DETERMINATION OF AMINO ACIDS AND SUGARS CONTENT IN ANTENNARIA DIOICA GAERTN

Objective: The purpose of our study was to determine sugars and amino acids content of Antennaria dioica Gaertn. herb. In continuation of the investigation of biologically active substances from Antennaria dioica Gaertn., it advisable to study the qualitative composition and quantitative contents of sugars and amino acids from the herb of this plant. Methods: The herb of Antennaria dioica Gaertn. was analyzed for the content of sugars by GC/MS. The amino acids were identified and quantified by HPLC method. Results: The results of GC/MS analysis showed that in Antennaria dioica Gaertn. herb D-glucose had the highest content (7.16±0.09) mg/g, followed by D-fructose (5.27±0.06) mg/g and D-saccharose (6.72±0.08) mg/g. In the raw material a large amount of monosaccharides derivative–Myoinositol was revealed, a content of which was (2.12±0.06) mg/g. We determined 17 bound and 16 free amino acids in the Antennaria dioica Gaertn. by HPLC method. Conclusion: The contents of primary metabolites provide opportunities for creating medicine and food supplements. The results show that Antennaria dioica Gaertn. is a rich source of these important biologically active substances. The resulting data will be used with the further purpose to produce new drugs of natural origin.


INTRODUCTION
The importance of medicinal plants did not reduce the annual increase in the number of synthetic medicines, which often model biologically active substances of plants and are their chemical equivalents. The most interesting are medicinal plants that have a long history of use and have been shown to possess low side effects and are well tolerated by patients, regardless of age [1].
The most representative plants for the treatment of diseases are plants of families Asteraceae, Rosaceae, Fabaceae, Lamiaceae, Boraginaceae, Apiaceae, Poaceae.
The Asteraceae family includes around 23,000 species in the world and is the largest family of flowering plants [2][3][4].
The diversity and heterogeneity of this family justify the great importance of its individual members, which are known and used from ancient times, not only as food sources or as spices, but also for medicinal purposes [5,6].
Antennaria dioica Gaertn. is a perennial plant in the family Asteraceae [7]. It grows in almost throughout the whole of Ukraine on the wastelands and sandy or stony places [8].
Antennaria dioica Gaertn. traditionally used to treat biliary and respiratory ailments [9,10], and also have astringent and hemostatic properties [9,11]. Scientific sources give few data regarding the chemical composition of this species. Merili et al. [10] studied the extracts obtained from the flowers of Antennaria dioica Gaertn. and isolated chlorogenic and ursolic acids, apigenin-7-O-glucoside, and luteolin-7-O-glucoside. This is the only scientific study that describes the flavonoid composition of this species, and it does not establish a connection between the potential bioactivities of the species and the isolated compounds [2,12].
However, there is no published information about some primary metabolites, such as polysaccharides and amino acids.
Thus, the aim of our research was to determine the content of amino acids and polysaccharides in aerial parts of Antennaria dioica Gaertn. collected from natural habitat.

Plant materials
Aerial parts of the Antennaria dioica Gaertn. were collected in Western Ukraine, Vyzhnytsya district, Chernivtsi region (N 48 °13'23.2" E 25 °11'42.0"), during a mass flowering period in 2016. The raw material was authenticated by prof. Svitlana Marchyshyn (TNMU, Ternopil, Ukraine). A voucher specimen no. 189 is kept at the Department of Pharmacognosy and Medical Botany, TNMU, Ternopil, Ukraine.  Sample preparation, gas chromatography coupled with mass spectrometry (GC/MS) analysis and high-performance liquid chromatography (HPLC) method

GC/MS determination of sugars
GC/MS analysis of sugars was performed using gas chromatograph Agilent 6890N with mass detector 5973 inert (Agilent Technologies, USA). The initially set up oven temperature at 160 °C and held for 8 min, then at the rate of 5 °C/min raised to 240 °C and finally kept at this point for 6 min. The detection was performed in the SCAN mode at the width range of 38-400 m/z. Helium was used as the carrier gas at a constant flow rate of 1.2 ml/min.
For the determination of sugars 500 mg of powdered Antennaria dioica Gaertn. was added to 500 micrograms of methanol solution with sorbitol (internal standard). Extraction was performed in the ultrasonic bath at 80 °C for 4 h. Then, 2 ml of the extract were evaporated to dryness and 0.3 ml of derivatization reagent (hydroxylamine hydrochloride in pyridine/methanol (4:1 v/v)) was added. For acetylation of aldonitrile derivatives, 1 ml of acetic anhydride was subsequently added to the samples and incubated at 75 °C for 15 min. 2 ml of dichloroethane was added, and the excess removed by the double extraction with water and 1 M hydrochloric acid. Dichloroethane layer was dried and dissolved in the mixture of ethyl acetate/heptane (1:1 v/v).
Identification was based on comparing their retention times with retention times of standards of the NIST 02 mass spectral library. Quantification was done using sorbitol (1 mg/ml) added to the sample [13][14][15].

HPLC determination of amino acids
The amino acids composition of Antennaria dioica Gaertn. herb is determined by HPLC method with a pre-column derivatization 9fluorenylmethyl chloroformate (FMOC) and o-phthalaldehyde (OPA). The pre-column derivatization was performed in an automatic programmable mode using OPA reagent and FMOC reagent. Identification of derivatized amino acids was done by using a fluorescence detector [16,17]. For the extraction of free amino acids to the 131 mg of powdered the raw material, placed in a vial, 0.1 mol/l aqueous solution of hydrochloric acid was added. The extraction was performed in the ultrasonic water bath at 50 °C for 3 h. Extraction of bound and free amino acids was carried out by adding 2 ml of an aqueous solution of 6 N hydrochloric acid to 136 mg of powdered the raw material. Hydrolysis was carried out for 24 h in a thermostat at 110 °C. 0.5 ml of centrifuged extract was evaporated on a rotary evaporator and then rinse three times with purified water to remove hydrochloric acid. The product obtained was resuspended in 0.5 ml water and filtered through membrane filters from regenerated cellulose with pores of 0.2 μm. Before entering the samples into the chromatographic column in the automatic program mode , fluorescence derivative amino acids were obtained.
Identification of amino acids was done according to their retention time (with using standards as a reference) at 265 nm. The quantitative content of amino acids is calculated from the value of the of the peak area of the amino acids.
The amount of amino acids in µg/mg was calculated by the following equation: Where: C -concentration, obtained from the chromatogram by calculating the reference solution and the test solution; V -the volume of solvent for extraction; m -is a mass of plant material [18].

Statistical analysis
All the assays were carried out in five times. The results were expressed as mean values and standard deviation. Values were determined using Statistica v 10.0 (StatSoft I nc.) program. The level of significance was set at *p<0.05 for all statistical analyses.

GC/MS chromatography analysis of sugars
The GC/MS method determined the qualitative composition and quantitative content of monosaccharides, their derivatives, and saccharose. The Antennaria dioica Gaertn. herb contains D-fructose, Dglucose, D-saccharose, L-rhamnose and Myo-inositol ( fig. 3, table 1). The predominant ones were D-glucose (7.16±0.09) mg/g, D-fructose (5.27±0.06) mg/g and D-saccharose (6.72±0.08) mg/g. Among the monosaccharides, D-glucose dominates in the studied raw material. The basic function of glucose is to provide energy for physiological processes. Of all tissues and organs, mammalian, neurons and the brain have the supreme energy demand. The processes requiring mental effort are deteriorated if the concentration of this aldohexose reduces [19,20]. In the Antennaria dioica Gaertn. herb, a large amount of monosaccharides derivative-Myo-inositol was revealed, a content of which was (2.12±0.06) mg/g. Myo-inositol decreases serum androgen concentrations, reduces circulating insulin, improves glucose tolerance and other metabolic values altered associated with insulin resistance in women affected by Polycystic ovary syndrome [21][22][23]. Table 2 presents the amino acids composition of the herb of Antennaria dioica Gaertn. The HPLC method we identified in the raw material sixteen free amino acids ( fig. 4) and seventeen bound amino acids ( fig. 5), of which nine were essential.    Note: ‫٭‬ -essential amino acid.

HPLC determination of amino acids
The dominant components of amino acids from Antennaria dioica Gaertn. with respect to total content were L-proline, L-aspartic acid, L-glutamic acid, L-alanine, and L-valine. Free L-proline was present of the Antennaria dioica Gaertn. in the greatest amount (3.06±0.07) μg/mg. Proline is a proteinogenic amino acid with an exceptional conformational rigidity, and is essential for primary metabolism [24,25]. Valine support the balance of branched chain amino acids, whereas alanine is involved on hepatic autophagy, transamination and gluconeogenesis [26][27][28]. The amounts of the other amino acids were less. Free amino acids in the herb of Antennaria dioica Gaertn. included 9 essential amino acids (His, Thr, Val, Met, Phe, Ile, Leu, and Lys) in addition to L-arginine, which is considered conditionally essential because it is extremely necessary for young people [29][30][31]. L-cystine was not found.
Seventeen bound amino acids were identified in the herb of Antennaria dioica Gaertn. The quantitative contents of all amino acids showed a tendency to increase significantly after hydrolysis. The contents of bound L-glutamic acid (7.38±0.20) μg/mg, L-aspartic acid (5.38±0.12) μg/mg, L-cystine (3.29±0.09) μg/mg and L-lysine (3.31±0.16) μg/mg were the greatest (table 2). Aspartic acid function is essential for pyrimidine, asparagine, purine and inositol synthesis. Glutamic acid take part of the synthesis of glutathione [26,28]. However, L-cystine was found only in bound form.

CONCLUSION
We determined the primary metabolites in the Antennaria dioica Gaertn. herb. Using the GC/MS method, we identified the monosaccharides, their derivatives, and saccharose. The main compounds identified in the herb of Antennaria dioica Gaertn. were D-glucose, D-saccharose, and Myoinositol. The amino acids composition of Antennaria dioica Gaertn. herb was determined by the HPLC method. We determined sixteen free and seventeen bound amino acids which were identified in the raw material. The content of bound L-glutamic acid increased compared with the other acids from (0.34±0.03) μg/mg to (7.38±0.20) μg/mg; the content of bound L-aspartic acid increased from (0.33±0.02) μg/mg to (5.38±0.12) μg/mg. The contents of primary metabolites provide opportunities for creating medicine and food supplements. The results show that Antennaria dioica Gaertn. is a rich source of these important biologically active substances.