Vilma Kaškonienė

Evaluation of phytochemical composition of fresh and dried raw material of introduced Chamerion angustifolium L. using chromatographic, spectrophotometric and chemometric techniques

Due to the wide spectrum of biological activities, Chamerion angustifolium L. as medicinal plant is used for the production of food supplements. However, it should be kept in mind that quality (biological activity) of the herb depends on its geographic origin, the way of raw material preparation or extraction and chemotype. The purpose of this study was to evaluate and compare the compositions of volatile, non-volatile compounds and antioxidant activities of C. angustifolium grown in Kaunas Botanical Garden after the introduction from different locations in Lithuania. The compositions of fresh and air-dried samples were compared. The profile of volatile compounds was analyzed using headspace solid phase microextraction coupled with GC/MS. trans-2-Hexenal (16.0-55.9% of all volatiles) and trans-anethole (2.6-46.2%) were determined only in the dried samples, while cis-3-hexenol (17.5-68.6%) only in fresh samples. Caryophyllenes (α- and β-) were found in all analyzed samples, contributing together from 2.4% to 52.3% of all volatiles according to the origin and preparation (fresh or dried) of a sample. Total amount of phenolic compounds, total content of flavonoids and radical scavenging activity (using 2,2-diphenyl-1-picrylhydrazyl (DPPH)) were determined using spectrophotometric assays. The variation of total phenolic compounds content was dependent on the sample origin, moreover, drying reduced amount of phenolics 1.5-3.5 times. The DPPH free radical scavenging activity was in the range of 238.6-557.1mg/g (expressed in rutin equivalents) in the fresh samples and drastically reduced to 119.9-124.8 mg/g after drying. The qualitative analysis of phenolic compounds in the aqueous methanolic extracts of C. angustifolium was performed by means of HPLC with UV detection. Oenothein B and rutin were predominant in the samples; also caffeic and chlorogenic acids, and quercetin were determined. Chemometric methods, namely principal component analysis, hierarchical cluster analysis and K-means clustering analysis, were applied for evaluation of the results. Chemometric analysis showed existence of different chemotypes of C. angustifolium L. and their relation to the geographic origin.

Fast screening of the main phenolic acids with antioxidant properties in common spices using on-line HPLC/UV/DPPH radical scavenging assay

Batch 1,1-diphenyl-2-picrylhydrazyl (DPPH˙) and on-line HPLC/UV/DPPH˙ radical scavenging assays were compared for the rapid screening of antioxidant properties of extracts of six Lamiaceae family spices, namely Origanum vulgare, Ocimum basilicum, Rosmarinus officinalis, Origanum majorana, Thymus vulgaris and Satureja hortensis. Generally, all spices showed good radical scavenging properties, which were in the following order: oregano > rosemary > savory ∼ thyme ∼ marjoram > basil. Rosmarinic acid and lithospermic acid B, as strong antioxidants, were identified according to their retention time, UV and mass spectra. Rosmarinic acid was found to be the main compound in all extracts and as it was measured by HPLC/UV/DPPH˙ it was responsible for more than 50% of the total extract radical scavenging capacity, except for oregano, when rosmarinic and lithospermic acid B were almost equally important radical scavengers. The highest amounts of rosmarinic and lithospermic acids were found in oregano extracts; lithospermic acid B was also found in reasonable concentrations in marjoram, savory and thyme extracts. Lithospermic acid B is reported as a constituent of marjoram, savory and thyme for the first time.

Volatile compounds composition and antioxidant activity of bee pollen collected in Lithuania

Characterization of volatile compounds composition and evaluation of antioxidant properties of bee pollen collected in Lithuania is presented in the paper, which is the first study on pollen of Lithuanian origin to our knowledge. Three polyfloral pollen samples collected by honey bees were analyzed. Characterization of volatile compounds was performed using solid phase microextraction (SPME) and GC-MS. Styrene was predominant in all samples contributing 19.6–27.0 mass %. Sample A distinguished by a high amount of limonene, 9.0 mass %, sample B differed from the rest by a high content of hexanal, 9.3 mass %, and nonanal, 12.3 mass %, while sample C showed the highest content of 1-tridecene, 43.3 mass %. Screening of antioxidant properties was carried out by spectrophotometric methods and liquid chromatography coupled with a post-column 1,1-diphenyl-2-picrylhydrazyl (DPPH) reaction detector and electrochemical detector. Total phenolic and flavonoid contents together with radical scavenging activity in the free radical (DPPH) model system were evaluated in the tested samples. Total phenolic content varied from 24.4 mg g−1 to 38.9 mg g−1, total flavonoid content was in the range of 7.3–10.0 mg g−1 and radical scavenging activity was found between 30.7 mg g−1 and 34.9 mg g−1, all data are expressed in rutin equivalents. To process the collected data statistically and classify the bee pollen samples to clusters according to their volatile composition and antioxidant activity, principal component analysis, hierarchical cluster analysis and non-linear discriminant analysis were applied.

Chemometric analysis of volatiles of propolis from different regions using static headspace GC-MS

Six samples of propolis were analyzed in the paper: a sample from Brazil, Estonia, China and three samples from different locations of Uruguay. Static headspace technique coupled with gas chromatography-mass spectrometry analysis has been applied for the determination of the characteristic volatile profile with the aim to differentiate the propolis from different regions. Monoterpenes (α- and β-pinenes) were predominant in all samples, except the sample from China. This sample separated itself by the alcohols 3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol, (40.33% and 11.57%, respectively) and ester 4-penten-1-yl acetate (9.04%). α-Pinene and β-pinene composed 64.59–77.56% of volatiles in Brazilian and Uruguayan propolis, and 29.43% in Estonian propolis. Brazilian propolis was distinguished by a high amount of β-methyl crotonaldehyde (10.11%), one of Uruguayan samples 3- by limonene (15.58%), and the Estonian sample — by eucalyptol (25.95%). Statistical investigation of the samples was made applying principal component, hierarchical cluster and K-Means cluster analyses. Various data pre-processing techniques were proposed and used to study and obtain the important volatile compounds contributed to the differentiation of the propolis samples from different regions to separate clusters.

Flavonoids of willow herb (Chamerion angustifolium (L.) Holub) and their radical scavenging activity during vegetation

PURPOSE Willow herb has been traditionally used in folk medicine and currently it is a potential raw material for production of phytopharmaceuticals. The aim of this work was to determine the highest amount of flavonoids and the highest radical scavenging activity of willow herb, which was collected in different vegetation phases (intensive growing, bud, massive blooming, ripening of fruits (seeds) and the end of vegetation) and in different parts of the plant (blooms, leaves and stems). MATERIAL/METHODS Raw material was collected at Kaunas Botanical garden of Vytautas Magnus University. Willow herb was extracted using methanol/water mixture (75/25 v/v, %). Methanolic extracts were purified using solid-phase extraction. For determination of the radical scavenging activity of compounds the HPLC system with the on-line post-column DPPH (1,1-diphenyl-2-picrylhydrazyl) radical reaction detection was used. RESULTS Five flavonoids were identified and their quantitative distribution and radical scavenging activity were evaluated. The highest total amount of flavonoids and radical scavenging activity were determined in willow herb collected during the massive blooming phase (11.12 ± 0.34 mg/g and 8.71 ± 0.29 mg/g, respectively). CONCLUSIONS The highest amount of flavonoids and radical scavenging activity was determined for raw material collected during the massive blooming phase. Evaluation of different parts of the plant during the massive blooming phase revealed that the highest amount of flavonoids and radical scavenging activity are characteristic for blooms of the plant.

Screening of antioxidant activity and volatile compounds composition of Chamerion angustifolium (L.) Holub ecotypes grown in Lithuania

Since biological activity of medicinal plants is dependent on cultivation area, climatic conditions, developmental stage, genetic modifications and other factors, it is important to study flora present in different growing sites and geographical zones. This study was focused on screening of antioxidant activity of C. angustifolium harvested in six different locations in Lithuania. The total contents of phenolic compounds, flavonoids and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity were evaluated by spectrophotometric methods. A correlation between radical scavenging activity and total phenolic compounds content was observed (correlation coefficient 0.98). HPLC with online post-column DPPH radical scavenging reaction detection was used for the separation of extracts. Oenothein B, rutin and one unidentified compound were predominant. Volatile compounds were analysed using solid-phase microextraction coupled with gas chromatography–mass spectrometry. Based on the analysis of volatiles, all samples were classified into two chemotypes: (I) with predominant α- and β-caryophyllenes and (II) with predominant anethole.

The impact of solid-state fermentation on bee pollen phenolic compounds and radical scavenging capacity

The aim of this study was to perform solid-state fermentation of bee collected pollen by lactic acid bacteria and evaluate the impact of fermentation to total phenolic compounds content, total flavonoid content and radical scavenging activity of fermented and non-fermented bee pollen. Natural fermentation of bee pollen in the hive, done by bees, results in well-known product—bee bread. This study was the first attempt to produce artificial “bee bread”. To our knowledge literature date about bee pollen artificial fermentation are scarce. Five different bee pollen samples were fermented with Lactobacillus rhamnosus GG and without bacteria. Obtained fermented samples were analyzed for total phenolic compound content, total flavonoid content and free radical (DPPH) scavenging capacity by spectrophotometric methods; phenolic profile was evaluated by HPLC. Fermentation revealed positive impact on the total flavonoid content (amount of flavonoids increased by 55–135%). The results were compared with the antioxidant activity of natural bee bread. Acquired data were processed with Matlab software carrying out calculations of various similarity measures between each pollen and fermented pollen sample and bee bread sample.

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 a-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. The hop samples analysed were separated into 3 and 5 clusters according to the data filtering scenario used. However, the targeted Lithuanian hop varieties were clustered identically in both cases and fell into the same group together with other cultivated hop varieties from Ukraine and Poland.

Analysis of antiproliferative effect of Chamerion angustifolium water extract and its fractions on several breast cancer cell lines

PURPOSE To evaluate the antiproliferative effect of the aerial part of Chamerion angustifolium (L.) Holub. (Onagraceae) extract and its fractions in vitro. This is the first study on the anti-proliferative effect of C. angustifolium on 3 distinct breast cancer cell lines. MATERIAL/METHODS Breast cancer cell lines MCF7, MDA-MB-468 and MDA-MB-231 were exposed to different concentrations of the water extract of C. angustifolium, where DPPH radical scavenging activity was 0.018-0.443mg/ml, expressed in rutin equivalents. Cell growth was analyzed after 24, 48 and 72h of incubation. Solid-phase extraction was applied for the fractionation of C. angustifolium water extract and MDA-MB-468 cell line growth was tested using different fractions. RESULTS The concentrations corresponding to radical scavenging activity of 0.117 and 0.266mg/ml caused MCF7 cells growth inhibition, while in the samples exposed to the highest concentration (0.355 and 0.443mg/ml) no proliferation was register, suggesting cell death. MDA-MB-468 cell analysis showed similar responses. MDA-MB-231 demonstrated cell growth inhibition following the exposure to all analyzed high extract doses (0.117-0.443mg/ml). MDA-MB-468 cells were selected to evaluate the effect of fractions. In the samples exposed to the fraction containing the highest amount (91%) of oenothein B, at the concentration of 0.117mg/ml a pronounced cell growth inhibition while at higher concentrations (0.266 and 0.443mg/ml) no cell proliferation was observed. CONCLUSIONS The consumption of C. angustifolium herb can be advantageous, alongside with conventional breast cancer treatment.

Optimization of a capillary zone electrophoresis-contactless conductivity detection method for the determination of nisin

Determination of natural preservatives using electrophoretic or chromatographic techniques in fermented milk products is a complex task due to the following reasons: (i) the concentrations of the analytes can be below the detection limits, (ii) complex matrix and comigrating/coeluting compounds in the sample can interfere with the analytes of the interest, (iii) low recovery of the analytes, and (iv) the necessity of complex sample preparation. The aim of this study was to apply capillary zone electrophoresis coupled with contactless conductivity detection for the separation and determination of nisin in fermented milk products. In this work, separation and determination of natural preservative–nisin in fermented milk products is described. Optimized conditions using capillary zone electrophoresis coupled with capacitance‐to‐digital technology based contactless conductivity detector and data conditioning, which filter the noise of the electropherogram adaptively to the peak migration time, allowed precise, accurate, sensitive (limit of quantification: 0.02 μg/mL), and most importantly requiring very minute sample preparation, determination of nisin. Sample preparation includes following steps: (i) extraction/dilution and (ii) centrifugation. This method was applied for the determination of nisin in real samples, i.e. fermented milk products. The values of different nisin forms were ranging from 0.056 ± 0.003 μg/mL to 9.307 ± 0.437 μg/g.