Inhibition of the CYP Enzymatic System Responsible of Heterocyclic Amines Bioactivation by an Asclepias subulata Extract.

Asclepias subulata plant extract has previously demonstrated antiproliferative activity and antimutagenicity against heterocyclic aromatic amines (HAAs) commonly found in cooked meat. The objective of this work was to evaluate the in vitro ability of an ethanolic extract from the medicinal plant Asclepias subulata extract (ASE), non-heated and heated (180 °C), to inhibit the activity of CYP1A1 and CYP1A2, which are largely responsible for HAAs bioactivation. Ethoxyresorufin and methoxyresorufin O-dealkylation assays were performed in rat liver microsomes exposed to ASE (0.002-960 µg/mL). ASE exerted an inhibitory effect in a dose-dependent manner. The half inhibitory concentration (IC50) for unheated ASE was 353.6 µg/mL and 75.9 µg/mL for heated ASE in EROD assay. An IC40 value of 288.4 ± 5.8 µg/mL was calculated for non-heated ASE in MROD assay. However, after heat treatment, the IC50 value was 232.1 ± 7.4 µg/mL. Molecular docking of corotoxigenin-3-O-glucopyranoside, one of the main components of ASE, with CYP1A1/2 structure, was performed. Results show that the interaction of corotoxigenin-3-O-glucopyranoside with CYP1A1/2s' α-helices, which are related with the active site and the heme cofactor, may explain the plant extract's inhibitory properties. Results showed that ASE inhibits CYP1A enzymatic subfamily and may potentially act as a chemopreventive agent by inhibiting bioactivation of promutagenic dietary HAAs.

ß-Carboline Alkaloids in Soy Sauce and Inhibition of Monoamine Oxidase (MAO).

Monoamine oxidase (MAO) oxidizes neurotransmitters and xenobiotic amines, including vasopressor and neurotoxic amines such as the MPTP neurotoxin. Its inhibitors are useful as antidepressants and neuroprotectants. This work shows that diluted soy sauce (1/3) and soy sauce extracts inhibited human MAO-A and -B isozymes in vitro, which were measured with a chromatographic assay to avoid interferences, and it suggests the presence of MAO inhibitors. Chromatographic and spectrometric studies showed the occurrence of the ß-carboline alkaloids harman and norharman in soy sauce extracts inhibiting MAO-A. Harman was isolated from soy sauce, and it was a potent and competitive inhibitor of MAO-A (0.4 µM, 44 % inhibition). The concentrations of harman and norharman were determined in commercial soy sauces, reaching 243 and 52 µg/L, respectively. Subsequently, the alkaloids 1,2,3,4-tetrahydro-ß-carboline-3-carboxylic acid (THCA) and 1-methyl-1,2,3,4-tetrahydro-ß-carboline-3-carboxylic acid (MTCA) were identified and analyzed in soy sauces reaching concentrations of 69 and 448 mg/L, respectively. The results show that MTCA was a precursor of harman under oxidative and heating conditions, and soy sauces increased the amount of harman under those conditions. This work shows that soy sauce contains bioactive ß-carbolines and constitutes a dietary source of MAO-A and -B inhibitors.

Effect of Different Roasting Conditions and Coreopsis Extract on Heterocyclic Amine Formation in Roast Lamb Products.

ABSTRACT: Heterocyclic amines (HCAs), which are known carcinogens in thermally processed foods, were investigated in roast lamb patties under various time and temperature conditions. HCAs in lamb products roasted at some temperatures increased with roasting time. An exponential model with a time factor fit well for the production of HCAs. The mean pH and cooking loss at various temperatures were also determined. The mean pH decreased as the temperature increased. Coreopsis extract was added to lamb patties roasted at 230°C for 15 min per side. The amount of coreopsis extract added had a significant effect on HCA development. A weak positive relationship was observed between the antioxidant activity of the lamb patty with the coreopsis extract and the inhibitory effect of coreopsis extract on various HCAs, with a correlation coefficient of 0.14 to 0.44 (P > 0.05). Coreopsis extract containing flavonoids can be a beneficial additive for production of barbecue meat.

The effect of onion and garlic on non-polar heterocyclic aromatic amines (α-, ß-, γ- and δ-carbolines) formation in pan-fried meat and gravy.

Thermal treatment of protein-rich food can lead to the formation of biologically active heterocyclic aromatic amines (HAAs). One of the methods to learn how to reduce the content as well as the influence of these compounds on heath is the study of factors inhibiting their synthesis. In the current investigation, the effect of onion and garlic on the formation of six possibly carcinogenic non-polar HAAs (α-, γ- and δ-carbolines) and two co-mutagenic ß-carbolines (harmane and norharmane) was evaluated by comparing their contents in meat and gravy samples obtained from pan-fried pork dishes prepared in the presence and absence of these vegetables. Carbolines were isolated from food samples by solid phase extraction. The quantitative analysis was performed by high-performance liquid chromatography with fluorescence detection. The concentrations of individual compounds in dishes prepared without added vegetables ranged from 0.02 ng g-1 (3-amino-1,4-dimethyl-5 H-pyrido(4,3-b)indole; Trp-P-1) to 10.1 ng g-1 of meat (2-amino-9 H-pyrido[2,3-b]indole; AαC). Onion (30 g/100 g of meat) and garlic (15 g/100 g of meat) lowered the total content (in meat and gravy) of the α-, δ- and γ-carbolines in the range from 52% to 87%. In contrast, onion caused an increase in the norharmane concentration both in meat and gravy. The percentage of carbolines in the gravies (assuming that their total content in meat and gravy is 100%) was higher in dishes prepared with onion and garlic than in dishes without these seasonings.

Mitigation of heterocyclic amines by phenolic compounds in allspice and perilla frutescens seed extract: The correlation between antioxidant capacities and mitigating activities.

The effect of different levels of allspice and perilla frutescens seed extract (ASE and PSE) on the formation of heterocyclic amines (HCAs) in pan-fried chicken meat patties and the bioactive components found in ASE and PSE that contribute to the mitigation of HCAs were investigated in this study. DPPH radical scavenging activity was evaluated and the results indicated that APSE (ASE + PSE) showed the highest capacity to scavenge free radicals, and the most effective inhibition of HCAs formation. Furthermore, Single and mixed phenolic compounds exhibited a positive effect in scavenging free radicals and mitigating HCAs. The radical scavenging activity and HCAs inhibition effect of single phenolic compounds were highly correlated, whereas mixed phenolic compounds exhibited poor correlation. PCA analysis indicated that phenolic compounds had the maximum inhibitory effect on IQ, followed by Norharman and harman and the minimal effect on PhIP and 7,8-DiMeIQx.

Effects of ten vegetable oils on heterocyclic amine profiles in roasted beef patties using UPLC-MS/MS combined with principal component analysis.

Soybean oil (SBO), rapeseed oil (RSO), peanut oil (PO), corn oil (CO), olive oil (OO), sunflower oil (SFO), rice germ oil (RGO), walnut oil (WO), torreya seed oil (TSO), and grapeseed oil (GSO) were used to investigate the formation of heterocyclic amines (HAs) in roasted beef patties. Seven HAs, including 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4-dimethyl-3H-imidazo[4,5-f]quinolone (MeIQ), 2-amino-3-methyl-3H-imidazo[4,5-f]quinoxaline (IQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 1-methyl-9H-pyrido[3,4-b]indole (harman), and 9H-pyrido[3,4-b]indole (norharman) were detected in control patties and patties with vegetable oils. GSO, SFO, and WO greatly reduced the content of PhIP and MeIQ. 1.25%TSO and 3.75% RGO showed higher inhibition effects on the more strongly mutagenic compounds (PhIP, MeIQ, IQx, 4,8-DiMeIQx, MeIQx). SBO, PO, and RSO promoted imidazoquin(ox)aline (MeIQ, MeIQx, 4,8-DiMeIQx, and IQx) and ß-carboline (harman and norharman); 1.25% SBO had the most significant promoting effect on total HA. This could be useful for the reduction of HA by selecting oils during cooking.

Exploring the relationship between potato components and Maillard reaction derivative harmful products using multivariate statistical analysis.

The correlation between potato components and Maillard reaction derivative harmful products (MRDHPs) formation during heat-processing was assessed in nine commercial potato varieties in China. Principal component analysis (PCA) combined with canonical correlation analysis (CCA) approach was performed to explore their relationships. The variables contributing most to the PCA results were extracted for CCA, and the results indicated that several amino acids, including lysine, tryptophan, alanine, phenylalanine, aspartate, and glutamate, have significant impacts on acrylamide and ß-carboline heterocyclic amine formation. Moreover, 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, α-solanine, and α-chaconine were also important factors associated with acrylamide and ß-carboline heterocyclic amine formation. Optimally using raw potato varieties based on the impacts of these factors can help control MRDHP formation during thermal processing. For the first time, such approach was applied, which may be a useful tool for discovering the correlation of food components and MRDHPs.

Phytochemical-Rich Antioxidant Extracts of Vaccinium Vitis-idaea L. Leaves Inhibit the Formation of Toxic Maillard Reaction Products in Food Models.

Thermal treatment of proteinaceous foods generates heat-induced Maillard reaction substances including toxic advanced glycation end products (AGEs) and heterocyclic amines (HAs). It is known that plant phenolic compounds may influence Maillard reaction. This study investigated the impact of lingonberry leaf extracts on the formation of Nε -(carboxymethyl)lysine (CML) and Nε -(2-furoylmethyl)-L-lysine (furosine) in milk model system and HAs in meat-protein and meat model systems. In addition, lingonberry leaf extracts obtained by different solvents were characterized by radical scavenging, Folin-Ciocalteu assays and ultrahigh pressure liquid chromatography quadruple-time-of flight mass spectrometry (UPLC-qTOF-MS). Water extract (WE) stronger suppressed CML than furosine formation in milk model system: CML levels were reduced by nearly 40%. Moreover, quinic acid and catechin, which were abundant in WE, were effective in inhibiting CML and furosine formation. WE and acetone extract (AE) at 10 mg/mL significantly inhibited HAs formation in both model systems. However, higher suppressing effect on HAs formation showed AE, which had lower antioxidant capacity and total phenolic content values than WE. WE contained higher amounts of hydroxycinnamic acids, proanthocyanidins and flavonols, while AE was richer in flavan-3-ols and arbutin derivatives. It indicates that the composition of phenolics might be a major factor for explaining different effect of extracts from the same plant on HAs formation. In general, the results suggest that lingonberry leaves is a promising source of phytochemicals for inhibiting toxic Maillard reaction products and enriching foods with plant bioactive compounds. PRACTICAL APPLICATION: The increased consumption in processed foods has been linked with the increased risks of various diseases, while thermal food processing is required to develop flavor, insure safety, and extend shelf life. Therefore, developing effective technological means for inhibiting the formation of heat-induced toxic substances is an important task. This study showed a potential of lingonberry leaf extracts containing health beneficial phytochemicals to suppress the formation of toxic Maillard reaction products during heating of milk and meat.

Inhibitory effect of the extract from Sonchus olearleu on the formation of carcinogenic heterocyclic aromatic amines during the pork cooking.

The aim of this study was to assess the inhibitory effects of Sonchus olearleu extract on the generation of heterocyclic amines in roasted pork patties cooked by pan-frying. All samples were cooked for two different durations (45 min and 105 min) under 200 °C and 230 °C. 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-ami- no-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinox-aline (4,8-DiMeIQx), harman, and norharman were detected and quantified. In patties cooked at 230 °C for 105 min, S. olearleu extract (0.5%) significantly inhibited the formation of IQ, harman, and norharman by 39%, 67%, and 63%, respectively. In contrast to IQ, the levels of harman and norharman were significantly reduced by the extracts tested. However, no such effects were observed for MeIQx and 4, 8-DiMeIQx. Notably, the inhibitory effect on heterocyclic amines is significantly correlated with the antioxidant potential and total phenolic content of S. olearleu extract.

Inhibitory effect of Chrysanthemum morifolium flower extract on the formation of heterocyclic amines in goat meat patties cooked by various cooking methods and temperatures.

The effect of Chrysanthemum morifolium flower extract (CME) was investigated on the formation of heterocyclic amines (HCAs) in goat meat patties cooked at different temperatures by different cooking methods. In patties without CME, the level of total HCAs increased analogously with the increase in cooking temperature. The inhibitory effect of CME on HCAs ranged from 14% to 82%. The total HCAs content were reduced by 46%, 40% and 35% in pan fried, deep fat fried and oven roasted goat meat patties, respectively, at 175 °C. While, at 225 °C, the amount of total HCAs was decreased by 52%, 47% and 32% in deep fat fried, pan fried and oven roasted patties respectively, whereas, in barbecue patties the reduction was 36%. Hence, it is concluded that the effect of CME was more prominent in deep fat frying and pan frying cooking methods and increase in temperature enhance the efficacy of CME in both cooking methods.

Inhibitory effect of mixture herbs/spices on formation of heterocyclic amines and mutagenic activity of grilled beef.

Natural antioxidants in spices and herbs have attracted considerable attention as potential inhibitors against the formation of mutagenic heterocyclic amines (HCAs) in heat-processed meat. In this study, the inhibitory activity of four spices/herbs and their mixtures on HCAs formation in grilled beef were examined. A simplex centroid mixture design with four components comprising turmeric, curry leaf, torch ginger and lemon grass in 19 different proportions were applied on beef samples before grilling at 240 ºC for 10 min. The HCAs were extracted from the samples using solid phase extraction (SPE) method and analysed using Liquid chromatography mass spectrometry LC-MS/MS. All spices/herbs in single or mixture forms were found to reduce total HCA concentrations in marinated grilled beef ranging from 21.2% for beef marinated with curry leaf to 94.7% for the combination of turmeric and lemon grass (50:50 w/w). At the optimum marinade formula (turmeric: lemon grass 52.4%: 47.6%), concentration of 2-amino-3-methylimidazo[4,5-f]quinolone (IQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), Harman, Norharman and AαC were 2.2, 1.4, 0.5, 2.8 and 1.2 ng/g, respectively. The results of the mutagenic activity demonstrated that this optimised marinade formula significantly (p < 0.05) diminished mutagenicity of marinated grilled beef in bacterial Ames test.

In vitro inhibition of food borne mutagens induced mutagenicity by cinnamon (Cinnamomum cassia) bark extract.

Cinnamon (Cinnamomum cassia) is an important spice which is widely consumed in the Indian subcontinent as well as in several other parts of the world. In the present study, NMR spectroscopy showed the presence of cinnamaldehyde to be the major component of the bark. The possible mutagenic effects of cinnamon bark ethanolic extract (CEE, 0.01-1 mg/plate) cinnamon oil (CNO, 0.125-1 mg/plate), and its active component cinnamadehyde (CLD, 0.125-1 mg/plate) were evaluated. Antimutagenic activity of CEE, CNO, and CLD was also tested against various food borne mutagens (heterocyclic amines and aflatoxin B1 (AFB1)) and sodium azide (SA) using Ames assay. Similarly, the antimicrobial activity was studied using agar well diffusion assay against various pathogens. CEE was non-mutagenic in any of the five tester strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102, and TA104) in Ames test. CEE exhibited antimutagenic activity against all the mutagens tested in the higher doses. Additionally, CEE, CNO, and CLD were effective against various pathogens such as Staphylococcus aureus, Proteus vulgaris, S. typhimurium, Klebsiella pneumoniae, and Escherichia coli in the agar well diffusion assay. Promising antimutagenic and antimicrobial properties were shown by the cinnamon bark ethanolic extract and cinnamaldehyde, respectively. Therefore, their role in cancer chemoprevention, as well as a natural antimicrobial agent must be exploited and studied in depth in in vivo conditions.

Inhibitory effects of apple peel polyphenol extract on the formation of heterocyclic amines in pan fried beef patties.

The efficacy of polyphenol-rich dried apple peel extract (DAPP) to inhibit the formation of heterocyclic aromatic amines (HCAs) during frying of beef patties was assessed after DAPP was applied at 0.1, 0.15 and 0.3% (w/w) either on the surface of the patties or mixed inside the patty prior to frying. 2-Amino-3,8-dimethylimidazo[4,5f]quinoxaline (MeIQx), 2-amino-1-ethyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,4,8-dimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) were quantified after frying. HCA concentrations decreased (p<0.05) upon both surface and mixed applications of DAPP at all of the tested doses. Surface application of 0.3% DAPP showed greater (p<0.05) inhibitory effects on HCA formation by 68% for MeIQx, 56% for 4,8-DiMeIQx and 83% for PhIP as opposed to 41%, 21% and 60% respectively, for the mixed DAPP application of 0.3%. The present study results indicate that surface application of DAPP in meat preparation prior to pan-frying can be a useful approach to minimize the formation of genotoxic HCAs in fried beef patties.

Inhibitory Effect of Rosa rugosa Tea Extract on the Formation of Heterocyclic Amines in Meat Patties at Different Temperatures.

In previous studies, heterocyclic amines (HCAs) have been identified as carcinogenic and a risk factor for human cancer. Therefore, the present study was designed to identify bioactive natural products capable of controlling the formation of HCAs during cooking. For this purpose we have evaluated the effect of Rosa rugosa tea extract (RTE) on the formation of HCAs in ground beef patties fried at 160 °C or 220 °C. RTE is rich in phenolic compounds and capable of inhibiting the formation of free radicals. The pyrido[3,4-b]indole (norharman) and 1-methyl-9H-pyrido[3,4-b]indole (harman) contents were significantly (p < 0.05) decreased in RTE-treated patties at 220 °C. 9H-3-Amino-1-methyl-5H-pyrido[4,3-b]indole acetate (Trp-P-2) and 3-amino-1,4-dimethyl-5H-pyrido-[4,3-b]indole acetate (Trp-P-1) were not detected at 160 °C and were statistically (p < 0.01) reduced at 220 °C compared to the control. RTE remarkably inhibited the formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) at 220 °C (p < 0.001) and at 160 °C (p < 0.05). 2-Amino-9H-pyrido[2,3-b]indole (AαC) and 2-amino-3-methyl-9H-pyrido[2,3-b]-indole (MeAαC) were only detected in the control group at 160 °C but were comparatively (p > 0.05) similar in the control and treated groups at 220 °C. 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-3,4,8-trimethylimidazo[4,5-f]-quinoxaline (4,8-DiMeIQx) were not detected in any sample. Total HCAs were positively correlated with cooking loss. In the RTE-treated groups, 75% of the total HCAs were decreased at 160 °C and 46% at 220 °C, suggesting that RTE is effective at both temperatures and can be used during cooking at high temperatures to lessen the amount of HCAs formed.

Detoxification of chlorella supplement on heterocyclic amines in Korean young adults.

Heterocyclic amines (HCAs) and polycyclic aromatic hydrocarbons (PAHs) have been established as carcinogenic chemicals in Western diet. This study was performed to estimate HCA exposure levels in Korean daily life and to assess the ability of Chlorella vulgaris to detoxify carcinogenic HCAs in a randomized, double blind, placebo-controlled crossover study with chlorella supplement (N=6, all females, age: 27.17±7.73yr) for 2 weeks. We analyzed HCAs in hydrolyzed urine specimens using LC/TOF-MS. As results, urinary levels of MeIQx, PhIP, and IQx-8-COOH were 323.36±220.11ng/L, 351.59±254.93ng/L, and 130.85±83.22ng/L, respectively. Effects of chlorella to reduce urinary MeIQx were marginally significant (before, 430±226.86pg/mL vs. after, 174.45±101.65pg/mL: 0.05

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is selectively toxic to primary dopaminergic neurons in vitro.

Parkinson's disease (PD) is the second most common neurodegenerative disease. Much data has linked the etiology of PD to a variety of environmental factors. The majority of cases are thought to arise from a combination of genetic susceptibility and environmental factors. Chronic exposures to dietary factors, including meat, have been identified as potential risk factors. Although heterocyclic amines that are produced during high-temperature meat cooking are known to be carcinogenic, their effect on the nervous system has yet to be studied in depth. In this study, we investigated neurotoxic effects of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a highly abundant heterocyclic amine in cooked meat, in vitro. We tested toxicity of PhIP and the two major phase I metabolites, N-OH-PhIP and 4'-OH-PhIP, using primary mesencephalic cultures from rat embryos. This culture system contains both dopaminergic and nondopaminergic neurons, which allows specificity of neurotoxicity to be readily examined. We find that exposure to PhIP or N-OH-PhIP is selectively toxic to dopaminergic neurons in primary cultures, resulting in a decreased percentage of dopaminergic neurons. Neurite length is decreased in surviving dopaminergic neurons. Exposure to 4'-OH-PhIP did not produce significant neurotoxicity. PhIP treatment also increased formation of oxidative damage markers, 4-hydroxy-2-nonenal (HNE) and 3-nitrotyrosine in dopaminergic neurons. Pretreatment with N-acetylcysteine was protective. Finally, treatment with blueberry extract, a dietary factor with known antioxidant and other protective mechanisms, prevented PhIP-induced toxicity. Collectively, our study suggests, for the first time, that PhIP is selectively toxic to dopaminergic neurons likely through inducing oxidative stress.