QUANTITATIVE DETERMINATION OF THE AMOUNT OF FLAVONOIDS IN THE HERB OF SALVIA FARINACEA BENTH

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Salvia L. genus is a big genus of perennial grassy plants and brushes from the Lamiaceae L. family. All species of this genus have essential oils; the whole range of them entered the cultivation as medicinal plants, for example Salvia officinalis L., Salvia sclarea L., Salvia aethiopis L. [5, 11]. Salvia farinacea Benth. is not a medicinal plant, there are small data about its chemical composition and quantitative content of some biologically active substances (BAS) in the literature. Decoctions and infusions of Salvia farinacea Benth. are widely used in folk medicine for headache treatment as well as for treatment of cold and toxications [12, 13] Salvia farinacea Benth. is an easy to keep, long blossom perennial grassy plant 1 m tall. Leaves are oviform, flexuose or smooth-edged, bare, with little fuzz along the veins. Inflorences are 15-20 cm long, on high flower stalks. Rhachis and cups have dense dark-blue, rarely light-gray short fuzz. Corolla is dark-blue, rarely white. Blossom is from the middle of August to the late autumn. Picturesque brushes with crumbly lilac-blue colored inflorences have a decorative value. They are a background for other annual plants in group beddings. In natural conditions they live several years, but they do not hibernate in more severe climate in an open ground, so they are cultivated as a summer plant. The long period of growing till blossom (about 100 days) conditions the necessity of germination method. Salvia farinacea Benth. native land are humid subtropics of America, Texas, New Mexico [5, 13, 15]. There are no data about the content of phenolic compounds in Salvia farinacea Benth. and their qualitative composition in scientific literature. Therefore the purpose for this study was to investigate a qualitative content of phenolic compounds in a herb of Salvia farinacea Benth. and to determine the quantitative content of flavonoids sum in raw materials [8, 11]. Experimental part Raw materials samples (herb) of Salvia farinacea Benth. was stocked in a botanical garden of the institute (PMPI) in 2010-20123 on the experimental grounds at blossom, where the plant was introduced from the seeds. The seeds were bought in flower stores of the Caucasus Mineral Water region (Pyatigorsk, Mineralnye Vody, Rassvet Company). Raw materials were dried out with air-shadow method. Preliminary qualitative reactions for the presence of flavonoids in the raw materials were carried out by the extraction, obtained in accordance with the method described in SP XI vol. 2, in “Herb of Hyperiaim” article [SP]. 1 ml of extracts was added with 2 ml of 2% solution of aluminum chloride in 95% ethanol and 7 ml of 95% ethanol; the solution was colored with greenish-yellow. 1 ml of extract were added with 0.5 g of zinc dust and 1 ml of concentrated hydrochloric acid. Gradually, after the heating on a water bath, pink color appeared (cyanidine probe) [3, 4, 6]. Preliminary qualitative reactions for the presence of flavonoids in the raw materials were carried out by the extraction, obtained in accordance with the method described in SP XI vol. 2, in “Herb of Hyperiaim” article [SP]. 1 ml of extracts was added with 2 ml of 2% solution of aluminum chloride in 95% ethanol and 7 ml of 95% ethanol; the solution was colored with greenish-yellow. 2 ml of extract were added with 0.5 g of zinc dust and 1 ml of concentrated hydrochloric acid. Gradually, after the heating on a water bath, pink color appeared (cyanidine probe) [3, 4, 6]. To establish a qualitative composition of phenolic compounds from the grass of Salvia farinacea Benth. we obtained extracts using 40% and 70% ethanol, raw materials and extragent relation was equal to 1:10. The process was carried out on a water bath at temperature 60-70ºС within one hour. Qualitative composition of phenolic compounds was studied using paper and thin-layer chromatography. 40% and 70% of alcohol extracts and 0.05% of alcohol solutions of rutin, quercetine, hyperoside, ferulic and gallic acids were placed as a point of 0.1 ml on the line of chromatographic paper with microsyringe. The paper with probes was dried out in the air and placed into a chamber with solvents blend: n-butanol - glacial acetic acid water (4:1:5), preliminary saturated within a year and chromatographed using ascending technique. Chromatography was done during 18-20 hours. After the solvents reached the fin- ish line the paper was took out, dried out, and examined in UV light. The results of chromatography are shown in the table 1. By using the paper chromatography we have identified rutin, quercetine, gallic and ferulic acids [1, 7, 10, 14]. For a thin-layer chromatography we used Sorbfil PTSH-AF-A-UF and Silufol UF plates. 0.05 ml spots of 40% and 70% alcohol extract of Salvia farinacea Benth. and alcohol solutions of standard samples were placed on a start line with a microsyringe. The plate with probes was dried out in the air during 10 minutes, then placed into a chamber with solvents blend butanol: glacial acetic acid: water (4:1:5), preliminary saturated during 40 minutes, and then it was chromatographed using ascending technique. After the reaching of 10 cm by the solvents, the plate was took out, dried out in the draught cupboard during 20 minutes and examined in UV-light. Chromatogramm showed spots (brown - rutin, yellow - quercetine). After the processing with 2% alcohol solution of aluminum chloride and heating during 3 minutes in a draught cupboard at temperature 100-105 ºС, the spots of fenolic compounds acquired yellow color in visible and yellow-green fluorescence in UV light [6, 7, 15]. Thus, using the methods of chromatographic analysis in alcohol extracts of Salvia farinacea Benth. we have found the presence of rutin, quercetine, heperoside, as well as gallic and ferulic acids. For more detailed investigation of phenolic compounds of the grass of Salvia farinacea Benth. we used HPLC [2, 9]. The study was carried out at the Department of Chemistry and High Technologies at Kuban State University. For this study we used an extract, obtained with 70% ethanol. Alcohol extract was diluted (1:10) with the blend of acetonithryl-phosphate buffer (1:1) before the chromatography. The study was carried out by using the reversed-phase chromatography, which provides a high definition and sensitivity. The conditions of chromatography were composed in respect to the physical and chemical properties of BAS in plant raw materials, changing the composition of the eluent and the detection wave length. The analysis was carried out in Shimadzu LC 20 Prominence chromatographer with the following computer processing using LC Solution program. Diode-array detection in UV spectrum allowed evaluation of spectral characteristics of divided components by the chromatography and to receive an additional information for their identification. Optimal results of elution were received in binary gradient system: acetonithryl-potassi- um dehydrophosphate water solution (0.04 М), acidified with phosphoric acid to pH 2.8. Chromatography was done on a Zorbax SB С18 column with sizes 150×2.1 мм, filled with sorbent particles 5 µm (Agilent). The eluent flow velocity was equal to 0.25 ml/min, thermostat temperature and detector cell amounted to 35 °С. Sample volume amounted to 5 µm. To carry out the analysis we used gradient stepped regime of eluent inflow (table 2). Detection of the substances was carried out at three wave lengths (280 nm, 322 nm, 370 nm). For their identification we used a retention time and absorption spectrums of standard samples, preliminary having chromatographed each of them separately. The results of the qualitative analysis of the alcohol extract from the grass of Salvia farinacea Benth. Are represented in the figure 1 and in the table 3. Using HPLC method we have discovered 20 substances of phenolic nature in the grass of Salvia farinacea Benth. Of this amount 4 phenolic compounds were identified (coffeic, p-coumaric, ferulic, and cinnamic), and three flavonoids (rutin, hyperoside, and quercetine). Qualitative determination of flavonoids was carried out following the method, described in SP XI vol. 2, in the “Herb of Hyperiaim” article [4]. The analytical sample of the raw materials was milled up to the sizes of particles, which pass through a sieve with 1 mm pores. About 1 g (precise weighing) of milled raw materials was placed into 150 ml flask with a slice, and then it was added with 30 ml of 40% alcohol. The flask was connected to the reversed condenser and heated on a boiling bath during 30 minutes, sometimes shaking to wash the raw materials particles from the flask walls. Hot extract was filtered through a cotton wool into a measuring flask 100 ml volume, so the particles of raw materials would not touch the filter. Cotton wool was placed into the flask for the extraction and added with 30 ml of 40% alcohol. Extraction was repeated two times in the conditions mentioned above, filtering the extract into the same measuring flask. After cooling, the volume of the extract was brought to the mark with 40% alcohol and mixed (solution A). One ml of the solution A was poured into a 25 ml flask, 1 ml of aluminum chloride in 95% alcohol was brought to the mark with 95% ethanol. In 40 minutes, an optical density of the solution was measured in the spectrophotometer at the wave length 415 nm in a cuvet with layer thickness of 10 mm. A solution composed from 1 ml of extract, 1 drop of acetic acid, and brought to the mark with 95% alcohol in the 25 ml measuring flask was used as a comparison solution. The calculation of flavonoid content was carried out in terms of rutin. At the same time we measured optical density of a solution of standard rutin sample. The content of total flavonoids in the grass of Salvia farinacea Benth. and absolutely dry materials in per cent (X%) was calculated using the following formula: Preparation of rutin standard sample solution: about 0.05 (accurate weighing) of rutin standard sample, previously dried out at 130- 135°C during 3 hours, are desolved in 85 ml of 95% ethanol in 100 ml measuring flask while heating in water bath, then cooled, quantatively relocated into 100 ml measuring flask, and the solution volume is brought to the mark by the same alcohol, and then blended [4]. The dynamics of the flavonoids accumulation was determined in the raw materials of Salvia farinacea Benth., gathered in 2011-2013. We have established that more intensive accumulation of flavonoids took place in the raw materials of Salvia farinacea Benth., gathered in more isolated experimental grounds. These plants had more leaves and the bush sizes. The content of total flavonoids in 2011 varied from 0.55% to 0.58%, in 2012 it was 0.56-0.60%, in 2013 there was 0.55-0.60%. Conclusions Thus, using the chromatographic analysis we have identified 9 substances of phenolic nature in the grass of Salvia farinacea Benth. There were flavonoids - rutin, quercetine, hyperoside; and 5 phenolcarbonic acids which included caffeic, p-coumaric, ferulic, gallic, and cinnamic acids. Quantitative content of total flavonoids in Salvia farinacea Benth. amounted to 0.55-0.60%. The studies conducted allowed broadening of scientifi data about phenolic compounds of Salvia farinacea Benth. and to offer the opportunity for the processing of Salvia farinacea Benth. in pharmacy and medicine.

About the authors

O. I Popova

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

Pyatigorsk

A. S Nikitina

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

Email: lina_nikitina@mail.ru
Pyatigorsk

E. A Azryakova

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

Pyatigorsk

References

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