Response surface methodology (RSM) was employed to optimize the conditions for the ultrasonic-assisted extraction (UAE) of polysaccharides from the flowers of
The genus
Conventionally, hot-water-infusion technology has been successfully used for polysaccharide extraction [
However, thus far, there are limited studies on the optimization of the extraction process of DDF polysaccharides (DDFPs) as well as their characterization and antioxidant activities. In this study, both UAE and RSM were used to optimize the extraction conditions of DDFPs based on a single-factor preliminary experiment. Three factors (temperature, time, and water-to-raw material ratio) affecting the extraction yield of the polysaccharides were investigated. Then, the
The flowers of
Extraction was performed according to a previously published method [
The polysaccharide extraction yield
The conditions for the UAE of polysaccharides from DDFs were optimized by RSM. To investigate these conditions, the three variables for extraction—temperature, extraction time, and water-to-raw material ratio—were denoted as labels
Response surface analysis program and results for the yield polysaccharides from
Run | Temperature |
Time |
Factor 3 |
Yield of polysaccharides |
---|---|---|---|---|
1 | 50 (−1) | 50 (0) | 25 |
17.30 |
2 | 60 (0) | 60 |
15 (−1) | 16.11 |
3 | 60 (0) | 40 (−1) | 25 |
16.68 |
4 | 60 (0) | 50 (0) | 20 (0) | 21.22 |
5 | 70 |
50 (0) | 15 (−1) | 16.22 |
6 | 50 (−1) | 50 (0) | 15 (−1) | 15.66 |
7 | 60 (0) | 40 (−1) | 15 (−1) | 15.41 |
8 | 60 (0) | 60 |
25 |
19.57 |
9 | 60 (0) | 50 (0) | 20 (0) | 19.4 |
10 | 50 (−1) | 60 |
20 (0) | 16.37 |
11 | 50 (−1) | 40 (−1) | 20 (0) | 15.42 |
12 | 60 (0) | 50 (0) | 20 (0) | 19.02 |
13 | 60 (0) | 50 (0) | 20 (0) | 20.00 |
14 | 60 (0) | 50 (0) | 20 (0) | 19.94 |
15 | 70 |
60 |
20 (0) | 18.65 |
16 | 70 |
40 (−1) | 20 (0) | 17.90 |
17 | 70 |
50 (0) | 25 |
18.58 |
The DDFPs (1.0 g) were dissolved in 100 mL of deionized water. Then, the solution was adjusted to the final ethanol concentration of 30% using 95% ethanol and stored at 4°C overnight. The residue obtained after the precipitation was centrifuged at 10000 ×g and 25°C for 20 min and freeze-dried to obtain a dry fraction, denoted as DDFPs30. Similar cycles were performed to prepare the fractions of DDFPs50 and DDFPs70 by adjusting the final precipitation ethanol concentration to 50% and 70%, respectively.
The DPPH radical-scavenging capacity assay was based on a 96-well microplate method [
The FRAP assay was also conducted according to our previous method using 96-well microplates [
Molecular weight and molecular size distribution were investigated using an HPSEC system coupled to an integrated detector array: a refractive index detector (Wyatt Technology, Santa Barbara, CA, USA), a UV L-2400 detector (Hitachi High Technologies America, Inc., Schaumburg, Illinois, USA), and a MALLS detector (Wyatt Technology, Santa Barbara, CA, USA). The chromatographic system consisted of an L-2130 pump (Hitachi Scientific Instruments Inc., Columbia, Maryland, USA) and a TOSOH TSKgel G4000PWXL column (300 mm × 7.8 mm i.d., Tokyo, Japan). Each polysaccharide (2 mg mL−1) of DDFPs30, DDFPs50, and DDFPs70 was subjected to the HPSEC system. The eluent consisted of a 0.1 mol L−1 NaNO2 solution and 0.5 g L−1 NaN3 at a flow rate of 0.5 mL min−1 with a run time of 25 min. The weight-average molecular weight (
DDFPs30, DDFPs50, and DDFPs70 (5 mg each) were hydrolyzed in 2 mL of 2 M trifluoroacetic acid at 120°C for 6 h. Excess trifluoroacetic acid was removed by codistillation [
The RSM optimization of the UAE conditions was based on the maximum DDFP yield of the sample. All the parameters (
The effect of various temperatures (30, 40, 50, 60, 70, and 80°C) on the extraction efficiency of DDFPs was investigated by maintaining the other two factors (extraction time and water-to-raw material ratio) constant at 60 min and 30 mL/g, respectively. As shown in Figure
Effects of the extraction temperature (a), extraction time (b), and water-to-material ratio (c) on yield of DDFPs.
Extraction time is also one of the important variables affecting the extraction efficiency of polysaccharides from natural products [
The yields of the DDFPs extracted by different water-to-raw material ratios (10, 15, 20, 25, 30, and 35 mL/g) are shown in Figure
Thus, the RSM experiments were conducted under the following conditions: an extraction temperature of 50–70°C, an extraction time of 40–60 min, and a water-to-raw material ratio of 15–25 mL/g.
A total of 17 runs were designed to evaluate the three independent variables
ANOVA for response surface quadratic model: analysis of variance table (partial sum of squares).
Source | Sum of squares | DF | Mean square |
|
|
Significant |
---|---|---|---|---|---|---|
Model | 50.10 | 9 | 5.566395 | 8.944987 | 0.0043 |
|
|
5.45 | 1 | 5.445 | 8.74991 | 0.0212 |
|
|
3.50 | 1 | 3.498013 | 5.621174 | 0.0495 |
|
|
9.53 | 1 | 9.526613 | 15.30891 | 0.0058 |
|
|
0.01 | 1 | 0.01 | 0.01607 | 0.9027 | |
|
0.1296 | 1 | 0.1296 | 0.208262 | 0.6620 | |
|
1.199025 | 1 | 1.199025 | 1.926788 | 0.2077 | |
|
8.451287 | 1 | 8.451287 | 13.5809 | 0.0078 |
|
|
8.421487 | 1 | 8.421487 | 13.53301 | 0.0079 |
|
|
10.23689 | 1 | 10.23689 | 16.45029 | 0.0048 |
|
Residual | 4.356045 | 7 | 0.622292 | |||
Lack-of-fit | 1.578925 | 3 | 0.526308 | 0.758064 | 0.5731 | |
Pure error | 2.77712 | 4 | 0.69428 | |||
Cor. total | 54.4536 | 16 |
Response surface plots (a, b, and c) and contour plots (d, e, and f) showing the effect of time, temperature, and water-to-material ratio on yield of DDFPs.
To further select the polysaccharide fraction with the highest antioxidant activity, three fractions (DDFPs30, DDFPs50, and DDFPs70) were prepared by the stepwise ethanol concentration precipitation method. Ethanol precipitation is an effective method for fractionation and purification of water-soluble DDFPs in aqueous solutions. In an aqueous solution, the DDFPs molecule exposes its charged and polar residues on the surface to maximize the contact with water molecules. Compared with other isolation methods for biopolymers such as chromatography and membrane, ethanol precipitation has the advantages of simple equipment and easy operation.
As shown in Figure
Antioxidant activity of three fractions from DDFPs. (a) Scavenging effect on DPPH radicals. (b) Reducing power evaluation.
The FRAP assay is a simple, reproducible, rapid, and inexpensive method to measure the reductive ability of an antiradical and is evaluated by the transformation of ferric ion (Fe3+) to ferrous ion (Fe2+), as a measure of the total antioxidant capacity [
To better explore the structural difference between three fractions (DDFPs30, DDFPs50, and DDFPs70) from DDFPs, HPSEC-MALLS was used to determine the molecular weight (
The physical properties of polysaccharides from
Fractions |
|
Polydispersity ( |
Slope |
---|---|---|---|
DDFPs30 | 5.41 ± 0.23 | 1.28 ± 0.15 | 0.33 ± 0.02 |
DDFPs50 | 3.78 ± 0.12 | 1.33 ± 0.09 | 0.35 ± 0.03 |
DDFPs70 | 5.63 ± 0.14 | 1.02 ± 0.05 | 0.32 ± 0.02 |
The HPAEC-PAD analysis profiles and data of the monosaccharide compositions of the three polysaccharides from the flowers of
HPAEC-PAD analysis profiles of monosaccharide composition in three fractions from DDFPs. Each peak was separated as described as 1:
In this study, RSM was applied for the first time to determine the optimal conditions for the extraction of DDFPs. The optimum conditions for the yield of DDFPs are as follows: an extraction temperature of 63.13°C, an extraction time of 53.10 min, and a water-to-raw material ratio of 22.11 mL/g. Furthermore, under the optimized conditions, the yield obtained from the verification experiments (20.25%) agreed well with the theoretical yield (20.38%), indicating that the regression model is efficient and successful for the extraction of DDFPs from DDFs. To further select the fraction with higher antioxidant activity, the stepwise ethanol precipitation method was used to separate the fractions from DDFPs as DDFPs30, DDFPs50, and DDFPs70. DDFPs50 exhibited the highest antioxidant activity in both the DPPH and FRAP assays.
The authors declare that they have no conflicts of interest.
This project was supported by the Agricultural Science and Technology Innovation Program (2014), China.