Natural Chinese herbal medicine hydrogel composite microneedle as well as preparation method and application thereof

Abstract

The invention provides a natural Chinese herbal medicine hydrogel composite microneedle and a preparation method and application thereof.The preparation method comprises the steps that S1, natural food-borne nutrients and glycyrrhizic acid are mixed in a solvent, and a glycyrrhizic acid / natural food-borne nutrient solution is obtained; the natural food-borne nutrient is one or more of astaxanthin, baicalein, berberine and curcumin; s2, dropwise adding a metal salt solution into the glycyrrhizic acid / natural food-borne nutrient solution, and performing ultrasonic treatment and stirring to obtain glycyrrhizic acid / food-borne active substance gel; s3, mixing the polyanionic polymer solution with the glycyrrhizic acid / food-borne active substance gel to obtain a mixed solution; and S4, adding the mixed solution into a microneedle mold, carrying out vacuum centrifugation, and drying to obtain the microneedle patch. Good biocompatibility of the natural Chinese herbal medicine extract glycyrrhizic acid is used as a carrier to increase the in-vivo utilization rate of natural food-borne nutrients, so that a better treatment effect is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biology and new medicine technology, and in particular to a natural Chinese herbal medicine hydrogel composite microneedle and a preparation method and application thereof. Background Art

[0002] Recurrent aphthous ulcers (RAU) are a common oral mucosal lesion characterized by recurrent ulcerative lesions. These ulcers are often accompanied by pain, particularly during eating or contact with the oral cavity, causing discomfort and impaired quality of life. Clinically, topical treatments such as glucocorticoids and immunosuppressants are administered via ointments, patches, and sprays. However, due to the moist oral environment, salivation, and muscle activity, drug utilization is low, resulting in limited efficacy. Long-term use of steroids may cause oral mucosal atrophy or secondary infection, while long-term use of immunosuppressants can lead to side effects such as immunosuppression and gastrointestinal disturbances. They are also prone to recurrence after discontinuation and are highly addictive. Current drug administration methods offer the highest drug utilization, but patient compliance is low. Treatment of RAU is plagued by inconsistent efficacy, low drug utilization, and the risk of side effects. New treatment options are needed to improve clinical outcomes and patient quality of life.

[0003] Soluble microneedle technology is a new drug delivery system with high biocompatibility and convenience, which uses a microneedle array to deliver drugs to specific areas under the skin. Microneedles are made of biodegradable or soluble materials. After releasing the drug in the skin, they dissolve or degrade on their own. They are painless, low-traumatic and highly efficient, and are widely used in vaccination, drug delivery and beauty. Common soluble microneedle components include gelatin, polyvinyl alcohol, polylactic acid-polycaprolactone and hyaluronic acid. Hyaluronic acid is a material with good biocompatibility. Ge et al. studied the preparation of multifunctional soluble hyaluronic acid (HA) microneedle (MN) patches to promote the healing of oral ulcers. The tip layer of the MN patch is loaded with triamcinolone acetonide and epidermal growth factor to inhibit inflammation and promote angiogenesis. Zeolite imidazole skeleton-8 is loaded onto the base layer of the MN patch, which effectively releases Zn 2+To mediate the antibacterial effect. Wang et al. studied a soluble microneedle patch based on hyaluronic acid, which is loaded with dexamethasone acetate, vitamin C and tetracaine hydrochloride for the treatment of recurrent oral ulcers. The Chinese patent application with publication number CN115252535A discloses a soluble microneedle loaded with glucocorticoids, which is integrally formed by a backing layer and a microneedle array, and contains glucocorticoid drugs and soluble polymers. The Chinese patent application with publication number CN115969770A discloses a soluble microneedle that mediates mesoporous polydopamine nanoparticles loaded with glucocorticoid drugs, which is prepared by an ultrasonic vibration method. It is composed of a neatly arranged microneedle array, containing mesoporous polydopamine nanoparticles loaded with glucocorticoids and two soluble polymers. The above studies have confirmed that HA has strong mechanical strength and good biocompatibility, and also has a certain protective effect on the mucosa. Microneedle technology has improved the utilization of oral ulcer drugs and achieved painless and minimally invasive drug delivery. However, the loaded drugs include hormone drugs or immunosuppressants, which have a greater risk of side effects.

[0004] In summary, reported delivery strategies primarily focus on improving water dispersibility, stability, and drug availability. Among them, most current reports on microneedle therapy for oral ulcers focus on the delivery of antibiotics, significantly improving drug availability but also associated with significant side effects. Treatment options for aphthous ulcers are diverse, but several limitations remain. Long-term use of certain medications (such as steroids and antibiotics) can lead to side effects such as immunosuppression and increased risk of infection. Furthermore, certain pathogens may develop drug resistance, reducing therapeutic efficacy. Topical application of some drugs has limited efficacy and difficulty penetrating deep into ulcer tissue, resulting in suboptimal efficacy. Natural food-derived nutrients, such as curcumin, lutein, and astaxanthin, are those derived from natural foods. These nutrients possess anti-inflammatory, antioxidant, and biocompatibility properties, but are often fat-soluble, resulting in poor intestinal permeability and low bioavailability, significantly limiting their direct application in biomedicine. Glycyrrhizic acid, derived from licorice root extract, has potent anti-inflammatory and antiviral properties. It can also stimulate and promote oral mucosal repair, helping patients recover oral health more quickly. Currently available clinical drug delivery technologies have not reported using glycyrrhizic acid loaded with natural nutrients to prepare soluble microneedles for the treatment of oral ulcers. Summary of the Invention

[0005] In order to solve the above problems of the prior art, the present invention provides a natural Chinese herbal medicine hydrogel composite microneedle and a preparation method and application thereof.

[0006] The present invention is achieved through the following technical solutions: In a first aspect, the present invention provides a method for preparing a natural Chinese herbal medicine hydrogel composite microneedle, comprising: S1, mixing natural food-derived nutrients and glycyrrhizic acid in a solvent to obtain a glycyrrhizic acid / natural food-derived nutrient solution; the natural food-derived nutrients are one or more of astaxanthin, baicalein, berberine, and curcumin; S2, dropping the metal salt solution into the glycyrrhizic acid / natural food-derived nutrient solution, ultrasonicating, and stirring to obtain a glycyrrhizic acid / food-derived active substance gel; S3, mixing the polyanionic polymer solution with the glycyrrhizic acid / food-derived active substance gel to obtain a mixed solution; S4, adding the mixed solution into the microneedle mold, vacuum centrifuging, and drying to obtain a microneedle patch.

[0007] Preferably, S1 specifically comprises: dissolving natural food-derived nutrients in an organic solvent to obtain a natural food-derived nutrient solution; dissolving glycyrrhizic acid in an organic solvent to obtain a glycyrrhizic acid solution; mixing the glycyrrhizic acid solution with the natural food-derived substance solution, removing the organic reagent after rotary evaporation, and redissolving in water to obtain a glycyrrhizic acid / natural food-derived nutrient solution.

[0008] Preferably, in S1, the mass ratio of glycyrrhizic acid to natural food-derived nutrients is 10:(0.1~5).

[0009] Preferably, in S2, the metal salt in the metal salt solution is one or more of calcium salt, sodium salt and magnesium salt.

[0010] Preferably, in S2, the mass ratio of the metal salt to glycyrrhizic acid in the metal salt solution is (1-2.2):1.

[0011] Preferably, in S3, the polyanionic polymer in the polyanionic polymer solution is hyaluronic acid, sodium alginate or sodium carboxymethyl cellulose.

[0012] Preferably, in S3, the solvent of the polyanionic polymer solution is water, phosphate buffer solution or physiological saline.

[0013] Preferably, in S3, the mass ratio of the polyanionic polymer solution to the glycyrrhizic acid / food-derived active substance gel is (1-8):1.

[0014] In a second aspect, the present invention provides natural Chinese herbal medicine hydrogel composite microneedles obtained by the preparation method described above.

[0015] In a second aspect, the present invention provides the use of the natural Chinese herbal medicine hydrogel composite microneedle in the preparation of a drug for treating recurrent aphthous ulcer.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention is the first to develop a natural Chinese herbal medicine hydrogel composite microneedle using glycyrrhizic acid, natural food-derived nutrients, and polyanionic polymers as raw materials. The microneedle uses glycyrrhizic acid as a carrier to significantly improve the solubility of natural nutrients. By forming a glycyrrhizic acid / nutrient composite gel and then mixing it with a polyanionic polymer, a soluble microneedle is prepared by a two-step casting method. The microneedle delivery system has the following advantages: (1) The good biocompatibility of glycyrrhizic acid, a natural Chinese herbal medicine extract, is used as a carrier to increase the in vivo utilization rate of natural food-derived nutrients, thereby achieving better therapeutic effects. (2) The two-step casting method for microneedle preparation achieves drug encapsulation, allowing the drug to better act on the target site and be released at the inflammatory site, achieving better therapeutic effects. (3) The natural Chinese herbal medicine extract is used in combination with the soluble microneedle transdermal drug delivery system, which has better biocompatibility. The drug comes from a natural product, has low toxicity, is safe for long-term use, and has a synergistic therapeutic effect. The present invention demonstrates, using a rat aphthous ulcer model, that the glycyrrhizic acid / nutrient microneedle treatment group significantly improved aphthous ulcers compared to an untreated group and a group treated with standard nutrients, demonstrating improved drug delivery efficiency and the ability to protect nutrients from saliva degradation. This invention has significant market potential in the medical field and holds broad prospects for new applications and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 This is the synthesis route of the natural Chinese herbal medicine hydrogel composite microneedle of the present invention.

[0019] Figure 2 These are cryo-electron microscopy images of glycyrrhizic acid and glycyrrhizic acid / baicalein nanomicelles at different magnifications.

[0020] Figure 3 Figure a is the dynamic light scattering (DLS) measurement analytical potential result of glycyrrhizic acid / baicalein nanomicelles, and figure b is the dynamic light scattering (DLS) measurement analytical potential result of glycyrrhizic acid / astaxanthin nanomicelles.

[0021] Figure 4 Figure a is a schematic diagram of the apparent solubility of glycyrrhizic acid / baicalein and free baicalein, and figure b is a schematic diagram of the apparent solubility of glycyrrhizic acid / astaxanthin and free astaxanthin.

[0022] Figure 5Optical microscope (ab), laser confocal microscope (c), and scanning electron microscope (df) images of glycyrrhizic acid / astaxanthin / baicalein composite microneedles.

[0023] Figure 6 The changes in the damage of the aphthous ulcer rat model in normal rats (a), model group (b), untreated group (c), baicalein / astaxanthin treated group (d), and glycyrrhizic acid / baicalein / astaxanthin treated group (e).

[0024] Figure 7 HE and Masson staining images of the untreated group (a) and glycyrrhizic acid / astaxanthin / baicalein treated group (b) in the aphthous ulcer rat model. DETAILED DESCRIPTION

[0025] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0026] It should be noted that the process equipment or devices not specifically specified in the following embodiments are all conventional equipment or devices in the art.

[0027] It should be noted that the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product, or apparatus comprising a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to these processes, methods, products, or apparatuses. Furthermore, unless otherwise specified, the numbering of each method step is merely a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to define the scope of the invention. Changes or adjustments to their relative relationships, without substantially changing the technical content, should also be considered within the scope of the invention.

[0028] The preparation method of the natural Chinese herbal medicine hydrogel composite microneedle of the present invention comprises: S1, mixing natural food-derived nutrients and glycyrrhizic acid in a solvent to obtain a glycyrrhizic acid / natural food-derived nutrient solution; the natural food-derived nutrients are one or more of astaxanthin, baicalein, berberine, and curcumin; S2, dropping the metal salt solution into the glycyrrhizic acid / natural food-derived nutrient solution, ultrasonicating, and stirring to obtain a glycyrrhizic acid / food-derived active substance gel; S3, mixing the polyanionic polymer solution with the glycyrrhizic acid / food-derived active substance gel to obtain a mixed solution; S4, adding the mixed solution into the microneedle mold, vacuum centrifuging, and drying to obtain a microneedle patch.

[0029] Glycyrrhizic acid, derived from licorice root extract, has potent anti-inflammatory and antiviral effects, thereby reducing the development of ulcers. Glycyrrhizic acid can also stimulate and promote the repair process of the oral mucosa, helping patients recover oral health more quickly. In the present invention, glycyrrhizic acid also acts as a carrier, significantly increasing the solubility of other poorly soluble natural food-derived nutrients and promoting the absorption and utilization of active substances. Astaxanthin, derived from a carotenoid, is typically extracted from shrimp, crab, and certain algae. As a natural antibacterial agent, astaxanthin can reduce the spread and deepening of ulcers by inhibiting bacteria in the mouth. It can also scavenge free radicals in the body, reduce damage, promote ulcer healing, and help repair damaged oral mucosa. Baicalein, derived from an extract of the Scutellaria baicalensis plant, has natural anti-inflammatory effects. It can stimulate the regeneration and proliferation of oral mucosal cells and promote the repair of damaged mucosa. It also has antioxidant properties, scavenging free radicals and protecting cells from oxidative damage, helping to reduce inflammation and promote cell repair, thus playing a supporting role in the treatment of oral ulcers. These natural active ingredients generally possess excellent antibacterial, anti-inflammatory, and antioxidant activities, and have the potential to improve aphthous ulcers. This invention utilizes glycyrrhizic acid as a carrier to enhance the solubility of natural food-derived nutrients such as baicalein and astaxanthin, thereby improving drug absorption, reducing pain, avoiding the first-pass effect in the liver, minimizing side effects, and enhancing their biocompatibility. This achieves a synergistic therapeutic effect, addressing the shortcomings of natural food-derived nutrients, such as poor water solubility, low utilization, and poor stability (due to the complex environment of the oral cavity).

[0030] In some specific embodiments of the present invention, S1 specifically comprises: dissolving natural food-derived nutrients in an organic solvent to obtain a natural food-derived nutrient solution; dissolving glycyrrhizic acid in an organic solvent to obtain a glycyrrhizic acid solution; mixing the glycyrrhizic acid solution with a natural food-derived substance solution, removing the organic reagent after rotary evaporation, and redissolving in water to obtain a glycyrrhizic acid / natural food-derived nutrient solution.

[0031] The organic solvent is any one of methanol, dichloromethane, acetonitrile, and tetrahydrofuran, or a mixed solvent in any proportion.

[0032] In some specific embodiments of the present invention, the mass ratio of glycyrrhizic acid to natural food-derived nutrients is 10:(0.1~5).

[0033] As a preferred embodiment, the natural food-derived nutrients of the present invention are: a combination of astaxanthin and baicalein, a combination of berberine and curcumin, a combination of berberine and baicalein, or a combination of curcumin and astaxanthin. The two natural food-derived nutrients are dissolved in an organic solvent and mixed with a glycyrrhizic acid solution to obtain two glycyrrhizic acid / natural food-derived nutrient solutions. These solutions are then reacted with a metal salt solution to obtain two glycyrrhizic acid / food-derived active substance gels. The two glycyrrhizic acid / food-derived active substance gels are used in combination at a mass ratio of 1:1.

[0034] In some specific embodiments of the present invention, in S2, the metal salt in the metal salt solution is one or more of a calcium salt, a sodium salt, and a magnesium salt, and the mass ratio of the metal salt to glycyrrhizic acid in the metal salt solution is (1-2.2):1. The concentration of the metal salt solution is 10-22.2 mg / mL.

[0035] In some specific embodiments of the present invention, in S3, the polyanionic polymer in the polyanionic polymer solution is hyaluronic acid (HA), sodium alginate (SA) or sodium carboxymethyl cellulose (CMC), and its molecular weight range is 100,000 to 1,000,000, and the solvent of the polyanionic polymer solution is water, phosphate buffer solution or physiological saline.

[0036] The preparation method of the polyanionic polymer solution is as follows: slowly adding a solvent to the polyanionic polymer under ice bath conditions, and stirring the polyanionic polymer with a magnetic stirrer to fully dissolve the polyanionic polymer to obtain the polyanionic polymer solution.

[0037] In some specific embodiments of the present invention, in S3, the mass ratio of the polyanionic polymer solution to the glycyrrhizic acid / food-derived active substance gel is (1-8):1.

[0038] In some specific embodiments of the present invention, in S3, the reaction temperature is 4°C and the centrifugal mixing is for more than 1 hour. In some specific embodiments of the present invention, in S4, the vacuum time is greater than 2 hours, the centrifugal time is greater than 1 hour, the drying temperature is 37°C, and the drying time is greater than 24 hours.

[0039] Taking glycyrrhizic acid, baicalein, astaxanthin and hyaluronic acid as examples, the reaction process of the present invention is described. Figure 1As shown, glycyrrhizic acid and baicalein are fully dissolved in anhydrous methanol and mixed thoroughly in a certain proportion, astaxanthin is fully dissolved in tetrahydrofuran, glycyrrhizic acid and astaxanthin are mixed thoroughly in a certain proportion, refluxed in the dark for a period of time, and then the organic solvents methanol and tetrahydrofuran are removed and redissolved in deionized water to obtain glycyrrhizic acid / baicalein solution and glycyrrhizic acid / astaxanthin solution, and calcium chloride solution is added respectively to form glycyrrhizic acid / baicalein and glycyrrhizic acid / astaxanthin gels; the two gels are fully mixed under ice bath conditions, and then mixed thoroughly with hyaluronic acid in a certain proportion, added to a polydimethylsiloxane mold, and dried after vacuum operation to obtain the final glycyrrhizic acid / baicalein / astaxanthin soluble microneedle patch.

[0040] The natural hydrogel composite microneedle patch pierces the ulcer surface and protects astaxanthin and baicalein when they are delivered to the complex environment of saliva in the oral cavity. When they are delivered to the deep tissue, the microneedles dissolve, thereby releasing them and exerting their biological activity.

[0041] The present invention is further described in detail below with reference to the embodiments.

[0042] Example 1 (1) Preparation of glycyrrhizic acid / baicalein / astaxanthin solution (GA / BA / AST) Glycyrrhizic acid (10 mg, 0.012 mmol) and baicalein (1 mg, 0.0045 mmol) were dissolved in 1 mL of anhydrous methanol, and astaxanthin (1 mg, 0.0017 mmol) was dissolved in 1 mL of tetrahydrofuran. The mixture was ultrasonically stirred to fully dissolve. The glycyrrhizic acid solution and the astaxanthin solution were mixed at a glycyrrhizic acid to astaxanthin mass ratio of 15:1, and the glycyrrhizic acid solution and the baicalein solution were mixed at a glycyrrhizic acid to baicalein mass ratio of 10:0.5. After rotary evaporation and reflux, the organic reagents were removed. The mixture was redissolved in 1 mL of deionized water and shaken thoroughly to obtain a glycyrrhizic acid / baicalein solution and a glycyrrhizic acid / astaxanthin solution, respectively.

[0043] (2) Preparation of glycyrrhizic acid / baicalein / astaxanthin gel and hyaluronic acid solution Dissolve anhydrous calcium chloride (11.1 mg, 0.1 mmol) in 1 mL of deionized water, vortex ultrasonically, and thoroughly mix to obtain a calcium chloride solution. Slowly dropwise add the calcium chloride solution to the glycyrrhizic acid / baicalein solution and the glycyrrhizic acid / astaxanthin solution, respectively, and thoroughly mix with ultrasonic agitation to obtain glycyrrhizic acid / baicalein gel and glycyrrhizic acid / astaxanthin gel (mass ratio of metal salt to glycyrrhizic acid is 1.1:1). Slowly add phosphate buffer solution to hyaluronic acid in an ice bath and magnetically stir to fully dissolve the hyaluronic acid solution.

[0044] (3) Preparation of glycyrrhizic acid / baicalein / astaxanthin hydrogel composite microneedles The hyaluronic acid solution, glycyrrhizic acid / baicalein gel, and glycyrrhizic acid / astaxanthin gel were mixed at a mass ratio of 4:1:1 by centrifugation at 4°C for 1 hour and further thoroughly mixed by ultrasonic agitation. The resulting mixed solution was added to a polydimethylsiloxane microneedle mold, vacuum pumped for 2 hours, centrifuged for 1 hour, and then dried in a constant temperature drying oven at 37°C for 24 hours to obtain the target dissolvable microneedle patch.

[0045] In addition, according to the method of this example, glycyrrhizic acid / baicalein gels with a mass ratio of glycyrrhizic acid to baicalein of 10:0.0125, 10:0.25, 10:1, 10:2, and 10:4 were prepared, and glycyrrhizic acid / astaxanthin gels with a mass ratio of glycyrrhizic acid to astaxanthin of 5:1, 10:1, and 20:1 were prepared for subsequent stability performance evaluation.

[0046] Example 2 (1) Preparation of glycyrrhizic acid / baicalein / astaxanthin solution (GA / BA / AST) Glycyrrhizic acid (10 mg, 0.012 mmol) and baicalein (1 mg, 0.0045 mmol) were dissolved in 1 mL of anhydrous methanol, and astaxanthin (1 mg, 0.0017 mmol) was dissolved in 1 mL of tetrahydrofuran. The mixture was ultrasonically stirred to fully dissolve. The glycyrrhizic acid solution and the astaxanthin solution were mixed at a glycyrrhizic acid to astaxanthin mass ratio of 10:1, and the glycyrrhizic acid solution and the baicalein solution were mixed at a glycyrrhizic acid to baicalein mass ratio of 10:1. After rotary evaporation and reflux, the organic reagents were removed. The mixtures were redissolved in 1 mL of deionized water and shaken thoroughly to obtain glycyrrhizic acid / baicalein solutions and glycyrrhizic acid / astaxanthin solutions.

[0047] (2) Preparation of glycyrrhizic acid / baicalein / astaxanthin gel and hyaluronic acid solution Dissolve anhydrous calcium chloride (22.2 mg, 0.2 mmol) in 1 mL of deionized water, vortex ultrasonically, and thoroughly mix to obtain a calcium chloride solution. Slowly add the calcium chloride solution dropwise to the glycyrrhizic acid / baicalein solution and the glycyrrhizic acid / astaxanthin solution, respectively, with ultrasonic stirring to thoroughly mix, to obtain a glycyrrhizic acid / baicalein gel and a glycyrrhizic acid / astaxanthin gel (mass ratio of metal salt to glycyrrhizic acid is 2.2:1). Slowly add deionized water dropwise to hyaluronic acid in an ice bath and magnetically stir to fully dissolve the hyaluronic acid solution.

[0048] (3) Preparation of glycyrrhizic acid / baicalein / astaxanthin hydrogel composite microneedles The hyaluronic acid solution, glycyrrhizic acid / baicalein gel, and glycyrrhizic acid / astaxanthin gel were mixed at a mass ratio of 2:1:1 by centrifugation at 4°C for 1 hour and further thoroughly mixed by ultrasonic agitation. The resulting mixed solution was added to a polydimethylsiloxane microneedle mold, vacuum pumped for 2 hours, centrifuged for 1 hour, and then dried in a constant temperature drying oven at 37°C for 24 hours to obtain the target dissolvable microneedle patch.

[0049] Example 3 (1) Preparation of glycyrrhizic acid / berberine / curcumin solution (GA / BE / CU) Dissolve glycyrrhizic acid (10 mg, 0.012 mmol) and berberine (1 mg, 0.03 mmol) in 1 mL of anhydrous methanol, and dissolve curcumin (1 mg, 0.0027 mmol) in 1 mL of dichloromethane. Dissolve thoroughly with ultrasonic stirring. Mix the glycyrrhizic acid solution with the berberine solution at a mass ratio of 10:1, and then mix the glycyrrhizic acid solution with the curcumin solution at a mass ratio of 10:4. After rotary evaporation and reflux, remove the organic reagents, redissolve each solution in 1 mL of deionized water, and shake thoroughly to obtain a glycyrrhizic acid / berberine solution and a glycyrrhizic acid / curcumin solution.

[0050] (2) Preparation of glycyrrhizic acid / berberine / curcumin gel and sodium alginate solution Sodium chloride (15 mg, 0.26 mmol) was dissolved in 1 mL of deionized water and thoroughly mixed by ultrasonic vortexing to obtain a sodium chloride solution. The sodium chloride solution was then slowly added dropwise to the glycyrrhizic acid / berberine solution and the glycyrrhizic acid / curcumin solution, respectively, with ultrasonic stirring to thoroughly mix. This yielded a glycyrrhizic acid / berberine gel and a glycyrrhizic acid / curcumin gel (with a mass ratio of metal salt to glycyrrhizic acid of 1.5:1). Normal saline was slowly added dropwise to the sodium alginate in an ice bath and magnetically stirred to fully dissolve the sodium alginate solution.

[0051] (3) Preparation of glycyrrhizic acid / berberine / curcumin hydrogel composite microneedles Sodium alginate solution, glycyrrhizic acid / berberine gel, and glycyrrhizic acid / curcumin gel in a mass ratio of 4:1:1 were mixed by centrifugation at 4°C for 1 hour and further thoroughly mixed by ultrasonic agitation. The resulting mixed solution was added to a polydimethylsiloxane microneedle mold, vacuum pumped for 2 hours, centrifuged for 1 hour, and then dried in a constant temperature drying oven at 37°C for 24 hours to obtain the target dissolvable microneedle patch.

[0052] Example 4 (1) Preparation of glycyrrhizic acid / berberine / curcumin solution (GA / BE / CU) Dissolve glycyrrhizic acid (10 mg, 0.012 mmol) and berberine (1 mg, 0.03 mmol) in 1 mL of acetonitrile, and dissolve curcumin (1 mg, 0.0027 mmol) in 1 mL of dichloromethane. Dissolve thoroughly with ultrasonic stirring. Mix the glycyrrhizic acid solution with the berberine solution at a mass ratio of 10:5, and the glycyrrhizic acid solution with the curcumin solution at a mass ratio of 10:2. After rotary evaporation and reflux, remove the organic reagents, and redissolve each in 1 mL of deionized water and shake thoroughly to obtain a glycyrrhizic acid / berberine solution and a glycyrrhizic acid / curcumin solution.

[0053] (2) Preparation of glycyrrhizic acid / berberine / curcumin gel and sodium alginate solution Sodium chloride (20 mg, 0.347 mmol) was dissolved in 1 mL of deionized water and thoroughly mixed by ultrasonic vortexing to obtain a sodium chloride solution. The sodium chloride solution was then slowly added dropwise to the glycyrrhizic acid / berberine solution and the glycyrrhizic acid / curcumin solution, respectively, with ultrasonic stirring to thoroughly mix, to obtain a glycyrrhizic acid / berberine gel and a glycyrrhizic acid / curcumin gel (the mass ratio of metal salt to glycyrrhizic acid was 2:1). Phosphate buffer solution was slowly added dropwise to the sodium alginate in an ice bath and magnetically stirred to fully dissolve the sodium alginate solution.

[0054] (3) Preparation of glycyrrhizic acid / berberine / curcumin hydrogel composite microneedles Sodium alginate solution, glycyrrhizic acid / berberine gel, and glycyrrhizic acid / curcumin gel in a mass ratio of 8:1:1 were mixed by centrifugation at 4°C for 1 hour and further thoroughly mixed by ultrasonic agitation. The resulting mixed solution was added to a polydimethylsiloxane microneedle mold, vacuum pumped for 2 hours, centrifuged for 1 hour, and then dried in a constant temperature drying oven at 37°C for 24 hours to obtain the target dissolvable microneedle patch.

[0055] Example 5 (1) Preparation of glycyrrhizic acid / berberine / baicalin solution (GA / BE / BA) Glycyrrhizic acid (10 mg, 0.012 mmol) and berberine (1 mg, 0.03 mmol) were dissolved in 1 mL of dichloromethane, and baicalein (1 mg, 0.0045 mmol) was dissolved in 1 mL of anhydrous methanol. The mixture was ultrasonically stirred to fully dissolve. The glycyrrhizic acid solution and berberine solution were mixed at a mass ratio of glycyrrhizic acid to berberine of 10:1, and the glycyrrhizic acid solution and baicalein solution were mixed at a mass ratio of glycyrrhizic acid to baicalein of 10:2. After rotary evaporation and reflux, the organic reagents were removed. The mixture was redissolved in 1 mL of deionized water and shaken thoroughly to obtain glycyrrhizic acid / berberine solution and glycyrrhizic acid / baicalein solution.

[0056] (2) Preparation of glycyrrhizic acid / berberine / baicalein gel and sodium carboxymethylcellulose solution Magnesium chloride (10 mg, 0.105 mmol) was dissolved in 1 mL of deionized water and thoroughly mixed by ultrasonic vortexing to obtain a magnesium chloride solution. The magnesium chloride solution was slowly added dropwise to the glycyrrhizic acid / berberine solution and the glycyrrhizic acid / curcumin solution, respectively, with ultrasonic stirring to thoroughly mix, to obtain glycyrrhizic acid / berberine gel and glycyrrhizic acid / curcumin gel (the mass ratio of metal salt to glycyrrhizic acid was 1:1). Phosphate buffer solution was slowly added dropwise to sodium carboxymethyl cellulose in an ice bath and magnetically stirred to fully dissolve the solution, to obtain a sodium carboxymethyl cellulose solution.

[0057] (3) Preparation of glycyrrhizic acid / berberine / baicalein hydrogel composite microneedles The sodium carboxymethylcellulose solution was mixed with glycyrrhizic acid / berberine gel and glycyrrhizic acid / baicalein gel in a mass ratio of 1:1:1 by centrifugation at 4°C for 1 hour and further thoroughly mixed by ultrasonic agitation. The resulting mixed solution was added to a polydimethylsiloxane microneedle mold, vacuum pumped for 2 hours, centrifuged for 1 hour, and then dried in a constant temperature drying oven at 37°C for 24 hours to obtain the target dissolvable microneedle patch.

[0058] Example 6 (1) Preparation of glycyrrhizic acid / curcumin / astaxanthin solution (GA / CU / AST) Glycyrrhizic acid (10 mg, 0.012 mmol) and curcumin (1 mg, 0.0027 mmol) were dissolved in 1 mL of anhydrous methanol, and astaxanthin (1 mg, 0.0017 mmol) was dissolved in 1 mL of tetrahydrofuran. The mixture was ultrasonically stirred to fully dissolve. The glycyrrhizic acid solution and the astaxanthin solution were mixed at a mass ratio of glycyrrhizic acid to astaxanthin of 10:0.1, and the glycyrrhizic acid solution and the curcumin solution were mixed at a mass ratio of glycyrrhizic acid to curcumin of 10:2. After rotary evaporation and reflux, the organic reagents were removed. The mixture was redissolved in 1 mL of deionized water and shaken thoroughly to obtain a glycyrrhizic acid / curcumin solution and a glycyrrhizic acid / astaxanthin solution.

[0059] (2) Preparation of glycyrrhizic acid / curcumin / astaxanthin gel and sodium carboxymethylcellulose solution Magnesium chloride (20 mg, 0.21 mmol) was dissolved in 1 mL of deionized water and thoroughly mixed by ultrasonic vortexing to obtain a magnesium chloride solution. The magnesium chloride solution was then slowly added dropwise to the glycyrrhizic acid / curcumin solution and the glycyrrhizic acid / astaxanthin solution, respectively, with ultrasonic stirring to thoroughly mix, to obtain glycyrrhizic acid / curcumin gel and glycyrrhizic acid / astaxanthin gel (the mass ratio of metal salt to glycyrrhizic acid was 2:1). Deionized water was slowly added dropwise to sodium carboxymethyl cellulose in an ice bath and magnetically stirred to fully dissolve the solution, to obtain a sodium carboxymethyl cellulose solution.

[0060] (3) Preparation of glycyrrhizic acid / curcumin / astaxanthin hydrogel composite microneedles The sodium carboxymethylcellulose solution, glycyrrhizic acid / curcumin gel, and glycyrrhizic acid / astaxanthin gel were mixed at a mass ratio of 2:1:1 by centrifugation at 4°C for 1 hour and further thoroughly mixed by ultrasonic agitation. The resulting mixed solution was added to a polydimethylsiloxane microneedle mold, vacuum pumped for 2 hours, centrifuged for 1 hour, and then dried in a constant temperature drying oven at 37°C for 24 hours to obtain the target dissolvable microneedle patch.

[0061] Comparative Example 1 (1) Preparation of baicalin / astaxanthin solution (BA / AST) Dissolve baicalein (1 mg, 0.0045 mmol) in 1 mL of anhydrous methanol and astaxanthin (1 mg, 0.0017 mmol) in 1 mL of tetrahydrofuran, respectively, and thoroughly dissolve under ultrasonic stirring. Remove the organic reagents by rotary evaporation and reflux. Redissolve each in 1 mL of deionized water and shake thoroughly to obtain a baicalein solution and an astaxanthin solution.

[0062] (2) Preparation of hyaluronic acid solution Phosphate buffer solution was slowly added dropwise to the hyaluronic acid under ice bath conditions, and magnetic stirring was performed to fully dissolve the hyaluronic acid to obtain a hyaluronic acid solution.

[0063] (3) Preparation of baicalin / astaxanthin composite microneedles The hyaluronic acid solution, baicalein solution, and astaxanthin solution were mixed at a mass ratio of 4:1:1 by centrifugation at 4°C for 1 hour and further thoroughly mixed by ultrasonic agitation. The resulting mixed solution was added to a polydimethylsiloxane microneedle mold, vacuum pumped under negative pressure for 2 hours, centrifuged for 1 hour, and then dried in a constant temperature drying oven at 37°C for 24 hours to obtain the target microneedle patch.

[0064] Example 7 (taking Example 1 as an example) Properties and characterization of glycyrrhizic acid / baicalein gel and glycyrrhizic acid / astaxanthin gel (1) Morphology of glycyrrhizic acid / baicalein (GA / BA) micelles Figure 2 Figures ac in the middle are images of GA micelles at different magnifications under cryo-electron microscopy. Figure 2 The middle part shows images of GA / BA micelles at different magnifications under a cryo-electron microscope. The photos show that GA is arranged in a regular linear pattern with an ultra-long diameter-to-length ratio, and BA is successfully loaded on GA.

[0065] (2) Evaluation of the stability of GA / BA and GA / AST micelles The micelles with different ratios of GA / BA and GA / AST were able to remain stable, and their sizes were measured at predetermined time intervals, and the potential was continuously monitored by DLS. Figure 3The results in middle a show that the absolute values of the potentials of different baicalein feeding ratios (the mass ratios of glycyrrhizic acid and baicalein were 10:0.0125, 10:0.25, 10:0.5, 10:1, 10:2, and 10:4, respectively) were all maintained above 40 mV, indicating that the formed structure was stable. Figure 3 The results in middle b show that the absolute values of the potential at different astaxanthin feed ratios (the mass ratios of glycyrrhizic acid and astaxanthin are 5:1, 10:1, 15:1, and 20:1, respectively) are all maintained above 50 mV, and the potential at the mass ratio of 15:1 is the largest, indicating that the formed structure is stable.

[0066] (3) Apparent solubility evaluation of GA / BA and GA / AST micelles The solubility of GA / BA and free BA (freeBA), as well as the solubility of GA / AST and free AST (freeAST) were compared using an ultraviolet spectrophotometer. Figure 4 Figure a is a schematic diagram of the apparent solubility of GA / BA and free BA. Figure 4 Figure b is a schematic diagram of the apparent solubility of GA / AST and free AST. The results show that glycyrrhizic acid can effectively improve the solubility of baicalein and astaxanthin.

[0067] Example 8 (taking Example 1 as an example) Properties and characterization of glycyrrhizic acid / baicalein / astaxanthin hydrogel microneedles (1) Characterization of glycyrrhizic acid / baicalein / astaxanthin hydrogel microneedles Figure 5 Figures 5a and 5b show the morphology of the glycyrrhizic acid / baicalein / astaxanthin hydrogel microneedles under an optical microscope. The microneedles are evenly distributed, each conical, uniformly sized, and with a pointed tip. The number of microneedle patches (MNs) in the array is 15 × 15, and the tips consist of a hyaluronic acid-based shell and a glycyrrhizic acid / baicalein / astaxanthin-based core. Each MN is 600 μm tall, with a base diameter of 300 μm and a center-to-center spacing of 600 μm. The patch base measures 10 × 10 mm.

[0068] (2) Visualization of glycyrrhizic acid / baicalin / astaxanthin hydrogel microneedle loading capacity Figure 5 Figure c shows that incorporating a blue fluorescent dye into hyaluronic acid facilitates visualization of the needle tip, demonstrating the microneedle's loading capacity. Furthermore, scanning electron microscopy revealed the presence of a rough surface and the conical structure of the MN was clearly visible ( Figure 5 in df).

[0069] Example 9 (taking Example 1 as an example) (1) In vivo therapeutic effects of glycyrrhizic acid / baicalin / astaxanthin hydrogel microneedles 6-8 week old SD rats weighing 170-200g were used for oral ulcer modeling using glacial acetic acid chemical cauterization. Typical oral ulcer pseudomembranes were induced in the rats' mouths. The rats were observed 24-48 hours later to establish an oral ulcer model. Furthermore, a seven-day treatment cycle was established, during which the microneedle patch of Example 1 was administered orally daily (glycyrrhizic acid / baicalein / astaxanthin treatment group). An untreated group served as a control. Regular observation and recording of changes in the rats throughout the treatment cycle revealed that the treated group was able to alleviate ulcer inflammation. Figure 6 Middle a is the normal rat oral mucosa, Figure 6 Middle b is a schematic diagram of the completion of oral ulcer modeling. Figure 6 The CEs are the untreated group, the baicalein / astaxanthin treated group (corresponding to the microneedle patch of proportion 1), and the glycyrrhizic acid / baicalein / astaxanthin treated group. Figure 6 It can be seen that the baicalein / astaxanthin treatment group and the glycyrrhizic acid / baicalein / astaxanthin treatment group had significant healing effects compared with the untreated group, and the treatment effect of the glycyrrhizic acid / baicalein / astaxanthin treatment group was significantly better than that of the baicalein / astaxanthin treatment group, indicating that glycyrrhizic acid as a microneedle carrier can significantly promote healing and relieve inflammation in the body.

[0070] (2) Healing effect of ulcer inflammation site The degree of ulcer healing is one of the important indicators for evaluating the degree of inflammation in the recurrent oral ulcer model. After the treatment cycle, the mice were sacrificed and dissected, and their oral tissues were obtained for comparison. The mucosal tissue was embedded in paraffin, cut into 5 μm sections, and stained with hematoxylin for 5 minutes. The tissue was then washed with PBS buffer, differentiated with 1% hydrochloric acid in ethanol, stained with eosin solution, dehydrated in ethanol, and sealed with neutral gel. After the tissue was sectioned, it was stained with Masson's stain to remove the floating color, and the sections were then examined using a light microscope.

[0071] pass Figure 7 As shown, H&E staining of the untreated group showed mucosal epithelial defects, necrosis and shedding of epithelial cells, and coagulation of cell nuclei. In addition, the formation of necrotic tissue was observed, the surface was covered with fibrin exudate, inflammatory cells were densely distributed, and the capillaries in the lamina propria were dilated and congested. H&E-stained mucosal sections showed that the tissue surface of the rats in the untreated group remained concave and was characterized by inflammatory cell infiltration. In contrast, the rats in the glycyrrhizic acid / baicalein / astaxanthin treatment group showed intact oral mucosa with sparse connective tissue at the base. In addition to the ulcer area, the thickness of the newly formed epithelium of the oral mucosal tissue is an important indicator of the treatment effect. The degree of re-epithelialization in the glycyrrhizic acid / baicalein / astaxanthin treatment group was significantly better than that in the untreated group. Masson staining results showed that the amount of blue collagen deposition in the glycyrrhizic acid / baicalein / astaxanthin treatment group was significantly better than that in the untreated group, further demonstrating the healing-promoting ability of the target microneedle patch of the present invention.

[0072] This invention, for the first time, develops a natural hydrogel composite microneedle material using glycyrrhizic acid, natural food-derived nutrients, and a polyanionic polymer as raw materials. The use of glycyrrhizic acid, a natural Chinese herbal extract, significantly increases the in vivo utilization of baicalein and astaxanthin, achieving enhanced therapeutic effects. The combination of this natural herbal extract with a soluble microneedle transdermal delivery system offers high biocompatibility, low toxicity, and relative safety for long-term use, avoiding the side effects of glucocorticoids and immunosuppressants.

[0073] Compared to a healthy oral cavity, the oral environment is more demanding under inflammatory conditions. This microneedle protects nutrients from being broken down by saliva, delivering medication to deeper tissues and avoiding the effects of the first-pass effect in the gastrointestinal tract. This invention offers painless, minimally invasive, safe, and highly effective treatment with excellent biocompatibility. This invention has enormous market potential in the medical field and holds broad prospects for new applications and development.

Claims

1. A method for preparing natural Chinese herbal medicine hydrogel composite microneedles, characterized in that: include: S1, mixing natural food-derived nutrients and glycyrrhizic acid in a solvent to obtain a glycyrrhizic acid / natural food-derived nutrient solution; The natural food-derived nutrients are one or more of astaxanthin, baicalein, berberine and curcumin; S2, dropping the metal salt solution into the glycyrrhizic acid / natural food-derived nutrient solution, ultrasonicating, and stirring to obtain a glycyrrhizic acid / food-derived active substance gel; S3, mixing the polyanionic polymer solution with the glycyrrhizic acid / food-derived active substance gel to obtain a mixed solution; S4, adding the mixed solution into the microneedle mold, vacuum centrifuging, and drying to obtain a microneedle patch.

2. The method for preparing the natural Chinese herbal medicine hydrogel composite microneedle according to claim 1, characterized in that: S1 specifically comprises: dissolving natural food-derived nutrients in an organic solvent to obtain a natural food-derived nutrient solution; dissolving glycyrrhizic acid in an organic solvent to obtain a glycyrrhizic acid solution; mixing the glycyrrhizic acid solution with a natural food-derived substance solution, removing the organic reagent after rotary evaporation, and redissolving in water to obtain a glycyrrhizic acid / natural food-derived nutrient solution.

3. The method for preparing the natural Chinese herbal medicine hydrogel composite microneedle according to claim 1, characterized in that: In S1, the mass ratio of glycyrrhizic acid to natural food-derived nutrients is 10:(0.1~5).

4. The method for preparing the natural Chinese herbal medicine hydrogel composite microneedle according to claim 1, characterized in that: In S2, the metal salt in the metal salt solution is one or more of calcium salt, sodium salt and magnesium salt.

5. The method for preparing the natural Chinese herbal medicine hydrogel composite microneedle according to claim 1, characterized in that: In S2, the mass ratio of the metal salt to glycyrrhizic acid in the metal salt solution is (1-2.2):

1.

6. The method for preparing the natural Chinese herbal medicine hydrogel composite microneedle according to claim 1, characterized in that: In S3, the polyanionic polymer in the polyanionic polymer solution is hyaluronic acid, sodium alginate or sodium carboxymethyl cellulose.

7. The method for preparing the natural Chinese herbal medicine hydrogel composite microneedle according to claim 1, characterized in that: In S3, the solvent of the polyanionic polymer solution is water, phosphate buffer solution or physiological saline.

8. The method for preparing the natural Chinese herbal medicine hydrogel composite microneedle according to claim 1, characterized in that: In S3, the mass ratio of the polyanionic polymer solution to the glycyrrhizic acid / food-derived active substance gel is (1-8):

1.

9. The natural Chinese herbal medicine hydrogel composite microneedle obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the natural Chinese herbal medicine hydrogel composite microneedle according to claim 9 in the preparation of a drug for treating recurrent aphthous ulcer.

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