CC BY
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THE STUDY OF PHENOLIC COMPOUNDS OF SANICULA EUROPAEA L.
Grytsyk L.,
PhD in Pharmaceutical Sciences, Associate Professor Legin N., Assistant Svirska S.,
PhD in Pharmaceutical Sciences, Associate Professor
Grytsyk A.
Dr. in Pharmaceutical Sciences, Professor
Ivano-Frankivsk National Medical University.
Ivano-Frankivsk, Ukraine
ABSTRACT
The results of detection of phenolic compounds of Sanicula europaea L. herb using the methods of paper chromatography, high performance liquid chromatography and spectrophotometry were presented.
The content of 14 phenolic compounds in Sanicula europaea L. herb was established. It was investigated that epicatechin gallate is the dominant component among the tannins (11058.63 mg/kg), quercetin - among flavonoids (119.00 mg/kg), rosemary acid - among hydroxycinnamic acids (4933.50 mg/kg). The content of the total polyphenols, tannins, flavonoids and hydroxycinnamic acids in Sanicula europaea L. herb was established.
Keywords: phenolic compounds, tannins, hydroxycinnamic acids, flavonoids, coumarins, herb, Sanicula eu-ropaea L.
Introduction. Phenolic compounds are one of the most common and numerous classes of natural substances that have a variety of biological effects. Hy-droxycinnamic acids have antimicrobial, anti-inflammatory, hepatoprotective, choleretic, antioxidant and immunotropic effects [1]. Flavonoids are used as anti-inflammatory, choleretic, antiulcer, diuretic, antispas-modic, antihemorrhoidal, antiviral and antitumor agents. Futhermore, flavonoids are strong antioxidants and provide protection against oxidation and free radical damage [2]. Tannins are a group of biologically active substances that directly affect cell membranes, enzyme proteins, improve the metabolism of adrenaline, ascorbic acid, acetylcholine. They have anti-inflammatory, antioxidant, astringent, antimicrobial effects. As well as all phenolic compounds, they strengthen capillaries, increase blood clotting and have radioprotective effect [3].
The significant attention is drawn to plants that are used in folk medicine for a long time. Sanicula europaea L. of the family Apiaceae belongs to them. Sanicula europaea L. is widely used in folk medicine of the
Czech Republic, Romania, Germany, Austria and Ukraine. In folk medicine, Sanicula europaea L. is used as a hemostatic, anti-inflammatory, wound healing, expectorant, choleretic, astringent, diuretic, sedative, antimicrobial and antifungal agent.
There is a lack of literature data on the content of phenolic compounds in the Sanicula europaea L. raw material. Therefore, it is noteworthy to study biologically active substances of phenolic structure in Sanicula europaea L.
The aim of the study was to determine the composition and the content of phenolic compounds in Sanicula europaea L. herb to predict its possible pharmacological effects and and develop new drugs.
Materials and methods. The object of the study was Sanicula europaea L. herb. It was harvested in 2015 in Ivano-Frankivsk region (near Viktoriv village) during the mass growing season.
In order to detect phenolic compounds, Sanicula europaea L. herb was extracted with purified water and 70% ethanol in 1:10 ratio of raw material and the ex-tractant. Phenolic compounds were investigated in the
obtained extracts using identification reactions with 10% alcohol solution of potassium hydroxide and 10% solution of hydrochloric acid, 10% alcohol solution of potassium hydroxide and diazotized sulfanilic acid; with a solution of iron (III) ammonium sulfate, 1% solution of gelatin, 1% solution of quinine hydrochloride; with magnesium powder and hydrochloric acid, 3% solution of iron (III) chloride, 5% alcohol solution of aluminum chloride.
Further study of the composition of flavonoids and hydroxycinnamic acids was performed using paper chromatography in the system of solvents: «-butanol-acetic acid-water (4:1:2) and 15% acetic acid. The chro-matograms were air dried, examined under UV light and treated with chromogenic reagents such as ammonia vapor, 3% aluminum (III) chloride solution.
HPLC was used to study the composition of phenolic compounds in detail. The analysis was performed using the high performance liquid chromatograph Agilent 1200 3 D LC System Technologies (USA) equipped with the photometric diode-array detector UV-Vis G1315C, followed by processing the results using personal computer with Agilent ChemStation software [4, 5].
Reverse phase chromatography was performed to isolate the phenolic compounds using a 250 x 4.6 mm Discovery C18 chromatographic column with sorbent: silica gel modified with octadecyl groups. 0.005 N or-thophosphoric acid (solvent A) and acetonitrile (solvent B) were used as the mobile phases to separate hy-droxycinnamic acids, flavonoids and coumarins; a mixture of 0.1% trifluoroacetic acid, 5% acetonitrile and
Using method of paper chromatography based on spot fluorescence in UV light and mobility of substances on chromatograms, 8 substances of phenolic nature were revealed in Sanicula europaea L. herb: 5 substances as spots with blue and blue-green fluorescence were classified as hydroxycinnamic acids and 3 substances as dark spots and spots with yellow fluorescence were classified as flavonoid compounds.
deionized water (eluent A) and 0.1% trifluoroacetic acid in acetonitrile (eluent B) was used to separate the tannins. The detection wavelength for hydroxycinnamic acids was 320 and 330 nm; for tannins -255 and 280 nm; for flavonoids and coumarins - 255 and 340 nm. The analysis was performed at room temperature. The feed rate of the eluent was 0.8 ml/min (for flavonoids), 0.7 ml/min (for hydroxycinnamic acids),
0.1 ml/min (for tannins) [6]. The identification of the components was performed by the retention time of the standards and spectral characteristics, the content was determined by calculating the area of the peaks on the chromatogram.
The determination of the content of the total polyphenols and tannins in Sanicula europaea L. herb in terms of pyrogallol was performed using spectrophoto-metric method at a wavelength of 760 nm, determination of hydroxycinnamic acids - in terms of rosmarinic acid at a wavelength of 325 nm, determination of fla-vonoids - in terms of rutin. The optical density of the solutions was measured using the spectrophotometer Specord M 40.
Results and discussion. According to the results of identification reactions, tannins were established in aqueous extracts of Sanicula europaea L. herb; flavonoids, hydroxycinnamic acids and coumarins were established in water-alcohol extracts of Sanicula europaea L. herb.
The results of the identification reactions of phenolic compounds in the raw material are given in table.
1.
Table 1
Using HPLC method the content of 14 phenolic compounds, represented by flavonoids, hydroxycinnamic acids (Fig. 1), tannins (Fig. 2) and coumarins, was established in Sanicula europaea L. herb. Gallocatechin, catechin, epicatechin, catechin gallate, epicatechin gallate, gallic, chlorogenic, rosmarinic, caf-feic and ferulic acids, apigenin, quercetin, kaempferol and coumarin were identified by the retention time of the standard solutions.
Identification reactions of phenolic compounds in Sanicula europaea L. herb
ReagentIdentification reaction Analytical effect The presence of the analytical effect *
Coumarins
Lactone test Turbidity of the solution ±
Diazotized sulfanilic acid Brown color of the solution ±
Tannins
Solution of iron (III) ammonium sulfate Black-green color +++
1% gelatin solution Amorphous precipitate, which disappears with excess gelatin +++
1% quinine hydrochloride solution Light brown amorphous precipitate +++
Flavonoids
Cyanidine reaction according to Bryant Red-brown color ++
5 % solution of iron (III) chloride Dirty green color ++
5% alcohol solution of aluminum chloride Yellow color ++
Notes: "+" - the reaction is positive, the number of signs indicates the intensity; "-" - the reaction is negative.
Fig. 1. Chromatograms of hydroxycinnamic compounds of Sanicula europaea L. herb: A - wavelength 330 nm,
B - wavelength 320 nm; 1 - chlorogenic acid, 2 - rosmarinic acid, 3 - apigenin, 4 - caffeic acid, 5 - ferulic acid.
Fig. 2. Chromatograms of tannins of Sanicula europaea L. herb: A - wavelength 280 nm, B - wavelength 255 nm; 1 - gallocatechin, 2 - catechin, 3 - epicatechin, 4 - catechin gallate, 5 - epicatechin gallate, 6 - ellagic acid.
The results of the study of composition and content of phenolic substances in Sanicula europaea L. herb using HPLC method are given in table 2.
Table 2
The content of phenolic compounds, mg/kg
Tannins Hydroxycinnamic acids
Gallocatechin 3129,25 Chlorogenic acid 324,38
Catechin 840,75 Rosmarinic acid 4933,50
Catechin gallate 969,13 Caffeic acid 257,50
Epicatechin 479,88 Ferulic acid 89,63
Epicatechin gallate 11058,63 Flavonoids
Ellagic acid 25,88 Quercetin 119,00
Coumarins Kaempferol 18,63
Coumarin | 97,38 Apigenin, 108,50
Among tannins, the highest content in Sanicula europaea L. herb was established for epicatechin gallate (11058.63 mg/kg), among flavonoids - for querce-tin (119.00 mg/kg), among hydroxycinnamic acids -
for rosmarinic acid (4933.50 mg/kg). Coumarins of Sanicula europaea L. herb are represented by coumarin with the content of 97.38 mg/kg.
The results of determining of phenolic compounds content in Sanicula europaea L. herb are given in table. 3.
Table 3
The content of phenolic compounds in Sanicula europaea L. herb
№ Group of phenolic compounds Method of analysis Wavelength, nm Content, %, x ± Ax n = 9
1. Total polyphenols Spectrophotometric in terms of pyrogalol 760 10,43±0,03
2. Tannins Spectrophotometric in terms of pyrogalol 760 7,28±0,03
3. Hydroxycinnamic acids Spectrophotometric in terms of rosma-rinic acid 325 1,26±0,01
4. Flavonoids Spectrophotometric in terms of rutin 408 2,37±0,01
Thus, it was found that the content of the total polyphenols in Sanicula europaea L. herb was 10.43±0.03%, tannins - 7.28±0.03%, hydroxycinnamic acids - 1.26±0.01%, flavonoids - 2.37±0.01%.
Conclusions.
1. The composition of phenolic compounds of Sanicula europaea L. herb was studied using paper chro-matography and HPLC methods.
2. The content of 14 phenolic compounds, which were represented by tannins, hydroxycinnamic acids, flavonoids and coumarins, was determined using HPLC method.
3. It was established that in Sanicula europaea L. herb the dominant compound among hydroxycinnamic acids was rosmarinic acid, among flavonoids -kaempferol, among tannins - epicatechin gallate, among coumarins - coumarin.
4. The content of total polyphenols, tannins, flavo-noids, hydroxycinnamic acids was established.
References
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MACROSCOPIC AND MICROSCOPIC MORPHOLOGICAL FEATURES OF VERBENA L. SPECIES
Grytsyk A.,
Doctor in Pharmaceutical Sciences, Professor
Posatska N., Master of Pharmacy, Assistant Svirska S.
PhD in Pharmaceutical Sciences, Associate Professor Ivano-Frankivsk National Medical University.
Ivano-Frankivsk, Ukraine
ABSTRACT
Verbena L. species are herbaceous plants or shrubs. They have strong root system and are able to grow up to 100 cm. Their stems are erect or branched, quadrangular and covered with hairs that are pressed to the surface. The leaves are small, opposite, oblong. They are located on the petioles. Inflorescences are at the top of the plants and are collected in panicles, long and spike-shaped, grow in the axils of the middle and upper leaves. Verbena L. flowers have different colors and shades. The fruits have a convex dihedral shape. They are linear, brown, slightly oblong and usually wrinkled.
The light microscopy and scanning electron microscopy are used in modern scientific and applied researches to perform in-depth study of the micromorphological structure of leaves, stems and flowers of plants. These methods give more accurate images of plant diagnostic features. Anatomical features of leaves, stems and flowers of Verbena officinalis L. were used to specify the microscopic characteristics of medicinal plant raw materials.
Key words: epidermis, leaf, stem, flower, Verbena officinalis L.
