Total Phenolic and Phytosterol Compounds and the Radical Scavenging Activity of Germinated Australian Sweet Lupin Flour Rumiyati, Vijay Jayasena & Anthony P. James
Plant Foods for Human Nutrition ISSN 0921-9668 Plant Foods Hum Nutr DOI 10.1007/s11130-013-0377-6
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Author's personal copy Plant Foods Hum Nutr DOI 10.1007/s11130-013-0377-6
ORIGINAL PAPER
Total Phenolic and Phytosterol Compounds and the Radical Scavenging Activity of Germinated Australian Sweet Lupin Flour Rumiyati & Vijay Jayasena & Anthony P. James
# Springer Science+Business Media New York 2013
Abstract In addition to their favourable nutritional profile, legumes also contain a range of bioactive compounds such as phenolic compounds and phytosterols which may protect against chronic diseases including cancer and cardiovascular disease. Germination of some legume seeds has been previously reported to increase the concentration of the bioactive compounds. In this study, the effect of germination of Australian Sweet Lupin (ASL) seeds for 9 days on the concentration of some bioactive compounds and the radical scavenging activity in the resulting flour was determined. The concentration of total phenolic compounds in methanolic extracts of germinated ASL flour was determined using Folin Ciocalteu reagent and phytosterols in oil extracts were analyzed by gas– liquid chromatography. The methanolic and oil extracts were also used to determine radical scavenging activity toward 2,2diphenyl-1-picrylhydrazyl. In the methanolic extracts of germinated ASL flour, phenolic contents and the antioxidant activity were significantly increased following germination (700 and 1400 %, respectively). Analysis of the oil extracts of germinated ASL flour revealed that the concentration of phytosterols and the antioxidant activity were also increased
Electronic supplementary material The online version of this article (doi:10.1007/s11130-013-0377-6) contains supplementary material, which is available to authorized users. Rumiyati Faculty of Pharmacy, Universitas Gadjah Mada, Sekip Utara, Yogyakarta, Indonesia 55281 V. Jayasena : A. P. James (*) School of Public Health, Faculty of Health Sciences, Curtin University of Technology, GPO Box U1987, Perth, Western Australia 6845, Australia e-mail:
[email protected] V. Jayasena Centre for Food & Genomic Medicine (CFGM), Western Australian Institute for Medical Research, 50 Murray Street, Perth, Western Australia 6000, Australia
significantly compared to ungerminated ASL flour (300 and 800 %, respectively). The relative proportion of phytosterols in germinated ASL flour was: β-sitosterol (60 %), stigmasterol (30 %) and campesterol (10 %). Germination increases the concentration of bioactive compounds and the radical scavenging activity in the germinated ASL flour. Keywords Phenolic compounds . Phytosterol . Radical scavenging activity . Australian Sweet Lupin (ASL) . Germination Abbreviations ASL DPPH DPPH-RSA DM FID GC GAE RT RH TPCs
Australian Sweet Lupin 2,2-diphenyl-1-picrylhydrazyl DPPH-radical scavenging activity Dry matter Flame ionization detection Gas chromatography Gallic acid equivalent Retention time Relative humidity Total phenolic compounds
Introduction Lupin is a legume that contains high protein, dietary fiber, micronutrients and bioactive compounds [1, 2]. Three main cultivated species of lupin are Lupinus angustifolius or Australian Sweet Lupin (ASL), Lupinus albus and Lupinus luteus. ASL contains a lower concentration of anti-nutritional factors than other lupin species and is consequently more favoured in the human diet [3]. There are a number of bioactive compounds in lupins, such as isoflavones [3, 4] and phytosterols
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[5]. Consumption of lupin containing these bioactive compounds may therefore have an important role in lowering cholesterol levels and antioxidant activity. Given the potential health benefits of consumption of these compounds, and the favourable nutritional profile of lupin, attempts have been made to incorporate lupin into various food products including noodles [6], pasta [7], bread [8] and muffins [9]. Germination of seeds involves a variety of reactions including synthesis, degradation and transformation of biomolecules during transformation of seeds into a plant. Germination process results in modifying the chemical composition of the seeds. Indeed the relative proportions of the macronutrients, such as protein, lipid and carbohydrate, in ASL are changed during germination [10]. Furthermore the concentration of antinutritional factors such as phytate, trypsin inhibition, α-galactoside and alkaloids in lupins and legumes are diminished by germination [3, 11, 12]. There is some limited evidence that the concentration of bioactive compounds increases during germination, with two studies reporting an increase in total polyphenol content of lupin during germination [2, 13]. However there is no information on the effect of lupin germination on the concentration and composition of other bioactive compounds such as phytosterols in ASL. An improvement in the concentration and activity of bioactive compounds in ASL after germination, in addition to the previously reported improvements in macronutrient composition, would be expected to further promote the advantages of the use of germinated ASL as an ingredient in a range of food applications. Germinated ASL flour could be used as an innovative food ingredient with more health benefits than the ungerminated ASL flour. The objective of this study is to determine whether germination affects the concentration of a range of bioactive compounds and the radical scavenging activity of the methanolic extract and oil in ASL flour.
Material and Methods Plant Material ASL (Lupinus angustifolius) seeds grown at Wongan Hills Research Station, Department of Agriculture and Food, Western Australia and harvested in 2005 were used for the study. Following harvest the seeds were stored between 9 and 11 °C prior to being used for experiments.
hypochlorite. The disinfected ASL seeds were soaked in 2 L of distilled water at room temperature, in the dark, for16 h. Following this the soaking water was decanted and the seeds were rinsed and spread on dishes lined with moistened paper towels. The dishes were then covered with wet paper towels and placed on trays in an incubator set at 25 °C and relative humidity (RH) 90–95 %. The seeds in each dish were germinated for various lengths of time ranging from 1 to 9 days. During the germination period, the seeds were watered twice per day with distilled water. At the appropriate time point, the seeds were harvested and separated by hand into dehulled sprouts, hulls, rotten sprouts and non germinated seeds, and then dried in an oven at 50 °C. The dried dehulled sprouts were then ground into powder using a coffee grinder (DeLonghi) and passed through a 0.5 mm sieve. The flour obtained was stored in the dark at 4 °C prior to analyses. This germination process was repeated three times for each of the time points in order to produce three separate batches of flour for analyses. Preparation of Methanolic and Oil Extracts of Germinated ASL Flour Methanolic and oil extracts were prepared to separate the bioactive compounds contained in the lupin based on their solubility. The methanolic extract which contains the majority of the phenolic compounds was prepared and used for total phenolic analysis and antiradical assay. The oil extracted was prepared for analysis of phyosterols and their radical scavenging activities. Methanolic extracts were prepared according to the method used by Zielinski [14] with some modifications. Samples were extracted with 80 % methanol by shaking in a waterbath for 2 h at room temperature. The mixture was then centrifuged at 4000 rpm for 15 min. The supernatant was filtered and then evaporated in a rotary evaporator to concentrate the content of bioactive compounds. The residue was cooled and frozen until further analyses. The oil was extracted by petroleum ether using a Buchi E816 Soxhlet extraction unit (Switzerland). Weighed samples were placed into a thimble, and put on pre-weighed extraction cups, placed on the extraction chamber in the soxhlet, and extracted by petroleum ether for 90 min. The extracted oil was kept for further analyses. Analysis of Total Phenolic Compounds
Seed Germination Prior to germination, lupin seeds (250 g) were soaked in 1 L of water containing 0.07 % (w/v) sodium hypochlorite for 30 min to limit microbial growth, followed by rinsing with distilled water until a neutral pH resulted. Any equipment used in the germination process was also treated with sodium
Total phenolic compounds (TPCs) in the methanol extract were analyzed using a Folin-Ciocalteau method [15]. A sample (0.5 mL) of extracted solutions was mixed with 2.5 mL of 10-fold diluted Folin-Ciocalteu reagents solution and the mixture was subsequently mixed with 2 mL of 7.5 % Na2CO3. After incubation at 45 °C for 15 min for blue colour
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development, the absorbance was determined at 765 nm. The TPCs were expressed as gallic acid equivalent (mg of GAE/g dried sample).
internal standard in each sample was applied to correct the calculation. Statistical Analysis
Analysis of DPPH Radical Scavenging Activity DPPH radical scavenging activity (DPPH-RSA) of extracts was estimated using the method of Brand-Williams et al. [16]. The methanolic extracts of 0.1 mL were added to 3.9 mL DPPH radical methanolic solution (25 mg/L). After incubation for 30 min, in the dark, and at room temperature, the absorbance at 515 nm was recorded using spectrophotometer. The blank reference was methanol. The ability of components in the oil extract to scavenge DPPH radicals was measured according to the method of Ramadan and Moersel [17]. Briefly oil samples (1 mg) were diluted in 100 μL toluene and then mixed with 390 μL tolueneic solution of DPPH radicals (25 mg/L). Absorbance was measured after incubation for 30 min, in the dark at room temperature using spectrophotometer at 515 nm against a blank of toluene. Phytosterol Analysis Phytosterol contents in sample were measured by a modified method of Jiang and Wang [18]. The protocol consists of three main steps including saponification of the oils, extraction of unsaponifiable matter and analysis using gas chromatography (GC). Saponification was done by mixing oil (approximately 0.1–1 g) with 3 mL of 50 % KOH, 3 mL of absolute ethanol and internal standards (5-α-cholestan-3β-ol) in a pre-weighed beaker. The mixture was then heated in a 90 °C in water bath for 2 h. The unsaponifiable matter was then extracted twice with 15 mL of diethyl ether using funnels and the pooled diethyl ether extracts were washed several times with distilled water until a neutral pH resulted. The diethyl ether containing phytosterols were then evaporated under a stream of nitrogen to dryness. The residue resulting from the unsaponifiable extract was dissolved with hexane for GC analysis using a Perkin-Elmer Autosystem XL GC with Phenomenex ZB-1, size 530 μm×30 m, 1.5 μm film thicknesses and equipped with a flame ionization detector (FID). Helium gas was used as carrier at a rate of 1 mL/min and the temperature of the detector and injector were set at 300 °C. The initial oven temperature was held at 70 °C for 4 min and at 270 °C for 30 min. Phytosterol standards (campesterol, sigmasterol and βsitosterol) were used to determine phytosterols present in the samples. For quantification, a calibration curve for each standard was created by plotting between concentrations (x) against area (y). The linear regression obtained from the plot of each standard was employed for calculation of phytosterol concentration present in the samples. Recovery of spiked
Data were analyzed using SPSS for Windows version 18. The effect of germination of the various parameters was assessed using one-way ANOVA. Following Levene’s homogeneity test of variance, the Tukey’s post hoc test was then used to identify which germination time points were different from each other. A 5 % level of significance was applied in the statistical tests, where P-value of less than 0.05 indicated the presence of significant differences.
Results and Discussion Effect of Germination on the Concentration of Total Phenolic Compounds and the Radical Scavenging Activity of the Methanolic Extract It has been previously reported that the nutritional profile of ASL was improved after germination [10]. In addition to the analysis of the changes in macronutrient composition in our previous study, the effect of germination on the concentration of bioactive compounds in ASL was examined in the present study. The concentration of total phenolic compounds was determined in methanolic extracts at various stages of the germination process. In the methanolic extract, germination of ASL for 9 days resulted in a significant increase in the concentration of total phenolic compounds (Table 1). The concentration of TPCs expressed as gallic acid equivalent (GAE) (mg GAE/100 g dried germinated flour) was 95.4±18.9 mg GAE/100 g in ungerminated flour. The concentration steadily increased during germination to a concentration of 788±147.3 mg GAE/100 g at 9 days of germination (700 %; P
