A nanoemulsion and a compound nanoemulsion containing active ingredients of traditional Chinese medicine and a preparation method thereof

Abstract

The application discloses a nanoemulsion and a composite nanoemulsion containing traditional Chinese medicine active ingredients and a preparation method of the nanoemulsion and the composite nanoemulsion, wherein the traditional Chinese medicine active ingredients are composed of berberine, ferulic acid and angelica polysaccharide; the composite nanoemulsion further contains allergen protein on the basis of the nanoemulsion containing traditional Chinese medicine active ingredients; and the oil phase and the emulsifier are isopropyl myristate, Span 80 and Tween 80. The application has the innovative advantages that the allergen and the multi-component traditional Chinese medicine active ingredients are loaded in a W / O / W nano delivery system, and a new intervention strategy for simultaneously improving the safety and effectiveness of OIT treatment is constructed by relying on the wrapping protection effect of the nano carrier and combining the synergistic pharmacological advantages of the anti-inflammatory and immune regulation of the traditional Chinese medicine ingredients, so that the risk of adverse reactions of OIT can be effectively reduced, the immune therapy response rate can be improved, and the remission phenomenon after treatment can be effectively improved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to nanoemulsions and composite nanoemulsions containing active ingredients of traditional Chinese medicine and their preparation methods, specifically to a nanoemulsion and composite nanoemulsion containing berberine, its preparation method and uses. Background Technology

[0002] Food allergy and atopic dermatitis are both common atopic diseases in clinical practice, belonging to the systemic atopic disease spectrum. The pathological mechanisms of these two diseases are highly homologous, both characterized by Th2-type immune hyperactivity, epithelial and mucosal barrier damage, and persistent activation of the inflammatory cascade. They often occur sequentially or concurrently, forming a typical atopic process that severely impacts patients' quality of life. In recent years, with changes in the environment and lifestyle, the incidence of allergic diseases has been increasing year by year, exhibiting characteristics of younger onset, chronicity, and recurrent attacks, becoming a prominent public health problem. Oral immunotolerance therapy (OIT) is currently the only core approach that can achieve etiological treatment of food allergy and gradually rebuild the body's allergen immune tolerance. It can improve systemic atopic immune dysregulation and has potential curative value for food allergy, atopic dermatitis, and allergic rhinitis, showing significant long-term clinical advantages compared to traditional symptomatic treatment.

[0003] Food allergies are primarily pathologically based on intestinal mucosal immune dysregulation, including both IgE-mediated and non-IgE-mediated types. Once food allergens enter the body, they disrupt intestinal immune homeostasis, inducing mucosal congestion and edema, inflammatory cell infiltration, and promoting the massive release of inflammatory mediators such as histamine and interleukins, leading to gastrointestinal symptoms such as abdominal pain, diarrhea, and vomiting. In severe cases, they can induce systemic anaphylactic shock. Currently, clinical intervention mainly relies on allergen avoidance, antihistamines, and glucocorticoids for symptomatic relief, which can only temporarily alleviate symptoms and cannot correct the abnormal immune state or repair the intestinal barrier, resulting in a high recurrence rate. OIT (Oral Intolerance Treatment), through continuous low-dose allergen stimulation, can gradually induce immune tolerance and is currently the only treatment that can cure food allergies. However, traditional OIT has significant drawbacks: direct contact of allergens with the mucosa easily induces local and systemic allergic adverse reactions, resulting in poor treatment safety and patient tolerability; furthermore, OIT alone has a slow onset of action and a long treatment cycle, with limited improvement on concurrent skin and respiratory allergic inflammations, greatly limiting its clinical application.

[0004] Atopic dermatitis is a chronic, relapsing inflammatory skin disease, with core pathological features including skin barrier deficiency and abnormal keratinocyte function. The damaged skin barrier cannot effectively resist external stimuli and allergens, continuously activating the Th2 inflammatory response, resulting in skin erythema, dryness, desquamation, and persistent itching. As the initial disease in the atopic process, atopic dermatitis can significantly increase the risk of food allergies and allergic rhinitis. Current treatments can only locally relieve inflammation and cannot regulate systemic immune disorders; long-term topical medications can easily lead to adverse reactions such as skin atrophy, pigmentation, and drug resistance, and relapse is common after discontinuation. While topical infiltration therapy (OIT) can improve allergic constitution to some extent, it is difficult to directly target skin lesions and cannot quickly repair the skin barrier or control local inflammation, thus its therapeutic effect on atopic dermatitis is limited. Summary of the Invention

[0005] The purpose of this invention is to provide a nanoemulsion containing active ingredients of traditional Chinese medicine and a composite nanoemulsion, as well as their preparation methods. Specifically, it relates to a nanoemulsion containing active ingredients of traditional Chinese medicine, its preparation method, and its application in combination with oral oxygen therapy (OIT) in the treatment of food allergies and atopic dermatitis. This invention solves the problems of easy degradation of allergen proteins in vivo, numerous adverse reactions during OIT treatment, and significant resensitization effects. This patent utilizes the "two-membrane, three-phase" structure of W / O / W nanoemulsions to load proteins and hydrophilic and hydrophobic drugs into different phases, achieving co-delivery of drugs and peanut protein allergens. By employing a strategy of combining anti-inflammatory drugs with OIT, the synergistic effect of maximizing bioavailability is amplified, aiming to treat food allergies and atopic dermatitis through both inhibiting the inflammatory response caused by allergies and inducing active tolerance.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A nanoemulsion containing active ingredients of traditional Chinese medicine is prepared from the following active ingredients of traditional Chinese medicine, an oil phase and an emulsifier in parts by weight: the active ingredients of traditional Chinese medicine are 1.5-2.5 parts of berberine, 0.8-2 parts of ferulic acid and 0.6-2 parts of Angelica polysaccharide; the oil phase and emulsifier are 174 parts of isopropyl myristate, 10-20 parts of Span 80 and 30-50 parts of Tween 80.

[0007] The nanoemulsion containing active ingredients of traditional Chinese medicine is prepared from the following parts by weight of active ingredients of traditional Chinese medicine, oil phase and emulsifier: the active ingredients of traditional Chinese medicine are 2.4 parts of berberine, 1.9 parts of ferulic acid and 1.5 parts of Angelica polysaccharide; the oil phase and emulsifier are 174 parts of isopropyl myristate, 10 parts of Span 80 and 38 parts of Tween 80.

[0008] The nanoemulsion containing the active ingredients of traditional Chinese medicine is prepared by the following steps: Step 1: Preparation of W1 / O colostrum: Dissolve the prescribed amount of Angelica polysaccharide in an appropriate amount of ultrapure water to obtain the inner aqueous phase W1; then dissolve berberine and ferulic acid in isopropyl myristate, add the lipophilic emulsifier Span 80, and mix with magnetic stirring to obtain the oil phase O; under high-speed stirring, slowly inject the inner aqueous phase W1 into the oil phase O, mix thoroughly, and then use an ultrasonic cell disruptor to perform ultrasonic emulsification with the set parameters to obtain the W1 / O type colostrum; Step 2: Preparation of W1 / O / W2 colostrum: Add the prescribed amount of hydrophilic emulsifier Tween 80 to an appropriate amount of ultrapure water and stir to mix well to obtain the external aqueous phase W2. Add the colostrum obtained in Step 1 dropwise to the external aqueous phase W2 while stirring. After mixing thoroughly, use an ultrasonic cell disruptor with the set parameters to emulsify, and finally form W1 / O / W2 nanoemulsion.

[0009] A composite nanoemulsion containing active ingredients of traditional Chinese medicine and allergen protein is prepared from the above-mentioned nanoemulsion containing active ingredients of traditional Chinese medicine and allergen protein, using the following parts by weight of raw materials, oil phase and emulsifier: the raw materials are 0.3-30 parts of allergen protein, 1.5-2.5 parts of berberine, 0.8-2 parts of ferulic acid and 0.6-2 parts of Angelica polysaccharide; the oil phase and emulsifier are 174 parts of isopropyl myristate, 10-20 parts of Span 80 and 30-50 parts of Tween 80.

[0010] The composite nanoemulsion containing active ingredients of traditional Chinese medicine and allergen protein is prepared from the following raw materials, oil phase and emulsifier in parts by weight: the raw materials are 0.3-30 parts of allergen protein, 2.4 parts of berberine, 0.8 parts of ferulic acid and 0.6 parts of Angelica polysaccharide; the oil phase and emulsifier are 174 parts of isopropyl myristate, 10 parts of Span 80 and 38 parts of Tween 80.

[0011] The composite nanoemulsion containing active ingredients of traditional Chinese medicine and allergen proteins is prepared by the following steps: Step 1: Preparation of W1 / O colostrum: Dissolve the prescribed amount of allergen protein in an appropriate amount of ultrapure water to obtain the inner aqueous phase W1; then dissolve berberine and ferulic acid in isopropyl myristate, add the lipophilic emulsifier Span 80, and mix with magnetic stirring to obtain the oil phase O; under high-speed stirring, slowly inject the inner aqueous phase W1 into the oil phase O, mix thoroughly, and then use an ultrasonic cell disruptor to perform ultrasonic emulsification with the set parameters to obtain the W1 / O colostrum; Step 2: Preparation of W1 / O / W2 colostrum: Add the prescribed amount of Angelica polysaccharide and hydrophilic emulsifier Tween 80 to ultrapure water and stir to obtain the external aqueous phase W2. Add the colostrum obtained in Step 1 dropwise to the external aqueous phase W2 while stirring. After thorough mixing, use an ultrasonic cell disruptor with the set parameters to emulsify, and finally form W1 / O / W2 nanoemulsion.

[0012] In the preparation method of the composite nanoemulsion, the ultrasonic time and power in step 1 are 2-10 minutes and 100W-300W, respectively; and in step 2 of the preparation method, the ultrasonic time and power are 4-8 minutes and 150W-300W, respectively.

[0013] The application of the nanoemulsion containing active ingredients of traditional Chinese medicine in the preparation of drugs for treating food allergies or atopic dermatitis.

[0014] The application of the composite nanoemulsion containing active ingredients of traditional Chinese medicine and allergen proteins in the preparation of drugs for treating food allergies or atopic dermatitis.

[0015] The application of the nanoemulsion containing active ingredients of traditional Chinese medicine in the preparation of drugs that enhance the efficacy of allergen proteins.

[0016] The composite nanoemulsion contains ovalbumin and / or peanut protein as the allergen protein.

[0017] Positive and beneficial effects: W / O / W dual nanoemulsions possess a two-membrane, three-phase structure, capable of co-loading water-soluble allergens and water-soluble and lipid-soluble active ingredients of traditional Chinese medicine. They offer advantages such as isolating and protecting the loaded substances, long-lasting sustained release, good biocompatibility, and strong mucosal penetration. Traditional oral irritation therapy (OIT) often triggers severe gastrointestinal allergic reactions due to allergens easily irritating the intestinal mucosa, and suffers from low response rates and easy relapse after discontinuation. This invention encapsulates allergen proteins and three traditional Chinese medicine components simultaneously in a W / O / W composite nanoemulsion: relying on the carrier to isolate allergens from mucosal contact, reducing adverse reactions to OIT; leveraging the multi-target regulation of traditional Chinese medicine to improve desensitization efficacy and prevent resensitization; and utilizing the nanocarrier to enhance drug absorption and prolong the duration of action in vivo, effectively solving the problems of high side effects and low response rates in OIT treatment.

[0018] The system co-loads berberine, ferulic acid, and angelica polysaccharide, which work synergistically through multiple pathways: berberine inhibits allergic inflammation and reduces tissue inflammatory infiltration; ferulic acid regulates gut microbiota and improves the gut immune microenvironment; and angelica polysaccharide balances Th1 / Th2 responses and corrects typical Th2 immune shifts in food allergies. These three components intervene in the allergic microenvironment from multiple dimensions, including anti-inflammation, barrier repair, microbiota regulation, and immune remodeling. They can synergistically enhance the effects of oral immunotolerance therapy (OIT), accelerating the formation of immune tolerance and reducing toxicity.

[0019] The preparation process of this invention is simple, stable, and highly reproducible. The resulting nanoemulsions have uniform particle size, high encapsulation efficiency, and excellent physicochemical properties and biological safety, making them suitable for industrial production. They also have good prospects for clinical translation in the long-term synergistic treatment of atopic dermatitis and food allergies. Attached Figure Description

[0020] Figure 1This is a scanning electron microscope image of the W / O / W type nanoemulsion No. 1 in Example 1 of the present invention; Figure 2 This refers to the type of W / O / W type No. 1 nanoemulsion in Example 1 of the present invention; Figure 3 The in vitro release curves of four active ingredients in nanoemulsion No. 1 in Example 1 of this invention are shown: (A) berberine; (B) ferulic acid; (C) angelica polysaccharide; and (D) peanut protein. Figure 4 The following are the drug concentration-time curves of three active ingredients in nanoemulsion No. 3 in Example 3 of this invention: (A) berberine; (B) ferulic acid; (C) Angelica polysaccharide. Figure 5 The effects of the composite nanoemulsion in this invention on peanut allergy are shown in the following images: (A) stool condition; (B) small intestine pathological section; (C) spleen pathological section. (Note: OIT group used nanoemulsion No. 2, NE group used nanoemulsion No. 3, and ON group used nanoemulsion No. 4.) Figure 6 This invention demonstrates the regulatory effect of the composite nanoemulsion on peanut-specific IgE (A), interleukin-5 (B), and interleukin-10 (C) inflammatory factors in peanut-allergic mice. (Note: Nanoemulsion No. 2 was used in the OIT group, nanoemulsion No. 3 in the NE group, and nanoemulsion No. 4 in the ON group.) Detailed Implementation

[0021] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any way. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention. The present invention will be further described below with reference to examples: In the following examples, berberine was purchased from Shanghai Maclean Biochemical Co., Ltd., batch number B832574; ferulic acid was purchased from Chengdu Ruifensi Biotechnology Co., Ltd., batch number A-023; and Angelica polysaccharide was purchased from Shanghai Yuanye Biotechnology Co., Ltd., batch number JS240753. Peanut protein was self-extracted, as described in Example 5.

[0022] Example 1: Process optimization of W / O / W composite nanoemulsion (1) Single-factor screening of preparation process The optimal ratio of aqueous phase W1 to oil phase O in colostrum: Using 10% peanut protein aqueous solution as aqueous phase W1 and oil phase O (IPM (isopropyl myristate): Span80 = 96:4), aqueous phase W1 and oil phase were mixed in mass ratios of 1:9, 2:8, 3:7, 4:6, and 5:5, respectively. Based on the nanoemulsion droplet size, polydispersity index, and whether the nanoemulsion was stratified, the optimal mass ratio of aqueous phase W1 to oil phase O was selected as 3:7.

[0023] The optimal ratio of colostrum to external aqueous phase W2 in the preparation of double emulsion: using a 2% (w / w) Angelica polysaccharide aqueous solution as the external aqueous phase, the colostrum and external aqueous phase were mixed in a mass ratio of 1:9, 2:8, 3:7, 4:6, and 5:5. The optimal ratio of colostrum to external aqueous phase was 3:7.

[0024] Optimal dosage of the lipophilic emulsifier Span80: The mass ratio of Span80 in the primary emulsion was set as follows: 2:98, 4:96, 6:94, 8:92, and 10:90. Nanoemulsions were prepared according to the above optimal mass ratios. The optimal mass ratio of the lipophilic emulsifier Span80 to the primary emulsion was determined to be 4:96.

[0025] Optimal dosage of hydrophilic emulsifier Tween80: Nanoemulsions were prepared according to the optimal process with mass ratios of hydrophilic emulsifier to the external aqueous phase W2 set at 2:98, 4:96, 6:94, 8:92, and 10:90. The optimal mass ratio of hydrophilic emulsifier to the external aqueous phase was determined to be approximately 6:94.

[0026] Optimal ultrasonic power and time in the preparation of colostrum and re-emulsion: During colostrum preparation, ultrasonic power was set to 100, 150, 200, 250, and 300 W to determine the optimal ultrasonic power for preparing the nanoemulsion colostrum. After determining the optimal power of 200 W, ultrasonic times were set to 2, 4, 6, 8, and 10 min, and the optimal ultrasonic time for preparing the nanoemulsion colostrum was determined to be 8 min. During re-emulsion preparation, ultrasonic power was set to 100, 150, 200, 250, and 300 W, and the optimal ultrasonic power for re-emulsion preparation was determined to be 250 W. After determining the optimal power, ultrasonic times were set to 2, 4, 6, 8, and 10 min, and the optimal ultrasonic time for preparing the nanoemulsion colostrum was determined to be 8 min.

[0027] (2) Response surface optimization of the preparation process

[0028] Based on the results of the single-factor screening, ANOVA one-way variance analysis was used to analyze the significance of single factors on particle size and PDI size. The factors with the highest significance were selected: W1:O mass ratio, W1 / O:W mass ratio and Tween 80 dosage (see Table 1). Then, a Box-Behnken response surface methodology experiment was conducted (each factor and level is shown in Table 2) to further optimize the preparation process.

[0029] Table 1. ANOVA One-Way Analysis of Variance: , Table 2. Three-factor, three-level table for response surface design. , Results of optimal processing conditions and physicochemical properties of composite nanoemulsion No. 2.0 (1) W1 / O nano-collagen process Using IPM as the oil phase and Span-80 as the emulsifier, 2.028 g of IPM, 0.118 g of Span-80, 28 mg of berberine, and 22 mg of ferulic acid were weighed and placed in a vial. The mixture was stirred magnetically in a 25°C water bath to obtain the oil phase. Separately, 15 mg of peanut protein was completely dissolved in 0.90 mL of ultrapure water to form the inner aqueous phase, which was then slowly added to the oil phase while stirring. After thorough stirring, emulsification was performed in an ultrasonic cell disruptor with an ultrasonic power of 200 W and an ultrasonic time of 8 min to form the primary emulsion.

[0030] (2) W1 / O / W2 nano-emulsion process 18 mg of Angelica sinensis polysaccharide was completely dissolved in 7 mL of ultrapure water as the external aqueous phase. 0.45 g of Tween 80 was added and mixed thoroughly. The pre-emulsion prepared in the previous step was slowly added to the external aqueous phase in a 25°C water bath while stirring continuously. After thorough stirring, emulsification was performed using an ultrasonic cell disruptor with an ultrasonic power of 250 W and an ultrasonic time of 8 min, forming a W1 / O / W2 nanoemulsion, named Nanoemulsion No. 1. This nanoemulsion was a pale yellow, homogeneous milky liquid, without visible layering, oil separation, precipitation, or flocculation. The bottle walls were clean with no oil droplets adhering to them, exhibiting the Tyndall effect.

[0031] (3) Physicochemical properties of W / O / W composite nanoemulsions Freshly prepared W / O / W composite nanoemulsions under the above-described process conditions were diluted with distilled water at a ratio of 1:10. With a scattering angle of 173°, the average droplet size, measured at 25°C, was 178.85 ± 2.81 nm, exhibiting a unimodal normal distribution. The average polydispersity index (PDI) was 0.14 ± 0.03, and the average zeta potential was -28.13 ± 1.31 mV. The morphology of the nanoemulsions was observed using transmission electron microscopy. Figure 1The droplet size was basically consistent with the droplet size measured by the nanoparticle size analyzer. The nanoemulsion had a clear outline and distinct boundaries. There was no mutual adhesion between the droplets, indicating good dispersibility and relatively uniform distribution.

[0032] The type of nanoemulsion was determined using a staining method: When the oil-soluble dye Sudan Red was added to the nanoemulsion, it did not diffuse significantly and quickly settled to the bottom of the vial. In contrast, the water-soluble dye methylene blue diffused rapidly within the nanoemulsion and integrated with it, indicating that the nanoemulsion was a W / O / W nanoemulsion. Figure 2 ).

[0033] Stability of nanoemulsions: The homogeneity of the nanoemulsions was observed over 15 days at room temperature, with particle size and PDI measured every 3 days. The results showed that the nanoemulsions exhibited good stability at room temperature, with no significant changes in particle size or PDI. The emulsions were homogeneous and showed no stratification. When the nanoemulsions were centrifuged at 12,000 rpm for 10 minutes, they remained in a homogeneous emulsion state, without stratification or oil separation.

[0034] 3. Drug loading capacity and in vitro release performance of W / O / W composite nanoemulsions (1) Encapsulation efficiency and drug loading of No. 1 W / O / W composite nanoemulsion The contents of berberine and ferulic acid in nanoemulsion were quantitatively determined by high performance liquid chromatography (HPLC). The fluorescence intensity of FITC-labeled Angelica polysaccharide and peanut protein was determined by fluorescence spectrophotometry, and the contents of the two macromolecular components were then quantitatively calculated.

[0035] Encapsulation efficiency determination method: The nanoemulsion was centrifuged at 5000 rpm for 15 min, and the drug content in the filtrate after filtration was measured; the encapsulation efficiency was calculated based on the total amount of drug added. The results showed that the nanoemulsion system had good encapsulation capacity for all four active substances, with berberine encapsulation efficiency of (84.90 ± 2.83)%, ferulic acid of (82.51 ± 2.81)%, peanut protein of (81.32 ± 2.30)%, and angelica polysaccharide of (79.57 ± 3.20)%.

[0036] Drug loading determination method: Accurately weigh the nanoemulsion sample, add methanol solution, and ultrasonically break up the emulsion to release all the encapsulated active ingredients. The content of each drug in the system is then determined using the corresponding detection methods described above. The drug loading calculation formula is as follows: Drug loading = (Total drug in nanoemulsion / Total mass of nanoemulsion) × 100%; The drug loadings of berberine, ferulic acid, angelica polysaccharide, and peanut protein in the nanoemulsion are 2.37 ± 0.26 mg / g, 1.791 ± 0.21 mg / g, 1.26 ± 0.033 mg / g, and 0.975 ± 0.11 mg / g, respectively.

[0037] (2) In vitro release of No. 1 composite nanoemulsion in simulated gastric and intestinal fluids The composite nanoemulsion was sealed in a dialysis bag and placed in a shaker at 37°C for in vitro release testing. Samples were taken at 0, 1, 2, 4, 6, 8, 12, and 24 hours. An equal volume of fresh release medium was added immediately after sampling. For the first 2 hours of the test, a pH 1.2 medium was used to simulate gastric fluid; after 2 hours, this was replaced with a pH 6.8 medium to simulate intestinal fluid. The content of the active ingredient in the samples at each time point was determined using the corresponding quantitative method, and the cumulative release rate was calculated.

[0038] Results of in vitro release ( Figure 3 The results showed that, under simulated highly acidic gastric conditions for the first 2 hours, the cumulative release amounts of berberine, ferulic acid, angelica polysaccharide, and peanut protein in the nanoemulsion system were significantly lower than those of their corresponding free active ingredients. The cumulative release amounts of the four components over 2 hours were 16.5 ± 2.52%, 9.85 ± 0.99%, 8.27 ± 2.27%, and 6.84 ± 1.56%, respectively. This phenomenon is attributed to the unique "two-membrane, three-phase" hierarchical structure of the W / O / W nanoemulsion, which can effectively inhibit the rapid dissolution and release of active ingredients in the acidic gastric environment, achieving sustained-release protection in the stomach. When the medium was switched to a pH 6.8 environment simulating intestinal fluid, the release rate of the four active ingredients encapsulated in the nanoemulsion gradually increased, and the total cumulative release over 24 hours was significantly higher than that of the free drug. The cumulative release rates of berberine, ferulic acid, angelica polysaccharide, and peanut protein over 24 hours were 74.19 ± 4.51%, 85.56 ± 3.42%, 65.44 ± 3.99%, and 55.21 ± 2.71%, respectively.

[0039] Example 2: 2.028 g IPM, 0.118 g Span 80, and 12 mg peanut protein were dissolved in 0.90 mL of ultrapure water, and the colostrum was prepared according to the optimal process conditions in Example 1. 0.448 g Tween 80 was dissolved in 7 mL of ultrapure water, and the nanoemulsion was prepared according to the optimal method in Example 1, named Nanoemulsion No. 2. Note: Ovalbumin can be used instead of peanut protein here.

[0040] Example 3: 2.028g IPM, 0.118g Span 80, 0.028g berberine, 0.009g ferulic acid, and 0.0075g Angelica polysaccharide were dissolved in 0.90mL of ultrapure water and the primary emulsion was prepared according to the optimal process conditions in Example 1; 0.448g Tween 80 and 7mL of ultrapure water were used to prepare a nanoemulsion, named Nanoemulsion No. 3, according to the optimal process conditions in Example 1.

[0041] Example 4: 2.028 g IPM, 0.118 g Span 80, 0.028 mg berberine, 0.009 mg ferulic acid, and 0.012 mg peanut protein were dissolved in 0.90 mL of ultrapure water. The colostrum was prepared according to the optimal process conditions in Example 1. 0.448 g Tween 80 and 75 mg Angelica sinensis polysaccharide were dissolved in 7 mL of ultrapure water. The nanoemulsion, named Nanoemulsion No. 4, was prepared according to the optimal method in Example 1. Note: Ovalbumin can be used instead of peanut protein. When the dosage of peanut protein exceeds 1 mg / animal / day, some peanut protein is loaded in the external aqueous phase.

[0042] Example 5: Peanut protein extraction: Fresh peanuts were dried in an oven and then peeled. The peanuts were then completely pulverized using a mortar and pestle. An appropriate amount of pre-cooled acetone was added for soaking, and the mixture was kept at 4°C overnight. After the acetone was air-dried, PBS buffer was added and soaked overnight at 4°C. The mixture was repeatedly centrifuged at 1500 rpm / min to remove the upper fat layer and the lower residue, retaining the middle layer, which is the peanut protein extract. After freeze-drying, the purity of the peanut protein was determined to be approximately 75% using a BCA protein quantification kit. The peanut protein powder was stored at -20°C.

[0043] Example 6: Pharmacodynamic Study 1. In vivo pharmacokinetics Twelve SPF-grade female SD rats, 6-8 weeks old and weighing 180-220g, were randomly divided into a nanoemulsion group and a drug-free group.

[0044] The free drug group was administered the following mixture at the following dosages: berberine 50 mg / kg, ferulic acid 15 mg / kg, and Angelica sinensis polysaccharide 12.5 mg / kg. The nanoemulsion group was prepared using the method described in Example 3, and the same dosages of the three drugs were administered orally. Blood samples were collected from the ocular venous plexus of rats at time points of 0, 0.25, 1, 2, 4, 6, 8, 12, and 24 h. Centrifuge tubes were placed in a centrifuge at 4°C and 4000 rpm for 15 min. The supernatant plasma was collected, and the fluorescence intensity of Angelica sinensis polysaccharide was measured to calculate its concentration. Then, an appropriate amount of methanol was added to remove protein, and the mixture was centrifuged again to collect the supernatant. The methanol was evaporated in a vacuum centrifuge, and the mixture was reconstituted with methanol. The concentrations of berberine and ferulic acid were then detected using high-performance liquid chromatography (HPLC). The results are shown in Figure 4.

[0045] Blood drug concentration-time curves of nanoemulsion group and free drug group ( Figure 4 The morphology is similar, and the time to peak concentration is basically the same, but the peak concentrations (C0) of each drug component in the nanoemulsion group are different. max ) and the area under the curve (AUC) 0-24 The concentrations of berberine, ferulic acid, and Angelica polysaccharide C in the nanoemulsion were all significantly higher than those in the free group.max The AUC increased by 2.07 times, 2.01 times, and 1.53 times respectively. 0-24 The concentrations increased by 2.22-fold, 2.86-fold, and 1.71-fold, respectively, and the mean retention time (MRT) was prolonged by 1.0 h, 1.5 h, and 0.2 h, respectively, as shown in Table 3. These results indicate that nanoemulsions may improve the drug solubility of each component based on a "two-membrane, three-phase" structure, and increase the retention time in the intestine, thereby promoting intestinal absorption and improving the in vivo absorption characteristics of the drug.

[0046] Table 3. Pharmacokinetic parameters of each active ingredient in nanoemulsions: , 2. In vivo pharmacodynamic experiments (1) Preparation of experimental animals and peanut allergy reagent Fifty SPF-grade female BALB / c mice, 6-8 weeks old and weighing 19-21g, were subjected to a pharmacodynamic evaluation of food allergy after one week of acclimatization. They were randomly divided into 5 groups: Naive (normal); Model (model); OIT (Original Injection Therapy) group (prepared as nanoemulsion No. 2 in Example 2); NE (Traditional Chinese Medicine Active Ingredient Nanoemulsion) group (prepared as nanoemulsion No. 3 in Example 3); and ON (Traditional Chinese Medicine Active Ingredient Nanoemulsion + Oral Immunotherapy) group (prepared as nanoemulsion No. 4 in Example 4).

[0047] Preparation of peanut protein basic sensitization solution: Weigh the crude peanut protein according to the protein quantification results, take an appropriate amount of aluminum hydroxide solution (original solution concentration 40 mg / mL), add an appropriate amount of PBS and mix thoroughly. Prepare and use immediately.

[0048] Preparation of peanut protein ointment mixture: Peanut protein and calcipotriol ointment are mixed in a certain mass ratio so that each 20 mg mixture contains 0.2 mg of peanut protein.

[0049] Preparation of peanut protein stimulation solution: Based on the results of BCA protein quantification, weigh an appropriate amount of peanut protein and dissolve it in PBS buffer to a final concentration of 25 mg / mL. Prepare fresh and use immediately for the subsequent stimulation and sensitization stage.

[0050] (2) Modeling method and administration route The establishment of a peanut allergy model consists of two stages: basic sensitization and challenge sensitization. Basic sensitization phase: Basic immunization was established on days 0, 7, 14, and 21. Except for the control group, mice in all other groups were intraperitoneally injected with 0.2 mL of basic sensitization solution at the corresponding time points, for a total of four times. Mice in the control group were intraperitoneally injected with an equal volume of PBS buffer. On days 1-7 and 21-28, except for the control group, mice in all other groups were applied to the backs of mice with a mixture of calcipotriol ointment containing peanut protein. Calcipotriol ointment was applied to the backs of mice in the control group once a day for 7 consecutive days.

[0051] Challenge and sensitization phase: One week after the last basal sensitization, mice were challenged and sensitized by gavage on days 28, 35, 42, 49, and 56 (a total of 5 times). Except for the Naïve control group, mice in all other groups were gavaged with 0.2 mL of a challenge solution containing peanut protein, while the Naïve group was gavaged with an equal volume of physiological saline.

[0052] Administration method and dosage: Treatment began on day 21, with each group receiving oral administration and sensitization stimulation once daily. The normal control group (Naïve) and the model group (Model) received the same volume of pure water. The oral immunotherapy group (OIT) used nanoemulsion No. 2 prepared in Example 2 (starting from 0.25 mg / day / animal and gradually increasing to 16 mg / day / animal, doubling the peanut protein dose every 3 days). The nanoemulsion group (NE) used nanoemulsion No. 3 prepared in Example 3 (based on berberine 50 mg / kg, ferulic acid 15 mg / kg, and Angelica polysaccharide 12.5 mg / kg). The nanoemulsion combined with oral immunotherapy group (ON) used nanoemulsion No. 4 prepared in Example 4 (based on berberine 50 mg / kg, ferulic acid 15 mg / kg, Angelica polysaccharide 12.5 mg / kg, with peanut protein dose starting from 0.25 mg / day / animal and gradually increasing to 16 mg / day / animal).

[0053] (3) Body temperature, allergy index and pathological evaluation On day 56, after the last challenge, the rectal temperature of mice was measured using a contact thermometer. The temperature at 0 min was used as the basal body temperature, and the temperature change over 60 min was recorded. Allergy index was also observed, and the specific allergy index scores were as follows: 0: no symptoms; 1: scratching of face and head; 2: erect hair, decreased activity frequency; 3: rapid breathing, abdominal panting, diarrhea or loose stools; 4: no obvious reaction to stimulation, tremors or mild spasms; watery stools; 5: death.

[0054] Mice were sacrificed on day 57. Before sacrifice, the mice were weighed, and the small intestine and spleen were removed and fixed with 4% paraformaldehyde. The specimens were then embedded in paraffin and sectioned. The pathological specimens were stained with hematoxylin and eosin (H&E) to observe histopathological changes.

[0055] (4) Measurement of relevant inflammatory cytokines and peanut protein-specific IgE The levels of peanut-specific IgE, IL-5, and IL-10 in mouse serum were determined using the ELISA method, strictly following the instructions of the ELISA kit.

[0056] (5) Statistical methods Statistical analysis was performed using SPSS 19.0 software. All data are expressed as mean ± standard deviation (mean ± SD). One-way ANOVA was used for comparisons among multiple groups, and t-tests were used for pairwise comparisons. p <0.05 indicates a statistically significant difference.

[0057] (6) Results Body temperature changes: After the last allergen exposure, except for the normal Naïve group, the body temperature of all other groups of mice showed a decreasing trend, indicating that the peanut allergy model was successfully established. Re-exposure to the allergen led to dysregulation of body temperature regulation. Compared with the Naïve group, the Model group mice experienced an average decrease in body temperature of 1.3℃. P <0.001). Drug and immunotherapy interventions could reverse the trend of decreasing body temperature in mice to some extent; the body temperature of mice in the OIT, drug, and ON groups decreased by 1.1, 0.7, and 0.3℃, respectively. Combined drug and OIT treatment could better improve the trend of decreasing body temperature in mice.

[0058] Results of allergy index and fecal status in mice: After the last allergen challenge, the allergy index of mice in the Model group increased significantly (P < 0.01). Compared with the Model group, the allergy index of mice in the OIT group showed a certain decreasing trend, but the difference between the groups was not statistically significant; the allergy index of mice in the NE group decreased significantly (P < 0.01), showing a good allergy intervention effect; while the allergy index of the ON group decreased the most significantly, showing the best intervention effect on peanut-allergic mice.

[0059] Stool form ( Figure 5 The above observations were consistent with changes in body temperature and allergy index. Mice in the Model group developed severe watery stools, suggesting that food allergies can damage the intestinal barrier function in mice. Among the intervention groups, the diarrhea symptoms in the OIT group mice were slightly improved compared to the Model group; the diarrhea severity in the NE group mice was significantly alleviated compared to the Model group; and the fecal morphology in the ON group mice was close to that of the normal group mice.

[0060] Small bowel pathological sections ( Figure 5(Middle row) Observation: In the normal group, Naïve mice showed neatly arranged and intact small intestinal villi without breakage, edema, or necrosis. Compared with the Naïve group, the Model group mice showed extensive inflammatory cell infiltration in the small intestinal tissue, shortened and broken villi with necrosis at the tips, accompanied by pathological damage such as villi edema, lamina propria, and epithelial cell division. Among the intervention groups, the ON group mice showed the most significant repair of small intestinal tissue structure damage and the most outstanding improvement in pathological morphology.

[0061] Spleen histopathology ( Figure 5 The following sections show that in the Naïve group mice, the red and white pulp boundaries of the spleen were clear, the tissue structure was intact, and there were no pathological changes such as congestion or hemorrhage. Compared with the Naïve group, the normal structure of the spleen in the Model group mice was severely damaged, the red pulp sinuses were dilated, and there was obvious congestion and hemorrhage in the splenic intersplenic spaces, indicating significant pathological damage to the spleen.

[0062] Expression levels of various inflammatory factors and peanut-specific IgE in serum ( Figure 6 A): Compared with the normal Naïve mice, the serum peanut-specific IgE level of the Model group mice was significantly increased, and the serum IgE level of each intervention group mice was downregulated to varying degrees. Among them, the IgE level of the ON group was reduced to an extremely significant degree (P<0.01), and the effect of inhibiting the allergic humoral immune response was the best.

[0063] Serum Th2 cytokine IL-5 levels in Model group mice ( Figure 6 B) The levels of IL-5 were significantly higher in the ON group than in the Naïve group, confirming that the Th2-type immune response-mediated inflammatory response synergistically participates in the progression of peanut allergy. Compared with the Model group, both the ON and NE groups showed better downregulation of IL-5 than the OIT group alone, with the ON group exhibiting the most significant decrease in the levels of these inflammatory factors (P < 0.01). Simultaneously, the serum IL-10 anti-inflammatory factor level in the Model group was significantly lower than that in the Naïve group (…). Figure 6 C, P<0.01). After intervention, all treatment groups were able to reverse the trend of decreasing serum IL-10 levels in mice to varying degrees. Among them, the ON group showed a significantly better improvement in the expression level of anti-inflammatory factors than other groups (P<0.01), which can effectively balance the body's inflammation and anti-inflammatory immune homeostasis.

[0064] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A nanoemulsion containing active ingredients of traditional Chinese medicine, characterized in that, It is prepared from the following parts by weight of active ingredients of traditional Chinese medicine, oil phase and emulsifier: active ingredients of traditional Chinese medicine are 1.5-2.5 parts of berberine, 0.8-2 parts of ferulic acid and 0.6-2 parts of Angelica polysaccharide; oil phase and emulsifier are 174 parts of isopropyl myristate, 10-20 parts of Span 80 and 30-50 parts of Tween 80.

2. The nanoemulsion containing active ingredients of traditional Chinese medicine as described in claim 1, characterized in that, It was prepared from the following parts by weight of active ingredients of traditional Chinese medicine, oil phase and emulsifier: active ingredients of traditional Chinese medicine are 2.4 parts of berberine, 1.9 parts of ferulic acid and 1.5 parts of Angelica polysaccharide; oil phase and emulsifier are 174 parts of isopropyl myristate, 10 parts of Span 80 and 38 parts of Tween 80.

3. The nanoemulsion containing active ingredients of traditional Chinese medicine as described in claim 1, characterized in that, The preparation method includes the following steps: Step 1: Preparation of W1 / O colostrum: Dissolve the prescribed amount of Angelica polysaccharide in an appropriate amount of ultrapure water to obtain the inner aqueous phase W1; then dissolve berberine and ferulic acid in isopropyl myristate, add the lipophilic emulsifier Span 80, and mix with magnetic stirring to obtain the oil phase O. Under high-speed stirring, the aqueous phase W1 is slowly injected into the oil phase O. After thorough mixing, ultrasonic emulsification is performed using an ultrasonic cell disruptor with set parameters to obtain the W1 / O type colostrum. Step 2: Preparation of W1 / O / W2 colostrum: Add the prescribed amount of hydrophilic emulsifier Tween 80 to an appropriate amount of ultrapure water and stir to mix well to obtain the external aqueous phase W2. Add the colostrum obtained in Step 1 dropwise to the external aqueous phase W2 while stirring. After mixing thoroughly, use an ultrasonic cell disruptor with the set parameters to emulsify, and finally form W1 / O / W2 nanoemulsion.

4. A composite nanoemulsion containing active ingredients of traditional Chinese medicine and allergen proteins, characterized in that, The nanoemulsion containing the active ingredients of traditional Chinese medicine as described in claim 1 or 2, and the allergen protein, are prepared from the following raw materials, oil phase, and emulsifier in parts by weight: the raw materials are 0.3-30 parts of allergen protein, 1.5-2.5 parts of berberine, 0.8-2 parts of ferulic acid, and 0.6-2 parts of Angelica polysaccharide; the oil phase and emulsifier are 174 parts of isopropyl myristate, 10-20 parts of Span 80, and 30-50 parts of Tween 80.

5. The composite nanoemulsion containing active ingredients of traditional Chinese medicine and allergen proteins as described in claim 4, characterized in that, It is prepared from the following raw materials, oil phase and emulsifier in parts by weight: the raw materials are 0.3-30 parts of allergen protein, 2.4 parts of berberine, 0.8 parts of ferulic acid and 0.6 parts of Angelica polysaccharide; the oil phase and emulsifier are 174 parts of isopropyl myristate, 10 parts of Span 80 and 38 parts of Tween 80.

6. The composite nanoemulsion containing traditional Chinese medicine active ingredients and allergen proteins as described in claim 4, characterized in that, The preparation method includes the following steps: Step 1: Preparation of W1 / O colostrum: Dissolve the prescribed amount of allergen protein in an appropriate amount of ultrapure water to obtain the inner aqueous phase W1; then dissolve berberine and ferulic acid in isopropyl myristate, add the lipophilic emulsifier Span 80, and mix with magnetic stirring to obtain the oil phase O. Under high-speed stirring, the aqueous phase W1 is slowly injected into the oil phase O. After thorough mixing, ultrasonic emulsification is performed using an ultrasonic cell disruptor with set parameters to obtain the W1 / O type colostrum. Step 2: Preparation of W1 / O / W2 colostrum: Add the prescribed amount of Angelica polysaccharide and hydrophilic emulsifier Tween 80 to ultrapure water and stir to mix well to obtain the external aqueous phase W2. Add the colostrum obtained in Step 1 dropwise to the external aqueous phase W2 while stirring. After mixing thoroughly, use an ultrasonic cell disruptor with the set parameters to emulsify, and finally form W1 / O / W2 nanoemulsion.

7. The composite nanoemulsion as described in claim 4, characterized in that, In step 1 of the preparation method, the ultrasonic time and power are 2-10 minutes and 100W-300W, respectively; in step 2 of the preparation method, the ultrasonic time and power are 4-8 minutes and 150W-300W, respectively.

8. The use of the nanoemulsion containing the active ingredients of traditional Chinese medicine as described in claim 1 in the preparation of drugs for treating food allergies or atopic dermatitis.

9. The application of the nanoemulsion containing the active ingredients of traditional Chinese medicine as described in claim 1 in the preparation of drugs that enhance the efficacy of allergen proteins.

10. The composite nanoemulsion as described in claim 4, characterized in that, The allergenic protein is ovalbumin and / or peanut protein.

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