Pickering emulsion with cinnamyl aldehyde as oil phase

Abstract

The invention discloses a Pickering emulsion with cinnamyl aldehyde as an oil phase and a preparation method of the Pickering emulsion. The Pickering emulsion is prepared by the following steps: carrying out a cross-linking reaction on anions released by sodium tripolyphosphate and protonated cations of chitosan to prepare chitosan nanoparticles as a water phase, and then taking cinnamyl aldehyde as an oil phase. The Pickering emulsion delivery system disclosed by the invention can effectively release cinnamyl aldehyde, shows the remarkable antibacterial property of cinnamyl aldehyde on a wound surface, promotes wound surface healing, and shows good treatment potential.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and in particular relates to a Pickering emulsion with cinnamaldehyde as an oil phase. Background Art

[0002] In daily life, there are many reasons for human skin injuries, and there are many types of wounds, such as chronic ulcers, cuts, burns, friction injuries, and surgical wounds. Moreover, skin injuries are very susceptible to bacterial infection, which will not only delay the healing process, but also cause tissue necrosis in severe cases, and even require amputation. Among them, bacterial infection is one of the important factors affecting wound healing, especially bacteria such as Staphylococcus aureus and Escherichia coli, which are easy to breed in the wound, making healing difficult.

[0003] Cinnamaldehyde is an aldehyde organic compound extracted from the traditional spice cinnamon. It can give food a unique smell and can be used as a natural preservative. Cinnamaldehyde is widely used in food, cosmetics, medicine and other fields. In addition, cinnamaldehyde has broad-spectrum antibacterial properties and can effectively inhibit a variety of bacteria such as Escherichia coli, Salmonella, Staphylococcus aureus, Clostridium botulinum and Bacillus. However, due to its poor water solubility, high volatility, poor oxidative stability, and sensitivity to environmental factors such as light and heat, and its irritating smell, the direct application of cinnamaldehyde essential oil in antibacterial treatment of wounds is limited.

[0004] Emulsions usually refer to two immiscible liquids, one of which is dispersed in the other; Pickering emulsions are emulsions that use solid particles as stabilizers instead of traditional surfactants. Compared with traditional emulsions, Pickering emulsions do not require the use of inorganic or polymer surfactants during the preparation process. Many solid particles have the potential to be Pickering emulsion stabilizers, such as polysaccharides, proteins, and inorganic particles, which are often used to stabilize Pickering emulsions. Therefore, Pickering emulsions have important application value and broad prospects in many fields such as food, medicine, papermaking, and cosmetics.

[0005] Chitosan is a renewable natural alkaline polysaccharide that is widely available and is formed by partial or complete deacetylation of chitin. Chitosan has the characteristics of low toxicity, good biocompatibility and biodegradability. Therefore, chitosan nanoparticles can not only be used as a carrier for controlled release of active substances, but also as a stabilizer for emulsions. However, chitosan is only soluble in acidic environments and insoluble in neutral and alkaline solutions. The formed nanoparticles have poor stability, which limits the encapsulation efficiency of some drugs.

[0006] Therefore, the present invention designs to prepare chitosan nanoparticles by ionic crosslinking reaction for stabilizing Pickering emulsion. Summary of the invention

[0007] One of the objects of the present invention is to provide a Pickering emulsion, which is prepared by the following steps:

[0008] Step 1, dissolving chitosan in an acetic acid solution to obtain a chitosan solution, dissolving sodium tripolyphosphate in water to obtain a sodium tripolyphosphate solution, adding the sodium tripolyphosphate solution dropwise to the chitosan solution, stirring and mixing, centrifuging, removing the supernatant, and adding pure water to obtain a chitosan particle suspension;

[0009] The mass percentage concentration of chitosan in the chitosan solution is 0.5% to 2%, and the mass ratio of chitosan to sodium tripolyphosphate is 1:1 to 5:1;

[0010] Step 2, mixing the chitosan particle suspension with the oil phase, and ultrasonically emulsifying the mixture to obtain the Pickering emulsion;

[0011] The oil phase is cinnamaldehyde, and the volume of the oil phase is 5% to 20% of the volume of the mixed liquid.

[0012] Furthermore, the mass ratio of acetic acid to chitosan in the acetic acid solution is 3:2.

[0013] Furthermore, the mass percentage concentration of chitosan in the chitosan solution is 0.5%, and the mass ratio of chitosan to sodium tripolyphosphate is 5:1.

[0014] Furthermore, the volume of the oil phase is 0.5% of the volume of the mixed liquid.

[0015] Furthermore, the ultrasonic emulsification conditions are 200W to 400W, 1 min to 10 min, preferably 2 min.

[0016] The second object of the present invention is to provide the use of the above Pickering emulsion in the preparation of external antibacterial products.

[0017] Furthermore, the bacteria are Escherichia coli and Staphylococcus aureus.

[0018] The present invention adopts a nanoparticle suspension with good biocompatibility as the aqueous phase, and selects cinnamaldehyde with significant antibacterial effect as the oil phase, constructs a Pickering emulsion delivery system suitable for treating bacterial infection on wounds, and achieves the purpose of cinnamaldehyde exerting excellent antibacterial effect on wounds. In order to improve the stability of the Pickering emulsion, the anions released by sodium tripolyphosphate are cross-linked with the protonated cations of chitosan to prepare chitosan nanoparticles that stabilize the Pickering emulsion; at the same time, cinnamaldehyde is used as the oil phase, because cinnamaldehyde can inhibit and kill pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, and Salmonella in and outside the body, so that the Pickering emulsion shows higher antibacterial activity.

[0019] The Pickering emulsion of the present invention is used as an antibacterial agent, can effectively inhibit pathogenic bacteria in vitro, can reduce the reproduction of bacteria, such as Escherichia coli, Staphylococcus aureus, etc., improves the therapeutic effect, and has high safety; it can greatly reduce the side effects of the emulsion and significantly enhance the antibacterial effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The particle size and PDI distribution diagram of the chitosan particle suspension prepared in Example 1;

[0021] Figure 2 is a SEM image of the chitosan particle suspension prepared in Example 1;

[0022] Figure 3 This is a graph showing the change in particle size of the Pickering emulsion prepared in Example 1 over storage time;

[0023] Figure 4 The Pickering emulsion prepared in Example 1 is a graph showing the change of different ultrasonic times over time;

[0024] Figure 5 This is a graph showing the change of the ratio of different oil phases in the Pickering emulsion over time in Example 1;

[0025] Figure 6 This is the SEM image of the Pickering emulsion in Example 1;

[0026] Figure 7 The Pickering emulsion inhibition zone diagram in Example 2;

[0027] Figure 8 The bacteria live and dead staining fluorescence imaging in Example 2;

[0028] Fig. 9 is the antibacterial ratio of the Pickering emulsion in Example 2. DETAILED DESCRIPTION

[0029] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0031] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0032] Example 1

[0033] 1. Preparation of chitosan particle suspension

[0034] Dissolve chitosan powder in acetic acid solution, the mass percentage concentration of chitosan is 0.5%, 1%, 1.5%, and 2%, respectively, wherein the mass ratio of chitosan powder to acetic acid is maintained at 2:3, and stir overnight until the chitosan powder is completely dissolved. Dissolve sodium tripolyphosphate powder in pure water, stir until the sodium tripolyphosphate powder is completely dissolved, and prepare a sodium tripolyphosphate solution with a concentration of 1 mg / mL. Slowly add the sodium tripolyphosphate solution to the above chitosan solution, the mass ratio of chitosan to sodium tripolyphosphate is 1:1 to 5:1, and the specific dosage ratio is shown in Table 1. The mixed solution is stirred for 30 minutes. The mixed solution after stirring is placed in a centrifuge tube, divided into 30 to 35 mL per tube, centrifuged at 10000xg for 35 minutes, the supernatant is removed, and 25 mL of pure water is added to each tube to disperse the precipitate to obtain chitosan particle suspensions with different concentration ratios.

[0035] Table 1 Preparation ratio of chitosan particle suspension

[0036]

[0037] Dilute the chitosan particle suspension with pure water and use a particle size analyzer to measure the particle size and PDI. Figure 1 It can be seen that the particle size of the chitosan particle suspension with a chitosan concentration of 0.5% and a mass ratio of chitosan to sodium tripolyphosphate of 5:1 is 165.2nm and the PDI is 0.24. Considering comprehensively, the chitosan suspension has the best ratio of particle size and PDI.

[0038] A scanning electron microscope (SEM) was used to take photos of a chitosan particle suspension with a chitosan concentration of 0.5% and a chitosan to sodium tripolyphosphate mass ratio of 5:1. SEM images are referenced Figure 2 , it can be seen that the surface of the particles is relatively smooth and flat.

[0039] 2. Preparation of Pickering emulsion

[0040] The chitosan particle suspension was mixed with cinnamaldehyde and ultrasonicated in an ice-water bath, wherein cinnamaldehyde accounted for 5% of the total liquid volume, the ultrasonic power was 300 W, the ultrasonic time was 2 min, and the Pickering emulsion particle size was measured with a laser particle size analyzer.

[0041] To evaluate the stability of the Pickering emulsion, the particle size of the Pickering emulsion was measured after storage at 4°C for 30 days. Figure 3 As shown, after 30 days, it can be seen that the Pickering emulsion with a chitosan concentration of 0.5% and a chitosan to sodium tripolyphosphate mass ratio of 5:1 has a smaller particle size and a higher overall stability.

[0042] In order to evaluate the stability of Pickering emulsions with different oil phases, Pickering emulsions with oil phase ratios of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%, and water phase ratios of 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, and 10% were prepared, and the particle size of the Pickering emulsions was measured using a laser particle size analyzer. Figure 4 It can be seen that the Pickering emulsion with 5% oil phase is relatively stable.

[0043] In order to evaluate the stability of Pickering emulsions under different ultrasonic times, Pickering emulsions were prepared at different ultrasonic times of 1 min to 10 min and an ultrasonic power of 300 W, and the particle size of the Pickering emulsions was measured using a laser particle size analyzer. Figure 5 As shown in the figure, when the ultrasonic time is 2 min, the particle size of the Pickering emulsion is smaller.

[0044] The morphology of Pickering emulsions prepared with 0.5% chitosan concentration, a chitosan to sodium tripolyphosphate mass ratio of 5:1, and 5% cinnamaldehyde was investigated using SEM to determine the characteristics of the Pickering emulsion. Figure 6 It can be seen that the Pickering emulsion is chitosan wrapped with cinnamaldehyde oil droplets.

[0045] Example 2

[0046] Evaluation of the Antibacterial Properties of Pickering Emulsions in Vitro

[0047] Prepare 100 mL of LB medium, including 2.5 g of LB, and sterilize the medium by wet heat. Add Escherichia coli and Staphylococcus aureus to the medium and incubate at 37 degrees overnight.

[0048] Prepare 100 mL of LB agar medium, including 2.5 g of LB and 1.5 g of agar, and sterilize the medium with wet heat. Cool the medium to 50-60 degrees, pour it into a dish, 25-30 mL of the medium per dish, and solidify it at room temperature.

[0049] After solidification, make 4 holes on the culture medium. On the agar plate with holes, use a bacterial spreading stick to evenly spread Escherichia coli (E.coli) and Staphylococcus aureus (S.aureus), and be careful to avoid the sample holes. After spreading, wait for 5 to 10 minutes, and add sample 1: 5 microliters of cinnamaldehyde, sample 2: 95 microliters of pure water, sample 3: 95 microliters of 0.5% chitosan concentration, chitosan and sodium tripolyphosphate The mass ratio of chitosan is 5:1. The suspension of chitosan particles, sample 4: 100 microliters of 0.5% chitosan concentration, chitosan and sodium tripolyphosphate The mass ratio is 5:1, and 5% cinnamaldehyde is prepared by Pickering emulsion. After adding, incubate the agar plate at 37 degrees for 24 hours. After 24 hours, take a picture to record the inhibition zone. The inhibition zone is referenced Figure 7 .like Figure 7 As shown, the Pickering emulsion prepared with 0.5% chitosan concentration, a mass ratio of chitosan to sodium tripolyphosphate of 5:1, and 5% cinnamaldehyde has a good antibacterial effect.

[0050] Treat Escherichia coli and Staphylococcus aureus with the four samples for 2 hours, and collect the bacteria by centrifugation at 3500xg for 5 minutes. Wash the precipitate with 0.9% sodium chloride solution. Stain with a live-dead staining kit in the dark. After staining, take photos with a fluorescent microscope and calculate the percentage of live and dead bacteria. Fluorescence imaging reference Figure 8 , live and dead bacteria ratio reference Fig. 9 .like Figure 8 As shown in the figure, CS is a chitosan suspension with a chitosan concentration of 0.5% and a chitosan to sodium tripolyphosphate mass ratio of 5:1, Cin is cinnamaldehyde, and CPE is a Pickering emulsion prepared with a chitosan concentration of 0.5%, a chitosan to sodium tripolyphosphate mass ratio of 5:1, and 5% cinnamaldehyde. It can be seen that CPE shows good antibacterial properties like Cin. The results are shown in Fig. 9 shown.

[0051] From the above results, it can be concluded that the Pickering emulsion prepared with 0.5% chitosan concentration, a mass ratio of chitosan to sodium tripolyphosphate of 5:1, and 5% cinnamaldehyde has excellent antibacterial properties.

Claims

1. A Pickering emulsion, characterized in that The preparation was carried out by the following steps: Step 1, dissolving chitosan in an acetic acid solution to obtain a chitosan solution, dissolving sodium tripolyphosphate in water to obtain a sodium tripolyphosphate solution, adding the sodium tripolyphosphate solution dropwise to the chitosan solution, stirring and mixing, centrifuging, removing the supernatant, and adding pure water to obtain a chitosan particle suspension; The mass percentage concentration of chitosan in the chitosan solution is 0.5% to 2%, and the mass ratio of chitosan to sodium tripolyphosphate is 1:1 to 5:1; Step 2, mixing the chitosan particle suspension with the oil phase, and ultrasonically emulsifying the mixture to obtain the Pickering emulsion; The oil phase is cinnamaldehyde, and the volume of the oil phase is 5% to 20% of the volume of the mixed liquid.

2. The Pickering emulsion according to claim 1, characterized in that The mass ratio of acetic acid to chitosan in the acetic acid solution is 3:

2.

3. The Pickering emulsion according to claim 1, characterized in that The mass percentage concentration of chitosan in the chitosan solution is 0.5%, and the mass ratio of chitosan to sodium tripolyphosphate is 5:

1.

4. The Pickering emulsion according to claim 3, characterized in that The volume of the oil phase is 0.5% of the volume of the mixed solution.

5. The Pickering emulsion according to claim 1, characterized in that The ultrasonic emulsification conditions are 200W to 400W and 1min to 10min.

6. Use of the Pickering emulsion according to claim 1 in the preparation of external antibacterial products.

7. The use according to claim 6, characterized in that: The bacteria are Escherichia coli and Staphylococcus aureus.

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