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Dec 31, 2011 - Keywords: quercetin, onion skin, microwave, ultrasound, optimization ..... Degree of .... Technology Development Program for Agriculture and.
Food Sci. Biotechnol. 20(6): 1727-1733 (2011) DOI 10.1007/s10068-011-0238-8

RESEARCH ARTICLE

Optimization of Various Extraction Methods for Quercetin from Onion Skin Using Response Surface Methodology Eun Young Jin, Seokwon Lim, Sang oh Kim, Young-Seo Park, Jae Kweon Jang, Myong-Soo Chung, Hoon Park, Kun-Sub Shim, and Young Jin Choi

Received: 2 September 2011 / Revised: 22 October 2011 / Accepted: 24 October 2011 / Published Online: 31 December 2011 © KoSFoST and Springer 2011

Abstract Quercertin and typical flavonoids present in onion skin draw interest due to bioactive properties. For utilizing quercetin from onion skin, conventional solvent extraction (CSE), microwave assisted extraction (MAE), and ultrasound assisted extraction (UAE) were employed. Statistic models of each method were proposed to estimate the best possible yield of quercertin employing response surface methodology (RSM). The effects of several independent variables including concentration of ethanol, provided power or the temperature, and reaction time were investigated. From 1 g sample of dried onion skin, the highest yield of each method could be achieved at 16.5 min of process time under 59.2oC for CSE with 59.3% ethanol, 117 s for MAE with 69.7% ethanol, and 21.7 min for UAE using power of 606.4 W with 43.8% ethanol. The most productive method was MAE, whose maximum yield was 20.3 and 30.8% higher than UAE and CSE, respectively. Eun Young Jin, Seokwon Lim, Sang oh Kim, Young Jin Choi ( ) Department of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, Seoul 151-742, Korea Tel: +82-2-880-4851; Fax: +82-2-873-5095 E-mail: [email protected] Young-Seo Park Department of Food Science and Biotechnology, Kyungwon University, Seongnam, Gyeonggi 461-701, Korea Jae Kweon Jang Department of Food Science, Chungkang College of Cultural Industries, Icheon, Gyeonggi 467-744, Korea Myong-Soo Chung Department of Food Science and Engineering, Ewha Womans University, Seoul 120-750, Korea Hoon Park Department of Food Science, Sunmoon University, Asan, Chungnam 336708, Korea Kun-Sub Shim Greenbio Co., Ltd., Icheon, Gyeonggi 513-46, Korea

Keywords: quercetin, onion skin, microwave, ultrasound, optimization

Introduction Recently, quercetin has drawn attention due to its beneficial effects for human health (1). Besides well-known antioxidant activity (2), such anticancer properties were revealed as to reduce the carcinogenic activity of several mutagens in cooked foods and to inhibit the enzymatic activities associated with several types of tumor cells (3). Quercetin is a typical flavonoid ubiquitously present in vegetables and fruits as the form of aglycon or glycosides. Moreover, by the preference trend in these days for natural compounds rather than synthesized products, plant origin flavonoids are drawing much interest (4). Consequently, there is an increasing demand on the practical method to obtain such bioactive compounds from diverse plant sources (3). To utilize such valuable bioactive compounds in the plants, methods to isolate such substances from the source materials are needed promptly. Therefore, novel techniques of extraction such as ultrasound assisted extraction (UAE), microwave assisted extraction (MAE), supercritical fluid extraction (SFE), and accelerated solvent extraction (ASE) are introduced using diverse solvents to increase the yield of extraction and the process efficiency by reducing the process time and the consumption of solvent and energy (5-9). Especially, MAE (10, 11) rapidly delivers the energy both to the overall volume of solvent and to solid matrix of plant. Subsequently, heating by MAE is efficient and homogeneous (12). Because water within the plant matrix absorbs microwave energy, the internal superheating promotes cell disruption, which facilitates desorption of chemicals from the matrix, improving the eruption of

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nutraceuticals (13). Since the ultrasound increases the rate of diffusion to allow more rapid penetration of solvent into the matrix of materials (14), UAE is known as the fast and efficient technique to extract chemicals from plant materials (15). Nonetheless, proper methods and conditions to optimize extraction process were known to be varied depend on the physiochemical properties of the plant sources (16). Onion is one of the most common and the richest natural source of flavonoids (17). Among flavonoids present in the onion, quercetin and its glycoside are well-known major substances (18). Kang et al. (19) investigated the yields of onion juice and the optimal conditions of various methods to extract quercetin and its related glycosides. Though several studies on this issue were progressed with whole onions (19-21), the retrieval of helpful bioactive substances from the onion skin also has been attended as the way to utilize or evaluate the abundant parts of the resources (22). Thus, the extraction methods of valuable components from the waste of onion should be worth enough to pursuit in economic point of view and environmental benefit. Turner et al. (21) performed SFE of onion waste using water and showed β-glucosidase-catalyzed hydrolysis of quercetin glycosides. And Kiassos et al. (20) optimized the extraction of phenolics from the solid waste of the onion. Here, practical methods for extracting quercetin and its glycosides from dried onion skin were investigated, because the most flavonoids in onion are distributed in the outer skin (23). The highest yield of several extraction processes was estimated and the effect of independent variables on each method was evaluated using response surface methodology (RSM), which has been employed for the optimization in industrial applications and other processes due to its practical convenience (24).

Materials and Methods Preparation of onion skin Onions were purchased from local market placed in Gwanak-gu, Seoul, Korea. As soon as peeled, onion skins were immediately frozen at −75oC for 48 h, and then dehydrated in a freeze drier (FD 5508; Ilshin, Yangju, Korea). After pulverized with the blender (BW-05G; Jeiotech, Seoul, Korea), onion skins were stored at an average temperature of 4oC. Conventional solvent extraction (CSE) As the solvent, certain concentration of ethanol solution was applied solely. Because the yield of quercetin and its glycoside using 60% ethanol was 5 times higher than that using pure distilled water, meanwhile, there is no significant difference from the case of methanol (25). Hence, there were 3 control factors subjected as the variables of CSE; the

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concentration of ethanol solution, the process time, and the temperature of extraction. Extraction was started by mixing 1 g of dried onion skins with 40 mL of diverse concentration of ethanol. The extraction process was performed in the water bath (BS-11; Jeiotech) at given temperature. Extract was separated by 30 min of centrifugation (KR/Union 55R; Hanil, Seoul, Korea) at 1,600×g under room temperature. The supernatant was sampled to analyze the chemical composition of the extract and the yield of quercetin. Microwave assisted extraction (MAE) The mixture of onion skin with the solvent, prepared as described previously, was microwaved in the closed falcon tube using a commercial microwave oven (RE-21CRN; Samsung, Seoul, Korea), which has 700 W of the power and irradiates at 10 s of the interval times. For MAE, the process time and the concentrations of ethanol were subjected as the variable. Ultrasound assisted extraction (UAE) To carry out ultrasound treatment, a closed extractor was applied to fit an ultrasonic horn transducer (750 W, VCX 750; Sonics & Materials Inc., Newtown, CT, USA). Previously described mixture for the extraction was treated using diverse power level of ultrasound during various process times. The temperature of process was controlled to maintain less than 30oC. Analysis of the extract HPLC (600S controller; Waters, Milford, MA, USA) was used to analyze the chemicals in the extract. Before injected to HPLC, the extract were diluted with methanol and filtered through a 0.2-µm filter (Milipore, Billerica, MA, USA). The content of quercetin was analyzed by the area of the peak at 375 nm, measured from the UV detector (SLC-200; Samsung) interfacing Nova-Pak C-18 column (3.9×150 mm, i.d. 4 µm) with 20µL loop HPLC system (25). Obtained data were collected and evaluated using the Autochro-2000 PC program (Young Rin, Ahnyang, Korea). Chromatography was performed at 1 mL/min of flow rate using 0.1% formic acid and methanol as the solvent. Sample peaks were quantified with the external standard methods. The quercetin standard was purchased from Sigma-Aldrich (St. Louis, MO, USA). Statistical analysis Each experiment was replicated more than 3 times. Acquired data were handled to calculate statistical values such as mean-square and standard deviation (SD) using Microsoft Excel (Microsoft Inc., Redmond, WA, USA). The statistical significant was evaluated and verified at the level of p1. The precise value of α depends on certain properties desired for the design and on the number of factors involved. CSE: Response surface analysis (RSA) was performed to estimate the effects of independent variables on the response within the range of investigation. Design of the response surface regression (RSREG) procedure of statistical software SAS (9.1 SAS Institute Inc., Cary, NC, USA) was used to design CCD (uniform precision) (27) and to analyze the experimental data with 3 independent variables [X1, ethanol concentration (%); X2, extraction temperature (oC); X3, extraction time (time)] at 5 levels was performed. Axial scaling of RSREG design is rotatable design and α for axial scaling is 1.68. Table 1 presented the range of Table 1. Central composite design by RSM program for optimization of conventional extraction conditions Experiment Ethanol conc. Temperature no. (X1, %) (X2, oC) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Time (X3, min)

25 (-1) 22 (-1) 9 (-1) 25 (-1) 22 (-1) 25 (1) 25 (-1) 50 (1) 9 (-1) 25 (-1) 50 (1) 25 (1) 81 (1) 22 (-1) 9 (-1) 81 (1) 22 (-1) 25 (1) 81 (1) 50 (1) 9 (-1) 81 (1) 50 (1) 25 (1) 5.9 (-1.68) 36 (0) 17 (0) 100 (1.68) 36 (0) 17 (0) 53 (0) 12.5 (-1.68) 17 (0) 53 (0) 59.5 (1.68) 17 (0) 53 (0) 36 (0) 3.5 (-1.68) 53 (0) 36 (0) 30.5 (1.68) 53 (0) 36 (0) 17 (0) 53 (0) 36 (0) 17 (0) 53 (0) 36 (0) 17 (0) 53 (0) 36 (0) 17 (0) 53 (0) 36 (0) 17 (0) 53 (0) 36 (0) 17 (0)

Quercetin (mg/g) 1.00 1.11 2.18 2.46 2.12 3.12 3.30 3.06 0.48 2.25 1.82 3.61 1.83 2.51 2.62 2.59 2.39 2.62 2.05 2.44

independent variables, their ranges and the whole design consisted of 20 experimental points carried out random order to optimize the extraction process (27). Experimental data were fitted to a second-order polynomial model and regression coefficients obtained. The generalized secondorder polynomial model used in the RSA was as following equation: 3

3

i=1

i=1

2

Y = β0 + ∑ βiXi + ∑ βiiXi +

3

∑ βijXiXj

i