Saffron, which has many kinds of biological activities, has been widely used in medicine, cosmetics, food, and other fields of health promotion industries. Crocins are the main component of saffron (
Saffron, which is the red stigma of
To our knowledge, extraction methods for crocins in addition to the chromatographic techniques [
Linear Relationship of Calibration Curve Regression Equation of crocin-1 was obtained by regression analysis of absorbance value of crocin-1 solution (
As shown in Figure
The effect of extraction voltage (a), extraction time (b), extraction temperature (c), and ethanol concentration (d) on the yields of crocins from saffron (
Figure
As shown in Figure
According to Figure
Design-Expert 8.0.6 Trial software was used to analyze the experimental data of response surface and the quadratic polynomial equation was presented in
According to the ANOVA results (Table
Results of the variance analysis of regression model.
Source | Sum of squares | df | Mean squares |
|
Prob |
---|---|---|---|---|---|
Model | 64.82 | 14 | 4.63 | 19.78 | <0.0001 |
|
0.25 | 1 | 0.25 | 1.05 | 0.3221 |
|
1.97 | 1 | 1.97 | 8.41 | 0.0116 |
|
3.29 | 1 | 3.29 | 14.04 | 0.0022 |
|
4.30 | 1 | 4.30 | 18.36 | 0.0008 |
|
0.01 | 1 | 0.01 | 0.043 | 0.8392 |
|
0.00 | 1 | 0.00 | 0.015 | 0.9031 |
|
1.44 | 1 | 1.44 | 6.15 | 0.0264 |
|
0.22 | 1 | 0.22 | 0.92 | 0.3528 |
|
0.01 | 1 | 0.00 | 0.03 | 0.8630 |
|
0.09 | 1 | 0.09 | 0.40 | 0.5386 |
|
8.20 | 1 | 8.20 | 35.05 | <0.0001 |
|
0.01 | 1 | 0.01 | 0.029 | 0.8683 |
|
1.54 | 1 | 1.54 | 6.58 | 0.0225 |
|
48.42 | 1 | 48.42 | 206.87 | <0.0001 |
Residual | 3.28 | 14 | 0.23 | ||
Lack of fit | 2.53 | 10 | 0.25 | 1.35 | 0.4149 |
Pure error | 0.75 | 4 | 0.19 | ||
Cor total | 68.09 | 28 | |||
|
Adj |
The correlation between predicted values obtained by the quadratic polynomial equations and actual experimental values.
The 3D response surface plots were made by Design-Expert software in order to get a better visualization between the relations of extraction variables and crocins yields (Figure
Response surface plot showing the effect of extraction voltage and extraction temperature on the yield of crocins from saffron.
The optimal values of extraction voltage, extraction time, ethanol concentration, and extraction temperature were also calculated by Design-Expert software, which were 111.2 V, 40 s, 70%, and 50°C, respectively. Under the above conditions, the maximum predicted yield of crocins was 22.84%.
In order to compare the predicted value with the actual result, an experiment was performed for three times and the extraction parameters were set at 111 V, 40 s, 70%, and 50°C for operational convenience. The mean value of these three experiments was 22.76%, which was slightly lower than the one predicted by equation. This result demonstrated that the optimized model adequately reflected the actual extraction process of crocins.
Homogenate extraction was compared with ultrasonic extraction for crocins from saffron. The ultrasonic extraction process was also optimized by RSM, and the optimal parameters of ultrasonic extraction were 41%, 40°C, and 29 min for ethanol concentration, extraction temperature, and extraction time, respectively. Under this condition, the extraction yield of crocins for ultrasonic extraction was 18.51%, which was 22.96% lower than that in homogenate extraction. In addition to the higher extraction yield of crocins, the extraction time of homogenate extraction was also significantly shorter than ultrasonic extraction. Hence, it was worth noticing that homogenate extraction was a good alternative to extract crocins from saffron.
Saffron, which was authenticated by Professor Ping Wang, was purchased from Jiande Sandu farm, Hangzhou, Zhejiang Province of China. Crocin-1 was isolated from saffron in our laboratory. Other reagents were all of analytical grade.
Homogenate extraction was carried out on JHBE-50A homogenate extractor (Golden Star Technology, Inc., Ltd., Zhengzhou, China). UV-Vis spectrophotometer (756 PC) was purchased from Shanghai Spectrum Instruments Co., Ltd. (Shanghai, China).
The total crocins content was determined using a standard curve with crocin-1. Firstly, the stock solution of standard crocin-1 (1.03 mg/ml) was prepared with ultrapure water, and a serial dilution of 2.57
Under the designed extraction voltage, extraction time, extraction temperature, and ethanol concentration, saffron powder (0.1 g) mixed with ethanol (100 ml) was extracted by homogenate extractor; then the sample was centrifuged at 4700 rpm for 10 min. After that, the supernatant was filtered and collected to determine the content of crocins by UV-Vis spectrophotometer at 440 nm. The whole extraction process was carried out in triplicate.
According to the results of single factor experiments, response surface methodology based on Box-Behnken design (BBD) was applied to identify the best extraction conditions for crocins by using four variables, the extraction voltage (
Coded levels of independent variables used in the RSM design.
Factor | Level | ||
---|---|---|---|
−1 | 0 | 1 | |
Extraction voltage |
100 | 110 | 120 |
Extraction time |
20 | 30 | 40 |
Ethanol concentration |
25 | 50 | 75 |
Extraction temperature |
40 | 60 | 80 |
As showed in Table
Experimental design and responses of the dependent variables to the extract parameters.
Numbers | Factors | Yield/% | |||
---|---|---|---|---|---|
|
|
|
|
||
1 | 0 | 1 | −1 | 0 | 20.80 |
2 | −1 | −1 | 0 | 0 | 20.48 |
3 | 1 | 0 | 0 | −1 | 19.09 |
4 | 0 | 0 | 0 | 0 | 22.62 |
5 | −1 | 0 | −1 | 0 | 19.55 |
6 | 0 | 0 | −1 | −1 | 19.25 |
7 | 1 | 1 | 0 | 0 | 21.77 |
8 | 0 | 0 | 1 | −1 | 20.67 |
9 | −1 | 0 | 0 | −1 | 18.24 |
10 | 1 | 0 | 0 | 1 | 16.92 |
11 | 0 | −1 | −1 | 0 | 20.88 |
12 | −1 | 1 | 0 | 0 | 21.53 |
13 | 0 | −1 | 0 | 1 | 18.53 |
14 | 0 | 1 | 0 | 1 | 19.34 |
15 | −1 | 0 | 1 | 0 | 20.28 |
16 | 0 | 0 | 0 | 0 | 21.61 |
17 | 1 | −1 | 0 | 0 | 20.52 |
18 | 0 | 0 | 1 | 1 | 18.47 |
19 | −1 | 0 | 0 | 1 | 18.47 |
20 | 0 | 0 | 0 | 0 | 21.97 |
21 | 0 | 0 | 0 | 0 | 22.50 |
22 | 0 | 0 | −1 | 1 | 17.66 |
23 | 0 | −1 | 1 | 0 | 21.77 |
24 | 0 | −1 | 0 | −1 | 19.17 |
25 | 1 | 0 | −1 | 0 | 20.68 |
26 | 0 | 1 | 0 | −1 | 20.15 |
27 | 0 | 0 | 0 | 0 | 21.85 |
28 | 1 | 0 | 1 | 0 | 21.29 |
29 | 0 | 1 | 1 | 0 | 22.62 |
In this article, we use crocin-1 as a standard substance to determine the content of crocins and decide which extraction process is better, because all the crocins such as trans-4-GG (crocin-1), trans-3-Gg (crocin-2), trans-2-gg, and crocetin have similar UV absorption wavelength. The test results of crocins in extraction solution were not interfered by safranal, picrocrocin, and flavonoids, because these compounds have no absorption at 440 nm except for crocins
In this study, an efficient homogenate extraction method was successfully applied to extract crocins from saffron. RSM was used to optimize the extraction conditions with high extraction yield. The optimum conditions were 111 V, 40 s, 70%, and 50°C for extraction voltage, extraction time, ethanol concentration, and extraction temperature, respectively. Under these conditions, extraction yield of crocins was much higher than ultrasonic extraction method, in a significantly shorter extraction time. All these data showed that homogenate extraction method was a more efficient procedure for extracting crocins from saffron.
The authors declare no conflicts of interest.
The authors are grateful to China Postdoctoral Science Foundation (no. 2016M592016) and Key Project of Science and Technology Department of Zhejiang Province (no. 2015C02032) for financial help.