Food Chemistry 154 (2014) 282–290

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Food Chemistry

journal homepage: www.elsevier .com/locate / foodchem

Analytical Methods

Preparation of curcumin microemulsions with food-grade soybeanoil/lecithin and their cytotoxicity on the HepG2 cell line

0308-8146/$ - see front matter � 2014 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2014.01.012

⇑ Corresponding author. Address: Department of Materials Science and Engi-neering, I-Shou University, No.1, Sec. 1, Syuecheng Rd., Dashu District, KaohsiungCity 84001, Taiwan. Tel./fax: +886 (7) 657 8228.

E-mail address: meihwalee@ntu.edu.tw (M.-H. Lee).

Chuan-Chuan Lin a, Hung-Yin Lin b, Ming-Hung Chi b, Chin-Min Shen c, Hwan-Wen Chen b,Wen-Jen Yang d, Mei-Hwa Lee c,⇑a Department of Food Science, China University of Science and Technology, Taipei 115, Taiwanb Department of Chemical and Materials Engineering, National University of Kaohsiung, Kaohsiung 811, Taiwanc Department of Materials Science and Engineering, I-Shou University, Kaohsiung 840, Taiwand Department of Life Sciences, National University of Kaohsiung, Kaohsiung 811, Taiwan

a r t i c l e i n f o a b s t r a c t

Article history:Received 25 April 2013Received in revised form 25 September 2013Accepted 6 January 2014Available online 14 January 2014

Keywords:CurcuminLecithinMicroemulsionsSoybean oilCytotoxicity

The choice of surfactants and cosurfactants for preparation of oral formulation in microemulsions is lim-ited. In this report, a curcumin-encapsulated phospholipids-based microemulsion (ME) using food-gradeingredients soybean oil and soybean lecithin to replace ethyl oleate and purified lecithin from our previ-ous study was established and compared. The results indicated soybean oil is superior to ethyl oleate asthe oil phase in curcumin microemulsion, as proven by the broadened microemulsion region withincreasing range of surfactant/soybean oil ratio (approx. 1:1–12:1). Further preparation of two formulawith different particle sizes of formula A (30 nm) and B (80 nm) exhibited differential effects on the cyto-toxicity of hepatocellular HepG2 cell lines. At 15 lM of concentration, curcumin-ME in formula A withsmaller particle size resulted in the lowest viability (approx. 5%), which might be explained by increasingintake of curcumin, as observed by fluorescence microscopy. In addition, the cytotoxic effect of curcumin-ME is exclusively prominent on HepG2, not on HEK293, which showed over 80% of viability at 15 lM. Theresults from this study might provide an innovative applied technique in the area of nutraceuticals andfunctional foods.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Curcumin (diferuloylmethane), a polyphenolic compoundderived from the natural product turmeric, possesses not onlychemopreventive but also anti-cancer activities for treating humandisease. A few studies in phase I clinical trials have reported curcu-min is safe and well tolerated in high daily doses of 12 g (Chenget al., 2001; Lao et al., 2006; Shoba et al., 1998). However, the oralbioavailability of curcumin is very low in vivo because it is insolu-ble in water and poorly soluble in the organic phase (Ravindranath& Chandrasekhara, 1981). Even though its bioavailability is lowerin human, the therapeutic efficacy of curcumin has been provenby several researchers, including cancer, cardiovascular diseases,Alzheimer’s disease and Crohn’s disease (Aggarwal & Harikumar,2009). In the past decade, a numerous approaches to enhance thebioavailability of curcumin have been documented, for exampleliposomal curcumin (Li, Braiteh, & Kurzrock, 2005), curcuminnanoparticles (Shaikh, Ankola, Beniwal, Singh, & Kumar, 2009)

and curcumin microemulsions (Lee, Lin, Chen, & Thomas, 2008;Lin, Lin, Chen, Yu, & Lee, 2009).

Microemulsions (ME), with droplet diameters less than 100 nm,are the spontaneous formation of stable vesicles consisting ofwater, oil and surfactants. They offer the advantages of opticaltransparency, thermodynamic stability, long-term stability andease of preparation. Microemulsions have been employed overthe past few years to improve the solubility of poorly water-solu-ble drugs in aqueous solution and either the penetration into orabsorption by cells in the pharmaceutical and functional foods field(Krishnaiah, 2010). Meanwhile, microemulsions have attractedmuch interest as potential drug delivery systems to prepare O/Wmicroemulsions as lipophilic/poorly soluble drug delivery vehicles(Bhatia, Zhou, & Banga, 2013; Fanun, 2012; Hsu, Cui, Mumper, &Jay, 2003; Kogan & Garti, 2006; Lee, Kao, & Lin, 2011; Lee, Yu,Kao, & Lin, 2009; Lin et al., 2009, 2012; Sahle, Metz, Wohlrab, &Neubert, 2012; Shaikh, Jadhav, Gide, Kadam, & Pisal, 2006; Solanki,Sarkar, & Dhanwani, 2012; Thanki, Gangwal, Sangamwar, & Jain,2013; Wang, Wu, et al., 2011; Yallapu, Jaggi, & Chauhan, 2012)

In general, microemulsions require higher surfactants/cosurfac-tant concentration to reduce the surface tension between oil phaseand water phase, but leading to increased toxicity. Some syntheticsurfactants are not permissible for food application in many

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countries. Consequently, the types of surfactants and cosurfactantsavailable for preparation of the oral formulation of microemulsionsare restricted. In recent years, phospholipids-based microemulsionshave received much attention as pharmaceutically acceptable for-mulaion (Attwood, Mallon, & Taylor, 1992; Constantinides, 1995;Lee et al., 2009; Lin et al., 2009; Patel, Schmid, & Lawrence, 2006)

Soybean oil does not contain much saturated fat and cholesterol.It is a popular and widely used health food supplement all over theworld. Garti’s group reported preparation of food-grade micro-emulsions using medium-chain triglycerides or essential oils asthe oil phase (Garti, Aserin, Wachtel, Gans, & Shaul, 2001). It wasexpected difficult to prepare microemulsions using triglyceridescontaining long-chain fatty acids, which are semi-polar comparedto hydrocarbons and too bulky to penetrate the interfacial film toassist in the formation of an optimal curvature (Gaonkar & Bagwe,2003). A pseudoternary phase diagrams of systems containing soy-bean oil and water, as well as the anionic and nonionic surfactantswas formulated and characterised (Mou et al., 2008; Polizelli, Telis,Amaral, & Feitosa, 2006). Retinoic acid microemulsions and lipo-philic drugs nanoemulsion were reported to be formulated usingsoybean oil and phospholipids, both of which were emulsified usinga high pressure homogenizer (Hwang, Lim, Park, & Kim, 2004).

In our previous study, a stable phospholipid-microemulsioncontaining curcumin, ethyl oleate (EO), purified lecithin and poly-sorbate 80 (Tween 80) with enhanced bioavailability of curcuminwas constructed. This study utilised food-grade ingredients soy-bean oil and soybean lecithin to replace ethyl oleate and purifiedlecithin from the previous system, respectively. Furthermore, theeffect of cell cytotoxicity of different curcumin-encapsulated soy-bean oil-based microemulsions in human hepatoma HepG2 cellline was evaluated.

2. Materials and Methods

2.1. Chemicals

Curcumin powder of 95% purity and tween 80 were purchasedfrom Fluka (Seelze, Germany) and Sigma–Aldrich Chemical Co. (St.Louis, MO), respectively. Soybean lecithin (L-a-phosphatidylcho-line and inositol phospholipids, purity >80%) from natural plantsoybean oil residue extraction as a food supplement and soybeanoil (SO) were obtained from Taiwan Sugar Corporation (Tainan,Taiwan) and Uni-President Corporation (Tainan, Taiwan), respec-tively. They are both food-grade products. The human liver hepato-cellular carcinomacell line (HepG2; BCRC#60025) was purchasedfrom the Bioresource Collection and Research Center (BCRC) ofthe Food Industry Research and Development Institute, Hsinchu,Taiwan. The culture medium for the HepG2 cell line contains 90%minimum essential medium (Eagle), 2 mM L-glutamine, Earle’sBSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids(NEAA, Invitrogen), 1.0 mM sodium pyruvateand 10% fetal bovine serum (FBS). The last three chemicals andMTT (3-(4-,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-mide; purity of 97.50%) were also from Sigma–Aldrich ChemicalCo. (St. Louis, MO). Dimethyl sulfoxide (DMSO, product #161954) was purchased from Panreac (Barcelona, Spain).

2.2. Preparation of curcumin microemulsion

The mixture was prepared by adding appropriate amounts ofsoybean oil, lecithin and Tween 80 of different mole ratios as sur-factants, and curcumin in a test tube, and was kept at 50 �C andwell mixed using a vortex mixer. Water was then added to themixture in a bath sonicator at 50 �C until a transparent and isotro-pic microemulsion was obtained. A ternary phase diagram of soy-

bean oil-based phospholipid microemulsion was constructedwhere the microemulsion region was identified by a clear andtransparent appearance of the solution. To find a stable micro-emulsion for encapsulation of curcumin in this study, a series offormula within the microemulsion region, at specific surfactant/cosurfactant (lecithin/Tween80) mole ratio of 0.3, were preparedand re-evaluated in a total of 10 g. The stability of microemulsionwas evaluated by measuring the turbidity using a UV/VIS (Thermospectronic, Madison, WI) at a wavelength of 502 nm.

2.3. Diameter distributions of curcumin microemulsions

The diameter distributions of curcumin microemulsions in dif-ferent formulations were determined using dynamic light scatter-ing (DLS) with a 90Plus particle size analyzer (BrookhavenInstruments Co., New York, U.S.A). The data of size distributionwas collected at the 90� scattering angle and calculated using theSoftware.

2.4. MTT (cytotoxicity) assay for HepG2 and HEK293 treated withcurcumin microemulsions

A simple colorimetric assay has been used to screen survival andproliferation of HepG2 and HEK 293 cells treated with curcuminmicroemulsions using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole) test. Ten microli-tres 106 cells/mL HepG2 and 190 lL medium were added in eachwell of the 96 wells cells plate (NalgeNunc International, Rochester,NY), and then incubated at 37 �C in 5% CO2 for 24 h. Thirty microli-tres of various concentration of curcumin microemulsion from 1 to15 lM was added to each well. Fifty microlitres MTT solution inphosphate buffered saline (PBS) was added to each well after24 h, and then incubated in 5% CO2 for 3 h at 37 �C. The solutionwas removed from each well and the cell morphologies were ob-served by microscopy (Nikon ECLIPSE TE2000-S; Japan). Then,100 lL dimethyl sulfoxide (DMSO) was added in each well andincubated at 37 �C for 30 min in the dark. The absorbance of thiscoloured solution can be quantified at a measuring wavelength of570 nm against 620 nm as reference by a the ELISA reader (PowerWave HT340, BioTek, Winooski, USA). Effective adsorption is de-fined as the subtraction of the absorption from the emission to ref-erence wavelengths. The percentage of cell viability was calculatedas the number of viable cells divided by the total from the ratio ofeffective absorption of experimental cells to controls. Experimentswere carried out in quadruplicate wells and repeated at least threetimes. The statistical analysis data were obtained by Student’s t-testusing SPSS statistics software (SPSS, Inc, Chicago, IL).

2.5. Commercial curcurmn exact granule

‘‘Curcumin Extract Granule’’ from company A contains curcu-min as main ingredient formulated with soybean lecithin and oth-ers which helps to dissolve curcumin. Two gram curcumin extractgranule containing 50 lg curcumin component was added to1500 g of ionic water, and then to form suspension using magneticstirring. The concentration of curcumin in the suspension was al-most 135 lM. The appropriate concentration (50, 25, 12,5, and6.25 lM) were obtained by dilution.

2.6. The cellular intake of curcumin in HepG2 cells

Forty microlitres of HepG2 (106 cells/mL) and 760 lL mediumwere added in each well of the 24-wells culture plate (Nunc, Ros-kilde, Denmark), and the plate was incubated at 37 �C in 5% CO2 for24 h. One hundred and twenty microliter of 1 to 15 lM of curcu-min microemulsion was added to each well. The 24-well culture

Fig. 1. The ternary phase diagrams of O/W microemulsion system (A) soybean oil/soybean lecithin and tween 80, (B) using ethyl oleate oil or soybean oil as oil phase.

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plate was incubated and the morphologies and the curcumin up-takes of HepG2 cells treated with various curcumin-MEs wereexamined using an inverted fluorescence microscope (CKX41,Olympus, Melville, NY).

2.7. The encapsulation efficacy of curcumin microemulsion

Various amounts of curcumin were added to microemulsionsformulated as described above. The resultant microemulsions werepassed through a 0.45 lm filter to remove excess curcumin andthen subjected to high pressure liquid chromatography (HPLC)for analysing the loading capacity. The separation was performedon a Cosmosil 5C 18 MS column (5 lm, 25 cm � 4.6 mm I.D., Nac-

alai Tesque, Kyoto, Japan). The sample (20 lL) was eluted with themobile phase composed of 0.1% H3PO4 (40%) and acetonitrile (60%).The flow rate and detection wavelength were set to be 1.0 mL/minand 420 nm, respectively. The standard curve of curcumin in 50% ofethanol ranging from 0.1 to 0.001 mg/mL was used for calculation.

3. Results and discussion

3.1. Preparation of curcumin-loaded soybean oil-basedmicroemulsions

In this study, a soybean oil-based curcumin microemulsion sys-tem was established and the ternary phase diagram is shown in

Fig. 2. (A) The particle size distributions of formula A and B by light scattering measurement (B) the transmission electron microscopy of curcumin-encapsulatedmicroemulsion formulated by A and B.

Fig. 3. The stability of formula A and B microemulsions with or without curcumin-ecncapsulated stored at 5 �C for over 6 months was assessed by measuring meanparticle sizes.

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Fig. 1A. In our previous published study, a ternary phase diagram ofan O/W microemulsion system, lecithin and Tween 80 as the sur-factants and ethyl oleate (EO) as the oil phase, was successfully

constructed for the encapsulation of curcumin (Lin et al., 2009).In Fig. 1B, two curves in the ternary phase diagram using EO or soy-bean oil as the oil phase were shown and compared. The range ofoil-phase (EO) weight percent in the formulation of microemulsionvaried from 2.1 to 10.3 wt.% while the surfactant/oil ratio waswithin the range of 3:1–6:1. Compared with the previously pub-lished microemulsion system using ethyl oleate as the oil phase,the stable microemulsion region was much broadened, as indi-cated from the range of the surfactant/soybean oil ratio (approx.1:1–12:1) and the percentage of soybean oil reaching over11.82%. To carry more curcumin in the microemulsion, the oil con-tent must be as high as possible, but increasing the concentrationof oil may cause instability and require more surfactants, increas-ing total cost. The present result indicates soybean oil is superior toethyl oleate as the oil phase in curcumin microemulsion for itsfood-grade property and enhancement of curcumin encapsulationin the oil phase.

3.2. Characterisation and stability of two different curcumin-loadedmicroemulsions

To further examine the effect of soybean oil content in the cur-cumin microemulsion, two formulas with different soybean oilcontents were prepared. As the weight of water was kept constant

Fig. 4. (A) The cell viability of HepG2 cells treated with various concentration ofcurcumin granule dissolved in water from commercial product. (B) The cell viabilityand microscopic imaging of HepG2 cells treated with various concentration ofcurcumin dissolved in DMSO (less than 0.5%). (C) The cell viability and microscopicimaging of HEK293 cells treated with various concentration of curcuminmicroemulsion.

Fig. 5. The cell viability of HepG2 cells treated with various concentration ofcurcumin microemulsions in (A) formula A (B) formula B. Comparing curcumin-freeand curcumin-containing at the same condition, the curcumin-containing ME atHepG2 cell lines were significantly different (⁄P < 0.05, ⁄⁄P < 0.001, ⁄⁄⁄P < 0.0001)when statistics analysed by Student’s t-test.

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(approx. 70%), the weight ratios of the curcuim-soybean oil towater were �0.102 (7%) and �0.192 (12%) in formulation A and

B and the ratio of surfactants/oil in A and B formulations were3.34 and 1.67. The wt ratios of oil/surfactant/water for formula Aand B were 7:23:70 and 12:19:69, respectively (Fig. 1). The meanparticle size of oil droplets of A and B with curcumin were29.95 ± 2.78 nm and 80.02 ± 3.29 nm by light scattering measure-ments (Fig. 2A). Formula B with 2-fold soybean oil resulted inthe droplets shifting to larger sizes. Formariz et al. showed the sizeof the oil droplets in microemulions was decreased significantlywhen the ratio of surfactant/oil phase increased (Formariz et al.,2006). In terms of the stability of the two formula, the mean diam-eters of formula A and B with or without curcumin encapsulatedwere measured at various times for over six months of storage at5 �C. The result in Fig. 3 indicated these formulas remained stablewithout any aggregation as observed from the measured constantdiameters during 6 months of storage. The calculated percentage ofincorporation efficiency was exceeded 95%. The maximum capacitywas obtained at 5.2 mg/mL.

3.3. Cytotoxicity of the two microemulsions on HepG2 and HEK 293cells

In the previous report, the remarkable cytotoxicity (about 20%of cell viability at 15 lM of curcumin concentration in microemul-sion) observed in both oral squamous cell carcinoma cell lines(OSCC-4 and OSCC-25) was surprising (Lin et al., 2012) Therefore,to further examine the mechanism, the effect of curucmin

Fig. 6. The microscopic images of HepG2 cells treated with various concentration of curcumin microemulsions in formula A and B.

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Fig. 7. (A) curcumin dissolved in soybean oil and (B) curcumin microemulsion in HepG 2 cells. (C) The cellular intake of curcumin for HepG2 cells treated with 15 lM ofcurcumin microemulsions in formula A and B using fluorescence microscopy.

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microemulsions with different formulas on HepG2 cells cytoxicitywas further examined. Due to its very poor solubility in water, cur-cumin alone has no statistically significant effect on cell survival orgrowth (data not shown). The toxicity of the commercial formula-tion of curcumin granule was shown in Fig. 4A. The result showedthe viability of HepG2 cell line was not affected while the concen-tration of curcumin ranging from 6.25 to 50 lM. When curcuminwas dissolved in less than 0.5% of DMSO and dispersed into cellcultures, a small reduction in cell growth (10%) was observed with15 lM of the highest curcumin concentration applied (Fig. 4B).Next, the viability (via MTT test) of HepG2 cells following treat-ment with a different formulation of curcumin-containing micro-emulsions was examined and compared. The toxicity ofcurcumin-MEs on human embryonic kidney cells HEK293 was alsoexamined. The results in Fig. 4C indicated that when the two cur-cmuin-MEs showed no significant reduction on HEK293 cellgrowth (over 80% of viability at 15 lM), the cytotoxicity on HepG2of both formula A and B were prominent.

The results of the dose response study by a series dilution of15 lM curcumin-ME with phosphate buffered saline indicatedthe cytoxicity was prominent even in low concentrations for bothformula A and B (Fig. 5). Meanwhile, microscopic imaging of thecells confirmed the toxic effects of the curcumin microemulsionsin both A and B formula, showing a gradual increase in damagedand ruptured cells after treatment with increased concentrationsof curcumin-ME (Fig. 6). The dose response effects of cytotoxicitywere observed in both cases where the curcumin-microemulsion

in formula A and B began to exhibit significant toxicity at the low-est concentrations of 5 lM and 2 lM, respectively. It is noted thatat 15 lM of the highest concentration, the curcumin-ME in for-mula A resulted in the lowest viability (approx. 5%), compared withthe one in formula B. The different particle sizes between formulaA (30 nm) and B (80 nm) might explain the different cytotoxicityobserved in this case. Our previous ex vivo transmembrane studyusing mouse skins indicated particle diameter in microemulsionis not a factor affecting the permeation coefficient if it is less than100 nm. However, this is the first report demonstrating the effectsof different microemulsion diameters on the cytotoxicity of hepa-tocellular carcinoma cell lines.

We further investigated the cellular uptake of the two curcu-min-MEs using fluorescence microscopy. The fluorescence imagesof the control group of curcumin dissolved in soybean oil onlyand curcumin microemulsion in HepG2 cell were shown inFig. 7A and B, respectively. The results indicated that the fluores-cence was observed in the curcumin dissolved in soybean oil(Fig. 7A), but did not appear on curcumin microemulsion in HepG2cell (Fig. 7B), which might be explained by the low resolution offluorescence microscopy that can only detect flurorcent curucmindissolved in soybean oil when aggregation of over 1 lm dropletwas formed. The droplet sizes of curcumin microemulsion of for-mulations A and B were below 1 lm in Fig. 2A, therefore, the fluo-rescent spots cannot be observed in Fig. 7B using an invertedfluorescence microscope. The time-course result in Fig. 7C indi-cated that after 8 h of incubation, the uptake of curcumin were

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observed in both formula A and B at 15 lM of concentration. It isalso noted that formula A-treated group caused much more curcuminentering into the cells, as indicated from fluorescent curcuminaggregation at 8 h. The intensity of fluorescence was graduallydiminished probably due to the curcumin metabolism. After 24 hof incubation, the death of the HepG2 cells was observed in for-mula A-treated group which possesses 30 nm of smaller particlesize, compared to formula B-treated one. Previous study indicatedcurcumin induced HepG2 cell apoptosis via changing the cell-sur-face morphology or disrupting mitochondrial membrane potential,which provided a plausible reason for the cytotoxic effect of curcu-min-phospholipid microemulsions in HepG2 cells (Wang, Ruan,et al., 2011). It is expected that the toxicity might be a consequenceof the increased delivery of curcumin to the target sites, perhapsvia fusion of microemulsion droplets with cellular membranes.From a literature search of previously published reports, two stud-ies were reported using curcumin nanoparticles (CURN) and curcu-min nanodisks (ND) to enhance anti-HepG2 activities (Ghosh et al.,2011; Yen, Wu, Tzeng, Lin, & Lin, 2010). The former curcuminnanoparticle (CURN) was formulated using a simple nanoprecipita-tion technique with polyvinylpyrrolidone (PVP) as the hydrophiliccarrier and the ND was constructed as apolipoprotein/phospho-lipid complexes. By comparing the dosage and viability, our curuc-min-ME system was superior to CURN (41%, 20 lg/mL) andcurcumin-ND (45%, 20 lM) for anti-HepG2 cytotoxicity.

4. Conclusions

Our previous reports demonstrated phospholipids-based micro-emulsions are good candidates for natural products carriers be-cause of their nanometer size, which enhances thetransmembrane permeation as well as increasing the loadingcapacity for lipophilic nutraceuticals. It is believed the small sizeallows passage through organ filtering and cellular membrane sys-tems, both of which are highly desirable characteristics. In this re-port, we focused on using food-grade ingredients soybean oil andsoybean lecithin to replace ethyl oleate and purified lecithin in aprevious system. The results indicated soybean oil is superior toethyl oleate as the oil phase in curcumin microemulsion as provenby the broadened microemulsion region and increased percentageof soybean oil for enhancing of curcumin encapsulation. This fur-ther study is the first to demonstrate the different effects of differ-ent curcumin microemulsion diameters on the cytotoxicity ofhepatocellular HepG2 cell lines, and is much more effective thanpreviously published results. When the concentrations of curcuminmicoremulsions(formulation A and B) were from 1 to 15 lM, thetoxicity was much lower in human embryonic kidney cells(HEK293) than in HepG2 cells. In addition, food-grade ingredientsto prepare microemulsions can reduce cost and help in scaling-up.The results from this study might provide an innovative appliedtechnique in the area of nutraceuticals and functional foods.

Acknowledgements

The authors would like to thank the National Science Councilof the Republic of China, Taiwan, for financially supporting thisresearch under Contract No. NSC 98-2622-E-214-010-CC3, NSC97-2320-B-390-001-MY3 and NSC100-2314-B-390-001-MY3.

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