Optimization of the purification process of polysaccharides from Polygonati Rhizoma by using response surface methodology with macroporous adsorbent resin and in vitro antioxidant study
Yaosong Yang1; Siyi Zhang1; Shengxin Cui1; Juan Pei1; Sijin Tao1; Peiyu He1; Fei Long1; Teng Peng1,*
1Chengdu University of Traditional Chinese Medicine, Chengdu 611630, China
Objective: To investigate the purification effect of six macroporous resins, NKA-9, D101, HPD600, HP20, AB-8 and HPD750, on the processed Polygonati Rhizoma(PR) polysaccharides, to optimize the optimal purification process, and to investigate and analyze the in vitro antioxidant activity of the processed PR.
Methods: The weighted composite scores of polysaccharide retention, decolorization and deproteinization rate were used as the indexes to investigate the effects of resin type, sample concentration, elution flow rate, sample volume, and elution volume on the purification results, and a response surface model was established to optimize the process parameters for the purification of the processed PR polysaccharides from the macroporous resins by Box-Behnken design. The structural characteristics of processed PR polysaccharides before and after purification were analyzed by UV and FT-IR, and the total reducing capacity and scavenging capacity of ABTS, DPPH, and hydroxyl radicals were determined before and after purification according to the proposed protocol.
Results: The optimal macroporous resin purification process for the processed PR polysaccharides was as follows: D101 macroporous resin was selected, with a sampling concentration of 21 mg/mL, an elution flow rate of 1.0 mL/min, a sampling volume of 10 mL, and an elution volume of 40 mL. After purification by this optimal process, the retention rate of the processed PR polysaccharides was 63.96%, the decolorization rate was 94.00%, and the deproteinization rate was 94.82%. The impurities of purified the processed PR polysaccharides were significantly reduced and the polysaccharide structure was not changed. The results of in vitro antioxidant activity studies showed that the processed PR polysaccharides had some scavenging ability against ABTS, DPPH and hydroxyl radicals.
Conclusion: The structure and purification process of the processed PR polysaccharides were stable and reliable, and the purification process of D101 macroporous resin was effective, and the processed PR polysaccharides have certain reducing ability and antioxidant activity.
Key words: the processed PR polysaccharides; response surface methodology; macroporous resin; external oxidation resistance
Huangjing (Polygonati Rhizoma, PR), the dried rhizome of Polygonatum kingianum Coll, Polygonatum sibiricum Red or Polygonatum cyrtonema Hua in the family Liliaceae1, has the effect of nourishing yin and moistening the lungs, benefiting the vital essence and brightening the eyes, tonifying the spleen and benefiting the kidneys, and so on. Domestic and foreign studies have found that PR contains a variety of active ingredients such as polysaccharides, flavonoids, amino acids, etc. Among them, polysaccharides are one of the higher active ingredients mainly contained in PR2, which has a variety of physiological activities such as antioxidant, hypoglycemic, antitumour, immunomodulatory and anti-fatigue. At present, polysaccharide ingredients have been used as natural raw materials for the research and development of traditional Chinese medicine functional food.
Raw PR is spicy, stimulates the throat and causes allergic reactions. In contrast, the processed PR is sweet and not irritating and sensitized, and its function of nourishing spleen, moistening lung and benefiting kidney will be significantly enhanced, so the processed PR is often used as medicine. The processing of PR has a long history, and there are more than 10 kinds of processing methods, among which “nine steaming and nine drying” is the traditional processing process in China. Through long-term moistening, steaming, stewing and drying, all the ingredients in the drug fully react, and finally achieve effect-enhancing and toxicity-reducing. In recent years, medicinal plant polysaccharides have been widely used in medicine, health food development and other fields with good antioxidant activity. Natural polysaccharides not only have strong antioxidant capacity, but also have few side effects, which shows a wide application prospect in health promotion and disease prevention. As one of the active ingredients, PR polysaccharide has clear antioxidant activity. It has been found that the processed PR water extract has obvious antioxidant activity than the unprocessed PR water extract, and the effect is more prominent3.
Response surface methodology (RSM) has a wide range of applications in the field of biochemicals and pharmaceuticals due to its high modelling4, and compared with orthogonal design, the results of RSM are more accurate and more suitable for scientific research. At the same time, some studies have also shown that the macroporous resin method has a more significant decolorization and deproteinization than Sevag, TCA, activated carbon adsorption, etc. At present, the purification of the processed PR polysaccharides is not well studied, and a feasible and detailed solution is yet to be produced. Therefore, on the basis of the previous research, this experiment optimized the parameters of enrichment and purification of the processed PR polysaccharides with macroporous resin by using one-factor and Box-Behnken RSM, so as to determine the optimal process for the purification of the processed PR polysaccharides with macroporous resin. Meanwhile, we also explored the in vitro antioxidant activity of the processed PR polysaccharides before and after purification, which provided some theoretical basis for the further study of the processed PR polysaccharides and their exploitation as antioxidants.
1 Experimental apparatus and materials
1.1 Instruments and reagents
MAPADA UV/Visible Spectrophotometer (Shanghai Mapada Instrument Co., Ltd); Electronic Analytical Balance (Sartorius Scientific Instruments (Beijing) Co., Ltd); Infinitely Adjustable Power Ultrasonic Cleaning Machine (Xiaomei Ultrasonic Instruments (Kunshan) Co., Ltd); EYELA Rotary Evaporator (Shanghai Ailang Instrument Co., Ltd); Electro Thermal Blast Drying Oven (Beijing Zhongxingweiye Instrument Co., Ltd); TDZ5-WS Benchtop Low Speed Centrifuge (Xiangyi Centrifuge Instrument Co., Ltd); Freeze Dryer (Beijing Yaxing Yike Technology Development Co. , Ltd).
NKA-9, D101, HPD600, HP20, AB-8, and HPD750 type macroporous resins were purchased from Hecheng New Material CO.,Ltd; coomassie brilliant blue was purchased from Shanghai Maikun Chemical Co., Ltd, production lot 20230316; Glucose control was purchased from Sichuan Vicky Biotechnology Co., Ltd, production lot wkq22111408; DPPH, ABTS, Vitamin C, phenanthroline, phenol, concentrated sulfuric acid, anhydrous ethanol, potassium persulfate, anhydrous sodium dihydrogen phosphate, potassium ferricyanide and trichloroacetic acid was all purchased from ChengDu Chron Chemicals Co,.Ltd; Sterile PBS was purchased from BOSTER Biological Technology Co.Ltd; 30% hydrogen peroxide purchased from Shanghai Wokai Biological Technology Co. Ltd; ferrous sulfate was purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd; anhydrous disodium hydrogen phosphate was purchased from Sinopharm Chemical Reagent Co., Ltd; anhydrous iron trichloride was purchased from Shanghai Macklin Biochemical Co., Ltd, all the above reagents are analytical reagents.
1.2 Medicinal materials
The herb used in this experiment was identified as the dried rhizome of Polygonatum cyrtonema Hua from the lily family by Associate Professor Fei Long of the School of Pharmacy, Chengdu University of Traditional Chinese Medicine.
According to the preparation method of Lin et al.5 with minor modifications, clean, remove, remove surface sediment, cut into thick slices, and dry in 60℃ oven. Take 200 g of dry raw PR and steam for 8 h, take out, put in a drying box for 60℃ drying for 6 h, get “one steaming and one drying” PR, continue to follow the above operation for 8 times, finally get the “nine steaming and nine drying” PR.
2 Methods
- Extraction of PR
The PR should be prepared quantitatively, cut into pieces and dissolved in water (material/liquid ratio 1:10). The solution should then be subjected to reflux extraction for four hours, repeating this process twice. At the end of the extraction process, the resulting filtrate should be filtered and combined before concentration at 60 °C under pressure. The concentrated solution should be diluted with 95 % ethanol to a final concentration of 80 %. This solution should then be stored at 4℃ for 24 hours. The alcohol precipitation should be repeated twice, after which the solution should be centrifuged at 4000 rpm for 10 minutes. The resulting precipitate should be dissolved in distilled water and freeze-dried to obtain the processed PR polysaccharides.
2.2 Determination of absorption wavelengths of measured pigments
According to the principle of colour complementarity6, the colour of a solution is determined by the wavelength it absorbs, and the orange-yellow colour of the processed PR polysaccharides indicates that it absorbs blue light at a wavelength of approximately 480 nm. Therefore, 480 nm was chosen as the wavelength for pigment determination. The decolourisation rate was calculated as (A before decolourisation – A after decolourisation) / A before decolourisation × 100%.
2.3 Determination of total protein content and polysaccharide content
Determination of total protein content in the processed PR polysaccharides using coomassie brilliant blue staining. The test solution was prepared according to the kit instructions and the dilution ratio of the polysaccharide solution was adjusted to ensure that the protein concentration was below 1.3 g/mL. The absorbance value was then determined by UV-visible spectrophotometry at a wavelength of 595 nm after a standing time of five minutes. The deproteinization rate was calculated as (D before deproteinization – D after deproteinization) / D before deproteinization × 100%. The phenol-sulphuric acid method was used to determine the sugar content in processed PR polysaccharides7.
2.4 Pre-treatment of resin
Resin samples were taken and soaked in anhydrous ethanol for 12 h. The column was loaded using the wet loading column method, the leachate was released and washing with ethanol continued until the wash solution was no longer turbid with water and there was no absorption peak under UV scanning8. The resin column was then washed with a large volume of distilled water as mobile phase until the eluate was free of ethanol odour and stored wet at low temperature.
2.5 Static adsorption experiments
6 different types of macroporous resins (NKA-9, D101, HPD600, HP20, AB-8, HPD750) were taken into 100 mL conical flasks, 6 copies of each, and 20 mL of crude PR polysaccharides at 30 mg/mL were added and adsorbed at 30 ℃ on a slow shaker for 12 h, and then filtered. The resin was then washed with 20 mL of deionised water, adsorbed again for 12 h, filtered and the filtrates were combined to 25 mL and the absorbance at 450 nm was measured. The composite score S was calculated from polysaccharide retention A (40% weight), protein removal B (30% weight) and decolourisation C (30% weight). The formula is S = 0.4A + 0.3B + 0.3C. One-way analysis of variance (ANOVA) was performed using SPSS 25.0 to determine the resin with the best purification results. In this process, purification was achieved by the macroporous resin by adsorbing the pigments and proteins in solution and retaining the polysaccharides in the eluate.
2.6 Dynamic adsorption experiments
The resins with excellent performance in static screening were selected for wet column loading (column height of 20 cm, diameter of 2 cm), and six copies of each resin were operated in parallel. About 30 mg/mL of crude PR polysaccharide solution was added to half of the volume of the resin bed, and the flow rate was controlled to be 1 min/mL, so that the polysaccharides, pigments and proteins were allowed to adsorb staticly inside the column for 30 min to reach saturation adsorption. Subsequently, the column was eluted with distilled water at a flow rate of 1 min/mL until the eluate was negative for Molish reaction and collection was stopped. ANOVA was performed using SPSS 25.0 according to the scoring criteria under 2.5 to determine the resin with the best results for the purification of the processed PR polysaccharides under dynamic conditions.
2.7 One-factor examination experiment
Based on the determination of the optimal resin type, a one-way experiment was conducted to investigate the effects of sample mass concentration, elution flow rate, sample volume and elution volume on the purification of crude polysaccharides from the processed PR. The column was loaded according to the operation under 2.6 and the results of each condition were analysed by the weighted values of polysaccharide retention rate, decolourisation rate and deproteinization rate and a comprehensive score was obtained to determine the optimal conditions for each point.
2.8 Box-Behnken experimental design
On the basis of the single factor test, Box-Behnken design test was used, sample mass concentration (A), flow rate (B), eluted liquid product (C) as the independent variables, the weighted composite score of polysaccharide retention rate, decolourisation rate and deproteinization rate as the dependent variables, the response surface optimization experiment, and analyzed with Design-Expert 8.0.6.
2.9 In vitro antioxidant activity assay
2.9.1 DPPH free radical scavenging
A series of concentrations (0.2-1.0 mg·mL-1) of the processed PR solutions were prepared before and after purified, in accordance with the methodology outlined by Tahidul Islam et al9. 2 mg of DPPH were weighed with precision, dissolved in anhydrous ethanol, and adjusted to 100 mL. The test tubes were taken separately, and 2 mL of extract and 2 mL of DPPH solution were added to determine the absorbance (Ai), 2 mL of extract and 2 mL of anhydrous ethanol were added to determine the absorbance (Aj), 2 mL of 50% ethanol and 2 mL of DPPH solution were added to determine the absorbance (Ac). Following a 30-minute reaction period under light protection, the absorbance was measured at 517 nm, and the clearance was calculated. Meanwhile, the same concentration of vitamin C solution was used as a positive control.
DPPH free radical scavenging rate (%) = [1-(Ai-Aj)/Ac]×100% (1)
2.9.2 ABTS+ free radical scavenging
ABTS+ working solutions were prepared according to the method of Sirivibulkovit et al10: 7 mmol·L-1 ABTS was mixed with 2.45 mmol·L-1 potassium persulfate in equal volume, and placed at room temperature for 12 hours in a light-protected environment, and then diluted with anhydrous ethanol until the absorbance at 734 nm was 0.700 ± 0.005. Next, 4 mL of ABTS solution with 1 mL of different concentrations (0.2-1.0 mg·mL-1) of the processed PR polysaccharides solutions before and after purification were taken from test tubes, and the absorbance Ai was measured. In addition, 4 mL of absolute ethanol and 1 mL of different concentration (0.2-1.0 mg·mL-1) of the processed PR polysaccharides solutions were processed before and after purification to determine the absorbance Aj. Add 4 mL of ABTS+ solution with 1 mL of ultrapure water and measure the absorbance as Ac. The absorbance was measured at 734 nm after 6 min of reaction at room temperature and protected from light to calculate the ABTS+ clearance. Meanwhile, the same concentration of vitamin C solution was used as a positive control.
ABTS+ free radical scavenging rate (%) = [1-(Ai-Aj)/Ac]×100% (2)
2.9.3 Hydroxyl radical scavenging
Using the assay method of Xiong et al11, 1.0 mL of 5×10-3 mol·L-1 phenanthroline solution, 0.5 mL of 7.5×10-3 mol·L-1 ferrous sulfate solution, 1.0 mL of PBS buffer, 1.0 mL of the processed PR polysaccharides solutions before and after purification at different concentrations (0.2-1.0 mg·mL-1), and 0.5 mL of 0.1% hydrogen peroxide were added to the test tubes in turn, which was fixed to 10 mL with distilled water. The absorbance was measured at 510 nm after a water bath at 37°C for 30 min and the clearance rate was calculated according to equation (2). Meanwhile, the same concentration of vitamin C solution was used as a positive control.
Hydroxyl radical scavenging rate (%) = (A1-A2)/(A3-A2)×100% (3)
In the formula, A1 is the extract, A2 is replacing the extract with water, A3 is replacing the extract and H2O2 with water.
2.9.4 Determination of total reducing capacity
Referring to the method of An et al12 with slight modification, the total reducing capacity of the processed PR polysaccharides solutions were determined by the reduction method of potassium ferricyanide. After the pre-test, 1.0 mL of the processed PR polysaccharides solutions before and after purification at different concentrations (0.2-1.0 mg·mL-1) was added to 1.0 mL of 0.2 mol·L-1 phosphate buffer (pH 6.6) and 1.0 mL of 1% potassium ferricyanide solution, mixed and reacted at 50℃ for 20 minutes. Then, 1.0 mL of 10% trichloroacetic acid solution and 1.0 mL of 0.1% iron trichloride solution were added, mixed and placed 10 min, and the absorbance at 700 nm was measured.Meanwhile, distilled water was used as a blank control and vitamin C solution of the same concentration was used as a positive control.
Total reducing capacity (H) = A1-A0 (4)
3 Results
3.1 Experimental results of static and dynamic adsorption
According to the static adsorption results (Table 1), D101 resin exhibited the strongest adsorption capacity (63.91%), while HPD750 had the weakest adsorption capacity (51.03%). The static desorption capacity of all the resins was generally low, probably due to the fact that the desorption equilibrium was not reached. HP20 resin had the highest desorption rate (53.45%) while AB-8 had the lowest rate (36.24%). In order to screen the best resin, weighted values (adsorption rate × 50% + desorption rate × 50%) were used for evaluation. Although HP20 has the highest weighted value, its higher cost does not show a significant advantage. Therefore, NKA-9 (52.91 ± 1.189), D101 (50.28 ± 1.001) and HPD600 (53.11 ± 0.902) were selected for further dynamic adsorption screening.
According to the dynamic adsorption results of different resins ( Table 2), the D101 macroporous resin had the highest comprehensive score (53.72%) for the purification of the processed PR polysaccharides, and considering the economic benefits, D101 resin was selected as the best dynamic adsorption resin and used for subsequent experiments.
3.2 Results of single-factor examination
3.2.1 Sample mass concentration
The effect of sampling mass concentration on the effect of D101 resin purification of the processed PR polysaccharides (Table 3), from the data in the table, when the sampling mass concentration of 0.02 g/mL, the composite score of the purification effect reaches the maximum value, so the up-sampling mass concentration of 0.02 g/mL was selected as the mid-point of the response surface star-point design.
3.2.2 Eluant velocity
The impact of eluant velocity on the efficacy of D101 resin in purification from the processed PR polysaccharides (Table 4) is illustrated. It can be observed that at a flow rate of 1.0 min/mL, the comprehensive score reached its maximum. Consequently, the flow rate of 1.0 min/mL was identified as the midpoint of the response surface star point design.
3.2.3 Sampling quantity
The Influence of different sampling quantity on the effect of D101 resin on the purification of the processed PR polysaccharides (Table 5). According to the data, the comprehensive score reached the maximum when the upper sample volume was 10 mL, so the sampling amount of 10 mL was selected as the midpoint of the response surface star design.
3.2.4 Elution volume
The impact of varying elution volumes on the efficacy of D101 resin on the purification of the processed PR polysaccharides (Table 6) demonstrated a peak integrated score at an elution volume of 40 mL. Consequently, this volume was identified as the midpoint of the response surface star point design.
3.3 RSM to optimize the results of D101 resin on the purification of the processed PR polysaccharides
Following the screening of both static and dynamic pre-experimental results, D101 resin was identified as the optimal choice for the purification of the processed PR polysaccharides. The response surface experimental design and results of dynamic adsorption are presented in Table 7.
The Design-Expert 8.0.6 software analysis revealed that the results of the response surface test were fitted to the results of the response surface test with sampling mass concentration (A), flow rate (B) and elution volume (C) as variables, with the composite score of the polysaccharide purification effect serving as the response value (Y). This led to the quadratic multinomial regression equation being obtained as follows: Y = 82.14 + 0.36A + 0.45B + 0.47C- 0.37AB+0.17AC+0.54BC-1.33A2-1.66B2-2.02C2. The results of the analysis of variance and significance test for the model regression equation (Table 8) demonstrate that the simulated regression F-value for the response surface test is 38.51, P < 0.0001. This indicates that the quadratic polynomial simulation is statistically significant. Additionally, the P-value of the misfit error is 0.5476, which further supports the validity and statistical significance of the regression equation.
The linear terms A, B and C (P < 0.05) have a significant effect on the regression equation. Similarly, the interaction term BC (P < 0.05) has a significant effect, while the quadratic terms A2, B2 and C2 (P < 0.0001) have a highly significant effect. The P-values of the interaction factors AB and AC on the model are both greater than 0.05, so they were subjected to optimisation. Following the removal of all interaction terms (AB and AC), the value of lack of fit increased from 0.82 to 1.38, with the P-value changed from 0.5476 to 0.3877. Following the removal of the interaction term AB, the value of lack of fit altered from 0.82 to 2.60, while the P-value shifted from 0.5476 to 0.1884. Following the removal of the interaction term AC, the value of lack of fit altered from 0.82 to 0.81, while the P-value shifted from 0.5476 to 0.5774. Accordingly, the optimal solution is chosen to remove only the interaction term AC, and the factorial ANOVA tables preceding and following the optimisation (Tables 8 and 9). The fitting equations for the quadratic polynomials are as follows: Y=82.14+0.36A+0.45B+0.47C-0.37AB+0.54BC-1.33A2-1.66B2-2.02C2.
A response surface is a three-dimensional spatial map comprising the values of factors A, B, and C, along with their contour maps on a two-dimensional plane. This enables the visualisation of the interactions between the factors. The software was employed to obtain the results of the response surface analysis, as illustrated in Figs 2 and 3.
Based on the obtained model, the optimal process conditions were predicted: sampling mass concentration of 21.23 mg/ml, flow rate of 1.07 min/ml, elution volume of 40.70 ml, under which the weighted value of extracted the processed PR polysaccharides could reach 82.22%, but considering the feasibility of the actual situation and the precision of the experimental apparatus, the optimal process was adjusted to use a column with a length of 50 cm and a diameter of 2 cm with a sampling volume of 10 ml, a sampling mass concentration of 21 mg/ml, a flow rate of 1.0 min/ml and an elution volume of 40 ml.
Validation experiments were conducted under optimised conditions, and the resulting data were analysed to obtain the following values: polysaccharide retention (63.96 ± 3.40%), decolourisation rate (94.00 ± 0.35%), deproteinization rate (94.82 ± 1.17%), and weighted value (82.23 ± 1.54%). These values were compared with the predicted values, which were 82.22%. The results demonstrated a good fit between the predicted and actual values.
3.4 In vitro antioxidant activity
3.4.1 Scavenging activity against DPPH radicals
The results of the scavenging rate of DPPH radicals by the processed PR polysaccharides before and after purification are shown in Fig 4. As illustrated in the figure, the scavenging capacity of the processed PR polysaccharides exhibited an increase with an elevated concentration of polysaccharides. Moreover, the purified processed PR polysaccharides demonstrated a more pronounced scavenging effect. At a polysaccharide concentration of 0.6 mg/mL, the clearance rate reached 91.03%. Thereafter, it increased steadily and was slightly lower than that of the positive control VC group, which reflected good antioxidant activity.
3.4.2 Scavenging activity against ABTS+ radicals
The results of scavenging rate of ABTS+ free radicals by concocted flavonoid polysaccharides before and after purification are shown in Fig. (5). The scavenging of ABTS+ by both before and after purified concocted Rhizoma Polygonati polysaccharides showed a dose-dependent effect on ABTS+ compared to VC. The scavenging rate of purified concocted Rhizoma Polygonati polysaccharides could reach 88.03% when the solution concentration was 1 mg/mL. This indicates a good ability to resist ABTS+ radicals.
The scavenging rate of ABTS+ free radicals by the processed PR polysaccharides, both before and after purification, is illustrated in Fig 5. The scavenging of ABTS+ by both the pre- and post-purified processed PR polysaccharides demonstrated a dose-dependent effect on ABTS+ in comparison to VC. The scavenging rate of the purified polysaccharides reached 88.03% at a solution concentration of 1 mg/mL. This suggests that the substance in question displays a notable capacity to resist ABTS+ radicals.
3.4.3 Scavenging activity against hydroxyl radicals
The scavenging rate of hydroxyl radicals by the processed PR polysaccharides is illustrated in Fig 6, which depicts the results obtained before and after purification. As illustrated in the figure, the scavenging effect of the polysaccharides of the processed PR polysaccharides, both before and after purification, on the hydroxyl radicals was found to be positively proportional to the mass concentration within the range of 0.2-1.0 mg/mL. Furthermore, the purified polysaccharides exhibited a more pronounced effect. At a concentration of 1.0 mg/mL, the scavenging rate was observed to reach 25.11%. Nevertheless, a discrepancy was observed between the results and those obtained with VC at the same concentration. This could be attributed to the fact that polysaccharides were not the primary substances responsible for scavenging hydroxyl radicals in the processed PR.
3.4.4 Total reducing capacity
The outcomes of the total reducing capacity of the processed PR polysaccharides, both prior to and following purification, are illustrated in Fig 7. A greater reducing capacity correlates with a stronger antioxidant property. From the figure, the elevated absorbance values observed with increasing concentrations of the polysaccharides suggest an enhanced total reducing power. It is postulated that the reduced efficacy of the purified PR may be attributed to the removal of phenolics during the purification process.
4 Discussion
Pigments and proteins are the main impurities in polysaccharide studies, which affect the structural analysis13. Macroporous resins are widely used in various fields due to their unique composition and structure, combining adsorption and screening functions, higher adsorption capacity, easy elution, high strength, and strong resistance to contamination compared to other adsorbents or gel-based resins14. It has been demonstrated that macroporous resins demonstrate superior decolourisation and deproteinisation capabilities in polysaccharide purification when compared to alternative methods, including the Sevag method and the activated carbon method15. Accordingly, the purification of the processed PR polysaccharides was conducted in the present study via the macroporous resin method. Through the analysis of three-dimensional effect and contour plots of Box-Behnken response surface method, the established quadratic polynomial model is excellent, the surface is close to the sphere, the contour orthorhombicity is good, the ellipticity is low, and the preferred process parameters are located in the centre of the design range, which ensures the accuracy and stability of the results.
In this experiment, in the process of optimising the selection process, the specifications of the analytical column were fixed to facilitate the experimental study.In other cases, the specification of the analytical column, the sampling volume, and the elution volume can be enlarged or reduced by the same proportion, while the preferred sampling concentration must not fluctuate too much to avoid affecting the purification effect.
It was also found that the processed PR polysaccharides were able to scavenge certain DPPH free radicals, ABTS+ free radicals and hydroxyl free radicals, and had better reducing ability and antioxidant activity.
5 Conclusion
In conclusion, our study provides a more reliable method for further purification of the processed PR polysaccharides, and a preliminary exploration for further research on the development and utilisation of the polysaccharides of the processed PR polysaccharides as a kind of antioxidant.The structure and purification process of the processed PR polysaccharides were stable and reliable, and the purification process of D101 macroporous resin was effective, and the processed PR polysaccharides have certain reducing ability and antioxidant activity.
Data availability statement
The original contributions presented in the study are included in the article, and the corresponding authors can be contacted directly for further inquiries.
Author contributions
YS conducted study conception, design and data analyses. SY conducted draft preparation and prepared figures. FL identified the PR. SX and JP performed the experiments. SJ and PY conducted supervision of the study. All authors reviewed the manuscript, participated in the article, and approved the submitted version.
Acknowledgments
We thank PubMed and CNKI for providing the publicly available paper database.
Conflict of interest
We declare that the study was conducted without any business or financial relationship that could be interpreted as a potential conflict of interest.
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| Resin type | Adsorption capacity (mg/g) | Adsorption rate (%) | Desorption capacity (mg/g) | Desorption rate (%) | Weighted value (%) |
| NKA-9 | 4.60±0.115 | 54.13±1.364 | 2.38±0.111 | 51.69±3.680 | 52.91±1.189 |
| D101 | 5.43±0.192 | 63.91±2.292 | 1.99±0.164 | 36.66±4.137 | 50.28±1.001 |
| HPD600 | 4.55±0.055 | 53.60±0.680 | 2.39±0.083 | 52.62±2.467 | 53.11±0.902 |
| HPD750 | 4.34±0.300 | 51.03±3.507 | 1.77±0.137 | 40.88±2.766 | 45.96±1.739 |
| HP20 | 4.65±0.150 | 54.79±1.765 | 2.48±0.211 | 53.45±6.119 | 54.12±2.217 |
| AB-8 | 5.10±0.158 | 59.97±1.905 | 1.84±0.176 | 36.24±4.581 | 48.11±1.339 |
Table 1
Comprehensive evaluation table of static adsorption of different macroreticular resins. (, n=3).
| Resin type | Comprehensive score |
| NKA-9 | 39.05±3.19 |
| D101 | 53.72±9.01 |
| HPD600 | 39.84±4.00 |
Table 2
Comprehensive evaluation table for dynamic adsorption of different macroreticular resins (, n=3).
| ρsampling (g·ml-1) | Polysaccharide retention rate (%) | Decolourisation rate (%) | Deproteinization rate (%) | Comprehensive score |
| 0.01 | 73.92 | 78.30 | 86.38 | 78.97 |
| 0.02 | 87.94 | 82.87 | 84.83 | 85.49 |
| 0.03 | 87.66 | 82.10 | 83.04 | 84.60 |
| 0.04 | 83.11 | 83.76 | 84.54 | 83.74 |
| 0.05 | 83.78 | 83.92 | 81.30 | 83.08 |
Table 3
The purification effects for the processed PR polysaccharides of different sample mass concentration of D101 resin.
| Eluant velocity (min/ml) | Polysaccharide retention rate (%) | Decolourisation rate (%) | Deproteinization rate (%) | Comprehensive score |
| 0.5 | 76.72 | 81.80 | 87.06 | 81.34 |
| 1 | 79.27 | 82.69 | 87.33 | 82.71 |
| 1.5 | 79.51 | 82.53 | 81.15 | 80.91 |
| 2 | 78.66 | 83.09 | 80.58 | 80.57 |
Table 4
The purification effects for the processed PR polysaccharides of different eluant velocity of D101 resin.
| Sampling quantity (ml) | Polysaccharide retention rate (%) | Decolourisation rate (%) | Deproteinization rate (%) | Comprehensive score |
| 5 | 85.43 | 81.00 | 79.45 | 82.31 |
| 10 | 89.50 | 83.32 | 90.72 | 88.01 |
| 15 | 78.57 | 88.18 | 90.32 | 84.98 |
| 20 | 80.18 | 85.38 | 79.10 | 81.41 |
Table 5
The purification effects for the processed PR polysaccharides of different sampling quantity of D101 resin.
| Eluant volume (ml) | Polysaccharide retention rate (%) | Decolourisation rate (%) | Deproteinization rate (%) | Comprehensive score |
| 35 | 59.24% | 90.72% | 90.32% | 78.01 |
| 40 | 65.97% | 93.36% | 94.79% | 82.84 |
| 45 | 64.82% | 87.01% | 89.52% | 78.89 |
Table 6
The purification effects for the processed PR polysaccharides of different eluant volume of D101 resin.
| No. | A Sample mass concentration (mg·ml-1) | B Flow rate (min·ml-1) | C Effluent volume （ml） | Y Comprehensive score |
| 1 | 10.00 | 0.50 | 40.00 | 77.70 |
| 2 | 30.00 | 0.50 | 40.00 | 79.33 |
| 3 | 10.00 | 1.50 | 40.00 | 79.70 |
| 4 | 30.00 | 1.50 | 40.00 | 79.87 |
| 5 | 10.00 | 1.00 | 35.00 | 78.26 |
| 6 | 30.00 | 1.00 | 35.00 | 78.45 |
| 7 | 10.00 | 1.00 | 45.00 | 78.79 |
| 8 | 30.00 | 1.00 | 45.00 | 79.66 |
| 9 | 20.00 | 0.50 | 35.00 | 78.23 |
| 10 | 20.00 | 1.50 | 35.00 | 77.68 |
| 11 | 20.00 | 0.50 | 45.00 | 78.17 |
| 12 | 20.00 | 1.50 | 45.00 | 79.77 |
| 13 | 20.00 | 1.00 | 40.00 | 81.86 |
| 14 | 20.00 | 1.00 | 40.00 | 82.38 |
| 15 | 20.00 | 1.00 | 40.00 | 81.70 |
| 16 | 20.00 | 1.00 | 40.00 | 82.64 |
| 17 | 20.00 | 1.00 | 40.00 | 82.10 |
Table 7
Box-Behnken response surface experimental design and results.
| Sources of variation | Quadratic sum | Degree of freedom | Mean square | F-value | P-value | Significance |
| Model | 46.42 | 9 | 5.16 | 38.51 | ＜0.0001 | ++ |
| A | 1.02 | 1 | 1.02 | 7.63 | 0.0280 | + |
| B | 1.61 | 1 | 1.61 | 12.03 | 0.0104 | + |
| C | 1.78 | 1 | 1.78 | 13.26 | 0.0083 | + |
| AB | 0.53 | 1 | 0.53 | 3.98 | 0.0863 | |
| AC | 0.12 | 1 | 0.12 | 0.86 | 0.3838 | |
| BC | 1.16 | 1 | 1.16 | 8.63 | 0.0218 | + |
| A2 | 7.44 | 1 | 7.44 | 55.54 | 0.0001 | ++ |
| B2 | 11.56 | 1 | 11.56 | 86.28 | ＜0.0001 | ++ |
| C2 | 17.13 | 1 | 17.13 | 127.86 | ＜0.0001 | ++ |
| Residual | 0.94 | 7 | 0.13 | |||
| Lack of fit | 0.36 | 3 | 0.12 | 0.82 | 0.5476 | |
| Pure error | 0.58 | 4 | 0.15 | |||
| Total | 47.36 | 16 |
Table 8
Variance analysis of unoptimized multiple regression model.
| Sources of variation | Quadratic sum | Degree of freedom | Mean square | F-value | P-value | Significance | |
| Model | 46.30 | 8 | 5.79 | 43.96 | ＜0.0001 | ++ | |
| A | 1.02 | 1 | 1.02 | 7.77 | 0.0237 | + | |
| B | 1.61 | 1 | 1.61 | 12.24 | 0.0081 | + | |
| C | 1.78 | 1 | 1.78 | 13.50 | 0.0063 | + | |
| AB | 0.53 | 1 | 0.53 | 4.05 | 0.0790 | ||
| BC | 1.16 | 1 | 1.16 | 8.78 | 0.0181 | + | |
| A2 | 7.44 | 1 | 7.44 | 56.51 | 0.0001 | ++ | |
| B2 | 11.56 | 1 | 11.56 | 87.79 | ＜0.0001 | ++ | |
| C2 | 17.13 | 1 | 17.13 | 130.08 | ＜0.0001 | ++ | |
| Residual | 0.94 | 7 | 0.13 | ||||
| Lack of fit | 0.36 | 3 | 0.12 | 0.81 | 0.5774 | ||
| Pure error | 0.58 | 4 | 0.15 | ||||
| Total | 47.36 | 16 | |||||
Table 9
Variance analysis of optimized multiple regression model.
Fig.1
The effect of sample mass concentration and flow rate on the purification weighted value of the processed PR polysaccharides.
Fig.2
The effect of flow rate and elution volume on the purification weighted value of the processed PR polysaccharides.
Fig.3
Scavenging capacity of the processed PR polysaccharides on DPPH free radicals.
Fig.4
Scavenging capacity of the processed PR polysaccharides on ABTS+ free radicals.
Fig.5
Scavenging capacity of the processed PR polysaccharides on hydroxyl radicals.
Fig.6
Total reducing capacity of the processed PR polysaccharides.