Optimization Of Decolorization Process And Antioxidant Activity Of Cistanche Deserticola Polysaccharide Extract By Response Surface Methodology Ⅱ
Mar 10, 2025
2 Results and Analysis
2.1 Single-Factor Experiments
2.1.1 Effect of Activated Carbon Dosage on Decolorization of Cistanche Polysaccharide Extract
Activated carbon decolorization is a type of adsorption decolorization. The particle surface of activated carbon contains numerous pores and channels, which can strongly adsorb pigment molecules. However, it can also adsorb polysaccharide molecules in the solution, causing polysaccharide loss [25]. As shown in Figure 1A:
With the increase in activated carbon dosage, the decolorization rate gradually increases. However, when the dosage exceeds 10%, the growth trend of the decolorization rate slows down.
When the dosage is below 20%, the polysaccharide recovery rate increases, but when it exceeds 20%, the recovery rate starts to decrease.
When the dosage is 20%, the decolorization effect is optimal.
Considering the operational and cost factors in practical production, 10%, 20%, and 30% were selected as the activated carbon dosage levels for the response surface experiment.
High-Level Polysaccharide Herbal Cistanche Supplements
2.1.2 Effect of Decolorization Time on Cistanche Polysaccharide Extract
The adsorption of pigment molecules by activated carbon requires a certain amount of time. As shown in Figure 1B:
Within 10–50 minutes, the decolorization rate gradually increases. However, when the decolorization time exceeds 50 minutes, the decolorization rate starts to decrease. This phenomenon occurs because decolorization via activated carbon involves a dynamic process of adsorption and desorption. As the desorption of pigment molecules accelerates with prolonged interaction time, the volume of desorbed pigment molecules increases, resulting in a decrease in the decolorization rate.
The polysaccharide recovery rate shows a gradual downward trend. This may be because the polysaccharide content in the solution is significantly higher than the adsorption capacity of the activated carbon, reducing the difference between the adsorption and desorption rates [26].
Although the decolorization rate and recovery rate during the first 30 minutes are acceptable, it is challenging to process large volumes of samples within such a short time in practical production.
Therefore, considering practical production conditions, 40, 50, and 60 minutes were selected as the decolorization time levels for the response surface experiment.
Fig.1 Influence of activated carbon concentration, decolorization time, decolorization temperature and pH on decolorization effects of Cistanche deserticola polysaccharide
2.1.3 Effect of Decolorization Temperature on the Decolorization of Cistanche Polysaccharide Extract
As shown in Figure 1C, within the range of 20–40 °C, the decolorization rate of Cistanche gradually increases with rising decolorization temperature. When the decolorization temperature reaches 40 °C, the decolorization rate and polysaccharide recovery rate are 64.39% and 84.49%, respectively. However, when the temperature exceeds 40 °C, the decolorization rate begins to decline. This may be because excessively high temperatures negatively affect the adsorption of pigments by activated carbon, thereby weakening the decolorization effect.
As the decolorization temperature increases, the polysaccharide recovery rate decreases slightly. However, minor variations in temperature have little impact on the adsorption of polysaccharides by activated carbon [11]. Within the range of 30–50 °C, the mass recovery rate remains relatively high. Considering energy consumption in practical production processes, 30 °C, 40 °C, and 50 °C were selected as the decolorization temperature levels for the response surface experiment.
2.1.4 Effect of pH on the Decolorization of Cistanche Polysaccharide Extract
The adsorption decolorization process using activated carbon requires the participation of H⁺ ions. Appropriately increasing the concentration of H⁺ ions in the solution can improve the decolorization rate [27]. As shown in Figure 1D, with the increase in pH, the decolorization rate of Cistanche generally shows a downward trend, while the polysaccharide recovery rate gradually increases. This may be because low pH levels affect the structure of the polysaccharides, reducing the recovery rate.
Within the range of pH 4–7, the decline in decolorization rate is not significant, and the polysaccharide recovery rate remains relatively high. Considering that the measured pH of the sample solution is 5.03, choosing pH 5.03 as the optimal pH in industrial production can avoid the use of acids and bases, reduce costs, and benefit environmental protection. Therefore, pH levels of 4, 5, and 6 were selected as the pH levels for the response surface experiment.
2.2 Optimization of Decolorization Process for Cistanche Polysaccharide Extract Using Response Surface Methodology
2.2.1 Results of Response Surface Optimization Experiments
The experimental results are shown in Table 2, and the variance analysis results are shown in Table 3. From Table 3:
In the response surface fitting model using decolorization rate (Y1) and polysaccharide recovery rate (Y2) as response values, the P-values of the models for Y1 and Y2 were both less than 0.01, indicating highly significant differences. Thus, the models are meaningful.
The P-values of the lack-of-fit terms were both greater than 0.05, indicating no significant differences. This suggests that the predicted values of the model fit well with the actual values, making them suitable for predicting experimental results and determining the optimal process conditions.
The correlation coefficients (R²) for Y1 and Y2 were 0.8633 and 0.9308, respectively, indicating that the model could predict 86.33% and 93.08% of the experimental results, with small errors and good predictive capability.
Through multiple regression fitting and variance analysis, the quadratic polynomial regression models for the decolorization rate (Y1) and polysaccharide recovery rate (Y2) regarding activated carbon dosage, time, temperature, and pH were established as follows:
Decolorization rate (Y1):
Y1 = 61.21 + 0.10A – 0.22B – 0.44C – 0.29D – 0.03AB + 0.10AC – 0.24AD + 0.07BC – 0.07BD – 3.08A² – 1.27B² – 1.09C² – 1.82D²
Polysaccharide recovery rate (Y2):
Y2 = 97.93 + 0.12A – 0.41B – 0.54C – 0.30D – 0.04AB – 0.08AC – 0.13AD + 0.08BC + 0.04BD + 0.04CD – 3.78A² – 1.15B² – 1.51C² – 2.10D²
Additionally, the quadratic models for both response values showed highly significant differences. Factors C, A², B², C², and D² had significant effects on the decolorization rate of the Cistanche polysaccharide extract (P < 0.05 or P < 0.01). Similarly, factors B, C, A², B², C², and D² had significant effects on polysaccharide recovery rate (P < 0.05 or P < 0.01). This indicates that the relationships between the factors and the response values are not simple linear relationships.
2.2.2 Interaction Between Two Factors on Decolorization Rate
Using Design Expert 8.0.6 software, the interaction effects between factors were analyzed, and 3D response surface plots were generated to visually analyze the interactions between pairs of factors and their effects on the response values.
As shown in Figures 2a–f:
As the levels of the interacting factors increased, the decolorization rate of the Cistanche polysaccharide extract showed a trend of initially increasing and then decreasing.
The changes between activated carbon dosage and other factors were more pronounced, indicating strong interactions between these factors.
In contrast, the changes between decolorization temperature and decolorization time, pH and decolorization temperature, and pH and decolorization time were less pronounced, indicating weaker interactions between these pairs of factors.
2.2.3 Interaction Between Two Factors on Polysaccharide Recovery Rate
As shown in Figures 3a–f:
As the levels of the interacting factors increased, the polysaccharide recovery rate of the Cistanche polysaccharide extract also showed a trend of initially increasing and then decreasing.
The changes between activated carbon dosage and other factors were more pronounced, indicating strong interactions between these factors.
In contrast, the changes between decolorization temperature and decolorization time, pH and decolorization temperature, and pH and decolorization time were less pronounced, indicating weaker interactions between these pairs of factors.
| Experiment No. | A Activated Carbon Dosage (%) | B Decolorization Time (min) | C Decolorization Temperature (°C) | D pH | Y₁ Decolorization Rate (%) | Y₂ Polysaccharide Recovery Rate (%) |
|---|---|---|---|---|---|---|
| 1 | 0 | 0 | 0 | 0 | 57.66 | 94.60 |
| 2 | 1 | 0 | 0 | 0 | 57.75 | 93.45 |
| 3 | -1 | 0 | 0 | 0 | 57.65 | 93.46 |
| 4 | 0 | 1 | 0 | 0 | 57.66 | 93.42 |
| 5 | 0 | -1 | 0 | 0 | 57.64 | 93.40 |
| 6 | 0 | 0 | 1 | 0 | 57.65 | 93.31 |
| 7 | 0 | 0 | -1 | 0 | 57.63 | 93.30 |
| 8 | 0 | 0 | 0 | 1 | 57.64 | 93.47 |
| 9 | 0 | 0 | 0 | -1 | 57.62 | 93.46 |
| 10 | 1 | 1 | 0 | 0 | 57.75 | 93.39 |
| 11 | 1 | -1 | 0 | 0 | 57.73 | 93.30 |
| 12 | 1 | 0 | 1 | 0 | 57.74 | 93.25 |
| 13 | 1 | 0 | -1 | 0 | 57.72 | 93.23 |
| 14 | 1 | 0 | 0 | 1 | 57.73 | 93.40 |
| 15 | 1 | 0 | 0 | -1 | 57.71 | 93.39 |
| 16 | -1 | 1 | 0 | 0 | 57.65 | 93.42 |
| 17 | -1 | -1 | 0 | 0 | 57.63 | 93.30 |
| 18 | -1 | 0 | 1 | 0 | 57.64 | 93.25 |
| 19 | -1 | 0 | -1 | 0 | 57.62 | 93.23 |
| 20 | -1 | 0 | 0 | 1 | 57.63 | 93.40 |
| 21 | -1 | 0 | 0 | -1 | 57.61 | 93.39 |
| 22 | 0 | 1 | 1 | 0 | 57.66 | 93.25 |
| 23 | 0 | -1 | -1 | 0 | 57.62 | 93.23 |
| 24 | 0 | 1 | -1 | 0 | 57.64 | 93.23 |
| 25 | 0 | -1 | 1 | 0 | 57.64 | 93.25 |
| 26 | 0 | 1 | 0 | 1 | 57.65 | 93.42 |
| 27 | 0 | -1 | 0 | -1 | 57.63 | 93.39 |
| 28 | 0 | 0 | 0 | 0 | 57.66 | 94.60 |
| 29 | 0 | 0 | 0 | 0 | 57.66 |
| Source | Coefficient | Y₁ | Y₂ | Sum of Squares | Y₁ | Y₂ | Mean Square | Y₁ | Y₂ | F Value | Y₁ | Y₂ | P Value | Y₁ | Y₂ |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Model | 13.64 | 73.29 | 78.39 | 14.55 | 10.88 | <0.0001 | <0.0001 | ||||||||
| A (Activated Carbon Dosage) | 0.10 | 0.72 | 0.98 | <0.05 | |||||||||||
| B (Time) | |||||||||||||||
| Note |
2.2.4 Determination of Optimal Process Conditions and Validation Experiments
Using the Box-Behnken design, the experimental scheme was developed, and the experimental data were analyzed using Design Expert 8.0.6 software. The optimal decolorization process conditions for the Cistanche polysaccharide extract with activated carbon were determined as follows:
Activated carbon dosage: 20.18%
Temperature: 37.74 °C
Time: 48.88 minutes
pH: 4.92
Under these conditions, the predicted results were:
Decolorization rate: 61.25%
Polysaccharide recovery rate: 98.02%
Considering practical production feasibility, the optimal process conditions were slightly adjusted to:
Activated carbon dosage: 20%
Temperature: 37 °C
Time: 49 minutes
pH: 5.03
Under these adjusted optimal conditions, three parallel validation experiments were conducted. The results were:
Decolorization rate: 62.66%
Polysaccharide recovery rate: 96.16%
The measured values showed deviations from the theoretical values of the regression model within a reasonable range, demonstrating that the model is valid [28].
2.3 Antioxidant Activity of Cistanche Polysaccharides
2.3.1 DPPH Radical Scavenging Ability of Cistanche Polysaccharides
Plant polysaccharides exhibit significant antioxidant activity, generally within the concentration range of 1.0–10.0 mg/mL. The decolorization process can influence the antioxidant activity of polysaccharides [16,29].
As shown in Figure 4, the DPPH radical scavenging ability of decolorized Cistanche polysaccharides was significantly improved. At a concentration of 1.0 mg/mL, the DPPH radical scavenging rate increased from 14.78% (before decolorization) to 38.38% (after decolorization).
This indicates that the decolorization process can enhance the DPPH radical scavenging ability of Cistanche polysaccharides. This improvement may be attributed to the removal of impurities during the decolorization process, which increases the purity of the polysaccharides and subsequently enhances their activity.
2.3.2 Total Reducing Power of Cistanche Polysaccharides
Under acidic conditions, antioxidants can reduce Fe³⁺-TPTZ (ferric-tripyridyltriazine) to Fe²⁺-TPTZ, which produces a blue color. The absorbance at 593 nm can be used to calculate the total reducing power of the sample. A higher FRAP value indicates stronger total reducing power [30].
As shown in Figure 5, the total reducing power of 1.0 mg/mL Cistanche polysaccharides showed no noticeable difference before and after decolorization. This may be due to the lack of substances in the extract capable of reacting with Fe³⁺-TPTZ, or the presence of substances that inhibit the conversion of Fe³⁺-TPTZ to Fe²⁺-TPTZ.
2.3.3 Hydroxyl Radical Scavenging Ability of Cistanche Polysaccharides
As shown in Figure 6, the hydroxyl radical scavenging rate of 1.0 mg/mL Cistanche polysaccharides increased from 84.37% (before decolorization) to 96.18% (after decolorization).
This indicates that Cistanche polysaccharides have significant hydroxyl radical scavenging ability, which was further enhanced after decolorization. This improvement may be attributed to the increase in polysaccharide purity after the decolorization process, which enhances the hydroxyl radical scavenging ability.
Similarly, Sun Mingli et al. [31] found that the hydroxyl radical scavenging ability of Scutellaria polysaccharides was significantly enhanced after decolorization, consistent with the results obtained in this study.
2.3.4 ABTS Cation Radical Scavenging Ability of Cistanche Polysaccharides
Studies have shown that decolorization treatment of polysaccharides can reduce their ABTS cation radical scavenging ability [32]. As shown in Figure 7, 1.0 mg/mL Cistanche polysaccharides exhibit significant ABTS cation radical scavenging activity. However, after decolorization, the activity decreased significantly, dropping from 7.85% (before decolorization) to 0.89% (after decolorization).
This indicates that the decolorization process weakens the ABTS cation radical scavenging ability of Cistanche polysaccharides. This reduction may be due to the removal of components within the Cistanche polysaccharides that react with ABTS cation radicals, leading to a decline in scavenging ability.
3 Conclusion
This study used activated carbon as a decolorizing agent, with decolorization rate and polysaccharide recovery rate as evaluation indicators. Based on single-factor experimental results, response surface experimental results, and practical production feasibility, the optimal process conditions were determined as follows:
Activated carbon dosage: 20%
Temperature: 37 °C
Time: 49 minutes
pH: 5.03
Under these conditions, the decolorization rate of the Cistanche polysaccharide extract was 62.66%, and the polysaccharide recovery rate was 96.16%. The resulting polysaccharides showed significantly improved DPPH radical scavenging ability and hydroxyl radical scavenging ability.
This decolorization process demonstrated good decolorization efficiency and high polysaccharide recovery rate, providing a reference for preparing high-purity, aesthetically superior Cistanche polysaccharide products.
