Eye-protecting DHA-xanthophyll ester, preparation method and application thereof

Abstract

The application belongs to the technical field of application of medicine / health products (functional food) in the medical field, and particularly relates to an eye-protecting DHA-xanthophyll ester and a preparation method and application thereof. The DHA-xanthophyll ester is xanthophyll monoester and / or xanthophyll diester, and can be applied in the preparation of stable and efficient eye-protecting and vision-improving products. The DHA-xanthophyll ester is synthesized by combining DHA and xanthophyll, which not only improves the stability and bioavailability of xanthophyll, but also comprehensively utilizes the advantages of DHA and xanthophyll, and DHA cooperates with xanthophyll to act in the macular area of the retina, so as to protect eye health and improve vision. The DHA-xanthophyll ester can be further prepared into oil suspension and microcapsule powder and applied in the fields of food additives, dietary supplements and pet food, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical / health product (functional food) application technology, specifically relating to an eye-protecting DHA-lutein ester, its preparation method, and its application. Background Technology

[0002] The retina, as a receptor for light signals, plays a crucial role in vision formation. Light reaching the retina forms visual images, and prolonged exposure to intense visible light can damage retinal photoreceptor cells. While visual cell damage can progress to apoptosis and vision loss, "retinal photodamage" may subside with functional recovery. With technological advancements, people are increasingly exposed to artificial light sources (e.g., LED lights, mobile phones, computers, and other devices). Photochemical damage caused by shorter wavelengths of visible light (400–550 nm, such as blue light) is the most significant and prevalent type of retinal photodamage and is currently a focus of research. Studies have shown that excessive exposure to blue light leads to a significant increase in reactive oxygen species production, resulting in photoreceptor loss, lipid peroxidation, and apoptosis. The synergistic effect of blue light and N-retinyl ethanolamine (A2E), along with the light reversal of bleaching, further exacerbates photochemical damage, causing activation of inflammatory responses, DNA damage, and inhibition of mitochondrial and lysosomal functions. In particular, blue light not only harms the retina, but also damages the ocular surface through oxidative stress and inflammation, significantly impacting age-related macular degeneration and myopia. Many eye diseases are the result of long-term combined effects of multiple factors. Besides conventional surgical treatments and developing good eye habits, improving diet is also a crucial way to improve vision and address eye diseases. Therefore, protecting eye health and improving vision through dietary supplements or functional foods is receiving increasing attention.

[0003] Lutein is a major carotenoid in the human macula and retina, widely found in plants such as marigolds, pumpkins, and cabbages. It possesses biological activities that help prevent brain aging, protect vision, relieve eye strain, and promote eye health. Lutein protects the retina primarily through two pathways: one is by utilizing its reducing properties to inactivate singlet oxygen and capture reactive oxygen species, thus protecting photosensitive cells; the other is by using its blue light filtering effect to prevent blue light from reaching the underlying structures of the retina, thereby reducing the risk of light-induced oxidative damage. Furthermore, some studies have shown that lutein intake through diet or as a nutritional supplement is beneficial for eye diseases, but its low bioavailability and instability under high temperature and strong light limit its application in functional foods. Lutein esters are a naturally occurring form of lutein, obtained by esterification of lutein with one or two fatty acids (such as myristic acid, lauric acid, and palmitic acid), and are one of the safest sources of lutein. Lutein esters are absorbed into the body and enter the small intestine where they are hydrolyzed by trypsin into free fatty acids and lutein. Under the emulsification of bile, lutein, fatty acids, bile salts, and other components form mixed micelles that enter the lymph and bloodstream.

[0004] Docosahexaenoic acid (DHA) is a marine-derived dietary omega-3 long-chain polyunsaturated fatty acid with bioactivities including anti-inflammatory, antioxidant, cardiovascular disease prevention, and neuronal development promotion. It is also a major structural lipid in the outer segment membrane of the retinal photoreceptor. The state of DHA in tissues affects retinal cell signaling mechanisms involved in phototransduction and can influence retinal function by altering permeability, fluidity, thickness, and lipid phase properties. Furthermore, DHA may play a role in signaling cascades, enhancing the activation of membrane-bound retinal proteins and participating in rhodopsin regeneration. In some cases, DHA supplementation can improve visual processing deficiencies; however, its susceptibility to oxidation affects product quality. Summary of the Invention

[0005] The technical problem to be solved by this invention is that lutein has biological activities such as protecting vision, relieving eye fatigue and promoting eye health, but its low bioavailability and instability under high temperature and strong light limit its application in functional foods.

[0006] To address the aforementioned issues, this invention provides an eye-protecting DHA-lutein ester, its preparation method, and its application. By synthesizing DHA and lutein into DHA-lutein ester, not only is the stability and bioavailability of lutein improved, but the advantages of both are also combined. DHA synergistically assists lutein in its function in the macular region of the retina, thereby protecting eye health and improving vision.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution: an eye-protecting DHA-lutein ester, the structural formula of which is shown below:

[0008]

[0009] Where R stands for DHA.

[0010] This invention introduces DHA into lutein compounds. After the phenolic hydroxyl groups in lutein are esterified, the stability of the compound increases, thereby forming a novel lutein ester derivative, which improves the problem of DHA's easy oxidation.

[0011] The combination of DHA and lutein is more effective than lutein alone in increasing macular pigment density, protecting retinal pigment epithelial cells (RPE cells) from reactive oxygen species, and increasing plasma lutein levels. DHA also promotes lutein accumulation in the blood and macula, working synergistically with lutein in protecting eye health. However, when lutein and DHA are supplemented alone, a large portion is excreted during ingestion, with only a small portion remaining to exert their physiological effects. Lutein derived from the breakdown of lutein esters has higher bioavailability and stability than free lutein and is more readily utilized by gut microbiota (e.g., Bifidobacteria and Lactobacillus), alleviating oxidative stress symptoms, significantly increasing macular pigment density, and improving eye function. The compounds of this invention, in monoester form, can deliver more lutein and DHA into the body, which are then further broken down into lutein and DHA to exert their effects, solving the problems of low activity, rapid metabolism, and low bioavailability of lutein in the body.

[0012] The diester form of the compound of this invention can alter the intestinal microbiome structure, increase the abundance of beneficial bacteria such as Bifidobacteria and Lactobacillus, regulate the metabolic products of the microbiome, and thus alleviate the symptoms of oxidative stress, thereby significantly improving the macular pigment density of the retina. Therefore, the compound of this invention can exert the synergistic effect of DHA and lutein in protecting eye health and improving vision, while avoiding the disadvantages of lutein's low activity in vivo, rapid metabolism, low bioavailability, and DHA's easy oxidation.

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

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

[0015] Furthermore, the DHA-lutein ester is used in the preparation of stable and efficient eye-protecting and vision-improving products.

[0016] Furthermore, the product contains the above-mentioned lutein ester at a pharmacologically effective concentration (1%-100%).

[0017] Furthermore, the product shown is a pharmaceutical or biological agent, and the method of administration is oral.

[0018] Furthermore, the product is a food, a special medical food, a health product, a functional food, or a dietary supplement. The functional food includes milk, beverages, or baked goods, etc.

[0019] A method for preparing the above-mentioned eye-protecting DHA-lutein ester includes the following steps:

[0020] (1) Weigh lutein and free DHA, add dichloromethane, 4-dimethylaminopyridine (DMAP) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) catalysts, purge with nitrogen, and shake in a water bath; after the reaction, transfer the reaction mixture to a beaker, add dichloromethane, and then wash with hydrochloric acid solution, saturated sodium bicarbonate solution and saturated sodium chloride solution in sequence; recover the organic phase, and rotary evaporate under reduced pressure until no dichloromethane is found to obtain crude DHA-lutein ester, which contains both monoesters and diesters, with a reaction degree of over 90%;

[0021] (2) After the silica gel is activated, it is fully dissolved in hexane and then packed into a silica gel column. The crude product obtained is then dissolved in hexane and slowly poured into the silica gel column. It is eluted sequentially with a mixture of hexane and hexane-acetone solution. The eluent is collected. During the elution process, the separation of lutein monoester and lutein diester is determined by thin-layer silica gel plate chromatography. The eluents of lutein monoester and diester are collected separately and concentrated under reduced pressure under low temperature and light-protected conditions. Lutein monoester and diester are collected separately.

[0022] Furthermore, step (1) involves shaking in a water bath at 25℃-30℃ for 1-8 hours.

[0023] Further, in step (2), the crude product was eluted sequentially with hexane, a hexane-acetone solution with a volume ratio of 9:1, and a hexane-acetone solution with a volume ratio of 8:2. A certain column volume of hexane eluent, five column volumes of hexane-acetone solution with a volume ratio of 9:1, and an hexane-acetone solution with a volume ratio of 8:2 were obtained. The hexane-acetone solution with a volume ratio of 9:1 contained lutein diester; the hexane-acetone solution with a volume ratio of 8:2 contained lutein monoester.

[0024] Furthermore, in step (1), the amount of DMAP and EDCI added is 0.2-2 g / g lutein, the amount of DHA added is 1.5-3 g / g lutein, and the concentration of lutein in the solvent is 5-100 mg / mL.

[0025] The beneficial effects of this invention are as follows:

[0026] (1) In this invention, DHA-lutein ester is prepared by using lutein and free DHA as raw materials, and further separated and purified to obtain a purity of over 90%.

[0027] (2) The preparation method provided by the present invention has low cost, simple steps, low amount of organic solvent used, and low environmental pollution from organic solvents.

[0028] (3) Animal experiments have verified that the lutein ester products prepared by the method of the invention have better eye protection effects than lutein alone. They can be further made into oil suspensions and microcapsule powders for use in food additives, dietary supplements, pet food and other fields, and have good application prospects. Attached Figure Description

[0029] Figure 1 This is a thin-layer chromatogram of the crude product components of the present invention;

[0030] Figure 2 This is a high-performance liquid chromatogram of the free lutein standard and the purified reaction product.

[0031] Among them, (a) is free lutein, (b) is DHA-lutein monoester, and (c) is DHA-lutein diester. Detailed Implementation

[0032] 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 will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0034] Example 1: Preparation of DHA-lutein ester

[0035] Weigh 500 mg lutein, 1200 mg DHA, 800 mg EDCI, and 200 mg DMAP, and add them to 5 mL of dichloromethane. Incubate at 25 °C with shaking for 1 h. Wash the mixture sequentially with 10 mL dichloromethane, 100 mL dilute hydrochloric acid, 100 mL sodium bicarbonate solution, and 100 mL saturated sodium chloride solution to separate the layers. Collect the organic phase. Remove the organic solvent and purify the product using silica gel column chromatography. Dissolve the crude product thoroughly in hexane and pack it into a silica gel column. Dissolve the crude product in hexane and slowly pour it into the silica gel column. Elute sequentially with hexane, a hexane / acetone (90 / 10), and a hexane / acetone (80 / 20) mixture. Collect the eluent and perform thin-layer silica gel plate chromatography. Remove the dichloromethane, dry the eluent, and store at -20 °C protected from light. The eluent collected in hexane / acetone (90 / 10) was lutein diester; the eluent collected in hexane / acetone (80 / 20) was lutein monoester.

[0036] Example 2: Detection of DHA-lutein esters

[0037] 1. Method

[0038] 1.1 Thin-layer chromatography detection

[0039] The crude product from the reaction in Example 1 was preliminarily detected by thin-layer chromatography using a hexane-acetone solution with a volume ratio of 4:1 as the developing solvent. 10 ml of the developing solvent was added to the chromatography tank, and the tank was sealed and equilibrated for 30 min. The sample was spotted using a 0.3 × 100 mm capillary tube, with the spotting line 1 cm from the lower edge of the silica gel plate. This process was repeated multiple times, with the solvent being dried promptly during the spotting process. When the solvent front was 1 cm from the upper edge of the silica gel plate, the plate was removed and allowed to develop naturally at room temperature.

[0040] 1.2 High Performance Liquid Chromatography Detection

[0041] Chromatographic conditions: Column: YMC C30 (4.6 mm × 250 mm, 3 μm); Mobile phase A: chromatographic grade methanol, B: chromatographic grade methyl tert-butyl ether; isogradient elution, flow rate 1.0 ml / min; detection wavelength 450 nm; column temperature: 30 ℃; injection volume: 10 μl.

[0042] 2. Experimental Results

[0043] 2.1 Results of Thin-Layer Chromatography

[0044] The developing solvent used in Example 2 effectively separated free lutein, DHA-lutein monoester, and DHA-lutein diester. The results of thin-layer chromatography under natural color development are as follows: Figure 1As shown, three bands appeared on the TLC plate with Rf values ​​of 0.90, 0.31, and 0.08, respectively. Referring to the bands of the free lutein standard, the band with an Rf value of 0.08 represents free lutein. After the hydroxyl groups at both ends of lutein react with DHA to form an ester, the polarity of the product decreases. The polarity of the DHA-lutein diester with substitutions on both sides is lower than that of the DHA-lutein monoester with substitution on one side. Therefore, it is preliminarily determined that the band with an Rf value of 0.31 represents DHA-lutein monoester, and the band with an Rf value of 0.90 represents DHA-lutein diester.

[0045] 2.2 Results of High Performance Liquid Chromatography

[0046] Figure 2 High-performance liquid chromatograms of DHA-lutein monoesters and diesters obtained by column chromatography purification in Example 1, which are lutein standards. Figure 2 (a) is the chromatographic peak of the free lutein standard, with an elution time of 3-4 min; Figure 2 (b) The peak time was 5-6 min, and combined with the first-order mass spectrum, it was determined to be DHA-lutein monoester with m / z of 897.4. Figure 2 (c) The peak time was 11-12 min, and based on the primary mass spectrum, it was identified as DHA-lutein diester, with an m / z of 1125.8. The YMC C30 column is a reversed-phase column. Compounds with higher polarity elute first, followed by compounds with lower polarity. The peak times of DHA-lutein monoester and diester were higher than those of free lutein, indicating that the polarity of the product decreased.

[0047] Example 3: The repair effect of DHA-lutein esters on blue light-induced retinal damage

[0048] 1. Method

[0049] 1.1 Preparation of Gavage Emulsion

[0050] Purified lutein esters and free lutein were added to a mixed solution containing 90% physiological saline and 10% corn oil (wt / wt), followed by 0.1% porcine bile salts in the total system. After sonication in an ice bath to promote dissolution, the cells were lysed. Finally, an emulsion with a lutein equivalent concentration of 10 mg / ml was prepared and administered to rats by gavage.

[0051] 1.2 Animal Experiments

[0052] Seventy SD rats were acclimatized for one week. Ten rats served as the control group and were not subjected to blue light exposure. Forty rats were anesthetized with sodium pentobarbital injection at a dose of 30 mg / kg body weight, and then given compound tropicamide eye drops to dilate their pupils, followed by strong blue light exposure. After exposure, the rats were randomly divided into six groups and administered physiological saline, lutein emulsion, physiological saline with added DHA, a mixed emulsion of DHA and lutein, DHA-lutein monoester emulsion, and DHA-lutein diester emulsion by gavage, respectively: model group, lutein group, DHA group, lutein + DHA group, DHA-lutein monoester group, and DHA-lutein diester group. Electroretinography (ERG) of the rats was measured after one week of continuous gavage.

[0053] 2. Results

[0054] Table 1. Amplitudes of wave a and b in rat electroretinograms

[0055]

[0056] Note: The letters a, b, c, d, e, and f indicate significant differences between the data (P < 0.05).

[0057] Table 1 shows the changes in the amplitude of electroretinogram (ERG) waves a and b in rats after one week of gavage. Wave a mainly originates from the receptor potential of photoreceptor cells; wave b has a larger amplitude and is mainly related to the activity of bipolar cells. As can be seen from the table above, the amplitude of waves a and b decreased significantly after strong blue light damage, indicating that the model was successfully constructed.

[0058] After administration of DHA and lutein alone, the amplitudes of a and b waves increased, but the increase was much smaller than that of the DHA+lutein combination and DHA-lutein ester, indicating that the combined use of DHA and lutein, or the synthesis of lutein ester, is significantly more effective than either of them alone. The increase in a and b wave amplitudes of DHA-lutein ester was significantly higher than that of the DHA+lutein combination, indicating that the DHA-lutein ester obtained in this invention has good blue light protection and eye health protection effects. Among them, the increase in a and b wave amplitudes of the DHA-lutein diester group was significantly higher than that of the DHA-lutein monoester group, indicating that the DHA-lutein diester group is more effective.

[0059] Example 4: The effect of DHA-lutein ester on improving vision in myopic animals.

[0060] 1. Method

[0061] 1.1 Preparation of Gavage Emulsion

[0062] Purified lutein esters and free lutein were added to a mixed solution containing 90% physiological saline and 10% corn oil (wt / wt), followed by 0.1% porcine bile salts in the total system. After sonication in an ice bath to promote dissolution, the cells were lysed. Finally, an emulsion with a lutein equivalent concentration of 10 mg / ml was prepared and administered to young rabbits by gavage.

[0063] 1.2 Animal Experiments

[0064] The experiment consisted of 7 groups, with 5 rabbits in each group: a control group, a model group, and 5 experimental groups (lutein group, DHA group, DHA+lutein group, DHA-lutein monoester group, and DHA-lutein diester group). The rabbits in the model group and the 5 experimental groups were housed in tightly sealed cages with the floral pattern facing inwards, each cage containing multiple squares made of thick floral fabric. The cages were further divided into grids using the fabric, each square initially large enough to accommodate one rabbit. The rabbits were allowed free movement, so initially their visual distance (depth) was generally no more than 5-10 cm. As the rabbits grew, the cage size and the number of squares were gradually increased, but the visual distance was always maintained at around 5-10 cm. To encourage close-up viewing, the food was finely chopped and mixed with twice the amount of gravel, allowing the rabbits to choose their food from the mixed feed. During the suckling rabbit stage, the milk bottles, food troughs, and water containers were placed outside the squares inside the cages covered with thick floral fabric, forcing the young rabbits to only stick their heads out to eat and drink. To provide multifaceted stimulation and force the young rabbits to use their eyes to observe more, the positions of the milk bottles, food troughs, and water containers were changed every 3-5 days to train the young rabbits in each experimental group to choose food visually. The control group of 5 young rabbits had their cages uncovered, and no measures were taken to stimulate their vision or restrict their viewing distance. When the experiment reached 25 weeks, the pupils were dilated with 1% atropine solution, and then the eyelids were widened using a self-made eyelid dilator. The refractive power (D) of the left and right eyes of each group of young rabbits was measured using computer refraction. At the same time, the naked eye foraging distance (cm) was measured with a tape measure, and the weight (g) was measured.

[0065] After confirming successful modeling, the rabbits were administered the emulsion via gavage for one month. During this period, the young rabbits in each group were kept in an enclosure under the same natural light and environment. The condition of both eyelids, conjunctiva, cornea, limbus, anterior chamber, and iris was observed daily, and a comprehensive examination was performed weekly using a slit lamp. The treatment results were then measured using computer-aided refraction as described above, and the efficacy was statistically analyzed.

[0066] result

[0067] Tables 2 and 3 show the results of the myopia rabbit model at 25 weeks of experimentation and the changes in refractive error, foraging distance, and weight of the myopia rabbits after one month of gavage. The tables show that the rabbits, living in a limited-distance, low-light environment during their growth period, could only see at close range and had no need to see at a distance. Furthermore, they needed to spend long periods selecting food at close range, which objectively forced their eyes to develop for adaptive near vision. This resulted in a decrease in the myopia refractive error (D) value during computer testing, indicating the successful establishment of the myopia model.

[0068] After administration, the refractive power of all experimental groups increased. With the improvement in visual acuity, coupled with the improved growth environment, the growth and development of young rabbits were promoted, and the distance of their foraging response without naked eyes was increased. The increase in refractive power in the DHA-lutein monoester group and the lutein diester group was much greater than that in other groups, indicating that DHA-lutein esters have a significant effect on improving visual acuity. Although there was no significant difference between the DHA-lutein monoester group and the DHA-lutein diester group, the refractive power (D) and foraging distance of the DHA-lutein diester group were higher than those of the DHA-lutein monoester group, indicating that the DHA-lutein diester group was more effective.

[0069] Table 2. Results of the myopia model in rabbit calves at 25 weeks of experimentation.

[0070]

[0071] Table 3 Comparative Study of the Effects of Treatment on Myopic Rabbits after 30 Days

[0072]

[0073] Note: The letters a, b, c, and d indicate significant differences between the data (P < 0.05).

[0074] In conclusion, DHA-lutein esters prepared from DHA and lutein can provide better protection against blue light, safeguard eye health, and improve vision, demonstrating promising application prospects.

[0075] 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, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. The application of an eye-protecting DHA-lutein ester in the preparation of drugs for improving myopia vision, characterized in that... Compounds containing the following structural formulas: Where R stands for DHA; The medication is to be taken orally.

2. The application as described in claim 1, characterized in that: The DHA-lutein ester has a DHA content of 30-60%.

3. The application as described in claim 1, characterized in that: The drug contains the above-mentioned lutein ester at a pharmacologically effective concentration of 1%-100%.

4. The application as described in claim 1, characterized in that, The preparation method of the DHA-lutein ester includes the following steps: (1) Weigh lutein and free DHA, add dichloromethane, DMAP and EDCI catalysts, purge with nitrogen, and shake in a water bath; after the reaction, transfer the reaction mixture to a beaker, add dichloromethane, and then wash with hydrochloric acid solution, saturated sodium bicarbonate solution and saturated sodium chloride solution in sequence; recover the organic phase, and rotary evaporate under reduced pressure until no dichloromethane is found to obtain crude DHA-lutein ester; (2) After the silica gel is activated, it is fully dissolved in hexane and then packed into a silica gel column. The crude product obtained is then dissolved in hexane and slowly poured into the silica gel column. It is eluted sequentially with a mixture of hexane and hexane-acetone solution. The eluent is collected. During the elution process, the separation of lutein monoester and lutein diester is determined by thin-layer silica gel plate chromatography. The eluents of lutein monoester and diester are collected separately and concentrated under reduced pressure under low temperature and light-protected conditions. Lutein monoester and diester are collected separately.

5. The application as described in claim 4, characterized in that: Step (1) Shake in a water bath at 30℃ for 1-8 hours.

6. The application as described in claim 4, characterized in that: Step (2) involves elution with n-hexane, a n-hexane-acetone solution with a volume ratio of 9:1, and a n-hexane-acetone solution with a volume ratio of 8:

2.

7. The application as described in claim 4, characterized in that: In step (1), the amount of DMAP and EDCI added is 0.2-2 g / g lutein, the amount of DHA added is 1.5-3 g / g lutein, and the concentration of lutein in the solvent is 5-100 mg / mL.

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