{"id":54542,"date":"2023-12-31T10:38:00","date_gmt":"2023-12-31T10:38:00","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=54542"},"modified":"2024-01-05T06:54:51","modified_gmt":"2024-01-05T06:54:51","slug":"effect-of-talatisamine-and-its-derivate-14-o-benzoyltalatisamine-on-functional-state-of-rat-liver-and-heart-mitochondria","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol16no4\/effect-of-talatisamine-and-its-derivate-14-o-benzoyltalatisamine-on-functional-state-of-rat-liver-and-heart-mitochondria\/","title":{"rendered":"Effect of Talatisamine and its Derivate 14-O-Benzoyltalatisamine on Functional State of Rat Liver and Heart Mitochondria"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Leading scientific centers, universities and\nlaboratories recognize that the dysfunction of mitochondria of various tissues\ncauses the development of many pathological processes, including ischemia1-5<\/sup>. Much attention is being paid to the use of local\nplant biologically active substances, including alkaloids, in the prevention\nand treatment of pathological processes related to mitochondrial dysfunction.\nThis is very relevant in relation to ischemic diseases and is of scientific and\npractical importance in the search for new pharmacological agents that correct\nthe functions of damaged mitochondria for their treatment. This is very relevant in relation to ischemic\ndiseases and is of scientific and practical importance in the search for new\npharmacological agents that correct the functions of damaged mitochondria for\ntheir treatment. Despite the achieved results, there are insufficient\nstudies aimed at determining the effects of diterpene alkaloids on cells and\nmitochondria, repairing membrane and molecular damage.<\/p>\n\n\n\n

In our country, in order to create effective drugs for\nthe prevention and treatment of liver, cardiovascular, neurodegenerative and\nother diseases based on plant compounds, scientific research is being conducted\nto determine their mechanisms of action6-11<\/sup>.<\/p>\n\n\n\n

Mitochondria are important\nintracellular structures that not only  produce energy necessary for\nthe cells to function, but also play a role in various pathological\nprocesses. In the double-membrane system of mitochondria of living organisms, there are mechanisms that ensure the transport of\nmetabolites, various ion channels, and transporters, critically involved in the\ndevelopment of pathological processes. Functional structures located in mitochondria and\nmembranes are the main targets for various pathological effects1-4<\/sup>. Mitochondrial functions that can be corrected by drugs\nare implemented by structures that form PTP, the activity of which is regulated\nby Ca2+<\/sup> ions, cyclophilin D, membrane potential, etc.\nPharmacons-modulators of the PTP state of mitochondria are known: cyclosporin\nA, ubiquinone, boncrecat, buterol, sodium hydrosulfide, progesterone, doxorubicin,\n1-O-benzoylnapelline, songorine etc5<\/sup>,9,11<\/sup>. The Ca2+<\/sup>-dependent transition of PTP to\nthe open state increases the permeability of the inner membrane of mitochondria,\nresulting in a decrease in the transmembrane potential (\u0394\u03c8m), swelling of the\norganelles, followed by tear\nand burst of the outer\nmembrane and the release of proapoptotic factors12<\/sup>.\nOne of\nthe mechanisms that leads to mitochondrial disorders is the critical increase\nin lipid peroxidation, which can disturb the integrity of cell membranes,\nincrease non-selective membrane permeability, uncouple oxidative\nphosphorylation and ATP hydrolysis. Diterpene alkaloids is a new class of compounds with fascinating\npharmacological properties. In\nparticular, talatisamine and its derivatives have antiarrhythmic, hypotensive,\nN-cholinoblocking, curare-like, spasmolytic,\nanti-inflammatory, anesthetic, vasoprotective, anticancer and other effects7,10,13<\/sup>.<\/p>\n\n\n\n

Since the mechanism of action of talatisamine and its\nderivative 14-O-benzoyltalatisamine on mitochondria has not been studied so far,\nin this study we have tested for the effect of these diterpene alkaloids on the\nLPO and mPTP state of rat liver and heart mitochondria.<\/p>\n\n\n\n

Materials and Methods<\/strong><\/p>\n\n\n\n

Animals<\/strong><\/p>\n\n\n\n

White outbred male rats, weighing 180-220 g, were housed under standard\nlaboratory conditions (20\u00b0C\u201324\u00b0C, natural light cycle and 65% humidity.  Food and water were available ad\nlibitum<\/em>).<\/p>\n\n\n\n

Isolation of intact mitochondria<\/strong><\/p>\n\n\n\n

Differential centrifugation was used to isolate intact\nrat liver mitochondria, as described previously14<\/sup>. Briefly, rat livers were homogenized in a following\nsolution (in mM): sucrose – 250, Tris-HCl – 10, EDTA – 1, pH 7.4, and centrifuged at 1500\u00b4<\/strong>g for seven min (0-2\u00b0C). Sedimentation of intact mitochondria\nwas achieved by supernatant centrifugation (6000\u00b4<\/strong>g) for 15 min (0-2\u00b0C). The supernatant\nwas discarded, and the resulting pellet was re-suspended in a small volume (10 mitochondria:1 solution) of medium\ncontaining: sucrose \u2013 250\nmM, Tris-HCl \u2013 10\nmM and placed on ice.<\/p>\n\n\n\n

Rat heart mitochondria were also isolated\nusing differential centrifugation. Rat hearts were homogenized in a solution\ncontaining (in mM): sucrose – 300, Tris-HCl – 10, EDTA – 2, albumin 0.2%, pH 7.4. To isolate a sufficient amount of\nmitochondria, 5 or 6 rat hearts were isolated and placed in a chilled isolation\nmedium. Laboratory\nequipment and petri dishes were stored in a freezer, and the procedure was\ncarried out on ice. Rat hearts were cleaned of adipose tissue, blood and other\nlarge blood vessels in a chilled 0.9% KCl solution.\nThe cleaned heart was washed\nagain, dried using filter paper, the heart mass was determined and cut into\nsmall pieces using scissors. After that, the minced heart tissue was placed in\na homogenizer and homogenized using a teflon pestle, poured with a 10:1 ratio\nof separation medium. Because the heart tissue is composed\nof transverse muscles, it was more homogenized than the liver tissue. The\nhomogenate was centrifuged at 1500 g for 7 min (0-2\u00b0C). At this stage, heavy aggregates\nsettle. In\nthe next step, centrifugation was performed at 6000 g for 20 min (0-2\u00b0C). Isolated mitochondria were washed in\nmedium without EDTA and albumin. Mitochondria isolation procedures were carried\nout under cold conditions. Mitochondria were kept on ice during the\nexperiments. The Lowry method by Peterson15<\/sup> was used to determine\nmitochondrial protein content.<\/p>\n\n\n\n

A method for determining the state of the mitochondrial permeable pore<\/strong><\/p>\n\n\n\n

Mitochondrial folding kinetics were investigated by\nstudying the state of the Ca2+<\/sup>-dependent permeable pore of the\nmitochondrial membrane. The optical density of swollen mitochondria was\nmeasured at 540 nm. The temperature of the incubation medium was 26\u00b0C, and the protein\ncontent was 0.3-0.4 mg\/ml. To determine PTP permeability in mitochondria, the following incubation solution was used (mM): sucrose –\n200, EGTA – 0.02, succinate – 5, Tris – 20, HEPES – 20, KH2<\/sub>PO4 – 1,\nrotenone – 0.002, oligomycin – 1 \u03bcg\/ml, pH 7.4 12<\/sup>.<\/strong><\/p>\n\n\n\n

LPO measurement<\/strong><\/p>\n\n\n\n

The LPO process induced by Fe2+<\/sup>\/ascorbate\nwas recorded by photometric inhibition of liver mitochondrial swelling in the\nsolution containing (mM): KCl – 125, Tris-HCl – 10, protein content 0.4 mg\/ml,\npH 7.4. <\/p>\n\n\n\n

10 \u03bcM FeSO4 <\/sub>and 200 \u03bcM ascorbate were\nadded to induce mitochondrial swelling. All experiments were carried out at the\ntemperature of 24\u201326\u00b0C to preserve the integrity of mitochondria during\nincubation. The\nantioxidant properties of the tested drugs were measured by inhibition of Fe2+<\/sup>\/ascorbate-induced mitochondrial swelling at 540 nm. This method was chosen because of the\nreported linear correlation\nbetween the LPO induced by the Fe2+<\/sup>\/ascorbic acid and swelling of\nmitochondria 16<\/sup>.<\/p>\n\n\n\n

The intensity of LPO was assessed by measuring the concentration of\nmalondialdehyde (MDA). LPO was\ninduced by adding 10 \u03bcM\nFeSO4<\/sub> and 200 \u03bcM\nascorbate to the incubation\nsolution containing (in mM): 125 KCl, 10  Tris-HCl, with \npH  adjusted to 7.4. LPO products were separated with thiobarbituric acid (TBA), and the reaction was stopped by 0.220 ml\nof 70% (w\/v) trichloroacetic acid (TCA). Next, the suspension containing mitochondria was centrifuged at 15000 g for 15 min. Then, 2 ml of supernatant was taken and poured in 1 ml of 75% TB\u0410 (w\/v). 2 ml of H2<\/sub>O\nand 1 ml of TB\u0410 were added to the control solution. The mixture was incubated\nin a water bath at\n300<\/sup>C for\n30 min and optical density was detected again at 540 nm. The final\nconcentration of MDA was calculated using the molar extinction\ncoefficient (e=1.56\u00d7105<\/sup> M-1<\/sup> cm-1<\/sup>) according\nto the following formula17<\/sup>: nmol\nMDA\/mg protein = D\/ 1.56\u00d730. 1 ml of incubation medium contained\n0.3\u20130.4 mg of mitochondrial\nprotein. <\/p>\n\n\n\n

Drugs and Reagents<\/strong><\/p>\n\n\n\n

EDTA was from Sandoz, Switzerland,\nTris-HCl was from Serva, Germany; KH2<\/sub>PO4<\/sub>, MgSO4<\/sub>, K2<\/sub>HPO4<\/sub>, succinate,\nsucrose, FeSO4, <\/sub>ascorbic acid, KCl, TBA and trichloroacetic acid were from Chemreaktivsnab, Russia; EGTA, HEPES, diazoxide, olygomycin<\/em>,\nrotenone, ATP, CaCl2<\/sub>\nwere from Sigma, USA; and cyclosporine A was from Wako, China. <\/p>\n\n\n\n

Diterpene alkaloid talatisamine (C24<\/sub>H39<\/sub>NO5<\/sub>) was isolated from Aconitum talassicum<\/em>18<\/sup>.<\/em> The structural formulas of talatisamine and its derivatives 14-O-benzoyltalatisamine (C31<\/sub>H43<\/sub>NO6<\/sub>) were drawn using the ChemOffice 2002 and Chem Draw Ultra 7.0 software  (Figure. 1).<\/p>\n\n\n\n\n\n
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Figure 1: Structural formulas of diterpene alkaloids<\/strong><\/p>\n

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Statistical Analysis<\/strong><\/p>\n\n\n\n

Statistical significance\nbetween groups was calculated based on the Student’s t-test using OriginPro 8.6\nsoftware (OriginLab, Northampton, Massachusetts, USA). P<0.05 was considered\nstatistically significant. <\/p>\n\n\n\n

Results and Discussion<\/strong><\/p>\n\n\n\n

In a number of pathologies, it has been found that the\nmPTP transitions to an open state, and the long duration of this process leads\nto a violation of cell function and structure. This, in turn, requires the\ncorrection of these pathological conditions with biologically active drugs.\nTherefore, it is necessary to search for local bioactive substances that\ninhibit mPTP and at the same time activate mechanisms that protect the cell\nfrom negative factors, namely ischemia, hypoxia, “overload” of Ca2+<\/sup>\nions, oxidative stress, etc. In order to study the membrane activity properties of\nthe studied alkaloids, the effects of talatisamine and 14-O-benzoyltalatisamine\nditerpene alkaloids in different concentrations on the PTP state of the rat\nliver and heart mitochondria were studied in vitro. A concentration of 10 \u00b5M of\nCa2+<\/sup> ions was used as an inducer of mitochondrial swelling. Due to the addition of Ca2+<\/sup> to the incubation\nmedium, mitochondrial swelling\nand the transition of mPTP to a high-conductance state were observed. This\ncondition was taken as 100% as a control (Figure 2A). In subsequent\nexperiments, the effects of talatisamine and its derivative\n14-O-benzoyltalatisamine alkaloids on the mPTP of rat liver and heart were studied. It was found that\na 10 \u03bcM concentration of talatizamine alkaloid\ninhibited rat liver mPTP by 24.5\u00b11.7% and rat heart mPTP by 18\u00b12.1%,\nrespectively, compared to control. When the concentration of talatisamine\nalkaloid was increased to 50, 100 and 200 \u03bcM incubation medium, the mPTP of rat liver and heart were 46\u00b12,1%, 52.8\u00b12,3%, 56.8\u00b11,9%, and 31\u00b13,0%,\n39\u00b11,7%, 44\u00b11,5% were reliably inhibited. The half-maximum inhibitory\nconcentration (IC50<\/sub>) of talatisamine was 78\u00b13.6\n\u03bcM in liver mitochondria (Figure 2A). <\/p>\n\n\n\n

In order to predict which component of the PTP of the rat liver and heart mitochondria of the studied diterpene alkaloids affect, it was compared with the half-maximal inhibitory concentration of CsA (0.2 \u03bcM) in the experiments. During the studies, the effect of talatisamine on the Ca2+<\/sup>-dependent swelling of the rat liver and heart mitochondria in the presence of CsA in the incubation medium was investigated (Figure 2B). Similar to the above experiments, in this study, mitochondrial inhibition by 10 \u03bcM Ca2+<\/sup> was taken as a control (100.0\u00b13.4%). A concentration of 10 \u00b5M of this alkaloid significantly inhibited the mPTP of the rat liver and heart by 86.5\u00b11.6% and 64.5\u00b12.1%, respectively. Talatisamine concentrations of 50, 100, and 200 \u03bcM reliably inhibited rat liver mPTP by 88.4%, 90.6%, 94.6%  and heart mPTP by 72%, 80%,  87.5%, respectively.<\/p>\n\n\n\n\n\n
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Figure 2: Effects of the alkaloid talatisamine on the Ca2+<\/sup>-dependent contraction of rat liver and heart mitochondria in the absence of CsA in the medium (A) and in the presence of CsA in the medium (B).<\/strong><\/p>\n

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To compare the effects of 14-O-benzoyltalatizamine, a\nderivative of talatisamine, on the mPTP of the rat liver and heart,\nconcentrations from 1 \u03bcM to 200 \u03bcM were investigated (Figure 3A). Compared to the control, liver mPTP was reliably\ninhibited from 10% to 81% and heart mPTP from 3.6% to 71.5% at these\nconcentrations. The half-maximum inhibitory concentration (IC50<\/sub>) of\n14-O-benzoyltalatisamine was 38.6\u00b13.2 and 85.6\u00b13.5 \u00b5M in liver and heart\nmitochondria (Figure 3A).<\/p>\n\n\n\n

It was found that the level of inhibition of this channel increased in the presence of the half-maximal inhibitory concentration of CsA in the medium. The effect of 14-O-benzoyltalatisamine on the Ca2+<\/sup>-dependent opening of the permeability pore of rat liver and heart mitochondria in the presence of CsA in the medium was studied. In the presence of 1, 10, 50, 100 and 200 \u00b5M concentrations of 14-O-benzoyltalatisamine in the medium, rat liver reliably inhibited mPTP by 32.2%, 90%, 92%, 95% and almost 100%, respectively, compared to the control. In the presence of the above concentrations of 14-O-benzoyltalatisamine in the medium, rat heart mPTP was reliably inhibited by 21%, 73.6%, 85%, 91%, and almost 96%, respectively, compared to the control (Figure 3B). Therefore, the increased inhibitory effect of 14-O-benzoyltalatisamine on mPTP in the presence of CsA in the incubation medium suggests that this alkaloid acts on this channel.<\/p>\n\n\n\n\n\n
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Figure 3: Effects of the alkaloid 14-O-benzoyltalatisamine on the Ca2+<\/sup>-dependent contraction of rat liver and heart mitochondria in the absence (A) and presence (B) of CsA.<\/strong><\/p>\n

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Talatisamine at a concentration of 200 \u03bcM inhibited\nliver mitochondrial mPTP by 56.8% compared to control, and heart mitochondrial\nmPTP by 44%. At this concentration, it was found that 14-O-benzoyltalatisamine\ninhibited liver mitochondrial mPTP by 81% and heart mitochondrial mPTP by 71.5%\ncompared to the control.\nThese alkaloids inhibited\nliver mitochondria more effectively than heart mitochondria. Talatisamine\nalkaloid was observed to be effective in the presence of CsA in the medium\nstarting from a concentration of 10 \u03bcM.<\/p>\n\n\n\n

 This shows that the inhibitory properties of these\nalkaloids are more strongly manifested in the presence of the half-maximal\ninhibitory concentration of CsA in the medium. Hence, these alkaloids can\nactivate cyclophilin D (CyP-D), a component of the PTP.<\/p>\n\n\n\n

Thus, it was found that 14-O-benzoyltalatisamine has a\nmore active effect on the Ca2+<\/sup>-dependent contraction of rat liver and heart\nmitochondria than talatisamine. Talatisamine had no effect at 1 \u03bcM\nconcentration, but 14-O-benzoyltalatisamine had an effect starting at 1 \u03bcM\nconcentration. The introduction of a benzoyl group into the talatisamine\nalkaloid may have increased its activity. By introducing a benzoyl group at the\nC-14 position, the relaxant activity of this alkaloid was found to increase\nsignificantly10<\/sup>.<\/p>\n\n\n\n

Several toxic gases, heavy metal salts, chemical reagents, inducers in the environment have a toxic effect not only on living organisms at the cellular level, but also at the level of mitochondria, destroying the barrier function of the membrane19<\/sup>. In this condition, the lability of the mitochondrial membranes increases and the membrane potential decreases. As a result, membrane damage accelerates, and the formation of free radicals increases, which causes various pathologies20<\/sup>.<\/p>\n\n\n\n

Oxidative stress, which disrupts normal cellular\nsignaling mechanisms, plays an important role in cells and tissues damage21<\/sup>.\nAmong the factors that damage the mitochondrial functional system, the\naccumulation of free radicals in the cell, as well as high concentrations of H2<\/sub>O2<\/sub>,\naccelerates the process of LPO in the mitochondrial membrane, causing the cellular\nantioxidant system to derail20<\/sup>.<\/p>\n\n\n\n

There is an increasing demand for biologically active\nsubstances extracted from plants to overcome various pathological processes\nthat cause LPO processes23<\/sup>. Detection of malondialdehyde (MDA), a product of LPO,\nwhich reacts with thiobarbituric acid in cells and tissues, is one of the ways\nto study LPO in biological systems24-25<\/sup>. During LPO, the amount of MDA directly represents the\nlevel of oxidative stress in cells and tissues. Therefore, in the next part of\nthe study, the effect of diterpene alkaloids and their derivatives on the LPO\nprocess was investigated.<\/p>\n\n\n\n

Addition of Fe2+<\/sup>\/ascorbate to incubation medium during the experiment\nresulted in a dramatic increase in MDA formation in mitochondria (3.15 nmol\nMDA\/mg protein), and this was taken as a control (100%) to the MDA formation\nprocess in rat liver mitochondria with the addition of LPO process inducers Fe2+<\/sup>\/ascorbate\nto the incubation medium the\neffect of talatisamine alkaloid and its derivative 14-O-benzoyltalatisamine at\ndifferent concentrations was studied (Figure 4). It was found that talatisamine alkaloid decreases MDA\nformation in rat liver mitochondria in a concentration-dependent manner, that\nis, MDA formation at a concentration of 50 \u03bcM was reduced by 8\u00b12.9% compared to\nthe control, and by 21.8\u00b13.5% at a concentration of 100 \u03bcM, 150 \u03bcM and reduced\nby 33\u00b13.7% and 40.5\u00b13.2% at 200 \u03bcM concentrations, respectively. When\n14-O-benzoyltalatisamine was also tested at these concentrations, it was found\nto reduce MDA formation by 25\u00b13.4%, 41.5\u00b13.7%, 70\u00b12.9% and 81\u00b13.4% compared to\nthe control.<\/p>\n\n\n\n

Thus, it was found that talatisamine alkaloid and its derivative at the studied concentrations, inducers of the LPO process, decrease the formation of MDA through the Fe2+<\/sup>\/ascorbate system in a concentration-dependent manner. The half-maximal inhibitory concentration of LPO in the membranes of liver mitochondria for 14-O-benzoyltalatisamine was IC50<\/sub>=115.2\u00b13.1 \u00b5M (Figure 4).<\/p>\n\n\n\n\n\n
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Figure 4: Influence of different concentrations of talatisamine and 14-O-benzoyltalatisamine, MDA accumulation on LPO in mitochondria induced by Fe2+<\/sup>\/ascorbate.<\/strong><\/p>\n

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The products formed during the LPO process in cells\nand tissues in physiological and pathological conditions are activated as a\nprotective signaling system in the body6<\/sup>. The LPO process leads to a\ndisruption of the membrane structure, a change in permeability, a decrease in\npotential, separation of oxidative phosphorylation and ATP hydrolysis, and a\ndecrease in the rate of electron transfer along the respiratory chain. Depending\non the elimination of these processes by biologically active substances, it is\npossible to show the level of their activity. Similarly, biologically active\nsubstances extracted from plants are antioxidant compounds.<\/p>\n\n\n\n

Therefore, according to the research results, it was\nfound that 14-O-benzoylatisamine alkaloid is twice as active as talatisamine\nalkaloid in inhibiting the formation of MDA.<\/p>\n\n\n\n

The products formed during the LPO process in cells\nand tissues in physiological and pathological conditions are activated as a\nprotective signal system in the body25<\/sup>. It is known that the LPO\nprocess leads to a change in the permeability of biomembranes in mitochondria,\na decrease in the membrane potential, the separation of oxidative\nphosphorylation processes, and the hydrolysis of ATP. Depending on the\nelimination of these processes by biologically active substances, it is\npossible to show the degree of their activity. Similarly, biologically active\nsubstances extracted from plants are antioxidant compounds.<\/p>\n\n\n\n

Therefore, in subsequent studies, the antioxidant activity of diterpene alkaloids was also studied in the liver mitochondria model. In subsequent in vitro<\/em> experiments, the antioxidant activity of the studied alkaloids was determined in the model of liver mitochondria contraction, where Fe2+<\/sup>\/ascorbate-induced mitochondrial contraction amplitude (t-5min, \u2206A540=0.330\u00b10.012) was taken as 100%. In this study, the effects of talatisamine alkaloid on the liver mitochondrial LPO process were examined at concentrations of 10, 25, 50, 75 and 100 \u00b5M, 7.0\u00b12.3%, 18.7\u00b11.9%, 34.3\u00b12.6% , was found to be inhibited by 49.9\u00b12.1% and 62\u00b11.8%. According to the results of the study, it was found that talatisamine had a relatively stronger inhibition at a concentration of 100 \u03bcM (Figure 5A). According to the results of the study, it was found that talatisamine had a relatively stronger inhibition at a concentration of 100 \u03bcM (Figure 5A). During the studies, the effect of 14-O-benzoyltalatisamine on the LPO process in the mitochondrial membrane was studied (Figure 5B). In contrast to talatisamine, 14-O-benzoyltalatisamine began to significantly affect the LPO process at a concentration of 10 \u03bcM. It was found to inhibit the LPO process at this concentration by 29\u00b12.5% compared to the control. As the concentration of 14-O-benzoyltalatisamine in the environment increased, its inhibitory effect on LPO began to be strongly manifested. Incubation medium concentrations of 25, 50, 75, and 100 \u03bcM of 14-O-benzoyltalatizamine inhibited LPO by 38\u00b12.1%, 59\u00b12.9%, 73\u00b11.7%, and 91\u00b12.3%, respectively, compared to control was found to inhibit (Figure 5B).<\/p>\n\n\n\n\n\n
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Figure 5:<\/strong> Concentration-dependent effects of the alkaloids talatisamine (A) and 14-O-benzoyltalatisamine (B) on the Fe2+<\/sup>\/ascorbate-induced LPO process in mitochondria.<\/strong><\/p>\n

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A concentration of 100 \u00b5M of 14-O-benzoyltalatisamine alkaloid was found to\nmaximally inhibit the LPO process in rat liver mitochondria membrane compared\nto the control. These\nmitochondrial suppression results were consistent with the results of\nexperiments in which diterpenoid alkaloids reduced mitochondrial MDA formation.\nFrom the studies, it should be noted that the antioxidant activity of\nditerpenoid alkaloids – talatisamine and 14-O-benzoyltalatisamine showed\nrelatively weaker antioxidant activity compared to 1-O-benzoylnapelline6<\/sup>.<\/p>\n\n\n\n

Today, diterpene\nalkaloids are gaining importance as one of the classes of promising substances\nfor the creation of new pharmacological drugs. Diterpene\nalkaloids have various physiological activities.\nFor example: antiarrhythmic26<\/sup>, antispasmodic7<\/sup>, regenerative27<\/sup>, antimetastatic28<\/sup>, antidepressant2<\/sup>9<\/sup>,\nantipyretic, anxiolytic, antioxidant, anti-inflammatory, wound healing30<\/sup>,\nrelaxant3<\/sup>1<\/sup> effects were studied. A number of mechanisms of\naction of diterpene alkaloids have been studied, but mechanisms of\naction at the membrane level of\na rat liver and heart\nmitochondria have not been studied.<\/p>\n\n\n\n

 In our\nresearch, it was found for the first time that talatisamine and its derivative\n14-O-benzoyltalatisamine inhibit the Ca2+<\/sup>-dependent opening of the mitochondrial permeable pore in rat liver and\nheart, Fe2+<\/sup>\/ascorbate-induced mitochondrial swelling (LPO process) and inhibit the formation of MDA in membranes. The\nantioxidant activity of talatisamine and 14-O-benzoyltalatisamine alkaloids and\nthe inhibition of Ca2+<\/sup>-dependent opening of the mitochondrial pore open the prospects for the\ncreation of new cytoprotective agents.<\/p>\n\n\n\n

Mitochondria are the target organelles that control apoptosis, while the\nmPTP is particularly noteworthy as it performs an important regulatory function\nin cell life. Therefore, the mPTP is a target for various pharmaceutical agents\nand biologically active substances1<\/sup>. Since mPTP inhibitors have the\nproperty of stabilizing mitochondrial membranes, it is one of the urgent tasks\nto study the effect of biologically active compounds on the state of mPTP. A\nsmall increase in Ca2+<\/sup> ions in mitochondria can cause a small amount\nof matrix degradation, which is observed with increased activity of oxidative\nphosphorylation and electron transport chain. An excess of Ca2+<\/sup> ions\nin the mitochondria leads to the opening of the mPTP, which causes pathological\nstrangulation32<\/sup>.<\/p>\n\n\n\n

Currently, several pharmacological agents that modulate mPTP activity,\nsuch as CsA and adriamycin (doxorubicin), have undergone clinical trials. The\nclassical inhibitor of mPTP, CsA, inhibits mitochondrial contraction caused by Ca2+<\/sup>\nions33<\/sup> and switches the pore to the closed state. The inhibitory\neffect of CsA on mPTP prevents cardiomyocyte damage and inhibits\npalmitate-induced apoptosis in them. Perhaps, due to the inhibition of mPTP, CsA\ncan prevent cell necrosis from the damaging effects of pH in\nischemia-reperfusion, the toxicity of Ca2+<\/sup>-ionophores, and oxidative\nstress. The PTP-inhibiting property of CsA can be used for cell preservation in\nnormothermic transplantation34<\/sup>. CsA has immunosuppressive properties\nand blocks transcription of cytokine genes by activated T-cells. CsA forms a\ncomplex with cyclophilin D by inhibiting the peptidylprolyl-cis-trans-isomerase\nactivity of cyclophilin D35<\/sup>. Inhibition of cyclophilin D by major\ninhibitors of PTP prevents Ca2+<\/sup>-induced shock in liver mitochondria\ndue to normalization of matrix Ca2+<\/sup>. CsA and its analogues have the effect\nof reducing the functional activity of mPTP, acting as an inhibitor by changing\nthe value of peptidyl-prolyl cis-trans isomerase enzyme activity against\ncyclophilin D in mitochondria36<\/sup>.<\/p>\n\n\n\n

The obtained results show that talatisamine and 14-O-benzoyltalatisamine\nalkaloid reliably inhibit mPTP. The inhibitory effect on rat liver and heart\nmPTP may be mediated by the activation of cyclophilin D. It can be explained\nthat the introduction of the benzoyl group into the structure increases the\ninhibitory property of cyclophilin D and leads to the inhibition of mPTP. Thus,\nthe above-mentioned alkaloids inhibit mPTP in vitro and have a stabilizing\neffect on the membranes of rat liver and heart mitochondria.<\/p>\n\n\n\n

Thus, in our studies, it was found that the degree of mitochondrial swelling\ncaused by the addition of a low concentration of Ca2+<\/sup> was higher in\nliver mitochondria than in heart mitochondria. This indicates that liver tissues with high\nregenerative properties are less resistant to apoptosis caused by induced Ca2+<\/sup>.\nStudies in this area show that brain37-38 <\/sup>and heart39<\/sup> mitochondria\nare more resistant to Ca2+<\/sup>-dependent permeability pore\nopening than liver cells. This process is explained by tissue specificity\nrather than the amount of Ca2+<\/sup> exposed. When exposed to low\nconcentrations of Ca2+<\/sup>, heart mitochondria are more resistant to the\ndeleterious effects of Ca2+<\/sup> than liver mitochondria, consistent with\nliterature data39<\/sup>.<\/p>\n\n\n\n

Thus, the obtained results confirm the existence of a\ntissue-specific feature of cell protection against the occurrence of Ca2+<\/sup>-dependent\napoptosis and necrosis processes.<\/p>\n\n\n\n

Pharmacological modulators that have an inhibitory\neffect on mPTP can be used in cases of hypoxia and ischemia, including in the\ntreatment of cardiovascular diseases. On the contrary, those that modulate the\nopen state of mPTP, that is, inducers that stimulate apoptosis, can be used in\nthe treatment of oncological diseases.<\/p>\n\n\n\n

Therefore, the researched diterpene alkaloids\nprevent the pathologies developed due to the dysfunction of mitochondria and\nhave a corrective effect on them. In this process, diterpene alkaloids\ninteract with receptors and signaling systems located in mitochondrial\nmembranes. These alkaloids reduced the negative effects of Fe2+<\/sup>\/ascorbate\nby protecting mitochondrial membranes to some extent from damage. These\nalkaloids lead to the recovery of the membrane potential, oxidative\nphosphorylation process and ATP synthesis as a result of the recovery of the\nmembrane structure to a certain extent during the LPO process, and the\nacceleration of the speed of electron transmission along the respiratory chain. <\/p>\n\n\n\n

The investigated alkaloids exhibited antioxidant\nproperties by inhibiting Fe2+\/ascorbate-induced mitochondrial suppression (LPO\nprocess) and MDA formation in membranes.<\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

Studies have shown that 14-O-benzoyltalatisamine inhibits the Ca2+<\/sup>-dependent permeable pore of rat liver and heart mitochondria more strongly than the diterpenoid alkaloid talatisamine. The half-maximum inhibitory concentration (IC50<\/sub>) of talatisamine in liver mitochondria is 78\u00b13.6 \u03bcM, the half-maximum inhibitory concentration (IC50<\/sub>) of 14-O-benzoyltalatisamine in liver mitochondria is 38.6\u00b13.2 and in heart mitochondria was 85.6\u00b13.5 \u03bcM. The sensitivity of the Ca2+<\/sup>-dependent conductive pore of liver mitochondria to diterpene alkaloids was found to be higher than that of heart mitochondria. These alkaloids exhibited antioxidant properties by inhibiting the LPO process and MDA formation in the mitochondrial membrane. Acylation of the hydroxyl group at the C-14 position of talatisamine in the presence of benzoyl chloride resulted in an increase in the overall activity of the alkaloid 14-O-benzoyltalatisamine. This diterpene alkaloid may be the basis for creating new effective cardio-, cyto- and hepatoprotective agents in the future.<\/p>\n\n\n\n

Therefore, these diterpene alkaloids inhibit the Ca2+<\/sup>-dependent permeable pore, prevent apoptosis, and exert a stabilizing effect on mitochondrial membranes, and in the process of LPO, reduce the harmful effects of Fe2+<\/sup>\/ascorbate and protect the membranes to some extent from damage.<\/p>\n\n\n\n

Ethical considerations <\/strong><\/p>\n\n\n\n

All experiments were\nconducted in accordance with the Guide for the Care and Use of Laboratory\nAnimals, and within the guidelines of the local ethics committee\u2018s \u201cBioethical regulations for the use of\nlaboratory animals in scientific research\u201d (BEC\/IBB-NUU\/2019\/02\/22), the Institute of Biophysics and biochemistry at the National university of Uzbekistan named\nafter Mirzo Ulugbek.<\/p>\n\n\n\n

Acknowledgments<\/strong><\/p>\n\n\n\n

We would like to express our\nappreciation to the scientific team of the alkaloids laboratory of the\nInstitute of Plant Substances Uzbek Academy of Sciences for kindly providing\nditerpene alkaloids for the research.<\/p>\n\n\n\n

Conflict of Interest<\/strong><\/p>\n\n\n\n

The authors declare that they\nhave no conflict of interest.<\/p>\n\n\n\n

Funding\nSources<\/strong><\/p>\n\n\n\n

There are no funding sources.<\/p>\n\n\n\n

References<\/strong><\/p>\n\n\n\n

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