CN113456831A - Method for preparing nano-selenium by using eucommia polysaccharide and prepared nano-selenium - Google Patents
Method for preparing nano-selenium by using eucommia polysaccharide and prepared nano-selenium Download PDFInfo
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Abstract
The invention discloses a method for preparing nano-selenium by utilizing eucommia polysaccharide, relating to the technical field of nano materials and comprising the following steps: (1) preparing a eucommia polysaccharide crude extract by taking eucommia leaves or eucommia bark as a raw material; (2) decolorizing the polysaccharide crude extract with activated carbon, purifying with macroporous resin, and drying to obtain Eucommiae cortex polysaccharide; (3) preparing eucommia polysaccharide nano selenium compound solution; (4) adding beta-cyclodextrin with the mass concentration of 0.5-1 per mill into the eucommia polysaccharide nano selenium compound solution, stirring at room temperature, centrifuging, and freeze-drying the precipitate to obtain the product. The invention also provides the nano-selenium prepared by the method. The invention has the beneficial effects that: the synthesized eucommia polysaccharide wraps the nano-selenium, so that the stability of the eucommia polysaccharide nano-selenium is improved, and the stability is further enhanced by loading the eucommia polysaccharide nano-selenium through beta-cyclodextrin.
Description
Technical Field
The invention relates to the technical field of nano materials, in particular to a method for preparing nano selenium by utilizing eucommia polysaccharide and the prepared nano selenium.
Background
Selenium (Se) is one of indispensable trace elements for human and animal life, has the effects of resisting oxidation, regulating immunity, promoting animal growth, detoxifying, relieving stress and the like, plays a vital role in maintaining body health and regulating diseases, and is known as 'vital fire species' and 'anticancer king'. Selenium entering the human body is mainly used as a structure or an active component to participate in forming selenoprotein. These selenoproteins have multiple biological functions, including excellent antioxidant and anti-inflammatory properties, as well as inducing the production of active thyroid hormones. Since selenoprotein is closely related to various diseases induced by factors such as oxidative stress and inflammation, proper selenium content is maintained in human body, and the selenoprotein has positive significance for improving autoimmunity and resisting oxidative stress and inflammatory diseases. At present, nutrition survey of 13 provinces and cities in China shows that the intake of adult selenium is only 26.63 micrograms per day and is only half of the recommended daily intake. Therefore, at the present stage, the selenium deficiency of the people in China is common, the selenium supplement is not slow, and the selenium industry also becomes a new entry point and a new explosion point for detonating the billions of health industries in China.
The existing forms of selenium in nature mainly include three types, inorganic selenium, organic selenium and nano selenium. Compared with inorganic selenium and organic selenium, nano selenium becomes the most safe and effective selenium supplementing mode due to excellent biological activity, good biocompatibility and lower toxicity, and the synthesis and application of nano selenium also becomes one of the hot directions of the current selenium industry research. The preparation method of nano-selenium comprises a physical and chemical synthesis method, a microbial reduction method and a plant extract synthesis method. The plant extract is rich in natural polysaccharide, protein and other components, and can be coated on the surface of the nano selenium in the process of reducing inorganic selenium to generate simple substance nano selenium, thereby playing double roles of stabilizing the nano selenium and enhancing the biological activity of the nano selenium. Patent application with publication number CN109650350A discloses a method for preparing polysaccharide modified nano-selenium in large scale and application thereof, which discloses that lentinan, chitosan and other polysaccharides are used to prepare nano-selenium, and the nano-selenium modified by lentinan has good stability, but the nano-selenium modified by lentinan has poor oxidation resistance.
Disclosure of Invention
The invention aims to solve the technical problems that the stability of the nano-selenium modified by lentinan is good, but the oxidation resistance of the nano-selenium modified by lentinan is poor, and provides a preparation method of the nano-selenium with uniform particle size, stable structure and good oxidation resistance and the prepared nano-selenium.
The invention solves the technical problems through the following technical means:
a method for preparing nano-selenium by using eucommia polysaccharide comprises the following steps:
(1) preparing a eucommia polysaccharide crude extract by taking eucommia leaves or eucommia bark as a raw material;
(2) decolorizing the polysaccharide crude extract with activated carbon, purifying with macroporous resin, and drying to obtain Eucommiae cortex polysaccharide;
(3) preparing an eucommia polysaccharide solution with the mass concentration of 1-2 per mill, adding 0.1-0.2% of sodium selenite and 0.8-1.6% of vitamin C into the eucommia polysaccharide solution, stirring, placing the reaction mixed solution into a water bath with the temperature of 40-50 ℃ for standing reaction for 4-8 hours, centrifuging, and dissolving precipitates with deionized water to obtain an eucommia polysaccharide nano selenium compound solution;
(4) and (4) adding beta-cyclodextrin with the mass concentration of 0.5-1 per mill into the eucommia polysaccharide nano selenium compound solution in the step (3), stirring at room temperature, centrifuging, and freeze-drying the precipitate to obtain a product.
Has the advantages that: the synthesized eucommia polysaccharide nano selenium has the advantages that the stability of the eucommia polysaccharide nano selenium is improved, the beta-cyclodextrin is loaded, the stability is further enhanced, the prepared nano selenium has uniform particle size and stable structure, the biological activity of the synthesized eucommia polysaccharide nano selenium is stronger, and the biological activity of the eucommia polysaccharide coated nano selenium is obviously superior to that of the nano selenium and the eucommia polysaccharide and superior to that of the nano selenium prepared by lentinan. The nano-selenium has double effects of eucommia polysaccharide and nano-selenium, and has good effects of inhibiting bacteria, diminishing inflammation, resisting oxidation, improving immunity and the like.
The invention adopts eucommia polysaccharide, which is an extract of a rare medicinal plant eucommia in China, to synthesize the nano-selenium, and solves the problems that the cost is high, the nano-selenium is easy to agglomerate, and residual chemicals in the synthesis process can cause pollution and potential safety hazards and the like in the traditional physical and chemical method for synthesizing the nano-selenium.
Preferably, the centrifugation rotating speed in the step (3) and the centrifugation rotating speed in the step (4) are both 10000rpm-15000rpm, and the centrifugation time is both 10-20 min.
Preferably, the preparation method of the crude extract of the eucommia ulmoides polysaccharide comprises the following steps: drying eucommia leaves or eucommia bark, crushing, adding the dried eucommia leaves or eucommia bark into a flash extractor according to the material-liquid ratio of 1:15-40, setting the voltage to be 220V, the extraction frequency to be 1-3 times, the extraction time to be 30-60 seconds, merging filtrate after the extraction is finished, concentrating under reduced pressure, centrifuging, collecting supernatant, adding absolute ethyl alcohol into the supernatant, keeping the final concentration of the ethyl alcohol to be 75-80%, standing overnight at 4 ℃, centrifuging, and obtaining a precipitate, namely the eucommia polysaccharide crude extract.
Preferably, the decolorization with activated carbon comprises the following steps: dissolving the crude extract of eucommia ulmoides polysaccharide in a phosphate buffer, adding active carbon, decoloring for 1-2h at 50-70 ℃, and collecting filtrate, namely the decolored crude extract of eucommia ulmoides polysaccharide.
Preferably, the eucommia polysaccharide crude extract is dissolved in a phosphate buffer solution, the mass concentration of the polysaccharide crude extract is adjusted to be 1-3g/L, and then activated carbon with the mass content of 0.4-0.8% of that of the eucommia polysaccharide crude extract is added.
Preferably, the decolorized eucommia polysaccharide crude extract is loaded to macroporous resin, then is eluted by 60% -75% ethanol solution, filtrate is collected, and freeze drying is carried out, so that the eucommia polysaccharide is obtained.
Has the advantages that: compared with the crude extract of the eucommia polysaccharide and the crude extract of the eucommia polysaccharide subjected to decoloration treatment, the same amount of sodium selenite is added, the adding amount of the extract is the same, and the more the crude extract of the eucommia polysaccharide is subjected to decoloration by active carbon and purification by macroporous resin, the more the amount of the synthesized eucommia polysaccharide nano-selenium is.
The invention extracts eucommia ulmoides plant polysaccharide through a flash extractor, and then prepares the high-purity eucommia ulmoides polysaccharide through activated carbon decoloration and macroporous resin adsorption purification.
Preferably, the sample loading speed in the step (3) is 1-2mL/min, and the elution speed is 1-2 mL/min.
Preferably, the macroporous resin is one of D-101, DM-130, AB-8, NKA-9 and HPD 600.
Preferably, the obtained eucommia ulmoides polysaccharide is dissolved in 1L of deionized water to prepare a eucommia ulmoides plant polysaccharide solution with the mass concentration of 1-2 permillage of polysaccharide, and then 1-2g of sodium selenite and 8-16g of vitamin C are sequentially added.
The invention also provides the nano-selenium prepared by the method.
The invention has the advantages that: the eucommia polysaccharide synthesized by the method is coated with the nano selenium, the stability of the eucommia polysaccharide nano selenium is improved, the stability is further enhanced by loading the eucommia polysaccharide nano selenium through beta-cyclodextrin, the synthesized eucommia polysaccharide nano selenium has stronger biological activity, and the biological activity of the eucommia polysaccharide coated nano selenium is obviously superior to that of the nano selenium and the eucommia polysaccharide and superior to that of the nano selenium prepared from lentinan. The nano-selenium has double effects of eucommia polysaccharide and nano-selenium, and has good effects of inhibiting bacteria, diminishing inflammation, resisting oxidation, improving immunity and the like.
The invention adopts eucommia polysaccharide, which is an extract of a rare medicinal plant eucommia in China, to synthesize the nano-selenium, and solves the problems that the cost is high, the nano-selenium is easy to agglomerate, and residual chemicals in the synthesis process can cause pollution and potential safety hazards and the like in the traditional physical and chemical method for synthesizing the nano-selenium.
Compared with the crude extract of the eucommia polysaccharide and the crude extract of the eucommia polysaccharide subjected to decoloration treatment, the same amount of sodium selenite is added, the adding amount of the extract is the same, and the more the crude extract of the eucommia polysaccharide is subjected to decoloration by active carbon and purification by macroporous resin, the more the amount of the synthesized eucommia polysaccharide nano-selenium is.
Drawings
FIG. 1 is a scanning electron microscope image of eucommia ulmoides polysaccharide nano selenium in example 1 of the present invention.
Fig. 2 shows the particle size distribution of the eucommia ulmoides polysaccharide nano selenium in example 1 of the present invention.
FIG. 3 shows the EDX spectrum analysis result of the eucommia ulmoides polysaccharide nano selenium in example 1 of the present invention.
Fig. 4 is a graph showing the comparison result of the antioxidant capacity of the eucommia ulmoides polysaccharide nano selenium and the eucommia ulmoides polysaccharide and the chemically synthesized nano selenium in example 1 of the present invention.
FIG. 5 is a graph showing the comparison of the antioxidant properties of nano-selenium synthesized by different methods according to the present invention.
FIG. 6 is a graph showing the results of comparing the stability of nano-selenium synthesized by different methods. In the figure, A is comparative example 1, B is comparative example 2, and C is example 1.
FIG. 7 is a graph showing the results of comparing the properties of synthesized nano-selenium before and after purification of eucommia ulmoides polysaccharide of the present invention. In the figure, A is comparative example 3, B is comparative example 4, and C is comparative example 5.
FIG. 8 is a graph showing the results of the measurement of the purification effect of crude extract of eucommia ulmoides polysaccharide by activated carbon treatment with different addition amounts.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Test materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The specific techniques or conditions not specified in the examples can be performed according to the techniques or conditions described in the literature in the field or according to the product specification.
Example 1
The method for preparing the nano-selenium by utilizing the eucommia polysaccharide specifically comprises the following steps:
1) preparing a eucommia ulmoides plant polysaccharide crude extract: picking folium Eucommiae, oven drying at 50 deg.C for 36 hr, pulverizing, and sieving with 60 mesh sieve. Taking a certain amount of water, and adding the water into a flash extractor according to the ratio of material to liquid of 1: 15. The extraction time was set to 30 seconds, the voltage was set to 100V, and the number of extractions was set to 1. After extraction, the filtrates are combined and concentrated under reduced pressure. Subsequently, the mixture was centrifuged at 4000rpm for 5 minutes, and the supernatant was collected. Adding anhydrous ethanol into the supernatant to make ethanol final concentration 75%, standing at 4 deg.C overnight, centrifuging at 10000rpm for 10 min, and collecting precipitate to obtain crude extract of Eucommiae cortex polysaccharide.
2) Active carbon decolorization of eucommia ulmoides plant polysaccharide crude extract: dissolving the crude extract of eucommia ulmoides polysaccharide prepared in the step 1) in a phosphoric acid buffer solution (pH 5.0) to make the mass concentration of the polysaccharide be 1 g/L. Adding active carbon with the mass equivalent to 0.4 percent of that of the crude polysaccharide, carrying out suction filtration at the decoloring temperature of 50 ℃ for 1 hour, and collecting filtrate, namely the decolored eucommia ulmoides plant polysaccharide crude extract.
3) Purifying a crude extract of eucommia ulmoides plant polysaccharide: 2) the eucommia ulmoides plant polysaccharide crude extract prepared in the step (1) is loaded into macroporous resin (D-101) at the speed of 1.0 mL/min, 60% ethanol solution is adopted, elution is carried out at the flow rate of 1.0 mL/min, filtrate is collected, freeze drying is carried out, purified eucommia ulmoides plant polysaccharide freeze-dried powder is obtained, and the polysaccharide yield is calculated.
4) Synthesis of eucommia polysaccharide nano selenium compound: dissolving the eucommia ulmoides plant polysaccharide freeze-dried powder prepared in the step 3) in 1L of deionized water to prepare an eucommia ulmoides plant polysaccharide solution with the mass concentration of 1 per mill of polysaccharide. And then, sequentially adding 1g of sodium selenite and 8g of vitamin C (Vc), stirring uniformly, placing the reaction mixed solution in a water bath at 40 ℃, standing for reaction for 4 hours, centrifuging at 10000rpm for 10 minutes, and dissolving the obtained precipitate with deionized water to obtain the eucommia polysaccharide nano selenium compound solution.
5) Further functionalization of the eucommia polysaccharide nano selenium compound: adding beta-cyclodextrin with the mass concentration of 0.5 per mill into the eucommia polysaccharide nano selenium compound solution obtained in the step 4), stirring for 2 hours at room temperature, centrifuging for 10 minutes at 10000rpm, and freeze-drying the obtained precipitate to obtain the beta-cyclodextrin functionalized eucommia polysaccharide nano selenium compound.
The experimental results are as follows: the yield of the eucommia ulmoides polysaccharide was 1.94% by using the conditions in this example.
Example 2
The method for preparing the nano-selenium by utilizing the eucommia polysaccharide specifically comprises the following steps:
1) preparing a eucommia ulmoides plant polysaccharide crude extract: picking folium Eucommiae, oven drying at 60 deg.C for 40 hr, pulverizing, and sieving with 60 mesh sieve. Taking a certain amount of water, and adding the water into a flash extractor according to the ratio of material to liquid of 1: 30. The extraction time was set to 40 seconds, the voltage was set to 160V, and the number of extractions was set to 2. After extraction, the filtrates are combined and concentrated under reduced pressure. Subsequently, the mixture was centrifuged at 4500rpm for 10 minutes, and the supernatant was collected. Adding anhydrous ethanol into the supernatant to make ethanol final concentration 75%, standing at 4 deg.C overnight, and centrifuging at 15000rpm for 20min to obtain precipitate as crude extract of Eucommiae cortex polysaccharide.
2) Active carbon decolorization of eucommia ulmoides plant polysaccharide crude extract: dissolving the eucommia ulmoides polysaccharide crude extract prepared in the step 1) in a phosphoric acid buffer solution (pH 5.0) to ensure that the mass concentration of the polysaccharide is 2 g/L. Adding active carbon with the mass equivalent to 0.5 percent of that of the crude polysaccharide, carrying out suction filtration at 60 ℃ for 2 hours, and collecting filtrate, namely the crude extract of the eucommia ulmoides plant polysaccharide subjected to the decolorization treatment.
3) Purifying a crude extract of eucommia ulmoides plant polysaccharide: 2) the eucommia ulmoides plant polysaccharide crude extract prepared in the step (1) is loaded into macroporous resin (AB-8) at the speed of 1.0 mL/min, 75% ethanol solution is adopted, elution is carried out at the flow rate of 1.0 mL/min, filtrate is collected, freeze drying is carried out, purified eucommia ulmoides plant polysaccharide freeze-dried powder is obtained, and the polysaccharide yield is calculated.
4) Synthesis of eucommia polysaccharide nano selenium compound: dissolving the eucommia ulmoides plant polysaccharide freeze-dried powder prepared in the step 3) in 1L of deionized water to prepare an eucommia ulmoides plant polysaccharide solution with the mass concentration of 1.5 per mill of polysaccharide. And then, sequentially adding 1.5g of sodium selenite and 12g of vitamin C (Vc), stirring uniformly, placing the reaction mixed solution in a water bath at 45 ℃, standing for reaction for 8 hours, centrifuging at 15000rpm for 20 minutes, and dissolving the obtained precipitate with deionized water to obtain the eucommia polysaccharide nano selenium compound solution.
5) Further functionalization of the eucommia polysaccharide nano selenium compound: adding beta-cyclodextrin with the mass concentration of 0.8 per mill into the eucommia polysaccharide nano selenium compound solution obtained in the step 4), stirring for 4 hours at room temperature, centrifuging for 20 minutes at 15000rpm, and freeze-drying the obtained precipitate to obtain the beta-cyclodextrin functionalized eucommia polysaccharide nano selenium compound.
The experimental results are as follows: the yield of the eucommia ulmoides polysaccharide was 3.26% by using the conditions in this example.
Example 3
The method for preparing the nano-selenium by utilizing the eucommia polysaccharide specifically comprises the following steps:
1) preparing a eucommia ulmoides plant polysaccharide crude extract: picking folium Eucommiae, oven drying at 70 deg.C for 48 hr, pulverizing, and sieving with 60 mesh sieve. Taking a certain amount of water, and adding the water into a flash extractor according to the ratio of material to liquid of 1: 40. The extraction time was set to 60 seconds, the voltage was set to 220V, and the number of extractions was set to 3. After extraction, the filtrates are combined and concentrated under reduced pressure. Subsequently, centrifugation was carried out at 5000rpm for 5 minutes, and the supernatant was collected. Adding anhydrous ethanol into the supernatant to make ethanol final concentration 80%, standing at 4 deg.C overnight, and centrifuging at 15000rpm for 10 min to obtain precipitate as crude extract of Eucommiae cortex polysaccharide.
2) Active carbon decolorization of eucommia ulmoides plant polysaccharide crude extract: dissolving the eucommia ulmoides polysaccharide crude extract prepared in the step 1) in a phosphoric acid buffer solution (pH 5.0) to ensure that the mass concentration of the polysaccharide is 3 g/L. Adding active carbon with the content of 0.8 percent of the crude polysaccharide, carrying out suction filtration at the decolorizing temperature of 70 ℃ for 1 hour, and collecting filtrate, namely the crude extract of the eucommia ulmoides plant polysaccharide subjected to decolorization treatment.
3) Purifying a crude extract of eucommia ulmoides plant polysaccharide: 2) the crude extract of the eucommia ulmoides plant polysaccharide prepared in the step (1) is loaded into macroporous resin NKA-9 at the speed of 1.0-2.0 mL/min, 60% ethanol solution is adopted for elution at the flow speed of 1.5 mL/min, filtrate is collected and is freeze-dried to obtain purified eucommia ulmoides plant polysaccharide freeze-dried powder, and the polysaccharide yield is calculated.
4) Synthesis of eucommia polysaccharide nano selenium compound: dissolving the eucommia ulmoides plant polysaccharide freeze-dried powder prepared in the step 3) in 1L of deionized water to prepare an eucommia ulmoides plant polysaccharide solution with the mass concentration of polysaccharide of 0.1%. And then sequentially adding 2g of sodium selenite and 16g of vitamin C (Vc), stirring uniformly, placing the reaction mixed solution in a water bath at 50 ℃ for standing for 6 hours, centrifuging at 10000rpm for 20 minutes, and dissolving the obtained precipitate with deionized water to obtain the eucommia polysaccharide nano selenium compound solution.
5) Further functionalization of the eucommia polysaccharide nano selenium compound: adding beta-cyclodextrin with the mass concentration of 1 per mill into the eucommia polysaccharide nano selenium compound solution obtained in the step 4), stirring for 4 hours at room temperature, centrifuging for 20 minutes at 15000rpm, and freeze-drying the obtained precipitate to obtain the beta-cyclodextrin functionalized eucommia polysaccharide nano selenium compound.
The experimental results are as follows: the yield of the eucommia ulmoides polysaccharide was 2.21% by using the conditions in this example.
Example 4
This embodiment is different from embodiment 1 in that: replacing folium Eucommiae with cortex Eucommiae.
Example 5
This embodiment is different from embodiment 1 in that: the addition amount of the activated carbon was adjusted to 0.6%.
Comparative example 1
The nano-selenium is synthesized by a chemical synthesis method. Cysteine with the concentration of 25mM is dropwise added into 10mL of 1M Na under the action of a magnetic stirrer2And (3) adding distilled water into the SeO solution to a constant volume of 25ml, reacting at room temperature for 30min, and centrifuging at 10000rpm for 20min to obtain red precipitate, namely the nano-selenium prepared by the chemical method.
Comparative example 2
This comparative example differs from example 1 in that: freeze-drying the eucommia polysaccharide nano selenium compound solution in the step 4).
Comparative example 3
1) Preparing a eucommia ulmoides plant polysaccharide crude extract: picking folium Eucommiae, oven drying at 50 deg.C for 36 hr, pulverizing, and sieving with 60 mesh sieve. Taking a certain amount of water, and adding the water into a flash extractor according to the ratio of material to liquid of 1: 15. The extraction time was set to 30 seconds, the voltage was set to 100V, and the number of extractions was set to 1. After extraction, the filtrates are combined and concentrated under reduced pressure. Subsequently, the mixture was centrifuged at 4000rpm for 5 minutes, and the supernatant was collected. Adding anhydrous ethanol into the supernatant to make ethanol final concentration 75%, standing at 4 deg.C overnight, centrifuging at 10000rpm for 10 min, and collecting precipitate to obtain crude extract of Eucommiae cortex polysaccharide.
2) Synthesis of eucommia polysaccharide nano selenium compound: dissolving the eucommia ulmoides polysaccharide crude extract prepared in the step 1) in 1L of deionized water to prepare an eucommia ulmoides plant polysaccharide solution with the mass concentration of 1 per mill of polysaccharide. And then, sequentially adding 1g of sodium selenite and 8g of vitamin C (Vc), stirring uniformly, placing the reaction mixed solution in a water bath at 40 ℃, standing for reaction for 4 hours, centrifuging at 10000rpm for 10 minutes, and dissolving the obtained precipitate with deionized water to obtain the eucommia polysaccharide nano selenium compound solution.
3) Further functionalization of the eucommia polysaccharide nano selenium compound: adding beta-cyclodextrin with the mass concentration of 0.5 per mill into the eucommia polysaccharide nano selenium compound solution obtained in the step 2), stirring for 2 hours at room temperature, centrifuging for 10 minutes at 10000rpm, and freeze-drying the obtained precipitate to obtain the beta-cyclodextrin functionalized eucommia polysaccharide nano selenium compound.
Comparative example 4
1) Preparing a eucommia ulmoides plant polysaccharide crude extract: picking folium Eucommiae, oven drying at 50 deg.C for 36 hr, pulverizing, and sieving with 60 mesh sieve. Taking a certain amount of water, and adding the water into a flash extractor according to the ratio of material to liquid of 1: 15. The extraction time was set to 30 seconds, the voltage was set to 100V, and the number of extractions was set to 1. After extraction, the filtrates are combined and concentrated under reduced pressure. Subsequently, the mixture was centrifuged at 4000rpm for 5 minutes, and the supernatant was collected. Adding anhydrous ethanol into the supernatant to make ethanol final concentration 75%, standing at 4 deg.C overnight, centrifuging at 10000rpm for 10 min, and collecting precipitate to obtain crude extract of Eucommiae cortex polysaccharide.
2) Active carbon decolorization of eucommia ulmoides plant polysaccharide crude extract: 1) the crude extract of eucommia ulmoides polysaccharide prepared in (1) is dissolved in a phosphoric acid buffer solution (pH 5.0) so that the mass concentration of the polysaccharide is 1 g/L. Adding active carbon with the mass equivalent to 0.4 percent of that of the crude polysaccharide, carrying out suction filtration at the decoloring temperature of 50 ℃ for 1 hour, collecting filtrate, and carrying out freeze drying to obtain the eucommia ulmoides plant polysaccharide crude extract subjected to decoloring treatment.
3) Synthesis of eucommia polysaccharide nano selenium compound: dissolving the eucommia ulmoides plant polysaccharide crude extract prepared in the step 2) in 1L of deionized water to prepare an eucommia ulmoides plant polysaccharide solution with the mass concentration of 1 per mill of polysaccharide. And then, sequentially adding 1g of sodium selenite and 8g of vitamin C (Vc), stirring uniformly, placing the reaction mixed solution in a water bath at 40 ℃, standing for reaction for 4 hours, centrifuging at 10000rpm for 10 minutes, and dissolving the obtained precipitate with deionized water to obtain the eucommia polysaccharide nano selenium compound solution.
4) Further functionalization of the eucommia polysaccharide nano selenium compound: adding beta-cyclodextrin with the mass concentration of 0.5 per mill into the eucommia polysaccharide nano selenium compound solution obtained in the step 3), stirring for 2 hours at room temperature, centrifuging for 10 minutes at 10000rpm, and freeze-drying the obtained precipitate to obtain the beta-cyclodextrin functionalized eucommia polysaccharide nano selenium compound.
Comparative example 5
The comparative example adopts chitosan to prepare nano-selenium, and replaces the eucommia polysaccharide with the chitosan with the same content (purchased from Beijing Sorley Tech Co., Ltd.) on the basis of the example 1.
Comparative example 6
The comparative example adopts lentinan to prepare nano-selenium, and replaces the eucommia ulmoides polysaccharide with the lentinan with the same content (purchased from Changsha Hejian biological technology limited company) on the basis of the example 1.
Comparative example 7
This comparative example differs from example 1 in that: the addition amount of the activated carbon was adjusted to 0.2%.
Comparative example 8
This comparative example differs from example 1 in that: the addition amount of the activated carbon was adjusted to 0.3%.
Experimental data and analysis:
and detecting the particle size of the eucommia polysaccharide nano selenium compound by using a Malvern laser particle size tester (DLS). And observing the size and the dispersion condition of the eucommia polysaccharide nano selenium compound by adopting a scanning electron microscope. And (3) characterizing the element content in the eucommia polysaccharide nano selenium sample by using an energy spectrometer (EDS). And (3) characterizing the functional group composition in the eucommia polysaccharide nano selenium sample by using a Fourier transform infrared spectrometer (FTIR). With reference to the national standard GB/T39100-2020, the antioxidant capacity of the eucommia polysaccharide nano-selenium is evaluated by adopting a DPPH free radical scavenging capacity test. Here, the measurement methods of the laser particle size analyzer, the scanning electron microscope, and the energy spectrometer are all the prior art.
In example 1, the eucommia ulmoides polysaccharide nano selenium has a particle size range of 20-50nm as shown in figure 1. Using a scanning electron microscope and an energy spectrometer (SEM-EDX), nanoparticles having a particle size of 20 to 50nm were observed as shown in fig. 2 and 3, and an EDX spectrum showed a characteristic peak of Se, indicating that the prepared nanoparticles were nano-selenium particles.
The particle size of the eucommia polysaccharide nano selenium compound detected by a Malvern laser particle size tester (DLS) shows that the particle size of the eucommia polysaccharide nano selenium synthesized in the embodiment 2 is 30-50 nm.
The particle size result of the eucommia polysaccharide nano selenium compound detected by a Malvern laser particle size tester (DLS) shows that the particle size of the eucommia polysaccharide nano selenium synthesized in the embodiment 3 is 50-80 nm.
As shown in fig. 4, due to the synergistic effect of the eucommia ulmoides polysaccharide and the nano-selenium, the DPPH radical scavenging ability of the eucommia ulmoides polysaccharide synthesized in example 1 is obviously higher than that of the eucommia ulmoides polysaccharide synthesized in example 1, and is higher than that of the nano-selenium synthesized by the chemical method in comparative example 1. Wherein the abscissa in fig. 4 represents the concentrations of the eucommia ulmoides polysaccharide, the eucommia ulmoides polysaccharide nano selenium and the chemically synthesized nano selenium in comparative example 1.
As shown in FIG. 5, when the concentration of the polysaccharide nano-selenium is 1mg/mL, the antioxidant performance of the eucommia polysaccharide nano-selenium is superior to that of chitosan nano-selenium and lentinan nano-selenium.
FIG. 6 is a comparison of the stability of nano-selenium synthesized in different ways. In the figure, A is comparative example 1, B is comparative example 2, and C is example 1. It can be seen that the nano-selenium aqueous solution has granular precipitates at the bottom of A, B after standing for 1 day, and the precipitates are formed by the agglomeration of nano-selenium. No nano-selenium agglomeration exists in the solution C, and the eucommia polysaccharide nano-selenium is uniformly distributed in the solution, which shows that the stability is obviously improved after the beta-cyclodextrin is loaded.
FIG. 7 is a comparison of the properties of synthesized nano-selenium before and after purification of eucommia ulmoides polysaccharide. In the figure, A is a comparative example 3, the eucommia polysaccharide crude extract is adopted to synthesize the nano selenium, B is a comparative example 4, the eucommia polysaccharide crude extract decolored by active carbon is adopted to synthesize the nano selenium, C is a comparative example 5, the eucommia polysaccharide decolored by the active carbon and purified by macroporous resin is adopted, the addition amount of the extract in A, B, C is 1g/L, and the addition amount of the sodium selenite is 2 mM. The darker the color, the more amount of synthesized eucommia ulmoides polysaccharide nano selenium.
Fig. 8 shows the effect of different amounts of activated carbon treatment on the purification effect of crude extract of eucommia ulmoides polysaccharide, and the polysaccharide content and the impurity content of the crude extract of eucommia ulmoides polysaccharide without purification treatment were both set to 100%. When the amount of the added activated carbon is 0.5%, the yield of the polysaccharide is higher, but when the amount of the added activated carbon is increased, the yield of the polysaccharide is decreased.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. A method for preparing nano selenium by using eucommia polysaccharide is characterized by comprising the following steps: the method comprises the following steps:
(1) preparing a eucommia polysaccharide crude extract by taking eucommia leaves or eucommia bark as a raw material;
(2) decolorizing the polysaccharide crude extract with activated carbon, purifying with macroporous resin, and drying to obtain Eucommiae cortex polysaccharide;
(3) preparing an eucommia polysaccharide solution with the mass concentration of 1-2 per mill, adding 0.1-0.2% of sodium selenite and 0.8-1.6% of vitamin C into the eucommia polysaccharide solution, stirring, placing the reaction mixed solution into a water bath with the temperature of 40-50 ℃ for standing reaction for 4-8 hours, centrifuging, and dissolving precipitates with deionized water to obtain an eucommia polysaccharide nano selenium compound solution;
(4) and (4) adding beta-cyclodextrin with the mass concentration of 0.5-1 per mill into the eucommia polysaccharide nano selenium compound solution in the step (3), stirring at room temperature, centrifuging, and freeze-drying the precipitate to obtain a product.
2. The method for preparing nano-selenium using eucommia ulmoides polysaccharide as claimed in claim 1, wherein: the centrifugation rotating speed in the step (3) and the step (4) is 10000rpm-15000rpm, and the centrifugation time is 10-20 min.
3. The method for preparing nano-selenium using eucommia ulmoides polysaccharide as claimed in claim 1, wherein: the preparation method of the eucommia polysaccharide crude extract comprises the following steps: drying eucommia leaves or eucommia bark, crushing, adding the dried eucommia leaves or eucommia bark into a flash extractor according to the material-liquid ratio of 1:15-40, setting the voltage to be 220V, the extraction frequency to be 1-3 times, the extraction time to be 30-60 seconds, merging filtrate after the extraction is finished, concentrating under reduced pressure, centrifuging, collecting supernatant, adding absolute ethyl alcohol into the supernatant, keeping the final concentration of the ethyl alcohol to be 75-80%, standing overnight at 4 ℃, centrifuging, and obtaining a precipitate, namely the eucommia polysaccharide crude extract.
4. The method for preparing nano-selenium using eucommia ulmoides polysaccharide as claimed in claim 1, wherein: the decoloring method by using the activated carbon comprises the following steps: dissolving the crude extract of eucommia ulmoides polysaccharide in a phosphate buffer, adding active carbon, decoloring for 1-2h at 50-70 ℃, and collecting filtrate, namely the decolored crude extract of eucommia ulmoides polysaccharide.
5. The method for preparing nano-selenium using eucommia ulmoides polysaccharide as claimed in claim 4, wherein the method comprises the following steps: dissolving the eucommia polysaccharide crude extract in a phosphate buffer solution, adjusting the mass concentration of the polysaccharide crude extract to be 1-3g/L, and then adding activated carbon with the mass content of 0.4-0.8% of that of the eucommia polysaccharide crude extract.
6. The method for preparing nano-selenium using eucommia ulmoides polysaccharide as claimed in claim 1, wherein: and (3) loading the decolorized eucommia polysaccharide crude extract to macroporous resin, eluting with 60-75% ethanol solution, collecting filtrate, and freeze-drying to obtain eucommia polysaccharide.
7. The method for preparing nano-selenium using eucommia ulmoides polysaccharide as claimed in claim 6, wherein the method comprises the following steps: and (3) the sampling speed is 1-2mL/min, and the elution speed is 1-2 mL/min.
8. The method for preparing nano-selenium using eucommia ulmoides polysaccharide as claimed in claim 6, wherein the method comprises the following steps: the macroporous resin is one of D-101, DM-130, AB-8, NKA-9 and HPD 600.
9. The method for preparing nano-selenium using eucommia ulmoides polysaccharide as claimed in claim 1, wherein: dissolving the obtained eucommia ulmoides polysaccharide in 1L of deionized water to prepare a eucommia ulmoides plant polysaccharide solution with the mass concentration of 1-2 per mill of polysaccharide, and then sequentially adding 1-2g of sodium selenite and 8-16g of vitamin C.
10. Nano-selenium obtainable by the method according to any one of claims 1 to 9.
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