DETERMINATION OF FLAVONOIDS OF WILLOW TRIANDRA (SALIX TRIANDRA L.), GROWING IN THE NORTH CAUCASUS

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Introduction. Flavonoids are phenolic compounds, which often consist of phenylchromon or phenylchroman found in all parts of higher plants as glycosides and/or aglycones. Their qualitative composition and quantitative content depends on the family and the species [1]. The works of V. A. Kompantsev shown that plants of the Salix genus are rich in various flavonoids. They found that leaves of Salix triandra contain up to 5% of rutin, in the cortex - salipurpozid and naringenin (about 1.5%) in the inflorescence of rutin and quercetin (about 1.5%) [2, 3]. Quantitative determination of rutin in the leaves of willow triandra (Salix triandra L.) was conducted in 1960s using spectrophotometry, which at that time required a long sample preparation (extraction of the raw material using TLC, the chromatographic layer extraction with methanol, removal of the solvent, dissolution in ethanol, optical density measurements). In parallel, in the same conditions, was used to determine optical density of the reference standard (RS) rutin [3]. Currently describes the available and simple methods for the determination of rutin in plant material. So in the cortex of the willow triandra by differential spectrophotometry found 0.41% of total flavonoids based on the rutin [4]. However, in the leaves and branches of willow triandra to date, such definitions have not been conducted. Given that leaves of willow triandra contain significant amounts of rutin and can be a potential raw material for the creation of medicines on their basis, it is essential to determine the amount of flavonoids in the leaves of willow triandra and its branches [5]. The purpose of this study is the determination of the amount of flavonoids in the Salix triandra, which grows in the North Caucasus, depending on the place and time of its collection. Methods. Leafy annual, not lignified up to 30 cm branches of willow triandra, growing in the North Caucasus, collected on the banks of the Podkumok river (Pyatigorsk, Stavropol Krai), Malka river (Kabardino-Balkarian Republic), Kuban river (Kochubeevsky region, Stavropol Krai) were the objects of the research. The raw materials were collected in different stages of the growing season in 2013, 2014 and 2015 from 20 to 30 of each month. Further, we compared the content of flavonoids in the branches and in leaves and branches without leaves separately. Determination of flavonoids. Obtaining extraction for identification of flavonoids: The raw material (equals 1.0 g) was placed in a conical flask, then added 20 ml of ethanol (70%). Than the flask was connected with reflux condenser, placed in a boiling water bath and since that moment heated for 30 min. The obtained extract was cooled, filtered through a paper filter and then we carried out the following reactions: cyanidin test; boric-citric reaction; the reaction with solution of lead acetate and solution of ammonia [6]. We used thin-layer chromatography for identification. For the mobile phase we used n-butanol-glacial acetic acid-water system (4:1:2). [1]. On the plate “Sorbfil” we put on the start line (on the side 15 cm width) samples of alcoholic Salix extract from branches, leaves and branches without leaves on 15 µl and samples of alcoholic reference standards on 5 µl (0.05%). The plates were placed in chromatography chamber. When the solvent front reaches the 12 cm the plate was removed, dried in a laboratory fume hood at r.t., viewed in visible and ultraviolet light at a wavelength of 365 nm. The substances detection was performed with ammonia vapors or 2% solution of aluminum chloride. For the quantitative determination the amount of flavonoids we used the method of differential spectrophotometry, based on the reaction of flavonoids with aluminum chloride [7]: the raw material (equals 1.0 g) was placed in a 100 ml conical flask, then added 30 ml of ethanol (70%) than the flask was connected with reflux condenser, placed in a boiling water bath and since that moment heated for 30 minutes. The hot extract obtained was filtered through a cotton swab into volumetric 100 ml flask, then placing the cotton swab into the jar with the raw material. Extraction was carried out twice. The solution was cooled at ambient temperature, filtering through a paper filter “blue tape” in the same flask, adjusted with alcohol to the mark., The re-extraction was filtered if necessary (solution A). 2 ml of solution A was placed in a 25 ml volumetric flask, add 10 ml of ethanol (95%) and 2 ml of alcoholic solution aluminium chloride (2%). After 10 minutes, was added 0.1 ml of acetic acid diluted and adjusted with alcohol (95%) to the mark. After 40 minutes, measured optical density in a cuvette with a layer thickness of 10 mm at SF-2000 spectrophotometer at a wavelength of 410 nm. The solution was used as a reference solution consisting of 2 ml of the appropriate extraction, 0.1 ml of acetic acid diluted and adjusted with alcohol (95%) to the mark in 25 ml volumetric flask. In parallel, measured optical density of the reference standard rutin. 1 ml (0.05%) alcoholic solution of rutin was placed in a 2 volumetric flask (25 ml), further we received as described above. The content of flavonoids amount was calculated as rutin. Validation of assessment methods for the quantitative determination of flavonoids was carried out in accordance with the requirements of the General Pharmacopoeia article.1.1.0012.15 “Validation of analytical procedures”[8]. For construction of the calibration curve were selected 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 ml of solution A (see methodology) and placed in a 25 ml volumetric flask was imported as described in the method. The content the amount of flavonoids based on the rutin defined in a specific aliquot in g/ml. According to the data obtained, we calculated the coefficient and free term of the regression equation in accordance with the requirements of the General Pharmacopoeia article.1.1.0013.15 “Statistical processing of results of chemical experiment” [9]. Results. The positive results of the following reactions indicate the presence of flavonoids (see table 1). Using thin-layer chromatography, we confirmed that leaves, branches and branches without leaves contain (on the chromatogram looked through the bright stain at the stain RS of rutin), rutin was also observed weakly colored stain at the stain RS of quercetin, but in the branches without leaves the stain of quercetin was observed.Further, using a differential spectrophotometric methodology adopted in SP XIII edition (2015) to determine the amount of flavonoids in medicinal plant in the raw materials, we determined the amount of flavonoids in the leaves, branches and branches without leaves of willow triandra. Given that all the samples contained rutin while the absorption maximum of the solutions of the samples was located in the region of maximum absorption of the complex of rutin with aluminum chloride - 410-412 nm, the content of the amount of flavonoids was calculated in terms of rutin. Since this method to determine the amount of flavonoids of Salix triandra is used for the first time, we have previously been carried out validation of methodology according to the criteria: specificity, precision, linearity and accuracy (see table 2) on the example of the amount of flavonoids in branches of willow triandra, collected in September 2013. The value of the correlation coefficient for this sample amounted to 0.994. The equation obtained allows making a conclusion about existence of linear dependence of optical density on the concentration of flavonoids in the concentration range from 0.0005 g/ml to 0.0026 g/ml. Accuracy of methodology viewed as a result of the study of linear validation of methodology: if a free term in the equation are statistically not significantly different from zero, then the use of such a method gives results free from systematic errors. According to the statistical processing of the obtained equation the coefficient of “a” has confidence interval 374±10, and free term of “b” not significantly different from zero, because its confidence interval is much longer, and this value is equal to 0.014 (0.002± 0.014). Since the free term b = 0, the equation becomes y = (375 ± 3)x and the proposed method is not burdened with systematic error [10]. Thus, based on the obtained results (see table. 2), we can conclude that the procedure used is one of the common characteristics in the following: specificity, linearity, accuracy, and repeatability. The validated method was used to determine the amount of flavonoids in the branches, leaves and branches without leaves, collected in 2013, 2014 and 2015, in different times and different parts of the North Caucasus. The analysis was conducted in the year of collection of raw materials (see table 3). Discussion. Thus, the results of the table 3 suggest that the greatest amount of flavonoids accumulated in leaves (from 2.661% to 3.676%) in branches this value is slightly smaller (from 1.73% to 3.26%). The branches without leaves flavonoid content is almost 10 times lower (0.117% to 3.266%). Due to the low content of flavonoids in raw materials, of harvested in 2015, the amount of flavonoids in the branches without leaves was conducted. The analysis on the example of raw materials harvested of alluvial lands have Podkumok over three years (September 2013, 2014 and 2015), suggests that the content of amount of flavonoids in raw materials of willow triandra, assembled in one place, but in different years exposed minor changes (2.14%, 2.80% and 2.61% in the branches and 3.19%, 3.23% and 3.49%, respectively, in the leaves). However, the flavonoid accumulation during the spring, summer and autumn in different years of collection is ambiguous. So, in 2014, the highest amount of flavonoids was accumulated in summer, while in 2015 it was the highest in autumn. It was also, found that the content of flavonoids depends on the place of collection of raw materials that should also, be considered when harvesting on an industrial scale. Conclusion. While studying the presence of flavonoids in the leaves and branches of Salix triandra, growing in the North Caucasus, we have found that the leaves and branches contain flavonoids: rutin and quercetin. Validation assessment of spectrophotometric methods for deter- mining the amount of flavonoids in raw materials of Salix triandra has confirmed the applicability. The error determination is within the range 2.5%. We found that the greatest quantity the amount of flavonoids accumulated in the leaves (from 2.6% to 3.7%), in the branches of this value is slightly smaller (from 1.7% to 3.3%). The branches without leaves the content of flavonoids is almost 10 times less (from 0.106% to 0.323%). It is shown, that the content of flavonoids is dependent on the place and time collecting of raw materials.

About the authors

E. G Sannikova

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

Email: je-je4ka2012@yandex.ru
Pyatigorsk, Russia

O. I Popova

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

Email: beegeeslover@mail.ru
Pyatigorsk, Russia

O. O Frolova

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

Pyatigorsk, Russia

A. U Airapetova

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

Email: asyapgfa@mail.ru
Pyatigorsk, Russia

References

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