Influence of Glycyrrhizic Acid, Menthol and Their Supramolecular Compounds on the Functional Activity of Rat Mitochondria in in-vitro Experiments

Obtaining a Supramolecular Complex. Supramolecular compounds were obtained by the following steps. Firstly, 1.68 g (0.002 M) of GA dissolved 25 ml distilled water and 25 ml ethanol. This solution widely mixed with a magnetic stirrer at 50–60 ℃. Then we added 0.156 g (0.001 M) Menthol and continued the mixing process for 5–6 hours. After then the reaction mixture was filtered. The rest of the ethanol was removed in a vacuum, and the aqueous part was dried in a lyophilic manner. Final product was a yellow powder: liquid = 205–210 ℃ R_f = 0.9 (System 2) Units: 85% IR spectrum:1042 (-O-) cm^-1; 1655 (CO) cm^-1 2948 (CH₃) cm^-1; 3600–3200 (OH) cm^-1. Other supramolecular complexes of GA with M were also obtained by this synthesis method:

The supramolecular complex of GA with menthol was obtained according to Fig. 1.

Figure1.

Reaction of the supramolecular complex between GA and Menthol.

Supramolecular complexes with molar ratio 2:1; 4:1; 9:1 of glycyrrhizic acid with menthol were obtained in water:etanol system.

The experiments were performed on purebred white rats. The feeding and keeping of the animals was carried out in vivary conditions and it was kept at a normal level. The experiments were performed on male white rats weighing 180–200 g.

In the first in vitro studies of the effect of supramolecular complexes obtained on the basis of menthol, GA and their different ratios on the functional parameters of rat liver mitochondria, the biological activity of GA: M (4: 1) supramolecular complex was more pronounced than other supramolecular compounds. Therefore, in the later stages of our study, it was selected to study the hypoglycemic property in in vivo experiments.

Mitochondria from rat liver to Schneider^10,11 were separated using the differential centrifugation method. By decapitating the animal, the liver was extracted from the abdominal cavity and then placed in a frozen separation medium in a beaker. Separation medium composition: 250 mM sucrose, 10 mM tris-chloride, 1 mM EDTA, pH 7.4. After the liver mass was measured, it was homogenized in a Teflon homogenizer in a 6-fold separation medium by mechanical pressing. For the first time, a 600 g rotation was centrifuged at 0 ± 1 ℃ in an angular-type rotor TsLR-1 for 7 min to separate the nucleus and cell fragments. The supernatant was centrifuged at 6000 g for 15 min at high temperature for 15 min. The precipitate was suspended in a separation medium at a ratio of 10:1 (the amount of the separation medium in ml per gram of liver mass). During the experiment, mitochondria were kept in an ice bath.

Mitochondrial protein biuret reaction.¹² To prepare the biuret reagent, we dissolved 750 mg of copper sulphate (CuSO₄ × 5H₂O) and 3 g of sodium potassium hydroxide (NaKC₄H₄O₆ × 4H₂O) in 500 ml of H₂O. To stop oxidation to the prepared solution, 150 ml of 10% NaOH and 1 g of KJ were added and stored in a polyethylene container to a volume of 1l.

To determine the protein in the mitochondrial suspension, we added 0.9 ml of 2N KOH and 10 mg of deoxycholic acid to 0.1 ml of the drug suspension to break down the membranes. After complete dissolution of the protein, add 4 ml of biuret reagent and leave at room temperature for 30 min. Simultaneously, we prepared a control probe (1 ml of 2N KOH + 10 mg of deoxycholate acid + 4 ml of biuret reagent). We then colorized at 540 nm in cuvettes with a thickness of 10 nm. We calculated the protein serum albumin standards according to the calibration curve obtained by colorimetry.

The swelling (swelling) kinetics of mitochondria (0.3-0.4 mg/mL) were determined by changing the optical density of the mitochondrial suspension at 26 ℃ in an open cell (volume 3 mL) at 540 nm with constant stirring. The following incubation medium (IM) was used to determine the permeability of PTP in mitochondria: 200 mM sucrose, 20 μM EGTA, 5 mM succinate, 2 μM rotenone, 1 μg / ml oligomycin, 20 mM Tris, 20 mM HEPES, and 1 mM KH₂-PO₄, pH 7.4.¹³ We calculated the concentration of ionized calcium in the medium using a computer program BAD4 in the presence of Ca²⁺-EGTA buffers.

Determination of Peroxidation Oxidation Products of Lipids in Mitochondria. Separation of LPO products was performed in the presence of thiobarbituric acid (TBA). The reaction stopped by adding 0.220 ml of 70% trichloroacetic acid to the IM. After this step, the mitochondrial suspension was centrifuged at 4,000 rpm for 15 min. Then 2 ml of sedimentary liquid was obtained and 1 ml of 75% TBA was infused. 2 ml of N2O and 1 ml of TBA were added to the control solution. The mixture was incubated for 30 min in a water bath. After cooling, a change in optical density at a wavelength of 540 nm was detected.

In determining the amount of MDA, the molar coefficient extinction (e = 1.56 × 105 M^-1 cm^-1) was used in the formula: nmol MDA / mg protein = D / 1.56 × 30.

A Fe²⁺ / ascorbate system was also used to study the LPO process in the mitochondrial membrane. Under the influence of this system, the mitochondrial membrane lost its barrier function, resulting in an increase in organelle size and the suppression of mitochondria. This volume change was detected photometrically. IM: KSI - 125 mM, tris-NSI - 10 mM, rN 7.4; Concentrations: FeSO₄ - 10 μM, ascorbate - 600 μM; mitochondrial volume 0.5 mg / ml; Statistical processing of the obtained results. Statistical processing of the obtained results and drawing of images was carried out using the computer program OriginPro 7.5 (Microsoft, USA). In experiments, the swelling kinetics of mitochondria were performed as a percentage of the maximum, in the form of calculating the arithmetic mean of 4–7 different experiments. The difference between the values obtained from the control, experiment, and experiment + research material was calculated on the t-test. In this case R <0.05; R <0.01; and values of R <0.001 represent statistical reliability

Initially, the effect of the compounds on rat-liver mitochondria Ca²⁺ - dependent megakanal (mPTP) was studied. Ionic channels located in the inner and outer membranes of the mitochondria serve as the main pharmacological and therapeutic targets in cell protection.^14,15 One such ion channel is the mitochondrial permeability transition poremPTP, which is a channel that physiologically regulates the release of Ca²⁺ ions from the matrix. Mitochondria play an important role in the homeostasis of Ca²⁺ ions between the PTP matrix and the cytosol, as well as in the Ca²⁺-signalization. This megapora ensures the transition of mitochondrial membranes to a highly permeable state in various pathologies. In pathological conditions, permeabilization of the mitochondrial membrane takes place in the open conformational state of mPTP. Some biologically active compounds exhibit the property of inhibiting the high permeability state of PTP resulting from mitochondrial dysfunction. To determine this property, the effect of supramolecular complexes obtained on the basis of menthol, GA and their different ratios on rat liver mPTP was studied.

Initially, the effect of menthol on the PTP status of rat liver mitochondria was studied in experiments. A concentration of 10 μM of Ca²⁺ ions was used as the inductor. It is known that Ca²⁺ ions increase the permeability of mPTP and convert it to the open state, resulting in mitochondrial swelling. Studies have shown that when 10 μM of menthol is exposed to energized mitochondria induced by Ca²⁺ ions, mitochondrial swelling decreases by 24 ± 2.1% and 30 and 50 μM by 48 ± 3.8% and 82 ± 3.7%, respectively (Fig. 2, A). Hence, the menthol monoterpenoid had an inhibitory effect on mPTP.

The effect of GA at 10, 30, and 50 μM concentrations on rat liver mitochondrial edema was studied. In the presence of Ca²⁺ ions in the incubation medium and in the absence of GA, mitochondrial swelling was assumed to be 100%. According to the results obtained, the concentrations of GA 10, 30 and 50 μM were 18 ± 3.1% of the control of mitochondrial swelling, respectively; It was found to inhibit 55 ± 4.3% and 85 ± 2.9% (Fig. 2, B).

Figure2.

Effect of menthol (A) and GA (B) on mitochondrial edema of rat liver (*R < 0.05; **R < 0.01; n = 5).

Thus, at concentrations of menthol 10–50 μM and GA 10–30 μM studied, hepatic mitochondria had an inhibitory effect on mPTP permeability and prevented mitochondrial suffocation. The identified property of these compounds can be used as a corrective agent for disorders of the mitochondrial membrane in pathological conditions.

In our next experiments, the effect of supramolecular compounds of GA and menthol in the ratios GA: M (2: 1), GA: M (4: 1) and GA: M (9: 1) on mPTP permeability of rat liver mitochondria was studied. Experimental results showed that the levels of GA: M (2: 1) at 20 and 50 μg / ml inhibited rat liver mitochondrial suffocation by 14 ± 2.3% and 66 ± 2.8%, respectively (Fig. 3, A).

The ratio of GA: M supramolecular complex (4: 1) was found to reduce mitochondrial swelling by 57 ± 4.1% and 87 ± 3.2% compared to control (Fig. 2, B). The ratio of GA: M supramolecular complex (9: 1) was found to inhibit mitochondrial permeability by 35 ± 4.4% and 71 ± 2.5% relative to control (Fig. 3С). Hence, supramolecular complexes of GA and menthol inhibited rat liver mitochondrial suffocation, leading to an open conformation of mPTP relative to control. Here, the effect of the supramolecular complex of GA and menthol (4:1) on mitochondrial mPTP permeability was found to be effective against its (2:1) and (9:1) ratio compounds.

Figure3.

Effect of supramolecular compounds of GA and menthol on the mitochondria of rat liver in the ratios GA: M (2:1) (A), GA: M (4:1) (B) and GA: M (9:1) (V) (* R < 0.05; ** R <0.01; n = 5).

These results suggest that various glyceric acid compounds.¹⁶ Corresponds to data from the literature on inhibitory activity. Studies require experiments to further study the pharmacological activity of the supramolecular complex in the ratio of GA and menthol (4:1).

It is known that many inhibitors of mitochondrial mPTP have antioxidant properties, which in turn effectively affect the process of ATF synthesis. In this regard, in order to determine the antioxidant properties of menthol, GA and their supramolecular complexes in rat liver mitochondria, the effect on the process of peroxidation of lipids in mitochondria caused by Fe²⁺ / ascorbate was studied. Initially, the effect of menthol monoterpenoid and GA triterpenoid on liver mitochondria LPO was determined. After Fe²⁺ / ascorbate was added to the incubation medium, the induced LPO process, i.e., mitochondrial tumor rate, was assumed to be 100%. In this experiment, the resulting products of lipid peroxidation disrupt the barrier function of the mitochondrial membrane, resulting in an increase in its swelling rate relative to control. Menthol monoterpenoid in experiments. At 10 μM, the effect on LPO in the mitochondrial membrane was negligible. However, concentrations of menthol 20, 30, and 40 μM were 76.5 ± 4.5%, respectively, relative to the control of peroxidation of hepatic mitochondria under Fe²⁺ / ascorbate; Decreases were found to be 89.4 ± 3.0% and 93.5 ± 1.7%, respectively (Fig. 4, A). This result indicates that menthol concentrations of 20, 30 and 40 μM have antioxidant properties. The antioxidant properties of menthol are also consistent with the literature.¹⁷ But it was first discovered that it exhibited this activity in the hepatic mitochondria.

In our next experiment, we studied the effect of GA concentrations in the range of 10–40 μM on the peroxidation of lipids induced by Fe²⁺ / ascorbate in rat liver mitochondria (Fig. 4, B). According to the results, the concentration of GA 10 μM did not affect the lipid peroxidation of the liver mitochondrial membrane. However, a concentration of 15 μM of GА was found to reduce the LPO formed by the action of Fe²⁺ / ascorbate on the mitochondrial membrane by 58.0 ± 5.0% relative to control. GA concentrations of 20, 30, and 40 μM were 87.5 ± 5.0%, respectively, relative to LPO rate control in hepatic mitochondria; Further reductions were found to be 89.1 ± 4.0% and 92.8 ± 3.0%, respectively (Fig. 3, B). Thus, a concentration of 10 μM of GА does not affect the LPO induced by the Fe²⁺ / ascorbate inducer of the mitochondrial membrane, but its high 15, 20, 30, and 40 μM levels may have a reliable inhibitory effect on LPO intensity. The inhibitory effect of GA and its supramolecular compounds synthesized with various flavonoids on lipid peroxidation in rat heart and brain tissue is consistent with the literature data.¹⁸ However, in our experiments, it was first identified that GA exhibited antioxidant properties in the hepatic mitochondria under the influence of the Fe²⁺ / ascorbate inducer.

Figure4.

Effect of menthol (A) and GA (B) on hepatic mitochondria in the LPO process induced by Fe²⁺+ ascorbate (control reliability *R < 0.05; **R < 0.01; n = 5).

In our next study, the effect of GA and menthol supramolecular complexes on the LPO process induced using the Fe²⁺ / ascorbate system of the rat liver mitochondrial membrane was studied. According to the results, the supramolecular compound of GA and menthol in the ratio GA: M (2: 1) to LPO induced by Fe²⁺ / ascorbate of rat liver mitochondria at 20 and 50 μg / ml, respectively, was 46.6 ± 5.5% and 57.7 ± A 2.8% inhibitory effect was found (Fig. 5, A).

In a subsequent experiment, the effect of GA and menthol on the rate of lipid peroxidation of the supramolecular complex of rat liver mitochondria in the ratio GA: M (4:1) was studied. According to the results of the experiment, the rates of peroxidation of lipids induced by Fe²⁺ / ascorbate in the liver mitochondria GA: M (4:1) supramolecular complex 20 and 50 μg / ml were 68.6 ± 4.4% and 94.6 ± 3, respectively, compared with controls. A 2% inhibitory effect was noted (Fig. 5, B).

Figure5.

Supramolecular compounds of GA and menthol in ratios GA: M (2:1) (A), GA: M (4:1) (B) and GA: M (9:1) (C) were induced by Fe²⁺+ ascorbate of rat liver mitochondria Effect on LPO process (reliability with control *R < 0.05; **R < 0.01; n = 5).