Methodological developments in evaluation of antioxidant activity of complex mixtures : towards miniaturization and coupling of analytical methods

Antioxidant compounds in food play an important role as a health-protecting factor. Highly reactive free radicals and oxygen species are present in biological systems. Antioxidant compounds like phenolic acids, polyphenols and flavonoids scavenge free radicals such as peroxide, hydroperoxide or lipid peroxyl and thus inhibit the oxidative mechanisms that lead to degenerative diseases. There is a number of the free radical scavenging assays for evaluation of plant extracts. The main task of this work was to create rapid, simple and less material and time consuming method to separate analytes and evaluate antioxidant activity within a single analytical run. The on-line capillary electrophoresis-reaction (antioxidant activity) detection method can be used for a rapid evaluation of individual antioxidants in complex mixtures, particularly plant extracts. Combined UV (positive signals of all UV absorbing separated compounds) and DPPH (2,2-diphenyl-1-picrylhydrazyl) reaction detector (negative signals for colored DPPH radical bleaching) signal profiles are registered. Electropherograms of the methanolic extracts of wild hops forms cones as well as antioxidant activity detection profiles will be presentedBiochemijos katedraVytauto Didžiojo universiteta

Coupling of capillary electrophoresis with reaction detection for the on-line evaluation of radical scavenging activity of analytes

5th Symposium by Nordic Separation Science Society (NoSSS 2009); Tallinn Univ Technol, Tallinn, ESTONIA, AUG 26-29, 2009The main task of this work was to create a rapid, simple and less material and time consuming method involving capillary electrophoretic separation in order to separate analytes and evaluate antioxidant activity within a single analytical run. Several interfaces were developed and used to couple CE to the reaction detector. The method developed enables simultaneous electrophoretic separation and evaluation of antioxidant activity of each separated compound in the mixture. The analysis was performed using DPPH (2,2-diphenyl-1-picrylhydrazyl) as synthetic radical reagent. The on-line capillary electrophoresis-reaction (antioxidant activity) detection method can be used for a rapid evaluation of individual antioxidants in complex mixtures, particularly extracts of natural products. Possibility to evaluate radical scavenging activity of extracts of natural products components is demonstrated on an example of aqueous propolis extract. Four phenolic acids where separated and their radical scavenging activity was on-line evaluatedBiochemijos katedraVytauto Didžiojo universiteta

Coupling of capillary electrophoresis with reaction detection for on-line evaluation of antioxidant activity of analytes

Biochemijos katedraVytauto Didžiojo universiteta

Electrophoresis of hop cones extract and beer

Vytauto Didžiojo universiteta

Capiliary electrophoresis of hop extracts

Vytauto Didžiojo universiteta

Xanthohumol content in Lithuanian hop (Humulus lupulus L.) varieties

This project was financed by Research Council of Lithuania project No. 09.3.3-LMT-K-712-03-0133Hop (Humulus lupulus L.) is a dioecious perennial plant which is probably the most frequently used in brewery. However recently hops are analyzed for their potential application in pharmaceutical industry, because hops have various compounds which have antibacterial, anticancer, radical scavenging, peroxide reducing and antioxidant properties [1, 2]. Xanthohumol is a flavonoid, which is found only in hops, recently gained a particular interest of scientists, because it has shown strong anticancer and antioxidant effects. Xanthohumol is mostly found in the cones of hops, but literature data, shows that there is a lower amount of this compound in the leaves of hops [3]. The studies of biologically active compounds in leaves of hops would probably allow to find more applications of hop harvest as a big amount of hop leaves that are harvested each year are assumed to be almost useless. The composition of biologically active compounds depends on the part of the plant, location of cultivar, variety and phase of vegetation. The composition of volatile compounds [4] and concentration of bitter α-acids of Lithuanian hop varieties were analyzed previously [5]. The aim of this study was to evaluate and compare concentration of xanthohumol in leaves and cones of H. lupulus L. plant growing in Kaunas Botanical Garden of Vytautas Magnus University, Lithuania. To our knowledge, there is no data about xanthohumol content in Lithuanian hop varieties. The concentration of xanthohumol was analyzed using high performance liquid chromatography method under isocratic conditions [6]. Results were expressed as percent of xanthohumol in dry weight of plant material. The concentration of xanthohumol in the samples of cones varied between 0.1181–0.2136 % (RSD did not exceed 3.8 %) while the concentration in the leaves was up to 84 times lowerBiochemijos katedraBotanikos sodasGamtos mokslų fakultetasInstrumentinės analizės APCVytauto Didžiojo universiteta

Clustering analysis of different hop varieties according to their essential oil composition measured by GC/MS

This study describes the analysis of total hops essential oils from 18 cultivated varieties of hops, five of which were bred in Lithuania, and 7 wild hop forms using gas chromatography-mass spectrometry. The study sought to organise the samples of hops into clusters, according to 72 semi-volatile compounds, by applying a well-known method, k-means clustering analysis and to identify the origin of the Lithuanian hop varieties. The bouquet of the hops essential oil was composed of various esters, terpenes, hydrocarbons and ketones. Monoterpenes (mainly β-myrcene), sesquiterpenes (dominated by β-caryophyllene and α-humulene) and oxygenated sesquiterpenes (mainly caryophyllene oxide and humulene epoxide II) were the main compound groups detected in the samples tested. The above compounds, together with α-muurolene, were the only compounds found in all the samples. Qualitative and quantitative differences were observed in the composition of the essential oils of the hop varieties analysed. For successful and statistically significant clustering of the data obtained, expertise and skills in employing chemometric analysis methods are necessary. The result is also highly dependent on the set of samples (representativeness) used for segmentation into groups, the technique for pre-processing the data, the method selected for partitioning the samples according to the similarity measures chosen, etc. To achieve a large and representative data set for clustering analysis from a small number of measurements, numerical simulation was applied using the Monte Carlo method with normal and uniform distributions and several relative standard deviation values. The grouping was performed using the k-means clustering method, employing several optimal number of clusters evaluation techniques (Davies-Bouldin index, distortion function, etc.) and different data pre-processing approaches. [...]Biochemijos katedraBotanikos sodasInstrumentinės analizės APCKauno technologijos universitetasMatematikos ir informatikos institutasVytauto Didžiojo universiteta

Quencher procedure for analysis of biologically active compounds of hop (Humuluslupulus L.) leaves and cones

This project was financed by Research Council of Lithuania project No. 09.3.3-LMT-K-712-03-0133Hop (Humulus lupulus L.) is a plant, which is probably the best known for being used in brewery for bittering. Bitter a- and p-acids found in cones of hops have antibacterial and antkmdant properties. However, it is knovvn that hops contain other biologically active compounds (i.e. xanthohumoį 8-prenylnaringenin and others) that have anti-cancer, radical scavenging, peroxide reducing and antioxidant properties [1,2]. It is very likely, that biologically active compounds can be found in different proportions across the whole plant. It is also possible that various parts of hop plant contain biologically active compounds that are insoluble in some solvents. There is lack of information about biologically active compounds of the leaves of hops. The QUENCHER procedure, standing for QUick, Easy, New, CHEap and Reproducible, is the method, which relies on the surface reaction phenomenon between solid (bound or trapped antioxidant compounds) and dissolved materials [3]. The purpose of this study was to apply QUENCHER procedure for determination of phenolic compounds, flavonoid compounds and radical scavenging activity of hop leaves and cones as well as to compare the results with that obtained using extract of raw materiaį which prepared by conventional liąuid extraction. Five different Lithuanian hop varieties (Raudoniai, Fredos taurieji, Fredos derlingieji, Kauno ankstyvieji, Kauno gražieji) growing in Kaunas Botanical Garden of Vytautas Magnus University were analyzed. To our knowledge, it is the first study on the application of QUENCHER procedure for the analysis of phenolic compounds of different parts of hops. The totai amount of phenolic compounds, the amount of flavonoids and radical scavenging activity using DPPH (l,l-diphenyl-2-picrylhydrazyl) were determined using spectrophotometric methods [4]. Results were expressed as rutin eąuivalents mg/g of plant. [...]Biochemijos katedraBotanikos sodasGamtos mokslų fakultetasVytauto Didžiojo universiteta

Analysis of the Leaves and Cones of Lithuanian Hops (Humulus lupulus L.) Varieties by Chromatographic and Spectrophotometric Methods

This work involves a comprehensive chemical composition analysis of leaf and cone samples of Lithuanian hop varieties. This study aimed to determine the chemometric properties of the leaves and cones of five Lithuanian hop varieties. Determined properties were the following: (a) xanthohumol content, (b) phenolic compounds, (c) flavonoids, (d) radical scavenging activity, and (e) the qualitative composition of volatile compounds. The total content of phenolic compounds in aqueous 75% methanolic extracts varied between 31.4–78.2 mg of rutin equivalents (RE)/g, and the concentration of flavonoids was between 11.0–23.3 mg RE/g. Radical scavenging activity varied between 34.4–87.2 mg RE/g. A QUENCHER analysis procedure showed 91.7–168.5 mg RE/g of the total phenolic compound content, 12.7–21.4 mg RE/g of flavonoids, and 48.4–121.0 mg RE/g of radical scavenging activity. ‘Fredos taurieji’ and ‘Fredos derlingieji’ varieties have shown maximum values of phenolic compounds and radical scavenging activity both in leaf and cone suspensions. These varieties accumulated a higher amount of xanthohumol in leaves. The concentration of xanthohumol in the samples varied between 0.0014–0.2136% of dry mass, with the highest concentration in the cones of ‘Kauno gražieji’. We identified 19 volatile compounds in leaves, and in cones, we identified 32. In both of them, α-humulene and β caryophyllene dominated. ‘Raudoniai’ leaves were exceptional in their aroma due to dominating compound nagina ketone (Kovats index 1306). The QUENCHER procedure has shown a great potential for the unextractable residue of hop raw material. Further investigation and valorization of different hop biomass components, not only cones, are essential