Preparation method of selenium xiazuo tea polysaccharide and application thereof

Abstract

This invention belongs to the field of tea polysaccharide extraction technology, specifically disclosing a method for preparing selenized summer and autumn tea polysaccharides and their application. The preparation method includes the following steps: S1, raw material pretreatment to obtain tea powder; S2, adding the tea powder to a eutectic solvent system for extraction, supplemented by ultrasonic extraction, to obtain a summer and autumn tea polysaccharide extract; S3, separation and purification to obtain summer and autumn tea polysaccharides; S4, selenization modification: dissolving the summer and autumn tea polysaccharides obtained in S3 in nitric acid solution, adding sodium selenite, stirring, and cooling after the reaction is complete, adjusting the pH value to 5-6 to obtain a selenized summer and autumn tea polysaccharide reaction solution; S5, product purification to obtain selenized summer and autumn tea polysaccharides. This invention uses the above-mentioned method for preparing selenized summer and autumn tea polysaccharides and their application, employing a eutectic solvent to extract summer and autumn tea polysaccharides and performing selenization modification to improve the bioactivity of summer and autumn tea polysaccharides, especially enhancing their inhibitory effect on glucose and lipid metabolism enzymes.

Description

Technical Field

[0001] This invention relates to the field of tea polysaccharide extraction technology, and in particular to a method for preparing selenized summer and autumn tea polysaccharides and their applications. Background Technology

[0002] Summer and autumn teas, accounting for approximately 60% of the total annual tea production, are a major component of tea output. However, due to environmental factors affecting tea trees during summer and autumn, these teas tend to have lower levels of amino acids and higher levels of polyphenols, resulting in a bitter taste and inferior quality compared to spring tea. This leads to a significant waste of tea resources as large quantities of summer and autumn tea are discarded. Optimizing summer and autumn tea processing techniques to improve their taste and nutritional value would greatly enhance resource utilization, increase tea farmers' income, extend the tea industry chain, and promote high-quality tea development.

[0003] Tea polysaccharides (TP) are a general term for acidic polysaccharides or acidic glycoproteins that are tightly bound to proteins. Various factors, such as tea variety, fermentation level, and tea processing techniques, can influence the monosaccharide composition of tea polysaccharides. Selenium (Se), an essential trace element for the human body, has multiple physiological functions, such as reducing cancer incidence, maintaining the normal state of cells and tissues, and promoting the functioning of the immune and nervous systems. In nature, selenium mainly exists in inorganic forms such as selenates, selenites, or elemental selenium, which have low bioavailability. Combining selenium with tea polysaccharides is safer and more easily absorbed than inorganic selenium or polysaccharides, and its effects in anti-tumor, antioxidant, and immune-enhancing properties are superior to polysaccharides, showing broad application prospects and research value. Therefore, exploring the optimal conditions for selenization processes has become a crucial step in the preparation of selenized polysaccharides.

[0004] In existing technologies, polysaccharide extraction methods mainly employ water extraction and alcohol precipitation, acid-base extraction, and enzymatic hydrolysis, most of which suffer from low extraction efficiency, high energy consumption, and significant loss of active ingredients. Selenium polysaccharide synthesis methods mainly include plant transformation, microbial transformation, and chemical synthesis. The first two methods, namely bioaccumulation, utilize the transformation of inorganic selenium by plants or fungi to store selenium in the form of polysaccharides. Chemical synthesis involves introducing inorganic selenium as a carrier into polysaccharides, controlling the location and amount of selenium introduction through different chemical forms and synthesis conditions to obtain specific selenium polysaccharides. However, the optimization of selenization modification process parameters is not yet perfect, affecting the yield and quality of selenized polysaccharides. Therefore, developing an efficient and environmentally friendly technology for the extraction and selenization modification of polysaccharides from summer and autumn tea is of significant practical importance. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing selenized summer and autumn tea polysaccharides and their application. The method involves extracting summer and autumn tea polysaccharides using a eutectic solvent and then modifying them with selenization to improve their bioactivity, especially enhancing their inhibitory effect on glucose and lipid metabolism enzymes.

[0006] To achieve the above objectives, the present invention provides a method for preparing selenized summer and autumn tea polysaccharides, comprising the following steps: S1. Raw material pretreatment: Select fresh summer and autumn tea leaves, wash, dry, crush, decolorize, and grind to obtain tea powder; S2. Add the tea powder obtained in S1 to a eutectic solvent system composed of choline chloride and urea in a molar ratio of 1:4, extract, and then perform ultrasonic extraction to obtain a summer and autumn tea polysaccharide extract. S3. Separation and purification: The extract obtained in S2 was centrifuged, adsorbed by macroporous resin, eluted with ethanol, concentrated and freeze-dried to obtain summer and autumn tea polysaccharides; among which, the macroporous resin was D101 type macroporous adsorption resin. S4, Selenization modification: Dissolve the summer and autumn tea polysaccharides obtained in S3 in nitric acid solution, add sodium selenite, stir, and after the reaction is completed, cool and adjust the pH value to 5-6 to obtain the selenized summer and autumn tea polysaccharide reaction solution. S5. Product purification: The selenized summer and autumn tea polysaccharide reaction solution obtained in S4 was subjected to dialysis, vacuum concentration and freeze drying to obtain selenized summer and autumn tea polysaccharide.

[0007] Preferably, in step S1, the pulverized tea leaves are decolorized with 4 times their volume of 95% ethanol for 12 hours.

[0008] Preferably, in S2, the solid-liquid ratio of tea powder to the eutectic solvent system is 1:40.

[0009] Preferably, in S2, the water content of the eutectic solvent system is 50%, and the extraction is performed at 70°C for 30 minutes, while simultaneously using 200W ultrasound.

[0010] Preferably, in S3, after centrifugation, the supernatant is collected, protein impurities are removed by the Sevage method, the filtrates are combined, and 4 times the volume of anhydrous ethanol is added for alcohol precipitation. The precipitate is centrifuged and freeze-dried to obtain summer and autumn tea polysaccharides.

[0011] The preferred method, Sevage, is as follows: Add Sevage reagent to the supernatant at a volume ratio of 1:5, place in a shaker and continue the reaction. Centrifuge, remove the lower organic phase and the intermediate denatured protein gel, retain the supernatant, repeat several times, and combine all supernatants. The Sevage reagent is a mixture of chloroform and n-butanol, with a volume ratio of chloroform to n-butanol of 4:1.

[0012] Preferably, in S4, the concentration of summer and autumn tea polysaccharides in nitric acid solution is 10 mg / mL, the reaction time is 4-8 h, the reaction temperature is 60-80 °C, the nitric acid concentration is 0.3-0.7%, and the mass ratio of sodium selenite to summer and autumn tea polysaccharides is 0.8-1.2:1.

[0013] Preferably, in S4, the concentration of summer and autumn tea polysaccharides in nitric acid solution is 10 mg / mL, the reaction time is 5.67 h, the reaction temperature is 64 °C, the nitric acid concentration is 0.41%, and the mass ratio of sodium selenite to summer and autumn tea polysaccharides is 0.90:1.

[0014] This invention also provides the application of selenized summer and autumn tea polysaccharides in the preparation of antioxidants.

[0015] This invention also provides the application of selenized summer and autumn tea polysaccharides in the preparation of glucose and lipid metabolism regulators.

[0016] Therefore, the present invention employs the above-mentioned method for preparing selenized summer and autumn tea polysaccharides and its application, with the following beneficial effects: High extraction efficiency: The deep eutectic solvent (DES) is combined with ultrasonic extraction technology. DES is a low-melting-point mixture formed by hydrogen bond donors and hydrogen bond acceptors in a certain proportion. It can destroy the hydrogen bond network and hydrophobic interaction of tea polysaccharides in tea cells, reduce its solubility barrier, achieve the purpose of full extraction, and significantly improve the yield of tea polysaccharides. Environmentally friendly and energy-saving: The eutectic solvent can be recycled and reused multiple times while maintaining high extraction efficiency, reducing solvent consumption costs and making the extraction process green and environmentally friendly. Excellent selenization effect: The optimal selenization process parameters were obtained through response surface methodology, and the selenium content in selenized summer and autumn tea polysaccharides can reach 6.85±0.07mg / g; Strong bioactivity: Selenization significantly enhances the antioxidant activity and glucose and lipid metabolism enzyme inhibition of summer and autumn tea polysaccharides. Selenized summer and autumn tea polysaccharides have significant scavenging effects on ABTS free radicals and DPPH free radicals, and have better inhibitory ability on α-amylase, α-glucosidase and pancreatic lipase. With broad application prospects, the prepared selenized summer and autumn tea polysaccharides can be used in functional foods, health products, pharmaceuticals and other fields, and have good market prospects and economic benefits.

[0017] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0018] Figure 1 This is a standard curve of selenium content from Embodiment 2 of the present invention; Figure 2This is a product diagram of Embodiment 2 of the present invention, wherein (a) is the dialysis stock solution, (b) is the freeze-dried finished product, and (c) is the freeze-dried and peeled finished product; Figure 3 This is a graph showing the effect of reaction time on selenium content and yield in Example 2 of the present invention; Figure 4 This is a graph showing the reaction temperature results of Embodiment 2 of the present invention; Figure 5 This is a graph showing the nitric acid concentration results of Example 2 of the present invention; Figure 6 This is a graph showing the mass ratio results of Embodiment 2 of the present invention; Figure 7 This is a graph showing the interaction between reaction time and nitric acid concentration in Example 2 of the present invention, where (a) is a response graph and (b) is a contour plot; Figure 8 This is a graph showing the interaction between reaction temperature and nitric acid concentration in Example 2 of the present invention, where (a) is a response graph and (b) is a contour plot; Figure 9 This is a graph showing the interaction between reaction temperature and mass ratio in Embodiment 2 of the present invention, where (a) is a response graph and (b) is a contour plot. Figure 10 This is the total sugar standard curve diagram of Example 3 of the present invention; Figure 11 This is a graph showing the ABTS free radical scavenging rate results of an embodiment of the present invention; Figure 12 This is a graph showing the DPPH free radical scavenging rate results of an embodiment of the present invention; Figure 13 This is a graph showing the α-glucosidase inhibition rate results of an embodiment of the present invention; Figure 14 This is a graph showing the α-amylase inhibition rate results of an embodiment of the present invention; Figure 15 This is a graph showing the pancreatic lipase inhibition rate results of an embodiment of the present invention. Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0021] Example 1 A polysaccharide from summer and autumn tea, the preparation method of which includes the following steps: Using dried fresh leaves of Zhonghuang No. 1 summer and autumn tea as raw material, the sample was accurately weighed and processed by a swing pulverizer for 30 seconds. Four times the volume of 95% ethanol was added for decolorization, and the mixture was allowed to stand at room temperature for 12 hours. After decolorization, the supernatant was removed, the precipitate was collected and dried at room temperature in a fume hood, ground into powder, and then sealed and stored at -20℃ for later use.

[0022] Choline chloride (HBA) and urea (HBD) were accurately weighed at a molar ratio of 1:4, mixed, and stirred continuously in a 90℃ constant temperature water bath for 40 min until a homogeneous and transparent liquid was formed, thus obtaining the DES system. Pretreated tea powder was added to the DES system at a solid-liquid ratio of 1:40, and extraction was performed at 70℃ for 30 min. The water content of the DES system was set to 50%, and extraction was enhanced by 200W ultrasonication. After centrifugation, the supernatant was collected, and protein impurities were removed using the Sevage method. The filtrates were combined, and 4 volumes of anhydrous ethanol were added for alcohol precipitation (standing at 4℃ for 12 h). The precipitate was centrifuged at 5000 r / min for 15 min, and then freeze-dried to obtain the summer and autumn tea polysaccharide product (denoted as TP).

[0023] The specific operation of the Sevage method is as follows: Sevage reagent (chloroform to n-butanol volume ratio of 4:1) was added to the crude polysaccharide extract at a ratio of 1:5 (v / v). The mixture was first placed in a shaker at 180 rpm for 60 min, followed by centrifugation at 6000 rpm for 15 min. The lower organic phase and intermediate denatured protein gel were removed, and the supernatant was retained. To thoroughly remove protein impurities, the above deproteinization steps were repeated 6 times until no flocculent precipitate was observed. Finally, all supernatants were combined as the purified polysaccharide solution.

[0024] Example 2 1. This embodiment provides a method for determining the selenium content of selenized summer and autumn tea polysaccharides: (1) Plotting the standard curve for selenium content: Precisely prepare 100 μg·mL -1 Sodium selenite standard solution was prepared by transferring 0, 0.25, 0.50, 0.75, 1.00, 1.25, and 1.50 mL of standard solution into 25 mL volumetric flasks, and then diluting to volume to obtain a range of 0–6.0 μg / mL. -1 Selenium standard series solutions. Take each of the above solutions into an Erlenmeyer flask, and add 2 mL of EDTA-2Na complexing agent (10 g·L⁻¹) sequentially. -1 ), using 1 mol·L -1 Adjust the pH of the solution to approximately 2.0 with hydrochloric acid, then add 2 mL of o-phenylenediamine colorimetric reagent (10 g·L⁻¹). -1After mixing, react in the dark for 40 min. Add 10 mL of toluene extractant, shake vigorously for 5 min, and let stand until the interface between the two phases is clear. Collect the upper organic phase and measure the absorbance at 334 nm. Plot a selenium content standard curve with selenium content as the x-axis and absorbance as the y-axis. Figure 1 As shown.

[0025] (2) Determination and calculation of selenium content in polysaccharides of fresh summer and autumn tea leaves: Accurately weigh 20.0 mg of selenized summer and autumn tea polysaccharide product sample and place it in a test tube. Add 2 mL of concentrated nitric acid and heat to digest (80℃) until the solution is clear and transparent. After cooling to room temperature, add 5 mL of 6 mol·L⁻¹. -1 The hydrochloric acid solution was concentrated to near dryness by heating. After the reaction solution cooled, it was quantitatively transferred to a 25 mL volumetric flask to obtain the test solution. The absorbance value of the test solution was obtained according to the standard curve determination method. The selenium content was calculated by substituting the absorbance value into the standard curve formula, and the yield was obtained by weighing.

[0026] 2. This embodiment provides a selenized summer and autumn tea polysaccharide, the preparation method of which includes the following steps: Accurately weigh 0.5g of the summer and autumn tea polysaccharide product prepared in Example 1 and place it in an Erlenmeyer flask. Then, add 50mL of nitric acid solution of different concentrations to the Erlenmeyer flask, and under heating and continuous stirring, allow the polysaccharide to fully dissolve, obtaining a 10mg / mL summer and autumn tea polysaccharide solution. Next, under different temperature and time conditions, add an appropriate amount of sodium selenite to the above solution, stirring to promote the reaction between the polysaccharide and the selenizing reagent. After the reaction is complete, cool to room temperature, and add 5% sodium bicarbonate solution to adjust the pH of the mixture to approximately 5-6. Then, centrifuge for 5 min (4000 r / min) and filter. The resulting filtrate is dialyzed for 48 h using a 3500 Da dialysis bag. During the dialysis process, every 8 h, a small amount of dialysate is mixed with ascorbic acid for colorimetric detection. If no red color appears, the dialysis is complete. The dialyzed solution was concentrated under reduced pressure and rotary evaporated to approximately 20 mL. It was then transferred to a petri dish, cooled, and refrigerated overnight at -20°C. After sufficient refrigeration, the sample was freeze-dried to obtain the selenized summer and autumn tea polysaccharide product (denoted as Se-TP) and weighed. Figure 2 As shown, the selenium content and the final yield of selenized polysaccharides were calculated.

[0027] The effects of reaction time, reaction temperature, nitric acid concentration, and sodium selenite content on the selenization modification of summer and autumn tea polysaccharides were determined through single-factor experiments. Then, based on selenium content and the final yield of selenized polysaccharides, response surface methodology was used to select experimental conditions for optimization. Reaction time, reaction temperature, nitric acid concentration, and the mass ratio of sodium selenite to summer and autumn tea polysaccharides were selected as analytical factors, with selenium content as the response value. A four-level, three-factor optimization experiment was designed to determine the optimal parameter combination.

[0028] (1) Effect of reaction time on the selenization modification of summer and autumn tea polysaccharides.

[0029] The preparation conditions were fixed as follows: reaction temperature 70℃, nitric acid concentration 0.5%, sodium selenite dosage (sodium selenite to summer and autumn tea polysaccharide mass ratio) 1:1. The reaction times were varied to 4h, 6h, 8h, 10h, and 12h. After preparation, the selenium content and yield were measured. Each experiment was performed in triplicate. The results are as follows: Figure 3 As shown.

[0030] Depend on Figure 3 It can be seen that with the increase of reaction time, both the selenium content and the yield show a trend of first increasing and then decreasing. During the first 4-6 hours of the reaction, the selenium content increases with the extension of reaction time, indicating that the binding reaction of selenium with polysaccharides requires a certain amount of time. Within this time range, the selenization reaction continues, and more and more selenium binds to the polysaccharides of *Tea suspensa* and *Tea japonica*. At 6 hours of reaction, the selenium content reaches its peak (6.94±0.11 mg / g), at which point the binding sites on the polysaccharide molecules are almost saturated. When the reaction time exceeds 6 hours, the binding efficiency and yield of selenium in the system show a significant decreasing trend. This may be because the continuous high temperature in the 70℃ acidic reaction environment causes hydrolytic breakage of the polysaccharide chains, damaging the structure of key binding sites, thereby weakening the ability of polysaccharides to bind selenium, ultimately resulting in a decrease in selenium content and yield. Therefore, 4-8 hours was selected as the experimental condition for subsequent response surface methodology analysis.

[0031] (2) Effect of reaction temperature on the selenization modification of summer and autumn tea polysaccharides.

[0032] The preparation conditions were fixed as follows: reaction time 8 h, nitric acid concentration 0.5%, sodium selenite dosage (sodium selenite to summer and autumn tea polysaccharide mass ratio) 1:1. The reaction temperatures were varied to 50℃, 60℃, 70℃, 80℃, and 90℃. After preparation, the selenium content and yield were measured. Each experiment was performed in triplicate. The results are as follows: Figure 4 As shown.

[0033] Depend on Figure 4 It can be seen that with the increase of reaction temperature, both selenium content and yield show a trend of first increasing and then decreasing. In the lower temperature range (50~70℃), selenium content and yield increase with increasing reaction temperature, reaching the highest value (6.83±0.14mg / g) at 70℃. Therefore, 60~80℃ was selected as the experimental conditions for subsequent response surface methodology.

[0034] (3) Effect of nitric acid concentration on the selenization modification of polysaccharides in summer and autumn tea.

[0035] The preparation conditions were fixed as follows: reaction time 8 h, reaction temperature 70 °C, sodium selenite dosage (sodium selenite to summer and autumn tea polysaccharide mass ratio) 1:1. The nitric acid concentration was varied to 0.3%, 0.5%, 0.7%, 0.9%, 1.1%, and 1.3%. After preparation, the selenium content and yield were measured. Each experiment was performed in triplicate. The results are as follows: Figure 5 As shown.

[0036] Depend on Figure 5 It was found that with increasing nitric acid concentration, both selenium content and yield showed a trend of first increasing and then decreasing. Within the range of 0.3%–0.5%, both selenium content and yield increased with increasing nitric acid concentration, reaching their maximum values ​​(6.73 ± 0.17 mg / g) at a nitric acid concentration of 0.5%. When the nitric acid concentration was below 0.5%, the catalytic effect could not be fully activated due to insufficient acidic environment. However, when the acid concentration continued to increase, excessive acid not only failed to catalyze the selenization reaction normally through acid hydrolysis, but also caused partial acidolysis of the polysaccharides in summer and autumn tea due to excessive acidity, destroying normal active groups and binding sites, which was detrimental to the selenization reaction. Therefore, a nitric acid concentration of 0.3%–0.7% was selected as the experimental condition for subsequent response surface methodology analysis.

[0037] (4) Effect of the mass ratio of sodium selenite to summer and autumn tea polysaccharides on the selenization modification of summer and autumn tea polysaccharides.

[0038] The preparation conditions were fixed as follows: reaction time 8 h, reaction temperature 70 °C, and nitric acid concentration 0.5%. The mass ratio of sodium selenite to summer and autumn tea polysaccharides was varied to 0.4:1, 0.6:1, 0.8:1, 1:1, 1.2:1, and 1.4:1. After preparation, the selenium content and yield were measured. Each experiment was performed in triplicate. The results are as follows: Figure 6 As shown.

[0039] Depend on Figure 6 It can be seen that as the mass ratio increases, both the selenium content and yield show a trend of first increasing and then decreasing. When the mass ratio is in the range of 0.4-1, the selenium content and yield continuously increase with the increase of the mass ratio, reaching a maximum value (6.85±0.12 mg / g) at a mass ratio of 1:1. This may be because as the amount of sodium selenite increases, the binding sites on the tea polysaccharides are continuously able to bind with selenium. However, when the amount of sodium selenite is further increased, the selenium content decreases with the increase of sodium selenite. It is inferred that under the condition of a mass ratio of 1:1, the bonding between selenium and polysaccharides has reached a saturated state. If the amount of sodium selenite is further increased, it may disrupt the original equilibrium state, thereby reducing the selenium content and yield. Therefore, a mass ratio of 0.8~1.2 was selected as the experimental conditions for subsequent response surface analysis.

[0040] (5) Response surface optimization experimental design, four-level three-factor table as shown in Table 1 below: Table 1 Factor Level Table

[0041] The design and results are shown in Table 2 below: Table 2 Box-Behnken Experimental Design and Results

[0042] The analysis of variance is shown in Table 3 below: Table 3 Analysis of Variance

[0043] Note: **The difference is extremely significant (P<0.01); * indicates a significant difference (P<0.05); not significant (P>0.05).

[0044] As shown in Tables 1-3, the model is highly significant (P<0.0001), indicating that the response surface regression equation can correctly and effectively describe the combined influence of four factors—reaction time (A), reaction temperature (B), nitric acid concentration (C), and mass ratio (D)—on the target response value. The P-value for lack of fit is 0.5904 > 0.05, indicating that the model does not omit any key factors, has a good fit, and possesses high predictive reliability. R 2 =0.9683, Adjusted R 2 =0.9362, indicating that the model can explain 93.62% of the response value variation, and the goodness of fit is high.

[0045] Further analysis of the influence and interactions of each factor revealed that: the mass ratio (D) had the largest F-value (25.53), followed by the reaction temperature (B) with an F-value of 9.78; both were highly significant (P<0.01). The effect of reaction time (A) was relatively weak, with an F-value of 4.18. The significance of nitric acid concentration (C) was close to the critical value, with the smallest F-value of 3.26. Therefore, the order of influence of the four different factors on the selenium content after polysaccharide selenization is: D (mass ratio) > B (reaction temperature) > A (reaction time) > C (nitric acid concentration). Among the interactions, BD and BC had more prominent effects, while AC, although significant, had a weaker effect. Based on the significance test results, the regression equation can be optimized by removing insignificant interaction terms such as AB, AD, and CD (all P>0.05). The regression equation is as follows: Y=-10.55144+0.719A+0.2532B+14.79375C+5.64375D-0.2125AC-0.055BC+0.0625BD-0.082938A 2 -0.00213B 2 -10.91875C 2-6.1375D 2 Response plots and contour plots were generated to calculate the effect of the interaction between reaction time and nitric acid concentration on selenium content, such as... Figure 7 As shown. The response plot and contour plot show the effect of the interaction between reaction temperature and nitric acid concentration on selenium content, as shown. Figure 8 As shown. The response plot and contour plot show the effect of the interaction between reaction temperature and mass ratio on selenium content, as shown. Figure 9 As shown.

[0046] Depend on Figure 7 As can be seen from the data, the contour lines are most densely packed in the region with a reaction time of 5–7 hours and a nitric acid concentration of 0.4–0.6%. The response curve is steep, indicating that within this range, the selenium content is sensitive to changes in reaction time and nitric acid concentration. The contour lines are generally elliptical, indicating a significant interaction between the two factors. The long axis extends roughly along the reaction time axis. This suggests that the effect of reaction time on selenium content is slightly greater than that of nitric acid concentration.

[0047] Depend on Figure 8 It can be seen that the contour lines are most dense in the region with a reaction temperature of 65-75℃ and a nitric acid concentration of 0.4-0.6%, and the response curve is steep, indicating that the selenium content is sensitive to changes in these two factors. The contour lines are generally elliptical, indicating a significant interaction. The long axis extends along the reaction temperature axis, indicating that the influence of reaction temperature is slightly greater than that of nitric acid concentration.

[0048] Depend on Figure 9 It can be seen that the contour lines are relatively dense in the region of reaction temperature 65~75℃ and mass ratio 0.9~1.1, and the surface of the response diagram is relatively steep, indicating that the selenium content is more sensitive to changes in reaction temperature and mass ratio within this range. The contour lines are generally clearly elliptical, indicating significant interaction. The long axis extends along the reaction temperature axis, indicating that the influence of reaction temperature is slightly greater than that of mass ratio.

[0049] The optimal preparation process for selenized summer and autumn tea polysaccharides was predicted using a regression model based on Design-Expert 13 as follows: reaction time 5.67 h, reaction temperature 64 ℃, nitric acid concentration 0.41%, and mass ratio 0.90.

[0050] Example 3 1. This embodiment provides a method for determining the total sugar content of selenium-enriched summer and autumn tea polysaccharides: Prepare 200 μg / mL glucose standard solution and 6% phenol. Accurately transfer 0–0.8 mL of the 200 μg / mL glucose standard solution into test tubes, and dilute to 1 mL with distilled water to prepare solutions with concentrations of 0, 20, 40, 60, 80, 100, 120, and 140 μg / mL. Shake well to mix thoroughly. Accurately transfer 200 μL of each of the above glucose standard solutions to another set of test tubes. Add 200 μL of 6% phenol solution to each test tube, mix immediately, and quickly add 1 mL of concentrated sulfuric acid, mixing again thoroughly. Heat the mixture in a boiling water bath for 30 min, then rapidly cool. Use the 0 μg / mL solution as a blank control and measure the absorbance of each solution at 490 nm. Finally, plot a standard curve with glucose concentration on the x-axis and absorbance on the y-axis, as shown below. Figure 10 As shown, the purity of the polysaccharide was calculated.

[0051] 2. This embodiment provides a selenized summer and autumn tea polysaccharide, the preparation method of which includes the following steps: Accurately weigh 0.5g of the *Tea japonica* polysaccharide product prepared in Example 1 and place it in an Erlenmeyer flask. Then, add 50mL of 0.41% nitric acid solution to the flask and heat while continuously stirring to fully dissolve the polysaccharide, obtaining a 10mg / mL *Tea japonica* polysaccharide solution. Next, add 0.45g of sodium selenite (sodium selenite to *Tea japonica* polysaccharide mass ratio 0.90) to the above solution. Stir to promote the reaction between the polysaccharide and the selenizing reagent at a reaction time of 5.67h and a reaction temperature of 64℃. After the reaction is complete, cool to room temperature and add 5% sodium bicarbonate solution to adjust the pH of the mixture to approximately 5-6. Then, centrifuge for 5min (4000r / min) and filter. The resulting filtrate is dialyzed for 48h using a 3500Da dialysis bag. During dialysis, a small amount of dialysate is mixed with ascorbic acid every 8h for colorimetric detection. If no red color appears, dialysis is complete. The dialyzed solution was concentrated under reduced pressure and rotary evaporated to approximately 20 mL. It was then transferred to a petri dish, cooled, and refrigerated overnight at -20°C. After sufficient refrigeration, the sample was freeze-dried to obtain selenized summer and autumn tea polysaccharide product. The selenium content of this selenized summer and autumn tea polysaccharide was calculated to be 6.85±0.07 mg / g, and the polysaccharide purity was 53.28±2.67%. The polysaccharide purity obtained by this extraction method was significantly higher than that obtained by traditional methods such as water extraction.

[0052] Test The products prepared in Examples 1 and 3 were subjected to bioactivity studies.

[0053] (1) ABTS free radical scavenging test: Prepare 7 mmol ABTS solution, 2.45 mmol potassium persulfate solution, 20 mg / mL *Tea japonica* polysaccharide solution, and 20 mg / mL selenized *Tea japonica* polysaccharide solution. Mix 10 mL of ABTS solution and 10 mL of potassium persulfate solution in the dark and react for 16 hours, measuring the absorbance. Continuously dilute with phosphate buffer (pH=7.4) until the absorbance of the mixed solution is 0.70 ± 0.1. Dilute the 20 mg / mL *Tea japonica* polysaccharide solution and the selenized *Tea japonica* polysaccharide solution with distilled water to concentrations of 3, 6, 9, 12, 15, and 18 mg / mL, respectively. Take 1 mL of each concentration sample solution in a test tube, add 2 mL of ABTS diluent to each dilution, and shake at room temperature for 5 minutes. After the reaction is complete, measure the absorbance of each solution at 734 nm. A i Simultaneously, the absorbance at 734 nm was measured under the same conditions, with 1 mL of phosphate buffer replacing the sample solution and 2 mL of phosphate buffer replacing the ABTS solution. A 3. A 4) Use absorbance measurements of solutions with vitamin C concentrations of 0.1–20 mg / mL under the same conditions as a positive control. Each experiment was performed in triplicate. The clearance rate was calculated using the formula shown below: Clearance rate (%) = ×100% Note: A i The absorbance represents the absorbance of polysaccharide samples with different concentrations.

[0054] like Figure 11 As shown, with Vc as the positive control group, within the given concentration range, the ABTS radical scavenging rate of both solutions increased with increasing solution concentration. This indicates that the higher the solution concentration, the stronger the radical scavenging ability. At the same concentration, the ABTS radical scavenging rate of Se-TP solution was consistently higher than that of TP solution. The slope of the Se-TP solution curve was steeper than that of TP solution, especially in the higher concentration region. This indicates that the scavenging rate of Se-TP solution is more sensitive to concentration changes, that is, increasing the concentration can significantly enhance its scavenging ability. At a concentration of 18 mg / mL, the scavenging rate of Se-TP solution reached 52.5 ± 2.1%, while that of TP solution was 45.3 ± 1.3%. The selenized *Tea japonica* polysaccharide solution (Se-TP) showed better performance in radical scavenging, which may be because selenium has a significant activating effect on the hydrogen atoms of isopropyl carbon. When polysaccharides combine with selenium, they can provide more available hydrogen atoms, thereby promoting the generation of more free radicals.

[0055] (2) Determination of DPPH free radical scavenging rate: Accurately weigh 10 mg of DPPH reagent and dilute to a 250 mL volumetric flask with 50% ethanol solution to prepare 20 mg / mL solutions of *Tea japonica* polysaccharides and selenized *Tea japonica* polysaccharides. Store these solutions in the dark for later use. Dilute the two solutions with distilled water to different concentrations of 3, 6, 9, 12, 15, and 18 mg / mL, respectively. Take 1 mL of each concentration solution, add 2 mL of DPPH solution, mix well, and react in the dark for 5 min. After the reaction is complete, measure the absorbance at a wavelength of 517 nm. A i Meanwhile, 1 mL of 50% ethanol solution was used to replace the sample solution, and 2 mL of 50% ethanol solution was used to replace the DPPH solution. Under the same conditions, the absorbance at 517 nm was measured. A 3. A 4) Use absorbance measurements of solutions with vitamin C concentrations of 0.1-20 mg / mL under the same conditions as a positive control. Each experiment was performed in triplicate. The clearance rate was calculated using the formula shown below: Clearance rate (%) = ×100% Note: A i The absorbance represents the absorbance of polysaccharide samples with different concentrations.

[0056] like Figure 12 As shown, with Vc as the positive control group, within the given concentration range, the DPPH radical scavenging rate of both solutions increased with increasing solution concentration. This indicates that the higher the solution concentration, the stronger the radical scavenging ability. At the same concentration, the ABTS radical scavenging rate of Se-TP solution was consistently higher than that of TP solution. The scavenging rate of TP solution increased slowly at first and then gradually accelerated, while the slope of the Se-TP solution curve was steeper than that of TP solution, especially in the higher concentration region. This indicates that the scavenging rate of Se-TP solution is more sensitive to concentration changes, meaning that increasing the concentration significantly enhances its scavenging ability. At a concentration of 18 mg / mL, the scavenging rate of Se-TP solution reached 57.7 ± 2.3%, while that of TP solution was 47.2 ± 1.7%. The selenized *Tea japonica* polysaccharide solution (Se-TP) exhibited better performance in radical scavenging, possibly because selenization may activate hydrogen donors in the anodic region, significantly improving the DPPH radical scavenging rate of selenium polysaccharide compared to natural polysaccharides.

[0057] (3) Determination of α-glucosidase inhibition rate: The inhibitory effect of each polysaccharide sample on α-glucosidase activity was determined by the PNPG method. A 0.1 U / mL α-glucosidase solution (diluted with phosphate buffer), a 1 mmol / L PNPG solution (phosphate buffer as solvent), a 0.2 mol / L Na₂CO₃ solution, and 20 mg / mL solutions of *Tea japonica* polysaccharide and selenized *Tea japonica* polysaccharide (all in distilled water) were prepared and stored at 4°C for later use.

[0058] Two polysaccharide solutions were diluted to concentrations of 4, 8, 12, 16, and 20 mg / mL. In the experiment, for the sample group, 500 μL of polysaccharide solution and 500 μL of α-glucosidase solution were mixed in a test tube and incubated at 37°C for 15 min. Then, 500 μL of PNPG was added, and incubation continued at the same temperature for 10 min. Finally, 500 μL of Na₂CO₃ solution was added, and the absorbance at 405 nm was measured using a microplate reader. For the control group, PBS was used instead of the α-glucosidase solution, and for the whole enzyme group, distilled water was used instead of the sample solution; all other steps were the same. The measured absorbance values ​​were recorded as follows: A 1 (Sample Group) A 2 (Control Group) A 3 (whole enzyme group): The absorbance of acarbose at the same concentration measured under the same conditions was used as a positive control. Each group of experiments was performed in triplicate. The inhibition rate was calculated using the following formula: Inhibition rate (%) = 100 × (1 - ) like Figure 13 As shown, within the given concentration range, the inhibition rates of both solutions increased with increasing solution concentration. The increase in inhibition rate of the Se solution slowed down after reaching 16 mg / mL, while the increase in the Se-TP solution remained more pronounced. At a concentration of 20 mg / mL, the inhibition rate of the Se-TP solution reached 45.2 ± 1.8%, while that of the TP solution was 36.1 ± 1.3%. The selenized *Tea japonica* polysaccharide solution (Se-TP) exhibited better performance in terms of α-glucosidase inhibition rate. This may be because the spatial structure of the polysaccharide may change after binding with selenium, which promotes a synergistic effect between the coordination moiety and the exposed active groups, thereby enhancing the bioactivity of the polysaccharide.

[0059] (4) Determination of α-amylase inhibition rate: Prepare DNS reagent, 5 μg / mL α-amylase solution (dissolved and diluted with distilled water), 0.5% starch solution (dissolved with distilled water), and 1.8 mg / mL Xiaqiu tea polysaccharide and selenized Xiaqiu tea polysaccharide solution (all in phosphate buffer solution). Store all solutions at 4°C for later use.

[0060] Two polysaccharide solutions were diluted to concentrations of 0.3, 0.6, 0.9, 1.2, 1.5, and 1.8 mg / mL. In the experiment, for the sample group, 1 mL of polysaccharide solution and 1 mL of α-amylase solution were mixed in a test tube and incubated at 37°C for 15 min. Then, 500 μL of 0.5% starch solution was added, and the mixture was incubated for another 10 min at the same temperature. Finally, 1 mL of DNS reagent was added, and the absorbance at 540 nm was measured using a microplate reader. For the control group, distilled water was used instead of the α-amylase solution, and for the whole enzyme group, distilled water was used instead of the sample solution; all other steps were the same. The measured absorbance was recorded as follows: A 1 (Sample Group) A 2 (Control Group) A 3 (whole enzyme group): The absorbance of acarbose at the same concentration measured under the same conditions was used as a positive control. Each group of experiments was performed in triplicate. The inhibition rate was calculated using the following formula: Inhibition rate (%) = 100 × (1 - ) like Figure 14 As shown, within the given concentration range, the inhibition rates of both solutions increased with increasing solution concentration. The inhibition rate of TP solution initially increased rapidly and then slowed down, while the inhibition rate of Se-TP solution remained relatively fast. At a concentration of 1.8 mg / mL, the inhibition rate of Se-TP solution reached 47.6 ± 1.9%, while that of TP solution was 34.2 ± 1.4%. The selenized *Tea japonica* polysaccharide solution (Se-TP) exhibited better performance in terms of α-amylase inhibition rate. This may be because the structure of selenized *Tea japonica* polysaccharide is more porous, thus increasing the exposure of active groups.

[0061] (5) Determination of pancreatic lipase inhibition rate: Prepare 0.1 mol Tris-HCl buffer, 0.5 mg / mL 4-nitrophenyl palmitate solution (dissolved in isopropanol first, then diluted to volume with Tris-HCl), 0.01 g / mL pancreatic lipase solution, and 2 mg / mL solutions of *Tea japonica* and selenized *Tea japonica* polysaccharides. Store the buffer, substrate working solution, and polysaccharide solution at 4°C, and the enzyme solution at -20°C. For the experiment, the polysaccharide solution was diluted to 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mg / mL for the sample groups. Take 50 μL of each concentration of sample solution in a test tube, add 150 μL of porcine pancreatic lipase solution and 350 μL of buffer, preheat at 37°C for 10 min, then add 450 μL of substrate solution, mix thoroughly, and react at 37°C for 2 h. After the reaction, the sample was centrifuged at 12000 rpm for 10 min, and the supernatant was collected. The absorbance was measured at 405 nm. For the blank control group, distilled water was used instead of the polysaccharide solution. For the blank group, buffer solution and distilled water were used instead of the enzyme solution and polysaccharide solution, respectively. For the sample control group, buffer solution was used instead of the enzyme solution. The measured absorbance values ​​were recorded as follows: A 1 (Blank control group) A 2 (Blank Group) A 3 (Sample Group) A 4 (Sample Control Group): The absorbance of orlistat at the same concentration measured under the same conditions was used as a positive control. Each group of experiments was performed in triplicate. The inhibition rate was calculated using the following formula: Inhibition rate (%) = 100 × like Figure 15 As shown, within the given concentration range, the inhibition rates of both solutions increased with increasing solution concentration, and the growth trends were roughly the same. The growth of the TP solution slowed significantly at a concentration of 1.0 mg / mL. At a concentration of 1.2 mg / mL, the inhibition rate of the Se-TP solution reached 49.7 ± 2.3%, while that of the TP solution was 42.9 ± 1.7%. The selenized *Tea japonica* polysaccharide solution (Se-TP) exhibited better performance in terms of pancreatic lipase inhibition, but overall, the difference in inhibition rates between the two solutions was not significant.

[0062] Therefore, the present invention adopts the above-mentioned method for preparing selenized summer and autumn tea polysaccharides and its application, using a eutectic solvent to extract summer and autumn tea polysaccharides and then performing selenization modification to improve the bioactivity of summer and autumn tea polysaccharides, especially to enhance their inhibitory effect on glucose and lipid metabolism enzymes.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing seleniumized polysaccharides of Herba lamiophlomis rotata, characterized in that, Includes the following steps: S1. Raw material pretreatment: Select fresh summer and autumn tea leaves, wash, dry, crush, decolorize, and grind to obtain tea powder; S2. Add the tea powder obtained in S1 to a eutectic solvent system composed of choline chloride and urea in a molar ratio of 1:4, extract, and then perform ultrasonic extraction to obtain a summer and autumn tea polysaccharide extract. S3. Separation and purification: The extract obtained in S2 was centrifuged, adsorbed by macroporous resin, eluted with ethanol, concentrated and freeze-dried to obtain summer and autumn tea polysaccharides. S4, Selenization modification: Dissolve the summer and autumn tea polysaccharides obtained in S3 in nitric acid solution, add sodium selenite, stir, and after the reaction is completed, cool and adjust the pH value to 5-6 to obtain the selenized summer and autumn tea polysaccharide reaction solution. S5. Product purification: The selenized summer and autumn tea polysaccharide reaction solution obtained in S4 was subjected to dialysis, vacuum concentration and freeze drying to obtain selenized summer and autumn tea polysaccharide.

2. The method for preparing selenium summer and autumn tea polysaccharide according to claim 1, characterized in that, In S1, the pulverized tea raw material is decolorized with 4 times its volume of 95% ethanol for 12 hours.

3. The method for preparing selenium summer tea polysaccharide according to claim 1, characterized in that, In S2, the solid-liquid ratio of tea powder to the eutectic solvent system is 1:

40.

4. The method for preparing selenized summer and autumn tea polysaccharides according to claim 1, characterized in that, In S2, the water content of the eutectic solvent system is 50%, and the extraction is carried out at 70°C for 30 minutes, while simultaneously being ultrasonicated at 200W.

5. The method for preparing selenized summer and autumn tea polysaccharides according to claim 1, characterized in that, In S3, after centrifugation, the supernatant was collected, and protein impurities were removed using the Sevage method. The filtrates were combined and precipitated with 4 times the volume of anhydrous ethanol. The precipitate was centrifuged and freeze-dried to obtain summer and autumn tea polysaccharides.

6. The method for preparing selenized summer and autumn tea polysaccharides according to claim 5, characterized in that, The Sevage method specifically refers to: Add Sevage reagent to the supernatant at a volume ratio of 1:5, place in a shaker and continue the reaction. Centrifuge, remove the lower organic phase and the intermediate denatured protein gel, retain the supernatant, repeat several times, and combine all supernatants. The Sevage reagent is a mixture of chloroform and n-butanol, with a volume ratio of chloroform to n-butanol of 4:

1.

7. The method for preparing selenized summer and autumn tea polysaccharides according to claim 1, characterized in that, In S4, the concentration of summer and autumn tea polysaccharides in nitric acid solution was 10 mg / mL, the reaction time was 4-8 h, the reaction temperature was 60-80 ℃, the nitric acid concentration was 0.3-0.7%, and the mass ratio of sodium selenite to summer and autumn tea polysaccharides was 0.8-1.2:

1.

8. The method for preparing selenized summer and autumn tea polysaccharides according to claim 7, characterized in that, In S4, the concentration of summer and autumn tea polysaccharides in nitric acid solution was 10 mg / mL, the reaction time was 5.67 h, the reaction temperature was 64 ℃, the nitric acid concentration was 0.41%, and the mass ratio of sodium selenite to summer and autumn tea polysaccharides was 0.90:

1.

9. The application of selenized summer and autumn tea polysaccharides prepared by the method according to any one of claims 1 to 8 in the preparation of antioxidants.

10. The application of selenized summer and autumn tea polysaccharides prepared by the method according to any one of claims 1 to 8 in the preparation of glucose and lipid metabolism regulators.

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