Composite polysaccharide nano-selenium compound as well as preparation method and application thereof

Abstract

The invention provides a compound polysaccharide nano-selenium compound and a preparation method and application thereof.The compound comprises polysaccharide-modified nano-selenium particles, the polysaccharide-modified nano-selenium particles are obtained through an oxidation-reduction reaction of a polysaccharide composition and a selenium solution, the polysaccharide composition comprises bolete polysaccharide, purslane polysaccharide and astragalus polysaccharide, and the selenium solution is a selenium solution. The mass ratio of the bolete polysaccharide to the purslane polysaccharide to the astragalus polysaccharide is (0.5-3): (0.5-3): (0.5-3). According to the compound polysaccharide nano-selenium compound disclosed by the invention, nano-selenium is synergistically modified through various polysaccharides, so that the nano-selenium is effectively prevented from being gathered and oxidized, the dispersity and stability of the nano-selenium are remarkably improved, the bioavailability and targeting property of the nano-selenium are improved, the antioxidant activity of the nano-selenium is enhanced, and the cytotoxicity of the nano-selenium is reduced; the multi-functionalization of the nano-selenium is realized, and the application potential of the nano-selenium in the fields of medicines, foods, cosmetics and the like is expanded.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological materials, and particularly relates to a composite polysaccharide nano-selenium compound as well as a preparation method and application thereof. BACKGROUND

[0002] Selenium is an essential trace element for human body, which has multiple physiological functions such as antioxidant, anti-tumor and immune regulation. However, the low bioavailability and narrow toxicity window of selenium limit its application. Nano-selenium (SeNPs) has become a research hotspot due to its high bioavailability and low toxicity, but the problems of poor stability and easy aggregation need to be solved. As a natural polymer material, polysaccharide has rich functional groups (such as hydroxyl and carboxyl groups), which can be used as a stabilizer and carrier for nano-selenium to improve its dispersibility and biological activity. At present, there are studies on nano-selenium compound stabilized by single polysaccharide, but there is no report on nano-selenium compound stabilized by multiple polysaccharides and its preparation method.

[0003] The polysaccharide nano-selenium compound in the prior art at least has the following problems:

[0004] (1) Narrow toxicity window: the safety window between the effective dose and the toxic dose of selenium is narrow, which limits its application in nutritional supplements and drugs; the nano-selenium modified by single polysaccharide may increase the risk of toxicity while improving the biological activity;

[0005] (2) Complex preparation process: the conditions are harsh, and it is difficult to realize large-scale production; the nano-selenium modified by single polysaccharide may have problems such as uneven particle size and poor dispersibility during preparation. The typical preparation techniques reported in the literature mainly include: ① physical mixing method, such as the Chinese invention patent with publication number CN119978159A, which discloses a preparation method of high-purity jujube polysaccharide nano-selenium, which simply mixes polysaccharide with pre-synthesized nano-selenium, but the interfacial bonding force is weak and it is easy to dissociate in physiological environment; ② reduction method, for example, Shi Menghua et al. disclosed in the article "Research Progress of Preparation, Characterization and Biological Activity of Polysaccharide Nano-selenium" that polysaccharide is used to reduce selenium salt (such as Na2SeO3) to generate nano-selenium, but the particle size distribution is wide (50-200 nm) and the loading rate is low (<5%); ③ fermentation method (such as commercial selenium yeast), although it realizes intracellular deposition of selenium, but the bioavailability is less than 30%;

[0006] (3) Limited application range: the existing nano-selenium compound is limited in the application in the fields of medicine, food, cosmetics, etc.; the nano-selenium modified by single polysaccharide has poor adaptability and stability in complex environment. SUMMARY

[0007] Therefore, the present application aims to provide a composite polysaccharide nano selenium compound, a preparation method and application thereof, so as to develop a nano selenium compound with low toxicity, high safety, strong adaptability and high stability, and provide a simple, efficient and controllable preparation method suitable for industrial production.

[0008] To achieve the above-mentioned purposes, the technical scheme of the present application is as follows:

[0009] In a first aspect, the present application provides a composite polysaccharide nano selenium compound, which comprises polysaccharide modified nano selenium particles, wherein the polysaccharide modified nano selenium particles are obtained by redox reaction of a polysaccharide composition and a selenium solution, and the polysaccharide composition comprises boletus polysaccharide, spilanthes polysaccharide and astragalus polysaccharide.

[0010] Further, the particle size of the polysaccharide modified nano selenium particles is 20-200 nm.

[0011] Further, the mass ratio of boletus polysaccharide, spilanthes polysaccharide and astragalus polysaccharide is 0.5-3:0.5-3:0.5-3; preferably, the mass ratio of boletus polysaccharide, spilanthes polysaccharide and astragalus polysaccharide is 0.8-1.2:0.8-1.2:0.8-1.2; further preferably, the mass ratio of boletus polysaccharide, spilanthes polysaccharide and astragalus polysaccharide is 1:1:1.

[0012] In a second aspect, the present application provides a preparation method of a composite polysaccharide nano selenium compound, which comprises the following steps:

[0013] S1, mixing a polysaccharide composition with water to obtain a polysaccharide mixed solution, wherein the polysaccharide composition comprises boletus polysaccharide, spilanthes polysaccharide and astragalus polysaccharide, and the mass ratio of boletus polysaccharide, spilanthes polysaccharide and astragalus polysaccharide is 0.5-3:0.5-3:0.5-3;

[0014] S2, stirring the selenium solution and the polysaccharide mixed solution uniformly to obtain a polysaccharide nano selenium suspension;

[0015] S3, mixing a stabilizer with the polysaccharide nano selenium suspension and stirring until the reaction is complete to obtain a reaction liquid;

[0016] S4, centrifuging, washing and drying the reaction liquid to obtain a composite polysaccharide nano selenium compound.

[0017] Further, the polysaccharide composition includes boletus polysaccharide, purslane polysaccharide, and astragalus polysaccharide, and the concentration of the polysaccharide composition in the polysaccharide mixed solution is 1-5 mg / mL, for example, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, or 5 mg / mL; preferably 1.5-3 mg / mL; and further preferably 2 mg / mL.

[0018] Preferably, the mass ratio of the boletus polysaccharide, the purslane polysaccharide, and the astragalus polysaccharide is 0.8-1.2:0.8-1.2:0.8-1.2; and further preferably, the mass ratio of the boletus polysaccharide, the purslane polysaccharide, and the astragalus polysaccharide is 1:1:1.

[0019] Further, the extraction and purification of the boletus polysaccharide, the purslane polysaccharide, and the astragalus polysaccharide includes the steps of hot water extraction, alcohol precipitation, deproteinization, ion exchange chromatography, and gel filtration chromatography, and is suitable for polysaccharides from various sources such as fungi and plants. Preferably, the extraction of the boletus polysaccharide, the purslane polysaccharide, or the astragalus polysaccharide is performed by hot water extraction, and the specific process is as follows:

[0020] (1) Extraction of crude polysaccharide

[0021] The raw material powder (boletus, purslane, and astragalus) is added to deionized water at a solid-liquid ratio of 1:20 (w / v), extracted in a water bath at 90°C for 3 h, centrifuged (6000 rpm, 10 min) to collect the supernatant, and the extraction is repeated for 3 times. The extraction solutions are combined, concentrated to 1 / 4 of the original volume, 4 volumes of 95% ethanol are added, and alcohol precipitation is performed at 4°C overnight. The precipitate is collected by centrifugation, resuspended in water, and a crude polysaccharide solution is obtained.

[0022] (2) Deproteinization

[0023] Sevage method (chloroform:n-butanol = 4:1, 5:1 v / v added to the sample) is used, and after oscillation for 20 min, centrifugation (4000 rpm, 10 min) is performed, and the process is repeated until there is no protein precipitate at the interface. After removing the organic solvent, the sample is dialyzed in a 3500 Da dialysis bag for 48 h, and freeze-drying is performed to obtain deproteinized crude polysaccharide.

[0024] (3) Ion exchange chromatography purification

[0025] The deproteinized crude polysaccharide is separated by DEAE-52 cellulose column (6.0x100.0 cm), eluted with 0-0.5 mol / L NaCl gradient, and the polysaccharide peak is detected by phenol-sulfuric acid method. The main peak is collected, dialyzed, and freeze-dried to obtain purified polysaccharide.

[0026] (4) Gel filtration chromatography verification

[0027] The purified polysaccharide (20 mg) was further separated by a Sephadex G-100 column, eluted with deionized water, and the main peak was combined and freeze-dried to obtain a homogeneous polysaccharide.

[0028] Further, the concentration of the selenium solution in step S2 is 30-80 mmol / L, for example, can be 30 mmol / L, 35 mmol / L, 40 mmol / L, 45 mmol / L, 50 mmol / L, 55 mmol / L, 60 mmol / L, 65 mmol / L, 70 mmol / L, 75 mmol / L, 80 mmol / L; preferably 50-70 mmol / L; further preferably 60 mmol / L.

[0029] Further, the volume ratio of the selenium solution and the polysaccharide mixed solution is 1:3-8, for example, can be 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8; preferably 1:4-7, further preferably 1:5.

[0030] Further, the stabilizer is selected from one of a sodium citrate solution and an ascorbic acid solution.

[0031] Further, the reaction time in step S3 is 24 hours or more.

[0032] Further, the concentration of the stabilizer is 30-80 mmol / L, for example, can be 30 mmol / L, 35 mmol / L, 40 mmol / L, 45 mmol / L, 50 mmol / L, 55 mmol / L, 60 mmol / L, 65 mmol / L, 70 mmol / L, 75 mmol / L, 80 mmol / L; preferably 50-70 mmol / L; further preferably 60 mmol / L;

[0033] Preferably, the volume ratio of the stabilizer and the polysaccharide nano-selenium suspension is 2-5:5-10; preferably 3-5:5-8; further preferably 4:6.

[0034] In a third aspect, the present application provides a use of the composite polysaccharide nano-selenium complex according to the first aspect or the preparation method according to the second aspect in the preparation of medicines, food, and cosmetics.

[0035] In a fourth aspect, the present application provides a use of a polysaccharide composition in the preparation of a composite polysaccharide nano-selenium complex, the polysaccharide composition comprising boletus polysaccharide, spilanthes polysaccharide, and astragalus polysaccharide, the mass ratio of the boletus polysaccharide, the spilanthes polysaccharide, and the astragalus polysaccharide being 0.5-3:0.5-3:0.5-3, and the use comprising at least one of the following:

[0036] (1) the application in improving the stability of nanometer selenium;

[0037] (2) the application in improving the antioxidant capacity of the composite polysaccharide nanometer selenium complex;

[0038] (3) the application in reducing the cytotoxicity of the composite polysaccharide nanometer selenium complex;

[0039] (4) the application in improving the bioavailability of the composite polysaccharide nanometer selenium complex.

[0040] Further, the antioxidant capacity includes the DPPH radical scavenging capacity, the ABTS radical scavenging capacity or the hydroxyl radical scavenging capacity.

[0041] Compared with the prior art, the composite polysaccharide nanometer selenium complex, the preparation method and the application thereof have the following advantages:

[0042] (1) The composite polysaccharide nanometer selenium complex in the application is obtained by modifying nanometer selenium with multiple polysaccharides, and is a high-efficiency and stable multiple polysaccharide nanometer selenium complex, which effectively prevents the aggregation and oxidation of nanometer selenium, significantly improves the dispersibility and stability of nanometer selenium, solves the technical problem of poor stability of nanometer selenium, and significantly improves the physicochemical properties (such as particle size, surface charge and solubility) of the polysaccharide nanometer selenium complex, so that the polysaccharide nanometer selenium complex has stronger adaptability and stability in a complex environment (such as the gastrointestinal tract and blood).

[0043] (2) The composite polysaccharide nanometer selenium complex in the application improves the bioavailability and targeting of nanometer selenium, the bioactivities of different polysaccharides are complementary, and the synergistic effect can enhance the antioxidant activity of nanometer selenium and enhance its physiological functions.

[0044] (3) The composite polysaccharide nanometer selenium complex in the application reduces the cytotoxicity of nanometer selenium, widens the safe application range of nanometer selenium, realizes the multifunctionalization of nanometer selenium, and multiple polysaccharides can endow nanometer selenium with more functional properties, such as antibacterial, anti-inflammatory and anti-aging, so as to meet the application requirements in different fields and expand the application potential of nanometer selenium in the fields of medicine, food and cosmetics.

[0045] (4) The preparation method of the composite polysaccharide nanometer selenium complex in the application has the advantages of simple preparation process, mild conditions, high efficiency and controllability, is suitable for large-scale production, can reduce the amount of single polysaccharide compared with single polysaccharide nanometer selenium complex, effectively reduces the production cost, reduces the use of stabilizers, and improves the purity and safety of the product. BRIEF DESCRIPTION OF DRAWINGS

[0046] The accompanying drawings, which form a part of this application, are included to provide a further understanding of the application and are incorporated in and constitute a part of this application. The embodiments of the application illustrated in the drawings are intended to explain the aspects of the present application and are not limiting of the present application. In the drawings:

[0047] Figure 1 The Fourier infrared spectrum of the polysaccharide nanoselem composite prepared for Example 1 and Comparative Example 1;

[0048] Figure 2 The stability test results of the polysaccharide nanoselem composite prepared for Example 1 are shown in the schematic diagram;

[0049] Figure 3 The antioxidant activity test results of the polysaccharide nanoselem composite prepared for Example 1 and Comparative Examples 1-3 are shown in the schematic diagram;

[0050] Figure 4 The cytotoxicity test results of the polysaccharide nanoselem composite prepared for Example 1 and Comparative Examples 1-3 are shown in the schematic diagram. DETAILED DESCRIPTION

[0051] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0052] The present application will be described in detail below with reference to the accompanying drawings and in conjunction with the embodiments.

[0053] The boletus polysaccharide, purslane polysaccharide and astragalus polysaccharide used in the present embodiment and comparative examples are extracted by hot water extraction method, and the specific process is as follows:

[0054] (1) Extraction of crude polysaccharide

[0055] The raw material powder (boletus, purslane, astragalus) is added with deionized water at a material to liquid ratio of 1:20 (w / v), and extracted in a water bath at 90°C for 3h. The supernatant is collected by centrifugation (6000rpm, 10min), and the extraction is repeated for 3 times. The extraction liquid is combined and concentrated to 1 / 4 of the original volume, and 4 volumes of 95% ethanol are added for alcohol precipitation at 4°C overnight. The precipitate is collected by centrifugation and redissolved in water to obtain a crude polysaccharide solution.

[0056] (2) Deproteinization treatment

[0057] Sevage method (chloroform: n-butanol = 4:1, 5:1 v / v added with sample) is adopted, and after oscillation for 20min, centrifugation (4000rpm, 10min) is performed, and the process is repeated until there is no protein precipitate at the interface. After removing the organic solvent, the deproteinized crude polysaccharide is obtained by dialysis in a 3500Da dialysis bag for 48h and freeze-drying.

[0058] (3) Purification by ion exchange chromatography

[0059] The deproteinized crude polysaccharide was separated by DEAE-52 cellulose column (6.0*100.0 cm) and eluted with 0-0.5 mol / L NaCl gradient, and the polysaccharide peak was detected by phenol-sulfuric acid method. The main peak was collected, dialyzed and freeze-dried to obtain the purified polysaccharide.

[0060] (4) Gel filtration chromatography verification

[0061] The purified polysaccharide (20 mg) was further separated by Sephadex G-100 column, eluted with deionized water, and the main peak was combined and freeze-dried to obtain the uniform polysaccharide.

[0062] Example 1

[0063] The preparation method of the composite polysaccharide nano selenium complex of the embodiment comprises the following steps:

[0064] (1) The boletus polysaccharide, the purslane polysaccharide and the astragalus polysaccharide were dissolved in deionized water at a ratio of 1:1:1 to prepare a polysaccharide mixed solution with a concentration of 2 mg / mL;

[0065] (2) Sodium selenite (Na2SeO3) was dissolved in deionized water to prepare a selenium source solution with a concentration of 60 mmol / L;

[0066] (3) Under stirring, 1 mL of the selenium source solution with a concentration of 60 mmol / L was added to 5 mL of the polysaccharide mixed solution and stirred for 1 h to obtain a polysaccharide nano selenium suspension;

[0067] (4) Freshly prepared 4 mL of ascorbic acid solution with a concentration of 60 mmol / L was added dropwise to the polysaccharide nano selenium suspension, and the reaction was continued for 24 h under stirring and light shielding to fully combine the polysaccharide and the nano selenium, and a reaction solution was obtained;

[0068] (5) The reaction solution was centrifuged to collect the complex, washed with deionized water for 3 times, and freeze-dried to obtain the polysaccharide nano selenium complex.

[0069] Example 2

[0070] The preparation method of the composite polysaccharide nano selenium complex of the embodiment comprises the following steps:

[0071] (1) The boletus polysaccharide, the purslane polysaccharide and the astragalus polysaccharide were dissolved in deionized water at a ratio of 0.8:1:1.2 to prepare a polysaccharide mixed solution with a concentration of 1.5 mg / mL;

[0072] (2) Sodium selenite (Na2SeO3) was dissolved in deionized water to prepare a selenium source solution with a concentration of 50 mmol / L;

[0073] (3) Under stirring, 1 mL of 50 mmol / L selenium source solution was added into 4 mL of polysaccharide mixed solution to stir for 1 h to obtain polysaccharide nano selenium suspension;

[0074] (4) Freshly prepared 3 mL of 50 mmol / L ascorbic acid solution was added dropwise into the polysaccharide nano selenium suspension, and the reaction was continued to stir for 24 h in the dark to make the polysaccharide fully combined with nano selenium to obtain a reaction solution;

[0075] (5) The reaction solution was centrifuged to collect the complex, which was washed with deionized water for 3 times and freeze-dried to obtain polysaccharide nano selenium complex.

[0076] Example 3

[0077] The preparation method of the complex polysaccharide nano selenium complex of the present example comprises the following steps:

[0078] (1) Boletus polysaccharide, purslane polysaccharide and astragalus polysaccharide were dissolved in deionized water at a ratio of 1:0.8:1.2 to prepare a polysaccharide mixed solution with a concentration of 3 mg / mL;

[0079] (2) Sodium selenite (Na2SeO3) was dissolved in deionized water to prepare a selenium source solution with a concentration of 70 mmol / L;

[0080] (3) Under stirring, 1 mL of 70 mmol / L selenium source solution was added into 7 mL of polysaccharide mixed solution to stir for 1 h to obtain polysaccharide nano selenium suspension;

[0081] (4) Freshly prepared 5 mL of 70 mmol / L ascorbic acid solution was added dropwise into the polysaccharide nano selenium suspension, and the reaction was continued to stir for 24 h in the dark to make the polysaccharide fully combined with nano selenium to obtain a reaction solution;

[0082] (5) The reaction solution was centrifuged to collect the complex, which was washed with deionized water for 3 times and freeze-dried to obtain polysaccharide nano selenium complex.

[0083] Example 4

[0084] The preparation method of the complex polysaccharide nano selenium complex of the present example comprises the following steps:

[0085] (1) Boletus polysaccharide, purslane polysaccharide and astragalus polysaccharide were dissolved in deionized water at a ratio of 1.2:1:0.8 to prepare a polysaccharide mixed solution with a concentration of 2 mg / mL;

[0086] (2) Sodium selenite (Na2SeO3) was dissolved in deionized water to prepare a selenium source solution with a concentration of 60 mmol / L;

[0087] (3) Under stirring, 1 mL of 60 mmol / L selenium source solution was added into 5 mL of polysaccharide mixed solution to obtain polysaccharide nano selenium suspension after stirring for 1 h;

[0088] (4) Freshly prepared 4 mL of 60 mmol / L ascorbic acid solution was added dropwise into the polysaccharide nano selenium suspension, and the reaction was continued under stirring for 24 h in the dark to fully combine the polysaccharide and nano selenium, to obtain a reaction solution;

[0089] (5) The reaction solution was centrifuged to collect the complex, which was washed with deionized water for 3 times, and freeze-dried to obtain polysaccharide nano selenium complex.

[0090] Comparative Example 1

[0091] The polysaccharide nano selenium complex preparation method of the present comparative example comprises the following steps:

[0092] (1) The boletus polysaccharide was dissolved in deionized water to prepare a polysaccharide mixed solution with a concentration of 2 mg / mL;

[0093] (2) Sodium selenite (Na2SeO3) was dissolved in deionized water to prepare a selenium source solution with a concentration of 60 mmol / L;

[0094] (3) Under stirring, 1 mL of 60 mmol / L selenium source solution was added into 5 mL of polysaccharide mixed solution to obtain polysaccharide nano selenium suspension after stirring for 1 h:

[0095] (4) Freshly prepared 4 mL of 60 mmol / L ascorbic acid solution was added dropwise into the polysaccharide nano selenium suspension, and the reaction was continued under stirring for 24 h in the dark to fully combine the polysaccharide and nano selenium, to obtain a reaction solution;

[0096] (5) The reaction solution was centrifuged to collect the complex, which was washed with deionized water for 3 times, and freeze-dried to obtain boletus polysaccharide nano selenium complex.

[0097] Comparative Example 2

[0098] The polysaccharide nano selenium complex preparation method of the present comparative example comprises the following steps:

[0099] (1) The boletus polysaccharide was dissolved in deionized water to prepare a polysaccharide mixed solution with a concentration of 2 mg / mL;

[0100] (2) Sodium selenite (Na2SeO3) was dissolved in deionized water to prepare a selenium source solution with a concentration of 60 mmol / L;

[0101] (3) Under stirring, 1 mL of 60 mmol / L selenium source solution was added into 5 mL of polysaccharide mixed solution to obtain polysaccharide nano selenium suspension after stirring for 1 h:

[0102] (4) Freshly prepared 4 mL of ascorbic acid solution with a concentration of 60 mmol / L was added dropwise to the polysaccharide nanometer selenium suspension, and the reaction was continued under stirring in the dark for 24 hours to allow the polysaccharide to fully combine with the nanometer selenium, obtaining a reaction liquid;

[0103] (5) The reaction liquid was centrifuged to collect the complex, washed with deionized water for 3 times, and freeze-dried to obtain the Portulaca oleracea polysaccharide nanometer selenium complex.

[0104] Comparative Example 3

[0105] The polysaccharide nanometer selenium complex preparation method of the present comparative example comprises the following steps:

[0106] (1) Astragalus polysaccharide was dissolved in deionized water to prepare a polysaccharide mixed solution with a concentration of 2 mg / mL;

[0107] (2) Sodium selenite (Na2SeO3) was dissolved in deionized water to prepare a selenium source solution with a concentration of 60 mmol / L;

[0108] (3) Under stirring, 1 mL of the selenium source solution with a concentration of 60 mmol / L was added to 5 mL of the polysaccharide mixed solution to stir for 1 h, obtaining a polysaccharide nanometer selenium suspension:

[0109] (4) Freshly prepared 4 mL of ascorbic acid solution with a concentration of 60 mmol / L was added dropwise to the polysaccharide nanometer selenium suspension, and the reaction was continued under stirring in the dark for 24 hours to allow the polysaccharide to fully combine with the nanometer selenium, obtaining a reaction liquid;

[0110] (5) The reaction liquid was centrifuged to collect the complex, washed with deionized water for 3 times, and freeze-dried to obtain the Astragalus polysaccharide nanometer selenium complex.

[0111] Performance Test Example 1 Fourier Infrared Spectroscopy

[0112] The polysaccharide nanometer selenium complexes prepared in Example 1 and Comparative Example 1 were subjected to Fourier infrared spectroscopy detection, and the results are shown in Figure 1 The infrared spectroscopy analysis of the single polysaccharide nanometer selenium complex and the complex polysaccharide nanometer selenium complex of Boletus showed that the spectrum of the Boletus single polysaccharide nanometer selenium complex prepared in Comparative Example 1 had multiple characteristic absorption peaks, which corresponded to different functional groups in the polysaccharide molecule. For example, the absorption peak at 3419 cm -1 was the stretching vibration of O-H, and the absorption peak at 2933 cm -1The absorption peaks of C-H stretching vibration are typical characteristic peaks of saccharides. The spectrum of the composite polysaccharide nano-selenium complex prepared in Example 1 shows similar absorption peaks to the single polysaccharide nano-selenium complex of boletus, indicating that the introduction of nano-selenium does not significantly change the basic structure of the polysaccharide. However, the position or intensity of some peaks changes, which may be due to the interaction between nano-selenium and polysaccharide molecules. When polysaccharide acts as a stable template to modify nano-selenium, the red shift degree of the infrared absorption peaks of the hydroxyl and carbonyl groups of polysaccharide is greater, indicating that the interaction between polysaccharide and nano-selenium is stronger. For example, the change in the intensity of the O-H stretching vibration absorption peak indicates that nano-selenium may interact with the hydroxyl group of polysaccharide. This interaction may help to improve the stability of nano-selenium and may have an impact on its biological activity.

[0113] Performance test example 2 stability test

[0114] Experimental method: The polysaccharide nano-selenium complexes prepared in Example 1, Comparative Examples 1-3 were prepared into a polysaccharide nano-selenium complex solution with a concentration of 1 mg / mL. 1 mL of polysaccharide nano-selenium complex solution with a concentration of 1 mg / mL was added to 5 mL of PBS (pH=5.6, 7.4) solution, water, DMEM solution and DMEM (containing 10% FBS) solution, respectively, and placed at room temperature for 4 days. The Zeta potential instrument was used to monitor the size change of BLPs-SeNPs in PBS (pH=5.6, 7.4) solution, water, DMEM solution and DMEM (containing 10% FBS) solution after incubation at different time points (0h, 12h, 24h, 36h, 48h, 60h, 72h, 84h and 96h).

[0115] The experimental results are shown in Table 1. Figure 2 As shown in Table 1, the composite polysaccharide nano-selenium complex prepared in the present application can maintain a stable state for nearly 72h in water, PBS solution (pH=5.6, 7.4), DMEM and DMEM containing 10% FBS, etc. It has more excellent stability than the single polysaccharide (boletus polysaccharide, polysaccharide of gynostemma pentaphyllum or astragalus polysaccharide) nano-selenium complex. This is because the polysaccharide composition can form a more stable three-dimensional network structure through synergistic effect, which can effectively prevent the aggregation of nano-selenium particles compared with single polysaccharide. At the same time, the functional groups (such as hydroxyl, carboxyl, etc.) of different polysaccharides form multiple coordination bonds with nano-selenium, enhancing the structural stability of the complex. Therefore, the composite polysaccharide nano-selenium complex has a longer shelf life and better dispersibility during storage and transportation.

[0116] The combination of the composite polysaccharide nanometer selenium complex can be designed according to the target application (such as medicine, food, cosmetics) to meet the needs of different fields; the physicochemical properties (such as particle size, surface charge, solubility) of the complex can be accurately regulated by adjusting the ratio of polysaccharides; in a complex environment (such as the gastrointestinal tract, blood), the composite polysaccharide nanometer selenium complex shows stronger adaptability and stability.

[0117] Performance test example 3 antioxidant activity test

[0118] Free radicals, as an important pathogenic factor of various acute and chronic diseases, can cause serious oxidative stress. The inherent antioxidant defense system in the body plays a key role in maintaining redox balance, removing excess free radicals, and protecting cells from oxidative damage. Excessive free radicals can cause oxidative reactions with biological macromolecules (including membrane lipids, proteins, carbohydrates, and nucleic acids, etc.), leading to damage to cell structure and function. This oxidative damage is considered an important mechanism leading to aging and related diseases. Studies have shown that the occurrence and development of various chronic diseases such as metabolic syndrome (diabetes, hypertension), liver disease, cardiovascular disease, and malignant tumor are closely related to free radical-mediated oxidative damage. In this test example, Vc was used as a positive control to evaluate the in vitro antioxidant activity of three single polysaccharide nanometer selenium complexes and composite polysaccharide nanometer selenium complexes by DPPH free radical, ABTS cationic free radical and hydroxyl radical scavenging experiments:

[0119] (1) DPPH free radical scavenging

[0120] Experimental method: 0.1 mmol / L DPPH-ethanol working solution was prepared in advance. 5.0 mg of polysaccharide nanometer selenium complex prepared in Example 1 and Comparative Examples 1-3 was accurately weighed and dissolved in 5 mL of anhydrous ethanol to prepare a 1 mg / mL stock solution, and serial dilutions were made to obtain 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL test solutions. Ascorbic acid (Vc) was used as a positive control to prepare a corresponding concentration of control solution.

[0121] The experimental groups are as follows:

[0122] Test group: Mix 1 mL of DPPH solution with different concentrations of sample solution in equal volumes;

[0123] Sample background group: Mix 1 mL of ethanol with 1 mL of different concentrations of sample solution;

[0124] Blank control group: Mix 1 mL of ethanol with 1 mL of DPPH solution.

[0125] After the above mixture was placed in the dark for 30 min, the absorbance value was measured at the maximum absorption wavelength of 517 nm using a UV-visible spectrophotometer. Three independent replicates were set for each concentration. The DPPH free radical scavenging rate was calculated according to formula (1):

[0126]

[0127] In the formula: A1 is the absorbance of the test group; A2 is the absorbance of the sample background group; A0 is the absorbance of the blank control group.

[0128] (2) ABTS free radical scavenging ability

[0129] Experimental method: 10 μL of polysaccharide nano selenium complex and Vc standard solution prepared by gradient concentration of example 1 and comparative examples 1-3 were taken respectively, and the corresponding reaction system was added in turn according to the ABTS free radical scavenging ability detection kit (Beijing Solabio Technology Co., Ltd.) operation manual.

[0130] The experimental groups are as follows:

[0131] Test group: 10 μL sample + ABTS working solution (according to the kit);

[0132] Sample background group: 10 μL sample + buffer (without ABTS);

[0133] Blank control group: 10 μL solvent (such as PBS) + ABTS working solution;

[0134] Standard curve group: gradient concentration Vc + ABTS working solution.

[0135] After the mixed reaction solution was mixed thoroughly, it was incubated at room temperature for 6 min in the dark. The absorbance values of each reaction system were measured at 405 nm characteristic wavelength using an enzyme marker, and three parallel replicates were set for each concentration gradient. The clearance rate was calculated according to the standard curve and formula (1).

[0136] (3) Hydroxyl radical scavenging ability

[0137] Experimental method: Gradient dilution method was used to prepare polysaccharide nano selenium complex sample solution and ascorbic acid (Vc) standard solution prepared by example 1 and comparative examples 1-3. According to the operation manual of the hydroxyl radical scavenging ability determination kit (Beijing Solabio Technology Co., Ltd.), the corresponding reaction system was added in turn.

[0138] The experimental groups are as follows:

[0139] Test group: sample + H2O2 + color developing agent;

[0140] Sample background group: sample + PBS (without H2O2 / color developing agent);

[0141] Blank control group: PBS + H2O2+ color reagent;

[0142] Standard curve group: gradient concentration Vc + PBS.

[0143] After the reaction system was fully shaken and mixed, it was incubated in a 37℃ constant temperature water bath for 15min. The absorbance value of each reaction system was detected at 510nm characteristic wavelength using ultraviolet-visible spectrophotometer, and 3 independent repeats were set for each sample. The clearance rate of the sample was calculated according to the standard curve and formula (2).

[0144]

[0145] In the formula: A1 is the absorbance value of the test group; A2 is the absorbance value of the sample background group; A0 is the absorbance value of the blank control group.

[0146] The experimental results are shown in Table 1. Figure 3 As shown in Table 1, compared with polysaccharide selenium nanocomposites prepared from single polysaccharide, the polysaccharide selenium nanocomposites prepared from combined polysaccharide showed stronger free radical scavenging ability, and showed obvious dose-effect relationship in a certain concentration range, which proved that the synergistic effect between polysaccharide compositions could enhance the antioxidant activity of selenium nanoparticles. In addition, the polysaccharide selenium nanocomposites prepared by the present application showed higher bioavailability and more significant therapeutic effect in cell experiments and animal models.

[0147] Performance test example 4 cytotoxicity test

[0148] CCK8 kit was used for detection. After adjusting the cell density of RAW 264.7 cells to 1×10 4 6 / mL, the cells were inoculated in a 96-well plate and cultured for 24h. The cells were treated with different concentrations of polysaccharide selenium nanocomposites (0, 12.5, 25, 50, 100, 200μg / mL) for 24h. After the treatment, the culture medium was replaced, 10% CCK8 solution prepared with RAW 264.7 special culture medium was added to each well, and the culture was continued for 2h. Finally, the OD value was measured at 450nm wavelength, and the experiment was repeated three times.

[0149] The experimental results are shown in Table 2. Figure 4 As shown in Table 2, compared with polysaccharide selenium nanocomposites prepared from single polysaccharide, the polysaccharide selenium nanocomposites prepared from combined polysaccharide showed higher cell survival rate, which was due to the following two reasons:

[0150] (1) The combined modification of polysaccharide selenium nanocomposites can reduce the surface energy of selenium nanoparticles, and reduce the toxicity of selenium nanoparticles to normal cells;

[0151] (2) The synergistic protection of polysaccharides can slow down the release rate of nano selenium in vivo, and avoid the toxicity caused by the local high concentration.

[0152] Therefore, in the safety evaluation, the damage of the composite polysaccharide nano selenium complex to normal cells is significantly lower than that of the single polysaccharide nano selenium complex.

[0153] In summary, the composite polysaccharide nano selenium complex prepared by the polysaccharide synergistic modification of nano selenium is significantly superior to the single polysaccharide nano selenium complex in stability, biological activity, safety, application adaptability and functional diversity, and is a new functional material with more development potential, which has wider application potential in the fields of multifunctional drug delivery, functional food additives and high-end cosmetic raw materials.

[0154] The above-described embodiments are only part of the embodiments of the present application, not all the embodiments, and other embodiments obtained by those skilled in the art based on the embodiments of the present application without creative labor are within the protection scope of the present application.

Claims

1. A complex polysaccharide nano-selenium complex, characterized in that: The complex comprises polysaccharide-modified nanoseelenium particles, which are obtained by a redox reaction of a polysaccharide composition and a selenium solution, the polysaccharide composition comprising boletus polysaccharide, purslane polysaccharide and astragalus polysaccharide, and the mass ratio of the boletus polysaccharide, the purslane polysaccharide and the astragalus polysaccharide being 0.5-3:0.5-3:0.5-3.

2. The complexed polysaccharide nano-selenium complex of claim 1, wherein: The mass ratio of the boletus polysaccharide, the purslane polysaccharide and the astragalus polysaccharide is 0.8-1.2:0.8-1.2:0.8-1.

2.

3. A method for preparing a complex polysaccharide nano-selenium complex, characterized in that, The preparation method comprises the following steps: S1, mixing a polysaccharide composition with water to obtain a polysaccharide mixed solution, the polysaccharide composition comprising boletus polysaccharide, purslane polysaccharide and astragalus polysaccharide, and the mass ratio of the boletus polysaccharide, the purslane polysaccharide and the astragalus polysaccharide being 0.5-3:0.5-3:0.5-3; S2, stirring the selenium solution and the polysaccharide mixed solution uniformly to obtain a polysaccharide nanoseelenium suspension; S3, mixing a stabilizer with the polysaccharide nanoseelenium suspension and stirring until the reaction is complete to obtain a reaction liquid; S4, centrifuging, washing and drying the reaction liquid to obtain a complex polysaccharide nanoseelenium complex.

4. The method of claim 3, wherein: The polysaccharide composition comprises boletus polysaccharide, purslane polysaccharide and astragalus polysaccharide, and the concentration of the polysaccharide composition in the polysaccharide mixed solution is 1-5 mg / mL.

5. The method of claim 3, wherein: The concentration of the selenium solution in step S2 is 30-80 mmol / L, and the volume ratio of the selenium solution to the polysaccharide mixed solution is 1:3-8.

6. The method of claim 3, wherein: The stabilizer is selected from one of sodium citrate and ascorbic acid.

7. The method of claim 3, wherein: The concentration of the stabilizer is 30-80 mmol / L.

8. Use of the complex polysaccharide nanoseelenium complex of claim 1 or 2 or the preparation method of any one of claims 3-7 in the preparation of medicines, food and cosmetics.

9. Use of a polysaccharide composition for the preparation of a complex polysaccharide nano-selenium complex, characterized in that: The polysaccharide composition comprises boletus polysaccharide, purslane polysaccharide and astragalus polysaccharide, and the mass ratio of the boletus polysaccharide, the purslane polysaccharide and the astragalus polysaccharide is 0.5-3:0.5-3:0.5-3, and the use comprises at least one of the following: (1) use in improving the stability of nanoseelenium; (2) use in improving the antioxidant capacity of the complex polysaccharide nanoseelenium complex; (3) use in reducing the cytotoxicity of the complex polysaccharide nanoseelenium complex; (4) use in improving the bioavailability of the complex polysaccharide nanoseelenium complex.

10. Use according to claim 9, characterized in that: The antioxidant capacity comprises DPPH radical scavenging capacity, ABTS radical scavenging capacity or hydroxyl radical scavenging capacity.

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