Application of baicalin combined with tetracycline antibiotics in preparation of products for resisting staphylococcus aureus

Abstract

The application discloses application of total flavones of radix scutellariae combined with tetracycline antibiotics in preparation of products against staphylococcus aureus, and belongs to the technical field of medicines. The application provides an antibacterial composition containing total flavones of radix scutellariae and tetracycline antibiotics, aiming at the problems of high drug resistance rate and great toxic side effects of tetracycline antibiotics in the treatment of staphylococcus aureus and methicillin-resistant staphylococcus aureus infection. The total flavones of radix scutellariae contain more than 89% of flavones, and contain various active ingredients such as baicalin and baicalein. The tetracycline antibiotics are selected from doxycycline or minocycline. In vitro experiments prove that the combination of the two has a significant synergistic antibacterial effect on staphylococcus aureus and drug-resistant staphylococcus aureus, and the dosage of doxycycline or minocycline can be reduced by 75%, thereby effectively reducing the toxic side effects.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of total flavonoids from Scutellaria baicalensis combined with tetracycline antibiotics in the preparation of antibacterial products, as well as pharmaceutical preparations and treatment methods containing this composition. Background Technology

[0002] Staphylococcus aureus ( Staphylococcus aureus Staphylococcus aureus (S. aureus) is a common Gram-positive pathogen that can cause a variety of infectious diseases, including skin and soft tissue infections, pneumonia, endocarditis, and sepsis. With the widespread and extensive use of antibiotics, the emergence of drug-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA) has posed a significant challenge to clinical treatment, and its drug resistance problem is becoming increasingly serious. According to data from the CHINET surveillance network in 2024, the tetracycline resistance rate of Staphylococcus aureus exceeded 10%, and the tetracycline resistance rate of methicillin-resistant Staphylococcus aureus (MRSA) was even higher. Tetracycline antibiotics (such as doxycycline and minocycline) are important drugs for treating the aforementioned bacterial infections, but the increasing resistance of bacteria has led to a gradual decline in their clinical efficacy. More seriously, tetracycline antibiotics have a variety of dose-related serious adverse reactions, limiting their clinical application. Minocycline, in particular, has received multiple black box warnings from the FDA, including: tooth discoloration and enamel hypoplasia (contraindicated in children under 8 years old), inhibition of bone growth, hepatotoxicity (potentially leading to fatal liver failure), central nervous system effects (dizziness, vertigo, affecting driving and operating machinery), idiopathic intracranial hypertension (potentially causing visual impairment or even blindness), autoimmune syndromes (drug-induced lupus-like syndrome, autoimmune hepatitis, vasculitis, etc.), and skin pigmentation. While doxycycline is relatively safe, it still has adverse reactions such as photosensitivity, gastrointestinal reactions, and liver and kidney damage.

[0003] Due to the aforementioned serious safety concerns, there is an urgent clinical need to reduce the dosage of tetracycline antibiotics while maintaining or enhancing their antibacterial activity. In recent years, the combined use of active ingredients from traditional Chinese medicine with antibiotics to enhance antibacterial effects, reverse bacterial resistance, and reduce toxic side effects has become a research hotspot. Scutellaria baicalensis (… Scutellaria baicalensis Scutellaria baicalensis is a traditional Chinese medicine for clearing heat and detoxifying. Modern research shows that its flavonoid components have significant antibacterial, anti-inflammatory, and antiviral activities. Therefore, exploring the combined use of Scutellaria baicalensis and antibiotics is a worthwhile direction to explore. However, in the current technology, there are no systematic research reports on the combined use of total flavonoids of Scutellaria baicalensis with doxycycline or minocycline, nor are there any related patents on reducing the dosage of doxycycline and minocycline antibiotics to reduce toxic side effects through combined drug use. Summary of the Invention

[0004] Technical issues In the current treatment of MRSA and other drug-resistant Staphylococcus aureus infections, tetracycline antibiotics have high resistance rates, declining efficacy, and various serious adverse reactions. Clinically, there is an urgent need to reduce their dosage to decrease toxic side effects while maintaining or enhancing their antibacterial activity.

[0005] Technical content To overcome the shortcomings of existing technologies, this invention provides the application of total flavonoids from Scutellaria baicalensis combined with tetracycline antibiotics in the preparation of antibacterial products. Through synergistic effects, it significantly reduces the clinical dosage of tetracycline antibiotics, thereby reducing their toxic side effects and lowering the risk of drug resistance, providing a safer and more effective new strategy for clinical anti-infective treatment.

[0006] This invention is the first to propose the combined use of total flavonoids from Scutellaria baicalensis with tetracycline antibiotics (doxycycline and minocycline) for antibacterial treatment. The checkerboard method experiment confirmed that the combination of the two has good in vitro synergistic antibacterial activity against Staphylococcus aureus and drug-resistant Staphylococcus aureus. The dosage of doxycycline and minocycline can be reduced to 1 / 4 of the original MIC dose of each, thereby significantly reducing the risk of dose-related toxic side effects.

[0007] The present invention provides an antibacterial composition comprising total flavonoids from Scutellaria baicalensis and tetracycline antibiotics.

[0008] Furthermore, the antibacterial composition comprises total flavonoids from Scutellaria baicalensis and tetracycline antibiotics.

[0009] Furthermore, the total flavonoids from Scutellaria baicalensis are high-content total flavonoids from Scutellaria baicalensis, wherein the flavonoid content is greater than 89%.

[0010] Furthermore, the flavonoids include baicalin, baicalein, wogonin, wogonin, scutellarin A-7-O-β-D-glucuronide, scutellarin A, and scutellarin.

[0011] Furthermore, the preparation method of the above-mentioned total flavonoids from Scutellaria baicalensis includes the following steps: S1. Scutellaria baicalensis slices are pulverized to obtain medicinal powder; S2. Soak the medicinal powder obtained in step S1 in ethanol, sonicate, and then reflux extract. S3. Dry the extract obtained in step S2 to obtain the crude extract; S4. Add water to the crude extract obtained in step S3 and dissolve it with ultrasonic assistance to obtain a suspension; S5. After the suspension in step S4 is adsorbed by macroporous resin, it is first eluted with water and then eluted with ethanol. S6. Collect the ethanol eluent from step S5, and dry it to obtain high content of total flavonoids from Scutellaria baicalensis.

[0012] Furthermore, in step S2, the ratio of medicinal powder to ethanol is 1:8~12 (w / w).

[0013] Furthermore, in step S2, the ethanol concentration is 80%~100% (v / v).

[0014] Furthermore, in step S2, the soaking, sonication, and reflux extraction are repeated three times, with ethanol concentrations of 80-85% (v / v), 90-95% (v / v), and 100% respectively.

[0015] Specifically, in step S2, soaking, sonication, and reflux extraction are repeated three times, with ethanol concentrations of 80% (v / v), 90% (v / v), and 100% respectively.

[0016] Furthermore, in step S2, the medicinal powder is soaked in ethanol for 1-2 hours and sonicated for 0.5-1 hour.

[0017] Furthermore, in step S2, the reflux time of the medicinal powder is 1.5-2 hours, and the temperature is 45-55℃.

[0018] Furthermore, in step S4, the ratio of crude extract to deionized water is 1:2~5 (w / w).

[0019] Furthermore, in step S5, the macroporous resin is AB-8 macroporous resin.

[0020] Furthermore, the elution process in step S5 is as follows: first, elute with 2-3 BV of deionized water at a flow rate of 2-3 BV / h to remove impurities; then, elute the resin column with 3-5 BV of 85-90% (v / v) ethanol at a flow rate of 1-2 BV / h; and finally, elute the resin column with 3-5 BV of anhydrous ethanol at a flow rate of 2-3 BV / h.

[0021] In the extraction of total flavonoids from Scutellaria baicalensis, a stepwise concentration reflux method was adopted, in which the ethanol concentration was sequentially increased during reflux extraction. Ultrasonic-assisted extraction was also used to enhance the extraction effect of flavonoids, and the total flavonoid content of Scutellaria baicalensis was more than 89%, resulting in a relatively high yield of total flavonoids.

[0022] Furthermore, the tetracycline antibiotic is selected from doxycycline or minocycline.

[0023] Furthermore, when the antibacterial composition exists in liquid form, the concentration of total flavonoids from Scutellaria baicalensis in the composition is not less than 0.0195 mg / mL, and the concentration of tetracycline antibiotics is not less than 0.03125 μg / mL.

[0024] Specifically, when the antibacterial composition is in liquid form, and when the antibacterial composition acts on Staphylococcus aureus, the concentration of total flavonoids from Scutellaria baicalensis in the composition is not less than 0.39 mg / mL, and the concentration of doxycycline is not less than 0.125 μg / mL; Alternatively, the total flavonoid concentration of Scutellaria baicalensis shall not be less than 0.39 mg / mL, and the concentration of minocycline shall not be less than 0.0625 μg / mL.

[0025] Specifically, when the antibacterial composition exists in liquid form, and when the antibacterial composition acts on drug-resistant Staphylococcus aureus, the concentration of total flavonoids of Scutellaria baicalensis in the composition is not less than 0.39 mg / mL, and the concentration of doxycycline is not less than 0.0625 μg / mL; Alternatively, the total flavonoid concentration of Scutellaria baicalensis shall not be less than 0.195 mg / mL, and the concentration of minocycline shall not be less than 0.03125 μg / mL.

[0026] Furthermore, when the antibacterial composition exists as a solid (powder, ointment), the ratio of total flavonoids from Scutellaria baicalensis to tetracycline antibiotics in the composition is 0.0195~0.5 mg : 0.03125~0.2 μg.

[0027] Furthermore, when the antibacterial composition exists as a solid (powder, ointment), the ratio of total flavonoids from Scutellaria baicalensis to tetracycline antibiotics in the composition is 0.0195~0.39 mg : 0.03125~0.125 μg.

[0028] Specifically, when the antibacterial composition exists in solid form, and when the antibacterial composition acts on Staphylococcus aureus, the ratio of total flavonoids from Scutellaria baicalensis to doxycycline in the composition is 0.39~0.5 mg : 0.125 μg; Alternatively, the ratio of total flavonoids from Scutellaria baicalensis to minocycline is 0.39~0.5 mg : 0.0625 μg.

[0029] Specifically, when the antibacterial composition exists in solid form, and when the antibacterial composition acts on Staphylococcus aureus, the ratio of total flavonoids from Scutellaria baicalensis to doxycycline in the composition is 0.39 mg : 0.125 μg; Alternatively, the ratio of total flavonoids from Scutellaria baicalensis to minocycline was 0.39 mg : 0.0625 μg.

[0030] Specifically, when the antibacterial composition exists in solid form, and when the antibacterial composition acts on drug-resistant Staphylococcus aureus, the ratio of total flavonoids from Scutellaria baicalensis to doxycycline in the composition is 0.39~0.5 mg : 0.0625 μg; Alternatively, the ratio of total flavonoids from Scutellaria baicalensis to minocycline is 0.195~0.3 mg : 0.03125 μg.

[0031] Specifically, when the antibacterial composition exists in solid form, and when the antibacterial composition acts on drug-resistant Staphylococcus aureus, the ratio of total flavonoids of Scutellaria baicalensis to doxycycline in the composition is 0.39 mg : 0.0625 μg; Alternatively, the ratio of total flavonoids from Scutellaria baicalensis to minocycline is 0.195 mg : 0.03125 μg.

[0032] This invention provides the application of the above-mentioned antibacterial composition in the preparation of medical products that are resistant to Staphylococcus aureus and drug-resistant Staphylococcus aureus.

[0033] Furthermore, the medical products include antibacterial ointments, antibacterial sprays, or antibacterial suppositories.

[0034] The present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the above-described antibacterial composition and pharmaceutically acceptable excipients.

[0035] Furthermore, the pharmaceutical composition can be administered in unit dose form; the dosage form can be a conventional dosage form, such as liquid dosage forms like emulsions, colloids, true solutions, microparticles, and suspensions; or other conventional dosage forms such as tablets, capsules, pellets, aerosols, pills, oral liquids, powders, injections, solutions, suspensions, emulsions, granules, inclusion complexes, and implants. These dosage forms can be prepared according to conventional pharmaceutical preparation methods, such as mixing, granulation, tableting, filling, dissolving, or dispersing.

[0036] The pharmaceutical compositions of this invention are suitable for clinical use in mammals, including humans and animals, and can be administered via intravenous injection, mouth, nose, skin, lungs, or gastrointestinal tract. Regardless of the method of administration, the optimal dosage for an individual should be determined based on the specific treatment. Generally, a small dose is started, and the dose is gradually increased until the most suitable dosage is found.

[0037] The present invention provides a treatment method for Staphylococcus aureus, drug-resistant Staphylococcus aureus or Escherichia coli infection, the method comprising using the above-mentioned pharmaceutical composition.

[0038] Beneficial effects Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Significantly Reduces Antibiotic Dosage and Minimizes Toxic Side Effects: This invention utilizes the synergistic effect of total flavonoids from Scutellaria baicalensis and tetracycline antibiotics to reduce the dosage of doxycycline and minocycline by 75%. Given that minocycline has serious adverse reactions such as hepatotoxicity, autoimmune syndromes, and idiopathic intracranial hypertension, some of which are dose-related, reducing its dosage can significantly reduce the risk of these toxic side effects and improve the safety of clinical medication.

[0039] 2. Effective against drug-resistant bacteria: This invention also shows good synergistic antibacterial effect against drug-resistant Staphylococcus aureus (FICI=0.31-0.38), providing a new approach for the treatment of refractory drug-resistant bacterial infections. Attached Figure Description

[0040] Figure 1 The checkerboard method results show the synergistic effect of total flavonoids from Scutellaria baicalensis and doxycycline against Staphylococcus aureus; where A is the result of a 96-well plate experiment and B is a heatmap generated based on the MIC data of the 96-well plate.

[0041] Figure 2 The checkerboard method results show the synergistic effect of total flavonoids from Scutellaria baicalensis and minocycline against Staphylococcus aureus; where A is the result of a 96-well plate experiment and B is a heatmap generated based on the MIC data of the 96-well plate.

[0042] Figure 3 The checkerboard method results show the synergistic effect of total flavonoids from Scutellaria baicalensis and doxycycline against methicillin-resistant Staphylococcus aureus; where A is the result of a 96-well plate experiment and B is a heatmap generated based on the MIC data of the 96-well plate.

[0043] Figure 4 The checkerboard method results show the synergistic effect of total flavonoids from Scutellaria baicalensis and minocycline against methicillin-resistant Staphylococcus aureus; where A is the result of a 96-well plate experiment and B is a heatmap generated based on the MIC data of the 96-well plate.

[0044] Figure 5 The results are from experiments involving the combined use of baicalein, minocycline, and doxycycline.

[0045] Figure 6 The results of the antibacterial experiment of Staphylococcus aureus using the paper disc method with total flavonoids from Scutellaria baicalensis and doxycycline or minocycline.

[0046] Figure 7 The results of the antibacterial experiment using the paper disc method on total flavonoids from Scutellaria baicalensis combined with doxycycline or minocycline to inhibit methicillin-resistant Staphylococcus aureus. Detailed Implementation

[0047] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0048] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0049] The resistance of Staphylococcus aureus to tetracycline antibiotics is becoming increasingly serious, and drugs such as minocycline have serious dose-related toxicities, including hepatotoxicity, autoimmune syndromes, and central nervous system toxicity, which poses great challenges to clinical treatment. This invention establishes, using a checkerboard method, that the combined in vitro antibacterial activity of total flavonoids from Scutellaria baicalensis with doxycycline and minocycline against the aforementioned bacteria exhibits a synergistic effect, significantly reducing antibiotic dosage and thus minimizing toxic side effects.

[0050] The total flavonoids from Scutellaria baicalensis used in the examples were prepared according to the method described in patent 202511502282X, specifically as follows: S1: 1005.14g of Scutellaria baicalensis slices were pulverized using a pulverizer to obtain medicinal powder; S2: The medicinal powder obtained in step S1 is first soaked in an 80% (v / v) ethanol aqueous solution at a material-to-liquid ratio of 1:10 (w / w) for 1 hour, then ultrasonically extracted for 0.5 hours, and then refluxed at 50°C for 2 hours. Then the extracted medicinal powder is soaked in a 90% (v / v) ethanol aqueous solution at a material-to-liquid ratio of 1:10 (w / w) for 1 hour, then ultrasonically extracted for 0.5 hours, and then refluxed at 50°C for 2 hours. After that, the extracted medicinal powder is soaked in pure ethanol at a material-to-liquid ratio of 1:10 (w / w) for 1 hour, then ultrasonically extracted for 0.5 hours, and then refluxed at 50°C for 2 hours. S3: Combine the extracts obtained in step S2 and concentrate them by rotary evaporation at 50 °C until dry to obtain 440.7 g of crude extract; S4: Add the crude extract obtained in step S3 to deionized water at a material-to-liquid ratio of 1:2 (w / w), and dissolve it with ultrasonic assistance to obtain a suspension; S5: After the suspension in step S4 is adsorbed by AB-8 macroporous resin, it is eluted with 3 BV of deionized water at a flow rate of 2 BV / h to remove impurities; then the resin column is eluted with 3 BV of 90% (v / v) ethanol at a flow rate of 1 BV / h, and then the resin column is eluted with 3 BV of anhydrous ethanol at a flow rate of 2 BV / h. S6: Collect the ethanol eluent from step S5, concentrate and dry under reduced pressure to obtain 120.69g of high-content total flavonoids from Scutellaria baicalensis. Example 1 Synergistic effect of total flavonoids of Scutellaria baicalensis Georgi combined with doxycycline on Staphylococcus aureus 1. Materials and methods 1.1 Reagents Doxycycline (HPLC≥98%), minocycline (HPLC≥98%), and baicalein (HPLC≥99%) were purchased from the National Institutes for Food and Drug Control; total flavonoids of Scutellaria baicalensis Georgi (self-made, total flavonoid content≥89%, containing main active ingredients such as baicalin, baicalein, wogonoside, and chrysin); TSB medium was purchased from Haibo Biotechnology Co., Ltd. Staphylococcus aureus (ATCC 25923) was purchased from Beijing Zhongke Quality Inspection Biotechnology Co., Ltd. Resazurin indicator was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0051] 1.2 Experimental methods The checkerboard assay was used to determine the antibacterial activity of the combination of total flavonoids of Scutellaria baicalensis Georgi and doxycycline against the standard strain of Staphylococcus aureus (ATCC 25923). The minimum inhibitory concentration (MIC) of total flavonoids of Scutellaria baicalensis Georgi and doxycycline alone and in combination was determined, and the FICI value (combination index) was calculated. FICI = MIC of drug A in combination / MIC of drug A alone + MIC of drug B in combination / MIC of drug B alone. The judgment criteria are as follows: FICI ≤ 0.5 indicates synergistic effect; 0.5 < FICI ≤ 1 indicates additive effect; 1 < FICI ≤ 2 indicates no relevant effect; FICI > 2 indicates antagonistic effect.

[0052] The specific operation of the checkerboard assay is as follows: Staphylococcus aureus was cultured in TSB broth medium. Total flavonoids of Scutellaria baicalensis Georgi (as drug A) and doxycycline (as drug B) were serially diluted with TSB broth medium with the highest concentrations of 25 mg / mL and 1 μg / mL, respectively. 100 μL of TSB broth medium containing different concentrations of the two drugs was added along the horizontal and vertical axes of a 96-well microtiter plate, and then 100 μL of the pathogenic bacterial solution was added to each well, so that the final number of bacteria per well was 2×10 5 CFU. It was cultured at 37℃ for 18 - 24 h, then 20 μL of resazurin at 20 μg / mL was added, and it was cultured at 37℃ for 4 - 5 h. The results were observed, and the lowest concentration at which the wells in the 96-well plate showed dark blue was taken as the MIC value. The minimum inhibitory concentration (MIC) of the two drugs alone and in combination was recorded, and the FICI value was calculated.

[0053] 2. Experimental results Results of the checkerboard assay ( Figure 1The results showed that the MIC of total flavonoids from Scutellaria baicalensis alone against Staphylococcus aureus was 3.125 mg / mL, while the MIC of doxycycline alone was 0.5 μg / mL. When used in combination, the MIC of total flavonoids from Scutellaria baicalensis decreased to 0.39 mg / mL, and the MIC of doxycycline decreased to 0.125 μg / mL (a 75% reduction), with a FICI value of 0.375, indicating a synergistic effect. This result suggests that the combined use can reduce the dosage of doxycycline to one-quarter of the original dose.

[0054] Example 2 Synergistic effect of total flavonoids from Scutellaria baicalensis and minocycline against Staphylococcus aureus The reagents and experimental methods were the same as in Example 1. The strain was Staphylococcus aureus (ATCC 25923).

[0055] Chessboard method experimental results ( Figure 2 The results showed that the MIC of total flavonoids from Scutellaria baicalensis (drug A) alone was 3.125 mg / mL, and the MIC of minocycline (drug B) alone was 0.25 μg / mL. When used in combination, the MIC of total flavonoids from Scutellaria baicalensis decreased to 0.195 mg / mL, and the MIC of minocycline decreased to 0.0625 μg / mL (a 75% reduction), with a FICI value of 0.3125, indicating a synergistic effect. These results suggest that combined use can reduce the dosage of minocycline to one-quarter of the original dose, significantly reducing its dose-related hepatotoxicity, central nervous system toxicity, and the risk of autoimmune syndromes.

[0056] Example 3 Synergistic effect of total flavonoids from Scutellaria baicalensis and doxycycline against drug-resistant Staphylococcus aureus The reagents and experimental methods were the same as in Example 1, and the strain was methicillin-resistant Staphylococcus aureus (ATCC 43300).

[0057] Chessboard method experimental results ( Figure 3 The results showed that the MIC of total flavonoids from Scutellaria baicalensis (drug A) alone was 3.125 mg / mL, and the MIC of doxycycline (drug B) alone was 0.25 μg / mL. When used in combination, the MIC of total flavonoids from Scutellaria baicalensis decreased to 0.39 mg / mL, and the MIC of doxycycline decreased to 0.0625 μg / mL (a 75% reduction), with a FICI value of 0.375, indicating a synergistic effect. This result suggests that even against resistant strains, combined use can reduce the dosage of doxycycline to one-quarter of the original dose.

[0058] Example 4 Synergistic effect of total flavonoids from Scutellaria baicalensis and minocycline against drug-resistant Staphylococcus aureus The reagents and experimental methods were the same as in Example 1, and the strain was methicillin-resistant Staphylococcus aureus (ATCC 43300).

[0059] Chessboard method experimental results ( Figure 4 The results showed that the MIC of total flavonoids from Scutellaria baicalensis (drug A) alone was 3.125 mg / mL, and the MIC of minocycline (drug B) alone was 0.125 μg / mL. When used in combination, the MIC of total flavonoids from Scutellaria baicalensis decreased to 0.195 mg / mL, and the MIC of minocycline decreased to 0.03125 μg / mL (a 75% reduction), with a FICI value of 0.375, indicating a synergistic effect. This result confirms that even against resistant strains, the combination therapy can reduce the dosage of minocycline to one-quarter of the original dose.

[0060] Example 5 Effects of baicalin and minocycline on Staphylococcus aureus The reagents and experimental methods were the same as in Example 1. The strain was Staphylococcus aureus (ATCC 25923).

[0061] Chessboard method experimental results ( Figure 5 A) indicates that the MIC of baicalein (drug A) alone is greater than 1.25 mg / mL, and the MIC of doxycycline (drug B) alone is 0.5 μg / mL. When used in combination, no synergistic effect was observed (FICI ≤ 0.5). This result suggests that there is no synergistic effect between baicalein and doxycycline against Staphylococcus aureus (ATCC 25923).

[0062] Example 6 Effects of baicalin and minocycline on Staphylococcus aureus The reagents and experimental methods were the same as in Example 1. The strain was Staphylococcus aureus (ATCC 25923).

[0063] Chessboard method experimental results ( Figure 5 B) indicates that the MIC of baicalein (drug A) alone is greater than 1.25 mg / mL, while the MIC of minocycline (drug B) alone is 0.25 μg / mL. When used in combination, no synergistic effect was observed. This result suggests that there is no synergistic effect between baicalein and minocycline in the treatment of Staphylococcus aureus (ATCC 25923).

[0064] Example 7 Effects of baicalin and doxycycline on drug-resistant Staphylococcus aureus The reagents and experimental methods were the same as in Example 1, and the strain was methicillin-resistant Staphylococcus aureus (ATCC 43300).

[0065] Chessboard method experimental results ( Figure 5C) indicates that the MIC of baicalein (drug A) alone is greater than 1.25 mg / mL, while the MIC of doxycycline (drug B) alone is 0.25 μg / mL. When used in combination, no synergistic effect was observed. This result suggests that there is no synergistic effect between baicalein and doxycycline against Staphylococcus aureus (ATCC 43300).

[0066] Example 8 Effects of baicalin and doxycycline on drug-resistant Staphylococcus aureus The reagents and experimental methods were the same as in Example 1, and the strain was methicillin-resistant Staphylococcus aureus (ATCC 43300).

[0067] Chessboard method experimental results ( Figure 5 D) indicates that the MIC of baicalein (drug A) alone is greater than 1.25 mg / mL, and the MIC of minocycline (drug B) alone is 0.25 μg / mL. When used in combination, no synergistic effect was observed. This result suggests that there is no synergistic effect between baicalein and doxycycline against Staphylococcus aureus (ATCC 43300).

[0068] Example 9 Staphylococcus aureus paper disc method antibacterial test Freshly cultured Staphylococcus aureus (ATCC 25923) colonies, cultured for 18-24 hours, were suspended in sterile physiological saline and adjusted to a McFarland turbidity standard concentration of 0.5 (approximately 1.5 × 10⁻⁶). 8 CFU / mL); then, using a sterile cotton swab, dip it in the bacterial solution and squeeze along the tube wall to remove excess liquid. Spread the solution evenly three times on the MH agar plate at a 60° angle, rotating the plate each time. Finally, spread the solution around the edge to form a uniform bacterial colony. Allow it to air dry at room temperature for 3-5 minutes. Then, using sterile forceps, gently press four 6mm diameter sterile blank paper discs onto the plate surface, with a spacing ≥24mm and a distance ≥15mm from the edge. Add 20μL of 0.39mg / mL Scutellaria baicalensis total flavonoid solution to each disc (…). Figure 6 A1), 3.125 mg / mL Scutellaria baicalensis total flavonoids solution ( Figure 6 A2), 0.125 μg / mL doxycycline solution ( Figure 6 A3), combined drug solution ( Figure 6 A4, 0.125 μg / mL doxycycline solution and 0.39 mg / mL total flavonoids of Scutellaria baicalensis). Let stand at room temperature for 1-2 hours until the liquid is completely absorbed, then incubate at 35℃ for 16-18 hours; finally, measure the diameter of the inhibition zone, including the diameter of the paper disc, with vernier calipers, and judge the antibacterial effect based on the results of the paper disc method.

[0069] Experimental Results: The paper disc method was used to detect the inhibitory effect of total flavonoids from Scutellaria baicalensis combined with minocycline on Staphylococcus aureus. Figure 6As shown in Figure A, the inhibition zone determination results indicate that the mixed solution of 0.125 μg / mL doxycycline solution and 0.39 mg / mL total flavonoids of Scutellaria baicalensis has the largest inhibition zone (16.3 mm), which is larger than the inhibition zones of 0.125 μg / mL doxycycline solution alone (13.4 mm) and 0.39 mg / mL total flavonoids of Scutellaria baicalensis solution alone (2.3 mm).

[0070] Example 10 Staphylococcus aureus paper disc method antibacterial test Freshly cultured Staphylococcus aureus (ATCC 25923) colonies, cultured for 18-24 hours, were suspended in sterile physiological saline and adjusted to a McFarland turbidity standard concentration of 0.5 (approximately 1.5 × 10⁻⁶). 8 CFU / mL); then, using a sterile cotton swab, dip it in the bacterial solution and squeeze along the tube wall to remove excess liquid. Spread the solution evenly three times on the MH agar plate at a 60° angle, rotating the plate each time. Finally, spread the solution around the edge to form a uniform bacterial colony. Let it air dry at room temperature for 3-5 minutes. Then, using sterile forceps, gently press four 6mm diameter sterile blank paper discs onto the plate surface, with a spacing ≥24mm and a distance ≥15mm from the edge. Add 20μL of 0.195mg / mL Scutellaria baicalensis total flavonoid solution to each disc (CFU / mL). Figure 6 B1), 3.125 mg / mL Scutellaria baicalensis total flavonoids solution ( Figure 6 B2), 0.0625 μg / mL minocycline solution ( Figure 6 B3), combined drug solution ( Figure 6 B4, 0.0625 μg / mL minocycline solution and 0.195 mg / mL total flavonoids of Scutellaria baicalensis). Let stand at room temperature for 1-2 hours until the liquid is completely absorbed, then incubate at 35℃ for 16-18 hours; finally, measure the diameter of the inhibition zone, including the diameter of the paper disc, with vernier calipers, and judge the antibacterial effect based on the results of the paper disc method.

[0071] Experimental Results: The paper disc method was used to detect the inhibitory effect of total flavonoids from Scutellaria baicalensis combined with minocycline on Staphylococcus aureus. Figure 6 As shown in Figure B, the inhibition zone determination results showed that the mixed solution of 0.0625 μg / mL minocycline solution and 0.39 mg / mL total flavonoids of Scutellaria baicalensis had the largest inhibition zone (16.6 mm), which was larger than the inhibition zones of 0.0625 μg / mL minocycline solution alone (13.5 mm) and 0.39 mg / mL total flavonoids of Scutellaria baicalensis solution alone (2.2 mm).

[0072] Example 11 Methicillin-resistant Staphylococcus aureus paper disc method antibacterial test Methicillin-resistant Staphylococcus aureus (ATCC 43300) colonies cultured for 18-24 hours were suspended in sterile physiological saline and adjusted to a McFarland turbidity standard concentration of 0.5 (approximately 1.5 × 10⁻⁶). 8CFU / mL); then, using a sterile cotton swab, dip it in the bacterial solution and squeeze along the tube wall to remove excess liquid. Spread the solution evenly three times on the MH agar plate at a 60° angle, rotating the plate each time. Finally, spread the solution around the edge to form a uniform bacterial colony. Allow it to air dry at room temperature for 3-5 minutes. Then, using sterile forceps, gently press four 6mm diameter sterile blank paper discs onto the plate surface, with a spacing ≥24mm and a distance ≥15mm from the edge. Add 20μL of 0.39mg / mL Scutellaria baicalensis total flavonoid solution to each disc (…). Figure 7 A1), 3.125 mg / mL Scutellaria baicalensis total flavonoids solution ( Figure 7 A2), 0.625 μg / mL minocycline solution ( Figure 7 A3), combined drug solution ( Figure 7 A4, 0.0625 μg / mL minocycline solution and 0.39 mg / mL total flavonoids of Scutellaria baicalensis). Let stand at room temperature for 1-2 hours until the liquid is completely absorbed, then incubate at 35℃ for 16-18 hours; finally, measure the diameter of the inhibition zone, including the diameter of the paper disc, with vernier calipers, and judge the antibacterial effect based on the results of the paper disc method.

[0073] Experimental Results: The paper disc method was used to detect the inhibitory effect of total flavonoids from Scutellaria baicalensis combined with minocycline on Staphylococcus aureus. Figure 7 As shown in Figure A, the inhibition zone determination results indicate that the mixed solution of 0.0625 μg / mL minocycline and 0.39 mg / mL total flavonoids from Scutellaria baicalensis has the largest inhibition zone (16.0 mm), which is larger than the inhibition zones of 0.0625 μg / mL minocycline alone (13.1 mm) and 0.39 mg / mL total flavonoids from Scutellaria baicalensis alone (1.9 mm).

[0074] Example 12 Methicillin-resistant Staphylococcus aureus paper disc method antibacterial test Methicillin-resistant Staphylococcus aureus (ATCC 43300) colonies cultured for 18-24 hours were suspended in sterile physiological saline and adjusted to a McFarland turbidity standard concentration of 0.5 (approximately 1.5 × 10⁻⁶). 8 CFU / mL); then, using a sterile cotton swab, dip it in the bacterial solution and squeeze along the tube wall to remove excess liquid. Spread the solution evenly three times on the MH agar plate at a 60° angle, rotating the plate each time. Finally, spread the solution around the edge to form a uniform bacterial colony. Allow it to air dry at room temperature for 3-5 minutes. Then, using sterile forceps, gently press four 6mm diameter sterile blank paper discs onto the plate surface, with a spacing ≥24mm and a distance ≥15mm from the edge. Add 20μL of 0.39mg / mL Scutellaria baicalensis total flavonoid solution to each disc (…). Figure 7 B1), 3.125 mg / mL Scutellaria baicalensis total flavonoids solution ( Figure 7 B2), 0.03125 μg / mL minocycline solution ( Figure 7 B3) and combination drug mixture ( Figure 7B4, 0.03125 μg / mL minocycline solution and 0.39 mg / mL total flavonoids of Scutellaria baicalensis). Let stand at room temperature for 1-2 hours until the liquid is completely absorbed, then incubate at 35℃ for 16-18 hours; finally, measure the diameter of the inhibition zone, including the diameter of the paper disc, with vernier calipers, and judge the antibacterial effect based on the results of the paper disc method.

[0075] Experimental Results: The paper disc method was used to detect the inhibitory effect of total flavonoids from Scutellaria baicalensis combined with minocycline on Staphylococcus aureus. Figure 7 As shown in Figure B, the inhibition zone determination results showed that the mixed solution of 0.0625 μg / mL minocycline solution and 0.39 mg / mL total flavonoids of Scutellaria baicalensis had the largest inhibition zone (16.3 mm), which was larger than the inhibition zones of 0.0625 μg / mL minocycline solution alone (13.3 mm) and 0.39 mg / mL total flavonoids of Scutellaria baicalensis solution alone (2.0 mm).

[0076] Table 1. Summary of synergistic antibacterial effects of total flavonoids from Scutellaria baicalensis combined with tetracycline antibiotics

[0077] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. An antibacterial composition, characterized in that, The antibacterial composition contains total flavonoids from Scutellaria baicalensis and tetracycline antibiotics; The total flavonoids in the Scutellaria baicalensis contain more than 89% flavonoids, including baicalin, baicalin, wogonin, wogonin, scutellarin A-7-O-β-D-glucuronide, scutellarin A, and scutellarin.

2. The antibacterial composition according to claim 1, characterized in that, When the antibacterial composition is in liquid form, the concentration of total flavonoids from Scutellaria baicalensis in the antibacterial composition is not less than 0.0195 mg / mL, and the concentration of tetracycline antibiotics is not less than 0.03125 μg / mL.

3. The antibacterial composition according to claim 1, characterized in that, When the antibacterial composition exists in solid form, the ratio of total flavonoids from Scutellaria baicalensis to tetracycline antibiotics in the composition is 0.0195~0.5 mg : 0.03125~0.2 μg.

4. The antibacterial composition according to claim 2, characterized in that, When the antibacterial composition is in liquid form, and when the antibacterial composition acts on Staphylococcus aureus, the concentration of total flavonoids from Scutellaria baicalensis in the composition is not less than 0.39 mg / mL, and the concentration of doxycycline is not less than 0.125 μg / mL. Alternatively, the total flavonoid concentration of Scutellaria baicalensis shall not be less than 0.39 mg / mL, and the concentration of minocycline shall not be less than 0.0625 μg / mL.

5. The antibacterial composition according to claim 2, characterized in that, When the antibacterial composition is in liquid form, and when the antibacterial composition acts on drug-resistant Staphylococcus aureus, the concentration of total flavonoids of Scutellaria baicalensis in the composition is not less than 0.39 mg / mL, and the concentration of doxycycline is not less than 0.0625 μg / mL. Alternatively, the total flavonoid concentration of Scutellaria baicalensis shall not be less than 0.195 mg / mL, and the concentration of minocycline shall not be less than 0.03125 μg / mL.

6. The antibacterial composition according to claim 3, characterized in that, When the antibacterial composition exists in solid form, and when the antibacterial composition acts on Staphylococcus aureus, the ratio of total flavonoids from Scutellaria baicalensis to doxycycline in the composition is 0.39~0.5 mg : 0.125 μg; Alternatively, the ratio of total flavonoids from Scutellaria baicalensis to minocycline is 0.39~0.5 mg : 0.0625 μg.

7. The antibacterial composition according to claim 3, characterized in that, When the antibacterial composition exists in solid form, and when the antibacterial composition acts on drug-resistant Staphylococcus aureus, the ratio of total flavonoids of Scutellaria baicalensis to doxycycline in the composition is 0.39~0.5 mg : 0.0625 μg; Alternatively, the ratio of total flavonoids from Scutellaria baicalensis to minocycline is 0.195~0.3 mg : 0.03125 μg.

8. The antibacterial composition according to any one of claims 1 to 7, characterized in that, The method for preparing the total flavonoids from Scutellaria baicalensis includes the following steps: S1. Scutellaria baicalensis slices are pulverized to obtain medicinal powder; S2. Soak the medicinal powder obtained in step S1 in ethanol, sonicate, and then reflux extract. S3. Dry the extract obtained in step S2 to obtain the crude extract; S4. Add water to the crude extract obtained in step S3 and dissolve it with ultrasonic assistance to obtain a suspension; S5. After the suspension in step S4 is adsorbed by macroporous resin, it is first eluted with water and then eluted with ethanol. S6. Collect the ethanol eluent from step S5, and dry it to obtain high content of total flavonoids from Scutellaria baicalensis. In step S2, soaking, sonication, and reflux extraction were repeated three times, with ethanol concentrations of 80-85% (v / v), 90-95% (v / v), and 100% respectively.

9. The use of the antibacterial composition according to any one of claims 1 to 7 in the preparation of medical products resistant to Staphylococcus aureus and drug-resistant Staphylococcus aureus.

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