Compound composition for treating inflammatory lesions of the breast and method for preparing same

Abstract

The application discloses a compound composition for treating mammary gland inflammatory injury and a preparation method thereof, and belongs to the technical field of pharmaceutical preparations, and solves the problems of poor stability of various active ingredients in the existing Chinese medicinal compound composition for treating mammary gland inflammation and difficulty in realizing synergistic effect. The compound composition is composed of active ingredients and nano-structured lipid carriers, the active ingredients contain baicalin, chlorogenic acid, astragalus polysaccharide, rosmarinic acid and rosemary oil, and the weight ratio is 1:4-6:20-30:0.5-1:20-40; the rosemary oil contains 10%-30% beta-caryophyllene and is used as a liquid lipid; a solid lipid is selected from glycerol monostearate or stearic acid; the average particle size of the carrier is 50-200 nm; the astragalus polysaccharide and the rosmarinic acid are loaded on the surface of the carrier to form a hydrophilic shell; and the baicalin and the chlorogenic acid are loaded in a lipid core. The compound composition can be applied to the preparation of a medicine for treating mammary gland inflammatory injury.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical preparations. More specifically, this invention relates to a compound composition for treating inflammatory lesions of the mastoid process and a method for preparing the same. Background Technology

[0002] Bovine mastitis is one of the most common peripartum diseases in dairy farming, severely impacting reproductive performance and milk production, and hindering the development of the dairy industry. Escherichia coli (E. coli) is a contributing factor. Escherichia coli, E. coli Escherichia coli (E. coli) is one of the main pathogens causing mastitis in dairy cows. Lipopolysaccharide (LPS), as a characteristic lipid component of the extracellular lobules of E. coli, is one of the most critical virulence factors of the bacterium and a core driving factor in triggering the host's inflammatory response. Moreover, its toxic effects are directly related to the pathogenicity of E. coli.

[0003] Currently, antibiotics remain the primary treatment for mastitis in dairy cows, but long-term use can lead to bacterial resistance and drug residues. Traditional Chinese medicine (TCM) active ingredients, due to their multi-target effects, are also attracting attention in the treatment of mastitis. Among them, Baicalin is the main active ingredient of the traditional Chinese medicine Scutellaria baicalensis, possessing biological functions such as antibacterial, anti-inflammatory, immunomodulatory, and antioxidant effects. However, baicalin suffers from structural instability and is easily oxidized; furthermore, due to its glycosyl group structure, it exhibits poor water and lipid solubility. This poor water and lipid solubility leads to poor oral absorption and low bioavailability, limiting its full clinical efficacy. Chlorogenic acid is the main active ingredient of Lonicera japonica, possessing antibacterial, antiviral, anti-inflammatory, and immune-enhancing effects. However, chlorogenic acid also faces the problem of poor stability, easily degrading during formulation and storage. The pharmacokinetic behavior of chlorogenic acid in vivo is not fully understood, making its development into a stable formulation suitable for mastitis treatment challenging. Astragalus polysaccharides are an important active ingredient of Astragalus membranaceus, exhibiting certain therapeutic effects on mastitis. However, due to their large molecular weight and volume, astragalus polysaccharides have low oral bioavailability. Currently, the clinical application of astragalus polysaccharides mainly relies on injection administration, which limits its convenience in certain therapeutic scenarios. Rosmarinic acid is a phenolic acid compound extracted from rosemary, possessing antioxidant, anti-inflammatory, and antibacterial biological functions. Studies have shown that rosmarinic acid can alleviate mastitis by inhibiting the TLR4 / MyD88 / NF-κB signaling pathway. However, when used as a single component, rosmarinic acid has relatively limited intervention targets for inflammatory damage in the breast, making it difficult to comprehensively address the multi-stage pathological process involving inflammation, oxidative stress, and tissue damage. Rosemary oil contains various terpenoids, among which β-caryophyllene possesses pharmacological activities such as anti-inflammatory, antibacterial, and antioxidant effects. However, as a volatile oil, rosemary oil suffers from high volatility, low water solubility, and poor stability, making it easy to lose active ingredients when applied directly, and difficult to maintain effective concentrations after administration.

[0004] Theoretically, it is reasonable to combine the above-mentioned active ingredients with different pharmacological effects to exert a synergistic effect. However, the physicochemical properties of each active ingredient differ significantly, such as solubility, stability, and molecular size, making it difficult to achieve synchronous delivery and synergistic effect when simply mixed. Summary of the Invention

[0005] One object of the present invention is to provide a compound composition for treating inflammatory lesions of the mastoid process and a method for preparing the same, so as to at least solve the above-mentioned problems.

[0006] To achieve the objectives and other advantages of this invention, a compound composition for treating inflammatory lesions of the mastoid process is provided, comprising an active ingredient and a nanostructured lipid carrier; the active ingredient comprises baicalin, chlorogenic acid, astragalus polysaccharide, rosmarinic acid, and rosemary oil, wherein the rosemary oil serves as both the active ingredient and the liquid lipid component of the nanostructured lipid carrier, constituting the lipid core matrix of the carrier, and the rosemary oil contains 10% to 30% β-caryophyllene; the nanostructured lipid carrier further comprises solid lipids and stearin. The solid lipid is at least one of glyceryl monostearate or stearic acid; the weight ratio of baicalin, chlorogenic acid, astragalus polysaccharide, rosmarinic acid and rosemary oil in the active ingredients is 1:4-6:20-30:0.5-1:20-40; the average particle size of the nanostructured lipid carrier is 50-200 nm, the astragalus polysaccharide and rosmarinic acid are loaded on the surface of the nanostructured lipid carrier to form a hydrophilic outer shell layer, and the baicalin and chlorogenic acid and rosemary oil are co-encapsulated in the lipid core of the nanostructured lipid carrier.

[0007] Preferably, the weight ratio of the solid lipid to rosemary oil is 1:1.5 to 1:2.5.

[0008] Preferably, the solid lipid is a mixture of glyceryl monostearate and stearic acid, wherein the weight ratio of glyceryl monostearate to stearic acid is 2:1 to 4:1, and the amount of stearamine added is 5%-8% of the solid lipid.

[0009] The present invention also provides a method for preparing the above-mentioned compound composition, comprising the following steps: Step 1: Weigh baicalin, chlorogenic acid, astragalus polysaccharide, rosmarinic acid and rosemary oil in a weight ratio of 1:4-6:20-30:0.5-1:20-40, wherein the β-caryophyllene content in the rosemary oil is 10% to 30%. Dissolve baicalin, chlorogenic acid and all the rosemary oil in anhydrous ethanol to obtain the oil phase. Step 2: Add at least one of the solid lipids, glyceryl monostearate or stearic acid, to the oil phase. The weight ratio of the solid lipids to rosemary oil is 1:1.5 to 1:2.5. Heat at 70°C to 80°C to completely melt the solid lipids and mix them evenly with the oil phase to obtain a molten oil phase. Step 3: The molten oil phase is dripped into a phosphate buffer containing an emulsifier, such as poloxamer 188 or Tween-80, at a temperature of 70°C to 80°C. The emulsifier concentration is 5-20 mg / mL. The mixture is then subjected to high-speed shearing at 10000-15000 r / min for 5-10 minutes to form a nanoemulsion. Step 4: The nanoemulsion is ultrasonically treated in a water bath at 0-2℃ for 5-15 minutes with an ultrasonic power of 200-400W to obtain a nanostructured lipid carrier suspension loaded with baicalin, chlorogenic acid and β-caryophyllene. Step 5: Dilute the nanostructured lipid carrier suspension with phosphate buffer to a solid lipid content of 5-10 mg / mL. Under stirring, alternately add phosphate buffer solution of astragalus polysaccharide and phosphate buffer solution of rosmarinic acid, stirring and incubating for 15-45 minutes after each addition, so that astragalus polysaccharide and rosmarinic acid are stepwise saturated and co-adsorbed on the surface of the nanostructured lipid carrier, forming a hydrophilic outer shell layer of astragalus polysaccharide / rosmarinic acid composite. Repeat the alternating loading operation 1-3 times to obtain the five-component co-loaded nanostructured lipid carrier suspension. Step 6: Centrifuge the pentagonal co-loaded nanostructured lipid carrier suspension at 30,000-45,000g at high speed for 30-60 minutes, controlling the centrifugation temperature at 2-8℃. Collect the precipitate and wash it 2 to 3 times with phosphate buffer to obtain the compound composition.

[0010] Preferably, in step one, when preparing the oil phase, baicalin is first added to anhydrous ethanol and heated and stirred at 40°C to 60°C until dissolved, then chlorogenic acid is added and stirred until dissolved, and finally the entire amount of rosemary oil is added and mixed well.

[0011] Preferably, in step two, the solid lipid is a mixture of glyceryl monostearate and stearic acid, with a weight ratio of glyceryl monostearate to stearic acid of 2:1 to 4:1, and the amount of stearamine added is 5%-8% of the solid lipid.

[0012] Preferably, in step three, the molten oil phase is kept at 70°C to 80°C and stirred at a constant speed of 200-400 r / min for 15-30 minutes, and then dropped into the phosphate buffer containing the emulsifier at a dropping rate of 2 ml / min.

[0013] Preferably, in step five, the concentration of the phosphate buffer solution of Astragalus polysaccharide is 0.5-5 mg / mL, and the concentration of the phosphate buffer solution of rosmarinic acid is 0.1-2 mg / mL; the temperature for stirring and incubation is 15-30℃, and the stirring speed is 100-300 rpm.

[0014] The present invention also provides the use of the above-mentioned compound composition in the preparation of a medicament for treating inflammatory damage of the mastoid gland.

[0015] The present invention has at least the following beneficial effects: First, the compound composition of the present invention forms a hydrophilic outer shell layer by loading the hydrophilic active ingredients astragalus polysaccharide and rosmarinic acid onto the surface of a nanostructured lipid carrier, while simultaneously encapsulating baicalin, chlorogenic acid, and rosemary oil within the lipid core. This achieves stable co-loading of multiple active ingredients, avoiding direct contact and mutual interference between the components, and thus helping to maintain their respective activities. Rosemary oil serves as both an active ingredient and a liquid lipid matrix, reducing the need for additional liquid lipid excipients and improving the drug loading efficiency of the carrier. The surface hydrophilic layer helps improve the dispersion stability of the carrier in an aqueous environment, and the core lipid can slow down the degradation of baicalin and chlorogenic acid, as well as the volatilization of β-caryophyllene in rosemary oil. This facilitates the synergistic effect of each component on intervening in mastitis-related damage after administration.

[0016] Secondly, in the preparation of this invention, baicalin, chlorogenic acid, and rosemary oil are dissolved together in anhydrous ethanol to form an oil phase, which is beneficial for the subsequent encapsulation of the three components in the lipid core. A heating temperature of 70-80℃ ensures complete melting of the solid lipids while avoiding damage to heat-sensitive components due to excessively high temperatures. The molten oil phase is dripped into a buffer solution containing emulsifier under a holding temperature of 70-80℃, coupled with high-speed shearing, to prevent premature solidification of the oil phase, forming nanoemulsions with uniform particle size. Ultrasonic treatment in a 0-2℃ water bath solidifies the lipids to form a nanostructure carrier. The combination of ultrasonic power and time allows for the acquisition of carriers with the target particle size, while avoiding lipid oxidation or drug leakage. After diluting the carrier suspension, astragalus polysaccharide solution and rosmarinic acid solution are added alternately and incubated with stirring. Astragalus polysaccharide and rosmarinic acid are gradually adsorbed and saturated on the carrier surface through electrostatic and hydrogen bonding interactions, forming a uniform and stable astragalus polysaccharide / rosmarinic acid composite hydrophilic outer shell layer. Centrifugation and washing effectively remove free components and residual emulsifiers, improving the purity of the final product. Throughout the preparation process, the parameters for each step are clearly defined and the operations are consistent, which helps to ensure the consistency of product quality between batches.

[0017] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0018] Figure 1 The results are HE staining of breast tissue from the blank control group, LPS model, Example 1, and Control Example 1. Detailed Implementation

[0019] The present invention will now be described in further detail with reference to the embodiments and accompanying drawings, so that those skilled in the art can implement it based on the description.

[0020] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0021] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0022] Example 1: The preparation method of the compound composition for treating inflammatory damage of the breast is as follows: Baicalin, chlorogenic acid, astragalus polysaccharide, rosmarinic acid, and rosemary oil were weighed in a weight ratio of 1:5:25:0.8:30, wherein the rosemary oil contained 20% β-caryophyllene (the rosemary oil was a blended oil, based on natural distilled rosemary oil, with the addition of exogenous β-caryophyllene). Baicalin was added to anhydrous ethanol and heated at 50°C with stirring until completely dissolved. Chlorogenic acid was then added and stirred until dissolved. Finally, the entire amount of rosemary oil was added and mixed thoroughly to obtain the oil phase. Solid lipids (a mixture of glyceryl monostearate and stearic acid in a weight ratio of 3:1) and stearamine were weighed and added to the oil phase. The weight ratio of solid lipids to rosemary oil was 1:2, and the amount of stearamine added was 6.5% of the solid lipids. The mixture was heated at 75°C to completely melt the solid lipids and stearamine and mix them evenly with the oil phase to obtain the molten oil phase. The molten oil phase was stirred at 300 rpm for 20 minutes at 75°C, and then added dropwise at 2 mL / min to a phosphate buffer (pH 7.0) containing the emulsifier poloxamer 188 (concentration 10 mg / mL). The mixture was then sheared at 12000 rpm for 8 minutes to form a nanoemulsion. The nanoemulsion was then sonicated in a 0-2°C water bath for 10 minutes at a power of 300 W to obtain a nanostructured lipid carrier suspension loaded with baicalin, chlorogenic acid, and β-caryophyllene. The nanostructured lipid carrier suspension was diluted with phosphate buffer to a solid lipid content of 5-10 mg / mL. Then, under stirring at 25℃ and 200 rpm, 2 mg / mL Astragalus polysaccharide phosphate buffer and 1 mg / mL Rosmarinic acid phosphate buffer were added alternately, with stirring and incubation for 30 minutes after each addition, allowing the Astragalus polysaccharide and Rosmarinic acid to be stepwise saturated and co-adsorbed onto the surface of the nanostructured lipid carrier. This alternating loading process was repeated twice, adding 1 / 3 of the total amount each time, to obtain the pentagonal co-loaded nanostructured lipid carrier suspension. The pentagonal co-loaded nanostructured lipid carrier suspension was centrifuged at 38000 rpm for 45 minutes, with the centrifugation temperature controlled at 2-8℃. The precipitate was collected and washed three times with phosphate buffer to obtain the compound composition.

[0023] Example 2: The preparation method of the compound composition for treating inflammatory damage of the breast is as follows: Baicalin, chlorogenic acid, astragalus polysaccharide, rosmarinic acid, and rosemary oil were weighed in a weight ratio of 1:4:20:0.5:20, wherein the rosemary oil contained 10% β-caryophyllene. Baicalin was added to anhydrous ethanol and heated and stirred at 40°C until dissolved. Chlorogenic acid was then added and stirred until dissolved. Finally, the entire amount of rosemary oil was added and mixed thoroughly to obtain the oil phase. Solid lipids, glyceryl monostearate and stearylamine, were weighed and added to the oil phase. The weight ratio of solid lipids to rosemary oil was 1:1.5, and the amount of stearylamine added was 5% of the solid lipids. The mixture was heated at 70°C to completely melt the solid lipids and stearylamine and mix them evenly with the oil phase to obtain the molten oil phase. The molten oil phase was stirred at 200 rpm for 15 minutes at 70°C, and then added dropwise at 2 ml / min to a phosphate buffer solution containing Tween-80 emulsifier (5 mg / mL). The mixture was then sheared at 10,000 rpm for 5 minutes to form a nanoemulsion. The nanoemulsion was then sonicated in a 0-2°C water bath for 5 minutes at 200 W to obtain a nanostructured lipid carrier suspension loaded with baicalin, chlorogenic acid, and β-caryophyllene. The nanostructured lipid carrier suspension was diluted with phosphate buffer to a solid lipid content of 5-10 mg / mL. Then, under stirring at 100 rpm and 15°C, 0.5 mg / mL astragalus polysaccharide phosphate buffer solution and 0.1 mg / mL rosmarinic acid phosphate buffer solution were added alternately, with stirring and incubation for 45 minutes after each addition. Repeat the alternating loading operation once, adding 1 / 2 of the total amount each time, to obtain a pentagonal co-loaded nanostructured lipid carrier suspension. Centrifuge the suspension at 30000g for 60 minutes at a controlled temperature of 2-8℃, collect the precipitate, and wash twice with phosphate buffer to obtain the compound composition.

[0024] Example 3: The preparation method of the compound composition for treating inflammatory damage of the breast is as follows: Baicalin, chlorogenic acid, astragalus polysaccharide, rosmarinic acid, and rosemary oil were weighed in a weight ratio of 1:6:30:1:40, wherein the rosemary oil contained 30% β-caryophyllene. Baicalin was added to anhydrous ethanol and heated and stirred at 60°C until dissolved. Chlorogenic acid was then added and stirred until dissolved. Finally, the entire amount of rosemary oil was added and mixed thoroughly to obtain the oil phase. Solid lipids (a mixture of glyceryl monostearate and stearic acid in a weight ratio of 4:1) and stearylamine were weighed and added to the oil phase. The weight ratio of solid lipids to rosemary oil was 1:2.5, and the amount of stearylamine added was 8% of the solid lipids. The mixture was heated at 80°C to completely melt the solid lipids and stearylamine and mix them evenly with the oil phase to obtain the molten oil phase. The molten oil phase was stirred at 400 rpm for 30 minutes at 80°C, and then added dropwise at 2 mL / min to a phosphate buffer containing poloxamer 188 (20 mg / mL emulsifier). The mixture was then sheared at 15000 rpm for 10 minutes to form a nanoemulsion. The nanoemulsion was sonicated in a 0-2°C water bath for 15 minutes at 400 W to obtain a nanostructured lipid carrier suspension loaded with baicalin, chlorogenic acid, and β-caryophyllene. The suspension was diluted with phosphate buffer to a solid lipid content of 5-10 mg / mL. Then, under stirring at 30°C and 300 rpm, 5 mg / mL astragalus polysaccharide phosphate buffer and 2 mg / mL rosmarinic acid phosphate buffer were added alternately, with stirring and incubation for 15 minutes after each addition. Repeat the alternating loading operation three times, adding 1 / 4 of the total amount each time, to obtain a pentagonal co-loaded nanostructured lipid carrier suspension. Centrifuge the suspension at 45000g for 30 minutes at a controlled temperature of 2-8℃, collect the precipitate, and wash it three times with phosphate buffer to obtain the compound composition.

[0025] Comparative Example 1: The preparation method of the compound composition for treating inflammatory damage of the breast is as follows: A nanostructured lipid carrier suspension loaded with baicalin, chlorogenic acid, and β-caryophyllene was prepared according to the same proportions and steps as in Example 1, with the β-caryophyllene content in rosemary oil being 20%. The suspension was diluted with phosphate buffer to a solid lipid content of 5-10 mg / mL. Then, under stirring conditions of 25°C and 200 rpm, a 2 mg / mL astragalus polysaccharide phosphate buffer solution and a 1 mg / mL rosmarinic acid phosphate buffer solution were simultaneously added to the suspension, and the mixture was stirred and incubated for 60 minutes to allow the astragalus polysaccharide and rosmarinic acid to be simultaneously loaded onto the surface of the nanostructured lipid carrier. The suspension was centrifuged at 38000 rpm for 45 minutes, with the centrifugation temperature controlled at 2-8°C. The precipitate was collected and washed three times with phosphate buffer to obtain the compound composition.

[0026] Comparative Example 2: The preparation method of the compound composition for treating inflammatory damage of the breast is as follows: Baicalin, chlorogenic acid, astragalus polysaccharide, rosmarinic acid, and rosemary oil were weighed in a weight ratio of 1:5:25:0.8:30, wherein the rosemary oil contained 20% β-caryophyllene. An equal weight of medium-chain triglycerides was weighed as an additional liquid lipid. The main fatty acids in the medium-chain triglycerides were caprylic acid and capric acid, with a ratio of approximately 60:40, and the total content of these two fatty acids was not less than 95.0% of the total fatty acid content. Baicalin was added to anhydrous ethanol and heated and stirred at 50°C until dissolved. Chlorogenic acid was then added and stirred until dissolved. Finally, the entire amount of rosemary oil and medium-chain triglycerides were added and mixed thoroughly to obtain the oil phase. Solid lipids (a mixture of glyceryl monostearate and stearic acid in a weight ratio of 3:1) and stearylamine were weighed and added to the oil phase. The weight ratio of solid lipids to rosemary oil was 1:2, i.e., the weight ratio of solid lipids to total liquid lipids (rosemary oil + medium-chain triglycerides) was 1:4. The amount of stearylamine added was 6.5% of the solid lipids. The mixture was heated to 75°C to completely melt the solid lipids and stearylamine and mix them evenly with the oil phase to obtain a molten oil phase. Subsequent steps were the same as in Example 1.

[0027] Comparative Example 3: The preparation method of the compound composition for treating inflammatory damage of the breast is as follows: Weigh each active ingredient according to the same proportions as in Example 1. The β-caryophyllene content in the rosemary oil is 20%. Dissolve baicalin, chlorogenic acid, and all the rosemary oil in anhydrous ethanol at a ratio of baicalin to anhydrous ethanol of 1 g:30 ml. Heat and stir at 50°C until dissolved. Add solid lipids (glyceryl monostearate and stearic acid mixed at a weight ratio of 3:1) and stearamine to the oil phase at a weight ratio of 1:2. The amount of stearamine added is 6.5% of the solid lipids. Heat at 75°C to completely melt the solid lipids and stearamine and mix them evenly with the oil phase. Add the molten oil phase dropwise at 2 ml / min to a phosphate buffer containing the emulsifier poloxamer 188 (10 mg / mL) while maintaining the temperature at 75°C. Shear at 5000 rpm for 2 minutes to form a crude emulsion. The crude emulsion was directly diluted with phosphate buffer to a solid lipid content of 5-10 mg / mL. The subsequent surface loading steps of astragalus polysaccharide and rosmarinic acid were the same as in Example 1.

[0028] Comparative Example 4: The preparation method of the compound composition for treating inflammatory damage of the breast is as follows: A nanostructured lipid carrier suspension loaded with baicalin, chlorogenic acid, and β-caryophyllene was prepared according to the same proportions and steps as in Example 1, with the β-caryophyllene content in rosemary oil being 20%. The suspension was centrifuged at 12000 r / min for 45 minutes, the precipitate was collected, and washed three times with phosphate buffer to obtain a compound composition containing only the core active ingredients and without astragalus polysaccharide and rosmarinic acid loaded on the surface.

[0029] Comparative Example 5: The preparation method of the compound composition for treating inflammatory damage of the breast is as follows: Baicalin, astragalus polysaccharide, rosmarinic acid, and medium-chain triglycerides were weighed in a weight ratio of 1:25:0.8:30. Baicalin was added to anhydrous ethanol and heated at 50°C with stirring until dissolved. The entire amount of medium-chain triglycerides was then added and mixed thoroughly to obtain the oil phase. Solid lipids (a mixture of glyceryl monostearate and stearic acid in a weight ratio of 3:1) and stearamine were weighed and added to the oil phase. The weight ratio of solid lipids to medium-chain triglycerides was 1:2, and the amount of stearamine added was 6.5% of the solid lipids. The mixture was heated at 75°C to completely melt the solid lipids and stearamine and mix them evenly with the oil phase to obtain the molten oil phase. Subsequent emulsification, low-temperature ultrasonic curing, and surface loading of astragalus polysaccharide and rosmarinic acid were performed as in Example 1.

[0030] Compare with Example 1: The preparation method of the compound composition for treating inflammatory damage of the breast is as follows: Baicalin, chlorogenic acid, astragalus polysaccharide, rosmarinic acid and rosemary oil were weighed in a weight ratio of 1:5:25:0.8:30, wherein the β-caryophyllene content of the rosemary oil was 20%. The five components were placed in a mortar and ground and mixed at room temperature for 15 minutes to ensure that they were fully physically mixed and uniform, thus obtaining the control sample 1.

[0031] Experimental Example 1: Physicochemical Characterization of Compound Compositions The following physicochemical properties were determined for the samples obtained in each example and Comparative Examples 1-3. Particle size determination: Appropriate amounts of each sample were diluted to a suitable concentration with phosphate buffer (pH 7.0), and the average particle size and polydispersity index (PDI) were determined using a dynamic light scattering particle size analyzer. Encapsulation efficiency determination: Appropriate amounts of each sample were taken, and the free drug and the drug encapsulated in the carrier were separated using ultrafiltration centrifugation. The contents of baicalin, chlorogenic acid, and β-caryophyllene were determined using high-performance liquid chromatography (HPLC), and the encapsulation efficiency of each component was calculated. Surface loading determination: Appropriate amounts of each sample were taken, and the surface-loaded astragalus polysaccharide and rosmarinic acid were separated from the drug encapsulated inside the carrier using dialysis. The content of astragalus polysaccharide was determined using the phenol-sulfuric acid method, and the content of rosmarinic acid was determined using HPLC. The loading amounts of both on the carrier surface were calculated. The results for each sample are shown in Table 1.

[0032] Table 1. Particle size, encapsulation efficiency, and surface loading of each embodiment and comparative example. As shown in Table 1, the average particle size of the carriers obtained in each embodiment is within the range of 50-200 nm, the PDI is less than 0.25, and the particle size distribution is uniform. The encapsulation efficiency of baicalin in each embodiment reaches over 82%, chlorogenic acid over 78%, and β-caryophyllene over 85%, indicating that all three are effectively encapsulated in the lipid core. The surface loading of astragalus polysaccharide and rosmarinic acid reaches a high level, and the surface loading of Example 1, due to alternating loading twice, is significantly higher than that of Comparative Example 1, which uses simultaneous addition, indicating that alternating loading is beneficial to improving the loading efficiency of surface active ingredients. Comparative Example 2, due to the additional addition of medium-chain triglycerides diluting the lipid core and the increase in total lipids reducing the ratio of emulsifier to lipids, resulted in a significant decrease in the encapsulation efficiency of the three core components to 65.4%, 58.7%, and 70.2%, respectively, far lower than in the embodiments. Comparative Example 3, without low-temperature ultrasonic treatment, has a carrier particle size far exceeding the nanometer range, reaching 685 nm, and insufficient lipid solidification, resulting in an encapsulation efficiency of less than 42%. The above results indicate that the preparation method of the present invention can form nanostructured lipid carriers with regular structure, high encapsulation efficiency, and large surface loading.

[0033] Experimental Example 2: Evaluation of the in vitro release behavior of the compound composition For the samples obtained in each example, comparative example, and control example 1, the in vitro release behavior of five active ingredients in each sample was determined using the dialysis bag method. An appropriate amount of each sample (equivalent to 1 mg of baicalin) was placed in a pretreated dialysis bag with a molecular weight cutoff of 80-100 kDa. The dialysis bag was placed in a release medium containing 0.5% Tween-80 phosphate buffer (pH 7.0) and oscillated at 37°C and 100 rpm. Samples were taken at 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 hours, and an equal amount of fresh release medium was added. The cumulative release amounts of baicalin, chlorogenic acid, rosmarinic acid, and β-caryophyllene in the release medium were determined using high-performance liquid chromatography (HPLC), and the cumulative release amount of astragalus polysaccharide was determined using the phenol-sulfuric acid method. The cumulative release percentage at each time point was calculated. The cumulative release rates at the key time points (2 hours and 48 hours) are shown in Table 2.

[0034] Table 2. Cumulative release rate (%) of detectable active ingredients in each sample. As shown in Table 2, the astragalus polysaccharides and rosmarinic acid loaded on the carrier surface in each embodiment exhibited rapid release characteristics, with cumulative release rates exceeding 80% within 2 hours. In contrast, baicalin, chlorogenic acid, and β-caryophyllene, encapsulated within the lipid core, showed significant sustained-release characteristics. In Example 1, the cumulative release rates after 48 hours were 62.3%, 58.7%, and 55.2%, respectively, with smooth release curves and no burst release observed. This differentiated pattern of rapid release of surface-active components and slow release of core active components is beneficial for rosmarinic acid and astragalus polysaccharides to initially exert their anti-inflammatory and immunomodulatory effects, followed by the sustained release of baicalin, chlorogenic acid, and β-caryophyllene to maintain a long-lasting anti-inflammatory effect. In Comparative Example 1, the release of astragalus polysaccharides and rosmarinic acid was asynchronous (rosmarinic acid release rate was 92.1% and astragalus polysaccharide release rate was 78.4% after 2 hours), indicating that simultaneous co-loading led to uneven distribution of the two on the carrier surface. In Comparative Example 2, the addition of medium-chain triglycerides diluted the core, accelerating the release of the core component; the release rate of baicalin reached 81.5% after 48 hours, but the sustained-release effect was weakened. In Comparative Example 3, due to the large particle size and irregular structure of the carrier, the core component was released in large quantities within 12 hours, with a baicalin release rate of 92.7% after 48 hours, and β-caryophyllene volatilized faster due to insufficient solidification. The sustained-release of the core component in Comparative Example 4 was comparable to that in Example 1. In Comparative Example 5, the surface component release was normal, but the release rate of the core component, baicalin, reached 75.2% after 48 hours. Control Example 1 was a physical mixture without carrier encapsulation; β-caryophyllene was in direct contact with the medium, and over 60% was rapidly released within 2 hours. Later, due to the decrease in concentration difference, the release rate slowed down, but the cumulative release continued to increase, and the final release amount was higher than that of the sustained-release system in the examples. The above results indicate that the compound composition prepared by the method of the present invention can achieve differentiated and ordered release behavior, and the absence of any core component or surface component will disrupt this ordered release pattern.

[0035] Experimental Example 3: Inhibitory effect of compound composition on LPS-induced inflammatory response in RAW264.7 cells A LPS-induced inflammation model was established using RAW264.7 cells to evaluate the anti-inflammatory activity of samples from each example, comparative example, and control example 1. RAW264.7 cells were cultured at 4 × 10⁻⁶ cells per cell line. 5Cells were seeded at 1 mL / well in 12-well plates and cultured at 37°C in a 5% CO2 incubator until adherence. A blank control group, an LPS model group (1 μg / mL LPS), each example group (equivalent to 25 μg / mL baicalin), each comparative group (equivalent to 25 μg / mL baicalin), and control group 1 (equivalent to 25 μg / mL baicalin) were set up. Each treatment group adopted a post-treatment mode: LPS was added for stimulation for 2 hours, followed by treatment with the corresponding sample for 24 hours. Cell supernatants were collected, and NO content was detected using the Griess method; the secretion levels of TNF-α, IL-1β, and IL-6 in the supernatant were detected using ELISA. The results are shown in Table 3. The LPS model group is considered as 100%, and each treatment group represents a percentage of the model group; lower values ​​indicate stronger inhibition.

[0036] Table 3. Effects of each sample on LPS-induced expression of inflammatory factors in RAW264.7 cells (%) Table 3 shows that all examples significantly inhibited LPS-induced protein secretion of NO, TNF-α, IL-1β, and IL-6 (P<0.001), with Example 1 showing the best inhibitory effect, reducing all indicators to 28%-35% of the model group. Examples 2 and 3 were slightly less effective than Example 1 (P<0.05), but still significantly better than the comparative examples and Control Example 1 (P<0.01). The inhibitory effect of Comparative Example 1 was significantly weaker than that of Example 1, demonstrating that the composite hydrophilic shell layer formed by alternating loading can better exert the synergistic anti-inflammatory effect of the surface components. The inhibitory effect of Comparative Example 2 was further weakened due to the low encapsulation rate of the core components. Comparative Example 3 had the weakest inhibitory effect due to the low drug delivery efficiency caused by carrier structural defects. Notably, the NO content of Comparative Example 4, without a surface hydrophilic layer, was still as high as 65.7%, significantly higher than the 32.6% of Example 1, indicating that even with intact core component encapsulation, the lack of surface-loaded astragalus polysaccharide and rosmarinic acid leads to a significant decrease in anti-inflammatory activity, proving that the hydrophilic shell layer is indispensable in synergistic therapy. The inhibitory effect of Comparative Example 5, lacking chlorogenic acid and β-caryophyllene, was also significantly weaker than that of Example 1, indicating that the absence of chlorogenic acid and β-caryophyllene in the core severely weakened the long-lasting anti-inflammatory effect. While the physical mixture in Control Example 1 had some inhibitory effect, it was far less effective than those in the examples. The above in vitro cell experiments demonstrate that the complete combination of the five active ingredients and its spatially ordered carrier structure of this invention exhibit significantly superior technical effects at the cellular level against LPS-induced inflammatory responses compared to any single-component deficiency or structural defect scheme.

[0037] Experimental Example 4: Therapeutic effect of compound composition on LPS-induced mastitis in mice A mouse mastitis model induced by LPS lactiferous duct perfusion was used to evaluate the in vivo anti-inflammatory activity of the samples from each example, comparative example, and control example 1. Six-week-old SPF-grade Balb / c pregnant mice were selected, and the experiment was conducted one week after delivery. Mice were randomly divided into a blank control group, an LPS model group, each example group, each comparative example group, and control example 1 group, with six mice in each group. Except for the blank control group, the other groups of mice were infused with 50 μL of 0.2 mg / mL LPS through the lactiferous ducts of the fourth pair of nipples to establish a mastitis model. Drug administration began 24 hours after model establishment. The example groups, comparative example groups, and control example 1 group were administered the drug (equivalent to 20 mg / kg body weight of baicalin) through lactiferous duct perfusion every 24 hours for 3 consecutive days. The blank control group and the LPS model group received an equal volume of phosphate buffer. Twenty-four hours after the last administration, the mice were sacrificed, and blood and fourth pair of mammary gland tissue were collected. Serum TNF-α, IL-1β, and IL-6 levels were measured using ELISA. The results are shown in Table 4 (serum inflammatory factor content, pg / mL).

[0038] Table 4. Effects of each sample on serum inflammatory factors in LPS-induced mouse mastitis. As shown in Table 4, the levels of the three inflammatory factors in the serum of the LPS model group were significantly higher than those in the blank control group (P<0.001). All examples significantly reduced serum inflammatory factor levels (P<0.001), with Example 1 showing the best effect, reducing serum TNF-α to 98.2 pg / mL. Comparative Examples 1 and 2 showed some improvement but still exhibited significant inflammation; Comparative Example 3 showed limited improvement; Comparative Examples 4, 5, and Control Example 1 all showed significantly higher serum inflammatory factor levels than Example 1 (P<0.01), but were still superior to Control Example 1. These results further validated the cell experiment conclusions in Table 3 in vivo: both the surface hydrophilic layer and the intact core components are indispensable for achieving the best anti-mastitis damage effect. The in vivo anti-inflammatory effects of all examples were significantly better than all comparative examples and Control Example 1 (P<0.01), completely consistent with the in vitro experimental results.

[0039] Furthermore, HE staining was performed on the fourth pair of breast tissues from the blank control group, LPS model, Example 1, and Control Example 1. The results are as follows: Figure 1 As shown.

[0040] Depend on Figure 1It was found that the mammary gland tissue of the control group mice had a clear structure, with neatly and tightly arranged alveolar and ductal epithelial cells, normal cell morphology, and loose stroma. Only a few fibroblasts were observed, and there was no inflammatory cell infiltration. The mammary lobules were clearly structured, with round or oval alveoli and complete outlines. The glandular epithelial cells were neatly arranged in a single layer of cuboidal cells, with rounded nuclei located at the base and abundant cytoplasm. A small amount of homogeneous eosinophilic secretion was observed in the alveolar lumen. There was no edema in the stroma, only a very small number of fibroblast nuclei were observed, and no inflammatory cell infiltration such as neutrophils or macrophages was observed. The overall tissue morphology was normal, and there were no pathological changes.

[0041] Mild to moderate inflammatory response was observed in the mammary tissue of mice in the LPS model group: some acinar structures were irregular, epithelial cells showed mild edema and vacuolar degeneration, the interstitium was slightly widened, and blood vessels were dilated and congested. A small number of inflammatory cells, mainly neutrophils, were scattered or focally infiltrated, and a small amount of exudate was occasionally seen in the acinar lumen. Compared with the blank control group, the lobular structure of the mammary glands in the LPS group was still identifiable, but some acinar outlines were irregular, the interacinar spaces were slightly widened, and the interstitium showed a certain degree of edema. Some acinar epithelial cells showed mild vacuolar degeneration, and a few cells were detached into the lumen. Inflammatory cell infiltration of varying numbers, mainly neutrophils, accompanied by a small number of macrophages and lymphocytes, was observed in the acinar interstitium and lumen, in a focal or scattered distribution. Aggregates of inflammatory cells and a small amount of cell debris were occasionally seen in the lumen. No obvious necrosis or abscess formation was observed.

[0042] In Example 1, the structure of the mammary lobules in mice was largely restored, with clear alveolar outlines and relatively neat arrangement. Compared with the LPS group, interstitial edema was significantly reduced, and the interapical space was close to normal width. Mammary epithelial cell degeneration was improved, with only a few cells showing vacuolar degeneration and rare shedding. Inflammatory cell infiltration was significantly reduced, with occasional scattered neutrophils and no focal aggregation. Secretions within the mammary lumen were clear, and the overall tissue morphology was similar to the blank control group, with results approximately equal to the blank control group and significantly better than Control Example 1.

[0043] In control case 1, the lobular structure of the breast was improved compared to the LPS group. Most acini were still discernible, but mild widening of the interacini and mild interstitial edema were observed in some areas. The degree of degeneration of glandular epithelial cells was reduced, with scattered individual cell shedding. Inflammatory cell infiltration was reduced compared to the LPS model group, with a small number of neutrophils scattered throughout the interstitium, occasionally showing small focal clusters, but both in extent and density were smaller than in the LPS model group. Overall, the inflammatory response was partially alleviated.

[0044] In summary, the preparation method of the five-element co-loaded nanostructured lipid carrier compound composition of the present invention forms a well-structured, highly encapsulated, and orderly released nanodelivery system by constructing a complete surface hydrophilic layer and an optimized lipid core, thereby achieving synergistic therapy of the five active ingredients.

[0045] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the compound composition of the present invention for treating inflammatory mastitis and its preparation method will be readily apparent to those skilled in the art.

[0046] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A compound composition for treating inflammatory lesions of the mastoid process, characterized in that, It is composed of active ingredients and a nanostructured lipid carrier. The active ingredients consist of baicalin, chlorogenic acid, astragalus polysaccharide, rosmarinic acid, and rosemary oil. Rosemary oil serves as both an active ingredient and a liquid lipid component of the nanostructured lipid carrier, forming the lipid core matrix of the carrier. The β-caryophyllene content in the rosemary oil is 10% to 30%. The nanostructured lipid carrier also contains solid lipids and stearamine. The solid lipids are at least one of glyceryl monostearate or stearic acid. The average particle size of the nanostructured lipid carrier is 50-200 nm. Astragalus polysaccharide and rosmarinic acid are loaded on the surface of the nanostructured lipid carrier to form a hydrophilic outer shell. Baicalin and chlorogenic acid, together with rosemary oil, are encapsulated in the lipid core of the nanostructured lipid carrier. The compound composition is prepared by a method comprising the following steps: Step 1: Weigh baicalin, chlorogenic acid, astragalus polysaccharide, rosmarinic acid, and rosemary oil in a weight ratio of 1:4-6:20-30:0.5-1:20-40. Dissolve baicalin, chlorogenic acid, and all the rosemary oil in anhydrous ethanol to obtain an oil phase; Step 2: Add at least one of the solid lipids, glyceryl monostearate or stearic acid, and stearamine to the oil phase. The weight ratio of the solid lipids to rosemary oil is 1:1.5 to 1:2.

5. Heat at 70°C to 80°C to completely melt the solid lipids and stearamine and mix them evenly with the oil phase to obtain a molten oil phase; Step 3: Dropwise add the molten oil phase to a phosphate buffer containing an emulsifier, poloxamer 188 or Tween-80, at a temperature of 70°C to 80°C. The emulsifier concentration is 5-20%. mg / mL, high-speed shearing at 10000-15000 r / min for 5-10 minutes to form a nanoemulsion; Step four, sonicate the nanoemulsion in a 0-2℃ water bath for 5-15 minutes at an ultrasonic power of 200-400 W to obtain a nanostructured lipid carrier suspension loaded with baicalin, chlorogenic acid, and β-caryophyllene; Step five, dilute the nanostructured lipid carrier suspension with phosphate buffer to a solid lipid content of 5-10%. mg / mL, under stirring conditions, phosphate buffer solution of astragalus polysaccharide and phosphate buffer solution of rosmarinic acid are added alternately in sequence, with stirring and incubation for 15-45 minutes after each addition, so that astragalus polysaccharide and rosmarinic acid are stepwise saturated and co-adsorbed on the surface of the nanostructured lipid carrier to form a hydrophilic outer shell layer of astragalus polysaccharide / rosmarinic acid composite; repeat the alternating loading operation 1-3 times to obtain a five-component co-loaded nanostructured lipid carrier suspension; step six, the five-component co-loaded nanostructured lipid carrier suspension is centrifuged at 30000-45000g for 30-60 minutes at high speed, with the centrifugation temperature controlled at 2-8℃, the precipitate is collected, and washed 2 to 3 times with phosphate buffer to obtain the compound composition.

2. The compound composition for treating inflammatory lesions of the mastoid process as described in claim 1, characterized in that, The amount of stearamine added is 5%-8% of the solid lipid.

3. The compound composition for treating inflammatory lesions of the mastoid process as described in claim 2, characterized in that, The solid lipid is a mixture of glyceryl monostearate and stearic acid, with a weight ratio of glyceryl monostearate to stearic acid of 2:1 to 4:

1.

4. The method for preparing the compound composition according to claim 1, characterized in that, Includes the following steps: Step 1: Weigh baicalin, chlorogenic acid, astragalus polysaccharide, rosmarinic acid and rosemary oil in a weight ratio of 1:4-6:20-30:0.5-1:20-40, wherein the β-caryophyllene content in the rosemary oil is 10% to 30%. Dissolve baicalin, chlorogenic acid and all the rosemary oil in anhydrous ethanol to obtain the oil phase. Step 2: Add at least one of the solid lipids, glyceryl monostearate or stearic acid, and stearamine to the oil phase. The weight ratio of solid lipids to rosemary oil is 1:1.5 to 1:2.

5. Heat at 70°C to 80°C to completely melt the solid lipids and stearamine and mix them evenly with the oil phase to obtain a molten oil phase. Step 3: The molten oil phase is dripped into a phosphate buffer containing an emulsifier, such as poloxamer 188 or Tween-80, at a temperature of 70°C to 80°C. The emulsifier concentration is 5-20 mg / mL. The mixture is then subjected to high-speed shearing at 10000-15000 r / min for 5-10 minutes to form a nanoemulsion. Step 4: The nanoemulsion is ultrasonically treated in a water bath at 0-2℃ for 5-15 minutes with an ultrasonic power of 200-400W to obtain a nanostructured lipid carrier suspension loaded with baicalin, chlorogenic acid and β-caryophyllene. Step 5: Dilute the nanostructured lipid carrier suspension with phosphate buffer to a solid lipid content of 5-10 mg / mL. Under stirring, alternately add phosphate buffer solution of astragalus polysaccharide and phosphate buffer solution of rosmarinic acid, stirring and incubating for 15-45 minutes after each addition, so that astragalus polysaccharide and rosmarinic acid are stepwise saturated and co-adsorbed on the surface of the nanostructured lipid carrier, forming a hydrophilic outer shell layer of astragalus polysaccharide / rosmarinic acid composite. Repeat the alternating loading operation 1-3 times to obtain the five-component co-loaded nanostructured lipid carrier suspension. Step 6: Centrifuge the pentagonal co-loaded nanostructured lipid carrier suspension at 30,000-45,000g at high speed for 30-60 minutes, controlling the centrifugation temperature at 2-8℃. Collect the precipitate and wash it 2 to 3 times with phosphate buffer to obtain the compound composition.

5. The method for preparing the compound composition according to claim 4, characterized in that, In step one, when preparing the oil phase, baicalin is first added to anhydrous ethanol and heated and stirred at 40°C to 60°C until dissolved. Then, chlorogenic acid is added and stirred until dissolved. Finally, the entire amount of rosemary oil is added and mixed well.

6. The method for preparing the compound composition according to claim 5, characterized in that, In step two, the solid lipid is a mixture of glyceryl monostearate and stearic acid, with a weight ratio of glyceryl monostearate to stearic acid of 2:1 to 4:1, and the amount of stearamine added is 5%-8% of the solid lipid.

7. The method for preparing the compound composition according to claim 6, characterized in that, In step three, the molten oil phase is kept at 70°C to 80°C and stirred at a constant speed of 200-400 r / min for 15-30 minutes, and then dropped into the phosphate buffer containing the emulsifier at a dropping rate of 2 ml / min.

8. The method for preparing the compound composition according to claim 7, characterized in that, In step five, the concentration of the phosphate buffer solution of Astragalus polysaccharide is 0.5-5 mg / mL, and the concentration of the phosphate buffer solution of rosmarinic acid is 0.1-2 mg / mL; the temperature for stirring and incubation is 15-30℃, and the stirring speed is 100-300 rpm.

9. The use of the compound composition as described in claim 1 in the preparation of a medicament for treating inflammatory lesions of the mastoid process.

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