Carotenoid Composition of Telekia speciosa

The carotenoid composition of the flower of Telekia speciosa was investigated for the first time by HPLC-DAD-MS. In addition to the main carotenoid lutein and its geometrical isomers, 5,6-epoxy-carotenoids, namely violaxanthin, lutein 5,6-epoxide and antheraxanthin, were detected in larger amounts. In addition, β-carotene 5,6-epoxide and β-carotene 5,6,5′,6′-diepoxide were found, which occurs very rarely in plants. For unambigous identification, β-carotene 5,6-epoxide and β-carotene 5,6,5′,6′-diepoxide were prepared semisynthetically, and they were characterized by 1H and 13C NMR and HPLC-CD methods.


Introduction
Telekia speciosa (Schreb.)Baumg.(basionym-Buphtalmum speciosum Schreb.) is the only species classified in the genus Telekia Baumg.(Asteraceae).It can mostly be found in Southeastern Europe and Asia Minor.It is the only species belonging to the genus Telekia Baumg.and, based on recent studies [1], is related to the resiniferous taxa of Inula L., including I. helenium.
To date, little phytochemical work has been performed on T. speciosa, known as "yellow oxeye" in English [1][2][3][4].Its leaves are triangular, doubly serrated, long and petiolated, and the flowers are yellow [5].The roots of T. speciosa contain essential oils with eudesmanetype sesquiterpene lactones as major constituents [6].The essential oils of different parts of Telekia speciosa contain more than 100 compound, which were determined by GC-MS-FID technique [7].From the flowers, ferulic acid and caffeic acid derivatives were isolated: 6-O-(E)-caffeoyl-glucopyranose, and 3-O-caffeoylquinic acid (chlorogenic acid).An analysis showed the presence of a flavonol glucoside (patulitrin) as well [8].However, the carotenoid composition of the Telekia speciosa flower has not been studied so far.
To aid in the identification of β-carotene-5,6-epoxide (10) and 5,6,5 ,6 -diepoxide (11), the compounds were prepared semisynthetically from β-carotene by epoxidation with monoperoxophthalic acid.The epoxidation reaction was carried out in our laboratory by Péter Molnár in the 1970s.The separation and purification of β-carotene mono-(10) and diepoxide (11) was achieved by open-column chromatography on Ca(OH) 2 adsorbent.A detailed NMR analysis of the isolated products was undertaken in the 1990s at the Department of Organic Chemistry of the University of Bern, by Andrea Steck.The purity of the materials used for our study, produced nearly 50 years ago, was checked with HPLC and proton and carbon-13 NMR tests.The results (purity >95%, HPLC) show that carotenoids stored under the right conditions (crystalline, in an ampoule under N 2 or Ar atmosphere, at −20 • C) do not decompose for a long time.
Péter Molnár in the 1970s.The separation and purification of β-carotene mono-(10) and diepoxide (11) was achieved by open-column chromatography on Ca(OH)2 adsorbent.A detailed NMR analysis of the isolated products was undertaken in the 1990s at the Department of Organic Chemistry of the University of Bern, by Andrea Steck.The purity of the materials used for our study, produced nearly 50 years ago, was checked with HPLC and proton and carbon-13 NMR tests.The results (purity >95%, HPLC) show that carotenoids stored under the right conditions (crystalline, in an ampoule under N2 or Ar atmosphere, at −20 °C) do not decompose for a long time.
As previously mentioned, the epoxidation of β-carotene with monoperoxyphthalic acid was not stereoselective, since the two diastereomeric mono-epoxides formed in equal amounts (Figure 2).The separation of diastereomeric β-carotene-5,6-epoxides (5R,6S)-10a and (5S,6R)-10b was achieved on a Chiralpak IA column using hexane/dichloromethane 85:15 as an eluent.Since online HPLC-UV and HPLC-ECD measurements had been used efficiently for the investigation stereoisomeric mixtures of natural products [25][26][27], this technique was utilized in the separation of diastereomers.The HPLC-ECD chromatogram recorded at 270 nm showed opposite Cotton effects (CEs) for the two diastereomers.Online HPLC-ECD spectra were recorded by stopping the flow of the eluent in the HPLC-ECD flow cell at the maximum concentration of the separated diastereomers.The diastereomers had nearmirror-image ECD curves above 220 nm, allowing for the configurational assignment of the synthetic diastereomers (Figure 3).The CEs of natural, and semisynthetic β-carotene-5,6-epoxides had opposite signs above 240 nm, reflecting the different configuration of the 5,6-epoxy end groups.The first eluting diastereomer was the semisynthetic (5S,6R) stereoisomer, while the second eluting diastereomer was the natural one (5S,6R).These data corroborated the reported values of (5 S,6 R)and (5 R,6 S)-β-cryptoxanthin-5 ,6epoxide [23].
Since online HPLC-UV and HPLC-ECD measurements had been used efficiently for the investigation stereoisomeric mixtures of natural products [25][26][27], this technique was utilized in the separation of diastereomers.The HPLC-ECD chromatogram recorded at 270 nm showed opposite Cotton effects (CEs) for the two diastereomers.Online HPLC-ECD spectra were recorded by stopping the flow of the eluent in the HPLC-ECD flow cell at the maximum concentration of the separated diastereomers.The diastereomers had near-mirrorimage ECD curves above 220 nm, allowing for the configurational assignment of the synthetic diastereomers (Figure 3).The CEs of natural, and semisynthetic β-carotene-5,6-epoxides had opposite signs above 240 nm, reflecting the different configuration of the 5,6-epoxy end groups.The first eluting diastereomer was the semisynthetic (5S,6R) stereoisomer, while the second eluting diastereomer was the natural one (5S,6R).These data corroborated the reported values of (5′S,6′R)-and (5′R,6′S)-β-cryptoxanthin-5′,6′-epoxide [23].The separation of diastereoisomers of β-carotene diepoxide (11) was successful on the Chiralpak IA-3 column using hexane/dichloromethane 75:25 as an eluent (Figure 4).Even though baseline separation could not be achieved, three peaks were observed and weak ECD spectra were recorded for the enantiomeric diepoxides, the first-and thirdeluting peaks.Due to the weak ECD signals, enantiomeric correction was used instead of correction with blank solvent as the background.Near-mirror-image ECD spectra were obtained after smoothing, and the assignment of the absolute configuration of the enantiomers was performed on the basis of our previous results [22,23] using the sign of the CE below 240 nm (Figure 5).The first-eluting peak was identified as the (5S,6R,5′S,6′R)-diepoxide, while the third-eluting peak belonged to the (5R,6S,5′R,6′S)-diepoxide. The second-eluting stereoisomer, with roughly twice the UV intensity of the other two and a baseline ECD curve, was identified as the meso compound.The separation of diastereoisomers of β-carotene diepoxide (11) was successful on the Chiralpak IA-3 column using hexane/dichloromethane 75:25 as an eluent (Figure 4).Even though baseline separation could not be achieved, three peaks were observed and weak ECD spectra were recorded for the enantiomeric diepoxides, the first-and third-eluting peaks.Due to the weak ECD signals, enantiomeric correction was used instead of correction with blank solvent as the background.Near-mirror-image ECD spectra were obtained after smoothing, and the assignment of the absolute configuration of the enantiomers was performed on the basis of our previous results [22,23] using the sign of the CE below 240 nm (Figure 5).The first-eluting peak was identified as the (5S,6R,5 S,6 R)-diepoxide, while the third-eluting peak belonged to the (5R,6S,5 R,6 S)-diepoxide.The second-eluting stereoisomer, with roughly twice the UV intensity of the other two and a baseline ECD curve, was identified as the meso compound.

Analysis of Telekia speciosa
The HPLC-DAD and HPLC-DAD-MS analyses were performed using a C30 phase.The carotenoids were identified by their elution order on the C30 HPLC column via spiking with authentic standards, UV-visible spectra (λmax, spectral fine structure (%III/II)), cis peak intensity (%AB/AII) and mass spectrum compared to standards and the data available in the literature [19,21].

Analysis of Telekia speciosa
The HPLC-DAD and HPLC-DAD-MS analyses were performed using a C30 phase.The carotenoids were identified by their elution order on the C30 HPLC column via spiking with authentic standards, UV-visible spectra (λmax, spectral fine structure (%III/II)), cis peak intensity (%AB/AII) and mass spectrum compared to standards and the data available in the literature [19,21].

Analysis of Telekia speciosa
The HPLC-DAD and HPLC-DAD-MS analyses were performed using a C 30 phase.The carotenoids were identified by their elution order on the C 30 HPLC column via spiking with authentic standards, UV-visible spectra (λ max , spectral fine structure (%III/II)), cis peak intensity (%A B /A II ) and mass spectrum compared to standards and the data available in the literature [19,21].
Peak 6 was found to be (13′Z)-lutein because of the hypsochromic shift of λmax, and the high intensity of the cis peak in the UV spectrum.Its m/z value was 551, co-chromatography with the iodine-catalyzed isomerization mixture of lutein (1) confirming to the the (13′Z)-isomer.The other cis isomer of lutein ( 1), (13Z)-lutein was covered by peak 5, and could be identified by EIC spectra at m/z 551.
The next major component, Peak 3, provided λmax at 415, 438, 467 nm in UV with a fine structure and a 567 m/z value ([M + H − H2O] + ) in the MS spectra.After spiking with a standard isolated from the petals of Chelidonium majus, it was confirmed to be all-translutein 5,6-epoxide (7).
Peak 4 had a UV-visible spectra similar to that of lutein (1).The molecular masses, detected at 584 seemed to correspond to (all-E)-antheraxanthin (5).This assumption was confirmed by co-elution with the authentic standard.1.
Peak 6 was found to be (13 Z)-lutein because of the hypsochromic shift of λ max , and the high intensity of the cis peak in the UV spectrum.Its m/z value was 551, co-chromatography with the iodine-catalyzed isomerization mixture of lutein (1) confirming to the (13 Z)-isomer.The other cis isomer of lutein ( 1), (13Z)-lutein was covered by peak 5, and could be identified by EIC spectra at m/z 551.
The next major component, Peak 3, provided λ max at 415, 438, 467 nm in UV with a fine structure and a 567 m/z value ([M + H − H 2 O] + ) in the MS spectra.After spiking with a standard isolated from the petals of Chelidonium majus, it was confirmed to be all-trans-lutein 5,6-epoxide (7).
Peak 4 had a UV-visible spectra similar to that of lutein (1).The molecular masses, detected at 584 seemed to correspond to (all-E)-antheraxanthin (5).This assumption was confirmed by co-elution with the authentic standard.
Peaks 14 and 16 provided a UV-visible spectrum similar to that of peak 7. The molecular masses ([M + H] + ), detected at m/z 553 for both compounds, seemed to correspond to (all-E)-α-cryptoxanthin (9), and (all-E)-β-carotene 5,6-epoxide (10).These assumptions were confirmed by spiking with the authentic standards.Peak 8 had a UV-visible spectra similar to that of peak 1 and 3 (λ max : 418, 437, 468 nm, (%III/II: 91)).The molecular mass ([M + H] + ), detected at m/z 569 for the compound, showed that the molecule contained two oxygen atoms.Based on the fine structure of the UV-visible spectrum and the retention time of this peak, it was identified as β-carotene-5,6,5 ,6 -diepoxide (11).These were confirmed by co-chromatography with the authentic standards produced in our laboratory.
Peak 18 had a UV-visible spectra similar to that of zeaxanthin (3).The m/z value of 537 suggested that this was (all-trans)-β-carotene (8).Peak 19 was the 9Z-isomer of β-carotene (8) based on its UV-VIS and MS spectra.

Discussion
Flowers were collected at two different places, but no significant differences were observed in the total carotenoid content.The petals contained slightly more carotenoids, while the carotenoid content of the floret did not even reach a tenth of the inflorescences.
One part of the work focused on the identification of carotenoids in fresh flowers of T. speciosa using HPLC.Similarly to some other flowers, lutein and its geometrical isomers were the major carotenoids.The amounts of (all-E)-lutein varied between 38% and 44% in inflorences, florets and petals.The proportion of (9Z)-and (9 Z)-lutein isomers was almost the same, except for floret, where the amount of the (9 Z)-isomer reached 11%, whereas in petals it was only approx.1%.Other hydroxy carotenoids are represented by zeaxanthin (3) and α-cryptoxanthin (9), as well as their cis-isomers.Epoxy-carotenoids, namely violaxanthin (4), lutein 5,6-epoxide (7) and antheraxanthin (5), were found in higher amounts (5-8%) in the petals and in the whole inflorescence.The co-occurrence of these three carotenoids in flowers is quite rare.Usually, besides violaxanthin (4), some neoxanthin can mainly be detected.In some cases, violaxanthin (4) can be found, in addition to larger amounts of lutein epoxide (7), but these flowers (Inula helenium, Cytisus nigricans) do not contain lutein (1) [unpublished].The floret did not contain the 5,6-epoxides, but contained their furanoids.Due to the complexity of the system, we could not clearly identify these peaks.To our surprise, we found a larger amount of β-carotene diepoxide (11) and a smaller amount of β-carotene monoepoxide (10).In petals, the amount of β-carotene diepoxide enriched to 10%, while in the tubular flower it could not detected.
The occurrence of the 5,6-epoxy and 5,6,5 ,6 -diepoxy β-carotene, besides the violaxanthin and lutein 5,6-epoxide, is rather rare in nature.The detection of these compounds indicates that not only β-carotene hydroxylase, but also the enzyme β-carotene epoxidase, works in the Telekia speciosa flower.In plants, generally, β-carotene hydroxylase produces zeaxanthin, which is further converted into antheraxanthin and violaxanthin by the enzyme zeaxanthin epoxidase.The hydroxylation of β-carotene usually takes place faster than epoxidation, so no β-carotene epoxides can be detected.
In order to clearly identify the occurrence of β-carotene 5,6-epoxide (10) and 5,6,5 ,6diepoxide (11), they were prepared semisynthetically from β-carotene (8) and their structure was confirmed by NMR studies.This is the first report of the complete 1 H and 13 C NMR data of these carotenoids.The resulting stereoisomers were successfully separated during a chiral stationary phase; the configuration assigned to the peaks was performed using the on-line HPLC-ECD method.

Plant Materials, Pigment Extraction, Determination of Carotenoid Content
The flowers of Telekia speciosa were collected from Szováta and Bálványosfürdő (Maros country, Transylvania, Romania) in summer, 2019.The wild plant material was collected and identified by Erzsébet Varga, and example vouchers were deposited in the Department of Pharmacognosy and Phytotherapy, George Emil Palade University of Medicine, Pharmacy, Science and Technology of Targu Mures.The voucher specimen number was FS/0107/2019.
Analytical-grade chemicals were used for the extractions.The fresh plant samples (20-40 g) were extracted twice with acetone and once with Et 2 O.After evaporation, the residue of the acetonic extracts was dissolved in Et 2 O.The ethereal solutions were combined, and this total extract was saponified in heterogeneous phase (30% KOH/MeOH) overnight.The reaction mixture was washed with water 10 times.The saponified pigments were stored in benzene at −20 • C under nitrogen.The HPLC sample was prepared immediately before measurement.The benzene solution was evaporated to dryness and dissolved in tert-butyl methyl ether (MTBE)-methanol (MeOH) mixture.

General Experimental Procedures
The UV-VIS spectra were recorded with a Jasco V-530 spectrophotometer in benzene.NMR spectra were recorded with a Bruker DRX4OO ( 1 H: 400.14 MHz; 13 C: 100.61MHz) and a Bruker Avance III Ascend 500 spectrometer (500/125 MHz for 1 H/ 13 C) in CDCl 3 .Chemical shifts are referenced as Me 4 Si ( 1 H), or the residual solvent signals ( 13 C).Solvents for the HPLC analysis (MeOH: methanol, MTBE: tert-butyl methyl ether, acetone) were of HPLC grade.

Equipment for HPLC-DAD Separations on a C 30 Stationary Phase
The HPLC analysis was performed by Dionex 3000 HPLC system (Thermo Fisher Scientific Inc., Waltham, MA, USA).Chromatograms were detected at 450 nm wavelength; data acquisition was performed by Chromeleon 7.20  Chiral HPLC separations were carried out with a Jasco HPLC system on Chiralpak IA column (0.46 cm × 25 cm, 5 µm) using n-hexane:dichloromethane = 85:15 at a flow rate of 1 mL/min for 5, 7 and 10, and Chiralpak IA column (0.46 cm × 25 cm, 3 µm) using n-hexane:dichloromethane = 75:25 for 11.HPLC-UV chromatograms were measured with a Jasco MD-910 multiwavelength detector (JASCO Corporation, 2967-5 Ishikawamachi Hachioji-shi, Tokyo, Japan).The baseline of the chromatograms was reduced to zero immediately after the start of each run; this allowed for the measurement of the relative absorbance.The HPLC-ECD traces were recorded at the specified wavelength with a Jasco J-810 ECD spectropolarimeter (JASCO Corporation, 2967-5 Ishikawamachi Hachioji-shi, Tokyo, Japan) equipped with a 1 cm path length HPLC flow cell, and the baseline was reduced to zero after the start of each run.The online ECD and UV spectra were recorded simultaneously by stopping the flow at the UV absorption maximum of each peak.ECD ellipticity values (φ) were not corrected for concentration.For an HPLC-ECD spectrum, three consecutive scans were recorded and averaged with 2 nm bandwidth, 1 s response, and standard sensitivity.The HPLC-ECD spectrum of the eluent, recorded in the same way, was used as background.The concentration of the injected sample was set so that the HT (voltage) value did not exceed 500 V in the HT channel.

Identification of the Peaks
The carotenoids were identified using the following data: elution order on the C 30 HPLC column, spiking with authentic standards, UV-visible spectrum (λ max , spectral fine structure (% III/II), cis peak intensity (% AB/II)) and mass spectrum (molecular ion and fragments) compared to standards and the data available in the literature [21].Authentic samples were taken from our collection.A total of 10 mL of 0.25 M concentration monoperoxyphthalic acid (Et 2 O solution) was added to a solution of β-carotene (8) (400 mg) in Et 2 O (800 mL) at room temperature.The mixture was kept under N 2 , in the dark.After 21 h, the mixture was washed with 5% aq.NaHCO 3 solution, the organic phase was dried (Na 2 SO 4 ) and the solvent was evaporated.
The residue was dissolved in hexane and submitted to open-column chromatography (Ca(OH) 2 , eluent: hexane).Picture after development:

Plants 2023 ,
12, x FOR PEER REVIEW 2 of 12 other xanthophylls impart deep yellow to orange colours to flowers, depending on the carotenoid content in the petals.Certain flowers show distinctive carotenoid profiles [10].
* Percentage of peak area in the HPLC chromatogram at 450 nm.