Saša Likić

Phenolic content and antioxidative capacity of green and white tea extracts depending on extraction conditions and the solvent used

The efficiencies of different solvents in the extraction of phenolics from bagged and loose leaves of white and green tea, after different extraction times, as well as the antioxidative capacity of the obtained extracts, were investigated. The developed HPLC method has the potential to separate and determinate 17 phenolics widely distributed in plants, but in investigated tea extracts only four catechins and traces of three flavonols and one flavone were separated and detected based on comparison with authentic standards. The extraction efficiency of phenolics depended strongly on the time of extraction and the solvents used. The extraction of catechins from green tea was significantly affected by the form (bagged or loose) of the tea, whereas this effect was shown not to be statistically significant for white tea. Green tea was a richer source of phenolics than was white tea. The extraction of phenolics from white tea by water could be accelerated by the addition of lemon juice. Aqueous ethanol (40%) was most effective in the prolonged extraction of catechins. The antioxidative capacity of the investigated tea extracts correlated with their phenolic content.

Antioxidant effects of flavonoid from Croatian Cystus incanus L. rich bee pollen.

Oxidant/antioxidant status, estrogenic/anti-estrogenic activity and gene expression profile were studied in mice fed with Cystus incanus L. (Cistaceae) reach bee pollen from location in Central Croatias Dalmatia coast and offshore islands. Seven phenolic compounds (out of 13 tested) in bee pollen sample were detected by high performance liquid chromatography (HPLC) analysis. Phenolics detected in C. incanus L. bee pollen belong to flavonol (pinocembrin), flavanols (quercetin, kaempferol, galangin, and isorhamnetin), flavones (chrysin) and phenylpropanoids (caffeic acid). Bee pollen as a food supplement (100mg/kgbw mixed with commercial food pellets) compared to control (commercial food pellets) modulated antioxidant enzymes (AOE) in the mice liver, brain and lysate of erythrocytes and reduced hepatic lipid peroxidation (LPO). Bee pollen induced 25% of anti-estrogenic properties while no estrogenic activity was found. Differential gene expression profile analyses after bee pollen enriched diet identify underexpressed gene Hspa9a, Tnfsf6 (liver) and down-regulated gene expression of Casp 1 and Cc121c (brain) which are important in the apoptosis pathway and chemotaxis. These results indicate that used bee pollen possess a noticable source of compounds with health protective potential and antioxidant activity.

Phenolic composition and antioxidant properties of some traditionally used medicinal plants affected by the extraction time and hydrolysis.

INTRODUCTION Polyphenolic phytochemicals in traditionally used medicinal plants act as powerful antioxidants, which aroused an increasing interest in their application in functional food development. OBJECTIVE The effect of extraction time (5 and 15 min) and hydrolysis on the qualitative and quantitative content of phenolic compounds and antioxidant capacity of six traditionally used medicinal plants (Melissa officinalis L., Thymus serpyllum L., Lavandula officinalis Miller, Rubus fruticosus L., Urtica dioica L., and Olea europea L.) were investigated. METHODOLOGY The content of total phenols, flavonoids, flavan-3-ols and tannins was determined using UV/Vis spectrophotometric methods, while individual phenolic acids, flavones and flavonols were separated and detected using HPLC analysis. Also, to obtain relevant data on the antioxidant capacity, two different assays, (2,2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical scavenging and ferric reducing/antioxidant power (FRAP) assays were used. RESULTS The extraction efficiency of phenolics, as well as the antioxidant capacity of plant extracts, was affected by both prolonged extraction and hydrolysis. The overall highest content of phenolic compounds was determined in hydrolyzed extract of blackberry leaves (2160 mg GAE/L), followed by the non-hydrolyzed extract of lemon balm obtained after 15 min of extraction (929.33 mg GAE/L). The above extracts also exhibited the highest antioxidant capacity, while extracts of olive leaves were characterized with the lowest content of phenolic compounds, as well as the lowest antioxidant capacity. The highest content of rosmarinic acid, as the most abundant phenolic compound, was determined in non-hydrolyzed extract of lemon balm, obtained after 15 min of extraction. Although the hydrolysis provided the highest content of polyphenolic compounds, longer extraction time (15 min) was more efficient to extract these bioactives than shorter extraction duration (5 min). CONCLUSION The distribution of detected phenolic compounds showed a wide variability with regard to their botanical origin. Examined medicinal plants showed to be a valuable supplement to a daily intake of bioactive compounds.

Antiestrogenic and antigenotoxic activity of bee pollen from Cystus incanus and Salix alba as evaluated by the yeast estrogen screen and the micronucleus assay in human lymphocytes.

The estrogenic/antiestrogenic activity and the genotoxicity/antigenotoxicity of bee pollen from Salix alba L. and Cystus incanus L. and its derivative extracts in yeast and human cells was investigated. All samples showed a marked inhibitory effect on the activity of the natural estrogen 17 beta-estradiol (higher than 90% for extracts 2) and failed to cause estrogenic activity and chromosome damage. At least one preparation from each species showed a marked antigenotoxic effect against the action of the anticancer drugs mytomicin C, bleomycin, and vincristine. Bee pollens from C. incanus and S. alba were found to be neither genotoxic nor estrogenic as well as effective estrogen inhibitors, and able to reduce the chromosome damage induced by the three cancer drugs used, thus supporting their use as a safe food supplement and future chemoprotective/chemopreventive agents.

Relations between western Balkan endemic Campanula L. (Campanulaceae) lineages: Evidence from chloroplast DNA

Abstract The relationships among western Balkan and some amphi-Adriatic endemic campanulas were studied using RFLP analysis of the 1.8-kb trnT – trnF chloroplast DNA region. The results were compared to those of a comparative floral morphometry study performed earlier on a similar sample. Maximum parsimony and distance methods produced very similar results to those of the floral morphometry, both of which point to a clear separation of the taxa into three main clades. The first isophyllous, or Garganicae series, clade comprised Campanula poscharskyana, C. portenschlagiana, C. garganica, C. reatina and two C. fenestrellata-subspecies. The second “isophylloid” clade consisted of C. pyramidalis agg., C. waldsteiniana agg. and C. carpatica. The third, heterophyllous clade was very uniform, comprising undistinguishable taxa of the C. rotundifolia complex and alpine endemic C. cochleariifolia. Interestingly the Ligurian stenoendemic C. isophylla was clearly separated from the members of the ser. Garganicae, C. reatina was the most distant taxon in the Garganicae clade, the controversial species Asyneuma pichleri was nested inside the Campanula core, while Campanula carpatica was closer to the “isophylloids” than to the sect. Rapunculus Boiss. In order to resolve the taxonomic uncertainty among the closely related heterophyllous taxa more refined molecular methods, e.g. AFLP or microsatellites, should be applied.

Separation of plant viral satellite double-stranded RNA using high-performance liquid chromatography

High-performance liquid chromatography was developed for further separation of double-stranded (ds) RNAs obtained by CF-11 cellulose chromatography from plants infected with satellite associated cucumber mosaic virus. Fractions separated by monolithic polymer column, especially applicable for nucleic acid analyses, were identified electrophoretically and confirmed with a polymerase chain reaction test. Once standardized, the method has revealed clear evidence of satellite presence without precipitation and electrophoresis. According to demonstrated sensitivity, its application in the preliminary diagnostics of field samples is also predictable. Principally, it can be used as a powerful preparative approach resulting in highly pure satellite dsRNA for further analyses.

Involvement of kaempferol in the defence response of virus infected Arabidopsis thaliana

The roles of several phenolic compounds in plant defence response have been extensively studied, yet little is known about the role of flavonoids in plant-virus interaction. Quantitative and qualitative changes of selected phenolics in Arabidopsis thaliana induced by Cucumber mosaic virus containing satellite RNA (CMVsat) infection were analysed accompanied by plant hormone, chalcone synthase and pathogenesis-related gene expression analysis. Lower leaves of infected plants had a lower concentration of total phenolics compared to control plants. The concentration of kaempferol in upper leaves of all infected plants was significantly lower compared to control plants, while the expression of the chalcone synthase gene in those leaves was in most cases upregulated. All infected plants had a higher concentration of indole-3-acetic acid in lower leaves, which was accompanied with a lower concentration of kaempferol in upper leaves. Our research demonstrates a correlation between kaempferol and indole-3-acetic acid in response to CMVsat infection in Arabidopsis. We demonstrated two different metabolic patterns in infected plants suggesting the activation of two different defence responses. We also propose kaempferol to be an important part of the auxin-dependent defence response which limits systemic movement of CMVsat and that this defence response is activated prior to the well-known salicylic acid dependent defence response. Further research on kaempferol and its role in Arabidopsis-CMVsat interaction will improve our understanding on the role of flavonoids in plant defence.

CHANGES IN PHENOLIC COMPOUNDS IN NICOTIANA SPECIES AS A RESPONSE TO WOUNDING AND VIRAL INFECTION

The quantitative and qualitative changes in phenolic compounds were compared in Nicotiana spp. plants that were either wounded or infected with Cucumber mosaic virus (CMV) containing satellite RNA. Significant changes in total phenolic (TP) content were analysed in N. megalosiphon, N. glutinosa and N. tabacum var. Samsun at four different time intervals. The highest decrease of TP was detected in wounded N. megalosiphon plants while the highest increase was detected in infected N. megalosiphon plants. Depending on treatment and time, a significant increase or decrease of caffeic acid, quercetin or kaempferol was detected in the investigated species. The strongest increase in quercetin concentration was observed in infected N. glutinosa. The largest number of significant changes of caffeic acid, quercetin and kaempferol concentration in the analysed time intervals was observed in infected N. megalosiphon plants. The defence response to wounding in all investigated Nicotiana species resulted in a significant decrease of detected phenolic content. The detected changes in phenolics in virus-infected plants were characteristic for each species, suggesting that each species activates a different defence response to viral infection. The observed changes of phenolics are obvious responses to wounding and viral infection, which could likely be involved in activating a distinct defence response to a specific stress.