Biological Activities and Phytochemical Composition of Arum rupicola var. rupicola: An In Vitro and In Silico Study

ABSTRACT

The phytochemical composition and biological activities of extracts obtained from the aerial parts (ARA), fruits (ARF), and rhizomes (ARR) of Arum rupicola var. rupicola (Araceae) were investigated using in vitro and in silico approaches. Phenolic and volatile profiles were characterized by HPLC and GC/MS. Antioxidant (DPPH), antimicrobial (MIC), and cytotoxic (MTT) activities were evaluated. Molecular docking, MM‐GBSA, and ADME analyses were performed using Schrödinger Maestro. HPLC revealed that rutin (35,001 µg/mL) and coumaric acid (13,145 µg/mL) were the dominant phenolics in ARA, while quercetin (18,256 µg/mL) predominated in ARF. ARA showed the strongest cytotoxic activity against HL‐60 cells with an IC₅₀ value of 141 µg/mL and a selectivity index of 3.8. The strongest antimicrobial activity was observed in ARR against Bacillus cereus (MIC: 90 µg/mL). Molecular docking indicated that rutin exhibited high binding affinity toward cancer‐related and antimicrobial targets, with docking scores of −9.24 and −9.32 kcal/mol against KRAS G12D and β‐lactamase, respectively. ADME analysis showed that most compounds complied with Lipinski's Rule of Five. These findings highlight the potential of A. rupicola var. rupicola as a source of anticancer and antimicrobial agents.

Phytochemical profiling and in vitro bioactivity evaluation of Arum rupicola var. rupicola extracts revealed rutin as the dominant phenolic compound in aerial parts, demonstrating high binding affinities against cancer‐related and antimicrobial target proteins via molecular docking. The most significant finding in the cytotoxicity study was observed in the aerial extract. The rhizome extract showed potent activity against Bacillus cereus.

1. Introduction

Cancer and antimicrobial resistance have emerged as major global health challenges, necessitating the discovery of novel therapeutic agents [1, 2]. Medicinal plants, owing to their vast repertoire of secondary metabolites, remain a primary resource for drug discovery [3, 4]. Specifically, natural compounds are actively investigated for their capacity to suppress antiapoptotic proteins (such as Bcl‐2 and Bcl‐xL) or oncogenic mutants (e.g., p53 Y220C, KRAS, G12D) in cancer pathogenesis, as well as to inhibit resistance‐conferring enzymes (like β‐lactamases, DHFR, and DNA GyrB) in pathogenic microorganisms [5, 6, 7, 8].

Within the rich biodiversity of the Eastern Mediterranean [9], the genus Arum L. (Araceae) holds remarkable ethnobotanical significance. Various Arum species are traditionally used in folk medicine for gastrointestinal and inflammatory disorders, or consumed as a processed traditional food locally known as “tirşik” [10]. Previous phytochemical studies on widespread species such as A. maculatum L., A. italicum Mill., and A. palaestinum Boiss. have demonstrated potent antioxidant and anticancer properties [11].

Despite the well‐documented medicinal potential of the genus Arum, studies on A. rupicola Boiss. var. rupicola—a taxon distributed across Eastern and Southeastern Anatolia—remain critically limited. The organ‐specific phytochemical signatures and corresponding multitarget bioactivities of this taxon have remained largely unexplored, representing a significant research gap. To address this gap, the present study provides the first comprehensive phytochemical profiling and bioactivity evaluation of the aerial parts, fruits, and rhizomes of A. rupicola var. rupicola. By identifying distinct chemotaxonomic features—such as an exceptionally high accumulation of rutin in the aerial parts—and integrating in vitro bioassays with advanced in silico approaches, this research aims to elucidate the mechanisms of action of its dominant compounds against critical cancer and microbial protein targets. Ultimately, this study provides a molecular foundation for future bioactivity‐guided drug discovery within the genus Arum.

2. Experimental Section

2.1. Plant Material Collection and Extraction

Samples of Arum rupicola var. rupicola used in this study were collected from Kıvrıklı (37°4'44“ N, 35°43'23” E and 37°5'2“ N, 35°42'44” E) and Günyazı (37°4'2“ N, 35°57'40” E), Ceyhan, Adana, Türkiye. Species identification was carried out by Prof. Dr. Tuna Uysal, and samples were deposited in the KNYA Herbarium under the accession number SCK‐001. The aerial parts, fruits, and rhizomes of the plant were shade‐dried, ground into powder, and extracted with 80% methanol in an ultrasonic bath (30 min, 45°C) [12]. The resulting extracts were coded as ARA (aerial parts), ARF (fruit), and ARR (rhizome), and stored at −20°C until further use. Extraction yields were calculated as the percentage ratio of the obtained extract mass to the initial dry plant material.

2.2. Phytochemical Composition Analyses

The phenolic and volatile compound profiles of the extracts were determined by HPLC and GC‐MS techniques, respectively. HPLC analysis was performed on a Shimadzu system equipped with an SPD‐M20A diode array detector (DAD) and an ACE 5 C18 column (250 × 4.6 mm, 5 µm). Three mobile phases were employed: 2% acetic acid in water (A), 50% acetonitrile/0.5% acetic acid (B), and pure acetonitrile (C). The flow rate was set to 1.2 mL/min, and compounds were identified by comparison with 12 reference standards: gallic acid, 4‐hydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, coumaric acid, rutin, benzoic acid, cinnamic acid, rosmarinic acid, and quercetin. For GC/MS analysis, volatile compounds were determined using a Shimadzu GC/MS‐QP2020 system equipped with an Rxi‐5Sil MS capillary column (30 m × 0.25 mm ID, 0.25 µm df). Helium was used as the carrier gas; the injector temperature was set to 250°C and the initial oven temperature to 40°C. Compound identification was carried out by matching mass spectra against the Wiley 9 mass spectral library.

2.3. In Vitro Bioactivity Studies

2.3.1. Total Phenolic Content

Total phenolic content was determined spectrophotometrically using the Folin–Ciocalteu method. Sample absorbances were measured at 765 nm, and results were expressed as milligrams of gallic acid equivalent per gram of extract (mg GAE/g extract) [13].

2.3.2. Antioxidant Activity

The free radical scavenging capacity of the extracts was assessed using the 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) assay in 96‐well plates. Samples were serially diluted over a concentration range of 0.0312–1 mg/mL, and absorbance measurements were performed at 490 nm using an ELISA reader following 30 min of incubation. The half‐maximal inhibitory concentration (IC₅₀) values were subsequently calculated [14, 15].

2.3.3. Antimicrobial Activity

The antimicrobial activities of the extracts were evaluated against the following bacterial strains: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 70603, Staphylococcus aureus (MRSA) ATCC 43300, Salmonella enteritidis ATCC 13076, Sarcina lutea ATCC 9341, and Bacillus cereus ATCC 11778, as well as the yeast strain Candida albicans NRRL Y‐417. Minimum inhibitory concentrations (MIC) were determined using the broth microdilution method in accordance with the procedure described by Abbasoğlu, Tosun [16]. Brain Heart Infusion Broth (BHIB) was used for organism culture, while Mueller–Hinton Broth (MHB) was employed for MIC testing. Extract concentrations ranged from 6.25 mg/mL to 6.12 µg/mL, and gentamicin was used as the positive control. Microbial growth was monitored visually following incubation with TTC (2,3,5‐triphenyltetrazolium chloride) solution, and the lowest concentration at which no visible growth was observed was recorded as the MIC value.

2.3.4. Cytotoxic Activity

Cytotoxicity studies were conducted using DLD‐1 (colon carcinoma; ATCC CVCL_0248), HUVEC (normal endothelial; ATCC CCVL_2959), and HL‐60 (leukemia; ATCC CVCL_0002) cell lines. The DLD‐1, HUVEC and HL‐60 cell lines were kindly provided by Dr. Ali Uğur Ural (DLD‐1 and HUVEC) and Dr. Zerrin Öztürk (HL‐60). Cells were maintained in RPMI‐1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin‐Streptomycin, and incubated at 37°C in a 5% CO₂ atmosphere. Experiments were carried out in 96‐well plates; following cell seeding, cultures were treated with serially diluted extracts for 24, 48, and 72 h. Cell viability was assessed using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay, with absorbance measurements taken at 570 nm, and cell viability curves were plotted accordingly [17]. Cisplatin was used as a positive control. The selectivity index (SI) of the extracts toward cancer cells relative to normal cells was also calculated. All in vitro bioassays were performed in three independent experiments (biological replicates), with each experiment carried out using at least three technical replicates. Statistical analyses were performed with GraphPad Prism 11 software (GraphPad Software, San Diego, CA, USA) on Windows. Normality and homogeneity of variances were assessed using the Shapiro–Wilk and Brown–Forsythe tests, respectively, before two‐way ANOVA. Dunnett's multiple comparisons test was used for post hoc comparisons with the control group. A p‐value <0.05 was considered statistically significant.

2.3.5. Molecular Docking Studies

All computational analyses were performed using the Schrödinger Maestro 2025‐1 software package developed by Schrödinger LLC [18]. Target protein structures were retrieved from the RCSB Protein Data Bank (PDB) [19]. The target proteins investigated in this study are listed in Table 1.

TABLE 1.

Target protein structures and their PDB codes used in molecular docking studies.

Protein Structures	PDB ID	References
Bcl‐2	4LVT	[20]
p53	2VUK	[6]
Bcl‐xL	2YXJ	[21]
Kras (G12D)	4LUC	[7]
DNA Gyrase B (DNA GyrB)	7C7N	[22]
Dihydrofolate reductase (DHFR)	3SRW	[8]
β‐Lactamase	1BC2	[23]

Protein structures were prepared for docking using the Protein Preparation Wizard module within the Maestro interface. This process involved completing missing atoms and amino acid residues, adding polar hydrogen atoms, removing crystallographic water molecules located more than 5 Å from the binding site, and optimizing the hydrogen bond network. Protein structures were adjusted to physiological pH (7.4 ± 2.0) and energy minimization was performed using the OPLS4 force field.

Ligands were selected from among the phenolic compounds identified by HPLC and the volatile compounds detected at higher relative abundances by GC/MS analysis. Their three‐dimensional structures were retrieved from the PubChem database [24]. Ligand preparation was carried out using the LigPrep module in the Maestro interface, which generated ionization states and possible tautomers appropriate for physiological pH (7.0 ± 2.0), followed by energy minimization using the OPLS4 force field. A grid box was defined based on the determined parameters, and docking calculations were performed using the Glide module. Docking scores were validated and refined through repeated docking runs. Ligands were treated as fully flexible during docking to allow sampling of their possible conformations. The resulting extra‐precision (XP) docking scores were further evaluated by MM‐GBSA (Molecular Mechanics/Generalized Born Surface Area) analysis using the Prime module, in order to obtain more accurate estimates of binding free energies and to ensure the reliability of the docking scores. Protein–ligand interactions were visualized through 2D interaction diagrams and 3D binding surface images.

2.3.6. ADME Properties and Drug‐likeness Analysis

The pharmacokinetic profiles and drug‐likeness characteristics of the compounds identified in this study were evaluated using the QikProp module within the Maestro software package. Key pharmacokinetic parameters were computed, including molecular weight (MW), lipophilicity (logP), number of hydrogen bond donors (HBD), number of hydrogen bond acceptors (HBA), and polar surface area (PSA). The oral absorption potential of the compounds was assessed using QikProp's HumanOralAbsorption score (1: low, 2: medium, 3: high). Drug‐likeness was examined in accordance with Lipinski's Rule of Five (MW ≤ 500 g/mol, logP ≤ 5, HBD ≤ 5, HBA ≤ 10) using Rule of Five parameter.

2.3.7. Toxicity Prediction

The toxicological profiles of the identified phytochemicals were predicted using the ProTox‐3.0 software, a robust platform for the in silico prediction of small molecule toxicity. This analysis enabled the determination of acute oral toxicity, expressed as the half‐maximal lethal dose (LD₅₀, mg/kg), and the assignment of compounds to specific toxicity classes (Class I–VI), with Class I representing the most toxic and Class VI the least toxic category. Furthermore, the compounds were evaluated for potential organ‐specific toxicities and endpoints, including hepatotoxicity, carcinogenicity, immunotoxicity, and mutagenicity, alongside blood‐brain barrier (BBB) permeability. The predictive models employed by ProTox‐III rely on molecular similarity and pharmacophore identification, providing a reliable safety assessment for potential drug candidates.

3. Results and Discussion

3.1. Extraction Yield

Based on the percentage yield values (% w/w) of the methanol extracts obtained from different parts of A. rupicola var. rupicola, the highest extraction yield was obtained from the fruit extract, followed by the aerial parts and rhizome extracts, respectively. The extraction yield chart for all extracts is presented in Figure 1.

FIGURE 1.

Extraction yield chart of methanol extracts obtained from different plant parts.

3.2. Phytochemical Analysis and Antioxidant Activity Results

3.2.1. HPLC Analysis

HPLC analysis revealed the distribution of 12 standard phenolic compounds across different parts of the plant at varying concentrations. In the aerial part extract, rutin and coumaric acid were quantitatively predominant over the other phenolic compounds by a considerable margin. The fruit extract exhibited a rich phenolic profile characterized by high levels of quercetin and benzoic acid, whereas the rhizome extract displayed a comparatively poor phenolic composition relative to the other extracts. Within the rhizome, chlorogenic acid, rutin, and quercetin were the relatively prominent compounds. These quantitative differences suggest that the synthesis and accumulation of phenolic compounds in this species are organ‐specific, and that different plant organs fulfil distinct bioactive roles. A comparative overview of all identified phenolic compounds across the different plant parts is provided in Table 2.

TABLE 2.

Comparison of phenolic compound contents in different parts of A. rupicola var. rupicola based on HPLC analysis.

Phenolic compounds	ARA (µg/mL	ARF (µg/mL)	ARR (µg/mL)
Gallic acid	0.156	0.268	0.298
4‐hydroxybenzoic acid	0.107	2.571	0.386
Chlorogenic acid	0.320	0.992	0.989
Vanillic acid	0.078	0.179	0.137
Caffeic acid	0.389	0.085	0.049
Syringic acid	0.496	1.488	0.235
Coumaric acid	13.145	2.552	0.195
Rutin	35.001	4.513	0.946
Benzoic acid	1.320	8.864	0.119
Cinnamic acid	0.92	0.166	0.009
Rosmarinic acid	0.549	0.87	0.160
Quercetin	1.352	18.256	0.504

3.2.2. GC‐MS Analysis

According to the GC‐MS analysis results of A. rupicola var. rupicola, the most abundant compound in the aerial part extract was identified as methyl oleate, followed by methyl palmitate and hexadecane, 2,6,10,14‐tetramethyl‐, an isomer of phytane. In the fruit extract, the compound with the highest relative abundance was benzene, 1,4‐bis(1,1‐dimethylethyl)‐, followed by tetradecane and hexadecane, 2,6,10,14‐tetramethyl‐. Similarly, in the rhizome extract, benzene, 1,4‐bis(1,1‐dimethylethyl)‐ emerged as the dominant constituent, while methyl oleate, squalene and methyl linoleate were among the other notable compounds. Although aliphatic hydrocarbons were detected in varying proportions across all three extracts, fatty acid esters were markedly predominant in the aerial part and rhizome extracts, suggesting that these plant parts may represent a significant source of lipophilic bioactive compounds. The most abundant compounds in each plant part are compared in Table 3.

TABLE 3.

Most abundant compounds identified by GC‐MS analysis and their relative abundances based on relative peak area (%) of A. rupicola var. rupicola.

Compound	Aerial parts (Area %)	Fruit (Area %)	Rhizome (Area %)	Ret. time	Ret. index (Avg.)
Benzene, 1,4‐bis(1,1‐dimethylethyl)	3.97	8.29	7.78	25.574	1248
Methyl oleate	24.72	—	7.58	51.473	2099
Tetradecane	3.53	7.46	6.20	31.038	1398
Hexadecane, 2,6,10,14‐tetramethyl	6.22	7.12	2.81	40.659	1698
Heptane	5.80	4.82	3.93	4.949	699
Methyl palmitate	7.08	—	4.88	46.972	1923
Hexadecane	3.84	4.96	4.36	37.627	1598
Dodecane	—	5.01	3.66	23.660	1198
1‐Octadecanol	2.56	2.69	—	43.366	1791
Squalene	—	—	7.18	51.714	2108
Methyl linoleate	—	—	5.28	51.312	2092
Nonane, 5‐(2‐methylpropyl)	—	3.7	—	17.755	1053
Hexadecane	—	3.43	—	26.507	1273
1‐Heptacosanol	2.56	—	—	53.866	2193

3.2.3. Antioxidant Potential and Total Phenolic Content

The total phenolic contents and DPPH radical scavenging activities of the aerial part, fruit, and rhizome extracts of A. rupicola var. rupicola were determined using the Folin–Ciocalteu method and DPPH assay, respectively. Total phenolic content was expressed as mg GAE/g, and IC₅₀ values were calculated for antioxidant capacity. The results are presented in Table 4.

TABLE 4.

Total phenolic content and DPPH antioxidant capacity of A. rupicola var. rupicola extracts.

Extracts	Total phenolic content (mg GAE/g)	DPPH IC50 (mg/mL)
ARA	32.9	0.37
ARF	75.77	0.22
ARR	13.21	nd

The total phenolic content followed the order: fruit > aerial parts > rhizome. The DPPH radical scavenging activity results were consistent with the phenolic content data, with the highest antioxidant capacity observed in the fruit extract. The aerial part extract exhibited lower activity compared to the fruit, while the rhizome extract failed to reach 50% inhibition even at the highest concentration tested. Accordingly, the antioxidant efficacy of the extracts ranked in the order: fruit > aerial parts > rhizome.

3.2.4. Antimicrobial Activity

When the antimicrobial potential of the aerial part, fruit, and rhizome extracts of Arum rupicola var. rupicola were evaluated against seven bacterial and one yeast strain, none of the extracts exhibited activity against the Gram‐negative bacteria E. coli and P. aeruginosa. Against Klebsiella pneumoniae, the aerial part and rhizome extracts demonstrated only weak inhibition. A broader inhibitory spectrum was observed against Gram‐positive bacteria; notably, the rhizome extract displayed the most remarkable activity against B. cereus with a remarkably low MIC value. The fruit extract was recorded as the most effective extract against Sarcina lutea and Salmonella enteritidis. In the assessment of antifungal potential, the highest inhibitory activity against C. albicans was again observed in the rhizome extract, while the aerial part and fruit extracts exhibited comparatively weaker MIC values. The detailed MIC values of the extracts and the positive control gentamicin against all tested strains are presented in Table 5.

TABLE 5.

MIC values (µg/mL) of Arum rupicola var. rupicola extracts and gentamicin as the positive control. *× = No antimicrobial activity was observed. **N/A = Gentamicin, an antibiotic, was not used for comparison against C. albicans as it is a yeast strain.

Microorganisms	Strains	Aerial parts	Fruit	Rhizome	Gentamicin
Escherichia coli (ATCC 25922)	Gram (−)	×	×	×	1.25
Pseudomonas aeruginosa (ATCC 27853)	Gram (−)	×	×	×	156
Klebsiella pneumoniae (ATCC 70603)	Gram (−)	6250	×	6250	5
Salmonella enteritidis (ATCC 13076)	Gram (−)	6250	3125	6250	1.25
Staphylococcus aureus (MRSA) (ATCC 43300)	Gram (+)	6250	×	6250	312
Sarcina lutea (ATCC 9341)	Gram (+)	6250	1560	6250	156
Bacillus cereus (ATCC 11778)	Gram (+)	780	780	90	156
Candida albicans (NRRL Y‐417)	Yeast	3125	3125	1560	N/A

3.2.5. Assessment of Cytotoxic Potential

The cytotoxic activities of A. rupicola var. rupicola extracts and cisplatin were systematically evaluated at 24, 48, and 72‐h intervals. The extracts showed mostly dose‐ and time‐dependent cytotoxic effects in all tested cell lines (Figure 2). Among the tested extracts, the aerial part extract consistently showed the strongest and most selective cytotoxic effect, particularly against the HL‐60 cell line. A significant temporal increase in activity was observed; this was evidenced by the gradual decrease in IC₅₀ values from 0.639 mg/mL at 24 h to 0.549 mg/mL at 48 h, and reaching a very low value of 0.141 mg/mL at 72 h. In contrast, the fruit extract showed minimal cytotoxic activity at earlier time points (IC₅₀ > 1 mg/mL at 24 and 48 h), while moderate activity only emerged after prolonged exposure (IC₅₀ = 0.638 mg/mL at 72 h). The rhizome extract showed moderate activity, reaching an IC₅₀ value of 0.512 mg/mL at 72 h. In the DLD‐1 cell line, the aerial parts extract similarly showed an increase in cytotoxicity over time; IC₅₀ values decreased from 0.783 mg/mL at 24 h to 0.567 mg/mL at 48 h and 0.467 mg/mL at 72 h. However, both fruit and rhizome extracts showed relatively limited antiproliferative effects in this cell line; Only the rhizome extract showed moderate activity at 72 h (IC₅₀ = 0.721 mg/mL), highlighting the lower susceptibility of DLD‐1 cells compared to HL‐60 cells. Importantly, cytotoxicity assessments in the noncancerous HUVEC cell line revealed significantly higher cell viability in all treatments, suggesting selective toxicity against malignant cells. The extract from the aerial parts of the plant exhibited IC₅₀ values exceeding 1 mg/mL at 24 h, while showing moderate reductions to 0.636 mg/mL and 0.539 mg/mL at 48 and 72 h, respectively. The fruit extract never caused a 50% reduction in cell viability. In contrast, the rhizome extract showed limited cytotoxicity, reaching only an IC₅₀ of 0.780 mg/mL at 72 h. The IC₅₀ doses of cisplatin, used as a positive control, were calculated as 78.125 micrograms/mL at 24 h and 10.81 micrograms/mL at 48 h in the DLD1 cell line, with the 72‐h dose being below the range examined. In the HL60 cell line, the 24‐h IC₅₀ dose was calculated as 10.8 micrograms/mL, and the 48 and 72‐h IC₅₀ values were found to be <6.25 micrograms/mL. In the HUVEC cell line, the 24‐h IC₅₀ dose was calculated as 38.09 micrograms/mL, and the 48 and 72‐h IC₅₀ values were found to be <6.25 micrograms/mL.

FIGURE 2.

Cell viability graphs of studied cell lines (A: DLD‐1; B: HL‐60 and C: HUVEC cell lines).

Taken collectively, these findings highlight that the extract obtained from the aerial parts of the plant is the most biologically active extract, exhibiting significant, time‐dependent cytotoxicity and remarkable selectivity, particularly against HL‐60 cancer cells (Table 6). The relatively low toxicity in HUVEC cells, as reflected in the calculated selectivity indices, further supports its potential therapeutic importance.

TABLE 6.

Selectivity indices of the extracts against the cell lines.

Selectivity index (SI)	ARA	ARF	ARR
DLD1 versus HUVEC 48h	1.12	nd	nd
DLD1 versus HUVEC 72h	1.15	nd	1.08
HL‐60 versus HUVEC 48h	1.16	nd	nd
HL‐60 versus HUVEC 72h	3.8	nd	1.5

In early‐stage drug development, the selectivity index (SI) is used as a crucial parameter to assess the potential safety range of a drug candidate. While an SI > 10 is generally desired for pure therapeutic agents, in natural product research, an SI > 3 is widely accepted as a promising threshold justifying the fractionation of crude plant extracts based on biological activity [25]. In this study, while fruit and rhizome extracts showed relatively poor selectivity (SI < 2), the extract obtained from aerial parts (ARA) achieved an SI value of 3.8 against HL‐60 cells at 72 h. Although this crude extract itself cannot be directly recommended as a therapeutic agent based solely on this value, an SI of 3.8 demonstrates a moderate but biologically significant degree of selective toxicity against leukaemia cells. This finding strongly supports the need for future isolation studies targeting biologically active compounds within this species. Pure isolated compounds generally exhibit significantly higher and safer selectivity profiles than their corresponding crude extracts. The strong cytotoxic activity observed in the extract obtained from the aerial parts of the plant is presumed to be related to the dominant phenolic components, particularly rutin and coumaric acid. However, since fractionation based on biological activity or isolation of specific active compounds was not performed in this study, this relationship currently remains a preliminary hypothesis rather than definitive proof. The literature reports that flavonoids such as rutin and quercetin exhibit anticancer effects by triggering apoptosis and causing cell cycle arrest, and molecular docking studies confirming these mechanisms have also been documented [25, 26, 27, 28]. The obtained data support the idea that the aerial parts of A. rupicola var. rupicola, in particular, may be a promising natural resource for anticancer drug research.

3.2.6. Molecular Docking and MM‐GBSA Analysis

As an initial validation step in the molecular docking study, the reliability and predictive accuracy of the molecular docking protocol were confirmed through a re‐docking procedure [26, 27]. For this purpose, the cocrystallized ligands were deleted from their respective target protein structures and redocked [28]. It should be noted that the 1BC2 structure is an apo form lacking a native ligand; therefore, its grid box was accurately centered using the active site coordinates detailed in the original crystallographic study. Furthermore, the redocking of the native ligand for 3SRW was challenging, likely due to its high conformational flexibility within the rigid‐receptor grid. The redocking results demonstrated that the calculated RMSD values for the validated targets were almost entirely below the universally accepted threshold of 2.0 Å [26, 27], confirming the robustness of the utilized grid parameters. The exact RMSD values and docking scores of the native ligands are summarized in Table 7.

TABLE 7.

Re‐docking scores and RMSD values of the native ligands for the validation of the molecular docking protocol.

Target protein	PDB ID	Native ligand	Docking scores (kcal/mol)	RMSD values (Å)
Bcl‐2	4LVT	1XJ 301	−10.0	1.19
Bcl‐xL	2YXJ	N3C 1001	−9.1	2.01
P53 (Y220C)	2VUK	P83 1291	−5.8	0.74
Kras (G12D)	4LUC	20G 203	−7.2	1.39
DNA GyrB	7C7N	FKR 301	−8.91	1.17
DHFR	3SRW	A2G 1	—	—
ß‐Lactamase	1BC2	—	—	—

In this study, molecular docking and MM‐GBSA analyses were performed to evaluate the binding affinities of compounds identified in Arum rupicola var. rupicola against proteins that are frequently targeted in drug discovery studies for cancer treatment — namely Bcl‐2, Bcl‐xL, p53 (Y220C mutant), and KRAS (G12D mutant) — as well as proteins playing critical roles in the survival of pathogenic microorganisms and the development of drug resistance. The docking scores of the investigated compounds are presented in detail in Table 8, while the binding free energies (ΔG) calculated by MM‐GBSA are provided in Table 9.

TABLE 8.

Molecular docking scores of compounds identified in A. rupicola var. rupicola against the target proteins.

Compounds and scores (kcal/mol)	Bcl‐2	Bcl‐xL	P53 (Y220C)	Kras (G12D)	DNA GyrB	DHFR	ß‐ Lactamase
Gallic acid	−3.85	−4.40	−4.94	−6.22	−4.27	−4.96	−6.19
4‐Hydroxybenzoic acid	−4.83	−2.8	−3.10	−4.12	−3.13	−4.27	−3.75
Chlorogenic acid	−6.23	−6.72	0.23	−6.74	−1.36	−8.17	−6.72
Vanillic acid	−3.43	−3.51	−3.45	−4.49	−3.28	−4.83	−4.14
Caffeic acid	−3.57	−4.35	−5.60	−4.93	−4.45	−6.01	−4.52
Syringic acid	−2.92	−3.73	−3.56	−5.90	−3.30	−4.80	−4.33
Coumaric acid	−4.74	−3.60	−3.95	−3.28	−3.50	−4.76	−3.79
Rutin	−7.50	−7.40	−7.27	−9.24	−6.90	−6.49	−9.32
Benzoic acid	−4.70	−2.4	−3.14	−3.65	−2.76	−3.78	−2.76
Cinnamic acid	−4.54	−4.05	−3.33	−3.46	−3.33	−4.93	−2.23
Rosmarinic acid	−6.95	−5.98	−8.31	−8.11	−5.87	−8.41	−6.46
Quercetin	−6.47	−6.58	−5.37	−6.57	−6.19	−8.81	−4.67
Squalene	−6.93	−6.07	−3.24	−4.79	0.75	−0.88	2.13

Note: The best docking scores for each target protein are indicated in bold and highlighted in green.

TABLE 9.

MM‐GBSA scores of compounds identified in A. rupicola var. rupicola against the target proteins.

Compounds and MM‐GBSA (ΔG) scores	Bcl‐2	Bcl‐xL	P53 (Y220C)	Kras (G12D)	DNA GyrB	DHFR	ß‐Lactamase
Gallic acid	−11.92	−14.04	−22.31	−30.04	−10.93	−15.51	−25.56
4‐Hydroxybenzoic acid	−20.36	−16.87	−7.08	−12.37	−9.92	−15.04	−14.97
Chlorogenic acid	−43.28	−32.63	−29.67	−33.98	−36.55	−46.89	−13.49
Vanillic acid	−19.11	−19.82	−20.12	−19.39	−14.96	−21.13	3.92
Caffeic acid	−22.47	−18.53	−25.22	−24.65	−22.38	−15.12	−13.30
Syringic acid	−20.97	−21.00	−12.77	−19.31	−11.28	−22.50	6.89
Coumaric acid	−22.75	−20.82	−18.40	−22.67	−16.86	−23.96	−8.74
Rutin	−58.54	−46.62	−60.80	−42.09	−40.73	−44.10	−15.12
Benzoic acid	−20.45	−16.07	−10.80	−19.07	−9.72	−15.04	−6.05
Cinnamic acid	−25.57	−24.42	−17.52	−29.76	−17.62	−27.24	−2.49
Rosmarinic acid	−48.42	−49.92	−43.85	−33.00	−50.44	−47.62	−21.59
Quercetin	−31.26	−36.38	−28.33	−49.17	−46.70	−52.29	−24.95
Squalene	−79.90	−75.01	−43.81	−44.67	−55.64	−43.60	−24.89

Note: The best docking scores for each target protein are indicated in bold.

According to molecular docking analysis results, rutin exhibits the highest binding affinity for most of the tested target proteins. Rutin showed high docking scores against Bcl‐2, Bcl‐xL, KRAS (G12D), and β‐lactamase proteins. The highest docking score against the p53 (Y220C) protein was obtained for rosmarinic acid, while rutin ranked second with a moderate to high score. In addition to rutin, the docking scores of other compounds associated with cytotoxicity and showing high binding affinity against cancer target proteins are also noteworthy. This study demonstrates that rutin, a naturally occurring flavonoid, exhibits a strong potential binding affinity against key target proteins associated with cancer and bacterial diseases. Molecular docking analyses reveal that rutin forms stable computational interactions with the antiapoptotic proteins Bcl‐2 and Bcl‐xL, suggesting a theoretical binding profile that could target mechanisms suppressing programmed cell death (Figures 3, 4, 5, 6). Furthermore, rutin appears to occupy the binding gap created by the p53 Y220C mutation, which may provide structural complementarity; however, this is a hypothesis, and definitive experimental verification is needed to confirm functional recovery of its tumor suppressor activity (Figures 7 and 8). Its high binding affinity and multiple interactions with the oncogenic KRAS G12D suggest that after purification, rutin could be a promising candidate for future in vitro testing against this challenging target (Figures 9 and 10). In addition, rutin was found to interact with the active sites of β‐lactamase and DNA GyrB in a computational setting; this suggests potential binding interactions that warrant future investigation related to antibiotic resistance and bacterial proliferation (Figures 11, 12, 13, 14). Overall, these findings highlight rutin as a promising multitarget agent candidate for both anticancer and antibacterial therapeutic strategies; however, further experimental and clinical validation is definitely needed.

FIGURE 3.

2D interaction diagram of the Bcl‐2/rutin complex. Examination of the 2D interaction diagram of rutin in the Bcl‐2 BH3 binding pocket reveals that the ligand forms three hydrogen bonds with Ala146, Asp108, and Asn140 residues via its hydroxyl groups. Hydrophobic residues such as Phe101, Phe109, Ala110, Met112, and Val130, which constitute the hydrophobic surface of the binding pocket, surround the ligand and stabilize the binding interaction. Additionally, a π–π stacking interaction was formed between Tyr105 and the aromatic ring of rutin. This combination of interactions suggests that rutin is strongly accommodated within the P2 pocket of the Bcl‐2 BH3 active binding site and may block apoptosis inhibition.

FIGURE 4.

3D surface representation of the Bcl‐2/rutin complex.

FIGURE 5.

2D interaction diagram of the Bcl‐xL/rutin complex. In the Bcl‐xL BH3 pocket, rutin formed six hydrogen bonds with negatively charged and polar residues including Gly138, Asp133, and Asp107. The hydrophobic pocket shaped by Leu108, Ala104, and Tyr101 surrounds the ligand and reinforces the binding conformation. Notably, the number of hydrogen bonds is higher compared to Bcl‐2, indicating that the more polar character of the Bcl‐xL binding pocket engages more extensively with the hydroxyl groups of rutin.

FIGURE 6.

3D surface representation of the Bcl‐xL/rutin complex.

FIGURE 7.

2D interaction diagram of the P53 (Y220C)/rutin complex. The interaction diagram within the p53 Y220C mutant binding pocket reveals that rutin forms multiple hydrogen bonds with key residues surrounding the mutation site, including Ser227, Asp228, Gly226, and Thr230. Residues such as Pro222, Pro223, and Glu221 hydrophobically encircle the ligand. The density of polar interaction lines suggests that rutin may fill the cavity created by the Y220C mutation, potentially enhancing the thermal stability of p53 and thereby partially restoring its tumor suppressor function.

FIGURE 8.

3D surface representation of the P53 (Y220C)/rutin complex.

FIGURE 9.

2D interaction diagram of the KRAS (G12D)/rutin complex. Within the KRAS G12D binding pocket, rutin notably forms seven hydrogen bonds and establishes a π–cation interaction with Lys88 via its aromatic ring. This π–cation interaction serves as the primary explanation for the exceptionally high docking score of −9.24 kcal/mol. This extensive network of interactions involving multiple polar and hydrophobic residues of varying charge suggests that rutin is positioned in close proximity to the GTP‐binding site of the KRAS G12D mutant protein and carries the potential to inhibit this constitutively active oncogenic protein.

FIGURE 10.

3D surface representation of the KRAS (G12D)/rutin complex.

FIGURE 11.

2D interaction diagram of the ß‐Lactamase/rutin complex. In the β‐lactamase binding pocket, rutin formed hydrogen bonds with Asn180 and Ser174 residues. A salt bridge formed with Lys171 is also noteworthy. The active site residues Ala175 and Leu178 complete the hydrophobic environment. Given that the β‐lactamase active site harbors catalytic residues responsible for hydrolyzing the β‐lactam ring, the high‐affinity binding of rutin to this site suggests that it may competitively inhibit enzyme activity.

FIGURE 12.

3D surface representation of the ß‐Lactamase/rutin complex.

FIGURE 13.

2D interaction diagram of the DNA GyrB/rutin complex. In the DNA GyrB binding pocket, rutin established hydrogen bonds with the negatively charged residues Glu50, Asp49, and Asp73, as well as with the polar residue Asn46, and formed peripheral interactions with hydrophobic, negatively charged, and polar residues. This positions the ligand in close proximity to the ATP‐binding site. Inhibition of this binding site, which is critical for bacterial DNA replication, represents a clinically significant target capable of arresting bacterial proliferation.

FIGURE 14.

3D surface representation of the DNA GyrB/rutin complex.

Chlorogenic acid stood out with moderate‐to‐high scores against the Bcl‐2 and Bcl‐xL proteins, while quercetin yielded significant scores for Bcl‐xL and KRAS mutant proteins. Rosmarinic acid displayed a high score against the KRAS (G12D) mutant protein, approaching that of rutin.

Comparative analysis of binding scores between naturally cocrystallised ligands and identified phytochemicals provides valuable preliminary information regarding relative binding affinities. Although in silico binding scores are estimates rather than absolute measures of in vitro efficacy, several phytochemicals exhibit stronger binding energies than their respective naturally cocrystallised ligands. For example, in structural models of KRAS (G12D) and the p53 (Y220C) mutant, rutin (−9.24 and −7.27 kcal/mol) and rosmarinic acid (−8.11 and −8.31 kcal/mol) showed higher negative binding scores than their naturally cocrystallized ligands (−7.20 and −5.80 kcal/mol). In contrast, for targets such as Bcl‐2, Bcl‐xL, and DNA GyrB, most of the evaluated phytochemicals exhibited moderate to weak interactions, while highly optimized naturally cocrystallized ligands maintained superior binding affinities. This observation is theoretically expected, since natural ligands are typically synthetic molecules specifically engineered for maximum receptor affinity, whereas plant‐derived secondary metabolites naturally interact with these binding pockets in varying capacities. Overall, these comparative computational findings suggest that specific components of A. rupicola var. rupicola can suitably occupy specific active site clefts, highlighting a target‐dependent competitive potential requiring further experimental validation. Considering that these compounds are also present in the aerial part extract — and particularly that rutin is most abundant in the aerial part extract —it is hypothesized that the observed cytotoxic activity may be associated with rutin, potentially acting synergistically with other co‐occurring compounds. Nonetheless, functional validation through targeted isolation and specific bioassays is strictly necessary to confirm these predictions. Specifically, although in silico docking profiles demonstrate favorable interactions with apoptosis‐regulating proteins such as Bcl‐2 and Bcl‐xL, the exact mechanisms of cell death induced by the extracts cannot be definitively established without advanced in vitro mechanistic assays. To translate these findings from computational hypotheses into validated pharmacological mechanisms, future studies incorporating Annexin V/PI flow cytometry, caspase activation assays, and Western blot analysis of target proteins are explicitly required. Despite having the highest total phenolic content, the fruit extract failed to match the activity of the aerial part extract, which further supports the prominent role of rutin.

Furthermore, quercetin exhibited a notable binding affinity against the DHFR protein with a score of −8.81 kcal/mol. However, the evaluation of antimicrobial target proteins did not reveal a compelling correlation between the docking results and the experimental antimicrobial data. The rhizome extract demonstrated potent activity against Bacillus cereus with a remarkably low MIC value of 90 µg/mL, yet this extract had the lowest total phenolic content of all extracts. This suggests that the strong antimicrobial activity of the rhizome extract might be hypothetically associated with lipophilic constituents such as fatty acid esters detected by GC‐MS analysis, or to synergistic interactions between these lipophilic compounds and phenolic constituents; however, specific isolation and bioactivity‐guided fractionation are strictly required to definitively confirm this mechanism. The molecular docking results of rutin — the compound yielding the highest docking scores — were visualized for each target protein in terms of docking scores, 2D interaction diagrams, and 3D binding surface representations, and are presented in figures.

The MM‐GBSA scores corroborated the docking scores and provided a more precise assessment of binding free energies. Rutin exhibited the lowest binding free energies among all compounds against Bcl‐2 and p53 (Y220C) proteins, with values of −58.54 and −60.80 kcal/mol, respectively (Table 8). These values indicate that rutin forms thermodynamically highly stable complexes with these proteins. Against Bcl‐xL and KRAS (G12D) proteins, rutin displayed a binding profile that can still be considered strong, with MM‐GBSA scores of −46.62 and −42.09 kcal/mol, respectively. This consistent pattern demonstrates that rutin possesses a broad‐spectrum binding capacity against cancer‐related target proteins.

Rosmarinic acid exhibited a binding energy comparable to that of rutin against the p53 (Y220C) protein with an MM‐GBSA score of −43.85 kcal/mol, while demonstrating notably strong interactions against DNA GyrB and DHFR proteins with scores of −50.44 and −47.62 kcal/mol, respectively. These results suggest that rosmarinic acid may exert considerable efficacy, particularly against antimicrobial target proteins. Quercetin achieved the strongest MM‐GBSA score among all compounds for the DHFR target with a value of −52.29 kcal/mol. Also, it demonstrated significant binding energies against Bcl‐xL and KRAS (G12D) proteins with scores of −36.38 and −49.17 kcal/mol, respectively. Chlorogenic acid, which was also identified among the phenolic compounds with notable docking scores, demonstrated strong MM‐GBSA binding free energies against Bcl‐2 (−43.28 kcal/mol) and KRAS (G12D) (−33.98 kcal/mol), as well as a particularly favorable interaction with DNA GyrB (−36.55 kcal/mol). These values suggest that chlorogenic acid, despite its moderate docking scores, forms thermodynamically stable complexes with both cancer‐related and antimicrobial target proteins, warranting further attention as a secondary bioactive constituent. Overall, these findings indicate that while rutin stands out as a promising multitarget anticancer agent, rosmarinic acid and quercetin may offer complementary advantages, particularly in antimicrobial and target‐specific therapeutic strategies.

When the chemical profiles of each extract are considered in terms of their dominant constituents, distinct activity‐composition relationships emerge. The aerial part extract (ARA), characterized by rutin (35.001 µg/mL) and coumaric acid (13.145 µg/mL) as the principal phenolic constituents, and by methyl oleate (24.72%) and methyl palmitate (7.08%) among its major volatile components, demonstrated the most potent cytotoxic activity (IC₅₀: 141 µg/mL at 72 h against HL‐60) alongside the highest selectivity index, suggesting that the combination of these compounds — particularly rutin — drives its anticancer potential. The fruit extract (ARF), dominated by quercetin (18.256 µg/mL) and benzene, 1,4‐bis(1,1‐dimethylethyl)‐ (8.29%) as the most abundant volatile, exhibited the highest total phenolic content and the strongest antioxidant capacity (DPPH IC₅₀: 0.22 mg/mL); the high binding affinity of quercetin against Bcl‐xL, KRAS (G12D), and DHFR further supports its therapeutic relevance. The rhizome extract (ARR), while comparatively poor in phenolic content, contained a chemically diverse volatile fraction comprising benzene, 1,4‐bis(1,1‐dimethylethyl)‐ (7.78%), methyl oleate (7.58%), squalene (7.18%), and methyl linoleate (5.28%), which is hypothesized to be associated with its remarkable antimicrobial activity against B. cereus (MIC: 90 µg/mL), although this correlation warrants further experimental validation. Collectively, these observations reinforce the notion that the bioactivity of each extract is shaped by the interplay of its dominant phenolic and lipophilic constituents rather than by a single compound.

3.2.7. ADME Assessment

Upon examination of the QikProp analysis results, the vast majority of the 13 compounds tested were found to comply with Lipinski's Rule of Five, thereby possessing favorable pharmacokinetic profiles with respect to oral bioavailability. Detailed ADME analysis results are presented in Table 10.

TABLE 10.

Drug‐likeness results obtained for each compound through ADME studies.

Compounds and ADME assessment	MW (g/mol)	logP	HBD	HBA	PSA (Å2)	Oral absorp.	Rule of Five
Gallic acid	170.121	−0.572	4	4.25	115.464	2	0
4‐Hydroxybenzoic acid	138.123	0.582	2	2.75	73.326	3	0
Chlorogenic acid	354.313	−0.302	6	9.65	187.374	1	1
Vanillic acid	168.149	1.029	2	3.5	79.697	3	0
Caffeic acid	180.160	0.545	3	3.5	90.863	2	0
Syringic acid	198.175	0.959	2	4.25	86.77	3	0
Coumaric acid	164.160	1.425	2	2.75	74.179	3	0
Rutin	610.524	−2.796	9	20.55	278.149	1	3
Benzoic acid	122.123	1.868	1	2	49.784	3	0
Cinnamic acid	148.161	1.9	1	2	51.641	3	0
Rosmarinic acid	360.320	1.162	5	7	171.176	1	0
Quercetin	302.24	0.367	4	5.25	143.337	2	0
Squalene	410.725	12.007	0	0	0	1	1

Rutin, the focal compound of the molecular docking analysis, has a molecular weight of 610.524 g/mol, 9 hydrogen bond donors, 20.55 hydrogen bond acceptors, and a PSA value of 278.149 Ų, violating three of Lipinski's rules and yielding a low oral absorption score. This indicates that the oral bioavailability of rutin in its free form may be limited. However, this is a frequently encountered characteristic in the literature for flavonoid glycosides; it is well established that bioavailability can be enhanced through formulation strategies such as nanoparticle encapsulation, liposomal encapsulation, or biocatalytic hydrolysis, or by utilizing rutin as a lead compound for the development of novel derivatives through medicinal chemistry approaches. Rosmarinic acid, another high‐scoring compound, does not violate Lipinski's rules; however, its oral absorption score remained low due to its high PSA value. Quercetin, while at borderline values, exhibited no Lipinski violations and demonstrated moderate oral absorption. Chlorogenic acid, with 6 hydrogen bond donors and a PSA of 187.374 Ų, was the only other compound showing a single Lipinski violation, and its oral absorption was found to be low. Squalene exhibited an excessively lipophilic profile and was calculated to have a low oral absorption score. In contrast, simple phenolic acids such as gallic acid, vanillic acid, caffeic acid, syringic acid, coumaric acid, benzoic acid, and cinnamic acid demonstrated full compliance with Lipinski's rules and exhibited drug‐like characteristics with high oral absorption scores.

3.2.8. Toxicity Assessment

In line with modern drug discovery protocols, the safety profiles of the identified compounds were comprehensively evaluated using the ProTox‐III webserver, which predicts various toxicological endpoints, including hepatotoxicity, carcinogenicity, immunotoxicity, mutagenicity, and blood–brain barrier (BBB) permeability. The toxicity prediction results are summarized in Table 11.

TABLE 11.

Toxicity and bood–brain barrier (BBB) permeability prediction results obtained using the ProTox‐3.0 software. Note: In the table, (+) indicates ‘Active’ (toxic or permeable) for the corresponding parameter, while (−) indicates ‘Inactive’ (non‐toxic or non‐permeable). LD₅₀ values are expressed in mg/kg.

Compounds	LD50 (mg/kg)	Tox. class	Hepato.	Carcino.	Immuno.	Muta.	BBB permeability
Gallic acid	2000	4	—	+	—	—	+
4‐Hydroxybenzoic acid	2200	5	—	—	—	—	+
Chlorogenic acid	5000	5	—	—	+	—	+
Vanillic acid	2000	4	—	—	—	—	+
Caffeic acid	2980	5	—	+	—	—	+
Syringic acid	1700	4	—	—	—	—	+
Coumaric acid	2850	5	—	+	—	—	+
Rutin	5000	5	—	—	+	—	—
Benzoic acid	290	3	+	—	—	—	+
Cinnamic acid	2500	5	+	—	—	—	+
Rosmarinic acid	5000	5	—	—	+	—	+
Quercetin	159	3	—	+	—	+	+
Squalene	5000	5	—	—	—	—	+

Most of the compounds were classified within toxicity classes 4 and 5, which are considered to carry a low risk of acute toxicity, with LD50 values ranging from 159 to 5000 mg/kg. Notably, key bioactive constituents—such as rutin, chlorogenic acid, and rosmarinic acid—were assigned to toxicity class 5, indicating a favorable safety profile. Although certain compounds were predicted to be ‘Active’ (+) for specific endpoints, such as carcinogenicity or hepatotoxicity, it should be noted that these represent preliminary in silico findings and do not necessarily imply definitive toxicity in biological systems. Regarding BBB permeability, most compounds exhibited the potential to cross the blood–brain barrier, suggesting potential applications for central nervous system targets. Overall, these computational toxicity assessments, when combined with favorable pharmacokinetic properties, reinforce the potential of the major constituents of A. rupicola var. rupicola as safe, lead candidates for further pharmacological development.

This study comprehensively elucidates the phytochemical profiles, biological activities, and potential mechanisms of action of the aerial part, fruit, and rhizome extracts of A. rupicola var. rupicola through integrated in vitro and in silico analyses.

Phytochemical analyses demonstrated that different parts of the species possess chemically distinct profiles. HPLC analysis revealed that the aerial part extract was rich in rutin and coumaric acid, the fruit extract in quercetin, while phenolic compound levels in the rhizome extract remained generally low. GC‐MS analysis highlighted the predominance of fatty acid esters in the aerial part and rhizome extracts, and aliphatic hydrocarbons in the fruit extract. It was also noted that all plant parts contained aliphatic hydrocarbons to varying degrees, which may contribute to secondary metabolite diversification. Bioactivity assays revealed a consistent relationship between total phenolic content and antioxidant activity, with the highest antioxidant capacity observed in the fruit extract, which also possessed the highest total phenolic content. Among the most striking findings of the antimicrobial activity tests was the potent MIC value of the rhizome extract against B. cereus, despite its having the lowest phenolic content. This suggests that the antimicrobial potential of the rhizome may originate from nonphenolic lipophilic constituents such as fatty acid esters, or from the synergistic effects of multiple compounds. In cytotoxicity studies, the aerial part extract demonstrated superior activity compared to the other extracts, exhibiting the lowest IC₅₀ value and the highest selectivity index against the HL‐60 cell line, thereby presenting the most promising anticancer potential.

Comparison with previously reported data on other Arum species reveals both conserved chemotaxonomic traits and notable species‐specific differences. Consistent with the fatty acid profiles reported for A. maculatum leaves [29], the present study detected abundant methyl oleate and methyl palmitate in the aerial and rhizome extracts. This parallel suggests that lipophilic constituents represent characteristic secondary metabolites across the genus. Regarding phenolic constituents, previous studies identified gallic acid, chlorogenic acid, and quercetin in A. maculatum [30], alongside ferulic acid, luteolin, and rutin in A. italicum tubers [31]. The presence of these phenolics in A. rupicola var. rupicola confirms a shared phenolic backbone within the genus. However, the exceptionally high accumulation of rutin in the aerial parts uniquely distinguishes A. rupicola var. rupicola from related species, highlighting a potential species‐specific chemotaxonomic marker.

When contextualized within the broader Arum genus, the fruit extract of A. rupicola var. rupicola demonstrated superior antioxidant capacity compared to the fruit extracts of A. maculatum [32]. Similarly, the aerial parts exhibited a richer total phenolic content than that documented for A. maculatum leaves [29]. Nevertheless, compared to highly active species like A. palaestinum and A. dioscoridis [33], A. rupicola var. rupicola displays an intermediate antioxidant potential. This variance is likely attributable to differences in specific molecular compositions, particularly the prominent quercetin content in the fruits, which appears to drive the observed free radical scavenging capability.

Regarding antimicrobial activity, the potent inhibitory effect of the rhizome extract against B. cereus aligns with findings from related species; for instance, A. maculatum and A. italicum extracts also demonstrated strong inhibition against Bacillus strains [29, 34]. This suggests that anti‐Bacillus efficacy may be a conserved bioactivity within the genus. Conversely, the limited efficacy against Gram‐negative bacteria observed in the present study contrasts with the stronger activities reported for A. maculatum essential oils and A. palaestinum flowers [34, 35]. This discrepancy underscores the critical influence of the extraction methodology on the antimicrobial spectrum, suggesting that the active principles against Gram‐negative pathogens might be highly volatile or strictly nonpolar.

In terms of cytotoxic potential, the selective activity of the aerial part extract against the HL‐60 cell line is comparable in magnitude to the effects reported for the nonpolar fractions of A. rupicola leaves against MCF‐7 cells [36]. This reinforces the consistent antiproliferative potential of this species across varying experimental conditions. Although earlier studies demonstrated potent cytotoxicity for A. italicum tubers and A. palaestinum fractions against lung and breast cancer cells [37, 38], the current study emphasizes the distinct sensitivity of leukemic cells to the rutin‐rich aerial extracts of A. rupicola var. rupicola. These interspecific differences in cytotoxicity likely reflect variations in intrinsic chemical compositions and the varying resistance mechanisms of the employed cell lines.

Molecular docking and MM‐GBSA analyses further supported the in vitro findings at a mechanistic level. The binding affinity of rutin against antiapoptotic proteins such as Bcl‐2 and Bcl‐xL is supported by literature, suggesting the potential of flavonoids to serve as potent inhibitors within these pathways [39]. Regarding the p53 (Y220C) target, earlier computational work demonstrated that the structural crevice created by the mutation constitutes a tractable binding site for small molecules capable of restoring the tumor suppressor function [40]. The present observation that rutin effectively occupies this cavity is in strong agreement with this mechanistic framework. Furthermore, the competitive binding of rutin against the KRAS (G12D) mutation aligns with recent virtual screening studies aiming to target this prevalent oncogenic driver. For the antimicrobial targets, the computational interactions of quercetin and rosmarinic acid with DNA gyrase and DHFR corroborate previous in silico evaluations of these polyphenols, substantiating their multitarget antibacterial potential [41, 42].

Finally, the pharmacokinetic profiling via ADME analysis revealed that the majority of the simple phenolic acids fully complied with Lipinski's Rule of Five, exhibiting high oral absorption scores. Although rutin demonstrated strong molecular interactions, its high molecular weight and hydrogen bond donors resulted in Lipinski violations—a common characteristic for flavonoid glycosides in the literature. However, it is well established that such bioavailability limitations can be effectively mitigated through appropriate nano‐formulation strategies, solidifying the potential of these extracts for future drug development.

4. Conclusion

In conclusion, this study presents the first comprehensive phytochemical profile and preliminary biological evaluation of A. rupicola var. rupicola extracts. Although the aerial parts extract of the plant showed significant in vitro cytotoxic effects against the HL‐60 cell line, the rhizome extract exhibited specific in vitro antimicrobial activity; however, these findings are strictly preliminary. The strong computational binding affinities observed for the dominant compounds, particularly rutin, suggest theoretical molecular mechanisms that require further investigation. However, it is crucial to emphasize that these results are based solely on in vitro and in silico models. Without subsequent in vivo and clinical validation, no definitive claims can be made about the true therapeutic or pharmacological potential of these extracts. Future research should prioritize isolating active compounds based on biological activity, conducting comprehensive Molecular Dynamics (MD) simulations to assess complex stability, and, most importantly, using rigorous in vivo animal models to determine actual physiological efficacy, bioavailability, and safety.

Author Contributions

Selim Can Kamalak: conceptualization, investigation, software, visualization, writing – original draft, resources, data curation, methodology, formal analysis. Ela Nur Şimşek Sezer: methodology, investigation, validation, writing – review & editing, project administration, funding acquisition, supervision, formal analysis. Erdoğan Güneş: Investigation, methodology. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that supports the findings of this study are available in the Supporting Information of this article.