Synthesis and Preliminary Biological Activity of Selenium Polysaccharides from Tussilago farfara L.

Tussilago farfara L. selenium polysaccharides (STFPs, similarly hereinafter) were prepared by Selenous acid salt method from Tussilago farfara L. polysaccharide (TFP, similarly hereinafter). Taking the selenium content of STFPs as an indicator, the synthesis process of STFPs was optimized using response surface methodology based on single factor experiments. The optimal synthesis process conditions obtained are: 90 °C, 12 hours, selenide reagent ratio of 1.2: 1, HNO3 volume fraction of 0.7%, and the average selenium content of STFPs measured under these conditions is 2.338 mg/g. Characterization was carried out by measuring the selenium content, molecular weight, infrared spectroscopy. The results of antioxidant experiments showed that the maximum clearance rate of DPPH· by the selenium polysaccharide from Tussilago farfara L. was 73.16%, which were higher than from Tussilago farfara L. polysaccharides.


Introduction
Selenium is an indispensable trace element in human and animal bodies, which is essential for maintaining normal life activities of the body, especially for cancer, diabetes, cardiovascular diseases and other diseases [1,2] .Selenium supplementation can effectively reduce the occurrence of these diseases, so it is necessary to supplement selenium.In recent years, research on selenium polysaccharides has just begun both domestically and internationally, and few natural selenium polysaccharides have been discovered so far.Only selenium polysaccharides such as garlic, astragalus, and konjac have been reported [3] .However, the content of selenium in Natural product is too small to meet the needs of clinical application.At present, chemical methods are mainly used internationally to synthesize selenium polysaccharides [4,5] .The dry flower buds of the Tusilago farfarfara possess lung-moistening, cough-relieving, and phlegm and asthma-soothing properties.Among these virtues, Tussilago farfara L. polysaccharides are one of the active ingredients.Studies have found that polysaccharides from Tussilago farfara L. can induce apoptosis of leukemia K562 cells and display good oxygen free radical activity.The study aims to improve the activity of polysaccharides through the selenization modification of polysaccharides, which has been shown to be effective.With this in mind, the proposed method involves using selenite reagent to prepare Tussilago farfara L. selenium polysaccharides.To enhance the medicinal potentials and facilitate the development of various functions of Tussilago farfara L. polysaccharides, food additives offer valuable theoretical insights.

Materials and reagents
Tussilago farfara L polysaccharide (TFPs) is self-made by our laboratory.

Synthesis process
Prepare STFPs according to the method described in reference [6] .Weigh an appropriate amount of TFP, add 100 ml of a certain volume fraction of HNO 3 aqueous solution, mix and heat at 70 ℃ with magnetic force, fully dissolve, and then add a certain proportion of Na 2 SeO 3 and anhydrous BaCl 2 2 g.React in a water bath at a certain temperature for 12 hours.After the reaction is completed, cool to room temperature and filter.Collect the supernatant, adjust the pH to neutral with anhydrous Na 2 CO 3 , and precipitate BaCl 2 3000 r/min in the reaction solution with an appropriate amount of anhydrous Na 2 SO 4 .Centrifuge for 15 min.Collect the supernatant and filter.Repeat this process several times to make the filtrate clear.After the filtrate is concentrated by rotary evaporation, it is dialyzed with tap water and distilled water for 24 hours each, until the ascorbic acid does not turn red.The product was precipitated with four times the volume of anhydrous ethanol, left to stand overnight, centrifuged and precipitated, and freeze-dried to obtain STFPs.

Optimization of STFPs synthesis process using response surface methodology
Using the selenium content of STFPs as an indicator, four factors were selected: reaction temperature (A), reaction time (B), selenide reagent ratio (C), and HNO 3 volume fraction (D).Box Behnken's design principle was applied to optimize the process parameters through response surface methodology.The experimental factor level design is shown in Table 1.

Production of standard curves
Take a series of standard Glucan as molecular weight standard, dissolve it in high-purity water to prepare 1.0 mg/ml solution, inject the sample, and make records.The logarithm value of standard molecular weight is the ordinate, and the retention time of corresponding chromatographic peak is the abscissa, so as to make linear regression.

Molecular weight determination of STFPs
Prepare the sample into a 1.0 mg/ml aqueous solution, passing 0.22 μl After the m microporous filter membrane is injected for analysis, the chromatographic conditions are consistent with 2.4.1.The molecular weight of TFP can be calculated by substituting the retention time of the chromatogram into the standard curve equation.

Infrared spectroscopy analysis
Sample processing: KBr compression method is used.Weigh a certain amount of TFP and STFPs separately, mix and grind them with dried and dehydrated KBr, press them into tablets, and measure them in an infrared spectrometer.The detection wavelength is between 4000 cm -1 and 400 cm -1 .

Antioxidant activity
According to the methods in literature [7,8] , the scavenging ability of crude polysaccharides, TFP, and STFPs on DPPH• was measured.Calculate according to the following formula: Clearance rate (%)=[1-(A 2 -A 1 )/A 0 ]×100% In the formula, A 0 : absorbance of blank control solution; A 1 : absorbance of the sample with 50% ethanol; A 2 : absorbance of the sample with DPPH ethanol.

Response Surface Optimization Analysis
According to the response surface experiment, the experimental combination and results obtained are shown in Table 2, and the analysis of variance is shown in Table 3 [9] .The regression equation obtained through Box Behnken test fitting: Y=2.47+0.0719A+0.0292B+0.0373C+0.0333D+0.2788AB-0.0482AC-0.0702AD+0.0183BC-0.0397BD-0.0618CD-0.3475A 2 -0.3875B 2 -0.3148C 2 +0.0192D 2 From Table 3, it can be seen that the significance level of the model fitted by Design Expert v 12 software is much lower than 0.05 (P<0.0001), and the response surface model obtained has high significance.Excluding the error is not significant (P=0.1495),unknown factors have little impact on the experimental results.The model R 2 =0.9929, the coefficient of variation CV=1.71%<5%, and the correction coefficient R2adj=0.9859,indicating that the predicted model results are reliable .From Table 4, it can be seen that single factor A is highly significant, and AB, AC, AD, BD, CD are significant in the interactive effects, with FA=50.86,FB=8.41,FC=13.64,FD=10.87,indicating the intensity of the influence of each factor on selenium content: reaction temperature>selenide reagent ratio>HNO 3 volume fraction>reaction time.

Response surface analysis
The regression optimization response surface graph is shown in Figure 1.The optimal synthesis process for STFPs predicted by the regression model can be obtained: reaction temperature of 90 ℃, reaction time of 12.2 hours, selenidation reagent ratio of 1.22: 1, HNO 3 volume fraction of 0.70%.Under these conditions, the selenium content in STFPs obtained from selenidation can theoretically reach 2.327 mg/g.Based on the experimental results and considering the convenience of practical application, the optimal process conditions for adjusting selenization are: reaction temperature of 90 ℃, reaction time of 12 hours, selenization reagent ratio of 1.2: 1, and HNO 3 volume fraction of 0.7%.The average selenium content in STFPs was measured to be 2.338 mg/g.As shown in Figure 2B, the absorption of STFPs, 3447 cm -1 represents the O-H stretching vibration, 2920 cm -1 represents the C-H stretching vibration, 1740 and 1602 cm -1 represent the stretching vibration absorption peak of carboxyl C=O, 1430 cm -1 represents the deformation absorption peak of CH 2 , and 1063 cm -1 represents the variable angle vibration absorption peak of alcohol hydroxyl groups, all of which are characteristic absorption peaks of polysaccharides [10] .Compared with TFP in the figure, the waveform, intensity, and peak width of its main absorption peaks are roughly the same as TFP, indicating that the main structure of STFPs obtained by selenization has not undergone significant changes.In addition, there is a characteristic peak at 664 cm -1 that is caused by the stretching vibration of the symmetric ring Se=O or Se-H.From this, it can be seen that the polysaccharides from winter flowers have successfully selenified [11] .

Antioxidant activity
The experimental results are shown in Figure 3.  3, when the polysaccharide is 5 mg/ml, the clearance rate of crude polysaccharides is the highest at 21.4%.TFP is the highest at 61.58%, and STFPs is the highest at 73.16%.It can be seen that under the same mass concentration of selenium polysaccharides and polysaccharides, selenium polysaccharides have stronger scavenging ability than polysaccharides.This indicates that modifying winter flower polysaccharides through selenization can help enhance their antioxidant activity.

Conclusion
This study utilized Na 2 SeO 3 and Tussilago farfara L. polysaccharides as primary sources, alongside nitric acid.The researchers optimized the preparation conditions for Tussilago farfara L. selenium polysaccharides and subsequently ascertained the optimal selenization process.The optimal synthesis process conditions obtained are 90 ℃, 12 hours, selenide reagent ratio of 1.2:1, HNO 3 volume fraction of 0.7%, and the average selenium content of STFPs measured under these conditions is 2.338 mg/g.This method of producing selenium polysaccharides, utilizing an nitric acid catalyst, has milder reaction conditions compared to other combinations and is effective.The results of antioxidant experiments showed that the maximum clearance rate of DPPH• by the selenium polysaccharide from Tussilago farfara L. was 73.16%,This research achievement provides a new approach for the comprehensive development and utilization of Tussilago farfara L., with certain practical and theoretical value, and can provide a certain reference for similar research, especially the development and utilization of polysaccharides.

Figure 1 .
Figure 1.Effect of reaction time to reaction temperature (A), selenium reagent ratio to reaction temperature (B), reaction temperature to HNO3 volume fraction (C), reaction time to selenium reagent ratio (D), HNO 3 volume fraction to reaction time (E), HNO 3 volume fraction to selenium reagent ratio (F) on selenium content sections, subsections and subsubsections

Figure 2 .
Figure 2. A. HPGPC.B. Infrared spectrum According to Figure 2A, the high performance gel liquid chromatography shows that the peak time of TFP is 9.035 min, and the peak time of STFPs is 9.031 min.By substituting it into the standard curve (y=-1.1613x+15.177,r=0.9993), the molecular weight of TFP is 48.37 kDa, and the molecular weight of STFPs is 48.89 kDa.Comparing the peak time of TFP, it can be concluded that the STFPs synthesized by TFP and selenide reagents have almost no effect on the structure of polysaccharides themselves.As shown in Figure2B, the absorption of STFPs, 3447 cm -1 represents the O-H stretching vibration, 2920 cm -1 represents the C-H stretching vibration, 1740 and 1602 cm -1 represent the stretching vibration absorption peak of carboxyl C=O, 1430 cm -1 represents the deformation absorption peak of CH 2 , and 1063 cm -1 represents the variable angle vibration absorption peak of alcohol hydroxyl groups, all of which are characteristic absorption peaks of polysaccharides[10] .Compared with TFP in the figure, the waveform, intensity, and peak width of its main absorption peaks are roughly the same as TFP, indicating that the main structure of STFPs obtained by selenization has not undergone significant changes.In addition, there is a characteristic peak at 664 cm -1 that is caused by the stretching vibration of the symmetric ring Se=O or Se-H.From this, it can be seen that the polysaccharides from winter flowers have successfully selenified[11] .

Table 2 .
Response surface analysis experimental scheme and experimental results

Table 3 .
Analysis of variance table