A brain-protecting composition containing high-absorption LPC-DHA and a preparation method thereof

Abstract

The present application relates to the technical field of medical materials and health products, and specifically discloses a brain-protecting composition containing high-absorption LPC-DHA and a preparation method thereof. The brain-protecting composition comprises raw materials such as LPC-DHA, algal oil DHA, phytosterol, GINKGOSELECT ginkgo leaf extract, N-acetylneuraminic acid, etc. The used LPC-DHA is obtained by dynamic enzymolysis of natural phospholipids by phospholipase A1 and purification by a core-shell structure composite adsorbent. The preparation method comprises the following steps: obtaining high-purity LPC-DHA by utilizing the synergistic effect of electrostatic anchoring and hydrophobic capture of the adsorbent; and then co-assembling the high-purity LPC-DHA and the remaining components into a targeted nanocomposite by microjet homogenization. The present application guarantees the active configuration of LPC-DHA through synergistic extraction, constructs a nanodelivery system with an N-acetylneuraminic acid-targeted crown layer through co-assembly, realizes cascade trans-barrier delivery, and significantly improves the brain absorption rate of DHA and the brain-protecting efficacy.

Description

Technical Field

[0001] This invention belongs to the field of medical materials and health products technology, and relates to a brain-protecting composition containing highly absorbable LPC-DHA and its preparation method. Background Technology

[0002] Docosahexaenoic acid (DHA) is a key structural lipid for brain and retinal development and function maintenance. However, traditional ethyl ester or triglyceride DHA is absorbed passively through diffusion in vivo, making it difficult to effectively cross the blood-brain barrier (BBB), blood-eye barrier, and placental barrier. This results in extremely low bioavailability in the brain, limiting its application in improving memory and alleviating neurodegenerative diseases such as Alzheimer's disease.

[0003] Lysophosphatidylcholine-coupled DHA (LPC-DHA) can be actively targeted and absorbed via the Mfsd2a transporter on the blood-brain barrier, making it a recognized highly efficient form of intracerebral delivery. However, the preparation and application of existing LPC-DHA face two major bottlenecks: First, in terms of enzymatic preparation, existing enzymatic hydrolysis reactions suffer from severe product feedback inhibition, where the generated LPC-DHA and free fatty acids hinder enzyme-substrate binding, resulting in low conversion rates. Simultaneously, LPC-DHA at the sn-2 position readily migrates to the sn-1 position, generating a low-activity isomer, and free fatty acids are difficult to separate efficiently from the product, thus limiting the production capacity and purity of highly active sn-2-LPC-DHA. Secondly, in terms of brain-protecting formula design, existing products mostly combine LPC-DHA with other brain-protecting ingredients in a simple physical way. This approach lacks molecular-level assembly synergy, which not only fails to shield LPC-DHA from degradation by phospholipases in the bloodstream, but also leads to the instability of the LPC-DHA choline head conformation, making it difficult for the Mfsd2a transporter to be efficiently recognized, resulting in reduced cross-barrier delivery efficiency and failing to truly unleash the potential of multi-component synergistic brain protection. Summary of the Invention

[0004] To address the above issues, this invention provides a brain-protective composition containing highly absorbable LPC-DHA and its preparation method. By constructing a composite adsorbent with a synergistic confinement effect of "electrostatic anchoring-hydrophobic capture," highly selective preparation of LPC-DHA with a high sn-2 content is achieved. Simultaneously, using a thermodynamic and kinetic-driven self-assembly technology, LPC-DHA, algal oil DHA, phytosterols, ginkgo biloba extract, and N-acetylneuraminic acid are assembled into a multi-receptor-mediated targeted nanocomposite, enabling multiple components to efficiently cross the blood-brain barrier and blood-eye barrier, and synergistically improving memory and concentration, delaying neuroaging, and alleviating Alzheimer's disease.

[0005] To achieve the above and other objectives, the technical solution adopted by the present invention is as follows:

[0006] This invention provides a highly absorbable LPC-DHA, which is prepared by a dynamic enzymatic hydrolysis-adsorption purification method. The raw materials for preparation include: natural phospholipids, phospholipase A1, and a composite adsorbent. The specific preparation method includes the following steps:

[0007] (1) Substrate pretreatment: Natural phospholipids were dispersed in a citrate-sodium citrate buffer solution at pH 6.0, and sodium taurocholate was added as an interfacial activator. The mixture was preheated at 40°C for 15 minutes and then subjected to high-speed shearing to form a substrate liposome dispersion.

[0008] (2) Dynamic enzymatic hydrolysis: Phospholipase A1 and composite adsorbent were added to the substrate liposome dispersion. The amount of phospholipase A1 added was 4% of the mass of the substrate liposome dispersion, and the amount of composite adsorbent added was 15% of the mass of the substrate liposome dispersion. The reaction was continuously stirred at 42℃ and 200rpm for 4-6 hours. During this process, phospholipase A1 specifically hydrolyzed the fatty acid at the sn-1 position of natural phospholipids to generate sn-2-LPC-DHA and free fatty acids. The silanol groups on the surface of mesoporous silica are negatively charged. They accurately anchor the positively charged sn-2-LPC-DHA choline head through electrostatic attraction and hydrogen bond network. At the same time, they electrostatically repel the negatively charged free fatty acids and drive them to the activated carbon region. The activated carbon firmly captures the free fatty acids through strong hydrophobicity, which restricts the migration of sn-2 to sn-1 acyl groups.

[0009] (3) Gradient elution and purification: After the reaction, the precipitate containing the product was collected by centrifugation. A pH 4.0 acetate-sodium acetate buffer solution was added to the precipitate and stirred at 30°C for 1 hour for desorption. During this process, a large number of protons provided by the acidic buffer competitively destroyed the hydrogen bond network between the silanol groups of mesoporous silica and the choline head of LPC-DHA, causing LPC-DHA to desorb rapidly into the aqueous phase. The free fatty acids were protonated under acidic conditions and their hydrophobic affinity with activated carbon reached its maximum and they were firmly retained. After centrifugation again, the supernatant containing high purity sn-2-LPC-DHA was collected. After ultrafiltration and freeze drying, LPC-DHA with a high sn-2 content was obtained.

[0010] Furthermore, the phospholipase A1 is derived from Aspergillus oryzae.

[0011] Furthermore, the natural phospholipid is a marine-derived phospholipid rich in DHA, which is either squid lecithin or phospholipid extracted from marine microalgae.

[0012] Furthermore, the composite adsorbent is a core-shell structured composite material, with activated carbon as the core and a mesoporous silica shell coating on the surface. The preparation method of the composite adsorbent is as follows: (a) 10g of activated carbon is dispersed in a mixed solvent of 100mL anhydrous ethanol and deionized water, and 2g of sucrose fatty acid ester is added as a template agent. The mixture is stirred at 300rpm for 30 minutes at 40℃ to obtain a dispersion; (b) 2.5g of tetraethyl orthosilicate is added to the dispersion, and ammonia is added dropwise to adjust the pH to 10. The mixture is refluxed at 60℃ for 6 hours. During this process, tetraethyl orthosilicate undergoes hydrolysis and condensation under the catalysis of ammonia. Under the template guidance of sucrose fatty acid ester, it grows in situ on the surface of activated carbon to form a mesoporous silica shell; (c) After the reaction is completed, the precipitate is collected by centrifugation at 4000rpm for 10 minutes, washed alternately with hot deionized water at 60℃ until no foam overflows, washed once with anhydrous ethanol, and then vacuum dried at 60℃ for 12 hours to obtain the composite adsorbent.

[0013] The present invention also provides a brain-protecting composition containing the above-mentioned highly absorbable LPC-DHA, wherein the brain-protecting composition comprises the following raw materials in parts by weight: 12-18 parts LPC-DHA, 5-10 parts algal oil DHA, 2-4 parts phytosterols, 3-6 parts GINKGOSELECT® Ginkgo biloba extract, 1-3 parts N-acetylneuraminic acid, 0.1-0.5 parts antioxidant, and 55-70 parts excipients, and its preparation method includes the following steps:

[0014] S1. Dissolve LPC-DHA, algal oil DHA, phytosterols and GINKGOSELECT® Ginkgo biloba extract in anhydrous ethanol, heat to 40°C to aid dissolution, and obtain a mixed solution;

[0015] S2. Dissolve the excipient and N-acetylneuraminic acid in deionized water to obtain an aqueous solution. Then heat the solution to 55°C and keep it warm. Under mechanical stirring at 800 rpm, add the mixed solution obtained in step S1 dropwise to the aqueous solution at a rate of 1-2 mL / min. After the addition is complete, continue stirring at a constant temperature for 20 minutes to allow the ethanol to evaporate and form a primary emulsion.

[0016] S3. The primary emulsion was homogenized three times using a microfluidic homogenizer at a pressure of 100 MPa, and then allowed to stand and mature at 25°C for 2 hours to obtain a multi-receptor-mediated targeted nanocomposite. Under strong shear force and thermodynamic drive, the components compounded to form nanoparticles. The hydrophobic tail chain of LPC-DHA aggregated to form a hydrophobic core, and its hydrophilic choline head was arranged on the outer surface to form a hydrophilic canopy. The rigid hydrophobic ring structure of phytosterols contracted the hydrophobic core. The hydrophobic flavonoids and lactones in GINKGOSELECT® Ginkgo biloba extract were embedded in the hydrophobic core, significantly improving its solubility and bioavailability. At the same time, N-acetylneuraminic acid crosslinked with gum arabic and LPC-DHA choline head through hydrogen bonds and anchored to the outermost layer of the nanoparticles to form a targeted functional canopy.

[0017] S4. Add an antioxidant to the targeted nanocomposite, mix evenly, and then spray dry to obtain the brain-protecting composition.

[0018] Furthermore, the antioxidant is either rosemary extract or natural vitamin E; the excipient is a mixture of maltodextrin and gum arabic in a mass ratio of 3:1.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] This invention designs a composite adsorbent with an "activated carbon@mesoporous silica" core-shell structure and constructs a multi-targeted nanocomposite system. In the composite adsorbent, the negatively charged silanol groups on the surface of the mesoporous silica precisely anchor the positively charged sn-2-LPC-DHA choline head, while electrostatically repelling negatively charged free fatty acids and driving them to the activated carbon region for strong hydrophobic capture. Through the synergistic effect of electrostatic anchoring of the mesoporous silica shell and hydrophobic capture of the activated carbon core, the bottleneck of low conversion rate caused by product inhibition in traditional enzymatic hydrolysis is overcome, and the problems of free fatty acid accumulation and sn-2 acyl group migration in LPC-DHA preparation are solved. At the same time, this invention utilizes the spontaneous association of polarity differences and intermolecular interactions between components to construct a nanoconformation, solving the problems of lack of cross-barrier delivery mechanism and extremely low absorption rate of ordinary DHA in conventional physical compounding.

[0021] The core synergistic effect of this invention lies in: (1) constructing a dual receptor cascade targeted cross-barrier delivery mechanism to achieve high absorption and utilization. Ordinary algal oil DHA is difficult to cross the blood-brain barrier and blood-eye barrier, while the LPC-DHA used in this invention is a specific substrate of the MFSD2A transporter protein on the barrier. During the self-assembly process, LPC-DHA constructs a hydrophilic canopy, and its choline head is exposed by eversion. N-acetylneuraminic acid is enriched in the outermost layer to form a targeted modification layer. After entering the body, N-acetylneuraminic acid first recognizes the Siglec receptor of endothelial cells to mediate endocytosis. After entering the cell, the exposed LPC-DHA choline head is accurately recognized by Mfsd2a to mediate transcellular extracellular discharge. The cascade synergistic effect achieves active and efficient crossing of the blood-brain barrier and blood-eye barrier. (2) Multi-component synergistic brain protection. Phytosterols densify the hydrophobic core to enhance stability, while co-encapsulation of algal oil DHA and ginkgo extract solves the problem of poor dissolution of ginkgo flavonoids. After release into the brain, LPC-DHA and algal oil DHA replenish nerve cell membrane lipids, repair neurons, and promote synapse formation. Ginkgo extract exerts antioxidant and anti-inflammatory effects, and N-acetylneuraminic acid promotes ganglioside synthesis and signal remodeling. The components work synergistically to show significant advantages in improving memory and concentration and alleviating Alzheimer's disease. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0023] Figure 1 The graph shows the results of measuring the purity of LPC-DHA and the proportion of the sn-2 site in the sample prepared in this invention. Detailed Implementation

[0024] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and 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.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0026] Unless otherwise specified, all materials used in the following implementations are new materials purchased from the market, and all quantities are parts by weight. The phospholipase A1 used is derived from Aspergillus oryzae; the DHA content in the algal oil DHA is 61g / 100g; the phytosterol is β-sitosterol; the key parameters of GINKGOSELECT® Ginkgo biloba extract are: ginkgo flavonoid glycoside content 24.3%, total content of ginkgolides A, B, C and ginkgolides 6.1%; the excipient is a mixture of maltodextrin and gum arabic at a mass ratio of 3:1.

[0027] Example 1: This example provides a composite adsorbent, which is a core-shell structured composite material with activated carbon as the core and a mesoporous silica shell on the surface. The preparation method is as follows: (a) 10g of activated carbon is dispersed in a mixed solvent composed of 50mL of anhydrous ethanol and 50mL of deionized water, and 2g of sucrose fatty acid ester is added as a template agent. The mixture is stirred at 300rpm for 30 minutes in a 40℃ water bath to obtain a uniform dispersion; (b) 2.5g of tetraethyl orthosilicate is slowly added dropwise to the dispersion under continuous stirring, followed by the addition of a 25% ammonia solution to adjust the pH of the system to 10.0. The reaction apparatus is moved to a 60℃ oil bath, a reflux condenser is installed, and the reaction is continuously stirred for 6 hours; (c) After the reaction is completed, the mixture is centrifuged at 4000rpm for 10 minutes, and the precipitate is collected. The precipitate was washed three times with hot deionized water at 60°C, and then washed once with anhydrous ethanol to remove unreacted template agent and byproducts. The washed solid was placed in a vacuum drying oven at 60°C and dried for 12 hours. After grinding, it was passed through a 200-mesh sieve to obtain the composite adsorbent.

[0028] Example 2: This example provides a brain-protecting composition containing highly absorbable LPC-DHA. The brain-protecting composition comprises the following raw materials in parts by weight: 12 parts LPC-DHA, 5 parts algal oil DHA, 2 parts phytosterols, 3 parts GINKGOSELECT® Ginkgo biloba extract, 1 part N-acetylneuraminic acid, 0.1 parts rosemary extract, and 55 parts excipients. The preparation method of the LPC-DHA is as follows:

[0029] (1) 100g of squid lecithin rich in DHA was dispersed in 1000mL of citrate-sodium citrate buffer solution with pH 6.0, and 10g of sodium taurocholate was added as an interface activator. The mixture was preheated at 40℃ for 15 minutes and then subjected to high-speed shearing to form a substrate liposome dispersion.

[0030] (2) Add 44.4g of phospholipase A1 and 166.5g of composite adsorbent to the substrate liposome dispersion, and stir continuously for 4 hours at 42℃ and 200rpm.

[0031] (3) After the reaction is completed, the precipitate containing the product is collected by centrifugation. Acetic acid-sodium acetate buffer solution with pH 4.0 is added to the precipitate. The mixture is stirred and desorbed at 30°C for 1 hour. After centrifugation again, the supernatant is collected and then subjected to ultrafiltration and freeze drying to obtain LPC-DHA with a high sn-2 content.

[0032] This embodiment also provides a method for preparing a brain-protective composition containing highly absorbable LPC-DHA, the specific steps of which are as follows:

[0033] S1. Dissolve 12 parts LPC-DHA, 5 parts algal oil DHA, 2 parts phytosterols and 3 parts GINKGOSELECT® Ginkgo biloba extract in 200 mL of anhydrous ethanol, heat to 40 °C and stir at 200 rpm for 20 minutes to aid dissolution, and obtain a mixed solution.

[0034] S2. Dissolve 55 parts of excipient and 1 part of N-acetylneuraminic acid in 500 mL of deionized water, heat to 55 °C and keep warm. Under mechanical stirring at 800 rpm, add the mixed solution obtained in step S1 dropwise to the above aqueous solution at a rate of 1 mL / min. After the addition is complete, continue stirring at 55 °C for 20 minutes to allow the ethanol to evaporate and form a primary emulsion.

[0035] S3. The obtained primary emulsion was homogenized three times by a microfluidic homogenizer at a pressure of 100 MPa, and then allowed to stand and mature at 25°C for 2 hours to obtain a multi-receptor-mediated targeted nanocomposite.

[0036] S4. Add 0.1 parts of rosemary extract to the obtained multi-receptor-mediated targeted nanocomposite, mix evenly, and then spray dry to obtain the brain-protecting composition.

[0037] Example 3: This example provides a brain-protecting composition containing highly absorbable LPC-DHA. The brain-protecting composition comprises the following raw materials in parts by weight: 15 parts LPC-DHA, 7.5 parts algal oil DHA, 3 parts phytosterols, 4.5 parts GINKGOSELECT® Ginkgo biloba extract, 2 parts N-acetylneuraminic acid, 0.3 parts natural vitamin E, and 62.7 parts excipients. The preparation method of the LPC-DHA is as follows:

[0038] (1) 100g of DHA-rich marine microalgae phospholipids were dispersed in 1000mL of pH6.0 citrate-sodium citrate buffer, and 10g of sodium taurocholate was added as an interface activator. The mixture was preheated at 40℃ for 15 minutes and then subjected to high-speed shearing to form a substrate liposome dispersion.

[0039] (2) Add 44.4g of phospholipase A1 and 166.5g of composite adsorbent to the substrate liposome dispersion, and stir continuously for 5 hours at 42℃ and 200rpm.

[0040] (3) After the reaction is completed, the precipitate containing the product is collected by centrifugation. Acetic acid-sodium acetate buffer solution with pH 4.0 is added to the precipitate. The mixture is stirred and desorbed at 30°C for 1 hour. After centrifugation again, the supernatant is collected and then subjected to ultrafiltration and freeze drying to obtain LPC-DHA with a high sn-2 content.

[0041] This embodiment also provides a method for preparing a brain-protective composition containing highly absorbable LPC-DHA, the specific steps of which are as follows:

[0042] S1. Dissolve 15 parts LPC-DHA, 7.5 parts algal oil DHA, 3 parts phytosterols and 4.5 parts GINKGOSELECT® Ginkgo biloba extract in 250 mL of anhydrous ethanol, heat to 40 °C and stir at 200 rpm for 20 minutes to aid dissolution, and obtain a mixed solution.

[0043] S2. Dissolve 62.7 parts of excipient and 2 parts of N-acetylneuraminic acid in 600 mL of deionized water, heat to 55 °C and maintain the temperature. Under mechanical stirring at 800 rpm, add the mixed solution obtained in step S1 dropwise to the above aqueous solution at a rate of 1.5 mL / min. After the addition is complete, continue stirring at 55 °C for 20 minutes to allow the ethanol to evaporate and form a primary emulsion.

[0044] S3. The primary emulsion was homogenized three times by a microfluidic homogenizer at a pressure of 100 MPa, and then allowed to stand and mature at 25°C for 2 hours to obtain a multi-receptor-mediated targeted nanocomposite.

[0045] S4. Add 0.3 parts of natural vitamin E to the targeted nanocomposite, mix evenly, and then spray dry to obtain the brain-protecting composition.

[0046] Example 4: This example provides a brain-protecting composition containing highly absorbable LPC-DHA. The brain-protecting composition comprises the following raw materials in parts by weight: 18 parts LPC-DHA, 10 parts algal oil DHA, 4 parts phytosterols, 6 parts GINKGOSELECT® Ginkgo biloba extract, 3 parts N-acetylneuraminic acid, 0.5 parts rosemary extract, and 70 parts excipients. The preparation method of the LPC-DHA is as follows:

[0047] (1) 100g of squid lecithin rich in DHA was dispersed in 1000mL of citrate-sodium citrate buffer solution with pH 6.0, and 10g of sodium taurocholate was added as an interface activator. The mixture was preheated at 40℃ for 15 minutes and then subjected to high-speed shearing to form a substrate liposome dispersion.

[0048] (2) Add 44.4g of phospholipase A1 and 166.5g of composite adsorbent to the substrate liposome dispersion, and stir continuously for 6 hours at 42℃ and 200rpm.

[0049] (3) After the reaction is completed, the precipitate containing the product is collected by centrifugation. Acetic acid-sodium acetate buffer solution with pH 4.0 is added to the precipitate. The mixture is stirred and desorbed at 30°C for 1 hour. After centrifugation again, the supernatant is collected and then subjected to ultrafiltration and freeze drying to obtain LPC-DHA with a high sn-2 content.

[0050] This embodiment also provides a method for preparing a brain-protective composition containing highly absorbable LPC-DHA, the specific steps of which are as follows:

[0051] S1. Dissolve 18 parts LPC-DHA, 10 parts algal oil DHA, 4 parts phytosterols and 6 parts GINKGOSELECT® Ginkgo biloba extract in 300 mL of anhydrous ethanol, heat to 40 °C and stir at 200 rpm for 20 minutes to aid dissolution, and obtain a mixed solution.

[0052] S2. Dissolve 70 parts of excipient and 3 parts of N-acetylneuraminic acid in 700 mL of deionized water, heat to 55 °C and keep warm. Then, under mechanical stirring at 800 rpm, add the mixed solution obtained in step S1 dropwise to the above aqueous solution at a rate of 2.0 mL / min. After the addition is complete, continue stirring at 55 °C for 20 minutes to allow the ethanol to evaporate and form a primary emulsion.

[0053] S3. The obtained primary emulsion was homogenized three times by a microfluidic homogenizer at a pressure of 100 MPa, and then allowed to stand and mature at 25°C for 2 hours to obtain a multi-receptor-mediated targeted nanocomposite.

[0054] S4. Add 0.5 parts of rosemary extract to the obtained targeted nanocomposite, mix evenly, and then spray dry to obtain the brain-protecting composition.

[0055] Example 5: This example provides a brain-protecting composition containing highly absorbable LPC-DHA. The brain-protecting composition comprises the following raw materials in parts by weight: 15 parts LPC-DHA, 7.5 parts algal oil DHA, 3 parts phytosterols, 4.5 parts GINKGOSELECT® Ginkgo biloba extract, 2 parts N-acetylneuraminic acid, 0.4 parts natural vitamin E, and 62.6 parts excipients. The preparation method of the LPC-DHA is as follows:

[0056] (1) 100g of DHA-rich marine microalgae phospholipids were dispersed in 1000mL of pH6.0 citrate-sodium citrate buffer, and 10g of sodium taurocholate was added as an interface activator. The mixture was preheated at 40℃ for 15 minutes and then subjected to high-speed shearing to form a substrate liposome dispersion.

[0057] (2) Add 44.4g of phospholipase A1 and 166.5g of composite adsorbent to the substrate liposome dispersion, and stir continuously for 5.5 hours at 42℃ and 200rpm.

[0058] (3) After the reaction is completed, the precipitate containing the product is collected by centrifugation. Acetic acid-sodium acetate buffer solution with pH 4.0 is added to the precipitate. The mixture is stirred and desorbed at 30°C for 1 hour. After centrifugation again, the supernatant is collected and then subjected to ultrafiltration and freeze drying to obtain LPC-DHA with a high sn-2 content.

[0059] This embodiment also provides a method for preparing a brain-protective composition containing highly absorbable LPC-DHA, the specific steps of which are as follows:

[0060] S1. Dissolve 15 parts LPC-DHA, 7.5 parts algal oil DHA, 3 parts phytosterols and 4.5 parts GINKGOSELECT® Ginkgo biloba extract in 250 mL of anhydrous ethanol, heat to 40 °C and stir at 200 rpm for 20 minutes to aid dissolution, and obtain a mixed solution.

[0061] S2. Dissolve 62.6 parts of excipient and 2 parts of N-acetylneuraminic acid in 600 mL of deionized water, heat to 55 °C and keep warm. Under mechanical stirring at 800 rpm, add the mixed solution obtained in step S1 dropwise to the above aqueous solution at a rate of 1.5 mL / min. After the addition is complete, continue stirring at 55 °C for 20 minutes to allow the ethanol to evaporate and form a primary emulsion.

[0062] S3. The obtained primary emulsion was homogenized three times by a microfluidic homogenizer at a pressure of 100 MPa, and then allowed to stand and mature at 25°C for 2 hours to obtain a multi-receptor-mediated targeted nanocomposite.

[0063] S4. Add 0.4 parts of natural vitamin E to the obtained targeted nanocomposite, mix evenly, and then spray dry to obtain the brain-protecting composition.

[0064] Comparative Example 1: This comparative example provides an LPC-DHA and a brain-protecting composition. The only difference from Example 3 is that the LPC-DHA is prepared by conventional column chromatography. Specifically, 100g of phospholipids extracted from marine microalgae are hydrolyzed with 44.4g of phospholipase A1, and the hydrolysate is loaded onto a silica gel column and eluted with chloroform:methanol:water (volume ratio 65:25:4). The LPC-DHA fraction is collected and dried by rotary evaporation. The formulation and preparation method of the subsequent brain-protecting composition are completely consistent with those of Example 3.

[0065] Comparative Example 2: This comparative example provides an LPC-DHA and a brain-protecting composition. The only difference from Example 3 is that in the LPC-DHA preparation step (2), the composite adsorbent is replaced with an equal mass of pure activated carbon (i.e., without a mesoporous silica shell). The remaining extraction steps and the formulation and preparation method of the subsequent brain-protecting composition are completely consistent with Example 3.

[0066] Comparative Example 3: This comparative example provides a brain-protecting composition, which differs from Example 3 only in that: no phytosterols are added to the brain-protecting composition formulation, and the mass is made up with an equal weight of excipients; the addition of phytosterols is omitted in the preparation method; and the remaining steps are completely consistent with Example 3.

[0067] Comparative Example 4: This comparative example provides a brain-protecting composition, which differs from Example 3 only in that the formulation is exactly the same as that of Example 3, but the co-assembly process of S1-S3 is not performed. Instead, 15 parts of LPC-DHA, 7.5 parts of algal oil DHA, 3 parts of phytosterols, 4.5 parts of GINKGOSELECT® Ginkgo biloba extract, 2 parts of N-acetylneuraminic acid, 0.3 parts of natural vitamin E and 62.7 parts of excipients are directly added to a three-dimensional mixer and physically mixed at 20 rpm for 30 minutes to obtain a physically mixed powder.

[0068] The performance of the LPC-DHA and brain-protecting compositions prepared in the above examples and comparative examples was tested:

[0069] 1. Determination of LPC-DHA purity and sn-2 site proportion: LPC-DHA samples prepared in Examples 2-5 and Comparative Examples 1-2 were analyzed using high-performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD). Chromatographic column: Silica column (4.6 × 250 mm, 5 μm); mobile phase: n-hexane-isopropanol-acetic acid-triethylamine (50:45:5:0.1, v / v); flow rate: 1.0 mL / min; ELSD drift tube temperature: 70℃; nitrogen flow rate: 2.0 L / min. Purity and sn-2 site LPC-DHA proportion were determined. The results are as follows: Figure 1 As shown.

[0070] 2. Performance testing of brain-protecting compositions: The brain-protecting compositions prepared in Examples 3, 5 and Comparative Examples 1-4 were used as samples for testing. 1g of each composition powder was taken and reconstituted with deionized water to 10mL. The average particle size, polydispersity index (PDI) and zeta potential were determined using a Malvern Zetasizer NanoZS90. The results are shown in Table 1.

[0071] Table 1 Performance test results of the brain-protecting composition

[0072]

[0073] 3. Validation of in vitro blood-brain barrier (BBB) ​​penetration capability: The targeted delivery capability was validated using an in vitro cultured bEnd.3 brain microvascular endothelial cell model. Cells were seeded in the upper layer of Transwell chambers and cultured until the transendothelial resistance (TEER) > 200 Ω·cm. 2 The compositions prepared in Examples 2-5 and Comparative Example 4 were added to the upper chamber at a DHA concentration of 50 μg / mL. Samples of the lower culture medium were taken at 1 h, 2 h, and 4 h, respectively. The DHA content in the lower culture medium was determined by GC-MS, and the apparent permeability coefficient was calculated. The results are shown in Table 2.

[0074] Table 2 Apparent permeability coefficients (Papp, ×10⁻¹⁰) of in vitro BBB model -6 cm / s)

[0075]

[0076] 4. Animal Experiments: SPF-grade APP / PS1 double transgenic AD model mice (6 months old) were randomly divided into a model control group, Example 3 group, Comparative Example 1 group, and Comparative Example 4 group. An additional age-matched C57BL / 6J mouse served as a blank control group. Each group consisted of 10 mice. The treatment groups were administered the corresponding combination (dose calculated as DHA 100 mg / kg) via gavage daily. The blank control group and model group were administered an equal volume of physiological saline via gavage for 8 consecutive weeks. After the last administration, behavioral tests were conducted using the Morris water maze: the pool diameter was 120 cm, and the platform diameter was 10 cm, placed in the target quadrant at a water temperature of 22±1℃. The orientation and navigation experiment lasted 5 days, with training twice daily. The escape latency of the mice in finding the platform was recorded. On day 6, a spatial exploration experiment was conducted. The platform was removed, and the time the mice spent in the original platform quadrant and the number of times they crossed the original platform location within 60 seconds were recorded. The results are shown in Table 3, which was used to evaluate the improvement in memory and concentration.

[0077] Table 3. Results of the Morris Water Maze Test

[0078]

[0079] observe Figure 1The data shows that the LPC-DHA prepared in the embodiments of this invention has significantly higher purity and sn-2 site ratio than the comparative example. Comparative example 1 uses conventional column chromatography, which lacks an in-situ adsorption purification mechanism, resulting in severe acyl group migration and a low sn-2 site ratio. Comparative example 2 replaces the composite adsorbent with pure activated carbon, losing the electrostatic anchoring and specific desorption functions of mesoporous silica, leading to a significant decrease in both purity and ratio. This proves that the extraction process of this invention can obtain LPC-DHA with a higher active configuration (based on targeted absorption channels).

[0080] Observation of Table 1 reveals that Examples 3 and 5 of this invention formed stable nanoparticles with small size, uniform distribution, and strong negative surface charge through a ternary co-assembly process. The high absolute value of the Zeta potential provided a strong electrostatic repulsion force, further ensuring the physical stability of the nanoparticles. Comparative Examples 1 and 2, due to the low sn-2 content of LPC-DHA, affected the assembly efficiency, resulting in increased particle size and decreased stability. Comparative Example 3, lacking phytosterols, could not form a dense hydrophobic core, resulting in a loose assembly and a significant increase in particle size and PDI. Comparative Example 4 was merely a physical mixture and could not form a nanostructure. This demonstrates the crucial role of a complete formulation and co-assembly process in constructing a stable nanodelivery system.

[0081] As shown in Table 2, the Papp value of the Example 1 was significantly higher than that of Comparative Example 4, which strongly demonstrates that the nanocomposite constructed by the co-assembly process with an outer layer modified by N-acetylneuraminic acid can more effectively cross the blood-brain barrier model. As shown in Table 3, compared with the model control group, the escape latency of mice in Example 3 was significantly shortened, the time spent in the target quadrant was significantly prolonged, and the number of platform crossings was significantly increased, with effects approaching those of the blank control group. Although Comparative Example 1 showed improvement, its effect was significantly inferior to that of Example 3. The results indicate that the brain-protective composition of the present invention can significantly improve the learning and memory abilities of AD model mice.

[0082] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0083] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A brain-protective composition containing highly absorbable LPC-DHA, characterized in that, The brain-protecting composition comprises the following raw materials in parts by weight: 12-18 parts LPC-DHA, 5-10 parts algal oil DHA, 2-4 parts phytosterols, 3-6 parts GINKGOSELECT® Ginkgo biloba extract, 1-3 parts N-acetylneuraminic acid, 0.1-0.5 parts antioxidant, and 55-70 parts excipients; The LPC-DHA is prepared by a dynamic enzymatic hydrolysis-adsorption purification method. The raw materials for its preparation include: natural phospholipids, phospholipase A1, and a composite adsorbent. The composite adsorbent is a core-shell structured composite material with activated carbon as the core and a mesoporous silica shell on the surface. The preparation method of LPC-DHA includes the following steps: (1) Substrate pretreatment: Natural phospholipids were dispersed in citrate-sodium citrate buffer, taurocholate sodium was added for preheating, and substrate liposome dispersion was formed by high-speed shearing; (2) Dynamic enzymatic hydrolysis: Phospholipase A1 and composite adsorbent were added to the substrate liposome dispersion and the mixture was stirred. (3) Gradient elution and purification: After the reaction is completed, the precipitate containing the product is collected by centrifugation. Acetic acid-sodium acetate buffer is added to the precipitate and stirred to desorb. Then, the precipitate is separated by centrifugation again. The supernatant containing high purity sn-2-LPC-DHA is collected. After ultrafiltration and freeze drying, LPC-DHA with high sn-2 content is obtained.

2. The brain-protective composition containing highly absorbable LPC-DHA according to claim 1, characterized in that, The preparation method of the composite adsorbent is as follows: (a) Activated carbon is dispersed in a mixed solvent of anhydrous ethanol and deionized water, and sucrose fatty acid ester is added and stirred to obtain a dispersion; (b) Tetraethyl orthosilicate is added to the dispersion, and then the pH is adjusted and the reaction is carried out by reflux; (c) After the reaction is completed, the precipitate is collected by centrifugation, washed and dried to obtain the composite adsorbent.

3. The brain-protective composition containing highly absorbable LPC-DHA according to claim 1, characterized in that, The phospholipase A1 is derived from Aspergillus oryzae.

4. The brain-protective composition containing highly absorbable LPC-DHA according to claim 1, characterized in that, The natural phospholipids are marine-derived phospholipids rich in DHA, and can be either squid lecithin or phospholipids extracted from marine microalgae.

5. The brain-protective composition containing highly absorbable LPC-DHA according to claim 1, characterized in that, The amount of phospholipase A1 added is 4% of the mass of the substrate liposome dispersion; the amount of the composite adsorbent added is 15% of the mass of the substrate liposome dispersion.

6. The brain-protective composition containing highly absorbable LPC-DHA according to claim 1, characterized in that, The antioxidant is either rosemary extract or natural vitamin E.

7. The brain-protective composition containing highly absorbable LPC-DHA according to claim 1, characterized in that, The excipient is a mixture of maltodextrin and gum arabic in a mass ratio of 3:

1.

8. A method for preparing a brain-protective composition containing highly absorbable LPC-DHA according to any one of claims 1-7, characterized in that, Includes the following steps: S1. Dissolve LPC-DHA, algal oil DHA, phytosterols and GINKGOSELECT® Ginkgo biloba extract in anhydrous ethanol, and heat to aid dissolution to obtain a mixed solution; S2. Dissolve the excipient and N-acetylneuraminic acid in deionized water to obtain an aqueous solution. Then, add the mixed solution dropwise to the aqueous solution and stir to obtain the primary emulsion. S3. Homogenize the primary emulsion by circulation, and then allow it to stand for aging to obtain the targeted nanocomposite. S4. Add an antioxidant to the targeted nanocomposite, mix and dry to obtain the brain-protecting composition.

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