Study on Lutein and Antioxidants

Lutein is a non-vitamin A active oxygen-containing carotenoid. The molecule contains two different chromone rings, each with a functional hydroxyl group at the third carbon atom. There are three asymmetric centers at C-3, C-3′ and C-6′, so there are theoretically eight stereoisomers. Due to the complexity of the process of preparing lutein, the chemical synthesis of a single isomer of lutein has not yet been successful. Currently, lutein can only be extracted from natural plants.

Lutein is a natural carotenoid widely found in vegetables, flowers, fruits and certain algae. It is a natural food colorant and food nutrient. In recent years, a large amount of epidemiological evidence has shown [1-2] that lutein has significant antioxidant, anti-aging and anti-mutagenic biological effects, and has a wide range of biological activities in preventing macular degeneration, cardiovascular disease, blocking tumorigenesis and development, and enhancing immunity. The US FDA approved lutein as a generally recognized as safe food in 2003, and at the end of 2006, China also approved the use of lutein as a coloring agent in food, with an addition amount of 50mg/kg to 150mg/kg. It also approved the addition of lutein as a nutritional supplement to infant or children's formula food, with an addition amount of 300mg/kg to 4230mg/kg [3].

1 Extraction method of lutein

Currently, only lutein extracted from natural plants has antioxidant biological activity. In recent years, with the increasing demand for lutein, researchers at home and abroad are striving to find extraction methods with higher yields. Extraction methods of lutein:

1.1 Extraction with organic solvents

Organic solvent extraction is by far the most widely used method for extracting lutein. Commonly used organic solvents include hexane, ethanol, ethyl acetate, and solvent oil No. 6. Zhou Yanfang et al. [4] used a Soxhlet extractor and reflux co-cooking method to extract pigment extracts from marigold particles, and found that the best conditions were a mixture of acetone and ethyl acetate (1:1), with a material-to-liquid ratio of 1:4 and an extraction time of 7.0 h. The extraction yield of marigold xanthophyll could reach 22.6%. Liu Shuo et al. [5] studied the extraction and purification of lutein from the heterotrophic Chlorella USTB-01 and concluded that the optimized process is as follows: (1) Use hexane-ethanol (volume ratio 6.7:1.0) as the extraction agent to extract lutein and esters from USTB-01 algae powder. with an extraction time of 1 h and a solid-liquid ratio of 1:50; (2) the optimized control conditions for the conversion of lutein esters to lutein were 20% NaOH-aqueous solution as the saponification agent, saponification temperature 50 °C, time 6 h, saponification agent dosage 1:40; (3) through the processes of extraction, saponification and purification, lutein with a purity of 80.6% was obtained, laying an important research foundation for the production of lutein from Chlorella cells.

1.2 Microwave and ultrasonic extraction

Most of the pigment components of natural plants are intracellular substances, and the plant cells often need to be broken during extraction. Mechanical crushing methods are less efficient, and chemical crushing methods can easily cause changes to the structure and properties of the extract. Ultrasonic waves are elastic mechanical vibration waves that can produce a high-speed, strong cavitation effect to destroy plant cells, allowing the solvent to penetrate the plant cells, thereby accelerating the release, diffusion and dissolution of substances in the cells, shortening the extraction time and improving the extraction efficiency. Microwave is a kind of instantaneous penetrating heating method. Under the action of the microwave field, the plant cells are broken, thereby accelerating the extraction rate and effectively improving the yield of the product.

Yang Maisheng et al. [6] used kale vacuum freeze-dried leaf powder as raw material, and the conditions for lutein extraction with tetrahydrofuran were ultrasonic power 400W, temperature 30°C, time 20min, and the extraction reached 317.25mg/kg. Dai Gang et al. [7] used marigold particles as raw material to extract lutein esters under the optimal conditions of 80% hexane, a material ratio of 1:30, an ultrasonic power of 800 W, a time of 20 min, and a temperature of 30 °C, with an extraction rate of up to 93.9%. Huang Xingxin et al. [8] studied the ultrasonic extraction of lutein from chlorella, and concluded that the optimal process parameters were a liquid-to-solid ratio of 40:1, an ultrasonic power of 561 W, a time of 16.5 min, an irradiation mode of 4 s/5 s, and an extraction temperature of 45 °C. The optimal yield of lutein was 1.511 mg/g.

1.3 Supercritical CO2 extraction

Supercritical fluid extraction technology makes use of the special properties of supercritical fluids. The mixture of solid or liquid to be separated is contacted under high pressure conditions, and the operating pressure and temperature of the system are adjusted to extract the desired substance. Subsequently, the density of the supercritical fluid is reduced by depressurization or heating to separate the extract. Supercritical CO2 extraction has a series of advantages, such as being non-toxic and harmless, having strong solubilizing ability, leaving little solvent residue, and the product having high purity. It is therefore attracting more and more attention and is widely used in the extraction of natural products.

Li Gaofeng [9] et al. extracted lutein from marigold dried flower particles by supercritical CO2 extraction and concluded that the optimal process was an extraction pressure of 45 MPa and a temperature of 50 °C; a separation I pressure of 8 MPa and a temperature of 55 °C, with the main extract collected in the separation I tank; and a separation II pressure of 4 MPa and a temperature of 20 °C, with the impurities collected in separation II. Under these conditions, the lutein extract has a high extraction rate. Liang Lin [10] used supercritical CO2 extraction technology to extract lutein from sea buckthorn pomace, and the optimal process conditions were determined to be a temperature of 48°C, a pressure of 35 MPa, a separation pressure of 15 MPa, with entraining agent dosage of 9 mL. A comparison of supercritical CO2 extraction with organic solvent extraction shows that supercritical CO2 extraction can compensate for the disadvantages of organic solvent extraction, such as complicated operation, long processing time, and low yield.

1.4 Enzyme-mediated organic solvent extraction

The enzymatic method destroys the integrity of the cell structure, exposing more substances inside the cells during extraction and increasing the oil permeability. Since the plant cell wall is mainly composed of polysaccharides, cellulase and hemicellulase have the highest activity in degrading polysaccharides and the best results. Enzymatic degradation of marigold flowers does not cause isomerization of lutein, and the enzymatically treated marigold powder has the highest content of all-trans lutein, reaching 25.1 g/kg dry weight (dw) [11]. However, due to the long reaction time of the enzymatic treatment method, the large amount of moisture in the enzymatic treatment process needs to be removed before solvent extraction, which limits the practical application of the above method.

BARZANA E et al. [12] proposed a method for simultaneous enzymatic reaction and organic solvent extraction, in which a series of hydrolases are used to degrade cell wall components in a medium with mainly organic solvents and low moisture content. In terms of the selection of hydrolases, NAVARRETE-BOLANOS JL et al. [13-14] studied the effect of a non-commercial enzyme preparation on the extraction of lutein from marigold flowers. This enzyme was synthesized by endophytic microorganisms produced during the ensiling process, and had high cellulase activity and good extraction results. Li Xiuxia et al. [15] studied the ultrasonic-enzymatic assisted extraction process of lutein from corn protein powder, and determined that the optimal process parameters for enzymatic extraction of lutein from corn protein powder were an enzyme concentration of 7682 U/g, a substrate concentration of 818%, and an enzymatic hydrolysis time of 212 h. The yield of this process was 65 μg/g, which is consistent with the predicted results.

1.5 Current new methods in use

There has been relatively little research in China on the use of new methods to prepare lutein. CISNEROS M et al. [16] extracted Chlorella vulgaris algaemudder with 30% ethanol by weight of Chlorella vulgaris, and then used this crude extract as a material to study the distribution behavior of lutein in a PEG-phosphate bicellar system. The results showed that In a two-phase system formed by 22.9% (m/m) PEG 8000 and 10.3% (m/m) phosphate at pH 7.0, most of the lutein was distributed in the upper phase, while the residue was distributed in the lower phase. The lutein yield was 81.0%.

Fan Jianfeng et al. [17] used marigold flowers as a raw material and used microwave-surfactant synergistic extraction to extract lutein. The optimal process was found to be the use of ethyl acetate-Tween-20 as the best extractant (0.03% by mass), an extraction solid-liquid ratio of 1:60 (g/mL), a microwave power of 400 W, a temperature of 60 °C, and a time of 2 min. The amount of lutein extracted was 3.209 mg/g. Under the same conditions, this method increased the yield by 65% compared to microwave radiation alone, and increased it by 37.14% compared to the traditional solvent extraction method, greatly reducing the time.

2 Functions and applications of lutein

2.1 Antioxidant effect of lutein

The numerous effects of lutein are inseparable from its antioxidant effect. The human body can produce a variety of reactive oxygen species, which all have the effect of breaking DNA, lipid peroxidation, changing enzyme activity, degrading polysaccharides and killing cells, thus causing tremendous damage to the human body. Lutein has multiple conjugated double bonds in its structure, which can transfer high energy to convert reactive oxygen species into stable oxygen molecules, thereby preventing singlet oxygen from attacking lipid double bonds or conjugated double bonds. It is an effective scavenger of peroxyl radicals, and is particularly effective at low oxygen pressures in living cell tissues.

BOSCH-MORELL F et al. [18] believe that free radical scavengers and antioxidants can alleviate inflammatory responses. Li Dajing et al. [19] used marigold flower supercritical extract (SEMF) for research. The in vitro antioxidant activity of SEMF was comprehensively examined in terms of its ability to resist lipid peroxidation, reduce energy, and scavenge hydroxyl radicals and superoxide anion radicals. The results showed that SEMF exhibited strong antioxidant capacity in these systems and is an excellent natural antioxidant and free radical scavenger. Lanfang et al. [20] showed through experiments that lutein can increase the activity of SOD and GPX in tissues, thereby increasing the antioxidant capacity index and inhibiting lipid peroxidation, providing some experimental basis for the application of lutein in the prevention and improvement of uveitis.

2.2 Lutein's role in protecting the eyes and preventing AMD

Lutein and zeaxanthin are both fat-soluble vitamins that selectively accumulate in the macular region of the retina and lens, where they act as light filters and powerful antioxidants. They are important antioxidants for the eyes. They are the yellow pigments that make up the macula. Since higher animals cannot synthesize carotenoids, the main source of macular pigment is dietary intake. Many studies have shown that a decrease in macular pigment function is related to the onset of age-related macular degeneration (AMD). A decrease in lutein and zeaxanthin levels can lead to AMD and nuclear cataracts. Appropriate supplementation can significantly increase the macular pigment density in the serum and macular region, delaying the occurrence and development of age-related eye diseases such as AMD [21]. RICHER S et al. [22-23] showed that lutein supplementation can improve macular pigment optical density (MPOD) and visual function in patients with ARMD.

OLMEDILLA B et al. [24] showed that long-term supplementation with antioxidants (lutein and vitamin E) can improve serum levels and visual function in patients with senile cataracts. Higher doses of lutein, either through lutein-rich fruits and vegetables or supplements, may be beneficial for visual function in patients with senile cataracts. Lutein supplementation can improve visual function and effectively relieve visual fatigue. The results of a study by Ma et al. [25] showed that lutein intervention significantly improved tear film break-up time, visual acuity, and simple reaction time in people with long-term screen light exposure. Due to the protective effect of lutein on the eyes, eye health products with lutein as the main ingredient have been developed in recent years, such as the Bausch & Lomb lutein tablets introduced and sold by Shandong Freda Sales and Medicine Group in January 2008, which have obtained the import product access certificate of the State Food and Drug Administration (Guo Shi Jian Zi J20070004). The global English trademark is OcuviteR.

2.3 The anticancer effect of lutein

Lutein powder is one of the main carotenoids in human blood and has a special biological function in inhibiting tumor growth. Recent research has shown that lutein has an inhibitory effect on a variety of cancers (such as breast cancer, prostate cancer, stomach cancer, skin cancer, etc.). According to a recent study by the College of Pharmacy at New York University, there is a close relationship between breast cancer incidence and lutein intake. The breast cancer incidence in the experimental group with low lutein intake was 2.08 to 2.21 times that of the high intake group. The research results of Fu Lei [26] and others show that lutein can inhibit the proliferation of human gastric cancer cell lines (SGC-7901) and induce their apoptosis. Pei Yingxin and others [27] showed that lutein induces apoptosis of human esophageal squamous cell carcinoma cells by regulating the expression of Bax and Bcl-2 proteins. According to a study on the proliferation of prostate cancer cells, lutein alone can reduce the growth rate of cancer cells by 25%, and if it acts in synergy with lycopene, the growth rate can be reduced by 32% [28-29].

2.4 Lutein's role in preventing cardiovascular and cerebrovascular diseases

Recent research results show that lutein has a delaying effect on the early stages of the atherosclerosis process. It has been found that lutein can prevent the arterial wall from thickening. A low lutein content in the blood can easily cause the arterial wall to thicken. As the lutein content gradually increases, the tendency for the arterial wall to thicken decreases, and arterial embolism is also significantly reduced. Meanwhile, lutein in the arterial wall cells can also reduce the oxidation of low-density lipoprotein (LDL). Medical experiments have shown that natural lutein and zeaxanthin can scavenge peroxynitrite, reduce adhesion molecules on the surface of aortic endothelial cells, and play an important role in preventing atherosclerosis. People who consume more lutein tend to have a lower incidence of coronary heart disease or stroke, indicating that lutein may have a therapeutic effect on heart disease. A study of the relationship between changes in the thickness of the intima of the main carotid artery and lutein levels in the blood found that after 18 months of testing, the thickness of the carotid artery wall in the group with higher lutein levels in the blood hardly changed, while the thickness of the blood vessel wall in the group with lower lutein levels in the blood increased significantly.

2.5 Lutein's role in preventing diabetes and its complications

Many studies have shown that diabetes is associated with increased oxidative stress and decreased antioxidant capacity in the body. Free radical-induced biochemical reactions play an important role in the development of diabetes. Oxidative stress can also be caused in the state of diabetes, which can lead to or promote damage to various tissues such as the heart, brain, and kidneys, as well as diabetes complications [30]. Zhang Qing et al. [31] showed that the serum antioxidant enzyme levels and serum lutein, lycopene and β2-carotene content of type 2 DM patients were significantly lower than those of the normal group, and the MDA content was higher, reflecting hyperoxidation stress and reduced antioxidant capacity of the body in diabetes.

COYNE T et al. [32] showed that serum carotenoid levels were inversely proportional to type 2 diabetes, which is consistent with the findings of ZHANG Q et al. ZHANG Q et al. [33] found in an earlier study that lutein can alleviate renal damage in diabetic rats. The mechanism may be related to lutein's ability to increase the activity of antioxidant enzymes, and reduce the expression of pro-inflammatory cytokines. Hu Bojie et al. [34] concluded from clinical observations that the serum lutein and zeaxanthin concentrations of patients with simple diabetic retinopathy (DR) were significantly lower than those of normal people, and that supplementation could improve this.

2.6 Lutein application in food and feed additives

As synthetic pigments are harmful to human health, lutein from biological sources has become a natural coloring agent. It has been listed as a food coloring agent and nutrient in Europe and the United States, and is used in eye-benefiting drinks and baby food [35]. The wide application of lutein in feed additives is mainly reflected in the following aspects: (1) coloring: studies on lutein have been reported in the literature, for example, on lutein-enriched chicken feed, which improves the color and quality of egg yolk and white [36-38]. Lutein can also give fish eggs yellow, orange and red colors.

Leng Xiangjun et al. [39] found that adding lutein to the feed can significantly improve the body color of goldfish, increase the lutein content in the scales, skin, muscles and tail fins, and make the goldfish body color more vibrant. In a farm in Hokkaido, Japan, the golden yellow carotenoid extracted from marigold petals is used as a coloring agent to feed rainbow trout, and the scales of the rainbow trout turn yellow, which is extremely attractive. This method not only improves the meat quality of rainbow trout, but also increases its nutritional value. (2) Improve the fertilization rate and hatchability of eggs: Studies have found that the circulatory system and vascular zone of embryos in eggs with high lutein content develop faster.

Lutein in egg yolks can also promote the accumulation of large amounts of vitamin A and glycogen in the embryo's liver, promote the absorption of lipids in the embryo's liver, and improve the fertilization rate and hatchability of eggs [40]. Aren et al. [41] showed that adding lutein to the feed can significantly improve the fertilization rate and hatchability of quail eggs, reduce the rate of dead embryos, and thus improve their reproductive performance. (3) Improves immunity: Lutein can enhance the reproduction, survival and immune capacity of livestock, fish and shrimp, and also protect lipids from oxidation. BEDECARRATS G Y et al. [42-43] found that lutein can stimulate the antibody response of laying hens to immunize against bronchitis virus. Tian Heshan et al. [41] showed that adding lutein can increase the activity of antioxidant enzymes in the liver of chicks and reduce the content of lipid peroxide MDA.

3 Summary

Lutein research has a history of more than 10 years, and some major foreign companies are at the forefront of research in the development and application of lutein. In recent years, universities and natural product research institutes in China have also carried out lutein research. However, compared with the research progress abroad, there is still a certain gap between the domestic research and production levels. Developing and producing high-purity lutein, broadening its application fields, and industrializing it will be an important task for Chinese researchers in the future. At present, research on the physiological effects of lutein lacks sufficient molecular biological evidence. There is also no obvious statistical evidence to support it. However, with the deepening of understanding of the relevant mechanisms and the rapid progress of technical means, the mechanism of action of lutein will gradually be revealed, laying a foundation for better application of lutein.

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