Preparation method and application of piperine-cinnamic acid self-assembled nanoparticles

Abstract

The present invention relates to the field of preparation of natural products, and specifically provides a method for preparing piperine-cinnamic acid self-assembled nanoparticles and applications. The method comprises the following steps: dissolving piperine and cinnamic acid, mixing, adjusting pH value, heating, concentrating, purifying, and drying. The method is simple to operate, and the prepared piperine-cinnamic acid particles can significantly inhibit Staphylococcus aureus, and have the advantages of high biocompatibility, low toxicity and adverse reactions, etc.

Description

Technical Field

[0001] The invention belongs to the field of preparation of natural products, and specifically relates to a preparation method and application of piperine-cinnamic acid self-assembled nanoparticles. Background Art

[0002] Antibiotic resistance is a major challenge facing modern medicine. The "Antibiotic Resistance Review" reports that by 2050, antibiotic resistance may cause at least 10 million deaths per year. Long-term use of antibiotics will destroy the human microbiome and threaten people's health. As one of the 12 high-priority pathogens of the World Health Organization, Staphylococcus aureus is a widely distributed pathogen that can cause serious clinical infections. Staphylococcus aureus has developed resistance to almost all antibiotics such as β-lactams, glycopeptides, tetracyclines, aminoglycosides and fluoroquinolones. Therefore, there is an urgent need to find non-toxic, non-residue and multi-pathway antimicrobial agents to inhibit Staphylococcus aureus infections.

[0003] 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. Piperine is the main active ingredient of pepper, which has multiple effects such as antibacterial, anticancer, antioxidant, and anticonvulsant. However, its poor water solubility and low bioavailability limit its efficient use. Nanoparticles have strong permeability and can be used as carriers to achieve targeted delivery of compounds and improve their utilization. Designing nanoparticles suitable for delivering piperine has become an urgent problem to be solved.

[0004] In recent years, the use of natural products as reactants or structures to construct functional nanoparticles has become a research hotspot. A large number of studies have found that some natural active ingredients such as terpenes, alkaloids, flavonoids, and polysaccharides have the ability to self-assemble or co-assemble to form delivery vectors. Compared with the inorganic metal-based delivery vectors currently in use, natural product self-assembled nanoparticles have the advantages of high biocompatibility, strong synergistic effects, low toxicity and side effects. However, there are few reports on piperine self-assembled nano delivery vectors 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 cinnamic acid to construct self-assembled nanoparticles to improve the ability of piperine to inhibit Staphylococcus aureus.

[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-cinnamic acid self-assembled nanoparticle, wherein the self-assembled nanoparticle is a self-assembled nanoparticle constructed by piperine and cinnamic acid; the preparation method thereof is as follows: 0.01mmol / L piperine and 0.01mmol / L cinnamic acid are mixed to obtain a mixed solution; 5 mg / mL sodium hydroxide solution is used to adjust the pH value of the mixed solution to 7.0-7.5, and then a phosphate buffer solution with a pH value of 7.4 equal to the mixed solution is added, and the mixture is reacted at 55°C and 180 r / min for 30 minutes to obtain a reaction mixture; the reaction mixture is ultrasonicated at 35°C and 100W for 10 minutes, and then 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, and the volume ratio of ethyl acetate: petroleum ether is 8:1, and the eluate is collected; the eluate is dialyzed in a 2.0 KDa filter membrane for 16 hours, concentrated, and vacuum freeze-dried to obtain piperine-cinnamic acid self-assembled nanoparticles;

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

[0009] A1: uniformly mixing citric acid and choline chloride in a molar ratio of 3:1 and heating until the mixture forms a clear and transparent liquid to obtain a citric acid-choline chloride deep eutectic solvent; the heating temperature is 55° C. and the heating time is 30 min;

[0010] A2: adding white pepper powder to the citric acid-choline chloride low eutectic solvent in step A1 to obtain a mixed solution, heating the mixed solution to 60° 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 piperine extract, wherein the solid-liquid ratio of the white pepper powder to the citric acid-choline chloride is 1 / 35 g / mL;

[0011] A3: adding the piperine extract described in step A2 to an 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 isopropanol and water, and the mass ratio of the piperine extract, isopropanol, and water is 2:2:3; the upper phase is an isopropanol phase containing piperine, and the lower phase is a low eutectic solvent phase containing a mixture of the piperine extract; the upper phase is distilled under reduced pressure to 1 / 8 of the original volume, and residual isopropanol is removed to obtain a piperine extract.

[0012] A4: Add 3 times the volume of anhydrous ethanol to the piperine extract in step A3, incubate for 24 h at 4°C for crystallization, collect the crystals, and freeze-dry them in vacuum to obtain high-purity piperine.

[0013] The cinnamic acid is commercially available with a purity of >90%.

[0014] Furthermore, the solvent for the 0.01 mmol / L piperine is a methanol solution; and the solvent for the 0.01 mmol / L cinnamic acid is a 45°C phosphate buffer solution.

[0015] Furthermore, the volume ratio of the 0.01 mmol / L piperine and the 0.01 mmol / L cinnamic acid is 1:1.

[0016] Furthermore, the particle size of the silica gel in the silica gel column is 200-300 meshes.

[0017] The second aspect of the present invention provides the use of the piperine-cinnamic acid self-assembled nanoparticles in the preparation of an antibacterial agent for Staphylococcus aureus.

[0018] Beneficial effects of the present invention:

[0019] 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 ability of piperine to inhibit Staphylococcus aureus. DETAILED DESCRIPTION

[0020] 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.

[0021] Example 1 Preparation of piperine

[0022] A1: uniformly mixing citric acid and choline chloride in a molar ratio of 3:1 and heating until the mixture forms a clear and transparent liquid to obtain a citric acid-choline chloride deep eutectic solvent; the heating temperature is 55° C. and the heating time is 30 min;

[0023] A2: adding white pepper powder to the citric acid-choline chloride low eutectic solvent in step A1 to obtain a mixed solution, heating the mixed solution to 60° 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 piperine extract, wherein the solid-liquid ratio of the white pepper powder to the citric acid-choline chloride is 1 / 35 g / mL;

[0024] A3: adding the piperine extract described in step A2 to an 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 isopropanol and water, and the mass ratio of the piperine extract, isopropanol, and water is 2:2:3; the upper phase is an isopropanol phase containing piperine, and the lower phase is a low eutectic solvent phase containing a mixture of the piperine extract; the upper phase is distilled under reduced pressure to 1 / 8 of the original volume, and residual isopropanol is removed to obtain a piperine extract.

[0025] A4: Add 3 times the volume of anhydrous ethanol to the piperine extract in step A3, incubate for 24 h at 4°C for crystallization, collect the crystals, and freeze-dry them in vacuum to obtain high-purity piperine.

[0026] Example 2 Cinnamic acid was purchased from Aladdin Reagent (Shanghai) Co., Ltd. (purity 99%).

[0027] Example 3 Preparation of piperine-cinnamic acid self-assembled nanoparticles

[0028] This embodiment provides a method for preparing piperine-cinnamic acid self-assembled nanoparticles, the method comprising the following steps:

[0029] (1) 0.01 mmol / L piperine and 0.01 mmol / L cinnamic acid were mixed to obtain a mixed solution. The pH of the mixed solution was adjusted to 7.0-7.5 using 5 mg / mL NaOH, and a pH 7.4 phosphate buffer solution of an equal volume to the mixed solution was added, and the mixture was reacted at 55°C and 180 r / min for 30 min to obtain a reaction mixture. The piperine was obtained in Example 1, and the cinnamic acid was obtained in Example 2.

[0030] (2) The reaction mixture was ultrasonically treated at 35°C and 100 W for 10 min, and then concentrated to 1 / 6 of the original volume to obtain a concentrated solution. The concentrated solution was eluted by normal phase silica gel column chromatography with ethyl acetate and petroleum ether as eluents, with a volume ratio of ethyl acetate:petroleum ether = 8:1, and the eluate was collected. The eluate was dialyzed on a 2.0 KDa filter membrane for 16 h, concentrated, and vacuum freeze-dried to obtain piperine-cinnamic acid self-assembled nanoparticles.

[0031] Example 4 Preparation of piperine-cinnamic acid self-assembled nanoparticles

[0032] This embodiment provides a method for preparing piperine-cinnamic acid self-assembled nanoparticles, the method comprising the following steps:

[0033] (1) 0.01 mmol / L piperine and 0.01 mmol / L cinnamic acid were mixed to obtain a mixed solution. The pH of the mixed solution was adjusted to 7.0-7.5 using 5 mg / mL NaOH, and a pH 7.4 phosphate buffer solution of an equal volume to the mixed solution was added, and the mixture was reacted at 55°C and 180 r / min for 30 min to obtain a reaction mixture. The piperine was obtained in Example 1, and the cinnamic acid was obtained in Example 2.

[0034] (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, using ethyl acetate and petroleum ether as eluents, with a volume ratio of ethyl acetate:petroleum ether = 8:1, and the eluate was collected; the eluate was dialyzed on a 2.0 KDa filter membrane for 16 h, concentrated, and vacuum freeze-dried to obtain piperine-cinnamic acid self-assembled nanoparticles.

[0035] Example 5 Preparation of piperine-cinnamic acid self-assembled nanoparticles

[0036] This embodiment provides a method for preparing piperine-cinnamic acid self-assembled nanoparticles, the method comprising the following steps:

[0037] (1) 0.01 mmol / L piperine and 0.01 mmol / L cinnamic acid were mixed to obtain a mixed solution. The pH of the mixed solution was adjusted to 7.0-7.5 using 5 mg / mL NaOH, and a pH 7.4 phosphate buffer solution of an equal volume to the mixed solution was added, and the mixture was reacted at 55°C and 180 r / min for 30 min to obtain a reaction mixture. The piperine was obtained in Example 1, and the cinnamic acid was obtained in Example 2.

[0038] (2) The reaction mixture was ultrasonically concentrated to 1 / 6 of the original volume at 35°C and 100 W for 10 min to obtain a concentrate. The concentrate was dialyzed on a 2.0 KDa filter membrane for 16 h, concentrated, and vacuum freeze-dried to obtain piperine-cinnamic acid self-assembled nanoparticles.

[0039] Example 6 Preparation of piperine-cinnamic acid self-assembled nanoparticles

[0040] This embodiment provides a method for preparing piperine-cinnamic acid self-assembled nanoparticles, the method comprising the following steps:

[0041] (1) 0.01 mmol / L piperine and 0.01 mmol / L cinnamic acid were mixed to obtain a mixed solution. The pH of the mixed solution was adjusted to 7.0-7.5 using 5 mg / mL NaOH, and a pH 7.4 phosphate buffer solution of an equal volume to the mixed solution was added, and the mixture was reacted at 55°C and 180 r / min for 30 min to obtain a reaction mixture. The piperine was obtained in Example 1, and the cinnamic acid was obtained in Example 2.

[0042] (2) The reaction mixture was ultrasonically treated at 35°C and 100 W for 10 min, and then concentrated to 1 / 6 of the original volume to obtain a concentrated solution. The concentrated solution was subjected to normal phase silica gel column chromatography using ethyl acetate and petroleum ether as eluents, with a volume ratio of ethyl acetate:petroleum ether = 8:1, and the eluate was collected. The solution was concentrated and freeze-dried in a vacuum to obtain piperine-cinnamic acid self-assembled nanoparticles.

[0043] Example 7 Preparation of piperine-cinnamic acid self-assembled nanoparticles

[0044] This embodiment provides a method for preparing piperine-cinnamic acid self-assembled nanoparticles, the method comprising the following steps:

[0045] (1) 0.01 mmol / L piperine and 0.01 mmol / L cinnamic acid were mixed to obtain a mixed solution. The pH of the mixed solution was adjusted to 7.0-7.5 using 5 mg / mL NaOH, and a pH 7.4 phosphate buffer solution of an equal volume to the mixed solution was added, and the mixture was reacted at 60°C and 180 r / min for 30 min to obtain a reaction mixture. The piperine was obtained in Example 1, and the cinnamic acid was obtained in Example 2.

[0046] (2) The reaction mixture was ultrasonically treated at 35°C and 100 W for 10 min, and then concentrated to 1 / 6 of the original volume to obtain a concentrated solution. The concentrated solution was eluted by normal phase silica gel column chromatography with ethyl acetate and petroleum ether as eluents, with a volume ratio of ethyl acetate:petroleum ether = 8:1, and the eluate was collected. The eluate was dialyzed on a 2.0 KDa filter membrane for 16 h, concentrated, and vacuum freeze-dried to obtain piperine-cinnamic acid self-assembled nanoparticles.

[0047] Example 8 Preparation of piperine-cinnamic acid self-assembled nanoparticles

[0048] This embodiment provides a method for preparing piperine-cinnamic acid self-assembled nanoparticles, the method comprising the following steps:

[0049] (1) 0.01 mmol / L piperine and 0.01 mmol / L cinnamic acid were mixed to obtain a mixed solution. The pH of the mixed solution was adjusted to 7.0-7.5 using 5 mg / mL NaOH, and a pH 7.4 phosphate buffer solution of an equal volume to the mixed solution was added, and the mixture was reacted at 50°C and 180 r / min for 40 min to obtain a reaction mixture. The piperine was obtained in Example 1, and the cinnamic acid was obtained in Example 2.

[0050] (2) The reaction mixture was ultrasonically treated at 35°C and 100 W for 10 min, and then concentrated to 1 / 6 of the original volume to obtain a concentrated solution. The concentrated solution was eluted by normal phase silica gel column chromatography with ethyl acetate and petroleum ether as eluents, with a volume ratio of ethyl acetate:petroleum ether = 8:1, and the eluate was collected. The eluate was dialyzed on a 2.0 KDa filter membrane for 16 h, concentrated, and vacuum freeze-dried to obtain piperine-cinnamic acid self-assembled nanoparticles.

[0051] Example 9 Preparation of piperine-cinnamic acid self-assembled nanoparticles

[0052] This embodiment provides a method for preparing piperine-cinnamic acid self-assembled nanoparticles, the method comprising the following steps:

[0053] (1) 0.01 mmol / L piperine and 0.01 mmol / L cinnamic acid were mixed to obtain a mixed solution. The pH of the mixed solution was adjusted to 7.0-7.5 using 5 mg / mL NaOH, and an equal volume of pH 7.4 phosphate buffer was added to the mixed solution. The mixture was reacted at 55°C and 180 r / min for 30 min to obtain a reaction mixture. The piperine was purchased from Aladdin Reagent (Shanghai) Co., Ltd. (purity>97%), and the cinnamic acid was obtained in Example 2.

[0054] (2) The reaction mixture was ultrasonically treated at 35°C and 100 W for 10 min, and then concentrated to 1 / 6 of the original volume to obtain a concentrated solution. The concentrated solution was eluted by normal phase silica gel column chromatography with ethyl acetate and petroleum ether as eluents, with a volume ratio of ethyl acetate:petroleum ether = 8:1, and the eluate was collected. The eluate was dialyzed on a 2.0 KDa filter membrane for 16 h, concentrated, and vacuum freeze-dried to obtain piperine-cinnamic acid self-assembled nanoparticles.

[0055] Experiment 1 Minimum inhibitory concentration determination experiment

[0056] The 9 samples of different mass concentrations were added to the MHB medium, shaken, and the growth of bacteria was determined by measuring the optical density at 600nm. 100 μL of samples of different concentrations (0.05 to 25.60 mg / mL) were added to 100 μL of bacterial suspension (OD600 = 0.1) and incubated in a constant temperature water bath at 37°C for 16 hours. Subsequently, the OD600 value was measured to evaluate the antibacterial effect. The MHB liquid medium containing bacteria was used as a blank bacterial control, and the MHB liquid medium was used as a solvent control. The inhibition rate was calculated according to the following formula:

[0057] Inhibition rate (%) = 1-(optical density of sample-optical density of solvent) / (optical density of blank bacteria-optical density of solvent) × 100%. The lowest concentration of the sample solution when the bacterial inhibition rate is 80% is the minimum inhibitory concentration (MIC).

[0058] The specific experimental results are shown in Table 1.

[0059] Table 1 Minimum inhibitory concentration of different groups against Staphylococcus aureus (mg / mL)

[0060]

[0061] Note: Different letters indicate significant differences among different groups ( p >0.05)

[0062] From Table 1, we can see that:

[0063] (1) Compared with Example 1-2, the minimum inhibitory concentration of the self-assembled particles of piperine and cinnamic acid in Example 3-9 is significantly reduced, indicating that the self-assembled nanoparticles of piperine and cinnamic acid can enhance the inhibitory effect of piperine on Staphylococcus aureus.

[0064] (2) Experimental Example 3 is compared with Example 9. The minimum inhibitory concentration of the self-assembled particles of piperine and cinnamic acid prepared by the piperine extracted by the method of the present invention is significantly reduced. It can be seen that the piperine extracted and crystallized by the citric acid-choline chloride deep eutectic solvent two-phase system can enhance the inhibitory effect of piperine on Staphylococcus aureus.

[0065] (3) In comparison between Experimental Example 3 and Examples 4, 5, and 6, changes in the preparation conditions of the self-assembled particles of piperine and cinnamic acid are not conducive to their ability to inhibit Staphylococcus aureus. Among them, the lack of the ultrasonic step is the most detrimental to the inhibition of Staphylococcus aureus by the self-assembled particles of piperine and cinnamic acid, followed by silica gel column purification.

[0066] (4) In comparison between Experimental Example 3 and Examples 7 and 8, changes in the reaction conditions of the reactants during the preparation of the piperine and cinnamic acid self-assembled particles are not conducive to the inhibition of Staphylococcus aureus. Among them, the change in reaction temperature is the most unfavorable for the inhibition of Staphylococcus aureus by the piperine and cinnamic acid self-assembled nanoparticles, and the reaction time is the second most influential.

[0067] 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-cinnamic acid self-assembled nanoparticle, characterized in that: The self-assembled nanoparticles are self-assembled nanoparticles constructed from piperine and cinnamic acid. The preparation method is as follows: 0.01 mmol / L piperine and 0.01 mmol / L cinnamic acid are mixed at a volume ratio of 1:1 to obtain a mixed solution, wherein the solvent of the 0.01 mmol / L piperine is a methanol solution; the solvent of the 0.01 mmol / L cinnamic acid is a 45°C phosphate buffer solution; the pH value of the mixed solution is adjusted to 7.0-7.5 using a 5 mg / mL sodium hydroxide solution, and then an equal volume of a phosphate buffer with a pH value of 7.4 is added to the mixed solution, and the mixture is reacted at 55°C and 180 r / min for 30 min to obtain a reaction mixture; the reaction mixture is ultrasonically heated at 35°C and 100W for 10 min. min later, the mixture was concentrated to 1 / 6 of the original volume to obtain a concentrated solution; the concentrated solution was subjected to normal phase silica gel column chromatography, eluted with ethyl acetate and petroleum ether, the volume ratio of ethyl acetate: petroleum ether = 8:1, and the eluate was collected, wherein the particle size of the silica gel in the silica gel column was 200-300 mesh; the eluate was dialyzed in a 2.0 KDa filter membrane for 16 h, concentrated, and vacuum freeze-dried to obtain piperine-cinnamic acid self-assembled nanoparticles; Wherein, the preparation method of piperine is: A1: uniformly mixing citric acid and choline chloride in a molar ratio of 3:1 and heating until the mixture forms a clear and transparent liquid to obtain a citric acid-choline chloride deep eutectic solvent; the heating temperature is 55° C. and the heating time is 30 min; A2: adding white pepper powder to the citric acid-choline chloride low eutectic solvent in step A1 to obtain a mixed solution, heating the mixed solution to 60° 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 piperine extract, wherein the solid-liquid ratio of the white pepper powder to the citric acid-choline chloride is 1 / 35 g / mL; A3: adding the piperine extract described in step A2 to an 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 isopropanol and water, and the mass ratio of the piperine extract, isopropanol, and water is 2:2:3; the upper phase is an isopropanol phase containing piperine, and the lower phase is a low eutectic solvent phase containing a mixture of the piperine extract; the upper phase is distilled under reduced pressure to 1 / 8 of the original volume, and residual isopropanol is removed to obtain a piperine extract; A4: Add 3 times the volume of anhydrous ethanol to the piperine extract in step A3, crystallize at 4°C for 24 hours, collect the crystals, and freeze-dry them in vacuum to obtain high-purity piperine; The cinnamic acid is commercially available with a purity of >90%.

2. Use of the piperine-cinnamic acid self-assembled nanoparticles according to claim 1 in preparing an antibacterial agent for Staphylococcus aureus.

Images