Research Article |
Corresponding author: Liliia Budniak ( stoyko_li@tdmu.edu.ua ) Academic editor: Paraskev Nedialkov
© 2022 Svitlana Marchyshyn, Yuriy Mysula, Vitalii Kishchuk, Liudmyla Slobodianiuk, Elina Parashchuk, Liliia Budniak.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Marchyshyn S, Mysula Yu, Kishchuk V, Slobodianiuk L, Parashchuk E, Budniak L (2022) Investigation of amino acids content in the herb and tubers of Stachys sieboldii. Pharmacia 69(3): 665-672. https://doi.org/10.3897/pharmacia.69.e86227
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The aim of this research was the comparative study of the content of the amino acids in the herb and tubers of Stachys sieboldii. The study of the amino acid composition of the raw materials was carried out using high-performance liquid chromatography (HPLC). The results obtained have shown that the aerial parts of plants investigated have higher amino acid content than the underground organs. Free and bound L-aspartic acid, L-proline, and L-phenylalanine were present in the analyzed samples in the greatest amount. Moreover, L-cysteine was found only in Stachys sieboldii tubers in amounts (8.11 mg/g). This research established that Stachys sieboldii herb and tubers have the most suitable amino acids composition and are prospective for further pharmacological studies.
Stachys sieboldii , herb, tubers, amino acids, HPLC
The world pharmaceutical industry uses herbal raw materials as a basis for the creation of drugs (
The genus Stachys L., a numerous member of Lamiaceae family, includes about 300 species, dispersing tropical and temperate regions of Asia, the Mediterranean, southern Africa, and America (
Most species of this genus were previously analyzed in numerous studies concerning their pharmacological properties, therapeutic uses, and chemical composition. Nevertheless, the literature data on Stachys sieboldii activity are scarce, and little is known about chemical components with this plant.
Stachys sieboldii Miq. is widely distributed in Asia, North America, and Europe, and has been used for the treatment of different gastrointestinal problems, ischemic stroke, and senile dementia (
Hyeon Kyung Cho et al. (2014) isolated two new triterpene saponins named sieboldii saponin B and C from the methanol extract of tubers of Stachys sieboldii Miq. It should be noted that the S. sieboldii contain a rare tetrasaccharide – stachyose, which is similar in composition and properties to inulin and has an insulin-like effect (
However, few studies have focused on Stachys sieboldii primary metabolites and their relationship with its therapeutic properties. Amino acids are building blocks of proteins. Proteins are a fundamental part of all living animals and take part in virtually every process within the cell. Identification of amino acid sequence in a protein is of utmost importance in synthesizing new drugs for the treatment of diseases such as diabetes, cancer, and many more related to genetic disorders (
Herb and tubers of the Stachys sieboldii were collected on research grounds of Educational and Scientific Centre “Institute of Biology and Medicine”, Taras Shevchenko National University of Kyiv in November 2017. The raw materials were then dried, crushed and stored according to the general GACP requirements (
Standards of amino acids were of analytical grade (> 99% purity). The chemicals were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA) and were: L-histidine, L-arginine, L-aspartic acid, L-proline, L-lysine, L-alanine, L-valine, L-isoleucine, L-tyrosine, L-glutamic acid, L-cystine, L-serine, L-methionine, L-leucine, L-threonine, L-phenylalanine, Glycine (
The amino acids composition in the Stachys sieboldii herb and tubers are determined by HPLC method with a pre-column derivatization FMOC and OPA (
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 (Table
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.
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 (
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 of the peak area of the amino acids. The content of bound amino acids was determined by subtracting the content of free amino acids from their total content (
The analytical procedure has been validated to confirm its reliability. 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 0.015–0.625 μmol/ml range, and the linearity range for which the correlation coefficient that characterizes the regression line R2 was obtained, was examined visually.
The method was validated for linearity, limit of detection (LOD), limit of quantitation (LOQ) and precision. 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 and are shown in Table
Amino acid | Correlation coefficient R2 | Limit of detection LOD, µmol/ml | Limit of quantification LOQ, µmol/ml | Retention time (SD±0.01) |
---|---|---|---|---|
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 |
Appropriate to the polar nature of amino acids, derivatization is required prior to HPLC analysis (
The amino acid profiles of the herb and tubers of Stachys sieboldii were evaluated using the HPLC method (Figs
Number of peak on chromatogram | Amino acid Name | Amino acids content of Stachys sieboldii, mg/g | |||
---|---|---|---|---|---|
Herb | Tubers | ||||
Free | Bound | Free | Bound | ||
1 | L-aspartic acid | 0.24±0.02 | 16.22±0.06 | 0.23±0.02 | 9.15±0.04 |
2 | L-serine | 0.33±0.02 | 14.39±0.05 | 0.48±0.02 | n/d |
3 | L-glutamic acid | 0.63±0.01 | 8.11±0.04 | 0.71±0.01 | 2.91±0.02 |
4 | L-histidine | 0.13±0.01 | 3.53±0.03 | 0.29±0.01 | 0.85±0.01 |
5 | Glycine | 0.05±0.01 | 7.10±0.06 | 0.20±0.01 | 2.48±0.02 |
6 | L-threonine | 0.36±0.02 | 5.88±0.05 | 0.96±0.02 | 2.48±0.01 |
7 | L-arginine | 0.12±0.01 | 6.48±0.05 | 0.67±0.02 | 8.44±0.05 |
8 | L-alanine | 0.24±0.03 | 7.15±0.06 | 0.87±0.01 | 2.87±0.03 |
9 | L-tyrosine | 0.16±0.02 | 4.33±0.03 | 0.51±0.02 | 1.66±0.01 |
10 | L-cysteine | n/d | n/d | n/d | 8.11±0.07 |
11 | L-valine | 0.54±0.02 | 6.10±0.04 | 0.52±0.02 | 4.09±0.03 |
12 | L-methionine | n/d | 0.73±0.02 | 0.07±0.01 | 0.33±0.02 |
13 | L-phenylalanine | 0. 73±0.02 | 8.13±0.04 | 0.84±0.02 | 2.04±0.03 |
14 | L-isoleucine | 0.21±0.03 | 6.27±0.03 | 0.27±0.02 | 2.55±0.02 |
15 | L-lysine | 0.17±0.01 | 15.31±0.07 | 0.50±0.02 | 3.46±0.04 |
16 | L-leucine | 0.06±0.01 | 6.21±0.04 | 0.21±0.03 | 4.42±0.03 |
17 | L-proline | 0.64±0.02 | 6.49±0.04 | 1.16±0.03 | 2.49±0.03 |
The results show that the herb of Stachys sieboldii has the highest concentration of certain free amino acids, which possibly explains the presence of a pronounced antioxidant effect in the herb (
Experimental data showed that the herb of S. sieboldii is characterized by the highest content among bound amino acids, L-aspartic acid (16.22 mg/g), L-serine (14.39 mg/g), and L-lysine (15.31 mg/g) (Fig.
The presented results prove that L-proline (1.16 mg/g), L-threonine (0.96 mg/g), L-alanine (0.87), and L-phenylalanine (0.84) were predominates among free amino acids in Stachys sieboldii tubers (Fig.
Among the bound amino acids, the content of L-aspartic acid (9.15 mg/g) and L-arginine (8.44 mg/g) were the highest (Fig.
As a result of this work, the comparative analyses of amino acids in the herb and tubers of Stachys sieboldii using a sensitive HPLC method were carried out for the first time. It should be noted that the amino acid content in the aboveground organs is higher compared to their underground organs. High concentrations of the free amino acids such as L-aspartic acid, L-proline, and L-phenylalanine predominate in the analyzed samples. Due to the fact that amino acids take part in different metabolic processes in the body, it is important to study the pharmacological activities of Stachys sieboldii herb and tubers. Moreover, the predominant hydrophobic amino acid group (proline, alanine, isoleucine, leucine, and phenylalanine) in the herb and tubers of Stachys sieboldii is an important factor, that affects the antioxidant properties of the plant. Attention should be paid to future pharmacological studies on the nootropic activity of the analyzed raw materials.