Sinomenine-ursolic acid nanoemulsion, and preparation method and application thereof

Abstract

The application relates to the technical field of medicines, in particular to a sinomenine-ursolic acid nanoemulsion as well as a preparation method and application thereof. The sinomenine-ursolic acid nanoemulsion formula is composed of coix seed oil, bletilla striata polysaccharide, glycyrrhizic acid, sinomenine, ursolic acid and water. The sinomenine-ursolic acid nanoemulsion can be efficiently targeted to arthritic inflammation sites, can strongly relieve rheumatoid arthritis inflammation and joint damage, can regulate the immune state of the body, can reduce drug toxicity, and simultaneously has excellent biological safety. The application realizes synergistic attenuation and combination of medicine and adjuvant, provides technical support for developing a novel anti-rheumatoid arthritis drug with high efficiency, low toxicity and active targeting capability and a delivery strategy, and has great development and application value.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a sinomenine-ursolic acid nanoemulsion, its preparation method, and its application. Background Technology

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by erosive joint inflammation. Its core pathological features include abnormal proliferation of synovial cells, widespread microangiogenesis, extensive infiltration of inflammatory cells, and progressive destruction of articular cartilage and bone. As the disease progresses, RA not only leads to joint deformities and loss of function but also frequently results in serious extra-articular complications affecting the cardiovascular and pulmonary systems, causing a decline in physical function and quality of life, and imposing a significant economic burden on patients, their families, and society.

[0003] Currently, the main drugs used clinically to treat rheumatoid arthritis (RA) include nonsteroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs, such as methotrexate MTX), glucocorticoids, and biologics. Although these drugs have achieved certain clinical efficacy, they still have limitations such as poor targeting and significant systemic toxicity. Therefore, finding novel antirheumatic drugs and delivery strategies that are highly effective, low in toxicity, and possess active targeting capabilities is a major scientific problem that urgently needs to be solved in this field.

[0004] In recent years, the active ingredients of natural Chinese medicine have shown great potential in the treatment of rheumatoid arthritis (RA) due to their synergistic anti-inflammatory advantages across multiple targets and pathways. Sinomenine (SIN), an alkaloid extracted from the Menispermaceae plant *Ipomoea quamoclit*, possesses significant anti-inflammatory and immunosuppressive effects. Ursolic acid (UA), a pentacyclic triterpenoid compound widely found in natural plants, shows great potential in combating oxidative stress and protecting cartilage. These two compounds are complementary in their pharmacological mechanisms, and their combined application can exert a significant synergistic effect, from blocking inflammatory mediators and inhibiting oxidative stress to repairing the synovial microenvironment. However, these two natural monomers have significant drawbacks in terms of drug-likeness: SIN has a short half-life, and high-dose free administration can easily trigger toxic side effects; UA has low permeability and is poorly soluble in water. Furthermore, neither can spontaneously accumulate in inflamed joints in vivo, resulting in significantly reduced efficacy when used alone or in combination.

[0005] Based on this, the present invention proposes a nanoemulsion system simultaneously loaded with sinomenine and ursolic acid, aiming to solve the above-mentioned problems in the prior art and provide technical support for the clinical targeted combination therapy of rheumatoid arthritis. Summary of the Invention

[0006] The present invention aims to provide a sinomenine-ursolic acid nanoemulsion and its preparation method, and another objective is to provide the application of the nanoemulsion in the preparation of drugs for treating rheumatoid arthritis.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The sinomenine-ursolic acid nanoemulsion formulation of this invention is composed of coix seed oil, Bletilla striata polysaccharide, glycyrrhizic acid, sinomenine, ursolic acid and water, wherein: the volume fraction of coix seed oil is 1%~5%; the mass ratio of Bletilla striata polysaccharide to glycyrrhizic acid is 1:9~9:1, and its total mass fraction in water is 0.1%~1.0%; the mass ratio of sinomenine to ursolic acid is 1:1, and the total addition amount is 5~15mg per 100mL of sinomenine-ursolic acid nanoemulsion.

[0008] Preferably, in the formulation of the sinomenine-ursolic acid nanoemulsion of the present invention: the volume fraction of coix seed oil is 2%~4%; the mass ratio of Bletilla striata polysaccharide to glycyrrhizic acid is 1:9~3:7, and its total mass fraction in water is 0.4%-0.6%; the total addition amount of sinomenine and ursolic acid is 10~12.5mg per 100mL of sinomenine-ursolic acid nanoemulsion.

[0009] In a further preferred embodiment, the formulation of the sinomenine-ursolic acid nanoemulsion of the present invention comprises: 3% by volume of coix seed oil; a mass ratio of Bletilla striata polysaccharide to glycyrrhizic acid of 1:9, with a total mass fraction of 0.5% in water; and a total addition amount of sinomenine and ursolic acid of 12.5 mg per 100 mL of sinomenine-ursolic acid nanoemulsion.

[0010] Preferably, in the formulation of the sinomenine-ursolic acid nanoemulsion of the present invention, the water is deionized water.

[0011] The specific steps of the preparation method of the sinomenine-ursolic acid nanoemulsion of the present invention are as follows: accurately weigh the prescribed amount of glycyrrhizic acid and Bletilla striata polysaccharide, add them to water, and dissolve them by sonication and heating to form the aqueous phase; accurately weigh the prescribed amount of sinomenine and ursolic acid, add them to the prescribed amount of coix seed oil to form the oil phase; pour the aqueous phase into the oil phase, stir evenly, and sonicate at 130-360W power for 2-10 minutes to obtain the nanoemulsion.

[0012] Preferably, in the preparation method of the sinomenine-ursolic acid nanoemulsion of the present invention, the power of the ultrasound is 260-360W.

[0013] In a further preferred embodiment, in the preparation method of the sinomenine-ursolic acid nanoemulsion of the present invention, the power of the ultrasound is 325W.

[0014] Preferably, in the preparation method of the sinomenine-ursolic acid nanoemulsion of the present invention, the ultrasonication time is 8-10 min.

[0015] In a further preferred embodiment, in the preparation method of the sinomenine-ursolic acid nanoemulsion of the present invention, the ultrasonication time is 10 min.

[0016] The application of the sinomenine-ursolic acid nanoemulsion described in this invention in the preparation of drugs for treating rheumatoid arthritis.

[0017] The beneficial effects of this invention are: 1. This invention successfully constructed a stable and controllable "drug-excipient integrated" dual-drug co-loaded nanoemulsion system, SIN-UA-NE, and determined its optimal formulation and process (3% coix seed oil, 0.5% composite emulsifier, BSP:GA mass ratio 1:9, and ultrasonic treatment at 325W for 10 min) through single-factor and orthogonal experiments. SIN-UA-NE prepared under the optimal formulation and process conditions exhibits a uniform O / W-type spherical structure with excellent thermodynamic and kinetic stability. At a drug loading of 12.5 mg, the encapsulation efficiency of both SIN and UA reaches over 85%, effectively overcoming the co-loading challenge caused by the differences in the physicochemical properties of the two drugs. Furthermore, the SIN-UA-NE of this invention demonstrates excellent sustained-release and controlled-release performance in an in vitro release model, which is beneficial for prolonging the circulation time of the drug in vivo and improving bioavailability.

[0018] 2. The SIN-UA-NE provided by this invention exhibits excellent synergistic anti-inflammatory effects at the in vitro cellular level. It can effectively inhibit the abnormal proliferation of synovial fibroblasts (inhibition rate up to 84.45%) and efficiently remove excess reactive oxygen species (ROS) accumulated in cells in a dose-dependent manner. At the same time, it can significantly downregulate the secretion levels of core pro-inflammatory factors such as TNF-α, IL-6 and IL-1β. Its regulatory efficacy is comprehensively superior to that of free single-agent physical mixtures.

[0019] 3. In vivo pharmacodynamic evaluation results show that SIN-UA-NE provided by this invention can effectively target the site of joint inflammation, strongly relieving inflammation and joint damage in rheumatoid arthritis. Furthermore, SIN-UA-NE can regulate the body's immune status, reduce drug toxicity, and possesses excellent biocompatibility. This invention achieves synergistic enhancement and toxicity reduction, as well as drug-excipient integration, providing technical support for the development of novel, highly effective, low-toxicity anti-rheumatoid arthritis drugs and delivery strategies with active targeting capabilities. Attached Figure Description

[0020] Figure 1 Appearance and particle size of nanoemulsions prepared from coix seed oil with different volume fractions; Figure 2 Nanoemulsion particle size and appearance prepared with composite surfactants of different mass fractions; Figure 3 Nanoemulsion particle size and appearance prepared for different BSP to GA mass ratios; Figure 4 The effect of different ultrasonic times on the appearance and particle size of nanoemulsions; Figure 5 The effect of different ultrasonic powers on the appearance and particle size of nanoemulsions; Figure 6 The appearance and particle size of nanoemulsions under different amounts of sinomenine and ursolic acid; Figure 7 Nanoemulsion particle size and appearance prepared with different surfactants (in the figure: A is single BSP; B is single GA; C is BSP-GA with a mass ratio of 1:9). Figure 8 Appearance of three batches of nanoemulsions prepared according to the optimal formulation; Figure 9 The physicochemical characterization results of the co-loaded sinomenine and ursolic acid nanoemulsion are shown in the figure (A is the type identification result of SIN-UA-NE; B is the Tyndall effect of SIN-UA-NE; C is the TEM image of SIN-UA-NE; D is the particle size distribution of SIN-UA-NE; E is the zeta potential distribution of SIN-UA-NE). Figure 10 The particle size, PDI and zeta potential changes, and appearance of SIN-UA-NE at different dilution factors; Figure 11 Particle size, PDI and zeta potential changes, and appearance of SIN-WA-NE after 30 days of storage; Figure 12 The changes in particle size, PDI and ζ potential, and appearance of SIN-UA-NE before and after centrifugation at different speeds are shown. Figure 13 The changes in particle size, PDI and zeta potential, and appearance of SIN-WA-NE after being stored at 60℃ for 10 days; Figure 14 The drug loading and encapsulation efficiency of nanoemulsions under different amounts of sinomenine and ursolic acid were determined. Figure 15 Cumulative release curves of sinomenine and ursolic acid in different formulations (in the figure: A is sinomenine; B is ursolic acid). Figure 16 The results of the establishment and validation of the MH7A cell inflammation model are shown in the figure (A shows the effect of different concentrations of LPS on the viability of MH7A cells; B and C show the effects of LPS on the secretion of IL-6 and TNF-α by MH7A cells, respectively). Figure 17 Results of cell viability screening experiments; Figure 18 The effect of SIN-UA-NE on LPS-induced abnormal proliferation of MH7A cells; Figure 19The effect of SIN-UA-NE on LPS-induced NO release and secretion of TNF-α, IL-6, and IL-1β in MH7A cells; Figure 20 The fluorescence intensity of MH7A cells from different groups at various time points; Figure 21 The effect of different concentrations of SIN-UA-NE on ROS levels in MH7A cells; Figure 22 The trends of toe thickness, AI score, and body weight changes in rats in each group during model establishment and drug administration (in the figure: A is the trend of toe thickness change; B is the trend of AI score change; C is the trend of body weight change). Figure 23 The morphological appearance of the paws of CIA rats in each group at different time points; Figure 24 The effects of different drug administration groups on the immune organ index of CIA rats; Figure 25 The effects of different drug administration groups on serum inflammatory factors (TNF-α, IL-6, IL-1β) in CIA rats; Figure 26 Micro-CT imaging scans of the ankle joints of the hind limbs of rats in each group; Figure 27 For Micro-CT bone microstructure parameter analysis; Figure 28 H&E staining was performed on the ankle joints of rats in each group. Detailed Implementation

[0021] The technical solution of the present invention will be described in detail below with reference to specific embodiments. The following embodiments are for explanation and illustration only, and do not constitute a limitation on the technical solution of the present invention.

[0022] Example 1

[0023] The formulation of the sinomenine-ursolic acid nanoemulsion consists of coix seed oil, Bletilla striata polysaccharide, glycyrrhizic acid, sinomenine, ursolic acid, and water. The volume fraction of coix seed oil is 3%; the mass ratio of Bletilla striata polysaccharide to glycyrrhizic acid is 1:9, and its total mass fraction in water is 0.5%; the mass ratio of sinomenine to ursolic acid is 1:1, and 12.5 mg of sinomenine-ursolic acid nanoemulsion is added for every 100 mL of sinomenine-ursolic acid nanoemulsion prepared.

[0024] Example 2

[0025] The formulation of the sinomenine-ursolic acid nanoemulsion consists of coix seed oil, Bletilla striata polysaccharide, glycyrrhizic acid, sinomenine, ursolic acid, and water. The volume fraction of coix seed oil is 1%; the mass ratio of Bletilla striata polysaccharide to glycyrrhizic acid is 1:9, and its total mass fraction in water is 0.1%; the mass ratio of sinomenine to ursolic acid is 1:1, and 5 mg of sinomenine-ursolic acid nanoemulsion is added for every 100 mL of sinomenine-ursolic acid nanoemulsion prepared.

[0026] Example 3

[0027] The formulation of the sinomenine-ursolic acid nanoemulsion consists of coix seed oil, Bletilla striata polysaccharide, glycyrrhizic acid, sinomenine, ursolic acid, and water. The volume fraction of coix seed oil is 5%; the mass ratio of Bletilla striata polysaccharide to glycyrrhizic acid is 9:1, and its total mass fraction in water is 1.0%; the mass ratio of sinomenine to ursolic acid is 1:1, and 15 mg of sinomenine-ursolic acid nanoemulsion is added for every 100 mL of sinomenine-ursolic acid nanoemulsion prepared.

[0028] Example 4

[0029] The sinomenine-ursolic acid nanoemulsion was prepared using the formulations of Examples 1-3. The specific steps were as follows: accurately weigh the prescribed amounts of glycyrrhizic acid and Bletilla striata polysaccharide, add them to water, and dissolve them by sonication and heating to form the aqueous phase; accurately weigh the prescribed amounts of sinomenine and ursolic acid, add them to the prescribed amounts of coix seed oil to form the oil phase; pour the aqueous phase into the oil phase, stir evenly, and sonicate at 325 W power for 10 min to obtain the final product.

[0030] Example 5

[0031] The sinomenine-ursolic acid nanoemulsion was prepared using the formulations of Examples 1-3. The specific steps were as follows: accurately weigh the prescribed amounts of glycyrrhizic acid and Bletilla striata polysaccharide, add them to water, and dissolve them by sonication and heating to form the aqueous phase; accurately weigh the prescribed amounts of sinomenine and ursolic acid, add them to the prescribed amounts of coix seed oil to form the oil phase; pour the aqueous phase into the oil phase, stir evenly, and sonicate at 130 W power for 10 min to obtain the final product.

[0032] Example 6

[0033] The formulations of Examples 1-3 were used to prepare sinomenine-ursolic acid nanoemulsions. The specific steps were as follows: accurately weigh the prescribed amounts of glycyrrhizic acid and Bletilla striata polysaccharide, add them to water, and dissolve them by sonication and heating to form the aqueous phase; accurately weigh the prescribed amounts of sinomenine and ursolic acid, add them to the prescribed amounts of coix seed oil to form the oil phase; pour the aqueous phase into the oil phase, stir evenly, and sonicate at 360 W power for 2 min to obtain the final product.

[0034] Example 7

[0035] The sinomenine-ursolic acid nanoemulsion prepared according to the preparation methods in Examples 4-6 was used as a treatment for rheumatoid arthritis.

[0036] To further verify the reliability of the present invention and select the optimal solution, the inventors conducted a series of experiments, as follows: 1. Formulation optimization and physicochemical characterization of nanoemulsions co-loaded with sinomenine and ursolic acid 1.1 Materials and Instruments The experimental instruments are shown in Table 1, and the experimental materials are shown in Table 2.

[0037] ; .

[0038] 1.2 Experimental Methods 1.2.1 Preparation method of nanoemulsion co-loaded with sinomenine and ursolic acid Using ultrasonic emulsification, precisely weighed amounts of glycyrrhizic acid and Bletilla striata polysaccharide were added to deionized water and dissolved by ultrasonication and heating to form the aqueous phase. Precise amounts of coix seed oil were placed in a glass bottle, and precisely weighed amounts of sinomenine and ursolic acid were added. The mixture was ultrasonicated to ensure uniform dispersion in the coix seed oil, forming the oil phase. The aqueous phase was then poured into the oil phase and stirred thoroughly with a magnetic stirrer. Subsequently, the mixture was ultrasonicated for 10 minutes using a cell disruptor to obtain SIN-UA-NE.

[0039] 1.2.2 Single-factor investigation experiment To obtain the optimal ratio and process of co-loaded sinomenine and ursolic acid nanoemulsion, the appearance, average particle size, and polydispersity index (PDI) of the emulsion were used as comprehensive evaluation indicators. The following were investigated: different volume fractions of coix seed oil (1%, 2%, 3%, 4%, 5%); different total mass fractions of composite surfactants in the aqueous phase (with a fixed BSP to GA mass ratio of 1:1, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%); different BSP to GA mass ratios (1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1); different ultrasonic times (2 min, 4 min, 6 min, 8 min, 10 min); and different ultrasonic powers (130 W, 195 W, 260 W, 325 W, 360 W). The effects of different total dosages (fixed SIN to UA mass ratio of 1:1, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg) on ​​the emulsification effect and system stability of nanoemulsions.

[0040] 1.2.3 Comparative Evaluation of the Performance of Composite Surfactants and Single Surfactants To verify the superiority of the compound system, the emulsifying performance and storage stability of single surfactants (BSP, GA) and compound surfactants (BSP-GA, 1:9) were further compared.

[0041] 1.2.4 Orthogonal experimental design to optimize the formulation and process of co-loaded sinomenine and ursolic acid nanoemulsion Based on the results of the single-factor investigation, the changes in the oil phase ratio, the total amount of composite surfactant, and the mass ratio of BSP to GA had a significant impact on particle size and PDI. Therefore, the oil phase content (A, 2%, 3%, 4%), the total amount of composite surfactant (B, 0.4%, 0.5%, 0.6%), and the mass ratio of BSP to GA (C, 3:7, 1:9, 2:8) were determined as the factors to be investigated, and an L9 (3 4 Orthogonal experiment. Particle size and PDI were normalized, and the overall score (CS) was calculated using weighted coefficients (particle size 0.7, PDI 0.3). The calculation formula is as follows: Particle size normalization = (current group particle size - minimum particle size) / (maximum particle size - minimum particle size); PDI normalization = (current group PDI - minimum PDI) / (maximum PDI - minimum PDI); CS = 0.7 × (normalized particle size) + 0.3 × (normalized PDI).

[0042] 1.2.5 Determination and Verification Experiment of Optimal Prescription Based on the results of single-factor screening, orthogonal experimental optimization, and drug loading investigation, the optimal formulation and process conditions for SIN-UA-NE were finally determined as follows: 3% coix seed oil, 0.5% total amount of composite emulsifier, BSP to GA mass ratio of 1:9, ultrasonic power of 325 W, ultrasonic treatment for 10 min, and drug dosage of 12.5 mg (SIN:UA=1:1). Three batches of nanoemulsions were prepared in parallel under the above optimal conditions for verification experiments.

[0043] 1.2.6 Physicochemical characterization of nanoemulsions co-loaded with sinomenine and ursolic acid Using methylene blue as a water-soluble dye and Sudan red as an oil-soluble dye, the emulsion type of SIN-UA-NE was identified by the staining-diffusion method. The macroscopic properties of SIN-UA-NE under natural light were observed and recorded. After appropriate dilution, the samples were irradiated with a red laser beam in a dark room to further verify the colloidal properties of the system. The microstructure of SIN-UA-NE was observed and recorded at a size of 200 nm using TEM. The average particle size and PDI of SIN-UA-NE were determined by dynamic light scattering (DLS), and its zeta potential was also measured. The pH of SIN-UA-NE was measured at room temperature (25℃) using a calibrated digital pH meter. The viscosity of SIN-UA-NE was measured using an NDJ-8S rotational viscometer (rotor 0, rotation speed 6.0 r / min). The turbidity of SIN-UA-NE was determined by ultraviolet-visible spectrophotometry.

[0044] 1.2.7 Stability Study of Nanoemulsions Co-loaded with Tendrine and Ursolic Acid Using appearance, average particle size, PDI, and zeta potential as comprehensive evaluation indicators, the dilution stability (diluted with deionized water at 50, 100, 200, and 1000 times), storage stability (stored at 25℃ in the dark for 30 days, with samples taken and measured on days 0, 7, 14, 21, 28, and 30), centrifugation stability (centrifugation speeds of 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, and 10000 r / min for 10 min), and thermal stability (stored at 60℃, with samples taken and measured on days 0, 3, 5, 7, and 10) were investigated.

[0045] 1.3 Experimental Results 1.3.1 Results of Single-Factor Experiment (1) Oil phase screening results See results Figure 1 Within the 1%-5% range observed, the nanoemulsion particle size and appearance showed significant variations. Both excessively low (<3%) and excessively high (>3%) oil phase concentrations were detrimental to system stability, leading to either a broadened particle size distribution or the presence of unemulsified free oil droplets, respectively. When the volume fraction of coix seed oil was 3%, the resulting nanoemulsion exhibited the smallest average particle size (191.90 nm), a narrow distribution, and a clear and uniform appearance. Therefore, 3% was selected as the optimal oil phase concentration for this system.

[0046] (2) Screening results of the amount of compound surfactant See results Figure 2 When the mass fraction of the composite surfactant was too low (0.1%-0.3%), the oil phase was not sufficiently emulsified, resulting in a large particle size (≥206.90 nm). As the dosage increased to 0.4%-0.5%, emulsification became more complete, and the particle size significantly decreased to 182.80-186.70 nm, while the PDI remained at a low level (approximately 0.141), indicating that the emulsion was uniform and stable. Further increasing the dosage to 0.6%-1.0% caused the particle size to increase again (189.00-221.10 nm), suggesting that excessive surfactant was actually detrimental to the formation of uniform nanoemulsions. Considering all factors, the optimal dosage of the composite surfactant was determined to be 0.5%.

[0047] (3) Effect of the ratio of Bletilla striata polysaccharide and glycyrrhizic acid on the performance of nanoemulsions See results Figure 3As the proportion of BSP in the composite emulsifier increases, the particle size of the nanoemulsion gradually increases, rising from 187.80 nm when the BSP to GA mass ratio is 1:9 to 248.00 nm when it is 9:1. Meanwhile, the PDI is lowest (0.121) at the BSP to GA mass ratio of 1:9, indicating the most uniform emulsion distribution under this condition. As the ratio deviates from this value, the PDI and particle size generally increase, and the nanoemulsion exhibits stratification, suggesting a decrease in system homogeneity and stability. Considering all factors, the optimal mass ratio of BSP to GA is determined to be 1:9.

[0048] (4) Screening of ultrasound time See results Figure 4 As the sonication time increased, both the particle size and PDI of the nanoemulsion gradually decreased. Increasing the sonication time from 2 min to 10 min significantly reduced the particle size from 262.80 nm to 144.80 nm, and the PDI also decreased from 0.114 to 0.075, indicating that the homogeneity and stability of the system improved with increasing sonication time. Shorter sonication times (e.g., 2 min) resulted in incomplete emulsification due to insufficient energy input, leading to stratification, larger particle sizes, and uneven distribution. When the sonication time was extended to 10 min, the system achieved sufficient dispersion, with the smallest particle size, narrowest distribution, and a uniform emulsion appearance. Considering all factors, the optimal sonication time was determined to be 10 min.

[0049] (5) Screening of ultrasonic power See results Figure 5 As the ultrasonic power increased from 130 W to 325 W, the nanoemulsion particle size gradually decreased from 219.40 nm to 181.90 nm, and the PDI also decreased from 0.164 to 0.122, indicating that appropriately increasing the power helps improve droplet uniformity and system stability. However, when the power was further increased to 360 W, both the particle size and PDI rebounded (to 193.90 nm and 0.164, respectively). Taking all factors into consideration, the optimal ultrasonic power was finally determined to be 325 W.

[0050] (6) Screening of drug dosage See results Figure 6 Particle size and PDI analysis showed that at a total dosage of 12.5 mg, the average particle size of the system reached a minimum of 143.70 nm, and the PDI was 0.105, exhibiting excellent dispersibility. However, when the dosage was further increased to 15 mg, the particle size rebounded to 180 nm, and the PDI increased dramatically to 0.447. At this point, the system distribution was extremely uneven, indicating that the drug loading had exceeded the solubility limit of the nanoemulsion, leading to droplet aggregation or drug precipitation. Therefore, this experiment preferred 12.5 mg as the total dosage of SIN and UA (i.e., 6.25 mg SIN and 6.25 mg UA).

[0051] 1.3.2 Comparative Evaluation of the Performance of Composite Surfactants and Single Surfactants See results Figure 7 Nanoemulsions prepared with a single surfactant had a large initial particle size, and after 7 days at room temperature, the particle size further increased and was accompanied by stratification, indicating poor stability. In contrast, nanoemulsions prepared using a BSP / GA composite surfactant at a mass ratio of 1:9 not only had a suitable particle size but also exhibited good storage stability, uniform appearance, and no stratification. Considering all factors, the optimal mass ratio of BSP to GA was determined to be 1:9.

[0052] 1.3.3 Results of Orthogonal Experiments The orthogonal experimental factors and levels, experimental design, and comprehensive scoring results are shown in Table 3. Range analysis revealed that the order of influence of each factor on the SIN-UA-NE comprehensive score was: A (oil phase dosage) > B (composite surfactant dosage) > C (BSP to GA mass ratio). The absolute value of the range R further confirmed that the oil phase ratio is the most critical factor affecting the physicochemical properties of this nanoemulsion system. According to K-value analysis, the optimal level for each factor was K², i.e., the optimal formulation combination was A²B²C² (oil phase dosage 3%, total composite surfactant dosage 0.5%, BSP to GA mass ratio 1:9). Further variance analysis was conducted, and the results are shown in Table 4. The data show that factors A, B, and C all have a significant impact on the comprehensive evaluation index of the nanoemulsion (P < 0.05). This result verifies the scientific validity of the single-factor screening conclusion, indicating that the selected parameter range is reasonable and has a clear regulatory effect on the system quality.

[0053] ; .

[0054] 1.3.4 Optimal Prescription Validation The three batches of nanoemulsions prepared according to the optimal formulation were uniform milky white liquids. Figure 8 The particles were similar in size, ranging from 141.20 to 150.30 nm, and the PDI was controlled below 0.15 (Table 3). The results show that the preparation process is stable, the formulation has good reproducibility, and the optimal formulation selected has high feasibility and scientific validity.

[0055] 1.3.5 Physicochemical characterization results of nanoemulsions co-loaded with sinomenine and ursolic acid The physicochemical characterization results of the co-loaded sinomenine and ursolic acid nanoemulsion are shown in the figure. Figure 9 .

[0056] Type identification results ( Figure 9A) The results show that the hydrophilic methylene blue diffuses rapidly to the surrounding area after being added, making the entire system a uniform blue color; while the lipophilic Sudan Red diffusion is hindered after being added, and it only remains on the liquid surface without obvious diffusion, indicating that SIN-UA-NE is an O / W type nanoemulsion.

[0057] Results of observation of appearance characteristics ( Figure 9 B) Results: SIN-UA-NE appears as a uniform, fine, milky-white liquid with good fluidity. After standing, there is no stratification, flocculation, or oil droplet precipitation. When the light beam passes through the emulsion, a bright and clear "path" appears, exhibiting a significant Tyndall effect, confirming the presence of a large number of nanoscale dispersed particles in the system, which is consistent with the characteristics of colloidal dispersions.

[0058] Microscopic morphology observation ( Figure 9 C) The results showed that SIN-UA-NE appeared as a regular, well-defined spherical or near-spherical entity under electron microscopy; the droplets were uniform in size and well-dispersed, with no obvious droplet aggregation, adhesion or breakage; the particle size observed by TEM was about 150 nm, which was basically consistent with the results measured by Malvern particle size analyzer, further confirming that the system constructed a uniform and stable nanodispersion system.

[0059] Particle size distribution results ( Figure 9 D) The average particle size of SIN-UA-NE is (145.30±2.35) nm, and the particle size distribution curve is normally distributed; the PDI value is 0.095±0.022, indicating that the particle size distribution of the system is extremely narrow and exhibits excellent monodispersity.

[0060] Figure 9 E shows that the zeta potential of the system is (-39.51±0.43) mV. This high negative charge value indicates that sufficient electrostatic repulsion has accumulated on the surface of the droplets, which can effectively prevent the aggregation and fusion of droplets, suggesting that the nanoemulsion has good physical stability.

[0061] pH measurement results showed that the pH value of the nanoemulsion was 3.990±0.033 (n=3). Considering that the surface of human skin is weakly acidic, this pH range meets the safety requirements of transdermal drug delivery formulations, which helps to reduce irritation to skin tissue while ensuring drug stability.

[0062] Viscosity measurements showed that the average viscosity of the nanoemulsion was (66.32±4.02) mPa·s (n=3), indicating that SIN-UA-NE is a low-viscosity fluid with good flowability. This low-viscosity characteristic allows the nanoemulsion to rapidly spread and form a uniform film upon contact with the skin, increasing the contact area between the drug and the skin and creating favorable conditions for subsequent transdermal drug absorption.

[0063] Turbidity measurements showed that the average turbidity of the nanoemulsion was (16.16 ± 2.29) cm⁻¹. -1 (n=3) quantitatively reflects the scattering effect of the nanoemulsion droplets on incident light, which is consistent with the translucent appearance and Tyndall effect observed in the appearance properties, indicating that the system is uniformly dispersed and the droplet size is controlled at the nanoscale.

[0064] 1.3.6 Results of the stability test of nanoemulsion co-loaded with sinomenine and ursolic acid The results of the dilution stability test are as follows: Figure 10 As shown, SIN-UA-NE remained clear and transparent even at high dilutions (1000-fold), exhibiting a characteristic pale blue opalescence. No demulsification, layering, or drug crystallization was observed to the naked eye. Quantitative data showed that the average particle size of the system fluctuated very little with increasing dilution factor, and the zeta potential remained at a high negative charge level below -40 mV. These results confirm that the composite interfacial film formed by glycyrrhizic acid and Bletilla striata polysaccharide is tightly bound and did not undergo desorption or micelle disintegration due to the increase in continuous phase volume, indicating that this nanoemulsion system has excellent anti-dilution stability.

[0065] The results of the storage stability test are as follows Figure 11 As shown in the figure, during the 30-day observation period, SIN-UA-NE maintained a uniform emulsion appearance, and no physical instability phenomena such as stratification, flocculation, or demulsification were observed. Particle size monitoring data showed that with the extension of storage time, the average particle size of the nanoemulsion only fluctuated slightly, the PDI remained below 0.3, and the zeta potential remained at a high negative charge level between -34 and -48 mV. These results confirm that the system has excellent kinetic stability.

[0066] The results of the centrifugal stability test are as follows Figure 12 As shown, after centrifugation at different speeds, the appearance of SIN-UA-NE remained uniform, without any stratification or precipitation. Measurement data showed that even at a high speed of 10,000 r / min, the average particle size, PDI, and zeta potential of the nanoemulsion only exhibited slight fluctuations compared to the uncentrifuged group. These results indicate that the system possesses excellent physical stability.

[0067] The results of the thermal stability test are as follows Figure 13 As shown in the figure. After being placed at 60℃ for 10 days, the appearance of SIN-UA-NE remained uniform, with no oil-water separation or demulsification. Particle size data showed that although the high temperature increased the kinetic energy of the system, the particle size, PDI, and zeta potential of the nanoemulsion did not change significantly, and the data at each time point were not significantly different from the initial values. The above results indicate that the composite interfacial film constructed from glycyrrhizic acid and Bletilla striata polysaccharide has high mechanical strength and thermal resistance, and can effectively resist the Ostwald ripening process induced by high temperature.

[0068] 2. Determination of SIN-UA-NE content, encapsulation performance, and in vitro release behavior. 2.1 Method for determining the content of sinomenine (1) Preparation of reference solution Accurately weigh an appropriate amount of sinomenine reference standard and place it in a 10 mL brown volumetric flask. Add an appropriate amount of anhydrous ethanol, sonicate to completely dissolve the solution, then dilute to the mark with anhydrous ethanol and shake well to obtain the SIN reference standard stock solution.

[0069] (2) Preparation of the test solution Accurately measure an appropriate amount of SIN-UA-NE sample and place it in a 10 mL volumetric flask. Add anhydrous ethanol and sonicate to break the emulsion, then dilute to the mark with anhydrous ethanol and shake well. Filter the mixture through a 0.22 μm microporous membrane and collect the filtrate to obtain the test solution.

[0070] (3) Drawing the standard curve Accurately pipette the SIN reference stock solution and prepare a series of standard working solutions with mass concentrations of 1.0, 1.25, 2.5, 5.0, 10.0, 25.0, 50.0, and 100.0 μg / mL using a serial dilution method. Measure the absorbance (A) of each concentration at 295 nm. Perform linear regression analysis with absorbance (A) as the ordinate (y) and SIN mass concentration as the abscissa (x, μg / mL).

[0071] 2.2 Method for determining the content of ursolic acid (1) Preparation of reference solution Accurately weigh an appropriate amount of UA reference standard, place it in a 10 mL brown volumetric flask, add an appropriate amount of anhydrous ethanol, sonicate to dissolve completely, dilute to the mark with anhydrous ethanol, and shake well to obtain the solution.

[0072] (2) Preparation of the test solution Accurately measure an appropriate amount of SIN-UA-NE sample and place it in a 10 mL volumetric flask. Add anhydrous ethanol and sonicate to break the emulsion, then dilute to the mark with anhydrous ethanol and shake well. Filter the mixture through a 0.22 μm microporous membrane and collect the filtrate to obtain the test solution.

[0073] (3) Chromatographic conditions The UA content was determined by HPLC. The chromatographic analysis conditions were as follows: the chromatographic column was C2000. 18 The column was 250 mm × 4.6 mm, 5 μm; the mobile phase was acetonitrile-0.5% phosphoric acid aqueous solution with a volume ratio of 85:15; the flow rate was 1.0 mL / min; the detection wavelength was 210 nm; the column temperature was 25 °C; and the injection volume was 20 μL.

[0074] (4) Drawing the standard curve Accurately pipette an appropriate amount of ursolic acid reference standard stock solution and serially dilute it with anhydrous ethanol to prepare a series of standard working solutions with mass concentrations of 5, 10, 25, 100, 250, and 500 μg / mL. Accurately pipette 20 μL of each solution into the high-performance liquid chromatograph (HPLC) for analysis, and record the corresponding peak area at each concentration. Perform linear regression analysis with the mass concentration of ursolic acid as the x-axis (x, μg / mL) and the corresponding peak area as the y-axis.

[0075] 2.3 Experimental Methods 2.3.1 Methods for determining encapsulation efficiency and drug loading The encapsulated and free drugs in the system were separated by ultrafiltration centrifugation, and the encapsulation efficiency and drug loading of sinomenine and ursolic acid were calculated.

[0076] Total drug amount (W) 总 Determination of SIN-UA-NE sample: Accurately measure 1.0 mL of SIN-UA-NE sample into a centrifuge tube, add 4.0 mL of anhydrous ethanol, and sonicate for 10 min to fully break the nanoemulsion; then centrifuge at 12000 r / min for 10 min, and filter the supernatant through a 0.22 μm microporous membrane as the total drug content test solution. Take appropriate amounts of the above filtrate: determine the absorbance of SIN at 295 nm using the UV spectrophotometric method under section "2.1"; determine the absorbance of UA using the HPLC method under section "2.2"; substitute the absorbance or peak area of ​​each into the corresponding standard curve equation to obtain the total mass of SIN and UA in 1.0 mL of nanoemulsion (denoted as W). SIN-总 W UA-总 ).

[0077] Free drug amount (W) 游离 Determination of SIN-UA-NE: Accurately measure 1.0 mL of the SIN-UA-NE sample and place it in the upper chamber of an ultrafiltration centrifuge tube (molecular weight cutoff of 30 kDa). Centrifuge at 12000 r / min for 10 min. Accurately pipette an appropriate amount of the lower ultrafiltrate, filter it through a 0.22 μm microporous membrane, and determine it in parallel using the aforementioned ultraviolet spectrophotometry and HPLC methods. Substitute the measured values ​​into their respective standard curve equations to obtain the masses of free SIN and UA (denoted as W). SIN-游离 With W UA-游离 ).

[0078] Calculate the encapsulation efficiency (EE) and drug loading (DL) using the following formulas respectively: ; ; In the formula: W 总W represents the total mass of the drug. 游离 For the quality of free drug, W 辅料 The total mass of the oil phase, emulsifiers, and other matrix excipients contained in 1.0 mL of nanoemulsion.

[0079] 2.3.2 Determination of encapsulation efficiency and drug loading of nanoemulsions prepared with different dosages SIN and UA were mixed at a 1:1 mass ratio, and the total dosage was set to 5, 7.5, 10, 12.5, and 15 mg to prepare drug-loaded nanoemulsions SIN-UA-NE. The EE and DL of both were determined according to the method in section "2.3".

[0080] 2.3.3 Investigation on the in vitro release rate of sinomenine The in vitro release behavior of SIN under simulated physiological conditions was investigated using the dialysis bag method. A PBS buffer solution with pH 7.4 was accurately prepared as the release medium, and 0.3% (w / v) of Tween 80 was added to meet the leakage conditions for the release of poorly soluble drugs. 1.0 mL of single-loaded sinomenine nanoemulsion (SIN-NE) sample and 1.0 mL of free sinomenine control solution (solvent: anhydrous ethanol, drug concentration consistent with the nanoemulsion) were precisely measured and placed in a pretreated dialysis bag (molecular weight cutoff: 10000 Da), and both ends were sealed with dialysis clips. The dialysis bag was completely immersed in a centrifuge tube containing 10.0 mL of release medium, and then placed in a constant-temperature shaking incubator at 37°C and a shaking frequency of 100 r / min. At 0, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, and 48 h, 1.0 mL of the release solution was taken at regular intervals, and an equal volume of fresh release medium at 37 °C was immediately added to the system. The released solution was filtered through a 0.22 μm microporous membrane, and the absorbance was measured using the previously established ultraviolet spectrophotometric method. The mass concentration of sinomenine was calculated by substituting the absorbance into the standard curve. The cumulative release rate (Q) at each time point was calculated using the following formula. n ), and plot the cumulative release curve.

[0081] ; In the formula: Q n V represents the cumulative release rate of the drug at different times, V0 represents the total volume of the release medium, and C represents the cumulative release rate of the drug at different times. t V represents the drug concentration measured at different times, V is the sampling volume, and W is the total mass of the drug added.

[0082] 2.3.4 In vitro release rate of ursolic acid The in vitro release behavior of UA under simulated physiological conditions was investigated using the dialysis bag method, the same as in "2.3.3". A single-loaded ursolic acid nanoemulsion (UA-SE) was used as the sample, and 1.0 mL of free ursolic acid solution (anhydrous ethanol, with the same drug concentration as the nanoemulsion) was used as a control. The concentration of UA was determined using the previously established HPLC method, the cumulative release rate at each time point was calculated, and a cumulative release curve was plotted.

[0083] 2.4 Experimental Results 2.4.1 Results of Encapsulation Efficiency and Drug Loading at Different Dosage Rates See results Figure 14 The results show that the encapsulation efficiency of both SIN and UA remained at a high level. When the total dosage of SIN and UA was 12.5 mg (i.e., 6.25 mg SIN and 6.25 mg UA), the encapsulation efficiency of SIN was 88.31% with a drug loading of 24.96 mg / g, and the encapsulation efficiency of UA was 87.36% with a drug loading of 28.28 mg / g. The drug loading capacity of the two was well balanced, indicating that the nanoemulsion has a high encapsulation efficiency for both SIN and UA.

[0084] 2.4.2 In vitro release study of sinomenine and ursolic acid The cumulative release curves of sinomenine and ursolic acid in different formulations are shown below. Figure 15 The results show that there are significant differences in the release behavior of free drugs and drugs encapsulated in nanoemulsions.

[0085] Free sinomenine exhibits typical rapid diffusion and burst release in PBS release medium, with extremely rapid release in the initial 6-8 hours, and a cumulative release rate exceeding 80% by 8 hours. It then rapidly stabilizes in the subsequent time, with a release rate >90% at 48 hours. In contrast, sinomenine nanoemulsion (SIN-NE) demonstrates excellent and gradual sustained release characteristics. Its release process can be divided into two stages: a slow release period of approximately 40% in the first 4 hours, possibly attributed to diffusion of the free drug within the nanoemulsion; and a stable sustained release period from 4 to 48 hours, with a cumulative release rate of approximately 62% at 24 hours, slowly increasing to around 70% by 48 hours.

[0086] Free ursolic acid diffuses rapidly in the release medium, with a release rate exceeding 50% within 2 hours, exhibiting a clear burst release characteristic. The release rate reaches 85.83% by 24 hours, then gradually stabilizes, with a cumulative release rate of 93.42% after 48 hours, indicating that the drug is essentially completely released. In contrast, the drug-loaded nanoemulsion (SIN-UA) exhibits a significantly delayed release process, displaying typical sustained-release characteristics: the release rate is only 31.90% at 2 hours, significantly lower than that of free drug; the release rate is 58.35% at 12 hours, still showing a significant lag; and the cumulative release rate after 48 hours is 74.05%, with approximately 26% of the drug remaining unreleased, indicating that the nanoemulsion has a significant delaying effect on the release of ursolic acid.

[0087] Further analysis using in vitro release kinetics models revealed that both SIN-NE and UA-NE exhibited more significant sustained-release effects and better model fitting performance compared to free drugs. This indicates that the nanoemulsion system has a good ability to regulate drug release, which helps maintain effective blood drug concentrations in vivo for a longer period of time, thereby reducing the frequency of drug administration and providing strong support for subsequent long-acting treatment.

[0088] 3. Study on the synergistic anti-inflammatory effect of SIN-UA-NE 3.1 Experimental Methods 3.1.1 Establishment and validation of LPS-induced MH7A cell inflammation model The effect of different concentrations of LPS on the viability of MH7A cells was investigated using the CCK-8 assay, and the expression levels of inflammatory factors (IL-6 and TNF-α) in the cell supernatant were detected by ELISA to verify the pro-inflammatory induction effect of LPS on MH7A cells.

[0089] 3.1.2 Effects of SIN-UA-NE on the activity of normal MH7A cells The MTT assay was used to investigate the effects of different drug-treated groups on the viability of normal MH7A cells and to screen for safe dosage ranges. After MH7A cells adhered to the culture medium, the experiment was divided into 6 groups: Control, NE, SIN, UA, SIN-UA, and SIN-UA-NE. The drug-treated groups (SIN, UA, SIN-UA, SIN-UA-NE) were all set with equivalent concentration gradients: 0, 50, 60, 70, 80, 90, and 100 μg / mL; the NE group was supplemented with corresponding blank nanoemulsion solutions at concentrations ranging from 1912, 2295, 2677, 3060, 3442, and 3825 μg / mL; and the Control group was supplemented with 100 μL of DMEM medium per well. After 24 h of drug treatment, 20 μL of MTT solution (0.5 mg / mL) was added to each well, and the cells were incubated for another 4 h. The supernatant of each well was then aspirated, and 100 μL of DMSO was added. The cells were shaken at low speed in the dark for 10 min until the crystals were fully dissolved. The absorbance at 490 nm was measured using an ELISA reader, and the cell viability of each group was calculated.

[0090] 3.1.3 Inhibitory effect of SIN-UA-NE on LPS-induced abnormal proliferation of MH7A cells After MH7A cells adhered to the culture medium, the experiment was divided into 6 groups: control group, model group, SIN, UA, SIN-UA, and SIN-UA-NE. Except for the control group, which received normal DMEM culture medium, the other groups received DMEM culture medium containing 1 μg / mL lipopolysaccharide. After incubation for 24 h, the supernatant of each well was discarded. The control and model groups received fresh DMEM culture medium; the other groups received their corresponding sample solutions containing 80 μg / mL of the drug. Each group was divided into 5 replicates, and incubation continued for 24 h. After drug administration, 20 μL of MTT solution (0.5 mg / mL) was added to each well, and incubation continued for 4 h. The supernatant of each well was then aspirated, and 100 μL of dimethyl sulfoxide (DMSO) was added. The cells were shaken slowly in the dark for 10 min until the crystals were fully dissolved. The absorbance was measured at 490 nm using a microplate reader, and the inhibition rate of abnormal MH7A cell proliferation in each drug administration group was calculated.

[0091] 3.1.4 Effect of SIN-UA-NE on NO release in RAW264.7 cells RAW264.7 cells were cultured in an adherent manner, and subsequent experimental methods and grouping were the same as in "3.1.3". After drug administration, the cell supernatant from each well was aspirated, and the NO inhibition rate was calculated according to the instructions of the nitric oxide (NO) detection kit.

[0092] 3.1.5 Effects of SIN-UA-NE on LPS-induced secretion of IL-6, IL-1β, and TNF-α in MH7A cells The effects of different drug-treated groups on the secretion of pro-inflammatory factors IL-6, IL-1β, and TNF-α by LPS-induced MH7A cells were investigated using enzyme-linked immunosorbent assay (ELISA). MH7A cells were cultured in adherent form, and subsequent experimental methods were the same as those for grouping in "3.1.3". After drug administration, the cell supernatant of each well was collected, and the expression levels of IL-6, IL-1β, and TNF-α were detected according to the ELISA kit instructions.

[0093] 3.1.6 SIN-UA-NE uptake experiment in MH7A cells After MH7A cells adhered to the culture medium, the experiment was divided into a normal cell group and an inflammatory cell group. The inflammatory cell group was incubated with DMEM medium containing 1 μg / mL LPS for 24 h to construct a cellular inflammation model; the normal cell group was incubated with an equal volume of DMEM medium. After 24 h, the supernatant was aspirated, and both groups were added with a coumarin-6 (C6)-labeled SIN-UA-NE sample solution. Different drug administration time gradients were set up (24, 12, 6, 2, 1 h). After drug administration at each time point, the drug solution was discarded, and the cells were gently washed three times with PBS buffer to thoroughly remove untaken fluorescent probes and serum components from the culture medium. 500 μL of PBS was added to each well, and the cells were observed under an inverted fluorescence microscope. The distribution of green fluorescence in each group was captured in photographs, and the fluorescence photographs were digitally processed using ImageJ software to calculate the average fluorescence intensity of each group for semi-quantitative analysis.

[0094] 3.1.7 Effects of SIN-UA-NE on intracellular ROS levels Intracellular ROS levels were determined using the DCFH-DA fluorescent probe method. After MH7A cells adhered to the culture medium, the culture medium was aspirated, and DCFH-DA probe diluted with serum-free medium was added. The cells were incubated at 37°C in the dark for 30 min, and then washed to remove any probe that had not entered the cells. The experiment consisted of five groups: Control, Rosup-induced group (positive group), and high, medium, and low dose SIN-UA-NE groups (80, 40, and 10 μg / mL, respectively). Except for the control group, all other groups were treated with Rosup reagent to induce ROS production, while simultaneously receiving the corresponding concentration of the drug solution.

[0095] 3.2 Experimental Results 3.2.1 Establishment and validation of LPS-induced MH7A cell inflammation model The results of establishing and validating the LPS-induced MH7A cell inflammation model are shown in [the table below]. Figure 16 .

[0096] Depend on Figure 16As shown in Figure A, low concentrations of LPS (0.1-1 μg / mL) significantly promoted the proliferation of MH7A cells. The cell viability was highest at an LPS concentration of 1 μg / mL, showing a significant difference compared to the control group (P < 0.05). This result indicates that this concentration of LPS can effectively activate MH7A cells, mimicking the pathological state of abnormal synovial fibroblast proliferation in rheumatoid arthritis. When the LPS concentration was increased to 5-20 μg / mL, cell viability began to decline and was slightly lower than the control group, indicating that excessively high concentrations of LPS may have caused slight toxic damage to the cells and are unsuitable as induction conditions for an inflammation model. Based on the above screening results, this study ultimately selected 1 μg / mL LPS stimulation for 24 h as the in vitro modeling condition for MH7A cells.

[0097] Depend on Figure 16 BC analysis revealed that under normal culture conditions, MH7A cells secreted IL-6 at a low level. After LPS induction, the IL-6 secretion level in the model group increased to 239.78±11.23 pg / mL, which was significantly different from the control group (P<0.01). TNF-α results showed that the TNF-α secretion level in the control group was low at 18.79±4.15 pg / mL, while the TNF-α level in the model group increased to 230.52±42.92 pg / mL under LPS stimulation, which was significantly different from the control group (P<0.01).

[0098] The above results indicate that stimulation with 1 μg / mL LPS for 24 h can induce an inflammatory response in MH7A cells and significantly upregulate the secretion level of pro-inflammatory factors, thus successfully establishing an MH7A cell inflammation model.

[0099] 3.2.2 Effects of SIN-UA-NE on the viability of normal MH7A cells See results Figure 17 The results showed that free UA exhibited a significant concentration-dependent inhibitory effect on MH7A cells, with cell viability decreasing as the concentration increased. At a concentration of 80 μg / mL, the survival rate of the UA group decreased to 80.12 ± 3.56%, indicating that high concentrations of free UA have potential toxicity to normal synovial cells. In contrast, free SIN, SIN-UA-NE, SIN-UA, and NE all showed excellent safety across the entire concentration range (0-100 μg / mL), with survival rates consistently above 90%. In cellular pharmacodynamic studies, the highest concentration with 80% cell viability is typically selected as the dosage. Therefore, this experiment ultimately chose 80 μg / mL as the dosage concentration for subsequent evaluation of the inhibitory effect of SIN-UA-NE on MH7A cell proliferation and its anti-inflammatory effects.

[0100] 3.2.3 Inhibitory effect of SIN-UA-NE on LPS-induced abnormal proliferation of MH7A cells See results Figure 18 The results showed that LPS could significantly induce abnormal proliferation of synovial cells. All treatment groups inhibited this process to varying degrees, with the SIN-UA-NE group showing the best inhibitory effect, reaching an inhibition rate of 84.45%.

[0101] 3.2.4 Effects of SIN-UA-NE on LPS-induced NO release and secretion of TNF-α, IL-6, and IL-1β in MH7A cells.

[0102] See results Figure 19 The results showed that after induction with 1 μg / mL LPS, the levels of NO, TNF-α, IL-6, and IL-1β secreted by cells in the normal group were significantly different from those in the model group (P < 0.01), indicating that the cell inflammation model was successfully constructed.

[0103] Depend on Figure 19 As shown in Figure A, compared with the single-drug groups, the inhibition rate of the free SIN group (51.38%) was better than that of the free UA group (32.07%), while the inhibition rate of the free combined group (SIN-UA) was 53.22%, showing a preliminary synergistic effect, indicating that the combination of the two drugs can more effectively inhibit the generation of NO; ​​the inhibition rate of the SIN-UA-NE group was 79.26%, which was significantly better than the free single-drug and free combined groups at the same concentration. This not only confirms the anti-inflammatory synergistic potential of the combination of SIN and UA, but also highlights the advantages of nanoemulsion as a drug delivery system in improving bioavailability.

[0104] Depend on Figure 19 B indicates that in the free single-drug group, SIN showed strong anti-inflammatory activity (inhibition rate 56.66%), while UA at this concentration had a weak inhibitory effect on TNF-α in MH7A cells; the average inhibition rate of the free combination group (SIN-UA) was 61.64%, which was better than the two single-drug groups, initially demonstrating the synergistic effect of SIN and UA in anti-inflammatory effects; the SIN-UA-NE group had the best anti-inflammatory performance, with an inhibition rate of 81.93±3.31%.

[0105] Depend on Figure 19 C indicates that in the free monotherapy group, SIN exhibited definite IL-6 inhibitory activity, with an inhibition rate of 48.04%, significantly superior to the UA group at the same dose. The inhibition rate in the free combination group (SIN-UA) increased to 53.73%, higher than the two monotherapy groups, demonstrating the synergistic anti-inflammatory effect of sinomenine and ursolic acid. The average inhibition rate of the SIN-UA-NE group reached 75.32%. Compared with the free combination group, the efficacy was significantly improved.

[0106] Depend on Figure 19 D shows that the inhibitory effect of free UA was relatively limited, with an inhibition rate of only 12.98%; free SIN showed a robust anti-inflammatory effect, with an inhibition rate of 36.50%; the average inhibition rate of the free combined group (SIN-UA) slightly increased to 39.71%, showing a preliminary synergistic blocking effect; the average inhibition rate of the SIN-UA-NE group was 52.80%, which was better than that of the free combined group.

[0107] 3.2.5 SIN-UA-NE uptake experiment in MH7A cells See results Figure 20 The results showed that the uptake of SIN-UA-NE by MH7A cells in both the normal group and the LPS-induced inflammation group was time-dependent. As the incubation time increased from 1 h to 24 h, the intracellular fluorescence intensity continuously increased, and at each observation time point, the uptake by MH7A cells in the inflammation group was greater than that in the normal group. These results indicate that SIN-UA-NE exhibits a higher affinity for LPS-induced inflammatory cells, can be efficiently absorbed by synovial fibroblasts, and the uptake increases linearly with time.

[0108] 3.2.6 Effects of SIN-UA-NE on intracellular ROS levels See results Figure 21 The results showed that the fluorescence intensity in the Rosup group was significantly higher than that in the control group, indicating that the reagent successfully induced intracellular oxidative stress, leading to an increase in ROS levels. SIN-UA-NE exhibited excellent ROS scavenging ability. As the drug concentration increased from 10 μg / mL to 80 μg / mL, the average intracellular fluorescence intensity decreased from 43.02 to 17.03, with the highest scavenging rate at 80 μg / mL, which reduced ROS to near normal cell levels, demonstrating the excellent antioxidant properties of SIN-UA-NE.

[0109] 4. Pharmacodynamic evaluation of SIN-UA-NE against rheumatoid arthritis 4.1 Experimental Methods 4.1.1 Experimental grouping and drug administration Adult female SD rats (weighing 180-200 g) were acclimatized for one week and then randomly divided into 6 groups (Control group, Model group, MTX group, SIN-UA group, and SIN-UA-NE group), with 6 rats in each group. The rats were initially immunized, recorded as day 0. The control group was injected with saline, while the other groups received a subcutaneous injection of 0.3 mL of bovine type II collagen emulsion at the base of the tail and the left hind paw. Seven days later, a second immunization was administered. The control group received saline, while the other groups received an injection of 0.1 mL of bovine type II collagen emulsion at the base of the tail. Seven days later, oral administration of the drug was initiated once daily at the following dosages: MTX 1 mg / kg / day, SIN-UA and SIN-UA-NE groups 12.4 mg / kg / day, and the control and model groups received the same volume of saline. This administration continued for 28 days.

[0110] 4.1.2 Index Measurement From the initial immunization (day 0) until the end of the experiment, the following indicators were measured every 3 days: the thickness of the rat paws was measured and recorded using vernier calipers, and the swelling of the rat paws was evaluated; the rat arthritis index (AI) score was calculated (the scoring criteria are shown in Table 5); the weight of the rats was weighed and recorded; and the hind paws of the experimental rats in each group were photographed for observation.

[0111] .

[0112] After the treatment cycle, the following indicators were measured: the final fasting body weight (g) of rats in each group was recorded; the thymus index and spleen index were measured; the levels of inflammatory factors (TNF-α, IL-6, IL-1β) in rat serum were measured according to the ELSA kit instructions; Micro-CT imaging scans and analysis were performed on the rat hind limb ankle joints, and bone parameters were analyzed based on the CT results, including BMD (bone mineral density), TMD (tissue mineral density), Tb.Th (trabecular bone thickness), Tb.N (trabecular bone number), Tb.Sp (avg) (trabecular bone separation), and BV / TV (bone volume fraction); histopathological sections of rat ankle joint tissue were prepared and evaluated by H&E staining.

[0113] 4.2 Experimental Results 4.2.1 Trends in paw thickness, AI score, and body weight in CIA rats The trends in paw thickness changes in each group of rats during model establishment and drug administration are as follows: Figure 22As shown in Figure A, the paw volume of rats in the control group remained stable throughout the experimental period, with no obvious signs of swelling. In contrast, the paw swelling volume of rats in the model group increased rapidly after modeling, showing a typical trend of inflammatory growth, peaking on day 18. This indicates that the collagen-induced arthritis (CIA) rat model was successfully established, exhibiting persistent and severe joint pathological changes. The swelling curve of the SIN-UA group overlapped with that of the model group in the early stage, and it almost failed to effectively inhibit paw swelling. Only in the middle and late stages of administration (days 27-42) did it show a slight trend of swelling reduction. This result suggests that when sinomenine and ursolic acid are administered orally in free form, their poor water solubility and first-pass effect result in low oral bioavailability, making it impossible to rapidly reach and maintain an effective therapeutic concentration in vivo. The SIN-UA-NE group demonstrated rapid and sustained anti-inflammatory and swelling-reducing advantages. From day 6 to 15, the foot swelling volume in the SIN-UA-NE group was consistently significantly lower than that in the model group and the free drug group. After day 18, the swelling reduction rate in the SIN-UA-NE group was significantly accelerated. By the end of the experiment on day 42, the foot swelling value had dropped to 6.11, which was more effective than the SIN-UA free drug combination group. Moreover, the overall swelling reduction trend and treatment effect were approaching those of the positive control drug methotrexate.

[0114] Changes in CIA rat arthritis scores over time are as follows: Figure 22 As shown in Figure B, no joint redness or deformation was observed in the control group rats throughout the observation period, and the AI ​​score remained at 0. In the model group rats, the AI ​​score progressively increased after initial immunization, reaching a peak of inflammation on day 21 and remaining at a high level for a prolonged period thereafter, indicating that the CIA model successfully induced arthritis. The AI ​​score curve of the free SIN-UA group from day 0 to 21 almost overlapped with that of the model group. In the later stages of disease regression, the score decreased extremely slowly, possibly due to the extremely low bioavailability of SIN and UA under conventional oral administration, preventing accumulation to an effective concentration for systemic antirheumatic effects. The SIN-UA-NE group showed significant therapeutic effects, with a decreasing AI score during the observation period from day 24 to 42, significantly better than the model group and the free drug group. By the experimental endpoint of day 42, the average AI score of the SIN-UA-NE group had decreased to 1.67, greatly improving joint redness and stiffness. Its overall therapeutic trend was comparable to that of the positive control drug methotrexate.

[0115] The results of body weight changes in CIA rats are as follows: Figure 22As shown in Figure C. Throughout the experimental period, the control group rats maintained stable weight gain, while the model group rats showed significantly limited weight gain. After day 15 post-modeling, weight gain almost stopped, and by day 42, their weight was significantly lower than that of the control group, reflecting the negative impact of joint inflammation on the rats' systemic metabolism and growth. The SIN-UA group showed similar weight changes to the model group in the early stages of the experiment, but weight gain stagnated from day 24 to day 42. This may be because the free sinomenine and ursolic acid have low oral bioavailability, failing to effectively inhibit inflammation. Additionally, direct oral administration may have caused some gastrointestinal irritation, both of which limited the rats' weight gain. The SIN-UA-NE group rats showed robust recovery in weight gain, with growth status superior to the free drug group and the model group, and slightly better than the first-line clinical drug methotrexate. This is presumably because the nanoemulsion encapsulation avoided direct contact with the gastrointestinal tract and improved the bioavailability of sinomenine and ursolic acid. The stagnant weight gain in the methotrexate group may be due to gastrointestinal side effects from long-term use.

[0116] 4.2.2 Macroscopic observation of rat paws See results Figure 23 Throughout the 42-day experimental period, the rats in the control group maintained normal paw morphology and skin color, with no signs of erythema, edema, or joint deformity. In the model group, rats began to show significant redness and swelling of the toes and ankle joints around days 12-18, which persisted in subsequent experiments. The paws exhibited severe diffuse edema, and the joints were severely stiff and deformed, indicating that the inflammatory response of the CIA rat model was successfully elicited. In the SIN-UA group, significant redness and swelling of the paws remained in the early stages of treatment. With prolonged treatment, the swelling lessened to some extent compared to the severe model group, but significant thickening and residual erythema were still visible on the dorsum of the foot and ankle joints. In the MTX group, paw erythema and edema were significantly suppressed. By day 42, the joint morphology had largely recovered, with only very slight swelling, visually similar to the control group. The SIN-UA-NE group exhibited excellent anti-inflammatory effects. After administration, the redness and swelling subsided rapidly starting from day 24. By day 36, the fineness of the toes and the skin color had almost returned to the level of the control group.

[0117] 4.2.3 Determination of thymus index and spleen index in rats The effects of different drug administration groups on the immune organ index of CIA rats are shown in the figure. Figure 24 .

[0118] Spleen index measurements showed that, compared to the control group, the spleen index of the model group rats increased, exhibiting significant splenomegaly, indicating systemic immune abnormal activation in CIA rats. Although the free SIN-UA group received intervention, it failed to effectively inhibit the excessive proliferation of immune cells, and the spleen index remained at a high level. In contrast, the SIN-UA-NE group significantly reduced the spleen index, effectively inhibiting compensatory splenomegaly, and its immunomodulatory capacity was significantly better than the free drug group, approaching the level of the positive control drug MTX. This indicates that the nanoemulsion delivery system effectively promoted the absorption of sinomenine and ursolic acid, exerting its core pharmacological effect of inhibiting peripheral immune cell activation.

[0119] The thymus index measurement results showed that, compared with the control group, the thymus index of rats in the model group was significantly decreased, confirming that rheumatoid arthritis induced thymic atrophy. Free SIN-UA had a very limited effect on improving thymic atrophy, while the thymus index in the SIN-UA-NE group showed a significant recovery, inhibiting the pathological atrophy of the thymus and restoring it to near the normal level of the control group.

[0120] 4.2.4 Determination of inflammatory factors in rat serum The effects of different drug administration groups on serum inflammatory factors (TNF-α, IL-6, IL-1β) in CIA rats are shown in the following results. Figure 25 The results showed that the levels of three pro-inflammatory factors in the serum of rats after modeling were significantly higher than those in blank rats, confirming a severe systemic inflammatory response. After different drug interventions, each treatment group showed varying degrees of inhibition of pro-inflammatory factors. SIN-UA-NE nanoemulsion exhibited excellent inhibitory effects on TNF-α and IL-6, with average inhibition rates of 56.56% and 91.63%, respectively, both higher than the free SIN-UA combined group. Regarding IL-1β regulation, SIN-UA-NE showed an average inhibition rate of 54.67%, slightly better than the positive control MTX group and the free SIN-UA combined group. The pharmacological efficacy of SIN-UA-NE at the molecular level confirms that the nanocarrier constructed from glycyrrhizic acid and Bletilla striata polysaccharide effectively overcomes the barriers of short half-life and low bioavailability of free drugs in vivo, achieving highly efficient targeted delivery of the two drugs to the inflammatory microenvironment.

[0121] 4.2.5 Micro-CT imaging and analysis of rat hindlimb ankle joint Micro-CT imaging results of the hind limb ankle joints of rats in each group are shown below. Figure 26 Micro-CT bone microstructure parameter analysis can be found in [link to article]. Figure 27 .

[0122] Depend on Figure 26It was found that the joint bones of the control group rats exhibited healthy physiological morphology. The model group rats showed severe diffuse bone resorption and erosion in the carpal / tarsal region, with rough, pitted bone surfaces, narrowing of multiple joint spaces, bony fusion, blurred joint edges of the fingers / toes, and some degree of structural deformity, resulting in irreversible bone destruction and exhibiting typical bone destruction characteristics of rheumatoid arthritis. The SIN-UA group showed a certain inhibitory effect on joint bone destruction in rats. Compared with the model group, the overall degree of bone erosion was reduced, and the morphology of some fingers / toes was maintained. However, in the densely populated areas of the carpal / tarsal bones, relatively obvious local bone defects and surface roughness were still observed, and some joint spaces remained blurred, indicating that the efficacy of simple drug-physical mixtures in preventing deep bone destruction is limited. The MTX group showed excellent anti-bone destruction efficacy, with imaging findings highly similar to the control group, proving that it can effectively inhibit the bone erosion process of RA. The SIN-UA-NE group exhibited excellent osteoprotective effects, superior to the SIN-UA group and comparable to the positive control drug MTX group. CT images showed excellent protection of the overall integrity of the rat skeleton. The carpal / tarsal joint contours were clear, with only minor bone irregularities visible at a few local edges, and most joint spaces were clearly preserved, effectively preventing extensive subchondral bone destruction and arthrodesis. These results strongly confirm that preparing SIN and UA into a nanoemulsion drug delivery system can significantly improve drug targeting or bioavailability, thereby playing a key role in blocking the core pathological step of RA bone destruction.

[0123] Depend on Figure 27 It was found that the Model group showed significantly reduced BMD, TMD, and BV / TV, and increased Tb.Sp, with thinning of trabeculae and increased gaps, consistent with the characteristics of a bone microstructure damage model. Compared with the Model group, the Control group showed significantly better bone mineral density, trabecular structure, and bone mass, indicating successful model establishment. All drug groups improved bone microstructure and bone mineral density, with the SIN-UA-NE group showing significantly better results than the SIN-UA group, indicating that nanoemulsion encapsulation of drugs can enhance osteoprotective activity. Quantitative analysis of rat joint bone microstructure using Micro-CT showed that compared with the Model group, SIN-UA, SIN-UA-NE, and MTX all significantly improved bone mineral density, trabecular morphology, and bone mass levels, with MTX showing the best intervention effect. SIN-UA-NE had a stronger osteoprotective effect than SIN-UA, suggesting that nanoemulsion encapsulation of sinomenine and ursolic acid can enhance their efficacy in improving bone microstructure.

[0124] 4.2.6 Evaluation of rat ankle joint histopathological sections and H&E staining See results Figure 28The control group showed healthy joint tissues. The model group exhibited significant synovial hyperplasia accompanied by dense inflammatory cell infiltration; the hyperplastic synovium formed obvious pannus and invaded the cartilage surface and joint cavity; the articular cartilage surface was rough, with large-area peeling, a significant reduction in chondrocytes, and obvious erosive bone destruction in the deep subchondral bone, presenting typical pathological features of rheumatoid arthritis. The MTX group effectively curbed severe destruction of articular cartilage and subchondral bone. The SIN-UA group showed good cartilage protection, with an overall intact cartilage surface, and significant improvement in synovial hyperplasia and inflammatory infiltration compared to the model group. The SIN-UA-NE group had a clear joint space and no obvious pannus formation; compared to the model group, synovial hyperplasia and inflammatory cell infiltration were significantly inhibited; the articular cartilage surface was smooth and intact, with no obvious bone erosion or cartilage destruction, and the histological morphology was close to the control group, demonstrating excellent therapeutic effect and cartilage protection.

[0125] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A sinomenine-ursolic acid nanoemulsion, characterized in that, The sinomenine-ursolic acid nanoemulsion formulation consists of coix seed oil, Bletilla striata polysaccharide, glycyrrhizic acid, sinomenine, ursolic acid, and water. The volume fraction of coix seed oil is 1%~5%; the mass ratio of Bletilla striata polysaccharide to glycyrrhizic acid is 1:9~9:1, and its total mass fraction in water is 0.1%~1.0%; the mass ratio of sinomenine to ursolic acid is 1:1, and the total addition amount is 5~15 mg per 100 mL of sinomenine-ursolic acid nanoemulsion.

2. The sinomenine-ursolic acid nanoemulsion according to claim 1, characterized in that: The volume fraction of the coix seed oil is 2%~4%; the mass ratio of the Bletilla striata polysaccharide to glycyrrhizic acid is 1:9~3:7, and its total mass fraction in water is 0.4%~0.6%; the total addition amount of sinomenine and ursolic acid is 10~12.5 mg per 100 mL of sinomenine-ursolic acid nanoemulsion.

3. The sinomenine-ursolic acid nanoemulsion according to claim 2, characterized in that: The volume fraction of the coix seed oil is 3%; the mass ratio of the Bletilla striata polysaccharide to glycyrrhizic acid is 1:9, and its total mass fraction in water is 0.5%; the total addition amount of sinomenine and ursolic acid is 12.5 mg per 100 mL of sinomenine-ursolic acid nanoemulsion.

4. The sinomenine-ursolic acid nanoemulsion according to claim 1, characterized in that, The water is deionized water.

5. The preparation method of the sinomenine-ursolic acid nanoemulsion as described in claim 1, characterized in that, The specific steps are as follows: accurately weigh the prescribed amount of glycyrrhizic acid and Bletilla striata polysaccharide, add them to water, and dissolve them by sonication and heating to form the aqueous phase; accurately weigh the prescribed amount of sinomenine and ursolic acid, add them to the prescribed amount of coix seed oil to form the oil phase; pour the aqueous phase into the oil phase, stir evenly, and sonicate for 2-10 minutes under a power of 130-360 W to obtain the final product.

6. The preparation method of the sinomenine-ursolic acid nanoemulsion according to claim 5, characterized in that, The power of the ultrasound is 260-360 W.

7. The preparation method of the sinomenine-ursolic acid nanoemulsion according to claim 6, characterized in that, The power of the ultrasound is 325 W.

8. The preparation method of the sinomenine-ursolic acid nanoemulsion according to claim 5, characterized in that, The ultrasound session lasted 8-10 minutes.

9. The preparation method of the sinomenine-ursolic acid nanoemulsion according to claim 8, characterized in that, The ultrasound session lasted for 10 minutes.

10. The use of the sinomenine-ursolic acid nanoemulsion as described in claim 1 in the preparation of a drug for treating rheumatoid arthritis.

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