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American Journal Of Biomedical Science & Pharmaceutical Innovation
(ISSN
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:
102-112
SJIF
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)
(2023:
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)
OCLC
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1121105677
Publisher:
Oscar Publishing Services
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ABSTRACT
Research was undertaken to examine the amino acid and elemental composition of “Hyposedaf”, a dry extract known
for its hypotensive properties. Quantitative analyses of both micro and macro elements were conducted using
Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Additionally, the amino acid content was quantitatively
analyzed through High-Performance Liquid Chromatography (HPLC). This study identified 20 different amino acids in
the “Hyposedaf” dry extract, 10 of which are essential. Notable amino acids, such as histidine, cysteine, tyrosine,
methionine, valine, asparagine, and leucine, amounted to a total of 29.15 mg. Of all detected amino acids, 46.5% were
classified as non-
essential, while 53.5% were essential. The elemental analysis of the “Hyposedaf” dry extract revealed
61 different elements, including 7 essential elements (Co, Cr, Cu, Fe, Mn, Se, Zn) and 4 conditionally necessary
elements. Key elements like potassium, calcium, sodium, zinc, iron, magnesium, and phosphorus, found in substantial
amounts, are crucial for human health. The concentrations of heavy metals and arsenic in the dry extract were within
safe limits according to the Sanitary-epidemiological rules and norms, complying with the quantitative standards State
Pharmacopoeia of the Republic of Uzbekistan (Volume 1, Issue 1) and State Pharmacopoeia of the Russian Federation
XIV (RF) [1,2,3]. The findings indicate that “Hyposedaf” dry extract possesses significant value, offering potential for
medical application and the development of other medicinal forms.
Research Article
“HYPOSEDAF” DRY EXTRACT’S AMINO ACID AND ELEMENTAL
COMPOSITION STUDY
Submission Date:
January 21, 2024,
Accepted Date:
January 26, 2024,
Published Date:
January 31, 2024
Crossref doi:
https://doi.org/10.37547/ajbspi/Volume04Issue01-16
Safarova Diyora Tolibovna
Assistant Of The Tashkent Pharmaceutical Institute, Uzbekistan
Maksudova Firuza Khurshidovna
Tashkent Pharmaceutical Institute, Doctor Of Pharmaceutical Sciences, Associate Professor, Uzbekistan
Karaeva Nargizakhan Yuldash Qizi
Assistant Of The Tashkent Pharmaceutical Institute, Uzbekistan
Journal
Website:
https://theusajournals.
com/index.php/ajbspi
Copyright:
Original
content from this work
may be used under the
terms of the creative
commons
attributes
4.0 licence.
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KEYWORDS
Dry extract, micro and macro elements, amino acids, HPLC method, mass spectrometry.
INTRODUCTION
Extensive research has established the critical role of
minerals and amino acids in the human div, acting as
fundamental nutrients for synthesizing all proteins.
Besides contributing to brain function, including
intellectual activity, motivation, and general mental
state, amino acids play a vital role in maintaining overall
well-being. These nutrients are key components of all
human tissues, such as muscles. Amino acids and
chemical elements not only exhibit specific
pharmacological effects but also demonstrate
synergism with various substances, paving the way for
the development of combined drugs with multiple
functional properties derived from plants. Importantly,
macro and micronutrients, as well as amino acids
sourced from plants, are more effectively absorbed by
the human div, owing to their “biological” quantities
present in plants. Furthermore, amino acids assist in
the optimal performance of mineral substances within
the human div. The study of amino acid and mineral
compositions
in
medicinal
plants
and
phytopreparations is vital for the creation of new
pharmaceutical drugs [4].
Currently, it's essential to understand the trace
element content in the entire div of medicinal plants,
including their separate generative and vegetative
organs - roots, stems, leaves, buds, flowers, fruits, and
seeds. This knowledge significantly expands the scope
of their utilization. In plants, bioelements are found in
forms that are absorbable by the human div, such as
amino acids, vitamins, proteins, etc., and they are
present in complex forms. This fact underscores the
importance of medicinal plants as sources of
biologically active substances and elements. The
investigation of the elemental composition of
medicinal plants and their based preparations is not
only necessary for determining their value but also for
preventing toxic and carcinogenic properties due to
increased levels of heavy metals and arsenic [5,6].
Considering the above, the feasibility of using
medicinal plant raw materials or extracts based on
them in healthcare practice involves studying their
amino acid and elemental composition. Amino acids
are crucial for plant development, occurring during
photosynthesis and participating in a wide range of
biochemical reactions that support optimal growth
and development [7,8,9].
Object of the Study:
The aim of this study is to
investigate the amino acid and elemental composition
of the dry extract of “Hyposedaf”.
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Experimental Part: The research focused on the dry
extract of “Hyposedaf”, conditionally named and
derived from various plant raw materials, including
peppermint leaves (Folia Menthae piperitae L.),
Turkistan lion's tail grass (Herba Leonuri turkistanicae),
dark-red hawthorn fruits (Fructus Crataegi sanguineae
Pall), and field horsetail grass (Herba Equiseti arvensis).
This extract complied with the specifications outlined
in the publications State Pharmacopoeia of the Russian
Federation and XIV State Pharmacopoeia (RF) [10,11].
In the first phase of our research, we concentrated on
the elemental composition of “Hyposedaf” analyzing
its macro and microelements through Inductively
Coupled Plasma Mass Spectrometry (ICP MS) as per
the methodological guidelines [12].
In the second phase, the amino acid composition of the
“Hyposedaf” dry extract was examined. This involved
isolating free amino acids and conducting their
quantitative analysis by comparing the retention times
and peak areas of a standard sample with those of
amino acids obtained from phenylthiocarbamyl.
Results and Dis
cussion: The “Hyposedaf” dry extract
was analyzed using the highly sensitive Inductively
Coupled Plasma Mass Spectrometry (ICP-MS) method.
This
technique
enabled
the
simultaneous
determination of numerous elements in a single
sample with very low detection limits. The mass-
spectral analysis was developed in Russia and validated
in Uzbekistan (MBI) UzO`U 0677:2015 (MBI №499
-
AEM/MS), facilitating the determination of 61 elements
(Na, Mg, Al, P, K, Ca, Ti, Mn, Fe, Li, Be, B, Sc, V, Cr, Co,
Ni, Cu, Zn, Ga, As, Se, Rb, Sr, Y, Zr, Nb, Mo, Ag, Cd, In,
Sn, Sb, Te, Cs, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho,
Er, Tm, Yb, Lu, Hf, Ta, W, Re, Pt, Au, Tl, Pb, Bi, Th, U).
This method utilized argon inductively coupled plasma
as an ion source. The test sample, weighing 0.1 g, was
introduced into the plasma in solution form. Prior to
measurement, the sample underwent solubilization.
The resulting solution, containing the test sample, was
aerosolized with a stream of argon and introduced into
the plasma. The core temperature of the plasma
reached up to 9000°C, at which the sample was
desiccated, and the molecular elements transitioned to
an atomic state, becoming ionized to form positive
ions. These positive ions, along with electrons and
neutral particles, were channeled through cone-
shaped interfaces into the vacuum segment of the
analyzer, passing through several electric ion lenses
that filtered out electrons and neutral particles from
the ions.
The test sample and a standard sample, each weighing
0.1 g, were placed in Teflon bags. Decomposition of the
test, standard, and control samples was performed in
a system involving Teflon bags with lids (Hot Block,
Environmental Express). The Hot Block, consisting of
36 or 40 cells depending on the model, allowed for the
heating of dissected samples in large volumes. This
decomposition process utilized a mixture of HF, HClO4,
and HNO3 acids in an open system to fully dissolve the
test sample. After removing the bags from the heating
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plate and allowing them to cool, 3 ml of concentrated
hydrochloric acid was added to each. The resultant
solutions were transferred to polyethylene tubes,
diluted with distilled water, mixed, and subsequently
analyzed. Elemental measurements in aqueous
solutions were conducted within a mass range of 5 to
240. The analysis of each sample, ranging from 36 to 40
samples, took approximately 2-3 minutes. Following
each measurement, the spray system was thoroughly
rinsed with a 2% nitric acid solution and water for 0.5-1
minute.
The analysis of the elemental composition of the
“Hyposedaf” dry extract is presented in Table 1.
Table 1.
The elemental composition of the “Hyposedaf” dry extract
Element
Quantity, mkg/g
Element
Quantity, mkg/g
Makroelementlar
Gallic, Ga
0,170
Potassium, K
65 000
Yttrium, Y
0,150
Calcium, Ca
10 000
Neodymium, Nd
0,130
Magnesium, Mg
9 100
Antimony, Sb
0,110
Millielements
Tin, Sn
0,076
Tory, Th
0,071
Phosphorus, P
4000
Zirconium, Zr
0,067
Sodium, Na
1300
Platinum, Pt
<0,05
Aluminum, Al
460
Gold, Au
<0,05
Iron, Fe
280
Praseodym, Pr
0,04
Manganese, Mn
53,0
Samari, Sm
0,028
Zinc, Zn
47,0
Nanoelements
Strontium, Sr
47,0
Gadolinium, Gd
0,018
Rubidium, Rb
39,0
Tantalum, Ta
<0,01
Boron, B
32,0
Thallium, Tl
<0,01
Microelements
European, Eu
0,01
Titan, Ti
13,0
Dysprosium, Dy
0,01
Copper, Cu
6,90
Ermiy, Er
0,01
Nickel, Ni
4,10
Ittermiy, Yb
0,01
Lithium, Li
2,10
Terbiy, Tb
<0,01
Molybdenum, Mo
1,40
Tuly, Tm
<0,01
Chrom, Cr
1,20
Gafni, Hf
<0,01
Silver, Ag
1,10
Berelli, Be
0,006
Selenium, Se
0,50
Golmi, Ho
0,006
Cerium, Ce
0,320
Indium, In
0,001
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The subsequent section details the quantitative
composition of elements in the dry extract, measured
in micrograms per gram (as classified by Polyanskaya
I.S., 2005):
-
Macroelements (К –
65 000, Ca
–
10 000, Mg-9100);
-
Millielements (Р –
4000, Na
–
4300, Al
–
460, Fe
–
280,
Mn
–
53, Zn
–
47, Sr-47, Rb-
39, В –
32);
- Microelements (Ti-13, Cu 6.9, Ni-4.10,Li-2.10, Mo-1.40,
Cr-1.20,Ag-1.10, Se-0.50, Ce-0.32, Ga-0.170, Y-0.150, Nd-
0.130, Sb-0.110, Sn-0.076, Th-0.071, Zr-0.067, Pt-<0.05,
Au-<0.05, Pr-0.04, Sm-0.028
- Nanoelements (Gd-0.18, Ta-<0.01, Tl-<0.01, Eu-0.01, Dy-
0.01, Er-0.01, Yb-0.01, Tb-<0.01, Tm-<0.01, Hf-<0.01, Be-
0.006, Ho-0.006, In-0.001)
The results indicate that the “Hyposedaf” dry extract
has a high biological value, containing substantial
amounts of elements crucial for human life, such as
potassium, calcium, phosphorus, magnesium, sodium,
aluminum, iron, etc. Literature suggests that
potassium compounds are vital for muscle contraction
and brain function, and their deficiency can lead to
fatigue and overall div weakness.
Magnesium is an essential element found in all div
tissues, crucial for the normal functioning of cells. It
holds particular importance for internal organs,
especially the heart and blood vessels.
Calcium compounds present in the analyzed dry
extract are key microelements for human life and
health. In medical applications, they serve as sedatives,
hemostatics, treatments for allergic diseases, and
blood pressure regulators. Calcium is key in
strengthening the skeletal system, forming tooth
enamel, and plays significant roles in the immune
system, skin, hair, nail health, and reproductive
function. It is also instrumental in enhancing metabolic
processes within the div.
The high concentration of sodium compounds in the
dry extract, combined with potassium, aids in
maintaining acid-base balance, osmotic concentration
of blood, activation of numerous enzymes, and
contributes to the formation of membrane potential.
Therefore, the “Hyposedaf” dry extract can be
regarded as a valuable source of both macro and
microelements.
According to normative documents, the content of
heavy metals and arsenic in plants and their derived
medicinal preparations is in compliance with the
standards of XIV State Pharmacopoeia and Sanitary-
epidemiological rules and norms. It has been verified
that these concentrations do not exceed established
limits.
The obtained results are presented in Table 2.
Table 2
Amount of heavy metals and arsenic in dry extract of “Hyposedaf” mkg/g
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Element
Quantity according to
the XIV State
Pharmacopoeia
Quantity as requested
by the World Health
Organization
Determined amount
Lead, Pb
6,0
10,0
0,750
Cadmium, Cd
1,0
0,3
0,012
Mercury, Hg
0,1
-
not identified
Arsenic, As
0,5
1,0
0,10
In the subsequent stage of the research, we focused
on analyzing the amino acid composition of the
“Hyposedaf” dry extract. Initially, the process involved
isolating free amino acids. For this, 1 ml of the test
sample was combined with an equal volume (1 ml) of
20% trichloroacetic acid. After a duration of 10 minutes,
the resultant precipitate was separated through
centrifugation at a speed of 8000 revolutions per
minute for 15 minutes. Subsequently, 0.1 ml of the
clarified liquid was isolated and subjected to
lyophilization. The hydrolyzate was then evaporated,
and the dry residue was dissolved in a triethylamine-
acetonitrile-water mixture (ratio 1:7:1) and dried again.
The amino acid phenylthiocarbamyl derivatives (FTC)
were synthesized using the phenylisothiocyanate
reaction, following the methodology of Steven A. and
Koen Daniel. The identification of these amino acid
derivatives was carried out via high-performance liquid
chromatography (HPLC). Employing an Agilent
Technologies 1200 HPLC column detector (75x4.6
mm), we utilized solution A: 0.14M CH3COONa with
0.05% TEA at pH 6.4, and solution B: CH3CN. The flow
rate was maintained at 1.2 ml/min, with detection at
269 nm. The gradient percentage per minute was
established as follows: 1-6%/0-2.5 min; 6-30%/2.51-40
min; 30-60%/40.1-45 min; 60-60%/45.1-50 min; 60-
0%/50.1-55 min [13].
The chromatographic analysis of both the standard
amino acid sample of the “Hyposedaf” dry extract and
the amino acids present in the extract under study are
depicted in Figures 1 and 2.
Table 3 presents the quantitative composition and
chemical structure classification of the amino acids.
According to the data in Table 3, the “Hyposedaf” dry
extract contains 20 amino acids, including 10 essential
amino acids, which highlight its substantial quantity
and pharmacological significance. The total amino acid
content is 29.151 µg/g, of which 15.605 µg/g are
essential amino acids, and the remaining 15.605 µg/g
are non-essential.
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1.Fig. Standard sample chromatogram of a mixture of amino acids
2.Fig. Chromatogram of dry extract of “Hyposedaf”
In the “Hyposedaf” dry extract, histidine stands out as
a crucial essential amino acid, present at a
concentration of 4.856877 µg/g. It is a key component
of hemoglobin
—
the vital protein in red blood cells
responsible for carrying oxygen from the lungs to div
cells, and for transporting carbon dioxide in the
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opposite direction. A deficiency in histidine can impede
the div’s ability to produce the necessary quantity of
hemoglobin, potentially leading to anemia. Histidine
plays an important role in the functioning of the
gastrointestinal tract, as well as in liver and kidney
operations. Furthermore, the div converts histidine
into histamine, a compound integral to the nervous
system. Histamine is involved in synthesizing various
hormones and is essential for regulating the heart's
rhythm [14].
Table 3
Analysis of amino acids of dry extract of “Hyposedaf”
№
Amino acid
Amount of amino
acids, mkg/g
Aliphatic amino acids
Monoaminocarbons
1.
Alanine
0,077593
2.
Glysine
0,8991
3.
Valin
2,49772
4.
Isoleucine
0,342585
5.
Leucine
1,502734
Oxysaminocarbons
6.
Serine
0,845982
7.
Threonine
0,310199
Monoaminodicarbons
8.
Aspartic acid
0,321104
9.
Glutamic acid
0,31053
Monoaminocarbon amides
10.
Asparagine
1,800129
11.
Glutamine
0,909695
Diaminocarbons
12.
Lysine
0,194589
13.
Arginine
0,472063
Sulfur preservative
14.
Cysteine
4,444809
15.
Methionine
2,508621
Aromatic amino acids
16.
Phenylalanine
0,341222
17.
Tyrosine
3,820187
18.
Tryptophan
0,517421
Heterocyclic amino acids
19.
Proline
2,178288
20.
Histidine
4,856877
Amount of non-essential amnio acids
13,544
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Amount of exchangeable amino acids
15,605
Total amount of amino acids
29,15145
Methionine, at a concentration of 2.508621 µg/g, ranks
second in abundance in the “Hyposedaf” dry extract.
This essential sulfur-containing amino acid is known for
its multiple unique functions [15]. Crucially involved in
protein synthesis, methionine's fat-soluble properties
also aid in preventing fatty liver disease. Furthermore,
it serves as a foundational component for several
hormones,
including adrenaline,
choline,
and
melatonin. Cysteine, present at 4.444809 µg/g, is
another sulfur-bearing amino acid. Classified as semi-
essential or conditionally essential, cysteine is unique
among the twenty amino acids for containing a thiol
group (-HS) capable of undergoing both oxidation and
reduction reactions. The oxidation process transforms
cysteine into cystine, and it also contributes
significantly to metabolic processes and the formation
of peptides and proteins [16].
Tyrosine, found at 3.820187 µg/g, plays roles in
reducing appetite, decreasing fat accumulation,
stimulating melanin production, and enhancing the
functionality of the adrenal, thyroid, and pituitary
glands [17].
CONCLUSION
In conclusion, the
“Hyposedaf” dry extract contains 61
micro and macro elements. This includes 7 essential
elements (Co, Cr, Cu, Fe, Mn, Se, Zn) and 4 conditionally
necessary ones. Key elements like potassium, calcium,
sodium, sulfur, iron, magnesium, and phosphorus are
crucial for human health and are prominently featured
in the extract. The concentrations of heavy metals and
arsenic in the extract were found to be within
acceptable limits as defined by the Sanitary-
epidemiological
rules
and
norms
of
State
pharmacopoeia of the Republic of Uzbeksitan (Volume
1, Issue 1) and XIV State Pharmacopoeia (RF).
Moreover, the amino acid analysis of the dry extract
revealed the presence of 20 amino acids, including 10
essential ones. The total amino acid content is 29.151
µg/g, with essential amino acids accounting for 15.605
µg/g and non-essential ones making up the remaining
15.605 µg/g. These findings underscore the
“Hyposedaf” dry extract’s richness in amino acids,
macro, and microelements, affirming its significant role
in the development of other effective pharmaceutical
formulations.
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