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 belongs to the applied technical field of drugs / health foods (functional foods) in the pharmaceutical field, and specifically relates to lutein esters for maintaining eye health, a preparation method thereof, and applications.

Background Art

[0002] The retina, as a receptor of light signals, plays an important role in visual formation. After light reaches the retina, a visual image is formed. However, due to the long-term stimulation of strong visible light, the photosensitive cells of the retina can be damaged. The damage of visual cells may progress to apoptosis or vision loss, but "light-induced retinal damage" can disappear with the recovery of function. With the development of science and technology, people are increasingly exposed to artificial light sources (such as LED lights, mobile phones, computers, and other devices), and photochemical damage caused by visible light with shorter wavelengths (such as blue light with a wavelength of 400 - 550 nm) is the most important and common among light-induced retinal damage and is currently the focus of research. Excessive exposure to blue light causes a significant increase in reactive oxygen species, and it has been pointed out in research that it can cause photoreceptor loss, lipid peroxidation, and apoptosis. The synergistic effect of blue light and N-retinylidene-N-retinylethanolamine (A2E) and bleached photoreversal further exacerbate photochemical damage, causing the activation of inflammatory reactions, DNA damage, and the suppression of the functions of mitochondria and lysosomes. In particular, blue light not only harms the retina but also damages the surface of the eyeball through oxidative stress and inflammatory reactions, and has a great impact on myopia. Many eye diseases are the result of the long-term effects of various factors. In addition to conventional surgical treatments and good eye-using habits, dietary improvement is also an important method for improving vision and dealing with eye diseases. Therefore, maintaining eye health and improving vision with nutritional supplements or functional foods are attracting more and more attention.

[0003] Lutein is a major carotenoid in the human macula and retina, but it is widely present in plants such as French marigold, pumpkin, and cabbage, and has biological activities such as preventing brain aging, maintaining vision, relieving visual fatigue, and promoting eye health. Lutein mainly protects the retina through two pathways. One is to utilize the reducibility of lutein to eliminate singlet oxygen and capture reactive oxygen free radicals, thereby exerting the effect of protecting photoreceptor cells. The other is to utilize the filtering effect on blue light to prevent blue light from reaching the underlying structure of the retina, thereby reducing the risk of light-induced oxidative damage. Furthermore, although the intake of lutein as a diet or dietary supplement is beneficial for eye diseases, several studies have pointed out that its low bioavailability and instability under high temperature and strong light limit the application of lutein as a functional food. Lutein ester is a form of lutein existing in nature and is one of the safe forms of lutein obtained by esterifying lutein with 1 or 2 fatty acids (such as myristic acid, lauric acid, palmitic acid, etc.). Lutein ester is 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 and enter the lymph and blood.

[0004] Docosahexaenoic acid (DHA) is a marine-derived dietary omega-3 long-chain polyunsaturated fatty acid, which has biological activities such as anti-inflammatory, antioxidant, cardiovascular disease prevention, and promotion of neuron development, and is also the main structural lipid of the outer segment of photoreceptor cells in the retina. The state of tissue DHA affects the retinal cell signal transduction mechanism involved in light information transmission and can also affect retinal function by changing permeability, fluidity, thickness, and lipid phase characteristics. Furthermore, DHA may promote the activation of membrane-bound retinal proteins and act on the signal cascade involved in the regeneration of rhodopsin. In some cases, visual processing defects can be improved by DHA supplementation, but the problem of easy oxidation of DHA affects the quality of the product.

Summary of the Invention

Problems to be Solved by the Invention

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

Means for Solving the Problems

[0006] To solve the above problems, the present invention provides a DHA-lutein ester for maintaining eye health, a preparation method thereof, and an application. By synthesizing DHA and lutein into DHA-lutein ester, not only the stability and bioavailability of lutein are improved, but also the advantages of both are combined. Through the synergistic effect of DHA and lutein in the macula of the retina, eye health is maintained and visual function is improved.

[0007] To achieve the above object, the present invention is achieved by the following technical solution, and is a compound represented by the following structural formula, namely, a DHA-lutein ester for maintaining eye health. Lutein monoester:

Chemical formula

Chemical formula

[0008] The present invention introduces DHA into lutein compounds, esterifies the phenolic hydroxyl groups of lutein, and then generates a new type of lutein ester derivative, thereby increasing the stability of the compound and improving the problem that DHA is easily oxidized.

[0009] The combination of DHA and lutein can further increase the optical density of macular pigment in the human body, protect human retinal pigment epithelial cells (RPE cells) from the attack of reactive oxygen species, and increase the lutein level in plasma, compared with using lutein alone. In addition, DHA can promote the accumulation of lutein in the blood and in the macula, and exert a synergistic effect with lutein in maintaining eye health. However, when lutein or DHA is ingested alone, most of lutein and DHA are excreted during the process of entering the body, and only a very small part is taken into the body to exert physiological effects. Lutein obtained by the decomposition of lutein ester has higher bioavailability and stability compared with free lutein, can be utilized by intestinal microorganisms (such as Bifidobacterium and Lactobacillus), relieve the symptoms of oxidative stress, significantly increase the optical density of retinal macular pigment, and is more advantageous for improving eye function.

[0010] After bringing more lutein and DHA into the body in the form of the monoester of the compound of the present invention, it decomposes into lutein and DHA in the body to exert effects, and solves the problems of low activity, excessive metabolism, and low bioavailability of lutein in the body.

[0011] In the form of the diester of the compound of the present invention, the composition of the intestinal microbiota is changed, the abundance of beneficial bacteria such as Bifidobacterium and Lactobacillus is increased, and the symptoms of oxidative stress are relieved by regulating the flora metabolites, so that the optical density of the retinal macular pigment is significantly increased.

[0012] Therefore, the compound of the present invention not only exerts the synergistic effect of DHA and lutein in maintaining eye health and improving eyesight, but also avoids the disadvantages such as low activity, excessive metabolism, low bioavailability of lutein in the body, and easy oxidation of DHA.

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

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

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

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

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

[0018] Furthermore, the preparation is a food, and is a food for special medical purposes, a health food, a functional food, or a dietary supplement. The functional foods include milk, beverages, baked foods, etc.

[0019] In addition, the present invention discloses a method for preparing the DHA-lutein ester for maintaining the eye health as described above, which includes the following steps.

[0020] (1) Weigh lutein and free DHA, add dichloromethane, N,N-dimethyl-4-aminopyridine (DMAP) catalyst, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), then fill with nitrogen and place in a water bath to shake. After the reaction, transfer the reaction mixture to a beaker, add dichloromethane, and wash successively with dilute hydrochloric acid solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution. Recover the organic phase, evaporate under reduced pressure while rotating until dichloromethane disappears, and a DHA-lutein ester crude product is obtained. The crude product contains two compounds, DHA-lutein monoester and DHA-lutein diester, and the reaction rate is 90% or more.

[0021] (2) Activate the silica gel, fully dissolve it in n-hexane, and then pack it into a column. Dissolve the crude product obtained in step (1) in n-hexane, and then gradually add it to the silica gel column. Elute it sequentially with a mixed reagent of n-hexane and n-hexane / acetone solution, and collect the eluate. During the elution process, confirm the separation state of DHA-lutein monoester and DHA-lutein diester in the eluate by silica gel substrate thin layer chromatography, and collect the DHA-lutein monoester eluate and DHA-lutein diester eluate respectively. Concentrate it under reduced pressure while avoiding light at low temperature, and recover DHA-lutein monoester and DHA-lutein diester respectively.

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

[0023] Furthermore, in step (1), place it in a water bath at 25°C to 30°C and shake it. The reaction time is 1 to 8 hours.

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

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

[0026] The present invention specifically includes the following: [1] A lutein monoester which is a compound of the following structural formula: [Chemical formula] and / or a lutein diester: [Chemical formula] [wherein, R is DHA] The 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] The following preparation method: Weigh lutein and free DHA, add dichloromethane, a DMAP catalyst and EDCI, and then react under an inert gas atmosphere to obtain The DHA-lutein ester according to [1], prepared by the method.

[0029] [4] The DHA-lutein ester according to [3], characterized in that the addition amounts of both DMAP and EDCI are about 0.2 to 2 g / g of lutein, the addition amount of DHA is about 1.5 to 3 g / g of 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 it in a 30°C water bath and shake it, and the reaction time is 1 to 8 hours.

[0031] [6] The following purification method: 1) Add dichloromethane to the reaction mixture, and then wash it successively with a dilute hydrochloric acid solution, a saturated sodium bicarbonate solution, and a saturated sodium chloride solution, and recover the organic phase. 2) Evaporate under reduced pressure while rotating until dichloromethane disappears to obtain a crude product of the DHA-lutein ester. 3) After the activated silica gel is sufficiently dissolved in n-hexane and packed into a column, the crude product obtained in step 2) is dissolved in n-hexane, gradually added to the silica gel column, and eluted sequentially with a mixed reagent of n-hexane and an n-hexane / acetone solution, and the eluate is collected. 4) Concentrate under reduced pressure while avoiding light at low temperature. The DHA-lutein ester according to [3], which is characterized by being purified by the above method.

[0032] [7] The purification method is as follows: In step 3), the DHA-lutein diester eluate and the DHA-lutein monoester eluate are respectively collected by eluting sequentially with n-hexane, an n-hexane / acetone solution with a volume ratio of 9:1, and an n-hexane / acetone solution with a volume ratio of 8:2. In step 4), DHA-lutein diester and DHA-lutein monoester are respectively obtained by concentrating under reduced pressure while avoiding light at low temperature. The DHA-lutein ester according to [6], which is characterized by the above method.

[0033] [8] A method for preparing the DHA-lutein ester according to [1], comprising the following steps: Weigh lutein and free DHA, add dichloromethane, a DMAP catalyst, and EDCI, and then react under an inert gas atmosphere to obtain. The above preparation method.

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

[0035]

[10] The reaction conditions are to place in a 30°C water bath and shake, and the reaction time is 1 - 8 hours. The preparation method of the DHA-lutein ester according to [8], which is characterized by the above method.

[0036]

[11] The purification method after the preparation includes the following steps: 1) After adding dichloromethane to the reaction mixture, it is successively washed with dilute hydrochloric acid solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution, and the organic phase is recovered. 2) After evaporating dichloromethane until it disappears while rotating under reduced pressure, a crude product of DHA-lutein ester is obtained. 3) After fully dissolving activated silica gel in n-hexane and filling it into a column, the crude product obtained in step 2) is dissolved in n-hexane and gradually added to the silica gel column, and eluted successively with a mixed reagent of n-hexane and n-hexane / acetone solution, and the eluate is recovered. 4) Concentrate under reduced pressure while avoiding light at low temperature. The preparation method of DHA-lutein ester according to [8], which includes the above steps.

[0037]

[12] The purification method is as follows: In step 3), by eluting successively with n-hexane, an n-hexane / acetone solution with a volume ratio of 9:1, and an n-hexane / acetone solution with a volume ratio of 8:2, an eluate of DHA-lutein diester and an eluate of DHA-lutein monoester are recovered respectively. In step 4), by concentrating under reduced pressure while avoiding light at low temperature, DHA-lutein diester and DHA-lutein monoester are obtained respectively. The preparation method of DHA-lutein ester according to

[11] , which is characterized by the above.

[0038]

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

[0039]

[14] The preparation according to

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

[0040]

[15] The preparation according to

[13] , characterized in that it contains DHA-lutein ester with 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 a biological preparation and is characterized in that the method of use is oral ingestion.

[0043]

[18] The preparation according to

[13] , which is a food and is characterized in that it is a food for special medical purposes, a health food, a functional food, a dietary supplement, a food additive, a pet food or a pet food additive.

[0044]

[19] Application of the DHA-lutein ester according to [1] in the preparation of a stable visual function improver that effectively maintains eye health.

[0045]

[20] The application according to

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

[0046]

[21] The application according to

[19] , characterized by containing DHA-lutein ester at an effective concentration of 1% to 100%.

[0047]

[22] The application according to

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

[0048]

[23] The application according to

[19] , which is a drug or a biological preparation and is characterized in that the method of use is oral ingestion.

[0049]

[24] The application according to

[19] , which is a food and is characterized in that it is a food for special medical purposes, a health food, a functional food, a dietary supplement, a food additive, a pet food or a pet food additive. [Advantages of the Invention]

[0050] The beneficial effects of the present invention are as follows. (1) The present invention is a product with a purity of 90% or more, obtained by synthesizing DHA-lutein ester using lutein and free DHA as starting materials, and further separating and purifying it. (2) The preparation method provided by the present invention has low cost, simple process, small amount of organic solvent used, and little environmental pollution caused by organic solvents. (3) In the verification of animal experiments, the DHA-lutein ester preparation prepared by the method of the present invention further improves the effect of maintaining eye health compared with lutein alone. DHA-lutein ester can be further processed into an oil suspension or microcapsule powder, and is expected to be well applied in the fields of food additives, dietary supplements, pet food, etc.

Brief Description of the Drawings

[0051]

Figure 1

Figure 2

Modes for Carrying Out 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 of the 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 creative labor belong to the protection scope of the present invention.

[0053] The materials and reagents used in the following examples were purchased from stores unless specifically stated.

Examples

[0054] [Example 1] Preparation of DHA-lutein ester Weighed 500 mg of lutein, 1200 mg of DHA, 800 mg of EDCI, and 200 mg of DMAP, put them into 5 mL of dichloromethane, filled with nitrogen, placed in a water bath at 25 °C and shaken, and reacted for 1 hour. After the reaction, the reaction mixture was transferred to a beaker, and 10 mL of dichloromethane, 100 mL of dilute hydrochloric acid solution, 100 mL of saturated sodium bicarbonate solution, and 100 mL of saturated sodium chloride solution were sequentially added for washing, and the layers were separated to recover the organic phase. Evaporated under reduced pressure while rotating until dichloromethane disappeared to remove the organic solvent and obtained a crude product of DHA-lutein ester. The crude product contained two compounds, DHA-lutein monoester and DHA-lutein diester, and the reaction rate was 90% or more.

[0055] The crude product was purified by silica gel column chromatography. The silica gel was activated, fully dissolved in n-hexane, and then filled into the column. The obtained crude product was dissolved in n-hexane, gradually added to the silica gel column, and eluted sequentially with n-hexane, a mixed reagent of n-hexane / acetone (90 / 10), and a mixed reagent of n-hexane / acetone (80 / 20) to recover the eluate. During the elution process, the separation state of DHA-lutein diester and DHA-lutein monoester in the eluate was confirmed by silica gel substrate thin layer chromatography, and the DHA-lutein diester eluate and the DHA-lutein monoester eluate were recovered respectively.

[0056] Concentrated at low temperature while avoiding light under reduced pressure, removed dichloromethane and dried, and stored at -20 °C while avoiding light. At that time, the recovered eluate of n-hexane / acetone (90 / 10) was a DHA-lutein diester solution, and the recovered eluate of n-hexane / acetone (80 / 20) was a DHA-lutein monoester solution.

[0057] [Example 2] Detection of DHA-lutein ester 1. Method 1.1 Detection by thin-layer chromatography An n-hexane / acetone solution with a volume ratio of 4:1 was used as the developing agent, and the crude reaction product of Example 1 was preliminarily detected by thin-layer chromatography. 10 mL of the developing agent was weighed and added to the tank, sealed, and equilibrated for 30 minutes. A 0.3×100 mm capillary was used to supply the sample in dots, with the sample line set 1 cm from the lower end of the silica gel substrate, and the sample supply was repeated multiple times. During the process of supplying the sample, the solvent was quickly blown dry. When the solvent front moved 1 cm to the upper end of the silica gel substrate, the silica gel substrate was taken out and presented 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: methanol of chromatographic purity, Mobile phase B: methyl tert-butyl ether of chromatographic purity, Gradient elution: flow rate 1.0 mL / min, Detection wavelength: 450 nm, Column temperature: 30 °C, Sample injection volume: 10 μL.

[0059] 2. Experimental results 2.1 Results of thin-layer chromatography The developing agent used in Example 2 was able to effectively separate free lutein, DHA-lutein monoester, and DHA-lutein diester. The detection results of naturally colored thin-layer chromatography are shown in Fig. 1. Three bands appeared on the TLC plate, and the Rf values were 0.90, 0.31, and 0.08 in order. By referring to the band of the free lutein standard, it was found that the band with an Rf value of 0.08 was free lutein. When the hydroxyl groups at both ends of lutein react with DHA and are esterified, the polarity of the product becomes lower. At this time, the polarity of DHA-lutein diester in which both hydroxyl groups at both ends of lutein are esterified is even lower than that of DHA-lutein monoester in which only one hydroxyl group at one end of lutein is esterified. Therefore, according to the 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 Results of High Performance Liquid Chromatography Fig. 2 is a high performance liquid chromatogram of lutein standard, DHA-lutein monoester and DHA-lutein diester purified by column chromatography of Example 1. Fig. 2(a) shows the chromatographic peak of free lutein standard, and the time when the peak appears is 3 - 4 min. The time when the peak in Fig. 2(b) appears is 5 - 6 min. Combining with the first mass spectrogram, it was judged to be DHA-lutein monoester, and its m / z is 897.4. The time when the peak in Fig. 2(c) appears is 11 - 12 min. Combining with the first mass spectrogram, it was judged to be DHA-lutein diester, and its m / z is 1125.8. The YMC C30 column is a reverse phase column, and the peak of the compound with a large polarity appears first, and the peak of the compound with a small polarity appears later. It is shown that the peaks of DHA-lutein monoester and DHA-lutein diester appear later than that of free lutein, indicating that the polarity of their products becomes smaller.

[0061] 〔Example 3〕Repair effect of DHA-lutein ester on blue light-induced retinal damage 1. Method 1.1 Preparation of Intragastric Administration Emulsion Purified DHA-lutein ester or free lutein was added to a mixed solution containing 90% normal saline and 10% corn oil (wt / wt). Then, 0.1% porcine bile extract was added to the whole solution. After placing it in an ice bath and promoting dissolution by ultrasonic waves, cells were disrupted. Finally, an emulsion with a lutein concentration of 10 mg / mL was prepared and administered intragastrically to rats.

[0062] 1.2 Animal Experiment 70 SD rats were allowed to adaptively feed for one week. 10 rats were used as the control group and were not subjected to light damage by blue light. 60 rats were anesthetized by injecting pentobarbital sodium injection at a dose of 30 mg / kg body weight. Then, the pupils of the rats were dilated with compound tropicamide eye drops and damaged by strong blue light. After the damage, the rats were randomly divided into 6 groups and intragastrically administered with normal saline, lutein emulsion, DHA-added normal saline, mixed emulsion of DHA and lutein, DHA-lutein monoester emulsion, and DHA-lutein diester emulsion, respectively, and were designated as the model group, lutein group, DHA group, lutein + DHA group, DHA-lutein monoester group, and DHA-lutein diester group. After continuous intragastric administration for one week, the electroretinogram of the rat retina was detected.

[0063] 2. Results

Table 1

[0064] Table 1 shows the changes in the a-wave amplitude and b-wave amplitude of the electroretinogram of the rat retina one week after intragastric administration. The a-wave is mainly due to the potential of the photoreceptors of photosensitive cells. The b-wave amplitude is larger and is mainly related to the activity of bipolar cells. From the previous table, it is shown that the a-wave amplitude and b-wave amplitude decreased significantly after strong blue light damage, indicating that the model structure was successful.

[0065] After administering DHA or lutein alone, the a-wave amplitude and b-wave amplitude increased to some extent, but the degree of increase was much less than that in the lutein + DHA group and the DHA-lutein ester group. Therefore, it is shown that the use effect of the combination of DHA and lutein and the use effect after synthesizing into DHA-lutein ester are significantly superior to the use effects of DHA or lutein alone. Since the degree of increase in the a-wave amplitude and b-wave amplitude in the DHA-lutein ester group was significantly higher than that in the lutein + DHA group, it is shown that the DHA-lutein ester obtained by the present invention effectively protects the eyes from blue light and has the effect of maintaining eye health. At that time, the degree of increase in the a-wave amplitude and b-wave amplitude in the DHA-lutein diester group was significantly higher than that in the DHA-lutein monoester group, and the effect of DHA-lutein diester was even better.

[0066] Example 4: Improvement effect of DHA-lutein ester on the vision of myopic animals 1. Method 1.1 Preparation of intragastric administration emulsion Purified DHA-lutein ester or free lutein was added to a mixed solution containing 90% physiological saline and 10% corn oil (wt / wt), and then 0.1% porcine bile extract was added to the whole. After placing it in an ice bath and promoting dissolution by ultrasonic waves, the cells were disrupted. Finally, an emulsion with a lutein concentration of 10 mg / mL was prepared and administered intragastrically to young rabbits.

[0067] 1.2 Animal experiment The experiment divided the animals into 7 groups of 5 each, namely a control group, a model group, and 5 experimental groups (lutein group, DHA group, lutein + DHA group, DHA - lutein monoester group, and DHA - lutein diester group). The young rabbits in the model group and the 5 experimental groups were each enclosed in an experimental cage where the flowered side of a thick flowered fabric was placed against the back and tightly covered. Inside the cage, multiple square compartments were formed by dividing it vertically and horizontally with thick flowered fabric. The size of each square compartment was initially set to be able to enclose one young rabbit. The young rabbits could move freely, and initially, the visual distance (depth) of the young rabbits was generally set to about 5 - 10 cm. As the young rabbits grew, the cage volume was gradually expanded, and accordingly, the square compartments were also expanded, but the visual distance needed to be constantly maintained at about 5 - 10 cm. Since the young rabbits were forced to use their eyes at close range, the food was cut into small pieces and mixed with twice the amount of gravel so that the young rabbits would select the food from the gravel. The feeding bottles, food boxes, and water tanks for the lactating young rabbits were all placed outside the enclosed square compartments surrounded by thick flowered fabric, and the young rabbits could only stretch their heads out to eat or drink. To stimulate them in many aspects, the young rabbits were made to observe many things with their eyes, and the positions of the feeding bottles, food boxes, and water tanks were changed every 3 - 5 days to train the young rabbits in each experimental group to select food with their eyes. The cages of the 5 young rabbits in the control group were not covered, and no measures were taken for visual stimulation or visual distance restriction. After the experiment had proceeded for 25 weeks, the pupils were dilated with a 1% atropine solution, and then a self - made eyelid retractor was used to expand the eyelids, and a computerized ophthalmoscope was used to measure the refractive index (D) values of the left and right eyes of the young rabbits in each group. At the same time, a measuring tape was used to measure the naked - eye food - gathering distance (cm) value and the body weight (g) value.

[0068] After confirming the success of the model structure, the emulsion was administered intragastrically to each, and intragastric administration was continued for 1 month. During the intragastric administration period, the young rabbits in each group were enclosed and released in the same indoor natural light and natural environment. Every day, at the same time, the state of the upper and lower eyelids, conjunctiva, cornea, limbus, anterior chamber of the eye, and iris was observed, and a comprehensive examination was performed once a week with a slit lamp. Also, computerized ophthalmoscopy was performed to measure the treatment results, and the treatment effect was statistically calculated according to the above - mentioned method.

[0069] Result Tables 2 and 3 respectively show the research results of the myopic young rabbit model at the 25th week of the experiment and the changes in the refractive power, feeding distance, and body weight of the myopic young rabbits after one month of intragastric administration. From Table 2, young rabbits raised in a weak light environment with restricted visual distance during the growth period can only see nearby objects with both eyes and do not need to see distant objects. In particular, by selecting food in the short-distance range for a long time, objectively, the eyeballs grow adaptively to see closer, and the myopic refractive power (D) value of computerized optometry decreases, indicating that the myopic model structure has been successful.

[0070] After drug administration, the refractive powers of all experimental groups increased. The improvement of vision and the improvement of the growth environment promoted the growth and development of young rabbits, and the feeding reaction distance with naked eyes increased. Since the increase in refractive power in the DHA-lutein monoester group and the DHA-lutein diester group was much larger than that in other groups, it is shown that DHA-lutein ester has a significant effect on improving vision. There is no significant difference in the results between the DHA-lutein monoester group and the DHA-lutein diester group, but the refractive power (D) and feeding distance in the DHA-lutein diester group are larger than those in the DHA-lutein monoester group, indicating that the effect of DHA-lutein diester is better.

[0071] [Table 2]

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

[0073] To sum up the above, by synthesizing DHA-lutein ester using DHA and lutein as starting materials, it can protect the eyes from blue light, maintain eye health, and better exert the effect of improving vision, showing good application prospects.

[0074] The specific embodiments 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 changes or modifications without affecting the substantial content of the present invention within the scope of the claims.

Claims

1. Lutein monoester represented by the following structural formula: 【Chemical 1】 and / or lutein diester: 【Chemical 2】 〔wherein, R is DHA〕 The DHA-lutein ester represented by the formula.

2. A method for preparing the DHA-lutein ester according to Claim 1, comprising the following steps: Weigh lutein and free DHA, add dichloromethane, DMAP catalyst and EDCI, and then react under an inert gas atmosphere. The method for preparing the DHA-lutein ester, comprising the above steps.

3. The purification method after the above preparation comprises the following steps: 1) Add dichloromethane to the reaction mixture, and then wash it successively with dilute hydrochloric acid solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution, and recover the organic phase. 2) Evaporate until dichloromethane disappears while rotating under reduced pressure to obtain a crude DHA-lutein ester product. 3) Thoroughly dissolve the activated silica gel in n-hexane, fill it into a column, dissolve the crude product obtained in step 2) in n-hexane, gradually add it to the silica gel column, and elute successively with a mixed reagent of n-hexane and n-hexane / acetone solution, and recover the eluate. 4) Concentrate under reduced pressure while avoiding light at low temperature. The method for preparing the DHA-lutein ester according to Claim 2, comprising the above steps.

4. A preparation comprising the DHA-lutein ester according to Claim 1.

5. The preparation according to Claim 4, which is a drug or a biological preparation, and the method of use is characterized by being orally ingested.

6. The preparation according to Claim 4, which is a food for special medical purposes, a health food, a functional food, a dietary supplement, a food additive, a pet food, or a pet food additive.

7. Application of the DHA-lutein ester according to Claim 1 in the preparation of a stable visual function improver for effectively maintaining eye health.

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