Antioxidant and Antifungal Activities and Characterization of Phenolic Compounds Using High-Performance Liquid Chromatography and Mass Spectrometry (HPLC-MS) in Empetrum rubrum Vahl ex Willd.

In searching for compounds with antioxidant and antifungal activity, our study focused on the subshrub species Empetrum rubrum Vahl ex Willd. (Ericaceae). We measured the antioxidant activity of its methanolic extract (MEE) obtained from the aerial parts (leaves and stems) and of its methanolic extract (MEF) obtained from the lyophilized fruits. The antioxidant activity of the MEE and MEF was evaluated in vitro via a 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical and 2,2'-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) cationic radical. The results were expressed in gallic acid and Trolox equivalents for the DPPH and ABTS assays, respectively. The antioxidant activities, for the DPPH and ABTS assays, were also evaluated by considering the IC50 values. Concerning the antioxidant activity, the total phenolic content (TPC) in the MEE and MEF was determined using the Folin-Ciocalteu method. Polyphenols contained in the leaves, stems, and fruits of E. rubrum were determined qualitatively by employing high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS) analysis. The antifungal activity of the MEE obtained from the aerial parts of E. rubrum was tested against Rhizoctonia solani. The results of IC50 values measured by the DPPH and ABTS methods with MEE were 0.4145 ± 0.0068 mg mL-1 and 0.1088 ± 0.0023 mg mL-1, respectively, and the IC50 values for MEF were 6.4768 ± 0.0218 mg mL-1 and 0.7666 ± 0.0089 mg mL-1 measured by the DPPH and ABTS methods, respectively. The HPLC-MS analysis revealed the presence of anthocyanins, phenolic acids derivatives, and flavonols. In vitro, mycelial growth of this fungus was reduced from 90% to nearly 100% in the presence of MEE. The observed antifungal effect is related to the presence of the abovementioned phenols, detected in the MEE.

Metabolic profile and antioxidant capacity of five Berberis leaves species: A comprehensive study to determine their potential as natural food or ingredient.

A comprehensive study of bioactive compounds was carried out in the leaves of the main Berberis species growing in Chile (Berberis microphylla, Berberis darwinii, Berberis empetrifolia, Berberis trigona, and the introduced Berberis vulgaris). We identified 117 compounds, by a detailed analysis of each molecule, including phenolic acids, alkaloids, flavonols, and other compounds, using high-performance liquid chromatography and high-resolution mass spectrometry. Quantitative analysis of main compound was also included for all species. Hydroxycinnamic acid derivatives were the main compounds in all the studied leaves, with the highest concentration in Berberis microphylla. Quercetin derivatives were the most relevant flavonols in all species, except in Berberis vulgaris, in which isorhamnetin-3-rutinoside was the most concentrated. The fatty acid profile, determined by gas chromatography mass spectrometry revealed the presence of linoleic and linolenic acids in all species studied. Berberis vulgaris showed higher levels of these fatty acids. The antioxidant capacity, explored by three in vitro methods, showed high values for all studied Berberis species. The obtained levels are higher than those of other prominent foods. The findings will inform novel approaches for the valorization of these leaves as natural food or ingredient.

Determination of the Chemical Stability of Dapagliflozin by LC/DAD and MS/MS Methods.

A simple and fast stability-indicating liquid chromatographic method with diode array detection (DAD) was developed and validated for the determination of dapagliflozin (DAPA) in bulk and tablets, in the presence of its major degradation products (DP). The drug was subjected to hydrolytic, oxidative, photolytic, thermal and humidity/thermal stress conditions, showing significant degradation under humidity/thermal with the formation of two DP, which were preliminarily identified by liquid chromatography with diode array detector coupled with electrospray ionization-tandem mass spectrometry (HPLC-DAD-ESI-MS/MS). Chromatographic separation of dapagliflozin and its DP was achieved with a core-shell RP-18 column, using acetonitrile and water as mobile phase in isocratic elution mode. The described method was linear over a range of 50-150 µg/mL. For precision, the relative standard deviation (RSD) was <1.3%, the recovery was 99.64-100.11%, and the assay demonstrated adequate selectivity. The degradation kinetics of dapagliflozin was evaluated corresponding to first-order under thermal and humidity/thermal stress conditions. Dapagliflozin was well resolved from its drug products showing the power of stability-indicating of the method. The results showed that the proposed method was found to be suitable for routine analysis, quantitative determination and the stability study of dapagliflozin in pharmaceutical samples.

Degradation studies of quetiapine fumarate by liquid chromatography-diode array detection and tandem mass spectrometry methods.

Quetiapine fumarate (QUE) is an antipsychotic agent with a chemical structure that is susceptible to degradation; therefore, it is important to study its stability using appropriate analytical tools. Knowledge of the stability profile of a drug is important because chemical degradation of its active component often results in a loss of potency, affecting its efficacy and safety. This current work reports degradation studies of QUE as drug substance, under different stress conditions such as oxidation, hydrolysis, heat, humidity and photolysis, by a stability-indicating LC method. The chemical stability was evaluated using a simple HPLC/diode array detection method, with a core-shell C18 column under isocratic conditions, which allows the separation of all primary degradation products (DPs) in a short run time. QUE was mainly degraded under oxidative and hydrolytic conditions, with the formation of three and two DPs, respectively, which were identified by electrospray ionization-tandem mass spectrometry. The method was properly validated in terms of linearity, accuracy, precision, selectivity, robustness and quantitation limit. Commercial tablets containing 25 mg of QUE were quantified, with results obtained within the United States Pharmacopeia limits. The proposed method is suitable to assess the stability and perform routine analysis of QUE in pharmaceutical samples.

Pharmacokinetics of low molecular weight phenolic compounds in gerbil plasma after the consumption of calafate berry (Berberis microphylla) extract.

Calafate is a berry with high concentration of anthocyanins and hydroxycinnamic acids that grows in South Patagonia. To date, no metabolism studies of phenolic compounds using calafate have been carried out. A calafate extract was characterized by HPLC-DAD-ESI-MS/MS. After extract administration (300 mg/kg), a pharmacokinetic study of phenolic compounds in gerbil plasma was performed by GC-MS/MS. Sixteen phenolic acids increased after intake. Phenylacetic acid derivatives exhibit the highest concentration, while main increase of phenolic catabolites was observed 2 h post-intake. 3-hydroxyphenylacetic and phenylacetic acids increased at 4-8 h post-intake. All catabolites found in gerbil plasma exhibit concentration peaks between 0.1 and 1 µM, however no parental anthocyanins were detected. Establish in vivo plasmatic concentration ranges of phenolic compounds derived from polyphenol consumption following WHO recommendations, plays a key role to carry out future in vitro assays in order to correctly assign biological benefits of calafate berry consumption.

Determination of vortioxetine and its degradation product in bulk and tablets, by LC-DAD and MS/MS methods.

Vortioxetine hydrobromide (VOR), is a novel antidepressant used for the treatment of major depressive disorder. It has a chemical structure susceptible to degradation, therefore it is important to have suitable analytical methods to determine VOR in presence of its main degradation products (DP), because if the compound degrades, this could result in diminution of the therapeutic activity and safety. A simple HPLC method with photodiode array detection was developed and validated for determination of VOR in bulk and tablets, in the presence of its major DP. The drug was subjected to oxidative, hydrolytic, and photolytic stress conditions, showing significant degradation under oxidation with the formation of one DP, which was identified by ESI-MS/MS. A C18 column was used, with mobile phase consisting of acetonitrile and water with acetic acid and triethylamine in isocratic elution mode, with detection at 228 nm and 1.0 mL/min flow rate. The assay was linear in the 25-125 µg/mL concentration range. For precision, the RSD was <1.8%, the recovery was 100.0-101.6%, and the method demonstrated adequate selectivity. The method was successfully applied to quantify VOR in tablets. The results showed that the method is useful for routine analysis and for quality control purposes.

Oligostilbenoids in Vitis vinifera L. Pinot Noir grape cane extract: Isolation, characterization, in vitro antioxidant capacity and anti-proliferative effect on cancer cells.

The following oligostilbenoids were isolated from extracts of Vitis vinifera L. Pinot Noir grape canes produced at a pilot-plant scale: (E)-ε-viniferin, (E)-resveratrol, (E)-piceatannol, ampelopsin A, vitisin B, pallidol, (E)-δ-viniferin, (E)-ω-viniferin, (E)-trans-cis-miyabenol C, isorhapontigenin, scirpusin A, and a new isomer named isoscirpusin A. The antioxidant capacity of the isolated stilbenoids was studied by three different assays, and their 50% inhibition concentration (IC50) against cancer cells was determined by MTT reduction assay. Besides (E)-resveratrol, stilbenoids have outstanding antioxidant capacity in the ORAC-FL assay. The strongest antiproliferative effect was observed for (E)-piceatannol and ampelopsin A against the bladder cancer cell line J82. (E)-Piceatannol has inhibitory effect on human lung cancer SK-MES-1 cells. Moreover, the whole extract has antiproliferative effect on all tested cell lines. In conclusion, beside (E)-resveratrol, grape cane extract contains oligostilbenoids with potential health benefits. This underexploited viticultural residue has the potential to produce valuable phytochemicals or ingredients in functional foods.

C18 core-shell column with in-series absorbance and fluorescence detection for simultaneous monitoring of changes in stilbenoid and proanthocyanidin concentrations during grape cane storage.

Grape canes, the residues from the annual pruning of vines, contain high levels of inducible (E)-resveratrol and also oligomeric stilbenoids and proanthocyanidins. These two families of phenolic compounds are bioactive, but to quantify them in a single chromatographic run using only ultraviolet detection is a difficult task. To overcome this limitation, a chromatographic method was developed using a core shell column for separation, an ultraviolet-visible diode array detector (DAD) and a fluorescence (FL) detector connected in series for quantification, with an electrospray ionization interface (ESI) and a triple quadrupole mass spectrometric detector (MS/MS) added for identification of the analytes. The proanthocyanidins (+)-catechin, (-)-epicatechin, procyanidins B1, B2, and C1, an unknown dimer and trimer, two prodelphinidin dimers, and monogallate procyanidin dimers were detected in the tested grape cane samples. The stilbenoids detected were (E)-resveratrol, (E)-piceatannol, (E)-piceid, (E)-ε-viniferin, vitisin B, a glycosylated monomer, three oxidized dimers, an unknown dimer and a tetramer, pallidol, hopeaphenol, (E)-δ-viniferin, and (E)-ω-viniferin. However, this method required 60min for each analysis. A faster and more efficient method for quantitative analysis was developed based on HPLC-DAD-FL, reducing the time required to 24min for the simultaneous quantification of proanthocyanidins and stilbenoids in Cabernet Sauvignon, Pinot Noir, and Tintorera grape canes stored at controlled temperatures and relativity humidities for 134days after pruning. To the best of our knowledge, this is the first time a prodelphinidin dimer has been quantified in grape canes. The incorporation of fluorescence detection in series with DAD not only allowed the quantification of proanthocyanidins, it also improved the detectability of some minor stilbenoids present in the canes, such as (E)-piceid. The (E)-resveratrol and (E)-piceatannol levels increased significantly during cane storage, while those of (E)-ε-viniferin and ampelopsin A did not show significant increases. The relative humidity had a determining effect on the levels of (E)-resveratrol and (E)-piceatannol in the canes of all varieties studied; their concentrations were higher at a relative humidity of 60% than at 70%. This is the first time that the proanthocyanidin profiles of canes stored after pruning were monitored under controlled conditions of temperature, time and relative humidity. The concentration of (-)-epicatechin decreased during storage under both relative humidities. Furthermore, the levels of proanthocyanidin B1 and the prodelphinidin dimer also decreased to a certain extent.

Stability-Indicating Liquid Chromatographic Methods with Photodiode Array Detection and Light Scattering Detection for Simultaneous Determination of Candesartan and Hydrochlorothiazide.

Development, validation and comparison of two stability-indicating LC methods, one with photodiode array detector (DAD) and the other with evaporative light scattering detector (ELSD), were performed for simultaneous determination of candesartan cilexetil (CANC) and hydrochlorothiazide (HCTZ), in pharmaceutical samples. A RP-18 column (125 mm × 4 mm, 5 µm) was used for separation of CANC, HCTZ and its major degradation products, using acetonitrile and phosphate buffer (pH 6.0) for DAD method and acetonitrile and water with acetic acid and triethylamine (pH 4.1) for ELSD method, as mobile phase in a gradient mode. The response with ELSD was fitted to a power function and the DAD response by a linear model over a range of 32-160 µg/mL for CANC and 25-125 µg/mL for HCTZ. The precision and accuracy of the methods were similar, with RSD below 3.0% and recovery between 98.1% and 103.9%. The drugs were subjected to stress conditions of hydrolysis, oxidation, photolysis, humidity and temperature. The degradation products were satisfactory separated from the main peaks and from each other. Both drugs mainly degrade by hydrolysis, showing the formation of one degradation product for HCTZ and two for CANC; its identification was conducted by LC/MS/MS. The methods were successfully applied to the analysis of CANC and HCTZ in combined commercial tablets. The performance of DAD and ELSD methods are comparable, therefore both methods are suitable for stability study and determination of CANC and HCTZ in pharmaceutical samples.

Evaluation of microextraction by packed sorbent, liquid-liquid microextraction and derivatization pretreatment of diet-derived phenolic acids in plasma by gas chromatography with triple quadrupole mass spectrometry.

Miniaturized sample pretreatments for the analysis of phenolic metabolites in plasma, involving protein precipitation, enzymatic deconjugation, extraction procedures, and different derivatization reactions were systematically evaluated. The analyses were conducted by gas chromatography with mass spectrometry for the evaluation of 40 diet-derived phenolic compounds. Enzyme purification was necessary for the phenolic deconjugation before extraction. Trimethylsilanization reagent and two different tetrabutylammonium salts for derivatization reactions were compared. The optimum reaction conditions were 50 µL of trimethylsilanization reagent at 90°C for 30 min, while tetrabutylammonium salts were associated with loss of sensitivity due to rapid activation of the inert gas chromatograph liner. Phenolic acids extractions from plasma were optimized. Optimal microextraction by packed sorbent performance was achieved using an octadecylsilyl packed bed and better recoveries for less polar compounds, such as methoxylated derivatives, were observed. Despite the low recovery for many analytes, repeatability using an automated extraction procedure in the gas chromatograph inlet was 2.5%. Instead, using liquid-liquid microextraction, better recoveries (80-110%) for all analytes were observed at the expense of repeatability (3.8-18.4%). The phenolic compounds in gerbil plasma samples, collected before and 4 h after the administration of a calafate extract, were analyzed with the optimized methodology.

Differences in Vvufgt and VvmybA1 Gene Expression Levels and Phenolic Composition in Table Grape (Vitis vinifera L.) 'Red Globe' and Its Somaclonal Variant 'Pink Globe'.

A novel 'Red Globe' (RG)-derived grape variety, 'Pink Globe' (PG), was described and registered as a new genotype, with earlier ripening and sweeter taste than those of RG. Microsatellite analysis revealed that PG and RG are undifferentiable; however, the PG VvmybA1c contains six single-nucleotide polymorphisms within the coding and noncoding region, possibly related to the reduced VvmybA1 expression levels. Conversely, HPLC-DAD-ESI-MS/MS analysis showed significantly lower anthocyanin content in PG skin than in RG skin, and PG had no detectable trihydroxylated anthocyanins. Total flavonols did not differ between the variants, although some quercetin derivate concentrations were lower in PG. HPLC-FLD analysis revealed slightly higher concentrations of epicatechin and a procyanidin dimer in PG seeds, although the antioxidant capacity of crude extracts from either variety did not differ significantly. These differences, particularly in monomeric anthocyanin content, can be attributed to altered activity of a MYB-type transcription factor, reducing Vvufgt expression.

Comparison of Stability-Indicating LC Methods Using Light Scattering and Photodiode Array Detection with Monolithic Column for Determination of Quinapril and Hydrochlorothiazide.

Rapid stability-indicating LC methods for simultaneous analysis of quinapril and hydrochlorothiazide were developed, validated and compared using evaporative light scattering detection (ELSD) and diode array detection (DAD). For the separation of quinapril, hydrochlorothiazide and its major degradation products, a monolithic column was used and the analytes were eluted within 7 min, applying gradient mobile phase in both methods. Quinapril was subjected to hydrolytic, oxidative, thermal, humidity and photolytic stress conditions. Degradation products were well resolved from main peaks and from each other, proving the stability-indicating power of the methods. The response with DAD was linear and the response with ELSD was fitted to a power function, for quinapril and hydrochlorothiazide concentrations of 20-160 and 12.5-100 µg mL(-1), respectively. DAD method achieved better precision than ELSD method, the LOQ of DAD was lower and the accuracy of the methods was similar. Quinapril degrade by hydrolysis and thermal stress, showing the formation of quinaprilat and quinapril diketopiperazine as degradants, which were identified by MS-MS. The methods were successfully applied to quantify quinapril and hydrochlorothiazide in commercial tablets. LC-DAD and LC-ELSD methods are suitable to assess the stability and routine analysis of quinapril and hydrochlorothiazide in pharmaceutical industry.

Sonophotocatalytic mineralization of Norflurazon in aqueous environment.

Norflurazon (4-chloro-5-(methylamino)-2-[3-(trifluoromethyl)phenyl]pyridazin-3(2H)-one; C12H9ClF3N3O) is an excellent weed controlling agent being practiced in the agricultural lands. The excessive addition or the undissolved Norflurazon (maximum solubility 28 mg/L at 25 °C) enters into the aquatic environment and causes the adverse effects associated with its high concentration. To avoid the perilous effects, visible light assisted photocatalysis set-up coupled with the 42 kHz ultrasound producing bath type sonicator is used to completely mineralize the Norflurazon. TiO2, ZnO and gold loaded zinc oxide nanocatalysts were utilized to study the mineralization of Norflurazon. Au-ZnO shows the greater efficiency for the sonophotocatalytic removal of Norflurazon among the various nanocatalysts employed to study the mineralization. The order of Norflurazon mineralization was sonophotocatalysis > sonocatalysis > photocatalysis. The additive effect was achieved for the sonophotocatalytic degradation. The high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometric (LCMS) analyses were employed to identify the various intermediates produced during the mineralization. The identification of four pseudo molecular ions and various intermediates using the LCMS analysis evidently suggests the sonophotocatalytic degradation was preceded in various decay pathways. A suitable mechanism has been proposed for the sonophotocatalytic mineralization of Norflurazon.

Hydroxycinnamic acids and flavonols in native edible berries of South Patagonia.

Diverse edible berries are native to the Patagonian region of Southern Chile. These berries are underused because their nutritional properties are relatively unknown. In this work, the profiles and concentrations of hydroxycinnamic acid derivatives and flavonols, and the antioxidant capacity of the berry extracts, were studied using HPLC-DAD-ESI-MS/MS and CUPRAC assays, respectively. In total, 46 compounds were identified, including 17 hydroxycinnamic acid derivatives and 26 flavonols. Caffeoylquinic acid isomers were the most abundant compounds, and quercetin and myricetin derivatives were the main flavonols found. The berries from Ribes genera showed a high diversity and concentration of these 2 families of compounds and contained 3-caffeoylquinic acid and quercetin-3-rutinoside at the highest concentrations. The Patagonian berries, especially the berries of Rubus and Ribes genera, had high cupric reducing antioxidant capacity, comparable with that described for berries from the Northern hemisphere. These results contribute to promote the nutritional study of these fruits.

Flavonols, alkaloids, and antioxidant capacity of edible wild berberis species from patagonia.

There are 20 species of the Berberidaceae family described in Chile, whose fruits are edible and show high anthocyanin and hydroxycinnamic acid levels. Berberis microphylla G. Forst, commonly known as calafate, is the most extensively distributed. Flavonols and alkaloids in seed, pulp, skin, and whole calafate berry extracts and other Berberis were studied using HPLC-DAD-ESI-MS/MS and HPLC with fluorescence detector. Berry samples from different locations in Chilean Patagonia, including different phenological stages, were systematically addressed. Results were compared with other organs of the plant and with other Berberis species. Total flavonol concentration in calafate (n = 65) was 1.33 ± 0.54 µmol/g. Glycosyl metabolites of quercetin and isorhamnetin were the most abundant. Similar profiles were observed in calafate from distinct locations, but important differences were observed for the other edible Berberis species. Calafate pulp and skin have higher flavonol concentrations than seeds, and the maturation process reduced its levels. TEACCUPRAC and TEACABTS of whole calafate extracts and fractions are also explored. Finally, only berberine was detected in the fruit (0.001%), mainly in seeds. Results contribute to the promotion of this berry as a superfruit from Patagonia.

Isolation and structural elucidation of anthocyanidin 3,7-ß-O-diglucosides and caffeoyl-glucaric acids from calafate berries.

Calafate (Berberis microphylla G. Forst) is an edible wild berry growing in South Patagonia that is very rich in anthocyanins and hydroxycinnamic acid derivatives. Calafate contains unusual phenolic compounds compared to other berries, such as anthocyanidin dihexosides, different from the common 3,5-diglucosides, and isomeric esters of caffeic acid with hexaric acids. After isolation, their structures have been elucidated by UV-vis, MS/MS, and NMR spectroscopies. The anthocyanidin dihexosides constitute the complete series of 3,7-ß-O-diglucosides of the five anthocyanidins usually found in calafate, the structures of which were completely elucidated in the cases of delphinidin, petunidin, and malvidin derivatives and tentatively suggested in the cases of cyanidin and peonidin, and their occurrence seems to be characteristic of calafate among other wild berries from South Patagonia. With regard to caffeoyl-hexaric acids, two of four isomers have been assigned as 3- and 4-trans-caffeoyl-glucaric acids, but the determination of the linkage position for each isomer was not possible. A third isomer was also isolated, but it easily degraded and was suggested to be the 2- or 5-trans-caffeoyl-glucaric acid. The caffeoyl-glucaric acids account for around half of the pool of hydroxycinnamic acid derivatives in calafate.

Influence of post-pruning storage on stilbenoid levels in Vitis vinifera L. canes.

Increasing evidence for the health benefits of E-resveratrol has triggered interest in stilbenoids in grapes, wine and by-products. Less attention has been paid to stilbenoid levels in viticulture residues. However, grape canes are a promising source of stilbenoids and have economic potential because they are a source of high-value phytochemicals. The objective of this research was to determine the effect of post-pruning storage on stilbenoid levels in grape canes. In most samples, the predominant stilbenoid was (E)-resveratrol, followed by (E)-ε-viniferin. In Pinot Noir canes stored after pruning at room temperature, the stilbenoid levels increased significantly after 8 months. The concentration was increased by up to fivefold, reaching 4,777 mg kg(-1)dw (dry weight). This effect did not occur in frozen, lyophilised or milled material. Branches collected directly from the plants after grape vintage and those remaining on the plant after pruning showed only a small increase in stilbenoid levels.

Analysis of hydroxycinnamic acids derivatives in calafate (Berberis microphylla G. Forst) berries by liquid chromatography with photodiode array and mass spectrometry detection.

Calafate (Berberis microphylla G. Forst) is a Patagonian barberry very rich in anthocyanins and one of the fruits with the highest levels of these polyphenols. Other phenolic compounds have also been described in calafate berries. However, to the best of our knowledge there is no available information on hydroxycinnamic acid derivatives. The complexity of hydroxycinnamic acids determination in calafate berries, due to their structure similarities and the interference of high anthocyanin concentration is addressed by means of solid liquid extraction, followed by solid phase extraction clean-up on MCX columns and HPLC-DAD-ESI-MS/MS. The optimized extraction, clean-up and HPLC separation method allowed the assignation of identity and quantification of 20 hydroxycinnamic acids from calafate fruits. 5-Caffeoylquinic acid was the main compound found in all the studied samples. Other 13 hydroxycinnamoyl quinic acids and 6 caffeic acid esters with aldaric acid derivatives assigned as glucaric acid were also identified. Moreover, the glucaric-based hydroxycinnamic acid derivatives accounted for almost the half of total content of this kind of phenolic compounds. The total concentration of hydroxycinnamic acids derivatives ranged between 0.32±0.00 µmol/g and 8.28±0.01 µmol/g. Effect of ripening and geographical location on hydroxycinnamic acid profiles and concentrations are also evaluated. The methodology allows the determination of hydroxycinnamic acids from calafate despite of the high anthocyanin concentrations, showing a much higher concentration of these acids than other widely consumed berries. Thus suggesting that calafate could be considered a very interesting fruit from the point of view of their nutraceutical composition. However, geographical location and ripening have incidence in levels of studied compounds.

Stimulation of cytosolic and mitochondrial calcium mobilization by indomethacin in Caco-2 cells: modulation by the polyphenols quercetin, resveratrol and rutin.

BACKGROUND: The effect of indomethacin (INDO) on Ca(2+) mobilization, cytotoxicity, apoptosis and caspase activation and the potential protective effect of quercetin (QUE), resveratrol (RES) and rutin (RUT) were determined in Caco-2 cells. METHODS: Caco-2 cells were incubated with INDO in the presence or absence of QUE, RES or RUT. The concentrations of Ca(2+) in the cytosol (Fluo-3 AM) and mitochondria (Rhod-2 AM) were determined as well as the cytotoxicity (MTT reduction and LDH leakage), apoptosis (TUNEL) and caspase-3 and 9 activities. RESULTS: INDO promoted Ca(2+) efflux from the endoplasmic reticulum (ER), resulting in an early, but transient, increment of cytosolic Ca(2+) at 3.5min, followed by a subsequent increment of intra-mitochondrial Ca(2+) at 24min. INDO also induced cytotoxicity, apoptosis, and increased caspase activities and cytochrome c release. All these alterations were prevented by the inhibitors of the IP3R and RyR receptors, 2-Aminoethoxydiphenyl borate (2-APB) and dantrolene. QUE was the most efficient polyphenol in preventing Ca(2+) mobilization induced by INDO and all of its consequences including cytotoxicity and apoptosis. CONCLUSIONS: In Caco-2 cells, INDO stimulates ER Ca(2+) mobilization, probably through the activation of IP3R and RyR receptors, and the subsequent entry of Ca(2+) into the mitochondria. Polyphenols protected the cells against the Ca(2+) mobilization induced by INDO and its consequences on cytotoxicity and apoptosis. GENERAL SIGNIFICANCE: These results confirm the possibility of using polyphenols and particularly QUE for the protection of the gastroduodenal mucosa in subjects consuming NSAIDs.

In vitro activity on human gut bacteria of murta leaf extracts (Ugni molinae turcz.), a native plant from Southern Chile.

Despite the fact that murta infusions have been used to treat gut/urinary infections by native Chileans for centuries, the mechanisms promoting such effects still remain unclear. As a first attempt to unravel these mechanisms, human fecal samples were incubated in a medium containing water extract of murta leaves (ML) and the growth of different bacterial groups was evaluated. Control incubations were made in media containing fructooligosaccharides (FOS) and glucose as a carbon source. Phenolic compounds in the ML extract, likely promoters of bioactivity, were identified by HPLC-DAD-MS(n) . Concentrations (log10 CFU/mL) of bifidobacteria and lactobacilli in media containing the extract and FOS were 7.33 ± 0.05/4.95 ± 0.20 and 6.44 ± 0.22/6.05 ± 0.06, respectively. Clostridia, anaerobes and Enterobacteriaceae grew to a similar extent in media containing murta extract and FOS. In vitro tests (disk diffusion) showed that Gram-positive (Bacillus and Paenibacillaceae) and Gram-negative (Enterobacteriaceae) bacteria isolated from fecal samples were sensitive to both water and 50/50 ethanol/water extracts of ML (28.4 µg gallic acid equivalents). At this concentration, the antimicrobial activity of ML extracts was significantly (P < 0.05) lower than that of penicillin (10 U), whereas the difference between activity of ML extracts and gentamicine (10 µg) was no significant (P > 0.05). No evidence of dependency between the antimicrobial activity of ML extracts and the enzymatic capability of the sensitive strains was found.