Isorhamnetin and Hispidulin from Tamarix Ramosissima Inhibit
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foods Article Isorhamnetin and Hispidulin from Tamarix ramosissima Inhibit 2-Amino-1-Methyl-6- Phenylimidazo[4,5-b]Pyridine (PhIP) Formation by Trapping Phenylacetaldehyde as a Key Mechanism Xiaopu Ren 1,2 , Wei Wang 1, Yingjie Bao 1, Yuxia Zhu 1, Yawei Zhang 1, Yaping Lu 3, Zengqi Peng 1,* and Guanghong Zhou 1 1 Key Laboratory of Meat Processing and Quality Control, Ministry of Education China, Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China; [email protected] (X.R.); [email protected] (W.W.); [email protected] (Y.B.); [email protected] (Y.Z.); [email protected] (Y.Z.); [email protected] (G.Z.) 2 Xinjiang Production & Construction Group Key Laboratory of Agricultural Products Processing in Xinjiang South, College of Life Science, Tarim University, Alar 843300, China 3 College of Life Science, Nanjing Agricultural University, Nanjing 210095, China; [email protected] * Correspondence: [email protected]; Tel.: +86-25-84396558 Received: 7 March 2020; Accepted: 18 March 2020; Published: 3 April 2020 Abstract: Tamarix has been widely used as barbecue skewers to obtain a good taste and a unique flavor of roast lamb in China. Many flavonoids have been identified from Tamarix, which is an important strategy employed to reduce the formation of heterocyclic amines (HAs) in roast meat. Isorhamnetin, hispidulin, and cirsimaritin from Tamarix ramosissima bark extract (TRE) effectively inhibit the formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP), the most abundant HAs in foods, both in roast lamb patties and in chemical models. According to the results of the GC-MS analysis, TRE and the three flavonoids significantly reduced the contents of phenylacetaldehyde, an important intermediate involved in PhIP formation at three levels. A subsequent ultra performance liquid chromatography-mass spectrometry (UPLC-MS) analysis revealed that these flavonoids trapped phenylacetaldehyde by forming interaction adducts. The formation of three postulated adducts, 8-C-(E-phenylethenyl)isorhamnetin, 6-C-(E-phenylethenyl)isorhamnetin and 8-C-(E-phenylethenyl)hispidulin, in the chemical models and roast lamb patties was further confirmed by a TOF-MS/MS analysis. Our results demonstrate that TRE and the corresponding flavonoids trap phenylacetaldehyde to form adducts and thus inhibit PhIP formation, suggesting their great potential beneficial effects on human health. Keywords: 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP); flavonoid; flavonoid- phenylacetaldehyde adducts; model system; roast lamb patties 1. Introduction Tamarix is considered as an interesting potential source of functional food due to its abundant natural flavonoids [1]. Many flavonoids have been identified from various species of Tamarix, such as quercetin, isoquercetin, apigenin, amentoflavone, (+)-catechin, rhamnocitrin, isorhamnetin, tamarixetin, chrysoeriol and rhamnazin [1–4]. Hispidulin and cirsimaritin were firstly identified among 13 flavonoids detected in the genus Tamarix from southern Xinjiang, along with isorhamnetin, which are the main three flavonoids present in T. ramosissima (Figure1)[ 5]. Tamarix has been widely used as barbecue Foods 2020, 9, 420; doi:10.3390/foods9040420 www.mdpi.com/journal/foods Foods 2020, 9, x FOR PEER REVIEW 2 of 16 used as barbecue skewers to obtain a good taste and a unique flavor of roast lamb in China, specifically in southern Xinjiang. However, the roasting of meat generates heterocyclic amines (HAs), and the International Agency for Research on Cancer (IARC) classified processed meat as “carcinogenic to humans” (group 1), based on more than 800 epidemiological studies that reported a link between meat consumption and cancer. Concerning roasted and barbecued meat, HAs were pointed out as components with high carcinogenic potential [6], of which 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine (PhIP) is well known as the most abundant HA in roast meat, with levels of up to 480 ng/g detected [7]. PhIP is also the largest contributor to the estimated average daily HA exposure in China (29.06 ng/day, 58%) [8]. PhIP is regarded as a possible human carcinogen (class 2B) by IARC [9] and is also listed as reasonably anticipated to be a human carcinogen by the National Toxicology Program [10]. The factors that affected the formation of PhIP in foods including the cooking methods, cooking temperature, cooking time, the amounts of precursors and the content of substances with enhancing or inhibiting effects on the formation of PhIP [11]. Many strategies have been employed to reduce the formation of PhIP. Many works have Foods 2020demonstrated, 9, 420 that HAs can be inhibited by natural antioxidants, such as spices and extracts rich in 2 of 15 polyphenols [12–14]. However, the mechanisms underlying the effect have been overlooked. In the present work, the mechanism of action was explored more in-depth. Several flavonoids exhibited skewerseffective to obtain inhibition a good on PhIP. taste According and a unique to Zhu flavor et al. [15], of roast apigenin, lamb luteolin, in China, kaempferol, specifically naringenin, in southern Xinjiang.quercetin, However, genistein, the roasting phlorizin of and meat epigallocatechin generates heterocyclic gallate (EGC aminesG) added (HAs), to roast and thebeef International patties significantly inhibited PhIP formation and reduced its concentration by 60–80%. Norartocarpetin, Agency for Research on Cancer (IARC) classified processed meat as “carcinogenic to humans” (group 1), quercetin and naringenin effectively inhibit PhIP formation in roast beef patties [16]. Naringenin and based on more than 800 epidemiological studies that reported a link between meat consumption and quercetin directly react with phenylacetaldehyde, an important intermediate involved in PhIP cancer.formation, Concerning to form roasted interaction and adduct, barbecued thus leading meat,HAs to a significant were pointed reduction out in as PhIP components levels in model with high carcinogenicsystems potentialand real meat [6], ofsamples. which Phenylacetaldehyde 2-amino-1-methyl-6-phenylimidazo[4,5- scavenging is considered ba]pyridine key mechanism (PhIP) of is well knownnaringenin as the most and abundant quercetin HA [15,17]. in roast Isorhamnetin meat, with is levels a 3′-O of-methylated up to 480 ngmetabolite/g detected of [quercetin.7]. PhIP is also the largestHispidulin contributor and cirsimaritin to the estimated exhibit similar average structural daily HA characteristics exposure into Chinanaringenin. (29.06 Based ng/day, on the 58%) [8]. PhIP isinhibitory regarded effects as a possibleof flavonoids human on carcinogenPhIP formation (class that 2B) depend by IARC upon [9 the] and structural is also listedcharacteristics, as reasonably anticipatedresearchers to be have a human hypothesized carcinogen that isorhamnetin, by the National hispidulin Toxicology and cirsimaritin Program from [10]. T.ramosissima The factors that effectively inhibit PhIP formation, which has rarely been reported in real meat systems. The present affected the formation of PhIP in foods including the cooking methods, cooking temperature, cooking study aimed to investigate the abilities of T.ramosissima bark extract (TRE), isorhamnetin, hispidulin time, theand amountscirsimaritin of to precursors inhibit the andformation the content of PhIP of in substances the model system with enhancing and roast lamb or inhibiting patties and e fftoects on the formationexplore the of probable PhIP [11 inhibitory]. mechanism. Isorhamnetin Hispidulin Cirsimaritin Figure 1. Structures of the three flavonoids derived from Tamarix ramosissima bark extract (TRE). Figure 1. Structures of the three flavonoids derived from Tamarix ramosissima bark extract (TRE). 2. Materials and Methods Many strategies have been employed to reduce the formation of PhIP. Many works have demonstrated2.1. Reagents that and HAs Materials can be inhibited by natural antioxidants, such as spices and extracts rich in polyphenols [12–14]. However, the mechanisms underlying the effect have been overlooked. Phenylalanine, glucose, creatinine, diethylene glycol and phenylacetaldehyde were purchased In thefrom present Yuanye work, Bio-Technology the mechanism Co., Ltd. of (Shanghai, action was China). explored Isorhamnetin, more in-depth.hispidulin and Several cirsimaritin flavonoids exhibitedwere effobtainedective inhibitionfrom Sigma-Aldr on PhIP.ich According(Shanghai, China). to Zhu The et al.PhIP [15 standard], apigenin, was luteolin,purchased kaempferol, from naringenin,Toronto quercetin, Research Chemicals genistein, (Downsview, phlorizin and Ontario, epigallocatechin Canada). Oasis gallate MCX (60 (EGCG) mg, 3 addedmL) cartridges to roast beef pattieswere significantly supplied inhibitedby Waters PhIP(Milford, formation MA, USA) and an reducedd diatomaceous its concentration earth was by purchased 60–80%. from Norartocarpetin, Merck quercetin and naringenin effectively inhibit PhIP formation in roast beef patties [16]. Naringenin and quercetin directly react with phenylacetaldehyde, an important intermediate involved in PhIP formation, to form interaction adduct, thus leading to a significant reduction in PhIP levels in model systems and real meat samples. Phenylacetaldehyde scavenging is considered a key mechanism of naringenin and quercetin [15,17]. Isorhamnetin is a 30-O-methylated metabolite of quercetin. Hispidulin and cirsimaritin