Chitosan oligosaccharide-anthocyanin nanocomposite and preparation method thereof

Abstract

The invention discloses a preparation method of a chitosan oligosaccharide-anthocyanin nanocomposite. The preparation method comprises the following steps: dissolving purple corn anthocyanin in distilled water; dissolving chitosan oligosaccharide in distilled water; the method comprises the following steps: adding an organic solvent into a reaction kettle, reacting at room temperature in a dark place for 4-48 hours, transferring a mixed solution into a centrifugal tube, centrifuging for 10-30 minutes to separate out nano-composite precipitates and uncombined supernatant, and then adding ultrapure water for repeated centrifugation. And finally, carrying out freeze drying treatment on the precipitate to obtain the final chitosan oligosaccharide-anthocyanin nano-composite. The nano-composite prepared by the method has high stability, good dispersibility and enhanced biological activity, and provides a new way for wide application of chitosan oligosaccharide and anthocyanin in the fields of food, cosmetics, biological medicine 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 chitosan oligosaccharide-anthocyanidin nanocomposite. Background Art

[0002] Anthocyanins are a class of water-soluble pigments widely found in plants. They belong to the flavonoids and have multiple pharmacological effects, including anti-inflammatory, antioxidant, antibacterial, antiproliferative and anti-tumor biological activities. Many clinical trials have shown that anthocyanins have significant benefits for human health. For example, taking anthocyanins can relieve edema symptoms, improve skin elasticity, enhance immunity, and alleviate premenstrual syndrome. In addition, anthocyanins have also been used to treat varicose veins, allergies, asthma, bronchitis and other diseases, all with good results. However, anthocyanins may degrade or discolor under conditions such as light, high temperature or changes in pH, affecting their stability and application effects. The bioavailability of anthocyanins is also relatively low, which limits their clinical application in the field of biomedicine.

[0003] At present, there have been many studies on improving the bioavailability of anthocyanins, including the construction of nano drug delivery systems, the preparation of emulsions, nano micelles, liposomes, gels, etc. Among them, the carrier materials in the nano drug delivery system, such as solid lipid nanoparticles, polylactic acid glycolic acid nanoparticles, etc., have improved the bioavailability of anthocyanins to a certain extent, but their biocompatibility and safety still need to be further evaluated. Long-term or large-scale use of these nanoparticles may cause potential toxicity or adverse reactions to the human body. Its safety is difficult to guarantee, and whether its biodegradability will have an impact on the body remains to be investigated.

[0004] Chitosan is a natural cationic polysaccharide with good biocompatibility, biodegradability, non-toxicity and other advantages. It also has multiple pharmacological effects such as anti-inflammatory, antibacterial, antioxidant and anti-tumor. However, due to its structure and molecular weight, it has poor water solubility, which limits its application. CN103536583B discloses a proanthocyanidin sustained-release nanosphere, characterized in that the proanthocyanidin sustained-release nanosphere comprises the following ingredients: 1 to 5 parts of proanthocyanidin; 1 to 12 parts of chitosan or its derivatives; 1 to 12 parts of phospholipids or its derivatives; 1 to 10 parts of excipients; the chitosan and its derivatives are one or more of high, medium and low molecular weight chitosan, chitosan oligosaccharides, chitosan quaternary ammonium salts, and carboxymethyl chitosan. Although the document mentions chitosan oligosaccharides as chitosan derivatives, the document actually uses chitosan to prepare nanospheres, and selects chitosan and its derivatives of high, medium and low molecular weight to prepare nanospheres, wherein the molecular weight of low molecular weight chitosan and its derivatives is 160kDa to 340kDa, and phospholipids are also required. The goal is to achieve the sustained release of proanthocyanidins, and anthocyanidins cannot be protected. In addition, anthocyanidins and proanthocyanidins are two different compounds. Proanthocyanidins are colorless polyphenols, while the anthocyanidins protected by this patent are dark purple flavonoid compounds. CN105105137A discloses a blueberry anthocyanidin and chitosan oligosaccharide composition and a preparation method thereof. The method only puts 30 to 50 parts by weight of blueberry anthocyanidin purified liquid, 30 to 50 parts by weight of chitosan oligosaccharide powder, 5 to 10 parts by weight of L-carnitine, and 1 to 3 parts by weight of cassia seed into a food material mixer and stirs, and no complex is formed. CN112891518B discloses a complex containing non-denatured type II collagen and chitosan oligosaccharide and a preparation method thereof, but the coupling steps are complicated and the effect directions are different.

[0005] The poor stability and low bioavailability of anthocyanins are technical issues that need to be solved urgently. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing a chitosan oligosaccharide-anthocyanidin nanocomposite in view of the shortcomings of the prior art. In order to achieve the above-mentioned purpose, the present invention adopts the following technical solution:

[0007] The steps include: (1) dissolving anthocyanidin in distilled water to obtain an anthocyanidin solution with a concentration of 5-30 mg / mL;

[0008] (2) dissolving chitosan oligosaccharide in distilled water to obtain a chitosan oligosaccharide solution with a concentration of 5-30 mg / mL;

[0009] (3) adding the anthocyanin solution to the chitosan oligosaccharide solution, stirring for reaction at a speed of 120-800 rpm to obtain a mixed solution;

[0010] (4) The mixed solution is centrifuged to obtain a precipitate.

[0011] (5) The precipitate in step (4) is vacuum freeze-dried to obtain the nanocomposite.

[0012] Furthermore, the degree of polymerization of chitosan oligosaccharide is 2-20. The molecular weight of chitosan oligosaccharide is less than 1000Da. 。

[0013] Furthermore, step (3) is carried out at room temperature in the dark for 4-48 hours.

[0014] 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.

[0015] The beneficial effects of the present invention relative to the prior art are:

[0016] (1) The preparation method of the present invention is simple and easy to implement, and does not require complicated equipment and cumbersome steps. The nanocomposite can be prepared by conventional means such as magnetic stirring and centrifugation, which reduces production costs and improves production efficiency.

[0017] (2) The chitosan oligosaccharide-anthocyanidin nanocomposite prepared by the method of the present invention has significantly improved stability. The nanoscale complex can effectively prevent the degradation of anthocyanidin in the environment, extend its shelf life, and make its performance in various applications more stable and reliable.

[0018] (3) Due to the high stability, good dispersibility and enhanced biological activity of the nanocomplex, its application field has been greatly broadened. It can be used not only in traditional fields such as food and cosmetics, but also in high-end fields such as biomedicine, providing a new way and idea for the comprehensive utilization of chitosan oligosaccharides and anthocyanins. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the ultraviolet spectrum change diagram of chitosan oligosaccharide-anthocyanidin nanocomplex and single chitosan oligosaccharide and anthocyanidin when the concentration ratio of anthocyanidin solution to chitosan oligosaccharide solution is 1:1.

[0020] Figure 2 This is the ultraviolet spectrum change diagram of chitosan oligosaccharide-anthocyanidin nanocomplex and single chitosan oligosaccharide and anthocyanidin when the concentration ratio of anthocyanidin solution to chitosan oligosaccharide solution is 1:2.

[0021] Figure 3 This is a graph showing the ultraviolet spectrum changes of the chitosan oligosaccharide-anthocyanidin nanocomposite of Example 1 and individual chitosan oligosaccharides and anthocyanidins.

[0022] Figure 4 The infrared spectra of chitosan oligosaccharide-anthocyanidin nanocomplex and individual chitosan oligosaccharide and anthocyanidin.

[0023] Figure 5 The particle size distribution diagram of the chitosan oligosaccharide-anthocyanidin nanocomposite of Example 1 and the chitosan oligosaccharide and anthocyanidin alone.

[0024] Figure 6 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.

[0025] Figure 7 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.

[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-anthocyanin nanocomposite of Example 1 on anthocyanin in a pH=3 environment.

[0031] Fig.13 This is the protective effect of the chitosan oligosaccharide-anthocyanin nanocomposite of Example 1 on anthocyanin in a pH=5 environment.

[0032] Fig.14 This is the protective effect of the chitosan oligosaccharide-anthocyanin nanocomposite of Example 1 on anthocyanin in a pH=7 environment.

[0033] Fig.15 This is the protective effect of the chitosan oligosaccharide-anthocyanin nanocomposite of Example 1 on anthocyanin in an environment of pH = 9.

[0034] Fig.16 This is the protective effect of the chitosan oligosaccharide-anthocyanin nanocomposite of Example 1 on anthocyanin in an environment of pH=11.

[0035] Fig.17This is the protective effect of the chitosan oligosaccharide-anthocyanidin nanocomplex of Example 1 on anthocyanidin at 36.5°C.

[0036] Fig.18 This is the protective effect of the chitosan oligosaccharide-anthocyanin nanocomposite of Example 1 on anthocyanin at 60°C.

[0037] Fig.19 This is the protective effect of the chitosan oligosaccharide-anthocyanin nanocomposite of Example 1 on anthocyanin at 105°C.

[0038] Fig. 20 This is an electron microscope image of the chitosan oligosaccharide-anthocyanin nanocomplex at a magnification of 100,000 times;

[0039] Fig.21 This is an electron microscope image of the chitosan oligosaccharide-anthocyanin nanocomplex at a magnification of 50,000 times;

[0040] Fig. 22 This is an electron microscope image of Comparative Example 2 at a magnification of 50,000 times. DETAILED DESCRIPTION

[0041] 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.

[0042] Example 1

[0043] A method for preparing a chitosan oligosaccharide-anthocyanidin nanocomposite is carried out according to the following steps:

[0044] (1) Dissolve anthocyanins in distilled water at a concentration of 20 mg / mL;

[0045] (2) Dissolve chitosan oligosaccharide in distilled water at a concentration of 10 mg / mL;

[0046] (3) taking the solution of step (1) and step (2) into a beaker, reacting at room temperature for 24 hours under a magnetic stirring speed of 150 rpm;

[0047] (4) The mixed solution was centrifuged at 12000 rpm for 30 min, then double distilled water was added and the centrifugation was repeated three times.

[0048] (5) The precipitate in step (4) is vacuum freeze-dried to obtain the nanocomposite.

[0049] The concentration ratio of the anthocyanin solution in step (1) to the chitosan oligosaccharide solution in step (2) is 2:1.

[0050] Example 2

[0051] 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 2:1, that is, 10 mg / mL and 20 mg / mL respectively. The other steps and parameters are the same as those in Example 1.

[0052] Table 1

[0053]

[0054]

[0055] As shown in Table 1, the zeta potential of chitosan oligosaccharide is positive (11.27±1.56mV), while 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 the chitosan oligosaccharide coating may change the surface charge properties of anthocyanin, making it more negatively charged. The enhancement of negative charge helps the stability of nanoparticles in the body and reduces nonspecific interactions with charged biomolecules, 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 particle size of chitosan oligosaccharide is small 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 RNP (dissolution method) was 637.00±0.35nm, which was significantly larger than that of chitosan oligosaccharides and anthocyanins.

[0056] Table 2

[0057]

[0058] 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 0.06 mg / mL, 0.12 mg / mL and 0.07 mg / mL respectively. Especially in terms of inhibiting ABTS free radicals, the chitosan oligosaccharide-anthocyanidin nanocomplex also showed a considerable effect. 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.

[0059] Comparative Example 1

[0060] Solvent evaporation method (HNP): 50-200 mg of polylactide-glycolide (PLGA) was dissolved in 1-20 mL of dichloromethane and then added dropwise to 1-30 mL of purple corn anthocyanin (PCA) solution. After ultrasonication in an ice-water bath for 3 min, vortex oscillation was performed and 1-30 mL of chitosan oligosaccharide (COS)-1% polyvinyl alcohol (PVA) solution was added dropwise, and ultrasonication was performed in an ice-water bath for 1-60 min. The pH of the solvent was gradually adjusted to 9.0 with 1-20% hydrochloric acid and sodium hydroxide, and magnetic stirring was performed for 1-48 h to evaporate the organic solvent. After removal, the mixture was transferred to centrifuge tubes respectively, and ultra-high-speed centrifugation was performed for 10-30 min at a speed of 11000 rpm to remove the supernatant solution. The precipitate was washed with ultrapure water 3 times, and the precipitate was freeze-dried to obtain a chitosan oligosaccharide-anthocyanin nanocomposite.

[0061] Comparative Example 2

[0062] The present embodiment is different from Example 1 in that the molecular weight of chitosan oligosaccharide is 4000Da, the degree of polymerization is about 30-60, and the other steps and parameters are the same as those in Example 1.

[0063] 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. Fig. 20 and 21 It can be seen that the chitosan oligosaccharide-anthocyanin nanocomposite is successfully compounded. However, when the degree of polymerization of chitosan oligosaccharide is high, a good compound cannot be formed.

[0064] Example 3

[0065] The preparation method of chitosan oligosaccharide-anthocyanidin nanocomposite adopts the following technical scheme:

[0066] The steps include: (1) dissolving anthocyanidin in distilled water to obtain anthocyanidin solution with a concentration of 5 mg / mL;

[0067] (2) dissolving chitosan oligosaccharide in distilled water to obtain a chitosan oligosaccharide solution with a concentration of 30 mg / mL;

[0068] (3) adding the anthocyanin solution to the chitosan oligosaccharide solution, stirring and reacting at a speed of 120 rpm to obtain a mixed solution;

[0069] (4) The mixed solution is centrifuged to obtain a precipitate.

[0070] (5) The precipitate in step (4) is vacuum freeze-dried to obtain the nanocomposite.

[0071] The degree of polymerization of chitosan oligosaccharide is 20. Step (3) is to react at room temperature for 4 hours.

[0072] 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.

[0073] Example 4

[0074] The preparation method of chitosan oligosaccharide-anthocyanidin nanocomposite adopts the following technical scheme:

[0075] The steps include: (1) dissolving anthocyanin in distilled water to obtain an anthocyanin solution with a concentration of 30 mg / mL;

[0076] (2) dissolving chitosan oligosaccharide in distilled water to obtain a chitosan oligosaccharide solution with a concentration of 5 mg / mL;

[0077] (3) adding the anthocyanin solution to the chitosan oligosaccharide solution, stirring for reaction at a speed of 800 rpm to obtain a mixed solution;

[0078] (4) The mixed solution is centrifuged to obtain a precipitate.

[0079] (5) The precipitate in step (4) is vacuum freeze-dried to obtain the nanocomposite.

[0080] The degree of polymerization of chitosan oligosaccharide is 2. Step (3) is to react at room temperature for 48 hours.

[0081] 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.

[0082] 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.

[0083] 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 chitosan oligosaccharide-anthocyanidin nanocomposite, characterized in that: The following technical solutions are adopted: The steps include: (1) dissolving anthocyanin in distilled water to obtain anthocyanin solution; (2) dissolving chitosan oligosaccharide in distilled water to obtain a chitosan oligosaccharide solution; (3) adding the anthocyanin solution to the chitosan oligosaccharide solution, stirring to react and obtaining a mixed solution; (4) Centrifuging the mixed solution to obtain a precipitate; (5) The precipitate in step (4) 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 4-48 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: Step (1) dissolving anthocyanidins in distilled water to obtain anthocyanidin solution with a concentration of 5-30 mg / mL; and / or (2) Dissolve chitosan oligosaccharide in distilled water to obtain a chitosan oligosaccharide solution with a concentration of 5-30 mg / mL.

6. A chitosan oligosaccharide-anthocyanidin nanocomposite prepared by the method of any one of claims 1 to 5.

7. Use of the chitosan oligosaccharide-anthocyanidin nanocomposite prepared by the method of any one of claims 1 to 5 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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