Application of curcumin-cinnamon essential oil nano-emulsion in preparation of preparation for inhibiting mycobacteria

Abstract

The invention relates to an application of curcumin-cinnamon essential oil nano-emulsion in preparation of a preparation for inhibiting mycobacteria. After the cinnamon essential oil and the curcumin are compounded according to the proportion, a subsequent emulsion preparation process is matched, and the stable emulsion with high inhibitory activity on mycobacteria can be obtained; the existence of a small amount of curcumin can significantly improve the mycobacterium inhibition activity of the emulsion. The nano-emulsion disclosed by the invention not only can reduce the volatility of the cinnamon essential oil, but also can reduce the instability of curcumin and improve the bioavailability, and basically has no influence and damage on beneficial active ingredients. The utilization values of the curcumin and the cinnamon essential oil are expanded.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to use of a curcumin-cinnamon essential oil nanoemulsion in preparing a mycobacterium inhibition preparation. Background Art

[0002] At present, the main method to inhibit mycobacteria is to use anti-tuberculosis drugs, such as isoniazid, rifampicin, ethambutol, etc. However, on the one hand, the problem of mycobacteria's resistance to antibiotics is becoming increasingly serious, including multidrug-resistant Mycobacterium tuberculosis and extremely drug-resistant Mycobacterium tuberculosis. Usually, several drugs need to be used in combination to prevent the development of resistance; second, antibiotics not only kill pathogenic bacteria, but also destroy beneficial flora in the intestine, such as probiotics and other microorganisms. This may lead to intestinal dysbiosis (intestinal flora disorder), which in turn causes digestive problems such as diarrhea and constipation, and even increases the risk of intestinal infections or other diseases. In addition, some antibiotics may have toxic effects on the liver or kidneys, especially when used in high doses or for a long time. The existing tuberculosis prevention and control system lacks effective food-grade anti-mycobacterial products that inhibit mycobacteria with little harm to the human body.

[0003] Curcumin is a polyphenol active ingredient with low molecular weight extracted and separated from the rhizome of Curcuma longa L., a plant of the ginger family. It is a very rare diketone compound in the plant kingdom. With the deepening of research on curcumin, it has been found that it has a wide range of pharmacological activities such as anti-inflammatory, antioxidant, anti-cancer, anti-viral, and anti-atherosclerotic, and has low toxicity and few adverse reactions. Curcumin is a food additive approved for use by the World Health Organization, the US Food and Drug Administration, and many countries, and is one of the world's largest-selling natural food pigments. What attracts researchers is not only curcumin as a non-steroidal anti-inflammatory drug, but also its chemopreventive properties. Curcumin has a wide range of preventive properties for diseases. In view of the fact that modern medical research has found that the occurrence of many diseases in the human body is related to the formation of free radicals and the participation of inflammatory reactions, the antioxidant activity and anti-inflammatory effects of curcumin have attracted widespread attention from scholars at home and abroad. At present, some scientists have found that curcumin helps to assist the health care of tuberculosis. However, curcumin has low solubility, poor stability, and low absorption rate. It is easily converted into complexes such as glucuronic acid and sulfonic acid in the intestine, has rapid metabolism, and a short half-life. The existence of these problems leads to its low bioavailability.

[0004] Cinnamon essential oil is made from fresh branches, leaves, stems and bark of the cinnamon tree of the Lauraceae family. It is extracted by steam distillation and refined by fumigation. The content of cinnamaldehyde is as high as 98.2%, followed by active substances such as eugenol, α-phellandrene, 1,8-cineole, p-cymene, and camphor. Cinnamaldehyde is the main component of cinnamon leaf oil and gives cinnamon a unique smell and taste. It has been found to have a wide range of medical values ​​such as anti-inflammatory, antioxidant, and anti-cancer. Studies have shown that cinnamaldehyde has a certain inhibitory effect on Mycobacterium tuberculosis, but the inhibitory activity is obviously insufficient, and a single cinnamaldehyde is also prone to drug resistance. The physical and chemical properties of cinnamon essential oil, such as poor water solubility and high volatility, are not conducive to its use. In addition, high-concentration cinnamon essential oil has a certain special smell, and excessive content may cause discomfort to people. Summary of the invention

[0005] In order to solve the lack of a safe and effective food-grade anti-mycobacterial product in the prior art, the present invention provides a method for preparing a curcumin-cinnamon essential oil nanoemulsion. Curcumin and cinnamon essential oil can play a synergistic role within a specific range and have significantly improved mycobacterial inhibition activity. The present invention also obtains a stable antibacterial emulsion through process optimization, which retains the active ingredients of cinnamon essential oil and curcumin while reducing the impact of sensory properties. The present invention forms a nanoemulsion by using water to include cinnamon essential oil and turmeric, thereby preventing the oxidation reaction of the active ingredients, reducing volatilization, and improving the utilization of biological activity. The present invention achieves the above-mentioned purpose through the following technical means: A use of a curcumin-cinnamon essential oil nanoemulsion in preparing a mycobacterium inhibition preparation, wherein the curcumin-cinnamon essential oil nanoemulsion is prepared by a preparation method comprising the following steps: (S1) placing cinnamon essential oil in a container, adding curcumin, and mixing uniformly to prepare an oil phase; the volume mass ratio of cinnamon essential oil to curcumin powder is 1 mL: 3-3.5 mg; (S2) adding a surfactant to the oil phase, making up to volume with water, mixing evenly, and ultrasonically treating at 0-10° C. to obtain a curcumin-cinnamon essential oil nanoemulsion; the surfactant includes Tween 80 and medium-chain triglycerides.

[0006] The inventor unexpectedly found that after compounding cinnamon essential oil and curcumin according to the above ratio, combined with the subsequent emulsion preparation process, a stable emulsion with high inhibitory activity against mycobacteria can be obtained; the presence of a small amount of curcumin can significantly improve the mycobacterial inhibitory activity of the emulsion. However, the inventor found that the ratio of cinnamon essential oil and curcumin needs to be strictly controlled, and too little or too much curcumin will not significantly improve the antibacterial activity. Too much curcumin content will lead to a decrease in encapsulation efficiency and unstable emulsion; too little content will not achieve the antibacterial effect.

[0007] Furthermore, in step (S1), the volume mass ratio of cinnamon essential oil to curcumin powder is 1 mL: 3-3.5 mg.

[0008] Furthermore, in step (S2), the surfactant is a compound of Tween 80 and medium-chain triglycerides in a mass ratio of 1-3:1-3. Tween 80 provides excellent emulsification effect and stability, while medium-chain triglycerides provide a stable carrier for fat-soluble substances and improve their bioavailability. The combination of the two can significantly enhance the effect of the emulsion and improve the release and absorption of active ingredients (under the same preparation conditions, the nanoemulsion prepared by replacing medium-chain triglycerides with glycerol will have obvious stratification).

[0009] Furthermore, in step (S2), the ratio of the volume of the surfactant to the volume of the cinnamon essential oil is 0.5-3:1, preferably 1-2:1.

[0010] Furthermore, in step (S2), the amount of water used is such that the concentration of cinnamon essential oil in the emulsion is 5-10 vol%.

[0011] Further, in step (S2), the ultrasonic treatment is carried out at an ultrasonic power of 300-500 W and an ultrasonic frequency of 20-60 kHz for 5-10 min. The ultrasonic treatment at low temperature can obtain a stable emulsion without destroying the effective active ingredients of cinnamon essential oil and curcumin.

[0012] The strong smell of cinnamon essential oil may cause discomfort and headaches. It is also highly irritating. Direct consumption may irritate the gastrointestinal tract and cause nausea, vomiting, abdominal pain and other discomfort symptoms. In contrast, after it is prepared into a nanoemulsion, its safety, compliance and bioavailability can be significantly improved. Nanoemulsion is a technology that increases its solubility and absorption efficiency by dispersing the oil phase into small nano-sized particles. This form of cinnamon essential oil can not only be better absorbed by the human body, but also greatly reduces the irritation to the gastrointestinal tract and avoids the side effects caused by direct intake of essential oils. In addition, nanoemulsion can provide more precise dosage control and reduce the risk of excessive intake, making it safer to consume cinnamon essential oil. In this way, the active ingredients of cinnamon essential oil can be released gently and continuously, exerting better health effects.

[0013] Nanoemulsions have high drug solubility and bioavailability, which can help drugs penetrate the respiratory tract and lungs more effectively and exert their efficacy. Due to its low viscosity, nanoemulsions can be used for aerosol inhalation. Compared with oral or injection, aerosol therapy can deliver drugs directly into the lungs, increase local concentrations, and thus enhance targeting, helping to reduce side effects and improve efficacy. Nanoemulsions can also have sustained or controlled release functions, which can prolong the duration of action in the lungs, thereby improving efficacy and reducing the frequency of dosing. As aerosol inhalation, it is particularly suitable for diseases with localized tuberculosis infections.

[0014] The invention also provides use of the curcumin-cinnamon essential oil nanoemulsion in preparing a bactericide, a spray or an atomized inhalant.

[0015] Preferably, the bactericide is used in the form of spraying, atomizing, or atomizing inhalation.

[0016] In offices, shopping malls, hospitals and other places, in order to avoid airborne diseases, ordinary disinfectants can be replaced with nanoemulsions. The efficient bactericidal properties of nanoemulsions can be used to continuously release antibacterial ingredients through the air circulation system, thereby reducing germs and viruses in the air and protecting people's health. In hospitals, especially in operating rooms and intensive care units, the use of nanoemulsions to sterilize the air can effectively reduce the risk of nosocomial infections and keep the air in the wards fresh.

[0017] In daily life, the essential oils and curcumin ingredients are combined and sprayed in the form of nanoemulsions, so that harmful microorganisms such as bacteria and viruses in the air can be quickly killed. Unlike traditional air disinfectants, the fine particles of nanoemulsions can be more evenly distributed in the air, covering a wide area and having a longer bactericidal effect. For example, some families use nanoemulsions as air antibacterial agents. Nanoemulsions can continuously release antibacterial ingredients, which can effectively refresh the air and eliminate bacteria and viruses in the air, especially during the flu season and during the peak of the epidemic, and can provide a safer living environment for family members.

[0018] The present invention obtains an optimal preparation process through process optimization. Mycobacteria are inoculated into nanoemulsion, which can significantly inhibit mycobacteria. The nanoemulsion prepared by the invention can not only reduce the volatility of cinnamon essential oil, but also reduce the instability of curcumin and improve the bioavailability, and has basically no effect or damage on its beneficial active ingredients. The utilization value of curcumin and cinnamon essential oil is broadened. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a SEM image of the curcumin-cinnamon essential oil nanoemulsion obtained in Example 1; Figure 2 This is the graph of the nanoemulsion after a 50°C high temperature stability test; Figure 3 This is the inhibitory effect of 10% cinnamon essential oil (a) and curcumin-cinnamon essential oil nanoemulsion (b) in Application Example 3 on Mycobacterium smegmatis. DETAILED DESCRIPTION

[0020] To make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be described in detail below. The following examples are convenient for better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified.

[0021] Example 1 (S1) Accurately weigh 3 mg of curcumin powder and add it to 1 mL of cinnamon essential oil and mix well to prepare an oil phase; (S2) 0.5 g of Tween 80 and 0.5 g of medium-chain triglyceride were added to the oil phase, the volume was adjusted to 10 mL with ultrapure water, and the mixture was mixed evenly. The mixture was ultrasonicated at 350 W and 40 kHz for 10 min under ice-water bath conditions (0-5°C) to obtain a curcumin-cinnamon essential oil nanoemulsion, and nanoparticles were formed inside.

[0022] Figure 1 This is the SEM image of the curcumin-cinnamon essential oil nanoemulsion obtained in Example 1. It can be seen that the nanoparticles adsorbed on the surface of the droplets form a strong film on the interface, resulting in the droplets attracting each other to form a dense network structure, further improving the strength and stability of the emulsion. The nanoparticles in the nanoemulsion can be observed to be regular spherical droplets with a diameter of about 100 nm, which is consistent with the size measured by the particle size.

[0023] Example 2 The other conditions were the same as those in Example 1, except that in step (S1), the amount of curcumin powder was 3.5 mg.

[0024] Example 3 The other conditions were the same as those in Example 1, except that in step (S1), 1.6 mg of curcumin powder was added with 0.5 mL of cinnamon essential oil, that is, the ratio of curcumin to cinnamon essential oil remained unchanged, but in the final emulsion, the concentrations of cinnamon essential oil and curcumin were halved.

[0025] Example 4 The other conditions were the same as those in Example 1, except that in step (S1), 4 mg of curcumin powder was added with 1.25 mL of cinnamon essential oil, that is, the ratio of curcumin to cinnamon essential oil remained unchanged, but in the final emulsion, the concentration of cinnamon essential oil and curcumin was 1.25 times that of the example.

[0026] Comparative Example 1 The other conditions were the same as those in Example 1, except that in step (S2), the surfactant was 1 g of Tween 80.

[0027] Comparative Example 2 The other conditions were the same as those in Example 1, except that in step (S2), the surfactant was 1 g of medium-chain triglyceride.

[0028] Comparative Example 3 Other conditions were the same as those in Example 1, except that curcumin powder was not added in step (S1).

[0029] Comparative Example 4 The other conditions were the same as those in Example 1, except that in step (S1), the amount of curcumin powder was 2 mg.

[0030] Comparative Example 5 The other conditions were the same as those in Example 1, except that in step (S1), the amount of curcumin powder was 4 mg.

[0031] Application Example 1 The stability of the nanoemulsion obtained in the above example was evaluated from three dimensions, namely, particle size, Zeta potential and 50°C stability (recording the time when the emulsion stratification, demulsification and flocculation occurred, and more than 10 days was considered passed). The results are shown in Table 1 below: Table 1 Evaluation of emulsion stability .

[0032] Figure 2 This is a graph of the nanoemulsion after a 50°C high temperature stability test. Figure 2 A and B are the comparison diagrams of the nanoemulsions of Example 1 and Example 2 before and after the 10-day 50°C high temperature stability test. Figure 2 C is a photograph of the nanoemulsion of comparative example 5 after a 10-day 50°C high temperature stability test. It can be seen that the nanoemulsions of Examples 1 and 2 remain stable after high temperature without stratification or demulsification. However, the nanoemulsion of comparative example 5 exhibits stratification.

[0033] Application Example 2 The antibacterial activity of the curcumin-cinnamon essential oil nanoemulsions of the above examples and comparative examples against mycobacteria was tested by the following method: (1) Traditional culture of mycobacteria: Use a pipette to draw 100ul of the bacterial suspension of the standard strain of Mycobacterium smegmatis in a biological safety cabinet, and drop it onto the neutral Roche culture medium. Gently rotate the Roche culture tube to evenly cover the inclined surface of the culture medium. Tighten the lid of the culture tube with the bacterial solution, place it in a 37℃ incubator and incubate it for 5 days to obtain pure and fresh Mycobacterium smegmatis colonies.

[0034] (2) Scrape the Mycobacterium smegmatis from the Roche solid medium, dissolve it in a sterile saline grinding tube containing saline, and grind the bacteria by shaking. Take the above ground bacterial suspension, dilute it with sterile saline, and adjust it with a BioMerieux electronic turbidimeter DENSIMAT to prepare a bacterial suspension with a turbidity of 1, i.e. 1.0 mg / mL.

[0035] (3) Dilute with 7H9 medium, the final concentration of mycobacterium inoculation is 1×10 3 CFU / mL.

[0036] (4) The nanoemulsion was diluted to 128 μg / mL as the initial concentration for inhibition, and double dilution was performed based on this concentration. 16 concentration gradients of curcumin-cinnamon essential oil nanoemulsions were added to the culture medium, and the culture medium containing different concentrations of emulsions was added to the bacterial suspension, and cultured in an incubator at 37°C for 6 days. Through observation, Mycobacterium smegmatis was significantly inhibited, indicating that curcumin-cinnamon essential oil nanoemulsion has a significant inhibitory effect on the growth of mycobacteria. The results are shown in Table 2 below.

[0037] Table 2 Antibacterial activity test of curcumin-cinnamon essential oil nanoemulsion against mycobacteria .

[0038] Application Example 3 The antibacterial activity of Example 1 in aerosol form was tested using the following method: (1) Traditional culture of mycobacteria: Use a pipette to draw 100ul of the bacterial suspension of the standard strain of Mycobacterium smegmatis in a biological safety cabinet, and drop it onto the neutral Roche culture medium. Gently rotate the Roche culture tube to evenly cover the inclined surface of the culture medium. Tighten the lid of the culture tube with the bacterial solution, place it in a 37℃ incubator and incubate it for 5 days to obtain pure and fresh Mycobacterium smegmatis colonies.

[0039] (2) Scrape the Mycobacterium smegmatis from the Roche solid medium, dissolve it in a sterile saline grinding tube containing saline, and grind the bacteria by shaking. Take the above ground bacterial suspension, dilute it with sterile saline, and adjust it with a BioMerieux electronic turbidimeter DENSIMAT to prepare a bacterial suspension with a turbidity of 1, i.e. 1.0 mg / mL.

[0040] (3) Dilute with 7H9 medium, the final concentration of mycobacterium inoculation is 1×10 3 CFU / mL.

[0041] (4) Take 1 mL of bacterial suspension and add it to a culture dish. Place the dish on a 40 cm 310 vol% pure cinnamon essential oil and the nanoemulsion of Example 1 were accurately drawn and placed in mesh atomizers, respectively. The atomizer was placed in a sealed box to atomize the emulsion and essential oil at an atomization rate of ≥ 0.15 mL / min. After the atomization was completed, the culture dish was incubated at 37°C for 6 days. The results were as follows: Figure 3 As shown. Through observation, the antibacterial effect of 10vol% pure cinnamon essential oil on Mycobacterium smegmatis after atomization was not very obvious (the bacteria grew almost all over the plate, Figure 3 a), but the curcumin-cinnamon essential oil nanoemulsion had a significant inhibitory effect on Mycobacterium smegmatis (the bacteria hardly grew, Figure 3 b). This indicates that although the active ingredients of 10 vol% cinnamon essential oil are the same, the aerosol application form of curcumin-cinnamon essential oil nanoemulsion has a more obvious inhibitory effect on the growth of mycobacteria.

[0042] Note: 10 vol% cinnamon essential oil is obtained by mixing cinnamon essential oil and water at a volume ratio of 9:1 by high-speed shearing.

[0043] Comparative application example 1 The following method was used to test the inhibitory activity of curcumin solution on mycobacteria: (1) Traditional culture of mycobacteria: Use a pipette to draw 100ul of the bacterial suspension of the standard strain of Mycobacterium smegmatis in a biological safety cabinet, and drop it onto the neutral Roche culture medium. Gently rotate the Roche culture tube to evenly cover the inclined surface of the culture medium. Tighten the lid of the culture tube with the bacterial solution, place it in a 37℃ incubator and incubate it for 5 days to obtain pure and fresh Mycobacterium smegmatis colonies.

[0044] (2) Scrape the Mycobacterium smegmatis from the Roche solid medium, dissolve it in a sterile saline grinding tube containing saline, and grind the bacteria by shaking. Take the above ground bacterial suspension, dilute it with sterile saline, and adjust it with a BioMerieux electronic turbidimeter DENSIMAT to prepare a bacterial suspension with a turbidity of 1, i.e. 1.0 mg / mL.

[0045] (3) Dilute with 7H9 medium, the final concentration of mycobacterium inoculation is 1×10 3 CFU / mL.

[0046] (4) The curcumin solution was prepared to 10 mg / mL as the initial concentration for inhibition, and double dilution was performed based on this concentration. 16 curcumin solutions with different concentration gradients were added to the culture medium, and the culture medium containing different curcumin solution concentrations was added to the bacterial suspension, and cultured in an incubator at 37°C for 6 days. Through observation, the minimum inhibitory concentration of curcumin solution for Mycobacterium smegmatis was 5000 μg / mL, indicating that the curcumin solution had little inhibitory effect on the growth of mycobacteria.

Claims

1. Use of a curcumin-cinnamon essential oil nanoemulsion in preparing a mycobacterium inhibition preparation, wherein the curcumin-cinnamon essential oil nanoemulsion is prepared by a preparation method comprising the following steps: (S1) placing cinnamon essential oil in a container, adding curcumin, and mixing uniformly to prepare an oil phase; the volume mass ratio of cinnamon essential oil to curcumin powder is 1 mL: 3-3.5 mg; (S2) adding a surfactant to the oil phase, making up to volume with water, mixing evenly, and ultrasonically treating at 0-10° C. to obtain a curcumin-cinnamon essential oil nanoemulsion; the surfactant includes Tween 80 and medium-chain triglycerides.

2. The use according to claim 1, characterized in that In step (S2), the surfactant is a compound of Tween 80 and medium chain triglycerides in a mass ratio of 1-3:1-3.

3. The use according to claim 1, characterized in that In step (S2), the ratio of the volume of the surfactant to the volume of the cinnamon essential oil is 0.5-3:

1.

4. The use according to claim 3, characterized in that In step (S2), the ratio of the volume of the surfactant to the volume of the cinnamon essential oil is 1-2:

1.

5. The use according to claim 1, characterized in that: In step (S2), the amount of water used is such that the concentration of cinnamon essential oil in the emulsion is 5-10 vol%.

6. The use according to claim 1, characterized in that: In step (S2), the ultrasonic treatment is carried out at an ultrasonic power of 300-500 W and an ultrasonic frequency of 20-60 kHz for 5-10 min.

7. The use according to any one of claims 1 to 6, wherein the preparation is a spray or an aerosol inhalation.

8. The use according to any one of claims 1 to 6, characterized in that: The preparation is used in the form of spray, atomizer and / or aerosol inhalation.

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