GC-MS analysis of volatiles from different Lycopodium species with acetylcholinesterase activity

Searching for natural inhibitors of different enzymes, e.g. acetylcholinesterase (AChE), is a modern approach in drug discovery. Lycopodium L. (Lycopodiaceae) species containing alkaloids appear to be a rich source of acetylcholinesterase inhibitors.                In our study, a GC-MS analysis of the extracts of L. clavatum, L. annotinum, and Huperzia serrata (syn. Lycopodium serratum Thunb.), containing volatile constituents with AChE-inhibitory activity, was performed. The detection of AChE-inhibitory activity of the investigated extracts was achieved by TLC-bioautography.                The GC-MS analysis was carried out on a Shimadzu GC-2010 Plus chromatograph coupled to a QP2010 Ultra mass spectrometric detector using a Phenomenex capillary column ZB-5MS (30 m; diameter of 0.25 mm and thickness of 0.25 µm). The initial column temperature was set to 50 °C, which was held for 3 min, and in the next step, the column was heated to 250 °C at a rate of 8 °C per min (and was then held at that temperature for 2 min). The split ratio after the injection of 1 µL was 1:20 and helium was used as the carrier gas (the flow rate was 1 mL per min). Ionization was performed by electron impact at 70 eV. The identification of compounds was based on a comparison of their mass spectra with those of MassFinder and NIST mass spectral libraries, as well as by retention index (calculated based on a homologous series of n-alkanes) comparison.                The GC-MS analysis allowed the identification of alkaloids belonging to the main classes of alkaloids characteristic for Lycopodium spp., e.g. the lycopodane group

Antioxidant activity of lemongrass essential oil and its constituents

Plants and their ingredients (essential oil, flavonoids, etc.) are considered to be functional food if specific health benefits are attained by their consumption. These benefits include mostly preventive and protective but sometimes curative properties against one or more diseases, usually the so-called civilization diseases (different types of cancer, cardiovascular and neurodegenerative diseases) [1]. The essential oil (EO) obtained from Cymbopogon citratus (DC.) Stapf. (Poaceae) is a perfect functional food ingredient. Its characteristic, pleasant and refreshing odor goes hand in hand with its antiinflammatory, diuretic and sedative activities, due to which the plant was used in folk medicine for the treatment of nervous and gastrointestinal illnesses [2].                The aim of this work was an assessment of the antioxidant activity of lemongrass essential oil and its constituents. EO was hydrodistilled from the leaves and also the stems of C. citratus grown in Poland, while its chemical composition was analyzed by GC-MS. Both essential oils had a very similar chemical composition, with the exception of the presence of a sesquiterpene alcohol, elemol, in the stems oil, as well as a different antioxidant activity, which was higher for the EO obtained from the stems. The next stage of our research work was the isolation of the major components by the use of high-performance counter-current chromatography (HPCCC). The isolated compounds, neral, geranial, and elemol, as well as citronellol and citronellal, were subjected to antioxidant activity testing by TLC-bioautography using DPPH as the detection reagent, as well as by the CUPRAC method. However, the performed studies did not confirm the antioxidant activity of the major components alone present in the lemongrass essential oil. In total, neral, geranial, and elemol constituted 84.2% of all components present in the EO from C. citratus stems but they were not responsible for the observed antioxidant effect. It can be concluded that the antioxidant activity of lemongrass oil is the result of a synergistic effect of all ingredients present in this EO

Volatile constituents of several Seseli species with acetylcholinesterase activity

Apiaceae is a well-known family of plants with characteristic umbels comprising a lot of aromatic plants important from the economic, medical and ecological points of view. Species from the Seseli L. genus are used in traditional medicine since ancient times from their antimicrobial, antiinflammatory and analgesic activities. Plants belonging to the genus Seseli are also a rich source of coumarin compounds of different type, which show the ability to inhibit several important enzymes, e.g. acetylcholinesterase (inhibitors of AChE are used in Alzheimer’s disease treatment).                Extracts containing volatile compounds were prepared from several Seseli representatives (S. elatum subsp. osseum (Crantz) P.W.Ball, S. andronakii Woronow ex Schischk., S. hartvigii Parolly & Nordt, and S. petraeum M. Bieb.). AChE-inhibitory activity of the tested extracts from Seseli species was determined by TLC-bioautography on TLC plates (0.2-mm thickness) covered with silica gel. All TLC plates were developed with the optimized the mobile phase system containing an optimal concentration of 2-naphthyl acetate (30 mg/20 mL).                The GC-MS analysis was carried out on a Shimadzu GC-2010 Plus chromatograph coupled to a QP2010 Ultra mass spectrometric detector using a Phenomenex capillary column ZB-5MS (30 m; diameter of 0.25 mm and thickness of 0.25 µm). The initial column temperature was set to 50 °C, which was held for 3 min, and in the next step, the column was heated to 250 °C at a rate of 8 °C per min (and was then held at that temperature for 2 min). The split ratio after the injection of 1 µL was 1:20 and helium was used as the carrier gas (the flow rate was 1 mL per min). Ionization was performed by electron impact at 70 eV. The identification of compounds was based on a comparison of their mass spectra with those of MassFinder and NIST mass spectral libraries, as well as by retention index (calculated based on a homologous series of n-alkanes) comparison

Essential oils from the Herba and fruits of Peucedanum luxurians and their antituberculosis activity

The Apiaceae family has been accompanying people for thousands of years, being present in the kitchen, as well as in the pharmacy. Plants belonging to this family are well known as sources of coumarins and essential oils.                Essential oils from the Herba, as well as fruits, of Peucedanum luxurians Tamamsch. (an endemic umbelliferous plant taxon from Armenia) were obtained by hydrodistillation in a Deryng apparatus for the first time. The GC-MS analyses showed the presence of trans-β-farnesene (16%) and germacrene D (13%) as the most abundant components of the essential oils.                One of the most valuable properties of essential oils is their antimicrobial activity. It is a very desirable feature, especially in the case of some bacteria, which cause huge health problems. A good example is Mycobacterium tuberculosis, one of the leading causes of human morbidity and mortality.                The activity of essential oils from different parts of P. luxurians was tested for antituberculosis activity. Minimal Inhibitory Concentrations (MIC) values for the essential oils were determined by a 96-well microplate method with alamarBlue (Invitrogen). The inoculum of the reference strain of Mycobacterium tuberculosis H37Ra in Middlebrook 7H9 broth (Difco) was 5 x 105 cfu/mL per well, according to CLSI standards. Serial twofold dilutions of essential oils ranged from 8 to 256 µg/mL. As the internal control of the method, serial twofold dilutions of four first-line antibiotics dedicated to tuberculosis treatment: isoniazid (INH), rifampicin (RMP), ethambutol (EMB), and streptomycin (SM) were used [1,2]

Neuropsychopharmacological profiling of scoparone in mice.

Scoparone (6,7-dimethoxycoumarin) is a simple coumarin from botanical drugs of Artemisia species used in Traditional Chinese Medicine and Génépi liquor. However, its bioavailability to the brain and potential central effects remain unexplored. We profiled the neuropharmacological effects of scoparone upon acute and subchronic intraperitoneal administration (2.5-25 mg/kg) in Swiss mice and determined its brain concentrations and its effects on the endocannabinoid system (ECS) and related lipids using LC-ESI-MS/MS. Scoparone showed no effect in the forced swimming test (FST) but, administered acutely, led to a bell-shaped anxiogenic-like behavior in the elevated plus-maze test and bell-shaped procognitive effects in the passive avoidance test when given subchronically and acutely. Scoparone rapidly but moderately accumulated in the brain (Cmax < 15 min) with an apparent first-order elimination (95% eliminated at 1 h). Acute scoparone administration (5 mg/kg) significantly increased brain arachidonic acid, prostaglandins, and N-acylethanolamines (NAEs) in the FST. Conversely, subchronic scoparone treatment (2.5 mg/kg) decreased NAEs and increased 2-arachidonoylglycerol. Scoparone differentially impacted ECS lipid remodeling in the brain independent of serine hydrolase modulation. Overall, the unexpectedly potent central effects of scoparone observed in mice could have toxicopharmacological implications for humans

Bioactivity-guided isolation of trypanocidal coumarins and dihydro-pyranochromones from selected Apiaceae plant species.

Bioactivity-guided isolation of natural products from plant matrices is widely used in drug discovery. Here, this strategy was applied to identify trypanocidal coumarins effective against the parasite Trypanosoma cruzi, the etiologic agent of Chagas disease (American trypanosomiasis). Previously, phylogenetic relationships of trypanocidal activity revealed a coumarin-associated antichagasic hotspot in the Apiaceae. In continuation, a total of 35 ethyl acetate extracts of different Apiaceae species were profiled for selective cytotoxicity against T. cruzi epimastigotes over host CHO-K1 and RAW264.7 cells at 10 μg/mL. A flow cytometry-based T. cruzi trypomastigote cellular infection assay was employed to measure toxicity against the intracellular amastigote stage. Among the tested extracts, Seseli andronakii aerial parts, Portenschlagiella ramosissima and Angelica archangelica subsp. litoralis roots exhibited selective trypanocidal activity and were subjected to bioactivity-guided fractionation and isolation by countercurrent chromatography. The khellactone ester isosamidin isolated from the aerial parts of S. andronakii emerged as a selective trypanocidal molecule (selectivity index ∼9) and inhibited amastigote replication in CHO-K1 cells, though it was significantly less potent than benznidazole. The khellactone ester praeruptorin B and the linear dihydropyranochromones 3'-O-acetylhamaudol and ledebouriellol isolated from the roots of P. ramosissima were more potent and efficiently inhibited the intracellular amastigote replication at < 10 μM. The furanocoumarins imperatorin, isoimperatorin and phellopterin from A. archangelica inhibited T. cruzi replication in host cells only in combination, indicative of superadditive effects, while alloimperatorin was more active in fractions. Our study reports preliminary structure-activity relationships of trypanocidal coumarins and shows that pyranocoumarins and dihydropyranochromones are potential chemical scaffolds for antichagasic drug discovery

Pharmacometabolic Effects of Pteryxin and Valproate on Pentylenetetrazole-Induced Seizures in Zebrafish Reveal Vagus Nerve Stimulation.

Zebrafish (Danio rerio) assays provide a versatile pharmacological platform to test compounds on a wide range of behaviors in a whole organism. A major challenge lies in the lack of knowledge about the bioavailability and pharmacodynamic effects of bioactive compounds in this model organism. Here, we employed a combined methodology of LC-ESI-MS/MS analytics and targeted metabolomics with behavioral experiments to evaluate the anticonvulsant and potentially toxic effects of the angular dihydropyranocoumarin pteryxin (PTX) in comparison to the antiepileptic drug sodium valproate (VPN) in zebrafish larvae. PTX occurs in different Apiaceae plants traditionally used in Europe to treat epilepsy but has not been investigated so far. To compare potency and efficacy, the uptake of PTX and VPN into zebrafish larvae was quantified as larvae whole-body concentrations together with amino acids and neurotransmitters as proxy pharmacodynamic readout. The convulsant agent pentylenetetrazole (PTZ) acutely reduced the levels of most metabolites, including acetylcholine and serotonin. Conversely, PTX strongly reduced neutral essential amino acids in a LAT1 (SLCA5)-independent manner, but, similarly to VPN specifically increased the levels of serotonin, acetylcholine, and choline, but also ethanolamine. PTX dose and time-dependent manner inhibited PTZ-induced seizure-like movements resulting in a ~70% efficacy after 1 h at 20 µM (the equivalent of 4.28 ± 0.28 µg/g in larvae whole-body). VPN treated for 1 h with 5 mM (the equivalent of 18.17 ± 0.40 µg/g in larvae whole-body) showed a ~80% efficacy. Unexpectedly, PTX (1-20 µM) showed significantly higher bioavailability than VPN (0.1-5 mM) in immersed zebrafish larvae, possibly because VPN in the medium dissociated partially to the readily bioavailable valproic acid. The anticonvulsive effect of PTX was confirmed by local field potential (LFP) recordings. Noteworthy, both substances specifically increased and restored whole-body acetylcholine, choline, and serotonin levels in control and PTZ-treated zebrafish larvae, indicative of vagus nerve stimulation (VNS), which is an adjunctive therapeutic strategy to treat refractory epilepsy in humans. Our study demonstrates the utility of targeted metabolomics in zebrafish assays and shows that VPN and PTX pharmacologically act on the autonomous nervous system by activating parasympathetic neurotransmitters

Verbascum nigrum : cytotoxicity evaluation in A431 epidermoid carcinoma cells and untargeted LC‐HR‐MS/MS metabolite profiling

The crude methanolic extract obtained from Verbascum nigrum aerial parts (VNE) and its six fractions (VNF1-VNF6) were initially screened regarding their effects on the viability of immortalized HaCaT keratinocytes and A431 epidermoid carcinoma cells (MTT assay, 24 h). None of the tested samples affected the viability of HaCaT cells in a concentration range of 25-150 μg/mL. VNE and VNF4 exhibited significant cytotoxic effects in A431 cells, with IC50 values of 81.92 and 12.27 μg/mL, respectively; the selectivity index was higher than 10 for VNF4. The untargeted LC/HR-MS/MS metabolite profiling led to the tentative annotation of a total number of 23 compounds. Of these, VNE comprised mainly iridoid glycosides (harpagoside, laterioside, acylated aucubin derivatives), whereas VNF4 showed a high abundance of triterpene saponin glycosides (ilwensisaponins A and C, songarosaponins A and B), constituents known for their selective cytotoxic potential