Ergothioneine oral soluble film with high absorption efficiency and preparation method of ergothioneine oral soluble film

Abstract

The invention discloses an ergothioneine oral soluble film with an efficient absorption function and a preparation method of the ergothioneine oral soluble film. The method comprises the following steps: preparing a first layer of coating liquid from ergothioneine, a substrate component, a copper ion chelating agent and a water-soluble polymer, and preparing a second layer of coating liquid from a polymer matrix, a tackifier and a temperature-sensitive adhesion polymer; and sequentially coating and drying to form a quick-release layer and an adhesive slow-release layer, thereby obtaining the double-layer orally-soluble film. The temperature-sensitive polymer disclosed by the invention adopts an LCST regulation and control type copolymer, the lowest critical solution temperature is 35-39 DEG C, and the temperature-sensitive polymer can be subjected to phase change at the oral cavity temperature to form a gel layer; and the tackifier is selected from sulfhydrylated chitosan, quaternized chitosan or dopamine modified polymer, and can form covalent or electrostatic adhesion with mucous membranes. Soluble microneedle arrays may also be integrated to enhance mucosal penetration. According to the invention, quick release and long-acting adhesion synergy of ergothioneine are realized, the oral retention time is effectively prolonged, and the mucous membrane absorption efficiency is improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically a highly efficient ergothioneine oral soluble membrane and its preparation method. Background Technology

[0002] L-Ergothioneine is a naturally occurring sulfur-containing amino acid derivative widely distributed in fungi such as mushrooms and certain bacteria, exhibiting excellent antioxidant, anti-inflammatory, and cell-protective activities. Since the human body cannot synthesize ergothioneine and it must be obtained through diet or supplements, the development of highly efficient ergothioneine delivery formulations has significant application value.

[0003] Ergothioneine is highly hydrophilic, exhibits good solubility in water, and demonstrates good chemical stability. However, cellular uptake of ergothioneine primarily relies on active transport mediated by the organic cation transporter OCTN1 (SLC22A4), as it is difficult for it to penetrate biological membranes via passive diffusion. Studies have shown that the OCTN1 transporter exhibits saturation kinetics, with absorption efficiency decreasing with increasing dosage. Furthermore, the SLC22A4 gene contains multiple single nucleotide polymorphisms, leading to significant differences in ergothioneine absorption capacity among individuals. Currently, commercially available ergothioneine formulations are mainly in capsule and tablet forms. These traditional oral dosage forms require absorption through the gastrointestinal tract before entering the portal circulation, thus limiting oral bioavailability.

[0004] Orally disintegrating films, as a novel oral drug delivery system, offer advantages such as no need for swallowing with water, convenient administration, and rapid onset of action. They are particularly suitable for the elderly, children, and other special patient groups with swallowing difficulties. Once placed in the oral cavity, the film rapidly disintegrates and dissolves, allowing some drugs to be directly absorbed through the oral mucosa into the systemic circulation, thus avoiding the first-pass effect of the liver and potentially improving drug bioavailability. The oral mucosa is richly vascularized, highly permeable, and has relatively low surface enzyme activity and a mild pH environment, making it an ideal site for drug delivery.

[0005] However, existing orally disintegrating film technologies still have several shortcomings when applied to ergothioneine delivery. First, traditional orally disintegrating films mostly employ a single-layer structure design, primarily aiming for rapid disintegration. This results in a short residence time in the oral cavity and limited contact time between the drug and the oral mucosa, making it difficult to achieve sufficient transmucosal absorption. Second, while the film-forming materials used in conventional orally disintegrating films, such as polyvinyl alcohol and hydroxypropyl methylcellulose, have good film-forming properties and fast dissolution rates, their mucosal adhesion is limited. They are easily dislodged and migrated under the influence of saliva secretion and swallowing movements, further shortening the effective drug delivery time. Furthermore, single-layer orally disintegrating films cannot simultaneously meet the synergistic functional requirements of rapid and sustained release, nor can they achieve targeted drug release towards the mucosa. Some of the drug released into the oral cavity is diluted by saliva or enters the gastrointestinal tract with swallowing, reducing the efficiency of transmucosal absorption.

[0006] To address the issue of insufficient mucosal adhesion of orally dissolving films, researchers have attempted to introduce mucosal adhesion polymers such as chitosan and its derivatives, and carbomer, to prolong the retention time of formulations in the oral cavity. Thermosensitive polymers, due to their ability to undergo a sol-gel phase transition in response to temperature changes, have also been used in oral drug delivery system research, aiming to form a gel layer in situ upon contact with oral temperatures, thereby enhancing adhesion to the mucosa. However, existing thermosensitive polymers, such as poly(N-isopropylacrylamide), typically have a minimum critical solution temperature (LCST) of around 32°C, lower than normal oral temperatures, making it difficult to achieve the expected thermosensitive response function in practical applications. Furthermore, the thionyl group in ergothioneine molecules can undergo coordination reactions with metal ions such as copper ions, potentially leading to oxidative degradation during storage. Therefore, appropriate stabilizers need to be added to the formulation to ensure product quality stability.

[0007] In conclusion, developing an ergothioneine orally disintegrating film formulation that can prolong oral retention time, enhance mucosal adhesion, and achieve synergistic effects of immediate and sustained release is of great significance for improving the oral mucosal absorption efficiency of ergothioneine. Summary of the Invention

[0008] To address the limitations of existing oral formulations of ergothioneine in terms of bioavailability, and the short retention time and insufficient mucosal adhesion of traditional orally dissolving films, this invention provides a method for preparing a highly efficient ergothioneine orally dissolving film and the resulting film product. This invention achieves a synergistic effect of rapid release and sustained absorption of ergothioneine in the oral cavity through a bilayer structure design, the introduction of a temperature-sensitive adhesive polymer, and optional microneedle array integration. This effectively prolongs the retention time of the formulation on the oral mucosa and improves the efficiency of drug absorption via the mucosa.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing a highly efficient ergothioneine oral soluble membrane includes the following steps: S1. Add ergothioneine, substrate components, copper ion chelating agent, and water-soluble polymer to deionized water, and stir at 50-80℃ for 1-3 hours until completely dissolved to prepare the first coating solution. In this step, heating and stirring help the water-soluble polymer to fully swell and dissolve, ensuring the coating solution system is uniform and stable. Controlling the temperature within the range of 50-80℃ ensures polymer dissolution efficiency while avoiding the adverse effects of excessively high temperatures on the activity of ergothioneine.

[0010] S2. Add the polymer matrix, tackifier, and thermosensitive adhesive polymer to deionized water and stir at 50-80℃ for 1-4 hours until completely dissolved to prepare the second coating solution. The tackifier and thermosensitive adhesive polymer introduced into the second coating solution are key components for achieving the adhesive membrane function, and need to be thoroughly stirred to ensure uniform dispersion in the polymer matrix.

[0011] S3. The first coating solution is uniformly coated onto the substrate with a thickness of 200-400 μm, and dried at 40-55℃ for 6-12 hours to form a quick-release layer containing ergothioneine. The substrate can be polyester film, silicone paper, or other suitable release substrate. The coating thickness and drying conditions directly affect the film quality. Too thin a coating may result in an uneven film layer, while too thick a coating will prolong the drying time and may cause bubbles or cracks. The drying temperature is controlled at 40-55℃, which can effectively remove moisture and prevent the film layer from shrinking and deforming or the ergothioneine from degrading due to excessive temperature.

[0012] S4. A second coating liquid is uniformly coated onto the immediate-release layer to a thickness of 150-350 μm. The coating is then dried at 40-55°C for 6-12 hours to form an adhesive sustained-release layer. The resulting film is then cut and packaged to obtain an ergothioneine oral soluble film. The double-layer coating process ensures good adhesion between the immediate-release layer and the adhesive sustained-release layer, preventing separation at the interlayer interface. The cutting size can be determined according to actual application requirements; typically, a single film area is 2-6 cm². 2 .

[0013] In a preferred embodiment of the present invention, the first coating solution in S1 is prepared by weight as follows: 20-40 parts polyvinyl alcohol, 10-25 parts hydroxypropyl methylcellulose, 3-15 parts ergothioneine, 0.5-5 parts substrate, 0.1-1 parts copper ion chelating agent, and 8-15 parts glycerol, and is prepared with deionized water to form a coating solution with a solid content of 15-20%. Polyvinyl alcohol and hydroxypropyl methylcellulose are the main film-forming materials, and their combined use can obtain good film-forming properties, mechanical strength, and water solubility. Glycerol, as a plasticizer, can improve the flexibility of the film layer and prevent the film layer from becoming brittle after drying. Its amount is adjusted according to the total amount of film-forming materials to obtain suitable film layer feel and mechanical properties.

[0014] In a preferred embodiment of the present invention, the second coating liquid in step S2 is formulated by weight as follows: 15-30 parts polyvinyl alcohol, 10-20 parts hydroxypropyl methylcellulose, 2-10 parts tackifier, 0.5-5 parts thermosensitive adhesive polymer, and 6-12 parts glycerin, and is prepared with deionized water to form a coating liquid with a solid content of 12-16%. The solid content of the second coating liquid is slightly lower than that of the first layer, which is beneficial for wetting and spreading on the surface of the immediate-release layer during coating, forming a uniform adhesive slow-release layer. The tackifier is selected from one or more of thiolated trimethyl chitosan, N-trimethyl chitosan, hydroxypropyl trimethylammonium chloride chitosan, thiolated chitosan, dopamine-modified polyvinyl alcohol, and dopamine-modified hyaluronic acid. The aforementioned thickeners exhibit excellent mucosal adhesion properties. Thiol-modified chitosans can form disulfide bonds with cysteine ​​residues in mucoproteins, achieving covalent adhesion. Quaternized chitosans carry a permanent positive charge, generating electrostatic attraction with negatively charged mucosal surfaces. Dopamine-modified polymers can form covalent bonds with mucosal proteins through Michael addition reactions or Schiff base reactions with catechol groups. All of the above thickeners can be obtained commercially or prepared using conventional methods in the art. Thiol-modified chitosan can be prepared by amidation of chitosan with thioglycolic acid or L-cysteine ​​under carbodiimide catalysis; thiolated trimethyl chitosan can be prepared by first quaternizing chitosan and then thiolated; dopamine-modified polyvinyl alcohol and dopamine-modified hyaluronic acid can be prepared by reacting dopamine hydrochloride with the corresponding polymers under weakly alkaline conditions (pH 8-9), utilizing the oxidative self-polymerization properties of dopamine to graft it onto the polymer backbone.

[0015] In a preferred embodiment of the present invention, the substrate component is one or more of L-carnitine, acetyl-L-carnitine, and carnitine hydrochloride. These substances are naturally occurring nutrients in the human body, and their use in combination with ergothioneine can enhance the functionality of the product.

[0016] In a preferred embodiment of the present invention, the copper ion chelating agent is selected from one or more of disodium ethylenediaminetetraacetate, citric acid, sodium citrate, and phytic acid. The thion group in ergothioneine molecules can undergo coordination reactions with transition metal ions such as copper ions, which may lead to oxidative degradation during storage. The addition of a copper ion chelating agent can effectively complex trace metal ions that may be present in the formulation, improving the storage stability of the preparation.

[0017] In a preferred embodiment of the present invention, the thermosensitive adhesive polymer is one or more of a thermosensitive polymer or a mucosal adhesive polysaccharide. The thermosensitive polymer is selected from one or more of poly(N-isopropylacrylamide-co-acrylic acid) copolymer, poly(N-isopropylacrylamide-co-acrylamide) copolymer, and poly(N-vinylcaprolactam-co-vinylimidazolium) copolymer, and its minimum critical solution temperature is 35-39°C. By introducing hydrophilic monomers through copolymerization, the LCST of thermosensitive polymers such as poly(N-isopropylacrylamide) can be adjusted from approximately 32°C to the range of 35-39°C, allowing it to remain hydrophilically dissolved at room temperature for easy processing. Upon contact with oral cavity temperature (approximately 37°C), a phase transition occurs to form a hydrophobic gel layer, enhancing adhesion to the mucosal surface. The above-mentioned LCST-regulated thermosensitive polymer can be prepared by free radical copolymerization. Taking poly(N-isopropylacrylamide-co-acrylic acid) copolymer as an example, copolymerizing N-isopropylacrylamide and acrylic acid in a molar ratio of 9:1 to 8:2 can yield copolymers with an LCST in the range of 35-39°C. The LCST value of the copolymer can be precisely controlled by adjusting the feeding ratio of the hydrophilic monomer (acrylic acid or acrylamide). The above-mentioned temperature-sensitive copolymer can also be custom-synthesized through commercial channels. The mucosal adhesive polysaccharide is selected from one or more of hyaluronic acid, chondroitin sulfate, and sodium alginate. These natural polysaccharides have good biocompatibility and mucosal adhesion ability, which can help prolong the retention time of the preparation in the oral cavity.

[0018] In a preferred embodiment of the present invention, the stirring speed of the first coating liquid and the second coating liquid is 500-1000 rpm. A suitable stirring speed ensures that the components are thoroughly and evenly mixed, avoiding the introduction of excessive air bubbles due to excessive stirring.

[0019] In a preferred embodiment of the present invention, step S5 is further included: dissolving and mixing one or more water-soluble polymer materials selected from polyvinylpyrrolidone, hyaluronic acid, and sodium carboxymethyl cellulose with ergothioneine, injecting the mixture into a microneedle mold, drying and molding it at 25-40°C, and then demolding it to obtain a soluble microneedle array; attaching and fixing the microneedle array to the back side of the ergothioneine orally dissolving film obtained in step S4; the height of the microneedles is 100-500 μm. The soluble microneedle array can penetrate the stratum corneum of the oral mucosa, delivering ergothioneine directly to the deep mucosa. At the same time, the mechanical anchoring effect of the microneedles can significantly enhance the fixation effect between the orally dissolving film and the mucosa, preventing the formulation from shifting and falling off during oral movement. The microneedle height is controlled within the range of 100-500 μm, which can effectively penetrate the surface layer of the mucosa without penetrating the entire mucosa and causing discomfort.

[0020] This invention also provides a highly efficient ergothioneine oral dissolving membrane prepared using the above method. The immediate-release layer of the ergothioneine oral dissolving membrane has a thickness of 30-60 μm, the adhesive sustained-release layer has a thickness of 20-50 μm, and the total thickness is 50-110 μm; the moisture content is 2-5%. This thickness range ensures sufficient drug loading without affecting the comfort of administration and disintegration performance due to excessive membrane thickness; controlling the moisture content of the finished product within the range of 2-5% helps maintain the flexibility of the membrane and ensures storage stability.

[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention adopts a double-layer structure design, organically combining an immediate-release layer containing ergothioneine with an adhesive sustained-release layer. The immediate-release layer rapidly disintegrates and releases the drug in the oral cavity, meeting the need for rapid onset of action; the adhesive sustained-release layer adheres tightly to the oral mucosa through the synergistic effect of a thickener and a temperature-sensitive adhesive polymer, continuously releasing the remaining drug and prolonging the oral retention time. This double-layer structure overcomes the shortcomings of traditional single-layer orofacial films, such as short retention time and insufficient drug absorption, and achieves synergy between immediate-release and sustained-release functions, effectively improving the transmucosal absorption efficiency and overall bioavailability of ergothioneine.

[0022] (2) This invention introduces LCST-regulated thermosensitive polymers and high-efficiency mucosal adhesion materials into the adhesive sustained-release layer. The minimum critical dissolution temperature of the thermosensitive polymer is controlled at 35-39℃, which matches the oral temperature. After the formulation is placed in the oral cavity, it can respond to body temperature and undergo a sol-gel phase transition, forming a gel layer in situ to enhance mucosal adhesion. Thiolized chitosan, quaternized chitosan and other thickeners can firmly bind to the oral mucosa through multiple mechanisms such as covalent bonding and electrostatic interaction. The synergistic effect of the two significantly improves the adhesion strength and adhesion persistence of the formulation, ensuring the effective retention and absorption of the drug in the oral cavity.

[0023] (3) The present invention can further enhance the drug delivery effect by integrating a soluble microneedle array. The microneedles penetrate the stratum corneum of the mucosa to deliver ergothioneine directly to the deep layer of the mucosa, while playing a mechanical anchoring role to prevent the formulation from migrating and falling off. In addition, the preparation process of the present invention is simple and controllable, using a solvent casting method to coat and dry layer by layer. The parameters of each step are clear, which is easy to scale up for industrial production. The product quality is stable and has good application prospects. Detailed Implementation

[0024] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to these embodiments. Those skilled in the art should recognize that the present invention covers all possible alternatives, improvements and equivalents included within the scope of the claims. Example 1

[0025] A highly efficient ergothioneine orally soluble membrane is prepared according to the following steps: S1. Preparation of the first coating solution: By weight, add 30 parts of polyvinyl alcohol and 18 parts of hydroxypropyl methylcellulose to deionized water and stir at 65°C until completely swollen. Then add 10 parts of ergothioneine, 3 parts of acetyl-L-carnitine, 0.5 parts of disodium EDTA and 12 parts of glycerol, and continue stirring until completely dissolved. The stirring speed is 750 rpm and the stirring time is 2 hours to prepare the first coating solution with a solid content of 18%.

[0026] S2. Preparation of the second coating solution: By weight, add 22 parts polyvinyl alcohol and 15 parts hydroxypropyl methylcellulose to deionized water and stir at 65°C until completely swollen. Then add 6 parts thiolated chitosan, 3 parts poly(N-isopropylacrylamide-co-acrylic acid) copolymer, and 9 parts glycerol, and continue stirring until completely dissolved. The stirring speed is 750 rpm, and the stirring time is 2.5 hours, to prepare a second coating solution with a solid content of 14%. The minimum critical dissolution temperature of the poly(N-isopropylacrylamide-co-acrylic acid) copolymer is 37°C.

[0027] S3. Preparation of the immediate-release layer: The first coating liquid is uniformly coated on the polyester film substrate with a coating thickness of 300 μm, and dried at 48°C for 9 hours to form an immediate-release layer containing ergothioneine. The thickness of the immediate-release layer after drying is 45 μm.

[0028] S4. Preparation of the adhesive sustained-release layer: A second coating liquid was uniformly coated onto the immediate-release layer with a thickness of 250 μm, and dried at 48℃ for 9 hours to form the adhesive sustained-release layer. The thickness of the adhesive sustained-release layer after drying was 35 μm. It was then cut into 2cm × 3cm pieces for packaging to obtain ergothioneine oral soluble films. The total thickness of the finished oral soluble films was 80 μm, and the moisture content was 3.5%. Example 2

[0029] A highly efficient ergothioneine orally soluble membrane is prepared according to the following steps: S1. Preparation of the first coating solution: By weight, add 20 parts of polyvinyl alcohol and 10 parts of hydroxypropyl methylcellulose to deionized water and stir at 50°C until completely swollen. Then add 3 parts of ergothioneine, 0.5 parts of L-carnitine, 0.1 parts of citric acid and 8 parts of glycerin, and continue stirring until completely dissolved. The stirring speed is 500 rpm and the stirring time is 3 hours to prepare the first coating solution with a solid content of 15%.

[0030] S2. Preparation of the second coating solution: By weight, add 15 parts of polyvinyl alcohol and 10 parts of hydroxypropyl methylcellulose to deionized water and stir at 50°C until completely swollen. Then add 2 parts of N-trimethyl chitosan, 0.5 parts of hyaluronic acid, and 6 parts of glycerin, and continue stirring until completely dissolved. The stirring speed is 500 rpm and the stirring time is 4 hours to prepare a second coating solution with a solid content of 12%.

[0031] S3. Preparation of the immediate release layer: The first coating liquid is uniformly coated on the silicone paper substrate with a coating thickness of 200 μm and dried at 40°C for 12 hours to form an immediate release layer containing ergothioneine. The thickness of the immediate release layer after drying is 30 μm.

[0032] S4. Preparation of the adhesive sustained-release layer: A second coating liquid was uniformly coated onto the immediate-release layer with a thickness of 150 μm. The coating was then dried at 40°C for 12 hours to form the adhesive sustained-release layer, which had a thickness of 20 μm after drying. The film was then cut into 2 cm × 3 cm pieces and packaged to obtain ergothioneine oral soluble films. The total thickness of the finished oral soluble films was 50 μm, and the moisture content was 5%. Example 3

[0033] A highly efficient ergothioneine orally soluble membrane is prepared according to the following steps: S1. Preparation of the first coating solution: By weight, add 40 parts of polyvinyl alcohol and 25 parts of hydroxypropyl methylcellulose to deionized water and stir at 80°C until completely swollen. Then add 15 parts of ergothioneine, 5 parts of carnitine hydrochloride, 1 part of phytic acid, and 15 parts of glycerin. Continue stirring until completely dissolved at a stirring speed of 1000 rpm for 1 hour to prepare the first coating solution with a solid content of 20%.

[0034] S2. Preparation of the second coating solution: Add 30 parts by weight of polyvinyl alcohol and 20 parts by weight of hydroxypropyl methylcellulose to deionized water, and stir at 80°C until completely swollen. Then add 10 parts by weight of hydroxypropyltrimethylammonium chloride chitosan, 5 parts by weight of poly(N-vinylcaprolactam-co-vinylimidazole) copolymer, and 12 parts by weight of glycerol, and continue stirring until completely dissolved. The stirring speed is 1000 rpm, and the stirring time is 1 hour, to prepare a second coating solution with a solid content of 16%. The minimum critical dissolution temperature of the poly(N-vinylcaprolactam-co-vinylimidazole) copolymer is 38°C.

[0035] S3. Preparation of the immediate-release layer: The first coating liquid is uniformly coated on the polyester film substrate with a coating thickness of 400 μm, and dried at 55°C for 6 hours to form an immediate-release layer containing ergothioneine. The thickness of the immediate-release layer after drying is 60 μm.

[0036] S4. Preparation of the adhesive sustained-release layer: A second coating liquid was uniformly coated onto the immediate-release layer to a thickness of 350 μm, and dried at 55°C for 6 hours to form the adhesive sustained-release layer, which had a thickness of 50 μm after drying. The film was then cut into 2 cm × 3 cm pieces and packaged to obtain ergothioneine oral soluble films. The total thickness of the finished oral soluble films was 110 μm, and the moisture content was 2%. Example 4

[0037] A highly efficient ergothioneine orally soluble membrane integrating a microneedle array is prepared according to the following steps: Steps S1-S4 are the same as in Example 1.

[0038] S5. Preparation of soluble microneedle array: 15 parts by weight of polyvinylpyrrolidone and 10 parts by weight of hyaluronic acid were dissolved in deionized water by stirring. Then, 5 parts by weight of ergothioneine were added and mixed thoroughly. The resulting solution was injected into a polydimethylsiloxane microneedle mold, dried at 35°C for 24 hours, and then demolded to obtain a soluble microneedle array. The microneedles were conical in shape, with a height of 300 μm, a base diameter of 150 μm, and a microneedle density of 196 needles / cm². The microneedle array was then bonded and fixed to the back side (i.e., the outer side of the sustained-release layer) of the ergothioneine oral dissolution membrane obtained in step S4 using medical pressure-sensitive adhesive, thus obtaining an ergothioneine oral dissolution membrane with an integrated microneedle array.

[0039] A highly efficient ergothioneine orally soluble membrane is prepared according to the following steps: S1. Preparation of the first coating solution: By weight, add 35 parts of polyvinyl alcohol and 20 parts of hydroxypropyl methylcellulose to deionized water and stir at 70°C until completely swollen. Then add 12 parts of ergothioneine, 2 parts of acetyl-L-carnitine, 0.8 parts of sodium citrate, and 10 parts of glycerin. Continue stirring until completely dissolved at a stirring speed of 800 rpm for 1.5 hours to prepare the first coating solution with a solid content of 17%.

[0040] S2. Preparation of the second coating solution: By weight, add 25 parts polyvinyl alcohol and 12 parts hydroxypropyl methylcellulose to deionized water and stir at 70°C until completely swollen. Then add 3 parts dopamine-modified polyvinyl alcohol, 2 parts dopamine-modified hyaluronic acid, 3 parts mercapto-trimethyl chitosan, 2 parts poly(N-isopropylacrylamide-co-acrylamide) copolymer, 0.5 parts chondroitin sulfate, 0.5 parts sodium alginate, and 8 parts glycerin. Continue stirring until completely dissolved at 800 rpm for 2 hours to obtain a second coating solution with a solid content of 15%. The minimum critical dissolution temperature of the poly(N-isopropylacrylamide-co-acrylamide) copolymer is 36°C.

[0041] S3. Preparation of the immediate-release layer: The first coating liquid is uniformly coated on the polyester film substrate with a coating thickness of 350 μm, and dried at 50°C for 7 hours to form an immediate-release layer containing ergothioneine. The thickness of the immediate-release layer after drying is 52 μm.

[0042] S4. Preparation of the adhesive sustained-release layer: A second coating liquid was uniformly coated onto the immediate-release layer to a thickness of 280 μm, and dried at 50°C for 7 hours to form the adhesive sustained-release layer, which had a thickness of 40 μm after drying. The film was then cut into 2 cm × 3 cm pieces and packaged to obtain ergothioneine oral soluble films. The total thickness of the finished oral soluble films was 92 μm, and the moisture content was 3%.

[0043] An ergothioneine oral dissolving film is prepared according to the following steps: By weight, 30 parts of polyvinyl alcohol and 18 parts of hydroxypropyl methylcellulose were added to deionized water and stirred at 65°C until completely swollen. Then, 10 parts of ergothioneine, 3 parts of acetyl-L-carnitine, 0.5 parts of disodium EDTA, and 12 parts of glycerol were added, and stirring continued until completely dissolved to prepare a coating solution with a solid content of 18%. The coating solution was uniformly coated onto a polyester film substrate to a thickness of 550 μm and dried at 48°C for 10 hours. The film was then cut into 2 cm × 3 cm pieces and packaged to obtain a single-layer ergothioneine oral soluble film with a thickness of 80 μm after drying.

[0044] The difference between this comparative example and Example 1 is that it adopts a single-layer structure, does not contain an adhesive slow-release layer, and does not contain tackifiers or temperature-sensitive adhesive polymers.

[0045] An ergothioneine oral dissolving film is prepared according to the following steps: S1. Preparation of the first coating liquid: Same as in Example 1.

[0046] S2. Preparation of the second coating solution: By weight, add 22 parts of polyvinyl alcohol and 15 parts of hydroxypropyl methylcellulose to deionized water and stir at 65°C until completely swollen. Then add 6 parts of thiolated chitosan and 9 parts of glycerol and continue stirring until completely dissolved to prepare a second coating solution with a solid content of 14%.

[0047] Steps S3-S4 are the same as in Example 1.

[0048] The difference between this comparative example and Example 1 is that the second coating liquid does not contain a temperature-sensitive adhesive polymer.

[0049] An ergothioneine oral dissolving film is prepared according to the following steps: S1. Preparation of the first coating liquid: Same as in Example 1.

[0050] S2. Preparation of the second coating solution: By weight, add 22 parts of polyvinyl alcohol and 15 parts of hydroxypropyl methylcellulose to deionized water and stir at 65°C until completely swollen. Then add 6 parts of thiolated chitosan, 3 parts of poly(N-isopropylacrylamide) (pure product, LCST about 32°C), and 9 parts of glycerin. Continue stirring until completely dissolved to prepare a second coating solution with a solid content of 14%.

[0051] Steps S3-S4 are the same as in Example 1.

[0052] The difference between this comparative example and Example 1 is that pure poly(N-isopropylacrylamide) is used instead of the LCST-regulated copolymer, which has a minimum critical dissolution temperature of approximately 32°C, lower than the oral temperature. Comparative Example 4

[0053] An ergothioneine oral dissolving film is prepared according to the following steps: S1. Preparation of the first coating liquid: Same as in Example 1.

[0054] S2. Preparation of the second coating solution: By weight, add 22 parts of polyvinyl alcohol and 15 parts of hydroxypropyl methylcellulose to deionized water and stir at 65°C until completely swollen. Then add 3 parts of poly(N-isopropylacrylamide-co-acrylic acid) copolymer and 9 parts of glycerol and continue stirring until completely dissolved to prepare a second coating solution with a solid content of 14%.

[0055] Steps S3-S4 are the same as in Example 1.

[0056] The difference between this comparative example and Example 1 is that the second coating liquid does not contain a thickener (thiolized chitosan). Performance testing

[0057] The following performance tests were performed on the ergothioneine oral dissolving films prepared in the above embodiments and comparative examples: 1. Disintegration time determination Referring to the disintegration time test method in General Chapter 0931 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, artificial saliva (pH 6.8 phosphate buffer, 37±0.5℃) was used as the test medium. The oral saliva film with the immediate release layer facing upwards was placed in the medium, and the time for the immediate release layer to completely disintegrate was recorded. Each group of samples was measured in parallel 6 times, and the average value was taken.

[0058] 2. Measurement of mucosal adhesion strength The mucosal adhesion strength of the ortholytic membrane was determined using a TA.XT Plus texture analyzer. Fresh porcine buccal mucosa was used as a model mucosa. The ortholytic membrane with the sustained-release layer facing down was attached to the mucosal surface. A pre-pressure of 50g was applied and held for 60 seconds, then the membrane was vertically pulled away at a speed of 1 mm / s, and the maximum pull-away force was recorded. Adhesion strength was expressed as the maximum pull-away force per unit area (N / cm²).2 Each group of samples was measured in parallel 5 times, and the average value was taken.

[0059] 3. In vitro mucosal permeation experiment In vitro osmosis experiments were conducted using a Franz diffusion cell. Fresh porcine buccal mucosa was used as the osmotic barrier, with the stratum corneum facing the supply chamber. The receiving solution was pH 7.4 phosphate buffer, and the temperature was maintained at 37 ± 0.5 °C. The effective diffusion area was 1.77 cm². 2 The orally dissolving membrane was attached to the supply chamber side with the adhesive sustained-release layer facing the mucosa. Samples of 1 mL were taken at 0.5, 1, 2, 3, and 4 hours, and an equal volume of fresh receiving fluid was added. Ergothioneine content was determined using high-performance liquid chromatography (HPLC), and the cumulative permeate volume per unit area (μg / cm²) was calculated. 2 ) and steady-state permeation flux (μg / cm) 2 ·h).

[0060] 4. Oral retention time measurement The retention performance of ortholytic membranes on mucosal surfaces was evaluated using a modified flow rinsing method. Fresh porcine buccal mucosa was fixed on a glass plate at a 45° angle. The ortholytic membrane was applied to the mucosal surface with an adhesive slow-release layer and gently pressed for 30 seconds. The mucosal surface was then continuously rinsed with artificial saliva (37°C, flow rate 2 mL / min). The time it took for the ortholytic membrane to completely detach was recorded as the oral retention time. Test Results

[0061] Table 1. Performance test results of the examples and comparative examples: .

[0062] Results Analysis As shown in Table 1, Example 1, using the intermediate ratio, exhibited the best performance across all indicators, with a cumulative permeation rate of 295 μg / cm³ over 4 hours. 2 Example 2 used the lower limit formulation; due to the relatively low amount of functional excipients, its performance was relatively lower but still better than the comparative examples. Example 3 used the upper limit formulation; the thicker membrane layer prolonged the disintegration time, and the permeation efficiency was lower than that of Example 1. These results indicate that there is an optimal ratio range for each component. In Example 4, after integrating the microneedle array, the permeation rate increased to 398 μg / cm³. 2 This represents a 35% improvement compared to Example 1.

[0063] Comparative Example 1, with its single-layer structure, had a retention time of only 6 minutes in the oral cavity, and its permeation volume was only 40% of that of Example 1, indicating that the bilayer structure is crucial for prolonging retention time and improving absorption efficiency. Comparative Examples 2-4, lacking a thermosensitive polymer, using a thermosensitive polymer with LCST mismatch, and lacking a thickener, respectively, all showed significantly lower performance than the examples, demonstrating the technical necessity of the synergistic effect of LCST-regulated thermosensitive polymers and thickeners.

[0064] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a highly efficient ergothioneine oral soluble membrane, characterized in that, Includes the following steps: S1. Add ergothioneine, substrate components, copper ion chelating agent and water-soluble polymer to deionized water, and stir at 50-80℃ for 1-3 hours until completely dissolved to prepare the first coating solution. S2. Add the polymer matrix, tackifier and temperature-sensitive adhesive polymer to deionized water and stir at 50-80℃ for 1-4 hours until completely dissolved to prepare the second coating liquid. S3. The first coating liquid is uniformly coated on the substrate with a coating thickness of 200-400μm, and dried at 40-55℃ for 6-12 hours to form an immediate-release layer containing ergothioneine. S4. The second coating liquid is uniformly coated on the immediate-release layer with a coating thickness of 150-350μm, and dried at 40-55℃ for 6-12 hours to form an adhesive sustained-release layer. Then, it is cut and packaged to obtain an ergothioneine oral soluble film.

2. The method for preparing ergothioneine oral soluble film according to claim 1, characterized in that, The first coating liquid in S1 is prepared by mass parts of: 20-40 parts of polyvinyl alcohol, 10-25 parts of hydroxypropyl methylcellulose, 3-15 parts of ergothioneine, 0.5-5 parts of substrate components, 0.1-1 parts of copper ion chelating agent, and 8-15 parts of glycerin, and is prepared with deionized water to form a coating liquid with a solid content of 15-20%.

3. The method for preparing ergothioneine oral soluble film according to claim 1, characterized in that, The second coating liquid in S2 is formulated by weight as follows: 15-30 parts polyvinyl alcohol, 10-20 parts hydroxypropyl methylcellulose, 2-10 parts tackifier, 0.5-5 parts thermosensitive adhesive polymer, and 6-12 parts glycerin, and is prepared with deionized water to form a coating liquid with a solid content of 12-16%; the tackifier is selected from one or more of thiolated trimethyl chitosan, N-trimethyl chitosan, hydroxypropyl trimethylammonium chloride chitosan, thiolated chitosan, dopamine-modified polyvinyl alcohol, and dopamine-modified hyaluronic acid.

4. The method for preparing ergothioneine oral soluble film according to claim 1, characterized in that, The substrate component is one or more of L-carnitine, acetyl-L-carnitine, and carnitine hydrochloride.

5. The method for preparing ergothioneine oral soluble film according to claim 1, characterized in that, The copper ion chelating agent is selected from one or more of disodium ethylenediaminetetraacetate, citric acid, sodium citrate, and phytic acid.

6. The method for preparing ergothioneine oral soluble film according to claim 1, characterized in that, The thermosensitive adhesive polymer is one or more of thermosensitive polymers or mucosal adhesive polysaccharides; the thermosensitive polymer is selected from one or more of poly(N-isopropylacrylamide-co-acrylic acid) copolymer, poly(N-isopropylacrylamide-co-acrylamide) copolymer, and poly(N-vinylcaprolactam-co-vinylimidazole) copolymer, and its minimum critical dissolution temperature is 35-39℃.

7. The method for preparing ergothioneine oral dissolving film according to claim 6, characterized in that, The mucosal adhesive polysaccharide is selected from one or more of hyaluronic acid, chondroitin sulfate, and sodium alginate.

8. The method for preparing ergothioneine oral soluble film according to claim 1, characterized in that, The stirring speed of both the first and second coating liquids is 500-1000 rpm.

9. The method for preparing ergothioneine oral soluble film according to claim 1, characterized in that, The method also includes step S5: dissolving and mixing one or more of polyvinylpyrrolidone, hyaluronic acid, and sodium carboxymethyl cellulose with ergothioneine, injecting the mixture into a microneedle mold, drying and molding it at 25-40°C, and then demolding it to obtain a soluble microneedle array; attaching and fixing the microneedle array to the back side of the ergothioneine oral soluble membrane obtained in step S4; the height of the microneedles is 100-500 μm.

10. A highly efficient ergothioneine-absorbing oral soluble membrane prepared by the method according to any one of claims 1-9, characterized in that, The ergothioneine oral soluble film has an immediate-release layer thickness of 30-60 μm, an adhesive sustained-release layer thickness of 20-50 μm, and a total thickness of 50-110 μm; the moisture content is 2-5%.