Synergistic Interactions between Tocol and Phenolic Extracts from Different Tree Nut Species against Human Cancer Cell Lines.

Tree nuts are rich in polar (phenolic compounds) and non-polar (tocols) antioxidants, with recognized effects in the prevention of diseases such as cancer. These biomolecules possess antiproliferative activity on cancer cells; however, the combined effect of both types of compounds has been scarcely studied, and this approach could give valuable information on the real anticancer potential of tree nuts. In the present study, the antiproliferative activity of pure tocols and phenolic compounds, tocol- and phenolic-rich extracts (TRE and PRE, respectively) from tree nuts and the extracts combinations, was evaluated in four cancer (HeLa, MCF7, PC3, A549) and one control (ARPE) cell lines. The most sensible cell lines were HeLa and MCF7. TRE and PRE from nuts were chemically characterized; γ and δ tocopherols, total tocols, total tocopherols and total phenolic compounds were negatively correlated with cell viability in MCF7 cells. In HeLa cells, only δ and total tocopherols were negatively correlated with cell viability. TRE and PRE had a low effect in reducing cell viability of the cancer cell lines, the most effective extracts were those of emory oak acorn (EOA), pecan nut (PEC) and walnut (WAL), and these were further studied for their pharmacological interactions, using the combination index and the isobologram methods. Combinations of both extracts showed a synergistic and strongly synergistic behavior in the three nuts (EOA, PEC and WAL), with combination indexes between 0.12 and 0.55. These results highlight the need to understand the interactions among components found in complex natural extracts or food products in order to fully understand their bioactivities.

Is Chickpea a Potential Substitute for Soybean? Phenolic Bioactives and Potential Health Benefits.

Legume seeds are rich sources of protein, fiber, and minerals. In addition, their phenolic compounds as secondary metabolites render health benefits beyond basic nutrition. Lowering apolipoprotein B secretion from HepG2 cells and decreasing the level of low-density lipoprotein (LDL)-cholesterol oxidation are mechanisms related to the prevention of cardiovascular diseases (CVD). Likewise, low-level chronic inflammation and related disorders of the immune system are clinical predictors of cardiovascular pathology. Furthermore, DNA-damage signaling and repair are crucial pathways to the etiology of human cancers. Along CVD and cancer, the prevalence of obesity and diabetes is constantly increasing. Screening the ability of polyphenols in inactivating digestive enzymes is a good option in pre-clinical studies. In addition, in vivo studies support the role of polyphenols in the prevention and/or management of diabetes and obesity. Soybean, a well-recognized source of phenolic isoflavones, exerts health benefits by decreasing oxidative stress and inflammation related to the above-mentioned chronic ailments. Similar to soybeans, chickpeas are good sources of nutrients and phenolic compounds, especially isoflavones. This review summarizes the potential of chickpea as a substitute for soybean in terms of health beneficial outcomes. Therefore, this contribution may guide the industry in manufacturing functional foods and/or ingredients by using an undervalued feedstock.

Mango phenolics increase the serum apolipoprotein A1/B ratio in rats fed high cholesterol and sodium cholate diets.

BACKGROUND: Serum lipoproteins are in dynamic equilibrium, partially controlled by the apolipoprotein A1 to apolipoprotein B ratio (APOA1/APOB). Freeze-dried mango pulp (FDM) is a rich source of phenolic compounds (MP) and dietary fiber (MF), although their effects on lipoprotein metabolism have not yet been studied. RESULTS: Thirty male Wistar rats were fed with four different isocaloric diets (3.4 kcal g-1 ) for 12 weeks: control diet, high cholesterol (8 g kg-1 ) + sodium cholate (2 g kg-1 ) diet either alone or supplemented with MF (60 g kg-1 ), MP (1 g kg-1 ) or FDM (50 g kg-1 ). MP and FDM reduced food intake, whereas MF and MP tended to increase serum APOA1/APOB ratio, independently of their hepatic gene expression. This suggests that lipoprotein metabolism was favorably altered by mango bioactives, MP also mitigated the non-alcoholic steatohepatitis that resulted from the intake of this diet. CONCLUSION: We propose that phenolics are the most bioactive components of mango pulp, acting as anti-atherogenic and hepatoprotective agents, with a mechanism of action tentatively based on changes to the main protein components of lipoproteins. © 2018 Society of Chemical Industry.

The Antidiabetic Mechanisms of Polyphenols Related to Increased Glucagon-Like Peptide-1 (GLP1) and Insulin Signaling.

Type-2 diabetes mellitus (T2DM) is an endocrine disease related to impaired/absent insulin signaling. Dietary habits can either promote or mitigate the onset and severity of T2DM. Diets rich in fruits and vegetables have been correlated with a decreased incidence of T2DM, apparently due to their high polyphenol content. Polyphenols are compounds of plant origin with several documented bioactivities related to health promotion. The present review describes the antidiabetic effects of polyphenols, specifically related to the secretion and effects of insulin and glucagon-like peptide 1 (GLP1), an enteric hormone that stimulates postprandial insulin secretion. The evidence suggests that polyphenols from various sources stimulate L-cells to secrete GLP1, increase its half-life by inhibiting dipeptidyl peptidase-4 (DPP4), stimulate ß-cells to secrete insulin and stimulate the peripheral response to insulin, increasing the overall effects of the GLP1-insulin axis. The glucose-lowering potential of polyphenols has been evidenced in various acute and chronic models of healthy and diabetic organisms. Some polyphenols appear to exert their effects similarly to pharmaceutical antidiabetics; thus, rigorous clinical trials are needed to fully validate this claim. The broad diversity of polyphenols has not allowed for entirely describing their mechanisms of action, but the evidence advocates for their regular consumption.

In vitro Inhibition of Pancreatic Lipase by Polyphenols:A Kinetic, Fluorescence Spectroscopy and Molecular Docking Study.

The inhibitory activity and binding characteristics of caffeic acid, p-coumaric acid, quercetin and capsaicin, four phenolic compounds found in hot pepper, against porcine pancreatic lipase activity were studied and compared to hot pepper extract. Quercetin was the strongest inhibitor (IC50=(6.1±2.4) µM), followed by p-coumaric acid ((170.2±20.6) µM) and caffeic acid ((401.5±32.1) µM), while capsaicin and a hot pepper extract had very low inhibitory activity. All polyphenolic compounds showed a mixed-type inhibition. Fluorescence spectroscopy studies showed that polyphenolic compounds had the ability to quench the intrinsic fluorescence of pancreatic lipase by a static mechanism. The sequence of Stern-Volmer constant was quercetin, followed by caffeic and p-coumaric acids. Molecular docking studies showed that caffeic acid, quercetin and p-coumaric acid bound near the active site, while capsaicin bound far away from the active site. Hydrogen bonds and π-stacking hydrophobic interactions are the main pancreatic lipase-polyphenolic compound interactions observed.

Modulation of PPAR Expression and Activity in Response to Polyphenolic Compounds in High Fat Diets.

Peroxisome proliferator-activated receptors (PPAR) are transcription factors that modulate energy metabolism in liver, adipose tissue and muscle. High fat diets (HFD) can negatively impact PPAR expression or activity, favoring obesity, dyslipidemia, insulin resistance and other conditions. However, polyphenols (PP) found in vegetable foodstuffs are capable of positively modulating this pathway. We therefore focused this review on the possible effects that PP can have on PPAR when administered together with HFD. We found that PP from diverse sources, such as coffee, olives, rice, berries and others, are capable of inducing the expression of genes involved in a decrease of adipose mass, liver and serum lipids and lipid biosynthesis in animal and cell models of HFD. Since cells or gut bacteria can transform PP into different metabolites, it is possible that a synergistic or antagonistic effect ultimately occurs. PP molecules from vegetable sources are an interesting option to maintain or return to a state of energy homeostasis, possibly due to an adequate PPAR expression and activity.

Cyanidin-3-O-glucoside: Physical-Chemistry, Foodomics and Health Effects.

Anthocyanins (ACNs) are plant secondary metabolites from the flavonoid family. Red to blue fruits are major dietary sources of ACNs (up to 1 g/100 g FW), being cyanidin-3-O-glucoside (Cy3G) one of the most widely distributed. Cy3G confers a red hue to fruits, but its content in raspberries and strawberries is low. It has a good radical scavenging capacity (RSC) against superoxide but not hydroxyl radicals, and its oxidative potential is pH-dependent (58 mV/pH unit). After intake, Cy3G can be metabolized (phases I, II) by oral epithelial cells, absorbed by the gastric epithelium (1%-10%) and it is gut-transformed (phase II & microbial metabolism), reaching the bloodstream (<1%) and urine (about 0.02%) in low amounts. In humans and Caco-2 cells, Cy3G's major metabolites are protocatechuic acid and phloroglucinaldehyde which are also subjected to entero-hepatic recycling, although caffeic acid and peonidin-3-glucoside seem to be strictly produced in the large bowel and renal tissues. Solid evidence supports Cy3G's bioactivity as DNA-RSC, gastro protective, anti-inflammatory, anti-thrombotic chemo-preventive and as an epigenetic factor, exerting protection against Helicobacter pylori infection, age-related diseases, type 2 diabetes, cardiovascular disease, metabolic syndrome and oral cancer. Most relevant mechanisms include RSC, epigenetic action, competitive protein-binding and enzyme inhibition. These and other novel aspects on Cy3G's physical-chemistry, foodomics, and health effects are discussed.

Structural Stability and Viability of Microencapsulated Probiotic Bacteria: A Review.

Probiotics are live microorganisms that confer a number of health benefits when consumed in adequate amounts, mostly due to improvement of intestinal microflora. Bacterial strains from the genera Lactobacillus, Bifidobacterium, and Bacillus have been widely studied and are used to prepare ready-to-eat foods. However, the physicochemical stability and bioavailability of these bacteria have represented a challenge for many years, particularly in nonrefrigerated foodstuffs. Microencapsulation (ME) helps to improve the survival of these bacteria because it protects them from harsh conditions, such as high temperature, pH, or salinity, during the preparation of a final food product and its gastrointestinal passage. The most common coating materials used in the ME of probiotics are ionic polysaccharides, microbial exopolysaccharides, and milk proteins, which exhibit different physicochemical features as well as mucoadhesion. Structurally, the survival of improved bacteria depends on the quantity and strength of the functional groups located in the bacterial cell walls, coating materials, and cross-linkers. However, studies addressing the role of these interacting groups and the resulting metabolic impacts are still scarce. The fate of new probiotic-based products for the 21st century depends on the correct selection of the bacterial strain, coating material, preparation technique, and food vehicle, which are all briefly reviewed in this article.

Enriched Red Wine: Phenolic Profile, Sensory Evaluation and In Vitro Bioaccessibility of Phenolic Compounds.

The beneficial health effect of red wine depends on its phenolic content and the phenolic content in red wines is affected by ecological, agricultural, and enological practices. Enriched wines have been proposed as an alternative to increase the phenolic content in wines. Nevertheless, phenolic compounds are related to the sensory characteristics of red wines, so enrichment of red wines requires a balance between phenolic content and sensory characteristics. In the present study, a Merlot red wine was enriched with a phenolic extract obtained from Cabernet Sauvignon grape pomace. Two levels of enrichment were evaluated: 4 and 8 g/L of total phenolic content (gallic acid equivalents, GAE). Wines were evaluated by a trained panel to determine their sensory profile (olfactive, visual, taste, and mouthfeel phases). The bioaccessibility of phenolic compounds from enriched red wines was evaluated using an in vitro digestive model and phenolic compounds were quantified by High Performance Liquid Chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). Enrichment increased mainly flavonols and procyanidins. Such an increase impacted astringency and sweetness perceived by judges. This study proposes an alternative to increase the phenolic content in wines without modifying other main sensory characteristics and offers a potential beneficial effect on the health of consumers.

Semi-Targeted Ultra-High-Performance Chromatography Coupled to Mass Spectrometry Analysis of Phenolic Metabolites in Plasma of Elderly Adults.

A group of 23 elderly persons was given functional meals (a beverage and a muffin) specially formulated for the prevention of sarcopenia (age-related loss of muscle mass). Plasma samples were taken at the beginning of the intervention and after 30 days of consuming the functional meals. A semi-targeted ultra-high-performance chromatography coupled with tandem mass (UPLC-MS/MS) analysis was carried out to identify phenolic compounds and their metabolites. Plasma proteins were precipitated with ethanol and the samples were concentrated and resuspended in the mobile phase (1:1 acetonitrile: water) before injection into the UPLC-MS/MS instrument. Separation was carried out with a C18 reverse-phase column, and compounds were identified using their experimental mass, isotopic distribution, and fragment pattern. Compounds of interest were compared to those of data banks and the internal semi-targeted library. Preliminary results showed that the major metabolites identified after the intervention were phenylacetic acid, glycitin, 3-hydroxyphenylvaleric acid, and gomisin M2.

Phytochemical Content and Potential Health Applications of Pecan [Carya illinoinensis (Wangenh) K. Koch] Nutshell.

BACKGROUND: The pecan nutshell contains phytochemicals with various biological activities that are potentially useful in the prevention or treatment of diseases, such as cancer, diabetes, and metabolic imbalances associated with heart diseases. OBJECTIVE: The aim of this study is to update this topic by means of a literature review and include those studies that contribute to the knowledge of the chemical composition and biological activities of pecan nutshell, particularly those related to the therapeutic potential against some chronic degenerative diseases associated with oxidative stress. METHODS: Exhaustive and detailed review of the existing literature was conducted using electronic databases. CONCLUSION: The pecan nutshell is a promising natural product with pharmaceutical uses in various diseases. However, additional research related to the assessment of efficient extraction methods and characterization, particularly the evaluation of the mechanisms of action in new in vivo models, is necessary to confirm these findings and development of new drugs with therapeutic use.

Cardioprotective effect of red wine and grape pomace.

Several studies have related moderate consumption of red wine with prevention of cardiovascular diseases (CVD). According to epidemiological studies, those regions with high consumption of red wine and a Mediterranean diet show a low prevalence of CVD. Such an effect has been attributed to phenolic compounds present in red wines. On the other hand, by-products obtained during winemaking are also a significant source of phenolic compounds but have been otherwise overlooked. The cardioprotective effect of red wine and its byproducts is related to their ability to prevent platelet aggregation, modify the lipid profile, and promote vasorelaxation. Phenolic content and profile seem to play an important role in these beneficial effects. Inhibition of platelet aggregation is dose-dependent and more efficient against ADP. The antioxidant capacity of phenolic compounds from red wine and its by-products, is involved in preventing the generation of ROS and the modification of the lipid profile, to prevent LDL oxidation. Phenolic compounds can also, modulate the activity of specific enzymes to promote NO production and vasorelaxation. Specific phenolic compounds like resveratrol are related to promote NO, and quercetin to inhibit platelet aggregation. Nevertheless, concentration that causes those effects is far from that in red wines. Synergic and additive effects of a mix of phenolic compounds could explain the cardioprotective effects of red wine and its byproducts.

Functionality of Bread and Beverage Added with Brosimum alicastrum Sw. Seed Flour on the Nutritional and Health Status of the Elderly.

Physiological changes in elderly individuals (EI) can contribute to nutritional deterioration and comorbidities that reduce their quality of life. Factors such as diet can modulate some of these effects. The aim was to evaluate the functionality of foods added with Brosimum alicastrum Sw. seed flour in EI. EI (n = 23) living in nursing home conditions agreed to participate. A control stage was carried out (30 days) and subsequently, an intervention stage (30 days) was realized in which a muffin and a beverage, designed for EI, were added to the participants' their usual diet. In both stages, anthropometric parameters, body composition, nutritional status, dietary intake, sarcopenic status, cognitive and affective states, biometric parameters, and total phenolic compounds (TPC), and antioxidant capacity in foods and plasma of EI were determined. The results showed that the consumption of the foods improved the energy intake and preserved the muscle reserves of the EI. The EI gained body weight (+1.1 kg), increased their protein (+18.6 g/day; 1.5 g/kg BW/day), dietary fiber (+13.4 g/day), iron (+4.4 mg/day), zinc (+1.8 mg/day), folic acid (+83.4 µg/day) consumption while reducing their cholesterol (-66 mg/day) and sodium (-319.5 mg/day) consumption. LDL-C lipoproteins reduced (14.8%) and urea (33.1%) and BUN (33.3%) increased. The TPC increased (7.8%) in the plasma, particularly in women (10.7%). The foods improve the EI nutritional status, and this has a cardiovascular protective effect that can benefit the health of the EI.

Daily consumption of apple, pear and orange juice differently affects plasma lipids and antioxidant capacity of smoking and non-smoking adults.

Epidemiological studies have shown an inverse correlation between a fruit and vegetable-rich diet and cardiovascular diseases; this beneficial effect of fruits and vegetables is probably due to the presence of antioxidant phytochemicals. In contrast, cigarette smoking is a high risk factor for lung and heart diseases, associated with chronic oxidative stress. In the present study, the effect of the consumption of a pear, an apple and 200 ml orange juice, during 26 days, on total plasma antioxidant capacity (TAC) and lipid profile of chronic smokers and non-smoking healthy adults was analyzed. Fruit consumption increased TAC in non-smokers, but not in smokers. In non-smokers, total cholesterol, high-density lipoprotein-cholesterol, and low-density lipoprotein-cholesterol increased significantly; while in smokers, total cholesterol and low-density lipoprotein-cholesterol decreased. We may conclude fruit/juice supplementation showed different effects, depending on the smoking habit: in non-smokers it increased TAC and cholesterol; in smokers it reduced cholesterol, without inducing a TAC increase.

Interaction of N-succinyl-diaminopimelate desuccinylase with flavonoids.

DapE is an enzyme that belongs to the meso-diaminopimelate/Lysine pathway. It is recognized as an antimicrobial target, hence compounds that inhibit its catalytic activity are required. The principal features considered in the selection of potential inhibitors for this enzyme are compounds containing metal binding groups that could block access of the substrate to the Zinc metal centers and/or block the assembly of the oxyanion hole. We show the interaction of DapE from Enterococcus faecium, Staphylococcus aureus, Klebsiella aerogenes, Pseudomonas aeruginosa and Escherichia coli with flavonoids: quercetin, catechin, luteolin, rutin and hesperidin. Flavonoids contain several oxygen atoms distributed along their structure in a pattern that may be considered for the development of new antibiotics. Docking experiments suggest that these compounds containing metal binding groups that interact with metal centers of DapE and binding experiments indicate that glycoside flavonoids are preferred by DapE.

Effect of Elicitation on Polyphenol and Carotenoid Metabolism in Butterhead Lettuce (Lactuca sativa var. capitata).

The effect of elicitation in butterhead lettuce on carotenoid and polyphenol metabolism was evaluated. Different concentrations of arachidonic acid (AA), salicylic acid (SA), methyl jasmonate (MJ) (15, 45, and 90 µM) and Harpin protein (HP) (30, 60, and 120 mg/L) were applied on red and green butterhead lettuces. Total phenolic and flavonoid content were incremented by MJ (90 µM) in green and red lettuce. Carotenoids were increased in red lettuce (AA; 45 µM). Green lettuce modifies their phenolic acid profile after elicitation with AA and MJ; meanwhile, red lettuce incremented mainly in hydroxycinnamic acids and flavonols, MJ being the elicitor with the highest effect. There was an impact on secondary metabolite enzyme gene transcript concentration. Phenylalanine ammonia-lyase (PAL) and lycopene beta cyclase (LBC) increased in both varieties after elicitation. A relationship between phytochemical increase and the activation of the metabolic pathways after elicitation in butterhead lettuce was observed.

Chemical Composition and In Vitro Bioaccessibility of Antioxidant Phytochemicals from Selected Edible Nuts.

The ultimate health benefits of peanuts and tree nuts partially depend on the effective gastrointestinal delivery of their phytochemicals. The chemical composition and in vitro bioaccessibility of tocopherols, tocotrienols and phenolic compounds from peanuts and seven tree nuts were evaluated by analytical and chemometric methods. Total fat and dietary fiber (g 100 g-1) ranged from 34.2 (Emory oak acorn) to 72.5 (pink pine nut; PPN) and from 1.2 (PPN) to 22.5 (pistachio). Samples were rich in oleic and linoleic acids (56-87 g 100 g-1 oil). Tocopherols and tocotrienols (mg·kg-1) ranged from 48.1 (peanut) to 156.3 (almond) and 0 (almond, pecan) to 22.1 (PPN) and hydrophilic phenolics from 533 (PPN) to 12,896 (Emory oak acorn); flavonoids and condensed tannins (mg CE.100 g-1) ranged from 142 (white pine nut) to 1833 (Emory oak acorn) and 14 (PPN) to 460 (Emory oak acorn). Three principal components explained 90% of the variance associated with the diversity of antioxidant phytochemicals in samples. In vitro bioaccessibility of tocopherols, tocotrienols, hydrophilic phenolics, flavonoids, and condensed tannins ranged from 11-51%, 16-79%, 25-55%, 0-100%, and 0-94%, respectively. Multiple regression analyses revealed a potential influence of dietary fiber, fats and/or unsaturated fatty acids on phytochemical bioaccessibility, in a structure-specific manner.

Brosimum alicastrum Sw. (Ramón): An Alternative to Improve the Nutritional Properties and Functional Potential of the Wheat Flour Tortilla.

The wheat flour tortilla (WFT) is a Mexican food product widely consumed in the world, despite lacking fiber and micronutrients. Ramón seed flour (RSF) is an underutilized natural resource rich in fiber, minerals and bioactive compounds that can be used to improve properties of starchy foods, such as WFT. The study evaluated the impact of partial replacement of wheat flour with RSF on the physicochemical, sensory, rheological and nutritional properties and antioxidant capacity (AC) of RSF-containing flour tortilla (RFT). Results indicated that RFT (25% RSF) had higher dietary fiber (4.5 times) and mineral (8.8%; potassium 42.8%, copper 33%) content than WFT. Two sensory attributes were significantly different between RTF and WFT, color intensity and rollability. RFT was soft and it was accepted by the consumer. Phenolic compounds (PC) and AC were higher in RFT (11.7 times, 33%-50%, respectively) than WFT. PC identification by ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-QTOF-MS) showed that phenolic acids esterified with quinic acid, such as chlorogenic and other caffeoyl and coumaroyl derivatives were the major PC identified in RSF, resveratrol was also detected. These results show that RSF can be used as an ingredient to improve nutritional and antioxidant properties of traditional foods, such as the WFT.

Proanthocyanidins with a Low Degree of Polymerization are Good Inhibitors of Digestive Enzymes Because of their Ability to form Specific Interactions: A Hypothesis.

Inhibition of target digestive enzymes is an accepted strategy to prevent diseases such as obesity and diabetes. Proanthocyanidins (PACs) are known for their ability to bind, inhibit, and precipitate enzymes, which makes them potential bioDrugs with an impact on the digestive process. PAC degree of polymerization (DP) is one of the structural features responsible for their differential inhibitory potency but the explanation for this phenomenon is still unclear. Pecan nut (Carya illinoinensis L.) kernels and nutshells are rich in oligomeric and polymeric PACs. We have used thiolysis and HPLC analyses to propose four theoretical model structures of PACs representative of four semipurified fractions obtained from pecan kernel and shell, which showed different inhibitory activity against intestinal lipases, amylases, and proteases. The noncovalent interactions between PACs and digestive enzymes were predicted by in silico methods through computational software. These observations are discussed in view of current literature on the biological effects of PACs with different DPs and allowed us to propose the hypothesis that "small oligomeric PACs could be digestive enzyme inhibitors due to their capacity to enter and bind the enzymes' specific cavities better than polymers and oligomers of medium and high molecular weight."

Effect of Different Elicitors and Preharvest Day Application on the Content of Phytochemicals and Antioxidant Activity of Butterhead Lettuce (Lactuca sativa var. capitata) Produced under Hydroponic Conditions.

The effect of four elicitors on phytochemical content in two varieties of lettuce was evaluated. The best preharvest day for application of each elicitor was chosen. Solutions of arachidonic acid (AA), salicylic acid (SA), methyl jasmonate (MJ), and Harpin protein (HP) were applied by foliar aspersion on lettuce leaves while cultivating under hydroponic conditions. Application of elicitors was done at 15, 7, 5, 3, or 1 day before harvest. Green lettuce showed the highest increase in phytochemical content when elicitors (AA, SA, and HP) were applied on day 7 before harvest. Similarly, antioxidant activity rose in all treatments on day 7. In red lettuce, the highest content of bioactive molecules occurred in samples treated on day 15. AA, SA, and HP were the elicitors with the highest effect on phytochemical content for both varieties, mainly on polyphenol content. Antioxidant activity also increased in response to elicitation. HPLC-MS showed an increase in the content of phenolic acids in green and red lettuce, especially after elicitation with SA, suggesting activation of the caffeic acid pathway due to elicitation.