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Review

Isolation and Analysis of Carotenoids in Hungary from Zechmeister until Today

by
Attila Agócs
1,
Erika Turcsi
1,
Veronika Nagy
1 and
József Deli
1,2,*
1
Department of Biochemistry and Medical Chemistry, Medical School, University of Pécs, Szigeti u. 12, H-7624 Pécs, Hungary
2
Department of Pharmacognosy, Faculty of Pharmacy, University of Pécs, Rókus u. 2, H-7624 Pécs, Hungary
*
Author to whom correspondence should be addressed.
Processes 2022, 10(4), 707; https://doi.org/10.3390/pr10040707
Submission received: 16 March 2022 / Revised: 30 March 2022 / Accepted: 31 March 2022 / Published: 5 April 2022
(This article belongs to the Special Issue Control and Optimization of Extractive Methodologies and Analysis of Natural Products)
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Abstract

:
László Zechmeister, one of the most important pioneers of carotenoid chemistry, died 50 years ago. He founded a carotenoid research group in Pécs (Hungary), which is the only place in the world where carotenoid research has been conducted continuously over the past 95 years. This review presents the life of Zechmeister and gives a summary about the evolution of the methods of analysis, isolation, and structure elucidation of carotenoids from the 1930s until today, based on the results of the research group founded by Zechmeister.

1. Introduction

Fifty years ago, on 28 February 1972, László Zechmeister (Figure 1), one of the most important pioneers of carotenoid chemistry and classical column chromatography, passed away. As members of the Hungarian carotenoid research group founded by Zechmeister, we consider it of great importance to commemorate him and his achievements. This paper deals with him as a dedicated scientist and his work on carotenoids. Further, we provide an overview of the progression of classical carotenoid research.
This review provides the possibility to show comprehensive details of the development of carotenoid isolation and analysis over the years. Despite the lack of modern analytical devices, Zechmeister’s team achieved many novel findings. With the evolution of instrumental techniques, the analytics evolved at a great pace, as well; however, some separation methods which were developed by Zechmeister and co-workers are still used in our laboratory in the same form as they were 90 years ago.

2. Historical Overview: The Life of László Zechmeister and His Research Group

Many scientists today know little about László Zechmeister or his work, yet his books [1,2,3,4] and scholarly publications were widely read and cited during the 1930s to the 1950s. He was one of the researchers who rediscovered chromatography and demonstrated its use for separating and isolating complex natural pigments.
László Zechmeister was born on 14 May 1889 in the city of Győr, a town in the north-west of Hungary. He received his primary education in Győr, and he finished high school in 1906. In 1907, Zechmeister commenced his studies in chemistry at the Swiss Federal Institute of Technology (ETH) in Zurich, where he followed lectures and enjoyed the guidance of the future Nobel Prize laureate Richard Willstätter. He received his degree as a chemical engineer in 1911. When Willstätter left for Berlin in 1912, Zechmeister followed him and worked as his assistant from 1912 to 1914. During this period, he wrote his doctorate thesis about cellulose and lignins [5].
It was Willstätter who sparked his interest in the investigations of natural products such as chlorophylls and carotenoids, a subject which he never gave up. Willstätter was the first to determine the correct molecular formulae of some plant carotenoids, thus placing a key in the hands of carotenoid researchers for further investigations. Zechmeister admired his professor (he kept a photo of him above his desk), and he often said, citing the work style of Willstätter, “to achieve the maximum possible result, we have to wish for the impossible”.
His scientific career was interrupted when World War I began. Zechmeister was enlisted in the Hungarian army and had to fight on the front line, where he was injured twice. He was taken captive and sent to a Russian prison camp in Siberia for three years. At the end of the war, he left the prison and came home to Hungary. Due to post-war chaos, he could only find temporary positions, such as the work of a chief scientific officer at the Chinoin pharmaceutical factory in Hungary, where he conducted industrial chemical research. At the same time, he worked at a veterinary college with György Hevesy. He was offered a teaching appointment at the Royal Danish Agriculture and Veterinary Academy in Copenhagen, where he worked as an instructor and scientific assistant of Niels Bjerrum in 1922–1923.
In 1923, Zechmeister was offered a professorship and directorship of a chemistry laboratory at a medical school in the newly established Erzsébet University in Pécs. This was, of course, a great honor, as Zechmeister was only 33 years old, and there had never been such a young person holding a position such as that in Hungary before [6]. The university, as an institution, had operated in earlier years in Pozsony (today Bratislava). In respect to the equipment, everything stayed in Bratislava, and only the name was transferred to the new university. Zechmeister was mainly involved in organizational work, such as the building process of the premises and the laboratories and the hiring of scientific staff. In a short time, he developed his department into an effective teaching and research institute. He had the ability to collect brilliant colleagues around him, who played an important role in educational and research work, and who later became prominent in academic circles. His colleagues in the department worked in different fields of chemistry, but he had the greatest passion for carotenoid research. In the preface of his book, “Carotinoide” [1], Zechmeister wrote: “It would be an understatement to say that carotenoids are just a special class of natural colorants and put them into a system. On the one hand, the peculiar structure poses new tasks to the organic chemist and on the other hand, the vast distribution of these polyene pigments in a large number of different organisms gives a strong indication that these compounds are essential for life”. Zechmeister soon became a respected teacher and a worldwide acknowledged authority in carotenoid research. By the end of the 1930s, he had gained several awards and a well-deserved reputation in Europe, as well as in the US. He was often invited to teach at various universities.
As an authority in the field of chromatography and a well-known expert of research on pigments and stereoisomers, Zechmeister was also the founder of the book series Progress in the Chemistry of Organic Natural Products and was its editor until 1969 [7]. The first volume was inaugurated in 1938, and, remarkably, the series has not yet completed with the recent publication of the 118th issue. Although in former times the series was simply referred to as “Fortschritte”, when German was still the language of science, it is now also known under the short form “Zechmeister”, in memory of its founder, or simply as “Progress”.
The prosperous years of scientific research were again interrupted by political events. The outbreak of the World War II in 1939 prompted Zechmeister to leave Hungary. He accepted an invitation by Linus Pauling to work at the Gates and Crellin Laboratories of Chemistry at the California Institute of Technology in Pasadena, where he continued his work on pigments and carotenoids. In 1959 László Zechmeister became professor emeritus at the Department of Organic Chemistry at CalTech. Until this time he had published 254 scientific papers with 87 co-authors. Nevertheless, he remained active even after his retirement. In 1962, he published a monograph on cis and trans isomeric carotenoids [4], which is very useful even today. Zechmeister received many honours during his life, including the Medaille Pasteur from the Biochemical Society (Paris), the Medaille Claude Bernard, the Grand Prize of the Hungarian Academy of Science, the Semmelweis Medal of the American-Hungarian Medical Association, and the American Chemical Society’s Award on Chromatography and Electrophoresis. He was an honorary member of the Hungarian Academy of Science and that of the Austrian Chemical Society. He was a foreign member of the Royal Dutch Academy of Science, and in 1971 the University of Pécs Medical School conferred on him an honorary M.D. degree.
He continued teaching and researching until the summer of 1971 when he fell ill and passed away on 28 February 1972 in Pasadena. As he requested, his ashes were scattered into the ocean near Los Angeles.
After Zechmeister left, the carotenoid research group in Pécs was led by László Cholnoky, head of the Chemistry Department since 1946. Cholnoky was ten years younger than Zechmeister, and he was enrolled as a student of pharmacy at the University of Budapest and showed a great interest in analytical chemistry already during his studies. He obtained his Ph.D. in pharmacy in 1924, and then he moved to Pécs and started to work as Zechmeister’s assistant. In 1940 he was promoted to a department professor. Prior to World War II he spent some months in Jena (Germany), where he studied the theoretical and practical questions of optical measurements, and later he stayed at Graz with F. Pregl to learn methods of organic microanalysis. Cholnoky also played a very active role in the university community; he was a vice dean, vice rector, and finally the rector of the university between 1964 and 1967. He was awarded the Kossuth Prize in 1959 and became a corresponding member of the board of the Hungarian Academy of Sciences in 1960. His sudden death on 12 June 1967 was a great shock for everybody.
After the death of Cholnoky, József Szabolcs became the head of the carotenoid research group and later also head of the department. He graduated as a chemist at the Loránd Eötvös University (Budapest) in 1951 and then started to work at the Department of Biophysics of the University of Pécs, and he joined the Department of Chemistry in 1954.
In 1990 József Szabolcs unexpectedly retired, and Gyula Tóth became the new head of the carotenoid research group. He completed his studies as a medical doctor at the University of Pécs in 1961. After his graduation he began to work with László Cholnoky, and, at his suggestion, the name of the department was changed in 1991 to the Department of Medical Chemistry. In 2002 the Department of Medical Chemistry was fused with the Department of Biochemistry to become the Department of Biochemistry and Medical Chemistry, hosting other groups for the research of analytical chemistry and biochemistry. In 2004 Gyula Tóth retired, and since then József Deli has been leading the carotenoid research group.

3. Advances in the Chromatographic Separation of Carotenoids

In this paper we give a summary on the evolution of the methods of analysis, isolation, and structure elucidation of carotenoids from the 1920s until today, based on the results of our research group. The field of analytical research of carotene pigments covers isolation and characterization. Determination of the carotenoid content of a biological sample requires first sampling and sample preparation, carotenoid extraction, separation, identification, quantification, and validation of the used methods. Today, for carotenoid analysis, high-performance liquid chromatography (HPLC) and spectrophotometry are the most frequently used methods.
To establish the identity of isolated or synthesized compounds and to characterize their structure (configuration and conformation), a versatile repertoire of techniques is available: UV/VIS spectrometry, infrared spectroscopy (IR), nuclear magnetic resonance spectroscopy (NMR), mass spectrometry (MS), and X-ray crystallography.
It should be noted here that most of the research of Zechmeister was conducted without the help of modern methods. For structure elucidation he had to rely only on the absorption spectra of carotenoids and the changes of those on simple chemical transformations, such as reduction, acylation, and reactions with acids. Zechmeister and co-workers developed the hydrogenation of carotenoids, determined the number of double bonds in the molecules, and confirmed the structures by catalytic hydrogenation of some carotenes (1928–1933). They realized the polyene structure, which is characteristic for carotenoid pigments, and introduced the term “Polyene” [8,9,10].

3.1. Open-Column Chromatography

It is difficult to determine the exact time when Zechmeister started using the column chromatography technique, but he certainly played a major role in the expansion of its use from the 1930s and onwards. Already in 1934 in his book Carotinoide [1], Zechmeister dealt in detail with the principles and applications of chromatography. The researchers in Pécs were the first to realize the importance and possibilities of the application of this method in organic chemistry. It is highly remarkable that, although Willstätter himself did not consider chromatography an important tool, it was his student who brought back this forgotten method.
In the 1930s Zechmeister and his colleagues developed chromatography into a high-performance preparative separation method for organic compounds (Figure 2). They applied calcium carbonate powder as a stationary phase, and the coloured carotenoids could be well-seen on the column. The relation between their chromatographic behaviour and their structure could easily be recognized [11,12,13]. There is a lot of information about the retention properties of carotenoids, especially on normal phases such as calcium carbonate, which still comes from the era of Zechmeister and Cholnoky.
In 1937 Zechmeister and Cholnoky published the book Die Chromatographische Adsorptionsmethode, which was the very first handbook on chromatography [2]. As Leslie L. Ettre, the former editor of the journal Chromatographia, stated: “This was the right book published by the right people at the right time, and it became an instant best-seller. In fact, it was such a success that within one year a second, greatly enlarged edition was published” [6]. It was later translated into English and Russian and reprinted many times. With this method at hand, Zechmeister and Cholnoky had great success in the isolation and the determination of the constitution of carotenoid dyes and their properties.
Based on their own methods, they reported many novel findings: the isolation and structure elucidation of paprika dyes capsanthin and capsorubin [14,15,16,17,18,19,20,21], lycophyll, and lycoxanthin [22,23]; alkaline degradation of capsanthin to β-citraurin [24] (Figure S1); photometric and colorimetric determination of carotenoids [25]; and the investigation of different flowers and fruits (Solanum dulcamara [26], Tamus communis [27], Lycium halimifolium [28], Calendula officinalis [29]). They established the occurrence of zeaxanthin dipalmitate (physalien) in Physalien berries, and the occurrence of different capsanthin esters in paprika [18].
After the leaving of Zechmeister in the 1940s, the intensity of publication slackened, and the next articles were published only in the 1950s [30]. However, these articles were the result of tedious work and presented important results: this was the first time when Cholnoky reported the physiological importance of carotenoids in plants and supported it with plant analytical data [30,31]. He emphasized again the importance of carotenoid epoxides as possible oxygen carriers in photosynthesis [32].
In the following years, using column chromatography Cholnoky and his co-workers discovered new carotenoids: α-cryptoxanthin (1958) [33,34] and cryptocapsin (1963) [35]. During those years the department had been lacking modern equipment. The determination of carotenoid spectra was carried out using a Löwe–Schumm grating spectroscope, and they measured the extinction rates of the different wavelengths one by one. They made a table of the results, and then they drew the spectrum on paper.
The fruitful organic microanalysis was based on this precise but tedious work, which led to the structural revision of some compounds. Cholnoky and his co-workers determined the structure of capsanthin and capsorubin in 1960 [36,37], at the same time as Paul Karrer (Zürich) [38] and Basil C. L. Weedon (London) [39]. These compounds possessed an interesting new feature among carotenoids, since instead of a trimethyl-cyclohexan ring they contain a trimethyl-cyclopentan ring (Figure S1). However, in the structure determination Karrer and Weedon enjoyed priority, as they already had IR equipment at their disposal.
The Hungarian research group was engaged in the Oppenauer oxidation in carotenoids, as well. They elaborated the LiAlH4 reduction of carotenoid-epoxides and furanoids [40]. In a collaboration with Weedon’s group, using the modern instruments of his department, they investigated the mass-spectrometry of carotenoid epoxides and furanoids [41]. In addition, they determined the structure of neoxanthin, carotenoids which can be found in higher plants containing allene bonds [42,43,44] (Figure S1).
In 1967 József Szabolcs led the carotenoid research group in Pécs. The group continued fruitful collaboration with a research group in London [45,46,47,48]. As a result of this important cooperation, a basic article about Optical Rotatory Dispersion of Carotenoids was published in 1969 [49], which allowed the determination of the absolute configuration of carotenoids. During this period of time the investigation of 5,6- and 5,8-epoxides, including their cis isomers, was continued [50,51,52,53,54,55,56,57,58,59,60]. While still using classical column chromatography, József Szabolcs, Gyula Tóth, and Péter Molnár isolated a huge number of naturally occurring carotenoid 5,6- and 5,8-epoxides (antheraxanthin, violaxanthin, lutein-5,6-epoxide, flavoxanthin, chrysanthemaxanthin, etc.), most of which derived from petals. They also studied the occurrence of cis-isomers in Nature. The main mono-cis isomers of some symmetric (zeaxanthin, capsorubin) and asymmetric (lutein, capsanthin, antheraxanthin, lutein 5,6-epoxide) molecules were prepared by thermal and iodine catalyzed photoisomerization for all-trans-carotenoids (Figure S1). For structure elucidation, they had 1H- and 13C-NMR- as well as CD-spectroscopies at their disposal. These results were presented as a plenary lecture on the 8th International Carotenoid Symposium in Boston by József Szabolcs [61].
In the 1990s the focus lay on the study on paprika carotenoids (see HPLC section) and on the further investigation of cis-trans isomeration [62,63,64,65,66,67,68,69]. Collaborations developed with the research group of Conrad H. Eugster in Zürich and later with the group of Hanspeter Pfander at the University of Bern.
As the many examples above demonstrate, chromatography on calcium carbonate is an efficient tool for the separation of diverse carotenoids, which is the reason that this type of open column chromatography has been used in our laboratory until today (Figure 2). Even very similar structures, such as configurational isomers, can be separated easily on calcium carbonate. The only disadvantage of this stationary phase is its very small capacity. Only 10–20 mg of carotenoid mixture can be separated on hundreds of grams of calcium carbonate, and a huge amount of eluent is needed, thus this method is of limited use for the preparative scale. To improve the capacity of column chromatography, silica gels can be an obvious choice as a stationary phase; however, their acidic character may induce some undesired transformations of sensitive samples.
A simple alkaline treatment of commercial silica gels can overcome this issue by converting silica to a basic form without lowering the capacity and without any change in retention properties. We demonstrated the affordability of such modified silica gels on acid-sensitive carotenoid epoxides [70]. The resolution of separation on silica is much less effective than on calcium carbonate, but it can be applied successfully for the separation of molecules of considerably different polarity. For instance, carotenoid glycosides from crude extracts of algae can be separated from other carotenoids and chlorophylls very efficiently, even in a gram-scale using modified silica (Figure 3) [71].
Now, we use and propose the combination of the two methods: modified silica gel can be used for the bulk pre-purification of extracts, and the classical calcium carbonate stationary phase is excellent for further fine purification of the gained fractions.

3.2. Thin-Layer Chromatography

Thin-layer chromatography, as a form of planar liquid-solid phase chromatography, dates back to the late 1950s, when it began to replace paper chromatography. Although in biochemical and analytical laboratories there is now a tendency to forget TLC and to try to solve all problems of carotenoid isolation and identification by HPLC, this simple method remains very useful. Practical application now focuses more on a combination of both TLC and HPLC, rather than TLC alone. For preliminary tests of biological extracts as well as for monitoring reactions, TLC remains a cheap and indispensable alternative [72,73,74]. On a TLC plate without any sample preparation, some information can be obtained in a few minutes about the approximate composition of the sample. Additionally, during a chemical reaction, if it is to be monitored quickly, thin-layer chromatography can solve this task. HPLC requires sample preparation, and in the case of a carotene mixture—such as a raw extract—the elution time can be long as well. On the other hand, preparative TLC can be used for the separation of carotenoid extracts or reaction mixtures, as well [75]. Occasionally, only small amounts are available from a natural isolate or reaction mixture, and therefore the isolation or purification cannot be made with column chromatography. In this case preparative TLC offers a fast and inexpensive alternative. For a precise as well as for a qualitative analysis, HPLC-UV is necessary.

3.3. High Performance Liquid Chromatography

The HPLC method was introduced to the research of carotenoids in the 1970s. Since then, a large number of applications of HPLC methods have been published along with the evolution of stationary phases and the detection method. Both normal- and reversed-phase systems are used, either in isocratic or gradient elution modes. Reversed-phase systems are generally preferred over normal-phase HPLC having, e.g., lower column stability and poorer reproducibility of the retention times. In present times, in reversed-phase systems C18 and C30 chemically bonded phases are used [76,77,78,79]. The thirty-carbon group at the stationary phase contacts a high surface area of the carotene molecules, which causes more secondary interactions. As a result, the retention times for C30 phases are considerably higher than for C18 phases [80].
We investigated and compared the separation of many carotenoids for both types of stationary phases [80]. Our results demonstrate that the C30 stationary phase seems to be ideal for the separation of nonpolar carotenes (hydrocarbons, mono-hydroxy compounds) and their cis-trans isomers, whereas the separation of these compounds for C18 is very poor. Although the C18 column did not achieve baseline separation of hydrocarbons, the C30 column shows well-resolved separation with retention time differences of several minutes (Figure 4 and Figure S1).
However, the C18 bonded silica gel phase is great at providing a total carotenoid profile of a natural isolate. The elution order for this stationary phase follows the polarity order of carotenoids, whereas C30 is not appropriate for the prediction of the structure of a carotenoid in a complex mixture.
The C18 phase gives better separation in the case of mixtures which contain larger amounts of polar carotenoids, for example, paprika extract. From the middle of the 1980s, using C18 HPLC methods, we investigated the carotenoid composition of different kinds and differently coloured varieties of paprika during ripening [81,82,83,84]. Our studies showed three characteristic carotenoid profiles. In all unripe (green) fruits of different kinds of paprika, the typical chloroplast pigments, lutein (without esterification) and β-carotene, are the main carotenoids. The ripe yellow paprika whose colour never turns red contains violaxanthin, antheraxanthin, zeaxanthin, lutein, α- and β-cryptoxanthin, and α- and β-carotene as main carotenoids. The red colour of ripe red paprika is due to carotenoids with κ-end group(s). The main carotenoids are capsanthin, zeaxanthin, cucurbitaxanthin A, β-cryptoxanthin, and β-carotene. All hydroxy carotenoids are present as esters (Figure S1).
Our laboratory revealed novel pigments from red paprika using a C18 stationary phase. These new carotenoids contained a 3,6-epoxy end group, such as cycloviolaxanthin, cucurbitaxanthin A and B, cucurbitachrome epimers, and capsanthin 3,6-epoxide [85]. We also found novel paprika carotenoids bearing a 3,5,6-trihydroxy end group, such as 6-epikarpoxanthin, 5,6-diepikarpoxanthin, 5,6-diepicapsokarpoxanthin, and 5,6-diepilatoxanthin [86]. During the isolation of these compounds, two carotenoids with interesting 6-hydroxy γ-end groups, namely nigroxanthin [87] and prenigroxanthin [88], were isolated (Figure S1).
Although all isolated compounds were carefully characterized by modern spectroscopic methods (UV-VIS and mass spectrometry, NMR and circular dichroism spectroscopy), they did not give any indication for the configuration at C(6′). Thus, we explained the configuration by the biosynthetic pathway of paprika carotenoids, and the proposed structures with the 6′S configuration for nigroxanthin and prenigroxanthin were strongly supported [88].
We have investigated or reinvestigated other plants which contain κ-carotenoids, such as the buds of Asparagus officinalis [89], Lilium tigrinum [90], Asparagus falcatus [91], and flowers of different species of Aesculus (horse chestnut) [92]. In this way, two other minor carotenoids were also isolated and, based on their spectral data, identified, namely capsoneoxanthin [93] and (9Z)-capsanthin 5,6-epoxide [64]. Capsoneoxanthin contains another interesting allenic end group (Figure S1). Later, capsoneoxanthin was isolated also from red mamey [94].
The C30 phase is recommended if the extract contains larger amounts of nonpolar carotenoids, for example, in the case of red mamey (Pouteria sapota). Using a C18 column, only few carotenoid peaks are visible, whereas on a C30 phase 47 peaks can be detected [95]. Based on the HPLC analytical results, we isolated a few interesting carotenoids which contain a non-hydroxylated kappa end group, for instance, sapotexanthin [96], deoxy-capsorubin and dideoxy-capsorubin [97], cryptocapsin epoxides [98], and sapotexanthin 5,6-epoxide [99] (Figure S1).

3.4. Detection Methods for HPLC

The most common method used in the analysis of carotenoids is HPLC, employing various detection techniques. The success of the determination of a compound depends equally on the separation and the detection steps.
Simple UV-VIS detection is by far the most common because carotenoids absorb strongly in the visible region between 400 and 500 nm (all-trans isomers), whereas cis isomers also exhibit absorption near the UV region, around 320 nm. The detectors are simple and cheap, and photometric detection is readily compatible with gradient elution. Diode array detection (DAD) provides certain possibilities for analyte identification, which however, should not be overestimated. Diode array detection yields spectra sometimes with fine structures. Although the spectra of many carotenoids are very similar (e.g., those of β-carotene and zeaxanthin), the retention time and the spectrum of the peak all together are informative. In the 1980s, we described a method for identification of aldehydes, ketones, 5,6-epoxides, and 5,8-epoxy groups in carotenoids using simple, known chemical reactions, such as the reduction of sodium borohydride or furanoid rearrangement on acid treatment. These transformations are followed by characteristic changes in the UV-VIS spectra, which are useful in the structure elucidation studies [100].
The main advantage of MS detection is that it enables not only the analyte quantification, but also the elucidation of its structure on the basis of molecular mass and fragmentation. The coupled diode array and mass detection with a known retention time may allow for complete structure elucidation in certain cases. However, there are exceptions, such as in the separation of antheraxanthin and cucurbitaxanthin A on C30. Both compounds have the same retention time, UV-VIS spectra, and molecular mass, thus they cannot be distinguished this way. In this case, a simple chemical derivatization, such as a reaction with acids, is very useful [100]. Antheraxanthin, being a 5,6-epoxide, rearranges with acids and mutatochromes to show a shift in retention time and spectra, whereas cucurbitaxanthin A does not (Figure 5).
In our laboratory, the detailed carotenoid analysis of red mamey (Pouteria sapota) was achieved by HPLC-DAD-MS, chemical tests, and co-chromatography with authentic samples. Altogether, 47 components were detected and 34 were identified from the total extract or after fractionation with classical column chromatography [95].
On-line HPLC-CD detection is a powerful tool which opens new perspectives in the identification of enantiomers or diastereomers of carotenoids. The separation of carotenoid epimers on chiral phases has been known for a long time, but CD (circular dichroism) detection is the newest technique.
During the investigation of mamey carotenoids, we isolated a new compound, cryptocapsin 5,6-epoxide (Figure 6) [98]. This carotenoid was characterized by its UV-Visible spectra, 1H-NMR, mass, and circular dichroism (CD) spectra. As the enantiomeric non-hydroxylated (5R,6S)- and (5S,6R)-5,6-epoxy β-end groups cannot be distinguished by the 1H-NMR spectra, the elucidation of the configuration at the cyclohexane ring was based on chiroptical data. To fully characterize the structure of the natural cryptocapsin 5,6-epoxide, it was semisynthetized from cryptocapsin by epoxidation with monoperoxyphthalic acid. The epoxidation reaction always yields two 5,6-epoxy diastereomers with (5R,6S, natural) and (5S,6R) absolute configurations. Generally, mainly in the case of an unsubstituted β-ring, the separation of such isomeric epoxides is not easy. In this case, the separation of the two stereoisomers is only possible on a chiral HPLC column. The chromatogram shows that the two epoxides form approximately in equal amounts during the reaction. The CD spectra of the natural (anti) and semisynthetic (syn) cryptocapsin 5,6-epoxide are opposite, demonstrating the different configuration of the 5,6-epoxy end groups [98].
Twenty five years ago, β-cryptoxanthin was epoxidized with monoperoxyphthalic acid [101]. By classical column chromatography, three monoepoxides and two diepoxides could be separated, which were characterized by NMR and CD spectroscopy as well as mass spectrometry. These compounds were identified as anti- and syn-5,6-monoepoxides, anti 5′,6′-monoepoxide, as well as anti,anti- and syn,syn-diepoxides of β-cryptoxanthin. We have recently reinvestigated the 5′,6′-monoepoxide, which was previously identified as the anti-compound. On a chiral stationary phase, we obtained two peaks with the same UV spectra. As in the case of cryptocapsin epoxides, the non-hydroxylated syn and anti 5,6-epoxy carotenoids could not been separated, neither on calcium carbonate nor C18 or C30; however, they could be separated on a chiral phase (Figure 7) [102].
NMR detection is the best method for the identification of unknown stereoisomers. It can be used either off-line or on-line. Measurement of NMR spectra requires pure components in greater amounts than those of UV/Vis or MS detection modes. Klaus Albert and co-workers identified all lutein [103] and anhydrolutein I [104] stereoisomers using HPLC-NMR. In our laboratory, we investigated the carotenoid composition of cooked vegetables, for example, sorrel [105]. We found that lutein converts to 3′-epilutein and anhydrolutein I and II under acidic conditions, and we also observed the formation of cis isomers of these compounds [106,107]. For full characterization of these compounds, the semisynthetic Z-isomers were prepared by thermal and I2-catalyzed photoisomerization [108]. By classical column chromatography, we could not separate the Z-isomers of anhydrolutein I, and thus for the separation and identification of these cis-isomers, combined HPLC-MS and HPLC-NMR on-line coupling techniques were used, in cooperation with Klaus Albert’s group [104].

4. In the Footsteps of Zechmeister

In the second half of the 20th century, the main research field of the Hungarian carotenoid group shifted to carotenoid analysis of natural extracts, isolation, and identification of carotenoids with interesting structures. However, we kept using chemical reactions for structure elucidation studies. Péter Molnár in particular investigated and fully characterized the transformation of geometrical isomers of diverse carotenoids, revealing their kinetic and thermodynamic aspects [69]. After László Zechmeister left for the US, aside from carotenoid analysis, he continued his investigation on the in vitro chemical transformations of carotenoids and described many interesting findings [108]. In the last 15 years, we hearkened back to these works and applied Zechmeister’s results in order to prepare novel derivatives of natural carotenoids. The synthesis with carotenoids became a new direction in our research aiming to create covalently modified derivatives with improved solubility and antioxidant properties [109]. Nevertheless, we continue fruitful carotenoid analysis to find new structures and to explore the biosynthetic pathways of carotenoids.

5. Conclusions

In the past 60–70, but mostly in the past 40 years, separation science and instrumental analysis underwent a huge development. As a result of this, isolation and structure elucidation of minor components became possible, helping the understanding of biosynthetic and metabolic pathways. The precision and the development of analytical tools make isolation and structure elucidation of new pigments possible. However, in some cases, old findings should also be revised with the help of new and powerful analytical methods.
Although most natural carotenoids have been discovered so far, tropical plants offer new sources for novel types of carotenoids, so there remains much to conduct in the field of isolation. Comprehensive chromatographic methods (such as supercritical chromatography) can help the scientific community to make the separation of natural products faster and more effective to be able to isolate previously unknown minor pigments.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/pr10040707/s1, Figure S1: Structure of carotenoids.

Author Contributions

Conceptualization, J.D.; formal analysis, E.T.; writing—original draft preparation, J.D.; writing—review and editing, A.A. and V.N.; visualization, A.A. and V.N.; funding acquisition, A.A. and J.D. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Hungarian Scientific Research Fund (NKFI), K 128253 and K 131493.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The authors are grateful to Erika Jámbor and Krisztina Sajti for their skillful assistance.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Zechmeister, L. Carotenoide Ein Biochemischer Bericht Über Pflanzliche und Tierische Polyenfarbstoffe; Springer: Berlin/Heidelberg, Germany, 1934. [Google Scholar]
  2. Zechmeister, L.; Cholnoky, L. Die Chromatographische-Adsorptionsmethode; Springer: Vienna, Austria, 1937. [Google Scholar]
  3. Zechmeister, L. Progress in Chromatography 1938–1947; Chapman & Hall: London, UK; J. Wiley & Sons: New York, NY, USA, 1950. [Google Scholar]
  4. Zechmeister, L. Cis-Trans Isomeric Carotenoids, Vitamins A and Arylpolyenes; Academic Press: New York, NY, USA, 1962. [Google Scholar]
  5. Zechmeister, L. Zur Kenntnis der Cellulose und des Lignins. Ph.D. Thesis, Ε.Τ.H., Zürich, Switzerland, 1913. [Google Scholar]
  6. Ettre, L. László zechmeister: A pioneer of chromatograpy. Anal. Chem. 1989, 61, 1315A–1322A. [Google Scholar] [CrossRef]
  7. Wirth, M.L. Zechmeister, His Life and Pioneering Work in Chromatography; Springer: Vienna, Austria, 2013. [Google Scholar]
  8. Zechmeister, L.; Cholnoky, L.; Vrabély, V. Über die katalytische hydrierung von carotin. Ber. Dtsch. Chem. Ges. 1928, 61, 566–568. [Google Scholar] [CrossRef]
  9. Zechmeister, L.; Cholnoky, L. Beitrag zum konstitutions-problem des carotins. Ber. Dtsch. Chem. Ges. 1928, 61, 1534–1539. [Google Scholar] [CrossRef]
  10. Zechmeister, L.; Cholnoky, L.; Vrabély, V. Zur bestimmung der doppelbindungen im carotin-molekül. Ber. Dtsch. Chem. Ges. 1933, 66, 123–124. [Google Scholar] [CrossRef]
  11. Zechmeister, L.; Cholnoky, L. Untersuchungen über den paprika-farbstoff VII. (Adsorptionsanalyse des pigments). Liebigs Ann. Chem. 1934, 509, 269–287. [Google Scholar] [CrossRef]
  12. Zechmeister, L.; Cholnoky, L. Dreissig jahre chromatographie. Mon. Chem. 1936, 68, 288–300. [Google Scholar] [CrossRef]
  13. Zechmeister, L.; Cholnoky, L.; Ujhelyi, E. Contribution a la chromatographie des substances incolores. Bull. Soc. Chim. Biol. 1936, 18, 1885–1887. [Google Scholar]
  14. Zechmeister, L.; Cholnoky, L. Untersuchungen über den paprika-farbstoff I. Liebigs Ann. Chem. 1927, 454, 54–71. [Google Scholar] [CrossRef]
  15. Zechmeister, L.; Cholnoky, L. Untersuchungen über den paprika-farbstoff II. Liebigs Ann. Chem. 1927, 455, 70–81. [Google Scholar] [CrossRef]
  16. Zechmeister, L.; Cholnoky, L. Untersuchungen über den paprika-farbstoff III. Katalytische hydrierung. Liebigs Ann. Chem. 1928, 465, 288–299. [Google Scholar] [CrossRef]
  17. Zechmeister, L.; Cholnoky, L. Untersuchungen über den paprika-farbstoff IV. Einige umwandlungen des capsanthins. Liebigs Ann. Chem. 1930, 478, 95–111. [Google Scholar] [CrossRef]
  18. Zechmeister, L.; Cholnoky, L. Untersuchungen über den paprika-farbtoff V. Natürliche und synthetische ester des capsanthins. Liebigs Ann. Chem. 1931, 487, 197–213. [Google Scholar] [CrossRef]
  19. Zechmeister, L.; Cholnoky, L. Untersuchungen über den paprika-farbstoff VI. Das pigment des japanischen paprikas. Liebigs Ann. Chem. 1931, 489, 1–6. [Google Scholar] [CrossRef]
  20. Zechmeister, L.; Cholnoky, L. Untersuchungen über den paprika-farbstoff VIII. Zur konstitution des capsanthins und capsorubins. Liebigs Ann. Chem. 1935, 516, 30–45. [Google Scholar] [CrossRef]
  21. Zechmeister, L.; Cholnoky, L. Untersuchungen über den paprika-farbstoff IX. Partieller abbau des capsanthins. Liebigs Ann. Chem. 1936, 523, 101–118. [Google Scholar] [CrossRef]
  22. Zechmeister, L.; Cholnoky, L. Über einen neunen farbstoff mit lycopin-spektrum. Naturwissenchaften 1935, 23, 407. [Google Scholar] [CrossRef]
  23. Zechmeister, L.; Cholnoky, L. Lycoxanthin und lycophyll. Ber. Dtsch. Chem. Ges. 1936, 69, 422–429. [Google Scholar] [CrossRef]
  24. Zechmeister, L.; Cholnoky, L. Untersuchungen über den paprika-farbstoff X. Citraurin aus capsanthin. Liebigs Ann. Chem. 1937, 530, 291–300. [Google Scholar] [CrossRef]
  25. Cholnoky, L. Stufenphotometrische bestimmung der paprikafarbstoffe. Z. Lebensm. Unters. 1939, 78, 401–403. [Google Scholar] [CrossRef]
  26. Zechmeister, L.; Cholnoky, L. Lycopin aus solanum dulcamara. Ber. Dtsch. Chem. Ges. 1930, 63, 787–790. [Google Scholar] [CrossRef]
  27. Zechmeister, L.; Cholnoky, L. Über das pigment der reifen beeren des tamus communis. Ber. Dtsch. Chem. Ges. 1930, 63, 422–427. [Google Scholar] [CrossRef]
  28. Zechmeister, L.; Cholnoky, L. Über den farbstoff der bocksdorn-beere und über das vorkommen von chemisch gebundenen carotinoiden in der natur. Liebigs Ann. Chem. 1930, 481, 42–56. [Google Scholar] [CrossRef]
  29. Zechmeister, L.; Cholnoky, L. Über den farbstoff der ringelblume (Calendula officinalis). Ein beitrag zur kenntnis des blüten-lycopins. Z. Phys. Chem. 1932, 208, 26–32. [Google Scholar] [CrossRef]
  30. Cholnoky, L.; Györgyfy, K.; Nagy, E.; Pánczél, M. Untersuchungen über carotinoid-farbstoffe. 1. Die farbstoffe des roten tomatenförmigen paprikas. Acta Chim. Hung. 1955, 6, 143–147. [Google Scholar]
  31. Cholnoky, L.; Györgyfy, K.; Nagy, E.; Pánczél, M. Untersuchungen über die carotenoid-farbstoffe. 3. Die farbstoffe des gelben tomatenförmigen paprikas (Capsicum annuum varietas lycopersiciforme flavum). Acta Chim. Hung. 1958, 16, 227–246. [Google Scholar]
  32. Cholnoky, L.; Györgyfy, K.; Nagy, E.; Pánczél, M. Function of carotenoids in chlorophyll-containing organs. Nature 1956, 178, 410–411. [Google Scholar] [CrossRef]
  33. Cholnoky, L.; Szabolcs, J.; Nagy, E. Untersuchungen über die carotinoid-farbstoffe. 4. α-Cryptoxanthin. Liebigs Ann. Chem. 1958, 616, 207–218. [Google Scholar] [CrossRef]
  34. Cholnoky, L.; Szabolcs, J. Die schmelzpunkte des α- und β-Kryptoxanthinacetates. Naturwissenschaften 1959, 46, 424–425. [Google Scholar] [CrossRef]
  35. Cholnoky, L.; Szabolcs, J.; Cooper, R.D.G.; Weedon, B.C.L. The structure of cryptocapsin. Tetrahedron Lett. 1963, 19, 1257–1259. [Google Scholar] [CrossRef]
  36. Cholnoky, L.; Szabolcs, J. Über die struktur des paprikafarbstoffes. Acta Chim. Hung. 1960, 22, 117–119. [Google Scholar]
  37. Cholnoky, L.; Szabolcs, J. Bemerkungen zur struktur des capsorubins. Experientia 1960, 16, 483–484. [Google Scholar] [CrossRef] [PubMed]
  38. Entschel, R.; Karrer, P. Zur konstitution des capsanthins und capsorubins. Helv. Chim. Acta 1960, 43, 89–94. [Google Scholar] [CrossRef]
  39. Barber, M.S.; Jackman, L.M.; Warren, C.K.; Weedon, B.C.L. The structures of the paprika ketones. Proc. Chem. Soc. London 1960, 1, 19–20. [Google Scholar]
  40. Cholnoky, L.; Szabolcs, J.; Tóth, G. Untersuchungen über carotinoidfarbstoffe. 7. Reduction von carotenoidoxiden mit lithiumalanat. Liebigs Ann. Chem. 1967, 708, 218–223. [Google Scholar]
  41. Baldas, J.; Porter, Q.N.; Cholnoky, L.; Szabolcs, J.; Weedon, B.C.L. Mass spectrometry of carotenoid epoxides and furanoid oxides. Chem. Commun. 1966, 23, 852–854. [Google Scholar] [CrossRef]
  42. Cholnoky, L.; Györgyfy, K.; Szabolcs, J.; Weedon, B.C.L.; Waight, E.S. Foliaxanthin. Chem. Commun. 1966, 13, 404–405. [Google Scholar] [CrossRef]
  43. Mallams, A.K.; Waight, E.S.; Weedon, B.C.L.; Cholnoky, L.; Györgyfy, K.; Szabolcs, J.; Krinsky, N.I.; Schimmer, B.P.; Chichester, C.O.; Katayama, T.; et al. The identity of neoxanthin and foliaxanthin. Chem. Commun. 1967, 10, 484–485. [Google Scholar] [CrossRef]
  44. Cholnoky, L.; Györgyfy, K.; Rónai, A.; Szabolcs, J.; Tóth, G.; Galasko, G.; Mallams, A.K.; Waight, E.S.; Weedon, B.C.L. Carotenoids and related compounds. Part XXI. Stucture of neoxanthin (Foliaxanthin). J. Chem. Soc. C 1969, 9, 1256–1263. [Google Scholar] [CrossRef]
  45. Cholnoky, L.; Szabolcs, J.; Waight, E.S. The structure of lycoxanthin and lycophyll. Tetrahedron Lett. 1968, 16, 1931–1933. [Google Scholar] [CrossRef]
  46. Baldas, I.; Porter, Q.N.; Leftwick, A.P.; Holzer, R.; Weedon, B.C.L.; Szabolcs, J. Mass spectrometry of carotenoid ketones. Chem. Commun. 1969, 8, 415–416. [Google Scholar] [CrossRef]
  47. Goodfellow, D.; Moss, G.P.; Szabolcs, J.; Tóth, G.; Weedon, B.C.L. Configuration of carotenoid epoxides. Tetrahedron Lett. 1973, 40, 3925–3928. [Google Scholar] [CrossRef]
  48. Bernhard, K.; Moss, G.P.; Tóth, G.; Weedon, B.C.L. Stereoisomers of fucoxanthin. Tetrahedron Lett. 1974, 44, 3899–3902. [Google Scholar] [CrossRef]
  49. Bartlett, L.; Klyne, W.; Mose, W.P.; Scopes, P.M.; Galasko, G.; Mallams, A.K.; Weedon, B.C.L.; Szabolcs, J.; Tóth, G. Optical rotatory dispersion of carotenoids. J. Chem. Soc. C 1969, 18, 2527–2544. [Google Scholar] [CrossRef] [PubMed]
  50. Tóth, G.; 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. An attempt to isolate taraxanthin. Acta Chim. Hung. 1970, 64, 393–406. [Google Scholar]
  51. Moss, G.P.; Szabolcs, J.; Tóth, G.; Weedon, B.C.L. The stereochemistry of the carotenoid violeoxanthin. Acta Chim. Hung. 1975, 87, 301–309. [Google Scholar]
  52. Baranyai, M.; Szabolcs, J.; Tóth, G.; Radics, L. Determination of the geometrical configuration of the polyen chain of mono-cis C40-carotenoids I. A 13C NMR study of mono-cis zeaxanthins and mono-cis capsorubins. Tetrahedron 1976, 32, 867–870. [Google Scholar] [CrossRef]
  53. Szabolcs, J. Some studies on the stereochemistry of carotenoids. Pure Appl. Chem. 1976, 47, 147–149. [Google Scholar] [CrossRef] [Green Version]
  54. Baranyai, M.; Kajtár, J.; Bujtás, G.; Szabolcs, J. The configuration of capsanthin epoxides. Acta Chim. Hung. 1977, 94, 67–74. [Google Scholar]
  55. Tóth, G.; Kajtár, J.; Molnár, P. Szabolcs, the stereochemistry of natural ‘cis-antheraxanthin’. (Preliminary communication). Acta Chim. Hung. 1978, 97, 359–361. [Google Scholar]
  56. Molnár, P.; Szabolcs, J. Occurence of 15-cis-violaxanthin in Viola tricolor. Phytochemistry 1980, 19, 623–627. [Google Scholar] [CrossRef]
  57. Molnár, P.; Szabolcs, J. β-Citraurin epoxyde, a new carotenoid from Valencia orange peel. Phytochemistry 1980, 19, 633–637. [Google Scholar] [CrossRef]
  58. Baranyai, M.; Molnár, P.; Szabolcs, J.; Radics, L.; Kajtár-Peredy, M. Determination of the geometric configuration of the polyene chain of mono-cis C40 carotenoids II. A 13C NMR study of mono-cis luteins and mono-cis capsanthins. Tetrahedron 1981, 37, 203–207. [Google Scholar] [CrossRef]
  59. Tóth, G.; Szabolcs, J. Occurrence of some mono-cis-isomers of asymmetric C40-carotenoids. Phytochemistry 1981, 20, 2411–2415. [Google Scholar] [CrossRef]
  60. Deli, J.; Molnár, P.; Tóth, G.; Szabolcs, J.; Radics, L. Determination of the geometrical configuration of naturally occurring mono-cis-lutein epoxides. Phytochemistry 1988, 27, 547–549. [Google Scholar] [CrossRef]
  61. Szabolcs, J. Plant carotenoids. In Carotenoids Chemistry and Biology; Krinsky, N.I., Mathews-Roth, M.M., Taylor, R.F., Eds.; Plenum Press: New York, NY, USA; London, UK, 1989; pp. 39–58. [Google Scholar]
  62. Molnár, P.; Deli, J.; Matus, Z.; Tóth, G.; Steck, A. Isomerisierung von cycloviolaxanthin herstellung und characterisierung von 9Z- und 13Z-cycloviolaxanthin. Helv. Chim. Acta 1996, 79, 1444–1450. [Google Scholar] [CrossRef]
  63. Molnár, P.; Deli, J.; Matus, Z.; Tóth, G.; Renneberg, G.; Pfander, H. Isomerization of (all-E)-cucurbitaxanthin A. Helv. Chim. Acta 2000, 83, 1535–1541. [Google Scholar] [CrossRef]
  64. Molnár, P.; Deli, J.; Tóth, G.; Haberli, A.; Pfander, H.; Bernhard, K. (9Z)-capsanthin 5,6-epoxide, a new carotenoid from the fruits of Asparagus falcatus. J. Nat. Prod. 2001, 64, 1254–1255. [Google Scholar] [CrossRef]
  65. Molnár, P.; Deli, J.; Tóth, G.; Haberli, S.; Pfander, H. Isomerization of (all-E)-5,6- diepikarpoxanthin. Helv. Chim. Acta 2002, 85, 1327–1339. [Google Scholar] [CrossRef]
  66. Molnár, P.; Deli, J.; Zsila, F.; Steck, A.; Pfander, H.; Tóth, G. Preparation and (E/Z)-isomerization of the diastereoisomers of violaxanthin. Helv. Chim. Acta 2004, 87, 11–27. [Google Scholar] [CrossRef]
  67. Molnár, P.; Deli, J.; Ősz, E.; Matus, Z.; Tóth, G.; Zsila, F. (E/Z)-isomerization of 3′-epilutein. Helv. Chim. Acta 2004, 87, 2169–2179. [Google Scholar] [CrossRef]
  68. Molnár, P.; Ősz, E.; Tóth, G.; Zsila, F.; Deli, J. Preparation and spectroscopic characterization of (9Z,9′Z)-lutein (neolutein C). Helv. Chim. Acta 2006, 89, 667–674. [Google Scholar] [CrossRef]
  69. Molnár, P. Research of the (E/Z)-isomerization of carotenoids in pécs since the 1970s (Review). Arch. Biochem. Biophys. 2009, 483, 156–164. [Google Scholar] [CrossRef] [PubMed]
  70. Nagy, V.; Agócs, A.; Turcsi, E.; Deli, J. Isolation and purification of acide-labile carotenoid 5,6-epoxides on modified silica gels. Phytochem. Anal. 2009, 20, 143–148. [Google Scholar] [CrossRef]
  71. Nagy, V.; Agócs, A.; Deli, J.; Gulyás-Fekete, G.; Illyés, T.-Z.; Kurtán, T.; Turcsi, E.; Béni, S.; Dékány, M.; Ballot, A.; et al. Carotenoid glycoside isolated and identified from cyanobacterium Cylindrospermopsis raciborskii. J. Food Compos. Anal. 2018, 65, 6–10. [Google Scholar] [CrossRef]
  72. Deli, J. Thin-layer chromatography of carotenoids. JPC-Mod. TLC 1998, 11, 311–312. [Google Scholar]
  73. Forgács, E.; Cserháti, T. Thin-layer chromatography of natural pigments: New advances. J. Liq. Chromatogr. 2002, 25, 1521–1541. [Google Scholar] [CrossRef]
  74. Cimpoiu, C.; Hosu, A.J. Thin layer chromatography for the analysis of vitamins and their derivatives. J. Liq. Chromatogr. 2007, 30, 701–728. [Google Scholar] [CrossRef]
  75. Schiedt, K. Chromatography. part III. thin-layer chromatography. In Carotenoids 1A: Isolation and Analysis; Britton, G., Liaaen-Jensen, S., Pfander, H., Eds.; Birkhauser: Basel, Switzerland, 1995; pp. 131–144. [Google Scholar]
  76. Emenhiser, C.; Sander, L.C.; Schwartz, S.J. Capability of a polymeric C30 stationary phase to resolve cis-trans carotenoid isomers in reversed-phase liquid chromatography. J. Chromatogr. A 1995, 707, 205–216. [Google Scholar] [CrossRef]
  77. Emenhiser, C.; Simunovic, N.; Sander, L.C.; Schwartz, S.J. Separation of geometrical carotenoid isomers in biological extracts using a polymeric C30 column in reversed-phase liquid chromatography. J. Agric. Food Chem. 1996, 44, 3887–3893. [Google Scholar] [CrossRef]
  78. Dachtler, M.; Kohler, K.; Albert, K. Reversed-phase high-performance liquid chromatographic identification of lutein and zeaxanthin stereoisomers in bovine retina using a C30 bonded phase. J. Chromatogr. B 1998, 720, 211–216. [Google Scholar] [CrossRef]
  79. Sander, L.C.; Sharpless, K.E.; Pursch, M. C30 stationary phases for the analysis of food by liquid chromatography. J. Chromatogr. A 2000, 880, 189–202. [Google Scholar] [CrossRef]
  80. Turcsi, E.; Nagy, V.; Deli, J. Study on the elution order of carotenoids on endcapped C18 and C30 reverse silica stationary phase. A rewiev of the database. J. Food Compos. Anal. 2016, 47, 101–112. [Google Scholar] [CrossRef]
  81. Matus, Z.; Deli, J.; Szabolcs, J. Carotenoid composition of yellow pepper during ripening: Isolation of β-cryptoxanthin 5,6-epoxide. J. Agric. Food Chem. 1991, 39, 1907–1914. [Google Scholar] [CrossRef]
  82. Deli, J.; Matus, Z.; Szabolcs, J. Carotenoid composition in the fruits of black paprika (Capsicum annuum variety longum nigrum) during ripening. J. Agric. Food Chem. 1992, 40, 2072–2076. [Google Scholar] [CrossRef]
  83. Deli, J.; Matus, Z.; Tóth, G. Carotenoid composition in the fruits of Capsicum annuum cv. szentesi kosszarvú during ripening. J. Agric. Food Chem. 1996, 44, 711–716. [Google Scholar] [CrossRef]
  84. Deli, J.; Molnár, P.; Matus, Z.; Tóth, G. Carotenoid composition in the fruits of red paprika (Capsicum annuum var. lycopersiciforme rubrum) during ripening; Biosynthesis of carotenoids in red paprika. J. Agric. Food. Chem. 2001, 49, 1517–1523. [Google Scholar] [CrossRef]
  85. Deli, J.; Molnár, P.; Matus, Z.; Tóth, G.; Steck, A. Reisolation of carotenoid 3,6-epoxides from red paprika (Capsicum annuum). Helv. Chim. Acta 1996, 79, 1435–1443. [Google Scholar] [CrossRef]
  86. Deli, J.; Molnár, P.; Matus, Z.; Tóth, G.; Steck, A.; Pfander, H. Isolation of carotenoids with 3,5,6-trihydroxy-5,6-dihydro-β-end groups from red paprika (Capsicum annuum). Helv. Chim. Acta 1998, 81, 1233–1241. [Google Scholar] [CrossRef]
  87. Deli, J.; Matus, Z.; Molnár, P.; Tóth, G.; Szalontai, G.; Steck, A.; Pfander, H. Nigroxanthin (3′,4′-didehydro-β,γ-carotene-3,6′-diol), a new carotenoid isolated from paprika (Capsicum annuum var. longum nigrum). Chimia 1994, 48, 102–104. [Google Scholar]
  88. Deli, J.; Molnár, P.; Matus, Z.; Tóth, G.; Traber, B.; Pfander, H. “Prenigroxanthin” [(all-E,3R,3′S,6′S)-β, γ-carotene-3,3′6′-triol], a novel carotenoid from red paprika (Capsicum annuum). Tetrahedron Lett. 2001, 42, 1395–1397. [Google Scholar] [CrossRef]
  89. Deli, J.; Matus, Z.; Tóth, G. Carotenoid composition in the fruits of Asparagus officinalis. J. Agric. Food Chem. 2000, 48, 2793–2796. [Google Scholar] [CrossRef] [PubMed]
  90. 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. Chromatographia 1998, 48, 27–31. [Google Scholar] [CrossRef]
  91. Deli, J.; Molnár, P.; Ősz, E.; Tóth, G. Analysis of the carotenoids of the fruits of Asparagus falcatus; Isolation of 5,6-diepikarpoxanthin. Chromatographia 2000, 51, S183–S187. [Google Scholar] [CrossRef]
  92. Deli, J.; Matus, Z.; Tóth, G. Comparative study on the carotenoid composition in the buds and flowers of different Aesculus species. Chromatographia 2000, 51, S179–S182. [Google Scholar] [CrossRef]
  93. Deli, J.; Molnár, P.; Ősz, E.; Tóth, G. Capsoneoxanthin, a new carotenoid, isolated from the fruits of Asparagus falcatus. Tetrahedron Lett. 2000, 41, 8153–8155. [Google Scholar] [CrossRef]
  94. Agócs, A.; Murillo, E.; Turcsi, E.; Béni, S.; Darcsi, A.; Deli, J. Isolation of allene carotenoids from mamey. J. Food Compos. Anal. 2018, 65, 1–5. [Google Scholar] [CrossRef]
  95. Murillo, E.; Turcsi, E.; Szabó, I.; Mosquera, Y.; Agócs, A.; Nagy, V.; Gulyás-Fekete, G.; Deli, J. Carotenoid compositon of the fruit of red mamey (Pouteria sapota). J. Agric. Food Chem. 2016, 64, 7148–7155. [Google Scholar] [CrossRef]
  96. Murillo, E.; McLean, L.; Britton, G.; Agócs, A.; Nagy, V.; Deli, J. Sapotexanthin, a new A-provitamin carotenoid isolated from mamey (Pouteria sapota). J. Nat. Prod. 2011, 74, 283–285. [Google Scholar] [CrossRef]
  97. Murillo, E.; Mosquera, Y.; Kurtán, T.; Gulyás-Fekete, G.; Nagy, V.; Deli, J. Isolation and characterization of two novel capsorubin like carotenoids from the red mamey (Pouteria sapota). Helv. Chim. Acta 2012, 95, 983–988. [Google Scholar] [CrossRef]
  98. Gulyás-Fekete, G.; Murillo, E.; Kurtán, T.; Papp, T.; Illyés, T.Z.; Drahos, L.; Visy, J.; Agócs, A.; Turcsi, E.; Deli, J. Cryptocapsinepoxide-type carotenoids from red mamey, Pouteria sapota. J. Nat. Prod. 2013, 76, 607–614. [Google Scholar] [CrossRef] [Green Version]
  99. Murillo, E.; Agócs, A.; Nagy, V.; Király, S.B.; Kurtán, T.; Toribio, E.M.; Lakey-Beitia, J.; Deli, J. Isolation and identification of sapotexanthin 5,6- and 5,8-epoxide from red mamey (Pouteria sapota). Chirality 2020, 32, 579–587. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  100. Matus, Z.; Baranyai, M.; Tóth, G.; Szabolcs, J. Identification of oxo, epoxy and some cis-carotenoids in high-performance liquid chromatography. Chromatographia 1981, 14, 337–340. [Google Scholar] [CrossRef]
  101. Molnár, P.; Deli, J.; Matus, Z.; Tóth, G.; Steck, A.; Pfander, H. Partial synthesis and characterization of the mono- and diepoxides of β-cryptoxanthin. Helv. Chim. Acta 1997, 80, 221–229. [Google Scholar] [CrossRef]
  102. Turcsi, E.; Murillo, E.; Kurtán, T.; Szappanos, Á.; Illyés, T.Z.; Gulyás-Fekete, G.; Agócs, A.; Avar, P.; Deli, J. Isolation of β-cryptoxanthin-epoxides, precursors of cryptocapsin and 3′-deoxycapsanthin, from red mamey (Pouteria sapota). J. Agric. Food Chem. 2015, 63, 6059–6065. [Google Scholar] [CrossRef]
  103. Dachtler, M.; Glaser, T.; Kohler, K.; Albert, K. Combined HPLC-MS and HPLC-NMR on-line coupling for the separation and determination of lutein and zeaxanthin stereoisomers in spinach and in retina. Anal. Chem. 2001, 73, 667–674. [Google Scholar] [CrossRef]
  104. Hentschel, P.; Grynbaum, M.D.; Molnár, P.; Putzbach, K.; Rehbein, J.; Deli, J.; Albert, K. Structure elucidation of deoxylutein II isomers by on-line capillary high-performance liquid chromatography—Nuclear magnetic resonance spectroscopy. J. Chromatogr. A 2006, 1112, 285–292. [Google Scholar] [CrossRef]
  105. Deli, J.; Molnár, P.; Ősz, E.; Tóth, G.; Zsila, F. Epimerisation of lutein to 3′-epilutein in processed foods. Bioorgan. Med. Chem. Lett. 2004, 14, 925–928. [Google Scholar] [CrossRef]
  106. Molnár, P.; Ősz, E.; Szabó, Z.; Oláh, P.; Tóth, G.; Deli, J. Separation and identification of lutein derivatives in processed foods. Chromatographia 2004, 60, S101–S105. [Google Scholar] [CrossRef]
  107. Molnár, P.; Ősz, E.; Zsila, F.; Deli, J. Isolation and structure elucidation of anhydroluteins from cooked sorrel (Rumex rugosus Campd.). Chem. Biodivers. 2005, 2, 928–935. [Google Scholar] [CrossRef]
  108. Zechmeister, L. Some in vitro conversions of naturally occurring carotenoids. Fortschr. Chem. Org. Nat. 1958, 15, 31–82. [Google Scholar]
  109. Háda, M.; Nagy, V.; Deli, J.; Agócs, A. Hydrophilic carotenoids: Recent progress. Molecules 2012, 17, 5003–5012. [Google Scholar] [CrossRef] [Green Version]
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Figure 1. László Zechmeister, 14 May 1889 (Győr, Hungary)—28 February 1972 (Pasadena, CA, USA).
Figure 1. László Zechmeister, 14 May 1889 (Győr, Hungary)—28 February 1972 (Pasadena, CA, USA).
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Figure 2. Column chromatography from around 1930 and from the present day.
Figure 2. Column chromatography from around 1930 and from the present day.
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Figure 3. Column chromatography of an alga extract on modified silica gel.
Figure 3. Column chromatography of an alga extract on modified silica gel.
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Figure 4. Separation of carotene mixtures for C18 and C30 reversed-phase columns. C18 Conditions: Technokroma C18 5 μm stat. phase (250 × 4.6 mm), 22 °C. Eluents: A: MeOH/H2O = 12/88 v/v%, B: MeOH, C: Acetone/MeOH = 50/50 v/v%. Gradient program: 0′ 80% A, 20% B, 8′ 50% A, 50% B, 17′ 100% B, 15–17′ 100% B, 24′ 100% C, 24–31′ 100% C, 33′ 100% B, 33–34′ 100% B, 35′ 80% A, 20% B. Flow rate: 1.25 cm3/min; λ = 450 nm; C30 condition: YMC C30 3 μm stat. phase (250 × 4.6 mm), 22 °C. Eluents: A: MeOH/TBME/H2O = 81/15/4 v/v%, B: MeOH/TBME/H2O = 6/90/4 v/v%. Gradient program: 0–90′ 100% B, 95′ 100% A. Flow rate: 1.00 cm3/min; λ = 450 nm.
Figure 4. Separation of carotene mixtures for C18 and C30 reversed-phase columns. C18 Conditions: Technokroma C18 5 μm stat. phase (250 × 4.6 mm), 22 °C. Eluents: A: MeOH/H2O = 12/88 v/v%, B: MeOH, C: Acetone/MeOH = 50/50 v/v%. Gradient program: 0′ 80% A, 20% B, 8′ 50% A, 50% B, 17′ 100% B, 15–17′ 100% B, 24′ 100% C, 24–31′ 100% C, 33′ 100% B, 33–34′ 100% B, 35′ 80% A, 20% B. Flow rate: 1.25 cm3/min; λ = 450 nm; C30 condition: YMC C30 3 μm stat. phase (250 × 4.6 mm), 22 °C. Eluents: A: MeOH/TBME/H2O = 81/15/4 v/v%, B: MeOH/TBME/H2O = 6/90/4 v/v%. Gradient program: 0–90′ 100% B, 95′ 100% A. Flow rate: 1.00 cm3/min; λ = 450 nm.
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Figure 5. Structures of antheraxanthin and cucurbitaxanthin A.
Figure 5. Structures of antheraxanthin and cucurbitaxanthin A.
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Figure 6. Structure of naturally occurring cryptocapsin 5,6-epoxide with a non-hydroxylated (5R,6S)-5,6-epoxy β-end group.
Figure 6. Structure of naturally occurring cryptocapsin 5,6-epoxide with a non-hydroxylated (5R,6S)-5,6-epoxy β-end group.
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Figure 7. HPLC separation of β-cryptoxanthin 5′,6′-monoepoxides. C30 Conditions: see Figure 4. Chiral chromatography conditions: Chiralcel OD C18 3 μm stat. phase (250 × 4.6 mm), 22 °C. Eluents: A: MeOH/EtOH = 50/50 v/v%, B: MeCN/EtOH = 50/50 v/v%. Gradient program: from 0% B to 100% B eluent in 30 min. Flow rate: 1.00 cm3/min; λ = 450 nm.
Figure 7. HPLC separation of β-cryptoxanthin 5′,6′-monoepoxides. C30 Conditions: see Figure 4. Chiral chromatography conditions: Chiralcel OD C18 3 μm stat. phase (250 × 4.6 mm), 22 °C. Eluents: A: MeOH/EtOH = 50/50 v/v%, B: MeCN/EtOH = 50/50 v/v%. Gradient program: from 0% B to 100% B eluent in 30 min. Flow rate: 1.00 cm3/min; λ = 450 nm.
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Agócs, A.; Turcsi, E.; Nagy, V.; Deli, J. Isolation and Analysis of Carotenoids in Hungary from Zechmeister until Today. Processes 2022, 10, 707. https://doi.org/10.3390/pr10040707

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Agócs A, Turcsi E, Nagy V, Deli J. Isolation and Analysis of Carotenoids in Hungary from Zechmeister until Today. Processes. 2022; 10(4):707. https://doi.org/10.3390/pr10040707

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Agócs, Attila, Erika Turcsi, Veronika Nagy, and József Deli. 2022. "Isolation and Analysis of Carotenoids in Hungary from Zechmeister until Today" Processes 10, no. 4: 707. https://doi.org/10.3390/pr10040707

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