Optimization of ionic liquid-based microwave-assisted extraction technique for curcuminoids from Curcuma longa L.

Accepted Manuscript Title: Optimization of ionic liquids-based microwave-assisted extraction technique for curcuminoids from Curcuma longa L. Authors: Hui Liang, Wenchao Wang, Jialin Xu, Qing Zhang, Zheluan Shen, Zhen Zeng, Qingyong Li PII: DOI: Reference:

S0960-3085(17)30044-5 http://dx.doi.org/doi:10.1016/j.fbp.2017.04.003 FBP 857

To appear in:

Food and Bioproducts Processing

Received date: Revised date: Accepted date:

17-10-2016 3-3-2017 19-4-2017

Please cite this article as: Liang, Hui, Wang, Wenchao, Xu, Jialin, Zhang, Qing, Shen, Zheluan, Zeng, Zhen, Li, Qingyong, Optimization of ionic liquids-based microwaveassisted extraction technique for curcuminoids from Curcuma longa L.Food and Bioproducts Processing http://dx.doi.org/10.1016/j.fbp.2017.04.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Optimization of ionic liquids-based microwave-assisted extraction technique for curcuminoids from Curcuma longa L. Hui Lianga,b, Wenchao Wanga,b, Jialin Xub, Qing Zhangc, Zheluan Shena,b, Zhen Zengb, Qingyong Lia,b* a

Collaborative Innovation Center of Yangtze River Region Green Pharmaceuticals,

Zhejiang University of Technology, Hangzhou, China b

College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou,

China c

College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China

*Corresponding author. No. 18 Chaowang Road, Hangzhou City 310014, China. Tel: +86-571-88320984; E-mail: [email protected]. Graphical abstract

Highlights The extraction conditions were optimized by response surface methodology. The model for optimization was precise and reliable. The optimum conditions were different according to yield or antioxidant value. Ionic liquid based microwave extraction is a highly efficient and fast method. 1

Abstract: The application of ionic liquid-based microwave-assisted extraction was successfully developed for extracting curcuminoids from Curcuma longa L., and the antioxidant value of extract was determined by DPPH and TRAP methods. The response surface methodology was employed to optimize experimental conditions for extraction yield and antioxidant value. The results indicated that the optimal extraction solvent was 1-octyl-3-methylimidazolium bromide of 0.3 mol/L. With extraction yield as reference index, the optimum conditions were extraction temperature of 55

, extraction time of 8 min and solid-liquid ratio of 0.5/30 (g/mL).

With antioxidant value as reference index, the optimum conditions were extraction temperature of 70

, extraction time of 2 min and solid-liquid ratio of 0.5/30 (g/mL).

Under optimum conditions (extraction yield as reference index), the average yield of three kinds of curcuminoids was 1.77%. Compared with ultrasonic-assisted extraction, microwave-assisted extraction is a highly efficient and pollution-free method, which takes shorter extraction time and saves energy consumption. Keywords: Microwave-assisted extraction, Ionic liquid, Extraction yield, Antioxidant value, Curcuminoids

1

1

Abbreviations: IL-based MAE, Ionic liquid-based microwave-assisted extraction; ILs, Ionic liquids; MAE, Microwave-assisted extraction; CUR, curcumin; DMC, demethoxycurcumin; BDMC, bisdemethoxycurcumin; AVE, average; RSM, response surface methodology; BBD, Box-Behnken design; DPPH, 1,1 diphenyl-2-trinitrobenzene hydrazine; ABTS, 2,2'-azobis[2-methylpropionamidine]dihydrochloride; AAPH, 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate); TRAP, ROO∙ radical scavenging activity. 2

1. Introduction Curcuma longa L. is a perennial herb, cultivated in tropical areas of Asia and central America (Riela et al. 2014). The dried rhizome of Curcuma longa L. is commonly known as Turmeric (Osorio-Tobón et al. 2016). The main active components of Turmeric

are

curcumin

(CUR),

demethoxycurcumin

(DMC)

and

bisdemethoxycurcumin (BDMC), together known as curcuminoids, which are natural hydrophobic polyphenol (Mandal et al. 2008). Due to their stable color and low toxicity, curcuminoids have been widely used as food addictive, flavoring and food dye (Kong et al. 2009; Caprioglio et al. 2016; Kwon et al. 2015). In addition, curcuminoids have been confirmed to decrease renal triglyceride accumulation and successfully applied in the treatment of Alzheimer's disease and cardiac disorders (Soetikno et al. 2013; Ahmed et al. 2010; Morimoto et al. 2010). There are several reports indicating a wide range of pharmacological activities of curcuminoids, such as antioxidant,

anti-inflammatory,

anti-tumor,

anti-bacterial,

anti-diabetic,

etc.

(Jayaprakasha et al. 2006; Ruby et al. 1995; De et al. 2009; Wickenberg et al. 2010) Hence, there has been an increasing research focus on CUR, DMC and BDMC (chemical structure were shown in Fig. 1(a)). The synthesis and extraction are the major sources of curcuminoids (Al-Wabli et al. 2012; Stankovic et al. 2004). According to the Joint FAO/WHO Expert Committee on Food Additives (JECFA) specifications, curcuminoids as food additives must be derived from extraction (Stankovic et al. 2004). Several techniques have been applied for extracting curcuminoids, such as conventional organic solvent extraction, alkaline 3

solution extraction, supercritical fluid extraction and ultrasonic-assisted extraction (UAE) (Braga et al. 2003; Stransky et al. 1979; Costa et al. 2012; Xu et al. 2015). However, these methods are time-consuming, laborious and not beneficial for thermo-sensitive

substances

(curcuminoids

are thermo-sensitive

substances),

requiring expensive instruments and apparatus (supercritical fluid), high energy consumption and large volume of organic solvent. Moreover, they may have lower extraction yield (Li et al. 2014; Zhan et al. 2011). Some applications of microwave-assisted extraction (MAE) for extraction have been reported. MAE has been shown to be promising and effictive for extraction, ensuring higher yields at much shorter time (Sinha et al. 2012; Heleno et al. 2016). MAE operates through cell bursting, which facilitates entry of the extracting solvent to solubilize out the target compounds, thus lead to faster extraction and prevent degradation of target compounds (Mandal et al. 2008). Ionic liquids (ILs) are defined as organic salts that are composed of a large organic cation and an organic or inorganic anion, and ILs are liquid at or near room temperature (Aguilera-Herrador et al. 2010). The replacement of organic solvents by ILs in diverse applications has been paid more attention due to their unique properties such as non-volatile, non-flammable, high thermal and chemical stabilities, broad liquid temperature range, high solubility and excellent microwave-absorbing ability (Aguilera-Herrador et al. 2010; Passos et al. 2014; Tana et al. 2016; Shah et al. 2016; Welton et al. 2004). Comparing to traditional organic solvents, ILs can not evaporate into the atmosphere and contaminate the environment in health-harming ways (Poolea 4

et al. 2010). Besides that, the chemical and physical properties of ILs (polarity, hydrophobicity, viscosity, etc.) can be determined by choosing the cationic or the anionic constituent (Aguilera-Herrador et al. 2010; Welton et al. 2004). Since ILs are considered as “green designer solvent”, increasing their potential uses. In this paper, we report a novel technology of ionic liquid-based microwave-assisted extraction of curcuminoids for exploring a green and easy-to-use route, which can extract curcuminoids quickly and the maximum to avoid destruction of curcuminoids. Experimental conditions were optimized using the response surface methodology (RSM). Finally, the ILs-based MAE experiment results were compared with other extraction methods. 2. Experimental 2.1 plant materials and reagents Dried rhizomes of Curcuma longa L. were purchased from local medicinal materials market (Hangzhou,China) in 2015. The dried rhizomes were ground to a powder and passed through 60 mesh sieve. Standards of CUR, DMC, BDMC (purity 98%) were bought from Shifeng biological technology Co, Ltd. (Shanghai, China). Acetic acid was purchased from Lingfeng chemical reagent Co, Ltd. (Shanghai, China). Acetonitrile and methanol of HLPC grade were bought from Tedia Company, Inc. All ILs ([BMIM][Br], [HMIM][Br], [OMIM][Br] and [OMIM][BF4]; purity 99%) shown in Fig. 1(c) were purchased from Chengjie chemical Co, Ltd. (Shanghai, China). 1,1 diphenyl-2-trinitrobenzene

hydrazine

2,2'-azobis[2-methylpropionamidine]dihydrochloride 5

(DPPH), (ABTS),

2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (AAPH) and Vitamin C of analytical grade were bought from J&K Chemical Ltd. (Beijing, China). All other chemicals were of analytical reagent grade and deionized water was used in all experiments. 2.2 Apparatus The microwave extractor applied to the experiment was a CEM Discover system equipped with a 2455 MHz magnetron and infrared fiber-optic probe. (CEM Corporation, Matthews, NC, Made in USA). The max current and microwave power were 6.3 A and 300 W, respectively. The HPLC analysis of curcuminoids was performed on an Agilent 1290 Infinity HPLC comprising a automatic column temperature control box, a automatic sampler, a quaternary pump and a DAD detector (Germany). Chromatographic separation was carried out with a Sino Chrom ODS-BP C18 reversed phase column (4.6 mm×250 mm, 5 μm, Elite-AAA, Dalian China). 2.3 HPLC analysis and quantification The mobile phase consisted of 60% acetonitrile and 40% acetic acid aqueous solution (0.5% v/v) was set at an isocratic mode with a flow rate of 0.9 mL/min. The chromatographic separation was performed at 30

and the injection volume was 5

μL. The detection wavelength was 420 nm. All the standard solutions and samples were filtered through 0.22 μm nylon membrane. The retention times of BDMC, DMC, CUR were 6.9, 7.5 and 8.2 min, respectively (Fig. 1(b)). Peak area was used for the quantification of curcuminoids in extracts using standard calibration curve equation. The equations curves are: 6

YBDMC  19632 X  169.40 (R2=0.9932, X: 0.01-1 mg/mL) YDMC  27579 X  78.648 (R2=0.9991, X: 0.01-1 mg/mL) Y CUR 19492 X  94.311

(R2=0.9996, X: 0.01-1 mg/mL)

X represents the concentration of BDMC, DMC and CUR standards, respectively. Y represents the peak area of BDMC, DMC and CUR standards, respectively. 2.4 Microwave-assisted extraction with ionic liquid A certain quality of turmeric powder (0.05-2 g) was added to a glass reaction tube with 30 mL ionic liquid aqueous solutions (concentration of 0.2-0.7 mol/L). Then the tube was immersed in the microwave cavity. In order to make turmeric powder completely disperse in ionic liquid aqueous solution, a magnetic stirrer was added and used for mixing. The extraction was performed under different temperature (30-80

)

for certain irradiation time (2-12 min). Finally, extract was filtered through 0.22 μm nylon membrane for subsequent HPLC analysis. The following equations were used to calculate the extraction yield of BDMC, DMC, CUR and average three kinds of curcuminoids (AVE). Yield

BDMC

(%)  C BDMC * V （ / m * 10）

Yield

DMC

(%)  C DMC * V （ / m * 10 ）

Yield

CUR

(%)  C CUR * V /( m * 10 )

Yield AVE (%)  Yield CUR (%)  Yield DMC (%)  Yield BDMC (%)  / 3

where CBDMC, CDMC, CCUR (mg/mL) are the concentration of BDMC, DMC and CUR in extract, respectively. V (mL) is the volume of the extraction solvent, and m (g) is the mass of turmeric powder. Yield AVE is the average extraction yield of three kinds of 7

curcuminoids. 2.5 Optimization ILs-based MAE by response surface methodology In order to select optimal experimental conditions, an optimization process was performed using RSM, which is a effective statistical method. Due to its simple and effective (Sharif et al. 2014), Box-Behnken design (BBD) was employed to optimize extraction process parameters and access the interactions between factors on the basis of the single-factor experiment results. Extraction temperature (X1), extraction time (X2) and solid-liquid rate (X3) were selected as independent variables. Response variables were the average extraction yield of curcuminoids and antioxidant value. The related data of independent variables and response variables were analyzed to get the second-order polynomial model as shown in following equation. 3

3

2

Y  b0  bi X i   bii X i  i 1

i 1

3

b

ij

XiX

j

i  j 1

Where Y expresses the response variable, b0, bi, bii, and bij mean the intercept, linear, quadratic and interaction coefficients, respectively. Xi and Xj are independent variables. 2.6 In vitro antioxidant assay 2.6.1 DPPH radical scavenging activity For DPPH assay, all the extracts were diluted 10 times in 80% ethanol solution (v/v), namely diluted solutions. As a reference antioxidant, vitamin C was dissolved in 80% ethanol solution (v/v), namely standard solution. 200 μL of diluted solutions and standard solutions (2.5-60 μg/mL) were placed in different tubes with 2 mL of DPPH 8

solution in ethanol (0.04 g/L). The mixture was thoroughly mixed and incubated at 25

in dark for 30 min. The absorbance of mixture was measured at 517 nm. All

tests and analyses were performed in triplicate. The antioxidant value was calculated by standard curve method, which was expressed as mg of trolox equivalent per g turmeric (mg TE/g sample). 2.6.2 ROO∙ radical scavenging activity (TRAP) For TRAP assay, all the extracts were diluted 10 times in 80% ethanol solution (v/v), namely diluted solutions. As a reference antioxidant, vitamin C was dissolved in 80% ethanol solution (v/v), namely standard solution. 0.05 g of ABTS and 0.675 g of AAPH dissolved with acetate buffer (PH=4.3) respectively, and then transferred to a 500mL volumetric flask together. The mixed solution was incubated at 45

in water

bath for 1 h and cooled to room temperature in the dark. 4 mL of radical solution was mixed with 100 μL of diluted solutions or standard solution (20-200 μg/mL) at different tubes. The mixture was incubated at 25

in dark for 30 min, and then the

absorbance of mixture was measured at 734 nm. The antioxidant value was calculated as above, which was expressed as mg of trolox equivalent per g turmeric (mg TE/g sample). 2.7 Conventional reference extraction method Ultrasonic-assisted extraction was selected as the reference method for extraction of curcuminoids from Curcuma longa L.. Ultrasonic-assisted extraction was performed at the optimum conditions (Xu et al. 2015), including extraction time of 90 min, ultrasonic power of 250 W, concentration of ILs of 0.42 mol/L and solid-liquid rate of 9

1/30 (g/mL). In addition, effect of different solvents on microwave-assisted extraction of curcuminoids from Curcuma longa L. was investigated. The solvents included water, ethanol(75%, 100%) and methanol(75%, 100%). The extraction experiment for MAE was operated at optimum conditions optimized by our work. 3. Result and discussion 3.1 Screening of types of ionic liquids The type of ILs had a considerable influence on the extracting. In order to find the optimum ionic liquid, the four kinds of ILs with different anions (BF4-, Br-) and carbon chain lengths (C-4–C-8) in the cation were investigated. The result was shown in Fig. 2(a) that [OMIM][BF4] was less efficient because of its poor water solubility, which was not conducive to facilitate analytes dissolving in solvent. Increasing alkyl chain length from butyl to octyl had significant influence on the extraction yield and the highest extraction yield of curcuminoids was [OMIM][Br]. This was owing to the lipophilicity of curcuminoids, and the lipophilicity of ILs was increased with increasing the alkyl chain length (Yang et al. 2011). As a result, [OMIM][Br] was chosen as extraction solvent and used for further extraction research. 3.2 Selection of optimal ionic liquid concentration Ionic liquid concentration was a crucial factor, which was studied to improve the extraction efficiency. The observations was given in Fig. 2(b). The extraction yield was increased with increasing concentration up to 0.3M. This can be explained by the fact that ILs can dissolve more plant fiber and promote solvent to seep into the cells (Usuki et al. 2011). As the same time, ILs of microwave absorption capacity and 10

transmission capacity were enhanced with increasing concentration (Zeng et al. 2010). Further increase of concentration showed negligible effect on the extraction yield due to the increasing viscosity thus solvent gets hardly inside the plant cells. From Fig. 2(b), the highest yield of target analytes was obtained in ILs concentration of 0.3 mol/L. Taking into consideration of extraction yield and economic energy-saving, 0.3M [OMIM][Br] was selected in the following experiments. 3.3 The effect of extraction temperature The extraction efficiency of curcuminoids affected by different extraction temperature was demonstrated in Fig. 3(a). The extraction yield increased as extraction temperature increased up to 50 50

, and then decreased as extraction temperature above

. This finding might be attributable to the solubility and thermal instability of

curcuminoids. A high temperature may improve the solubility of curcuminoids and diffusion of ILs. However, there was a risk of thermal degradation. The interpretation was consistent with our experiment data. Considering supreme extraction yield, 50 was adopted in subsequent single-factor experiment. In the process of employing RSM to optimize extraction conditions, extraction temperature of 30, 40, 50, 60 and 80

were used for further analysis.

3.4 The effect of extraction time To evaluate the effect of extraction time, a series of experiments were carried out at different extraction time. The result shown in Fig. 3(b) indicated extraction yield was increased with increasing extraction time. There are advantages and disadvantages to prolong extraction time. On the one hand, the cell structure could break down, which 11

is beneficial to the dissolution of curcuminoids. On the other hand, thermo-sensitive substances are damaged and the biological activity of materials also declined. It could be seen from Fig. 3(b), extraction time had no remarkable impact on extraction yield. Thinking about extraction yield and economic energy-saving, 6 min was employed in following single-factor experiment. In the process of employing RSM to optimize extraction conditions, extraction time of 2, 4, 6, 8, 10 and 12 min were used for further analysis. 3.5 The effect of solid-liquid rate The effect of solid-liquid rate on extraction yield was shown in Fig. 3(c). When the solid-liquid rate changed from 0.05/30 to 2/30, extraction yield was increased. This phenomenon could be explained that turmeric power played a role of a maxing and promoted transmission of microwave energy, which led to the effective dissolution of curcuminoids. Beyond solid-liquid rate of 0.5/30, there was a drastic decrease in extraction yield due to a saturation state of solvent. 0.5/30 (g/mL) was regarded as the most suitable solid-liquid rate. In the process of employing RSM to optimize extraction conditions, solid-liquid rate of 0.5/30, 1/30 and 2/30 (g/mL) were used for further analysis. 3.6 Optimization variables by response surface methodology According to the above experiment results, the interactions between response variables and independent variables were investigated by the BBD combined with RSM. In the BBD method, a model with 17 experimental runs and 5 replicates at center point was used to optimize extraction temperature, extraction time and 12

solid-liquid rate. The experimental design matrix and the results of corresponding response variables were presented in Table 1. According to a regression analysis of experiment data, second-order polynomial equations were obtained to express response values as the following equations:

Yield CUR  2.36  4.411 *10 4 X 1  8.114 * 10 4 X 2  0.23 X 3  0.028 X 1 X 2  2

2

0.6054 * 10 3 X 1 X 3  7.165 * 10 3 X 2 X 3  0.037 X 1  0.027 X 2  0.065 X 3

2

Yield DMC  0.81  3.897 *10 3 X 1  1.257 * 10 3 X 2  0.080 X 3  0.015 X 1 X 2  2

2

2.073 *10 3 X 1 X 3  3.986 *10 3 X 2 X 3  0.016 X 1  0.016 X 2  0.037 X 3

Yield

BDMC

2

 1 .56  0 .012 X 1  0 .015 X 2  0 .23 X 3  0 .044 X 1 X 2  2

2

4 .443 * 10  3 X 1 X 3  0 .013 X 2 X 3  0 .054 X 1  0 .046 X 2  0 . 073 X 3 2

2

 0 .015 X 1 X 2  0 .022 X 1 X 3  0 .016 X 1 X 2

2

2

Yield AVE  1.58  0.01 X 1  3.015 * 10 3 X 2  0.17 X 3  0.029 X 1 X 2  2

2

7.848 * 10  3 X 1 X 3  7.928 * 10 3 X 2 X 3  0.037 X 1  0.028 X 2  0 .06 X 3

2

YDPPH  0.4  5.41* 10 4 X 1  0.016 X 2  0.12 X 3  7.432 * 10 3 X 1 X 2  2

2

3.825 *10 4 X 1 X 3  0.022 X 2 X 3  5.877 *10 3 X 1  5.971*10 4 X 2  0.024X 3

2

YTRAP  1.33  0.018 X 1  0.015 X 2  0.43 X 3  0.022 X 1 X 2  0.013 X 1 X 3 2

2

 0.035 X 2 X 3  0.029 X 1  0.025 X 2  0.14 X 3

2

The three-dimensional images of response surface (Fig. 4) were drawn to illustrate the interactions among extraction temperature (X1), extraction time (X2) and solid-liquid rate (X3). The results of variance (ANOVA) for the quadratic model were shown in Table 2, Table 3. The model was highly significant when its “p-value” less than 0.01 and “Lack of it” more than 0.05. A small p-value for model (p<0.01) and an insignificant Lack of it (p>0.05) were obtained from Table 2 and Table 3, which indicated that the model was significant and applicable to optimize extraction conditions. The corresponding variables would be more significant with smaller p-value and greater F-value. The sequence of the influence of three factors on yield 13

was solid-liquid rate (p<0.0001), extraction temperature (p=0.1146) and extraction time (p=0.5275). Howerver, the rank order of the influence of factors on antioxidant value was solid-liquid rate (p<0.0001), extraction time (p=0.0196) and extraction temperature (p=0.9193). In this model, all determination coefficients (R2) were above 0.98, indicating a effective correlation between the observed value and predicted value. The low value of pure error and CV (CVAVE=0.85%, CVDPPH=3.52%, under 10%) indicated that the model was precise and reliable. With extraction yield as reference index, the optimum conditions were obtained by RSM analysis as follows extraction temperature of 55

, extraction time of 8 min,

and solid-liquid ratio of 0.5/30 (g/mL). However, with antioxidant value as reference index, the optimum conditions were obtained by RSM analysis as follows extraction temperature 70

, extraction time 2 min, and solid-liquid ratio 0.5/30 (g/mL). The

difference can be explained that the biological activity and the antioxidant ability of curcuminoids would be declined with increasing extraction time (Liyana-Pathirana et al. 2005). Furthermore, the antioxidant ability is dependent on the synergistic effect of extracted polyphenol. But synergistic effect will decrease when the content of polyphenol are too high (Thoo et al. 2010). In the experiment, total extract was adopted to determine antioxidant value. There might be other biological active ingredients that produced the difference. Therefore, further studies are still needed to investigate these assumptions. 3.7 Verification tests Verification tests were used to verify the reliability of the model by comparing the 14

actual value from experiment with the predictive value from RSM analysis. Verification tests were performed under the optimum conditions: extraction temperature of 55

, extraction time of 8 min and solid-liquid ratio of 0.5/30 (g/mL).

Experiment was carried out in triplicate, the average extraction yield of curcuminoids was 1.77% which was close to the predictive value of 1.69%. The results revealed that the model was reliable and reasonable. The RSD of 0.23% indicated the experimental condition was more stable with good reproducibility. 3.8 Comparison of different extraction procedures The purpose of this comparison was to further confirm that ILs-based MAE is a optimal method of extraction of curcuminoids from Curcuma longa L.. The results was presented in Table 4. Compared with ILs-based UAE, ILs-based MAE not only can dramatically improve yield of curcuminoids, but also shorten the extraction time greatly (from 90 min to 8 min) and save energy. Such differences in efficiency and time can be mainly caused by unique extraction mechanism of MAE. MAE operate through cell bursting that facilitate entry of the extracting solvent to solubilize out the target compounds. Another one is MAE can produce electromagnetic fields, which can accelerate dissolution of target compounds. Under our optimum conditions, the effect of ethanol(75%, 100%) and methanol(75%, 100%) on microwave-assisted extraction of curcuminoids was similar to [OMIM][Br]. Curcuminoids could not be extracted completely with water, which was compliant with the principle of the dissolution in the similar material structure. On the contrary, ionic liquid aqueous solution was propitious to extract curcuminoids. This illuminated that the polarity of 15

water can be changed by ionic liquid. Even though the yield with organic solvents was slightly higher than that with ILs, organic solvents are volatile and added pollution to the environment. However, ILs are non-volatile and can not evaporate into the atmosphere to prevent the environment from being polluted. Moreover, ILs have the potential to be recycled. The above data indicated ILs-based MAE is a better method for the extraction of curcuminoids from Curcuma longa L.. 4.Conclusions In the present research, microwave-assisted extraction of curcuminoids from Curcuma longa L. with ionic liquid was proved to be a fast and effective method. The extraction conditions were optimized by response surface methodology. With extraction yield as reference index, the optimum conditions were determined as follows extraction temperature of 55

, extraction time of 8 min and solid-liquid ratio

of 0.5/30 (g/mL). Another result, with antioxidant value as reference index, the optimum conditions were determined as follows extraction temperature 70

,

extraction time 2 min and solid-liquid ratio 0.5/30 (g/mL). The high temperature and short time contributed to maintain antioxidant activity of extract, and there might be other biological active ingredients that produced the difference. Under optimized conditions (extraction yield as reference index), The yield of AVE was 1.77%. As compared with ultrasonic-assisted extraction, microwave-assisted extraction could improve the yield, shorten extraction time and save energy consumption. Acknowledgments This work was financially supported by Qianjiang talents project in Zhejiang 16

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Tables Table 1. Experimental parameters of Box-Benhnken design and the extraction yield and the antioxidant value for ILs-based microwave-assisted extraction. Table 2. Analysis of variance (ANOVA) of the fitted second-order polynomial model for AVE. Table 3. Analysis of variance (ANOVA) of the fitted second-order polynomial model for DPPH. Table 4. Comparative study of extraction yield using different extraction methods

Figures: Figure 1. (a) Chemical structures for curcuminoids of Curcumin (CUR), Demethoxycurcumin (DMC) and Bisdemethoxycurcumin (BDMC). (b) HPLC chromatogram of raw extract of Curcuma longa L.. (c) Chemical structures for ILs. Figure 2. (a) Effect of types of ILs. Extraction parameters were as follows: 0.6 g turmeric powder, ILs concentration of 0.3 mol/L, extraction temperature of 40

and

extraction time of 5 min. (b) Effect of the concentration of [OMIM][Br]. The extraction experiment was performed at different concentrations (0.2, 0.3, 0.4, 0.5, 0.6 and 0.7 mol/L), other extraction parameters were set as following: 0.5 g turmeric powder, extraction temperature of 50

and extraction time of 5 min.

Figure 3. (a) Effect of the extraction temperature. The extraction procedure was carried out at different extraction temperature (30, 40, 50, 60 and 80

), other

extraction parameters were set as following: 0.5 g turmeric powder, ILs concentration 22

of 0.3 mol/L and extraction time of 5 min. (b) Effect of the extraction time. The extraction procedure was performed at different time (2, 4, 6, 8, 10 and 12 min), other extraction parameters were set as following: 5 g turmeric powder, ILs concentration of 0.3 mol/L and extraction temperature of 50

. (c) Effect of the solid-liquid rate.

The extraction procedure was carried out at different solid-liquid rate of 0.05/30, 0.1/30, 0.3/30, 0.5/30, 1/30 and 2/30 (g/mL), other extraction parameters were set as following: ILs concentration of 0.3 mol/L, extraction temperature of 50

and

extraction time of 5 min. Figure 4. Response surface for the interactions of independent variables on extraction yield of AVE and antioxidant value of DPPH and TRAP.

23

Table 1. Experimental parameters of Box-Benhnken design and the extraction yield and the antioxidant value for ILs-based microwave-assisted extraction. Antioxidant capacity Factor

Yield(%) (mg TE/g sample)

Run X1

X2

X3

( )

(min)

(g/mL)

1

80

7

2

55

3

CUR

DMC

BDMC

AVE

DPPH

TRAP

0.50

2.49±0.045

0.83±0.051

1.66±0.010

1.66±0.012

16.11±0.023

56.82±0.061

2

2.00

2.04±0.081

0.67±0.083

1.21±0.009

1.31±0.067

9.34±0.091

31.49±0.018

55

7

1.25

2.33±0.012

0.79±0.081

1.53±0.052

1.55±0.084

12.08±0.045

40.81±0.075

4

55

7

1.25

2.36±0.056

0.80±0.050

1.55±0.027

1.57±0.039

12.69±0.057

41.38±0.016

5

55

2

0.50

2.50±0.071

0.83±0.017

1.64±0.031

1.66±0.073

17.95±0.060

60.44±0.028

6

30

7

0.50

2.49±0.024

0.83±0.049

1.62±0.085

1.65±0.047

16.22±0.033

54.50±0.081

7

55

7

1.25

2.36±0.064

0.80±0.021

1.56±0.094

1.58±0.097

12.13±0.064

40.03±0.038

8

80

12

1.25

2.28±0.028

0.77±0.072

1.44±0.021

1.50±0.023

11.64±0.079

39.67±0.062

9

80

7

2.00

2.02±0.059

0.68±0.048

1.24±0.045

1.31±0.034

9.09±0.038

31.31±0.052

10

55

7

1.25

2.36±0.086

0.81±0.061

1.56±0.039

1.58±0.081

12.13±0.082

40.03±0.027

11

55

12

0.50

2.51±0.046

0.85±0.027

1.70±0.076

1.68±0.073

15.02±0.065

56.24±0.042

12

55

7

1.25

2.36±0.051

0.81±0.071

1.56±0.082

1.58±0.054

12.13±0.087

40.03±0.048

13

55

12

2.00

2.03±0.011

0.67±0.058

1.22±0.034

1.30±0.037

8.99±0.026

31.55±0.073

14

30

7

2.00

2.04±0.026

0.68±0.042

1.22±0.053

1.32±0.028

9.24±0.078

30.52±0.062

15

80

2

1.25

2.33±0.039

0.80±0.091

1.53±0.059

1.55±0.063

12.30±0.085

40.72±0.029

16

30

2

1.25

2.26±0.027

0.75±0.048

1.39±0.064

1.47±0.016

11.79±0.025

38.85±0.026

17

30

12

1.25

2.32±0.018

0.78±0.091

1.48±0.048

1.53±0.024

12.02±0.064

40.42±0.075

X1 for extraction temperature, X2 for extraction time and X3 for solid-liquid rate.

24

Table 2. Analysis of variance (ANOVA) of the fitted second-order polynomial model for AVE. Sum of Mean F p-value Df Squares Square Value Prob > F Model 0.27 9 0.030 185.17 <0.0001 significant X1 5.344E-004 1 5.344E-004 3.25 0.1146 X2 7.274E-005 1 7.274E-005 0.44 0.5275 X3 0.25 1 0.25 1508.12 <0.0001 X1X2 3.356E-003 1 3.356E-003 20.39 0.0027 X1X3 7.025E-005 1 7.025E-005 0.43 0.5344 X2X3 2.514E-004 1 2.514E-004 1.53 0.2563 2 X1 4.252E-003 1 4.252E-003 25.83 0.0014 2 X2 2.893E-003 1 2.893E-003 17.58 0.0041 2 X3 0.013 1 0.013 76.78 <0.0001 Residual 1.152E-003 7 1.646E-004 Not Lack of Fit 5.607E-004 3 1.869E-004 1.26 0.3991 significant Pure Error 5.914E-004 4 1.479E-004 Cor Total 0.28 16 C.V. % 0.85 R-Squared 0.9958 X1 for extraction temperature, X2 for extraction time and X3 for solid-liquid rate.

Table 3. Analysis of variance (ANOVA) of the fitted second-order polynomial model for DPPH. Sum of Mean F p-value Df Squares Square Value Prob > F Model 107.98 9 12.00 62.87 <0.0001 significant X1 2.107E-003 1 2.107E-003 0.011 0.9193 X2 1.73 1 1.73 9.08 0.0196 X3 102.38 1 102.38 536.48 <0.0001 X1X2 0.20 1 0.20 1.04 0.3414 X1X3 5.267E-004 1 5.267E-004 2.760E-003 0.9596 X2X3 1.67 1 1.67 8.74 0.0212 2 X1 0.22 1 0.22 1.13 0.3229 2 X2 0.019 1 0.019 0.10 0.7588 2 X3 1.84 1 1.84 9.63 0.0172 Residual 1.34 7 0.19 Not Lack of Fit 1.07 3 0.36 5.49 0.0668 significant Pure Error 0.26 4 0.065 Cor Total 109.32 16 C.V. % 3.52 R-Squared 0.9878 X1 for extraction temperature, X2 for extraction time and X3 for solid-liquid rate. 25

Table 4. Comparative study of extraction yield using different extraction methods Methods Water MAE Ethanol MAE 75% Ethanol MAE Methanol MAE 75% Methanol MAE [OMIM][Br] MAE [OMIM][Br] UAE

Extraction time (min) 8 8

Extraction yield (mean + SD, %) CUR DMC BDMC AVE 0 0 0 0 2.71±0.076 0.93±0.054 1.69±0.042 1.78±0.057

8

2.78±0.057

1.02±0.039

1.97±0.064

1.92±0.053

8

2.71±0.035

0.94±0.045

1.74±0.081

1.80±0.054

8

2.69±0.053

0.97±0.071

1.89±0.060

1.85±0.062

8

2.67±0.041

0.91±0.055

1.72±0.051

1.77±0.049

90

1.38±0.068

0.50±0.041

0.91±0.047

0.93±0.052

Fig. 1 26

Fig. 2

Fig. 3

27

Fig. 4

28