Chemical Compositions and Antibacterial Activity of Extracts Obtained from the Inflorescences of Cirsium canum (L.) All.

The aim of 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: 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, kaempferol. The antimicrobial activity of tested extracts was determined in vitro against reference microorganisms, including bacteria or fungi, belonging to yeasts. The our data showed that the tested extracts had no influence on the growth of the reference strains of Gram-negative bacteria and of yeasts belonging to Candida spp. Among them, the fractions possessed the highest activity against Gram-positive bacteria, especially S. aureus and S. pneumoniae belonging to pathogens and S. epidermidis, B. cereus and B. subtilis belonging to opportunistic microorganisms.


Extraction in boiling point
One part aqueous solution was extracted with diethyl ether, then washed with 5% aqueous sodium bicarbonate solution. After acidification and extraction with diethyl ether, fractions containing free phenolic acids (fraction FA) were obtained. Hydrolysis was performed by the Schmidtlein and Herrmann method (Schmidtlein & Hermann, 1975;Świątek, 1997;Polish Pharmacopoeia VI). The fractions containing bonded phenolic acids liberated after acid hydrolysis (fraction FB) and analysis of bonded phenolic acids liberated by alkaline hydrolysis (fraction FC).
The second part aqueous solution was extracted sequentially with diethyl ether obtaining fractions: (fraction F1), ethyl acetate (fraction F2), n-butanol (fraction F3). Then the acidic hydrolysis of and total acid hydrolysis of 5% HCl has been performed on diethyl ether extract for 2 h (fraction F4). Extracts residues were evaporated to dryness under reduced pressure. Afterwards respectively they were investigated for their microbiological activity.
For HPLC analysis fractions were dissolved in methanol, passed through 0.45 μm PTFE membrane filters (Cronus Syringe Filter, PTFE 25mm, 0.2µm) and transferred to a 10 mL calibrated vials.

RP-LC analysis
LC was performed with an Agilent 1100 (Agilent Technologies, USA) system coupled with an auto-sampler, a column thermostat; a diode -array detector (DAD). Compounds were separated on 250 x 4.6 mm stainless-steel column packed with 5 μm Hypersil XDB-C18 (Zorbax, Eclipse), using a stepwise mobile phase gradient prepared from 1% v/v aqueous acetic acid (component A) and methanol (component B) (v/v). The gradient was: 0 min, 2% B in A; 8 min 5% B in A; 12 min 10% B in A; 20 min 25% B in A; 35 min 45% B in A; 40 min 60% B in A, 45 min 75% B in A. The mobile phase flow-rate was 1 mL min -1 , the sample injection volume was 10 μL, and was performed at 25ºC. The LC autosampler, column oven, and DAD were monitored and controlled using of ChemStation rev.10.0 software (Agilent Technologies). Compounds were identified by comparison of retention times and UV spectra with those of appropriate standards analyzed under the same conditions. Qualitative and quantitative determination were performed at the 320 nm wavelength of maximum absorption of flavonoids and for cinnamic acid derivatives (ferulic, chlorogenic, p-coumaric, transcinnamic and caffeic acids) and 254 nm for benzoic acid derivatives (protocatechuic, phydroxybenzoic, vanillic, and syringic acids).
Each extract was injected in triplicate on the same day. The method of precision was evaluated by use of intra-day tests. Intra-day experiments were performed by replicate analysis of three aliquots of the same sample on the same day. Peak area of each of the extract components was measured. The RSD (relative standard deviation, %) of peak areas were used. Quantifications were performed by comparing the peak areas of the compounds present in the extracts M1-M6 with those of the external standards.

In vitro antimicrobial assay:
The Appropriate DMSO, growth and sterile controls were carried out. The medium with no tested extracts was used as control.

The MBC (Minimal Bactericidal Concentration) or MFC (Minimal Fungicidal
Concentration) are defined as the lowest concentration of the compounds that is required to kill a particular bacterial or fungal species. MBC or MFC was determined by removing of the culture using for MIC determinations from each well and spotting onto appropriate agar medium. After incubation the lowest compounds concentrations with no visible growth observed was assessed as a bactericidal/fungicidal concentration. All the experiments were repeated three times and representative data are presented (Łączkowski et al., 2013).
In this study, no bioactivity was defined as a MIC > 1000 µg/ml, mild bioactivity as a MIC in the range 501 -1000 µg/ml, moderate bioactivity with MIC from 126 to 500 µg/ml,  -fraction FC (the bonded phenolic acids liberated by alkaline hydrolysis).