PERSPECTIVE ASPECTS OF THE APPLICATION OF THE CRYO-ACTIVATED POWDER OF ARONIA
MELANOCARPA
L.V. Savchenkova, O.D. Nemyatykh, M.S. Akimova,V.V. Rokotyanska
Lugansk State Medical University
The growth rate of technological progress, environmental degradation,
increases the tension of life and strengthening the influence of factor’s that
exert pathogenic effects on the human body (e.g.- stress, AIDS, the
consequences of the Chernobyl accident, intake of Xenobiotics, etc.), determine
the actual problem of modern medicine in development of new drugs, especially
of plant origin, which would more benefit than
that of the different synthetic analogues with biological affinity for
the body tissue, with good action and low toxicity [1,2,3].
Unfortunately today, the range
of modern hepatoma, stressprotector
and drugs
that have immunostimulatory effects
were increased, especially synthetic
agents on the background of which the
probable excessive intervention in almost all
functions and systems of the body and, consequentl,
higher risk of side effects.That is
why in recent
years, attention has struck to pharmacists, and technologists to draw
out the Aronia
extracts, the growing interest is related primarily to its
high and multidirectional biological
activity [1,4].
Therefore, the
aim of this work was to study hepato- and
stress-protector activity of cryo-activated powder Aronia and determination of the tablet form of this medical
plant material.
Materials and methods. The
study was carried by 256
adult mongrel rats of both sexes, weighing 160-180 g.
The animals were in a placed in a vivarium DZ "LSMU"
and received a standard diet in the form of granulated fodder for the
established norms. All studies or tests
were performed in accordance with international rules to animals.
Immunodeficiency in
immature rats intraperitoneally modeled
by a single introduction of hydrocortisone acetate (Hydrocortison - Richter,
Budapest, Hungary) at a dose of 250 mg/kg. The functional activity of humoral
immunity in the rat with normal immune status was evaluated by the Level of hemagglutinin (HA) in serum and the number of antibody-forming cells
(AUC) in the spleen. Rat immunized with the antigen antibody
forming, namely, sheep erythrocytes (SE, 3% solution at a dose
of 1 ml/100g rat weight intraperitoneally, once). On the fifth day after
immunization titers were determined hemaglyutinіn in the serum of experimental
animals by serial dilutions in polystyrene plates [21].
Effect of nonspecific resistance of
animals was evaluated in a test of the
phagocytic activity of
neutrophils (FAN) of blood with latex for phagocytic index (Fi), phagocytic number (Fn) and index of
phagocytic activity (IFA)
[22].
In determining the level of
implementation іmmunotropnic properties of the
original drug against
the background of immunodeficiency, drug was
given for 5 days befor
playing the simulated pathology and during all duration of the
experiment untill the day of the
indicators of immune response. The total
period of administration was
11 day. Immunized animals
were on the diagram
above, a day after the administration
of hydrocortisone acetate (HA). To determine
the degree of immune deficiency assessment on a
5-day were determined by control after
immunization titers and a mass
coefficients (MC) of immunogenesis (thymus and spleen). As the
reference drug used
tincture exinacea purpure at a dose of 1 ml/100kg.
Experimental model was for the Toxic
hepatitis (TH), pathological
process that develops in animals after intraperitoneal injection of carbon
tetrachloride in a dose 4ml/kg within 4 days [5]. Assessment of energy
homeostasis in rats with TH after application of cryo-activated Aronia melanocarpa was performed by determining the
content of ATP, ADP and AMP in erythrocyte membranes of rats by thin layer
chromatography of the company «Merk» (Germany) on plates [11].
In the
study of hepatoprotector Aronia
melanocarpa, reference preparation Silіbor
(Pharmaceutical company «Health»,
m.Kharkіv, Ukraine) was used in doses, respectively 258 mg/kg and 165 mg/kg in
a 5% aqueous suspension daily for 10 days 1 hour after administration of carbon
tetrachloride. All studies were performed in dynamics: before treatment, at 7
and 14 day study.
As indicators for
evaluating the effectiveness of stressprotector Aronia melanocarpa we used
classical psychopharmacological test «open field» [9]. It is important to note
that in the previous step, the evaluation of behavioral responses to the test
«open field», all the animals were divided into stress stable ones and
unstable. Hypokinetic stress (HS) simulated by placing rats
in a close cage-penal for 24 hours [10]. The Cryo-activated powder
of Aronia melanocarpa and the reference drug were administered orally phenibut
at doses of respectively 149 mg/kg and 25 mg/kg daily for 10 days after the
formation of this pathology. All studies
were performed in dynamics: 1, 10 and 15 day for experiment
Statistical processing of the
results was performed using t-criterion
of Student. Data processing was
performed using the software
packages Microsoft Excel XP, Statistica
6.0 [23].
Results
and discussion.
Investigation
of the effect of tablets containing cryo-activated powder
of Aronia melanocarpa
on humeral immunity of immature rats with normal immune
status showed that the introduction of the
drug increased hem agglutinin titers in the serum
and increasing the number of AUC in the spleens
of immunized animals. This indicates pronounced immune
stimulatory properties of the drug. The greatest ability to enhance
antibody formation of tablets in doses of Aronia melanocarpa implement 3,0 and
4,0g/kg, significantly increasing the titers in the serum of immunized animals
(tabl.1).
Tablet 1
The
influence of Aronia melanocarpa pills on humeral
immunity of young
rats
with normal immune status
|
Groups of animals |
Dose |
Titles, Log2, (Ме (LQ; UpQ) |
AUK on spleen |
|
Intact |
- |
13 (13; 13) |
18022±1404 |
|
Reference |
1,0 мл/кг |
13 (13; 13) |
15678±957* |
|
Research |
2,0 г/кг |
14 (13;17) */** |
24622±2193** |
|
3,0 г/кг |
15 (14;17) */** |
29493±2359*/** |
|
|
4,0 г/кг |
15 (14; 16) */** |
29485±2341*/** |
Notes: * - Difference significant in comparison with a group of intact animals;
**
- Difference significant in comparison with the reference group
The
study of the spleen cells of saturation showed
that under the influence of drug, injected in doses of 2,0; 3,0
and 4,0 g/kg there is an increase
in the number of splenocytes at 36, 64, 64%, respectively,
suggesting a pronounced activation of immune-competent cells in the experimental group as compared
with intact rats. At the same time, draws
attention to the negative correlation between the
reference and experimental series of experiments.
Comparative
analysis of the phagocyte activity of blood
neutrophils as a key indicator of the functional state of
the immune system found that a drug developed based on a
combination of plant bioactive substances in doses of 3,0
and 4,0 g/kg in two weeks promotes a significant increase
in phagocyte number and index realized an increase
of IFA by 21 and 28% compared with an intact set
of experiments, respectively. In this study drug is active at
the level of the reference drugs.
Thus,
the results of this series of experiments give reason to believe
that the studied drug implements the ability to
stimulate the humeral and cellular immunity in healthy animals.
It
is important to note that the introduction of hydrocortisone
acetate to rats of the control group shows the
development of a pathological condition that affects a
significant decrease in A titer and is accompanied by a
clear tendency to decrease in MC spleen and thymus. The
latter is a very natural result of the cytotoxic effect of
large doses of hydrocortisone. Prophylactic administration of tablets
of melanocarpa helps to restore immune response in animals. The
effectiveness of the original drug in the context of the conservation
title antitilogen manifested on the level of A
titer rat’s intact group. At the same time increasing the
dose has a positive effect on the value of the
estimated figure for the studied experimental conditions.
It
should be noted that under the influence of the original drug prevention also
notes changes in the MC spleen and thymus.Thus,during treatment with tablets at
a dose of Aronia melanocarpa,
at dose of 3,0 g/ kg compared with the values of the coefficients the control resumed on 40 and 20%, while increasing the dose by 25 and 80% respectively.
Next data
suggest that the formation of TH accompanied
by a substantial and long-term violation
of relationships in the system ATP-ADP-AMP.
Yes, the formation of toxic liver damage leads to a
decrease in ATP content already on the
first observation time by 50%
compared with intact rats. Application
of the same with the treatment
and prevention by Aronia
melanocarpa prevents the
formation of energy-deficient state,
helping to improve the content of ATP at 7 and 14 day observation at 56% and 50%, respectively. The
efficacy comparison of the
experimental conditions mentioned in the folded 30-37%
(fig.1,A).
|
|
|
ВFigure 1. The
influence of Aronia melanocarpa adenine nucleotide content in
in toxic liver injury tetrachloroethane (A – ATP, B – ADP)
A similar dynamic is noted in the
study of the content in
erythrocytes of rats triglyceride levels of ADP. From Table 1 shows
that the formation of
hepatitis reduces
the content of the
marked component of adenine nucleotides to 28-41% in different
periods of observation. In
animals that received powder of Aronia recorded level of ADP,
which is 46% and 39% higher than in the controls at the
appropriate time study at the
same time, against the background of the
contents of the last silіbor increased only by 10-25%
(fig.1,B).
An analysis of AMP found
that when the simulated form of triglycerides
in the erythrocytes of rats significantly increases the level
of the marked component of adenine nucleotides
(in 1,6-1,8 times relative
to performance in the
intact group),
which is conditioned, in our opinion,
the first amplification process dephosphorylation of ATP and
ADP. In addition, aronia
melanocarpa significantly more effective than silіbor, prevents
the degradation of ATP, which
decrease in the concentration of AMP (by
12-17%) in a series of experimental
animals, compared with the reference group (fig.2).
Figure 2. The
influence of Aronia melanocarpa AMP in
in toxic liver injury tetrachloroethane
For more correct estimatation the
energy profile in animals with TH we have analyzed the
parameters of energy. It is
established that under the conditions of
formation of TH indicated a sharp
decline in the degree of filling of the
ATP-ADP-AMP with
high-energy phosphate ligaments. This is
evidenced by the magnitude of the
energy charge (EC),
which in the control series of animals throughout
the experiment was
significantly (p< 0,01-0,001) less than in the
intact group of animals. Against
the background of the same
application Aronia melanocarpa marked
increase in the value EC (on average 28-38%) compared with control rats,
approaching the performance of intact rats. And Aronia melanocarpa in
their ability to increase the degree of filling adenil nucleotide
of phosphate bonds by 10-12% higher than the comparator drug
silibor.
In studying the rate of energy potential (EP) cells, which indicates the
rate of mitochondrial respiration revealed that TH statistically significant
declining trend throughout the experiment at 19-30%. At the same time, the
application of Aronia melanocarpa in a
simulated form of TH implemented prevention to reduce the rate of mitochondrial
respiration, as evidenced by the increase of the EP for all the duration of the
study. It was also established that the group of rats with toxic liver lesions
which received the experienced drug, the value of EP is close to those in the
intact and the reference groups during all periods of the study (p<0,05).
Othere informative parameter is
the index of energy phosphorylation (EPR), which is defined as the ratio of ATP
to the sum of ADP and AMP. Experimentally proved that in the control group, the
values of EPR are reduced in 1,8-2,3 times with respect to performance,
recorded in intact animals. Against the background of the introduction of the
same powder of Aronia melanocarpa to rats with toxic liver lesion there is a
significant (65-70%) increase in EPR compared with the control group. Moreover,
a group of animals that received the drug aronia, on 7th and 14th from time of
the study analyte index values close to the level of intact animals
(p<0,05). The effectiveness of Aronia melanocarpa to exceed the reference
drug for 14-23%.
The maximum is adequate, in accordance with the intended purpose, an
indicator of oxidative phosphorylation is a thermodynamic control of breathing
(TCB), which indicates the dependence of the rate of respiration, not only on
the concentration of individual components
adenilnucleotide system, but also on the intensity of phosphorylation as
a whole. The values of TCB in control animals significantly (by 52-70%) and
significantly (p <0,001) reduced compared with the intact group of animals
for all the duration of the study. The same time, the rats with TH taking
Aronia powder, the rate of TCB during all periods of observation are
significantly higher than in controls. It is also important to note that the
effectiveness of Aronia melanocarpa under
conditions of toxic liver damage exceeded
for a comparator to 30-37%.
Thus, the studies found that aronіya,
which is used in the form of cryo-activated powder exhibits pronounced hepatoprotective effect, contributing to the
resumption of oxidative and energy homeostasis in liver in case of induced
hepatitis.
To study the action stressprotector of Aronia
melanocarpa used classical test «open fly». It is
proved that the formation of HS stress- animals leads to
a significant decrease in horizontal locomotor
activity in rats by an
average of 60-87% compared
with the intact group of rats at
different periods of the investigation
(fig.3).
Course assignment Aronia powder leads to
a resumption of the horizontal
activity of animals, where stressprotector action largely realized with a 5-th
time of study, contributing to the
improvement of this indicator as much
as 144-201% at
different periods of observation.
|
|
B |
Figure 3. Influence of Aronia melanocarpa at horizontal locomotor
activity in rats with stress response (A)
and stress unstabletypes (B) types in the hypokinetic stress
Similar dynamics of the orienting-exploratory
behavior of animals observed in
animals stress type of response when the
level of horizontal activity of the
control group during the first 10 days remains the
same and shows 4,0-5,6% of
the rate in intact rats. Conduct drugs also contributed
to the increase in horizontal
activity of rats 70-169%
compared with controls. In
contrast, animals in the
stress stable and stress unstable rats stressprotector
maximum effect was only during 15 day
of the study, and for the
severity of less than
26%. The efficacy of
preparation in comparison on both groups was much smaller.
One of the most important indicators that characterize the orientation and
exploratory behavior of animals in the HS is the definition of the latent time
of transition from the central square of the rat. It should be noted that the
simulated pathology occurs in all animals, a significant increase in the marked
indicator, describing anxiety-depression state. It should be noted that the
application of Aronia is the early time
of the study observed almost complete renewal of the indicator, and the
effectiveness of the latter is kept until the end of the period of the
experiment. It should be noted also that the efficiency of powder Aronia
melanocarpa on animals with stress type
response to stress was almost identical with the animals of stress unstable response
type.
In further studies it was found
that the formation of HS in rats with
stress response and stress unstabletypes type the response is a
significant decrease in vertical activity, «mink
reflex», acts grooming and
autonomic balance. It is important to note that in all groups of animals of
different types of responses that received
aronia in the 10 days to 15th days of study, a complete restoration of studied parameters or
indicators, so you can judge the characteristic of high motivational
constituent of these animals, the resumption of the orienting-investigative
activity that clearly indicates the ability of Aronia to renew orienting-investigative activity of the
animals with different types of responses to stress, helping to reduce anxiety
and emotional animals. Comparator drug has not shown similar efficacy.
CONCLUSIONS:
1.
The use
of tablets Aronia melanocarpa realized the enhance of
immune response of humeral and cellular immunity, in both
healthy animals and in rats with immunodeficiency.
2.
Course application
of cryo-activated powder of Aronia melanocarpa with medical
prophylaxis against toxic liver marked protective
effect with regard to energy reactions in the
mitochondria in a study of pathology, which is implemented
by normalizing levels of all parameters of energy.
2. The cryo-activated powder
of Aronia melanocarpa leads to the restoration of
orienting-exploratory and motivational behavior of animals
with hypokinetic stress, helping to
reduce emotional animals, and such manifestations
of stress, like anxiety and depression.
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