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:

X=Ах⋅m0

A0⋅25⋅5⋅100 ,

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.

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 96%0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

Концентрація екстрагенту

Вміст

ам

інок

исло

т в п

ерер

ахун

ку н

а гл

іцин

, %

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

1. Hrushchenko L. Thyme paradise. High Technology Research Station

Lubenski herbs Agroecology Institute of Agrarian Sciences / L. Hrushchenko, B.

Cancer // Pharmacist - practices: scientific- popu . and estate- home journal. - 2010

. - № 9. - P. 26-28 .

2 . F. Zani, G. Massimo, S. Benvenuti [ et al.] // Planta Med. - 1991 . - Vol.

57 , № 3 . - P. 237-241.

3 . Zarivna N. O, Logoyda L. S Development of methods of identification

aminoacids in herb of wild thyme // The role and the place of medicine in the

provision of health in modern society: an international scientific conference. ,

January 31 - February 1, 2014: abstracts of scientific papers. - Lviv, 2014. - P. 96-

98 .

4. Kovalev S. Amino acid and mineral composition of some species of

Phaseolus L. / S.V. Kovalev , V. M Kovalev , A. M. Bezugla // Journal of

Pharmacy. - 2011. - № 2 ( 66). - P. 41-44.

5. Borisova D. A. Amino acids of raw materials Primula veris / D. A

Borisova // Pharmacy. - 2011 . - № 8. - P. 11-13.

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.

7. Quantification of determination of amino acids in pollen / I. V Duhanyna,

A. J. Ayrapetova, G. D. Lazaryan. [ and others] // Hym. - farm. Zh. - 2006. - Vol

40, № 2. - P. 22-23.

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.