FEMA GRAS assessment of natural flavor complexes: Sage oil, Orris Root Extract and Tagetes Oil and related flavoring ingredients.

In recent years, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) has conducted a program to re-evaluate the safety of natural flavor complexes (NFCs) used as flavor ingredients. This publication, twelfth in the series, details the re-evaluation of NFCs whose constituent profiles are characterized by alicyclic or linear ketones. In its re-evaluation, the Expert Panel applies a scientific constituent-based procedure for the safety evaluation of NFCs in commerce using a congeneric group approach. Estimated intakes of each congeneric group of the NFC are evaluated using the well-established and conservative Threshold of Toxicological Concern (TTC) approach. In addition, studies on the toxicity and genotoxicity of members of the congeneric groups and the NFCs under evaluation are reviewed. The scope of the safety evaluation of the NFCs contained herein does not include added use in dietary supplements or any products other than food. Thirteen (13) NFCs derived from the Boronia, Cinnamomum, Thuja, Ruta, Salvia, Tagetes, Hyssopus, Iris, Perilla and Artemisia genera are affirmed as generally recognized as safe (GRAS) under conditions of their intended use as flavor ingredients based on an evaluation of each NFC and the constituents and congeneric groups therein.

FEMA GRAS assessment of natural flavor complexes: Allspice, anise, fennel-derived and related flavoring ingredients.

The FEMA Expert Panel program to re-evaluate the safety of natural flavor complexes (NFCs) used as flavoring ingredients in food has resulted in the publication of an updated constituent-based procedure as well as publications on the safety evaluation of many botanical-derived NFCs. This publication, ninth in the series and related to the ninth publication, describes the affirmation of the generally recognized as safe (GRAS) status for NFCs with propenylhydroxybenzene and allylalkoxybenzene constituents under their conditions of intended use as flavoring ingredients added to food. The Panel's procedure applies the threshold of toxicological concern (TTC) concept and evaluates relevant data on absorption, metabolism, genotoxic potential and toxicology for the NFCs themselves and their respective constituent congeneric groups. For NFCs containing allylalkoxybenzene constituent(s) with suspected genotoxic potential, the estimated intake of the individual constituent is compared to the TTC for compounds with structural alerts for genotoxicity and if exceeded, a margin of exposure is calculated using BMDL10 values derived from benchmark dose analyses using Bayesian model averaging, as presented in the tenth article of the series. Safety evaluations for NFCs derived from allspice, anise seed, star anise, sweet fennel seed and pimento leaves were conducted and their GRAS status was affirmed for use as flavoring ingredients. The scope of the safety evaluation contained herein does not include added use in dietary supplements or any products other than food.

FEMA GRAS assessment of natural flavor complexes: Asafetida oil, garlic oil and onion oil.

The Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) applies its procedure for the safety evaluation of natural flavor complexes (NFCs) to re-evaluate the safety of Asafetida Oil (Ferula assa-foetida L.) FEMA 2108, Garlic Oil (Allium sativum L.) FEMA 2503 and Onion Oil (Allium cepa L.) FEMA 2817 for use as flavoring in food. This safety evaluation is part of a series of evaluations of NFCs for use as flavoring ingredients conducted by the Expert Panel that applies a scientific procedure published in 2005 and updated in 2018. Using a group approach that relies on a complete chemical characterization of the NFC intended for commerce, the constituents of each NFC are organized into well-defined congeneric groups and the estimated intake of each constituent congeneric group is evaluated using the conservative threshold of toxicological concern (TTC) concept. Data on the metabolism, genotoxic potential and toxicology for each constituent congeneric group are reviewed as well as studies on each NFC. Based on the safety evaluation, Asafetida Oil (Ferula assa-foetida L.), Garlic Oil (Allium sativum L.) and Onion Oil (Allium cepa L.) were affirmed as generally recognized as safe (GRASa) under their conditions of intended use as flavor ingredients.

FEMA GRAS assessment of natural flavor complexes: Eucalyptus oil and other cyclic ether-containing flavoring ingredients.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) initiated a program for the re-evaluation of the safety of over 250 natural flavor complexes (NFCs) used as flavor ingredients. This publication, the sixth in the series, will summarize the re-evaluation of eight NFCs whose constituent profiles are characterized by significant amounts of eucalyptol and/or other cyclic ethers. This re-evaluation was based on a procedure first published in 2005 and subsequently updated in 2018 that evaluates the safety of naturally occurring mixtures for their intended use as flavoring ingredients. The procedure relies on a complete chemical characterization of the NFC intended for commerce and the organization of its chemical constituents into well-defined congeneric groups. The safety of the NFC is evaluated using the well-established and conservative threshold of toxicological concern (TTC) concept in addition to data on absorption, metabolism and toxicology of the constituents of the congeneric groups and the NFC under evaluation. Eight NFCs derived from the Eucalyptus, Melaleuca, Origanum, Laurus, Rosmarinus and Salvia genera were affirmed as generally recognized as safe (GRAS) under their conditions of intended use as flavor ingredients based on an evaluation of each NFC and the constituents and congeneric groups therein.

FEMA GRAS assessment of natural flavor complexes: Origanum oil, thyme oil and related phenol derivative-containing flavoring ingredients.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) initiated a re-evaluation of the safety of over 250 natural flavor complexes (NFCs) used as flavor ingredients, mostly consisting of a variety of essential oils and botanical extracts. This publication, seventh in the series, re-evaluates NFCs with constituent profiles dominated by phenolic derivatives including carvacrol, thymol and related compounds using a constituent-based procedure first published in 2005 and updated in 2018. The procedure is based on the chemical characterization of each NFC as intended for commerce and the estimated intake of the constituent congeneric groups. The procedure applies the threshold of toxicological concern (TTC) concept and evaluates relevant data on absorption, metabolism, genotoxic potential and toxicology of the constituent congeneric groups and the NFC under evaluation. Herein, the FEMA Expert Panel affirmed the generally recognized as safe (GRAS) status of seven phenolic derivative-based NFCs, Origanum Oil (Extractive) (FEMA 2828), Savory Summer Oil (FEMA 3013), Savory Summer Oleoresin (FEMA 3014), Savory Winter Oil (FEMA 3016), Savory Winter Oleoresin (FEMA 3017), Thyme Oil (FEMA 3064) and Thyme White Oil (FEMA 3065) under their conditions of intended use as flavor ingredients.

FEMA GRAS assessment of natural flavor complexes: Clove, cinnamon leaf and West Indian bay leaf-derived flavoring ingredients.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association initiated the safety re-evaluation of over 250 natural flavor complexes (NFCs) used as flavor ingredients. This publication, 4th in a series focusing on the safety evaluation of NFCs, presents an evaluation of NFCs rich in hydroxyallylbenzene and hydroxypropenylbenzene constituents using a procedure initially published in 2005 and updated in 2018 that evaluates the safety of naturally occurring mixtures for their intended use as flavoring ingredients. The procedure requires the characterization of the chemical composition for each NFC and subsequent organization of the constituents into defined congeneric groups. The safety of each NFC is evaluated using the conservative threshold of toxicological concern (TTC) approach together with studies on absorption, metabolism and toxicology of the NFC and its constituent congeneric groups. By the application of this procedure, seven NFCs, derived from clove, cinnamon leaf and West Indian bay leaf were affirmed as "generally recognized as safe (GRAS)" under their conditions of intended use as flavor ingredients. An eighth NFC, an oleoresin of West Indian bay leaf, was affirmed based on its estimated intake, which is below the TTC of 0.15 µg/person per day for compounds with structural alerts for genotoxicity.

FEMA GRAS assessment of natural flavor complexes: Lavender, Guaiac Coriander-derived and related flavoring ingredients.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) initiated a program for the re-evaluation of the safety of over 250 natural flavor complexes (NFCs) used as flavor ingredients. This publication, fifth in the series, evaluates the safety of NFCs containing linalool and/or other characteristic mono- and sesquiterpenoid tertiary alcohols and esters using the safety evaluation procedure published by the FEMA Expert Panel in 2005 and updated in 2018. The procedure relies on a complete chemical characterization of the NFC intended for commerce and organization of the chemical constituents of each NFC into well-defined congeneric groups. The safety of each NFC is evaluated using the well-established and conservative threshold of toxicological concern (TTC) concept in addition to data on absorption, metabolism and toxicology of both the constituent congeneric groups and the NFCs. Sixteen NFCs, derived from the Lavandula, Aniba, Elettaria, Daucus, Salvia, Coriandrum, Ribes, Guaiacum/Bulnesia, Citrus, Pogostemon, Melaleuca and Michelia genera, were affirmed as generally recognized as safe (GRAS) under their conditions of intended use as flavor ingredients based on an evaluation of each NFC and the constituents and congeneric groups therein.

Revisiting the evidence for genotoxicity of acrylamide (AA), key to risk assessment of dietary AA exposure.

The weight of evidence pro/contra classifying the process-related food contaminant (PRC) acrylamide (AA) as a genotoxic carcinogen is reviewed. Current dietary AA exposure estimates reflect margins of exposure (MOEs) < 500. Several arguments support the view that AA may not act as a genotoxic carcinogen, especially not at consumer-relevant exposure levels: Biotransformation of AA into genotoxic glycidamide (GA) in primary rat hepatocytes is markedly slower than detoxifying coupling to glutathione (GS). Repeated feeding of rats with AA containing foods, bringing about uptake of 100 µg/kg/day of AA, resulted in dose x time-related buildup of AA-hemoglobin (Hb) adducts, whereas GA-Hb adducts remained within the background. Since hepatic oxidative biotransformation of AA into GA was proven by simultaneous urinary mercapturic acid monitoring it can be concluded that at this nutritional intake level any GA formed in the liver from AA is quantitatively coupled to GS to be excreted as mercapturic acid in urine. In an oral single dose-response study in rats, AA induced DNA N7-GA-Gua adducts dose-dependently in the high dose range (> 100 µg/kg b w). At variance, in the dose range below 100 µg/kg b.w. down to levels of average consumers exposure, DNA N7 -Gua lesions were found only sporadically, without dose dependence, and at levels close to the lower bound of similar human background DNA N7-Gua lesions. No DNA damage was detected by the comet assay within this low dose range. GA is a very weak mutagen, known to predominantly induce DNA N7-GA-Gua adducts, especially in the lower dose range. There is consensus that DNA N7-GA-Gua adducts exhibit rather low mutagenic potency. The low mutagenic potential of GA has further been evidenced by comparison to preactivated forms of other process-related contaminants, such as N-Nitroso compounds or polycyclic aromatic hydrocarbons, potent food borne mutagens/carcinogens. Toxicogenomic studies provide no evidence supporting a genotoxic mode of action (MOA), rather indicate effects on calcium signalling and cytoskeletal functions in rodent target organs. Rodent carcinogenicity studies show induction of strain- and species-specific neoplasms, with MOAs not considered likely predictive for human cancer risk. In summary, the overall evidence clearly argues for a nongenotoxic/nonmutagenic MOA underlying the neoplastic effects of AA in rodents. In consequence, a tolerable intake level (TDI) may be defined, guided by mechanistic elucidation of key adverse effects and supported by biomarker-based dosimetry in experimental systems and humans.

FEMA GRAS assessment of natural flavor complexes: Cinnamomum and Myroxylon-derived flavoring ingredients.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) initiated a program for the re-evaluation of the safety of over 250 natural flavor complexes (NFCs) used as flavor ingredients. This publication, third in the series, considers NFCs composed primarily of constituents with the 3-phenyl-2-propenyl or a cinnamyl functional group, using the procedure outlined in 2005 and updated in 2018 to evaluate the safety of naturally-occurring mixtures for their intended use as flavor ingredients. The procedure relies on a complete chemical characterization of the NFC intended for commerce and organization of each NFC's chemical constituents into well-defined congeneric groups. The safety of the NFC is evaluated using the well-established and conservative threshold of toxicological concern (TTC) concept in addition to data on absorption, metabolism and toxicology of members of the congeneric groups and the NFC under evaluation. Six NFCs from the Myroxylon and Cinnamomum genera, Balsam Oil, Peru (FEMA 2117), Tolu Balsam Extract (FEMA 3069), Cassia Bark Extract (FEMA 2257), Cassia Bark Oil (FEMA 2258), Cinnamon Bark Extract (FEMA 2290) and Cinnamon Bark Oil (FEMA 2291) were evaluated and affirmed as generally recognized as safe (GRAS) under their conditions of intended use as flavor ingredients.

FEMA GRAS assessment of natural flavor complexes: Mint, buchu, dill and caraway derived flavoring ingredients.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) initiated a re-evaluation of the safety of over 250 natural flavor complexes (NFCs) used as flavor ingredients. NFC flavor materials include a variety of essential oils and botanical extracts. The re-evaluation of NFCs is conducted based on a constituent-based procedure outlined in 2005 and updated in 2018 that evaluates the safety of NFCs for their intended use as flavor ingredients. This procedure is applied in the re-evaluation of the generally recognized as safe (GRAS) status of NFCs with constituent profiles that are dominated by alicyclic ketones such as menthone and carvone, secondary alcohols such as menthol and carveol, and related compounds. The FEMA Expert Panel affirmed the GRAS status of Peppermint Oil (FEMA 2848), Spearmint Oil (FEMA 3032), Spearmint Extract (FEMA 3031), Cornmint Oil (FEMA 4219), Erospicata Oil (FEMA 4777), Curly Mint Oil (FEMA 4778), Pennyroyal Oil (FEMA 2839), Buchu Leaves Oil (FEMA 2169), Caraway Oil (FEMA 2238) and Dill Oil (FEMA 2383) and determined FEMA GRAS status for Buchu Leaves Extract (FEMA 4923), Peppermint Oil, Terpeneless (FEMA 4924) and Spearmint Oil, Terpeneless (FEMA 4925).

Alkylpyrazines from Coffee are Extensively Metabolized to Pyrazine Carboxylic Acids in the Human Body.

SCOPE: Coffee is a complex mixture of over 1000 compounds, including diverse heteroaromatic compounds such as alkylpyrazines. Little is known about the intake, metabolism, and bodily distribution of these compounds. Therefore, a human intervention study is conducted to investigate the excretion of alkylpyrazine metabolites in urine after the ingestion of brewed coffee containing alkylpyrazines. METHODS AND RESULTS: After consuming a diet without heat-processed food, ten volunteers consumed 500 mL of freshly brewed coffee prepared from coffee pads, providing intakes of 2-methylpyrazine (2-MeP), 2,5-dimethylpyrazine (2,5-DMeP), and 2,6-dimethylpyrazine (2,6-DMeP) amounting to 17.2, 4.4, and 4.9 µmol, respectively. These alkylpyrazines are metabolized into the corresponding pyrazine carboxylic acids, namely pyrazine-2-carboxylic acid (PA), 5-hydroxypyrazine-2-carboxylic acid (5-OHPA), 5-methylpyrazine-2-carboxylic acid (5-MePA), and 6-methylpyrazine-2-carboxylic acid (6-MePA). In total, 64% of the ingested 2-MeP is excreted as PA, as well as 26% as 5-OHPA, while 91% and 97% of the ingested 2,5-DMeP and 2,6-DMeP are recovered as 5-MePA and 6-MePA, respectively, in urine samples collected after coffee consumption. CONCLUSION: This study provides evidence that alkylpyrazines are rapidly metabolized into the corresponding carboxylic acids and excreted via urine by humans, which is consistent with earlier rodent studies.

Urinary Excretion of Niacin Metabolites in Humans After Coffee Consumption.

SCOPE: Coffee is a major natural source of niacin in the human diet, as it is formed during coffee roasting from the alkaloid trigonelline. The intention of our study was to monitor the urinary excretion of niacin metabolites after coffee consumption under controlled diet. METHODS AND RESULTS: We performed a 4-day human intervention study on the excretion of major niacin metabolites in the urine of volunteers after ingestion of 500 mL regular coffee containing 34.8 µmol nicotinic acid (NA) and 0.58 µmol nicotinamide (NAM). In addition to NA and NAM, the metabolites N1 -methylnicotinamide (NMNAM), N1 -methyl-2-pyridone-5-carboxamide (2-Py), and nicotinuric acid (NUA) were identified and quantified in the collected urine samples by stable isotope dilution analysis (SIVA) using HPLC-ESI-MS/MS. Rapid urinary excretion was observed for the main metabolites (NA, NAM, NMNAM, and 2-Py), with tmax values within the first hour after ingestion. NUA appeared in traces even more rapidly. In sum, 972 nmol h-1 of NA, NAM, NMNAM, and 2-Py were excreted within 12 h after coffee consumption, corresponding to 6% of the ingested NA and NAM. CONCLUSION: The results indicate regular coffee consumption to be a source of niacin in human diet.

Coffee consumption rapidly reduces background DNA strand breaks in healthy humans: Results of a short-term repeated uptake intervention study.

SCOPE: Intervention studies provide evidence that long-term coffee consumption correlates with reduced DNA background damage in healthy volunteers. Here, we report on short-term kinetics of this effect, showing a rapid onset after normal coffee intake. METHODS AND RESULTS: In a short-term human intervention study, we determined the effects of coffee intake on DNA integrity during 8 h. Healthy male subjects ingested coffee in 200 mL aliquots every second hour up to a total volume of 800 mL. Blood samples were taken at baseline, immediately before the first coffee intake and subsequently every 2 h, prior to the respective coffee intake. DNA integrity was assayed by the comet assay. The results show a significant (p < 0.05) reduction of background DNA strand breaks already 2 h after the first coffee intake. Continued coffee intake was associated with further decrements in background DNA damage within the 8 h intervention (p < 0.01 and p < 0.001, respectively). Mean tail intensities (TIs%) decreased from 0.33 TI% (baseline, 0 h) to 0.22 TI% (within 8 h coffee consumption). CONCLUSION: Repeated coffee consumption was associated with reduced background DNA strand breakage, clearly measurable as early as 2 h after first intake resulting in a cumulative overall reduction by about one-third of the baseline value.

Modulation of 3',5'-cyclic AMP homeostasis in human platelets by coffee and individual coffee constituents.

3',5'-Cyclic AMP (cAMP) is one of the most important second messengers in mammalian cells, mediating a multitude of diverse cellular signalling responses. Its homeostasis is primarily regulated by adenylate cyclases and phosphodiesterases (PDE), the activities of which are partially dependent on the downstream events of adenosine receptor signalling. The present study was conducted to determine whether coffee constituents other than caffeine can influence the homeostasis of intracellular cAMP in vitro and in vivo by evaluating the effects of selected constituents present in coffee, coffee brews and coffee extracts on platelet PDE activity. In addition, to evaluate the potential effects of these constituents on platelet cAMP concentrations and PDE activity in humans, a 7-week pilot intervention study with eight subjects was conducted. The subjects consumed a regular commercial coffee and a low-caffeine coffee at a rate of 750 ml/d for 2 weeks each. The in vivo results revealed a highly significant inhibition of PDE activity (P< 0·001) after coffee intervention that was not directly dependent on the caffeine content of coffee. Although our in vitro and in vivo findings suggest that caffeine plays some role in the modulation of platelet cAMP status, other natural and roasting-associated compounds such as pyrazines and other currently unidentified species also appear to contribute significantly. In conclusion, moderate consumption of coffee can modulate platelet PDE activity and cAMP concentrations in humans, which may contribute to the putative beneficial health effects of coffee. Further detailed mechanistic investigations will be required to substantiate these beneficial effects and to elucidate the underlying mechanisms.

Assessment of dietary phytoestrogen intake via plant-derived foods in China.

The potential influence of dietary phytoestrogen exposure on human health during different life phases including early childhood is a matter of scientific debate. In order to improve the risk-benefit assessment of exposure to dietary phytoestrogen, reliable and age-stratified exposure data are desirable. For contributing to the database on phytoestrogen exposure, in the present study plant-derived foods from the Chinese market were analysed by LC-MS/MS for their contents of phytoestrogens, including daidzein, genistein, secoisolariciresinol, glycitein and coumestrol. The analytical data showed the presence of phytoestrogens in a concentration range of less than 0.1 to about 50 µg g(-1). Dietary intake was assessed on the basis of average food intake data obtained from interviewing 1000 randomly selected people with the help of food frequency questionnaires. Based on the overall population sampled, the average total phytoestrogen intake was estimated at 232 µg kg(-1) day(-1). Genistein contributed to about 66%, secoisolariciresinol and glycitein to about 10% each, and daidzein to about 7% of the overall intake. Coumestrol was present only in trace amounts. Age-related exposure assessment indicated that pre-pubertal children (aged 0-14 years) were exposed at the highest level with an average total phytoestrogen intake of 621 µg kg(-1) day(-1). The substantially higher average exposure of children as compared with adults should trigger further research into the potential health effects of early life exposure to phytoestrogen.

Physicochemical characterization and in vitro permeation of an indirubin derivative.

The active component of the traditional Chinese medicine, indirubin, exerts anticancer effect on different cancer cell lines. E804, a potent derivative of indirubin inhibits the activation of Stat3 and Stat5 in chronic myelocytic leukaemia (CML) cells. However, physicochemical properties and permeation rate of the compound relevant to the drug formulation have never been reported. Therefore, the ionization constant (pK(a)), lipophilicity (logD/P), aqueous and organic solubility of E804 and its permeation across Caco-2 cells were investigated. Both high throughput and traditional determinations were used in this study. The Caco-2 cell permeation assay was carried out in Poloxamer 188/HBSS++ solution in order to maintain the solubility of drug. The potential P-gp (P-glycoprotein) interaction for E804 was determined through Calcein-AM uptake assay. The results showed that E804 did not have a detectable pK(a) in the range of pH 2-11. Log D (distribution coefficient) and Log P (partition coefficient) were determined to be 3.54 ± 0.03. Aqueous solubility test revealed that E804 is practically insoluble in water. Among organic solvents E804 showed the highest solubility in DMSO. The P(app A→B) and P(app B→A) across Caco-2 cell monolayer were 2.0 ± 0.25 × 10(-6)cm/s and 1.14 ± 0.12 × 10(-6)cm/s respectively, and the calculated efflux ratio (ER) was 0.57. Calcein-AM uptake assay showed that E804 was not a strong substrate for P-gp. The results indicate that solubility is the major rate limiting step for the drug permeation. The high membrane permeability makes E804 promising for the oral delivery. Therefore, further investigation on solubility of E804 in lipid vehicles is needed to determine an appropriate formulation for the drug.

Enhancement of oral bioavailability of E804 by self-nanoemulsifying drug delivery system (SNEDDS) in rats.

Indirubin and its derivatives have been shown to interrupt the cell cycle by inhibiting cyclin-dependent kinases, explaining their long-time use in traditional Chinese medicine for the treatment of chronic myelocytic leukemia. A potent derivative of indirubin, indirubin-3'-oxime 2,3-dihydroxypropyl ether (E804), has been shown to block the Src-Stat3 and Src-Stat5 signaling pathway in human cancer cells, inducing apoptosis. The anticancer effects of E804, however, cannot be easily examined in vivo because of its poor water solubility and low absorption. The aim of this study was to develop and evaluate a self-nanoemulsifying drug delivery system (SNEDDS) containing E804 for enhancing its solubility and bioavailability. Solubility of E804 was determined in various vehicles, and pseudoternary phase diagram was used to evaluate the self-emulsifying existence area. The SNEDDS composed of Capmul MCM (oil), Solutol HS 15 (surfactant), and polyethylene glycol 400 (cosurfactant) on the ratio of 20.5:62.5:16 loaded 1.5% of E804. The particle size of droplets was found to be 16.8 and 140 nm, and SNEDDS was stable after freeze-thaw cycles and upon dilution in HCl 0.1 N and pH 7.4 HBSS++. The ability of formulation for absorption enhancement was studied in rats in vivo after oral administration. The results showed that the developed SNEDDS increased the E804 bioavailability 984.23% compared with the aqueous suspension. Our studies for the first time show that the developed SNEDDS can be used as a possible formulation for E804 to improve its solubility and oral bioavailability.

Profiling of mercapturic acids of acrolein and acrylamide in human urine after consumption of potato crisps.

SCOPE: Acrolein (AC) and acrylamide (AA) are food contaminants generated by heat treatment. We studied human exposure after consumption of potato crisps by monitoring excretion of mercapturic acids (MAs) in urine. METHODS AND RESULTS: MA excretion was monitored in human urine collected up to 72 h after ingestion of a test meal of experimental (study 1: 1 mg AA/150 g) or commercially available (study 2: 44 µg AA plus 4.6 µg AC/175 g) potato crisps. MA contents were analysed after purification via SPE using HPLC-ESI-MS/MS. On the basis of the area under the curve values of MAs excreted in urine, the total excretion of AC-related MAs exceeded that of AA-related MAs up to 12 times in study 1 and up to four times in study 2. Remarkably, AC content of potato crisps of study 2 was found to be only about 1/10 the AA content, as determined by isotope dilution headspace GC/MS. CONCLUSION: Our results indicate substantially higher exposure to AC from potato crisps than to AA. Total AC in such foods may encompass bioavailable AC forms not detected by headspace GC/MS. Both findings may also apply to other heat processed foods.

Effect of coffee combining green coffee bean constituents with typical roasting products on the Nrf2/ARE pathway in vitro and in vivo.

This study investigated Nrf2-activating properties of a coffee blend combining raw coffee bean constituents with 5-O-caffeoylquinic acid (CGA) as a lead component with typical roasting products such as N-methylpyridinium (NMP). In cell culture (HT29) the respective coffee extract (CN-CE) increased nuclear Nrf2 translocation and enhanced the transcription of ARE-dependent genes as exemplified for NAD(P)H:quinone oxidoreductase and glutathione-S-transferase (GST)A1, reflected in the protein level by an increase in GST enzyme activity. In a pilot human intervention study (29 healthy volunteers), daily consumption of 750 mL of CN-coffee for 4 weeks increased Nrf2 transcription in peripheral blood lymphocytes on average. However, the transcriptional response pattern of Nrf2/ARE-dependent genes showed substantial interindividual variations. The presence of SNPs in the Nrf2-promoter, reported recently, as well as the detection of GSTT1*0 (null) genotypes in the study collective strengthens the hypothesis that coffee acts as a modulator of Nrf2-dependent gene response in humans, but genetic polymorphisms play an important role in the individual response pattern.

Indirubin derivatives induce apoptosis of chronic myelogenous leukemia cells involving inhibition of Stat5 signaling.

Indirubin is the major active anti-tumor component of a traditional Chinese herbal medicine used for treatment of chronic myelogenous leukemia (CML). While previous studies indicate that indirubin is a promising therapeutic agent for CML, the molecular mechanism of action of indirubin is not fully understood. We report here that indirubin derivatives (IRDs) potently inhibit Signal Transducer and Activator of Transcription 5 (Stat5) protein in CML cells. Compound E804, which is the most potent in this series of IRDs, blocked Stat5 signaling in human K562 CML cells, imatinib-resistant human KCL-22 CML cells expressing the T315I mutant Bcr-Abl (KCL-22M), and CD34-positive primary CML cells from patients. Autophosphorylation of Src family kinases (SFKs) was strongly inhibited in K562 and KCL-22M cells at 5 µM E804, and in primary CML cells at 10 µM E804, although higher concentrations partially inhibited autophosphorylation of Bcr-Abl. Previous studies indicate that SFKs cooperate with Bcr-Abl to activate downstream Stat5 signaling. Activation of Stat5 was strongly blocked by E804 in CML cells. E804 down-regulated expression of Stat5 target proteins Bcl-x(L) and Mcl-1, associated with induction of apoptosis. In sum, our findings identify IRDs as potent inhibitors of the SFK/Stat5 signaling pathway downstream of Bcr-Abl, leading to apoptosis of K562, KCL-22M and primary CML cells. IRDs represent a promising structural class for development of new therapeutics for wild type or T315I mutant Bcr-Abl-positive CML patients.