Application of cryptotanshinone in preparation of anti-mycobacterium tuberculosis drugs

Abstract

The invention discloses an application of cryptotanshinone in preparation of an anti-mycobacterium tuberculosis drug, and relates to the technical field of bioengineering. The molecular formula of the valerianin disclosed by the invention is C19H20O3. The mycobacterium tuberculosis is a mycobacterium tuberculosis H37Rv strain. Cryptotanshinone is disclosed as a natural-source compound for the first time, has the dual advantages of high efficiency and low toxicity, has an obvious effect of resisting mycobacterium tuberculosis, provides a brand new candidate drug scheme with development potential for solving the difficulty of tuberculosis treatment, and has great scientific value and application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering, in particular to the application of cryptotanshinone in the preparation of anti-mycobacterium tuberculosis drugs. BACKGROUND

[0002] Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis. According to the World Health Organization (WHO) report, the global number of new tuberculosis cases reached 8.2 million in 2023, and 1.25 million people died of tuberculosis, including 400,000 new cases of multidrug-resistant / rifampicin-resistant tuberculosis. However, the global treatment success rate for multidrug-resistant / rifampicin-resistant tuberculosis is still only 68%. Therefore, it is urgent to develop new anti-tuberculosis drugs to solve the problems of tuberculosis treatment and drug resistance.

[0003] The difficulty in curing tuberculosis and the severity of drug resistance are the bottlenecks of tuberculosis prevention and control. The main reasons are: (1) the existing anti-tuberculosis standard short-course treatment regimen has a long treatment time (at least 6 months) and a large number of drug combination chemotherapy, resulting in poor patient compliance and insufficient treatment effect; (2) the relapse rate of initial treatment sensitive tuberculosis is as high as 3%-9%; (3) the cure success rate of multidrug-resistant tuberculosis is insufficient. However, the current first-line anti-tuberculosis drugs were developed more than 40 years ago, and the development of new effective anti-mycobacterium tuberculosis drugs is slow. This leads to serious challenges in tuberculosis treatment. Therefore, the development of effective new tuberculosis drugs / treatment regimens is a research hotspot in current tuberculosis prevention and control.

[0004] Cryptotanshinone is a diterpene quinone compound extracted from the roots of Salvia miltiorrhiza. There are more than 80 structurally elucidated components in Salvia miltiorrhiza, of which more than half are diterpenes. Most of these compounds are isolated from the roots of Salvia miltiorrhiza. The two major components in Salvia miltiorrhiza are lipophilic diterpenes and water-soluble polyphenolic acids, both of which have pharmacological activities. Previous studies have found that cryptotanshinone has anti-tumor, anti-inflammatory, and antibacterial activities. However, this study found that cryptotanshinone has a significant direct killing effect on Mycobacterium tuberculosis. The minimum inhibitory concentration (MIC) of cryptotanshinone in vitro has no cytotoxic effect on human monocytic leukemia cell THP-1 differentiated macrophages, and cryptotanshinone can significantly inhibit the growth of intracellular Mycobacterium tuberculosis in THP-1 macrophages. Therefore, this compound has good value for the development of anti-tuberculosis drugs.

[0005] Cryptotanshinone has a molecular formula of C 19 H 20 O3 and a molecular weight of 296.36. Its chemical substance accession number (CAS number) is 35825-57-1, and its structural formula is as follows:

[0006] . SUMMARY

[0007] The technical problem solved by the present application is to provide an application of cryptotanshinone in preparing an anti-mycobacterium tuberculosis drug. 37 Rv.

[0008] The technical problem solved by the present application is achieved by the following technical solutions.

[0009] One purpose of the present application is to provide an application of cryptotanshinone in preparing an anti-mycobacterium tuberculosis drug.

[0010] Preferably, in the above technical solution, the mycobacterium tuberculosis is mycobacterium tuberculosis H 37 Rv.

[0011] Another purpose of the present application is to provide an application of cryptotanshinone in preparing an anti-tuberculosis drug.

[0012] Preferably, in the above technical solution, the tuberculosis is caused by mycobacterium tuberculosis H 37 Rv.

[0013] Still another purpose of the present application is to provide an anti-tuberculosis drug, which contains cryptotanshinone as an effective component.

[0014] Preferably, in the above technical solution, the drug further contains a pharmaceutically acceptable excipient.

[0015] The above technical solution of the present application has the following beneficial effects.

[0016] (1) The present application first discovers that cryptotanshinone has obvious anti-mycobacterium tuberculosis effect.

[0017] (2) The present application first proposes an application of cryptotanshinone in preparing an anti-tuberculosis drug. BRIEF DESCRIPTION OF DRAWINGS

[0018] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the present application and, together with the description, serve to explain the principles of the present application.

[0019] Figure 1 Direct killing effect of different concentrations of cryptotanshinone on mycobacterium tuberculosis in Example 1;

[0020] Figure 2 Bacteriostatic curve of different concentrations of cryptotanshinone on mycobacterium tuberculosis in Example 1;

[0021] Figure 3 Antimicrobial effect of different concentrations of Cryptotanshinone on Mycobacterium tuberculosis in Example 1;

[0022] Figure 4 Effect of different concentrations of Cryptotanshinone on macrophage cytotoxicity in Example 2;

[0023] Figure 5 Effect of Cryptotanshinone on intracellular Mycobacterium tuberculosis survival rate in macrophages in Example 3. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present application will now be described in detail with reference to the accompanying drawings. It should be noted that the relative arrangement of the components and steps, numerical expressions, and numerical values set forth in these embodiments are not limiting to the scope of the present application unless otherwise specifically stated.

[0025] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials and reagents used are commercially available unless otherwise specified, and the equipment used in the experiments is well known to those skilled in the art unless otherwise specified.

[0026] The OD 600 measurement method, AlamarBlue cell viability detection reagent, and colony-forming unit (CFU) were used to determine the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of Cryptotanshinone on Mycobacterium tuberculosis standard strain H 37 Rv, and the results showed that Cryptotanshinone had a direct killing effect on Mycobacterium tuberculosis H 37 Rv.

[0027] At the same time, CCK8 was used to detect the cytotoxicity of Cryptotanshinone-treated Mycobacterium tuberculosis-infected THP-1 macrophages and non-infected cells, and the results showed that Cryptotanshinone had no toxicity to THP-1 macrophages at the minimum inhibitory concentration.

[0028] Further, in the Mycobacterium tuberculosis-infected THP-1 macrophage model, colony-forming unit (CFU) was used to detect the number of Mycobacterium tuberculosis in macrophages under Cryptotanshinone treatment and non-treatment conditions, and the results showed that Cryptotanshinone could effectively kill Mycobacterium tuberculosis in macrophages.

[0029] Example 1

[0030] In vitro bactericidal activity determination of Cryptotanshinone

[0031] 1. Preparation and dilution of Cryptotanshinone

[0032] Cryptotanshinone (purchased from Selleck Company, item number: E2354) was dissolved in DMSO (purchased from Sigma Company, item number: D2650) to prepare a solution with a final concentration of 20 mM under sterile conditions. The dissolved stock solution was stored at -80°C for later use.

[0033] The prepared cryptotanshinone stock solution was diluted to 160 mM using 7H9 medium (product of BD Company, item number: 271310) containing OADC (product of BD Company, item number: 212351) and then diluted 1:1 to obtain a total of 8 concentrations, with the final concentration of cryptotanshinone being 80 mM, 40 mM, 20 mM, 10 mM, 5 mM, 2.5 mM, 1.25 mM, and 0.625 mM, respectively.

[0034] 2. Culture and cryptotanshinone treatment of Mycobacterium tuberculosis (H 37 Rv)

[0035] Mycobacterium tuberculosis H 37 Rv was inoculated into H9-OADC medium and cultured to the logarithmic growth phase (OD 600 = 0.6-0.8). After the bacteria were dispersed by a bacterial disperser (purchased from Tbkang Biotech (Guangdong) Co., Ltd.), the McFarland turbidity was measured, and the turbidity of the bacteria was diluted to OD 600 = 0.3 using 7H9-OADC medium and diluted 20 times. The diluted bacterial solution was added to 96-well cell culture plates containing different concentrations of cryptotanshinone at 100 μL / well, with the final volume being 200 μL per well. The plates were placed in a 37°C incubator for 7 days.

[0036] 3. Detection of the killing effect of cryptotanshinone on Mycobacterium tuberculosis

[0037] On the 7th day of culture, 70 μl of color developing agent (containing 20 μl of Alamar Blue + 50 μl of 5% Tween 80) was added to each culture well. After 24 hours of incubation at 37°C, the color change of the solution in the culture plate was observed.

[0038] The results showed that pink indicated bacterial growth, and blue indicated no bacterial growth. The drug concentration corresponding to the last blue well was read as the minimum inhibitory concentration (MIC), as shown in Table 1. Figure 1

[0039] At the same time, 100 ul of the bacterial solution treated with different concentrations of cryptotanshinone was gradient diluted and plated, and the number of colonies was counted to calculate the inhibitory activity of the drug on bacterial growth. The formula was as follows: inhibition rate (%) = 1 - (experimental group CFU / control group CFU) x 100%.

[0040] ​The inhibitory rate of different concentrations of cryptotanshinone on the growth of Mycobacterium tuberculosis was determined after treatment with different concentrations of cryptotanshinone, and a dose-inhibition rate curve was drawn as shown in Figure 2 Each drug concentration was repeated twice, and the results were expressed as mean ± standard deviation.

[0041] The experimental results showed that in 7H10-OADC medium, the inhibitory rate of cryptotanshinone at a concentration of 10 μM and above on Mycobacterium tuberculosis reached 61% to 100%, and had a significant direct killing effect on Mycobacterium tuberculosis.

[0042] Table 1 Inhibitory rate of different drug concentrations on Mycobacterium tuberculosis

[0043]

[0044] Example 2

[0045] Effect of cryptotanshinone on the survival rate of macrophages

[0046] 1. Differentiation of THP-1 macrophages: THP-1 cells in suspension were cultured in RPMI-1640 containing 10% FBS and placed in a cell incubator at 37°C, 5% CO2, and 20 ng / ml of PMA was added to a final concentration of 1×10 5 cells / well in a 96-well cell culture plate, and cultured for 48 h to differentiate into macrophages;

[0047] 2. Determination of cell survival rate: the above THP-1 macrophages were infected with Mycobacterium tuberculosis H 37 Rv at a multiplicity of infection of 5 (MOI=5), and after 6 h of infection, they were washed with PBS three times to remove extracellular unphagocytosed Mycobacterium tuberculosis. Then, different concentrations of cryptotanshinone were added to the uninfected and infected macrophages, respectively, to a final drug concentration of 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, each concentration was repeated 4 times; the same volume of DMSO as the drug was used as a control, and the THP-1 macrophages were cultured for another 48 h, then 10 μl of CCK8 solution (purchased from TargetMol Company, item number: C0005-1 mL) was added to the cell culture well and placed in a 37°C CO2 incubator for 2-4 h, and the OD450nm absorbance value was determined by using a microplate reader;

[0048] The cell survival rate was calculated according to the following formula: cell survival rate%=[A / A0]×100, where A0 is the absorbance value of the DMSO control, and A is the absorbance value of the different concentrations of drug treatment.

[0049] The experimental results show that there is no statistical difference in the cytotoxic effect of different concentrations of cryptotanshinone on macrophages in Mycobacterium tuberculosis infected and uninfected cells Figure 4 ).

[0050] Example 3

[0051] 1. Differentiation of THP-1 macrophages: THP-1 cells in suspension were cultured in RPMI-1640 containing 10% FBS, and the cells were inoculated into a 24-well cell culture plate at a concentration of 3x10 5 cells / well, and 20 ng / ml of PMA was added, and the cells were incubated in a cell incubator at 37°C and 5% CO2for 48 h to differentiate into macrophages, and then the cells were washed twice with PBS;

[0052] 2. Intracellular bactericidal activity assay: the THP-1 macrophages washed with PBS were infected with Mycobacterium tuberculosis H 37 Rv at a multiplicity of infection (MOI) of 5, and after 6 h of infection, the cells were washed three times with PBS to remove extracellular Mycobacterium tuberculosis that was not phagocytosed, and then fresh complete medium was added to the cell culture plate, and cryptotanshinone was added to a final concentration of 10 μM, with 3 replicates for each concentration, and the same volume of DMSO as the drug was added as a control group, and the cells were incubated in a cell incubator for 48 h and 72 h, and then 500 μl of 0.05% triton X-100 was added to each well to lyse the cells, and the cells were repeatedly blown. The cell lysate was diluted by 10 times in gradient, and 4 concentrations were plated on 7H10 plates, and the plates were incubated in a constant temperature incubator at 37°C for 3 weeks, and the colonies were counted.

[0053] The results are shown in Table 1. Figure 3 After 48 h of treatment with 10 μM cryptotanshinone, the average number of Mycobacterium tuberculosis colonies in the macrophages decreased from 48.67x10 4 to 4.93x10 4 , indicating that the intracellular bactericidal rate of cryptotanshinone reached 89.86%; and after 72 h of treatment with 10 μM cryptotanshinone, the average number of Mycobacterium tuberculosis colonies in the macrophages decreased from 73.33x10 4 to 13.33x10 4 , indicating that the intracellular bactericidal rate of cryptotanshinone reached 81.82%; and cryptotanshinone had no obvious cytotoxicity at this concentration. Therefore, cryptotanshinone can be used to develop highly effective and low-toxicity anti-tuberculosis drugs.

[0054] Although the present application has been disclosed in the above embodiments, it is not intended to limit the present application, and any person skilled in the art can make various modifications and changes without departing from the spirit and scope of the present application, and the scope of protection of the present application is defined by the claims and their equivalents.

Claims

1. Application of cryptotanshinone in the preparation of anti-tuberculosis drugs.

2. The application according to claim 1, characterized in that, The tuberculosis mycobacterium is Mycobacterium tuberculosis H. 37 Rv.

3. Application of cryptotanshinone in the preparation of anti-tuberculosis drugs.

4. The application according to claim 3, characterized in that, The tuberculosis is caused by Mycobacterium tuberculosis H. 37 Tuberculosis caused by Rv.

5. An anti-tuberculosis drug, characterized in that, The drug contains cryptotanshinone as an active ingredient.

6. The anti-tuberculosis drug according to claim 5, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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