Audrius Pukalskas

Antioxidant activity of horehound (Marrubium vulgare L.) grown in Lithuania

Acetone extracts (AE), deodorised acetone extracts (DAE), and deodorised water extracts (DWE) from leaves of horehound (Marrubium vulgare L. ) were tested for their antioxidant activity in rapeseed ( Brassica napus L.) oil at 80 °C. Well investigated antioxidants containing extracts (AE and DAE) of sage (Salvia officinalis L.) were used for comparison purposes. The effect of the extracts on the edible oil stability was assessed by measuring weight gain, peroxide value, and the UV absorption. The antioxidant activity of AE and DAE of horehound were comparable to the antioxidant activity of AE and DAE of sage. For both plants AE was shown to have better antioxidant properties than DAE. The volatile components of horehound were hydrodistilled and analysed by GC and GC/MS. Totally, 47 components were identified in the essential oil (EO), the main ones being (Z)- β-farnesene, βcaryophyllene, (E)-hex-2-enal, α-humulene, and germacrene D. Thirtynine constituents are reported in M. vulgare for the first time.

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.

Antioxidant properties, phenolic composition and potentiometric sensor array evaluation of commercial and new blueberry (Vaccinium corymbosum) and bog blueberry (Vaccinium uliginosum) genotypes.

Antioxidant properties of juices of newly bred and known blueberry (Vaccinium corymbosum) genotypes and wild bog blueberry (Vaccinium uliginosum) were evaluated by ABTS(+) scavenging capacity (RSC), ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC), total phenolic content (TPC) and total anthocyanin content (TAC) assays. TPC varied in the range of 0.85-2.81 mg gallic acid equiv./mL, RSC, FRAP and ORAC values were 6.38-20.9, 3.07-17.8 and 4.21-45.68 μmol Trolox equiv./g, respectively. New blueberry genotypes and bog blueberry demonstrated stronger antioxidant properties and TAC than other studied genotypes. The content of quinic (203-3614 μg/mL), chlorogenic (20.0-346.8 μg/mL) acids and rutin (0.00-26.88 μg/mL) measured by UPLC/ESI-QTOF-MS varied depending on the genotype. Juices were evaluated by electronic tongue; PCA score plot showed that the method discriminates different genotypes although some juice samples were located very closely and overlapping. Significant differences were observed between L(∗), a(∗), b(∗) colour parameters of some genotypes.

Antioxidant properties and polyphenolic compositions of fruits from different European cranberrybush (Viburnum opulus L.) genotypes

Antioxidant properties of fruit juices of six Viburnum opulus genotypes were evaluated by DPPH, ABTS(+) radical scavenging capacity (RSC), ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC) and Folin-Ciocalteu total phenolic content (TPC) assays. TPC varied in the range of 5.4-10.6 mg gallic acid equivalents/g, RSC (ABTS(+)), FRAP and ORAC values were 31.9-109.8, 32.3-61.8 and 141.6-260.4 μmol trolox equivalents/g, respectively. V. opulus var. sargentii fruit juice was a remarkably stronger antioxidant than the other five V. opulus genotypes. The content of chlorogenic acid (the main phenolic compound in berry juices) depending on plant cultivar varied in the range of 0.54-6.93 mg/ml. The RSC of individual constituents was measured by the on-line HPLC-UV-DPPH method: chlorogenic acid was the dominant radical scavenger in V. opulus P3 (74%), while epicatechin and catechin (the main antioxidants in V. opulus var. sargentii) contributed to 40% and 23% of the total RSC for the sargentii genotype. Nine constituents were identified in V. opulus juice by using ultra high performance liquid chromatography coupled to quadruple and time-of-flight mass spectrometers (UPLC-QTOF-MS). In general, the study demonstrated that V. opulus var. sargentii followed by V. opulus P3 and V. opulus var. americanum possessed the highest antioxidant capacity. The obtained results may assist in selecting the most valuable V. opulus genotypes for the production of fruits possessing strong antioxidant capacity and containing beneficial phenolic constituents.

Phytochemical and antioxidant profiles of leaves from different Sorbus L. species

Leaves of Sorbus L. have been used in various traditional medicine systems. Phenolic compounds determine the main pharmacological effects of Sorbus L. In this study, phytochemical and antioxidant profiles of Sorbus anglica, Sorbus aria, Sorbus arranensis, Sorbus aucuparia, Sorbus austriaca, Sorbus caucasica, Sorbus commixta, Sorbus discolor, Sorbus gracilis, Sorbus hostii, Sorbus semi-incisa and Sorbus tianschanica were determined. Twenty four constituents were identified in Sorbus L. species using ultra high performance liquid chromatography coupled to quadruple and time-of-flight mass spectrometers. Post-column FRAP assay identified compounds with reducing activity and revealed significantly greatest total antioxidant activity of 175.30 μmol TE/g DW, 169.20 μmol TE/g DW and 148.11 μmol TE/g DW in S. commixta, S. discolor and S. gracilis leaf samples, respectively, with neochlorogenic and chlorogenic acids being most significant contributors. Characteristic fingerprints of phytochemical and antioxidant profiles could be applied for the quality evaluation of various raw materials of Sorbus L. species.

Variations in antioxidant capacity and phenolics in leaf extracts isolated by different polarity solvents from seven blueberry (Vaccinium L.) genotypes at three phenological stages

Antioxidant potential of blueberry leaves of seven genotypes (Vaccinium corymbosum, V. angustifolium, and hybrids) harvested at three phenological stages from three different bushes was evaluated by total phenolic content (TPC), ABTS and DPPH radical scavenging capacity, ferric reducing antioxidant power, and oxygen radical absorbance capacity (ORAC). The leaves were consecutively extracted with hexane (He), acetone (Ac), and methanol/water (Me/W). The extracts isolated with higher polarity solvents were stronger antioxidants. Remarkable variations in antioxidant capacity were observed depending on harvesting stage, genotype, and individual bushes. TPC was in the ranges of 29–143 (He), 87–312 (Ac), 85–333 (Me/W) mg GAE/g, ORAC—2633–10170 (Ac), 1517–13302 (Me/W) μmol trolox eqv/g. In general Putte, Northland and Gila genotypes demonstrated higher antioxidant capacity than Bluecrop, Dixi, Gretha, and Northblue. Phytochemical composition of selected samples was screened by UPLC/MS2; six previously not reported in blueberries leaves compounds were identified. In conclusion, this study demonstrated that blueberry leaves are a good source of natural antioxidants and valuable polyphenolic compounds, which could find various applications.

Redox Properties of Novel Antioxidant 5,8-Dihydroxycoumarin: Implications for its Prooxidant Cytotoxicity

The aim of this work was to characterize the redox properties of the new antioxidant 5,8- dihydroxycoumarin (5,8-DHC), isolated from sweet grass (Hierochloë odorata L.), and to determine its impact on its cytotoxic action. Reversible electrochemical oxidation of 5,8- DHC at pH 7.0 was characterized by the midpoint potential (Ep/2) of 0.23 V vs. the normal hydrogen electrode. 5,8-DHC was slowly autoxidized at pH 7.0, and it was active as a substrate for peroxidase (POD, EC 1.11.1.7) and tyrosinase (TYR, EC 1.14.18.1). Oxidation of 5,8-DHC by POD/H2O2 yielded the product(s) which reacted with reduced glutathione and supported the oxidation of NADPH by ferredoxin:NADP+ reductase (FNR, EC 1.18.1.2) and NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2). The concentration of 5,8-DHC for 50% survival of bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) during a 24-h incubation was (60 ± 5.5) μm. Cytotoxicity of 5,8-DHC was decreased by desferrioxamine, catalase, the antioxidant N,N’-diphenyl-p-phenylene diamine, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea and dicumarol, an inhibitor of NQO1. This shows that 5,8-DHC possesses the oxidative stress-type cytotoxicity, evidently due to the action of quinodal oxidation product(s). The protective effect of isoniazide, an inhibitor of cytochrome P-450 2E1, points to hydroxylation of 5,8-DHC as additional toxification route, whereas the potentiating effect of 3,5-dinitrocatechol, an inhibitor of catechol-omethyltransferase (COMT, EC 2.1.1.6), points to the o-methylation of hydroxylation products as the detoxification route.

Variety-based research on the phenolic content in the aerial parts of organically and conventionally grown buckwheat.

The aim of this study was to evaluate the impact of different farming types-organic and conventional-on phenolic content in buckwheat varieties grown in Lithuania. Rutin was identified as the dominant phenolic compound in contrast to both phenolic acids (chlorogenic and neochlorogenic acids) and other flavonoids (quercetin and quercitrin). It was determined that variety had the highest impact (p<0.05) on the phenolic content of various aerial parts of buckwheat. In most cases, farming practice significantly (p<0.05) affected the accumulation of phenolics in buckwheat. Organically grown plants usually contained higher amounts of phenolics than those grown under conventional farming conditions. According to a cluster analysis, varieties Panda, Zaleika, and VB Nojai were found to accumulate the highest amounts of phenolics.

Evaluation of the biological activity of naturally occurring 5,8-dihydroxycoumarin.

5,8-Dihydroxycoumarin (5,8-DHC) was isolated from aerial parts of sweet grass (Hierochloë odorata L.) and screened for antioxidant and genotoxic activities. A clear linear dependency of radical scavenging capacity in DPPH• and ABTS•+ assays was determined. 5,8-DHC was very efficient in retarding rapeseed oil oxidation (Oxipress test). TPC (total phenols content) and FRAP (the ability to reduce ferric ion to ferrous ion) assays revealed a somewhat lower antioxidant capacity of 5,8-DHC as compared with gallic acid. Genotoxic activity was tested using different genetic end-points: chromosome aberrations (CAs) and micronuclei (MN) in Wistar rat bone marrow in vivo, CAs and sister chromatid exchanges (SCEs) in human lymphocytes in vitro, and somatic mutations and recombination in Drosophila melanogaster wing cells in vivo. 5,8-DHC did not increase frequency of CAs in rat bone marrow cells, but induced a significant increase of MN. It was slightly mutagenic in the Drosophila melanogaster assay after 120 h of treatment, but not after 48 h of treatment. 5,8-DHC induced both CAs and SCEs in vitro in human lymphocytes in a clear dose-dependent manner. Thus, 5,8-DHC may be classified as weakly genotoxic both in vivo and in vitro.