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Bioaccessibility of polyphenols from wheat (Triticum aestivum), sorghum (Sorghum bicolor), green gram (Vigna radiata) and chickpea (Cicer arietinum) as influenced by domestic food processing Gavirangappa Hithamani, and Krishnapura Srinivasan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf503450u • Publication Date (Web): 23 Oct 2014 Downloaded from http://pubs.acs.org on November 4, 2014
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Journal of Agricultural and Food Chemistry
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Bioaccessibility of polyphenols from wheat (Triticum aestivum), sorghum (Sorghum bicolor), green gram (Vigna radiata) and chickpea (Cicer arietinum) as influenced by domestic food processing
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Gavirangappa Hithamani and Krishnapura Srinivasan*
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Department of Biochemistry and Nutrition, CSIR – Central Food Technological Research Institute, Mysore – 570 020, India
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Running Title: Bioaccessibility of polyphenols from cereals and legumes
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-----------------------------*Corresponding author E-mail:
[email protected]; Fax # +91-821-2517233; Tel # +91-821-2514876
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--------------------------------------------------------------------------------------------------------------------ABSTRACT
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Cereals (wheat and sorghum) and legumes (green gram and chickpea) commonly consumed in
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Asia and Africa were evaluated for the polyphenolic content. Bioaccessibility of polyphenols
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from these grains as influenced by domestic processing was also estimated. Total polyphenol
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content of wheat and sorghum was 1.20 and 1.12 mg/g respectively, which was increased by
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49% and 20% respectively, on roasting. In contrary, a significant reduction of the same was
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observed in both the cereals after pressure-cooking, open-pan boiling and microwave heating.
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Total flavonoids that was 0.89 mg/g in native sorghum, reduced drastically after processing.
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Tannin content of both the cereals significantly increased on sprouting as well as roasting. Total
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polyphenol content reduced by 31% on sprouting but increased to 24% on roasting in green
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gram. Pressure-cooking (53%), open-pan boiling (64%) and microwave heating (>2-fold
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increase) significantly increased total polyphenol content in chickpea, while drastic reduction
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was observed in the total flavonoid content. Bioaccessible total polyphenols from these grains
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were in the order: green gram > chickpea > wheat > sorghum. Domestic processing of these
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grains had minimal/ no effect on the bioaccessible total flavonoid content. Not all the phenolic
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compounds present in them were bioaccessible. Concentration of bioaccessible phenolic
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compounds increased especially on sprouting and roasting of these grains, except chickpea,
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where sprouting significantly reduced the same (476 to 264 µg/g). Microwave heating
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significantly enhanced the concentration of bioaccessible polyphenols especially from chickpea.
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Thus, sprouting and roasting provided more bioaccessible polyphenols from the cereals and
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legumes studied.
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KEYWORDS: Bioaccessibility, Polyphenols, Domestic processing, Wheat, Sorghum, Green
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gram, Chickpea 2
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Journal of Agricultural and Food Chemistry
INTRODUCTION
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Phenolic compounds are popular phytochemicals found in plants known for their potential
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health effects. Indeed, polyphenols are the most abundant antioxidants in diet. Challenges for
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research on polyphenols from food with respect to bioavailability, metabolism, and cellular and
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molecular mechanisms have been recently reviewed.1
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Cereals and legumes, which form staple foods for the majority of Asian and African
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population, contain these phenolic compounds and have found immense applications in
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functional foods.2,3 Wheat (Triticum aestivum) being the most widely cultivated cereal in the
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world contributing to 27% of total cereal production,4 serve as one of the important protein
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source for human population. Wheat flour and wheat bran are extensively used as ingredients in
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dietary fiber rich ready-to-eat breakfast cereals. Phytochemical content and antioxidant activity
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of different varieties of wheat have been reported.5 Some varieties of sorghum (Sorghum
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bicolor), which are drought-resistant staple food crop of semi-arid regions of Africa and Asia are
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found to contain high amounts of phenolic compounds compared to other cereals.6,7 Many
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epidemiological studies have correlated consumption of whole-grain cereals with a reduced risk
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of developing colonic and breast cancer, type-2 diabetes and coronary heart disease.8-10
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Legumes, though secondary to cereals in terms of consumption, are important as protein
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supplements to cereals, play an important role in human nutrition. Chickpea (Bengal gram, Cicer
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arietinum) is the second most important legume in the world.4 Green gram (Mung bean; Vigna
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radiata), is reported to be a good source of carbohydrates, proteins and minerals.
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Antiproliferative effects of legumes have been associated with the presence of phenolic
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compounds.11-13
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In order to exert their health beneficiary effects, the polyphenols from these cereals and
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legumes should be bioavailable. The bioavailability depends on the release of these compounds
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from the food matrix which is referred as bioaccessibility. It is suggested that the gastro-
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intestinal tract may act as an extractor where polyphenols are progressively released from the
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solid matrix and made available for the absorption or to exert their biological effects in the
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gastro-intestinal tract.14 Cereal grains and legumes generally undergo different types of
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processing during food preparation, depending on the food culture and taste preferences.
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Information on the effect of food processing on the polyphenol content as well as their
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bioaccessibility is very scarce. We have recently reported the effect of domestic processing on
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the polyphenol content and bioaccessibility in finger millet (Eleusine coracana) and pearl millet
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(Pennisetum glaucum).15 Most common forms of domestic processing are sprouting, roasting,
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pressure-cooking, open-pan boiling and microwave heating, which may bring about several
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changes in the nutritional quality of food. Hence, the present investigation was carried out to
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determine the extent to which domestic processing influence phenolic profile and
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bioaccessibility of polyphenols from commonly consumed cereals – wheat and sorghum and
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legumes – green gram and chickpea.
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MATERIALS AND METHODS
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Materials.
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Sorghum (Sorghum bicolor L. Moench), wheat (Triticum aestivum L.), green gram (Vigna
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radiata L.), and chickpea (Cicer arietinum L.) were procured from the National Seeds
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Corporation (Mysore, Karnataka, India). Standard phenolic compounds, pepsin, pancreatin and
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bile extract (porcine origin), were procured from Sigma-Aldrich Chemical Co. (St. Louis, MO,
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USA). HPLC grade solvents were from Qualigens Chem. Co. (Mumbai, India). All other
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chemicals and reagents used were of analytical grade.
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Processing of grain samples. A known amount of grain samples was subjected to various types of domestic processing in
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triplicates as described below:
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Sprouting: After soaking the grains overnight (30 g in 90 mL), water was decanted; samples
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were allowed to germinate for a period of 48 h under ambient conditions (25 ºC), while keeping
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them wet by intermittently spraying water. The sprouted grains were dried under shade,
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powdered and used for extraction.
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Heat processing:
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(i) Pressure cooking: Powdered grain samples were pressure-cooked (15 p.s.i.) in triple distilled water (10 g in 30 mL) for 15 min.
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(ii) Open-pan boiling: Powdered grain samples were boiled in triple distilled water (10 g in 50 mL) on a hot plate for 10 min.
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(iii) Microwave heating: Powdered grain samples were microwave heated in triple distilled water
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(10 g in 50 mL) for 4 min in a household microwave system (SAMSUNG Trio, Combi-
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CE1031LAT; Samsung Electronics Co. Ltd., Suwon, Korea) at 450 W. Cooked samples
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were used for further studies.
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(iv) Roasting: Known quantity of each sample (30 g) was roasted on preheated acid washed sand
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at 150 ºC ± 2 ºC for 5 min (until the sample gave a characteristic aroma and color). Roasted
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sample was cooled to room temperature, powdered and stored in air-tight pouches in dark at
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4 ºC until future studies.
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Analysis of Polyphenols.
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Extraction of polyphenols
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Extraction of polyphenols was carried out by refluxing the grain samples (2 g) in acidified
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methanol for 2 h.16 Total polyphenols, total flavonoids and tannins were analyzed in the filtered
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extracts as described below.
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Total polyphenols Total polyphenol content was estimated by Folin-Ciocalteu method as described by Singleton
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et al.,17 with slight modifications. Briefly, an aliquot of acidified methanolic extract was
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appropriately diluted with water to 3.0 mL, mixed with 1 mL each of 1N Folin–Ciocalteu reagent
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and 20% sodium carbonate and incubated for 30 min at room temperature. The absorbance was
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recorded in a spectrophotometer (Model UV-1800, Shimadzu Corporation, Kyoto, Japan) and
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compared with those of known standard gallic acid concentrations (R2 = 0.999). The total
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polyphenol content was computed as mg equivalent gallic acid per g of sample.
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Total flavonoids Total flavonoid content was determined according to the method of Zhishen et al.18 Acidified
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methanolic extract (0.1 mL) was diluted with 4.9 mL of distilled water and mixed with 0.3 mL of
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(5% w/v) NaNO2. After 5 min, 0.3 mL of (10% w/v) AlCl3 and 2 mL of 1 M NaOH were added,
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and diluted with distilled water to a total volume of 10 mL. The mixture was vortexed and the
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absorbance was read at 510 nm. Standard catechin was used to prepare a calibration curve (R2 =
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0.999). The flavonoid concentration was expressed as mg catechin equivalents per g of sample. 6
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Tannins Tannin content was determined by the modified vanillin–HCl method.19 An aliquot of
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acidified methanolic extract was appropriately diluted to 1 mL with distilled water and was
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mixed with 5 mL of vanillin–HCl reagent. The samples were allowed to stand at room
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temperature for 20 min and the colour developed was recorded at 500 nm. The absorbance was
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also recorded for standard catechin solution (R2 = 0.998) and the tannin concentration was
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expressed as mg catechin equivalents per g of sample.
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HPLC analysis
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Phenolic extract of native and processed samples were analyzed by HPLC to obtain a profile
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of individual phenolic compounds.20 Analysis was carried out in a HPLC system (Agilent 1200
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Series; Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a Diode Array detector.
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Filtered samples (20 µL) were analyzed for polyphenols using C18 analytical column (250 × 4.6
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mm; 5 µm; Agilent Technologies Inc., USA) with the mobile phase consisting of 0.1%
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trifluoroacetic acid as solvent A and 100% methanol as solvent B. The total run time was 60 min
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with a flow rate of 1.0 mL/min with the gradient programme as follows: initial B concentration
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of 20% to 40% in 40 min which was maintained for 10 min and then again to 20% B in the next
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five min and 5 min of post-run for reconditioning. Peaks were recorded simultaneously at 280
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and 320 nm.
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Bioaccessibility of polyphenols.
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Bioaccessibility of polyphenols was determined by an in vitro method as described by Luten
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et al.,21 involving simulated gastrointestinal digestion with suitable modifications. Initially,
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simulated gastric digestion was carried out by incubating the powdered samples (10 g) with 7
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pepsin at pH 2.0 and a temperature of 37 °C for 2 h. At the end of gastric digestion, titratable
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acidity was determined in an aliquot of gastric digest as the amount of 0.2M sodium hydroxide
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required to attain a pH of 7.5 in the presence of a mixture of pancreatin and bile extract dissolved
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in 0.1 M sodium bicarbonate (4 g pancreatin and 25 g bile extract per liter).
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Subsequently, intestinal digestion was simulated by suspending segments of dialysis tubing
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(molecular mass cut-off: 10 kDa) containing 25 mL aliquots of sodium bicarbonate solution,
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being equivalent in moles to the titratable acidity (sodium hydroxide needed to neutralize the
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gastric digest) in Erlenmeyer flasks containing the gastric digest and incubated at 37 °C with
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shaking until the pH of the digest reached 5.0. Pancreatin–bile extract mixture (5 mL) was then
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added and incubation was continued for 2 h or longer until the pH of the digest reached 7.0. At
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the end of this simulated gastrointestinal digestion, the dialyzate was analyzed for polyphenols
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by both spectrophotometry and HPLC as described in previous paragraphs. The bioaccessible
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polyphenol present in the grain sample is the dialyzable portion of the total polyphenol which is
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expressed as percent bioaccessibility.
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Statistical analysis.
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All determinations were made in three replicate, and the average values are reported.
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Statistical analysis was carried out using Graphpad INSTAT, Version 3.06, Graphpad software.
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Data was analyzed by applying one-way analysis of variance (ANOVA) and the differences
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between means were assessed by Dunnet’s test and considered significant when P < 0.05.
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RESULTS
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Phenolic extracts of native and variously processed cereals and legumes as well as of
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dialysates obtained after simulated gastric digestion of these samples were analyzed for total
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polyphenols, flavonoids, tannins and individual polyphenol profile.
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Total polyphenol, flavonoid and tannin content of cereal grains.
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Total polyphenol content, total flavonoid content and tannin content of wheat are given in
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Table 1. Total polyphenol content of unprocessed wheat was 1.20 mg/g, which significantly
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increased on sprouting (by 19%) and roasting (by 20%) (P
