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Rumex
obtusifolius
*
Rumex obtusifolius
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
1Research Institute of Biology, Yerevan State University, 1 Alex Manoogian, 0025 Yerevan, RA,
Armenia. 2Laboratory of Immunology and Tissue Engineering, L.A. Orbeli Institute of Physiology NAS RA, Yerevan,
Armenia. 3Department of Genetics and Cytology, Yerevan State University, Yerevan, Armenia. 4Denovo Sciences
Inc, Yerevan, Armenia. 5Center for Developmental Health, Knight Cardiovascular Institute, Oregon Health &
Science University, Portland, USA. *email: nv.avtandilyan@ysu.am
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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
Nrf2 e transcription factor nuclear factor erythroid 2-related factor 2
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
e continuous increase in cancer rates, failure of conventional chemotherapies to control the disease, and exces-
sive toxicity of chemotherapies (in some cases, including immunotherapy) clearly demand alternative approaches.
Natural products contain many constituents that can act on various bodily targets to induce pharmacodynamic
responses1–5. Modulating biochemical and immune functions using medicinal plants and their products com-
bined with chemotherapeutic agents has recently become an accepted therapeutic approach6. 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).
Targeting the PI3K/Akt signaling pathway and related extracellular and intracellular components is an essen-
tial goal for several reasons. Correctly regulating the activity and quantity of these components can have a strong
anticancer eect on cancer cells. Several pro-tumorigenic processes converge on hyperactive PI3K/Akt signal-
ing, and it is becoming increasingly evident that reactive oxygen species (ROS) metabolism is no exception to
this7. Metabolic reconguration 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. is occurs by directly inuencing mitochondrial energy production and activating NADPH oxi-
dases (NOXs) or indirectly generating ROS as a metabolic byproduct8. Comprehending the intricate relationship
between ROS and PI3K/Akt signaling is vital for devising eective therapeutic approaches to combat tumors
reliant on this pathway. is signicance is underscored by recent clinical trials showing limited ecacy of
PI3K/Akt pathway inhibitors and the emergence of resistance. ese ndings 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 specically target PI3K-driven tumors8,9. 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 inuencing the
electron transport chain. e heightened ROS levels then trigger cascades, such as caspase activation, release of
cytochrome C, and DNA damage, ultimately leading to apoptosis8. In this study, we incorporated a chemothera-
peutic compound alongside 5-FU. is approach allows for the concurrent inhibition of the PI3K/Akt pathway
while maintaining or increasing signicant levels of ROS and reactive nitrogen species (RNS). Such elevation
eectively promotes the induction of apoptosis.
e PI3K pathway stimulates 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 by activating the PI3K/Akt pathway10. e PI3K/Akt signaling
pathway also promotes TNF-induced endothelial cell migration and regulates tumor angiogenesis. TNF-α plays
a signicant role in promoting the survival and metastasis of lung cancer. e levels of TNF-α in tumor tissues
and serum collected from patients with NSCLC substantially increase with the clinical stage of the tumor. Addi-
tionally, matrix metalloproteinases (MMPs) and cyclooxygenase-2 (COX-2) contribute to tumor angiogenesis.
COX-2 stimulates endothelial angiogenesis primarily through upregulating the antiapoptotic protein Bcl-2 and
activating the PI3K/Akt signaling pathway7,11–13.
e literature reveals that TNF-α, VEGF-α, COX-2, MMP-2, Caspase-3, and NOS are interconnected in
various cancer processes. eir interrelations are mediated through the PI3K/Akt signaling pathway. is study
focused on employing a combination of natural compounds and a chemotherapeutic agent to target these specic
components to attain potent anticancer eects.
We hypothesized that phytoextracts, either single or in combination with chemotherapy compounds, may
eectively 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. Our previous research
showed the promising anticancer eect of Rumex obtusifolius (RO) in an invivo experimental breast cancer
rat model; in parallel, its cytotoxic eect was elucidated against two cell cultures: MCF-7 and HT2914. e
combined anticancer eects of inhibitors targeting the metabolic pathway of L-arginine were also investigated.
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e ndings demonstrate signicant anti-tumor properties, including tumor size, number, and mortality reduc-
tions. Changes in various blood biochemical parameters associated with L-arginine metabolic participants were
observed. However, alterations in key factors that would provide a detailed understanding of the exact molecular
mechanisms underlying the anticancer eects were not noted15. e current study elucidated the mechanisms of
the anticancer eects of the R. obtusifolius extract, both independently and in combination with the conventional
chemotherapeutic compound 5-FU. Specically, 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
identied by HPLC/MS/MS of the eect of RO on PI3K/Akt in the active site pocket was also elucidated by an
in silico study. Uncovering the molecular mechanisms of their anticancer eects, identifying the most active
phytochemicals, and clarifying the specic targets of these compounds create a potential to inhibit, prevent, or
delay cancer development with minimal side eects.
R. obtusifolius
e growth-inhibiting properties of the RO seed ethanol extract on A549 cancer cells were evaluated using an
invitro MTT assay. e RO extract, even at the highest tested concentration (0.5mg DW/mL) and at any of the
tested exposure times (4, 24, and 72h), did not show any statistically signicant impact on the growth of the
A549 cells (Fig.1A).
Further modulating activity of the non-inhibitory concentrations of the RO extract (0.25mg DW/mL) with
uorouracil (5-FU) on A549 cells (Fig.1B–D) was investigated using invitro MTT assay. ere was a statistically
signicant strong modulation with 5-FU at 24h exposure time with all the tested 5-FU concentrations (Fig.1C).
Considerable modulation was also detected at 4h of exposure time (Fig.1B). However, at 72h, no modulation
was observed (Fig.1D).
R. obtusifolius
Using ELISA, changes in TNFα-related COX2 and VEGF-related MMP2 were assessed. e RO extract decreased
TNFα (Fig.2A) and VEGFα (Fig.2B) in the cell medium by 40% and 33%, respectively, and the quantities of
COX-2 and MMP2 by 31.5% and 33%, respectively.
Figure1. Growth rate of A549 cells treated with the RO extract for 4, 24, and 72h (A). e growth-inhibiting
eect of 5-uorouracil separately and in combination with the none-inhibitory concentration of RO extract
(0.25mg DW/mL) on A549 cells at 4 (B), 24 (C), and 72h (D). e results represent the means ± SD from three
independent experiments; SD values did not exceed 15%.
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5-FU alone does not aect TNFa/COX-2, but when combined with the plant extract, the eect was greater
than with the plant extract alone or 5-FU alone (p ≤ 0.05). e most visible modulatory eects were TNFα (the
combined eect decreased it close to the value of RO), VEGFa (33% and 50% decrease compared to 5-FU and
RO alone, respectively, p ≤ 0.01), and COX-2 (the reduction was about 90% for 5-FU and 80% for RO). In the
case of COX-2, a synergistic phenomenon was documented.
e downstream signaling pathway was elucidated to understand further the cause of decreased TNFα, VEGFa,
COX-2, and MMP-2 levels and their eect on the cell.
RO acts as an inhibitor of the Pi3K/Akt pathway. In particular, total and phosphorylated PI3K and Akt were
reduced aer treating the A549 cells with the RO extract (Fig.3A). e 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 did not aect these two forms of PI3K, and 5-FU only aected the total amount of PI3K. RO and 5-FU
reduced both the phosphorylated and total Akt levels, and the combination increased this reduction several-fold
showing a synergistic eect (Fig.3B). We assumed that RO works through the TNFa-PI3K-Akt cascade. us,
changes in the amount and activity of NO, MDA, Arginase, and NOS participants were elucidated to understand
the mechanism further.
Since, in most cases, COX-2 interacts with arginase and NOS16, 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).
By reducing VEGF, the plant extract also inhibited NOS activity (p ≤ 0.001) but increased the quantity of
NO (p ≤ 0.05, Fig.4B,C). Since the activity of NOS was depressed, but the amount of nitrite ions increased, the
increase in RNS may be due to the increase in ROS. ROS and generated NO most likely lead to RNS generation
and ROS/NO-mediated apoptosis. e latter was conrmed by the high quantity of MDA, which was promoted
by the plant (p ≤ 0.01, Fig.4D). Typically, in cancer, activation of the PI3K/Akt pathway leads to increased
Figure2. e inuence of the R. obtusifolius extract alone and in combination with 5-FU on TNFa (A), COX-2
(B), VEGFa (C), and MMP-2 (D) in A549 cells. Control—A549C, 5-Fluorouracil—5-FU (40μM), Rumex
obtusifolius—RO (0.25mg/mL), ROFU—RO + 5-FU (0.25mg/mL + 40μM). Each test sample was added to three
dierent passages in triplicate (n = 3, *—p ≤ 0.05, **—p ≤ 0.01, ns—non-signicant).
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ROS17,18; conversely, increased ROS leads to activation of this pathway. However, this phenomenon was not
observed in our study. In this case, the plant adjusted so that suppressing the PI3K/Akt pathway increased
the amount of ROS and RNS. is indicated a multi-target eect of the compounds contained in the herb. To
verify that increased RNS and ROS quantities, as well as inhibition of the PI3K/Akt pathway, leads to apoptosis,
Caspase-3 activity was assessed, and chromatin staining was performed with Hoechst 33258 dye to observe
segmentation and condensation.
Hoechst 33258 staining allows discrimination of apoptotic and non-apoptotic cells based on morphological
changes of the nuclei (Fig.5). e nuclei in normal cells exhibited evenly dispersed and weak uorescence and
smooth edges (Fig.5A). Apoptotic cells were distinguished by condensed chromatin (Fig․ 5B, marked with
arrows) and the rough edges of their nuclei (Fig.5C, marked with arrows), as well as signs of nuclear fragmen-
tation (Fig.5D, marked with arrow). e results indicated that incubating A549 cells with 5-FU, RO, and their
combination for 24h signicantly increased the rate of apoptotic cells (Fig․ 5E). In the control cells, the apoptosis
rate was 2.40 ± 0.56%. Treatment with 5-FU or RO alone signicantly 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). us, the apoptosis of A549 cells induced by the combination of
5-FU + RO was signicantly 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 a colorimetric assay to determine
whether it was activated following treatment with 5-FU, RO, and their combination 5-FU + RO for 24h (Fig․
5F). e spectrophotometric analysis revealed a signicant increase in caspase-3 activity in all treatment variants,
which was more pronounced in the cells treated with 5-FU + RO (p < 0.01).
e 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) (Figs.6, 7). Each crys-
tallographic structure’s binding pocket contained a bound ligand, which was extracted and rocked as a control.
Figure3. Eect of RO, 5-FU, and their combination on the PI3K/Akt pathway in A549 cells (A-PI3K, B—Akt).
Total and phospho-kinases were 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).
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e docking results are presented in Table1. Based on the average score of the 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. erefore, the extracted compounds of interest may have a better anity toward AKT.
Understanding the absorption, distribution, metabolism, and excretion (ADME) properties of drug candi-
dates is essential due to their profound inuence on therapeutic agents’ pharmacokinetics, ecacy, and potential
side eects19. For this reason, we computationally analyzed the ADME characteristics of the 17 leading com-
pounds using the SwissADME online service, and the key features are outlined in Table2. A variety of physical
and chemical properties, such as molecular weight (MW), partition coecient (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 eective oral medication when it
does not violate more than one of these rules: no more than 5 hydrogen bond donors, 10 hydrogen bond accep-
tors, a molecular mass less than 500 Daltons, and a LogP not exceeding 520. e results illustrate that 10 of the
17 compounds did not violate Lipinski’s rule of 5, indicating their potential to be drug candidates. Remarkably,
Figure4. Stimulation of RNS and ROS by RO regulates arginase (A) and NOS (B) activity, nitrite anions (C),
and MDA (D) quantity in A549 cells. Each condition was added to ve dierent passages in triplicate (n = 5,
*—p ≤ 0.05, **—p ≤ 0.01, ***—p ≤ 0.001, ****—p ≤ 0.0001).
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these 10 compounds included the top 4 with the best docking scores (namely, endocrocin, emodin, luteolin,
and quercetin).
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,
7). Based on the analysis, emodin formed hydrogen bonds with Asp293 and Lys160 amino acids of AKT. e
interactions with 9 other amino acids were hydrophobic (Fig.6A). With PI3K, emodin formed hydrogen bonds
with Tyr867 and Asp 964. e interactions of emodin and Glu880, Val882, Trp812, Ile831, Ile963, and Ile879
interactions were hydrophobic (Fig.7A). Regarding endocrocin and AKT interactions, there were hydrogen
bonds with Glu230, Ala232, Lys160, and Asp293. In addition, there were hydrophobic interactions with 8 other
amino acids (Fig.6B). Endocrocin formed only one hydrogen bond, namely with Asp963, with PI3K. Further-
more, there were hydrophobic interactions with 12 additional amino acids (Fig.7B).
Luteolin formed hydrogen bonds with the Lys181 and Asp293 amino acids of AKT, complemented by 8 other
hydrophobic interactions (Fig.6C). Regarding PI3K, luteolin formed one hydrogen bond with Tyr867 and Ala885
and two hydrogen bonds with Val882. Furthermore, there were hydrophobic interactions with 8 other amino
acids (Fig.7C). Finally, Quercetin formed four hydrogen bonds with Asp293, Lys181, Ala232, and Glu230 of
AKT. In addition, there are 8 hydrophobic interactions (Fig.6D). Regarding PI3K, quercetin formed hydrogen
bonds with Ser806 and Val882. is was complemented by hydrophobic interactions with 10 other amino acids
(Fig.7D). e specic interactions between emodin, endocrocin, luteolin, and quercetin with the target pro-
teins AKT and PI3K highlight their potential to modulate the function of these proteins. Notably, while all four
compounds formed hydrogen bonds with key residues in AKT, the variety and number of interactions diered,
which may inuence their anity and specicity. e prevalence of the hydrophobic interactions alongside
hydrogen bonds, particularly with PI3K, underscores the potential of these compounds to anchor rmly within
the binding pockets, possibly conferring stable interactions and eective inhibition. Such dierential binding
patterns could translate into varying degrees of therapeutic ecacy and selectivity among these compounds.
Although chemotherapy is the most commonly used treatment, it also kills normal cells, causing many side
eects. erefore, it is urgent to develop novel alternative therapeutic strategies to overcome these problems.
Many phytochemicals have been isolated from various plants that have regulatory eects on the targets con-
sidered in our study. We hypothesized that the RO extract, either alone or in combination with chemotherapy
compounds, may eectively modulate the immune system (TNFa/COX-2/Arginase), inhibit angiogenesis and
progression of metastasis (VEGFa/NOS/NO/MMP-2) via regulation of the PI3K/Akt signaling pathway.
Figure5. Hoechst 33258 staining (blue) assay to assess apoptosis in A549 cells. Apoptotic cells are indicated
with white arrows. e scale bar is 100μm. (A) Untreated cells (A549C) have smooth edges and dispersed
uorescence (B,E). 5-FU-induced apoptosis can be seen by the occurrence of pyknotic cells with condensed
chromatin (C,E). Incubation with RO elevated the number of apoptotic cells with rough edges of nuclei (D,E).
e combined treatment of cells with 5-FU + RO resulted in cells with signs of nuclei fragmentation and
chromatin condensation. (E) Apoptosis rate evaluated by the Hoechst 33258 staining, *p < 0.05—compared
with the RO, **p < 0.01—compared with the control. (F)—caspase-3 activity evaluated by the colorimetric assay,
*p < 0.05—compared with the 5-FU, **p < 0.01—compared with the control.
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e study examined the eect of RO on 5-FU-induced apoptosis in A549 cells. An MTT assay showed that
RO alone did not induce noticeable inhibition of the growth of A549 cells. is is interesting as, according to our
previous research, the RO seed extract expressed strong cytotoxic activity on two tested cancer cell lines (HT29
and MCF-7) at even 0.125mg DW/mL concentration14. Although the RO extract did not inhibit the growth of
A549 cells, we assumed that acting synergically would increase the cytotoxic properties of chemotherapeutic
agents like Fluorouracil. e speculations were made based on earlier studies, where the RO extract, when
combined with NG-nitro-L-arginine methyl ester (NOS inhibitor) and NG-hydroxy-nor-L-arginine (arginase
inhibitor), increased their therapeutic eects probably by regulating redox homeostasis14. e experiments were
done in an in vivo rat mammary carcinogenesis model, and based on the obtained data, the RO extract possessed
a modulating eect on 5-FU. It is essential to point out that RO had a synergic eect rather than an additive, as
Figure6. 2D binding analysis and interaction types for AKT in combination with 3D visualization. 3D
Visualizations: (A): Emodin interaction diagram. Panel (a) shows the surface representation of the binding
pocket with Emodin in green sticks. (B): Endocrocin interaction diagram. Panel (b) displays the surface
representation of the binding pocket with Endocrocin in magenta sticks. (C) Luteolin interaction diagram.
Panel (c) presents the surface representation of the binding pocket with Luteolin in cyan sticks. (D) Quercetin
interaction diagram. Panel (d) illustrates the surface representation of the binding pocket with Quercetin in
yellow sticks. In each 2D interaction diagram, the atoms are colored as follows: carbon (black), oxygen (red),
nitrogen (blue), and hydrogen (not shown for clarity). Amino acids forming hydrogen bonds with the ligands
are labeled, and their interactions are shown with green dotted lines and the bond distances in angstroms.
Hydrophobic interactions are represented by red semicircles around the interacting amino acids. Amino acids
involved in hydrophobic interactions are labeled in red, and those involved in hydrogen bonding are labeled in
green.
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the RO extract did not show any growth-inhibiting eect at the concentration used in the combined treatment.
e modulating properties of the RO seed extract could have great importance, considering that modulation
of the anticancer eects of chemotherapy drugs through plant extracts or derived compounds is a promising
strategy to overcome drug resistance and reduce side eects21.
Dierent biochemical parameters were explored to understand further biochemical mechanisms underly-
ing modulating properties RO on 5-FU, 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
because it is a major signaling pathway in various types of cancer. It controls the hallmarks of cancer, including
cell survival, angiogenesis, inammation, metastasis, and metabolism. According to the literature, the VEGF is
the most potent stimulant of angiogenesis and activates NOX isoforms directly or indirectly through PI3K/Akt
Figure7. 2D binding analysis and interaction types for PI3K in combination with 3D visualization. 3D
Visualizations: (A): Emodin interaction diagram. Panel (a) shows the surface representation of the binding
pocket with Emodin in green sticks. (B): Endocrocin interaction diagram. Panel (b) displays the surface
representation of the binding pocket with Endocrocin in magenta sticks. (C): Luteolin interaction diagram.
Panel (c) presents the surface representation of the binding pocket with Luteolin in cyan sticks. (D): Quercetin
interaction diagram. Panel (d) illustrates the surface representation of the binding pocket with Quercetin in
yellow sticks. In each 2D interaction diagram, the atoms are colored as follows: carbon (black), oxygen (red),
nitrogen (blue), and hydrogen (not shown for clarity). Amino acids forming hydrogen bonds with the ligands
are labeled, and their interactions are shown with green dotted lines and the bond distances in angstroms.
Hydrophobic interactions are represented by red semicircles around the interacting amino acids. Amino acids
involved in hydrophobic interactions are labeled in red, and those involved in hydrogen bonding are labeled in
green.
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induction8. Aer activation by VEGF, Akt promotes the proliferation, migration, and survival of endothelial
cells, thus aecting angiogenesis. is nding also supports the conclusion that endothelial nitric oxide syn-
thase (eNOS), which controls vascular tone, is a specic substrate of Akt1 in endothelial cells7. e subsequent
production of superoxide and hydrogen peroxide is necessary to regulate 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. e overexpres-
sion of COX-2 eectively potentiates the cisplatin and other chemotherapy drug resistance of NSCLC cells by
promoting EMT22.
Our data showed that RO extracts signicantly decreased the quantities of TNFα, VEGFa, COX-2, and MMP2
in A549 cancer cells in combination with 5-FU. Inammatory 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
pathway7. ese factors also play an important role in various immune system regulation processes. Researchers
found that neoadjuvant immunotherapy for NSCLC, immune checkpoint inhibitors for melanoma, and adjuvant
immunotherapy for melanoma and hepatocellular carcinoma are extremely relevant but still underdeveloped
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
Table 2. ADME properties of the 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
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directions for this eld warrant further investigation22. Models like the one we propose (herb + chemotherapeutic
compound) can be further incorporated into any neoadjuvant and adjuvant immunotherapy phases. PI3K/Akt
signaling blocks the expression of proapoptotic proteins, reduces tissue apoptosis, and increases the survival
rate of cancer cells7. 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 resist-
ance to increased apoptosis induced by TNF. During routine chemotherapy, no treatment interval exists, allowing
resistant cells to be generated and leading to tumor regeneration. e PI3K/Akt signaling pathway is important
for the drug resistance of dierent types of cancer, such as lung cancer and esophageal cancer. For NSCLC cells
with high Akt expression, PI3K/Akt signaling pathway inhibitors increase chemotherapy-induced apoptosis and
reduce their resistance to chemotherapy23. erefore, 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 treatment7.
is work is important because the obtained results touch on the question of 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 Akt8. In addition, the change in Akt activity also aects
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. e possibility of chromatin
segmentation and condensation under the eect of herb and combination was also studied by Hoechst stain to
elucidate the stimulation of apoptosis further. Hoechst staining revealed an increase in the rate of apoptotic cells
aer treatment with 5-FU (40 uM) or RO alone. e combination of 5-FU + RO synergistically evoked Caspase-3
activity; thus, RO elevated the frequency of 5-FU-induced apoptosis. e results obtained in the case of combi-
nations of herbs and chemotherapeutic agents showed a decrease in TNFa and VEGFa and an increase in NO
and MDA quantity. e latter indicated ROS/RNS-mediated cytotoxicity of herbs in the tumor microenviron-
ment. Many factors can damage DNA, proteins, or lipids in cells directly or indirectly, such as exogenous drugs,
endogenous reactive oxygen species, or free radicals. During this process, the transcription factor nuclear factor
erythroid 2-related factor 2 (Nrf2) is considered a signicant modulator, maintaining the cellular redox balance
by expressing antioxidant proteins24,25. Several cancer chemopreventive compounds targeting Nrf2 have been
reported, such as Oltipraz, Sulforaphane, Curcumin, Resveratrol, and Luteolin24. e increase in MDA and NO
amount of RO + 5-FU combination is possibly promoted by the modulation of the Nrf2 pathway itself, which
will be claried in future studies.
Decreased COX-2, Arginase, and MMP-2 were observed in the A549 cells under the inuence of the herb
extracts and combinations. e work is also highlighted by considering the herb and the classical chemothera-
peutic compound 5-uorouracil, which has a broad spectrum eect and is used in chemotherapeutic cocktails26.
It was important to observe the herb-drug interaction and identify whether there was a synergistic eect between
this herb and 5-FU. Even though several studies show the anticancer eect of various herbs, and our invivo model
showed the eective use of this herb against breast cancer in combination with L-arginine metabolic pathway
inhibitors, few studies have revealed the mechanisms by which this eect occurs. is 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 claried by a parallel in silico study. ere are 3 main ndings. Elucidated
the mechanisms of the anticancer eect of an unexplored herb by looking at the TNFa/PI3K/Akt/COX-2/ARG/
NOS/ROS/RNS/Caspase-3 pathway demonstrated an herb-drug synergistic interaction aected by dierent
compounds, which were revealed based on in silico studies. ese compounds also had the greatest anity for
PI3K/Akt, which may play a key role in RO extracts with promising anticancer properties. In our previous work
in another cell culture (MCF-7), RO has also been shown to have a down-regulating eect on total and phospho-
rylated amounts of PI3K27. Another important nding of the work is that the quantitative data of the MDA and
nitrite anions diered from our previous studies invivo 14. During earlier invivo studies on the rat mammary
carcinogenesis model, a decrease in the amount of malondialdehyde and nitrite ions was observed in the blood.
At the same time, an increment of their quantity was detected in the cell culture. e circumstance of selective
eect is also seen here, thanks to which it is possible to deliver these active compounds to the tumor environment
through delivery systems and to leave a point eect on the targets presented28,29. en, we elucidated the main
compounds of the RO extract that might have promising anticancer properties. In our previous research, more
than 200 phytochemicals were identied in the ethanol extract of RO ethanol extract based on LC-Q-Orbitrap-
HRMS analysis. e full list of identied compounds in RO ethanol extract is presented in earlier work14. During
this study, the in silico analyses revealed that 4 of these compounds (namely, endocrocin, emodin, luteolin, and
quercetin) had a high anity for PI3K and Akt, indicating that the downregulation of the PI3K/Akt pathway
by the herbs may be responsible for their benecial eects on the quantitative changes in the explored factors
and enzymes. e results demonstrated that all 4 compounds formed at least 2 hydrogen bonds and at least 6
hydrophobic interactions with amino acids of the binding pockets of both AKT and PI3K. e only exception
was the endocrine-PI3K interaction, with only one hydrogen bond. Nevertheless, this was amply compensated
with an additional 12 hydrophobic interactions. e analysis indicated strong interactions in the case of all 8
ligand–protein pairs, which can potentially change both proteins’ function and achieve biological modulation
of physiological pathways. ese ndings imply that the unique binding patterns of these compounds may con-
tribute to varying therapeutic ecacies and selectivities, highlighting their promising potential for modulating
the functions of AKT and PI3K.
e literature partially conrms the results obtained from in silico studies. Particularly, luteolin, a bioac-
tive avone 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 receptor28. 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 pathway30. Emodin, another selected compound based on in silico experiments, inhibits cancer growth
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by suppressing the expression of MMP7, MMP9, VEGF, EMT, N-cadherin, b-catenin, and Snail based on the
literature. It also inhibits the Wnt/b-catenin signaling pathway by downregulating target genes, including c-Myc,
Cyclin-D1, and TCF4. In addition, endocrocin is reported to have anticancer properties, although there is a lack
of available data about the possible mechanisms of its action31. Based on the in silico studies, we assumed that
these 4 compounds could have important contributions to the overall promising anticancer properties of RO
extract. Further invitro and invivo evaluation of their anticancer potential, both separate and with dierent
combinations, are needed to conrm their role in the RO extract’s anticancer properties and elucidate the role
of combined treatment.
In conclusion, this study revealed the potential of the R. obtusifolius seed alcoholic extract as an adjunct
therapy in cancer treatment, specically in combination with the classical chemotherapeutic agent 5-uorouracil.
ese ndings 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.
1. e current research primarily investigated herbal decoction. Future work will explore individual phyto-
chemicals, especially those identied in silico studies, with the highest anity for the PI3K/Akt pathway.
2. is study focused on the PI3K/Akt pathway. Future studies will expand the investigation to include other
important signaling pathways, such as MAPK, Nrf2, and JAK/STAT.
3. e observed changes in the PI3K/Akt pathway have yet to be conrmed through invivo studies. Future
research will aim to validate these results using experimental models of dierent types of cancer in rats/mice.
4. e study did not elucidate the key members of the cell signaling pathways in dierent cell lines. Future
studies will address this by examining these pathways across various cell lines to provide a more compre-
hensiveunderstanding.
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.
e Rumex obtusifolius L. seeds were harvested from the Tavush region of Armenia (1400–1600m height above
mean sea level) according to the protocol described before32. Dr. Narine Zakaryan identied plant material at
the YSU Department of Botany and Mycology. Plant materials were deposited at the Herbarium of YSU, where
the Voucher specimen serial number was given (ERCB 13208). e 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:// world rainf orests. co m/ b iodi v er si ty/ en/ a rmen ia/ EN. h tml
/https:// www . iucnr edlist. org/ search? query= Rumex% 20obt usifo lius% 20& searc hType= speci es/https:// cites. org/
eng/ search? search_ api_ fullt ext= Rumex+ obtus ifoli us +). e plant is prevalent in natural settings and routinely
collected by local populations for culinary purposes. ere is no specic 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 aer being
gathered from the wild. is 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 specically collected only the seeds of Rumex obtusifolius. is collection method 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. Our study strictly adheres to general ethical guidelines for botanical
research despite the lack of specic regulations surrounding the collection of Rumex obtusifolius in Armenia.
e grounded seeds were extracted by maceration with 96% ethanol at a 10:1 solvent-to-sample ratio (v/w).
Stock solutions of 50mg DW/mL crude ethanol extract were prepared as described earlier33. e percent yield
was 10.60 ± 2.31%.
Human lung adenocarcinoma A549 cells were obtained from ATCC (cat # CCL-185) and maintained in
DMEM medium supplemented with L-glutamine (2mmol/L), sodium pyruvate (200mg/L), fetal bovine serum
(100mL/L), and antibiotics (100 U/mL penicillin and 100µg/L streptomycin). e cells were grown at 37°C
under a humidied atmosphere with 5% CO2 in a Biosmart (Biosan, Latvia) as described before34. Cultured cells
were regularly examined for mycoplasma contamination using the Universal Mycoplasma Detection Kit from
ATCC (Manassas, Virginia, USA).
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e growth-inhibiting properties of the R. obtusifolius ethanol extract and its combination with 5-FU were
assessed in A549 cells using the MTT assay, as described previously35. For combination studies, the cells were
seeded in 96-well plates and exposed to dierent concentrations of 5-FU in the absence or presence of 0.25mg
DW/mL of RO extract for 4, 24, or 72h. Cell growth was assessed as described previously36. e results were
calculated as the percentage of cell growth in the presence of the tested compounds, extracts, or their combina-
tions, compared to control cells treated with the corresponding volume of solvent alone (1% EtOH in the nal
culture), whose growth was considered 100%. ree independent replicates of each treatment were performed
with three technical replicates.
A549 cells (2 × 105) were cultured in 12-well plates and incubated for 24h. Aer 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 24h and then the culture medium was harvested.
TNFa, VEGFa, and MMP-2 in the supernatant were quantied according to the manufacturer’s instructions. Cells
from each group were collected (trypsinized, neutralized, centrifuged), lysed on ice with lysis buer, collected in
a centrifuge tube, and further lysed for 10min. e supernatant was collected aer centrifugation at 13,000 × g
for 10min at 4°C. Changes in the levels of COX-2 and Akt were measured using ELISA kits, according to the
manufacturer’s instructions. e protein concentration in the cell culture medium and lysates were measured
using the Bradford method. Each test sample (70 μL) was added to three dierent passages in triplicate.
A549 cells were seeded in 24-well (5 × 104 cells per well) plates and incubated for 24h. Aer incubation, the
medium in the wells (450 μL) was refreshed. e cells were treated with 50 μL of the control or test compounds
at the following nal concentrations: PBS, 1% ethanol (Control, A549C), 5-FU (40μM), RO (0.25mg/mL), and
RO + 5-FU (0.25mg/mL + 40μM). Aer 24h, the supernatant was discarded. Cells from each group were col-
lected (trypsinized, neutralized, centrifuged), lysed on ice with lysis buer, collected in a centrifuge tube, and
further lysed for 10min. e supernatant was collected aer centrifugation at 13,000 × g for 10min at 4°C. e
levels of Nitrite anions, MDA, Arginase, and NOS were quantied according to the methods described below14.
Each test sample (50 μL) was added to ve passages in triplicate.
NO levels in the cell culture medium were determined as nitrite anions. e Griess assay was used as described
before37. A total of 100 μL Griess reactant was added to 100 μL of each sample. e supernatants were transferred
to the tubes containing pellets of cadmium and incubated at room temperature for 12h to convert nitrate to
nitrite. e samples’ absorbance was measured at λ = 550nm, and the NO quantity was calculated based on a
standard curve prepared with NaNO2.
MDA quantity in the cell culture medium was determined by a colorimetric assay using the Ohkawa thiobarbi-
turic acid-malondialdehyde method38.
e modied Diacetyl Monoxime colorimetric method assessed the arginase activity in A549 cell lysates39.
Nitric oxide synthase activity (µmol citrulline/mg protein) in A549 cell lysates was measured by convert-
ing L-arginine to L-citrulline40. A total of 100µl of the cell lysate was added to 200mL of reaction mixture
(50mmol/L Tris buer, pH 7.4, containing 10mmol/L dithiothreitol (DTT), 10µmol/L tetrahydrobiopterin
(THB4), 10µg/mL calmodulin, 1mmol/L NADPH, 4µmol/L avin adenine dinucleotide (FAD), 4µmol/L avin
mononucleotide (FMN), and 2µmol/L L-arginine). e assay was carried out at 37°C, and it was terminated
with 2mL of ice-cold stop buer (20mmol/L CH3COONa, pH 5.5, containing 2mmol/L EDTA and 1mmol/L
L-citrulline). Assays were systematically performed with Ca2+ (1mmol/L CaCl2) or without Ca2+ (0mmol/L
CaCl2) to measure total versus Ca2+-independent NOS activities. e Ca2+-dependent NOS activity was cal-
culated as total NOS activity minus Ca2+-independent NOS activity. All assays were performed in triplicate on
aliquoted samples (to avoid freezing/thawing cycles). e results were normalized for protein content.
A549 cells (1.5 × 104 cells per well) were seeded in the 96-well plates treated for tissue culture. Aer 24h incuba-
tion, the cell medium (180 μL) was refreshed. e cells were treated with 20μL of the control or test compounds
at the following nal concentrations: PBS, 1% ethanol solution (Control, A549C), 5-FU (40μM), RO (0.25mg/
mL), and RO + 5-FU (0.25mg/mL + 40μM). e calculations during the seeding of the cells were done in a way
that reached approximately 80% conuency at xation time. Aer 24h, the medium was discarded, and the cells
were xed 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. e OD450 readings were corrected for cell number by dividing
the OD450 reading for a given well by the OD595 reading. is 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
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Kinase p85 antibodies included in the PI 3 Kinase Kit. According to the manufacturer’s instructions, phospho-PI
3 kinase p85 and Total PI3K levels were measured using an In-Cell ELISA kit (ab207484).
A549 cells (5 × 105 cells per well) were cultured in 6-well plates and incubated for 24h. en, 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 at the following nal concentrations: 5-FU (40μM), RO (0.25mg/mL), and RO + 5- FU (0.25mg/
mL + 40μM). Aer 24h, the cells were harvested. Each test sample (100 μL) was added to three dierent passages
in triplicate. e cells were resuspended in 50 µL of chilled Cell Lysis Buer and incubated on ice for 10min.
en, the cell lysate was centrifuged for 1min (10,000 × g). Next, the supernatant (cytosolic extract) was trans-
ferred 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, tak-
ing into account the number of cells. All the steps were performed according to the protocol presented in the
Caspase-3/CPP32 Colorimetric Assay Kit (K106, BioVision) instructions.
e percentage of apoptotic cells was evaluated as previously described41. A549 cells (2 × 105 cells/mL) were
treated with vehicle or 5-FU (40μM), RO (0.25mg/mL), and RO + 5- FU (0.25mg/mL + 40μM) for 24h, respec-
tively. Aer the treatment, the cells were washed with PBS and xed with 4% paraformaldehyde in PBS for 10min.
en, the cells were washed twice with PBS for 5min and stained with Hoechst 33258 reagent (10μg/mL) for
10min at room temperature in the dark. en, the cells were washed with PBS and analyzed under a uorescence
microscope (× 250 magnication) (Zeiss, Germany). e Hoechst 33258 staining identies apoptotic cells based
on nuclear morphology. Cells with typical morphological changes, such as karyopyknosis, hyperuorescence,
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.
e crystallographic structures of PI3K and AKT were procured from the Protein Data Bank (PDB) database
(https:// www. rcsb. org/), using the identiers 6AUD and 2JDO, respectively. Visualization and preliminary assess-
ment of these structures were performed with the PyMOL Molecular Graphics System (Schrödinger, LLC). e
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. e resulting streamlined protein structures were then used for docking explorations. e extracted
ligands, on the other hand, were reserved for ensuing redocking studies as controls.
Ligand docking and binding site analysis with PyMOL and Autodock/Vina were used for docking42. e protein
and ligand structures were prepared using Autodock Tools43. During a typical procedure, the "exhaustiveness"
parameter was calibrated to 8, and standard parameters suggested by the program creators were used to ensure
the delity of the results. e compounds were sorted based on their binding strengths. e 2D binding mode
analysis of best docking scores was performed using LigPlot + soware (EMBL-EBI).
All the results are presented as the 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,
which was used to evaluate the statistical signicance of the TNFa, VEGFa, MMP-2, COX-2, arginase, NOS,
MDA, nitrite anions, Caspase-3, and apoptosis rate results. e signicance of the results obtained for PI3K and
Akt was assessed using two-way ANOVA and Tukey’s multiple comparisons tests. e statistical analyses were
performed using GraphPad Prism 8 soware (San Diego, CA, USA), and a signicance level of p < 0.05 was
deemed statistically signicant.
e data used to support the ndings of this study are included in the articles.
Received: 11 April 2024; Accepted: 24 June 2024
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Content courtesy of Springer Nature, terms of use apply. Rights reserved
16
Vol:.(1234567890)
| (2024) 14:14676 | https://doi.org/10.1038/s41598-024-65816-5
www.nature.com/scientificreports/
Plant materials were identied by Dr. Narine Zakaryan from the Department of Botany and Mycology at Yerevan
State University (YSU). is work was supported by the Science Committee of MESCS RA through research
projects numbered 21T-1F283, 21AG-1F068, and 23LCG-1F010.
e study’s conception and design were the results of collective contributions from all authors. e 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. e 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 nal version of the manuscript.
e authors declare no competing interests.
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 024- 65816-5.
Correspondence and requests for materials should be addressed to N.A.
Reprints and permissions information is available at www.nature.com/reprints.
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