Phytochemical Composition and Antioxidant Activity of Various Extracts of Fibre Hemp (Cannabis sativa L.) Cultivated in Lithuania.

The phytochemistry of fibre hemp (Cannabis sativa L., cv. Futura 75 and Felina 32) cultivated in Lithuania was investigated. The soil characteristics (conductivity, pH and major elements) of the cultivation field were determined. The chemical composition of hemp extracts and essential oils (EOs) from different plant parts was determined by the HPLC/DAD/TOF and GC/MS techniques. Among the major constituents, ß-caryophyllene (≤46.64%) and its oxide (≤14.53%), α-pinene (≤20.25%) or α-humulene (≤11.48) were determined in EOs. Cannabidiol (CBD) was a predominant compound (≤64.56%) among the volatile constituents of the methanolic extracts of hemp leaves and inflorescences. Appreciable quantities of 2-monolinolein (11.31%), methyl eicosatetraenoate (9.70%) and γ-sitosterol (8.99%) were detected in hemp seed extracts. The octadecenyl ester of hexadecenoic acid (≤31.27%), friedelan-3-one (≤21.49%), dihydrobenzofuran (≤17.07%) and γ-sitosterol (14.03%) were major constituents of the methanolic extracts of hemp roots, collected during various growth stages. The CBD quantity was the highest in hemp flower extracts in pentane (32.73%). The amounts of cannabidiolic acid (CBDA) were up to 24.21% in hemp leaf extracts. The total content of tetrahydrocannabinol (THC) isomers was the highest in hemp flower pentane extracts (≤22.43%). The total phenolic content (TPC) varied from 187.9 to 924.7 (average means, mg/L of gallic acid equivalent (GAE)) in aqueous unshelled hemp seed and flower extracts, respectively. The TPC was determined to be up to 321.0 (mg/L GAE) in root extracts. The antioxidant activity (AA) of hemp extracts and Eos was tested by the spectrophotometric DPPH● scavenging activity method. The highest AA was recorded for hemp leaf EOs (from 15.034 to 35.036 mmol/L, TROLOX equivalent). In the case of roots, the highest AA (1.556 mmol/L, TROLOX) was found in the extracts of roots collected at the seed maturation stage. The electrochemical (cyclic and square wave voltammetry) assays correlated with the TPC. The hydrogen-peroxide-scavenging activity of extracts was independent of the TPC.

In Vitro Antioxidant and Prooxidant Activities of Red Raspberry (Rubus idaeus L.) Stem Extracts.

Leaves and stems of red raspberry (Rubus idaeus) are used in Lithuanian folk medicine. Healing properties of raspberry are related to the content of bioactive compounds, mainly polyphenols. Extracts of raspberry leaves contained higher total phenolic content (TPC) (1290 mg/L, expressed in gallic acid equivalent) compared to that in extracts of stems or peeled bark (up to 420 mg/L and 598 mg/L, respectively). To find out whether the collection time of herbal material was critical for the properties of the extracts, the stems were collected at different times of the year. TPC in the extracts depended more on extraction conditions rather than on the sampling time. Antioxidant activity of raspberry stem and bark extracts tested by spectrophotometric (DPPH● scavenging) and electrochemical (cyclic and differential pulse voltammetry) assays correlated with TPC. DPPH radical scavenging activity values for stem, leaf, and bark extracts were as follows: ≤1.18 ± 0.07, 1.63 ± 0.10, and ≤1.90 ± 0.04 (mmol/L, TROLOX equivalent), respectively. Assessed electrochemically, hydrogen peroxide-scavenging activity of extracts was independent on TPC. The latter activity was related to the presence of some protein in the extract as revealed by gel electrophoresis. Prooxidant activity of raspberry stem extracts was dependent on solution pH and temperature.

Antioxidant and Toxic Activity of Helichrysum arenarium (L.) Moench and Helichrysum italicum (Roth) G. Don Essential Oils and Extracts.

Helichrysum arenarium (L.) Moench (sandy everlasting) is the only species from genus Helichrysum Mill that grows spontaneously in Lithuania. The chemical composition of the essential oils (EOs) from inflorescences and leaves of H. arenarium wild plants was analysed by GC-MS. Palmitic (≤23.8%), myristic (≤14.9%) and lauric (6.1%) acids, n-nonanal (10.4%), and trans-ß-caryophyllene (≤6.5%) were the major constituents in the EOs. For comparison, the main components in EO from flowers (commercial herb material) of H.italicum were γ-curcumene (21.5%), ß-selinene (13.6%), α-selinene (8.1%), ß-eudesmol (8.3%), and α-pinene (6.5%). Composition of H. arenarium methanolic extracts was investigated by HPLC-DAD-TOF. The main compounds were the following: luteolin-7-O-glucoside, naringenin and its glucoside, apigenin, chlorogenic acid, arenol, and arzanol. Antioxidant activity of EOs and extracts was tested by DPPH● and ABTS●+ assays. Sandy everlasting extracts exhibited significantly higher radical scavenging activities (for leaves 11.18 to 19.13 and for inflorescences 1.96 to 6.13 mmol/L TROLOX equivalent) compared to those of all tested EOs (0.25 to 0.46 mmol/L TROLOX equivalent). Antioxidant activity, assayed electrochemically by cyclic and square wave voltammetry correlated with total polyphenolic content in extracts and radical scavenging properties of EOs and extracts. The toxic activity of EOs of both Helichrysum species was evaluated using a brine shrimp (Artemia salina) bioassay. H. italicum inflorescence EO was found to be toxic (LC50 = 15.99 µg/mL) as well as that of H. arenarium (LC50 ≤ 23.42 µg/mL) oils.

Features of iron accumulation at high concentration in pulcherrimin-producing Metschnikowia yeast biomass.

In previous studies it was found that the antimicrobial properties of pulcherrimin-producing Metschnikowia species are related to the formation of a red pigment-pulcherrimin and sequestration of free iron from their growth medium. For strains of Metschnikowia pulcherrima, M. sinensis, M. shaxiensis, and M. fructicola, at a high, ≈80 mg/kg, elemental Fe concentration in agar growth media we observed the essentially different (metal luster, non-glossy rust like, and colored) yeast biomass coatings. For the studied strains the optical and scanning electron microscopies showed the increased formation of chlamydospores that accumulate a red pigment-insoluble pulcherrimin rich in iron. The chlamydospore formation and decay depended on the iron concentration. In this study pulcherrimin in biomass of the selected Metschnikowia strains was detected by Mössbauer spectroscopy. At ≈80 mg/kg elemental Fe concentration the Mössbauer spectra of biomass of the studied strains were almost identical to these of purified pulcherrimin. Iron in pulcherrimin reached ≈1% of biomass by weight which is very high in comparison with elemental Fe percentage in growth medium and is not necessary for yeast growth. The pulcherrimin in biomass was also observed by Mössbauer spectroscopy at lower, ≈5 mg/kg, elemental Fe concentration. Through chemical binding of iron pulcherrimin sequestrates the soluble Fe in the growth media. However, at high Fe concentrations, the chemical and biochemical processes lead to the pulcherrimin accumulation in biomass chlamydospores. When soluble iron is sequestrated or removed from the growth media in this way, it becomes inaccessible for other microorganisms.

Mediated amperometry as a prospective method for the investigation of electroporation.

Pulsed electric field effects induced in a membrane, as well as intracellular structures, depend on cell type, field and media parameters. To achieve desired outcomes, membranes should be permeabilized in a controlled manner, and thus efficiency of electroporation should be investigated in advance. Here, we present a framework for using mediated amperometry as a prospective method for the investigation of electroporation and its effects on cellular machinery. Whole-cell sensors with single mediator systems comprised of hydrophilic or lipophilic mediators were successfully employed to investigate membrane permeability as well as cellular responses. Exposure of yeast cells to single electric field pulse (τ = 300 µs, E = 16 kV/cm) resulted in up to tenfold increase of current strength mediated with hydrophilic mediators. Exposure to PEF resulted in decrease of menadione mediated current strength (from 138 ± 15 to 32 ± 15 nA), which could be completely compensated by supplementing electrolyte with NADH.

In vitro inhibition of Saccharomyces cerevisiae growth by Metschnikowia spp. triggered by fast removal of iron via two ways.

Simple and convenient innovative assays in vitro demonstrating Metschnikowia spp. competition with Saccharomyces cerevisiae for an essential nutrient iron are presented. The tested Metschnikowia strains possess a common genetically determined property of secreting a pulcherriminic acid which in the presence of iron (III) ions forms an insoluble red pigment pulcherrimin. Both initial accumulation in growing Metschnikowia cells and subsequent precipitation in the form of pulcherrimin in the media contribute to iron removal by functioning cells. The predominant way depends on the strain. Due to fast elimination of iron, the growth of S. cerevisiae can be inhibited by tested Metschnikowia strains at concentrations of elemental iron in the media not exceeding 12 mg kg-1. Inhibition can be regulated by additional supply of microquantities of iron onto the surface of the solid medium within 20-24 h. At relatively low concentrations of elemental iron (below 1 mg kg-1), additional supplements of iron onto the surface provide an advancement in understanding the inhibition possibilities and enable the assay control. Microscopy observations revealed that Metschnikowia chlamydospores are involved in iron removal at relatively high iron concentrations. The results may find application in development of new methodologies and strategies for biocontrol or inhibition of pathogenic microorganisms.

Shell-isolated nanoparticle-enhanced Raman spectroscopy for characterization of living yeast cells.

Studying the biochemistry of yeast cells has enabled scientists to understand many essential cellular processes in human cells. Further development of biotechnological and medical progress requires revealing surface chemistry in living cells by using a non-destructive and molecular structure sensitive technique. In this study shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS) was applied for probing the molecular structure of Metschnikowia pulcherrima yeast cells. Important function of studied cells is the ability to eliminate iron from growth media by precipitating the insoluble pigment pulcherrimin. Comparative SERS and SHINERS analysis of the yeast cells in combination with bare Au and shell-isolated Au@SiO2 nanoparticles were performed. It was observed that additional bands, such as adenine ring-related vibrational modes appear due to interaction with bare Au nanoparticles; the registered spectra do not coincide with the spectra where Au@SiO2 nanoparticles were used. SHINERS spectra of M. pulcherrima were significantly enhanced comparing to the Raman spectra. Based on first-principles calculations and 830-nm excited Raman analysis of pulcherrimin, the SHINERS signatures of iron pigment in yeast cells were revealed. Being protected from direct interaction of metal with adsorbate, Au@SiO2 nanoparticles yield reproducible and reliable vibrational signatures of yeast cell wall constituents.

Toxic, Radical Scavenging, and Antifungal Activity of Rhododendron tomentosum H. Essential Oils.

The chemical composition of eight (seven shoot and one inflorescence) essential oils (EOs) of Rh. tomentosum H. plants growing in Eastern Lithuania is reported. The plant material was collected during different phases of vegetation (from April to October). The oils were obtained by hydrodistillation from air-dried aerial parts (leaves and inflorescences). In total, up to 70 compounds were identified by GC-MS and GC (flame-ionization detector, FID); they comprised 91.0 ± 4.7%-96.2 ± 3.1% of the oil content. Sesquiterpene hydrocarbons (54.1 ± 1.5%-76.1 ± 4.5%) were found to be the main fraction. The major compounds were palustrol (24.6 ± 2.6%-33.5 ± 4.4%) and ledol (18.0 ± 2.9%-29.0 ± 5.0%). Ascaridol isomers (7.0 ± 2.4%-14.0 ± 2.4% in three oils), myrcene (7.2 ± 0.3% and 10.1 ± 1.3%), lepalol (3.3 ± 0.3% and 7.9 ± 3.0%), and cyclocolorenone isomers (4.1 ± 2.5%) were determined as the third main constituents. The toxic activity of marsh rosemary inflorescence and shoot oils samples was evaluated using a brine shrimp (Artemia sp.) bioassay. LC50 average values (11.23-20.50 µg/mL) obtained after 24 h of exposure revealed that the oils were notably toxic. The oil obtained from shoots gathered in September during the seed-ripening stage and containing appreciable amounts of palustrol (26.0 ± 2.5%), ledol (21.5 ± 4.0%), and ascaridol (7.0 ± 2.4%) showed the highest toxic activity. Radical scavenging activity of Rh. tomentosum EOs depended on the plant vegetation stage. The highest activities were obtained for EOs isolated from young shoots collected in June (48.19 ± 0.1 and 19.89 ± 0.3 mmol/L TROLOX (6-hydroxy-2,5,7,8-tetra-methylchromane-2-carboxylic acid) equivalent obtained by, respectively, ABTS•+ (2,2'-amino-bis(ethylbenzothiazoline-6-sulfonic acid) diammonium salt) and DPPH•(2,2-diphenyl-1-picrylhydrazyl) assays). Agar disc diffusion assay against pathogenic yeast Candida parapsilosis revealed the potential antifungal activity of EOs. An alternative investigation of antifungal activity employed mediated amperometry at yeast Saccharomyces cerevisiae-modified electrodes. The subjection of yeast cells to vapors of EO resulted in a three to four-fold increase of electrode responses due to the disruption of yeast cell membranes.

Formation of self-organized periodic patterns around yeasts secreting a precursor of a red pigment.

Formation of self-organized regular patterns (Liesegang patterns) due to reaction-diffusion process in the gel medium and related to vital activity of yeasts is presented. Two different yeast strains (Candida pulcherrima and non-Candida pulcherrima) possess a common characteristic feature to secrete a precursor which in the presence of iron(III) ions forms an insoluble red pigment. During yeast cultivation onto solid agar media, periodic spontaneous distinctly spaced red-colored patterns around the yeasts can are formed if the concentration of elemental iron in the growth media is in the range 4-12mg/L. By changing the composition yeast growth media (YEPD or minimal), growth time and temperature, the mode of yeast inoculation, a variety of red-pigmented patterns around live and proliferating yeasts can be obtained.

Variability, toxicity, and antioxidant activity of Eupatorium cannabinum (hemp agrimony) essential oils.

CONTENT: Eupatorium cannabinum L. (Asteraceae) is as a potential source of biologically active compounds. The plant is used in traditional medicine for the treatment of diarrhea and livers diseases. OBJECTIVE: The present study provides investigation on pharmacological properties (antioxidant and toxic activities) of essential oils of E. cannabinum, collected from 11 wild populations in Lithuania. MATERIALS AND METHODS: Twenty-two hemp agrimony essential oil samples were prepared by hydrodistillation according to the European Pharmacopoeia, and their chemical composition was determined by GC-FID and GC-MS. Compositional data were subjected to principal components analysis (PCA). Instead of conventional spectrophotometric methods, cyclic voltammetry (CV) and square wave voltammetry (SWV) techniques were applied to determine antioxidant activity of hemp agrimony essential oils. Meanwhile, toxicity of the oils was determined using brine shrimp (Artemia sp.) assay. RESULTS: Chemical profiles of E. cannabinum oils were described according to the first predominant components: germacrene D (≤22.0%), neryl acetate (≤20.0%), spathulenol (≤27.2%), and α-terpinene (11.5%). For the first time, α-zingiberene (≤7.8%) was found to be among three major constituents (as the second one) for hemp agrimony oils. SWV measurements revealed that oxidation potentials of compounds present in the oils are lower (below 0.1 V) compared with that of well-known antioxidant quercetin (0.15 V). Toxicity tests evaluated that hemp agrimony oils containing predominant amounts of germacrene D and neryl acetate were notably toxic (LC50 value 16.3-22.0 µg/mL). CONCLUSION: The study provided some new data concerning chemical composition and pharmaceutical properties of E. cannabinum essential oils.

Mediated amperometry reveals different modes of yeast responses to sugars.

Menadione-mediated amperometry at carbon paste electrodes modified with various yeasts (Saccharomyces cerevisiae, Candida pulcherrima, Pichia guilliermondii and Debaryomyces hansenii) was employed to monitor redox activity inside the yeast cells induced by glucose, fructose, sucrose, maltose or galactose. Continuous measurements revealed distinct modes (transient or gradually increasing) of the current development during the first 2 to 3 min after subjection to glucose, fructose and sucrose at electrodes containing S. cerevisiae and non-Saccharomyces strains. Different modes (increasing or decreasing) of the current development after yeast subjection to galactose at electrodes with S. cerevisiae or D. hansenii and at electrodes with C. pulcherrima and P. guilliermondii suggested different mechanisms of galactose assimilation.

Compositional Variability and Toxic Activity of Mugwort (Artemisia vulgaris) Essential Oils.

The compositional variability of the essential oils of aerial parts of mugwort (Artemisia vulgaris L.), collected from fifteen wild populations in Lithuania is detailed. The most predominant components were davanones (13.8-45.5%, six oils), germacrene D (9.1-30.5%, four oils), 1,8-cineole (16.4%, one oil), camphor (18.9%, one oil), trans-thujone (8.9 and 10.9%, two oils) and cis-chrysanthenyl acetate (10.4%, one oil). To the best of our knowledge, the davanone chemotype for A. vulgaris oils is described for the first time. The toxicity of the mugwort essential oils was determined using brine shrimp (Anemia sp.) assay. LC0 values (10.3-23.1 µg/mL) obtained for the oils after 24 h of exposure revealed that the oils containing appreciable amounts of germacrene D, 1,8-cineole, camphor and davanone were notably toxic.