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PREVENTIVE ACTIVITY OF THE EXTRACT OF THE DARKLING BEETLE ULOMOIDES DERMESTOIDES IN THE DIET OF C57BL/6JSTO MICE IN A NEUROTOXIC MODEL OF PARKINSON'S DISEASE

: The effect of aqueous extracts of the biomass of the adult beetle Ulomoides dermestoides on the delayed effects of defoliant paraquat causing parkinsonism in experimental mice was evaluated. The motor activity of the animals was analyzed in behavioral tests using a rotarod and a vertical pole. The number of tyrosine hydroxylase-immunopositive neurons in the ventral part of the substantia nigra of the midbrain of experimental and control mice were studied by immunohistochemistry. In the model in vitro system with SH-SY5Y – human neuroblastoma, the effect of the extracts on cell proliferation was examined in the absence and in the presence of the neurotoxin MPP + . The isolation of biologically active substances from raw biomass using cavitation effects made it possible to obtain extracts with protective properties in the model of an early stage of Parkinson's disease used.

number of tyrosine hydroxylase (TH)-immunopositive neurons in the ventral part of the substantia nigra of the midbrain of mice were studied using immunohistochemistry [48]. In a model system with SH-SY5Y-human neuroblastoma, we examined the effect of the extracts on cell proliferation without and in the presence of the neurotoxin MPP + [49].

Results
The rotarod test, assessing the biological effect of beetle extracts in the diet of mice under conditions of a neurotoxic model of an early stage of Parkinson's disease, revealed (Fig. 2, Table 1) that continuous administration of "extract-cavitation" with food caused a slight insignificant increase in the mean rotation speed which the mouse could sustain before falling down compared with the control-toxin group (from 12.5±1.2 to 15±1.6 rpm; M±S.E.M.; p>0.1).
The mean time of holding the mouse on the rotarod before falling increased significantly (from 701 to 825 sec, p<0.05, n=12/24, U-test).
The preparation "extract-lightning" caused a sharp increase in the ability of the animal to hold on the rotarod rotating both with constant and increasing speed. Improvement occurred on both parameters: rotarod rotation speed and mouse retention time (Fig. 2, Table 1). The indicators in this group did not differ from those of the intact control, with a significant difference from the control-toxin group (for both indicators, p <0.001).  The results of intergroup comparisons of the maximum. minimum and mean duration of exposition on the vertical pole are presented in Table 2. Comparison of the maximum, minimum and mean times (out of three attempts) spent by mice on the pole using the Kruskal-Wallis test showed that there were significant differences between the groups in maximal and mean times only (maximum time: H3=10.19, P=0.017; mean time: H3=11.10, P=0.011), therefore, further. in statistical analysis, the minimal time is excluded from the account.
Pairwise a posteriori comparisons using the Steel-Dvas-Critchloo-Flyiner test showed that the maximal time spent on the pole was significantly higher in the "extract-cavitation" group than in the control-toxin group. And the mean time spent on the pole was significantly higher in the "extract-lightning" group than in the control-toxin group (Table 3). The ratio of the maximum in three attempts to the maximum possible time spent on the pole (Table 4) in the "extract-lightning" group was significantly higher than in the other groups. The same relation was significantly higher in the control group without toxin, as well as in the "extract-cavitation" group, compared to the control-toxin group.
There were no significant differences in this parameter between the "extract-cavitation" and toxin-free control intact groups. The relation of the mean exposure time on the pole to the maximal possible in three attempts in the "extract-lightning" group was also significantly higher than in the other groups, and in the "extract-cavitation" groupcompared with the control-toxin group. There were no significant differences in this parameter between the other groups. In the middle brain samples of 4 animals from the intact control, toxin control and two "extract" groups. The number of TH-immunopositive cells was counted. The number of TH-immunopositive cells in animals that received an injection of paraquat was significantly lower than in intact control animals, as well as in animals in the "extract-cavitation" and the "extract-lightning" groups (H3=12.794, P=0.005, Kruskal-Wallis test). At the same time, the number of TH-immunopositive cells in animals from the "extract-cavitation" group was significantly lower than in control intact animals and animals from the "extract-lightning" group (Table 5 and 6). Representative slices of the substantia nigra of the midbrain of the intact control animal, the toxin-control one, and the one from the "extract-lightning" group, stained for TH, are shown in Fig

Discussion
The use of methods for the extraction of biologically active substances from the raw biomass of the darkling beetle of animals after intoxication with paraquat compared with the control group, which was intoxicated with paraquat, but did not receive the antidote. Intake of the preparation "extract-lightning" with food almost completely eliminated the toxic effect of paraquat in the rotarod test. In the vertical pole test, the mice from both "extract" groups could hold on to an upright pole longer without sliding or jumping off. That is why the proportion of the maximum and the mean (in three attempts) times spent on the pole (from the maximal possible) in mice of these groups were higher than in mice from the toxin-control group. This is probably due to a higher muscle tone and better coordination of movements, both during locomotion and maintaining a stationary position on the supporting surface of the vertical pole. The mice from the "extract-lightning" group demonstrated the best results.
The behavioral tests results were consistent with the results of histochemical examination of mouse brain slices. The
Adult sexually mature beetles of both sexes separated from the substrate were immobilized by cooling at -18°C and the resulting biomass was divided into two parts. One part was crushed in distilled water using cavitation on a rotary-pulsation unit as part of RPA 0.8-55A-5.5/2 "Stalpischeprom, Ltd.", Russia, followed by separation of the sediment by centrifugation in an Ohaus FRONTER 5706 for 15 min at 5000 g. Received extract we named as "extract-cavitation". Beetle substances were extracted from the second part of the biomass by the method of electro-pulse plasma-dynamic extraction (EPPDE) in distilled water at 23 0 C. EPPDE extraction was carried out on a "KorolevFarm LLC" setup, Russia [36]. Extraction parameters: the power of the transmitted electric discharge is 38,000 Volts, the pulse frequency is one pulse per second, the distance between the electrodes is 5 mm, the extraction time is 7 min. After exposure, the extract was separated from the solid fraction by centrifugation at 5000 g for 15 min.
Received extract we named as "extract-lightning". Antibacterial processing of extracts was carried out by radiation decontamination using a beam of accelerated electrons using a compact radiation sterilization unit with local biosecurity (CRSU) of the Moscow Radiotechnical Institute, Russian Academy of Sciences, at 15 KGy. The energy of the accelerated electrons is 5 MeV, the power of the electron beam is 1.5 kW.
"Extract-lightning" contained 20 mg/ml, and "extract-cavitation"-12 mg/ml of dry matter. The obtained extracts were immobilized on sterile food wheat bran; the final moisture content of the mass was 8%. The preparations were stored in a refrigerator and fed to experimental mice, for which dry preparations were added to the main food mixture for mice by fractional and thorough mixing (at the rate of 4 g of the "preparation-lightning" and 8 g of the "preparation-cavitation" per 1 kg of the food mixture). The main food mixture consisted of porridge, including boiled oats and peas, with the addition of vegetable oil.
To study the biological activity of the extracts, we used a neurotoxic model of an early stage of Parkinson's disease in male C57Bl/6jsto mice administrated with paraquat toxin [19,[21][22][23][24][25][26]. The animals were divided into 4 groups. Group 1 (n=12) -toxin+"extract-cavitation"; the animals were injected i.p. twice (with an interval of 1 week) with 10 mg/kg of paraquat, dissolved in 0.3 ml of saline; an extract obtained by cavitation was added to the food as an antidote as described above; the supplement began one week before the 1 st injection of the toxin and continued throughout the entire experiment. Group 2 (n=11) -toxin+"extract-lightning": the animals were injected with paraquat and received the beetle extract with the food -everything according to the same regimen as Group 1. Group 3 (n=12) -intact control; the animals were not injected with paraquat and did not receive anything additional to the food. Group 4 (n=24)toxin-control; the animals were injected with paraquat as groups 1 and 2, but they did not receive any antidote. Four days after the 2 nd injection of the toxin, the motor activity of all the animals was tested using a rotarod and then a vertical pole.
The mice were placed on a rod rotating at a constant speed of 6 rpm for a period of 600 sec; then the rotation speed was increased automatically by 1 rpm every 30 sec up to 20 rpm. The entire duration of the test was 990 sec. It was recorded how long each mouse can hold out on the rod without falling at a constant speed, and at what maximum speed of rotation. If the mouse was able to stay on the rotarod for 990 seconds, and do not fall at 20 rpm within 30 sec, then the test was considered 100% complete. Statistical analysis was performed using the nonparametric U test (Mann-Whitney).
The test for locomotor activity on a vertical pole was used in accordance with the previously described method [12,18,46,47].  After that, 20 μl of MTT preparation (diluted in saline solution 5 μg/ml) was added to each well for 3 hours. Then the solution was removed from the wells and 60 μl of dimethyl sulfoxide (DMSO) was added to each well. Shake thoroughly until the formazan crystals are completely dissolved. The quantitative determination of formazan was carried out on a brand multichannel photometer with a 530 nm filter. Cell viability was assessed by the ratio of optical density in the control wells without the tested extract and in the wells with the extract [51].

Patent
Zagorinsky Funding: This study was funded by a private investor.

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Ethics Committee of the Severtsov Institute of Ecology and Evolution of the Russian Academy of Sciences (protocol code 009, date of approval 26.10.2020).