Countenance and implication of Β-sitosterol, Β-amyrin and epiafzelechin in nickel exposed Rat: in-silico and in-vivo approach

The detrimental impact of reactive oxygen species on D.N.A. repair processes is one of the contributing factors to colon cancer. The idea that oxidative stress may be a significant etiological element for carcinogenesis is currently receiving more and more support. The goal of the current study is to evaluate the anti-inflammatory and anticancer activity of three powerful phytocompounds—sitosterol, amyrin, and epiafzelechin—alone and in various therapeutic combinations against colon cancer to identify the critical mechanisms that mitigate nickel's carcinogenic effect. To evaluate the ligand–protein interaction of four selected components against Vascular endothelial growth factor (VEGF), Matrix metalloproteinase-9 (MMP9) inhibitor and Interleukin-10 (IL-10) molecular docking approach was applied using PyRx bioinformatics tool. For in vivo analysis, hundred albino rats were included, divided into ten groups, each containing ten rats of weight 160–200 g. All the groups were injected with 1 ml/kg nickel intraperitoneally per week for three months, excluding the negative control group. Three of the ten groups were treated with β-sitosterol (100 mg/kg b wt), β-amyrin (100 mg/kg b wt), and epiafzelechin (200 mg/kg b wt), respectively, for one month. The later four groups were fed with combinatorial treatments of the three phyto compounds for one month. The last group was administered with commercial drug Nalgin (500 mg/kg b wt). The biochemical parameters Creatinine, Protein carbonyl, 8-hydroxydeoxyguanosine (8-OHdG), VEGF, MMP-9 Inhibitor, and IL-10 were estimated using ELISA kits and Glutathione (G.S.H.), Superoxide dismutase (S.O.D.), Catalase (C.A.T.) and Nitric Oxide (NO) were analyzed manually. The correlation was analyzed through Pearson’s correlation matrix. All the parameters were significantly raised in the positive control group, indicating significant inflammation. At the same time, the levels of the foresaid biomarkers were decreased in the serum in all the other groups treated with the three phytocompounds in different dose patterns. However, the best recovery was observed in the group where the three active compounds were administered concomitantly. The correlation matrix indicated a significant positive correlation of IL-10 vs VEGF (r = 0.749**, p = 0.009), MMP-9 inhibitor vs SOD (r = 0.748**, p = 0.0 21). The study concluded that the three phytocompounds β-sitosterol, β-amyrin, and epiafzelechin are important anticancer agents which can target the cancerous biomarkers and might be used as a better therapeutic approach against colon cancer soon.


Molecular docking
To evaluate the ligand-protein interaction of four selected components against VEGF, MMP9 inhibitor, and IL-10 molecular docking approach was applied using the PyRx bioinformatics tool (https:// pyrx.sourc eforge.io/) 33 .VEGF, MMP9 inhibitor, and IL-10 enzyme as receptor proteins were uploaded in PyRx individually and converted into a macromolecule.All ligand molecules were uploaded using the Open Babel window; the energy

Bioactivities
Orally administrated drugs need to be compatible with the drug-likeness properties to be finalized as pharmaceutically consistent with their bioactivity score, and that's why we used Molinspiration Toolkit to predict selected compounds (https:// www.molin spira tion.com/ cgi-bin/ prope rties).

In vivo analysis
Adult Wister male albino rats were taken from the Institute of Molecular Biology and Biotechnology, Lahore, Pakistan, with body weights ranging from 160 to 200 g.The Ethics Committee approved a study for Scientific Research at the University of Lahore (Approval No: U.S.M./Animal Ethics approval/2009/45/140).All animal experiments followed the Institutional Guidelines for the Care and Use of Animals for Scientific Purposes.A p-value of < 0.05 was considered statistically significant.

Experimental design
Hundred (100) albino rats were divided into ten groups.Every group had ten rats.The Group A rats were fed a normal diet and distilled water.For 12 weeks in a row, albino rats (Rattus norvegicus) of all groups excluding group A received an intraperitoneal injection of nickel chloride (1:1 v/v) along with saline (1 mL/kg b wt. per week) to cause colon damage, which was verified by biochemical tests.To assess the protective effects, Β-sitosterol (2.5 mg/ml/kg b wt), β-amyrin(2.5 mg/ml/kg b wt), and epiafzelechin (2.5 mg/ml/kg b wt)were fed orally to groups C, D, E, F, G, H and I for one month in different doses.Group J was treated orally with NALGIN (100 mg) 1tab/kg body weight that contains Silybum marianum (200 mg/ml/kg), Picrorhiza kurroa (50 mg/ml/kg), Glycyrrhiza glabra (50 mg/ml/kg) and Cichorium intybus (75 mg/mg/ml) (Table 1) 37 .

Tissue and serum separation
After the treatments, the animals were sacrificed by an intraperitoneal injection of ketamine (Arevipharma GmbH, Radebeul, Germany) (1 mL/g) as an anaesthesia.5 mL blood was collected from the rat heart via cardiac Table 1.Experimental Design.Doses: Ni@ (1 mg/kg B.Wt. per week for three months).β-Sitosterol,β-Amyrin, and Epiafzelechin @ (2.5 mg/Kg B.Wt./for six months).NALGIN © @ (500 mg/Kg B.Wt/day) for six months.www.nature.com/scientificreports/puncture procedure into ethylene diamine tetra acetic acid (EDTA) treated sample bottles, and serum was separated by centrifugation at 1500 rpm for 10 min.After the serum separation, it was stored at -60 °C until further biochemical analysis.The colon portions were separated and collected in falcon tubes containing 5% formalin for histopathological examination.

Ethics approval
It has been confirmed that the experimental data collection complied with relevant institutional, national, and international guidelines and legislation with appropriate permissions from authorities of the Ethical Review Committee of the Institute of Molecular Biology and Biotechnology (IMBB), The University of Lahore-Pakistan.

ADMET analysis
ADMET study was carried out using Swiss ADMET to identify different parameters like physical properties, lipophilicity, Lipinski, and solubility of bioactive constituents 42 .ADMET has a key role in drug development, as many drug candidates fail a clinical trial.No more than two violations are acceptable for oral drug candidates 43 .Lipinski rule of five (≤ 5 H-bond donor, ≤ 10 H-bond acceptors, Mol.weight ≤ 500 Da, Molar Refractivity 40-130 and ≤ 5logP) 44 was followed for selected compounds to analyze whether these may act as drugs or not.The results in Table 1 show that all selected compounds pass the Lipinski rule of five, in which Epiafzelechin and Malondialdehyde have no single violation.In contrast, Beta-Amyrin and Beta-Sitosterol have one violation.Gastrointestinal absorption shows that two compounds, i.e., Epiafzelechin and Malondialdehyde, are highly soluble.For water solubility, Logs values range from − 10 to 0, indicating different solubility categories from − 10 to 0, i.e., − 10, − 6, − 4, − 2, and 0 for insoluble, poorly soluble, soluble, very soluble, and highly soluble, respectively 45 .Table 2 shows that all compounds are in the range of solubility, and no one is insoluble in the selected candidates.

Toxicity
AdmetSAR Online Protox-II server was used to analyze all constituents which may behave like a drug candidate to check their toxicity like hepatotoxicity, carcinogenicity, mutagenicity, and cytotoxicity.Today, toxicity prediction is a key step in drug designing to determine the unfavorable impacts of the compound on humans, plants, and the environment.Conventional approaches include different animal tests to assess the compound's toxicity, which is costly and time-consuming and includes serious ethical concerns.Computer-aided toxicity prediction is less time-consuming and inexpensive in comparison, provides results more frequently, and reduces the number of experimental biological tests.Table 3 shows that all selected candidates, i.e., Beta-Sitosterol, Beta-Amyrin, and Epiafzelechin, are non-toxic and may act as drug candidates.To validate and cross-check the toxicity of selected compounds, they were analyzed using admetSAR, which shows (Table 4) that three compounds with high docking scores are AMES non-toxic and non-carcinogenic.Further, all compounds were weak inhibitors for the human ether-a-go-go-related gene (hERG) and showed weak rat acute toxicity with a median lethal dose (LD50) of 2.26 mol/kg.As per the predicted acute oral toxicity, Table 2. ADMET analysis and drug-likeness results of selected compounds.

Bioactivities
One of the greatest tools for assessing the bioactivity scores of natural chemicals for therapeutic targets is Molinspiration, as demonstrated in (Table 5).The likelihood that a compound has notable biological activity depends on its bioactivity score.Compounds with bioactivity values above 0.00 are most likely noteworthy, while those with scores between -0.50 and 0.00 are moderately dynamic, and those below − 0.50 are inactive.Our analysis demonstrates that many routes may be implicated in the chemical compounds' physiological actions.Interactions with nuclear receptor ligands, GPCR ligands, ion channel modulator ligands, enzyme inhibitors, and protease inhibitors might also bring it on.The bioactivity scores (Table 5) show that Beta-Amyrin, Beta-Sitosterol, and Epiafzelechin are highly active ligands of Nuclear Receptor and Beta-Amyrin Epiafzelechin act as highly active ligands of Enzyme inhibitors.Furthermore, the results show that Beta-Amyrin, Beta-Sitosterol, and Epiafzelechin are moderately dynamic toward GPCR ligand and Protease inhibitors.Beta-Sitosterol and Epiafzelechin are moderately dynamic toward Ion Channel, while Enzyme inhibitor Epiafzelechin shows moderate dynamics.

In vivo biochemical response of β-sitosterol, β-amyrin and epiafzelechin in nickel-exposed rat
Depending on the colon microenvironment, several cytokines are responsible for the modulation of cellular transformation, their relative concentrations in the serum, cytokine receptor expression levels, and the surrounding cells' commencement status during chronic inflammation.Table 7 depicts the tumoral response of Creatinine, Protein carbonyl, and 8-hydroxydeoxyguanosine (8OHdG) during the progression of colon carcinogenesis in the intestine.The renal parameters Protein carbonyl and Creatinine were 39% and 12.2%, respectively, higher in rats receiving nickel (group B) than the normal group of rats (group A), indicating the malignant potential of the colon.Similarly, other inflammatory parameters' levels of 8OHdG (3.1%) were also significantly higher in nickel-intoxicated rats than in the negative control group (group A).However, the sole treatment of the standardized active constituents β-sitosterol, β-amyrin, and epiafzelechin showed antitumor effects with 77%, 93%, and 90% recovery compared to the positive control group (group B).Different combinations of these active phytocompounds show significant (p ≥ 0.05) restoration with 57.9% in 8OHdG.The increased production of 8OHdG induces a tumour phenotype based on generating reactive oxygen species and reactive nitrogen species, which induce DNA damage, facilitating tumorigeneses.Raised serum 8OHdG is a hallmark of DNA.damage because 8oxodG can pair with adenine, leading to the transversion of G: C to T: A (GT transversion).The best www.nature.com/scientificreports/restoration was obtained through the combinatorial regimen of three phytocompounds (group I) with 72% 8OHdG, 64% creatinine, and 67% A.L.T. compared to the commercially used anti-inflammatory drug Nalgin containing silymarin, Glycyrrhizin, and Cichorium minibus.Table 8 portrays the picture of the antioxidative status of superoxide dismutase (S.O.D.), glutathione (G.S.H.), and catalase (C.A.T.) and the carcinogenic status of Matrix metalloproteinase (MMP-9 inhibitor) in rats intoxicated with nickel and groups treated with different combinations of the three phytocompounds β-sitosterol, β-amyrin, and epiafzelechin.S.O.D., G.S.H., and C.A.T. were lower in rats administered with nickel (52%, 30.9%, and 17.25%, respectively) than in the negative control group.However, the levels of MMP-9 inhibitor were raised by 45% in the serum compared to the normal control group.The sole treatments of three bioactive compounds triggered significant (p ≥ 0.05) improvement in the antioxidative status (CAT.67%, SOD.56%, and GSH.45%) and MMP-9 inhibitor, 78%, as compared to the positive control group (Group B).Matrix metalloproteinases (MMPs) comprise a multigene family of zincdependent extracellular matrix (ECM) remodelling endopeptidases implicated in pathological processes, such as carcinogenesis.In this regard, their activity is pivotal in tumour growth and the multistep processes of invasion and metastasis, including proteolytic degradation of ECM, alteration of the cell-cell and cell-ECM interactions, migration and migration, and angiogenesis.The best recovery was obtained in the group of rats treated with the combinatorial regimen of the three active antitumor compounds (group I), with an increase of 78% in SOD, 89% in GSH, 93% in CAT and a decrease of 58% in NO and 83% in MMP-9 inhibitor protein as compared to the commercially used standard drug NALGIN.Table 7 shows the variation of crucial colon cancer vascular endothelial growth factor (VEGF) and interleukins (IL-10) biomarkers.There is significant (p ≥ 0.05) elevation found in the levels of VEGF (59%) and IL-10 (44.2%) in the group of rats intoxicated with nickel as compared to the normal healthy rats.Higher levels of IL-10 are associated with cellular stress and macromolecule modification, thus regulating the signaling pathways of cellular proliferation through Akt, Erk1/2, and hypoxia-inducible factor-1 (HIF-1) activation.In this concept, the best restoration in the levels of foresaid biomarkers (VEGF) and IL-10 (76.8%) was obtained by the co-treatment of β-sitosterol, β-amyrin, and epiafzelechin as compared to NALGIN-treated rats (Fig. 10).
IL-10 is also an angiogenic factor highly correlated with VEGF in colon cancer (r = 0.749, p = 0.009), as IL-10 induces VEGF expression dose-dependently.This means that the increase in VEGF value will also cause an increase in IL-10 in a dose-dependent manner.Similarly, a significant correlation was also depicted in Table 9 between IL-10 and 8-OHdG (r = 0.648, p = 0.035), VEGF, and 8-OHdG (r = 0.648, p = 0.032).Both are the hallmarks of cellular stress and inflammation (Table 9).The direct relationship between MMP-9 and CAT.(r = 0.679,p = 0.041) and with SOD.(0.748, p = 0.021) was observed, which means that when colon inflammation increased, the increased levels of MMP-9 caused the elevation of SOD. and CAT.levels in the serum.

Histopathological analysis
Evaluation of disease activity and severity, as well as reporting treatment outcomes in clinical trials, are essential for the clinical management of colon cancer.The treatment's most obvious goal is to control inflammation and achieve complete symptom remission.The histopathological analysis depicted the typical normal colonic mucosa composed of many goblet cells that release mucin.There is a submucosa underneath.Tiny lymphoid nodules linked with the intestines could also be observed (Fig. 11A).The presence of neutrophils indicates the existence of inflammatory activity.Active inflammation is characterized by neutrophilic cryptitis, crypt abscesses, bleeding, erosions, ulceration, and necrosis.The architectural distortion is more pronounced, characterized by the irregular spacing of the crypts resulting from the inflammatory cells' lamina propria expansion and basal lymphoid aggregates (Fig. 11B).However, inflamed coli are reduced in the right side figure with 30 to 40% restoration of rectal mucosa can be observed due to the treatment with a combinatorial dose of β-sitosterol, β-amyrin, and epiafzelechin (Fig. 11C).The epithelial cellular proliferation was also reduced due to the combinatorial treatment.www.nature.com/scientificreports/These histopathological results also agree with our in vivo results (Table 7).This indicated the best colon necrosis and ulceration recovery due to the synergistic dosage regimen of the foresaid phytocompounds.

Discussion
Nickel contamination in large quantities of food or water can be extremely hazardous 46 .Cancer initiation and development are frequently linked to nickel-induced apoptosis inhibition in colon cells 47 .Consequently, a thorough understanding of apoptotic signalling pathways is related to discovering target-selective therapeutic www.nature.com/scientificreports/medicines.The current study aimed to assess how the phytoactive compounds β-sitosterol, β-amyrin, and epiafzelechin in different dosage combinations combat the nickel-affected adult Wistar rats' ascending colons.The two main apoptotic events are morphological and metabolic changes in the cancerous colon cells 48 .Cell morphological changes, including cell shrinkage, chromatin condensation, membrane blebbing, and nuclear disintegration, which later result in the formation of apoptotic bodies, arise after triggering biochemical signals in cell 49 .In this study, analysis of the Creatinine, protein carbonyl, 8-OHdG, VEGF, S.O.D., G.S.H., C.A.T., NO, MMP-9 Inhibitor, and IL-10 was carried out to investigate the nickel-induced biochemical changes in colon cancer cells.The intraperitoneal induction of NiCl 2 in rats caused significant elevation to the serum levels of Creatinine and P.C. in a manner reminiscent of compromised colon integrity.However, the intraperitoneal administration of different concentrations of β-sitosterol, β-amyrin, and epiafzelechin in rats prevented the elevation of the indices for colon dysfunction.These findings support previous studies showing that β-sitosterol, β-amyrin, and epiafzelechin offered colon and hepatoprotection 50,51 .Also, in a recent study, the protective effect of β-sitosterol, β-amyrin, and epiafzelechin against lead-induced colorectal toxicity has been demonstrated 52 .The evidence supports the notion that including the above-mentioned phytoactive compounds in diets may enhance the health of the inflamed or cancerous colon.8-hydroxydeoxyguanosine (8OHdG) has been the hallmark of cancer 53 and oxidative D.N.A. damage 54 .The nickel administered in the rats in our study caused D.N.A. methylation, which is a post-translational modification during the synthesis of cancerous colon protein and D.N.A. adducts 8OHdG and 8-oxodG, both are also the major products of D.N.A. oxidation.Nalgin tablets used as a standard commercial drug in the study couldn't combat the inflammation caused by D.N.A. damage due to the formation of D.N.A. adducts 8OHdG and 8-oxodG (Table 7).The increased levels of 8OHdG (31.32 pg/ml) as compared to normal 0.98 pg/ml in rats (Table 7) is the main risk factor for developing colon cancer which is subsequently decreased by the co-administration of the foresaid bioactive compounds (5.16 pg/ml).This is supported by Pearson's correlation (Table 9) between COX-2 and 8-OHdG (r = 0.648**, p = 0.035).Similarly, VEGF, IL-10, and MMP-9 inhibitor proteins are all increased due to free radical generation in the pathogenesis of colon cancer 55 .The combination regimen of the three phytocompounds in group I lowers the levels of these biomarkers of D.N.A. oxidation compared to the isolated treatment.The correlation matrix shows the significant and direct relationship of IL-10 and VEGF (r = 0.749**, p = 0.009), MMP-9, and C.A.T. (r = 0.679*, p = 0.041).These results are also in accordance with the in silico analysis of VEGF with the three bioactive compounds showing strong binding affinity of − 8.7 with β-amyrin, − 6.6 with epiafzelechin, and − 7.2 with β-sitosterol (Table 6) which means that these bioactive compounds used as drugs in the current study bind strongly in the active pockets of VEGF protein, consequently controlling its expression to control the disease.This suggests that these phytoactive compounds are more potent drugs than others used in chemotherapy.The significant correlation between the foresaid parameters depicts that all these parameters are the prominent biomarkers of colon tumorigeneses which were significantly restored by the synergistic treatment of the three bioactive compounds.The matrix metalloproteinases are the most potent proteinases associated with tissue homeostasis in normal cell circumstances 56 .However, the over-expression of the MMP-9 inhibitor protein causes a change in proteolysis due to angiogenesis resulting in metastatic colon tumor invasion and inflammation 57 .In our study, we also analyzed the data statistically through Pearson's correlation matrix to know the relationship of the said variables, which helped us to identify and visualize the large dataset pattern of the severity of the disease.The correlation matrix shows the significant and indirect relationship between MMP-9 inhibitor protein and S.O.D. (r = 0.748**, p = 0.021).In our study, the modulation of MMP-9 inhibitor protein is inhibited and downregulated via cell cycle control at G1 and S phase by epiqafzelechin (Fig. 1), β-sitosterol (Fig. 2) and β-amyrin (Fig. 3), thus can be used as an anticancer therapy for colon tumors.Β-amyrin controls the transcription and proteolytic cleavage of their NH 2 -terminal domain by plasmin and furin proteases and maintains the mRNA stability, thus inhibiting the overexpression of MMPs 58 .The literature reported that HepG2 was first treated with different concentrations of β-amyrin.Then it was exposed to DAPI staining, which depicted that β-amyrin induced apoptosis in Hep-G2 cells, shown by the increased number of cells with white color nuclei at 50 µM β-amyrin concentration 59 .The effect of β-amyrin on the phosphorylation of J.N.K. and p38 was observed, and the results showed increased phosphorylation of both J.N.K. and p38 in a concentration-dependent manner 60 .The levels of antioxidants S.O.D., C.A.T., and G.S.H., which were decreased in the serum due to over-expression of tumor biomarkers, were restored gradually due to targeted therapy of β-sitosterol, β-amyrin, and epiafzelechin.Much research has been conducted on the effect of epiafzelechin-rich extracts on cancer cell lines.Findings demonstrated that epiafzelechin inhibited the proliferation of cancer cell lines in a dose-dependent manner.Epiafzelechin extracted from acacia tundra at a concentration of 100 µM revealed the inhibition against the cervical Cancer Hela cells and gastric adenocarcinomas MKN-45 cells 61 .However, 140 mg/g of epiafzelechin demonstrated 20% inhibition of the human bladder cancer TCCUP cell line 62 .Epiafzelechin also depicted necrotic cell death at 130 mg/g concentration in human dermal microvascular endothelial cells.Thus, epiafzelechin at high doses shows antiproliferative activity 63 .However, IL-10 showed a significant indirect relationship with VEGF (r = 0.749*, p = 0.009).Rats exposed to NiCl 2 had disrupted epithelial barrier, as seen by the histopathology of colon sections.In the groups injected phytocompounds containing combinatorial regimen of β-sitosterol, β-amyrin, and epiafzelechin, overall mucosal architecture and graded from the presence of ulcerations, granulation tissue, irregular crypts or the crypt produced by NiCl 2 were strikingly absent or attenuated (Figs. 10, 11).The histology presentations support the biochemical results, which further demonstrate the therapeutic protection provided by the combinatorial treatment of β-sitosterol, β-amyrin, and epiafzelechin against nickel-induced tissue damage.
To our knowledge, this is the first study to show that adding β-sitosterol, β-amyrin, and epiafzelechin in combination dose as described in the current work to the diet shielded the rat colon from the harmful effects brought on by exposure to NiCl 2 .The data support the medicinal and nutritional potential of the above-discussed phytocompounds with more scientific evidence.Consuming a diet high in β-sitosterol, β-amyrin, and epiafzelechin Vol:.( 1234567890

Conclusion
The current study concluded through in silico and in vivo analysis that the standardized phytoconstituents β-sitosterol, β-amyrin and epiafzelechin in combination dose regimen offered more potent anti-carcinogenic activity in nickel-induced colon inflammation in rats as compared to the drugs used in chemotherapy with minimum side effects.Therefore, these bioactive compounds in synergism would be more useful as antitumor agents than the individual phytocompounds as well as commercial drugs of chemotherapy and can be used as a better theranostic approach towards treating colon cancer in the future. https://doi.org/10.1038/s41598-023-48772-4

Figure 1 .
Figure 1.(A) shows the interaction of Epiafzelechin with receptor protein MMP9-inhibitor, while (B) shows the 2D interaction and bonding.Discover Studio version 4.5 was used to develop the structure (https:// www.discn gine.com/ disco very-studio).

Figure 2 .
Figure 2. (A) shows the interaction of Beta-Sitosterol with receptor protein MMP9-inhibitor, while (B) shows the 2D interaction and bonding.Discover Studio version 4.5 was used to develop the structure (https:// www.discn gine.com/ disco very-studio).

Figure 5 .
Figure 5. (A) shows the interaction of Beta-Sitosterol with receptor protein IL-10, while (B) shows the 2D interaction and bonding.Discover Studio version 4.5 was used to develop the structure (https:// www.discn gine.com/ disco very-studio).

Figure 6 .Figure 7 .
Figure 6.(A) shows the interaction of Epiafzelechin with receptor protein IL-10, while (B) shows the 2D interaction and bonding.Discover Studio version 4.5 was used to develop the structure (https:// www.discn gine.com/ disco very-studio).

Figure 8 .
Figure 8. (A) shows the interaction of Beta-Sitosterol with receptor protein VEGF, while (B) shows the 2D interaction and bonding.Discover Studio version 4.5 was used to develop the structure (https:// www.discn gine.com/ disco very-studio).

Figure 9 .
Figure 9. (A) shows the interaction of Epiafzelechin with receptor protein VEGF, while (B) shows the 2D interaction and bonding.Discover Studio version 4.5 was used to develop the structure (https:// www.discn gine.com/ disco very-studio).

Table 3 .
Toxicity results of selected compounds using Protox-II online server.

Table 4 .
Toxicity profiling of selected compounds (with high binding affinity) using admetSAR server.

Table 6 .
Molecular docking results of selected compounds.