Mass Spectrometry and 1H-NMR Study of Schinopsis lorentzii (Quebracho) Tannins as a Source of Hypoglycemic and Antioxidant Principles.

The ethyl acetate extract of the commercial tannin Tan'Activ QS-SOL (from Schinopsis lorentzii wood), employed for the production of red wine, was subjected to chromatography on Sephadex LH-20, providing nine fractions (A-1-A-9), which were estimated for total phenols content (GAE), antioxidant activity (DPPH, ORAC), and hypoglycemic activity (α-glucosidase and α-amylase inhibition). All the fractions were analyzed by means of HPLC/ESI-MS/MS and 1H-NMR to identify the principal active constituents. Fractions A-1 and A-3 showed the highest antioxidant activity and gallic acid (1), pyrogallol (3), eriodictyol (6), catechin (12), and taxifolin (30) were identified as the major constituents. The highest α-glucosidase and α-amylase inhibitory activity was observed in fractions A-7-A-9 containing condensed (9', 15, 18, 19, 23, and 27) hydrolysable tannins (13 and 32) as well as esters of quinic acid with different units of gallic acid (5, 11, 11', 14, and 22). This last class of gallic acid esters are here reported for the first time as α-glucosidase and α-amylase inhibitors.

A mass spectrometry and 1H NMR study of hypoglycemic and antioxidant principles from a Castanea sativa tannin employed in oenology.

The ethanol extract of the commercial tannin Tan'Activ C, (from Castanea sativa wood), employed in oenology, was subjected to chromatography on XAD-16 affording fractions X1-X5, evaluated for total phenols content (GAE), antioxidant activity (DPPH, ORAC), and hypoglycemic activity (α-glucosidase inhibition). Fraction X3 showed GAE, radical scavenging activity, and α-glucosidase inhibition higher than those of the Castanea sativa extract, and was subjected to chromatography on Sephadex LH-20 to obtain fractions S1-S7, analyzed by HPLC/ESI-MS/MS and 1H NMR to identify the main active constituents. In fractions with higher antioxidant activity, gallic acid (4), grandinin (5), valoneic acid dilactone (9), 1,2,3-tri-O-galloyl-ß-glucose (14), 3,4,6-tri-O-galloyl-ß-glucose (15) and galloyl derivative of valoneic acid dilactone (21) were identified as the major constituents. The highest hypoglycemic activity was found in fractions S6 and especially S7; the major constituents of these fractions are valoneic acid dilactone (9), three tetragalloyl glucose isomers (16-18) and 1,2,3,4,6-penta-O-galloyl-ß-glucose (23), previously reported as α-glucosidase inhibitors.

Identification by Inverse Virtual Screening of magnolol-based scaffold as new tankyrase-2 inhibitors.

The natural product magnolol (1) and a selection of its bioinspired derivatives 2-5, were investigated by Inverse Virtual Screening in order to identify putative biological targets from a panel of 308 proteins involved in cancer processes. By this in silico analysis we selected tankyrase-2 (TNKS2), casein kinase 2 (CK2) and bromodomain 9 (Brd9) as potential targets for experimental evaluations. The Surface Plasmon Resonance assay revealed that 3-5 present a good affinity for tankyrase-2, and, in particular, 3 showed an antiproliferative activity on A549 cells higher than the well-known tankyrase-2 inhibitor XAV939 used as reference compound.

Biological effects of polyphenol-rich extract and fractions from an oenological oak-derived tannin on in vitro swine sperm capacitation and fertilizing ability.

Although excessive ROS levels induce sperm damage, sperm capacitation is an oxidative event that requires low amounts of ROS. As the antioxidant activity of the ethanol extract (TRE) of a commercial oenological tannin (Quercus robur toasted oak wood, Tan'Activ R®) and its four fractions (FA, FB, FC, FD) has been recently reported, the present study was set up to investigate the biological effects of TRE and its fractions in an in vitro model of sperm capacitation and fertilization. Boar sperm capacitation or gamete coincubation were performed in presence of TRE or its fractions (0, 1, 10, 100 µg/ml). TRE at the concentration of 10 µg/ml (TRE10) stimulated sperm capacitation, as it increased (p < .001) the percentage of spermatozoa with tyrosine-phosphorylated protein positivity in the tail principal piece (B pattern) (67.0 ±â€¯10.6 vs. 48.6 ±â€¯9.0, mean ±â€¯SD for TRE10 vs. Ctr respectively). Moreover T10 significantly (p < .001) increased oocyte fertilization rate (91.9 ±â€¯4.0 vs. 69.0 ±â€¯14.8, TRE10 vs. Ctr respectively). An opposite effect of TRE at the concentration of 100 µg/ml (TRE100) on both sperm capacitation (B pattern cell percentage 33.3 ±â€¯29.2) and fertilizing ability (fertilization rate 4.9 ±â€¯8.3), associated with a higher sperm viability (66.9 ±â€¯9.3 vs. 35.4 ±â€¯10.8, TRE100 vs. Ctr respectively) (p < .001), was recorded. The potency of the TRE fractions seems to be highest in FB followed by FC, faint in FD and nearly absent in FA. Our results show that TRE and its fractions, in a different extent, exert a powerful biological effect in finely modulating capacitation and sperm fertilizing ability.

Quantification of the resveratrol analogs trans-2,3-dimethoxy-stilbene and trans-3,4-dimethoxystilbene in rat plasma: application to pre-clinical pharmacokinetic studies.

trans-2,3-Dimethoxystilbene (2,3-DMS) and trans-3,4-dimethoxystilbene (3,4-DMS) are two synthetic resveratrol (trans-3,5,4'-trihydroxystilbene) analogs. In this study, a simple HPLC method was developed and validated to determine 2,3-DMS and 3,4-DMS in rat plasma. Chromatographic separation was obtained with a reversed-phase HPLC column through a 12.5-min gradient delivery of a mixture of acetonitrile and water at the flow rate of 1.5 mL/min at 50 °C. The lower limit of quantification was 10 ng/mL. After successful validation, the pharmacokinetic profiles of 2,3-DMS and 3,4-DMS were subsequently studied in Sprague-Dawley rats. Upon single intravenous administration (4 mg/kg), 2,3-DMS had a medium volume of distribution of the central compartment (Vc = 2.71 ± 0.51 L/kg), quite rapid clearance (Cl = 52.0 ± 7.0 mL/min/kg), moderate mean transit time (MTT0→last = 131.0 ± 4.5 min) but a fairly long terminal elimination half-life (t1/2 λZ = 288.9 ± 92.9 min). Interestingly, 3,4-DMS displayed a pharmacokinetic profile apparently distinct from 2,3-DMS and it had more extensive distribution (Vc = 5.58 ± 1.73 L/kg), faster clearance (Cl = 143.4 ± 40.5 mL/min/kg) and shorter residence (MTT0→last = 61.4 ± 27.1 min). Following single oral administration (10 mg/kg), 2,3-DMS had low and erratic plasma exposure (Cmax = 37.5 ± 23.7 ng/mL) and poor oral bioavailability (2.22% ± 2.13%) while the oral bioavailability of 3,4-DMS was even poorer than 2,3-DMS. Clearly, the location of the methoxy groups had a significant impact on the pharmacokinetics of resveratrol analogs. This study provided useful information for the design of resveratrol derivatives in future study.

Biological effects on granulosa cells of hydroxylated and methylated resveratrol analogues.

Several resveratrol analogues have been designed to improve bioactivity: among these polymethoxystilbenes appear to be particularly promising. The present study was set up to investigate the biological functions of polymethoxystilbenes 2 and 3, recently found in our lab as antiangiogenic agents, on a well-defined swine granulosa cell model. Proliferative activity and effects on steroidogenesis were evaluated, as well as the effect on granulosa cell vascular endothelial growth factor (VEGF) production, since these cells in basic conditions synthesize the main proangiogenic peptide. Moreover, we considered the effect of these two resveratrol analogues on granulosa cell redox status. Analogue 3 inhibited granulosa cell growth, while it stimulated steroidogenesis. A similar effect was displayed by 2 on estradiol 17beta production and cell proliferation at the highest concentration tested. On the other hand, at the same dosage 2 decreased progesterone levels. Both analogues inhibited VEGF output. Granulosa cell redox status was unaffected by resveratrol analogue 2 while the highest concentration of 3 stimulated free radicals generation and scavenging enzyme activities. The overall results indicate that analogue 3 is the more powerful compound, thus suggesting that a slight modification in the structure markedly increases effectiveness. These data could be useful to develop more active resveratrol analogues for therapeutic use.

A simple and sensitive HPLC-UV method for the quantification of piceatannol analog trans-3,5,3',4'-tetramethoxystilbene in rat plasma and its application for a pre-clinical pharmacokinetic study.

A simple and sensitive HPLC-UV method was developed and validated for the quantification of piceatannol analog trans-3,5,3',4'-tetramethoxystilbene (M-PIC) in rat plasma. Following protein precipitation with three volumes of acetonitrile, the analytes were separated on a RP-HPLC column, which was protected by a guard column through gradient delivery of a mixture of acetonitrile-water at 40 degrees C. The UV absorbance at 325nm was recorded to quantify M-PIC. The retention time of M-PIC and trans-3,5-dimethoxystilbene (internal standard) was 7.4 and 8.4min, respectively. The calibration curves were linear (R(2)>0.9989) with a lower limit of quantification of 15ng/ml. The intra- and inter-day precisions, in terms of RSD, were all lower than 7.5%. The average analytical recovery ranged from 97.0 to 104.3% while the average absolute recovery ranged from 101.8 to 105.0%. This reliable HPLC method was subsequently applied to assess the pharmacokinetic profile of M-PIC in Sprague-Dawley rats using 2-hydroxypropyl-beta-cyclodextrin as a dosing vehicle. The terminal elimination half-life (t(1/2lambdaz)) and clearance (Cl) of M-PIC were 313+/-20min and 33.1+/-3.9ml/min/kg, respectively; and its absolute oral bioavailability was as high as 50.7+/-15.0%. M-PIC appeared to have a favorable pharmacokinetic profile and further pharmacological investigation on this phyto-stilbene was warranted.

Effects of resveratrol analogs on steroidogenesis and mitochondrial function in rat Leydig cells in vitro.

Resveratrol and its analogs are considered to be a promising drug candidate for treatment of cancer and different age-associated diseases. In the present study we have investigated the effects of resveratrol and its synthetic analogs on steroidogenesis and mitochondrial function in primary cultures of rat Leydig cells. Our findings indicate that resveratrol and its analogs structure-dependently attenuated hCG-activated steroidogenesis in Leydig cells through suppression of the expression of steroidogenic acute regulatory protein and cytochrome P450c17. 3,5-Diacetyl resveratrol was observed to modulate mitochondrial function in Leydig cells, suppressing polarization of inner mitochondrial membrane, and 3,4,4'-trimethoxystilbene stimulated the overall activity of intracellular reductases involved in the reduction of WST-1 to formazan. Thus, the inhibitory actions of resveratrol analogs on steroidogenesis in Leydig cells indicate novel mechanisms of action of these compounds, which may be of potential therapeutic interest, where suppression of androgen action is needed.

Bioassay-guided isolation of antiproliferative compounds from grape (Vitis vinifera) stems.

The fractionation, guided by cell-growth inhibition assay, of the EtOAc crude extract from grape stems of the Sicilian Vitis vinifera variety 'Nerello Mascalese' allowed identification often constituents, isolated either as pure compounds (1, 3-5, 7-10) or inseparable mixtures (2a-d and 6a-e). The pure constituents were: two triterpenoid acids, oleanolic (1) and betulinic acids (5); daucosterol (7); a stilbenoid, E-resveratrol (3) and its dimer E-epsilon-viniferin (4); the simple phenol gallic acid (8); and the flavanols catechin (9) and gallocatechin (10). Four 6'-O-acyldaucosterols (2a-d) and five 1,2-di-O-acyl-3-O-beta-D-galactopyranosyl glycerols (6a-e) were also identified as inseparable mixtures. All the isolated compounds were subjected to spectroscopic analysis and MTT bioassay on MCF-7 human breast cancer cells. The majority showed growth-inhibitory activity, 5 being the most active (GI50 = 0.57 microM). Compounds 3-5 were also tested on HT-29, U-87-MG and U-373-MG cell lines.

Antiangiogenic resveratrol analogues by mild m-CPBA aromatic hydroxylation of 3,5-dimethoxystilbenes.

A mild treatment of the resveratrol analogue 3,5,4'-trimethoxystilbene 2 with m-CPBA afforded two hydroxylated methoxystilbenes 5 and 6 by direct aromatic hydroxylation. A similar protocol was applied to other stilbenes bearing a 3,5-dimethoxy moiety, namely tetramethoxystilbenes 7 and 10 to obtain respectively the hydroxylated analogues 8, 9 and 11, 12. The substrate 2 and the new compounds 5, 8 and 11 were evaluated as anti-angiogenic agents and proved significantly active in the range 1-100 microM.

Citrus limonoids and their semisynthetic derivatives as antifeedant agents against Spodoptera frugiperda larvae. A structure-activity relationship study.

The antifeedant activity of Citrus-derived limonoids limonin (1), nomilin (2), and obacunone (3) and their semisynthetic derivatives 4-26 was evaluated against a commercially important pest, Spodoptera frugiperda. Simple chemical conversions were carried out on the natural limonoids obtained from seeds of Citrus limon. These conversions focused on functional groups considered to be important for the biological activity, namely the C-7 carbonyl and the furan ring. In particular, reduction at C-7 afforded the related alcohols, and from these their acetates, oximes, and methoximes were prepared. Hydrogenation of the furan ring was also performed on limonin and obacunone. The known antifeedant properties of the Citrus limonoids are confirmed. Comparison with previously reported data shows that insect species vary in their behavioral responses to these structural modifications. Highly significant antifeedant activity (P < 0.01) for two natural (1 and 3) and three semisynthetic limonoids (4, 8, and 10) was observed against S. frugiperda.