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In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. www.rsc.org/foodfunction Page 1 of 30 Food & Function Inhibitory effects of selected dietary flavonoids on the formation of total heterocyclic amines and 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) in roast beef patties and in chemical models Qin Zhu,a Shuang Zhang,b Mingfu Wang,c Jie Chenb,d,, and Zong-Ping Zhengb, aCollege of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, Zhejiang, 310036, People’s Republic of China bState Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Manuscript Jiangsu 214122, People’s Republic of China cSchool of Biological Sciences, The University of Hong Kong, Hong Kong, People’s Republic of China d Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Accepted Jiangsu, 214122, People’s Republic of China * To whom correspondence should be addressed: Function Zong-Ping Zheng & Tel./Fax:+86 510 85327850 E-mail address: [email protected] Food Jie Chen Tel.: +86-51085329032. Fax: +86-51085329065. E-mail address: [email protected] Food & Function Page 2 of 30 1 Abstract 2 In this study, the inhibitory effects of eight kinds of dietary flavonoids on the formation of 3 heterocyclic amines (HAs) were investigated in roast beef patties. The results showed that 4 most of them exhibited significant inhibition on both total HAs and 2-amino-1-methyl-6- 5 phenylimidazo[4,5-b]pyridine (PhIP) as one of the most abundant HAs. Among them, 6 phlorizin, epigallocatechin gallate (EGCG), and qucercetin were found to be the most 7 effective in both the reduction of total HAs (55~70%) and PhIP (60~80%). The reaction 8 activities between flavonoids and phenylacetaldehyde (key intermediate of PhIP formation) Manuscript 9 showed a good correlation with the inhibition of flavonoids PhIP formation in aqueous 10 model system (R2 = 0.8904) and di(ethylene) glycol reaction system (R2 = 0.6514). 11 However, no significant correlation was found between the antioxidant capacities of Accepted 12 flavonoid and PhIP formation (R2 = 0.2359). The postulated adducts of flavonoids- 13 phenylacetaldehyde were further confirmed by LC-MS analysis in chemical models. Since 14 phenylacetaldehyde was the chief intermediate of PhIP formation, these results combined 15 suggested that the inhibitory effects of flavonoids on PhIP formation were mainly depended Function & 16 on their abilities to trap phenylacetaldehyde, instead of their antioxidant capacities. 17 Keywords: heterocyclic amines, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine Food 18 (PhIP), dietary flavonoids, phenylacetaldehyde, phenylacetaldehyde-flavonoid adducts 19 20 21 Introduction Page 3 of 30 Food & Function 22 The treatment of heating on meat and fish products generates a class of heterocyclic amines 23 (HAs).1,2 These HAs are mainly formed with amino acids, sugars and creatin(in)e or other 24 aldehydes as precursors during food processing.3 Although they present at very low 25 concentration (at ppb range) in foods, the safety issues regarding their mutagenicity have 26 been known in bacteria mutagenicity4 and in animal carcinogenicity.5 Their potential health 27 risks for human beings have been further recognized by people during the recent years. 28 Many evidences have proved that the intake of meat rich in HAs is associated with an 6 7 29 increased risk for several kinds of cancer, such as pancreatic cancer, colon cancer, prostate Manuscript 30 cancer,8 breast cancer,9 and lung cancer.10 Up to now, more than 25 HAs have been 31 identified from the food samples with structures elucidation, and 2-amino-1-methyl-6- 32 phenylimidazo[4, 5-b]pyridine (PhIP) is one of the most abundant HAs in addition to 2- Accepted 33 amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3,4,8- 34 trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx).11,12 Because the precursors of HAs are 35 food components and HAs are hard to are hard to be removed once they are already formed, 36 it is significant to find out potent inhibitors in their formation pathways to reduce their Function & 37 amount in foods. 38 The identification of possible inhibitors from dietary plants is one of the effective Food 39 strategies for their easy incorporation in daily cuisine. Plant extracts, including fruit 40 extracts, spices, and essential oils are regarded as health-promoting foods with anti-oxidant 41 and anti-tumor effects.13,14 They were also reported to have the ability to effectively inhibit 42 the formation of HAs.3,15-20 Certain dietary phenolic compounds from the plant extracts Food & Function Page 4 of 30 43 were also identified as powerful inhibitors.18-20 44 The popular mechanism involved in the inhibition of HA formation by these food- 45 derived phenolic compounds is proposed as free radical scavenging (antioxidant) activity 46 in previous studies21. Recently, another possible mechanism was proposed that these 47 substance may inhibit HAs formation through trapping the precursors or intermediates in 48 HA formation pathways.18,19,22 The postulated inhibition mechanism of dietary flavonoids 49 (naringenin and EGCG) on PhIP formation was the direct reaction with the intermediate 50 phenylacetaldehyde. However, there has been debate on the HA inhibition mechanism Manuscript 51 because of complexity of the reaction matrix, and it is significant to clarify the principal 52 mechanism responsible for dietary flavonoids’ intervention on HA formation. 53 The aims of present study were to evaluate the inhibitory effects of dietary phenolic Accepted 54 compounds on HA formation; to investigate the inhibitory mechanism of total HAs and 55 PhIP formation by different structure types of flavonoids (Figure 1); and find out the 56 correlation of possible mechanism involved. Effects of flavonoids on total HA formation 57 is firstly evaluated in roast beef patties UPLC/MS analysis. For the mechanism elucidation, Function & 58 the intermediates-scavenging ability was investigated in chemical models and PhIP’s 59 precursor phenylacetaldehyde was chosen as the typical intermediates in HA formation Food 60 with further confirmation of these adducts in roast beef patties. The antioxidant capacity of 61 these flavonoids was accessed by ABTS radical scavenging assay. Finally, the relationship 62 between the two mechanism in HA formation was calculated in order to clarify their 63 contribution. Page 5 of 30 Food & Function 64 Materials and methods 65 Chemicals 66 Phenylacetaldehyde, diethylene glycol, ethyl acetate, methanol and acetonitrile were 67 purchased from Sinopharm Chemical Reagent Co, Ltd. (PR China). Apigenin, luteolin, 68 kaempferol, quercetin, genistein, phlorizin, and epigallocatechin gallate (EGCG) were 69 purchased from Nanjing Ze Lang Medical Technology CO, Ltd. (PR China). The standards 70 of HAs 2-amino-1-methyl-6-phenylimidazo [4,5-b]-pyridine (PhIP), 2-amin-1, 6- 71 dimethylimidazo[4,5-b]pyridine (DMIP), 2-amino-3,8-dimethylimidazo[4,5-f]- Manuscript 72 quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo [4,5-f]-quinoxaline4,8-(DiMeIQx), 73 1-methyl-9H-pyrido [3,4-b]indole (Harman), 9H-pyrido[3,4-b]indole (Norharman), and 2- 74 amino-1,5,6-trimethylimidzao[4,5-b]-pyridine (1,5,6-TMIP) were purchased from Toronto Accepted 75 Research Chemicals (Toronto, Canada). Oasis MCX cartridges (3 mL/60 mg) were 76 purchased from Waters (Milford, Massachusetts, USA). 2,2'-Azinobis-(3- 77 ethylbenzothiazoline-6-sulfonic acid) (ABTS) and naringenin were purchased from 78 Sigma-Aldrich Co. (St. Louis, MO). Caffeine was provided by J&K Chemical Ltd. Function & 79 (Shanghai, China). Fresh ground beef (hindquarter) was purchased from local supermarket. 80 LC/MS analysis of HAs in roast beef patties Food 81 The ground beef (40 g) was thoroughly mixed with different kinds of powdered flavonoids 82 (0.2 mM), and then was formed into beef patties with a Petri dish (6 × 1.5 cm). The patties 83 were covered by a Petri dish and were allowed to incubate under 4 °C conditions for 8 h in 84 the refrigerator before frying. The beef patties were roasted in an electric oven for 20 min Food & Function Page 6 of 30 85 (10 min per side) at 230 °C . Three samples were taken for each treatment in each 86 experiment. 87 Beef patties were homogenized after cooling according to the previous method.23 An 88 amount of 5 g roast beef was dissolved into 30 mL 1 M NaOH and homogenized for 2 min. 89 After that, the viscous solution was mixed with diatomaceous earth and poured into empty 90 extrelut columns with ethyl acetate as the eluent. The concentrated eluent was then 91 transferred into the Oasis MCX cartridges. The cartridges were washed with 3 mL 0.1 M 92 HCl and 3 mL MeOH twice, respectively. The analytes were eluted with 2 mL of MeOH- Manuscript 93 aqueous ammonia mixture (19:1) and concentrated under a nitrogen flushing and the 94 residue was dissolved in 300 μL MeOH and subject to UPLC/MS analysis.20,23-25 95 Fresh working standard solutions were prepared daily by mixing and diluting each stock Accepted 96 standard solution in methanol to obtain different concentrations with similar peak areas.