Pickering emulsion gel based on drug-adjuvant combination and preparation method and application thereof

Abstract

This invention discloses a Pickering emulsion gel based on drug-excipient integration, its preparation method, and its application. The method includes: adding ethylene glycol chitosan to a whey protein solution, stirring until homogeneous, then adding glycyrrhizin and stirring to obtain a whey protein-ethylene glycol chitosan-glycyrrhizin composite solution; mixing Gastrodia elata volatile oil and clove oil, and ultrasonically homogenizing to obtain an oil phase; adding the oil phase to the whey protein-ethylene glycol chitosan-glycyrrhizin composite solution, and emulsifying by high-speed shearing to obtain an emulsion; then refrigerating the emulsion to induce whey protein denaturation and gelation, forming a Pickering emulsion gel. Based on the concept of "drug-excipient integration," this invention uses whey protein as a particle stabilizer and the main active ingredients, Gastrodia elata volatile oil and clove oil, as the mixed oil phase, reducing the amount of excipients. Through screening the aqueous phase of the emulsion gel, the prepared Pickering emulsion gel has a dense texture, high stability, and a sustained-release effect on the active ingredients, achieving a long-lasting sleep-improving effect.

Description

Technical Field

[0001] This invention belongs to the field of emulsion gel biomaterials, and particularly relates to a Pickering emulsion gel based on drug-excipient combination, its preparation method and application. Background Technology

[0002] In recent years, with the accelerating pace of work and life and increasing competitive pressure, the incidence of insomnia has gradually increased due to various factors. Insomnia is the most common initial symptom of depression and one of the high-risk factors for relapse; insomnia is also a symptom of anxiety disorders, and sleep disturbances are often a precursor; severe insomnia during perimenopause can significantly affect mood and quality of life, and is a very common clinical manifestation in menopausal women. Insomnia often induces adverse symptoms such as irritability, fear, anxiety, and depression, and also increases the probability of cardiovascular diseases and mental illnesses such as hypertension, coronary heart disease, and memory decline. A study by the World Health Organization (WHO) shows that about 27% of people worldwide suffer from insomnia, and about 40% of adults and 35% of the elderly in my country experience insomnia. Therefore, insomnia should be given high priority and actively prevented and treated. Currently, Western medicines for treating insomnia mainly consist of benzodiazepines and some newer sedative-hypnotic drugs, but long-term use can lead to aftereffects, drug dependence, and even addiction. Traditional Chinese medicine has unique advantages in improving sleep. Therefore, screening for active ingredients in traditional Chinese medicine that improve sleep with minimal toxic side effects and developing them into sedative and sleep-aiding products is an effective strategy for preventing and treating insomnia.

[0003] Gastrodia elata is a plant belonging to the Orchidaceae family. (Gastrodia eLata BL., GE The dried tuber of Gastrodia elata, entering the liver meridian, possesses the effects of calming wind and stopping spasms, suppressing liver yang, and promoting sedation and sleep. It is mainly used to treat internal liver wind, convulsions, and dizziness. Modern research shows that Gastrodia elata contains abundant volatile components, including aromatic hydrocarbons, terpenes, heterocyclic compounds, esters, aromatic ethers, phenols, acids, aldehydes, alcohols, ketones, and alkanes. Pharmacological studies have shown that Gastrodia elata volatile oil can promote the calming of brain nerves, prevent excessive neuronal activity, and alleviate discomfort symptoms such as difficulty falling asleep, early awakening, and anxiety caused by insomnia. However, the volatile oil suffers from problems such as easy oxidation, volatility, degradation, and poor solubility, which severely limit its clinical application.

[0004] clove( Eugeniacaryophyllata Clove is a plant of the Myrtaceae family, widely cultivated in tropical and subtropical countries. Clove oil is a volatile oil obtained by distillation of its dried flower buds or stems and leaves, mainly containing volatile components such as eugenol, eugenol acetate, and β-eugenolene. Clove oil has anti-inflammatory, analgesic, antibacterial, and antiseptic effects, and is widely used in the perfume, cosmetic, medical, and food industries. However, clove oil also has disadvantages such as high volatility, poor water solubility, and low bioavailability, which greatly limits its clinical application.

[0005] Pickering emulsion gels can immobilize emulsified oil droplets within a network structure, thereby inhibiting droplet flocculation and aggregation. Simultaneously, emulsion gels can stabilize unstable bioactive substances. Compared to emulsions or gels alone, emulsion gels offer advantages such as structural stability and tunable viscoelasticity. Currently, the commonly used oil phases for preparing Pickering emulsion gels include soybean oil, sesame oil, rapeseed oil, and peanut oil. However, these methods suffer from problems such as soft texture and low cross-linking density, severely limiting the application of oil phases like soybean oil in emulsion gels. Aqueous phases often utilize naturally derived chitosan and sodium alginate, but chitosan has low solubility in water, requiring the addition of dilute acid to increase its solubility, which can compromise the stability of the gel system. Furthermore, the cross-linking mechanism of commonly used emulsion gel matrices is often electrostatic cross-linking to form a gel network structure. This structure is highly susceptible to electrostatic effects, resulting in a soft gel texture, low cross-linking density, and susceptibility to degradation. Summary of the Invention

[0006] To address the aforementioned issues, the present invention aims to provide a method for preparing a Pickering emulsion gel based on the integration of pharmacology and excipients. This method uses the main active ingredients, Gastrodia elata volatile oil and clove oil, as a mixed oil phase, reducing the amount of excipients. Through screening the aqueous phase of the emulsion gel, the prepared Pickering emulsion gel has a dense texture, high stability, and a sustained-release effect on the active ingredients, thus achieving a long-lasting effect of improving sleep.

[0007] This invention is achieved through the following technical solution:

[0008] A method for preparing a Pickering emulsion gel based on drug-excipient combination includes the following steps:

[0009] (1) Add ethylene glycol chitosan to whey protein solution, stir evenly, then add glycyrrhizin, stir and mix to obtain whey protein-ethylene glycol chitosan-glycyrrhizin composite solutions with different mass ratios.

[0010] (2) Gastrodia elata volatile oil and clove oil were mixed and homogenized by ultrasonication to obtain the oil phase;

[0011] (3) The oil phase is added to the whey protein-ethylene glycol chitosan-glycyrrhizin composite solution and emulsified by high-speed shearing to obtain an emulsion. The emulsion is then refrigerated to induce the denaturation and gelation of whey protein to form Pickering emulsion gel.

[0012] The whey protein solution of the present invention can be prepared by the following method: a certain amount of whey protein powder and purified water are heated and stirred with a magnetic stirrer at a speed of 300-400 r / min for 3-5 hours under constant temperature conditions of 40-60℃ to fully dissolve and hydrate the whey protein.

[0013] Preferably, in step (1), the mass concentration of the whey protein solution is 10-20%.

[0014] This invention optimizes the selection of ethylene glycol chitosan (GC) and glycyrrhizin (AGA) as the aqueous phase of the Pickering emulsion gel. The amino groups (-NH2) in the ethylene glycol chitosan (GC) molecule and the aldehyde groups (-CHO) in the glycyrrhizin (AGA) molecule can undergo a Schiff base reaction to generate imine bonds. The gel network structure prepared by chemical crosslinking has a denser texture and higher stability.

[0015] Preferably, in step (1), the mass ratio of ethylene glycol chitosan to glycyrrhizin is 1:2 to 2:1.

[0016] Preferably, in step (1), the mass ratio of whey protein, ethylene glycol chitosan and glycyrrhizin in the whey protein-ethylene glycol chitosan-glycyrrhizin composite solution is 4:1:2 to 4:2:1.

[0017] Based on the concept of "medicine and excipient integration", this invention uses gastrodia elata volatile oil, which has sedative and hypnotic effects, and clove oil, which has anti-inflammatory, analgesic, antiseptic, and flavoring effects, as a mixed oil phase to replace traditional non-medicinal soybean oil, sesame oil, rapeseed oil, and peanut oil, thereby reducing the amount of excipients used. In addition, ultrasonic homogenization is used to completely encapsulate gastrodia elata volatile oil with clove oil, thereby reducing the irritation of gastrodia elata volatile oil.

[0018] The gastrodia elata volatile oil of the present invention can be obtained by conventional ultrasonic-assisted steam distillation extraction.

[0019] Preferably, in step (2), the volume ratio of the gastrodia elata volatile oil to the clove oil is 1:3~5.

[0020] Preferably, in step (3), the volume ratio of the oil phase to the whey protein-ethylene glycol chitosan-glycyrrhizin composite solution is 1~2:8~9.

[0021] Preferably, in step (3), the high-speed shearing speed is 1000~1500 rpm and the time is 3min~5min.

[0022] Preferably, in step (3), the refrigeration conditions are: refrigerated at 4°C for 24~48h.

[0023] The present invention also provides a Pickering emulsion gel based on drug-excipient combination, which is prepared by the above-described preparation method.

[0024] This invention also provides the application of the above-mentioned Pickering emulsion gel based on drug-excipient combination in the preparation of drugs for the prevention or treatment of insomnia.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] This invention is based on the concept of "drug and excipient integration," using whey protein as a particle stabilizer and the main active ingredients, Gastrodia elata volatile oil and clove oil, as a mixed oil phase. This reduces the amount of excipients used and improves the stability of Gastrodia elata volatile oil and clove oil. Simultaneously, it utilizes the imine bond product generated by the Schiff base reaction of ethylene glycol chitosan (GC) and glycyrrhizin (AGA), which has better water solubility, as the aqueous phase, and prepares a Pickering emulsion gel through chemical cross-linking. This gel has a denser cross-linked network structure, stronger binding effect on emulsion droplets, smaller and more stable particle size, and can sustainably release clove oil and Gastrodia elata volatile oil. Pharmacological verification shows that the Pickering emulsion gel prepared by this invention can achieve a long-lasting effect in improving sleep. Attached Figure Description

[0027] Figure 1 The images show the appearance of the whey protein-ethylene glycol chitosan-glycyrrhizic acid composite solution prepared in Example 1 (A) and the whey protein-chitosan-glycyrrhizic acid composite solution prepared in Comparative Example 1 (B).

[0028] Figure 2 The appearance of the Pickering emulsion gel prepared for Comparative Example 1 is shown in Figure (A) and scanning electron microscope (SEM) image (B), and the appearance of the Pickering emulsion gel after 7 days of storage is shown in Figure (C).

[0029] Figure 3 Appearance (A) and particle size (B) of Pickering emulsion when the volume ratio of Gastrodia elata volatile oil to clove oil is 1:2.

[0030] Figure 4 When the volume ratio of Gastrodia elata volatile oil to clove oil is 1:4, the appearance (A), particle size (B), appearance of Pickering emulsion gel after 7 days of storage (C), and appearance of Pickering emulsion gel after 28 days of storage (D) are compared.

[0031] Figure 5 The appearance of Pickering emulsion when the volume ratio of hemp volatile oil to clove oil is 1:6;

[0032] Figure 6Upright and inverted images of Pickering emulsion gels (PEG) prepared for different proportions of gel matrix;

[0033] Figure 7 Optical microscope images of Pickering emulsion gels prepared with different proportions of gel matrix;

[0034] Figure 8 Particle size distribution of Pickering emulsion gels prepared with different proportions of gel matrix;

[0035] Figure 9 Rheological properties of Pickering emulsion gels prepared with different proportions of gel matrix (strain scan curves; frequency scan curves; time scan curves; viscosity as a function of shear rate).

[0036] Figure 10 The proton NMR spectrum of glycyrrhizin (AGA) 1 H NMR spectrum;

[0037] Figure 11 The proton NMR spectrum of ethylene glycol chitosan (GC) 1 H NMR spectrum;

[0038] Figure 12 The 1H NMR spectrum of Pickering emulsion gel ( 1 H NMR spectrum;

[0039] Figure 13 FTIR spectra of whey protein (WPC), ethylene glycol chitosan (GC), glycyrrhizin (AGA), and Pickering emulsion gel (PEG);

[0040] Figure 14 Thermogravimetric analysis (TGA) curves, differential thermogravimetric analysis (DTG) curves, and differential thermogravimetric analysis (DGA) curves of Pickering emulsion and Pickering emulsion gel.

[0041] Display the scanning calorimetry (DSC) lines;

[0042] Figure 15 SEM image (A) and EDS-Mapping image (B) of Pickering emulsion gel;

[0043] Figure 16 To investigate the effects of Pickering emulsion gel on the pathological state of the hypothalamus in insomnia mice, hypothalamic sections were stained with H&E (50X, 100X).

[0044] Figure 17 To investigate the effect of Pickering emulsion gel on the pathological state of the hippocampus in insomnia mice, hippocampal sections were stained with H&E (50X). Detailed Implementation

[0045] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. The described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] Preparation of Gastrodia elata volatile oil:

[0047] Accurately weigh 50 g of Gastrodia elata powder and place it in a 1000 mL round-bottom flask, then add 450 mL of distilled water. Sonicate for 45 min at 280 W. After sonication, allow to stand and soak for 2 h. After soaking, transfer the round-bottom flask to a constant-temperature magnetic stirrer, and connect the volatile oil analyzer and spherical condenser to form a complete distillation reflux apparatus. Turn on the heating function and raise the system temperature to a state of slight boiling. Maintain this slight boiling condition for 8 h to ensure complete distillation and separation of the volatile oil components. After heating, allow the mixture to stand for 1 h to allow the oil and water phases to fully separate, then collect the Gastrodia elata volatile oil.

[0048] Preparation of whey protein solution:

[0049] A certain amount of whey protein powder and purified water were heated and stirred at 350 r / min for 4 h under constant temperature of 50℃ to fully dissolve and hydrate the whey protein, thus obtaining a whey protein solution. By adjusting the amount of whey protein powder added, whey protein solutions of different mass concentrations were obtained.

[0050] Example 1: Preparation of Pickering emulsion gel based on drug-excipient combination

[0051] (1) Add ethylene glycol chitosan to whey protein solution, stir evenly, then add glycyrrhizin, stir and mix to obtain whey protein-ethylene glycol chitosan-glycyrrhizin composite solution; wherein, the mass concentration of whey protein solution is 15%, and the mass ratio of whey protein, ethylene glycol chitosan and glycyrrhizin in whey protein-ethylene glycol chitosan-glycyrrhizin composite solution is 4:2:1;

[0052] (2) Gastrodia elata volatile oil and clove oil were mixed and homogenized by ultrasonication to obtain the oil phase; the volume ratio of Gastrodia elata volatile oil to clove oil was 1:4.

[0053] (3) The oil phase was added to the whey protein-ethylene glycol chitosan-glycyrrhizin composite solution (the volume ratio of the oil phase to the whey protein-ethylene glycol chitosan-glycyrrhizin composite solution was 2:8), and the emulsion was obtained by high-speed (1200 rpm) shear emulsification. The emulsion was then refrigerated at 4°C for 48 h to induce whey protein denaturation and gelation, forming Pickering emulsion gel.

[0054] Comparative Example 1: Preparation of Pickering Emulsion Gel from Conventional Gel Matrix

[0055] (1) Add chitosan to whey protein solution, stir evenly, then add glycyrrhizic acid, stir and mix to obtain whey protein-chitosan-glycyrrhizic acid composite solution; wherein, the mass concentration of whey protein solution is 15%, and the mass ratio of whey protein, chitosan and glycyrrhizic acid in whey protein-chitosan-glycyrrhizic acid composite solution is 4:2:1.

[0056] (2) Gastrodia elata volatile oil and clove oil were mixed and homogenized by ultrasonication to obtain the oil phase; the volume ratio of Gastrodia elata volatile oil to clove oil was 1:4.

[0057] (3) The oil phase was added to the whey protein-chitosan-glycyrrhizic acid composite solution (the volume ratio of the oil phase to the whey protein-ethylene glycol chitosan-glycyrrhizic acid composite solution was 2:8), and the emulsion was obtained by high-speed (1200 rpm) shear emulsification. The emulsion was then refrigerated at 4°C for 48 h to induce whey protein denaturation and gelation, forming Pickering emulsion gel.

[0058] Appearance of the whey protein-ethylene glycol chitosan-glycyrrhizin composite solution prepared in Comparative Example 1 (Figure) Figure 1 Appearance images of the whey protein-chitosan-glycyrrhizic acid composite solutions prepared in Comparative Example A and Comparative Example 1 (Figure A) Figure 1 (Comparative Example B) The results showed that the chitosan in the composite solution of Comparative Example 1 was not completely dissolved. Therefore, the present invention uses ethylene glycol chitosan, which has better solubility.

[0059] Figure 2 The image shows the appearance and electron micrograph of the Pickering emulsion gel prepared in Comparative Example 1. Figure 2 As shown in Figure A, Comparative Example 1 uses chitosan and glycyrrhizic acid as the gel matrix. The cross-linking mechanism is electrostatic cross-linking to form a gel network structure. This gel has a soft texture and low cross-linking density. Figure 2 The same situation was observed during B-scanning electron microscopy, showing that the formed gel was unstable and easily degraded. Figure 2 The C-value shows that after being stored at room temperature for 7 days, the Pickering emulsion gel has a softer texture and some of it has degraded, indicating poor stability.

[0060] Example 2: Optimization of the volume ratio of Gastrodia elata volatile oil to clove oil

[0061] The effect of the mixing ratio of Gastrodia elata volatile oil and clove oil on the formation of Pickering emulsion gel was investigated. The mass ratio of ethylene glycol chitosan to glycyrrhizin was controlled at 2:1. This invention investigated three cases where the volume ratio of Gastrodia elata volatile oil to clove oil was 1:2, 1:4 and 1:6.

[0062] When the volume ratio of volatile oil from Gynostemma pentaphyllum to clove oil is 1:2, Figure 3 Image A shows the appearance of a Pickering emulsion. Visually, it appears to form a Pickering emulsion, but... Figure 3 B represents the particle size of the Pickering emulsion, which was measured to be around 2300 μm. It is speculated that the aggregation may have occurred due to the instability of the Pickering emulsion.

[0063] When the volume ratio of volatile oil from Gastrodia elata to clove oil is 1:4, Figure 4 Image A shows the appearance of a Pickering emulsion. Visual observation confirms the formation of a Pickering emulsion. Figure 4 B represents the particle size of the Pickering emulsion, which was measured to be around 700 μm. The Pickering emulsion gel did not show significant changes after being stored at room temperature for 7 and 28 days, indicating that the Pickering emulsion gel is stable.

[0064] When the volume ratio of volatile oil from Gynostemma pentaphyllum to clove oil is 1:6, Figure 5 The image shows the appearance of a Pickering emulsion. Visual observation reveals that the emulsion exhibits layering and cannot form a Pickering emulsion.

[0065] Example 3: Screening of Gel Matrix Ratio

[0066] The effect of the mass ratio of ethylene glycol chitosan to glycyrrhizin on the formation of Pickering emulsion gel was investigated. The volume ratio of Gastrodia elata volatile oil to clove oil was controlled at 1:4. The mass ratios of ethylene glycol chitosan to glycyrrhizin were investigated at 1:1, 2:1 and 1:2.

[0067] Figure 6Upright and inverted images of Pickering emulsion gels (PEG) prepared with different proportions of gel matrix. Visual characterization revealed that the Pickering emulsion gels (PEG) all exhibited a uniform and consistent milky-white appearance. No visible oil phase layer, water phase sedimentation, or solid particle aggregation was observed, indicating that the oil phase (Gastrodia elata volatile oil) was highly dispersed in the aqueous phase, with good uniformity in droplet size, consistent with the macroscopic morphological characteristics of a stable O / W emulsion. Inverted stability verification of the Pickering emulsion gels showed no significant flow deformation, maintaining its initial formed state stably. The gel exhibited a dense overall texture, a smooth surface, and no localized depressions, cracks, or component separation.

[0068] Figure 7 Optical micrographs of Pickering emulsion gels (PEG) prepared with different proportions of gel matrix. Microscopic observation reveals relatively dispersed spherical droplets of relatively uniform size, with no obvious aggregation, indicating good dispersion of the oil phase in the aqueous phase of the emulsion system. PEG (1:1) exhibits a denser granular structure, with droplets encapsulated by the gel matrix, resulting in a more compact particle distribution. However, some dispersion is still observed, indicating that when the mass ratio of ethylene glycol chitosan to glycyrrhizin is 1:1, the gel matrix begins to crosslink and encapsulate and fix the emulsion droplets. However, the density of the gel network can still be improved at this point. The granular structure of PEG (1:2) is further denser, with a tendency for particle aggregation in some areas. This may be due to the relatively high proportion of glycyrrhizin, which affects the growth of the gel network and the encapsulation of droplets during the crosslinking process, leading to local particle aggregation. This reflects a decrease in the microscopic uniformity of the gel system at this ratio. PEG (2:1) exhibits a relatively uniform and dense network structure, with droplets well embedded in the gel network and no obvious large particle aggregation.

[0069] from Figure 8The particle size analysis data showed significant differences in the particle size of Pickering emulsion gels (PEG) with different mass ratios of ethylene glycol chitosan to glycyrrhizin. At a mass ratio of 2:1, the average particle size was 722.7 nm; at 1:1, the average particle size increased to 980.07 nm; and at 1:2, the average particle size further increased to 1108 nm. This indicates that the particle size of the gel system increases with the increase of the relative proportion of glycyrrhizin. Ethylene glycol chitosan and glycyrrhizin undergo a Schiff base reaction with the aldehyde groups. When the glycyrrhizin ratio is low (e.g., 2:1), the network structure formed by the cross-linking reaction is denser, resulting in stronger binding of emulsion droplets and thus smaller particle size. However, as the glycyrrhizin ratio increases, the degree of cross-linking or the uniformity of the network structure may change, leading to droplet aggregation or increased network porosity, and consequently, an increase in particle size. Comprehensive analysis shows that PEG (2:1) has a higher relative content of ethylene glycol chitosan, and the cross-linked gel network is more conducive to the uniform dispersion and stable encapsulation of droplets, with better uniformity and density of microstructure.

[0070] Figure 9 Rheological properties of Pickering emulsion gels (PEG) prepared with different ratios of gel matrix (ethylene glycol chitosan: glycyrrhizin = 1:1, 1:2, 2:1). In strain scanning, the elastic modulus (G') of all samples was greater than the viscous modulus (G''), reflecting the elastic-dominant characteristics of a solid-like gel. This is closely related to the network effect formed by particles at the interface in the Pickering emulsion. The "PEG 2:1" group had the highest G', while the "PEG 1:2" group had the lowest. Furthermore, the "PEG 2:1" group had a larger critical strain, indicating that a higher proportion of ethylene glycol chitosan results in a denser gel network and stronger resistance to deformation, while an increased proportion of glycyrrhizin leads to a relatively looser network. Frequency scanning revealed different growth trends for G' and G" in samples with different ratios. The G' of the "PEG 2:1" group showed a more significant increase with frequency, indicating that its network is more sensitive to frequency and exhibits greater elasticity under rapid external force. Time scanning showed that the G' of the "PEG 2:1" group remained consistently higher and more stable, demonstrating good long-term stability, while the G' of the "PEG 1:2" group tended to fluctuate or decrease over time. The viscosity-shear rate curves reflected shear thinning behavior. The "PEG 2:1" group had higher viscosity and weaker shear thinning, indicating strong shear resistance in its network. The "PEG 1:2" group, on the other hand, had lower viscosity and more significant thinning, making its network more susceptible to shear damage.

[0071] The Pickering emulsion gel based on drug-excipient combination prepared in Example 1 was subjected to the following tests.

[0072] Experimental Example 1:

[0073] Figure 10 Glycyrrhizin (AGA) 1In the 1H NMR spectrum, the characteristic resonance peak of the hydrogen atom on the aldehyde group (-CHO) appears around 5.0 ppm. This peak is the signature proton signal of the aldehyde group in the AGA molecule.

[0074] Figure 11 GC-based ethylene glycol chitosan 1 The H NMR spectrum shows characteristic resonance peaks of protons in the sugar ring and hydroxyethyl-substituted protons in the chitosan molecular chain. These characteristic peaks correspond to the typical chemical structure of GC.

[0075] Figure 12 For Pickering emulsion gel (PEG 2:1) 1 Compared to the spectra of GC and AGA, the 1H NMR spectrum shows a new proton resonance signal at 8.75 ppm. Simultaneously, the characteristic peak of the aldehyde hydrogen in the AGA spectrum disappears, and the proton peaks associated with the amino group (-NH2) in the GC molecule show changes in chemical shift or integrated intensity. This phenomenon is highly consistent with the Schiff base reaction mechanism, where the aldehyde group (-CHO) in glycyrrhizin (AGA) reacts with the amino group (-NH2) in ethylene glycol chitosan (GC) to form an imine bond.

[0076] Figure 13 The images show the FTIR spectra of whey protein (WPC), ethylene glycol chitosan (GC), glycyrrhizin (AGA), and Pickering emulsion gel (PEG). The FTIR spectrum of whey protein (WPC) exhibits typical functional group vibration peaks in the CO stretching vibration region of the amide bond, reflecting the vibrational information of its intramolecular peptide bonds and side chain groups. The FTIR spectrum of ethylene glycol chitosan (GC) shows characteristic vibrational peaks of functional groups such as hydroxyl (-OH) and amino (-NH2) groups (e.g., OH stretching vibration peaks, NH bending vibration peaks), which match the functional group structure of GC as a polysaccharide molecule. The FTIR spectrum of glycyrrhizin (AGA) shows a peak at 1724 cm⁻¹. -1 The presence of characteristic stretching vibration peaks of the aldehyde group (C=O) nearby indicates the presence of the aldehyde functional group in the molecule. The FTIR spectrum of the Pickering emulsion gel (PEG) not only includes some characteristic peaks of WPC, GC, and AGA (reflecting the presence of each component in the gel system), but also shows new characteristic peaks or shifts and intensity changes of existing characteristic peaks. For example, in the 1630-1650 cm⁻¹ range... -1Vibrational peaks related to the imine bonds (-N=CH) formed by the Schiff base reaction appeared nearby, while the intensity of the amino characteristic peak of GC and the aldehyde characteristic peak of AGA decreased. This is consistent with the consumption process of amino and aldehyde groups in the Schiff base reaction, further confirming the cross-linking effect between GC and AGA from the perspective of functional group vibration, and the process by which WPC, GC, and AGA form Pickering emulsion gel in the gel network through chemical and physical interactions such as Schiff base reaction, hydrogen bonding, and hydrophobic interactions.

[0077] Experimental Example 2: Thermal Stability Analysis of Pickering Emulsion Gel

[0078] The thermogravimetric curves of the Pickering emulsion gel (PEG 2:1) sample are as follows: Figure 14 As shown, weight loss begins at 30℃, reaching 22.54% by 243℃. Slight weight loss occurs before 100℃, due to moisture absorbed from the air after removal of the lyophilized sample. According to the DTG curve, the weight loss rate accelerates around 243℃, possibly due to the thermal decomposition of volatile oils. As shown in both the TG and DTG curves, further thermal decomposition begins around 350℃, caused by the decomposition of whey proteins and the gel matrix in the Pickering emulsion gel. Specifically, the weight loss is 58.22% when the temperature rises from 350℃ to 500℃, with the maximum weight loss rate at 389℃.

[0079] Differential scanning calorimetry (DSC) is a widely used technique for characterizing inclusion compounds, which can examine in detail the thermal behavior of each component and the inclusion compounds they form. Figure 14 The thermal behavior of Pickering emulsion (PE) was demonstrated. The DSC thermogram of PE shows a distinct endothermic peak in the range of approximately 30℃ to 336℃, with a peak temperature of 104℃, which represents the melting point (Tm) of the sample at 104℃. Figure 14 The thermal behavior of Pickering emulsion gel (PEG 2:1) was demonstrated. The DSC thermogram of PEG (2:1) shows two distinct endothermic peaks. The first endothermic peak is located between 50℃ and 300℃, with a melting point Tm of 88℃ at the peak. The second endothermic peak is located between 385℃ and 389℃, with a melting point Tm of 387℃ at the peak.

[0080] The DSC curve also exhibits a corresponding absorption peak during the weight loss phase of the TG curve. At around 88℃, a broad and gentle endothermic peak appears on the DSC curve of PEG (2:1), indicating the evaporation stage of water, which corresponds to its TG curve. In the 385~389℃ range, a narrow endothermic peak appears, corresponding to its TG curve, presumably due to the decomposition reaction of some substances with low content during this stage. Based on the peak shape, it can be seen that the content of this substance in PEG (2:1) is relatively low. It is worth noting that by comparing the DSC thermogram data of PE and PEG (2:1), it was found that PEG has one more endothermic peak than PE, proving that incorporating the gel matrix into the Pickering emulsion has a significant impact on its thermal stability. The sample with the addition of the gel matrix has a better stabilizing effect.

[0081] Experimental Example 3: Microstructure of Pickering Emulsion Gel

[0082] Figure 15 Image A shows a SEM image of the Pickering emulsion gel (PEG 2:1). From the SEM images at different magnifications (a1-a4), it can be observed that the Pickering emulsion gel (PEG 2:1) loaded with Gastrodia elata volatile oil exhibits a porous structure. This porous structure provides the spatial basis for the loading and subsequent release of Gastrodia elata volatile oil, and also reflects the microstructural construction of the gel system. Figure 15 Image B is the EDS-Mapping diagram of the Pickering emulsion gel (PEG 2:1), showing the distribution of C, O, and N elements. The image shows that these elements are uniformly distributed on the gel surface, indicating that the components constituting the gel (such as ethylene glycol chitosan, glycyrrhizic acid, whey protein, and the loaded Gastrodia elata volatile oil) are uniformly mixed in the gel system. This elemental distribution provides evidence for the successful formation of the emulsion gel, demonstrating that the components are well integrated through chemical or physical processes, forming a uniform gel network.

[0083] Experiment 4: Pharmacodynamic results of Pickering emulsion gel in treating insomnia in rats

[0084] This invention establishes a rat model of insomnia and studies the therapeutic effect of Pickering emulsion gel on insomnia rats.

[0085] (1) Construction and experimental grouping of insomnia model rats:

[0086] C57 / BL mice were divided into 6 groups, with 10 mice in each group: A) normal control group (administered only 0.9% saline), B) model group (PCPA group) (PCPA concentration 400 mg / kg, dosage 10 mL / kg / day), C) positive control group (administered diazepam concurrently with modeling, concentration 1.3 mg / kg), D) low-dose PEG group (administered concurrently with modeling, concentration 5 mg / kg), Z) medium-dose PEG group (administered concurrently with modeling, concentration 10 mg / kg), and G) high-dose PEG group (administered concurrently with modeling, concentration 15 mg / kg). Except for the normal control group, all other groups of mice were intraperitoneally injected with PCPA once a day for 4 consecutive days. The control group received an equal volume of saline. Within 28–30 h after PCPA induction, the circadian rhythm and sleep duration of the rats changed significantly compared to the control group without PCPA injection, indicating successful induction of the insomnia model.

[0087] (2) Drug administration and sample processing:

[0088] Mice were given the drug on day 4 of modeling and continued to be given the drug for 5 days.

[0089] (3) Righting Reflection Experiment:

[0090] After administration on day 5, mice were injected intraperitoneally with chloral hydrate. The time interval from chloral hydrate administration to the mice remaining upright for 60 seconds was recorded as sleep latency, and sleep duration was also recorded. The sleep latency and sleep duration of the six groups of mice were compared, and the results are shown in Table 1.

[0091] Table 1. Sleep latency and sleep duration in different groups of mice

[0092]

[0093] Table 1 shows that the sleep latency of mice in each model group was prolonged compared to the normal group, and the sleep duration, except for the low-dose PEG group (D), was shortened compared to the blank group. The sleep latency of mice in the positive drug group, low-, medium-, and high-dose PEG groups was shorter than that of mice in model group B, and the sleep duration was prolonged in all groups except for the medium-dose PEG group (E). The righting reflex test further confirmed the successful establishment of the mouse insomnia model, and data comparison showed that the sleep duration of mice in the medium-dose PEG group (E) and the high-dose PEG group (F) was greater than that of the positive drug group (C).

[0094] (4) Pathological examination of brain tissue:

[0095] HE staining: Oral mucosal tissue from the ulcer site was routinely embedded in paraffin, sectioned, dewaxed, washed, stained with hematoxylin, washed, differentiated, blued, and soaked in water, then placed in eosin solution. Routine dehydration, clearing, and mounting were then performed. Changes in the mucosal tissue and the infiltration of inflammatory cells in the epithelial connective tissue were observed under a light microscope.

[0096] Figure 16 The H&E staining of the hypothalamus in insomnia mice with Pickering emulsion gel (PEG) shows the results. In the normal control group (A), the hypothalamus was rich in neurons, evenly distributed, with intact cell structure, neat arrangement, and clearly defined round or oval nuclei. Compared to the normal control group, the model group (B) showed fewer hypothalamic neurons, deformed structure, sparse arrangement, vacuolation, and inconsistent nuclei size. Compared to the model group, the positive drug group (C), low-dose PEG group (D), medium-dose PEG group (E), and high-dose PEG group (F) significantly improved the aforementioned pathological damage, with increased hypothalamic neuron numbers, reduced vacuolation, and generally normal morphology.

[0097] H&E staining results showed significant structural damage to hypothalamic neurons in the sleep deprivation model animals, including neuronal cell body atrophy, nuclear condensation, increased cytoplasmic eosinophilia, and vacuolar degeneration. The use of diazepam as a positive control significantly improved this damage, validating the reliability of the model and the effectiveness of the detection method.

[0098] Compared with the normal group, all dose groups showed varying degrees of neuroprotective effects after intervention with Pickering emulsion gel, with the medium and high dose groups showing significant improvement. In the high-dose group, neuronal structure remained intact, with a significant reduction in necrotic or apoptotic cells, and tissue morphology similar to the normal group.

[0099] The results show that the Pickering emulsion gel of the present invention can significantly reduce hypothalamic neuronal damage caused by sleep deprivation and has good neuroprotective, antioxidant and anti-inflammatory effects.

[0100] Figure 17The image above shows the H&E staining of the hippocampus of insomniac mice with Pickering emulsion gel (PEG). As can be seen, in the normal group (A), the hippocampal neurons were intact, neatly arranged, and evenly distributed, with clearly visible nuclei and no obvious pathological damage. However, in the model group (B, PCPA group), the hippocampal tissue showed disordered arrangement, even loss of normal neuronal morphology, darker color, cell atrophy, and numerous necrotic neurons. Compared with the model group, the positive control group, the low-dose PEG group (D), the medium-dose PEG group (E), and the high-dose PEG group (F) significantly improved hippocampal pathological damage. The hippocampal neurons in these groups showed relatively normal morphology and clear structure. The hippocampal neurons in the positive control group and the low-dose PEG group (D) showed slight improvement compared to the PCPA group, but neuronal morphology still varied, the color was darker, and damage remained. The H&E staining results indicate that Pickering emulsion gel has a certain repair effect on hippocampal neuronal damage.

[0101] H&E staining results showed that sleep deprivation caused significant structural damage to hippocampal neurons in mice, with a marked reduction in the number of neurons, extensive neuronal cell atrophy, vacuolar degeneration, and widening of interstitial spaces. Pickering emulsion gel exhibited a significant dose-dependent neuroprotective effect. Medium and high doses effectively improved neuronal morphology, reduced cell degeneration and necrosis, and restored the integrity of the hippocampal hierarchical structure, with a protective effect comparable to melatonin. This suggests that the Pickering emulsion gel of this invention may alleviate sleep deprivation-induced neurological damage through antioxidant, anti-inflammatory, and energy metabolism regulation pathways, and has potential neuroprotective application value.

[0102] In summary, histopathological examination showed that all dosage groups exhibited varying degrees of neuroprotective effects after intervention with the Pickering emulsion gel of this invention. The medium and high dosage groups effectively improved neuronal morphology, restored the integrity of tissue hierarchical structure, reduced cell degeneration and necrosis, and the tissue morphology was similar to that of the normal group. The Pickering emulsion gel of this invention (10%) and high dosage (15%) of volatile oil can exert a neuroprotective effect and has potential application value in preventing and treating sleep disorders and neurological function damage.

Claims

1. A method for preparing a Pickering emulsion gel based on drug-excipient combination, characterized in that, Includes the following steps: (1) Add ethylene glycol chitosan to whey protein solution, stir evenly, then add glycyrrhizin, stir and mix to obtain whey protein-ethylene glycol chitosan-glycyrrhizin composite solution; the mass ratio of ethylene glycol chitosan to glycyrrhizin is 2:

1. (2) Gastrodia elata volatile oil and clove oil are mixed and homogenized by ultrasonication to obtain the oil phase; the volume ratio of Gastrodia elata volatile oil to clove oil is 1:3~5; (3) The oil phase is added to a whey protein-ethylene glycol chitosan-glycyrrhizin composite solution and emulsified by high-speed shearing to obtain an emulsion. The emulsion is then refrigerated to induce the denaturation and gelation of whey protein to form Pickering emulsion gel.

2. The method for preparing Pickering emulsion gel based on drug-excipient combination according to claim 1, characterized in that: In step (1), the mass concentration of the whey protein solution is 10-20%.

3. The method for preparing Pickering emulsion gel based on drug-excipient combination according to claim 1, characterized in that: In step (1), the mass ratio of whey protein, ethylene glycol chitosan and glycyrrhizin in the whey protein-ethylene glycol chitosan-glycyrrhizin composite solution is 4:2:

1.

4. The method for preparing Pickering emulsion gel based on drug-excipient combination according to claim 1, characterized in that: In step (3), the volume ratio of the oil phase to the whey protein-ethylene glycol chitosan-glycyrrhizin composite solution is 1~2:8~9.

5. The method for preparing Pickering emulsion gel based on drug-excipient combination according to claim 1, characterized in that: In step (3), the high-speed shearing speed is 1000~1500 rpm and the time is 3 min~5 min.

6. The method for preparing Pickering emulsion gel based on drug-excipient combination according to claim 1, characterized in that: In step (3), the refrigeration conditions are: refrigerated at 4°C for 24 h to 48 h.

7. A Pickering emulsion gel based on drug-excipient combination, prepared by the preparation method according to any one of claims 1-6.

8. The use of the Pickering emulsion gel based on drug-excipient combination according to claim 7 in the preparation of drugs for the prevention or treatment of insomnia.

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