Carotenoids and Their Antioxidant Power

Abstract

Carotenoids are natural products found in photosynthetic organisms such as plants, algae, and some bacteria species. Humans and animals cannot synthesize carotenoids, and they obtain these molecules through their diet. The common structure of carotenoids contains conjugated double bonds that provide color formation in the visible spectrum, at 400–500 nm. In photosynthetic organisms, carotenoids contribute to color formation for various purposes, such as sex selection, protection from predators, and light-harvesting to increase the spectral range of photosynthesis. The conjugated double bonds not only provide color formation but also provide antioxidant properties to carotenoid molecules. Studies have shown that carotenoids are capable of scavenging free radicals and reactive oxygen species, as well as quenching singlet oxygen molecules. The antioxidant power of carotenoids results in several health benefits. These include anticancer, neuroprotective, and anti-atherosclerotic activities. This chapter aims to review the antioxidant activities and health benefits of major carotenoids, beginning with their structure and synthesis, and also discussing their natural sources.

1. Introduction

Carotenoids are a natural group of lipid-soluble pigments [1]. They consist of polyunsaturated isoprene units (C5H8) and have 30–50 carbons in their structure with conjugated double bonds [2]. These conjugated double bonds provide the color formation in carotenoid molecules [3]. The most naturally abundant carotenoids are C40 carotenoids and the functional groups at the end of the chain diversify the carotenoids, leading to the formation of more than 750 carotenoid molecules [2, 3]. Depending on oxygen presence, carotenoids are divided into two groups: carotenes consisting of only carbon and hydrogen, and xanthophylls containing oxygen in their structure [3]. Carotenoids are mainly biosynthesized in photosynthetic organisms such as plants, algae, some bacteria, and fungi species [3]. However, animals and humans cannot synthesize carotenoids; thus they must obtain them from dietary sources [4].

Carotenoids play a significant role in photosynthesis due to two main functions: as photoprotective agents and as light harvesting pigments [5]. In addition, carotenoids have exhibited various biological activities such as antioxidant, anticancer, anti-atherosclerotic properties [6]. Also, it was shown that increased carotenoid consumption correlates with a lower risk of chronic diseases [4]. Another important aspect of carotenoids is their role as provitamin A. Vitamin A is involved in various processes, including vision, reproduction, cell differentiation and proliferation, and immunity. Vitamin A deficiency can cause several symptoms, such as xerophthalmia, increased susceptibility to severe infections, increased mortality, and detrimental effects on growth and fetal development [7]. β-carotene, α-carotene, and β-cryptoxanthin exhibit provitamin-A activity and thus, are useful to combat vitamin A deficiency [7]. Apart from all these health benefits, a study revealed that long-term supplementation of β-carotene has increased the cognitive abilities of the men subjects [8]. Overall, carotenoids play a significant role in human health due to their biological activities.

2. Biosynthesis of carotenoids

Carotenoids are mainly found in photosynthetic organisms and synthesized from plastids [9]. The most abundant carotenoids are the C40 carotenoids, and their synthesis starts from 5-carbon isoprenoid units as isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) [9]. These two precursors are synthesized from one of the two non-homologous pathways: the mevalonate (MVA) pathway and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway [2, 10]. In the first step of MVA pathway, three acetyl-CoA molecules are condensed to yield 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), which is subsequently reduced to MVA via the catalysis of HMG-CoA reductase. The phosphorylation of MVA twice and subsequent decarboxylation result in the formation of IPP, and through the catalysis of IPP isomerase, IPP is converted to DMAPP. On the other hand, the starting point of MEP pathway is the synthesis of 1-deoxy-D-xylulose 5-phosphate (DXP) from pyruvate and D-glyceraldehyde-3-phosphate (G3P) by head-to-head condensation of these two molecules, catalyzed via DXP synthase. Intramolecular rearrangement of DXP results in formation of 2-C-methyl-D-erythritol (ME) and the subsequent reduction of ME generates MEP. Finally, MEP is converted to IPP and DMAPP via four intermediates [11].

The complete biosynthesis of certain carotenoids is shown in Figure 1. The two precursors, IPP and DMAPP, first form the C10 molecule geranyl pyrophosphate (GPP) via head-to-tail condensation. GPP and IPP yields C15 molecule farnesyl pyrophosphate (FPP) by addition, and further addition of one more IPP molecule to FPP results in the formation of C20 intermediate precursor geranylgeranyl pyrophosphate (GGPP). The head-to-head condensation of two GGPP molecules leads to prephytoene diphosphate (PPPP), which forms phytoene via the removal of the diphosphate group and stereospecific proton abstraction. Phytoene is a colorless molecule with three conjugated double bonds and provides the C40 skeleton of carotenoids. It is the primary precursor to synthesize C40 carotenoids. For the formation of phytoene from IPP, there are four enzymes to catalyze the steps. These enzymes are IPP isomerase for isomerization of IPP to DMAPP, GPP synthase to convert IPP and DMAPP into GPP, GGPP synthase to catalyze conversion of IPP and DMAPP to GGPP, and lastly, phytoene synthase that catalyzes the formation of phytoene from GGPP [9].

Figure 1.

Biosynthesis pathway of carotenoids.

Conjugated carbon-carbon double bonds provide the required chromophore structure for carotenoid coloration. Desaturation of phytoene by phytoene desaturase (PDS) in higher plants and carotene isomerase (CrtI) in bacteria and fungi species leads to formation of lycopene at four steps by introducing one desaturation at each step [9]. Increased number of conjugated carbon-carbon double bonds results in red colored lycopene [2, 9]. Phytoene is found as 15-cis isomer in higher plants while lycopene is observed as all-trans, which indicates the isomerization that is facilitated by light and catalyzed by plant desaturases at some point [9].

Lycopene is the precursor for the synthesis of cyclic and bicyclic carotenoids, which is catalyzed by lycopene cyclases [2]. At first, cyclization of lycopene by lycopene-β-cyclase (LYC-b) by the introduction of β-rings and lycopene-ε-cyclase (LYC-e) by the introduction of ε-rings yields β-carotene and α-carotene. Further modifications of carotenes via β-carotene ketolase and β-carotene hydroxylase generate several C40 carotenoids that belong to the xanthophylls group [2]. The first xanthophylls formed from β-carotene are β-cryptoxanthin and zeaxanthin, which are obtained via hydroxylation on C-3 and C-3′ positions of the β-rings by β-carotene hydroxylase. Zeaxanthin epoxidase (ZEP) modifies 3-hydroxy β-rings of zeaxanthin by introducing 5,6-epoxy groups and yields antheraxanthin and violaxanthin. Finally, capsanthin/capsorubin synthase (CCS) forms cyclopentane rings (κ-rings) from 3-hydroxy-5,6-epoxy β-rings of antheraxanthin and violaxanthin, and results in capsanthin and capsorubin, which are the ketolated carotenoids [9]. Astaxanthin is synthesized by β-carotene ketolase, catalyzing the conversion of zeaxanthin into astaxanthin [12]. C50 and C30 carotenoids are rare, and C50 carotenoids are synthesized through the addition of two DMAPP molecules on C40 carotenoids [13], while C30 carotenoids are synthesized by two ways: by the condensation of FPP molecules or from the oxidative cleavage of C40 carotenoids [2, 14].

3. Dietary sources of carotenoids

Carotenoids cannot be synthesized by humans and must be obtained through daily dietary intake. Most important sources of carotenoids in the human diet are fruits and vegetables, which provide almost 50 different carotenoids. Various carotenoids can be detected in human blood plasma, including zeaxanthin, β-cryptoxanthin, lutein, lycopene, β-carotene, and α-carotene [15]. A study revealed that the highest carotenoid-containing fruits and vegetables are red peppers, carrots, apricots, nectarines, plums, peaches, and the lowest in cherries [16]. Table 1 summarizes some of the fruits and vegetables and their carotenoid contents as μ/100 g vegetable or fruit.

Carotenoid	Description	Carotenoid per 100 g (μg)
Lycopene	Tomato powder	46,300
	Tomato, sun-dried	45,900
	Catsup	12,100
	Rose Hips, wild (Northern Plains India)	6800
	Guavas, common, raw	5200
β-Carotene	Peppers, sweet, red, freeze-dried	42,900
	Carrot, dehydrated	34,000
	Grape leaves, raw	16,200
	Sweet potato, frozen, cooked, baked, without salt	12,500
	Tomato powder	10,300
α-Carotene	Carrot, dehydrated	14,300
	Peppers, sweet, red, freeze-dried	6930
	Pumpkin, raw	4020
β-Cryptoxanthin	Species, pepper, red or cayenne	6250
	Species, paprika	6190
	Papayas, raw	589
	Peppers, sweet, red, raw	490
Zeaxanthin	Eggs, Grade A, large, egg yolk	546
	Spinach, mature	466
	Spinach, baby	191
Lutein	Spinach, mature	7450
	Spinach, baby	5830
	Eggs, Grade A, large, egg yolk	612

Table 1.

Carotenoid contents of different fruits and vegetables according to information from [17].

Due to its provitamin A activity, β-carotene is considered one of the most important carotenoids. Mainly, apricot, mango, and pumpkin species are rich in β-carotene and also contain a portion of α-carotene [18]. Sixty to seventy percent of total carotenoids of apricot is determined as β-carotene [19]. Orange vegetables such as carrots, and dark green leafy vegetables like lettuce and spinach, are other sources rich in β-carotene [20, 21]. According to the database (https://fdc.nal.usda.gov/fdc-app.html#/?component=1122), lycopene is found in tomato (46 mg/100 g FW (fresh weight)), watermelon (1.6–3.5 mg/100 g FW), papaya (1.8–4.2 mg/100 g FW), and guava (3.2–7.0 mg/100 g FW) [17]. Eighty to ninety percent of the total pigments in tomatoes are determined to be lycopene, making tomatoes and tomato-based products such as ketchup and sauce significant sources of lycopene [22, 23]. Gac fruit aril is not included in the database, yet. However, gac fruit aril is determined as the highest β-carotene (20.5 mg/100 g FW) and lycopene (146.6 mg/100 g FW) containing fruit [24].

Corn seeds and egg yolks are good sources of lutein and zeaxanthin due to their high content of these carotenoids [23]. In egg yolk, 21.8 μg/g FW lutein and 13.4 μg/g FW zeaxanthin were determined [25]. The lutein and zeaxanthin content of egg yolk is coming from corn seeds; hence chicken diet is primarily depending on corn seeds [23]. Total lutein and zeaxanthin content from four different corn cultivars was determined between 947 and 2758 μg/100 g [26]. Spinach (11.93 mg/100 g) and kale (39.55 mg/100 g) are found as high-level lutein and zeaxanthin containing vegetables [27]. Carotenoids can exist in esterified form among plants. For instance, the high level of zeaxanthin dipalmitate (35.7 mg/g FW) was determined in goji berry at fully ripe stage, while 5% of total carotenoids was estimated as β-cryptoxanthin monopalmitate (2.2 mg/g FW) [28]. β-Cryptoxanthin is provitamin A xanthophyll and the major carotenoid found in mandarins and oranges [15]. β-Cryptoxanthin was determined in both skin (283–1254 μg/g FW) and pulp (76.5–287 μg/g FW) of the persimmon with different levels depending on the cultivar [29].

In addition to plants, carotenoids are also synthesized via various microorganisms, including certain algae, bacteria, and fungi species. Algae are pigment-producing photosynthetic organisms and perceived as a good source of bioactive compounds including carotenoids [30]. Astaxanthin is a red carotenoid with an antioxidant activity 10 times higher than other carotenoids [31]. Main microalgae species producing astaxanthin are Haematococcus pluvialis, Chlorella zofingiensis, and Chlorococcum sp. [32]. Richest source of astaxanthin is H. pluvialis with 80% (relative to biomass) of the total carotenoids and H. pluvialis is the primary industrial source for the production of astaxanthin [30, 32]. Halotolerant green algae Dunaliella salina synthesizes carotenoids such as β-carotene, α-carotene, lutein, and lycopene [33]. Relatively, 86% of total carotenoids from D. salina was determined as β-carotene that makes D. salina the richest source of β-carotene [33]. Lutein and zeaxanthin are synthesized via algal species such as Scenedesmus spp., Chlorella spp., Rhodophyta spp., or Spirulina spp. Chlorella genus is the best source for industrial level lutein production. β-carotene, β-cryptoxanthin, and zeaxanthin are synthesized by Spirulina platensis [30]. Generally, algae species produce various carotenoids, and they are the most suitable organisms for industrial scale-up processes to synthesize carotenoids.

Despite the high yield of microalgal carotenoid biosynthesis, the cost of carotenoid biosynthesis via microalgae in an industrial process is higher than that of bacteria since the microalgae require longer cultivation time and depend on light to produce carotenoids [34]. Some bacterial species are also natively capable of biosynthesizing carotenoids and gain attention for carotenoid biosynthesis due to shorter cultivation time [34]. In literature, Brevibacterium linens was reported as high β-cryptoxanthin (0.3 mg/mL) producing bacteria [35], while Paracoccus zeaxanthinifaciens was reported as high zeaxanthin (13.8 mg/L) producer [36] and Paracoccus carotinfaciens (E-396) was reported as high astaxanthin producer (19.9 mg/L) [37]. Besides, some endophytic bacteria species are identified as native carotenoid producers. For instance, Pseudomonas sp. 102,515 was isolated from the leaves of Taxus chinensis and capable of producing an antioxidant natural product, zeaxanthin diglucoside (380 ± 12 mg/L) [38].

4. Antioxidant potential and health benefits of carotenoids

4.1 Functional roles of different carotenoid molecules

Carotenoids have various functions in nature, including light-harvesting, photoprotection, coloration for sexual purposes, and protection in animals, as well as provitamin A activity in vertebrates [39]. All carotenoids possess light-harvesting and photoprotection properties due to conjugated double bonds, which can exhibit π → π* transition following light-absorption and subsequent high-energy excitation [39]. This light-harvesting property of carotenoids increases the spectral range of photosynthesis in plants [40]. Besides, light absorption and excitation result in color formation and carotenoids exhibit yellow- orange or red color, which are in the visible spectrum with wavelengths 400–500 nm [39].

In the presence of at least 9 conjugated double bonds, molecules exhibit singlet oxygen quenching activity and radical scavenging activity that provides carotenoids’ antioxidant properties. In addition, the linear system of conjugated C-C double bonds renders carotenoids potent antioxidants via high reducing potential in lipid formation via oxidation. When carotenoid molecules interact with membranes or dissolve in lipid structures, they can protect these structures from oxidative damage caused by aggressive radical species, thereby preventing irreversible destruction [40]. The effective neutralization of reactive oxygen species and other free radicals by carotenoids in both photosynthetic and non-photosynthetic organisms provides protection from oxidative damage. This makes carotenoids excellent candidates as natural antioxidants [41].

4.2 Role of carotenoids in reducing oxidative stress via antioxidant properties

Reactive oxygen species (ROS) are formed by cells for normal cellular functions, such as intracellular signaling and redox regulation. ROS molecules are derived from oxygen and can be extremely reactive in different forms such as hydroxyl radical, or less reactive like superoxide and hydrogen peroxide. Intracellular free radicals, which have unpaired electrons, are often considered as ROS. The excessive formation of ROS is called oxidative stress and causes severe damage in cellular structures [42, 43]. ROS and free radicals start a chain reaction by interacting with biomolecules like lipids, proteins, and DNA, and form free radicals readily [42, 43]. Newly formed free radicals further react with another free radical to eliminate the unpaired electron or with a free radical scavenger, a primary antioxidant, to break the chain [42]. Antioxidant is defined as “any substance that delays, prevents or removes oxidative damage to a target molecule” [44]. Antioxidants are investigated under two groups as enzymatic antioxidants and non-enzymatic antioxidants [43] or as preventative and chain-breaking antioxidants [45].

Carotenoids are non-enzymatic, chain-breaking, and lipid soluble antioxidants due to their hydrophobic nature [45, 46]. Hydrophobic scavengers are located on the cellular membrane or found in lipoproteins, preventing lipid peroxidation by interrupting chain reactions of ROS or free radicals [47]. Chain-breaking antioxidants interfere with the chain reaction via trapping the chain-carrying radicals [45]. Chain-breaking antioxidants, such as α-tocopherol, tend to donate hydrogen atom to trap free radicals [48]. However, a study investigated the radical scavenging activity of four carotenoids, astaxanthin, β-carotene, canthaxanthin, and zeaxanthin, to reveal the radical scavenging mechanism of carotenoids and concluded that carotenoids demonstrated the antioxidant activity via the addition of radicals to conjugated polyene chain in their structure unlike the other natural antioxidants. The conjugated polyene chain stabilizes the free radicals by the resonance of conjugated double bonds under low oxygen pressure similar to physiological tissue environment [48].

Carotenoids are secondary free radical scavengers and physical singlet oxygen quenchers [47]. Singlet oxygen (¹O₂) is an excited state of oxygen where an electron changes spin and yields two unpaired electrons with opposite spins. It is responsible for ¹O₂ dependent lipid peroxidation and DNA damage [49, 50]. Vitamin-E (α-tocopherol) is the most efficient peroxyl radicals’ scavenger in cellular membrane phospholipid bilayer structure [47]. Importantly, the inhibition of ¹O₂ dependent lipid peroxidation was also achieved by carotenoids including β-carotene, astaxanthin, and lycopene, due to their ¹O₂ scavenging activity as great as that of α-tocopherol [49]. An in vitro study to show the protection of cells from lipid peroxidation via singlet oxygen quenching activity of carotenoids concluded that the highest protection was provided by lycopene, followed by astaxanthin and then, β-carotene [51]. Basically, carotenoids prevent lipid peroxidation and cellular damage through radical scavenging or singlet oxygen quenching activities, rendering carotenoids effective natural antioxidants and important dietary supplements for health.

4.3 Potential health benefits associated with carotenoid consumption

In human plasma, 12 carotenoids have been detected, including lutein, zeaxanthin, lycopene, α-carotene, β-carotene, and β-cryptoxanthin [52]. It has been hypothesized that dietary carotenoids could reduce cancer rate, and several studies have been conducted to test this hypothesis. These studies considered different types of cancer, and revealed that a high consumption of carotenoid-rich vegetables and fruits was associated with a decreased risk of cancer [53]. Epidemiological studies investigated the mechanism behind reduced risk of cancer associated with carotenoids and showed that the supplementation of vitamin A does not decrease the risk of cancer, unlike carotenoids. This suggests that carotenoids may exert their anticancer effects through mechanisms other than those related to vitamin A activity [54]. The antioxidant activity of carotenoids is well-known for their ability to scavenge radicals and quench singlet oxygen. Both radicals and singlet oxygen can damage biomolecules in cells through mechanisms such as lipid peroxidation or DNA damage. Since the genetic changes caused by DNA damage can led to cancer formation, carotenoids can decrease the risk of cancer through their antioxidant activities [34, 53]. For instance, a study showed that there was an inverse relationship between developing prostate cancer and the consumption of lycopene rich tomato and tomato-based products. This suggests that lycopene has a significant impact to reduce the prostate cancer development risk [55]. In an in vitro study on breast cancer, it was demonstrated that carotenoids, including astaxanthin, β-carotene, and lutein, exhibited synergistic effects with anticancer drug doxorubicin. These carotenoids increased the ROS-mediated apoptosis of cancer cells while showing no toxicity to healthy cells [56]. Overall, various carotenoids exhibit anticancer activity attributed to their radical scavenging and singlet oxygen quenching capabilities. The supplementation of carotenoids holds promise for the treatment and prevention of several cancer types.

Oxidative stress may contribute to the pathogenesis of Alzheimer’s disease. Astaxanthin was reported with its antioxidant activity as well as in vivo and in vitro neuroprotective effects. In a study investigating the neuroprotective effect of astaxanthin for Alzheimer’s disease using HT22 cells, it was found that astaxanthin could be beneficial for the treatment of neurological diseases such as Alzheimer’s disease, since it significantly suppressed the accumulation of ROS in HT22 cells and was involved in the regulation of other biomolecules implicated in the progression of the disease [57]. In the case of subarachnoid hemorrhage, neuronal apoptosis plays a significant role in the development of pathogenesis. The administration of astaxanthin after subarachnoid hemorrhage was shown to reduce the neuronal apoptosis and mitigate secondary brain injury, thereby alleviating brain dysfunction. This neuroprotective effect of astaxanthin involves modulation of the Akt/Bad pathway [58]. In addition, oxidative stress is also involved in aging and reduces cognitive abilities such as memory and language [59]. In 2013, a study revealed that serum lutein levels were significantly associated with better cognitive abilities in the subjects over 80 years old. Additionally, serum levels of lutein, zeaxanthin, and β-carotene were all correlated with better cognitive abilities in subjects over 100 years old. Besides, it was found that brain lutein and β-carotene levels are associated with better cognitive abilities [59]. It may be concluded that carotenoids are also beneficial for improving cognitive abilities and preventing neurodegenerative diseases.

Macula is a part of the eye that contains high density of cone cells and provides vision. It is exposed to light and oxidative stress on a regular basis. Age-related macular degeneration (AMD) is a chronic disease that progresses to blindness and oxidative stress aids the manifestation of AMD. Zeaxanthin and lutein are accumulating on the human macula to protect the macula through scavenging light-induced radicals and reducing oxidative stress. Previous studies indicated that the antioxidant activity of carotenoids in macula reduces the ROS levels and prevents damage caused from oxidative stress, which ultimately helps to reduce the risk of AMD. It was concluded that the supplementation of lutein and zeaxanthin is a preventative treatment against AMD [60]. Skin is the largest organ in the human body and is exposed continuously to light and solar radiation, like eyes. The solar radiation and light exposure cause the generation of wrinkles, pigmentation, and premature aging as well as ROS formation [34]. Lycopene and β-carotene supplementation was reported to provide protection against UV-induced skin damage while elevated dietary intake of these carotenoids protected the skin from UV-induced erythema in humans [61].

Lutein is mainly suggested for eye-related disorders, but it has a higher antioxidant potential than β-carotene and lycopene, which makes it a suitable target for the prevention and treatment of cardiovascular diseases. It has been showed that lutein exhibits anti-atherosclerotic activity. Lutein is involved in the regulation of certain pathways related to antioxidant enzyme production and reduces ROS via both direct antioxidant activity and expression of antioxidant enzymes. Low density lipoproteins (LDL) play a key role in the development of atherosclerosis, and lutein reduces the distribution of LDL particles in plasma and accumulation of LDL particles in the aorta. There are several other mechanisms that lutein involves and prevents cardiovascular disorders and thus, it is a good candidate to prevent or delay atherosclerosis [62]. Several studies demonstrated that lycopene exhibits similar mechanisms of action to other carotenoids, including the modulation of oxidative stress through the regulation of different pathways. Additionally, various cleavage products of lycopene were shown to interact with transcription factors and regulate the overexpression of antioxidants [63]. Lycopene was also considered as a promising molecule for the treatment and prevention of cardiovascular diseases, neurodegenerative diseases, and various cancer types due to its high antioxidant activity [63].

5. Conclusion

Carotenoids are secondary metabolites synthesized mainly by photosynthetic organisms such as plants, algae, and certain bacterial species. They are synthesized from IPP and DMAPP through the catalysis of several enzymes and have a conjugated polyene chain in their structure. The conjugated polyene chain provides carotenoids with antioxidant activities to scavenge free radicals and ROS and quench singlet oxygen. Since animals and humans cannot synthesize carotenoids, they must obtain them through their diet. Fruits and vegetables contain different types of carotenoids, some of which have been detected in human blood plasma, indicating the absorption and distribution of dietary carotenoids in the human body. Carotenoids exhibit various biological activities such as anticancer, neuroprotective, and anti-atherosclerotic properties due primarily to their antioxidant activity. Therefore, carotenoids are beneficial to human health for both the prevention and treatment of various diseases. However, the supply chain of carotenoids depends on extraction from plants, which is not sufficient for industry, is affected by climate change, and also requires longer time for plant growth. Currently, biotechnological processes are gaining attention for the microbial synthesis of carotenoids. There are various microbial species that can synthesize carotenoids. Carotenoid producing microorganisms can be used for industrial scale-up production as well as genetically modified microorganisms. In the industrial process of microbial carotenoid biosynthesis, synthetic biology and metabolic engineering approaches are useful to increase the yield. However, high amounts of production might be toxic to microorganisms. Further studies should investigate alternative methods for the high-yield and low-cost production of carotenoids.

Conflict of interest

The authors declare no conflict of interest.