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UDK 615.014
DETERMINATION OF PROTEIN AND AMINO ACID COMPOSITION OF THE
DEVELOPED DRY EXTRACT "IMMUNASHIP"
1
Zuparova Zulfiya Ahror kizi ,
2
Khudayshukurova Aziza Arslanbek kizi
3
Ismoilova Guzaloy Mukhutdinovna
1
Tashkent Medical Academy, Tashkent
2
Termez branch of the Tashkent Medical Academy , Termez
3
Tashkent Pharmaceutical Institute, Tashkent
e-mail: zazulfiya@gmail.com tel (number) +998935206696
Abstract.
Herbal preparations that enhance immunity, due to the presence of a variety of
biologically active substances in them, gently affect the div and restore impaired functions of
the immune response. For the prevention of immune system disorders, preparations based on
purple echinacea (
Echinacea purpurea )
, as they have a positive effect on the div's immunity to
infectious agents and reduce the manifestations of allergic diseases. This article defines the
protein and amino acid composition of the dry extract "Immunaship". The protein in the dry
extract " Immunaship " was determined by the Kjeldahl method for the quantitative content of
nitrogen. The amino acid composition of the dry extract was studied and 20 free amino acids were
identified, with a quantitative content of 8.14969 mg/g, 10 of which are essential, which indicates
the biological value of the dry extract " Imunaship " based on purple echinacea herb and rose hips.
Key words:
Protein, amino acid composition, dry extract, Immunaship, HPLC, Kjeldahl method,
purple echinacea, rose hip, immunity.
Introduction.
Understanding the role of drugs that enhance immunity in the biosystems of a
living organism is the key to creating new classes of effective dosage forms. The use of
environmentally friendly plant materials is based on the deep relationship of natural components
with the human div [1,2,3]. The healing properties of the plant are due to the action of a
complex of biologically active substances contained in the plant, their harmonious interaction and
optimal ratio when acting on the human div. Research into herbal medicines with the aim of
introducing them into medical practice, developing optimal technologies for obtaining dry extract
and studying the biologically active substances in them that determine their pharmacological
action is very relevant[4,5,6,7].
Herbal preparations that enhance immunity, due to the presence of a variety of biologically active
substances in them, gently affect the div and restore impaired functions of the immune response.
To prevent immune system disorders, preparations based on purple echinacea (
Echinacea
purpurea)
, as they have a positive effect on the div's immunity to pathogens of infectious
diseases and reduce the manifestations of allergic diseases,[8,9,10,11,12].
The effectiveness of purple echinacea can be enhanced by combining it with medicinal plants
with antioxidant action, as well as by optimizing the dosage form [13,14,15,16,17].
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Purpose of the study
Determination of protein and amino acid composition of the developed dry
extract " Immunoship " based on purple echinacea herb and rose hips.
Materials and methods
The object of the study was the extract from the soup “Immuna ship”
based on the herb Echinacea purpurea and rose hips.
Identification of amino acid derivatives in the dry extract was carried out by HPLC using an
Agilent chromatograph. Technologies 1200 with DAD detector, with 75 x 4.6 mm column
Discovery H S C
18
. The mobile phase consists of solution (A): 0.14 M sodium acetate and 0.05%
triethylamino, at a solution pH of 6.4; Solution (B) consists of acetonitrile. Chromatographic
analysis was carried out in the following mode: I - 1-6% / 0-2.5 min; II - 6-30% / 2.51-40 min; III
- 30-60% / 40.1-45 min; IV - 60-60% / 45.1-50 min; V - 60-0% / 50.1-55 min, flow rate 1.2
ml/min, at UV detector absorption of 269 nm , at room temperature.
Protein was determined by the Kjeldahl method.
Experimental part
In plants, amino acids are found in a free and protein-bound state, and during extraction, both free
and bound amino acids pass into the extract. When determining free amino acids in a dry extract,
protein-bound amino acids and peptides interfering with the analysis were precipitated by
centrifugation. For this, an exact volume of 1 ml of 20% trichloroacetic acid was added to 1 ml of
the sample being studied . The sediment was precipitated in centrifuge cups for 10 min at a
rotation speed of 8000 rpm. 0.1 ml was collected from the supernatant liquid and dried in an air-
lyophilic dryer. The hydrolyzate was evaporated, the dry residue was dissolved in a mixture
consisting of triethylamine - acetonitrile - water in a ratio of (1:7:1) and dried. In order to
neutralize the acids, this procedure was repeated twice. Phenylthiocarbamyl derivatives (PTC) of
amino acids were obtained with a reaction with phenylthioisocyanate according to the Steven
method. A ., Cohen Daviel . The obtained data on the composition and quantitative content of
amino acids are given in Table 1, the chromatograms of the standard sample and dry extract are
shown in Fig . 1 and Fig . 2, respectively.[18,19,20,21,22]
Table 1
Composition of free amino acids in the dry extract " Immunaship "
No. Name of amino acids
Content, mg/g
1
Aspartic acid
0.083098
2
Glutamic acid
0.274905
3
Serene, Ser
0.458546
4
Glycine, Gly
0.121465
5
Asparagine,
Asn
0.241324
6
Glutamine,
Gln
0.816401
7
Cysteine, Cys
2.396721
8
Threonine, Thr
*
0.930596
9
Arginine, Arg
*
0.658713
10
Alanine, Ala
0.104805
11
Proline, Pro
0.910415
12
Tyrosine, Tyr
0.401187
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The composition of the dry extract was studied and 20 free amino acids were identified, with a
quantitative content of 8.14969 mg/g, 10 of which are essential, which indicates the biological
value of the dry extract " Imunaship ".
Fig. 1. Chromatogram of standard samples of free amino acids
Fig. 2. Chromatogram of the amino acid composition of the dry extract "Imunaship"
13
Valin, Val
*
0.212527
14
Methionine, Met
*
0.105826
15
Histidine, His
*
0.106568
16
Isoleucine, Ile
*
0.040607
17
Leucine, Leu
*
0.08202
18
Tryptophan, Trp
*
0.079156
19
Phenylalanine, Phe
*
0.058455
20
Lysine HCl , Lys
*
0.066333
Total
8.149669
Total content of essential amino
acids
2.34
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Determination of the amount of total protein. The method consists of determining nitrogen
according to Kjeldahl with subsequent recalculation into protein. The essence of the method
consists of decomposing the organic substance of the sample with boiling concentrated sulfuric
acid to form ammonium salts, converting ammonium into ammonia, distilling it into an acid
solution, quantitatively accounting for ammonia using the titrimetric method and calculating the
nitrogen content in the material being studied.[23,24,25]
From the averaged crushed homogeneous sample of the test specimen, an exact sample was
weighed in a test tube for analysis, with an error of no more than 0.1%. The sample was
quantitatively transferred to a Kjeldahl flask. Then the experiments were carried out according to
the methodological instructions [1].
Processing of results:
The mass fraction of nitrogen (X) in the test sample as a percentage of its
mass during the distillation of ammonia into sulfuric acid was calculated using the formula
Vо
– the volume of 0.1 mol/ l sodium hydroxide solution used for titration of 0.05 mol/l sulfuric
acid in the control experiment, ml.
V1
– the volume of 0.1 mol/l sodium hydroxide solution used
for titration of sulfuric acid in the test solution, ml;
TO
– correction to the titer of 0.1 mol/l sodium
hydroxide solution;[24,25]
0.0014 – the amount of nitrogen equivalent to 1 ml of 0.05 mol/l sulfuric acid solution;
M
– weight of sample, g. The final test result was the arithmetic mean of the results of five
parallel tests. The results were calculated to the third decimal place and rounded to the second
decimal place.
The mass fraction of nitrogen in terms of dry matter of the product (X3), in percent, was
calculated using the formula:
X 1
– mass fraction of nitrogen in the test sample, %;
W
– humidity of the test sample, %.
The mass fraction of protein (Y) in percent was calculated using the formula:
Y
=
K
Ч
X
, where
TO
– nitrogen to protein conversion factor: with moderate lipid content – 6.38;
Conclusion
The protein in the dry extract "Immunaship" was determined using the Kjeldahl
method based on the quantitative nitrogen content. The amino acid composition of the dry extract
was studied and 20 free amino acids were identified, with a quantitative content of 8.14969 mg/g,
10 of which are essential, which indicates the biological value of the dry extract "Imunaship".
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