Beta vulgaris L. beetroot protects against iron-induced liver injury by restoring antioxidant pathways and regulating cellular functions | Scientific Reports

Scientific Reports volume 14, Article number: 25205 (2024) Cite this article

Metrics details

Beta vulgaris L. is a root vegetable that is consumed mainly as a food additive. This study aimed to describe the protective effect of B. vulgaris on Fe2+-mediated oxidative liver damage through in vitro, ex vivo, and in silico studies to establish a strong rationale for its protective effect. To induce oxidative damage, we incubated the livers of healthy male rats with 0.1 mM FeSO4 to induce oxidative injury and coincubated them with an aqueous extract of B. vulgaris root (BVFE) (15–240 µg/mL). Induction of liver damage significantly (p < .05) decreased the levels of GSH, SOD, CAT, and ENTPDase activities, with a corresponding increase in MDA and NO levels and Na+/K+ ATPase, G6 Pase, and F-1,6-BPase enzyme activities. BVFE treatment (p < .05) reduced these levels and activities to almost normal levels, with the most prominent effects observed at 240 µg/mL BVFE. An HPLC investigation revealed sixteen compounds in BVFE, with quercetin being the most abundant. Chlorogenic acid and iso-orientation showed the highest binding affinities for G6 Pase and Na+/K + ATPase, respectively. These findings suggest that B. vulgaris can protect against Fe2+-mediated liver damage by suppressing oxidative stress and cholinergic and purinergic activities while regulating gluconeogenesis. Overall, the hepatoprotective activity of this extract might be driven by the synergistic effect of the identified compounds and their probable interactions with target proteins.

The role of phytochemicals in food, health, and pharmaceuticals is steadily becoming a ‘big bang’, as there is much current research and published scientific findings supporting the medicinal efficacy of phytochemicals in food or isolation1. Scientists have reported that beet (Beta vulgarisL.) originated from northern Africa and eastern Europe and is a member of the Chenopodiaceae family of Amaranthaceae. It is a rich source of various phytochemical and biologically functional compounds that improve health, such as phenolics, epicatechin, catechin hydrate, betalains, ferulic acid, caffeic acid, and dietary fiber, and has proven health benefits for humans2,3. Roots are considered the most significant part of the beet plant and are commonly eaten and widely used both industrially and domestically. Due to its attractive color and variety of nutrients and phytochemicals, beetroot has received increasing attention from researchers and the food industry. We refer to it as a superfood because of its abundant antioxidant and vitamin contents, which confer enormous health-promoting benefits to plants2,3. Studies have shown that beetroot harvested at optimal maturity has a high antioxidant capacity, with variations in betalain content influencing this property4,5,6. Furthermore, beetroot has been ranked among the top ten most potent antioxidant vegetables, with research confirming its high total antioxidant capacity when evaluated using extraction-based methods5,6. Beet consumption has been considered an adjunct therapy in the management of several disease conditions associated with inflammation and oxidative stress7. Some common uses of beetroot include the formulation of functionalized products such as wine, juices, gels, and dehydrated powders (capsules or tablets). Researchers have also used it in folkloric medicine for blood pressure regulation2. Beetroot ranks among the top ten most powerful vegetables for its total phenolic and antioxidant capacity7. This is because direct comparisons of antioxidant activity in beetroot-based products have been conducted, revealing correlations between antioxidant potential and nitrite and nitrate content, which are markers of beetroot’s health-promoting properties8,9. These findings provide a more detailed context for understanding the health benefits of beetroot and support the claims made about its powerful antioxidative effects. Betalains and large amounts of phenolics in beet have been linked to its therapeutic potential in the treatment of diseases, showing outstanding chemopreventive, anti-inflammatory, and antioxidant activities in both in vivo and in vitro models2.

The liver plays an essential role in the metabolic function of the body because it is involved in detoxification and excretion functions10. It is also a key component in the metabolism of macromolecules such as proteins, fats, and carbohydrates because it regulates the homeostasis of key molecules and nutrients in the body10.

Iron is an essential micromineral that performs several functions, including being involved in various metabolic reactions and serving as a cofactor for several enzymes involved in most biological functions, due to its ability to assume both distinct ferric and ferrous ionic states10. This ability to exist in two ionic states makes iron a key player in maintaining redox balance. The liver is involved in iron homeostasis, which, when impaired, results in iron imbalance, which is the root cause of iron-related disorders such as anemia and hemochromatosis. These two iron-related conditions are the predominant iron disorders and affect more than one billion people worldwide11. In the case of iron overdose, the liver protects other vital organs, such as the heart and pancreas, from iron-induced cellular damage by increasing iron storage. This chronic elevation in iron storage in the liver (iron overdose) could result in liver injury by increasing the generation of free radicals that can overwhelm the antioxidant defense system, leading to liver oxidative damage. Oxidative damage has been linked to the onset and progression of numerous liver diseases, including fibrosis, liver cancer, and cirrhosis11. Previous studies have demonstrated that beetroot extract has potent antioxidant properties and can protect against oxidative stress-associated liver damage12,13, Beta vulgaris L.) protect Against High-Fat Diet-Induced oxidative damage in the liver in mice. Nutrients. 10 (7), E872 (2018)." href="#ref-CR14" id="ref-link-section-d8508453e827_2">14,15. For instance, beetroot has been reported to have hepatoprotective effects in animal models fed high-cholesterol diets, suggesting its role in modulating metabolic pathways related to liver health12. In addition, beetroot extracts have been reported to exhibit anti-inflammatory and antimicrobial activities, which may contribute to their protective effects against hepatic oxidative damage15,16,17. However, these studies have not fully explored the specific metabolic pathways involved in oxidative liver injury, such as gluconeogenesis, cholinergic signaling, and purinergic signaling pathways. Despite the few pharmacological activities reported on beetroot, there is little information on its effects on metabolites and pathways involved in oxidative-mediated injury in liver tissues. The current study aimed to bridge this knowledge gap by providing a detailed analysis of how beetroot extract influences these pathways and modulates their specific functions in the context of iron-induced liver damage. This is achieved through a combination of in vitro, ex vivo, and in silico approaches, offering a novel approach to understanding the protective mechanisms of beetroot against liver toxicity by restoring antioxidative metabolic pathways, modulating gluconeogenesis, and reducing cholinergic and purinergic dysfunction in oxidative liver injury.

The antioxidant activity of the aqueous Beta vulgaris root extract (BVFE) was determined as the mean percentage of inhibition and compared to that of standard ascorbic acid (Fig. 1). The scavenging potential of BVFE for hydroxyl (OH-), nitric oxide (NO), and DPPH radicals and its ferric (Fe3+)-reducing antioxidant ability compared favorably with those of the standard antioxidant (ascorbic acid), showing that BVFE effectively scavenged free radicals.

BVFE significantly scavenged (p < .05) the hydroxyl radical (•OH) with the best activity at 240 µg/mL (Fig. 1A), with the •OH scavenging activity of the extract having a higher IC50 value (108.42 µg/mL) than that of the standard ascorbic acid with a lower IC50 (88.94 µg/mL). BVFE significantly scavenged (p < .05) •NO radicals in a concentration-dependent manner (Fig. 1B). The NO scavenging potential of standard ascorbic acid was lower (IC50 = 22.21 µg/mL) than that of BVFE (IC50 = 90.88 µg/mL) (Fig. 1B). Figure 1C shows the DPPH scavenging activity of BVFE at different concentrations. BVFE revealed a concentration-dependent increase in DPPH free radical scavenging activity, with an IC50 value of 115.50 µg/mL compared to ascorbic acid, with an IC50 value of 82.29 µg/mL. The Fe3+ reducing antioxidant power (FRAP) of BVFE showed a gradient increase (p < .05) at concentrations up to 240 µg/mL, with an IC50 value of 41.22 µg/mL, compared with that of ascorbic acid, which had a lower IC50 (24.60 µg/mL) (Fig. 1D).

Antioxidant effects of BVFE on (A) hydroxyl, (B) NO, (C) DPPH, and (D) ferric reducing power. The data are expressed as the means ± SDs (n = 3). Legends: BVFE: Beta vulgaris root extract; NO: nitric oxide; DPPH: 1,1-diphenyl-2-picrylhydrazyl.

The iron chelating activity is dependent on the absorbance of the iron(II)–ferrozine (Fe(II)(FZ)3) complex, which produces a red chromophore with a maximum absorbance at 562 nm. Chelating agents can capture iron II (Fe2+) before the ferrozine complex is formed. The Fe2+ chelating activity and total antioxidant capacity of aqueous BVFE using ethylene diamine tetraacetic acid (EDTA) and α-tocopherol as the respective standards are presented as % inhibition, as shown in Fig. 2. As the concentration of BVFE increased (IC50 = 80.65 µg/mL), the ability to scavenge metal ion-chelating radicals increased (p < .05) to a degree similar to but slightly lower than that observed for standard EDTA (IC50 = 10.49 µg/mL) (Fig. 2A). We also observed a similar trend for the total antioxidant scavenging activity of BVFE, in that the extract exhibited concentration-dependent antioxidant activity, as did standard α-tocopherol (Fig. 2B). The inhibition of TAC was greatest for the extract with a lower IC50 of 171.82 µg/mL than for the standard with an IC50 value of 182.37 µg/mL.

Iron chelation ability and total antioxidant capacity of B. vulgaris root extracts. The data are expressed as the means ± SDs (n = 3). Legends: BVFE: Beta vulgaris root extract.

As shown in Fig. 3A-D, FeSO4 treatment caused oxidative liver injury, which resulted in a significant (p < .05) decrease in CAT activity, SOD activity, and GSH levels, with a corresponding increase in MDA levels. However, treatment with BVFE resulted in a considerable reversal (p < .05) of enzyme activities and close to normal levels of GSH and MDA.

Effect of B. vulgaris roots on (A) catalase (B) superoxide dismutase, (C) reduced glutathione, and (D) malondialdehyde levels in oxidative hepatic damage. The data are expressed as the mean ± SD of triplicate samples. # Values are significantly different ( p  < .05) from those of normal liver tissues; * values are significantly different ( p  < .05) from those of FeSO 4 -induced liver tissues.

Figure 4 shows the inhibitory impact of BVFE on the generation of nitric oxide (NO) in FeSO4-induced hepatotoxicity. Unlike normal tissues, animals treated with FeSO4 produced no nitric oxide; however, BVFE effectively reduced nitric oxide production at different doses in treated animals. However, the inhibitory effect was not concentration dependent (p < .05), with the highest levels of inhibition observed at 15, 30, and 240 µg/ml.

Effect of B. vulgaris roots on nitric oxide levels in oxidative hepatic damage. The data are expressed as the mean ± SD of triplicate samples. # Values are significantly different ( p  < .05) from those of normal liver tissues; * values are significantly different ( p  < .05) from those of FeSO 4 -induced liver tissues.

The inhibitory effect of aqueous BVFE on the activity of acetylcholinesterase (AChE) in oxidative hepatotoxicity was evaluated, and the results are shown in Fig. 5. The experiment showed that the untreated rats had increased AChE activity (p < .05). However, we observed a significant decrease in the number of animals treated with various doses of plant extract (p < .05). The inhibitory effect of the extract on enzyme activity was dose dependent, as shown in the figure; it increased as the concentration of the extract decreased, with the greatest inhibitory effect recorded at 240 µg/ml.

Effect of B. vulgaris on acetylcholinesterase activity in oxidative hepatic injury. The data are expressed as the mean ± SD of triplicate samples. # Values are significantly different ( p  < .05) from those of normal liver tissues; * values are significantly different ( p  < .05) from those of FeSO 4 -induced liver tissues.

We studied the effect of BVFE on the activities of ATPase and ENTPDase in oxidative liver injury, as shown in Fig. 6. Following the induction of oxidative hepatotoxicity, there was a significant (p < .05) increase in liver ATPase activity (Fig. 6A), with a corresponding decrease in ENTPDase activity (Fig. 6B). Treatment with BVFE resulted in a substantial (p < .05) reversal of enzyme activity to near-normal levels, indicating an inhibitory effect.

Effect of B. vulgaris on (A) adenylpyrophosphatase and (B) ectonucleoside triphosphate diphosphohydrolase in oxidative hepatic damage. The values are expressed as the means ± SDs of triplicate samples. # Values are significantly different ( p  < .05) from those of normal liver tissues; * values are significantly different ( p  < .05) from those of FeSO 4 -induced liver tissues.

Figure 7 shows the effect of aqueous BVFE on the activities of G6 Pase and F-1,6-BPase in hepatic injury. FeSO4-induced hepatotoxicity (Fig. 7A and B) significantly increased G6 Pase and F-1,6-BPase activities, indicating that hyperglycemia occurred via increased gluconeogenesis. When these enzymes were treated with the extract, their activity was considerably restored (p < .05) to near normal levels. The extract became more potent as its concentration increased, with the maximum reversal ability documented at 240 g/ml.

Effect of B. vulgaris on (A) G6 Pase and (B) F-1,6-BPase activities in hepatic injury. The values are expressed as the means ± SDs of triplicate samples. # Values are significantly different ( p  < .05) from those of normal liver tissues; * values are significantly different ( p  < .05) from those of FeSO 4 -induced liver tissues.

Chromatograms obtained at different retention times from HPLC analysis of the aqueous extract of B. vulgaris root (Figure S1) revealed several constituents: chlorogenic acid, orientin, kaempferol, cryptochlorogenic acid, taxiphyllin, quercetin, apigenin, schaftoside, luteolin, catechin, iso-orientationin, 6-glucosylapigenin, alpha-amyline, ferulic acid, p-coumaric acid, and benzoic acid (Table 1).

The reliability of both predictions was quite reliable, as evidenced by the local distance difference test (pIDDT) values of up to 95 (out of 100, not shown). The α-helical elements were predicted with very high precision, as shown by the color coding of the predicted structure (from red to blue) (Fig. 8a and b). Blue (corresponding to pIDDT scores of 90 and higher) shows very good precision, equal to experimentally established structures, allowing us to study the details of individual side chains.

Homology modeling of 3D structures of (a) glucose-6-phosphatase and (b) Na+/K+ ATPase.

Using the Jupyter notebook method to construct the Ramachandran plot ensured the quality of these models. The results of the Ramachandran plots revealed 96.8% and 85.9% of residues in the most favorable regions of glucose-6 phosphatase and Na+/K+ ATPase, respectively, showing that the models are of good quality (Fig. 9a and b).

Ramachandran plots of the prepared proteins indicating (a) glucose-6-phosphatase and (b) Na+/K+ ATPase.

We molecularly docked the bioactive chemicals found with G6 Pase and Na+/K + ATPase to better understand the role of B. vulgaris extracts in moderating liver toxicity. We then subjected the lead compounds of the docked compounds to further studies via molecular dynamics simulation. The binding site of the modeled G6 Pase comprises catalytic residues, as presented in Table 2.

The hit compound, chlorogenic acid, has a binding energy of -9.3 kcal/mol, which is the highest of the docked ligands (Table S1). This high binding energy (-9.3 kcal/mol) is believed to result from the chemical interactions of chlorogenic acid at the active site of the receptor (Table S2; Fig. 10), which include three (3) hydrogen bonds involving residues LYS-76 and LYS-263; three (3) hydrophobic interactions involving residues TYR-44, ARG-40, and LYS-263; and one (1) electrostatic interaction involving the LYS-263 residue.

(a) 3D and (b) 2D interactions within the binding site of G6 Pase.

The binding site of the modeled Na+/K+ ATPase comprises a catalytic residue, as presented in Table 3.

The hit compound, isoorientin, has a binding energy of -8.5 kcal/mol, which is the highest of the docked ligands (Table S3). This binding energy is the result of chemical interactions at the active site of the receptor (Table S4; Fig. 11), which includes six (6) hydrogen bonds involving the ARG-184, TYR-239, LYS-109, and THR-240 residues and five (5) hydrophobic interactions involving the LYS-112, PHE-113, and PRO116 residues.

(a) 3D and (b) 2D interactions within the binding site of the Na+/K+ ATPase.

The first step in the analysis of 30 ns molecular dynamics (MD) simulations was to assess conformational stability based on the backbone root mean square deviation (RMSD). We assessed the stability of the hit compound (chlorogenic acid) along with G6 Pase using MD studies. We carried out MD simulations for 30 ns for this system. The RMSD was calculated using C-alpha atoms, and the RMSF graphs are shown in Fig. 12a and b. These analyses showed that the simulation returned less than 3 Å RMSD fluctuations (Fig. 12a), indicating conformational stability. As a result, we decided to conduct more detailed analyses using the simulation’s stable frames.

RMSD and RMSF plots of C-alpha atoms in G6 Pase.

Figure 13a and b shows the molecular interaction between the protein and the compound. After the 30 ns simulations, we observed an increase in both the number of hydrogen bonds (hydrogen bond donors) and the number of pi-cations.

Molecular interaction between G6 Pase and chlorogenic acid.

We computed the free binding energy of the ligand in the docked complex using the entire trajectory obtained during the MD simulations. The free binding energies were calculated and are shown in Table S5. The complex had negative total solvation energies (ΔG (Solvation)) and negative total gas phase molecular mechanics energies (ΔG (Gas)), showing that they were favorable (Table S5). For the chlorogenic acid binding energies, the sum of the total polar contribution (ΔE (EEL) + ΔE (EPB)) remained numerically negative, indicating a favorable polar contribution to the relative binding energy in the complex (-28438.99 kcal/mol) (Table S5). Distance analyses were performed between the center of mass of selected key residues and the center of mass of chlorogenic acid to support the MM-PBSA observations (Figure S2a and b).

The evaluation of the conformational stability of the RMSD-based backbone was the first step in the analysis of the 30 ns MD simulations (Fig. 14). These analyses revealed that in the last 3 ns of MD simulations, all the simulations returned to approximately 10 RMSD fluctuations (Fig. 14), indicating a stable conformation. As a result, we were able to use the stable frames of the simulations for more detailed analyses.

RMSD graphs of the C-alpha atoms of Na+/K+ ATPase.

The last 3 ns of the stable conformation of the MD simulation were used to estimate the binding energy between the hit compound (isoorientin) and Na+/K+ ATPase following the determination of conformational stability. In the current system studied, the loop regions (residues 0–9, 22–33, 66–81, 86–91, and 97–99) and the helical regions (residues 10–21 and 34–65) with a few beta sheet regions (residues 82–85 and 92–96) were mobile and facilitated ligand binding (Fig. 15a and b).

RMSF graphs of the C-alpha Na+/K+ ATPase.

We computed the free binding energy of the ligand in the docked complex using the entire trajectory obtained during the MD simulations. The AMBER MMPBSA tool computes the binding energy using the MM/PBSA approach. Poisson-Boltzmann calculations were performed employing the internal PBSA solver in MMPBSA_py_energy, as shown in Table S6. The complex showed negative total solvation energies (ΔG (Solvation)) and negative total gas phase molecular mechanics energy (ΔG (Gas)), showing that they were favorable (Table S6). For the isoorientin binding energies, the sum of the total polar contribution (E (EEL) + E (EPB)) remained numerically negative, indicating a favorable polar contribution to the relative binding energy in the complex (-23520.2923 kcal/mol) (Table S6). In contrast, when the total nonpolar contribution was analyzed, the isoorientin-Na+/K+ ATPase complex showed a clear ~ 18-fold decrease compared to the total polar contribution (-1280.1755 kcal/mol) (Table S6). Distance analyses were performed between the center of mass of selected key residues and the center of mass of isoorientin in support of the MM-PBSA observations (Figure S3).

The results of various assays used to measure the antioxidant activities of plant extracts show a direct relationship between plant bioactive compounds and antioxidant activities18,19,20,21. BVFE was found to successfully scavenge DPPH, hydroxyl, and nitric oxide radicals in this study. Furthermore, the strong dose-dependent FRAP activity of BVFE observed in this work demonstrated the ability of BVFE to decrease Fe3+ to Fe2+ in vitro. This finding is consistent with previous research on the antiradical and antioxidant properties of Beta vulgaris22,23,24.

Nitric oxide (NO) is a cellular signaling molecule that plays a significant role in physiological and pathological processes. When it reacts with superoxide (O2−) in the epithelium, it can cause high blood pressure and oxidative damage to nucleic acids because of the formation of peroxynitrite (ONOO−), which has the propensity to cause severe damage to the supercoiled DNA helix25. In this study, BVFE showed a strong tendency to inhibit the production of NO, with concentrations having little or no effect on this activity. This is most likely because of the high concentrations of bioactive constituents in B. vulgaris21,26.

Ferrous ions (Fe2+) contribute to the formation of reactive oxygen species (ROS), which can catalyze lipid peroxidation through the Fenton and Haber-Weiss reactions, in which Fe2+ reacts with H2O2 to produce highly reactive •OH. These •OH radicals can adversely affect important macromolecules, including nucleic acids, lipids, and proteins. Chelating potential is regarded as an important measure of antioxidant activity. The reducing power of an antioxidant can be assayed by the absorbance of the red color produced by the reduction of Fe2+ in the Fe(II)(FZ)3 complex as a result of the coexisting chelator capturing the Fe2+before the formation of the ferrozine complex27. In this study, BVFE was able to chelate Fe2+in a dose-dependent manner, with the highest chelating power recorded at 240 µg/ml. Similarly, BVFE showed a strong total antioxidant capacity in a dose-dependent manner, competing favorably with the standard. This chelating ability and antioxidant activity could be attributable to the presence of some phytochemicals, including phenolics, in the extract. Hence, the potential of phenolics, especially flavonoids, to chelate powerful pro-oxidant metal ions, including iron, enhances their antioxidant capabilities. Flavonoids can form stable metal complexes through their multiple hydroxyl groups and carbonyl moieties28,29. Our findings correlate with an earlier report by Babagil et al23. on B. vulgaris. It is apparent from this study that B. vulgaris possesses chelating and antioxidant activities and that this could play a significant role in protecting against oxidative damage stimulated by metal-catalyzed decomposition reactions30,31,32,33. This could be due to the presence of flavonoid compounds in the extract.

The liver, as the main detoxifying organ, helps to maintain metabolic homeostasis. The organ metabolizes various compounds that produce ROS, that is, oxygen-containing free radicals34, causing the liver, especially liver parenchymal cells, to be more vulnerable to oxidative stress. Disequilibrium in the levels of pro-oxidants and antioxidants causes oxidative stress, which has been linked to the pathogenesis and development of hepatic toxicity and thus to ailments such as liver fibrosis and cirrhosis and ultimately to liver cancer35,36,37,38. In this study, treatment of the liver with FeSO4 caused oxidative injury and significantly reduced CAT and SOD activities, as well as GSH levels, with an accompanying increase in MDA levels, indicating the development of oxidative stress and inflammation. When BVFE was administered, significant reversion of these alterations caused by FeSO4 was detected, confirming the resolving effect of the extract on hepatic oxidative stress. This finding supports earlier studies on the hepatoprotective activity of B. vulgaris39,40,41,42.

Free radicals are molecular species that have unpaired electrons in their atomic orbitals. This feature makes them unstable and highly reactive, and it also gives them the ability to either donate or accept electrons from other molecules, allowing them to operate as oxidants or reductants. They play an important role in a variety of biological processes that are essential for life. Excessive generation of free radicals such as O2•− and •OH can cause oxidative stress in cells and has been linked to the development of a variety of metabolic and chronic diseases, including stroke, cancer, and diabetes43,44. Medicinal herbs have been shown to inhibit the development of oxidative stress by scavenging free radicals45,46,47.

An impairment in hepatic cholinergic function, as shown by increased AChE activity, has been linked to the pathogenesis of hepatic toxicity because of the ability of Fe2+to induce an inflammatory response in hepatocytes48. We regard hepatic inflammation as the primary cause of hepatic injury, causing the disease to progress from nonalcoholic fatty liver disease (NAFLD) to severe fibrogenesis and, ultimately, to hepatocellular carcinoma49. The relationship between the elevated AChE activity observed in this study and the activation of oxidative damage confirms the development of oxidative stress and a higher level of NO because an increase in oxidative stress and inflammation is indicative of enhanced AChE activity48,49. Treatment with BVFE significantly repressed AChE activity in a concentration-dependent manner, showing that the extract has anti-inflammatory and protective effects against cholinergic dysfunction in oxidative hepatic toxicity. This is probably because of the ability of BVFE to enhance AChE phosphorylation at serine residues50. The increasing activity in hepatic tissues treated with increasing concentrations of BVFE revealed that the extract is very active at relatively high concentrations. Our results are consistent with earlier reports on the hepatoprotective effect of Cannabis sativa48. This could be due to the presence of flavonoid compounds reported to possess anticholinergic properties.

Purines and purine nucleotides are biological molecules that play crucial roles in a variety of metabolic processes, including nucleotide synthesis, cell proliferation, differentiation, and death, and serve as ligands for purinergic receptors51,52. Adenosine triphosphate (ATP) and its hydrolytic products, comprising adenosine, adenosine monophosphate (AMP), and adenosine diphosphate (ADP), serve as general intracellular energy-carrying molecules for various biological reactions as well as extracellular signaling molecules that, with other nucleotides and nucleotide sugars, help to regulate several processes, such as neurotransmission, blood coagulation, neurological diseases, and diabetes48,53,54.

The liver is a vital organ in the body that performs different functions, including carbohydrate metabolism, lipid and protein synthesis, bile secretion, and detoxification55. It is also where nucleotide synthesis occurs (salvage or de novosynthesis). Hepatic toxicity has been linked to disruptions in purinergic enzyme activity in the liver56. The induction of oxidative injury resulted in a considerable increase in ATPase activity and a decrease in ENTPDase activity in the animals, as reported in this study. Elevated ATPase activity was associated with a reduction in hepatic ATP activity, indicating altered bioenergetic activity, since ATP is hydrolyzed to adenosine, whereas decreased ENTPDase activity indicates adenosine depletion57. After treatment with BVFE, we observed a significant reversal of these activities, with decreased ATPase activity and elevated ENTPDase activity and improved bioenergetics. This is probably because of the regulatory effect of the extract on the activities of purinergic enzymes in oxidative-mediated hepatic toxicity. The levels of ATPase and ENTPDase activities in the hepatic tissues of untreated animals corroborate an earlier report on altered purinergic activities in oxidative injuries48.

FeSO4-stimulated oxidative hepatic injury in the animal substantially heightened the activities of the carbohydrate-metabolizing enzymes G6 Pase and F-1,6-BP, resulting in increased gluconeogenesis, which can lead to hepatic hyperglycemia because of an elevated concentration of glucose in the liver58,59. Hepatic hyperglycemia can induce chronic damage and injury to the liver since excess glucose can be transferred and used as fuel for other pathogenic pathways, including advanced glycation end-product (AGE) formation, polyol, or protein kinase C (PKC) pathways48,60. Hence, it is crucial to achieve good glycemic control to prevent or delay hepatic injury and disease complications. Treatment with BVFE significantly (p< .05) reduced both G6 Pase and F-1,6-BPase activities, probably through the endogenous production of glucose and the alteration of gluconeogenic flux61. The hypoglycemic effect of the extract implies a reduction in glucose for oxidation and the oxidative pathways mentioned above45,62.

HPLC analysis of BFE confirmed sixteen bioactive compounds. Quercetin had the highest concentration, while alpha-amyline had the lowest. The antioxidant and hepatoprotective activities of phenolics have been documented in the literature21,47,63.

We employed an artificial intelligence approach, alphafold264, which is available as a Google Colab notebook, to predict the respective structures since mouse model 3D structures of glucose-6 phosphatase and Na+/K+ATPase are not available. Several assessment approaches, including a Ramachandran plot, were utilized to examine the quality and dependability structure of the models65. We checked the stereochemical quality of each protein using the Procheck server66. This is achieved by examining the residue-by-residue geometry and overall structure geometry. A model with more than 85% residues in the most favorable region is regarded as an excellent quality model67.

Over the years, much biological understanding has been gathered concerning the involvement of G6 Pase in the enzyme complex involved in both glucose homeostasis and type 2 diabetes. G6 Pase, which is mostly found in the liver, catalyzes the final step in both gluconeogenesis and glycogenolysis68. The absence of G6 Pase, which regulates the release of the simple sugar glucose from glycogen stored in the liver, results in an aberrant accumulation of glycogen in the liver, causing the liver to expand and generating symptoms of hypoglycemia and hyperuricemia (gout)69. It is therefore reasonable to think that enhancing the activity of G6 Pase would be a sound pharmacological approach. The LYS-263 residue appears to be central to the molecular catalytic mechanism of G6 Pase48. This residue is one of the catalytic residues identified in the G6 Pase binding site. Lysine is required for normal growth and plays a vital role in the production of carnitine, a nutrient that aids in the conversion of fatty acids into energy and lowers cholesterol70. In addition, chlorogenic acids (CGAs) have been shown to lower blood pressure and body fat while also improving fat metabolism71. Therefore, we predicted that it would have a greater binding energy than the other studied ligands and would serve as a potent activator of G6 Pase.

The sodium-potassium pump (Na+/K+ ATPase) establishes Na+ and K+concentration gradients across the biomembrane and thus plays an important role in the generation of action potentials72,73,74.

The RMSD of G6 Pase in association with chlorogenic acid reached equilibrium at approximately 25 ns when it reached 18. Å and remained constant throughout the 30 ns simulation time. Residues with RMSF values greater than 0.2 nm (2Ao) are deemed mobile75. The N-terminus (residues 0–10) and C-terminus (residues 340–357) are mobile in the current system investigated and aid in ligand binding. Based on theoretical and experimental correlations between H-bond pairings and their effects on ligand binding affinity, it was shown that H-bonds enhance receptor‒ligand interactions when both the donor and acceptor have either significantly stronger or significantly weaker H-bonding capabilities than the hydrogen and oxygen atoms in water76. Therefore, chlorogenic acid has a stronger binding affinity for G6 Pase than for the other ligands that are docked in its binding site.

We computed the free binding energy of the ligand in the docked complex using the entire trajectory obtained during MD simulations. Overall, polar interactions were the major contributors to the binding energy between chlorogenic acid and the G6 Pase active site. The amino acid residues at the first 10 ns of the MD simulation through the MM-PBSA method remained almost 1-fold greater than those of chlorogenic acid during the last 20 ns of the MD simulations. It is worth mentioning that the nonbonded interaction, which determines the strength of the ligand‒protein bonding, was computed. We found that the total interaction energy was greater for the first 10 ns than for the remaining 20 ns. We may associate this with the bond distance between the chlorogenic acid and the amino acid residue. However, further study is required to characterize this bond.

Following the assessment of conformational stability, the last 3 ns of the stable conformation of the MD simulations were utilized to estimate the binding energy between the hit compound (isoorientin) and the Na+/K+ ATPase. Residues with RMSF values greater than 0.2 nm (2Ao) are thought to be mobile75.

MD simulations were used to compute the free energy of the ligand. The amino acid residues obtained in the first 20 ns of the MD simulation remained almost half a-fold greater than those of iso-orientin during the last 10 ns. Notably, the nonbonded interaction, which determines the strength of the bonding interaction between the ligand and the protein, was computed. We found the total interaction energy to be the same throughout the 30 ns of the MD simulation.

Finally, the findings suggest that B. vulgaris protects against Fe+-induced liver damage by suppressing oxidative stress, modulating gluconeogenesis, and enhancing cholinergic and purinergic enzyme activity. In addition, in vivo investigations to elucidate the mechanism of action of this plant are now being conducted in our laboratory.

In December 2021, beetroot plants were purchased from the Jos Terminal Market, Plateau State, Nigeria. Experimental research and field studies on plants (either cultivated or wild), including the collection of plant material, complied with relevant institutional, national, and international guidelines and legislation. The plant was verified at the Forestry Research Institute of Nigeria in Ibadan with herbarium number FHI 114,105.

The plants were thoroughly cleaned, cut into small pieces, and allowed to dry before being ground with an industrial miller into powder. A total of 400 mL of water was steeped for 48 h with 50 g of beetroot powder. Muslin cloth was used to sieve the solution, which was concentrated in a steam bath at 45 °C. A yield of 34.83 g was obtained.

The NO scavenging ability of B. vulgaris root extract (BVFE) was assessed using the procedure described by Alam et al.[77]. The percentage inhibition was calculated as follows:

The DPPH activity of BVFE was assessed following the procedure described by Mzoughi et al.78. Ascorbic acid was used as a standard in this experiment. Ascorbic acid was used as the standard in this study. The percentage inhibition was calculated as follows:

The FRAP of BVFE was determined by the procedure described by Mzoughi et al.78. In brief, 250 µL of various concentrations of the plant sample were added to 0.625 mL of 0.2 M phosphate buffer (pH 6.6) and 0.625 µL of 1% K3Fe(CN)6. Following incubation at 500 °C for 20 min and subsequent cooling of the reaction mixture at room temperature, 0.625 mL of 10% trichloroacetic acid was added to halt the reaction. Afterwards, the mixture was centrifuged at 2000 × g for 10 min; 0.625 mL of the supernatant was pipetted into a clean test tube containing 0.625 ml of distilled water and 125 µl of 1% FeCl3. The mixture was allowed to stand for 10 min, and the absorbance was read at 700 nm against a blank.

The iron (Fe) chelating ability of BVFE was determined using the methods described by Alam et al.77 with slight modifications. Approximately 500 µL of 0.2 mM FeCl3 was added to 100 µL of varying concentrations of the beet extract and standard (EDTA). The reaction was activated by adding 200 µL of 5 mM ferrozine to the mixture, which was subsequently incubated at 25 °C for 10 min. The absorbance was measured at 562 nm.

This study was carried out using the method described by Olofinsan et al.79 The reaction mixture was prepared by sequentially reacting 100 µL of beet extract with 150 µL of 20 mM deoxyribose, 250 µL of phosphate-buffered saline, 100 µL of 500 µM FeSO4 and 100 µL of 1% H2O2. The mixtures were incubated for 30 min at 37 °C, after which 200 µL of 10% TCA and 600 µL of 0.25% TBA were added. Afterwards, the mixture was boiled for 20 min and then left to cool at room temperature. Ascorbic acid was used as the control solution. The absorbance was read at 532 nm, and the radical scavenging activity was calculated using the following formula:

The TAC of the BVFE was assessed following the procedure described by Rahman et al.80 A total of 250 µL of varying concentrations of beet samples and standard (α-tocopherol) were mixed in test tubes with 1.5 mL of a reaction mixture containing 0.6 M H2SO4, 0.028 M sodium phosphate, and 1% ammonium molybdate. The test tubes were then incubated at 95 °C for 10 min to complete the reaction. After cooling at room temperature, the absorbance was read at 695 nm against a blank solution containing 250 µL of the solvent used for the samples and 1.5 mL of the reaction mixture.

Five healthy male Wistar rats weighing 150–200 g were obtained from the Department of Biochemistry, Landmark University. The animals’ liver tissue was harvested and washed in 0.9% normal saline to remove blood. They were homogenized in 1% Triton X-100 in 50 mM potassium phosphate buffer (pH 7.2) and then centrifuged at 15,000 rpm at 4 °C. The supernatants were collected for ex vivo experiments. The study was conducted under the approved procedures of the Animal Ethics Committee of Landmark University, Nigeria (LUAC/BCH/2022/0001A), and reported according to the ARRIVE guidelines. We confirm that all methods were performed and approved by the institution’s ethical committee and reported according to the ARRIVE guidelines.

Liver toxicity was induced ex vivo using Fe2+, as defined by Erukainure et al.81 Briefly, 100 µL of 0.1 mM FeSO4 was mixed with 200 µL of tissue lysate containing different concentrations (15–240 µg/mL) of plant extract. The samples were incubated for 30 min at 37 °C before being used for biochemical evaluations. Reaction mixtures comprising only tissue supernatant were used as normal controls, while a solution of only tissue supernatant and FeSO4 was defined as FeSO4-induced.

The liver tissue was analyzed for catalase activity81, reduced glutathione levels by Salau et al.82, superoxide dismutase by Ajiboye et al.83, and the level of lipid peroxidation using the method of Erukainure et al.81.

The methods outlined by Olofinsan et al.79 and Erukainure et al.84 were used to measure acetylcholinesterase, ATPase, and ectonucleoside triphosphate diphosphohdrolase (ENTPDase) activities.

The liver tissues were analyzed for glucose-6-phosphatase (G6 Pase) activity following the method described by Balogun and Ashafa85 and F-1,6-BPase activity following the procedures described by Balogun and Ashafa85 and Olofinsan et al.79 in the supernatants.

The HPLC method described by47 was used. Beta vulgaris roots at 10 mg/ml were injected with a SIL-20 A Shimadzu Auto model sampler, which produced a chromatogram with a specified peak area. We performed all chromatography operations at 25 °C in triplicate.

The G6 Pase and Na+/K+ATPase sequences were obtained from the NCBI database using the accession numbers AAC52122.1 and sp|P14231.2|AT1B2, respectively. Threading, ab initio, and homology modeling are used to predict the 3D structure of proteins86. We used the Jupyter notebook to run homology modeling using the AlphaFold2 pipeline. Quality assessments were performed using the PROCHECK server.

The chemical structures of sixteen (16) phytochemicals (from aqueous extracts of B. vulgaris roots) were recovered from the PubChem compound database (https://pubchem.ncbi.nlm.nih.gov). To create atomic coordinates, we converted the MOL SDF format of these ligands to a PDBQT file using the PyRx program. On PyRx, the energy was minimized using the optimization procedure with the force field set to mmff94 (required) on PyRx.

After the receptor and ligands were prepared, we used PyRx’s AutoDock Vina feature to perform molecular docking analysis based on the scoring functions. To determine the binding site, the grid box resolution was centered at −2.7445 × −0.957 × −0.6017 (for G6 Pase) and 3.5902 × 3.7892 × −0.9628 (for Na+/K+ATPase) along the x, y, and z axes, respectively, at a grid dimension of 25 × 25 × 25 Å. Pymol was used to view the best docked complexes, and Accelrys Discovery Studio Visualizer was used to access and visualize their interactions87.

To understand the structural behavior of the docked complexes, a Jupyter notebook was used that uses the OpenMM engine and AMBER force field for protein models from the AlphaFold2 pipeline88to perform molecular dynamics simulations of the docked complexes. The steepest descent algorithm was used to minimize the energy of the docked complexes. For each simulation, 50,000 steps were employed to minimize energy. The LINCS algorithm89was used to limit bond lengths, while the PME method90 was utilized to compute long-range electrostatics. Finally, the production of an MD run was accomplished for 30 ns with a time step of 2 fs. The protein RMSD and RMSF were calculated using the built-in AMBER notebook. Thereafter, the binding free energy calculation was carried out.

The data are expressed as the means ± SDs of three determinations. GraphPad Prism 9 (GraphPad Software Inc., San Diego, California, USA) was used to statistically analyze the data via one-way analysis of variance (ANOVA) and the Duncan multiple range test (DMRT). The data were considered statistically significant at p < .05.

The data are available upon request from the corresponding author.

Aqueous extract of B. vulgaris root

Catalase

Ectonucleoside triphosphate diphosphohydrolase

Iron II sulfate

Fructose-1,6-bisphosphatase

Glucose-6-phosphatase

Glutathione

High-performance liquid chromatography

Malondialdehyde

Nitric oxide

Sodium potassium adenosine triphosphatase

Root mean square deviation

Superoxide dismutase

Molecular dynamics

Dillard, C. J. & German, J. B. Phytochemicals: nutraceuticals and human health. J. Sci. Food. Agric. 80 (12), 1744–1756. https://doi.org/10.1002/1097-0010(20000915) (2000).

Article CAS Google Scholar

de Oliveira, S. P. A., do Nascimento, H. M. A., Sampaio, K. B. & de Souza, E. L. A review on bioactive compounds of beet (Beta vulgaris L. subsp. Vulgaris) with special emphasis on their beneficial effects on gut microbiota and gastrointestinal health. Crit. Rev. Food Sci. Nutr. 61 (12), 2022–2033. https://doi.org/10.1080/10408398.2020.1768510 (2021).

Article CAS PubMed Google Scholar

Edziri, H. et al. Phytochemical analysis, antioxidant, anticoagulant and in vitro toxicity and genotoxicity testing of methanolic and juice extracts of Beta vulgaris L. South. Afr. J. Bot. 126, 170–175. https://doi.org/10.1016/j.sajb.2019.01.017 (2019).

Article CAS Google Scholar

Abarna, S. et al. Beetroot betalains and antioxidant potential: a function of Maturity Stage. Natl. Acad. Sci. Lett. 46, 457–460 (2023).

Article CAS Google Scholar

Carrillo, C. et al. Antioxidant capacity of Beetroot: traditional vs novel approaches. Plant. Foods Hum. Nutr. 72, 266–273 (2017).

Article CAS PubMed Google Scholar

Abarna, S. et al. Betalains Stability and antioxidant activity of Beetroots: as a function of Maturity Stage. Sugar Tech. 26 (1), 77–86 (2024).

Article Google Scholar

Erukainure, O. et al. Cannabis sativa L. (var. indica) Exhibits Hepatoprotective Effects by Modulating Hepatic Lipid Profile and Mitigating Gluconeogenesis and Cholinergic Dysfunction in Oxidative Hepatic Injury. Front. Pharmacol. Frontiers in pharmacology, https://doi.org/10.3389/fphar.2021.705402 (2021).

Brzezińska-Rojek, J. et al. Antioxidant capacity, Nitrite and Nitrate content in beetroot-based dietary supplements. Foods. 12, 1017 (2023).

Article PubMed PubMed Central Google Scholar

Mirmiran, P. et al. Functional properties of beetroot (Beta vulgaris) in management of cardio-metabolic diseases. Nutr. Metab. (Lond). 17, 3 (2020).

Article PubMed Google Scholar

Anderson, E. R. & Shah, Y. M. Iron homeostasis in the liver. Compr. Physiol. 3 (1), 315–330 (2013).

Article PubMed PubMed Central Google Scholar

Bendary, E., Francis, R. R., Ali, H. M. G., Sarwat, M. I., Hady, E. & S Antioxidant and structure–activity relationships (SARs) of several phenolic and aniline compounds. Annals Agricultural Sci. 58 (2), 173–181. https://doi.org/10.1016/j.aoas.2013.07.002 (2013).

Article Google Scholar

Al-Harbi, L. N. et al. Beta vulgaris L. (Beetroot) Methanolic Extract Prevents Hepatic Steatosis and Liver Damage in T2DM Rats by Hypoglycemic, Insulin-Sensitizing, Antioxidant Effects, and Upregulation of PPARα. Biology, 10, 1306. (2021).

El-Gendy, H. F. et al. Trial for decreasing ifosfamide-induced beetroot extract-induced hematological toxicity, oxidative stress, inflammation, and hepatotoxicity in male albino rats. Comp. Clin. Pathol. 31, 699–712 (2022).

Article CAS Google Scholar

Lorizolam, I. M. et al. Beet stalks and leaves (< i > Beta vulgaris L.) protect Against High-Fat Diet-Induced oxidative damage in the liver in mice. Nutrients. 10 (7), E872 (2018).

Article Google Scholar

Bashir, R. et al. Bioactive profile, pharmacological attributes and potential application of Beta vulgaris. Food Measure. 18, 3732–3743 (2024).

Article Google Scholar

Rehman, S., Mufti, I. U., Ain, Q. U. & Ijaz, B. in Bioactive Compounds and Biological Activities of Red Beetroot (Beta vulgaris L). Bioactive Compounds in the Storage Organs of Plants. Reference Series in Phytochemistry (eds Murthy, H. N., Paek, K. Y. & Park, S. Y.) (Springer, 2024). https://doi.org/10.1007/978-3-031-29006-0_42-1

Asgary, S. et al. Improvement of hypertension, endothelial function and systemic inflammation following short-term supplementation with red beet (Beta vulgaris L.) juice: a randomized crossover pilot study. J. Hum. Hypertens. 30, 627–632. https://doi.org/10.1038/jhh.2016.34 (2016).

Article CAS PubMed Google Scholar

Tavarini, S. & Angelini, L. G. Stevia rebaudiana Bertoni as a source of bioactive compounds: the effect of harvest time, experimental site and crop age on steviol glycoside content and antioxidant properties. J. Sci. Food Agricµlture. 93 (9), 2121–2129 (2013).

Article CAS Google Scholar

Zhao, H. X., Zhang, H. S. & Yang, S. F. Phenolic compounds and its antioxidant activities in ethanolic extracts from seven cultivars of Chinese jujube. Food Sci. Hum. Wellness. 3 (3–4), 183–190. https://doi.org/10.1016/j.fshw.2014.12.005 (2014).

Article Google Scholar

Ojo, O. A. et al. Antidiabetic activity of avocado seeds (Persea americana Mill.) In diabetic rats via activation of PI3K/AKT signaling pathway. Sci. Rep. 12, 2919. https://doi.org/10.1038/s41598-022-07015-8 (2022a).

Article ADS CAS PubMed PubMed Central Google Scholar

Zielińska-Przyjemska, M. et al. The beetroot component betanin modulates ROS production, DNA damage and apoptosis in human polymorphonuclear neutrophils. Phytother. Res. 26 (6), 845–852. https://doi.org/10.1002/ptr.3649 (2012).

Article CAS PubMed Google Scholar

Babagil, A., Tasgin, E., Nadaroglu, H. & Kaymak, H. C. Antioxidant and antiradical activity of beetroot (Beta vulgaris L. var. conditiva Alef.) grown using different fertilizers. Journal of Chemistry, 2018, Article ID 7101605. Doi: (2018). https://doi.org/10.1155/2018/7101605

Spiegel, M., Gamian, A. & Sroka, Z. Antiradical activity of beetroot (Beta vulgaris L.) Betalains. Molecules, 26(9), p.2439. (2021). https://doi.org/10.3390/molecules26092439

Jimoh, M. O., Afolayan, A. J. & Lewu, F. B. Suitability of Amaranthus species for alleviating human dietary deficiencies. South. Afr. J. Bot. 115, 65–73. https://doi.org/10.1016/j.sajb.2018.01.004 (2018).

Article CAS Google Scholar

Taira, J., Tsuchida, E., Katoh, M. C., Uehara, M. & Ogi, T. Antioxidant capacity of betacyanins as radical scavengers for peroxyl radical and nitric oxide. Food Chem. 166, 531–536. https://doi.org/10.1016/j.foodchem.2014.05.102 (2015).

Article CAS PubMed Google Scholar

Santos, J. S., Brizola, V. R. A. & Granato, D. High-throughput assay comparison and standardization for metal chelating capacity screening: a proposal and application. Food Chem. 214, 515–522. https://doi.org/10.1016/j.foodchem.2016.07.091 (2017).

Article CAS PubMed Google Scholar

Canabady-Rochelle, L. L. et al. Determination of reducing power and metal chelating ability of antioxidant peptides: revisited methods. Food Chem. 183, 129–135. https://doi.org/10.1016/j.foodchem.2015.02.147 (2015).

Article CAS PubMed Google Scholar

Cherrak, S. A. et al. In vitro antioxidant versus metal ion chelating properties of flavonoids: A structure-activity investigation. PloS One, 11(10), p.e0165575. (2016). https://doi.org/10.1371/journal.pone.0165575

Peng, Y., Wang, C., Xu, H. H., Liu, Y. N. & Zhou, F. Binding of α-synuclein with Fe (III) and with Fe (II) and biological implications of the resultant complexes. J. Inorg. Biochem. 104 (4), 365–370 (2010).

Article CAS PubMed Google Scholar

Cassagnes, L. E. et al. The catalytically active copper-amyloid‐beta state: coordination site responsible for reactive oxygen species production. Angew. Chem. Int. Ed. 52 (42), 11110–11113. https://doi.org/10.1002/anie.201305372 (2013).

Article CAS Google Scholar

Miotto, M. C. et al. Site-specific copper-catalyzed oxidation of α-synuclein: tightening the link between metal binding and protein oxidative damage in Parkinson’s disease. Inorg. Chem. 53 (9), 4350–4358. https://doi.org/10.1021/ic4031377 (2014).

Article CAS PubMed Google Scholar

Cheignon, C., Faller, P., Testemale, D., Hureau, C. & Collin, F. Metal-catalyzed oxidation of Aβ and the resulting reorganization of Cu binding sites promote ROS production. Metallomics. 8 (10), 1081–1089. https://doi.org/10.1039/c6mt00150e (2016).

Article CAS PubMed Google Scholar

Hasanuzzaman, M. et al. Regulation of ascorbate-glutathione pathway in mitigating oxidative damage in plants under abiotic stress. Antioxidants, 8(9), p.384. (2019).

Rector, R. S. et al. Mitochondrial dysfunction precedes insµLin resistance and hepatic steatosis and contributes to the natural history of nonalcoholic fatty liver disease in an obese rodent model. J. Hepatol. 52 (5), 727–736 (2010).

Article CAS PubMed PubMed Central Google Scholar

Cichoż-Lach, H. & Michalak, A. Oxidative stress as a crucial factor in liver diseases. World Journal of Gastroenterology: WJG, 20(25), p.8082. (2014).

Aseervatham, G. S. B., Ananth, D. A. & Sivasudha, T. The liver: oxidative stress and dietary antioxidants. In The Liver (239–246). Academic. (2018).

Grohmann, M. et al. Obesity drives STAT-1-dependent NASH and STAT-3-dependent HCC. Cell. 175 (5), 1289–1306. https://doi.org/10.1016/j.cell.2018.09.053 (2018).

Article CAS PubMed PubMed Central Google Scholar

Ul-Haq, I., Butt, M. S., Randhawa, M. A. & Shahid, M. Hepatoprotective effects of red beetroot-based beverages against CCl4‐induced hepatic stress in Sprague Dawley rats. Journal of Food Biochemistry, 43(12), p.e13057. (2019). https://doi.org/10.1111/jfbc.13057

Albrahim, T. & Alonazi, M. A. Role of beetroot (Beta vulgaris) juice on chronic nanotoxicity of silver nanoparticle-induced hepatotoxicity in male rats. International Journal of Nanomedicine, 15, p.3471. (2020). https://doi.org/10.2147/IJN.S248078

Shaban, N. Z. et al. The synergistic hepatoprotective potential of Beta vulgaris juice and 2,3-dimercaptosuccinic acid in lead-intoxicated rats by improving the hepatic oxidative and inflammatory stress. BMC Complementary Medicine and Therapies, 20(1), pp.1–15. (2020). https://doi.org/10.1186/s12906-020-03056-6

Al-Harbi, L. N. et al. Beta vulgaris L. (Beetroot) methanolic extract prevents hepatic steatosis and liver damage in T2DM rats by hypoglycemic, insulin-sensitizing, antioxidant effects, and upregulation of PPARα. Biology, 10(12), p.1306. (2021). https://doi.org/10.3390/biology10121306

Reuter, S., Gupta, S. C., Chaturvedi, M. M. & Aggarwal, B. B. Oxidative stress, inflammation, and cancer: how are they linked? Free Radic. Biol. Med. 49 (11), 1603–1616. https://doi.org/10.1016/j.freeradbiomed.2010.09.006 (2010).

Article CAS PubMed PubMed Central Google Scholar

Tsatsakis, A. M. et al. Hormetic neurobehavioral effects of low dose toxic chemical mixtures in real-life risk simulation (RLRS) in rats. Food Chem. Toxicol. 125, 141–149. https://doi.org/10.1016/j.fct.2018.12.043 (2019).

Article CAS PubMed Google Scholar

Erukainure, O. L., Matsabisa, M. G., Salau, V. F., Erhabor, J. O. & Islam, M. Cannabis sativa L. mitigates oxidative stress and cholinergic dysfunction; and modµLates carbohydrate metabolic perturbation in oxidative testicular injury. Comp. Clin. Pathol. 30 (2), 241–253. https://doi.org/10.1007/s00580-021-03200-9 (2021a).

Article CAS Google Scholar

Ojo, O. A. et al. Chromatographic fingerprint analysis, antioxidant properties, and inhibition of cholinergic enzymes (acetylcholinesterase and butyrylcholinesterase) of phenolic extracts from Irvingia gabonensis (aubry-Lecomte ex O’Rorke) baill bark. J. Basic Clin. Physiol. Pharmacol. 29 (2), 217–224. https://doi.org/10.1515/jbcpp-2017-0063 (2018).

Article CAS PubMed Google Scholar

Ojo, O. A. et al. Antidiabetic activity of elephant grass (Cenchrus purpureus (Schumach.) Morrone) via activation of PI3K/AkT signaling pathway, oxidative stress inhibition, and apoptosis in Wistar rats. Front. Pharmacol. 651. https://doi.org/10.3389/fphar.2022.845196 (2022).

Erukainure, O. et al. Cannabis sativa L. (var. indica) Exhibits hepatoprotective effects by modulating hepatic lipid profile and mitigating gluconeogenesis and cholinergic dysfunction in oxidative hepatic injury. Frontiers in Pharmacology, 12. (2021). https://doi.org/10.3389/fphar.2021.705402

Del Campo, J. A., Gallego, P. & Grande, L. Role of inflammatory response in liver diseases: Therapeutic strategies. World Journal of Hepatology, 10(1), p.1. (2018).

Shaked, I. et al. MicroRNA-132 potentiates cholinergic anti-inflammatory signaling by targeting acetylcholinesterase. Immunity. 31 (6), 965–973. https://doi.org/10.1016/j.immuni.2009.09.019 (2009).

Article CAS PubMed Google Scholar

Wang, T. et al. Oral intake of hydrogen-rich water ameliorated chlorpyrifos-induced neurotoxicity in rats. Toxicol. Appl. Pharmcol. 280 (1), 169–176. https://doi.org/10.1016/j.taap.2014.06.011 (2014).

Article CAS Google Scholar

Huang, Z. et al. From purines to purinergic signaling: molecular functions and human diseases. Signal. Transduct. Target. Therapy. 6 (1), 1–20. https://doi.org/10.1038/s41392-021-00553-z (2021).

Article CAS Google Scholar

Gnad, T. et al. Adenosine/A2B receptor signaling ameliorates the effects of aging and counteracts obesity. Cell Metabol. 32 (1), 56–70. https://doi.org/10.1016/j.cmet.2020.06.006 (2020).

Article CAS Google Scholar

Jain, S. & Jacobson, K. A. Purinergic signaling in diabetes and metabolism. Biochemical Pharmacology, 187, p.114393. (2021). https://doi.org/10.1016/j.bcp.2020.114393

Rui, L. Energy metabolism in the liver. Comprehensive Physiology, 4(1), p.177. (2014).

Sun, X. et al. Disordered purinergic signaling and abnormal cellular metabolism are associated with development of liver cancer in Cd39/ENTPD1 null mice. Hepatology. 57 (1), 205–216 (2013).

Article CAS PubMed Google Scholar

Salau, V. F. et al. FerµLic acid modµLates dysfunctional metabolic pathways and purinergic activities, while stalling redox imbalance and cholinergic activities in oxidative brain injury. Neurotox. Res. 37 (4), 944–955. https://doi.org/10.1007/s12640-019-00099-7 (2020).

Article CAS PubMed Google Scholar

Sunny, N. E., Parks, E. J., Browning, J. D. & Burgess, S. C. Excessive hepatic mitochondrial TCA cycle and gluconeogenesis in humans with nonalcoholic fatty liver disease. Cell Metabol. 14 (6), 804–810. https://doi.org/10.1016/j.cmet.2011.11.004 (2011).

Article CAS Google Scholar

Erukainure, O. L., Oyebode, O. A., Ibeji, C. U., Koorbanally, N. A. & Islam, M. Vernonia Amygdalina Del. stimulated glucose uptake in brain tissues enhances antioxidative activities; and modµLates functional chemistry and dysregulated metabolic pathways. Metab. Brain Dis. 34 (3), 721–732. https://doi.org/10.1007/s11011-018-0363-7 (2019).

Article CAS PubMed Google Scholar

Luo, X., Wu, J., Jing, S. & Yan, L. J. Hyperglycemic stress and carbon stress in diabetic glucotoxicity. Aging Disease. 7 (1), 90. https://doi.org/10.14336/AD.2015.0702 (2016).

Article PubMed PubMed Central Google Scholar

Gutiérrez, R. M., Muñiz-Ramirez, A., Garcia-Campoy, A. H. & Mota Flores, J. M. Evaluation of the antidiabetic potential of extracts of Urtica dioica, Apium graveolens, and Zingiber officinale in mice, zebrafish, and pancreatic β-Cell. Plants. 10, 1438. https://doi.org/10.3390/plants10071438 (2021).

Article CAS Google Scholar

Aljamal, J. A. & Badawneh, M. Vitro vitro inhibition of human erythrocyte hexokinase by various hyperglycemic drugs. J. Biochem. Mol. Toxicol. 31 (8), e21910. https://doi.org/10.1002/jbt.21910 (2017).

Article CAS Google Scholar

Erukainure, O. L., Sanni, O. & Islam, M. M. Clerodendrum Volubile:phenolics and applications to Health, in: (eds Watson, R., Preedy, V. & Zibadi, S.) Polyphenols: Mechanisms of Action in Human Health and Disease. Elsevier, Amsterdam, Netherlands. Doi:https://doi.org/10.1016/b978-0-12-813006-3.00006-4. (2018).

Chapter Google Scholar

Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature. 596, 583–589. https://doi.org/10.1038/s41586-021-03819-2 (2021).

Article ADS CAS PubMed PubMed Central Google Scholar

Idrees, S. et al. In silico sequence analysis, homology modeling and function annotation of Ocimum basilicum hypothetical protein G1CT28_OCIBA. International Journal Bioautomation, 16(2), 111. (2012).

Rost, B., Yachdav, G. & Liu, J. The predictprotein server. Nucleic Acids Res. 32 (suppl_2), W321–W326. https://doi.org/10.1093/nar/gkh377 (2004).

Article CAS PubMed PubMed Central Google Scholar

Ho, B. K. & Brasseur, R. The Ramachandran plots of glycine and preproline. BMC Struct. Biol. 5 (1), 1–11. https://doi.org/10.1186/1472-6807-5-14 (2005).

Article CAS Google Scholar

Westergaard, N. & Madsen, P. Glucose-6-phosphatase inhibitors for the treatment of type 2 diabetes. Expert Opin. Ther. Pat. 11 (9), 1429–1441 (2001).

Article Google Scholar

Baginski, E. S., Foà, P. P. & Zak, B. Glucose-6-phosphatase. In Methods of Enzymatic Analysis (876–880). Academic. (1974).

Singh, M., Pande, S. & Battu, S. Medicinal uses of L-lysine: past and future. (2011).

Katada, S., Watanabe, T., Mizuno, T., Kobayashi, S., Takeshita, M., Osaki, N., … Katsuragi,Y. (2018). Effects of chlorogenic acid-enriched and hydroxyhydroquinone-reduced coffee on postprandial fat oxidation and antioxidative capacity in healthy men: A randomized,double-blind, placebo-controlled, crossover trial. Nutrients, 10(4), 525.

Ogawa, H., Shinoda, T., Cornelius, F. & Toyoshima, C. Crystal structure of the sodium-potassium pump (Na+, K+-ATPase) with bound potassium and ouabain. Proc. Natl. Acad. Sci. 106 (33), 13742–13747 (2009).

Article ADS CAS PubMed PubMed Central Google Scholar

Ziqubu, K. et al. Isoorientin: a dietary flavone with the potential to ameliorate diverse metabolic complications. Pharmacol. Res. 158, 104867 (2020).

Article CAS PubMed Google Scholar

Ojo, A. B. et al. Syzygium aromaticum (L.) Merr. & L.M.Perry mitigates iron-mediated oxidative brain injury by modulation of redox imbalance, cholinergic and purinergic dysfunctions, and glucose metabolizing enzymes activities. Journal of Molecular Structure 1268 C (2022) 133675 (2022). https://doi.org/10.1016/j.molstruc.2022.133675

Sundar, S., Thangamani, L., Manivel, G., Kumar, P. & Piramanayagam, S. Molecular docking, molecular dynamics and MM/PBSA studies of FDA approved drugs for protein kinase a of Mycobacterium tuberculosis; application insights of drug repurposing. Inf. Med. Unlocked. 16, 100210 (2019).

Article Google Scholar

Chen, D. et al. Regulation of protein–ligand binding affinity by hydrogen bond pairing. Sci. Adv., 2(3), e1501240. (2016).

Alam, M. N., Bristi, N. J. & Rafiquzzaman, M. Review on in vivo and in vitro methods evaluation of antioxidant activity. Saudi Pharm. J. 21, 143–152 (2013).

Article PubMed Google Scholar

Mzoughi, Z. et al. Wild edible Swiss chard leaves (Beta vulgaris L. var. cicla): Nutritional, phytochemical composition and biological activities. Food Res. Int. 119, 612–621. https://doi.org/10.1016/j.foodres.2018.10.039 (2019).

Article CAS PubMed Google Scholar

Olofinsan, K. A., Salau, V. F., Erukainure, O. L. & Islam, M. S. Ocimum tenuiflorum mitigates iron-induced testicµLar toxicity via modulation of redox imbalance, cholinergic and purinergic dysfunctions, and glucose metabolizing enzymes activities. Andrologia. 53, e14179. https://doi.org/10.1111/and.14179 (2021).

Article CAS PubMed Google Scholar

Rahman, M. M., Islam, M. B., Biswas, M. & Khurshid Alam, A. H. In vitro antioxidant and free radical scavenging activity of different parts of Tabebuia pallida growing in Bangladesh. BMC Res. Notes. 8, 621. https://doi.org/10.1186/s13104-015-1618-6 (2015).

Article CAS PubMed PubMed Central Google Scholar

Erukainure, O. L. et al. Buddleja saligna Willd (Loganiaceae) inhibits angiotensin-converting enzyme activity in oxidative cardiopathy with concomitant modµLation of nucleotide hydrolyzing enzymatic activities and dysregµLated lipid metabolic pathways. J. Ethnopharmacol. 248, 112358. https://doi.org/10.1016/j.jep.2019.112358 (2020).

Article CAS PubMed Google Scholar

Salau, V. F., Erukainure, O. L. & Islam, M. Caffeic acid protects against iron-induced cardiotoxicity by suppressing angiotensin-converting enzyme activity and modulating lipid spectrum, gluconeogenesis and nucleotide hydrolyzing enzyme activities. Biol. Trace Elem. Res. 199, 1052–1061. https://doi.org/10.1007/s12011-020-02227-3 (2021).

Article CAS PubMed Google Scholar

Ajiboye, B. O., Oyinloye, B. E., Agboinghale, P. E. & Ojo, O. A. Cnidoscolus aconitifolius (Mill.) IM Johnst leaf extract prevents oxidative hepatic injury and improves muscle glucose uptake ex vivo. J. Food Biochem. 43, e13065. https://doi.org/10.1111/jfbc.13065 (2019).

Article PubMed Google Scholar

Erukainure, O. L., Mopuri, R., Oyebode, O. A., Koorbanally, N. A. & Islam, M. S. Dacryodes edµLis enhances antioxidant activities, suppresses DNA fragmentation in oxidative pancreatic and hepatic injuries; and inhibits carbohydrate digestive enzymes linked to type 2 diabetes. Biomed. Pharmacother. 96, 37–47. https://doi.org/10.1016/j.biopha.2017.09.106 (2017).

Article CAS PubMed Google Scholar

Balogun, F. O. & Ashafa, A. O. T. Aqueous root extracts of Dicoma anomala (Sond.) Extenuates postprandial hyperglycemia in vitro and its modulation on the activities of carbohydrate-metabolizing enzymes in streptozotocin-induced diabetic Wistar rats. South. Afr. J. Bot. 112, 102–111. https://doi.org/10.1016/j.sajb.2017.05.014 (2017).

Article CAS Google Scholar

Ambrose, G. O., Afees, O. J., Oluwaseun, A. S., Terkuma, C. & Iorwuese, M. Silico Sequence Analhomologymmodelingdelinfunctionaltannotationtation of Farnesyl Diphossynthasenthase (FDS) of Azadirachta indica. J. Appl. Bioinforma Comput. Biol. 7, 2 (2018).

Article Google Scholar

BIOVIA Discovery Studio. : a comprehensive predictive science application for life sciences. San Diego, CA, USA. (2017). R2 http://accelrys.com/products/collaborativescience/biovia-discovery-studio

Abraham, M. J. et al. (2015).

GROMACS. High-performance molecular simulations through mµLti-level parallelism from laptops to supercomputers. SoftwareX, 1, 19–25 .

Hess, B., Bekker, H., Berendsen, H. J. & Fraaije, J. G. LINCS: a linear constraint solver for molecular simulations. J. Comput. Chem. 18 (12), 1463–1472 (1997).

3.0.CO;2-H" data-track-item_id="10.1002/(SICI)1096-987X(199709)18:123.0.CO;2-H" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1002%2F%28SICI%291096-987X%28199709%2918%3A12%3C1463%3A%3AAID-JCC4%3E3.0.CO%3B2-H" aria-label="Article reference 89" data-doi="10.1002/(SICI)1096-987X(199709)18:123.0.CO;2-H">Article CAS Google Scholar

Essmann, U. et al. A smooth particle mesh via the Ewald method. J. Chem. Phys. 103 (19), 8577–8593 (1995).

Article ADS CAS Google Scholar

Download references

No funding was received.

Phytomedicine, Molecular Toxicology, and Computational Biochemistry Research Laboratory (PMTCB-RL), Department of Biochemistry, Bowen University, Iwo, 232101, Nigeria

Oluwafemi Adeleke Ojo

Department of Biochemistry, Landmark University, Omu-Aran, Nigeria

Temiloluwa Rhoda Adeyemo, Matthew Iyobhebhe, Rotdelmwa Maimako Asaleye, Jadesola Abdurrahman, Tobiloba Christiana Maduakolam-Aniobi & Charles Obiora Nwonuma

Clinical Biochemistry, Phytopharmacology and Biochemical Toxicology Research Laboratory (CBPBT-RL), Department of Biochemistry, Baze University, Abuja, Nigeria

Moses Dele Adams

Department of Food Science and Microbiology, Landmark University, Omu-Aran, Nigeria

Ikponmwosa Owen Evbuomwan

Department of Environmental Management and Toxicology, University of Ilesa, Ilesa, Nigeria

Olalekan Elijah Odesanmi & Adebola Busola Ojo

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

OAO, TRA, and ABO designed and conceptualized the study. Analysis, interpretation of data, and drafting the manuscript: TRA, JA, MI, TCM-A, IOE, MDA, OED, CON, and RMA performed and analyze the in silico studies. Manuscript draft: OAO, RMA, IOE, MI, OED, and ABO. Revise for intellectual content: OAO, ABO, OO, AOA, AIO, and SAE. All author approved the final version.

Correspondence to Oluwafemi Adeleke Ojo.

The authors declare no competing interests.

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Below is the link to the electronic supplementary material.

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

Ojo, O.A., Adeyemo, T.R., Iyobhebhe, M. et al. Beta vulgaris L. beetroot protects against iron-induced liver injury by restoring antioxidant pathways and regulating cellular functions. Sci Rep 14, 25205 (2024). https://doi.org/10.1038/s41598-024-77503-6

Download citation

Received: 06 April 2022

Accepted: 23 October 2024

Published: 24 October 2024

DOI: https://doi.org/10.1038/s41598-024-77503-6

Anyone you share the following link with will be able to read this content:

Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative