Valdas Jakštas

Comparative evaluation of post-column free radical scavenging and ferric reducing antioxidant power assays for screening of antioxidants in strawberries.

ABTS and FRAP post-column techniques evaluate the antioxidant characteristics of HPLC separated compounds with specific reagents. ABTS characterize their ability to scavenge free radicals by electron-donating antioxidants, resulting in the absorbance decrease of the chromophoric radical. FRAP - is based on the reduction of Fe(III)-tripyridyltriazine complex to Fe(II)-tripyridyltriazine at low pH by electron-donating antioxidants, resulting in an absorbance increase. Both post-column assays were evaluated and compared according to the following validation parameters: specificity, precision, limit of detection (LoD), limit of quantitation (LoQ) and linearity. ABTS and FRAP post-column assays were specific, repeatable and sensitive and thus can be used for the evaluation of antioxidant active compounds. Antioxidant active compounds were quantified according to TEAC for each assay and ABTS/FRAP ratio was derived. No previous records of antioxidative activity of leaves and fruits of strawberries (Fragaria viridis, Fragaria moschata) research have been found. The research results confirm the reliability of ABTS and FRAP post-column assays for screening of antioxidants in complex mixtures and the determination of radical scavenging and ferric reducing ability by their TEAC values.

Optimization and validation of post-column assay for screening of radical scavengers in herbal raw materials and herbal preparations

On-line method, which combines HPLC distribution and post-column reaction, was designed for the search of individual antioxidants. Optimization of the assay was performed evaluating optimal ABTS(+) radical cation concentration in the reactor, reaction time, impact of flow rate, reaction coil length. HPLC-ABTS assay validation in this work was performed by assessing reference antioxidant negative peak areas in radical scavenging chromatogram. Sample free radical scavenging activity is expressed as trolox equivalent antioxidant capacity (TEAC). Optimized and validated method was applied in detection of compounds possessing free radical scavenging ability in complex mixtures. Antioxidant compounds were studied in perilla (Perilla frutescens (L.) Britton var. crispa f. viridis) herbal raw material and its preparations. The HPLC-separated antioxidant compounds were identified using HPLC-photodiode array coupled to mass spectrometer, using a reference mass for determining accurate masses. Radical scavenging characteristics of rosmarinic acid, which is the dominant phenolic compound in medicinal herbal raw material of perilla and its preparations, were confirmed by the calculated TEAC values. Compounds responsible for antioxidant effect in herbal raw materials and herbal preparations were identified, evaluated and compared.

The quantitative analysis of biologically active compounds in Lithuanian honey

Different sorts of Lithuanian honey were analysed by quantitative determination of eight free phenolic acids and phenylpropanoid vanillin using high performance liquid chromatography (HPLC). Aqueous ethanol 60% was used as a solvent for preparation of honey samples. Amounts of phenolic acids honey solutions were in the range from 7.176 to 125.624mg/ml. Free phenolic acids and vanillin had been detected in all samples of honey, but their composition and amount depended on its botanic origin. p-Coumaric and ferulic acids proved to be the main components in the acacia and buckwheat honey. Raspberry and polyfloral forest honey contained the highest amounts of chlorogenic acid. Gallic acid is a principal phenolic acid in eucalyptus honey. The highest amount of vanillin was identified in buckwheat honey. The results of the analysis revealed that free phenolic acids and vanillin were detected in all honey samples analysed, and their range and amounts varied.

Antioxidant effects of Camellia sinensis L. extract in patients with type 2 diabetes

The prevalence of diabetes mellitus (DM) has dramatically increased in the past decade. Furthermore, increasing evidence from research shows that oxidative stress (OS) plays a crucial role in the pathogenesis of diabetes and in its complications. A search for ways to reduce oxidative damage has become the focus of interest for the majority of scientists. In this study, we determined the radical scavenging activity of single green tea constituents by using an on-line high performance liquid chromatography (HPLC)-2,2-diphenyl-1-picrylhydrazyl (DPPH) method and evaluated the antioxidant effects on type 2 diabetic patients by performing a double-blind, placebo-controlled study. Epigallocatechin gallate was identified as the most potent antioxidant, contributing approximately 50% of the total antioxidant capacity of green tea extract. We also found a statistically significant decrement of lipid peroxidation markers in patients treated with green tea extract after 9 months or after 18 months of follow-up. Overall, these findings are attractive for diabetic patients, helping them to keep a high level of performance and well-being, which ultimately may delay the time of disability and reduce mortality.

Effect of farming systems on the yield, quality parameters and sensory properties of conventionally and organically grown potato (Solanum tuberosum L.) tubers.

The objectives of this two-year research were to study the impact of two different farming types, conventional and organic, on the yield and sensory properties of five Lithuanian varieties of potato tuber. The parameters and properties examined were: phenolic acids; dry matter and starch content; and the spread and intensity of Phytophthora infestans growth. It was determined that potato yield fluctuates with the variety, but for conventional farming it is significantly (p<0.05) higher than that obtained by organic farming. The farming type has no significant effect (p>0.05) on the content of phenolic acids. No significant effect (p>0.05) of farming type on dry matter and starch content, or sensory properties was found. No significant relation (p>0.05) was found between the content of phenolic acids and P. infestans spread. The spread of P. infestans was faster and infection was heavier in organically grown potatoes.

Complexes of dextran sulfate and anthocyanins from Vaccinium myrtillus: Formation and stability.

To improve the stability and antioxidant activity of anthocyanins (ATC), complexes of dextran sulfate (DESU) and ATC extracted from Vaccinium myrtillus were formed during electrostatic interaction between sulfo groups of DESU and cationic moieties of ATC. At the optimal weight ratio DESU/ATC=0.4 g/g, the amount of ATC introduced into a complex depended on the total concentration of the reagents. About 1.7 g of ATC per g of DESU could be incorporated into a complex. The formation of DESU/ATC complexes was confirmed by HPLC and FT-IR spectroscopy. According to HPLC analysis, the amount of individual ATC incorporated into a complex varied from 73.7% in the case of malvidin-3-O-glucoside to 90.8% in the case of delphinidin-3-arabinoside.

Source of variation of isoflavone concentrations in perennial clover species.

Introduction: Clover has attracted considerable interest not only as a valuable livestock forage plant, but also as an important source of isoflavones. The current study was aimed to assess the variation of concentration of three isoflavones in clover species grown under a cool temperate climate environment in Lithuania. Materials and Methods: Isoflavone contents were quantified in the plant parts of 21 accessions belonging to five perennial species of genus Trifolium (T. pratense, T. repens, T. medium, T. rubens, and T. pannonicum). Daidzein, formononetin, and genistein concentrations were quantified in separate plant parts by reversed-phase high-performance liquid chromatography. The analyte extraction was performed from dried and ground leaves, stems, flowers, and roots. The procedure included acid hydrolysis of isoflavone glycosides to aglycones. Results: According to the averaged sum of the three isoflavones quantified in leaves-stems-flowers, the five clover species ranked as follows: T. medium (7.54-3.62-2.31 mg/g) >T. pratense> T. rubens> T. pannonicum> T. repens (0.191-0.204-0.171 mg/g). The contribution of individual compound to the total isoflavone content depended on the species, accession, and plant part. The major part of the isoflavones is concentrated in leaves or stems; however, there is a great variation also. Conclusion: There exists a large variation in the total as well as in individual concentration of isoflavones among the clover species and plant parts and within species. With regard to isoflavone concentration and variability within species, some accessions of T. medium and T. pratense can be considered a highly promising source of phytoestrogens.

Optimization of carvacrol, rosmarinic, oleanolic and ursolic acid extraction from oregano herbs (Origanum onites L., Origanum vulgare spp. hirtum and Origanum vulgare L.)

The aim of our study was to increase the extraction efficiency of carvacrol, rosmarinic, oleanolic and ursolic acid from the different species of oregano herbs (Origanum onites L., Origanum vulgare spp. hirtum and Origanum vulgare L.). Various extraction methods (ultrasound-assisted, heat-reflux, continuous stirring, maceration, percolation) and extraction conditions (different solvent, material:solvent ratio, extraction temperature, extraction time) were used, and the active substances were determined by HPLC. The lowest content of carvacrol, rosmarinic, oleanolic and ursolic acid was obtained by percolation. During heat-reflux extraction, the content of active substances depended on the solvent used: ethanol/non-aqueous solvent (glycerol or propylene glycol) mixture was more effective compared with ethanol alone. The results showed that for each species of oregano the most optimal extraction method should be selected to maximize the content of biologically active substances in the extracts.

Development of an RP‐HPLC Method for the Analysis of Phenolic Compounds in Achillea millefolium L.

Abstract Several major phenolic constituents present in yarrow (Achillea millefolium L. s.l.) were determined for a homogenized plant sample. In order to optimize the conditions for sample preparation of the botanical matrix two different solvent extraction methods (maceration and ultrasonic agitation) were assayed. The preliminary maceration studies were performed to determine the influence of extracting solvents on the recovery of phenolics by using a different concentration of aqueous ethanol (40–96%, v/v) as extractant. On the basis of these results, sonication extractions were carried out at different time intervals (5, 10, 20, 30, and 60 min) using optimum extractant concentration. Levels of seven constituents (chlorogenic acid, vicenin‐2, luteolin‐7‐O‐glucoside, rutin, apigenin‐7‐O‐glucoside, luteolin, and apigenin) were measured by means of high performance liquid chromatography (HPLC) with ultraviolet and photodiode array detection systems. The main validation steps of the HPLC method were evaluated to demonstrate its selectivity, linearity, and precision. In addition, the method was applied to characterization of the flavonoid and phenolcarbonic acid complex in different samples of yarrow.

Method Development for Determination of Anthocyanidin Content in Bilberry (Vaccinium myrtillus L) Fruits

Abstract The optimal conditions for blueberry fruits extraction were chosen for better sample preparation. The biggest amount of anthocyanins was found in methanolic extracts. Ultrasonic extraction was performed for comparison with the maceration method. Ten minutes of ultrasonic extraction has shown the best recovery of anthocyanins in all analysed samples. The optimization of acid hydrolysis conditions was determined as well. At various times and concentrations of HCl, the hydrolysis of anthocyanins was conducted. Five major anthocyanidins (delphinidin, cyanidin, petunidin, peonidin, and malvidin) were estimated by high performance liquid chromatography (HPLC) in bilberry fruit extracts. A simple HPLC method has been developed for the analysis of anthocyanidins in bilberry extracts. The main steps of HPLC method validation were estimated, respectively.