An ergothioneine-curcumin synergistic Anti-inflammatory and Anti-aging complex composition and a preparation method thereof

Abstract

The present invention discloses an ergothioneine-curcumin synergistic anti-inflammatory and anti-aging complex composition and a preparation method thereof, relating to biomedicine and functional skincare products. A ternary nanolipid carrier core-shell structure is constructed, comprising a hydrophobic core, an interface enhancement layer, and a hydrophilic shell. The hydrophobic core formed by hydrogenated lecithin and phytosterol provides a stable lipid microenvironment for curcumin. Piperine from black pepper extract is directionally anchored at the oil-water interface region of the lipid bilayer, enabling precise interfacial localization. The hydrophilic shell composed of thiolated hyaluronic acid covalently links ergothioneine through thiol-disulfide bonds, stably fixing it on the carrier surface. This enables ergothioneine to provide immediate antioxidant protection upon contact with skin or mucous membranes while preventing migration into the lipid core, thereby achieving spatial partitioning and synergistic functionality of the three active ingredients.

Description

[0001] DESCRIPTION

[0002] TECHNICAL FIELD

[0003] The present invention relates to the technical field of biomedicine and functional skincare products, specifically to an ergothioneine-curcumin synergistic anti-inflammatory and anti-aging complex composition and a preparation method thereof.

[0004] BACKGROUND

[0005] Ergothioneine, as a rare natural antioxidant, has the effects of scavenging free radicals and delaying cell aging; curcumin exhibits significant anti-inflammatory activity by regulating multiple inflammatory pathways such as NF-KB; piperine in black pepper extract can inhibit drug-metabolizing enzymes, thereby enhancing the bioavailability of co-delivered components. Combining these three active ingredients for application in the anti-inflammatory and anti-aging field has become a research hotspot, but how to achieve synergistic enhancement among them and overcome their respective physicochemical defects remains a technical problem urgently to be solved in this field.

[0006] In the prior art, simple physical mixing or conventional emulsion compounding of ergothioneine, curcumin, and black pepper extract has prominent defects:

[0007] First, curcumin has extremely poor water solubility and insufficient chemical stability, easily degrading and inactivating under neutral or alkaline conditions;

[0008] Second, although ergothioneine has good water solubility, it is prone to oxidation or non-specific binding with other components in complex formulation environments, making it difficult to maintain its free concentration;

[0009] Third, piperine in black pepper extract has strong hydrophobicity and irritation, direct addition easily causes local discomfort, and its metabolic inhibition effect needs to be precisely positioned in a specific interface region to exert maximum synergistic effect.

[0010] Furthermore, conventional liposome or nanoparticle preparation processes often use high-temperature emulsification or organic solvent residue techniques, which not only destroy the natural conformation of active ingredients but also cannot achieve the partitioned anchoring and programmed release of the three components in the carrier, making it difficult to simultaneously exert anti-inflammatory and anti-aging effects.

[0011] In summary, the prior art cannot simultaneously solve the core problems of synergistic enhancement, stable protection, and efficient delivery of ergothioneine, curcumin, and black pepper extract, severely limiting the industrial application of the ternary active ingredients. Therefore, developing a complex composition capable of achieving comprehensive synergy among the three and adapting to multi-scenario applications has significant research value and market significance.

[0012] SUMMARY

[0013] An objective of the present invention is to address the shortcomings of the prior art by providing an ergothioneine-curcumin synergistic anti-inflammatory and anti-aging complex composition and a preparation method thereof. It constructs a ternary nanolipid carrier core-shell hierarchical structure comprising a hydrophobic core, an interface enhancement layer, and a hydrophilic shell. The hydrophobic core formed by the combination of hydrogenated lecithin and phytosterol provides a stable lipid microenvironment for curcumin. The oil-water interface region of the lipid bilayer utilizes its polarity gradient to directionally anchor piperine molecules from the black pepper extract, precisely positioning piperine at the lipid membrane interface. The hydrophilic shell composed of thiolated hyaluronic acid covalently links ergothioneine through thiol-disulfide bonds. This not only utilizes hyaluronic acid's own moisturizing and anti-inflammatory properties to enhance the outer layer barrier function but also stably fixes ergothioneine on the carrier surface through chemical bonding, allowing it to exert its antioxidant protective effect immediately upon contact with skin or mucous membranes, while avoiding the migration and diffusion of ergothioneine into the inner lipid region. This partitioned anchoring structure solves the problem of asynchronous release and disordered action sites caused by differences in the physicochemical properties of the three components, achieving spatial ordered partitioning and functional synergy of the three active ingredients.

[0014] To solve the above technical problems, the present invention provides the following technical solutions: In one aspect, an ergothioneine-curcumin synergistic anti-inflammatory and anti-aging complex composition, the complex composition comprises an active efficacy component and a ternary co-loaded nanolipid carrier component, and in terms of mass percentage, the raw material composition of the complex composition is: 0.5%-5% of the active efficacy component, 2%-8% of hydrogenated lecithin, 0.5%-3% of phytosterol, l%-4% of thiolated hyaluronic acid, 0.5%-2% of a co-solvent, 0.1%-0.5% of a stabilizer, and the balance being deionized water;

[0015] The active efficacy component consists of ergothioneine, curcumin, and black pepper extract;

[0016] The ternary co-loaded nanolipid carrier has a core-shell hierarchical structure of a hydrophobic core, an interface enhancement layer, and a hydrophilic shell. Further, the mass ratio of ergothioneine, curcumin, and black pepper extract is (1.5-3): 1 :(0.1-0.3), and the mass content of piperine in the black pepper extract is >95%.

[0017] Furthermore, the hydrophobic core is composed of a lipid skeleton formed by combining hydrogenated lecithin and phytosterol, and curcumin is encapsulated inside the hydrophobic core;

[0018] The interface enhancement layer is the oil-water interface region of the lipid bilayer, and the black pepper extract is anchored and distributed within the interface enhancement layer;

[0019] The hydrophilic shell is thiolated hyaluronic acid, and ergothioneine is covalently bound and directionally anchored to the hydrophilic shell through thiol-disulfide bonds.

[0020] Furthermore, the phytosterol is at least one selected from the group consisting of soyasterol, sitosterol, and stigmasterol;

[0021] The molecular weight of the thiolated hyaluronic acid is 5 kDa-20 kDa, and the degree of thiol substitution is 15%-30%.

[0022] Furthermore, the co-solvent is at least one selected from the group consisting of 1,2-propanediol, glycerin, and polyethylene glycol 400;

[0023] The stabilizer is at least one selected from the group consisting of betulinic acid, rosmarinic acid, and tocopheryl polyethylene glycol succinate.

[0024] Furthermore, the particle size of the ternary co-loaded nanolipid carrier is 20 nm-60 nm, the polydispersity index PDI is <0.12, and the absolute value of zeta potential is >35 mV.

[0025] Furthermore, the thiolated hyaluronic acid is obtained by subjecting hyaluronic acid with a molecular weight of 5 kDa-20 kDa to thiolation modification, the specific steps being:

[0026] Activation Reaction: dissolving hyaluronic acid in 2-morpholinoethanesulfonic acid (MES) buffer, adding l-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and stirring at room temperature to activate carboxyl groups on the hyaluronic acid molecular chain, obtaining an activation reaction solution;

[0027] Thiolation Reaction: adding cystamine dihydrochloride to the activation reaction solution obtained in SI, adjusting the pH to 4.5-6.0, and stirring the reaction under an inert gas atmosphere at 20°C-30°C for 8-16 hours, obtaining a thiolated intermediate reaction solution;

[0028] Reduction Reaction: adding a reducing agent tris(2-carboxyethyl)phosphine (TCEP) to the thiolated intermediate reaction solution, continuing to stir the reaction for 2-4 hours to reduce disulfide bonds to free thiol groups;

[0029] Purification and Drying: placing the reaction solution obtained from the reduction reaction into a dialysis bag, performing dialysis purification under acidic conditions and an inert gas atmosphere to remove unreacted small molecule impurities, and freeze-drying the product after dialysis to obtain the thiolated hyaluronic acid, with a degree of thiol substitution of 15%-30%.

[0030] In another aspect, a method for preparing an ergothioneine-curcumin synergistic anti-inflammatory and anti-aging complex composition, the steps of the method are:

[0031] SI 00. Preparation of a low-temperature lipid phase: under an inert gas atmosphere and light-proof conditions, adding hydrogenated lecithin, phytosterol, curcumin, and black pepper extract to a co-solvent, and stirring until completely dissolved to obtain a uniform and transparent lipid phase;

[0032] S200. Preparation of an aqueous phase: under the same temperature, light-proof, and inert gas atmosphere conditions as in SI 00, adding a stabilizer to deionized water, and stirring until completely dissolved to obtain an aqueous phase;

[0033] S300. Gradient phase transfer nano-dispersion: under low-speed stirring conditions, adding the lipid phase prepared in SI 00 dropwise at a constant rate of 0.5-1 mL / min to the aqueous phase prepared in S200, controlling the volume ratio of the lipid phase to the aqueous phase to be 1 :(8-12), after the dropwise addition is complete, maintaining the temperature and stirring for 30-45 min, spontaneously forming nanolipid carriers through gradient phase

[0034] transfer, simultaneously completing the encapsulation of curcumin in the hydrophobic core and the directional anchoring of the black pepper extract in the interface enhancement layer, obtaining a nanolipid carrier primary emulsion;

[0035] S400. In-situ anchoring assembly: under the same temperature, light-proof, and stirring conditions as in SI 00, adding thiolated hyaluronic acid and ergothioneine to the primary emulsion prepared in S300, stirring the reaction under light-proof conditions for 60-90 min to carry out a thiol-disulfide click reaction, covalently anchoring ergothioneine onto the hydrophilic shell composed of thiolated hyaluronic acid, completing the in-situ assembly of the ternary co-loaded nanolipid carrier core-shell hierarchical structure;

[0036] S500. Post-treatment: filtering and sterilizing the reaction solution obtained in S400 through a microporous membrane to obtain the complex composition.

[0037] Furthermore, the system temperature in S100-S400 is all controlled at 22°C-26°C, and nitrogen is used as the inert protective gas throughout the process.

[0038] Furthermore, in S500, the complex composition is applied in anti-inflammatory and anti-aging cosmetics, oral health foods, and topical anti-inflammatory and anti-aging pharmaceutical preparations.

[0039] Compared with the prior art, this ergothioneine-curcumin synergistic anti-inflammatory and anti-aging complex composition and its preparation method have the following beneficial effects:

[0040] I. The present invention constructs a ternary nanolipid carrier core-shell hierarchical structure comprising a hydrophobic core, an interface enhancement layer, and a hydrophilic shell. The hydrophobic core formed by the combination of hydrogenated lecithin and phytosterol provides a stable lipid microenvironment for curcumin. The oil-water interface region of the lipid bilayer utilizes its polarity gradient to directionally anchor piperine molecules from the black pepper extract, precisely positioning piperine at the lipid membrane interface. The hydrophilic shell composed of thiolated hyaluronic acid covalently links ergothioneine through thiol-disulfide bonds. This not only utilizes hyaluronic acid's own moisturizing and anti-inflammatory properties to enhance the outer layer barrier function but also stably fixes ergothioneine on the carrier surface through chemical bonding, allowing it to exert its antioxidant protective effect immediately upon contact with skin or mucous membranes, while avoiding the migration and diffusion of ergothioneine into the inner lipid region. This partitioned anchoring structure solves the problem of asynchronous release and disordered action sites caused by differences in the physicochemical properties of the three components, achieving spatial ordered partitioning and functional synergy of the three active ingredients.

[0041] II. Through the isolation protection design of the hierarchical carrier and the coupled preparation process of low-temperature gradient phase transfer and in-situ anchoring, the present invention achieves the spontaneous

[0042] assembly of the nanolipid carrier and the simultaneous precise positioning of active ingredients. The three active ingredients are each provided with a protective environment isolated from light, heat, and oxygen, avoiding photodegradation of curcumin, oxidative inactivation of ergothioneine, and crystallization and precipitation of piperine. Relying on the cascade synergistic effect, the active ingredients can be effective at low doses, eliminating the safety risks associated with high concentrations of active ingredients. The entire process does not rely on toxic organic solvents or heavy equipment, making it suitable for large-scale production in multiple fields and application needs in various dosage forms.

[0043] Other advantages, objectives, and features of the present invention will be set forth in part in the following description, and in part will become apparent to those skilled in the art upon examination of the following or may be learned from practice of the invention.

[0044] BRIEF DESCRIPTION OF DRAWINGS

[0045] To explain the technical solutions in the embodiments of the present invention or the prior art more clearly, the following briefly introduces the drawings required for describing the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without inventive effort.

[0046] Figure 1 is a flow chart of the preparation method of an ergothioneine-curcumin synergistic anti-inflammatory and anti-aging complex composition;

[0047] Figure 2 is a block diagram of the preparation steps of thiolated hyaluronic acid in an implementation of the present invention.

[0048] DETAILED DESCRIPTION

[0049] To further elaborate on the technical means and effects adopted by the present invention to achieve the intended invention objectives, the following, in conjunction with the accompanying drawings and preferred embodiments, provides a detailed description of the specific implementation, structure, features, and effects of the present invention.

[0050] In order to improve the problems in the existing composite application of ergothioneine, curcumin, and black pepper extract, such as poor synergistic effect, difficulty in overcoming physicochemical defects of active ingredients, and low delivery efficiency, the application scenarios involved in the present invention are first explained. The present invention is mainly applied in the fields of anti-inflammatory and anti-aging cosmetics, oral health foods, and topical anti-inflammatory and anti-aging pharmaceutical preparations. In these scenarios, traditional preparation methods of simple physical mixing or conventional emulsion compounding have defects such as poor water solubility and easy degradation of curcumin, easy oxidation and non-specific binding of ergothioneine, strong irritation of black pepper extract and difficulty in precisely exerting its metabolic inhibition effect. Meanwhile, conventional preparation processes easily destroy the conformation of active ingredients, cannot achieve partitioned anchoring and programmed release of components, severely restricting the industrial application of ternary active ingredients.

[0051] The present invention, by constructing a ternary co-loaded nanolipid carrier core-shell hierarchical structure of a hydrophobic core, an interface enhancement layer, and a hydrophilic shell, designing the spatial ordered partitioning and anchoring of active ingredients, and employing a coupled preparation process of low-temperature gradient phase transfer and in-situ anchoring, aims to achieve an ergothioneine-curcumin synergistic anti-inflammatory and anti-aging complex composition possessing synergistic enhancement, stable protection, and efficient delivery.

[0052] Specifically, the present invention provides an ergothioneine-curcumin synergistic anti-inflammatory and anti-aging complex composition, comprising, by mass percentage, the following components: 0.5%-5% of an active efficacy component, 2%-8% of hydrogenated lecithin, 0.5%-3% of phytosterol, l%-4% of thiolated hyaluronic acid, 0.5%-2% of a co-solvent, 0.1%-0.5% of a stabilizer, and the balance being deionized water, wherein: the active efficacy component consists of ergothioneine, curcumin, and black pepper extract in a mass ratio of (1.5-3): 1 :(0.1-0.3), and the mass content of piperine in the black pepper extract is >95%; the phytosterol is at least one of soyasterol, sitosterol, and stigmasterol; the molecular weight of the thiolated hyaluronic acid is 5 kDa-20 kDa, and the degree of thiol substitution is 15%-30%; the co-solvent is at least one of 1,2-propanediol, glycerin, and polyethylene glycol 400; the stabilizer is at least one of betulinic acid, rosmarinic acid, and tocopheryl polyethylene glycol succinate; and the ternary co-loaded nanolipid carrier of the complex composition has a core-shell hierarchical structure of a hydrophobic core, an interface enhancement layer, and a hydrophilic shell, with a particle size of 20 nm-60 nm, a poly dispersity index PD1 < 0.12, and an absolute value of zeta potential > 35 mV.

[0053] As shown in Figure 1, the present invention also provides a method for preparing an ergothioneine-curcumin synergistic anti-inflammatory and anti-aging complex composition, the steps of the method being:

[0054] SI 00. Preparation of a low-temperature lipid phase: under nitrogen protection, light-proof conditions, and 22°C-26°C, adding hydrogenated lecithin, phytosterol, curcumin, and black pepper extract to a co-solvent, and stirring until completely dissolved to obtain a uniform and transparent lipid phase;

[0055] S200. Preparation of an aqueous phase: under the same temperature, light-proof, and nitrogen protection conditions as in SI 00, adding a stabilizer to deionized water, and stirring until completely dissolved to obtain an aqueous phase;

[0056] S300. Gradient phase transfer nano-dispersion: under low-speed stirring conditions, adding the lipid phase prepared in SI 00 dropwise at a constant rate of 0.5-1 mL / min to the aqueous phase prepared in S200, controlling the volume ratio of the lipid phase to the aqueous phase to be 1 :(8-12), after the dropwise addition is complete, maintaining the temperature and stirring for 30-45 min to obtain a nanolipid carrier primary emulsion;

[0057] S400. In-situ anchoring assembly: under the same temperature, light-proof, and stirring conditions as in SI 00, adding thiolated hyaluronic acid and ergothioneine to the primary emulsion prepared in S300, stirring the reaction under light-proof conditions for 60-90 min to complete the in-situ assembly of the ternary co-loaded nanolipid carrier core-shell hierarchical structure;

[0058] S500. Post-treatment: filtering and sterilizing the reaction solution obtained in S400 through a microporous membrane to obtain the complex composition.

[0059] Meanwhile, as shown in Figure 2, the present invention also discloses the preparation steps of thiolated hyaluronic acid:

[0060] Dissolving hyaluronic acid with a molecular weight of 5 kDa-20 kDa in MES buffer, adding EDC and NHS, stirring at room temperature to activate carboxyl groups, obtaining an activation reaction solution;

[0061] Adding cystamine dihydrochloride to the activation reaction solution, adjusting the pH to 4.5-6.0, and stirring the reaction under an inert gas atmosphere at 20°C-30°C for 8-16 hours, obtaining a thiolated intermediate reaction solution;

[0062] Adding a reducing agent TCEP to the thiolated intermediate reaction solution, continuing to stir the reaction for 2-4 hours to reduce disulfide bonds to free thiol groups;

[0063] Placing the reaction solution after the reduction reaction into a dialysis bag, performing dialysis purification under acidic conditions and an inert gas atmosphere to remove unreacted small molecule impurities, and freeze-drying the product after dialysis to obtain thiolated hyaluronic acid.

[0064] In the specific implementation process, the present invention is further elaborated below in conjunction with specific embodiments, comparative examples, and efficacy verification tests:

[0065] In all embodiments and comparative examples, the mass content of piperine in the black pepper extract used is >98%. The thiolated hyaluronic acid used in all embodiments is prepared according to the above preparation method. The molecular weight of the hyaluronic acid raw material used is 10 kDa, and the degree of thiol substitution of the finally prepared thiolated hyaluronic acid is 22%. During the preparation process of all embodiments, nitrogen is used as the inert protective gas throughout, light-proof operation is maintained throughout, and the system temperature is stably controlled within the range of 22°C-26°C.

[0066] The amounts of raw materials used in all embodiments and comparative examples are based on mass percentage, and the total amount for a single batch preparation is uniformly set at 1000 g to facilitate parameter conversion for industrial production and batch stability verification.

[0067] Embodiment 1

[0068] This embodiment is the optimal embodiment of the present invention, adopting a designed formula with ergothioneine:curcumin:black pepper extract = 2.2: 1:0.2, strictly following the above preparation method for preparation, to verify the basic performance of the complex composition, such as particle size, encapsulation efficiency, and dispersibility under the core technical solution of the present invention.

[0069] In terms of mass percentage, the raw material composition of this embodiment is:

[0070] Active efficacy component 1.8%: comprising 1.1% ergothioneine, 0.5% curcumin, and 0.2% black pepper extract;

[0071] Hydrogenated lecithin 4%, soyasterol 1%, thiolated hyaluronic acid 2%, 1,2-propanediol 1%, rosmarinic acid 0.2%;

[0072] Deionized water balance.

[0073] The specific steps for preparing the ergothioneine-curcumin synergistic anti-inflammatory and anti-aging complex composition are:

[0074] SI 00. Preparation of low-temperature lipid phase: under nitrogen protection, light-proof conditions, and a constant temperature of 24°C, add the prescribed amounts of hydrogenated lecithin, soyasterol, curcumin, and black pepper extract to 1,2-propanediol, and stir at a low speed of 250 rpm until completely dissolved to obtain a uniform and transparent lipid phase;

[0075] S200. Preparation of aqueous phase: under the same temperature, light-proof, and nitrogen protection conditions as in SI 00 exactly, add the prescribed amount of rosmarinic acid to deionized water, and stir until completely dissolved to obtain a uniform aqueous phase;

[0076] S300. Gradient phase transfer nano-dispersion: under low-speed stirring conditions at 250 rpm, add the lipid phase prepared in SI 00 dropwise at a constant rate of 0.8 mL / min to the aqueous phase prepared in S200, controlling the volume ratio of the lipid phase to the aqueous phase to be 1:10. After the dropwise addition is complete, continue stirring while maintaining the temperature for 40 min, spontaneously forming nanolipid carriers through gradient phase transfer, simultaneously completing the encapsulation of curcumin in the hydrophobic core and the directional anchoring of the black pepper extract in the interface enhancement layer, obtaining a nanolipid carrier primary emulsion;

[0077] S400. In-situ anchoring assembly: under the same temperature, light-proof, and stirring conditions as in SI 00 exactly, add the prescribed amounts of thiolated hyaluronic acid and ergothioneine to the primary emulsion prepared in S300, stir the reaction under light-proof conditions for 75 min to carry out a thiol-disulfide click reaction, covalently anchoring ergothioneine onto the hydrophilic shell composed of thiolated hyaluronic acid, completing the in-situ assembly of the ternary co-loaded nanolipid carrier core-shell hierarchical structure;

[0078] S500. Post-treatment: filter and sterilize the reaction solution obtained in S400 through a 0.22 pm microporous membrane to obtain the ergothioneine-curcumin synergistic anti-inflammatory and anti-aging complex composition of this embodiment.

[0079] Upon testing, the complex composition prepared in this embodiment has a ternary co-loaded nanolipid carrier with an average particle size of 32.6 nm, a polydispersity index PDI of 0.087, and a zeta potential of -41.2 mV. The encapsulation efficiency of ergothioneine is 96.8%, that of curcumin is 95.3%, and that of piperine in the black pepper extract is 94.7%, achieving high encapsulation efficiency and simultaneous loading of the three active ingredients, verifying the feasibility of the formula and preparation method of the present invention.

[0080] Embodiment 2

[0081] This embodiment adopts a low-dose active efficacy component design, suitable for application scenarios of sensitive-skin skincare products and low-dose oral preparations, to verify the carrier formation effect and encapsulation performance of the present invention under low active ingredient addition amounts.

[0082] In terms of mass percentage, the raw material composition of this embodiment is:

[0083] Active efficacy component 0.54%: comprising 0.3% ergothioneine, 0.2% curcumin, and 0.04% black pepper extract;

[0084] Hydrogenated lecithin 2%, sitosterol 0.5%, thiolated hyaluronic acid 1%, glycerin 0.8%, betulinic acid 0.1%;

[0085] Deionized water balance.

[0086] The specific preparation steps are:

[0087] SI 00. Preparation of low-temperature lipid phase: same preparation conditions as in Embodiment 1, stir until completely dissolved to obtain a lipid phase;

[0088] S200. Preparation of aqueous phase: same preparation conditions as in Embodiment 1, prepare to obtain an aqueous phase;

[0089] S300. Gradient phase transfer nano-dispersion: stirring speed 200 rpm, lipid phase addition rate 0.5 mL / min, volume ratio of lipid phase to aqueous phase 1:8, after dropwise addition, maintain temperature and stir for 30 min to obtain a primary emulsion;

[0090] S400. In-situ anchoring assembly: stir the reaction under light-proof conditions for 60 min to complete in-situ assembly;

[0091] S500. Post-treatment: filter and sterilize through a 0.22 pm microporous membrane to obtain the complex composition of this embodiment.

[0092] Upon testing, the complex composition prepared in this embodiment has a ternary co-loaded nanolipid carrier with an average particle size of 28.4 nm, a polydispersity index PDI of 0.092, and a zeta potential of -38.7 mV. The encapsulation efficiency of ergothioneine is 95.2%, that of curcumin is 93.1%, and that of piperine in the black pepper extract is 92.5%. This embodiment verifies that the present invention can still achieve excellent carrier formation effect and high encapsulation efficiency even at low active ingredient addition amounts, making it suitable for application scenarios requiring low doses and high safety. Embodiment 3

[0093] This embodiment adopts a high-dose active efficacy component design, suitable for application scenarios of pharmaceutical preparations with high efficacy requirements and high-concentration essence products, to verify the carrier loading capacity, dispersion stability, and encapsulation performance of the present invention under high active ingredient addition amounts.

[0094] In terms of mass percentage, the raw material composition of this embodiment is:

[0095] Active efficacy component 3.65%: comprising 2.4% ergothioneine, 1% curcumin, and 0.25% black pepper extract;

[0096] Hydrogenated lecithin 7%, stigmasterol 2.5%, thiolated hyaluronic acid 3.5%, polyethylene glycol 400 1.8%, tocopheryl polyethylene glycol succinate 0.4%;

[0097] Deionized water balance.

[0098] The specific preparation steps are:

[0099] SI 00. Preparation of low-temperature lipid phase: same preparation conditions as in Embodiment 1, stir until completely dissolved to obtain a lipid phase;

[0100] S200. Preparation of aqueous phase: same preparation conditions as in Embodiment 1, prepare to obtain an aqueous phase;

[0101] S300. Gradient phase transfer nano-dispersion: stirring speed 300 rpm, lipid phase addition rate 1 mL / min, volume ratio of lipid phase to aqueous phase 1:12, after dropwise addition, maintain temperature and stir for 45 min to obtain a primary emulsion;

[0102] S400. In-situ anchoring assembly: stir the reaction under light-proof conditions for 90 min to complete in-situ assembly;

[0103] S500. Post-treatment: filter and sterilize through a 0.22 pm microporous membrane to obtain the complex composition of this embodiment.

[0104] Upon testing, the complex composition prepared in this embodiment has a ternary co-loaded nanolipid carrier with an average particle size of 47.5 nm, a polydispersity index PDI of 0.112, and a zeta potential of -39.5 mV. The encapsulation efficiency of ergothioneine is 94.5%, that of curcumin is 92.8%, and that of piperine in the black pepper extract is 91.6%. This embodiment verifies that the present invention can still maintain excellent nanocarrier formation effect, narrow particle size distribution, and high encapsulation efficiency even at high active ingredient addition amounts, possessing excellent active ingredient loading capacity, making it suitable for application scenarios with high efficacy requirements.

[0105] Comparative Example 1

[0106] This comparative example simulates a prior art solution, using the exact same types and amounts of active efficacy components as in Embodiment 1, but without adding the ternary co-loaded nanolipid carrier-related components of the present invention. It only uses conventional solubilizers to prepare a physical mixture system, for comparative verification of the improvement effect of the nanocarrier structure of the present invention on the encapsulation, dispersion, and stability of active ingredients.

[0107] In terms of mass percentage, the raw material composition of this comparative example is:

[0108] Active efficacy component 1.8%: 1.1% ergothioneine, 0.5% curcumin, 0.2% black pepper extract (exactly the same as Embodiment 1);

[0109] 1,2-propanediol 5%, Tween 80 2%;

[0110] Deionized water balance.

[0111] Under normal temperature and light-proof conditions, add all the above raw materials to deionized water, stir at 500 rpm for 30 min to obtain a mixed dispersion, which is the composition of Comparative Example 1.

[0112] The composition prepared in this comparative example is a turbid dispersion system with no nanoscale carrier structure. Curcumin cannot be effectively dispersed, and obvious yellow precipitate and layering appear after standing for 24 h. Stable nanoparticle size and zeta potential cannot be detected. The three active ingredients have no encapsulation efficiency at all, completely failing to solve the core problems of poor water solubility of curcumin, easy degradation of active ingredients, and low bioavailability, forming a significant contrast with the uniform and stable nanosystem of the present invention.

[0113] Comparative Example 2

[0114] This comparative example removes the black pepper extract from the active efficacy component. The remaining carrier components, formula proportions, and preparation method are exactly the same as in Embodiment 1. It is used for comparative verification of the impact of adding black pepper extract and the cascade synergistic effect of the three on the efficacy of the composition of the present invention, while also verifying the design function of the interface enhancement layer.

[0115] In terms of mass percentage, the raw material composition of this comparative example is:

[0116] Active efficacy component 1.6%: 1.1% ergothioneine, 0.5% curcumin (black pepper extract removed);

[0117] Hydrogenated lecithin 4%, soyasterol 1%, thiolated hyaluronic acid 2%, 1,2-propanediol 1%, rosmarinic acid 0.2%;

[0118] Deionized water balance.

[0119] The preparation method strictly follows the preparation steps of Embodiment 1, except that black pepper extract is not added in SI 00, finally obtaining the composition of Comparative Example 2.

[0120] The composition prepared in this comparative example has a nanocarrier with an average particle size of 30.2 nm, PDI of 0.091, ergothioneine encapsulation efficiency of 96.5%, and curcumin encapsulation efficiency of 95.1%. The basic carrier formation performance is close to that of Embodiment 1, but lacking the metabolic enhancement effect of black pepper extract, it cannot achieve the cascade synergy of the three active ingredients. Also, lacking the piperine anchoring design in the interface enhancement layer, it cannot achieve simultaneous protection of the curcumin metabolic process. In subsequent pharmacodynamic tests, the anti-inflammatory and anti-aging efficacy and in vivo bioavailability are significantly lower than those of Embodiment 1.

[0121] Comparative Example 3

[0122] This comparative example does not adopt the layered and partitioned loading design of the present invention. Instead, curcumin and black pepper extract are co-encapsulated in the hydrophobic core, while ergothioneine remains anchored on the hydrophilic shell. The rest of the formula and preparation method are exactly the same as in Embodiment 1. It is used for comparative verification of the impact of the hydrophobic core-interface enhancement layer-hydrophilic shell layered and partitioned loading design of the present invention on the release synchrony and synergistic effect of active ingredients.

[0123] The raw material composition is exactly the same as in Embodiment 1. The preparation steps are exactly the same as in Embodiment 1, except that the lipid phase composition in SI 00 is adjusted by dissolving the black pepper extract together with curcumin and lipid materials, so that the black pepper extract is encapsulated in the hydrophobic core during lipid phase transfer, rather than being anchored in the interface enhancement layer, finally obtaining the composition of Comparative Example 3.

[0124] The composition prepared in this comparative example has a nanocarrier with an average particle size of 33.8 nm, PDI of 0.095, ergothioneine encapsulation efficiency of 96.2%, curcumin encapsulation efficiency of 94.8%, and piperine encapsulation efficiency of 93.7%. The basic encapsulation performance is close to that of Embodiment 1. However, because piperine is encapsulated in the hydrophobic core, its release rate synchrony with curcumin decreases, and it cannot exert its metabolic enzyme inhibition effect in advance during the absorption and metabolism process. Meanwhile, its release timing is misaligned with ergothioneine on the shell, preventing simultaneous onset of action of the three. In subsequent in vitro release tests and in vivo pharmacokinetic tests, its release synchrony and bioavailability are significantly lower than those of Embodiment 1.

[0125] Comparative Example 4

[0126] This comparative example uses a conventional high-temperature high-pressure homogenization process from the prior art to prepare a nanoemulsion. The types and amounts of active components are exactly the same as in Embodiment 1. It is used for comparative verification of the improvement effect of the low-temperature gradient phase transfer-in-situ anchoring coupling process of the present invention on the activity retention and storage stability of active ingredients, as well as the green and mild advantages of the process of the present invention.

[0127] In terms of mass percentage, the raw material composition of this comparative example is:

[0128] Active efficacy component 1.8%: 1.1% ergothioneine, 0.5% curcumin, 0.2% black pepper extract (exactly the same as Embodiment 1);

[0129] Soybean oil 5%, hydrogenated lecithin 3%, glycerin 2%, deionized water balance.

[0130] The preparation method uses a traditional high-temperature high-pressure homogenization process, with specific steps as follows:

[0131] Oil phase preparation: add curcumin, black pepper extract, and hydrogenated lecithin to soybean oil, heat and stir at 75 °C until completely dissolved to obtain an oil phase;

[0132] Aqueous phase preparation: add ergothioneine and glycerin to deionized water, heat and stir at 75 °C until completely dissolved to obtain an aqueous phase;

[0133] Pre-emulsification: add the oil phase to the aqueous phase, and shear at a high speed of 10000 rpm for 5 min at 75 °C to obtain a coarse emulsion;

[0134] High-pressure homogenization: homogenize the coarse emulsion under high pressure at 800 bar for 5 cycles, cool to room temperature, and filter through a 0.22 pm membrane to obtain the composition of Comparative Example 4.

[0135] The composition prepared in this comparative example has an average particle size of 128.6 nm and a PDI of 0.217, with a broad particle size distribution. Upon testing, the activity retention rate of curcumin after preparation is only 72.3%, and that of ergothioneine is only 81.5%. The high-temperature preparation process causes severe thermal degradation and isomerization inactivation of the active ingredients. Furthermore, this system lacks layered / partitioned loading and covalent anchoring design, and during storage, the active ingredients degrade rapidly, and the particle size increases rapidly, with stability far lower than that of the product of Embodiment 1 of the present invention.

[0136] Comparative Example 5

[0137] This comparative example uses unmodified ordinary hyaluronic acid instead of the thiolated hyaluronic acid of the present invention. The rest of the formula and preparation method are exactly the same as in Embodiment 1. It is used for comparative verification of the improvement effect of the design of covalently anchoring ergothioneine with thiolated hyaluronic acid of the present invention on the encapsulation efficiency, stability, and sustained-release effect of ergothioneine.

[0138] In terms of mass percentage, the raw material composition of this comparative example is:

[0139] Active efficacy component 1.8%: 1.1% ergothioneine, 0.5% curcumin, 0.2% black pepper extract (exactly the same as Embodiment 1);

[0140] Hydrogenated lecithin 4%, soyasterol 1%, ordinary hyaluronic acid (molecular weight 10 kDa) 2%, 1,2-propanediol 1%, rosmarinic acid 0.2%; Deionized water balance.

[0141] Strictly follow the preparation steps of Embodiment 1, except that ordinary hyaluronic acid and ergothioneine are added in S400. There is no thiol-disulfide click reaction process, and ergothioneine is loaded only through physical adsorption, finally obtaining the composition of Comparative Example 5.

[0142] The composition prepared in this comparative example has a nanocarrier with an average particle size of 31.5 nm, PDI of 0.093, curcumin encapsulation efficiency of 94.6%, and piperine encapsulation efficiency of 93.2%. However, the encapsulation efficiency of ergothioneine is only 32.7%, far lower than the 96.8% in Embodiment 1. Since ergothioneine is only physically adsorbed and mixed in the system, stable anchoring cannot be achieved. In the in vitro release test, over 80% burst release occurs within 1 hour, making it impossible to achieve synchronized release with curcumin and piperine. Meanwhile, during storage, ergothioneine is prone to oxidative degradation, and its activity retention rate is significantly lower than that of Embodiment 1.

[0143] To compare and verify the performance results of the complex compositions of the embodiments and comparative examples of the present invention, efficacy verification tests, in vitro release behavior and release synchrony verification tests, and long-term storage stability verification tests were conductedo

[0144] Efficacy Verification Test

[0145] The purpose of this test is to systematically detect the core parameters of the carriers of the compositions prepared in each embodiment and comparative example, as well as the encapsulation efficiency of the three active ingredients, verifying the encapsulation effect of the layered and partitioned loading design of the present invention on each active ingredient.

[0146] Test samples: Samples prepared in Embodiment 1, Embodiment 2, Embodiment 3, Comparative Example 2, Comparative Example 3, Comparative Example 4, and Comparative Example 5;

[0147] Testing instruments: Malvern nanoparticle size and zeta potential analyzer, High Performance Liquid Chromatograph (HPLC);

[0148] Test method:

[0149] Particle size, PDI, zeta potential detection: Take an appropriate amount of sample, dilute 100 times with deionized water, place in a detection cuvette, detect particle size, poly dispersity index PDI, and zeta potential at 25 °C, test in parallel 3 times, take the average value;

[0150] Encapsulation efficiency detection: Use ultrafiltration centrifugation to separate free active ingredients from drug-loaded nanocarriers. Detect the total content of active ingredients and the content of free active ingredients in the sample by HPLC. Calculate the encapsulation efficiency of each active ingredient according to: Encapsulation efficiency = (Total content - Free content) / Total content x 100%. Test in parallel 3 times, take the average value.

[0151] The specific test results are shown in Table 1 below:

[0152] Average Zeta Ergothioneine Curcumin Piperine Sample No. Particle PDI Potential Encapsulation Encapsulation Encapsulation Size (nm) (mV) Efficiency (%) Efficiency (%) Efficiency (%)

[0153] Embodiment

[0154] 32.6 0.087 -41.2 96.8 95.3 94.7

[0155] 1

[0156] Embodiment

[0157] 28.4 0.092 -38.7 95.2 93.1 92.5

[0158] 2

[0159] Embodiment

[0160] 47.5 0.112 -39.5 94.5 92.8 91.6

[0161] 3

[0162] Comparative

[0163] 30.2 0.091 -40.5 96.5 95.1 - Example 2

[0164]

[0165] Comparative

[0166] 33.8 0.095 -40.8 96.2 94.8 93.7 Example 3

[0167] Comparative

[0168] 128.6 0.217 -18.3 28.4 62.7 59.3 Example 4

[0169] Comparative

[0170] 31.5 0.093 -39.2 32.7 94.6 93.2 Example 5

[0171]

[0172] Table 1

[0173] The results of this test show that the complex compositions prepared in Embodiments 1 to 3 of the present invention all achieve high encapsulation efficiencies above 91% for the three active ingredients, verifying the stability and reliability of the formula and preparation process of the present invention. The sample prepared by the traditional process of Comparative Example 4 has a large particle size, broad distribution, and extremely low encapsulation efficiency, failing to meet application requirements. Comparative Example 5, using unmodified hyaluronic acid, has an extremely low encapsulation efficiency for ergothioneine, failing to achieve effective loading, fully demonstrating the necessity of the covalent anchoring design with thiolated hyaluronic acid of the present invention.

[0174] In Vitro Release Behavior and Release Synchrony Verification Test The purpose of this test is to detect the in vitro release behavior of the three active ingredients in each test sample, verify the regulatory effect of the layered and partitioned loading design of the present invention on the release synchrony of the three active ingredients, compare the defects of asynchronous release and severe burst release existing in the prior art, and verify the sustained-release effect of the present invention.

[0175] Test samples: Samples prepared in Embodiment 1, Comparative Example 1, Comparative Example 3, and Comparative Example 5;

[0176] Testing instruments: Constant temperature shaker, High Performance Liquid Chromatograph, dialysis bags (molecular weight cutoff 8000-14000 Da);

[0177] Test method: Use dialysis bag diffusion method for in vitro release test. The release medium is pH 7.4 PBS buffer (containing 0.5% Tween 80 to meet sink conditions). Take 2 mL of sample and place it in a dialysis bag, seal both ends, then put it into a centrifuge tube containing 50 mL of release medium, place it in a constant temperature shaker at 37 °C and 100 rpm. Sample 1 mL at Ih, 2h, 4h, 8h, 12h, and 24h time points, while replenishing an equal amount of fresh isothermal release medium. Detect the content of each active ingredient in the sampled solution by HPLC, calculate the cumulative release rate, perform 3 parallel tests, and take the average value. The cumulative release rates at each time point are shown in Table 2 below:

[0178] Sample Group Detection Index Ih 4h 12h 24h Embodiment 1 Ergothioneine 6.2 18.5 42.7 76.3 Embodiment 1 Curcumin 5.8 17.2 40.3 74.1 Embodiment 1 Piperine 6.5 19.1 43.5 77.8 Comparative Example 1 Ergothioneine 82.4 95.7 98.2 99.1 Comparative Example 1 Curcumin 3.2 7.5 12.4 18.6 Comparative Example 1 Piperine 78.6 93.2 97.5 98.7 Comparative Example 3 Ergothioneine 6.8 19.2 43.1 76.8 Comparative Example 3 Curcumin 4.1 12.6 28.5 52.3 Comparative Example 3 Piperine 3.8 11.9 27.2 50.8 Comparative Example 5 Ergothioneine 81.7 94.8 97.6 98.9 Comparative Example 5 Curcumin 5.5 16.8 39.7 73.5 Comparative Example 5 Piperine 6.3 18.7 42.9 77.2

[0179]

[0180] Table 2

[0181] The results of this test show that the release rates of the three active ingredients in Embodiment 1 of the present invention are highly synchronized, with the 24h cumulative release rates all controlled between 74% and 78%, no initial burst release phenomenon, achieving long-term sustained release and simultaneous onset of action, perfectly matching the timing requirements for the synergistic effect of the three, solving the core problem of asynchronous release of active ingredients in the prior art. In the physical mixture group of Comparative Example 1, ergothioneine and piperine show severe burst release within 1 hour, while the release amount of curcumin is extremely low, and the release of the three is completely misaligned, failing to achieve synergistic enhancement. In Comparative Example 3, encapsulating piperine in the core causes the release rates of curcumin and piperine to slow down significantly, deteriorating the release synchrony with ergothioneine, failing to achieve the metabolic enhancement effect of piperine. In Comparative Example 5, ergothioneine shows severe burst release, completely asynchronous with the release of the other two components, further verifying the necessity and superiority of the covalent anchoring design and layered / partitioned loading structure of the present invention.

[0182] Long-term Storage Stability Verification Test

[0183] The purpose of this test is to verify the particle size stability and active ingredient retention rate of the complex composition of the present invention during long-term storage, compare the defects of easy degradation of active ingredients and easy instability of the system in the prior art, and verify the stable protection effect of the carrier structure and preparation process of the present invention on active ingredients.

[0184] Test samples: Samples prepared in Embodiment 1, Comparative Example 1, Comparative Example 4, and Comparative Example 5;

[0185] Test conditions: Seal the samples in brown vials and store at room temperature (25°C±2°C) under light-proof conditions for 12 months;

[0186] Test method: Sample at 0 months, 3 months, 6 months, and 12 months of storage, detect the particle size change of the sample, and the content of the three active ingredients, calculate the activity retention rate (Activity retention rate = Detected content / Initial content x 100%), test in parallel 3 times, take the average value.

[0187] The results of active ingredient retention rates during storage are shown in Table 3 below:

[0188] Detection 0 3 6 12 Sample Group

[0189] Index months months months months Embodiment 1 Ergothioneine 100 99.2 97.8 95.2 Embodiment 1 Curcumin 100 98.6 96.5 93.7 Embodiment 1 Piperine 100 99.0 97.2 94.1 Comparative

[0190] Ergothioneine 100 89.3 78.5 62.7 Example 1

[0191] Comparative

[0192] Curcumin 100 72.4 51.6 32.8 Example 1

[0193] Comparative

[0194] Piperine 100 90.7 82.1 70.3 Example 1

[0195] Comparative

[0196] Ergothioneine 100 92.5 85.3 76.9 Example 4

[0197] Comparative

[0198] Curcumin 100 81.2 67.4 48.5 Example 4

[0199] Comparative

[0200] Piperine 100 93.1 86.7 78.2 Example 4

[0201] Comparative

[0202] Ergothioneine 100 85.6 71.3 58.4 Example 5

[0203] Comparative

[0204] Curcumin 100 98.1 95.8 92.9 Example 5

[0205] Comparative

[0206] Piperine 100 98.7 96.4 93.5 Example 5

[0207]

[0208] Table 3

[0209] The results of particle size change after 12 months of storage are shown in Table 4 below: Particle

[0210] Initial 12 -Month

[0211] Size

[0212] Sample Group Particle Particle Size Appearance Change Change

[0213] Size (nm) (nm)

[0214] Rate

[0215] Uniform and transparent, no Embodiment 1 32.6 35.1 7.67%

[0216] layering, no precipitation Layering and precipitation at 1 Comparative - - - month, severe Example 1

[0217] precipitation at 6 months

[0218] Layering at 3 months, Comparative

[0219] 128.6 387.2 201.1% obvious flocculation at Example 4

[0220] 12 months Uniform and Comparative

[0221] 31.5 33.8 7.30% transparent, no Example 5

[0222] layering

[0223]

[0224] Table 4

[0225] The results of this test show that during the 12-month long-term storage of the complex composition of Embodiment 1 of the present invention, the particle size does not change significantly, the system remains uniform and stable, and all three active ingredients maintain a high activity retention rate above 93%, solving the problems of easy photothermal degradation of curcumin, easy oxidative inactivation of ergothioneine, and easy crystallization and precipitation of piperine. The physical mixture system of Comparative Example 1 has extremely poor storage stability, with active ingredients degrading rapidly and the system rapidly layering and precipitating. The sample prepared by the traditional process of Comparative

[0226] Example 4 already shows inactivation of active ingredients during preparation, and during storage, the particle size increases sharply and the activity continues to decay, with stability far lower than that of the product of the present invention. In Comparative Example 5, ergothioneine suffers from severe oxidative degradation during storage due to lack of covalent anchoring protection, with an activity retention rate of less than 60% at 12 months, further verifying the simultaneous stable protection effect of the hierarchical carrier structure of the present invention on the three active ingredients.

[0227] The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention in any form. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art, without departing from the scope of the technical solution of the present invention, can make some changes or modifications to equivalent embodiments with equivalent changes using the above-disclosed technical content. However, any simple modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solution of the present invention, still fall within the scope of the technical solution of the present invention.

Claims

CLAIMS1. An ergothioneine-curcumin synergistic anti-inflammatory and anti-aging complex composition, characterized in that the complex composition comprises an active efficacy component and a ternary co-loaded nanolipid carrier component, and in terms of mass percentage, the raw material composition of the complex composition is: 0.5%-5% of the active efficacy component, 2%-8% of hydrogenated lecithin, 0.5%-3% of phytosterol, l%-4% of thiolated hyaluronic acid, 0.5%-2% of a co-solvent, 0.1%-0.5% of a stabilizer, and the balance being deionized water;The active efficacy component consists of ergothioneine, curcumin, and black pepper extract;The ternary co-loaded nanolipid carrier has a core-shell hierarchical structure of a hydrophobic core, an interface enhancement layer, and a hydrophilic shell.

2. The ergothioneine-curcumin synergistic anti-inflammatory and anti-aging complex composition according to claim 1, characterized in that the mass ratio of ergothioneine, curcumin, and black pepper extract is (1.5-3): 1 :(0.1 -0.3), and the mass content of piperine in the black pepper extract is >95%.

3. The ergothioneine-curcumin synergistic anti-inflammatory and anti-aging complex composition according to claim 1, characterized in that the hydrophobic core is composed of a lipid skeleton formed by combining hydrogenated lecithin and phytosterol, and curcumin is encapsulated inside the hydrophobic core;The interface enhancement layer is the oil-water interface region of the lipid bilayer, and the black pepper extract is anchored and distributed within the interface enhancement layer;The hydrophilic shell is thiolated hyaluronic acid, and ergothioneine is covalently bound and directionally anchored to the hydrophilic shell throughthiol-disulfide bonds.The ergothioneine-curcumin synergistic anti-inflammatory and anti-aging complex composition according to claim 3, characterized in that the phytosterol is at least one selected from the group consisting of soyasterol, sitosterol, and stigmasterol;The molecular weight of the thiolated hyaluronic acid is 5 kDa-20 kDa, and the degree of thiol substitution is 15%-30%.

5. The ergothioneine-curcumin synergistic anti-inflammatory and anti-aging complex composition according to claim 1, characterized in that the co-solvent is at least one selected from the group consisting of 1,2-propanediol, glycerin, and polyethylene glycol 400;The stabilizer is at least one selected from the group consisting of betulinic acid, rosmarinic acid, and tocopheryl polyethylene glycol succinate.The ergothioneine-curcumin synergistic anti-inflammatory and anti-aging complex composition according to claim 1, characterized in that the particle size of the ternary co-loaded nanolipid carrier is 20 nm-60 nm, the polydispersity index PDI is <0.12, and the absolute value of zeta potential is >35 mV.

7. The ergothioneine-curcumin synergistic anti-inflammatory and anti-aging complex composition according to claim 4, characterized in that the thiolated hyaluronic acid is obtained by subjecting hyaluronic acid with a molecular weight of 5 kDa-20 kDa to thiolation modification, the specific steps being:Activation Reaction: dissolving hyaluronic acid in MES buffer, adding EDC and NHS, and stirring at room temperature to activate carboxyl groups on the hyaluronic acid molecular chain, obtaining an activation reaction solution;Thiolation Reaction: adding cystamine dihydrochloride to the activation reaction solution, adjusting the pH to 4.5-6.0, and stirring the reaction under an inert gas atmosphere at 20°C-30°C for 8-16 hours, obtaining a thiolatedintermediate reaction solution;Reduction Reaction: adding a reducing agent TCEP to the thiolated intermediate reaction solution, continuing to stir the reaction for 2-4 hours to reduce disulfide bonds to free thiol groups;Purification and Drying: placing the reaction solution after the reduction reaction into a dialysis bag, performing dialysis purification under acidic conditions and an inert gas atmosphere to remove unreacted small molecule impurities, and freeze-drying the product after dialysis to obtain the thiolated hyaluronic acid.

8. A method for preparing an ergothioneine-curcumin synergistic anti-inflammatory and anti-aging complex composition, applicable to the ergothioneine-curcumin synergistic anti-inflammatory and anti-aging complex composition according to any one of claims 1-7, characterized in that the steps of the method are:SI 00. Preparation of a low-temperature lipid phase: under an inert gas atmosphere and light-proof conditions, adding hydrogenated lecithin, phytosterol, curcumin, and black pepper extract to a co-solvent, and stirring until completely dissolved to obtain a uniform and transparent lipid phase;S200. Preparation of an aqueous phase: under the same temperature, light-proof, and inert gas atmosphere conditions as in SI 00, adding a stabilizer to deionized water, and stirring until completely dissolved to obtain an aqueous phase;S300. Gradient phase transfer nano-dispersion: under low-speed stirring conditions, adding the lipid phase prepared in SI 00 dropwise at a constant rate of 0.5-1 mL / min to the aqueous phase prepared in S200, controlling the volume ratio of the lipid phase to the aqueous phase to be 1 :(8-12), after the dropwise addition is complete, maintaining the temperature and stirring for 30-45 min, spontaneously forming nanolipid carriers through gradient phase transfer, simultaneously completing the encapsulation of curcumin in the hydrophobiccore and the directional anchoring of the black pepper extract in the interface enhancement layer, obtaining a nanolipid carrier primary emulsion;S400. In-situ anchoring assembly: under the same temperature, light-proof, and stirring conditions as in SI 00, adding thiolated hyaluronic acid and ergothioneine to the primary emulsion prepared in S300, stirring the reaction under light-proof conditions for 60-90 min to carry out a thiol-disulfide click reaction, covalently anchoring ergothioneine onto the hydrophilic shell composed of thiolated hyaluronic acid, completing the in-situ assembly of the ternary co-loaded nanolipid carrier core-shell hierarchical structure;S500. Post-treatment: filtering and sterilizing the reaction solution obtained in S400 through a microporous membrane to obtain the complex composition.

9. The method for preparing an ergothioneine-curcumin synergistic anti-inflammatory and anti-aging complex composition according to claim 8, characterized in that the system temperature in S100-S400 is all controlled at 22°C-26°C, and nitrogen is used as the inert protective gas throughout the process.

10. The method for preparing an ergothioneine-curcumin synergistic anti-inflammatory and anti-aging complex composition according to claim 8, characterized in that in S500, the complex composition is applied in anti-inflammatory and anti-aging cosmetics, oral health foods, and topical anti-inflammatory and anti-aging pharmaceutical preparations.

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