A method for preparing self-assembled particles for improving the antibacterial activity of piperine

Abstract

The present invention relates to the field of preparation of natural products, and specifically provides a method for preparing self-assembled particles for improving the antibacterial property of piperine. The method comprises the following steps: dissolving piperine and sweet orange flavonoids, mixing, heating, concentrating, purifying, and drying. The method is simple to operate, and the prepared piperine particles have the advantages of high biocompatibility, low toxicity and adverse reactions, and can significantly improve the antibacterial property of piperine.

Description

Technical Field

[0001] The invention belongs to the field of preparation of natural products, and particularly relates to a method for preparing self-assembly particles for improving the antibacterial property of piperine. Background Art

[0002] In recent years, the use of antibiotics has become common in daily life. Even in meat products and eggs, antibiotics can be detected. The abuse of traditional antibiotics can cause bacterial resistance. Long-term use of antibiotics can also destroy the human microbial community and threaten people's health. Therefore, the search for non-toxic and specific antibiotics to inhibit bacterial infections has attracted more and more attention. Plants are one of the potential sources of natural antibiotics. The use of natural products to inhibit bacteria has the characteristics of multi-pathway, multi-target, and high safety, and has become a hot topic of research.

[0003] Piperine is the main extract of pepper, which has antibacterial, antioxidant, neuroprotective, anticonvulsant and anticancer effects. However, due to its poor water solubility and low bioavailability, it is not conducive to its efficient use. Therefore, designing highly biocompatible and biodegradable nanocarriers is an urgent problem to improve the effectiveness and safety of piperine.

[0004] In recent years, the use of natural biomass as reactants or structures to construct functional nanostructures has become a research hotspot. A large number of studies have found that some natural active ingredients such as terpenes, alkaloids, flavonoids, anthraquinones, and polysaccharides have the ability to self-assemble or co-assemble to form delivery carriers. Compared with the inorganic metal-based delivery carriers currently in use, natural ingredients as nanocarriers have the advantages of high biocompatibility, low toxicity and adverse reactions, and strong synergistic effects. However, there are few reports on stable piperine self-assembled nano delivery carriers in the prior art. Summary of the invention

[0005] Based on the structural characteristics of piperine and previous research foundation, the present invention uses piperine and sweet orange flavonoids to construct self-assembled nanoparticles to improve the antibacterial property and biocompatibility of piperine.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The first aspect of the present invention provides a piperine self-assembled nanoparticle, wherein the self-assembled nanoparticle is a self-assembled nanoparticle constructed of piperine and sweet orange flavonoids; the preparation method thereof comprises the following steps: 0.04 mmol / L piperine and 0.04 mmol / L sweet orange flavonoids are mixed, reacted at 75-85°C for 50-70 min to obtain a reaction mixture, the reaction mixture is concentrated to 1 / 7-1 / 5 of the original volume to obtain a concentrated solution, the concentrated solution is subjected to normal phase silica gel column chromatography, eluted with ethyl acetate and petroleum ether as eluents, the volume ratio of ethyl acetate: petroleum ether being 10:1, and the eluate is collected; the eluate is concentrated and dried to prepare a 5 mg / mL solution, the solution is then loaded onto a column using NKA-9 resin at a flow rate of 1.5 mL / min, eluted with 70% V / V ethanol at a flow rate of 2.0 mg / mL, the eluate is collected, concentrated, and vacuum freeze-dried to obtain the piperine self-assembled particles;

[0008] Wherein, the preparation method of piperine is:

[0009] A1: white pepper is crushed through a 40-mesh sieve to obtain pepper powder, and 80% V / V edible alcohol is added to obtain a mixed solution, and the mixed solution is heated to 60° C. and then heated at a constant temperature for 60 min, and subjected to leaching treatment to obtain a piperine extract, wherein the solid-liquid ratio of the pepper powder to 80% V / V edible alcohol is 0.04 g / mL;

[0010] A2 concentrated the piperine extract, dried it, and prepared a 2 mg / mL piperine crude extract. The piperine crude extract was loaded onto the column using HPD22 resin at a flow rate of 1.5 mL / min, and eluted with 90% V / V ethanol at a flow rate of 2.0 mg / mL to obtain a crude piperine product.

[0011] A3: The crude piperine was concentrated and dried, and 100% V / V edible alcohol was used as a solvent to prepare a 200 mg / mL piperine extract. The piperine extract was crystallized at 4°C for 24 h, and then recrystallized twice. The crystals were collected and freeze-dried in vacuum to obtain high-purity piperine.

[0012] The preparation method of the sweet orange flavonoids is:

[0013] B1. Mixing betaine and maltose in a molar ratio of 1:1 and heating until the mixture forms a clear and transparent liquid to obtain a betaine-maltose eutectic solvent, wherein the heating temperature is 80° C. and the heating time is 30-60 min;

[0014] B2: adding orange peel to the betaine-maltose low eutectic solvent in step B1 to obtain a mixed solution, heating the mixed solution to 40° C. and then continuously heating at a constant temperature for 30 min, extracting the mixed solution, centrifuging and filtering the extracted mixed solution, collecting the filtrate, removing the filter residue to obtain a clarified sweet orange flavonoid extract, wherein the solid-liquid ratio of the orange peel to the betaine-maltose low eutectic solvent is 1 / 35 g / mL;

[0015] B3: adding the sweet orange flavonoids extract described in step B2 to the extraction solvent, oscillating at room temperature and then ultrasonically treating, standing for phase separation, and obtaining an upper phase and a lower phase; the extraction solvent is composed of ethyl acetate and water, and the mass ratio of the sweet orange flavonoids extract, ethyl acetate, and water is 2:2:3; the upper phase is an ethyl acetate phase containing sweet orange flavonoids, and the lower phase is a low eutectic solvent phase containing a mixture of the sweet orange flavonoids extract, and the upper phase is distilled under reduced pressure to remove residual ethyl acetate to obtain high-purity sweet orange flavonoids.

[0016] Furthermore, the solvent of the 0.04 mmol / L piperine is a hydrochloric acid solution; the solvent of the 0.04 mmol / L sweet orange flavonoids is a phosphate buffer solution.

[0017] Furthermore, the volume ratio of the 0.04 mmol / L piperine and the 0.04 mmol / L sweet orange flavonoids is 1:1.

[0018] Furthermore, the particle size of the silica gel in the silica gel column is 200-300 meshes.

[0019] Furthermore, in step B3, the standing temperature is 25° C. and the standing time is 4 h.

[0020] Furthermore, in step B3, the temperature of the reduced pressure distillation is 50-70°C.

[0021] The second aspect of the present invention provides the use of the above-mentioned piperine self-assembled nanoparticles in inhibiting Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Pseudomonas aeruginosa.

[0022] Beneficial effects of the present invention:

[0023] The method is simple to operate, and the prepared piperine particles have the advantages of high biocompatibility, low toxicity and adverse reactions, and can significantly improve the antibacterial property of piperine. DETAILED DESCRIPTION

[0024] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0025] Example 1 Preparation of piperine

[0026] A1: white pepper is crushed through a 40-mesh sieve to obtain pepper powder, and 80% V / V edible alcohol is added to obtain a mixed solution, and the mixed solution is heated to 60° C. and then heated at a constant temperature for 60 min, and subjected to leaching treatment to obtain a piperine extract, wherein the solid-liquid ratio of the pepper powder to 80% V / V edible alcohol is 0.04 g / mL;

[0027] A2 concentrated the piperine extract, dried it, and prepared a 2 mg / mL piperine crude extract. The piperine crude extract was loaded onto the column using HPD22 resin at a flow rate of 1.5 mL / min, and eluted with 90% V / V ethanol at a flow rate of 2.0 mg / mL to obtain a crude piperine product.

[0028] A3 concentrates and dries the crude piperine, uses 100% V / V edible alcohol as solvent to prepare a 200 mg / mL piperine extract, crystallizes the piperine extract at 4°C for 24 h, recrystallizes it twice, collects the crystals, and freeze-dries them in vacuum to obtain high-purity piperine.

[0029] Example 2 Preparation of sweet orange flavonoids

[0030] B1. Mixing betaine and maltose in a molar ratio of 1:1 and heating until the mixture forms a clear and transparent liquid to obtain a betaine-maltose eutectic solvent, wherein the heating temperature is 80° C. and the heating time is 30-60 min;

[0031] B2: adding orange peel to the betaine-maltose low eutectic solvent in step B1 to obtain a mixed solution, heating the mixed solution to 40° C. and then continuously heating at a constant temperature for 30 min, extracting the mixed solution, centrifuging and filtering the extracted mixed solution, collecting the filtrate, removing the filter residue to obtain a clarified sweet orange flavonoid extract, wherein the solid-liquid ratio of the orange peel to the betaine-maltose low eutectic solvent is 1 / 35 g / mL;

[0032] B3: Add the sweet orange flavonoids extract described in step B2 to the extraction solvent, oscillate at room temperature and then perform ultrasonic treatment, stand for phase separation, and obtain an upper phase and a lower phase. The standing temperature is 25°C and the standing time is 4 h. The extraction solvent is composed of ethyl acetate and water, and the mass ratio of the sweet orange flavonoids extract, ethyl acetate and water is 2:2:3. The upper phase is an ethyl acetate phase containing sweet orange flavonoids, and the lower phase is a low eutectic solvent phase containing a mixture of the sweet orange flavonoids extract. The upper phase is distilled under reduced pressure at a temperature of 60°C to remove residual ethyl acetate to obtain high-purity sweet orange flavonoids.

[0033] Example 3 Preparation of self-assembled particles for improving the antibacterial activity of piperine

[0034] This embodiment provides a method for preparing self-assembled particles with improved antibacterial properties of piperine, the method comprising the following steps:

[0035] (1) 0.04 mmol / L piperine and 0.04 mmol / L sweet orange flavonoids were mixed and reacted at 80°C for 60 min to obtain a reaction mixture, wherein the solvent for the 0.04 mmol / L piperine was a hydrochloric acid solution, and the solvent for the 0.04 mmol / L sweet orange flavonoids was a phosphate buffer solution. The piperine was obtained in Example 1, and the sweet orange flavonoids was obtained in Example 2;

[0036] (2) The reaction mixture was concentrated to 1 / 6 of the original volume to obtain a concentrated solution, and the concentrated solution was subjected to normal phase silica gel column chromatography, eluted with a volume ratio of ethyl acetate: petroleum ether = 10:1, the eluate was collected, and the eluate was concentrated and dried to prepare a 5 mg / mL solution. The solution was then loaded onto the column using NKA-9 resin at a flow rate of 1.5 mL / min, and eluted with 70% V / V ethanol at a flow rate of 2.0 mg / mL. The eluate was collected, concentrated, and vacuum freeze-dried to obtain piperine self-assembled particles.

[0037] Example 4 Preparation of self-assembled particles for improving the antibacterial activity of piperine

[0038] This embodiment provides a method for preparing self-assembled particles with improved antibacterial properties of piperine, the method comprising the following steps:

[0039] (1) 0.04 mmol / L piperine and 0.04 mmol / L sweet orange flavonoids were mixed and reacted at 85°C for 60 min to obtain a reaction mixture, wherein the solvent for the 0.04 mmol / L piperine was a hydrochloric acid solution, and the solvent for the 0.04 mmol / L sweet orange flavonoids was a phosphate buffer solution. The piperine was obtained in Example 1, and the sweet orange flavonoids was obtained in Example 2.

[0040] (2) The reaction mixture was concentrated to 1 / 6 of the original volume to obtain a concentrated solution, and the concentrated solution was subjected to normal phase silica gel column chromatography, eluted with a volume ratio of ethyl acetate: petroleum ether = 10:1, the eluate was collected, and the eluate was concentrated and dried to prepare a 5 mg / mL solution. The solution was then loaded onto the column using NKA-9 resin at a flow rate of 1.5 mL / min, and eluted with 70% V / V ethanol at a flow rate of 2.0 mg / mL. The eluate was collected, concentrated, and vacuum freeze-dried to obtain piperine self-assembled particles.

[0041] Example 5 Preparation of self-assembled particles for improving the antibacterial activity of piperine

[0042] This embodiment provides a method for preparing self-assembled particles with improved antibacterial properties of piperine, the method comprising the following steps:

[0043] (1) 0.04 mmol / L piperine and 0.04 mmol / L sweet orange flavonoids were mixed and reacted at 85°C for 60 min to obtain a reaction mixture, wherein the solvent for the 0.04 mmol / L piperine was a hydrochloric acid solution, and the solvent for the 0.04 mmol / L sweet orange flavonoids was a phosphate buffer solution. The piperine was obtained in Example 1, and the sweet orange flavonoids was obtained in Example 2.

[0044] (2) The reaction mixture was concentrated to 1 / 6 of the original volume to obtain a concentrated solution, and the concentrated solution was subjected to normal phase silica gel column chromatography, eluted with a volume ratio of ethyl acetate: petroleum ether = 10:1, the eluate was collected, and the eluate was concentrated and vacuum freeze-dried to obtain piperine self-assembled particles.

[0045] Example 6 Preparation of self-assembled particles for improving the antibacterial activity of piperine

[0046] This embodiment provides a method for preparing self-assembled particles with improved antibacterial properties of piperine, the method comprising the following steps:

[0047] (1) 0.04 mmol / L piperine and 0.04 mmol / L sweet orange flavonoids sold on the market were mixed and reacted at 80°C for 60 min to obtain a reaction mixture, wherein the solvent for the 0.04 mmol / L piperine was a hydrochloric acid solution, and the solvent for the 0.04 mmol / L sweet orange flavonoids was a phosphate buffer solution. The piperine was obtained in Example 1, and the sweet orange flavonoids were obtained from the market.

[0048] (2) The reaction mixture was concentrated to 1 / 6 of the original volume to obtain a concentrated solution, and the concentrated solution was subjected to normal phase silica gel column chromatography, eluted with a volume ratio of ethyl acetate: petroleum ether = 10:1, the eluate was collected, and the eluate was concentrated and dried to prepare a 5 mg / mL solution. The solution was then loaded onto the column using NKA-9 resin at a flow rate of 1.5 mL / min, and eluted with 70% V / V ethanol at a flow rate of 2.0 mg / mL. The eluate was collected, concentrated, and vacuum freeze-dried to obtain piperine self-assembled particles.

[0049] Experiment 1: Determination of the diameter of the inhibition zone

[0050] Prepare 4 concentrations with a range of 10 6 ~10 8 cfu / mL bacterial solution (Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa), take 0.10 mL of bacterial solution respectively, and evenly spread it on LB medium. Soak a sterilized 6 mm diameter circular filter paper in 2 mg / mL sample solution for 30 s, take it out, and place it on the culture medium. The sample solution is prepared by Examples 1, 2, 3, 4, 5, and 6 respectively. Sterile water is used as a control, and the culture is kept at 37 ° C for 24 h, and the diameter of the inhibition zone is measured.

[0051] The specific results of the experiment are shown in Table 1. In addition, the result of the sterile water control is 0, indicating no contamination.

[0052] Experiment 2: Minimum inhibitory concentration determination experiment

[0053] Add 6 samples of different mass concentrations to LB medium and shake well. The 6 samples are prepared by Examples 1-6. Each sample is prepared into a medium with a final concentration of 25.60, 12.80, 6.40, 3.20, 1.60, 0.80, 0.40, 0.20, 0.10, 0.05, and 0.025 mg / mL. Take 0.20 mL of 10 6 ~10 7 cfu / mL bacterial solution (Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa) was cultured in the culture medium at 37 °C for 24 h. The LB culture medium without sample solution was used as the blank control, and the lowest concentration of the sample solution without bacterial growth was used as the minimum inhibitory concentration (MIC).

[0054] The specific results of the experiment are shown in Table 2. In addition, the result of the blank control is 0, which indicates no contamination.

[0055] Table 1 Diameters of inhibition zones of different groups against the tested bacteria (mm)

[0056]

[0057] Note: Different letters indicate significant differences among different groups ( p >0.05)

[0058] Table 2 Minimum inhibitory concentration of different groups against the test bacteria (mg / mL)

[0059]

[0060] Note: Different letters indicate significant differences among different groups ( p >0.05)

[0061] From Table 1 and Table 2, we can see that:

[0062] (1) Compared with Example 1-2, the diameter of the antibacterial zone of the self-assembled particles of piperine and sweet orange flavonoids in Example 3-6 increased significantly, and the minimum inhibitory concentration decreased significantly. It can be seen that the self-assembled compound of piperine and sweet orange flavonoids can enhance the antibacterial activity of piperine.

[0063] (2) Compared with Example 6, the diameter of the antibacterial zone of the self-assembled particles of piperine and sweet orange flavonoids prepared by using the sweet orange flavonoids extracted by the method of the present invention increased significantly, and the minimum inhibitory concentration decreased significantly. It can be seen that the flavonoids extracted by the betaine-maltose low eutectic solvent can enhance the antibacterial activity of the self-assembled particles of piperine and sweet orange flavonoids.

[0064] (3) In comparison between Experimental Example 3 and Examples 4 and 5, changes in the preparation conditions of the self-assembled particles of piperine and sweet orange flavonoids are not conducive to the antibacterial activity of the particles. Among them, the lack of NKA-9 and re-purification is the most detrimental to the antibacterial effect of the self-assembled particles of piperine and sweet orange flavonoids, followed by the reaction temperature.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A piperine self-assembled nanoparticle, characterized in that: The self-assembled nanoparticles are self-assembled nanoparticles constructed of piperine and sweet orange flavonoids; the preparation method thereof comprises the following steps: 0.04 mmol / L piperine and 0.04 mmol / L sweet orange flavonoids are mixed, reacted at 80-85°C for 60 min to obtain a reaction mixture, the reaction mixture is concentrated to 1 / 6 of the original volume to obtain a concentrated solution, the concentrated solution is subjected to normal phase silica gel column chromatography, eluted with ethyl acetate and petroleum ether as eluents, the volume ratio of ethyl acetate: petroleum ether being 10:1, and the eluate is collected; the eluate is concentrated and dried to prepare a 5 mg / mL solution, the solution is then loaded onto the column at a flow rate of 1.5 mL / min using NKA-9 resin, eluted with 70% V / V ethanol at a flow rate of 2.0 mg / mL, the eluate is collected, concentrated, and vacuum freeze-dried to obtain piperine self-assembled particles; Wherein, the preparation method of piperine is: A1: white pepper is crushed through a 40-mesh sieve to obtain pepper powder, and 80% V / V edible alcohol is added to obtain a mixed solution, and the mixed solution is heated to 60° C. and then heated at a constant temperature for 60 min, and subjected to leaching treatment to obtain a piperine extract, wherein the solid-liquid ratio of the pepper powder to 80% V / V edible alcohol is 0.04 g / mL; A2 concentrated the piperine extract, dried it, and prepared a 2 mg / mL piperine crude extract. The piperine crude extract was loaded onto the column using HPD22 resin at a flow rate of 1.5 mL / min, and eluted with 90% V / V ethanol at a flow rate of 2.0 mg / mL to obtain a crude piperine product. A3: The crude piperine was concentrated and dried, and 100% V / V edible alcohol was used as a solvent to prepare a 200 mg / mL piperine extract. The piperine extract was crystallized at 4°C for 24 h, and then recrystallized twice. The crystals were collected and freeze-dried in vacuum to obtain high-purity piperine. The preparation method of the sweet orange flavonoids is: B1. Mixing betaine and maltose in a molar ratio of 1:1 and heating until the mixture forms a clear and transparent liquid to obtain a betaine-maltose eutectic solvent, wherein the heating temperature is 80° C. and the heating time is 30-60 min; B2: adding orange peel to the betaine-maltose low eutectic solvent in step B1 to obtain a mixed solution, heating the mixed solution to 40° C. and then continuously heating at a constant temperature for 30 min, extracting the mixed solution, centrifuging and filtering the extracted mixed solution, collecting the filtrate, removing the filter residue to obtain a clarified sweet orange flavonoid extract, wherein the solid-liquid ratio of the orange peel to the betaine-maltose low eutectic solvent is 1 / 35 g / mL; B3: adding the sweet orange flavonoids extract described in step B2 to the extraction solvent, oscillating at room temperature and then ultrasonically treating, standing for phase separation, and obtaining an upper phase and a lower phase; the extraction solvent is composed of ethyl acetate and water, and the mass ratio of the sweet orange flavonoids extract, ethyl acetate, and water is 2:2:3; the upper phase is an ethyl acetate phase containing sweet orange flavonoids, and the lower phase is a low eutectic solvent phase containing a mixture of the sweet orange flavonoids extract, and the upper phase is distilled under reduced pressure to remove residual ethyl acetate to obtain high-purity sweet orange flavonoids.

2. The piperine self-assembled nanoparticles according to claim 1, characterized in that: The solvent of the 0.04 mmol / L piperine is a hydrochloric acid solution; the solvent of the 0.04 mmol / L sweet orange flavonoids is a phosphate buffer solution.

3. The piperine self-assembled nanoparticles according to claim 1, characterized in that: The volume ratio of the 0.04 mmol / L piperine and the 0.04 mmol / L sweet orange flavonoids is 1:

1.

4. The piperine self-assembled nanoparticles according to claim 1, characterized in that: The particle size of the silica gel in the silica gel column is 200-300 meshes.

5. The piperine self-assembled nanoparticles according to claim 1, characterized in that: In step B3, the standing temperature is 25° C. and the standing time is 4 h.

6. The piperine self-assembled nanoparticles according to claim 1, characterized in that: In step B3, the temperature of the reduced pressure distillation is 50-70°C.

7. Use of the piperine self-assembled nanoparticles according to claim 1 in preparing an antibacterial agent for Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Pseudomonas aeruginosa.

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