmanspectrophotometer (model DU).

RESULTS

Carotenoi4s from petalsCarotene hydrocarbons. Carotene hydrocarbons

isolated from petals of D. regia are listed in Table 1.Two phytofluene bands (blue-green fluorescence),

with similar absorption spectra, have been observedduring the chromatography of extracts of tomatoesand petals of Calendula officinalis (Koe & Zech-moister, 1953; Goodwin, 1954). Only one phyto-fluene band was present in the extracts of petals ofD. regia and this was identical with the lower phyto-fluene band from tomatoes. Petracek & Zech -

Table 1. Chromoatographic-adsorption analysis and identifIcation of carotene hydrocarbonsfrom petalsof Delonix regia

The pigments were chromatographed on alumina (deactivated by treatment with 1% of water) by using lightpetroleum containing varyg amounts of ether as the developing solvent. The pigments are listed in the order inwhich they were eluted from the column.

(Absorption maxima

in lightpetroleum

Band (mP)1 275, 285, 296

2 332, 348, 367

3 425, 450, 4784 380, 405, 425, 4505 380, 400, 425

6 427, 455, 4857 432, 460, 490

8 440, 4689 440, 465, 490

10 445, 470, 503

Concn. (%, v/v)of ether

required forelution ofband

2

3-5

5-78

9

10-13

15-17

20

Iodine-isomerization

test Co-chromatography with authentic sample- From tomatoes (Rabourn,

Quackenbush & Porter, 1954)- From tomatoes (Koe & Zechmeister,

1953)tranm Synthetic (Hoffmann-La Roche)transtrans From tomatoes (Porter & Zscheile,

1946)transtrane From carrots (Kuhn & Brockmann,

1933)ciscia From tomatoes (Porter & Zscheile,

1946)trans From tomatoes (Porter & Zscheile,

1946)

IdentificationPhytoene

Phytofluene

,8-CarotenePigment XC-Carotene

8-Caroteney-Carotene

ProlycopeneNeolycopene

Lycopene

2 1962

CAROTENOIDS IN DELONIX REGIA FLOWERmeister (1952) have suggested that the normalnaturally occurring phytofluene is probably a ci8-isomer.- f-Carotene formed the major hydrocarbonof petals and was crystallized (m.p. 181-182°) fromlight petroleum. Pigment X isolated from petals ofD. regia had chromatographic and light-absorptionproperties (Fig. 1A) similar to those of a pigmentisolated from Phycomycea blakealeeanu8 (Goodwin &Osman, 1953) and C. officinalia (Goodwin, 1954).Petzold, Quackenbush & McQuistan (1959) haveisolated two similar pigments, a- and ,B-zeacarotene,from corn, and pigment X may be identical withf-zeacarotene. Lycopene occurred as a mixture ofci-trana isomers, and tran8-lycopene was crystal-lized (m.p. 168-169°) from light petroleum. Pro-lycopene and neolycopene, after isomerization withiodine in light, showed a bathochromic shift, and anew peak at about 500 m, appeared with pro-lycopene (Goodwin, 1956).Monohydroxy xanthophyUs8 and epoxide8 of caro-

tene hydrocarborw and of monohydroxy xanthophyla8.These carotenoids isolated from the petals ofD. regia are listed in Table 2. 5,6-Monoepoxy-p-carotene and 5,6-diepoxy-fl-carotene were charac-terized by their conversion into mutatochrome(Am, 402, 425, 452 m,) and aurochrome (A.380, 400, 425 mbt) respectively on the addition oftraces of ethanolic hydrochloric acid (Karrer &Jucker, 1945 b). Mutatochrome itself was presentin the extracts and differs from flavochrome inhaving slightly higher A. values. Crypto-xanthin formed a very faint band on the column.Unidentified-428, situated above cryptoxanthin onalumina columns (Table 2, band 5), showed a singlebroad band at 428 m,u (Fig. 1B) and its spectrumdid not change on the addition of ethanolic hydro-chloric acid. However, it gave a faint blue colouron the addition of ether and hydrochloric acid,indicating the presence of a 5,8-epoxy group. Thepartition ratio between hexane and 95% (v/v)methanol (88:12) suggests that it may contain asingle hydroxyl group. Unidentified-420 (Table 2,band 6) was found to be the cia-isomer of unidenti-fied-425 (Table 2, band 7); the absorption spectrumof unidentified-425 is given (Fig. 1,C). Chromato-graphic behaviour, partition ratio, absorptionspectrum and the ethanolic hydrochloric acid testsuggest that unidentified-425 is a 5,8-epoxy-monohydroxy carotenoid, perhaps tran8-crypto-flavin (Karrer & Jucker, 1946). The absorptionspectrum oftrane-rubixanthin was indistinguishablefrom that of y-carotene (Mackinney, 1935) and thusdiffered from rubixanthin-like pigments reportedby Goodwin (1954) and Taha (1954) in flower petalsof C. officinalia and Tecoma capenai8 respectively.Unidentified-425a (Table 2, band 10; Fig. 2,A),present in petals, showed the presence ofa 5,8-epoxygroup with a single hydroxyl group as suggested by

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Vol. 85 3

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F. B. JUNGALWALA AND H. R. CAMA

chromatographic behaviour, partition ratio, absorp-tion spectrum and ethanolic hydrochloric acid test.

Di- and poly-hydroxy xanthophyU8 and theirepoxides. Di- and poly-hydroxy xanthophylls andtheir epoxides characterized are given in Tables 3and 4. Though the absorption spectra of 5,6-monoepoxylutein, antheraxanthin, violaxanthin,lutein, zeaxanthin and neoxanthin are very similar,these pigments could be differentiated easily bytheir chromatographic behaviour on differentadsorbants (magnesium oxide-Celite and methanol -deactivated alumina) and by their reactions withethanolic hydrochloric acid (Tables 3 and 4). Thesignificant difference in the partition ratios observedfor members of this group of carotenoids (Table 4)also aided their identification.

Chrysanthemaxanthin and flavoxanthin are c8-tran8 isomers. Auroxanthin occurred as a mixtureof ci&-trans isomers. Neoxanthin, a leaf carotenoid,tentatively identified as 5,6-epoxy-6'(or 5')-hydro-3,3',5'(or 6')-trihydroxy-p-carotene (Goldsmith &Krinsky, 1960), was present in the petals of D.regia.The adsorption affinity on alumina columns of

these carotenoids increases with the increase intheir hypophasic character (Tables 3 and 4). Nosuch relationship was found between partitionratios and adsorption affinity on magnesium oxide-Celite columns.Xanthophylls having one or more 5,6-epoxy

groups (e.g. 5,6-monoepoxylutein, antheraxanthinand violaxanthin) are less strongly adsorbed on mag-nesium oxide-Celite columns than the correspond-ing parent xanthophylls (lutein and zeaxanthin),and an increase in the number of 5,6-epoxy groupsalso results in decreased adsorption [in the order:violaxanthin, antheraxanthin, zeaxanthin (Table3)]. Similar observations were made by Tsukida &Zechmeister (1958) on the adsorption of ,-carotene,

5,6- monoepoxy., - carotene and 5,6 - diepoxy-, -carotene on magnesium oxide-Hyflo-Supercelcolumns. Further, neoxanthin, with three hydroxylgroups and one 5,6-epoxy group, is adsorbed lessstrongly than chrysanthemaxanthin, which hasonly two hydroxyl groups and one 5,8-epoxy group(Table 3).

Carotenoids from sepal8Carotenoids present in sepals of D. regia were

characterized according to the methods describedfor petals and are listed in Table 6.

Phytofluene in sepals was identical with thelower phytofluene band from tomatoes (Petracek &Zechmeister, 1952). a.-Carotene (A,, 420, 443 and474 m,u) could be distinguished from ,-caroteneand its ci8-isomer by its absorption spectrum andthe iodine-isomerization test. Unidentified-428was identical with that from petals.

Carotenoids from filamentsCarotenoids present in filaments of D. regia are

given in Table 6. The unidentified-428 (Fig. 2, B)was identical with that from petals and sepals.

Carotenoids from anthersCarotenoids in anthers of D. regia were few in

number compared with other parts of the flower(Table 6). p-Carotene and its ci8-isomer, neo-fl-carotene, were the only carotene hydrocarbonspresent in anthers. Two unknown carotenoids(unidentified-430 and unidentified-435), with ad-sorption affinities slightly greater than that ofcryptoxanthin on alumina columns, had Am.. at410, 430 and 455 m,t and at 410, 435 and 460 m,u(Fig. 2, C) respectively. Chromatographic behaviour,partition ratio, absorption spectra and the etha-nolic hydrochloric acid test suggest that unidenti-

Table 3. Chromatographic-ad8orption analyai8 of di- and poly-hydroxy xanthophyll8 and their epoxidesfrom Delonix regia

The developing solvent used was light petroleum containing varying amounts of acetone. The pigments arelisted in the order in which they were eluted from the column.

Order of adsorption of pigments on methanol-deactivatedalumina column

Lutein (3,3'-dihydroxy-oc-carotene)Zeaxanthin (3,3'-dihydroxy-,-carotene)5,6-MonoepoxyluteinChrysanthemaxanthin (5,8-monoepoxylutein)Flavoxanthin (5,8-monoepoxylutein)Antheraxanthin (5,6-monoepoxyzeaxanthin)Violaxanthin (5,6-diepoxyzeaxanthin)Auroxanthin (5,8-diepoxyzeaxanthin)Neoxanthin (5,6-monoepoxy-6'-hydro-5'-hydroxyzeaxanthin?)

Conen.(%, v/v) of

acetone toelute bandfrom thecolumn10-1515-1717-2020-2320-2325-3030-4050-6070-80

Order of adsorption ofpigments on magnesium

oxide-Celite (3:2)column

5,6-MonoepoxyluteinViolaxanthinLuteinAntheraxanthinZeaxanthinNeoxanthinChrysanthemaxanthinFlavoxanthinAuroxanthin

Concn.(%, v/v) ofacetone toelute bandfrom thecolumn5-1010-1515-2020-2525-3030-3535-4035-4040-50

4

CAROTENOIDS IN DELONIX REGIA FLOWER

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Vol. 85 5

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F. B. JUNGALWALA AND H. R. CAMAfled-435 is a 5,8-epoxy-monohydroxy carotenoidand unidentified-430 is its ci8-isomer. Zeaxanthinwas crystallized from light petroleum containingmethanol and had m.p. 2050 unchanged by ad-miixture with an authentic sample supplied byHoffmann-La Roche. All other carotenoids, except$-carotene and its derivatives, were absent inanthers.

Quantitative experimentsTotal carotenoids present in petals, sepals, fila-

ments and anthers of D. regia are given in Table 5.Relative amounts of the individual carotenoidspresent in these parts of the flower are given inTable 6.

In petals, carotene hydrocarbons predominateover 'oxygenated' carotenoids (hydroxy and epoxycarotenoids) (7:3). #-Carotene is present in thelargest amount. y-Carotene, rubixanthin and lyco-pene isomers are other major pigments in petals.The ratio of carotene hydrocarbons to 'oxygen-

ated' carotenoids is higher in sepals than in petals(21:4). Sepals contain large amounts of colourlesscarotenoids (phytoene and phytofluene) togetherwith f-carotene.

Filaments contain considerable amounts of'oxygenated' carotenoids (65% of total caro-tenoids), mainly lutein, zeaxanthin and epoxycarotenoids. Phytoene and fl-carotene are the majorcarotene hydrocarbons present.The total carotenoids present in anthers of

D. regia (570 mg./100 g. dry wt.) are significantlyhigher than values recorded for anthers of otherflowers. The bulk of the total carotenoids presentare 'oxygenated' carotenoids (98 %), mainly zea-xanthin (90 %). fl-Carotene and its ci-isomer arepresent in very small anounts.

DISCUSSION

Although many investigators have studied thedistribution of carotenoids in petals and anthers ofdifferent flowers, no systematic qualitative andquantitative data are available on carotenoids indifferent parts of the same flower. The presentinvestigation describes a detailed study of caro-tenoids in different parts of D. regia flower.The distribution of carotenoids in flower petals

varies considerably; some contain few or no caro-

E 0-6

0-4 -/A~~~~~A360 400 440 480 500

Wavelength (mg)Fig. 1. Absorption spectra in light petroleum of caro-tenoids from petals. A, Pigment X, ; B, unidentified-428 (Table 1, band 5),----- ; C, unidentified-425 (Table 1,band 7), *-*-.

)0 440Wavelength (m,)

Fig. 2. Absorption spectra in light petroleum of caro-tenoids. A, Unidentified-425a (from petals, Table 1, band10), ; B, unidentified-428 (from filaments), e-e-* ;C, unidentified-435 (from anthers),-----.

Table 5. Quantitative di8tribution of-total carotenoid8 in Delonix regia

Total carotenoids

mg./100 g. mg./100 g.fresh wt. dry wt.

18 8 129-015-5 97.55-1 36-0

190-4 570-1

EXAm at445 mu aftersaponification

315-075-242-5

408-0

Ratio of carotenehydrocarbons to

' oxygenated'carotenoids70:3084:1635:652:98

PetalsSepalsFilamentsAnthers

19626

CAROTENOIDS IN DELONIX REGIA FLOWER

Table 6. RekWive amounts of individual carotenoid8 in petal8, 8epalI,filaments and anthers of Delonix regia

A dash indicates the absence of the carotenoid.

Percentage by wt. of total carotenoids present

Carotenoid In petals In sepals In filaments In anthersCarotene hydrocarbonsPhytoene 7-66 36-64 11-79Phytofluene 2-48 7-59 1-35ox-Carotene 0-54Neo-,-carotene - 1-49 0-12$-Carotene 34-05 32-85 12-55 1-73Pigment X 0-88c-Carotene 1-49 1-34 1-618-Carotene 1-65y-Carotene 11-21 1.64 3-03Prolycopene 4-61 0-76Neolycopene 2-07 1.65 1-6Lycopene 4-27 1-42 2-0

Monohydroxy xanthophylls and epoxides of carotene hydrocarbons and of monohydroxy xanthophylls5,6-Monoepoxy-,B-carotene 0-27 - 0-11Mutatochrome 1-0 0-5 0-155,6-Diepoxy-p-carotene 0-17 - 1-16Cryptoxanthin 0-07 1-21 5-7 2-21Unidentified-428 0-19 0-45 0-9Unidentified-420 0-8Unidentified-425 1-13Unidentified-430 - 0-05Unidentified-435 0-22ci-Rubixanthin 5-02Rubixanthin 10-05Unidentified-425a 1-60

Di- and poly-hydroxy xanthophylls and their epoxidesLutein 2-48Zeaxaanthin 1-825,6-Monoepoxylutein 0-82Chrysanthemaxanthin + flavoxanthin 1-5Antheraxanthin 0-44Violaxanthin 0-85cu-Auroxanthin GC28Auroxanthin 0-94Neoxanthin 0-2

3-362-59

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2-470-4

21-715-23.73-956-154-43

1-08

89-82

5-240-29

0-18

tenoids, whereas others contain mainly epoxy caro-tenoids specific to petals (Goodwin, 1952; Kaxrer,Jucker & Krause-Voith, 1947). The petals ofD. regia contain nine well-characterized epoxycarotenoids and a few unidentified carotenoidswhich appear to be epoxides, in addition to variousother carotenoids. The role of epoxy carotenoids inpetals is not yet known. However, they may beintermediates in the transfer of oxygen and forma-tion of xanthophylls (Cholnoky, Gyorgyfy, Nagy &P6ncz6l, 1955).

Certain carotenoids are present only in a particu-lar part of D. regia flower, e.g. pigment X, 8-caro-tene and rubixanthin are only found in petals,whereas m-carotene is confined to sepals.

Vivino & Palmer (1944) observed that mixedpollen gathered by bees contains traces of carotene(4-15 mg./100 g.), but considerable amounts ofxanthophylls (14-41 mg./100 g.). Karrer, Eugster& Faust (1950), while studying the distribution of

carotenoids in pollen and anthers, found that esteri-fled lutein is the principal carotenoid component ofpollen, whereas p-carotene is present in traces. Inagreement with these observations, xanthophyllsrepresent 98% ofthe pigments present in anthers ofD. regia. However, instead of lutein, zeaxanthin isthe principal carotenoid and represents 90% of thetotal carotenoids. Large amounts of 'oxygenated'carotenoids, all having the P,ionone ring structure,are present in anthers of D. regia, whereas com-paratively snall quantities of carotenoids of variedring structure (oc-, ,- and y-ionone) are present inpetals, sepals and filaments. P-Carotenoids may besynthesized from ,-carotene by oxidation andhydroxylation in situ in anthers of D. regia or theymay be formed from various carotenoids present inother parts of the flower, only being transferred tothe anthers after synthesis is completed.

Indirect evidence indicates that carotenoids playa part in reproduction of cryptogams and various

Vol. 85 7

8 F. B. JUNGALWALA AND H. R. CAMA 1962

animal species (Goodwin, 1952). It is not yetknown whether they play a similar role in thephanerogams. The constituents of anthers aretransferred in part to seeds, but petal, sepal andfilament constituents are discarded by plants. Thepresence of considerable amounts of specific caro-tenoids in the anthers of D. regia suggests that'oxygenated' carotenoids may have some role inthe reproduction of this plant.

SUMMARY

1. The qualitative and quantitative distributionof carotenoids in different parts of Delonix regia(Gul Mohr) flower was studied by chromatographic,spectrophotometric and other methods. The parti-tion ratios, hitherto not reported, of a number ofcarotenoids between different solvents are recorded.

2. The petals ofD. regia contained 29 carotenoids.The major pigments identified are: phytoene,phytofluene, $-carotene, y-carotene, lycopeneisomers, rubixanthin, lutein, zeaxanthin andseveral epoxy carotenoids.

3. The sepals contained 18 carotenoids. Phyto-ene, phytofluene, ,-carotene, y-carotene, lycopeneisomers, lutein, zeaxanthin and carotenoid epoxideswere the major carotenoids.

4. Filaments contained 20 carotenoids, includingphytoene, ,-carotene, y-carotene, cryptoxanthin,lutein, zeaxanthin, antheraxanthin, violaxanthin,chrysanthemaxanthin, flavoxanthin and otherepoxy carotenoids.

5. The highest concentrations of total caro-tenoids were found in anthers. Of the total caro-tenoids, 90% is zeaxanthin. The only other caro-tenoids present were ,-carotene derivatives withtraces of two uncharacterized carotenoids.

6. The probable physiological significance ofsuch a complex mixture of carotenoids in differentparts of the flower is discussed.

Sincere thanks are due to Professor P. Karrer, Dr 0.Isler and Professor T. W. Goodwin for generous gifts ofauthentic samples of carotenoids and for providing usefuldata.

REFERENCES

Cholnoky, L., Gyorgyfy, C., Nagy, E. & Pancz6l, M. (1955).Acta chim. Acad. Sci. hung. 6, 143.

Curl, A. L. & Bailey, G. F. (1954). J. agric. Fd Chem. 2,685.Gillam, A. E. & Heilbron, I. M. (1935). Biochem. J. 29, 1064.Goldsmith, T. H. & Krinsky, N. I. (1960). Nature, Lond.,

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