1. One- and Multidimensional Thin-Layer Chromatography for the Analysis of Phenolic Acids and Flavonoids from the Different Salvia Species
M. Sajewicz1, Ł. Cieśla2, D. Staszek1, Ł. Wojtal1*, M. Waksmundzka-Hajnos2,
and T. Kowalska1
1Institute of Chemistry, Silesian University, 9 Szkolna Street, Katowice, Poland
2Department of Inorganic Chemistry, Medical University of Lublin, 6 Staszica Street, Lublin, Poland
Introduction
Salvia (sage) encompasses about 900 species, widespread throughout the world, and includes several ornamental, culinary and medical species. Plants belonging to this genus have been commonly used in traditional medicine, due to a wide spectrum of their biological activities, including antioxidant, anti-platelet, anti-tumor, antiviral, etc. In recent years, much attention has been focused on biologically active, water-soluble components, including polyphenolic flavonoids and phenolic acids. Polar phenolic acids constitute major part of the water-soluble components of the Salvia decoction. Different sage species contain few simple benzoic acids, however, the majority of the phenolic acids are the caffeic acid derivatives. Rosmarinic acid was established as the most abundant caffeic acid dimer in the Salvia genus and it has been reported as the main phenolic contributor to the confirmed high antioxidant activity of the sage samples [1]. Flavonoids are also widely distributed in the different species of Salvia, mostly as flavones, flavonols and the glycosides thereof. The 6-hydroxylated flavones have been reported as the taxonomically most important compounds [1]. The majority of flavonoids are flavones (apigenin and luteolin) and flavonols, mostly kaempferol and quercetin methyl esters.
Over the years, classification of different Salvia species has become very confusing. Many species look similar to one another, and many of them appear in the varieties that have been given different specific names. Studying chemical composition of Salvia extracts will hopefully help to understand the biological potential, as well as the taxonomic relationship among the different investigated species.
Aim
The aim of this study was to compare the phenolic acid and flavonoid composition in twenty different sage species. Extracts of the dried Salvia herbs were obtained from the plants collected in the course of the 2007 vegetation period. Botanical material has been authenticated and voucher specimens were deposited at the Department of Pharmacognosy, Medical University of Lublin, Poland.
Extraction
For the experiment, extracts were obtained from the dried leaves originating from twenty different sage species and collected in 2007 and Extracts were also made from the fresh sage leaves collected in Plant material was dried at 35-40oC for 40 h in an oven with a forced air flow. The dry and fresh plant material was stored until the analysis in the deep-freeze compartment of refrigerator. Extraction was performed in a Dionex (Sunnyvale, CA, USA) model ASE 200 accelerated solvent extraction unit. In metal cells, 5-g samples of plant material were placed. Detailed working parameters are given in the below table. Hexane extracts were discarded, as this step was meant to remove chlorophyll and the less polar constituents from the samples. Methanol extracts were evaporated to dryness and then the solid residue was dissolved in 5 mL methanol. Methanol solutions were ultrasonicated for 15 min in ultrasonic bath (model RK 255H Sonorex Super, manufactured by Bandelin, Berlin, Germany). Finally, each sample was condensed to the volume of 1 mL by solvent evaporation and filtered with use of a syringe filtre Anotop (diameter 25 mm, average particle diameter 0.02 μm, alumina membrane; cat. # 11320, Merck, Darmstadt, Germany). These samples were ready for the chromatographic analysis.
Thin-Layer Chromatography, TLC
One-dimensional thin-layer chromatography
On to the chromatographic plates, 10-μL aliquots of the methanol sage extracts were spotted. Initially, the least polar fractions were compared. The investigated extracts were developed on the chromatographic plates pre-coated with silica gel. As mobile phase, toluene + ethyl acetate (5:95, v/v) was used. Chromatograms were visualized by spraying them with 5% methanol solution of conc. sulphuric acid.
APOLAR FRACTION
Fig.5. Concentration profiles of the sage (Salvia L.) extracts in methanol from the dried plant material (recorded in the day light)
Eluent
Temperature
[°C]
Pressure
[atm]
Period of preliminary heating the sample
[min]
Period of heating the sample after adding the solvent
Period of static heating the sample
Solvent volume
[mL]
Number of cycles
n-Hexane 40 68 10 5 2
Methanol
100 65
Fig.6. Concentration profiles of the sage (Salvia L.) extracts in methanol from the dried plant material (recorded in the UV light, at the wavelength λ = 366 nm)
Special thin-layer chromatogram developing techniques
GMD
(Gradient Multiple Development) 1 2 3 4 5 6 7 8 9 10
S. lavandulifolia
S. sclarea
S. tesquicola
S. staminea
S. amplexicaulis
S. cadmica
S. pratensis
S. canariensis
S. nemorosa
S. jurisicii 11 12 13 14 15 16 17 18 19 20
S. stepposa
S. hians
S. officinalis
S. forskohlei
S. azurea
S. verticillata
S. triloba
S. deserta
S. glutinosa
S. atropatana
Fig.7. An example of the GMD technique applied to developing the chromatograms for the selected sage species. As stationary phase, the chromatographic plates pre-coated with silica gel were used. As eluents, the following binary mixtures were applied:
a) isopropanol : n-heptane, 5 : 95 (v/v)
b) isopropanol : n-heptane, 10 : 90 (v/v)
c) isopropanol : n-heptane, 40 : 60 (v/v)
d) isopropanol : n-heptane, 80 : 20 (v/v)
Visualization was carried out by means of the UV lamp (Camag, Muttenz, Switzerland) at the wavelength λ = 366nm.
MGD
(Multiple Gradient Development)
Fig.1. Videoscans of the chromatograms registered at the wavelength λ = 366 nm
On to the chromatographic plates, again the 10-μL aliquots were spotted of the methanol sage extracts. This time, the more polar fractions were compared. Again, extracts were developed on the chromatographic plates pre-coated with silica gel. This time the mobile phase of higher elution strength was used, composed of ethyl acetate + toluene + formic acid (70:30:1, v/v). Chromatograms were visualized by spraying them with 1% methanol solution of aluminium chloride.
Fig.8. An example of the MGD technique applied to construction of the „fingerprint” with the selected sage species. The chromatographic plate with the sage extracts spotted on to it, underwent the three-stage developing with use of the following eluents:
Stage I: isopropanol : n-heptane, 40 : 60 (v/v)
Stage II: isopropanol : n-heptane, 60 : 40 (v/v)
Stage III: methanol : acetic acid : water, 78:2:20 (v/v)
Visualization was carried out by means of the UV lamp (Camag, Muttenz, Switzerland) at the wavelength λ = 366nm
Fig.2. Videoscans of the chromatograms registered at the wavelength λ = 366 nm
Binary chromatographic fingerprints
POLAR FRACTION
As stationary phase, silica gel was used. The eluent applied for polar fraction was ethyl acetate + water + acetic acid + formic acid (100:26:11:11, v/v), and for the non-polar fraction, it was toluene + ethyl acetate + formic acid (70:30:1, v/v). Chromatograms were developed in the horizontal sandwich DS chambers (Chromdes, Lublin, Poland), and visualized with aluminium chloride. Pictures of the chromatograms were taken in the day light and in the UV light at the wavelength λ = 366nm.
Two-dimensional thin-layer chromatography a) b) c) F L L K K 1D 1D 1D 2D 2D 2D
Fig.9. Videoscans of the chromatograms obtained from the two-dimensional separation of the Salvia officinalis methanol extract.
Adsorbents: a, b) CN-silica, c) Diol-silica.
The applied eluents:
1D: isopropanol : n-heptane (2x) 30 : 70 (v/v) for the entire plate length,
isopropanol + acetic acid + n-heptane, 78 : 2 : 20 (v/v) do ostatniej rozdzielonej w poprzednim kroku plamki (as observed in UV light),
2D: methanol : water : acetic acid, 10 : 88 : 2 (v/v)
Symbols: F – ferulic acid, K- kaempferol, L – luteolin.
Fig.3. Concentration profiles of the sage (Salvia L.) extracts in methanol from the dried plant material (recorded in the day light)
Conclusions
A traditional one-dimensional isocratic development of the chromatograms proves insufficient, when a comparative analysis of chemical composition is carried out with closely related botanical species, containing substances within a wide polarity range.
Construction of the binary chromatographic fingerprints allows a comparison of the fractions with different chromatographic properties (e.g., of the polar and non-polar fraction, etc.)
Application of the GMD tchnique for the fingerprint construction is particularly advantageous with complex samples, as it allows a comparison of the numerous fractions differing in their respective properties.
The MGD technique allows separation of substances differing with the polarity parameters; however, its basic limitation with very complex samples – in comparison with the GMD technique – is a relatively low number of spots which can be separated from one another.
Application of the two-dimensional separation of the chromatograms on the chemically bonded CN-silica adsorbent allows obtaining of characteristic fingerprints for the individual sage species. However, the low-concentration components can prove difficult to detect in the case of the 2D-TLC mode.
References
[1] E. Reich, A. Schibli, High-Performance Thin-Layer Chromatography for the Analysis of Medicinal Plants. Thieme, New York, 2008.
[2] X.H. Fan, Y.Y. Cheng, Z.L. Ye, R.C. Lin, Z.Z., Qian, Anal. Chim. Acta, 555 (2006) 217.
[3] Ł. Cieśla, A. Bogucka-Kocka, M. Hajnos, A. Petruczynik, M. Waksmundzka-Hajnos, J. Chromatogr. A, 1207 (2008) 160.
Fig.4. Concentration profiles of the sage (Salvia L.) extracts in methanol from the dried plant material (recorded in the UV light, at the wavelength λ = 366 nm)