DETERMINATION OF RUTIN IN LEAVES OF SALIx TRIANDRA USING THE METHOD OF PLANAR CHROMATOGRAPHY

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Salix triandra L. is widely spread in the North Caucasus. It is a tall growing shrub or a small tree from 5 to 6 meters. Salix triandra has a spreading dense crown, long yellow-green shoots and long leaves [3]. Salix triandra refers to section Amygdalinae. The plants in this section contain rutin in leaves up to 5%. Rutin is a specific for a taxon of this section of willow [2]. Quantitative determination of rutin in leaves of Salix triandra was carried out 60’s of the twentieth century by the chromato-spectro-photometry. This method required a long preparation of the analyte. Nowadays literature describes available and simple methods for the determination of rutin in plant materials are. The rutin analysis in the leaves of Salix triandra was not described. The leaves of Salix triandra can be a potential raw material for the creation of drugs. The development of improved and reliable analytical methods of rutin analysis in leaves of Salix triandra remains actual. The aim of this work is to develop methods for identification and quantitative determination of rutin in the leaves of Salix triandra by using high performance thin-layered chromatography. To obtain the extract from leaves we used different methods. Method 1. The raw material (equals 3.0 g) (accurately weighed) was placed in a 100 ml conical flask, then added 50 ml of ethanol (70%) and left for 30 minutes at r.t. Then the flask was connected with reflux condenser, placed in a boiling water bath and since that moment was heated for 2 hours. The obtained extract was filtered through a paper filter into volumetric 50 ml flask, adjusted with alcohol to the mark and mixed well. Method 2. The raw material (equals 3.0 g) (accurately weighed) is treated with chloroform at room temperature (3 times×50 ml) to remove lipophilic substances. The chloroform is filtered, raw materials is dried in a laboratory fume hood for 15 minutes and transferred quantitatively into a 250 ml flask. Added 50 ml of ethanol (70%) and connected with reflux con- denser, placed in a boiling water bath and since that moment heated for 2 hours. The obtained extract was filtered through a paper filter into volumetric 50 ml flask, adjusted with alcohol to the mark and mixed well. Preparation of the reference solution (RS) rutin: 0.1 g rutin (Pharmacopoeia article 42- 2508-96) (accurately weighed) was placed into a volumetric 100 ml flask, dissolved in ethanol (95%) at heating on water bath. The solution was cooled to r.t. and adjusted to the mark with ethanol. The concentration of rutin in a solution 1.0 µg/µсl. To identify and quantify we used thin-layer chromatography with registration of analytical signal by densitometry. We used «Sorbfil» PTLC-P-A-UV plates 10x15 cm. For the mobile phase we used n-butanol glacial acetic acid- water system (4:1:1). The total distance from start to finish line is 9 cm. The stains detection was performed with ammonia vapors. We put on the start line (on the side 15 cm width) several samples of rutin RS 1.0; 2.0; 3.0; 4.0; 5.0 µl with corresponding rutin consentration equals 1.0; 2.0; 3.0; 4.0; 5.0 µg/µl. Between the samples on the start line we put samples of alcoholic willow extract from leaves equals 2.0; 3.0; 4.0; 5.0 µl. The samples were applied using a microsyringe MSH-10 (Agate). The plates were placed in chromatography chamber capacity 2000 cm3. After finishing of the chromatography process from chromatography chamber the plate was removed, dried in a laboratory fume hood at r.t. until all solvent vapors removed. We observed yellow stains on the dried plate. To enhance the color of the stains we put plate into concentrated ammonia vapors. The plates were scanned using a flatbed scanner «HP Scanjet 3670» (resolution 100 dpi), and their digital processing carried out by the computer program «video-densitometry Sorbfil» (Krasnodar). Quantitative determination was performed by absolute calibration (external standard) for the calibration curve «mass of matter - the area of the peak» (the linear approximation). Statistical analysis was performed in accordance with the General Pharmacopoeia article «Statistical analysis of the results of the experiment of chemical and biological testing». [1] On the control sample tracks we visually detected yellow stains Rf (0.64 ± 0.02) for all concentration. This correlates with color and Rf for standard samples. On the chromatogram of the alcohol willow extract we detected a peak corresponding to the peaks rutin of standard samples (Fig. 1). Plate efficiency is about 2000 theoretical plates. The limit of detection is 0.5 µg. Rutin calibration curve is shown in Fig. The regression equation and the correlation coefficient were calculated in the Microsoft Excel. The regression equation is: S = 4.75×103 m (free term «a» is insignificant). By using a thin-layer chromatography we carried out quantitative determination of rutin in leaves of Salix triandra. The research results for air-dry raw materials are shown in Table 1. The extraction of rutin from skimmed raw materials most effectively. Conclusions 1. We propose reliable method of qualitative and quantitative determination of rutin in the plant material. The method does not require the use of complex high-cost equipment. 2. The most complete rutin recovery occurs from leaves by the method 2. The results are significantly different (ttest - 25.7; ttab - 2.3). 3. The content of rutin in the leaves of Salix triandra, performed by described procedure, was 1.81 ± 0.06% for air dry raw materials and equals 1.91±0.06% at taking into account the moisture content of raw materials (5%).

About the authors

E. G Sannikova

Pyatigorsk Medical and Pharmaceutical Institute - branch of Volgograd State Medical University of the Ministry of Health of Russia

Pyatigorsk

T. D Mezenova

Pyatigorsk Medical and Pharmaceutical Institute - branch of Volgograd State Medical University of the Ministry of Health of Russia

Email: mezenova@yandex.ru
Pyatigorsk

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