Corresponding author: Alona Savych ( alonasavych@gmail.com ) Academic editor: Plamen Peikov
© 2022 Alona Savych, Svitlana Marchyshyn, Liudmila Mosula, Oksana Bilyk, Ihor Humeniuk , Alexandra Davidenko.
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, Mosula L, Bilyk O, Humeniuk I, Davidenko A (2022) Analysis of amino acids content in the plant components of the antidiabetic herbal mixture by GC-MS. Pharmacia 69(1): 69-76. https://doi.org/10.3897/pharmacia.69.e77251
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Medicinal plants and their combinations due to the wide range of biologically active substances can influence on various links of the pathogenetic mechanism of development of DM type 2 and its complications. One of such combinations is an antidiabetic herbal mixture (Urticae folia, Rosae fructus, Myrtilli folia, Menthae folia and Taraxaci radices) with established hypoglycaemic, hypolipidemic, antioxidant, hepatoprotective, pancreatoprotective activity in previous pharmacological studies in vivo and in vitro and defined phytochemical composition. Thus, the aim of this study was to identify and establish the content of amino acids in the plant components of antidiabetic herbal mixture. The amino acids were separated by GC-MS method with pre-column derivatization. The calibration curves of twenty CRS of amino acids were linear (R2 > 0.98) over the range of 1–100 µg/mL, the LODs and the LOQs were in the range of 0.01–0.07 µg/mL and 0.02–0.20 µg/mL, respectively. The results of analysis showed that the predominant essential amino acid was L-proline in Taraxaci radices, Urticae folia, Rosae fructus and Menthae folia, its total content was 101.46 mg/g, 25.31 mg/g, 23.04 mg/g and 19.30 mg/g, respectively. In addition, it was established total content of essential amino acid – L-leucine that can stimulate insulin secretion in β-cells of the pancreas. Its total content was 58.51 mg/g in Taraxaci radices, 9.58 mg/g in Myrtilli folia, 4.68 mg/g in Rosae fructus, 2.99 mg/g in Urticae folia and 0.79 mg/g in Menthae folia. Chromatographic examination also revealed L-phenylalanine, an essential amino acid important for antidiabetic therapy that can increase insulin secretion, stimulate proliferation and neogenesis of β-cells of the pancreas and reduce insulin resistance. Its total content was 13.42 mg/g in Myrtilli folia, 2.23 mg/g in Rosae fructus, 1.478 mg/g in Urticae folia, 1.46 mg/g in Taraxaci radices and 0.52 mg/g in Menthae folia. This phytochemical study shows, which plant material forms the amino acid composition and content in the finished herbal mixture and due to which biologically active substances the antidiabetic activity of this phytocomposition is manifested.
antidiabetic herbal mixture, amino acids, gas chromatography-mass spectrometry, diabetes mellitus, L-proline, L-leucine, L-phenylalanine
Diabetes mellitus (DM) is a global social problem in the field of health care, due to rapid spread of this disease and the development of serious complications such as micro- and macroangiopathies, which significantly reduce the quality and life expectancy of patients (
One of these areas is phytotherapy, as it has several advantages over traditional therapy, namely, it is low-toxic, has a mild pharmacological effect and possibility to be used for long periods of time without significant side effects, is well combined with synthetic drugs, has a complex activity through several biologically active compounds (
One of such combinations is an antidiabetic herbal mixture (Urticae folia, Rosae fructus, Myrtilli folia, Menthae folia and Taraxaci radices) with established hypoglycaemic, hypolipidemic, antioxidant, hepatoprotective, pancreatoprotective activity in pharmacological study in vivo (
Biologically active substances of plant origin cause a large list of pharmacodynamics, including antidiabetic activity, which is manifested by different effects on the pathogenesis of DM type 2 and its complications (
Thus, the aim of this study was to identify and establish the content of amino acids in Urticae folia, Rosae fructus, Myrtilli folia, Menthae folia, Taraxaci radices as the plant components of antidiabetic herbal mixture.
It was used the herbal raw materials of Urticae folia, Rosae fructus, Myrtilli folia, Menthae folia and Taraxaci radices harvested from June to October 2020 in Ternopil region and Charpathians (Myrtilli folia) (Ukraine) during the study. The raw materials were then dried, crushed and stored according to the general GACP requirements (
All applied reagents were of analytical grade (≥ 99% purity). Chemical reference substances (CRS) of amino acids 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-glutamine, 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 flask with 2.0 mL of 0.1 M 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 M 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, and then 333 μL of methanol and 67 μL of pyridine were added 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 5% Phenyl Methyl Siloxane (30 m × 0.25 mm × 0.25 mm, Agilent Technologies) (
Amino acid identification was performed by comparing the retention times (tR) 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-glutamine | 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. A standard calibration solution containing 200 µg/mL for each CRS of amino acids was prepared (0.5 g of each CRS of amino acid was dissolved in 250 mL of 0.1 M aqueous solution of hydrochloric acid). From this solution, six decreasing dilutions were made (100, 50, 25, 10, 5, 1 μg/mL). A stock solution of internal standard (Nor-valine) at 5 μg/mL was prepared in 0.1 M aqueous solution of hydrochloric acid. Linearity was performed by injecting a series of standard solutions with a threefold derivatization procedure and a single injection for each CRS of amino acids. 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 removal and re-installation of the column. Repeatability precision was determined by six-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, six standard preparations of each CRS of amino acids 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 six 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 analytical procedure has been validated to confirm its reliability. All the peaks of CRS of amino acids showed good linearity (R2 > 0.98) in a wide concentration range (1–100 µg/mL). The results showed that the LODs and the LOQs of amino acids were in the range of 0.01–0.07 µg/mL and 0.02–0.20 µg/mL, respectively, indicating that the sensitivity of the method was satisfactory (Table
Results of linearity data obtained for CRS of amino acids after GC-MS analysis.
CRS of amino acids | Regression equations | R2 | LOD, µg/mL | LOQ, µg/mL |
---|---|---|---|---|
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-glutamine | 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 |
According to the results of the GC-MS analysis, it was identified eleven amino acids in free form in Taraxaci radices (Fig.
The results of the quantitative study showed that the predominant amino acid in free form was L-proline in Taraxaci radices (17.33±0.13 mg/g), in Menthae folia (7.35±0.15 mg/g), in Urticae folia (6.49±0.07 mg/g), in Rosae fructus (1.96±0.13 mg/g) and L-isoleucine in Myrtilli folia (3.46±0.17 mg/g). As for amino acids after hydrolysis, the predominant compound was L-proline in four plant components of antidiabetic herbal mixture. Its content was 84.13±0.23 mg/g in Taraxaci radices, 21.08±0.19 mg/g in Rosae fructus, 18.82±0.21 mg/g in Urticae folia and 11.95±0.17 mg/g in Menthae folia. However, Myrtilli folia contained the largest amount of L-isoleucine, its content was 28.55±0.22 mg/g (Table
The results of the GC-MS analysis of amino acids in the plant components of antidiabetic herbal mixture.
tR, min (SD±0.02) | Identified substance | Content in the herbal raw materials, mg/g | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Urticae folia | Myrtilli folia | Rosae fructus | Taraxaci radices | Menthae folia | |||||||
Free | Bound | Free | Bound | Free | Bound | Free | Bound | Free | Bound | ||
14.77 | Glycine | n/d | n/d | n/d | n/d | n/d | n/d | 1.34±0.12 | 21.45±0.19 | n/d | n/d |
14.85 | L-alanine | n/d | 0.65±0.12 | n/d | 2.83±0.16 | n/d | 0.76±0.08 | n/d | n/d | n/d | 0.217±0.05 |
18.56 | L-valine* | n/d | 1.95±0.17 | 1.19±0.16 | 5.76±0.19 | n/d | 2.45±0.12 | 1.63±0.11 | 33.45±0.21 | 0.16±0.08 | 0.381±0.03 |
19.57 | Nor-valine | Internal standard | |||||||||
20.77 | L-leucine* | n/d | 2.99±0.15 | n/d | 9.57±0.21 | n/d | 4.68±0.16 | 1.17±0.14 | 57.34±0.22 | n/d | 0.785±0.04 |
21.11 | L-serine | n/d | 1.49±0.17 | 0.10±0.04 | 4.79±0.19 | n/d | n/d | n/d | 23.15±0.15 | n/d | n/d |
21.31 | L-threonine* | n/d | n/d | 0.43±0.11 | 3.19±0.16 | 0.28±0.09 | 1.39±0.12 | 0.63±0.03 | 2.86±0.12 | 0.85±0.07 | 0.437±0.02 |
21.87 | L-isoleucine* | 0.26±0.08 | 0.22±0.04 | 3.46±0.17 | 28.54±0.22 | 0.93±0.11 | 18.95±0.17 | 0.15±0.02 | 1.86±0.11 | 4.76±0.13 | 2.915±0.12 |
21.97 | L-proline* | 6.49±0.07 | 18.82±0.21 | 2.73±0.18 | 18.92±0.23 | 1.96±0.13 | 21.07±0.19 | 17.33±0.13 | 84.13±0.23 | 7.35±0.15 | 11.951±0.17 |
23.90 | L-asparagine | n/d | n/d | 0.09±0.02 | 0.65±0.03 | n/d | n/d | n/d | n/d | n/d | n/d |
24.02 | L-aspartic acid | 0.86±0.11 | 2.58±0.12 | 0.74±0.04 | 3.79±0.12 | 1.37±0.08 | 20.44±0.18 | 0.13±0.05 | 53.64±0.22 | 4.16±0.13 | 5.849±0.15 |
26.86 | L-glutamic acid | n/d | 0.96±0.11 | n/d | 2.07±0.13 | n/d | 0.99±0.05 | 1.24±0.12 | 31.75±0.17 | n/d | 0.451±0.05 |
27.14 | L-methionine* | n/d | n/d | n/d | n/d | n/d | n/d | 1.01±0.11 | 6.85±0.15 | n/d | n/d |
29.18 | L-cysteine | n/d | n/d | 0.13±0.06 | 0.18±0.08 | n/d | n/d | n/d | 19.32±0.14 | n/d | n/d |
29.74 | L-phenylalanine* | 0.10±0.12 | 1.49±0.11 | 1.52±0.14 | 11.89±0.16 | n/d | 2.23±0.14 | 0.32±0.03 | 1.13±0.11 | n/d | 0.519±0.03 |
31.90 | L-glutamine | n/d | n/d | n/d | n/d | n/d | n/d | n/d | n/d | n/d | n/d |
35.91 | L-lysine* | 1.02±0.13 | 1.38±0.14 | 0.45±0.03 | 6.04±0.15 | n/d | 1.12±0.12 | n/d | 21.01±0.11 | n/d | 0.167±0.02 |
37.24 | L-histidine* | n/d | n/d | n/d | n/d | n/d | 1.36±0.11 | n/d | 1.13±0.08 | n/d | n/d |
38.91 | L-tyrosine | n/d | 0.41±0.05 | n/d | 1.97±0.11 | n/d | 0.49±0.06 | 1.23±0.12 | 16.74±0.16 | n/d | n/d |
42.01 | L-tryptophan | n/d | n/d | n/d | n/d | n/d | n/d | n/d | 0.13±0.04 | n/d | n/d |
In addition, some plant components of antidiabetic herbal mixture contained an important essential amino acid – L-leucine. During chromatographic analysis, it was detected 57.34±0.22 mg/g and 1.17±0.14 mg/g of L-leucine in bound and free form, respectively in Taraxaci radices, 9.57±0.21 mg/g of L-leucine in bound form in Myrtilli folia, 4.678±0.16 mg/g – in Rosae fructus, 2.99±0.15 mg/g – in Urticae folia and 0.79±0.04 mg/g – in Menthae folia (Table
Another extremely important essential amino acid for antidiabetic therapy is L-phenylalanine. Chromatographic examination revealed this amino acid in the studied objects, it was established that Myrtilli folia contains 1.52±0.14 mg/g and 11.89±0.16 mg/g of L-phenylalanine in free and bound form, respectively, Rosae fructus – 2.23±0.14 mg/g of L-phenylalanine in bound form, Urticae folia – 0.10±0.12 mg/g and 1.49±0.11 mg/g of L-phenylalanine in free and bound form, respectively, Taraxaci radices – 0.32±0.03 mg/g and 1.13±0.11 mg/g of L-phenylalanine in free and bound form, respectively and Menthae folia – 0.52±0.03 mg/g of L-phenylalanine in bound form (Table
The results show that all plant components, such as Urticae folia, Rosae fructus Myrtilli folia, Menthae folia and Taraxaci radices, of the antidiabetic herbal mixture have a high content of amino acids, which provide numerous pharmacological properties of this phytomixture. Amino acids obtained from plants are very important active substances for the prevention and treatment of DM type 2 and diabetic angiopathies as they have hypoglycaemic effects by different pathogenic mechanisms.
This phytochemical study of amino acid content in Urticae folia, Rosae fructus, Myrtilli folia, Menthae folia and Taraxaci radices shows, which plant material forms the amino acid composition and content in the finished herbal mixture that was established in previous studies and shows due to which biologically active substances is manifested the antidiabetic activity of this phytocomposition. The results of GC-MS analysis indicate the need to include each plant component in the antidiabetic herbal mixture to form the hypoglycaemic activity required for the treatment of DM type 2 (
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We identified and established the quantity content of amino acids in free and bound form in Urticae folia, Rosae fructus, Myrtilli folia, Menthae folia and Taraxaci radices, which are plant components of antidiabetic herbal mixture with hypoglycaemic, hypolipidemic, antioxidant, hepatoprotective, pancreatoprotective activity and defined phytochemical composition. The results of GC-MS analysis showed that the predominant essential amino acid was L-proline in Taraxaci radices, Urticae folia, Rosae fructus and Menthae folia, its total content was 101.46 mg/g, 25.31 mg/g, 23.04 mg/g and 19.30 mg/g, respectively and L-isoleucine in Myrtilli folia, its total content was 32.01 mg/g. In addition, it was established total content of essential amino acids, as L-leucine that can stimulate insulin secretion in β-cells of the pancreas and L-phenylalanine that can increase insulin secretion, stimulate proliferation and neogenesis of β-cells of the pancreas and reduce insulin resistance, which are important factors in the treatment of DM type 2. This phytochemical study shows, which plant material forms the amino acid composition and content in the finished herbal mixture and due to which biologically active substances is manifested the antidiabetic activity of this phytocomposition.