3.1 Effect of enzymatic and non - enzymatic antioxidants
The impact of A. purpurata on enzymatic antioxidants in the ovaries of both control and experimental animals is shown in Fig. 1. Rats in group II that were exposed to 4-vinyl cyclohexane had considerably lower levels of enzymatic antioxidants. However, compared to group II, animals who had received medication treatment (groups III and IV) experienced a return to normal levels of enzymatic activity. Between Group V and the control group (Group I), that just received the plant extract, there was no discernible difference.
Free radicals have the potential to induce lipid peroxidation and harm the macromolecules and cellular structures within an organism, such as the endothelium and erythrocytes. This process of lipid peroxidation can alter membrane permeability and lead to tissue damage. An essential protective mechanism against free radical-induced damage is provided by the antioxidant system [21]. The cell membrane is easily penetrated by hydrogen peroxide, which then attacks various places and damages DNA by breaking both single and double strands. The levels of antioxidant status in the ethyl acetate extract of A. purpurata were examined in this study in relation to ovarian tissue.
SOD, functioning to dismutase two superoxide radicals into H2O2 and O2, plays a pivotal role as the body's initial defense against superoxide radicals. Furthermore, CAT and GPx, by converting H2O2 into H2O, serve as complementary antioxidant enzymes, safeguarding cells against reactive oxygen species (ROS) damage. The endogenous antioxidant selenoprotein GPx actively participates in this protective mechanism and is located within both the cytosol and the mitochondrial matrix [22]. A rise in radical generation during DMBA metabolism may be to blame for the decline in enzymatic activity. According to the findings of the current investigation, the rise in MDA formation is probably due to an increase in reactive oxygen species (ROS), which is consistent with the finding that these free radicals tend to decrease SOD function. Based on the biochemical data, it was found that 4-vinyl cyclohexane-exposed rats' SOD, CAT, and GPx activity was lower than that of control rats. This reduction in enzymatic activity may be attributed to the excessive production of radicals, which could be a consequence of ovarian cancer [23].
In the investigation of 4-vinylcyclohexane-induced ovarian cancer, Fig. 1 shows the effect of A. purpurata on non-enzymatic antioxidant levels. When compared to the control group, group II mice's ovarian tissue showed a considerable decline in non-enzymatic antioxidant activity. But when compared to mice induced with 4-vinyl cyclohexane, the A. purpurata and cisplatin therapy increased GSH, vitamin C, and vitamin E levels (p < 0.05) in these groups. Following treatment, animals in group V that had only been exposed to the plant extract did not vary significantly from those in group I (p < 0.05) in any way.
When ascorbic acid and carotenoids are present, two other cooperative antioxidants, vitamin E functions well as a lipid-soluble, chain-breaking antioxidant. This cooperative effect strengthens its capacity to guard against oxidative damage. It has been demonstrated that altering non-enzymatic antioxidants including total reduced glutathione (GSH), vitamin C, and vitamin E during cancer therapy can lower the risk of cancer recurrence and lessen side effects linked to elevated free radical levels [24].
Vitamins E and C, which are non-enzymatic antioxidants, are found in healthy cells and can be consumed through diet. In addition to reducing H2O2 and hydro peroxide levels, antioxidants can also trap metal ions, halt chain reactions by scavenging reactive free radicals, and even encourage the repair and/or removal of cell damage. Additionally, some antioxidants promote the manufacture of defense enzymes or additional antioxidants [25].
Various amounts of these antioxidants were found, but they were not enough to offset the rise in reactive oxygen stress, which damages cells and molecules and encourages the cancer cell cycle [26]. The finding that GSH, vitamin C, and vitamin E levels were decreased in cancer-bearing mice was another indication of oxidative stress, which contributes to damage to cells and a breakdown of cell membrane working stability.
3.2 Effect of membrane bound enzymes and tumor markers
The enzyme activity of membrane-bound ATPase enzymes in the ovarian tissue of experimental and non-experimental mice is shown in Fig. 2. When 4-vinylcyclohexane was used to stimulate mice, it was shown that these enzymes' activity considerably decreased (p < 0.05) when compared to control animals. However, during pharmaceutical therapy, their levels showed a noticeable dose-dependent rise. Comparing the group receiving only the plant extract and the group receiving a healthy control, there were no noticeable distinction.
Among the many variables that can affect ATPase levels are peroxidation of lipids and modifications in membrane fluidity. A decrease in ATPase activity under ischemia conditions can cause irreversible necrotic alterations in the myocardial cells that are damaged, as well as a loss of function. The oxidation of "SH" groups inside the binding sites of Na+/K+ ATPase and Ca2+ ATPase, that is caused by lipid peroxidation in the cellular membrane, is the main cause of this drop-in ATPase activity. This shift in enzyme conformation is frequently linked to this decrease in ATPase activity [27]. In fact, an increase in intracellular levels of calcium ions that are free (Ca2+) can be brought on by a reduction in Ca2+ ATPase activity. This can have a variety of effects, such as alterations in signal transduction systems and adjustments to cellular fluidity. Numerous cellular activities depend on the control of intracellular calcium levels, and any disturbance in this equilibrium can have a major influence on cell functioning and communication [28]. The functionality of different cell types, particularly platelets and the nuclear cells in peripheral blood, can be affected by changes in Na+/K+-ATPase activity. The rate of signal transmission is impacted by this enzyme's critical involvement in catalyzing the Ca2+-dependent interchange of hydride ions with calcium ions. This procedure plays a key role in the rapid dissolution of secretory granules and the creation of actino-myosin compounds in platelet surfaces during adhesion, which regulate cellular activities and reactions.
According to reports, ATPase activity is inhibited in cancerous situations. As these enzymes are membrane-bound, any disruption with the cell membrane, especially adjustments to the solubility and ionic concentration of the membrane, can in fact affect how they function. Because ATPase plays a part in malignant conditions, its decreased activity may also be linked to cellular harm. As a result, any changes to the cell membrane may result in changes to the functions of membrane-bound ATPase, which may indicate how serious the underlying disease is. These findings highlight the complex interplay between ATPase activity, membrane integrity, and the health or illness of cells [29].
The plant medicine's bioactive ingredients are known for their capacity to affect the permeability of cellular membranes. They can interact with animal cell Na+/K-ATPase pumps and may even have the ability to promote electron transport. Due to these characteristics, ATPase activity may be brought back to normal levels, which would then allow the cell to function normally. These bioactive components are essential for controlling cellular processes and may be able to reverse cellular malfunction [30].
Following administration of A. purpurata extract, experimental and control rats, ovarian tissue range of 5'-nucleotidase and γ-glutamyl transpeptidase altered, as indicated in Fig. 2. The ovarian tissues of group II mice treated with 4-vinylcyclohexane showed a significant (p < 0.05) increase in the quantity of a cancer marker compared to the control animals in group I. These marker enzyme levels were dramatically lowered and matched those in the cancer-bearing animals in group II in group III and IV, when mice were given A. purpurata and cisplatin (p < 0.05). Notably, the marker activity of the enzyme in group V, which animals received only the plant extract as a treatment, did not differ significantly from that of the group I control animals.
At the sinusoidal and bile canalicular sides of hepatocytes' plasma membranes, there is an enzyme called 5'-nucleotidase (5'NT). It works by hydrolyzing nucleotides with phosphate groups linked to the carbon atoms of the ribose sugar. It's interesting that mice with cancer were shown to have increased activity. 5'NT is also used as an indicator to evaluate liver damage. The presence and activity of this enzyme can be used to monitor liver health and function as a marker for specific liver diseases [31]. In the current study, rats that were carrying ovarian cancer showed increased 5'NT activity. The emergence of the marker enzyme may be connected to the growth of the tumor and hepatic cell damage, which may cause 5'-nucleotidase spillage into the bloodstream. In this sense, treatment with A. purpurata significantly decreased the activity of the enzyme 5'NT. This might be connected to the plant extract's ability to quench free radicals, which would compensate for the hepatic cell damage.
Strongly secretory or detoxifying cells have gamma glutamyl transferase (γ-GT), a membrane-bound enzyme, on their surface. Because it has been demonstrated that disorders such bile duct necrosis and cholestasis cause the enzyme level to be raised in serum and the liver, it is one of the important indicators of liver damage. There have been reports of a variety of substances, notably xenobiotic, forming a substrate of γ-GT following their attachment to GSH, which mainly occurs in the liver. The loss of GSH may also result in a rise in hepatic γ-GT activity through an elevation in the synthesis of its mRNA. It was found in the present study that rats with cancer had increased levels of γ-GT activity. This is consistent with the earlier observation that [32]. However, ethyl acetate extract restores the level of the γ-GT enzyme throughout treatment. This might be as a result of the extract's cytoprotective properties, which might have assisted in stabilizing the cellular membranes of the hepatocytes and other bodily tissues and preventing the loss of the functional properties of the cell membrane.
3.3 Effect of nucleic acid constituents in ovary, serum liver marker enzymes and renal markers of control and experimental animals
In a variety of tumor settings, DNA content is an important growth indicator that is more relevant to the pharmacological and functional aspects of the tumor. According to [33], the amount of DNA is a trustworthy predictor and frequently coincides with the DNA composition of the tumor. The effects of A. purpurata ethyl acetate extract on the levels of nucleic acids (DNA and RNA) in the ovarian tissues of experimental and control mice are shown in Fig. 3. Rats treated with 4-vinylcyclohexane in group II showed noticeably larger amounts of nucleic acid constituents than the control animals in group I (p < 0.05). When contrasted to the cancer-bearing rats in group II, however, these elevated levels were noticeably decreased in group III and group IV animals administered A. purpurata along with cisplatin (p < 0.05).
In contrast, when comparing the group V animals administered exclusively using the plant extract to the group I control animals, no appreciable variations in nucleic acid quantities were found.
In the current study, there was an observed increase in the amount of RNA in animals afflicted with ovarian cancer. This increase could be attributed to the heightened DNA content associated with cancerous conditions, potentially leading to increased transcription and, consequently, elevated RNA levels. Conversely, the rats treated with A. purpurata, both DNA and RNA levels returned to normal. Consequently, the application of the ethyl acetate extract from A. purpurata not only inhibited tumor growth but also regulated nucleic acid biosynthesis.
These findings align with earlier research studies on the subject [34]. Marker enzyme analysis is a useful tool for locating tumors and directing medical decisions. The effect of A. purpurata extract on transaminases and alkaline phosphatase blood levels in both experimental and control animals is shown in Fig. 3. Marker enzyme levels were significantly (p < 0.05) lower in the 4-vinylcyclohexane-induced animals in group II compared to the control animals in group I, indicating the impact of this induction. On the other hand, these enzyme levels were markedly decreased in the A. purpurata and cisplatin-treated animals in groups III and IV and were comparable to the levels seen in the cancer-bearing animals in group II (p < 0.05).
However, there were no discernible variations in the marker enzyme activity in animals that were treated exclusively using the plant extract in group V as compared to the untreated animals in group I. In particular, biochemical marker enzymes are used to screen for cancer disorders to aid in differential diagnosis, tracking of advancement, and therapy response [35]. These enzymes have become more distinct, and changes in their activity reveal the effects of metabolic cycle and cell division on proliferating cells. The amount of changed cells present in malignant conditions has been discovered to be highly correlated with their greater activity. The transport processes of cell organelles, such as hepatocytes, are disturbed in malignant situations. Due to modifications in the plasma membrane's permeability, this disruption results in the release of enzymes. As a result, the quantities of these enzyme markers in the serum or blood increase while their concentration within the cells declines. According to reports, when animals are exposed to toxicity, the skeletal framework of their cells is weakened, which causes enzymes to diffuse from the cell cytoplasmic into the blood stream [36]. It is thought that the intracellular enzyme concentrations are over three orders of magnitude greater than external fluid concentrations. As a result, it is a sensitive indication since even a small amount of cellular damage can cause a considerable increase in enzyme activity in the extracellular fluids or plasma. Due to its clinical importance, measuring enzyme activity in both serum and tissues is a popular diagnostic tool in human medicine. Due to the major clinical consequences of both aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, research on serum transaminase activities as a measure of tissue injury has mostly concentrated on human subjects. In the current study, ALT levels showed the opposite tendency to AST levels, which were shown to be high and dropping in the blood and tissues of mice with cancer. The increased enzyme activity in the serum may be caused by a number of things, including the release of enzymes into the bloodstream from cancerous cells, the presence of tumors causing the emit of enzymes from normal tissue, or a possible impact of a tumor on distant tissue that results in a breakdown of an enzyme and its discharge into the bloodstream [37].
Figure 3 displays the concentrations of uric acid, creatinine and urea. The animals in group II that had been exposed to 4-vinylcyclohexane had considerably higher levels of urea and creatinine. Animals treated with extract and cisplatin had lower levels (group III and IV). The above amounts of treatment for rats using the group V medication alone were comparable to the control groups. However, group II cancer-induced animals had lower uric acid levels. Animals treated with extract and cisplatin had considerably higher levels (group III and IV). The above amounts of treatment for rats using the group V medication alone were comparable to the control groups.
Urea, also known as carbamide, serves as the primary nitrogen-containing compound in the urine of mammals. It is an organic chemical with a crucial role in the metabolism of nitrogen-containing substances in animals. The rate of glomerular filtration determines how much urea is eliminated, and as production outpaces excretion, plasma levels rise. Renal function is measured by serum creatinine. It is created endogenously by tissue creatinine breakdown and an increase in serum creatinine may be attributable to the tissue damage. The glomerular filtration rate determines how much creatinine is expelled; when this excretion does not equal the production, serum creatinine increases [38].
The enhanced usage of uric acid against lipid peroxide, a hallmark of cancer conditions, may be the cause of the lower level of uric acid in ovarian cancer. Higher uric acid levels seen following therapy for A. purpurata could be caused by a lower tumor burden, which is consistent with earlier observations of [39].
3.4 Estimation the level of tissue protein and Lipid peroxidation
The levels of protein activity in the ovarian tissue of experimental and control mice are shown in Fig. 4 (B). Induced with 4-vinylcyclohexane, the ovarian tissues of Group II mice displayed a conspicuous drop in protein levels when compared to the control group (Group I) mice (p < 0.05). The protein levels in Group III and IV animals were, however, noticeably higher than in Group II rats (p < 0.05). The protein levels in the control and drug-treated animals (Group V) did not differ noticeably from one another.
Because they are macromolecular structures, proteins are essential for preserving the structural integrity of cells. Any protein level alterations can cause metabolic imbalances, especially in cancer situations, which can result in severe cellular abnormalities. The greatest amount of protein synthesis occurs in tissues, wherein the metabolism of proteins is a critical activity. Protein wastage is characterized by an apparent rise in the rate of protein breakdown while the visible rate of protein production appears to be relatively steady. This indicates an underlying metabolic imbalance. A drop in ovarian protein levels in 4-vinylcyclohexane-induced cancer-bearing mice can be related to an increased efflux of amino acids from tissue and a reduction in the recycling of amino acids, both of which happen under tumor conditions [40]. Degradation of tissue proteins results from the host's reaction to the tumor load. However, therapy with A. purpurata may have stopped this protein deterioration, returning protein levels to a more typical range. The modulation of protein synthesis during this therapy may have also affected the overall protein level, indicating that the plant extract significantly regulates both protein production and maintenance. These results are consistent with past studies from the reference [41].
Figure 4 (A) shows changes in the amount of lipid peroxidation (LPO) in the ovarian tissue of experimental and control rats. The 4-vinylcyclohexane-induced rats (Group II) showed a noticeable rise in LPO levels. On the other hand, animals receiving the plant extract plus cisplatin (Group III and Group IV) as compared to those with cancer showed a substantial (p < 0.05) reduction in malondialdehyde, employed as an indicator of peroxidation of lipids levels. This implies that using these medications to correct the aberrant variations in LPO levels was successful.
When compared to the control group, animals administered only with A. purpurata did not show any statistically significant variations (p < 0.05) in LPO levels, showing that the plant extract did not significantly affect LPO levels on its own.
Free radicals can react with polyunsaturated fatty acids to create malondialdehyde. It is well known for its effect on lipid peroxidation as well as its impact on the control of radicals during the development of cancer. Naturally, there is a delicate balance between the body's creation of free radicals and the antioxidant defense system's defensive mechanisms, which defend the body from disease [42]. It has been proposed that elevated lipid peroxidation seen in erythrocytes may be caused by a compromised antioxidant system. As a result, the amount of MDA is frequently employed as a meter to evaluate the severity of harm brought on by lipid peroxidation triggered by free radicals.