Study on Antioxidant Beta Carotene

Oxidative stress (OS) is a state of imbalance in which the production of reactive oxygen species (ROS) and the clearance of protective mechanisms cannot be offset [1]. An appropriate amount of free radicals helps maintain normal life activities in animals, but when the endogenous clearance system cannot remove excessive free radicals in time, oxidative stress occurs in the body [2]. Oxidative stress can lead to a decline in immune function, chronic inflammation and even organ damage [3]. It can also adversely affect production and reproductive performance, seriously affecting economic efficiency. Beta-carotene is abundant in nature and has extremely high research value for its biological activity. Its function is similar to that of vitamin A and it is a precursor to vitamin A [4].

It is abundant in green to red fruits and vegetables, and the pure product is a shiny dark red hexahedron or crystalline powdery substance. To date, more than 600 natural carotenoids have been discovered. β-Carotene has good antioxidant properties and is a good antioxidant additive. It can reduce organ damage caused by oxidative stress by inhibiting lipid oxidation and promoting the cell's antioxidant defense system [5]. This article mainly outlines the structure, types, sources and metabolism of β-carotene, as well as its antioxidant mechanism of action and application in livestock and poultry production.

1 Structure, types and sources of β-carotene

1.1 Structure of β-carotene

The molecular formula of β-carotene is C40 H56, with a relative molecular weight of about 537. It contains 15 conjugated unsaturated double bonds and two β-ionone rings at both ends. The structure is shown in Figure 1.

The structure of carotene contains multiple conjugated double bonds, which allows it to form multiple cis-trans isomers, including all-trans β-carotene, 9-cis β-carotene, 13-cis β-carotene and 15-cis β-carotene. All-trans β-carotene, 9-cis β-carotene, 13-cis β-carotene and 15-cis β-carotene are some of the more common isomers in nature, with all-trans β-carotene having the highest bioavailability among all isomers [6].

1.2 Types and sources of β-carotene

Beta-carotene can be divided into three main categories based on its source: natural extraction, chemical preparation, and biosynthesis [7]. Extracting β-carotene from natural plants was one of the earliest methods of obtaining β-carotene and played a key role in the early extraction of β-carotene [8]. Natural β-carotene is mainly extracted from fruits and vegetables such as carrots, and microalgae such as Dunaliella.

The chemical synthesis of β-carotene involves the use of precursors with a similar chemical structure to β-carotene, and the synthesis of new β-carotene through multiple chemical reactions under suitable conditions. The chemical formula of the chemically synthesized β-carotene is the same as that of the natural β-carotene, i.e., the chemical formula of the synthetic product is C40 H56, but the structures of the two are not necessarily identical. Carotenoids with different structures have different absorption rates, conversion rates, and solubility in the body, and there are potential, unknown toxic side effects. Therefore, the chemical synthesis of β-carotene has not been widely promoted [9]. The biosynthesis method is mainly used to prepare β-carotene through bacteria, yeasts and other fungi, as well as other genetically engineered bacteria, such as the filamentous fungus Blakeslea trispora [10] and genetically engineered bacteria such as Escherichia coli.

For β-carotene, different preparation methods can affect the color, isomers, particle size in water dispersion and microstructure of β-carotene [11]. Natural carotenoids are safer and more effective during use, but they have problems such as complex processes and low biological activity; the structure and content of each component of chemically prepared β-carotene are different from those of natural substances, and it is unstable during the synthesis process, which leads to greater functional instability [12]; the biosynthesis of β-carotene has the advantages of a short production cycle and easy cultivation, and it is expected that large-scale production of β-carotene can be achieved through microbial synthesis. For a detailed comparison of the three extraction methods, see Table 1.

2 The metabolic mechanism of β-carotene

Beta-carotene is also known as provitamin A because it has the structure of two vitamin A molecules. Due to the existence of a negative feedback regulation mechanism, beta-carotene is converted into vitamin A in livestock and poultry according to their own needs, thus effectively avoiding the problem of excessive accumulation of vitamin A in livestock and poultry [13].

Most of the β-carotene in living organisms exists in the form of a protein complex. The intestine is the main organ for absorbing and converting β-carotene in mammals[14], while the liver is the main organ for storing β-carotene in livestock[15]. There are currently two known metabolic mechanisms for β-carotene in the body. One is the symmetric cleavage reaction that occurs under the action of β-carotene-15,15'-monooxygenase (BCMO1), This reaction is the main pathway for the conversion of β-carotene to vitamin A; the other is an asymmetric cleavage reaction mediated by β-carotene-9',10'-monooxygenase (BCMO1). BCM1 and BCO2 are found in the liver cells and mucosal epithelial cells of the body [14].

BCMO1 has a high degree of selectivity for the chemical bond at the 15,15' position of the carotenoid [16]. Its mechanism of action is to break the double bond at the 15,15' position of β-carotene to form two molecules of retinaldehyde (RAL). Retinaldehyde is then oxidized to retinoic acid (RA) by aldehyde dehydrogenase (ALDH1) or retinal dehydrogenase (RALDH). RA is one of the key metabolites that can reflect the activity of vitamin A [17]. Meanwhile, retinoaldehyde can also be reduced to retinol by alcohol dehydrogenase (ADH) and retinol dehydrogenase (RDH), and some of it is esterified to retinoic acid by retinoic acid ligase (LRAT) and retinoic acid hydroxylase (REH) [14]. The mechanism of action of BCO2 is to cause β-carotene to break at double bonds other than the 15,15' double bond, producing β-ionone and apocarotenoids. Apocarotenoids can be further converted into retinal molecules, but the mechanism is not yet clear. The metabolic process of β-carotene in the body is shown in Figure 2 [18].

BCMO1 also has a unique regulatory mechanism: tissue-specific negative feedback regulation [19]. When the body ingests excess β-carotene or endogenous β-carotene exceeds the scope of its own metabolism, the intestinal epithelial cells secrete a large amount of RA. RA binds to the retinoic acid receptor (RAR) bound to the retinoic acid-specific response element (RARE) in the promoter region of the BCMO1 gene, activating the small intestine-specific homeobox transcription factor (ISX) [20]. ISX suppresses the expression of the BCMO1 gene [21], and can also indirectly suppress the expression of the BCMO1 gene through the high-density lipoprotein receptor (SR-BI) [20, 22], preventing the occurrence of excessive vitamin A accumulation and poisoning [23]. The specific process is shown in Figure 3.

3 Antioxidant activity and mechanism of action of β-carotene

β-carotene has good antioxidant capacity, which can reduce the production of intracellular lipid oxidation and alleviate oxidative stress [24]. It has been found that β-carotene, as a feed additive, has a significant effect on the antioxidant and anticancer properties of livestock and poultry. The current stage of research has found that the antioxidant mechanism of β-carotene may include the following: ① the double bond structure binds to free radicals, reducing their activity or removing them; ② quenching singlet oxygen through electron transfer; ③ promoting Nrf2 mRNA expression, which activates Nrf2 expression and promotes the expression of antioxidant enzymes.

3.1 Scavenging free radicals

Beta-carotene has the ability to scavenge free radicals due to its multiple conjugated polyene double bonds, and is an ideal free radical quencher [25]. Excess free radicals in the body can disrupt the balance of the redox system [26], induce oxidative stress in the animal body, damage cell membranes, cause damage to proteins and DNA, and induce a series of diseases [2]. At present, three reaction pathways have been discovered in which β-carotene eliminates or reduces the activity of free radicals: [27] ① hydrogen atom transfer (HAT), in which the hydrogen atom on β-carotene is transferred to the free radical, thereby weakening the oxidizing effect of the free radical [28]; ② radical addition reaction (RAF), carotenoids directly undergo addition reactions with free radicals to form very stable free radicals [29-30]; and ③ is the electron transfer mechanism (ET), in which the hydroxyl group becomes activated under the influence of β-carotene, promoting hydrogen transfer to the peroxide radical [31]. Wang Ying et al. [32] extracted β-carotene from the fermentation of Trichophora brasiliensis and measured the free radical scavenging rate. The results showed that, at the same temperature, the higher the concentration of β-carotene, the stronger the antioxidant activity of the added substance. Yuan Lei et al. [33] used the DPPH method, salicylic acid method and hydroquinone autoxidation method to determine the ability of four similar but different carotenoids to scavenge free radicals. The analysis showed that β-carotene has the ability to reduce or eliminate free radicals.

3.2. Quenching singlet oxygen

It has been experimentally proven that one molecule of β-carotene can eliminate more than 1,000 singlet oxygen molecules [34]. Since β-carotene is very similar in structure to lycopene and has a similar arrangement of outer electrons, it is possible that β-carotene can quench singlet oxygen by electron exchange energy transfer, that is, electrons from β-carotene and singlet oxygen exchange with electrons from β-carotene with opposite spin directions [35], but this has not been confirmed. By observing the quantitative relationship between β-carotene and singlet oxygen in a single complex, that is, a decrease of 1 to 2 β-carotene in the complex, and a significant increase in the number of singlet oxygen, Telfer et al. concluded that β-carotene can act as an effective quencher of singlet oxygen [36].

3.3 Promoting the expression of antioxidant enzymes

Beta-carotene is a type of non-enzymatic antioxidant in the body that can promote the process by which antioxidant enzymes function [37]. The Keap1-Nrf2-ARE pathway is a key pathway that has been noted in recent years to resist oxidative stress in cells. Nrf2 acts as a switch to activate this signaling pathway [38]. Nrf2 concentration decreases after a period of oxidative stress in the body [39]. β-Carotene has an upregulatory effect on the relative expression of Nrf2 mRNA. The mechanism of action of β-carotene as an antioxidant may be to trigger the Nrf2 “switch” thereby increasing the expression of antioxidant enzyme genes [40]. Rocha et al. [37] demonstrated that β-carotene can change the activity of antioxidant enzymes such as glutathione-S-transferase by analyzing β-carotene nano-dispersions. Sowmya et al. [41] isolated β-carotene from spinach β-carotene can exert an antioxidant effect by observing the expression of antioxidant marker proteins in breast cancer cells (MCF-7), which further explains the anticancer activity of β-carotene.

4 Application of β-carotene's antioxidant properties in the production of livestock and economic animals

In actual production, the antioxidant properties of β-carotene are mainly used to improve animal immunity, improve meat quality, improve growth and production performance, and alleviate the harm of lung and respiratory tract damage and damage to other organs [42]. In addition, β-carotene is non-genotoxic and is recognized as a non-toxic, safe, and nutritious feed additive. When the external environment changes rapidly or when animals are in a special physiological condition, such as pregnancy, they are prone to a series of stress reactions such as oxidative stress. Oxidative stress can cause livestock and poultry to eat less, have decreased resistance, induce various inflammations, and even die, causing certain economic losses to the aquaculture industry. Zhang Xianglun et al. [43] added β-carotene to the diet of beef cattle, and found that the total antioxidant capacity, glutathione content, and total superoxide dismutase activity in the serum of the test cattle were significantly increased, and the malondialdehyde content was reduced, which proved that β-carotene as a feed additive can significantly improve the antioxidant function of beef cattle. When treating bovine infectious keratitis, the therapeutic effect can be significantly improved by supplementing the diet with green forage high in carotene content [44].

Pre-treating porcine intestinal epithelial cells (IPEC-J2) with different doses of β-carotene (25, 50, 100, 150, 200 μmol/L) was found to stimulate an inflammatory response, with the highest cell viability and transmembrane resistance in the treatment group. indicating that β-carotene has the special effect of enhancing the communication between intestinal cells and reducing intestinal damage, and plays a role in maintaining the intestinal health of livestock and poultry [45]. The antioxidant properties of β-carotene, its ability to enhance the body's immunity and strengthen the gap junctions between cells are one of the reasons why β-carotene is widely accepted as an anti-tumor agent. Previous studies have shown that as the concentration of β-carotene increases, the inhibition rate of lipid peroxidation significantly increases [46]. β-carotene can be transformed to be compatible with the lipids in cell membranes, and it can quench free radicals before they cause damage to the body, thus playing a key role in protecting membrane lipids [41].

Bi Yulin et al. [47] added different levels of β-carotene to the feed of beef cattle and quantitatively measured the concentrations of serum glutathione (GSH) and serum malondialdehyde (MDA). They found that as the level of β-carotene added increased, the measured indicators showed significant linear and quadratic changes, proving that β-carotene can greatly affect antioxidant function, change blood physiological indicators and meat quality. Wang Bo [48] found that β-carotene can significantly shorten the time to estrus after calving in dairy cows, promote estrus, increase the conception rate, not only improve the quality of milk, but also reduce the incidence of udder disease in dairy cows. Elomda et al. [49] verified that retinol, a decomposition product of β-carotene, has the ability to promote embryonic development during in vitro culture of rabbit embryos.

5 Summary

As a provitamin A, β-carotene can not only resist oxidation, but also improve the body's immunity, regulate glycolipid metabolism, or be used as a natural coloring agent in feed additives, which can play a certain role in preventing cancer. It also plays an important role in maintaining the normal growth and reproduction of livestock and poultry. Research on the absorption, metabolism and biological activity of β-carotene has become a research hotspot and focus at this stage, and relatively outstanding results have been achieved. However, there are still many issues that require further research and exploration, such as the technology and procedures for the artificial synthesis of β-carotene, the cultivation and breeding of high-yield β-carotene yeast strains, etc. In addition, the potential biological activity of β-carotene and the amount to be added to the feed also need to be further explored.

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