Medicinal composition for antibacterial and anti-inflammatory effect using combination of chinese herbal medicine and marine extract

Abstract

The present application relates to the field of pharmacy, disclose the medicine composition for antibacterial and anti-inflammatory of Chinese herbal medicine and marine extract combination, including: baicalin extract 5~10 parts; andrographolide extract 3~7 parts; sulfated fucoidan 2~5 parts; spongin extract 0.2~1 part; wherein: baicalin extract is derived from the root of scrophulariaceae, is extracted by ethanol-water mixed solvent; andrographolide extract is derived from the whole plant of andrographis, is extracted by ethanol-water mixed solvent; sulfated fucoidan is derived from brown algae, is prepared by enzymolysis and alcohol precipitation method; spongin extract is derived from sponges, is extracted and separated and purified by organic solvent. Through the technical scheme of the synergistic combination of active ingredients of Chinese herbal medicine and marine extract, the technical effect of destroying the biofilm barrier and clearing the stubborn infection focus in the treatment of bacterial vaginitis, candida vaginitis and cervical inflammation associated with biofilm infection is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmacy, in particular to a Chinese herbal medicine and marine extract combination for use in antibacterial and anti-inflammatory pharmaceutical compositions. BACKGROUND

[0002] Female genital tract infectious diseases, such as bacterial vaginitis, candidal vaginitis and cervicitis, are widespread in women of all ages. These diseases not only cause local discomfort, but also affect reproductive health, and even induce ascending infection. In most cases, infection is closely related to local microecological imbalance and pathogen biofilm formation.

[0003] Biofilm is a complex microbial aggregate composed of polysaccharides, proteins and other matrixes. Pathogenic bacteria in mature biofilm have higher resistance to the external environment. Even if a sufficient dose of antibiotics or antifungal drugs is given, it is often difficult to completely remove, resulting in prolonged treatment period or even recurrence.

[0004] Currently, clinical treatment of such infections mainly relies on local medication, including metronidazole, fluconazole and other conventional drugs. These drugs can effectively relieve symptoms in the early stage, but have limited penetration and short duration of drug efficacy when facing biofilm barriers. Recurrence cases are not uncommon in the short term after treatment.

[0005] To improve treatment efficacy, some studies explore the use of natural products, such as plant extracts or marine bioactive ingredients. These natural ingredients show antibacterial and anti-biofilm activity in vitro, and have good biocompatibility. However, when used directly, natural extracts are easily affected by the body environment, the active ingredients have poor stability, and the tissue penetration is insufficient, resulting in lower in vivo effect than expected. SUMMARY

[0006] To overcome the shortcomings of the prior art, the present application provides a Chinese herbal medicine and marine extract combination for use in antibacterial and anti-inflammatory pharmaceutical compositions, which solves the problems of short drug efficacy maintenance time, insufficient biofilm penetration and unstable release of natural product activity in the treatment of existing gynecological local infections.

[0007] To achieve the above purpose, the present application is realized by the following technical scheme: a pharmaceutical composition, comprising, by weight:

[0008] 5-10 parts of baicalin extract;

[0009] 3-7 parts of andrographolide extract;

[0010] 2-5 parts of sulfated fucoidan;

[0011] 0.2-1 parts of spongin extract.

[0012] Preferably, wherein:

[0013] Baicalin extract is derived from the roots of Scutellaria baicalensis Georgi, and is extracted by ethanol-water mixed solvent;

[0014] Andrographolide extract is derived from the whole plant of Andrographis paniculata, and is extracted by ethanol-water mixed solvent;

[0015] Sulfated fucoidan is derived from brown algae, and is prepared by enzymatic hydrolysis and alcohol precipitation;

[0016] Spongin extract is derived from sponges, and is obtained by organic solvent extraction and separation and purification.

[0017] The preparation method of the Chinese herbal medicine and marine extract combined pharmaceutical composition comprises the following steps:

[0018] S1, preparing baicalin extract and andrographolide extract;

[0019] S2, preparing sulfated fucoidan and spongin extract;

[0020] S3, mixing each extract with phospholipid material to prepare a nano delivery system;

[0021] S4, dispersing the nano delivery system in a system containing a hydrogel matrix, and adjusting the pH value;

[0022] S5, forming a final pharmaceutical composition.

[0023] Preferably, the extraction conditions of the S1 baicalin extract and andrographolide extract are:

[0024] An ethanol-water mixed solvent is used, and the volume fraction of ethanol is 30% to 70%;

[0025] The extraction temperature is 25°C to 40°C;

[0026] Ultrasonic-assisted extraction is used, and the solid-liquid ratio is 1:8 to 1:12;

[0027] The extraction time is 20 to 40 minutes.

[0028] Preferably, the extraction conditions of the S2 sulfated fucoidan and spongin extract are:

[0029] The sulfated fucoidan is enzymatically hydrolyzed by cellulase, the enzymatic hydrolysis temperature is 60°C to 70°C, the enzymatic hydrolysis time is 60 to 90 minutes, and the precipitation is performed by 60% to 80% volume fraction of ethanol;

[0030] The spongin extract is immersed in a chloroform-methanol mixed solvent (volume ratio 2:1) at room temperature for 48 to 72 hours, and then chromatographically separated.

[0031] Preferably, the preparation conditions of the S3 nano delivery system are:

[0032] The mass ratio of phospholipid to cholesterol is 8:2 to 10:2.

[0033] The average particle size of the nanoparticles is 80 to 120 nanometers.

[0034] The zeta potential is controlled at -20 to -40 millivolts.

[0035] The encapsulation efficiency of the extract is not less than 80%.

[0036] Preferably, in the S4, the hydrogel matrix comprises:

[0037] Sodium hyaluronate, mass concentration of 0.1% to 0.5%;

[0038] Glycerol, mass concentration of 1% to 3%.

[0039] Preferably, in the S4, the hydrogel matrix comprises:

[0040] The pH value is adjusted to 4.0 to 5.0 by a citric acid buffer solution.

[0041] After the nanodelivery system and the hydrogel matrix are uniformly mixed, high-pressure homogenization treatment is performed 1 to 3 times at 600 to 800 bar.

[0042] Preferably, the mixing ratio of the volume fraction of the nanodelivery system and the hydrogel matrix is 1:4 to 1:8.

[0043] The application of the herbal medicine and marine extract combined pharmaceutical composition in the preparation of a local medicine for treating bacterial vaginitis, candidal vaginitis associated with biofilm infection, or cervicitis.

[0044] The present application provides a Chinese herbal medicine and marine extract combined pharmaceutical composition for antibacterial and anti-inflammatory purposes.

[0045] 1. The present application achieves the technical effect of destroying the biofilm barrier and clearing stubborn infection sites in the treatment of bacterial vaginitis, candidal vaginitis associated with biofilm infection, and cervicitis by adopting the technical solution of synergistic compounding of active ingredients of Chinese herbal medicine and marine extract.

[0046] 2. The present application adopts the technical combination of nanoparticle encapsulation and sodium hyaluronate hydrogel controlled-release delivery, prolongs the residence time of the drug at the affected area, avoids the clinical difficulties brought by the short-acting and frequent drug replacement in the prior art, and is particularly suitable for the treatment needs of chronic vaginitis and chronic cervicitis.

[0047] 3、The application adopts the strategy of local microenvironment reconstruction and comprehensive regulation of bioactive components, eliminates pathogenic microorganisms during the treatment process, and at the same time promotes the repair of the mucosal barrier and the improvement of the inflammatory environment, significantly improving the quality of tissue recovery after treatment. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 The preparation process of the application is shown in the schematic diagram. DETAILED DESCRIPTION

[0049] The technical solutions of the application will be described clearly and completely below in combination with the drawings of the application. Obviously, the described embodiments are only a part of the embodiments of the application, not all. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the application.

[0050] Please refer to the accompanying Figure 1 The pharmaceutical composition provided by the embodiments of the application comprises, by weight:

[0051] 5-10 parts of baicalin extract, which is derived from the roots of Scutellaria baicalensis Georgi and obtained by extraction with an ethanol-water mixed solvent;

[0052] 3-7 parts of andrographolide extract, which is derived from the whole grass of Andrographis paniculata (Burm. f.) Nees and obtained by extraction with an ethanol-water mixed solvent;

[0053] 2-5 parts of sulfated fucoidan, which is derived from brown algae and prepared by enzymatic hydrolysis and alcohol precipitation;

[0054] 0.2-1 parts of spongin extract, which is derived from sponges and obtained by extraction with an organic solvent and separation and purification.

[0055] The preparation method of the combined pharmaceutical composition of Chinese herbal medicine and marine extract comprises the following steps:

[0056] S1, preparing baicalin extract and andrographolide extract; the extraction conditions of S1 baicalin extract and andrographolide extract are:

[0057] An ethanol-water mixed solvent is used, and the volume fraction of ethanol is 30%-70%;

[0058] The extraction temperature is 25-40 DEG C;

[0059] Ultrasonic-assisted extraction is used, and the solid-liquid ratio is 1:8-1:12;

[0060] The extraction time is 20-40 minutes.

[0061] S2, preparing sulfated fucoidan and spongin extract; the extraction conditions of sulfated fucoidan and spongin extract are as follows:

[0062] The sulfated fucoidan is subjected to cellulase enzymolysis, the enzymolysis temperature is 60-70 DEG C, the enzymolysis time is 60-90 minutes, and the sulfated fucoidan is precipitated by 60%-80% volume fraction ethanol;

[0063] The spongin extract is soaked in chloroform-methanol mixed solvent (volume ratio 2:1) at room temperature for 48-72 hours, and then chromatographically separated.

[0064] S3, mixing each extract with phospholipid material to prepare a nano delivery system; the preparation conditions of the nano delivery system are as follows:

[0065] The mass ratio of phospholipid to cholesterol is 8:2-10:2;

[0066] The average particle size of the nano particles is 80-120 nm;

[0067] The Zeta potential is controlled at -20 to -40 mV;

[0068] The extract encapsulation rate is not less than 80%.

[0069] S4, dispersing the nano delivery system in a system containing a hydrogel matrix, and adjusting the pH value; the hydrogel matrix comprises:

[0070] Sodium hyaluronate, mass concentration 0.1%-0.5%;

[0071] Glycerol, mass concentration 1%-3%. The pH value is adjusted to 4.0-5.0 by citric acid buffer solution;

[0072] After the nano delivery system and the hydrogel matrix are uniformly mixed, they are subjected to high pressure homogenization treatment 1-3 times at 600-800 bar. The volume fraction mixing ratio of the nano delivery system and the hydrogel matrix is 1:4-1:8.

[0073] S5, forming a final pharmaceutical composition.

[0074] In order to facilitate the understanding of the technical solution, based on the same inventive concept, the following embodiments are proposed:

[0075] Example 1: preparation of a standard formula:

[0076] Formula composition: baicalin extract: 7 parts;

[0077] Andrographolide extract: 5 parts;

[0078] Sulfated fucoidan: 3 parts;

[0079] Spongin extract: 0.5 parts;

[0080] Preparation method:

[0081] Extraction process: Scutellaria baicalensis root and Andrographis paniculata whole plant were extracted using ethanol-water mixture (50% ethanol concentration) by ultrasonic extraction. The ratio of extraction solvent to medicinal material was 1:10 (g / mL), ultrasonic power was 200 W, frequency was 40 kHz, and extraction time was 30 minutes. Sulfated fucoidan was enzymatically digested using cellulase at a temperature of 65 °C for 80 minutes, followed by precipitation using 60% ethanol by volume to obtain the polysaccharide component. Spongin extract was obtained by soaking in chloroform-methanol (2:1, volume ratio) for 72 hours, followed by separation and purification using silica gel column chromatography.

[0082] Phospholipid nanoparticle preparation: Phospholipid and cholesterol were dissolved in chloroform at a mass ratio of 9:1, followed by thin film formation using the solvent evaporation method. A buffer solution containing water-soluble drugs was then added, and ultrasonic treatment (power 300 W, frequency 40 kHz) was used to form nanoparticles with a particle size of 100 nm, a Zeta potential of -30 mV, and an encapsulation efficiency of 85%.

[0083] Hydrogel matrix preparation: The hydrogel matrix was composed of 0.3% (w / v) sodium hyaluronate and 2% (w / v) glycerol. The sodium hyaluronate solution was dissolved by stirring, and glycerol was added to the solution. The pH was adjusted to 4.5, and the gel matrix was formed by freeze-drying.

[0084] Final composition formation: The phospholipid nanoparticles and the hydrogel matrix were mixed at a ratio of 1:6, and homogenized (500 bar, 2 times) to obtain the final pharmaceutical composition.

[0085] Example 2: Preparation of high-concentration marine extract formula

[0086] Formula composition: Scutellaria baicalensis glycosides extract: 8 parts;

[0087] Andrographolide extract: 6 parts;

[0088] Sulfated fucoidan: 4 parts;

[0089] Spongin extract: 1 part;

[0090] Preparation method:

[0091] Extraction process: Baicalin and Andrographolide extracts were extracted using ethanol-water mixture. The extraction was carried out using 60% ethanol solution, solid-liquid ratio of 1:12, ultrasonic power of 250 W, frequency of 40 kHz, and extraction time of 40 minutes. Sulfated fucoidan was prepared by cellulase enzymatic hydrolysis at 70 °C for 90 minutes and precipitated using 80% ethanol. Spongin extract was obtained by soaking in chloroform-methanol solution (2:1, v / v) for 48 hours and purified by extraction and chromatography.

[0092] Phospholipid nanoparticle preparation: The mass ratio of phospholipid to cholesterol was 8:2, thin film formation was carried out using solvent evaporation method, followed by the addition of drug solution (containing Chinese herbal medicine extract and marine extract), and nanoparticles were formed using ultrasonic treatment (300 W, frequency 40 kHz), with a particle size of 110 nm, a Zeta potential of -35 mV, and an encapsulation efficiency of 88%.

[0093] Hydrogel matrix preparation: The hydrogel matrix was composed of 0.4% (w / v) sodium hyaluronate and 1.5% (w / v) glycerol. After dissolving the sodium hyaluronate in water, glycerol was added, the pH value was adjusted to 4.2, and the hydrogel was prepared by freeze-drying method.

[0094] Final composition formation: The nanoparticles and hydrogel matrix were mixed in a ratio of 1:7, and high-pressure homogenization technology (700 bar, 3 times) was used to uniformly disperse them to obtain the drug composition.

[0095] Example 3: Preparation of low-concentration formula

[0096] Formula composition: Baicalin extract: 6 parts;

[0097] Andrographolide extract: 4 parts;

[0098] Sulfated fucoidan: 2 parts;

[0099] Spongin extract: 0.3 parts;

[0100] Extraction process: Baicalin and Andrographolide were extracted using 50% ethanol-water solution. The solid-liquid ratio was 1:8, the ultrasonic power was 220 W, the frequency was 40 kHz, and the extraction time was 30 minutes. Sulfated fucoidan was treated by cellulase enzymatic hydrolysis at 60 °C for 60 minutes and precipitated using 70% ethanol. Spongin extract was obtained by soaking in chloroform-methanol (3:1, v / v) solution for 48 hours and separated by chromatography.

[0101] Phospholipid Nanoparticle Preparation: The mass ratio of phospholipid to cholesterol is 10:0, using solvent evaporation method to form a film, then adding a drug solution (including herbal extract and marine extract), using ultrasonic treatment (power 200W, frequency 40kHz) to form nanoparticles, the particle size is about 90nm, the Zeta potential is-25mV, and the encapsulation efficiency is 82%.

[0102] Hydrogel Matrix Preparation: The hydrogel matrix is composed of 0.2% (w / v) sodium hyaluronate and 2% (w / v) glycerol. After dissolving the sodium hyaluronate, glycerol is added, the pH value is adjusted to 4.5, and the hydrogel is prepared by freeze-drying method.

[0103] Final Composition Formation: The nanoparticles are mixed with the hydrogel matrix at a ratio of 1:5, and are uniformly treated by high-pressure homogenization (600bar, 2 times), and finally a drug composition is obtained.

[0104] Comparative Example 1-1: Follow the preparation procedure of Example 1, but do not add spongin extract, and the remaining steps and parameters remain unchanged.

[0105] That is, the composition only includes baicalin, andrographolide and sulfated fucoidan, without the intervention of marine active ingredients.

[0106] Comparative Example 1-2: According to the formulation ratio (containing marine extract) of Example 1, but do not prepare phospholipid nanoparticles, directly dissolve the extract in the hydrogel matrix, and omit the nanoparticle formation step.

[0107] The drug components are directly loaded in the hydrogel as stock solution, without nanoparticle encapsulation.

[0108] Comparative Example 1-3: During the extraction of baicalin and andrographolide, the traditional hot reflux extraction method is used, the temperature is 85℃, the extraction time is 2 hours, and the ultrasonic extraction process is not used.

[0109] The subsequent preparation steps remain unchanged from Example 1.

[0110] Comparative Example 2-1: Based on Example 2, but the amount of spongin extract added is reduced to 0.2 parts, and the rest of the medicinal material ratio and preparation process remain unchanged.

[0111] Form a composition with a significantly reduced marine component.

[0112] Comparative Example 2-2: Prepare the extract according to the formulation ratio of Example 2, but directly mix the extract solution with the hydrogel matrix, omit the nanoparticle formation step.

[0113] Directly form a coarse particle mixture, without forming a nanoparticle structure.

[0114] Comparative Example 2-3: The preparation process is the same as Example 2, but the hydrogel matrix is changed to carboxymethylcellulose (0.5%, w / v) + glycerol 1% (w / v), and no sodium hyaluronate is used.

[0115] The pH value is adjusted to 5.5, and the gel is formed using conventional stirring, without freeze-drying treatment.

[0116] Comparative Example 3-1: When extracting baicalin and andrographolide, the extraction temperature is increased to 90°C, the extraction time is maintained for 30 minutes, and the ultrasonic extraction conditions remain unchanged.

[0117] The rest of the preparation process is consistent with Example 3.

[0118] Comparative Example 3-2: Based on the preparation of Example 3, the mass ratio of phospholipid to cholesterol is adjusted to 7:3 (i.e., the proportion of cholesterol is increased).

[0119] The preparation method of the nanoparticles remains unchanged, and the other process steps are consistent.

[0120] Comparative Example 3-3: According to Example 3, but no sulfated fucoidan is added at all, and the rest of the formulation ratio and preparation process is the same.

[0121] Only contains Chinese herbal medicine extract and spongin extract, and the polysaccharide protective layer component is missing.

[0122] Experiment 1: In vitro antibacterial activity test:

[0123] Experimental procedure: Take Staphylococcus aureus (ATCC25923) and Candida albicans (ATCC10231) standard strains, place them on nutrient agar medium, and incubate at 37°C for 24 hours for standby.

[0124] According to the preceding Examples 1, 2, 3, and corresponding Comparative Examples 1-1, 1-2, 2-1, 2-2, 3-3, respectively, prepare the drug solution so that the final effective ingredient concentration is consistent (100 μg / mL).

[0125] Dilute the bacterial suspension to 0.5 McFarland standard concentration (about 1 × 10 8 CFU / mL).

[0126] Use a sterile cotton swab to evenly apply the bacterial solution to the surface of a freshly prepared nutrient agar plate.

[0127] Use a sterile puncher to punch holes in the plate, and add 50 μL of drug solution to each hole.

[0128] Set three holes for each treatment, and perform three parallel experiments.

[0129] Place the treated culture dishes in a 37°C incubator for 24 hours.

[0130] After taking out, the diameter of the inhibition zone was measured with a vernier caliper, the data was recorded, and the average value was calculated.

[0131] Table 1: Statistical results of the diameter of the inhibition zone (unit: mm)

[0132]

[0133]

[0134] The composition of the present application exhibits significantly better antibacterial activity than the comparative examples, especially against Staphylococcus aureus, the diameter of the inhibition zone of Example 2 is much higher than all the comparative examples. It can be speculated that this effect not only comes from the antibacterial effect of a single component, but also from the synergistic mechanism after the combination of marine extracts and Chinese herbal medicine. As an antibacterial active factor from marine sources, spongin produces a combined bactericidal effect with traditional medicinal ingredients such as baicalin and andrographolide, destroys the integrity of the bacterial cell membrane, and exacerbates cell metabolic disorders, thereby greatly improving the antibacterial efficacy.

[0135] When comparing the nanoparticle carrier with the direct mixing system, the difference is also significant. The inhibition zone of Comparative Examples 1-2 and 2-2, which are not prepared into nanoparticles, is generally small. This is likely due to the insufficient stability of the active ingredients in the unwrapped state, the drug release speed is too fast, and the local concentration is not enough to sustain the inhibition of bacterial population expansion. The introduction of phospholipid nanoparticles not only improves the enrichment of drugs in the lesion area, but also prolongs the release time of active ingredients, making the antibacterial effect more persistent and powerful. This is exactly the same as the mechanism we proposed for the nanoparticle delivery system.

[0136] It is worth mentioning that different combinations of ratios also show interesting changes in the inhibition effect on Candida albicans. In Example 2, the content of sulfated fucoidan is higher, which seems to promote the destruction of the extracellular matrix of fungi to some extent, indirectly enhancing the bactericidal effect. This result to some extent confirms our assumption of the multi-pathway synergistic antibacterial mechanism. That is, the present application does not simply rely on the effect of a single component, but through the interaction of multiple source components, a network type of antibacterial barrier is formed.

[0137] Experiment 2: Inhibition of biofilm formation experiment

[0138] Staphylococcus aureus biofilm high expression strain (laboratory preserved strain) was selected.

[0139] The strain was inoculated in TSB medium and cultured at 37°C in a shaking incubator overnight to prepare fresh bacterial suspension.

[0140] The concentration of the bacterial suspension was adjusted to 1×10 7 CFU / mL for standby.

[0141] Take 96-well plates, add 100 μL of bacterial suspension to each well.

[0142] The treatment group was added with the following samples: Example 1, Example 2, Comparative Example 1-1, 1-2, 2-1, 2-2, respectively.

[0143] The drug concentration was set to 100 μg / mL final concentration.

[0144] The control group was only added with bacterial solution and medium, without adding drugs.

[0145] Place the 96-well plate at 37°C and incubate for 24 hours to promote biofilm formation.

[0146] After incubation, discard the supernatant and gently rinse each well twice with PBS.

[0147] Add 0.1% crystal violet solution, 100 μL per well, and stain at room temperature for 15 minutes.

[0148] Discard the staining solution and wash with PBS three times, then air dry.

[0149] Add 200 μL of 95% ethanol to dissolve the stained biofilm.

[0150] Measure the absorbance (OD450) at 450 nm, with 3 wells in parallel for each group.

[0151] Table 2 Comparison of biofilm formation (OD450):

[0152]

[0153]

[0154] As can be seen from the experimental data, Examples 1 and 2 significantly reduce the amount of bacterial biofilm formation, especially Example 2, with a more obvious decrease in OD450 value. This result confirms the synergistic effect of the combined extract and nano-delivery system in the technical solution of the present application. The spongin in the marine extract plays an important role in inhibiting the initial adhesion and polymerization of bacteria in this experiment, thereby blocking the construction process of the biofilm. This phenomenon is highly consistent with the previously proposed mechanism of interfering with biofilm formation, verifying the innovative design of the drug composition.

[0155] The performance of the comparative sample further highlights the advantages of the present application. Comparative examples 1-2, 2-2, which do not use phospholipid nanoparticle encapsulation, have a significantly inferior biofilm inhibition effect to the examples. This shows that the directly released drug ingredients are easily inactivated or shielded in the complex biofilm environment, resulting in limited therapeutic effect. In contrast, the slow-release effect of the phospholipid nanoparticles ensures the long-term maintenance of active ingredients in the microenvironment, thereby continuously resisting bacterial adhesion and expansion. The presence of nanoparticles is not just simple delivery, but also an important guarantee for improving local drug efficacy and delaying the formation of drug-resistant biofilms.

[0156] In addition, by observing the data between different examples and comparative examples, it can be inferred that sulfated fucoidan plays a key role in hindering the development of biofilms. In Example 2, the proportion of polysaccharide ingredients is increased, and the amount of biofilm formation is significantly reduced, suggesting that it has a synergistic enhancement effect in disrupting the polymerization bridge between bacteria and weakening the stability of the extracellular matrix.

[0157] Experiment 3: Cytokine inhibition experiment (in vitro anti-inflammatory activity detection):

[0158] Take mouse macrophage cell line RAW264.7 cells, place them in RPMI-1640 culture medium, add 10% fetal bovine serum, and incubate at 37°C in a 5% CO2 atmosphere.

[0159] Seed the cells in a 96-well culture plate at a seeding density of 1 x 10 5 cells / well, and incubate for 24 hours to allow them to adhere.

[0160] Replace the ordinary culture medium with the drug treatment group: Example 1, Example 2, Example 3, Comparative Example 1-1, 2-1, 3-3;

[0161] The final concentration of the drug in the treatment group is uniformly adjusted to 50 μg / mL.

[0162] Add LPS (lipopolysaccharide, final concentration 1 μg / mL) to each well to stimulate cells and induce inflammation.

[0163] Set up a negative control group (no drug added, add LPS) and a positive control group (dexamethasone 5 μg / mL).

[0164] Continue to incubate the cells for 24 hours.

[0165] Collect the supernatant and detect the cytokine levels using IL-6 and TNF-α ELISA kits according to the kit instructions.

[0166] Set up 3 replicate wells for each group, measure the absorbance, and convert the cytokine concentration according to the standard curve.

[0167] Table 3: IL-6 and TNF-a level detection results (unit: pg / mL)

[0168]

[0169]

[0170] The experimental results clearly show that the drug of the examples can significantly inhibit the secretion of IL-6 and TNF-a in cells, and the inhibition effect of the comparative examples is obviously insufficient. In particular, Example 2, the decrease in the level of the two cytokines is most obvious. This is highly consistent with our previous mechanism speculation: under the combined regulation of multiple sources, the bioactive molecules in the marine extract not only directly act on the pro-inflammatory signaling pathway, but also inhibit key inflammatory transcription factors such as NF-κB, thereby achieving the weakening of the overall anti-inflammatory response. Thus, the present application forms a multi-target intervention in the anti-inflammatory mechanism, with obvious advantages.

[0171] The comparative examples particularly expose the importance of the drug delivery system. The samples not using nanoparticle encapsulation, such as Comparative Example 2-1, have IL-6 and TNF-a levels close to or even higher than one time more than other examples. It can be reasonably inferred that the active ingredients in the unloaded state are easily inactivated in the cell microenvironment, or cannot effectively enter the cell interior, resulting in attenuation of the effect. The protection and uptake-promoting effect of the nanoparticles obviously provides a basic guarantee for the anti-inflammatory effect of the present application, which is closely related to the previously emphasized sustained-release and controlled-release characteristics.

[0172] Another notable phenomenon is the potential contribution of polysaccharide components in synergistic regulation. The anti-inflammatory effect of Example 2 is slightly better than that of Examples 1 and 3, suggesting that sulfated fucoidan may play a certain auxiliary role in inhibiting the release of cytokines. This mechanism speculation is consistent with the known role of polysaccharides in regulating the extracellular matrix and stabilizing the cell signal microenvironment. It shows that the present application is not a single chemical intervention, but achieves a more comprehensive anti-inflammatory effect through multi-dimensional regulation.

[0173] Experiment 4: Nanoparticle particle size and stability detection:

[0174] Sample preparation: According to the aforementioned Examples 1, 2, 3 and Comparative Examples 1-2, 2-2, prepare nanoparticle solutions.

[0175] The drugs in Examples 1, 2, and 3 are wrapped by phospholipid nanoparticles;

[0176] Comparative Examples 1-2 and 2-2 directly dissolve the drugs without nanoparticle encapsulation.

[0177] Particle size test: Dynamic light scattering (DLS) instrument is used for particle size and PDI (polydispersity index) determination.

[0178] Each sample was taken an appropriate amount of solution, diluted 10 times, and the particle size (unit: nm) and PDI value were tested by DLS.

[0179] Zeta potential test: The Zeta potential of each group of drug solution was measured to evaluate its stability.

[0180] The Zeta potential of the sample was measured using ZetaSizer (unit: mV).

[0181] Particle size stability test: Each sample solution was placed at room temperature for 7 days, and the particle size and Zeta potential were measured every day. Particle size and Zeta potential were detected once a day to observe the change trend. Data recording: The particle size, PDI, Zeta potential and stability data of each sample were recorded.

[0182] Table 4 Nanoparticle particle size and Zeta potential change data

[0183]

[0184]

[0185] From the experimental data, it can be seen that the nanoparticle particle size of Example 1, Example 2 and Example 3 is relatively small, and the stability is good. In particular, Example 2 has smaller particle size change and Zeta potential change, indicating that its particles have higher stability in suspension. This is closely related to the design of the nanoparticles. The encapsulation effect of phospholipid nanoparticles significantly improves the stability of the drug, avoiding the rapid release of the drug in the body. The nanoparticle system can ensure the long-term effect of the drug in the body through a slow release mechanism, thereby enhancing the targeting and therapeutic effect of the drug.

[0186] Compared with the examples, Comparative Examples 1-2 and 2-2 show larger particle size change and poorer stability. This indicates that samples without nanoparticle encapsulation have drug particles that are prone to aggregation, precipitation or polymerization. The system that simply dissolves the drug lacks a stable carrier structure, resulting in rapid or uneven drug release, which affects the therapeutic effect. This result verifies the key role of nanoparticles in improving drug stability and biocompatibility.

[0187] Furthermore, the Zeta potential data also revealed the relationship between particle surface charge and stability. Example 2 showed a higher Zeta potential (-30.2 mV), indicating strong electrostatic repulsion and preventing particle aggregation and precipitation. In contrast, Comparative Example 2-2 showed a lower Zeta potential (-18.7 mV), highlighting the instability of the drug system in solution without the use of nanoparticle carriers, making it impossible to form an effective drug delivery system. Therefore, the introduction of nanoparticles not only improves drug stability but also optimizes clinical efficacy by delaying drug release time.

[0188] Experiment 5: Detection of in vitro drug release performance of hydrogels:

[0189] Sample preparation: Drug-loaded hydrogels were prepared according to the formulations of Example 2 and Comparative Examples 2-3 described above.

[0190] Example 2 uses sodium hyaluronate (0.4%, w / v) as a hydrogel matrix, and mixes the drug solution with nanoparticles after preparation.

[0191] Comparative Examples 2-3 used carbomer (0.5%, w / v) as a hydrogel matrix to directly dissolve the drugs.

[0192] Hydrogel drug embedding: The drug solution and hydrogel matrix are mixed in a specific ratio and then freeze-dried into a gel block. This ensures that the drug is uniformly distributed within the hydrogel.

[0193] Drug release test: The hydrogel samples were placed in PBS buffer (pH 7.4) and kept at 37°C.

[0194] Samples were taken every 1 hour, 4 hours, 8 hours, 12 hours, 24 hours, and 48 hours, and the drug concentration in the solution was detected using a UV-Vis spectrophotometer.

[0195] The amount of drug released is calculated using a standard curve.

[0196] Data recording and processing: Three replicate experiments were set up for each sample, and the drug release at each time point was calculated and the release curve was plotted.

[0197] Table 5. In vitro drug release curves (unit: mg)

[0198]

[0199]

[0200] The experimental results show that the drug release amount of Example 2 is significantly higher than that of Comparative Example 2-3 within 48 hours, and the release curve is relatively smooth, indicating that the sodium hyaluronate matrix plays a key role in the controlled release of the drug. The slow release of the drug in Example 2 conforms to the aforementioned nanometer particle slow release mechanism. The sodium hyaluronate hydrogel matrix can gradually release the drug to the external environment through the affinity between the drug molecules, which provides an effective slow release platform for the present application. In contrast, the release curve of Comparative Example 2-3 is more irregular, the release speed is faster, and the release amount gradually decreases in the later stage, indicating that the embedding and release control of the drug by the carbomer matrix is weak, which may lead to the rapid initial release of the drug and the decrease over time.

[0201] The properties of the hydrogel are closely related to the matrix used. In the sodium hyaluronate system, the drug gradually penetrates and releases through the network structure of the hydrogel, slowly and stably releasing the drug, while the carbomer may cause the drug release process to be unstable due to its lower hydration and larger particle size, resulting in a deviation in the release amount. This phenomenon is highly consistent with the matrix selective release mechanism mentioned in the present application, i.e., by optimizing the hydrogel matrix, the release rate and duration of the drug can be adjusted to maximize the therapeutic effect of the drug.

[0202] It is worth noting that the drug release amount in Example 2 tends to be stable after 24 hours, showing the advantage of sodium hyaluronate hydrogel in long-term drug release. In contrast, due to the physical properties of the carbomer hydrogel, the sustained release of the drug is limited, showing a relatively fast release peak. In the later release stage, the drug release of the sodium hyaluronate matrix hardly decreases, fully demonstrating the stability of the slow release carrier and the long-term effectiveness of the drug. This further proves the synergistic effect of the matrix material and the drug carrier, optimizing the bioavailability of the drug.

[0203] Experiment 6: In vitro biofilm disruption and antibacterial activity detection experiment:

[0204] Bacterial and fungal strain preparation: Select standard bacterial strains (such as Gardnerella vaginalis, Escherichia coli) and standard fungal strains (Candida albicans) to construct a biofilm model.

[0205] The strains were resuscitated in a suitable liquid medium and cultured to the logarithmic growth phase for standby use.

[0206] Biofilm formation:

[0207] Inoculate 1x10 7 CFU / mL of bacterial solution into a 96-well plate, 200 μL per well.

[0208] Incubate at 37°C for 24 hours to promote the formation of mature biofilm at the bottom of the well.

[0209] Treatment and intervention:

[0210] Remove supernatant and wash with PBS for 3 times to remove unattached cells.

[0211] Add drug treatment solution (Example 2 hydrogel treatment group, Comparative Example 2-3 hydrogel treatment group, positive control dexamethasone group, negative control PBS group).

[0212] The drug concentration is unified to 50 μg / mL, and incubate for 24 hours.

[0213] Biofilm destruction detection:

[0214] After incubation, rinse with PBS to remove planktonic bacteria.

[0215] Add crystal violet staining solution to stain the biofilm (0.1%, 20 min), and wash to remove excess dye.

[0216] Add ethanol solution (95%) to dissolve crystal violet, and measure the OD595 absorbance value of each well to reflect the amount of biofilm.

[0217] Antibacterial activity detection: Set another parallel route, dilute and spread the liquid in the well plate on solid medium after treatment. Count CFU after 24 hours of culture to reflect the amount of residual live bacteria in the biofilm.

[0218] Table 6 Biofilm destruction and live bacteria residual detection results:

[0219]

[0220] The experimental results show that the drug composition of Example 2 has obvious destructive effect on bacterial and fungal biofilms, and the number of surviving bacteria and fungi is greatly reduced after destroying the biofilm. In practical application, this effect is particularly crucial for the treatment of vaginitis, because once the biofilm is formed, it not only blocks the antibacterial agent, but also becomes the main source of repeated infection. The present application plays a bactericidal effect by synergistic action of herbal medicine and marine extract in destroying the membrane, compared with the traditional single bactericidal scheme, which truly solves the problem of difficult penetration of stubborn biofilm barrier of bacteria and fungi.

[0221] In local treatments of bacterial vaginosis and candidal vaginitis with accompanying infections, the effectiveness of the medication in disrupting the biofilm determines the completeness of the treatment. Experiments show that while traditional comparative hydrogel formulations have some effect on free-floating bacteria, they are almost ineffective against mature biofilms. In contrast, this invention, through nanoparticle delivery and controlled release via hydrogel, allows the active ingredients to penetrate deep into the biofilm, continuously disrupting the microbial defense layer and significantly improving the success rate of local treatment.

[0222] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition, characterized in that, Included by weight: 7-8 parts of baicalin extract; 5-6 parts of andrographolide extract; 3-4 parts of sulfated brown algae polysaccharide; Spongin extract 0.5-1 part; Baicalin extract is derived from the root of Scutellaria baicalensis and is obtained by extraction with a mixed solvent of ethanol and water. Andrographolide extract is obtained from the whole herb of Andrographis paniculata by extraction with a mixed solvent of ethanol and water. Sulfated brown algae polysaccharides are derived from brown algae and prepared by enzymatic hydrolysis and alcohol precipitation. Spongin extract is derived from sponges and obtained through organic solvent extraction and purification. The method for preparing the pharmaceutical composition includes the following steps: S1. Preparation of baicalin extract and andrographolide extract; The extraction conditions for the S1 baicalin extract and andrographolide extract are as follows: An ethanol-water mixed solvent is used, with an ethanol volume fraction of 30%–70%. The extraction temperature is 25℃~40℃; Ultrasonic-assisted extraction, with a material-to-liquid ratio of 1:8 to 1:12; Extraction time is 20–40 minutes; S2. Preparation of sulfated brown algae polysaccharide and spongin extract; S3. Prepare a nanodelivery system by mixing the extracts with phospholipid materials; The preparation conditions for the S3 nanodelivery system are as follows: The mass ratio of phospholipids to cholesterol is 8:2 to 10:2; The average particle size of the nanoparticles is 80–120 nanometers; The zeta potential is controlled between -20 and -40 millivolts; The encapsulation rate of the extract is not less than 80%; S4. Disperse the nanodelivery system in a system containing a hydrogel matrix and adjust the pH value; in S4, the hydrogel matrix includes: Sodium hyaluronate, with a mass concentration of 0.1% to 0.5%; Glycerin, with a mass concentration of 1% to 3%; In S4: Adjust the pH to 4.0–5.0 using a citric acid buffer solution; After the nano-delivery system is uniformly mixed with the hydrogel matrix, it is subjected to high-pressure homogenization treatment at 600-800 bar 1-3 times; S5. Form the final pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, characterized in that, The extraction conditions for the S2 sulfated brown algae polysaccharide and spongin extract are as follows: Sulfated brown algae polysaccharides were hydrolyzed by cellulase at a temperature of 60℃~70℃ for 60~90 minutes, and then precipitated with 60%~80% volume fraction ethanol. The spongin extract was obtained by chromatographic separation after soaking in a chloroform-methanol mixed solvent at room temperature for 48–72 hours.

3. The pharmaceutical composition according to claim 1, characterized in that, The volume fraction mixing ratio of the nanodelivery system to the hydrogel matrix is ​​1:4 to 1:8.

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