Phenolic Compounds in Foods and Natural Health Products

Chapter 4

Antioxidant Activity of Sesame Fractions 1,2

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Fereidoon Shahidi and Chandrika M. Liyana-Pathirana

Downloaded by CORNELL UNIV on October 12, 2016 | http://pubs.acs.org Publication Date: July 21, 2005 | doi: 10.1021/bk-2005-0909.ch004

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Departments of Biology and Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland A1B 3X9, Canada

The total phenolic content (TPC), total antioxidant activity (TAA), free radical scavenging capacity, metal chelation capacity and inhibition of copper-mediated oxidation of low density lipoprotein (LDL) cholesterol of ethanolic extracts of black and white sesame seeds and their hull fractions demonstrated that both the seeds and hulls of black sesame possessed greater antioxidant potential than those of white sesame. The TPC and TAA were highest for black sesame hulls while white sesame seeds were lowest. The results obtained for free radical scavenging, metal chelation and inhibition of LDL oxidation corresponded well with total phenolic content and total antioxidant activity. All the extracts exhibited dose-dependent activity in each antioxidant assay. However, among all the differentfractionsexamined, the black sesame hulls exhibited considerable antioxidant properties and may serve as an excellent source of natural antioxidants for the food and nutraceutical industries.

© 2005 American Chemical Society Shahidi and Ho; Phenolic Compounds in Foods and Natural Health Products ACS Symposium Series; American Chemical Society: Washington, DC, 2005.


34 Currently there is much interest in the phytochemicals such as phenylpropanoids and polyphenols compounds because of their potential health benefits related to their antioxidant, anti-inflammatory and anti-aggregatory properties (7,2). In general, increased consumption of foods of plant origin has been associated with a reduced risk of a variety of chronic diseases (3,4). This has, in fact, been attributed to the presence of phytochemicals such as vitamins, polyphenols and carotenoids, among others that may possess antioxidant and free radical scavenging properties that play a significant role in the etiology of chronic diseases via modulating oxidative damage to cells and biological molecules (5). Different methods have been developed to measure the efficiency of dietary antioxidants either as pure compounds or in food/plant extracts (6). These methods focus on different antioxidant mechanisms including scavenging of superoxide anion and hydroxyl radicals, reduction of lipid peroxyl radicals, inhibition of lipid peroxidation or chelation of metal ions, among others (6). Most assays involve a pro-oxidant, which is usually a free radical and an oxidizable substrate. Thus, the pro-oxidant induces oxidative damage to the substrate that may be inhibited in the presence of an antioxidant. The prooxidants concerned in these methods, in general, are of pathologic importance. Hence, the existence of various harmful pro-oxidants such as 0 '*, H 0 ROO* and *OH in vivo makes antioxidants crucial for the maintenance of a healthy life (7). Thus an antioxidant may efficiently reduce a pro-oxidant subsequently giving rise to products with no or low toxicity (8). In particular, polyphenols compounds of higher plants may act as antioxidants contributing to anticarcinogenic or cardioprotective actions (2). The reactive species, in general, possess the ability to alter chemically, all major biomolecules such as lipids, proteins and nucleic acids with subsequent changes in structure and function leading to various pathologic conditions and/or diseases (9). Dietary antioxidants may exert protection in the body against these reactive species thereby preventing the incidence of these diseases (70). Recently much effort has been paid in the preparation of antioxidants from natural sources by extraction, purification and fractionation. Plants synthesize the well known antioxidants such as tocopherols, ascorbic acid and carotenoids. In addition, plants synthesize substantial amounts of phenolic and polyphenols compounds. Plants use these chemicals to protect themselves against oxidative damage by inhibiting or quenching free radicals and reactive species (77). It has been reported that phenolic and polyphenols compounds in fruits and vegetables are better antioxidants than other common antioxidants such as vitamin C and Ε and contribute more than 80% to the total antioxidant activity (12,13). Sesame (Sesamum indicum L.) is an important crop which provides an excellent source of edible oil (14). Sesame is cultivated on a worldwide basis for both oil and protein where the seed is composed of 55% lipid and 20% protein. Sesame seed hulls contain large amounts of oxalic acid and fiber (75). In general, seed hulls play a major role in physical and chemical defense of the seed 2

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Shahidi and Ho; Phenolic Compounds in Foods and Natural Health Products ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

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(16) and serve a good source of antioxidants (16-18% Chang et al. (19) have demonstrated that sesame hulls possess considerable antioxidant activity, in part due to the presence of phenolic compounds. Kuo et al (16) reported that seeds of the medicinal plant adlay had a moderate antioxidant activity and their hulls exhibited greater antioxidant capacity than other parts of the adlay seed such as testa, bran, and polished seed (20). In oat, most of the phenolic compounds were located in the seed coat/aleurone/sub-aleurone layers. Thus, there was a decreasing concentration of phenolics toward the interior of the seed (20). This study was designed to determine total phenolic content of whole seed and the hull fractions of white and black sesame seeds and to compare the antioxidant activity using different antioxidative assays.

Materials and Methods

Materials The samples of black sesame as such were obtained from a supermarket in Singapore while white sesame seeds were from Egypt. Their hulls were separated by a combined mechanical and aspiration method. The chemicals used were obtained from Sigma Chemical Co. (St. Louis, MO) or Aldrich Chemical Co. (Milwaukee, WI). Solvents used in this study were ACS-grade or better and were purchased from Fischer Scientific Co. (Nepean, ON).

Methods

Preparation ofsamples Sesame seeds were ground in an electric coffee grinder for 10 min. Ground samples were then defatted by blending with hexane (1:5 w/v, 5 min X 3) in a Waring blender at ambient temperature. The resulting slurry was filtered under suction and the residue was air dried for 12 h. The dried defatted meal was stored in vacuum packed polyethylene pouches and kept at -20 °C prior to analysis. Sesame hulls were also defatted in the same manner prior to analysis.


36 Preparation of crude phenolic extracts Phenolic constituents were extracted from both sesame meal and hulls under reflux condition in a thermostated water bath. Phenolic compounds of sesame samples (6 g) were extracted into 80% aqueous ethanol (100 mL) at 70 °C for 30 min. The resulting slurries were centrifuged for 5 min at 4000 X g. The supernatants were collected and the residues re-extracted under the same conditions. The solvent from the combined supernatants was removed under vacuum at 40 °C; the resulting concentrated solutions were lyophilized for 72 h at -49 °C and 46 Χ 10* mbar. Downloaded by CORNELL UNIV on October 12, 2016 | http://pubs.acs.org Publication Date: July 21, 2005 | doi: 10.1021/bk-2005-0909.ch004

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Determination of total phenolic content ofsesame extracts The content of total phenolics was determined according to a modified version of the procedure described by Singleton and Rossi (21). Extracts were dissolved in methanol to obtain a 1 mg/mL solution. Folin-Ciocalteu's reagent (0.5 mL) was added to centrifuge tubes containing 0.5 mL of the extracts. Contents were mixed and 1 mL of a saturated solution of sodium carbonate was added to each tube followed by adjusting the volume to 10 mL with distilled water. The contents in the tubes were thoroughly mixed by vortexing. Tubes were allowed to stand at ambient temperature for 45 min until the characteristic blue color appeared and then centrifuged for 5 min at 4000 X g. Absorbance of the supernatants was recorded at 725 nm. The content of total phenolics in each extract was reported as mg catechin equivalents per gram of extract.

Measurement of total antioxidant activity by Trolox equivalent antioxidant capacity (TEAC) assay Total antioxidant activity was determined according to the procedure described by van den Berg et al (22). The extracts and reagents were prepared in a 100 mM phosphate buffered saline (pH 7.4, 150 mM NaCl) solution (PBS buffer). A solution of 2,2-azinobis-(3-ethylbenzthiazoline-6-sulfonate) radical anion (ABTS* ) was prepared by mixing 2.5 mM 2,2-azobis-(2methylpropionamidine) dihydrochloride (AAPH) with 2.0 mM A B T S in a 1:1 (v/v) ratio, and heating at 60 °C for 12 min. The radical solution was stored at room temperature and protected from light. A standard curve was prepared using different concentrations of Trolox. The reduction in absorbance of the ABTS** solution (1960 μ ί ) at different concentrations of Trolox (40 μ ί ) over a 6 min period was measured and plotted. TEAC values of the extracts (1 mg/mL) were determined in the same way and expressed as μΜ Trolox equivalents. +

2+


37 Scavenging of 1, l-diphenyl-2-picrylhydrazyl (DPPH) radical The method described by Kitts et al. (23) was used to assess the DPPH radical scavenging capacity of sesame extracts. A 100 μΜ DPPH solution in 95% ethanol was mixed with various amounts of sesame extracts (5, 10, 20, 40 μg/mL) and vortexed thoroughly. The mixture was allowed to stand at ambient temperature for 30 min. The absorbance was measured at 519 nm. The scavenging percentage was calculated according to the equation:


Scavenging % = {(Abs

contro

, - Abs

samp

i ) / Abs e

contro

i} X 100.

Determination of iron (II) chelating capacity Solutions of ferrous sulfate (400 μΜ), extracts/ standard were prepared in a 10 mM hexamine-HCl buffer containing 10 mM KC1 (pH 5.0). One milliliter of ferrous sulfate was mixed with 1 mL of extracts/ standard followed by the addition of 0.1 mL of a 1 mM solution of tetramethylmurexide prepared in the same buffer. The final concentration of extracts/ standard in the assay medium was 50 or 100 ppm based on catechin equivalents. Absorbance of the reaction mixture was recorded at 460 and 530 nm and the ratio of A o to A calculated. A standard curve of absorbance ratio versus free iron (II) was prepared. The difference between the total iron (II) and the free iron (II) indicates the concentration of chelated iron (II). Iron (II) chelating capacities of extracts/ standard were calculated using the following equation. 46

5 3 0

Iron (II) chelating capacity, % = {Concentration of chelated iron (II)/ Concentration of total iron (II)} X 100

Inhibition of copper-mediated human low density lipoprotein (LDL) cholesterol Human LDL cholesterol was dialyzed in 10 mM PBS (pH 7.4, 150 mM NaCl) at 4 °C in the dark for 24 h. Human LDL (0.2 mg protein/mL) was mixed with different amounts of sesame extracts (25-100 ppm phenolics). Catechin was used as the reference antioxidant compound. Reaction was initiated by adding a solution of CuS0 (10 μΜ); samples were then incubated at 37 °C for 22 h. The formation of conjugated dienes was measured at 234 nm as described by Hu and Kitts (24). 4


38 Statistical analysis All experiments were carried out in triplicate and the significance of differences among mean values determined at p

Phenolic Compounds in Foods and Natural Health Products

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