5-Fluorouracil and Rumex obtusifolius extract combination trigger A549 cancer cell apoptosis: Uncovering PI3K/Akt inhibition by in vitro and in silico approaches

5-Fluorouracil and Rumex obtusifolius extract combination trigger A549 cancer cell apoptosis: Uncovering PI3K/Akt inhibition by in vitro and in silico approaches | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article 5-Fluorouracil and Rumex obtusifolius extract combination trigger A549 cancer cell apoptosis: Uncovering PI3K/Akt inhibition by in vitro and in silico approaches Mikayel Ginovyan, Hayarpi Javrushyan, Svetlana Hovhannisyan, Edita Nadiryan, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4254380/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Jun, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract In this study, the objective was to explore novel strategies for improving the efficacy of anticancer therapy. The focus was on investigating the antiproliferative effects of combining Rumex obtusifolius extract (RO) with the chemotherapeutic agent 5-Fluorouracil (5-FU) in non-small A549 lung cancer cells (NSCLC). Key factors such as the PI3K/Akt cell signaling system, cytokines, growth factors (TNFa, VEGFa), and enzymes (Arginase, NOS, COX-2, MMP-2) were analyzed to assess the impact of the combination treatment. Results revealed that the combined treatment of 5-FU and RO demonstrated a significant reduction in TNFa levels, comparable to the effect observed with RO alone. RO was found to modulate the PI3K/Akt pathway, influencing the phosphorylated and total amounts of these proteins during the combined treatment. Notably, COX-2, a key player in inflammatory processes, substantially decreased with the combination treatment. Caspase-3 activity, indicative of apoptosis, increased by 1.8 times in the combined treatment compared to separate treatments. In addition, in silico analyses explored the binding affinities and interactions of RO's major phytochemicals with intracellular targets, revealing a high affinity for PI3K and Akt. These findings suggest that the combined treatment induces apoptosis in A549 cells by regulating the PI3K/Akt pathway. Biological sciences/Biochemistry Biological sciences/Cancer Biological sciences/Computational biology and bioinformatics Biological sciences/Plant sciences Rumex obtusifolius 5-Fluorouracil lung adenocarcinoma phytochemicals PI3K/Akt pathway apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction The continuous increase in cancer rates, failure of conventional chemotherapies to control the disease, and excessive toxicity of chemotherapies (in some cases including immunotherapy) clearly demand alternative approaches. Natural products contain many constituents that can act on various targets in the body to induce pharmacodynamic responses 1–3 . Modulating biochemical and immune functions using medicinal plants and their products combined with chemotherapeutic agents has recently become an accepted therapeutic approach 4 . Lung cancer is one of the most common causes of cancer-related deaths worldwide. According to the American Cancer Society, approximately 85% of all lung cancer deaths were the result of non-small-cell lung cancer (NSCLC). There are several reasons for targeting the PI3K/Akt pathwaysignaling pathway and related extracellular and intracellular components. Correctly regulating the activity and quantity of these components can have a strong anticancer effect on cancer cells. Several pro-tumorigenic processes converge on hyperactive PI3K/Akt signaling, and it is becoming increasingly evident that reactive oxygen species (ROS) metabolism is no exception to this 5 . Metabolic reconfiguration and the resulting generation of ROS are vital for facilitating the development of tumors. Dysregulated PI3K/Akt signaling is crucial in regulating numerous molecular processes that elevate ROS levels. This occurs either by directly influencing mitochondrial energy production and activating NADPH oxidases (NOXs), or indirectly by generating ROS as a metabolic byproduct 6 . Comprehending the intricate relationship between ROS and PI3K/Akt signaling is important for devising effective therapeutic approaches to combat tumors reliant on this pathway. This significance is underscored by recent clinical trials showing limited efficacy of PI3K/Akt pathway inhibitors and the emergence of resistance. It could lead to the discovery of new biomarkers and metabolic vulnerabilities, as well as the development of more potent therapeutic combinations that disrupt redox balance and specifically target PI3K-driven tumors 6,7 . Chemotherapy and radiotherapy stand as primary treatments for cancer patients, and they induce apoptosis in cancer cells partly by increasing ROS levels. Agents like 5-FU, cisplatin, and other chemotherapeutic drugs trigger ROS production by influencing the electron transport chain. The heightened ROS levels then trigger cascades such as caspase activation, release of cytochrome C, and DNA damage, ultimately leading to apoptosis 6 . Given the potential pro-oxidative properties of herbs, we incorporated a chemotherapeutic compound alongside 5-FU in our model. This approach allows for the concurrent inhibition of the PI3K/Akt pathway while maintaining or increasing significant levels of ROS and reactive nitrogen species (RNS). Such elevation can effectively promote the induction of apoptosis. PI3K pathway promotes metastasis by promoting tumor neovascularization, which is required for the metastatic spread of tumors. PI3K forms a complex with E-cadherin, β-catenin, and VEGFR-2 and is involved in endothelial signaling mediated by VEGF through the activation of the PI3K/Akt pathway 8 . The PI3K/Akt signaling pathway also promotes TNF-induced endothelial cell migration and regulates tumor angiogenesis. TNF-α plays a significant role in promoting the survival and metastasis of lung cancer. The levels of TNF-α in tumor tissues and serum collected from patients with NSCLC substantially increase with the clinical stage of the tumor. Additionally, matrix metalloproteinases (MMPs) and cyclooxygenase-2 (COX-2) contribute to tumor angiogenesis. COX-2 stimulates endothelial angiogenesis primarily through the upregulation of the antiapoptotic protein Bcl-2 and activation of the PI3K/Akt signaling pathway 5 . A recent study indicates that COX-2 inhibition protects against hypoxia/reoxygenation-induced cardiomyocyte apoptosis via AKT-dependent enhancement of iNOS expression 9 . Preconditioning the cells with the COX-2 inhibitor NS398 resulted in decreased expressions of TNFα, prostaglandin E2 (PGE2), and interleukin-6 (IL-6) pro-inflammatory factors. It was observed that inhibiting COX-2 could mitigate the increased release of nitric oxide (NO) and expression of inducible nitric oxide synthase (iNOS) induced by Giardia. Studies unveiled a crucial role of COX-2 in modulating the pro-inflammatory response and defense-related NO production in interactions between Giardia and macrophages 10 . MMP-2 transcriptional suppression decreased VEGF, PI3K protein levels, and AKT phosphorylation in lung cancer cells. MMP-2 suppression disrupted phosphatidylinositol 3-kinase (PI3K) dependent VEGF expression; ectopic expression of myr-AKT restored VEGF inhibition. Studies with either function blocking integrin-αVβ3 antibody or MMP-2 specific inhibitor (ARP-100) indicate that suppression of MMP-2 decreased integrin-αVβ3-mediated induction of PI3K/AKT leading to decreased VEGF expression. A549 xenograft tissue sections from mice that were treated with MMP-2 siRNA showed reduced expression of VEGF 11 .Numerous studies provide evidence of the influence of natural compounds on the components within this pathway. For instance, the natural ubiquinone Coenzyme Q0 (CoQ0) has shown efficacy against the proliferation of various cancer cell lines such as HepG2, A549, and SW480, promoting apoptosis by elevating ROS levels. Treatment with LY249002, which blocks the PI3K/AKT pathway, significantly reduced NFκB activation and MMP-9 levels . The analysis of the literature reveals that TNF-α, VEGF-α, COX-2, MMP-2, Caspase-3, and NOS are interconnected in various cancer processes. Their interrelations are mediated through the PI3K/Akt signaling pathway. This study focused on employing a combination of natural compounds and a chemotherapeutic agent to target these specific components to attain potent anticancer effects. We hypothesized that phytoextracts, either single or in combination with chemotherapy compounds, may effectively modulate the immune system (TNFa/COX-2/Arginase), inhibit angiogenesis and progression of metastasis (VEGFa/NOS/NO/MMP-2) via regulation of PI3K/Akt signaling pathway. In our previous research works we showed the promising anticancer effect of Rumex obtusifolius (RO) in an in vivo experimental breast cancer rat model, in parallel, its cytotoxic effect was elucidated against two cell cultures: MCF-7, and HT29 12 . The combined anticancer effects of inhibitors targeting the metabolic pathway of L-arginine have also been investigated. The findings demonstrate significant anti-tumor properties, including reductions in tumor size, number, and mortality. Changes in various biochemical parameters in the blood, associated with participants in the metabolic pathway of L-arginine, were observed. However, alterations in key factors that would provide a detailed understanding of the exact molecular mechanisms underlying the anticancer effects were not noted 13 . This research elucidated the mechanisms of the anticancer effects of R. obtusifolius extract, both independently and in combination with the conventional chemotherapeutic compound 5-FU. Specifically, the impact of the herbal extract of R. obtusifolius (0.25mg/mL) on the TNFα-VEGFα/PI3K/Akt/NOS/COX-2-MMP-2 pathway was assessed separately and in combination with 5-FU (40µM) in non-small lung adenocarcinoma A549 cells. In addition, the possible interaction of compounds identified by HPLC/MS/MS of RO on PI3K/Akt in the active site pocket was also elucidated by in silico study in comparison with its ligand care. By uncovering the molecular mechanisms of anticancer effects, identifying the most active phytochemicals, and clarifying the specific targets of these compounds, there is potential to inhibit, prevent, or delay cancer development with minimal side effects. 2. RESULTS 2.1. Modulation of growth-inhibiting properties of 5-Fluorouracil with R. obtusifolius extract tested by MTT assay Growth inhibiting properties of RO seed ethanol extract on A549 cancer cells were tested with in vitro MTT assay. Based on the obtained data the RO extract even at the highest tested concentration (0.5 mg DW/mL) and at any of the tested exposure times (4, 24, and 72 h) did not show any statistically significant impact on the growth of A549 cells (Fig. 1 A). Further modulating activity of none-inhibitory concentrations of RO extract (0.25 mg DW/mL) toward fluorouracil (5-FU) on A549 cells (Fig. 1 B, C, D) was investigated using in vitro MTT assay. Based on the obtained data, statistically significant strong modulation of 5-FU was observed at 24 h exposure time on all tested 5-FU concentrations (Fig. 1 C). Considerable modulation was detected on 4 h exposure time as well (Fig. 1 B). However, during 72 h exposure time, no modulation was observed (Fig. 1 D). 2.2. R. obtusifolius extract alone and in combination with 5-FU downregulate quantitative changes of TNFa, VEGFa, COX-2, and MMP-2. Quantitative changes in TNFα-related COX2 and VEGF-related MMP2 were assessed in the next step using ELISA. RO extract decreased the quantities of TNFα (Fig. 2 , A) and VEGFα (Fig. 2 , B) in the cell medium by 40% and 33%, respectively, and the quantities of COX-2 and MMP2, respectively by 31.5% and 33%. 5-FU alone does not affect TNFa/COX-2, but when combined with the plant, the effect is greater than with the plant alone or 5-FU alone (p ≤ 0.05). The most visible modulatory effect on each other is TNFα (combined effect decreases it close to the value of RO), VEGFa (33% and 50% decrease, respectively, compared to 5-FU and RO alone, p ≤ 0.01), and in the case of COX-2 (the reduction is about 90% for 5-FU and 80% for RO). In the case of COX-2, a synergism phenomenon can be documented. 2.3. Regulation of PI3K/Akt pathway To further understand the cause of decreased TNFα, VEGFa, COX-2, MMP-2 and its effect on the cell, the downstream signaling pathway from TNFα and VEGFa to PI3K/Akt, whose dysregulation is characteristic of cancer, was elucidated. We demonstrated that RO acts as an inhibitor of the Pi3K/Akt pathway. Particularly, quantitative reduction of total and phosphorylated PI3K and Akt was observed after the treatment of A549 cells with RO extract (Fig. 3 , A). The combination of RO and 5-FU reduced the amount of total and phosphorylated PI3K by about 2.5-fold compared to the control cells (p ≤ 0.01). RO alone does not affect these two forms of PI3K, and 5-FU only affects the total amount of PI3K. RO and 5-FU reduce both phosphorylated and total Akt amounts, and the combination can increase this effect several-fold, showing a synergistic effect (Fig. 3 , B). We assumed one of the ways the effects of RO is the TNFa-PI3K-Akt cascade. Changes in the amount and activity of NO, MDA, Arginase, and NOS participants were elucidated to understand where the effect goes next. 2.4. Stimulation of RNS and ROS by regulation of Arginase and NOS activity Since in most cases COX-2 interacts with arginase and NOS 14 , changing the concentration of VEGFa with NO and NOS and TNFa with ROS and RNS was valuable and necessary to observe the activities of arginase, NOS, and quantitative changes of NO and MDA (Fig. 4 ). The results showed that by reducing the amount of VEGF, the plant also inhibited the activity of NOS (p ≤ 0.001), but increased the quantity of NO ( p ≤ 0.05 , Fig. 4 , B and C ). Since the activity of NOS is depressed, but the amount of nitrite ions is increased, the increase in the amount of RNS may be due to the increase in ROS. ROS and generated NO most likely lead to RNS generation and ROS/NO-mediated apoptosis. The latter is confirmed by the high quantity of MDA, which is promoted by the plant ( p ≤ 0.01 , Fig. 4 , D). Normally in cancer, activation of the PI3K/Akt pathway leads to increased ROS 15 , 16 , and conversely, increased ROS leads to activation of this pathway, but this phenomenon was not observed in our experiments. In this case, the plant adjusted in such a way that by suppressing the PI3K/Akt pathway, it increases the amount of ROS and RNS. This indicates a multi-target effect of the compounds contained in the herb. To verify that increased RNS and ROS quantities, as well as inhibition of the PI3K/Akt pathway, should lead to apoptosis, Caspase-3 activity was assessed and chromatin staining was performed with Hoersch 3328 dye to observe segmentation and condensation. 2.5. Assessment of apoptosis rate by Hoechst 33258 staining Hoechst 33258 staining allows discrimination of apoptotic and non-apoptotic cells based on morphological changes of the nuclei (Fig. 5 ). The nuclei in normal cells exhibited evenly dispersed and weak fluorescence, as well as smooth edges (Fig. 5 , A). Apoptotic cells can be distinguished by the condensed chromatin ( Fig․ 5, B, marked with arrows ) and rough edges of their nuclei (Fig. 5 , C, marked with arrows), as well as signs of nuclear fragmentation (Fig. 5 , D, marked with arrow ). The results indicate that incubating A549 cells with 5-FU, RO, and their combination 5-FU + RO for 24 h significantly increased the rate of apoptotic cells ( Fig․ 5, E ). In the control, the rate of apoptosis was 2.40 ± 0.56%. Treatment with 5-FU or RO alone significantly increased the rate of apoptotic cells up to 14.70 ± 1.83% and 11.30 ± 0.98%, respectively. At the same time, the combination of 5-FU + RO elevated the rate of apoptotic cells up to 29.5 ± 4.94% (p < 0.01). Thus, the apoptosis of A549 cells induced by the combination of 5-FU + RO was significantly higher when compared with that of 5-FU or RO alone (p < 0.05). Since Caspase-3 is active in the execution phase of apoptosis, we next used colorimetric assay to determine whether it was activated following treatment with 5-FU, RO, and their combination 5-FU + RO for 24 h ( Fig․ 5, F ). The spectrophotometric analysis revealed a significant increase in caspase-3 activity in all treatment variants which was more pronounced in cells treated with 5-FU + RO (p < 0.01). 2.6. The interaction of potential compounds with PI3K and Akt The docking of the top compounds present in the RO ethanolic extract ( Suppl. Table ) by Autodock Vina was performed on the binding pockets of AKT (PDB ID: 2JDO) and PI3K (PDB ID: 6 AUD) ( Fig, 6 and 7 ). Each crystallographic structure’s binding pocket contained a bound ligand, which was extracted and rocked as a control. The docking results are presented in Table 1 . Based on the average score on two target proteins, none of the compounds showed a better docking score (the lower, the better) than the redocking score of the PI3K control. However, 14 out of 17 investigated compounds demonstrated better docking scores than the redocking score of the AKT control ligand. Therefore, this shows that the extracted compounds of interest may have a better affinity toward AKT. Table 1 Docking results of RO on PI3K and Akt. Compound AKT (2JDO) PI3K (6AUD) Average PI3K-control ligand -9.6 -9.6 Endocrocin -9.1 -9.2 -9.15 Emodin -8.8 -9.2 -9 Luteolin -8.5 -8.8 -8.65 Quertecin -8.5 -8.4 -8.45 Epicatechin-gallate -8.5 -8.3 -8.4 Eriodictol -8.3 -8.5 -8.4 Quercetin-3-D-galactoside -8.3 -8.5 -8.4 Hamamelofuranose -8.4 -8.2 -8.3 Isorhamnetin-3-O-glucoside -8 -8.5 -8.25 Catechin -7.8 -8.5 -8.15 Epicatechin -8.1 -7.8 -7.95 Apigenin-sulfate -8.1 -7.6 -7.85 Qurecetin-diglucoside -7.5 -7.8 -7.65 4-glucogallic acid -7.5 -6.9 -7.2 AKT-control ligand -7 -7 Procyanidin-dimer -7.1 -6.3 -6.7 Protocatechuic-acid -5.7 -5.6 -5.65 Hydroxybenzoic-acid -5.5 -5.5 -5.5 Understanding the absorption, distribution, metabolism, and excretion (ADME) properties of drug candidates is essential due to their profound influence on the pharmacokinetics, efficacy, and potential side effects of therapeutic agents 17 . For this reason, we computationally analyzed the ADME characteristics of the 17 leading compounds using the SwissADME online service, and key features were outlined in Table 2 . A variety of physical and chemical properties such as molecular weight (MW), partition coefficient (LogP), hydrogen bond acceptors (HBA), and hydrogen bond donors (HBD) were assessed. Based on these calculations, we examined how the compounds adhere to Lipinski’s rule of 5 which is a set of rule-of-thumb guidelines in medicinal chemistry that predict oral bioavailability in drugs, stating that a molecule will likely be an effective oral medication when it does not violate more than one of these rules: no more than 5 hydrogen bond donors, 10 hydrogen bond acceptors, a molecular mass less than 500 Daltons, and a LogP not exceeding 5 18 . The results illustrate that 10 out of the 17 compounds did not violate Lipinski’s rule of 5, indicating their potential to be drug candidates. Remarkably, these 10 compounds included the top 4 ones having the best docking score (namely: endocrocin, emodin, luteolin, and quercetin). Table 2 ADME properties of RO extract phytochemicals. Compounds MW HBA HBD LogP Num of Viol Endocrocin 314 7 4 1.43 0 Emodin 270 5 3 1.87 0 Luteolin 284 6 4 1.73 0 Quercetin 299 7 5 0.17 0 Epicatechin gallate 442 10 7 1.25 1 Erodictol 280 6 4 0.84 0 Quercetin 3-D-galactoside 464 12 8 -0.25 2 Hamamelofuranose 180 6 5 -1.94 0 Isorhamnetin 3-O-glucoside 478 12 7 -0.15 2 Catechin 290 6 5 0.85 0 Epicatechin 290 6 5 0.85 0 Apigenin sulfate 364 9 3 1.28 0 Qurecetin diglucoside 607 17 11 -2.8 3 4 glucogallic acid 332 10 7 1.9 1 Procyanidin dimer 562 12 10 0.54 3 Protocatechuic acid 151 4 3 0.4 0 Hydroxybenzoic acid 134 3 2 0.72 0 Accordingly, we decided to examine further the 4 compounds mentioned above. In particular, we analyzed the binding mode of their best docking scores for the two proteins in addition to a 3D visual inspection (Figs. 6 and 7 ). Based on the analysis, emodin forms hydrogen bonds with Asp293 and Lys160 amino acids of AKT. The interactions with 9 other amino acids are hydrophobic (Fig. 6 , A). With PI3K, emodin forms hydrogen bonds with Tyr867 and Asp 964. The interactions of emodin and Glu880, Val882, Trp812, Ile831, Ile963, and Ile879 interactions are hydrophobic (Fig. 7 , A). In the case of endocrocin and AKT interactions, there are hydrogen bonds with Glu230, Ala232, Lys160, and Asp293. In addition, there are hydrophobic interactions with 8 other amino acids (Fig. 6 , B). With PI3K, endocrocin forms only one hydrogen bond, namely with Asp963. Furthermore, there are hydrophobic interactions with 12 additional amino acids (Fig. 7 , B). Luteolin forms hydrogen bonds with Lys181 and Asp293 amino acids of AKT, complemented by 8 other hydrophobic interactions (Fig. 6 , C). With PI3K, luteolin forms one hydrogen bond with Tyr867 and Ala885 and two hydrogen bonds with Val882. Furthermore, there are hydrophobic interactions with 8 other amino acids (Fig. 7 , C). Finally, Quercetin forms four hydrogen bonds with Asp293, Lys181, Ala232, and Glu230 of AKT. In addition, there are 8 hydrophobic interactions (Fig. 6 , D). With PI3K, quercetin forms hydrogen bonds with Ser806 and Val882. This is complemented by hydrophobic interactions with 10 other amino acids (Fig. 7 , D). The specific interactions between emodin, endocrocin, luteolin, and quercetin with the target proteins AKT and PI3K highlight their potential to modulate the function of these proteins. It is noteworthy that while all four compounds formed hydrogen bonds with key residues in AKT, the variety and number of interactions differ, which may influence their affinity and specificity. The prevalence of hydrophobic interactions alongside hydrogen bonds, particularly with PI3K, underscores the potential of these compounds to anchor firmly within the binding pockets, possibly conferring stable interactions and effective inhibition. Such differential binding patterns could translate into varying degrees of therapeutic efficacy and selectivity among these compounds. 3. DISCUSSION Although chemotherapy is the most commonly used treatment, it also kills normal cells, causing many side effects. Therefore, it is urgent to develop novel alternative therapeutic strategies to overcome these problems. Many phytochemicals have been isolated from various plants that have regulatory effects on the targets that are considered in our study. We hypothesized that RO extract, either single or in combination with chemotherapy compounds, may effectively modulate the immune system (TNFa/COX-2/Arginase), inhibit angiogenesis and progression of metastasis (VEGFa/NOS/NO/MMP-2) via regulation of PI3K/Akt signaling pathway. During the study, the effect of RO on 5-FU-induced apoptosis in A549 cells was examined. MTT assay showed that RO alone did not induce noticeable inhibition of growth of A549 cells. This is interesting as according to our previous research, RO seed extract expressed strong cytotoxic activity on two tested cancer cell lines (HT29 and MCF-7) at even 0.125 mg DW/mL concentration 12 . Although RO extract did not possess growth-inhibiting activity in A549 cells, we assumed that acting synergically could increase the cytotoxic properties of chemotherapeutic agents like Fluorouracil. The speculations made based on earlier studies, where RO extract when combined with NG-nitro-L-arginine methyl ester (NOS inhibitor) and NG-hydroxy-nor-L-arginine (arginase inhibitor) increased their therapeutic effects probably by regulating redox homeostasis 12 . The experiments were done in an in vivo rat mammary carcinogenesis model and based on the obtained data RO extract possessed a modulating effect on 5-FU. It is important to point out that RO expressed a synergic effect rather than an additive as RO extract did not show any growth-inhibiting effect at the concentration used in combined treatment. The modulating properties of RO seed extract could have great importance, taking into account that modulation of the anticancer effects of chemotherapy drugs through plant extracts or derived compounds is a promising strategy to overcome drug resistance and reduce side effects 19 . To further understand biochemical mechanisms underlying modulating properties RO on 5-FU different biochemical parameters were explored including quantitative changes of TNFa, VEGFa, COX-2, and MMP-2, regulation of PI3K/Akt pathway, assessment of apoptosis, etc. We considered the PI3K/Akt signaling pathway taking into account that it is a major signaling pathway in various types of cancer. It controls the hallmarks of cancer, including cell survival, angiogenesis, inflammation, metastasis, and metabolism. According to the literature, the vascular endothelial growth factor (VEGF) is the most potent stimulant of angiogenesis and can activate NOX isoforms either directly or indirectly through PI3K/Akt induction 6 . After activation by VEGF, Akt promotes the proliferation, migration, and survival of endothelial cells, thus affecting angiogenesis. This finding also provides lateral support for the conclusion that endothelial nitric oxide synthase (eNOS), which controls vascular tone, is a specific substrate of Akt1 in endothelial cells 5 . The subsequent production of superoxide and hydrogen peroxide is necessary for the regulation of transcription factors, which promote angiogenesis, including NF-κB, MMPs, COX-2, and HIF-1α. COX-2 is up-regulated in many malignant cancers, including gastric, colon, breast, esophagus, pancreas, hepatocellular carcinoma, and NSCLC. The overexpression of COX-2 effectively potentiates the cisplatin and other chemotherapy drug resistance of NSCLC cells by promoting EMT. NS398, a COX-2 inhibitor, induced apoptosis and additionally potentiated chemosensitivity to cisplatin-mediated apoptosis in human non-small cell lung cancer by targeting the AKT 20 . Studies indicate MMP-2 siRNA inhibited lung cancer cell-induced tube formation of endothelial cells in vitro ; the addition of recombinant human-MMP-2 restored angiogenesis. Our research obtained results showed that RO extracts significantly decreased the quantities of TNFα, VEGFa, COX-2, and MMP2 in A549 cancer cells in combination with 5-FU. Inflammatory cytokines, growth factors, and their receptors, such as TNF, TNFR, VEGF, and VEGFR, act as positive regulators to transmit signals to mTOR through the PI3K/Akt pathway 5 . PI3K/Akt signaling blocks the expression of proapoptotic proteins reduces tissue apoptosis and increases the survival rate of cancer cells. Akt inhibits the proapoptotic factors Bad and procaspase-9 through phosphorylation and induces the expression of the proapoptotic factor Fas ligand. In addition, Akt activation is associated with resistance to increased apoptosis induced by TNF. During routine chemotherapy, no treatment interval exists, allowing resistant cells to be generated and leading to tumor regeneration. The PI3K/Akt signaling pathway is important for the drug resistance of different types of cancer, such as lung cancer and esophageal cancer. For NSCLC cells with high Akt expression, the use of PI3K/Akt signaling pathway inhibitors increases their cell apoptosis induced by chemotherapy and reduces their resistance to chemotherapy 21 . Therefore, inhibition of the PI3K/Akt signaling pathway, which has been shown to regulate cancer cell apoptosis can serve as a new direction for future research on cancer treatment 5 . The importance of this work is also that the obtained results touch on such a question as plant pro-oxidation. Increased malondialdehyde and nitrite ions are present in the cellular environment, indicating increased ROS and RNS. According to the literature, the latter is also regulated by Akt 6 . In addition, the change in Akt activity also affects the regulation of Caspase-3 activity and therefore apoptosis. Given that cell leakage may be a factor in RNS- and ROS-mediated apoptosis, the alteration of Caspase-3 activity was observed. The possibility of chromatin segmentation and condensation under the effect of herb and combination was also studied by Hoechst stain to further elucidate the stimulation of apoptosis. Hoechst staining revealed an increase in the rate of apoptotic cells after treatment with 5-FU (40 uM) or RO alone. The combination of 5-FU + RO synergistically evoked Caspase-3 activity, thus RO elevated the frequency of 5-FU-induced apoptosis. The results obtained in the case of combinations of herbs and chemotherapeutic agents showed a decrease in TNFa and VEGFa and an increase in NO and MDA quantity. The latter is indicative of ROS/RNS-mediated cytotoxicity of herbs in the tumor microenvironment. A decrease in COX-2, Arginase, and MMP-2 was observed in the A549 under the influence of herb extracts and combinations. The work is also highlighted by considering the herb together with a classical chemotherapeutic compound. As a classic chemotherapeutic compound, 5-fluorouracil was used, which has a broad spectrum effect and is used in chemotherapeutic cocktails 22 . It was important to observe the herb-drug interaction and identify whether there is a synergistic effect between this herb and 5-FU. Even though several works show the anticancer effect of various herbs, and our in vivo model showed the effective use of this herb against breast cancer in combination with L-arginine metabolic pathway inhibitors, there are few works, which revealed the mechanisms by which this effect occurs. The work is also valuable in that, by using a multi-component decoction of the medicinal plant, the possible protection of these compounds against PI3K and Akt enzymes was also clarified by parallel in silico research. The research has 3 main findings. Elucidated the mechanisms of the anticancer effect of an unexplored herb by looking at the TNFa/PI3K/Akt/COX-2/ARG/NOS/ROS/RNS/Caspase-3 pathway, demonstrated herb-drug synergistic interactions affected by different compounds, which were revealed based on in silico studies. These compounds had also the greatest affinity for PI3K/Akt, which may play a key role in RO extracts with promising anticancer properties. An important finding of the work is also the fact that the quantitative images of MDA and nitrite anions differ from our previous studies in vivo 12 . During earlier in vivo studies on the rat mammary carcinogenesis model, a decrease in the amount of malondialdehyde and nitrite ions was observed in the blood, while an increment of their quantity was detected in the cell culture. The circumstance of selective effect is also seen here, thanks to which it is possible to deliver these active compounds to the tumor environment itself with the use of delivery systems and to leave a point effect on the targets presented 23 . The purpurin (naturally occurring anthraquinone) could effectively kill A549 cancer cell lines and lead to cell death, thus conforming to increased cytotoxicity, production of ROS-mediated enhancement of lipid peroxidation, nuclear fragmentation, and apoptosis. The study demonstrates that purpurin inhibits the phosphorylated PI3K/AKT molecules mediated cyclin-D1, thereby inducing apoptosis by observing increased proapoptotic mediators Bax cleaved PARP, cytochrome-c, caspase-9, and caspase-3; and decreased Bcl-2 expression in the lung cancer cell lines 24 . Later we tried to elucidate the main compounds that contribute promising anticancer properties of RO extract. In our previous research works more than 200 phytochemicals were identified in the ethanol extract of RO ethanol extract based on LC-Q-Orbitrap-HRMS analysis. The full list of identified compounds in RO ethanol extract is presented in earlier work 12 . Further in silico analyses revealed that 4 of these compounds (namely: endocrocin, emodin, luteolin, and quercetin) have a high affinity for PI3K and Akt, indicating that the downregulation of the PI3K/Akt pathway by the herbs may be responsible for their beneficial effects on the quantitative changes in the explored factors and enzymes. The results demonstrate that all 4 compounds form at least 2 hydrogen bonds and at least 6 hydrophobic interactions with amino acids of the binding pockets of both AKT and PI3K. The only exception is the endocrine-PI3K interaction, where there is only one hydrogen bond. Nevertheless, this is amply compensated with an additional 12 hydrophobic interactions. The analysis indicates strong interactions in the case of all 8 ligand-protein pairs, which have the potential to change both proteins’ function and achieve biological modulation of physiological pathways. These findings imply that the unique binding patterns of these compounds may contribute to varying therapeutic efficacies and selectivities, highlighting their promising potential for modulating the functions of AKT and PI3K. Literature data partially confirms the obtained results based on in silico studies. Particularly, luteolin, a bioactive flavone derivative present mainly in its shell, exerts breast cancer-inhibiting properties through an anti-angiogenesis mechanism by inhibiting VEGF production and its binding with the receptor 23 . In addition, it also downregulates epithelial-mesenchymal transition markers and lowers metastatic activity. Studies have shown that another compound quercetin reduces tumor weight by targeting VEGFR2 through the Akt/mTOR/P70S6K signaling pathway 25 . Emodin, which is another selected compound based on in silico experiments, inhibits cancer growth by suppressing the expression of MMP7, MMP9, VEGF, EMT, N-cadherin, b-catenin, and Snail based on literature data. It also inhibits the Wnt/b-catenin signaling pathway by downregulating target genes, including c-Myc, Cyclin-D1, and TCF4. According to the literature, endocrocin is reported to have anticancer properties, although there is a lack of available data about the possible mechanisms of its action 26 . Based on in silico studies, we assumed that these 4 compounds could have important contributions to the overall promising anticancer properties of RO extract. Further in vitro and in vivo evaluation of their anticancer potential combined and in different combinations could have great importance. In conclusion, this study revealed the potential of R. obtusifolius seed alcoholic extract as an adjunct therapy in cancer treatment, specifically in combination with the classical chemotherapeutic agent 5-fluorouracil. The study allowed us to find several key insights into the mechanisms underlying the anticancer effects of RO and its synergy with 5-FU. We extensively explored the TNFa/VEGFa//PI3K/Akt/COX-2/ARG/NOS/ROS/RNS/Caspase-3 pathway during the study, revealing a complex interplay of factors influenced by RO and 5-FU. The combination of RO and 5-FU demonstrated a synergistic effect on various cellular components. This finding suggests that RO, while not directly inhibiting the growth of A549 cells on its own, can enhance the cytotoxic properties of 5-FU, potentially leading to more effective cancer cell eradication. The in silico analysis identified specific compounds within RO with high affinities for PI3K and Akt, hinting at their potential role in mediating the observed therapeutic effects. This computational approach deepens our understanding of the molecular interactions involved. Notably, the study revealed the selective effects of RO on MDA and nitrite ions in different environments. This selective action suggests the possibility of targeted drug delivery systems to achieve localized therapeutic effects while minimizing systemic side effects. Overall, this research contributes significantly to the field of cancer therapeutics by unraveling the complex molecular mechanisms underpinning the anticancer effects of RO and its synergistic relationship with 5-FU. The detailed molecular analysis reveals that emodin, endocrocin, luteolin, and quercetin present in RO extract exhibit distinctive interaction profiles with the target proteins AKT and PI3K․ These findings pave the way for further investigations into the development of novel, targeted cancer treatment strategies that harness the potential of medicinal plants like R. obtusifolius in the research. 4. Methods 4.1. Chemicals and reagents All chemicals were purchased from Sigma-Aldrich (USA) and Abcam (UK). Antibodies against TNFa (ab46087), VEGFa (ab193555), MMP-2 (ab92536), COX-2 (ab38898), PI3K and phosphorylated (p)-PI3K (ab191606), as well as ELISA kits for AKT and p-AKT (ab179463) were purchased from Abcam. 4.2. Plant material The seeds of Rumex obtusifolius L. were harvested from the Tavush region of Armenia (1400–1600 m height above mean sea level) according to the protocol described before 27 . Identification of plant material was carried out at the YSU Department of Botany and Mycology by Dr. Narine Zakaryan. Plant materials were deposited at the Herbarium of YSU, where the Voucher specimen serial number was given (ERCB 13208). The collection of plant material complied with relevant institutional, national, and international guidelines and legislation. Rumex obtusifolius L., commonly known as broad-leaved dock, is an edible plant widely distributed and commonly found throughout Armenia. It is not on the list of Endangered species in Armenia( https://worldrainforests.com/biodiversity/en/armenia/EN.html / https://www.iucnredlist.org/search?query=Rumex%20obtusifolius%20&searchType=species / https://cites.org/eng/search?search_api_fulltext=Rumex+obtusifolius+ ). The plant is not only prevalent in natural settings but also routinely collected by local populations for culinary purposes. There is no specific prohibition or regulatory constraint on the collection of this plant in Armenia, and it is a common sight at local markets, where it is sold after being gathered from the wild. This widespread availability and cultural integration into local diets supports the ethical sourcing and utilization of Rumex obtusifolius for research purposes under the conditions described in our study. For our research, we specifically collected only the seeds of Rumex obtusifolius . This method of collection ensures minimal impact on the natural populations of the plant, as it does not involve uprooting or damaging the plants themselves. We ensure our research practices are sensitive to ecological and conservation concerns, even in cases where no formal collection restrictions exist. As such, our study strictly adheres to general ethical guidelines for botanical research, despite the lack of specific regulations surrounding the collection of Rumex obtusifolius in Armenia. 4.3. Plant crude extract The grounded seeds were extracted by maceration with 96% ethanol at a 10:1 solvent-to-sample ratio (v/w). Stock solutions of 50 mg DW/mL crude ethanol extract were prepared as described earlier 28 . The percent yield was 10.60 ± 2.31%. 4.4. Cell cultures Human lung adenocarcinoma A549 cell culture was obtained from ATCC (cat # CCL-185) and maintained in DMEM medium supplemented with L-glutamine (2 mmol/L), sodium pyruvate (200 mg/L), fetal bovine serum (100 mL/L), and antibiotics (100 U/mL penicillin and 100 µg/L streptomycin). Cells were grown at 37°C under a humidified atmosphere with 5% CO 2 in a Biosmart (Biosan, Latvia) as described before 29 . Cultured cells were regularly examined for the presence of mycoplasma contamination using the Universal Mycoplasma Detection Kit from ATCC (Manassas, Virginia, USA). 4.5. MTT cytotoxicity test The MTT test was performed as described previously 30 to assess the growth inhibition of A549 cells exposed to different concentrations of the R. obtusifolius extract for 4, 24, or 72 h. 4.6. ELISA of TNFa, VEGFa, COX-2, MMP-2, and Akt. A549 cells (2 × 10 5 ) were cultured in 12-well plates and incubated for 24 h. After incubation, the cell medium (630 𝜇L) was replaced and the cells were treated with PBS and 1% Ethanol solution (Control, A549C), 5-FU (40𝜇M), RO (0.25mg/mL), and RO + 5- FU (0.25mg/mL + 40𝜇M) for 24 h and then the culture medium was harvested. TNFa, VEGFa, and MMP-2 in the supernatant were quantified according to the manufacturer's instructions. Cells from each group were collected (trypsinized, neutralized, centrifuged), lysed on ice with Lysis buffer, collected in a centrifuge tube, and further lysed for 10 min. After centrifugation at 13,000 × g for 10 min at 4°C, the supernatant was collected. Changes in the levels of COX-2 and Akt were measured using ELISA kits, according to the manufacturer's instructions. Protein concentration in cell culture medium and lysates were measured with a Bradford method. Each test sample (70 𝜇L) was added to three different passages, which were triplicated. 4.7. Cells preparation for Arginase, NOS, and NO activity, MDA analysis A549 cells were seeded in 24-well (5 × 10 4 cells per well) plates and incubated for 24 h. After incubation, the medium in wells (450 𝜇L) was refreshed. The cells were treated with 50 𝜇L control or test compounds with the following final concentrations: PBS, 1% ethanol (Control, A549C), 5-FU (40𝜇M), RO (0.25mg/mL), and RO + 5-FU (0.25mg/mL + 40𝜇M). After 24 h incubation, the supernatant without cells was discarded. Cells from each group were collected (trypsinized, neutralized, centrifuged), lysed on ice with Lysis buffer, collected in a centrifuge tube, and further lysed for 10 min. The supernatant was collected after centrifugation at 13,000 × g for 10 min at 4°C. The levels of Nitrite anions, MDA, Arginase, and NOS were quantified according to the methods described below 12 , 31 . Each test sample (50 𝜇L) was added to five different passages, which were triplicated. 4.8. NO quantity measurement NO levels in the cell culture medium were determined as nitrite anions. Griess assay was used for measurement as described before 32 . 100 𝜇L Griess reactant was added to 100 𝜇L of each sample. The supernatants were transferred to the tubes containing pellets of cadmium and incubated at room temperature for 12 h to convert nitrate to nitrite. The samples’ absorbance was measured at λ = 550 nm and the NO quantity was calculated based on a standard curve prepared with NaNO 2 . 4.9. MDA assay MDA quantity in the cell culture medium was determined with a colorimetric assay using the Ohkawa thiobarbituric acid-malondialdehyde method 33 . 4.10. Arginase activity The modified Diacetyl Monoxime colorimetric method assessed the arginase activity in A549 cell lysates 34 . 4.11. NOS activity Nitric oxide synthase activity (µmol citrulline/mg protein) in A549 cell lysates was measured by the conversion of L-arginine to L-citrulline 35 . 100µl of cell lysates was added to 200 mL of reaction mixture (50 mmol/L Tris buffer, pH 7.4, containing 10 mmol/L dithiothreitol (DTT), 10 µmol/L tetrahydrobiopterin (THB4), 10 µg/mL calmodulin, 1 mmol/L NADPH, 4 µmol/L flavin adenine dinucleotide (FAD), 4 µmol/L flavin mononucleotide (FMN), and 2 µmol/L L-arginine). The assay was carried out at 37 o C, and it was terminated with 2 mL of ice-cold stop buffer (20 mmol/L CH3COONa, pH 5.5, containing 2 mmol/L EDTA, and 1 mmol/L L-citrulline). Assays were systematically performed with Ca 2+ (1 mmol/L CaCl2) or without Ca 2+ (0 mmol/L CaCl2) to measure total versus Ca 2+ -independent NOS activities. The Ca 2+ -dependent NOS activity was calculated as total NOS activity minus Ca 2+ -independent NOS activity. All assays were performed in triplicate on aliquoted samples (to avoid freezing/thawing cycles). The results were normalized for protein content. 4.12. Phospho-PI 3 kinase p85 + Total In-Cell ELISA assay A549 cells (1.5x10 4 cells per well) were seeded in the 96-well plates treated for tissue culture. After 24 h incubation, the cell medium (180 𝜇L) was refreshed and the cells were treated with 20𝜇L control or test compounds with the following final concentrations: PBS, 1% ethanol solution (Control, A549C), 5-FU (40𝜇M), RO (0.25mg/mL), and RO + 5-FU (0.25mg/mL + 40𝜇M). The calculations during the seeding of the cells were done in a way to reached approximately 80% confluency at fixation time. After 24 h exposure, the medium was discarded and cells were fixed with 100 µL of 4% formaldehyde in PBS. Crystal Violet was used to stain cells for normalizing readings in 450nm for Phospho-PI 3 kinase p85 + Total. The measured OD450 readings were corrected for cell number by dividing the OD450 reading for a given well by the OD595 reading for that well. This relative cell number was then used to normalize each reading. Total and phospho-PI 3 kinase p85 were each assayed in triplicate using the phospho- and total PI 3 Kinase p85 antibodies included in the PI 3 Kinase Kit. Phospho-PI 3 kinase p85 and Total PI3K levels were measured using an In-Cell ELISA kit (ab207484), according to the manufacturer's instructions. 4.13. Caspase-3/CPP32 Colorimetric assay A549 cells (5 × 10 5 cells per well) were cultured in 6-well plates and incubated for 24 h. Then, the cell medium (900 𝜇L) was refreshed and the cells were treated with 10 𝜇L of PBS + 1% ethanol solution (control, A549C) or test compounds with the following final concentrations: 5-FU (40 𝜇M), RO (0.25 mg/mL), and RO + 5- FU (0.25 mg/mL + 40 𝜇M). After 24 h the cells were harvested. Each test sample (100 𝜇L) was added to three different passages, which were triplicated. Cells were resuspended in 50 µL of chilled Cell Lysis Buffer and incubated on ice for 10 minutes. Then, cell lysate was centrifuged for 1 min (10,000 x g). After that supernatant (cytosolic extract) was transferred to a fresh tube and put on ice for immediate assay. Fold-increase in CPP32 activity has been determined by comparing these results with the level of the uninduced control. Optical density values were corrected taking into account the number of cells. All steps were performed according to the protocol presented in the Caspase-3/CPP32 Colorimetric Assay Kit (K106, BioVision) instructions. 4.14. Analysis of apoptosis by Hoechst 33258 staining The percentage of apoptotic cells was evaluated as previously described 36 . A549 cells (2×10 5 cells/mL) were treated with vehicle or 5-FU (40 𝜇M), RO (0.25 mg/mL), and RO + 5- FU (0.25 mg/mL + 40 𝜇M) for 24 hours, respectively. After treatment cells were washed with PBS and fixed with 4 %paraformaldehyde in PBS for 10 min. Then cells were washed twice with PBS for 5 min and stained with Hoechst 33258 reagent (10 𝜇g/mL) for 10 mins at room temperature in the dark. Then cells were washed with PBS and analyzed under a fluorescence microscope (x250 magnification) (Zeiss, Germany). The Hoechst 33258 staining allows the identification of apoptotic cells based on nuclear morphology. Cells with typical morphological changes, such as karyopyknosis, hyperfluorescence, nuclear fragmentation, and apoptotic bodies, were considered apoptotic. All variants were examined in duplicate. For each treatment variant, 500 cells were scored and the percentage of apoptotic cells was calculated as follows: % apoptotic cells = (the number of apoptotic cells/500 cells)*100. 4.16. Preparation of Protein structures The crystallographic structures of PI3K and AKT were procured from the Protein Data Bank (PDB) database ( https://www.rcsb.org/ ), using the identifiers 6AUD and 2JDO, respectively. Visualization and preliminary assessment of these structures were performed with the PyMOL Molecular Graphics System (Schrödinger, LLC). The retrieved crystallographic structures were subject to preprocessing, which involved removing extraneous entities such as water molecules, ions, and other non-protein moieties contained within the structures. Simultaneously, the ligands co-crystallized with each protein structure were separated and retained for redocking validation experiments. The resulting streamlined protein structures were then used for docking explorations. The extracted ligands, on the other hand, were reserved for ensuing redocking studies as controls. 4.17. D ocking Ligand docking and binding site analysis with PyMOL and Autodock/Vina were used for docking 37 . The protein and ligand structures were prepared using Autodock Tools 38 . During a typical procedure, the "exhaustiveness" parameter was calibrated to 8 and standard parameters suggested by the program creators were used to ensure the fidelity of the results. The compounds were sorted based on their binding strengths. The 2D binding mode analysis of best docking scores was performed using LigPlot + software (EMBL-EBI). 4.18. Statistic analysis All results are presented as means ± SEM. We analyzed the data either by one-way ANOVA or by its non-parametric analog Kruskal-Wallis test based on the normality test performed followed by Dunn's test was used to evaluate the statistical significance of the TNFa, VEGFa, MMP-2, COX-2, arginase, NOS, MDA, nitrite anions, Caspase-3, and apoptosis rate results. The significance of the results obtained for PI3K and Akt was assessed using two-way ANOVA and Tukey's multiple comparisons tests. Statistical analyses were performed using GraphPad Prism 8 software (San Diego, CA, USA), and a significance level of p < 0.05 was deemed statistically significant. Abbreviations Akt, protein kinase B; ANOVA, analysis of variance; Bcl-2, B-cell lymphoma-2; COX-2, cyclooxygenase-2; CoQ0, Coenzyme Q0; ELISA, enzyme-linked immunosorbent assay; EMT, Epithelial-mesenchymal transition; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; HBA, hydrogen bond acceptors; HBD, hydrogen bond donors, IκBα, an inhibitor of NF-κBα; IL-6, interleukin-6; MAPK, mitogen-activated protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; MMP-2, matrix metalloproteinase-2; MW, molecular weight; NOXs, NADPH oxidases; NF-κB, nuclear factor-κB; NOS, nitric oxide synthase; NSCLC, non-small cell lung cancer; PI3K, phosphoinositol-3-kinase; PG E2, prostaglandin E2; RO, Rumex obtusifolius extract; ROS, reactive oxygen species; TNF-α, tumor necrosis factor alpha; THB4, tetrahydrobiopterin; VEGF, vascular endothelial growth factor. Declarations 5. Availability of data and materials The data used to support the findings of this study are included in the articles. 6. Declaration of interest The authors declare no conflicts of interest in this article. 7. Authors' contributions The study's conception and design were the results of collective contributions from all authors. The investigations and analysis of results were carried out by MG, NA, HJ, SH, EN, GS, and TH. MG and NA wrote the manuscript. Assessment of apoptosis rate by Hoechst 33258 staining and analysis of apoptosis performed by TH. The docking and ADME of the top compounds present in the RO ethanolic extract were performed by SG. NA, MG, HJ, ZK, and AM directed the project, corrected, and edited the manuscript. All authors participated in the revision and approval of the final version of the manuscript. 8. Acknowledgments Plant materials were identified by Dr. Narine Zakaryan from the Department of Botany and Mycology at Yerevan State University (YSU).This work was supported by the Science Committee of MESCS RA through research projects numbered 21T-1F283, 21AG-1F068, and 23LCG-1F010. References Gordaliza, M. Natural products as leads to anticancer drugs. Clinical and Translational Oncology 9, 767–776 (2007). Cragg, G. M. & Pezzuto, J. M. Natural Products as a Vital Source for the Discovery of Cancer Chemotherapeutic and Chemopreventive Agents. Medical Principles and Practice 25, 41–59 (2016). Hou, X. L. et al. Suppression of Inflammatory Responses by Dihydromyricetin, a Flavonoid from Ampelopsis grossedentata, via Inhibiting the Activation of NF-κB and MAPK Signaling Pathways. J Nat Prod 78, 1689–1696 (2015). Yin, S. Y., Wei, W. C., Jian, F. Y. & Yang, N. S. Therapeutic applications of herbal medicines for cancer patients. Evidence-based Complementary and Alternative Medicine vol. 2013 Preprint at https://doi.org/10.1155/2013/302426 (2013). He, Y. et al. Targeting PI3K/Akt signal transduction for cancer therapy. Signal Transduction and Targeted Therapy vol. 6 Preprint at https://doi.org/10.1038/s41392-021-00828-5 (2021). Koundouros, N. & Poulogiannis, G. Phosphoinositide 3-Kinase/Akt signaling and redox metabolism in cancer. Frontiers in Oncology vol. 8 Preprint at https://doi.org/10.3389/fonc.2018.00160 (2018). Fruman, D. A. et al. The PI3K Pathway in Human Disease. Cell vol. 170 605–635 Preprint at https://doi.org/10.1016/j.cell.2017.07.029 (2017). Jiang, B. H. & Liu, L. Z. Chapter 2 PI3K/PTEN Signaling in Angiogenesis and Tumorigenesis. Advances in Cancer Research vol. 102 19–65 Preprint at https://doi.org/10.1016/S0065-230X(09)02002-8 (2009). Pang, L. et al. Cox-2 Inhibition Protects against Hypoxia/Reoxygenation-Induced Cardiomyocyte Apoptosis via Akt-Dependent Enhancement of iNOS Expression. Oxid Med Cell Longev 2016, (2016). Zhao, Y. et al. COX-2 is required to mediate crosstalk of ROS-dependent activation of MAPK/NF-κB signaling with pro-inflammatory response and defense-related NO enhancement during challenge of macrophage-like cell line with Giardia duodenalis. PLoS Negl Trop Dis 16, (2022). Chetty, C., Lakka, S. S., Bhoopathi, P. & Rao, J. S. MMP-2 alters VEGF expression via αVβ3 integrin-mediated PI3K/AKT signaling in A549 lung cancer cells. Int J Cancer 127, 1081–1095 (2010). Ginovyan, M. et al. Anti-cancer effect of Rumex obtusifolius in combination with arginase/nitric oxide synthase inhibitors via downregulation of oxidative stress, inflammation, and polyamine synthesis. Int J Biochem Cell Biol 158, 106396 (2023). Choudhari, A. S., Mandave, P. C., Deshpande, M., Ranjekar, P. & Prakash, O. Phytochemicals in cancer treatment: From preclinical studies to clinical practice. Front Pharmacol 10, 1–18 (2020). Abd El-Aleem, S. A., Abdelwahab, S., AM-Sherief, H. & Sayed, A. Cellular and physiological upregulation of inducible nitric oxide synthase, arginase, and inducible cyclooxygenase in wound healing. J Cell Physiol 234, 23618–23632 (2019). Wang, S., Yan, Y., Cheng, Z., Hu, Y. & Liu, T. Sotetsuflavone suppresses invasion and metastasis in non-small-cell lung cancer A549 cells by reversing EMT via the TNF-α/NF-κB and PI3K/AKT signaling pathway. Cell Death Discov 4, (2018). Yang, H. L. et al. Anti-EMT properties of CoQ0 attributed to PI3K/AKT/NFKB/MMP-9 signaling pathway through ROS-mediated apoptosis. Journal of Experimental and Clinical Cancer Research 38, (2019). Alqahtani, S. In silico ADME-Tox modeling: progress and prospects. Expert Opinion on Drug Metabolism and Toxicology vol. 13 1147–1158 Preprint at https://doi.org/10.1080/17425255.2017.1389897 (2017). Lipinski, C. A., Lombardo, F., Dominy, B. W. & Feeney, P. J. Experimental and Computational Approaches to Estimate Solubility and Permeability in Drug Discovery and Development q Settings . Advanced Drug Delivery Reviews vol. 46 www. elsevier.com/locate/drugdeliv (2001). Bazrafshani, M. S. et al. The prevalence and predictors of herb-drug interactions among Iranian cancer patients during chemotherapy courses. BMC Complement Med Ther 23, 1–11 (2023). Alhamed, A. S. et al. Blockade of store-operated calcium entry sensitizes breast cancer cells to cisplatin therapy via modulating inflammatory response. Saudi Pharmaceutical Journal 31, 245–254 (2023). Sanaei, M. J., Razi, S., Pourbagheri-Sigaroodi, A. & Bashash, D. The PI3K/Akt/mTOR pathway in lung cancer; oncogenic alterations, therapeutic opportunities, challenges, and a glance at the application of nanoparticles. Translational Oncology vol. 18 Preprint at https://doi.org/10.1016/j.tranon.2022.101364 (2022). Sundaram, M. K. et al. Combinational Use of Phytochemicals and Chemotherapeutic Drugs Enhance Their Therapeutic Potential on Human Cervical Cancer Cells. Int J Cancer Manag 12, (2019). Parveen, A. et al. Phytochemicals targeting VEGF and VEGF-related multifactors as anticancer therapy. Journal of Clinical Medicine vol. 8 Preprint at https://doi.org/10.3390/jcm8030350 (2019). Bo, S. et al. Purpurin, a anthraquinone induces ROS-mediated A549 lung cancer cell apoptosis via inhibition of PI3K/AKT and proliferation. Journal of Pharmacy and Pharmacology 73, 1101–1108 (2021). Lotfi, N. et al. The potential anti-cancer effects of quercetin on blood, prostate and lung cancers: An update. Frontiers in Immunology vol. 14 Preprint at https://doi.org/10.3389/fimmu.2023.1077531 (2023). Khaliq, T., Akhter, S., Sultan, P. & Hassan, Q. P. Critical review on Rumex dentatus L. a strong pharmacophore and the future medicine: Pharmacology, phytochemical analysis and traditional uses. Heliyon 9, e14159 (2023). Ginovyan, M., Petrosyan, M. & Trchounian, A. Antimicrobial activity of some plant materials used in Armenian traditional medicine. BMC Complement Altern Med 17, 1–9 (2017). Ginovyan, M., Hovhannisyan, S., Javrushyan, H. & Sevoyan, G. Screening revealed the strong cytotoxic activity of Alchemilla smirnovii and Hypericum alpestre ethanol extracts on different cancer cell lines. 10, 12–22 (2022). Koss-Mikołajczyk, I., Kusznierewicz, B., Namieśnik, J. & Bartoszek, A. Juices from non-typical edible fruits as health-promoting acidity regulators for food industry. LWT - Food Science and Technology 64, 845–852 (2015). Koss-Mikołajczyk, I., Kusznierewicz, B., Wiczkowski, W., Sawicki, T. & Bartoszek, A. The comparison of betalain composition and chosen biological activities for differently pigmented prickly pear (Opuntia ficus-indica) and beetroot (Beta vulgaris) varieties. Int J Food Sci Nutr 70, 442–452 (2019). Avtandilyan, N., Javrushyan, H., Petrosyan, G. & Trchounian, A. The Involvement of Arginase and Nitric Oxide Synthase in Breast Cancer Development: Arginase and NO Synthase as Therapeutic Targets in Cancer. Biomed Res Int 2018, 1–9 (2018). Vodovotz, Y. Modified microassay for serum nitrite and nitrate. Biotechniques 20, 390–394 (1996). Zeb, A. & Ullah, F. A Simple Spectrophotometric Method for the Determination of Thiobarbituric Acid Reactive Substances in Fried Fast Foods. J Anal Methods Chem 1–5 (2016) doi: 10.1155/2016/9412767 . Avtandilyan, N., Javrushyan, H., Petrosyan, G. & Trchounian, A. The Involvement of Arginase and Nitric Oxide Synthase in Breast Cancer Development: Arginase and NO Synthase as Therapeutic Targets in Cancer. Biomed Res Int 2018, (2018). Combet, S., Balligand, J.-L., Lameire, N., Goffin, E. & Devuyst, O. A Specific Method for Measurement of Nitric Oxide Synthase Enzymatic Activity in Peritoneal Biopsies . Kidney International vol. 57 (2000). Yang, X., Wang, Y., Luo, J., Liu, S. & Yang, Z. Protective effects of YC-1 against glutamate induced PC12 cell apoptosis. Cell Mol Neurobiol 31, 303–311 (2011). Seeliger, D. & De Groot, B. L. Ligand docking and binding site analysis with PyMOL and Autodock/Vina. J Comput Aided Mol Des 24, 417–422 (2010). Trott, O. & Olson, A. J. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31, 455–461 (2010). Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Published Journal Publication published 24 Jun, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 27 May, 2024 Reviews received at journal 13 May, 2024 Reviews received at journal 02 May, 2024 Reviewers agreed at journal 23 Apr, 2024 Reviewers agreed at journal 23 Apr, 2024 Reviewers invited by journal 23 Apr, 2024 Editor assigned by journal 23 Apr, 2024 Editor invited by journal 22 Apr, 2024 Submission checks completed at journal 22 Apr, 2024 First submitted to journal 11 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4254380","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":294479736,"identity":"a4b30ed5-ace2-43f7-a6cb-adcde33e5bc7","order_by":0,"name":"Mikayel Ginovyan","email":"","orcid":"","institution":"Yerevan State University","correspondingAuthor":false,"prefix":"","firstName":"Mikayel","middleName":"","lastName":"Ginovyan","suffix":""},{"id":294479737,"identity":"a5722021-8bca-47b9-9b5b-74bc85dcffed","order_by":1,"name":"Hayarpi Javrushyan","email":"","orcid":"","institution":"Yerevan State University","correspondingAuthor":false,"prefix":"","firstName":"Hayarpi","middleName":"","lastName":"Javrushyan","suffix":""},{"id":294479738,"identity":"40fde4ad-6f3a-469d-8539-156657d7e2ac","order_by":2,"name":"Svetlana Hovhannisyan","email":"","orcid":"","institution":"Yerevan State University","correspondingAuthor":false,"prefix":"","firstName":"Svetlana","middleName":"","lastName":"Hovhannisyan","suffix":""},{"id":294479739,"identity":"f29f5b53-14bc-4b79-a8cb-1b271894d853","order_by":3,"name":"Edita Nadiryan","email":"","orcid":"","institution":"Yerevan State University","correspondingAuthor":false,"prefix":"","firstName":"Edita","middleName":"","lastName":"Nadiryan","suffix":""},{"id":294479751,"identity":"57933443-ae19-46ed-b74a-459a59ef551c","order_by":4,"name":"Gohar Sevoyan","email":"","orcid":"","institution":"L.A. Orbeli Institute of Physiology NAS RA","correspondingAuthor":false,"prefix":"","firstName":"Gohar","middleName":"","lastName":"Sevoyan","suffix":""},{"id":294479766,"identity":"df511f3d-2cb6-4235-85e3-1e18f273f07c","order_by":5,"name":"Tigran Harutyunyan","email":"","orcid":"","institution":"Yerevan State University","correspondingAuthor":false,"prefix":"","firstName":"Tigran","middleName":"","lastName":"Harutyunyan","suffix":""},{"id":294479767,"identity":"736c26ad-a89e-46e5-a943-0b896ef21c1a","order_by":6,"name":"Smbat Gevorgyan","email":"","orcid":"","institution":"Denovo Sciences Inc","correspondingAuthor":false,"prefix":"","firstName":"Smbat","middleName":"","lastName":"Gevorgyan","suffix":""},{"id":294479768,"identity":"ad12ae40-23d0-4207-bf37-7c28faa78bb7","order_by":7,"name":"Zaruhi Karabekian","email":"","orcid":"","institution":"L.A. Orbeli Institute of Physiology NAS RA","correspondingAuthor":false,"prefix":"","firstName":"Zaruhi","middleName":"","lastName":"Karabekian","suffix":""},{"id":294479769,"identity":"45a38416-59e8-4f85-90cf-d11cc57036a9","order_by":8,"name":"Alina Maloyan","email":"","orcid":"","institution":"Knight Cardiovascular Institute, Oregon Health \u0026 Science University","correspondingAuthor":false,"prefix":"","firstName":"Alina","middleName":"","lastName":"Maloyan","suffix":""},{"id":294479770,"identity":"390263e0-6f76-46bc-8371-96262b7c0efe","order_by":9,"name":"Nikolay Avtandilyan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYFCDw8wNQNKGWOUJIC2MIC1ppGg5ANZymLBic4kExs+8P+zy+I4ztkn83HE+cf6MBMZPN/BosZyRwCzNk5BcLHmYsU2y98ztxA03gCI5eLQYnDnAIAnUlrgBqEWCtw2oRSKBgZAW5p8zEurBWiT/tp0DOYz5N14txxvYJD4kHAZrkeZtO5DYcCOBDb8txxvbLD6kHU+ceZix2Vq2Ldl4w5mHbdZ4tRxmPnwjwaY6se/84YM337bZyc5vTz58G58WBgZwdIABiwSQcGxAEiEImD8ACXuilY+CUTAKRsGIAQAi01UFl8ZOAQAAAABJRU5ErkJggg==","orcid":"","institution":"Yerevan State University","correspondingAuthor":true,"prefix":"","firstName":"Nikolay","middleName":"","lastName":"Avtandilyan","suffix":""}],"badges":[],"createdAt":"2024-04-11 21:29:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4254380/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4254380/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-65816-5","type":"published","date":"2024-06-25T00:29:50+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":55290977,"identity":"119742f7-fa2a-4008-bc6b-353cfc568690","added_by":"auto","created_at":"2024-04-25 09:10:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":173456,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGrowth rate of A549 cells treated with RO extract for 4, 24, and 72h (A). The growth-inhibiting effect of 5-fluorouracil separately and in combination with the none-inhibitory concentration of RO extract (0.25 mg DW/mL) on A549 cells for 4 (B), 24 (C), and 72h (D). \u0026nbsp;Results represent means ± SD from three independent experiments; SD values did not exceed 15%.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4254380/v1/adb916f8e27882373a112b18.png"},{"id":55290972,"identity":"2f613511-ac45-4739-a9c0-b6454a9be06b","added_by":"auto","created_at":"2024-04-25 09:10:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":92638,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe influence of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eR. obtusifolius\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eextract alone and in combination with 5-FU on quantitative changes of TNFa (A), COX-2 (B), VEGFa (C) and MMP-2 (D) in A549 cells. Control - A549C, 5-Fluorouracil - 5-FU (40𝜇M), \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eRumex obtusifolius\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e - RO (0.25mg/mL), ROFU - RO+5-FU (0.25mg/mL + 40𝜇M). Each test sample was added to three different passages, which were triplicated (n=3, * - p≤0.05, ** - p≤0.01, ns – non-significant).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4254380/v1/4ca4a0c8532182ca90e63011.png"},{"id":55290516,"identity":"2dabf8af-c162-4288-a24d-2884f2d53b2c","added_by":"auto","created_at":"2024-04-25 09:02:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":55817,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of RO, 5-FU, and their combination on PI3K/Akt pathway in A549 cells (A-PI3K, B - Akt). Total and phospho-kinases were each assayed in triplicate using the phospho- and total Kinase antibodies included in the PI 3 Kinase and Akt kits (n=3, * - p≤0.05, ** - p≤0.01, *** - p≤0.001, **** - p≤0.0001). p85-PI3K - Phospho-PI 3 kinase p85, pS473AKT - phospho-Akt (Ser473).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4254380/v1/c7a92e9ff7ef1c2847f2c426.png"},{"id":55290518,"identity":"119f0621-977c-49ba-bae7-2e4af74b5374","added_by":"auto","created_at":"2024-04-25 09:02:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":208032,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStimulation of RNS and ROS by RO regulation of arginase (A) and NOS (B) activity, nitrite anions (C), and MDA (D) quantity in A549 cells. Each test sample was added to five different passages, which were triplicated (n=5, * - p≤0.05, ** - p≤0.01, *** - p≤0.001, **** - p≤0.0001).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4254380/v1/2e3e91481a294a18166f7061.png"},{"id":55290975,"identity":"67d06fb8-2332-444b-a8cc-9a37e5728eae","added_by":"auto","created_at":"2024-04-25 09:10:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":302375,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHoechst 33258 staining (blue) assay of apoptosis in A549 cells. Apoptotic cells are indicated with white arrows. The scale bar is 100 μm. (A) Untreated cells (A549C) have smooth edges and dispersed fluorescence. (B and E) 5-FU induced apoptosis as can be seen by the occurrence of pyknotic cells with condensed chromatin. (C and E) Incubation with RO elevated the number of apoptotic cells with rough edges of nuclei. (D and E) The combined treatment of cells with 5-FU+RO resulted in the occurrence of cells with signs of nuclei fragmentation and chromatin condensation. (E) Apoptosis rate evaluated by the Hoechst 33258 staining, *p\u0026lt;0.05 - compared with the RO, \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e**\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003ep\u0026lt;0.01 - compared with the control. (F) – caspase-3 activity evaluated by the colorimetric assay, \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003ep\u0026lt;0.05 - compared with the 5-FU, **p\u0026lt;0.01 - compared with the control.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4254380/v1/850c4d9cad685f2d47ea26a8.png"},{"id":55290974,"identity":"a5a83ae1-9461-48d1-a2bc-f18091f2d09b","added_by":"auto","created_at":"2024-04-25 09:10:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1152256,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e2D binding analysis and interaction types on AKT in combination with 3D visualization. A – Emodin (a), B – Endocrocin (b), C – Luteolin (c), D – Quercetin (d). Hydrogen bonding is indicated by green dotted lines, while the remaining interactions are hydrophobic.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-4254380/v1/90e50c42bf8a9bb9b669cb04.png"},{"id":55290520,"identity":"9689960d-2635-4a6c-abce-6fa6e5da19d1","added_by":"auto","created_at":"2024-04-25 09:02:49","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":997844,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e2D binding analysis and interaction types on Pi3K in combination with 3D visualization. A – Emodin (a), B – Endocrocin (a), C – Luteolin (c), D – Quercetin (d). Hydrogen bonding is indicated by green dotted lines, while the remaining interactions are hydrophobic.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-4254380/v1/0cac3dcb1dcb5fb086a08c7d.png"},{"id":59060142,"identity":"4c80818e-fe3d-4782-a9fd-91d8c74d04b6","added_by":"auto","created_at":"2024-06-26 00:30:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5007483,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4254380/v1/0fb0bf82-0d43-4a7c-96a0-56a93418ca9f.pdf"},{"id":55290519,"identity":"0a4f9ccf-af2e-4aaa-8dca-10ca1ba7297a","added_by":"auto","created_at":"2024-04-25 09:02:49","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":745090,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4254380/v1/65396ee580e53faeaa1b870b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"5-Fluorouracil and Rumex obtusifolius extract combination trigger A549 cancer cell apoptosis: Uncovering PI3K/Akt inhibition by in vitro and in silico approaches","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe continuous increase in cancer rates, failure of conventional chemotherapies to control the disease, and excessive toxicity of chemotherapies (in some cases including immunotherapy) clearly demand alternative approaches.\u0026nbsp;Natural products contain many constituents that can act on various targets in the body to induce pharmacodynamic responses\u0026nbsp;\u003csup\u003e1\u0026ndash;3\u003c/sup\u003e. Modulating biochemical and immune functions using medicinal plants and their products combined with chemotherapeutic agents has recently become an accepted therapeutic approach\u0026nbsp;\u003csup\u003e4\u003c/sup\u003e.\u0026nbsp;Lung cancer is one of the most common causes\u0026nbsp;of cancer-related deaths worldwide. According to the American Cancer Society, approximately\u0026nbsp;85% of all lung cancer deaths were the\u0026nbsp;result of non-small-cell lung cancer (NSCLC).\u003c/p\u003e\n\u003cp\u003eThere are several reasons for targeting the PI3K/Akt pathwaysignaling pathway and related extracellular and intracellular components. Correctly regulating the activity and quantity of these components can have a strong anticancer effect on cancer cells.\u0026nbsp;Several pro-tumorigenic processes converge on hyperactive PI3K/Akt signaling, and it is becoming increasingly evident that reactive oxygen species (ROS) metabolism is no exception to this\u0026nbsp;\u003csup\u003e5\u003c/sup\u003e.\u0026nbsp;Metabolic reconfiguration and the resulting generation of ROS are vital for facilitating the development of tumors. Dysregulated PI3K/Akt signaling is crucial in regulating numerous molecular processes that elevate ROS levels. This occurs either by directly influencing mitochondrial energy production and activating NADPH oxidases (NOXs), or indirectly by generating ROS as a metabolic byproduct\u0026nbsp;\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u0026nbsp;Comprehending the intricate relationship between ROS and PI3K/Akt signaling is important for devising effective therapeutic approaches to combat tumors reliant on this pathway. This significance is underscored by recent clinical trials showing limited efficacy of PI3K/Akt pathway inhibitors and the emergence of resistance. It could lead to the discovery of new biomarkers and metabolic vulnerabilities, as well as the development of more potent therapeutic combinations that disrupt redox balance and specifically target PI3K-driven tumors\u0026nbsp;\u003csup\u003e6,7\u003c/sup\u003e.\u0026nbsp;Chemotherapy and radiotherapy stand as primary treatments for cancer patients, and they induce apoptosis in cancer cells partly by increasing ROS levels. Agents like 5-FU, cisplatin, and other chemotherapeutic drugs trigger ROS production by influencing the electron transport chain. The heightened ROS levels then trigger cascades such as caspase activation, release of cytochrome C, and DNA damage, ultimately leading to apoptosis \u003csup\u003e\u003cspan lang=\"EN-US\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Given the potential pro-oxidative properties of herbs, we incorporated a chemotherapeutic compound alongside 5-FU in our model. This approach allows for the concurrent inhibition of the PI3K/Akt pathway while maintaining or increasing significant levels of ROS and reactive nitrogen species (RNS). Such elevation can effectively promote the induction of apoptosis.\u003c/p\u003e\n\u003cp\u003ePI3K pathway promotes metastasis by promoting tumor neovascularization, which is required for the metastatic spread of tumors. PI3K forms a complex with E-cadherin, \u0026beta;-catenin, and VEGFR-2 and is involved in endothelial signaling mediated by VEGF through the activation of the PI3K/Akt pathway \u003csup\u003e8\u003c/sup\u003e. The PI3K/Akt signaling pathway also promotes TNF-induced endothelial cell migration and regulates tumor angiogenesis.\u0026nbsp;TNF-\u0026alpha; plays a significant role in promoting the survival and metastasis of lung cancer. The levels of TNF-\u0026alpha; in tumor tissues and serum collected from patients with NSCLC substantially increase with the clinical stage of the tumor. Additionally, matrix metalloproteinases (MMPs) and cyclooxygenase-2 (COX-2) contribute to tumor angiogenesis. COX-2 stimulates endothelial angiogenesis primarily through the upregulation of the antiapoptotic protein Bcl-2 and activation of the PI3K/Akt signaling pathway \u003csup\u003e\u003cspan lang=\"HY\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u0026nbsp;A recent study indicates that\u0026nbsp;COX-2\u0026nbsp;inhibition\u0026nbsp;protects against\u0026nbsp;hypoxia/reoxygenation-induced\u0026nbsp;cardiomyocyte\u0026nbsp;apoptosis \u003cem\u003evia\u003c/em\u003eAKT-dependent enhancement of iNOS expression \u003csup\u003e9\u003c/sup\u003e. Preconditioning the cells with the COX-2 inhibitor NS398 resulted in decreased expressions of TNF\u0026alpha;, prostaglandin E2 (PGE2), and interleukin-6 (IL-6) pro-inflammatory factors. It was observed that inhibiting COX-2 could mitigate the increased release of nitric oxide (NO) and expression of inducible nitric oxide synthase (iNOS) induced by Giardia. Studies unveiled a crucial role of COX-2 in modulating the pro-inflammatory response and defense-related NO production in interactions between Giardia and macrophages\u0026nbsp;\u003csup\u003e10\u003c/sup\u003e. MMP-2 transcriptional suppression\u0026nbsp;decreased VEGF, PI3K protein levels, and AKT phosphorylation in lung cancer cells. MMP-2 suppression disrupted phosphatidylinositol 3-kinase (PI3K) dependent VEGF expression; ectopic\u0026nbsp;expression of myr-AKT restored VEGF inhibition. Studies\u0026nbsp;with either function blocking integrin-\u0026alpha;V\u0026beta;3 antibody or MMP-2 specific inhibitor (ARP-100) indicate that suppression of MMP-2 decreased integrin-\u0026alpha;V\u0026beta;3-mediated induction of PI3K/AKT leading to decreased VEGF expression. A549 xenograft tissue sections from mice that\u0026nbsp;were\u0026nbsp;treated with MMP-2 siRNA showed reduced expression of VEGF\u0026nbsp;\u003csup\u003e11\u003c/sup\u003e.Numerous studies provide evidence of the influence of natural compounds on the components within this pathway. For instance, the natural ubiquinone Coenzyme Q0 (CoQ0) has shown efficacy against the proliferation of various cancer cell lines such as HepG2, A549, and SW480, promoting apoptosis by elevating ROS levels. Treatment with LY249002, which blocks the PI3K/AKT pathway, significantly reduced NF\u0026kappa;B activation and MMP-9 levels .\u003c/p\u003e\n\u003cp\u003eThe analysis of the literature reveals that TNF-\u0026alpha;, VEGF-\u0026alpha;, COX-2, MMP-2, Caspase-3, and NOS are interconnected in various cancer processes. Their interrelations are mediated through the PI3K/Akt signaling pathway. This study focused on employing a combination of natural compounds and a chemotherapeutic agent to target these specific components to attain potent anticancer effects.\u003c/p\u003e\n\u003cp\u003eWe hypothesized that phytoextracts, either single or in combination with chemotherapy compounds, may effectively modulate the immune system\u0026nbsp;(TNFa/COX-2/Arginase),\u0026nbsp;inhibit angiogenesis and\u0026nbsp;progression of\u0026nbsp;metastasis\u0026nbsp;(VEGFa/NOS/NO/MMP-2)\u0026nbsp;via regulation of PI3K/Akt\u0026nbsp;signaling pathway. \u0026nbsp;In our previous research\u0026nbsp;works\u0026nbsp;we showed\u0026nbsp;the\u0026nbsp;promising\u0026nbsp;anticancer effect of\u0026nbsp;\u003cem\u003eRumex obtusifolius\u003c/em\u003e (RO) in\u0026nbsp;an \u003cem\u003ein vivo\u003c/em\u003e experimental breast cancer rat model, in parallel, its cytotoxic effect was elucidated against two cell cultures: MCF-7, and HT29\u0026nbsp;\u003csup\u003e12\u003c/sup\u003e.\u0026nbsp;The combined anticancer effects of inhibitors targeting the metabolic pathway of L-arginine have also been investigated. The findings demonstrate significant anti-tumor properties, including reductions in tumor size, number, and mortality. Changes in various biochemical parameters in the blood, associated with participants in the metabolic pathway of L-arginine, were observed. However, alterations in key factors that would provide a detailed understanding of the exact molecular mechanisms underlying the anticancer effects were not noted\u0026nbsp;\u003csup\u003e13\u003c/sup\u003e. This research elucidated the mechanisms of the anticancer effects of \u003cem\u003eR. obtusifolius\u003c/em\u003e extract, both independently and in combination with the conventional chemotherapeutic compound 5-FU. Specifically, the impact of the herbal extract of R. obtusifolius (0.25mg/mL) on the TNF\u0026alpha;-VEGF\u0026alpha;/PI3K/Akt/NOS/COX-2-MMP-2 pathway was assessed separately and in combination with 5-FU (40\u0026micro;M) in non-small lung adenocarcinoma A549 cells.\u0026nbsp;In addition, the possible interaction of compounds identified by HPLC/MS/MS of RO on PI3K/Akt in the active site pocket was also elucidated by \u003cem\u003ein silico\u003c/em\u003e study in comparison with its ligand care. By uncovering the molecular mechanisms of anticancer effects, identifying the most active phytochemicals, and clarifying the specific targets of these compounds, there is potential to inhibit, prevent, or delay cancer development with minimal side effects.\u003c/p\u003e"},{"header":"2. RESULTS","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Modulation of growth-inhibiting properties of 5-Fluorouracil with \u003cem\u003eR. obtusifolius\u003c/em\u003e extract tested by MTT assay\u003c/h2\u003e \u003cp\u003eGrowth inhibiting properties of RO seed ethanol extract on A549 cancer cells were tested with \u003cem\u003ein vitro\u003c/em\u003e MTT assay. Based on the obtained data the RO extract even at the highest tested concentration (0.5 mg DW/mL) and at any of the tested exposure times (4, 24, and 72 h) did not show any statistically significant impact on the growth of A549 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther modulating activity of none-inhibitory concentrations of RO extract (0.25 mg DW/mL) toward fluorouracil (5-FU) on A549 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C, D) was investigated using \u003cem\u003ein vitro\u003c/em\u003e MTT assay. Based on the obtained data, statistically significant strong modulation of 5-FU was observed at 24 h exposure time on all tested 5-FU concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Considerable modulation was detected on 4 h exposure time as well (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). However, during 72 h exposure time, no modulation was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.2.\u003c/b\u003e \u003cb\u003eR. obtusifolius\u003c/b\u003e \u003cb\u003eextract alone and in combination with 5-FU downregulate quantitative changes of TNFa, VEGFa, COX-2, and MMP-2.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eQuantitative changes in TNFα-related COX2 and VEGF-related MMP2 were assessed in the next step using ELISA. RO extract decreased the quantities of TNFα (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, A) and VEGFα (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, B) in the cell medium by 40% and 33%, respectively, and the quantities of COX-2 and MMP2, respectively by 31.5% and 33%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e5-FU alone does not affect TNFa/COX-2, but when combined with the plant, the effect is greater than with the plant alone or 5-FU alone (p\u0026thinsp;\u0026le;\u0026thinsp;0.05). The most visible modulatory effect on each other is TNFα (combined effect decreases it close to the value of RO), VEGFa (33% and 50% decrease, respectively, compared to 5-FU and RO alone, p\u0026thinsp;\u0026le;\u0026thinsp;0.01), and in the case of COX-2 (the reduction is about 90% for 5-FU and 80% for RO). In the case of COX-2, a synergism phenomenon can be documented.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Regulation of PI3K/Akt pathway\u003c/h2\u003e \u003cp\u003eTo further understand the cause of decreased TNFα, VEGFa, COX-2, MMP-2 and its effect on the cell, the downstream signaling pathway from TNFα and VEGFa to PI3K/Akt, whose dysregulation is characteristic of cancer, was elucidated.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe demonstrated that RO acts as an inhibitor of the Pi3K/Akt pathway. Particularly, quantitative reduction of total and phosphorylated PI3K and Akt was observed after the treatment of A549 cells with RO extract (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, A). The combination of RO and 5-FU reduced the amount of total and phosphorylated PI3K by about 2.5-fold compared to the control cells (p\u0026thinsp;\u0026le;\u0026thinsp;0.01). RO alone does not affect these two forms of PI3K, and 5-FU only affects the total amount of PI3K. RO and 5-FU reduce both phosphorylated and total Akt amounts, and the combination can increase this effect several-fold, showing a synergistic effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, B). We assumed one of the ways the effects of RO is the TNFa-PI3K-Akt cascade. Changes in the amount and activity of NO, MDA, Arginase, and NOS participants were elucidated to understand where the effect goes next.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Stimulation of RNS and ROS by regulation of Arginase and NOS activity\u003c/h2\u003e \u003cp\u003eSince in most cases COX-2 interacts with arginase and NOS \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, changing the concentration of VEGFa with NO and NOS and TNFa with ROS and RNS was valuable and necessary to observe the activities of arginase, NOS, and quantitative changes of NO and MDA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results showed that by reducing the amount of VEGF, the plant also inhibited the activity of NOS (p\u0026thinsp;\u0026le;\u0026thinsp;0.001), but increased the quantity of NO (\u003cb\u003ep\u0026thinsp;\u0026le;\u0026thinsp;0.05\u003c/b\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, B \u003cb\u003eand C\u003c/b\u003e). Since the activity of NOS is depressed, but the amount of nitrite ions is increased, the increase in the amount of RNS may be due to the increase in ROS. ROS and generated NO most likely lead to RNS generation and ROS/NO-mediated apoptosis. The latter is confirmed by the high quantity of MDA, which is promoted by the plant (\u003cb\u003ep\u0026thinsp;\u0026le;\u0026thinsp;0.01\u003c/b\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, D). Normally in cancer, activation of the PI3K/Akt pathway leads to increased ROS \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, and conversely, increased ROS leads to activation of this pathway, but this phenomenon was not observed in our experiments. In this case, the plant adjusted in such a way that by suppressing the PI3K/Akt pathway, it increases the amount of ROS and RNS. This indicates a multi-target effect of the compounds contained in the herb. To verify that increased RNS and ROS quantities, as well as inhibition of the PI3K/Akt pathway, should lead to apoptosis, Caspase-3 activity was assessed and chromatin staining was performed with Hoersch 3328 dye to observe segmentation and condensation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Assessment of apoptosis rate by Hoechst 33258 staining\u003c/h2\u003e \u003cp\u003eHoechst 33258 staining allows discrimination of apoptotic and non-apoptotic cells based on morphological changes of the nuclei (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The nuclei in normal cells exhibited evenly dispersed and weak fluorescence, as well as smooth edges (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, A). Apoptotic cells can be distinguished by the condensed chromatin (\u003cb\u003eFig․ 5, B, marked with arrows\u003c/b\u003e) and rough edges of their nuclei (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, C, marked with arrows), as well as signs of nuclear fragmentation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, D, \u003cb\u003emarked with arrow\u003c/b\u003e). The results indicate that incubating A549 cells with 5-FU, RO, and their combination 5-FU\u0026thinsp;+\u0026thinsp;RO for 24 h significantly increased the rate of apoptotic cells (\u003cb\u003eFig․ 5, E\u003c/b\u003e). In the control, the rate of apoptosis was 2.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56%. Treatment with 5-FU or RO alone significantly increased the rate of apoptotic cells up to 14.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.83% and 11.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98%, respectively. At the same time, the combination of 5-FU\u0026thinsp;+\u0026thinsp;RO elevated the rate of apoptotic cells up to 29.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.94% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Thus, the apoptosis of A549 cells induced by the combination of 5-FU\u0026thinsp;+\u0026thinsp;RO was significantly higher when compared with that of 5-FU or RO alone (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eSince Caspase-3 is active in the execution phase of apoptosis, we next used colorimetric assay to determine whether it was activated following treatment with 5-FU, RO, and their combination 5-FU\u0026thinsp;+\u0026thinsp;RO for 24 h (\u003cb\u003eFig․ 5, F\u003c/b\u003e). The spectrophotometric analysis revealed a significant increase in caspase-3 activity in all treatment variants which was more pronounced in cells treated with 5-FU\u0026thinsp;+\u0026thinsp;RO (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.6. The interaction of potential compounds with PI3K and Akt\u003c/h2\u003e \u003cp\u003eThe docking of the top compounds present in the RO ethanolic extract (\u003cb\u003eSuppl. Table\u003c/b\u003e) by Autodock Vina was performed on the binding pockets of AKT (PDB ID: 2JDO) and PI3K (PDB ID: 6 AUD) (\u003cb\u003eFig, 6 and 7\u003c/b\u003e). Each crystallographic structure\u0026rsquo;s binding pocket contained a bound ligand, which was extracted and rocked as a control. The docking results are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Based on the average score on two target proteins, none of the compounds showed a better docking score (the lower, the better) than the redocking score of the PI3K control. However, 14 out of 17 investigated compounds demonstrated better docking scores than the redocking score of the AKT control ligand. Therefore, this shows that the extracted compounds of interest may have a better affinity toward AKT.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDocking results of RO on PI3K and Akt.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAKT (2JDO)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePI3K (6AUD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAverage\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePI3K-control ligand\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-9.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-9.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEndocrocin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-9.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-9.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-9.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEmodin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-8.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-9.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLuteolin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-8.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-8.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-8.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQuertecin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-8.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-8.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-8.45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEpicatechin-gallate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-8.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-8.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-8.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEriodictol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-8.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-8.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-8.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQuercetin-3-D-galactoside\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-8.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-8.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-8.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHamamelofuranose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-8.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-8.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-8.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsorhamnetin-3-O-glucoside\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-8.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-8.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCatechin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-7.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-8.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-8.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEpicatechin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-8.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-7.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-7.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eApigenin-sulfate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-8.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-7.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-7.85\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQurecetin-diglucoside\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-7.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-7.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4-glucogallic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-6.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-7.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAKT-control ligand\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProcyanidin-dimer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-7.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-6.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-6.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProtocatechuic-acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-5.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-5.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-5.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHydroxybenzoic-acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-5.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eUnderstanding the absorption, distribution, metabolism, and excretion (ADME) properties of drug candidates is essential due to their profound influence on the pharmacokinetics, efficacy, and potential side effects of therapeutic agents \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. For this reason, we computationally analyzed the ADME characteristics of the 17 leading compounds using the SwissADME online service, and key features were outlined in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. A variety of physical and chemical properties such as molecular weight (MW), partition coefficient (LogP), hydrogen bond acceptors (HBA), and hydrogen bond donors (HBD) were assessed. Based on these calculations, we examined how the compounds adhere to Lipinski\u0026rsquo;s rule of 5 which is a set of rule-of-thumb guidelines in medicinal chemistry that predict oral bioavailability in drugs, stating that a molecule will likely be an effective oral medication when it does not violate more than one of these rules: no more than 5 hydrogen bond donors, 10 hydrogen bond acceptors, a molecular mass less than 500 Daltons, and a LogP not exceeding 5 \u003csup\u003e18\u003c/sup\u003e. The results illustrate that 10 out of the 17 compounds did not violate Lipinski\u0026rsquo;s rule of 5, indicating their potential to be drug candidates. Remarkably, these 10 compounds included the top 4 ones having the best docking score (namely: endocrocin, emodin, luteolin, and quercetin).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eADME properties of RO extract phytochemicals.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompounds\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMW\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHBA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHBD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLogP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNum of Viol\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEndocrocin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e314\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEmodin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e270\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLuteolin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e284\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQuercetin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e299\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEpicatechin gallate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e442\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eErodictol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e280\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQuercetin 3-D-galactoside\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e464\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHamamelofuranose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-1.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsorhamnetin 3-O-glucoside\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e478\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCatechin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e290\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEpicatechin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e290\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eApigenin sulfate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e364\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQurecetin diglucoside\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e607\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4 glucogallic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e332\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProcyanidin dimer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e562\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProtocatechuic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e151\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHydroxybenzoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e134\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAccordingly, we decided to examine further the 4 compounds mentioned above. In particular, we analyzed the binding mode of their best docking scores for the two proteins in addition to a 3D visual inspection (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Based on the analysis, emodin forms hydrogen bonds with Asp293 and Lys160 amino acids of AKT. The interactions with 9 other amino acids are hydrophobic (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, A). With PI3K, emodin forms hydrogen bonds with Tyr867 and Asp 964. The interactions of emodin and Glu880, Val882, Trp812, Ile831, Ile963, and Ile879 interactions are hydrophobic (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, A). In the case of endocrocin and AKT interactions, there are hydrogen bonds with Glu230, Ala232, Lys160, and Asp293. In addition, there are hydrophobic interactions with 8 other amino acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, B). With PI3K, endocrocin forms only one hydrogen bond, namely with Asp963. Furthermore, there are hydrophobic interactions with 12 additional amino acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLuteolin forms hydrogen bonds with Lys181 and Asp293 amino acids of AKT, complemented by 8 other hydrophobic interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, C). With PI3K, luteolin forms one hydrogen bond with Tyr867 and Ala885 and two hydrogen bonds with Val882. Furthermore, there are hydrophobic interactions with 8 other amino acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, C). Finally, Quercetin forms four hydrogen bonds with Asp293, Lys181, Ala232, and Glu230 of AKT. In addition, there are 8 hydrophobic interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, D). With PI3K, quercetin forms hydrogen bonds with Ser806 and Val882. This is complemented by hydrophobic interactions with 10 other amino acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, D). The specific interactions between emodin, endocrocin, luteolin, and quercetin with the target proteins AKT and PI3K highlight their potential to modulate the function of these proteins. It is noteworthy that while all four compounds formed hydrogen bonds with key residues in AKT, the variety and number of interactions differ, which may influence their affinity and specificity. The prevalence of hydrophobic interactions alongside hydrogen bonds, particularly with PI3K, underscores the potential of these compounds to anchor firmly within the binding pockets, possibly conferring stable interactions and effective inhibition. Such differential binding patterns could translate into varying degrees of therapeutic efficacy and selectivity among these compounds.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. DISCUSSION","content":"\u003cp\u003eAlthough chemotherapy is the most commonly used treatment, it also kills normal cells, causing many side effects. Therefore, it is urgent to develop novel alternative therapeutic strategies to overcome these problems. Many phytochemicals have been isolated from various plants that have regulatory effects on the targets that are considered in our study. We hypothesized that RO extract, either single or in combination with chemotherapy compounds, may effectively modulate the immune system (TNFa/COX-2/Arginase), inhibit angiogenesis and progression of metastasis (VEGFa/NOS/NO/MMP-2) via regulation of PI3K/Akt signaling pathway.\u003c/p\u003e \u003cp\u003eDuring the study, the effect of RO on 5-FU-induced apoptosis in A549 cells was examined. MTT assay showed that RO alone did not induce noticeable inhibition of growth of A549 cells. This is interesting as according to our previous research, RO seed extract expressed strong cytotoxic activity on two tested cancer cell lines (HT29 and MCF-7) at even 0.125 mg DW/mL concentration \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Although RO extract did not possess growth-inhibiting activity in A549 cells, we assumed that acting synergically could increase the cytotoxic properties of chemotherapeutic agents like Fluorouracil. The speculations made based on earlier studies, where RO extract when combined with NG-nitro-L-arginine methyl ester (NOS inhibitor) and NG-hydroxy-nor-L-arginine (arginase inhibitor) increased their therapeutic effects probably by regulating redox homeostasis \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The experiments were done in an in \u003cem\u003evivo\u003c/em\u003e rat mammary carcinogenesis model and based on the obtained data RO extract possessed a modulating effect on 5-FU. It is important to point out that RO expressed a synergic effect rather than an additive as RO extract did not show any growth-inhibiting effect at the concentration used in combined treatment. The modulating properties of RO seed extract could have great importance, taking into account that modulation of the anticancer effects of chemotherapy drugs through plant extracts or derived compounds is a promising strategy to overcome drug resistance and reduce side effects \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo further understand biochemical mechanisms underlying modulating properties RO on 5-FU different biochemical parameters were explored including quantitative changes of TNFa, VEGFa, COX-2, and MMP-2, regulation of PI3K/Akt pathway, assessment of apoptosis, \u003cem\u003eetc.\u003c/em\u003e We considered the PI3K/Akt signaling pathway taking into account that it is a major signaling pathway in various types of cancer. It controls the hallmarks of cancer, including cell survival, angiogenesis, inflammation, metastasis, and metabolism. According to the literature, the vascular endothelial growth factor (VEGF) is the most potent stimulant of angiogenesis and can activate NOX isoforms either directly or indirectly through PI3K/Akt induction \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. After activation by VEGF, Akt promotes the proliferation, migration, and survival of endothelial cells, thus affecting angiogenesis. This finding also provides lateral support for the conclusion that endothelial nitric oxide synthase (eNOS), which controls vascular tone, is a specific substrate of Akt1 in endothelial cells \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The subsequent production of superoxide and hydrogen peroxide is necessary for the regulation of transcription factors, which promote angiogenesis, including NF-κB, MMPs, COX-2, and HIF-1α. COX-2 is up-regulated in many malignant cancers, including gastric, colon, breast, esophagus, pancreas, hepatocellular carcinoma, and NSCLC. The overexpression of COX-2 effectively potentiates the cisplatin and other chemotherapy drug resistance of NSCLC cells by promoting EMT. NS398, a COX-2 inhibitor, induced apoptosis and additionally potentiated chemosensitivity to cisplatin-mediated apoptosis in human non-small cell lung cancer by targeting the AKT \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Studies indicate MMP-2 siRNA inhibited lung cancer cell-induced tube formation of endothelial cells \u003cem\u003ein vitro\u003c/em\u003e; the addition of recombinant human-MMP-2 restored angiogenesis.\u003c/p\u003e \u003cp\u003eOur research obtained results showed that RO extracts significantly decreased the quantities of TNFα, VEGFa, COX-2, and MMP2 in A549 cancer cells in combination with 5-FU. Inflammatory cytokines, growth factors, and their receptors, such as TNF, TNFR, VEGF, and VEGFR, act as positive regulators to transmit signals to mTOR through the PI3K/Akt pathway \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. PI3K/Akt signaling blocks the expression of proapoptotic proteins reduces tissue apoptosis and increases the survival rate of cancer cells. Akt inhibits the proapoptotic factors Bad and procaspase-9 through phosphorylation and induces the expression of the proapoptotic factor Fas ligand. In addition, Akt activation is associated with resistance to increased apoptosis induced by TNF. During routine chemotherapy, no treatment interval exists, allowing resistant cells to be generated and leading to tumor regeneration. The PI3K/Akt signaling pathway is important for the drug resistance of different types of cancer, such as lung cancer and esophageal cancer. For NSCLC cells with high Akt expression, the use of PI3K/Akt signaling pathway inhibitors increases their cell apoptosis induced by chemotherapy and reduces their resistance to chemotherapy \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Therefore, inhibition of the PI3K/Akt signaling pathway, which has been shown to regulate cancer cell apoptosis can serve as a new direction for future research on cancer treatment \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The importance of this work is also that the obtained results touch on such a question as plant pro-oxidation. Increased malondialdehyde and nitrite ions are present in the cellular environment, indicating increased ROS and RNS. According to the literature, the latter is also regulated by Akt \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. In addition, the change in Akt activity also affects the regulation of Caspase-3 activity and therefore apoptosis. Given that cell leakage may be a factor in RNS- and ROS-mediated apoptosis, the alteration of Caspase-3 activity was observed. The possibility of chromatin segmentation and condensation under the effect of herb and combination was also studied by Hoechst stain to further elucidate the stimulation of apoptosis. Hoechst staining revealed an increase in the rate of apoptotic cells after treatment with 5-FU (40 uM) or RO alone. The combination of 5-FU\u0026thinsp;+\u0026thinsp;RO synergistically evoked Caspase-3 activity, thus RO elevated the frequency of 5-FU-induced apoptosis. The results obtained in the case of combinations of herbs and chemotherapeutic agents showed a decrease in TNFa and VEGFa and an increase in NO and MDA quantity. The latter is indicative of ROS/RNS-mediated cytotoxicity of herbs in the tumor microenvironment. A decrease in COX-2, Arginase, and MMP-2 was observed in the A549 under the influence of herb extracts and combinations. The work is also highlighted by considering the herb together with a classical chemotherapeutic compound. As a classic chemotherapeutic compound, 5-fluorouracil was used, which has a broad spectrum effect and is used in chemotherapeutic cocktails \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. It was important to observe the herb-drug interaction and identify whether there is a synergistic effect between this herb and 5-FU. Even though several works show the anticancer effect of various herbs, and our \u003cem\u003ein vivo\u003c/em\u003e model showed the effective use of this herb against breast cancer in combination with L-arginine metabolic pathway inhibitors, there are few works, which revealed the mechanisms by which this effect occurs. The work is also valuable in that, by using a multi-component decoction of the medicinal plant, the possible protection of these compounds against PI3K and Akt enzymes was also clarified by parallel \u003cem\u003ein silico\u003c/em\u003e research. The research has 3 main findings. Elucidated the mechanisms of the anticancer effect of an unexplored herb by looking at the TNFa/PI3K/Akt/COX-2/ARG/NOS/ROS/RNS/Caspase-3 pathway, demonstrated herb-drug synergistic interactions affected by different compounds, which were revealed based on \u003cem\u003ein silico\u003c/em\u003e studies. These compounds had also the greatest affinity for PI3K/Akt, which may play a key role in RO extracts with promising anticancer properties. An important finding of the work is also the fact that the quantitative images of MDA and nitrite anions differ from our previous studies \u003cem\u003ein vivo\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. During earlier \u003cem\u003ein vivo\u003c/em\u003e studies on the rat mammary carcinogenesis model, a decrease in the amount of malondialdehyde and nitrite ions was observed in the blood, while an increment of their quantity was detected in the cell culture. The circumstance of selective effect is also seen here, thanks to which it is possible to deliver these active compounds to the tumor environment itself with the use of delivery systems and to leave a point effect on the targets presented \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The purpurin (naturally occurring anthraquinone) could effectively kill A549 cancer cell lines and lead to cell death, thus conforming to increased cytotoxicity, production of ROS-mediated enhancement of lipid peroxidation, nuclear fragmentation, and apoptosis. The study demonstrates that purpurin inhibits the phosphorylated PI3K/AKT molecules mediated cyclin-D1, thereby inducing apoptosis by observing increased proapoptotic mediators Bax cleaved PARP, cytochrome-c, caspase-9, and caspase-3; and decreased Bcl-2 expression in the lung cancer cell lines \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Later we tried to elucidate the main compounds that contribute promising anticancer properties of RO extract. In our previous research works more than 200 phytochemicals were identified in the ethanol extract of RO ethanol extract based on LC-Q-Orbitrap-HRMS analysis. The full list of identified compounds in RO ethanol extract is presented in earlier work \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Further \u003cem\u003ein silico\u003c/em\u003e analyses revealed that 4 of these compounds (namely: endocrocin, emodin, luteolin, and quercetin) have a high affinity for PI3K and Akt, indicating that the downregulation of the PI3K/Akt pathway by the herbs may be responsible for their beneficial effects on the quantitative changes in the explored factors and enzymes. The results demonstrate that all 4 compounds form at least 2 hydrogen bonds and at least 6 hydrophobic interactions with amino acids of the binding pockets of both AKT and PI3K. The only exception is the endocrine-PI3K interaction, where there is only one hydrogen bond. Nevertheless, this is amply compensated with an additional 12 hydrophobic interactions. The analysis indicates strong interactions in the case of all 8 ligand-protein pairs, which have the potential to change both proteins\u0026rsquo; function and achieve biological modulation of physiological pathways. These findings imply that the unique binding patterns of these compounds may contribute to varying therapeutic efficacies and selectivities, highlighting their promising potential for modulating the functions of AKT and PI3K.\u003c/p\u003e \u003cp\u003eLiterature data partially confirms the obtained results based on \u003cem\u003ein silico\u003c/em\u003e studies. Particularly, luteolin, a bioactive flavone derivative present mainly in its shell, exerts breast cancer-inhibiting properties through an anti-angiogenesis mechanism by inhibiting VEGF production and its binding with the receptor \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In addition, it also downregulates epithelial-mesenchymal transition markers and lowers metastatic activity. Studies have shown that another compound quercetin reduces tumor weight by targeting VEGFR2 through the Akt/mTOR/P70S6K signaling pathway \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Emodin, which is another selected compound based on \u003cem\u003ein silico\u003c/em\u003e experiments, inhibits cancer growth by suppressing the expression of MMP7, MMP9, VEGF, EMT, N-cadherin, b-catenin, and Snail based on literature data. It also inhibits the Wnt/b-catenin signaling pathway by downregulating target genes, including c-Myc, Cyclin-D1, and TCF4. According to the literature, endocrocin is reported to have anticancer properties, although there is a lack of available data about the possible mechanisms of its action \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Based on \u003cem\u003ein silico\u003c/em\u003e studies, we assumed that these 4 compounds could have important contributions to the overall promising anticancer properties of RO extract. Further in vitro and in vivo evaluation of their anticancer potential combined and in different combinations could have great importance.\u003c/p\u003e \u003cp\u003eIn conclusion, this study revealed the potential of \u003cem\u003eR. obtusifolius\u003c/em\u003e seed alcoholic extract as an adjunct therapy in cancer treatment, specifically in combination with the classical chemotherapeutic agent 5-fluorouracil. The study allowed us to find several key insights into the mechanisms underlying the anticancer effects of RO and its synergy with 5-FU. We extensively explored the TNFa/VEGFa//PI3K/Akt/COX-2/ARG/NOS/ROS/RNS/Caspase-3 pathway during the study, revealing a complex interplay of factors influenced by RO and 5-FU. The combination of RO and 5-FU demonstrated a synergistic effect on various cellular components. This finding suggests that RO, while not directly inhibiting the growth of A549 cells on its own, can enhance the cytotoxic properties of 5-FU, potentially leading to more effective cancer cell eradication. The \u003cem\u003ein silico\u003c/em\u003e analysis identified specific compounds within RO with high affinities for PI3K and Akt, hinting at their potential role in mediating the observed therapeutic effects. This computational approach deepens our understanding of the molecular interactions involved. Notably, the study revealed the selective effects of RO on MDA and nitrite ions in different environments. This selective action suggests the possibility of targeted drug delivery systems to achieve localized therapeutic effects while minimizing systemic side effects. Overall, this research contributes significantly to the field of cancer therapeutics by unraveling the complex molecular mechanisms underpinning the anticancer effects of RO and its synergistic relationship with 5-FU. The detailed molecular analysis reveals that emodin, endocrocin, luteolin, and quercetin present in RO extract exhibit distinctive interaction profiles with the target proteins AKT and PI3K․ These findings pave the way for further investigations into the development of novel, targeted cancer treatment strategies that harness the potential of medicinal plants like \u003cem\u003eR. obtusifolius\u003c/em\u003e in the research.\u003c/p\u003e"},{"header":"4. Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Chemicals and reagents\u003c/h2\u003e \u003cp\u003eAll chemicals were purchased from Sigma-Aldrich (USA) and Abcam (UK). Antibodies against TNFa (ab46087), VEGFa (ab193555), MMP-2 (ab92536), COX-2 (ab38898), PI3K and phosphorylated (p)-PI3K (ab191606), as well as ELISA kits for AKT and p-AKT (ab179463) were purchased from Abcam.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Plant material\u003c/h2\u003e \u003cp\u003eThe seeds of \u003cem\u003eRumex obtusifolius\u003c/em\u003e L. were harvested from the Tavush region of Armenia (1400\u0026ndash;1600 m height above mean sea level) according to the protocol described before \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Identification of plant material was carried out at the YSU Department of Botany and Mycology by Dr. Narine Zakaryan. Plant materials were deposited at the Herbarium of YSU, where the Voucher specimen serial number was given (ERCB 13208). The collection of plant material complied with relevant institutional, national, and international guidelines and legislation. \u003cem\u003eRumex obtusifolius\u003c/em\u003e L., commonly known as broad-leaved dock, is an edible plant widely distributed and commonly found throughout Armenia. It is not on the list of Endangered species in Armenia( \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://worldrainforests.com/biodiversity/en/armenia/EN.html\u003c/span\u003e\u003cspan address=\"https://worldrainforests.com/biodiversity/en/armenia/EN.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e / \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.iucnredlist.org/search?query=Rumex%20obtusifolius%20\u0026amp;searchType=species\u003c/span\u003e\u003cspan address=\"https://www.iucnredlist.org/search?query=Rumex%20obtusifolius%20\u0026amp;searchType=species\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e / \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cites.org/eng/search?search_api_fulltext=Rumex+obtusifolius+\u003c/span\u003e\u003cspan address=\"https://cites.org/eng/search?search_api_fulltext=Rumex+obtusifolius+\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ). The plant is not only prevalent in natural settings but also routinely collected by local populations for culinary purposes. There is no specific prohibition or regulatory constraint on the collection of this plant in Armenia, and it is a common sight at local markets, where it is sold after being gathered from the wild. This widespread availability and cultural integration into local diets supports the ethical sourcing and utilization of \u003cem\u003eRumex obtusifolius\u003c/em\u003e for research purposes under the conditions described in our study. For our research, we specifically collected only the seeds of \u003cem\u003eRumex obtusifolius\u003c/em\u003e. This method of collection ensures minimal impact on the natural populations of the plant, as it does not involve uprooting or damaging the plants themselves. We ensure our research practices are sensitive to ecological and conservation concerns, even in cases where no formal collection restrictions exist. As such, our study strictly adheres to general ethical guidelines for botanical research, despite the lack of specific regulations surrounding the collection of \u003cem\u003eRumex obtusifolius\u003c/em\u003e in Armenia.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.3. Plant crude extract\u003c/h2\u003e \u003cp\u003eThe grounded seeds were extracted by maceration with 96% ethanol at a 10:1 solvent-to-sample ratio (v/w). Stock solutions of 50 mg DW/mL crude ethanol extract were prepared as described earlier \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. The percent yield was 10.60\u0026thinsp;\u0026plusmn;\u0026thinsp;2.31%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.4. Cell cultures\u003c/h2\u003e \u003cp\u003eHuman lung adenocarcinoma A549 cell culture was obtained from ATCC (cat # CCL-185) and maintained in DMEM medium supplemented with L-glutamine (2 mmol/L), sodium pyruvate (200 mg/L), fetal bovine serum (100 mL/L), and antibiotics (100 U/mL penicillin and 100 \u0026micro;g/L streptomycin). Cells were grown at 37\u0026deg;C under a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e in a Biosmart (Biosan, Latvia) as described before \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Cultured cells were regularly examined for the presence of mycoplasma contamination using the Universal Mycoplasma Detection Kit from ATCC (Manassas, Virginia, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.5. MTT cytotoxicity test\u003c/h2\u003e \u003cp\u003eThe MTT test was performed as described previously \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e to assess the growth inhibition of A549 cells exposed to different concentrations of the \u003cem\u003eR. obtusifolius\u003c/em\u003e extract for 4, 24, or 72 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.6. ELISA of TNFa, VEGFa, COX-2, MMP-2, and Akt.\u003c/h2\u003e \u003cp\u003eA549 cells (2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) were cultured in 12-well plates and incubated for 24 h. After incubation, the cell medium (630 \u0026#120583;L) was replaced and the cells were treated with PBS and 1% Ethanol solution (Control, A549C), 5-FU (40\u0026#120583;M), RO (0.25mg/mL), and RO\u0026thinsp;+\u0026thinsp;5- FU (0.25mg/mL\u0026thinsp;+\u0026thinsp;40\u0026#120583;M) for 24 h and then the culture medium was harvested. TNFa, VEGFa, and MMP-2 in the supernatant were quantified according to the manufacturer's instructions. Cells from each group were collected (trypsinized, neutralized, centrifuged), lysed on ice with Lysis buffer, collected in a centrifuge tube, and further lysed for 10 min. After centrifugation at 13,000 \u0026times; g for 10 min at 4\u0026deg;C, the supernatant was collected. Changes in the levels of COX-2 and Akt were measured using ELISA kits, according to the manufacturer's instructions. Protein concentration in cell culture medium and lysates were measured with a Bradford method. Each test sample (70 \u0026#120583;L) was added to three different passages, which were triplicated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.7. Cells preparation for Arginase, NOS, and NO activity, MDA analysis\u003c/h2\u003e \u003cp\u003eA549 cells were seeded in 24-well (5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well) plates and incubated for 24 h. After incubation, the medium in wells (450 \u0026#120583;L) was refreshed. The cells were treated with 50 \u0026#120583;L control or test compounds with the following final concentrations: PBS, 1% ethanol (Control, A549C), 5-FU (40\u0026#120583;M), RO (0.25mg/mL), and RO\u0026thinsp;+\u0026thinsp;5-FU (0.25mg/mL\u0026thinsp;+\u0026thinsp;40\u0026#120583;M). After 24 h incubation, the supernatant without cells was discarded. Cells from each group were collected (trypsinized, neutralized, centrifuged), lysed on ice with Lysis buffer, collected in a centrifuge tube, and further lysed for 10 min. The supernatant was collected after centrifugation at 13,000 \u0026times; g for 10 min at 4\u0026deg;C. The levels of Nitrite anions, MDA, Arginase, and NOS were quantified according to the methods described below \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Each test sample (50 \u0026#120583;L) was added to five different passages, which were triplicated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.8. NO quantity measurement\u003c/h2\u003e \u003cp\u003eNO levels in the cell culture medium were determined as nitrite anions. Griess assay was used for measurement as described before \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. 100 \u0026#120583;L Griess reactant was added to 100 \u0026#120583;L of each sample. The supernatants were transferred to the tubes containing pellets of cadmium and incubated at room temperature for 12 h to convert nitrate to nitrite. The samples\u0026rsquo; absorbance was measured at λ\u0026thinsp;=\u0026thinsp;550 nm and the NO quantity was calculated based on a standard curve prepared with NaNO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.9. MDA assay\u003c/h2\u003e \u003cp\u003eMDA quantity in the cell culture medium was determined with a colorimetric assay using the Ohkawa thiobarbituric acid-malondialdehyde method \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.10. Arginase activity\u003c/h2\u003e \u003cp\u003eThe modified Diacetyl Monoxime colorimetric method assessed the arginase activity in A549 cell lysates \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.11. NOS activity\u003c/h2\u003e \u003cp\u003eNitric oxide synthase activity (\u0026micro;mol citrulline/mg protein) in A549 cell lysates was measured by the conversion of L-arginine to L-citrulline \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. 100\u0026micro;l of cell lysates was added to 200 mL of reaction mixture (50 mmol/L Tris buffer, pH 7.4, containing 10 mmol/L dithiothreitol (DTT), 10 \u0026micro;mol/L tetrahydrobiopterin (THB4), 10 \u0026micro;g/mL calmodulin, 1 mmol/L NADPH, 4 \u0026micro;mol/L flavin adenine dinucleotide (FAD), 4 \u0026micro;mol/L flavin mononucleotide (FMN), and 2 \u0026micro;mol/L L-arginine). The assay was carried out at 37\u003csup\u003eo\u003c/sup\u003eC, and it was terminated with 2 mL of ice-cold stop buffer (20 mmol/L CH3COONa, pH 5.5, containing 2 mmol/L EDTA, and 1 mmol/L L-citrulline). Assays were systematically performed with Ca\u003csup\u003e2+\u003c/sup\u003e (1 mmol/L CaCl2) or without Ca\u003csup\u003e2+\u003c/sup\u003e (0 mmol/L CaCl2) to measure total versus Ca\u003csup\u003e2+\u003c/sup\u003e-independent NOS activities. The Ca\u003csup\u003e2+\u003c/sup\u003e-dependent NOS activity was calculated as total NOS activity minus Ca\u003csup\u003e2+\u003c/sup\u003e-independent NOS activity. All assays were performed in triplicate on aliquoted samples (to avoid freezing/thawing cycles). The results were normalized for protein content.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.12. Phospho-PI 3 kinase p85\u0026thinsp;+\u0026thinsp;Total In-Cell ELISA assay\u003c/h2\u003e \u003cp\u003eA549 cells (1.5x10\u003csup\u003e4\u003c/sup\u003e cells per well) were seeded in the 96-well plates treated for tissue culture. After 24 h incubation, the cell medium (180 \u0026#120583;L) was refreshed and the cells were treated with 20\u0026#120583;L control or test compounds with the following final concentrations: PBS, 1% ethanol solution (Control, A549C), 5-FU (40\u0026#120583;M), RO (0.25mg/mL), and RO\u0026thinsp;+\u0026thinsp;5-FU (0.25mg/mL\u0026thinsp;+\u0026thinsp;40\u0026#120583;M). The calculations during the seeding of the cells were done in a way to reached approximately 80% confluency at fixation time. After 24 h exposure, the medium was discarded and cells were fixed with 100 \u0026micro;L of 4% formaldehyde in PBS. Crystal Violet was used to stain cells for normalizing readings in 450nm for Phospho-PI 3 kinase p85\u0026thinsp;+\u0026thinsp;Total. The measured OD450 readings were corrected for cell number by dividing the OD450 reading for a given well by the OD595 reading for that well. This relative cell number was then used to normalize each reading. Total and phospho-PI 3 kinase p85 were each assayed in triplicate using the phospho- and total PI 3 Kinase p85 antibodies included in the PI 3 Kinase Kit. Phospho-PI 3 kinase p85 and Total PI3K levels were measured using an In-Cell ELISA kit (ab207484), according to the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e4.13. Caspase-3/CPP32 Colorimetric assay\u003c/h2\u003e \u003cp\u003eA549 cells (5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well) were cultured in 6-well plates and incubated for 24 h. Then, the cell medium (900 \u0026#120583;L) was refreshed and the cells were treated with 10 \u0026#120583;L of PBS\u0026thinsp;+\u0026thinsp;1% ethanol solution (control, A549C) or test compounds with the following final concentrations: 5-FU (40 \u0026#120583;M), RO (0.25 mg/mL), and RO\u0026thinsp;+\u0026thinsp;5- FU (0.25 mg/mL\u0026thinsp;+\u0026thinsp;40 \u0026#120583;M). After 24 h the cells were harvested. Each test sample (100 \u0026#120583;L) was added to three different passages, which were triplicated. Cells were resuspended in 50 \u0026micro;L of chilled Cell Lysis Buffer and incubated on ice for 10 minutes. Then, cell lysate was centrifuged for 1 min (10,000 x g). After that supernatant (cytosolic extract) was transferred to a fresh tube and put on ice for immediate assay. Fold-increase in CPP32 activity has been determined by comparing these results with the level of the uninduced control. Optical density values were corrected taking into account the number of cells. All steps were performed according to the protocol presented in the Caspase-3/CPP32 Colorimetric Assay Kit (K106, BioVision) instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e4.14. Analysis of apoptosis by Hoechst 33258 staining\u003c/h2\u003e \u003cp\u003eThe percentage of apoptotic cells was evaluated as previously described \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. A549 cells (2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/mL) were treated with vehicle or 5-FU (40 \u0026#120583;M), RO (0.25 mg/mL), and RO\u0026thinsp;+\u0026thinsp;5- FU (0.25 mg/mL\u0026thinsp;+\u0026thinsp;40 \u0026#120583;M) for 24 hours, respectively. After treatment cells were washed with PBS and fixed with 4 %paraformaldehyde in PBS for 10 min. Then cells were washed twice with PBS for 5 min and stained with Hoechst 33258 reagent (10 \u0026#120583;g/mL) for 10 mins at room temperature in the dark. Then cells were washed with PBS and analyzed under a fluorescence microscope (x250 magnification) (Zeiss, Germany). The Hoechst 33258 staining allows the identification of apoptotic cells based on nuclear morphology. Cells with typical morphological changes, such as karyopyknosis, hyperfluorescence, nuclear fragmentation, and apoptotic bodies, were considered apoptotic. All variants were examined in duplicate. For each treatment variant, 500 cells were scored and the percentage of apoptotic cells was calculated as follows: % apoptotic cells = (the number of apoptotic cells/500 cells)*100.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e4.16. Preparation of Protein structures\u003c/h2\u003e \u003cp\u003eThe crystallographic structures of PI3K and AKT were procured from the Protein Data Bank (PDB) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), using the identifiers 6AUD and 2JDO, respectively. Visualization and preliminary assessment of these structures were performed with the PyMOL Molecular Graphics System (Schr\u0026ouml;dinger, LLC). The retrieved crystallographic structures were subject to preprocessing, which involved removing extraneous entities such as water molecules, ions, and other non-protein moieties contained within the structures. Simultaneously, the ligands co-crystallized with each protein structure were separated and retained for redocking validation experiments. The resulting streamlined protein structures were then used for docking explorations. The extracted ligands, on the other hand, were reserved for ensuing redocking studies as controls.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e4.17.\u003c/b\u003e D\u003cb\u003eocking\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eLigand docking and binding site analysis with PyMOL and Autodock/Vina were used for docking \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. The protein and ligand structures were prepared using Autodock Tools \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. During a typical procedure, the \"exhaustiveness\" parameter was calibrated to 8 and standard parameters suggested by the program creators were used to ensure the fidelity of the results. The compounds were sorted based on their binding strengths. The 2D binding mode analysis of best docking scores was performed using LigPlot\u0026thinsp;+\u0026thinsp;software (EMBL-EBI).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e4.18. Statistic analysis\u003c/h2\u003e \u003cp\u003eAll results are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. We analyzed the data either by one-way ANOVA or by its non-parametric analog Kruskal-Wallis test based on the normality test performed followed by Dunn's test was used to evaluate the statistical significance of the TNFa, VEGFa, MMP-2, COX-2, arginase, NOS, MDA, nitrite anions, Caspase-3, and apoptosis rate results. The significance of the results obtained for PI3K and Akt was assessed using two-way ANOVA and Tukey's multiple comparisons tests. Statistical analyses were performed using GraphPad Prism 8 software (San Diego, CA, USA), and a significance level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was deemed statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAkt, protein kinase B; ANOVA, analysis of variance;\u0026nbsp;Bcl-2, B-cell lymphoma-2; COX-2, cyclooxygenase-2;\u0026nbsp;CoQ0, Coenzyme Q0; ELISA, enzyme-linked immunosorbent assay; EMT, Epithelial-mesenchymal transition; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; HBA, hydrogen bond acceptors; HBD, \u0026nbsp;hydrogen bond donors, I\u0026kappa;B\u0026alpha;, an inhibitor of NF-\u0026kappa;B\u0026alpha;;\u0026nbsp;IL-6, interleukin-6; MAPK, mitogen-activated protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; MMP-2, matrix metalloproteinase-2;\u0026nbsp;MW, molecular weight; NOXs,\u0026nbsp;NADPH oxidases;\u0026nbsp;NF-\u0026kappa;B, nuclear factor-\u0026kappa;B; NOS, nitric oxide synthase; NSCLC, non-small cell lung cancer; PI3K, phosphoinositol-3-kinase; PG E2, prostaglandin E2;\u0026nbsp;RO, \u003cem\u003eRumex obtusifolius\u003c/em\u003e extract; ROS, reactive oxygen species; TNF-\u0026alpha;, tumor necrosis factor alpha; THB4, tetrahydrobiopterin; VEGF, vascular endothelial growth factor.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e5. \u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe data used to support the findings of this study are included in the articles.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eDeclaration of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7. Authors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study\u0026apos;s conception and design were the results of collective contributions from all authors. The investigations and analysis of results were carried out by MG, NA, HJ, SH, EN, GS, and TH. MG and NA wrote the manuscript.\u0026nbsp;Assessment of apoptosis rate by Hoechst 33258 staining and analysis of apoptosis performed by TH. The docking and ADME of the top compounds present in the RO ethanolic extract were performed by SG. NA, MG, HJ, ZK, and AM directed the\u0026nbsp;project,\u0026nbsp;corrected, and edited the manuscript. All authors participated in the revision and approval of the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlant materials were identified by Dr. Narine Zakaryan from the Department of Botany and Mycology at Yerevan State University (YSU).This work was supported by the Science Committee of MESCS RA through research projects numbered 21T-1F283, 21AG-1F068, and 23LCG-1F010.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGordaliza, M. Natural products as leads to anticancer drugs. Clinical and Translational Oncology 9, 767\u0026ndash;776 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCragg, G. M. \u0026amp; Pezzuto, J. M. Natural Products as a Vital Source for the Discovery of Cancer Chemotherapeutic and Chemopreventive Agents. Medical Principles and Practice 25, 41\u0026ndash;59 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHou, X. L. \u003cem\u003eet al.\u003c/em\u003e Suppression of Inflammatory Responses by Dihydromyricetin, a Flavonoid from Ampelopsis grossedentata, via Inhibiting the Activation of NF-κB and MAPK Signaling Pathways. J Nat Prod 78, 1689\u0026ndash;1696 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYin, S. Y., Wei, W. C., Jian, F. Y. \u0026amp; Yang, N. S. Therapeutic applications of herbal medicines for cancer patients. \u003cem\u003eEvidence-based Complementary and Alternative Medicine\u003c/em\u003e vol. 2013 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2013/302426\u003c/span\u003e\u003cspan address=\"10.1155/2013/302426\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe, Y. \u003cem\u003eet al.\u003c/em\u003e Targeting PI3K/Akt signal transduction for cancer therapy. \u003cem\u003eSignal Transduction and Targeted Therapy\u003c/em\u003e vol. 6 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41392-021-00828-5\u003c/span\u003e\u003cspan address=\"10.1038/s41392-021-00828-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoundouros, N. \u0026amp; Poulogiannis, G. Phosphoinositide 3-Kinase/Akt signaling and redox metabolism in cancer. \u003cem\u003eFrontiers in Oncology\u003c/em\u003e vol. 8 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fonc.2018.00160\u003c/span\u003e\u003cspan address=\"10.3389/fonc.2018.00160\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFruman, D. A. \u003cem\u003eet al.\u003c/em\u003e The PI3K Pathway in Human Disease. \u003cem\u003eCell\u003c/em\u003e vol. 170 605\u0026ndash;635 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cell.2017.07.029\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2017.07.029\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang, B. H. \u0026amp; Liu, L. Z. Chapter 2 PI3K/PTEN Signaling in Angiogenesis and Tumorigenesis. \u003cem\u003eAdvances in Cancer Research\u003c/em\u003e vol. 102 19\u0026ndash;65 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0065-230X(09)02002-8\u003c/span\u003e\u003cspan address=\"10.1016/S0065-230X(09)02002-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePang, L. \u003cem\u003eet al.\u003c/em\u003e Cox-2 Inhibition Protects against Hypoxia/Reoxygenation-Induced Cardiomyocyte Apoptosis via Akt-Dependent Enhancement of iNOS Expression. \u003cem\u003eOxid Med Cell Longev\u003c/em\u003e 2016, (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao, Y. \u003cem\u003eet al.\u003c/em\u003e COX-2 is required to mediate crosstalk of ROS-dependent activation of MAPK/NF-κB signaling with pro-inflammatory response and defense-related NO enhancement during challenge of macrophage-like cell line with Giardia duodenalis. PLoS Negl Trop Dis 16, (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChetty, C., Lakka, S. S., Bhoopathi, P. \u0026amp; Rao, J. S. MMP-2 alters VEGF expression via αVβ3 integrin-mediated PI3K/AKT signaling in A549 lung cancer cells. Int J Cancer 127, 1081\u0026ndash;1095 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGinovyan, M. \u003cem\u003eet al.\u003c/em\u003e Anti-cancer effect of Rumex obtusifolius in combination with arginase/nitric oxide synthase inhibitors via downregulation of oxidative stress, inflammation, and polyamine synthesis. Int J Biochem Cell Biol 158, 106396 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoudhari, A. S., Mandave, P. C., Deshpande, M., Ranjekar, P. \u0026amp; Prakash, O. Phytochemicals in cancer treatment: From preclinical studies to clinical practice. Front Pharmacol 10, 1\u0026ndash;18 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbd El-Aleem, S. A., Abdelwahab, S., AM-Sherief, H. \u0026amp; Sayed, A. Cellular and physiological upregulation of inducible nitric oxide synthase, arginase, and inducible cyclooxygenase in wound healing. J Cell Physiol 234, 23618\u0026ndash;23632 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, S., Yan, Y., Cheng, Z., Hu, Y. \u0026amp; Liu, T. Sotetsuflavone suppresses invasion and metastasis in non-small-cell lung cancer A549 cells by reversing EMT via the TNF-α/NF-κB and PI3K/AKT signaling pathway. Cell Death Discov 4, (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang, H. L. \u003cem\u003eet al.\u003c/em\u003e Anti-EMT properties of CoQ0 attributed to PI3K/AKT/NFKB/MMP-9 signaling pathway through ROS-mediated apoptosis. Journal of Experimental and Clinical Cancer Research 38, (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlqahtani, S. In silico ADME-Tox modeling: progress and prospects. \u003cem\u003eExpert Opinion on Drug Metabolism and Toxicology\u003c/em\u003e vol. 13 1147\u0026ndash;1158 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/17425255.2017.1389897\u003c/span\u003e\u003cspan address=\"10.1080/17425255.2017.1389897\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLipinski, C. A., Lombardo, F., Dominy, B. W. \u0026amp; Feeney, P. J. \u003cem\u003eExperimental and Computational Approaches to Estimate Solubility and Permeability in Drug Discovery and Development q Settings\u003c/em\u003e. \u003cem\u003eAdvanced Drug Delivery Reviews\u003c/em\u003e vol. 46 www.\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003eelsevier.com/locate/drugdeliv\u003c/span\u003e\u003cspan address=\"http://elsevier.com/locate/drugdeliv\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBazrafshani, M. S. \u003cem\u003eet al.\u003c/em\u003e The prevalence and predictors of herb-drug interactions among Iranian cancer patients during chemotherapy courses. BMC Complement Med Ther 23, 1\u0026ndash;11 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlhamed, A. S. \u003cem\u003eet al.\u003c/em\u003e Blockade of store-operated calcium entry sensitizes breast cancer cells to cisplatin therapy via modulating inflammatory response. Saudi Pharmaceutical Journal 31, 245\u0026ndash;254 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSanaei, M. J., Razi, S., Pourbagheri-Sigaroodi, A. \u0026amp; Bashash, D. The PI3K/Akt/mTOR pathway in lung cancer; oncogenic alterations, therapeutic opportunities, challenges, and a glance at the application of nanoparticles. \u003cem\u003eTranslational Oncology\u003c/em\u003e vol. 18 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.tranon.2022.101364\u003c/span\u003e\u003cspan address=\"10.1016/j.tranon.2022.101364\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSundaram, M. K. \u003cem\u003eet al.\u003c/em\u003e Combinational Use of Phytochemicals and Chemotherapeutic Drugs Enhance Their Therapeutic Potential on Human Cervical Cancer Cells. Int J Cancer Manag 12, (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParveen, A. \u003cem\u003eet al.\u003c/em\u003e Phytochemicals targeting VEGF and VEGF-related multifactors as anticancer therapy. \u003cem\u003eJournal of Clinical Medicine\u003c/em\u003e vol. 8 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/jcm8030350\u003c/span\u003e\u003cspan address=\"10.3390/jcm8030350\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBo, S. \u003cem\u003eet al.\u003c/em\u003e Purpurin, a anthraquinone induces ROS-mediated A549 lung cancer cell apoptosis via inhibition of PI3K/AKT and proliferation. Journal of Pharmacy and Pharmacology 73, 1101\u0026ndash;1108 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLotfi, N. \u003cem\u003eet al.\u003c/em\u003e The potential anti-cancer effects of quercetin on blood, prostate and lung cancers: An update. \u003cem\u003eFrontiers in Immunology\u003c/em\u003e vol. 14 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fimmu.2023.1077531\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2023.1077531\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhaliq, T., Akhter, S., Sultan, P. \u0026amp; Hassan, Q. P. Critical review on Rumex dentatus L. a strong pharmacophore and the future medicine: Pharmacology, phytochemical analysis and traditional uses. Heliyon 9, e14159 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGinovyan, M., Petrosyan, M. \u0026amp; Trchounian, A. Antimicrobial activity of some plant materials used in Armenian traditional medicine. BMC Complement Altern Med 17, 1\u0026ndash;9 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGinovyan, M., Hovhannisyan, S., Javrushyan, H. \u0026amp; Sevoyan, G. Screening revealed the strong cytotoxic activity of Alchemilla smirnovii and Hypericum alpestre ethanol extracts on different cancer cell lines. 10, 12\u0026ndash;22 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoss-Mikołajczyk, I., Kusznierewicz, B., Namieśnik, J. \u0026amp; Bartoszek, A. Juices from non-typical edible fruits as health-promoting acidity regulators for food industry. LWT - Food Science and Technology 64, 845\u0026ndash;852 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoss-Mikołajczyk, I., Kusznierewicz, B., Wiczkowski, W., Sawicki, T. \u0026amp; Bartoszek, A. The comparison of betalain composition and chosen biological activities for differently pigmented prickly pear (Opuntia ficus-indica) and beetroot (Beta vulgaris) varieties. Int J Food Sci Nutr 70, 442\u0026ndash;452 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAvtandilyan, N., Javrushyan, H., Petrosyan, G. \u0026amp; Trchounian, A. The Involvement of Arginase and Nitric Oxide Synthase in Breast Cancer Development: Arginase and NO Synthase as Therapeutic Targets in Cancer. \u003cem\u003eBiomed Res Int\u003c/em\u003e 2018, 1\u0026ndash;9 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVodovotz, Y. Modified microassay for serum nitrite and nitrate. Biotechniques 20, 390\u0026ndash;394 (1996).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeb, A. \u0026amp; Ullah, F. A Simple Spectrophotometric Method for the Determination of Thiobarbituric Acid Reactive Substances in Fried Fast Foods. J Anal Methods Chem 1\u0026ndash;5 (2016) doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1155/2016/9412767\u003c/span\u003e\u003cspan address=\"10.1155/2016/9412767\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAvtandilyan, N., Javrushyan, H., Petrosyan, G. \u0026amp; Trchounian, A. The Involvement of Arginase and Nitric Oxide Synthase in Breast Cancer Development: Arginase and NO Synthase as Therapeutic Targets in Cancer. \u003cem\u003eBiomed Res Int\u003c/em\u003e 2018, (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCombet, S., Balligand, J.-L., Lameire, N., Goffin, E. \u0026amp; Devuyst, O. \u003cem\u003eA Specific Method for Measurement of Nitric Oxide Synthase Enzymatic Activity in Peritoneal Biopsies\u003c/em\u003e. \u003cem\u003eKidney International\u003c/em\u003e vol. 57 (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang, X., Wang, Y., Luo, J., Liu, S. \u0026amp; Yang, Z. Protective effects of YC-1 against glutamate induced PC12 cell apoptosis. Cell Mol Neurobiol 31, 303\u0026ndash;311 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeeliger, D. \u0026amp; De Groot, B. L. Ligand docking and binding site analysis with PyMOL and Autodock/Vina. J Comput Aided Mol Des 24, 417\u0026ndash;422 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTrott, O. \u0026amp; Olson, A. J. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31, 455\u0026ndash;461 (2010).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Rumex obtusifolius, 5-Fluorouracil, lung adenocarcinoma, phytochemicals, PI3K/Akt pathway, apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-4254380/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4254380/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this study, the objective was to explore novel strategies for improving the efficacy of anticancer therapy. The focus was on investigating the antiproliferative effects of combining \u003cem\u003eRumex obtusifolius\u003c/em\u003e extract (RO) with the chemotherapeutic agent 5-Fluorouracil (5-FU) in non-small A549 lung cancer cells (NSCLC). Key factors such as the PI3K/Akt cell signaling system, cytokines, growth factors (TNFa, VEGFa), and enzymes (Arginase, NOS, COX-2, MMP-2) were analyzed to assess the impact of the combination treatment. Results revealed that the combined treatment of 5-FU and RO demonstrated a significant reduction in TNFa levels, comparable to the effect observed with RO alone. RO was found to modulate the PI3K/Akt pathway, influencing the phosphorylated and total amounts of these proteins during the combined treatment. Notably, COX-2, a key player in inflammatory processes, substantially decreased with the combination treatment. Caspase-3 activity, indicative of apoptosis, increased by 1.8 times in the combined treatment compared to separate treatments. In addition, \u003cem\u003ein silico\u003c/em\u003e analyses explored the binding affinities and interactions of RO's major phytochemicals with intracellular targets, revealing a high affinity for PI3K and Akt. These findings suggest that the combined treatment induces apoptosis in A549 cells by regulating the PI3K/Akt pathway.\u003c/p\u003e","manuscriptTitle":"5-Fluorouracil and Rumex obtusifolius extract combination trigger A549 cancer cell apoptosis: Uncovering PI3K/Akt inhibition by in vitro and in silico approaches","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-25 09:02:44","doi":"10.21203/rs.3.rs-4254380/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-27T10:01:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-13T22:38:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-02T16:44:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"bf4d4031-b05b-4232-96c1-348bd9cdd26b","date":"2024-04-23T09:43:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"0bfeafe3-2826-43f6-a1b1-df2ddfa988e9","date":"2024-04-23T09:29:08+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-23T09:26:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-23T09:24:39+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-04-22T15:04:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-22T15:01:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-04-11T21:28:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"61003b28-e75d-465d-84ab-bc575919fa23","owner":[],"postedDate":"April 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":31041599,"name":"Biological sciences/Biochemistry"},{"id":31041600,"name":"Biological sciences/Cancer"},{"id":31041601,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":31041602,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2024-06-26T00:29:50+00:00","versionOfRecord":{"articleIdentity":"rs-4254380","link":"https://doi.org/10.1038/s41598-024-65816-5","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-06-25 00:29:50","publishedOnDateReadable":"June 25th, 2024"},"versionCreatedAt":"2024-04-25 09:02:44","video":"","vorDoi":"10.1038/s41598-024-65816-5","vorDoiUrl":"https://doi.org/10.1038/s41598-024-65816-5","workflowStages":[]},"version":"v1","identity":"rs-4254380","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4254380","identity":"rs-4254380","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}