Application of rifampicin-cinnamon essential oil emulsion

Abstract

The invention relates to an application of a rifampicin-cinnamon essential oil emulsion in preparing a medicine for inhibiting rifampicin drug-resistant mycobacterium tuberculosis, which is characterized in that each milliliter of the emulsion contains the following components: 0.4 to 0.7 mg of cinnamon essential oil, 3 to 5 mg of rifampicin, 300 to 500 microliters of Tween 80, 300 to 500 microliters of medium chain triglyceride and the balance of water. Through process optimization, rifampicin and cinnamon essential oil form a new structure and stable emulsion, rifampicin plays a role again, rifampicin drug-resistant bacteria are obviously inhibited, the emulsion prepared from rifampicin not only can reduce the volatility of cinnamon essential oil, but also can increase the inhibition effect of rifampicin on rifampicin drug-resistant bacteria, so that the rifampicin drug-resistant bacteria can be effectively inhibited. And the beneficial active components are basically not influenced and damaged. The invention widens the clinical value of reutilization of rifampicin in rifampicin drug-resistant bacteria.

Description

Technical Field

[0001] The present invention belongs to the field of antibacterial drugs for Mycobacterium tuberculosis, and particularly relates to the use of a rifampicin-cinnamon essential oil emulsion. Background Art

[0002] Rifampicin-resistant pulmonary tuberculosis (RR-PTB) refers to Mycobacterium tuberculosis that has developed resistance to rifampicin, covering all tuberculosis cases resistant to this drug. Studies have shown that the number of deaths caused by RR-PTB accounts for a relatively large proportion. Among newly diagnosed tuberculosis patients, the proportion of RR-PTB is 3-4%, while in patients who have received anti-tuberculosis treatment, this proportion is as high as 18-21%. RR-PTB has strong infectivity and a low cure rate, seriously threatening public health. In addition, due to reasons such as a long course of disease and high treatment costs, the treatment compliance of RR-PTB patients is generally low. Previous studies have shown that the treatment compliance of tuberculosis patients is an important factor affecting the treatment effect. Currently, there are many studies on the treatment compliance of ordinary pulmonary tuberculosis, but relatively few studies on the influencing factors of RR-PTB compliance.

[0003] With the increasing problem of antibiotic resistance, it is usually necessary to use several drugs in combination to prevent the emergence of resistance. Second, antibiotics not only kill pathogenic bacteria but also destroy beneficial bacteria in the intestine, such as probiotics and other microorganisms. This may lead to intestinal flora imbalance (intestinal flora disorder), which in turn causes digestive problems such as diarrhea and constipation, and even increases the risk of developing intestinal infections or other diseases. Moreover, certain antibiotics may have toxic effects on the liver or kidneys, especially at high doses or with long-term use. In the existing tuberculosis prevention and control system, once Mycobacterium tuberculosis becomes resistant to rifampicin, the clinical treatment will replace the drug. Many first-line drugs with less harm to the human body will have to be replaced by second-line drugs with greater harm to the human body due to the drug resistance of Mycobacterium tuberculosis to the product.

[0004] Rifampicin, with the chemical name of 3-[[(4-methyl-1-piperazinyl)imino]methyl]rifamycin, has a strong antibacterial effect on Mycobacterium tuberculosis and is one of the main anti-tuberculosis drugs. Rifampicin achieves bactericidal effects by binding to the bacterial RNA polymerase subunit to inhibit transcription initiation. The drug resistance of rifampicin has been confirmed to be related to mutations in the core region of 81 bases in the rpoB RRDR region, the coding gene of the β subunit of RNA polymerase. Rifampicin resistance means that after a patient takes rifampicin, the drug components and pharmacodynamics in the body change, resulting in a weakened or even ineffective treatment effect of the drug on tuberculosis. The cure rate of this drug resistance is generally low. It is necessary to replace other drugs or use combination drugs, making rifampicin lose its advantage in the treatment of tuberculosis.

[0005] Rifampicin is a poorly soluble drug. There are reports in the prior art of preparing rifampicin into a microemulsion. Using water, liquid paraffin as the oil phase, Tween-80 as the surfactant, and Span-80 as the co-surfactant, mixing them in a certain proportion to prepare a relatively stable microemulsion with an average particle size of 30 nm. The prepared microemulsion matrix has a solubilizing effect on rifampicin and good stability. However, it cannot enhance the inhibitory ability against rifampicin-resistant bacteria. Summary of the Invention

[0006] In order to make rifampicin reusable against rifampicin-resistant bacteria, the present invention provides the use of a rifampicin-cinnamon essential oil emulsion in inhibiting Mycobacterium tuberculosis. The inventors unexpectedly found that in the rifampicin-cinnamon essential oil emulsion, when the cinnamon essential oil is controlled at a specific ratio, the obtained emulsion has significantly improved antibacterial activity against rifampicin-resistant bacteria, and for the resistant bacteria that rifampicin has been powerless against, a satisfactory antibacterial effect is re-produced. The present invention achieves the above object through the following technical means:

[0007] The use of a rifampicin-cinnamon essential oil emulsion in the preparation of a drug for inhibiting rifampicin-resistant Mycobacterium tuberculosis, wherein each milliliter of the emulsion contains the following components: 0.4 - 0.7 mg of cinnamon essential oil, 3 - 5 mg of rifampicin, 300 - 500 μL of Tween 80, 300 - 500 μL of medium-chain triglycerides, and the balance is water.

[0008] Currently, due to the extensive use of rifampicin, many Mycobacterium tuberculosis have developed resistance to rifampicin, and this kind of bacteria is called rifampicin-resistant Mycobacterium tuberculosis. Since its introduction, rifampicin has always been a first-line drug in the tuberculosis treatment regimen, which can shorten the tuberculosis treatment course. Drug resistance means an extended treatment course or even the loss of treatment effect, resulting in poor prognosis for patients. If other second-line drugs are used instead, they may be more expensive or have greater side effects. The treatment cycle of rifampicin-resistant tuberculosis (RR-TB) may be extended from 6 months to 18 - 24 months. This greatly increases the economic burden and pain of patients. In addition, rifampicin is the core drug for the treatment of tuberculosis (TB), and drug resistance will lead to the failure of the treatment regimen, and patients will carry and transmit resistant strains for a long time. Solving drug resistance can effectively cut off the transmission chain and prevent community and regional outbreaks of the epidemic. Rifampicin resistance may develop into more severe extensively drug-resistant tuberculosis (XDR-TB), resulting in "no drug available for treatment". Controlling rifampicin resistance is the key defense line to avoid the emergence of super-resistant bacteria. Rifampicin is the "cornerstone drug" of the tuberculosis combination therapy, and its failure will force the reliance on more expensive and more side-effect second-line drugs (such as bedaquiline). Solving drug resistance can extend the service life of existing drugs and gain time for the research and development of new drugs. How to improve the antibacterial activity of rifampicin against rifampicin-resistant bacteria has become an urgent problem to be solved in tuberculosis at present and has important significance.

[0009] The inventor originally intended to formulate rifampicin, a poorly soluble drug, into an emulsion to improve the bioavailability of rifampicin. However, the inventor unexpectedly found that compounding cinnamon essential oil and rifampicin in a certain ratio to form an emulsion could significantly improve the antibacterial activity of rifampicin against rifampicin-resistant Mycobacterium tuberculosis. This discovery has greatly alleviated the phenomenon of original rifampicin resistance. Moreover, cinnamon essential oil is a mature drug system, and its safety can be guaranteed. After treatment with the rifampicin-cinnamon essential oil emulsion of the present invention, the growth of rifampicin-resistant bacteria has been significantly inhibited, while the active ingredients in rifampicin and cinnamon essential oil are basically not affected.

[0010] Further, each milliliter of the emulsion contains the following components: 0.5 - 0.6 mg of cinnamon essential oil, 3 - 3.5 mg of rifampicin, 400 - 500 μL of Tween 80, 400 - 500 μL of medium-chain triglycerides, and the balance is water.

[0011] Further, the emulsion is prepared by a preparation method including the following steps: dissolving rifampicin powder in cinnamon essential oil, adding Tween 80 and medium-chain triglycerides thereto, and making up the volume with ultrapure water, and performing ultrasonic treatment under ice bath conditions to obtain the rifampicin-cinnamon essential oil emulsion.

[0012] Further, the ultrasonic treatment is carried out at a power of 300 - 500 W and a frequency of 60 - 180 kHz for 5 - 10 min. The ultrasonic process is carried out under ice bath conditions to avoid the instability of the emulsion caused by the increase in the system temperature.

[0013] Further, the preparation method of the emulsion includes the following steps:

[0014] (S1) Absorb cinnamon essential oil into a container;

[0015] (S2) Weigh rifampicin powder and add it to the cinnamon essential oil, mix evenly to form an oil phase;

[0016] (S3) Respectively absorb Tween 80 and medium-chain triglycerides and add them to the oil phase, make up the volume with ultrapure water, and mix evenly;

[0017] (S4) Under ice bath conditions, ultrasonically mix the mixture solution evenly to obtain the rifampicin-cinnamon essential oil emulsion.

[0018] In a preferred technical solution of the present invention, the preparation method of the emulsion includes the following steps:

[0019] (S1) Absorb 0.5 - 0.6 mg of cinnamon essential oil into a 10 mL centrifuge tube;

[0020] (S2) Weigh 3 - 3.5 mg of rifampicin powder and add it to the cinnamon essential oil, mix evenly to form an oil phase;

[0021] (S3) Respectively aspirate 400 - 500 μL of Tween 80 and medium-chain triglyceride and add them to the oil phase, make up the volume to 10 mL with ultrapure water, and mix evenly;

[0022] (S4) Under ice bath conditions, ultrasonicate the evenly mixed solution at 300 - 350 W and a frequency of 60 - 180 kHz for 5 - 10 min to obtain rifampicin-cinnamon essential oil emulsion.

[0023] The present invention also provides a rifampicin-cinnamon essential oil emulsion, and each milliliter of the emulsion contains the following components: 0.4 - 0.7 mg of cinnamon essential oil, 3 - 5 mg of rifampicin, 300 - 500 μL of Tween 80, 300 - 500 μL of medium-chain triglyceride, and the balance is water.

[0024] Preferably, the rifampicin-cinnamon essential oil emulsion, each milliliter of the emulsion contains the following components: 0.5 - 0.6 mg of cinnamon essential oil, 3 - 3.5 mg of rifampicin, 400 - 500 μL of Tween 80, 400 - 500 μL of medium-chain triglyceride, and the balance is water.

[0025] Through process optimization, the present invention inoculates rifampicin-resistant bacteria into the emulsion, which can significantly inhibit rifampicin-resistant bacteria. Preparing it into an emulsion can not only reduce the volatility of cinnamon essential oil, but also reduce the concentration of rifampicin to improve bioavailability, and basically has no influence or damage on its beneficial active ingredients. It broadens the utilization value of rifampicin and cinnamon essential oil. Brief Description of the Drawings

[0026] Figure 1 is the Fourier transform infrared spectrum of rifampicin (a), cinnamon essential oil (b), and rifampicin-cinnamon essential oil emulsion (c).

[0027] Figure 2 is the inhibitory activity of the pure rifampicin emulsion of Comparative Example 1 and the rifampicin-cinnamon essential oil emulsion of Example 1 against Mycobacterium tuberculosis 587 and Mycobacterium tuberculosis 632. Detailed Embodiments [[ID=??]]

[0028] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0029] The rifampicin-resistant bacteria 587 (M. tuberculosis 587) and rifampicin-resistant bacteria 632 (M. tuberculosis 632) are both preserved in the Tuberculosis Laboratory of the Beijing Center for Disease Control and Prevention.

[0030] It should be noted that there seems to be a mistake in the original text where "??" is used in the ID in line 26. It should probably be a correct ID number. Also, the English translation is based on the best understanding of the context, and any professional-specific terms might need further verification depending on the actual field knowledge.By the Xpert molecular probe method, rifampicin-resistant bacteria of two different mutation types, M. tuberculosis strain 587, were determined by molecular detection to have mutations at the 512-518, 518-523, and 523-529 sites of the rpoB gene; M. tuberculosis strain 632 had mutations at the 512-518, 518-523, and 529-533 sites of the rpoB gene. These related sites are clinical test sites for rifampicin resistance detection and are highly related sites for rifampicin resistance recognized by the World Health Organization (WHO).

[0031] Example 1

[0032] S1. Weigh 3 mg of rifampicin powder and add it to 0.5 mg of cinnamon essential oil, mix well to form an oil phase. Then, respectively pipette 500 μL of Tween 80 and medium-chain triglycerides into the oil phase, and make up the volume to 10 mL with ultrapure water, and mix well.

[0033] S2. Under ice bath conditions, ultrasonicate the well-mixed solution at 350 W for 7.5 min to obtain a rifampicin-cinnamon essential oil emulsion.

[0034] Figure 1 are the Fourier transform infrared spectra of rifampicin (a), cinnamon essential oil (b), and the rifampicin-cinnamon essential oil emulsion (c). The phenolic peaks of cinnamon essential oil and rifampicin shifted from 3061 cm -1 and 2969 cm -1 to 3322 cm -1 accompanied by a significant increase in intensity, which is direct evidence of hydrogen bond formation. In the rifampicin-cinnamon essential oil emulsion, the 1241 cm -1 peak of rifampicin and the 1121 cm -1 peak of cinnamon essential oil shifted together to 1125 cm -1 . This is not only proof of the binding of rifampicin to cinnamon essential oil but also a sign of the formation of a new structure through their interaction. At the same time, the significant enhancement of the OH stretching band at 3322 cm -1 can also be attributed to the synergistic effect of hydrogen bonding and electrostatic interaction, further consolidating the structural model of the stable binding of rifampicin and cinnamon essential oil in the emulsion. It can be seen that a chemical reaction occurred between rifampicin and cinnamon essential oil and hydrogen bonds were formed. The synergistic effect of hydrogen bonding and electrostatic interaction not only helps to form a new structure of rifampicin and cinnamon essential oil and maintain its stability but may also promote the release of active ingredients, thereby further improving the bioavailability.

[0035] Example 2

[0036] Other conditions are the same as in Example 1, with the difference that in step S1, the amount of cinnamon essential oil used is 0.6 mg.

[0037] Comparative Example 2

[0038] Other conditions were the same as in Example 1, except that in step S1, the cinnamon essential oil was replaced with an equal volume of peppermint essential oil.

[0039] Application Example

[0040] 1. Traditional culture of rifampicin-resistant bacteria: Using a pipette in a biosafety cabinet, aspirate 100 μL of the bacterial suspension of the rifampicin-resistant bacteria standard strain and drop it onto the neutral Lowenstein-Jensen medium. Gently rotate the Lowenstein-Jensen culture tube to evenly spread the bacterial liquid over the inclined surface of the medium. Tighten the lid of the culture tube with the dropped bacterial liquid and place it in an incubator at 37 °C for 14 days to obtain pure and fresh rifampicin-resistant bacterial colonies.

[0041] 2. Scrape the rifampicin-resistant bacteria on the Lowenstein-Jensen solid medium and dissolve them in a sterile grinding tube containing physiological saline, and shake and grind the bacteria. Aspirate the ground bacterial suspension prepared above, dilute it with sterile physiological saline, and adjust it with the bioMérieux electronic turbidimeter DENSIMAT to prepare a 1 McFarland turbidity, that is, a 1.0 mg / mL bacterial suspension.

[0042] 3. Dilute with 7H9 medium and inoculate the rifampicin-resistant bacteria at a final concentration of 1×10 3 CFU / mL.

[0043] 4. Respectively inoculate the activated rifampicin-resistant bacteria 587 and 632 into the rifampicin-cinnamon essential oil emulsion, pure rifampicin solution (3 mg rifampicin + 341.3 μL DMSO, finally made up to 10 mL with deionized water), and cinnamon essential oil (0.5 mg essential oil + 341.3 μL DMSO, finally made up to 10 mL with deionized water) prepared in Example 1 at different concentrations, and culture in an incubator at 37 °C for 14 days.

[0044] 5. Results: After culturing in an incubator at 37 °C for 14 days, the results are shown in Table 1. In the rifampicin-cinnamon essential oil emulsion of the present invention, the rifampicin-resistant bacteria were significantly inhibited, indicating that the rifampicin-cinnamon essential oil emulsion has a significant inhibitory effect on the growth of rifampicin-resistant bacteria. This significant enhancement of the inhibitory activity of cinnamon essential oil against rifampicin-resistant bacteria has not been reported before. Moreover, cinnamon essential oil has proven its safety and does not belong to a drug, so it can be added as an additive without the need for a strict drug approval process. This characteristic of cinnamon essential oil helps rifampicin regain its use in inhibiting Mycobacterium tuberculosis.

[0045] Figure 2 It shows the inhibitory activities of the pure rifampicin emulsion of Comparative Example 1 and the rifampicin-cinnamon essential oil emulsion of Example 1 against Mycobacterium tuberculosis 587 and Mycobacterium tuberculosis 632. The final inoculation concentrations of rifampicin-resistant bacteria 587 and 632 were 1×10 3 CFU / mL. Among themFigure 2 A is the antibacterial activity of pure rifampicin emulsion against Mycobacterium tuberculosis 587 (from top to bottom, the rifampicin concentrations in the pure rifampicin emulsion are 32 μg / mL, 16 μg / mL, and 8 μg / mL in sequence), Figure 2 B is the antibacterial activity of the rifampicin-cinnamon essential oil emulsion of Example 1 against Mycobacterium tuberculosis 587 (from top to bottom, the rifampicin concentrations in the rifampicin-cinnamon essential oil emulsion are 0.6 μg / mL, 0.3 μg / mL, and 0.15 μg / mL in sequence); Figure 2 C is the antibacterial activity of pure rifampicin emulsion against Mycobacterium tuberculosis 632 (from top to bottom, the rifampicin concentrations in the pure rifampicin emulsion are 32 μg / mL, 16 μg / mL, and 8 μg / mL in sequence), Figure 2 D is the antibacterial activity of the rifampicin-cinnamon essential oil emulsion of Example 1 against Mycobacterium tuberculosis 632 (from top to bottom, the rifampicin concentrations in the rifampicin-cinnamon essential oil emulsion are 1.2 μg / mL, 0.6 μg / mL, and 0.3 μg / mL in sequence). It can be seen that the rifampicin emulsion without adding cinnamon essential oil has lost the inhibitory ability against rifampicin-resistant bacteria 587 and 632 and still grows normally at the rifampicin concentration of 32 μg / mL; while the rifampicin-cinnamon essential oil emulsion of the present invention has a significantly improved antibacterial ability against rifampicin-resistant bacteria.

[0046] Table 1 MIC (μg / mL) of rifampicin-cinnamon essential oil emulsion against rifampicin-resistant bacteria

[0047]

[0048] The dose of rifampicin in the rifampicin-cinnamon essential oil emulsion for inhibiting rifampicin-resistant bacteria decreases from 128 μg / mL, which has no effect on the growth of resistant bacteria, to a large extent to 0.15 - 0.3 μg / mL, and effectively inhibits highly rifampicin-resistant bacteria. Moreover, cinnamon essential oil does not belong to drugs and has no safety risks brought by combined medication. By utilizing the cooperative effect of cinnamon essential oil and rifampicin and the characteristics of the emulsions after encapsulation, the clinical value of rifampicin for reuse in rifampicin-resistant bacteria is broadened.

Claims

1. Use of rifampicin-cinnamon essential oil emulsion in the preparation of a drug for inhibiting rifampicin-resistant Mycobacterium tuberculosis, characterized in that, Each milliliter of the emulsion contains the following ingredients: 0.4-0.7 mg of cinnamon essential oil, 3-5 mg of rifampicin, 300-500 μL of Tween 80, 300-500 μL of medium-chain triglycerides, and the balance is water.

2. The use according to claim 1, characterized in that, Each milliliter of the emulsion contains the following ingredients: 0.5-0.6 mg of cinnamon essential oil, 3-3.5 mg of rifampicin, 400-500 μL of Tween 80, 400-500 μL of medium-chain triglycerides, and the balance is water.

3. The use according to claim 1, wherein The rifampicin-cinnamon essential oil emulsion is prepared by a preparation method comprising the following steps: dissolving rifampicin powder in cinnamon essential oil, adding Tween 80 and medium-chain triglycerides thereto, fixing the volume with ultrapure water, and performing ultrasonic treatment under ice bath conditions to obtain the rifampicin-cinnamon essential oil emulsion.

4. The use according to claim 3, characterized in that, The ultrasonic treatment is carried out at a power of 300-500 W and a frequency of 60-180 kHz for 5-10 minutes.

5. The use according to claim 3, wherein The preparation method of the rifampicin-cinnamon essential oil emulsion comprises the following steps: (S1) collecting cinnamon essential oil into a container; (S2) Weighing rifampicin powder and adding it to cinnamon essential oil, mixing them evenly to prepare an oil phase; (S3) Tween 80 and medium-chain triglycerides were added to the oil phase, fixed to volume with ultrapure water, and mixed evenly; (S4) The mixture solution is ultrasonically mixed in an ice bath to obtain a rifampicin-cinnamon essential oil emulsion.

6. The use according to claim 3, characterized in that, The preparation method of the rifampicin-cinnamon essential oil emulsion comprises the following steps: (S1) Pipette 0.5-0.6 mg of cinnamon essential oil into a 10 mL centrifuge tube; (S2) Weighing 3-3.5 mg of rifampicin powder and adding it to cinnamon essential oil, mixing well to prepare an oil phase; (S3) 400-500 μL of Tween 80 and medium-chain triglycerides were added to the oil phase, the volume was adjusted to 10 mL with ultrapure water, and mixed well; (S4) Under ice bath conditions, the mixed solution is sonicated at 300-350 W and a frequency of 60-180 kHz for 5-10 min to obtain a rifampicin-cinnamon essential oil emulsion.

7. A rifampicin-cinnamon essential oil emulsion, characterized in that, Each milliliter of the emulsion contains the following ingredients: 0.4-0.7 mg of cinnamon essential oil, 3-5 mg of rifampicin, 300-500 μL of Tween 80, 300-500 μL of medium-chain triglycerides, and the balance is water.

8. A rifampicin-cinnamon essential oil emulsion, characterized in that, Each milliliter of the emulsion contains the following ingredients: 0.5-0.6 mg of cinnamon essential oil, 3-3.5 mg of rifampicin, 400-500 μL of Tween 80, 400-500 μL of medium-chain triglycerides, and the balance is water.

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