Comparative assessment of phenolic bioaccessibility from 100% grape juice and whole grapes.

Juicing of grapes includes contact with phenolic rich seeds and skins that otherwise rely on maceration for phenolic release. To understand if 100% grape juice can provide a matrix with highly bioaccessible phenolics relative to whole fruit, differences in phenolic content and bioaccessibility from commonly consumed table, Concord (CG) and Niagara (NG) grapes and their 100% juices were compared. Phenolic contents in whole grapes and 100% juices were assayed by LC-MS prior to in vitro digestion to determine phenolic bioaccessibility. Phenolic compounds were concentrated in CG and NG seeds as flavan-3-ols (222.2-285.5 mg per 100 g fw). CG skins were rich in anthocyanins (201.4 mg per 100 g fw) and flavonols (15.5 mg per 100 g fw). Product form had a significant impact on content (p < 0.01), relative bioaccessibility, and absolute bioaccessibility (p < 0.01). CG had a higher total phenolic content (21.9-50.7 mg per 100 g fw) compared to CGJ (5.8 mg per 100 g fw), though NG (4.9-10.8 mg per 100 g fw) was similar in phenolic content to NGJ (9.4-10.8 mg per 100 g fw). Absolute bioaccessibility of total phenolics from CGJ (5.2 mg per 100 g fw) was similar to CG (2.6-9.6 mg per 100 g fw), while NGJ (5.1-5.7 mg per 100 g fw) had higher bioaccessible phenolic content than NG (0.8-1.1 mg per 100 g fw). Differences in bioaccessible content were driven by high relative bioaccessibility of anthocyanins in CGJ (86-135%) compared to CG (14-39%) as well as for flavan-3-ols and phenolic acids from CGJ/NGJ (48-101; 39-85%) compared to CG/NG (0-3; 9-67%). Comparisons between juices and table grapes followed similar trends. A greater fraction of skin and seed phenolics was extracted through juicing and made bioaccessible, making 100% grape juice and whole fruit similar in phenolic delivery to consumers.

A simple LC-MS/MS method for the simultaneous quantification of resveratrol and its major phase II metabolites: Assessment of their urinary and biliary excretions in rats.

Trans-resveratrol (Res) is rapidly metabolized, extensively distributed into various tissues and mainly excreted by urine. The present study aimed to establish a simple LC-MS/MS method to simultaneously quantify Res and its major phase II metabolites (Res-3-O-ß-d-glucuronide, R3G; Res-4'-O-ß-d-glucuronide, R4'G; Res-resveratrol 3-sulfate, R3S; and Res-4'-sulfate, R4'S), and apply this method to assess their urinary and biliary excretions in rats. A simplified salting-out assisted liquid-liquid extraction (SALLE) strategy was developed to prepare samples with acetonitrile-methanol mixture (8:2, v/v) as extractant and ammonium acetate solution (10M) as salting-out reagent. The method validation demonstrated an acceptable recovery (>80%), good accuracy (85-115%), low deviation of detection (<15%) and no obvious matrix effect (<20%). Then the validated method was successfully applied to analyze the excretion of Res and its metabolites after intragastric administration of Res at 50mg/kg in rats. Only a minor proportion of Res (0.51nmol) and its metabolites (R3S, 35.8nmol; R4S, 0.25nmol; R3G, 142.3nmol; R4'G, 0.19nmol) were eliminated via bile, while the majority of Res (1670.2nmol), R3G (14,089.0nmol) and R3S (2975.6nmol) were excreted through urine. The major forms found in feces were Res and R3S, which were accumulated up to 241.8 and 250.8nmol, respectively. In summary, the SALLE technique simplified the samples preparation and could be well popularized, especially for those highly polar compounds in biosamples like urine, bile and feces, where various endogenous substances could significantly affect the extraction recovery and detection response.

In vivo assessment of the vascular disrupting effect of M410 by DCE-MRI biomarker in a rabbit model of liver tumor.

The present study aimed to prospectively monitor the vascular disrupting effect of M410 by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in rabbits with VX2 liver tumors. Twenty-eight rabbits bearing VX2 tumors in the left lobe of the liver were established and randomly divided into treatment and control groups, intravenously injected with 25 mg/kg M410 or sterile saline, respectively. Conventional and DCE-MRI data were acquired on a 3.0-T MR unit at pretreatment, 4 h, 1, 4, 7 and 14 days post-treatment. Histopathological examinations [hematoxylin and eosin (H&E) and CD34 immunohistochemisty staining] were performed at each time point. The dynamic changes in tumor volume, kinetic DCE-MRI parameter [volume transfer constant (Ktrans)] and histological data were evaluated. Tumors grew slower in the M410 group 4-14 days following treatment, compared with rapidly growing tumors in the control group (P<0.05). At 4 h, 1 and 4 days, Ktrans significantly decreased in the M410 group compared with that in the control group (P<0.05). However, Ktrans values were similar in the two groups for the other time points studied. The changes in DCE-MRI parameters were consistent with the results obtained from H&E and CD34 staining of the tumor tissues. DCE-MRI parameter Ktrans may be used as a non-invasive imaging biomarker to monitor the dynamic histological changes in tumors following treatment with the vascular targeting agent M410.

Longitudinal assessment of cerebral ß-amyloid deposition in mice overexpressing Swedish mutant ß-amyloid precursor protein using 18F-florbetaben PET.

UNLABELLED: The progression of ß-amyloid deposition in the brains of mice overexpressing Swedish mutant ß-amyloid precursor protein (APP-Swe), a model of Alzheimer disease (AD), was investigated in a longitudinal PET study using the novel ß-amyloid tracer (18)F-florbetaben. METHODS: Groups of APP-Swe and age-matched wild-type (WT) mice (age range, 10-20 mo) were investigated. Dynamic emission recordings were acquired with a small-animal PET scanner during 90 min after the administration of (18)F-florbetaben (9 MBq, intravenously). After spatial normalization of individual PET recordings to common coordinates for mouse brain, binding potentials (BPND) and standardized uptake value ratios (SUVRs) were calculated relative to the cerebellum. Voxelwise analyses were performed using statistical parametric mapping (SPM). Histochemical analyses and ex vivo autoradiography were ultimately performed in a subset of animals as a gold standard assessment of ß-amyloid plaque load. RESULTS: SUVRs calculated from static recordings during the interval of 30-60 min after tracer injection correlated highly with estimates of BPND based on the entire dynamic emission recordings. (18)F-florbetaben binding did not significantly differ in APP-Swe mice and WT animals at 10 and 13 mo of age. At 16 mo of age, the APP-Swe mice had a significant 7.9% increase (P < 0.01) in cortical (18)F-florbetaben uptake above baseline and at 20 mo there was a 16.6% increase (P < 0.001), whereas WT mice did not show any temporal changes in tracer uptake during the interval of follow-up. Voxelwise SPM analyses revealed the first signs of increased cortical binding at 13 mo and confirmed progressive binding increases in both the frontal and the temporal cortices (P < 0.001 uncorrected) to 20 mo. The SUVR strongly correlated with percentage plaque load (R = 0.95, P < 0.001). CONCLUSION: In the first longitudinal PET study in an AD mouse model using the novel ß-amyloid tracer (18)F-florbetaben, the temporal and spatial progression of amyloidogenesis in the brain of APP-Swe mice were sensitively monitored. This method should afford the means for preclinical testing of novel therapeutic approaches to the treatment of AD.

Antioxidant cosmeto-textiles: skin assessment.

Resveratrol, a natural product, has been reported to have antioxidant activities such as the scavenging of free radicals. This compound could be used in the dermocosmetic field to protect the skin from oxidative stress. In this work, the percutaneous profile of resveratrol in ethanol solutions through pig skin was determinated by an in vitro methodology. The percutaneous absorption of resveratrol was measured and compared with trolox, an analogous of Vitamin E. Both antioxidants were found in all skin sections (stratum corneum, epidermis, and dermis). Besides, the free radical scavenging activity of resveratrol and trolox has been evaluated using DPPH method. The effective dose (ED50) of compounds and DPPH radical inhibition in each skin layer were evaluated. Under the conditions used for these experiments, it can be deduced that resveratrol is more efficient than trolox as an antioxidant, also in the inner skin layers. The cosmeto-textiles with an active substance incorporated into their structure are increasingly used in the cosmetics and pharmaceutical industries. The action of several cosmeto-textiles on the skin was assessed by in vitro and in vivo methodologies. Samples of these cosmeto-textiles were prepared with resveratrol incorporated into cotton and polyamide fabrics. An in vitro percutaneous absorption was used to demonstrate the delivery of the resveratrol from the textile to the different skin layers (stratum corneum, epidermis, and dermis). Additionally, these cosmeto-textiles containing the antioxidant were applied onto the forearms of volunteers to evaluate the textiles' efficacy in skin penetration. The antioxidant's antiradical capacity was evaluated using the DPPH method. Results showed that resveratrol could be detected in the dermis, epidermis, and stratum corneum (SC) by an in vitro percutaneous absorption method and was also detected in the outermost layers of the SC by an in vivo method (stripping). A smaller amount of resveratrol was penetrated through the skin layers when cosmeto-textiles were used compared to direct topical application of the antioxidant solution. The cosmeto-textiles investigated can act as a reservoir system capable of progressively deliver the active substance to the skin layers. From the skin penetration profiles and the antioxidant efficacy assessment of resveratrol, it is possible to ameliorate the inherent antioxidant capacity of skin.

Development and validation of a rapid and sensitive liquid chromatography-tandem mass spectrometry method for benvitimod quantification in human plasma.

Benvitimod is a newly synthesized non-steroid small molecule being developed as a candidate drug for the treatment of inflammatory skin diseases. Here a rapid, sensitive and specific high performance liquid chromatography-tandem mass spectrometry (LC/ESI/MS/MS) method was developed for the determination of benvitimod in human plasma. The samples were alkalified with disodium tetraborate firstly, and then extracted by methyl tert-butyl ether. Fluorophenyl-benvitimod was used as internal standard (I.S.). Chromatographic separation was performed on an Ultra C(18) column (150mm×2.1mm, 5.0µm). The mixed mobile phase delivered at 300µl/min was CH3CN/H2O, 76.65:23.35 (v/v), containing 0.2mmol/L NH(4)COOH. Detection and quantitation was performed by electrospray ionization (ESI) and multiple reaction monitoring (MRM) in the negative ion mode. The most intense [M-H](-) MRM transition of benvitimod at m/z 253.1→211.0 was used for benvitimod quantitation and the transition at m/z 270.9→229.2 was used to monitor I.S. The calibration curve was linear within the concentration range of 0.1-10.0ng/mL (r>0.99). The lower limit of quantification (LLOQ) was 0.1ng/mL. The extraction recovery was above 80%. The accuracy expressed as relative error (RE) was less than 1.03%. The intra- and inter-day precisions were less than 11.81%. The freeze-thaw stability was also investigated and it was found that both benvitimod and the I.S. were quite stable. This method is especially useful for the pharmacokinetic study of benvitimod.

Impact of glutaraldehyde on in vivo colon-specific release of resveratrol from biodegradable pectin-based formulation.

Despite potential therapeutic efficacy of resveratrol on colitis and colorectal cancer, rapid absorption and metabolism at the upper gastro-intestinal (GI) tract prevent its clinical application. To overcome this, we attempted to develop colon-specific multi-particulate calcium-pectinate (Ca-pectinate) formulations of resveratrol. However, they were unable to prevent premature drug release at the upper GI tract. Thus, glutaraldehyde (Glu) was used for further cross-linking of the pectin chains. The formulation conditions and procedure were optimized from the in vitro drug release study. The optimized formulation was subjected to in vivo pharmacokinetic study in rats and compared with the unmodified Ca-pectinate and suspension formulation of resveratrol. Spherical particles (∼1 mm diameter) with high drug encapsulation were produced. Low cross-linking solution pH (1.5), minimum Glu concentration (2.5%) and cross-linking time (2 h) were crucial to exhibit colon-specific drug release. As Glu was added in the cross-linking solution, cross-linking between pectin chains and Glu occurred simultaneously during Ca-pectinate network formation, which appeared as a cost-effective formulation technique. Most importantly, the pharmacokinetic study demonstrated in vivo colon-specific drug release from the optimized formulation, while faster drug release was observed from the unmodified and suspension formulations. Hence, the developed formulation has potential to be used as colon-specific delivery system of resveratrol.

Assessment of pharmacodynamic vascular response in a phase I trial of combretastatin A4 phosphate.

PURPOSE: Clinical evaluation of novel agents that target tumor blood vessels requires pharmacodynamic end points that measure vascular damage. Positron emission tomography (PET) was used to measure the effects of the vascular targeting agent combretastatin A4 phosphate (CA4P) on tumor and normal tissue perfusion and blood volume. PATIENTS AND METHODS: Patients with advanced solid tumors were enrolled onto part of a phase I, accelerated-titration, dose-escalation study. The effects of 5 to 114 mg/m2 CA4P on tumor, spleen, and kidney were investigated. Tissue perfusion was measured using oxygen-15 (15O)-labeled water and blood volume was measured using 15O-labeled carbon monoxide (C15O). Scans were performed immediately before, and 30 minutes and 24 hours after the first infusion of each dose level of CA4P. All statistical tests were two sided. RESULTS: PET data were obtained for 13 patients with intrapatient dose escalation. Significant dose-dependent reductions were seen in tumor perfusion 30 minutes after CA4P administration (mean change, -49% at >or= 52 mg/m2; P =.0010). Significant reductions were also seen in tumor blood volume (mean change, -15% at >or= 52 mg/m2; P =.0070). Although by 24 hours there was tumor vascular recovery, for doses >or= 52 mg/m2 the reduction in perfusion remained significant (P =.013). Thirty minutes after CA4P administration borderline significant changes were seen in spleen perfusion (mean change, -35%; P =.018), spleen blood volume (mean change, -18%; P =.022), kidney perfusion (mean change, -6%; P =.026), and kidney blood volume (mean change, -6%; P =.014). No significant changes were seen at 24 hours in spleen or kidney. CONCLUSION: CA4P produces rapid changes in the vasculature of human tumors that can be assessed using PET measurements of tumor perfusion.

Assessment of resveratrol bioavailability in the perfused small intestine of the rat.

Resveratrol, which is present in grapes, wine and peanuts, is believed to possess chemoprotective properties such as anticarcinogenic effects and to provide protection against cardiovascular diseases. Little is known, however, about its intestinal absorption. We investigated the absorption and metabolism of resveratrol by using an isolated preparation of luminally and vascularly perfused rat small intestine. A synthetic perfusate free from blood components was used as vascular medium with a perfluorocarbon as oxygen carrier. Luminal media consisted of a bicarbonate buffered sodium chloride solution spiked with resveratrol in physiological, nutritionally relevant concentrations (28, 34 and 57 micromol/l, respectively). Viability was maintained during the entire perfusion and no significant differences between resveratrol and control perfusions for oxygen consumption, arterial pressure, lactate-pyruvate ratio and acid-base homeostasis were observed. Vascular uptake of luminally administered resveratrol was 20.5%. The majority of the absorbed resveratrol was conjugated to yield resveratrol glucuronide (16.8%), which was also the main luminal metabolite (11.2%). Lesser amounts of resveratrol sulfate, 3.0% and 0.3%, were found on the luminal and vascular side, respectively, while only minute amounts of resveratrol and resveratrol conjugates (1.9%) were found in the intestinal tissue. The structures of the resveratrol conjugates were verified by liquid chromatography coupled with mass spectometry (LC-MS). The results demonstrate an ample uptake and metabolic conversion of resveratrol. The proposed perfusion model serves as a tool to evaluate intestinal absorption and metabolic handling of phytochemicals, a pertinent input to the ongoing discussion about their health benefits.