How do metabolites differ from their parent molecules and how are they excreted?

Understanding which physicochemical properties, or property distributions, are favorable for successful design and development of drugs, nutritional supplements, cosmetics, and agrochemicals is of great importance. In this study we have analyzed molecules from three distinct chemical spaces (i) approved drugs, (ii) human metabolites, and (iii) traditional Chinese medicine (TCM) to investigate four aspects determining the disposition of small organic molecules. First, we examined the physicochemical properties of these three classes of molecules and identified characteristic features resulting from their distinctive biological functions. For example, human metabolites and TCM molecules can be larger and more hydrophobic than drugs, which makes them less likely to cross membranes. We then quantified the shifts in physicochemical property space induced by metabolism from a holistic perspective by analyzing a data set of several thousand experimentally observed metabolic trees. Results show how the metabolic system aims to retain nutrients/micronutrients while facilitating a rapid elimination of xenobiotics. In the third part we compared these global shifts with the contributions made by individual metabolic reactions. For better resolution, all reactions were classified into phase I and phase II biotransformations. Interestingly, not all metabolic reactions lead to more hydrophilic molecules. We were able to identify biotransformations leading to an increase of logP by more than one log unit, which could be used for the design of drugs with enhanced efficacy. The study closes with the analysis of the physicochemical properties of metabolites found in the bile, faeces, and urine. Metabolites in the bile can be large and are often negatively charged. Molecules with molecular weight >500 Da are rarely found in the urine, and most of these large molecules are charged phase II conjugates.

Nutritional Quality of Dry Vegetable Soups.

Dry soups with vegetables are often perceived as having low nutritional quality, but there are only limited data on the nutritional value of dry soups. Therefore, we measured the nutritional composition of dry vegetable powders used in dry soups and compared the results with published data on fresh and cooked vegetables. We also analyzed the nutritional composition of dry vegetable soups and compared these with published data on home-made and other soups. Dietary fiber, minerals, vitamins, and carotenoids in dry vegetables powders and soups were analyzed. Based on these data, a nutrient density score was calculated as measure of overall nutritional quality. Nutrient density scores for fresh and cooked vegetables, as well as home-made and other soups, were calculated based on the United Stated Department of Agriculture (USDA) and "Bundeslebensmittelschlüssel" (BLS) food composition data. The nutrient density scores of dry vegetable powders did not systematically differ from cooked vegetables. Nutrient contributions to European Food and Safety Authority (EFSA) dietary reference intakes per 250 mL serving of soup ranged from 11-45% for fiber; 3-23% for iron, magnesium, and zinc; 8-22% for potassium; 11-15% for vitamin A; 2-17% for B-vitamins; and 2-15% for vitamin K. The nutrient density scores of dry vegetable soups were in the same order of those of home-made and other soups. These data indicate that dry vegetable soups, like home-made soups, can deliver a significant part of recommended daily nutrient and vegetable intake.

Dietary Impact of Adding Potassium Chloride to Foods as a Sodium Reduction Technique.

Potassium chloride is a leading reformulation technology for reducing sodium in food products. As, globally, sodium intake exceeds guidelines, this technology is beneficial; however, its potential impact on potassium intake is unknown. Therefore, a modeling study was conducted using Dutch National Food Survey data to examine the dietary impact of reformulation (n = 2106). Product-specific sodium criteria, to enable a maximum daily sodium chloride intake of 5 grams/day, were applied to all foods consumed in the survey. The impact of replacing 20%, 50% and 100% of sodium chloride from each product with potassium chloride was modeled. At baseline median, potassium intake was 3334 mg/day. An increase in the median intake of potassium of 453 mg/day was seen when a 20% replacement was applied, 674 mg/day with a 50% replacement scenario and 733 mg/day with a 100% replacement scenario. Reformulation had the largest impact on: bread, processed fruit and vegetables, snacks and processed meat. Replacement of sodium chloride by potassium chloride, particularly in key contributing product groups, would result in better compliance to potassium intake guidelines (3510 mg/day). Moreover, it could be considered safe for the general adult population, as intake remains compliant with EFSA guidelines. Based on current modeling potassium chloride presents as a valuable, safe replacer for sodium chloride in food products.

Snooker structure-based pharmacophore model explains differences in agonist and blocker binding to bitter receptor hTAS2R39.

The human bitter taste receptor hTAS2R39 can be activated by many dietary (iso)flavonoids. Furthermore, hTAS2R39 activity can be blocked by 6-methoxyflavanones, 4'-fluoro-6-methoxyflavanone in particular. A structure-based pharmacophore model of the hTAS2R39 binding pocket was built using Snooker software, which has been used successfully before for drug design of GPCRs of the rhodopsin subfamily. For the validation of the model, two sets of compounds, both of which contained actives and inactives, were used: (i) an (iso)flavonoid-dedicated set, and (ii) a more generic, structurally diverse set. Agonists were characterized by their linear binding geometry and the fact that they bound deeply in the hTAS2R39 pocket, mapping the hydrogen donor feature based on T5.45 and N3.36, analogues of which have been proposed to play a key role in activation of GPCRs. Blockers lack hydrogen-bond donors enabling contact to the receptor. Furthermore, they had a crooked geometry, which could sterically hinder movement of the TM domains upon receptor activation. Our results reveal characteristics of hTAS2R39 agonist and bitter blocker binding, which might facilitate the development of blockers suitable to counter the bitterness of dietary hTAS2R39 agonists in food applications.

The antioxidant activity of regularly consumed fruit and vegetables reflects their phenolic and vitamin C composition.

Recent studies are emphasising the importance and putative modes of action of specific flavonoids as bioactive components of the diet in in vivo and in vitro models. Thus, it is important to have a clear idea of the major phenolic families of which fruit and vegetables are comprised and the levels contained therein. Regularly consumed fruit and vegetables of mixed varieties available on the UK market were analysed for the composition of the major individual phenolic components. The total phenolic content (applying the Folin assay) and the vitamin C levels were also determined. The antioxidant capacities of aqueous/methanolic extracts were comparatively assessed using the TEAC (Trolox Equivalent Antioxidant Capacity), the FRAP (Ferric Reducing Ability of Plasma) and ORAC (Oxygen Radical Absorbance Capacity) assays, which comprise contributions from polyphenols, simple phenols and the ascorbate component. The results were calculated in terms of 100 g fresh weight (FW) uncooked portion sizes. Fruit and vegetables rich in anthocyanins (e.g. strawberry, raspberry and red plum) demonstrated the highest antioxidant activities, followed by those rich in flavanones (e.g. orange and grapefruit) and flavonols (e.g. onion, leek, spinach and green cabbage), while the hydroxycinnamate-rich fruit (e.g. apple, tomato, pear and peach) consistently elicited the lower antioxidant activities. The TEAC, FRAP and ORAC values for each extract were relatively similar and well-correlated with the total phenolic and vitamin C contents. The antioxidant activities (TEAC) in terms of 100 g FW uncooked portion size were in the order: strawberry>> raspberry = red plum >> red cabbage >>>grapefruit = orange > spinach > broccoli > green grape approximately/= onion > green cabbage > pea > apple > cauliflower tomato approximately/= peach=leek > banana approximately/= lettuce.

6-methoxyflavanones as bitter taste receptor blockers for hTAS2R39.

Many (dietary) bitter compounds, e.g. flavonoids, activate bitter receptor hTAS2R39 in cell-based assays. Several flavonoids, amongst which some flavanones, are known not to activate this receptor. As certain flavanones are known to mask bitter taste sensorially, flavanones might act as bitter receptor antagonists. Fourteen flavanones were investigated for their potential to reduce activation of hTAS2R39 by epicatechin gallate (ECG), one of the main bitter compounds occurring in green tea. Three flavanones showed inhibitory behavior towards the activation of hTAS2R39 by ECG: 4'-fluoro-6-methoxyflavanone, 6,3'-dimethoxyflavanone, and 6-methoxyflavanone (in order of decreasing potency). The 6-methoxyflavanones also inhibited activation of hTAS2R14 (another bitter receptor activated by ECG), though to a lesser extent. Dose-response curves of ECG at various concentrations of the full antagonist 4'-fluoro-6-methoxyflavanone and wash-out experiments indicated reversible insurmountable antagonism. The same effect was observed for the structurally different agonist denatonium benzoate.

Bitter taste receptor activation by flavonoids and isoflavonoids: modeled structural requirements for activation of hTAS2R14 and hTAS2R39.

Many flavonoids and isoflavonoids have an undesirable bitter taste, which hampers their use as food bioactives. The aim of this study was to investigate the effect of a large set of structurally similar (iso)flavonoids on the activation of bitter receptors hTAS2R14 and hTAS2R39 and to predict their structural requirements to activate these receptors. In total, 68 compounds activated hTAS2R14 and 70 compounds activated hTAS2R39, among which 58 ligands were overlapping. Their activation threshold values varied over a range of 3 log units between 0.12 and 500 µM. Ligand-based 2D-fingerprint and 3D-pharmacophore models were created to detect structure-activity relationships. The 2D models demonstrated excellent predictive power in identifying bitter (iso)flavonoids and discrimination from inactive ones. The structural characteristics for an (iso)flavonoid to activate hTAS2R14 (or hTAS2R39) were determined by 3D-pharmacophore models to be composed of two (or three) hydrogen bond donor sites, one hydrogen bond acceptor site, and two aromatic ring structures, of which one had to be hydrophobic. The additional hydrogen bond donor feature for hTAS2R39 ligands indicated the possible presence of another complementary acceptor site in the binding pocket, compared to hTAS2R14. Hydrophobic interaction of the aromatic feature with the binding site might be of higher importance in hTAS2R14 than in hTAS2R39. Together, this might explain why OH-rich compounds showed different behaviors on the two bitter receptors. The combination of in vitro data and different in silico methods created a good insight in activation of hTAS2R14 and hTAS2R39 by (iso)flavonoids and provided a powerful tool in the prediction of their potential bitterness. By understanding the "bitter motif", introduction of bitter taste in functional foods enriched in (iso)flavonoid bioactives might be avoided.

Molecular structures of citrus flavonoids determine their effects on lipid metabolism in HepG2 cells by primarily suppressing apoB secretion.

This study investigated the underlying mechanisms of action for blood lipid lowering effects of citrus flavonoids and their methoxylated analogues (n = 19; dose range: 0-100 µM) in HepG2 cells. Cholesterol (CH) and triglyceride (TG) syntheses were assessed by measuring the incorporation of (14)C-acetate and (14)C-glycerol, respectively, whereas apoB secretion was determined by ELISA. Results show that two polymethoxylated citrus flavonoids (PMFs), tangeretin and nobiletin, potently inhibited apoB secretion (IC(50) = 13 and 29 µM, respectively) and modestly inhibited CH synthesis (IC(50) = 49 and 68 µM) and TG synthesis (IC(50) = 14 and 73 µM), without effecting LDL-receptor activity. Other PMFs (e.g., sinensetin) and non-PMFs (e.g., hesperetin and naringenin) had only weak effects on CH and TG syntheses and apoB secretion (IC(50) > 100 µM). The structure-activity analysis indicated that a fully methoxylated A-ring of the flavonoid structure was associated with a potent inhibitory activity on hepatic apoB secretion. In conclusion, this study using HepG2 cells indicates that citrus flavonoids with a fully methoxylated A-ring may lower blood CH and TG concentrations primarily by suppressing hepatic apoB secretion as a main underlying mode of action.

Soy isoflavones and other isoflavonoids activate the human bitter taste receptors hTAS2R14 and hTAS2R39.

The aim of this study was to identify the bitter receptor(s) that recognize the bitter taste of the soy isoflavone genistein. Screening of all 25 human bitter receptors revealed genistein as agonist of hTAS2R14 and hTAS2R39. Genistein displayed threshold values of 4 and 8 µM on hTAS2R14 and hTAS2R39 and EC(50) values of 29 and 49 µM, respectively. In addition, the behavior of structurally similar isoflavonoids was investigated. Although the two receptors are not closely related, the results for hTAS2R14 and hTAS2R39 were similar toward most isoflavonoid aglycones. By trend, threshold values were slightly lower on hTAS2R14. Glucosylation of isoflavones seemed to inhibit activation of hTAS2R14, whereas four of five glucosylated isoflavones were agonists of hTAS2R39, namely, glycitin, genistin, acetylgenistin, and malonylgenistin. A total of three hydroxyl substitutions of the A- and B-rings of the isoflavonoids seemed to be more favorable for receptor activation than fewer hydroxyl groups. The concentration of the trihydroxylated genistein in several soy foods exceeds the determined bitter receptor threshold values, whereas those of other soy isoflavones are around or below their respective threshold value. Despite its low concentration, genistein might be one of the main contributors to the bitterness of soy products. Furthermore, the bioactive isoflavonoids equol and coumestrol activated both receptors, indicating that their sensory impact should be considered when used as food ingredients.

Modeling of the relationship between dipeptide structure and dipeptide stability, permeability, and ACE inhibitory activity.

Selected di- and tripeptides exhibit angiotensin-I converting enzyme (ACE) inhibitory activity in vitro. However, the efficacy in vivo is most likely limited for most peptides due to low bioavailability. The purpose of this study was to identify descriptors of intestinal stability, permeability, and ACE inhibitory activity of dipeptides. A total of 228 dipeptides were synthesized; intestinal stability was obtained by in vitro digestion, intestinal permeability using Caco-2 cells and ACE inhibitory activity by an in vitro assay. Databases were constructed to study the relationship between structure and activity, permeability, and stability. Quantitative structure-activity relationship (QSAR) modeling was performed based on computed models using partial least squares regression based on 400 molecular descriptors. QSAR modeling of dipeptide stability revealed high correlation coefficients (R > 0.65) for models based on Z and X scales. However, amino acid (AA) clustering showed the best results in describing stability of dipeptides. The N-terminal AA residues Asp, Gly, and Pro as well as the C-terminal residues Pro, Ser, Thr, and Asp stabilize dipeptides toward luminal enzymatic peptide hydrolysis. QSAR modeling did not reveal significant correlation models for intestinal permeability. 2D-fingerprint models were identified describing ACE inhibitory activity of dipeptides. The intestinal stability of 12 peptides was predicted. Peptides were synthesized and stability was confirmed in simulated digestion experiments. Based on the results, specific dipeptides can be designed to meet both stability and activity criteria. However, postabsorptive ACE inhibitory activities of dipeptides in vivo are most likely limited due to the very low intestinal permeability of dipeptides.