UDC 582
UDC 582.929.4-035.22:547.466
N.O. ZARIVNA
Ternopil State Medical University named after I.
Gorbachevski
STUDING OF AMINO ACID COMPOSITION IN HERB OF THYMUS
SERPYLLUM
The results of the study of the qualitative and quantitative
analysis of amino acids in the grass creeping thyme . Established
that quantitative content of amino acids in the studied raw
materials should be less than 0.6% in terms of glycine.
Key words: creeping thyme, amino acids, thin layer
chromatography, spectrophotometry.
INTRODUCTION
Creeping Thyme (Thymus serpyllum), mint family (Lamiaceae),
naturally distributed in Ukraine in wild and cultivated extensively
[1, 2]. According to the literature creeping thyme contains
flavonoids, tannins, phenol carbonic acid, essential oil , etc.,
which in combination determine the pharmacological activity of
diversified materials and substances derived on the basis of [2 ].
In a previous study we found that in addition to flavonoids and
polysaccharides (last we found the first time) thyme herb is also
rich in amino acids [3 ]. It is known that amino acids serve
important functions in the body. In addition to protein synthesis ,
amino acids perform many other important biological functions [4 ]
, and the goal of our research was to identify and determine the
form of amino acids in the test material.
MATERIALS AND METHODS
To analyze the use of wild raw western regions of Ukraine,
namely Ternopil and Lviv oblasts and Designs raw materials used by
domestic producers of medical products ( FF Zhitomir "Liktravy" and
Kiev FF). The study of the quality of the amino acids was performed
by thin layer chromatography (TLC) [5]. Chromatographic studies
were performed on chromatographic plates Silica gel 60 F254 («
Mersk ", Germany ) using a mobile phase of isopropanol P - P formic
acid - water P ( 40:2:10 ). Substances discovered were: glycine ,
alanine , aspartic acid, leucine, glutamic acid, tyrosine. For
chromatogram Film development using ninhydrin solution of 2 g / l (
5 ml of dilute acetic acid , 95 ml of butanol). As a result of TLC
- studies, we propose the following method of identifying amino
acids in herb of creeping thyme.
Methods of identification of amino acids in raw creeping
thyme.
Test solution: 1,000 g of minced raw creeping thyme, placed in a
conical flask 100 ml, add 50 ml of alcohol (60% (v / v )) and
heated in a water bath under reflux for 45 min. The solution was
cooled and filtered through a filter "blue ribbon" in a conical
flask of 100 ml.
Reference solution: 0.1 g of standard sample of glycine (Sigma),
0,1 g of standard sample of tyrosine (Fluka), 0,1 g of standard
sample glutamic acid (Sigma), 0,1 g of standard sample alanine
(Fluka), 0,1 g standard sample aspartic acid (Fluka), 0,1 g of
standard sample leucine, dissolved in 25 ml of water R. At the
starting line chromatographic plate Silica gel F254 size 20x20 cm
with a layer thickness of 0.25 mm is applied in the form of strips,
length 10 mm, 20 ml of the test solution and 5 ml reference
solution. The plate was dried in air for 10 min, placed in a
chamber with mobile phase isopropanol P - formic acid P - P water
(40:2:10) and ascending chromatography method. When the solvent
front will be held 15 cm from the starting line, it is removed from
the chamber and dried in air. The plates were sprayed with a
solution of 2 g / L ninhydrin R and heat at 100 ⁰ C for 3-5 min and
viewed in daylight.
Fig 1. Scheme chromatogram of the test solution (7) and solution
(1 - 6) , where 1 - glycine; 2 - alanine; 3 - aspartic acid; 4 -
leucine ; 5 - glutamic acid; 6 – tyrosine
In the chromatogram obtained with the test solution was observed
occurrence of zones of glycine, alanine, tyrosine, leucine,
glutamic and aspartic acids at a level appropriate zones in the
chromatogram obtained with reference solution (Fig. 1).
Quantitative determination of amino acids was performed by
spectrophotometry in the visible range of the known reaction of
amino acids colored product with ninhydrin [6, 7].
In order to select the best extractant for the extraction of
amino acids from raw material determine their quantitative content
in the water-alcohol extract, obtained by extractants with
different concentration of alcohol (0 - 96%). Extract obtained by
boiling in a water bath at reflux sample material from ethanol-
water solution of appropriate concentration. Method of quantitative
determination of amino acid content are shown below.
Method of quantitative determination of amino acid content in
grass creeping thyme.
Test solution: 1,000 g of raw thyme placed in a conical flask
with a ground stopper add 80 ml of alcohol corresponding
concentration, boil 45 min at reflux. The resulting extract was
cooled, filtered into a volumetric flask of 100 ml and dilute
solution of alcohol corresponding to the mark. 5.0 ml of the
extract obtained is placed in a test tube with a capacity of 15 ml,
add 5.5 ml of freshly prepared 0.2% ninhydrin solution R and heat
in a boiling water bath for 20 min. After complete cooling, the
solution was quantitatively transferred to a volumetric flask and
diluted with 25 ml of water R to the mark, mix. After 1 h measured
optical density of the resulting solution on a spectrophotometer at
a wavelength of 567 nm 2 cell with a layer thickness of 10 mm.
Reference solution: 0.100 g of glycine CRS placed in a
volumetric flask of 100 ml, add 70 ml of water R, dissolved within
5-10 min by heating in a water bath at 50 C. Cool the solution and
dilute with water R to 100 ml and mix. 1 ml of the resulting
solution is placed in a flask of 25 mL and dilute the solution with
water R to the mark, mix. To 1.0 ml of the resulting solution,
placed in a test tube with a capacity of 15 ml, add 1.1 ml of 0.2%
ninhydrin solution R and heat in a boiling water bath for 20 min.
After complete cooling solution quantitatively transferred to a
volumetric flask of 100 mL and diluted with water R to the mark,
mix.
The compensation solution. 1.0 ml of water R is placed in a test
tube, add 1.1 ml of 0.2 % ninhydrin solution R and heat in a
boiling water bath for 20 min. After complete cooling solution
quantitatively transferred to a volumetric flask of 100 ml and
diluted with water R to the mark, mix
0.2% ninhydrin solution R. 0.1 g ninhydrin placed in a
volumetric flask of 50 mL, dissolved in 30 ml of water R, dilute
solution in the same solvent to the mark and mix
After 1 h measured optical density of the resulting solution on
a spectrophotometer at a wavelength of 567 nm 2 cell with a layer
thickness of 10 mm. The content of amino acids in the grass
creeping thyme , as a percentage, calculated as glycine, calculated
by the formula:
where Ah - optical density of the test solution ;
A0 - absorbance reference solution ;
m0 - mass of sample standard sample of glycine, g
In selected conditions, assay of electronic absorption spectrum,
obtained in photometric colored reaction product, there is the
appearance of two absorption bands at 400 ± 2 nm and 567 ± 2 nm
(Fig. 2). Since at 400 nm absorbing more hydroxycinnamic acids that
are present in the recovery, so to measure absorbance otometric or
compounds should be at a wavelength of 567 nm.
Fig. 2. Electronic absorption spectra of solutions of amino
acids with ninhydrin in terms of quantification of 1 - test
solution. obtained for 30 % of the extractant; 2 - test solution
obtained for 40% of extractant
The results of the quantitative determination of amino acid
content of grass creeping thyme in terms of glycine are presented
in Figure 3.
Fig. 3. Dependence of amino acids converted to glycine in
alcohol withdrawal of grass creeping thyme, the concentration of
alcohol in the extractant
For a comparative analysis of amino acid content in different
samples of raw materials used thyme quantification of results are
presented in Table.
Table
AMINO ACID CONTENT, IN TERMS OF GLYCINE IN MEDICINAL PLANTS
CREEPING THYME (P = 0,95; n = 5)
Sample of raw material
Amino acid content, %
Zhitomir FF "Liktravy" with. 61010
0,69 ± 0,01
Zhitomir FF "Liktravy" with. 40810
0,66 ± 0,02
Zhitomir FF "Liktravy" with. 2011
0,71 ± 0,01
Wild stuff Ternopil oblast, district Husyatyn 2009
harvesting
0,65 ± 0,02
Wild stuff Ternopil oblast, district Husyatyn 2010
harvesting
0,62 ±0,02
As seen from the results, the quantitative content of amino
acids correlates for different samples, it is possible to explain
the conditions of growth of MCQ. RESULTS AND DISCUSSION
As a result, TLC - analysis of the studied material we first
identified 6 amino acids that are completely in color zones and
factor mobility coincide with the corresponding zones SPS. As seen
from the results presented quantitative determination of the most
complete removal of amino acids from grass creeping thyme treated
using 30 % ethanol, indicating the feasibility of its use in the
preparation of standard solutions for the quantitative
determination of amino acids in raw materials. The study of
qualitative and quantitative analysis of amino acids shown creeping
thyme that stuff is really rich in amino acids, which requires
further study of their biological activity to improve approaches to
standardization of thyme herb and possible expansion of indications
for use.
CONCLUSIONS
1. The first time studied the composition of amino acids grass
creeping thyme, hereby detected leucine, glycine, tyrosine,
alanine, aspartic and glutamic acid.
2. Developed and tested a method of quantitative determination
of amino acids grass creeping thyme spectrophotometric method in
terms of glycine.
3. Selected 30 % ethanol, as the best extractant for extracting
amino acids from raw materials.
4. Determined that for different samples of raw amino acid
content is 0.6 - 0.7 %.
5. Results of the study of amino acid composition of grass
creeping thyme indicate promising depth study of its
pharmacological action.
REFERENCES
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1991 . - Vol. 57 , № 3 . - P. 237-241.
3 . Zarivna N. O, Logoyda L. S Development of methods of
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place of medicine in the provision of health in modern society: an
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2014: abstracts of scientific papers. - Lviv, 2014. - P. 96-98
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4. Kovalev S. Amino acid and mineral composition of some species
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5. Borisova D. A. Amino acids of raw materials Primula veris /
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6. Spectrophotometric determination of glycine in tablets
containing glycine and some plant extracts/ L. S Logoyda, L. V.
Vronska, A. E Demid, M. M. Mykhalkiv// Pharmaceutical Journal. -
2010. - № 4. - P. 50-54.
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UDK 582.929.4-035.22:547.466
N. O. Zarivna
THE STUDING OF AMINO ACID COMPOSITION OF WILD THYME
The results of studying the qualitative and quantitative
composition of the amino acid in the herb wild thyme has been
developed. Quantitative amino acid content in the feed, which
should be no less than 0.6 at% based on glycine.Key words: wild
thyme, amino acids, thin layer chromatography,
spectrophotometry.
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