Effect of the Number of Conjugated Double Bonds on the Wavenumber Position of CC Stretching Vibration
Three naturally occurring carotenoids, crocetin, β-carotene, and lycopene, were chosen as examples to investigate the relationship between v1 wavenumber and the number of conjugated carbon–carbon double bonds present in the polyene chain. For this purpose, FT-Raman measurements were taken from those fresh plant tissues, which are known to contain these pigments as main carotenoids.
Crocetin is a unique 7-conjugated CC carotenoid substance occurring in stigmas of crocus (Crocus sativus L.) flower, which are used as saffron spice. Its golden-yellow coloring matter has long been employed for a food flavoring, whereas, in the Middle Ages, saffron was employed as an artist's colorant.12, 24 In fact, the bright coloring power of saffron comes from the crocins, glycosyl esters of crocetin.25, 26 As can be seen in Figure 2 A, the ν1 band in the FT-Raman spectrum of saffron appears at 1536 cm−1. This band of crocetin has been previously reported at 1537 cm−1 in the Resonance Raman spectrum12 and at 1540 cm−1 when excited in a near infrared region.25 The second intense band in the FT-Raman spectrum of saffron is seen in Figure 2A at 1165 cm−1 and can be attributed to a CC stretching vibration of the carotenoid skeleton. The in-plane rocking mode of CH3 groups attached to the polyene chain is observed as a peak of medium intensity at 1020 cm–1.
The principal pigment of red tomato fruits is lycopene, an acyclic 11-conjugated carotene.29, 30 Figure 2C presents the FT-Raman spectrum of tomato; the three most intense bands are seen at 1510 (ν1), 1156 (ν2), and 1004 cm−1 (ν3). The signal at 1510 cm−1 looks asymmetrical and it can be assumed that the shoulder to be seen at higher wavenumbers (about 1520 cm−1) is due to β-carotene, which is also present in tomato but in lower amounts. The assignment of the band registered at 1510 cm−1 is confirmed by Raman measurement of tomato puree rich in lycopene (see Table I). Furthermore, spectra measured in orange tomatoes have shown a higher intensity band near 1520 cm−1 with a shoulder at 1510 cm−1 that corresponds to higher amounts of β-carotene (1520 cm−1) in comparison to lycopene (1510 cm−1), which is reflected also in the color of this vegetable (spectra not presented).
Based on Resonance Raman spectra of retinal, crocetin, β-carotene, lycopene, decapreno-β-carotene, and dodecapreno-β-carotene it has already been shown that the wavenumber of the ν1 band decreases with the extent of the conjugation length of the central polyene chain due to an electron–phonon coupling.12 Our experiment revealed that this relationship also occurs for carotenoids (i.e., crocetin, β-carotene, and lycopene) measured in situ by FT-Raman spectroscopy. The observed ν1 shift toward red is correlated with an increasing number of conjugated double bonds in the carotenoid chain, i.e., 1536 cm−1 (7) 1524 cm−1 (9) 1510 cm−1 (11). Contrary to that, no correlation is observed for v2 and v3 modes (see Table I). A similar relationship is observed for other carotenoids as shown in Table I.
Other Agents Influencing the Wavenumber Location of CC Stretching Vibration
Cyclization and other modifications during the biosynthetic pathway (hydrogenation, dehydrogenation, isomerization, introduction of hydroxyl groups, rearrangements, etc.) result in a variety of carotenoid structures. Most of them have 9 conjugated double bonds in the central chain. In this study we analyzed three examples of such carotenoids: β-carotene, α-carotene, and lutein with different side groups. Bicyclic β- and α-carotenes are formed in two separate branches of the biosynthetic pathway and differ only in the position of the double bond in one β-ionone ring whereas dihydroxylation of the latter results in lutein formation (see Figure 1).
In Figure 3 the FT-Raman spectra of the pure standards (β-carotene, α-carotene, and lutein) are presented. The wavenumber positions of CC stretching vibrations are different for the above-mentioned compounds, the lowest is seen for β-carotene at 1515 cm−1, for α-carotene at 1521 cm−1, and at 1522 cm−1 for lutein. All these carotenoids contain the same number of conjugated double bonds, so obviously the regarded wavenumber shift is not correlated with the length of the polyene chain. It can be therefore concluded that the side groups also influence the wavenumber location of ν1.
It was observed that the wavenumber position of the CC stretching of β-carotene in a pure standard (1515 cm−1) is moved in comparison to that obtained at in situ measurement of carrot roots (1520 cm−1). This phenomenon can be explained by the fact that carotenoids in carrot roots are bonded to proteins,31 but the presence of other carotenoids such as α-carotene or lutein may also affect the band shift toward higher wavenumbers and therefore cannot be neglected in this context. Generally, chromatographic analyses of carotenoids (TLC, HPLC) in plant tissues include solvent extraction of the pigment. However, this procedure causes the destruction of the natural carotene–protein complexes. Contrary to that, FT-Raman spectroscopy allows the investigation of the carotenoids nondestructively in their natural environment.
In the next step, five carotenoids characterized by 9 conjugated double bonds in the main chain were measured in situ. Figure 4 shows the FT-Raman spectra of red pepper fruit (main carotenoid:capsanthin) (Figure 4A),32–34 nectarine fruit (main carotenoid: β-cryptoxanthin) (Figure 4B),1 yellow carrot root (main carotenoid:lutein) (Figure 4C),35 pumpkin fruit (main carotenoid:β-carotene) (Figure 4D),27, 29 and corn (main carotenoid:zeaxanthin) (Figure 4E).1, 36 For all of these pigments, the characteristic CC stretching vibration can be found in the wavenumber range between 1517 cm−1 (capsanthin) and 1527 cm−1 (lutein and β-cryptoxanthin). The dispersion of the ν1 wavenumbers of 9-conjugated systems is significant, however this wide range is still distinct and is located below 1536 cm−1 (characteristic of the 7-conjugated system of crocetin) and above 1510 cm−1 (characteristic of the 11-conjugated lycopene). These in situ measurements of carotenoids confirm the above-mentioned observation that the side groups of the central polyene chain also influence the position of the ν1 wavenumber. The ν1 shift can also be attributed to the fact that carotenoids usually bond to other compounds in plants. It is known that green leaves and vegetables contain unesterified hydroxy carotenoids, mainly lutein, whereas carotenols in fruit are esterified with fatty acids.1, 37–39 In our results lutein can be seen at 1527 cm−1 in yellow carrot root, in green leaves at 1525–1526 cm−1, and in green vegetables at 1524 cm−1 (see Table I). A similar situation is observed for β-carotene in orange carrot root (1520 cm−1) and pumpkin or apricot (1524 cm−1).
Figure 4. FT-Raman spectra of red pepper fruit (A), nectarine fruit (B), yellow carrot root (C), pumpkin fruit (D), and corn seed (E).
Download figure to PowerPoint
These examples show that the wavenumber location of CC stretching is influenced not only by the length of the polyene chain and the molecular structure of the terminal groups of carotenoids but also significantly by their interaction with other plant constituents (proteins, fatty acids, etc.). In this context FT-Raman spectroscopy is a potential tool to perform more detailed investigations of these molecular interactions in situ.
Bixin (see Figure 1) is a unique carotenoid compound present in annatto seeds (Bixa orellana L.) and it is used in food industry as a natural colorant. Unlike most of the other carotenoids, bixin occurs in nature in the cis form, but after extraction in organic solvent it converts to the more stable trans form.20 This experimental observation has already been confirmed by theoretical calculation.19
The spectra of bixin obtained directly from seeds as well as related chloroform extracts are presented in Figure 5 A and 5B, respectively. As can be seen, the v1 position of bixin occurring in the natural environment is found at 1518 cm−1 whereas in organic solution a band at a higher (1523 cm−1) wavenumber is detected that can be assigned to its two conformational forms, cis and trans, respectively. However, some influence of plant constituents bonding to bixin (cis isomer) and the chloroform solvent (trans isomer) on the v1 position should also be taken into consideration.
Figure 5. FT-Raman spectra of cis-bixin measured directly from annatto seeds (A) and trans-bixin in chloroform extract (B).
Download figure to PowerPoint
FT-Raman spectra of cis- and trans-crocetin, extracted from Crocus sativus L. stigmas and separated by an HPLC method have been previously reported.26 Stretching vibrations of CC bonds were observed at 1535 and 1547 cm−1 for trans and cis isomers, respectively. The assignment of these bands is opposite of that discussed above for bixin. Thus, the lower wavenumber of conjugated CC stretching vibrations is observed for naturally occurring cis-bixin in annatto and trans-crocetin in crocus.
The difference in the v1 position between cis and trans isomers in both cases is significant and can be easily detected using FT-Raman spectroscopy. It has already been reported that carotenoids in fruit and vegetables undergo isomerization during processing and/or storage and, as a consequence, a decrease in their color intensity and a reduction of their bioactivity occurs.1 Heat, light, acids, and adsorption on the metal surfaces promote trans–cis isomerization, e.g., an increase of cis-β-carotene can be observed in cooked carrot.40, 41 Therefore, FT-Raman spectroscopy can be efficiently applied for the investigation of conformational changes of carotenoids, e.g., in the field of quality control.
Raman 2-D Maps of Carotenoid Distribution in Plants
FT-Raman spectroscopy can also be successfully applied to characterize the carotenoid distribution in the plant tissue at cellular level. The use of a horizontal stage with automatically controlled motion provides the opportunity to perform measurements from a specified area of living tissue. As a consequence, a detailed distribution of individual analytes within the measured region in a 2-D map can be visualized.42–44
Euonymus fortunei Turcs. ‘Canadale Gold’ is a chlorophyll mutant with light green/yellow edge on leaf blades. The presence of yellow carotenoids, mainly lutein, and β-carotene is masked by green chlorophyll, therefore it is not possible to evaluate the carotenoid distribution without analysis. This is why we used Raman mapping to determine the carotenoid distribution in a leaf of Euonymus; the relative concentration of carotenoids was determined according to the intensity of the band at 1525 cm−1 (Figure 6). As can be seen, the higher level of carotenoids is observed in dark green leaf regions, which corresponds to a higher amount of chlorophyll in that tissue. A more detailed view was obtained from a second Raman mapping performed over a smaller area of the same leaf but with a higher resolution (Figure 6D). Comparing this map with the related microscopic image supplies a rapid overview of the individual carotenoid level corresponding to the anatomical structure of the leaf. This example confirms also the nondestructive feature of NIR-FT-Raman spectroscopy: repeated measurements can be taken from the same sample area several times without perceptible changes with regard to quality or reliability.
Figure 6. Picture of Euonymus fortunei ‘Canadale Gold’ leaf (A), a microscopic image of the defined area (C), and corresponding Raman maps colored according to the intensity of the band at 1525 cm−1, which represents the total carotenoid content (B and D).
Download figure to PowerPoint
On the basis of HPLC investigations it is known that petals and pollen of marigold (Calendula officinalis L.) contain mostly flavoxanthin and luteoxanthin (see Figure 1).45 Luteoxanthin is formed from violaxanthin in the process of epoxidefuranoxide rearrangement, resulting in shortening of the chain to 8 conjugated double bonds. Further epoxidation results in the formation of auroxanthin with a 7-conjugated chain.1 Auroxanthin is present in marigold petals in high amounts whereas, in the pollen, lutein and antheraxanthin (both pigments are 9-conjugated carotenoids) are additionally detected.45 The spectra presented in Figure 7 A are taken from marigold flower; they show the different location of the carotenoid v1 bonds. The stretching vibration of CC bonds of the diepoxycarotenoid auroxanthin is observed at 1536 cm−1, exactly at the same wavenumber as for crocetin, the other 7-conjugated system (Figure 7A, upper spectrum). Lutein and anthraxanthin signals are seen at about 1524 cm−1, which is the characteristic range for 9-conjugated chains (Figure 7A, bottom spectrum). However, the most interesting carotenoids are flavoxanthin and luteoxanthin, as they possess 8 double bonds in the central chain, and, until now, Raman spectra of such systems were not reported. As expected, these carotenoids give strong Raman signals in the range between 1529 and 1531 cm−1 (Figure 7A, middle spectrum). In Figure 7 additionally a photo of marigold flower (Figure 7B) and three Raman maps (Figures 7C, 7D, and 7E) showing the distribution of different carotenoids are presented. The maps were obtained according to the intensities of the characteristic CC stretching vibrations for 7-, 8-, and 9-conjugated systems, respectively. The presented Raman maps reveal much higher accumulation of auroxanthin in the outer petals and its lack in the inner part of the flower, which is composed mainly of stigmas filled with pollen (Figure 7C). The distribution of lutein and antheraxanthin is opposite, which is in agreement with the performed HPLC measurements (Figure 7E).45 The derived luteoxanthin with 8-conjugated bonds can be allocated to all flower parts (Figure 7D). However, it is important to stress that the Raman mapping results show the concentration of carotenoids both at the surface and in the surface layer of the flower. The application of NIR laser excitation and lack of confocal arrangement implicate that Raman measurements of the sample may be recorded by penetrating a relatively thick outer layer. This is the reason why, in some parts of Calendula flower, where several petals are lying one on the other, a high concentration of carotenoids can be seen (see red spots in Figures 7C and 7D).
Figure 7. FT-Raman spectra of Calendula officinalis L. measured in three different points showing the presence of 7-, 8-, and 9-conjugated carotenoids (A). Picture of Calendula officinalis flower (B) and corresponding Raman maps colored according to the band intensity at 1536 (C), 1530 (D), and 1524 cm−1 (E) related to the content of 7-, 8-, and 9-conjugated double bond carotenoids, respectively.
Download figure to PowerPoint
The spectrum of Calendula pollen with a strong band at about 1530 cm−1 is similar to that measured before42 in chamomile pollen where the ν1 mode was observed at 1529 cm−1 (see Table I). Therefore it is most likely that both flowers contain 8-conjugated epoxycarotenoids in their pollen.
In conclusion, the presented Raman maps performed in situ allow the precise localization of various carotenoids in different parts of the plant tissue and principally a semiquantitative characterization of these pigments can be achieved. Raman spectroscopy also confirms the presence of 8- and 7-conjugated carotenoids in the Calendula flower, most likely flavoxanthin, luteoxanthin, and auroxanthin. These data also show the possibility that FT-Raman spectroscopy can be applied for the measurement of epoxycarotenoids in situ, which can be very helpful as these compounds have the tendency to easily undergo a degradation and therefore are often underestimated in conventional food or plant analysis. Their occurrence in nature is also often questioned as some of them can be formed as artefacts during sample extraction or analysis.1