Homogenate Extraction of Crocins from Saffron Optimized by Response Surface Methodology

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 themain component of saffron (Crocus sativus L.). At present, most of the extractionmethods for crocins require long time or special instruments to complete the process and some of them are not suitable for industrial production at present. In this article, homogenate extraction technology which is a convenient and efficient method was developed for crocins extraction from saffron. Firstly, the influences of extraction voltage, extraction time, ethanol concentration, and temperature on crocins yield were studied by single factor experiments; and then response surface methodology (RSM) was used to optimize levels of four variables based on the result of single factor experiments. Results showed that the optimum extraction process conditions for crocins were as follows: extraction voltage, 110V; ethanol concentration, 70%; extraction temperature, 57C; and extraction time, 40 s. Based on these conditions, the extraction yield of crocins can reach 22.76% which is higher than ultrasonic extraction method. Therefore, homogenate extraction is an effective way to extract crocins from saffron with higher extraction yield and shorter extraction time.


Introduction
Saffron, which is the red stigma of Crocus sativus L., is widely cultivated in Iran, India, Pakistan, Greece, Italy, China, Japan, Azerbaijan, and so on [1].It is considered to be one of the world's most expensive spices.The main components in saffron are carotenoids like crocins and crocetin, monoterpene aldehydes like picrocrocin and safranal, and flavonoids.Crocin, which is responsible for the color of saffron, is the most abundant compound in saffron [2].It has been known that saffron is useful to cure various illnesses, for example, neuronal diseases like depressive disorder [3]; it is also useful for preventing diabetes complication [4], metabolic syndrome [5], and cancer [6] in both clinical trials and animal model studies.It also has various biological activities extensively reviewed by Bathaie's [7] and Hosseinzadeh's research groups [8].It is also safe and nontoxic in both human and animal studies [7,9].
To our knowledge, extraction methods for crocins in addition to the chromatographic techniques [10] are including ultrasonic-assisted extraction [11][12][13], molecularly imprinted polymer solid-phase extraction [14], high-voltage pulsed electric field-assisted extraction [15], solid-liquid dynamic extraction [13], and supercritical fluid extraction [16].These procedures give good extraction yields, but most of them require long time or special instruments to complete the process and some of them are not suitable for industrial production at present.Because of the instability of crocins in solution and in the light, the new extraction technology used in crocins extraction from saffron needs to shorten the extraction time and procedure.Meanwhile the homogenate extraction method can just meet the above requirements.Under the action of high-speed shear machine and cutting fluid, it can extract chemical compounds from material in solvent within a very short time without heating and pressure.Moreover, the process of homogenate extraction is carried out in stainless steel tanks; it can also effectively reduce the impact of light on crocins.It has been used to extract camptothecin and hydroxycamptothecin from Camptotheca acuminata leaves [17] and isoflavones from soybean meal [18], which has been proved to be an effective extraction method.However, as far as we know, there was no report about its application on crocins extraction from saffron.In this study, it was applied to extract crocins from saffron and optimized by response surface method for the first time.(%) at 50%.Then, it was also relatively stable with slight decrease when ethanol concentration increased to 75%.But the crocins yield sharply decreased to 6.49 ± 0.11 (%) when the ethanol concentration increased from 75 to 100%.Thus, ethanol concentration was suitable to set at 25-75% in the Box-Behnken design process.

Response Surface
( According to the ANOVA results (Table 1), the  values of  2 ,  3 ,  4 ,  1  4 ,  1 2 , and  4 2 were smaller than 0.05, indicating they had a significant influence on the yield of crocins from saffron.The  2 and Adj  2 values of (1) were 0.9519 and 0.9038, respectively, which meant the predicted values of (1) had great correlation with real experimental values between the chosen variables including extraction voltage, extraction time, ethanol concentration, and extraction temperature.The correlation between predicted values and real experimental values was showed in Figure 2. As a result of lack of fit test, the fitness of the model was good, because the  value of lack of fit was 0.4149, which was greater than 0.05.
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 3).Among the plots, the interaction of extraction voltage and extraction temperature had significant effects on crocins yield at a fixed time and ethanol concentration and the highest extract yield was achieved when the voltage and temperature were set nearly 110 V and 50 ∘ C, respectively.From other plots, the yield variations of crocins can also be seen, but they were not statistically significant.
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.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.Inc., Ltd., Zhengzhou, China).UV-Vis spectrophotometer (756 PC) was purchased from Shanghai Spectrum Instruments Co., Ltd.(Shanghai, China).

Total Crocins Determination.
The total crocins content was determined using a standard curve with crocin-1.Firstly, where  is the coded value of a variable,   is the real value of a variable,  0 is the actual value of a variable at the center point, and Δ is the step change value.As showed in Table 3, a total of 29 experimental runs for BBD were carried out.The independent variables and the crocins yield were correlated by the following quadratic polynomial model: where  is the predicted response;  0 ,   ,   , and   are the coefficients for constant, linear, squared, and interaction terms, respectively.

Conclusions
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.

Figure 2 :ABCFigure 3 :
Figure 2: The correlation between predicted values obtained by the quadratic polynomial equations and actual experimental values.

Table 1 :
Results of the variance analysis of regression model.
showed that the crocins yield reached 20.64 ± 0.16 (%) at 30 s. From 10 to 20 s and 30 to 40 s, the crocins yield decreased with extraction time increasing while it increased with extraction time increasing from 20 to 30 s.So in the Box-Behnken design process extraction time was set from 20 to 40 s.2.2.3.Effect of ExtractionTemperature on Crocins Yield.As shown in Figure1(c), the crocins yield increased slowly with extraction temperature from 0 to 60 ∘ C to reach 21.96 ± 0.13 (%) at 60 ∘ C; then it was relatively stable with slight decrease when temperature increased from 60 to 80 ∘ C. Therefore, 40-80 ∘ C was selected as the extraction temperature in the Box-Behnken design test.2.2.4.Effect of Ethanol Concentration on CrocinsYield.According to Figure1(d), the crocins yield increased with ethanol concentration from 0 to 50% and reached 21.65±0.08*  < 0.05: significant; * *  < 0.01: highly significant; df: degree of freedom.

Table 2 :
Coded levels of independent variables used in the RSM design.

Table 3 :
Experimental design and responses of the dependent variables to the extract parameters.