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Water-soluble extracts from defatted sesame seed flour show antioxidant activity in vitro
Food Chemistry 175 (2015) 306–314
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Water-soluble extracts from defatted sesame seed flour show antioxidant activity in vitro Sana Ben Othman a, Nakako Katsuno b,1, Yoshihiro Kanamaru a, Tomio Yabe a,⇑ a b
United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu, Gifu 501-1193, Japan Shinsei Co. Ltd., 29 Katabashinmachi, Kitanagoya, Aichi 481-8526, Japan
a r t i c l e
i n f o
Article history: Received 21 July 2013 Received in revised form 25 November 2014 Accepted 29 November 2014 Available online 4 December 2014 Keywords: Sesamum indicum L. Water-soluble extracts DPPH radical scavenging ORAC Hydrophilic antioxidants
a b s t r a c t Defatted white and gold sesame seed flour, recovered as a byproduct after sesame oil extraction, was extracted with 70% ethanol to obtain polar-soluble crude extracts. The in vitro antioxidant activity of the extract was evaluated by DPPH free radical scavenging activity and oxygen radical absorbing capacity (ORAC). The polar-soluble crude extracts of both sesame seed types exhibited good antioxidant capacity, especially by the ORAC method with 34,720 and 21,700 lmol Trolox equivalent/100 g of white and gold sesame seed extract, respectively. HPLC, butanol extraction, and UPLC–MS analyses showed that different compounds contributed to the antioxidant activity of the polar-soluble crude extracts. Sesaminol glycosides were identified in the butanol-soluble fractions; whereas, purified water-soluble fraction contained ferulic and vanillic acids. This study shows that hydrophilic antioxidants in the purified water-soluble fraction contributed to the antioxidant activity of white and gold sesame seed polar-soluble crude extracts. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Sesame (Sesamum indicum L.) is an important oil seed crop containing 45% to 50% lipids. In fact, around 70% of the world’s production of sesame is used for the extraction of oil for different utilizations as a food product and in the formulation of multiple cosmetic and pharmaceutical products. In addition, sesame seeds are consumed as a paste or incorporated as an ingredient of different baked goods and confectionery products. In Asian countries, there is an ancient belief in the use of sesame as a nutritious food beneficial for human health (Kumazawa, Koike, Usui, Nakayama, & Fukuda, 2003; Moazzami, Haese, & Kamal-Eldin, 2007; Xu, Chen, & Hu, 2005). Sesame oil is characterized by its resistance to oxidative rancidity despite its high content of unsaturated fatty acids (about 85%); this oxidative stability has been attributed to the presence of ctocopherol and lignans, mainly sesamin, sesamolin, and sesamol (Kansoula & Liakopoulou-Kyriakides, 2010). Recent years, researches were mainly focused on the sesame seed oil fraction which is rich in lignans. Sesame lignans are reported to exhibit a
⇑ Corresponding author. Tel./fax: +81 58 293 2913. E-mail address: [email protected] (T. Yabe). Present address: Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, Gifu 501-1193, Japan. 1
http://dx.doi.org/10.1016/j.foodchem.2014.11.155 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
number of beneficial effects on human health, including cholesterol- and blood pressure-lowering capacity, protection of the liver against ethanol-induced injury (Chen et al., 2005; Kang, Kawai, Naito, & Osawa, 1999), as well as anti-cancer and neuroprotective effects in vivo and in vitro (Hamada et al., 2011; Sheng et al., 2007). On the other hand, the antioxidant components contained in defatted sesame seed flour obtained as a byproduct after oil extraction have attracted less interest. Lignan glycosides have been purified from defatted sesame seeds and identified as sesaminol glucosides, pinoresinol glycosides, and sesamolinol glycosides; however, they have been reported to possess low antioxidant capacity compared to the corresponding aglycones (Kang et al., 1999; Katsuzaki, Kawakishi, & Osawa, 1994). A recent study also reported that black sesame seed meal exhibited anti-hypertensive effect in pre-hypertensive human subjects; this activity has been attributed to lignans (sesamin and sesamolin) and tocopherol remaining in black sesame seed meal after oil extraction (Wichitsranoi et al., 2011). Moreover, some reports suggested that defatted sesame seed flour contains antioxidant compounds other than lignan glycosides (Kansoula & Liakopoulou-Kyriakides, 2010; Mohdaly, Smetanska, Ramadan, Sarhan, & Mahmoud, 2011; Xu et al., 2005). Antioxidants are attracting a great deal of interest because of their potential to protect cells against oxidative damage caused by reactive oxygen species (ROS) and thus prevent several diseases,
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such as cancer, aging-related diseases, and cardiovascular diseases. In particular, water-soluble antioxidants are predicted to have a significant impact as they play an important role in assisting water-insoluble antioxidants in protection against ROS-induced oxidative damage (Ajisaka et al., 2009). In this study, we investigated the antioxidant potential of water-soluble extracts from defatted sesame seed flour of two different varieties of sesame, white and gold sesame seeds, recovered as byproducts after oil extraction. 2. Materials and methods 2.1. Materials Defatted sesame seed flour recovered as a sesame oil extraction byproduct was provided by Shinsei Co., Ltd. (Kitanagoya, Japan). Two different colored varieties were used: white sesame (imported from Paraguay) and gold sesame (imported from Turkey), which are used for oil extraction by the company. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was obtained from Wako Pure Chemical Industries (Osaka, Japan) and 6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), catechin, fluorescein sodium salt (FL), and 2,20 -azobis(2-amidinopropane) dihydrochloride (AAPH) were purchased from Sigma–Aldrich (St. Louis, MO). Vanillic acid was purchased from Nacalai Tesque Inc. (Kyoto, Japan) and ferulic acid was from LKT Laboratories Inc. (St. Paul, MN). Methanol used was of HPLC grade and all other solvents used in this study were of analytical grade. 2.2. Extraction of polar-soluble crude extract from defatted sesame seeds Both white and gold sesame seed powders (100 g) were defatted using acetone as a solvent in which fats were dissolved. Briefly, acetone was added (1:4, w/v) to the sesame powder, which was extracted for 24 h at room temperature. Next, acetone–sesame powder mixture was filtered and the residue was washed with diethyl ether. The obtained dried defatted powder was extracted with 70% ethanol (1:10, w/v) for 1 h at room temperature; the extraction was performed three times to obtain a better yield. The polar-soluble extract obtained was concentrated by ethanol evaporation using a rotary evaporator and remaining aqueous solution was lyophilized to obtain white or gold sesame polar-soluble crude extract powder. 2.3. DPPH free radical scavenging assay DPPH dissolved in ethanol gives a purple colored solution. Free radical scavenging capacity is determined by the decrease in color intensity of the solution, which was determined by measuring the absorbance at 540 nm. Measurements were made in 96-well microplates; each well contained 240 ll of methanol, 30 ll of DPPH ethanol solution (1 mM), and 30 ll of distilled water (blank), catechin 10–80 lg/ml (standard) or sample (polar-soluble crude extract 2.5–15 mg/ml, HPLC fractions F1 2 mg/ml and F2 5 mg/ ml, butanol-soluble fractions 1–4 mg/ml, or purified water-soluble fraction 2–10 mg/ml). The microplates were then left for 30 min at room temperature and the absorbance was measured at 540 nm using a plate reader (Immuno-Mini NJ-2300; Inter Med, Tokyo, Japan). DPPH free radical scavenging activity was calculated as follows:
DPPH radical scavenging activity ð%Þ ¼
ðA0 Asample Þ 100 A0
307
A0: absorbance at 540 nm of DPPH solution in absence of sample. Asample: absorbance at 540 nm of DPPH solution in presence of sample. 2.4. Oxygen radical absorbance capacity (ORAC) assay The ORAC method consists of measuring the decrease in fluorescence of a protein, fluorescein (FL), as a result of the loss of its conformation by oxidative damage. This method measures the ability of an antioxidant to protect FL from oxidative damage. There are two types of ORAC method according to the reaction environment: - Hydrophilic using phosphate buffered saline (PBS) (H-ORAC). - Lipophilic using 50% acetone (L-ORAC). Considering the nature of our sample, the H-ORAC method was used to investigate antioxidant capacity in the present study. The ORAC method used was as described by Zulueta, Esteve, and Frigola (2009). Briefly, measurements were made in 96-well microplates. Each well contained 50 ll of FL (0.78 lM) and plates were incubated for 15 min at 37 °C with 50 ll of PBS (75 mM, pH 7) as a blank, Trolox (20 lM) as a standard, or different concentrations of white and gold sesame seed samples (polar-soluble crude extract 10–100 lg/ml, butanol-soluble fractions 20–100 lg/ml, or purified water-soluble fraction 100–500 lg/ml). Oxidation was started by adding 25 ll of AAPH radical (221 lM) to each well. The fluorescence was measured immediately after addition of AAPH and measurements were then taken every 5 min for 55 min with a plate reader (InfiniteÒ M200; Tecan Inc., Männedorf, Switzerland) using fluorescence filters for an excitation wavelength of 485 nm and an emission wavelength of 535 nm. ORAC values, expressed as lM Trolox equivalent, were calculated by the following formula:
ORAC ðlM TEÞ ¼
C Trolox ðAUCSample AUCBlank Þ ðAUCSample AUCBlank Þ
where CTrolox is the concentration (lM) of Trolox and AUC is the area below the fluorescence decay curve of the sample, Trolox, or blank. 2.5. Separation of polar-soluble crude extract by gel filtration HPLC Polar-soluble crude extracts of white and gold sesame were dissolved in water (10 mg/ml) and filtered using 0.45 lm filters. Samples were analyzed by an HPLC system equipped with an Intelligent HPLC pump system (880-PU; Jasco, Tokyo, Japan), and an Intelligent Refractive Index Detector (830 RI; Jasco). White and gold sesame polar-soluble crude extracts were separated using a YMC-PACK Diol 60 gel filtration column (5 lm, 300 8.0 mm; YMC Co., Ltd., Kyoto, Japan). MilliQ water was used as the mobile phase at a flow rate of 0.5 ml/min. A refractive index detector was used to detect the different saccharide fractions. 2.6. Purification of water-soluble fraction by butanol extraction White and gold sesame seed polar-soluble crude extracts (1 g) were dissolved in 50 ml of distilled water, the same volume of butanol was added, and subjected to extraction and phase separation using a separating funnel. This extraction process was repeated 3 times to obtain the butanol-soluble fraction. The remaining water-soluble fraction was extracted according to the same process with butanol to obtain 3 more supplementary
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butanol extracts designated as +1, +2, and +3 butanol extracts. The final purified water-soluble fraction was recovered with a yield of 46.7% and 51.8% of polar-soluble crude extract for white and gold sesame seed, respectively. The butanol-soluble fraction and butanol extracts were concentrated by evaporation using a rotary evaporator, and dried samples were dissolved in water. All recovered fractions were then lyophilized and stored at 20 °C until further use. 2.7. UPLC–MS analysis of purified water-soluble fraction UPLC–TOF MS (WatersÒ Xevo™ QTof MS; Waters, Milford, MA) was performed using a C18 column (Ø 2.1 mm 100 mm; Waters). The eluents used were water (A) and methanol (B); the elution conditions were 0–3 min, 5% B; 3–18 min, linear gradient to 100% B; 18–20 min, 100% B; the flow rate was 0.3 ml/min and the injection volume was 5 ll. The mass data were collected in negative ionization mode ([MH]). The capillary voltage was 3.0 kV. Cone and desolvation gas flow rates were set at 50 and 1000 l/h, respectively, and the source and desolvation temperature was 150 °C and 500 °C, respectively. Samples used for analysis were purified water-soluble fractions (1 mg/ml) and supplementary butanol extracts (1 mg/ml) from white and gold sesame seeds. The injected sample was first separated by UPLC and then MS of each peak was performed. To identify detected compounds, we referred to MassBank (URL = ‘‘http:// www.massbank.jp’’), a database of mass spectral data, as well as data about antioxidant compounds from sesame seeds reported by Katsuzaki et al. (1994) and Moazzami, Andersson, and KamalEldin (2006). 2.8. Statistical analysis All data were obtained from at least three independent experiments and reported as means ± standard deviation (SD). Statistical analyses were performed using Dunnett’s test for multiple data comparison or Student’s T test. 3. Results and discussion 3.1. Extraction of polar-soluble crude extract from defatted sesame seeds White and gold sesame seed flours recovered as byproducts from sesame oil extraction were completely defatted and then extracted with 70% ethanol to obtain polar-soluble crude extracts. Extraction yields from defatted sesame seed powders were 11.7% and 11.0% for white and gold sesame seeds, respectively. 3.2. Antioxidant activities of white and gold sesame seed polar-soluble crude extracts Methods for measuring the antioxidant capacity in vitro can be divided in two categories according to the chemical reaction involved. The first type is electron transfer (ET) reaction, which includes the DPPH free radical scavenging method. The second type is based on hydrogen atom transfer (HAT) reactions, such as the ORAC method (Zulueta et al., 2009). Both methods were used to evaluate the antioxidant capacities of sesame seed polar-soluble crude extracts in vitro. 3.2.1. DPPH radical scavenging activity DPPH radical scavenging assay, considered as one of the standard and easy colorimetric methods (Mishra, Ojha, & Chaudhury,
2012), is routinely used to evaluate the free radical scavenging potentials of antioxidant molecules. White and gold sesame seed polar-soluble crude extracts were assessed for their DPPH scavenging activity, with catechin taken as a standard antioxidant. As shown in Fig. 1A, catechin exhibited a minimum activity of 18.6% at 10 lg/ml, and its activity increased in a concentration-dependent manner. White and gold sesame seed polar-soluble crude extracts also exhibited concentration-dependent scavenging activity with sample concentration ranges between 2.5 and 15 mg/ml (Fig. 1B). The results also showed that white sesame seed polarsoluble crude extract exhibited higher scavenging activity with 88.18% at 15 mg/ml, whereas gold sesame polar-soluble crude extract exhibited only 64.16% scavenging activity at the same concentration. 3.2.2. Oxygen radical absorbance capacity (ORAC) The ORAC method is attracting interest as a method for evaluation of antioxidant activity as it uses a biologically relevant free radical, i.e., the AAPH-derived peroxyl radical that mimics lipid peroxyl radicals involved in lipid peroxidation chain reaction in vivo (Tai, Sawano, Yazama, & Ito, 2011). In addition, the ORAC result integrates the degree and duration of antioxidant reaction in the same value (Zulueta et al., 2009). Antioxidant capacities of white and gold sesame seed polar-soluble crude extracts were evaluated by the hydrophilic ORAC (H-ORAC) method. Fig. 2A shows the fluorescence intensity decay due to the oxidation of fluorescein (FL) in the presence or absence of sesame seed polarsoluble crude extracts (100 lg/ml). Both white and gold sesame polar-soluble crude extracts delayed the decrease in fluorescence intensity compared to the blank curve suggesting that sesame seed extracts protected FL from oxidative damage by scavenging AAPH free radicals. Using the fluorescence intensity decay curves, the ORAC value is calculated and expressed as lM Trolox equivalent (lM TE). ORAC values for different sample concentrations, 10–100 lg/ml, are illustrated in Fig. 2B. Both seed type extracts exhibited concentration-dependent activity. White sesame seed polar-soluble crude extract still exhibited ORAC of 4.22 lM TE at 10 lg/ml, whereas gold sesame seed extract exhibited no activity at the same concentration. Therefore, as previously observed in the DPPH assay, ORAC assay results showed that white sesame seed polar-soluble crude extract exhibits higher antioxidant activity than gold sesame seed extract. The U.S. Department of Agriculture published a database for the ORAC of selected foods in 2010, in which both hydrophilic and lipophilic ORAC as well as total ORAC are expressed as lmol of Trolox equivalent (TE) per 100 g (U.S. Department of Agriculture, 2010). To compare our samples with some selected foods, we calculated the average ORAC values as lmol TE/100 g of polar-soluble crude extracts, which were 34,720 and 21,700 lmol TE/100 g for white and gold sesame seed polar-soluble crude extracts, respectively. H-ORAC values of white and gold sesame seed polar-soluble crude extracts were compared to H-ORAC values of selected foods reported in the USDA database. The results showed that sesame seed polar-soluble crude extract had a higher H-ORAC value than blueberries (4633 lmol TE/100 g), almond nuts (4282 lmol TE/ 100 g), soybean seeds (5409 lmol TE/100 g), and also rice bran (8817 lmol TE/100 g). Thus, sesame seed polar-soluble crude extract contains strong antioxidant compounds. On the other hand, H-ORAC values of our extracts were lower than those of some spices that showed high ORAC values, such as ground cinnamon (143,264 lmol TE/100 g) and cumin seeds (47,600 lmol TE/ 100 g), while other spices had ORAC values close to our samples, such as curry powder (24,981 lmol TE/100 g) and mustard seeds (28,759 lmol TE/100 g). This comparison suggests that the
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A % DPPH scavenging activity
100 90
**
80 70
**
60 50
**
40 30 20 10 0 10
20
40
80
Catechin concentration (µg/ml)
B
Fig. 1. DPPH radical scavenging activities of catechin (A), and the white and gold sesame seed polar-soluble crude extracts (B). Results are averages of 5 independent experiments ± standard deviation (SD). Statistical analysis was done by Dunnett’s test, ** or ##: p < 0.01 compared to each sample lowest concentration.
obtained polar-soluble crude extracts recovered from defatted sesame seeds possess good ORAC activity. Unfortunately, sesame seeds and derivative products were not reported in the database, which would have been interesting for comparison with our samples. Ishiyama, Nagashima, Yasumoto, and Fukuda (2006) reported ORAC measurement of two sesame seed varieties from Japan Kanto No. 1 and Gomazou. ORAC values of methanol extracts from both sesame seeds were 658.3 and 827 mg TE/100 g of sesame seed for Kanto No. 1 and Gomazou, respectively. These values correspond to 2630 and 3304 lmol TE/100 g of sesame seed. According to these results, we can conclude that hydrophilic antioxidant compounds remaining in defatted sesame seed after oil extraction exhibit as important antioxidant capacity as methanol extracts from non-defatted sesame seeds. 3.3. Separation of saccharides fraction by gel filtration HPLC 3.3.1. HPLC analysis In this study, we focused on hydrophilic antioxidants potentially present in the polar-soluble crude extracts from defatted sesame seeds. Therefore, we attempted to separate the polar-soluble crude extracts into different saccharide fractions. Extracts were analyzed by HPLC gel filtration column chromatography (YMCPack Diol 60) and the eluted saccharide fractions were identified using a refractive index detector (RID). Chromatograms of white and gold sesame seed extracts are illustrated in Fig. 3A and B. Both extracts were separated into two main saccharide fractions. The eluted peaks were identified by comparison with standard oligosaccharides separated under the same HPLC conditions (Fig. 3C). The first fraction, designated as F1 was obtained with 17.1% and 17.5% yield from white and gold sesame seed polar-soluble crude extracts, respectively. This fraction corresponded to relatively high molecular weight saccharides, such as polysaccharides or other
complex molecules. The second fraction (F2) was obtained with 73.8% and 75.2% yield from white and gold sesame seed polar-soluble crude extracts, respectively. This fraction consisted of different oligosaccharides ranging from di- to hexasaccharides consistent with the oligosaccharide composition of sesame seeds reported previously as sucrose, raffinose, stachyose, planteose, sesamose, as well as penta- and hexasaccharides (Wankhede & Tharanathan, 1976). 3.3.2. Antioxidant capacity of saccharide fractions F1 and F2 To determine if separated saccharide fractions are involved in the detected antioxidant activity of polar-soluble crude extracts, both F1 and F2 were collected and investigated for their antioxidant capacities using both DPPH radical scavenging and ORAC assays. In the case of DPPH radical scavenging assay (Fig. 4A), samples were collected from HPLC analysis from the same amount of injected polar-soluble crude white and gold sesame seed extracts. The collected F1 fractions (2 mg/ml) exhibited good DPPH scavenging activity with values of 38.62% and 27.57% for white and gold sesame seeds, respectively, whereas the oligosaccharide fraction F2 (5 mg/ml) had very low activity with values of only 6.33% and 2.91% for white and gold sesame seeds, respectively. For ORAC assay, different concentrations of F1 and F2 fractions were investigated (Fig. 4B). F1 fraction exhibited high ORAC values at 50 and 100 lg/ml, corresponding to average values of 50,953 and 54,420 lmol TE/100 g for white and gold sesame seed extracts, respectively. The ORAC values of the F2 fraction were measured at 250 and 500 lg/ml, corresponding to 5347 and 6068 lmol TE/ 100 g for white and gold sesame seed extracts, respectively. Both DPPH radical scavenging and ORAC methods showed that F1 exhibited around 10-fold higher antioxidant activity than the oligosaccharide fraction F2. F1 from both seed types was
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Relative Fluorescence Intensity (%)
A 100 blank standard 80
WS (100 µg/ml) GS (100 µg/ml)
60
40
20
0 0
10
20
30
40
50
60
Time (min)
B
Fig. 2. ORAC of white and gold sesame seed polar-soluble crude extracts. (A) Fluorescence intensity decay curves of fluorescein in the presence of 100 lg/ml of white and gold sesame seed polar-soluble crude extracts. Standard is 20 lM Trolox. (B) ORAC of white and gold sesame seed polar-soluble crude extracts at different concentrations. Results are expressed as mean values ± SD. All results are averages of 4 independent experiments. Statistical analysis was done by Dunnett’s test, * or #: p < 0.05, and ** or ##: p < 0.01 compared to each sample lowest concentration.
characterized by a dark brown color compared to F2, which had a yellowish white color. Xu et al. (2005) isolated brown pigment from the ethanolic extract of black sesame seeds that showed antioxidant activity in vitro. Furthermore, melanoidin-like browning compounds produced during roasting of sesame seeds are also believed to be involved in the antioxidant activity of roasted sesame seed oil (Kumazawa et al., 2003). Thus, the F1 fraction may be related to such compounds. However, neither the structure nor antioxidant mechanisms of such compounds have yet been elucidated. The oligosaccharide fraction F2 showed very low DPPH radical scavenging activity, with values of 6.33% and 2.91% for white and gold sesame seeds, respectively. On the other hand, it had relatively high ORAC values of 5347 and 6068 lmol TE/100 g for white and gold sesame seeds, respectively. These results may be explained by the difference in reaction medium, as the ORAC method uses PBS, which is favorable for hydrophilic compounds such as oligosaccharides. In addition, DPPH free radicals are not similar to the highly reactive peroxyl radicals usually involved in lipid peroxidation in vivo (Huang, Ou, & Prior, 2005). Whereas, AAPH-derived radicals used in the ORAC assay mimic peroxyl radical-induced oxidation (Tai et al., 2011). Therefore, the ORAC assay is a more accurate method for quantifying peroxyl radical scavenging capacity in vitro.
3.4. Purification of sesame seed water-soluble fraction by butanol extraction Defatted sesame seed flour is an important source of oil-insoluble lignan glycosides, such as pinoresinol glucosides and sesaminol glucosides (Moazzami et al., 2006). Such compounds may be contained in the polar-soluble crude extract obtained by extraction with 70% ethanol. This is why polar-soluble crude extracts from white and gold sesame seeds were purified by butanol extraction to remove relatively hydrophobic compounds contained in the crude extract. 3.4.1. Butanol extraction Sesame seed polar-soluble crude extract contained a nonhydrophilic fraction that was solubilized in butanol; 456.2 and 320.7 mg/1 g of crude extract were obtained from white and gold sesame seeds, respectively. Small amounts ranging between 19 and 42 mg/1 g of crude extract were still extracted by butanol in +1, +2, and +3 butanol extracts. The final purified water-soluble fraction consisted of approximately 50% of the crude extract with 467.2 and 517.7 mg/1 g of crude extract from white and gold sesame seeds, respectively. Consistent with our hypothesis, these results indicated that polar-soluble crude extract contained relatively non-hydrophilic
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Fig. 3. HPLC chromatograms of white (A) and gold (B) sesame seed polar-soluble crude extracts and oligosaccharides standard solution (C) analyzed by YMC-Pack Diol 60 gel filtration column using RID. 1: polysaccharides, 2: hexa-, pentasaccharides, 3: tetrasaccharides, 4: trisaccharides, 5: disaccharides, 6: monosaccharides, a: stachyose, b: raffinose, and c: sucrose.
compounds that might be involved in the observed antioxidant activity. Thus, the antioxidant activities of all recovered fractions were assessed using both DPPH radical scavenging and ORAC assays. 3.4.2. Antioxidant capacity of the different fractions recovered from butanol extraction The recovered fractions from butanol extraction of both white and gold sesame seed polar-soluble crude extract were investigated for their antioxidant potential using DPPH radical scavenging and ORAC assays (Fig. 5). DPPH radical scavenging activity (Fig. 5A) was determined for sample concentrations between 1 and 4 mg/ml for the butanol-
soluble fraction as well as supplementary butanol extracts. The butanol-soluble fraction (4 mg/ml) exhibited scavenging activity of 61.66% and 46.54% for white and gold sesame seeds, respectively. The +1 butanol extract exhibited comparable activity with value of 62.12% and 32.29% for white and gold sesame seeds, respectively. However, DPPH scavenging activity gradually decreased for +2 and +3 butanol extracts for both seed types. Butanol-soluble fraction also exhibited the highest activity in the ORAC assay (Fig. 5B) with value of 45.01 and 27.83 lM TE at 100 lg/ml for white and gold sesame seeds, respectively. Supplementary butanol extracts also exhibited activity at similar sample concentration ranges (50 and 100 lg/ml) and activity gradually decreased as observed in DPPH scavenging assay. These results suggest that
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% DPPH scavenging activity
A
100 90 80 70 60 50 40 30 20 10 0 F1 (2 mg/ml)
F2 (5 mg/ml)
F1 (2 mg/ml)
White sesame
F2 (5 mg/ml)
Gold sesame
B
Fig. 4. Antioxidant capacities of HPLC fractions of white and gold sesame seed polar-soluble crude extracts. (A) DPPH radical scavenging activity (%). (B) ORAC at different concentrations of F1 and F2 from white and gold polar-soluble crude extracts. Experiments were performed in triplicate; results are expressed as mean values ± SD. Statistical analysis was done by Student’s T test, * or #: p < 0.05, and ** or ##: p < 0.01 compared to each sample lowest concentration.
100
A
White Sesame
Gold Sesame
% DPPH scavenging activity
90 80 70 60 50 40 30 20 10 0 1 mg/ml 2 mg/ml 4 mg/ml 1 mg/ml 2 mg/ml 4 mg/ml 1 mg/ml 2 mg/ml 4 mg/ml 1 mg/ml 2 mg/ml 4 mg/ml 2 mg/ml 5 mg/ml 10 mg/ml Butanol-soluble fraction
60
+1 butanol extract
B
+2 butanol extract
White Sesame
+3 butanol extract
Water-soluble fraction
Gold Sesame
ORAC (µM TE)
50 40 30 20 10 0 20 µg/ml
50 µg/ml
100 µg/ml
Butanol-Soluble fraction
50 µg/ml
100 µg/ml
+1Butanol extract
50 µg/ml
100 µg/ml
+2 Butanol extract
50 µg/ml
100 µg/ml
+3 Butanol extract
100 µg/ml
200 µg/ml
500 µg/ml
Water-Soluble fraction
Fig. 5. Antioxidant capacities of different fractions recovered from butanol extraction of both white and gold sesame seed polar-soluble crude extracts. (A) DPPH radical scavenging activity (%), (B) ORAC at different concentrations. Results are expressed as mean values ± SD. All results are averages of 4 independent experiments. Statistical analysis was done by Dunnett’s test, all data showed statistical significant difference (p < 0.05) compared to each sample lowest concentration.
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sesame seed polar-soluble crude extracts contained non-hydrophilic antioxidant compounds that were extracted in the butanol-soluble fraction. Such non-hydrophilic antioxidants were still extracted by further butanol extractions, and the decreasing antioxidant activity of these extracts suggested that the remaining purified water-soluble fraction probably contained only hydrophilic components. Antioxidant activity of the purified water-soluble fraction was detected at sample concentrations between 2 and 10 mg/ml for DPPH scavenging assay (Fig. 5A) and between 100 and 500 lg/ml for ORAC assay (Fig. 5B). Both assays showed that the water-soluble fraction exhibited concentration-dependent antioxidant activity, although relatively high sample concentrations were required compared to different butanol extracts. Moreover, similar ORAC assay results were observed for the oligosaccharide fraction F2 (Fig. 4B) suggesting that the purified water-soluble fraction, which is mainly composed of oligosaccharides, also contains some hydrophilic antioxidant compounds. 3.4.3. Identification of potential antioxidant compounds in purified fractions by UPLC–MS White and gold sesame seed purified water-soluble fraction and supplementary butanol extracts (+1, +2, and +3 butanol extracts) were analyzed by UPLC–TOF MS. The mass data was collected in negative ionization mode ([MH]) and we referred to MassBank, a database for mass spectral data, as well as the available literature about antioxidant compounds from sesame seeds reported in previous studies to identify the detected compounds. The results are summarized in Table 1; white and gold sesame seed extracts were found to have similar compositions, so the results are presented independently from seed type. Purified water-soluble fraction analyzed by UPLC was separated into two fractions (Supplementary data 1). The major fraction was eluted between 0.8 and 1.6 min and contained most hydrophilic constituents. According to MS data, the eluted compounds corresponded to oligosaccharides (di- to pentasaccharides), in accordance with previous HPLC analysis of the crude extract (Fig. 3). In addition to oligosaccharides, a peak corresponding to vanillic acid was detected at around 1.5 min. The second fraction was eluted around 5.9 min, and was greater in white than gold sesame seed extracts. Mass data showed that this fraction contained another phenolic acid, ferulic acid. Jeong et al. (2004) reported the presence of both vanillic and ferulic acids in defatted sesame seed meal methanolic extract. However, they specified that ferulic acid was only detected in unroasted sesame seed extract, while vanillic acid appeared in roasted sesame seed extract. As our samples were obtained from sesame oil extraction byproducts, they come from roasted sesame seeds because roasting is a key operation in sesame oil preparation influencing its organoleptic properties as well as its oxidative stability. Our results demonstrated the
presence of both ferulic and vanillic acids in the water-soluble fraction; however, ferulic acid content seemed to be lower than that of vanillic acid. The difference from previous studies could have been due to differences in sample preparation and/or analysis method used. To confirm the contributions of both phenolic acids to the antioxidant activity of purified water-soluble fraction, pure compounds were purchased and their antioxidant activities were evaluated by DPPH free radical scavenging and ORAC assays. Vanillic and ferulic acids exhibited good antioxidant capacities with ORAC assay (Supplementary data 2), and exhibited comparable ORAC values (33.16 and 34.44 lM TE for vanillic and ferulic acid, respectively, at a sample concentration of 2.5 lg/ml) in agreement with the results obtained by Tai et al. (2011); they reported that vanillic and ferulic acids exhibited comparable ORAC results; however, they did not determine their ORAC values and only fluorescence decay curves were compared. On the other hand, vanillic acid showed almost no DPPH radical scavenging effect compared to ferulic acid (10.25% and 77.36% for vanillic and ferulic acid, respectively, at a sample concentration of 500 lg/ml). In fact, both phenolic acids have the same structure involved in radical scavenging reaction. However, ferulic acid, which is relatively more hydrophobic, reacts better in apolar medium compared to vanillic acid. Again, we concluded that ORAC assay, which is more relevant to oxidation chain reaction in vivo, is more appropriate to quantify the antioxidant potential of hydrophilic compounds in vitro. Moreover, sesame seeds were reported to contain both free phenolic acids esters and bound insoluble forms (Neveu et al., 2010), and bound phenolic acids are believed to be cleaved and liberated during roasting of seeds (Jeong et al., 2004). Therefore, vanillic and ferulic acids are more likely to be the bioactive components in the purified water-soluble fractions from both white and gold sesame seeds. When we examined the antioxidant activities of different fractions after butanol extraction, we found that supplementary butanol extracts exhibited decreasing antioxidant activity (+1 butanol > +2 butanol > +3 butanol extract). To better understand this observation, we investigated the compositions of all 3 extracts with UPLC–MS analysis. Several peaks were eluted between 0.8 and 1.8 min; these peaks correspond to oligosaccharides (di- and trisaccharides) and vanillic acid. These hydrophilic compounds were present as minor fractions; a main peak was eluted at 8.93 min corresponding to sesaminol triglucoside; a sesaminol diglucoside peak was also detected at 9.55 min. In addition, UPLC–MS analysis of +2 and +3 butanol extracts showed that sesaminol triglucoside peak intensity decreased gradually and sesaminol diglucoside was not detected in the two extracts (Supplementary data 3). These results suggest that sesaminol glycoside content in supplementary butanol extracts gradually decreased, in a manner that was correlated with the decrease in antioxidant activity.
Table 1 UPLC–MS analysis data of purified water-soluble fraction and +1 butanol extract. Sample
Peak elution time (min)
Molecular ion [MH] (m/z)
Peak assignment
References
Purified water-soluble fraction
0.8–1.6
341.1066 503.1564 665.2094 833.2536 167.0323 193.0488
Disaccharide (sucrose) Trisaccharide (raffinose) Tetrasaccharide Pentasaccharide Vanillic acid Ferulic acid
MassBank MassBank MassBank MassBank MassBank MassBank
341.1036 503.1601 167.0332 855.2600 693.2013
Disaccharide Trisaccharide Vanillic acid Sesaminol triglucoside Sesaminol diglucoside
MassBank database MassBank database MassBank database Katsuzaki et al. (1994) Moazzami et al. (2006)
5.9 +1 Butanol extract
0.8–1.8
8.93 9.55
database database database database database database
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Previous studies indicated that lignan glycosides possess lower radical scavenging activity compared to their corresponding aglycones (Kang et al., 1999). Shyu and Hwang (2002) also reported that sesaminol triglucoside did not show good antioxidant activity as assessed by DPPH radical scavenging and LDL oxidation. However, our results showed that supplementary butanol extracts ORAC was correlated to sesaminol triglucoside content (Fig. 5B and Supplementary data 3). Therefore, sesaminol triglucoside, which is more hydrophilic than sesaminol, apparently will not react appropriately in hydrophobic medium, such as that used for DPPH radical scavenging assay, while it will react well with AAPH-induced radicals in the ORAC method. Finally, the different assays used in the present study showed that extracts from white sesame seed had relatively higher antioxidant capacity than extracts from gold sesame seeds. This was most likely due to the differences in the contents of antioxidants as both seeds types had the same composition. In fact, Moazzami et al. (2007) quantified sesaminol glucosides in 65 different sesame seed samples and found that their contents varied depending on seed sample; however, there were no significant differences between black and white seeds, indicating that sesaminol glucosides contents did not necessarily depend on seed color. Wankhede and Tharanathan (1976) noted that composition of sesame seeds could vary according to growing conditions of sesame plants; as our samples were imported from two different countries (Turkey and Paraguay), this would explain the differences between white and gold sesame seed extracts observed here. 4. Conclusions In this study we showed that purified water-soluble fraction contributed to the polar-soluble crude extract antioxidant activity. Several studies reported that lignan glycosides are the main antioxidants in defatted sesame seeds; here we showed that purified water-soluble fraction contained ferulic and vanillic acids as bioactive antioxidants. As the use of polar solvents such as ethanol and ethyl acetate is considered to secure the dietary safety of the obtained extract, sesame seed polar-soluble crude extracts may be suitable for use as dietary antioxidants. Therefore, further studies on the physiological functions of these extracts are needed to determine the most appropriate use of these extracts. Acknowledgments The authors would like to acknowledge the help with UPLC–MS analysis provided by Professor Tohru Mitsunaga (Gifu University) and the members of his laboratory. We thank Professor Ryo Yamauchi (Gifu University) for helpful discussion and comments. We also thank the members of the Division of Genomics Research, Life Science Research Center, Gifu University, for their support in conducting the experiments. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem. 2014.11.155.
References Ajisaka, K., Agawa, S., Nagumo, S., Kurato, K., Yokoyama, K., Arai, K., et al. (2009). Evaluation and comparison of the antioxidative potency of various carbohydrates using different methods. Journal of Agricultural and Food Chemistry, 57, 3102–3107. Chen, P. R., Chien, K. L., Su, T. C., Chang, C. J., Liu, T. T., Cheng, H., et al. (2005). Dietary sesame reduces serum cholesterol and enhances antioxidant capacity in hypercholesterolemia. Nutrition Research, 25, 559–567. Hamada, N., Tanaka, A., Fujita, Y., Itoh, T., Ono, Y., Kitagawa, Y., et al. (2011). Involvement of heme-oxygenase-1 induction via Nrf2/ARE activation in protection against H2O2-induced PC12 cell death by a metabolite of sesamin contained in sesame seeds. Bioorganic & Medicinal Chemistry, 19, 1959–1965. Huang, D., Ou, B., & Prior, R. L. (2005). The chemistry behind antioxidant capacity assays. Journal of Agricultural and Food Chemistry, 53, 1841–1856. Ishiyama, K., Nagashima, M., Yasumoto, T., & Fukuda, Y. (2006). Changes in amounts of lignan compounds and radical scavenging activities in improved high-lignan breed of sesame, ‘‘Gomazou’’, during germination. Nippon Shokuhin Kagaku Kogaku Kaishi, 53, 8–16. Jeong, S. M., Kim, S. Y., Kim, D. R., Nam, K. C., Ahn, D. U., & Lee, S. C. (2004). Effect of seed roasting conditions on the antioxidant activity of defatted sesame meal extracts. Journal of Food Science: Food Chemistry and Toxicology, 69, 377–381. Kang, M. H., Kawai, Y., Naito, M., & Osawa, T. (1999). Dietary defatted sesame flour decreases susceptibility to oxidative stress in hypercholesterolemic rabbits. Journal of Nutrition, 129, 1885–1890. Kansoula, Z., & Liakopoulou-Kyriakides, M. (2010). Effect of endogenous antioxidants of sesame seeds and sesame oil to the thermal stability of edible vegetable oils. LWT – Food Science and Technology, 43, 1379–1386. Katsuzaki, H., Kawakishi, S., & Osawa, T. (1994). Sesaminol glucosides in sesame seeds. Phytochemistry, 35, 773–776. Kumazawa, S., Koike, M., Usui, Y., Nakayama, T., & Fukuda, Y. (2003). Isolation of sesaminols as antioxidative components from roasted sesame seed oil. Journal of Oleo Science, 52, 303–307. Mishra, K., Ojha, H., & Chaudhury, N. K. (2012). Estimation of antiradical properties of antioxidants using DPPH.assay: A critical review and results. Food Chemistry, 130, 1036–1043. Moazzami, A. A., Haese, S. L., & Kamal-Eldin, A. (2007). Lignan contents in sesame seeds and products. European Journal of Lipid Science and Technology, 109, 1022–1027. Moazzami, A. A., Andersson, R. E., & Kamal-Eldin, A. (2006). HPLC analysis of sesaminol glucosides in sesame seeds. Journal of Agricultural and Food Chemistry, 53, 633–638. Mohdaly, A. A. A., Smetanska, I., Ramadan, M. F., Sarhan, M. A., & Mahmoud, A. (2011). Antioxidant potential of sesame (Sesamum indicum) cake extract in stabilization of sunflower and soybean oils. Industrial Crops and Products, 34, 952–959. Neveu, V., Perez-Jiménez, J., Vos, F., Crespy, V., Chaffaut, L., Mennen, L., Knox, C., Eisner, R., Cruz, J., Wishart, D., & Scalbert, A. (2010). Phenol-Explorer: An online comprehensive database on polyphenol contents in foods. Database, http:// dx.doi.org/10.1093/database/bap024 [Version 1.5.2, available at http:// www.phenol-explorer.eu]. Sheng, H. Q., Hirose, Y., Hata, K., Zheng, Q., Kuno, T., Asano, N., et al. (2007). Modifying effect of dietary sesaminol glucosides on the formation of azoxymethane-induced premalignant lesions of rat colon. Cancer Letters, 246, 63–68. Shyu, Y. S., & Hwang, L. S. (2002). Antioxidative activity of the crude extract of lignan glycosides from unroasted Burma black sesame meal. Food Research International, 35, 357–365. Tai, A., Sawano, T., Yazama, F., & Ito, H. (2011). Evaluation of antioxidant activity of vanillin by using multiple antioxidant assays. Biochimica et Biophysica Acta, 1810, 170–177. U.S. Department of Agriculture, Agricultural Research Service (2010). Oxygen Radical Absorbance Capacity (ORAC) of Selected Foods, Release 2. Nutrient Data Laboratory Home Page: http://www.ars.usda.gov/nutrientdata/orac. Wankhede, D. B., & Tharanathan, R. N. (1976). Sesame (Sesamum indicum) carbohydrates. Journal of Agricultural and Food Chemistry, 24, 655–659. Wichitsranoi, J., Weerapreeyakul, N., Boonsiri, P., Settasatian, C., Settasatian, N., Komanasin, N., et al. (2011). Antihypertensive and antioxidant effects of dietary black sesame meal in pre-hypertensive humans. Nutrition Journal, 10, 82–89. Xu, J., Chen, S., & Hu, Q. (2005). Antioxidant activity of brown pigment and extracts from black sesame seed (Sesamum indicum L.). Food Chemistry, 91, 79–83. Zulueta, A., Esteve, M. J., & Frigola, A. (2009). ORAC and TEAC assays comparison to measure the antioxidant capacity of food products. Food Chemistry, 114, 310–316.
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Water-soluble extracts from defatted sesame seed flour show antioxidant activity in vitro Sana Ben Othman a, Nakako Katsuno b,1, Yoshihiro Kanamaru a, Tomio Yabe a,⇑ a b
United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu, Gifu 501-1193, Japan Shinsei Co. Ltd., 29 Katabashinmachi, Kitanagoya, Aichi 481-8526, Japan
a r t i c l e
i n f o
Article history: Received 21 July 2013 Received in revised form 25 November 2014 Accepted 29 November 2014 Available online 4 December 2014 Keywords: Sesamum indicum L. Water-soluble extracts DPPH radical scavenging ORAC Hydrophilic antioxidants
a b s t r a c t Defatted white and gold sesame seed flour, recovered as a byproduct after sesame oil extraction, was extracted with 70% ethanol to obtain polar-soluble crude extracts. The in vitro antioxidant activity of the extract was evaluated by DPPH free radical scavenging activity and oxygen radical absorbing capacity (ORAC). The polar-soluble crude extracts of both sesame seed types exhibited good antioxidant capacity, especially by the ORAC method with 34,720 and 21,700 lmol Trolox equivalent/100 g of white and gold sesame seed extract, respectively. HPLC, butanol extraction, and UPLC–MS analyses showed that different compounds contributed to the antioxidant activity of the polar-soluble crude extracts. Sesaminol glycosides were identified in the butanol-soluble fractions; whereas, purified water-soluble fraction contained ferulic and vanillic acids. This study shows that hydrophilic antioxidants in the purified water-soluble fraction contributed to the antioxidant activity of white and gold sesame seed polar-soluble crude extracts. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Sesame (Sesamum indicum L.) is an important oil seed crop containing 45% to 50% lipids. In fact, around 70% of the world’s production of sesame is used for the extraction of oil for different utilizations as a food product and in the formulation of multiple cosmetic and pharmaceutical products. In addition, sesame seeds are consumed as a paste or incorporated as an ingredient of different baked goods and confectionery products. In Asian countries, there is an ancient belief in the use of sesame as a nutritious food beneficial for human health (Kumazawa, Koike, Usui, Nakayama, & Fukuda, 2003; Moazzami, Haese, & Kamal-Eldin, 2007; Xu, Chen, & Hu, 2005). Sesame oil is characterized by its resistance to oxidative rancidity despite its high content of unsaturated fatty acids (about 85%); this oxidative stability has been attributed to the presence of ctocopherol and lignans, mainly sesamin, sesamolin, and sesamol (Kansoula & Liakopoulou-Kyriakides, 2010). Recent years, researches were mainly focused on the sesame seed oil fraction which is rich in lignans. Sesame lignans are reported to exhibit a
⇑ Corresponding author. Tel./fax: +81 58 293 2913. E-mail address: [email protected] (T. Yabe). Present address: Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, Gifu 501-1193, Japan. 1
http://dx.doi.org/10.1016/j.foodchem.2014.11.155 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
number of beneficial effects on human health, including cholesterol- and blood pressure-lowering capacity, protection of the liver against ethanol-induced injury (Chen et al., 2005; Kang, Kawai, Naito, & Osawa, 1999), as well as anti-cancer and neuroprotective effects in vivo and in vitro (Hamada et al., 2011; Sheng et al., 2007). On the other hand, the antioxidant components contained in defatted sesame seed flour obtained as a byproduct after oil extraction have attracted less interest. Lignan glycosides have been purified from defatted sesame seeds and identified as sesaminol glucosides, pinoresinol glycosides, and sesamolinol glycosides; however, they have been reported to possess low antioxidant capacity compared to the corresponding aglycones (Kang et al., 1999; Katsuzaki, Kawakishi, & Osawa, 1994). A recent study also reported that black sesame seed meal exhibited anti-hypertensive effect in pre-hypertensive human subjects; this activity has been attributed to lignans (sesamin and sesamolin) and tocopherol remaining in black sesame seed meal after oil extraction (Wichitsranoi et al., 2011). Moreover, some reports suggested that defatted sesame seed flour contains antioxidant compounds other than lignan glycosides (Kansoula & Liakopoulou-Kyriakides, 2010; Mohdaly, Smetanska, Ramadan, Sarhan, & Mahmoud, 2011; Xu et al., 2005). Antioxidants are attracting a great deal of interest because of their potential to protect cells against oxidative damage caused by reactive oxygen species (ROS) and thus prevent several diseases,
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such as cancer, aging-related diseases, and cardiovascular diseases. In particular, water-soluble antioxidants are predicted to have a significant impact as they play an important role in assisting water-insoluble antioxidants in protection against ROS-induced oxidative damage (Ajisaka et al., 2009). In this study, we investigated the antioxidant potential of water-soluble extracts from defatted sesame seed flour of two different varieties of sesame, white and gold sesame seeds, recovered as byproducts after oil extraction. 2. Materials and methods 2.1. Materials Defatted sesame seed flour recovered as a sesame oil extraction byproduct was provided by Shinsei Co., Ltd. (Kitanagoya, Japan). Two different colored varieties were used: white sesame (imported from Paraguay) and gold sesame (imported from Turkey), which are used for oil extraction by the company. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was obtained from Wako Pure Chemical Industries (Osaka, Japan) and 6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), catechin, fluorescein sodium salt (FL), and 2,20 -azobis(2-amidinopropane) dihydrochloride (AAPH) were purchased from Sigma–Aldrich (St. Louis, MO). Vanillic acid was purchased from Nacalai Tesque Inc. (Kyoto, Japan) and ferulic acid was from LKT Laboratories Inc. (St. Paul, MN). Methanol used was of HPLC grade and all other solvents used in this study were of analytical grade. 2.2. Extraction of polar-soluble crude extract from defatted sesame seeds Both white and gold sesame seed powders (100 g) were defatted using acetone as a solvent in which fats were dissolved. Briefly, acetone was added (1:4, w/v) to the sesame powder, which was extracted for 24 h at room temperature. Next, acetone–sesame powder mixture was filtered and the residue was washed with diethyl ether. The obtained dried defatted powder was extracted with 70% ethanol (1:10, w/v) for 1 h at room temperature; the extraction was performed three times to obtain a better yield. The polar-soluble extract obtained was concentrated by ethanol evaporation using a rotary evaporator and remaining aqueous solution was lyophilized to obtain white or gold sesame polar-soluble crude extract powder. 2.3. DPPH free radical scavenging assay DPPH dissolved in ethanol gives a purple colored solution. Free radical scavenging capacity is determined by the decrease in color intensity of the solution, which was determined by measuring the absorbance at 540 nm. Measurements were made in 96-well microplates; each well contained 240 ll of methanol, 30 ll of DPPH ethanol solution (1 mM), and 30 ll of distilled water (blank), catechin 10–80 lg/ml (standard) or sample (polar-soluble crude extract 2.5–15 mg/ml, HPLC fractions F1 2 mg/ml and F2 5 mg/ ml, butanol-soluble fractions 1–4 mg/ml, or purified water-soluble fraction 2–10 mg/ml). The microplates were then left for 30 min at room temperature and the absorbance was measured at 540 nm using a plate reader (Immuno-Mini NJ-2300; Inter Med, Tokyo, Japan). DPPH free radical scavenging activity was calculated as follows:
DPPH radical scavenging activity ð%Þ ¼
ðA0 Asample Þ 100 A0
307
A0: absorbance at 540 nm of DPPH solution in absence of sample. Asample: absorbance at 540 nm of DPPH solution in presence of sample. 2.4. Oxygen radical absorbance capacity (ORAC) assay The ORAC method consists of measuring the decrease in fluorescence of a protein, fluorescein (FL), as a result of the loss of its conformation by oxidative damage. This method measures the ability of an antioxidant to protect FL from oxidative damage. There are two types of ORAC method according to the reaction environment: - Hydrophilic using phosphate buffered saline (PBS) (H-ORAC). - Lipophilic using 50% acetone (L-ORAC). Considering the nature of our sample, the H-ORAC method was used to investigate antioxidant capacity in the present study. The ORAC method used was as described by Zulueta, Esteve, and Frigola (2009). Briefly, measurements were made in 96-well microplates. Each well contained 50 ll of FL (0.78 lM) and plates were incubated for 15 min at 37 °C with 50 ll of PBS (75 mM, pH 7) as a blank, Trolox (20 lM) as a standard, or different concentrations of white and gold sesame seed samples (polar-soluble crude extract 10–100 lg/ml, butanol-soluble fractions 20–100 lg/ml, or purified water-soluble fraction 100–500 lg/ml). Oxidation was started by adding 25 ll of AAPH radical (221 lM) to each well. The fluorescence was measured immediately after addition of AAPH and measurements were then taken every 5 min for 55 min with a plate reader (InfiniteÒ M200; Tecan Inc., Männedorf, Switzerland) using fluorescence filters for an excitation wavelength of 485 nm and an emission wavelength of 535 nm. ORAC values, expressed as lM Trolox equivalent, were calculated by the following formula:
ORAC ðlM TEÞ ¼
C Trolox ðAUCSample AUCBlank Þ ðAUCSample AUCBlank Þ
where CTrolox is the concentration (lM) of Trolox and AUC is the area below the fluorescence decay curve of the sample, Trolox, or blank. 2.5. Separation of polar-soluble crude extract by gel filtration HPLC Polar-soluble crude extracts of white and gold sesame were dissolved in water (10 mg/ml) and filtered using 0.45 lm filters. Samples were analyzed by an HPLC system equipped with an Intelligent HPLC pump system (880-PU; Jasco, Tokyo, Japan), and an Intelligent Refractive Index Detector (830 RI; Jasco). White and gold sesame polar-soluble crude extracts were separated using a YMC-PACK Diol 60 gel filtration column (5 lm, 300 8.0 mm; YMC Co., Ltd., Kyoto, Japan). MilliQ water was used as the mobile phase at a flow rate of 0.5 ml/min. A refractive index detector was used to detect the different saccharide fractions. 2.6. Purification of water-soluble fraction by butanol extraction White and gold sesame seed polar-soluble crude extracts (1 g) were dissolved in 50 ml of distilled water, the same volume of butanol was added, and subjected to extraction and phase separation using a separating funnel. This extraction process was repeated 3 times to obtain the butanol-soluble fraction. The remaining water-soluble fraction was extracted according to the same process with butanol to obtain 3 more supplementary
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butanol extracts designated as +1, +2, and +3 butanol extracts. The final purified water-soluble fraction was recovered with a yield of 46.7% and 51.8% of polar-soluble crude extract for white and gold sesame seed, respectively. The butanol-soluble fraction and butanol extracts were concentrated by evaporation using a rotary evaporator, and dried samples were dissolved in water. All recovered fractions were then lyophilized and stored at 20 °C until further use. 2.7. UPLC–MS analysis of purified water-soluble fraction UPLC–TOF MS (WatersÒ Xevo™ QTof MS; Waters, Milford, MA) was performed using a C18 column (Ø 2.1 mm 100 mm; Waters). The eluents used were water (A) and methanol (B); the elution conditions were 0–3 min, 5% B; 3–18 min, linear gradient to 100% B; 18–20 min, 100% B; the flow rate was 0.3 ml/min and the injection volume was 5 ll. The mass data were collected in negative ionization mode ([MH]). The capillary voltage was 3.0 kV. Cone and desolvation gas flow rates were set at 50 and 1000 l/h, respectively, and the source and desolvation temperature was 150 °C and 500 °C, respectively. Samples used for analysis were purified water-soluble fractions (1 mg/ml) and supplementary butanol extracts (1 mg/ml) from white and gold sesame seeds. The injected sample was first separated by UPLC and then MS of each peak was performed. To identify detected compounds, we referred to MassBank (URL = ‘‘http:// www.massbank.jp’’), a database of mass spectral data, as well as data about antioxidant compounds from sesame seeds reported by Katsuzaki et al. (1994) and Moazzami, Andersson, and KamalEldin (2006). 2.8. Statistical analysis All data were obtained from at least three independent experiments and reported as means ± standard deviation (SD). Statistical analyses were performed using Dunnett’s test for multiple data comparison or Student’s T test. 3. Results and discussion 3.1. Extraction of polar-soluble crude extract from defatted sesame seeds White and gold sesame seed flours recovered as byproducts from sesame oil extraction were completely defatted and then extracted with 70% ethanol to obtain polar-soluble crude extracts. Extraction yields from defatted sesame seed powders were 11.7% and 11.0% for white and gold sesame seeds, respectively. 3.2. Antioxidant activities of white and gold sesame seed polar-soluble crude extracts Methods for measuring the antioxidant capacity in vitro can be divided in two categories according to the chemical reaction involved. The first type is electron transfer (ET) reaction, which includes the DPPH free radical scavenging method. The second type is based on hydrogen atom transfer (HAT) reactions, such as the ORAC method (Zulueta et al., 2009). Both methods were used to evaluate the antioxidant capacities of sesame seed polar-soluble crude extracts in vitro. 3.2.1. DPPH radical scavenging activity DPPH radical scavenging assay, considered as one of the standard and easy colorimetric methods (Mishra, Ojha, & Chaudhury,
2012), is routinely used to evaluate the free radical scavenging potentials of antioxidant molecules. White and gold sesame seed polar-soluble crude extracts were assessed for their DPPH scavenging activity, with catechin taken as a standard antioxidant. As shown in Fig. 1A, catechin exhibited a minimum activity of 18.6% at 10 lg/ml, and its activity increased in a concentration-dependent manner. White and gold sesame seed polar-soluble crude extracts also exhibited concentration-dependent scavenging activity with sample concentration ranges between 2.5 and 15 mg/ml (Fig. 1B). The results also showed that white sesame seed polarsoluble crude extract exhibited higher scavenging activity with 88.18% at 15 mg/ml, whereas gold sesame polar-soluble crude extract exhibited only 64.16% scavenging activity at the same concentration. 3.2.2. Oxygen radical absorbance capacity (ORAC) The ORAC method is attracting interest as a method for evaluation of antioxidant activity as it uses a biologically relevant free radical, i.e., the AAPH-derived peroxyl radical that mimics lipid peroxyl radicals involved in lipid peroxidation chain reaction in vivo (Tai, Sawano, Yazama, & Ito, 2011). In addition, the ORAC result integrates the degree and duration of antioxidant reaction in the same value (Zulueta et al., 2009). Antioxidant capacities of white and gold sesame seed polar-soluble crude extracts were evaluated by the hydrophilic ORAC (H-ORAC) method. Fig. 2A shows the fluorescence intensity decay due to the oxidation of fluorescein (FL) in the presence or absence of sesame seed polarsoluble crude extracts (100 lg/ml). Both white and gold sesame polar-soluble crude extracts delayed the decrease in fluorescence intensity compared to the blank curve suggesting that sesame seed extracts protected FL from oxidative damage by scavenging AAPH free radicals. Using the fluorescence intensity decay curves, the ORAC value is calculated and expressed as lM Trolox equivalent (lM TE). ORAC values for different sample concentrations, 10–100 lg/ml, are illustrated in Fig. 2B. Both seed type extracts exhibited concentration-dependent activity. White sesame seed polar-soluble crude extract still exhibited ORAC of 4.22 lM TE at 10 lg/ml, whereas gold sesame seed extract exhibited no activity at the same concentration. Therefore, as previously observed in the DPPH assay, ORAC assay results showed that white sesame seed polar-soluble crude extract exhibits higher antioxidant activity than gold sesame seed extract. The U.S. Department of Agriculture published a database for the ORAC of selected foods in 2010, in which both hydrophilic and lipophilic ORAC as well as total ORAC are expressed as lmol of Trolox equivalent (TE) per 100 g (U.S. Department of Agriculture, 2010). To compare our samples with some selected foods, we calculated the average ORAC values as lmol TE/100 g of polar-soluble crude extracts, which were 34,720 and 21,700 lmol TE/100 g for white and gold sesame seed polar-soluble crude extracts, respectively. H-ORAC values of white and gold sesame seed polar-soluble crude extracts were compared to H-ORAC values of selected foods reported in the USDA database. The results showed that sesame seed polar-soluble crude extract had a higher H-ORAC value than blueberries (4633 lmol TE/100 g), almond nuts (4282 lmol TE/ 100 g), soybean seeds (5409 lmol TE/100 g), and also rice bran (8817 lmol TE/100 g). Thus, sesame seed polar-soluble crude extract contains strong antioxidant compounds. On the other hand, H-ORAC values of our extracts were lower than those of some spices that showed high ORAC values, such as ground cinnamon (143,264 lmol TE/100 g) and cumin seeds (47,600 lmol TE/ 100 g), while other spices had ORAC values close to our samples, such as curry powder (24,981 lmol TE/100 g) and mustard seeds (28,759 lmol TE/100 g). This comparison suggests that the
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A % DPPH scavenging activity
100 90
**
80 70
**
60 50
**
40 30 20 10 0 10
20
40
80
Catechin concentration (µg/ml)
B
Fig. 1. DPPH radical scavenging activities of catechin (A), and the white and gold sesame seed polar-soluble crude extracts (B). Results are averages of 5 independent experiments ± standard deviation (SD). Statistical analysis was done by Dunnett’s test, ** or ##: p < 0.01 compared to each sample lowest concentration.
obtained polar-soluble crude extracts recovered from defatted sesame seeds possess good ORAC activity. Unfortunately, sesame seeds and derivative products were not reported in the database, which would have been interesting for comparison with our samples. Ishiyama, Nagashima, Yasumoto, and Fukuda (2006) reported ORAC measurement of two sesame seed varieties from Japan Kanto No. 1 and Gomazou. ORAC values of methanol extracts from both sesame seeds were 658.3 and 827 mg TE/100 g of sesame seed for Kanto No. 1 and Gomazou, respectively. These values correspond to 2630 and 3304 lmol TE/100 g of sesame seed. According to these results, we can conclude that hydrophilic antioxidant compounds remaining in defatted sesame seed after oil extraction exhibit as important antioxidant capacity as methanol extracts from non-defatted sesame seeds. 3.3. Separation of saccharides fraction by gel filtration HPLC 3.3.1. HPLC analysis In this study, we focused on hydrophilic antioxidants potentially present in the polar-soluble crude extracts from defatted sesame seeds. Therefore, we attempted to separate the polar-soluble crude extracts into different saccharide fractions. Extracts were analyzed by HPLC gel filtration column chromatography (YMCPack Diol 60) and the eluted saccharide fractions were identified using a refractive index detector (RID). Chromatograms of white and gold sesame seed extracts are illustrated in Fig. 3A and B. Both extracts were separated into two main saccharide fractions. The eluted peaks were identified by comparison with standard oligosaccharides separated under the same HPLC conditions (Fig. 3C). The first fraction, designated as F1 was obtained with 17.1% and 17.5% yield from white and gold sesame seed polar-soluble crude extracts, respectively. This fraction corresponded to relatively high molecular weight saccharides, such as polysaccharides or other
complex molecules. The second fraction (F2) was obtained with 73.8% and 75.2% yield from white and gold sesame seed polar-soluble crude extracts, respectively. This fraction consisted of different oligosaccharides ranging from di- to hexasaccharides consistent with the oligosaccharide composition of sesame seeds reported previously as sucrose, raffinose, stachyose, planteose, sesamose, as well as penta- and hexasaccharides (Wankhede & Tharanathan, 1976). 3.3.2. Antioxidant capacity of saccharide fractions F1 and F2 To determine if separated saccharide fractions are involved in the detected antioxidant activity of polar-soluble crude extracts, both F1 and F2 were collected and investigated for their antioxidant capacities using both DPPH radical scavenging and ORAC assays. In the case of DPPH radical scavenging assay (Fig. 4A), samples were collected from HPLC analysis from the same amount of injected polar-soluble crude white and gold sesame seed extracts. The collected F1 fractions (2 mg/ml) exhibited good DPPH scavenging activity with values of 38.62% and 27.57% for white and gold sesame seeds, respectively, whereas the oligosaccharide fraction F2 (5 mg/ml) had very low activity with values of only 6.33% and 2.91% for white and gold sesame seeds, respectively. For ORAC assay, different concentrations of F1 and F2 fractions were investigated (Fig. 4B). F1 fraction exhibited high ORAC values at 50 and 100 lg/ml, corresponding to average values of 50,953 and 54,420 lmol TE/100 g for white and gold sesame seed extracts, respectively. The ORAC values of the F2 fraction were measured at 250 and 500 lg/ml, corresponding to 5347 and 6068 lmol TE/ 100 g for white and gold sesame seed extracts, respectively. Both DPPH radical scavenging and ORAC methods showed that F1 exhibited around 10-fold higher antioxidant activity than the oligosaccharide fraction F2. F1 from both seed types was
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Relative Fluorescence Intensity (%)
A 100 blank standard 80
WS (100 µg/ml) GS (100 µg/ml)
60
40
20
0 0
10
20
30
40
50
60
Time (min)
B
Fig. 2. ORAC of white and gold sesame seed polar-soluble crude extracts. (A) Fluorescence intensity decay curves of fluorescein in the presence of 100 lg/ml of white and gold sesame seed polar-soluble crude extracts. Standard is 20 lM Trolox. (B) ORAC of white and gold sesame seed polar-soluble crude extracts at different concentrations. Results are expressed as mean values ± SD. All results are averages of 4 independent experiments. Statistical analysis was done by Dunnett’s test, * or #: p < 0.05, and ** or ##: p < 0.01 compared to each sample lowest concentration.
characterized by a dark brown color compared to F2, which had a yellowish white color. Xu et al. (2005) isolated brown pigment from the ethanolic extract of black sesame seeds that showed antioxidant activity in vitro. Furthermore, melanoidin-like browning compounds produced during roasting of sesame seeds are also believed to be involved in the antioxidant activity of roasted sesame seed oil (Kumazawa et al., 2003). Thus, the F1 fraction may be related to such compounds. However, neither the structure nor antioxidant mechanisms of such compounds have yet been elucidated. The oligosaccharide fraction F2 showed very low DPPH radical scavenging activity, with values of 6.33% and 2.91% for white and gold sesame seeds, respectively. On the other hand, it had relatively high ORAC values of 5347 and 6068 lmol TE/100 g for white and gold sesame seeds, respectively. These results may be explained by the difference in reaction medium, as the ORAC method uses PBS, which is favorable for hydrophilic compounds such as oligosaccharides. In addition, DPPH free radicals are not similar to the highly reactive peroxyl radicals usually involved in lipid peroxidation in vivo (Huang, Ou, & Prior, 2005). Whereas, AAPH-derived radicals used in the ORAC assay mimic peroxyl radical-induced oxidation (Tai et al., 2011). Therefore, the ORAC assay is a more accurate method for quantifying peroxyl radical scavenging capacity in vitro.
3.4. Purification of sesame seed water-soluble fraction by butanol extraction Defatted sesame seed flour is an important source of oil-insoluble lignan glycosides, such as pinoresinol glucosides and sesaminol glucosides (Moazzami et al., 2006). Such compounds may be contained in the polar-soluble crude extract obtained by extraction with 70% ethanol. This is why polar-soluble crude extracts from white and gold sesame seeds were purified by butanol extraction to remove relatively hydrophobic compounds contained in the crude extract. 3.4.1. Butanol extraction Sesame seed polar-soluble crude extract contained a nonhydrophilic fraction that was solubilized in butanol; 456.2 and 320.7 mg/1 g of crude extract were obtained from white and gold sesame seeds, respectively. Small amounts ranging between 19 and 42 mg/1 g of crude extract were still extracted by butanol in +1, +2, and +3 butanol extracts. The final purified water-soluble fraction consisted of approximately 50% of the crude extract with 467.2 and 517.7 mg/1 g of crude extract from white and gold sesame seeds, respectively. Consistent with our hypothesis, these results indicated that polar-soluble crude extract contained relatively non-hydrophilic
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Fig. 3. HPLC chromatograms of white (A) and gold (B) sesame seed polar-soluble crude extracts and oligosaccharides standard solution (C) analyzed by YMC-Pack Diol 60 gel filtration column using RID. 1: polysaccharides, 2: hexa-, pentasaccharides, 3: tetrasaccharides, 4: trisaccharides, 5: disaccharides, 6: monosaccharides, a: stachyose, b: raffinose, and c: sucrose.
compounds that might be involved in the observed antioxidant activity. Thus, the antioxidant activities of all recovered fractions were assessed using both DPPH radical scavenging and ORAC assays. 3.4.2. Antioxidant capacity of the different fractions recovered from butanol extraction The recovered fractions from butanol extraction of both white and gold sesame seed polar-soluble crude extract were investigated for their antioxidant potential using DPPH radical scavenging and ORAC assays (Fig. 5). DPPH radical scavenging activity (Fig. 5A) was determined for sample concentrations between 1 and 4 mg/ml for the butanol-
soluble fraction as well as supplementary butanol extracts. The butanol-soluble fraction (4 mg/ml) exhibited scavenging activity of 61.66% and 46.54% for white and gold sesame seeds, respectively. The +1 butanol extract exhibited comparable activity with value of 62.12% and 32.29% for white and gold sesame seeds, respectively. However, DPPH scavenging activity gradually decreased for +2 and +3 butanol extracts for both seed types. Butanol-soluble fraction also exhibited the highest activity in the ORAC assay (Fig. 5B) with value of 45.01 and 27.83 lM TE at 100 lg/ml for white and gold sesame seeds, respectively. Supplementary butanol extracts also exhibited activity at similar sample concentration ranges (50 and 100 lg/ml) and activity gradually decreased as observed in DPPH scavenging assay. These results suggest that
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% DPPH scavenging activity
A
100 90 80 70 60 50 40 30 20 10 0 F1 (2 mg/ml)
F2 (5 mg/ml)
F1 (2 mg/ml)
White sesame
F2 (5 mg/ml)
Gold sesame
B
Fig. 4. Antioxidant capacities of HPLC fractions of white and gold sesame seed polar-soluble crude extracts. (A) DPPH radical scavenging activity (%). (B) ORAC at different concentrations of F1 and F2 from white and gold polar-soluble crude extracts. Experiments were performed in triplicate; results are expressed as mean values ± SD. Statistical analysis was done by Student’s T test, * or #: p < 0.05, and ** or ##: p < 0.01 compared to each sample lowest concentration.
100
A
White Sesame
Gold Sesame
% DPPH scavenging activity
90 80 70 60 50 40 30 20 10 0 1 mg/ml 2 mg/ml 4 mg/ml 1 mg/ml 2 mg/ml 4 mg/ml 1 mg/ml 2 mg/ml 4 mg/ml 1 mg/ml 2 mg/ml 4 mg/ml 2 mg/ml 5 mg/ml 10 mg/ml Butanol-soluble fraction
60
+1 butanol extract
B
+2 butanol extract
White Sesame
+3 butanol extract
Water-soluble fraction
Gold Sesame
ORAC (µM TE)
50 40 30 20 10 0 20 µg/ml
50 µg/ml
100 µg/ml
Butanol-Soluble fraction
50 µg/ml
100 µg/ml
+1Butanol extract
50 µg/ml
100 µg/ml
+2 Butanol extract
50 µg/ml
100 µg/ml
+3 Butanol extract
100 µg/ml
200 µg/ml
500 µg/ml
Water-Soluble fraction
Fig. 5. Antioxidant capacities of different fractions recovered from butanol extraction of both white and gold sesame seed polar-soluble crude extracts. (A) DPPH radical scavenging activity (%), (B) ORAC at different concentrations. Results are expressed as mean values ± SD. All results are averages of 4 independent experiments. Statistical analysis was done by Dunnett’s test, all data showed statistical significant difference (p < 0.05) compared to each sample lowest concentration.
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sesame seed polar-soluble crude extracts contained non-hydrophilic antioxidant compounds that were extracted in the butanol-soluble fraction. Such non-hydrophilic antioxidants were still extracted by further butanol extractions, and the decreasing antioxidant activity of these extracts suggested that the remaining purified water-soluble fraction probably contained only hydrophilic components. Antioxidant activity of the purified water-soluble fraction was detected at sample concentrations between 2 and 10 mg/ml for DPPH scavenging assay (Fig. 5A) and between 100 and 500 lg/ml for ORAC assay (Fig. 5B). Both assays showed that the water-soluble fraction exhibited concentration-dependent antioxidant activity, although relatively high sample concentrations were required compared to different butanol extracts. Moreover, similar ORAC assay results were observed for the oligosaccharide fraction F2 (Fig. 4B) suggesting that the purified water-soluble fraction, which is mainly composed of oligosaccharides, also contains some hydrophilic antioxidant compounds. 3.4.3. Identification of potential antioxidant compounds in purified fractions by UPLC–MS White and gold sesame seed purified water-soluble fraction and supplementary butanol extracts (+1, +2, and +3 butanol extracts) were analyzed by UPLC–TOF MS. The mass data was collected in negative ionization mode ([MH]) and we referred to MassBank, a database for mass spectral data, as well as the available literature about antioxidant compounds from sesame seeds reported in previous studies to identify the detected compounds. The results are summarized in Table 1; white and gold sesame seed extracts were found to have similar compositions, so the results are presented independently from seed type. Purified water-soluble fraction analyzed by UPLC was separated into two fractions (Supplementary data 1). The major fraction was eluted between 0.8 and 1.6 min and contained most hydrophilic constituents. According to MS data, the eluted compounds corresponded to oligosaccharides (di- to pentasaccharides), in accordance with previous HPLC analysis of the crude extract (Fig. 3). In addition to oligosaccharides, a peak corresponding to vanillic acid was detected at around 1.5 min. The second fraction was eluted around 5.9 min, and was greater in white than gold sesame seed extracts. Mass data showed that this fraction contained another phenolic acid, ferulic acid. Jeong et al. (2004) reported the presence of both vanillic and ferulic acids in defatted sesame seed meal methanolic extract. However, they specified that ferulic acid was only detected in unroasted sesame seed extract, while vanillic acid appeared in roasted sesame seed extract. As our samples were obtained from sesame oil extraction byproducts, they come from roasted sesame seeds because roasting is a key operation in sesame oil preparation influencing its organoleptic properties as well as its oxidative stability. Our results demonstrated the
presence of both ferulic and vanillic acids in the water-soluble fraction; however, ferulic acid content seemed to be lower than that of vanillic acid. The difference from previous studies could have been due to differences in sample preparation and/or analysis method used. To confirm the contributions of both phenolic acids to the antioxidant activity of purified water-soluble fraction, pure compounds were purchased and their antioxidant activities were evaluated by DPPH free radical scavenging and ORAC assays. Vanillic and ferulic acids exhibited good antioxidant capacities with ORAC assay (Supplementary data 2), and exhibited comparable ORAC values (33.16 and 34.44 lM TE for vanillic and ferulic acid, respectively, at a sample concentration of 2.5 lg/ml) in agreement with the results obtained by Tai et al. (2011); they reported that vanillic and ferulic acids exhibited comparable ORAC results; however, they did not determine their ORAC values and only fluorescence decay curves were compared. On the other hand, vanillic acid showed almost no DPPH radical scavenging effect compared to ferulic acid (10.25% and 77.36% for vanillic and ferulic acid, respectively, at a sample concentration of 500 lg/ml). In fact, both phenolic acids have the same structure involved in radical scavenging reaction. However, ferulic acid, which is relatively more hydrophobic, reacts better in apolar medium compared to vanillic acid. Again, we concluded that ORAC assay, which is more relevant to oxidation chain reaction in vivo, is more appropriate to quantify the antioxidant potential of hydrophilic compounds in vitro. Moreover, sesame seeds were reported to contain both free phenolic acids esters and bound insoluble forms (Neveu et al., 2010), and bound phenolic acids are believed to be cleaved and liberated during roasting of seeds (Jeong et al., 2004). Therefore, vanillic and ferulic acids are more likely to be the bioactive components in the purified water-soluble fractions from both white and gold sesame seeds. When we examined the antioxidant activities of different fractions after butanol extraction, we found that supplementary butanol extracts exhibited decreasing antioxidant activity (+1 butanol > +2 butanol > +3 butanol extract). To better understand this observation, we investigated the compositions of all 3 extracts with UPLC–MS analysis. Several peaks were eluted between 0.8 and 1.8 min; these peaks correspond to oligosaccharides (di- and trisaccharides) and vanillic acid. These hydrophilic compounds were present as minor fractions; a main peak was eluted at 8.93 min corresponding to sesaminol triglucoside; a sesaminol diglucoside peak was also detected at 9.55 min. In addition, UPLC–MS analysis of +2 and +3 butanol extracts showed that sesaminol triglucoside peak intensity decreased gradually and sesaminol diglucoside was not detected in the two extracts (Supplementary data 3). These results suggest that sesaminol glycoside content in supplementary butanol extracts gradually decreased, in a manner that was correlated with the decrease in antioxidant activity.
Table 1 UPLC–MS analysis data of purified water-soluble fraction and +1 butanol extract. Sample
Peak elution time (min)
Molecular ion [MH] (m/z)
Peak assignment
References
Purified water-soluble fraction
0.8–1.6
341.1066 503.1564 665.2094 833.2536 167.0323 193.0488
Disaccharide (sucrose) Trisaccharide (raffinose) Tetrasaccharide Pentasaccharide Vanillic acid Ferulic acid
MassBank MassBank MassBank MassBank MassBank MassBank
341.1036 503.1601 167.0332 855.2600 693.2013
Disaccharide Trisaccharide Vanillic acid Sesaminol triglucoside Sesaminol diglucoside
MassBank database MassBank database MassBank database Katsuzaki et al. (1994) Moazzami et al. (2006)
5.9 +1 Butanol extract
0.8–1.8
8.93 9.55
database database database database database database
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Previous studies indicated that lignan glycosides possess lower radical scavenging activity compared to their corresponding aglycones (Kang et al., 1999). Shyu and Hwang (2002) also reported that sesaminol triglucoside did not show good antioxidant activity as assessed by DPPH radical scavenging and LDL oxidation. However, our results showed that supplementary butanol extracts ORAC was correlated to sesaminol triglucoside content (Fig. 5B and Supplementary data 3). Therefore, sesaminol triglucoside, which is more hydrophilic than sesaminol, apparently will not react appropriately in hydrophobic medium, such as that used for DPPH radical scavenging assay, while it will react well with AAPH-induced radicals in the ORAC method. Finally, the different assays used in the present study showed that extracts from white sesame seed had relatively higher antioxidant capacity than extracts from gold sesame seeds. This was most likely due to the differences in the contents of antioxidants as both seeds types had the same composition. In fact, Moazzami et al. (2007) quantified sesaminol glucosides in 65 different sesame seed samples and found that their contents varied depending on seed sample; however, there were no significant differences between black and white seeds, indicating that sesaminol glucosides contents did not necessarily depend on seed color. Wankhede and Tharanathan (1976) noted that composition of sesame seeds could vary according to growing conditions of sesame plants; as our samples were imported from two different countries (Turkey and Paraguay), this would explain the differences between white and gold sesame seed extracts observed here. 4. Conclusions In this study we showed that purified water-soluble fraction contributed to the polar-soluble crude extract antioxidant activity. Several studies reported that lignan glycosides are the main antioxidants in defatted sesame seeds; here we showed that purified water-soluble fraction contained ferulic and vanillic acids as bioactive antioxidants. As the use of polar solvents such as ethanol and ethyl acetate is considered to secure the dietary safety of the obtained extract, sesame seed polar-soluble crude extracts may be suitable for use as dietary antioxidants. Therefore, further studies on the physiological functions of these extracts are needed to determine the most appropriate use of these extracts. Acknowledgments The authors would like to acknowledge the help with UPLC–MS analysis provided by Professor Tohru Mitsunaga (Gifu University) and the members of his laboratory. We thank Professor Ryo Yamauchi (Gifu University) for helpful discussion and comments. We also thank the members of the Division of Genomics Research, Life Science Research Center, Gifu University, for their support in conducting the experiments. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem. 2014.11.155.
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