A curcumin-protein-zinc nanocomplex, its pH-driven preparation method and application

Abstract

The present invention discloses a curcumin-protein-zinc nanocomplex and its pH-driven preparation method and application. The preparation method includes: adding a protein dispersion into a curcumin solution to obtain a curcumin-protein mixture, adjusting the mixture to a certain pH condition, further adding a zinc salt solution, adjusting the pH again after sufficient reaction, and collecting the supernatant after centrifugation to obtain the curcumin-protein-zinc nanocomplex. Compared with traditional chemical methods, the method for preparing the curcumin-protein-zinc nanocomplex of the present invention does not use organic reagents, is green and safe, has simple operation, and is easy for industrial production; moreover, the prepared nanocomplex has uniform scale, stable properties, and good biocompatibility, and has broad application potential in the fields of functional foods and biomedicines, etc.

Description

Technical Field

[0001] The present invention belongs to the technical fields of nano co-loading technology and functional food development technology, and particularly relates to a curcumin-protein-zinc nanocomposite, a pH-driven preparation method thereof, and applications thereof. Background Art

[0002] In modern society, people increasingly focus on the promotion of active ingredients and functional factors in food to human health and nutritional balance, which has gradually become a hot spot and breakthrough point in the research and development of new foods. As a natural plant polyphenol with anti-inflammatory, antioxidant, hypoglycemic, anti-tumor and other properties, curcumin is limited in its large-scale production and application due to disadvantages such as poor water solubility, fast metabolism rate, poor stability, and low bioavailability. Currently, researchers have tried to avoid the above problems through structural modification and encapsulation delivery, and common ones include liposomes, micelles, nanoemulsions, casein complexes, metal complexes, and saponin complexes. Zinc is an essential mineral for the human body, is a cofactor of various enzymes and regulates various cellular processes. Zinc deficiency can cause adverse effects such as loss of appetite, memory loss, and decreased immunity. However, zinc is difficult to be absorbed due to the influence of intestinal contents. Research shows that the combination of curcumin and metal is one of the effective ways to solve the problems of curcumin, and at the same time, the combination with polyphenols can promote the absorption of zinc by the human body. Therefore, curcumin-metal complexes have good prospects in the application of functional foods.

[0003] Currently, most researchers use traditional chemical methods to prepare curcumin-zinc complexes. CN101205234A discloses a preparation method and application of a curcumin-zinc compound and its solid dispersion, which is prepared by curcumin, zinc acetate, and polyvinylpyrrolidone-K30. The specific preparation method is as follows: Dissolve curcumin and acetic acid in an organic solvent, add an organic solution containing zinc acetate under nitrogen protection, stir and reflux, cool and filter, wash and dry to obtain a curcumin-zinc compound; Mix this compound and PVP-k30 and dissolve them in absolute ethanol, homogenize and filter under nitrogen protection, and spray dry to successfully prepare a solid dispersion of the curcumin-zinc compound. However, the use of organic reagents therein limits its application in food, and the complex process flow is not conducive to the popularization and application of this method in industrial production. Therefore, seeking a green, safe and simple preparation method is an urgent problem to be solved.

[0004] In recent years, the pH-driven method has attracted much attention in the research on the construction of biopolymer-based nanoparticles. This method mainly regulates the acidity and alkalinity of the microenvironment where the solute is located in the solution, enabling intermolecular interactions among solute molecules and forming a new stable system during this process. Compared with traditional chemical preparation methods, this method has good application potential in the large-scale production of the food industry due to its advantages such as low cost, low energy consumption, simple operation, and green safety. As a commonly used amphiphilic molecule, protein has been proven that some amino acid residues in its structure are the binding sites for curcumin and zinc, so it is expected to achieve the co-loading of curcumin and zinc.

[0005] Based on this, exploring the preparation of curcumin-protein-zinc nanocomposites using the pH-driven method has broad development and application prospects. Summary of the Invention

[0006] In order to overcome the above deficiencies existing in the prior art, the purpose of the present invention is to provide a ternary composite for achieving the co-loading of curcumin and zinc through protein, as well as its pH-driven preparation method and application

[0007] The primary purpose of the present invention is to improve the biochemical activities of curcumin and zinc, and to provide a method for preparing curcumin-protein-zinc composites by pH driving.

[0008] Another purpose of the present invention is to provide the curcumin-protein-zinc composite prepared by the above method.

[0009] Another purpose of the present invention is to provide the application of the above curcumin-protein-zinc composite.

[0010] The purpose of the present invention is achieved by at least one of the following technical solutions.

[0011] A pH-driven preparation method of a curcumin-protein-zinc nanocomposite provided by the present invention includes the following steps:

[0012] (1) Add protein to water and mix evenly to obtain a protein dispersion;

[0013] (2) Add curcumin to an alkaline solution and mix evenly to obtain a curcumin solution;

[0014] (3) Add the protein dispersion obtained in step (1) to the curcumin solution obtained in step (2), stir evenly to obtain a curcumin-protein mixture;

[0015] (4) Adjust the pH value of the curcumin-protein mixture obtained in step (3), then add a zinc salt solution, adjust the pH again after sufficient reaction, and centrifuge to take the supernatant to obtain the curcumin-protein-zinc nanocomposite.

[0016] Preferably, the protein in step (1) is one or more of mung bean protein, rice protein, potato protein, pea protein, and soybean protein, and the protein concentration of the protein dispersion is 1-50 mg / mL.

[0017] Preferably, the lye in step (2) is one or more of sodium hydroxide solution and potassium hydroxide solution; the concentration of the lye is 0.05-0.5 mol / L; the curcumin concentration of the curcumin solution is 0.5-2 mg / mL.

[0018] Preferably, the zinc salt solution in step (4) is one or more of zinc sulfate solution, zinc chloride solution, and zinc gluconate solution; the concentration of Zn 2+ in the zinc salt solution in step (4) is 0.35-14 mM.

[0019] Preferably, the mass ratio of curcumin in the curcumin solution to the protein in the protein dispersion in step (3) is 1:50-1:2.

[0020] Preferably, the mass ratio of curcumin in the curcumin-protein mixture to Zn 2+ in the zinc salt solution in step (4) is 1:0.01-1:0.23.

[0021] Preferably, the stirring rate in step (3) is 100-600 rpm, and the stirring time is 10-50 min.

[0022] Preferably, adjusting the pH value of the curcumin-protein mixture obtained in step (3) in step (4) makes the pH of the adjusted curcumin-protein solution 7.0-13.0.

[0023] Preferably, the sufficient reaction time in step (4) is 0.5-2.5 h.

[0024] Preferably, readjusting the pH after the sufficient reaction in step (4) makes the final pH 6.9-8.2.

[0025] Preferably, the centrifugation rate in step (4) is 3000-10000 g, and the centrifugation time is 10-50 min.

[0026] The present invention provides a curcumin-protein-zinc nanocomplex prepared by any of the above pH-driven preparation methods.

[0027] The present invention also provides the application of the curcumin-protein-zinc nanocomplex in the preparation of functional foods or drugs.

[0028] Furthermore, the functional foods or drugs refer to functional foods or drugs with antioxidant, immune-enhancing, or neuroprotective effects.

[0029] Curcumin, a natural plant polyphenol with multiple pharmacological properties, has been found to have anti-inflammatory, antioxidant, hypoglycemic, antibacterial, anti-tumor, neuroprotective and immunomodulatory effects. Zinc is one of the essential minerals for the human body. Zinc is involved in the functions of more than 2,000 proteins and is a cofactor for more than 300 enzymes, participating in the regulation of multiple cellular processes. Proteins have become a commonly used embedding carrier in food research for the embedding and delivery of active substances due to their wide sources, low price and amphiphilic structure.

[0030] The curcumin-protein-zinc nanocomposite prepared by the present invention can achieve the delivery and targeted release of curcumin. In addition to being used for the development of new functional foods, it can also be added to food systems as a nutritional supplement with special physiological activities.

[0031] The present invention discloses a curcumin-protein-zinc nanocomposite particle, a pH-driven preparation method thereof and applications. The present invention uses an amphiphilic protein as a carrier and utilizes the solubility characteristics of curcumin under specific pH conditions to prepare a curcumin-protein-zinc nanocomposite by pH-driven technology.

[0032] The preparation method provided by the present invention includes: adding a protein dispersion into a curcumin solution, adjusting the mixture to a certain pH condition after stirring for a certain time, then adding a zinc salt solution to react fully, and then adjusting the pH of the solution. After centrifugation, the supernatant is collected to obtain the curcumin-protein-zinc composite. The prepared composite has a nanoscale size.

[0033] Compared with traditional preparation methods, the ternary composite prepared by the present invention has the advantages of small size, good stability and uniformity, and biodegradability; and no organic reagents are used in the preparation process, which is green, safe, simple to operate and easy to industrialize.

[0034] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0035] (1) The pH-driven preparation method of a curcumin-protein-zinc nanocomposite provided by the present invention enables the stable coexistence of curcumin and zinc under neutral conditions with a protein, improves the solubility and stability of curcumin, and obtains a uniform ternary composite with a nanoscale size, having good application prospects.

[0036] (2) The preparation method provided by the present invention has a simple process, safe and pollution-free raw materials, does not involve organic reagents, is green and environmentally friendly, biodegradable, and can be used for large-scale production. Description of the Drawings

[0037] Figure 1Appearance diagrams after centrifugation of the curcumin-zinc mixture obtained in step (3) of the comparative example and the curcumin-protein-zinc mixture obtained in step (5) of Examples 1-5.

[0038] Figure 2 Particle size analysis results diagrams of the curcumin-protein-zinc nanocomposites prepared in Examples 1-5.

[0039] Figure 3 Uptake rates of the curcumin-protein-zinc nanocomposites prepared in Examples 1-5 in the Caco-2 monolayer cell model. Detailed implementation manners

[0040] The following further illustrates the specific implementation of the present invention in conjunction with the drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that for the processes not specifically described in detail below, those skilled in the art can implement or understand them with reference to the prior art. Reagents or instruments without indicating the manufacturer are regarded as conventional products that can be obtained through commercial purchase.

[0041] Comparative example

[0042] (1) Curcumin was added to a 0.05 M NaOH solution to make the final concentration of curcumin 1 mg / mL, and the mixture was stirred evenly to obtain a curcumin solution.

[0043] (2) Zinc sulfate was added to water to make the final concentration of Zn 2+ in the solution 0.7 mM, and the mixture was stirred evenly to obtain a zinc sulfate solution.

[0044] (3) 8 mL of the curcumin solution was taken, and the zinc sulfate solution in step (2) was added according to the mass ratio of curcumin:Zn 2+ = 1:0.01 to obtain a curcumin-zinc mixture. After stirring at 400 rpm for 10 min, the pH was adjusted back to pH 7.3 with a 2 M HCl solution, and then the mixture was centrifuged at 3000 g for 50 min to take the supernatant to obtain a curcumin-zinc complex.

[0045] Example 1

[0046] A method for preparing curcumin-protein-zinc nanocomposites by a pH-driven method, comprising the following steps:

[0047] (1) Curcumin was added to a 0.05 M NaOH solution to make the final concentration of curcumin 0.5 mg / mL, and the mixture was stirred evenly to obtain a curcumin solution.

[0048] (2) Soybean protein was added to water and stirred evenly to obtain a soybean protein dispersion, and the protein concentration of the soybean protein dispersion was 1 mg / mL.

[0049] (3) Add zinc sulfate to water to make the final concentration of Zn in the solution 2+ 0.35 mM, and stir evenly to obtain a zinc sulfate solution.

[0050] (4) Take 8 mL of the curcumin solution, and add a soy protein dispersion to it according to the mass ratio of curcumin:soy protein = 1:2 to obtain a curcumin-protein mixture, and stir at 600 rpm for 10 min.

[0051] (5) Take the curcumin-protein mixture obtained in step (4), adjust the pH of the mixture to 12.0 with 2M HCl solution, and then add the zinc sulfate solution in step (3) according to the mass ratio of curcumin:Zn 2+ = 1:0.01, react for 0.5 h to obtain a curcumin-protein-zinc mixture, further adjust the pH back to 6.9 with 2M HCl, and then centrifuge at 3000 g for 50 min, discard the precipitate, and the supernatant obtained is the curcumin-protein-zinc nanocomposite prepared by the pH-driven method.

[0052] Example 2

[0053] A method for preparing a curcumin-protein-zinc nanocomposite by a pH-driven method, comprising the following steps:

[0054] (1) Add curcumin to 0.05M KOH solution to make its final concentration 0.5 mg / mL, and stir evenly to obtain a curcumin solution.

[0055] (2) Add a certain amount of rice protein to water, and stir evenly to obtain a rice protein dispersion, and the protein concentration of the rice protein dispersion is 2 mg / mL.

[0056] (3) Add zinc chloride to water to make its final concentration 0.73 mM, and stir evenly to obtain a zinc chloride solution.

[0057] (4) Take 8 mL of the curcumin solution, and add a rice protein dispersion to it according to the mass ratio of curcumin:rice protein = 1:4 to obtain a curcumin-protein mixture, and stir at 100 rpm for 25 min.

[0058] (5) Take the curcumin-protein mixture obtained in step (4), adjust the pH of the mixture to 7.0 with 2M HCl solution, and then add the zinc chloride solution in step (3) according to the mass ratio of curcumin:Zn 2+ = 1:0.024, react for 2.5 h to obtain a curcumin-protein-zinc mixture, further adjust the pH back to 7.1 with 1M NaOH, and then centrifuge at 10000 g for 10 min, discard the precipitate, and the supernatant obtained is the curcumin-protein-zinc nanocomposite prepared by the pH-driven method.

[0059] Example 3

[0060] A method for preparing curcumin-protein-zinc nanocomposites by pH-driven method, comprising the following steps:

[0061] (1) Add curcumin to 0.2M NaOH solution to make its final concentration 1mg / mL, and stir evenly to obtain curcumin solution.

[0062] (2) Add a certain amount of mung bean protein to water, and stir evenly to obtain mung bean protein dispersion, and the protein concentration of the mung bean protein dispersion is 50mg / mL.

[0063] (3) Add zinc gluconate to water to make its final concentration 8.78mM, and stir evenly to obtain zinc gluconate solution.

[0064] (4) Take 8mL of curcumin solution, and add mung bean protein dispersion thereto according to the mass ratio of curcumin:mung bean protein = 1:50 to obtain curcumin-protein mixture, and stir at 400rpm for 30min.

[0065] (5) Take the curcumin-protein mixture obtained in step (4), adjust the pH of the mixture to 8.0 with 2M HCl solution, then add the zinc gluconate solution in step (3) according to the mass ratio of curcumin:Zn 2+ = 1:0.14, react for 2h to obtain curcumin-protein-zinc mixture, further adjust the pH back to 7.5 with 2M HCl, then centrifuge at 10000g for 10min, discard the precipitate, and the supernatant obtained is the curcumin-protein-zinc nanocomposites prepared by pH-driven method.

[0066] Example 4

[0067] A method for preparing curcumin-protein-zinc nanocomposites by pH-driven method, comprising the following steps:

[0068] (1) Add curcumin to 0.5M KOH solution to make its final concentration 1mg / mL, and stir evenly to obtain curcumin solution.

[0069] (2) Add a certain amount of potato protein to water, and stir evenly to obtain potato protein dispersion, and the protein concentration of the potato protein dispersion is 50mg / mL.

[0070] (3) Add zinc sulfate to water to make its final concentration 14mM, and stir evenly to obtain zinc sulfate solution.

[0071] (4) Take 8 mL of the curcumin solution, and add the potato protein dispersion thereto according to the mass ratio of curcumin:potato protein = 1:50 to obtain a curcumin-protein mixture, and stir at 200 rpm for 40 min.

[0072] (5) Take the curcumin-protein mixture obtained in step (4), adjust the pH of the mixture to 13.0 with 6M NaOH solution, and then add the zinc sulfate solution in step (3) according to the mass ratio of curcumin:Zn 2+ = 1:0.23, react for 1 h to obtain a curcumin-protein-zinc mixture, further adjust the pH back to 8.2 with 2M HCl, then centrifuge at 5000 g for 20 min, discard the precipitate, and the obtained supernatant is the curcumin-protein-zinc nanocomposite prepared by the pH-driven method.

[0073] Example 5

[0074] A method for preparing a curcumin-protein-zinc nanocomposite by a pH-driven method, comprising the following steps:

[0075] (1) Add curcumin to 0.5M NaOH solution to make its final concentration 2 mg / mL, and stir evenly to obtain a curcumin solution.

[0076] (2) Add a certain amount of pea protein to water, and stir evenly to obtain a pea protein dispersion, and the protein concentration of the pea protein dispersion is 20 mg / mL.

[0077] (3) Add zinc gluconate to water to make its final concentration 4.39 mM, and stir evenly to obtain a zinc gluconate solution.

[0078] (4) Take 8 mL of the curcumin solution, and add the pea protein dispersion thereto according to the mass ratio of curcumin:pea protein = 1:10 to obtain a curcumin-protein mixture, and stir at 200 rpm for 50 min.

[0079] (5) Take the curcumin-protein mixture obtained in step (4), adjust the pH of the mixture to 9.0 with 2M HCl solution, and then add the zinc gluconate solution in step (3) according to the mass ratio of curcumin:Zn 2+ = 1:0.036, react for 1 h to obtain a curcumin-protein-zinc mixture, further adjust the pH back to 7.8 with 2M HCl, then centrifuge at 5000 g for 20 min, discard the precipitate, and the obtained supernatant is the curcumin-protein-zinc nanocomposite prepared by the pH-driven method.

[0080] The curcumin-zinc composite prepared in the comparative example and the curcumin-protein-zinc nanocomposites prepared in Examples 1-5 were evaluated as follows, and the results are shown in Table 1 and Figures 1-3 .

[0081] Table 1

[0082]

[0083] Determination of particle size and polymer dispersity index (PDI): The average particle size and PDI of the obtained nanoparticles were determined using a nanoparticle size and zeta potential analyzer (Malvern Nano-ZS).

[0084] Determination of Caco-2 simulated small intestinal endothelial uptake rate: The human colon adenocarcinoma cell line Caco-2 was cultured in DMEM medium containing 10% fetal bovine serum and 1.1% antibiotics in an incubator at 37 °C and 5% CO2, and the culture medium was changed every 2 days to construct a Caco-2 monolayer cell model. The complexes obtained in Examples 1-5 were prepared into 400 μg / ml and added to Caco-2 cells. After 2 h, the cells were collected and lysed, and the uptake rate of curcumin was determined by HPLC. 50 μL of the sample was loaded onto a C18 column, and the mobile phase A (methanol), mobile phase B (0.1% formic acid in water), mobile phase C (water), and mobile phase D (acetonitrile). The detection wavelength was 420 nm. The uptake rate of curcumin was expressed as the ratio of the integrated area of the absorption peak of curcumin in the cells to the integrated area of the absorption peak of the initially added curcumin.

[0085] It is known from Figure 1 that curcumin and zinc cannot coexist under neutral conditions. After centrifugation, the supernatant was light in color, and there was a large amount of red precipitate at the bottom. When samples with different proteins were added, the precipitate at the bottom was less, and the supernatant was orange, indicating that curcumin and zinc were co-loaded with proteins by the pH-driven method. It is known from Figure 2 that the particle sizes of the complexes obtained by reacting soy protein, rice protein, mung bean protein, pea protein, and potato protein with curcumin solution and zinc solution showed a single-peak distribution with a narrow peak width, indicating that the complexes were of uniform size and stably dispersed. It is known from Figure 3 that the curcumin-protein-zinc ternary complexes in Examples 1-5 could be taken up by Caco-2 simulated small intestinal endothelial cells and had different uptake rates, indicating that this complex form had the potential to enable curcumin to be absorbed by the intestine in a stably dispersed state.

[0086] It can be seen from Table 1 that the average particle sizes of the curcumin-protein-zinc ternary complexes in Examples 1-5 were in the range of 93-133 nm, and the PDI was in the range of 0.19-0.52, with good uniformity and dispersibility. In the comparative example, the average particle size of the product obtained by reacting curcumin solution with zinc solution was 4600 nm, and the PDI was significantly greater than that of the examples, indicating poor uniformity of the system and being unfavorable for its stable dispersion in water. The above results show that by using the pH-driven method with proteins as intermediates, curcumin-protein-zinc ternary complexes with small and uniform particle sizes, stable dispersion under neutral conditions, and the ability to be taken up by Caco-2 simulated small intestinal endothelial cells can be prepared.

[0087] The preparation method provided by the present invention enables curcumin and zinc to combine to form a nanoscale homogeneous complex through a protein as an intermediate, which can well improve the solubility and stability of curcumin under neutral conditions. Its process is simple, green and safe, and has great potential for industrialization. The above embodiments are only the preferred embodiments of the present invention, and are only used to explain the present invention, rather than limiting the present invention. Any changes, substitutions, modifications, etc. made by those skilled in the art without departing from the spirit and essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A pH-driven preparation method of curcumin-protein-zinc nanocomposite, characterized in that, It includes the following steps: (1) Add protein into water and mix evenly to obtain a protein dispersion; (2) Add curcumin into an alkali solution and mix evenly to obtain a curcumin solution; (3) Add the protein dispersion obtained in step (1) into the curcumin solution obtained in step (2), stir evenly to obtain a curcumin-protein mixture; (4) Adjust the pH value of the curcumin-protein mixture obtained in step (3) so that the pH of the adjusted curcumin-protein solution is 7.0 - 13.0, then add a zinc salt solution, after fully reacting, adjust the pH again to 6.9 - 8.2, centrifuge and take the supernatant to obtain a curcumin-protein-zinc nanocomposite.

2. The pH-driven preparation method of a curcumin-protein-zinc nanocomplex according to claim 1, wherein The protein described in step (1) is one or more of rice protein, mung bean protein, potato protein, pea protein and soybean protein.

3. The pH-driven preparation method of a curcumin-protein-zinc nanocomplex according to claim 1, characterized in that, The protein concentration of the protein dispersion described in step (1) is 1 - 50 mg / mL.

4. A pH-driven preparation method of a curcumin-protein-zinc nanocomplex according to claim 1, characterized in that, The alkali solution described in step (2) is one or more of sodium hydroxide solution and potassium hydroxide solution; the concentration of the alkali solution is 0.05 - 0.5 mol / L; the curcumin concentration of the curcumin solution is 0.5 - 2 mg / mL.

5. A pH-driven preparation method of a curcumin-protein-zinc nanocomplex according to claim 1, characterized in that, The zinc salt solution described in step (4) is one or more of zinc sulfate solution, zinc chloride solution and zinc gluconate solution; The concentration of Zn in the zinc salt solution described in step (4) 2+ is 0.35 - 14 mM.

6. The pH-driven preparation method of a curcumin-protein-zinc nanocomplex according to claim 1, characterized in that, The mass ratio of curcumin in the curcumin solution described in step (3) to the protein in the protein dispersion is 1:50 - 1:2; Zn in the zinc salt solution and curcumin in the curcumin-protein mixture described in step (4) 2+ The mass ratio is 1:0.01 - 1:0.

23.

7. A pH-driven preparation method of a curcumin-protein-zinc nanocomplex according to claim 1, characterized in that, The stirring rate described in step (3) is 100 - 600 rpm, and the stirring time is 10 - 50 min.

8. A pH-driven preparation method of a curcumin-protein-zinc nanocomplex according to claim 1, characterized in that, The full reaction time described in step (4) is 0.5 - 2.5 h; the centrifugation rate is 3000 - 10000 g, and the centrifugation time is 10 - 50 min.

9. A curcumin-protein-zinc nanocomposite prepared by the pH-driven preparation method of the curcumin-protein-zinc nanocomposite according to any one of claims 1 - 7.

10. Use of the curcumin-protein-zinc nanocomposite according to claim 9 in the preparation of functional foods or drugs.

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