Analysis of Influence Factors on Extraction Rate of Lutein from Marigold and Optimization of Saponification Conditions

After lutein esters extracted by ultrasonic-assisted organic solvent from marigold powder, saponification conditions such as saponification solution concentration, saponification lipuid dosage, saponification temperature and saponification time were optimized by response surface analysis. The results showed that the optimal saponification conditions are saponification solution concentration 10%, saponification lipuid dosage 200 mL, saponification temperature 50°C, saponification time 2 h. Under the optimal condition, the content of lutein is the highest with nalue of 15.64%.


INTRODUCTION
Marigold is commonly known as stink hibiscus, marigold, Tagetes Asteraceae and annual herb.Native to Mexico and it has been widely introduced throughout the world (Wang et al., 2004).In addition, as a general ornamental flower, it is not only rich in mineral elements, vitamins and other nutrients but also contains a variety of biologically active substances, to be fully utilized in various fields.
Lutein is a non-vitamin A active carotenoids (Mei, 1989), the system is named 3, 3-dihydroxy-a-carotene formula C 40 H 56 O 2 , molecular weight 568.85 (Gou, 2002).As a natural pigment, with bright color, strong coloring, safe, non-toxic, strong antioxidant capacity characteristics, but also has to protect the eyes, cancer, modified UV damage, strengthen the immune system (Park et al., 1998), anti-atherosclerosis and other physiological functions.Widely used in feed additives, food additives, pharmaceutical, industrial dyes, aquatic products and other industries (Zhang et al., 2006).The study found mainly marigold lutein lutein esters (>90%) of the state of existence and lutein esters cannot be directly absorbed by the body metabolism, you must first be converted into free lutein can be absorbed by the body, so marigold extract lutein ester saponification, it is necessary to prepare a free lutein (Molldrem et al., 2004).
The author ultrasonic assisted extraction of petroleum ether on the basis of lutein esters, saponification with KOH methanol solution, based on the best conditions on the single factor experiment with SAS 9.1 software design response surface experiments, the results of response surface regression and analysis of variance of saponification conditions were optimized.

Materials:
Marigold particles are provided by Lindian Qi Xin natural products.

Method:
Marigold lutein basic saponification process: After freeze-drying marigold pulverized particles passed through a 60 mesh sieve.Accurately weighed 30 g frozen brown powder into 500 mL volumetric flask, petroleum ether as the extraction agent, according to a certain ratio of solid to liquid extraction agent added, using ultrasonic assisted extraction method, filtered and the filtrate was concentrated under vacuum to obtain lutein ester solvent recovery Baptist paste; weighed accurately extract lutein esters 5 g, added mass of nbutanol dissolved in a certain amount of methanol was added a solution of KOH concentration saponification reaction was completed, filtration and the filtrate was measured lutein.The remaining filtrate was washed with HCI solution, pH adjusted 2.5 mol/L value to neutral and the filtrate was filtered with a separatory funnel, Single factor experimental design: According to the basic flow, first determine the type of saponification liquid, then set each saponification concentration, dosage, saponification temperature and saponification time for the single factor experiments and they were repeated three times.Saponification concentration were 5, 7.5, 10, 12.5 and 15%, respectively a saponification liquid dosage is 100 mL, saponification temperature is 40°C, saponification time for the screening of optimum concentration of saponification conditions is for 2 h.At the optimum screening concentration of saponification, the amount of the saponification solution was 50, 100, 150, 200 and 250 mL, respectively saponification temperature was 40°C, saponification time was added for the screening of the optimal amount of the saponification conditions for 2 h.Under optimal screening concentration and amount of the saponification solution saponification temperatures were 35, 40, 45, 50 and 55°C, respectively saponification time optimal filter conditions for 2 h saponification temperature.Under the optimum screening concentration of saponification, the amount of temperature and saponification, saponification time optimal filter saponification time was 1, 1.5, 2, 2.5 and 3 h conditions, respectively.

Quadratic
regression rotation combination experimental design: On the basis of single factor experiments, using SAS 9.1 software for response surface design of experiments, KOH methanol concentration selected, KOH methanol addition, saponification temperature, saponification time four factors multivariate cross combination experiments, respectively, X1, X2, X3 and X4 representatives, low, medium and high levels of each of the experimental variables, respectively -1, 0, 1 self-encoded.And the relationship between the true values encoded in line with the following equation: where, i is Encoded value of the argument, X i is The actual value of the independent variable experimental level, X 0 is The level of the center of the actual value of the experiment, ∆X i is The increase value of a single variable and Y is The lutein content of the response value, The experimental factor level coding is shown in Table 1.Data processing and analysis: We use the SAS 9.2 software for quadratic regression rotation design process and analyze the experimental results.

RESULT ANALYSIS
Univariate results: Saponification solvent selection and concentration of the experimental results, the addition amount, the temperature of the saponification, the saponification time four factors obtained under different conditions lutein content results shown in Fig. 1.
Seen from Fig. 1A: A saturated solution of KOH in methanol was significantly better than the saturated ethanol solution of KOH in methanol and a saturated solution of NaOH, so the experiment was saponified select KOH solution in methanol solution.
Figure 1B shows: KOH methanol solution with increasing concentrations of lutein content after the first increase decrease.When the saponification solution concentration of 10%, the lutein content reaches the maximum value.Considering the various economic factors, etc., to determine the optimal concentration of 10% saponification.
Figure 1C shows: KOH methanol solution with increasing amount of lutein content increased.When the amount of KOH added 150 mL of methanol reaches a maximum, higher than 150 mL, lutein content of trans decreased and therefore to determine the optimal amount of methanol KOH 150 mL.
Figure 1D show: Saponification temperature increases, the lutein content increased.Saponification temperature is 50°C; the lutein content reaches the maximum and significantly higher than that of lutein other temperatures.When the saponification temperature is higher than 50°C, as the temperature rises, the content decreased.Therefore, to determine the optimum temperature of saponification 50°C.
Figure 1E shows: With the increase of the saponification time, lutein content increased.When the saponification time is 2 h we get the highest content of lutein.When the saponification time is greater than 2 h, lutein also declined.Therefore, we determine the best time for saponification is 2 h.

Results of quadratic regression rotation design:
Quadratic regression rotation design experiments to  2.

Significance test of secondary regression equation:
By F test to show the effectiveness of the regression equation, t test for regression model coefficients significant test, data processing system using SAS 9.2 on the experimental results of multiple regression analysis.The results are shown in Table 3.

Analysis of variance from Table 3 can be drawn:
In a key, KOH concentration, KOH methanol addition, saponification temperature and saponification time were significantly affected (p<0.05) on the results.In the interaction term, the results between the four factors of significant influence.The quadratic regression model F is 16.52, greater than the F (14, 12) 0.01 = 4.05 and p<0.0001, while the lack of fit of F is 24.94, less than F (10, 2) 0.01 = 99.40,coefficient of determination R 2 = 0.9507, description and results of the model to be extremely significant.A term, the second term F values are greater than the level of F = 0.01, so this regression equation is valid, it can be predicted within the scope of the design used.
Table 4 shows the t-test analysis of variance: Factors affecting the interaction between X34 significant (p<0.05),X14 affect significantly (p<0.01).The degree of influence of various factors on the results in descending order of: X3, X1, X4, X2.The experimental data were SAS 9.2 statistical analysis, we can get four quadratic regression equation is as follows:   According to the interaction equation, using SAS software can draw Y14, Y34 interaction response surface and contour equation.
Figure 2 shows: When KOH methanol addition 150 mL, when the saponification temperature 45°C, KOH concentration is increased to 10% from 6%, while the saponification time from 1.2 to 2.1 h also increased, along with the experimental results KOH concentration and increasing the saponification time increases.When KOH concentration of 10%, the saponification time is 2.1 h, the maximum synergistic effect between the two, i.e., the result is maximized.When the KOH concentration from 10 to 14%, the extraction time from 2.1 to 3 h, the marigold lutein yield increases with the concentration and time of KOH saponification reduced, indicating that the two obvious antagonism.
Figure 3 shows: When the KOH concentration of 10%, KOH methanol addition is when 150 mL, saponification temperature increases from 36 to 50°C, extraction time increased from 1.2 to 2.1 h, the experimental results show with the saponification temperature increasing, the saponification time increases.When the saponification temperature is 50°C, saponification time is at 2.1 h, the synergy between the two maximum, optimal values of the experimental results.When the saponification temperature is 50 to 54°C, the saponification time from 2.1 to 3 h, the experimental results along with saponification time and increasing the temperature to reduce the saponification, indicating there existed significant antagonism.

Main effects analysis:
The influence of various factors on the experimental results is shown in  factors influence the sequence of: X3>X1>X4>X2, the impact on the experimental results of saponification temperature, followed by concentration of KOH and then is saponification time.KOH in methanol was added affect the amount of the minimum of the experimental results.

Optimal value selection and inspection:
To find the optimal values of the regression equation, the results is shown in Table 6.
From Table 6, in response surface method predicted theoretical optimum extraction conditions do three validation experiments, the average content of lutein extract obtained was 15.53%.
In summary, the optimal combination of: KOH concentration of methanol is 10%, KOH methanol addition is 150 mL, saponification temperature is 50°C and saponification time is 2 h.

CONCLUSION
In this study, we took marigold particles as raw material, investigated the impacts of KOH methanol concentration, KOH methanol addition, saponification temperature and saponification time on the content of lutein.Through single factor experiments and four factors-three levels secondary rotation combination experiments to optimize the saponification conditions: KOH concentration of methanol is 10%, KOH methanol addition is 150 mL, saponification temperature is 50°C, saponification time is 2 h, lutein content of this condition is 15.64%.Verified experimentally derived results are consistent with the model predictions, indicating that this model can rely on the stability of the saponification conditions and good reproducibility.

Fig. 3 :
Fig. 3: Response surface and contour plot of KOH methanol addition and the saponification time

Table 1 :
Factors and their coded levels in central composite table

Table 2 :
Quadratic regression orthogonal rotation combination design and test results

Table 5 :
Analysis on the importance of key factor

Table 6 :
The results of some selected medium by flask-cultured experiments