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Mamajanov Mukhtorjon Murodullaevich, Namangan State University, Namangan E-mail: [email protected] Mirzakulov Sobit Oltinovich, Almatov Karim Tojibaevich, National University of Uzbekistan named after M. Ulugbek, Tashkent.
Botirov Erkin Khojiakbarovich, Head of Chemistry department, professor Surgut State University
CORRECTION OF PHYSICAL-BIOCHEMICAL PROCESSES IN THE ORGANISM BY FLAVOSAN
Abstract: Flavosan leads to a decrease in oxygen consumption, calcium transport, the formation of reactive oxygen species, the hydrolytic activity of phospholipase A2, but the indicators that determine the effectiveness of oxidative phosphorylation are almost unchanged. These changes reduce the consumption of oxygen in the body, do not cause drowsiness and do not lead to death. Hence, flavosan increasing the "bilayer" areas in membranes that increase stability translates the organism from the active metabolic state into a passive state.
Keywords: rat, mouse, flavosan, oxygen, heart, brain, liver, mitochondria, oxidative phosphoryla-tion, calcium, reactive oxygen species, phospholipase A2.
Introduction homeostasis, without the consumption of oxygen
Flavonoids are medicinal food substances that en- [20; 21; 22]. Each gram of food involved in the prosure the normal functioning of organs and tissues of the cess of cellular metabolism under aerobic conditions body and possess different clinical and pharmaceutical gives approximately 20 times more energy than in properties. Currently, flavonoids are widely used in the an anaerobic condition [23]. Because, the amount of prevention and treatment of widespread diseases of the energy released during the oxidation of nutrients is cardiovascular system, the mayor of cerebral circulation, several times higher than the energy released during neurodegenerative processes, cancer [1; 2; 3; 4; 5], hepatic the oxidation of pyruvic acid in widespread fermen-pathologies [6; 7; 8; 9; 10; 11] and many other diseases. tation or glycolysis.This ratio, i. e. "Oxygen/water
A number of works have been published on the (+ 0.82 V) and pyruvic acid/lactic acid (- 0.19 V)"
connection of the structure of flavonoids with their is determined by the difference between conjugation
antioxidant activity [12; 13; 14; 15; 16; 17; 18; 19], of oxidation-reduction potential [24]. If we take into
only in isolated works these relationships were quanti- account that the main substrates of respiration and tative. According to these studies, the antioxidant activ- fermentation potential is slightly greater than or equal
ity depended on the number and position of hydroxyl to - 0.7 V, the maximum difference in the oxidation-
groups in flavonoid molecules, the presence of two hy- reduction potential of respiration is 0.7 V + 0.82 V=
droxyl groups in the ortho position in the benzene ring. = 1.52, and for fermentation is 0.7 A - 0.19 C = 0.51 V.
Many living organisms cannot perform their vital Oxidative phosphorylation in the mitochondrial
functions - metabolic functions that require energy, system is considered one of the main factors determin-
generate energy-rich substrates, in other words, as- ing the energy state of adenine nucleotides in the cell.
similation and dissimilation reactions to maintain Mitochondria in addition to the production of ATP,
directly involved in the regulation of the concentration of free Ca2 + in the cell cytoplasm [25; 26; 27; 28; 29; 30].
Mitochondria take calcium at low speed and store, but in volume far exceeds the endo- and sarcoplasmic reticulum and directly participates in the regulation of free calcium in the cytoplasm. In a calm state of the cell, the mitochondria are also in a steady state and the calcium content around them is lower (10-7-10-8 M), because the proximity of free calcium is significantly lower than the calcium transport system of mitochondria (10-510-3 M). However, with an increase in the calcium content in the cytoplasm (especially in the areas between the mitochondria and the endoplasmic reticulum), a high content of free calcium makes it possible to accumulate calcium in the mitochondrial matrix (10-3-10-2M).
Under such conditions, an increase in the calcium content in the mitochondrial matrix affects the mito-chondrial states, in particular, stimulates an increase in ATP production [31]. However, the further accumulation of calcium ions in the mitochondrial matrix leads to the generation of oxygen radicals and an increase in the hydrolytic activity of endogenous phospholipases, the discovery of nonspecific pores in the mitochondrial membrane, the rupture of the outer membrane and, as a result, the loss of mitochondrial function [32; 33; 34].
Various cellular organelles contain many enzymes that catalyze the reduction of oxygen to H2O2 (substrate-oxygen-oxidoreductase). For example, back in the 70s of the last century it was known that 30% of the hydrogen peroxide formed in the liver appears due to mitochondria. It was found that in intact mitochondria (external, internal membranes and matrix), there are several enzymes that produce H2O2 and their activity is associated with tissues. For any tissue, the common and most active "enzymes" is the respiratory chain. The rate of production of H2O2 in intact mitochondria depends on their functional state and (V 3or V 4 states) on the nature of the oxidation substrates (NAD + - dependent substrates and succinate).
Submithochondrial particles (preparations from connected intramitochondrial membranes) have the ability to produce hydrogen peroxide with the participation of NADH or succinate. The immediate precursor of hydrogen peroxide, which is produced in the respiratory chain, is the superoxide radical (O2 • ) [35]. One of the products of lipid peroxidation is malonicdialdehyde.
The flavonoid preparation (luteolin, chrysoeriol, apigenin, cinaroside, formonononetin) isolated from thermopsis (Thermopsis alterniflora, legumes - Fabaceae) has very low toxicity [36]. At oral administration of flavo-san into the body of an animal at a dose of 5000 mg/kg, no adverse effect was observed [37]. Flavosan markedly increases the endurance of animals under hypoxic conditions [38]. The antihypoxic property of flavosan is explained by the careful consumption of oxygen in conditions of hypoxia [38].
Proceeding from this, it is important to consider the study of the effect of flavosan on oxygen consumption, mitochondrial respiration, oxidative phosphorylation, and calcium transport, formation of free radicals and hydrolytic activity of phospholipases.
Detection of the effect of flavosan on oxygen consumption and body temperature, oxidative phosphori-nation, accumulation of calcium and the formation of reactive oxygen species and the hydrolytic activity of phospholipase A2 was the goal of our study.
Materials and methods
In experimental studies, white male rats weighing an average of 180-200 g and white laboratory mice weighing 30-35 grams were used. The food and water of rats and mice were obtained without restriction. Experimental animals were divided into 6 groups: the first group was control, and the remaining groups orally received different doses of flavosan (the second - 100 mg/kg, the third - 200 mg/kg, the fourth - 300 mg/kg, the fifth -400 mg/kg and sixth - 500 mg/kg).
To determine the respiration of small animals, the oxygen content in the canister in which the animal was mixed was determined by the method of polarography [39]. A separate chamber was prepared according to the magnitude of each animal to determine the oxygen consumption by the animals. This equipment has an outgoing tube connected to the micropump. For this purpose, an improved micropump used in aquariums was used. The advantage of the equipment lies in the fact that in addition to measuring the content of oxygen absorbed through the pump, you can determine the time by the minute. For example, if an animal consumed 40 ml of oxygen per minute, multiplying this figure by 60 minutes, you can get that the consumed oxygen content is equal to 2400 ml. After this, the value obtained is divided by the mass of the animal. For example, if rats with a body
weight of200 g were used for the experiment, here from one kilogram it will be 5 times less. Correspondingly, the value obtained above, i. E. 2400 ml oxygen/hour is multiplied by 5 and as a result we get 12000 ml oxygen/kg. This value can be expressed in mole oxygen/hour. And this requires additional calculations. It is known that 1 mole of oxygen occupies 22.4 volumes. 1 liter of oxygen corresponds to 45 mM. Now the value obtained above, i. e. 12000 ml oxygen/kg per hour convert mM, we get 266 mM oxygen/kg hour. Determining the rate of oxygen consumption by the whole organism is exactly this.
Lipid peroxidation process in mitochondria was determined by micromethod developed by Yu. A. Vladi-mirov and A. I. Archakov.
All experiments were conducted in strict compliance with the principles of the Ethics Committee on animal experiments.
Mitochondria of their heart, brain and liver of rats were isolated using the differential centrifugation method of Hogeboom O. N. et al., [1948] with some modifications [40].
Lipid peroxidation process in mitochondria was determined by micromethod developed by Yu. A. Vladimirov and A. I. Archakov. 20 ^M FeS04 + 0.2 ascorbate was added to the mitochondria and the lipid peroxidation was measured after 20 min. Accumulation of malonic dialdehyde was determined at 532 nm. The measurement medium consisted of KCl - 115 mM, NaH2PO4-1 mM, Tris-HCl-5 mM (pH 7.4). The rate of the reaction of lipid peroxidation was expressed in nm malondialdehyde/mg min.
Hydrolysis of phosphatidylethanolamine under the influence of endogenous phospholipase A2 in mitochondria was determined by the formation of lyso-phosphatidylethanolamines in an incubation medium containing 0.25 M sucrose, 10 mM Tris-HCl, pH 9.5 for an hour at 37 °C [41]. The hydrolytic activity of phospholipase A2 was expressed in ^g/hr mg.
Calcium transport through the mitochondrial membranes is determined by the pH-metric method, based on the exchange of 2H/Ca 2+ in mitochondria. The medium used to accumulate calcium was 120 mM tris-HCl, 10 mM Tris-HCl, 5 mM succinate, pH 7.4, rotenone (1 mkg/ml) and 1 mM phosphate, pH 7.4.
To determine the effect of flavosanan respiration and oxidative phosphorylation of the mitochondria, 500 mg flavosan was introduced per 1 kg ofbody weight and after
20 minutes the mitochondria were isolated from the tissues of the heart, liver and brain, their changes in respiration and oxidative phosphorylation were revealed. The mitochondrial respiration and phosphorylation parameters were determined by the method of Chance B., Williams G. L. [42]. The following rates of respiration rate were determined: V2 - oxidation state of the oxidation substrate; V3 is the active phosphorylated state after the addition ofADP, V4 is the state after the consumption of ADP in the cell. Respiratory index according to Chance (V3/V4 ratio) and the ratio ofADP/O during oxidation of various substrates (10 mM succinate or glutamate). The rate of respiration in all metabolic states of the mitochondria was expressed in nanograms atom/min per mg of protein. The protein content in the mitochondria was determined by the method [43].
Flavosan was isolated from thermopsis (Thermop-sis alterniflora, legumes - Fabaceae) in the Institute of Chemistry of Plant Substances of the Academy of Sciences of the Republic of Uzbekistan.
Results and discussion
The change in oxygen consumption (as the value of the standard exchange) and body temperature after administration of a different concentration of flavosan in the body of rats and mice is shown in Table 1.
From the data obtained it was revealed that under the influence of flavosan the standard exchange and body temperature decreased and this process increased with increasing concentration of flavosan. It was found that a decrease in the standard exchange is slightly faster than a decrease in body temperature. If the standard exchange was measured after 60 min of administration of flavosan at concentrations of 100, 200, 300 and 500 mg/kg, it decreased by 22.2, 29.5, 38.5 and 44.4%, respectively. The same changes were observed in mice. At the same time, the standard exchange decreased by 22.8, 30.1, 41.2 and 47.3%. Hence, flavosan, depending on the reduction of heart rate, minute volume of blood and oxygen capacity of blood, significantly reduces the transport of oxygen into the body. But these changes do not affect the state of sleep and do not lead to the death of animals. It transfers from an active metabolic state to a passive metabolic state. From this it becomes evident that the influence of flavosan at the organism level reduces energy consumption, i. e. transfers oxygen to a system of economic consumption.
Table 1. - Influence of flavosan on oxygen consumption in animals (M ± m; n = 6-8)
Flavosan, mg/kg Rat Mouse
Oxygen consumption, мМ О2/min
0 1369.2 ± 136.8 100 3895.8 ± 608.9 100
100 1037.8 ± 129.5* 75.8 3007.5 ± 578.4 77.2
200 965.3 ± 118.2*** 70.5 2723.1 ± 565.6* 69.9
300 842.0 ± 109.9**** 61.5 2290.7 ± 440.8** 58.8
500 761.3 ± 102.3**** 55.6 2053.1 ± 378.5*** 52.7
Note: here and in other tables S. E.M.:*P < 0,05; **P < 0,02; "*P < 0,01; ""P < 0,001.
The stability of warm-blooded organisms to hypothermia and their recovery after prolonged cooling have long been the subject ofresearch [44; 45]. The central issue of hypothermia is the exchange of oxygen in chilled warm-blooded organisms. Until now, scientists have not come to a common opinion on the significance and order of the influence of various factors, leading to changes in the oxygen-oxygen metabolism of organisms in different stages of hypothermia. Until now, the question of the effect of reducing oxygen consumption in the system of its
transport in the body on the metabolic processes in the body remains open and whether it is the result of various changes in the system ofoxygen transfer in hyperthermia. In our opinion, respiration and oxidative phosphoryla-tion of mitochondria can play a major role in the transition ofwarm-blooded animals from the active metabolic state to the passive metabolic state.
To confirm this hypothesis, the following changes in respiration and oxidative phosphorylation of mitochondria isolated their heart, liver and brain tissues (Table 2).
Table 2. - Alteration of respiration and oxidative phosphorylation of mitochondria of various organs of rats under the influence of flavosan (M ± m; n = 6-8)
Organs and readings Respiration rate, Nano gram atom ofö^min/mg of protein
Glutamate Succinate
Control Flavosan Control Flavosan
Heart
V2 45.6 ± 4.0 29.0 ± 3.2** 214.1 ± 16.4 161.8 ± 7.5***
V3 161.8 ± 7.6 118.2 ± 5.4** 426.3 ± 23.6 321.0 ± 10.4***
V4 40.1 ± 4.7 27.9 ± 3.6** 203.6 ± 17.8 143.1 ± 8.6***
V 161.8 ± 9.5 121.8 ± 6.8** 477.3 ± 32.5 385.2 ± 13.8***
RCch 4.03 ± 0.23 4.23 ± 0.19 2.09 ± 0.09 2.24 ± 0.08*
ADP/O 2.76 ± 0.12 2.98 ± 0.14 1.75 ± 0.07 1.89 ± 0.07*
Brain
V2 13.54 ± 1.36 8.80 ± 1.22** 24.57 ± 2.56 18.55 ± 2.05**
V3 42.00 ± 3.23 29.64 ± 2.34**** 76.12 ± 4.17 47.21 ± 3.46**
V4 12.70 ± 1.49 8.57 ± 1.31** 23.31 ± 2.89 16.32 ± 2.12*
V 44.41 ± 3.86 31.62 ± 2.39**** 77.49 ± 4.46 65.94 ± 2.37*
RCch 3.31 ± 0.11 3.46 ± 0.12 3.26 ± 0.09 2.89 ± 0.09*
ADP/O 2.69 ± 0.08 2.84 ± 0.10 1.70 ± 0.07 1.85 ± 0.06*
Liver
V2 18.8 ± 2.2 12.7 ± 1.1*** 33.0 ± 3.1 25.5 ± 2.2**
V3 59.9 ± 5.7 43.6 ± 3.8**** 97.7 ± 7.5 79.1 ± 4.7***
V4 19.0 ± 2.5 13.0 ± 1.3*** 31.3 ± 3.3 24.5 ± 2.4**
V 61.0 ± 6.4 45.4 ± 4.2*** 160.0 ± 9.8 137.9 ± 6.0***
RCch 3.15 ± 0.10 3.35 ± 0.12 3.12 ± 0.12 3.23 ± 0.13
ADP/O 2.57 ± 0.09 2.87 ± 0.09 1.72 ± 0.08 1.83 ± 0.09
Flavosan slowed down the oxidation of glutamate in V2, V3, V4 and VDNP states compared to the control at 36.4; 27.0; 30.4 and 24.7%, but changes in the indices determining the efficacy of oxidative phosphorylation-the Chance respiratory rate and the ADP/O ratio-were almost not observed. Precisely the same changes were observed in the oxidation of suc-cinate in various metabolic states. Flavosan reduced the oxidation of succinate in the V2, V3, V4 and VDNP states as compared to the control at 24.4; 24.7; 29.7 and 19.4%, and the Chance coefficient of respiration and the ADP/O coefficient increased slightly.
When flavosan rats are introduced into the body in mitochondria of the liver, the oxidation of glutamate in the states V2, V3, V4 and VDNP is decreased as compared to the control by 22.1; 27.1; 31.6 and 25.5% and succinate by -22.7; 19.0; 21.6 and 13.8%. If the respiration rate for Chance and the ADP/O ratio increased slightly with glutamate, then the succinate did not change much. In the mitochondria of the brain, the oxidation of glutamate in the V2, V3, V4 and VDNP states decreased by 35.0 in comparison with the con-
Hence flavosan reduces the formation of active forms of oxygen in the mitochondria of various organs. This process is particularly noticeable in the mitochondria of the brain.
The plasma membrane, mitochondria and endoplasmic reticulum of eukaryotes contain a calcium transport system [47]. Typically, the plasma membrane contains three systems: Ca2 + channels, specific ATPase and Na+ - Ca 2+ exchange [48; 49]. If the calcium content in the cytoplasm increases markedly (especially in the region between the mitochondria and the endoplasmic reticulum), a high concentration of calcium ions in the mitochondrial matrix results in the accumulation of a certain amount of calcium (10-3-10-2 M). Under
trol; 29.4; 32.5 and 28.8%, and with succinate at 24.5; 18.0; 30.0 and 14.9%. The respiratory rate for Chance and the ADP/O ratio slightly increased.
As the effect of flavosan in the animal body results in an economical consumption of oxygen and a decrease in body temperature, a decrease in the respiration of the mitochondria without affecting the process of oxidative phosphorylation-the Chancerespiration coefficient and the ADP/O ratio and maintaining normal vital activity, i. e. translates the body from one metabolic state to another metabolic state? In our opinion, such changes are achieved by flavosan reduction in the formation of active forms of oxygen. Therefore, the purpose of the following experiments was to study the effect of flavosan on the formation of reactive oxygen species in mitochondria.
After preliminary administration of flavosan to the animals at doses of 100, 200, 300 and 500 mg/kg and mitochondrial release after 20 minutes from the heart, brain and liver, the content of malonaldehyde decreased by 9.3, respectively; 19.8; 28.4 and 44.3%; - 13.6; 34.0; 40.6 and 59.4%; 14.4; 25.1; 36.0 and 54.4% (3 - table).
these conditions, calcium accumulated in the mitochondrial matrix leads to increased respiration and ATP synthesis in mitochondria. But a further increase in the concentration of calcium ions in the mitochondrial matrix leads to an increase in the generation of oxygen radicals and the hydrolytic activity of phospholipases, the activation of nonspecific pores in the mitochondrial membrane, the rupture of the outer mitochondrial membrane, and the loss of the synthesizing function of ATP in the mitochondria [50; 51; 52; 53]. Therefore, our next goal was to elucidate the effect of flavosan on the amount of calcium accumulation and the hydrolytic activity of liver phospholipase A2 in liver mitochondria (Table 4).
Table 3. - Alteration of reactive oxygen species formation in mitochondria under the influence of flavosan (M ± m, n = 8-10)
Flavosan, mg/kg Rate of LPO reaction, nmol, malonicdialdeh yde/mg of protein
Heart % Brain % Liver %
0 0.420 ± 0.057 100 0.288 ± 0.034 100 0.300 ± 0.050 100
100 0.381 ± 0.048 90.7 0.220 ± 0.032* 76.4 0.257 ± 0.042* 85.6
200 0.337 ± 0.045* 80.2 0.192 ± 0.034* 66.0 0.223 ± 0.044** 74.9
300 0.301 ± 0.055** 71.6 0.171.0 ± 0.032*** 59.4 0.192 ± 0.038*** 64.0
500 0.234 ± 0.056**** 55.7 0.117 ± 0.027**** 40.6 0.137 ± 0.039**** 45.6
Table 4. - Alteration of calcium accumulation in liver mitochondria under the influence of flavosan (M ± m, n = 8-10)
Flavosan, mg/kg Volume of calcium accumulation in mitochondria, nmol/mg of protein PLA2 activity mkg/hour mg of protein
Control 87.9 ± 5.8 100 27.2 ± 3.4 100
100 77.8 ± 4.4* 88.5 24.6 ± 2.9 90.4
200 69.5 ± 4.3**** 79.0 22.4 ± 2.5 82.6
300 58.9 ± 4.0**** 67.0 20.0 ± 2.7** 73.7
500 39.7 ± 3.5**** 45.1 16.6 ± 2.9*** 60.9
After preliminary administration of flavosan to the animal organism at doses of 100, 200, 300 and 500 mg/kg and mitochondrial release after 20 minutes, a decrease in the calcium-accumulating volume of liver mitochondria was revealed in liver tissue compared to the control animals by 11.5; 20.9; 33.0 and 54.8%. Almost exactly the same changes were observed in the activity of phospho-lipase A2 mitochondria (decreased by 9.6, 17.4, 26.3 and 39.1%). Hence flavosan leads to a decrease in calcium transport and the activity of the hydrolytic activity of calcium-dependent endogenous phospholipases. As a result, "bilayer" areas increase in the membrane and "monolayer" areas decrease [54]. Phospholipases almost do not work in the «bilayer» areas. From this it follows that by reducing the influx of calcium into the mitochondria, the cell passes into a metabolically active state. At rest, the calcium content around the mitochondria is low (10-7 -- 10-8 M) and is kept in a calmly steady state, because the calcium content is lower than in the calcium of the mitochondrial transport system (10-5 - 10-3 M) [55; 56].
Conclusion
From the results obtained, it can be concluded that a decrease in the consumption of flavosan oxygen can be associated with a decrease in the formation of active forms of oxygen, calcium accumulation and phospholi-pase activity.
Breathing and oxidative phosphorylation of mitochondria of organs and tissues in conditions of invivo determines the oxygen-oxygen exchange of the organism. Heldt (1969) found that the rate of phosphorylation of exogenous ADP is directly proportional to the concentration of endogenous ATP. Hence, the speed of the mitochondria (the speed of the electron and proton
fluxes) is determined by the content of adenine nucleotides in the mitochondria - in the control animals above, and in animals that have received flavosan below. In our opinion, the influence of flavosan on the influx of calcium ions, the formation of free radicals and the activity of phospholipases of mitochondria leads to an increase in "bilayer" and a decrease in "monolayer" areas, increases the density and compactness of membranes, and as a result of reducing the transport of substrates to the active center of membrane-dependent enzymes their activity and transport of ions through special channels decreases. At the same time, the influence of endogenous and exogenous toxic substances decreases. From a functional point of view, the channels are more 103-105 compared to Na pump. Therefore, with an insignificant decrease in their density in the membrane, the need for the energy required to maintain the ion concentration gradient in the membrane is sharply reduced. Such processes take place in intracellular organelles. From this it follows that poikilothermic animal to adapt to unfavorable conditions should regulate the number of open channels and the associated metabolic rate. The goal of this strategy is to maintain at the minimum required level of energy, nutrients and various biologically active substances.
Hence flavosan reduces the transfer of calcium, the formation of free radicals and the hydrolytic activity of phospholipases, i. e. leads to an increase in "bilayer" areas in the membrane and an increase in the stability of the membrane. These changes reduce the respiration of mitochondria, the consumption of oxygen and substrates by the body, but they almost do not affect the indices determining the effectiveness of oxidative phosphoryla-tion - the Chance respiratory rate and the ADP/O ratio.
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