Corresponding author: Alona Savych ( alonasavych@gmail.com ) Academic editor: Plamen Peikov
© 2021 Alona Savych, Svetlana Marchyshyn, Myroslava Harnyk, Victoria Kudria, Anna Ocheretniuk.
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:
Savych A, Marchyshyn S, Harnyk M, Kudria V, Ocheretniuk A (2021) Determination of amino acids content in two samples of the plant mixtures by GC-MS. Pharmacia 68(1): 283-289. https://doi.org/10.3897/pharmacia.68.e63453
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Due to the wide range of biologically active substances, the plant mixtures can influence the development of diabetes mellitus and its complications. Amino acids attract particular attention due to their ability to stimulate insulin secretion, reduce hyperglycemia and regulate metabolic processes in patients with diabetes. The aim of this study was to investigate the content of amino acids in the plant mixture samples: 1) Cichorium intybus roots, Elymus repens rhizome, Helichrysum arenarium flowers, Rosa majalis fruits, Zea mays columns with stigmas, 2) Urtica dioica leaf, Taraxacum officinale roots, Vaccinium myrtillus leaf, Rosa majalis fruits, Mentha piperita herb, which have proven antidiabetic activity in studies in vivo. The amino acids were separated by validated method of gas chromatography-mass spectrometry with pre-column derivatisation. Quantitative analyses of amino acids showed that the predominant components were L-proline in the sample 1 and L-leucine and L-proline in the sample 2 of the plant mixtures.
plant mixtures, amino acids, gas chromatography-mass spectrometry, diabetes mellitus
Diabetes mellitus is one of the priority problems of the WHO, which requires immediate solution, as the epidemiological situation is alarming – the number of patients is growing rapidly each year, leading to increased disability and mortality due to the development of macro- and microangiopathies (
One of these areas is using phytomedicines in the form of monotherapy in the mild stages of the disease and for its prevention, and in combination with traditional therapy for more severe forms of the disease. Phytotherapy is a promising and reasonable method, as it has a number of advantages – relatively low toxicity of herbal medicines, mild pharmacological effect and the ability to use for a long time without significant side effects, the ability to combine well with synthetic drugs (
Amino acids, in addition to their main function as precursors of protein synthesis, play a key role in many metabolic processes, because they have a powerful secretolytic activity – stimulate the secretion of insulin, glucagon, cortisol and insulin-like growth factor-1 (IGF-1) (
Therefore, the aim of study was to investigate the content of amino acids in some plant mixtures with previously studied antidiabetic activity in vivo (
The herbal raw materials harvested from June to August 2019 in the Ternopil region region and Charpathians (Vaccinium myrtillus leaf) (Ukraine) were used. After harvesting, the raw materials were dried, crushed and stored according to the general GACP requirements (
Plant mixture | Plant drug component | Portion in the mixture, % | Relative ratio |
---|---|---|---|
Sample 1 | Cichorium intybus roots | 26.32 | 5 |
Elymus repens rhizome | 26.32 | 5 | |
Helichrysum arenarium flowers | 21.05 | 4 | |
Rosa majalis fruits | 15.79 | 3 | |
Zea mays columns with stigmas | 10.52 | 2 | |
Sample 2 | Urtica dioica leaf | 20.0 | 1 |
Taraxacum officinale roots | 20.0 | 1 | |
Vaccinium myrtillus leaf | 20.0 | 1 | |
Rosa majalis fruits | 20.0 | 1 | |
Mentha piperita herb | 20.0 | 1 |
All applied reagents were of analytical grade (≥ 99% purity). Standard reagents including glycine, L-alanine, L-valine, L-leucine, L-serine, L-threonine, L-isoleucine, L-proline, L-asparagine, L-aspartic acid, L-glutamic acid, L-methionine, L-cysteine, L-phenylalanine, L-glytamine, L-lysine, L-histidine, L-tyrosine, L-tryptophan were purchased from Sigma-Aldrich Chemical Co. (USA), as well as hydrochloric acid, sodium hydroxide, methanol, pyridine, methyl chloroformate, chloroform, sodium bicarbonate. Water used in the studies was produced by MilliQ Gradient water deionizaton system (USA).
For the extraction of free amino acids the samples of the herbal raw material were grinded into a powder by laboratory mill, then about 0.1 g (accurately weighed) was selected and placed into vial with 2.0 mL of 0.1 N aqueous solution of hydrochloric acid. The extractions were carried out in the ultrasonic water bath at 50 °C for 3 hours.
Extraction of bound amino acids was carried out by adding 2 mL of 6 N an aqueous solution of hydrochloric acid to 0.03 g (accurately weighed) of powdered herbal raw materials. Hydrolysis was carried out for 24 hours in a thermostat at 110 °C.
The resulting extracts were centrifuged at 3000 rpm and the supernatants were evaporated to dryness on a rotary evaporator washing three times with distilled water to remove hydrochloric acid.
The dry samples of plant mixtures were dissolved in 390 μL of 1 M sodium hydroxide, then were added 333 μL of methanol and 67 μL of pyridine and mixed thoroughly for 5 seconds. To the resulting mixtures was added 80 μL of methyl chloroformate, stirred thoroughly for 60 seconds. The amino acid derivatives were extracted with 400 μL of chloroform followed by the addition of 400 μL of 50 mM sodium bicarbonate. The chloroform phase was used for future analysis (
The amino acids composition in the samples of the herbal raw materials was studied by gas chromatography-mass spectrometry (GC-MS) method using the Agilent Technologies (USA) system, model 6890N/5973inert (6890 gas chromatography with mass spectrometry detector 5973) and capillary column HP-5ms (30 m × 0.25 mm × 0.25 mm, Agilent Technologies) (
Amino acid identification was performed by comparing the retention times of amino acid standards and the presence of representative molecular and fragment ions (Table
Amino acids | tR, min | Molecular ion, m/z | Main fragmentary ions, m/z |
---|---|---|---|
Glycine | 14.77 | 147 | 88 |
L-alanine | 14.85 | 161 | 102, 88 |
L-valine | 18.56 | 189 | 146, 130, 115, 98 |
L-leucine | 19.57 | 203 | 144, 115, 102, 88 |
L-serine | 20.77 | 191 | 176, 144, 114, 100, 88 |
L-threonine | 21.11 | 205 | 147, 115, 100, 88 |
L-isoleucine | 21.31 | 203 | 144, 115, 101, 88 |
L-proline | 21.87 | 187 | 128, 84 |
L-asparagine | 21.97 | 262 | 146, 127, 95 |
L-aspartic acid | 23.90 | 219 | 160, 128, 118, 101 |
L-glutamic acid | 24.02 | 233 | 201, 174, 142, 114 |
L-methionine | 26.86 | 221 | 147, 128, 115 |
L-cysteine | 27.14 | 192 | 192, 176, 158, 146, 132 |
L-phenylalanine | 29.18 | 237 | 178, 162, 146, 131, 103, 91 |
L-glytamine | 29.74 | 276 | 141, 109, 82 |
L-lysine | 31.90 | 276 | 244, 212, 142, 88 |
L-histidine | 35.91 | 285 | 254, 226, 210, 194, 140, 81 |
L-tyrosine | 37.24 | 296 | 252, 236, 220, 192, 165, 146, 121 |
L-tryptophan | 38.91 | 276 | 130 |
The method was validated for linearity, limit of detection (LOD), limit of quantitation (LOQ) and precision. Linearity was performed by injecting a series of standard solutions (0.1–10.0 mg/100 g) with a threefold derivatization procedure and a single injection for each reference standard. The mean value and standard deviation, as well as regression analysis were calculated using Microsoft Excel software package 2016 (USA). The values for LOD and LOQ were calculated based on the data obtained during linearity testing in the low concentration range of the working in the test solution, using the following formulas: LOD = 3.3 * s / Slope; LOQ = 10 * s / Slope. Linearity testing was repeated with the same samples after a complete restart of the system with removement and re-installation of the column. Repeatability precision was determined by five-fold injection of the same sample in a row. For the resulting relative peak area of the quantifier ions the relative standard deviation (RSD) was calculated. To determine intra-day precision, five standard preparations of each reference standard with the same concentration were single injected and the resulting relative peak areas were used to calculate the RSD. Inter-day precision for the day of sample preparation and the two following days was specified by injecting five standard sample of each reference standard preparations once each on all three days. The RSD of the samples on that day together with the previous samples were calculated as above (
The results of qualitative and quantitative analyses of free and bound amino acids in the plant mixtures are represented in Figures
During GC-MS analysis, 5 amino acids in free form and 14 in bound form were identified in the sample 1 of the plant mixture (Figs
Number of peak on chromato-gram | Name of amino acid | tR, min | Content of amino acids, mg/g | |||
---|---|---|---|---|---|---|
Sample 1 | Sample 2 | |||||
Free | Bound | Free | Bound | |||
1. | Glycine | 14.77 | n/d | 2.066±0.12 | 0.559±0.01 | 6.147±0.05 |
2. | L-alanine | 14.85 | n/d | n/d | n/d | n/d |
3. | L-valine* | 18.56 | 0.268±0.01 | 2.721±0.01 | 0.277±0.01 | 6.049±0.07 |
4. | Nor-valine | 19.57 | Internal standart | |||
5. | L-leucine* | 20.77 | n/d | 3.336±0.02 | n/d | 8.707±0.16 |
6. | L-serine | 21.11 | n/d | 1.720±0.07 | 0.132±0.01 | 4.928±0.08 |
7. | L-threonine* | 21.31 | n/d | 0.515±0.01 | 0.400±0.01 | 0.561±0.03 |
8. | L-isoleucine* | 21.87 | 5.136±0.09 | 1.661±0.04 | 4.612±0.08 | 6.479±0.12 |
9. | L-proline* | 21.97 | 19.885±0.12 | 13.856±0.09 | 4.794±0.19 | 4.617±0.16 |
10. | L-asparagine | 23.90 | 0.170±0.03 | 0.609±0.05 | 0.254±0.02 | 0.242±0.07 |
11. | L-aspartic acid | 24.02 | 4.592±0.05 | 7.065±0.06 | 4.070±0.03 | 5.772±0.11 |
12. | L-glutamic acid | 26.86 | n/d | 1.462±0.03 | n/d | 1.270±0.08 |
13. | L-methionine* | 27.14 | n/d | 0.101±0.01 | n/d | 0.840±0.01 |
14. | L-cysteine | 29.18 | n/d | n/d | n/d | 0.427±0.01 |
15. | L-phenylalanine* | 29.74 | n/d | 2.008±0.08 | n/d | 5.822±0.09 |
16. | L-glytamine | 31.90 | n/d | n/d | n/d | 0.169±0.01 |
17. | L-lysine* | 35.91 | n/d | 0.966±0.03 | n/d | 3.676±0.09 |
18. | L-histidine* | 37.24 | n/d | n/d | n/d | n/d |
19. | L-tyrosine | 38.91 | n/d | 0.681±0.01 | n/d | 2.368±0.11 |
20. | L-tryptophan | 42.01 | n/d | n/d | n/d | n/d |
The analytical procedure has been validated to confirm its reliability. All the peaks of reference standards showed good linearity (R2 > 0.98) in a wide concentration range (0.1–10.0 mg/100 g). The results showed that the LODs and the LOQs of amino acids were in the range of 0.01–0.07 mg/100 g and 0.02–0.20 mg/100 g, respectively, indicating that the sensitivity of the method was satisfactory. The repeatability of the subsequent derivatization and GC-measurement of five standard samples of each reference standard with the same concentration resulted in precision values for the derivatization procedure. For intra- and inter-day precision, the RSD was in a range of 1.24% to 8.10%, which is acceptable (Table
Results of linearity data obtained for individual amino acids after GC-MS analysis.
Amino acids | Regression Curve | R 2 | LOD, mg/100 g | LOQ, mg/100 g |
---|---|---|---|---|
Glycine | y = 95.25x + 4.308 | 0.992 | 0.01 | 0.03 |
L-alanine | y = 81.03x + 2.372 | 0.996 | 0.01 | 0.04 |
L-valine | y = 108.40x – 1.502 | 0.996 | 0.02 | 0.06 |
L-leucine | y = 44.24x + 2.285 | 0.984 | 0.01 | 0.03 |
L-serine | y = 110.90x – 0.241 | 0.998 | 0.01 | 0.03 |
L-threonine | y = 77.24x + 3.222 | 0.990 | 0.01 | 0.04 |
L-isoleucine | y = 44.24x + 2.285 | 0.984 | 0.01 | 0.03 |
L-proline | y = 124.50x + 0.359 | 0.998 | 0.01 | 0.02 |
L-asparagine | y = 80.84x + 2.885 | 0.990 | 0.01 | 0.03 |
L-aspartic acid | y = 154.40x + 2.375 | 0.999 | 0.01 | 0.03 |
L-glutamic acid | y = 65.30x + 3.934 | 0.992 | 0.06 | 0.20 |
L-methionine | y = 198.80x + 0.203 | 0.999 | 0.01 | 0.03 |
L-cysteine | y = 189.40x + 2.673 | 0.994 | 0.01 | 0.03 |
L-phenylalanine | y = 149.50x + 9.568 | 0.990 | 0.01 | 0.04 |
L-glytamine | y = 44.24x + 2.285 | 0.984 | 0.06 | 0.20 |
L-lysine | y = 127.80x + 5.598 | 0.984 | 0.07 | 0.20 |
L-histidine | y = 69.28x + 1.579 | 0.992 | 0.03 | 0.10 |
L-tyrosine | y = 124.90x + 2.897 | 0.995 | 0.01 | 0.05 |
L-tryptophan | y = 189.40x + 2.673 | 0.994 | 0.01 | 0.04 |
The results of chromatographic examination indicate a sufficient content of amino acids, in particular essential, which have the ability to stimulate insulin secretion, reduce hyperglycemia and regulate metabolic processes in patients with diabetes. The predominant amino acids components were L-proline (19.885 mg/g in free form and 13.856 mg/g in bound form) in the sample 1 of the plant antidiabetic mixture and L-leucine (8.707 mg/g in bound form) and L-proline (4.794 mg/g in free form) in the sample 2 of the plant antidiabetic mixture, an essential amino acids with the most pronounced insulin secretolytic activity. The obtained data testify to the expediency of using the studied plant mixtures in order to optimize antidiabetic pharmacotherapy.