Use of aconite extract

Abstract

The application provides an application of a grass Wu extract, relates to the field of bacteriostatic technology, and is used for inhibiting growth of escherichia coli; the minimum bacteriostatic concentration of the grass Wu extract is 2.5 mg / mL; the grass Wu extract can directly destroy the cell wall and the cell membrane of bacteria, down-regulate key genes of a flagellum assembly path, interfere with a core metabolic path of bacteria, and down-regulate related genes such as a two-component system and ABC transporter proteins; the MIC of the grass Wu extract on escherichia coli is determined as 2.5 mg / mL for the first time, which provides an accurate dose basis for developing the grass Wu extract into an antibacterial preparation, proves that the grass Wu extract has a definite and quantifiable direct bacteriostatic effect; meanwhile, through a multi-target attack strategy of 'breaking a membrane'-'breaking a leg'-'breaking food'-'causing blindness', the bacteria are difficult to produce drug resistance through a single mutation, and a new idea is provided for solving the problem of antibiotic drug resistance.

Description

Technical Field

[0001] This invention relates to the field of antibacterial technology, specifically to the application of an extract of Aconitum carmichaelii. Background Technology

[0002] Escherichia coli is a Gram-negative, flagellated, motile, non-spore-forming bacterium found in the small intestine of mammals. It is also an opportunistic pathogen, with various pathogenic Escherichia coli causing many zoonotic diseases. Currently, Escherichia coli causing human and animal diseases are classified into five categories: enterotoxigenic Escherichia coli (ETEC), invasive Escherichia coli (EIEC), enteropathogenic Escherichia coli (EPEC), enterohemorrhagic Escherichia coli (EHEC), and enteroaggregative Escherichia coli (EAggEC). In Mongolian medicine, one of the six basic diseases, "sticky" disease, includes intestinal stinging pain caused by sticky intestines, which is enteritis and bacillary dysentery caused by Escherichia coli or other bacteria. Aconitum carmichaelii has functions such as killing phlegm, drying yellow fluid, and relieving pain. It is mainly used to treat diphtheria, anthrax, intestinal pain, lung pain, carbuncles, measles, erysipelas, and other diseases.

[0003] Escherichia coli is highly susceptible to drug resistance due to its ability to acquire resistance genes from external sources and through horizontal gene transfer. Furthermore, resistance mutations occur rapidly, resistance rates gradually increase, multidrug-resistant strains are rapidly proliferating, and the resistance spectrum is further expanding, leading to broad-spectrum resistance to commonly used antibiotics. For example, Qi Haoyu et al. found that multidrug-resistant diarrheal Escherichia coli exhibited resistance rates of 87.1%, 79.2%, 64.4%, and 38.6% to ampicillin, tetracycline, nalidixic acid, and cefotaxime, respectively, and also displayed high genetic diversity. Developing novel antibacterial agents using multi-target strategies and methods that inhibit resistance mechanisms is one of the important measures for controlling drug resistance, aiming to delay or avoid its development. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides an application of Aconitum carmichaelii extract for inhibiting the growth of Escherichia coli, wherein the minimum inhibitory concentration (MIC) of the Aconitum carmichaelii extract is 2.5 mg / mL; the preparation method of Aconitum carmichaelii extract is as follows:

[0005] The preparation method of Aconitum carmichaelii extract is as follows:

[0006] a) Ammonia fumigation activation: After crushing the raw aconite, it is fumigated with ammonia to stabilize the ester bonds of aconitine in the aconite and make them easier to release;

[0007] b) Organic solvent reflux extraction: Dichloromethane was used as the solvent to perform at least three heating reflux extractions on the activated Aconitum carmichaelii powder;

[0008] c) Acid-base purification and enrichment:

[0009] c1) After concentrating the combined extracts, extract them with a dilute sulfuric acid solution with a concentration of 0.04-0.06 mol / L, and collect the acidic aqueous layer;

[0010] c2) Adjust the pH of the acidic water layer to an alkaline range of 9-10 using ammonia water;

[0011] c3) Extraction was performed using dichloromethane on an alkaline aqueous solution. The dichloromethane layer was collected, concentrated, and dried to obtain aconite extract powder enriched with alkaloids.

[0012] C4) Dissolve the Aconitum carmichaelii extract powder in DMSO to prepare a 20 mg / ml stock solution. When using, dilute with LB liquid medium to 2.5 mg / mL to obtain Aconitum carmichaelii extract.

[0013] Preferably, the Aconitum carmichaelii extract inhibits the growth of Escherichia coli by disrupting the cell wall and / or cell membrane of Escherichia coli.

[0014] Preferably, the Aconitum carmichaelii extract inhibits Escherichia coli growth by downregulating the expression of genes related to the flagellar assembly pathway in Escherichia coli.

[0015] Preferably, the flagellar assembly pathway-related genes are selected from at least one of flgB, flgC, flgD, flgF, flgG, flgH, flgI, flhA, flhB, fliD, fliS, fliE, fliF, fliG, fliH, fliI, fliJ, fliK, fliM, fliN, fliO, and fliP.

[0016] Preferably, the Aconitum carmichaelii extract inhibits Escherichia coli growth by downregulating the expression of the two-component system signaling gene and / or ABC transporter signaling gene in Escherichia coli, thereby interfering with its environmental response and substance transport functions.

[0017] Preferably, the Aconitum carmichaelii extract inhibits the growth of Escherichia coli by disrupting its metabolic pathways; the metabolic pathways are at least one of the tricarboxylic acid cycle, sugar metabolism, energy metabolism, ethanolamine metabolism, and folic acid metabolism.

[0018] Preferably, it is used to prepare a drug that inhibits the growth of Escherichia coli.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] 1. This invention utilizes ammonia fumigation to specifically release the alkaloid active ingredients in Aconitum carmichaelii, employs dichloromethane reflux extraction to efficiently dissolve fat-soluble active substances, and finally undergoes acid-base purification and enrichment to remove impurities, ultimately obtaining a high-purity, high-activity Aconitum carmichaelii extract, laying the foundation for the antibacterial effect of Aconitum carmichaelii extract.

[0021] 2. This invention, through microbiological methods, for the first time clearly determined that the minimum inhibitory concentration (MIC) of Aconitum carmichaelii extract against Escherichia coli is 2.5 mg / mL, providing a precise dosage basis for the development of Aconitum carmichaelii extract into antibacterial agents, and proving that it has a clear and quantifiable direct antibacterial effect.

[0022] 3. This invention transcends the traditional single-target antibiotic model. Through scanning electron microscopy and transcriptomics analysis, it reveals that Aconitum carmichaelii extract synergistically attacks Escherichia coli through multiple pathways, including:

[0023] Damages cell structure: It can directly damage the cell wall and cell membrane of bacteria, leading to leakage of bacterial contents and cell death;

[0024] Inhibition of motility and adhesion: Significant downregulation of key genes in the flagellar assembly pathway weakens bacterial motility and initial adhesion to the host surface, thereby potentially inhibiting biofilm formation, which is the root cause of many chronic and drug-resistant infections.

[0025] Disrupting core metabolism: Interfering with multiple core metabolic pathways of bacteria, such as the tricarboxylic acid cycle (TCA), energy metabolism, and folic acid metabolism, cutting off their energy sources and the synthesis of life substances, thus "starving" the bacteria;

[0026] Interfering with environmental responses: Downregulating genes related to the two-component system and ABC transporter weakens the bacteria's ability to sense environmental stress and expel harmful substances (including antibiotics), making them easier to eliminate;

[0027] This invention employs a multi-target attack strategy involving "membrane disruption," "leg severance," "food deprivation," and "blinding," making it difficult for bacteria to develop drug resistance through a single mutation, thus providing a new approach to solving the problem of antibiotic resistance. Attached Figure Description

[0028] Figure 1 Image showing the inhibition zone of Aconitum carmichaelii extract against Escherichia coli BW25113;

[0029] Figure 2 The results show the determination of the minimum inhibitory concentration (MIC) of Aconitum carmichaelii extract against Escherichia coli BW25113.

[0030] Figure 3 To observe the effects of Aconitum carmichaelii extract on the ultrastructure of Escherichia coli using scanning electron microscopy; Figure 3 a is a scanning electron microscope image of E. coli in the untreated control group. Figure 3 b is a scanning electron microscope image of Escherichia coli in the group treated with Aconitum carmichaelii extract;

[0031] Figure 4 The distribution of differentially expressed genes in the Escherichia coli transcriptome by Aconitum carmichaelii extract;

[0032] Figure 5 A pathway diagram of Escherichia coli K-12 MG1655 flagella assembly based on the KEGG database;

[0033] Figure 6 GO functional enrichment analysis of differentially expressed genes in Escherichia coli after treatment with Aconitum carmichaelii extract;

[0034] Figure 7 KEGG pathway enrichment analysis of differentially expressed genes in Escherichia coli after treatment with Aconitum carmichaelii extract. Detailed Implementation

[0035] The strains and reagents used in the following examples: Escherichia coli BW25113 was stored in our laboratory at −80 °C. The rapid total RNA extraction reagent for bacteria (TRIzol® Reagent) was purchased from Shanghai Kexing Trading Co., Ltd.; dimethyl sulfoxide (DMSO), LB agar medium and LB liquid medium were purchased from Beijing Solarbio Science & Technology Co., Ltd.; Escherichia coli (BW25113), Staphylococcus aureus (ATCC 25923), Staphylococcus aureus (ATCC26101), Shigella dysenteriae (ATCC51285), Pseudomonas aeruginosa (ATCC10102), Proteus mirabilis (ATCC49001), Bacillus anthracis (ATCC62001), Candida albicans (ATCC1405); dried aconite root, pulverizer (FW-100 high-speed pulverizer), round-bottom flask, MH-200 heating mantle, SC-3614 centrifuge, RE-5210A rotary evaporator, separatory funnel, dichloromethane (Tianjin Ford Chemical Technology Co., Ltd.), ammonia, and sulfuric acid.

[0036] Example 1 Preparation of Aconitum carmichaelii extract

[0037] The processed aconite root was prepared according to the processing method of dried aconite root in the "Inner Mongolia Mongolian Medicine Processing Standard" (2020 edition). 1.940 kg of dried aconite root was pulverized into coarse powder (no more than 40% of the powder passed through a No. 4 sieve). It was fumigated with ammonia water for 1 hour, then transferred to a round-bottom flask and extracted three times by reflux with dichloromethane: first, reflux with 8 times the volume of dichloromethane for 2 hours; second, reflux with 6 times the volume of dichloromethane for 1 hour; third, reflux with 6 times the volume of dichloromethane for 1 hour. The mixture was filtered through gauze, centrifuged at 4500 rpm for 5 minutes, and the supernatant was collected and concentrated to 1345 ml using a rotary evaporator. Extraction: Extraction was performed twice with an equal volume of 0.05 mol / L H₂SO₄, and the supernatant was collected. pH adjustment: The pH was adjusted to 9-10 with ammonia water. Extraction: Extraction was performed three times with an equal volume of dichloromethane, and the lower supernatant was collected and concentrated to obtain aconite root extract powder.

[0038] Example 2: Antibacterial detection and determination of minimum inhibitory concentration (MIC) of Aconitum carmichaelii extract

[0039] 1. Preparation of Escherichia coli BW25113 bacterial culture

[0040] Escherichia coli was revived on LB plates and incubated overnight at 37 °C. The next day, a single clone was picked from 5 mL of LB medium and cultured overnight at 37 °C with a shaker at 220 rpm / min. The following day, the OD of E. coli was measured using LB liquid medium as a blank control. 600 The value.

[0041] 2. Preparation of Aconitum carmichaelii extract solution

[0042] Take the Aconitum carmichaelii extract powder prepared in Example 1, dissolve it in DMSO to prepare a 20 mg / ml stock solution. Dilute with LB liquid medium to the required concentration before use.

[0043] Prepare solutions of 10 mg / ml, 5 mg / ml, 2.5 mg / ml, 1.25 mg / ml, 0.625 mg / ml, 0.3125 mg / ml, 0.15625 mg / ml, and 0 mg / ml respectively, filter through a 0.22 μm filter membrane, and set aside for later use.

[0044] 3. Antibacterial test of Aconitum carmichaelii extract

[0045] Prepare Escherichia coli bacterial suspension using the method described above, and adjust the bacterial suspension concentration to achieve its OD value. 600=1; and after diluting the bacterial solution 100, 5, and 15 times in sequence, take 750 μL and spread it evenly on an LB plate, make a small slit and place it flat in a 37° incubator, incubate for 2 hours and then air dry; place 3 clean paper strips of the same size on the LB plate covered with bacterial solution, add 20 μL of antibiotic, water and aconite solution to the 3 paper strips respectively, invert the LB plate in a 37° incubator and incubate overnight, and observe the inhibition zone the next day.

[0046] 4. Determination of the minimum inhibitory concentration (MIC) of Aconitum carmichaelii extract

[0047] Figure 1 This study presents the experimental results of detecting the antibacterial effect of Aconitum kusnezoffii extract against Escherichia coli using the disc method. Three different treatment groups were set up, with discs containing different substances placed on the surface of a culture medium coated with E. coli. The discs labeled "Extract of Aconitum kusnezoffii radix" contained Aconitum kusnezoffii extract, the discs labeled "Antibiotic" contained antibiotics as a positive control, and the discs labeled "Water" contained only water as a negative control. As can be observed in the figure, a clear inhibition zone appeared around the discs containing Aconitum kusnezoffii extract, with relatively clear edges and an approximately circular shape, clearly indicating that Aconitum kusnezoffii extract can significantly inhibit the growth of E. coli. Comparison with the positive and negative controls further confirmed the effectiveness and specificity of the antibacterial effect of Aconitum kusnezoffii extract.

[0048] Enterobacter bacillus bacterial suspensions were prepared at concentrations of 10 mg / ml, 5 mg / ml, 2.5 mg / ml, 1.25 mg / ml, 0.625 mg / ml, 0.3125 mg / ml, 0.15625 mg / ml, and 0 mg / ml, respectively. The minimum inhibitory concentration (MIC) of the aconite extract against Escherichia coli at different concentrations was determined using the OD value assay. Diluted bacterial suspensions were inoculated into 96-well plates, with three replicates for each concentration. 100 μL of different concentrations of Aconitum carmichaelii extract (10 mg / ml, 5 mg / ml, 2.5 mg / ml, 1.25 mg / ml, 0.625 mg / ml, 0.3125 mg / ml, 0.15625 mg / ml, 0 mg / ml) was added to each well, followed by 100 μL of diluted Escherichia coli bacterial suspension (10⁴ CFU / mL). The plates were inverted at 37°C and incubated overnight. OD₆₀₀ was measured after incubation. E. coli bacterial suspension served as a positive control, and LB medium served as a blank control. The MIC was determined after 24 h of treatment with different concentrations of Aconitum carmichaelii extract on E. coli. The results are as follows: Figure 2 As shown, the MIC of Aconitum carmichaelii extract against Escherichia coli is 2.5 mg / mL, indicating that Aconitum carmichaelii extract has good antibacterial activity against Escherichia coli.

[0049] 5. Determination of the antibacterial activity of Aconitum carmichaelii extract against the tested bacteria

[0050] Escherichia coli, Staphylococcus aureus, Staphylococcus, Shigella, Pseudomonas aeruginosa, Proteus, Bacillus anthracis, and Candida albicans were revived and passaged twice. Fresh bacterial cells were then inoculated into nutrient agar plates using the streak method. Sterile small steel cups were placed in each cup, and 200 μL of Aconitum carmichaelii extract at concentrations of 2.5 mg / mL, 1.25 mg / mL, and 0.625 mg / mL were added respectively. After 24 hours of incubation, the diameter of the inhibition zone was observed and measured using calipers. The results are shown in Table 1. Different concentrations of Aconitum carmichaelii extract showed inhibitory effects against Escherichia coli, Staphylococcus aureus, and Shigella, with the 2.5 mg / mL concentration showing the strongest inhibitory effect against Escherichia coli.

[0051] Table 1. Antibacterial test results of Aconitum carmichaelii extract

[0052]

[0053] Example 3: Scanning electron microscopy (SEM) observation of the morphology of Escherichia coli after treatment with Aconitum carmichaelii extract.

[0054] Six-mm cell slides were placed at the bottom of a 12-well plate. The aconite extract-treated group received 300 µL of LB medium containing 1 / 2 MIC aconite extract and 300 µL of bacterial culture. The untreated control group received no aconite extract. Incubation was performed at 37 °C for 24 h. After incubation, the slides were washed with PBS, fixed with 2.5% glutaraldehyde at 4 °C for 24 h, and dehydrated using ethanol fractionation. After critical point drying, the samples were sputter-coated with gold and observed under a scanning electron microscope.

[0055] After Escherichia coli was treated with Aconitum carmichaelii extract for 48 hours, changes in cell morphology were observed using an electron microscope. The results are as follows: Figure 3 As shown, the control group of E. coli cells were intact, short rod-shaped, plump, with a relatively smooth surface and intact cell walls. Figure 3 a); E. coli cells treated with Aconitum carmichaelii extract showed damage, blurred cell boundaries, and disruption of the cell membrane and cell wall. Figure 3 (b) The results showed that treatment of Escherichia coli with Aconitum carmichaelii extract significantly disrupted the normal morphology of bacterial cells, leading to cell damage.

[0056] Example 4: Transcriptomic Analysis of Aconitum carmichaelii Extract

[0057] Escherichia coli was cultured overnight in LB medium, with two groups: an untreated control group and a group treated with Aconitum carmichaelii extract (with 1 / 2 MIC Aconitum carmichaelii extract added), and cultured at 37 °C until OD. 6001. The sample was sent to Shanghai Ling'en Biotechnology Co., Ltd. for total RNA extraction and transcriptome sequencing.

[0058] Based on the transcriptome sequencing results, using the *E. coli* K-12 MG1655 genome as a reference sequence, differential gene expression analysis was performed on the untreated control group and the *Aconitum carmichaelii* extract-treated group (1 / 2 MIC *Aconitum carmichaelii* extract) using Trimmomatic software, Bowtie 2 (Bowtie 2-2.3.5.1) alignment software, and the DESeqR package (1.12.0). The threshold for screening differentially expressed genes was |log2|logFC|≥ 1 and FDR≤ 0.05. GO and KEGG enrichment analyses were performed on the differentially expressed genes.

[0059] The obtained data were analyzed using Graphpad Prism 8.0, and the results were plotted. The data were obtained as mean ± standard deviation (SD). (P < 0.05 indicates a significant difference compared to the untreated control group; **P < 0.01 indicates an extremely significant difference compared to the untreated control group.)

[0060] 4.1 Differential Gene Expression Analysis

[0061] EdgeR analysis was performed on the samples. The screening criteria were |logFC| ≥ 1 and FDR ≤ 0.05 to identify genes with significant differential expression. The results showed a total of 720 differentially expressed genes, of which 60 genes were upregulated by *Aconitum carmichaelii* extract, and 660 genes were downregulated by *Aconitum carmichaelii* extract. Figure 4 ).

[0062] 4.2 GO and KEGG enrichment analysis of differentially expressed genes

[0063] After treating E. coli with Aconitum carmichaelii extract, GO analysis was performed on differentially expressed genes, such as... Figure 6As shown, the top 30 differentially expressed genes with the highest enrichment levels were classified using the GO database. The molecular functions primarily enriched in these differentially expressed genes include binding, catalytic activity, transporter activity, transcription regulator activity, molecular function regulator, protein folding chaperone, toxin activity, antioxidant activity, molecular carrier activity, structural molecule activity, small molecule sensor activity, and molecular transducer activity. In terms of biological processes, they are mainly involved in cellular processes, metabolic processes, response to stimulus, biological regulation, localization, intraspecies interaction between organisms, interspecies interaction between organisms, and locomotion. In terms of cellular components, they are mainly involved in cellular anatomical entities, protein-containing complexes, and intracellular processes. KEGG analysis of the differentially expressed genes yielded the following results: Figure 7As shown, 93 pathways were differentially enriched in *E. coli* after treatment with aconite extract. The top 30 most significantly enriched pathways mainly involved flagellar assembly, nitrogen metabolism, exopolysaccharide biosynthesis, degradation of aromatic compounds, phenylalanine metabolism, thiamine metabolism, aminobenzoate degradation, ascorbate and aldarate metabolism, sulfur metabolism, two-component system, dioxin degradation, xylene degradation, histidine metabolism, ABC transporters, pyrimidine metabolism, and O-antigen nucleotide sugar biosynthesis. These pathways may be directly or indirectly related to the pathogenicity and drug resistance of *E. coli*. Among them, the flagellar assembly pathway was significantly enriched, and its differentially expressed genes mainly included flgB, flgC, and flgD (Table 2). This pathway may significantly affect the motility and adhesion of *E. coli* by disrupting flagellar structure. Figure 5 (The genes in pink are those in Table 2).

[0064] Table 2. Differentially expressed proteins in the flagellar assembly pathway.

[0065]

[0066] 4.3 The regulatory effect of Aconitum carmichaelii extract on Escherichia coli metabolism

[0067] The energy required for bacterial growth and host parasitism is generated by cellular metabolism and respiration. The proton dynamics generated by these processes drive cellular mechanisms, including redox balance, membrane potential, motility, acid tolerance, and substrate import / export. Aconitum carmichaelii extract can disrupt amino acid metabolism, lipid metabolism, and energy metabolism in Escherichia coli. Transcriptome analysis revealed 145 differentially expressed genes involved in multiple metabolic pathways, including carbohydrate metabolism, coenzyme and vitamin metabolism, nucleic acid metabolism, exogenous biodegradation and metabolism, glycogen biosynthesis and metabolism, and the metabolism of terpenes and polyketides. The genes involved include eutG (encoding ethanolamine utilization protein EutG), bglB (encoding phosphorylated β-glucosidase within the implicit bgl operon), fumB (encoding fumarate), rpiB (encoding ribose-5-phosphate isomerase B), scpA (encoding cold shock protein CspA), metF (codes for 5,10-methylenetetrahydrofolate reductase), argC (encoding N-acetyl-γ-glutamyl phosphate reductase), argB (encoding N-acetylglutamate kinase), and paoC (encodes the large, molybdenum cofactor-containing subunit of a heterotrimeric periplasmic aldehyde). The following enzymes were significantly downregulated: oxidoreductase (encoding the large subunit of the heterotrimeric periplasmic aldehyde oxidoreductase containing a molybdenum cofactor), carA (encoding the small subunit of carbamoyl phosphate synthase II), mhpD (encoding 2-keto-4-pentenoate hydratase), lacZ (encoding β-galactosidase), and thiL (encoding thiamine-monophosphate kinase); and hcxB and torC were significantly upregulated. Among these, the eutG gene encodes the ethanolamine utilization protein EutG, a key enzyme in the ethanolamine metabolic pathway in *E. coli*, whose functions include carbon and nitrogen source utilization, cell membrane homeostasis maintenance, and antibiotic resistance regulation. The metF gene encodes 5,10-methylenetetrahydrofolate reductase, a core enzyme in the folate metabolic pathway in *E. coli*, and the fumB gene encodes a fumarate enzyme in *E. coli*, involved in the tricarboxylic acid cycle (TCA) and energy metabolism. The RpiB gene encodes ribose-5-phosphate isomerase B, which is involved in the metabolism of rare sugars, allose, and the more common ribose. Differential expression of these genes suggests that Aconitum carmichaelii extract can affect the tricarboxylic acid cycle, carbohydrate metabolism, energy metabolism, ethanolamine metabolism, and folic acid metabolism in Escherichia coli.

[0068] 4. Molecular regulation of the environmental response of Aconitum carmichaelii extract to Escherichia coli

[0069] Aconitum carmichaelii extract significantly regulates multiple environmental response mechanisms in Escherichia coli, including the two-component system, ABC transport system, biofilm formation, quorum sensing, and bacterial chemotaxis. Transcriptome analysis revealed differential expression of 42 genes regulating the two-component system and 41 genes regulating ABC transporters (Figure 7). Among these, 80 genes, including fhuB, afuC, tauA, rcsF, phoR, and appY, were significantly downregulated, while 3 genes, including xylF, torC, and evgA, were significantly upregulated. Nine genes related to the phosphotransferase system (PTS) and four genes related to the bacterial secretion system were significantly downregulated, including mngA, srlA, cmtA, agaC, yghF, gspD, and gspE. Among them, the RCSF gene is an important signaling component in the RCS signaling system and may participate in E. coli-specific physiological processes such as aggregation, biofilm formation, and quorum sensing; gspD and gspE are components of the type II secretion system (T2SS) and are involved in the secretion of toxins and enzymes; T2SS deficiency significantly reduces bacterial pathogenicity; PhoR is a histidine kinase of the PhoBR two-component regulatory system, responsible for sensing phosphate concentration and activating PhoB transcription factors; the evgA and evgS genes together constitute the EvS-EvA two-component system, where evgS is a transmembrane sensor kinase and evgA is an intracellular response regulatory protein. When E. coli is exposed to an acidic environment, evgS senses the low pH signal and activates autophosphorylation, subsequently transferring a phosphate group to evgA, causing it to phosphorylate and bind to DNA, regulating the expression of downstream genes. This indicates that Aconitum carmichaelii extract can affect the acid resistance, iron uptake, flagellar motility, and biofilm formation of E. coli, and reduce the adaptability of E. coli to the environment.

[0070] In summary, the experimental data above indicate that Aconitum carmichaelii extract has significant antibacterial activity against Escherichia coli. At a concentration of 2.5 mg / ml, it can completely inhibit bacterial growth and significantly inhibit bacterial biofilm formation. RNA-seq analysis after treating E. coli with Aconitum carmichaelii extract showed that it regulates the metabolism and drug resistance gene expression levels of E. coli.

[0071] Bacterial biofilm formation involves four stages: adhesion and aggregation, growth, maturation, and detachment. Bacterial adhesion is the first step in biofilm formation. Flagella are the primary motility organs of *E. coli*, mediating adhesion, motility, and chemotaxis, helping bacteria attach to the host and migrate to nutrient-rich locations. KEGG enrichment analysis showed that *Aconitum carmichaelii* extract significantly enriched the anti-*E. coli* and flagellar assembly pathways, with significantly downregulated differentially expressed genes including flgB, flgC, flgD, flgF, flgG, flgH, flgI, flhA, flhB, fliD, fliS, fliE, fliF, fliG, fliH, fliI, fliJ, fliK, fliM, fliN, fliO, and fliP.

[0072] The flagellum consists of three parts: the basal body, the hook, and the filament. The basal body, embedded in the cell wall and cell membrane, comprises four coaxial loops (MS, P, L, and C) and a rotation axis, and is the most complex structure within the flagellum. The MS loop, embedded in the cytoplasmic membrane, is composed of a single FliF protein. Within the MS loop is the C loop, composed of FliG, FliM, and FliN proteins, which connect to the MS loop via FliG to form a transition complex. The FliM protein inserts into the filament via an N-terminal helical structure, forming a curved helical structure that allows the flagellum to generate thrust on the bacterial surface, thus enabling cell movement. Flagellum rotation is driven by the ATPase activity of FliG, enabling the bacteria to move back and forth. The P loop, located in the peptidoglycan layer, is composed of FlgI protein, while the L loop, located in the outer membrane layer, is composed of FlgH protein. FlgB, FlgC, and FliE are located in the proximal portion of the basal body rod (between the P ring and MS ring), and are flagellar components of the proximal portion of the basal body rod. Furthermore, FliE acts as a bridge between the flagellum and the cell membrane; its C-terminus inserts into the cell membrane, and its N-terminus connects to the flagellar axon. Its insertion and exit from the cell membrane are crucial steps in flagellar rotation. FlgD is a flagellar assembly protein. FlgF is also located in the proximal portion of the basal body rod (between the L ring and P ring), and is another flagellar component of the proximal portion of the basal body rod. FlgG is located in the cell-distal portion of the basal body rod, and is the flagellar component of the cell-distal portion of the basal body rod.FlhA, FlhB, FliH, FliI, FliO, and FliP are spherical handles located on the cytoplasmic side of the center of the C ring. FlhA, FlhB, and FliH are putative flagellar export pore proteins involved in the flagellar assembly process. FliI is a flagellum-specific ATP synthase. FliO and FliP are flagellar biosynthesis proteins, belonging to the Type III secretion system. FliD is located at the distal tip of the flagellar filament; it is a capping protein that caps the filament, preventing flagellar subunits from leaking into the medium. FliJ and FliS are flagellar synthesis and assembly chaperones involved in flagellar synthesis and assembly. FliK is a flagellar assembly protein responsible for controlling the length of the flagellar hook. Furthermore, FlhA, FlhB, FliO, and FliP are four of the six proteins that make up the core structure of the flagellar secretion system. They form hollow tubular exit channels within the MS loop, allowing this secretion system to secrete virulence factors into the host cell.

[0073] In other words, Aconitum carmichaelii extract downregulates differentially expressed genes in the flagellar assembly pathway, and has a significant impact on Escherichia coli flagellar assembly and disassembly, ATPase activity, flagellar bending and rotation, flagellar protein-cell membrane interaction, and flagellar protein-cell movement regulation.

[0074] Furthermore, transcriptome results showed that Aconitum carmichaelii extract could cause disorders in Escherichia coli's amino acid metabolism, lipid metabolism, energy metabolism, carbohydrate metabolism, coenzyme and vitamin metabolism, nucleic acid metabolism, exogenous biodegradation and metabolism, glycogen biosynthesis and metabolism, and metabolism of terpenes and polyketides. It also had a significant regulatory effect on multiple environmental response mechanisms in Escherichia coli, including the two-component system, ABC transport system, biofilm formation, quorum sensing, and bacterial chemotaxis.

[0075] This invention uses transcriptomics to analyze the target genes and molecular signaling pathways regulated by Aconitum carmichaelii extract. It is preliminarily believed that Aconitum carmichaelii extract may inhibit Escherichia coli translocation, adhesion, chemotaxis and secretion of virulence factors by inhibiting the flagellar assembly pathway, while affecting the formation and development of Escherichia coli biofilm and controlling the development of drug resistance. This can provide a theoretical basis for subsequent drug research and development.

Claims

1. Use of aconite extract, characterized in that, The minimum inhibitory concentration of the aconite extract for inhibiting E. coli is 2.5 mg / mL; The preparation method of the aconite extract is as follows: a) ammonia fumigation activation: after the aconite raw material is crushed, ammonia is used for fumigation; b) organic solvent reflux extraction: using dichloromethane as the solvent, the activated aconite powder is extracted by heating reflux for at least three times; c) acid-base purification and enrichment: c1) after the combined extract after reflux extraction is concentrated, it is extracted with a dilute sulfuric acid solution with a concentration of 0.04-0.06 mol / L, and the acid water layer is collected; c2) the pH value of the acid water layer is adjusted to an alkaline range of 9-10 by using ammonia water; c3) the alkaline aqueous solution is extracted using dichloromethane, the lower layer is collected and concentrated and dried to obtain aconite extract powder; C4) the aconite extract powder is dissolved in DMSO to prepare a 20 mg / ml mother liquor, which is diluted to 2.5 mg / mL using LB liquid medium to obtain the aconite extract.

2. Use according to claim 1, characterized in that, The aconite extract inhibits the growth of E. coli by destroying the cell wall and / or cell membrane of E. coli.

3. Use according to claim 1, characterized in that, The aconite extract inhibits the growth of E. coli by down-regulating the expression of flagellum assembly pathway genes in E. coli.

4. Use according to claim 1, characterized in that, The aconite extract inhibits the growth of E. coli by down-regulating the expression of two-component system genes and / or ABC transporter genes in E. coli.

5. The use according to claim 1, characterized in that, The aconite extract inhibits the growth of E. coli by disrupting its metabolic pathways; the metabolic pathways are at least one of the tricarboxylic acid cycle process, sugar metabolism, energy metabolism, ethanolamine metabolism, and folate metabolism.

6. The use according to claim 1, characterized in that, A medicine for inhibiting the growth of E. coli.

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