Preparation method of high-purity red date polysaccharide nano-selenium

Abstract

The invention discloses a preparation method of high-purity red date polysaccharide nano-selenium, and belongs to the field of biological material preparation. The preparation method of the high-purity red date polysaccharide nano-selenium comprises the following steps: adding water to dissolve red date polysaccharide to obtain a red date polysaccharide solution, then mixing the red date polysaccharide solution with a nano-selenium solution, reacting to obtain a mixed solution, and separating and purifying to obtain the red date polysaccharide nano-selenium. According to the preparation method disclosed by the invention, high-purity red date polysaccharide nano-selenium can be efficiently and stably prepared, the product has huge application potential in the fields of biomedicine, functional food and the like, and powerful technical support is provided for the development of related industries. Compared with a conventional process in the prior art, the preparation process for preparing the red date polysaccharide and the nano-selenium first and then compounding the red date polysaccharide and the nano-selenium to obtain the final product has obvious performance advantages in the aspects of product purity, storage stability and the like, and powerful technical support is provided for further application and development of the red date polysaccharide nano-selenium.

Description

Technical Field

[0001] The invention relates to the field of biomaterial preparation, and in particular to a method for preparing high-purity red date polysaccharide nano-selenium. Background Art

[0002] As a high molecular compound, red date polysaccharide has attracted much attention due to its multiple biological functions such as anti-oxidation and immunomodulation. Red date polysaccharide can not only effectively scavenge free radicals and protect cells from oxidative damage, but also enhance the body's immune function and improve the body's resistance. These characteristics make red date polysaccharide have a wide range of application potential in the fields of functional foods and biomedicine. As a new type of nanomaterial, nano-selenium has shown great application prospects in many fields such as medicine, food, and agriculture in recent years due to its unique physicochemical properties and significant biological activity. Compared with conventional selenium forms, nano-selenium has higher bioavailability and lower toxicity, and can more effectively exert the biological effects of selenium. Combining nano-selenium with red date polysaccharide is expected to develop composite biomaterials with higher biological activity and application value.

[0003] However, the current research on the preparation of high-purity red date polysaccharide nano-selenium faces many technical challenges. At present, although some methods for preparing red date polysaccharide nano-selenium have been reported, these methods generally have problems such as complex preparation process, low product purity, and poor stability. The complex preparation process not only increases the production cost, but also limits the possibility of its large-scale industrial application; while the low purity and poor stability of the product seriously affect the biological activity and application effect of red date polysaccharide nano-selenium, limiting its further application and development in the fields of biomedicine and functional food.

[0004] Therefore, it is particularly important to develop a new method that is efficient, stable and capable of preparing high-purity red jujube polysaccharide nano-selenium. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing high-purity red date polysaccharide nano-selenium to solve the above-mentioned problems in the background technology. Through the preparation method of the present invention, high-purity red date polysaccharide nano-selenium can be prepared efficiently and stably, and the product has great application potential in the fields of biomedicine, functional food, etc., and provides strong technical support for the development of related industries.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] One of the technical solutions of the present invention is to provide a method for preparing high-purity red date polysaccharide nano-selenium, comprising the following steps:

[0008] The red date polysaccharide is dissolved in water to obtain a red date polysaccharide solution, which is then mixed with a nano-selenium solution to react to obtain a mixed solution, which is separated and purified to obtain the red date polysaccharide nano-selenium.

[0009] Furthermore, the concentration of red jujube polysaccharide in the red jujube polysaccharide solution is 1-2 mg / mL; the concentration of nano-selenium in the nano-selenium solution is 1-2 mg / mL.

[0010] Furthermore, the volume ratio of the red date polysaccharide solution to the nano-selenium solution is 1:1-1:2.

[0011] Furthermore, the preparation method of the nano-selenium solution comprises the following steps: mixing ascorbic acid and sodium selenite in a molar ratio of 2:1-3:1 in water, and then reacting at 30-40°C for 3-5 hours to obtain a crude nano-selenium solution, and then separating and purifying with an ultrafiltration membrane with a molecular weight cutoff of 10-20 kDa to obtain the nano-selenium solution.

[0012] Furthermore, the reaction time is 2-4 hours.

[0013] Furthermore, the separation and purification is as follows: the mixed solution is separated and purified by gel filtration chromatography to obtain an elution solution, and the elution solution is freeze-dried to obtain the red date polysaccharide nano-selenium;

[0014] The gel medium of the gel filtration chromatography is Sephadex G-50, and the eluent is a phosphate buffer with a pH value of 7.0 and a concentration of 0.1 mol / L.

[0015] Furthermore, the preparation method of the red date polysaccharide comprises the following steps:

[0016] The red dates are mixed with water, heated and extracted, and then the solid and liquid are separated to obtain a supernatant, which is precipitated with alcohol to obtain crude red date polysaccharides, and then separated and purified to obtain the red date polysaccharides.

[0017] Furthermore, the temperature of the heating extraction is 80-90°C and the time is 2-3 hours; the temperature of the alcohol precipitation is 4°C and the time is 12-24 hours; the separation and purification is first separated by DEAE-cellulose column chromatography, then gradient eluted with water and sodium chloride solution respectively, and then dialyzed with a dialysis bag with a molecular weight cutoff of 3500-5000Da.

[0018] The second technical solution of the present invention is to provide a red date polysaccharide nano-selenium obtained according to the above preparation method.

[0019] The beneficial technical effects of the present invention are as follows:

[0020] Through the preparation method of the present invention, high-purity red date polysaccharide nano-selenium can be prepared efficiently and stably, and the product has great application potential in the fields of biomedicine, functional food, etc., and provides strong technical support for the development of related industries. Compared with the conventional process in the prior art, the preparation process designed by the present invention, which first prepares red date polysaccharide and nano-selenium and then compounds to obtain the final product, has obvious performance advantages in terms of product purity, storage stability, etc., and provides strong technical support for the further application and development of red date polysaccharide nano-selenium. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 This is the SEM image of red date polysaccharide nano-selenium in Example 1.

[0023] Figure 2 This is a performance characterization of the red date polysaccharide nano-selenium in Example 1; wherein A is the atomic absorption spectrum, B is the standard curve graph, C is the high performance liquid chromatography, and D is the infrared spectrum. DETAILED DESCRIPTION

[0024] With the rapid advancement of nanotechnology, nano-selenium, as a new form of selenium, has shown its potential for wide application in many fields such as medicine, animal husbandry and food industry due to its unique physical and chemical properties and significant biological activity. It is particularly noteworthy that after nano-selenium is combined with polysaccharides to form a nanocomposite, it not only significantly improves its stability, but also greatly broadens its application prospects in various industries.

[0025] Polysaccharide is a macromolecular substance that is very important for the normal life activities of the human body. Most polysaccharides such as Lycium barbarum polysaccharide, Lentinan polysaccharide, Polyporus umbellatus polysaccharide, Laminaria japonica polysaccharide, Red date polysaccharide, Astragalus membranaceus polysaccharide, etc. have the function of regulating human physiology. Polysaccharides have anti-inflammatory, antioxidant, anti-tumor, diabetes improvement and immune regulation activities. They can be used as immune promoters, control cell division and differentiation, regulate cell growth and aging; and can effectively remove oxygen free radicals in the human body. In addition, it also has physiological functions such as anti-cancer and anti-AIDS, and can be widely used in medicine, health food and functional food.

[0026] Nano-selenium (Nano-Se) is a new form of selenium at the nanoscale. Due to the particle size of nano-selenium, it is easier to be absorbed and utilized by humans or other organisms, and exhibits strong biological activity. Despite this, the stability of nano-selenium is poor, and further improvements through polysaccharides are needed to enhance its stability. The research on polysaccharides and nano-selenium has always been a research focus worldwide, especially the preparation technology of polysaccharide nano-selenium is crucial.

[0027] Selenium polysaccharides in nature have the disadvantages of few types, low content and complex structure. Artificial preparation of polysaccharide nano-selenium effectively solves the above problems. Red jujube polysaccharides with antioxidant, bioadhesive and non-toxic properties can be used as carriers of nano-selenium to form active composite materials, effectively enhance the stability of nano-selenium, and reduce the limitations of selenium and polysaccharide applications. The preparation methods of nano-selenium include chemical reduction, physical generation and biological transformation. Among them, the chemical reduction method has lower requirements for equipment, mainly redox reaction, and the product quality particle size is uniform and controllable; the physical generation method has higher requirements for equipment, often using microwave radiation, gamma ray radiation, laser burning, etc., and the product quality particle size is relatively uniform and controllable; the biological transformation method mainly uses the biological transformation of microorganisms / plants and the reduction of microorganisms / plant extracts, and the particle size controllability is poor.

[0028] Polysaccharide nano-selenium has unique surface characteristics and physical and chemical properties, including nano-size effect, high specific surface area and quantum size effect, which enable it to show excellent performance in catalysis, optics and electronics. In addition, red date polysaccharide nano-selenium also has good biocompatibility and stability, making significant contributions in the fields of food, medicine and agriculture.

[0029] As a new type of nanocomposite, polysaccharide nano-selenium has good biological activities and functions such as antioxidant, anti-tumor, anti-inflammatory, antibacterial, immunomodulatory, and anti-diabetic. Both red date polysaccharides and nano-selenium have good antioxidant activity. Compared with natural polysaccharides and nano-selenium, the red date polysaccharide nano-selenium system shows stronger antioxidant and anti-inflammatory activity, which can effectively remove free radicals inside and outside the body, protect cells from oxidative stress damage, and play a positive role in delaying aging and preventing various chronic diseases. Sugar and selenium have important effects on the specific and non-specific immunity of animals. Polysaccharide nano-selenium can effectively promote the proliferation of T cells in the body, enhance the release of related immune factors, and enhance the activity of immune cells in the body.

[0030] In biomedicine, nano-selenium polysaccharides are used for anti-cancer treatment, radiochemotherapy sensitization, and targeted drug delivery. In green agriculture, nano-selenium complexes can be used for the preparation of green pesticides and pollution and environmental protection treatment, as well as improving the growth of crops through soil microbial regulation. In modern animal husbandry, polysaccharide nano-selenium can be used as a feed additive to improve animal growth and development and enhance the quality of livestock products. It can be seen that nano-selenium polysaccharides have shown wide application potential in many fields.

[0031] Now, various exemplary embodiments of the present invention are described in detail, and this detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention.

[0032] In addition, for the numerical range in the present invention, it is understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0033] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in the present invention are open-ended terms, meaning including but not limited to.

[0035] The invention discloses a method for preparing high-purity red date polysaccharide nano-selenium, comprising the following steps:

[0036] (1) Extraction of red date polysaccharides: Select red dates that are free of mold and insect infestation, wash and remove the core, cut into small pieces to obtain red date raw materials, add distilled water at a material-liquid ratio of 1g:10-15mL, and perform hot water extraction at 80-90°C for 2-3 hours, stirring continuously during the process to promote the dissolution of polysaccharides; after the extraction is completed, the extract is cooled to room temperature, and then centrifuged at a speed of 4000-6000r / min for 15-20 minutes to remove precipitated impurities to obtain a supernatant;

[0037] Add 3-5 times the volume of 95 vol% ethanol to the supernatant, stir thoroughly, place in a 4°C refrigerator for 12-24 hours to allow the red date polysaccharide to fully precipitate, then collect the precipitate by centrifugation, wash with anhydrous ethanol 2-3 times, and then place in a vacuum drying oven and dry at 40-50°C to constant weight to obtain crude red date polysaccharide;

[0038] The crude red date polysaccharide is dissolved in distilled water, separated and purified by DEAE-cellulose column chromatography, then eluted with distilled water to remove impurities, and then gradient eluted with sodium chloride solution to collect the polysaccharide eluate; the polysaccharide eluate is dialyzed, the molecular weight cutoff of the dialysis bag is 3500-5000Da, the dialysis time is 24-48 hours, small molecular impurities are removed, and finally the dialyzed solution is freeze-dried to obtain high-purity red date polysaccharide;

[0039] (2) Preparation of nano-selenium: Weigh sodium selenite and prepare a sodium selenite solution with a concentration of 0.05-0.1 mol / L with distilled water, then add ascorbic acid (the molar ratio of ascorbic acid to sodium selenite is 2:1-3:1), stir evenly, and react at 30-40° C. for 3-5 hours, during which the color of the solution gradually changes from colorless to red, to obtain a crude nano-selenium solution; separate and purify the crude nano-selenium solution through an ultrafiltration membrane with a molecular weight cutoff of 10-20 kDa, remove unreacted raw materials and impurities, and obtain a high-purity nano-selenium solution;

[0040] (3) Preparation of jujube polysaccharide and nano-selenium complex: The jujube polysaccharide prepared in step (1) is prepared into a jujube polysaccharide solution with a concentration of 1-2 mg / mL with distilled water, and then the jujube polysaccharide solution and the nano-selenium solution are mixed at a volume ratio of 1:1-1:2, and the mixture is stirred and reacted at room temperature for 2-4 hours to allow the jujube polysaccharide and the nano-selenium to fully combine to obtain a mixed solution, and the mixed solution is further separated and purified by gel filtration chromatography to remove unbound polysaccharides and nano-selenium, and the eluted solution containing the jujube polysaccharide and nano-selenium complex is collected and freeze-dried to obtain a high-purity jujube polysaccharide and nano-selenium powder.

[0041] Furthermore, the variety of red dates is a variety with high sugar content and rich polysaccharide content, preferably gray dates or Jun dates.

[0042] The sodium selenite, sodium hydroxide, hydrochloric acid and ethanol used in the present invention are of analytical grade.

[0043] The "room temperature" in the present invention is 10-30°C unless otherwise specified.

[0044] All raw materials used in the following examples and comparative examples of the present invention are commercially available products.

[0045] Example 1

[0046] A method for preparing high-purity red date polysaccharide nano-selenium, the steps are as follows:

[0047] (1) Extraction of polysaccharides from red dates: Select gray dates without mildew or insect infestation, wash and remove the core, cut into small pieces, and obtain 500 g of red dates raw material. Add 5 L of distilled water and perform hot water extraction at 85° C. for 2.5 hours. Stir continuously during the extraction to promote the dissolution of polysaccharides. After the extraction, cool the extract to room temperature, and then centrifuge at 5000 r / min for 18 minutes to remove precipitated impurities and obtain a supernatant.

[0048] Add 4 times the volume of 95 vol% ethanol to the supernatant, stir thoroughly, place in a 4°C refrigerator for 18 hours to allow the red date polysaccharide to fully precipitate, then collect the precipitate by centrifugation, wash with anhydrous ethanol, and then place in a vacuum drying oven at 45°C to dry to constant weight to obtain crude red date polysaccharide;

[0049] The crude red jujube polysaccharide is dissolved in distilled water, separated and purified by DEAE-cellulose column chromatography, and then the DEAE-cellulose column is fully eluted with distilled water to remove unbound impurities, and then 0.1M, 0.2M, 0.3M, 0.4M and 0.5M sodium chloride solutions are sequentially set for gradient elution (the elution volume of each concentration of the solution is 2-3 times the column volume), and during the elution process, the eluate is collected by an automatic fraction collector to obtain a polysaccharide eluate; the polysaccharide eluate is dialyzed, the dialysis bag has a molecular weight cutoff of 4800Da, and the dialysis time is 48 hours to remove small molecular impurities, and finally the dialyzed solution is freeze-dried to obtain a high-purity red jujube polysaccharide;

[0050] (2) Preparation of nano-selenium: Weigh 5 g of sodium selenite and prepare a sodium selenite solution with a concentration of 0.08 mol / L with distilled water, then add 10.2 g of ascorbic acid, stir evenly, and react at 35° C. for 4 hours, during which the color of the solution gradually changes from colorless to red, to obtain a crude nano-selenium solution; separate and purify the crude nano-selenium solution through an ultrafiltration membrane with a molecular weight cutoff of 15 kDa, remove unreacted raw materials and impurities, and obtain a high-purity nano-selenium solution;

[0051] (3) Preparation of jujube polysaccharide-nanoselenium complex: The jujube polysaccharide prepared in step (1) is prepared into a jujube polysaccharide solution with a concentration of 1.5 mg / mL with distilled water, and then the jujube polysaccharide solution and the nanoselenium solution are mixed at a volume ratio of 1:1.5, and the mixture is stirred and reacted at room temperature for 3 hours to allow the jujube polysaccharide and the nanoselenium to fully combine to obtain a mixed solution. The mixed solution is further separated and purified by gel filtration chromatography (the gel medium is Sephadex G-50, and the eluent is a phosphate buffer solution with a pH value of 7.0 and a concentration of 0.1 mol / L) to remove unbound polysaccharides and nanoselenium, and the eluted solution containing the jujube polysaccharide-nanoselenium complex is collected and freeze-dried to obtain a high-purity jujube polysaccharide-nanoselenium complex.

[0052] Comparative Example 1 (Conventional method for preparing red date polysaccharide nano-selenium in the prior art)

[0053] A method for preparing red date polysaccharide nano-selenium, the steps are as follows:

[0054] 1. Raw material preparation

[0055] Preparation of sodium selenite solution: Accurately weigh sodium selenite and use distilled water to prepare a sodium selenite solution with a concentration of 0.1 mol / L.

[0056] 2. Mixed reaction

[0057] The jujube polysaccharide obtained in step (1) of Example 1 is prepared into a jujube polysaccharide solution with distilled water at a concentration of 1.5 mg / mL, the jujube polysaccharide solution is mixed with a sodium selenite solution (the molar ratio of the sugar unit in the jujube polysaccharide to the sodium selenite is 3:1), stirred evenly, and then a reducing agent ascorbic acid solution (the concentration is 0.3 mol / L, and the added amount is 1.2 times the mass required for complete reaction with sodium selenite) is slowly added dropwise, and the reaction is stirred at room temperature in the dark for 3 hours to reduce the sodium selenite to nano-selenium and combine with the jujube polysaccharide to form a complex to obtain a mixed solution.

[0058] 3. Product separation and purification

[0059] The mixed solution was further separated and purified by gel filtration chromatography (the gel medium was Sephadex G-50, and the eluent was a phosphate buffer solution with a pH value of 7.0 and a concentration of 0.1 mol / L), and the eluted solution containing the red date polysaccharide nano-selenium complex was collected and freeze-dried to obtain the red date polysaccharide-nano-selenium complex.

[0060] Comparative Example 2

[0061] A method for preparing nano-selenium, the steps are as follows:

[0062] Ascorbic acid is used as a reducing agent and konjac flour is used as a template: sodium selenite, ascorbic acid and konjac flour are mixed in water, the molar ratio of sodium selenite to ascorbic acid is n(Na2SeO3):n(Vc)=1:3, and the mass fraction of konjac flour is 3.0×10 -3 , reacted at 40°C for 1.5 hours to obtain uniform and stable spherical selenium nanoparticles with an average particle size of about 60-150nm.

[0063] Comparative Example 3

[0064] A method for preparing nano-selenium, the steps are as follows:

[0065] Sodium borohydride is used as a reducing agent: sodium borohydride solution (concentration is 0.1 mol / L) is slowly added dropwise to sodium selenite solution (concentration is 0.05 mol / L) (the volume ratio of sodium borohydride solution to sodium selenite solution is 2:1), and the mixture is reacted at 25°C for 1 hour to prepare nano-selenium.

[0066] Effect verification

[0067] 1. Product characterization and detection

[0068] (1) Scanning electron microscopy (SEM) was used to observe the morphology of red date polysaccharide nanoselenium and determine its particle size and distribution. The test results are as follows: Figure 1 shown.

[0069] Figure 1 This is the SEM image of red date polysaccharide nano-selenium in Example 1.

[0070] In Example 1, it can be seen from the SEM image that the particle size of the product is between 120-180 nm.

[0071] (2) Atomic absorption spectroscopy (AAS) was used to determine the selenium content in the product to ensure that it was within the appropriate range to ensure its biological activity and safety; high performance liquid chromatography (HPLC) was used to determine the content of red date polysaccharides in the product to ensure that its purity reached more than 95%; the chemical structure of the product was analyzed by Fourier transform infrared spectroscopy (FT-IR) to determine the binding mode between red date polysaccharides and nano-selenium; the test results are as follows Figure 2 shown.

[0072] The specific steps for determining the selenium content in the product by atomic absorption spectroscopy (AAS) are as follows:

[0073] Sample pretreatment: accurately weigh the red jujube polysaccharide-nanoselenium complex product, add appropriate amount of nitric acid and perchloric acid (volume ratio 4:1) for digestion, heat on a heating plate at low temperature until the sample is completely digested and the solution becomes colorless and transparent; then transfer the digestion solution to a volumetric flask and dilute to 50 mL with deionized water to obtain a sample solution.

[0074] Standard curve drawing: Prepare a series of selenium standard solutions of different concentrations (e.g. 0, 1, 2, 5, 10 μg / mL). Preheat the atomic absorption spectrometer and adjust it to the optimal working conditions for measuring selenium (wavelength 196.0 nm, lamp current, slit width, etc. are set according to the instrument model). Measure the absorbance of the standard solutions in turn, and draw a standard curve with absorbance as the ordinate and selenium concentration as the abscissa.

[0075] Sample determination: Inject the treated sample solution into the atomic absorption spectrometer to determine its absorbance. Calculate the selenium content in the sample based on the standard curve.

[0076] The specific steps of determining the content of red date polysaccharides in the product by high performance liquid chromatography (HPLC) are as follows:

[0077] Sample pretreatment: Weigh 0.1g of red date polysaccharide-nanoselenium complex product, add appropriate amount of distilled water, and perform ultrasonic extraction at 80°C for 360 minutes to fully dissolve the red date polysaccharide. Then centrifuge the extract (5000r / min, 10 minutes), take the supernatant and filter it through a 0.45μm microporous filter membrane to obtain the sample solution for later use.

[0078] Standard curve drawing: accurately weigh the red date polysaccharide standard and prepare a series of standard solutions of different concentrations (e.g. 0.1, 0.2, 0.5, 1.0 mg / mL) with distilled water. Preheat the high performance liquid chromatograph and adjust it to the best working conditions (chromatographic column is TSK-GEL G3000PWXL; mobile phase is 0.1 mol / L sodium nitrate solution; flow rate is 0.5-1.0 mL / min; column temperature is 30-40°C). Inject the standard solutions sequentially to determine the peak area, and draw the standard curve with the peak area as the ordinate and the polysaccharide concentration as the abscissa.

[0079] Sample determination: Inject the treated sample solution into a high performance liquid chromatograph to determine its peak area. Calculate the content of red jujube polysaccharides in the sample according to the standard curve, and calculate the purity.

[0080] The specific steps of analyzing the chemical structure of the product by Fourier transform infrared spectroscopy (FT-IR) are as follows:

[0081] Sample preparation: Using the potassium bromide tableting method, the dried red jujube polysaccharide-nanoselenium complex product was mixed with dried potassium bromide at a mass ratio of 1:100, ground evenly in an agate mortar, and then transferred to a tableting mold and pressed into a transparent thin sheet at a pressure of 10-15 MPa.

[0082] Spectral measurement: Put the pressed slice into Fourier transform infrared spectrometer at 4000-400cm -1 Scan within the range and record the infrared absorption spectrum.

[0083] Figure 2 This is a performance characterization of the red date polysaccharide nano-selenium in Example 1; wherein A is the atomic absorption spectrum, B is the standard curve graph, C is the high performance liquid chromatography, and D is the infrared spectrum.

[0084] FT-IR analysis results show that the characteristic absorption peak of red jujube polysaccharide is at 3400 cm -1 Around (-OH stretching vibration), 2930cm -1 Around (-CH stretching vibration), 1600-1700cm -1 Around (C=O stretching vibration), etc., indicating that red date polysaccharides and nano-selenium are successfully combined (nano-selenium may cause the displacement or intensity change of certain absorption peaks. By comparing the spectra of red date polysaccharides and the complex, the binding mode between red date polysaccharides and nano-selenium can be determined, such as hydrogen bonding, coordination bonding, etc.).

[0085] The purity of the red date polysaccharide in Example 1 was determined by HPLC to be 96.5% (ensuring that the purity of the product reaches more than 95% can ensure the quality and effectiveness of the product. High-purity red date polysaccharides have better biological activities, such as immunomodulation and anti-tumor effects. At the same time, high purity also helps to reduce the impact of impurities on product safety and stability, and is more suitable for applications in the fields of biomedicine and functional foods). In Comparative Example 1, due to the simultaneous reaction of multiple substances during its preparation process, the reaction system is relatively complex, it is difficult to accurately control the reaction process, and more impurities will be introduced, resulting in a red date polysaccharide purity of only about 85%. This shows that the preparation method of Example 1 of the present invention has significant advantages in improving product purity.

[0086] The selenium content determined by AAS was 7.6 mg / g (the selenium content in the red date polysaccharide-nanoselenium complex can be controlled between 1-10 mg / g; if the selenium content is too low, it may not be able to exert its due biological activity, such as antioxidant, immunomodulatory and other effects; and if the content is too high, it may cause selenium poisoning and have adverse effects on the human body).

[0087] (3) The yield calculation method of the present invention is as follows:

[0088] Yield (%) = (actual mass of red jujube polysaccharide-nanoselenium complex obtained / theoretically obtainable mass of complex) × 100%

[0089] The theoretically obtainable mass of the composite needs to be calculated based on the amount of the reaction raw materials used and the stoichiometric relationship of the reaction.

[0090] Under the preparation conditions of Example 1, the yield of the red jujube polysaccharide-nano-selenium complex is about 71.34%, while the yield of the red jujube polysaccharide-nano-selenium complex prepared by the conventional method of Comparative Example 1 is only about 49.78%, and the performance advantage is obvious. The present method uses red jujube polysaccharide as a stabilizer and carrier, which may improve the generation efficiency and stability of nano-selenium, thereby improving the yield.

[0091] (4) Storage stability comparison of the red jujube polysaccharide nano-selenium complex prepared in Example 1 and Comparative Example 1:

[0092] Experimental design: The red date polysaccharide nano-selenium complex was placed in an environment with a storage temperature of 25°C and a relative humidity of 60%, and various performance tests were performed regularly (once every month), including observing the morphological changes using a scanning electron microscope (SEM), determining the selenium content by atomic absorption spectroscopy (AAS), and determining the red date polysaccharide content by high performance liquid chromatography (HPLC) to determine its stability. At the same time, the stability of the sample in a refrigerated environment was tested by only changing the above storage environment to a 4°C refrigerated environment.

[0093] Experimental results: After being placed in a room temperature environment for 3 months, the product of comparative example 1 was observed by SEM, and its nanoparticles showed obvious agglomeration, and the particle size distribution became wider; AAS detection showed that the selenium content decreased by about 15%, which may be because the agglomeration caused some nano-selenium to be oxidized or other chemical reactions occurred, resulting in the loss of selenium; HPLC determined that the purity of its red date polysaccharide dropped to about 80%, indicating that the product decomposed or other changes occurred during the storage process, resulting in a decrease in the content of red date polysaccharide. While the product of Example 1 was placed under the same conditions for 3 months, SEM observation found that the nanoparticles still maintained a relatively uniform dispersion state, and the particle size changed little; AAS detected that the selenium content only decreased by about 5%; HPLC determined that the purity of red date polysaccharide remained above 94%, showing good stability. After being placed in a refrigerated environment at 4°C for 6 months, the product of comparative example 1 was partially precipitated, and the uniformity of the solution deteriorated after re-dissolution; AAS detected that the selenium content decreased by about 20%, and HPLC determined that the purity of red date polysaccharide decreased to about 75%. After refrigeration for 6 months, the product of Example 1 had no obvious change in appearance, the solution remained uniform, the selenium content detected by AAS decreased by about 8%, and the purity of red date polysaccharides determined by HPLC was maintained at about 92%.

[0094] Result analysis: In comparative example 1, since the preparation process is to directly reduce sodium selenite in a mixed solution to generate nano-selenium and combine it with red date polysaccharide at the same time, this method makes the product structure unstable and easily affected by environmental factors during storage, resulting in changes such as agglomeration and decomposition. In Example 1, high-purity red date polysaccharide and nano-selenium are first prepared separately, and then compounded, which reduces the influence of impurities, and at the same time, a relatively stable combination mode is formed between red date polysaccharide and nano-selenium, thereby significantly improving the storage stability of the product.

[0095] (5) Particle size: The average particle size of the red jujube polysaccharide-nanoselenium complex of Example 1 was measured by dynamic light scattering (DLS), and was found to be between 120-180 nm, and the particle size distribution was narrow, indicating that the nanoselenium particles were evenly dispersed and had good stability.

[0096] (6) Antioxidant properties: The antioxidant properties of the red jujube polysaccharide-nano-selenium complex of Example 1 were measured by DPPH free radical scavenging method and ABTS free radical cation scavenging method. It was found that the scavenging rates of the red jujube polysaccharide-nano-selenium complex for DPPH free radicals and ABTS free radical cations were 90% and 85%, respectively.

[0097] (7) Biocompatibility: The biocompatibility of the red jujube polysaccharide-nano-selenium complex of Example 1 and the nano-selenium of Comparative Examples 2 and 3 was evaluated by cytotoxicity test (MTT method). It was found that within a certain concentration range (10-100 μg / mL), the red jujube polysaccharide-nano-selenium complex had little effect on the cell survival rate, and the cell survival rate was between 80% and 95%, indicating that it had good biocompatibility.

[0098] The pure nano-selenium (concentration 10-100 μg / mL) in Comparative Examples 2 and 3 may have certain toxicity to cells due to the lack of biological molecule modification on the surface. Within the same concentration range, the cell survival rate is 60%-80%.

[0099] The preparation method of the present invention can efficiently and stably prepare high-purity red date polysaccharide nano-selenium. The product has great application potential in the fields of biomedicine, functional food, etc., and provides strong technical support for the development of related industries.

[0100] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing high-purity red date polysaccharide nano-selenium, characterized in that: The following steps are involved: The red date polysaccharide is dissolved in water to obtain a red date polysaccharide solution, which is then mixed with a nano-selenium solution to react to obtain a mixed solution, which is separated and purified to obtain the red date polysaccharide nano-selenium.

2. The preparation method according to claim 1, characterized in that: The concentration of red jujube polysaccharide in the red jujube polysaccharide solution is 1-2 mg / mL; the concentration of nano-selenium in the nano-selenium solution is 1-2 mg / mL.

3. The preparation method according to claim 2, characterized in that: The volume ratio of the red date polysaccharide solution to the nano-selenium solution is 1:1-1:

2.

4. The preparation method according to claim 1, characterized in that: The preparation method of the nano-selenium solution comprises the following steps: mixing ascorbic acid and sodium selenite in a molar ratio of 2:1-3:1 in water, then reacting at 30-40°C for 3-5 hours to obtain a crude nano-selenium solution, and then separating and purifying with an ultrafiltration membrane with a molecular weight cutoff of 10-20 kDa to obtain the nano-selenium solution.

5. The preparation method according to claim 1, characterized in that: The reaction time is 2-4 hours.

6. The preparation method according to claim 1, characterized in that: The separation and purification comprises: separating and purifying the mixed solution through gel filtration chromatography to obtain an elution solution, and freeze-drying the eluted solution to obtain the red date polysaccharide nano-selenium; The gel medium of the gel filtration chromatography is Sephadex G-50, and the eluent is a phosphate buffer with a pH value of 7.0 and a concentration of 0.1 mol / L.

7. The preparation method according to claim 1, characterized in that: The preparation method of the red date polysaccharide comprises the following steps: The red dates are mixed with water, heated and extracted, and then the solid and liquid are separated to obtain a supernatant, which is precipitated with alcohol to obtain crude red date polysaccharides, and then separated and purified to obtain the red date polysaccharides.

8. The preparation method according to claim 7, characterized in that: The temperature of the heating extraction is 80-90°C and the time is 2-3 hours; the temperature of the alcohol precipitation is 4°C and the time is 12-24 hours; the separation and purification is firstly separated by DEAE-cellulose column chromatography, then gradient eluted with water and sodium chloride solution respectively, and then dialyzed with a dialysis bag with a molecular weight cutoff of 3500-5000Da.

9. Red jujube polysaccharide nano-selenium obtained according to the preparation method according to any one of claims 1 to 8.

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