Isolation, identification and evaluation of natural antioxidants from aromatic herbs cultivated in Lithuania

Oxidative spoilage of lipid-rich foods decreases their shelf-life and leads to undesirable chemical and physical changes. Nowadays natural antioxidants are generally preferred. The major part of industrially used antioxidants consists of radical scavengers, which inhibit the oxidative chain reaction by inactivating free radicals formed during peroxidation of lipids. Aromatic and medicinal herbs are rich sources of natural radical scavenging compounds. The research described in this thesis focuses on the evaluation of several aromatic plants grown in Lithuania as a possible source of food antioxidants. Various aspects of lipid oxidation, of antioxidative mechanisms and of natural sources of radical scavenging antioxidants are outlined in Chapter 1.Employment of model systems for antioxidant studies is often preferred over lipid stability tests due to their speed and the simplified comparison and interpretation of the obtained results. Preliminary screening of extracts from aromatic plants was carried out using theβ-carotene-linoleic acid model system (β-CLAMS). The method is based on a competition between the plant antioxidant and an oxidisable indicator (β-carotene) for free radicals, which are generated during temperature-accelerated oxidation of linoleic acid. The agar-diffusion and spectrophotometricβ-CLAMS tests singled out thyme ( Thymus vulgaris L.) and sage ( Salvia officinalis L.) as the most promising sources of natural antioxidant compounds (Chapter 2). The screening results also revealed that supercritical fluid extraction (SFE) as well as solvent extraction with acetone and methanol-water were the two most efficient techniques for isolation of antioxidant constituents from sage and thyme (Chapter 2). In further antioxidant activity tests, the spectrophotometricβ-CLAMS technique was adapted to microtiter plates, which gave a better reproducibility and a shorter analysis time. The modified and improved spectrophotometricβ-CLAMS confirmed the preliminary screening results. Sage and thyme acetone extracts again demonstrated the highest radical scavenging activity with relative antioxidant activities similar to those of 2,6-di- tert -butyl-4-methylphenol (BHT) (Chapter 3).The activity of primary antioxidants can be evaluated directly by monitoring the reduction of free radicals. Such experiments can be carried out off-line, i.e. by the introduction of previously isolated antioxidant compound(s) into an equilibrated free radical containing model system, or on-line, by adding a solution of a free radical to the eluate of an HPLC column. The on-line techniques allow for a rapid and selective detection of radical scavenging substances in the presence of many inactive constituents with a minimum of preparatory manipulations. Two on-line model systems based on the reduction of radical intermediates of the luminol chemiluminescence (CL) reaction and on the reduction of the stable 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH·) were used for the detection of antioxidant compounds in thyme and sage isolates. Ten compounds in the thyme acetone oleoresin and six in sage gave an inhibition signal on the CL detector (Chapter 4).An adequate HPLC separation and a sensitive detection of radical scavenging are two key-elements that determine the success of on-line experiments. Improved separation efficiency especially for polar analytes was observed after an acetonitrile/water gradient program was introduced and after lowering of the pH of the HPLC solvents (Chapter 5). A stable, pulse-free flow, appropriate instrumental set-up and optimised compositions of the CL and the DPPH·reagents improved the sensitivity of the on-line detection. The minimum detectable amounts registered in this study varied from 0.06 ng for rosmarinic acid to 2400 ng for carvacrol (HPLC-CL detection) and from 0.2 ng for eugenol to 19 ng for BHT (HPLC-DPPH·detection) (Chapter 5). It was found that the HPLC-CL system was on average more sensitive, however the HPLC-DPPH·method was more robust.The on-line HPLC-DPPH·method as well as TLC plates sprayed with DPPH·solution were used for the activity-guided isolation of radical scavenging compounds from a leaf extract of thyme. Nine active compounds were isolated from the methanolic and the acidic aqueous alcoholic extract by means of solvent partitioning, normal phase chromatography, size exclusion chromatography and reversed phase MPLC or HPLC (Chapter 6). Rosmarinic acid, eriodictyol, taxifolin, luteolin 7-glucuronide, p -cymene 2,3-diol and p -cymene 2,3-diol 6-6´-dimer were identified by a combination of UV, CD, mass spectrometry and 1H and 13C NMR. Two weakly active, volatile compounds, thymol and carvacrol, were identified by GC-MS and their structures confirmed by GC and TLC analysis with reference compounds. The structure of a highly active new phenylpropanoid trimer 3'-O-(8''-Z-caffeoyl)-rosmarinic acid was elucidated from its UV and CD spectra, FD mass spectrum, and 1H-, 13C-, COSY and direct and long range 1H-13C NMR spectra (Chapter 6). In off-line Trolox Equivalent Antioxidant Capacity (TEAC) and DPPH·assays, this compound was a weaker and stronger radical scavenger than rosmarinic acid, respectively.The presence in T. vulgaris of radical scavenging compounds of different polarity and with distinctive reactivity to different radicals offers the possibility to use extracts from this plant as natural antioxidants for various lipid-containing food systems.</p

Recent developments in the rapid analysis of plants and tracking their bioactive constituents

Natural products chemistry has witnessed many new developments in the last 5 years like extractions with subcritical water and ionic liquids, LC/HRMS and LC/SPE/cryo-NMR, UHPLC, TLC/MS, MS-based preparative HPLC, comprehensive chromatography (GC × GC, LC × LC), high-throughput screening, introduction of monolithic columns, miniaturisation, and automated structure identification. Nevertheless identifying bioactive constituents in complex plant extracts remains a tedious process. The classical approach of bioassay guided fractionation is time-consuming while off-line screening of extracts does not provide information on individual compounds and sometimes suffers from false positives or negatives. One way out of this is by coupling chromatography with chemical or biochemical assays, so called high resolution screening. An example is the development of HPLC on-line assays for antioxidants. By the post-column addition of a relatively stable coloured radical like DPPH¿ or ABTS¿+, radical scavengers are detected as negative peaks because in a reaction coil they reduce the model radical to its reduced, non-coloured form. When combined with LC/DAD/MS and LC/SPE/NMR, reliable identification of active constituents becomes possible without the necessity of ever isolating them in a classical sense. Also for finding leads for new drugs, combining HPLC with biochemical assays is interesting but technically more difficult. Most enzymes do not work at the organic modifier concentrations commonly encountered in RP-HPLC and the reaction time is often longer requiring dilution and lengthy coils respectively. Therefore, new techniques have to be implemented to gain the required sensitivity for on-line enzyme assays. For stable analytes, high temperature LC offers a solution to the organic modifier problem. When enzymes are highly expensive, like those used in the screening for Cytochrome P450 inhibitors, miniaturisation to chip format may offer a way out. Microreactors (chips) are not only useful for miniaturising larger assays but also offer completely new prospects in phytochemical analysis. One such application is in the sample clean-up of acids and bases like alkaloids. In a lay-out of three parallel channels of 100 ¿m width with the middle one containing organic phase and the two outer ones water of high pH (feed phase) and low pH (trapping phase) such a chip replaces two classical LLE steps but is much faster and requires less solvents and less manpower input

Actinobacterial Diversity in Volcanic Caves and Associated Geomicrobiological Interactions

16 páginas.-- 8 figuras.-- 2 tablas.-- 66 referencias.-- Material suplementario http://dx.doi.org/10.3389/fmicb.2015.01342Volcanic caves are filled with colorful microbial mats on the walls and ceilings. These volcanic caves are found worldwide, and studies are finding vast bacteria diversity within these caves. One group of bacteria that can be abundant in volcanic caves, as well as other caves, is Actinobacteria. As Actinobacteria are valued for their ability to produce a variety of secondary metabolites, rare and novel Actinobacteria are being sought in underexplored environments. The abundance of novel Actinobacteria in volcanic caves makes this environment an excellent location to study these bacteria. Scanning electron microscopy (SEM) from several volcanic caves worldwide revealed diversity in the morphologies present. Spores, coccoid, and filamentous cells, many with hair-like or knobby extensions, were some of the microbial structures observed within the microbial mat samples. In addition, the SEM study pointed out that these features figure prominently in both constructive and destructive mineral processes. To further investigate this diversity, we conducted both Sanger sequencing and 454 pyrosequencing of the Actinobacteria in volcanic caves from four locations, two islands in the Azores, Portugal, and Hawai'i and New Mexico, USA. This comparison represents one of the largest sequencing efforts of Actinobacteria in volcanic caves to date. The diversity was shown to be dominated by Actinomycetales, but also included several newly described orders, such as Euzebyales, and Gaiellales. Sixty-two percent of the clones from the four locations shared less than 97% similarity to known sequences, and nearly 71% of the clones were singletons, supporting the commonly held belief that volcanic caves are an untapped resource for novel and rare Actinobacteria. The amplicon libraries depicted a wider view of the microbial diversity in Azorean volcanic caves revealing three additional orders, Rubrobacterales, Solirubrobacterales, and Coriobacteriales. Studies of microbial ecology in volcanic caves are still very limited. To rectify this deficiency, the results from our study help fill in the gaps in our knowledge of actinobacterial diversity and their potential roles in the volcanic cave ecosystems.The authors acknowledge the Spanish Ministry of Economy and Competitiveness (project CGL2013-41674-P) and FEDER Funds for financial support. AM acknowledges the support from the Marie Curie Intra-European Fellowship of the European Commission's 7th Framework Programme (PIEF-GA-2012-328689). CR was funded by the Regional Fund for Science and Technology and Pro-Emprego program of the Regional Government of the Azores, Portugal [M3.1.7/F/013/2011, M3.1.7/F/030/2011]. Her work was partly supported by National funds from the Foundation for Science and Technology of the Portuguese Government, [Understanding Underground Biodiversity: Studies in Azorean Lava Tubes (reference PTDC/AMB/70801/2006]. The authors would like to thank the TRU Innovation in Research Grant, TRU UREAP Fund, Western Economic Diversification Canada Fund, Kent Watson (assisted with the Helmcken Falls Cave sample collection), Derrick Horne (UBC BioImaging Facility for the SEM work). We acknowledged the Canadian Ministry of Forests, Lands, and Natural Resource Operations for Park Use Permit#102172. This work was also supported by the Cave Conservancy of the Virginias, the Graduate Research Allocation Committee at UNM Biology, UNM Biology Grove Scholarship, the Student Research Allocation Committee at UNM, the National Speleological Society, the New Mexico Space Grant Consortium, the New Mexico Alliance for Minority Participation Program, the New Mexico Geological Society, and Kenneth Ingham Consulting. We acknowledge support from the UNM Molecular Biology Facility, which is supported by NIH grant number P20GM103452. The authors also wish to thank Fernando Pereira, Ana Rita Varela, Pedro Correia, Berta Borges, and Guida Pires for help during field and lab work in the Azores. The authors gratefully acknowledge the photographic contributions of Kenneth Ingham and Pedro Cardoso and Michael Spilde (SEM images). The authors would like to thank Dr. Steven Van Wagoner (TRU) and Drs. Julian Davies and Vivian Miao (UBC) for their invaluable comments in manuscript preparation. We gratefully acknowledge the help and collecting permits granted by the staff of El Malpais National Monument and Hawai'i Volcanoes National Park (USA).Peer reviewe

The antioxidant activity of some curcuminoids and chalcones

The antioxidant properties of the synthetic compound (C1)–(C8), which comprised 7 curcuminoids and a chalcone, were evaluated by two complementary assays, DPPH and β-carotene/linoleic acid. It was found that, in general, the free radical scavenging ability of (C1)–(C8) was concentration-dependent. Compounds (C1) and (C4), which contained (4-OH) phenolic groups, were found to be highly potent antioxidants with higher antioxidant values than BHT suggesting that synthetic curcuminoids are more potent antioxidants than standard antioxidants like BHT. Using β-carotene-linoleic acid assay, only the water-soluble 2, 4,6-trihydroxyphenolic chalcone (C5) showed 85.2 % inhibition of the formation of conjugated dienes reflecting on its potent antioxidant activity

Potential use of deodorised water extracts: polyphenol-rich extract of Thymus pannonicus All. as a chemopreventive agent

Deodorised water extracts of aromatic plants are obtained as by-products of essential oil isolation and usually discarded as waste. However, phytochemical composition of these extracts encourages their further utilization as food additives or functional food ingredients. In this study we investigated phytochemical composition, antioxidant and in vivo antiproliferative activity of deodorised water extract of Thymus pannonicus All. (DWE). HPLC analysis revealed rosmarinic acid (RA) (71.11 +/- 1.54 mg/g) as the most abundant constituent of the extract, followed by salvianolic acid H (14.83 +/- 0.79 mg/g, calculated as RA). DWE exhibited pronounced antioxidant activity in vitro, in FRAP and DPPH tests (FRAP value: 7.41 mmol Fe/g and SC50: 3.80 mu g/g, respectively). Using the model of Ehrlich carcinoma cells in mice that were treated with DWE prior, at the time, and after tumour cells implantation, the tumour growth suppression and redox status of malignant cells (i.e., activities of antioxidant enzymes, level of glutathione and intensity of lipid peroxidation) were followed. DWE applied as pretreatment caused disturbance of antioxidant equilibrium as well as apoptosis/necrosis of up to 90% EAC cells. Results obtained in the present study revealed chemopreventive potential and possibility of T. pannonicus DWE usage. High content of RA and other phenolic compounds explains, at least in part, the observed effects

Evaluation and comparison of two improved techniques for the on-line detection of antioxidants in liquid chromatography eluates

Two methods for the on-line detection in HPLC eluates of analytes possessing radical scavenging activity were improved and compared. The instrumental set-up of the method that is based on on-line inhibition of luminol chemiluminescence (CL) by antioxidants was improved using better quality syringe pumps, employing a diode array detector, and introducing a mixing/neutralisation coil and a pulse damper. Sensitivity of the HPLC-CL detection increased by a factor of 4. Post-column neutralisation of eluates improved compatibility of this detection method with acidified HPLC eluents. The second method, which is based on the post-column quenching of 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH.), was improved by readjusting composition and flow-rate of the reagent, mounting an additional pulse damper and detecting unreacted DPPH. with a detector equipped with a tungsten lamp. Purging of the DPPH. solution with He gas prior to analysis was introduced. This led to 30-fold better detection limits. The improved methods were compared with respect to limits of detection, the radical scavenging mechanism involved, compatibility with common HPLC solvents and pH range, and some technical aspects. The techniques described have high potential for the rapid identification of radical scavengers in complex samples like plant extracts. (C) 2001 Elsevier Science BN. All rights reserved