Optimization of Ionic Liquid 1-Butyl-3-Methylimidazolium Hexafluorophosphate ( [ Bmim ] PF 6 )-Based Microwave Assisted Extraction Method for Gamma Oryzanol from Rice Bran ( Oryza Sativa L . )

Article history: Received on: 13/07/2017 Accepted on: 26/08/2017 Available online: 30/12/2017 The effect of IL solvent and the microwave-assisted extraction (MAE) including concentration IL, the ratio of liquid/solid, time extraction and microwave power in the result of gamma oryzanol extract in rice bran (Oryza Sativa L.) were investigated. The application of IL [Bmim] PF6 as a selected solvent in the MAE method was developed for the extraction of gamma oryzanol from rice bran. The Box–Behnken design on four factors with response surface methodology version 10 was used to optimize experimental conditions. The optimum process for the MAE using ionic liquids ([Bmim]PF6) concentration 0.7 M [Bmim]PF6 solution, the ratio of liquid/solid 15mL/g, extraction time 10 min, and 30% microwave power with the gamma oryzanol value of 0.27 mg/g.The IL-MAE method is more efficient for the extraction of gamma oryzanol compounds from rice bran.


INTRODUCTION
Currently, Indonesia produces a lot amount of paddy which results an increasing by-product rice milling and rice bran is one of such by product.Some researchers have used rice bran as raw materials in the extraction process because rice bran has high nutrition and contains antioxidants such as tocopherol, oryzanol, tocotrienol, squalene, and polyphenol.Gamma oryzanol is a mixture compound of at least 10 components of ferulic acid esters and alcohols triterpene (Liazid et al., 2007).The various pharmacological activities of gamma oryzanol including anti-inflammatory, antineoplastic, hypoallergic, antidiabetic, antiulcerogenic, hypolipidemic (Islam et al., 2011) andanti-cancer (Forster et al., 2013).Suitable of solvents is needed in increasing the levels of gamma oryzanol in the extract.Some previous studies have been done in the extraction of rice bran oil by using conventional solvents such as isopropanol and hexane.Some disadvantages of conventional solvents such as the amount of solvent used, long extraction time and environmentally unfriendly (Ballard et al., 2010).Therefore, the use of anionic liquid solvent as the application of green chemistry principles can increase the active compounds from natural materials (Espino et al., 2016).Ionic liquids are known as safe, environmentally friendly, non-toxic solvents, vapor ignored pressure, good thermal stability, and non-flammable.IL-MAE approach is a green, simple, rapid, efficient extraction method for extracting bioactive compounds from plant materials (Xu et al., 2012).But in our knowledge, until now, IL-MAE has not yet been reported for extraction of gamma oryzanol from rice bran (Oryza Sativa L.) In this experiment, IL 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim]PF 6 ) was used as a hydrophobic solvent in microwave-assisted extraction (MAE) method in gamma oryzanol extraction from rice bran.The microwaveassisted extraction (MAE) method is a faster extraction method than conventional extraction methods such as Soxhlet (Du et al., 2007).The experimental results were analyzed by response surface methodology (RSM).Response surface methodology is a technique that can be used in the process of studying parameters or variables used in the study.Therefore, the purpose of this experiment was to investigate the effect of ionic liquid concentration, the ratio of liquid/solid, time extraction and microwave power in the level of gamma oryzanol extract in rice bran.The optimum conditions for extraction are determined by response surface methodology (RSM) version 10 so that the highest gamma oryzanol levels can be obtained.

Stabilization of rice bran
Sample rice bran fresh with IR-64 varieties was stabilized using an oven at 110° C for 15 minutes.After that rice bran sample was cooled in a container for 30 minutes to reach room temperature.The stabilized rice bran was put in clear plastic and stored at room temperature (Thanonkaew et al., 2012;Nasir et al., 2009).

Extraction method conventional solvent-MAE
The stabilized samples (1 g) were placed into a flatbottom flask, then was added isopropanol with the appropriate experimental design (w/v).The boiling flask was placed into a microwave by the treatment of variations of the ratio of liquid/solid (10-20 mL/g), extraction time (5, 10, 15 minutes) and the microwave power (10%, 30%, 50%).The results of the extract were cooled at room temperature for 10 min and diluted to 10 mL with isopropanol.Then 20 μL of the extract solution was injected using a syringe into the HPLC system.Optimization condition on extraction was employed by response surface methodology (RSM) version 10, so the extraction conditions were varied to some factors as described in Table 1.

Optimization IL-MAE by response surface methodology (RSM)
The Box-Behnken design on four factors with response surface methodology with Design-Expert® Software Version 10 (Stat-Ease, Inc., Minneapolis, USA) and it was used to optimize experimental conditions (Table 2) and analysis the interaction between some factors and optimize experimental conditions to get 25 sample of treatment.The factors for microwaved-assisted extraction of gamma oryzanol included IL [Bmim]PF 6 concentration (0.4-0.7 M), the ratio of liquid/solid (10-20 mL/g ) extraction time was varied on several conditions (5-15 minute), microwave power of 10, 30 and 50% of 900 W for 100% microwave power supply.The response value of this study gamma oryzanol was a yield (mg/g) with ANOVA analysis for the significance of the model.

Extraction method IL-MAE
The stabilized rice bran (1 g) was put in a flat-bottom flask.The ionic liquid solution with different concentration and volume was added into the flask.The boiling flask was placed into a microwave.The extraction was performed under different power level and time.After the extraction process was completed, the sample was cooled to room temperature ± 10 minutes, then was filtered using a filter paper to obtain the desired filtrate as a result of extraction MAE.Furthermore, the filtrate was added to 1 ml of solvent N-hexane and 1 ml KH 2 PO 4 0.01mol.Then was vortexed for 10 seconds and followed centrifuged at 3000 rpm for 15 minutes to separate the residue with the supernatant.

Determination of gamma oryzanol levels with HPLC
Calibration standard gamma oryzanol curve was constructed with correlating concentration in the range of concentration of 1 ppm-45 ppm and area under curve value (Figure 1).The supernatant (0.5 ml) was added isopropanol and put in a flask up to 10 ml.Then 20 µL was injected into the HPLC system (Shimadzu Corp., Japan).A mixture of methanol (50%), acetonitrile (40%), and isopropanol (10%) was used as mobile phase under isocratic conditions.UV detector wavelength set at 327 nm and the flow rate was set 1 ml/ min.Each sample was measured with 3 repetitions.

Optimum conditions of conventional solvent-MAE
Based on the results of the statistical analysis using RSM version 10 retrieved the value F of 5.07 and value P of 0.1042 thus the model was not significant in influencing levels of gamma oryzanol.Several factors indicated a significant relationship with the levels of gamma oryzanol included model AC (the ratio of liquid/solid and power) showed of P-value (0.0392), A 2 (the ratio of liquid/solid) of 0.0359 and B 2 (extraction time) of 0.0226.based on the analysis of the optimum conditions acquired RSM in increasing levels of gamma oryzanol on sample RUN 2 (the ratio of liquid/solid 15 mL/g, 10 min, and 30% power) and acquired the levels of gamma oryzanol of 0.25 mg/g.MAE method was using microwave radiation to rapid selective extraction through solvent warming quickly and efficiently (Jain et al., 2009).The selection of a suitable solvent on extraction process by MAE method will affect the solubility of targeted analytes, the penetration of solvents and interactions with the matrix of the sample.Furthermore the ratio between solvents and samples that will be concerned in the extraction process.The MAE methods were used in extraction rice bran oil can increase the amount of oil and antioxidant content with isopropanol solvent (Duvernay et al., 2005).
Optimum conditions of solvent and sample ratio affected the process of warming on vessels to be effective and were distributed evenly.Over of solvents caused microwave warming became worse so that microwave radiation was absorbed by solvent and energy required when warming became too much (Chan et al., 2011).On the research of conventional solvent was used in the method of MAE is isopropanol caused gamma oryzanol was due to a combination of alcohol triterpene and ferulate acid slightly polar, thus easy to dissolve in the polar solvent like isopropanol (Xu and Godber, 1999).Isopropanol solvent used in the extraction process because of the polar solvent can absorb microwave energy better than nonpolar solvents (Duvernay et al., 2005).
According to previous research explains that a polarity compound was affected by the solubility of a solvent, it is shown in the ability of the extraction of oryzanol better-using isopropanol and methanol than hexane solvent.This was caused due to an interaction of the hydroxyl group on the benzene ring of ferulate esters part of gamma oryzanol compound with alcohol functional group of isopropanol solvent (Chen and Bergman, 2005).
Time is an important parameter in the extraction.Extraction time is affected by the value of dielectric solvent.The exposure the solvents such as water, ethanol, and methanol were too long at risk on the thermolabile of the target compound (Mandal et al., 2007).Microwave power was chosen precisely to avoid the degradation temperature in target compounds and over pressure in the extraction process.The breakdown of cells at low power happened gradually.Microwave power mutually influenced by temperature and time in extraction.The combination of lowpower medium and long extraction time was the best extraction conditions approach.High temperature and power caused breakdown cell walls (Mandal et al., 2007)

Optimum Conditions of IL-MAE
Research on the optimum extraction condition of the specified IL-MAE with the application of RSM, that are included in the application Design Expert 10.03.Quadratic models vs-2 had a value of Prob > F and all samples normally distributed, it can be seen in Figure 2 below.Based on the analysis of ANOVA for the optimization of the IL-MAE concluded that the value of F 1.46 and value of P 0.27.This means that this model showed the relationship was not significant in the levels of gamma oryzanol.In this case, there were three models that show a significant relationship with the levels of gamma oryzanol (value of Prob> F under 0.05) include AC (concentration of IL and extraction time) of 0.0444, A 2 (concentration) of 0.0324 and C 2 (extraction time) of 0.0288.
Mathematical equation from a model that was used to show the relationship between the independent variables and the variable response was: Y = 0.11 + 9.167 E-003 A -E-003 6.667 B + E 6.667-003 C + 2,500 E-003 D -0.023 0.023 AB + AC + BC AD-0.013 0000 + 0000 BD -0.022 CD-0034-0045 A2 B2 -C2 D2 0037-0046, Y represented the level of gamma oryzanol (mg/g), A was IL concentration (M), B was the ratio of liquid/solid (mL/g), C was a microwave power (%), and D was extraction time (minute).Figure 3 showed the image coordinates of the hot areas under one condition on extraction by the concentration of 0.7 M, the ratio of liquid/solid 15 (mL/g) in the extraction of 10 minutes, microwave power of 30% and obtained the levels of gamma oryzanol of 0.27 mg/g.It can be concluded that this condition had the highest response.Information of hot coordinate area was very useful for optimization.
It can also be due to the concentration of 0.7 M has closed to the level of polarity γ-oryzanol compound compared with the concentration of 0.4 M and 1 M.The condition of the extraction time of 15 minutes and microwave power of 30% was optimum condition due to exposure to the temperatures was not too hot so that the potential damage to the compound gamma oryzanol was decreased.Determination of optimum conditions can be done by showing a 3D surface with response peaks in the central point is the location instructions of optimum conditions (Figure 4).Levels of gamma oryzanol with conventional Soxhlet method obtained 0.047 mg/g (Marriod et al., 2014).This is compared with the results of the isopropanol-MAE 0.25 mg/g and IL-MAE of 0.27 mg/g, therefore be concluded that the MAE method was more effective in increased levels of gamma oryzanol as compared to the conventional method.IL-MAE is very suitable to extract the unstable compounds on heating.The IL-MAE method showed high extraction results with using less of solvents and energy (Bogdanov, 2014).In this research, the utilizing of eco-friendly solvents such as ionic liquids are more effective as solvents in the extraction of gamma oryzanol than organic solvents because of the flexibility of combinations ions in the customize properties of physicochemical in the target compounds.In addition of ionic liquid had the potential to replace organic solvents which characteristic are flammable, toxic and volatile (Jessop et al., 2012).
The efficiency of extraction with the ionic liquid can be increased with decreasing alkyl groups, where the alkyl residues can be reduced by lengthening alkyl group (Ying etal., 2007).Ionic liquid [Bmim]PF 6 was used as an absorption medium and applied to the extraction of essential oil from dried fruits of Illicium verum hook.F. and Cuminum cyminum L. by microwaveassisted extraction (Zhai et al., 2009).Ionic liquids as solvents can be used to extract the compounds from Peperomia pellucida (L.) Kunth plants and also the extraction process becomes faster, easier, and efficient compared conventional methods in general (Ahmad et al., 2017).

Table 1 :
The experimental conditions of isopropanol-MAE method with RSM.

Table 2 :
The experimental conditions of IL-MAE method with RSM.