All spectral image analysis was performed in Matlab 2012 or 2015 (Mathworks) equipped with the statistics and machine learning, signal processing, image processing, and curve fit‐ ting toolboxes leveraging in‐house written software, functions, and scripts. Hyperspectral confocal Raman images were preprocessed to remove cosmic spikes [37]. When applicable, images from the same sample were compiled into a composite image data set. The use of composite images, rather than analyzing every image independently, increases the number of pixels for the MCR analysis and thus serves to improve spectral signature identification by adding additional variance [38]. The spectral region was trimmed to exclude the excitation laser line but still include about 5–10 pixels of the spectrum where the signal was blocked by the Rayleigh filter. This technique assists in the analysis by providing a "zero‐signal region" for assessing baseline contributions and is discussed in detail by Jones et al. [38]. In most cases, an image mask was created that excluded pixels outside the area of the cells from the analysis as they contain predominantly background signal.

Principal components analysis (PCA) was performed on the composite image and the Scree plot was inspected to determine the number of independent components present in the image data set (measured as being before the bend in the elbow of the Scree plot [29]). Multivariate curve resolution was then performed using a constrained alternating least‐ squares algorithm and employing robust constraints for equality (offset/baseline) and non‐ negativity (all true spectral components) to develop a spectral model that described the spectral variance within the data set. A PCA analysis of the residuals was used to confirm the appropriateness of the spectral model for describing the data and identify any unmod‐ eled spectral signatures. The multivariate curve resolution algorithm [39–43] and specific approaches for success with biological images have been described in detail elsewhere [29, 38]. The details of the spectral models developed are presented during the results and dis‐ cussion for each application in this chapter. The MCR‐identified spectra were then used in a classical least‐squares (CLS) analysis to predict the concentrations of each spectral compo‐ nent in each image pixel. Lastly, the resulting concentration maps were exported as 16‐bit grayscale tiffs such that they could be subject to traditional image analysis. Color image overlays and simple image cropping and scaling for visualization purposes was performed in Fiji [44].

#### **3. Results and discussion**

*Synechocystis* 6803 cells were obtained from the Pakrasi lab and cultured in BG‐11 media (UTEX recipe) in baffled flasks in a temperature‐controlled incubator under continuous light (∼30 µmol m−2 s−1, cool white fluorescent lights) with shaking (60 rpm). Prior to imaging, a 4‐µl sample was directly pipetted onto a BG‐11 agar‐coated slide and covered with a glass cover‐

Etiolated maize protoplasts were isolated from *Z. mays* var. B73 leaves as described previ‐ ously [36]. Released protoplasts were collected, washed, and shipped in a buffer solution overnight for Raman imaging. Upon arrival, protoplasts were concentrated by centrifugation at 500 × g and removal of all but 100 µl of the supernatant. Concentrated sample (25 µl) was loaded into an *in situ* frame (Gene Frame, ThermoFisher) previously adhered to a gridded microscope slide (Lovins Micro‐Slide Field Finder, Electron Microscopy Sciences) and cov‐ ered with a glass coverslip.  Light was incident at 532 nm and focused using a 50×/0.55 NA objective (*Synechocystis* 6803), 20×/0.45 NA objective (maize protoplasts), and a 100×/0.9 NA objective (*H. pluvialis*). Laser powers were chosen such that the spectral character was nearly invariant with time and did not cause a visible change in the sample during collec‐ tion. In most cases, power incident on the surface was held to less than 1 mW. The acquisition time per spectrum was chosen to provide adequate signal to noise to perform the multivariate analysis and varied for the different samples analyzed ranging from 4 to 10 ms per spectrum. The spectral response was 3 cm−1 per pixel, which through fitting resulted in the ability to specify peak position to an absolute accuracy of ±1 cm−1. All measurements were performed in an unpolarized back‐scattering arrangement. Images were acquired by scanning the sample using a piezo‐stage possessing a lateral resolution of <5 nm. Raman‐scattered light is collected continuously across the sample such that every spectrum is the average convolution of the beam intensity and sample response over the distance defining an individual voxel. Images were collected by acquiring a spectrum every 333 nm (*Synechocystis* 6803 and *H. pluvialis*) or 500 nm (maize protoplasts). Raman shifts were calibrated using the well‐established positions

All spectral image analysis was performed in Matlab 2012 or 2015 (Mathworks) equipped with the statistics and machine learning, signal processing, image processing, and curve fit‐ ting toolboxes leveraging in‐house written software, functions, and scripts. Hyperspectral confocal Raman images were preprocessed to remove cosmic spikes [37]. When applicable, images from the same sample were compiled into a composite image data set. The use of composite images, rather than analyzing every image independently, increases the number of pixels for the MCR analysis and thus serves to improve spectral signature identification by adding additional variance [38]. The spectral region was trimmed to exclude the excitation

microscopy for a separate experiment. Protoplasts were immediately imaged.

slip. Cells were immediately imaged.

40 Carotenoids

**2.2. Confocal Raman microscopy**

of silicon, 6H‐SiC, graphite, and 4‐acetameniphol.

**2.3. Image data analysis**

#### **3.1. Resolving different carotenoids in living cells:** *H. pluvialis*

*H. pluvialis* is a freshwater green microalga that can synthesize and accumulate astaxanthin, a high‐value nutraceutical, under stress conditions such as nutrient deprivation. Though some aspects of carotenogenesis leading to astaxanthin production are well understood, spatial‐temporal details of key carotenoids and enzymes have yet to be fully elucidated. In previous work, Collins and coworkers used confocal Raman spectroscopic imaging to provide *in vivo* resolution and localization of beta‐carotene and astaxanthin, along with chlorophyll throughout the life cycle of *H. pluvialis* cells [27]. While this work will not be reproduced in detail, here, some key elements will be presented to illustrate the poten‐ tial of the technique for carotenoids localization in living cells. **Figure 2** demonstrates the advantage of performing MCR analysis on the Raman spectroscopic image data as com‐ pared to simplistic band integration. While the image produced by integrating the area under the *ν*1 carotenoid vibration provides a highly resolved view of carotenoid location within the cell, the image is a superposition of two different carotenoids found in the cell. By contrast, MCR analysis resulted in a four‐component model consisting of two carotenoid spectra (astaxanthin and beta‐carotene), chlorophyll, and an autofluorescence component. (Note: A background component was also part of the model, but is omitted here for simplic‐

ity.) MCR analysis also generates a corresponding concentration map (i.e., image) for each of these species. This results in an exquisite degree of chemical resolution that allows for quantitative assessment via traditional image analysis methods. In **Figure 2D**, the images for astaxanthin, beta‐carotene, and chlorophyll are pseudocolored and overlaid for qualita‐ tive comparison. Importantly, in this example, the MCR analysis reveals that astaxanthin is located only in the cytosol. Beta‐carotene, meanwhile, is both co‐located with astaxanthin in the cytosol, as well as with the chlorophyll in the chloroplast. Together, these Raman images provide spatial‐temporal details of astaxanthin production and accumulation in this organism.

#### **3.2. Carotenoids in high‐chlorophyll backgrounds: maize protoplasts**

Similar to algae, plant carotenoids have roles in light harvesting and protection against light and heat stress. Metabolic engineering of carotenoids in plants has the potential to create variet‐ ies exhibiting increased adaptation to climate change as well as additional nutritional value. To develop plant cultivars with these enhanced properties, it is critical to understand the detailed spatial‐temporal arrangement of both carotenoids and chlorophyll *in situ*. While chlorophyll is straightforward to detect with a standard confocal fluorescence microscope, carotenoids have little to no detectable fluorescence signal and are not amenable to exogenous labeling.

**Figure 2.** Raman‐spectroscopic imaging of *H. pluvialis*: the advantage of MCR analysis. (A) Raw spectra from Raman‐ spectroscopic image of *H. pluvialis*. (B) Integrated Raman image created by traditional integration over the area of the *ν*1 band of the carotenoid (1480–1530 cm−1) for the image data in A. C. MCR identified spectral component from image data in A. RGB image created by overlaying the independent component maps generated in the MCR analysis. Scale bar *= 10* µ*m*. (note: **Figure 2** is best viewed in color. Please refer to the online version of this chapter for the color version of **Figure 2**).

Confocal Raman spectroscopic imaging is capable of detecting carotenoids without the use of any exogenous labels and does so by detecting the resonance Raman signal of carotenoids. This signal can be observed also within the background of other pigments, such as the chlo‐ rophyll precursor, protochlorophyllide, which is found in cells of etiolated tissue [45] such as plant protoplasts from etiolated maize (**Figure 3**). Plant protoplasts are plant cells that have their cell wall removed through enzyme treatment and thus are an excellent experimental system for plant biologists because they improve the ease of introducing new genes.

ity.) MCR analysis also generates a corresponding concentration map (i.e., image) for each of these species. This results in an exquisite degree of chemical resolution that allows for quantitative assessment via traditional image analysis methods. In **Figure 2D**, the images for astaxanthin, beta‐carotene, and chlorophyll are pseudocolored and overlaid for qualita‐ tive comparison. Importantly, in this example, the MCR analysis reveals that astaxanthin is located only in the cytosol. Beta‐carotene, meanwhile, is both co‐located with astaxanthin in the cytosol, as well as with the chlorophyll in the chloroplast. Together, these Raman images provide spatial‐temporal details of astaxanthin production and accumulation in this

Similar to algae, plant carotenoids have roles in light harvesting and protection against light and heat stress. Metabolic engineering of carotenoids in plants has the potential to create variet‐ ies exhibiting increased adaptation to climate change as well as additional nutritional value. To develop plant cultivars with these enhanced properties, it is critical to understand the detailed spatial‐temporal arrangement of both carotenoids and chlorophyll *in situ*. While chlorophyll is straightforward to detect with a standard confocal fluorescence microscope, carotenoids have

little to no detectable fluorescence signal and are not amenable to exogenous labeling.

**Figure 2.** Raman‐spectroscopic imaging of *H. pluvialis*: the advantage of MCR analysis. (A) Raw spectra from Raman‐ spectroscopic image of *H. pluvialis*. (B) Integrated Raman image created by traditional integration over the area of

 band of the carotenoid (1480–1530 cm−1) for the image data in A. C. MCR identified spectral component from image data in A. RGB image created by overlaying the independent component maps generated in the MCR analysis. Scale bar *= 10* µ*m*. (note: **Figure 2** is best viewed in color. Please refer to the online version of this chapter for the color

**3.2. Carotenoids in high‐chlorophyll backgrounds: maize protoplasts**

organism.

42 Carotenoids

the *ν*1

version of **Figure 2**).

**Figure 3** highlights the spatial location and levels of carotenoids and protochlorophyllide in plastids (immature chloroplasts) of etiolated maize protoplasts as observed by confocal Raman microscopy. Previous high‐performance liquid chromatography data have shown that the dominant carotenoid in maize‐etiolated protoplasts is lutein with a minor amount of violaxanthin (data not shown). The peak positions (*ν*1 = 1525 cm−1 and *ν*2 = 1157 cm−1) in the isolated Raman spectrum shown in **Figure 3C** are consistent with the purified lutein spectrum in **Figure 1** and by Collins et al. [27]. The slight deviation is likely a result of the violaxanthin contribution as the *ν*1 vibration of violaxanthin is typically 1528–1530 cm−1 [46]. For compari‐ son, Panels A and B show the correlated bright‐field and confocal fluorescence chlorophyll images, respectively, for the cell shown in **Figure 3D**. Individual component images are shown in grayscale as well as the red‐green merged image (please refer to the online version for color figure). Green corresponds to the carotenoid and red corresponds to the protochloro‐ phyllide pigments in these figures. Localization is confined to the plastids (bright foci ∼1 µm in size within the protoplast). Interestingly, a high degree of variation in carotenoid and chlo‐ rophyll composition within individual plastids is observed. An example of this heterogeneity is indicated by the yellow arrows in the merged image of **Figure 3D**. These arrows highlight plastids that have predominantly carotenoid (green), predominantly protochlorophyllide (red), or roughly equal amounts of both (yellowish‐orange). These differences in relative abundance could arise from differences in the developmental phase or carotenoid abundance. The observed heterogeneities raise important scientific questions about carotenoid biogenesis that Raman‐spectroscopic imaging is well poised to answer in future research.

#### **3.3. Approaching the limits of spatial resolution:** *Synechocystis* **6803**

Cyanobacteria and closely related organisms are thought to be the evolutionary ancestors to chloroplasts found in plants. Cyanobacteria perform photosynthesis to convert light energy to chemical energy and play key roles in the ecology of the earth. Light is harvested by photo‐ synthetic antennae, which are pigment‐protein complexes composed of pigments with vary‐ ing absorption and emission properties in order to "funnel" the energy to the reaction center where it is converted to chemical energy [47]. Carotenoids are integrated into the membrane‐ associated photosynthetic antennae complexes as well as the photosystems of all cyanobac‐ teria and fundamentally coupled to the processes of light harvesting and photosynthesis. Unfortunately, carotenoids in cyanobacteria are often quite difficult to localize *in situ* because of the very small size of many of the cyanobacteria (1.5–4 µm for the most species) and the co‐localization in complexes with highly fluorescent light‐harvesting pigments, like phyco‐ bilins and chlorophylls. Previous work with hyperspectral confocal fluorescence microscopy has demonstrated sensitivity for localization carotenoids via resonance Raman signatures in

**Figure 3.** Confocal Raman‐spectroscopic imaging of carotenoids and protochlorophyllide in etiolated maize protoplasts. (A) Bright‐field image of several protoplasts with red square highlighting single protoplast imaged in B and D. (B) Confocal fluorescence image of cell highlighted in A (chlorophyll emission channel). (C) Spectral components of MCR model from the analysis of confocal Raman image of maize protoplasts. (D) Left panel: protochlorophyllide concentration map. Center panel: carotenoid concentration map. Right panel: Merged red and green color overlay, where the protochlorophyllide image is assigned to the red channel and carotenoid image is assigned to the green channel. Arrows highlight examples of plastid heterogeneity. (E) and (F). Two additional protoplasts. Image panels for E and F are the same as in D. Scale bars = 5 µm. (note: **Figure 3** is best viewed in color. Please refer to the online version of this chapter for the color version of **Figure 3**).

*Synechocystis* 6803 [48]; however, the spectral resolution of such a system is not sufficient to resolve different carotenoids.

*Synechocystis* 6803 has emerged as a model cyanobacterium due to the ease of genetic manip‐ ulation. Although *Synechocystis* 6803 cells are quite small (1.5–2 µm), Raman spectroscopic imaging can provide information about subcellular carotenoid localization. **Figure 4** shows the results of Raman spectroscopic imaging of wild‐type *Synechocystis* 6803 cells. The carot‐ enoid spectrum isolated in **Figure 4A** has *ν*1 vibration appearing at 1515 cm−1 and *ν*2 vibration at 1155 cm−1, identifying that carotenoid most likely corresponds to beta‐carotene, one of the major carotenoid species in *Synechocystis* 6803. The phycobilin spectrum peak is centered at ∼3250 cm−1 (equivalent to 643.2 nm) and thus corresponds to phycocyanin, the primary pig‐ ment found in the antenna structure. The fluorescence emission of phycocyanin is ∼645 nm. While subcellular details are somewhat limited given the small number of pixels in each cell, cell‐to‐cell differences in localization patterns are clearly evident. For example, in **Figure 4**, the carotenoids in the dividing cell on the left are concentrated into one small foci on each cell, whereas in the other, two cells have carotenoids localized more uniformly through the thylakoid membranes that house the light‐harvesting complexes, similar to the phycocyanin.

**Figure 4.** Results from confocal Raman‐spectroscopic imaging of wild‐type *Synechocystis* 6803 cells. (A) MCR identified pure component spectra. A fourth component consisting of a tail at ∼3500 cm−1 was isolated as well and corresponds to chlorophyll. It was omitted for simplicity. (B) MCR concentration maps indicating carotenoid (bottom row) and phycobilin (top row) abundance for three different cells. Image intensities have been independently scaled between for maximum visibility; however, all intensity scales are within 20% of each other. A concentration map for the offset component is not shown. Scale bar = 1 µm.

Cell‐to‐cell heterogeneity and population dynamics are important to understand cyanobacte‐ rial adaptation to changes in the abiotic or biotic environment and could be addressed with Raman spectroscopic imaging in the future. It is also important to note that the images in **Figure 4** were collected under slightly less than diffraction‐limited conditions. Improvements in spatial resolution are possible with a more optimal arrangement.

#### **4. Conclusions**

*Synechocystis* 6803 [48]; however, the spectral resolution of such a system is not sufficient to

**Figure 3.** Confocal Raman‐spectroscopic imaging of carotenoids and protochlorophyllide in etiolated maize protoplasts. (A) Bright‐field image of several protoplasts with red square highlighting single protoplast imaged in B and D. (B) Confocal fluorescence image of cell highlighted in A (chlorophyll emission channel). (C) Spectral components of MCR model from the analysis of confocal Raman image of maize protoplasts. (D) Left panel: protochlorophyllide concentration map. Center panel: carotenoid concentration map. Right panel: Merged red and green color overlay, where the protochlorophyllide image is assigned to the red channel and carotenoid image is assigned to the green channel. Arrows highlight examples of plastid heterogeneity. (E) and (F). Two additional protoplasts. Image panels for E and F are the same as in D. Scale bars = 5 µm. (note: **Figure 3** is best viewed in color. Please refer to the online version

*Synechocystis* 6803 has emerged as a model cyanobacterium due to the ease of genetic manip‐ ulation. Although *Synechocystis* 6803 cells are quite small (1.5–2 µm), Raman spectroscopic imaging can provide information about subcellular carotenoid localization. **Figure 4** shows the results of Raman spectroscopic imaging of wild‐type *Synechocystis* 6803 cells. The carot‐

at 1155 cm−1, identifying that carotenoid most likely corresponds to beta‐carotene, one of the major carotenoid species in *Synechocystis* 6803. The phycobilin spectrum peak is centered at ∼3250 cm−1 (equivalent to 643.2 nm) and thus corresponds to phycocyanin, the primary pig‐ ment found in the antenna structure. The fluorescence emission of phycocyanin is ∼645 nm. While subcellular details are somewhat limited given the small number of pixels in each cell, cell‐to‐cell differences in localization patterns are clearly evident. For example, in **Figure 4**, the carotenoids in the dividing cell on the left are concentrated into one small foci on each cell, whereas in the other, two cells have carotenoids localized more uniformly through the thylakoid membranes that house the light‐harvesting complexes, similar to the phycocyanin.

vibration appearing at 1515 cm−1 and *ν*2

vibration

resolve different carotenoids.

44 Carotenoids

of this chapter for the color version of **Figure 3**).

enoid spectrum isolated in **Figure 4A** has *ν*1

Localizing carotenoids in living cells and tissues is challenging due to the complex biological matrices of the living cells and intense sometimes colocated interfering fluorescence. Raman spectroscopic imaging coupled with multivariate curve resolution analysis provides the nec‐ essary spatial and chemical resolution to identify highly similar carotenoids even in the midst of highly overlapping, strong chlorophyll emission and complex backgrounds. Three exam‐ ples were presented that highlight different advantages of the methodology for investigating photosynthetic organisms. Recent technological advances in detector speed and sensitivity will most likely catalyze future investigations of carotenoid biogenesis in single cells, includ‐ ing population dynamics and response to changing environmental conditions, by facilitating time‐course studies that were previously prohibitive given the long scan times required for Raman spectroscopic imaging. These capabilities are anticipated to impact a variety of research areas including carbon partitioning and utilization, microbial ecology, and crop analytics.

#### **Acknowledgements**

The authors are grateful to the following people for their assistance with the research pre‐ sented in this chapter: Anthony McDonald for assistance with the collection of Raman spec‐ troscopic image data; Meghan Dailey for culturing the *Synechocystis* 6803; Stephen Anthony for the current implementation of the MCR analysis software used to deconvolve spectral components; Howland Jones, Mark Van Benthem, David Melgaard, Mike Keenan, and David Haaland for original development of the MCR algorithm and software; Wim Vermaas, Arizona State University, for the gift of the purified carotenoid standards, Himadri Pakrasi, Washington University, St. Louis, for the gift of *Synechocystis sp*. PCC 6803; Qiang Hu for the gift of the *H. pluvialis.*

This research was primarily supported as part of the Photosynthetic Antenna Research Center (PARC), an Energy Frontier Research Center funded by the U.S. Department of Energy (DOE), Office of Science, Basic Energy Sciences (BES), under Award # DE‐SC 0001035 (Maize proto‐ plasts imaging, Synechocystis imaging, writing), by Sandia National Laboratories' Laboratory Directed Research and Development (LDRD) Program under Award # 141528 (*H. pluvialis* imaging), and by funding from the National Institutes of Health (GM081160) (to ETW) for research on carotenoids in maize protoplasts. Sandia National Laboratories is a multimis‐ sion laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy's National Nuclear Security Administration under contract DE‐AC04‐94AL85000.

#### **Author details**

Jerilyn A. Timlin1 \*, Aaron M. Collins2 , Thomas A. Beechem1 , Maria Shumskaya3,5 and Eleanore T. Wurtzel3,4

#### **References**

[4] Lewis IR, Edwards HGM, editors. Handbook of Raman Spectroscopy. New York: Marcel Dekker, Inc.; 2001.

for the current implementation of the MCR analysis software used to deconvolve spectral components; Howland Jones, Mark Van Benthem, David Melgaard, Mike Keenan, and David Haaland for original development of the MCR algorithm and software; Wim Vermaas, Arizona State University, for the gift of the purified carotenoid standards, Himadri Pakrasi, Washington University, St. Louis, for the gift of *Synechocystis sp*. PCC 6803; Qiang Hu for the

This research was primarily supported as part of the Photosynthetic Antenna Research Center (PARC), an Energy Frontier Research Center funded by the U.S. Department of Energy (DOE), Office of Science, Basic Energy Sciences (BES), under Award # DE‐SC 0001035 (Maize proto‐ plasts imaging, Synechocystis imaging, writing), by Sandia National Laboratories' Laboratory Directed Research and Development (LDRD) Program under Award # 141528 (*H. pluvialis* imaging), and by funding from the National Institutes of Health (GM081160) (to ETW) for research on carotenoids in maize protoplasts. Sandia National Laboratories is a multimis‐ sion laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy's National Nuclear Security

, Thomas A. Beechem1

3 Department of Biological Sciences, Lehman College, The City University of New York

5 Department of Biology, School of Natural Sciences, Kean University, Union, NJ, USA

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## **The Biochemistry and Antioxidant Properties of Carotenoids**

### Oguz Merhan

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50 Carotenoids

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67592

#### **Abstract**

Carotenoids are one of the most widespread pigment groups distributed in nature, and more than 700 natural carotenoids have been described so far, and new carotenoids are introduced each year. Carotenoids are derived from 4 terpenes, including totally 40 carbon atoms. Carotenoids are naturally synthesized by cyanobacteria, algae, plants, some fungi, and some bacteria, but not made by mammals. Lately, the beneficial properties of α-carotene, β-carotene, γ-carotene, lycopene, phytoene, phytofluene, lutein, zeaxanthin, β-cryptoxanthin, astaxanthin, and fucoxanthin carotenoids in prevention of various diseases, such as tumor formation, cardiovascular, and vision, have been documented due to their roles as antioxidants, activation in certain gene expression associated with cell-to-cell communication, provitamin A activity, modulation of lipoxygenase activity, and immune response. In this chapter, in addition to biochemical properties of carotenoids, how the structure of these molecules influences the oxidative stress in health and reducing the risk of formation of various diseases will be described.

**Keywords:** carotenoids, antioxidants, biochemistry, health, toxicity

#### **1. Introduction**

Overproduction of reactive oxygen and nitrogen species, such as superoxide and peroxide radicals, plays important roles in the formation of breast, cervical, ovarian, and colorectal cancer in addition to some other malignancies in the cardiovascular system and eye. The antioxidant property of carotenoids may result from its double carbon-carbon bonds interacting with each other via conjugation and causing electrons in the molecule to move freely across these areas of the molecule. Carotenoid intake from food sources reduces the risk of breast, lung, head and neck, cervical, ovarian, colorectal, and prostate cancers and cardiovascular

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

or eye diseases due to their roles as antioxidants. It has been reported that carotenoids can directly interact with some free radical species.

#### **2. The biochemistry and metabolism of main carotenoids**

#### **2.1. Terpenes**

Terpenes are structures derived from isoprene chains. The chemical formula of the isoprene molecule is CH2 ═C(CH3 )─CH═CH2 (2-methyl-1,3-butadiene) [1, 2]. There are two double bonds in the molecule and these bonds are conjugated (**Figure 1**). When 5-carbon containing isoprene molecules are polymerized, compounds called terpenes are formed [3]. This group includes biologically very important molecules. Some among these are lycopene, β-carotene, vitamin A, and squalene [4].

Terpenes are biosynthetically derived from isoprene units, the chemical formula of this unit is C5 H8 , and the basic molecular formulas of terpenes are multiplied, that is, (C5 H8 ) n (n is the number of joined isoprene units). The isoprene units can be connected from head to tail to form straight chains or rings. The isoprene unit is a building brick that is widely used in nature [5, 6].

Terpenes can be classified according to the number of isoprene units used, such as hemiterpenes (prenol and isovaleric acid); one isoprene unit (5C), monoterpenes (geraniol and limonene); two isoprene units (10C), sesquiterpenes (farnesol); three isoprene units (15C), diterpenes (quinagolides, sembren, and taxadiene); four isoprene units (20C), sesterterpenes; five isoprene units (25C), triterpenes (squalene); six isoprene units (30C), tetraterpenes (carotenoids); and eight isoprene units (40C), politerpenes, which include great number of isoprene units (natural rubber) [6–9].

Monoterpenes, sesquiterpenes, diterpenes, and sesterterpenes are formed by head-to-tail association of isoprene units, and triterpenes and tetraterpenes (carotenoids) are formed by head-to-head association [9].

The most important group of terpenes is carotenoids with the C40H64 molecular formula which is formed by a tetraterpene containing eight isoprene units [8]. Carotenoids are terpene

**Figure 1.** Chemical formula for isoprene.

group materials which are formed by binding of 5-carbon-containing isoprene molecules, and they have straight chain structure formed by the condensation of isoprene molecules [1]. Carotenoids which colors vary from light yellow to dark red stemming from the double bonds are soluble in organic solvents and oils like other lipids [10].

#### **2.2. Biosynthesis of terpenes and carotenoids**

or eye diseases due to their roles as antioxidants. It has been reported that carotenoids can

Terpenes are structures derived from isoprene chains. The chemical formula of the isoprene

bonds in the molecule and these bonds are conjugated (**Figure 1**). When 5-carbon containing isoprene molecules are polymerized, compounds called terpenes are formed [3]. This group includes biologically very important molecules. Some among these are lycopene, β-carotene,

Terpenes are biosynthetically derived from isoprene units, the chemical formula of this unit

the number of joined isoprene units). The isoprene units can be connected from head to tail to form straight chains or rings. The isoprene unit is a building brick that is widely used in

Terpenes can be classified according to the number of isoprene units used, such as hemiterpenes (prenol and isovaleric acid); one isoprene unit (5C), monoterpenes (geraniol and limonene); two isoprene units (10C), sesquiterpenes (farnesol); three isoprene units (15C), diterpenes (quinagolides, sembren, and taxadiene); four isoprene units (20C), sesterterpenes; five isoprene units (25C), triterpenes (squalene); six isoprene units (30C), tetraterpenes (carotenoids); and eight isoprene units (40C), politerpenes, which include great number of isoprene

Monoterpenes, sesquiterpenes, diterpenes, and sesterterpenes are formed by head-to-tail association of isoprene units, and triterpenes and tetraterpenes (carotenoids) are formed by

The most important group of terpenes is carotenoids with the C40H64 molecular formula which is formed by a tetraterpene containing eight isoprene units [8]. Carotenoids are terpene

, and the basic molecular formulas of terpenes are multiplied, that is, (C5

(2-methyl-1,3-butadiene) [1, 2]. There are two double

H8 ) n (n is

**2. The biochemistry and metabolism of main carotenoids**

)─CH═CH2

directly interact with some free radical species.

═C(CH3

**2.1. Terpenes**

52 Carotenoids

molecule is CH2

is C5 H8

nature [5, 6].

vitamin A, and squalene [4].

units (natural rubber) [6–9].

head-to-head association [9].

**Figure 1.** Chemical formula for isoprene.

Carotenoids which are tetraterpenes and biological pigments including eight isoprene units are found in plants and some other photosynthetic microorganisms (naturally occurring cyanobacteria, algae, plants, some fungi, and some bacteria) [11].

Acetyl CoA, which is synthesized directly from free acetate as well as the oxidation of sugars and condensation of pyruvic acid or fatty acids, is used as a precursor in the synthesis of mevalonic acid in addition to synthesis of many natural compounds [9, 12]. First, two acetyl CoA molecules enter the reaction to give acetoacetyl-CoA, and then β-hydroxy-βmethylglutaryl-CoA (HMG-CoA) is obtained via HMG-CoA synthase with another acetyl CoA. The reduction of the ester group in HMG-CoA by NADPH and HMG-CoA reductase (the pathway's rate limiting enzyme) results in mevalonic acid formation. Mevalonic acid later forms mevalonate 5-phosphate using a total of two molecules of ATP by mevalonate kinase, and then mevalonate 5-phosphate forms mevalonate 5-diphosphate by phosphomevalonate kinase (mevalonate phosphate kinase). Isopentenyl diphosphate (IPP) building blocks from decarboxylation of mevalonate diphosphate via mevalonate diphosphate carboxylase produce isoprene chains. IPP:dimethylallyl-PP isomerase converts IPP to dimethylallyl diphosphate (DMAPP) which is an acceptor by successive transfer of isopentenyl residues [9, 13]. After the release of the diphosphate, the hemiterpenes are produced from DMAPP. Upon condensation of DMAPP and IPP via geranyl-PP synthase (dimethylallyl transferase), geranyl-PP forms. Monoterpenes are produced by geranyl-PP, which is the precursor of volatile oils. Likewise, farnesyl-PP is formed by condensation of geranyl-PP with IPP via farnesyl-PP synthase (geranyl transferase). Sesquiterpenes are produced by condensation of farnesyl-PP. In addition, two molecules of farnesyl-PP give rise to triterpenes [13, 14]. Squalene, a triterpenic compound, is the precursor of sterols that participate in membrane structure. Geranylgeranyl-PP is formed from head-to-tail condensation of farnesyl-PP and IPP by geranylgeranyl-PP synthase (farnesyl transferase). Geranylgeranyl-PP also causes the formation of diterpenes (**Figure 2**) [9].

Tetraterpenes (carotenoids) are formed by the condensation of two molecules of geranylgeranyl-PP [9, 15]. The first carotenoid formed by prephytoene diphosphate in this step is phytoene which is found in green plants in association with chlorophyll. This first stable carotenoid synthesis takes place via the phytoene synthase. The second product of carotenoid biosynthesis, phytofluene, forms as a result of desaturation (catalyzed by the enzyme phytoene desaturase), which leads to the formation of saturated double bonds. Phytofluene, after having a series of dehydrogenation reactions, forms a symmetric molecule, lycopene, containing 13 double bonds. The next step, α-carotene, β-carotene, and γ-carotene are produced from lycopene via ring formation in the end groups by lycopene cyclase. α- and β-carotenes are the xanthophylls as a result of hydroxylation by β-carotene hydroxylase (**Figure 2**) [16, 17].

**Figure 2.** Biosynthesis of terpenes and carotenoids.

#### **2.3. Carotenoids**

More than 700 natural carotenoids have been identified, and new carotenoids have been added to this number every year [18–20]. Carotenoids are found in red-, yellow-, and orangecolored fruits and vegetables as well as in all green leafy vegetables [10].

Carotenoids are found in plant tissues as free forms (crystalline or amorphous) are dissolved form in an oily solvent. They may also be esterified with fatty acids or complexed with sugars and proteins [17]. The conjugate double-bond structure found in carotenoids also determines biological functions, such as absorption of light during photosynthesis, energy transfer, and protection from harmful effects of light on the cells during the photosynthesis. The presence of carotenoids also determines the characteristic color of these compounds [21].

Carotenoids are present in large quantities in the Leydig cells, which produce steroid hormones, and in the outer layer of the adrenal gland [22]. Carotenoids are also found in fresh broccoli, vegetables, butter, egg yolks, and animal-based foods [23].

#### **2.4. Classification of carotenoids according to their structures**

According to structures of carotenoids, two classes are distinguished as hydrocarbon carotenoids and xanthophylls (**Figure 3**) [24]. The apolar characteristics of hydrocarbon carotenoids are also called carotenes which include α-carotene, β-carotene, γ-carotene, lycopene, phytoene, and phytofluene. Xanthophylls are more polar and contain oxygen in the form of methoxy, hydroxy, keto, carboxy, and epoxy positions. Examples of xanthophylls include lutein, zeaxanthin, β-cryptoxanthin, astaxanthin, and fucoxanthin [25, 26]. Carotenoids are also grouped as carotenoids with or without ring groups at the ends of the chain [26].

#### *2.4.1. Hydrocarbon carotenoids*

**Figure 2.** Biosynthesis of terpenes and carotenoids.

54 Carotenoids

Carotenoids with hydroaromatic rings are called carotenes [23]. These rings are located at the both ends of the four isoprene molecules. Thus, there are two hydroaromatic rings in each carotene molecule. These hydrocarbons are called ionospheric rings and have three ionone rings. These are α-, β-, and pseudo-ionone rings. α- and β-ionone rings are closed rings and include one double bond. The position of the double bond α is different in the β-ionone rings and pseudo rings. The pseudo-ionone ring includes two double bonds [3].

Carotene is a biochemically synthesized terpene from eight isoprene units [4, 8]. There are three main types, α-carotene, β-carotene, and γ-carotene. The carotenoids are precursors of vitamin A and converted into vitamin A in the body. From α-carotene and γ-carotene, one molecule of vitamin A while from β-carotene two molecules of vitamin A are synthesized [27–29].

α-Carotene molecule has β-ionone ring at one end and α-ionone ring at the other end. There are four molecules of polymerized isoprene molecules between them. The structure of vitamin A is like half of an α-carotene. Vitamin A has a β-ionone ring at one end and two isoprene molecules attached to it [27]. The second most common form of carotene, α-carotene, is found mostly in carrots, sweet potatoes, squash, tomatoes, red peppers [30], and dark green vegetables [26].

**Figure 3.** Classification of carotenoids according to their structures.

β-Carotene is a fat-soluble provitamin. Its active form is vitamin A [31]. The difference of β from α is that it carries the β-ionone ring at both ends. When β-carotene is divided into two molecules, vitamin A is synthesized [29]. It is found in fruits, cereals, vegetables (carrots, green plants, pumpkin, spinach), and oils [32].

γ-Carotene includes β-ionone ring at one end and the pseudo-ionone ring at the other end, and when the molecule is split, one molecule of vitamin A is synthesized [3].

Lycopene is an aliphatic carotenoid [33]. Lycopene is found among tomatoes, watermelons, pink grapefruit, and rosehip. Since lycopene dissolves in the oil, the presence of oils greatly increases its absorption by the digestive system [34].

Phytoene is a 40-carbon intermediate in the biosynthesis of phytoene carotenoids. Phytoene is a symmetric molecule containing three conjugated double bonds [35].

Phytofluene is an orange-colored carotenoid pigment found naturally in tomatoes and other vegetables [36].

#### *2.4.2. Xanthophylls*

β-Carotene is a fat-soluble provitamin. Its active form is vitamin A [31]. The difference of β from α is that it carries the β-ionone ring at both ends. When β-carotene is divided into two molecules, vitamin A is synthesized [29]. It is found in fruits, cereals, vegetables (carrots,

γ-Carotene includes β-ionone ring at one end and the pseudo-ionone ring at the other end,

Lycopene is an aliphatic carotenoid [33]. Lycopene is found among tomatoes, watermelons, pink grapefruit, and rosehip. Since lycopene dissolves in the oil, the presence of oils greatly

Phytoene is a 40-carbon intermediate in the biosynthesis of phytoene carotenoids. Phytoene is

Phytofluene is an orange-colored carotenoid pigment found naturally in tomatoes and other

and when the molecule is split, one molecule of vitamin A is synthesized [3].

a symmetric molecule containing three conjugated double bonds [35].

green plants, pumpkin, spinach), and oils [32].

**Figure 3.** Classification of carotenoids according to their structures.

increases its absorption by the digestive system [34].

vegetables [36].

56 Carotenoids

Xanthophylls contain oxygen atoms and are yellow pigments commonly found in nature [37].

Lutein is a dihydroxy-carotene formed from carotenoids with an alcohol group containing hydroaromatic α structure [38]. Its both ionone rings carry hydroxyl groups [39].  That yellowcolored lutein found in plants is an organic colorant on leaves of green vegetables, such as spinach and black pepper. It is generally found in covalent interactions within fatty acids [20].

The chemical formulas of lutein and zeaxanthin are the same; in other words, they are isomers, but they are not stereoisomers. The difference between the two is the position of a double bond in the end ring [17, 39].

Zeaxanthin is one of the most common carotenoid alcohols found in the nature. It is a pigment that gives its color to maize, saffron, and many other plants. When the zeaxanthin breaks down, picrocrocin, which is responsible for the taste and aroma of the saffron, forms [41].

β-Cryptoxanthin carotenoid has an alcohol group having a hydroaromatic structure including an OH group in one of its ionic rings. Since the other ring is the β-ionone ring, one molecule of vitamin A can be synthesized from it [27].

β-Cryptoxanthin is a natural carotenoid pigment. β-Cryptoxanthin is found in fruits and vegetables, such as mandarin, red pepper, and zucchini, and has important functions for human health. β-Cryptoxanthin is closely related to β-carotene. Only one OH group was added to β-cryptoxanthin. Although β-carotene is present in large quantities in a large number of fruits and vegetables, β-cryptoxanthin is present in small number of food sources but at high concentrations [42].

Astaxanthin is a keto-carotenoid and is a zeaxanthin metabolite, containing both hydroxyl and ketone functional groups [17, 43]. Astaxanthin is found in microalgae, yeast, salmon, trout, shrimp, shellfish, and some birds' feathers [44].

Fucoxantin, found in the chloroplasts of algae and mosses, gives them their brown or olive green color [45].

### **3. Antioxidant functions of carotenoids at molecular level for health and toxicity**

The interest in carotenoids found in plants over the last years is not only due to their A provitamin activity but also due to their reduction of oxidative stress in the organism by capturing oxygen radicals, that is, their antioxidant effects [46]. Free oxygen radicals play an important role in the stress-related tissue damage and pathogenesis of inflammation. The imbalance between protective and damaging mechanisms results in acute inflammation accompanied by neutrophil infiltration [47, 48]. Superoxide radicals formed by neutrophils react with lipids and cause lipid peroxidation [49–51]. Carotenoids can inhibit active radicals by transferring electrons, giving hydrogen atoms to radicals or attaching to radicals [16].

The number of conjugate double bonds in their structure is closely related to the superoxide inhibitory effect of carotenoids [52, 53]. Carotenoids could remove singlet oxygen and peroxyl radicals from reaction medium and also prevent their formation [53–55]. It is stated that carotenoids can inhibit cell renewal and transformation and regulate gene expression that plays a role in the formation of certain types of cancer. On the other hand, in some studies it has been shown that carotenoids could stimulate cancer in some cases. For example, it has been reported that in smokers, synthetic β-carotene does not create protective activity against lung cancer and cardiovascular diseases and even it fastens the progression of an aforementioned diseases [56–58].

The activity of carotenoids as antioxidants also depends on their interaction with other antioxidants, such as vitamins E and C [58]. In addition, some carotenoids and their metabolites activate the nuclear factor-erythroid 2-related factor-2 (Nrf2) transcription factor, which triggers antioxidant gene expression in certain cells and tissues [58, 59]. Thus, a number of chronic diseases characterized by oxidative stress, inflammation, and impaired mitochondrial function have been reported to reduce Nrf2 expression in some animal models. Elevation of Nrf2 has been shown to be effective in the prevention and treatment of many chronic inflammatory diseases, including various cardiovascular, renal, or pulmonary diseases; toxic liver damage; metabolic syndrome; sepsis; autoimmune disorders; inflammatory bowel disease; and HIV infection [58]. Prevention of low-density lipoproteins (LDL) oxidation by carotenoids has been suggested to be the basis of carotenoids' protective activity against coronary vascular disease [26].

The signaling pathways and molecules influenced by carotenoids to prevent various diseases, such as cancer and cardiovascular diseases, involve growth factor signaling members, cell cycle-associated proteins, differentiation-related proteins, retinoid-like receptors, antioxidant response element, nuclear receptors, AP-1 transcriptional complex, the Wnt/β-catenin pathway, angiogenic proteins, and inflammatory cytokines. During the treatment of cardiovascular and eye diseases and cancer, the dose and the exposure time of β-carotene, lycopene, lutein, and zeaxanthin have been reported to be crucial [37].

α-Carotene intake might decrease the development of non-Hodgkin lymphoma [60]. α-Carotene concentration in the blood may be associated with the development of various cancers [30].

β-Carotene, which is accepted as a provitamin of vitamin A (retinol) which is required for the fulfillment of visual functions [61], effects the oxidative damage that formed on cellular lipids, proteins, and DNA as a result of sunlight and causes the formation of erythema, premature aging of the skin, development of photodermatitis, and skin cancer. β-Carotene protects the skin from harmful effects of UV light by its prevention of reactive oxygen species formation and anti-inflammatory properties [62, 63].

Studies have shown that β-carotene's provitaminase activity or antioxidant properties prevent diseases, such as arthrosclerosis, cataracts, multiple sclerosis, and some types of cancers [64]. Moreover in recent studies, β-carotene has been proposed to decrease cell proliferation and induce apoptosis of various cancer cell lines by inhibiting calcium-/calmodulin-dependent protein kinase IV [65]. In another study, β-carotene has been reported to have anticancer stem cell actions on neuroblastoma, and this anticancer action is enhanced by retinoic acid receptor β [66].

The number of conjugate double bonds in their structure is closely related to the superoxide inhibitory effect of carotenoids [52, 53]. Carotenoids could remove singlet oxygen and peroxyl radicals from reaction medium and also prevent their formation [53–55]. It is stated that carotenoids can inhibit cell renewal and transformation and regulate gene expression that plays a role in the formation of certain types of cancer. On the other hand, in some studies it has been shown that carotenoids could stimulate cancer in some cases. For example, it has been reported that in smokers, synthetic β-carotene does not create protective activity against lung cancer and cardiovascular diseases and even it fastens the progression of an aforementioned

The activity of carotenoids as antioxidants also depends on their interaction with other antioxidants, such as vitamins E and C [58]. In addition, some carotenoids and their metabolites activate the nuclear factor-erythroid 2-related factor-2 (Nrf2) transcription factor, which triggers antioxidant gene expression in certain cells and tissues [58, 59]. Thus, a number of chronic diseases characterized by oxidative stress, inflammation, and impaired mitochondrial function have been reported to reduce Nrf2 expression in some animal models. Elevation of Nrf2 has been shown to be effective in the prevention and treatment of many chronic inflammatory diseases, including various cardiovascular, renal, or pulmonary diseases; toxic liver damage; metabolic syndrome; sepsis; autoimmune disorders; inflammatory bowel disease; and HIV infection [58]. Prevention of low-density lipoproteins (LDL) oxidation by carotenoids has been suggested to be the basis of carotenoids' protective activity against coronary

The signaling pathways and molecules influenced by carotenoids to prevent various diseases, such as cancer and cardiovascular diseases, involve growth factor signaling members, cell cycle-associated proteins, differentiation-related proteins, retinoid-like receptors, antioxidant response element, nuclear receptors, AP-1 transcriptional complex, the Wnt/β-catenin pathway, angiogenic proteins, and inflammatory cytokines. During the treatment of cardiovascular and eye diseases and cancer, the dose and the exposure time of β-carotene, lycopene,

α-Carotene intake might decrease the development of non-Hodgkin lymphoma [60]. α-Carotene concentration in the blood may be associated with the development of various

β-Carotene, which is accepted as a provitamin of vitamin A (retinol) which is required for the fulfillment of visual functions [61], effects the oxidative damage that formed on cellular lipids, proteins, and DNA as a result of sunlight and causes the formation of erythema, premature aging of the skin, development of photodermatitis, and skin cancer. β-Carotene protects the skin from harmful effects of UV light by its prevention of reactive oxygen species formation

Studies have shown that β-carotene's provitaminase activity or antioxidant properties prevent diseases, such as arthrosclerosis, cataracts, multiple sclerosis, and some types of cancers [64]. Moreover in recent studies, β-carotene has been proposed to decrease cell proliferation and induce apoptosis of various cancer cell lines by inhibiting calcium-/calmodulin-dependent

lutein, and zeaxanthin have been reported to be crucial [37].

and anti-inflammatory properties [62, 63].

diseases [56–58].

58 Carotenoids

vascular disease [26].

cancers [30].

Lycopene, synthesized by many plants and microorganisms, is an antioxidant that cannot be synthesized by animals and humans [67]. Conjugated dienes are active in creating antioxidant activity [46], and lycopene is reported to have a higher antioxidant capacity than other carotenoids, and in particular among carotenoids, lycopene inhibits the risk of prostate cancer [52, 68].

The use of lycopene can reduce the risk of cardiovascular disease, diabetes, osteoporosis, and prostate, esophagus, colorectal, and mouth cancer risk. Recent studies indicate that lycopene intake has protective functions against cardiovascular diseases by lowering high-density lipoprotein (HDL)-associated inflammation [69]. It was proposed that there is an inverse association between the occurrence of pancreatic cancer and dietary lycopene intake together with vitamin A and β-carotene [70]. β-Carotene 9′,10′-oxygenase which is a key enzyme for the metabolism of lycopene has been proposed to have an important roles to prevent prostate cancer progression by inhibiting NF-κB signaling [71].

In neurodegenerative diseases, lycopene has been reported to increase permeability of bloodbrain barrier, and a significant reduction in lycopene levels in diseases, such as Parkinson's disease and vascular dementia, has been observed. It has also been suggested that lycopene bestow protection against amyotrophic lateral sclerosis (ALS) impairment in humans [67].

The phytoene and phytofluene found in the diet accumulate in the human skin [72]. The dehydration of these carotenoids has a protective effect on the skin due to its UV absorber, antioxidant, and anti-inflammatory properties [73].

Zeaxanthin is one of two carotenoids found in the retina. Zeaxanthin mostly found at the center of the macula and lutein mostly found at the peripheral retina [39, 74]. Lutein and zeaxanthin are responsible for the formation of yellow pigment in the retina. Yellow pigments play an active role in protecting the eye from light and can prevent retinal damage [56].

β-Cryptoxanthin acts as a chemopreventive agent against lung cancer. β-Cryptoxanthin has been reported to decrease lung cancer through downregulating neuronal nicotinic acetylcholine receptor α7/PI3K signaling pathway [75]. In addition to lung cancer, β-cryptoxanthin enhances the action of a chemotherapeutic agent, oxaliplatin, to treat colon cancer [76]. Moreover, high lycopene and β-cryptoxanthin including diet might protect against aggressive prostate cancer [77].

Astaxanthin has an important role in the treatment and prevention of certain diseases by its antitumor properties and protection against free radicals, oxidation of basic polyunsaturated fatty acids, and UV light effect on cell membranes [20, 78]. For example, high concentrations of astaxanthin may suppress mammary carcinoma [79]. In addition, astaxanthin has beneficial health effects against the formation of prostate carcinogenesis and tumor progression by reactivating the expression of Nrf2 and Nrf2-target genes through epigenetic modification and chromatin remodeling [80]. Astaxanthin has been shown to have antitumorigenic and anti-inflammatory effects on human lung cancer cell lines by inhibiting ERK1/2 activity [81].

In vitro studies indicate that fucoxanthin stimulates apoptosis and decreases proliferation and migration in glioma cancer cell lines U87 and U251 through Akt/mTOR and p38 pathway inhibition [82]. Fucoxanthin intake may have health benefits for the treatment of Alzheimer's disease by inhibiting acetylcholinesterase and enhancing brain-derived neurotrophic factor expression [83].

Supplementation of carotenoids by humans may have some beneficial biological actions against the formation of numerous diseases, such as cancer, cardiovascular diseases, or their skin. In addition to nutrient supplements in humans, carotenoids have applications in animal feed. Optimal health promoting actions of carotenoids depend on their proper doses, lengths of treatment, and combinations of carotenoids to maximize their effects. In this book chapter, latest findings on the biochemical and antioxidant activities of main carotenoids and their possible mechanisms of action will be presented.

#### **Author details**

#### Oguz Merhan

Address all correspondence to<EMAIL_ADDRESS>
Department of Biochemistry, Faculty of Veterinary, Kafkas University, Kars, Turkey

#### **References**

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In vitro studies indicate that fucoxanthin stimulates apoptosis and decreases proliferation and migration in glioma cancer cell lines U87 and U251 through Akt/mTOR and p38 pathway inhibition [82]. Fucoxanthin intake may have health benefits for the treatment of Alzheimer's disease by inhibiting acetylcholinesterase and enhancing brain-derived neurotrophic factor

Supplementation of carotenoids by humans may have some beneficial biological actions against the formation of numerous diseases, such as cancer, cardiovascular diseases, or their skin. In addition to nutrient supplements in humans, carotenoids have applications in animal feed. Optimal health promoting actions of carotenoids depend on their proper doses, lengths of treatment, and combinations of carotenoids to maximize their effects. In this book chapter, latest findings on the biochemical and antioxidant activities of main carotenoids and their

expression [83].

60 Carotenoids

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Oguz Merhan

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## **β‐Carotene and Free Radical Reactions with Nitrogen Oxides**

Sara N. Mendiara and Luis J. Perissinotti

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67683

#### **Abstract**

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The following presentation is based on experimental work we have already developed and published. We investigated the nitrogen oxides in different solvents and analyzed their reaction with β‐carotene. The electron paramagnetic resonance spectroscopy (EPR) and ultraviolet and visible (UV‐vis) spectroscopy were applied to investigate the reaction of β‐carotene with nitrogen dioxide and nitric oxide in both pure dioxane and dioxane/ water solvent. Free radicals were detected and evaluated with the EPR technique, which is highly selective and sensitive. A reaction mechanism was proposed on the basis of the experimental kinetic and EPR results. The validity of the mechanism was checked by applying simulation set up conditions that reproduced the results achieved. The radical intermediates proposed in the reaction: the β‐carotene neutral radicals and the cyclic nitroxide neutral radicals were theoretically studied. For that purpose, the density functional theory (DFT) level was applied, selecting the most suitable method, the unre‐ stricted Becke‐style 3‐parameter with the Lee‐Yang‐Parr correlation functional (UB3LYP) and the 6‐31G(d) basis sets (d orbital functions). We developed an appropriate discussion on the importance of carotenoids compounds and their reactions in biological media. Also, we evaluated the role and the possible reactions of nitroxide intermediates.

**Keywords:** β‐carotene, EPR, neutral radicals, nitrogen dioxide, nitroxides

#### **1. Introduction**

The following presentation is based on experimental work we have already developed and published. We investigated the behavior of nitrogen oxides in different solvents and analyzed the reaction of nitrogen oxides with β‐carotene.

The reaction of free radicals with carotenoids and the properties of the carotenoid‐free radicals formed are of widespread interest because of their potential role in biological systems. We have

carried out our work only with β‐carotene, an unsaturated and extensively conjugated hydro‐ carbon. In vitro studies had shown the potential of carotenoids to act as free‐radical scaven‐ gers. Nitrogen oxides, such as nitric oxide (NO) and the nitrogen dioxide (NO2 ), constitute a source of free radicals; they are species that have an unpaired electron. It is expected that nitrogen oxides may react in some important way with carotenoids and their radical inter‐ mediates. It is important to note that in our preliminary investigations, when mixtures of NO and NO2 were added to certain organic purified compounds, compounds of the type of the nitroxides were detected using the technique of EPR [1].

 However, humans and primates accumulate them in several tis‐ sues. Carotenoids are absorbed in the intestinal mucosa like other lipophilic components. β‐carotene molecule is geared in the lipophilic membranes interacting, through van der Waals forces, with the hydrocarbon chains of the lipids [2–5]. Carotenoids can be traced in the cel‐ lular cytoplasm where they can interact with different components, including the nitrogen oxides. It is also important to take into account the metabolism of carotenoids; β‐carotene is particularly hydrophobic and it is reasonable to hypothesize that it would need to be trans‐ formed to more polar compounds in order to be excreted via urine [6]. As well, lutein and β‐carotene were measured in human brain tissue and related to better cognition in octogenar‐ ians. The protective effect may not merely be an antioxidant effect given that α‐tocopherol was less related to cognition than carotenoids [7]. The first report of the presence of β‐carotene in the brain was in 1976, the patient was taking a high‐dose β‐carotene as a treatment and the carotenoid was measured within whole sections of the cerebrum [8].

We understood that the research might be quite profitable. The purpose of our work was to study and to enlighten the following problems:

#### **2. Development: (a), (b), and (c): conclusions and perspective**

#### **2.1. (a) The behavior of the nitrogen oxides NO and NO2 in some solvents**

Luckily, we had started the research developing a detailed study of solutions of nitrogen oxides in several solvents [1]. Those solutions were monitored with the following spectro‐ scopic techniques: ultraviolet and visible (UV‐vis) and electron paramagnetic resonance (EPR). The research allowed us to know what solvents can be used because they do not react with nitrogen oxides. We learned how to develop a careful control of the solvents. The knowl‐ edge acquired in this first work allowed us to follow, understand, and manage to clear up the results later finally achieved from the β‐carotene assays. Therefore, the solvent dioxane and other later used in our research were adequately tested. It was also verified that they did not react with the nitrogen oxides, NO2 and NO, even after prolonged times of observation in EPR (unlike what happened with hexane) [9].

carried out our work only with β‐carotene, an unsaturated and extensively conjugated hydro‐ carbon. In vitro studies had shown the potential of carotenoids to act as free‐radical scaven‐

a source of free radicals; they are species that have an unpaired electron. It is expected that nitrogen oxides may react in some important way with carotenoids and their radical inter‐ mediates. It is important to note that in our preliminary investigations, when mixtures of NO

Carotenoids are a family of pigmented compounds that are synthesized by plants and micro‐ organisms but not animals. However, humans and primates accumulate them in several tis‐ sues. Carotenoids are absorbed in the intestinal mucosa like other lipophilic components. β‐carotene molecule is geared in the lipophilic membranes interacting, through van der Waals forces, with the hydrocarbon chains of the lipids [2–5]. Carotenoids can be traced in the cel‐ lular cytoplasm where they can interact with different components, including the nitrogen oxides. It is also important to take into account the metabolism of carotenoids; β‐carotene is particularly hydrophobic and it is reasonable to hypothesize that it would need to be trans‐ formed to more polar compounds in order to be excreted via urine [6]. As well, lutein and β‐carotene were measured in human brain tissue and related to better cognition in octogenar‐ ians. The protective effect may not merely be an antioxidant effect given that α‐tocopherol was less related to cognition than carotenoids [7]. The first report of the presence of β‐carotene in the brain was in 1976, the patient was taking a high‐dose β‐carotene as a treatment and the

We understood that the research might be quite profitable. The purpose of our work was to

(c) Theoretical evaluation of the intermediates proposed: the acyclic β‐carotene neutral radi‐ cals, the acyclic nitrous β‐carotene neutral radicals, and the cyclic nitroxide neutral radi‐

Luckily, we had started the research developing a detailed study of solutions of nitrogen oxides in several solvents [1]. Those solutions were monitored with the following spectro‐ scopic techniques: ultraviolet and visible (UV‐vis) and electron paramagnetic resonance (EPR). The research allowed us to know what solvents can be used because they do not react with nitrogen oxides. We learned how to develop a careful control of the solvents. The knowl‐ edge acquired in this first work allowed us to follow, understand, and manage to clear up the

(b) The interaction and reaction among nitrogen oxides and β‐carotene (2009) [9].

**2. Development: (a), (b), and (c): conclusions and perspective**

in some solvents (2001) [1].

 **in some solvents**

were added to certain organic purified compounds, compounds of the type of the

), constitute

gers. Nitrogen oxides, such as nitric oxide (NO) and the nitrogen dioxide (NO2

nitroxides were detected using the technique of EPR [1].

carotenoid was measured within whole sections of the cerebrum [8].

study and to enlighten the following problems:

cals (2015) [10].

(a) The behavior of nitrogen oxides: NO and NO2

**2.1. (a) The behavior of the nitrogen oxides NO and NO2**

and NO2

68 Carotenoids

NO2 solutions were very carefully followed. In this chapter, we show only highlights from the developed research. Our first manuscript on nitrogen oxides, carried out in 2001, showed experimental and technical research in detail. **Figure 1** displays a set of EPR spectral records of the radical NO2 at environmental temperature. Solutions of NO2 were prepared in different

**Figure 1.** EPR spectra of NO2 , at 290 K, in: (1) gas‐phase; (2) hexane; (3) carbon tetrachloride; and (4) acetone. General instrumental settings: microwave power, 18,000 mW; attenuation, 9 dB; modulator frequency, 100 kHz; modulation amplitude, 2.5 G; time constant or response time, 0.2 s; scan rate, 200 s. The recipient was previously flushed with argon.

media: hexane, benzene, carbon tetrachloride, acetone, and water. You can see that each spectrum do not differ considerably from the others. The signal is not resolved, due to the movement of the free radical and to the collisions that usually take place at the working tem‐ perature. Noticed that our research was always carried out under argon atmosphere [1, 9].

In **Table 1**, we can see the values of g, the spectroscopic factor and of ΔB, the total spectrum line width. Those data were extracted or calculated from **Figure 1**. We can also observe that the val‐ ues in non‐polar media are near the result measured in the gaseous phase. The general instru‐ mental settings were microwave power, 18.000 mW; attenuation, 9 dB; modulator frequency, 100 kHz; modulation amplitude, 2.5 G; time constant or response time, 0.2 s; scan rate, 200 s.

We also prepared NO solutions. The preparation and handling of solutions of NO were per‐ formed under strict inert atmosphere. Even though precautions were employed to exclude oxygen from the system, a fingerprint UV‐vis spectrum assigned to the presence of NO2 /N2 O4 / HNO2 as impurity was sometimes observed. UV‐vis spectra of solutions of NO/NO2 in different solvents were analyzed. Those Spectra shown splits in some solvents. Some authors had inter‐ preted them as the spectrum of NO [11]. However, it was confirmed that those UV‐vis spectra were obtained in polar solvents and could be attributed to the formation of HNO2 [1, 12].

Usually, one has to control nitrogen dioxide samples, recording the UV‐vis spectrum and the corresponding EPR spectrum. The EPR spectrum of NO2 is looked for in the following field interval: 1000–2000 Gauss, as you can observe in **Figure 1**. If we want to search for nitroxides, we must work in the following scan range: 50–200 Gauss. The EPR spectrum of NO was not detected in our field interval work.

When NO was dissolved in hexane, nitroxide intermediates were detected after 20 minutes or more, the radical was persistent. Probably no reaction took place when pure NO was present, but a trace amount of NO2 initiated the reaction [13, 14].

In 2003, we carried out the measurement of the N2 O4 dissociation constant (N2 O4 /2 NO2 ) in some solvents [15]. The N2 O4 dissociation constant measured in hexane, carbon tetrachloride,

\*Results obtained in the present work. Instrumental settings are shown in **Figure 1**.

a g values were determined with reference to a diphenylpicrylhydrazyl standard, g = 2.0036. The absolute errors are 0.002–0.004.

**Table 1.** EPR spectroscopic data of NO2 radical at 290 K: spectroscopic factor, g, and total line width, ΔB\* . and chloroform compares approximately with the values calculated by some previous authors [16–18]. The techniques usually applied were colorimetric and spectrophotometric ones. As the absorption along the appropriate wavelength range was small, the errors in mea‐ surements at low concentrations were considerably large. The EPR technique has also quan‐ tification errors; however, the method is more reliable and the radical is directly detected. As far as we were aware, this was the first attempt to measure this equilibrium with the EPR technique.

#### **2.2. (b) The interaction and reaction among nitrogen oxides and β‐carotene: kinetic analysis and the corresponding modeling work**

#### *2.2.1. UV*‐*vis measurements*

media: hexane, benzene, carbon tetrachloride, acetone, and water. You can see that each spectrum do not differ considerably from the others. The signal is not resolved, due to the movement of the free radical and to the collisions that usually take place at the working tem‐ perature. Noticed that our research was always carried out under argon atmosphere [1, 9].

In **Table 1**, we can see the values of g, the spectroscopic factor and of ΔB, the total spectrum line width. Those data were extracted or calculated from **Figure 1**. We can also observe that the val‐ ues in non‐polar media are near the result measured in the gaseous phase. The general instru‐ mental settings were microwave power, 18.000 mW; attenuation, 9 dB; modulator frequency, 100 kHz; modulation amplitude, 2.5 G; time constant or response time, 0.2 s; scan rate, 200 s. We also prepared NO solutions. The preparation and handling of solutions of NO were per‐ formed under strict inert atmosphere. Even though precautions were employed to exclude

oxygen from the system, a fingerprint UV‐vis spectrum assigned to the presence of NO2

as impurity was sometimes observed. UV‐vis spectra of solutions of NO/NO2

were obtained in polar solvents and could be attributed to the formation of HNO2

initiated the reaction [13, 14].

corresponding EPR spectrum. The EPR spectrum of NO2

In 2003, we carried out the measurement of the N2

O4

**Medium g a ΔB, Gauss** Gas phase 1.999 1190 ± 58 Hexane 1.981 1194 ± 40 Benzene 1.985 563 ± 20 Carbon tetrachloride 1.978 897 ± 34 Acetone 2.185 499 ± 23 Water 2.176 502 ± 20 \*Results obtained in the present work. Instrumental settings are shown in **Figure 1**.

detected in our field interval work.

but a trace amount of NO2

some solvents [15]. The N2

**Table 1.** EPR spectroscopic data of NO2

solvents were analyzed. Those Spectra shown splits in some solvents. Some authors had inter‐ preted them as the spectrum of NO [11]. However, it was confirmed that those UV‐vis spectra

Usually, one has to control nitrogen dioxide samples, recording the UV‐vis spectrum and the

interval: 1000–2000 Gauss, as you can observe in **Figure 1**. If we want to search for nitroxides, we must work in the following scan range: 50–200 Gauss. The EPR spectrum of NO was not

When NO was dissolved in hexane, nitroxide intermediates were detected after 20 minutes or more, the radical was persistent. Probably no reaction took place when pure NO was present,

g values were determined with reference to a diphenylpicrylhydrazyl standard, g = 2.0036. The absolute errors are

radical at 290 K: spectroscopic factor, g, and total line width, ΔB\*

O4

HNO2

70 Carotenoids

a

0.002–0.004.

/N2 O4 /

in different

[1, 12].

O4 /2 NO2

.

) in

is looked for in the following field

dissociation constant (N2

dissociation constant measured in hexane, carbon tetrachloride,

β‐Carotene, a non‐polar molecule, is insoluble in polar solvents like water. We desired to investigate the reaction of β‐carotene with the nitrogen oxides generated in situ from aqueous solutions of sodium nitrite and sulfuric acid. In order to achieve our purpose, we prepared solutions of β‐carotene in pure and adequately checked dioxane solvent, under argon atmosphere and constant temperature. The dioxane‐water mixtures remained homogeneous and so the UV‐vis spectroscopy studies could be applied. Other aprotic sol‐ vents easily cause cloudiness when water is added, then UV‐vis measurements cannot be carried out.

#### *2.2.2. EPR measurements*

EPR measurements were carried out with the β‐carotene solution in pure dioxane plus an aliquot of NO and NO2 in pure dioxane. The solutions in dioxane had low concentration of the persistent radical, besides the triplet‐type signal was recorded during 5 days. In pure dioxane, we only developed a qualitative approach. On the other hand, we could design a quantitative study carried out with β‐carotene solution in pure dioxane plus an aliquot of the dioxane/ water solution with the nitrogen oxides formed in situ from sodium nitrite in an acid medium. Also the formation and decay of the persistent intermediates was monitored and shown in **Figure 2**. Dioxane solvent was tested, and no EPR signal was detected even after prolonged times of observation.

#### *2.2.3. Kinetic measurements*

Kinetic measurements were developed in dioxane/water solvent at 298 K. All the measure‐ ments were successfully simulated with the software for chemical kinetics, following the reac‐ tion path proposed in **Figure 3**. In **Table 2**, each reaction was described and the corresponding kinetic constant assigned. Unfortunately, in our 2009 manuscript, the sixth equation had one mistake, we must write *P*1 instead of *P*2 [9]. In the 2015 manuscript, the rate constants *k*i (i = 1 to 7) were not written above the arrows [10].

In **Figure 3** and **Table 2**, we observed that whenever both NO and NO2 were present, abstrac‐ tion took place and nitroxides formed. Furthermore, it has already been shown that NO is less

**Figure 2.** Formation and decay of a persistent type radical generated from a solution of β‐carotene, 1 × 10−2M, and nitrite anion, 1 × 10−1M in dioxane:water (xdioxane = 0.65) and in the presence of an acid medium, pH = 2 at 298 K. The gray curve and symbols represent the values obtained from the kinetic simulation process; the black square symbols represent our experimental values. After 20 days, we were still able to measure a radical concentration of 3.5 × 10−5 M, not shown in the graph. The reactions were carried out under argon atmosphere.

**Figure 3.** General reaction pathway proposed. The figure shows, in first place, the abstraction reactions of allylic hydrogens from β‐carotene by nitrogen dioxide radicals and the formation of the products *P*1 . *P*1 represents different possible β‐carotene neutral radicals. *P*2 represents the nitrous β‐carotene formed with nitric oxide radicals. *P*3 represents the products formed with nitrogen dioxide radicals, not studied in this work. *P*4 represents the neutral radicals that are formed after the reaction of *P*2 with nitrogen dioxide radicals, another abstraction reaction of allylic hydrogens has taken place. Finally, when *P*4 generates an internal ring, a radical *P*4(nitroxide) comes up.

#### **Kinetic analysis in acid mediuma**

a T = 298K. *k*1 , *k*−1, and *k*2 were already known. The rate constants *k*4 and *k*5 were considered diffusion controlled. The rate constants *k*3 , *k*6 , and *k*7 were deduced from our modeling work. The kinetic simulation reproduced successfully the experimental kinetics results [9].

*P*1 represents a possible allylic‐type radical. *P*2 represents the possible isomers molecules of nitrous carotene. *P*4 represents the possible allylic‐type radicals obtained from *P*2 . When a radical *P*4 cycles internally a radical *P*4 *(nitroxide)* is formed. *P*3 represents the products formed with nitrogen dioxide, not studied in this work.

**Table 2.** Reaction of β‐carotene and nitrite in dioxane/water solution.

reactive and less efficient in the abstraction of a hydrogen atom than NO2 [9]. We can compare the following bond dissociation energies (BDE) values at 298 K:

• BDE (H‐NO) = 195.35 ± 0.25 kJ.mol−1

**Figure 2.** Formation and decay of a persistent type radical generated from a solution of β‐carotene, 1 × 10−2M, and nitrite anion, 1 × 10−1M in dioxane:water (xdioxane = 0.65) and in the presence of an acid medium, pH = 2 at 298 K. The gray curve and symbols represent the values obtained from the kinetic simulation process; the black square symbols represent our experimental values. After 20 days, we were still able to measure a radical concentration of 3.5 × 10−5 M, not shown in the

**Figure 3.** General reaction pathway proposed. The figure shows, in first place, the abstraction reactions of allylic

represents the nitrous β‐carotene formed with nitric oxide radicals. *P*3

with nitrogen dioxide radicals, another abstraction reaction of allylic hydrogens has taken

. *P*1

represents the neutral radicals that are

represents different

represents

hydrogens from β‐carotene by nitrogen dioxide radicals and the formation of the products *P*1

generates an internal ring, a radical *P*4(nitroxide) comes up.

the products formed with nitrogen dioxide radicals, not studied in this work. *P*4

graph. The reactions were carried out under argon atmosphere.

possible β‐carotene neutral radicals. *P*2

formed after the reaction of *P*2

place. Finally, when *P*4

72 Carotenoids

• BDE (H‐NO2 or H‐ONO) = 327.6 ± 2.1 kJ.mol−1

We proposed the formation of β‐carotene neutral radicals that in the presence of nitrogen oxides, originated persistent radical intermediates. The reactions were carried out under argon atmosphere because of the reactivity of triplet oxygen; otherwise oxygenated com‐ pounds would be formed with the carotenoid neutral radicals.

We followed and measured the difficult kinetic of the mechanism proposed. The kinetic of the reaction depended on β‐carotene, nitrite, and acid concentrations. We experimentally verified that the β‐carotene followed a first‐order decay and we measured the corresponding pseudo‐ first‐order‐constant [9].

In **Table 2**, a set of reactions represents the mechanism of the reaction. It is assumed that the system always behaves as if it has reached the acid‐base balance.

*P*4 was considered as *P*4 *(nitroxide)*. We ran a non‐commercial simulation program of chemi‐ cal kinetics. The validity of the proposed mechanism was therefore tested by numerical integration.

The values of *k*1 *, k*−1*, and k*2 were extracted from the literature*.* The rate constant *k*3 was adjusted following the UV‐vis decay of β‐carotene. The rate constants *k*6 and *k*7 were involved in the formation of the persistent radicals and were adjusted to the EPR results. The rate con‐ stants *k*4 and *k*5 were the recombination reactions between *P*1 radicals and nitrogen diox‐ ide and nitric oxide radicals. We calculated the following values: *k*4 = 1.4×1010M−1s−1 and *k*5 = 1.1×1010M−1s−1, by applying the equations of Smoluchoski, Stokes, and Einstein [19]. The calculations were developed taking into account the viscosity of the mixture dioxane‐water, η25°C = 1.42 × 10−3 Pa.s and the molecular size of the NO, NO2 and β‐carotene neutral radi‐ cals [20]. Actually, these constants were smaller because the nitrogen oxides reacted at some selected regions of the β‐carotene neutral radicals. It was confirmed that changes of *k*4 and *k*5 in the order of 107 to 1011 had no significant effect upon the simulation results, which match very well with our experimental results. So the value of 1.0 × 109 M−1s−1 was selected and shown for *k*4 and *k*5 in **Table 2**. *P*3 represents the products formed with nitrogen dioxide, not studied in this work.

We considered that the nitrous acid was proportional to the nitrite anion through the acid‐ base equilibrium [9]. The simulation results at 298 K reproduced quite well the experimental decay of β‐carotene in the range near the half‐life at pH = 5.3. The formation and the decay of the persistent radical intermediate at pH = 2.2 were also very well reproduced. For example, we showed *Case (a)* and *Case (b)* through which we tested the experiments carried out.

*Case (a)* Initial concentrations: β‐carotene, 8.0 × 10−6M; nitrite anion, 9.3 × 10−3M; pH, 5.3

The simulation program showed that the system achieved approximately the following steady state order of concentrations: [NO2 ] ≅ 10−8 to 10−9M [NO] ≅ 10−6 to 10−7M [*P*1 ] ≅ 10−10M [*P*2 ] ≅ 10−6M

The pH achieved in the simulation run remained almost constant: 5.30 ± 0.02.

The maximum radical concentration reached was: [*P*4 + *P*4(nitroxide)] ≅ 10−11M, in agreement with our EPR results.

The persistent radicals were not detected; evidently, our equipment had not enough sensibility.

*Case (b)* Initial concentrations: β‐Carotene, 1.0 × 10−2M; nitrite anion, 1.0 × 10−1M; pH, 2.2.

The system achieved approximately the following steady state concentrations order: [NO2 ] ≅ 10−7M [NO] ≅ 10−2M [*P*1 ] ≅ 0M [*P*2 ] ≅ 10−3M.

The pH achieved in the simulation run remained almost constant: 2.20 ± 0.03.

The maximum radical concentration reached was: [*P*4 + *P*4 *(nitroxide)*]=1.5x10−4M.

In agreement with our EPR results, 1.4 × 10−4M, see **Figure 2**.

After 20 days, the kinetics simulation delivered a radical concentration of 3.5 × 10−5M in per‐ fect agreement with our experimental results.

We can observe that the examples have quite different experimental conditions; however, they both work with the same set of rate constants.

In **Figure 2**, the experimental measurement of the formation and decay of the radical interme‐ diates was represented with black square symbols and the results from the kinetic simulation process were represented with gray symbols. **Figure 4** presents the recorded EPR spectrum of the radical intermediates. Although the spectrum may fit with a nitroxide‐type radical show‐ ing a hyperfine coupling constant, aN = 12.7 G, the spectrum exhibits an increment in the cen‐ tral line. This central increment could be attributed to the contribution of a related allylic‐type radical superimposed or to a set of related allylic‐type radicals. However, the kinetic analysis generated poor concentrations for *P*1 , which represented the allylic radical intermediates. In addition, finally, with the aid of the computational methods, the hypothesis of the formation of cyclic nitroxides was favored [10].

The values of *k*1

74 Carotenoids

stants *k*4 and *k*5

in the order of 107

studied in this work.

and *k*5

shown for *k*4

[*P*2

[NO2

] ≅ 10−6M

our EPR results.

*, k*−1*, and k*2

following the UV‐vis decay of β‐carotene. The rate constants *k*6

η25°C = 1.42 × 10−3 Pa.s and the molecular size of the NO, NO2

very well with our experimental results. So the value of 1.0 × 109

in **Table 2**. *P*3

steady state order of concentrations: [NO2

] ≅ 10−7M [NO] ≅ 10−2M [*P*1

The maximum radical concentration reached was: [*P*4

The maximum radical concentration reached was: [*P*4

fect agreement with our experimental results.

they both work with the same set of rate constants.

In agreement with our EPR results, 1.4 × 10−4M, see **Figure 2**.

ide and nitric oxide radicals. We calculated the following values: *k*4

were extracted from the literature*.* The rate constant *k*3

to 1011 had no significant effect upon the simulation results, which match

represents the products formed with nitrogen dioxide, not

] ≅ 10−8 to 10−9M [NO] ≅ 10−6 to 10−7M [*P*1

+ *P*4(nitroxide)] ≅ 10−11M, in agreement with

*(nitroxide)*]=1.5x10−4M.

formation of the persistent radicals and were adjusted to the EPR results. The rate con‐

= 1.1×1010M−1s−1, by applying the equations of Smoluchoski, Stokes, and Einstein [19]. The calculations were developed taking into account the viscosity of the mixture dioxane‐water,

cals [20]. Actually, these constants were smaller because the nitrogen oxides reacted at some

We considered that the nitrous acid was proportional to the nitrite anion through the acid‐ base equilibrium [9]. The simulation results at 298 K reproduced quite well the experimental decay of β‐carotene in the range near the half‐life at pH = 5.3. The formation and the decay of the persistent radical intermediate at pH = 2.2 were also very well reproduced. For example,

The simulation program showed that the system achieved approximately the following

The persistent radicals were not detected; evidently, our equipment had not enough sensibility.

The system achieved approximately the following steady state concentrations order:

] ≅ 10−3M.

After 20 days, the kinetics simulation delivered a radical concentration of 3.5 × 10−5M in per‐

We can observe that the examples have quite different experimental conditions; however,

+ *P*4

*Case (b)* Initial concentrations: β‐Carotene, 1.0 × 10−2M; nitrite anion, 1.0 × 10−1M; pH, 2.2.

we showed *Case (a)* and *Case (b)* through which we tested the experiments carried out. *Case (a)* Initial concentrations: β‐carotene, 8.0 × 10−6M; nitrite anion, 9.3 × 10−3M; pH, 5.3

The pH achieved in the simulation run remained almost constant: 5.30 ± 0.02.

] ≅ 0M [*P*2

The pH achieved in the simulation run remained almost constant: 2.20 ± 0.03.

selected regions of the β‐carotene neutral radicals. It was confirmed that changes of *k*4

were the recombination reactions between *P*1

and *k*7

was adjusted

and *k*5

] ≅ 10−10M

were involved in the

= 1.4×1010M−1s−1 and *k*5

M−1s−1 was selected and

radicals and nitrogen diox‐

and β‐carotene neutral radi‐

#### **2.3. (c) Theoretical evaluation of the intermediates proposed: the acyclic β‐carotene neutral radicals, the acyclic nitrous β‐carotene neutral radicals, and the cyclic nitroxide neutral radicals**

The purpose of the following research was to unravel the structures of the related persistent intermediate or intermediates, we called them *P*4  *(nitroxide)* or cyclic nitroxide neutral radical or radicals.

**Figure 4.** The figure shows a first‐derivative X‐band EPR spectrum of the intermediate called *P*4(nitroxide), one or more type of persistent intermediate formed in the reaction of β‐carotene with nitrite anion in acid medium (dioxane/water) at 298 K, under argon atmosphere. Spectrometer settings: microwave frequency, 9.92 GHz; modulation frequency, 100 kHz; microwave power, 11.3 mW; attenuation, 10 dB; field modulation, 0.125 G; receiver gain, 2×105 ; time constant, 0.05 s; scan range, 50 G; scan time, 5 s. The peak‐to‐peak value is of 12.7 Gauss and g = 1.994 [9].

On the basis of the kinetic studies, it was reasonable to propose intermediates of the nitroxide type. In a biological medium, one can expect that species react with each other. Or hope that radicals from β‐carotene react with neighboring molecules of nitrous carotenes generating acy‐ clic nitroxides. However, in the dilute solutions of β‐carotene, where the reactions with the nitro‐ gen oxides were carried out, the radicals kept away and intra‐radical reactions would preferably take place. Thus, on that account, we considered it suitable to propose the formation of the rings.

We looked for the help of the computational and theoretical methods in order to study and compare the characteristics of the proposed structures and found out that the density func‐ tional theory (DFT) met the best conditions, given that it included the effects of electron correlation. DFT used very suitable approaches for energy exchange and for the correlation. The main conclusion was that for neutral radicals we could obtain good results with the approximate method Unrestricted Becke‐style 3‐parameter density functional theory using the Lee‐Yang‐Parr correlation functional (UB3LYP), both for the calculation of the energy of the system and for the calculation of the hyperfine coupling constants (hfccs). The selected basis sets, that used *d orbital functions*, allowed a good resolution, with an adequate cost of time, with a personal computer. The software used allowed to obtain the values of energy of each structure, which permitted to evaluate the relative stability of the radicals. Also, the software provided us the isotropic hfccs of each nucleus of the radical intermediates pro‐ posed [10].

Finally, the hfccs of the nuclei of hydrogen (1 H) and nitrogen (14N) obtained with the theo‐ retical methods allowed us to develop the simulation of the spectra that were experimentally recorded, like that shown in **Figure 4**.

Different possible rings with 3, 5, 6, 7, or 8 atoms were built and tested. However, only with some of them the corresponding geometric optimizations were achieved. The radicals with cycles of five atoms were found to be the most favored and those of lower energy.

In the General Reaction Pathway proposed in **Figure 3**, the following intermediates were appreciated:

With the aim of avoiding confusion in the interpretations described, the process of formation of the radical intermediates follows.

• *Allylic‐type radicals, P*1

**Figure 5** helps to visualize the possible allylic hydrogens of β‐carotene (the characteristic atomic symbols (C, H) are usually not shown in this type of molecular representation). The set of neutral allylic radicals from β‐carotene is symbolized by *P*1 in **Figures 3** and **6**.

The molecule of β‐carotene has allylic hydrogens, which are hydrogen atoms of the methyl groups at positions 5 and 5'; 9 and 9'; 13 and 13'. Allylic‐type radicals are obtained by the loss, by abstraction, of those hydrogen atoms:

On the basis of the kinetic studies, it was reasonable to propose intermediates of the nitroxide type. In a biological medium, one can expect that species react with each other. Or hope that radicals from β‐carotene react with neighboring molecules of nitrous carotenes generating acy‐ clic nitroxides. However, in the dilute solutions of β‐carotene, where the reactions with the nitro‐ gen oxides were carried out, the radicals kept away and intra‐radical reactions would preferably take place. Thus, on that account, we considered it suitable to propose the formation of the rings. We looked for the help of the computational and theoretical methods in order to study and compare the characteristics of the proposed structures and found out that the density func‐ tional theory (DFT) met the best conditions, given that it included the effects of electron correlation. DFT used very suitable approaches for energy exchange and for the correlation. The main conclusion was that for neutral radicals we could obtain good results with the approximate method Unrestricted Becke‐style 3‐parameter density functional theory using the Lee‐Yang‐Parr correlation functional (UB3LYP), both for the calculation of the energy of the system and for the calculation of the hyperfine coupling constants (hfccs). The selected basis sets, that used *d orbital functions*, allowed a good resolution, with an adequate cost of time, with a personal computer. The software used allowed to obtain the values of energy of each structure, which permitted to evaluate the relative stability of the radicals. Also, the software provided us the isotropic hfccs of each nucleus of the radical intermediates pro‐

retical methods allowed us to develop the simulation of the spectra that were experimentally

Different possible rings with 3, 5, 6, 7, or 8 atoms were built and tested. However, only with some of them the corresponding geometric optimizations were achieved. The radicals with

In the General Reaction Pathway proposed in **Figure 3**, the following intermediates were

With the aim of avoiding confusion in the interpretations described, the process of formation

**Figure 5** helps to visualize the possible allylic hydrogens of β‐carotene (the characteristic atomic symbols (C, H) are usually not shown in this type of molecular representation). The

The molecule of β‐carotene has allylic hydrogens, which are hydrogen atoms of the methyl groups at positions 5 and 5'; 9 and 9'; 13 and 13'. Allylic‐type radicals are obtained by the loss,

cycles of five atoms were found to be the most favored and those of lower energy.

• *Cyclic nitroxides formed by internal cyclization of the nitrous allylic radicals, P*4(nitroxide)

set of neutral allylic radicals from β‐carotene is symbolized by *P*1

H) and nitrogen (14N) obtained with the theo‐

in **Figures 3** and **6**.

posed [10].

76 Carotenoids

appreciated:

• *Allylic‐type radicals, P*1

• *Allylic‐type radicals, P*1

• *Nitrous allylic radicals, P*4

of the radical intermediates follows.

by abstraction, of those hydrogen atoms:

Finally, the hfccs of the nuclei of hydrogen (1

recorded, like that shown in **Figure 4**.

**Figure 5.** The molecule of β‐carotene has allylic hydrogens, that are hydrogen atoms of the methyl groups at positions 5 and 5'; 9 and 9'; 13 and 13'. Vinylic hydrogen atoms are attached to the polyene chain at carbons: 7,8,10,11,12,14,15,15', 14',12',11',10',8', and 7'. Methyl groups with primary hydrogens, but not allylic type are represented with only a line or a bar in positions 1 and 1'.

*P*10(5 or 5'): The allylic‐type radical is formed by the loss of a hydrogen atom of the methyl attached to carbon *5 or 5'.*

*P*10(9 or 9'): The allylic‐type radical is formed by the loss of a hydrogen atom of the methyl attached to carbon *9 or 9'*.

*P*10(13 or 13'): The allylic‐type radical is formed by the loss of a hydrogen atom of the methyl attached to carbon *13 or 13'.*

The molecule of β‐carotene has also methyl groups (represented by a line or a bar) at posi‐ tions 1 and 1'; they are primary hydrogen atoms. Those primary hydrogen atoms are not abstracted, they have higher bond energy than the allylic ones. In **Figure 5**, you can also appreciate the vinylic hydrogen atoms in the polyene chain. Those vinylic hydrogen atoms are attached to carbons: 7,8,10,11,12,14,15,15',14',12',11',10',8', and 7'; they have even higher bond energy than the primary hydrogen atoms. Observe that the radicals formed by the loss of allylic hydrogens are indeed stabilized by resonance.

In **Figure 6**, we followed the formation of the neutral carotenoid allylic radicals described below:

*P*10(5 or 5'): The β‐carotene loses an allylic hydrogen atom from the methyl group attached to position 5 or 5'. The conjugation effect formed a contributing resonance structure, an allylic tertiary radical on carbon 6 (see **Figure 5**), it is *P*11(5 or 5')

*P*12(5 or 5'): It is another contributing resonance structure with the unpaired electron in carbon 13'.

*P*10(5 or 5') *;P*11(5 or 5') and *P*12(5 or 5') are contributing resonance structures of the *same resonance hybrid*.

**Figure 6.** Several radicals of the allylic type are shown, *P*1 . There are three possibilities of allylic hydrogens: *P*10 (5 or 5'); *P*10 (9 or 9'), and *P*10 (13 or 13'). The second subscript indicates different contributing resonance structures, for example, *P*11 (5 or 5') *and P*12 (5 or 5')*.* The radical formed by the loss of a methyl hydrogen atom in position 9 or 9' is *P*10 (9 or 9'), observe that the electronic movements are shown in three related structures. *P*1 0(13 or 13') is the radical formed by the loss of a methyl hydrogen atom in position 13 or 13'. For this last case, only one possible structure is designed.

In the case of *P*10(9 or 9'), the resonance or electronic movements are also shown. For *P*10(13 or 13')*,* only one structure is designed.

• *Nitrous allylic radicals, P*4

**Figure 6.** Several radicals of the allylic type are shown, *P*1

78 Carotenoids

. There are three possibilities of allylic hydrogens: *P*10 (5 or 5');

*P*10 (9 or 9'), and *P*10 (13 or 13'). The second subscript indicates different contributing resonance structures, for example, *P*11 (5 or 5') *and P*12 (5 or 5')*.* The radical formed by the loss of a methyl hydrogen atom in position 9 or 9' is *P*10 (9 or 9'), observe that the electronic movements are shown in three related structures. *P*1 0(13 or 13') is the radical formed by the loss of a methyl

hydrogen atom in position 13 or 13'. For this last case, only one possible structure is designed.

The *P*4 radicals were also investigated with the theoretical methods:

In **Figure 7**, we appreciate that *P*12 (5 or 5') is the intermediate that react with nitric oxide generat‐ ing *P*22, the nitrous allylic compound, see **Figure 3**.

*P*22 loses by abstraction an allylic hydrogen atom from the methyl in position 9' generating *P*42, the corresponding nitrous allylic radical and finally the formation of *P*42 (nitroxide).

(**Figure 6** in our work of 2015 has an error; the nitroxide displayed is not generated from the listed precursors. The precursors designed would actually lead to another nitroxide not shown. The optimization of that nitroxide was completed on the basis of negligible forces. The stationary point was found but the convergence criteria were reached by only three of the required four cases [10]. In the present work, we have the opportunity of showing and solving the mistake. In **Figure 7**, we appreciate the precursors that actually lead to *P*42*(nitroxide).*)

**Figure 7.** Formation of the cyclic *P*42*(nitroxide)* from the allylic precursor. The tertiary allylic radical *P*12 (5 or 5') is a contributing resonance structure of high weight.

In **Figure 8**, the optimized structure of *P*42 (nitroxide) is displayed. Each atom is labeled with a number.

In order to carry out the simulation, three selected nitroxides were chosen, those of lower energy and those that best met the requirements for optimization. We selected two rings of five atoms (*P*41 (nitroxide); *P*42 (nitroxide) ) and one of eight atoms (*P*43 (nitroxide)).

We simulated quite satisfactorily the experimental recorded EPR spectrum in **Figure 4**, by adding the theoretical spectra of *P*42 (nitroxide) from *P*12 (5 or 5') and *P*41 (nitroxide) from *P*11 (5 or 5'), both cyclic nitroxides of five atoms. Also we built another simulation by adding *P*42(nitroxide), *P*41 (nitroxide), and *P*43 (nitroxide) from *P*10 (9 or 9'), a cyclic nitroxide of eight atoms [10].

**Figure 9** shows the theoretical spectra and simulation of the persistent EPR recorded spec‐ trum of **Figure 4**. In part (a), we may appreciate the theoretical EPR spectra of the nitroxides *P*41*(nitroxide), P*42*(nitroxide), and P*43*(nitroxide)*. In order to simulate the experimental recorded spectra, one must add up the spectra of nitroxides, multiplying the values of *P*41*(nitroxide)* by the factor 1 and *P*42*(nitroxide)* by the factor 0.4 and multiply the values of *P*43*(nitroxide)* by the factor 0.2. Part (b) shows the sum that simulates quite well the experimental spectrum shown in **Figure 4**.

Simulations were also carried out considering the contribution of the β‐carotene neutral radi‐ cals or allylic neutral radicals. It was observed that with less than 0.1 order factors, there were no appreciable changes, as is expected due to the result of the kinetic modeling. Although the β‐carotene neutral radicals could be persistent, the nitroxide‐type signal obtained would not be modified.

The theoretical spectra were built from the calculated isotropic hyperfine coupling constants. The hfccs values larger than 0.4 Gauss are shown in **Table 3**.

**Figure 8.** Visualization of the optimized geometric structure of the intermediate radical proposed in **Figure 7**: *P*42*(nitroxide)*.

In **Figure 8**, the optimized structure of *P*42 (nitroxide) is displayed. Each atom is labeled with a

In order to carry out the simulation, three selected nitroxides were chosen, those of lower energy and those that best met the requirements for optimization. We selected two rings of

We simulated quite satisfactorily the experimental recorded EPR spectrum in **Figure 4**, by adding the theoretical spectra of *P*42 (nitroxide) from *P*12 (5 or 5') and *P*41 (nitroxide) from *P*11 (5 or 5'), both cyclic nitroxides of five atoms. Also we built another simulation by adding *P*42(nitroxide), *P*41 (nitroxide),

**Figure 9** shows the theoretical spectra and simulation of the persistent EPR recorded spec‐ trum of **Figure 4**. In part (a), we may appreciate the theoretical EPR spectra of the nitroxides *P*41*(nitroxide), P*42*(nitroxide), and P*43*(nitroxide)*. In order to simulate the experimental recorded spectra, one must add up the spectra of nitroxides, multiplying the values of *P*41*(nitroxide)* by the factor 1 and *P*42*(nitroxide)* by the factor 0.4 and multiply the values of *P*43*(nitroxide)* by the factor 0.2. Part (b) shows the sum that simulates quite well the experimental spectrum shown

Simulations were also carried out considering the contribution of the β‐carotene neutral radi‐ cals or allylic neutral radicals. It was observed that with less than 0.1 order factors, there were no appreciable changes, as is expected due to the result of the kinetic modeling. Although the β‐carotene neutral radicals could be persistent, the nitroxide‐type signal obtained would

The theoretical spectra were built from the calculated isotropic hyperfine coupling constants.

**Figure 8.** Visualization of the optimized geometric structure of the intermediate radical proposed in **Figure 7**:

five atoms (*P*41 (nitroxide); *P*42 (nitroxide) ) and one of eight atoms (*P*43 (nitroxide)).

and *P*43 (nitroxide) from *P*10 (9 or 9'), a cyclic nitroxide of eight atoms [10].

The hfccs values larger than 0.4 Gauss are shown in **Table 3**.

number.

80 Carotenoids

in **Figure 4**.

not be modified.

*P*42*(nitroxide)*.

**Figure 9.** EPR theoretical spectra and simulation. In the part (a) the EPR theoretical spectra of the nitroxides are recorded: *P41(nitroxide) in gray, P*42**(***nitroxide)* in black, and *P43(nitroxide)* in pale gray color. The values of *P41(nitroxide)* are multiplied by a factor of 1, *P*42*(nitroxide)* are multiplied by a factor of 0.4 and the values of *P43(nitroxide)* by a factor of 0.2. The part (b) shows the sum of the spectra in order to simulate the experimental spectrum. The peak‐to‐peak value is 12.1 Gauss, (10 Gauss ≡ 1 mT). Data from **Table 3** are used.

a The hfccs values were obtained by applying the method of calculation B3LYP/6‐31G (d) to the optimized geometries. The hfccs values larger than 0.4 Gauss are shown. Gaussian offers results in Gauss, 10 G ≡ 1 mT.

```
P41(nitroxide) and P42(nitroxide) are rings of five atoms. P43(nitroxide) corresponds to a ring of eight atoms.
```
Each atom is labeled with a number, as in **Figure 8** for *P*42*(nitroxide)*.

**Table 3.** Isotropic hyperfine coupling constants (hfccs), cyclic *P*4 *(nitroxides)*, (Gauss)a .

#### **3. Conclusions and perspective**

We have provided considerable information about the behavior of nitrogen dioxide in some solvents. Also, we followed the action of both NO2 and NO over some organic compounds. Most relevant was the study of the reaction of both oxides with the β‐carotene molecule.

It is important to highlight that two spectroscopic techniques were used: UV‐vis and EPR. UV‐vis is fundamental for the tracking of reagents, and EPR is essential for the monitoring of the radical intermediates.

Finally, we used the theoretical and computational methods that provided support to the characteristics of the intermediates. Calculations for β‐carotene cyclic nitroxide intermedi‐ ates, β‐carotene allyl radicals, and nitrous β‐carotene radical showed that the combination of the 6‐31G(d,p) basis set and the B3LYP exchange‐correlation functional provided a quite accurate description of the hfccs of the nuclei of hydrogen and nitrogen. The calculation of the hfcc for nitrogen nuclei (14N, nucleus) in the case of the nitroxide persistent radicals was particularly difficult. This problem with the nitroxides was investigated and finally deduced that the choice of the basis sets was very important. Especially the number and nature of *d orbital functions* must be taken into account [10].

β‐Carotene or other carotenoids compounds would be in the lipid and anaerobic part of cells where the investigated reactions could take place. The β‐carotene could be a convenient scav‐ enger, acting as a protective agent in biological media. The cyclic persistent nitroxides may react by disproportionation giving rise to hydroxylated carotenoid compounds and nitrones. Hydroxylated compounds can pass into the lymphatic system and be replaced by new mol‐ ecules of β‐carotene. The nitrone is a new scavenger that will lead to a continuously replace‐ ment process [10]. It is important to remember that the persistent nitroxides are still present in solutions after 20 days with a concentration of 3.5·10−5 M [9].

On the other hand, the nitroxides may react differently. For example:

Moreover, we can see that nitric oxide is generated in different types of cells. Neuronal nitric oxide synthase (nNOS) is constituvely expressed in specific neurons of the brain. In addi‐ tion to brain tissue, nNOS has been identified in adrenal glands, in epithelial cells of various organs. In mammalians, the largest source of nNOS in terms of tissue mass is in the skeletal muscle [23].

It might be expected that nitrogen oxides and non‐polar carotenoids compounds have together an important biological function. Take into account that in carotenoids with a hydroxyl group, the reaction path may be surely different.

The isolation of the cyclic nitroxides described would also be very important.

#### **Author details**

**3. Conclusions and perspective**

the radical intermediates.

36 H −0.42026

a

82 Carotenoids

solvents. Also, we followed the action of both NO2

Each atom is labeled with a number, as in **Figure 8** for *P*42*(nitroxide)*.

**Table 3.** Isotropic hyperfine coupling constants (hfccs), cyclic *P*4

*orbital functions* must be taken into account [10].

We have provided considerable information about the behavior of nitrogen dioxide in some

The hfccs values were obtained by applying the method of calculation B3LYP/6‐31G (d) to the optimized geometries.

*(nitroxides)*, (Gauss)a

.

The hfccs values larger than 0.4 Gauss are shown. Gaussian offers results in Gauss, 10 G ≡ 1 mT. *P*41*(nitroxide)* and *P*42*(nitroxide)* are rings of five atoms. *P*43*(nitroxide)* corresponds to a ring of eight atoms.

It is important to highlight that two spectroscopic techniques were used: UV‐vis and EPR. UV‐vis is fundamental for the tracking of reagents, and EPR is essential for the monitoring of

Finally, we used the theoretical and computational methods that provided support to the characteristics of the intermediates. Calculations for β‐carotene cyclic nitroxide intermedi‐ ates, β‐carotene allyl radicals, and nitrous β‐carotene radical showed that the combination of the 6‐31G(d,p) basis set and the B3LYP exchange‐correlation functional provided a quite accurate description of the hfccs of the nuclei of hydrogen and nitrogen. The calculation of the hfcc for nitrogen nuclei (14N, nucleus) in the case of the nitroxide persistent radicals was particularly difficult. This problem with the nitroxides was investigated and finally deduced that the choice of the basis sets was very important. Especially the number and nature of *d*

β‐Carotene or other carotenoids compounds would be in the lipid and anaerobic part of cells where the investigated reactions could take place. The β‐carotene could be a convenient scav‐ enger, acting as a protective agent in biological media. The cyclic persistent nitroxides may react by disproportionation giving rise to hydroxylated carotenoid compounds and nitrones.

Most relevant was the study of the reaction of both oxides with the β‐carotene molecule.

**Nuclei** *P***41***(nitroxide)* **Nuclei** *P***42***(nitroxide)* **Nuclei** *P***43***(nitroxide)* 3 H 0.73171 49 H 0.42705 28 H 0.94710 10 H 0.91335 51 H 2.99147 31 H 0.45231 14 H 0.56426 53 H −0.53599 53 H 2.00175 18 H 0.57683 55 H −0.55714 54 H 9.42852 25 H −0.53235 57 **N** 10.95771 56 H 19.03785 27 H −0.58125 60 H −0.45979 57 H 2.93589 29 **N** 11.68685 62 H −0.60386 88 **N** 11.40254 33 H −0.57914 64 H 18.74614 92 H 2.15932 34 H −0.47031 96 H 1.59845

and NO over some organic compounds.

Sara N. Mendiara1 \* and Luis. J Perissinotti1,2

\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Chemistry, Faculty of Exact and Natural Sciences, National University of Mar del Plata, Mar del Plata, Buenos Aires, Argentina

2 Commission of Scientific Research of the Province of Buenos Aires (CIC), La Plata, Buenos Aires, Argentina

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