Determination of amino acids of some plants from Gentianaceae family

Liliia Budniak; Liudmyla Slobodianiuk; Svitlana  Marchyshyn; Olha Demydiak; Iryna Dakhym

doi:10.3897/pharmacia.68.e67052

Abstract

Medicines from plants are widely used in the complex treatment of different diseases every day. Therefore, theoretical and practical interest is the in-depth study of the herb of perspective plants like Centaurium erythraea Rafn. and Gentiana cruciata L. These plants have a long history of usage and interest of people. The aim of the study was to determine the content of amino acids in these plants. The amino acids composition and content in the herb of study species of the family Gentianaceae determined by the HPLC method. The results of the research revealed that the raw material of Centaurium erythraea Rafn. and Gentiana cruciata L. contains free and bound amino acids. Sixteen free and seventeen bound amino acids were identified in the herb of Centaurium erythraea Rafn. The herb of Gentiana cruciata L. contained thirteen free and fifteen bound amino acids. L-glutamic acid, L-arginine, L-aspartic acid, and L-cystine were predominant of Centaurium erythraea Rafn. herb. Amino acids L-lysine, L-serine, L-aspartic acid, and L-phenylalanine were present in the herb of Gentiana cruciata L. in the greatest amount. The metabolic processes in which these amino acids are involved connected to the medicinal properties of the study plants according to their use in official or nontraditional medicine.

Keywords

Centaurium erythraea Rafn., Gentiana cruciata L., herb, amino acids, HPLC

Introduction

The use of medicinal plants with therapeutic properties is as old as human civilization. Herbal, mineral and animal products have been the main source of drugs for the treatment and prevention of various diseases for a long time (Rates 2001; Budniak et al. 2020; Marchyshyn et al. 2021b). The use of plant raw materials is one of the areas of modern pharmaceutical science in the production of herbal medicines (Slobodianiuk et al. 2020). The main effect of using the medicinal plant is the regulation of metabolic disorders, as plant metabolites are close to the metabolites of the human body (Darzuli et al. 2019; Slobodianiuk et al. 2021a). Nowadays, the search for plants with a history of constant use and small side effects is of interest to our society (Huzio et al. 2020; Kurylo et al. 2020; Darzuli et al. 2021).

Species belonging to the Gentianaceae family are important because of their pharmacological use, namely the formation of very rich and specific secondary metabolites (Rybczyński and Wójcik 2019). The Gentianaceae family consists of 99 genera and about 1736 species, and it is distributed worldwide (Tomiczak et al. 2019). Many Gentians species are widespread in the Pyrenees, Himalayas and alpine mountains throughout Eurasia. The Gentianaceae family includes the following genera: Centaurium Hill., Gentiana L., Swertia L., Menyanthes L., etc (Budniak et al. 2021a). Plants belonging to these genera are a source of important pharmacologically active phytochemicals that have antimicrobial, antipyretic, anti-inﬂammatory, cyto- and hepatoprotective, stomachic, analgesic, and gastroprotective properties (Singh 2008; Tomiczak 2016). Many species of Gentianaceae family are very widely used for pharmaceutical purposes, namely Centaurium erythraea Rafn. and Gentiana cruciata L.

Centaurium erythraea Rafn., commonly known as centaury, is a pharmacologically important medicinal plant from the Gentianaceae family. Centaury is a biennial, sometimes annual, herb that grows in humid and semi-arid areas throughout the northern hemisphere (Guedes et al. 2019; Simonovic et al. 2021). Centaurium erythraea Rafn., an official plant that is part of the Ukrainian Pharmacopoeia, the European Pharmacopoeia, the British Herbal Pharmacopoeia, and many others (Stoiko and Kurylo 2018).

The aboveground parts of common centaury are a abundant source of various bioactive specialized metabolites, including phenolic compounds, terpenoids, steroids and alkaloids (Šiler and Mišić 2016). The most abundant bioactive specialized metabolites, secoiridoids and xanthones, exhibit antidiabetic, antioxidative, hepatoprotective, antibacterial and antifungal activities, and are commonly used for gastrointestinal disorders treating, appetite stimulation, digestion issues regulation, against fever, anemia, anorexia, rheumatism, hypertension and many other conditions. The prevailing monoterpenoids of the iridoid type in Centaurium erythraea Rafn. are secoiridoid glycosides sweroside, gentiopicrin and swertiamarin (Filipovic et al. 2019).

Centaurium erythraea is a plant used in traditional medicine for some cardiovascular disorders, namely hypertension (Chda et al. 2020). It has been used in traditional human medicine as a gastric, digestive, sedative, antipyretic and depurative agent (Mihaylova et al. 2019). Centaury is traditionally given as a bitter tonic to increase gastric secretions, to relieve dyspeptic discomfort and loss of appetite. Centaurium erythraea is part of some cosmetics and toiletries due to its soothing and astringent properties for allergies (Stoiko et al. 2017).

Some Gentiana species are endangered plants. They include Gentiana cruciata L. But modern biotechnological methods offer alternative approaches to traditional cultivation methods, leading to rapid micropropagation of Gentiana cruciata L (Budniak et al. 2021a). The Gentiana cruciata herb and roots are a source of secoiridoid glycosides, such as sweroside, swertiamarine and gentiopicroside (Szucs et al. 2002; Tomiczak 2020). Some pharmacological applications have been identified in Gentiana cruciata L., namely anticholinesterase, antigenotoxic, antioxidant and antimicrobial activities (Olennikov et al. 2019). Its root has been used in folk medicine for stomachic and sedative effects. In addition, Gentiana cruciata roots stimulate production of white blood cells (Hayta et al. 2011).

However, few studies have focused on plants’ primary metabolites and the relationship with their therapeutic properties. It was observed that the experimental content of amino acids in Centaurium erythraea Rafn. and Gentiana cruciata L. is absent. Studies of the compounds formed by plants as a result of defense mechanisms allow us to understand the molecular mechanism involved in their medicinal properties. Thus, the aim of our study was to determine the amino acid content of Centaurium erythraea Rafn. and Gentiana cruciata L.

Material and method

Plant materials

Centaurium erythraea Rafn. herb was collected in Ukraine, on the outskirts of Zboriv (Ternopil region) during the flowering period in July 2017. The herb of Gentiana cruciata L. was collected in Western Ukraine, at the territory of Volove, Ternopil region during the flowering period in 2017. The herbs of the study objects was authenticated by professor Svitlana Marchyshyn (TNMU, Ternopil, Ukraine). A vouchers specimens of Centaurium erythraea Rafn. no. 135 and Gentiana cruciata L. no. 133 are kept at the Department of Pharmacognosy and Medical Botany, TNMU, Ternopil, Ukraine. The study plants materials were dried using the conventional method and stored in paper bags in a dry place (Husak et al. 2018; Marchyshyn et al. 2021a; Slobodianiuk et al. 2021b).

Chemicals and standards

Only reagents of known analytical grade and distilled water or demineralized water or equivalent purity water are used. Standards of amino acids, including L-aspartic acid, L-histidine, L-arginine, L-lysine, L-alanine, L-proline, L-isoleucine, L-tyrosine, L-valine, L-glutamic acid, L-cystine, L-methionine, L-serine, L-threonine, L-leucine, L-phenylalanine, Glycine all these amino acid standards were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA), were of analytical grade (> 99% purity).

Derivatizing agents o-phthalaldehyde (OPA) and 9-fluorenylmethyl chloroformate (FMOC) were purchased in Merck. Acetonitrile (ACN) and hydrochloric acid (HCl) were from Sigma-Aldrich.

Sample preparation, HPLC determination of amino acids

The amino acids composition of Centaurium erythraea Rafn. and Gentiana cruciata L. herbs are determined by HPLC method with a pre-column derivatization FMOC and OPA.

Reference solutions of free amino acids have been made with distilled water at 0.03 M concentrations of each (weighed with analytical accuracy), stored in the refrigerator and further diluted before use, in every second day.

HPLC analysis of аmino acids was conducted using Agilent 1200 (Agilent Technologies, USA). Samples were analyzed using a column length Zorbax AAA – 150 mm, inner diameter – 4.6 mm, the diameter of sorbent grain 3 µ (Hypersil ODS (prepared by BST, Budapest, Hungary)). Mobile phase А – 40 mM Na₂HPO₄, pH 7.8; mobile phase В – CH₃CN:CH₃OH:H₂O (45:45:10, v/v/v). Gradient separation regime with a constant mobile flow rate of 1.5 mL/min. The temperature of the thermostat column is 40 °C.

The pre-column derivatization was conducted with a help of an automatic programmable regulations using OPA reagent and FMOC reagent. Identification of derivatized amino acids was done by a fluorescence detector (Jambor and Molnar-Perl 2009; Vons et al. 2018; Slobodianiuk et al. 2019). For the extraction of free amino acids of powdered the raw material (to the 130 mg of Centaurium erythraea Rafn.; to the 131 mg of Gentiana cruciata L.), put in a test flask, 0.1 mol/L water solution of hydrochloric acid was added. The extraction was performed in the ultrasonic water bath at 50 °C for 3 hours. Extraction of bound and free amino acids was performed by adding 2 mL of a water solution of 6 M hydrochloric acid to the powdered of the raw material (to the 131 mg of Centaurium erythraea Rafn.; to the 132 mg of Gentiana cruciata L.). Hydrolysis was conducted for 24 hours in a thermostat at 110 °C (Slobodianiuk et al. 2021d).

0.5 mL of centrifuged extract was vaporized on a rotary evaporator and then rinse three times with purified water to eliminate hydrochloric acid. The product received was resuspended in 0.5 mL water and filtered through membrane filters from restored cellulose with pores of 0.2 μm. Before recording the samples into the chromatographic column in the automatic software mode, fluorescence derivative amino acids were obtained.

Identification of amino acids was performed according to their hold-up time (using standards as a reference) at 265 nm. The quantitative content of amino acids is calculated from the value of the peak area of the amino acids (Slobodianiuk et al. 2021c).

Validation of the method

The validation method and the analysis procedure of the amino acid content were performed according to validation guides for EURACHEM analytical methods.

To evaluate the sensitivity and linearity of the signal in relation to the concentration, 8 linear calibrations were generated for each amino acid. The calibration curves of each amino acid were plotted in the 0.625–5 μmol/mL range, and the linearity range for which the correlation coeﬃcient that characterizes the regression line R2 was obtained, was examined visually.

The mass spectrometer operated in automatic scanning mode (SCAN). The performance parameters of the reference amino acid method, concentrations, limit of detection (LOD), limit of quantiﬁcation (LOQ) and calibration curves were statistically calculated using Statistica v 10.0 (StatSoft I nc.) program (Budniak et al. 2021b) and are shown in Table 1. All statistical tests were performed at a conﬁdence level of 95% and k = 2.

Table 1.

Download as

CSV

XLSX

Performance parameters of the amino acid determination method.

Amino acid	Correlation coefficient R²	Limit of detection LOD, µmol/mL	Limit of quantiﬁcation LOQ, µmol/mL	Retention time
L-aspartic acid	0.9999	0.005437	0.01779	2.46
L-glutamic acid	0.9997	0.001589	0.005342	4.78
L-serine	0.9999	0.004365	0.014549	7.35
L-histidine	0.9989	0.001235	0.005238	8.19
Glycine	0.9994	0.002345	0.004895	8.58
L-threonine	0.9996	0.01817	0.060565	8.75
L-arginine	0.9998	0.010724	0.035745	9.41
L-alanine	0.9987	0.003456	0.013567	9.97
L-tyrosine	0.9996	0.004678	0.014356	11.06
L-cystine	0.9995	0.001592	0.005308	12.19
L-valine	0.9999	0.002622	0.00874	12.96
L-methionine	0.9996	0.01785	0.06543	13.15
L-phenylalanine	0.9995	0.004532	0.01356	14.33
L-isoleucine	0.9999	0.01235	0.05426	14.51
L-leucine	0.9989	0.002897	0.018652	15.12
L-lysine	0.9999	0.096521	0.321737	15.39
L-proline	0.9998	0.003978	0.013261	18.91

Results and discussion

The amino acids profiles of the herb of Gentiana cruciata L. and Centaurium erythraea Rafn. were determined using HPLC method (Figs 1–4; Table 2).

Sixteen free amino acids were determined in the Centaurium erythraea Rafn. herb. Among them, seven amino acids are essential namely L-histidine, L-threonine, L-valine, L-phenylalanine, L-isoleucine, L-leucine, and L-lysine. One acid (L-arginine) is a semi-essential amino acid. The other seven amino acids are nonessential amino acids.

Figure 1.

HPLC chromatogram of free amino acids of Centaurium erythraea Rafn.

Figure 2.

HPLC chromatogram of bound amino acids of Centaurium erythraea Rafn.

Table 2.

Download as

CSV

XLSX

The content of the amino acids in the herb of Gentiana cruciata L. and Centaurium erythraea Rafn.

The name of the amino acid	The content of the amino acid
	Centaurium erythraea Rafn. herb				Gentiana cruciata L. herb
	Free		Bound		Free		Bound
	μg/mg	%	μg/mg	%	μg/mg	%	μg/mg	%
Laspartic acid	0.92±0.02	0.76	12.99±0.2	10.73	0.08±0.002	0.05	18.28±0.3	11.01
Lglutamic acid	1.56±0.05	1.29	16.0±0.1	13.22	0.06±0.001	0.04	12.13±0.4	7.31
L-serine	0.54±0.01	0.45	4.40±0.01	3.64	0.06±0.002	0.04	19.28±0.4	11.61
Lhistidine	0.13±0.004	0.11	1.83±0.05	1.52	—	—	4.48±0.03	2.70
Glycine	0.13±0.005	0.11	5.68±0.06	4.69	0.01±0.0004	0.01	11.18±0.3	6.73
Lthreonine	0.37±0.01	0.31	4.52±0.1	3.73	0.03±0.0009	0.02	8.64±0.2	5.20
L-alanine	0.57±0.02	0.47	5.80±0.1	4.79	0.09±0.004	0.05	10.62±0.3	6.40
Ltyrosine	0.15±0.004	0.12	1.78±0.06	1.47	0.02±0.0007	0.01	5.20±0.07	3.13
Lvaline	0.58±0.01	0.48	4.77±0.07	3.94	0.05±0.001	0.03	8.62±0.2	5.19
Lmethionine	—	—	1.03±0.03	0.85	—	—	—	—
Lphenylalanine	0.41±0.01	0.34	5.48±0.05	4.52	0.18±0.007	0.11	9.38±0.2	5.64
Lisoleucine	0.34±0.01	0.28	5.21±0.07	4.30	0.03±0.001	0.02	8.93±0.3	5.37
Lleucine	0.43±0.01	0.35	8.16±0.05	6.74	—	—	9.30±0.1	5.60
Llysine	0.46±0.02	0.38	7.53±0.06	6.22	0.03±0.0008	0.02	19.61±0.4	11.81
Lproline	0.51±0.01	0.42	5.21±0.05	4.30	0.07±0.002	0.04	10.65±0.3	6.41
Larginine	1.16±0.03	0.96	4.86±0.05	4.01	0.02±0.0007	0.01	9.04±0.1	5.44
L-cystine	0.28±0.01	0.24	17.30±0.3	14.29	—	—	—	—

Free Lglutamic acid, Larginine and Laspartic acid were present in the herb of Centaurium erythraea Rafn. in the greatest amount, their content was 1.56 μg/mg (1.29% from total content amino acids), 1.16 μg/mg (0.96%) and 0.92 μg/mg (0.76%), respectively. L-glutamic acid participates in many metabolic reactions, is a source of glucose, and holds its level in the blood (Okasha et al. 2019). Also, this acid is used as a conjugate because its effectiveness of anticancer medicine is better and reductions of its toxicity in attitude to normal cells. The synthetic amides of this acid show activity concerning Ehrlich ascites carcinoma (Dutta et al. 2013; Slobodianiuk et al. 2021). L-arginine is used by all cells and constitutes 5–7% of the total amino acids in normal human nutrition. This amino acid is used by the body for tissue repairing, protein synthesis, immune cell function, and the urea cycle. Also, L-arginine is converted to citrulline and operates as a vasodilator (Pahlavani et al. 2014). L‐aspartic acid is an amino acid present in endocrine glands, nervous tissues, and used as a stock for the food and pharmaceutical industries (D’aniello et al. 2000; Slobodianiuk et al. 2021).

Seventeen bound amino acids were identified of the Centaurium erythraea Rafn. herb. Eight amino acids are essential like L-lysine, L-phenylalanine, L-valine, L-methionine, L-isoleucine, L-histidine, L-leucine, L-threonine, and one acid, L-arginine is a semi-essential amino acid. The other amino acids are nonessential amino acids. L-methionine was found only in bound form. Among the bound amino acids L-cystine, L-glutamic acid, L-aspartic acid were present in the Centaurium erythraea Rafn. herb in the greatest amount, their content were 17.30 μg/mg (14.29%), 16.00 μg/mg (13.22%), 12.99 μg/mg (10.73%), respectively. L-cystine ensures elasticity of keratin, and therefore, it is a part of complexes of vitamins for an advance of appearance of the hair, and skin, different additives, and shampoos. L-cystine is also used in complex therapy for the treatment of bronchitis, Alzheimer’s disease, diabetes, as well at joint diseases (Blazheyevskiy et al. 2020).

The herb of Gentiana cruciata L. contained thirteen free amino acids and fifteen bound amino acids. L-phenylalanine dominates among free amino acids in the studied raw material, its content was 0.18 μg/mg (0.11%). L-phenylalanine is one of several essential amino acids. Human hemoglobin is one of the richest sources of phenylalanine, it’s content 9.6% by weight (Britannica, The Editors of Encyclopaedia 2018). Bound L-lysine, L-serine, and L-aspartic acid were present of the herb in Gentiana cruciata L. in the greatest amount, which was 19.61 μg/mg (11.81%), 19.28 μg/mg (11.61%), and 18.28 μg/mg (11.01%), respectively. L-lysine plays a great role in the output of carnitine, a nourisher responsible for converted fatty acids into energy and helps to reduce cholesterol (Ghoreyshi et al. 2019; Tufarelli et al. 2020; Slobodianiuk et al. 2021). L-serine plays a major role in cellular proliferation, brain development and function, and necessary for specific functions in the central nervous system (Koning et al. 2003).

Figure 3.

HPLC chromatogram of free amino acids of Gentiana cruciata L.

Figure 4.

HPLC chromatogram of bound amino acids of Gentiana cruciata L.

Conclusions

The amino acids, present in the herb of Centaurium erythraea Rafn. and Gentiana cruciata L., were studied by HPLC analysis. The results revealed that the raw material of the study species plants contains significant amounts of free and bound amino acids. Sixteen free and seventeen bound amino acids were identified of the Centaurium erythraea Rafn. herb. The content of L-glutamic and L-aspartic acids are high in the herb of Centaurium erythraea Rafn. and Gentiana cruciata L. allowing these amino acids to be considered differential markers of the study species plants. Also, the dominant free amino acid in the Centaurium erythraea Rafn herb is L-arginine (0.96% from total content amino acids); among the bound amino acids is L-cystine (14.29%). Among bound amino acids in the herb of Gentiana cruciata L., the dominant ones are L-lysine (11.81%) and L-serine (11.61%). Among free amino acids, essential amino acid phenylalanine (0.11%) prevails. Character metabolic processes in which these amino acids participate are associated with the therapeutic properties of research plants.

References

Blazheyevskiy MYe, Bondarenko NYu, Serdiukova YuYu, Yaremenko VD (2020) Determination of L-Cystine in Tablets Using the Chemiluminescence Method. News of Pharmacy 1: 15–20. https://doi.org/10.24959/nphj.20.23

Britannica, The Editors of Encyclopaedia (2018) “Aspartic acid”. Encyclopedia Britannica. https://www.britannica.com/science/aspartic-acid

Budniak L, Slobodianiuk L, Marchyshyn S, Demydiak O (2020) Determination of Arnica foliosa Nutt. fatty acids content by GC/MS method. ScienceRise: Pharmaceutical Science 6(28): 14–18. https://doi.org/10.15587/2519-4852.2020.216474

Budniak L, Slobodianiuk L, Marchyshyn S, Klepach P, Honcharuk Ya (2021a) Determination of carbohydrates content in Gentiana cruciata L. by GC/MS method. International Journal of Applied Pharmaceutics 13(1): 124–128. https://doi.org/10.22159/ijap.2021v13i1.39820

Budniak L, Slobodianiuk L, Marchyshyn S, Kostyshyn L, Horoshko O (2021b) Determination of composition of fatty acids in Saponaria officinalis L. ScienceRise: Pharmaceutical Science 1(21): 25–30. https://doi.org/10.15587/2519-4852.2021.224671

Chda A, El Kabbaoui M, Fresco P, Silva D, Gonçalves J, Oliveira AP, Andrade PB, Valentão P, Tazi A, El Abida K, Bencheikh R (2020) Centaurium erythraea extracts exert vascular effects through endothelium- and fibroblast-dependent pathways. Planta Medica 86: 121–131. https://doi.org/10.1055/a-1023-8918

D’aniello G, Tolino A, D’aniello A, Errico F, Fisher GH, Di Fiore MM (2000) The role of D-aspartic acid and n-methyl-D-aspartic acid in the regulation of prolactin release. Endocrinology 141(10): 3862–3870. https://doi.org/10.1210/endo.141.10.7706

Darzuli N, Budniak L, Hroshovyi T (2019) Selected excipients in oral solid dosage form with dry extract of Pyrola rotundifolia L. International Journal of Applied Pharmaceutics 11(6): 210–216. https://doi.org/10.22159/ijap.2019v11i6.35282

Darzuli N, Budniak L, Slobodianiuk L (2021) Investigation of the antibacterial and antifungal activity of the Pyrola rotundifolia L. leaves dry extract. PharmacologyOnLine 1: 395–403.

Dutta S, Ray S, Nagarajan K (2013) Glutamic acid as anticancer agent. Saudi Pharmaceutical Journal 21(4): 337–343. https://doi.org/10.1016/j.jsps.2012.12.007

Filipovic B, Šiler B, Nestorovic J, Banjanac T, Skoric M, Božunovic J, Mišic D (2019) Diploid vs. tetraploid Centaurium erythraea Rafn: a comparative study of regenerative in vitro potential and biosynthetic capacity. Lekovite Sirovine 39: 53–60. https://doi.org/10.5937/leksir1939052F

Ghoreyshi SM, Omri B, Chalghoumi R, Bouyeh M, Seidavi A, Dadashbeiki M, Lucarini M, Durazzo A, van den Hoven R, Santini A (2019) Effects of Dietary Supplementation of L-Carnitine and Excess Lysine-Methionine on Growth Performance, Carcass Characteristics, and Immunity Markers of Broiler Chicken. Animals (Basel) 9(6): e362. https://doi.org/10.3390/ani9060362

Guedes L, Reis PBPS, Machuqueiro M, Ressaissi A, Pacheco R, Serralheiro ML (2019) Bioactivities of Centaurium erythraea (Gentianaceae) decoctions: Antioxidant activity, enzyme inhibition and docking studies. Molecules 24: e3795. https://doi.org/10.3390/molecules24203795

Hayta S, Gurel A, Akgun IH, Altan F, Ganzera M, Tanyolac B, Bedir E (2011) Induction of Gentiana cruciatahairy roots and their secondary metabolites. Biologia 66(4): 618–625. https://doi.org/10.2478/s11756-011-0076-4

Husak L, Dakhym I, Marchyshyn S, Nakonechna S (2018) Determination of sugars and fructans content in Stachys sieboldii. International Journal of Green Pharmacy 12: 70–74. https://doi.org/10.22377/ijgp.v12i01.1527

Huzio N, Grytsyk A, Slobodianiuk L (2020) Determination of carbohydrates in Agrimonia eupatoria L. herb. ScienceRise: Pharmaceutical Science 6(28): 35–40. https://doi.org/10.15587/2519-4852.2020.221661

Jambor A, Molnar-Perl I (2009) Quantitation of amino acids in plasma by high performance liquid chromatography: Simultaneous deproteinization and derivatization with 9-fluorenylmethyloxycarbonyl chloride. Journal of Chromatography A 1216: 6218–6223. https://doi.org/10.1016/j.chroma.2009.06.083

Koning TJ, Snell K, Duran M, Berger R, Poll-The BT, Surtees R (2003) L-serine in disease and development. The Biochemical journal 371(Pt3): 653–661. https://doi.org/10.1042/bj20021785

Kurylo Kh, Budniak L, Volska A, Zablotskyy B, Klishch I (2020) Influence of phytocompositions on dynamics of change in basic glycemia and glycemia in oral glucose tolerance test in rats with streptozotocin-nicotinamide-induced diabetes mellitus type 2. Georgian medical news 300(3): 112–116.

Marchyshyn S, Budniak L, Slobodianiuk L, Ivasiuk I (2021a) Determination of carbohydrates and fructans content in Cyperus esculentus L. Pharmacia 68(1): 211–216. https://doi.org/10.3897/pharmacia.68.e54762

Marchyshyn S, Slobodianiuk L, Budniak L, Skrynchuk O (2021b) Analysis of carboxylic acids of Crambe cordifolia Steven. Pharmacia 68(1): 15–21. https://doi.org/10.3897/pharmacia.68.e56715

Mihaylova D, Vrancheva R, Popova A (2019) Phytochemical profile and in vitro antioxidant activity of Centaurium erythraea Rafn. Bulgarian Chemical Communications 51: 95–100.

Okasha RM, Al-Shaikh NE, Aljohani FS, Naqvi A, Ismail EH (2019) Design of Novel Oligomeric Mixed Ligand Complexes: Preparation, Biological Applications and the First Example of Their Nanosized Scale. International journal of molecular sciences 20(3): e743. https://doi.org/10.3390/ijms20030743

Olennikov DN, Gadimli AI, Isaev JI, Kashchenko NI, Prokopyev AS, Kataeva TN, Chirikova NK, Vennos C (2019) Caucasian Gentiana species: untargeted lc-ms metabolic profiling, antioxidant and digestive enzyme inhibiting activity of six plants. Metabolites 9(11): e271. https://doi.org/10.3390/metabo9110271

Pahlavani N, Jafari M, Sadeghi O, Rezaei M, Rasad H, Rahdar HA, Entezari MH (2014) L-arginine supplementation and risk factors of cardiovascular diseases in healthy men: a double-blind randomized clinical trial. F1000Research 3: e306. https://doi.org/10.12688/f1000research.5877.2

Rates SMK (2001) Plants as source of drugs. Toxicon 39(5): 603–613. https://doi.org/10.1016/S0041-0101(00)00154-9

Rybczyński JJ, Wójcik AI (2019) The effect of L-glutamine on the genetic transformation of embryogenic cell suspensions of gentian species (Gentiana lutea L., Gentiana cruciata L., and Gentiana kurroo Royle) using Agrobacterium tumefaciens. BioTechnologia 100(1): 5–18. https://doi.org/10.5114/bta.2019.83207

Šiler B, Mišić D (2016) Biologically Active Compounds from the Genus Centaurium s.l. (Gentianaceae). Studies in Natural Products Chemistry 49: 363–397. https://doi.org/10.1016/B978-0-444-63601-0.00011-9

Simonovic AD, Trifunovic-Momcilov MM, Filipovic BK, Markovic MP, Bogdanovic MD, Subotic AR (2021) Somatic Embryogenesis in Centaurium erythraea Rafn—Current Status and Perspectives: A Review. Plants 10(1): e70. https://doi.org/10.3390/plants10010070

Singh A (2008) Phytochemicals of Gentianaceae: a review of pharmacological properties. International Journal of Pharmaceutical Sciences and Nanotechnology 1(1): 33–36. https://doi.org/10.37285/10.37285/ijpsn.2008.1.1.4

Slobodianiuk L, Budniak L, Marchyshyn S, Basaraba R (2019) Determination of amino acids and sugars content in Antennaria dioica Gaertn. International Journal of Applied Pharmaceutics 11: 39–43. https://doi.org/10.22159/ijap.2019v11i5.33909

Slobodianiuk L, Budniak L, Marchyshyn S, Basaraba R (2020) Investigation of the hepatoprotective effect of the common cat’s foot herb dry extract. PharmacologyOnLine 3: 310–318.

Slobodianiuk L, Budniak L, Marchyshyn S, Kostyshyn L, Zakharchuk O (2021a) Analysis of carbohydrates in Saponaria officinalis L. using GC/MS method. Pharmacia 68(2): 339–345. https://doi.org/10.3897/pharmacia.68.e62691

Slobodianiuk L, Budniak L, Marchyshyn S, Parashchuk E, Levytska L (2021b) Experimental studies on expectorant effect of extract from Pimpinella saxifraga L. PharmacologyOnLine 1: 404–410.

Slobodianiuk L, Budniak L, Marchyshyn S, Sinichenko A, Demydiak O (2021c) Determination of Amino Acids of Cultivated Species of the Genus Primula L. Biointerface Research in Applied Chemistry 11: 8969–8977. https://doi.org/10.33263/BRIAC112.89698977

Slobodianiuk L, Budniak L, Marchyshyn S, Skrynchuk O, Kudria V (2021d) HPLC analysis of amino acids content in Crambe cordifolia and Crambe koktebelica leaves. International Journal of Applied Pharmaceutics 13(4): In press. https://doi.org/10.22159/ijap.2021v13i4.41265

Stoiko L, Dakhym I, Pokotylo O, Marchyshyn S (2017) Polysaccharides in Centaurium erythraea RAFN. International Journal of Research in Ayurveda and Pharmacy 8(Suppl 2): 252–255. https://doi.org/10.7897/2277-4343.082122

Stoiko L, Kurylo Kh (2018) Development of optimal technology of alcohol extract Centaurium erythraea rafn. herb. Archives of the Balkan Medical Union 53(4): 523–528. https://doi.org/10.31688/ABMU.2018.53.4.06

Szucs Z, Danos B, Nyiredy S (2002) Comparative analysis of the under-ground parts of Gentiana species by HPLC with diode-array and mass spectrometric detection. Chromatographia 56: 19–23. https://doi.org/10.1007/BF02494108

Tomiczak K, Mikuła A, Niedziela A, Wójcik-Lewandowska A, Domzalska L, Rybczyński JJ (2019) Somatic embryogenesis in the family Gentianaceae and its biotechnological application. Frontiers in Plant Science 10: e762. https://doi.org/10.3389/fpls.2019.00762

Tomiczak K (2020) Molecular and cytogenetic description of somatic hybrids Between Gentiana cruciata L. and G. tibetica King. Journal of Applied Genetics 61: 13–24. https://doi.org/10.1007/s13353-019-00530-x

Tomiczak K, Sliwinska E, Rybczynski JJ (2016) Comparison of the morphogenic potential of ﬁve Gentiana species in leaf mesophyll protoplast culture and ploidy stability of regenerated calli and plants. Plant Cell, Tissue and Organ Culture 126: 319–331. https://doi.org/10.1007/s11240-016-1000-y

Tufarelli V, Mehrzad-Gilmalek H, Bouyeh M, Qotbi A, Amouei H, Seidavi A, Paz E, Laudadio V (2020) Effect of Different Levels of L-carnitine and Excess Lysine-Methionine on Broiler Performance, Carcass Characteristics, Blood Constituents, Immunity and Triiodothyronine Hormone. Agriculture 10(4): e138. https://doi.org/10.3390/agriculture10040138

Vons B, Tryhubchak O, Grochovuy T, Chubka M, Bihunyak V (2018) Research of powders of the cryolyophilized xenoderm of porcine skin. International Journal of Applied Pharmaceutics 12(3): 657–664.