Application of valerian in preparation of anti-mycobacterium tuberculosis drugs

Abstract

The invention discloses application of valerian in preparation of a medicine for resisting mycobacterium tuberculosis, and relates to the technical field of bioengineering. The molecular formula of the valerianin disclosed by the invention is C22H30O8. The mycobacterium tuberculosis is a mycobacterium tuberculosis H37Rv strain. The invention discloses valerian as a natural-source compound for the first time, the valerian has the dual advantages of high efficiency and low toxicity, the valerian has an obvious effect of resisting mycobacterium tuberculosis, a brand new candidate drug scheme with development potential is provided for solving the difficulty of tuberculosis treatment, and the valerian has great scientific value and application prospect.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to the application of valerin in the preparation of anti-tuberculosis drugs. Background Technology

[0002] Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis. However, the global treatment success rate for multidrug-resistant / rifampicin-resistant TB remains only 68%. Therefore, there is an urgent need to develop new anti-TB drugs to address the challenges of TB treatment and drug resistance.

[0003] The difficulty in curing tuberculosis and its severe drug resistance are bottlenecks in tuberculosis control. The main reasons are: (1) the existing standard short-course anti-tuberculosis treatment regimens have a long treatment time (at least 6 months), and the toxic side effects of combined chemotherapy with multiple drugs are significant, leading to poor patient compliance and insufficient treatment efficacy; (2) the relapse rate of newly diagnosed sensitive tuberculosis is as high as 3%-9%; and (3) the cure rate of multidrug-resistant tuberculosis is insufficient. However, the current first-line anti-tuberculosis drugs were all developed more than 40 years ago, and the development of new and effective anti-tuberculosis drugs has been slow. This has led to severe challenges in tuberculosis treatment. Therefore, the development of effective new drugs / regimens for tuberculosis treatment is a current research hotspot in tuberculosis control.

[0004] Valepotriate, also known as valeric acid ester or valeric triester, is a natural compound isolated from *Valeriana jatamansi*, with a chemical structure of iridoid ether. This study found that valeric acid exhibits significant direct cytotoxic activity against *Mycobacterium tuberculosis*. Its minimum inhibitory concentration (MIC) in vitro showed no cytotoxic effect on macrophages differentiated from human monocytic leukemia cells (THP-1), and valeric acid significantly inhibited the growth of intracellular *Mycobacterium tuberculosis* within THP-1 macrophages. Therefore, this compound suggests significant potential for the development of anti-tuberculosis drugs.

[0005] Valerianin, with the molecular formula C 22 H 30 O8, with a molecular weight of 422.47 and a CAS (Chemical Registry Number) of 18296-44-1, has the following structural formula:

[0006] . Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide an application of valerin in the preparation of anti-tuberculosis drugs. Studies have found that valerin can kill Mycobacterium tuberculosis in vitro and in vivo, and has good value for the development of anti-tuberculosis drugs. The Mycobacterium tuberculosis in this study is specifically Mycobacterium tuberculosis H. 37 Rv.

[0008] The technical problem to be solved by the present invention is achieved through the following technical solution:

[0009] One object of this application is to provide the use of valerin in the preparation of anti-tuberculosis drugs.

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

[0011] Another object of this application is to provide the use of valerin in the preparation of anti-tuberculosis drugs.

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

[0013] Another object of this application is to provide an anti-tuberculosis drug containing valerin as an active ingredient.

[0014] Preferably, in the above technical solution, the drug further includes pharmaceutically acceptable excipients.

[0015] The above-described technical solution of the present invention has the following beneficial effects:

[0016] (1) This invention is the first to discover that valerin has a significant anti-tuberculosis effect;

[0017] (2) This invention first proposed the application of valerin in the preparation of anti-tuberculosis drugs. Attached Figure Description

[0018] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments of the invention and, together with their description, serve to explain the principles of the invention.

[0019] Figure 1 This illustrates the direct killing effect of different concentrations of valerin on Mycobacterium tuberculosis in Example 1.

[0020] Figure 2 The antibacterial curves of different concentrations of valerin against Mycobacterium tuberculosis in Example 1 are shown.

[0021] Figure 3 Example 1 illustrates the antibacterial effect of different concentrations of valerin on Mycobacterium tuberculosis;

[0022] Figure 4 The effect of different concentrations of valerin on macrophage cytotoxicity in Example 2;

[0023] Figure 5This is the effect of valerin on the survival rate of intracellular Mycobacterium tuberculosis in macrophages in Example 3. Detailed Implementation

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

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

[0026] This invention uses OD 600 Assay method: AlamarBlue cell viability assay and colony count (CFU) were used to determine the effect of valerin on Mycobacterium tuberculosis standard strain H. 37 The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of valerin were determined, showing that valerin has the ability to directly kill Mycobacterium tuberculosis H. 37 The function of Rv;

[0027] Meanwhile, the cytotoxicity of valerin in THP-1 macrophages infected with Mycobacterium tuberculosis and uninfected cells was detected using CCK8 assay. The results showed that valerin had no cytotoxicity to THP-1 macrophages at the minimum inhibitory concentration.

[0028] Further, in a THP-1 macrophage model infected with Mycobacterium tuberculosis, the number of Mycobacterium tuberculosis colonies in macrophages under valerin treatment and no treatment was detected by colony clonal count (CFU). The results showed that valerin could effectively kill Mycobacterium tuberculosis in macrophages.

[0029] Example 1

[0030] In vitro bactericidal activity assay of valerin:

[0031] 1. Preparation and dilution of valerin

[0032] Under aseptic conditions, valerin (purchased from Selleck, catalog number: E2354) was dissolved in DMSO (purchased from Sigma, catalog number: D2650) to prepare a solution with a final concentration of 20 mM. The mother solution was stored at -80°C for later use.

[0033] Using 7H9 medium (BD Biosciences, Inc., Catalog No. 271310) containing enrichment broth OADC (BD Biosciences, Inc., Catalog No. 212351), the prepared valerine stock solution was diluted to 160 μM. Then, it was diluted 1:1 for a total of 8 concentrations, resulting in final concentrations of valerine of 80 μM, 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, and 0.625 μM.

[0034] 2. Mycobacterium tuberculosis (H) 37 Rv) cultivation and valerin treatment

[0035] Mycobacterium tuberculosis H 37 Rv was inoculated into H9-OADC medium and cultured until the logarithmic growth phase (OD200). 600 = 0.6~0.8), after dispersing the bacteria using a bacterial dispersion instrument (purchased from Tibicon Biotechnology (Guangdong) Co., Ltd.), the McFarland turbidity was measured, and the turbidity was calculated according to the instrument. The bacterial turbidity was then adjusted to OD using 7H9-OADC medium. 600 =0.3 and diluted 20 times. Add 100 μL / well of the diluted bacterial solution to 96-well cell culture plates containing different concentrations of valerin, making the final volume of each well 200 μL. Incubate at 37°C for 7 days.

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

[0037] On the 7th day of incubation, 70 μl of colorimetric reagent (containing 20 μl AlamarBlue + 50 μl 5% Tween 80) was added to each culture well; after incubation at 37℃ for 24 days, 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 the minimum inhibitory concentration (MIC). Figure 1 As shown.

[0039] Simultaneously, 100 μL of bacterial suspensions treated with different concentrations of valerin were serially diluted and plated, the number of colonies was counted, and the inhibitory activity of the drug on bacterial growth was calculated. The formula is as follows: Inhibition rate (%) = [1 − (Experimental group CFU / Control group CFU)] × 100%

[0040] The inhibition rate of different concentrations of valerine on the growth of Mycobacterium tuberculosis was determined after treatment with Mycobacterium tuberculosis, and the dose-inhibition rate curves were plotted as follows: Figure 2 As shown, each drug concentration was repeated twice.

[0041] Table 1. Inhibition rate of different drug concentrations on Mycobacterium tuberculosis

[0042]

[0043] Experimental results showed that in 7H10-OADC medium, valerin at concentrations of 10 μM and above inhibited Mycobacterium tuberculosis by 88% to 100%, demonstrating a significant direct killing effect on Mycobacterium tuberculosis.

[0044] Example 2

[0045] Effects of valerin on macrophage survival:

[0046] 1. Differentiation of THP-1 macrophages: Suspended THP-1 cells were cultured in RPMI-1640 containing 10% FBS and incubated in a cell culture incubator at 37°C and 5% CO2. PMA was added to a final concentration of 20 ng / ml at a concentration of 1×10⁻⁶. 5 The cells were seeded into 96-well cell culture plates and cultured for 48 hours to differentiate them into macrophages.

[0047] 2. Cell viability determination: The above-mentioned THP-1 macrophages were infected with Mycobacterium tuberculosis H at a multiplicity of infection (MOI) of 5. 37 Rv, after 6 hours of infection, was washed three times with PBS to remove extracellular unphagocytosed Mycobacterium tuberculosis. Then, different concentrations of valerin were added to uninfected and infected macrophages to achieve final drug concentrations of 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, and 1.25 μM, with each concentration repeated four times. Using the same volume of DMSO as a control, THP-1 macrophages were cultured for another 48 hours. Then, 10 μl of CCK8 solution (purchased from TargetMol, catalog number: C0005-1mL) was added to the cell culture wells, and the cells were incubated at 37°C in a CO2 incubator for another 2-4 hours. The absorbance at OD450nm was measured using a microplate reader.

[0048] Cell viability was calculated using the following formula: Cell viability % = [A / A0] × 100, where A0 is the absorbance value of the DMSO control and A is the absorbance value of different drug concentrations.

[0049] Experimental results showed that there was no statistically significant difference in the cytotoxic effects of different concentrations of valerin on macrophages between Mycobacterium tuberculosis-infected and uninfected cells. Figure 4 It has good biosafety.

[0050] Example 3

[0051] 1. Differentiation of THP-1 macrophages: Suspended THP-1 cells were cultured in RPMI-1640 containing 10% FBS at a rate of 6 × 10⁻⁶ cells / year. 5 Cells were seeded into 12-well cell culture plates, and PMA was added to a final concentration of 20 ng / ml. The plates were then cultured in a cell culture incubator at 37°C and 5% CO2 for 48 hours to differentiate them into macrophages. The cells were washed twice with PBS.

[0052] 2. Intracellular bactericidal activity assay: THP-1 macrophages washed with PBS were infected with Mycobacterium tuberculosis H at a multiplicity of infection (MOI) of 5. 37 Six hours after infection with Rv, the cells were washed three times with PBS to remove extracellular, unphagocytosed Mycobacterium tuberculosis. Fresh complete culture medium was then added to cell culture plates, with valerin added to final concentrations of 5 μM and 10 μM, repeated three times for each concentration. An equal volume of DMSO was added as a control. After 48 and 72 hours of incubation, 500 μl of 0.05% Triton X-100 solution was added to each well to lyse the cells, and the cells were repeatedly pipetted. The cell lysates were serially diluted 10-fold to four different concentrations and plated onto 7H10 plates. The plates were incubated at 37°C, and colonies were counted after three weeks.

[0053] The results are as follows Figure 5 As shown, after treating THP-1 macrophages with 5 μM and 10 μM valerin for 48 h, the average number of Mycobacterium tuberculosis colonies in the macrophage cells increased from 48.67 × 10⁻⁶ to 48.67 × 10⁻⁶. 4 The number of each decreased to 24.5 × 10. 4 The sum is 18 × 10 4 The results showed that the bactericidal rate within macrophages reached 49.66% and 63.02%, respectively; while after treatment of THP-1 macrophages with 5 μM and 10 μM valerine for 72 h, the average number of Mycobacterium tuberculosis colonies in macrophage cells decreased from 73.33 × 10⁻⁶. 4 The number of each decreased to 22.67×10. 4 The sum is 18.67 × 10 4 The results showed that valerin achieved a bactericidal rate of 69.08% and 74.54% in macrophages; and at this concentration, valerin did not have significant cytotoxicity. Therefore, valerin can be used to develop highly effective and low-toxicity anti-tuberculosis drugs.

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

Claims

1. Application of valerin 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 valerin 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 valerin as its 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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