Compound leprosy treatment drug and preparation method and application thereof

Abstract

This invention provides a compound drug for treating melioidosis, its preparation method, and its application. The drug comprises active components of traditional Chinese medicine, a bioactive preparation, and a pharmaceutically acceptable carrier. The active components of traditional Chinese medicine are primarily extracts of Scutellaria baicalensis, Coptis chinensis, Fagopyrum dibotrys, and Polygonum cuspidatum. The bioactive preparation includes polymyxin B, homoserine lactonease, and recombinant human β-defensin 3. The components work synergistically to construct a comprehensive intervention system covering all pathological stages, including biofilm disruption, sterilization, anti-endotoxin activity, intracellular bacterial clearance, immune regulation, and tissue repair. This system can effectively disrupt bacterial biofilms, kill multidrug-resistant strains, neutralize endotoxins, and eliminate latent intracellular bacteria, addressing the core pain points of existing melioidosis treatments, such as strong drug resistance, significant toxic side effects, and high recurrence rates. The preparation process of this invention is carried out under low-temperature and sterile conditions throughout, which fully preserves the stability of the active ingredients, making it suitable for industrial production. The drug can be prepared in various dosage forms, covering all clinical subtypes of melioidosis, and possesses clinical application value and promising prospects for widespread application.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a compound melioidosis treatment drug, its preparation method, and its application. Background Technology

[0002] Melioidosis is a tropical medically transmitted disease caused by Burkholderia pseudomallei (B. pseudomallei). It is prevalent in tropical and subtropical regions, including northern Australia and many Southeast Asian countries. In my country, cases are mainly distributed in Hainan, Guangdong, Guangxi, Fujian, and Taiwan, with Hainan Province having the highest incidence rate. Increasing evidence suggests that melioidosis is a spreading zoonotic disease, with local cases reported in some non-traditional endemic areas, including South Africa, West Africa, South America, and the Middle East. It is estimated that in 2015, the global number of people suffering from melioidosis may have exceeded 160,000, with 89,000 deaths, a case fatality rate as high as 53.9%. The incidence rate in Australia and Southeast Asia reached 50 per 100,000. Global climate change and extreme environmental events also significantly impact the environmental distribution and microecological balance of Burkholderia pseudomallei, and outbreaks of melioidosis due to severe climate change occur frequently. The situation regarding the prevention and control of melioidosis in my country is not optimistic, and basic and clinical research related to melioidosis needs to be strengthened. With the construction of the Hainan Free Trade Zone and the international tourism island, exchanges and cooperation between Hainan and inland provinces are becoming increasingly frequent. There have been reports of imported cases of melioidosis from Beijing, Yunnan, and other places, posing new threats and challenges to public health.

[0003] The clinical symptoms of melioidosis are diverse. Based on the severity of clinical manifestations and the rate of disease progression, it can be classified into latent infection, acute infection, and chronic infection. Melioidosis infection can affect a variety of tissues and organs, including the orbit, face, central nervous system, lungs, liver, kidneys, prostate, and bones. Its clinical manifestations are complex and varied, often described as "resembling a hundred diseases," with melioidosis pneumonia and melioidosis sepsis being the most common clinical presentations. Imaging findings of melioidosis pneumonia often show multiple consolidations, abscesses, or cavitations, and it is prone to septicemia and organ abscesses. The mortality rate of acute melioidosis can reach 20%-60%.

[0004] Currently, first-line clinical treatment for melioidosis primarily relies on antibiotics, with core drugs including ceftazidime, meropenem, and polymyxins. However, existing treatments suffer from insurmountable technological limitations: First, Burkholderia melioidosis exhibits natural multidrug resistance, is inherently insensitive to most antibiotics, and readily forms bacterial biofilms. Pathogens within these biofilms exhibit resistance 100-1000 times higher than those in planktonic bacteria, making it difficult for antibiotics to penetrate the biofilm barrier for complete eradication, a core reason for clinical treatment failure. Second, as a Gram-negative bacterium, Burkholderia melioidosis releases endotoxins (LPS) upon lysis, which can induce sepsis, septic shock, and multiple organ dysfunction syndrome. The core factors contributing to organ dysfunction include: 1) Existing antibiotics can only kill bacteria, but cannot neutralize endotoxins, block the inflammatory cascade of sepsis, or reduce the mortality rate of acute severe cases; 2) Burkholderia melioides can parasitize host macrophages, evading host immune clearance and the effects of antibiotics, which is the root cause of chronic persistent infection and recurrent flare-ups. Existing antibiotics are difficult to penetrate intracellularly for complete eradication, resulting in clinical treatment cycles lasting several months and poor patient compliance; 3) Long-term use of high doses of antibiotics can easily cause adverse reactions such as liver and kidney damage and intestinal flora imbalance. Among these, the nephrotoxicity and neurotoxicity of polymyxins severely limit their clinical application.

[0005] Traditional Chinese medicine (TCM) possesses advantages in the field of anti-infection due to its multi-target, multi-pathway, and low resistance to drug resistance. While some heat-clearing and detoxifying TCMs are used as adjunctive treatments for melioidosis, they suffer from slow onset of action, poor efficacy against acute severe infections, insufficient targeting, and inability to effectively break down biofilm barriers. Antimicrobial peptides and quorum sensing inhibitors, while offering advantages in targeted bactericidal action and membrane-breaking, suffer from poor in vivo stability, short half-life, easy induction of drug resistance when used alone, and inability to cover all pathological stages. Currently, no existing technology targets the core pathological characteristics of melioidosis, failing to fundamentally address the core pain points of current melioidosis treatments. Summary of the Invention

[0006] In view of this, the present invention proposes a compound melioidosis treatment drug, its preparation method and application, to solve the above problems.

[0007] The technical solution of the present invention is implemented as follows: a compound medicament for treating melioidosis, comprising active components of traditional Chinese medicine, bioactive preparations, and a pharmaceutically acceptable carrier; the active components of traditional Chinese medicine include Scutellaria baicalensis extract, Coptis chinensis extract, Andrographis paniculata extract, Sophora flavescens extract, Forsythia suspensa extract, Patrinia scabiosaefolia extract, Astragalus membranaceus extract, Angelica dahurica extract, and borneol; each component works synergistically to clear heat and detoxify, promote blood circulation and drain pus, and invigorate qi and strengthen the body.

[0008] The bioactive preparation comprises polymyxin B, homoserine lactonease, and recombinant human β-defensin 3. Polymyxin B, as the core bactericidal component, directly disrupts the bacterial outer membrane. Homoserine lactonease degrades bacterial quorum sensing signaling molecules, interfering with biofilm formation and enhancing antibiotic penetration. Recombinant human β-defensin 3 is an endogenous antimicrobial peptide that can disrupt bacterial membrane structure and has immunomodulatory effects. When used in combination with the traditional Chinese medicine components, these three components produce significant synergistic bactericidal, anti-biofilm, and toxicity-reducing effects.

[0009] Preferably, the active components of the medicine, by weight, include 8-16 parts of Scutellaria baicalensis extract, 6-12 parts of Coptis chinensis extract, 7-14 parts of Fagopyrum dibotrys extract, 5-10 parts of Polygonum cuspidatum extract, 4-8 parts of Andrographis paniculata extract, 3-7 parts of Sophora flavescens extract, 3-6 parts of Paeonia suffruticosa extract, 2-5 parts of Forsythia suspensa extract, 2-5 parts of Patrinia scabiosaefolia extract, 3-8 parts of Astragalus membranaceus extract, 2-6 parts of Artemisia annua extract, 2-4 parts of Angelica dahurica extract, 3-7 parts of Bletilla striata extract, and 1-3 parts of borneol.

[0010] Preferably, the active components of the medicine, by weight, include 12.0 parts of Scutellaria baicalensis extract, 9.0 parts of Coptis chinensis extract, 10.5 parts of Fagopyrum dibotrys extract, 7.5 parts of Polygonum cuspidatum extract, 6.0 parts of Andrographis paniculata extract, 5.0 parts of Sophora flavescens extract, 4.5 parts of Paeonia suffruticosa extract, 3.5 parts of Forsythia suspensa extract, 3.5 parts of Patrinia scabiosaefolia extract, 5.5 parts of Astragalus membranaceus extract, 4.0 parts of Artemisia annua extract, 3.0 parts of Angelica dahurica extract, 5.0 parts of Bletilla striata extract, and 2.0 parts of borneol.

[0011] Preferably, the bioactive preparation comprises, by weight, 0.5-2.0 parts of polymyxin B, 0.5-2.0 parts of homoserine lactonease, and 0.3-1.2 parts of recombinant human β-defensin 3.

[0012] Preferably, the bioactive preparation comprises, by weight, 1.3 parts polymyxin B, 1.5 parts homoserine lactonease, and 0.7 parts recombinant human β-defensin 3.

[0013] Furthermore, the present invention provides a method for preparing the above-mentioned compound melioidosis treatment drug, comprising the following steps:

[0014] S1. Weigh each Chinese herbal raw material according to the ratio, and extract it through low temperature ultrasonic-negative pressure dual circulation, solid-liquid separation by disc centrifugation, low temperature concentration by nanofiltration membrane classification, and vacuum freeze drying to obtain ultrafine powder of Chinese herbal active components with a particle size ≤10μm.

[0015] S2. Pretreatment of stock solution: The ultrafine powder of active components of traditional Chinese medicine is reconstituted with sterile buffer solution and sterilely filtered through a 0.22μm filter membrane to obtain sterile traditional Chinese medicine stock solution; each component of bioactive preparation is weighed according to the ratio and reconstituted with sterile buffer solution containing Bletilla striata polysaccharide in a sterile environment of 4~8℃ to obtain sterile bioactive stock solution.

[0016] S3. Formulation: According to the target dosage form, under Class 100 sterile environment and light-protected conditions at 15~25℃, sterile traditional Chinese medicine stock solution, sterile bioactive stock solution and pharmaceutically acceptable carrier are mixed and homogenized, and then sterilely filled / vacuum freeze-dried / tableted to obtain the compound melioidosis treatment drug.

[0017] Preferably, in step S1, the temperature of the low-temperature ultrasonic-negative pressure dual-cycle extraction is 28~35℃, the ultrasonic power is 250~400W, the vacuum degree is -0.07~-0.09MPa, the extraction solvent is an ethanol aqueous solution with a volume fraction of 40~60%, the extraction time is 25~45min, and the extraction is performed 2~3 times; the temperature of the nanofiltration membrane fractional low-temperature concentration is ≤35℃, the molecular weight cutoff of the first-stage nanofiltration membrane is 1000Da, and the molecular weight cutoff of the second-stage nanofiltration membrane is 200Da.

[0018] Preferably, in step S3, the mixing and homogenization speed is 600~1200 r / min, the mixing time is 15~30 min, and the pH value of the system is controlled at 6.0~7.4.

[0019] This invention also claims protection for the use of the aforementioned compound melioidosis treatment drug in the preparation of a medicament for treating infectious diseases caused by Burkholderia pseudomallei. The infectious diseases caused by Burkholderia pseudomallei include acute septicemic melioidosis, pulmonary melioidosis, cutaneous and soft tissue melioidosis, chronic persistent melioidosis, and multidrug-resistant Burkholderia pseudomallei infection.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] This invention, for the first time, targets four core pathological features of melioidosis: drug resistance to biofilms, endotoxin sepsis, intracellular parasitic recurrence, and difficulty in lesion repair. It constructs a synergistic formulation system of "active components of traditional Chinese medicine + bioactive preparations" to achieve precise intervention across all pathological stages. Through the dual-target synergistic disintegration of biofilms and breaking down bacterial resistance barriers using homoserine lactonease and buckwheat extract, low-dose polymyxin B combined with recombinant human β-defensin 3 and active components of traditional Chinese medicine such as Coptis chinensis, Scutellaria baicalensis, and Andrographis paniculata synergistically kills bacteria and significantly reduces antibiotic dosage and the risk of resistance induction. Polygonum cuspidatum... The extracts, including Artemisia annua extract and polymyxin B, neutralize endotoxins through a dual pathway, thereby blocking the TLR4 / NF-κB inflammatory pathway and the sepsis cascade. Astragalus extract and Artemisia annua extract can regulate the host's innate immunity and clear intracellular latent bacteria to reduce the recurrence rate. Meanwhile, borneol and Angelica dahurica can improve the efficiency of drug penetration and accumulation in lesions. Bletilla striata extract and recombinant human β-defensin 3 synergistically promote fibroblast proliferation and epithelial repair. Ultimately, this approach comprehensively solves the core technical challenges of high mortality, high recurrence rate, and difficult lesion repair in the current treatment of melioidosis.

[0022] This invention significantly reduces the clinical dosage of polymyxin B through the synergistic effect of traditional Chinese medicine and biological agents, thereby reducing adverse reactions such as nephrotoxicity and neurotoxicity from the root cause. The traditional Chinese medicine components are selected from medicinal and edible materials or classic clinical herbs, while the biological agents are selected from recombinant human proteins and natural enzymes. They have good biocompatibility, high safety, and no obvious toxic side effects, making them suitable for the long-term treatment needs of chronic melioidosis that can last for several months, and significantly improving patient medication compliance.

[0023] This invention addresses the industry pain point of easily losing activity of heat-sensitive active ingredients in traditional Chinese medicine and enzymes and peptides in biological agents at high temperatures. It develops a fully low-temperature aseptic preparation process: low-temperature ultrasonic-negative pressure dual-cycle extraction efficiently extracts the full spectrum of active ingredients from traditional Chinese medicine under low-temperature conditions, avoiding degradation of heat-sensitive components; nanofiltration membrane fractionation at low temperatures replaces traditional high-temperature vacuum concentration, further reducing activity loss; phase-separated aseptic reconstitution and low-temperature light-protected formulation processes completely preserve the enzyme and protein activity of biological agents; and buffer solutions containing Bletilla striata polysaccharides significantly improve the storage stability of biological agents and extend product shelf life. The entire process is controllable, highly repeatable, requires no complex equipment, and is suitable for large-scale industrial production. Detailed Implementation

[0024] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0025] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0026] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0027] Example 1: Compound melioidosis topical gel

[0028] Prescription composition (by weight, total 100 parts)

[0029] Active components of traditional Chinese medicine: Scutellaria baicalensis extract 12.0 parts, Coptis chinensis extract 9.0 parts, Fagopyrum dibotrys extract 10.5 parts, Polygonum cuspidatum extract 7.5 parts, Andrographis paniculata extract 6.0 parts, Sophora flavescens extract 5.0 parts, Paeonia suffruticosa extract 4.5 parts, Forsythia suspensa extract 3.5 parts, Patrinia scabiosaefolia extract 3.5 parts, Astragalus membranaceus extract 5.5 parts, Artemisia annua extract 4.0 parts, Angelica dahurica extract 3.0 parts, Bletilla striata extract 5.0 parts, Borneol 2.0 parts, totaling 81.0 parts;

[0030] Bioactive preparations: Polymyxin B 1.3 parts, homoserine lactonease 1.5 parts, recombinant human β-defensin 30.7 parts, totaling 3.5 parts;

[0031] Pharmaceutically acceptable carrier: Carbomer 940 2.0 parts, glycerol 5.0 parts, propylene glycol 4.0 parts, triethanolamine 0.3 parts, sterile water for injection 4.2 parts.

[0032] Preparation method

[0033] Preparation of active components of S1 traditional Chinese medicine: Scutellaria baicalensis, Coptis chinensis, Fagopyrum dibotrys, Polygonum cuspidatum, Andrographis paniculata, Sophora flavescens, Paeonia suffruticosa, Forsythia suspensa, Patrinia scabiosaefolia, Astragalus membranaceus, Artemisia annua, Angelica dahurica, and Bletilla striata were weighed according to the prescription ratio, pulverized and passed through a 20-mesh sieve, and 50% (v / v) ethanol aqueous solution was added at a material-to-liquid ratio of 1:15. The mixture was placed in a low-temperature ultrasonic extraction device, with the temperature set at 30℃, ultrasonic power at 300W, and vacuum at -0.08MPa. Extraction was performed twice, 30 min each time. After extraction, the mixture was centrifuged at 8000 r / min using a disc centrifuge. Solid-liquid separation was performed, and the supernatants were combined. The supernatants were then concentrated using nanofiltration membranes, with the first-stage nanofiltration membrane having a molecular weight cutoff of 1000 Da and the second-stage nanofiltration membrane having a molecular weight cutoff of 200 Da. The concentration temperature was controlled at 32℃ to obtain a concentrated traditional Chinese medicine solution. The concentrated traditional Chinese medicine solution was then freeze-dried under vacuum and pulverized through a 300-mesh sieve to obtain ultrafine powder of the active components of the traditional Chinese medicine with a particle size ≤10μm. Borneol was weighed according to the prescription, and ultrafinely pulverized at low temperature through a 200-mesh sieve to obtain fine borneol powder. This fine borneol powder was then mixed with the above-mentioned ultrafine powder of the active components of the traditional Chinese medicine to obtain the final powder of the active components of the traditional Chinese medicine.

[0034] S2 stock solution pretreatment: The final active component powder of traditional Chinese medicine was reconstituted with sterile pH 6.8 phosphate buffer and filtered through a 0.22μm filter membrane for two stages of sterile filtration to obtain sterile traditional Chinese medicine stock solution; Each component of the bioactive preparation was weighed according to the prescription ratio and reconstituted with sterile phosphate buffer containing 0.5% Bletilla striata polysaccharide in a Class 100 sterile environment at 6℃ to obtain sterile bioactive stock solution;

[0035] S3 Formulation: Carbomer 940, glycerin, and propylene glycol were weighed according to the prescription, added to sterile water for injection, and after full swelling, triethanolamine was added to adjust the pH to 6.5 to obtain a sterile gel matrix. After moist heat sterilization at 121℃, the temperature was lowered to 22℃. Under Class 100 sterile environment and light-protected conditions at 22℃, sterile traditional Chinese medicine stock solution and sterile bioactive stock solution were added to the sterile gel matrix in sequence, stirred and homogenized at 800r / min for 25min, and the pH of the system was adjusted to 6.5. After aseptic filling, the compound melilothorax external gel was obtained.

[0036] Example 2: Compound melioidosis injection powder

[0037] Prescription composition (by weight, total 100 parts)

[0038] Active components of traditional Chinese medicine: Scutellaria baicalensis extract 12.0 parts, Coptis chinensis extract 9.0 parts, Fagopyrum dibotrys extract 10.5 parts, Polygonum cuspidatum extract 7.5 parts, Andrographis paniculata extract 6.0 parts, Sophora flavescens extract 5.0 parts, Paeonia suffruticosa extract 4.5 parts, Forsythia suspensa extract 3.5 parts, Patrinia scabiosaefolia extract 3.5 parts, Astragalus membranaceus extract 5.5 parts, Artemisia annua extract 4.0 parts, Angelica dahurica extract 3.0 parts, Bletilla striata extract 5.0 parts, Borneol 2.0 parts, totaling 81.0 parts;

[0039] Bioactive preparations: Polymyxin B 1.3 parts, homoserine lactonease 1.5 parts, recombinant human β-defensin 30.7 parts, totaling 3.5 parts;

[0040] Pharmaceutically acceptable carriers: 10.0 parts mannitol, 4.0 parts lactose, and 1.5 parts human serum albumin.

[0041] Preparation method

[0042] Preparation of S1 active components of traditional Chinese medicine: Same as step S1 in Example 1, to prepare the final active component powder of traditional Chinese medicine;

[0043] S2 stock solution pretreatment: The final active component powder of traditional Chinese medicine was reconstituted with sterile pH 7.0 phosphate buffer, and then filtered through a 0.22μm filter membrane for two stages of sterile filtration to obtain sterile traditional Chinese medicine stock solution; each component of the bioactive preparation was weighed according to the prescription ratio, and reconstituted with sterile phosphate buffer containing 0.3% Bletilla striata polysaccharide in a Class 100 sterile environment at 4℃ to obtain sterile bioactive stock solution;

[0044] S3 Formulation: Under Class 100 sterile conditions and at 20°C in the dark, sterile traditional Chinese medicine stock solution, sterile bioactive stock solution, and the prescribed amounts of mannitol, lactose, and human serum albumin were mixed and homogenized at 1000 rpm for 20 min. The pH of the system was adjusted to 7.0, and the mixture was aseptically filtered through a 0.22 μm filter membrane. The mixture was dispensed into sterile vials and partially sealed with rubber stoppers. The vials were then placed in a vacuum freeze dryer and pre-frozen at -45°C for 5 h. The first stage of sublimation drying was carried out at -10°C and a vacuum of 10 Pa for 24 h. The second stage of desorption drying was carried out at 25°C and a vacuum of 5 Pa for 6 h. After freeze-drying, the vials were vacuum-sealed and capped to obtain the compound melilothorax injection powder.

[0045] Example 3: Compound melioidosis oral capsules

[0046] Prescription composition (by weight, total 100 parts)

[0047] Active components of traditional Chinese medicine: Scutellaria baicalensis extract 12.0 parts, Coptis chinensis extract 9.0 parts, Fagopyrum dibotrys extract 10.5 parts, Polygonum cuspidatum extract 7.5 parts, Andrographis paniculata extract 6.0 parts, Sophora flavescens extract 5.0 parts, Paeonia suffruticosa extract 4.5 parts, Forsythia suspensa extract 3.5 parts, Patrinia scabiosaefolia extract 3.5 parts, Astragalus membranaceus extract 5.5 parts, Artemisia annua extract 4.0 parts, Angelica dahurica extract 3.0 parts, Bletilla striata extract 5.0 parts, Borneol 2.0 parts, totaling 81.0 parts;

[0048] Bioactive preparations: Polymyxin B 1.3 parts, homoserine lactonease 1.5 parts, recombinant human β-defensin 30.7 parts, totaling 3.5 parts;

[0049] Pharmaceutically acceptable carriers: 8.0 parts microcrystalline cellulose, 5.0 parts crospovidone, 1.0 part magnesium stearate, and 1.5 parts micronized silica.

[0050] Preparation method

[0051] Preparation of S1 active components of traditional Chinese medicine: Same as step S1 in Example 1, to prepare the final active component powder of traditional Chinese medicine;

[0052] S2 Aseptic Mixing: Under Class 100 aseptic environment and 25°C light-protected conditions, the final active ingredient powder of traditional Chinese medicine, the various components of bioactive preparation, and the prescribed amount of microcrystalline cellulose and cross-linked polyvinylpyrrolidone are mixed evenly, dry granulation is performed, and the granules are sieved through a 20-mesh sieve. The prescribed amount of magnesium stearate and micronized silica gel are added, and the mixture is mixed for 15 minutes to obtain the drug mixture powder.

[0053] S3 Formulation: The drug mixture powder is filled into No. 1 pharmaceutical gelatin empty capsules using a fully automatic capsule filling machine, and then polished and packaged with aluminum-plastic blister packs to obtain the compound melioidosis oral capsules.

[0054] Comparative Example 1: Antibiotic-only control group

[0055] This comparative example is the polymyxin B monotherapy group, which contains only 1.3 parts of polymyxin B in the formulation, with the remainder being the gel matrix from Example 1, totaling 100 parts. The preparation method is the same as in Example 1.

[0056] Comparative Example 2: Pure Traditional Chinese Medicine Control Group

[0057] This comparative example is a pure traditional Chinese medicine group. The prescription contains only 81.0 parts of the active traditional Chinese medicine components in Example 1, without any bioactive preparations. The rest is the gel matrix in Example 1, with a total of 100 parts. The preparation method is the same as in Example 1.

[0058] Comparative Example 3: Control group of homoserine lactonease

[0059] Except for the absence of homoserine lactonease, the other components, proportions, and preparation methods in this comparative formulation are completely consistent with those in Example 1.

[0060] Test Example 1: In vitro antibacterial activity test

[0061] This experiment verified the antibacterial and bactericidal activity of the drug of the present invention against Burkholderia melioides. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Burkholderia melioides standard strain ATCC 23343 and clinical multidrug-resistant strain in Example 1 and each comparative example were determined. The experimental results are shown in Table 1.

[0062]

[0063] As shown in Table 1, the MIC and MBC of the standard strain and clinically resistant strain of Burkholderia melioides in Example 1 of this invention are much lower than those of the comparative examples. The MIC values ​​are 16-64 times lower than those of the pure antibiotic group (Comparative Example 1) and 64-128 times lower than those of the pure traditional Chinese medicine group (Comparative Example 2). This proves that the synergistic combination of traditional Chinese medicine and biological agents in this invention can produce a significant synergistic bactericidal effect, and has extremely strong killing activity against multidrug-resistant strains, completely reversing bacterial drug resistance.

[0064] Experimental Example 2: Biofilm Removal Efficacy Test

[0065] This experiment verifies the effect of the drug of the present invention on the removal of mature biofilm of Burkholderia melioides. Using the crystal violet staining method, the standard strain of Burkholderia melioides was cultured for 72 hours to form a mature biofilm. The drugs of Example 1 and each comparative example were added respectively. After 24 hours of treatment, the biofilm removal rate was determined by the crystal violet staining method. The experimental results are shown in Table 2.

[0066]

[0067] As shown in Table 2, Example 1 of the present invention has a very strong removal effect on mature biofilm of Burkholderia melioides. The removal rate at a concentration of 8 μg / mL is close to 100%, which is much higher than that of the comparative examples. This proves that the dual-target membrane-breaking system formed by the homoserine lactonease and the traditional Chinese medicine components in the present invention can efficiently break down mature biofilms and solve the core problem that existing drugs cannot penetrate the biofilm barrier.

[0068] Experimental Example 3: Endotoxin Neutralization Activity Test

[0069] This experiment was conducted in accordance with the 2025 edition of the Pharmacopoeia of the People's Republic of China, Part IV, General Chapter 1143, Bacterial Endotoxin Test Method (Dynamic Turbidity Method), with no pyrogen source throughout the process, to verify the neutralizing activity of the drug against endotoxins.

[0070] 1. Preparation before the experiment

[0071] (1) Core reagents and instruments: national standard for bacterial endotoxins, Limulus amebocyte lysate (LAL) reagent for dynamic turbidity method (sensitivity λ=0.005EU / mL), pyrogen-free ultrapure water / phosphate buffer (PBS), pyrogen-free consumables, dynamic turbidity detector, and constant temperature incubation equipment;

[0072] (2) Establishment of standard curve: Prepare a series of endotoxin standard solutions of 0.008~10 EU / mL, mix with an equal volume of Limulus amebocyte lysate (LAL) reagent, detect at 37℃ for 60 min, fit the standard curve, and require a correlation coefficient |r|≥0.980, with no positive reaction in the negative control;

[0073] (3) Sample pretreatment and interference verification: Take the samples from Example 1 and each comparative example, disperse them with pyrogen-free PBS, centrifuge and take the supernatant, and after gradient dilution, conduct an interference test to determine that 8 times dilution is the concentration without interference (endotoxin recovery rate 50%~200%).

[0074] 2. Formal test operation

[0075] (1) Group setup: Set up a blank control group, a 1 EU / mL endotoxin positive control group, Example 1 group, and Comparative Examples 1-3 groups, with 6 replicates in each group; Add endotoxin with a final concentration of 1 EU / mL and the corresponding test sample (final concentration of 5 μg / mL and 20 μg / mL) to the test group and mix the total system in equal volumes;

[0076] (2) Incubation and detection: Incubate at 37℃±0.2℃ in the dark for 2 hours, and detect the concentration of residual endotoxin in each group of solutions according to the validated dynamic turbidity method.

[0077] 3. Data Calculation and Statistics

[0078] The endotoxin neutralization rate was calculated using the following formula. Results are expressed as mean ± standard deviation. Differences between groups were analyzed using t-tests.

[0079] Endotoxin neutralization rate (%) = (Measured endotoxin concentration in the positive control group - Measured endotoxin concentration in the experimental group) / Measured endotoxin concentration in the positive control group × 100%

[0080] 4. Determination of Experimental Validity

[0081] The results must simultaneously meet the following conditions: the correlation coefficient of the standard curve |r| ≥ 0.980; no positive reaction in the negative control; the deviation between the measured and theoretical values ​​of endotoxin in the positive control ≤ 20%; and no interference from the detection concentration of the test sample. The experimental results are shown in Table 3.

[0082]

[0083] As shown in Table 3, Example 1 of the present invention has extremely strong endotoxin neutralizing activity, with a neutralization rate of over 90% at a concentration of 5 μg / mL, which is much higher than that of the pure antibiotic group. This proves that the dual-pathway endotoxin neutralization system formed by the traditional Chinese medicine and biological agent of the present invention can effectively neutralize endotoxins, block the inflammatory cascade reaction of sepsis from the root, and solve the core problem that existing antibiotics cannot reduce the mortality rate of acute severe melioidosis.

[0084] Experiment Example 4: In vivo animal protection experiment

[0085] 1. Test materials

[0086] 1.1 Experimental animals: SPF grade BALB / c mice, male, 6-8 weeks old, weighing 18-22g, a total of 80 mice.

[0087] 1.2 Test strain: Burkholderia pseudomallei standard strain ATCC23343.

[0088] 1.3 Test drugs: Example 1: Compound melioidin topical gel (prepared as an injection based on the active ingredients), Comparative Example 1: Polymyxin B monotherapy preparation, Comparative Example 2: Pure traditional Chinese medicine preparation.

[0089] 1.4 Main reagents and instruments: Brain Heart Infusion (BHI) medium, Columbia blood agar plates, sterile physiological saline, high-speed refrigerated centrifuge, biosafety cabinet, CO2 incubator, electronic balance, tissue homogenizer, etc.

[0090] 2. Test Methods

[0091] 2.1 Preparation of bacterial culture

[0092] Frozen Burkholderia melioides standard strains were streaked onto Columbia blood agar plates and incubated aerobically at 37°C for 24 h. Single colonies were picked and inoculated onto BHI liquid medium and cultured at 37°C with shaking at 180 rpm until the logarithmic growth phase. The culture was centrifuged at 4°C and 5000 rpm for 10 min, the supernatant was discarded, and the culture was resuspended in sterile physiological saline and washed twice. The bacterial concentration was adjusted to 1 × 10⁻⁶. 6 CFU / mL (2×LD) 50 Dosage), prepare and use immediately.

[0093] 2.2 Animal grouping and model establishment

[0094] After acclimatizing for 3 days, 80 mice were randomly divided into 4 groups of 20 mice each:

[0095] (1) Model group; (2) Example 1 group; (3) Comparative example 1 group; (4) Comparative example 2 group.

[0096] Except for normal animals, mice in each group were intraperitoneally injected with 0.2 mL of the above-mentioned bacterial solution to establish an acute septicemic melioidosis infection model.

[0097] 2.3 Dosing regimen

[0098] Administer medication 2 hours after infection and continue for 7 days, once daily.

[0099] Example 1 group: The active ingredient injection solution of Example 1 was injected via the tail vein at a dose of 20 mg / kg;

[0100] Comparative Example 1: The formulation of Comparative Example 1 was injected via tail vein, with the same dosage as the active ingredient in Example 1;

[0101] Comparative Example 2: The preparation of Comparative Example 2 was injected via the tail vein, with the same dosage as the herbal components in Example 1;

[0102] Model group: An equal volume of sterile saline was injected into the tail vein.

[0103] 2.4 Survival rate observation

[0104] From the day of infection, mice were observed continuously for 14 days. The number of surviving mice, the time of death, and their general condition were recorded daily, and the 14-day survival rate of each group of mice was calculated.

[0105] 2.5 Liver bacterial load determination

[0106] At the end of the observation period (day 14), surviving mice in each group were removed, liver tissue was aseptically dissected and removed, accurately weighed, and 9 times the volume of sterile physiological saline was added. A 10% tissue homogenate was prepared under ice bath conditions. The homogenate was serially diluted 10 times, and 100 μL of each dilution was spread on Columbia blood agar plates and incubated at 37°C for 24 h for colony counting.

[0107] Calculate the bacterial load in the liver using the following formula:

[0108] Liver bacterial load (lg CFU / g) = lg [(plate colony count × dilution factor × homogenate volume) / liver tissue mass]

[0109] 3. Test Results

[0110] 3.1 Comparison of 14-day survival rates among different groups of mice

[0111]

[0112] 3.2 Comparison of bacterial load in the livers of mice in different groups

[0113]

[0114] 4. Experimental Conclusions

[0115] The compound melioidosis treatment drug of this invention can significantly improve the 14-day survival rate of mice with acute septicemia melioidosis, greatly reduce the bacterial load in the liver, and has a significantly better in vivo anti-infection effect than the single-drug antibiotic group and the pure traditional Chinese medicine group. It has excellent in vivo therapeutic effect on acute severe melioidosis infection.

[0116] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A compound melioidosis treatment drug, characterized in that, It includes active components of traditional Chinese medicine, bioactive preparations, and pharmaceutically acceptable carriers; the active components of traditional Chinese medicine include Scutellaria baicalensis extract, Coptis chinensis extract, Andrographis paniculata extract, Sophora flavescens extract, Forsythia suspensa extract, Patrinia scabiosaefolia extract, Astragalus membranaceus extract, Angelica dahurica extract, and borneol; the bioactive preparations include polymyxin B, homoserine lactonease, and recombinant human β-defensin 3.

2. The compound melioidosis treatment drug as described in claim 1, characterized in that, The drug, by weight, comprises the following active ingredients: 8-16 parts of Scutellaria baicalensis extract, 6-12 parts of Coptis chinensis extract, 7-14 parts of Fagopyrum dibotrys extract, 5-10 parts of Polygonum cuspidatum extract, 4-8 parts of Andrographis paniculata extract, 3-7 parts of Sophora flavescens extract, 3-6 parts of Paeonia suffruticosa extract, 2-5 parts of Forsythia suspensa extract, 2-5 parts of Patrinia scabiosaefolia extract, 3-8 parts of Astragalus membranaceus extract, 2-6 parts of Artemisia annua extract, 2-4 parts of Angelica dahurica extract, 3-7 parts of Bletilla striata extract, and 1-3 parts of borneol.

3. A compound melioidosis treatment drug as described in claim 2, characterized in that, The drug, by weight, comprises the following active ingredients: 12.0 parts of Scutellaria baicalensis extract, 9.0 parts of Coptis chinensis extract, 10.5 parts of Fagopyrum dibotrys extract, 7.5 parts of Polygonum cuspidatum extract, 6.0 parts of Andrographis paniculata extract, 5.0 parts of Sophora flavescens extract, 4.5 parts of Paeonia suffruticosa extract, 3.5 parts of Forsythia suspensa extract, 3.5 parts of Patrinia scabiosaefolia extract, 5.5 parts of Astragalus membranaceus extract, 4.0 parts of Artemisia annua extract, 3.0 parts of Angelica dahurica extract, 5.0 parts of Bletilla striata extract, and 2.0 parts of borneol.

4. A compound melioidosis treatment drug as described in claim 1, characterized in that, The drug, by weight, comprises polymyxin B 0.5-2.0 parts, homoserine lactonease 0.5-2.0 parts, and recombinant human β-defensin 3 0.3-1.2 parts.

5. A compound melioidosis treatment drug as described in claim 4, characterized in that, The drug, by weight, comprises 1.3 parts polymyxin B, 1.5 parts homoserine lactonease, and 0.7 parts recombinant human β-defensin 3.

6. A compound melioidosis treatment drug as described in claim 1, characterized in that, The drug is in a pharmaceutically acceptable dosage form, including topical preparations, oral preparations, or injectable preparations; the topical preparations are ointments, gels, sprays, or wet compresses; the oral preparations are tablets, capsules, or granules; and the injectable preparations are powder for injection or injection solutions.

7. A compound melioidosis treatment drug as described in claim 1, characterized in that, The pharmaceutically acceptable carriers include one or more of the following: matrix, penetration enhancer, humectant, buffer, lyophilization protectant, disintegrant, and lubricant.

8. A method for preparing a compound melioidosis treatment drug according to any one of claims 1 to 7, characterized in that, Includes the following steps: S1. Weigh each Chinese herbal raw material according to the ratio, and extract it through low temperature ultrasonic-negative pressure dual circulation, solid-liquid separation by disc centrifugation, low temperature concentration by nanofiltration membrane classification, and vacuum freeze drying to obtain ultrafine powder of Chinese herbal active components with a particle size ≤10μm. S2. Pretreatment of stock solution: The ultrafine powder of active components of traditional Chinese medicine is reconstituted with sterile buffer solution and sterilely filtered through a 0.22μm filter membrane to obtain sterile traditional Chinese medicine stock solution; each component of bioactive preparation is weighed according to the ratio and reconstituted with sterile buffer solution containing Bletilla striata polysaccharide in a sterile environment of 4~8℃ to obtain sterile bioactive stock solution. S3. Formulation: According to the target dosage form, under Class 100 sterile environment and light-protected conditions at 15~25℃, sterile traditional Chinese medicine stock solution, sterile bioactive stock solution and pharmaceutically acceptable carrier are mixed and homogenized, and then sterilely filled / vacuum freeze-dried / tableted to obtain the compound melioidosis treatment drug.

9. A compound melioidosis treatment drug as described in claim 8, characterized in that, In step S1, the temperature of the low-temperature ultrasonic-negative pressure dual-cycle extraction is 28~35℃, the ultrasonic power is 250~400W, the vacuum degree is -0.07~-0.09MPa, the extraction solvent is an ethanol aqueous solution with a volume fraction of 40~60%, the extraction time is 25~45min, and the extraction is performed 2~3 times; the temperature of the nanofiltration membrane fractional low-temperature concentration is ≤35℃, the molecular weight cutoff of the first-stage nanofiltration membrane is 1000Da, and the molecular weight cutoff of the second-stage nanofiltration membrane is 200Da. In step S3, the mixing and homogenization speed is 600~1200 r / min, the mixing time is 15~30 min, and the pH value of the system is controlled at 6.0~7.

4.

10. The use of the compound melioidosis treatment medicament according to any one of claims 1 to 7 in the preparation of a medicament for treating infectious diseases caused by Burkholderia melioides.

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