ACTIVITY OF NEWLY SYNTHESIZED SALICYLATES AT VARIOUS DOSES
Cheretayev I.V., Yakovchyuk T.V., Koreniuk I.I.,
Khusainov D.R., Gamma T.V., Epishkin I.V.
Simferopol, Тaurida National I.I. Vernadsky
Abstract: It is shown that psychotropic activity of
newly synthesized salicylates at various doses is manifested as a nonmonotonic
dependence having its peaks at therapeutic and ultra-low dose zones.
Key words: psychotropic activity, salicylates, therapeutic
doses, ultra-low doses.
The effectiveness of most pharmaceutical products is noted to rise as the
dose is increased, in some cases reaching its maximum at high dosage rate.
However, the administration of pharmaceutical products at therapeutic doses and
at high doses, a fortiori, is usually accompanied by the development of a broad
spectrum of side-effects. Thus, for acetylsalicylic acid (Asp), along with the
anti-inflammatory and analgesic action, the manifestation of psychotropic
effects is observed at the upper limit of therapeutic dose (TD) . Aspirin,
in spite of having the great number of positive properties, also provokes
stomach ulceration, negatively influences the cells of liver and of blood .
It should be noted that the spectrum of side-effects enlarges as the dose is
increased. It was shown pharmacological activity of
aspirin at ultra-low doses (ULD) with
decreasing side-effects recently, and it was found anti-stress, anxiolytic,
antidepressant effects at TD and ULD . No exceptions, that newly synthesized
aspirin's derivatives – acetylsalicylate
cobalt (ASC) and acetylsalicylate zinc (ASZ), which manifested psychotropic
activity at TD, is probable to preserve the
same properties at ULD, but without side-effects. Due to the strictly
individual nature of effect manifestation at ULD for each pharmaceutical
product , this work is aimed to reveal the specificity of psychotropic
effects of ASС
and ASZ at
various doses and to compare them with the action of aspirin.
The research was conducted at 250 white outbred
male rats having 200-250 gr of weight that were divided into 25 groups, 10
animals in each group. The studied substances (Asp, acetylsalicylate cobalt
(ASC) and acetylsalicylate zinc (ASZ)) were diluted in normal saline and
injected intraperitoneally (i.p.) 0.2 ml at a time, at high doses (120 and 80
mg/kg), at TD (40, 15 mg/kg), “dead zone” dose (40·10-4 mg/kg) and
at the three ULD (40·10-8, 40·10-10, 40·10-13
mg/kg). The animals of the control group were administered to the equivalent
volume of normal saline solution. The TD and ULD zones are separated by the
“dead zone” in dosage range 10-12-10-8 M where the
psychotropic effects are absent (Table 1). The control point at 40·10-4
mg/kg is taken to provide better illustration.
1. The ratio of dose/concentration.
30 minutes after the i.p. injection we studied the
behavioral response of the rats using open field test  and Porsolt’s test .
The reliability of disparity between the control group and the experimental
group was estimated with the use of non-parametric U-criterion by Mann-Whitney
(p ≤ 0,05, р ≤ 0,01). The data is represented in relative units
(%), values of the control group being taken for 100%.
The standard Open Field test is commonly used to
assess locomotor, exploratory and anxiety-like behavior in laboratory animals
(rats/mice). This test is particularly useful in evaluating the effects of
anxiolytic and anxiogenic drugs, locomotor responses to drug and as well as
behavioral responses to novelty. The Open Field area generally consists of an
empty and bright square arena, surrounded by walls to prevent animal from
The animal is usually place in the center of the
arena and its behavior recorded for 3 min. We are registered horizontal motor
activity and research activity.
Porsolt’s test is the most commonly used test for assessment
of depressive status in animals. The experimental setup is a transparent
cylinder filled with water of room temperature. A rat is placed in the water;
its behavior is observed for 3 min. The time of active swimming and the time of
immobility (when a rat just weakly moves the paws to keep afloat) are
registered. The latter is interpreted as “hope loss behavior” and is longer in
animals with depressive-like status. This immobility time is decreased by
The reliability of disparity between the control
group and the experimental group was estimated with the use of nonparametric
U-criterion by Mann-Whitney (p ≤ 0.05, р ≤ 0.01). The data is
represented in relative units (%), values of the control group being taken for
100%. During the experiments all the ethic norms were observed.
The Figure 1, A shows psychotropic effects
of Asp in dependence on the dose in the Open Field test. It is obvious that Asp
at high doses significantly raised the horizontal movement activity (HMA) and
investigative activity (IA) which is the evidence of its marked anti-stress and
anxiolytic action (р ≤ 0.01) . At the upper limit of TD of Asp (40
mg/kg) IA was decreased reliably and HMA—unreliably, that is, anxiogenic effect
was produced instead of anxiolytic one, anti-stress action neutralized. At 40∙10–8
mg/kg Asp dose the effects of TD (40 mg/kg) were repeated. At 40∙10–10
and 40∙10–13 mg/kg the anxiogenic action of Asp became
obsolete, but anti-stress action was revealed against this background.
Figure 1. The curves
“dose-effect” of aspirin (A), acetylsalicylate zinc (B) and acetylsalicylate
cobalt (C) in open field test.
ASZ at high doses, as well, as Asp, increased HMA
and IA in the animals, which is the evidence of its anti-stress and anxiolytic
action (Figure 1, B). The dose being decreased, ASZ shows the growth of
its anxiolytic effect along with the reduction of anti-stress one. At the upper
limit of TD (40 mg/kg) anxiolytic effect reaches its maximum, while anti-stress
effect is not manifested at TD Zone at all. Further decrease of the dose causes
neutralization of psychotropic effects of ASZ. However, at 40∙10–8
mg/kg dose ASZ makes anxiolytic action, but it is less marked than that one at
TD. At other ULD (40∙10–10 and 40∙10–13
mg/kg) anti-stress action of ASZ is well seen in the absence of anxiolytic
effect. ASC at high doses produced toxic effect. Under these conditions 70% of
the sampling administered to 120 mg/kg dose, and 40% of the sampling
administered to 80 mg/kg died. The surviving animals were excluded from further
experiment. At the upper limit of TD anxiolytic action of ASC was manifested (Figure
1, C). The dose being decreased, the effect grew neutralized. At ULD Zone a
certain regularity of effect development is discovered: the dose being
decreased, anxiolytic action of ASC grows 2 times as much as that at TD, then
at 40∙10–10 mg/kg dose the effect is ne utralized, to be
manifested again at 40∙10–13 mg/kg dose, but with less
intensity. It must be noted that anxiolytic effect of ASC at ULD is accompanied
by anti-stress effect development, reaching its maximum at 40∙10–13
Thus, judging by the results of the Open Field test
we can see that the same way of psychotropic effects development is typical for
both Asp and ASC. It must be noted that ASC has the most intensive anxiolytic
action at ULD Zone (40∙10–13 mg/kg) compared with Asp and ASC,
where substances preserve their biologically active properties, but
side-effects are absent.
At Figure 2, A we can see that in Porsolt
test Asp at all doses extended the time of active swimming and reduced the time
of immobility in experimental group rats as compared with the control group
animals (р ≤ 0,05), which is the evidence of its antidepressant action
. At 120 mg/kg dose Asp demonstrated development of the maximum
antidepressant effect that decreased at TD (40 mg/kg). The dose being
decreased, antidepressant effect was neutralized, to be manifested again at ULD
Zone, repeating the TD effect.
ASZ at high doses showed antidepressant action. At
the upper limit of TD it was reduced insignificantly (Figure 2, B). The
decrease of the dose caused neutralization of the effect. At ULD Zone ASZ at 40∙10–8
mg/kg dose significantly raised the depression level in animals, which was
manifested by reduction of active swimming time and, correspondingly, extension
of immobility time. However, at 40∙10–10 and 40∙10–13
mg/kg ASZ demonstrated antidepressant effect. This fact indicates the inversion
of substance operational sign at 40∙10–8 mg/kg dose. ASC
at the upper limit of TD had antidepressant action. The dose brought down to 15
mg/kg, this effect was neutralized, the same way as at the dead zone (Figure
2, C). At ULD Zone, all doses of ASC extended the time of active
swimming and, correspondingly, reduced the time of immobility. This is the
evidence of antidepressant effect of ASC at ULD. Consequently, of all the
studied substances maximum antidepressant effect at ULD was demonstrated by
ASC, compared with Asp and ASZ.
Figure 2. The curves
“dose-effect” of aspirin (A), acetylsalicylate zinc (B) and acetylsalicylate
cobalt (C) in Porsolt’s test.
Thus, we have shown that in case of salicylates
administration at various doses their psychotropic action is manifested as a
nonmonotonic dependence having its peaks at both TD and ULD zones. It must be
noted that the development of psychotropic effect at ULD is accompanied by
manifestation of the new properties. On summing up the data we discovered that
ASC, causing acute toxicity at high doses, resulted in maximum intensity
effects of all the tested compounds when used at ULD.
Generally, we have shown that administration of Asp
and its salts at high doses causes maximum psychotropic effects to occur.
However, the use of both Asp and its derivatives  at the studied dosage
range is often accompanied by side-effects; moreover, for ASC at high doses the
acute toxicity and, consequently, lethality is typical. So, the high doses of
Asp must be administered with caution, while its newly synthesized derivatives
require to be carefully studied as to having biological effectiveness at lower
According to the results of this research, ULD zone
has shown psychotropic action which repeats the effects of high doses and TD,
at that approaching the maximum intensity at the lowest doses. The use of Asp
at ULD offers a number of advantages: e.g., no side effects, a more pronounced
effectiveness compared with TD, etc. The results give us grounds for
recommending the use of Asp at ULD in clinical testing as a psychotropic
The absence of biological effects of Asp and its
salts at 10–12-10–8 range is explained by the “dead zone”
existence. This phenomenon has been demonstrated in almost all experimental
works. Formation of the “dead zone” is explained in literature by the
hypothesis about the existence of the systems with negative feedback .
The material collected in the field of ULD study
prevents us from skepticism towards ULD effects of biologically active
substances, and, undoubtedly, the phenomenon of ULD is worth serious scientific
The presence of psychotropic effects of Asp and its
salts at ULD range has its prerequisites justified by the data from the
literature on the subject. Asp at low doses has indicated effectiveness in
treatment of the vascular heart diseases, prevention of Alzheimer disease.
Also, scientists have conducted a complex research on blood coagulability under
aspirin influence at various doses . First they studied the effects of the
medication at therapeutic doses, after that – at low doses, then – to extremely
low ones, and finally – to apparent concentrations. At that, they discovered an
interesting phenomenon while studying the role of aspirin in blood clotting.
The scientists decided to test the effectiveness of the medication made “after
Hahnemann”, that is, as a homeopathic preparation. It was found that
“ultra-low-dose aspirin” (ULDA-H) created antithrombotic effect by the mechanism
other than that one at TD. While aspirin at TD knock out cyclooxygenase (COX-1),
ULDA-H does not produce any effect on that, but it arrests COX-2. It is
possible that Asp at ULD influences COX-2 getting to brain through
hematoencephalic barrier. Moreover, it was shown that it is in brain where COX-2
is mainly contained, so it is only possible for non-steroidal anti-inflammatory
drugs to reach anti-inflammatory effect in absence of side-effects on
gastrointestinal tract and other tissues involved in COX-1 activity if they
inhibit COX-2 activity electively .
However, no theory has been formulated yet in
literature to explain to the fullest all the effects of extremely low doses of
biologically active substances. Some efforts have been made to explain the
observed effects by following hypotheses: concoction of the medical substance,
existence of highly effective systems of receptor signaling transduction and
enhancement, “adaptation” of receptors towards the action of extremely low
doses, existence of supra-high-affinity receptors . Also, it must be borne
in mind that highly diluted solutions of Asp and its salts were prepared not by
usual diluting method, but by the homeopathic potentiating method. Still,
within the frame of this experiment the dosing frequency and the duration of
salicylates treatment were not taken into consideration depending on the
potency and dose.
Thus, there are a number of advantages in use
derivatives of Asp at ULD in mental illnesses treatment. Upon that, the
research has a perspective of further analyzing distinctive features of course
treatment with aspirin’s salts and studying possible mechanisms of their
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