Use of genistein in the preparation of a medicine against mycobacterium tuberculosis

Abstract

The application belongs to the technical field of medicine, and discloses application of genistein in preparation of medicine for resisting Mycobacterium tuberculosis. The application discloses application of genistein or a pharmaceutically acceptable derivative thereof in inhibition of Mycobacterium tuberculosis or preparation of products for resisting Mycobacterium tuberculosis. Experiments show that the MIC of genistein to M. tb H37Rv ΔleuΔpan and M. tb M. tb M. tb M. tb M. tb H37Ra is 0.03 mg / mL, and under the action of 30 μg / mL genistein, the minimum inhibitory concentration of a rifampicin-resistant strain is reduced from 50 μg / mL to 6.25 μg / mL, which greatly improves the sensitivity of the drug-resistant strain to rifampicin. Therefore, genistein can be used in preparation of medicine for resisting Mycobacterium tuberculosis or resisting tuberculosis alone, and can also be used as an auxiliary medicine for other anti-tuberculosis medicines for combined use, that is, the types of anti-tuberculosis medicines are widened and the course of treatment of tuberculosis is expected to be shortened.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of genistein in the preparation of drugs against Mycobacterium tuberculosis. Background Technology

[0002] Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mycobacterium tuberculosis). Mycobacterium tuberculosis , M. tb Chronic infectious diseases caused by drug resistance are among the deadliest infectious diseases worldwide. M. tb The emergence of tuberculosis, especially the spread of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB), has made the treatment of TB extremely difficult. According to the World Health Organization's "Global Tuberculosis Report 2024" published in 2025, there were 10.7 million new cases of tuberculosis globally in 2024, and approximately 1.2 million people died from tuberculosis. Although more than 164,000 patients with drug-resistant tuberculosis received treatment, the success rate was only 71%, indicating that existing therapies remain ineffective against drug-resistant tuberculosis.

[0003] With the emergence of drug-resistant Mycobacterium tuberculosis, the therapeutic effect of existing anti-tuberculosis drugs in clinical practice has been greatly reduced, which suggests that we urgently need to develop new anti-tuberculosis drugs. Among various types of drug-resistant tuberculosis, rifampicin-resistant tuberculosis (RR-TB) poses a particularly severe clinical challenge. Rifampicin is a key component in the current standard short-course chemotherapy regimen. Its failure not only renders the entire first-line treatment regimen ineffective, forcing patients to switch to second-line treatment regimens with longer treatment courses, more significant drug toxicity, and lower success rates, but also significantly increases the public health burden. Therefore, innovation in treatment strategies for RR-TB, especially the development of new therapeutic agents that can "rescue" the efficacy of rifampicin (i.e., synergistic effect with rifampicin or restore the sensitivity of drug-resistant bacteria to it), has become an urgent and valuable topic in the research and development of anti-tuberculosis drugs. Existing technologies (including reported natural products with anti-tuberculosis activity) mainly have the following shortcomings: (1) limited activity. The discovered active compounds (such as daidzein) have limited in vitro anti-tuberculosis activity and insufficient potential for clinical translation; (2) the core drug resistance problem has not been solved. Current research mainly focuses on testing the bactericidal effect of the compounds themselves, but has not provided an effective solution for the most important reason for current treatment failure—clinical drug resistance, especially resistance to key drugs such as rifampin; (3) lack of synergistic effect: current technology has not revealed that any isoflavone compounds can produce a synergistic effect with existing anti-tuberculosis drugs (such as rifampin) to improve the efficacy of existing therapies and overcome drug resistance.

[0004] Genistein is an isoflavone compound derived from the rhizome of the legume *Gynostemma pentaphyllum* (also known as *Gynostemma pentaphyllum*). It is a phytoactive flavonoid with the chemical formula 5,7-dihydroxy-3-(4-hydroxyphenyl)chromen-4-one (molecular formula C). 15 H 10 O5 (molecular weight 270.24), CAS number 446-72-0. Its structural formula is as follows:

[0005] Previous research has primarily focused on anti-cancer and prevention of chronic diseases such as osteoporosis. Notably, genistein, as a multi-target phytochemical, has also been reported to have the potential to regulate host immune responses and influence various microbial life processes, providing a biological basis for its potential anti-infective applications. However, its role in anti-tuberculosis, particularly against drug-resistant Mycobacterium tuberculosis, has not yet been systematically reported. Summary of the Invention

[0006] The present invention aims to at least solve one of the technical problems existing in the prior art. It provides the application of genistein in the preparation of drugs that effectively inhibit Mycobacterium tuberculosis, thereby achieving effective anti-tuberculosis effects.

[0007] The first aspect of the present invention aims to provide the use of genistein or a pharmaceutically acceptable derivative thereof in inhibiting Mycobacterium tuberculosis or in the preparation of products that inhibit Mycobacterium tuberculosis.

[0008] A second aspect of the present invention aims to provide the use of genistein or a pharmaceutically acceptable derivative thereof in the preparation of medicaments for the prevention and / or treatment of tuberculosis.

[0009] A third aspect of the present invention aims to provide the use of genistein or a pharmaceutically acceptable derivative thereof in the preparation of products that enhance the susceptibility of Mycobacterium tuberculosis to rifampin.

[0010] The fourth aspect of this invention is to provide a drug.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the invention provides the use of genistein or a pharmaceutically acceptable derivative thereof in the inhibition of Mycobacterium tuberculosis or in the preparation of products that inhibit Mycobacterium tuberculosis.

[0012] In some embodiments of the present invention, the Mycobacterium tuberculosis includes M. tb H37Rv DleuΔpan and / or M. tb H37Ra.

[0013] In some embodiments of the present invention, the Mycobacterium tuberculosis includes drug-resistant Mycobacterium tuberculosis.

[0014] In some embodiments of the present invention, the drug-resistant Mycobacterium tuberculosis includes any one of rifampicin-resistant strains, levofloxacin-resistant strains, and bedaquiline-resistant strains.

[0015] In some embodiments of the present invention, the effective dose of the genistein or its pharmaceutically acceptable derivative is 0.01-1 mg / mL, more specifically 0.03-0.5 mg / mL.

[0016] In some embodiments of the present invention, the pharmaceutically acceptable derivatives include pharmaceutical salts, pharmaceutical esters, pharmaceutical ethers, pharmaceutical amides, glycosides, solvates, and eutectics of genistein.

[0017] In some embodiments of the present invention, the pharmaceutical salt of the genistein includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

[0018] In some embodiments of the present invention, the metal salt includes alkali metal salts and alkaline earth metal salts.

[0019] In some embodiments of the present invention, the alkali metal salt includes at least one of sodium salt and potassium salt.

[0020] In some embodiments of the present invention, the alkaline earth metal salt includes at least one of calcium salt, magnesium salt, barium salt, and aluminum salt.

[0021] In some embodiments of the present invention, the salt formed with an organic base includes at least one of the following organic bases: trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine.

[0022] In some embodiments of the present invention, the salt formed with the inorganic acid includes at least one of the following inorganic acids: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid.

[0023] In some embodiments of the present invention, the salt formed with the organic acid includes at least one of the following organic acids: formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0024] In some embodiments of the present invention, the salt formed with the basic amino acid includes at least one of the following basic amino acids: arginine, lysine, and ornithine.

[0025] In some embodiments of the present invention, the salt formed with the acidic amino acid includes at least one of the following acidic amino acids: aspartic acid and glutamic acid.

[0026] In some embodiments of the present invention, the product includes reagents and drugs.

[0027] In vitro experiments showed that genistein can effectively inhibit... M. tb H37Rv DleuΔpan and M. tb H37Ra proliferation M. tb H37Rv DleuΔpan and M. tbThe MIC of H37Ra is 0.03 mg / mL. Genistein can also improve the sensitivity of drug-resistant Mycobacterium tuberculosis (especially rifampicin-resistant bacteria) to rifampicin, directly addressing the limitation of existing technologies in reversing clinical drug resistance.

[0028] A second aspect of the invention provides the use of genistein or a pharmaceutically acceptable derivative thereof in the preparation of medicaments for the prevention and / or treatment of tuberculosis.

[0029] In some embodiments of the present invention, the tuberculosis includes tuberculosis caused by… M. tb H37Rv DleuΔpan and / or M. tb Tuberculosis caused by H37Ra.

[0030] In some embodiments of the present invention, the pharmaceutically acceptable derivatives include pharmaceutical salts, pharmaceutical esters, pharmaceutical ethers, pharmaceutical amides, glycosides, solvates, and eutectics of genistein.

[0031] In some embodiments of the present invention, the pharmaceutical salt of the genistein includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

[0032] In some embodiments of the present invention, the metal salt includes alkali metal salts and alkaline earth metal salts.

[0033] In some embodiments of the present invention, the alkali metal salt includes at least one of sodium salt and potassium salt.

[0034] In some embodiments of the present invention, the alkaline earth metal salt includes at least one of calcium salt, magnesium salt, barium salt, and aluminum salt.

[0035] In some embodiments of the present invention, the salt formed with an organic base includes at least one of the following organic bases: trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine.

[0036] In some embodiments of the present invention, the salt formed with the inorganic acid includes at least one of the following inorganic acids: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid.

[0037] In some embodiments of the present invention, the salt formed with the organic acid includes at least one of the following organic acids: formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0038] In some embodiments of the present invention, the salt formed with the basic amino acid includes at least one of the following basic amino acids: arginine, lysine, and ornithine.

[0039] In some embodiments of the present invention, the salt formed with the acidic amino acid includes at least one of the following acidic amino acids: aspartic acid and glutamic acid.

[0040] In some embodiments of the present invention, the effective dose of the genistein or its pharmaceutically acceptable derivative is 0.01-1 mg / mL, more specifically 0.03-0.5 mg / mL.

[0041] A third aspect of the invention provides the use of genistein or a pharmaceutically acceptable derivative thereof in the preparation of products that enhance the susceptibility of Mycobacterium tuberculosis to rifampin.

[0042] In some embodiments of the present invention, the Mycobacterium tuberculosis includes drug-resistant Mycobacterium tuberculosis.

[0043] In some embodiments of the present invention, the drug-resistant Mycobacterium tuberculosis includes any one of rifampicin-resistant strains, levofloxacin-resistant strains, and bedaquiline-resistant strains; preferably, rifampicin-resistant strains.

[0044] In some embodiments of the present invention, the product includes a drug.

[0045] A fourth aspect of the invention provides a medicament comprising genistein or a pharmaceutically acceptable derivative thereof, and a combination medicament.

[0046] In some embodiments of the present invention, the combined drugs include at least one of rifampin, isoniazid, ethambutol, streptomycin, pyrazinamide, bedaquiline, levofloxacin, moxifloxacin, linezolid, clofazimine, cycloserine, terizone, clofazimine, prothionamide, delamani, putomani, para-aminosalicylic acid, imipenem, meropenem, amikacin, capreomycin, rifabutin, and rifapentine; preferably rifampin.

[0047] This invention provides a drug for the synergistic treatment of tuberculosis by combining genistein and rifampin, overcoming the problems of insufficient activity of existing compounds and their inability to synergize with core drugs, thereby improving the success rate of treatment, reducing the dosage and toxic side effects.

[0048] In some embodiments of the present invention, the medicament further includes pharmaceutically acceptable excipients.

[0049] In some embodiments of the present invention, the pharmaceutically acceptable excipient is selected from at least one of fillers, binders, disintegrants, lubricants, flavoring agents, colorants, masking agents, pH adjusters, buffers, preservatives, stabilizers, antioxidants, wetting agents, humidity regulators, surfactants, suspending agents, and absorption enhancers.

[0050] In some embodiments of the present invention, for ease of administration, genistein or its pharmaceutically acceptable salts can be processed with one or more pharmaceutically acceptable excipients into a specific dosage form. These excipients may be diluents (e.g., starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, and microcrystalline cellulose), absorbents (e.g., calcium sulfate, dicalcium phosphate, light magnesium oxide, and calcium carbonate), wetting agents (e.g., water and ethanol), binders (e.g., hydroxypropyl methylcellulose, povidone, starch paste, and syrup), disintegrants (e.g., dry starch, sodium hydroxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrants, and crospovidone), and lubricants (magnesium stearate, talc, hydrogenated vegetable oil, polyethylene glycol, and micronized powders). The following are examples of agents: silica gel, colorants (such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide), coating materials (such as acrylic resin, hydroxypropyl methylcellulose, and povidone), solvents (such as water for injection, ethanol, propylene glycol, and glycerin), acid-base regulators (such as hydrochloric acid, lactic acid, sodium hydroxide, tartaric acid, and sodium tartrate), antioxidants (such as sodium sulfite, sodium metabisulfite, and sodium thiosulfate), antibacterial agents (such as phenol, benzyl alcohol, and thimerosal), and isotonic regulators (such as sodium chloride and glucose).

[0051] In some embodiments of the present invention, the dosage form of the drug includes a gastrointestinal dosage form or a non-gastrointestinal dosage form.

[0052] In some embodiments of the present invention, the gastrointestinal dosage form includes at least one of powder, tablet, granule, capsule, sustained-release, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet.

[0053] In some embodiments of the present invention, the gastrointestinal dosage forms include, but are not limited to, enteric-coated tablets, coated tablets, film-coated tablets, sugar-coated tablets, dispersible tablets, sucking tablets, chewable tablets, effervescent tablets, scratch tablets, sustained-release and controlled-release dosage forms, sustained-release tablets, sustained-release coated tablets, controlled-release tablets, orally disintegrating tablets, lozenges, and oral patches.

[0054] In some embodiments of the present invention, the non-gastrointestinal drug delivery dosage form includes at least one of injection dosage form, respiratory dosage form, skin dosage form, mucosal dosage form, and cavity dosage form.

[0055] In some embodiments of the present invention, the injectable dosage forms include, but are not limited to, injection solutions, solutions for injection, injection solutions for intravenous infusion, suspensions for injection, sterile powders for injection, intravenous injections, water injections, emulsions for injection, powder injections, injections, sterile powder injections, lyophilized powder injections, etc.

[0056] The beneficial effects of this invention are: This invention discloses that genistein can be used to prepare drugs against Mycobacterium tuberculosis and other tuberculosis-related pathogens. In vitro experiments show that genistein... M. tb H37Rv DleuΔpan and M. tb The MIC of H37Ra is 0.03 mg / mL. With the action of 30 μg / mL genistein, the minimum inhibitory concentration of rifampicin-resistant strains decreased from 50 μg / mL to 6.25 μg / mL, significantly improving the sensitivity of resistant strains to rifampicin. Therefore, genistein can be used alone to prepare drugs against Mycobacterium tuberculosis or other tuberculosis agents, or it can be used as an adjunct to other anti-tuberculosis drugs in combination therapy. In other words, this invention broadens the types of anti-tuberculosis drugs and has the potential to shorten the course of tuberculosis treatment. Furthermore, this invention represents a "repurposing of an existing drug," thus saving drug development and treatment costs, and has significant economic benefits and clinical application value. Attached Figure Description

[0057] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 To detect the effect of genistein on the almar blue assay (MABA) using micropores. M. tb H37Rv and M. tb The minimum inhibitory concentration (MIC) of H37Ra.

[0058] Figure 2 To test the effects of different concentrations of genistein M. tb H37Rv DleuΔpan Time-inhibition curve.

[0059] Figure 3 For different concentrations of genistein M. tb H37Rv DleuΔpan Plate graph showing antibacterial effect.

[0060] Figure 4 For genistein and rifampin M. tb H37Rv DleuΔpan The combined effect heat map.

[0061] Figure 5 For genistein and rifampin M. tbNilefuping H37Rv DleuΔpan The combined effect heat map.

[0062] Figure 6 After treatment with genistein M. tb H37Rv DleuΔpan Scanning electron microscope image at 15,000x magnification. Detailed Implementation

[0063] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0064] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0065] Oleic acid and BSA were purchased from Simga, Inc., USA, with catalog numbers O1383-1G and V900933, respectively; glucose was purchased from Maclean’s, Inc., catalog number D823520; and catalase was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number C163049.

[0066] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0067] Example 1: Determination of genistein-p-genistein pair by MABA method M. tb H37Rv DleuΔpan and M. tb H37Ra's MIC This invention provides an embodiment of the determination of the effect of genistein on thymol using a turbidimetric method. M. tb H37Rv and M. tb The antibacterial curve of H37Ra specifically includes the following steps: (1) Preparation and dilution of genistein Under sterile conditions, genistein was prepared into a solution with a final concentration of 200 mg / mL using DMSO. The fully dissolved mother liquor was stored at -80°C for later use.

[0068] Using 7H9 medium (BD Biosciences Inc., catalog number 271310) containing 10 v / v% OADC enrichment broth (including oleic acid, BSA, glucose, and catalase) (denoted as 7H9-OADC medium), dilute the prepared genistein stock solution to 1 mg / mL. Add 200 μL of the 1 mg / mL genistein solution to column 11 of a 96-well plate per well. Add 100 μL of 7H9-OADC medium to columns 2-10. Then, take 100 μL of the solution from column 11 and add it to column 10 for dilution. After mixing, take 100 μL of the solution and add it to column 9 for dilution. Repeat this process until column 3. Then, discard 100 μL of the solution. Column 2 serves as a negative control. Use an equivalent volume of DMSO as a negative control. Set up three replicates for each concentration gradient.

[0069] (2) M. tb H37Rv DleuΔpan and M. tb Cultivation of H37Ra and treatment of genistein Will M. tb H37Rv DleuΔpan and M. tb H37Ra was inoculated into 7H9-OADC medium and cultured until the logarithmic growth phase (OD200). 600 =0.6-0.8), and then the bacterial solution was added to 7H9-OADC medium at a volume ratio of 1:100. 100 μL was added to the 96-well cell culture plate containing different concentrations of genistein in (1), so that the final concentrations of genistein were 0.003 mg / mL, 0.007 mg / mL, 0.015 mg / mL, 0.03 mg / mL, 0.06 mg / mL, 0.12 mg / mL, 0.25 mg / mL, and 0.5 mg / mL, respectively, so that the final volume of each well was 200 μL. The 96-well plate was then sealed and placed in a 37°C 5% CO2 incubator for 7 days. After 7 days, 32.5 μL of a freshly prepared mixture of 8:5 volume ratio of Alamar Blue and 20% Tween 80 was added to each well, and the plate was incubated again at 37°C for 24 h. The color change of each well was then observed. The MIC was defined as the lowest drug concentration at which no color change (blue) occurred.

[0070] (3) Results Analysis The resulting color patterns at different concentrations are shown in the following figures. Figure 1 As shown, from Figure 1 It can be seen from this that genistein has an effect on M. tb H37Rv DleuΔpan and M. tb The MIC of H37Ra was 0.03 mg / mL.

[0071] Example 2: Determination of genistein-p-genistein pair by CFU method M. tb H37Rv DleuΔpan Time-Inhibition Curve This embodiment uses the CFU method to determine the genistein pair. M. tb H37Rv DleuΔpan The time-antibacterial curve is as follows: Under aseptic conditions, genistein was prepared into a solution with a final concentration of 200 mg / mL using DMSO, and the dissolved stock solution was stored at -80°C for later use. The above genistein stock solution was diluted to 100 μg / mL and 50 μg / mL using 7H9-OADC medium (same as in Example 1).

[0072] Will M. tb H37Rv DleuΔpan Inoculate into 7H9-OADC medium and culture until the logarithmic growth phase (OD200). 600 =0.6-0.8), and then the bacterial suspension was added to 7H9-OADC medium containing 100 μg / mL and 50 μg / mL genistein respectively at a volume ratio of 1:100. The same volume of DMSO was added as a negative control. Each treatment was set up in triplicate and incubated statically at 37℃ in a 5% CO2 incubator. On days 0, 3, 6 and 9, 50 μL of bacterial suspension was taken and serially diluted 10-fold to obtain 5 concentrations. After shaking and mixing, 5 μL of bacterial suspension of each concentration was dropped onto Middle Brook 7H10 agar plates and incubated statically at 37℃ in a 5% CO2 incubator. After 3 weeks, the colony count was calculated and a time-inhibition curve was plotted.

[0073] lignin pair M. tb H37Rv DleuΔpan Time-antibacterial curve as follows Figure 2 As shown, from Figure 2 As can be seen, after 9 days of inhibition, 50 μg / mL genistein can inhibit the growth of approximately one-third of bacteria; 100 μg / mL genistein can significantly inhibit... M. tb H37Rv DleuΔpan Regenerative growth. The CFU plate at day 9... Figure 3 As shown, compared with DMSO, the CFU was significantly reduced after 9 days of treatment with 100 μg / mL genistein.

[0074] Example 3: Determination of the synergistic effect of genistein and rifampin using the checkerboard method This embodiment uses the checkerboard method to determine the synergistic effect of genistein and rifampin, as detailed below: (1) M. tb H37Rv DleuΔpan Culture and drug treatment Under aseptic conditions, genistein was prepared into a solution with a final concentration of 200 mg / mL using DMSO, and the mother liquor was stored at -80°C for later use. Similarly, rifampin was prepared into a solution with a final concentration of 10 mg / mL using DMSO, and the mother liquor was stored at -80°C for later use.

[0075] Will M. tb H37Rv DleuΔpan Inoculate into 7H9-OADC medium (same as in Example 1) and culture until the logarithmic growth phase (OD200). 600 =0.6-0.8), and then the bacterial suspension was added to fresh 7H9-OADC medium at a volume ratio of 1:100, and dispensed into six 1.5 mL centrifuge tubes (labeled tubes 1-6), 500 μL of diluted bacterial suspension in each tube. The rifampicin stock solution was then diluted to 12.5 μg / mL using 7H9-OADC medium, and 1 mL was transferred to a 1.5 mL centrifuge tube (tube 7). After vortexing, 500 μL was transferred to tube 6, and this process was repeated twofold until the rifampicin concentration in tube 1 was 0.1 mg / mL. Similarly, the genistein stock solution was diluted to 200 μg / mL as the highest concentration and then twofold diluted again. Subsequently, 100 μL of genistein dilution was added to the BG row of a 96-well plate, and 100 μL of rifampicin dilution containing the bacterial suspension was added to columns 3-9. The 96-well plates were then placed in a 37°C, 5% CO2 incubator for static incubation, and the FICI values ​​were calculated after two weeks.

[0076] Calculate the FICI index of the two drugs using the following formula. Here, gen represents genistein and rif represents rifampin. When FICI ≤ 0.5, the two drugs are considered to have a synergistic effect; when 0.5 < FICI < 1, the two drugs are considered to have an additive effect.

[0077] (2) M. tb Culture and drug treatment of rifampicin-resistant H37Rv Nerifampin M. tb H37Rv DleuΔpan Right now M. tb H37Rv DleuΔpan (RIF) R Inoculate into 7H9-OADC medium and culture until the logarithmic growth phase (OD200). 600=0.6-0.8), then the bacterial suspension was added to fresh 7H9-OADC medium at a ratio of 1:100 and dispensed into nine 1.5 mL centrifuge tubes (tubes 1-9), 500 μL of bacterial suspension in each tube. The rifampicin stock solution was diluted to 50 μg / mL using 7H9-OADC medium, and 1 mL was transferred to a 1.5 mL centrifuge tube (tube 10). After vortexing, 500 μL was transferred to tube 9, and this process was repeated twofold until the rifampicin concentration in tube 1 was 0.1 mg / mL. Similarly, the genistein stock solution was diluted to 63 μg / mL as the highest concentration and then twofold diluted again. Subsequently, 100 μL of genistein dilution was added to the BG row of a 96-well plate, and 100 μL of rifampicin dilution containing the bacterial suspension was added to columns 3-11. The 96-well plates were then placed in a 37°C, 5% CO2 incubator for static incubation, and the FICI values ​​were calculated after two weeks.

[0078] (3) Results Analysis Genistein and rifampin are sensitive to M. tb H37Rv DleuΔpan The results of the combined use are as follows Figure 4 As shown in the figure, the calculated FICI of genistein and rifampin was 0.5, indicating that the combined effect was significantly stronger than the sum of the effects of the two drugs alone. That is, genistein can significantly enhance the bactericidal effect of rifampin, and the two have a synergistic effect.

[0079] Figure 5 The image shows the reaction of lignin with rifampin against rifampin resistance. M. tb H37Rv DleuΔpan The combined use results showed a FICI of 0.625 (0.5 < FICI < 1), indicating that the two drugs had an additive effect. It was also evident that under the action of 30 μg / mL genistein, the minimum inhibitory concentration of rifampicin-resistant strains decreased from 50 μg / mL to 6.25 μg / mL, which greatly improved the sensitivity of the resistant strains to rifampicin.

[0080] Example 4: Scanning electron microscopy observation of the morphology of Mycobacterium tuberculosis after genistein treatment. To explore the unique mechanism of action of genistein in this invention, this embodiment uses scanning electron microscopy to observe the changes in cell morphology of Mycobacterium tuberculosis after treatment with genistein.

[0081] (1) M. tb H37Rv DleuΔpan Cultivation and treatment of genistein Will M. tb H37Rv DleuΔpan Inoculated into 7H9-OADC medium and cultured until the early logarithmic growth phase (OD100). 600=0.3-0.4), and then the bacteria were treated overnight with a final concentration of 30 μg / mL genistein, with the same volume of DMSO used as a control group.

[0082] (2) Dehydration, drying and observation of bacterial samples After treatment, the bacterial cells were collected by centrifugation at 6000g for 5 minutes and then subjected to a gradient dehydration treatment using 50%-70%-85%-100%-100% ethanol, with each concentration treated for 10 minutes. This was followed by replacement with acetone-amyl acetate. After overnight treatment, the samples were dried using a critical point desiccator to preserve the original cell morphology to the greatest extent possible. The dried samples were then mounted on the sample stage, and a nanometer-thick layer of platinum was sputtered onto the sample surface using an ion sputtering coating system to increase conductivity and image clarity. The prepared samples were then observed under a scanning electron microscope (Zeiss Crossbeam 550) at 3kV.

[0083] (3) Results Analysis The results are as follows Figure 6 As shown, the bacterial surface morphology showed obvious shrinkage after treatment with genistein, suggesting that the antibacterial mechanism of genistein is different from that of known isoflavones, and it is speculated that it may target the cell wall of Mycobacterium tuberculosis.

[0084] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. The use of genistein or a pharmaceutically acceptable derivative thereof in the inhibition of Mycobacterium tuberculosis or in the preparation of products that inhibit Mycobacterium tuberculosis.

2. The application according to claim 1, characterized in that, The tuberculosis mycobacteria include M. tb H37Rv Δ leuΔpan and / or M. tb H37Ra.

3. The application according to claim 1, characterized in that, The tuberculosis mycobacteria include drug-resistant tuberculosis mycobacteria; Preferably, the drug-resistant Mycobacterium tuberculosis includes any one of rifampicin-resistant strains, levofloxacin-resistant strains, and bedaquiline-resistant strains.

4. Use of genistein or pharmaceutically acceptable derivatives thereof in the preparation of medicaments for the prevention and / or treatment of tuberculosis.

5. Use of genistein or pharmaceutically acceptable derivatives thereof in the preparation of products that enhance the susceptibility of Mycobacterium tuberculosis to rifampin.

6. The application according to any one of claims 1-5, characterized in that, The pharmaceutically acceptable derivatives include pharmaceutical salts, pharmaceutical esters, pharmaceutical ethers, pharmaceutical amides, glycosides, solvates, and cocrystals of genistein; Preferably, the medicinal salt includes at least one of the following: metal salt, ammonium salt, salt formed with inorganic acid, salt formed with organic base, salt formed with organic acid, salt formed with basic amino acid, and salt formed with acidic amino acid.

7. The application according to claim 6, characterized in that, The metal salt includes alkali metal salts and alkaline earth metal salts; and / or, the alkali metal salt includes at least one of sodium salts and potassium salts; and / or, the salt formed with an inorganic acid includes at least one of the following inorganic acids: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid; and / or, the salt formed with an organic acid includes at least one of the following organic acids: formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid; and / or, the salt formed with a basic amino acid includes at least one of the following basic amino acids: arginine, lysine, and ornithine; and / or, the salt formed with an acidic amino acid includes at least one of the following acidic amino acids: aspartic acid and glutamic acid.

8. A drug comprising genistein or a pharmaceutically acceptable derivative thereof, and a combination drug.

9. The medicament according to claim 8, characterized in that, The combination drugs include at least one of rifampin, isoniazid, ethambutol, streptomycin, pyrazinamide, bedaquiline, levofloxacin, moxifloxacin, linezolid, clofazimine, cycloserine, terizone, clofazimine, prothionamide, delamani, putomani, para-aminosalicylic acid, imipenem, meropenem, amikacin, capreomycin, rifabutin, and rifapentine.

10. The medicament according to claim 9, characterized in that, The drug also includes pharmaceutically acceptable excipients; Preferably, the pharmaceutically acceptable excipient is selected from at least one of fillers, binders, disintegrants, lubricants, flavoring agents, colorants, flavor masking agents, pH adjusters, buffers, preservatives, stabilizers, antioxidants, wetting agents, humidity regulators, surfactants, suspending agents, and absorption enhancers; Preferably, the dosage form of the drug includes a gastrointestinal dosage form or a non-gastrointestinal dosage form.

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