Drug-loaded stable curcumin composite nanoparticles and a preparation method thereof

Abstract

The present application relates to the technical field of drug-loaded particles, and discloses a kind of drug-loaded stable curcumin composite nanoparticles and preparation method thereof, including rice protein enzymatic hydrolysate as carrier, load curcumin, and using chondroitin sulfate to stabilize the rice protein enzymatic hydrolysate load the curcumin, the curcumin composite nanoparticle encapsulation efficiency is 70%~90%, the mass ratio of the contained rice protein enzymatic hydrolysate and chondroitin sulfate is 1~5:1, the mass ratio of the contained rice protein enzymatic hydrolysate and curcumin is 8~12:1.In dissolved in gastric acid with pH 0.9~1.5, the protein in nanoparticle still keeps stable, and still can keep larger curcumin retention rate, particle size also keeps in nanometer, and nanometer particle size also makes nanoparticle easy to be taken up by intestinal cells, effectively improves bioavailability.

Description

Technical Field

[0001] This invention relates to the field of drug-loaded particle technology, and in particular to a drug-loaded and stable curcumin composite nanoparticle and its preparation method. Background Technology

[0002] Curcumin possesses a wide range of physiological activities, including antioxidant, anti-inflammatory, anti-cancer, anti-tumor, and anti-lipid metabolism disorders. It has been approved by relevant institutions and countries such as the European Union and China for use as a functional ingredient in health foods. However, due to the limitations of curcumin, such as its low water solubility (only 11 ng / mL in water), poor stability (easily degraded or loses its physiological activity due to processing conditions and environmental factors such as pH, temperature, oxygen, and light), and rapid metabolic rate (difficult to penetrate physiological barriers in vivo and easily metabolized or cleared by the liver), its bioavailability and potential health effects are greatly limited.

[0003] Complex nanocarrier matrices formed by the interaction of proteins and polysaccharides may help further improve the encapsulation capacity, protective effect, and bioavailability of bioactive substances. Rice protein (RP) is a recognized high-quality plant protein with advantages such as balanced amino acid composition, high bioavailability, high protein utilization, and low allergenicity. However, its low solubility is limited by the large number of disulfide crosslinks and hydrophobic interactions in gluten, the main component of rice protein, which restricts its large-scale commercial application in the food industry. Enzymatic modification of rice protein can effectively improve its solubility, and the resulting rice protein hydrolysate (RPH) has been shown to have the potential to be used as a nanocarrier for bioactive substances. However, the stability of rice protein hydrolysate nanocarriers in response to environmental changes is poor; the addition of polysaccharides is expected to improve its performance and stability. Chondroitin sulfate (Cs) is a sulfated glycosaminoglycan composed of acetylgalactosamine and glucuronic acid. It possesses various physiological activities, including anti-inflammatory, antioxidant, and immunomodulatory effects, and is used as a nutritional supplement and over-the-counter medication for treating osteoarthritis. Currently, chondroitin sulfate has been shown to effectively improve the encapsulation and protection of bioactive substances by protein-based nanocarriers such as zein and gliadin.

[0004] Chinese patent application number 202110432458.4 discloses a high-loading, high-stability protein-based curcumin product and its preparation method and application. It uses basic protein as a carrier to load curcumin to improve the solubility, suspension stability and biocompatibility of curcumin in water. However, this patent does not take into account that curcumin is absorbed by the gastrointestinal tract after being ingested by the human body. When the particle size of the curcumin product is too large, the absorption rate of gastrointestinal cells will be significantly reduced. In addition, many basic proteins are easily denatured under the action of gastric acid and are difficult to remain stable, which leads to the premature release of the loaded curcumin, resulting in unsatisfactory bioavailability of curcumin. Summary of the Invention

[0005] To address the issue that existing technologies for improving curcumin solubility do not consider bioavailability, this invention provides drug-loaded and stable curcumin composite nanoparticles, characterized by small particle size, high solubility, good dispersibility, and easy absorption by intestinal cells. This invention also provides a method for preparing drug-loaded and stable curcumin composite nanoparticles, resulting in curcumin composite nanoparticles with small particle size, high solubility, good dispersibility, and easy absorption by intestinal cells.

[0006] This invention is achieved through the following technical solution:

[0007] A drug-loaded and stabilized curcumin composite nanoparticle comprises using rice protein hydrolysate as a carrier to load curcumin, and using chondroitin sulfate to stabilize the rice protein hydrolysate loaded with curcumin. The encapsulation rate of the curcumin composite nanoparticle is 70% to 90%, the mass ratio of rice protein hydrolysate to chondroitin sulfate is 1 to 5:1, and the mass ratio of rice protein hydrolysate to curcumin is 8 to 12:1.

[0008] Nanoparticles can remain suspended and dispersed in water for extended periods, resisting aggregation and precipitation. This allows for greater dissolution of curcumin, facilitating absorption through the gastrointestinal tract. Furthermore, the stability of nanoparticles in acidic to neutral environments means that even when dissolved in gastric acid at pH 0.9–1.5, the cross-linking effect of chondroitin sulfate ensures the stability of rice protein hydrolysates within the nanoparticles, maintaining a high curcumin retention rate and nanoscale particle size. This nanoscale size also facilitates uptake by intestinal cells, effectively enhancing bioavailability. Because chondroitin sulfate exhibits relatively ideal stability to environmental changes and can inhibit the aggregation and precipitation of rice protein hydrolysates through electrostatic repulsion and steric hindrance, the rice protein hydrolysate-chondroitin sulfate binary composite nanocarrier exhibits lower environmental sensitivity and higher stability. The presence of chondroitin sulfate further stabilizes the morphology of the nanoparticles, preventing denaturation caused by changes in the folding state of the rice protein hydrolysates under the influence of gastric acid and pepsin, thus preventing premature leakage of the loaded curcumin.

[0009] Preferably, the nanoparticles are stable in dissolution and have a curcumin retention rate of greater than 85% in an environment of room temperature to 80°C; and the nanoparticle size is less than or equal to 300 nm when the pH value is below 2.

[0010] Preferably, the nanoparticles have a particle size of 100–190 nm and a zeta potential of -20–-30 mV.

[0011] Rice protein, primarily composed of gluten, suffers from low solubility due to numerous disulfide cross-links and hydrophobic interactions, limiting its large-scale commercial application in the food industry. Enzymatic modification of rice protein not only effectively improves its solubility but also yields rice protein hydrolysate (RPH), which has been shown to have potential as a nanocarrier for bioactive substances. However, RPH nanocarriers exhibit poor stability in response to environmental changes; the addition of polysaccharides is expected to improve their performance and stability. Chondroitin sulfate, an acidic mucopolysaccharide extracted from animal tissues, is hygroscopic. Chondroitin sulfate aqueous solution is viscous and does not coagulate upon heating. It is readily soluble in water but insoluble in organic solvents such as ethanol, acetone, and ether. Its salts are relatively stable to heat and are not destroyed even when heated to 80°C. However, chondroitin sulfate aqueous solution becomes unstable at higher temperatures or in acidic environments, mainly due to deacetylation or degradation into monosaccharides or polysaccharides with smaller molecular weights. However, when combined with rice protein hydrolysate to form a rice protein hydrolysate-chondroitin sulfate binary composite nanocarrier, it can maintain a stable state.

[0012] A method for preparing drug-loaded and stabilized curcumin composite nanoparticles includes the following steps:

[0013] 1) Prepare an aqueous solution of rice protein hydrolysate and prepare an aqueous solution of chondroitin sulfate, and mix and stir to form an aqueous solution of rice protein hydrolysate-chondroitin sulfate binary composite nanocarrier;

[0014] 2) Add curcumin ethanol solution dropwise into the aqueous solution of rice protein hydrolysate-chondroitin sulfate binary composite nanocarrier prepared in step 1), and adjust the pH value to 3-7 to obtain a mixed solution;

[0015] 3) The mixture prepared in step 2) is rotary evaporated, diluted to a fixed volume, and centrifuged to remove the precipitate. The supernatant is then refrigerated to obtain curcumin composite nanoparticles.

[0016] Preferably, in step 1), the mass concentration of the rice protein hydrolysate aqueous solution is 0.8–1.5 mg / mL, the mass concentration of the chondroitin sulfate aqueous solution is 0.8–1.5 mg / mL, and the mixing mass ratio of the rice protein hydrolysate and chondroitin sulfate is controlled at 1–5:1.

[0017] Preferably, in step 2), the mass concentration of curcumin ethanol solution is 0.8~1.5 mg / mL, and the mass ratio of rice protein hydrolysate to curcumin is controlled at 8~12:1.

[0018] Preferably, in step 3), the rotary evaporation temperature is 35~40℃, the centrifugation speed is 7000~10000×g, and the refrigeration temperature is 3~5℃.

[0019] An oral drug made from curcumin composite nanoparticles prepared according to the above-described drug-loaded stable curcumin composite nanoparticles or the above-described method for preparing drug-loaded stable curcumin composite nanoparticles.

[0020] The beneficial effects of this invention are:

[0021] (1) In this invention, rice protein hydrolysate and curcumin are self-assembled together to form composite nanoparticles and cross-linked with chondroitin sulfate, which cleverly avoids imine cross-linking agents and aldehyde cross-linking agents. The solubility and dispersibility of rice protein hydrolysate and curcumin are significantly improved. The nanoparticles have high curcumin loading rate and high stability. Even if the pH value becomes very small, the curcumin can still be kept in the carrier and it is difficult to leak. The overall preparation process is environmentally friendly and the toxicity is controllable.

[0022] (2) The curcumin nanoparticles of the present invention have high stability and high dispersibility in the physiological environment, so that they can remain relatively stable when passing through the low pH environment of the stomach until they are taken up by intestinal cells, effectively exerting antioxidant properties and having high bioavailability.

[0023] (3) The present invention uses an antisolvent method to encapsulate curcumin in rice protein hydrolysate, with an encapsulation rate of 70% to 90%.

[0024] (4) The preparation process of the present invention is simple and low in cost, which is conducive to industrial promotion. Attached Figure Description

[0025] Figure 1 The preparation process of rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles. Figure 2 X-ray diffraction patterns of rice protein hydrolysate (RPH), chondroitin sulfate, curcumin, and rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles (RPH-Cs-Cur NPs).

[0026] Figure 3 Scanning electron microscope image of rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles.

[0027] Figure 4The particle size and polydispersity index (PDI) of rice protein hydrolysate-curcumin nanoparticles (A Comparative Example 1) and rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles (B Example 2) were determined under different pH conditions.

[0028] Figure 5 The particle size and polydispersity index (PDI) of rice protein hydrolysate-curcumin nanoparticles (A Comparative Example 1) and rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles (B Example 2) under different NaCl concentrations were compared.

[0029] Figure 6 The retention rate of curcumin in rice protein hydrolysate-curcumin nanoparticles (RPH-Cur NPs, Comparative Example 1) and rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles (RPH-Cs-Cur NPs, Example 2) after treatment at 35~80℃ for 30 min was determined.

[0030] Figure 7 Qualitative (A) and quantitative (B) uptake of free curcumin, rice protein hydrolysate-curcumin nanoparticles (RPH-Cur NPs, Comparative Example 1), and rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles (RPH-Cs-Cur NPs, Example 2) by Caco-2 cells. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Unless otherwise specified, the means used in the embodiments are conventional means in the art. The terms "comprising," "including," or any other variations thereof used herein are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not explicitly listed or elements inherent to such a composition, step, method, article, or apparatus. Furthermore, the technical features involved in the various embodiments of the invention described below can be combined with each other as long as they do not conflict with each other. The experimental materials used in the embodiments and comparative examples of this invention are all commercially available products.

[0032] Example 1

[0033] A method for preparing drug-loaded and stabilized curcumin composite nanoparticles includes the following steps:

[0034] (1) Mix rice protein hydrolysate at a ratio of 1 mg / mL and stir until the solution is uniform and transparent to prepare an aqueous solution of rice protein hydrolysate;

[0035] (2) Mix chondroitin sulfate at a ratio of 1 mg / mL and stir until the solution is uniform and transparent to prepare an aqueous solution of chondroitin sulfate;

[0036] (3) Under light-protected conditions, curcumin and anhydrous ethanol were mixed at a ratio of 1 mg / mL and stirred until the solution was uniform and clear to prepare curcumin ethanol solution.

[0037] (4) Add the chondroitin sulfate aqueous solution from step (2) to the rice protein hydrolysate aqueous solution from step (1). The mass ratio of rice protein hydrolysate to chondroitin sulfate is 1:1. Stir at room temperature for 60 min to allow the rice protein hydrolysate and chondroitin sulfate to fully self-assemble and obtain a rice protein hydrolysate-chondroitin sulfate binary composite nanocarrier solution.

[0038] (5) Using a syringe pump at a rate of 1 mL / min, add the curcumin solution from step (3) to the rice protein hydrolysate-chondroitin sulfate binary composite nanocarrier solution from step (4) to obtain a rice protein hydrolysate-chondroitin sulfate-curcumin dispersion with a mass ratio of rice protein hydrolysate to curcumin of 10:1. Adjust the pH of the rice protein hydrolysate-chondroitin sulfate-curcumin dispersion to 4.0 using 0.1 mol / L HCl or NaOH solution to obtain crude rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles.

[0039] (6) Rice protein hydrolysate-chondroitin sulfate-curcumin crude composite nanoparticles were rotary evaporated at 40℃ for 10 min to remove ethanol, and then the volume was adjusted. The nanoparticles were centrifuged at 8000×g for 10 min to precipitate free curcumin. The supernatant was stored at 4℃ in the dark or freeze-dried for later use to obtain rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles.

[0040] Example 2

[0041] A method for preparing drug-loaded and stabilized curcumin composite nanoparticles includes the following steps:

[0042] (1) Mix rice protein hydrolysate at a ratio of 1 mg / mL and stir until the solution is uniform and transparent to prepare an aqueous solution of rice protein hydrolysate;

[0043] (2) Mix chondroitin sulfate at a ratio of 1 mg / mL and stir until the solution is uniform and transparent to prepare an aqueous solution of chondroitin sulfate;

[0044] (3) Under light-protected conditions, curcumin and anhydrous ethanol were mixed at a ratio of 1 mg / mL and stirred until the solution was uniform and clear to prepare curcumin ethanol solution.

[0045] (4) Add the chondroitin sulfate aqueous solution from step (2) to the rice protein hydrolysate aqueous solution from step (1). The mass ratio of rice protein hydrolysate to chondroitin sulfate is 2:1. Stir at room temperature for 60 min to allow the rice protein hydrolysate and chondroitin sulfate to fully self-assemble and obtain a rice protein hydrolysate-chondroitin sulfate binary composite nanocarrier solution.

[0046] (5) Using a syringe pump at a rate of 1 mL / min, add the curcumin solution from step (3) to the rice protein hydrolysate-chondroitin sulfate binary composite nanocarrier solution from step (4) to obtain a rice protein hydrolysate-chondroitin sulfate-curcumin dispersion with a mass ratio of rice protein hydrolysate to curcumin of 10:1. Adjust the pH of the rice protein hydrolysate-chondroitin sulfate-curcumin dispersion to 4.0 using 0.1 mol / L HCl or NaOH solution to obtain crude rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles.

[0047] (6) Rice protein hydrolysate-chondroitin sulfate-curcumin crude composite nanoparticles were rotary evaporated at 40℃ for 10 min to remove ethanol, and then the volume was adjusted. The nanoparticles were centrifuged at 8000×g for 10 min to precipitate free curcumin. The supernatant was stored at 4℃ in the dark or freeze-dried for later use to obtain rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles. Example

[0048] A method for preparing drug-loaded and stabilized curcumin composite nanoparticles includes the following steps:

[0049] (1) Mix rice protein hydrolysate at a ratio of 1 mg / mL and stir until the solution is uniform and transparent to prepare an aqueous solution of rice protein hydrolysate;

[0050] (2) Mix chondroitin sulfate at a ratio of 1 mg / mL and stir until the solution is uniform and transparent to prepare an aqueous solution of chondroitin sulfate;

[0051] (3) Under light-protected conditions, curcumin and anhydrous ethanol were mixed at a ratio of 1 mg / mL and stirred until the solution was uniform and clear to prepare curcumin ethanol solution.

[0052] (4) Add the chondroitin sulfate aqueous solution from step (2) to the rice protein hydrolysate aqueous solution from step (1). The mass ratio of rice protein hydrolysate to chondroitin sulfate is 3:1. Stir at room temperature for 60 min to allow the rice protein hydrolysate and chondroitin sulfate to fully self-assemble and obtain a rice protein hydrolysate-chondroitin sulfate binary composite nanocarrier solution.

[0053] (5) Using a syringe pump at a rate of 1 mL / min, add the curcumin solution from step (3) to the rice protein hydrolysate-chondroitin sulfate binary composite nanocarrier solution from step (4) to obtain a rice protein hydrolysate-chondroitin sulfate-curcumin dispersion with a mass ratio of rice protein hydrolysate to curcumin of 10:1. Adjust the pH of the rice protein hydrolysate-chondroitin sulfate-curcumin dispersion to 4.0 using 0.1 mol / L HCl or NaOH solution to obtain crude rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles.

[0054] (6) Rice protein hydrolysate-chondroitin sulfate-curcumin crude composite nanoparticles were rotary evaporated at 40℃ for 10 min to remove ethanol, and then the volume was adjusted. The nanoparticles were centrifuged at 8000×g for 10 min to precipitate free curcumin. The supernatant was stored at 4℃ in the dark or freeze-dried for later use to obtain rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles. Example

[0055] A method for preparing drug-loaded and stabilized curcumin composite nanoparticles includes the following steps:

[0056] (1) Mix rice protein hydrolysate at a ratio of 1 mg / mL and stir until the solution is uniform and transparent to prepare an aqueous solution of rice protein hydrolysate;

[0057] (2) Mix chondroitin sulfate at a ratio of 1 mg / mL and stir until the solution is uniform and transparent to prepare an aqueous solution of chondroitin sulfate;

[0058] (3) Under light-protected conditions, curcumin and anhydrous ethanol were mixed at a ratio of 1 mg / mL and stirred until the solution was uniform and clear to prepare curcumin ethanol solution.

[0059] (4) Add the chondroitin sulfate aqueous solution from step (2) to the rice protein hydrolysate aqueous solution from step (1). The mass ratio of rice protein hydrolysate to chondroitin sulfate is 4:1. Stir at room temperature for 60 min to allow the rice protein hydrolysate and chondroitin sulfate to fully self-assemble and obtain a rice protein hydrolysate-chondroitin sulfate binary composite nanocarrier solution.

[0060] (5) Using a syringe pump at a rate of 1 mL / min, add the curcumin solution from step (3) to the rice protein hydrolysate-chondroitin sulfate binary composite nanocarrier solution from step (4) to obtain a rice protein hydrolysate-chondroitin sulfate-curcumin dispersion with a mass ratio of rice protein hydrolysate to curcumin of 10:1. Adjust the pH of the rice protein hydrolysate-chondroitin sulfate-curcumin dispersion to 4.0 using 0.1 mol / L HCl or NaOH solution to obtain crude rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles.

[0061] (6) Rice protein hydrolysate-chondroitin sulfate-curcumin crude composite nanoparticles were rotary evaporated at 40℃ for 10 min to remove ethanol, and then the volume was adjusted. The nanoparticles were centrifuged at 8000×g for 10 min to precipitate free curcumin. The supernatant was stored at 4℃ in the dark or freeze-dried for later use to obtain rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles. Example

[0062] A method for preparing drug-loaded and stabilized curcumin composite nanoparticles includes the following steps:

[0063] (1) Mix rice protein hydrolysate at a ratio of 1 mg / mL and stir until the solution is uniform and transparent to prepare an aqueous solution of rice protein hydrolysate;

[0064] (2) Mix chondroitin sulfate at a ratio of 1 mg / mL and stir until the solution is uniform and transparent to prepare an aqueous solution of chondroitin sulfate;

[0065] (3) Under light-protected conditions, curcumin and anhydrous ethanol were mixed at a ratio of 1 mg / mL and stirred until the solution was uniform and clear to prepare curcumin ethanol solution.

[0066] (4) Add the chondroitin sulfate aqueous solution from step (2) to the rice protein hydrolysate aqueous solution from step (1). The mass ratio of rice protein hydrolysate to chondroitin sulfate is 5:1. Stir at room temperature for 60 min to allow the rice protein hydrolysate and chondroitin sulfate to fully self-assemble and obtain a rice protein hydrolysate-chondroitin sulfate binary composite nanocarrier solution.

[0067] (5) Using a syringe pump at a rate of 1 mL / min, add the curcumin solution from step (3) to the rice protein hydrolysate-chondroitin sulfate binary composite nanocarrier solution from step (4) to obtain a rice protein hydrolysate-chondroitin sulfate-curcumin dispersion with a mass ratio of rice protein hydrolysate to curcumin of 10:1. Adjust the pH of the rice protein hydrolysate-chondroitin sulfate-curcumin dispersion to 4.0 using 0.1 mol / L HCl or NaOH solution to obtain crude rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles.

[0068] (6) Rice protein hydrolysate-chondroitin sulfate-curcumin crude composite nanoparticles were rotary evaporated at 40℃ for 10 min to remove ethanol, and then the volume was adjusted. The nanoparticles were centrifuged at 8000×g for 10 min to precipitate free curcumin. The supernatant was stored at 4℃ in the dark or freeze-dried for later use to obtain rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles.

[0069] Comparative Example 1

[0070] A method for preparing drug-loaded and stabilized curcumin composite nanoparticles includes the following steps:

[0071] (1) Mix rice protein hydrolysate at a ratio of 1 mg / mL and stir until the solution is uniform and transparent to prepare an aqueous solution of rice protein hydrolysate;

[0072] (2) Under light-protected conditions, curcumin and anhydrous ethanol were mixed at a ratio of 1 mg / mL and stirred until the solution was uniform and clear to prepare curcumin ethanol solution.

[0073] (3) Using a syringe pump at a rate of 1 mL / min, add the curcumin solution from step (2) to the rice protein hydrolysate aqueous solution from step (1) to obtain a rice protein hydrolysate-curcumin dispersion with a mass ratio of rice protein hydrolysate to curcumin of 10:1. Adjust the pH of the rice protein hydrolysate-curcumin dispersion to 6.0 using 0.1 mol / L HCl or NaOH solution to obtain rice protein hydrolysate-curcumin crude composite nanoparticles.

[0074] (4) Rice protein hydrolysate-curcumin crude composite nanoparticles were rotary evaporated at 40℃ for 10 min to remove ethanol, and then the volume was adjusted. The mixture was centrifuged at 8000×g for 10 min to precipitate free curcumin. The supernatant was stored at 4℃ in the dark or freeze-dried for later use to obtain rice protein hydrolysate-curcumin composite nanoparticles.

[0075] The encapsulation efficiency and loading rate of the composite nanoparticles prepared in Examples 1-5 were determined by ethanol extraction. The specific process included:

[0076] Take 1 mL of the composite nanoparticle sample, add 4 mL of anhydrous ethanol, vortex for 30 s to mix, and then determine the curcumin content in the composite nanoparticles at 426 nm. Encapsulation efficiency (EE) and loading capacity (LC) are calculated using the following formulas:

[0077] [ Curcumin content (mg) in composite nanoparticles / amount of curcumin added (mg) ]

[0078] [ Curcumin content (mg) / Total protein content (mg) in composite nanoparticles ]

[0079] The particle size, zeta potential, and polydispersity index of the composite nanoparticles prepared in Examples 1-5 and Comparative Example 1 were determined using a particle size analyzer. The specific process included: diluting the sample by an appropriate factor, and then using a laser particle size analyzer to determine the particle size, zeta potential, and polydispersity index. The measurement temperature was 25°C, and the equilibration time was 5 min.

[0080] The results are shown in Tables 1-3 below:

[0081] Table 1. Encapsulation efficiency, loading rate, particle size, zeta potential, and polydispersity index of rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles prepared under different conditions.

[0082]

[0083] Table 2. Particle size, zeta potential, and polydispersity index of rice protein hydrolysate-curcumin nanoparticles (RPH-Cur NPs, Comparative Example 1) and rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles (RPH-Cs-Cur NPs, Example 2) before and after redispersion.

[0084]

[0085] Table 3. DPPH and ABTS free radical scavenging activities of free curcumin, rice protein hydrolysate-curcumin nanoparticles (RPH-Cur NPs, Comparative Example 1), and rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles (RPH-Cs-Cur NPs, Example 2).

[0086]

[0087] Table 1 shows that, under the condition of fixed amounts of curcumin and rice protein hydrolysate, the encapsulation efficiency of the rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles gradually increased with the increase of chondroitin sulfate mass, while the loading rate showed the opposite trend. Adding chondroitin sulfate provides additional binding sites for curcumin, or strengthens the binding with rice protein hydrolysate and curcumin through non-covalent interactions such as hydrogen bonding and electrostatics, thus improving the encapsulation efficiency of the composite nanoparticles. Simultaneously, the increase in chondroitin sulfate mass is much greater than the increase in the mass of the loaded curcumin, therefore the loading rate of the composite nanoparticles shows a decreasing trend. At a rice protein hydrolysate:chondroitin sulfate mass ratio of 2:1, the rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles simultaneously achieved a large encapsulation efficiency and loading rate. Compared with rice protein hydrolysate-curcumin nanoparticles, the composite nanoparticles had smaller particle size, higher absolute Zeta potential, and lower polydispersity index, indicating that adding chondroitin sulfate helps improve the performance of the nanoparticles.

[0088] As shown in Table 2, compared with the newly prepared nanoparticles, the rice protein hydrolysate-curcumin nanoparticles redispersed after freeze-drying exhibit larger particle size, smaller zeta potential, and larger polydispersity index, indicating poor resolution stability. The particle size, zeta potential, and polydispersity index of the rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles redispersed after freeze-drying are not significantly different from those of the newly prepared nanoparticles, indicating that they have excellent resolution stability. This may be because the shell material formed by chondroitin sulfate contains a sufficient number of charged or hydrophilic groups, which makes the composite nanoparticles exhibit excellent redispersibility.

[0089] As shown in Table 3, compared with free curcumin, rice protein hydrolysate-curcumin nanoparticles exhibit significantly enhanced DPPH and ABTS free radical scavenging abilities, indicating that curcumin in nanoparticle form is more effective than free curcumin in scavenging free radical activity. Curcumin encapsulated in nanoparticles with a hydrophilic surface has higher water solubility, better dispersibility, and a larger surface area, thus increasing the contact area / probability with free radicals and enhancing its ability to donate hydrogen atoms to free radicals. The chondroitin sulfate shell coating the surface of the rice protein hydrolysate further enhances these effects. Therefore, rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles have higher antioxidant activity than rice protein hydrolysate-curcumin nanoparticles.

[0090] like Figure 1 The diagram shows the preparation process of rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles; as shown. Figure 2The X-ray diffraction (XRD) patterns of rice protein hydrolysate (RPH), chondroitin sulfate, curcumin, and rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles (RPH-Cs-Cur NPs) are shown. The characteristic peaks of pure curcumin at 8.9°, 12.2°, 14.5°, 17.3°, 21.1°, 23.3°, 24.7°, and 25.5° indicate its highly crystalline state. The broad peaks of rice protein hydrolysate and chondroitin sulfate at 21.6° and 20.2°, respectively, indicate their amorphous state. The XRD pattern of the rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles does not show obvious curcumin characteristic peaks, indicating that curcumin is encapsulated in an amorphous state within the rice protein hydrolysate-chondroitin sulfate binary composite nanocarrier. Studies have shown that the oral bioavailability of bioactive substances in their amorphous state is higher than that in their crystalline state. Therefore, loading curcumin onto a rice protein hydrolysate-chondroitin sulfate binary composite nanocarrier may help improve its bioavailability. Figure 3 The image shown is a scanning electron microscope image of rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles. The rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles exhibit a compact, ordered, and nearly spherical structure.

[0091] like Figure 4 The figures show the particle size and polydispersity index (PDI) of rice protein hydrolysate-curcumin nanoparticles (A: Comparative Example 1) and rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles (B: Example 2) under different pH conditions. Compared with the control group, the particle size of the rice protein hydrolysate-curcumin nanoparticles did not change significantly only at pH values ​​of 6 and 7, indicating poor stability. In contrast, the particle size and polydispersity index of the rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles increased significantly only under extremely acidic conditions of pH values ​​of 1-2, while at pH values ​​of 3-7, there was no significant difference in particle size and polydispersity index compared with the control group, indicating that the addition of chondroitin sulfate can effectively improve the pH stability of the nanoparticles. This is because, compared with rice protein hydrolysate-curcumin nanoparticles, the rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles have a higher surface charge, so the electrostatic repulsion between particles is sufficient to resist the electrostatic attraction caused by pH changes.

[0092] like Figure 5As shown, the particle size and polydispersity index (PDI) of rice protein hydrolysate-curcumin nanoparticles (A Comparative Example 1) and rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles (B Example 2) under different NaCl concentrations were as follows: When the NaCl concentration was 0–50 mM, the particle size and PDI of the rice protein hydrolysate-curcumin nanoparticles did not change significantly; however, when the NaCl concentration was 100–250 mM, both the particle size and PDI increased significantly with increasing NaCl concentration. This is because the addition of NaCl caused an electrostatic shielding effect, leading to the aggregation of nanoparticles under high salt ion concentrations. Within the NaCl concentration range of 0–100 mM, the particle size and polydispersity index of the rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles did not change significantly, and the change trends of particle size and polydispersity index were relatively small within the NaCl concentration range of 150–250 mM, indicating that the rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles possess superior ionic strength stability.

[0093] like Figure 6 As shown, the retention rates of curcumin in rice protein hydrolysate-curcumin nanoparticles (RPH-Cur NPs, Comparative Example 1) and rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles (RPH-Cs-Cur NPs, Example 2) after treatment at 35–80 °C for 30 min were compared. After treatment at 65 °C and 80 °C for 30 min, the curcumin retention rate in the rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles was significantly higher than that in the rice protein hydrolysate-curcumin nanoparticles. This indicates that the rice protein hydrolysate-chondroitin sulfate binary composite nanocarrier can more effectively protect curcumin from thermal degradation. This may be due to the compact and ordered structure and small particle size of the rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles, thus reducing the exposure of the composite nanoparticles to environmental stress.

[0094] like Figure 7As shown, Caco-2 cell nuclei stained and labeled with 4',6-diamidin-2-phenylindole (DPAI) exhibited red fluorescence. After Caco-2 cells were co-incubated with curcumin or nanoparticles for 4 h, the red fluorescence of Caco-2 cell nuclei overlapped with the green fluorescence of curcumin, indicating that free curcumin and nanoparticles could be effectively taken up by Caco-2 cells (A). Under the same conditions, curcumin in nanoparticle form had a higher cellular uptake efficiency than free curcumin, indicating that both rice protein hydrolysate and the rice protein hydrolysate-chondroitin sulfate binary composite nanocarrier helped promote the uptake of curcumin by Caco-2 cells. In addition, the cellular uptake rate of the rice protein hydrolysate-chondroitin sulfate-curcumin composite nanoparticles was significantly higher than that of the rice protein hydrolysate-curcumin nanoparticles, which may be related to its smaller particle size and more regular spherical structure (B).

[0095] The above embodiments are merely examples of several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent.

[0096] For those skilled in the art, various modifications and improvements can be made without departing from the concept of this invention, and these all fall within the scope of protection of this invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A drug-loaded and stabilized curcumin composite nanoparticle, characterized in that, The invention includes using rice protein hydrolysate as a carrier to load curcumin, and using chondroitin sulfate to stabilize the rice protein hydrolysate loaded with curcumin. The encapsulation rate of the curcumin composite nanoparticles is 70% to 90%, the mass ratio of rice protein hydrolysate to chondroitin sulfate is 1 to 5:1, and the mass ratio of rice protein hydrolysate to curcumin is 8 to 12:

1. At room temperature to 80°C, the curcumin composite nanoparticles exhibit stable suspension and a curcumin retention rate greater than 85%; when the pH value is below 2, the particle size of the curcumin composite nanoparticles is less than or equal to 300 nm. The nanoparticles have a particle size of 100–190 nm and a zeta potential of -20–-30 mV.

2. The method for preparing drug-loaded and stabilized curcumin composite nanoparticles according to claim 1, characterized in that, Includes the following steps: 1) Prepare an aqueous solution of rice protein hydrolysate and prepare an aqueous solution of chondroitin sulfate, and mix and stir to form an aqueous solution of rice protein hydrolysate-chondroitin sulfate binary composite nanocarrier; 2) Add curcumin ethanol solution dropwise into the aqueous solution of rice protein hydrolysate-chondroitin sulfate binary composite nanocarrier prepared in step 1), and adjust the pH value to 3-7 to obtain a mixed solution; 3) The mixture prepared in step 2) is rotary evaporated, diluted to a fixed volume, and centrifuged to remove the precipitate. The supernatant is then refrigerated to obtain curcumin composite nanoparticles.

3. The method for preparing drug-loaded and stabilized curcumin composite nanoparticles according to claim 2, characterized in that, In step 1), the mass concentration of the rice protein hydrolysate aqueous solution is 0.8–1.5 mg / mL, the mass concentration of the chondroitin sulfate aqueous solution is 0.8–1.5 mg / mL, and the mixing mass ratio of the rice protein hydrolysate and chondroitin sulfate is controlled at 1–5:

1.

4. The method for preparing drug-loaded and stabilized curcumin composite nanoparticles according to claim 2, characterized in that, In step 2), the mass concentration of curcumin ethanol solution is 0.8~1.5 mg / mL, and the mass ratio of rice protein hydrolysate to curcumin is controlled at 8~12:

1.

5. The method for preparing drug-loaded and stabilized curcumin composite nanoparticles according to claim 2, characterized in that, In step 3), the rotary evaporation temperature is 35~40℃, the centrifugation speed is 7000~10000×g, and the refrigeration temperature is 3~5℃.

6. An oral drug made from curcumin composite nanoparticles prepared according to the method of preparing drug-loaded stable curcumin composite nanoparticles according to any one of claims 1 or 2 to 5.

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