Preparation method of nano-composite for improving stability of anthocyanin

Abstract

The invention discloses a preparation method of a nano-composite for improving stability of anthocyanin. The preparation method comprises the following steps: dissolving purple corn anthocyanin in distilled water; dissolving chitosan oligosaccharide in distilled water; the preparation method comprises the following steps: dropwise adding a chitosan oligosaccharide solution into an anthocyanin solution, uniformly stirring and mixing by magnetic force, dropwise adding a polyphosphate solution, reacting, transferring a mixed solution into a centrifugal tube, centrifuging for 10-30 minutes to separate out a nano-composite precipitate and an uncombined supernatant solution, adding ultrapure water, and repeatedly centrifuging. And finally, carrying out vacuum freeze drying treatment on the precipitate to obtain the nano-composite for improving the stability of the anthocyanin. The nano-composite prepared by the method has higher stability, good dispersibility and enhanced biological activity, can form a stable three-dimensional network structure, has small particle size and uniform distribution, and provides a new way for wide application of chitosan oligosaccharide and anthocyanin in the fields of food, cosmetics, biological medicines and the like.

Description

Technical Field

[0001] The invention relates to the field of pharmaceutical preparations, and in particular to a method for preparing a nanocomposite for improving the stability of anthocyanins. Background Art

[0002] The poor stability and low bioavailability of anthocyanins are technical problems that need to be solved urgently. The applicant prepared a nanocomposite of anthocyanins, with patent application number 2024118893387, which greatly improved the stability of anthocyanins; however, it is still necessary to further improve its stability and bioavailability. Summary of the invention

[0003] The purpose of the present invention is to provide a method for preparing a nanocomposite that improves the stability of anthocyanins in view of the shortcomings of the prior art. In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions:

[0004] The following technical solutions are adopted:

[0005] The steps include: (1) dissolving anthocyanidin in water to obtain anthocyanidin solution;

[0006] (2) dissolving chitosan oligosaccharide in water to obtain a chitosan oligosaccharide solution;

[0007] (3) the anthocyanin solution and the chitosan oligosaccharide solution are uniformly mixed, and a polyphosphate solution is added to obtain a mixed solution;

[0008] (4) centrifuging the mixed solution to obtain a precipitate;

[0009] (5) The precipitate is freeze-dried in vacuum to obtain the nanocomposite.

[0010] Further, (1) anthocyanidin is dissolved in distilled water to obtain an anthocyanidin solution with a concentration of 5-30 mg / mL.

[0011] Further, (2) dissolving chitosan oligosaccharide in distilled water to obtain a chitosan oligosaccharide solution with a concentration of 5-30 mg / mL.

[0012] Furthermore, a polyphosphate solution is slowly added; and / or the polyphosphate is sodium tripolyphosphate.

[0013] Furthermore, the anthocyanin solution is added to the chitosan oligosaccharide solution, mixed evenly, and the stirring speed is 120-800 rpm, and 1-24 mL of 0.01-5 mg / mL sodium tripolyphosphate solution is added dropwise to obtain a mixed solution;

[0014] Furthermore, the degree of polymerization of chitosan oligosaccharide is 2-20. The molecular weight of chitosan oligosaccharide is less than 1000Da. 。

[0015] Furthermore, step (3) is carried out at room temperature in the dark for 0.5-24 hours.

[0016] Furthermore, in step (4), the mixed solution is centrifuged at 6000-20000 rpm for 10-30 minutes, and then water is added and the centrifugation is repeated several times. For example, in step (4), the mixed solution is centrifuged at 12000 rpm for 10-30 minutes, and then double distilled water is added and the centrifugation is repeated 3 times.

[0017] The beneficial effects of the present invention relative to the prior art are:

[0018] The stability of the chitosan oligosaccharide-anthocyanidin nanocomposite prepared by the method of the present invention is more significantly improved. The nanoscale complex can effectively prevent the degradation of anthocyanidins in the environment, prolong its shelf life, and make its performance in various applications more stable and reliable. The addition of polyphosphate solution is to allow the positively charged chitosan oligosaccharide and the negatively charged polyphosphate ions to fully and evenly attract each other through electrostatic action, and an ionic cross-linking reaction occurs, thereby forming a stable three-dimensional network structure. LNP (i.e., the complex obtained in this application) has a smaller particle size than RNP (the complex obtained by patent application No. 2024118893387) and is more evenly distributed. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The UV spectra of chitosan oligosaccharide-anthocyanidin nanocomplex and individual chitosan oligosaccharide and anthocyanidin are changed when the concentration ratio of anthocyanin solution to chitosan oligosaccharide solution is 1:1, 1:2 and 2:1.

[0020] Figure 2 The infrared spectra of chitosan oligosaccharide-anthocyanidin nanocomplex and individual chitosan oligosaccharide and anthocyanidin are changed when the concentration ratio of anthocyanin solution to chitosan oligosaccharide solution is 1:1, 1:2 and 2:1.

[0021] Figure 3 The particle size distribution diagram of the chitosan oligosaccharide-anthocyanidin nanocomposite and the chitosan oligosaccharide and anthocyanidin alone in Example 1

[0022] Figure 4 The Zeta potential distribution diagram of the chitosan oligosaccharide-anthocyanidin nanocomposite of Example 1 and the chitosan oligosaccharide and anthocyanidin alone

[0023] Figure 5 (a) is an electron micrograph of the chitosan oligosaccharide-anthocyanidin nanocomplex at a magnification of 100,000 times; (b) is an electron micrograph of the chitosan oligosaccharide-anthocyanidin nanocomplex at a magnification of 50,000 times;

[0024] Figure 6 This is a graph showing the DPPH free radical scavenging rate of the chitosan oligosaccharide-anthocyanidin nanocomplex of Example 1 and individual chitosan oligosaccharides and anthocyanidins.

[0025] Figure 7 This is a graph showing the ABTS free radical scavenging rate of the chitosan oligosaccharide-anthocyanidin nanocomplex of Example 1 and individual chitosan oligosaccharides and anthocyanidins.

[0026] Figure 8 This is a graph showing the OH radical scavenging rate of the chitosan oligosaccharide-anthocyanidin nanocomposite of Example 1 and individual chitosan oligosaccharides and anthocyanidins.

[0027] Fig. 9 This is the protective effect of the chitosan oligosaccharide-anthocyanin nanocomposite of Example 1 on anthocyanin under light-proof conditions.

[0028] Fig.10 This is the protective effect of the chitosan oligosaccharide-anthocyanin nanocomplex of Example 1 on anthocyanin under incandescent light conditions.

[0029] Fig.11 This is the protective effect of the chitosan oligosaccharide-anthocyanin nanocomposite of Example 1 on anthocyanin under ultraviolet light conditions.

[0030] Fig.12 This is the protective effect of the chitosan oligosaccharide-anthocyanidin nanocomplex of Example 1 on anthocyanidin at 36.5°C.

[0031] Fig.13 This is the protective effect of the chitosan oligosaccharide-anthocyanin nanocomposite of Example 1 on anthocyanin at 60°C.

[0032] Fig.14 This is the protective effect of the chitosan oligosaccharide-anthocyanin nanocomposite of Example 1 on anthocyanin at 105°C.

[0033] Fig.15 This is the protective effect of the chitosan oligosaccharide-anthocyanin nanocomposite of Example 1 on anthocyanin in a pH=3 environment.

[0034] Fig.16 This is the protective effect of the chitosan oligosaccharide-anthocyanin nanocomposite of Example 1 on anthocyanin in a pH=5 environment.

[0035] Fig.17 This is the protective effect of the chitosan oligosaccharide-anthocyanin nanocomposite of Example 1 on anthocyanin in a pH=7 environment.

[0036] Fig.18 This is the protective effect of the chitosan oligosaccharide-anthocyanin nanocomposite of Example 1 on anthocyanin in an environment of pH = 9.

[0037] Fig.19 This is the protective effect of the chitosan oligosaccharide-anthocyanin nanocomposite of Example 1 on anthocyanin in an environment of pH=11. DETAILED DESCRIPTION

[0038] In order to facilitate the understanding of those skilled in the art, the present invention is further described below in conjunction with embodiments, and the contents mentioned in the implementation modes are not intended to limit the present invention.

[0039] Example 1

[0040] A method for preparing a chitosan oligosaccharide-anthocyanidin nanocomposite is carried out according to the following steps:

[0041] (1) Dissolve anthocyanins in distilled water at a concentration of 20 mg / mL;

[0042] (2) Dissolve chitosan oligosaccharide in distilled water at a concentration of 10 mg / mL;

[0043] (3) The solutions of step (1) and step (2) were placed in a beaker, mixed evenly, and then 2 mL of 0.5 mg / mL sodium tripolyphosphate solution was added dropwise, and the mixture was stirred at 150 rpm at room temperature for 2 hours;

[0044] (4) The mixed solution was centrifuged at 12000 rpm for 30 minutes, the supernatant was discarded, and double distilled water was added and the centrifugation was repeated three times.

[0045] (5) The precipitate in step (4) is vacuum freeze-dried to obtain the nanocomposite.

[0046] The concentration ratio of the anthocyanin solution in step (1) to the chitosan oligosaccharide solution in step (2) is 2:1.

[0047] Example 2

[0048] The difference between this embodiment and Example 1 is that the concentration ratio of the anthocyanidin solution in step (1) to the chitosan oligosaccharide solution in step (2) is 1:2, that is, 10 mg / mL and 20 mg / mL respectively. The other steps and parameters are the same as those in Example 1.

[0049] Example 3

[0050] The present embodiment is different from Example 1 in that the concentration ratio of the anthocyanidin solution in step (1) to the chitosan oligosaccharide solution in step (2) is 1:1, i.e., 15 mg / mL and 15 mg / mL, respectively. The other steps and parameters are the same as those in Example 1.

[0051] Table 1

[0052]

[0053] As can be seen from Table 1 (the COS / PCA / RNP data in this table are the same as those in Patent 2024118893387 and are listed again for reference), the zeta potential of chitosan oligosaccharide is positive (11.27±1.56mV), and the potential of anthocyanin is also relatively high, which is positive (21.80±0.21). However, after the chitosan oligosaccharide-anthocyanin nanocomposite is constructed, the zeta potential becomes negative, indicating that after the chitosan oligosaccharide and anthocyanin are cross-linked by sodium tripolyphosphate, the surface charge properties of anthocyanin are changed, making it more negatively charged. The enhancement of negative charge contributes to the stability of nanoparticles in vivo and reduces nonspecific interactions with charged biological molecules, thereby improving bioavailability and targeting. Secondly, from the perspective of particle size, the average particle size distribution of chitosan oligosaccharide (COS) is 72.25±0.55nm, which is a relatively small value, indicating that the chitosan oligosaccharide has a smaller particle size and may have better solubility and bioavailability. The average particle size distribution of anthocyanin (PCA) is 305.50±0.77nm, which is larger than chitosan oligosaccharide but still within the nanometer range. The average particle size distribution of LNP (ion crosslinking method) is 103.6±3.14nm, which significantly reduces the particle size of the complex compared to RNP, which is conducive to its digestion and absorption by organisms.

[0054] Table 2

[0055]

[0056] As shown in Table 2, the chitosan oligosaccharide-anthocyanidin nanocomposite obtained in Example 2 has a detailed list of the antioxidant properties of anthocyanidin, chitosan oligosaccharide and chitosan oligosaccharide-anthocyanidin nanocomposite against different free radicals such as ABTS, DPPH and ·OH. Anthocyanidin exhibits a very strong antioxidant capacity, and its IC 50 The values ​​were 0.08mg / mL, 0.16mg / mL and 0.05mg / mL respectively, especially the inhibitory effect on OH free radicals. In contrast, the antioxidant capacity of chitosan oligosaccharide was slightly inferior, and its IC 50 The values ​​were 0.21 mg / mL, 0.26 mg / mL and 0.11 mg / mL respectively. IC values ​​of chitosan oligosaccharide-anthocyanidin nanocomplexes for ABTS, DPPH and ·OH 50The values ​​were 0.03 mg / mL, 0.04 mg / mL and 0.05 mg / mL, respectively. Especially in terms of inhibiting ABTS free radicals, the chitosan oligosaccharide-anthocyanidin nanocomplex also showed a considerable effect. In addition, it is also better than RNP. In summary, the preparation process and technology of the chitosan oligosaccharide-anthocyanidin nanocomplex prepared in this study are innovative. Anthocyanidins are encapsulated in chitosan oligosaccharides by a specific method to form stable nanoparticles, which show good application prospects in the field of antioxidants.

[0057] Depend on Figure 9-19 It can be seen that the chitosan oligosaccharide-anthocyanidin nanocomposite obtained by the technical solution of the present application has better stability, can effectively prevent the degradation of anthocyanidin in the environment, and prolong its shelf life. Figure 5 It can be seen that the chitosan oligosaccharide-anthocyanin nanocomposite was successfully compounded. However, when the degree of polymerization of chitosan oligosaccharide was high, a good compound could not be formed.

[0058] Example 4

[0059] The preparation method of chitosan oligosaccharide-anthocyanidin nanocomposite adopts the following technical scheme:

[0060] The steps include: (1) dissolving anthocyanidin in distilled water to obtain anthocyanidin solution with a concentration of 5 mg / mL;

[0061] (2) dissolving chitosan oligosaccharide in distilled water to obtain a chitosan oligosaccharide solution with a concentration of 30 mg / mL;

[0062] (3) adding the anthocyanin solution to the chitosan oligosaccharide solution, adding 1 mL of a 5 mg / mL sodium tripolyphosphate solution dropwise while stirring at a rotation speed of 120 rpm to obtain a mixed solution;

[0063] (4) The mixed solution is centrifuged to obtain a precipitate.

[0064] (5) The precipitate in step (4) is vacuum freeze-dried to obtain the nanocomposite.

[0065] The degree of polymerization of chitosan oligosaccharide is 20. Step (3) is to react at room temperature for 2 hours.

[0066] In step (4), the mixed solution was centrifuged at 6000 rpm for 30 minutes, and then water was added and the centrifugation was repeated several times.

[0067] Example 5

[0068] The preparation method of chitosan oligosaccharide-anthocyanidin nanocomposite adopts the following technical scheme:

[0069] The steps include: (1) dissolving anthocyanin in distilled water to obtain an anthocyanin solution with a concentration of 30 mg / mL;

[0070] (2) dissolving chitosan oligosaccharide in distilled water to obtain a chitosan oligosaccharide solution with a concentration of 5 mg / mL;

[0071] (3) adding the anthocyanin solution to the chitosan oligosaccharide solution, and adding 24 mL of a 0.01 mg / mL sodium tripolyphosphate solution dropwise while stirring at a rotation speed of 800 rpm to obtain a mixed solution;

[0072] (4) The mixed solution is centrifuged to obtain a precipitate.

[0073] (5) The precipitate in step (4) is vacuum freeze-dried to obtain the nanocomposite.

[0074] The degree of polymerization of chitosan oligosaccharide is 2. Step (3) is to react at room temperature for 2 hours.

[0075] In step (4), the mixed solution was centrifuged at 20,000 rpm for 10 minutes, and then water was added and the centrifugation was repeated three times.

[0076] It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as generally understood by those skilled in the art in the art to which the present invention belongs. It should also be understood that terms such as those defined in general dictionaries should be understood to have meanings consistent with the meanings in the context of the prior art, and will not be interpreted with idealized or overly formal meanings unless specifically defined as herein.

[0077] It should be understood that the detailed description of the technical solutions of the present invention by means of the preferred embodiments is illustrative rather than restrictive. A person skilled in the art may modify the technical solutions described in the embodiments, or replace some of the technical features by equivalents, based on reading the specification of the present invention; and these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a nanocomposite for improving the stability of anthocyanins, characterized in that: The following technical solutions are adopted: The steps include: (1) dissolving anthocyanin in water to obtain anthocyanin solution; (2) dissolving chitosan oligosaccharide in water to obtain a chitosan oligosaccharide solution; (3) The anthocyanin solution and the chitosan oligosaccharide solution are mixed evenly, and the polyphosphate solution is added to obtain a mixed solution; (4) Centrifuging the mixed solution to obtain a precipitate; (5) The precipitate is freeze-dried in vacuum to obtain the nanocomposite.

2. The method according to claim 1, characterized in that The degree of polymerization of chitosan oligosaccharide is 2-20; the molecular weight of chitosan oligosaccharide is less than 1000 Da.

3. The method according to claim 1, characterized in that Step (3) is to react at room temperature in the dark for 2-24 hours.

4. The method according to claim 1, characterized in that: In step (4), the mixed solution is centrifuged at 12000 rpm for 10-30 minutes, and then water is added and the centrifugation is repeated several times; for example, in step (4), the mixed solution is centrifuged at 12000 rpm for 10-30 minutes, and then double distilled water is added and the centrifugation is repeated 3 times.

5. The method according to claim 1, characterized in that The polyphosphate solution is added slowly; and / or the polyphosphate is sodium tripolyphosphate.

6. The method according to claim 1, characterized in that The concentration of anthocyanin solution is 5-30 mg / mL.

7. The method according to claim 1, characterized in that The concentration of chitosan oligosaccharide solution is 5-30 mg / mL.

8. The method according to claim 1, characterized in that 1-24 mL of 0.01-5 mg / mL sodium tripolyphosphate solution.

9. A nanocomposite prepared by the method according to any one of claims 1 to 8.

10. Use of the anthocyanin nanocomposite prepared by the method of any one of claims 1 to 8 in preparing drugs for treating eye diseases, or preparing common foods or health foods for relieving visual fatigue, or preparing cosmetics.

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