Metal salicylates Co2+, Zn2+, Ni2+, Mn2+, Li+ and Mg2+: properties and effect on pain sensitivity

The paper presents the results of evaluating the effect of salicylates Co2+, Zn2+, Ni2+, Mn2+, Li+ and Mg2+ at doses of 5, 10 and 20 mg/kg on the pain sensitivity of male rats. Experiments were carried out on 119 male Wistar laboratory rats in test models of acute thermal pain (“tail-flick” and “hot plate”). It has been shown that acetylsalicylic acid (ASA) reduces the pain sensitivity with the participation of spinal and supraspinal mechanisms of regulation. The introduction of complexing metals (Co2+, Zn2+, Ni2+, Mn2+, Li+ and Mg2+) mainly leads to a decrease in the analgesic effect of ASA.


Introduction
Acetylsalicylic acid (ASA) has been used for many years not only as a universal analgesic, antipyretic and anti-inflammatory agent [1], but also as a unique source for the production of a wide coordination compounds variety, allowing the synthesis of new derivatives with complexing metals and various biologically active molecules and ligands [2]. The new compounds based on ASA obtained in this way are devoid of its negative side effects and are successfully used as antitumor, anti-inflammatory and antimicrobial substances [3,4].
Positive results of studies ASA derivatives biological effectiveness suggest that complexation based on divalent metals and salicylates is promising [4], since such derivatives with bimetals (Fe, Zn, Co, Cu, etc.) have fewer side effects and more pronounced effects than standard aspirin.
In this regard, the aim of this study was to identify the analgesic effects of salicylates (AS) Co 2+ , Zn 2+ , Ni 2+ , Mn 2+ , Li + and Mg 2+ on the thresholds of pain sensitivity (PS) in rats.

Animals
The experimental part of the work was performed in the Center for Collective Use of Scientific Equipment "Experimental Physiology and Biophysics" of the Department of Human and Animal Physiology and Biophysics of the Taurida Academy (structural division of the V. I. Vernadsky Crimean Federal University). The animals participating in the experiment were kept in standard vivarium conditions at a temperature of 18-22 °C on the "Rehofix MK 2000" litter (based on corn cobs) with a natural 12-hour light-dark cycle, free access to water (State Standard 33215-2014 2 "Guidelines for the maintenance and care of laboratory animals. Rules for the equipment of premises and the organization of procedures") and full-fledged granulated feed State Standard R-50258-92. The study was conducted in accordance with State Standard R-53434-2009 "Principles of Good Laboratory Practice" and the rules set out in Directive 2010/63/EU of the European Parliament and of the Council of 22.09.2010 on the protection of animals used for scientific purposes.

Design of research
The experiments were performed on male Wistar laboratory rats weighing 200-250 g. They were divided into 22 groups of 7 individuals each.
Group 1 -control (K) -male rats received intraperitoneal (i. p.) injections of 0.2 ml of saline solution (0.9 % NaCl) and were in standard vivarium conditions.
The studied substances were synthesized under the guidance of Professor A. N. Gusev at the Department of General and Inorganic Chemistry (Faculty of Biology and Chemistry) of Taurida Academy (structural division), V. I. Vernadsky Crimean Federal University. The chemical purity of tested compounds was not less than 98.0 %.
Testing of PS thresholds in rats was performed 20 minutes after injection in the "tail-flick" and "hot plate" models of acute pain stress. Before the "tail-flick" test, animals were placed in special retainers for rats (AE1001-R0, Open Science, Russia).
In the "tail-flick" test evaluated the perceptual component of pain. The main indicator of this test was the tail-flick latency (TFL) in response to light-thermal irritation. TFL was determined by the value of the time (s) of the tail withdrawal reaction. TFL was measured on the device LE7106 Tailflick Meter (Pan Lab Harvard Apparatus, Spain). On the tail of each rat, sitting in the fixator, three presentations of a thermal stimulus were performed, followed by the calculation of the average value of TFL in seconds for each animal. This test is based on a spinal flexor reflex that occurs in response to a local force on the tail by high temperature, and allows us to judge the PS of animals mainly at the spinal level [5][6][7][8].
In the "hot plate" test on experimental apparatus Cold and hot plate CHP (Bioseb, France), the hotplate test latency (HPTL) of the animal was recorded, which was determined by the value of the time (s) of the reaction manifestation of withdrawal and licking of the limbs and (or) vocalization from the heated surface. The test allows to judge the PS of animals with the participation of supraspinal mechanisms [8-11].

Statistical methods and analisys AS biological action with metals
Calculations, statistical processing and graphic design of the data obtained in the work were carried out using the Microsoft Excel program and the Graph Pad Prism 7.0 software package. The reliability of statistical differences between the control and experimental groups with different doses of ASA and metal salicylates was determined by using a one-way analysis of variance (ANOVA) with a posteriori Tukey test and Dunn's test of multiple comparisons. The EC approaches zero if the efficiency of the studied compound (АSmet) in relation to the studied indicator corresponds to that for the ASA.

Results and discussion
The results of the study showed that the administration of ASA at doses of 5, 10 and 20 mg/kg significantly reduces PS in rats (Table 1). This is evidenced by a significant increase in TFL in the "tail-flick" test by 58.7 % (p<0.05), 80.4 % (p<0.05) and 114.4 % (p<0.05), respectively, and HPTL in the "hot plate" test by 61.1 % (p<0.05) at a dose of 10 mg/kg and 78.8 % (p<0.05) at a dose of 20 mg/kg relative to these parameters in the control group of rats. a Significance of differences compared to the control group (р≤0.05). b Significance of differences compared to the control group (р≤0.01). c Significance of differences compared to the control group (р≤0.001). d Significance of differences compared to group 2 (р≤0.05). e Significance of differences compared to group 3 (р≤0.05). f Significance of differences compared to group 3 (р≤0.001). g Significance of differences compared to group 4 (р≤0.05). h Significance of differences compared to group 4 (р≤0.01). i Significance of differences compared to group 4 (р≤0.001).