Comparing extraction methods for the determination of tocopherols and tocotrienols in seeds and germinating seeds of soybean transformed with OsHGGT

Journal of Food Composition and Analysis 27 (2012) 70–80

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Original Research Article

Comparing extraction methods for the determination of tocopherols and tocotrienols in seeds and germinating seeds of soybean transformed with OsHGGT Yu Young Lee a,1, Hyang Mi Park a,1, Choon Ki Lee a, Sun Lim Kim a, Tae-Young Hwang a, Man Soo Choi a, Young-Up Kwon a, Wook Han Kim a, Si Ju Kim a, Sang Chul Lee b, Yul Ho Kim a,* a b

National Institute of Crop Science, RDA, Suwon 441-857, Republic of Korea School of Applied Bioscience, College of Agriculture and Life Sciences, Kyungpook National University, Daegu 702-701, Republic of Korea

A R T I C L E I N F O

A B S T R A C T

Article history: Received 18 October 2011 Received in revised form 26 February 2012 Accepted 6 March 2012

Previously, transgenic soybeans were generated and reported to produce tocotrienols (a-, g- and dtocotrienols), compounds not normally found in soybean. Three procedures were evaluated to optimize the extraction method for transgenic soybean seeds and germinating seeds. Significant differences were observed among the extraction methods in seeds and germinating seeds. In seeds, the highest analytical values (tocopherols, 37.11 mg 100 g1; and tocotrienols, 1.54 mg 100 g1) were observed by using rapid Soxhlet extraction. In germinating seeds, the content of transgenic soybean (B20 and C5) total vitamin E (tocopherols, 18.04, 20.73 mg 100 g1; and tocotrienols, 0.82 and 0.84 mg 100 g1) by direct extraction was approximately 16% and 9% greater than the amount obtained by saponification. In addition, 1,1diphenyl-2-picrylhydrazyl (DPPH) and 2,20 -azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) tests demonstrated a direct correlation between the radical-scavenging capacity and the total content of natural tocopherols and tocotrienols. Therefore, these results suggest that an optimal extraction method will provide a fast, simple, reproducible procedure for analyzing tocotrienols and tocopherols. Furthermore, this method may be used to determine novel minor functional compounds such as tocotrienols for the evaluation of biological activity. ß 2012 Elsevier Inc. All rights reserved.

Keywords: Tocotrienols Tocopherols Extraction Saponification Soxhlet extraction Direct extraction Rice homogentisate geranylgeranyl transferase (OsHGGT) Antioxidant activity Soybean Food analysis Food composition

1. Introduction Tocopherols and tocotrienols (vitamin E), crucial lipid-soluble antioxidants, are essential to human health. These molecules play important roles in scavenging free radicals and inhibiting lipid peroxidation in biological membranes (Folk and Munne-Bosch, 2010). In recent years, tocotrienols have received greater attention than tocopherols. Apart from vitamin E activity, tocotrienols have shown promise in numerous treatment areas. These include reducing blood cholesterol levels and preventing stroke-induced brain damage, as well as exhibiting anti-inflammatory and anti-angiogenesis properties (Aggarwal et al., 2010; Lee et al., 2008; Schaffer et al., 2005; Sen et al., 2006; Serbinova et al., 1991; Theriault et al., 1999). Tocotrienols also exhibit potent anti-cancer activity in various human cancer cells from tissues including the prostate, breast and colon (Constantinou et al., 2008; Miyazawa et al., 2009). These biological properties have resulted in the inclusion of tocotrienols in a broad spectrum of

* Corresponding author. Tel.: +82 31 290 6751; fax: +82 32 290 6742. E-mail address: [email protected] (Y.H. Kim). 1 These authors contributed equally to this work. 0889-1575/$ – see front matter ß 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jfca.2012.03.010

dietary supplements and functional foods (as well as nutra- and cosmeceutical applications). The extraction procedure for vitamin E is traditionally performed by enzymatic hydrolysis, saponification and Soxhlet extraction (Ruperez et al., 2001; Slover et al., 1969; Wrolstad, 2002). Among these methods, saponification has been widely used for the quantification of vitamin E in cereals (Chun et al., 2006; Lerma-Garcia et al., 2009; Piironen et al., 1986; Ruperez et al., 2001). Following saponification, the unsaponifiable components, including vitamin E, are extracted into an organic solvent, while fatty acid salts, glycerols and other potentially interfering substances remain in the alkaline aqueous phase. Nevertheless, saponification is time-consuming, complex and not always necessary. In previous studies, the saponification results did not vary from the traditional extraction methods (Ruperez et al., 2001). Residue oil from crude palm oil has been processed classically using Soxhlet extraction by screw press (Ollanketo et al., 2002). Supercritical carbon dioxide (SC-CO2) (Lau et al., 2006, 2008) and pressurized liquid extraction (PLE) (Sanagi et al., 2005) are currently used to extract from palm mesocarp. In rice bran, saponification is normally used for the determination of vitamin E (Diack and Saska, 1994), and the effects of solvent, extraction temperature and time have been studied (Chen and Bergman,

Y.Y. Lee et al. / Journal of Food Composition and Analysis 27 (2012) 70–80

2005; Duvernay et al., 2005; Devi and Arumughan, 2007; Hu et al., 1996; Proctor et al., 1994; Proctor and Bowen, 1996). One-step equilibrium direct solvent extraction and SC-CO2 are extraction methods used for the determination of tocopherols and tocotrienols from rice bran (Imsanguan et al., 2008; Lerma-Garcia et al., 2009; Sarmento et al., 2006). Soybean represents a good source of protein, polyunsaturated fats and fat-soluble vitamin E, especially tocopherol. Recently, tocopherols from deodorizer distillate of soybean oil (DDSO) were concentrated using SC-CO2, thus increasing the tocopherol value (Clark and Synder, 1989; Mendes et al., 2005). Soxhlet extraction, in comparison to saponification and direct solvent extraction, has been the best method for quantifying tocopherols in soybeans (Lim et al., 2007). The quantity of vitamin E in cereals, as well as vegetables, is influenced by the species, variety, maturity and growing conditions (Tsochatzis et al., 2012). Moreover, variations in vitamin E values result from many factors, including sample preparation, processing procedures and the conditions of the analytical methods (Clark and Synder, 1989; Lee et al., 2006; Peres et al., 2006; Tasai et al., 2007). Previous studies have demonstrated the production of transgenic soybeans producing tocotrienols, compounds not normally found in soybean, by metabolic engineering of the biosynthesis pathways. Transgenic soybean over-expressed rice HGGT (OsHGGT) using two different promoters produced four tocopherol isomers, two new g- and d-tocotrienols. Moreover, in transgenic plants, significantly higher antioxidant activities were detected in germinated than in non-germinated seeds. In particular, after 3 days of germination, the DPPH and ABTS radical-scavenging activities of seed extracts from transgenic plants were up to 17% and 35.3% higher, respectively, than extracts from un-germinated wild-type seeds. In addition, germinating seeds from transgenic lines exhibited dramatically lower MDA contents than intact and germinating wild-type seeds. Increased tocotrienol levels correlated with significant improvements in antioxidant activity (Kim et al., 2008, 2011). An optimum extraction method is desirable for various crop materials such as seeds and roots. To numerous plant-derived extracts and phytochemicals a variety of potentially healthpromoting biological activities have been ascribed (Ollanketo et al., 2002). Therefore, the purpose of this study was to optimize the extraction procedures for the quantitative determination of vitamin E, especially novel minor functional compounds such as tocotrienols, in transgenic soybean. In addition, an evaluation of the health-promoting biological activity of the extracted compounds should be included when determining the most effective extraction method.

2. Materials and methods 2.1. Chemicals and instruments Analytical grade ethyl acetate (EtOAc), n-hexane, isopropanol and water were purchased from J.T. Baker (Phillipsburg, NJ, USA).

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Ethanol, butylated hydroxytoluene (BHT), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,20 -azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), and tocopherols were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Tocotrienols were supplied by Davos Life Science (Tuas, Singapore). The tocopherol and tocotrienol content of soybeans were determined by HPLC. HPLC was performed with a normal-phase HPLC system comprising a solvent delivery pump (515; Waters, USA) equipped with a spectrofluorometric detector (2475; Waters, USA) and a Lichrosorb Si60 column (4.6 mm  250 mm, 5 mm; Hibar, Darmstadt, Germany). Extracts (10 mL) were injected into an analytical Lichrosorb Si60 column with an isocratic phase of nhexane:isopropanol (99:1, v/v) and a flow rate of 1.5 mL min1. The excitation and emission spectra were 290 and 330 nm, respectively. The injection volume was 10 mL, and the column temperature was regulated at 30 8C. 2.2. Samples Transgenic soybeans stably expressing the OsHGGT gene were obtained from the T4 generation of two lines: a homozygous line expressing OsHGGT under the control of the seed-specific rice globulin promoter (Glb-HGGT) (B20) and a homozygous line expressing OsHGGT under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter (35S-HGGT) (C5). Wild type soybean (WT, cv. Iksannamulkong) was included in these experiments. All the soybeans were harvested on 24 October 2010 at National Institute of Crop Science, the Rural Development Administration (RDA) farm, Suwon, Korea. The soybeans were stored at 4 8C before the experiment. The conditions for germination were as follows: seeds were soaked in 75% ethanol for 1 min and rinsed twice with distilled water. The seeds were then soaked in 20% sodium hypochlorite for 12 min at ambient temperature and thoroughly rinsed three times with distilled water. Seeds were germinated in plastic dishes containing 15 mL of distilled water at 27 8C for 72 h. They were stored at 70 8C for safe preservation until laboratory extraction and analysis. 2.3. Extraction method in soybean seeds All of the conditions used in the sample pre-treatment procedures were shown in Table 1. The saponification method was modified (Lim et al., 2007). Soybean seeds (WT, B20 and C5) (100 g) were ground at 260  g for 200 s in an auto-mill disintegrator (Tokken, Japan). Ground soybeans (2 g) were added to a 15 mL aliquot of ethanol containing pyrogallol (PG, 6 g 100 mL1, w/v) in a saponification vessel and vortexed for 30 s. After sonication for 5 min, 5 mL of potassium hydroxide (33.6 g L1) was added and the vessel was flushed with nitrogen gas for 1 min. An air condenser was attached and the contents were digested at 70 8C for 50 min in a shaking water bath. After the samples had been cooled for 5 min in an ice bath, 20 mL of sodium chloride (20 g L1) was added, and then vortexed for 30 s. The mixture was extracted three times with 20 mL n-hexane:EtOAc

Table 1 The conditions used in the sample pretreatment procedures.

Ratio of sample weight/solvent Solvent extractant Number of extraction step Total amount of solvent (mL) Extraction temperature (8C) Total time (min)

Saponification

Rapid Soxhlet

Ultrasonic

Direct

2 g/100 mL EtOH (with 60 g L1 PG), KOH (33.6 g L1), NaOH (20 g L1), n-hexane:EtOAc (with 0.1 g L1 BHT) 8 600 70 164

2 g/140 mL n-Hexane:EtOAc (with 0.1 g L1 BHT) 3 840 180/120 110/192

2 g/10 mL n-Hexane (with 0.1 g L1 BHT) 3 60 40–50 60

1 g/5 mL EtOH (10 g L1 PG) 3 30 – 10

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Table 2 Contents (mg 100 g1) of tocopherols and tocotrienols according to extraction solvents in ultrasonic extraction. Hexane

Isomers

a

a-Tb b-Tc g-Td g-T3e d-Tf d-T3g Th T3i Vitamin Ej

Hexane:EtOAc (85:15, v/v)

WT

B20

C5

WT

B20

C5

0.80  0.04 0.04  0.0 7.62  0.42 ND 0.11  0.01 ND 8.57  0.51 ND 8.57  0.51

1.38  0.45 0.11  0.04 11.82  0.42 0.39  0.04 0.17  0.06 0.19  0.07 13.48  0.46 0.58  0.13 14.06  0.43

0.87  0.04 0.04  0.01 82.1  7.5 trl 0.11  0.0 ND 9.23  0.75 tr 9.23  0.75

0.27  0.39 NDk 4.82  0.24 ND 0.73  0.37 ND 5.81  0.45 ND 5.87  0.45

0.13  0.18 ND 3.11  0.0 tr 0.41  0.01 ND 3.77  0.14 0.41  0.01 4.19  0.14

0.15  0.21 ND 3.13  0.53 tr 0.48  0.08 ND 3.75  0.81 tr 3.75  0.81

The limits of detection (LOD) were 0.02, 0.04 and 0.02 mg mL1 for b-tocopherol, g-tocotrienol and d-tocotrienol. Data represent the means of three replicates  SD. a Wild type. b a-Tocopherol. c b-Tocopherol. d g-Tocopherol. e g-Tocotrienol. f d-Tocopherol. g d-Tocotrienol. h Sum of tocopherols. i Sum of tocotrienols. j Sum of T and T3. k Not detected. l Below the limit of reliable quantification.

extraction solvent (85:15, v/v containing 0.1 g L1 butylated hydroxytoluene). The extracts were collected, diluted to a final volume of 50 mL, and filtered through a 0.2 mm filter. The rapid Soxhlet extraction method was conducted using automatic Soxhlet extraction. Ground soybeans (2 g) were extracted with 140 mL of n-hexane:EtOAc (85:15, v/v containing 0.1 g L1 BHT) using a Soxtherm automatic extraction unit (Gerhardt, Germany) at 180 8C for 110 min and 120 8C for 192 min under dim light conditions. Crude lipid extracts of 50 mL were passed through a 0.2 mm filter unit prior to HPLC analysis. For ultrasonic extraction, the procedure was as follows: ground seeds (2 g) were extracted with 10 mL of n-hexane (containing 0.1 g L1 BHT) for 50 min in an ultrasonic apparatus (Bransonic, USA) (Table 2). The temperature of the bath was controlled at a temperature between 40–50 8C. The supernatant was collected three times and 10 mL of extract was analyzed using a normalphase HPLC system. All analyses were performed with three independent samples.

2.4. Extraction method in germinating soybean seeds For germinating seeds, the saponification and rapid Soxhlet extraction methods were applied in the same manner as with the soybean seeds. The direct extraction method was modified to extract from germinating soybeans (Tavva et al., 2007). We modified ratios of solvent volume:sample weight according to seeds or germinating seeds. In case of germinating seeds, sample weight was reduced remarkably during germination. Therefore, we used 1 g of sample weight for germinating seed (Table 1). Germinating seeds (1 g) were ground in liquid nitrogen and the resultant powder was transferred to 5 mL of pyrogallol in ethanol (1 g 100 mL1). The extract was vortexed 30 s and centrifuged at 6225  g for 2 min to remove large pieces of debris. The supernatant was collected three times in a 10 mL flask. Aliquots (1 mL) of the supernatant were evaporated with nitrogen gas. Dried samples were resuspended in n-hexane and filtered through a 0.2 mm filter. To confirm the complete extraction, the tocopherol and tocotrienol contents of the re-extracts were analyzed (Table 3).

Table 3 Tocopherol and tocotrienol contents (mg mL1) of re-extracts from the remaining samples of rapid Soxhlet extraction, saponification extraction, ultrasonic extraction, and direct extraction in B20. Isomers a

a-T a-T3b b-Tc g-Td g-T3e d-Tf d-T3g a b c d e f g h i

Rapid Soxhlet extraction h

ND ND ND ND ND ND ND

Saponification

Ultrasonic extraction

Direct extraction

LODi (mg mL1)

ND ND ND ND ND ND ND

ND ND ND ND ND ND ND

ND ND ND ND ND ND ND

0.04 0.03 0.02 0.03 0.04 0.03 0.02

a-Tocopherol. a-Tocotrienol. b-Tocopherol. g-Tocopherol. g-Tocotrienol. d-Tocopherol. d-Tocotrienol. Not detected. Limits of detection. All values were lower values of LOD.

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Table 4 Maximum wavelengths, extinction coefficientsa and quantity ranges of tocopherol and tocotrienol standards.

2.5. Antioxidant activities The antioxidant activities of the extracts from soybean seeds and germinated seeds were determined from the scavenging activity of stable DPPH free radicals and the trolox equivalent antioxidant capacity. DPPH radical-scavenging activity was determined via the modification of a previously described method (Mensor et al., 2001). In a 96-well microtiter plate, samples containing 100 mL of soybean extract were added to 150 mL of DPPH in ethanol solution (1.5  104 mol). After incubation at 37 8C for 30 min, the absorbance of each solution was determined at 518 nm using a microplate reader (Tecan’s Infinite1 200, Madison, WI, USA). The ABTS assay is based upon the ability of antioxidants to scavenge the radical cation ABTS+ relative to a standard Trolox curve (Re et al., 1999). The radical cation was prepared by mixing ABTS stock solution (7.0  103 mol) with 2.45  103 mol potassium persulfate. The reaction mixture was diluted with ethanol and the absorbance was determined at 734 nm. For the photometric assay, 200 mL of ABTS+ solution and 100 mL of sample were mixed for 45 s. The absorbance was measured immediately after incubation at 30 8C for 5 min in a microplate reader. All samples were assessed at a final concentration of 25 mg mL1. Linear ranges varied from 0.0078 to 0.125  103 mol for Trolox solution. The linear regression coefficient (R2) was 0.999. DPPH and ABTS radical scavenging activity were expressed in Trolox equivalents antioxidant capacity (TEAC) based on mg 100 g1 of EtOH extract.

Isomers

Wavelength (nm)

Extinction coefficient, 1%, 1 cm

Quantity range of standard curve (g mL1)

a-Tocopherol b-Tocopherol g-Tocopherol d-Tocopherol a-Tocotrienol b-Tocotrienol g-Tocotrienol d-Tocotrienol

292 296 298 298 292 295 298 292

75.8 89.4 91.4 87.3 91.0 87.3 90.5 89.1

0.4–10.0 0.1–6.0 2.0–16.0 0.4–10.0 0.1–6.0 0.1–6.0 0.1–6.0 0.4–10.0

a

Extinction coefficients are given for ethanol solutions.

the extraction procedure and study the accuracy of the proposed method, the soybean sample of B20 was spiked with known quantities of tocopherols and tocotrienols. The accuracy was evaluated by analyzing 3 samples to which known concentrations of vitamin E isomers were added prior to extraction; 1 mL of the stock standard solution was added to the soybean sample of B20 with the 140 mL of extraction solvent. The results were statistically evaluated using ANOVA with SAS version 9.2 (SAS Institute, Cary, NC, USA). The means were compared using Duncan tests with a significance level of <0.05. 3. Results and discussion

2.6. Preparation of standards

3.1. Tocopherol and tocotrienol contents using three extraction methods

For quantification and identification purposes, standard stock solutions of four tocopherol isomers (a-, b-, g-, and d-tocopherol) and four tocotrienol isomers (a-, b-, g-, and d-tocotrienol) were prepared in n-hexane (containing 0.1 g L1 BHT) and stored under nitrogen at 20 8C in the dark. These solutions were diluted in hexane:isopropanol (99:1, v/v). Purity and stability were confirmed using the known extinction coefficient of each isomer by spectrophotometer (Hitachi High Technologies, Tokyo, Japan) (Tables 4 and 5). The working solution was prepared by diluting to a concentration ranging from 0.1 to 10 mg mL1 for each compound. Tocopherol and tocotrienol peaks were identified by comparing the sample retention times with the standards (Fig. 1). Concentrations were calculated from the peak areas determined by linear regression.

Saponification, rapid Soxhlet extraction and ultrasonic extraction were evaluated to ascertain their usefulness in determining the amount of tocopherols and tocotrienols in transgenic soybean (B20 and C5) and wild type (WT) (Table 6). Among the three extraction methods, significant differences were found in the content and composition of tocotrienol and tocopherol isomers. The highest analytical values were observed using rapid Soxhlet extraction, while the lowest values were obtained using ultrasonic extraction. The amount of g-tocotrienol and d-tocotrienol using rapid Soxhlet extraction in B20 increased by 1.2- and 1.5-fold, respectively, in comparison to saponification. Additionally, the content of g-tocotrienol and d-tocotrienol using rapid Soxhlet extraction in C5 increased 2.2- and 2.4-fold, respectively, in comparison to saponification. Saponification and rapid Soxhlet extraction produced similar quantities of a-tocopherol and btocopherols in WT, B20 and C5. However, the g-tocopherol and dtocopherol quantities obtained via rapid Soxhlet extraction in WT, B20 and C5 were greater than those obtained with saponification (1.3-, 1.4- and 1.2-fold, and 3.0-, 3.1- and 2.9-fold, respectively).

2.7. Method validation and statistical analysis The extraction methods were validated by determining the accuracy, precision and linearity. Limits of detection (LOD) were calculated as the concentrations (mg mL1) corresponding to three times the signal-to-noise ratio, respectively. To standardize

Table 5 The evaluation of peak purity by fluorescence ratio in standards, seeds (RSE) and 3-day-old germinating seeds (DE) of B20. Ratioa

a-Tocopherol RSEb

280/290 nm 270/290 nm 280/270 nm

0.54 0.19 2.78

DEc 0.54 0.22 2.75

a-Tocotrienol STd 0.57 0.21 2.76

RSE ND ND ND

e

b-Tocopherol

g-Tocopherol

g-Tocotrienol

d-Tocopherol

d-Tocotrienol

DE

ST

RSE

DE

ST

RSE

DE

ST

RSE

DE

ST

RSE

DE

ST

RSE

DE

ST

0.53 0.18 2.84

0.56 0.20 2.85

0.47 0.15 3.12

0.49 0.14 3.14

0.50 0.16 3.11

0.46 0.15 3.07

0.48 0.15 3.10

0.48 0.16 3.10

0.48 0.14 3.23

0.48 0.13 3.21

0.49 0.15 3.23

0.42 0.13 3.26

0.43 0.12 3.25

0.44 0.13 3.27

0.03 0.14 0.28

0.04 0.15 0.26

0.04 0.16 0.28

The peak purity (specificity) was determined using the procedures described by Lee et al. (2000). The limit of detection (LOD) of a-tocotrienol was 0.03 mg mL1. a Fluorescence ratios were calculated by dividing the values for the two peak heights for each isomers obtained from separate chromatographic runs at two different excitation wavelengths (270, 280 and 290 nm), with the emission wavelength constant kept at 330 nm. b Rapid Soxhlet extraction. c Direct extraction. d Standard. e Not detected.

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Fig. 1. HPLC profiles for tocopherol and tocotrienol isomers in a standard mixture (A), saponification extraction (B), rapid Soxhlet extraction (C), and direct extraction (D) of 3day-old germinating seeds in B20. All chromatograms were followed spectrofluorometric detector (excitation at 290 nm, emission at 330 nm). 1, a-Tocopherol (1.54 mg mL1); 2, a-tocotrienol (0.32 mg mL1); 3, b-tocopherol (0.31 mg mL1); 4, g-tocopherol (7.65 mg mL1); 5, b-tocotrienol (0.32 mg mL1); 6, g-tocotrienol (1.46 mg mL1); 7, d-tocopherol (1.27 mg mL1); and 8, d-tocotrienol (2.13 mg mL1). Retention time of all chromatogram were followed: 1, a-tocopherol (6.048 min); 2, atocotrienol (6.782 min); 3, b-tocopherol (10.88 min); 4, g-tocopherol (11.436 min); 5, b-tocotrienol (12.672 min); 6, g-tocotrienol (13.241 min); 7, d-tocopherol (16.466 min); and 8, d-tocotrienol (19.360 min).

Saponification has been widely used for the analysis of tocopherols and tocotrienols in cereals for food and nutritional studies. Notably, saponification changes acetate forms of tocopherols and tocotrienols into free forms. It is commonly acknowledged that the sample purity increases following saponification. However, saponification can create problematic emulsion formations when the samples contain a high fat content and the saponification parameters and conditions are not properly controlled. In this case, rapid analyte degradation can occur (Lee et al., 2006; Lim et al., 2007). Moreover, saponification can be timeconsuming. In contrast, rapid Soxhlet extraction produces simple, rapid results and the highest analytical values. Therefore, rapid

Soxhlet extraction may represent the best extraction method for transgenic soybean samples for the determination of minor functional compounds such as tocotrienols. 3.2. Tocopherol and tocotrienol contents according to the solvent and temperature used for rapid Soxhlet extraction Using rapid Soxhlet extraction, the effects of extraction solvent and temperature on the tocopherol and tocotrienol extracted from soybean seeds were measured. The quantities of tocopherol and tocotrienol in B20 and C5 and the WT were determined using hexane alone and a mixture of hexane and EtOAc (85:15) (Fig. 2). In

Table 6 Tocopherol and tocotrienol contents (mg 100 g1 dry weight) using three extraction methods in the seeds from transgenic soybeans (B20 and C5) and the wild type (WT). Rapid Soxhlet extraction

Saponification

a-Tb RSD% a b-Tc RSD% g-Td RSD% g-T3e RSD% d-Tf RSD% d-T3g RSD%

Ultrasonic extraction

WT

B20

C5

WT

B20

C5

WT

B20

C5

3.27bc 9.5 0.32a 9.4 18.03d 1.1 NDh – 1.45c 2.1 ND –

3.45b 2.6 0.26a 7.7 20.05c 1.5 0.99b 4.0 1.41c 7.8 0.27ab 29.6

3.00bc 5.0 0.27a 3.7 18.77cd 2.8 0.10e 10.0 1.43c 1.4 0.05c –

3.08bc 0.7 0.31a 3.3 23.97b 0.3 ND – 4.41a 0.5 ND –

3.84a 2.6 0.31a 3.2 28.54a 2.1 1.14a 2.6 4.42a 1.6 0.40a 2.5

2.95c 7.5 0.28a 3.6 22.71b 0.7 0.22d 4.6 4.17b 0.5 0.12c 8.3

0.80e 5.0 0.04c – 7.62f 5.5 ND – 0.11d 9.1 ND –

1.38d 32.6 0.11b 36.4 11.82e 3.6 0.39c 10.3 0.17d 35.3 0.19b 36.8

0.87e 4.6 0.04c 25.0 8.21f 9.1 ND – 0.11d – ND –

Different letters represent significant (p < 0.05) differences between means according to ANOVA combined with Duncan’s multiple range test. The limits of detection (LOD) were 0.04 and 0.02 mg mL1 for g-tocotrienol and d-tocotrienol. a The relative standard deviation. b a-Tocopherol. c b-Tocopherol. d g-Tocopherol. e g-Tocotrienol. f d-Tocopherol. g d-Tocotrienol. h Not detected.

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Fig. 2. Tocopherol (T) and tocotrienol (T3) content (mg g1 dry weight) by rapid Soxhlet extraction using different solvents (hexane and hexane mixed with EtOAc) in transgenic soybeans (B20 and C5) and the WT. Mixture of hexane mixed with EtOAc was prepared 85:15 (v/v) containing BHT (0.1 g L1). The HPLC conditions for quantification were as follows: solid phase: Lichrosorb Si60 column (4.6 mm  250 mm, 5 mm; Hibar, Darmstadt, Germany); mobile phase: n-hexane:isopropanol (99:1, v/ v); detector (FLD: excitation at 290 nm, emission at 330 nm); flow rate: 1.5 mL min1; injection volume: 10 mL; and the column temperature was regulated at 30 8C.

B20 and C5, the concentration of g-tocotrienol and d-tocotrienol extracted with hexane mixed with EtOAc produced higher values (1.4-, 2.2-, 0.9-, and 1.2-fold) in comparison to hexane alone. Additionally, hexane mixed with EtOAc extracted more tocopherol isomers in WT, B20 and C5. Specifically, 1.4–1.6-fold more a-tocopherol, 1.9–2.0-fold more b-tocopherol, 2.4-fold more g-tocopherol, and 2.3-fold more d-tocopherol were extracted by hexane mixed with EtOAc than with hexane alone. When comparing the total vitamin E, hexane mixed with EtOAc extracted 2.2-fold more than hexane alone. Hexane is the most common solvent for fat-soluble extraction, although hexane poses potential fire, health and environmental hazards. Furthermore, the efficiency of extraction with hexane varied for each tocopherol and tocotrienol. While a-tocopherol is completely extracted by hexane alone, b-, g- and d-tocopherol are not (Ueda and Igarashi, 1987). The results presented here are consistent with previous studies, which is that mixing hexane and EtOAc improves the recovery of b-, g- and d-tocopherol (Wrolstad, 2002). The tocopherol and tocotrienol concentrations in B20 and C5 and the WT were investigated using different temperature conditions (Fig. 3). At 180 8C, the content of d-tocotrienol in the C5 line increased 2.4-fold, 1.2-fold higher than all of the tocopherols. The content of g-tocotrienol and d-tocotrienol in

B20 was detected 1.14 and 0.39 mg 100 g1 at 180 8C and increased 1.3-fold compared to 120 8C. The total vitamin E content in B20, C5 and WT was greater (1.3-, 1.1- and 1.1-fold, respectively) at 180 8C than at 120 8C. The effects of heat on the extraction of atocopherol and other tocopherols and tocotrienols (Arora et al., 2010; Moreau and Hicks, 2006; Sabliov et al., 2009) have been previously reported. In the absence of oxygen, a-tocopherol is stable at high temperatures. However, under normal atmospheric conditions, the rate of a-tocopherol oxidation increases, leading to increased a-tocopherol degradation (Shin et al., 1997). In addition, several other factors such as alkali, light, minerals, and hydroperoxides affect a-tocopherol degradation (Zigoneanu et al., 2008). When samples were extracted under nitrogen, the atocopherol losses were insignificant (0.3%). The highest loss of atocopherol (13.7%) was observed at 260 8C for 80 min at 0.04 kg m1 s2 (Verleyen et al., 2001). When using Soxhlet extraction in this study, the samples were extracted under nitrogen and dim light. The temperature conditions of automatic Soxhlet extraction are related to the extraction time. Extraction at 180 8C required less time (110 min) compared to extraction (192 min) at 120 8C. Therefore, the degradation of tocopherol and tocotrienol during the extraction procedure can be decreased by shortening the extraction time and increasing the saturation of nitrogen.

Fig. 3. Tocopherol (T) and tocotrienol (T3) content (mg g1 dry weight) by rapid Soxhlet extraction at different temperatures (120 8C and 180 8C) in transgenic soybeans (B20 and C5) and the WT. Extraction solvent was used hexane mixed with EtOAc (85:15, v/v containing 0.1 g L1 of BHT). The HPLC conditions for quantification were as follows: solid phase: Lichrosorb Si60 column (4.6 mm  250 mm, 5 mm; Hibar, Darmstadt, Germany); mobile phase: n-hexane:isopropanol (99:1, v/v); detector (FLD: excitation at 290 nm, emission at 330 nm); flow rate: 1.5 mL min1; injection volume: 10 mL; and the column temperature was regulated at 30 8C.

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Table 7 Tocopherol and tocotrienol contents (mg 100 g1 dry weight) using three extraction methods in 3-day-old germinating seeds from transgenic soybeans (B20 and C5) and the wild type (WT). Rapid Soxhlet extraction

Saponification

a-T RSD% a-T3a RSD% b-T RSD% g-T RSD% g-T3 RSD% d-T RSD% d-T3 RSD%

Direct extraction

WT

B20

C5

WT

B20

C5

WT

B20

C5

0.75c 12.0 NDc – 0.35b 14.3 11.12c 1.8 ND – 2.87b 5.6 ND –

1.15b 7.0 trb – 0.36b 5.6 13.27b 2.0 0.54a 20.4 3.02b 4.0 0.10c 10.0

1.05b 4.8 tr – 0.36b 19.4 13.13b 1.1 tr – 3.81a 2.4 0.03d –

0.15d 6.7 ND – ND – 6.14d 7.3 ND – 1.79c 8.4 ND –

0.14d 7.1 ND – ND – 5.32d 7.0 ND – 1.08d 10.2 ND –

0.13d 15.4 ND – ND – 6.21d 2.1 ND – 1.84d 1.1 ND –

1.38a 4.4 ND – 0.44a 2.3 13.47b 7.1 ND – 2.75b 6.6 ND –

1.37a 2.9 0.36a 2.8 0.47a 4.3 15.9a 5.7 0.33b 3.0 2.99b 5.4 0.13b 7.7

1.39a 4.3 0.36a 2.8 0.47a 4.3 14.33ab 5.7 0.31b 9.7 3.09b 4.5 0.17a 11.8

The limits of detection (LOD) were 0.03, 0.02, 0.04 and 0.02 mg mL1 for a-tocotrienol, b-tocopherol, g-tocotrienol and d-tocotrienol. Different letters represent significant (p < 0.05) differences between means according to ANOVA combined with Duncan’s multiple range test. a a-Tocotrienol. b Below the limit of reliable quantification. c Not detected.

3.3. Content and composition of tocopherol and tocotrienol in germinating seeds using three extraction methods Three extraction methods were used to determine the content and composition of tocopherol and tocotrienol in germinating seeds of B20 and C5 and the WT (Table 7). Significant differences were observed depending upon the extraction method. From the rapid Soxhlet extraction method, only a-, g- and d-tocopherol were identified in B20 and C5 and the WT. With saponification, atocotrienol was detected in trace amounts, while a-, b-, g- and dtocopherol, g-tocotrienol, and d-tocotrienol were detected in transgenic seeds. Using direct extraction, all of the vitamin E isomers, except b-tocotrienol, were detected in the B20 and C5 lines. The total vitamin E (tocopherols, 180.4, 207.3 mg g1; and tocotrienols, 8.2 and 8.4 mg g1) contents of the B20 and C5 lines with direct extraction were approximately 16% and 9% higher than the level obtained with saponification. This elevated level was due to an increment increase in all of the vitamin E isomers. The

relative standard deviation (RSD%) of the vitamin E isomers obtained by direct extraction was less than 10%. The higher RSD% of tocotrienol, especially d-tocotrienol, may have been caused by its lower content in the samples. Germination of seeds is one processing method used to increase the nutritive value and health qualities of foods (Bau et al., 1997, 2000; Plaza et al., 2003). This process has been used with soybeans for a very long time, primarily in Eastern countries. Interest in this germination process is currently increasing in Western countries due to consumer demands for minimally processed, additive-free, more natural, nutritional and healthy foods. Thus, researchers have evaluated the chemical composition of soybeans during germination (Lee and Chang, 2003; Mostafa et al., 1987). However, little information is available concerning the extraction methods used to determine the tocopherol content in germinating soybeans. In addition to the extraction procedures used for seed, direct extraction is useful for determining tocotrienols as well as tocopherols in germinating seeds.

Table 8 Intra-day repeatability and inter-day precision for the rapid Soxhlet extraction method. Isomers

Parameter

Intra-day repeatability (n = 6)b WTe

a-Tocopherol

Mean RSD%

b-Tocopherol

a

B20

Inter-day precision (n = 6)c C5

WT

B20

C5

1.65  0.04 2.5

1.73  0.49 2.8

1.46  0.05 3.4

1.33  0.15 11.2

1.38  0.18 13.2

1.34  0.14 10.5

Mean RSD%

0.30  0.0 –

0.27  0.01 2.8

0.25  0.01 3.3

0.25 0.02 6.5

0.26  0.02 6.0

0.26  0.01 4.3

g-Tocopherol

Mean RSD%

16.16  0.58 3.6

16.21  0.41 2.5

14.71  0.41 2.8

16.08  0.49 3.1

14.99  0.58 3.9

g-Tocotrienol

Mean RSD%

NDd –

0.74  0.03 4.1

0.11  0.0 –

ND –

0.74  0.03 4.5

0.13  0.01 9.1

d-Tocopherol

Mean RSD%

6.91  0.34 5.0

6.99  0.17 2.5

6.82  0.20 2.9

6.08  0.2 3.2

6.97  0.24 3.5

6.94  0.27 4.0

d-Tocotrienol

Mean RSD%

ND –

0.41  0.02 3.8

0.18  0.01 2.9

ND –

0.42  0.02 5.1

0.14  0.03 18.3

13.72  0.10 7.5

The limits of detection (LOD) were 0.04 and 0.02 mg mL1 for g-tocotrienol and d-tocotrienol. Data represent the means of six replicates  SD. a mg 100 g1 sample on dry weight basis. b Intra-day repeatability was evaluated using six independent analyses of replicate samples for 1 day. c Inter-day precision was evaluated using six independent analyses of replicate samples performed for three days. d Not detected. e WT (wild type plant).

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Table 9 Intra-day repeatability and inter-day precision for the direct extraction method in germinating soybean seeds. Intra-day repeatability (n = 6)b

Isomers

Parameter

a-Tocopherol

Meana RSD%

1.40  0.05 3.6

a-Tocotrienol

Mean RSD%

b-Tocopherol

WT

B20

Inter-day precision (n = 6)c C5

1.47  0.05 3.6

1.52  0.07 4.8

NDd –

0.35  0.04 11.4

0.30  0.09 25.0

Mean RSD%

0.21  0.04 19.1

0.27  0.04 14.8

g-Tocopherol

Mean RSD%

12.60  0.40 3.2

g-Tocotrienol

Mean RSD%

ND –

d-Tocopherol

Mean RSD%

d-Tocotrienol

Mean RSD%

WT

B20

1.51  0.09 6.0

C5

1.76  0.07 4.0

1.99  0.18 9.0

ND –

0.35  0.04 11.4

0.42  0.03 7.1

0.23  0.03 13.0

0.21  0.02 9.8

0.26  0.05 19.7

0.29  0.03 10.6

13.85  0.23 1.7

14.70  0.4 2.7

12.88  0.75 5.8

14.57  0.85 5.9

14.47  0.97 6.5

0.32  0.04 10.6

0.39  0.05 11.5

ND –

0.38  0.02 5.5

0.39  0.02 6.0

2.86  0.13 4.6

3.24  0.10 3.1

3.79  0.04 1.1

3.32  0.31 9.4

3.44  0.30 8.8

3.76  0.42 11.2

ND –

0.13  0.01 7.7

0.16  0.00 –

ND –

0.13  0.01 7.7

0.13  0.01 7.7

The limits of detection (LOD) were 0.03, 0.04 and 0.02 mg mL1 for a-tocotrienol, g-tocotrienol and d-tocotrienol. Data represent the means of six replicates  SD. a mg 100 g1 sample on dry weight basis. b Intra-day repeatability was evaluated using six independent analyses of replicate samples for 1 day. c Inter-day precision was evaluated using six independent analyses of replicate samples performed for two days. d Not detected.

3.4. Precision of the developed extraction method The analytical method validation parameters such as precision, recovery and linearity were calculated using rapid Soxhlet extraction and direct extraction. These parameters were compared to saponification. Precision, expressed as a percentage of the relative standard deviation (RSD%), and accuracy, expressed as a percentage of recovery (recovery%) of the method, were determined from the different extraction procedures. The reproducibility of the results indicated precision under the same operating conditions over both short (intra-assay precision) and long time interval (inter-assay precision). In rapid Soxhlet extraction, the RSD% of the intra-assay was usually less than 5%, while that of the inter-assay for determining vitamin E isomers was less than 20% (Table 8). Throughout the 3 days used for the inter-assay, the RSD% values of a-tocopherol in WT, B20 and C5 and of d-tocotrienol in C5 were 11.5, 13.2, 10.4 and 18.3, respectively. As temperatures increased, the rate of a-tocopherol oxidation also increased. This led to increased a-tocopherol degradation. The higher RSD% of d-tocotrienol may have been due to its lower concentration in the samples. With the direct extraction method, the RSD% values of all of the examined tocopherols and tocotrienols in the intra-assay were less than 10%, with the exception of b-tocopherol (Table 9). The interassay for the direct extraction method was measured for 2 days, while the inter-assay of the rapid Soxhlet extraction was measured for 3 days. This was because the content of g-tocotrienol and dtocotrienol in B20 and C5 was not detected on day 3 (data not shown). The RSD% of the inter-assay in all of the tocopherols from WT was less than 10%. The RSD% of those from B20, with the exception of a-tocotrienol and b-tocopherol, was less than 10%. In C5, all of the tocotrienol and tocopherol, except b- and d-tocopherol, was less than 10%. The higher values may have been caused by a lower content in the samples, similar to the Soxhlet extraction. To determine analytical method accuracy, different concentrations (lower concentration (LC), intermediate concentration (IC) and higher concentration (HC)) of vitamin E isomers (LC (0.4 mg mL1), IC (5.0 mg mL1) and HC (10.0 mg mL1) for a-, d-tocopherol and d-tocotrienol; LC (0.1 mg mL1), IC (1.0 mg mL1) and HC (6.0 mg mL1) for b-tocopherol, a-, b- and g-tocotrienol;

LC (2.0 mg mL1), IC (10.0 mg mL1) and HC (16.0 mg mL1) for gtocopherol) of vitamin E isomers were prepared from independent stock solutions and analyzed (n = 3). Excellent mean recovery % values, close to 100%, along with low standard deviation values (RSD% < 20.0), indicate high accuracy of the analytical methods (Savic et al., 2009). The analytical recovery was good, with the ranging from 86.0 to 107.6% and the maximum RSD being 18.09% (Table 10). The linearity test for quantification was conducted over the ranges of 0.4–10.0, 0.1–6.0, 2.0–16.0, and 0.4–10.0 mg mL1 for a-, b-, g-, and d-tocopherol, respectively. The range of a-, b- and Table 10 Analytical recovery for the developed method. Isomers

Theoretical (mg mL1) Founda (mg mL1) RSD%b Recovery%

a-Tocopherol

0.4 5.0 10.0

0.35  0.06 4.46  0.1 10.13  0.24

16.07 2.22 2.41

87.50 89.20 101.30

a-Tocotrienol

0.1 1.0 6.0

0.09  0.01 0.88  0.09 5.76  0.52

1.06 10.28 9.07

89.76 88.00 96.00

b-Tocopherol

0.1 1.0 6.0

0.09  0.01 1.08  0.02 5.68  0.91

11.03 1.64 16.08

90.00 107.60 94.67

g-Tocopherol

2.0 10.0 16.0

1.96  0.22 9.87  0.47 16.39  0.05

11.39 4.72 0.28

98.00 98.70 102.44

b-Tocotrienol

0.1 1.0 6.0

0.1  0.01 0.86  0.02 5.78  0.1

9.34 2.73 1.70

98.00 86.00 96.33

g-Tocotrienol

0.1 1.0 6.0

0.09  0.01 0.96  0.09 5.87  0.09

11.24 9.04 1.57

89.00 96.09 97.91

d-Tocopherol

0.4 5.0 10.0

0.41  0.07 5.14  0.08 9.77  0.01

18.09 1.60 0.11

102.75 102.74 97.68

d-Tocotrienol

0.4 5.0 10.0

0.38  0.03 4.91  0.13 10.58  0.56

8.47 2.56 5.32

94.73 98.25 105.77

a b

Values are mean  standard deviation. The relative standard deviation.

78

Y.Y. Lee et al. / Journal of Food Composition and Analysis 27 (2012) 70–80

Table 11 Statistical data of the regression equations and validation parameters for tocopherols and tocotrienols. Isomers

Linearity

R2 a

Linear range (mg mL1)

LOD (mg mL1)b

a-Tocopherol a-Tocotrienol b-Tocopherol g-Tocopherol b-Tocotrienol g-Tocotrienol d-Tocopherol d-Tocotrienol

y = 3,707,772x  10,429 y = 7,864,725x  258,478 y = 9,847,296x  185,641 y = 5,484,907x  1,928,957 y = 8,723,666x  1,359,346 y = 6,756,329x  631,699 y = 14,721,170x  818,204 y = 11,703,560x  774,846

0.9988 0.9981 0.9988 0.9986 0.9982 0.9992 0.9980 0.9988

0.4–10 0.1–6 0.1–6 2.0–16 0.1–6 0.1–6 0.4–10 0.4–10

0.04 0.03 0.02 0.03 0.04 0.04 0.03 0.02

a

Linear correlation coefficient. Limits of detection (LOD): LOD was estimated based on the lowest concentration giving signal intensity equal to or greater than the sum of the blank signal plus three times. b

Fig. 4. Antioxidant activities of the extracts of soybean seeds and 3-day-old germinating seeds as determined with DPPH and ABTS. The rapid Soxhlet extract of soybean seeds of WT and transgenic lines (B20 and C5) was compared to the saponification extract (A, B). The direct extract of 3-day-old germinating seeds from transgenic soybeans (B20 and C5) and WT was compared to the saponification extract (C, D). DPPH and ABTS radical scavenging activity were expressed in Trolox equivalents based on mg 100 g1 of EtOH extract. The DPPH and ABTS assays of soybean seed used crude extract. The DPPH and ABTS assays of 3-day-old germinating seeds used 25 mg mL1 extracts. The values are expressed as the mean  SD of 3 replicates. Differences between the extraction methods were statistically significant at p < 0.01 by the Duncan’s multiple-range test. **Significant at 1% level; *significant at 5% level.

g-tocotrienol was 0.1–6.0 mg mL1. The range of d-tocotrienol was 0.4–10.0 mg mL1 (Table 11). Regression analysis showed an excellent linear relationship (R2 = 0.998). The validation parameter results were determined to be reliable and satisfactory for the determination of tocotrienols and tocopherols in transgenic soybean. 3.5. Antioxidant activities of the extract using different extraction methods The results of the DPPH and ABTS assays revealed significant differences according to the extraction methods in seeds and germinating extracts (Fig. 4). In soybean seed, the DPPH and ABTS radical-scavenging activities of the rapid Soxhlet extract were higher than those of the saponification extract. In germinating seed, increased antioxidant activities (DPPH and ABTS assays) were observed with the direct extraction method compared to saponification. When the DPPH test was conducted on different refined vegetable oils, a direct correlation was shown between the radical-scavenging capacity of the oils and the total contents of

natural tocopherol and tocotrienol (Rossi et al., 2007). The increased antioxidant activities based on different extraction procedures may be associated with enhanced tocopherol and tocotrienol contents. The choice of extraction method is critical for the extraction of valuable minor functional components, such as tocopherols and tocotrienols, and for the determination of quality characteristics of oil from soybean, palm oil and rice bran (Othaman et al., 2010). This study demonstrates that the extraction method can affect the measurable antioxidant activity and thus impact health benefits and nutrition. To select raw cereals with high antioxidant activity and thus with greater health benefits, the antioxidant activity of the samples should be compared. 4. Conclusion In conclusion, the described rapid Soxhlet extraction method for transgenic soybean seeds and direct extraction for germinating seeds provide fast, relatively inexpensive, and reproducible extraction procedures to analyze vitamin E isomers. These results

Y.Y. Lee et al. / Journal of Food Composition and Analysis 27 (2012) 70–80

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