Volume 04 Issue 02-2024
68
International Journal of Advance Scientific Research
(ISSN
–
2750-1396)
VOLUME
04
ISSUE
02
Pages:
68-74
SJIF
I
MPACT
FACTOR
(2021:
5.478
)
(2022:
5.636
)
(2023:
6.741
)
OCLC
–
1368736135
A
BSTRACT
To evaluate the hepatoprotective activity in the serum of experimental toxic hepatitis model rats treated
with polyphenolic compounds, total protein, alkaline phosphatase, AST, ALT were determined by Cypress
Diagnostica (Belgium) test kit. Blood was collected from animals, centrifuged at 3000 revolutions/min for
12 minutes, serum was isolated and biochemical indicators studied.
K
EYWORDS
Toxic hepatitis, ALT, AST, alkaline phosphatase, protein content, effect of polyphenols, catalase,
spectrophotometric, colorometric.
I
NTRODUCTION
Materials and research methods: the most
effective and classic method of studying the
changes in physiological processes occurring in
liver cells, especially mitochondria, and the
mechanisms of action of various biologically
active compounds on it in the conditions of toxic
hepatitis is SSI4 intoxication of animals. These
research methods are mainly conducted in vivo.
There are 2 types of animal intoxication: acute
and chronic toxic hepatitis. In acute toxic
Journal
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Research Article
DETERMINATION OF THE EFFECT OF POLYPHENOLS ON
CERTAIN BIOCHEMICAL CHANGES (ALT, AST, ALKALINE
PHOSPHOTASE AND TOTAL PROTEIN) IN THE CONDITIONS
OF TOXIC HEPATITIS
Submission Date:
February 11, 2024,
Accepted Date:
February 16, 2024,
Published Date:
February 21, 2024
Crossref doi:
https://doi.org/10.37547/ijasr-04-02-12
Mallayeva Mavjudaxon Maxramovna
Teacher At The Department Of General Hygiene And Ecology At Samarkand State Medical University,
Uzbekistan
Volume 04 Issue 02-2024
69
International Journal of Advance Scientific Research
(ISSN
–
2750-1396)
VOLUME
04
ISSUE
02
Pages:
68-74
SJIF
I
MPACT
FACTOR
(2021:
5.478
)
(2022:
5.636
)
(2023:
6.741
)
OCLC
–
1368736135
hepatitis, the toxicant selected for intoxication is
injected subcutaneously twice a day in a relatively
high dose.
In this case, acute toxic hepatitis is quickly called
and studied using several methods of studying
changes in the mitochondria of animal livers.
Researches were conducted in male white rats
(Rattus vulgaris L.) weighing 140-200 g. The
experiments were carried out in accordance with
the "Rules for the use of experimental animals", as
well as the rules adopted by the European
Convention for the Protection of Vertebrate
Animals Used for Experimental Research or for
Other Scientific Purposes.
Tetrachloromethane (SCl4) solution in 50% olive
oil was challenged by parenteral administration
at a dose of 2 ml/kg 2 times in 1 day [17].
Medicines Slimarin (pharmacological trade name
Karsil) 50 mg/kg, rutan 10 mg/kg, gossitan 10
mg/kg were administered orally in a dose of 10
mg/kg within 7 days after hepatitis was induced.
The experimental animals were divided into 5
groups: 1) healthy group, 2) experimental toxic
hepatitis (SCl4-infected toxic hepatitis), 3) group
Slimarin (Carsil) 50 mg/kg, 4) group rutan 10
mg/kg, 5) group gossitan The amount of total
protein, alkaline phosphatase, alanine and
aspartate aminotransferase (ALT, AST) in serum
after 10 mg/kg. 7 days was determined using the
test kits produced by Cupress Diognostica
Biochemical Tests (Belgium).
At the end of the experiment, the animals were
anesthetized with chloroform, decapitated and
studied to study their pathological changes.
Extraction of liver tissue homogenate We isolated
150-200 grams of rat liver tissue homogenate by
differential centrifugation [54]. The rat was first
immobilized, then the liver was removed from the
div and placed in an ice-cold isolation medium.
The composition of the separation medium is as
follows: sucrose 250 mM, tris-NSI 10 mM, eDTA 1
mM, pH 7.4. After washing in chilled saline to
obtain the homogenate, a 5-g sample of rat liver
tissue was placed in 5-10 ml medium containing
0.85% NaCl and 50 mM KH2PO4 (pH 7.4 at 4°C).
Mechanical pressed through the press. It is
homogenized with a Polytron type homogenizer
for 90 seconds. The homogenate was centrifuged
at 3000 g for 15 min and stored at minus 4°. In
order to evaluate the hepatoprotective activity in
the serum of rats treated with polyphenolic
compounds in an experimental toxic hepatitis
model, total protein, alkaline phosphatase, AST,
ALT were determined by Cypress Diagnostica
(Belgium) test kit. Blood was collected from
animals, centrifuged at 3000 rpm for 12 minutes,
serum
was
separated
and
biochemical
parameters were studied.
Determination of total protein concentration:
Total protein concentration in blood serum was
determined by the biuret method. The protein
forms a colored complex with copper ions in an
alkaline medium. (Table 2.1.1).
1.1- table.
Volume 04 Issue 02-2024
70
International Journal of Advance Scientific Research
(ISSN
–
2750-1396)
VOLUME
04
ISSUE
02
Pages:
68-74
SJIF
I
MPACT
FACTOR
(2021:
5.478
)
(2022:
5.636
)
(2023:
6.741
)
OCLC
–
1368736135
Add to test tubes
Sample
experience, ml
Calibration sample, ml Blank sample, ml
Working reactive
5,0
5,0
5,0
Serum
0,1
–
–
Calibrator
–
0,1
–
Distilled water
–
–
0,1
Alanine aminotransferase activity in serum was determined by the single Reitman-Frenkel method [5]. As
a result of transamination under the influence of alanine aminotransferase (ALT) enzyme, amino acids are
transferred from alanine to α
-ketoglutarate. ALT activity is proportional to the amount of pyruvate
dinitrophenylhydrazones formed in an alkaline medium and was determined by colorometric method
(Table 1.2).
1.2-table.
Sample composition
Sample experience
Blank sample
Substrate buffer solution, ml
0,25
0,25
Blood serum, ml
50
–
Incubate in a water bath at 37ºC for 60 minutes
Solution 2,4- DNFG, ml
0,25
0,25
Serum, ml
–
50
Aspartate aminotransferase activity in serum was determined by the single Reitmann-Frenkel method.
1.3-table.
Volume 04 Issue 02-2024
71
International Journal of Advance Scientific Research
(ISSN
–
2750-1396)
VOLUME
04
ISSUE
02
Pages:
68-74
SJIF
I
MPACT
FACTOR
(2021:
5.478
)
(2022:
5.636
)
(2023:
6.741
)
OCLC
–
1368736135
Sample composition
Sample experience
Blank sample
Substrate buffer solution, ml
0,25
0,25
Blood serum, ml
50
–
Incubate in a water bath at 37ºC for 60 minutes
Solution 2,4- DNFG, ml
0,25
0,25
Serum, ml
–
50
Alkaline phosphatase activity in blood serum was determined by the unique Bassey method [59]. The
amount of p-nitrophenol formed is proportional to the activity of the enzyme and is determined
photometrically. (Table 1.4)
1.4-table.
Composition of samples
Sample experience
Blank sample
Working reagent, ml
0,2
0,2
Serum, ml
20
–
Incubate at 37°C for 30 minutes
Diluted reagent No. 2 (Sodium hydroxide
solution), ml
2,0
2,0
Serum, mkl
–
20
Reagent 1 (glycine buffer) and Reagent 3 (p-
Nitrophenylphosphate) are mixed in a ratio of
4:1. The samples were mixed and photometered
at a wavelength of 405 nm. Enzyme activity is
calculated using a calibration curve. The activity
Volume 04 Issue 02-2024
72
International Journal of Advance Scientific Research
(ISSN
–
2750-1396)
VOLUME
04
ISSUE
02
Pages:
68-74
SJIF
I
MPACT
FACTOR
(2021:
5.478
)
(2022:
5.636
)
(2023:
6.741
)
OCLC
–
1368736135
of superoxide dismutase and catalase was
determined in the liver tissue homogenate.
Determination of SOD enzyme activity (KF
1.15.1.1) Misra and J. Fridovich [62]. conducted
according to the method. The principle of the
method is based on nitrotetrazolium blue (NTK)
for superoxide anions, which are formed as a
result of aerobic action and reduce the amount of
NADN phenosine metasulfate (FMS). (Table 2.1)
1.5-table.
Control
Experience
Reminder
TRIS-EDTAbuffer. rN=7.4
0.05 ml
-
Homogenate
-
0.05 ml
Reagent 1
2.0 ml
2.0 ml
10 minutes 37
0
S
Reagent 2
0.1 ml
0.1 ml
5 minutes 25
0
S
The
principle
of
spectrophotometric
measurement of catalase activity is based on the
ability of hydrogen peroxide to form a stable
colored complex with molybdenum salts [3] The
reaction was started by adding 0.1 ml of
homogenate to 2 ml of 0.03% hydrogen peroxide
solution. 0.1 ml of distilled water was added to the
blank sample instead of serum. The reaction was
stopped after 10 min by adding 1 mL of 4%
ammonium molybdate. Color intensity was
measured in a spectrophotometer at a
wavelength of 410 nm relative to a control sample
in which 2 mL of distilled water was added
instead of hydrogen peroxide.
Table 1.6.
Control
Experience
Reminder
N
2
O
2
2 ml
2 ml
-
Homogenate
-
0,1 ml
10 minutes 37
0
S
N
2
O
0,1ml
-
(NH
4
)
6
Mo
7
O
24
1ml
1ml
-
The amount of protein in the samples was
determined by the biuret reaction [6]. In order to
disrupt the mitochondrial membrane, 0.9 ml of 2
N KOH and 10 ml of deoxycholic acid were added
Volume 04 Issue 02-2024
73
International Journal of Advance Scientific Research
(ISSN
–
2750-1396)
VOLUME
04
ISSUE
02
Pages:
68-74
SJIF
I
MPACT
FACTOR
(2021:
5.478
)
(2022:
5.636
)
(2023:
6.741
)
OCLC
–
1368736135
to 0.1 ml of the mitochondrial suspension. After
the protein was completely dissolved, 4 ml of
biuret reagent was added to the solution and left
for 30 minutes at room temperature. At the same
time, control samples were prepared (1 ml of 2 n
KOH+10 ml of DOX+4 ml of biuret reagent). We
carried out calorimetry in cuvettes with a
thickness of 10 mm at a wavelength of 540 nm.
We determined the amount of protein by
calorimetry of bovine serum albumin (BSA) 10
mg/ml standards and using a calibrated curve.
Obtained results and comments: statistical
analysis of obtained experimental results and
drawing of pictures were carried out using the
computer program OriginPro 8.6 (Microsoft,
USA) in Hypothesis Testing one-sample t-test.
The results of 5 experiments were calculated as
the arithmetic mean value based on ± standard
deviation. The difference between the values
obtained from control, experiment and
experiment+study material was calculated by t-
test. Statistical reliability was calculated
according to Student's criterion. In this case,
values of R<0.05 represent statistical reliability.
Summary. In the course of our research, we used
modern biochemistry and molecular biology
methods, namely - the method of creating a toxic
hepatitis model, the method of isolating animal
tissue
homogenate
and
mitochondria,
determining the amount of LPO product MDA in
the liver tissue homogenate, determining
biochemical indicators in blood serum, protein
quantitative determination, antioxidant system
enzyme activity determination methods were
used.
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