Tautomers of the Orotate Anion Have Anti-Inflammatory Effects in the Correction of Drug-Induced Hepatitis in Rats

The purpose of this paper was to conduct a comparative experimental study of the anti-inflammatory effects of tautomeric forms of orotic acid in the correction of drug-induced hepatitis in rats. Materials and Methods. A total of 40 rats (Rattus norvegicus Berk.) were randomly divided into 5 groups: control group (n = 10); intervention group with drug-induced hepatitis (n = 15); animals with drug-induced hepatitis who were injected with orotic acid (ОА) tautomers: initial oxo-form (n = 5), hydroxy-form (n = 5) and dihydroxyform (n = 5) at a dose of 0.5 g/kg body weight a day in the course of 14 days. The tautomers were obtained by mechanical activation in a planetary ball mill AGO-2C for 1 (hydroxy-form) and 6 (dihydroxyform) hours. In the blood of animals of all experimental groups, the content of leukocytes, granulocytes, lymphocytes, and monocytes was determined. Liver sections were stained with hematoxylin and eosin to assess the histo- and cytostructure of the tissues, and immunohistochemically using a set of monoclonal antibodies to detect the expression of the CD68+ macrophage marker. Results. It was found that when correcting drug-induced hepatitis with the hydroxy-form of OA, the severity of leuko- and monocytosis decreased and the number of lymphocytes was restored. The number of CD68+ macrophages with a pro-inflammatory phenotype decreased in the group receiving the hydroxy-form of OA (by a factor of 1.32; p = 0.019), but remained unchanged at the administration of oxo- and dihydroxy-forms of OA. The intensity of reaction product expression decreased by a factor of 1.5 in the group administered with the initial drug and by a factor of 1.9 in animals administered with mechanically activated drugs (p = 0.0001). Thus, the obtained data indicate a pronounced anti-inflammatory activity of the hydroxyform of OA, which can substantiate its use as a hepatoprotective agent. For citation: Pazinenko K.A., Chuchkova N.N., Smetanina M.V. Tautomers of the Orotate Anion Have Anti-Inflammatory Effects in the Correction of Drug-Induced Hepatitis in Rats. Journal of Medical and Biological Research, 2021, vol. 9, no. 4, pp. 366–373. DOI: 10.37482/2687-1491-Z074

The Improved Method for Determination of Orotic Acid in Milk by Ultra-Fast Liquid Chromatography with Optimized Photodiode Array Detection

Ultra-fast liquid chromatography (UFLC) with a photodiode array detector (DAD) for simple and rapid determination of orotic acid (OAc) in milk of sheep and cows is described. Milk samples are treated with acetonitrile (1:1, v/v) and then centrifuged at 4 °C. To 1 mL of the obtained supernatant 9 mL of ultrapure water was added. Subsequently, 0.5–6 µL of the resulting solution was injected into the UFLC-DAD system. Separation and quantification of OAc in milk samples was achieved using two Kinetex C18 columns (1.7 µm, 150 mm × 2.1 mm, i.d., 100 Å; Phenomenex) fitted with a pre-column of 4 mm × 2 mm, i.d. (Phenomenex) containing C18 packing material. All separations were performed at a column temperature of 35 °C while the ambient temperature was 21–24 °C. Satisfactory separation of OAc from endogenous species of milk can be achieved using the binary gradient elution program and UV detection at wavelengths 278 nm. Our original procedure resulted in suitable separation and quantification of OAc in milk samples; OAc eluted at 6.44 ± 0.03 min. The total run time of OAc analysis (including re-equilibration) was 27 min. As expected, the OAc peak was absent from the blank when the proposed gradient elution program and UV detection at 278 nm was used. The average recoveries of OAc standards added to milk samples were satisfactory (96.7–105.3%). The low inter-and intra-assay coefficient of variation derived from the measurements of OAc in cow and ovine milk samples (i.e., 0.784%, 1.283% and 0.710%, 1.221%, respectively) and in O-Ac standards (i.e., 0.377% and 0.294%, respectively), as well as high recoveries of OAc added to ovine and cows’ milk (~100%) and the low detection (0.04 ng) and quantification (0.12 ng) limits point to satisfactory accuracy, precision and sensitivity of the reported method. OAc concentrations in ovine milk samples were within the range from 25 to 36 mg/L, while OAc levels in cows’ milk samples was found in the range of 32–36 mg/L. Our original procedure is suitable for routine quantification of OAc in milk of ewes and cows.

SORPTION-LUMINESCENT DETERMINATION OF OROTIC ACID IN MEDICINAL DRUGS

Luminescent properties of orotic acid (vitamin B13), known bioactive substance, have been evaluated in this research. It has been shown that inside a coordination complex of orotic acid with terbium (III) an intermollecular excitation energy transfer from a ligand to a lanthanide ion occurs which causes sensitization of terbium (III) ion luminescence. Intensity of the terbium (III) ion luminescence (ILum) increases in the presence of anionic and cationic surfaceactive agents as a result of displacement of water molecules from coordination sphere of the complex. As a consequence of this process, a non-radiative deactivation of excitation energy decreases and more efficient excitation energy transfer from a ligand to a lanthanide ion occurs. It has been established that after absorption of analyte of interest onto a polymer matrix of a solid phase extraction sorbent with subsequent increasing of its molecular rigidity more efficient excitation energy transfer is observed followed by elevation of luminescence (ILum) intensity of the coordination complex being studied. Intensity of luminescence of coordination complex of orotic acid with terbium (III) in the form of a sorbate salt has been estimated involving different types of sorbents (Phosphalugel, SFG-75, Calflo E, Celite 545, Silicagel Merck). Most suitable results have been achieved by the use of Silica gel Merck as a solid-phase matrix (sorbent). The removal rate of the complex from the solution in this case reaches to 97%. The most effective conditions for determination of orotic acid have been specified. The highest values of luminescence intensity is obtained by conducting of sorption using 5 mL of solution and 100 mg of sorbent. Luminescence intensity of the complex in the form of a sorbate salt, during a sorption process, increases in presence of ethonium, cationic dimeric surface-active agent. Decrease in luminescence intensity of the complexes in the form of a sorbate salt in presence of other surface-active agents may be caused by solubilization of the complex in the solution and thereby reduced level of its sorption. On the basis of presented research work a new analytical procedure has been developed for the solid-phase luminescence determination of orotic acid and orotic acid salts in pharmaceutical drugs. Determination of orotic acid salts has been performed in pharmaceutical drugs ‘Magnerot’ (magnesium orotate) and ‘Potassium orotate’. The proposed analytical procedure for determination of orotic acid salts is remarkable by good metrological characteristics and relative simplicity of performance. The relative standard deviation, Sr, is 3,6 - 4,3%.

Microcirculatory Liver Bloodstream in Hyperhomocysteinemia and Administration of Tautomeric Forms of Orotic Acid

The aim of the study was to analyze the microvasculation of the liver in administration of tautomeric forms of orotic acid for a methionine-induced liver pathology to assess the efficacy of the modified drugs and the potentialof their clinical use.Material and methods. The study included 30 white outbred rats, that were simulated methionine induced hyperhomocysteinemia by the administration of methionine, dosage 0.15 g/100 g of the body we ight, via food during 4 weeks. Animals with hyperhomocysteinemia were divided into 4 groups: animals of group 1 received orotic acid (OA) in the initial form (oxo-tautomer), animals of group 2 received hydroxy–tautomer OA after mechanomodification for 1 hour, animals of group 3 received dihydroxy-tautomer OA after mechanomodification for 6 hours. Histological preparations of the liver were used to evaluate the area of hepatocytes; diameters and areas of the central vein, the interlobular vein and artery, the bile duct, and the Visotto coefficient were calculated.Results. Morphometric data of the hepatic microvascular bloodstream with simulated hyperhomocysteinemia evidenced a general increase in the diameter and area of blood vessels, changes affected the system of blood inflow and outflow at the organ level. Administration of OA had a normalizing effect on the liver bloodflow, but the effects were different: the most pronounced effect was detected in administration of the hydroxy-form of the preparation, this can be explained by a modificationin the dispersion of the preparation (without changing the crystal lattice), an increase in the rate of dissolution in water and aqueous solutions, an increasein the number of functionally active groups in the heterocycle of the hydroxy-form of OA. Thus, the hydroxy-tautomer of orotic acid had the greatest efficacy relating to vasodilation of microvessels of the liver bloodstream in hyperhomocysteinemia; the fact suggesting feasibility of its further study in the clinical environment.

Importance of Bioactive Substances in Sheep’s Milk in Human Health

Sheep’s milk is an important source of bioactive substances that have health-promoting functions for the body. The valuable composition of sheep’s milk is due to the high content of fatty acids, immunoglobulins, proteins, hormones, vitamins and minerals. Many biopeptides found in milk have antibacterial, antiviral and anti-inflammatory properties. The bioactive substances of sheep’s milk also show anticancer properties. Sheep’s milk, thanks to its content of CLA and orotic acid, prevents the occurrence of type 2 diabetes, Alzheimer’s disease and cancer. Sheep’s milk, as a product rich in bioactive substances, can be used as a medical aid to support the body in the fight against neurological and cancer diseases.