Esters with pronounced antimicrobial and antifungal activity were synthesized via acylation by benzoyl chloride of the natural hydroxyl-containing compounds 1-octanol, thymol, and 4-bromothymol (thymol benzoate and 4-bromothymol benzoate for the first time). The antimicrobial activity of the synthesized compounds was determined experimentally on three strains of bacteria (Bacillus subtilis 10986 and 4419 and B. licheniformis 537) and the yeast fungus Yarrowia lipolytica W29. Chloramphenicol was chosen as the reference drug for testing the antibacterial activity of the synthesized compounds. Clotrimazole was used as the reference drug in mycostatic tests. Antimicrobial and antifungal tests of the synthesized compounds showed positive results, which opened up possibilities for further research of these compounds as antifungal and antibacterial drugs devoid of the toxicity inherent in benzoic acid.

Ацилированием хлорангидридом бензойной кислоты природных гидроксилсодержащих соединений 1-октанола и тимола, а также 4-бромтимола получены сложные эфиры (тимолбензоат и 4-бромтимолбензоат впервые), обладающие выраженной антимикробной и противогрибковой активностью. Экспериментально противомикробная активность синтезированных соединений определялась на 3 штаммах бактерий сенной палочки (Bacillus subtilis 10986, 4419 и Bacillus licheniformis 537) и дрожжевых грибах Yarrowia lipolytica W29. При испытании антибактериальной активности синтезированных соединений в качестве препарата сравнения был выбран хлорамфеникол. При микостатических испытаниях в качестве препарата сравнения применялся клотримазол. Антимикробные и противогрибковые испытания синтезированных соединений показали положительный результат, что открывает возможные перспективы дальнейшего исследования этих соединений в качестве противогрибковых и антибактериальных препаратов, лишенных токсичности, присущей бензойной кислоте.

An analysis is given of the dynamics of the activity of the phytase strain Yarrowia lipolytica that was transformed with the phytase genome from Obesumbacterium proteus based on the pUVLT2 vector with the VDAC (Voltage Dependent Anion Channel) mitochondrial porin promoter and induced under stress conditions when growing on low-value plant substrates. In the case of cultivation both in flasks and in a fermenter, an increase in the activity of intracellular phytase was observed when sunflower cake was used as a source of phytates. During the growth of the Y. lipolytica Po1f transformant under alkaline conditions, the VDAC promoter was activated as part of the pUV3-Op genetic construct, which was accompanied by an increase in the activity of intracellular phytase. The enzyme retained a high level of activity in Y. lipolytica pUV3-Op transformant cells after high-temperature treatment of the biomass in a spray dryer, which makes its use in the composition of feed additives for farm animals more effective.

The study presents the results of optimizing the medium components for the cultivation of the recombinant yeast of Yarrowia lipolytica Po1f pUV3-Op to increase the biomass concentration and its phytase activity. Glycerol, yeast extract, and the broth from low-value raw waste of the poultry farms were used as the substrates in the nutrient medium. During the cultivation on the developed nutrient medium in a 10-litre laboratory fermenter under the pilot conditions the optimal fermentation parameters were tested. The yield biomass under the optimized conditions reached 292 g of dried preparation with a phytase activity of 100.1 ± 1.07 mmol P i × min –1 × g –1 of dry biomass.

The study of the redox and mitochondria state, as well as some other hallmarks of cell aging of extremophilic yeast Yarrowia lipolytica W29, was performed during long-term cultivation for 120 hours. An increase in the ROS level upon the transition to the stationary growth phase was accompanied by a decrease in the cell respiratory rates, an increase in the alternative oxidase share. Moreover, the activities of superoxide dismutase and catalase increased. During stationary growth phase, the ratio of glutathione/glutathione disulfide increased two to three times. Cytometric studies of the populations in vivo stained with a potentiometric mitotracker showed a decrease in mitochondrial potential as the culture gets old. At the late stationary growth stage, some lysosomal processes in the Y. lipolytica cells were induced that apparently increased the resistance to oxidative stress and allowed the culture keep the survival rate at the level of 80%.

Using the extremophilic yeast of Yarrowia lipolytica, a new model has been proposed to study the protective properties of stilbene polyphenols, namely resveratrol and pinosylvin, under heat shock. It was shown that a short-term thermal exposure of yeast cells (55 C, 25 min) led to a 40% decrease in the colony-forming ability of the population, a fivefold decrease in the respiration rate, and a growth of cyanide resistance and catalase activity, which indicated the adaptive yeast response to heat stress. Under these conditions, natural biologically active stilbenes, resveratrol and pinosylvin, at a concentration of 10 μM each increased yeast survival by 28% and 13%, respectively. In heat shock, resveratrol additionally raised catalase activity, while pinosylvin increased the cell respiration rate and decreased cyanide resistance and catalase activity. The results obtained indicate that resveratrol acts as a mild pro-oxidant inducing antioxidant protection during the adaptive response of the yeast to heat shock. Unlike resveratrol, pinosylvin increases cell survival stabilizing mitochondrial function and preserving the ATP-generating component of respiration. Yarrowia lipolytica yeast, polyphenols, stilbenoids, resveratrol, pinosylvin, cellular respiratory activity, heat shock, superoxide dismutase, catalase

Effects of natural polyphenols resveratrol, dihydromyricetin, pinosylvin, epigallocatechin, myricetin, and dihydroquercetin on the peroxide generation in rat liver mitochondria in the conditions of toxic damage induced by hepatotrophic poison thiacetoamide were studied. In liver mitochondria of rats with toxic hepatitis all polyphenols at a concentration of 10 mu M served as antioxidants and lowered the signal of the fluorescent probe Amplex Red with the efficiency decreasing in the following order: resveratrol > myricetin > dihydroquercetin > pinosylvin > dihydromyricetin. In liver mitochondria isolated from the control animals the polyphenols studied affected the generation of free radicals insignificantly. In the conditions of the nonspecific Ca2+-dependent permeabilization of mitochondrial membrane in the presence of Ca2+ ions and inorganic phosphate, the polyphenols exhibited an antioxidant activity and notably decreased the peroxide generation rate in the mitochondria of the control animals. Mitochondria isolated from the liver of the animals with induced pathology showed more than a 20-fold increase in the peroxide level, which significantly decreased in the presence of polyphenols; most effective among the polyphenols were dihydroquercetin, pinosylvin, and dihydromyricetin, which decreased the level of peroxide production 7-13-fold. The presented data bring the clarity to the ambivalent effect of the polyphenols in living model systems and suggest that under the conditions of cell pathology natural polyphenols should produce a powerful antioxidant effect, while under physiological conditions the polyphenols can exert a neutral or mild pro-oxidant effect. The obtained data may be applied to the development of new potential pharmacological natural preparations to treat common diseases based on mitochondrial pathologies.

The effect of stilbene polyphenols (resveratrol and pinosilvin) and flavonoids (dihydromyricetin, epigallocatechin, and dihydroquercetin) on producing the reactive oxygen species (ROS) due to cell respiration using the isolated mitochondria of the fungus Dipodascus (Endomyces) magnusii was studied. It was shown that the inhibition of the ROS generation with stilbenes is related to both the oxygen consumption inhibition and their antioxidant properties. The degree of manifestation of polyphenol antioxidant properties depended on the number of hydroxyl groups in a polyphenol molecule. Of the flavonoids tested, dihydromyricetin was most active in reducing the ROS generation, but its inhibitory effect on oxygen consumption by mitochondria was significantly lower as compared to that of the stilbenes. The data obtained show that the mechanism of reducing the ROS generation with polyphenols is associated with both the respiratory inhibition and their antioxidant properties.

The characteristics of recombinant phytase from Obesumbacterium proteus have been studied after its integration into Yarrowia lipolytica with a Po1f (pUV3-Op) plasmid. Phytase activity was observed in Y. lipolytica Po1f transformants in a wide pH range (3.5–7.5) and remained at 80% of the initial value after 3-min heating at 95°C, which made it possible to add the enzyme to a fodder produced with heat treatment. In the case of transformant cultivation on low-grade vegetable substrates, they also possessed confirmed phytase activity, as well as an increase in the content of intracellular phosphates as compared to cells of the wild-type Y. lipolytica strain.

First, the effects of the biologically active polyphenols resveratrol and dihydroquercetin (DHQ) on morpho-physiological parameters and the antioxidant status of polyextremophilic yeast Yarrowia lipolytica at optimal and elevated temperatures were studied. Cultivation in the presence of 30 μM resveratrol at the optimal temperature caused a twofold increase in superoxide dismutase activity. It was shown that cultivation at a high temperature and high DHQ concentration stimulated a decrease in the alternate respiration level. High resveratrol concentrations and low dihydroquercetin concentrations were shown to have an inhibitory effect on the survival rate of Y. lipolytica yeast cells, while no visible inhibition effect of yeast growth was recorded at the optimal growth temperature. Polyphenols were shown to have a moderate stimulatory effect on the oxygen absorption rate by yeast cells.

Non-alcoholic fatty liver disease is associated with a number of disorders (diabetes, obesity, cardiovascular diseases), and can also be induced by drugs or toxic compounds. Recently the important branch of medicine is the search for effective means of prevention and treatment of fatty hepatosis. Our work was aimed to study the effect of some biologically active natural polyphenols (resveratrol and pinosylvin stilbenes as well as dihydromyricetin dihydroflavonol) on the function and histologic features of the liver. In the experimental model of thioacetamide-induced toxic hepaptitis, the male rats of the Wistar line daily received the effective doses of polyphenols intragastically by gavage together with 0.05% thioacetamide added to drinking water. All studied polyphenols contributed to stabilization of rat weight and a two-fold significant (p < 0.05) decrease in the level of direct bilirubin in the blood serum of animals treated with thioacetamide. Histological analysis of the liver confirmed a decrease in inflammation and hemorrhage in animals treated with polyphenols amid continued administration of thioacetamide for 30 days. Based on the data obtained, it can be concluded that the natural polyphenols which belong to the classes of dihydroflavonols (dihydromyricetin) and stilbenes (resveratrol and pinosylvin) have a positive effect on liver function in the experimental model of toxic hepatosis. The studied polyphenols can be considered as potential hepatoprotective drugs used as a part of the liver diseases complex therapy.

Неалкогольная жировая дисфункция печени сопровождает целый ряд заболеваний (диабет, ожирение, кардиоваскулярные болезни), а также может развиваться под действием лекарственных препаратов или токсических соединений. Актуальным направлением современной медицины является поиск эффективных средств профилактики и лечения жирового гепатоза. Целью работы было исследовать действие некоторых биологически активных природных полифенолов — стильбенов ресвератрола и пиносильвина, а также дигидрофлавонола дигидромирицетина — на функциональное состояние и гистологическую картину печени. В модели токсического гепатита, вызываемого введением гепатотоксиканта тиоацетамида, самцам крыс линии Wistar внутрижелудочно ежедневно вводили зондом эффективные дозы полифенолов на фоне добавления тиоацетамида к питьевой воде в концентрации 0,05%. Все исследованные полифенолы способствовали стабилизации веса крыс и двухкратному достоверному (p < 0,05) снижению уровня прямого билирубина в сыворотке крови животных, получавших тиоацетамид. Гистологическое исследование печени подтвердило замедление воспалительных процессов и уменьшение кровоизлияний у животных, получавших полифенолы на фоне продолжающегося введения тиоацетамида в течение 30 дней. На основании полученных данных можно сделать выводы, что природные полифенолы класса дигидрофлавонолов (дигидромирицетин) и стильбенов (ресвератрол и пиносильвин) оказывают положительное воздействие на функции печени в модели экспериментального токсического гепатоза; исследованные полифенолы можно рассматривать в качестве потенциальных гепатопротекторов в составе комплексной терапии при заболеваниях печени.

This review examines the main features of natural phytoalexines of flavonoid and stilbenoid natures, which are secondary metabolism products in numerous plants widely used as biologically active substances in the medicine, pharmacology, and agricultural plants protection. We considered the role of flavonoids and stilbenes in phytoimmune and antistress responses in plants, bactericide antifungul, and antiviral effects towards microorganisms, and the wide medical application for a number of mammalian pathologies. The main achievements in the metabolic engineering of flavonoids in microbial biotechnologies are discussed.