Phytochemical Profiling and Biological Activity of the Methanolic Extracts of Cirsium Monspessulanum (L.) Hill.

The study of new plant species and the identification of their chemical composition may contribute to the discovery of a new breakthrough substances for pharmacotherapeutical applications. For the first time, we examined antioxidant and antimicrobial activity of 70 % v/v methanolic extracts from inflorescences and roots of Cirsium monspessulanum (L.) Hill. obtained by the ASE method. In the (2,2-diphenyl-1-picrylhydrazyl) DPPH analysis, tested extract of inflorescences showed antioxidant activity with an EC50=0.223±0.0479 mg/mL, and (Cupric Ion Reducting Antioxidant Capacity) CUPRAC test assessed the antiradical activity on 14.95±0.13 mgTE/g and for roots the values were EC50=0.307±0.0554 mg/mL and 11.18±0.49 mgTE/g, respectively. Furthermore, extract from the inflorescences possessed the highest antimicrobial activity against Staphylococcus aureus, Staphylococcus epidermidis and Micrococcus luteus with MIC=1.25 mg/mL for each. HPLC/ESI-QTOF-MS/MS method identified 7 phenolic acids and 14 flavonoids in inflorescences extract and only 7 phenolic acids in roots extract. To the best of our knowledge, this is the first qualitative analysis of Cirsium monspessulanum (L.) Hill. and all substances were described for the first time.

Phenolic Composition of Inflorescences of Carduus nutans L.

Carduus nutans L. (Compositae) has been extensively used for medicinal purposes. As other representatives of the genus it is implemented in the treatment of liver disorders and used as diuretic and digestive agent. Previous studies have determined the predominant classes of secondary metabolites in Carduus species. Among the major representatives of their extracts flavonoids, phenolic acids, coumarins, sterols and terpenes were identified. The antiradical capacity of three extracts assessed in the DPPH test revealed the highest radical scavenging properties of methanol extract (the EC50 618±10.03 µg/mL) and based on these results it was selected for phenolic content determination. (TPC=61.49 mg/g). To understand better the induced pharmacological effects of the plant the aim of the study was to determine the composition of the methanol extract from the inflorescences of C. nutans and to study their phenolic composition. In order to determine the composition in a more efficient way, the crude extract was fractionated and subjected to hydrolyses. As a result more than twenty phenolic acids and flavonoids were identified in the extract and fractions by HPLC-DAD and/or HPLC-ESI-TOF-MS. The total extract was later subjected to fractionation by centrifugal partition chromatography using the Arizona system composed of hexane/ethyl acetate/methanol/water (0.7 : 4 : 0.8 : 4 v/v/v/v) to produce fractions enriched in flavonoids that are of high pharmacological significance.

ANALYSIS OF PHENOLIC ACIDS AND ANTIBACTERIAL ACTIVITY OF EXTRACTS OBTAINED FROM THE FLOWERING HERBS OF CARD UUS ACANTHOIDES L.

In this work the phenolic acids in crude methanol extracts from the flowering herbs of Carluus acanthoides L. were identified. The samples containing free phenolic acids and those released after acid and alka- line hydrolyses were investigated by 2D TLC on cellulose. After purification by SPE, samples were also analyzed by RP-HPLC. Chlorogenic, protocatechuic, p- coumaric, caffeic, syringic, p-hydroxybenzoic, ferulic, vanillic, gentisic and gallic acids were detected in fractions of the methanolic extract obtained from the flow- ering herb of C. acanthoides. This is the first study concerning the qualitative analysis of phenolic compounds and antibacterial activity of fractions and aqueous, ethyl acetate, dichloromethane, acetone and methanol (50, 80 and 100% v/v) extracts of flowering herbs of C. acanthoides L. The antimicrobial activity of tested extracts was determined in vito against reference microorganisms of Gram-positive bacteria, Gram-negative bacteria and fungi belonging to yeasts. The results of this study support the medical usage of C. acanthoides L. due to its antimicrobial properties.

Comparative HPLC-DAD-ESI-QTOF/MS/MS Analysis of Bioactive Phenolic Compounds Content in the Methanolic Extracts from Flowering Herbs of Monarda Species and Their Free Radical Scavenging and Antimicrobial Activities.

Comparative analysis of flavonoids and phenolic acids composition, in plants of six species of Monarda from family Lamiaceae was carried out. The 70% (v/v) methanolic extracts of flowering herbs of Monarda citriodora Cerv. ex Lag., Monarda bradburiana L.C. Beck, Monarda didyma L., Monarda media Willd., Monarda fistulosa L. and Monarda punctata L. were analyzed for their polyphenol composition as well as antioxidant capacity and antimicrobial effect. Liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-DAD-ESI-QTOF/MS/MS) was used to identify phenolic compounds. The in vitro antioxidant activity was assessed using a DPPH radical scavenging assay, while antimicrobial activity was measured by the broth microdilution method allowing for MIC (minimal inhibitory concentration) determination. The total polyphenol content (TPC) was assayed by the Folin-Ciocalteu method. The results showed the presence of eighteen different components including phenolic acids and flavonoids together with their derivatives. The presence of six constituents (gallic acid, hydroxybenzoic acid glucoside, ferulic acid, p-coumaric acid, luteolin-7-glucoside and apigenin-7-glucoside) was found to be dependent on the species. To differentiate the samples, the antioxidant activity of 70% (v/v) methanolic extracts was studied and expressed as a percent of DPPH radical inhibition and in EC50 values (mg/mL). The latter values were as follows: M. media (EC50 = 0.090 mg/mL), M. didyma (EC50 = 0.114 mg/mL), M. citriodora (EC50 = 0.139 mg/mL), M. bradburiana (EC50 = 0.141 mg/mL), M. punctata (EC50 = 0.150 mg/mL) and M. fistulosa (EC50 = 0.164 mg/mL). Moreover, all extracts indicated bactericidal activity against reference Gram-positive (MIC = 0.07-1.25 mg/mL) and Gram-negative bacteria (MIC = 0.63-10 mg/mL) as well as fungicidal effect towards yeasts (MIC = 1.25-10 mg/mL). Staphylococcus epidermidis and Micrococcus luteus were the most sensitive to them. All extracts showed promising antioxidant properties and noteworthy activity against the reference Gram-positive bacteria. Antimicrobial effect of the extracts against the reference Gram-negative bacteria as well as fungi (yeasts) from Candida spp. was slight. All extracts showed bactericidal and fungicidal effect. The obtained results indicated that the investigated extracts from Monarda spp. could be potential sources of natural antioxidants and antimicrobial agents, especially with activity towards Gram-positive bacteria. The differences in the composition and properties of the studied samples may influence the pharmacological effects of the studied species.

Cytotoxic activity of methanolic fractions of different Marrubium spp. against melanoma cells is independent of antioxidant activity and total phenolic content.

The Marrubium genus (horehound) has proved to be an abundant source of biologically active compounds, but there is little knowledge about its potential anticancer activity. Moreover, some Marrubium species have not been the subject of study in this regard. In this study, we performed comparative analysis of phenolic acid (PhA) content and total phenolic content in fractions obtained from methanolic extracts of Marrubium vulgare L. (common horehound), Marrubium cylleneum Boiss. & Heldr. and Marrubium friwaldskyanum Boiss herbs. We examined the cytotoxicity of these fractions against a human melanoma cancer cell line (A375) and normal human skin fibroblasts (BJ) using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide test, cell cycle analysis and real-time monitoring of cell viability. We detected caffeic, p-coumaric, ferulic and gentisic acids among the PhAs. Although the extracts obtained demonstrated low total phenolic content and did not show significant antioxidative properties, the nonhydrolyzed PhA fraction exhibited cytotoxic activity against a human melanoma cancer cell line, without affecting normal fibroblasts. Both acidic and alkaline hydrolysis abolished this activity, indicating that the esterified forms of phenolic compounds caused the observed cytotoxic effects. Further investigation of these compounds may facilitate the development of novel drugs for cancer treatment.

Comparison of Phenolic Compound Separations by HPTLC and PPEC with SDS as the Mobile Phase Component.

The application of the surfactant (sodium dodecyl sulphate, SDS) as the component of the water-organic mobile phase in thin-layer chromatography and pressurized planar electrochromatography is presented. The influence of various variables on the separation of various phenolic compounds (flavonoids and phenolic acids) as model compounds with systems containing surfactant is discussed. The effect of concentration of butanol and SDS as well as pH of the mobile phase buffer on migration distance of the solute zones is investigated. The presence of SDS in the eluent affects the butanol solubility in the mobile phase. It allows using higher organic solvent concentration systems compared with the mode without surfactant. The amount of SDS in the eluent has the effect on the solute retention, whereas the eluent buffer pH affects the migration distances of ionisable phenolic acids both in HPTLC and PPEC. The migration distances of flavonoid glycosides are considerably longer than those of pure flavonoids. Considering second group of investigated solutes, derivatives of the benzoic acid migrate longer distances in comparison with the cinnamic acid ones. In addition, in the majority of experiments, ionisable compounds (phenolic acids) migrate longer distances in PPEC than nonionisable compounds (flavonoids). Additionally, the order of solutes differs in the PPEC and HPTLC system.

Chemical composition and variability of the volatile components from inflorescences of Cirsium species.

The present study aimed to investigate the chemical composition of the essential oils of inflorescences Cirsium spp. (Asteraceae) by GC/MS method. Essential oils were extracted from the inflorescences of Cirsium pannonicum (Link), Cirsium ligulare Boiss., Cirsium heterophyllum (L.) Hill., Cirsium acaule (L.) Scop., Cirsium oleraceum (L.) Scop., Cirsium dissectum (L.) Hill., Cirsium decussatum (Janka) and Cirsium eriophorum (L.) Scop., using the steam distillation method. A gas chromatography-mass spectrometry method was employed for the analysis of essential oils. Our study shows the differences in chemical composition of volatile oils in the inflorescences of Cirsium spp. The main components of the essential oil were ketones and aldehydes with a long carbon side-chain. Volatile oils also contained small amounts of terpenes: thymol, ß-linalool, eugenol, carvacrol and fatty acids with odd number of carbon atoms-waxes. The compounds in the essential oils obtained from inflorescences Cirsium L. species have been identified for the first time.

Chemical compositions and antibacterial activity of extracts obtained from the inflorescences of Cirsium canum (L.) all.

The aim of this study was to investigate phenolic acids and flavonoids in methanolic, dichloromethane, acetone and ethyl acetate extracts and fractions from inflorescences of Cirsium canum (L.). RP-HPLC analysis enabled identification of the following: chlorogenic acid, caffeic acid, p-coumaric acid, protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, syringic acid, trans-cinnamic acid, luteolin-7-glucoside, apigenin-7-glucoside, kaempferol-3-glucoside, linarin, apigenin, rutoside, luteolin and kaempferol. The antimicrobial activity of tested extracts was determined in vitro against reference microorganisms, including bacteria or fungi, belonging to yeasts. Our data showed that the tested extracts had no influence on the growth of the reference strains of Gram-negative bacteria and yeasts belonging to Candida spp. Among them, the fractions possessed the highest activity against Gram-positive bacteria, especially Streptococcus aureus and Streptococcus pneumoniae belonging to pathogens and Streptococcus epidermidis, Bacilluscereus and Bacillus subtilis belonging to opportunistic microorganisms.