Selenized polygonatum polysaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of plant polysaccharide chemical modification, and particularly relates to selenized polygonatum polysaccharide as well as a preparation method and application thereof. The preparation method of the selenized polygonatum polysaccharide comprises the following steps: mixing polygonatum polysaccharide, dilute acid, barium chloride and sodium selenite, and carrying out selenylation reaction to obtain the selenized polygonatum polysaccharide, the mass fraction of the dilute acid is 0.5 to 1.75 percent; the mass ratio of the polygonatum polysaccharide to the barium chloride is 0.1: (0-0.15), and the mass of the barium chloride is not 0. The preparation method provided by the invention has the advantages that the operation is simple, the bioavailability of the polysaccharide can be improved, and the prepared selenized polygonatum sibiricum polysaccharide is high in biological activity; in-vitro experiment results show that the oxidation resistance of polygonatum cyrtonema polysaccharide is improved through selenylation.

Description

Technical Field

[0001] This invention belongs to the field of chemical modification technology of plant polysaccharides, specifically relating to a selenized Polygonatum polysaccharide, its preparation method, and its application. Background Technology

[0002] Polygonatum is the dried rhizome of *Polygonatum kingianum* Coll. et Hemsl., *Polygonatum sibiricum* Red., or *Polygonatum cyrtonema* Hua., all belonging to the Liliaceae family. It is neutral in nature and sweet in taste; it enters the spleen, lung, and kidney meridians; it has the effects of tonifying qi and nourishing yin, strengthening the spleen, moistening the lungs, and benefiting the kidneys; it is used for spleen and stomach qi deficiency, fatigue, stomach yin deficiency, dry mouth and poor appetite, lung deficiency and dry cough, consumptive cough with hemoptysis, deficiency of essence and blood, soreness and weakness of the waist and knees, premature graying of hair, and internal heat and thirst. Polygonatum is a traditional Chinese medicine used both as food and medicine. Modern research shows that the main chemical components of Polygonatum are polysaccharides, steroidal saponins, and flavonoids, etc., which have pharmacological effects such as anti-inflammatory, antibacterial, antitumor, antioxidant, anti-aging, anti-fatigue, hypoglycemic, and hypolipidemic effects. Among them, polysaccharides, a type of biomolecule, are considered to be the most important active ingredients.

[0003] Polysaccharides are natural biological macromolecules with excellent properties (such as bioactivity, biocompatibility, and structural diversity), and are widely used in the food industry. However, natural polysaccharides cannot meet the requirements of the food industry. Chemical modification is an effective, novel, and direct strategy to alter the structure of natural polysaccharides, which can endow them with ideal properties and enhance their potential application value in the food industry.

[0004] Selenium is an essential trace element for humans and animals, playing a vital role in maintaining normal bodily functions and health. It possesses antibacterial, antioxidant, and immune-regulating properties. However, selenium cannot be synthesized by the body and must be absorbed from external sources. Therefore, supplementing with a certain amount of selenium is crucial for humans and animals to prevent various diseases caused by selenium deficiency. However, the selenium content of Polygonatum polysaccharides extracted from Polygonatum is very low, failing to meet human needs, and Polygonatum polysaccharides also exhibit poor antioxidant activity. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a selenized Polygonatum sibiricum polysaccharide, its preparation method, and its applications. The preparation method provided by this invention yields a selenized Polygonatum sibiricum polysaccharide with high selenium content and high antioxidant activity.

[0006] This invention provides a method for preparing selenized Polygonatum polysaccharide, comprising the following steps: Selenization reaction was carried out by mixing Polygonatum polysaccharide, dilute acid, barium chloride and sodium selenite to obtain selenized Polygonatum polysaccharide; The mass fraction of acid in the dilute acid is 0.5-1.75%; The mass ratio of Polygonatum polysaccharide to barium chloride is 0.1:0~0.15, and the mass of barium chloride is not 0.

[0007] Preferably, the mass fraction of the dilute acid is 1~1.5%; the dilute acid is dilute nitric acid.

[0008] Preferably, the mass ratio of Polygonatum polysaccharide to barium chloride is 0.1:0.05~0.1.

[0009] Preferably, the mass ratio of Polygonatum polysaccharide to sodium selenite is 2:1~2.

[0010] Preferably, the selenization reaction is carried out at a temperature of 60-80°C for 5-9 hours.

[0011] Preferably, the Polygonatum polysaccharide is Polygonatum multiflorum polysaccharide.

[0012] Preferably, the preparation method of the Polygonatum polysaccharide includes the following steps: The crude polysaccharide of Polygonatum was loaded onto a DEAE-52 anion exchange column for separation and eluted with water to obtain the Polygonatum polysaccharide.

[0013] Preferably, the preparation method of the crude polysaccharide from Polygonatum odoratum includes the following steps: Polygonatum powder and ethanol solution were mixed for pretreatment, followed by solid-liquid separation to obtain the pretreated solid. The pretreated solid and water are mixed and extracted to obtain an aqueous extract; After concentrating the aqueous extract, starch removal, alcohol precipitation, and protein removal were performed sequentially to obtain crude polysaccharide from Polygonatum odoratum.

[0014] The present invention also provides selenized Polygonatum polysaccharide obtained by the preparation method described above, wherein the selenium content of the selenized Polygonatum polysaccharide is 0.8~1 mg / g.

[0015] The present invention also provides the application of the selenized Polygonatum polysaccharide described in the above technical solution in the preparation of antioxidant drugs.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for preparing selenized Polygonatum polysaccharide, comprising the following steps: mixing Polygonatum polysaccharide, dilute acid, barium chloride and sodium selenite to carry out a selenization reaction to obtain selenized Polygonatum polysaccharide; the mass fraction of the dilute acid is 0.5~1.75%; the mass ratio of Polygonatum polysaccharide to barium chloride is 0.1:0~0.15, and the mass of barium chloride is not 0.

[0017] This invention chemically modifies Polygonatum polysaccharides by adding dilute acid (such as HNO3) to provide an acidic environment, ensuring rapid and complete dissolution of the raw materials and forming a homogeneous reaction system. BaCl2 catalyzes the selenization reaction between Polygonatum polysaccharides and sodium selenite, resulting in selenized polysaccharides with high selenium content. The preparation method provided by this invention has the advantages of simple and stable process and improved polysaccharide bioavailability. The obtained selenized Polygonatum polysaccharides have a high selenium content (0.91±0.0677 mg / g) and strong antioxidant capacity. The main reason may be that during the selenization reaction, the hydroxyl groups on the polysaccharide chains are replaced by selenic acid groups, forming selenate bonds. These selenium-containing groups have stronger reducing and free radical scavenging abilities, introducing new and highly efficient "antioxidant attack sites" for the polysaccharide molecules. The introduction of selenium may also change the folding pattern and three-dimensional structure of the polysaccharide chains, exposing the previously encapsulated active hydroxyl groups, making them more easily contacted by free radicals, thereby enhancing the overall antioxidant activity.

[0018] Test data showed that, through in vitro experiments involving resuscitation, passage, seeding, and drug administration, selenization modification of Polygonatum polysaccharide (PCP) enhanced its protective effect against D-Gal-induced oxidative damage in Caco-2 cells. The optimal concentrations for enhancing the protective effect of selenized PCP were 25, 50, and 100 μg / mL. SOD and MDA were key indicators of oxidative stress levels. The study found that at a concentration of 100 μg / mL, selenized PCP had a stronger effect on the production of MDA and SOD in D-Gal-induced Caco-2 cells than unmodified PCP. Furthermore, at a concentration of 100 μg / mL, selenized PCP exhibited higher antioxidant activity than unmodified PCP. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 DEAE elution curve of crude polysaccharide from Polygonatum odoratum; Figure 2 A standard curve for selenium content determination; Figure 3 and Figure 4 The graph shows the effect of dilute HNO3 concentration on the selenization of Polygonatum cyrtonema polysaccharides. Figure 3 For selenium content, Figure 4 For yield; Figure 5 and Figure 6The graph shows the effect of BaCl2 dosage on the selenization of Polygonatum odoratum polysaccharides. Figure 5 For selenium content, Figure 6 For yield; Figure 7 Infrared spectra of selenized Polygonatum multiflorum polysaccharide and Polygonatum multiflorum polysaccharide; Figure 8 Scanning electron microscope images of selenized Polygonatum polysaccharide and Polygonatum polysaccharide; Figure 9 Figure showing the experimental results of selenized Polygonatum multiflorum polysaccharide Congo red; Figure 10 Figure showing the effect of different concentrations of selenized Polygonatum multiflorum polysaccharide on cell viability; Figure 11 Figure showing the effect of different concentrations of Polygonatum multiflorum polysaccharide on cell viability; Figure 12 Figure showing the protective effect of different concentrations of selenized Polygonatum cyrtonema polysaccharide against D-Gal-induced oxidative damage in Caco-2 cells; Figure 13 and Figure 14 The image shows the results of the antioxidant activity determination of selenium-enriched Polygonatum polysaccharides; among which... Figure 13 For SOD, Figure 14 For MDA. Detailed Implementation

[0021] This invention provides a method for preparing selenized Polygonatum polysaccharide (Se-PCP), comprising the following steps: Selenization reaction was carried out by mixing Polygonatum polysaccharide, dilute acid, barium chloride and sodium selenite to obtain selenized Polygonatum polysaccharide; The mass fraction of the dilute acid is 0.5~1.75%; The mass ratio of Polygonatum polysaccharide to barium chloride is 0.1:0~0.15, and the mass of barium chloride is not 0.

[0022] Unless otherwise specified, all materials and equipment used in this invention are commercially available products in the field.

[0023] In this invention, the Polygonatum polysaccharide is preferably Polygonatum multiflorum polysaccharide.

[0024] In this invention, the preparation method of Polygonatum polysaccharide (PCP) preferably includes the following steps: The crude polysaccharide of Polygonatum was loaded onto a DEAE-52 anion exchange column for separation and eluted with water to obtain Polygonatum polysaccharide (PCP, i.e., water-washed sugar).

[0025] In this invention, the crude polysaccharide of Polygonatum is preferably used in the form of an aqueous solution of crude polysaccharide of Polygonatum, and the concentration of the aqueous solution of crude polysaccharide of Polygonatum is preferably 40 mg / mL; the column volume of the DEAE-52 anion exchange column is about 100 mL; the component obtained by water elution is water-washed sugar PCP. The specific steps for separating Polygonatum polysaccharide from crude polysaccharide of Polygonatum include: 1) adjusting the flow rate to 5 mL / min; 2) dissolving 200 mg of crude polysaccharide in 5 mL of pure water and loading the sample; 3) eluting sequentially with ultrapure water, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L NaCl solutions; 4) after loading the sample, rinsing the column with 1 mol / L NaCl solution to remove residual pigments and other impurities; 5) rinsing with pure water to remove residual NaCl in the column.

[0026] The present invention does not have special requirements for the preparation method of the crude polysaccharide of Polygonatum odoratum. In the specific implementation of the present invention, it is preferred to include the following steps: Polygonatum powder and ethanol solution were mixed for pretreatment, followed by solid-liquid separation to obtain the pretreated solid. The pretreated solid and water are mixed and extracted to obtain an aqueous extract; After concentrating the aqueous extract, starch removal, alcohol precipitation, and protein removal were performed sequentially to obtain crude polysaccharide from Polygonatum odoratum.

[0027] In this invention, the preferred ratio of Polygonatum sibiricum powder to ethanol solution is 1g:10mL; the preferred concentration of the ethanol solution is 85%; the pretreatment is preferably carried out under shaking; the preferred temperature for the pretreatment is room temperature (25°C); and the preferred time is 24 hours. This invention removes low-polarity substances such as fats, pigments, and polyphenols through pretreatment.

[0028] In this invention, the preferred ratio of Polygonatum odoratum powder to water during extraction is 1g:30mL; the preferred extraction temperature is 80℃; the preferred number of extractions is 2; and the preferred extraction time for each extraction is 2h.

[0029] In this invention, the volume of the concentrated solution obtained by concentration is preferably 1 / 6 to 1 / 5 of the volume of the water extract (approximately 6000 mL to 500-600 mL). The starch removal process is preferably performed by mixing the concentrated solution and an α-amylase solution, stirring in a water bath, separating the solids from the liquid, and removing the solids. The preferred ratio of the concentrated solution to the α-amylase solution is 500-600 mL: 200 μL. The α-amylase solution is preferably purchased from Aladdin, CAS number: 9000-90-2. The preferred temperature for the water bath stirring is 65°C, and the preferred time is 1 hour.

[0030] In this invention, after removing starch, a supernatant is obtained. Preferably, the supernatant is mixed with ethanol for alcohol precipitation, and the volume fraction of ethanol in the system obtained by mixing the supernatant and ethanol is preferably 80%. This invention does not have special requirements for the removal of protein; chloroform and n-butanol can be used, and the volume ratio of chloroform to n-butanol is preferably 5:1.

[0031] In this invention, the mass fraction of the dilute acid is preferably 1-1.5%; the dilute acid is preferably dilute nitric acid. The preferred ratio of Polygonatum polysaccharide to dilute nitric acid is 100 mg: 10 mL. The dilute nitric acid concentration described in this invention results in a high yield and high selenium content of selenized Polygonatum polysaccharide without damaging the polysaccharide. Test data shows that the yield and selenium content reach their highest values ​​when the mass fraction of HNO3 is 1%.

[0032] In this invention, barium chloride is used as a catalyst; the preferred mass ratio of Polygonatum polysaccharide to barium chloride is 0.1:0.05~0.1. The amount of barium chloride used in this invention results in a high yield and high selenium content of selenized Polygonatum polysaccharide. Test data shows that, for 100 mg of Polygonatum polysaccharide, the highest selenium content and yield are achieved when the amount of barium chloride is 0.1 g.

[0033] In this invention, the preferred mass ratio of Polygonatum polysaccharide to sodium selenite is 2:1 to 2, more preferably 2:2 (i.e., 1:1). The mass ratio of Polygonatum polysaccharide to sodium selenite described in this invention results in a high yield and high selenium content of the selenized Polygonatum polysaccharide.

[0034] In this invention, the selenization reaction temperature is preferably 60-80℃, specifically 70℃ or 80℃, and the reaction time is preferably 5-9 hours, specifically 7 hours or 8 hours. The selenization reaction temperature and time described in this invention result in high yield and high selenium content of the selenized Polygonatum polysaccharide.

[0035] In this invention, the selenization reaction preferably further includes: terminating the reaction, adjusting the pH of the resulting system to 7-8, and adding sulfate to precipitate Ba. 2+ Solid-liquid separation is performed, and the resulting liquid is dialyzed and then freeze-dried. The pH adjuster is preferably Na₂CO₃; the sulfate is preferably Na₂SO₄, specifically a 0.5 mol / mL Na₂SO₄ solution can be used, preferably in excess; the dialysis is preferably performed using a dialysis bag with a molecular weight cutoff of 500 Da, with ultrapure water, until free selenium ions are undetectable by the ascorbic acid method.

[0036] This invention provides a chemical modification method for plant polysaccharides, which selenizes Polygonatum polysaccharides to improve their biological activity. The resulting selenized Polygonatum polysaccharides have excellent antioxidant activity.

[0037] The present invention also provides selenized Polygonatum polysaccharide obtained by the preparation method described above, wherein the selenium content of the selenized Polygonatum polysaccharide is 0.8~1mg / g, specifically 0.91±0.0677mg / g.

[0038] Compared with inorganic selenium and polysaccharides, the selenized Polygonatum polysaccharide of the present invention has better biological activity.

[0039] The present invention also provides the application of the selenized Polygonatum polysaccharide described in the above technical solution in the preparation of antioxidant drugs.

[0040] In this invention, when the concentration of the selenized Polygonatum polysaccharide is 100 μg / mL, it exhibits excellent antioxidant activity.

[0041] To further illustrate the present invention, the selenized Polygonatum polysaccharide, its preparation method, and its application provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0042] In this embodiment of the invention, the preparation method of crude polysaccharide from Polygonatum odoratum includes the following steps: Weigh 100g of dried Polygonatum odoratum powder into an Erlenmeyer flask, add 10 times the volume (mL / g) of 85% ethanol solution, seal the flask, shake in a constant temperature (room temperature) shaker for 24 h, filter and centrifuge, collect the precipitate and evaporate the ethanol.

[0043] Water extraction: Distilled water was added at a mass:volume ratio of 1:30 (g / mL), and the mixture was extracted twice with distilled water at 80°C, each time for 2 hours (using an electric stirrer). The mixture was filtered, and the supernatant was concentrated under reduced pressure using a rotary evaporator.

[0044] Starch removal: Add 200 μL of α-amylase solution (Aladdin, CAS No.: 9000-90-2) with a concentration of 46 U / mL to the concentrated solution (500~600mL) using a pipette, stir in a water bath at 65℃ for 1h to remove starch, centrifuge to remove precipitate (immediately place in boiling water for 10min after the reaction is complete to inactivate enzyme), and obtain supernatant.

[0045] Alcohol precipitation: Add ethanol to the supernatant until the final ethanol concentration is 80% (v / v), and precipitate for 24 h. Collect the precipitate by centrifugation, air dry the precipitate, and then reconstitute it with water (heat in a water bath at 65°C and stir until dissolved). After reconstitution, let it stand until the precipitate precipitates, centrifuge to remove the precipitate, and obtain the polysaccharide solution.

[0046] Protein removal (Sevag method): The polysaccharide solution, chloroform, and n-butanol were mixed at a volume ratio of 30:5:1 and stirred at room temperature for 2 hours. After centrifugation, the supernatant was retained. Subsequently, the mixture was dialyzed (molecular weight cutoff 3500 Da) and lyophilized to obtain crude polysaccharide of Polygonatum odoratum.

[0047] Example 1: Preparation of Selenized Polygonatum Polysaccharide The crude polysaccharide of Polygonatum odoratum was loaded onto a DEAE-52 anion exchange column (200 mg of crude polysaccharide was prepared into a solution with a concentration of 40 mg / mL for loading). The column was eluted in stages with 200 mL of ultrapure water, 0.1 M NaCl solution, 0.2 M NaCl solution, 0.3 M NaCl solution, 0.4 M NaCl solution, and 0.5 M NaCl solution. The washed sugar was collected, which is Polygonatum odoratum polysaccharide (denoted as PCP).

[0048] 100 mg of washed sugar (PCP) was added to 10 mL of 1% (w / w) dilute HNO3 solution, followed by 0.1 g of BaCl2. After dissolution, Na2SeO3 was added at a mass ratio of 1:1 (PCP to Na2SeO3). The selenization reaction was carried out at 80 °C for 7 h. After terminating the reaction, the mixture was cooled, and the pH was adjusted to 7-8 with anhydrous sodium carbonate. An excess of 0.5 mol / mL Na2SO4 solution was added to remove BaCl2. 2+ The reaction solution was centrifuged, filtered, dialyzed (with a molecular weight cutoff of 500 Da), and freeze-dried to obtain selenized Polygonatum polysaccharide (denoted as Se-PCP).

[0049] Caco-2 cell culture (1) Cell resuscitation Following the "slow freeze, fast thaw" principle, Caco-2 cells preserved in liquid nitrogen were rapidly placed in a 37°C water bath and thawed by rotating clockwise. The thawed cell culture was then quickly transferred to 1.5 mL EP tubes and centrifuged at 1000 rpm and 4°C for 5 min. The supernatant was discarded, and the cells were resuspended in fresh complete culture medium in a cell culture flask and cultured in a cell culture incubator at 37°C, 5% CO2, and 80% relative humidity.

[0050] (2) Cell passage Once the cells have grown to approximately 80% confluence, discard the complete culture medium. Rinse three times with PBS to remove cell debris and secretions. Add 2 mL of trypsin digestion solution and digest in an incubator for 4–6 min. When the cells become rounded and begin to detach, quickly add 1 mL of complete culture medium to stop the digestion. Gently pipette the cells to disperse them completely. Transfer the digested cells to centrifuge tubes and centrifuge at 1000 rpm and 4°C for 5 min. Discard the supernatant, resuspend the cells in fresh complete culture medium, and transfer them to new culture flasks at a 1:3 ratio for further culture.

[0051] (3) Cell cryopreservation Once the cells have grown to approximately 90% and are in good condition, discard the complete culture medium. Rinse three times with PBS to remove cell debris and secretions. Add 2 mL of trypsin digestion solution and digest in an incubator for 4–6 min. When the cells become rounded and begin to detach, quickly add 1 mL of complete culture medium to stop digestion. Gently pipette the cells to disperse them completely. Transfer the digested cells to centrifuge tubes and centrifuge at 1000 rpm and 4°C for 5 min. Discard the supernatant, add 1 mL of serum-free cryopreservation buffer, resuspend the cells, transfer to cryovials, and store in a -80°C temperature-programmed freezer for 24 h. The following day, transfer to a liquid nitrogen container for storage.

[0052] Optimization, identification, and performance analysis of selenized Polygonatum multiflorum polysaccharide processing. Experimental methods 1. Preparation of Polygonatum polysaccharide water-washed sugar (PCP) The crude polysaccharide of Polygonatum odoratum was initially separated using an anion exchange column. Separate elution was performed sequentially with ultrapure water, 0.1 M NaCl solution, 0.2 M NaCl solution, 0.3 M NaCl solution, 0.4 M NaCl solution, and 0.5 M NaCl solution to obtain the washed polygonatum odoratum sugar fraction and elution curve. After dialyzing with running water using a dialysis bag with a molecular weight cutoff of 3500 Da, the solution was further dialyzed with deionized water. The concentrate was then freeze-dried under vacuum.

[0053] 2. Orthogonal optimization of the preparation process of selenized Polygonatum multiflorum polysaccharide Step 1: Preparation of selenized Polygonatum multiflorum polysaccharide (Se-PCP) The crude polysaccharide from *Polygonatum cyrtonema* was loaded onto a DEAE-52 anion exchange column for separation, and the washed sugar was collected. 100 mg of the washed sugar was added to 10 mL of 1% (w / w) dilute HNO3 solution, followed by 0.1 g of BaCl2. After dissolution, Na2SeO3 was added at a mass ratio of washed sugar to Na2SeO3 of 1:1. A selenization reaction was carried out at 80℃ for 7 h. After terminating the reaction, the mixture was cooled, and the pH was adjusted to 7-8 with anhydrous sodium carbonate. An excess of 0.5 mol / mL Na2SO4 solution was added to remove BaCl2. 2+ The reaction solution is centrifuged, filtered, dialyzed (with a molecular weight cutoff of 500 Da), and freeze-dried to obtain the final product.

[0054] Step 2: Determination of selenium content (1) Drawing the standard curve Accurately weigh 10 mg of Na₂SeO₃ and dissolve it in 25 mL of deionized water to obtain a standard solution with a selenium content of 182.6 μg / mL. Accurately pipette 0.05, 0.1, 0.2, 0.4, and 0.8 mL of the standard solution into 20 mL volumetric flasks, add 2 mL of 2wt% o-phenylenediamine solution, add deionized water, adjust the pH to approximately 2, and then dilute to the mark. Incubate in the dark for approximately 1 hour, then extract with 5 mL of toluene. Remove the organic phase (toluene) and measure the absorbance of each standard reaction solution at 334 nm. Plot a standard curve with selenium content (μg / mL) on the x-axis and absorbance on the y-axis.

[0055] (2) Determination of selenium content in the test sample Take 20 mg of Se-PCP and place it in a test tube. Slowly add 2 mL of concentrated HNO3 (68% by mass) and heat with an alcohol lamp to digest. When no orange (yellow) fumes are released, cool the solution and add 5 mL of concentrated hydrochloric acid with a concentration of 6 mol / L. Continue heating with an alcohol lamp until no large amount of white fumes are emitted and the solution is nearly colorless. Cool the reaction solution and pour it into a volumetric flask to make up to 10 mL. This gives you the test solution.

[0056] Step 3: Single-factor experimental design (1) Effect of dilute HNO3 concentration on selenization of Polygonatum polysaccharides The selenization reaction was carried out by adding a catalyst (0.1 g BaCl2) to dilute HNO3 at mass fractions of 0.5%, 0.75%, 1%, 1.5%, and 1.75%. A concentration gradient reagent was used to systematically investigate the effect of dilute HNO3 concentration on the selenization of polysaccharides.

[0057] (2) Effect of BaCl2 amount on selenization of Polygonatum polysaccharides The amounts of BaCl2 were set to 0 g, 0.05 g, 0.1 g, 0.125 g, and 0.15 g. The effect of BaCl2 amount on polysaccharide selenization was investigated by controlling the gradient amount.

[0058] Step 4: Orthogonal experimental design The mass ratio of Polygonatum polysaccharide to Na2SeO3, reaction time, and reaction temperature were used as factors to investigate the study. An orthogonal design was used, with three factors and three levels selected. Detailed factor and level codes are shown in Table 1.

[0059] Table 1. Factors and Levels in Orthogonal Design Analysis

[0060] Step 5: Verification Experiment The results of the orthogonal experiment were verified by repeating the experiment three times.

[0061] 3. Characterization and analysis of selenium-enriched Polygonatum polysaccharides (1) Infrared spectroscopy analysis The characteristic absorption peaks of Polygonatum cyrtonema polysaccharide (washed sugar) and selenized Polygonatum cyrtonema polysaccharide were determined by Fourier transform infrared spectroscopy (FT-IR). 2 mg of Polygonatum cyrtonema polysaccharide and 200 mg of selenized Polygonatum cyrtonema polysaccharide were dried in an oven at 105℃ for 6 h. After drying, the samples were ground into powder, compressed into tablets using KBr, and then pressed into 4000–400 cm⁻¹. -1 Perform infrared detection.

[0062] (2) Scanning electron microscopy observation of polysaccharide microstructure Carbon conductive double-sided tape was securely adhered to the metal support of the microscope. The sample to be tested was then evenly laid on the surface of the fixed carbon conductive double-sided tape. A clean airflow was used to blow away any excess sample particles that were not firmly bonded to the tape surface. Subsequently, a thin gold coating was uniformly deposited on the conductive tape using an ion sputtering system for observation.

[0063] (3) Congo Red Experiment Two mL (2.5 mg / mL) of Polygonatum multiflorum polysaccharide and two mL (80 μmol / L) of selenized Polygonatum multiflorum polysaccharide were thoroughly mixed with two mL of Congo red solution. Different volumes of 1 mol / L NaOH solution were gradually added to adjust the mixture to different NaOH concentrations (0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, and 0.5 mol / L). The maximum absorption wavelength of the solution at different NaOH concentrations was recorded by ultraviolet scanning (200–600 nm).

[0064] 4. Screening of antioxidant activity of different concentrations of selenium-enriched Polygonatum polysaccharides D-galacturonic acid (D-Gal) can induce oxidative stress in cells, causing cell damage. Cells were treated with different concentration gradients of D-Gal for 24 h, and the modeling concentration was determined with the amount of MDA produced as a reference. According to previous studies, the modeling concentration of D-Gal was determined to be 300 mM, and the concentration of the positive control drug VC (ascorbic acid) was 2 mM.

[0065] Step 1: Procedure for operating the Caco-2 cell oxidative damage model Take cells in the logarithmic growth phase and adjust the cell density to 6 × 10⁻⁶. 4Cells were seeded at a density of 100 μL / well in 96-well plates and cultured for 24 h until cell adhesion was achieved. Experimental groups: Blank group: complete culture medium was added to each well; Control group: Caco-2 cells were cultured normally in complete culture medium without any treatment; D-Gal group: D-Gal was prepared to a final concentration of 300 mM. Each group had 6 replicates, with 100 μL of the respective culture medium added to each well. After completing the above steps, the plates were placed in a cell culture incubator and cultured for 24 h. 10 μL of CCK-8 was added to each experimental well, and the plates were incubated for another two hours. The OD value was measured at 450 nm using a microplate reader. Cell viability was calculated using the following formula: ; Among them, OD (dosage): has absorbance values ​​for wells containing cells, culture medium, CCK-8 solution and D-Gal; OD (blank): The absorbance value of the well containing culture medium, CCK-8 solution, and no cells; OD (0 drug): The absorbance value of wells containing cells, culture medium, CCK-8 solution, but no drug solution.

[0066] Step 2: Effects of selenized Polygonatum polysaccharide (Se-PCP) and different concentrations of Polygonatum polysaccharide on cell viability Control group: Caco-2 cells were cultured normally in complete culture medium without any treatment; Polysaccharide group: Selenized Polygonatum polysaccharide and Polygonatum polysaccharide were cultured at final concentrations of 25, 50, 100, 200, and 400 μg / mL for 24 h, with 6 replicates for each group. Follow the procedure in step 1 and calculate cell viability.

[0067] Step 3: Protective effect of Se-PCP and different concentrations of PCP on D-Gal-induced oxidative damage in Caco-2 cells. Cells were treated using the same modeling method as in step 1. After incubation for 24 h, the D-Gal-containing medium in the Model group was discarded, and complete medium was added for another 24 h of culture. In the polysaccharide group, selenized Polygonatum odoratum polysaccharide and Polygonatum multiflorum polysaccharide were treated at concentrations of 25, 50, 100, 200, and 400 μg / mL, respectively, and cultured for 24 h. Six replicates were performed for each group, and cell viability was calculated following the procedure in step 1.

[0068] Step 4: Effects of different concentrations of Se-PCP on SOD and MDA in D-Gal-induced Caco-2 cells To further evaluate the protective effects of different concentrations of selenized Polygonatum sibiricum polysaccharide and Polygonatum multiflorum polysaccharide (25, 50, 100 μg / mL) against D-Gal-induced oxidative damage, the contents of SOD and MDA in the culture medium were measured. Cells were treated with the positive control concentration (2 mM) and as described in step 3, and the contents of SOD and MDA were measured using commercial SOD and MDA kits according to the manufacturer's instructions.

[0069] 5. Statistical methods The data was organized using Excel 2019, and plotted using Origin 2024 and GraphPad 8.0, followed by one-way ANOVA. P Values ​​less than 0.05 are considered statistically significant and plotted.

[0070] Experimental results 1. Extraction of Polygonatum odoratum from washed sugar Figure 1 The elution curve for DEAE-52.

[0071] like Figure 1 The elution results shown indicate that the elution solution obtained by the DEAE-52 cellulose separation column showed a clear peak when eluted with ultrapure water. Therefore, the eluent corresponding to the number of tubes with the peak was collected, combined, concentrated, and freeze-dried to obtain Polygonatum polysaccharide, which was named PCP.

[0072] 2. Determination of selenium content Standard curve results are as follows Figure 2 Plotting selenium content on the x-axis and absorbance on the y-axis, the linear regression equation is y = 0.5984x + 0.0413, R0. 2 = 0.9906, which is quite accurate.

[0073] 3. Results of single-factor experimental design (1) Effect of dilute HNO3 concentration on selenization of Polygonatum polysaccharides Figure 3 The effect of HNO3 concentration on selenium content. Figure 4 The effect of HNO3 concentration on yield (weight after freeze-drying / weight of raw sugar).

[0074] like Figure 3 , 4 As shown, with the increase of dilute nitric acid concentration, the yield and selenium content of selenized Polygonatum polysaccharide generally showed a trend of first increasing and then decreasing. When the dilute nitric acid concentration was 1%, the selenium content reached its highest value, and the yield was also the highest. Therefore, a dilute nitric acid concentration of 1% was selected.

[0075] (2) Effect of barium chloride content on selenization of Polygonatum polysaccharides Figure 5The effect of BaCl2 weight on selenium content, Figure 6 The effect of BaCl2 weight on yield.

[0076] like Figure 5 , 6 The results showed that as the amount of barium chloride increased, the selenium content reached its highest level and the yield was also the highest when the amount of barium chloride was 0.1g. Therefore, the amount of barium chloride selected was 0.1g.

[0077] 4. Orthogonal experimental design and data analysis As shown in Tables 2, 3, and 4, R1B > R1C > R1A, and the p-values ​​for yields of B and C are <0.05, indicating a significant difference, while the p-value for A is >0.05. However, A, B, and C have no significant effect on selenium content. The comparison shows that the third level of temperature is the best, as is the third level of factor C. The differences among the three levels of factor A are not significant; too low a temperature leads to incomplete reaction, while too high a temperature may cause some polysaccharide chains to break and disrupt the stable structure of organoselenium. Therefore, the second level of A and the third level of B were selected.

[0078] Table 2 Results of the orthogonal experiment

[0079] Note: After retesting, combination 3 (A1B3C3) showed poor process stability (see Table 5 for verification experiments), therefore it was not selected as the preferred condition.

[0080] Table 3. Analysis of variance of yield

[0081] Table 4. Analysis of variance of selenium content

[0082] Analysis of range and variance showed that reaction temperature (B) and material ratio (C) had a significant impact on product yield (P<0.05), making them key factors; while reaction time (A) had no significant impact, making it a non-key factor. Factors B (reaction temperature) and C (material ratio) were statistically significant. This means that temperature and material ratio are key factors affecting yield. Factor A was not significant, indicating that the impact of time on yield was relatively small at the 5h, 7h, and 9h levels, and the differences may be due to experimental error. Therefore, time is not a key factor and can be selected within a certain range.

[0083] Orthogonal range analysis yielded a theoretical combination A1B3C3 (5 h, 80℃, 2:2), but the verification results (Table 5) showed extremely large yield fluctuations (RSD = 18.2%) and insufficient process stability. Under the optimal levels of the key factors (80℃, 2:2), the reaction time was optimized to a more robust 7 h, resulting in the final process A2B3C3. As confirmed by the verification experiments in Table 6, this combination exhibits stable yields (RSD = 1.46%), small selenium content fluctuations, and good process reproducibility. Appropriately extending the reaction time (from 5 h to 7 h) helps ensure a more complete and thorough reaction, improving process reproducibility and stability, and reducing batch-to-batch fluctuations caused by insufficient reaction time. After locking in the optimal levels of the key factors (B and C), the selection of non-key factor (A) should prioritize process robustness. The final optimized conditions of this invention are: a mass ratio of Polygonatum odoratum polysaccharide to Na2SeO3 of 2:2, a reaction time of 7 h, and a reaction temperature of 80℃.

[0084] Table 5. Results of the A1B3C3 verification experiment

[0085] Table 6. Results of the A2B3C3 Verification Experiment

[0086] 5. Infrared spectral analysis of selenized Polygonatum polysaccharides Figure 7 Infrared spectra of Se-PCP (selenized polysaccharide) and PCP (washed sugar).

[0087] Infrared spectral analysis of selenized Polygonatum polysaccharides is shown in [reference needed]. Figure 7 PCP 3380 cm -1 The strong absorption peak at 2938 cm⁻¹ is mainly due to the stretching vibration of OH groups. -1 These are the absorption peaks of CH, and both are characteristic absorption peaks of the polysaccharides in them. 1637cm -1 1373 cm -1 1132 cm -1 and 1027 cm -1 The absorption peaks at 933 and 811 cm⁻¹ are attributed to stretching vibrations of C=O, COOH, C-OH, and COC, respectively, indicating the presence of uronic acid and pyranose rings. Furthermore, the absorption peaks at 933 and 811 cm⁻¹ are also significant. -1 The peak at 597 cm⁻¹ is attributed to the furanose ring with a β-glycosidic bond. -1 The absorption peak at that point indicates mannose.

[0088] 620 cm -1 881 cm -1 and 1029 cm -1At these locations, the stretching vibrations are observed in O-Se-C, Se=O, and O-Se-O, respectively. Furthermore, the peak of the pyran ring undergoes a redshift, from 1027 cm⁻¹. -1 Moved to 1145 cm -1 This indicates that the selenization modification of Polygonatum polysaccharides was successful.

[0089] 6. Scanning electron microscopy analysis Figure 8 Scanning electron microscope images of PCP (A) and Se-PCP (B) at different magnifications.

[0090] like Figure 8 The images show the microstructure of PCP and Se-PCP observed under scanning electron microscopy at different magnifications. As can be seen from the images, natural PCP has a smooth surface with many pores. Selenization treatment of PCP (Se-PCP) disrupts the original smooth and compact surface of the polysaccharide, and selenization also degrades PCP into smaller fragments.

[0091] 7. Results of the Congo Red Experiment Figure 9 The graph shows the maximum absorption wavelength of Se-PCP under different concentrations of NaOH.

[0092] like Figure 9 As shown, the maximum absorption wavelength of the polysaccharide sample red-shifted as the NaOH solution concentration increased from 0 to 0.5 M. This indicates the presence of a triple helix conformation in the polysaccharide sample. The selenized polysaccharide showed a higher degree of red-shift than the original polysaccharide sample, indicating that selenization makes the polysaccharide structure more stable. Furthermore, a certain number of selenic acid groups may lead to a highly ordered chain conformation, which remains stable even in a strongly alkaline environment.

[0093] 8. Effects of different concentrations of selenized Polygonatum polysaccharide (Se-PCP) and Polygonatum polysaccharide (PCP) on cell viability Figure 10 This is a graph showing the effect of Se-PCP on Caco-2 cell viability. Figure 11 The figure shows the effect of Polygonatum multiflorum polysaccharide on the viability of Caco-2 cells.

[0094] like Figure 10 and 11 As shown, within the concentration range of 25–400 μg / mL for selenized Polygonatum polysaccharide and Polygonatum multiflorum polysaccharide, polysaccharide intervention did not significantly inhibit cell viability.

[0095] 9. Protective effects of different concentrations of Se-PCP and PCP on D-Gal-induced oxidative damage in Caco-2 cells. Figure 12 Figure 1 shows the protective effects of different concentrations of Se-PCP and PCP against D-Gal-induced oxidative damage in Caco-2 cells and a comparative figure.

[0096] like Figure 12 As shown, both selenized Polygonatum polysaccharide and Polygonatum polysaccharide can promote cell viability compared with the model group, and the effect of selenization is better than that of Polygonatum polysaccharide. Therefore, considering all factors, the dosage of selenized Polygonatum polysaccharide was determined to be 25, 50, and 100 μg / mL in subsequent experiments, and this dosage was used to affect MDA and SOD.

[0097] 10. Effects of different concentrations of Se-PCP and PCP on SOD (superoxide dismutase) and MDA (malondialdehyde) in D-Gal-induced Caco-2 cells. Figure 13 The effect of different concentrations of Se-PCP and PCP on D-Gal-induced SOD in Caco-2 cells; Figure 14 The effects of different concentrations of Se-PCP and PCP on D-Gal-induced MDA in Caco-2 cells were investigated; among them, compared with the blank control group, p< 0.05, p< 0.01, p< 0.001, p< 0.0001.

[0098] like Figure 13 and 14 As shown, compared with the control group, D-Gal significantly reduced SOD levels and increased MDA levels, indicating that D-Gal caused oxidative damage to Caco-2 cells. Selenized Polygonatum polysaccharide at a concentration of 100 μg / mL showed high antioxidant activity (mean SOD: 26.06 U / mgprot, MDA: 8.16 nmol / mgprot), which was superior to Polygonatum polysaccharide (mean SOD: 21.55 U / mgprot, MDA: 8.58 nmol / mgprot).

[0099] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on the present invention without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing selenized Polygonatum polysaccharide, characterized in that, Includes the following steps: Selenization reaction was carried out by mixing Polygonatum polysaccharide, dilute acid, barium chloride and sodium selenite to obtain selenized Polygonatum polysaccharide; The mass fraction of acid in the dilute acid is 0.5-1.75%; The mass ratio of Polygonatum polysaccharide to barium chloride is 0.1:0~0.15, and the mass of barium chloride is not 0.

2. The preparation method according to claim 1, characterized in that, The mass fraction of the dilute acid is 1~1.5%; the dilute acid is dilute nitric acid.

3. The preparation method according to claim 1 or 2, characterized in that, The mass ratio of Polygonatum polysaccharide to barium chloride is 0.1:0.05~0.

1.

4. The preparation method according to claim 1 or 2, characterized in that, The mass ratio of Polygonatum polysaccharide to sodium selenite is 2:1~2.

5. The preparation method according to claim 1, characterized in that, The selenization reaction is carried out at a temperature of 60-80°C for 5-9 hours.

6. The preparation method according to claim 1, characterized in that, The Polygonatum polysaccharide mentioned is Polygonatum multiflorum polysaccharide.

7. The preparation method according to claim 1 or 6, characterized in that, The preparation method of the Polygonatum polysaccharide includes the following steps: The crude polysaccharide of Polygonatum was loaded onto a DEAE-52 anion exchange column for separation and eluted with water to obtain the Polygonatum polysaccharide.

8. The preparation method according to claim 7, characterized in that, The preparation method of the crude polysaccharide from Polygonatum odoratum includes the following steps: Polygonatum powder and ethanol solution were mixed for pretreatment, followed by solid-liquid separation to obtain the pretreated solid. The pretreated solid and water are mixed and extracted to obtain an aqueous extract; After concentrating the aqueous extract, starch removal, alcohol precipitation, and protein removal were performed sequentially to obtain crude polysaccharide from Polygonatum odoratum.

9. The selenized Polygonatum polysaccharide obtained by the preparation method according to any one of claims 1 to 8, characterized in that, The selenium content of the selenized Polygonatum polysaccharide is 0.8~1 mg / g.

10. The use of the selenized Polygonatum polysaccharide according to claim 9 in the preparation of antioxidant drugs.

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