An innovative aluminium foil electrode modified with Al nanoparticles and EDTA for lead detection in biological samples.

This study presents a novel Aluminium foil-based electrode characterized by its affordability, flexibility, and ease of modification with carboxylic moiety-containing organic molecules. Upon foil modification with Aluminium nanoparticles and EDTA (AlNP-EDTA/AlE), the modified electrode exhibits remarkable activity in the oxidation of lead at potentials around -0.4 V. The lead signal is derived from the oxidation of lead deposited on the electrode surface using anodic stripping voltammetry (ASV). The addition of EDTA to AlNP/AlE increased the anodic peak current of lead by more than 500 %. The surface characterization of the electrode was performed by scanning electron microscopy (SEM) and infrared spectroscopy (IR), while its electroactive properties were evaluated by cyclic voltammetry (CV) and electrical impedance spectroscopy (EIS). Optimal operating parameters include pH 2.1, square-wave voltammetry (SWV) with an accumulation time of 60 s and an accumulation potential of -0.8 V. A low detection limit of 0.20 µmol/L and a relative standard deviation (RSD) of 3.0 % were achieved using five different electrodes. The effectiveness of AlNP-EDTA/AlE was further demonstrated with consistent results in biological samples spiked with Pb.

Electrochemical Determination of Morin in Natural Food Using a Chitosan-Graphene Glassy Carbon Modified Electrode.

This report presents a new application for the chitosan-graphene glassy carbon electrode (Ch-G/GCE) system in the determination of the hydroxyflavonoid morin (MR), one of the flavonoids with the highest favorable activity for people, due to its natural properties by square-wave voltammetry (SWV). The anodic peak current for MR was observed at 0.50 V with an increase of 73% compared with the glassy carbon electrode unmodified. The surface areas of Ch-G/GCE, Ch/GCE and GCE evaluated by cyclic voltammetry were 0.140, 0.053 and 0.011 cm2, respectively. Additionally, an increase greater than 100% compared to the electrode without modification was observed. The detection limit was 0.30 µmol/L for MR, and the relative standard deviations (RSDs) were 1.8% (n = 6). Possible interferences as quercetin, rutin, and applications in real samples were also evaluated with very acceptable results.

Electrochemical determination of paracetamol in a pharmaceutical dose by adsorptive voltammetry with a carbon paste/La2O3 microcomposite.

This paper presents a new application for microcomposites based on carbon paste (CP) and La2O3 (LaOX). This simple and versatile microcomposite (LaOX/CPE) was applied toward the determination of paracetamol (PCM) through proton oxidation by square wave adsorptive voltammetry (SWAdV). The anodic peak current for PCM increased by nearly 70.0% compared with an unmodified CP, and the detection limit was 0.020 µmol L-1. The relative standard deviations (RSDs) were 1.0% (n = 7). The accuracy of the new method was evaluated with tap water spiked with known quantities of PCM, while ascorbic acid, caffeine, and acetylsalicylic acid were used for interference studies. Finally, the usefulness of the microcomposite was shown to have acceptable results when applied to detect and quantify PCM in various forms of a pharmaceutical dose, such as solid tablets, fruit-flavored powders for colds and syrups for children.

Development of a microcomposite with single-walled carbon nanotubes and Nd2O3 for determination of paracetamol in pharmaceutical dosage by adsorptive voltammetry.

This study presents for the first time a new composite of carbon paste (CP), single-walled carbon nanotubes (SWCNTs) and Nd2O3 (NdOX). This versatile composite (NdOX-SWCNT/CPE) was applied to the oxidation of paracetamol (PCM). The newly formed surface was characterized by scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The results showed greater conductivity and a higher surface area for the composite than those of the carbon paste alone. Moreover, the anodic peak currents for PCM increased from 1.6 to 3.6 µA with CPE and NdOX-SWCNT/CPE, indicating an increase of nearly 51.0% for the anodic peak current. On the other hand, the anodic peak potentials shifted from 0.67 to 0.57 V. The detection limits were 0.05 µmol/L with NdOX-SWCNT/CPE and 0.50 µmol/L with SWCNT/CPE. The relative standard deviations (RSDs) were 1.5% (n = 7). The accuracy and interference of the methods were evaluated with a urine chemistry control spiked with known quantities of PCM, uric acid, dopamine, ascorbic acid, caffeine, acetylsalicylic acid, tartrazine, sunset yellow, allure red, rutin, morin and metal ions. Finally, the novelty and usefulness of the composite were evaluated to quantify PCM in pharmaceutical dosage forms such as tablets, powders and syrups for children.

Development of a microcomposite with single-walled carbon nanotubes and Nd2O3 for determination of paracetamol in pharmaceutical dosage by adsorptive voltammetry / 药物分析学报

This study presents for the first time a new composite of carbon paste (CP), single-walled carbon na-notubes (SWCNTs) and Nd2O3 (NdOX). This versatile composite (NdOX-SWCNT/CPE) was applied to the oxidation of paracetamol (PCM). The newly formed surface was characterized by scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The re-sults showed greater conductivity and a higher surface area for the composite than those of the carbon paste alone. Moreover, the anodic peak currents for PCM increased from 1.6 to 3.6 mA with CPE and NdOX-SWCNT/CPE, indicating an increase of nearly 51.0％ for the anodic peak current. On the other hand, the anodic peak potentials shifted from 0.67 to 0.57 V. The detection limits were 0.05 mmol/L with NdOX-SWCNT/CPE and 0.50 mmol/L with SWCNT/CPE. The relative standard deviations (RSDs) were 1.5％(n =7). The accuracy and interference of the methods were evaluated with a urine chemistry control spiked with known quantities of PCM, uric acid, dopamine, ascorbic acid, caffeine, acetylsalicylic acid, tartrazine, sunset yellow, allure red, rutin, morin and metal ions. Finally, the novelty and usefulness of the composite were evaluated to quantify PCM in pharmaceutical dosage forms such as tablets, powders and syrups for children.

Determination of Allura Red in the Presence of Cetylpyridinium Bromide by Square-wave Adsorptive Stripping Voltammetry on a Glassy Carbon Electrode.

A simple method to determine Allura Red (AR) in cherry gelatin, chili sauce and strawberry juice by square-wave adsorptive stripping voltammetry (SWAdV) in the presence of cetylpyridinium bromide (CPB) is reported. Using a glassy carbon electrode (GCE), Amaranth (AM), Ponceau 4R (P-4R) and AR were oxidized to very close potential values in medium acid pH 3.0 with phosphate buffer solution (PBS), making selective AR detection impossible. Under these conditions AM, P-4R and AR were oxidized at 0.80, 0.77 and 0.81 V, respectively. When small amounts of CPB were added, an AR-CPB aggregate was formed. This change displaced the oxidation almost 0.14 V in the direction of the most positive potential and increased the current almost 50%, while AM and P-4R were oxidized at almost the same potential. Under these conditions, the selective determination of AR in food was possible. Detection limit was 0.032 µmol L-1. Finally, the method was successfully applied to the determination of AR in these foods.

Electrocomposite Developed with Chitosan and Ionic Liquids Using Screen-Printed Carbon Electrodes Useful to Detect Rutin in Tropical Fruits.

This work reports the development of a composite of the ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM]BP4) and chitosan (CS) described in previous reports through a new method using cyclic voltammetry with 10 cycles at a scan rate of 50.0 mV s-1. This method is different from usual methods such as casting, deposition, and constant potential, and it allows the development of an electroactive surface toward the oxidation of rutin by stripping voltammetry applied to the detection in tropical fruits such as orange, lemon, and agraz (Vaccinium meridionale Swartz), with results similar to those reported in previous studies. In addition, the surface was characterized by electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM), and Raman spectroscopy. The limit of detection was 0.07 µmol L-1 and the relative standard deviation (RSD) of 10 measurements using the same modified electrode was 0.86%. Moreover, the stability of the sensor was studied for six days using the same modified electrode, where the variation of the signal using a known concentration of rutin (RT) was found to be less than 5.0%. The method was validated using a urine chemistry control spiked with known amounts of RT and possible interference was studied using ten substances including organic and biological compounds, metal ions, and dyes. The results obtained in this study demonstrated that this electrodeveloped composite was sensitive, selective, and stable.

Development of Antibacterial and Antifungal Triazole Chromium(III) and Cobalt(II) Complexes: Synthesis and Biological Activity Evaluations.

In this work, six complexes (2⁻7) of Cr(III) and Co(II) transition metals with triazole ligands were synthesized and characterized. In addition, a new ligand, 3,5-bis(1,2,4-triazol-1-ylmethyl)toluene (1), was synthesized and full characterized. The complexes were obtained as air-stable solids and characterized by melting point, electrical conductivity, thermogravimetric analysis, and Raman, infrared and ultraviolet/visible spectroscopy. The analyses and spectral data showed that complexes 3⁻7 had 1:1 (M:L) stoichiometries and octahedral geometries, while 2 had a 1:2 (M:L) ratio, which was supported by DFT calculations. The complexes and their respective ligands were evaluated against bacterial and fungal strains with clinical relevance. All the complexes showed higher antibacterial and antifungal activities than the free ligands. The complexes were more active against fungi than against bacteria. The activities of the chromium complexes against Candida tropicalis are of great interest, as they showed minimum inhibitory concentration 50 (MIC50) values between 7.8 and 15.6 µg mL-1. Complexes 5 and 6 showed little effect on Vero cells, indicating that they are not cytotoxic. These results can provide an important platform for the design of new compounds with antibacterial and antifungal activities.

UHPLC-ESI-ORBITRAP-MS analysis of the native Mapuche medicinal plant palo negro (Leptocarpha rivularis DC. - Asteraceae) and evaluation of its antioxidant and cholinesterase inhibitory properties.

UHPLC/ESI/MS identification of organic compounds is the first step in the majority of screening techniques for the characterization of biologically active metabolites in natural sources. This paper describes a method for the fast identification and characterisation of secondary metabolites in Leptocarpha rivularis DC. (Palo negro) extracts by HPLC/UV (DAD)-Mass Spectrometry (HPLC/MS). The plant is used for the treatment of several diseases since pre-hispanic Mapuche times. Thirty-seven compounds were detected in the aqueous edible extract for the first time including 4 sesquiterpenes, 10 flavonoids, 9 oxylipins, 2 organic acids, and 11 phenolic acids. In addition, phenolic content antioxidant and cholinesterase inhibitory activities were measured for the first time using the edible infusion. The total polyphenol content of the infusion was 230.76 ± 2.5 mmol GAE/kg dry weight, while the antioxidant activity was 176.51 ± 28.84; 195.28 ± 4.83; and 223.92 ± 2.95 mmol TE/kg dry weight, for the DPPH, ABTS, and FRAP assays, respectively. The cholinesterase inhibitory activity was 7.38 ± 0.03 and 5.74 ± 0.06 mmol GALAE/kg, for the inhibition of acetylcholinesterase AChE and BChE, respectively, showing that this plant is a candidate for the isolation of compounds that can be useful for the treatment of neurodegenerative diseases. Furthermore, this plant could serve also as a raw material for the production of dietary supplements, due to its content of polyphenolic compounds.

Detection of SO2 derivatives using a new chalco-coumarin derivative in cationic micellar media: application to real samples.

A new probe (E)-7-(diethylamino)-3-(3-(thiophen-2-yl)acryloyl)-2H-chromen-2-one (ChC16) was synthesized and studied as a turn-on fluorescent probe, based on a Michael addition mechanism for sensing SO2 derivatives, which is favored in the presence of cationic micellar media such as cetylpyridinium bromide (CPB). The probe showed high selectivity and sensitivity toward bisulfite over other anions and biothiols, including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), with a detection limit of 240 nM. Moreover, the probe showed great potential for its practical application in the detection of bisulfite in real samples, such as dry white wine, and in bioimaging.

Development of an iron-selective antioxidant probe with protective effects on neuronal function.

Iron accumulation, oxidative stress and calcium signaling dysregulation are common pathognomonic signs of several neurodegenerative diseases, including Parkinson´s and Alzheimer's diseases, Friedreich ataxia and Huntington's disease. Given their therapeutic potential, the identification of multifunctional compounds that suppress these damaging features is highly desirable. Here, we report the synthesis and characterization of N-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)-2-(7-hydroxy-2-oxo-2H-chromen-4-yl)acetamide, named CT51, which exhibited potent free radical neutralizing activity both in vitro and in cells. CT51 bound Fe2+ with high selectivity and Fe3+ with somewhat lower affinity. Cyclic voltammetric analysis revealed irreversible binding of Fe3+ to CT51, an important finding since stopping Fe2+/Fe3+ cycling in cells should prevent hydroxyl radical production resulting from the Fenton-Haber-Weiss cycle. When added to human neuroblastoma cells, CT51 freely permeated the cell membrane and distributed to both mitochondria and cytoplasm. Intracellularly, CT51 bound iron reversibly and protected against lipid peroxidation. Treatment of primary hippocampal neurons with CT51 reduced the sustained calcium release induced by an agonist of ryanodine receptor-calcium channels. These protective properties of CT51 on cellular function highlight its possible therapeutic use in diseases with significant oxidative, iron and calcium dysregulation.

Secondary Metabolite Profiling of Species of the Genus Usnea by UHPLC-ESI-OT-MS-MS.

Lichens are symbiotic associations of fungi with microalgae and/or cyanobacteria, which are considered among the slowest growing organisms, with strong tolerance to adverse environmental conditions. There are about 400 genera and 1600 species of lichens and those belonging to the Usnea genus comprise about 360 of these species. Usnea lichens have been used since ancient times as dyes, cosmetics, preservatives, deodorants and folk medicines. The phytochemistry of the Usnea genus includes more than 60 compounds which belong to the following classes: depsides, depsidones, depsones, lactones, quinones, phenolics, polysaccharides, fatty acids and dibenzofurans. Due to scarce knowledge of metabolomic profiles of Usnea species (U. barbata, U. antarctica, U. rubicunda and U. subfloridana), a study based on UHPLC-ESI-OT-MS-MS was performed for a comprehensive characterization of their secondary metabolites. From the methanolic extracts of these species a total of 73 metabolites were identified for the first time using this hyphenated technique, including 34 compounds in U. barbata, 21 in U. antarctica, 38 in U. rubicunda and 37 in U. subfloridana. Besides, a total of 13 metabolites were not identified and reported so far, and could be new according to our data analysis. This study showed that this hyphenated technique is rapid, effective and accurate for phytochemical identification of lichen metabolites and the data collected could be useful for chemotaxonomic studies.

Adsorptive Stripping Voltammetric Determination of Amaranth and Tartrazine in Drinks and Gelatins Using a Screen-Printed Carbon Electrode.

A fast, sensitive, and selective method for the simultaneous determination of one pair of synthetic colorants commonly found mixed in food products, Amaranth (AM) and Tartrazine (TZ), based on their adsorption and oxidation on a screen-printed electrode (SPE) is presented. The variation of peak current with pH, supporting electrolyte, adsorption time, and adsorption potential were optimized using square wave adsorptive voltammetry. The optimal conditions were found to be: pH 3.2 (PBS), Eads 0.00 V, and tads 30 s. Under these conditions, the AM and TZ signals were observed at 0.56 and 0.74 V, respectively. A linear response were found over the 0.15 to 1.20 µmol L-1 and 0.15 to 0.80 µmol L-1 concentrations, with detection limits (3σ/slope) of 26 and 70 nmol L-1 for AM and TZ, respectively. Reproducibility for 17.7 µmol L-1 AM and TZ solutions were 2.5 and 3.0% (n = 7), respectively, using three different electrodes. The method was validated by determining AM and TZ in spiked tap water and unflavored gelatin spiked with AM and TZ. Because a beverage containing both AM and TZ was not found, the method was applied to the determination of AM in a kola soft drink and TZ in an orange jelly and a soft drink powder.

Metabolomic Analysis of Two Parmotrema Lichens: P. robustum (Degel.) Hale and P. andinum (Mull. Arg.) Hale Using UHPLC-ESI-OT-MS-MS.

Lichens are symbiotic associations of fungi with microalgae and/or cyanobacteria. Lichens belonging to the Parmeliaceae family comprise 2700 species of lichens, including the Parmotrema genus which is composed of 300 species. The metabolites of this genus include depsides, depsidones, phenolics, polysaccharides, lipids, diphenylethers and dibenzofurans, which are responsible for the biological activities reported including antidiabetic, antihelmintic, anticancer, antioxidant, antibacterial, anti-inflammatory, antimitotic, antitumoral, antifungal, and antioxidant enzyme inhibitory. Due to scarce knowledge of metabolomic profiles of Parmotrema species (P. andinum and P. robustum), a full metabolome study based on ultra-high performance liquid chromatography- diode array detector-electrospray ionization-quadrupole-orbitrap-mass-spectrometry (UHPLC-DAD-ESI-Q-orbitrap MS) was performed for a comprehensive characterization of their substances. From the methanolic extracts of these species, a total of 54 metabolites were identified for the first time using this hyphenated technique, including thirty compounds in P. andinum, and thirty-seven in P. robustum. Moreover, two compounds were not identified as known compounds, and could be new structures, according to our data. This report shows that this technique is effective and accurate for rapid chemical identification of lichen substances and the compounds identified could serve as chemotaxonomic markers to differentiate these ruffle lichens.

Determinación de hidroquinona usando un electrodo de carbono vítreo modificado con quitosano, nanotubos de carbono de pared múltiple y líquido iónico. Posible uso como sensor / Determination of hydroquinone using a glassy carbon electrode modified with chitosan, multi-wall carbon nano-tubes and ionic liquid. Possible use as sensor

La propiedad electroactiva de hidroquinona (HQ) se estudió sobre un electrodo de carbono vítreo modificado con quitosano, nanotubos de carbono de pared múltiple (Ch-MWCNT-GCE). La HQ se depositó a un potencial controlado sobre la superficie del electrodo y la oxidación de la HQ se midió por voltametría de onda cuadrada (SWV, por sus siglas en inglés). Se observó una corriente de oxidación a 0,39 V y una corriente de reducción a 0,21 V con un ΔV de 0,18 V; ello indicó un proceso reversible. Un aumento en las corrientes de oxidación y de reducción de casi 50% se observó cuando el Ch-MWCNT-GCE se recubrió con líquido iónico (LI). El electrodo se caracterizó por voltamperometría cíclica (CV, por sus siglas en inglés) en presencia y sin la presencia de diferentes líquidos iónicos con distinto anión, siendo el más óptimo el líquido iónico (LI) 1-butil-3-metilimidazolio hexafluorofosfato (BMIMPF6). Se estudiaron las variables experimentales como pH, tiempo de adsorción (t ads) y potencial de adsorción (Eads), así como también las potenciales interferencias. Bajo las óptimas condiciones (pH 3,0; t acc 60s; Eacc 0,10 V), el pico de la corriente es proporcional a la concentración de HQ entre 4,20 * 10-6 y 30,0 * 10-6 mol L-1, con un límite de detección de 2,45 * 10-7 mol L-1. La desviación estándar relativa para una solución que contiene 1,0 * 10-4 mol L-1 de HQ, fue de 1,5% para siete medidas iguales. El método se validó con una muestra de agua dopada con HQ.

Property of the electroactive hydroquinone (HQ) was studied on a glassy carbon electrode modified chitosan, multi wall carbon nanotubes (Ch-MWCNT-GCE). HQ is deposited at a controlled potential to the electrode surface and oxidation of HQ is measured by square wave voltammetry (SWV). An oxidation current to 0.39 V and a reduction current to 0.21 V with a 0.18 ΔV indicating a reversible process were observed. An increase in the flow of oxidation and reduction of almost 50% was observed when the Ch-MWCNT-GCE was coated with ionic liquid (LI). The electrode was characterized by cyclic voltammetry (CV) in the presence and without the presence of different ionic liquids with different anion being the most optimal ionic liquid (LI) 1-butyl-3-methyllimidazolio hexafluorophosphate (BMIMPF6). Experimental variables such as pH, adsorption time (TADs) and adsorption potential (Eads), as well as potential interference, were studied. Under optimal conditions (pH 3.0; t acc 60s; Eacc 0.10 V) the peak current is proportional to the concentration of HQ between 4.20 * 10-6 and 30.0 * 10-5 mol L-1 with a detection limit of 2.45 * 10-7 mol L-1. The relative standard deviation for a solution containing 1.0 * 10-4 mol L-1 HQ was 1.5% for seven equal measures. The method was validated with a water sample doped with HQ.

Nafion-mercury coated film electrode for the adsorptive stripping voltammetric determination of lead and cadmium in the presence of pyrogallol red.

An adsorptive stripping voltammetric (AdSV) method is presented for the simultaneous determination of Pb(II) and Cd(II) at trace levels in natural waters, based on metal complexation with pyrogallol red (PR) and subsequent adsorptive deposition on a Nafion-mercury coated glassy carbon electrode (NHgFE). Pyrogallol red forms complexes with a metal:ligand stoichiometry of 1:1 with Pb(II) and of 1:2 with Cd(II). Optimal analytical conditions were pH 4.0 (acetate buffer); C(PR)=2.8 µmol L(-1); E(ads)=-0.40 V vs. Ag/AgCl; t(ads)=100 s. The linear calibration curves ranged from 1.0 µg L(-1) to 16.0 µg L(-1) for Pb(II) and from 1.0 µg L(-1) to 13.0 µg L(-1) for Cd(II). The detection limits (S/N=3) were 0.05 µg L(-1) for Pb(II) and 0.01 µg L(-1) for Cd(II). The relative standard deviation was 1.0% and 2.0% (n=7), respectively, for a solution containing 5.0 µg L(-1) Pb(II) and Cd(II). The method was validated by determining Pb(II) and Cd(II) in certified reference waste water (SPS-WW1). Finally, the method was applied to the determination of Pb(II) and Cd(II) in commercial mineral water samples after UV digestion.

Adsorptive stripping voltammetry of nickel with 1-nitroso-2-napthol using a bismuth film electrode.

A sensitive procedure is presented for the voltammetric determination of nickel. The procedure involves an adsorptive accumulation of nickel 1-nitroso-2-napthol (NN) complex on a bismuth film electrode prepared ex situ by electrodeposition. The most suitable operating conditions and parameters such as pH, ligand concentration (C(NN)), adsorptive potential (E(ads)), adsorptive time (t(ads)), scan rate and others were selected and the determination of nickel in aqueous solutions using the standard addition method was possible. The adsorbed Ni-NN complex gives a well defined cathodic stripping peak current at -0.70 V, which was used for the determination of nickel in the concentration range of 10.0-70.0 µg L(-1) (pH 7.5; C(NN) 6.5 µmol L(-1); E(ads) -0.30 V; t(ads) 60s) with a detection limit of 0.1 µg L(-1). The relative standard deviation for a solution containing 10.0 µg L(-1) of Ni(II) was 3.5% (n=4). The proposed method was validated determining Ni(II) in certified reference waste water (SPS-WW1) and Certified Reference Water for Trace Elements (TMDA 51.3) with satisfactory results. Then lake water samples were analyzed.

Determination of lead in the presence of morin-5'-sulfonic acid and sodium dodecyl sulfate by adsorptive stripping voltammetry.

A simple and sensitive electroanalytical method is developed for the determination of lead by adsorptive stripping voltammetry (AdSV) in the presence of morin-5'-sulfonic acid (MSA) and sodium dodecyl sulfate (SDS). The Pb-MSA complex accumulates on the surface of a hanging mercury drop electrode (HMDE) and peak current is measured by square wave voltammetry (SWV). The complex is reduced at -0.48 V and peak current increases when low concentrations of SDS are added to the sample solution. The experimental variables pH, MSA concentration (C(MSA)); accumulation time (t(acc)); accumulation potential (E(acc)), and SDS concentration (C(SDS)), as well as potential interferences, are investigated. Under the optimized conditions (pH 3.2; C(MSA): 0.5 micromol L(-1); t(acc): 60s; E(acc): -0.35 V, and C(SDS): 20 micromol L(-1)), peak current is proportional to the concentration of Pb(II) over the 0.1-32.0 microg L(-1) range, with a detection limit of 0.04 microg L(-1). The relative standard deviation for a solution containing 5.0 microg L(-1) of Pb(II) solution was 1.5% for seven successive assays. The method was validated by determining Pb(II) in synthetic sea water (ASTM D665) spiked with ICP multi-element standard solution and in certified reference water (GBW08607). Finally, the method was successfully applied to the determination of Pb(II) in tap water and sea water after UV digestion.