ABSTRACT _Psoralea bituminosa_ L. (Fabaceae) is a medicinal plant traditionally used for its antimicrobial, antihyperglycemic, and antioxidant effects. This study investigated its anticancer

and antioxidant potential using aqueous and methanol extracts. The methanol extract exhibited higher total phenol (81.57 mg/g) and total flavonoid (39.06 mg/g) contents compared to the

aqueous extract. Antioxidant activity, assessed via the DPPH assay, showed moderate potency (IC₅₀: 330.77 µg/mL for aqueous- and 348.27 µg/mL for methanol extracts). Notably, the methanol

extract demonstrated significant cytotoxicity against multiple cancer cell lines (IC₅₀: 27.73–53.90 µg/mL) particularly against A549, MDA-MB231, and PC3. Liquid chromatography-mass

distinct structural groups with differing drug-likeness scores. Bioinformatics analysis further identified transcriptomic signatures enriched in oxidative phosphorylation and key

cancer-related pathways (e.g., TP53, PI3K, NRF2, and MYC), offering mechanistic insight. This integrative approach combining LC-MS, cheminformatics, and bioinformatics provides a

cost-effective framework for decoding phytochemical bioactivity and guiding natural product-based drug discovery. SIMILAR CONTENT BEING VIEWED BY OTHERS POTENTIAL ANTIOXIDANT AND CYTOTOXIC

IMPACTS OF DEFATTED EXTRACT RICH IN FLAVONOIDS FROM_ STYPHNOLOBIUM JAPONICUM_ LEAVES GROWING IN EGYPT Article Open access 12 August 2024 IN VITRO ANTIOXIDANT, ANTICANCER AND IN SILICO

STUDIES OF POLYPHENOL ENRICHED LEAF EXTRACT OF _ASYSTASIA GANGETICA_ Article Open access 17 November 2024 IN VITRO AND COMPUTATIONAL INVESTIGATION OF ANTIOXIDANT AND ANTICANCER PROPERTIES OF

_STREPTOMYCES COERULEOFUSCUS_ SCJ EXTRACT ON MDA-MB-468 TRIPLE-NEGATIVE BREAST CANCER CELLS Article Open access 24 October 2024 INTRODUCTION The genus _Psoralea_ (family Fabaceae)

encompasses over 100 species distributed worldwide1. _Psoralea bituminosa_ L., also known as _Bituminaria bituminosa_ (L.) C. H. Stirt. (Fabaceae), is a perennial herbaceous plant, found in

South Africa, America, Australia, and in the Mediterranean region. This species is commonly referred to as the Arabian pea, pitch trefoil, or scurf pea2. _P. bituminosa_ thrives in a variety

of soil types, ranging from shallow, stony soils to deep, sandy substrates. It requires full sunlight and well-drained soil, conditions that allow the plant to remain green throughout the

summer, making it one of the few species capable of such resilience3,4. The plant’s leaves are composed of three unequal leaflets that transition in color from green to grey. Its violet,

pea-like flowers bloom between January and June, though the plant can flower year-round in milder climates5. Traditionally, the leaves and legumes of _P. bituminosa_ are used as feed for

goats and cattle. In herbal medicine, the plant is employed to treat urinary infections, fever, spasms, and epilepsy6,7. In Ayurvedic practices, it serves as an anthelmintic and diuretic8. A

decoction of the leaves is also applied a remedy for hair loss1. _P. bituminosa_ has attracted considerable pharmacological interest due to its diverse secondary metabolites. Among them are

two natural furanocoumarins, psoralen and its angular isomer angelicin, known for their antibacterial and antifungal activity, as well as their applications in the treatment skin diseases9.

Other notable bioactive compounds include plicatin B, genistein, daidzin, 8-prenyldaidzein, iso-orientin, apigenin, phenolic acids, lignans and saponins7. These constituents contribute to

the plant’s potential for pharmaceutical development. The present study aimed to investigate phytochemically the aqueous and methanol extracts of _P. bituminosa_, which grows wild in Jordan,

and to evaluate their antiproliferative activity against a panel of cancer cell lines. Additionally, bioinformatics tools were applied to analyze the gene expression profiles of the cancer

cell lines and to uncover the associated signaling pathways involved in their response. By assessing the chemical composition, antioxidant activity, and cytotoxic effects of the aqueous and

methanol extracts, this study will provide insights into the bioactive properties of the plant’s phytochemicals and their potential as therapeutic agents for cancer treatment. RESULTS TOTAL

PHENOL, TOTAL FLAVONOID CONTENT AND ANTIOXIDANT ACTIVITY The total phenol content (TPC), total flavonoid content (TFC), and antioxidant activity of the aqueous and methanol extracts of _P.

bituminosa_ are presented in Table 1. Antioxidant activity was evaluated using the DPPH assay, and the IC₅₀ values indicate the extract concentration required to scavenge 50% of the DPPH

radicals. Earlier Ayoubi et al., (2018) reported higher TPC and TFC values for the methanol extract of _P. bituminosa_, collected in Israel10. These discrepancies may be explained by the

differences in biogeography, seasonal variation, and experimental protocols, all of which influence the phytochemical composition of plant extracts11,12. ANTIPROLIFERATIVE EVALUATION The

antiproliferative effects of the aqueous and methanol extracts were evaluated against eight cancer cell lines: A549, MCF7, HCT116, DU145, PC3, MDA-MB231, T47D, and one fibroblast cell line

(Table 2; Fig. 1). At a screening concentration of 100 µg/mL, the methanol extract demonstrated notable cytotoxicity, particularly against A549 (13.7 ± 3.97%), MCF7 (27.35 ± 2.94%), and

MDA-MB231 (41.83 ± 2.58%) cell lines. In contrast, the aqueous extract exhibited minimal cytotoxic effects, with cell viability remaining relatively high in A549 (83.47 ± 2.39%) and MCF7

(94.81 ± 1.93%) cell lines. Cell viability assay results (Table 2) revealed that the methanol extract of _P. bituminosa_ induced significantly greater cytotoxicity across all tested cancer

cell lines compared to the aqueous extract, indicating a stronger antiproliferative effect of the methanol fraction. Based on the cytotoxic effect observed with the methanol extract, IC50

values were determined only for this extract, which exhibited varying potency across the all-tested cancer cell lines tested (Fig. 2). The IC50 values ranged from 27.73 µg/mL for A549 cells

to over 150 µg/mL for T47D cells, indicating differential cellular sensitivity. These variations may reflect differences in cancer cell type, passage number, and intrinsic resistance

mechanisms. A549 cells were the most sensitive to the methanol extract, while T47D cells exhibited the highest resistance. LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY (LC-MS) ANALYSIS The LC-MS

analysis of the aqueous and methanol extracts revealed a diverse array of bioactive compounds potentially responsible for the observed biological activities (Table 3). The LC-MS analysis of

both aqueous and methanol extracts revealed a diverse profile of bioactive phytochemicals, including phenolic acids, flavonoids, coumarins, and isoflavones, which may underlie the extracts’

anticancer activity. The aqueous extract was particularly rich in flavonoid glycosides, with high levels of iso-orientin (26.89%), vitexin (25.62%), and saponarin (8.51%), as well as notable

amounts of caffeic acid (13.36%) and the hydroxycoumarin aesculetin (5.71%). In contrast, the methanol extract exhibited elevated concentrations of isoflavones, including daidzein (20.15%),

biochanin A (19.28%), and 7,3’-dimethoxy-5,6,4’-trihydroxyisoflavone (13.70%), in addition to benzoic acid (9.98%) and catechol (10.46%). COMPUTATIONAL CHEMICAL BIOLOGY STUDIES

CHEMINFORMATICS ANALYSIS OF P. BITUMINOSA PHYTOCHEMICALS The cheminformatics analysis conducted on 47 molecular structures corresponding to the phytochemicals identified in the aqueous and

methanol extracts of _P. bituminosa_ by LC-MS (Table 3). First, the structural information of all compounds in SMILES format was retrieved from PubChem. The structures were standardized

using RDKit26 by applying the following procedures: aromatization, adding explicit hydrogens, generating 2D coordinates, cleaning the molecule for correct stereochemistry, and removing

stereochemistry information. 3,885 2D molecular descriptors were generated using alvaDesc27, out of 5,305 available descriptors. Finally, Principal Component Analysis (PCA) was performed

using 1,873 molecular descriptors, -after filtering out constant, near-constant, or missing-value descriptors, - to visualize the clustering and distribution of phytochemicals identified in

_P. bituminosa_ (Fig. 3). This figure presents four PCA-derived clusters based on the molecular descriptors of _P. bituminosa_ phytochemicals, highlighting their distribution and potential

bioactivity. The bottom-left (negative PC1, PC2) includes flavonoid-rich compounds from the methanol extract such as daidzein (20.15%), biochanin A (19.28%), and

7,3’-dimethoxy-5,6,4’-trihydroxyisoflavone (13.70%). This cluster likely drives the observed methanolic extract’s strong anticancer activity. The top left cluster (negative PC1 and positive

PC2) consists of glycosylated flavonoids and polyphenols including iso-orientin, vitexin, and saponarin. These are more abundant in the aqueous extract and are associated with antioxidant

activity but lower cytotoxicity, possibly due to reduced lipid permeability (as has been reported for glycosides extracted from other plants28. The bottom right cluster (positive PC1 and

negative PC2) is enriched with phenolic acids, caffeic acid and aesculetin, along with minor constituents like caffeine, scopoletin, and resveratrol, which have known antioxidant,

anti-inflammatory and anticancer properties. The top right cluster (positive PC1 and PC2) contains organic acids, where aromatic acids such as gallic acid and 2,5-dihydroxybenzoic acid have

moderate PC2 values, and aliphatic acids such as succinic-, pivalic-, and ascorbic acids with higher PC2 values. Benzoic acid (9.98%), which is abundant in the methanol extract, lies at the

boundary between the last two clusters. In the PCA plot, coordinates representing phytochemicals were color-coded based on their consensus drug-likeness scores (DLS_cons), which reflect the

average of seven drug-likeness scoring functions included in the alvaDesc descriptor software. The color gradient ranges from blue (lower DLS) to red (higher DLS), visually indicating the

relative drug-likeness of each compound. The DLS_cons values span from 0.0 to 1.0, and all phytochemicals analyzed in this study exhibited scores of 0.50 or higher, suggesting moderate to

high drug-like potential. BIOINFORMATICS ANALYSIS OF TESTED CANCER CELL LINES A bioinformatics analysis of seven cancer cell lines (A549, MCF7, HCT116, DU145, PC3, MDA-MB231, and T47D) was

conducted using transcriptomic data from CCLE29 and pathway enrichment data from cBioPortal30. PCA of RNA-seq data revealed that A549 had a distinct gene expression profile, clustering more

closely with DU145, HCT116, MCF7, and MDA-MB231, and was most dissimilar to PC3 and T47D (Fig. 4A). This aligned with cytotoxicity results, where A549 was the most sensitive and T47D the

most resistant to _P. bituminosa_ methanol extract, prompting further exploration of cancer pathway involvement. PATHWAY ANALYSIS OF TESTED CANCER CELL LINES Pathway perturbation data for

the cancer cell lines were mined from cBioPortal30 using CCLE_2019 dataset of the Cancer Cell Line Encyclopedia (CCLE). A similarity matrix based on the number of mutated cancer genes in 10

key cancer pathways was generated and plotted as a heatmap shown in Fig. 4B. The analyzed 10 cancer pathways included: RTK-RAS, HIPPO, PI3K, WNT, NOTCH, TGF-Beta, MYC, NRF2, cell cycle, and

TP53. According to the number of mutated genes in these 10 cancer pathways, DU145 and MCF7 shared highest similarity, and these two cell lines were more similar to HCT than the rest of cell

lines consisting of T47D, A549, PC3 and MDA-MB231. MECHANISTIC INSIGHTS The top genes exhibiting the most significant differential expression between T47D and other cell lines, particularly

A549, included ATP8, ND3, ND4L, COX2, and COX3. To explore the functional implications of these genes, their protein-protein interaction (PPI) networks were constructed, incorporating both

the genes themselves and their nearest neighbor proteins/genes. Subsequently, biological pathway enrichment analysis was performed to identify key pathways associated with these network

genes, as illustrated in Fig. 5. The pathway enrichment analysis revealed an association with oxidative phosphorylation (OXPHOS), a critical process that couples the electron transport chain

(ETC) to ATP generation. OXPHOS plays a pivotal role in cancer biology, as cancer cells exploit ETC and OXPHOS to meet their heightened energy demands, resist oxidative stress, and support

proliferation, metastasis, and apoptosis resistance. Beyond cancer, OXPHOS has been also linked to neurodegenerative diseases (e.g., Parkinson’s, Huntington’s, Alzheimer’s, and ALS),

thermogenesis, and prion diseases. Notably, OXPHOS intersects with several other critical pathways such as MYC, NRF2, TP53, PI3K, and the cell cycle, which may collectively contribute to the

anticancer activity of the methanol extract of _P. bituminosa_. MINING THE BIOMEDICAL LITERATURE FOR SUPPORTIVE EVIDENCE These findings were in line with previous studies on _Psoralea_

species, highlighting their cytotoxic potential. For example, a study in Korea revealed that ripe fruits of _P. corylifolia_ inhibited several colorectal cancer cell lines including SW480

(IC50: 37.9 µg/mL), HT-29 (IC50: 40.7 µg/mL), and HCT116 (IC50: 45.3 µg/mL), by downregulating cyclin D1 and CDK4 expression31. Another study from Ireland demonstrated antiproliferative

activity of _P. corylifolia_ ethanol extract against HEp-2 cells (IC50: 22 µg/mL), A549 cells (IC50: 68 µg/mL)32. Similarly, a study in China revealed that _P. corylifolia_ methanol extract

exhibited potent growth inhibition in KB, KBv200, K562, and K562/ADM cells, with IC50 values ranging from 10.0 to 26.9 µg/mL. In India, _P. corylifolia_ seed extract was found to induce

apoptosis in human breast cancer MCF-7 cells33. In Italy, a chloroform extract of _P. bituminosa_ leaves, shoots, and roots demonstrated cytotoxic activity against HeLa cells, with the

highest cytotoxicity observed in the shoots34. Daidzein and biochanin A, the two most abundant phytochemicals in the methanol extract, are known for their cytotoxic effects against

hormone-dependent cancers, which align with the observed potent activity against breast and prostate cancer cell lines. Biochanin A has demonstrated cytotoxicity against A549 lung cancer

cells35,36,37,38. Additionally, ferulic acid and benzoic acid, both present in significant concentrations, may contribute to the extract’s pro-apoptotic and antioxidant properties39,40. On

the other hand, the aqueous extract exhibited weaker cytotoxicity, as evidenced by its higher IC₅₀ values, likely due to the prevalence of flavonoid glycosides, which may have reduced

cellular uptake and bioavailability compared to their aglycone counterparts. The limited anticancer effects of the aqueous extract suggest that glycosylation plays a role in modulating the

bioactivity of these compounds. One study provided indirectly supported the mechanistic insights identified in this study linking OXPHOS to the cytotoxic effects of _P. bituminosa._ It

revealed that 7,3’,4’-trihydroxyisoflavone (THIF), a metabolite of daidzein (the major phytochemical in the methanol extract of _P. bituminosa_), inhibits UVB-induced cyclooxygenase-2

(COX-2) expression by suppressing NF-κB transcriptional activity41. THIF directly inhibits Cot and MKK4 kinase activities, reducing the phosphorylation of mitogen-activated protein kinases

(MAPKs). This result aligns well with the finding that COX2 was among the top differentially expressed genes in cancer cell lines most responsive to the methanolic extract, further

supporting its association with OXPHOS pathways. DISCUSSION _P. bituminosa_, a medicinal plant traditionally used for various ailments, has recently gained considerable attention due to its

broad pharmacological potential. In this study, we comprehensively evaluated the antioxidant and anticancer activities of both methanolic and aqueous extracts of _P. bituminosa_, using an

integrated experimental and computational approach. The antioxidant assays revealed that both extracts exhibit moderate to strong activity, in line with their high total phenolic and

flavonoid content, as quantified by Folin-Ciocalteu and aluminum chloride assays. These findings are corroborated by LC-MS analysis, which confirmed the presence of potent antioxidant

constituents, including flavonoids, phenolic acids, and coumarins. Evidence from biomedical literature reaffirmed the putative antioxidant potential of the aqueous extract of _P.

bituminosa_42,43. The methanolic extract, however, demonstrated superior cytotoxicity, with the most potent effects observed against the A549 lung cancer cell line, followed by MDA-MB231

(breast) and PC3 (prostate). The enhanced activity is likely due to the presence of lipophilic isoflavones (daidzein, biochanin A) and phenolic acids, which can cross cellular membranes more

efficiently and exert intracellular effects. These compounds are known to modulate multiple cancer-related pathways, including estrogen receptor signaling, apoptosis induction, and cell

cycle arrest, possibly through ROS generation and mitochondrial depolarization45. The redox properties of phenolics and flavonoids not only confer antioxidant protection but also play a dual

role in cancer, where at higher concentrations they may induce oxidative stress selectively in tumor cells. This pro-oxidant mechanism is particularly relevant in flavonoid-rich methanolic

extracts, supporting their selective cytotoxicity. Such biphasic behavior underscores the therapeutic promise of flavonoids as redox modulators in cancer. The computational chemical biology

studies unveiled the structural diversity and physicochemical properties of _P. bituminosa_ phytochemicals. Using cheminformatics tools, a total of 47 phytochemicals with drug-like

properties were analyzed. The drug-likeness of these compounds was quantified using consensus DLSs generated by alvaDesc. Notably, most of the analyzed phytochemicals exhibited high DLSs,

suggesting favorable absorption, distribution, metabolism, and excretion profiles. In contrast, flavonoid glycosides, which contain sugar moieties, scored lower, likely due to their

increased polarity, higher molecular weight, and reduced membrane permeability. This highlights how structural differences, such as the presence of glycosidic groups/sugar molecules, can

influence drug-like behavior and potentially modulate biological activity a finding consistent with the reduced cytotoxicity often observed for highly glycosylated flavonoids. Principal

Component Analysis (PCA) further underscored the physicochemical diversity of these compounds, emphasizing their potential bioactivity and aligning with their putative therapeutic

applications. The cheminformatics characterization of _P. bituminosa_ phytochemicals using 2D alvaDesc molecular descriptors and PCA-based clustering identified four unique clusters and

results were well-aligned with the experimental findings. The anticancer properties of the methanolic extract are likely driven by flavonoids as evidenced by the clustering of the three

major phytochemicals—daidzein, biochanin A, and 7,3’-Dimethoxy-5,6,4’-trihydroxyisoflavone—within a distinct group. These features are commonly associated with enhanced cellular uptake and

interaction with intracellular targets such as estrogen receptors and apoptotic regulators. Meanwhile, the antioxidant effects of the aqueous extract primarily arise from glycosylated

flavonoids and polyphenolic compounds (e.g., hydroxylated coumarins and phenolic acids) which are generally more hydrophilic and exert their effects through extracellular radical scavenging

and modulation of redox-sensitive pathways. Interestingly, the majority of phytochemical constituents of _P. bituminosa_ exhibit acceptable drug-like properties, as indicated by DLS above

0.7. However, the lowest scores were observed for flavonoid glycosides, including apiin, isoorientin, and saponarin, which had scores closer to 0.5—the midpoint of the 0.0 to 1.0 DLS_cons

range. This reduction in drug-likeness is likely due to their increased hydrophilicity, decreased membrane permeability, and reduced interaction with lipophilic binding pockets in protein

targets. These findings provide a structural/molecular basis for further research and the therapeutic exploration of bioactive phytochemicals. Bioinformatics analyses of seven cancer cell

lines (A549, MCF7, HCT116, DU145, PC3, MDA-MB231, and T47D) indicated distinct transcriptomic profiles that correlated with differential sensitivity to _P. bituminosa_ extracts. Lung cancer

cell line A549 exhibited strong sensitivity to the methanolic extract, while breast cancer cell line T47D (breast cancer) demonstrated resistance. Enrichment analysis of key genes identified

from the bioinformatics analysis of cancer cell lines revealed pathways centered on oxidative phosphorylation (OXPHOS), a key metabolic process that supports cancer proliferation and

survival. The findings underscore how _P. bituminosa_’s phytochemicals may target cancer pathways associated with NRF2, TP53, and PI3K, with potential effects on MYC, suggesting their

promise for therapeutic development. Evidence from the biomedical literature indicates that phytochemicals, generally, have been shown to influence cancer pathways associated with genes such

as NRF2, TP53, and PI3K. However, while MYC is a well-known modulator of cell proliferation and metabolism in cancer, there is limited direct evidence demonstrating that phytochemicals

specifically target MYC-driven pathways. Thus, this gap presents an opportunity for experimental investigation by interested researchers including pharmacologists, cancer biologists,

molecular biologists, biochemists, and other researchers exploring the therapeutic potential of phytochemicals in MYC-driven cancer pathways. These findings align with previous studies on

_Psoralea_ species, demonstrating cytotoxic effects against various cancer cell lines, including colorectal (IC50: 37.9–45.3 µg/mL)31, HEp-2 (IC50: 22 µg/mL), and A549 (IC50: 68 µg/mL)32, as

well as MCF-7 and HeLa cells33,34. Key compounds like daidzein, biochanin A, ferulic acid, and benzoic acid contribute to pro-apoptotic and antioxidant activities35,36,37,38,39,40. These

results further highlight the role of glycosylation in modulating bioactivity and the therapeutic potential of _Psoralea_ species. In conclusion the integrative approach combining

computational chemical biology and experimental pharmacological studies characterized the anticancer and antioxidant properties of _P. bituminosa_ and identified this medicinal plant as a

promising source of anticancer compounds, particularly against lung cancer. The presence of bioactive flavonoids such as daidzein, Biochanin A, and 7,3’-Dimethoxy-5,6,4’-trihydroxyisoflavone

is likely responsible for these effects. In contrast, the aqueous extract, which is rich in glycosylated flavonoids and polyphenolics, showed strong antioxidant activity but lower

cytotoxicity, suggesting a protective role rather than a therapeutic one in cancer. Cheminformatics and bioinformatics analyses further linked the anticancer effects to key metabolic and

signaling pathways, including oxidative phosphorylation (OXPHOS), NRF2, TP53, PI3K, and MYC. These findings emphasize the importance of solvent selection in maximizing the extraction of

bioactive compounds and highlight _P. bituminosa_ as a promising source of therapeutic agents for cancer and potentially other diseases related to oxidative stress and metabolism. Future

research should prioritize isolating active compounds, elucidating their mechanisms of action, and optimizing bioavailability for clinical applications, particularly in cancer therapy.

MATERIALS AND METHODS PREPARATION OF PLANT EXTRACTS The aerial parts of _P. bituminosa_ were collected from surroundings of Amman, the capital of Jordan (31°N, 35°E), located in the

Mediterranean biogeographic zone of Jordan44. The plant was identified by Prof. Fatma Afifi using descriptive references and by comparison with the herbarium species from the School of

Science, The University of Jordan5,45. A voucher specimen of _P. bituminosa_ has been deposited in the Department of Pharmaceutical Sciences, School of Pharmacy, The University of Jordan,

under the deposition number FMJ-PB-02. The fresh plant material was finely chopped and divided into two portions, then subjected to maceration using water and methanol, respectively, at room

temperature in the dark for one week. Following solvent evaporation using a rotary evaporator, the resulting crude extracts were stored in refrigerator until further analysis. DETERMINATION

OF TOTAL PHENOL A one mL aliquot of each extract (1 mg/mL) was diluted with 10 mL of distilled water. After five minutes, 200 µL of Folin-Ciocalteu reagent was added, followed by 800 µL of

20% sodium carbonate. The volume was adjusted to 5 mL with distilled water, and the mixture was incubated in the dark at room temperature for one hour. Absorbance was then measured at 765 

nm. Gallic acid was used as the standard at concentrations of 100, 50, 25, 12.5, and 6.125 µg/mL. All measurements were performed in triplicate46. DETERMINATION OF FLAVONOIDS CONTENT Two

milliliters of the extract (1 mg/mL) were mixed with 0.1 mL of 10% aluminum chloride (AlCl3), 100 µL of 1 M sodium acetate, and 2.8 mL of distilled water. The mixture was incubated at room

temperature for 30 min, and the absorbance was measured at 415 nm. Rutin was used as the standard, and methanol served as the blank47. ANTIOXIDANT ACTIVITY DETERMINATION The antioxidant

activity of the extracts was assessed using the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay. A 0.1 mM DPPH solution in methanol (40 µL) was added to 250 µL of plant

extract at serial concentrations (1–500 µg/mL) in a 96-well plate. The control consisted of methanol. After 30 min of incubation at room temperature, absorbance was measured at 517 nm.

Trolox was used as the standard, and DPPH inhibition was calculated using the following formula: $${\text{I}}\% = {\text{ }}\left( {{\text{O}}{{\text{D}}_{{\text{control}}}} -

{\text{O}}{{\text{D}}_{{\text{sample}}}}} \right)/{\text{O}}{{\text{D}}_{{\text{control}}}}~ \times { { 1}}00\%$$ where ODcontrol and ODsample are the optical densities of the control and

the sample, respectively. ANTIPROLIFERATIVE ASSAY The antiproliferative activity of the _P. bituminosa_ extracts was evaluated using the MTT assay, following the previously described

method44. Briefly, cells were seeded in 96-well plates. Treated with varying concentrations (3.9–500 µg/mL) of the extract for 72 h. Cell viability was calculated using the following

formula: $${\text{Cell viability }}\left( \% \right){\text{ }} = \left( {{\text{O}}{{\text{D}}_{{\text{treated cells}}}}/{\text{O}}{{\text{D}}_{{\text{control cells}}}}} \right){\text{ }}

\times {\text{ 1}}00\%$$ The IC50 values were calculated by plotting cell viability against extract concentration. Doxorubicin (50 mg/25 mL) was used as a positive control. LC-MS ANALYSIS

The chemical composition of the methanolic extract was analyzed using a Bruker Daltonik Impact II ESI-Q-TOF mass spectrometer (Bremen, Germany) coupled with a Bruker Daltonik Elute UPLC

system. Chromatographic separation was achieved using a Bruker Solo 2.0_C18 UHPLC column (100 mm × 2.1 mm, 2.0 μm particle size) maintained at 40 °C. The flow rate was set to 0.51 mL/min

with a total run time of 30 min. The injection volume was 3 µL. The mobile phases consisted of solvent A (water containing 0.1% formic acid) and solvent B (methanol). Gradient elution was

used for optimal separation. The ionization was performed using an Apollo II ion funnel electrospray (ESI) source operated in positive mode. The MS parameters were as follows: capillary

voltage, 2500 V; nebulizer gas pressure, 2.0 bar; dry gas (nitrogen) flow, 8 L/min; dry temperature, 200 °C; mass resolution, 50,000 FSR; mass accuracy, < 1 ppm; and TOF repetition rate,

up to 20 kHz. For identification, standard compounds were used to determine both the exact m/z values and retention times48. All solvents used (acetonitrile, methanol, water, and formic

acid) were LC/MS grade. SAMPLE PREPARATION Stock solutions of reference compounds were prepared by dissolving appropriate amounts in DMSO (analytical grade) and diluting with acetonitrile.

For analysis, unknown samples were dissolved in 2.0 mL of methanol and centrifuged at 4000 rpm for 2.0 min. A 1.0 mL aliquot of the supernatant was transferred to the autosampler vial, and

3.0 µL was injected for LC-MS analysis. COMPUTATIONAL METHODS INTEGRATIVE CHEMICAL BIOLOGY INFORMATICS APPROACH An integrative informatics workflow adapted from Hajjo et al.49,50,51,52, was

applied to investigate the network pharmacology and anticancer mechanisms of _P. bituminosa_. This workflow includes: (1) a cheminformatics module to assess the chemical diversity and

predicted activities of its phytochemicals; (2) a network-mining module to identify genetic perturbations in relevant cancer cell lines; and (3) a pathway enrichment module to elucidate

biological processes underlying its anticancer effects. MOLECULAR DESCRIPTORS Molecular descriptors quantify the physicochemical properties of molecules. In this study, 2D alvaDesc27

molecular descriptors were generated for 47 _P. bituminosa_ phytochemicals, whose structures were retrieved from PubChem in SMILES format and standardized following Hajjo et al.49,50,51,52.

Out of 5305 descriptors, 3885 2D descriptors were calculated, and 1873 were retained for PCA after removing invariant, incomplete, or low-variance descriptors49,50,51,52. PROTEIN-PROTEIN

INTERACTIONS Functional protein-protein interaction networks for key genes identified from studying the genetic perturbations in cancer lines were generated in Cytoscape version 3.10.1 using

the StringApp53. ENRICHMENT ANALYSIS Pathway enrichment analysis was conducted using Cytoscape StringApp53.The genes/proteins of the generated functional protein-protein interaction network

were used as query seeds for the enrichment analysis. The significance of the enrichment results was assessed by calculating the false discovery rates (FDRs) of the hypergeometric

_p_-values. Pathways with FDR values below the threshold of 0.05 were classified as statistically significant and relevant to the gene list under investigation. CANCER CELL LINE ENCYCLOPEDIA

(CCLE) DATABASE The CCLE, a joint effort between the Broad Institute and the Novartis Institutes for Biomedical Research, provides comprehensive molecular profiles for 1,072 cell lines

derived from diverse lineages and ethnic backgrounds. It serves as a robust resource for studying genetic alterations, identifying drug targets, and exploring small-molecule and biologic

therapies. Herein, gene-level RNA-seq data in TPM (Transcripts Per Million) format were retrieved for A549, MCF7, HCT116, DU145, PC3, MDA-MB231, and T47D cell lines. The TPM normalization

accounts for both gene length and sequencing depth, similarly to RPKM, to ensure that the total expression levels across samples are comparable. These data were used for the PCA analysis of

cancer cells lines, as well as pathway enrichments mined from cBioPortal30. STATISTICAL ANALYSIS Data were expressed as mean ± standard deviation (SD) of three independent experiments.

Statistical analyses were performed using SPSS (version 22). One sample t-tests, one-way analysis of variance (ANOVA), and Tukey’s test were used to assess the statistical significance

between the groups. A p-value \(\:\le\:\) 0.05 was considered statistically significant. DATA AVAILABILITY All data generated or analysed during this study are included in this published

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and visualization of proteomics data. _J. Proteome Res._ 18, 623–632 (2019). Article  CAS  PubMed  Google Scholar  Download references FUNDING This work was funded by the Deanship of

Scientific Research and Innovation at Al-Zaytoonah University of Jordan (2022–2023/07/23, 2022–2023/17/50 and 2024–2025/06/29). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of

Pharmaceutical Sciences, Faculty of Pharmacy, Al-Zaytoonah University of Jordan, Amman, Jordan Nour H. Aboalhaija, Rima Hajjo & Heba Syaj * Laboratory for Molecular Modeling, Division of

Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Rima Hajjo * Board Member, Jordan CDC, Amman,

Jordan Rima Hajjo * Department of Pharmaceutics and Pharmaceutical Technology, School of Pharmacy, The University of Jordan, Amman, Jordan Rana Abu-Dahab * Department of Pharmaceutical

Sciences, School of Pharmacy, The University of Jordan, Amman, Jordan Fatma Afifi Authors * Nour H. Aboalhaija View author publications You can also search for this author inPubMed Google

Scholar * Rima Hajjo View author publications You can also search for this author inPubMed Google Scholar * Fatma Afifi View author publications You can also search for this author inPubMed 

Google Scholar * Heba Syaj View author publications You can also search for this author inPubMed Google Scholar * Rana Abu-Dahab View author publications You can also search for this author

inPubMed Google Scholar CONTRIBUTIONS N.H. conceptualization, chemistry and antiproliferative methodology, software, validation, prepared Figs. 1 and 2, writing, review and editing; R.H.

computational analysis, software, validation, prepared Figs. 3, 4 and 5, writing, review and editing; F.A. conceptualization, writing, review and editing; H.S. chemistry methodology; R.A.

antiproliferative methodology. CORRESPONDING AUTHOR Correspondence to Nour H. Aboalhaija. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL

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characterization and bioinformatics guided evaluation of antioxidant and cytotoxic effects of _Psoralea bituminosa_. _Sci Rep_ 15, 19075 (2025). https://doi.org/10.1038/s41598-025-04195-x

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