Antibacterial compositions, methods of making and use thereof

Abstract

The application discloses antibacterial compositions, and a preparation method and application thereof, and relates to the technical field of biotechnology. The antibacterial composition is antibacterial composition 1 or antibacterial composition 2; the antibacterial composition 1 comprises forsythoside and betulinic acid; the mass ratio of forsythoside and betulinic acid is (6-2):4; the antibacterial composition 2 comprises isoheteroside and betulinic acid; the mass ratio of isoheteroside and betulinic acid is (3-7):(3-7). The application researches and finds that betulinic acid has a clear inhibitory effect on baumanii. Further research finds that the two antibacterial compositions (forsythoside+betulinic acid, isoheteroside+betulinic acid) show a clear synergistic inhibitory effect by reasonable proportioning, the use concentration of each component can be significantly reduced compared with single component, the inhibitory effect on baumanii (including multiple drug-resistant strains) is improved, and the cost increase or potential toxicity risk caused by high-concentration use of single component is reduced.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to antibacterial compositions, their preparation methods, and applications. Background Technology

[0002] Acinetobacter baumannii ( Acinetobacter baumannii Acinetobacter baumannii is a Gram-negative, non-fermenting bacterium widely found in hospital environments, soil, water bodies, and on the surface of human skin and mucous membranes. It is an opportunistic pathogen. With the development of medical technology, especially the widespread use of broad-spectrum antibiotics and invasive medical procedures (such as the use of ventilators and indwelling central venous catheters), Acinetobacter baumannii has become one of the main pathogens causing hospital-acquired infections. It can cause a variety of diseases, including pneumonia, bloodstream infections, urinary tract infections, and skin and soft tissue infections. Its infection rate remains high, especially in intensive care units (ICUs), posing a more significant threat to immunocompromised individuals (such as elderly patients, burn patients, and patients with malignant tumors).

[0003] Acinetobacter baumannii possesses extremely strong environmental adaptability and the ability to acquire drug resistance genes, making it highly susceptible to developing multidrug-resistant strains and even pan-drug-resistant strains. Currently, the efficacy of commonly used clinical antibiotics such as carbapenems, cephalosporins, and aminoglycosides against multidrug-resistant Acinetobacter baumannii is gradually declining, and some pan-drug-resistant strains even lack effective antibiotics, significantly increasing the difficulty of infection treatment.

[0004] Current control measures against Acinetobacter baumannii mainly rely on antibiotic treatment and environmental chemical disinfection (such as chlorine-containing disinfectants and peracetic acid). However, the irrational use of antibiotics has further exacerbated the development of drug resistance. Although chemical disinfectants can quickly kill Acinetobacter baumannii in the environment, long-term use can easily cause environmental pollution and cannot effectively meet the control needs of local human infections (such as skin and mucous membrane infections).

[0005] Plant-derived active ingredients have attracted widespread attention in the field of antibacterial applications due to their advantages such as wide availability, high biocompatibility, and diverse mechanisms of action. Therefore, it is necessary to develop compound antibacterial compositions based on plant-derived ingredients to achieve highly effective inhibition of Acinetobacter baumannii while reducing the concentration of single ingredients and minimizing the risk of drug resistance. Summary of the Invention

[0006] The purpose of this invention is to provide an antibacterial composition, its preparation method, and its application, in order to solve the problems existing in the prior art. The antibacterial composition provided by this invention can effectively inhibit Acinetobacter baumannii and has significant practical application value.

[0007] To achieve the above objectives, the present invention provides the following solution: This invention provides betulinic acid in the preparation of a drug that inhibits Acinetobacter baumannii ( ). Acinetobacter baumannii Applications in products.

[0008] Furthermore, the product is a medicine or disinfectant.

[0009] The present invention also provides an antibacterial composition 1 for inhibiting Acinetobacter baumannii, comprising forsythoside and betulinic acid; wherein the mass ratio of forsythoside to betulinic acid is (6-2):4.

[0010] Preferably, the mass ratio of forsythoside and betulinic acid is 1:1.

[0011] The present invention also provides an antibacterial composition 2 for inhibiting Acinetobacter baumannii, comprising isopropionyl glycoside and betulinic acid; wherein the mass ratio of isopropionyl glycoside to betulinic acid is (3-7):(3-7).

[0012] Preferably, the mass ratio of isopropargyl glycoside to betulinic acid is 3:7.

[0013] The present invention also provides a method for preparing an antibacterial composition that inhibits Acinetobacter baumannii, comprising the step of mixing forsythoside or isohythoside with betulinic acid to obtain the antibacterial composition; The mass ratio of forsythoside and betulinic acid is (6-2):4; The mass ratio of isopropargyl glycoside to betulinic acid is (3-7):(3-7).

[0014] The present invention also provides the use of the antimicrobial compositions described below (1) and / or (2) in the preparation of products that inhibit Acinetobacter baumannii: (1) Antibacterial composition 1; (2) Antibacterial composition 2; The product is a medicine or disinfectant.

[0015] The present invention also provides a drug for inhibiting Acinetobacter baumannii, the active ingredient comprising the antibacterial composition described in (1) and / or (2) below: (1) Antibacterial composition 1; (2) Antibacterial composition 2.

[0016] The present invention also provides a disinfectant for inhibiting Acinetobacter baumannii, wherein the active ingredient comprises the antibacterial composition described in (1) and / or (2) below: (1) Antibacterial composition 1; (2) Antibacterial composition 2.

[0017] The present invention discloses the following technical effects: This invention has revealed that betulinic acid has a clear inhibitory effect on Acinetobacter baumannii (including multidrug-resistant strains). Further research showed that two antibacterial compositions (forsythoside + betulinic acid, isopropanol + betulinic acid), when rationally formulated, exhibited a clear synergistic inhibitory effect (FICI ≤ 0.5). Compared to single-component formulations, this significantly reduced the required concentration of each component, thereby improving the inhibitory effect on Acinetobacter baumannii (including multidrug-resistant strains) while minimizing the increased costs or potential toxicity risks associated with high concentrations of single-component formulations.

[0018] The forsythoside, isohypoglycine, and betulinic acid in the antibacterial composition of this invention are all plant-derived active ingredients with good biocompatibility and no obvious toxic side effects. The prepared antibacterial agent does not contain toxic chemical disinfectant components, is environmentally friendly, and has low irritation to human skin and mucous membranes, making it suitable for disinfection in medical environments and prevention and control of local human infections. This antibacterial composition can be prepared into various dosage forms such as solutions, gels, and sprays, which can be used for surface and air disinfection in hospital environments to reduce the risk of cross-infection with Acinetobacter baumannii, and can also be used locally on human skin and mucous membranes to assist in the prevention or treatment of local infections caused by Acinetobacter baumannii, especially for infections caused by multidrug-resistant Acinetobacter baumannii, which has important clinical application value.

[0019] Compared to single antibiotics or antibacterial components, the antibacterial composition provided by this invention, through the synergistic effect of multiple components, can reduce the probability of bacteria escaping inhibition through a single drug resistance mechanism, thereby delaying the emergence and development of drug resistance. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Betulinic acid, also known as birch acid, is a natural lupin-type pentacyclic triterpenoid compound with the chemical name 3β-hydroxy-lup-20(29)-ene-28-oic acid and the molecular formula C. 30 H 48 O3, molecular weight 456.71, CAS number 472-15-1, boiling point 550.0±33.0℃ (Predicted), acidity coefficient (pKa) 4.61±0.70 (Predicted), density 1.065±0.06 g / cm³ 3 (Predicted), its structure is as follows: .

[0026] Betulinic acid was first discovered in the 1990s in the bark of evergreen plants of the Rhamnaceae family growing in East Africa. Betulinic acid has several biological sources, including extraction from the leaves of Syzygium aromaticum, birch bark, and jujube kernels; it can also be chemically synthesized from betulinol. In the mid-1990s, research revealed that betulinic acid can selectively kill human melanoma cells without harming healthy cells; it also has an inhibitory effect on HIV-1 infection, and further studies have shown that it inhibits malignant tumor cells such as brain tumors, neuroectodermal tumors, and leukemia.

[0027] Forsythin, also known as phillyrin or forsythin, has the CAS number 487-41-2 and the molecular formula C. 27 H 34 O 11Its molecular weight is 534.55, and its structural formula is as follows: .

[0028] Forsythoside is a natural active ingredient extracted from the dried fruit of Forsythia suspensa, a plant in the Oleaceae family. Forsythoside has the effects of clearing heat and detoxifying, reducing swelling and dissipating nodules, and can be used to treat various diseases such as carbuncles, scrofula, mastitis, erysipelas, and wind-heat colds.

[0029] Isopropanol, CAS number 4261-42-1, molecular formula C 21 H 20 O 11 It has a molecular weight of 448.377, a melting point of 237-239℃, and the following structural formula: .

[0030] Isohexin is a natural flavonoid compound extracted from plants such as buckwheat, sage, and angelica, and it has the following biological activities: (1) Antioxidant stress: By scavenging free radicals and increasing the activity of superoxide dismutase (SOD) and catalase (CAT), isopropionol can effectively alleviate oxidative stress damage. Its effect is closely related to the activation of the Nrf2 signaling pathway.

[0031] (2) Anti-inflammatory effects: Isorhizonine can significantly inhibit the release of inflammatory factors such as IL-1β and TNF-α, and the mechanism involves blocking the NF-κB pathway and regulating the phosphorylation process of MAPK. Studies have confirmed that isorhizonine has biological activities such as anti-oxidative stress, anti-inflammation, anti-tumor, improvement of diabetes and liver protection by regulating signaling pathways such as Nrf2 / NF-κB / MAPK.

[0032] (3) Antitumor activity: In in vitro experiments, isoharonidine can induce apoptosis in HepG2 liver cancer cells and inhibit tumor angiogenesis. Its anticancer effect is related to the regulation of Bcl-2 family protein expression and changes in mitochondrial membrane potential.

[0033] (4) Metabolic disease intervention: By activating the Akt / AMPK pathway, isopropionate can reduce blood glucose levels and improve insulin resistance. Studies have shown that it can alleviate renal fibrosis and pancreatic β-cell damage in diabetic model animals.

[0034] Example 1: Inhibitory effect of betulinic acid on Acinetobacter baumannii 1. Experimental Materials Strains: The standard strain of Acinetobacter baumannii (ATCC 19606) and the clinically isolated multidrug-resistant Acinetobacter baumannii strain (number AB-01, resistant to carbapenems) were both provided by the microbiology laboratory of a tertiary hospital.

[0035] Culture medium: MH medium (Mueller-Hinton Agar), purchased from Beijing Luqiao Biotechnology Co., Ltd.

[0036] Reagents: Betulinic acid (98% purity), purchased from Shanghai Yuanye Biotechnology Co., Ltd.; Dimethyl sulfoxide (DMSO, analytical grade), purchased from Sinopharm Chemical Reagent Co., Ltd.

[0037] 2. Experimental Methods Drug solution preparation: Using a small amount of DMSO as a solvent, betulinic acid was prepared into a series of solutions with concentrations of 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL and 4 μg / mL for later use (final DMSO concentration ≤1%, preliminary experiments have verified that this concentration has no inhibitory effect on Acinetobacter baumannii).

[0038] Antibacterial assay: The minimum inhibitory concentration (MIC) of betulinic acid against Acinetobacter baumannii was determined using the agar dilution method. A series of betulinic acid solutions were added to melted MH medium, mixed thoroughly, and poured into plates to prepare drug-containing plates; MH plates containing an equal amount of DMSO but without betulinic acid served as blank controls. Colonies of Acinetobacter baumannii in the logarithmic growth phase were picked, and the bacterial concentration was adjusted to ×10⁻⁶ with physiological saline. 6 The bacterial suspension was inoculated at CFU / mL onto drug-containing plates and blank control plates, and then incubated at 37°C for 24 hours.

[0039] Result determination: Observe the bacterial growth on the plate. The lowest concentration of drug solution that completely inhibits bacterial growth is the MIC value.

[0040] 3. Experimental Results Betulinic acid showed a MIC of 16 μg / mL against Acinetobacter baumannii ATCC 19606 and 32 μg / mL against the clinically multidrug-resistant strain AB-01. The bacteria grew well on the blank control plate. These results indicate that betulinic acid has a clear inhibitory effect on Acinetobacter baumannii (including multidrug-resistant strains).

[0041] Example 2: Synergistic inhibitory effect of composition 1 (forsythoside + betulinic acid) on Acinetobacter baumannii 1. Experimental Materials Strains: Acinetobacter baumannii ATCC 19606, the same strain as in Example 1.

[0042] Reagents: Forsythoside (purity 98%), purchased from Shanghai Yuanye Biotechnology Co., Ltd.; betulinic acid as in Example 1.

[0043] Culture medium: Same as in Example 1.

[0044] 2. Experimental Methods Single-agent MIC determination: The MIC values of phillyrin and betulinic acid against Acinetobacter baumannii ATCC19606 were determined by the agar dilution method, and the method was the same as that in Example 1.

[0045] Compound liquid preparation: According to the single-agent MIC values, a series of concentrations of compound liquids were prepared according to the mass ratios of phillyrin to betulinic acid of 8:4, 6:4, 4:4, 2:4, and 1:4.

[0046] Synergy evaluation: The MIC value of the compound composition was determined by the checkerboard dilution method, and the fractional inhibitory concentration index (FICI) was calculated to evaluate the synergy.

[0047] FICI = (MIC of phillyrin in the compound group / MIC of single-agent phillyrin) + (MIC of betulinic acid in the compound group / MIC of single-agent betulinic acid).

[0048] Judgment criteria: FICI ≤ 0.5 indicates synergy, 0.5 < FICI ≤ 1 indicates additive effect, 1 < FICI ≤ 2 indicates no effect, and FICI > 2 indicates antagonistic effect.

[0049] 3. Experimental results Single-agent MIC: The MIC value of phillyrin against the standard strain of Acinetobacter baumannii was 32 μg / mL, and the MIC value of betulinic acid was 16 μg / mL.

[0050] Compound synergy effect: The FICI values of the compound compositions with different mass ratios are shown in Table 1. When the mass ratio of phillyrin to betulinic acid was 4:4, the FICI value was the smallest (0.33), and the synergy was the strongest; when the mass ratio was within the range of (6 - 2):4, the FICI values were all ≤ 0.5, showing synergy; when the mass ratio was 8:4 or 1:4, the FICI values were 0.92 and 0.84 respectively, showing additive effect.

[0051] Table 1 FICI values of different ratios of Composition 1 (phillyrin + betulinic acid) Example 3 Synergistic inhibitory effect of Composition 2 (isoorientin + betulinic acid) on Acinetobacter baumannii 1. Experimental materials Strains: The same clinical multi-drug resistant Acinetobacter baumannii strain AB-01 as in Example 1.

[0052] Reagents: Isoorientin (purity 98%), purchased from Chengdu Pufeide Biotechnology Co., Ltd.; betulinic acid was the same as in Example 1.

[0053] Culture medium: The same as in Example 1.

[0054] 2. Experimental methods Single-agent MIC determination: The MIC values ​​of isopropargyl glycoside and betulinic acid against strain AB-01 were determined by the agar dilution method, the same as in Example 1.

[0055] Preparation of compound drug solutions: Prepare compound drug solutions of various concentrations according to the mass ratio of isopropargyl glycoside to betulinic acid of 9:1, 7:3, 5:5, 3:7, and 1:9, using the same method as in Example 2.

[0056] Synergistic effect evaluation: The FICI value was determined by the checkerboard dilution method, and the judgment criteria were the same as in Example 2.

[0057] 3. Experimental Results Single-agent MIC: The MIC value of isopropargyl glycoside against AB-01 strain was 64 μg / mL, and the MIC value of betulinic acid against AB-01 strain was 32 μg / mL.

[0058] Synergistic effect of compound: The FICI values ​​of compound compositions with different mass ratios are shown in Table 2. When the mass ratio of isopropargyl glycoside to betulinic acid is 3:7, the FICI value is the smallest (0.37), and the synergistic effect is the strongest. When the mass ratio is in the range of 7:3 to 1:9, the FICI values ​​are all ≤0.5, and all show synergistic effects. When the mass ratio is 9:1, it shows an additive effect.

[0059] Table 2. FICI values ​​of different ratios of Composition 2 (isoscarpicrin + betulinic acid) Example 4: Preparation and Application Effect of Antibacterial Composition Spray 1. Preparation of spray Formula (per 100 mL): Composition 1 (forsythoside: betulinic acid = 4:4) 2 g, ethanol 30 mL, propylene glycol 10 mL, Tween 80 1 mL, purified water added to 100 mL.

[0060] Preparation steps: Add forsythoside and betulinic acid to ethanol and stir until completely dissolved; add propylene glycol and Tween 80 and stir to mix well; slowly add purified water while stirring until the solution is clear and transparent; filter the solution (0.22μm microporous membrane) and dispense into spray bottles to obtain antibacterial spray.

[0061] 2. Application effect test Experimental Methods: Frequently touched surfaces in the school clinic (such as bed rails and IV stands) were selected, with three test points (each test point measuring 10cm × 10cm) for each type of surface. First, samples were collected using a sterile cotton swab method to determine the Acinetobacter baumannii colony count at each test point before disinfection. Then, the aforementioned antibacterial spray was applied to the test points (0.5mL / 100cm²). 2After 30 minutes of action, samples were taken again to measure the number of colonies and calculate the sterilization rate.

[0062] Sterilization rate (%) = (number of colonies before disinfection - number of colonies after disinfection) / number of colonies before disinfection × 100%.

[0063] Experimental results: Before disinfection, the colony count of Acinetobacter baumannii at each test point was 15–32 CFU / 100cm². 2 After disinfection, the colony count was 0 CFU / 100cm³. 2 The sterilization rate reached 100%, indicating that the antibacterial spray has an excellent killing effect on Acinetobacter baumannii on environmental surfaces.

[0064] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Betulinic acid in the preparation of preparations inhibiting Acinetobacter baumannii ( Acinetobacter baumannii Applications in products.

2. The application according to claim 1, characterized in that, The product is a medicine or disinfectant.

3. An antibacterial composition 1 for inhibiting Acinetobacter baumannii, characterized in that, It includes forsythoside and betulinic acid; the mass ratio of forsythoside and betulinic acid is (6-2):

4.

4. The antibacterial composition 1 according to claim 3, characterized in that, The mass ratio of forsythoside and betulinic acid is 1:

1.

5. An antibacterial composition 2 for inhibiting Acinetobacter baumannii, characterized in that, It includes isopropionyl glycoside and betulinic acid; the mass ratio of isopropionyl glycoside to betulinic acid is (3-7):(3-7).

6. The antibacterial composition 2 according to claim 3, characterized in that, The mass ratio of isopropargyl glycoside to betulinic acid is 3:

7.

7. A method for preparing an antibacterial composition that inhibits Acinetobacter baumannii, characterized in that, The method includes the step of uniformly mixing forsythoside or isohythoside with betulinic acid to prepare the antibacterial composition; The mass ratio of forsythoside and betulinic acid is (6-2):4; The mass ratio of isopropargyl glycoside to betulinic acid is (3-7):(3-7).

8. The use of the antimicrobial composition described below (1) and / or (2) in the preparation of a product that inhibits Acinetobacter baumannii: (1) The antibacterial composition 1 as described in claim 3 or 4; (2) The antibacterial composition 2 as described in claim 5 or 6; The product is a medicine or disinfectant.

9. A drug for inhibiting Acinetobacter baumannii, characterized in that, The active ingredients include the antimicrobial compositions described in (1) and / or (2) below: (1) The antibacterial composition 1 as described in claim 3 or 4; (2) The antibacterial composition 2 as described in claim 5 or 6.

10. A disinfectant that inhibits Acinetobacter baumannii, characterized in that, The active ingredients include the antimicrobial compositions described in (1) and / or (2) below: (1) The antibacterial composition 1 as described in claim 3 or 4; (2) The antibacterial composition 2 as described in claim 5 or 6.

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