Pickering emulsion gel based on medicine-adjuvant integration as well as preparation method and application of Pickering emulsion gel

Abstract

The invention discloses Pickering emulsion gel based on medicine-adjuvant integration and a preparation method and application thereof.The preparation method comprises the steps that ethylene glycol chitosan is added into a whey protein solution and stirred to be uniform, then glycyrrhizic aldehyde is added, stirring and mixing are conducted, and a whey protein-ethylene glycol chitosan-glycyrrhizic aldehyde composite solution is obtained; mixing the gastrodia elata volatile oil with clove oil, and performing ultrasonic homogenization to obtain an oil phase; adding the oil phase into a whey protein-ethylene glycol chitosan-glycyrrhizic aldehyde composite solution, carrying out high-speed shearing emulsification to obtain an emulsion, then refrigerating the emulsion, and inducing whey protein to denature and gelatinize to form the Pickering emulsion gel. Based on the idea of integration of medicines and auxiliary materials, the whey protein is used as a particle stabilizer, the main medicinal components of gastrodia elata volatile oil and clove oil are used as a mixed oil phase, the dosage of the auxiliary materials is reduced, and through screening of an emulsion gel aqueous phase, the prepared Pickering emulsion is compact in gel texture and high in stability, has a slow release effect on the medicinal components, and can be used for preparing the medicinal composition. The long-acting sleep improvement effect can be realized.

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 gel can fix emulsified oil droplets in the network structure, thereby playing a role in inhibiting oil droplet flocculation and coalescence. At the same time, the emulsion gel can also stabilize unstable bioactive substances. Compared with the emulsion or gel alone, the emulsion gel has the advantages of stable structure, adjustable viscoelasticity, etc. At present, the oil phase commonly used for preparing Pickering emulsion gel is soybean oil, sesame oil, rapeseed oil and peanut oil, etc., but there are problems such as soft texture, low crosslinking density, etc. of the prepared emulsion gel, which seriously restricts the application of soybean oil and other oil phases in emulsion gel. The water phase commonly uses natural chitosan, sodium alginate, etc., but there are disadvantages such as low solubility of chitosan in water, which needs to be added with dilute acid to increase its solubility, and the stability of the gel system is destroyed. At the same time, the commonly used emulsion gel matrix crosslinking mechanism is mainly electrostatic interaction crosslinking to form a gel network structure, which is greatly affected by electrostatic interaction, and has problems such as soft texture, low crosslinking density, easy degradation, etc. SUMMARY

[0006] To solve the above problems, the purpose of the present application is to provide a preparation method of Pickering emulsion gel based on drug-auxiliary integration, which uses the main effective component of Gastrodia elata volatile oil and clove oil as a mixed oil phase, reduces the amount of auxiliary materials, and through the screening of the water phase of the emulsion gel, the prepared Pickering emulsion gel has dense texture, high stability, and has a sustained-release effect on the effective component, which can achieve the effect of long-acting sleep improvement.

[0007] The present application is realized by the following technical solutions: A preparation method of Pickering emulsion gel based on drug-auxiliary integration, comprising the following steps: (1) Add ethylene glycol chitosan to the whey protein solution, stir uniformly, then add glycyrrhetin, stir and mix to obtain a whey protein-ethylene glycol chitosan-glycyrrhetin composite solution with different mass ratios; (2) Mix Gastrodia elata volatile oil and clove oil, and ultrasonically homogenize to obtain an oil phase; (3) Add the oil phase to the whey protein-ethylene glycol chitosan-glycyrrhetin composite solution, high-speed shear emulsify to obtain an emulsion, then refrigerate the emulsion, induce whey protein denaturation gelation, and form a Pickering emulsion gel.

[0008] The whey protein solution described in the present application can be prepared by the following method: a certain amount of whey protein powder is added to purified water under constant temperature conditions of 40-60℃, and heated and stirred with a magnetic stirrer at a speed of 300-400 r / min for 3-5h to fully dissolve and hydrate the whey protein, and then obtained.

[0009] Preferably, in step (1), the mass concentration of the whey protein solution is 10-20%.

[0010] The present application optimizes screening to take glycol chitosan (GC) and glycyrrhetin aldehyde (AGA) as the water phase of the Pickering emulsion gel. The Schiff base reaction between the amino group (-NH2) in the glycol chitosan (GC) molecule and the aldehyde group (-CHO) in the glycyrrhetin aldehyde (AGA) molecule can generate imine bond products, and the gel network structure prepared by chemical crosslinking has a more compact texture and higher stability.

[0011] Preferably, in step (1), the mass ratio of the glycol chitosan to the glycyrrhetin aldehyde is 1:2-2:1.

[0012] Preferably, in step (1), the mass ratio of the whey protein, the glycol chitosan and the glycyrrhetin aldehyde in the whey protein-glycol chitosan-glycyrrhetin aldehyde composite solution is 4:1:2-4:2:1.

[0013] Based on the concept of "drug and adjuvant in one", the present application takes the Gastrodia elata volatile oil with sedative and hypnotic effects and the clove oil with anti-inflammatory, analgesic, antiseptic and fragrance regulating effects as the mixed oil phase, replaces the traditional soybean oil, sesame oil, rapeseed oil and peanut oil without drug effects, reduces the amount of adjuvant, and uses ultrasonic homogenization to completely wrap the Gastrodia elata volatile oil with the clove oil, so that the irritability of the Gastrodia elata volatile oil can be reduced.

[0014] The Gastrodia elata volatile oil of the present application can be obtained by conventional ultrasonic-assisted steam distillation extraction.

[0015] Preferably, in step (2), the volume ratio of the Gastrodia elata volatile oil to the clove oil is 1:3-5.

[0016] Preferably, in step (3), the volume ratio of the oil phase to the whey protein-glycol chitosan-glycyrrhetin aldehyde composite solution is 1-2:8-9.

[0017] Preferably, in step (3), the speed of the high-speed shearing is 1000-1500 rpm, and the time is 3 min-5 min.

[0018] Preferably, in step (3), the refrigeration condition is 4℃ for 24-48 h.

[0019] The present application also provides a Pickering emulsion gel based on the drug and adjuvant in one, which is prepared by the above preparation method.

[0020] The present application also provides the application of the above Pickering emulsion gel based on the drug and adjuvant in one in the preparation of a medicine for preventing or treating insomnia.

[0021] Compared with the prior art, the present application has the following advantages: The present application is based on the concept of "drug and adjuvant in one", using whey protein as a particle stabilizer, and using the main effective components of Gastrodia elata volatile oil and clove oil as a mixed oil phase, thereby reducing the amount of adjuvant and improving the stability of Gastrodia elata volatile oil and clove oil. Meanwhile, an imine bond product generated by Schiff base reaction of glycol chitosan (GC) with glycyrrhetinyl (AGA) is used as an aqueous phase, and a Pickering emulsion gel is prepared by chemical crosslinking. The crosslinking network structure of the gel is more compact, the binding effect on emulsion droplets is stronger, the particle size is smaller and more stable, and the clove oil and Gastrodia elata volatile oil can be released slowly. Through pharmacodynamic verification, the Pickering emulsion gel prepared by the present application can achieve the effect of long-acting sleep improvement. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Appearance diagram of the whey protein-glycol chitosan-glycyrrhetinyl composite solution prepared in Example 1 (A) and appearance diagram of the whey protein-chitosan-glycyrrhizic acid composite solution prepared in Comparative Example 1 (B); Figure 2 Appearance diagram (A) and scanning electron microscope diagram (B) of the Pickering emulsion gel prepared in Comparative Example 1, and appearance diagram (C) of the Pickering emulsion gel after 7 days of storage; Figure 3 Appearance (A) and particle size (B) of the Pickering emulsion when the volume ratio of Gastrodia elata volatile oil to clove oil is 1:2; Figure 4 Appearance (A) and particle size (B) of the Pickering emulsion when the volume ratio of Gastrodia elata volatile oil to clove oil is 1:4, appearance (C) of the Pickering emulsion gel after 7 days of storage, and appearance (D) of the Pickering emulsion gel after 28 days of storage; Figure 5 Appearance of the Pickering emulsion when the volume ratio of Gastrodia elata volatile oil to clove oil is 1:6; Figure 6 Upright and inverted diagrams of the Pickering emulsion gels (PEG) prepared by different proportions of gel matrix; Figure 7 Optical microscope diagrams of the Pickering emulsion gels prepared by different proportions of gel matrix; Figure 8 Particle size distribution diagrams of the Pickering emulsion gels prepared by different proportions of gel matrix; Figure 9 Rheological properties (strain sweep curve; frequency sweep curve; time sweep curve; viscosity change with shear rate diagram) of the Pickering emulsion gels prepared by different proportions of gel matrix; Figure 10 H NMR) spectrum of glycyrrhetinic acid (AGA); 1 H NMR) spectrum of glycyrrhetinic acid (AGA); Figure 11 H NMR) spectrum of glycyrrhetinic acid (AGA); 1 H NMR) spectrum of glycyrrhetinic acid (AGA); Figure 12 H NMR) spectrum of glycyrrhetinic acid (AGA); 1 H NMR) spectrum of glycyrrhetinic acid (AGA); Figure 13 FTIR spectrum of whey protein (WPC), glycol chitosan (GC), glycyrrhetinic acid (AGA), Pickering emulsion gel (PEG); Figure 14 TGA curve, DTG curve and DSC curve of Pickering emulsion and Pickering emulsion gel; TGA curve, DTG curve and DSC curve of Pickering emulsion and Pickering emulsion gel; Figure 15 SEM image (A) and EDS-Mapping image (B) of Pickering emulsion gel; Figure 16 Effect of Pickering emulsion gel on the pathological state of hypothalamus of insomnia mice, and the hypothalamus section was stained with H&E (50X, 100X); Figure 17 Effect of Pickering emulsion gel on the pathological state of hippocampus of insomnia mice, and the hippocampus section was stained with H&E (50X); DETAILED DESCRIPTION

[0023] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments of the present application. The described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0024] Preparation of Gastrodia elata volatile oil:

[0025] Accurately weigh 50 g of Gastrodia elata powder into a 1000 mL round-bottom flask, and add 450 mL of distilled water. Then, ultrasonic for 45 min at 280 W. After the ultrasonic treatment, soak for 2 h. After the soaking is completed, move the round-bottom flask to a constant-temperature heating magnetic stirrer, and sequentially connect a volatile oil tester and a spherical condenser to form a complete distillation reflux device. Turn on the heating function, and raise the temperature of the system to a weak boiling state of the solution, and maintain the weak boiling condition for 8 h to ensure that the volatile oil components are fully distilled and separated. After the heating is completed, let the mixture stand for 1 h, and collect the Gastrodia elata volatile oil after the oil and water phases are fully separated.

[0026] Preparation of a whey protein solution:

[0027] A certain amount of whey protein powder is heated and stirred at 50°C under constant-temperature conditions with a magnetic stirrer at a rotation speed of 350 r / min for 4 h to fully dissolve and hydrate the whey protein in water, thereby obtaining a whey protein solution. By adjusting the amount of whey protein powder added, whey protein solutions with different mass concentrations are obtained.

[0028] Example 1: Preparation of a Pickering emulsion gel based on a combination of medicine and adjuvant (1) Add glycol chitosan to the whey protein solution, stir until uniform, and then add glycyrrhetin, stir and mix to obtain a whey protein-glycol chitosan-glycyrrhetin composite solution; wherein the mass concentration of the whey protein solution is 15%, and the mass ratio of whey protein, glycol chitosan, and glycyrrhetin in the whey protein-glycol chitosan-glycyrrhetin composite solution is 4:2:1; (2) Mix the Gastrodia elata volatile oil and clove oil, and ultrasonically homogenize to obtain an oil phase; the volume ratio of Gastrodia elata volatile oil to clove oil is 1:4; (3) Add the oil phase to the whey protein-glycol chitosan-glycyrrhetin composite solution (the volume ratio of the oil phase to the whey protein-glycol chitosan-glycyrrhetin composite solution is 2:8), and high-speed (1200 rpm) shear emulsification to obtain an emulsion, and then store the emulsion at 4°C for 48 h to induce whey protein denaturation and gelation, thereby forming a Pickering emulsion gel.

[0029] Comparative Example 1: Preparation of a Pickering emulsion gel using a conventional gel matrix (1) Add chitosan to the whey protein solution, stir until uniform, and then add glycyrrhizic acid, stir and mix to obtain a whey protein-chitosan-glycyrrhizic acid composite solution; wherein the mass concentration of the whey protein solution is 15%, and the mass ratio of whey protein, chitosan, and glycyrrhizic acid in the whey protein-chitosan-glycyrrhizic acid composite solution is 4:2:1; (2) Mix the Gastrodia elata volatile oil and clove oil, and ultrasonically homogenize to obtain an oil phase; the volume ratio of Gastrodia elata volatile oil to clove oil is 1:4; (3) The oil phase is added to the whey protein-chitosan-glycyrrhizic acid complex solution (the volume ratio of the oil phase to the whey protein-ethylene glycol chitosan-glycyrrhetinic acid complex solution is 2:8), and high-speed (1200 rpm) shear emulsification is performed to obtain an emulsion, and then the emulsion is refrigerated at 4°C for 48 h to induce whey protein denaturation and gelation, so as to form a Pickering emulsion gel.

[0030] The whey protein-ethylene glycol chitosan-glycyrrhetinic acid complex solution prepared in Comparative Example 1 is shown in FIG. Figure 1 A) and the whey protein-chitosan-glycyrrhizic acid complex solution prepared in Comparative Example 1 is shown in FIG. Figure 1 B), and the results show that the chitosan in the complex solution of Comparative Example 1 is not completely dissolved, and therefore, the ethylene glycol chitosan with better solubility is selected in the present application.

[0031] Figure 2 The appearance and electron microscope image of the Pickering emulsion gel prepared in Comparative Example 1 are shown in FIG. Figure 2 A), and the results show that the chitosan in the complex solution of Comparative Example 1 is not completely dissolved, and therefore, the ethylene glycol chitosan with better solubility is selected in the present application. Figure 2 B) is also the same case, and the formed gel is unstable and easy to degrade. Figure 2 C) shows the Pickering emulsion gel after being stored at room temperature for 7 days, and the texture is more soft, and a part of it has been degraded, and the stability is poor.

[0032] Example 2: Optimization of the volume ratio of Gastrodia elata volatile oil and clove oil

[0033] The effect of the mixing ratio of Gastrodia elata volatile oil and clove oil on the formation of Pickering emulsion gel is investigated, and the mass ratio of ethylene glycol chitosan to glycyrrhetinic acid is controlled to be 2:1, and the volume ratio of Gastrodia elata volatile oil to clove oil is investigated to be 1:2, 1:4 and 1:6.

[0034] When the volume ratio of Gastrodia elata volatile oil to clove oil is 1:2, Figure 3 A is the appearance of the Pickering emulsion, and it can be observed from the appearance that the Pickering emulsion can be formed, but Figure 3 B is the particle size of the Pickering emulsion, which is about 2300 μm, and it is speculated that the Pickering emulsion may be unstable and agglomerated.

[0035] When the volume ratio of Gastrodia elata volatile oil to clove oil is equal to 1:4, Figure 4 A is the appearance of the Pickering emulsion, and it can be observed from the appearance that the Pickering emulsion can be formed, Figure 4The particle size of the Pickering emulsion was about 700 μm, and the Pickering emulsion gel did not change obviously after being stored at room temperature for 7 days and 28 days, indicating that the Pickering emulsion gel was stable in property.

[0036] When the volume ratio of Gastrodia elata volatile oil to clove oil was 1:6, Figure 5 The appearance of the Pickering emulsion was layered, and the Pickering emulsion could not be formed.

[0037] Example 3: Screening of gel matrix ratio

[0038] The effect of the mass ratio of glycol chitosan to glycyrrhetin on the formation of the Pickering emulsion gel was investigated, the volume ratio of Gastrodia elata volatile oil to clove oil was controlled to be 1:4, and the mass ratio of glycol chitosan to glycyrrhetin was 1:1, 2:1 and 1:2, respectively.

[0039] Figure 6 The upright and inverted pictures of the Pickering emulsion gel (PEG) prepared by different ratios of gel matrix were as follows. Through appearance characterization observation, the Pickering emulsion gel (PEG) all presented uniform and milky white appearance, and the system had no visible oil phase floating layer, water phase sinking bottom or solid particle aggregation phenomenon, indicating that the oil phase (Gastrodia elata volatile oil) was highly dispersed in the water phase, the dispersion phase droplet size uniformity was good, and the macroscopic morphological characteristics of the stable O / W type emulsion were met. The inverted stability of the Pickering emulsion gel was verified, and the results showed that the sample had no obvious flow deformation, could stably maintain the initial forming state, and the gel as a whole had dense texture, smooth surface, and no local depression, cracking or component separation phenomenon.

[0040] Figure 7The optical microscope images of Pickering emulsion gels (PEG) prepared with different ratios of gel matrix. Microscopic observation can see more dispersed round droplets, the droplet size is relatively uniform, no obvious aggregation phenomenon shows that the dispersion of oil phase in the emulsion system in the water phase is good. PEG (1:1) field presents more dense granular structure, the droplets are wrapped by the gel matrix, the formed particles are more closely distributed, but a certain dispersion can still be observed, which shows that when the mass ratio of glycol chitosan to glycyrrhetin is 1:1, the gel matrix begins to crosslink and package the emulsion droplets. However, the density of the gel network still has room for improvement. The granular structure of PEG (1:2) is further dense, and part of the area appears a trend of particle aggregation, which may be due to the relatively high proportion of glycyrrhetin, which affects the growth of the gel network and the wrapping of the droplets during the crosslinking process, leading to local particle aggregation, reflecting that the micro-uniformity of the gel system at this ratio has decreased. PEG (2:1) presents a relatively uniform and dense network structure, and the droplets are well embedded in the gel network without obvious large particle aggregation.

[0041] From the particle size detection data of Figure 8 The particle size of Pickering emulsion gels (PEG) with different mass ratios of glycol chitosan to glycyrrhetin shows significant differences. When the mass ratio is 2:1, the average particle size is 722.7 nm; when the mass ratio is 1:1, the average particle size increases to 980.07 nm; when the mass ratio is 1:2, the average particle size further increases to 1108 nm. This shows that with the increase of the relative proportion of glycyrrhetin, the particle size of the gel system shows an increasing trend. Glycol chitosan reacts with aldehyde group of glycyrrhetin to form a network structure, when the proportion of glycyrrhetin is low (such as 2:1), the network structure formed by crosslinking reaction is more dense, and the binding effect on emulsion droplets is stronger, so the particle size is smaller; when the proportion of glycyrrhetin increases, the crosslinking degree or the uniformity of the network structure may change, leading to the aggregation of droplets or the increase of network pore, and the particle size rises. Comprehensive analysis, the relative content of glycol chitosan is higher under PEG (2:1), and the gel network formed by crosslinking is more conducive to the uniform dispersion and stable packaging of droplets, and the uniformity and density of the microstructure are better.

[0042] Figure 9Rheological properties of Pickering emulsion gels (PEG) prepared with different ratios of gel matrix (glycol chitosan: glycyrrhetinic acid = 1:1, 1:2, 2:1). In strain sweep, the elastic modulus (G') of all samples was greater than the viscous modulus (G''), which embodied the elastic dominant characteristics of solid-like gel, which was closely related to the network formed by particles at the interface in Pickering emulsion, where the G' of the "PEG 2:1" group was the highest, the G' of the "PEG 1:2" group was the lowest, and the critical strain of the "PEG 2:1" group was larger, indicating that the higher the ratio of glycol chitosan, the denser the gel network, the stronger the anti-deformation ability, while the increase of the proportion of glycyrrhetinic acid would make the network relatively loose. Frequency sweep showed that the growth trend of G', G" of different ratio samples was different, the G' of the "PEG 2:1" group grew more significantly with the increase of frequency, indicating that its network was more sensitive to frequency, and the elasticity was more prominent when the external force acted quickly. Time sweep showed that the G' of the "PEG 2:1" group was always higher and more stable, with good long-term stability, and the G' of the "PEG 1:2" group was easy to fluctuate or decrease with time. The viscosity-shear rate curve reflects the shear thinning behavior, the viscosity of the "PEG 2:1" group is higher and the degree of shear thinning is weaker, the network has strong shear resistance, and the viscosity of the "PEG 1:2" group is low and the thinning is more significant, the network is easy to be destroyed by shear.

[0043] The Pickering emulsion gel based on drug-assisted combination prepared in Example 1 was subjected to the following tests Test Example 1:

[0044] Figure 10 For glycyrrhetinic acid (AGA) 1 The H NMR spectrum showed that the characteristic resonance peak of hydrogen atoms on the aldehyde group (-CHO) appeared near 5.0 ppm, which was the characteristic proton signal of the aldehyde group in AGA molecules.

[0045] Figure 11 For glycol chitosan (GC) 1 The H NMR spectrum showed that the characteristic resonance peaks of sugar ring protons and hydroxyethyl substituent protons on the chitosan molecular chain were presented, which corresponded to the typical chemical structure of GC.

[0046] Figure 12 For Pickering emulsion gel (PEG 2:1) 1HNMR spectrum, compared with the spectrum of GC, AGA, a new proton resonance signal appears at 8.75 ppm, while the characteristic peak of aldehyde hydrogen in AGA spectrum disappears, and the proton peak related to amino group (-NH2) in GC molecule changes in chemical shift or integral intensity. This phenomenon is highly consistent with the reaction mechanism of Schiff base reaction, that is, the aldehyde group (-CHO) in the glycyrrhetinic acid (AGA) molecule reacts with the amino group (-NH2) in the glycol chitosan (GC) molecule to form an imine bond.

[0047] Figure 13 FTIR spectra of whey protein (WPC), glycol chitosan (GC), glycyrrhetinic acid (AGA), Pickering emulsion gel (PEG). The FTIR spectrum of whey protein (WPC) shows typical functional group vibration peaks of protein molecules in the C-O stretching vibration region of amide bond, reflecting the vibration information of intramolecular peptide bond and side chain group. The FTIR spectrum of glycol chitosan (GC) has characteristic vibration peaks of functional groups such as hydroxyl (-OH) and amino (-NH2) (such as O-H stretching vibration peak, N-H bending vibration peak, etc.), which is consistent with the functional group structure of GC as a polysaccharide molecule. The FTIR spectrum of glycyrrhetinic acid (AGA) shows a characteristic stretching vibration peak of aldehyde group (C=O) near 1724 cm -1 The FTIR spectrum of Pickering emulsion gel (PEG) not only contains partial characteristic peaks of WPC, GC, AGA (reflecting the existence of each component in the gel system), but also appears new characteristic peaks or shifts and intensity changes of original characteristic peaks. For example, near 1630-1650 cm -1 The vibration peak related to the imine bond (-N=CH) generated by the Schiff base reaction appears, and the intensity of the amino characteristic peak of GC and the aldehyde characteristic peak of AGA is weakened. This is consistent with the consumption process of amino and aldehyde groups in the Schiff base reaction, further confirming the cross-linking action between GC and AGA, and the process of WPC, GC, AGA forming Pickering emulsion gel through Schiff base reaction, hydrogen bond, hydrophobic interaction and other chemical and physical interactions in the gel network.

[0048] Test Example 2: Thermal stability analysis of Pickering emulsion gel

[0049] The thermal gravimetric curve of Pickering emulsion gel (PEG 2:1) sample is as follows Figure 14The weight loss started at 30℃ and the weight loss at 243℃ was 22.54%. There was a slight weight loss before 100℃, which was due to the water absorbed from the air after the lyophilized sample was removed. According to the DTG curve, the weight loss rate accelerated at about 243℃, which was probably due to the thermal decomposition of volatile oil. As shown in the TG curve and the DTG curve, more thermal decomposition started at about 350℃, which was due to the decomposition of whey protein and gel matrix in the Pickering emulsion gel. When the temperature was increased from 350℃ to 500℃, the weight loss was 58.22% and the maximum weight loss rate temperature was 389℃.

[0050] Differential scanning calorimetry (DSC) analysis is a widely used technique for characterizing inclusion complexes, which can provide detailed information on the thermal behavior of each component and the inclusion complex formed. Figure 14 The thermal behavior of the Pickering emulsion (PE) was demonstrated. The DSC thermogram of the PE had one obvious endothermic peak, with a range of 30℃ to about 336℃ and a peak top position temperature of 104℃, which represented the melting point Tm value of the sample as 104℃. Figure 14 The thermal behavior of the Pickering emulsion gel (PEG 2:1) was demonstrated. The DSC thermogram of the PEG (2:1) had two obvious endothermic peaks, with the first endothermic peak having a range of 50℃ to about 300℃ and a peak top position melting point Tm value of 88℃, and the second endothermic peak having a range of 385℃ to 389℃ and a peak top position melting point Tm value of 387℃.

[0051] The DSC curve also had corresponding absorption peaks in the weight loss stage of the TG curve. At about 88℃, a broad and slow endothermic peak appeared on the DSC curve of the PEG (2:1), which was the evaporation stage of water and corresponded to its TG curve. In the range of 385~389℃, a narrow endothermic peak appeared, which corresponded to the TG curve and was speculated to be a decomposition reaction of a small amount of substance in this stage. According to the peak shape, it can be seen that the content of this substance in the PEG (2:1) is small. It is worth noting that by comparing the DSC thermogram data of PE and PEG (2:1), it was found that PEG had one more endothermic peak than PE, which proved that the addition of gel matrix to the Pickering emulsion had a significant impact on its thermal stability, and the sample after adding the gel matrix had good stability.

[0052] Test Example 3: Microstructure of Pickering Emulsion Gel

[0053] Figure 15Figure 6A is a SEM image of the Pickering emulsion gel (PEG 2:1) of Gastrodia elata volatile oil, and Figure 6B is an EDS-Mapping image of the Pickering emulsion gel (PEG 2:1) of Gastrodia elata volatile oil. As can be observed from the SEM images (a1-a4) of different magnifications, the Pickering emulsion gel (PEG 2:1) of Gastrodia elata volatile oil exhibits a morphology characterized by a porous structure. This porous structure provides a spatial basis for the loading and subsequent release of Gastrodia elata volatile oil, and also reflects the construction of the gel system in the microstructure. Figure 15 Figure 6A is a SEM image of the Pickering emulsion gel (PEG 2:1) of Gastrodia elata volatile oil, and Figure 6B is an EDS-Mapping image of the Pickering emulsion gel (PEG 2:1) of Gastrodia elata volatile oil. As can be observed from the SEM images (a1-a4) of different magnifications, the Pickering emulsion gel (PEG 2:1) of Gastrodia elata volatile oil exhibits a morphology characterized by a porous structure. This porous structure provides a spatial basis for the loading and subsequent release of Gastrodia elata volatile oil, and also reflects the construction of the gel system in the microstructure.

[0054] Test Example 4: Pharmacodynamic results of the Pickering emulsion gel in the treatment of insomnia rats

[0055] The present application studies the therapeutic effect of the Pickering emulsion gel on insomnia rats by establishing an insomnia rat model.

[0056] (1) Construction of insomnia model rats and experimental grouping: The C57 / BL mice were divided into 6 groups, 10 mice in each group: A normal control group (only 0.9% normal saline was given), B model group (p-chlorophenylalanine (PCPA) group) (the drug concentration was 400 mg / kg, the drug dose was 10 mL / kg / d), C positive drug group (the model was established at the same time, and the positive drug diazepam was given, the drug concentration was 1.3 mg / kg), D low-dose PEG group (the model was established at the same time, and the drug was given, the drug concentration was 5 mg / kg), Z medium-dose PEG group (the model was established at the same time, and the drug was given, the drug concentration was 10 mg / kg), and G high-dose PEG group (the model was established at the same time, and the drug was given, the drug concentration was 15 mg / kg). Except for the normal control group, the mice in the other groups were injected intraperitoneally with PCPA once a day for 4 consecutive days. At the same time, the control group was given an equal amount of normal saline. The circadian rhythm and sleep duration of the rats within 28-30 h after p-chlorophenylalanine (PCPA) induction changed significantly compared with the control group without PCPA injection, indicating that the insomnia model was successfully induced.

[0057] (2) Drug administration and sample processing: The mice were administered on the 4th day of modeling, and the administration was continued for 5 consecutive days.

[0058] (3) Righting reflex test: After the end of administration on the 5th day, the mice were injected intraperitoneally with chloral hydrate. The time interval from chloral hydrate administration to the time the mice could not turn over for 60 seconds was recorded as sleep latency, and the sleep duration was also recorded. The sleep latency and sleep duration of the 6 groups of mice were compared, and the results are shown in Table 1.

[0059] Table 1 Sleep latency and sleep duration of different groups of mice

[0060] As shown by the results in Table 1, the sleep latency of the mice in each model group was longer than that of the normal group, and the sleep duration was shorter than that of the blank group except for the D low-dose PEG group. The sleep latency of the mice in the positive drug group, the low-dose, medium-dose and high-dose PEG groups was shorter than that of the mice in the B model group, and the sleep duration was longer than that of the mice in the B model group except for the E medium-dose PEG group. Through the righting reflex experiment, it can be further proved that the insomnia model of mice is successfully established, and through data comparison, it is shown that the sleep duration of the mice in the E medium-dose PEG group and the F high-dose PEG group is greater than that of the mice in the C positive drug group.

[0061] (4) Brain histopathology examination: HE staining: The ulcerated oral mucosa tissue was routinely paraffin-embedded, sectioned, dewaxed, washed with water, stained with hematoxylin, washed with water, differentiated, returned to blue, immersed in water, and placed in an eosin solution. Routine dehydration, transparency, and mounting were performed. The changes in the mucosa tissue and the inflammatory cell infiltration in the epithelial connective tissue were observed under an optical microscope.

[0062] Figure 16 The results of H&E staining of the hypothalamus of the insomnia mice for the Pickering emulsion gel (PEG) are shown in the figure. A: The hypothalamus of the normal control mice has abundant nerve cells, uniform distribution, complete cell structure, and orderly arrangement. The cell nucleus is round or oval and clear. Compared with the normal control group, the hypothalamus of the mice in the B model group has fewer nerve cells, deformed structure, sparse arrangement, vacuoles, and uneven cell nucleus size. Compared with the model group, the C positive drug group and the D low-dose PEG group, the E medium-dose PEG group, and the F high-dose PEG group significantly improved the above pathological damage, increased the number of hypothalamic nerve cells, reduced vacuoles, and had normal morphology.

[0063] The results of H&E staining show that the hypothalamic neurons of the sleep-deprived model animals have obvious structural damage, including neuron atrophy, nuclear condensation, enhanced cytoplasmic acidophilia, and vacuolar degeneration. The use of diazepam as a positive drug can significantly improve the damage, verifying the reliability of the model and the effectiveness of the detection method.

[0064] Compared with the normal group, after intervention of the Pickering emulsion gel, each dose group showed different degrees of neuroprotective effect, and the middle and high dose groups showed significant improvement effect.

[0065] The results show that the Pickering emulsion gel can significantly reduce the hypothalamic neuron damage caused by sleep deprivation, and has good neuroprotective and antioxidant and anti-inflammatory effects.

[0066] Figure 17 For H&E staining of the hippocampus of the insomnia mice by the Pickering emulsion gel (PEG), it can be seen from the upper figure that the hippocampal neurons of the normal group mice are complete, arranged in order, evenly distributed, and the cell nucleus is clear, and there is no obvious pathological damage. But the hippocampal tissue of the model group (PCPA group) is arranged in disorder, and even the normal morphology of the neurons disappears, the color deepens, the cells atrophy, and a large number of necrotic neurons can be seen. Compared with the model group, the hippocampal neurons of the positive drug group and the D low-dose PEG group, the E middle-dose PEG group and the F high-dose PEG group can be obviously improved. The hippocampal neurons of the positive drug group and the D low-dose PEG group are slightly relieved compared with the PCPA group, but the morphology of the neurons is still changed, the color is deeper, and there is still damage. The H&E staining results show that the Pickering emulsion gel has a certain repair effect on the hippocampal neuron damage.

[0067] The H&E staining results show that sleep deprivation causes obvious structural damage to the hippocampal neurons of the mice, and a large number of neuron cell bodies atrophy, appear vacuolar degeneration and tissue space expansion, and the Pickering emulsion gel has a significant dose-dependent neuroprotective effect. The middle and high dose groups can effectively improve the morphology of neurons, reduce cell degeneration and necrosis, and restore the integrity of the hippocampal structure, and the protection effect is comparable to that of melatonin, which suggests that the Pickering emulsion gel of the application can relieve the nerve damage caused by sleep deprivation through antioxidant, anti-inflammatory and energy metabolism pathway regulation, and has potential neuroprotective application value.

[0068] In summary, after intervention of the Pickering emulsion gel of the application, each dose group showed different degrees of neuroprotective effect. Among them, the middle and high dose groups can effectively improve the morphology of neurons, restore the integrity of the tissue structure, reduce cell degeneration and necrosis, and the tissue morphology is close to that of the normal group. The Pickering emulsion gel of the application (10%) and high dose (15%) volatile oil can play a neuroprotective role, and has potential application value in preventing and treating sleep disorders and nerve function damage.

Claims

1. A method for the preparation of a Pickering emulsion gel based on drug-in-adjuvant-one, characterized in that, The method comprises the following steps: (1) adding glycol chitosan into a whey protein solution, stirring uniformly, and then adding glycyrrhetin, stirring and mixing to obtain a whey protein-glycol chitosan-glycyrrhetin complex solution; (2) mixing Gastrodia elata volatile oil and clove oil, and ultrasonically homogenizing to obtain an oil phase; (3) adding the oil phase into the whey protein-glycol chitosan-glycyrrhetin complex solution, high-speed shearing emulsification to obtain an emulsion, and then cold storage of the emulsion to induce whey protein denaturation gelation, thereby forming a Pickering emulsion gel.

2. The method of preparing a co-processed Pickering emulsion gel according to claim 1, characterized in that: In step (1), the mass concentration of the whey protein solution is 10-20%.

3. The method of preparing a co-processed Pickering emulsion gel based on drug according to claim 1, characterized in that: In step (1), the mass ratio of the glycol chitosan to the glycyrrhetin is 1:2-2:

1.

4. The method of preparing a co-processed Pickering emulsion gel according to claim 1, characterized in that: In step (1), the mass ratio of the whey protein, the glycol chitosan and the glycyrrhetin in the whey protein-glycol chitosan-glycyrrhetin complex solution is 4:1:2-4:2:

1.

5. The method of preparing a co-processed Pickering emulsion gel according to claim 1, characterized in that: In step (2), the volume ratio of the Gastrodia elata volatile oil to the clove oil is 1:3-5.

6. The method of preparing a co-processed Pickering emulsion gel of claim 1, wherein: In step (3), the volume ratio of the oil phase to the whey protein-glycol chitosan-glycyrrhetin complex solution is 1-2:8-9.

7. The method of preparing a co-processed Pickering emulsion gel according to claim 1, characterized in that: In step (3), the speed of the high-speed shearing is 1000-1500 rpm, and the time is 3 min-5 min.

8. The method of preparing a co-processed Pickering emulsion gel of claim 1, wherein: In step (3), the cold storage condition is 4℃ for 24 h-48 h.

9. A Pickering emulsion gel based on medicine and adjuvant in one, which is prepared by the preparation method of any one of claims 1-8.

10. Application of the Pickering emulsion gel based on medicine and adjuvant in one of claim 9 in preparation of a medicine for preventing or treating insomnia.

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