Effect of Domestic Cooking on Carotenoids, Tocopherols, Fatty Acids

The phytochemicals and antioxidant activity in lipophilic and hydrophilic (extractable and bound) fractions of lentils before and after domestic cooki...
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Effect of Domestic Cooking on Carotenoids, Tocopherols, Fatty Acids, Phenolics, and Antioxidant Activities of Lentils (Lens culinaris) Bing Zhang,†,§,∥ Zeyuan Deng,*,† Yao Tang,§,#,∥ Peter X. Chen,§,⊥ Ronghua Liu,§ D. Dan Ramdath,§ Qiang Liu,§ Marta Hernandez,§ and Rong Tsao*,§ †

State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China Guelph Food Research Centre, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ontario N1G 5C9, Canada # Key Laboratory of Food Nutrition and Safety, Ministry of Education, Tianjin University of Science and Technology, Tianjin 300457, China ⊥ Department of Food Science, Ontario Agricultural College, University of Guelph, Guelph, Ontario N1G 2W1, Canada

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S Supporting Information *

ABSTRACT: The phytochemicals and antioxidant activity in lipophilic and hydrophilic (extractable and bound) fractions of lentils before and after domestic cooking were investigated. The hydrophilic fractions in lentils contributed much more to the antioxidant activity than the lipophilic fraction. The phenolic content of lentils was mainly composed of extractable compounds. Significant changes (P < 0.05) in carotenoid, tocopherol, total phenolic, and condensed tannin contents of both extractable and bound phenolics fractions, as well as in antioxidant activities, were found in lentils before and after cooking. More specifically, cooking was found to favor the release of carotenoids and tocopherols and flavonols (kaempferol glycosides), but led to losses of flavanols (monomeric and condensed tannin). Whereas reduced flavanols and other phenolic compounds may have negatively affected the antioxidant activity, other components, especially the lipophilic antioxidants, were increased. The present study suggests that incorporation of cooked lentils into the diet will not cause significant loss to the phytochemical antioxidants and thus will retain the potential health benefits. KEYWORDS: lentil, domestic cooking, lipophilic, phenolic, antioxidant activities

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diseases, diabetes, and cancer.15−17 Phytochemicals in legumes could combat oxidative stress in the body by maintaining the balance between oxidants and antioxidants, thus exerting beneficial effects to human health.18 Incorporation of legumes including lentils into Western diets is uncommon but has been often recommended.19,20 There has been a steady increase, however, in lentil consumption in recent years.21 Canada is by far the world’s largest lentil producer, accounting for 25% of the total world lentil output.22 There is therefore strong interest in acquiring a comprehensive understanding of the bioactive phytochemicals including lipophilic (such as fatty acids, carotenoids, and tocopherols) and hydrophilic components (such as extractable and bound phenolics) in lentils to improve and maximize their nutritive value. There are many studies that have reported bioactive components in raw lentils and attributed potential health benefits to the antioxidant activities of hydrophilic phytochemicals such as phenolics.6,23,24 Our recent study also reported the contribution of lipophilic bioactive components such as carotenoids and tocopherols in Canadian lentil cultivars.12 In addition, lentils are usually cooked by a domestic boiling process before consumption. The cooking process not only improves the flavor and palatability of lentils but also affects the bioaccessibility and bioavailability of nu-

INTRODUCTION Legumes are one of the most extensively cultivated crops throughout the world and are consumed as a staple food in many countries, providing ideal protein, carbohydrates (including dietary fibers), fatty acids, minerals, and vitamins complementary to cereal-based diets.1,2 Furthermore, legumes are rich in bioactive phytochemicals, including carotenoids, tocopherols, and phenolic compounds, that impart diverse physiological properties such as antimicrobial, antioxidant, anti-inflammatory, antitumor, and anticarcinogenic effects.3−7 Findings from epidemiological and interventional studies have indicated that legume consumption is positively associated with decreased risk of several chronic diseases, including coronary heart disease, type II diabetes mellitus, cardiovascular diseases, and cancer.8−10 Among legumes, lentils (Lens culinaris) have attracted great interest for their nutritive value in the diets of millions in developed countries due to the demand for healthy foods.11 Lentils are considered to be an excellent dietary source of phytochemicals (including lipophilic and hydrophilic compounds) possessing high antioxidant capacity.7,12 It is well recognized that the health-promoting effects of legumes including lentils are generally associated with the antioxidant activities or free radical scavenging properties of phytochemicals.13,14 Oxidative stress, defined as an imbalance between the production of oxidants or reactive oxygen species (ROS) and their elimination, is considered to be one of the inducing factors involved in various human diseases including cardiovascular diseases, neural disorders such as Alzheimer’s and Parkinson’s © 2014 American Chemical Society

Received: Revised: Accepted: Published: 12585

August 31, 2014 December 1, 2014 December 4, 2014 December 4, 2014 dx.doi.org/10.1021/jf504181r | J. Agric. Food Chem. 2014, 62, 12585−12594

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publication.12 Fatty acid methyl esters (FAME) were prepared using base-catalyzed reactions. Briefly, 10 mg of lipophilic extract was placed in a 15 mL glass tube equipped with Teflon-lined screw cap and dissolved in 80 μL of toluene. One milliliter of NaOCH3/methanol (0.5 N) was then added for methylation at 50 °C for 30 min. After cooling to room temperature, 1 mL of water was added to the solution, and the esters were extracted with 2 mL of hexane. Total Phytochemical Contents by Colorimetric Assays. Total Carotenoid Content (TCC). TCC in the raw and cooked lentil extract was determined according to the method described in our previous publication.12 In brief, the absorbance of extract diluted appropriately and serial dilutions of lutein standard (0.5−10 μg/mL) were measured at 450 nm using a UV−vis plate reader (EL 340, Bio-Tek Instruments Inc., Winooski, VT, USA). The purity of the lutein standard was based on the manufacturer’s information and has not been measured in the present study. The TCC was calculated on the basis of authentic lutein, which is the main carotenoid in lentil, and expressed as micrograms of lutein equivalent per gram of dry weight (DW) of sample. Total Phenolic (TPC), Flavonoid (TFC), and Condensed Tannin Content (CTC). The contents of total phenolics, flavonoids, and condensed tannins in raw and cooked lentil extracts were determined according to our previously reported method.28 Briefly, TPC was measured using Folin−Ciocalteu’s phenol reagent and expressed as milligrams of gallic acid equivalent (GAE) per gram of dry weight of lentil (mg GAE/g DW). TFC was measured by the aluminum chloride (AlCl3) assay by reading the absorbance at 510 nm, and result was expressed as milligrams of catechin equivalents (mg CAE) per gram of DW. CTC was determined according to the vanillin−HCl procedure by reading the absorbance at 500 nm, and the result was expressed as milligrams of catechin equivalents (mg CAE) per gram of DW. Phytochemical Composition by HPLC. Carotenoids and Tocopherols. All-trans-carotenoids, cis-isomers, and tocopherols were separated by HPLC using the method described in our recent publication.12 Identification of these lipophilic compounds was based on matching retention time, UV−vis spectra, and similarity in elution order with the available commercial standards, as well as by comparison with reported data in the literature. Phenolic Compounds. The phenolic compositions of hydrophilic extracts (extractable and bound) in raw and cooked lentils were analyzed according to a previously reported HPLC method.28 Briefly, HPLC separation was done in a Kinetex phenyl-hexyl column (100 × 4.60 mm, 2.6 μm) (Phenomenex Inc., Torrance, CA, USA) with a binary mobile phase consisting of 5% formic acid in water (v/v) (solvent A) and 95% methanol/5% acetonitrile (v/v) (solvent B). The gradient program was set as follows: 0−12 min, 0−20% B; 12−25 min, 20% B; 25−50 min, 20−80% B; 50−55 min, 80−100% B; 55−60 min, 100−0% B. The flow rate was 0.7 mL/min, and the data were collected at 280 nm for phenolic compounds. At least 21 phenolic compounds were identified in the lentil hydrophilic extract, all of which were quantified using individual standard curves of external standards or similar compounds of the same phenolic subgroup generated by plotting HPLC peak areas against the concentrations. For instance, catechin glucoside, catechin gallate, procyanidins, and related flavonoid derivatives were expressed as catechin equivalents; epicatechin glucoside and epicatechin gallate were expressed as epicatechin equivalents. Kaempferol glycosides and quercetin glycosides were quantified with the calibration curve of kaempferol and quercetin, respectively. Antioxidant Assays. DPPH Assay. The antioxidant activities of both lipophilic and hydrophilic extracts (including extractable and bound phenolics) were estimated by DPPH assay.12,28 Briefly, aliquots of 200 μL of methanolic solution of DPPH (350 μM) were mixed with 25 μL of the lipophilic lentil extract diluted 5-fold with methanol or the hydrophilic lentil extract or with a series of Trolox standard solutions (62.5−1000 μM) in a 96-well plate and allowed to react for 6 h at room temperature before the absorbance was recorded at 517 nm. Both lipophilic (DPPH-L) and hydrophilic (DPPH-H) DPPH antioxidant activities were calculated as micromoles of Trolox equivalent per gram of dry weight of lentil (μmol TE/g DW). Oxygen Radical Absorption Capacity Assay for Lipophilic Extract (ORAC-L). The ORAC-L assay was conducted according to existing

trients.25,26 However, available information on cooking and the effects on phytochemicals and antioxidant properties of lentil is rather scarce. Hence, the present study was conducted to deliver a comprehensive investigation on the effects of domestic boiling without presoaking, as is recommended by Pulse Canada,27 on phytochemical compositions (in both lipophilic and hydrophilic fractions) and antioxidant activities of lentils.

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MATERIALS AND METHODS

Plant Materials. Four lentil cultivars and breeding lines received from Saskatchewan Pulse Growers (Canada) on September 26, 2012, were selected for this study. The four selected lentils included two red lentil cultivars, Blaze and Maxim, and two green cultivars, Greenland and Sovereign. The four cultivars used in the present study were cultured at the same time and location. The whole raw lentil samples were used for cooking or ground into fine powder and stored in sealed plastic bags at −4 °C prior to analysis. Domestic Cooking Process. Whole raw lentils (150 g) were rinsed in a strainer with cold running water for 1 min and then shaken for 30 s to remove excess water. The rinsed lentils were transferred to a pot and boiled with 450 mL of deionized water (seed/water, 1:3, w/v) on a Cimarec hot plate (Fischer Scientific Inc., Waltham, MA, USA). The hot plate was set at 500 °C until boiling and then lowered to 280 °C to simmer the lentils with the pot covered. The lentils were tested for firmness after 30 min of cooking by squeezing lentils between two fingers. Lentils are considered cooked when the seeds have little or no resistance to squeezing. The cooked lentils were allowed to cool and then lyophilized with the boiling water. The cooked lentil samples were ground into fine powder and stored in sealed plastic bags at −4 °C prior to analysis. Chemicals and Reagents. 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH), fluorescein disodium (FL), 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH), Folin−Ciocalteu reagent, gallic acid (GA), catechin, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were purchased from Sigma (St. Louis, MO, USA). All standards used in this study were purchased from Sigma. Sodium acetate, ferric chloride hexahydrate, sodium phosphate monobasic, sodium phosphate dibasic, and HPLC grade solvents, including methanol (MeOH), methyl tert-butyl ether (MTBE), hexane, isopropanol, formic acid, and hydrochloric acid (HCl) were purchased from Caledon Laboratories (Georgetown, ON, Canada). All other chemical reagents used were of analytical grade. Sequential Extraction of Raw and Cooked Lentils. The lipophilic fraction of lentils was extracted from 1 g of the finely ground raw or cooked samples by using hexane/isopropanol (3:2, v/v) as described in our recent publication.12 The lipophilic extract was then evaporated under nitrogen stream at room temperature, redissolved in 1 mL of DMSO, and stored at −20 °C before use for different analyses. Subsequently, the residue from lipophilic extraction was air-dried under room temperature and extracted with aqueous methanol to obtain the hydrophilic extractable fraction. In brief, the dried residue was extracted with 20 mL of 70% methanol in 0.1% HCl (v/v) overnight (ca. 15 h) at room temperature with constant rolling on a rotary shaker (Scientific Industries Inc., Bohemia, NY, USA) at 150 rpm and then re-extracted two more times each with 10 mL of the same solvent. The supernatants were combined after centrifugation (Eppendorf centrifuge 5810R, Brinkman Instruments Inc., Westbury, NY, USA), topped up to 40 mL, filtered through a 0.2 μm PTFE membrane filter (VWR International, ON, Canada), and analyzed for extractable hydrophilic phenolics. The resulting residue from this extraction was dried under nitrogen and then hydrolyzed with 2 M NaOH (20 mL) at room temperature overnight on a rotary shaker at 150 rpm. The solution was then acidified to pH 2 with concentrated HCl (ca. 37%, w/w; 12 M) and extracted three times each with 20 mL of ethyl acetate/ethyl ether (1:1, v/v). The organic layer was combined, evaporated to dryness under nitrogen, dissolved in 1 mL of methanol, and analyzed for bound phenolics. Fatty Acid Analysis by Gas Chromatography. The fatty acid compositions of the lipophilic fraction of both raw and cooked lentils were analyzed according to the method described in our recent 12586

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2.01 ± 0.12 d 60.56 ± 2.13 c 1.42 ± 0.09 c 63.99 ± 2.34 cd

0.67 ± 0.04 b 54.96 ± 1.07 b 1.59 ± 0.13 c 57.22 ± 1.24 b

cooked 0.53 ± 0.06 a 0.77 ± 0.04 cd 0.48 ± 0.02 a 7.61 ± 0.25 c 0.35 ± 0.01 a 0.57 ± 0.02 a 0.54 ± 0.04 a 0.09 ± 0.00 a 10.94 ± 0.44 d 12.26 ± 0.14 d

Blaze

0.49 ± 0.03 a 0.73 ± 0.06 bc tr 5.12 ± 0.14 b 0.44 ± 0.02 b 0.61 ± 0.05 ab 0.63 ± 0.03 ab tr 8.02 ± 0.33 b 8.34 ± 0.26 b

raw

1.30 ± 0.08 c 47.94 ± 1.52 a 0.50 ± 0.06 a 49.74 ± 1.66 a

tr 0.66 ± 0.07 ab tr 7.11 ± 0.16 c 0.80 ± 0.04 d 0.83 ± 0.09 c 0.96 ± 0.05 d 0.13 ± 0.02 b 9.69 ± 0.43 c 11.56 ± 0.21 c

raw

Maxim

3.74 ± 0.21 f 57.45 ± 1.74 bc 0.58 ± 0.10 ab 61.77 ± 2.05 c

0.60 ± 0.04 ab 0.81 ± 0.03 cd 0.55 ± 0.04 ab 9.19 ± 0.26 d 1.10 ± 0.05 e 1.19 ± 0.08 d 0.84 ± 0.05 c 0.13 ± 0.00 b 12.71 ± 0.55 e 15.88 ± 0.16 f

cooked

0.81 ± 0.05 b 65.19 ± 2.33 d 0.67 ± 0.08 b 66.67 ± 2.46 d

cooked

1.59 ± 0.11 c 83.04 ± 4.16 e 0.63 ± 0.05 b 85.26 ± 4.32 e

0.65 ± 0.06 b 0.75 ± 0.02 bc 0.77 ± 0.05 d 10.33 ± 0.31 e 0.99 ± 0.08 e 1.29 ± 0.07 d 1.26 ± 0.10 e 0.29 ± 0.02 e 16.33 ± 0.61 f 24.50 ± 0.32 g

Greenland

0.51 ± 0.03 a 0.61 ± 0.01 a 0.62 ± 0.04 bc 9.22 ± 0.19 d 0.67 ± 0.05 c 0.91 ± 0.02 c 1.00 ± 0.04 d 0.23 ± 0.01 d 12.77 ± 0.39 e 16.28 ± 0.28 f

raw

0.53 ± 0.03 a 45.68 ± 1.98 a 0.59 ± 0.03 ab 46.80 ± 2.04 a

cooked

2.57 ± 0.18 e 55.60 ± 1.61 b 0.66 ± 0.09 b 58.83 ± 1.88 b

0.55 ± 0.02 a 0.84 ± 0.02 d 0.65 ± 0.04 c 6.91 ± 0.13 c 0.36 ± 0.02 a 0.69 ± 0.05 b 0.84 ± 0.05 c 0.19 ± 0.01 c 11.03 ± 0.34 d 14.71 ± 0.24 e

Sovereign

tr 0.70 ± 0.05 b tr 4.30 ± 0.11 a 0.33 ± 0.02 a 0.69 ± 0.07 b 0.70 ± 0.03 b 0.16 ± 0.00 b 6.88 ± 0.28 a 7.24 ± 0.10 a

raw

a

Values are the mean ± SD, n = 3. tr, trace, below detection limit. Values followed by different letters in the same row are significantly different (P < 0.05). bSum of concentrations of all detected carotenoids. cValues are from spectrophotometric analysis.

carotenoids 15-cis-lutein 13-cis-lutein 13′-cis-lutein all-trans-lutein all-trans-zeaxanthin 9-cis-lutein 9′-cis-lutein 9-cis-zeaxanthin total carotenoids index (TCI)b total carotenoids content (TCC)c tocopherols α-tocopherol γ-tocopherol δ-tocopherol total tocopherols

lipophilic phytochemical

lentil cultivars

Table 1. Effect of Domestic Cooking on the Lipophilic Phytochemical Contents (μg/g DW) of Lentil Cultivarsa

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Table 2. Effect of Domestic Cooking on the Fatty Acid Composition (Relative Percent) of the Lipophilic Extracts from Lentil Cultivarsa lentil cultivars Blaze

Maxim

Greenland

Sovereign

fatty acid

raw

cooked

raw

cooked

raw

cooked

raw

cooked

14:0 15:0 16:0 16:1 17:0 17:1 18:0 18:1 19:0 18:2n-6 20:0 20:1 18:3n-3 21:0 20:2n-6 22:0 20:3n-6 22:1 23:0 24:0 20:5n-3 24:1n-9 22:3n-6 26:0 22:5n-3 Σ SFA Σ MUFA Σ PUFA Σ UFA

0.28 ± 0.01 a 0.19 ± 0.01 ab 14.12 ± 0.12 d 0.34 ± 0.03 b 0.15 ± 0.01 b 0.16 ± 0.00 b 1.87 ± 0.05 d 15.36 ± 0.09 a 0.05 ± 0.00 b 49.30 ± 0.85 e 0.58 ± 0.04 bc 0.91 ± 0.04 a 14.00 ± 0.21 e 0.27 ± 0.01 b 0.14 ± 0.01 b 0.60 ± 0.05 c 0.03 ± 0.00 b 0.24 ± 0.05 b 0.23 ± 0.01 c 0.31 ± 0.03 bc 0.22 ± 0.01 d 0.07 ± 0.00 c 0.08 ± 0.00 c 0.43 ± 0.01 b 0.08 ± 0.01 c 19.08 ± 0.35 e 17.08 ± 0.21 a 63.85 ± 1.09 d 80.93 ± 1.30 ab

0.39 ± 0.02 bc 0.22 ± 0.01 b 14.19 ± 0.14 d 0.42 ± 0.05 c 0.15 ± 0.01 b 0.25 ± 0.01 c 1.97 ± 0.06 d 15.78 ± 0.21 b 0.08 ± 0.00 c 48.65 ± 1.03 de 0.60 ± 0.01 c 0.90 ± 0.02 a 12.61 ± 0.10 d 0.32 ± 0.02 c 0.12 ± 0.01 b 0.65 ± 0.05 c 0.04 ± 0.00 b 0.19 ± 0.01 b 0.25 ± 0.03 c 0.35 ± 0.03 c 0.03 ± 0.00 b 0.05 ± 0.01 b 0.11 ± 0.02 d 1.27 ± 0.08 c 0.21 ± 0.02 e 20.44 ± 0.46 f 17.59 ± 0.31 a 61.77 ± 1.18 d 79.36 ± 1.49 a

0.48 ± 0.03 d 0.17 ± 0.01 a 12.21 ± 0.10 b 0.34 ± 0.01 b 0.20 ± 0.01 c 0.17 ± 0.00 b 1.59 ± 0.07 c 25.30 ± 0.51 c 0.03 ± 0.00 a 44.64 ± 0.78 c 0.63 ± 0.05 c 0.88 ± 0.03 a 11.84 ± 0.24 c 0.25 ± 0.01 ab 0.08 ± 0.01 a 0.33 ± 0.01 a 0.01 ± 0.00 a 0.14 ± 0.01 a 0.13 ± 0.01 a 0.21 ± 0.04 a 0.04 ± 0.01 b 0.04 ± 0.00 a 0.01 ± 0.00 a 0.25 ± 0.02 a 0.02 ± 0.00 a 16.48 ± 0.36 ab 26.87 ± 0.56 b 56.64 ± 1.04 c 83.51 ± 1.60 b

0.34 ± 0.02 b 0.17 ± 0.01 a 12.19 ± 0.09 ab 0.17 ± 0.01 a 0.20 ± 0.01 c 0.16 ± 0.01 b 1.68 ± 0.04 c 26.30 ± 0.23 d 0.04 ± 0.01 ab 43.46 ± 1.21 bc 0.53 ± 0.05 ab 0.91 ± 0.07 a 10.64 ± 0.18 b 0.22 ± 0.02 a 0.08 ± 0.02 a 0.45 ± 0.06 b 0.01 ± 0.00 a 0.14 ± 0.01 a 0.16 ± 0.01 b 0.26 ± 0.01 a 0.07 ± 0.00 c 0.03 ± 0.00 a 0.03 ± 0.00 c 1.61 ± 0.10 d 0.15 ± 0.01 d 17.85 ± 0.42 cd 27.71 ± 0.33 b 54.44 ± 1.42 bc 82.15 ± 1.75 ab

0.48 ± 0.05 d 0.19 ± 0.01 ab 11.87 ± 0.22 a 0.15 ± 0.01 a 0.09 ± 0.00 a 0.06 ± 0.01 a 0.96 ± 0.06 a 26.29 ± 0.71 d 0.02 ± 0.00 a 46.21 ± 1.54 cd 0.48 ± 0.04 a 1.02 ± 0.04 b 10.29 ± 0.17 b 0.27 ± 0.05 b 0.08 ± 0.01 a 0.44 ± 0.03 b 0.01 ± 0.00 a 0.16 ± 0.01 a 0.14 ± 0.01 a 0.23 ± 0.01 a 0.01 ± 0.00 a 0.04 ± 0.01 a 0.02 ± 0.00 b 0.46 ± 0.03 b 0.04 ± 0.00 b 15.63 ± 0.51 a 27.72 ± 0.79 b 56.66 ± 1.72 c 84.38 ± 2.51 b

0.44 ± 0.02 cd 0.19 ± 0.01 ab 12.22 ± 0.18 ab 0.14 ± 0.01 a 0.09 ± 0.01 a 0.08 ± 0.01 a 1.00 ± 0.08 ab 28.24 ± 0.46 e 0.04 ± 0.00 b 42.75 ± 1.18 ab 0.49 ± 0.02 a 1.02 ± 0.06 b 9.38 ± 0.20 a 0.30 ± 0.01 bc 0.10 ± 0.02 ab 0.42 ± 0.01 b 0.01 ± 0.00 a 0.16 ± 0.02 a 0.15 ± 0.02 ab 0.26 ± 0.03 ab 0.03 ± 0.01 b 0.04 ± 0.00 a 0.05 ± 0.02 b 2.23 ± 0.15 e 0.18 ± 0.07 de 17.83 ± 0.54 cd 29.68 ± 0.56 c 52.50 ± 1.50 ab 82.18 ± 2.06 ab

0.42 ± 0.01 cd 0.20 ± 0.01 b 12.64 ± 0.13 c 0.17 ± 0.01 a 0.10 ± 0.01 a 0.06 ± 0.01 a 1.11 ± 0.07 b 29.24 ± 0.59 f 0.02 ± 0.00 a 41.82 ± 1.15 ab 0.51 ± 0.02 a 0.93 ± 0.04 a 10.12 ± 0.22 b 0.27 ± 0.01 b 0.09 ± 0.01 a 0.41 ± 0.03 b 0.01 ± 0.00 a 0.15 ± 0.01 a 0.15 ± 0.02 ab 0.27 ± 0.02 ab 0.04 ± 0.00 b 0.03 ± 0.00 a 0.08 ± 0.00 c 1.08 ± 0.12 c 0.08 ± 0.01 c 17.18 ± 0.45 bc 30.58 ± 0.66 cd 52.24 ± 1.39 ab 82.82 ± 2.05 ab

0.38 ± 0.02 b 0.18 ± 0.01 a 12.40 ± 0.17 bc 0.15 ± 0.02 a 0.11 ± 0.01 a 0.09 ± 0.01 a 1.13 ± 0.08 b 29.86 ± 0.65 f 0.03 ± 0.01 a 40.76 ± 1.06 a 0.49 ± 0.02 a 1.04 ± 0.06 b 9.10 ± 0.14 a 0.29 ± 0.02 b 0.09 ± 0.01 a 0.41 ± 0.02 b 0.01 ± 0.00 a 0.14 ± 0.01 a 0.14 ± 0.01 a 0.27 ± 0.03 ab 0.04 ± 0.00 b 0.03 ± 0.00 a 0.09 ± 0.01 c 2.58 ± 0.16 e 0.17 ± 0.03 de 18.41 ± 0.55 d 31.31 ± 0.75 d 50.26 ± 1.25 a 81.57 ± 2.00 ab

a

Data are expressed as the mean ± SD, n = 3. Values followed by different letters in the same row are significantly different (P < 0.05).

protocols described by Li et al.29 Briefly, samples and Trolox standard were prepared in 7% (w/v) randomly methylated β-cyclodextrin (RMCD) to ensure the solubility of the lipophilic antioxidants in the reaction mixture. Twenty-five microliters of blank, Trolox standard, or the lipophilic extract was mixed with 200 μL of fluorescein (0.0868 nM) and incubated for 30 min at 37 °C and then added to 25 μL of AAPH (153 mM). The fluorescence (excitation wavelength, 485 nm; emission wavelength, 528 nm) was measured every minute for about 120 min until it reached zero in a Bio-Tek fluorescence spectrophotometer equipped with an automatic thermostatic holder (PLX 800, Bio-Tek Instruments, Inc.). The results were expressed as micromoles of Trolox equivalent (TE) per gram of dry weight of sample (μmol TE/g DW). ORAC Assay for Hydrophilic Extract (ORAC-H). The ORAC assay for the hydrophilic extracts (both extractable and bound fractions) of lentil (ORAC-H) was conducted according to the method of Li et al.29 The protocol was the same as that described above for the ORAC-L assay except that a phosphate buffer (75 mM, pH 7.4) instead of 7% (w/v) RMCD was used. Statistical Analysis. All assays or tests were conducted in triplicate, and data are expressed as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to compare the means. Differences were considered significant at p < 0.05. All statistical analyses were performed using Statistix for Windows version 9.0 (Analytical Software, Tallahassee, FL, USA).

lentils before and after cooking are summarized in Table 1. The TCC varied from 7.24 to 16.28 μg/g DW in raw lentils and significantly increased (P < 0.05) to 12.26−24.50 μg/g DW after cooking (increases of 37.37−103.18%). The cooking process also caused significant increases in individual carotenoids; for example, increases of 12.04−60.70% were found for the major carotenoid all-trans-lutein among the four varieties after cooking. Another measure for total carotenoids is total carotenoid index (TCI), which is the sum of the concentrations of all detected carotenoids by HPLC. Similar to TCC, TCI also had a 21.80− 37.62% increase in cooked lentils as compared with all tested raw samples (Table 1). These results are the first to be reported for lentils but consistent with findings in other carotenoid-rich foods. For instance, the carotenoid content of tomato was found to be markedly higher after thermal processing.30 Because tocopherols are considered as one of the most effective lipophilic natural antioxidants, changes in concentration of individual and total tocopherols in lentils before and after cooking were determined by analyzing all tocopherol isomers by HPLC. γ-Tocopherol was the predominant isomer in lentils, which accounted for 96.38−97.78% of the total tocopherol content, followed by α- and δ-tocopherols among all tested raw lentils. Cooking caused the largest increase in α-tocopherol level by 187.52−381.78%. Significant increases (P < 0.05) in γtocopherol and total tocopherols, but not in δ-tocopherol level, were also observed after cooking (Table 1). This observation,

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RESULTS AND DISCUSSION Effect of Domestic Cooking on Lipophilic Phytochemicals. Concentrations of total and individual lipophilic antioxidants, namely, carotenoids and tocopherols, in tested 12588

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Table 3. Changes of TPC, TFC, and CTC in the Hydrophilic Extractable and Bound Phenolic Extracts of Lentil before and after Domestic Cookinga TPCb cultivar

TFCb

CTCb

sample

extractable (TPC-E)

bound (TPC-B)

extractable (TFC-E)

bound (TFC-B)

extractable (CTC-E)

bound (CTC-B)

Blaze

raw cooked

4.26 ± 0.18 bc 3.78 ± 0.12 a

0.29 ± 0.01 f 0.16 ± 0.00 c

0.76 ± 0.04 ab 0.68 ± 0.06 a

0.22 ± 0.01 e 0.05 ± 0.00 b

2.76 ± 0.16 c 1.62 ± 0.12 a

0.10 ± 0.00 e 0.01 ± 0.00 a

Maxim

raw cooked

6.96 ± 0.36 e 5.58 ± 0.18 d

0.27 ± 0.02 f 0.17 ± 0.01 cd

1.24 ± 0.08 c 1.16 ± 0.04 c

0.18 ± 0.01 d 0.10 ± 0.01 c

5.82 ± 0.48 e 3.42 ± 0.28 d

0.08 ± 0.01 d 0.03 ± 0.00 c

Greenland

raw cooked

7.80 ± 0.42 f 7.02 ± 0.30 e

0.18 ± 0.00 d 0.11 ± 0.00 a

1.72 ± 0.08 d 1.72 ± 0.12 d

0.03 ± 0.01 a 0.02 ± 0.00 a

6.24 ± 0.42 f 5.52 ± 0.16 e

0.02 ± 0.00 b nd

Sovereign

raw cooked

4.56 ± 0.30 c 4.04 ± 0.12 ab

0.20 ± 0.01 e 0.14 ± 0.01 b

0.80 ± 0.04 b 0.84 ± 0.04 b

0.06 ± 0.01 b 0.05 ± 0.00 b

2.52 ± 0.24 c 1.86 ± 0.08 b

0.03 ± 0.00 c nd

Values are the mean ± SD, n = 3. Values followed by the different letter in the same column are significantly different (P < 0.05). nd, not detected. TPC, total phenolic content, expressed as mg GAE/g DW; TFC, total flavonoid content, expressed as mg CAE/g DW; CTC, condensed tannin content, expressed as mg CAE/g DW. a b

however, is contrary to what was found by others. For instance, Kalogeropoulos et al. reported a 1−2 orders of magnitude decrease in α- and γ-tocopherols realtive to uncooked legumes including lentils.31 These discrepancies could be attributed to different cooking protocols. Kalogeropoulos et al. removed soaking water before cooking and drained excess water after cooking, which is believed to cause significant loss of tocopherols. However, presoaking is not a normally followed cooking practice for lentil. The present study was carried out according to the cooking protocol recommended by Pulse Canada,27 that is, one without presoaking and draining of the boiling water. This suggests that the recommended protocol retains the majority of tocopherols and other bioactive phytochemicals. Results of the present study also suggest that thermal processing such as domestic cooking by boiling can significantly enhance the bioaccessibility of lipophilic bioactives of lentil. Heating is considered to cause cell membranes and cell walls to disrupt, releasing the lipophilic components such as carotenoids and tocopherols from the lentil matrix, thus leading to increased extraction efficiency.30 Effect of Domestic Cooking on Fatty Acid Compositions. Although lentils contain only 1.5−3% oil, its fatty acid composition had a very favorable omega-3 to omega-6 ratio (1:4).12 Heating can potentially cause degradation of fatty acids, especially the essential fatty acids.32,33 The effect of boiling on the fatty acid composition of lentils was investigated, and the results are summarized in Table 2. The lipophilic extracts from raw lentils mainly contained unsaturated fatty acids (UFA) (80.93− 84.38%) including monounsaturated fatty acids (MUFA) (17.08−30.58%) and polyunsaturated fatty acids (PUFA) (52.24−63.85%), and an insignificant amount of saturated fatty acids (SFA). Cooking caused significant increases (P < 0.05) in SFA in all tested lentils, but had no significant effect on MUFA and PUFA. In terms of individual fatty acids, however, the predominant fatty acid 18:2n-6 decreased significantly (P < 0.05), whereas 18:3n-3 decreased only slightly without any statistical significance (P > 0.05) upon cooking. The concentration of the major MUFA 18:1 was markedly higher in cooked lentils (P < 0.05). These results indicate that the majority of PUFA in lentils remain relatively stable after cooking. Possible explanations may include the relatively low temperature of boiling as compared to other thermal processing techniques that

can cause lipid oxidation. It may also be due to the coexistence of tocopherols, which can prevent lipid peroxidation by acting as peroxyl radical scavengers that terminate chain reactions in membranes and lipoprotein particles.34 Effect of Domestic Cooking on Hydrophilic Phytochemicals. TPC, TFC, and CTC in Extractable and Bound Phenolic Extracts. Table 3 shows the total phenolic content, total flavonoid content, and condensed tannin content in the extractable (TPC-E, TFC-E, and CTC-E) and bound phenolic (TPC-B, TFC-B, and CTC-B) fractions from raw and cooked lentils. Significant (P < 0.05) decreases in TPC-E and CTC-E were found in the extractable fraction of cooked lentils, whereas no significant change in TFC-E was observed in the same sample. Decreased TPC-E and CTC-E contents in cooked lentils suggest that losses of condensed tannin might be the contributing factor. The losses of condensed tannin could be well explained by the interactions between tannins and starch molecules. Barros et al. recently reported that condensed tannins could interact with starch (forming insoluble complexes) during cooking, particularly with amylose, thus resulting in significant decreases in extractable phenolic content.35 Interactions between condensed tannins and starch molecules could, however, increase resistant starch content and decrease starch digestibility, thus providing benefits to human health. Bound phenolics in lentils, both raw and cooked, were low compared to extractable phenolics. As shown in Table 3, bound phenolics (TPC-B, TFC-B, and CTC-B) represented only roughly 5% of the respective extractable phenolic fraction. TPCB, TFC-B, and CTC-B varied significantly among tested lentils, ranging from 0.18 to 0.29 mg GAE/g DW, from 0.03 to 0.22 mg CE/g DW, and from 0.02 to 0.10 mg CE/g DW in cooked lentils, respectively. Bound phenolics, especially in TFC-B and CTC-B, were significantly higher in tested red lentil cultivars (Blaze and Maxim) than in the tested green cultivars (Greenland and Sovereign), despite the similar concentrations of extractable phenolics. Results that lentils had only a small portion of bound phenolics do not agree with those reported by Han et al., who found concentrations of bound phenolics were higher than extractable phenolics and the former to be the main contributor to total antioxidant activity in pulse seeds including lentils.20 The discrepancy could be attributed to the different extraction protocol, as well as the genotype of tested lentils. Similar to the extractable phenolics, cooking also significantly decreased the 12589

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19.18 ± 0.59 f 11.09 ± 0.34 g 71.04 ± 2.35 h 7.70 ± 0.21 d 2.03 ± 0.11 b 7.05 ± 0.32 e 41.29 ± 1.65 e 35.41 ± 1.12 e 17.74 ± 0.62 e 4.72 ± 0.23 d 217.25 ± 7.54 f

1.05 ± 0.14 a 3.48 ± 0.36 b 124.13 ± 5.11 bc 63.19 ± 1.98 d 39.42 ± 2.05 b 0.53 ± 0.07 a 4.54 ± 0.22 d 2.51 ± 0.41 ab 0.78 ± 0.05 ab 5.69 ± 0.62 bc 1.69 ± 0.05 c 214.72 ± 4.18 a 94.58 ± 2.54 a 0.84 ± 0.04 a 3.95 ± 0.26 a 9.38 ± 0.61 c 14.11 ± 0.97 c 30.16 ± 1.44 a 1.97 ± 0.49 a 4.52 ± 0.23 e 5.94 ± 0.21 bc 627.18 ± 22.03 a

raw

Blaze

5.21 ± 0.26 b 7.24 ± 0.31 e 9.12 ± 0.28 c 3.40 ± 0.15 a 1.95 ± 0.13 b 5.37 ± 0.21 d 31.82 ± 1.56 c 26.24 ± 1.14 c 9.68 ± 0.36 c 0.41 ± 0.06 a 100.44 ± 4.46 c

0.94 ± 0.08 a 3.69 ± 0.25 b 108.45 ± 3.46 a 55.74 ± 2.04 c 30.27 ± 1.56 a 0.71 ± 0.05 bc 3.85 ± 0.16 c 3.08 ± 0.11 c 0.69 ± 0.07 a 6.92 ± 0.59 d 1.88 ± 0.05 d 231.52 ± 3.98 b 102.26 ± 2.04 b 0.95 ± 0.06 a 5.18 ± 0.51 b 6.44 ± 0.32 b 10.38 ± 0.86 b 32.21 ± 2.13 a 2.16 ± 0.36 a 4.07 ± 0.31 e 5.43 ± 0.28 b 616.82 ± 19.27 a

cooked

11.56 ± 0.34 e 8.67 ± 0.18 f 58.05 ± 3.42 g 9.00 ± 0.41 e 2.82 ± 0.09 e 7.94 ± 0.27 f 45.49 ± 2.06 f 38.46 ± 1.33 f 16.82 ± 0.52 e 6.64 ± 0.14 e 205.45 ± 8.76 f

cooked

4.70 ± 0.22 a 5.47 ± 0.15 c 19.51 ± 0.69 e 6.45 ± 0.21 c 2.19 ± 0.10 bc 4.52 ± 0.16 c 24.00 ± 1.09 b 31.62 ± 1.64 d 8.17 ± 0.31 b 1.06 ± 0.04 b 107.69 ± 4.61 cd

2.62 ± 0.19 cd 4.58 ± 0.38 c 120.65 ± 4.26 b 60.88 ± 2.65 d 43.26 ± 2.14 b 0.87 ± 0.06 d 2.63 ± 0.15 b 3.75 ± 0.20 d 2.06 ± 0.12 f 8.23 ± 0.48 e 1.68 ± 0.08 c 310.24 ± 6.05 e 146.53 ± 2.59 f 1.56 ± 0.18 c 9.45 ± 0.33 e 16.17 ± 1.15 d 21.34 ± 1.27 d 40.12 ± 2.98 b 13.58 ± 1.06 e 3.97 ± 0.24 d 7.23 ± 0.21 c 821.40 ± 26.77 b

Maxim

2.41 ± 0.11 c 4.11 ± 0.28 bc 133.15 ± 6.14 cd 69.54 ± 2.59 e 51.09 ± 3.01 c 0.62 ± 0.05 ab 3.78 ± 0.24 c 3.24 ± 0.34 cd 1.55 ± 0.11 d 6.12 ± 0.10 c 2.42 ± 0.18 f 289.55 ± 3.34 d 137.49 ± 2.58 de 1.27 ± 0.08 b 8.14 ± 0.25 d 20.19 ± 1.31 e 25.28 ± 1.29 e 37.66 ± 1.86 b 11.53 ± 0.54 d 3.43 ± 0.12 c 6.09 ± 0.25 c 818.66 ± 24.77 b

raw

8.54 ± 0.34 d 6.39 ± 0.25 d 14.19 ± 0.51 d 9.72 ± 0.33 e 2.28 ± 0.14 cd 2.41 ± 0.11 b 31.78 ± 1.15 c 23.74 ± 1.06 b 11.56 ± 0.75 d 5.11 ± 0.21 d 115.72 ± 4.85 d

cooked

4.83 ± 0.16 ab 4.48 ± 0.20 b 2.16 ± 0.11 a 5.23 ± 0.16 b 2.11 ± 0.06 bc 1.34 ± 0.03 a 18.96 ± 1.07 a 15.23 ± 0.82 a 8.30 ± 0.26 b 1.75 ± 0.11 c 64.39 ± 2.98 a

3.12 ± 0.22 d 6.22 ± 0.31 e 128.30 ± 5.26 bc 71.02 ± 3.14 e 70.68 ± 2.95 d 1.05 ± 0.03 e 5.91 ± 0.32 e 4.26 ± 0.17 e 1.78 ± 0.08 e 14.11 ± 1.21 g 1.64 ± 0.14 c 331.20 ± 5.93 f 143.86 ± 3.67 e 2.13 ± 0.23 d 12.06 ± 1.35 g 21.08 ± 1.76 e 31.51 ± 2.07 f 62.33 ± 1.62 c 11.25 ± 1.22 d 2.37 ± 0.11 b 9.32 ± 0.43 e 935.20 ± 32.22 c

Greenland

2.76 ± 0.21 cd 5.41 ± 0.30 d 141.72 ± 4.52 d 78.15 ± 1.96 f 82.48 ± 2.24 e 0.82 ± 0.05 d 7.48 ± 0.51 f 4.85 ± 0.39 f 1.12 ± 0.04 c 12.16 ± 0.55 f 2.28 ± 0.08 e 302.14 ± 5.09 e 132.88 ± 2.79 d 1.57 ± 0.11 c 10.16 ± 0.23 f 35.56 ± 2.65 f 40.58 ± 1.54 g 59.13 ± 3.09 c 11.96 ± 1.12 d 2.17 ± 0.08 ab 8.10 ± 0.24 d 943.48 ± 27.79 c

raw

10.96 ± 0.43 e 7.59 ± 0.27 e 22.17 ± 1.13 f 9.25 ± 0.35 e 2.48 ± 0.11 d 4.62 ± 0.16 c 38.16 ± 1.69 d 31.44 ± 1.23 d 12.24 ± 0.62 d 7.14 ± 0.22 f 146.05 ± 6.21 e

cooked

6.64 ± 0.27 c 3.68 ± 0.19 a 4.19 ± 0.25 b 3.25 ± 0.16 a 1.09 ± 0.07 a 2.31 ± 0.11 b 26.94 ± 1.56 b 22.14 ± 1.33 b 6.18 ± 0.24 a 1.06 ± 0.06 b 77.48 ± 4.24 b

1.58 ± 0.12 b 3.64 ± 0.24 b 102.33 ± 3.41 a 39.60 ± 2.16 a 40.23 ± 1.94 b 0.79 ± 0.05 cd 2.11 ± 0.14 a 2.54 ± 0.20 b 0.85 ± 0.06 b 5.24 ± 0.28 b 0.43 ± 0.02 a 251.52 ± 4.21 c 109.34 ± 3.64 c 1.88 ± 0.13 d 7.36 ± 0.42 c 5.27 ± 0.29 a 7.06 ± 0.35 a 38.69 ± 2.44 b 4.28 ± 0.27 b 2.41 ± 0.21 b 4.02 ± 0.38 a 631.17 ± 20.96 a

Sovereign

1.24 ± 0.28 ab 2.33 ± 0.12 a 119.46 ± 4.58 b 50.26 ± 2.65 b 48.69 ± 1.55 c 0.71 ± 0.07 bc 3.04 ± 0.34 b 2.12 ± 0.19 a 0.71 ± 0.05 a 4.15 ± 0.13 a 1.08 ± 0.06 b 230.15 ± 5.02 b 91.42 ± 3.57 a 1.21 ± 0.08 b 6.05 ± 0.55 b 10.82 ± 1.12 c 11.32 ± 0.79 b 32.15 ± 1.54 a 5.09 ± 0.11 c 1.94 ± 0.18 a 3.47 ± 0.22 a 627.41 ± 23.20 a

raw

Values are the mean ± SD, n = 3. Values followed by different letters in the same column are significantly different (P < 0.05). bUnknown peaks were quantified according to the calibration curve of garlic acid standard and expressed as μg GAE/g DW.

a

extractable phenolics dihydroxybenzoic acid p-hydroxybenzolc acid catechin-3-glucoside catechin gallate epicatechin-3-glucoside sinapic derivative procyanidin dimer 1 syringic acid trans-p-coumaroyl-malic acid trans-p-coumaric acid epicatechin gallate kaempferol tetraglycoside kaempferol triglycoside kaempferol-3-robinoside-7-rhamnoside quercetin-3-xyloside procyanidin dimer 2 procyanidin dimer 3 quercetin-3-glucoside flavonoid derivative flavonoid derivative kaempferol-3-glucoside total phenolic index (TPI-E) bound phenolics gallic acid protocatachuic acid catechin epicatechin 3-hydroxycinnamic acid UK1b UK2b UK3b UK4b UK5b total phenolic index (TPI-B)

phenolic compound

lentil cultivar

Table 4. Effects of Cooking on the Hydrophilic Extractable and Bound Phenolic Contents (μg/g DW) of Lentil Cultivarsa

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bound phenolics including TPC-B, TFC-B, and CTC-B in all tested lentils (Table 3). Oxidative degradation of phenolic compounds, including enzymatic browning during cooking process, may promote losses of these phenolics.36 Individual Phenolic Compounds in Extractable and Bound Phenolic Extracts and Effect of Cooking. Twenty-one phenolic compounds were found in the extractable phenolic fraction, with the majority being flavonoids including kaempeferol glycosides and catechin/epicatechin glucosides. These phenolic compounds were identified by matching the retention times, UV spectra, and mass spectrometric data with those of the corresponding standards as described in our recent paper.28 Concentrations of the individual phenolic compounds in the extractable fraction of raw and cooked lentils are summarized in Table 4. Flavonols, mainly kaempferol glycosides, were the major extractable phenolic compounds of the tested raw lentils, with the tetraglycoside and triglycoside having the highest concentrations ranging from 214.72 to 302.14 μg/g DW and from 91.42 to 137.49 μg/g DW, respectively, followed by flavanols including catechin glucoside (119.46− 141.72 μg/g DW), catechin gallate (50.26−78.15 μg/g DW), and epicatechin glucoside (39.42− 82.48 μg/g DW). Several phenolic acids including trans-pcoumaric acid, syringic acid, and p-hydroxybenzoic acid were also detected in the extractable fraction of raw lentils, but in much lower amounts. These results indicate that the majority of extractable phenolics in tested raw lentils are flavonoid glycosides. The total phenolic indices (TPI-E) defined as the sum of concentrations of all phenolic compounds detected in extractable fraction of lentils were between 627.18 and 943.48 μg/g DW, much lower than the corresponding TPC-E indices (Table 3). This can be attributed to incomplete quantification of all peaks in the HPLC method and to the extractable but conjugated phenolic compounds, which may be bound to soluble components such as small peptides or oligosaccharides,28,37,38 but not separated and detected in HPLC. Potential interferences from other nonphenolic substances such as vitamin C and proteins, as well as Maillard reaction (nonenzymatic browning) and caramelization products, may also cause positive reactions in the Folin−Ciocalteu assay.39,40 Cooking led to significant increases in the concentrations of individual flavonols such as kaempferol and quercetin glycosides, as well as in phenolic acids, but significant losses in flavanols (catechin/epicatechin glucoside, catechin gallate, and oligomeric procyanidins) (P < 0.05). The net result as expressed in TPI-E suggests no significant overall change by cooking, probably due to cancellation between the gains and losses of these two groups of extractable phenolics (Table 4). The exact reason as to how certain subgroups of polyphenols are increased and others are decreased during cooking is unclear, although it is well-known that heat-induced breakdown of cellular constituents and cell walls can lead to enhanced release of certain food bioactives41 and that oxidative degradation and reaction with starch molecules may reduce the release.35 From the bound TPC-B, TFC-B, and CTC-B data, it was clear the phenolic contents in both raw and cooked lentils were extremely low compared to the extractable TPC-E (Table 3). This was also shown by the HPLC profile of the individual phenolic compounds. Figure 1 shows typical HPLC chromatograms of the bound phenolic extracts of raw and cooked lentil (Maxim). Five phenolic compounds (peaks 1, 2, 4, 7, and 9) including gallic acid, protocatechuic acid, catechin, epicatechin, and 3-hydroxycinnamic acid, respectively, were positively identified in both raw and cooked extracts by comparing their

Figure 1. HPLC chromatograms of bound phenolics extract of a typical lentil cultivar (Maxim) before and after cooking and a standard mixture monitored at 280 nm. Peaks: UK, unknown; 1, garlic acid; 2, protocatechuic acid; 3, p-hydroxybenzoic acid; 4, catechin; 5, caffeic acid; 6, syringic acid; 7, epicatechin; 8, ferulic acid; 9, 3-hydroxycinnamic acid.

retention time and UV spectra with those of the corresponding commercial standards. The identified bound phenolic compounds (peaks 1, 2, 4, 7, and 9) were mainly flavonoids and phenolic acids, among which catechin at 14.19−71.04 μg/g DW in raw lentils, was the major phenolic compound, followed by gallic acid (8.54−19.18 μg/g DW), protocatechuic acid (6.39− 11.09 μg/g DW), epicatechin (7.70−9.72 μg/g DW), and 3hydroxycinnamic acid (2.03−2.82 μg/g DW). These bound phenolic compounds were all aglycones, and their concentrations in cooked lentils were significantly lower individually and collectively (P < 0.05), suggesting cooking negatively affected the release of bound phenolic compounds (Table 4). In addition, catechin levels in the tested red lentil cultivars, both raw and cooked, again were significantly higher than those of the respective tested green lentils (Table 4). This is consistent with the above result on bound TFC-B and CTC-B (Table 3). Apart from the above five identified bound phenolic compounds, several unknown peaks (UK1−5) were detected from bound phenolic extracts of both raw and cooked lentils (Figure 1). None of these peaks matched the retention time or UV−/vis spectrum of the aglycones or glycosides detected in the extractable fraction or any known phenolics commonly found in lentils (Figure 1). Literature on phenolic contents (particularly bound phenolics) in both raw and cooked lentils is scarce, and data are sometimes contradictory. Phenolic acids (sinapic acid, chlorogenic acid) in the extractable fraction and 2,3,4trihydroxybenzoic acid as bound fraction were found to be the main phenolics in lentils by Xu and Chang,42 as opposed to the flavonols (kaempferol glycosides) in the extractable and flavanol (catechin) in the bound fractions found in the present study (Table 4). Whereas genetics and environmental conditions may affect the phytochemical compositions, other factors such as extraction and analytical methods used in different laboratories may also cause misidentification of targeted compounds and thus lead to such discrepancies. Currently, there is no available information on the composition and identity of bound phenolics of either raw or cooked lentils. Further studies using LC-MS or NMR are required to accurately report on the concentration and identity of the bound phenolics in lentils. However, what is clear from the present study is that these unknown bound phenolics were at much lower concentrations in cooked lentils. The UV− vis absorption spectra of the five unknown bound phenolics are shown in Figure S1 (Supporting Information), and quantification of these five unknown peaks was done using the calibration 12591

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Figure 2. Antioxidant activities of the lipophilic fractions (A, B) and extractable (C, D) and bound (E, F) phenolic fractions of raw and cooked lentils as measured by DPPH and ORAC assays. DPPH and ORAC values are expressed as micromoles Trolox equivalent per gram DW lentil (μmol TE/g DW). Values are means ± SD, n = 3. Values followed by the same letter in the same assay are not significantly different (p < 0.05).

were 3.74−4.67 and 8.32−9.89 μmol TE/g DW as assessed by DPPH and ORAC-L assays, respectively (Figure 2A,B). The antioxidant activities of hydrophilic extractable and bound fractions of raw lentils ranged from 28.40 to 35.94 μmol TE/g DW and from 12.80 to 14.36 μmol TE/g DW by the DPPH assay, respectively (Figure 2C,E) and from 108.78 to 169.27 μmol TE/g DW and from 27.29 to 48.52 μmol TE/g DW by ORAC-H, respectively (Figure 2D,F). These results are similar to those found by Xu et al. in lentils, but significantly higher than

curve of gallic acid standard and expressed as micrograms of GAE per gram of DW in Table 4. Effect of Domestic Cooking on Antioxidant Activities. The total antioxidant activities of lentil extracts were evaluated using DPPH and ORAC assays. Whereas DPPH was used for both the lipophilic and hydrophilic extracts, two protocols, ORAC-L and ORAC-H, were used for the two types of extracts, respectively.43 The antioxidant activities of the lipophilic extracts (containing mainly carotenoids and tocopherols) of raw lentils 12592

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in other pulses.25,44,45 From the Trolox equivalent antioxidant activities, it was clear that the hydrophilic extracts containing mainly phenolics, especially the extractable phenolics, were stronger antioxidants and are perhaps the main contributors to the overall antioxidant activities of raw lentils (Figure 2). The same can be said for the cooked lentils; however, cooking itself showed different effects on the antioxidant activities of the lipophilic and hydrophilic extracts. Cooked lentils showed significantly higher antioxidant activity in both DPPH and ORAC-L assays for the lipophilic extract (P < 0.05), but significantly lower activity in both DPPH and ORAC-H for both extractable and bound phenolics (P < 0.05) (Figure 2). This is in agreement with the changes in concentration of the antioxidants of lentils; that is, the increased antioxidant activity of the lipophilic extract after cooking may be due to the increased carotenoid and tocopherol contents, whereas reduction in the antioxidant activity of the hydrophilic extracts may be due to significantly lowered phenolic contents in both extractable and bound forms as a result of cooking (Table 3). In conclusion, lentils are an excellent source of phytochemical antioxidants such as carotenoids, tocopherols, and phenolics. The domestic cooking process significantly affects not only the phytochemical contents of both lipophilic and hydrophilic compounds but also the antioxidant activities associated with these components. Cooking caused significant increases in total and individual carotenoid and tocopherol levels and led to elevated antioxidant activity in the lipophilic fraction, whereas minimum effect was found on the fatty acid composition including PUFA. Total and individual phenolics were mainly in the extractable form as opposed to bound phenolics, and they are the main contributors to the antioxidant activities. Cooking significantly reduced the antioxidant activity of both extractable phenolic and bound fractions due to lowered total phenolic contents, whereas no or less effect was found in individual phenolic concentrations. More specifically, cooking was found to favor the release of flavonols (kaempferol glycosides) but led to losses of flavanols (monomeric and condensed tannin). Whereas reduced flavanols and other phenolic compounds may have negatively affected the antioxidant activity, other components, especially the lipophilic antioxidants, were increased. In general, the present study suggests that a properly designed cooking protocol will not cause significant loss of the phytochemical antioxidants and thus will retain the potential health benefits of lentils when incorporated into the diet.

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Funding

This project is supported by the A-Base research (RBPI 1004; 2834) of Agriculture and Agri-Food Canada and an AgriInnovation Program Grant with Pulse Canada (AIP#068). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Pulse Canada for providing the lentil samples.

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ASSOCIATED CONTENT

* Supporting Information S

UV absorption spectra of five unknown peaks (UK1−5) in bound phenolics extract of lentil and standards of all known phenolic compounds identified. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(Z.-Y.D.) Phone/fax: +86 791 88304402. E-mail: [email protected]. *(R.T.) Phone: +1 226-217-8108. Fax: +1 226-217-8183. E-mail: [email protected]. Author Contributions ∥

REFERENCES

B.Z. and Y.T. contributed equally to this work. 12593

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Journal of Agricultural and Food Chemistry

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dx.doi.org/10.1021/jf504181r | J. Agric. Food Chem. 2014, 62, 12585−12594