Cinnamaldehyde and arginine self-assembled nano particle as well as preparation method and application thereof

Abstract

The invention relates to a self-assembled nanoparticle formed by cinnamyl aldehyde and arginine through Schiff base reaction as well as a preparation method and application of the self-assembled nanoparticle. The method comprises the following steps: reacting cinnamyl aldehyde with arginine according to a molar ratio of (1: 2)-(2: 1) in absolute methanol to generate cinnamyl aldehyde-arginine Schiff base containing-C = N-bonds; the nano-particles are dissolved in dimethyl sulfoxide to prepare a stock solution, the stock solution is slowly and dropwise added into deionized water, and the nano-particles are formed through self-assembly. The average particle size of the obtained nanoparticles is 230-350nm, the polydispersity index (PDI) is less than or equal to 0.1, the critical aggregation concentration is as low as 10mu g / mL, and the nanoparticles show excellent self-assembly capability. The cinnamyl aldehyde and arginine self-assembled nano particles have good water solubility and pH response characteristics, active ingredients can be released through-C = N-bond breakage under the acidic condition, and the antibacterial property is remarkably superior to that of free cinnamyl aldehyde. The method is simple in process, environmentally friendly and suitable for the fields of drug delivery systems, food preservation, antibacterial coatings, cosmetics and the like.

Description

Technical Field

[0001] The present invention relates to the field of material technology, and in particular to cinnamaldehyde and arginine self-assembled nanoparticles, a preparation method and application thereof. Background Art

[0002] Cinnamaldehyde, a natural active ingredient found widely in plants like cinnamon, has a unique aroma and significant biological activities, including antibacterial, antioxidant, anti-inflammatory, and anti-cancer properties. It holds significant application value and promise in areas such as food preservation, pharmaceutical research and development, cosmetics, and biomaterials. In the food industry, cinnamaldehyde has been shown to effectively inhibit the growth of various microorganisms, significantly extending the shelf life of food, making it a highly sought-after natural preservative.

[0003] However, despite the many advantages of cinnamaldehyde, its practical application is limited by some physical and chemical properties. The main problems include low water solubility, high volatility and potential instability, which greatly limit its effective dispersion and delivery in water-based systems such as pharmaceutical preparations or biocompatible coatings. In addition, since it is prone to degradation, volatilization or inactivation during storage and use, this further reduces its bioavailability and duration of action. In order to address the above challenges, the existing technology generally adopts two strategies: one is to chemically modify cinnamaldehyde to improve its solubility or stability, but this often requires complex synthesis steps and may change its original active structure or introduce potential toxicity; the other is to use nanocarrier encapsulation technology to achieve sustained release, protection and targeted delivery through physical encapsulation, but this method has problems such as complex preparation process, high cost and unstable encapsulation efficiency.

[0004] Therefore, there is an urgent need in this field to develop a simple, efficient, low-cost, and biocompatible method that can simultaneously overcome the problem of poor solubility / stability of cinnamaldehyde. Summary of the Invention

[0005] The present invention addresses the existing problems of cinnamaldehyde, such as poor water solubility, low stability, and susceptibility to oxidative degradation. It provides cinnamaldehyde-arginine self-assembled nanoparticles and a method for preparing the same. Through a Schiff base condensation reaction, cinnamaldehyde and arginine can chemically combine under mild conditions. The product's amphiphilic structure then spontaneously assembles in water to form nanoparticles with uniform particle size. This method not only simplifies the preparation process but also, due to the use of economically available raw materials, enables large-scale production. More importantly, this nanoaggregate structure significantly improves the water solubility of cinnamaldehyde and provides a new approach to enhancing its stability. These nanoparticles not only significantly increase the water solubility and stability of cinnamaldehyde but also effectively enhance its antibacterial activity, making them suitable for a variety of applications, including drug delivery systems, food preservation, and antibacterial coatings.

[0006] To achieve the above object, the present invention provides a method for preparing self-assembled nanoparticles of cinnamaldehyde and arginine, which is characterized by comprising the following steps:

[0007] S1. Add cinnamaldehyde (CA) and arginine (Arg) in a molar ratio of 1:2 to 2:1 to anhydrous methanol and stir at 45-75°C for 12 hours to produce a yellow solution.

[0008] S2. After the reaction is completed, the solvent is removed by rotary evaporation. The residue is dissolved in a small amount of anhydrous methanol and precipitated by adding acetone, wherein the volume ratio of anhydrous methanol to acetone is 1:3 to 1:10. The product is filtered and vacuum dried to obtain a yellow powdery product, cinnamaldehyde-arginine complex (CA-Arg).

[0009] S3. Dissolve the above product in dimethyl sulfoxide to prepare a stock solution with a concentration of 10–40 mg / mL;

[0010] S4. Under continuous stirring, slowly add the DMSO stock solution dropwise to deionized water to a volume ratio of DMSO to water of 1:20 to 1:50. Let it stand for 1 hour to promote self-assembly to form uniformly dispersed nanoparticles (CANPs).

[0011] In the present invention, cinnamaldehyde reacts with arginine through Schiff base to generate CA-Arg molecules with -C=N-bonds. The CA-Arg molecules can spontaneously form nanoparticles with uniform particle size and narrow distribution in water at a specific concentration.

[0012] Furthermore, the nanoparticles are spontaneously formed by the cinnamaldehyde-arginine complex in water and have a uniform spherical structure with a particle size of 230-350 nm and a polydispersity index (PDI) of ≤0.1.

[0013] Furthermore, the average particle size of the nanoparticles is 301±11.6 nm.

[0014] Furthermore, the nanoparticles are self-assembled from a Schiff base structure formed by connecting cinnamaldehyde and arginine via a -C=N- bond.

[0015] Furthermore, the critical aggregation concentration of the nanoparticles is 10 μg / mL.

[0016] Furthermore, the nanoparticles are pH responsive and have long-term stability in a pH 7.4 environment.

[0017] Furthermore, under acidic conditions, the nanoparticles increase in size and release active ingredients.

[0018] The present invention also provides an application of the cinnamaldehyde and arginine self-assembled nanoparticles in a drug delivery system, food preservation, antibacterial coating or cosmetics.

[0019] The beneficial effects of the present invention are:

[0020] 1. Novel structure and simple process: The reaction process is mild and requires economical raw materials. CA-Arg is synthesized through the Schiff base reaction of cinnamaldehyde and arginine, which is common natural products. It is further self-assembled into nanoparticles (CANPs) in a DMSO / water system to efficiently generate the target product without the need for complex catalysts or harsh conditions, making it suitable for large-scale production.

[0021] 2. Excellent performance and strong stability: The obtained CANPs have uniform particle size (301±11.6nm), PDI≤0.1, long-term stability in water (no obvious change within 28 days), critical aggregation concentration as low as 10μg / mL, and have good self-assembly ability and application potential.

[0022] 3. pH responsiveness and high antibacterial activity: CANPs can release active ingredients by cleaving the -C=N- bond under acidic conditions. Compared with free cinnamaldehyde, it has stronger antibacterial ability per unit mole and exhibits higher antibacterial efficiency.

[0023] 4. Broad application prospects: The self-assembled nanoparticles have good water dispersibility and potential biocompatibility, providing a new and simple technical path for applying cinnamaldehyde and arginine in drug delivery systems, food preservation, antibacterial coatings or cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0025] Figure 1 3. It is the ultraviolet-visible (UV-Vis) absorption spectrum of the Schiff base product (CA-Arg) formed by cinnamaldehyde (CA) and arginine (Arg) prepared according to Example 1 of the present invention and cinnamaldehyde.

[0026] Figure 2 The H NMR spectrum of the Schiff base product (CA-Arg) formed by cinnamaldehyde (CA) and arginine (Arg) prepared according to Example 1 of the present invention in DMSO-d6 ( 1 1H NMR).

[0027] Figure 33 is the mass spectrum (Mass) of the Schiff base product (CA-Arg) formed by cinnamaldehyde (CA) and arginine (Arg) prepared according to Example 1 of the present invention.

[0028] Figure 4 This is a comparison chart of the particle size distribution of self-assembled nanoparticles at different CA / Arg molar ratios according to Examples 4-6 of the present invention.

[0029] Figure 5 3 is a particle size distribution diagram of self-assembled nanoparticles (CANPs) prepared according to Example 4 of the present invention.

[0030] Figure 6 The critical aggregation concentration (CAC) of the self-assembly prepared in Example 4 was determined by fluorescence method.

[0031] Figure 7 Graph showing the particle size change of the self-assembled nanoparticles (CANPs) prepared in Example 4 within 28 days.

[0032] Figure 8 The scanning electron microscope (SEM) morphology of the self-assembled nanoparticles (CANPs) prepared in Example 4 under different pH environments (A: pH = 7.4, B: pH = 5.5).

[0033] Figure 9 2 is the zeta potential diagram of the self-assembled nanoparticles (CANPs) prepared in Example 4 at pH values ​​of 7.4 and 5.5.

[0034] Figure 10 The minimum inhibitory concentration (MIC) of cinnamaldehyde and the self-assembled nanoparticles (CANPs) prepared in Example 4 against Escherichia coli in vitro was compared. DETAILED DESCRIPTION

[0035] The present invention will now be described in further detail with reference to the accompanying drawings, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0036] Example 1

[0037] This embodiment provides a method for preparing cinnamaldehyde-arginine Schiff base (CA-Arg), which specifically comprises the following steps:

[0038] Arginine (174.2 mg, 1 mmol) and cinnamaldehyde (132.16 mg, 1 mmol) were placed in a 100 mL round-bottom flask and 50 mL of anhydrous methanol was added to form a yellow solution. The reaction was stirred at 45°C for 12 h. The solvent was then removed by rotary evaporation. 0.5 mL of anhydrous methanol was then added to fully dissolve the solution. 5 mL of acetone (anhydrous methanol:acetone = 1:10) was then added. The solution was allowed to stand until a precipitate formed. The solution was then filtered and dried under vacuum to obtain a yellow powder.

[0039] Example 2

[0040] This example provides a method for preparing cinnamaldehyde-arginine Schiff base (CA-Arg), which differs from Example 1 only in that the ratio of cinnamaldehyde to arginine is replaced from 1:1 to 1:2. The method specifically includes the following steps:

[0041] Arginine (174.2 mg, 1 mmol) and cinnamaldehyde (264.32 mg, 2 mmol) were placed in a 100 mL round-bottom flask and added with 50 mL of anhydrous methanol to produce a yellow solution. The reaction was stirred at 45°C for 12 h. The solvent was then removed by rotary evaporation. 0.5 mL of anhydrous methanol was then added to fully dissolve the solution. 5 mL of acetone (anhydrous methanol:acetone = 1:10) was then added. The solution was allowed to stand until a precipitate formed. The solution was then filtered and dried under vacuum to obtain a yellow powder.

[0042] Example 3

[0043] This example provides a method for preparing cinnamaldehyde-arginine Schiff base (CA-Arg), which differs from Example 1 only in that the ratio of cinnamaldehyde to arginine is replaced from 1:1 to 2:1. The method specifically includes the following steps:

[0044] Arginine (348.4 mg, 2 mmol) and cinnamaldehyde (132.16 mg, 1 mmol) were placed in a 100 mL round-bottom flask and 50 mL of anhydrous methanol was added to produce a yellow solution. The reaction was stirred at 45°C for 12 h. The solvent was then removed by rotary evaporation. 0.5 mL of anhydrous methanol was then added to fully dissolve the solution. 5 mL of acetone (anhydrous methanol:acetone = 1:10) was then added. The solution was allowed to stand until a precipitate formed. The solution was then filtered and dried under vacuum to obtain a yellow powder.

[0045] Example 4

[0046] This example is based on the cinnamaldehyde-arginine Schiff base product (CA-Arg) of Example 1, and describes in detail the preparation and characterization methods of its self-assembled nanoparticles (CANPs):

[0047] First, 20 mg of the yellow powder obtained in Example 1 was dissolved in 0.5 mL of dimethyl sulfoxide (DMSO) to prepare a 40 mg / mL stock solution. The powder was then sonicated to ensure complete dissolution. Next, the DMSO stock solution was slowly added dropwise to 39 mL of deionized water under continuous stirring, achieving a 1:39 volume ratio of DMSO to water. After the addition, the mixture was allowed to stand for 1 hour to promote self-assembly, ultimately forming a dispersion of Arg-Cin nanoparticles.

[0048] Example 5

[0049] This example is based on the cinnamaldehyde-arginine Schiff base product (CA-Arg) of Example 2, and describes in detail the preparation and characterization methods of its self-assembled nanoparticles (CANPs):

[0050] First, 20 mg of the yellow powder obtained in Example 2 was dissolved in 0.5 mL of dimethyl sulfoxide (DMSO) to prepare a 40 mg / mL stock solution. The powder was then sonicated to ensure complete dissolution. Next, the DMSO stock solution was slowly added dropwise to 39 mL of deionized water under continuous stirring, achieving a 1:39 volume ratio of DMSO to water. After the addition, the mixture was allowed to stand for 1 hour to promote self-assembly, ultimately forming a dispersion of Arg-Cin nanoparticles.

[0051] Example 6

[0052] This example is based on the cinnamaldehyde-arginine Schiff base product (CA-Arg) of Example 3, and describes in detail the preparation and characterization methods of its self-assembled nanoparticles (CANPs):

[0053] First, 20 mg of the yellow powder obtained in Example 3 was dissolved in 0.5 mL of dimethyl sulfoxide (DMSO) to prepare a 40 mg / mL stock solution. The powder was then sonicated to ensure complete dissolution. Next, the DMSO stock solution was slowly added dropwise to 39 mL of deionized water under continuous stirring, achieving a 1:39 volume ratio of DMSO to water. After the addition, the mixture was allowed to stand for 1 hour to promote self-assembly, ultimately forming a dispersion of Arg-Cin nanoparticles.

[0054] like Figure 1To preliminarily verify the possibility of a Schiff base product (CA-Arg) formed from the reaction of cinnamaldehyde (CA) and arginine (Arg) in Example 1, the resulting yellow powder was dissolved in methanol and characterized by UV-visible absorption spectroscopy (UV-Vis). The results revealed a strong absorption peak at 265 nm, primarily attributable to the π→π* electronic transition of the benzene ring. Simultaneously, a distinct absorption shoulder was observed around 340 nm, attributed to the n→π* transition of the imine bond (-C=N-), suggesting the formation of a Schiff base structure. These spectral characteristics were consistent with the expected structure, supporting the successful synthesis of CA-Arg.

[0055] like Figure 2 As shown, in order to further confirm the successful preparation of the Schiff base product (CA-Arg) formed by cinnamaldehyde (CA) and arginine (Arg) in Example 1, the prepared yellow powder was dissolved in dimethyl sulfoxide-d6 (DMSO-d6) and subjected to nuclear magnetic resonance spectroscopy ( 1 H NMR) characterization. The characteristic peak observed at 7.97 ppm represents the formation of a Schiff base bond (-C=N-), which clearly indicates that the target molecule CA-Arg has been successfully synthesized.

[0056] like Figure 3 As shown, to further confirm the successful preparation of the Schiff base product (CA-Arg) formed by cinnamaldehyde (CA) and arginine (Arg) in Example 1, the prepared yellow powder was dissolved and subjected to mass spectrometry analysis. The mass spectrum shows that the accurate mass of the molecule is 288.16 Da. In the mass spectrum, the peak at 289.1669 m / z represents the molecular ion peak with an extra proton [M+H] + The presence of this characteristic peak indicates that the prepared compound has the expected molecular weight and structure, which further verifies the successful synthesis of the target molecule CA-Arg.

[0057] In summary, UV-visible absorption spectroscopy, H-NMR spectroscopy ( 1 The successful preparation of the Schiff base product (CA-Arg) formed from cinnamaldehyde (CA) and arginine (Arg) in Example 1 was confirmed by various characterization methods, including H NMR and mass spectrometry. The characteristic absorption shoulder at 340 nm in the UV spectrum indicated the formation of an imine bond (-C=N-); the characteristic peak observed at 7.97 ppm in the H NMR spectrum further confirmed the presence of the Schiff base structure; mass spectrometry analysis showed a molecular weight of 288.16 Da, and [M+H] + The ion peak (289.1669 m / z) is consistent with the theoretical value. The above results fully verify the structural correctness and synthetic feasibility of the target molecule CA-Arg from different perspectives.

[0058] like Figure 4 As shown in Figure 2, the effects of different CA / Arg molar ratios in Examples 4-6 on the particle size and morphology of self-assembled nanoparticles were investigated. The results showed that with the increase in the proportion of arginine (Arg), the particle size of the nanoparticles gradually increased, and the polydispersity index (PDI) also became wider, indicating that the uniformity of the system decreased. After system optimization, it was found that when the molar ratio of CA to Arg was 1:1, the nanoparticles formed had the best size distribution and morphological characteristics. The average particle size was 301±11.6nm, and the PDI value was 0.090±0.002, showing good uniformity ( Figure 4 B, Figure 5 ). Therefore, this ratio was selected for subsequent experimental studies.

[0059] like Figure 6 As shown in the figure, the critical aggregation concentration of the self-assembly was determined by fluorescence. The experimental results showed that at a concentration of 10 μg / mL, the I1 / I3 ratio showed a clear inflection point, indicating that the self-assembly began to transition from a monomer state to an aggregate state. Therefore, 10 μg / mL can be determined as the critical aggregation concentration of the self-assembly. This result further verifies that CANPs can effectively form stable nanostructures at a specific concentration, which is crucial for its application as a cinnamaldehyde stabilizer and antibacterial material. Below the critical aggregation concentration, the molecules mainly exist in the form of monomers, but above this concentration, they spontaneously form nanoaggregates with excellent physicochemical properties. This concentration-dependent self-assembly behavior not only helps to understand the formation mechanism of CANPs, but also provides an important reference for its concentration regulation in practical applications.

[0060] like Figure 7 As shown, we monitored the particle size changes of the self-assembled nanoparticles (CANPs) prepared in Example 4 over 28 days to study their stability in a pH 7.4 environment. The results showed that throughout the observation period, the average particle size of the nanoparticles remained stable at approximately 301±11.6nm, with a PDI value of approximately 0.090, indicating that the particle size distribution remained essentially unchanged, showing good long-term stability.

[0061] like Figure 8 As shown in Figure 3, the morphological characteristics of CANPs under different pH environments were observed by scanning electron microscopy (SEM). Figure 8 A shows that under pH 7.4, CANPs present a uniform and regular spherical structure with a particle size of approximately 301±11.6nm, indicating that the nanoparticles have good dispersibility and stability under this condition and are suitable as efficient antibacterial materials. It is worth noting that Figure 8Figure B shows that at pH 5.5, the CANPs exceed 1 μm in size, exhibiting significant size changes and morphological irregularities. This suggests that as the pH decreases, the dynamic acid-sensitive imine bonds may cleave, leading to structural changes in the nanoparticles. Nevertheless, even under acidic conditions, these aggregates effectively protect the internal cinnamaldehyde molecules, preventing rapid degradation and maintaining high water solubility.

[0062] like Figure 9 As shown in the figure, as the pH value decreases from 7.4 to 5.5, the zeta potential of CANPs increases significantly, from -9.86mV to +11.2mV. This change further confirms the reactivity of CANPs in acidic environments. Specifically, when the pH value decreases, the charge properties of the nanoparticle surface change significantly, indicating that the dynamic acid-sensitive imine bond in its internal structure may have been cleaved, resulting in the exposure of the positively charged arginine group, thereby causing a significant increase in the zeta potential. This pH-dependent charge change not only affects the stability of CANPs, but also has an important impact on their antibacterial properties. Under acidic conditions, positively charged CANPs can more effectively interact with the negatively charged bacterial cell membrane, enhancing their antibacterial effect. Therefore, by adjusting the pH value, the antibacterial activity of CANPs can be precisely controlled, so that they can perform optimally in different application scenarios.

[0063] like Figure 10 As shown in Figure 2, the antibacterial activity of cinnamaldehyde and its derivatives, the self-assembled nanoparticles (CANPs) prepared in Example 4, was evaluated by in vitro minimum inhibitory concentration (MIC) experiments. The standard microdilution method was used to treat Escherichia coli in a 96-well plate with a concentration gradient of 1000, 500, 250, 125, and 62.5 μg / mL. The OD was measured after incubation at 37°C for 16–20 hours. 600 The results showed that the minimum inhibitory mass concentration (MIC) of CANPs and free cinnamaldehyde was 500 μg / mL. However, considering that the molecular weight of CA-Arg is 288.16 g / mol, which is significantly higher than that of cinnamaldehyde (132.16 g / mol), its molar concentration is only about 46% of that of cinnamaldehyde at the same mass concentration. Therefore, based on the molar concentration, CANPs exhibit stronger antibacterial ability per unit mole, indicating that they have higher antibacterial efficiency.

[0064] In summary, the present invention provides a simple and efficient method for preparing cinnamaldehyde and arginine self-assembled nanoparticles (CANPs), which not only significantly improves the water solubility and stability of cinnamaldehyde, but also effectively enhances its antibacterial activity. Through a series of detailed characterization methods, including UV-visible absorption spectroscopy, nuclear magnetic hydrogen spectrum, mass spectrometry analysis, and fluorescence determination of critical aggregation concentration, we confirmed the successful synthesis of the target molecule and its self-assembly behavior. The experimental results showed that CANPs exhibited excellent stability and response characteristics under different pH environments, and its minimum inhibitory concentration was significantly lower than that of cinnamaldehyde used alone, showing a significant synergistic antibacterial effect. This provides new ideas and methods for the development of efficient and stable natural antibacterial materials, and has important application prospects.

[0065] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A method for preparing self-assembled nanoparticles of cinnamaldehyde and arginine, characterized in that: The following steps are involved: S1. Add cinnamaldehyde and arginine in a molar ratio of 1:2 to 2:1 to anhydrous methanol and stir at 45 to 75°C for 12 hours to produce a yellow solution; S2. After the reaction is completed, the solvent is removed by rotary evaporation, the residue is dissolved with a small amount of anhydrous methanol, and acetone is added to precipitate, wherein the volume ratio of anhydrous methanol to acetone is 1:3 to 1:10; filtered and vacuum dried to obtain a yellow powder product cinnamaldehyde-arginine complex; S3. Dissolve the above product in dimethyl sulfoxide to prepare a stock solution with a concentration of 10–40 mg / mL; S4. Slowly add the DMSO stock solution dropwise to deionized water under continuous stirring to a volume ratio of DMSO to water of 1:20 to 1:

50. Let the mixture stand for 1 hour to promote self-assembly to form uniformly dispersed nanoparticles.

2. The nanoparticles prepared by the preparation method according to claim 1, characterized in that: The nanoparticles are spontaneously formed by the cinnamaldehyde-arginine complex in water and have a uniform spherical structure with a particle size of 230-350 nm and a polydispersity index (PDI) of ≤0.

1.

3. The nanoparticles prepared by the preparation method according to claim 2, characterized in that: The nanoparticles are self-assembled from a Schiff base structure formed by connecting cinnamaldehyde and arginine via a -C=N- bond.

4. The cinnamaldehyde and arginine self-assembled nanoparticles according to claim 2, characterized in that The critical aggregation concentration of the nanoparticles is 10 μg / mL.

5. The cinnamaldehyde and arginine self-assembled nanoparticles according to claim 2, characterized in that The nanoparticles are pH responsive and have long-term stability in a pH 7.4 environment.

6. The cinnamaldehyde and arginine self-assembled nanoparticles according to claim 2, characterized in that Under acidic conditions, the nanoparticles increase in size and release active ingredients.

7. Use of the cinnamaldehyde and arginine self-assembled nanoparticles according to any one of claims 2 to 6 in drug delivery systems, food preservation, antibacterial coatings or cosmetics.

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