Prospective analysis of circulating metabolites and endometrial cancer risk.

BACKGROUND: Endometrial cancer is strongly associated with obesity and dysregulation of metabolic factors such as estrogen and insulin signaling are causal risk factors for this malignancy. To identify additional novel metabolic pathways associated with endometrial cancer we performed metabolomic analyses on pre-diagnostic plasma samples from 853 case-control pairs from the European Prospective Investigation into Cancer and Nutrition (EPIC). METHODS: A total of 129 metabolites (acylcarnitines, amino acids, biogenic amines, glycerophospholipids, hexoses, and sphingolipids) were measured by liquid chromatography-mass spectrometry. Conditional logistic regression estimated the associations of metabolites with endometrial cancer risk. An analysis focusing on clusters of metabolites using the bootstrap lasso method was also employed. RESULTS: After adjustment for body mass index, sphingomyelin [SM] C18:0 was positively (OR1SD: 1.18, 95% CI: 1.05-1.33), and glycine, serine, and free carnitine (C0) were inversely (OR1SD: 0.89, 95% CI: 0.80-0.99; OR1SD: 0.89, 95% CI: 0.79-1.00 and OR1SD: 0.91, 95% CI: 0.81-1.00, respectively) associated with endometrial cancer risk. Serine, C0 and two sphingomyelins were selected by the lasso method in >90% of the bootstrap samples. The ratio of esterified to free carnitine (OR1SD: 1.14, 95% CI: 1.02-1.28) and that of short chain to free acylcarnitines (OR1SD: 1.12, 95% CI: 1.00-1.25) were positively associated with endometrial cancer risk. Further adjustment for C-peptide or other endometrial cancer risk factors only minimally altered the results. CONCLUSION: These findings suggest that variation in levels of glycine, serine, SM C18:0 and free carnitine may represent specific pathways linked to endometrial cancer development. If causal, these pathways may offer novel targets for endometrial cancer prevention.

Astrocyte adenosine deaminase loss increases motor neuron toxicity in amyotrophic lateral sclerosis.

As clinical evidence supports a negative impact of dysfunctional energy metabolism on the disease progression in amyotrophic lateral sclerosis, it is vital to understand how the energy metabolic pathways are altered and whether they can be restored to slow disease progression. Possible approaches include increasing or rerouting catabolism of alternative fuel sources to supplement the glycolytic and mitochondrial pathways such as glycogen, ketone bodies and nucleosides. To analyse the basis of the catabolic defect in amyotrophic lateral sclerosis we used a novel phenotypic metabolic array. We profiled fibroblasts and induced neuronal progenitor-derived human induced astrocytes from C9orf72 amyotrophic lateral sclerosis patients compared to normal controls, measuring the rates of production of reduced nicotinamide adenine dinucleotides from 91 potential energy substrates. This approach shows for the first time that C9orf72 human induced astrocytes and fibroblasts have an adenosine to inosine deamination defect caused by reduction of adenosine deaminase, which is also observed in induced astrocytes from sporadic patients. Patient-derived induced astrocyte lines were more susceptible to adenosine-induced toxicity, which could be mimicked by inhibiting adenosine deaminase in control lines. Furthermore, adenosine deaminase inhibition in control induced astrocytes led to increased motor neuron toxicity in co-cultures, similar to the levels observed with patient derived induced astrocytes. Bypassing metabolically the adenosine deaminase defect by inosine supplementation was beneficial bioenergetically in vitro, increasing glycolytic energy output and leading to an increase in motor neuron survival in co-cultures with induced astrocytes. Inosine supplementation, in combination with modulation of the level of adenosine deaminase may represent a beneficial therapeutic approach to evaluate in patients with amyotrophic lateral sclerosis.

Evaluation of Dual 5-Lipoxygenase/Microsomal Prostaglandin E2 Synthase-1 Inhibitory Effect of Natural and Synthetic Acronychia-Type Isoprenylated Acetophenones.

Among the pathways responsible for the development of inflammatory responses, the cyclooxygenase and lipoxygenase pathways are among the most important ones. Two key enzymes, namely, 5-LO and mPGES-1, are involved in the biosynthesis of leukotrienes and prostaglandins, respectively, which are considered attractive therapeutic targets, so their dual inhibition might be an effective strategy to control inflammatory deregulation. Several natural products have been identified as 5-LO inhibitors, with some also being dual 5-LO/mPGES-1 inhibitors. Here, some prenylated acetophenone dimers from Acronychia pedunculata have been identified for their dual inhibitory potency toward 5-LO and mPGES-1. To gain insight into the SAR of this family of natural products, the synthesis and biological evaluation of analogues are presented. The results show the ability of the natural and synthetic molecules to potently inhibit 5-LO and mPEGS-1 in vitro. The potency of the most active compound (10) has been evaluated in vivo in an acute inflammatory mouse model and displayed potent anti-inflammatory activity comparable in potency to the drug zileuton used as a positive control.

Rapid identification of coumarins from Micromelum falcatum by UPLC-HRMS/MS and targeted isolation of three new derivatives.

Micromelum falcatum, a medicinal plant of the Rutaceae family, has been used in the Traditional Chinese Medicine (TCM) mainly against colds and rheumatoid arthritis. Despite its traditional use the association of its constituents with possible anti-inflammatory activity has not been explored. During this study, a rapid UPLC-ESI(+)-HRMS method was developed for the profiling of M. falcatum leave extracts and the targeted isolation of coumarin constituents. Based on chromatographic, spectroscopic and spectrometric features several 7-oxygenated coumarin derivatives were detected. After targeted isolation, eight coumarins, among them three new natural products, namely microfalcrin, microcoumaririn and micromelosidester, were purified using semi-preparative HPLC and unambiguously identified by 1 and 2D NMR. Furthermore, important spectrometric characteristics were revealed based on the HRMS and HRMS/MS spectra of the isolated 7-oxygenated coumarins facilitating their identification in complex mixtures. Finally, the anti-inflammatory properties of the extracts and representative compounds were evaluated by measuring the inhibition of the pro-inflammatory mediator NF-κB induction and nitric oxide (NO) production.

Safety of ashitaba sap as a Novel food pursuant to Regulation (EU) 2015/2283.

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on ashitaba sap as a novel food (NF) pursuant to Regulation (EU) 2015/2283. Ashitaba sap is collected from harvested stems of Angelica keiskei plants. The principal constituents of the sap with regard to the safety assessment are chalcones (1%-2.25%) and furanocoumarins (< 0.01%). The applicant proposed to use the NF in food supplements at a maximum dose of 780 mg per day. The target population is adults excluding pregnant and lactating women. Taking into consideration the composition of the NF and the proposed uses, the composition of the NF is not nutritionally disadvantageous. There are no concerns regarding genotoxicity of the NF. Based on a 90-day oral toxicity study performed with the product as intended to be placed on the market (30% ashitaba sap powder and 70% cyclodextrins), the Panel establishes a safe dose of 0.5 mg/kg body weight (bw) per day for the product as it is intended to be placed on the market. For the target population, i.e. adults, this safe dose corresponds to 35 mg per day of the product as it is intended to be placed on the market and 137 mg per day of the NF, which is lower than the use level proposed by the applicant. The Panel concludes that the NF is safe for the target population at intake levels up to 137 mg per day.

Safety of water lentil protein concentrate from a mixture of Lemna gibba and Lemna minor as a novel food pursuant to Regulation (EU) 2015/2283.

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on water lentil protein concentrate from a mixture of Lemna gibba and Lemna minor as a novel food (NF) pursuant to Regulation (EU) 2015/2283. Water lentil protein concentrate is produced from two water lentil species (L. gibba and L. minor) by separation of the protein fraction of the plant material from fibres, followed by pasteurisation and spray drying. The NF consists mainly of protein, fibre, fat and ash. The applicant proposed to use the NF as a food ingredient in a variety of food categories and as a food supplement. The target population is the general population when used as a food ingredient and exclusively adults when used as a food supplement. The Panel considers that taking into account the composition of the NF and the proposed conditions of use, the consumption of the NF is not nutritionally disadvantageous. There are no concerns regarding genotoxicity of the NF. The Panel considers that the risk of the NF triggering allergic reactions is low. The Panel concludes that the NF, water lentil protein concentrate from a mixture of L. gibba and L. minor, is safe under the proposed conditions of use.

Cytotoxic prenylated acetophenone dimers from Acronychia pedunculata.

Three new acetophenone dimers or Acronychia-type acetophenones, acropyrone (1), acropyranol A (2), and acropyranol B (3), were isolated from the trunk bark of Acronychia pedunculata and structurally characterized, together with four known acetophenone dimers, acrovestone (4), acrovestenol (5), acrofolione A (6), and acrofolione B (7), the acetophenone monomer acronyline (8), and four furoquinoline alkaloids. The chemical structures of the new isolated compounds were elucidated unambiguously by spectroscopic data analysis. The cytotoxic activities of the isolated acetophenone dimers were evaluated against the DU145 prostate and A2058 melanoma human cancer cell lines as well as the NHDF normal cell line. Acrovestone (4) and acrovestenol (5) exhibited substantial cytotoxicity, with IC(50) values of 0.38 and 2.8 µM against A2058 melanoma cells as well as 0.93 and 2.7 µM against DU145 prostate cancer cells, respectively.

Library-based discovery of DYRK1A/CLK1 inhibitors from natural product extracts.

The dual specificity tyrosine-phosphorylation-regulated kinase DYRK1A possesses diverse roles in neuronal development and adult brain physiology, and increased activity has been linked to neurodegenerative diseases. Very few inhibitors of this kinase have been reported up to now. Screening of a library of > 900 plant and fungal extracts afforded 25 extracts with IC50s < 10 µg/mL against DYRK1A. To identify the active constituents, the extracts were submitted to a process integrating physicochemical data with biological information, referred to as HPLC-based activity profiling. Follow-up investigation of four extracts led to the targeted isolation of harmine (1, IC50 0.022 µM) from Peganum harmala, emodin (3, IC50 4.2 µM) from Cassia nigricans, kaempferol (4, IC50 0.91 µM) from Cuscuta chinensis, and 3,8-di-O-methylherbacetin (11, IC50 8.6 µM), 3,3',4'-tri-O-methylmyricetin (12, IC50 7.1 µM) and ombuin (15, IC50 1.7 µM) from Larrea tridentata as the active constituents. Active extracts and compounds were also tested on the closely related cdc2-like kinase CLK1. Finally, the selectivity profile of compounds was evaluated by including other members of the DYRKs and CLKs families. While the flavonoids and emodin did not show significant differences in the potency of their activities, harmine (1) was most active against DYRK1A, CLK1, and CLK4, and less potent against the other kinases, with selectivity ranging from 2- to 20-fold.

Safety of Lemna minor and Lemna gibba whole plant material as a novel food pursuant to Regulation (EU) 2015/2283.

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on Lemna minor and Lemna gibba whole plant material as a novel food (NF) pursuant to Regulation (EU) 2015/2283. Lemna minor and Lemna gibba are aquatic plants commonly named water lentils. The NF is produced by cultivation of Lemna minor and Lemna gibba plants, washing with water and heat treatment. The main constituents of the NF are water, protein and fibre. The Panel notes that the concentration of trace elements and contaminants in the NF is highly dependent on the conditions of cultivation of the plant and the fertiliser composition. The NF is intended to be used as a vegetable, similar to other leafy vegetables. The target population is the general population. The Panel considers that, with the exception of concerns related to the manganese intake, taking into account the composition of the NF and the proposed conditions of use, the consumption of the NF is not nutritionally disadvantageous. The Panel considers that the risk that the consumption of the NF may trigger allergic reactions in humans is low. The Panel concludes that the NF, in consideration of its proposed uses and the concentration of manganese as compared to the normally present concentration of manganese in other leafy vegetables, may be of safety concern, therefore, the safety of the NF cannot currently be established.

Statement on safety of cannabidiol as a novel food: data gaps and uncertainties.

The European Commission has determined that cannabidiol (CBD) can be considered as a novel food (NF), and currently, 19 applications are under assessment at EFSA. While assessing these, it has become clear that there are knowledge gaps that need to be addressed before a conclusion on the safety of CBD can be reached. Consequently, EFSA has issued this statement, summarising the state of knowledge on the safety of CBD consumption and highlighting areas where more data are needed. Literature searches for both animal and human studies have been conducted to identify safety concerns. Many human studies have been carried out with Epidyolex®, a CBD drug authorised to treat refractory epilepsies. In the context of medical conditions, adverse effects are tolerated if the benefit outweighs the adverse effect. This is, however, not acceptable when considering CBD as a NF. Furthermore, most of the human data referred to in the CBD applications investigated the efficacy of Epidyolex (or CBD) at therapeutic doses. No NOAEL could be identified from these studies. Given the complexity and importance of CBD receptors and pathways, interactions need to be taken into account when considering CBD as a NF. The effects on drug metabolism need to be clarified. Toxicokinetics in different matrices, the half-life and accumulation need to be examined. The effect of CBD on liver, gastrointestinal tract, endocrine system, nervous system and on psychological function needs to be clarified. Studies in animals show significant reproductive toxicity, and the extent to which this occurs in humans generally and in women of child-bearing age specifically needs to be assessed. Considering the significant uncertainties and data gaps, the Panel concludes that the safety of CBD as a NF cannot currently be established.

Safety of Wolffia globosa powder as a Novel food pursuant to Regulation (EU) 2015/2283.

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on Wolffia globosa powder as a novel food (NF) pursuant to Regulation (EU) 2015/2283. Wolffia globosa is an aquatic plant, one out of the 38 species of the water lentil family which is composed by five genera (i.e. Spirodela, Landoltia, Lemna, Wolffiella and Wolffia). The NF is produced by cultivation of Wolffia globosa plants under controlled conditions, washing with hot water and drying. The main constituents of the NF are protein, fibre and fat. The Panel notes that the concentration of trace elements and contaminants in the NF is highly dependent on the conditions of cultivation of the plant and the fertiliser composition. The NF is intended to be used as food ingredient in a variety of food categories and as food supplement. The target population is the general population except for food supplements which are intended to be consumed exclusively by adults. The Panel considers that with the exception of concerns related to the manganese intake, taking into account the composition of the NF and the proposed conditions of use, the consumption of the NF is not nutritionally disadvantageous. The subchronic toxicity study provided with the NF revealed a number of significant findings and the Panel considers the middle dose (6.5 g/kg body weight (bw) per day) as the no observed adverse effect level (NOAEL). Based on the protein concentration, the Panel considers that the consumption of the NF may trigger allergic reactions. The Panel concluded that an increase in manganese intake from the NF used as food ingredient or food supplements is of safety concern and the safety of the NF cannot be established.

Safety of water lentil powder from Lemnaceae as a Novel Food pursuant to Regulation (EU) 2015/2283.

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on the safety of water lentil powder as a novel food (NF) pursuant to Regulation (EU) 2015/2283. Water lentils refer to aquatic plants belonging to the Araceae family and represented by five genera (Lemna, Wolffia, Wolffiella, Landoltia and Spirodela). The NF is thermally washed and dried water lentils, which are produced as a polyculture crop consisting of species from the Lemna genus (70-100%) and the Wolffia genus (0-30%). The main constituents of the NF are protein, fibre and fat. The Panel notes that the concentration of trace elements and contaminants in the NF is highly dependent on the conditions of cultivation of the plant and the fertiliser composition. The NF is expected to be stable and to comply with the specifications during the suggested shelf life. The NF is intended for human consumption as a food ingredient in herbs, spices and seasonings, sauces, soups and broths, protein products, dietary food for weight control and as a food supplement. The target population is the general population, except for food supplements which are exclusively intended for consumption by adults. The Panel considers that based on the composition of the NF and the proposed intended uses, the NF is not nutritionally disadvantageous, except for the concerns regarding intake of manganese from the NF. No adverse effect was observed in the submitted 90-day subchronic study, at the highest dose, 1,000 mg/kg body weight (bw) per day of NF. The Panel considers that, based on the protein concentration, the consumption of the NF may trigger allergic reactions. The Panel concluded that an increase in manganese intake from the NF used as food ingredient or food supplements is of safety concern and the safety of the NF cannot be established.

Safety of tetrahydrocurcuminoids from turmeric (Curcuma longa L.) as a novel food pursuant to Regulation (EU) 2015/2283.

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on tetrahydrocurcuminoids from turmeric (Curcuma longa L.) as a novel food (NF) pursuant to Regulation (EU) 2015/2283. Tetrahydrocurcuminoids are derivatives of curcuminoids, produced chemically by hydrogenation of curcuminoids extracted from the rhizomes of C. longa L. The NF consists of more than 95% of tetrahydrocurcuminoids. The applicant proposed to use the NF in food supplements at a maximum dose of 300 mg/day for adults excluding pregnant and lactating women. Taking into account the composition of the NF and the proposed conditions of use, consumption of the NF is not nutritionally disadvantageous. There are no concerns regarding genotoxicity of the NF. Based on a 90-day oral toxicity study and a reproduction/developmental toxicity screening test performed with the NF, the Panel derives a safe level of 2 mg/kg body weight per day. For the target population this level corresponds to 140 mg/day, which is lower than the use level as proposed by the applicant. The Panel concludes that the NF, tetrahydrocurcuminoids from turmeric (C. longa L.), is safe for the target population at 140 mg/day.

Novel foods in the European Union: Scientific requirements and challenges of the risk assessment process by the European Food Safety Authority.

The European Food Safety Authority (EFSA) has been involved in the risk assessment of novel foods since 2003. The implementation of the current novel food regulation in 2018 rendered EFSA the sole entity of the European Union responsible for such safety evaluations. The risk assessment is based on the data submitted by applicants in line with the scientific requirements described in the respective EFSA guidance document. The present work aims to elaborate on the rationale behind the scientific questions raised during the risk assessment of novel foods, with a focus on complex mixtures and whole foods. Novel foods received by EFSA in 2003-2019 were screened and clustered by nature and complexity. The requests for additional or supplementary information raised by EFSA during all risk assessments were analyzed for identifying reoccurring issues. In brief, it is shown that applications concern mainly novel foods derived from plants, microorganisms, fungi, algae, and animals. A plethora of requests relates to the production process, the compositional characterization of the novel food, and the evaluation of the product's toxicological profile. Recurring issues related to specific novel food categories were noted. The heterogeneous nature and the variable complexity of novel foods emphasize the challenge to tailor aspects of the evaluation approach to the characteristics of each individual product. Importantly, the scientific requirements for novel food applications set by EFSA are interrelated, and only a rigorous and cross-cutting approach adopted by the applicants when preparing the respective application dossiers can lead to scientifically sound dossiers. This is the first time that an in-depth analysis of the experience gained by EFSA in the risk assessment of novel foods and of the reasoning behind the most frequent scientific requests by EFSA to applicants is made.

Ion tree-based structure elucidation of acetophenone dimers (AtA) from Acronychia pedunculata and their identification in extracts by liquid chromatography electrospray ionization LTQ-Orbitrap mass spectrometry.

Acronychia-type acetophenones (AtA) is a chemical group of compounds of important structural and biological interest, abundant in Acronychia species. However, there are no data available for their characterization using mass spectrometry. In the current work, AtA have been investigated by multistage high resolution mass spectrometry and both electrospray ionization and atmospheric pressure chemical ionization, in positive and negative mode, were utilized for their structure elucidation and identification. The analysis of AtA using a linear ion trap-Orbitrap analyzer enabled the structural determination of key fragment ions and cleavages, which can be used for the structural characterization thereof. A systematic nomenclature based on protonated and deprotonated fragment ions under collision-induced dissociation conditions and decision trees for the structural determination of AtA are proposed. Furthermore, taking advantage of the characteristic fragmentation patterns, a selective Ultra High Performance Liquid Chromatography Electrospray Ionization multistage Mass Spectrometry (UHPLC-ESI(-)-MS(n)) method was developed and successfully applied for the dereplication of known AtA and the identification of potentially new ones in Acronychia extracts. Despite the structure similarity and the presence of isomers, accurate characterization of known and unknown AtA derivatives was possible.