Identification of the major metabolites of resveratrol in rat urine by HPLC-MS/MS

To identify the major metabolites of resveratrol in rat, rat urine samples were pretreated by using solid-phase extraction technique (SPE) with polyamide cartridges. And a LC-MS/MS method with electrospray ionisation (ESI), negative ion mode and collision induced dissociation (CID), was used to elucidate the structures of the major metabolites of resveratrol. According to the results of our experiment, we found that the main metabolites of resveratrol were resveratrol monoglucuronide (M1), dihydroresveratrol monosulfate (M2), resveratrol monosulfate (M3) and dihydroresveratrol (M4).     

Comparision of Piceid and Resveratrol in Antioxidation and Antiproliferation Activities In Vitro

Background

The clinic therapeutic effect of resveratrol is limited due to its low oral bioavailability. Piceid, a precursor of resveratrol, is the most abundant form of resveratrol in nature. A number of studies have hypothesized that piceid may have the same bioactivities like those of resveratrol. The aim of this work is to compare piceid with resveratrol in antioxidation and antiproliferation activities in vitro.

Methods

The antioxidative effects of resveratrol and piceid were evaluated by phenanthroline-Fe²⁺ method and H₂O₂-induced oxidative injury cell model. The antiproliferation effects were determined by MTT method in human liver tumor HepG2 cells, human breast cancer MDA-MB-231 cells and MCF-7 cells. The effects of resveratrol and piceid on the cell cycle and the apoptosis were evaluated by flow cytometry. Additionally, the uptake profiles of resveratrol and piceid in cancer cells were observed using fluorescence microscopy and clarified by LC-MS/MS.

Conclusion

Piceid exhibited higher scavenging activity against hydroxyl radicals than resveratrol in vitro. Resveratrol showed a significant protective effect against H₂O₂-induced cell damage. What is more, resveratrol had biphasic effects on tumor cells. Resveratrol and piceid only showed significant cytotoxicity on tumor cells at high concentration (≥50 µmol/L), while low concentration of resveratrol (<30 µmol/L) increased the cell viability. The principal effect of resveratrol and piceid on the viability of tumor cells was caused by the cell cycle arrest, while the effect on apoptosis was relatively minor. The reason that piceid showed lower biological activity than resveratrol at the same concentration was probably because piceid was more difficult in being uptaken by cells.     

Metabolism of [6,7-3H, 35S] estradiol 17-sulfate in rats

To confirm whether or not the sulfo group of estradiol 17-sulfate (ES) is removed during in vivo metabolism in rats, the doubly labeled conjugate [6,7-3H, 35S] ES was injected into rats, and its biliary and urinary metabolites were determined by reverse isotope dilution method (RIDM). In male rats, the major radioactivity was detected in biliary disulfate fraction, which was composed of mainly ES and its two minor metabolites, 2-hydroxyestradiol 17-sulfate (2-OH-ES) and 2-methoxyestradiol 17-sulfate (2-MeO-ES). In female rats, in contrast, the radioactivity was dispersed into three fractions:biliary monosulfate, biliary disulfate, and urinary monosulfate fractions (Frs.) In both monosulfate Frs., 7beta-hydroxyestradiol 17-sulfate was detected as the major metabolite followed by 6alpha-, 6beta-, and 15beta-hydroxyestradiol 17-sulfates. Like male rats, 2-OH-ES and 2-Meo-ES as the minor products were detected in biliary disulfate fraction. The isotope ratios of ES and its metabolites in both sexes were essentially the same as that of the dose except that of 6alpha-hydroxylated metabolite, which may be derived from the loss of the tritium labeled at C6. These results confirm the occurrence of the direct metabolism of ES in rats.     

白藜芦醇和白藜芦醇苷的测定方法研究进展

白藜芦醇和白藜芦醇苷具有抗肿瘤、保护心血管系统等多种显著的生物活性,国内外已经开始以其为指标性成分评定葡萄酒及相关中药的质量。本文综述了白藜芦醇和白藜芦醇苷等二苯乙烯类化合物的测定方法。     

Bioconversion of piceid to resveratrol by selected probiotic cell extracts

Resveratrol exerts several pharmacological activities, including anti-cancer, anti-inflammatory, cardioprotective, or antioxidant effects. However, due to its occurrence in plants more in glycosidic form as piceid, the bioavailability and bioactivity are limited. The enzymatic potential of probiotics for the transformation of piceid to resveratrol was elucidated. Cell extract from Bifidobacteria (B.) infantis, B. bifidum, Lactobacillus (L.) casei, L. plantarum, and L. acidophilus was evaluated for their effect in this bioconversion using high-performance liquid chromatography (HPLC) as analytical tool. Cell extract of B. infantis showed the highest effect on the deglycosylation of piceid to resveratrol, already after 30 min. Cell extracts of all other tested strains showed a significant biotransformation with no further metabolization of resveratrol. The conversion of piceid to resveratrol is of importance to increase bioavailability and bioactivity as shown for anti-inflammation in this study. Cell extracts from probiotics, especially from B. infantis, may be added to piceid containing products, for achieving higher biological effects caused by the bioactivity of resveratrol or by health promoting of the probiotics. These findings open a new perspective of novel combination of cell extracts from probiotics and piceid, in health-promoting pharmaceutical and food products.     

Inhibitory activity of stilbenes on Alzheimer's beta-amyloid fibrils in vitro

Polymerization of the amyloid beta-peptide (Abeta) has been identified as one of the major characteristics of Alzheimer's disease (AD). Thus, finding molecules to prevent the aggregation of Abeta could be of therapeutic value in AD. We describe an original routine in vitro assay to search for inhibitors of Abeta(25-35) fibril formation which uses UV-visible measurements and electron microscopy (EM). In particular, this routine assay was used to examine the effects of stilbenes, a well-known polyphenol class, as inhibitors of Abeta fibril formation. The inhibitory properties of resveratrol (RES), piceid (PIC), resveratrol diglucoside (DIG), piceatannol (PIA), astringine (AST), and viniferin (VIN) were characterized and compared. RES and PIC effectively and dose-dependently inhibited Abeta polymerization while other polyphenols exerted less inhibition. Although the mechanism of anti-amyloidogenic activity is still unknown, these results support the hypothesis that stilbenes could be of therapeutic value in AD.     

RP-HPLC法同时测定虎杖中白藜芦醇和白藜芦醇苷的含量

建立了国内首次用RP-HPLC同时测定虎杖中白藜芦醇和白藜芦醇苷含量的方法,并详细考察了2个指标成分的稳定性。对不同产地的虎杖药材、不同采收期的虎杖根及根茎以及虎杖的不同部位共25个样品中的白藜芦醇和白藜芦醇苷进行了含量测定,考察了两种活性成分的含量与产地、采收期及部位的关系,为虎杖资源的合理采收利用及进一步的研究提供了理论依据。     

Oligostilbenoids in stem bark of Vatica rassak

Three resveratrol oligomers, vaticanols. A, B and C, as well as three known stilbenoids, resveratrol, piceid and epsilon-viniferin were isolated from the stem bark of Vatica rassak (Dipterocarpaceae). Their structures were determined by the analysis of NMR spectral data including the application of 2D methods.     

Acetophenone C-glucosides and stilbene O-glucosides in Upuna borneensis

Three acetophenone C-glycosides; 2,4,6-trihydroxyacetophenone 3-C-beta-(2'-O-p-hydroxybenzoyl)-glucopyranoside, 2,4,6-trihydroxyacetophenone 3-C-beta-(2'-O-E-coumaroyl)-glucopyranoside, 2,4,6-trihydroxyacetophenone 3-C-beta-(2'-O-E-cinnamoyl)-glucopyranoside, and two resveratrol O-glycosides; piceid 2'-O-p-hydroxybenzoate and, piceid 2'-O-E-ferulate, together with three known compounds were isolated from the acetone soluble part of stem of Upuna borneensis (Dipterocarpaceae). The structures of isolates were determined by spectral analysis including extensive 2D-NMR spectral analyses.     

Biotransformation of piceid in Polygonum cuspidatum to resveratrol by Aspergillus oryzae

Biotransformation of piceid in Polygonum cuspidatum to resveratrol by Aspergillus oryzae was investigated in this study. Resveratrol is widely used in medicine, food, and cosmetic because of its pharmacological properties. However, it has a much lower content in plants compared with its glucoside piceid, which has a much lower bioavailability. Traditionally, the aglycone is acquired by acid or enzymatic hydrolysis of its glucoside, but the violent condition and the acid pollution in hydrolytic reaction and the high cost of the enzyme limit their industrial development. In this paper, fermentation of P. cuspidatum by A. oryzae was successfully performed, during which, piceid was converted to resveratrol with the highest yield of trans-resveratrol 1.35%, 3.6 times higher than that obtained from raw herb by microwave-assisted extraction. Scale-up production was also performed and the yield of trans-resveratrol was 3.1 times higher after 24 h incubation. Therefore, biotransformation is a better method to increase the yield of resveratrol because of its high yield and mild conditions.     

Isoflavones and stilbenes from Juniperus macropoda

An ethanolic extract of Juniperus macropoda, after chromatography, yielded three isoflavones, viz.irigenin, iridin and a new compound. In addition two stilbenes, resveratrol and piceid (resveratrol-3-O-β-D-glucoside), were isolated and characterized.     

Distinct sulfonation activities in resveratrol-sensitive and resveratrol-insensitive human glioblastoma cells

Glioblastoma multiforme (GBM) cells show different responses to resveratrol, for unknown reasons. Our data from human medulloblastoma cells and primary cultures of rat brain cells revealed an inverse correlation of sulfonation activity with resveratrol sensitivities, providing a clue to the underlying mechanisms of the variable sensitivities of GBM cells to resveratrol. In this study, we found that U251 cells were sensitive and LN229 cells were insensitive to resveratrol. Thus, these two cell lines were taken as comparable models for elucidating the influence of sulfonation activities on resveratrol sensitivity. HPLC showed identical resveratrol metabolic patterns in both cell lines. LC/MS and high-resolution mass MS analyses further demonstrated that resveratrol monosulfate generated by sulfotransferases (SULTs) was the major metabolite of human GBM cells. The levels of brain-associated SULT (SULT1A1, SULT1C2, and SULT4A1) expression in U251 cells were lower than those in LN229 cells, suggesting the inverse relationship of SULT-mediated sulfonation activity with high intracellular resveratrol bioavailability and resveratrol sensitivity of human GBM cells. Furthermore, immunohistochemical staining revealed reductions in expression of the three brain-associated SULTs in 72.8%, 47.5% and 66.3% of astrocytomas, respectively. Therefore, the levels of brain-associated SULTs and sulfonation activity mediated by them could be important parameters for evaluating the potential response of human GBM cells to resveratrol, and may have value in the personalized treatment of GBMs with resveratrol.     

Resveratrol affects in vitro rumen fermentation,methane production and prokaryotic community composition in a time- and diet-specific manner

This study aimed to investigate the effect of resveratrol on methane production, rumen fermentation and microbial composition under high-concentrate (HC) and high-forage (HF) diets using the in vitro fermentation system. A total of 25 mg of resveratrol was supplemented into 300 mg of either HC or HF diet. Methane production, total volatile fatty acid (VFA) concentration, molar proportion of VFA, metabolites of resveratrol and prokaryotic community composition were measured after 12 and 24 h of in vitro fermentation. Resveratrol reduced methane production (ml per mg of dry matter degraded) by 41% and 60% under both HC and HF diets (P < 0.001), respectively, and this result could be associated with the lower abundance of Methanobrevibacter (P < 0.001) in response to resveratrol. The molar proportion of propionate was significantly higher in the resveratrol group only under the HC diet (P = 0.045). The relative abundance of 10 bacterial genera was affected by the three-way interaction of treatment, diet and time (P < 0.05). Resveratrol was partly converted to dihydroresveratrol after 24 h of fermentation, and its degradation could be associated with microbes belonging to the order Coriobacteriales. Our results suggest that multiple factors (e.g. diet and time) should be considered in animal experiments to test the effect of polyphenol or other plant extracts on rumen fermentation, methane emission and microbial composition.     

Stilbene glucosides in the bark of Picea sitchensis

The fresh bark of Picea sitchensis contains astringin as the major stilbene; isorhapontin and piceid are present in minor amounts. The aglycones astringenin, isorhaponhgenin and resveratrol are absent in samples of fresh bark. Prenylation reactions on 5,3′,4′-tri-O-methylastringenin are described.     

Microwave-assisted aqueous two-phase extraction of piceid, resveratrol and emodin from Polygonum cuspidatum by ethanol/ammonium sulphate systems

Microwave-assisted, aqueous two-phase extraction was investigated to obtain effective constituents, including piceid, resveratrol and emodin in Polygonum cuspidatum. An aqueous two-phase system consisting of 25% (w/w) ethanol 21% (w/w) (NH₄)₂SO₄ gave equal yields of piceid, and 1.1- and 1.9-times higher yields of resveratrol and emodin, respectively, than that achieved by microwave-assisted extraction and heat reflux extraction. Three-separate operations, extraction, clarification and concentration, are hereby integrated into a single step to get higher yields at lower cost. This is therefore a potentially useful method for the extraction and purification of target products.     

Expression of the Stilbene Synthase (StSy) Gene from Grapevine in Transgenic White Poplar Results in High Accumulation of the Antioxidant Resveratrol Glucosides

When present, stilbene synthase leads to the production of resveratrol compounds, which are major components of the phytoalexin response against fungal pathogens of the plant and are highly bioactive substances of pharmaceutical interest. White poplar (Populus alba L.) was transformed with a construct containing a cDNA insert encoding stilbene synthase from grapevine (Vitis vinifera L.), under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and a chimeric kanamycin resistance gene. Southern blot hybridization analysis demonstrated the presence and integration of exogenous DNA sequences in the poplar genome. Expression of the stilbene synthase-encoding gene in different transgenic lines was confirmed by Western blot and Northern analyses. Compared to the controls, in the transgenic plants two new compounds were detected and were identified as the trans- and cis-isomers of resveratrol-3-glucoside (piceid) by high-pressure liquid chromatography (HPLC), UV spectrophotometry, electrospray mass spectrometry (HPLC-ESI-MS) and enzymatic hydrolysis. Since poplar is a good biomass producer and piceids are accumulated in substantial amounts (up to 615.2 microg/g leaf fresh weight), the transgenic plants represent a potential alternative source for the production of these compounds with high pharmacological value. Despite the presence of piceid, in our experimental conditions no increased resistance against the pathogen Melampsora pulcherrima, which causes rust disease, was observed when in vitro bioassays were performed.     

Resveratrol glucoside (Piceid) synthesis in seeds of transgenic oilseed rape (Brassica napus L.)

Resveratrol is a phytoalexin produced in various plants like wine, peanut or pine in response to fungal infection or UV irradiation, but it is absent in members of the Brassicaceae. Moreover, resveratrol and its glucoside (piceid) are considered to have beneficial effects on human health, known to reduce heart disease, arteriosclerosis and cancer mortality. Therefore, the introduction of the gene encoding stilbene synthase for resveratrol production in rapeseed is a tempting approach to improve the quality of rapeseed products. The stilbene synthase gene isolated from grapevine (Vitis vinifera L.) was cloned under control of the seed-specific napin promotor and introduced into rapeseed (Brassica napus L.) by Agrobacterium-mediated co-transformation together with a ds-RNA-interference construct deduced from the sequence of the key enzyme for sinapate ester biosynthesis, UDP-glucose:sinapate glucosyltransferase (BnSGT1), assuming that the suppression of the sinapate ester biosynthesis may increase the resveratrol production in seeds through the increased availability of the precursor 4-coumarate. Resveratrol glucoside (piceid) was produced at levels up to 361 μg/g in the seeds of the primary transformants. This value exceeded by far piceid amounts reported from B. napus expressing VST1 in the wild type sinapine background. There was no significant difference in other important agronomic traits, like oil, protein, fatty acid and glucosinolate content in comparison to the control plants. In the third seed generation, up to 616 μg/g piceid was found in the seeds of a homozygous T3-plant with a single transgene copy integrated. The sinapate ester content in this homozygous T3-plant was reduced from 7.43 to 2.40 mg/g. These results demonstrate how the creation of a novel metabolic sink could divert the synthesis towards the production of piceid rather than sinapate ester, thereby increasing the value of oilseed products.     

Role of peptide primary sequence in polyphenol-protein recognition: an example with neurotensin

Polyphenols are known for their impact on health and one of their major properties is the formation of complexes with proteins. To investigate the involvement of polyphenol-protein complexes in health, the interactions between bioactive polyphenols and neurotensin were examined by structural NMR and molecular modeling. Neurotensin is a linear bioactive tridecapeptide and polyphenols seem to affect the NT metabolism. We studied the polyphenols resveratrol and its glucoside the piceid in order to observe the possible role of glucose group and the penta-O-galloyl-D-glucopyranose (PGG). NMR data and molecular modeling showed that interaction occurred with the three polyphenols involving hydrophobic stacking and hydrogen bonds. Moreover, the peptide primary sequence plays a role in the specificity of complex formation.     

白藜芦醇代谢物的研究进展

白藜芦醇是一些植物在受到生物和非生物胁迫时产生的一种植物抗毒素.它在植物体内会迅速被代谢而生产紫檀芪、云杉新苷、viniferins等代谢物.这些代谢物与白藜芦醇一样具有抗菌、消炎、抗血小板凝集、防止细胞癌变等一系列的生物活性,且有些代谢物的选择性、稳定性远远大于白藜芦醇.本文对白藜芦醇代谢物中几种主要代谢物的合成途径、生物活性、代谢调控、检测方法等进行了综述.     

Role of peptide primary sequence in polyphenol–protein recognition: An example with neurotensin

Polyphenols are known for their impact on health and one of their major properties is the formation of complexes with proteins. To investigate the involvement of polyphenol–protein complexes in health, the interactions between bioactive polyphenols and neurotensin were examined by structural NMR and molecular modeling. Neurotensin is a linear bioactive tridecapeptide and polyphenols seem to affect the NT metabolism. We studied the polyphenols resveratrol and its glucoside the piceid in order to observe the possible role of glucose group and the penta-O-galloyl-d-glucopyranose (PGG). NMR data and molecular modeling showed that interaction occurred with the three polyphenols involving hydrophobic stacking and hydrogen bonds. Moreover, the peptide primary sequence plays a role in the specificity of complex formation.