Application of stephania root in preparation of medicine for treating mycoplasma pneumoniae infection

Abstract

The application provides application of stepholidine, a pharmaceutically acceptable salt or a prodrug thereof in preparation of a medicine for relieving and / or treating mycoplasma pneumoniae infection. Cell experiments and animal experiments show that stepholidine has inhibitory effect on mycoplasma pneumoniae, and stepholidine has certain biological safety. Therefore, stepholidine can be used for preparation of the medicine for relieving and / or treating mycoplasma pneumoniae infection.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of cepharanthine in the preparation of drugs for relieving and treating Mycoplasma pneumoniae infection and the respiratory inflammation it causes. Background Technology

[0002] Mycoplasma pneumoniae (MP) is one of the main pathogens of community-acquired pneumonia (CAP), causing acute upper and lower respiratory tract inflammation and extrapulmonary complications. Its genome is 816,394 bp long, consisting of 687 genes. It is the smallest prokaryote capable of independent survival in vitro. Due to its limited metabolic capacity, it requires complex culture components for in vitro propagation. MP was initially discovered in patients with atypical pneumonia. It has only a cell membrane and lacks a cell wall for support; the outermost cell membrane is composed of protein-lipid-protein. Because its structure is completely different from that of traditional bacteria, it cannot be observed with a regular optical microscope and requires an electron microscope. Under an electron microscope, MP cells are mainly short, thin filaments or dumbbell-shaped, with one end of the cell membrane extending outward to form a special bottle-shaped structure. A single MP cell is 0.1-0.2 μm in size and can pass through a 0.45 μm filter membrane. The pneumonia caused by it is called Mycoplasma pneumoniae pneumoniae (MPP), and its clinical manifestations are usually mild and self-limiting. However, life-threatening pneumonia and even acute respiratory distress syndrome requiring extracorporeal membrane oxygenation have been reported. In addition, some extrapulmonary symptoms, such as mucositis, hepatitis, encephalitis, hemolysis, or erythema multiforme, are associated with Mycoplasma pneumoniae infection and the formation of autoimmune or immune complexes, and are also closely related to refractory asthma. As a prokaryote, MP lacks a cell wall, making antibiotics that act on the cell wall ineffective in treating MPP. However, it is highly sensitive to antibiotics that act on bacterial DNA and protein synthesis, such as macrolides, quinolones, and tetracyclines, and can be used clinically to treat MPP. In clinical practice, tetracycline administration to children can directly irritate the gastrointestinal tract, causing significant intestinal reactions, and is hepatotoxic, potentially leading to enamel hypoplasia. Quinolone antibiotics can affect cartilage and bone development in children. Macrolide antibiotics, in contrast, do not have these side effects. They have low MICs (microinhibitory quotients) against bacteria, low toxicity, no contraindications in young children, high oral bioavailability, and a once-daily formulation, making them first-line drugs for treating mycoplasma pneumoniae (MP) infections in children. However, the incidence of macrolide-resistant MP is gradually increasing and has been reported worldwide, particularly in Asian countries, primarily due to the overuse of macrolides. It has been reported that 69%-95% of mycoplasma pneumoniae isolates in China are resistant. MP resistance has become an increasingly serious problem, with macrolide-resistant MP strains emerging globally. These strains can cause inflammation and refractory pneumonia, along with various extrapulmonary complications such as erythema nodosum, liver damage, and central nervous system injury. Therefore, it is urgent to understand their resistance mechanisms and develop corresponding anti-resistant MP drugs targeting these mechanisms.

[0003] Current research on anti-Mycoplasma pneumoniae drugs mainly focuses on improving existing antibiotics, so there is an urgent need to develop more alternative drugs. Summary of the Invention

[0004] This invention provides the use of senna extract, a pharmaceutically acceptable salt or prodrug thereof, in the preparation of a medicament for relieving and / or treating Mycoplasma pneumoniae infection.

[0005] According to embodiments of the present invention, pharmaceutically acceptable salts of senna are selected from acid addition salts having a sufficiently basic nitrogen atom in their structure, said acid addition salts comprising senna with inorganic acids such as hydrochloric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid, or nitric acid; or organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, and 3-hydroxy-2-naphthoic acid. Salts formed from nicotinic acid, pyruvic acid, pectinic acid, persulfate, 3-phenylpropionic acid, picric acid, tervaline, 2-hydroxyethanesulfonic acid, itaconic acid, aminosulfonic acid, trifluoromethanesulfonic acid, dodecyl sulfate, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucohepanoic acid, glycerophosphate, aspartic acid, sulfosalicylic acid, hemisulfonic acid, or thiocyanate.

[0006] According to an embodiment of the present invention, the Mycoplasma pneumoniae infection can lead to respiratory diseases and extrapulmonary complications.

[0007] According to an embodiment of the present invention, the respiratory diseases include: Mycoplasma pneumoniae pneumonia (primary atypical pneumonia) and upper respiratory tract infection.

[0008] According to an embodiment of the present invention, the upper respiratory tract infection includes pharyngitis, tonsillitis, sinusitis, and otitis media.

[0009] According to embodiments of the present invention, the extrapulmonary complications include: myocarditis, pericarditis, pericardial effusion, aseptic meningitis, aseptic encephalitis, Guillain-Barré syndrome (GBS), hemolytic anemia, thrombocytopenic purpura, hepatitis, gastroenteritis, erythema multiforme, Stevens-Johnson syndrome (SJS), arthritis or arthralgia.

[0010] According to an embodiment of the present invention, the MIC value of senna extract against Mycoplasma pneumoniae is 39.0625 μg / ml.

[0011] According to an embodiment of the present invention, the IC50 of *Tetrapanax papyriferus* is effective against *Mycoplasma pneumoniae*. 50 The value is 21 μM.

[0012] According to an embodiment of the present invention, senna extract, a pharmaceutically acceptable salt or prodrug, can directly inhibit the P1 protein of Mycoplasma pneumoniae in vitro.

[0013] According to embodiments of the present invention, senna extract, a pharmaceutically acceptable salt or prodrug thereof, can inhibit cytokine storms in lung tissue, such as reducing the expression of TNF-α, IL-6 and IL-1β.

[0014] The present invention also provides a method for alleviating and / or treating Mycoplasma pneumoniae infection, comprising: administering at least one of styrax extract, a pharmaceutically acceptable salt thereof, or a prodrug to an individual (e.g., a human) in need.

[0015] The inventors demonstrated through in vitro anti-Mycoplasma pneumoniae experiments that the MIC value of *Cynanchum paniculatum* extract against *Mycoplasma pneumoniae* was 39.0625 μg / ml, and the IC50 value was [missing information]. 50 The concentration was 21 μM, and gentian extract directly inhibited the P1 protein of Mycoplasma pneumoniae in vitro. Furthermore, the inventors established an animal model of Mycoplasma pneumoniae nasal infection to evaluate the efficacy of gentian extract against Mycoplasma pneumoniae. Experimental results showed that high (20 mg / kg), medium (15 mg / kg), and low (10 mg / kg) doses of gentian extract all inhibited the cytokine storm (a decrease in indicators such as TNF-α, IL-6, and IL-1β). Therefore, gentian extract, its pharmaceutically acceptable salts or prodrugs, can be used to prepare drugs for alleviating and / or treating Mycoplasma pneumoniae infection. Attached Figure Description

[0016] Figure 1 The graph shows the half-maximal inhibitory concentration (IC50) test results of different concentrations of styrax extract against Mycoplasma pneumoniae.

[0017] Figure 2 The graph shows that at the protein level, styrax extract inhibits the expression of Mycoplasma pneumoniae P1 protein.

[0018] Figure 3 The figure shows the effect of feroxetine on the inhibition of the expression levels of cytokines TNF-α, IL-6 and IL-1β in the lung tissue of mice infected with Mycoplasma pneumoniae (*** indicates P<0.001; **** indicates P<0.0001). Detailed Implementation

[0019] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0020] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0021] Example 1: Test of the anti-mycoplasma pneumoniae activity of senna extract

[0022] 1. Culture of Mycoplasma pneumoniae

[0023] The international standard strain of Mycoplasma pneumoniae (ATCC29342 / M129) was kindly provided by Beijing Children's Hospital. PPLO-Medium (Pleuropneumonia-Like Organism Medium) was used, which required the addition of serum (usually 20%–30% horse or bovine serum) to provide cholesterol and lipid components to maintain the stability of its membrane structure. The medium also needed to contain yeast extract, glucose (or other carbon source), phenol red (pH indicator), and necessary antibiotics (such as penicillin) to inhibit contamination by other bacteria. It was then incubated at 37°C in a 5% CO2 incubator.

[0024] 2. Determination of the minimum inhibitory concentration (MIC) of senna extract against Mycoplasma gondii in vitro

[0025] (1) Mycoplasma pneumoniae CCU count

[0026] To dilute the Mycoplasma pneumoniae strain 10-fold, prepare a 1.5 mL sterile EP tube, add 900 μL of culture medium, then add 100 μL of the original Mycoplasma pneumoniae, and continue dilution to 10-fold. 10 Using 1 mL of pure culture medium as a negative control, the culture was carried out in a 37°C incubator for 2 weeks. When the color did not change, the last EP tube that turned from red to yellow was the quantity of Mycoplasma pneumoniae [the bacterial load in liquid culture can be confirmed by the color change of the culture medium (phenol red turning from red to yellow).

[0027] (2) Preparation of MP working bacterial solution

[0028] The cryopreserved Mycoplasma pneumoniae strain was continuously passaged using liquid culture. The third passage was used, and the MP concentration was determined using the Colour Change Units (CCU) method. The culture was quantitatively aliquoted into small tubes and cryopreserved as seed culture. For subsequent experiments, one tube was taken and diluted to 10⁻⁶ with MP liquid medium.5 CCU / ml was used as the working bacterial solution.

[0029] (3) Drug gradient setting

[0030] Dissolve styrax extract in DMSO to prepare working solutions of different concentrations (1.25, 0.625, 0.3125, 0.15625, 0.078125, 0.0390625, 0.01953125, 0.00976562, 0.00488281 mg / ml), ensuring that the final concentration of DMSO is ≤0.1% (volume fraction).

[0031] (4) Determination of Minimum Inhibitory Concentration (MIC)

[0032] MIC determination was performed using the microdilution method to determine the MIC value of the experimental drug against clinical isolates of *MP*. The bacterial culture was pre-diluted to 10⁻⁶. 5 After incubating at CCU / ml, the culture was placed in a 37℃, 5% CO2 incubator for 2 hours. 175 μL of the incubated bacterial solution was added to a 96-well plate, followed by different concentrations of the drug. Each plate included a positive control, a negative control, and a drug control. Each sample was replicated in two wells, and the plates were sealed with paraffin oil. The plates were then incubated at 37℃, 5% CO2, and the results were recorded daily. Results were observed after 48–96 hours of incubation. The growth status of *Pseudomonas aeruginosa* (MP) was determined by the fact that its growth metabolites can alter the pH of the culture medium, causing a color change in the indicator. The minimum drug concentration at which the positive control wells showed a color change (i.e., the phenol red indicator in the culture medium changed from red to orange-yellow) without turbidity, and the drug-added wells showed no further color change, was defined as the minimum inhibitory concentration (MIC).

[0033] MIC determination results: The MIC value of styrax tinctoriae against MP standard strain M129 was 39.0625 μg / ml.

[0034] 3. In vitro inhibition of Mycoplasma pneumoniae IC50 by *Cyclocarya paliurus* extract. 50 Measurement

[0035] (1) Preparation of MP working bacterial solution

[0036] The cryopreserved Mycoplasma pneumoniae strain was continuously passaged using liquid culture. The third passage was used, and the MP concentration was determined using the Colour Change Units (CCU) method. The culture was quantitatively aliquoted into small tubes and cryopreserved as seed culture. For subsequent experiments, one tube was taken and diluted to 10⁻⁶ with MP liquid medium. 5 CCU / ml was used as the working bacterial solution.

[0037] (2) Drug gradient setting

[0038] Tinospora sinensis extract was dissolved in DMSO to prepare working solutions of different concentrations (0.19, 0.39, 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100 μM), ensuring that the final concentration of DMSO was ≤0.1% (volume fraction). A negative control group (without the drug) was set up.

[0039] (3) Infection and Treatment

[0040] The working solution of Mycoplasma pneumoniae strain (diluted to 10 with MP liquid medium) 5 CCU / ml) was inoculated into 1.5 mL sterile EP tubes and co-cultured with various concentrations of gentian root extract for 72 hours.

[0041] (4) Detection indicators

[0042] Using qPCR: quantitatively detect the expression level of specific genes (such as P1 adhesin) to reflect the number of mycoplasma.

[0043] (5) Data Analysis

[0044] After normalizing the relative growth inhibition rates at each drug concentration, nonlinear fitting (four-parameter logistic curve) was performed using GraphPad Prism software to calculate IC. 50 Value (the drug concentration that achieves a growth inhibition rate of 50%).

[0045] Test results are as follows Figure 1 As shown. Figure 1 The results of the half-maximal inhibitory concentration (IC50) experiment showed that different concentrations of gentianin could inhibit Mycoplasma pneumoniae, and its IC50 value was [not specified]. 50 The value was 21 μM. The test results indicate that senna extract has an anti-Mycoplasma pneumoniae infection effect.

[0046] IC 50 The half-maximal inhibitory concentration (WMC) represents the drug concentration required to inhibit pathogen growth by 50%. The lower the WMC, the better the inhibitory effect of the drug on the virus.

[0047] 4. In vitro inhibition of Mycoplasma pneumoniae P1 protein expression by senna extract

[0048] The treatment methods for *Tetrapanax papyriferus* and *Mycoplasma pneumoniae* are as described in Section 3 above. *Tetrapanax papyriferus* in vitro inhibition of *Mycoplasma pneumoniae* IC50... 50 As described in the assay, the cells were incubated at 37°C for 72 hours, then centrifuged to collect them for protein extraction.

[0049] Lysis: Add RIPA lysis buffer, which should be pre-mixed with protease and phosphatase inhibitor (1:100). Lyse on ice for 10 min. (RIPA 1960 μL + PMSF (Phenylmethylsulfonyl fluoride) 20 μL + protease inhibitor 20 μL), 200 μL / well. Shake on ice for 2 min. Collect the sample and centrifuge at 4°C, 12000 rpm / 10 min. Collect the supernatant (total protein) and transfer it to a 1.5 ml EP tube. The entire process must be performed on ice.

[0050] BCA quantification: Protein concentration was determined using a BCA kit, adjusted to 2 μg / μL, and 5×SDS-PAGE loading buffer was added. The protein was then denatured at 100℃ for 10 min.

[0051] SDS-PAGE electrophoresis: Prepare an 8% separating gel (for P1 protein, ~190kDa) and a 5% stacking gel.

[0052] Load 20 μg protein and 5 μL marker into each well. Electrophoresis is performed at a constant voltage of 80V (stacking gel) to 120V (separating gel) until the bromophenol blue reaches the bottom of the gel.

[0053] Transfer (wet transfer): PVDF membrane pretreatment, methanol activation for 1 min, immersion in transfer buffer, transfer conditions constant current 200 mA, 4℃ transfer for 90 min (to ensure complete transfer of P1 macromolecules).

[0054] Blocking and antibody incubation: Block with 5% skim milk (prepared with TBST) in a shaker at room temperature for 1 hour.

[0055] Primary antibody incubation: Anti-P1 monoclonal antibody (1:1000, diluted with 5% BSA / TBST), incubated overnight at 4°C.

[0056] Secondary antibody incubation: HRP-labeled secondary antibody (1:10000), shaker at room temperature for 1 hour.

[0057] ECL Development and Data Analysis: ECL A / B solutions were mixed in proportion and uniformly added to the membrane for imaging using a chemiluminescence analyzer.

[0058] Test results are as follows Figure 2 As shown. Figure 2 The test results showed that gentian extract effectively inhibited the expression of P1 protein in Mycoplasma pneumoniae. This indicates that gentian extract can inhibit Mycoplasma pneumoniae infection, and the above results strongly demonstrate the anti-Mycoplasma pneumoniae activity of gentian extract.

[0059] 5. Trial of Thousand-Gold-Teng Extract in Mice Treating Mycoplasma pneumoniae Infection

[0060] SPF-grade BALB / c mice were randomly divided into five groups: normal group, model group (MP), low-dose group (10 mg / kg), medium-dose group (15 mg / kg), and high-dose group (20 mg / kg), with five mice in each group. Except for the normal group, all other groups of mice were inoculated intranasally with 50 μL of 1 × 10⁻⁶ mol / L extract. 7 Mice were modeled with CCU / ml Mycoplasma pneumoniae strain, administered via nasal drops once daily for three consecutive days. After successful modeling, mice in the low-dose group (10 mg / kg), medium-dose group (15 mg / kg), and high-dose group (20 mg / kg) of *Tetrandrine* were administered 10 mg / kg, 15 mg / kg, and 20 mg / kg, respectively, via gavage. The normal control group and the model group were administered 100 μL of 0.9% sodium chloride injection via gavage. All groups were administered the solution for 3 days. Pathological changes in lung tissue were observed, and the expression of inflammatory factors TNF-α, IL-6, and IL-1β in the mouse lung tissue was detected.

[0061] Experimental results are as follows Figure 3 . Figure 3 The results showed that nasal injection of MP could induce the production of inflammatory factors TNF-α, IL-6 and IL-1β, and their expression levels in mouse lung tissue were significantly higher than those in the normal control group (P<0.001), indicating successful model establishment. In contrast, the three dose groups of senna administered to the model mice by gavage for 3 days significantly inhibited the production of TNF-α, IL-6 and IL-1β in the lung tissue of the model mice (P<0.001).

[0062] In summary, both cell and animal experiments demonstrate that gentianin has an inhibitory effect on Mycoplasma pneumoniae, and it also exhibits a certain degree of biocompatibility. Therefore, it can be used to prepare drugs for alleviating and / or treating Mycoplasma pneumoniae infections.

[0063] The exemplary embodiments of the present invention have been described above. However, the scope of protection of the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of styracil or a pharmaceutically acceptable salt thereof in the preparation of medicaments for relieving and / or treating Mycoplasma pneumoniae infection.

2. The application according to claim 1, wherein, Pharmaceutically acceptable salts of zephyranthesin are selected from acid addition salts of nitrogen atoms with sufficient basicity in their structure, including salts formed by zephyranthesin with inorganic or organic acids.

3. The application according to claim 2, wherein, The inorganic acid is hydrochloric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid, persulfuric acid, aminosulfonic acid, thiocyanate, or nitric acid.

4. The application according to claim 2, wherein, The organic acids are formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pyric acid, pectinic acid, 3-phenylpropionic acid, picric acid, tervaline, 2-hydroxyethanesulfonic acid, itaconic acid, trifluoromethanesulfonic acid, dodecyl sulfate, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucohepanoic acid, glycerophosphate, aspartic acid, and sulfosalicylic acid.

5. The application according to claim 1, wherein, Mycoplasma pneumoniae infection can lead to respiratory diseases and extrapulmonary complications.

6. The application according to claim 5, wherein, The respiratory diseases mentioned include: Mycoplasma pneumoniae pneumonia and upper respiratory tract infection.

7. The application according to claim 6, wherein, The upper respiratory tract infections include pharyngitis, tonsillitis, sinusitis, and otitis media.

8. The application according to claim 5, wherein, The extrapulmonary complications include: myocarditis, pericarditis, pericardial effusion, aseptic meningitis, aseptic encephalitis, Guillain-Barré syndrome (GBS), hemolytic anemia, thrombocytopenic purpura, hepatitis, gastroenteritis, erythema multiforme, Stevens-Johnson syndrome (SJS), arthritis or arthralgia.

9. The application according to claim 1, wherein, The MIC value of senna extract against Mycoplasma pneumoniae was 39.0625 μg / ml.

10. The application according to claim 1, wherein, Chrysanthemum indicum IC50 against Mycoplasma pneumoniae 50 The value is 21 μM.

11. The application according to claim 1, wherein, Tinospora sinensis extract or its pharmaceutically acceptable salts directly inhibit the P1 protein of Mycoplasma pneumoniae in vitro.

12. The application according to claim 1, wherein, Tinospora or its pharmaceutically acceptable salts inhibit cytokine storms in lung tissue.

13. The application according to claim 12, wherein, Tinospora or its pharmaceutically acceptable salts reduce the expression of TNF-α, IL-6 and IL-1β.

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