DHA-lutein ester for maintaining eye health, its preparation method and application

Abstract

The present invention discloses DHA-lutein ester, its preparation method and application. The DHA-lutein ester is DHA-lutein monoester and / or DHA-lutein diester, and can be applied to the preparation of a stable visual function improver that effectively maintains eye health. According to the present invention, synthesizing DHA and lutein into DHA-lutein ester not only improves the stability and bioavailability of lutein, but also combines the advantages of both, and DHA assists lutein to have a synergistic effect on the macula of the retina, thereby maintaining eye health and improving visual function. Furthermore, the DHA-lutein ester can be processed into an oily suspension or microencapsulated powder, and is expected to be well applied in the fields of food additives, dietary supplements, pet food, etc.

Description

[Technical Field]

[0001] The present invention relates to the applied technical field of pharmaceuticals / health foods (functional foods), and more particularly to lutein esters for maintaining eye health, their preparation methods and applications. [Background technology]

[0002] The retina, as a receptor for light signals, plays an important role in visual formation. After light reaches the retina, visual images are formed. However, prolonged exposure to strong visible light can cause damage to the retinal photosensitive cells. Damage to visual cells can progress to apoptosis and vision loss, but "light-induced retinal damage" usually resolves with functional recovery. With the development of science and technology, people are increasingly exposed to artificial light sources (e.g., LED lights, mobile phones, computers, and other devices). Photochemical damage from shorter wavelength visible light (e.g., blue light in the 400-550 nm range) is the most important and common form of light-induced retinal damage and is currently the focus of research. Research has shown that excessive exposure to blue light causes a significant increase in reactive oxygen species, leading to photoreceptor loss, lipid peroxidation, and apoptosis. The synergistic effects of blue light and N-retinylidene-N-retinylethanolamine (A2E) and bleaching light reversal further exacerbate photochemical damage, leading to the activation of inflammatory responses, DNA damage, and suppression of mitochondrial and lysosomal function. Blue light, in particular, not only harms the retina but also damages the ocular surface through oxidative stress and inflammatory responses, significantly affecting myopia. Many eye diseases are the result of the long-term effects of various factors. In addition to traditional surgical treatments and good eye habits, dietary modification is also an important method for improving vision and managing eye diseases. Therefore, there is increasing interest in maintaining eye health and improving vision through nutritional supplements and functional foods.

[0003] Lutein, the major carotenoid in the human macula and retina, is widely distributed in plants such as French marigold, pumpkin, and indigo. Its biological activities include preventing brain aging, maintaining vision, alleviating visual fatigue, and promoting eye health. Lutein protects the retina through two main pathways. First, it utilizes its reducing properties to quench singlet oxygen and scavenge reactive oxygen free radicals, thereby protecting light-sensitive cells. Second, it utilizes its blue light filtering properties to prevent blue light from reaching underlying retinal structures, thereby reducing the risk of light-induced oxidative damage. Furthermore, while lutein intake through dietary intake or dietary supplementation has been shown to be beneficial for ocular diseases, several studies have pointed out that its low bioavailability and instability at high temperatures and strong light conditions limit its application as a functional food. Lutein esters are a form of lutein found in nature and are a safe form of lutein obtained by esterifying lutein with one or two fatty acids (myristic acid, lauric acid, palmitic acid, etc.). Lutein esters are absorbed into the body, taken up by the small intestine, and hydrolyzed by trypsin into free fatty acids and lutein. Due to the emulsifying action of bile, components such as lutein, fatty acids, and cholate form mixed micelles, which are then absorbed into lymph and blood.

[0004] Docosahexaenoic acid (DHA), a marine-derived dietary omega-3 long-chain polyunsaturated fatty acid, possesses biological activities such as anti-inflammatory, antioxidant, cardiovascular disease prevention, and neuronal development promotion. It is also the major structural lipid in the outer segments of retinal photoreceptors. Tissue DHA status can affect retinal function by altering permeability, fluidity, thickness, and lipid phase properties, as well as by influencing retinal cell signaling mechanisms involved in phototransduction. Furthermore, DHA may promote the activation of membrane-bound retinal proteins and affect the signaling cascade involved in the regeneration of rhodopsin. While DHA supplementation can improve visual processing defects in some cases, the susceptibility of DHA to oxidation affects product quality. Summary of the Invention [Problem to be solved by the invention]

[0005] The problem to be solved by the present invention is that although lutein has biological activities such as maintaining visual acuity, relieving visual fatigue, and promoting eye health, its low bioavailability and instability under high temperatures and strong light limit its application in functional foods. [Means for solving the problem]

[0006] To solve the above problems, the present invention provides a DHA-lutein ester for maintaining eye health, its preparation method and application, and combines DHA and lutein to form DHA-lutein ester, which not only improves the stability and bioavailability of lutein, but also combines the advantages of both, thereby maintaining eye health and improving visual function through the synergistic action of DHA and lutein in the macula of the retina.

[0007] To achieve the above object, the present invention provides the following technical solution, which is a compound represented by the following structural formula, DHA-lutein ester, which maintains eye health: Lutein monoester: [ka] and / or Lutein diesters: [ka] [Wherein R is DHA] is esterified with lutein is]

[0008] The present invention increases the stability of the compound by introducing DHA into a lutein-related compound and esterifying the phenolic hydroxyl group of lutein to produce a new type of lutein ester derivative, thereby alleviating the problem of DHA's susceptibility to oxidation.

[0009] The combination of DHA and lutein increases the optical density of macular pigment in humans more than lutein alone, protecting human retinal pigment epithelial cells (RPE cells) from attack by reactive oxygen species while increasing plasma lutein levels. DHA also promotes lutein accumulation in the blood and macula, exerting a synergistic effect with lutein in maintaining eye health. However, when lutein or DHA is ingested alone, most of the lutein or DHA is excreted during absorption, while only a small portion is absorbed into the body to exert its physiological effects. Lutein obtained by the degradation of lutein esters is more bioavailable and stable than free lutein, and is more advantageous for utilization by intestinal microorganisms (e.g., bifidobacteria and lactobacilli), alleviating symptoms of oxidative stress, significantly increasing the optical density of macular pigment, and improving eye function.

[0010] The monoester form of the compound of the present invention allows more lutein and DHA to be introduced into the body, where they are decomposed into lutein and DHA to exert their effects, thereby solving the problems of low activity, excessive metabolism, and low bioavailability of lutein in the body.

[0011] The diester form of the compound of the present invention alters the composition of the intestinal microbial community, increasing the abundance of beneficial bacteria such as bifidobacteria and lactobacilli, and alleviating the symptoms of oxidative stress by modulating flora metabolites, thereby significantly increasing the optical density of retinal macular pigment.

[0012] Therefore, the compound of the present invention not only exhibits the synergistic effects of DHA and lutein in maintaining eye health and improving vision, but also avoids the drawbacks of lutein, such as low activity in the body, excessive metabolism, low bioavailability, and DHA's susceptibility to oxidation.

[0013] Furthermore, the DHA-lutein ester is one of DHA-lutein monoester and DHA-lutein diester, or a mixture of two or more of them.

[0014] Furthermore, the DHA-lutein ester has a DHA content of 30 to 60%.

[0015] Furthermore, the DHA-lutein ester can be applied to prepare a stable visual function improving agent that effectively maintains eye health.

[0016] Furthermore, the formulation contains the DHA-lutein ester at a pharmacologically effective concentration (1% to 100%).

[0017] Furthermore, the preparation is a drug or biological preparation, and the method of use of the drug is to be taken orally.

[0018] Furthermore, the preparation is a food product, such as a special medical food, a health food, a functional food, or a dietary supplement, including milk, beverages, baked goods, etc.

[0019] The present invention also discloses a method for preparing the eye health-maintaining DHA-lutein ester, comprising the steps of:

[0020] (1) Lutein and free DHA were weighed, and dichloromethane, N,N-dimethyl-4-aminopyridine (DMAP) catalyst, and ethyldimethylaminopropylcarbodiimide hydrochloride (EDCI) were added. The mixture was then flushed with nitrogen and placed in a water bath for shaking. After the reaction, the reaction mixture was transferred to a beaker, dichloromethane was added, and the mixture was washed sequentially with dilute hydrochloric acid, saturated sodium bicarbonate, and saturated sodium chloride. The organic phase was recovered and evaporated under reduced pressure while rotating until all the dichloromethane was gone, yielding the crude DHA-lutein ester product. The crude product contained two compounds, DHA-lutein monoester and DHA-lutein diester, with a conversion rate of over 90%.

[0021] (2) The silica gel is activated and thoroughly dissolved in n-hexane, then packed into a column. The crude product obtained in step (1) is dissolved in n-hexane and gradually added to the silica gel column. The column is eluted sequentially with n-hexane and a mixed reagent of n-hexane and acetone, and the eluate is collected. During the elution process, the separation of DHA-lutein monoester and DHA-lutein diester in the eluate is confirmed by silica gel thin-layer chromatography, and the DHA-lutein monoester eluate and the DHA-lutein diester eluate are collected, respectively. The mixture is concentrated under reduced pressure at low temperature, away from light, and the DHA-lutein monoester and DHA-lutein diester are collected, respectively.

[0022] Furthermore, under the reaction conditions of nitrogen filling in step (1), other inert gases can be used instead of nitrogen.

[0023] Furthermore, in step (1), the mixture is shaken in a water bath at 25°C to 30°C, and the reaction time is 1 to 8 hours.

[0024] In step (2), the crude product obtained in step (1) is sequentially eluted with n-hexane, a 9:1 volumetric ratio n-hexane / acetone solution, and an 8:2 volumetric ratio n-hexane / acetone solution. A certain column volume of n-hexane eluate, a 5 column volume of n-hexane / acetone eluate with a 9:1 volumetric ratio n-hexane / acetone solution, and a 5 column volume of n-hexane / acetone eluate with a 8:2 volumetric ratio n-hexane / acetone solution are obtained, respectively. The 9:1 volumetric ratio n-hexane / acetone eluate contains DHA-lutein diester, while the 8:2 volumetric ratio n-hexane / acetone eluate contains DHA-lutein monoester.

[0025] Furthermore, in step (1), the amounts of DMAP and EDCI added are both about 0.2 to 2 g / g lutein, the amount of DHA added is about 1.5 to 3 g / g lutein, and the concentration of lutein in the solvent is 5 to 100 mg / mL.

[0026] Specifically, the present invention provides the following. [1] Lutein monoester, a compound with the following structural formula: [ka] and / or lutein diesters: [ka] [Wherein R is DHA] is esterified with lutein is] DHA-lutein ester represented by the formula:

[0027] [2] The DHA-lutein ester according to [1], characterized in that the DHA content is 30 to 60%.

[0028] [3] Preparation method: Lutein and free DHA are weighed, and dichloromethane, DMAP catalyst, and EDCI are added, followed by reaction under an inert gas atmosphere. The DHA-lutein ester according to [1], which is prepared by

[0029] [4] The DHA-lutein ester according to [3], characterized in that the amounts of DMAP and EDCI added are both approximately 0.2 to 2 g / g lutein, the amount of DHA added is approximately 1.5 to 3 g / g lutein, and the lutein concentration in the solvent is 5 to 100 mg / mL.

[0030] [5] The DHA-lutein ester according to [3], characterized in that the reaction conditions for preparing the DHA-lutein ester are to place the mixture in a water bath at 30°C and shake it, and the reaction time is 1 to 8 hours.

[0031] [6] Purification methods including: 1) Add dichloromethane to the reaction mixture, then wash with dilute hydrochloric acid solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution in that order, and recover the organic phase; 2) Evaporate under reduced pressure while rotating until no dichloromethane remains, and obtain the crude DHA-lutein ester product. 3) After the activated silica gel is thoroughly dissolved in n-hexane and packed into a column, the crude product obtained in step 2) is dissolved in n-hexane and gradually added to the silica gel column, and the column is eluted successively with n-hexane and a mixed reagent of n-hexane / acetone solution, and the eluate is collected. 4) Concentrate at low temperature, away from light, and under reduced pressure. The DHA-lutein ester according to [3], characterized in that it is purified by

[0032] [7] The purification method comprises the steps of: In step 3), the DHA-lutein diester eluate and the DHA-lutein monoester eluate are respectively collected by sequentially eluting with n-hexane, an n-hexane / acetone solution in a volume ratio of 9:1, and an n-hexane / acetone solution in a volume ratio of 8:2. In step 4), the mixture is concentrated under reduced pressure at low temperature and away from light to obtain DHA-lutein diester and DHA-lutein monoester, respectively. The DHA-lutein ester according to [6],

[0033] [8] A method for preparing DHA-lutein ester according to [1], comprising the steps of: Lutein and free DHA are weighed out, and dichloromethane, DMAP catalyst, and EDCI are added, followed by reaction under an inert gas atmosphere. The preparation method comprising:

[0034] [9] The method for preparing DHA-lutein ester according to [8], characterized in that the amounts of DMAP and EDCI added are both approximately 0.2 to 2 g / g lutein, the amount of DHA added is approximately 1.5 to 3 g / g lutein, and the lutein concentration in the solvent is 5 to 100 mg / mL.

[0035]

[10] The method for preparing DHA-lutein ester according to [8], characterized in that the reaction conditions are to place the mixture in a water bath at 30°C and shake it, and the reaction time is 1 to 8 hours.

[0036]

[11] The post-preparation purification method comprises the steps of: 1) Add dichloromethane to the reaction mixture, then wash with dilute hydrochloric acid solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution in that order, and recover the organic phase; 2) Evaporate under reduced pressure while rotating until no dichloromethane remains, and obtain the crude DHA-lutein ester product. 3) After the activated silica gel is thoroughly dissolved in n-hexane and packed into a column, the crude product obtained in step 2) is dissolved in n-hexane and gradually added to the silica gel column, and the column is eluted sequentially with n-hexane and a mixed reagent of n-hexane / acetone solution, and the eluate is collected. 4) Concentrate at low temperature, away from light, and under reduced pressure. The method for preparing DHA-lutein ester according to [8], comprising:

[0037]

[12] The purification method comprises: In step 3), the DHA-lutein diester eluate and the DHA-lutein monoester eluate are respectively collected by sequentially eluting with n-hexane, an n-hexane / acetone solution in a volume ratio of 9:1, and an n-hexane / acetone solution in a volume ratio of 8:2. In step 4), the mixture is concentrated under reduced pressure at low temperature and away from light to obtain DHA-lutein diester and DHA-lutein monoester, respectively. The method for preparing DHA-lutein ester according to

[11] ,

[0038]

[13] A formulation containing the DHA-lutein ester described in [1].

[0039]

[14] The formulation according to

[13] , characterized in that the formulation is an oil suspension or a microcapsule powder.

[0040]

[15] The formulation according to

[13] , characterized in that it contains DHA-lutein ester at an effective concentration of 1% to 100%.

[0041]

[16] The preparation according to

[13] , characterized in that the purity of DHA-lutein ester is 90% or more.

[0042]

[17] The preparation according to

[13] , which is a drug or biological preparation and is used by oral administration.

[0043]

[18] The formulation according to

[13] , which is a food product and is a special medical food, health food, functional food, nutritional supplement, food additive, pet food, or pet food additive.

[0044]

[19] Application of the DHA-lutein ester described in [1] in the preparation of a stable visual function improving agent that effectively maintains eye health.

[0045]

[20] The application according to

[19] , characterized in that the formulation is an oil suspension or a microcapsule powder.

[0046]

[21] The application according to

[19] , characterized in that the effective concentration of DHA-lutein ester is 1% to 100%.

[0047]

[22] The application according to

[19] , characterized in that the purity of the DHA-lutein ester is 90% or more.

[0048]

[23] The application according to

[19] , characterized in that the drug or biological preparation is taken orally.

[0049]

[24] The application according to

[19] , characterized in that the food is a special medical food, a health food, a functional food, a nutritional supplement, a food additive, a pet food, or a pet food additive. [Effects of the Invention]

[0050] The beneficial effects of the present invention are as follows: (1) The present invention provides a product with a purity of 90% or more, which is obtained by synthesizing DHA-lutein esters using lutein and free DHA as starting materials, followed by separation and purification. (2) The preparation method provided by the present invention is low cost, has simple steps, uses a small amount of organic solvent, and causes less environmental pollution due to organic solvents. (3) Animal experiments have shown that the DHA-lutein ester preparation prepared by the method of the present invention has a more effective effect on maintaining eye health than lutein alone. DHA-lutein ester can be further processed into an oil suspension or microencapsulated powder, and is expected to be useful in fields such as food additives, nutritional supplements, and pet food. [Brief explanation of the drawings]

[0051] [Figure 1] FIG. 1 is an explanatory diagram showing a thin layer chromatograph of the reaction crude product components of the present invention. [Figure 2] High-performance liquid chromatographs of the free lutein standard and the purified reaction products, where (a) shows free lutein, (b) shows DHA-lutein monoester, and (c) shows DHA-lutein diester. DETAILED DESCRIPTION OF THE INVENTION

[0052] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described in the following examples. It is obvious that the embodiments described in the present invention are only some embodiments, not all of the embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without any creative work fall within the protection scope of the present invention.

[0053] Materials and reagents used in the following examples were purchased commercially unless specifically stated. [Example]

[0054] Example 1: Preparation of DHA-lutein ester 500 mg of lutein, 1200 mg of DHA, 800 mg of EDCI, and 200 mg of DMAP were weighed and added to 5 mL of dichloromethane. The mixture was then flushed with nitrogen and placed in a 25°C water bath with shaking for 1 hour. After the reaction, the reaction mixture was transferred to a beaker and washed with 10 mL of dichloromethane, 100 mL of dilute hydrochloric acid, 100 mL of saturated sodium bicarbonate, and 100 mL of saturated sodium chloride, followed by separation and recovery of the organic phase. The dichloromethane was evaporated under reduced pressure while rotating, and the organic solvent was removed to yield a crude DHA-lutein ester product. The crude product contained two compounds, DHA-lutein monoester and DHA-lutein diester, with a conversion rate of over 90%.

[0055] The crude product was purified by silica gel column chromatography. Silica gel was activated, thoroughly dissolved in n-hexane, and then packed into a column. The resulting crude product was dissolved in n-hexane and slowly added to the silica gel column. The column was sequentially eluted with n-hexane, a 90 / 10 n-hexane / acetone mixture, and an 80 / 20 n-hexane / acetone mixture, and the eluate was collected. During the elution process, the separation of DHA-lutein diester and DHA-lutein monoester in the eluate was confirmed by silica gel thin-layer chromatography, and the DHA-lutein diester eluate and DHA-lutein monoester eluate were collected, respectively.

[0056] The mixture was concentrated under reduced pressure at low temperature, avoiding light, to remove dichloromethane and dry, and then stored at -20°C, avoiding light. The recovered n-hexane / acetone (90 / 10) eluate was a DHA-lutein diester solution, and the recovered n-hexane / acetone (80 / 20) eluate was a DHA-lutein monoester solution.

[0057] Example 2: Detection of DHA-lutein esters 1. Method 1.1 Detection by thin-layer chromatography The crude reaction product of Example 1 was preliminarily detected by thin layer chromatography using a 4:1 volumetric ratio n-hexane / acetone solution as the developer. 10 mL of developer was weighed out and added to the tank, which was then sealed and allowed to equilibrate for 30 minutes. A 0.3 x 100 mm capillary was used to dispense the sample in a spot-like fashion, with the sample line positioned 1 cm from the bottom of the silica gel substrate. The sample was dispensed multiple times. During the sample dispensing process, the solvent was quickly blown dry. When the solvent front had moved 1 cm from the top of the silica gel substrate, the silica gel substrate was removed and allowed to retain its natural color at room temperature.

[0058] 1.2 Detection by high-performance liquid chromatography Chromatography conditions Chromatography column: YMC C30 (4.6 mm × 250 mm, 3 μm), Mobile phase A: chromatographically pure methanol, Mobile phase B: chromatographically pure methyl tert-butyl ether, Gradient elution: flow rate 1.0 mL / min, Detection wavelength: 450 nm, Column temperature: 30°C, Specimen injection volume: 10 μL.

[0059] 2. Experimental Results 2.1 Thin-layer chromatography results The developer used in Example 2 effectively separated free lutein, DHA-lutein monoester, and DHA-lutein diester. The results of the natural color thin-layer chromatography are shown in Figure 1. Three bands appeared on the TLC plate, with Rf values ​​of 0.90, 0.31, and 0.08, respectively. Based on the bands of the free lutein standard, the band with an Rf value of 0.08 was determined to be free lutein. When the hydroxyl groups at both ends of lutein react with DHA to form an ester, the resulting product becomes less polar. In this case, DHA-lutein diester, in which both hydroxyl groups at both ends of lutein are esterified, is even less polar than DHA-lutein monoester, in which only one hydroxyl group at one end of lutein is esterified. Therefore, based on initial judgment, the band with an Rf value of 0.31 is DHA-lutein monoester, and the band with an Rf value of 0.90 is DHA-lutein diester.

[0060] 2.2 High-performance liquid chromatography results Figure 2 shows high-performance liquid chromatographs of the lutein standard, DHA-lutein monoester, and DHA-lutein diester purified by column chromatography in Example 1. Figure 2(a) shows the chromatographic peaks of the free lutein standard, with the peak appearing between 3 and 4 minutes. The peak in Figure 2(b) appears between 5 and 6 minutes. The peak in Figure 2(c) appears between 11 and 12 ...

[0061] [Example 3] Repairing effect of DHA-lutein ester on retinal damage caused by blue light 1. Method 1.1 Preparation of emulsion for intragastric administration Purified DHA-lutein ester or free lutein was added to a mixture of 90% saline and 10% corn oil (wt / wt), followed by the addition of 0.1% pig bile extract. The mixture was placed in an ice bath and sonicated to promote dissolution. Finally, a 10 mg / mL lutein emulsion was prepared and administered intragastrically to rats.

[0062] 1.2 Animal experiments Seventy SD rats were fed a dietary adaptation regimen for one week. Ten rats served as a control group and were not subjected to blue-light damage. Sixty rats were anesthetized with a 30 mg / kg body weight injection of pentobarbital sodium. Subsequently, the rats' pupils were dilated with compound tropicamide eye drops, and the injury was induced by intense blue light. After injury, the rats were randomly divided into six groups and intragastrically administered saline, lutein emulsion, DHA-added saline, a mixed DHA and lutein emulsion, a DHA-lutein monoester emulsion, and a DHA-lutein diester emulsion. These groups were designated the model group, lutein group, DHA group, lutein + DHA group, DHA-lutein monoester group, and DHA-lutein diester group. After one consecutive week of intragastric administration, electrograms of the rat retina were obtained.

[0063] 2. Results [Table 1] NOTE: a, b, c, d, e, f are different letters and indicate significant differences (P < 0.05) between the data.

[0064] Table 1 shows the changes in the a-wave and b-wave amplitudes of the electrographs of rat retina one week after intragastric administration. The a-wave is mainly due to the photoreceptor potential of photosensitive cells. The b-wave amplitude is larger and is mainly related to the activity of bipolar cells. From the table, the a-wave and b-wave amplitudes significantly decreased after strong blue light damage, indicating the success of the model construction.

[0065] After administration of DHA or lutein alone, the a-wave and b-wave amplitudes increased to some extent, but the degree of increase was much less than that of the lutein + DHA group and the DHA-lutein ester group, indicating that the combined effects of DHA and lutein, and the effects of using DHA and lutein combined to form DHA-lutein ester, are significantly better than those of DHA or lutein alone. The DHA-lutein ester group showed significantly higher increases in a-wave and b-wave amplitudes than the lutein + DHA group, indicating that the DHA-lutein ester obtained by the present invention effectively protects the eyes from blue light and maintains eye health. The DHA-lutein diester group showed significantly higher increases in a-wave and b-wave amplitudes than the DHA-lutein monoester group, demonstrating the even better effects of DHA-lutein diester.

[0066] [Example 4] Effect of DHA-lutein ester on improving visual acuity in myopic animals 1. Method 1.1 Preparation of emulsion for intragastric administration Purified DHA-lutein ester or free lutein was added to a mixture of 90% saline and 10% corn oil (wt / wt), followed by the addition of 0.1% pig bile extract. The mixture was placed in an ice bath and sonicated to promote dissolution, followed by cell disruption. A final emulsion with a lutein concentration of 10 mg / mL was prepared and administered intragastrically to young rabbits.

[0067] 1.2 Animal experiments The animals were divided into seven groups of five each: a control group, a model group, and five experimental groups (lutein group, DHA group, lutein + DHA group, DHA-lutein monoester group, and DHA-lutein diester group). The model group and five experimental groups were housed in experimental cages tightly enclosed with thick floral-patterned cloth, with the floral side facing downwards. The cage was divided vertically and horizontally by the thick floral-patterned cloth to form multiple square compartments. Each square was initially large enough to accommodate one infant rabbit. The infant rabbits were allowed free movement, and their visual distance (depth) was generally set to approximately 5–10 cm initially. As the infant rabbits grew, the cage volume was gradually expanded, and the square compartments were accordingly enlarged; however, the visual distance should always be maintained at approximately 5–10 cm. To force the young rabbits to use their eyes for close distances, the food was cut into small pieces and mixed with twice the amount of gravel as the food, allowing the young rabbits to select food from the gravel. The feeding bottles, feeders, and water containers for the nursing young rabbits were all placed outside the rectangular enclosure, covered with thick floral fabric, so the young rabbits could only reach out to eat or drink. To provide multiple sources of stimulation, the young rabbits were required to observe multiple objects with their eyes. The positions of the feeding bottles, feeders, and water containers were constantly changed every 3–5 days, training the young rabbits in each experimental group to select food with their eyes. The cages of the five young rabbits in the control group were left uncovered, and no visual stimulation or visual distance restriction measures were used. After the experiment had progressed for 25 weeks, the pupils were dilated with 1% atropine solution, and the eyelids were dilated using a homemade eye dilator. The refractive index (D) of the left and right eyes of the young rabbits in each group was measured using a computerized ophthalmometer. At the same time, the naked eye foraging distance (cm) and body weight (g) were measured using a tape measure.

[0068] After confirming the success of the model construction, the emulsion was administered intragastrically to each group, and continued for one month. During the intragastrical administration period, the infant rabbits in each group were housed in a room with the same natural lighting and natural environment. The upper and lower eyelids, conjunctiva, cornea, sclerocorneal junction, anterior chamber, and iris were observed at the same time every day, and comprehensive examinations were performed using a slit lamp once a week. In addition, computerized ophthalmoscopy was used to measure the treatment results, and the treatment effects were statistically calculated according to the above method.

[0069] result Tables 2 and 3 show the results of the myopic infant rabbit model at 25 weeks of experimentation, and the changes in refraction, feeding distance, and weight of the myopic infant rabbits after one month of intragastric administration. Table 2 shows that infant rabbits raised in a weak light environment with limited visual distance during their growth period can only see close objects with both eyes and do not need to see distant objects. By selecting food in the close range, especially for long periods of time, the eyes objectively develop to adaptively see close objects, and the myopic refraction (D) values ​​measured by computer ophthalmology decrease, indicating that the construction of the myopic model was successful.

[0070] After drug administration, the refraction of the eyes in all experimental groups increased. The improved or enhanced visual acuity, combined with an improved growth environment, promoted the growth and development of the young rabbits and increased their foraging distance with the naked eye. The increase in refraction of the DHA-lutein monoester and DHA-lutein diester groups was much greater than that of the other groups, indicating that DHA-lutein ester had a significant effect on improving visual acuity. While there was no significant difference between the results of the DHA-lutein monoester and DHA-lutein diester groups, the refraction (D) and foraging distance of the DHA-lutein diester group were greater than those of the DHA-lutein monoester group, indicating the superior effect of DHA-lutein diester.

[0071] [Table 2]

[0072] [Table 3] NOTE: Different letters in a, b, c, d indicate significant differences (P < 0.05) between the data.

[0073] In summary, by synthesizing DHA-lutein ester using DHA and lutein as starting materials, it can effectively protect eyes from blue light, maintain eye health, and improve vision, and has good application prospects.

[0074] Although specific examples of the present invention have been described above, it should be understood that the present invention is not limited to the specific embodiments described above. Those skilled in the art can make various modifications and alterations within the scope of the claims without affecting the essential content of the present invention.

Claims

1. Lutein monoesters of the following structural formula: 【Chemistry 1】 and / or lutein diester: 【Chemistry 2】 [wherein R is a residue of DHA esterified with lutein] A method for preparing a DHA-lutein ester represented by the formula: Lutein and free DHA were weighed out, and dichloromethane, DMAP catalyst, and EDCI were added, followed by reaction under an inert gas atmosphere. A method for preparing the DHA-lutein ester, comprising:

2. Additionally, the following steps: 1) adding dichloromethane to the reaction mixture obtained after the reaction under an inert gas atmosphere, and then washing the mixture with a dilute hydrochloric acid solution, a saturated sodium bicarbonate solution, and a saturated sodium chloride solution in that order, and recovering the organic phase; 2) Evaporate under reduced pressure while rotating until dichloromethane is gone, and obtain the crude DHA-lutein ester product. 3) After the activated silica gel is thoroughly dissolved in n-hexane and packed into a column, the crude product obtained in step 2) is dissolved in n-hexane and gradually added to the silica gel column, and the column is eluted successively with n-hexane and a mixed reagent of n-hexane / acetone solution, and the eluate is collected.

2. A method for preparing the DHA-lutein ester of claim 1, comprising:

3. Lutein monoesters of the following structural formula: 【Chemistry 1】 and / or lutein diester: 【Chemistry 2】 [wherein R is a residue of DHA esterified with lutein] A preparation for increasing myopic refraction of an eye, comprising a DHA-lutein ester represented by the formula:

4. A preparation for increasing the myopic refraction of the eye as described in claim 3, which is a drug or biological preparation, characterized in that the method of use is to take it orally and the concentration of the DHA-lutein ester is 10 mg / mL lutein concentration.

5. The preparation for increasing myopic refraction of the eye according to claim 3, characterized in that it is a special medical food, health food, functional food, nutritional supplement, food additive, pet food or pet food additive.

6. Lutein monoesters of the following structural formula: 【Chemistry 1】 and / or lutein diester: 【Chemistry 2】 [wherein R is a residue of DHA esterified with lutein] The use of DHA-lutein ester represented by the formula (I) in the preparation of a stable visual function improving agent which increases myopic refraction of the eye and effectively maintains eye health.

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