The authors have declared that no competing interests exist.
Response surface methodology (RSM) was used to investigate the extraction condition of polysaccharide from cup plant (
Cup plant (
Polysaccharide, widely distributing in animal, plant and microorganism, is a kind of polymeric carbohydrate [
In addition, the physicochemical properties and bioactivities of polysaccharide were significantly affected by its drying methods. Hot air drying, vacuum drying and freeze drying have been widely used for the drying process of polysaccharide. The most common drying method used in food processing is hot air drying due to its easy accessibility and lower cost. However, serious physicochemical changes of the dried polysaccharide may appear during hot air drying process [
The aim of this study was to discover the potential of cup plant in production of CPP and to research the optimum extraction condition (water to raw material ratio, extraction time and extraction temperature) of CPP using RSM. In addition, the effects of three drying methods (hot air drying, vacuum drying and freeze drying) on the physicochemical properties and antioxidant activities of CPP were determined to seek the potential drying techniques. The
Fresh cup plant was harvested in Jilin Agricultural University (Changchun, China). The plants were in bloom stage. The entire crop was cut into 5 cm segments before drying at 50°C, then the dried cup plant segments were ground into powders, and passed through a 1 mm sieve prior to extraction of the polysaccharide. The reagents including DPPH radical, ABTS radical and vitamin C were obtained from Sigma-Aldrich (St. Louis, USA). All other chemicals used were analytical grade and bought from local suppliers.
The lipids of cup plant powder were taken out with 85% (v/v) ethanol for 24 h. The insoluble residue was dried in a vacuum freeze dryer and prepared for the extraction of polysaccharide with hot distilled water according to the designed water to raw material ratio, extraction time and extraction temperature. The extract was filtered with six layers gauze, and then the filtrate was concentrated to a 1/4 volume of the primary volume using a rotary evaporator under reduced pressure at 60°C. Subsequently, the concentrated solution was precipitated with 4 volumes of absolute ethanol. After being left for 12 h at 4°C, the precipitate was collected by centrifugation (3000 rpm for 15 min), and washed three times with absolute ethanol. Then the precipitate was dissolved with deionized water, removed protein by the Seveg reagent (4:1 of chloroform: normal butanol, v/v), and dialyzed in a dialysis bag (MWCO 1400 Da, Union Carbide). CPP was obtained by freeze-dried method. The extraction yield (
In the single-factor experiment, three variables including water to raw material ratio (10, 15, 20, 25 and 30 mL/g), extraction time (40, 50, 60, 70 and 80 min), and extraction temperature (60, 70, 80, 90 and 100°C) were taken into consideration to investigate their effects on the extraction yield of CPP. Each sample was extracted with hot distilled water according to the polysaccharide extraction procedure mentioned above. The CPP yields of different conditions were compared by one-way analysis of variance (ANOVA) using SPSS 17.0 (SPSS Inc., Chicago, IL).
After the appropriate ranges of water to raw material ratio (
CPP was extracted under the optimum extraction condition, and dried using three different methods including hot air drying (HD), vacuum drying (VD) and freeze drying (FD). HD was carried out in an electric heating air-blowing drier (101-2-BS, Shanghai Yuejin Medical Instrument co., LTD, Shanghai, China) at 50°C. VD was done in a vacuum drying oven (DZF, Shanghai Longyue Instrument Equipment co., LTD, Shanghai, China) at 50°C. FD was carried out in a vacuum freeze dryer (SCIENTZ-12N, Ningbo Scientz Biotechnology co., LTD, Ningbo, China) at −70°C. The CPP obtained by HD, VD and FD were named as HD-CPP, VD-CPP and FD-CPP, respectively.
The total polysaccharide content of CPP was determined by phenol-sulfuric acid method using D-glucose as standard [
The moisture content of the CPP was measured according to the literature [
The solubility of CPP was determined in distilled water. Briefly, the CPP sample (0.1 g) was placed into distilled water (50 mL) in a beaker, and then the beaker was placed in a water bath of different temperature (20, 40, 60, 80, 100°C). The CPP solution was stirred (150 rpm/min) with a digital mixer (JJ-5, Medical Instrument Factory of Jintan City, Jintan, China) until it was completely dissolved, and the length of dissolved time was recorded.
The CPP sample (1.0 mg) was dissolved in deionized water (2.0 mL), and then aqueous Congo red (2.0 mL, 80 μmol/L) in 0.001 M NaOH was added to the CPP solution. Aqueous solution of NaOH (4.0 mol/L) was added to the mixture till the final concentration of NaOH in the mixture was in the range from 0 to 0.5 mol/L. The maximum absorption wavelength (λ max) of the reaction solution was measured using a spectrophotometer (752, Shanghai Xianke Spectrophotometer Instrument co., LTD, Shanghai, China).
DPPH radical scavenging activity of CPP was measured according to a previous method with some modifications [
ABTS radical scavenging activity of CPP was determined with a previous method with minor modifications [
The ferric reducing power of CPP was evaluated with the reported method [
All experiments were performed at least three times. Analyses of all samples were run in triplicate and averaged. All values were presented as mean±standard deviation (SD). The statistical analysis was performed using one-way ANOVA in SPSS 17.0 for Windows (SPSS Inc., Chicago, IL). Duncan post hoc tests were performed when significant differences were found.
To evaluate the influence of water to raw material ratio on the extraction yield of CPP (
Extraction time is an important parameter affecting the extraction yield of polysaccharide. To investigate the influence of extraction time on the extraction yield of CPP (
To evaluate the influence of extraction temperature (60, 70, 80, 90 and 100°C) on the yield of CPP (
Base on the single-factor experiments, 17 runs were performed to optimize the extraction conditions of CPP. The designed matrix and the corresponding CPP yields (%) of the 17 runs to evaluate the 3 independent variables including water to raw material ratio (
Run | Water to raw material ratio ( |
Extraction time ( |
Extraction temperature ( |
CPP yields (%) | |
---|---|---|---|---|---|
Actual value | Predicted value | ||||
-1(10) | -1(50) | 0(90) | 5.26 | 5.32 | |
1(20) | -1(50) | 0(90) | 5.63 | 5.68 | |
-1(10) | 1(70) | 0(90) | 5.70 | 5.65 | |
1(20) | 1(70) | 0(90) | 5.97 | 5.91 | |
-1(10) | 0(60) | -1(80) | 5.16 | 5.17 | |
1(20) | 0(60) | -1(80) | 5.70 | 5.72 | |
-1(10) | 0(60) | 1(100) | 5.74 | 5.73 | |
1(20) | 0(60) | 1(100) | 5.81 | 5.80 | |
0(15) | -1(50) | -1(80) | 5.32 | 5.25 | |
0(15) | 1(70) | -1(80) | 5.45 | 5.50 | |
0(15) | -1(50) | 1(100) | 5.58 | 5.53 | |
0(15) | 1(70) | 1(100) | 5.78 | 5.85 | |
0(15) | 0(60) | 0(90) | 6.75 | 6.81 | |
0(15) | 0(60) | 0(90) | 6.81 | 6.81 | |
0(15) | 0(60) | 0(90) | 6.71 | 6.81 | |
0(15) | 0(60) | 0(90) | 6.93 | 6.81 | |
0(15) | 0(60) | 0(90) | 6.85 | 6.81 |
CPP, polysaccharide from cup plant.
The result of ANOVA analysis was shown in
Source | Sum of squares | df | Mean square | ||
---|---|---|---|---|---|
5.88 | 9 | 0.65 | 79.97 | < 0.0001 |
|
0.20 | 1 | 0.20 | 23.87 | 0.0018 |
|
0.16 | 1 | 0.16 | 19.65 | 0.0030 |
|
0.20 | 1 | 0.20 | 24.84 | 0.0016 |
|
2.554E-003 | 1 | 2.554E-003 | 0.31 | 0.5937 | |
0.058 | 1 | 0.058 | 7.11 | 0.0322 |
|
1.341E-003 | 1 | 1.341E-003 | 0.16 | 0.6976 | |
1.28 | 1 | 1.28 | 156.30 | < 0.0001 |
|
1.62 | 1 | 1.62 | 197.66 | < 0.0001 |
|
1.82 | 1 | 1.82 | 222.48 | < 0.0001 |
|
0.057 | 7 | 8.176E-003 | |||
0.027 | 3 | 9.093E-003 | 1.21 | 0.4126 | |
0.030 | 4 | 7.489E-003 | |||
5.942 | 16 | ||||
0.9904 | |||||
0.9780 | |||||
1.52 | |||||
23.709 |
*
**
CPP, polysaccharide from cup plant.
The determination coefficient (
The visual interpretation of individual and interaction effects of variables on the CPP yield could be presented with 3-dimensional (3D) response surface plots and their corresponding 2-dimensional (2D) contour plots. A variable was set at the 0 level, and anther two variables were described in the 3D and 2D surface plots. The patterns of the 2D contour plots could indicate the mutual interactions significance between two tested variables. The contour plot of circular shape indicates insignificant mutual interactions between the two variables, while the contour plot of elliptical or saddle shapes suggests significant mutual interactions between the two variables [
Response surface and contour plots for the polysaccharide yield of cup plant: effects of (A and B) water to raw material ratio and extraction time; (C and D) water to raw material ratio and extraction temperature on the polysaccharide yield; (E and F) extraction time and extraction temperature on the polysaccharide yield.
By analyzing the regression equation, the optimal conditions for the extraction of CPP were obtained as follow: water to raw material ratio 15.29 mL/g, extraction time 61.25 min, and extraction temperature 97.39°C. A maximum predicted CPP yield of 6.58% was obtained under these conditions. Considering the convenience during actual operation, the actual extraction conditions were modified slightly: water to raw material ratio 15 mL/g, extraction time 61 min, and extraction temperature 97°C. To verify the adequacy of the optimal condition, 3 verification tests were carried out and the extraction yield of CPP was found to be 6.49±0.08% (n = 3), which was in good agreement with the predicted CPP yield of 6.58%. Therefore, the regression model was adequate for predicting the extraction conditions of CPP.
The yields and bioactivities of polysaccharides are significantly influenced by the type of drying and drying temperature used. The yields and chemical composition of the three CPP were shown in
Samples | HD-CPP | VD-CPP | FD-CPP |
---|---|---|---|
5.87 ± 0.06b | 5.92 ± 0.05b | 6.46 ± 0.04a | |
57.54 ± 0.26c | 58.64 ± 0.23b | 59.41 ± 0.12a | |
0.58 ± 0.04c | 0.72 ± 0.04b | 1.14 ± 0.07a | |
2.47 ± 0.07c | 2.87 ± 0.06b | 3.57 ± 0.08a | |
1.28 ± 0.12b | 1.78 ± 0.09a | 1.81 ± 0.10a | |
10.73 ± 0.44a | 10.28 ± 0.32a | 10.25 ± 0.27a | |
7.22 ± 0.02a | 7.23 ± 0.02a | 7.23 ± 0.03a | |
1.03 ± 0.01b | 1.14 ± 0.05b | 1.42 ± 0.10a |
Values are expressed as mean ± standard deviation (n = 3).
Means within a row with different superscripts (a-c) differ significantly (
CPP, polysaccharide from cup plant; HD-CPP, polysaccharide obtained by hot air drying; VD-CPP, polysaccharide obtained by vacuum drying; FD-CPP, polysaccharide obtained by freeze drying.
The moisture contents, pH and relative viscosity of the three CPP were shown in
The solubility of CPP was shown in
CPP, polysaccharide from cup plant; HD-CPP, polysaccharide obtained by hot air drying; VD-CPP, polysaccharide obtained by vacuum drying; FD-CPP, polysaccharide obtained by freeze drying.
The conformation information of polysaccharide can be simply and rapidly analyzed by the Congo red assay. A biopolymer can be formed between Congo red and polysaccharide with the triple-helix conformation and exhibit a large red shift in λmax compare to the Congo red solution. When the biopolymer is exposed in the alkaline condition of enough strength, the helix conformation of polysaccharide is destroyed and the λmax of the biopolymer declined. The influences of NaOH concentrations on the λ max of the Congo red-CPP complexes were shown in
CPP, polysaccharide from cup plant; HD-CPP, polysaccharide obtained by hot air drying; VD-CPP, polysaccharide obtained by vacuum drying; FD-CPP, polysaccharide obtained by freeze drying.
The scavenging activity on DPPH radical assay has been widely used to determine the antioxidant capacity of samples due to its easy accessibility and the stability of DPPH radical [
Antioxidant activities of the polysaccharide from cup plant: (A) DPPH radical scavenging activity; (B) ABTS radical scavenging activity; (C) Ferric reducing power.
The ABTS radical scavenging activity assay was shown to be simple and quick in operation, and has been extensively used to evaluate the antioxidant activity of biological samples [
Ferric reducing power could be recognized as a significant symbol to evaluate the potential antioxidant activity of natural products [
The
The extractive condition of the polysaccharide from cup plant was optimized by performing RSM. The results indicated that the test variables of water to raw material ratio, extraction time and extraction temperature had significant effects on the extraction yield of CPP (
(XLS)
(XLS)
This work was supported by the Project funded by China Postdoctoral Science Foundation and the National Natural Science Foundation of China (No. 31601972). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.