2.1. Plant Material and Extraction
The extracts of Geranium sanguineum
L. were isolated, purified, and analyzed, as previously described [14
]. Briefly, the plant was collected between June and August in the flowering stage in the Lyulin Mountain, Bulgaria. A specimen was deposited in the Herbarium of the Institute of Botany at the Bulgarian Academy of Sciences, Sofia, Bulgaria (SOM-5/96). For all in vivo studies, we used a combined (total) ethanol extract from Geranium sanguineum
L. Briefly, air-dried aerial roots (500 g) were extracted by repeated extraction with ethanol (3 × 600 mL) using Soxhlet apparatus for 24 h at room temperature. The ethanol extracts were combined and lyophilized to obtain combined yields of 16.7% (flow chart A, Figure 1
). In order to analyze the constituents of the standardized ethanol extract of Geranium sanguineum
L., a subsequent extraction was performed with diethyl ether (Et2
O; fraction F1), chloroform (CHCl3
; fraction F2), dichloromethane (CH2
; fraction F3), ethyl acetate (EtOAc; fraction F4), n-
-ButOH; fraction F5), and water (fraction F6). Finally, each fraction was purified by column chromatography and analyzed by reverse phase high-performance liquid chromatography RP-HPLC (flow chart B, Figure 1
2.2. Used Chemicals
The reference compounds quercetin, kaempferol, myricetin, morin, apigenin, retusin, quercetin-3-O-galactoside (hyperoside), (−)-catechin, (+)-catechin, (−)-epicatechin, chlorogenic, ellagic, quinic, and caffeic acids were purchased from Sigma-Aldrich Chemie GmbH (Deisenhofen, Germany). Dr. N. Mahmood and Dr. A.J. Hay (NIMR, Mill Hill, London, UK) provided some of the polyphenol compounds. All solvents, reagents, and other chemicals were obtained from different suppliers (VWR, Sigma-Aldrich, Carl Roth, Merck, and others).
2.3. Extracts Purification and Analysis
(a) Column chromatography (CC) was performed using different sorbents and eluent systems as follows: (i) Sephadex LH-20—eluent system MeOH; (ii) Silica gel—eluent system EtOAc/MeOH in different ratio; and (iii) Polyamide S—eluent system MeOH/water 70:30.
(b) Thin layer chromatography (TLC) was achieved using different sorbents and suitable eluent systems as follows: (i) Polygram Silica gel UV254 (Merck)—eluent system EtOAc/formic acid/AcOH/water 100:11:11:27; (ii) Kieselgel 60 F254 (Merck) with eluent systems EtOAc/formic acid/AcOH 100:11:11 or CHCl3/MeOH 50:50, 75:25 or 85:15; (iii) Polyamide 11 F254 (Merck) with eluent systems EtOAc/formic acid/water 10:2:3 or CHCl3/MeOH 8:1; and (iv) Kieselgel 60 (without a fluorescent indicator) with eluent systems CHCl3/MeOH 10:50 or 85:15. For determination of the different constituents were used the following spray reagents: (i) natural substance-polyethylene glycol (NST-PEG, as a 1.0% solution of diphenylborate aminoethanol in MeOH) for flavonoids and (ii) 0.5% Echtblausalz in MeOH for tannins. The individual compounds were identified by comparison with authentic samples on different sorbents using diverse eluent systems and observed at UV364 before and after spray with NST-PEG for flavonoids and Echtblausalz for catechins. Authentic samples were provided by Prof. E. Wollenweber (Institute of Botany, Darmstadt, Germany).
(c) Reverse phase high-performance liquid chromatography (RP-HPLC) was performed using the following solvents, standards, and chemicals: acetonitrile (HPLC-gradient grade), MeOH (HPLC-grade), formic acid and ortho-phosphoric acid (analytical grade), and bi-distilled water (HPLC-grade). All standards and chemicals were purchased from Merck (Darmstadt, Germany). The reference standards of the flavone aglycones were supplied by Carl Roth GmbH & Co. KG (Karlsruhe, Germany).
The samples were prepared as previously described [14
]. Briefly, 0.2 g dried extracts from plant were dissolved in 20 mL MeOH and treated in an ultrasonic bath for 30 min. After re-dissolving in 1.5 M HCl (20 mL), hydrolysis was achieved on a water bath under reflux for 20 min at a temperature of 100 °C. The samples were then cooled at room temperature and diluted with MeOH (1/50).
The chromatographic analysis was performed on a Shimadzu 4A (Schimadzu Corporation, Kyoto, Japan) chromatographic system equipped with a tertiary pump and a Rheodyne injector (100 µL sample loop) coupled with an UV–Vis detector. Knauer Chromatography software and workstation were used for controlling the system and collecting the data. Preparative reversed phase chromatography was carried out on an Atlima C18 Rocket column (57 mm × 7 mm, particle size 3.0 μm) from Alltech Inc. (Lancashire, UK) at room temperature.
For the extract analysis, were used two RP-HPLC methods. A gradient elution (analytical method I) was carried out using bi-distilled water/methanol/formic acid (74.7:25:0.3; v
) as a mobile phase A and acetonitrile/formic acid (99.7:0.3; v
) as a mobile phase B. The linear gradient elution and run setting were as follows: 100% mobile phase A for 5 min to 100% mobile phase B after an additional 10 min, standing at 100% B for 5 min, and returning to 100% A after another 5 min. The flow rate was 1.0 mL/min and the quantification of catechins was performed at 270 nm. The samples were sonicated until completely dissolved and filtered through a Chromafil filter (0.45 µm from Millipore Ireland B.V., County Cork, Ireland) into a 2.0 mL HPLC glass vial in order to separate the dissolved from particulate material directly prior to injection. Then, 20 µL of each sample was injected into the Atlima C18 Rocket analytical HPLC column. The results obtained from the linear gradient elution are presented in Figure 2
For isocratic RP-HPLC separation (separation method II), a mobile phase of water/acetonitrile in a ratio of 65:35 (v
) adjusted to pH 2.3 with ortho-
phosphoric acid was used. The mobile phase was filtered through a Chromafil filter (0.45 μm) and de-gassed before sonication. The flow rate was 1.0 mL/min and the chromatograms were recorded at 254 nm selected on the specific UV absorption maximum of the assayed compounds. The results from the isocratic HPLC separation are shown in Figure 2
Identification of flavanols and phenolic acids was carried out by comparison of the retention times (tr
) and the respective UV absorbance (UVmax
in arbitrary units, a.u.) of the unknown peaks with those of the standards [14
]. For this purpose, a standard mixture containing ten reference compounds diluted in bi-distilled water/formic acid 99.7:0.3 (v
) was prepared and analyzed. As references, we used the following compounds: (+)-catechin (0.14 mg/mL), (D)-epicatechin (0.38 mg/mL), chlorogenic acid (0.6 mg/mL), caffeic acid (0.14 mg/mL), rutin (0.8 mg/mL), hyperoside (0.8 mg/mL), myricetin (0.9 mg/mL), morin (0.8 mg/mL), apigenin (0.7 mg/mL), and kaempferol (0.8 mg/mL)). Then, calibration curves were obtained using standard solutions in concentrations between 25% and 125%. All sample preparations from dried extracts and standard solutions were prepared in duplicate and analyzed by HPLC-C18 in triplicate [14