Milica Atanacković

Isoflavone composition, total polyphenolic content, and antioxidant activity in soybeans of different origin.

Twenty soybean cultivars, originating from the United States, Russia, Serbia, and China, were analyzed for their isoflavone composition, total polyphenolic content, and antioxidant activity. Isoflavones were extracted by aqueous methanol (80%) and analyzed by high-performance liquid chromatography/diode array detection. Precision and linearity of the applied method were within the standard limits of validation. The highest and the lowest total isoflavone contents were 4.59 and 1.45 mg/g of dried soybean weight, respectively. A significant difference (P < .05) was found in isoflavone concentration among the different cultivars, but it was observed that origin is not a significant factor that could influence isoflavone content in soybeans. Total polyphenolic content varied between 2.13 and 3.45 mg of gallic acid equivalents/g of dried soybean weight. The free radical scavenging activity of soybean extracts assayed by 2,2-diphenyl-1-picrylhydrazyl in terms of 50% inhibitory concentration (IC(50)) ranged from 1.40 to 3.35 mg/mL. Negative correlation between total polyphenolic content and IC(50) was observed, but there was no correlation between total isoflavone content and IC(50). On the basis of this study, soybean cultivars with larger potency for biological activity could be recognized.

Effect of potassium channel opener pinacidil on the contractions elicited electrically or by noradrenaline in the human radial artery.

In order to discover an agent that can prevent spasm of the human radial artery, the aim of our study was to evaluate the effect of the K(+) channel opener, pinacidil, on contractions in the radial artery. Contractions of the radial artery were evoked by exogenously applied noradrenaline or by electrical field stimulation (EFS, 20Hz, neurogenic). Pinacidil induced concentration-dependent inhibition of both EFS- and noradrenaline-evoked contractions of the radial artery. Glibenclamide, a selective blocker of ATP-sensitive K(+) channels (Kir6.x containing subunit) antagonized in the same manner the pinacidil-induced inhibition of neurogenic contractions and contractions evoked by exogenous noradrenaline. The inhibition of pinacidil relaxation by tetraethylammonium (TEA), a blocker of Ca-sensitive K(+) (K(Ca)) channels, was more pronounced in EFS-contracted preparations. A blocker of voltage-sensitive K(+) (K(V)) channels, 4-aminopyridine (4-AP), inhibited pinacidil relaxation only in EFS-contracted preparations. In order to test the presence of different K(+) channels, immunohistochemistry of K(+) channels expression in the radial artery was performed. The vascular wall of the human radial artery showed variable positivity with the following applied antibodies: Kv1.2, Kv1.3, Kir6.1, and K(Ca)1.1. The antibodies against Kv1.6, Kv2.1, and Kir6.2 channel subunits were completely negative. These results suggest that the inhibitory effect of pinacidil on contractions of the human radial artery might be postsynaptic and associated with opening of smooth muscle Kir6.1-containing K(ATP) channels. TEA- and 4-AP-sensitive K(+) channels may also contribute to pinacidil effect in the human radial artery.

Chapter 1 – Phytoestrogens: “Estrogene-Like” Phytochemicals

Phytoestrogens, also called “dietary estrogens,” are nonsteroidal phytochemicals structurally similar to female hormone estradiol (17-β-estradiol). According to the chemical structure, this group of compounds consists of the following subgroups: isoflavones, coumestans, lignans, as well as stilbenes and prenylated flavonoids. Isoflavones are the most investigated class of phytoestrogens. Soybeans (Glycine max) and red clover (Trifolium pratense) are the most important plant sources of these bioactive nonnutrients. The major isoflavone aglycones in soybeans are genistein, daidzein, and glycitein, which are also present as corresponding glycosides (β-glycoside, acetyl-β-glycoside, malonyl-β-glycoside). Major isoflavones present in red clover include biochanin A and formononetin, while genistein and daidzein are also present. It has been shown that different factors influence the content of isoflavones in plant sources. Various studies are focused on determination of these factors. The aim is to produce and/or select a raw material for further processing into food ingredients or dietary supplements which, due to their favorable characteristics, may enhance health benefits of these products. As bioactive compounds structurally similar to estrogen, phytoestrogens have the ability to bind to estrogen receptors either activating or blocking them, depending on the type of estrogen receptors. Their estrogenic and/or antiestrogenic effects are also shown to be tissue specific. Beside the estrogenic and antiestrogenic activity, phytoestrogens possess capacity for other biological activities. Numerous potential health benefits of these compounds have been investigated, including effects on cancer, vascular diseases, osteoporosis, and menopausal symptoms. Epidemiologic evidence and collected experimental data from animal studies highly suggest beneficial effects of isoflavones on health, but the clinical data in favor of such effects are still to be obtained. Due to their assumed beneficial effects, use of products and supplements containing isoflavones has significantly increased. These products are widely commercialized in many countries as alternative therapy for alleviating menopausal discomforts and for the prevention of diseases such as osteoporosis or breast cancer. Dietary supplements, containing phytoestrogens, are often a blend of extracts from different plants, and usually contain extracts from soy and red clover. Results obtained from analysis of phytoestrogens content in dietary supplements showed no uniformity in the profile and concentration of these compounds among the products of a particular category. The phytoestrogen levels also vary from batch to batch and product to product, causing more difficulties in interpretation of results. Current results indicate that the beneficial effects of phytoestrogens are strongly supported by synergistic effect with other phytochemicals in plants.

The differential effect of resveratrol on the renal artery of normal and diabetic rats

Results Resveratrol relaxed RA of normal rats more potently than RA of rats with diabetes (EC50 8 and 50 μM, respectively). L-NAME and methylene blue partly antagonized the relaxation of RA of normal animals only. A nonselective blocker of voltage-gated K (KV) channels, 4-aminopyridine (4-AP) partly inhibited the relaxation of RA of normal as well as of diabetic rats. However, margatoxin, a selective antagonist of KV1.x channels, completely antagonized the relaxation of RA of diabetic rats only. Glibenclamide, a highly selective blocker of ATP-sensitive K channels, did not block resveratrolinduced relaxation in both experimental models. Conclusions In conclusion, we have shown that resveratrol induces a strong endothelium-dependent relaxation of RA of normal rats, and that 4-AP-sensitive K channels are involved in this relaxation. In diabetic rats, resveratrol induced NO-independent relaxation and maragtoxinsensitive K channels are involved.

Effect of Wine Production Techniques on Wine Resveratrol and Total Phenolics

Abstract Resveratrol and phenolics are mainly responsible for the beneficial effects of wine consumption on human health. Their amount in wine depends on various factors such as grape variety, environmental factors, and extraction parameters. The winemaking process is one among these factors that can be controlled, and mainly includes a range of modifications during fermentation and maceration. An overview of available observations concerning this topic is presented in this chapter. A trend towards increasing concentrations of resveratrol and total phenolics is observed for thermovinification, increased fermentation temperature, must freezing, and extended maceration during the winemaking process. For some factors, e.g., yeast selection, juice run-off, or cold-soak treatments, consistent data were not obtained. Considering the importance of these natural compounds and their potential health benefits, understanding the factors influencing their preservation is essential for final product quality.

Improving the low solubility of resveratrol

Background Resveratrol, a polyphenol mainly present in grapes and red wine, demonstrated interesting biomedical properties for its cardioprotective action due to inhibition of the oxidation of low-density lipoprotein (LDL) and of platelet aggregation, inhibitory effects on cancer promotion and propagation and anti-inflammatory activities. These potential therapeutic and prophylactic applications are limited by the low bioavailability caused by its physical properties. Additionally, resveratrol has low water solubility and stability making its clinical success a formidable technological and medical challenge. The aim of this work is to present results of improvement of solubility of resveratrol through micellar and liposomal incorporation.

Comparison of Solubilization Capacity of Resveratrol in Sodium 3α,12α-Dihydroxy-7-oxo-5β-cholanoate and Sodium Dodecyl Sulfate

In this study we investigated resveratrol (trans-3,5,4′-trihydroxystilbene) solubilization with sodium 3α,12α-dihydroxy-7-oxo-5β-cholanoate (S7-OD) and sodium dodecyl sulfate (SDS). The investigation was aimed at determining whether large spherical micelles (SDS) or small longitudinal micelles (S7-OD) are more convenient for incorporation of resveratrol. Also, we studied resveratrol behavior in mixed micelles with mentioned surfactants using spectroflourimetric method as well as the effects of sodium chloride and urea on resveratrol solubilization capacity in the applied surfactants. Resveratrol solubilization curve was different in the investigated surfactants. Resveratrol solubilization curve for sodium 3α,12α-dihydroxy-7-oxo-5β-cholanoate at concentration 0.9 CMC reached saturation level of 60% dissolved resveratrol. The curve for sodium dodecyl sulfate was linear within the whole range of the investigated concentration; resveratrol solubilization rate reached 13% at 2 CMC. In S7-OD, NaCl increased capacity of resveratrol solubilization up to 1.4 CMC surfactant concentration, whilst maximum level of dissolved resveratrol (90%) was observed at 0.9 CMC. In SDS, NaCl decreased resveratrol solubilization capacity. Urea reduced resveratrol solubilization rate in sodium 3α,12α-dihydroxy-7-oxo-5β-cholanoate, whereas it had inverse effect in sodium dodecyl sulfate. The obtained results strongly suggest that structure, that is, shape, of the surfactant micelles significantly affects their capacity of resveratrol solubilization. Also, presence of NaCl and urea influences solubilization capacities of investigated surfactants.