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Abstract:

1. The phytoestrogenic compound trans-resveratrol (trans-3,5,4'-trihydroxystilbene) is found in appreciable quantities in grape skins and wine. It has been shown that both products rich in trans-resveratrol and pure trans-resveratrol inhibit platelet aggregation both in vivo and in vitro. However the mechanism of this action still remains unknown.
2. An essential component of the aggregation process in platelets is an increase in intracellular free Ca2+ ([Ca2+]i). Ca2+ must enter the cell from the external media through specific and tightly regulated Ca2+ channels in the plasma membrane. The objective of this study was to characterize what effect trans-resveratrol had on the Ca2+ channels in thrombin stimulated platelets.
3. In this study we showed that trans-resveratrol immediately inhibited Ca2+ influx in thrombin-stimulated platelets with an IC50 of 0.5 M. trans-Resveratrol at 0.1, 1.0 and 10.0 M produced 20 6, 37 6 and 57 4% inhibition respectively of the effect of thrombin (0.01 u ml-1) to increase [Ca2+]i.
4. trans-Resveratrol also inhibited spontaneous Ba2+ entry into Fura-2 loaded platelets, with 0.1, 1.0 and 10.0 Mtrans-resveratrol producing 10 5, 30 5 and 50 7% inhibition respectively. This indicated that trans-resveratrol directly inhibited Ca2+ channel activity in the platelets in the absence of agonist stimulation.
5. trans-Resveratrol also inhibited thapsigargin-mediated Ca2+ influx into platelets. This suggests that the store-operated Ca2+ channels are one of the possible targets of trans-resveratrol. These channels rely on the emptying of the internal Ca2+ stores to initiate influx of Ca2+ into the cell.
6. The phytoestrogens genistein, daidzein, apigenin and genistein-glucoside (genistin) produced inhibitory effects against thrombin similar to those seen with trans-resveratrol.
7. We conclude that trans-resveratrol is an inhibitor of store-operated Ca2+ channels in human platelets. This accounts for the ability of trans-resveratrol to inhibit platelet aggregation induced by thrombin.

Keywords:

trans-Resveratrol, platelets, calcium, barium, channels, thapsigargin,thrombosis, thrombin

Abbreviations:

ADP, adenosine diphosphate; [Ca2+]i, intracellular free calcium; EGTA, ethylene glycol bis( -aminoethyl ether) N,N,N',N'-tetraacetic acid; IP3, myo-inositol 1,4,5-trisphosphate; Me2SO, dimethylsulphoxide.

RESULTS AND DISCUSSION

Regardless of the separation technique, its analysis in grapes and wines requires the use of preconcentration and/or multisolvent extraction techniques because of the complexity of the matrices and the low concentration of the analyte. The combination of LD followed by REMPI-TOFMS detection can overcome these error sources.

Figure 1. TOFMS spectrum of a grape skin sample obtained at normal

experimental conditions. See text for comments.

Figure 1 shows a time-of-flight spectrum obtained from a sample of grape skin corresponding to a desorption area with 48 mg of grape skin and 79 mg of Zn. The trans-resveratrol peak is clearly noticeable. For this sample, the trans-resveratrol content has been determined using the standard additions method, i.e. adding known quantities of trans-resveratrol to several identical samples of grape skin; the value obtained for the intercept with the concentration axis gives the quantity of analyte in the blank. A value of 16.0 ± 0.5 µg trans-resveratrol g 1 grape skin was obtained, which corresponds to 16 µL L 1 of trans-resveratrol. The trans-resveratrol content in grape flesh was also investigated, however no significant signal was found (i.e. content below 2 nL L 1). This finding proves that the main content of trans-resveratrol selectively accumulates in grape skin, which it is consistent with previous.

investigations

Figure 4. Ethylene production by grapes infected with Botrytis cinerea (5 µL of the suspension at 103 conidia mL 1 per grape). At 0 h, grapes were inoculated and immediately placed into cuvettes under continuous air flow of 2 L h 1. The insets show the ethylene emission from untreated fruits compared with trans-resveratrol-treated ones for the first 4 d.

One of the major impediments to faster progress in anti-aging research is the time required to prove that a substance has life-prolonging effects. Consequently, one of the major goals of biomedical gerontology is to develop a set of biomarkers that can be used to measure biological age to determine a reasonably short period hether a substance has an age-retarding effect. Recently, two research teams have developed such biomarkers

About Resveratrol

Resveratrol is extracted from polygonum cuspidatum, and it has strong antioxidant effect,which can prolong the coming of old. Resveratrol can lower the cholesterol level in human body, and cease the conglomeration of haematoblast. So it is used to treat those people who have high blood grease diseases.Another important efficacy of resveratrol is preventing the growth of tumer cell or decreasing tumor promotion activity. It is recognized in the use for medication, cosmetics, and health food industries.

Parts Used: Root

Molecular Formula: C14H12O3

Molecular Weight: 228.24

CAS Reg.： 501-36-0

ontent Assay(the by HPLC):>98%

Appearance Appearance: White and fine powder White Fine Powder

Flavor Odor: Special flavor Characteristic

The ash a Sulphated Ash:<1%

The melting agent remains the Residual Solvents: Eur.Pharm.

Heavy metals Heavy metals: <10PPM

The agrochemical remains the Pesticides: Negative

The a Mesh Size: 80Mesh

The aridity loses the heavy Loss on Drying:<2%

The total germ falls the few Total Plate Count:<1000 CFUs/gm

The yeast fungus and the fungi Yeast& Mold:<100 CFUs/gm

Salmonella: Negative

Escherichia coliform E.Coli: Negative

Trans-resveratrol and piceid were first reported in the peel of grape berries for disease prevention and later in wines to benefit health. They were also found to be the major polyphenols in the root of Polygonum cuspidatum Sied. Et Zucc.3 Their contents were much higher in Polygonum cuspidatum Sied. et Zucc. than in grape.4 The root of this plant was traditionally used in China and Japan as a folk medicine for the treatment of atherosclerosis and for other therapeutic purposes. Trans-resveratrol and piceid elicited great interest after they were found to have therapeutic effects in inhibiting the copper-catalyzed oxidation of low-density lipoprotein (LDL),5 inhibiting platelet clotting and arachidonate

metabolism, reducing liver injury from peroxidized oil,6 and having cancer-chemopreventive activities. It has been suggested that trans-resveratrol, a common constituent of the human diet, merits investigation as a potential cancerchemopreventiveagent in .

For the analysis of trans-resveratrol and piceid, several analytical methods including HPLC8–10 and high-speed countercurrent chromatography were reported,11 but these methods are usually time consuming and require large amounts of reagent. Capillary zone electrophoresis (CZE) is a relatively new separation technique that is an

alternative for the analysis of a variety of compounds,including active ingredients in traditional Chinese medicines.12–14 CZE has unique advantages that make it an excellent candidate for the analysis of these compounds,including a very small sample size requirement, Experimental

Apparatus

A CZE-ED system

was constructed that is similar to that described previously.15 A ±30-kV

high-voltage dc power supply (Shanghai Institute of Nuclear Research, Shanghai, China) provided a voltage between the ends of the capillary. The inlet of the

capillary was held at a positive potential, and the outlet end of the capillary was maintained at ground potential. The separations were performed in a 70-cmlength,

25-µm-i.d., 360-µm-o.d. fused-silica capillary (Polymicro Technologies, Phoenix, AZ).A three-electrode electrochemical cell consisting of a 300-µm-diam carbon disk working electrode, a platinum auxiliary electrode, and a saturated calomel electrode

(SCE) as the reference electrode was used in combination with a BAS LC-3D amperometric detector (Biochemical System, West Lafayette, IN).

Sample preparation

The sample was dried at 60 °C for 4 hr in an oven and then ground into powder. An accurate weight (10,000g) of the powder was extracted with 50 mL anhydrous

ethanol (analytical reagent grade) and the running buffer (1:1) for 2 hr in an ultrasonic bath. The sample was then filtered, first through a filter paper, and then

through a 0.22-µm nylon filter, and made up to 50 mL in volume. The sample solution was stored in the dark at 4 °C.

Results and discussion

Optimum conditions for determination of

trans-resveratrol and piceid

1. Hydrodynamic voltammograms (HDVs). The potential applied to the working electrode directly affects the sensitivity and detection limits; therefore, it is necessary to determine the HDVs of the analytes to find the optimum potential.Figure 2 illustrates the HDVs of trans-resveratrol and

piceid. When the applied potential reaches 0.60 V (vs SCE), the peak current increases rapidly. However, when the potential exceeds 0.85–0.90 V (vs SCE), this current levels off. Although an applied potential greater than +0.90.

2. Effects of pH and concentration of buffer. The acidity and concentration of the running buffer play an important role in CZE because of their effect on zeta potential (z); electroosmotic flow (EOF);as well as the overall charge of analytes, which in turn affect the migration time and separation of the analytes.Therefore, it is important to study their influences

on CZE to obtain optimum separations. Based on experiments, 100 mmol/L borate buffer (pH 9.2) was chosen as the running buffer in consideration of the peak current, resolution, and the analysis time.

Chemopreventive Properties of trans-Resveratrol Are Associated with Inhibition of Activation of the I B Kinase1

trans-Resveratrol (Res), a phytoalexin found at high levels in grapes and in grape products such as red wine, has been shown to have anti-inflammatory and antioncogenic properties. Because the transcription factor nuclear factor B (NF- B) is involved in inflammatory diseases and oncogenesis, we tested whether Res could modulate NF- B activity. Res was shown to be a potent inhibitor of both NF- B activation and NF- B-dependent gene expression through its ability to inhibit I B kinase activity, the key regulator in NF- B activation, likely by inhibiting an upstream signaling component. In addition, Res blocked the expression of mRNA-encoding monocyte chemoattractant protein-1.

DESCRIPTION

Resveratrol is a naturally occurring phytoalexin produced by some higher plants in response to injury or fungal infection. Phytoalexins are chemical substances produced by plants as a defense against infection by pathogenic microorganisms, such as fungi. Alexin is from the Greek, meaning to ward off or to protect. Resveratrol may also have alexin-like activity for humans. Epidemiological, in vitro and animal studies suggest that a high resveretrol intake is associated with a reduced incidence of cardiovascular disease, and a reduced risk for cancer.

Resveratrol is found in grapevines (Vitis vinifera L). It occurs in the vines, roots, seeds and stalks, but its highest concentration is in grape skins. Wine also contains resveratrol. The concentration of resveratrol in red wine is much higher than that of white wine. The main difference between red and white wine production, besides the grapes used, is that for red wine the skins and seeds are involved in the process, while white wine is mainly prepared from the juice, essentially avoiding the use of grape skins and seeds. During the wine making process, resveratrol, as well as other polyphenols, including quercetin, catechins, gallocatechins, procyanidins and prodelphidins (condensed tannins), are extracted from the grape skins via a process called maceration.

Resveratrol, which is also known as 3,4',5 trihydroxystilbene and 3,4',5-stilbenetriol, exists in cis- and trans-stereoisomeric forms. Resveratrol is the parent molecule of a family of polymers called viniferins. Cis- and trans-resveratrol occur naturally as do their glucosides. Resveratrol-3-O-beta-D-glucoside is also known as piceid, and the respective cis- and trans-glucosides are called cis-piceid and trans-piceid. The molecular formula of resveratrol is C14H12O3 and its molecular weight is 228.25 daltons. It is represented by the following structural formula:

Resveratrol

The stereoisomer of resveratrol found in grapes and peanuts is the trans-form. Both cis- and trans-resveratrol are found in Polygonium cuspidatum. Therefore, dietary supplements containing resveratrol, which are principally derived from this plant, contain both stereoisomers. The amount of resveratrol (trans-resveratrol) in peanuts ranges from 0.02 to 1.79 micrograms per gram. Red wine contains from 0.6 to 0.8 micrograms per milliliter, and fresh grape skin, approximately 50 to 100 micrograms per gram. A glass of red wine delivers on the average, between 600 to 700 micrograms of resveratrol.