Composition for resisting mycobacterium tuberculosis and use thereof

Abstract

Provided is a composition of artemisinin or a derivative thereof in combination with clofazimine. The composition can be used for resisting pathogenic bacteria in the genus Mycobacterium, or pathogenic bacteria or parasites having a type II NADH dehydrogenase. The composition has high antimicrobial efficiency and overcomes drug resistance. It can be used in combination with an additional electron transport chain inhibitor to acquire a stronger antimicrobial effect, and reduce the doses of the combination or shorten the course of treatment.

Description

Anti-mycobacterium tuberculosis composition and application thereof Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to an anti-mycobacterium tuberculosis composition, and also relates to the application of the anti-mycobacterium tuberculosis composition. Background Art

[0002] Tuberculosis is one of the three major infectious diseases recognized by the World Health Organization (WHO). In 2020, approximately 1.5 million people died of the disease, most of whom were in low- and middle-income countries. It is an important infectious cause of death among adults worldwide. Tuberculosis is a chronic progressive mycobacterial infection that usually has an asymptomatic latent period after the initial infection. Its causative agent, Mycobacterium tuberculosis, is a type of slow-growing microaerophilic bacillus. They are acid-fast because their cell membranes are rich in lipids (i.e., they are resistant to acid decolorization after staining with calcineurin) and are relatively resistant to Gram staining. The most common mycobacterial infection is Mycobacterium tuberculosis. Others include leprosy and various environmental non-tuberculous mycobacterial infections, such as those caused by the Mycobacterium avium complex. Currently, commonly used anti-TB drugs consist of three major categories: antibiotics, synthetic drugs, and traditional Chinese medicines. Over 20 Western medicines are designated as essential treatments in China. First-line treatments primarily include isoniazid, rifampicin, streptomycin, ethambutol, pyrazinamide, and rifamycins. In recent years, fluoroquinolones have also been used to treat TB, playing an important role in combination therapy. However, rapid diagnosis and timely implementation of effective treatment options, including effective management of adverse reactions to second-line drugs, remain challenging for rifampicin-resistant TB and multidrug-resistant TB. This has led to continued epidemics, low cure rates, and the spread of drug-resistant TB. Patients with highly drug-resistant TB experience poorer treatment outcomes, including higher mortality rates, particularly among those co-infected with HIV, even those receiving antiretroviral therapy. Therefore, there is an urgent need for newer, shorter, and more effective (non-injectable) treatment options.

[0003] Artemisinin (ART) and its derivatives are good antimalarial drugs with the advantages of strong species specificity, rapid drug action, and low toxic and side effects. The double oxygen bridge in the structure of artemisinin is a key pharmacophore. The opening or breaking of the double oxygen bridge, that is, the reduction of artemisinin, is a prerequisite for its efficacy. Deoxyartemisinin obtained by replacing the double oxygen bridge with a single oxygen bridge will lose its antimalarial activity. In addition to antimalarial effects, artemisinin drugs also have the effect of partially inhibiting other types of parasites and antivirals, and are reported to have certain therapeutic effects on diabetes and the autoimmune disease lupus erythematosus. Although there are reports on the efficacy of artemisinin against tuberculosis bacteria, because researchers are unclear about the mechanism of action of artemisinin, the drug is used alone at extremely high concentrations, and the effect is not ideal, and it has no clinical application significance. Therefore, artemisinin has not been used in the treatment of tuberculosis.

[0004] Currently, tuberculosis (TB) suffers from a low cure rate, long treatment cycles, increasingly severe drug resistance, and significant adverse drug side effects. Treatment requires the oral administration of large quantities of tablets and long-term daily injections, placing significant financial and physical burdens on patients. The present invention aims to introduce clinically safe artemisinin-based drugs as anti-TB agents. Combining these drugs with clofazimine can more rapidly and effectively kill pathogens, thereby improving TB treatment efficacy or shortening treatment courses, overcoming drug resistance, and reducing the dosage of combined treatment drugs to minimize side effects on patients.

[0005] Summary of the Invention

[0006] The present invention provides an anti-tuberculosis mycobacterium tuberculosis composition, which solves the shortcomings of the prior art anti-tuberculosis drugs, such as easy drug resistance, low antibacterial efficiency and high dosage.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] An anti-tuberculosis mycobacterium composition comprises artemisinin and its derivatives and clofazimine.

[0009] Furthermore, the dosage of artemisinin and its derivatives is ≤50 μM, and the dosage of clofazimine is ≤5 μM.

[0010] Furthermore, the dosage of artemisinin is 1 μM-25 μM, and the dosage of clofazimine is 0.001-2.5 μM.

[0011] In the present invention, the artemisinin derivatives include one or more of artesunate, dihydroartemisinin, artemether, and arteether.

[0012] Furthermore, the dosage of the artemisinin derivative is 0.1-5 μM, and the dosage of clofazimine is 0.01-5 μM.

[0013] In some embodiments of the present invention, the artemisinin derivative is artesunate. Artesunate has better water solubility and stronger activity, which is about 5 times that of artemisinin.

[0014] In the present invention, the anti-Mycobacterium tuberculosis composition further comprises an anti-tuberculosis drug.

[0015] Furthermore, the anti-tuberculosis drug is an electron transport chain inhibitor or interferor.

[0016] In the present invention, the electron transport chain inhibitor includes one or more of Bedaquiline, Q203 (Telacebec), Lansoprazole and Aurachin D.

[0017] In some embodiments of the present invention, the composition comprises artemisinin and its derivatives, clofazimine and bedaquiline, the dosage of artemisinin and its derivatives is ≤25 μM, the dosage of clofazimine is ≤2.5 μM, and the dosage of bedaquiline is ≤5 μM.

[0018] In some embodiments of the present invention, the composition comprises artemisinin and its derivatives, clofazimine and Q203, the dosage of artemisinin and its derivatives is ≤25 μM, the dosage of clofazimine is ≤2.5 μM, and the dosage of Q203 (Telacebec) is ≤10 nM.

[0019] In some embodiments of the present invention, the composition comprises artemisinin and its derivatives, clofazimine and lansoprazole, the dosage of artemisinin and its derivatives is ≤25 μM, the dosage of clofazimine is ≤2.5 μM, and the dosage of lansoprazole is ≤10 μM.

[0020] In some embodiments of the present invention, the composition comprises artemisinin and its derivatives, clofazimine and Aurachin D, the dosage of artemisinin and its derivatives is ≤25 μM, the dosage of clofazimine is ≤2.5 μM, and the dosage of Aurachin D is ≤10 μM.

[0021] In the present invention, the anti-Mycobacterium tuberculosis composition is used under hypoxic conditions.

[0022] The invention relates to the use of artemisinin and its derivatives in combination with clofazimine in the preparation of products against Mycobacterium tuberculosis pathogens or products against pathogens containing type II NADH dehydrogenase or products against parasites.

[0023] Furthermore, the Mycobacterium tuberculosis pathogen is Mycobacterium tuberculosis.

[0024] The present invention has the following beneficial effects:

[0025] (1) The anti-tuberculosis composition of the present invention is used to fight Mycobacterium tuberculosis by combining artemisinin and its derivatives with clofazimine, which can kill pathogens with high antibacterial efficiency and overcome the problem of drug resistance.

[0026] (2) The composition of the present invention reduces the dosage of combined drugs and the side effects of drugs in the process of treating Mycobacterium tuberculosis. Compared with the existing technology, it is safer and has fewer toxic and side effects. At the same time, it helps to shorten the course of treatment, reduce treatment costs, and alleviate the burden on patients and the medical system. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The technical solution of the present invention will be further described below in conjunction with the accompanying drawings and specific implementation methods.

[0028] FIG1 is a schematic diagram of the mechanism of action of clofazimine and artemisinin according to the present invention;

[0029] Figures 2 and 3 show the inhibition of the growth of Saccharomyces cerevisiae by the combined use of artemisinin and clofazimine according to the present invention;

[0030] FIG4 shows the inhibition of Mycobacterium smegmatis growth by the combined use of artemisinin and clofazimine according to the present invention;

[0031] FIG5 shows the inhibition of Mycobacterium tuberculosis growth by the combination of artemisinin and clofazimine according to the present invention;

[0032] FIG6 shows the inhibition of Mycobacterium tuberculosis growth by the combination of artesunate and clofazimine according to the present invention;

[0033] FIG7 shows the inhibition of Mycobacterium tuberculosis growth by artesunate and clofazimine combined with bedaquiline, Q203, Aurachin D or lansoprazole of the present invention;

[0034] FIG8 shows the inhibition of the growth of Mycobacterium tuberculosis in macrophages by the combination of artemisinin and clofazimine according to the present invention. DETAILED DESCRIPTION

[0035] The present invention provides a combined application and composition of artemisinin and clofazimine. Through extensive experimental research, the inventors discovered that artemisinin contains peroxide bridges in its molecular structure, which can act as a strong oxidant by accepting electrons leaked from the electron transport chain and being reduced and activated. Its metabolites then act to kill cells by disrupting cell membrane potential (see Figure 1). Clofazimine, on the other hand, competes with coenzyme Q in the electron transport chain for the electrons generated by NADH dehydrogenase type II oxidation, converting itself from an oxidized state to a reduced state. Clofazimine can cycle between oxidized and reduced states, generating large amounts of reactive oxygen species, which kill cells (see Figure 1). The present invention conducted extensive in vitro and in vivo antibacterial experiments using a variety of cell systems, including yeast, Mycobacterium smegmatis, Mycobacterium bovis, and Mycobacterium tuberculosis. The results consistently demonstrated that the combination of artemisinin and clofazimine significantly enhances cytotoxicity, resulting in a superior antibacterial effect. To this end, the present invention proposes the use of artemisinin and its derivatives as a component of a multidrug combination for the preparation of anti-tuberculosis drugs.

[0036] Example 1

[0037] A composition comprises artemisinin and clofazimine, wherein the dosage of artemisinin is 1 μM and the dosage of clofazimine is 0.1 μM.

[0038] Example 2

[0039] A composition comprises artemisinin and clofazimine, wherein the dosage of artemisinin is 2.5 μM and the dosage of clofazimine is 0.1 μM.

[0040] Example 3

[0041] A composition comprises artemisinin and clofazimine, wherein the dosage of artemisinin is 5 μM and the dosage of clofazimine is 0.1 μM.

[0042] Example 4

[0043] A composition comprises artemisinin and clofazimine, wherein the dosage of artemisinin is 1 μM and the dosage of clofazimine is 0.001 μM.

[0044] Example 5

[0045] A composition comprises artemisinin and clofazimine, wherein the dosage of artemisinin is 1 μM and the dosage of clofazimine is 0.01 μM.

[0046] Example 6

[0047] A composition comprises artemisinin and clofazimine, wherein the dosage of artemisinin is 1 μM and the dosage of clofazimine is 1 μM.

[0048] Example 7

[0049] A composition comprises artemisinin and clofazimine, wherein the dose of artemisinin is 2.5 μM and the dose of clofazimine is 1 μM.

[0050] Example 8

[0051] A composition comprises artemisinin and clofazimine, wherein the dosage of artemisinin is 1 μM and the dosage of clofazimine is 0.25 μM.

[0052] Example 9

[0053] A composition comprises artemisinin and clofazimine, wherein the dosage of artemisinin is 2.5 μM and the dosage of clofazimine is 0.25 μM.

[0054] Example 10

[0055] A composition comprises artemisinin and clofazimine, wherein the dose of artemisinin is 5 μM and the dose of clofazimine is 1 μM.

[0056] Example 11

[0057] A composition comprises artemisinin and clofazimine, wherein the dosage of artemisinin is 20 μM and the dosage of clofazimine is 0.0625 μM.

[0058] Example 12

[0059] A composition comprises artemisinin and clofazimine, wherein the dosage of artemisinin is 5 μM and the dosage of clofazimine is 0.125 μM.

[0060] Example 13

[0061] A composition comprises artemisinin and clofazimine, wherein the dosage of artemisinin is 25 μM and the dosage of clofazimine is 1 μM.

[0062] Example 14

[0063] A composition comprises artesunate and clofazimine, wherein the dosage of artesunate is 0.1 μM and the dosage of clofazimine is 0.01 μM.

[0064] Example 15

[0065] A composition comprises artesunate and clofazimine, wherein the dosage of artesunate is 0.5 μM and the dosage of clofazimine is 0.0625 μM.

[0066] Example 16

[0067] A composition comprises artesunate and clofazimine, wherein the dosage of artesunate is 0.5 μM and the dosage of clofazimine is 0.1 μM.

[0068] Example 17

[0069] A composition comprises artesunate and clofazimine, wherein the dosage of artesunate is 2 μM and the dosage of clofazimine is 0.5 μM.

[0070] Example 18

[0071] A composition comprises artesunate and clofazimine, wherein the dosage of artesunate is 2 μM and the dosage of clofazimine is 1 μM.

[0072] Example 19

[0073] A composition comprises artesunate and clofazimine, wherein the dosage of artesunate is 2 μM and the dosage of clofazimine is 5 μM.

[0074] Example 20

[0075] A composition comprises artesunate and clofazimine, wherein the dosage of artesunate is 5 μM and the dosage of clofazimine is 0.125 μM.

[0076] Example 21

[0077] A composition comprises artemisinin, clofazimine and bedaquiline, wherein the dosage of artemisinin is 0.1 μM, the dosage of clofazimine is 2.5 nM and the dosage of bedaquiline is 0.2 μM.

[0078] Example 22

[0079] A composition comprises artemisinin, clofazimine and bedaquiline, wherein the dosage of artemisinin is 1.25 μM, the dosage of clofazimine is 25 nM and the dosage of bedaquiline is 0.1 μM.

[0080] Example 23

[0081] A composition comprises artemisinin, clofazimine and bedaquiline, wherein the dosage of artemisinin is 5 μM, the dosage of clofazimine is 100 nM and the dosage of bedaquiline is 0.2 μM.

[0082] Example 24

[0083] A composition comprises artemisinin, clofazimine and bedaquiline, wherein the dosage of artemisinin is 0.3125 μM, the dosage of clofazimine is 6.25 nM and the dosage of bedaquiline is 2 μM.

[0084] Example 25

[0085] A composition comprises artemisinin, clofazimine and Q203, wherein the dosage of artemisinin is 0.1 μM, the dosage of clofazimine is 2.5 nM and the dosage of Q203 is 0.1 nM.

[0086] Example 26

[0087] A composition comprises artemisinin, clofazimine and Q203, wherein the dose of artemisinin is 1.25 μM, the dose of clofazimine is 25 nM, and the dose of Q203 is 0.2 nM.

[0088] Example 27

[0089] A composition comprises artemisinin, clofazimine and Q203, wherein the dose of artemisinin is 5 μM, the dose of clofazimine is 100 nM and the dose of Q203 is 1 nM.

[0090] Example 28

[0091] A composition comprises artemisinin, clofazimine and Q203, wherein the dose of artemisinin is 0.3125 μM, the dose of clofazimine is 6.25 nM and the dose of Q203 is 5 nM.

[0092] Example 29

[0093] A composition comprises artemisinin, clofazimine and lansoprazole, wherein the dosage of artemisinin is 0.1 μM, the dosage of clofazimine is 2.5 nM and the dosage of lansoprazole is 1 μM.

[0094] Example 30

[0095] A composition comprises artemisinin, clofazimine and lansoprazole, wherein the dosage of artemisinin is 1.25 μM, the dosage of clofazimine is 25 nM and the dosage of lansoprazole is 2 μM.

[0096] Example 31

[0097] A composition comprises artemisinin, clofazimine and lansoprazole, wherein the dosage of artemisinin is 5 μM, the dosage of clofazimine is 100 nM and the dosage of lansoprazole is 5 μM.

[0098] Example 32

[0099] A composition comprises artemisinin, clofazimine and lansoprazole, wherein the dosage of artemisinin is 0.3125 μM, the dosage of clofazimine is 6.25 nM and the dosage of lansoprazole is 10 μM.

[0100] Example 33

[0101] A composition comprises artemisinin, clofazimine and aurachin D, wherein the dosage of artemisinin is 0.1 μM, the dosage of clofazimine is 2.5 nM and the dosage of aurachin D is 1 μM.

[0102] Example 34

[0103] A composition comprises artemisinin, clofazimine and aurachin D, wherein the dosage of artemisinin is 1.25 μM, the dosage of clofazimine is 25 nM and the dosage of aurachin D is 2 μM.

[0104] Example 35

[0105] A composition comprises artemisinin, clofazimine and aurachin D, wherein the dosage of artemisinin is 5 μM, the dosage of clofazimine is 100 nM and the dosage of aurachin D is 5 μM.

[0106] Example 36

[0107] A composition comprises artemisinin, clofazimine and aurachin D, wherein the dosage of artemisinin is 0.3125 μM, the dosage of clofazimine is 6.25 nM and the dosage of aurachin D is 10 μM.

[0108] (1) Effects of artemisinin and clofazimine combined on the growth of Saccharomyces cerevisiae

[0109] a. Wild-type Saccharomyces cerevisiae BY4742 and mutant gpd1Δgpd2Δ were grown in respiratory medium (YPGE) to OD 600 = 0.2, and 10-fold serial dilutions were performed and then spotted onto YPGE agar media supplemented with different drug concentrations. In Figure 2A, Group 1: The control group was a blank group, and the experimental group was artemisinin, with artemisinin concentrations of 2.5 μM, 5 μM, and 10 μM, respectively; Group 2: The control group was a blank group, and the experimental group was clofazimine, with clofazimine concentrations of 2.5 μM, 5 μM, and 10 μM, respectively. In Figure 2C, Group 1: The control group was a blank group, and the experimental group was artemisinin; Group 2: The control group was a blank group, and the experimental group was artemisinin plus 0.1 μM clofazimine; Group 3: The control group was a blank group, and the experimental group was artemisinin plus 1 μM clofazimine, with artemisinin concentrations of 1 μM, 2.5 μM, and 5 μM, respectively. After 4 days of growth in a 30°C incubator, photographs were taken. The results are shown in Figures 2A and 2C.

[0110] Wild-type Saccharomyces cerevisiae BY4742 and mutant gpd1Δgpd2Δ were grown in fermentation medium (YPD) to OD 600= 0.2, and 10-fold serial dilutions were performed and spotted onto YPD agar media with different concentrations of drugs as shown in the figure. In Figure 2B, the first group: the control group was a blank group, and the experimental group was artemisinin, with artemisinin concentrations of 5μM, 10μM, and 20μM, respectively; the second group: the control group was a blank group, and the experimental group was clofazimine, with clofazimine concentrations of 5μM, 10μM, and 20μM, respectively. In Figure 2D, the first group: the control group was a blank group, and the experimental group was artemisinin; the second group: the control group was 0.1μM clofazimine, and the experimental group was 0.1μM clofazimine + artemisinin, with artemisinin concentrations of 1μM, 2.5μM, and 5μM, respectively; the third group: the control group was 1μM clofazimine, and the experimental group was 1μM clofazimine + artemisinin, with artemisinin concentrations of 1μM, 2.5μM, and 5μM, respectively. After 2 days of growth in a 30°C incubator, photos were taken. The results are shown in Figures 2B and 2D.

[0111] The results in Figure 2 show that both artemisinin and clofazimine can inhibit the growth of Saccharomyces cerevisiae in respiration medium, but have limited inhibitory effects on the growth of Saccharomyces cerevisiae in fermentation medium, indicating that the inhibitory effects of artemisinin and clofazimine on Saccharomyces cerevisiae are related to the electron transport chain, and the inhibitory effect increases with increasing concentration. When artemisinin and clofazimine act together, the inhibitory effect is better, and the two have a synergistic effect on the growth inhibition of Saccharomyces cerevisiae.

[0112] b. Grow wild-type Saccharomyces cerevisiae BY4742 in respiratory medium (YPGE) to OD 600 = 0.2, then add different concentrations of drugs shown in Figure 3A, culture at 30 ° C shaker (200 rpm) for 48 hours and measure the OD600 value; Saccharomyces cerevisiae mutant gpd1Δgpd2Δ is grown in fermentation medium (YPD) to OD 600 =0.2, then different concentrations of drugs as shown in FIG3B were added and cultured at 30°C in a shaker (200 rpm) for 24 hours, and the OD600 value was measured. The relative growth rate of the experimental group without drug addition was set to 1, and the relative growth rates of the other experimental groups were calculated based on this. The results are shown in FIG3 .

[0113] The results in Figure 3 show that both artemisinin and clofazimine inhibited the growth rate of Saccharomyces cerevisiae in both respiratory and fermentation media. The inhibitory effect of artemisinin increased with increasing concentration from 1 μM to 2.5 μM, while that of clofazimine increased with increasing concentration from 0.1 μM to 0.25 μM. The inhibitory effects of both on the growth rate of Saccharomyces cerevisiae were synergistic.

[0114] (2) Effect of artemisinin and clofazimine combined on the growth of Mycobacterium smegmatis

[0115] Grow Mycobacterium smegmatis in LB liquid medium to an OD of600 =0.4, and then 10-fold serial dilutions were performed and spotted on LB agar medium with different concentrations of drugs. Among them, the first group: the control group was a blank group, the experimental group was clofazimine, and the clofazimine concentrations were 0, 0.001 μM, 0.005 μM, 0.01 μM, 0.05 μM, and 0.1 μM, respectively; the second group: the control group was 1 μM artemisinin, the experimental group was 1 μM artemisinin + clofazimine, and the clofazimine concentrations were 0.001 μM, 0.005 μM, 0.01 μM, 0.05 μM, and 0.1 μM, respectively; the third group: the control group was 1 μM artemisinin, the experimental group was 1 μM artemisinin + clofazimine, and the clofazimine concentrations were 0.001 μM CFZ, 0.005 μM, 0.01 μM, 0.05 μM, and 0.1 μM, respectively. The cells were grown in a 37°C incubator for 4 days and photographed. The results are shown in Figure 4 .

[0116] The results in Figure 4 show that both artemisinin and clofazimine inhibit the growth of Mycobacterium smegmatis. For artemisinin, the inhibitory effect increases as the concentration increases from 1 μM to 5 μM, while for clofazimine, the inhibitory effect increases as the concentration increases from 0.001 μM to 0.1 μM. The two inhibitory effects on the growth of Mycobacterium smegmatis are synergistic.

[0117] (3) Effect of artemisinin and clofazimine combined on the growth of Mycobacterium tuberculosis

[0118] Mycobacterium tuberculosis was grown in Middlebrook 7H9 broth to an OD of 600 =0.003, different concentrations of drugs were added, among which, the first group: the control group was the blank group, the experimental group was clofazimine, and the clofazimine concentrations were 0.0625μM, 0.125μM, 0.25μM, 0.5μM, and 1μM respectively; the second group: the control group was 5μM artemisinin, the experimental group was 5μM artemisinin + clofazimine, and the clofazimine concentrations were 0.0625μM, 0.125μM, 0.25μM, 0.5μM, and 1μM respectively; the third group: the control group was 20μM artemisinin, the experimental group was 20μM artemisinin + clofazimine, and the clofazimine concentrations were 0.0625μM, 0.125μM, 0.25μM, 0.5μM, and 1μM respectively. After culturing at 37° C. for 6 days, 0.02% resazurin was added. After further incubation for 2 days, the fluorescence intensity was measured using a microplate reader and the survival rate was calculated. The results are shown in FIG5 .

[0119] The results in Figure 5 show that both artemisinin and clofazimine inhibit the growth of Mycobacterium tuberculosis. The inhibitory effect of artemisinin increases with increasing concentration from 5 μM to 20 μM, while the inhibitory effect of clofazimine increases with increasing concentration from 0.0625 μM to 1 μM. The two exhibit a synergistic inhibitory effect on the growth of Mycobacterium tuberculosis.

[0120] (4) Effect of artesunate and clofazimine combined on the growth of Mycobacterium tuberculosis

[0121] Mycobacterium tuberculosis was grown in Middlebrook 7H9 broth to an OD of 600 =0.003, different concentrations of drugs were added, among which, the first group: the control group was a blank group, the experimental group was clofazimine, and the clofazimine concentrations were 0.0625μM, 0.125μM, 0.25μM, 0.5μM, and 1μM, respectively; the second group: the control group was 0.5μM artesunate, the experimental group was 0.5μM artesunate + clofazimine, and the clofazimine concentrations were 0.0625μM, 0.125μM, 0.25μM, 0.5μM, and 1μM, respectively; the third group: the control group was 2μM artesunate, the experimental group was 2μM artesunate + clofazimine, and the clofazimine concentrations were 0.0625μM, 0.125μM, 0.25μM, 0.5μM, and 1μM, respectively. After culturing at 37° C. for 6 days, 0.02% resazurin was added. After further incubation for 2 days, the fluorescence intensity was measured using a microplate reader and the survival rate was calculated. The results are shown in FIG6 .

[0122] The results in Figure 6 show that both artesunate and clofazimine inhibited the growth of Mycobacterium tuberculosis. For artesunate, the inhibitory effect increased as the concentration increased from 0.5 μM to 2 μM, while for clofazimine, the inhibitory effect increased as the concentration increased from 0.0625 μM to 1 μM. The two exhibited a synergistic inhibitory effect on the growth of Mycobacterium tuberculosis.

[0123] (5) Effects of artemisinin and clofazimine combined with bedaquiline, Q203, Aurachin D, or lansoprazole on the growth of Mycobacterium tuberculosis

[0124] Mycobacterium tuberculosis was grown in Middlebrook 7H9 broth to an OD of 600=0.003, different concentrations of drugs were added, among which the control groups of group 1 to group 7 were blank group, 5 μM artemisinin + 100 nM clofazimine, 2.5 μM artemisinin + 50 nM clofazimine, 1.25 μM artemisinin + 25 nM clofazimine, 0.625 μM artemisinin + 12.5 nM clofazimine, 0.3125 μM artemisinin + 6.25 nM clofazimine, 0.15625 μM artemisinin + 3.125 nM clofazimine, and the experimental groups were artemisinin + clofazimine + 0.2 μM bedaquiline, artemisinin + clofazimine + 0.2 nM Q203, artemisinin + clofazimine + 5 μM Aurachin D, and artemisinin + clofazimine + 5 μM lansoprazole. After culturing at 37° C. for 6 days, 0.02% resazurin was added. After further incubation for 2 days, the fluorescence intensity was measured using a microplate reader and the survival rate was calculated. The results are shown in FIG7 .

[0125] The results in Figure 7 demonstrate that artemisinin and clofazimine, when combined with bedaquiline, Q203, aurachin D, or lansoprazole, inhibited the growth of Mycobacterium tuberculosis, with the inhibitory effect increasing with increasing concentrations of artemisinin and clofazimine. The combination of bedaquiline, Q203, aurachin D, and lansoprazole with artemisinin and clofazimine exhibited a synergistic effect in inhibiting the growth of Mycobacterium tuberculosis.

[0126] (6) Effect of artemisinin and clofazimine on the growth of Mycobacterium tuberculosis in macrophages

[0127] RAW264.7:4E5 cells (mouse mononuclear macrophage leukemia cells) that have grown well were cultured at 2×10 6 The number of cells / well was spread into a six-well plate, and 1x10 7 Cells were infected with a large number of H37Ra tuberculosis bacteria for 4 hours. Uninfected bacteria were washed with PBS, and then various drug concentrations were added. The control group was a blank group, while the experimental groups included 25 μM artemisinin, 1 μM clofazimine, and 25 μM artemisinin plus 1 μM clofazimine. After incubation for 4 days, the cells were washed twice with PBS and lysed with 0.5% Triton X-100. The supernatant was centrifuged and serially diluted 10-fold before being plated on 7H10 agar. The cells were cultured at 37°C for 3-4 weeks and the number of colonies was counted. The vertical axis shows the increase in pathogen counts 4 days after infection relative to day 0 (see Figure 8).

[0128] The results in Figure 8 show that artemisinin and clofazimine have no inhibitory effect on the growth of Mycobacterium tuberculosis in macrophages when used alone, but the inhibitory effect on the growth of Mycobacterium tuberculosis in macrophages when artemisinin and clofazimine are very obvious when used together. The two have a synergistic effect on the inhibition of the growth of Mycobacterium tuberculosis in macrophages.

[0129] The above disclosure is merely a preferred embodiment of the present invention and certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. An anti-Mycobacterium tuberculosis composition, characterized in that: The invention comprises artemisinin and its derivatives and clofazimine; wherein the dosage of the artemisinin and its derivatives is ≤50 μM, and the dosage of the clofazimine is ≤5 μM.

2. The anti-Mycobacterium tuberculosis composition according to claim 1, characterized in that The dosage of artemisinin is 1 μM-25 μM, and the dosage of clofazimine is 0.001-2.5 μM.

3. The anti-Mycobacterium tuberculosis composition according to claim 1, characterized in that The artemisinin derivatives include one or more of artesunate, dihydroartemisinin, artemether, and arteether; wherein the dosage of the artemisinin derivatives is 0.1-5 μM, and the dosage of clofazimine is 0.01-5 μM.

4. The anti-Mycobacterium tuberculosis composition according to any one of claims 1 to 3, characterized in that The anti-mycobacterium tuberculosis composition further comprises an anti-tuberculosis drug.

5. The anti-Mycobacterium tuberculosis composition according to claim 4, characterized in that The anti-tuberculosis drug is an electron transport chain inhibitor or interfering agent.

6. The anti-Mycobacterium tuberculosis composition according to claim 5, characterized in that The electron transport chain inhibitor includes one or more of bedaquiline, Q203, lansoprazole and Aurachin D.

7. The anti-Mycobacterium tuberculosis composition according to claim 6, characterized in that The composition comprises artemisinin and its derivatives, clofazimine and bedaquiline, wherein the dosage of the artemisinin and its derivatives is ≤25 μM, the dosage of the clofazimine is ≤2.5 μM, and the dosage of the bedaquiline is ≤5 μM.

8. The anti-Mycobacterium tuberculosis composition according to claim 6, characterized in that The composition comprises artemisinin and its derivatives, clofazimine and Q203, wherein the dosage of the artemisinin and its derivatives is ≤25 μM, the dosage of the clofazimine is ≤2.5 μM, and the dosage of the Q203 is ≤10 nM.

9. The anti-Mycobacterium tuberculosis composition according to claim 6, characterized in that The composition comprises artemisinin and its derivatives, clofazimine and lansoprazole or Aurachin D, wherein the dosage of artemisinin and its derivatives is ≤25 μM, the dosage of clofazimine is ≤2.5 μM, and the dosage of lansoprazole or Aurachin D is ≤10 μM.

10. Use of artemisinin and its derivatives in combination with clofazimine in the preparation of products against Mycobacterium tuberculosis pathogens or products against pathogens containing type II NADH dehydrogenase or antiparasitic products.

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