Plant Drug Analysis by Planar Chromatography

Journal of Chromatographic Science, Vol. 40, November/December 2002 Plant Drug Analysis by Planar Chromatography Nicole Galand, Jacques Pothier*, and Claude Viel* Laboratoire de Pharmacognosie, Faculté de Pharmacie, 31, Avenue Monge, 37200—Tours, France Abstract Optimal performance laminar chromatography and automated multiple development chromatography are relatively recent techniques of planar chromatography that can be applied with success in plant material analysis. Therefore, these methods are used to study plant extracts and constituents belonging to different chemical classes of secondary metabolism: heterocyclic oxygen compounds (coumarins, flavonoids, and anthocyanins), alkaloids and quaternary ammonium salts, cannabinoids, essential oils, ginsenosides, and cardiac heterosides. Generally, the results obtained with these methods are good, and in most cases they compare with those of thin-layer chromatography. Introduction Besides classical thin-layer chromatography (TLC), two other techniques of planar chromatography appeared in the 1980s: overpressured layer chromatography, or optimal performance laminar chromatography (OPLC) (1–4), and automated multiple development (AMD) (5–8). Few studies concerning OPLC and AMD had been reported in the literature at the beginning of this study. These techniques were applied to the analysis and characterization of numerous natural compounds belonging to various chemical classes found in plant material, and the results were compared with those of TLC. All of the analyses were performed on crude plant extracts versus reference substances. Numerous compounds found in plants have pharmacological and, for some of them, therapeutic activity. The principal classes of compounds include nitrogen-containing heterocycles such as alkaloids and derivatives; heterosides and aglycones such as those of cardiotonic steroids; and phenolic compounds such as coumarins, flavonoids, and anthocyanins, which embrace a wide range of plant substances and possess in common an aromatic ring bearing one or more hydroxyl groups or phenolic ether substituents (9). All of the results with heterocyclic oxygen compounds, alkaloids and ammonium quaternary salts, cannabinoids, essential * Authors to whom correspondence should be addressed: emails and . oils, ginsenosides, and cardiac heterosides are reported in this work. Experimental A Linomat IV (Camag, Muttenz, Switzerland) was used for sample applications. A TLC–MAT (automated development in TLC) Desaga (Bionisis, Le Plessis-Robinson, France) was also used. A Chrompres 25 (OPLC-NIT Engineering Company, Budapest, Hungary) OPLC (Bionisis) was used. For AMD, a Densitometer Camag Model 76510 TLC–high-performance (HP) TLC scanner was used. All solvents and reagents were analytical grade and obtained from Merck (Darmstadt, Germany). Before use, solvents were filtered through a 0.45-µm Millipore membrane after sonication. All standards were commercially pure products. Heterocyclic oxygen compounds aglycones (10) Plant material samples Powdered plant material (5 g) was mixed with 50 mL of methanol, and then the dry sample residue was hydrolyzed with 2M hydrochloric acid for 1 h at 100°C. Once cool, the solution was extracted three times with 50 mL of ethyl acetate, dried on anhydrous sodium sulfate, and the solvent removed under reduced pressure whereupon the residue was taken up with 1 mL of methanol. Apparatus The apparatus used was a Chrompres 25. Plates Silica gel F254 20- ¥ 20-cm glass TLC plates (Merck, Art. 5715) were used. For OPLC, the chromatographic plates required a special preparation mode: three edges were obliquely scratched off and impregnated with a suitable polymer suspension Impress II (OPLC-NIT Engineering Company). Chromatographic conditions Three mobile phases were employed: ethyl acetate–chloroform (60:40, v/v) for coumarins and flavonoids, ethyl acetate–chloroform (90:10, v/v) for furanochromones, and ethyl acetate–methyl ethyl ketone–formic acid–2M hydrochloric acid (65:10:6:9, v/v) in Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission. 1 Journal of Chromatographic Science, Vol. 40, November/December 2002 the case of anthocyanins. During chromatography, the external pressure of the water cushion was 16 bars. The starting mobile phase pressure was 20 bars, and plates were developed on a distance of 17 cm with a mobile phase flow rate of 0.40 mL/min. The development time was between 12 and 25 min depending upon the eluent. The separations were checked first by visual observation under UV illumination (l = 365 nm) for coumarins and furanochromones, then by spraying with 1% methanolic diphenylboric acid b-ethylamino ester, followed by 5% ethanolic polyethyleneglycol 400 [NP/PEG reagent (11,12)]. Then, observation occurred at l = 365 nm for flavonoids and by direct visual observation without reagent treatment for anthocyanins. Alkaloids and quaternary ammonium salts Plant material samples Powdered plant material (1 g) was mixed thoroughly with 60% ethanol (10 mL) by shaking for 4 h, followed by filtration through a No. 2 glass frit and adjustment of the volume to 10 mL. The quantity of the sample that was applied to the layer was calculated from the average content of the drug in the plant material extracted. Plates The plates were prepared with the special mode for OPLC (see Phenolic compounds section). For alkaloids, aluminum oxide 60 F254 type E 20- ¥ 20-cm glass plates (Merck, Art. 5713) were used. For quaternary ammonium salts, silica gel 60 F254 20- ¥ 20-cm glass plates (Merck, Art. 5715) were used. For semipreparative, aluminum oxide 60 F254 20- ¥ 20cm glass plates (1.5-mm thickness for preparative layer chromatography) (Merck, Art. 5788) were used. Chromatographic conditions Analytical OPLC of alkaloids. Two mobile phases were employed: ethyl acetate (eluent A) for almost all of the alkaloids and methylene chloride–ethyl acetate (80:20, v/v) (eluent B) for Rauwolfia alkaloids. During chromatography, the external pressure of the water cushion was 15 bars, the starting mobile phase pressure was 7 bars, and plates were developed on a distance of 16 cm at a mobile phase flow rate of 0.40 mL/min. The development time was 10 min. The separations were checked by visual observation under UV illumination (l = 365 nm) and after being sprayed with either Dragendorff’s or iodoplatinate reagent (11). Densitograms were recorded at 540 nm after visualization with Dragendorff’s reagent. Semipreparative OPLC of alkaloids. Sample applications were performed by successive deposits in the line (8 times). For chromatography, the water cushion pressure was 12 bars and the flow rate 0.6 mL/min. For each plant extract, the alkaloids are listed in the following elution order. For Strychnos nux vomica [ethyl acetate–isopropanol (4:1, v/v)], it was 1-brucine then 2-strychnine. In the case of opium and Datura stramonium, a gradient of elu (...truncated)