Ionic liquid based ultrasonic-assisted extraction (ILUAE) was successfully applied to extract Oleanolic acid (OA) from grape seeds. Different kinds of 1-alkyl-3-methylimidazolium ionic liquids with different kinds of cations and anions were studied and 1-butyl-3-methylimidazolium chloride ([C 4 mim]Cl) solution was selected as extractants. The optimal conditions of ILUAE were determined by single factor experiments and Box-Be hnken design as follows: [C 4 mim]Cl concentration of 0.7 mol/L, extraction time of 4 h, liquid-solid ratio of 15:1 mL/g, ultrasonic power of 195 W, the ultrasonication time of 13 min, and extraction temperature of 48℃·min. The extraction yield of OA was 13.4 mg/g, which is well in close agreement with the value predicted by the model. Compared with the regular ultrasonic-assisted extraction (UAE), the proposed approach exhibited 1.64 times higher efficiency, which indicated that ILUAE has a broad prospect for sample preparation of the bioactive compounds.
The total production of grapes worldwide is about 60 million・tons per year, particularly in France, Italy, the USA and China. Approximately 80% of this harvest is used in wine production, with the remaining 20% of the processed grape being waste [
Different techniques have been employed for the extraction of OA such as heat reflux, hydrotropic extraction, microwave assisted extraction, and supercritical fluid extraction [
In this study, we report an ionic liquid based ultrasonic-assisted extraction (ILUAE) as an alternative method for the recovery of OA. An ionic liquid (IL) as a design green solvent is regarded as an attractive alternative or replacement to conventional volatile organic solvent. Ionic liquids (ILs) are low melting point salts composed entirely of ions. They have negligible volatility, low flammability, chemical stability, good environmental benignity, good solubility for organic compounds, and they are miscible with water. Moreover, a large number of possible variations in cation and anion features allowing the fine-tuning of the IL properties [
compared to conventional organic solvents. IL-assisted sample pretreatment techniques such as aqueous biphasic systems [
Ultrasound-assisted extraction has been successfully used for the extraction of the bioactive constituents from the plant materials [
Response surface methodology (RSM) is an effective statistical technique for optimizing complex processes because it allows more efficient and easier arrangement and interpretation of experiments compared to other methods. In addition, it is less laborious and time-consuming than other approaches that applied to optimize a process. It is widely used for optimization of the extraction process of the bioactive ingredients.
To the best of our knowledge, extraction of OA from grape seeds with ILs as the solvent has not yet been reported in the literature. Ultrasound-assisted extraction parameters, such as ionic liquids with different cations and anions concentration, solvent to solid ratio, ultrasonic power, ultrasonication time, and extraction temperature for enhancing the extraction efficiency of OA were systematically optimized. The aim of this work was to develop a rapid, effective, validated and environmental friendly ionic liquid based ultrasound-assisted approach for extraction of OA from grape seeds, and to compare it with conventional solvent extraction.
Grape seeds were obtained from the Bin He food industry Co, Ltd. (Gansu, China). OA (purity > 98% by HPLC) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). All the ionic liquids ([C4mim]Cl, [C4mim]Br, [C4mim]BF4, [C4mim]NO3, [C4mim]HSO4, [C4mim]ClO4) purchased from Lanzhou institute of chemical physics, Lanzhou, China. Other materials used in this study were of analytical grade.
Grape seeds powder (5 g) was dispersed in 50 ml of IL (0.5 mol/L) prepared deionized water by gentle stirring using magnetic stirrer. The extraction was carried out for 1 h by gentle stirring with magnetic stirrer at a stirring rate of 150 r/min at 35˚C. The extract obtained after IL extraction was filtered using filter paper to remove solid particles and quantified for the content of OA. The extraction yield of OA was defined as follow:
Y ( mg / g ) = C V / M
where C (mg/mL) is the concentration of OA in extract, V(mL) is the volume of the extract, and M(g) is the mass of sample weighed.
Six different ILs ([C4mim]Cl, [C4mim]Br, [C4mim]BF4, [C4mim]NO3, [C4mim]HSO4, [C4mim]ClO4) were used to screen the most suitable anion to extract OA from grape seeds at 0.5 mol/L, The selected ILs Cl− and BF 4 − with different cations (C2mim+, C4mim+, C6mim+, C8mim+) were used at 0.5 mol/L to investigate the effect of the cation on the extraction efficiency of OA. For maximum extraction of OA, concentration of [C4mim]Cl (0.1, 0.3, 0.5, 0.7 and 0.9 mol/L), time of extraction (1 - 5 h), solvent to solid ratio (10:1 - 30:1, mL/g), ultrasonic power (100 - 300 W), ultrasonication time (5 - 25 min), and temperature of extraction (30˚C - 70˚C) were optimized. The process of extraction was done as described above in this section. The IL extracts were then estimated for the content of OA. All the analyses were done in triplicates.
The conventional UAE was selected as the reference method for extraction of the target compounds [
The OA concentration in the top phase and bottom phase was estimated using the colorimetric method of vanillin perchloric acid under the conditions of OA as a standard. To evaporate sample solvent, the sample residue was reacted with 0.3 mL 5% (w/v) vanillin in glacial acetic acid and 1.0 mL perchloric acid at 70˚C in water bath for 25 minutes, the optical density was measured at 548 nm.
RSM was employed for experimental design, data analysis and model building with software Design Expert (Design-Expert 7.0). A Box behnken Design (BBD) with three variables and three levels was used to determine the response pattern and then to establish a model [
Y i = β 0 + ∑ β i i X i 2 + ∑ β i j X i X j
where Yi is the predicted response, XiXj are input varuables which influence the response variable Y. βo is the offset term; Bi is the ith linear coefficient; Bii is the iith quadratic coefficient and Bij is the ijth interaction coefficient. Design-expert package (version 7.0, Stat-Ease, Inc., Minneapolis, MN, USA) was used for the experimental design and regression analysis of the data obtained.
The experimental design consisted of twelve factorial points in
Factors | Unit | Notation | Range and levels | ||
---|---|---|---|---|---|
−1 | 0 | 1 | |||
Ultrasonic power | W | X1 | 150 | 200 | 250 |
Ultrasonication time | min | X2 | 10 | 15 | 20 |
Extraction temperature | ˚C | X3 | 40 | 50 | 60 |
Run | X1 | X2 | X3 | Y(mg/g) |
---|---|---|---|---|
1 | −1 | −1 | 0 | 11.2 |
2 | 1 | −1 | 0 | 10.1 |
3 | −1 | 1 | 0 | 9.65 |
4 | 1 | 1 | 0 | 10.21 |
5 | −1 | 0 | −1 | 10.21 |
6 | 1 | 0 | −1 | 9.36 |
7 | −1 | 0 | 1 | 9.87 |
8 | 1 | 0 | 1 | 8.56 |
9 | 0 | −1 | −1 | 12.3 |
10 | 0 | 1 | −1 | 10.21 |
11 | 0 | −1 | 1 | 10.23 |
12 | 0 | 1 | 1 | 10.23 |
13 | 0 | 0 | 0 | 13.51 |
14 | 0 | 0 | 0 | 13.24 |
15 | 0 | 0 | 0 | 13.25 |
16 | 0 | 0 | 0 | 13.56 |
17 | 0 | 0 | 0 | 13.26 |
significant. The interaction among the different independent variables and their corresponding effect on the response were studied by analyzing the response surface 3D plots.
The extraction yield of target compounds might be obviously affected by the physical and chemical properties of ionic liquids, while the two properties can be significantly influenced by their structure [
The anion identity is considered an obvious factor which can influence the characteristic of ionic liquids, especially for water miscibility [
Six ionic liquids with different cations (C2mim+, C4mim+, C6mim+, C8mim+) combined with BF 4 − and Cl− were also screened to obtain the optimal extraction yield of OA. The results are shown in
The concentration of [C4mim]Cl is an important factor that could influence the polarity of solution. The OA concentration affected by [C4mim]Cl amount was shown in
yield was observed. This may be due to increased viscosity of IL solution at high concentration. High viscosity of IL does not permit its penetration into the plant tissue due to which active compounds cannot be extracted out completely and quickly [
To extract target compounds from the cellular structure, a solvent must have access to the cellular compartments of the target compounds. Effect of extraction time was evaluated by varying the extraction time from 2 to 10 h. As shown in
The solvent volume must be sufficient to ensure that the entire sample is immersed, especially when having a matrix that will well during the extraction process. In this study, five different solvent to solid ratios (10:1, 15:1, 20:1, 25:1 and 30:1 (mL/g)) with 0.7 mol/L [C4mim]Cl, were selected to evaluate the influence of solvent to solid ratio on the extraction field of OA from grape seeds.
During ionic liquid-based ultrasound assisted extraction (ILUAE), optimization of the ultrasonic power is very important to ensure efficient extraction of the target compounds. The mechanical effects of ultrasound induce a greater penetration of solvent into cellular materials and improve mass transfer. Efficient cell disruption and effective mass transfer are reported as major factors leading to the enhancement of extraction with ultrasonic power [
To optimize the ultrasonication time, experiments were carried out for 5 - 25 min with an interval of 5 min, while the other four factors (extraction temperature, ultrasonic power, solvent to solid ratio and the concentration of [C4mim]Cl) were fixed at 50˚C, 200 W, 15:1 and 0.7 mol/L, respectively. It showed that the field of OA increased with the ultrasonication time ascended from 5 to 15 min the maximum concentration of OA was observed when the ultrasonication time was 15 min, after this point, the concentration of OA started to maintain a dynamic equilibrium with the increasing of ultrasonication time, and OA concentration was growing slowly when the ultrasonication time exceeded 15 min (
The yield of OA was increased with the increase of the extraction temperature due to the increase of the mass transfer rate and the solubility of OA. In order to study the effect of different temperatures on the yield of OA, extraction process was carried out by using different temperatures of 20˚C, 30˚C, 40˚C, 50˚C, 60˚C, and 70˚C, while the other extraction conditions were: ultrasonication time 15 min, ultrasonic power 200 W and solvent to solid ratio of 15:1. The yield of OA was increased with enhancing of extraction temperature increased from 20˚C to 50˚C. As shown in
In this work, the input variables (factors) and their levels were selected, based on single factor experiments mentioned above. The factors and their actual and coded levels were presented in
Y = + 13 . 36 − 0. 34A − 0. 44B − 0. 4 0 C + 0. 41AB − 0. 11AC + 0. 52BC − 2 . 16A2 − 0. 92B2 − 1 . 71C2
The statistical significance of the regression model was checked by F-test and p-value, and the analysis of variance (ANOVA) for the response surface quadratic model was shown in
After the response surface regression procedure, the coefficient of determination (R2) of the predicted model was 0.9704, suggesting a good fit, the predicted model (0.8249) seemed to reasonably represent the observed values. Thus, the response was sufficiently explained by the model. “Adeq Precision” measures the signal to noise ratio. A ratio greater than 4 is desirable. The ratio of 21.29 indicates an adequate signal. This model can be used to navigate the design
Source | Sum of Squares | df | Mean Square | F Value | p-value Prob > F | |
---|---|---|---|---|---|---|
Model | 44.57512853 | 9 | 4.952792059 | 59.43741059 | <0.0001 | significant |
X1 | 0.91125 | 1 | 0.91125 | 10.93571863 | 0.0130 | |
X2 | 1.5576125 | 1 | 1.5576125 | 18.69257837 | 0.0035 | |
X3 | 1.2720125 | 1 | 1.2720125 | 15.26515314 | 0.0058 | |
X1X2 | 0.6889 | 1 | 0.6889 | 8.267343283 | 0.0238 | |
X1X3 | 0.0529 | 1 | 0.0529 | 0.634841718 | 0.4518 | |
X2X3 | 1.092025 | 1 | 1.092025 | 13.1051612 | 0.0085 | |
X 1 2 | 19.61281289 | 1 | 19.61281289 | 235.3692219 | <0.0001 | |
X 2 2 | 3.530939211 | 1 | 3.530939211 | 42.37405511 | 0.0003 | |
X 3 2 | 12.25087605 | 1 | 12.25087605 | 147.020174 | <0.0001 | |
Residual | 0.583295 | 7 | 0.083327857 | |||
Lack of Fit | 0.484375 | 3 | 0.161458333 | 6.528844858 | 0.0508 | not significant |
Pure Error | 0.09892 | 4 | 0.02473 | |||
Cor Total | 45.15842353 | 16 |
R2 = 0.9871. adj R2 = 0.9705.
space. The coefficient of variation (CV) is the ratio of the standard error of estimate to the mean value of observed response expressed as a percentage. It is a measure of reproducibility of the models. The CV of the model was calculated as 2.60%. As a general rule, a model can be considered reasonably reproducible if its CV is not greater than 10%.
The 3D response surface plots were drawn to illustrate the main and interactive effects of the independent variables on the dependent one in
By prediction with computing program, the optimal conditions to obtain the highest yield of OA were determined as follows: the ultrasonic power of 195 W, the ultrasonication time of 13.5 min, and extraction temperature of 48.4˚C. At last because of the limit of instruments, the optimal conditions were chosen at the ultrasonic power of 195 W, the ultrasonication time of 13 min, and extraction temperature of 48˚C. The extraction yield of OA was 13.4 mg/g with a relative error about 0.6%.
In order to compare the extraction field of the [C4mim]Cl solution with organic solvent, ethanol was used to extract OA from grape seeds under the respective optimal condition. Under the optimal conditions of acetone extraction [
In this study, we have come up with a novel ILUAE for extraction of OA from grape seeds. In consideration of the influence of both the anion and cation of ionic liquids, [C4mim]Cl was selected for the subsequent analysis. The ultrasonic-assisted extraction conditions were optimized using single factor experiments and BBD in detail. Under these optimal conditions (e.g. with 0.7 mol/L [C4mim]Cl, solvent to solid ratio of 15:1, extraction time 4 h, ultrasonic power 195 W, ultrasonication time 13 min, and extraction temperature of 48˚C), this approach gained the highest extraction yield of OA 13.4 mg/g. Compared with conventional regular ultrasonic extraction, the proposed approach provides higher extraction efficiency, and obviously reduced extraction time. The proposed ILUAE has abroad prospect in sample preparation of bioactive compounds.
The authors would like to acknowledge the financial support by National undergraduate training programs for innovation and entrepreneurship (No. 201510740005).
The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.
Zhang, X.F., Li, X., He, Q., Zhang, B. and Zheng, X. (2017) Ionic Liquid Based Ultrasonic-Assisted Ex- traction of Oleanolic Acid from Grape Seeds. Open Access Library Journal, 4: e4148. https://doi.org/10.4236/oalib.1104148