Spent Material Extractives from Hemp Hydrodistillation as an Underexplored Source of Antimicrobial Cannabinoids.

Hemp (Cannabis sativa L.) has been used for millennia as a rich source of food and fibers, whereas hemp flowers have only recently gained an increased market interest due to the presence of cannabinoids and volatile terpenes. Currently, the hemp flower processing industry predominantly focuses on either cannabinoid or terpene extraction. In an attempt to maximize the valorization of hemp flowers, the current study aimed to evaluate the phytochemical composition and antimicrobial properties of several extracts obtained from post-distillation by-products (e.g., spent material, residual distillation water) in comparison to the essential oil and total extract obtained from unprocessed hemp flowers. A terpene analysis of the essential oil revealed 14 monoterpenes and 35 sesquiterpenes. The cannabinoid profiling of extracts showed seven acidic precursors and 14 neutral derivatives, with cannabidiol (CBD) reaching the highest concentration (up to 16 wt.%) in the spent material extract. The antimicrobial assessment of hemp EO, cannabinoid-containing extracts, and single compounds (i.e., CBD, cannabigerol, cannabinol, and cannabichromene) against a panel of 20 microbial strains demonstrated significant inhibitory activities against Gram-positive bacteria, Helicobacter pylori, and Trichophyton species. In conclusion, this work suggests promising opportunities to use cannabinoid-rich materials from hemp flower processing in functional foods, cosmetics, and pharmaceuticals with antimicrobial properties.

Phytochemical Profile and Biological Activity of the Ethanolic Extract from the Aerial Part of Crocus alatavicus Regel & Semen Growing Wildly in Southern Kazakhstan.

The composition of the ethanolic extract from the aerial parts of Crocus alatavicus Regel & Semen from southern Kazakhstan spontaneous flora was analyzed together with the determination of its antibacterial, antifungal, antiviral and anticancer activity. The phytochemical profile analysis by high-performance liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (HPLC/ESI-QTOF-MS) revealed the presence of multiple kaempferol derivatives. High-performance reverse-phase liquid chromatography combined with a photodiode-array detection (RP-HPLC/PDA) found that kaempferol 3-O-dihexoside and kaempferol 3-O-acyltetrahexoside accounted for 70.5% of the kaempferol derivatives. The minimum inhibitory concentration (MIC) values of the extract for all the tested reference microorganisms were high, reaching 10 mg/mL for yeasts and 20 mg/mL for bacteria. In contrast, antiviral activity was observed at 2 mg/mL, resulting in the inhibition of the HSV-1-induced cytopathic effect and the reduction in virus infectious titer by 1.96 log, as well as the viral load by 0.85 log. Among the tested prostate cancer cell lines, significant cytotoxic activity of the extract was noted only on the LNCaP cell line, with an IC50 value of 1.95 mg/mL. The LNCaP cell line treated with 2 mg/mL of the extract showed a noticeably reduced number of spindle-shaped cells with longer cellular projections, a significant increase in the peak corresponding to the population of apoptotic cells in the sub-G1 phase and a decreased intracellular glutathione (GSH) level, suggesting the prooxidative properties of the extract. The obtained data provide novel information about the flavonoids present in the aerial part of C. alatavicus and suggest its potential application as a source of the compounds active against HSV-1 and metastatic, androgen-sensitive prostate cancer.

Comparison of Phenolic Metabolites in Purified Extracts of Three Wild-Growing Herniaria L. Species and Their Antioxidant and Anti-Inflammatory Activities In Vitro.

The work is aimed at phytochemical characterization and In Vitro evaluation of antioxidant actions, anti-inflammatory effects, and cytotoxicity of purified extracts from three rupturewort (Herniaria L.) species, i.e., Herniaria glabra (HG), H. polygama (HP), and H. incana herb (HIh). The total phenolic content established in the purified extracts (PEs) of HIh, HP, and HG was 29.6, 24.0, and 13.0%, respectively. Thirty-eight non-saponin metabolites were identified using LC-HR-QTOF-ESI-MS; however, only 9 were common for the studied Herniaria species. The most abundant phenolic compound in HG-PE was narcissin (7.4%), HP-PE shared 3 major constituents, namely cis-2-hydroxy-4-methoxycinnamic acid 2-O-ß-glucoside (cis-GMCA, 5.8%), narcissin (5.4%), and rutin (5.3%). Almost half of HIh phenolic content (14.7%) belonged to oxytroflavoside A (7-O-methylkaempferol-3-O-[3-hydroxy-3-methylglutaryl-(1→6)]-[α-rhamnopyranosyl-(1→2)]-ß-galactopyranoside). Antioxidant properties of the Herniaria PEs were evaluated employing an experimental model of human blood plasma, exposed to the peroxynitrite-induced oxidative stress. The assays demonstrated significant reduction of oxidative damage to protein and lipid plasma components (estimated by measurements of 3-nitrotyrosine, protein thiol groups, thiobarbituric acid-reactive substances), and moderate protection of its non-enzymatic antioxidant capacity. Anti-inflammatory properties of the Herniaria PEs were evaluated In Vitro as inhibitory effects against cyclooxygenases (COX-1 and -2) and concanavalin A-induced inflammatory response of the peripheral blood mononuclear cells (PBMCs). None of the studied plants showed inhibitory effects on COXs but all purified extracts partly reduced the release of interleukin 2 (IL-2) and tumor necrosis factor-alpha (TNF-α) from PBMCs, which suggested their prospective ability to up-regulate inflammatory response of the cells. The purified extract from H. glabra turned out to be the most efficient suppressor of PBMCs' inflammatory response. Additionally, cytotoxicity of purified Herniaria extracts on PBMCs was ruled out based on In Vitro studies.

Fractionation of Lycopodiaceae Alkaloids and Evaluation of Their Anticholinesterase and Cytotoxic Activities.

In view of the abundant evidence that Lycopodiaceae alkaloids, including the well-known huperzine A (HupA), are among the potent acetylcholinesterase (AChE) inhibitors, an attempt was made to search for new compounds responsible for this property. For this purpose, three plant species belonging to the Lycopodiaceae family, commonly found in the Euro-Asia region, were subjected to the isolation of bioactive compounds, their identification and subsequent evaluation of their anticholinesterase and cytotoxic activities. Methanolic extracts of two Lycopodium and one Hupezia species were obtained via optimized pressurized liquid extraction (PLE) and then pre-purified using innovative gradient vacuum liquid chromatography (gVLC). For the first time, three sorbents of different porosity packed in polypropylene cartridges and mobile phase systems of different polarity were used to elute the target compounds. This technique proved to be a rapid tool for the obtainment of alkaloid fractions and allowed one to select the appropriate process conditions to yield potent AChE inhibitors in each of the species studied. More than 100 collected fractions were analyzed via HPLC/ESI-QTOF-MS, which enabled one to detect more than 50 compounds, including several new ones previously unreported. Some of them were present in high purity fractions (60-90% of the established purity). TLC bioautography assays proved that the analyzed species are rich sources of AChE inhibitors, but H. selago showed the highest anti-AChE activity. Additionally, the modified silanized silica gel sorbent used allowed one to isolate L. clavatum alkaloids more efficiently using an aqueous reversed-phase solvent system. Furthermore, the tested extracts from the three plant extracts were found to be safe, as they did not exhibit cytotoxicity to skin fibroblasts.

Phytochemical Fingerprinting and In Vitro Antimicrobial and Antioxidant Activity of the Aerial Parts of Thymus marschallianus Willd. and Thymus seravschanicus Klokov Growing Widely in Southern Kazakhstan.

The chemical composition of the hydroethanolic extracts (60% v/v) from the aerial parts of Thymus marschallianus Willd (TM) and Thymus seravschanicus Klokov (TS) from Southern Kazakhstan flora was analyzed together with their hexane fractions. Determination of antibacterial, antifungal and antioxidant activities of both extracts was also performed. RP-HPLC/PDA and HPLC/ESI-QTOF-MS showed that there were some differences between the composition of both extracts. The most characteristic components of TM were rosmarinic acid, protocatechuic acid, luteolin 7-O-glucoside, and apigenin 7-O-glucuronide, while protocatechuic acid, luteolin 7-O-glucoside, luteolin 7-O-glucuronide, and eriodictyol predominated in TS. The content of polyplenols was higher in TS than in TM. The GC-MS analysis of the volatile fraction of both examined extracts revealed the presence of thymol and carvacrol. Additionally, sesquiterpenoids, fatty acids, and their ethyl esters were found in TM, and fatty acid methyl esters in TS. The antioxidant activity of both extracts was similar. The antibacterial activity of TS extract was somewhat higher than TM, while antifungal activity was the same. TS extract was the most active against Helicobacter pylori ATCC 43504 with MIC (minimal inhibitory concentration) = 0.625 mg/mL, exerting a bactericidal effect. The obtained data provide novel information about the phytochemistry of both thyme species and suggest new potential application of TS as a source of bioactive compounds, especially with anti-H. pylori activity.

Optimization of Pressurized Liquid Extraction of Lycopodiaceae Alkaloids Obtained from Two Lycopodium Species.

Alkaloids of the Lycopodiaceae family are of great interest to researchers due to their numerous properties and wide applications in medicine. They play a very important role mainly due to their potent antioxidant, antidepressant effects and a reversible ability to inhibit acetylcholinesterase (AChE) enzyme activity. This property is of immense importance due to the growing problem of an increasing number of patients with neurodegenerative diseases in developed countries and a lack of effective and efficient treatment for them. Numerous studies have shown that Lycopodiaceae alkaloids are a rich source of AChE inhibitors. In the obtaining of new therapeutic phytochemicals from plant material, the extraction process and its efficiency is crucial. Therefore, the aim of this work was to optimize the conditions of modern PLE to obtain bioactive alkaloids from two Lycopodium species: L. clavatum L. and L. annotinum L. Five different solvents of different polarity were used for prepared plant extracts in order to compare the alkaloid content in and thereby effectiveness of the entire extraction. PLE parameters were used based on multiple studies conducted that gave the highest alkaloids recovery. Crude extracts were purified using solid-phase extraction (SPE) on Oasis HLB cartridge and examined by HPLC/ESI-QTOF-MS of the highly abundant alkaloids. To the best of our knowledge, this is the first time such high recoveries have been obtained for known Lycopodiaceae alkaloids. The best extraction results of alkaloid-lycopodine were detected in the dichloromethane extract from L. clavatum, where the yield exceeded 45%. The high recovery of annotinine above 40% presented in L. annotinum was noticed in dichloromethane and ethyl acetate extracts. Moreover, chromatograms were obtained with all isolated alkaloids and the best separation and quality of the bands in methanolic extracts. Interestingly, no alkaloid amounts were detected in cyclohexane extracts belonging to the non-polar solvent. These results could be helpful for understanding and optimizing the best conditions for isolating potent AChE inhibitors.

Phytochemical Profile and Biological Activities of the Extracts from Two Oenanthe Species (O. aquatica and O. silaifolia).

This study presents the evaluation of biological activities and chemical profiling of Oenanthe aquatica (L.) Poir. and Oenanthe silaifolia M. Bieb. The phytochemical profile, antioxidant, enzyme inhibitory, cytotoxic and antiviral activities of the methanolic and aqueous extracts were investigated. The aqueous extract of O. aquatica possessing the highest content of phenolics (60.85 mg gallic acid equivalent/g extract), also exhibited the strongest radical scavenging potential against 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (79.46 and 148.66 mg Trolox equivalent/g extract, respectively), the highest reducing ability (207.59 and 107.27 mg Trolox equivalent/g extract, for cupric reducing antioxidant capacity and ferric reducing antioxidant activity, respectively), metal chelating potential (33.91 mg ethylenediaminetetraacetic acid equivalent/g extract) and total antioxidant ability (1.60 mmol Trolox equivalent/g extract). Liquid chromatography-electrospray ionization-quadrupole time-of-flight-mass spectrometry (LC-ESI-QTOF-MS/MS) permitted tentative identification of compounds from simple organic acids, phenolic acids, coumarins, flavonoids and their glycosides in O. aquatica and O. silaifolia extracts. The methanolic extract of O. aquatica substantially depressed acetylcholinesterase (3.67 mg galantamine equivalent/g extract), tyrosinase (126.66 mg kojic acid equivalent/g extract), and α-amylase (0.83 mmol acarbose equivalent/g extract) enzymes. The methanolic extract of O. silaifolia showed highest enzymatic inhibitory property against butyrylcholinesterase, and its aqueous extract depressed α-glucosidase activity (0.26 mmol acarbose equivalent/g extract). All tested extracts exerted selective toxicity towards cancer cell lines, and the highest anticancer potential was found for O. aquatica aqueous extract on FaDu and HeLa cells with CC50 of 57.36 and 47.16 µg/mL, respectively. Significant antiviral activity against HSV-1 (HHV-1) was found for both aqueous extracts in concentrations of 1000 µg/mL, which inhibited the HSV-1 cytopathic effect (CPE) in virus infected VERO cells and reduced the virus infective titer by more than 3 log (logCCID50/mL). This study has produced critical scientific data on O. aquatica and O. silaifolia, which are potential contenders for the development of novel phyto-pharmaceuticals.

The Bioassay-Guided Fractionation and Identification of Potent Acetylcholinesterase Inhibitors from Narcissus c.v. 'Hawera' Using Optimized Vacuum Liquid Chromatography, High Resolution Mass Spectrometry and Bioautography.

Bioassay-guided isolation of bioactive compound is a modern and efficient technique in metabolites screening. It may shorten the total time of the entire process and reduce some costs of it. The aim of this paper was to fractionate and isolate alkaloids by developing an innovative vacuum liquid chromatography method for a species of Narcissus c.v. 'Hawera' rarely investigated so far and establishing the inhibitory activity of acetylcholinesterase (AChE). The studies consisted of the extraction of plant material by modern pressurized liquid extraction (PLE), followed by the isolation of alkaloidal fractions. For this purpose, the pioneering gradient vacuum liquid chromatography (gVLC) technique was employed by using two sorbents in various proportions packed in polypropylene cartridges for the first time. This step was performed in order to pre-clean the samples but also to establish the best combination of sorbents which permits obtaining potentially strong AChE inhibitors. The collected fractions were examined by HPLC/ESI-QTOF-MS in order to compare which combination of sorbents would allow us to obtain the highest concentration of alkaloids. The combination of these techniques confirmed the presence of the alkaloids and enabled the development of a modern method for the fractionation and isolation of the compounds with strong anti-AChE activity.

Determination of Cytisine and N-Methylcytisine from Selected Plant Extracts by High-Performance Liquid Chromatography and Comparison of Their Cytotoxic Activity.

Quinolizidine alkaloids exhibit various forms of biological activity. A lot of them were found in the Leguminosae family, including Laburnum and Genista. The aim of the study was the optimization of a chromatographic system for the analysis of cytisine and N-methylcytisine in various plant extracts as well as an investigation of the cytotoxic activities of selected alkaloids and plant extracts obtained from Laburnum anagyroides, Laburnum anagyroides L. quercifolium, Laburnum alpinum, Laburnum watereri, Genista germanica, and Genista tinctoria against various cancer cell lines. The determination of investigated compounds was performed by High Performance Liquid Chromatography with Diode Array Detection (HPLC-DAD), while High Performance Liquid Chromatography coupled with Quadrupole Time-of-Flight-Mass Spectrometry (HPLC-QTOF-MS) was applied for the qualitative analysis of plant extracts. The retention, separation selectivity, peaks shape, and systems efficiency obtained for cytisine and N-methylcytisine in different chromatographic systems were compared. The application of columns with alkylbonded and phenyl stationary phases led to a very weak retention of cytisine and N-methylcytisine, even when the mobile phases containing only 5% of organic modifiers were used. The strongest retention was observed when hydrophilic interaction chromatography (HILIC) or especially when ion exchange chromatography (IEC) were applied. The most optimal system in terms of alkaloid retention, peak shape, and system efficiency containing an strong cation exchange (SCX) stationary phase and a mobile phase consisted of 25% acetonitrile and formic buffer at pH 4.0 was applied for investigating alkaloids analysis in plant extracts. Cytotoxic properties of the investigated plant extracts as well as cytisine and N-methylcytisine were examined using human tongue squamous carcinoma cells (SCC-25), human pharyngeal squamous carcinoma cells (FaDu), human triple-negative breast adenocarcinoma cell line (MDA-MB-231), and human breast adenocarcinoma cell line (MCF-7). The highest cytotoxic activity against FaDu, MCF-7, and MDA-MB cancer cell lines was observed after applying the Genista germanica leaves extract. In contrast, the highest cytotoxic activity against SCC-25 cell line was obtained after treating with the seed extract of Laburnum watereri. The investigated plant extracts exhibit significant cytotoxicity against the tested human cancer cell lines and seem to be promising for further research on its anticancer activity.

Antimicrobial activity of Apis mellifera L. and Trigona sp. propolis from Nepal and its phytochemical analysis.

In this study, we evaluated antimicrobial activity, antimicrobial activity in combination with antibiotics, and chemical composition of Nepalese propolis 70% ethanolic extracts. Propolis originated from two genera of bees - Apis mellifera L. and Trigona sp. HPLC-DAD-MS/MS analyses revealed that the composition of both extracts was almost the same and the main components were flavonoid aglycones (mainly neoflavonoids, isoflavonoids) and pterocarpans. The highest antibacterial activity (disc diffusion test) was observed against Helicobacter pylori, Staphylococcus aureus and Shigella flexneri. Antibiotics exhibited synergism with Apis mellifera L. and Trigona sp. propolis against S. aureus and the strongest effect was observed for the combination with amikacin and tetracycline. Moreover, Nepalase propolis inhibited filamentation of C. albicans and caused oxidative stress by production of the superoxide anion radical (O2-) and a lower concentration of the hydroxyl radical (OH). Propolis extracts are potent antibacterial agents and may be used in combination with antibiotics.

Comparison of Various Chromatographic Systems for Identification of Vortioxetine in Bulk Drug Substance, Human Serum, Saliva, and Urine Samples by HPLC-DAD and LC-QTOF-MS.

Background: Determination of psychotropic drugs in clinical study is significant, and the establishment of methodologies for these drugs in biological matrices is essential for patients' safety. The search for new methods for their detection is one of the most important challenges of modern scientific research. The methods for analyzing of psychotropic drugs and their metabolites in different biological samples should be based on combining a very efficient separation technique including high-performance liquid chromatography (HPLC), with a sensitive detection method and effectively sample preparation methods. Objective: Retention, peaks symmetry and system efficiency of vortioxetine on Hydro RP, Polar RP, HILIC A (with silica stationary phase), HILIC-B (with aminopropyl stationary phase), and ACE HILIC-N (with polyhydroxy stationary phase and SCX columns were investigated. Various mobile phases containing methanol or acetonitrile as organic modifiers and different additives were also applied to obtained optimal retention, peaks shape, and systems efficiency. The best chromatographic procedure was used for simultaneous analysis of vortioxetine and its metabolites in human serum, urine and saliva samples. Methods: Analysis of vortioxetine was performed in various chromatographic systems: Reversed phase (RP) systems on alkylbonded or phenyl stationary phases, hydrophilic interaction liquid chromatography (HILIC), and ion-exchange chromatography (IEC). Based on the dependence of log k vs the concentration of the organic modifier, log kw values for vortioxetine in various chromatographic systems were determined and compared with calculated log P values. Solid phase extraction (SPE) method was applied for sample pre-treatment before HPLC analysis. HPLC-QTOF-MS method was applied for confirmation of presence of vortioxetine and some its metabolites in biological samples collected from psychiatric patient. Conclusions: Differences were observed in retention parameters with a change of the applied chromatographic system. The various properties of stationary phases resulted in differences in vortioxetine retention, systems' efficiency, and peaks' shape. Lipophilicity parameters were also determined using different HPLC conditions. The most optimal systems were chosen for the analysis of vortioxetine in biological samples. Both serum and urine or saliva samples collected from patients treated with vortioxetine can be used for the drug determination. For the first time, vortioxetine was detected in patient's saliva. Obtained results indicate on possibility of application of saliva samples, which collection are non-invasive and painless, for determination and therapeutic drug monitoring in patients.

Phytochemical composition of wormwood (Artemisia gmelinii) extracts in respect of their antimicrobial activity.

BACKGROUND: Extracts from medicinal plants with phytochemicals with known antimicrobial properties can be an effective adjunct in the complex treatment of infectious diseases. This study aimed to evaluate the antimicrobial activity of wormwood extracts collected in Kazakhstan (Artemisia gmelinii Weber ex Stechm.), along with their phytochemical analysis. METHODS: The ethanolic and chloroform extracts were subjected to HPLC combined with quadrupole time-of-flight mass spectrometry method. For quantitative assessment of antimicrobial activity, minimal inhibitory concentration (MIC) of the tested extracts was determined by micro-dilution broth method for the panel of the reference microorganisms. Minimal bactericidal concentration (MBC) or minimal fungicidal concentration (MFC) were also determined. RESULTS: LC/MS analysis showed the presence of 13 compounds in the tested extracts, including flavonoids: apigenin, luteolin, rutin, two O-methylated flavonols (isorhamnetin, rhamnazine), coumarin compounds (umbelliferone, scopoletin and scopolin (scopoletin 7-glucoside), 3-hydroxycoumarin and 4-hydroxycoumarin), chlorogenic acid and two dicaffeoylquinic acid isomers. Quantitative HPLC analysis showed that umbelliferone was dominant in the chloroform extract while chlorogenic acid was identified as a main compound in the ethanolic extract. The antibacterial and antifungal activity of chloroform and ethanolic extracts was comparable. The most sensitive were the Gram-positive bacteria represented by staphylococci, Micrococcus luteus and Bacillus spp. (MIC = 1.25-5 mg/ml) and yeasts represented by Candida spp. (MIC = 2.5-5 mg/ml), irrespective of the assayed extract. CONCLUSIONS: Extracts of wormwood Artemisia gmelinii have shown a wide spectrum of antibacterial and antifungal activity. Luteolin, rutin, isorhamnetin and scopolin were identified in A. gmelinii species for the first time. The determining of the most potential compounds of Artemisia gmelinii can be used to develop effective antibacterial and antifungal agents.

Determination of chemical stability of sitagliptin by LC-UV, LC-MS and FT-IR methods.

Sitagliptin was stored at high temperature/high humidity, dry hot air, UV/VIS light and different pH. Then, a selective LC-UV method was developed for determination of sitagliptin in the presence of degradation products and for estimation of degradation kinetics. Because parent drugs can react with excipients in final pharmaceutical formulations, stability of sitagliptin was also examined in the presence of excipients of different reactivity, using FT-IR and LC-UV methods. Finally, LC-MS method was used for identification of degradation products of sitagliptin. High degradation of sitagliptin, following the first order kinetics, was observed in strongly acidic, alkaline and oxidative media. The quickest degradation was found in 2 M HCl and 2 M NaOH. In addition, all excipients used in the present study, i.e. fumaric acid, lactose, mannitol and magnesium stearate showed potent interactions with sitagliptin. Some of these interactions were shown without any stress while others were accelerated by high temperature/high humidity and dry hot air, and less by UV/VIS light. Some mechanisms for the observed changes were proposed, i.e. the Michael addition in the presence of fumaric acid and the Maillard reaction in the presence of lactose. In addition, degradation of sitagliptin together with the occurrence of its impurities was stated in a broad range of stress conditions.

Kinetics and Characterization of Degradation Products of Dihydralazine and Hydrochlorothiazide in Binary Mixture by HPLC-UV, LC-DAD and LC-MS Methods.

Dihydralazine and hydrochlorothiazide were stored at high temperature and humidity, under UV/Vis light and different pH, as individual drugs and the mixture. Then, a sensitive and selective HPLC-UV method was developed for simultaneous determination of dihydralazine and hydrochlorothiazide in presence of their degradation products. Finally, the degradation products were characterized through LC-DAD and LC-MS methods. Dihydralazine was sensitive to high temperature and humidity, UV/Vis light and pH ≥ 7. At the same time, it was resistant to acidic conditions. Hydrochlorothiazide was sensitive to high temperature and humidity, UV/Vis light and changes in pH. Its highest level of degradation was observed in 1 M HCl. Degradation of the drugs was higher when they were stressed in the mixture. In the case of dihydralazine, the percentage degradation was 5-15 times higher. What is more, dihydralazine became sensitive to acidic conditions. Hydrochlorothiazide was shown to be more sensitive to UV/Vis light and pH > 4. Degradation of dihydralazine and hydrochlorothiazide followed first-order kinetics. The quickest degradation of dihydralazine was found to be in 1 M NaOH while of hydrochlorothiazide was in 1 M HCl (individual hydrochlorothiazide) or at pH 7-10 (hydrochlorothiazide in the mixture). A number of new degradation products were detected and some of them were identified by our LC-DAD and LC-MS methods. In the stressed individual samples, (phenylmethyl)hydrazine and 1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide were observed for the first time. Interactions between dihydralazine and hydrochlorothiazide in the mixture were confirmed by additional degradation products, e.g., 2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1,4-trioxide.

Anticancer effect of the water extract of a commercial Spirulina (Arthrospira platensis) product on the human lung cancer A549 cell line.

Spirulina is a well-described and popular dietary supplement derived from Arthrospira algae. In the present study, the anticancer potential of a water extract of a commercial Spirulina product (SE) against the human non-small-cell lung carcinoma A549 cell line was evaluated. After qualitative analysis, we investigated the effect of SE on cell viability, proliferation, and morphology. Furthermore, the influence of SE on regulation of the cell cycle, induction of apoptosis in lung cancer cells, and expression of cell cycle/apoptosis-related proteins was evaluated. Additionally, we examined the cytotoxic effect of SE on normal human skin fibroblasts (HSF). Our studies revealed that SE significantly reduced cancer cell viability and proliferation, which was accompanied by cell cycle inhibition in the G1 phase, induction of apoptosis, and prominent morphological changes. Moreover, we detected no cytotoxic effect of the tested Spirulina extract on normal skin fibroblasts. Our molecular studies demonstrated that SE reduced the phosphorylation of Akt and Rb proteins, reduced the expression of cyclin D1 and CDK4, and increased the Bax to Bcl-2 ratio in the A549 cells. In conclusion, the results obtained provide evidence of the anti-cancer activity of the commercial Spirulina product against lung cancer cells and strongly support the knowledge of the chemopreventive properties of Spirulina.

Study of a complex secondary metabolites with potent anti-radical activity by two dimensional TLC/HPLC coupled to electrospray ionization time-of-flight mass spectrometry and bioautography.

The high performance liquid chromatography (HPLC) coupled to electrospray ionization (ESI) octopole orthogonal acceleration time-of-flight (TOF) mass spectrometry (MS) was developed to separate secondary metabolites belonging to different groups of compounds with a wide range of polarity, i.e. phenolic acids, flavonoids, sesquiterpenes and others present in minute amounts, in a single run. Moreover, application of ESI-TOF-MS with a simple in source type of fragmentation and high mass accuracy measurements (around 2 ppm) in both positive and negative ionization modes with different and optimized fragmentor voltages, enabled the identification of each single compound based on proposed fragmentation patterns. In this way a total number of 14 compounds in methanolic and water-methanolic (1:1, v/v) extract from fruits of Eryngium amethystinum and 6 compounds in extract from fruits of E. planum have been identified for the first time. Additionally, two dimensional thin layer chromatography/high performance liquid chromatography/electrospray-ionization-time-of-flight mass spectrometry (TLC/HPLC/ESI-TOF-MS) system was developed for identification of compounds with the highest anti-radical activity revealed in TLC-DPPH bioauthographic test. This on-line procedure enables significant improvement in terms of efficiency and faster structural analysis of the potential antioxidant natural compounds directly in the plant extracts screened for bioactivity tests. This effect - directed analysis (EDA) method enables separation, identification and preliminary screening of plant extracts for anti-radical properties and can be easily adopted for other bioactivities.

Chemical stability and interactions in a new antihypertensive mixture containing indapamide and dihydralazine using FT-IR, HPLC and LC-MS methods.

Indapamide and dihydralazine can be combined in fixed-dose formulations because of their complementary actions against hypertension. On the other hand, combined formulations present the problem of chemical interactions between the active ingredients, e.g. accelerated degradation of constituents or generation of quite new degradation products. Therefore, the main goal of the present study was to examine the chemical stability of indapamide and dihydralazine, as individuals and as a mixture, to detect potent interactions between both constituents, using FT-IR, HPLC and LC-MS methods. It was clearly shown that both drugs degraded more when they were in the mixture, i.e. indapamide was degraded more under high temperature/high humidity while dihydralazine was more sensitive to UV/VIS light. In solutions, indapamide was sensitive to strong acidic and strong alkaline conditions while dihydralazine degraded at pH ≥ 7. Generally, the process of degradation of indapamide and dihydralazine followed first order kinetics. The fastest degradation of both indapamide and dihydralazine was found at pH ≥ 10. Several degradation products of indapamide and dihydralazine were detected and identified by our LC-MS method. Interactions between both drugs were confirmed by detection of new degradation products of indapamide, i.e. 4-chloro-3-sulfamoylbenzamide and 4-chloro-3-(formylsulfamoyl)-N-(2-methyl-2,3-dihydro-1H-indol-1-yl)benzamide, only in the presence of dihydralazine.

Correction: Chemical stability and interactions in a new antihypertensive mixture containing indapamide and dihydralazine using FT-IR, HPLC and LC-MS methods.

[This corrects the article DOI: 10.1039/C8RA06707D.].

Metabolic Profile of and Antimicrobial Activity in the Aerial Part of Leonurus turkestanicus V.I. Krecz. et Kuprian. from Kazakhstan.

An HPLC quadrupole time-of-flight (Q-TOF) MS method was developed for the identification of secondary metabolites in Leonurus turkestanicus V.I. Krecz. et Kuprian. Ethanolic and chloroform extracts from the plant's aerial parts were tested. A total of 16 compounds (iridoid glycosides, phenylpropanoids, flavonoids, and nitrogen-containing compounds, as well as diterpene acetate derivatives) were identified and tentatively characterized based on (or using) their retention times and UV and Q-TOF-MS data. Previously reported aucubin (1), 6-deoxy-8-acetylharpagid (2), and stachydrine (13, 15) and homostachydrine isomers (14, 16) were identified, along with a lavandulifolioside isomer (3), verbascoside (4), rutin (5), 3-O-kaempferol rutinoside (6), and an unknown diterpene acetate (8). Compounds 3-6 were detected for the first time in this plant. Additionally. antimicrobial activity was evaluated. No significant differences were found between ethanolic and chloroform extracts of L. turkestanicus; however, the alcoholic extract showed stronger antifungal activity [minimal inhibitory concentration (MIC) of 2.5-5 mg/mL], whereas the chloroform extract showed stronger activity against the tested spore-forming Bacillus species (MIC 1.25-2.5 mg/mL).

LC-QTOF-MS Analysis and Activity Profiles of Popular Antioxidant Dietary Supplements in Terms of Quality Control.

The dietary supplements with claimed antioxidant activity constitute a substantial part of the dietary supplement market. In this study, we performed the LC-QTOF-MS analysis and investigated the activity profiles of popular antioxidant dietary supplements from different chemical groups in terms of quality control. The commonly used antioxidant tests and statistical analysis revealed that substantial part of the results was comparable if 1 g sample was considered, but while comparing single and daily doses, significant differences in antioxidant values were noticed in all assays. The best antioxidant activity was obtained in ORAC assay (from 142 to 13814 µM of Trolox equivalents per 1 g of sample), and the strongest correlation occurred between TPC and ORAC. The LC-QTOF-MS analysis revealed that catechins were present in samples having the best antioxidant activity and that dietary supplements showing the weakest activity contained very small amount of any chemical constituents.