PHENOLIC COMPOUNDS of COCHLEARIA OFFICINALIS, GROWING ON THE SOLOVETSKY ARCHIPELAGO

Strusovskaya O.G., Artemyeva A.P., Afonina S.A.

The Northern State Medical University, Arkhangelsk, Russia

PHENOLIC COMPOUNDS of COCHLEARIA OFFICINALIS, GROWING ON THE SOLOVETSKY ARCHIPELAGO

Cochlearia officinalis belongs to the Cruciferous family (Crucifereae L.) and is a wide-spread plant in the European North. Cochlearia officinalis has long been used in traditional medicine as an antiscorbutic drug and also in treating gastro-intestinal diseases. However, the chemical composition of the plant has not been sufficiently studied.

The purpose of this study was to determine the polyphenolic substances of Cochlearia officinalis. The subject of the research was air-dried raw material plants of second growing year, harvested during flowering period in summer 2011 on the islands of the Solovetsky archipelago and dried by air-cured in a well ventilated area at 22-24°C. Preliminary analysis of the qualitative composition of phenolic compounds was based on the method of one-dimensional chromatography in a thin layer of sorbent (TLC). For this purpose, air-dried raw material of Cochlearia officinalis was crashed and sieved through laboratory sieves S30/50 (State standart (GOST) 3826-82) with an aperture size of 1 mm. Ethyl alcohol 70% was used as an extractant (State standart (GOST) 51652-2000). The exact mass of crushed plant material (about 1.0 g) was placed in a round bottom flask from a heat-resistant glass with ground joint, then 10 ml of alcohol was added, and the flask was attached to a reflux condenser and placed on a boiling water bath. The heating was performed for 45 min. The resulting alcoholic extract was cooled, filtered through a blue ribbon filter (Technological conditions 11.3.03) and evaporated to a volume of approximately 0.5 ml. In order to select solvents system for optimal separating components of the extract, the following mixtures were used: chloroform-ethyl alcohol-water (26:14-3) [1], n-butanol-acetic acid-water (BAW) (4:1:5); BAW (4:1:2) [2] BAW (5:1:1) [3]. The resulting extract (10 μl) and 10 μl of 1% solutions of tracking substances (routine (Sigma-Aldrich 207671-50-9), quercetin (Sigma-Aldrich 117-39-5), kaempferol (Sigma-Aldrich 520-18-3 ) quercitin (Fluka 522-12-3); isoquercitin (Fluka 482-35-9); hyperoside (Sigma-Aldrich 482-36-0) and luteolin (Sigma-Aldrich 491-70-3)) were spread on the starting line of the plate «Silica gel 60 F254» (Merk). The plate then was placed in a chromatographic chamber, which had previously been saturated with eluent vapor for 6 hours. Chromatogram was made in the ascending manner. When the eluent front reached 10 cm, the plate was removed from the chromatographic chamber, air dried and viewed in the visible and ultraviolet light at a wavelength of 254 nm before and after treating the chromatograms with alcoholic solution 5% of aluminum chloride [4].

During the studies it was found out that the most complete separation of the components of the mixture is observed in the solvent system BAW (4:1:5). Under the selected conditions, on the chromatogram of alcoholic extract of Cochlearia officinalis four spots were detected with the values of Rf = 0,17; 0,43; 0,68 and 0.90. The spots with the values of Rf = 0,43; 0,68 and 0.90 corresponded to the values of Rf of the spots of standard samples of rutin, hyperoside, and quercetin, respectively. The spot corresponding to the routine was dominant one. To identify the spot with the value of Rf = 0,17, the chlorogenic acid (Sigma-Aldrich 327-97-9) and tannin (Sigma-Aldrich 1401-55-4) were used as tracking substances. In the course of the studies it was found that the spot with the value of Rf = 0,17 corresponded to chlorogenic acid.

Amount of flavonoids in the material studied was determined by the method of spectrophotometry, by reaction with the solution of aluminum chloride [1].

Optimal conditions for the quantitative analysis were selected experimentally by varying concentration of the eluent, the time of extraction, concentration and the amount of added solutions of aluminum chloride 2% and acetic acid, exposure time after adding of reagent solutions.

In the course of the research, a methodology was developed for determining the quantitative content of flavonoids in the tested plant materials: about 1.0 g (exact mass) of crushed (to a size of 1 mm) air-dried raw material of Cochlearia officinalis is placed in a round bottom flask from heat-resistant glass with ground joint, then 50 ml of ethyl alcohol 70% is added. The flask is attached to a reflux condenser and placed in a boiling water bath. The heating is carried out for 15 min, the extraction obtained is filtered through a paper filter into a volumetric flask with a capacity of 100 ml, preventing ingress of raw material particles to the filter. The extraction is carried out three times. The volume of the combined extracts is adjusted in a volumetric flask with ethyl alcohol 70% to the mark (solution A).

1 ml of solution A is placed in a flask with a capacity of 25 ml, 2 ml of 2% solution of aluminum chloride in ethyl alcohol 95%, 0.1 ml of diluted acetic acid is added, the volume of the resulting solution is adjusted with 95% ethyl alcohol to the mark (solution B). After 40 min, in quartz cells with thickness of layer 1 cm, the optical density of the solution is measured relative to the reference solution which is solution B without adding the solution of aluminum chloride. The reference solution of rutin 0.05% (RIS) prepared similarly to that under study is used as the standard. Differential spectra of the investigated and standard solutions are presented in Fig. 1.

The quantitative content of flavonoids content (X%) in air-dried raw material Cochlearia officinalis is calculated using the formula:

A – optical density of the solution under study;
A_s- optical density of the routine reference solution;
a – weigh of raw material, g;
a_s –weigh of standard routine sample, g;
b - moisture level in raw material,%.

The results of determination of the quantitative content of flavonoids in air-dried raw material of Cochlearia officinalis are presented in Table 1.

During the studies of the UV absorption spectrum of the alcoholic extract of air-dried raw material of Cochlearia officinalis, it was found out that the maximum optical density is observed at a wavelength of 270 nm ± 2 nm. Gallic acid 0.001% solution has the similar maximum of optical density (Fig. 2).

Quantitative determination of the amount of phenol carbonic acids was performed by direct UV spectrophotometry, using as a reference solution alcohol solution 0.001% of gallic acid [5]. For this purpose, approximately 1 g (exact mass) of crushed (to a particle size of 1 mm) air-dried raw Cochlearia officinalis was placed in a flask from a heat-resistant glass with ground joint with a capacity of 100 ml, then 50 ml of ethyl alcohol 70% was added. The flask was attached to a reflux condenser and heated on a water bath for 1 hour. After cooling, the extract was filtered into a volumetric flask of 100 ml. The process was repeated. The combined extract was adjusted with ethyl alcohol 70% to the mark (solution A).

5 ml of solution A was placed in a flask with a capacity of 100 ml, volume of solution in the flask was adjusted with ethyl alcohol 70% to the mark (solution B). Optical density of solution B was measured by a spectrophotometer in a cuvette with a layer thickness of 10 mm, using ethyl alcohol 70% as a reference solution. The optical density of the reference solution of gallic acid was measured simultaneously. To prepare the reference solution, about 0.05 g of gallic acid was placed in a flask with a capacity of 100 ml, 30 ml of ethyl alcohol 70% was added and the mixture was stirred until complete dissolution. The amount of solution in the flask was adjusted with the same solvent to the mark. 2 ml of the resulting solution was transferred into a volumetric flask of 100 ml. The amount of solution in the flask was adjusted to the mark with ethyl alcohol 70%.

The content of the sum of phenol carbonic acids (%) in terms of gallic acid and absolutely dry raw material was carried out using the formula:

D_x - optical density of the solution;
D₀ - optical density of the reference solution of gallic acid;
a_s - weight of gallic acid, g;
a - weight of air-dried raw Cochlearia officinalis, g
b - loss in weight when drying raw material,%.

The results of the quantitative determination of the amount of phenol carbonic acid in air-dry raw material Cochlearia officinalis are presented in Table 1.

Since the chromatogram of alcoholic extract of raw material of Cochlearia officinalis had a spot corresponding to chlorogenic acid, and during the study of the UV spectrum the maximum optical density was observed at a wavelength of 330 nm ± 2 nm (Fig. 4), which is the characteristic of the alcoholic solution of this substance, the amount of phenylpropanoids in the material under study was determined by direct UV spectrophotometry in terms of chlorogenic acid [6]. For that, about 1.0 g (exact mass) of air-dried raw material of Cochlearia officinalis was crushed (to a particle size of 1 mm) and placed in a flask from a heat-resistant glass with a ground joint with a capacity of 100 ml, then 50 ml of ethyl alcohol 70% was added. The flask was attached to a reflux condenser and heated on a boiling water bath for 1 hour. After cooling, the extract was filtered into a volumetric flask 100 ml through a blue ribbon filter and the volume of the solution in the flask was adjusted with ethyl alcohol 70% to the mark (solution A).

5 ml of solution A was placed in a flask with a capacity of 100 ml, volume of the solution in the flask was adjusted with ethyl alcohol 70% to the mark (solution B). Optical density of solution B was measured by a spectrophotometer in a cell with a layer thickness of 10 mm, using ethyl alcohol 70% as a reference solution. The optical density of the reference solution of chlorogenic acid was measured simultaneously (Fig. 3). To prepare the reference solution, about 0.05 g of chlorogenic acid was placed in a flask with a capacity of 100 ml, then 30 ml of 70% ethyl alcohol was added, and the mixture was stirred until complete dissolution. The amount of the solution in the flask was adjusted with the same solvent to the mark. 2 ml of the resulting solution was transferred into a volumetric flask of 100 ml. The amount of the solution in the flask was adjusted with ethyl alcohol 70% to the mark.
The content of phenylpropanoids (%) in terms of chlorogenic acid and absolutely dry raw material was calculated using the formula:

The results of the quantitative determination of the amount of phenylpropanoids in air-dry raw material of Cochlearia officinalis are presented in Table 1.

Thus, in the course of study it has been found out that air-dry raw material of Cochlearia officinalis contains chlorogenic acid and the following flavonoids: rutin, quercetin, and hyperoside, with routine as the dominant component. The quantitative content of biologically active substances in the studied materials was as follows: the amount of flavonoids - 0.66%, the amount of phenol carbonic acids - 1.62%, the amount of phenylpropanoids in terms of chlorogenic acid - 1.54%.

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