Antioxidant activity of polyphenols from Ontario grown onion varieties using pressurized low polarity water technology

Journal of Functional Foods 31 (2017) 52–62

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Antioxidant activity of polyphenols from Ontario grown onion varieties using pressurized low polarity water technology Cynthya Maria Manohar a, Jun Xue b, Abdul Murayyan a, Suresh Neethirajan a,⇑, John Shi b a b

BioNano Laboratory, School of Engineering, 50 Stone Road East, University of Guelph, Guelph, ON N1G 2W1, Canada Guelph Food Research Centre, 93 Stone Road West, Agriculture and Agri-Food Canada, N1G 5C9, Canada

a r t i c l e

i n f o

Article history: Received 26 September 2016 Received in revised form 12 January 2017 Accepted 18 January 2017

Keywords: Ontario onions Flavonoids Antioxidants Phenolic compounds Polyphenols

a b s t r a c t Natural by-products, especially flavonoids, are in great demand in the nutra-pharmaceutical and biomedical industries. In this study, Ontario-grown onion varieties, namely Stanley, Safrane, Fortress, Lasalle and Ruby Ring, were screened for their antioxidant properties. Pressurized low polarity water technology, an environmentally friendly technique, was employed to extract the flavonoids from the onion varieties, followed by quantification and analysis using High Performance Liquid Chromatography. The antioxidant activities in the extracted samples were determined using various antioxidant assays, such as 2,2diphenyl-1-picrylhydrazyl, 2,20 -azino-bis (3-ethylbenzothiazoline-6-sulphonic acid), ferric reducing ability of plasma, lipid peroxidation, total antioxidant capacity and oxygen radical absorbance capacity assays. The total phenolic content extracted from the Ruby Ring variety was the highest when compared to all the other yellow onion varieties tested. Our results indicate that Ruby Ring may be chosen as a preferred variety over other onion varieties to develop functional and health food products. Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction Cancer is one of the leading causes of death of people in both developed and developing nations. Several epidemiological studies have indicated that consuming phytochemical rich fruits and vegetables helps in reducing many chronic diseases, such as diabetes, cancer, coronary heart disease, Alzheimer’s disease, neurodegenerative disorder and cataract formation (Hertog, 1996). Cases of cancer, diabetes and cardio-vascular disease could be reduced by one third by consuming a proper diet. Free radicals in our bodies are created when cells use oxygen to generate energy. The formation and the activity of reactive oxygen species (ROS) leads to potentially harmful effects. Therefore, in order to limit the levels of ROS, antioxidants from fruits and vegetables are required. Compounds such as phenolics, thiols and acids, which are present naturally in plants, exhibit antioxidant activity. They scavenge free radical species and inhibit the production of ROS. Antioxidants prevent proteins, lipids, DNA and cellular damage (Saija et al., 1995) and are widely used in dietary supplements to prevent chronic deadly diseases. Antioxidants are also currently used as preservatives in the food and cosmetic industries.

⇑ Corresponding author. E-mail address: [email protected] (S. Neethirajan). http://dx.doi.org/10.1016/j.jff.2017.01.037 1756-4646/Ó 2017 Elsevier Ltd. All rights reserved.

Cohort studies show that consumption of dietary rich food decreases the risk of cancer, immune dysfunctions and cardiovascular disease. Naturally occurring flavonoids have received global attention, since they are found in abundance in our daily diet as well as imparting several health benefits. Among vegetables, onions are considered the richest source of flavonoids and anthocyanins. Onions contain a number of phytochemicals and have been valued for their various health benefits, due to the presence of flavonoids. Flavonoids have potential antioxidant, antibacterial, anti-inflammatory and anti-cancerous properties. In addition, flavonoids have also been used in developing anti-AIDS drugs by inhibiting the function of viral protein (Yamazaki, Miyoshi, Kawabata, Yasuda, & Shimoi, 2014; Shimura et al., 1999). Flavonoids are a set of naturally occurring polyphenolic plant secondary metabolites. Researchers have found that flavonoids, especially quercetin mono- and di-glucosides, are widely present in the human diet. Onions have been ranked the highest in quercetin content among 28 vegetables and 9 fruits (Hertog, Hollman, & Katan, 1992). Epidemiological and in vitro and in vivo experimental data indicate that regular intake of onions is connected with a decreased risk of degenerative ailments. Onions (Allium cepa L.), are also a rich source of organosulfur compounds and most of the sulfur compounds present in them are in the form of cysteine derivatives, e.g. S-allyl cysteine sulfoxide. These compounds are also reported to have several potential health benefits, including

C.M. Manohar et al. / Journal of Functional Foods 31 (2017) 52–62

preventing tumors and cancers. Polymerization of flavonoids in foods into large molecules, called tannins, is either done by the plants themselves or during food processing. The major flavonoid present in onions is quercetin and its derivatives. Glycosides that constitute 80% of flavonoids in onions are quercetin 4-O-bglucoside and quercetin 3,4-O-b-diglucoside. Recent studies have suggested that quercetin as a pro-oxidant induces apoptosis in cancerous cells by preventing tumor proliferation and improves normal cell survival (Søltoft, Christensen, Nielsen, & Knuthsen, 2009). Other major flavonoids that are present in onions are kaempferol, myricetin and isorhamnetin derivatives. The glycosyl unit in these is identified as glucose. The bulb color and type depend on the quantity of flavonoids present in them (Søltoft et al., 2009). Onions are one of the most important agricultural crops produced in Canada (mostly in the eastern provinces of Ontario and Quebec), with an annual production of 210,000 tons at a market value of $74 million (Gianessi, 2013). Although onions are a major source of dietary flavonoids, due to genetic and environmental differences, thorough screening of the onions for flavonoid and phenolic contents is needed. At present there is a mounting interest in the promotion of horticultural food crops with high amounts and a desired composition of flavonoids. The differences in the phenolic and flavonoid content and their individual concentration amongst onion types will be useful knowledge to farmers or breeders, when selecting a variety with specific anticipated potential health benefits. Identification, enhancement and development of market quality traits, such as the antioxidant properties of onions, will enhance their marketability as nutraceutical products, as a natural antibiofilm coating agent for improving the shelf life of packaged food and as a preservative in processed food (Yang, Meyers, van der Heide, & Liu, 2004). In Ontario, 17 varieties of onions are grown and out of these we chose five varieties, since they are the most commonly used ones. Globally, the nutraceutical market is expected to grow at a rate of 6.4% to reach US $204.8 billion by 2017. The demand for antioxidants and antimicrobial coatings and films for food industries is estimated to reach US $2.7 billion by the year 2018 (King, 2014). There is not enough scientific evidence to back up any nutraceutical claims of Ontario-grown onions. Till date, the health effects of Ontario-grown onion extract have not been extensively explored. The unique properties of onion-based flavonoids, as well as their biocompatibility and biodegradability, are that they are safe to eat and this absence of toxicity make them more appealing than synthetic polymers or chemical-based compounds to develop as useful products for various nutraceutical and functional food applications. Previously, a well-established extraction process, based on organic solvents, was used to extract naturally occurring plant compounds, such as polyphenols. Nowadays there has been an increase in demand for environmentally friendly technologies capable of providing high-quality and high-activity extracts without any solvent toxicity. The pressurized low polarity water (PLPW) technology technique is an environmentally friendly method, which caters to a range of applications. Solvent extraction techniques are generally toxic and their commercial use requires explosion-proof facilities, along with storage and handling apparatus certified for use with flammable chemicals. In addition, solvents might remain in final products as residues, which raises safety concerns for consumption by humans. PLPW uses water heated under pressure to temperatures above boiling point, resulting in the change of its key properties, such as pH and polarity; and the dielectric constant of the water polarity can be decreased to those of solvents, such as ethanol or methanol, thus extracting different classes of phytochemical compounds. Therefore, an ecofriendly extraction technique is

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essential to extract functional compounds from natural products. In our study, we screened Ontario-grown onion varieties, namely Stanley, Safrane, Fortress, Lasalle and Ruby Ring, for their antioxidant properties, and isolated the flavonoids present in them. Investigation of the mechanisms behind the antioxidant properties of the volatile compounds of onions will provide new strategies for developing novel health products in the food industry. Understanding the chemistry behind bioactive compounds and their impact on health through linkage between onion flavonoids and their antioxidant properties will help to design and develop novel onion-based nutraceutical and health products for food and pharmaceutical industries. Furthermore, studying traits such as the antioxidant content of Ontario-grown onions will increase the marketability of these onions. 2. Materials and methods Onion varieties, namely Stanley, Safrane, Fortress, Lasalle and Ruby Ring, were received as gifts from Holland Marsh Growers’ Association, Ontario, Canada and used for the extraction of polyphenol compounds. 2.1. Chemicals and reagents Gallic acid, sodium carbonate, potassium hexacyanoferrate, Folin-Ciocalteu reagent (FCR), ABTS (2,20 -azino-bis (3-ethylbenzo thiazoline-6-sulphonic acid)), potassium acetate, aluminum chloride, DDPH (2,2-diphenyl-1-picryl-hydrazyl), potassium persulfate, ferric chloride and ascorbic acid were purchased from Sigma Aldrich (Burlington, Canada). 10% trichloroacetic acid was purchased from Labchem (Zelienpole, USA). Methanol, HPLC grade water, 96-well polystyrene microtiter plates and formic acid were purchased from Fisher Scientific (Burlington, Canada). Lipid Peroxidation (MDA) Assay Kits were purchased from Abbexa (Cambridge, United Kingdom). Total antioxidant capacity (TAC) colorimetric assay kits were purchased from BioVision, California, USA. Deionized water was procured from Barnstead Nanopure Diamond lab water system (APS Water Services Corporation, USA). Quercetin, quercetin 3,40 -diglucoside, isorhamnetin, kaempferol, quercetin 3-b-D-glucoside, kaempferol-3-glucoside, myricetin and quercetin 3-glucoside standards were purchased from Sigma Aldrich Canada. 2.2. Sample preparation and storage Medium sized onions of the same variety were peeled, cleaned and chopped. Then the onion samples were ground and placed at 30 °C overnight. The sample pastes from the five varieties were placed in a freeze drier for 72 h. The freeze- dried samples were then ground to fine powder and stored in air tight containers at 20 °C. The fine powders from the five varieties were further stored for analysis. All the experiments were carried out in triplicate and the results were recorded as means ± standard deviation. 2.3. Extraction of flavonoid compounds The flavonoids from the onion samples were extracted by PLPW technology. This extraction was performed at the facilities of the Guelph Food Research Centre of Agriculture and Agri-Food Canada, using an automated Speed SFE NP model 7100 pressurized low polarity instrument (Applied Separation Inc., Allentown, PA, USA) equipped with a pump (Module 7100) and a 10 mL thick-walled stainless cylindrical extractor vessel. This type of extraction is ecofriendly, ideal for extracting flavonoid-rich onion ingredients, and

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scalable for commercial use. Approximately 5 g of the freeze-dried onion powder was mixed with 80 mL of 0.1% formic acid in milli-Q water (v/v), and injected into the extractor vessel; the temperature was set at 60 °C, pressure at 150 bar and the total extraction time was 60 min.

2.4. High performance liquid chromatography analysis of phenolic compounds The extracted samples were identified and analyzed with photodiode array detection (DAD) using an Agilent 110 series High Performance Liquid Chromatography (HPLC) system. The flavonoids were separated using a reverse phase C18 Luna column (Phenomenex, USA, 250  4.6 mm; 5 m). The mobile and stationary phases’ solvent was A-0.1% formic acid: methanol (9:1; v/v) and solvent B-methanol. A continuous flow of mobile phase at 0.8 mL/min was set up in the column at 254 nm. The gradient program was: 5% B for 1 min, a linear gradient to 50% B for 34 min, then to 100% B for 5 min, the isocratic elution for 4 min, followed by a min ramp back to 5% B and re-equilibration for 6 min. The k Absorbance Detector used for identification of peaks was set at 254 nm (sampling rate: 1.0/s) (Søltoft et al., 2009).

2.5. Flavonoid standard quantification Each flavonoid standard compound was determined by HPLC with a standard run of every 6 samples. The standard curves were established to determine the ranges of flavonoid concentration in Ontario-grown onion varieties as reported by others (De Nardo, Shiroma-Kian, Halim, Francis, & Rodriguez-Saona, 2009; Formica & Regelson, 1995; Karakaya, 2004). Dilution of the flavonoid standards ranged in concentrations from 6.25 ppm (part per million) to 200 ppm. Quercetin, quercetin 3,40 -diglucoside, isorhamnetin, kaempferol, quercetin 3-b-D glucoside, kaempferol-3-glucoside, myricetin and quercetin 3-glucoside were used as standard controls. The flavonoids were then quantified by integrating the peak areas.

2.6. Estimation of total polyphenolic content The total phenolic content (TPC) was determined by FolinCicocalteu assay. The onion extracts were oxidized with FCR, and the reaction was then neutralized with 7.5% sodium carbonate. After 30 min incubation at room temperature in dark conditions, the optical density (OD) was measured at 760 nm. The OD values were measured by a plate reader (ELISA plate reader, Amersham Biosciences Corp., USA). The content of the total polyphenols in each of the extracted samples was determined using a standard curve for gallic acid and the results were expressed as mg of gallic acid equivalents (GAE) per gram of dried onion samples (Singleton, Orthofer, & Lamuela-Raventos, 1999).

2.7. Determination of total flavonoid amount Total flavonoid content was measured by aluminum chloride colorimetric assay. A total of 500 lL of the extracted onion samples were mixed with 100 lL of 10% aluminum chloride and 100 lL of 1 M potassium acetate. Then the sample reaction was diluted with 2.8 mL of deionized water and the samples were incubated for 30 min at room temperature. The OD was measured at 415 nm, using a 96-well plate, by the plate reader (ELISA plate reader, Amersham Biosciences Corp., USA). The total flavonoid content (TFC) was expressed as mg quercetin equivalent (QE)/g dry plant.

2.8. Antioxidant assays 2.8.1. DPPH radical scavenging activity assay (2,2-diphenyl-1-picrylhydrazyl-hydrate) The DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) assay was carried out using the method reported by Kedare and Singh (2011) with slight modifications. A total of 0.5 mL of the extracted sample was mixed with 3 mL of absolute ethanol followed by the addition of 50 lM of DPPH and incubation for 30 min in dark conditions. The DPPH reacted with the antioxidant compounds to induce color changes from deep violet to light yellow, which were read in a UV visible spectrophotometer at 517 nm. This assay was performed to determine the free radical scavenging activity of the extracted onion samples. Quercetin and ascorbic acid were used as standard controls and distilled water was used as blank. Experiments were performed in triplicate and the percent inhibition (%) was calculated. 2.8.2. ABTS radical scavenging assay (2,20 -azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)) ABTS (2,20 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) radical scavenging assay was performed using the method explained by Rael et al. (2004) with some modifications. The ABTS radical cation (blue/green ABTS+ chromophore) is generated through the reaction between ABTS and potassium persulfate. The stock solution was 7.4 mM ABTS+ solution and 2.6 mM potassium persulfate. The two stock solutions were added in equal amount to obtain a working solution, which was allowed to stand in dark conditions for 12–16 h at room temperature. This resulted in the ABTS radical cation solution. After the incubation period, the ABTS solution was diluted with 80% ethanol to an absorbance of 0.70 at 734 nm and equilibrated at 30 °C. 1 mL of the ABTS reagent solution prepared was added to 0.1 mL of the extracted samples. The diluted mixture was then incubated in dark conditions for 10 min at room temperature. The absorbance was measured at 734 nm using a microplate reader. Distilled water was used as blank. Each sample was performed in triplicate and the percent inhibition (%) was calculated. Ascorbic acid and quercetin were used as standard controls. Distilled water served as blank. 2.8.3. Ferric reducing antioxidant power assay The ferric reducing antioxidant power (FRAP) assay is based on the reduction of FeIII+ to FeII+ due to the action of antioxidants present in the sample, and was determined according to the method described by Szydłowska-Czerniak, Dianoczki, Recseg, Karlovits, and Szłyk (2008). A solution of 0.25 lL of the different onion sample extracts was mixed with 0.25 mL of 0.2 M of phosphate buffer and 0.25 mL of potassium hexacyanoferrate (K3Fe (CN)6). The solution was mixed well and was incubated in a hot water bath for 20 min. The reaction was stopped by the addition of 0.25 mL of trichloroacetic acid solution (10% w/v). The tubes were then centrifuged at 15,000g for 10 min. The supernatant was taken and mixed with ferric chloride (FeCl3) solution and 0.5 mL of distilled water. Absorbance of the solution was measured at 593 nm against blank to determine the reducing power. Ascorbic acid (0–200 lg/mL) acid was used as a standard and the OD values were analyzed and calculated with standards. 2.8.4. Total antioxidant capacity colorimetric assay The TAC assay was performed using a TAC calorimetric assay kit. A total of 100 ll of sample was diluted with 100 ll of protein mask (1:1). Dilution of one part of Cu2+ reagent with 49 parts of assay diluent was followed by mixing with the working reagent solution. Addition of a total of 100 ll of Cu2+ of working solution to the well containing the protein mask and the samples was followed by covering the plates and incubating at room temperature

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for 1.5 h. Samples were then read using the absorbance at 570 nm by a microplate reader. Trolox was used as a standard. All the samples were measured in Trolox equivalents (mM Trolox equivalent). 2.8.5. Lipid peroxidation (MDA) assay Lipid peroxidation forms malondialdehyde (MDA) and 4hydroxynonenal (4-HNE) as natural by products. This assay was determined by using a lipid peroxidation assay kit. 100 ll of the sample was added to 300 ll of reagent in centrifuge tubes. The mixture was incubated at 90 °C for 30 min. After incubation, the mixture was cooled in ice and centrifuged at 10,000g at 25 °C for 10 min. A total of 200 ll of the supernatant was added to the 96well microplate and the absorbance was read at 532 nm and 600 nm. The Lipid peroxidation (MDA) nmol/g was calculated. 2.8.6. Oxygen radical absorbance capacity assay Oxygen radical absorbance capacity (ORAC) assay kit (Cell bio labs Inc., USA) was employed for measuring the antioxidant capacity extracted from the flavonoids of Ontario-grown onion varieties. 25 lL of the samples were added into the 96-well microplates, followed by the addition of 150 lL of 1 fluorescein solution. The solution was mixed thoroughly and incubated for 30 min at 37 °C. Then 25 lL of free radical initiator solution was added into each well using a multichannel pipette. The reaction mixture was mixed thoroughly and immediately read with a fluorescent microplate reader at 37 °C, with an excitation wavelength of 480 nm and an emission wavelength of 520 nm. The plates were read in increments between 1 and 5 min for a total of 60 min. The lMole Trolox equivalents (TE) of the samples were calculated by comparing the standard curve and the results were expressed as TE per L or g of sample. The area under the curve (AUC) for each sample and standard were calculated using the final assay values and the linear regression formula. 2.9. Statistical analysis All the experiments were repeated thrice on three different samples and the data was reported as mean ± standard errors. One-tailed t-tests were performed using an R statistical programming software. Correlations between antioxidants and total phenolic and flavonoid content were also determined.

Table 1 Total phenolic and flavonoid contents of the five Ontario grown onion varieties. Variety names

Total phenolic content (mg of gallic acid equivalent (GAE)/g of dried onion sample)

Total flavonoid content (mg of quercetin equivalent/g of dried onion sample)

Ruby Ring Stanley Safrane Lasalle Fortress

1.95 ± 0.05 1.69 ± 0.13 1.62 ± 0.1 1.28 ± 0.2 1.27 ± 0.15

0.29 ± 0.02 0.33 ± 0.03 0.13 ± 0.03 0.12 ± 0.02 0.15 ± 0.02

extraction solution used influences the recovery of phenolic compounds due to its polarity (Patil, Pike, & Yoo, 1995). Lu, Ross, Powers, and Rasco (2011) reported that the total quercetin content present in the onion varieties ranged from 367.81 to 653.19 mg/kg fresh weight and represented >85% of TFC. A typical HPLC chromatogram showing the flavonoid peaks between 10 and 40 min appears in Fig. 1; it represents the total flavonol compounds in onions and agrees with previous research (Lee et al., 2014; Lu et al., 2011; Patil et al., 1995; Søltoft et al., 2009). The major peak observed in the yellow onion varieties was quercetin and in the red onion variety it was cyanidin 3-(600 malonylglucoside) (Table 1). The TFC by HPLC analysis is a summation of quercetin, quercetin 3,40 -diglucoside, quercetin 3-b-D-glucoside, quercetin 3-Oglucoside, quercetin 7,40 -diglucoside, quercetin 4-O-glucoside, myricetin, isorhamnetin 3,40 -diglucoside, isorhamnetin 40 glucoside, kaempferol and kaempferol 3-O-glucoside ranged from 323.2 to 819 mg/kg dried onion sample and represented >80% of the TFC (Table 1). Our results are significantly lower than those reported by Lu et al. (2011) and Rhodes and Price (1997). The variations in the total flavonoid concentrations may be due to various factors. Cultivation conditions play a major role in determining the flavonoid concentrations in onions. The biosynthesis of flavonoids may be due to the different cultivation conditions, such as weather conditions, harvest period and plant location. The quercetin content is mainly a relation to numerous factors, such as cultivation condition, genetic factors controlling localization, storage and also processing conditions. Therefore, it is always important to select onions with high quercetin contents that are stable and bioavail-

3. Results and discussion 3.1. Extraction and quantification of polyphenols by HPLC analysis The identity of phenolic compounds was ascertained using HPLC-DAD analysis and by comparison of retention time with those of the standards present. The predominant flavonoids present in the onion samples were identified and quantified (mg/kg dried onion sample) using HPLC and are listed in Fig. S1, Tables S1 and S2. HPLC chromatograms revealed 11 flavonoids and 5 anthocyanin peaks in the tested extracted samples, which is similar to those reported in Italian onion varieties (Tedesco, Carbone, Spagnuolo, Minasi, & Russo, 2015). Quercetin and its glucosides were predominant in free form in all the onion samples tested, with quercetin glucoside being the most abundant. The major flavonoids found in the onion type were quercetin and its glucosides which accounted for >80% of the TFC. The results obtained from HPLC-UV/vis of the onion samples tested revealed that the yellow onion variety (Stanley) showed the highest TFC (819 mg/kg dried onion sample) followed by the other varieties (Table 1). Due to the presence of anthocyanins, Ruby Ring variety showed the second highest in TFC among the tested varieties. This is in agreement with the previously reported literature, which indicated that the

Fig. 1. The % inhibition of DPPH radical scavenging activity for the five Ontariogrown onion varieties Lasalle (LAS), Fortress (FOR), Ruby Ring (RR), Stanley (STA) and Safrane (SAF) (mean ± SD, n = 5); p < 0.05.

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able for improving the nutritional properties of onions for benefits for consumers’ health. Flavonoids are generally present in the outer layer and concentrations are found to be higher than in the edible bulb portion, hence peeling results in great losses. The lower quercetin contents determined in the present study compared to previous reports could be due to the removal of the inedible outer layers. The skins of onions serve to protect the plant from ultraviolet radiation damage. The main flavonoids present in the outer skin are mainly aglycones, due to the flavonoid glucoside hydrolysis, which occurs during the peel formation. Flavonoids may degrade or evaporate or be washed out during handling; hence it is better to extract flavonoids from the bulb of the onions. In general, the onion bulb (edible portion) contains a broad range of quercetin contents with increasing concentrations of quercetin glucosides from the inner to the outer scales. These results also agree with Patil et al. (1995) who reported that the red and yellow onion varieties have higher amounts of quercetin. 3.2. Total polyphenolic and flavonoid contents The low polarity water extraction process increased the concentration of total phenolics and flavonoids in the onion bulb extracts. The total phenolic content of the onion varieties is shown in Table 1. The phenolic content of the red onion variety (Ruby Ring) showed the highest (p < 0.05) at 1.95 ± 0.05 mg of GAE/g of sample, followed by the yellow onion varieties Stanley (1.69 ± 0.13), Safrane (1.62 ± 0.1), Lasalle (1.28 ± 0.2) and Fortress (1.27 ± 0.15). A statistically significant difference occurred in the total phenolic content in various onion samples (p < 0.05). There was a 0.3-fold difference observed in the total phenolic content between the red onion variety Ruby Ring (highest ranked) and the yellow onion variety Fortress (lowest ranked) (p < 0.05). The different levels of antioxidant properties in plants may be due to the presence of phenolic acid components. In addition, presence of hydroxyl groups in the phenolic compounds may directly contribute to the antioxidant activity and play a major role in scavenging free radicals. The TFC of the five onion varieties is shown in Table 1. The TFC in the yellow onion variety (Stanley) showed the highest flavonoid content (p < 0.05) at 0.33 ± 0.03 mg of quercetin equiv/g of sample, followed by the other varieties Ruby Ring (0.29 ± 0.02), Fortress (0.15 ± 0.02), Safrane (0.13 ± 0.03) and Lasalle (0.12 ± 0.02). A statistically significant difference was seen in the TFC in various onion samples (p < 0.05). A 2-fold difference in the TFC between Stanley (highest ranked) and Lasalle (lowest ranked) varieties (p < 0.05) was observed. Lee et al. (2014) reported that the phenolic compounds extracted from onions by sub critical water extraction at 110 °C and 165 °C were found to be 218.73 ± 5.68 and 56.68 ± 2.28 (mg of gallic acid/g of the extract) respectively. Rodríguez Galdón, Rodríguez Rodríguez, and Díaz Romero (2008) reported that the total phenolic contents in the onion samples from Tenerife (Texas, Masca, Guayonje, San Juan, Carrizal Bajo and Carrizal Alto) ranged between 25.2 and 75.9 mg/100 g. Our present study indicated that the total polyphenol content ranged between 127.9 and 195.1 mg/100 g, which is higher than the reported Spanish onion varieties. Lu et al. (2011) observed that the total polyphenol content of white, yellow, red, sweet onions and Shallot (USA) varied between 142 and 428 mg/100 g. A study reported by Siddiq, Roidoung, Sogi, and Dolan (2013) suggested that, at 60 °C, heat treatment of the yellow onions resulted in a significant increase in total polyphenol content, from 44.92 to 52.32 mg GAE/100 g. Yang et al. (2004) suggested that the TFC of the Western Yellow onion variety was the highest, with a 11-fold difference compared to the other varieties tested. A study reported by Rhodes and Price (1997) found that the TFC were 91.8, 71.1, and 80.3 mg/100 g for

red, pink, and brown onion cultivars respectively. The flavonoid content of Ontario-grown onions in this present study was found to be 15.6, 12.2, 29.0, 13.6 and 33.7 mg/100 g for Fortress, Lasalle, Ruby Ring, Safrane and Stanley respectively. Rodríguez Galdón et al. (2008) reported that the TFC of Spanish onion varieties ranged between 7 and 9 mg/100 g. The lack of flavonoid contents found in some cultivars may reduce their importance as a health protection food product. Majid, Dhatt, Sharma, Nayik, and Nanda (2016) studied four Indian onion varieties, namely Punjab White, Punjab Naroya, PRO-6 and a commercial variety, and observed that the TFC ranged from 10.0 to 13.2 mg/100 g, which appeared to be less than some of the American and European varieties. Lee et al. (2014) reported that the total flavonoid compounds extracted from onions by sub critical water extraction at 110 °C and 165 °C were found to be 119.5 ± 10.43 and 27.1 ± 2.05 (mg of quercetin/g of the extract). The difference between the total phenolic and flavonoid contents could be due to genetic differences and variations in growing conditions, climate, maturity and harvest season. It is commonly known that the genetic and environmental factors play a vital role in the phenolic composition. The Ruby Ring and Stanley varieties showed the highest phenolic and flavonoid content and they also exhibited the greatest antioxidative activities. 3.3. Antioxidant properties The radical scavenging activities of the different onion samples were determined by DPPH, ABTS, and TAC Colorimetric and FRAP assays. Oxidative stress is widely known for its pathological effects in various chronic diseases such as diabetes, heart disease and cancer. It occurs when the formation of free radicals increases. In oxidative stress, the balance between the antioxidants and ROS is destroyed. In addition, oxidative stress causes damage to cell components, such as lipids, proteins and nucleic acids. Lipid peroxidation, a well-established mechanism of cellular injury in plants, is used as an indicator of oxidative stress in cells and tissues. Lipid peroxides are unstable and decompose to form a complex series of compounds, including reactive carbonyl compounds. Polyunsaturated fatty acid peroxides generate malondialdehyde (MDA) and 4-hydroxyalkenals (HAE) upon decomposition, and the measurement of MDA and HAE has been used as an indicator of lipid peroxidation. However, there is a wide range of model systems for determining the antioxidant activities and the choice depends upon the chemical structure of the constituents. Previous studies have reported that there are discrepancies in antioxidant activities of substances when tested in different model systems (Wettasinghe & Shahidi, 2002). Considering this fact, in the present study the antioxidant activities were tested using various assays. The DPPH method is preferred because it is fast, easy, reliable and does not require a special reaction or device. The stable free radical does not disintegrates in water or methanol/ethanol solvents. The free radical scavenging activities of the plant extracts depend on the ability of antioxidant compounds to lose hydrogen and on the structural confirmation of these compounds. The DPPH free radical at wavelength 517 nm receives an electron or hydrogen atom from antioxidant molecules to become a stable diamagnetic molecule. The DPPH radical ability to bind H is considered to be a radical scavenging property. The reason for dissolving DPPH in methanol is that it is converted to DPPH-H (diphenylhydrazine) molecules in the presence of an antioxidant. The color change is due to the decreasing quantity of DPPH radicals in the environment and reflects the radical scavenging activity of the plant extract.

DPPH þ A  H ! DPPH  H þ A

ð1Þ

The red onion variety (Ruby Ring) showed the highest DPPH radical scavenging activity (Fig. 1) when compared to the yellow onion varieties. The data is similar to those obtained for the total

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polyphenolic and flavonoid contents of the onion samples, which clearly indicates that higher phenolic content shows higher antioxidant activity. Shahidi and Naczk (2004) reported that the antioxidant activity of a food product depends on the chemical nature of its constituents and always not their quantities, as the efficiency of the compounds present varies considerably. In this assay, the onion extracts were able to reduce the stable DPPH radical to yellow-colored diphenylpicrylhydrazine. Thus this antioxidant assay is mostly related to their phenolic hydroxyl group. Ruby Ring, the red onion variety, showed the highest percentage of inhibition 21.52 ± 1.30% (p < 0.05), followed by Lasalle (15.46 ± 3.88%), Fortress (13.44 ± 4.19%), Stanley (11.38 ± 1.96%) and Safrane (11.1 ± 2.89%). In addition, the DPPH radical scavenging activity was tested for ascorbic acid and quercetin and were found to be 94.24 ± 0.37% and 93.55 ± 0.14%. A statistically significant difference was observed in the DPPH radical scavenging activities of the various onion samples (p < 0.05). Albishi, John, Al-Khalifa, and Shahidi (2013) have found that the skins of a red onion variety showed a higher DPPH value (0.152 ± 0.004) than yellow onions’ skin. Cheng et al. (2013) reported that the red onion variety showed higher DPPH radical scavenging activity (varying from 41.22 ± 3.0% to 81.96 ± 1.10%) than the yellow onion Chinese varieties. A ferryl myoglobin radical is formed from hydrogen peroxide and metmyoglobin. The ferryl myoglobin radical can oxidize ABTS to generate a radical cation, ABTS+, which is green in color. The antioxidants present in the plant extracts suppress this reaction by electron donation radical scavenging and also inhibit the formation of the colored ABTS radical. The concentration of antioxidants present in the test samples is inversely proportional to the ABTS radical formation. ABTS radical scavenging activities of different onion sample varieties are shown in Fig. 2. The ABTS radical scavenging activity of the red onion variety (Ruby Ring) was the highest of the varieties tested. Generally, samples with higher polyphenolic content are most effective as free radical scavengers. This assay is tested for screening antioxidant activities. Ruby Ring showed the highest percentage of inhibition at 95.8 ± 0.3%, followed by the other varieties namely Stanley (85.7 ± 4.7%), Safrane (84.19 ± 1.67%), Fortress (76.32 ± 3.63%) and Lasalle (72.7 ± 0.72%). ABTS radical scavenging activities was also determined for ascorbic acid and quercetin and found to be

96.06 ± 0.2% and 95.93 ± 0.18%. Statistical difference is observed in all the five varieties (p < 0.05). Fidrianny, Permatasari, and Wirasutisna (2013) reported that ethyl acetate extract of onion bulbs showed the highest ABTS capacity (49.03%). The increase in radical scavenging activity may have been due to the increase in total phenolic compounds. The compounds which have reduction potential react with potassium ferricyanide (Fe3+) to form potassium ferrocyanide (Fe2+), which then reacts with ferric chloride to form a complex which has an absorption max at 593 nm. The reducing power activity of the various onion samples are presented in Fig. 3. Ruby Ring showed the highest reducing power, 0.20 ± 0.002 as mM (ascorbic acid equivalent), followed by Stanley (0.18 ± 0.003), Fortress (0.08 ± 0.005), Safrane (0.06 ± 0.003) and Lasalle (0.04 ± 0.005). The data from these assays indicate that the red onion variety showed the highest radical scavenging activity compared to the yellow onion varieties. These results suggest that there is a possible relationship between the antioxidant activity and total flavonoid and total phenolic contents of the extracted samples. Lu et al. (2011) reported that the antioxidant capacity of onions measured by FRAP assay showed that the red onion variety had the highest (5.76 ± 0.47 lmol Trolox/g) ferric reducing power. Santas, Carbo, Gordon, and Almajano (2008) studied two Spanish onion varieties, namely white onion and Calcot de Valls, and observed that the total phenolic and antioxidant content was high. White onion extracts had the highest antioxidant activity (86.6 ± 2.97 and 29.9 ± 2.49 lmol Trolox/g DW for Trolox equivalent antioxidant capacity (TEAC) and FRAP assays. These results indicated that the antioxidant activity of plant extracts is mainly due to the presence of phenolic compounds. Antioxidants are capable of inhibiting the oxidation processes that occurs due to the presence of ROS. The total antioxidant is measured to investigate the relationship between dietary antioxidants and pathologies induced by oxidative stress. The assessment of the TAC is used to determine the additive antioxidant properties of plant foods. The TAC for the five onion varieties tested is shown in Fig. 4. The red onion variety was found to be the highest (0.382 ± 0.036 mM TE) followed by the yellow onion varieties namely Fortress (0.364 ± 0.018 mM TE), Stanley (0.333 ± 0.007), Lasalle (0.313 ± 0.003) and Safrane (0.312 ± 0.01). The natural antioxidants are multifunctional, possibly due to the presence of

Fig. 2. The % inhibition of ABTS radical scavenging activity for the five Ontariogrown onion varieties Lasalle (LAS), Fortress (FOR), Ruby Ring (RR), Stanley (STA) and Safrane (SAF) (mean ± SD, n = 5); p < 0.05.

Fig. 3. The reducing power activity (mM ascorbic acid equivalent) for the five Ontario-grown onion varieties tested namely Lasalle (LAS), Fortress (FOR), Ruby Ring (RR), Stanley (STA) and Safrane (SAF) (mean ± SD, n = 5); p < 0.05.

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polyphenols. There is no statistically significant difference observed in all the five varieties (p > 0.05). Venkatachalam, Rangasamy, and Krishnan (2014) reported that Indian red onion varieties showed the highest total antioxidant capacity (48.78 mg/100 g FW). These results indicate that plant compounds containing high phenolics may provide a source of dietary antioxidants. Fig. 5 shows the results for the lipid peroxidation assay tested for the five varieties. The quantification of lipid peroxidation is essential to assess oxidative stress in pathophysiological processes. The MDA (nmol/g) was calculated according to the weight of the sample. Ruby Ring, showed the highest (987.52 ± 25.491 MDA (nmol/g)) followed by Safrane (674.40 ± 9.406 MDA (nmol/g)), Lasalle (629.84 + 8.43 MDA (nmol/g)), Fortress (611.78 + 2.4 MDA (nmol/g)) and Stanley (578.06 + 59.62 MDA (nmol/g)). A statistically significant difference was observed in all the five varieties (p < 0.05). The increased formation of free radicals is associated

with the increase in lipid peroxidation (Rezaeizadeh et al., 2011). Therefore, one of the major roles of antioxidants is to inhibit the chain reaction of lipid peroxidation. Rosa et al. (2002) suggested that flavonoids reduced the amount of MDA formed in a dosedependent manner. Lee et al. (2014) reported that the inhibition of lipid peroxidation (%) of onion peel extract was higher in ethanol extraction (13.40 ± 2.41), followed by subcritical water extraction at 110 °C (5.34 ± 0.22) and they are statically significant. Nuutila, Puupponen-Pimiä, Aarni, and Oksman-Caldentey (2003) reported that concentrations of onion peel extract (yellow onions) at 80 mg/mL (methanol extraction) resulted in 81% inhibition of lipid peroxidation in rat hepatocytes. The ORAC assay measures the time-dependent decrease in the fluorescence intensity of the b-PE (beta-phycoerythrin) indicator protein, resulting from oxygen radical damage. Natural antioxidants protect the fluorescent molecule from oxidative degeneration. Each point reflects the level of antioxidant protection at that time, which many different antioxidants contribute to. This assay is beneficial because it takes samples with and without lag phases of their antioxidant capacities. This is beneficial while measuring foods and supplements which contain complex ingredients with slow and fast acting antioxidants. The antioxidant capacity of the sample correlates to the fluorescence decay curve, which is usually represented as the AUC. The net AUC was determined by subtracting the AUC of a blank from that of the antioxidative samples, which indicates the TAC of the samples tested. The ORAC radical scavenging activities for the five varieties is shown in Fig. 6. The Ruby Ring showed the highest Net AUC (35.2 ± 1.34 lM TE/g of the sample), followed by the yellow onion varieties Stanley (32 ± 0.98 lM TE/g of the sample), Safrane (28 ± 0.5 lM TE/g of the sample), Fortress (26.5 ± 0.8 lM TE/g of the sample) and Lasalle (25.8 ± 1.2 lM TE/g of the sample). A statistically significant difference was observed in the ORAC radical scavenging activities of various onion samples tested (p < 0.05). The main reason for the differences might be the variations in the concentration of flavonoids in various layers of different onion varieties. Vian, FabianoTixier, Elmaataoui, Dangles, and Chemat (2011) reported that the red onion variety showed the highest ORAC value of 201 lmol TE/g, followed by the other onion varieties. A study Siddiq et al. (2013) reported that the ORAC value for freshly cut onions was found to be 62.49 lM TE/g of the sample. In addition, the peroxyl

Fig. 5. The lipid peroxidation malondialdehyde (MDA) (nmol/g) for the onion varieties Lasalle (LAS), Fortress (FOR), Ruby Ring (RR), Stanley (STA) and Safrane (SAF) (mean ± SD, n = 5); p < 0.05.

Fig. 6. The ORAC (lM TE) for the onion varieties Lasalle (LAS), Fortress (FOR), Ruby Ring (RR), Stanley (STA) and Safrane (SAF) (mean ± SD, n = 5); p < 0.05.

Fig. 4. The total antioxidant assay (mM TE) for the five onion varieties Lasalle (LAS), Fortress (FOR), Ruby Ring (RR), Stanley (STA) and Safrane (SAF) (mean ± SD, n = 5); p < 0.05.

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radical scavenging capacity determined by ORAC assay strongly correlated positively with the total flavonoid and total phenolic contents (R2 = 0.77, p = 0.04; R2 = 0.89, p = 0.01). 3.4. Correlation study between TPC, TFC and total antioxidant activity of the onion varieties tested The correlations between TPC and TFC and total antioxidant activities are shown in Fig. 7. Poor correlation was observed between TFC in onions and their DPPH activity and TPC in onions and their DPPH activity (0.23 and 0.43 respectively, p > 0.0.5). On the other hand, positive correlation was observed between TFC in onions and their FRAP activity (0.96), indicating increased amounts of TFC in onions lead to increased FRAP activity. Also, excellent correlation was observed between TPC and ABTS activity (0.98), indicating increased amounts of TPC in onions lead to increased ABTS activity. Two linear regression relationships are developed for these activities as shown below and both the relations are statistically significant.

FRAP activityðas mM ascorbic acid equivalentÞ ¼ 0:705  TFCðas mg=g of onion as quercetin equivalentÞ  0:032; R2 ¼ 0:92; p < 0:05

ð2Þ

ABTS activityðas a %inhibitionÞ ¼ 34:94 þ 30:64 TPCðas mg=gÞ; R2 ¼ 0:97; p < 0:05

ð3Þ

Although there was a good correlation between TFC and ABTS activity (0.72), the linear regression relation between these two parameters were not statistically significant.

ABTS activityðas %inhibitionÞ ¼ 69:19 þ 66:16 TPCðas mg=gÞ; R2 ¼ 0:53; p < 0:05

ð4Þ

Similarly, although there was a good correlation between TPC and FRAP activity (0.82), the linear regression relation between these two parameters was not statistically significant.

FRAP activityðas mM ascorbic acid equivalentÞ ¼ 0:205  TPCðas mg=g of onion as quercetin equivalentÞ  0:207; R2 ¼ 0:67; p < 0:05:

ð5Þ

Poor correlation was observed between lipid peroxidation activity of onions and their TFC (correlation coefficient = 0.35, p > 0.05) and lipid peroxidation and TPC (correlation coefficient = 0.18, p > 0.05). Similarly, poor correlation was observed between the onions’ TAC and TFC (correlation coefficient = 0.455, p > 0.05) and the onions’ TAC and TPC (correlation coefficient = 0.398, p > 0.05). There was a good correlation observed between TPC and ORAC radical scavenging activity and the linear regression relation between these two parameters was statistically significant.

ORAC radical scavenging activityðas lM TE =g of sampleÞ ¼ 13:152  TPCðas mg=g of onion as quercetin equivalentÞ þ 8:9317; R2 ¼ 0:8955; p  value < 0:05: ORAC radical scavenging activityðas

ð6Þ

lM TE=g of sampleÞ

¼ 35:986  TFCðas mg=g of onion as quercetin equivalentÞ þ 22:115; R2 ¼ 0:7745; p  value < 0:05

ð7Þ

Good correlation was observed between TFC and ORAC radical scavenging activity and the linear regression relation between these two parameters was statistically significant. Our results are in agreement with other related work which indicated that a very good correlation was observed between total

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polyphenols, total flavonoids and antioxidant activities (Gheldof & Engeseth, 2002; Holasova et al., 2002; Rezaeizadeh et al., 2011). The quality of flavonoid extracts from onions depends on the technological processes involved in their extraction. In our study, the chemical characterization of polyphenolic content present in the five varieties suggested that phenolic-enriched extracts exhibit strong antioxidant activity. It also specified that synergistic combination of uncharacterized molecules explains the differences in quantification in the biological activities measured from the extracts of different varieties of onion. Successful screening and characterization of Ontario-grown onion varieties will allow the producers to grow the best and preferred variety. 3.5. Structural activity The radical scavenging activity of flavonoids depends on the substitution pattern and number of hydroxyl groups. Flavonoids are made up of fifteen-carbon skeletons which consist of two benzene rings (A and B rings) that are linked via a hetrocyclic pyrane ring (C ring) (Rice-Evans, Miller, & Paganga, 1996). The individual flavonoids such as quercetin, kaempferol, and myricetin differ in the pattern of substitution in the A and B rings. Quercetin has five OH groups, two each in A and B rings and one in the C ring. Kaempferol has 4 OH groups, namely two and one in A and B rings respectively. Myricetin has 6 OH groups and hence it is a potent anti-oxidant. It is known that three structural features determine the antioxidant potential (Wolfe & Liu, 2008) of flavonoids: (i) the o-dihydroxyl (OH) (catechol) structure in the B ring; (ii) the 2,3-double bond in conjugation with a 4-oxo function in the C ring; and (iii) presence of both 3- and 5-hydroxyl groups. The flavonoid quercetin consists of these three structural features and has shown itself to be a potent antioxidant. The function of antioxidants as scavengers of free radicals is that they can donate a hydrogen atom to the radicals (Carocho & Ferreira, 2013). Hydroxylation of the flavonoids’ A-ring, especially the 5- and 7-hydroxylations, is beneficial for antioxidant activity. The structural requirement considered essential for effective radical scavenging is 30 ,40 -dihydroxy groups, i.e., an o-dihydroxystructure (catechol structure) of the B ring, which possesses electron donating properties and is a radical target. The 3-OH moiety of the C ring is also beneficial for the antioxidant activity of flavonoids (Van Acker et al., 1996). The C2AC3 double bond conjugated with a 4-ketogroup in the C ring, which is responsible for electron delocalization from the B ring, further enhances the radical-scavenging capacity. The saturation of the 2,3-double bond is one of the causes of loss of activity (Rice-Evans et al., 1996). The presence of both 3-OH and 5-OH groups, in combination with a 4-carbonyl function and C2AC3 double bond, increases the radical scavenging activity when compared to the basic structure (Heijnen, Haenen, Van Acker, Van der Vijgh, & Bast, 2001). In the absence of an o-dihydroxy structure of the B ring, hydroxyl substituents in a catechol structure on the A-ring were able to compensate and become a larger determinant of the flavonoid antiradical activity (Arora, Nair, & Strasburg, 1998). The presence of the C2AC3 double bond, the OH-linked to the C3 position can lead to electronic oxidation to produce a hydroxyl radical, and unpaired electrons can delocalize in C2 and B ring. Several studies acknowledged the importance of the C2AC3 double bond, since it contributes to the antioxidant activity of flavonoids (Liyana-Pathirana & Shahidi, 2005). Flavonoids that contain more hydroxyl groups (one to six OH groups) have higher free radical and superoxide anion radical scavenging abilities, such as kaempferol, quercetin, myricetin and quercetin. The relationship between the number of hydroxyl groups and ORAC activity (Peroxyl radical absorbing activity) in flavones as observed in our study is linear. Flavones with a single OH had

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Fig. 7. (A) Correlation between TPC and percentage of antioxidant activity (ABTS scavenged effect). (B) Correlation between TFC and percentage of antioxidant activity (ABTS scavenged effect). (C) Correlation between TPC and percentage of antioxidant activity (FRAP activity). (D) Correlation between TFC and percentage of antioxidant activity (FRAP activity). (E) Correlation between TPC and oxygen radical absorbance capacity (ORAC activity). (F) Correlation between TFC and oxygen radical absorbance capacity (ORAC activity).

a low ORAC. ORAC activities of flavones with three to five OH substitutions were 2.6–3.9-fold greater than that of Trolox. Kamepferol exhibited lower ORAC activity than quercetin while myricetin exhibited the highest ORAC activity. More OH

substitutions lead to stronger ORAC Peroxyl radical absorbing activity. The activities of flavonoids in absorbing hydroxyl radicals produced by Cu2/-H2O2 increased proportionally to flavonoid concentration at low concentrations, but declined after reaching

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a maximum ORAC. The flavonoids that contain multiple OH substitutions have very strong antioxidant activities against peroxyl radicals. The order of free radical scavenging activities observed by Sim et al. (2007) in their DPPH assay was myricetin (six OH) > quercetin (five OH) > kaempferol (four OH), which suggests that an increase in the number of OH groups enhances scavenging of free radicals. Film-forming solutions containing onion flavonoids could potentially act as a sustained release of active molecules to enhance bioavailability of antioxidants in the food matrix. Powder phase ingredients from onions could be one kind of by-product that could be easily incorporated as a functional food antioxidant agent. The flavonoid solution could be directly applied or mixed with food or various dilutions upon consideration of the flavor and odor for enhancing the delivery of antioxidants. Integration of antioxidants, such as flavonoids, from onions into a food product must be carefully addressed by taking into account the onion variety, the type of extraction process and the flavor and odor. The flavors and odors from onions can be either desirable or highly undesirable as per the consumer perception. Due to its intrinsic properties, the type of target food system will decide the incorporation method and form (size, shape) of onion-based flavonoids to be added. Bakery products (for example: bread with 2% onion extracts) could be suitable for adding onion based flavonoids as it would significantly enhance the bioavailability of antioxidants. Fruit juices and soft drinks can be easily incorporated with the natural onion-based flavonoids, as the dispersion of the flavonoids would be rapid due to the low pH and low lipid contents. In addition, meat, poultry, and seafood products, along with dairy products, pasta sauce bases, snacks, alcoholic drinks and vegetable by-products could also benefit from the addition of flavonoids from onions. In addition to food, they can also be incorporated into health products such as creams and lotions. Driven by an increase in consumers’ health awareness, there is a robust and rapid growth of the market for natural antioxidants in Ontario and across North America. The global market for functional foods is estimated to be worth more than $60 billion. Over 750 Canadian companies specializing in the functional foods and natural health products area are garnering about $11 billion in revenues (Agricultural and Agri-Food Canada, 2016), of which Ontario is a major supplier of functional foods and natural health products. Successful screening and characterization will allow the producers to grow the best and preferred candidates of onion varieties with a defined set of criteria. Bioactive components from Ontario-grown onions include volatile sulfur containing compounds and health enhancers (antioxidants such as quercetin). As a result of this research, it will be possible to select optimum quality varieties and develop processing techniques to extract active components. Nutraceutical manufacturers and food processors would eventually benefit as they would enhance their profits by selling new antioxidant products from Ontario-grown onions. Bakers and other food manufacturers will also benefit from the availability of Ontario onion antioxidants by introducing new food products.

4. Conclusions The Ruby Ring variety showed the highest antioxidant activity and total polyphenolic content compared to all the tested varieties. Quercetin was identified as the major component in the yellow onion varieties. A strong correlation was found between total polyphenolic and total flavonoid content of onions against the observed antioxidant activities. Our current research work can provide consumers with increased awareness of the health benefits of different varieties of Ontario-grown onions. Flavonoids from Ontario-grown onions serve as a promising source of natural

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antioxidants for nutraceuticals or value added products. The knowledge of specific differences in the flavonoid and phenolic contents among the various types of onions may be of potential value to farmers, as they can select the best onion variety to cultivate or grow that has useful health benefits. Conflict of interest The authors declare no conflicts of interest. Acknowledgments The authors sincerely thank the Ontario Ministry of Research and Innovation (0520512), Natural Sciences and Engineering Research Council of Canada (400929) and the Ontario Ministry of Agriculture, Food and Rural Affairs (500135) for funding this study. The authors also thank the Holland Marsh Growers Association for donating onion samples. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jff.2017.01.037. References Agriculture and Agri-Food Canada (2016). Functional foods and natural health products (Accessed on August 11, 2016) http://www.agr.gc.ca/eng/industrymarkets-and-trade/statistics-and-market-information/by-productsector/functional-foods-and-natural-health-products/?id=1170856376710. Albishi, T., John, J. A., Al-Khalifa, A. S., & Shahidi, F. (2013). Antioxidative phenolic constituents of skins of onion varieties and their activities. Journal of Functional Foods, 5, 1191–1203. Arora, A., Nair, M. G., & Strasburg, G. M. (1998). Structure–activity relationships for antioxidant activities of a series of flavonoids in a liposomal system. Free Radical Biology and Medicine, 24(9), 1355–1363. Carocho, M., & Ferreira, I. C. (2013). A review on antioxidants, prooxidants and related controversy: Natural and synthetic compounds, screening and analysis methodologies and future perspectives. Food and Chemical Toxicology, 51, 15–25. Cheng, A., Chen, X., Jin, Q., Wang, W., Shi, J., & Liu, Y. (2013). Comparison of phenolic content and antioxidant capacity of red and yellow onions. Czech Journal of Food Sciences, 31(5), 501–508. De Nardo, T., Shiroma-Kian, C., Halim, Y., Francis, D., & Rodriguez-Saona, L. E. (2009). Rapid and simultaneous determination of lycopene and b-carotene contents in tomato juice by infrared spectroscopy. Journal of Agricultural and Food Chemistry, 57(4), 1105–1112. Fidrianny, I., Permatasari, L., & Wirasutisna, K. R. (2013). Antioxidant activities from various bulbs extracts, of three kinds allium using DPPH, ABTS assays and correlation with total phenolic, flavonoid, carotenoid content. International Journal Research of Pharmaceutical Science, 4, 438–444. Formica, J. V., & Regelson, W. (1995). Review of the biology of quercetin and related bioflavonoids. Food and Chemical Toxicology, 33, 1061–1080. Gheldof, N., & Engeseth, N. J. (2002). Antioxidant capacity of honeys from various floral sources based on the determination of oxygen radical absorbance capacity and inhibition of in vitro lipoprotein oxidation in human serum samples. Journal of Agricultural and Food Chemistry, 50, 3050–3055. Gianessi, L. (2013). Canadian onion production depends on fungicides. Washington DC: Crop Life Foundation (Report 77, www.croplife.org). Heijnen, C. G. M., Haenen, G. R. M. M., Van Acker, F. A. A., Van der Vijgh, W. J. F., & Bast, A. (2001). Flavonoids as peroxynitrite scavengers: The role of the hydroxyl groups. Toxicology In Vitro, 15(1), 3–6. Hertog, M. G. (1996). Epidemiological evidence on potential health properties of flavonoids. Proceedings of the Nutrition Society, 55(1B), 385–397. Hertog, M. G. L., Hollman, P. C. H., & Katan, M. B. (1992). Content of potentially anticarcinogenic flavonoids of 28 vegetables and 9 fruits commonly consumed in the Netherlands. Journal of Agricultural and Food Chemistry, 40, 2379–2383. Holasova, M., Fiedlerova, V., Smrcinova, H., Orsak, M., Lachman, J., & Vavreinova, S. (2002). Buckwheat—the source of antioxidant activity in functional foods. Food Research International, 35, 207–211. Karakaya, S. (2004). Bioavailability of phenolic compounds. Critical Reviews in Food Science and Nutrition, 44, 453–464. Kedare, S. B., & Singh, R. P. (2011). Genesis and development of DPPH method of antioxidant assay. Journal of Food Science and Technology, 48, 412–422. King, M. (2014). Food preservative market is shifting towards natural preservatives to align with consumer demands Retrieved from http://uk.finance.yahoo.com/ news/food-preservative-market-shifting-towards-000000922.html.

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