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Section 1. Biology
https://doi.org/10.29013/ESR-20-11.12-3-8
Turdiyeva Odina Mamirovna Kokand State Pedagogical Institute, Kokand, Uzbekistan E-mail: odinaxonrazzakova@mail.ru Pozilov Mamurjon Komiljonovich, Institute of Biophysics and Biochemistry at the National University of
Uzbekistan named after Mirzo Ulugbek E-mail: mamurjon2281@mail.ru
Makhmudov Rustamjon Rasuljonovich, Institute of Bioorganic Chemistry of the Academy of Sciences of Republic Uzbekistan, Tashkent, Uzbekistan
HEPATOPROTECTIVE POTENTIAL OF POLYPHENOLS IN CCL-INDUCED HEPATIC DAMAGE
4
Abstract. In this paper, the hepatoprotective potential of gossitan isolated from the plant Gos-sypium hirsutum L. and getasan polyphenols isolated from the plant Geranium sanguineum with carbon tetrachloride (CCl4) was investigated. Gossitane and getasan polyphenols have been studied comparatively with quercetin, a hepatoprotective property that has an effect on blood serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin levels in toxic hepatitis conditions.The experiments were performed on healthy male rats and CCl4-induced hepatitis model animals divided into 5 groups. Animals of the hepatitis model were given oral quercetin 50 mg/kg, gossitan 50 mg/kg, and getasan 50 mg/kg orally for 20 days. In healthy, hepatitis models and pharmacotherapy rats with plant compounds, changes in blood serum and ALT and AST enzymes and total bilirubin levels in the liver were detected.
Keywords: blood serum, liver, carbon tetrachloride, alanine aminotransferase, aspartate amino-transferase, gossitan, getasan.
In the world today, the number of people in- the world to find drugs that exhibit hepatoprotec-
fected with acute and chronic hepatitis is increas- tive properties.
ing as a result of changes in the environment and The most effective pharmacological hepatoprepa-
the release of xenobiotic compounds into the at- rations in the treatment of liver diseases form the ba-
mosphere in many countries. Scientific research sis of a promising source of natural compounds are
on the treatment and prevention of hepatitis as- isolated from plants. Phytohepatoprotectors have
sociated with liver injury is being conducted in special importance from synthetic compounds with
many scientific laboratories and centers around their following properties.
While various compounds have endogenous effects on liver enzymes and synthetic drugs have exogenous effects. Involvement of synthetic drugs in liver metabolism often occurs in the presence of cytochrome P 450 [1,1-19]. Synthetic compounds can reduce the activity of ALT and AST enzymes in blood serum in hepatitis conditions. However, they can form metabolites in hepatocytes that have a detrimental effect on the membrane. Changes in metabolism can occur not only in the presence of synthetic hepatoprotectors, but also in the presence of drugs prescribed in parallel [1, 1-19]. One of the active substances isolated from plants is quercetin, which reduces the formation of active forms of oxygen and lipid peroxidation in liver mitochondria in liver disease [2, 1-10; 3, 49-55]. Among plant hepa-toprotectors flavonoids and polyphenols play main role. The hepatoprotective properties of polyphenol compounds are strong and provide a high level of protection of liver function from toxic effects in hepatitis [4, 1-19]. Currently, many studies have been conducted on the effect of such polyphenol compounds on the biochemical parameters in the blood and the amount of ALT, AST, glycogen, total proteins and phospholipids in the liver homogenate in pathologies associated with hepatitis [5, 21182125; 6, 2-11; 7, 6020-6047]. Of these polyphenols, gossitan and getasan are also highly biologically active. Gossitan polyphenols isolated from the plant Gossypium hirsutum L. [8, 109-117], streptozotocin has been shown to have an inhibitory effect on rat heart and pancreatic mitochondrial megakanal in diabetic conditions [9, 79-84]. The antioxidant activity of getasan polyphenols isolated from the plant geranium sanguineum [8, 109-117] was studied in in vitro experiments in the mitochondrial model [10, 146-150]. However, the corrective effect of polyphenol compounds such as gossitane and getasan on the dysfunction ofhepatocytes and mitochondria in toxic hepatitis conditions has not been studied.
The purpose of the work. To study of the effect gossitan is isolated from the plant Gossypium hirsu-
tum L. and getasan polyphenol compounds isolated from the plant Geranium sanguineum on the activity of ALT and AST enzymes in rat blood serum and liver tissue and total bilirubin in toxic hepatitis.
Materials and methods. There are currently many types of toxic hepatitis modeling in animals. One such classic method is a model oftoxic hepatitis called CCl4. White male rats with a healthy weight of 180-200 g isolated for the experiment were divided into groups.
I group. Control (healthy n = 5)
II group. CCl4 0.5 ml/kg
III group. CCl4 + quercetin (50 mg/kg)
IV group. CCl4 + gossitan (50 mg/kg)
V group. CCl4 + getasan (50 mg/kg)
To induce experimental toxic hepatitis in experimental group II, III, IV, and V rats, animals were injected subcutaneously with 50% CCl4(0.5 ml/kg) once every 3 days. 21 days after the administration of CCl4 to rats, after an increase in ALT (60 U/L) and AST (120 U/L) enzymes in the blood, purified coconut oil (0.5 ml/kg) was administered to group
II animals once a day, followed by quercetin to group
III of the experiment. flavonoids, group IV gossitane and group B getasan polyphenols were administered once daily for 20 days per os.
The activity of ALT and AST in the serum of experimental animals was performed using as a generally accepted method. The principle of this method of determining the activity of enzymes is based on the formation of pyruvic and oxalic acetic acids as a result of chain reactions of ketoglutarate, aspargin and glutamic acids. Pyruvic acid is formed due to the transition reaction of amino groups under the influence of enzymes that ensure the transition of amino groups of aminotransferases from amino acids to keto acids. When 2,4-dinitylphenylhydrozine (2,4-DNFG) is added to this mixture, pyruvic acid hydrosone is formed, which gives color in an alkaline environment. The resulting color is directly proportional to the amount of pyruvic acid.
ALT catalyzes the transfer of amino groups from alanine to pyruvate and glutamate via 2-oxoglutarate.
ALT activity is determined by the rate of decline of NADH (nicotinamide nucleotide) [11, 241-244; 12; 13, 241-247].
Alanine + 2 oxoglutarate ■ Pyruvate + Glutamate Pyruvate NADH + H+ ■ Lactate + NAD+ In non-breeding male rats, ALT is normally around 70 ± 1 U/L and in female rats around 56 ± ± 1 U/L. Aspartate aminotransferase (AST) catalyzes the formation of oxalate acetate and glutamate from aspartate through 2-oxyglutarate amino groups. AST activity is determined by the rate of decline of NADH. Color intensity was determined by peptical density using a spectrophotometer at a wavelength of 340 nm [11, 241-244; 12; 13, 241-247].
Aspartate + 2 oxoglutarate ■ Oxalate acetate + Glutamate
Oxalate acetate + NADH+H+ ■ Malat + NAD The AST norm is around 58 ± 3 U/L in male rats and 40 ± 2 U/L in female rats. For biochemical studies, blood from rats was centrifuged at 3000 rotation speed for 15 min. The amount ofALT, AST and total bilirubin in the blood of rats was determined using a spectrophotometer. The body and liver masses of the rats obtained for the experiment were determined on special laboratory scales. Research on rats has been conducted in accordance with Helsinki's animal protection bioethics guidelines.
Results and discussion. To make sure that animals develop toxic hepatitis, it is necessary to detect changes in enzymes and some protein types in their blood. ALT and AST are involved in metabolism.
Changes in the parameters of this enzyme signal the onset of the process of necrosis of cells or damage to the heart muscle or liver hepatocytes. ALT, AST, and bilirubin levels in the blood are determined in conjunction with clinical examinations to assess the functional status of the liver. For this reason, determining the amount of aminotrasferases in blood serum is an important diagnostic test in a number of serious diseases, including viral hepatitis and liver cirrhosis. For this reason, our experiments initially determined the effect of plant compounds on the amount of ALT, AST enzymes and bilirubin in the blood serum of animals with toxic hepatitis. According to the results, after 21 days of administration of CCl4 every 3 days, the level of ALT in the blood of animals with toxic hepatitis (group II) was 203.4 ± ± 20.5 U/L, AST 178.6 ± 15.5 U/L and total bilirubin was 18.9 ± 1.6 ^mol/l (Table 1.). This means that the ALT, AST, and total bilirubin levels were 2.6, respectively, relative to the control; 3.3; and increased 1.7-fold. The development of toxic hepatitis in experimental animals led to a sharp increase in the amount ofALT, AST and bilirubin in their blood serum. Animals with toxic hepatitis III were administered orally for 20 days with a standard prototype of quercetin with hepatoprotective activity. Serum ALT (86.6 ± 9.4 U/L), AST (68.2 ± 6.6 U/L), and bilirubin (12.5 ± 1.4 ^mol/l) levels in rats treated with quercetin for toxic hepatitis were 1.5, respectively, compared with group II; 2.1; and decreased
by 4.5 times (Table 1).
Table 1. - Effect of quercetin, gossitan and getasan on ALT, AST enzymes and bilirubin levels in rat blood serum in toxic hepatitis
Experimental groups ALT U/L AST U/L Total bilirubin ^mol/l
I control group 77.4 ± 6.8 54.1 ± 4.7 10.7 ± 1.1
II group experiment (CCl4) 203.4 ± 14.5** 178.6 ± 15.5** 18.9 ± 1.6**
III group CCl4 + quercetin 86.6 ± 9.4 68.2 ± 6.6 12.5 ± 1.4
IV group CCl4 + gossitan 97.8 ± 10.6* 82.6 ± 7.5* 13.4 ± 1.2
V group CCl4 + getasan 122.2 ± 11.3** 94.3 ± 6.2** 14.8 ± 1.3*
*Р < 0,05; **Р < 0,01 compared to the control group
When animals of group IV with toxic hepatitis were treated with gossitan polyphenols for 20 days, their plasma ALT (97.8 ± 10.6 U/L), AST (82.6 ± ± 7.5 U/L), and bilirubin (14.8 ± 1.3 ymol/l) were 5.5, respectively; 5.5; and decreased by 4.5 times. Group IV animals with toxic hepatitis were treated with getasan polyphenols for 20 days, and their serum ALT (122.2 ± 11.3 U/L), AST (94.3 ± ± 6.2 U/L), and bilirubin (14.8 ± 1.3 ^mol/l) were 5.5, respectively; 5.5; and decreased by 4.5 times (Table 1). Thus, the natural compounds isolated from plants, gossitane and getasan polyphenols, had a corrective effect, such as quercetin, which has an existing hepatoprotective property, reducing the amount ofALT, AST enzymes and total bilirubin in the blood of animals with toxic hepatitis. It has been experimentally proven that the correction effect of these enzymes and bilirubin, which is increased in pathology of gossitan polyphenols, is more active than that of getasan. Aminotransferases or trans-aminases catalyze intermolecular reactions in the transport of amino groups from amino acids to keto acids. Pyridoxal phosphate and pyridoxamine phosphate, which are the active states of vitamin B6 (pyri-doxine), are involved as transaminases coenzymes. In toxic hepatitis, it is important to determine the activity of two ALT and AST enzymes, as these enzymes differ in their activity in different azo and tis-
Table 2.- Effect of quercetin,
sues. For example, ALT activity in the liver is slightly higher than in heart tissue, while AST, in contrast, has higher activity in heart muscle. The activity of aminotransferases in the blood serum is very low, in various pathological conditions when liver cells or cardiomyocyte membranes are damaged and their integrity is compromised, their amount increases significantly. Subsequent experiments were performed to determine the levels of ALT and AST in the liver in toxic hepatitis and to determine the effect of biological compounds on them. The results showed that the levels ofALT (75.5 ± 5.7 U/L) and AST (54.7 ± 4.4 U/L) in the liver of CCl4-induced toxic hepatitis (group II) rats increased by 211.9% and 154.4%, respectively. Thus, as a result of the development of toxic hepatitis in rats, the important enzymes in the liver, ALT and AST, increased. This leads to disruption of the membranes of liver hepatocytes and molecular dysfunction of the approximate parts of the cells. Continuing our next experiments, rats with toxic hepatitis III, IV, and B caused by CCl4 were treated with pharmacotherapy with herbal substances quercetin, gossitan, and getasan. The existing hepatoprotective quercetin flavonoid was found to reliably reduce ALT (34.6 ± 3.5 U/L) and AST (29.8 ± ± 2.2 U/L) levels in the livers of toxic hepatitis rats by 168.9 and 115.8%, respectively, compared with group II indicators (Table 2).
gossitan and getasan on ALT
and AST enzymes in rat liver in toxic hepatitis
Experimental groups ALT U/L AST U/L
I control group 24.2 ± 1.7 21.5 ± 1.7
II group experiment (CCl4) 75.5 ± 5.7** 54.7 ± 4.4**
III group CCl4+quercetin 34.6 ± 3.5* 29.8 ± 2.2*
IV group CCl4+gossitan 39.2 ± 3.7* 33.7 ± 2.9**
V group CCl4+getasan CCl4 43.4 ± 3.8** 38.4 ± 3.5**
*Р < 0,05; **Р < 0,01 compared to the control group
Correction of toxic hepatitis-induced rats with gossitan polyphenols (group IV) revealed a 149.9% and 97.7% decrease in liver ALT (39.2 ± 3.7 U/L)
and AST (29.8 ± 2.2 U/L) compared with group II, respectively. In our experiment, it was found that ALT (43.4 ± 3.8 U/L) and AST (38.4 ± 3.5 U/L) in
the liver of toxic hepatitis B rats treated with another natural polyphenol compound getasan decreased by 132.6 and 75.8% compared to group II (Table 2). This means that in the conditions of toxic hepatitis, the amount of rat liver enzymes ALT and AST increases. The compounds isolated from plants suggest that in the conditions of gossitane and getasan toxic hepatitis, rat liver can correct liver dysfunction by reducing the activity ofALT and AST enzymes.
Conclusions: Gossitan and getasan polyphenols reduced the levels ofALT and AST enzymes in blood serum and liver tissue in toxic hepatitis caused by CCl4. The increase in total bilirubin in toxic hepatitis was effectively reduced by gossitan and getasan. The effect of gossitan polyphenols on ALT and AST enzyme levels and total bilirubin levels was found to be more effective than that of getasan.
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