Chinese herbal medicine and marine extract combined antibacterial and anti-inflammatory pharmaceutical composition

Abstract

The invention relates to the field of pharmacy, and discloses a Chinese herbal medicine and marine extract combined antibacterial and anti-inflammatory pharmaceutical composition which comprises the following components in parts by weight: 5-10 parts of baicalin extract; 3 to 7 parts of an andrographolide extract; 2-5 parts of sulfated brown algae polysaccharide; 0.2 to 1 part of sponge extract; wherein the baicalin extract is derived from roots of scutellaria baicalensis and is obtained by extracting with an ethanol-water mixed solvent; the andrographolide extract is derived from the whole herb of andrographis paniculata and is obtained by extracting with an ethanol-water mixed solvent; the sulfated fucoidan is derived from fucoidan and is prepared by enzymolysis and alcohol precipitation methods; the sponge extract is derived from sponge and is obtained by organic solvent extraction, separation and purification. By adopting the technical scheme of synergistically compounding the Chinese herbal medicine active ingredients and the marine extract, the technical effects of destroying a biological membrane barrier and removing stubborn infection lesions in treatment of bacterial vaginitis, candida vaginitis associated biological membrane infection and cervicitis are achieved.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, in particular to a pharmaceutical composition comprising Chinese herbal medicine and marine extracts for antibacterial and anti-inflammatory purposes. Background Art

[0002] Female reproductive tract infections, such as bacterial vaginosis, candidal vaginitis, and cervicitis, are widespread among women of all ages. These conditions not only cause local discomfort but can also impact reproductive health and even induce ascending infections. In many cases, infections are closely linked to an imbalance in the local vaginal microbiome and the formation of pathogen biofilms.

[0003] Biofilms are complex microbial aggregates composed of a matrix consisting of polysaccharides, proteins, and other components. Pathogens within mature biofilms possess increased resistance to external environments. Even with adequate doses of antibiotics or antifungal drugs, complete eradication is often difficult, resulting in prolonged treatment and even recurrence.

[0004] Current clinical treatment for these infections primarily relies on topical medications, including conventional drugs such as metronidazole and fluconazole. While these drugs can effectively alleviate symptoms initially, they have limited permeability across biofilm barriers and their efficacy is short-lived. Relapses shortly after treatment are not uncommon.

[0005] To enhance therapeutic efficacy, some studies have explored the use of natural products, such as plant extracts or marine bioactive ingredients. These natural ingredients have demonstrated antibacterial and anti-biofilm activity in vitro and exhibit good biocompatibility. However, when applied directly, natural extracts are susceptible to environmental influences, resulting in poor stability of the active ingredients and insufficient tissue penetration, often resulting in lower-than-expected in vivo efficacy. Summary of the Invention

[0006] In response to the deficiencies of the existing technology, the present invention provides a pharmaceutical composition of Chinese herbal medicine and marine extracts for antibacterial and anti-inflammatory purposes, which solves the problems of short duration of efficacy, insufficient biomembrane penetration ability and unstable release of natural product activity in the existing treatment of local gynecological infections.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions: 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 sponge extract.

[0012] Preferably, wherein:

[0013] Baicalin extract is obtained from the root of Scutellaria baicalensis and extracted with an ethanol-water mixed solvent;

[0014] Andrographolide extract is derived from the whole herb of Andrographis paniculata and is extracted with an ethanol-water mixed solvent;

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

[0016] Sponge extract is derived from sponges and is obtained through organic solvent extraction and separation and purification.

[0017] The preparation method of the combined medicinal composition of Chinese herbal medicine and marine extract 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 a phospholipid material to prepare a nano delivery system;

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

[0022] S5. Forming the 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, with the ethanol volume fraction being 30% to 70%;

[0025] The extraction temperature is 25℃~40℃;

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

[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 at a temperature of 60° C. to 70° C. for 60 to 90 minutes, and then precipitated by 60% to 80% volume fraction ethanol.

[0030] The sponge extract was obtained by soaking in a chloroform-methanol mixed solvent (volume ratio 2:1) at room temperature for 48 to 72 hours and then separating by chromatography.

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

[0032] The mass ratio of phospholipids to cholesterol is 8:2 to 10:2;

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

[0034] Zeta potential was controlled at −20 to −40 mV;

[0035] The extract encapsulation rate is not less than 80%.

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

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

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

[0039] Preferably, in S4:

[0040] Adjust the pH value to 4.0-5.0 with citric acid buffer solution;

[0041] After the nano-delivery system is uniformly mixed with the hydrogel matrix, it is subjected to a high-pressure homogenization treatment at 600 to 800 bar for 1 to 3 times.

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

[0043] The invention relates to the use of a combined pharmaceutical composition of herbal and marine extracts, and the use of the pharmaceutical composition in the preparation of a topical medication for treating bacterial vaginosis, candidal vaginitis associated with biofilm infection or cervicitis.

[0044] The present invention provides a pharmaceutical composition comprising Chinese herbal medicine and marine extracts for antibacterial and anti-inflammatory effects. The composition has the following beneficial effects:

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

[0046] 2. The present invention adopts a combination of nanoparticle encapsulation and sodium hyaluronate hydrogel controlled release delivery technology to prolong the residence time of the drug in the affected area, avoiding the clinical problems caused by the short-term efficacy and frequent medication in the existing technology, and is particularly suitable for the treatment of chronic vaginitis and chronic cervicitis.

[0047] 3. The present invention adopts a strategy of comprehensive regulation of local microenvironment reconstruction and bioactive ingredients. During the treatment process, it not only eliminates pathogenic microorganisms, but also promotes mucosal barrier repair and improvement of the inflammatory environment, significantly improving the quality of tissue recovery after treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Schematic diagram of the preparation process steps of the present invention. DETAILED DESCRIPTION

[0049] The following will clearly and completely describe the technical solution of the present invention in conjunction with the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0050] Please see the attached Figure 1 , the embodiment of the present invention provides a pharmaceutical composition, which comprises, by weight:

[0051] 5-10 parts of baicalin extract, which is obtained from the root of Scutellaria baicalensis and extracted with an ethanol-water mixed solvent;

[0052] 3-7 parts of andrographolide extract, which is obtained from the whole herb of Andrographis paniculata 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 to 1 parts of sponge extract, which is derived from sponge and is obtained through organic solvent extraction, separation and purification.

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

[0056] S1. Preparation of baicalin extract and andrographolide extract; the extraction conditions of S1 baicalin extract and andrographolide extract are as follows:

[0057] An ethanol-water mixed solvent is used, with the ethanol volume fraction being 30% to 70%;

[0058] The extraction temperature is 25℃~40℃;

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

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

[0061] S2. Preparation of sulfated fucoidan and spongin extracts; the extraction conditions of the sulfated fucoidan and spongin extracts are as follows:

[0062] The sulfated fucoidan is enzymatically hydrolyzed by cellulase at a temperature of 60° C. to 70° C. for 60 to 90 minutes, and then precipitated by 60% to 80% volume fraction ethanol.

[0063] The sponge extract was obtained by soaking in a chloroform-methanol mixed solvent (volume ratio 2:1) at room temperature for 48 to 72 hours and then separating by chromatography.

[0064] S3. Mixing the extracts with the phospholipid material to prepare a nano-delivery system; the preparation conditions of the nano-delivery system are:

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

[0066] The average particle size of the nanoparticles is 80 to 120 nanometers;

[0067] Zeta potential was 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 includes:

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

[0071] Glycerol, mass concentration is 1% to 3%. Adjust the pH value to 4.0 to 5.0 with citric acid buffer solution;

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

[0073] S5. Forming the final pharmaceutical composition.

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

[0075] Example 1: Preparation of standard formula:

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

[0077] Andrographolide extract: 5 parts;

[0078] Sulfated fucoidan: 3 parts;

[0079] Sponge extract: 0.5 parts;

[0080] Preparation method:

[0081] Extraction process: The root of Scutellaria baicalensis and the whole herb of Andrographis paniculata were ultrasonically extracted using an ethanol-water mixed solvent (ethanol concentration of 50%). The material-liquid ratio of the extraction solvent to the medicinal material was 1:10 (g / mL), the ultrasonic power was 200W, the frequency was 40kHz, and the extraction time was 30 minutes. The sulfated brown algae polysaccharide was enzymatically hydrolyzed by cellulase at a hydrolysis temperature of 65°C and a hydrolysis time of 80 minutes, and then precipitated with 60% volume fraction ethanol to obtain the polysaccharide component. The spongin extract was soaked in a chloroform-methanol (2:1, volume ratio) solvent for 72 hours, and then separated and purified by silica gel column chromatography to obtain spongin.

[0082] Preparation of phospholipid nanoparticles: Phospholipids and cholesterol were dissolved in chloroform at a mass ratio of 9:1. A film was then formed by solvent evaporation. A buffer solution containing a water-soluble drug was then added. 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] Preparation of the hydrogel matrix: The hydrogel matrix consisted of 0.3% (w / v) sodium hyaluronate and 2% (w / v) glycerol. The sodium hyaluronate solution was dissolved by stirring, glycerol was added to the solution, the pH was adjusted to 4.5, and the gel matrix was formed by freeze-drying.

[0084] Formation of the final composition: Phospholipid nanoparticles and hydrogel matrix were mixed in 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: Baicalin extract: 8 parts;

[0087] Andrographolide extract: 6 parts;

[0088] Sulfated fucoidan: 4 parts;

[0089] Sponge extract: 1 part;

[0090] Preparation method:

[0091] Extraction process: Baicalin and andrographolide extracts were extracted using an ethanol-water mixed solvent. A 60% ethanol solution was used with a solid-liquid ratio of 1:12, an ultrasonic power of 250W, a frequency of 40kHz, and an extraction time of 40 minutes. Sulfated fucoidan was prepared by cellulase hydrolysis at 70°C for 90 minutes, followed by precipitation with 80% ethanol by volume to obtain pure polysaccharides. The spongin extract was soaked in a chloroform-methanol solution (2:1, volume ratio) for 48 hours and purified by extraction and chromatography.

[0092] Preparation of phospholipid nanoparticles: The mass ratio of phospholipid to cholesterol was 8:2. The film was formed by solvent evaporation, and then the drug solution (containing Chinese herbal extracts and marine extracts) was added. Ultrasonic treatment (300W, frequency 40kHz) was used to form nanoparticles with a particle size of 110nm, a zeta potential of -35mV, and an encapsulation efficiency of 88%.

[0093] Preparation of hydrogel matrix: The hydrogel matrix consists of 0.4% (w / v) sodium hyaluronate and 1.5% (w / v) glycerol. Sodium hyaluronate is dissolved in water and glycerol is added. The pH value is adjusted to 4.2, and the hydrogel is prepared by freeze drying.

[0094] The final composition was formed by mixing the nanoparticles and the hydrogel matrix in a ratio of 1:7 and uniformly dispersing them using a high-pressure homogenization technique (700 bar, 3 times) to obtain a pharmaceutical 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] Sponge extract: 0.3 parts;

[0100] Extraction process: Baicalin and andrographolide were extracted using a 50% ethanol-water solution. The solid-liquid ratio was 1:8, the ultrasonic power was 220W, the frequency was 40kHz, and the extraction time was 30 minutes. The sulfated fucoidan was enzymatically hydrolyzed with cellulase at 60°C for 60 minutes, followed by precipitation with 70% ethanol. The sponge extract was obtained by soaking in a chloroform-methanol (3:1, volume ratio) solvent for 48 hours and then chromatographically separating it.

[0101] Preparation of phospholipid nanoparticles: The mass ratio of phospholipid to cholesterol was 10:0. A thin film was formed by solvent evaporation. Then, drug solutions (including herbal extracts and marine extracts) were added. Ultrasonic treatment (power 200 W, frequency 40 kHz) was used to form nanoparticles with a particle size of approximately 90 nm, a zeta potential of -25 mV, and an encapsulation efficiency of 82%.

[0102] Preparation of hydrogel matrix: 0.2% (w / v) sodium hyaluronate and 2% (w / v) glycerol were used to form the hydrogel matrix. After dissolving the sodium hyaluronate, glycerol was added and the pH value was adjusted to 4.5. The hydrogel was prepared by freeze drying.

[0103] The final composition was formed by mixing the nanoparticles and the hydrogel matrix in a ratio of 1:5, and homogenizing them by high-pressure homogenization (600 bar, 2 times) to obtain a pharmaceutical composition.

[0104] Comparative Example 1-1: The preparation process of Example 1 was followed, but the sponge extract was not added, and the other steps and parameters remained the same.

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

[0106] Comparative Example 1-2: The formulation was prepared according to the ratio of Example 1 (containing marine extract), but phospholipid nanoparticles were not prepared. The extract was directly dissolved in the hydrogel matrix and mixed, and the nanoparticle formation step was omitted.

[0107] The drug components are directly loaded into the hydrogel in the form of stock solution without nanoparticle encapsulation.

[0108] Comparative Example 1-3: When extracting baicalin and andrographolide, the traditional hot reflux extraction method was adopted at a temperature of 85° C. and an extraction time of 2 hours, without using an ultrasonic extraction process.

[0109] The subsequent preparation steps were consistent with those in Example 1.

[0110] Comparative Example 2-1: Prepared on the basis of Example 2, but the addition amount of sponge extract was reduced to 0.2 parts, and the proportions of other medicinal materials and the preparation process remained unchanged.

[0111] A composition with significantly reduced marine content is formed.

[0112] Comparative Example 2-2: The extract was prepared according to the formulation ratio of Example 2, but the extract solution was directly mixed with the hydrogel matrix and the nanoparticle formation step was omitted.

[0113] A coarse particle mixture is directly formed without forming a nanoparticle structure.

[0114] Comparative Example 2-3: The preparation process was the same as that of Example 2, except that the hydrogel matrix was changed to carbomer (0.5%, w / v) + glycerol 1% (w / v), and sodium hyaluronate was not used.

[0115] The pH value was adjusted to 5.5 and the mixture was stirred into a gel using conventional methods without freeze-drying.

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

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

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

[0119] The nanoparticle preparation method remains unchanged, and other process steps are the same.

[0120] Comparative Example 3-3: Prepared according to Example 3, but without adding sulfated fucoidan, the remaining formula proportions and preparation process are the same.

[0121] It only contains Chinese herbal extracts and sponge extracts, and lacks the polysaccharide protective layer.

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

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

[0124] According to the aforementioned Example 1, Example 2, Example 3, and corresponding Comparative Examples 1-1, 1-2, 2-1, 2-2, and 3-3, drug solutions were prepared respectively to make the final active ingredient concentration consistent (100 μg / mL).

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

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

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

[0128] Three wells were set for each treatment, and the experiment was repeated three times.

[0129] The treated culture dish was placed 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 inhibition zone diameter (unit: mm)

[0132]

[0133]

[0134] The composition of the present invention exhibited significantly better antibacterial activity than the comparative examples, especially against Staphylococcus aureus, where the diameter of the inhibition zone of Example 2 was much higher than that of all the comparative examples. It can be speculated that this effect is not only due to the antibacterial effect of a single component, but also to the synergistic mechanism after the marine extract and the Chinese herbal medicine are compounded. Spongin, as an antibacterial active factor of marine origin, produces a combined bactericidal effect with traditional medicinal ingredients such as baicalin and andrographolide, destroying the integrity of bacterial cell membranes and exacerbating cellular metabolic disorders, thereby significantly improving the antibacterial efficacy.

[0135] When comparing nanoparticle carriers with direct mixing systems, the gap is also significant. The inhibition zones of comparative examples 1-2 and 2-2, which did not prepare nanoparticles, are generally small. This is likely due to the insufficient stability of the active ingredient in the unencapsulated state, the drug release rate is too fast, and the local concentration is insufficient to continuously inhibit the expansion of the bacterial population. The introduction of phospholipid nanoparticles not only improves the enrichment of the drug in the lesion area, but also prolongs the release time of the active ingredient, making the antibacterial effect more lasting and powerful. This is completely consistent with the description of the mechanism of the nano delivery system we proposed earlier.

[0136] It's worth noting that the inhibitory effects of different combinations on Candida albicans also showed interesting variations. The higher content of sulfated fucoidan in Example 2 appears to have promoted the destruction of the fungal extracellular matrix to a certain extent, indirectly enhancing the bactericidal effect. This result, to a certain extent, confirms our proposed multi-pathway synergistic antibacterial mechanism. In other words, the present invention does not rely solely on a single component to exert its effect, but rather forms a network-like antibacterial barrier through the interaction of multiple components.

[0137] Experiment 2: Biofilm formation inhibition experiment:

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

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

[0140] Adjust the concentration of bacterial suspension to 1×10 7 CFU / mL is reserved.

[0141] Take a 96-well plate and add 100 μL of bacterial suspension to each well.

[0142] The following samples were added to the treatment groups: Example 1, Example 2, Comparative Examples 1-1, 1-2, 2-1, and 2-2.

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

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

[0145] The 96-well plate was placed at 37°C for 24 hours to promote biofilm formation.

[0146] After the incubation period, the supernatant was discarded and each well was gently rinsed twice with PBS.

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

[0148] The staining solution was discarded, and the sections were washed three times with PBS and air-dried.

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

[0150] The absorbance (OD450) was measured at a wavelength of 450 nm, and three parallel wells were set up in each group.

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

[0152]

[0153]

[0154] As can be seen from the experimental data, Examples 1 and 2 significantly reduced the amount of bacterial biofilm formation, especially Example 2, where the OD450 value decreased more significantly. This result confirms the synergistic effect of the combined extract and the nanodelivery system in the technical solution of the present invention. The spongin in the marine extract played an important role in inhibiting the initial adhesion and aggregation of bacteria in this experiment, thereby blocking the biofilm construction process. This phenomenon is highly consistent with the mechanism of interference with biofilm formation proposed in the previous mechanism analysis, verifying the innovative design of the pharmaceutical composition.

[0155] The performance of the comparative samples further highlights the advantages of the present invention. Comparative Examples 1-2 and 2-2, which do not use phospholipid nanoparticle encapsulation, have significantly inferior biofilm inhibition effects than the examples. This indicates that directly released drug components are easily inactivated or shielded in a complex biofilm environment, resulting in limited therapeutic effects. In contrast, the sustained-release effect of phospholipid nanoparticles ensures that the active ingredients are maintained in the microenvironment for a long time, thereby continuously combating bacterial adhesion and expansion. The presence of nanoparticles is not only a simple delivery, but also an important guarantee for enhancing local drug efficacy and delaying the formation of drug-resistant biofilms.

[0156] Furthermore, observing the data from the different Examples and Comparative Examples demonstrates the key role of sulfated fucoidan in inhibiting biofilm development. In Example 2, the increased proportion of the polysaccharide component significantly reduced biofilm formation, suggesting a synergistic effect in disrupting interbacterial bridges and weakening the stability of the extracellular matrix.

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

[0158] Mouse macrophage cell line RAW264.7 cells were obtained and placed in RPMI-1640 medium supplemented with 10% fetal bovine serum and cultured at 37°C in a 5% CO2 atmosphere.

[0159] Cells were seeded in 96-well culture plates at a density of 1×10 5 cells / well and cultured for 24 hours to allow them to adhere.

[0160] The ordinary culture medium was replaced by the drug-containing treatment group: Example 1, Example 2, Example 3, Comparative Examples 1-1, 2-1, and 3-3;

[0161] The final drug concentration of the treatment groups was uniformly adjusted to 50 μg / mL.

[0162] At the same time, LPS (lipopolysaccharide, final concentration 1 μg / mL) was added to each well to stimulate the cells and induce an inflammatory response.

[0163] A negative control group (no drug, LPS) and a positive control group (dexamethasone 5 μg / mL) were set up.

[0164] The cells were cultured for an additional 24 hours.

[0165] The supernatant was collected and the cytokine levels were detected using ELISA kits for IL-6 and TNF-α according to the kit instructions.

[0166] Three replicate wells were set up in each group, the absorbance was measured, and the cytokine concentration was calculated based on the standard curve.

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

[0168]

[0169]

[0170] The experimental results clearly show that the example drugs can significantly inhibit the secretion of IL-6 and TNF-α in cells. In contrast, the inhibitory effect of the control group is obviously insufficient. In particular, in Example 2, the decrease in the levels of the two cytokines is the most obvious. This is highly consistent with the mechanism speculation we proposed before: under the joint regulation of multi-source components, the bioactive molecules in the marine extract not only directly act on the pro-inflammatory signaling pathway, but also achieve the weakening of the overall anti-inflammatory response by synergistically inhibiting key inflammatory transcription factors such as NF-κB. As a result, the present invention has formed a multi-target intervention in the anti-inflammatory mechanism, with obvious advantages.

[0171] The comparative examples particularly reveal the importance of drug delivery systems. Samples that do not use nanoparticle encapsulation, such as comparative example 2-1, have IL-6 and TNF-α levels close to or even more than double those of other examples. It can be reasonably inferred that the active ingredients in the unloaded state are prone to rapid inactivation in the cell microenvironment or are unable to effectively enter the cell interior, resulting in attenuation of the action force. The protective and uptake-promoting effects of the nanoparticles clearly provide a basic guarantee for the anti-inflammatory effect of the present invention, which is closely related to the sustained-release and controlled-release properties emphasized previously.

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

[0173] Experiment 4: Nanoparticle size and stability test:

[0174] Sample preparation: According to the above-mentioned Example 1, Example 2, Example 3 and Comparative Examples 1-2 and 2-2, a nanoparticle solution was prepared.

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

[0176] In Comparative Examples 1-2 and 2-2, the drug was directly dissolved without nanoparticle encapsulation.

[0177] Particle size measurement: Dynamic light scattering (DLS) instrument was used to measure particle size and PDI (polydispersity index).

[0178] Take an appropriate amount of solution from each sample, dilute it 10 times, and use DLS to measure the particle size (unit: nm) and PDI value.

[0179] Zeta potential test: The zeta potential of each group of drug solutions was measured to evaluate their stability.

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

[0181] Particle Size Stability Testing: Each sample solution was placed at room temperature for 7 days. Samples were taken daily for particle size and zeta potential measurements. Particle size and zeta potential were measured once daily to observe their changing trends. Data Recording: Particle size, PDI, zeta potential, and stability data for each sample were recorded.

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

[0183]

[0184]

[0185] As can be seen from the experimental data, the nanoparticles of Example 1, Example 2 and Example 3 have relatively small particle sizes and good stability. Especially in Example 2, its particle size change and Zeta potential change are small, indicating that the stability of its particles in the suspension is higher. This is closely related to the design of the nanoparticles. The encapsulation effect of the phospholipid nanoparticles significantly improves the stability of the drug and avoids the excessively fast release of the drug in vivo. The nanoparticle system can ensure that the drug plays a role in the body for a long time through a slow release mechanism, thereby enhancing the targeting and therapeutic effect of the drug.

[0186] Compared to the examples, Comparative Examples 1-2 and 2-2 exhibited significant particle size variation and poor stability. This suggests that drug particles in samples without nanoparticle encapsulation were prone to aggregation, precipitation, or polymerization. Systems that simply dissolve the drug lack a stable carrier structure, leading to rapid or uneven drug release, thus compromising therapeutic efficacy. These results demonstrate the key role of nanoparticles in improving drug stability and biocompatibility.

[0187] Further, the Zeta potential data also reveals the relationship between particle surface charge and stability. The Zeta potential of Example 2 is higher (-30.2mV), showing a strong electrostatic repulsion, which prevents particle aggregation and precipitation. In contrast, the Zeta potential of Comparative Example 2-2 is lower (-18.7mV), which highlights the instability of the drug system in solution when nanoparticle carriers are not used, and it is impossible to form an effective drug delivery system. Therefore, the introduction of nanoparticles not only improves the stability of the drug, but also optimizes the clinical effect of the drug by delaying the drug release time.

[0188] Experiment 5: In vitro drug release performance testing of hydrogels:

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

[0190] In Example 2, sodium hyaluronate (0.4%, w / v) was used as the hydrogel matrix, and the drug solution was prepared and then mixed with the nanoparticles.

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

[0192] Drug-encapsulated hydrogels: The drug solution is mixed with the hydrogel matrix in appropriate proportions and processed into gel blocks using freeze-drying to ensure that the drug is evenly distributed throughout the hydrogel.

[0193] Drug release test: The hydrogel samples were placed in PBS buffer (pH 7.4) and maintained 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 measured by UV-Vis spectrophotometer.

[0195] The drug release amount was calculated using the standard curve.

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

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

[0198]

[0199]

[0200] The experimental results show that the drug release of Example 2 within 48 hours is significantly higher than that of Comparative Examples 2-3, and its release curve is relatively stable, 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 nanoparticle sustained-release mechanism. The sodium hyaluronate hydrogel matrix may gradually release the drug to the external environment through the affinity between the drug molecules, which provides an effective sustained-release platform for the present invention. In contrast, the release curve of Comparative Examples 2-3 appears more irregular, with a faster release rate, and the release amount in the later stage gradually decreases, indicating that the embedding and release control of the carbomer matrix to the drug is weak, which may cause the drug to be released too quickly in the initial stage and then decrease over time.

[0201] The properties of hydrogels are closely related to the matrix they use. In sodium hyaluronate systems, drugs are gradually released through the hydrogel's network structure, resulting in slow and stable drug release. However, carbomer, due to its lower hydration and larger particle size, may result in a more unstable drug release process, resulting in deviations in the released amount. This phenomenon is highly consistent with the matrix-selective release mechanism described in the present invention, namely, by optimizing the hydrogel matrix, the rate and duration of drug release can be adjusted, thereby maximizing the therapeutic effect of the drug.

[0202] It is noteworthy that the drug release in Example 2 stabilized after 24 hours, demonstrating the advantages of sodium hyaluronate hydrogel in long-term drug release. In contrast, carbomer hydrogel exhibited a relatively rapid release peak due to its physical properties, which limited the sustained release of the drug. In the later release phase, the drug release of the sodium hyaluronate matrix showed almost no decrease, fully demonstrating the stability of the sustained-release carrier and the long-term effectiveness of the drug. This further demonstrates the synergistic effect of the matrix material and the drug carrier to optimize the bioavailability of the drug.

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

[0204] Preparation of bacterial and fungal strains: Standard bacterial strains (such as Gardnerella vaginalis and Escherichiacoli) and standard fungal strains (Candida albicans) were selected for constructing biofilm models.

[0205] The strain is revived in a suitable liquid culture medium and cultured to the logarithmic growth phase for later use.

[0206] Biofilm formation:

[0207] The bacterial solution (1×10 7 CFU / mL) were inoculated into 96-well plates, with 200 μL per well.

[0208] Incubate at 37°C for 24 hours to promote the formation of mature biofilms of bacteria or fungi at the bottom of the wells.

[0209] Treatment and intervention:

[0210] The supernatant was removed and the cells were gently washed three times with PBS to remove unattached cells.

[0211] Each group of drug treatment solutions (hydrogel treatment group of Example 2, hydrogel treatment group of Comparative Examples 2-3, positive control dexamethasone group, negative control PBS group) was added.

[0212] The drug concentration was unified at 50 μg / mL and incubated for 24 hours.

[0213] Biofilm disruption detection:

[0214] After incubation, the cells were washed with PBS to remove floating bacteria.

[0215] Crystal violet staining solution was added to stain the biofilm (0.1%, 20 min), and the excess dye was removed by washing.

[0216] Ethanol solution (95%) was added to dissolve crystal violet, and the OD595 absorbance of each well was measured to reflect the amount of biofilm.

[0217] Antimicrobial activity testing: Set up another parallel circuit. After treatment, dilute the liquid in the well plate and spread it onto solid culture medium. After 24 hours of incubation, count the CFU to reflect the number of viable bacteria remaining in the biofilm.

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

[0219]

[0220] The experimental results show that the pharmaceutical composition of Example 2 has a significant destructive effect on bacterial and fungal biofilms, and after destroying the biofilm, the number of surviving bacteria and fungi is greatly reduced. In practical applications, this effect is particularly critical for treating vaginitis, because once the biofilm is formed, it not only blocks antibacterial agents but also becomes the main source of repeated infection. The present invention uses the synergistic effect of herbs and marine extracts to exert a bactericidal effect while breaking the biofilm. Compared with traditional single bactericidal solutions, it truly solves the problem that the stubborn biofilm barrier of bacteria and fungi is difficult to penetrate.

[0221] In localized treatments for infections associated with bacterial vaginosis and candidal vaginitis, the effectiveness of the drug in disrupting the biofilm determines the thoroughness of the treatment. Experiments have shown that while the conventional comparative hydrogel formulation has some effect against free-living bacteria, it is virtually ineffective within mature biofilms. In contrast, the present invention, through nanoparticle delivery and controlled hydrogel release, allows the active ingredient to penetrate deep into the biofilm, continuously destroying the microbial defense layer and significantly improving the success rate of localized treatment.

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

Claims

1. A pharmaceutical composition, characterized in that In parts by weight: 5-10 parts of baicalin extract; 3-7 parts of andrographolide extract; 2-5 parts of sulfated fucoidan; 0.2-1 parts of sponge extract.

2. The pharmaceutical composition according to claim 1, characterized in that in: Baicalin extract is obtained from the root of Scutellaria baicalensis and extracted with an ethanol-water mixed solvent; Andrographolide extract is derived from the whole herb of Andrographis paniculata and is extracted with an ethanol-water mixed solvent; Sulfated fucoidan is derived from brown algae and is prepared by enzymatic hydrolysis and alcohol precipitation. Sponge extract is derived from sponges and is obtained through organic solvent extraction and separation and purification.

3. A method for preparing a pharmaceutical composition of Chinese herbal medicine and marine extracts, the pharmaceutical composition according to any one of claims 1-2, characterized in that: The following steps are involved: S1. preparing baicalin extract and andrographolide extract; S2, preparing sulfated fucoidan and spongin extract; S3, mixing each extract with a phospholipid material to prepare a nano delivery system; S4, dispersing the nanodelivery system in a system containing a hydrogel matrix and adjusting the pH value; S5. Forming the final pharmaceutical composition.

4. The method for preparing the combined pharmaceutical composition of Chinese herbal medicine and marine extract according to claim 3, characterized in that: The extraction conditions of the S1 baicalin extract and andrographolide extract are: An ethanol-water mixed solvent is used, with the ethanol volume fraction being 30% to 70%; The extraction temperature is 25℃~40℃; Ultrasonic-assisted extraction, solid-liquid ratio is 1:8-1:12; The extraction time is 20 to 40 minutes.

5. The method for preparing the combined pharmaceutical composition of Chinese herbal medicine and marine extract according to claim 3, characterized in that: The extraction conditions of the S2 sulfated fucoidan and spongin extract are: The sulfated fucoidan is enzymatically hydrolyzed by cellulase at a temperature of 60° C. to 70° C. for 60 to 90 minutes, and then precipitated by 60% to 80% volume fraction ethanol. The sponge extract is soaked in a chloroform-methanol mixed solvent at room temperature for 48 to 72 hours and then separated by chromatography.

6. The method for preparing the combined pharmaceutical composition of Chinese herbal medicine and marine extracts according to claim 3, characterized in that: The preparation conditions of the S3 nano delivery system are: The mass ratio of phospholipids to cholesterol is 8:2 to 10:2; The average particle size of the nanoparticles is 80 to 120 nanometers; Zeta potential was controlled at −20 to −40 mV; The extract encapsulation rate is not less than 80%.

7. The method for preparing the combined pharmaceutical composition of Chinese herbal medicine and marine extracts according to claim 3, characterized in that: In said S4, the hydrogel matrix comprises: Sodium hyaluronate, mass concentration is 0.1% to 0.5%; Glycerol, mass concentration is 1% to 3%.

8. The method for preparing the combined pharmaceutical composition of Chinese herbal medicine and marine extracts according to claim 3, characterized in that: In said S4: Adjust the pH value to 4.0-5.0 with citric acid buffer solution; After the nano-delivery system is uniformly mixed with the hydrogel matrix, it is subjected to a high-pressure homogenization treatment at 600 to 800 bar for 1 to 3 times.

9. The method for preparing the combined pharmaceutical composition of Chinese herbal medicine and marine extracts according to claim 3, characterized in that: The volume fraction mixing ratio of the nano delivery system and the hydrogel matrix is ​​1:4 to 1:

8.

10. Use of a combined pharmaceutical composition of herbal medicine and marine extracts, the pharmaceutical composition according to any one of claims 1-2, characterized in that: The pharmaceutical composition is used in preparing a local medicine for treating bacterial vaginosis, candidal vaginitis associated with biofilm infection or cervicitis.

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