Selenium modified pteridium aquilinum polysaccharide as well as preparation method and application thereof

Abstract

The invention relates to the field of polysaccharide preparation, and discloses a selenium-modified pteridium aquilinum polysaccharide and a preparation method and application thereof.The preparation method comprises the steps that firstly, pteridium aquilinum is sequentially subjected to smashing, impurity removal, filtration, extraction and vacuum concentration treatment, then a concentrated solution is subjected to deproteinization through a Sevag method, then the solution is subjected to decoloration and filtration, precipitation treatment is conducted through ethyl alcohol, a precipitate is obtained, and the selenium-modified pteridium aquilinum polysaccharide is obtained. The method comprises the following steps: dissolving pteridium aquilinum in water to obtain a precipitate, centrifugally separating the precipitate, freeze-drying to obtain pteridium aquilinum polysaccharide, selenizing the pteridium aquilinum polysaccharide to obtain selenized pteridium aquilinum polysaccharide, and finally, carrying out ultrafiltration separation on the selenized pteridium aquilinum polysaccharide to separate the selenized pteridium aquilinum polysaccharide 100 kda, 100 to 300 kDa, and gt; 3 components having a molecular weight of 300 kDa; the selenium-modified pteridium aquilinum polysaccharide provided by the invention has antioxidant activity, immunoregulation activity and alpha-glucosidase inhibitory activity superior to those of PAP, and the selenium-modified pteridium aquilinum polysaccharide provided by the invention can become a healthy food supplement.

Description

Technical Field

[0001] The present invention relates to the field of polysaccharide preparation, and more specifically, to a selenium-modified bracken polysaccharide, its preparation method and application. Background Art

[0002] Selenium (Se) is an essential trace element for human health as it is involved in the synthesis of enzymes, including types I, II, and III iodothyronine deiodinases, glutathione peroxidase, and thioredoxin reductase. These enzymes play a key role in protecting cell lipids, lipoproteins, and DNA from oxidative damage. Appropriate levels of selenium confer a range of health benefits to the human body, such as protecting the liver, repairing cell damage, antioxidant capacity, inhibiting cancer, enhancing immune regulatory functions, and preventing diabetes, while selenium deficiency will lead to poor health conditions in humans and accelerate the onset of diseases. For example, insufficient selenium intake can lead to an excess of reactive oxygen species, thereby increasing the risk of diabetes and affecting the body's immune function. Selenium exists in inorganic and organic forms. Compared with inorganic selenium, organic selenium has better bioavailability and lower toxicity, making it suitable for long-term use. Therefore, organic selenium supplements have attracted the interest of researchers as a promising health agent.

[0003] Selenium-polysaccharides have become a new source of organic selenium-rich supplements due to the dual benefits of selenium and polysaccharides. Incorporating selenium into polysaccharides can further improve the delivery efficiency of this trace element in the body.

[0004] However, even in selenium-rich areas, the selenium content in natural selenium polysaccharides is very low and cannot meet the selenium supplementation needs of humans and animals. Selenium polysaccharides obtained by artificial selenization not only serve as an effective source of selenium supplementation but also enhance the biological activity of the original polysaccharides. Artificial selenization modification has become an emerging hot topic in current polysaccharide science research.

[0005] Bracken, as one of the most widely distributed fern plants in the world, has been used as a food resource and is rich in various chemical components such as polysaccharides, flavonoids, and terpenoids, with various biological activities. Among them, bracken polysaccharide (PAP) has been reported to have antioxidant, immunomodulatory, and anti-inflammatory properties and has broad application prospects as a candidate sugar donor for selenium polysaccharides. So far, there has been no report on the successful construction of a bracken polysaccharide selenization system.

[0006] The present invention selenizes and modifies bracken polysaccharide by the HNO3-Na2SeO3 method, and uses ultrafiltration separation to clarify the molecular weight distribution of the most effective components in the selenized bracken polysaccharide. The obtained selenized bracken polysaccharide has characteristics such as high selenization efficiency and strong immunological activity of selenium polysaccharide, and is expected to provide ideas for solving the technical bottlenecks faced in the synthesis of artificial selenium polysaccharides and provide a reference for the further application of bracken selenium polysaccharide as a new selenium supplement preparation in the pharmaceutical and food fields. Summary of the Invention

[0007] To solve the above technical problems, the present invention provides a selenium-modified bracken polysaccharide, a preparation method thereof, and an application thereof.

[0008] A preparation method of a selenium-modified bracken polysaccharide includes first performing crushing, impurity removal, filtration, extraction, and vacuum concentration on bracken in sequence, then performing deproteinization on the concentrated solution by the Sevag method, then performing decolorization and filtration on the solution, and obtaining a precipitate through ethanol precipitation treatment. The precipitate is freeze-dried after centrifugal separation to obtain bracken polysaccharide, and then the bracken polysaccharide is selenized to obtain selenium-modified bracken polysaccharide. Finally, ultrafiltration separation is performed on the selenium-modified bracken polysaccharide:

[0009] Specifically: Continuously ultrafiltering the selenium-modified bracken polysaccharide using 100 kDa and 300 kDa ultrafiltration tubes to separate it into three components with molecular ranges of <100 kDa, 100 - 300 kDa, and >300 kDa.

[0010] Preferably: The specific process of performing crushing, impurity removal, filtration, extraction, and vacuum concentration on bracken in sequence is as follows:

[0011] First, use a pulverizer to crush dry bracken, sieve it through a 50-mesh sieve to obtain fine powder, and remove lipids, pigments, and other fat-soluble impurities;

[0012] Then filter, air-dry the residue, then mix it with water for extraction, and then concentrate the extract under reduced pressure.

[0013] Preferably: The fine powder is refluxed with 95% ethanol at 70 °C twice for 3 h to remove lipids, pigments, and other fat-soluble impurities.

[0014] Preferably: The residue is air-dried at 50 °C, then mixed with water at a ratio of 1:30 g / mL, then extracted at 80 °C for 2 h, and the extract is concentrated under reduced pressure at 55 °C.

[0015] Preferably: After performing deproteinization on the concentrated solution by the Sevag method, macroporous resin decolorizes the above solution at a ratio of resin:solution of 1:25.

[0016] Preferably: The specific process of ethanol precipitation treatment is: Add absolute ethanol to adjust the solution to an ethanol concentration of 76%, and store it overnight at 4 °C. The precipitate is freeze-dried after centrifugal separation to obtain bracken polysaccharide.

[0017] Preferably: The process of selenizing the bracken polysaccharide is as follows:

[0018] Take 500 mg of purified bracken polysaccharide dry powder and add it to 50 mL of HNO3, stir at room temperature until completely dissolved;

[0019] Divided into two portions, 700 mg of Na2SeO3 and 800 mg of Na2SeO3 were added respectively, stirred in a 70 °C water bath for 8 hours. After the reaction was completed, the solution was cooled to room temperature, adjusted to pH 7 with 5.6% NaHCO3 solution, and finally centrifuged to remove it;

[0020] Dialysis was carried out using a dialysis bag until sodium selenite could not be detected by the ascorbic acid method. The polysaccharide solution was concentrated and precipitated with ethanol, and then freeze-dried to obtain selenium-modified bracken polysaccharide.

[0021] Preferably: the concentration of HNO3 is 0.05%.

[0022] The present invention provides a selenium-modified bracken polysaccharide prepared by the above preparation method.

[0023] The present invention provides an application of the selenium-modified bracken polysaccharide in the preparation of food supplements.

[0024] The beneficial effects of the present invention are as follows: The selenium-modified bracken polysaccharide proposed by the present invention has antioxidant, immunomodulatory and α-glucosidase inhibitory activities superior to those of PAP. The selenium-modified bracken polysaccharide proposed by the present invention can become a healthy food supplement. Description of the Drawings

[0025] Figure 1 It is the ultraviolet spectrum diagram and FT-IR spectrum diagram in Example 4 of the present invention;

[0026] Figure 1 Among them: A is the ultraviolet spectrum diagram of the polysaccharide sample; B is the FT-IR spectrum diagram of the polysaccharide sample;

[0027] Figure 2 It is the XPS diagram and the spectral fitting result diagram in Example 4 of the present invention;

[0028] Figure 2 Among them: A is the XPS diagram of the polysaccharide sample; B is the 3d spectral fitting result diagram of Se;

[0029] Figure 3 It is the EDS diagram of SePAP1 and SePAP2 in Example 4 of the present invention;

[0030] Figure 4 It is the scanning electron microscope diagram of PAP, SePAP1 and SePAP2 in Example 4 of the present invention;

[0031] Figure 5 It is the test result diagram in Example 5 of the present invention; Figure 5 Among them: Figure 5 A is the test result diagram of the DPPH free radical scavenging activity of PAP, SePAP1 and SePAP2; Figure 5Figure B shows the test results of the DPPH free radical scavenging activity of the ultrafiltration fraction of SePAP1; Figure 5 Figure C shows the test results of the DPPH free radical scavenging activity of the ultrafiltration fraction of SePAP2; Figure D shows the test results of the ABTS free radical scavenging activity of PAP, SePAP1 and SePAP2; Figure 5 Figure E shows the test results of the ABTS free radical scavenging activity of the ultrafiltration fraction of SePAP1; Figure 5 Figure F shows the test results of the ABTS free radical scavenging activity of the ultrafiltration fraction of SePAP2;

[0032] Figure 6 Figure shows the test results of the effect of the polysaccharide sample in Example 6 of the present invention on the viability of RAW264.7 cells and on the secretion of NO by RAW264.7; Figure 6 In: Figure 6 Figure A shows the test results of the effect of PAP, SePAP1 and SePAP2 on the viability of RAW264.7 cells; Figure 6 Figure B shows the test results of the effect of the ultrafiltration fractions of SePAP1 and SePAP2 on the viability of RAW264.7 cells; Figure 6 Figure C shows the test results of the effect of PAP, SePAP1 and SePAP2 on the secretion of NO by RAW264.7; Figure 6 Figure D shows the test results of the effect of the ultrafiltration fractions of SePAP1 and SePAP2 on the secretion of NO by RAW264.7;

[0033] Figure 7 Figure shows the test results of the effect of the polysaccharide sample in Example 6 of the present invention on the secretion of TNF-α by RAW264.7 and on the proliferation index of mouse splenic lymphocytes; Figure 7 In: Figure 7 Figure E shows the test results of the effect of PAP, SePAP1 and SePAP2 on the secretion of TNF-α by RAW264.7; Figure 7 Figure F shows the test results of the effect of the ultrafiltration fractions of SePAP1 and SePAP2 on the secretion of TNF-α by RAW264.7; Figure 7 Figure G shows the test results of the effect of PAP, SePAP1 and SePAP2 on the proliferation index of mouse splenic lymphocytes; Figure 7 Figure H shows the test results of the effect of the ultrafiltration fractions of SePAP1 and SePAP2 on the proliferation index of mouse splenic lymphocytes

[0034] Figure 8 Figure shows the detection results of immunoblot analysis in Example 6 of the present invention;

[0035] Figure 9 Figure shows the results of protein quantitative analysis in Example 6 of the present invention; Figure 9In: Figure 9 A is the quantitative analysis result diagram of p-JNK / JNK protein; Figure 9 B is the quantitative analysis result diagram of p-ERK1 / 2 / ERK1 / 2 protein; Figure 9 C is the quantitative analysis result diagram of p-p38 / p38 protein; Figure 9 D is the quantitative analysis result diagram of p-p65 / p65 protein;

[0036] Figure 10 It is the test result diagram of α-glucosidase inhibition in Example 7 of the present invention; Figure 10 In: Figure 10 A is the test result diagram of the inhibitory activity of PAP, SePAP1 and SePAP2 on α-glucosidase; Figure 10 B is the test result diagram of the inhibitory activity of the ultrafiltration fraction of SePAP1 on α-glucosidase; Figure 10 C is the test result diagram of the inhibitory activity of the ultrafiltration fraction of SePAP2 on α-glucosidase;

[0037] Figure 11 It is the linear Lineweaver-Burk diagram on α-glucosidase in Example 7 of the present invention; Figure 11 In: Figure 11 A is the linear Lineweaver-Burk diagram of the inhibition of α-glucosidase by PAP; Figure 11 B is the linear Lineweaver-Burk diagram of the inhibition of α-glucosidase by SePAP1; Figure 11 C is the linear Lineweaver-Burk diagram of the inhibition of α-glucosidase by SePAP2; Figure 11 D is the linear diagram of the slope of PAP, SePAP1 and SePAP2 versus their concentrations;

[0038] Figure 12 It is the fluorescence quenching analysis result diagram of α-glucosidase loaded with PAP and SePAP1 at different concentrations in Example 7 of the present invention; Figure 12 In: Figure 12 A is the fluorescence emission spectrum of α-glucosidase at different concentrations under the action of PAP at 303.15 K (0 - 5 mg / mL); Figure 12 B is the fluorescence emission spectrum of α-glucosidase at different concentrations under the action of PAP at 313.15 K (0 - 5 mg / mL); Figure 12 C is the Stern-Volmer diagram of α-glucosidase quenched by PAP at 303.15 K and 313.15 K; Figure 12 D is the fluorescence emission spectrum of α-glucosidase at different concentrations under the action of SePAP1 at 303.15 K (0 - 5 mg / mL);Figure 12 E is the fluorescence emission spectra of α-glucosidase at different concentrations under the action of SePAP1313.15K (0 - 5 mg / mL); Figure 12 F is the Stern-Volmer plot of α-glucosidase quenched by SePAP1 at 303.15 K and 313.15 K;

[0039] Figure 13 It is the fluorescence quenching analysis result diagram of α-glucosidase loaded with SePAP2 at different concentrations in Example 7 of the present invention; Figure 13 In: Figure 13 G is the fluorescence emission spectra of α-glucosidase at different concentrations under the action of SePAP2 at 303.15 K (0 - 5 mg / mL); Figure 13 H is the fluorescence emission spectra of α-glucosidase at different concentrations under the action of SePAP2 at 313.15K (0 - 5 mg / mL); Figure I is the Stern-Volmer plot of α-glucosidase quenched by SePAP2 at 303.15 K and 313.15 K. Detailed implementation manners

[0040] Now, the subject matter described herein will be discussed with reference to exemplary implementation manners. It should be understood that discussing these implementation manners is only to enable those skilled in the art to better understand and thus implement the subject matter described herein. Without departing from the protection scope of the content of this specification, changes can be made to the functions and arrangements of the elements discussed. Each example can omit, substitute, or add various processes or components as needed. Additionally, the features described in some examples can also be combined in other examples.

[0041] Example 1

[0042] In this example, a preparation method of selenium-modified bracken polysaccharide is proposed, including the following steps:

[0043] S1: Dry bracken is crushed with a pulverizer and sieved through a 50-mesh sieve to obtain fine powder. The powder is refluxed with 95% ethanol at 70°C for 2 times, with a reflux time of 3h, to remove lipids, pigments, and other fat-soluble impurities.

[0044] S2: Then filter, air-dry the residue at 50°C, and then extract it with water at a ratio of 1:30 g / mL at 80°C for 2h. The extract is concentrated under reduced pressure at 55°C.

[0045] S3: Use the Sevag method to deproteinize the concentrated solution several times until no more flocculation occurs, and then decolorize the above solution with macroporous resin at a ratio of resin:solution 1:25 (v / v), and then filter;

[0046] S4: Finally, add absolute ethanol to adjust the solution to an ethanol concentration of 76%, and store it overnight at 4 °C. The precipitate is separated by centrifugation and then freeze-dried to obtain bracken polysaccharide;

[0047] S5: Selenize the bracken polysaccharide by the nitric acid-sodium selenite method;

[0048] Specifically: Take 500 mg of purified bracken polysaccharide dry powder and add it to 50 mL of HNO3 with a concentration of 0.05%, and stir at room temperature until completely dissolved;

[0049] Divide it into two parts, add 700 mg of Na2SeO3 and 800 mg of Na2SeO3 respectively, stir in a water bath at 70 °C for 8 hours. After the reaction is completed, cool the solution to room temperature, add 5.6% NaHCO3 solution to adjust the pH to 7, and finally centrifuge to remove it;

[0050] Dialyze with a dialysis bag until sodium selenite cannot be detected by the ascorbic acid method. Concentrate the polysaccharide solution and precipitate it with ethanol, and then freeze-dry to obtain selenized bracken polysaccharide. The obtained selenized bracken polysaccharide is of two types, named Se-PAP1 and Se-PAP2 respectively;

[0051] S6: Ultrafiltration separation of selenized bracken polysaccharides SePAP1 and SePAP2;

[0052] Specifically: Continuously ultrafilter the bracken selenized polysaccharide solution using 100 kDa and 300 kDa ultrafiltration tubes. Separate SePAP1 into three components with molecular ranges of <100 kDa, 100 - 300 kDa, and >300 kDa, named: SePAP1-I, SePAP1-II, and SePAP1-III respectively; Separate SePAP2 into three components with molecular ranges of <100 kDa, 100 - 300 kDa, and >300 kDa, named: SePAP2-I, SePAP2-II, and SePAP2-III respectively. The above samples are freeze-dried and stored.

[0053] Example 2

[0054] In this example, a selenium-modified bracken polysaccharide is proposed, which is obtained according to the preparation method in Example 1.

[0055] Example 3

[0056] In this example, an application of a selenium-modified bracken polysaccharide in the preparation of food supplements is proposed.

[0057] Example 4

[0058] This example is used for the characterization of the bracken polysaccharide PAP prepared in Example 1 and the selenium-enriched bracken polysaccharide SePAP.

[0059] 1. Method

[0060] 1.1. Determination of composition and selenium content

[0061] Using glucose as the standard, the total carbohydrate content was determined by the phenol-sulfuric acid method. Using bovine serum albumin as the standard, the protein content was determined by the Coomassie brilliant blue G-250 method.

[0062] The selenium content was measured by quantitative analysis using an inductively coupled plasma mass spectrometer (iCAP RQ 7000, Thermo Fisher Scientific) and a MARS microwave digester (MARS 200, Shanghai Metash Instruments Co., Ltd.).

[0063] 1.2. Monosaccharide composition

[0064] The monosaccharide composition was determined by the pre-column derivatization method with 1-phenyl-3-methyl-5-pyrazolone (PMP). First, 3 mL of 1.5 mol / L trifluoroacetic acid was used to hydrolyze for 1 hour at 120 °C, then the mixture was neutralized with 0.3 mol / L sodium hydroxide, and then derivatized with 0.5 mol / L PMP at 70 °C for 100 min. After neutralizing and removing the excess PMP with 0.3 mol / L hydrochloric acid, the derivatized sample was analyzed using an Agilent 1260 HPLC system connected to a C18 column (2.6 mm × 250 mm, 5 μm) at 249 nm. The mobile phase consisted of 0.1 mol / L ammonium acetate solution and acetonitrile, with a flow rate of 1 mL / min. The monosaccharides were identified by comparison with standards (rhamnose, mannose, glucose, galactose, arabinose).

[0065] 1.3. Ultraviolet

[0066] For each sample, 10 mg was taken and digested with 2 mL of sulfuric acid and nitric acid (v / v, 1:4) at 100 °C for 1.5 hours. After cooling, 1 mL of 0.1 g / mL o-phenylenediamine solution was added, and the reaction was stirred in the dark at room temperature for 2 h. Then, the mixture was extracted with 3 mL of toluene, and the extracted upper layer solution was scanned on a TU-1950 double-beam ultraviolet-visible spectrophotometer in the wavelength range of 300 - 500 nm.

[0067] 1.4. Fourier transform infrared spectroscopy (FT-IR) analysis

[0068] The dried sample (1 mg) was mixed with dried KBr (100 mg), pressed into a tablet, and analyzed using a Fourier transform mid-infrared spectrometer (Nicolet iS10, Thermo Fisher Scientific) in the range of 4000 - 400 cm -1Measure its absorption spectrum within the wavenumber range.

[0069] 1.5. The valence state of selenium was studied using an X-ray photoelectron spectrometer (ESCALAB 250Xi, Thermo Fisher Scientific, USA). The elemental composition of the sample was evaluated using an energy-dispersive X-ray spectrometer on a scanning electron microscope (JSM-6480 a, JEOL Ltd, Tokyo, Japan).

[0070] 1.6. Surface morphology observation

[0071] The dried sample powder was sputter-coated with gold under reduced pressure and measured using a field emission scanning electron microscope (SEM) (SU8010)). The dried powder sample was sputter-coated with gold under reduced pressure and measured by SEM at an acceleration voltage of 5 kV.

[0072] 2. Results

[0073] The changes in total carbohydrate, selenium content, and monosaccharide composition among PAP, SePAP1, and SePAP2 are shown in Table 1.

[0074]

[0075] The results showed that the carbohydrate content in both SePAP1 and SePAP2 was higher than that in PAP, indicating that the polysaccharide in bracken was further purified during the selenization process. In addition, the selenium contents in SePAP1 and SePAP2 were determined to be 7035.48 µg / g and 13505.26 µg / g, respectively. The above results showed that inorganic selenium atoms were successfully linked to the polysaccharide molecules, and PAP was successfully selenized; increasing the dosage of Na2SeO3 could significantly increase the selenium content in the polysaccharide of bracken. The study found that there were obvious compositional differences in the monosaccharide chromatograms of PAP, SePAP1, and SePAP2. PAP was composed of rhamnose, mannose, glucose, galactose, and arabinose, with a molar ratio of 0.281:0.128:1.171:4.966:1.126. It is worth noting that galactose, glucose, and arabinose were the main monosaccharides in PAP. The molar ratios of rhamnose, mannose, glucose, galactose, and arabinose in SePAP1 and SePAP2 were 0.499:0.288:4.183:0.771:2.694 and 0.568:0.316:2.701:0.8497:2.862, respectively (Table 1). After modification, the monosaccharide composition of PAP changed, showing a significant decrease in galactose content and a significant increase in glucose content. It has been reported that polysaccharide molecules can depolymerize under strong acid and long-term incubation conditions. Structurally, galactose and glucose are C-4 diastereoisomers, and these two sugars can isomerize, transform into each other, and produce other reaction products under certain conditions. The decrease in galactose and the increase in glucose content after selenization indicated the existence of a potential isomerization reaction and the phenomenon of sugar isomerization during the selenization modification process.

[0076] The UV analysis of the polysaccharide samples after acid digestion is as ​ shown in A. Compared with PAP, an obvious absorption peak appeared at around 346 nm in SePAP1 and SePAP2. This observed absorption peak was determined to be the characteristic absorption peak of selenium.

[0077] FT-IR spectra are usually used to identify characteristic groups and chemical bonds within polysaccharides. As ​ shown in B, the significant absorption peaks near 3413 and 2931 cm -1 were caused by the stretching vibrations of O-H and C-H, respectively. These are all characteristic peaks of polysaccharides. The bands observed at approximately 1611 cm -1 and 1416 cm -1 corresponded to the stretching vibration of the carboxyl C=O and the angular C-H vibration, respectively. In addition, at 1256 cm -1Signals in the vicinity indicate the presence of sulfate groups. The main absorption peaks of SePAP1 and SePAP2 are similar to those of PAP, indicating that the basic skeletal structure of the polysaccharide remains unchanged after selenization. However, three new absorption peaks were detected in the spectra around 1025 cm -1 , 806 cm -1 and 626 cm -1 , which belong to O-Se-O, Se=O, and Se-O-C vibrations, respectively. Notably, SePAP1 and SePAP2 also have distinct peaks around 1732 cm -1 , which may be related to the reaction products during the monosaccharide isomerization process. Similarly, a new absorption peak appeared in the FT-IR of selenized bracken polysaccharide at 675 cm -1 , indicating an asymmetric Se-O-C stretching vibration. Generally, hydroxyl groups in polysaccharides play a crucial role in providing many binding sites for the binding of selenium to polysaccharide molecules. These results indicate that the selenization modification has a good effect.

[0078] XPS analysis was performed on PAP, SePAP1, and SePAP2 to verify the presence of selenium and analyze its valence state. As ​ shown in A, PAP, SePAP1, and SePAP2 mainly consist of three elements, with peaks at 532.44 eV for oxygen, 400.06 eV for nitrogen, and 285.24 eV for carbon. In contrast, a new Se 3d peak appeared in the spectra of SePAP1 and SePAP2 around 56 eV, indicating the successful integration of Se into the polymer of PAP. In addition, the fitting results of the 3d spectrum of Se are shown in ​ B, showing a characteristic peak of Se at 55.6 eV, which may be zero-valent selenium. The EDS analysis results of SePAP1 and SePAP2, as shown in ​ , further confirmed the successful introduction of Se into the polymer structure.

[0079] The morphological differences between these polysaccharides were analyzed using scanning electron microscopy (SEM). At magnifications of ×500 or ×1200, the surfaces of PAP, SePAP1, and SePAP2 exhibited similar flaky structures, while the fragments of SePAP1 and SePAP2 were smaller and some of the flaky structures were pierced, indicating that glycosidic bonds were broken during the selenization process and the molecular weight of PAP decreased. After further magnification (×25000), the structures of SePAP1 and SePAP2 showed an obvious rough surface morphology, rather than the smooth surface observed in PAP. In particular, a large number of irregular small holes were observed in both SePAP1 and SePAP2 ( ​), which is the result of acid hydrolysis during the reaction. This selenium-induced surface morphology transformation is consistent with previous reports, indicating that chemical modification can profoundly affect the monosaccharide composition, molecular weight, and conformation of polysaccharides, thereby influencing their morphological characteristics.

[0080] Example 5

[0081] This example was used to determine the antioxidant activity of the selenium-enriched bracken polysaccharide prepared in Example 1.

[0082] 1. Antioxidant Activity Determination

[0083] 1.1 DPPH Radical Scavenging Activity

[0084] 100 μL of the polysaccharide solution was mixed with 100 μL of 0.05 mmol / L DPPH solution (dissolved in alcohol), and incubated in the dark at 25 °C for 45 minutes. Vc was used as the positive control, the polysaccharide solution and the alcohol solution without DPPH were used as blanks, and the DPPH alcohol solution and the alcohol solution without the polysaccharide solution were used as the background group. The absorbance at 517 nm was measured. The DPPH radical scavenging rate was calculated by the following formula:

[0085]

[0086] where A1 is the absorbance of the sample in each group, A2 is the absorbance of the control group, and A0 is the absorbance of the blank group.

[0087] 1.2 ABTS Radical Scavenging Activity

[0088] 20 μL of polysaccharide solutions with different concentrations were added to a 96-well plate, and 20 μL of PBS solution was added to the control group. Then, 100 μL of ABTS+· measuring solution was added, and the mixture was shaken at room temperature for 6 minutes, and then the absorbance value of the reaction solution was measured at a wavelength of 734 nm. Ascorbic acid (Vc) as the positive control was measured. All measurements were tested in triplicate. The ABTS radical scavenging rate was calculated as follows:

[0089]

[0090] where A1 is the absorbance of the sample in each group, A2 is the absorbance of the control group, and A0 is the absorbance of the blank group.

[0091] 2. Results

[0092] The antioxidant activity differences between PAP and SePAP1-2 were evaluated by the scavenging ability of DPPH and ABTS on free radicals.

[0093] ​Figure A shows that PAP, SePAP1, and SePAP2 exhibit significant dose-dependent scavenging ability against DPPH radicals, with IC50 values of 2.667, 0.7135, and 0.2674 mg / mL, respectively. Notably, compared with natural PAP, SePAP1 and SePAP2 show significantly enhanced DPPH radical scavenging rates. Similarly, in ​ Figure D, for all samples, the scavenging activity of ABTS radicals is dose-dependent within the tested concentration range. The IC50 values of the ABTS radical scavenging rates of PAP, SePAP1, and SePAP2 are 2.157, 1.219, and 0.8927 mg / mL, respectively, indicating that the scavenging activity of SePAP1-2 is superior to that of PAP. Compared with SePAP2, SePAP1 has stronger DPPH and ABTS radical scavenging activities.

[0094] SePAP1 and SePAP2 were continuously ultrafiltered to obtain molecular weight (Mw) ranges of <100 KDa, 100 - 300 KDa, and >300 KDa, and then the scavenging characteristics of DPPH and ABTS radicals were detected. For SePAP1-I (<100 KDa), SePAP1-II (100 - 300 KDa), and SePAP1-III (>300 KDa), the IC50 values for DPPH radicals are 0.7608, 0.1786, and 2.841 mg / mL ( ​ Figure B), among which SePAP1-II has the highest activity. Similarly, for SePAP2-I, SePAP2-II, and SePAP2-III scavenging DPPH radicals, the IC50 values are 0.2744, 0.2017, and 0.6652 mg / mL ( ​ Figure C), and the activity of the medium Mw fraction (SePAP2-II, 100 - 300 KDa) is the best. In terms of scavenging ABTS radicals, the IC50 values of SePAP1-I to III are 0.5427, 0.5815, and 1.718 mg / mL ( ​ Figure E), while the half-inhibitory concentration values of SePAP2-I to III are 0.4747, 0.4361, and 1.083 mg / mL ( ​ Figure F), indicating that small or medium Mw fractions exhibit stronger ABTS radical scavenging ability. The results show that the 100 - 300 KDa or <100 KDa fractions of SePAP1-2 have strong effects on DPPH and ABTS radicals, while the higher Mw fraction (>300 KDa) has weaker scavenging ability for radicals.

[0095] The biological activities of polysaccharides are related to their molecular structure, water solubility, molecular weight, monosaccharide composition, glycosidic bonds in the main chain, type and degree of substitution (DS), degree of branching, and conformation of the main chain. Selenium modification can enhance the antioxidant capacity of polysaccharides by increasing their molecular weight, reducing particle size, raising zeta potential, and influencing the conformation of polysaccharides. In addition, the scavenging activity of polysaccharides towards free radicals is positively correlated with the hydrogen-donating ability of their hydroxyl groups. The presence of selenium groups or selenate groups in selenized polysaccharides can activate the hydrogen atoms on vicinal carbons, thereby enhancing the hydrogen-donating potential of hydroxyl groups. Based on the above findings, SePAP1 and SePAP2 demonstrate that selenium plays a key role in enhancing the antioxidant activity of PAP.

[0096] Example 6

[0097] This example is used to determine the immunomodulatory activity of the selenized bracken polysaccharide prepared in Example 1.

[0098] 1. Determination of immunomodulatory activity

[0099] 1.1 Cell culture

[0100] The culture conditions for RAW 264.7 cells are as follows: DMEM medium supplemented with fetal bovine serum (10%), 1% penicillin and streptomycin (100 U / mL), and cultured in an incubator at 37 °C and 5% CO2. RAW264.7 cells at the logarithmic growth phase are cryopreserved as backups.

[0101] 1.2 Cell viability

[0102] The CCK-8 method is used to detect the cell viability of RAW264.7 macrophages. First, the cells are seeded at a density of 5×104 cells / mL and cultured for 24 h. Subsequently, they are treated with different concentrations of PAP or SePAP 1-2 (1.95 - 250 μg / mL) for another 24 h. Equal amounts of medium and LPS (10.0 μg / mL) are used as blank control and positive control, respectively. The absorbance is measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader (Perkinmar, USA).

[0103]

[0104] 1.3 Effects of PAP and SePAP 1-2 on the production of NO and TNF-α by macrophages

[0105] RAW264.7 cells are seeded in 96-well plates at a density of 4×105 cells / mL for the NO production assay and 5×104 cells / mL for the TNF-α production assay. After culturing for 24 h, the cells are treated with PAP or SePAP1-2 (1.95 - 62.5 μg / mL) respectively. The supernatant is collected and measured using a detection kit.

[0106] 1.4 Effect of Selenium Polysaccharide on the Proliferation of Mouse Spleen Cells

[0107] The CCK-8 method was used to evaluate the effects of PAP and SePAP1-2 on the proliferation of mouse spleen cells. Kunming mice at 5 weeks of age were purchased from Hubei Medical Experimental Animal Center. They were adaptively cultured for one week in a circadian cycle environment with a temperature of 22 - 24 °C and a relative humidity of 50% - 70%. All animal experimental procedures followed the published guidelines.

[0108] Spleen cell suspensions were prepared with RPMI1640 medium containing 10% FBS and inoculated on 96-well culture plates at a density of 2×106 cells / mL, 200 μL per well. Different concentrations of PAP or SePAP1-2 (1.95 - 62.5 μg / mL) were used to treat the cells at 37 °C for 24 hours. Then, 20 μL of CCK-8 solution was added and the cells were cultured for another 2 h. The absorbance value was measured at 540 nm. Con A (5 μg / mL) was used as the positive control. The calculation formula for the proliferation rate is as follows:

[0109]

[0110] In the formula, A0 is the absorbance of the blank group, and A1 is the absorbance of the sample solution group.

[0111] 1.5 Western Blot Analysis

[0112] First, RAW264.7 cells (2×106 cells / mL) were inoculated into 6-well plates. Subsequently, the cells were treated with polysaccharide (62.5 μg / mL) or LPS (10 μg / mL) and incubated for 24 h. After incubation, proteins in RAW264.7 cells were extracted using RIPA lysis buffer, which included 50 mM Tris-HCl (pH 7.4), 150 mM sodium chloride, 1% Nonidet P-40, and 0.1% sodium dodecyl sulfate (SDS). 30 μg of protein samples were loaded and separated by 12% SDS-PAGE. The separated proteins were then transferred onto PVDF membranes. Then, the membranes were blocked with 5% skim milk in Tris-buffered saline containing Tween 20 (TBST) for 1.5 hours. For immunodetection, the membranes were incubated with primary antibodies overnight at 4 °C, washed three times with TBST, and then incubated with secondary antibodies for 1 hour at room temperature. The expressed proteins were detected using ECL solution.

[0113] 2. Results

[0114] 2.1 Proliferation of RAW264.7 Cells

[0115] This example detected the effects of PAP, SePAP1, and SePAP2 on the proliferation of RAW264.7 cells. Cell viability assays showed that PAP and SePAP1-2 at different concentrations (1.95 - 62.5 μg / mL) had no cytotoxic effects, and all cell viability values exceeded 100% ( ​ A). After ultrafiltration separation, all fractions of SePAP1 and SePAP2 showed similar non-cytotoxic effects ( ​ B). Previous studies have shown that selenium polysaccharides generally have lower cytotoxicity to RAW264.7 macrophages compared to inorganic selenium. Consistent with these findings, our experimental results further confirmed the non-cytotoxicity of selenium-polysaccharides at all tested concentrations. To better compare the immunomodulatory effects of PAP, SePAP1, and SePAP2 on RAW264.7 cells, the maximum concentration was set at 62.5 μg / mL for the next test.

[0116] 2.2 Effects of selenium polysaccharides on NO release from RAW264.7 cells

[0117] Nitric oxide (NO) is a multifaceted regulatory factor that participates in various molecular and biological pathways and plays a key role in pathological and physiological activities. When tissues and the immune system are invaded by pathogenic microorganisms and cancer cells, macrophages can produce large amounts of cytokines and NO to resist pathogens. Therefore, the amount of NO released by stimulated macrophages can be used as an indicator to reflect their activation level and evaluate immune activity.

[0118] Initially, PAP showed moderate activation of RAW264.7 cells, and NO release was measured at 1.82 μM at a concentration of 62.5 μg / mL. After treatment with seleniumized products, NO production increased significantly at all tested concentrations. Under the condition of 62.5 μg / mL, the NO production levels in the SePAP1 and SePAP2 treatment groups reached 7.236 ± 0.41 μM and 7.459 ± 0.17 μM, respectively ( ​ C), showing a significant enhancement in immune activity compared to the PAP treatment group (p < 0.001). For the fractions of SePAP1 and SePAP2, compared with other fractions, the SePAP1-III and SePAP2-III fractions with the highest molecular weight (>300 KDa) had a stronger effect on enhancing NO secretion ( ​D), which is the opposite of antioxidant activity. Many studies have emphasized the relationship between the immunostimulatory activity of polysaccharides and their molecular weight. Polysaccharides with an appropriate molecular weight have been shown to effectively enhance the immune response. Studies have confirmed that polysaccharides with a molecular weight exceeding 1000 KDa may affect their diffusion and cellular uptake and limit their immune efficacy. At the same time, polysaccharides with a molecular weight below 10 KDa lose their structural integrity, thus affecting their biological activity. In our study, polysaccharides with a relatively high molecular weight (>300 KDa) showed significant immunomodulatory activity.

[0119] 2.3 Effects of selenium polysaccharides on the release of NO by RAW264.7 cells

[0120] Activated macrophages secrete various cytokines that are crucial for mediating and regulating the immune response. Among them, TNF-α plays an indispensable role in regulating the immune response, especially in antigen presentation, phagocytosis, and innate immune regulation of macrophages. Therefore, measuring the secretion of TNF-α is crucial for elucidating the immunomodulatory effects of polysaccharides and their selenized derivatives.

[0121] Both SePAP1 and SePAP2 stimulated the TNF-α production of RAW264.7 cells in a concentration-dependent manner, and the production of these two selenium polysaccharides at each concentration was significantly higher than that of PAP ( ​ E), when the concentration reached 62.5 μg / mL, the TNF-α secretion levels of PAP, SePAP1, and SePAP2 were 253.70 ± 15.74, 27565.75 ± 652.73, and 38397.48 ± 468.48 pg / mL, respectively. This result indicates that compared with PAP, SePAP1-2 showed significantly stronger immune effects. Among SePAP1 and SePAP2, SePAP2 with a higher selenium content had significantly stronger immune activity than SePAP1. ​ Figure F shows TNF-α secreted by the stimulation of SePAP1 and SePAP2. As the molecular weight increased, Mw > 300 KDa (SePAP1-III or SePAP2-III) showed the highest TNF-α secretion.

[0122] 2.4 Lymphocytes are key effector cells in the mammalian immune system. Our study showed that polysaccharides activated lymphocyte subsets to varying degrees. As ​As shown in Figure G, PAP, SePAP1, and SePAP2 can all promote the proliferation of splenocytes. At 62.5 μg / mL, the splenocyte proliferation indices of the PAP, SePAP1, and SePAP2 treatment groups were 1.18-fold, 1.33-fold, and 1.70-fold that of the control group, respectively. This suggests that SePAP1 and SePAP2 are more effective than PAP in stimulating splenocyte proliferation. Moreover, the proliferation index of the SePAP2 group was greater than that of the SePAP1 group. In addition, the splenocyte proliferation index was the highest in the detected components of Mw > 300 KDa, SePAP1-III, and SePAP2-III ( ​ H). This is consistent with the results of RAW264.7 macrophages producing NO and TNF-α.

[0123] In summary, selenization endows the original polysaccharide with greater ability to increase the production of NO and TNF-α by macrophages. Selenized derivatives with higher selenium content (SePAP2) stimulate the production of higher levels of NO and TNF-α, indicating its excellent immunomodulatory effect in RAW264.7 cells. According to the reasons for the immune enhancement of selenized polysaccharides, the interaction between selenium functional groups promotes the aggregation of polysaccharide chains, resulting in a decrease in steric hindrance. This change enables the polysaccharide to enter the cell and bind smoothly to the macrophage surface receptor, thereby triggering the release of related cytokines. In other studies, it has also been observed that polysaccharides with increased selenium content exhibit stronger immunomodulatory effects.

[0124] 2.5, Western Blot Analysis

[0125] The immunomodulatory effect of polysaccharides on the immune system may be promoted through multiple intracellular signaling pathways. Among them, the MAPK signaling pathway is a well-established pathway for regulating immune responses. To investigate this, we analyzed the levels of the total and phosphorylated forms of ERK1 / 2, JNK, and p38 in RAW264.7 cells treated with the three polysaccharides using Western blotting. As ​ shown, the expression levels of each total protein remained basically unchanged. Compared with the control group, after treatment with the polysaccharides, the expression level ratios of p-JNK / JNK, p-ERK1 / 2 / ERK1 / 2, and p-p38 / p38 were significantly increased ( ​ A, 9B, and 9C), indicating an increase in the levels of phosphorylated proteins (p-JNK, p-ERK1 / 2, and p-p38). After selenization, compared with the PAP treatment group, the expression rates of the SePAP1 and SePAP2 treatment groups were significantly increased (p < 0.05).

[0126] Activation of the NF-κB pathway is another key signaling event induced by immunostimulants. Therefore, we detected the expression of phosphorylated p65 in the nucleus. The results showed that the content of phosphorylated p65 in RAW264.7 cells treated with polysaccharides increased compared with that in cells without polysaccharide treatment ( ​ and 9 D). In addition, compared with the cells treated with PAP, the cells exposed to SePAP1 and SePAP2 showed an increased expression of phosphorylated p65 (p < 0.05).

[0127] Taken together, these results indicate that selenium polysaccharides activate the MAPK and NF-κB signaling pathways by upregulating the phosphorylation levels of ERK1 / 2, JNK, p38, and p65, and stimulate macrophages to produce a large amount of NO and cytokines. Our findings are consistent with the previously reported mechanisms involving the immunological activity of chemically modified polysaccharides. For example, sulfated cycloglycan polysaccharides have also been shown to induce the immunological effects of RAW264.7 cells by activating the NF-κB and MAPK pathways.

[0128] Example 7

[0129] This example is used to determine the hypoglycemic activity of the selenium-enriched bracken polysaccharide prepared in Example 1.

[0130] 1. Determination of hypoglycemic activity

[0131] 1.1. Determination of the inhibitory rate activity of α-glucosidase

[0132] The inhibitory activities of PAP and Se-PAP on α-glucosidase were determined. The specific operation steps were as follows: α-glucosidase and PNPG were dissolved in phosphate buffer solution (PBS, 0.1 mol / L, pH 6.8). 80 μL of polysaccharide solutions with different mass concentrations (0.156, 0.3125, 0.625, 1.25, 2.5 mg / mL) and 20 μL of α-glucosidase solution (2 U / mL) were mixed, and after pre-incubation at 37°C for 10 min to balance the reaction, 40 μL of 8 mmol / L PNPG solution was added as a substrate to the pre-incubated mixture system to initiate the reaction. After reacting at 37°C for 30 min, 60 μL of 1 mol / L sodium carbonate solution was immediately added to stop the reaction. Finally, the absorbance at 405 nm was measured using an enzyme-linked immunosorbent assay reader. Acarbose was used as a positive control, and the α-glucosidase inhibition rate was calculated according to the following formula: The α-glucosidase inhibition rate was calculated according to the following formula:

[0133]

[0134] A1, A2, A3, and A4 represent the absorbance values of the polysaccharide solution and enzyme added, the absorbance value of the polysaccharide solution added, the absorbance value of the enzyme added, and the absorbance value when neither the polysaccharide solution nor the enzyme is added, respectively.

[0135] 1.2. Inhibition Kinetics Determination

[0136] The following method was used to detect the inhibition type of bracken polysaccharide on α-glucosidase. When the α-glucosidase concentration was 2 U / mL, the initial reaction rates of the enzyme were measured at different substrate PNPG concentrations (0.5, 1, 2, 4, 6, 8 mM) for different mass concentrations of bracken polysaccharide (0.156, 0.3125, 0.625, 1.25, 2.5 mg / mL). A plot of 1 / V against 1 / [S] was made to obtain the Lineweaver-Burk curve diagram, and the inhibition kinetic constants Michaelis constant (K m ) and maximum reaction rate (V max ) were calculated to determine the inhibition type of the purified protein on α-glucosidase. Through secondary plotting, the dissociation constant K i of the inhibitor and enzyme and the dissociation constant K i ' of the inhibitor and enzyme-substrate complex were obtained. According to the Lineweaver-Burk curve and the magnitudes of K i and K i ', the specific inhibition type was determined and its inhibition mechanism was analyzed.

[0137]

[0138] In the formula, [S] and [I] represent the concentrations of the substrate and inhibitor, respectively, in mmol / L; V represents the initial reaction rate; K m and V max represent the Michaelis constant and the maximum reaction rate, respectively; K i and K i ' are both dissociation constants.

[0139] 1.3. Fluorescence Measurement

[0140] α-Glucosidase, DHP, and Se-DHP were all prepared with phosphate buffer (0.1 mol / L, pH 6.8). Under the condition that the fluorescence excitation wavelength (λex) was 280 nm, 3 mL of 2 U / mL α-glucosidase solution was mixed with 300 μL of polysaccharide solutions with different mass concentrations ((0.156, 0.3125, 0.625, 1.25, 2.5 mg / mL)) and incubated at different temperatures (303.15 K, 310.15 K) for 10 min, and the fluorescence spectra were recorded. The emission wavelength (λem) was set to 300 - 400 nm, and the slit width was 5 nm.

[0141] When the quenching mode belongs to a single quenching mode, i.e., static or dynamic quenching, the curve between F0 / F and [Q] tends to a linear relationship. The fluorescence quenching mechanism can be analyzed by the following linear Stern-Volmer:

[0142]

[0143] where F0 and F are the fluorescence intensities of α-glucosidase before and after polysaccharide treatment, respectively; [Q] is the concentration of the polysaccharide; K SV is the quenching constant;

[0144] In some cases, the Stern-Vlomer curve significantly deviates from the linear correlation, and K sv can be measured by an adjusted "Stern-Vlomer" equation:

[0145]

[0146] 1.4. Data processing

[0147] Data are expressed as the mean ± standard deviation (SD) of three independent experiments and analyzed using SPSS 17.0 software for one-way analysis of variance (ANOVA) and independent tests. When p < 0.05, it is considered statistically significant.

[0148] 2. Results

[0149] 2.1. In vitro inhibitory activity of α-glucosidase and enzyme inhibition mechanism

[0150] 2.1.1. Inhibition of α-glucosidase

[0151] Diabetes is a complex syndrome characterized by carbohydrate metabolism disorders. α-Glucosidase plays a key role in the conversion of carbohydrates to glucose, so inhibiting the activity of this enzyme can significantly reduce or delay postprandial hyperglycemia.

[0152] ​Figure A shows that within the tested concentration range, the α-glucosidase inhibitory activities of all polysaccharide samples increased in a concentration-dependent manner. The half-inhibitory concentration values of PAP, SePAP1, and SePAP2 against α-glucosidase were 1.388, 0.7615, and 0.7726 mg / mL, respectively, indicating that selenium polysaccharides had higher α-glucosidase inhibitory activities than natural polysaccharides. Although SePAP2 had a higher selenium content, it did not show better α-glucosidase inhibitory effects than SePAP1, suggesting that the α-glucosidase inhibitory activity of selenium-enriched polysaccharides in this study might be positively correlated with selenium content. The IC50 values of SePAP1-I to III against α-glucosidase were 0.7003, 0.7072, and 0.6303 mg / mL, respectively, indicating that SePAP1-III (> 300KDa) had the strongest α-glucosidase inhibitory ability ( ​ B). Similarly, the IC50 values of SePAP2-I to III against α-glucosidase were 0.6653, 0.7809, and 0.3530 mg / mL, respectively, and SePAP2-III (> 300 KDa) had the highest α-glucosidase inhibitory activity ( ​ C). These results indicated that the fractions of SePAP1-2 with a molecular weight exceeding 300 KDa were responsible for α-glucosidase inhibition.

[0153] 2.1.2, α-Glucosidase Inhibition Kinetics

[0154] The inhibition modes of PAP, SePAP1, and SePAP2 against α-glucosidase were studied using the Lineweaver-Burk equation (L-B plot). As shown in ​ A, 11B, and 11C, the L-B plots of PAP, SePAP1, and SePAP2 intersected with the control group in the third quadrant. In addition, the K m and V max both decreased with the increase in polysaccharide concentration, as shown in Table 2:

[0155]

[0156] Therefore, all PAP, SePAP1, and SePAP2 showed a mixed inhibition type against α-glucosidase, including non-competitive and uncompetitive inhibition, further indicating that PAP, SePAP1, and SePAP2 could bind to free α-glucosidase or the α-glucosidase-substrate complex.

[0157] The inhibition constants K i and K i', corresponding to the dissociation of the inhibitor - enzyme complex and the inhibitor - enzyme - substrate complex, respectively. The calculation results are shown in Table 2. The lower the values of these constants, the stronger the binding affinity, and thus the stronger the inhibitory effect. For all PAP, SePAP1, and SePAP2, K i values are greater than K i ' values, indicating that compared with free α - glucosidase, their affinity for the α - glucosidase - substrate complex is slightly stronger. In addition, the order of the inhibition constants of PAP, SePAP1, and SePAP2 for α - glucosidase is shown as K i PAP > K i SePAP1 > K i SePAP2 and K i ' PAP >K i ' SePAP1 > K i 'SePAP2. This indicates that compared with PAP, SePAP1 and SePAP2 both show higher binding affinity for α - glucosidase, which is consistent with the results of the α - glucosidase inhibitory activity assay. In addition, there is only one binding site between each polysaccharide and α - glucosidase, and its slope is linearly correlated with its concentration ( ​ D).

[0158] 2.1.3. Fluorescence Quenching Analysis of α - Glucosidase

[0159] Fluorescence quenching refers to the phenomenon that when a fluorescent substance interacts with other molecules, its fluorescence intensity decreases. α - Glucosidase contains aromatic amino acids such as tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe), which emit fluorescence when excited at a wavelength of 280 nm. The addition of a specific quencher can reduce the fluorescence signal emitted by the enzyme. Various molecular interactions contribute to this quenching effect, including the formation of ground - state complexes, collision quenching, excited - state reactions, molecular rearrangement, and energy - transfer mechanisms. To explore the interaction between α - glucosidase and three different polysaccharides, we measured the fluorescence intensity of α - glucosidase.

[0160] After interacting with PAP and SePAP1 - 2, the maximum fluorescence intensity of α - glucosidase decreased in a dose - dependent manner with the increase in polysaccharide concentration ( ​A, 12B, 12D, 12E, 13G and 13H). Compared with PAP, the fluorescence quenching effect of SePAP1-2 on α-glucosidase was significantly greater than that of PAP, indicating that the inhibitory activity of the selenized product was stronger than that of PAP, further verifying the results of the enzyme inhibition assay. Notably, after adding PAP, the wavelength of the maximum fluorescence intensity shifted to a longer wavelength (redshifted from 340 nm to 357 nm). Similarly, a slight redshift was also observed for SePAP1 and SePAP2. This indicates that α-glucosidase is unfolded when interacting with these three polysaccharides.

[0161] To clarify the fluorescence quenching mechanism of polysaccharides on α-glucosidase, the quenching constants (Ksv) at two different temperatures (303.15 K and 313.15 K) were calculated using the Stern-Volmer equation or its modified form. The fluorescence quenching mechanism generally can be divided into static quenching, dynamic quenching, and a combination of both. Static quenching is due to the formation of a non-fluorescent complex that cannot emit light or has a significantly reduced luminescence efficiency; usually, in this case, the Ksv value decreases with increasing temperature. In contrast, dynamic quenching results from the energy transfer collision between the quencher and the fluorophore, leading to an increase in Ksv with increasing temperature.

[0162] The Stern-Volmer plot showed a linear relationship for PAP ( ​ C), indicating the existence of a single quenching mechanism, either static or dynamic. As the temperature increased, the Ksv of PAP on α-glucosidase increased from 0.2636 to 0.4376 L / g, indicating a dynamic quenching process caused by collisions. However, as the concentration increased, the inhibition plots of SePAP1 and SePAP2 against α-glucosidase showed a concave slope towards the y-axis ( ​ F and 13I). This indicates that the fluorescence quenching in the interaction of these two selenized polysaccharides with α-glucosidase may be a combined quenching mechanism.

[0163] In this invention, the selenization of PAP was carried out by the HNO3-Na2SeO3 method to obtain SePAP1 and SePAP2 with high selenium content. This chemical modification changed the monosaccharide molar ratio of PAP, while the primary backbone structure did not change significantly. Compared with the unmodified polysaccharide, the selenized products had stronger antioxidant, immunomodulatory and α-glucosidase inhibitory activities. The content of added selenium was positively correlated with the antioxidant and immunomodulatory functions, but did not affect the α-glucosidase inhibitory effect. Ultrafiltration of the selenized products gave fractions in different molecular weight ranges. The fractions <100 KDa and 100 - 300 KDa had better antioxidant effects, while the fraction >300 KDa was more effective in immunomodulation and α-glucosidase inhibitory activity. The inhibition mechanism of α-glucosidase differed among PAP, SePAP1 and SePAP2, showing dynamic quenching for PAP, while SePAP1 and SePAP2 showed a mixed mode. The enhanced immunomodulatory effect of the selenized products in RAW264.7 cells was related to the increased phosphorylation levels of ERK1 / 2, JNK, p38 and p65, which were involved in the activation of the MAPK and NF-κB pathways. Overall, the selenization process increased the selenium content of the polysaccharide and improved various biological activities of PAP.

[0164] The embodiments of the present invention have been described above, but these embodiments are not limited to the above specific implementation manners. The above specific implementation manners are merely illustrative rather than restrictive. Under the inspiration of this embodiment, those of ordinary skill in the art can also make more forms of equivalent embodiments, all of which fall within the protection scope of this embodiment.

Claims

1. A preparation method of selenium-modified bracken polysaccharide. First, the bracken is successively subjected to crushing, impurity removal, filtration, extraction, and vacuum concentration. Then, the Sevag method is used to deproteinize the concentrated solution. Then, the solution is decolorized and filtered, and ethanol precipitation treatment is carried out to obtain a precipitate. The precipitate is freeze-dried after centrifugal separation to obtain bracken polysaccharide, and then the bracken polysaccharide is selenized to obtain selenium-modified bracken polysaccharide, characterized in that Ultrafiltration separation is carried out on the selenium-modified bracken polysaccharide: Specifically: Ultrafiltration tubes with 100 kDa and 300 kDa are used to continuously ultrafilter the selenium-modified bracken polysaccharide, and it is separated into three components with molecular ranges of <100 kDa, 100 - 300 kDa, and >300 kDa.

2. The preparation method of selenium-modified bracken polysaccharide according to claim 1, characterized in that, The specific process of successively crushing, impurity removing, filtering, extracting, and vacuum concentrating the bracken is as follows: First, a crusher is used to crush the dry bracken, and it is sieved through a 50-mesh sieve to obtain fine powder, removing lipids, pigments, and other fat-soluble impurities; Then, it is filtered, the residue is air-dried, then mixed with water for extraction, and then the extract is concentrated under reduced pressure.

3. The preparation method of a selenium-modified bracken polysaccharide according to claim 2, characterized in that, The fine powder is refluxed with 95% ethanol at 70 °C twice for 3 h to remove lipids, pigments, and other fat-soluble impurities.

4. The preparation method of selenium-modified bracken polysaccharide according to claim 2, characterized in that, The residue is air-dried at 50 °C, then mixed with water at a ratio of 1:30 g / mL, then extracted at 80 °C for 2 h, and the extract is concentrated under reduced pressure at 55 °C.

5. The preparation method of selenium-modified bracken polysaccharide according to claim 1, characterized in that, After deproteinizing the concentrated solution by the Sevag method, macroporous resin decolorizes the above solution at a ratio of resin:solution 1:

25.

6. The preparation method of a selenium-modified bracken polysaccharide according to claim 1, wherein The specific process of ethanol precipitation treatment is: anhydrous ethanol is added to adjust the ethanol concentration of the solution to 76%, and it is stored overnight at 4 °C. The precipitate is freeze-dried after centrifugal separation to obtain bracken polysaccharide.

7. A preparation method of selenium-modified bracken polysaccharide according to claim 1, characterized in that, The process of selenizing the bracken polysaccharide is: Take 500 mg of purified bracken polysaccharide dry powder and add it to 50 mL of HNO3, stir at room temperature until completely dissolved; It is divided into two portions, 700 mg of Na2SeO3 and 800 mg of Na2SeO3 are added respectively, stirred in a 70 °C water bath for 8 hours. After the reaction is completed, the solution is cooled to room temperature, and 5.6% NaHCO3 solution is added to adjust the pH to 7, and finally centrifuged to remove; Dialysis is carried out with a dialysis bag until sodium selenite cannot be detected by the ascorbic acid method. The polysaccharide solution is concentrated and precipitated with ethanol, and then freeze-dried to obtain selenium-modified bracken polysaccharide.

8. A preparation method of selenium-modified bracken polysaccharide according to claim 7, characterized in that, The concentration of HNO3 is 0.05%.

9. A selenium-modified fern polysaccharide, characterized in that, Prepared by the preparation method described in claim 1.

10. Application of selenium-modified bracken polysaccharide, characterized in that, Application in the preparation of food supplements.

Images