Abstract
In this study, phytochemical fingerprint and biological activities of Punica granatum L. seed extract (Pugex) were investigated. The phytochemical constituents were determined by LC-MS /MS and GC-MS analyses. The biological activity of the extract was investigated by anti-microbial, anti-genotoxic, anti-proliferative, anti-cholinesterase and anti-diabetic activity. All biological activities experimentally demonstrated in vitro were also investigated by molecular docking. The drug-likeness properties of selected phytochemicals were evaluated using the Molinspiration tool. The highest amounts of 2-oxatricyclo[4.3.1.0(3,8)]decane, 2-heptenal, 2-propyl- and 1 H-indene-1-one, gallic acid and ellagic acid were detected as major constituents in LC-MS/MS and GC-MS. Pugex showed a broad spectrum of anti-microbial activity by forming inhibition zones against all tested bacteria, with anti-genotoxic activity ranging from 53.31% to 74.48% against various chromosomal aberrations and anti-proliferative activity by reducing cell proliferation by 15.6%. Pugex inhibited α-glucosidase activity between 30.2% and 61.3% and α-amylase activity between 27.4% and 56.7%. The anti-cholinesterase activity of the seeds, which showed AChE inhibition up to 67.8% and BChE inhibition up to 79.8%, was associated with the essential oils they contained. The biological activities of the seeds were also confirmed by in silico interactions, and possible mechanisms were predicted, and drug likeness data provide preliminary information on the availability of phytochemicals for drugs. Drug-likeness features of the major components—2-Oxatricyclo [4.3.1.0(3,8)]decane, ellagic acid, gallic acid, and pulegone—were investigated. The logP values of all phytochemicals were found to be below 5, indicating compliance with Lipinski’s rule of five and suggesting their potential to cross biological membranes. Furthermore, phytochemicals with a total polar surface area below 140 Å are expected to exhibit improved intestinal absorption. Based on these findings, the phenolic acid derivatives evaluated in this study are anticipated to be well absorbed through the intestinal tract.
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Introduction
Plants are an important source of nutrients necessary for the survival of the organism, and wild plants have been used as food throughout history. Plants containing many active substances are also used in various industrial fields such as medicine, cosmetics, pharmaceuticals, agriculture and textiles. Various parts of plants and phytochemicals obtained from these parts are used internally or externally in the treatment of diseases. For this reason, the active components and biological activities of plants should be investigated in detail and these research results should be taken into consideration when using them for phytotherapy purposes1,2. In this study, phytochemical fingerprinting and biological activities of Punica granatum L. seeds were investigated. P. granatum is widely grown in East Asia, especially in Iran, Turkey, Tunisia, Egypt, Spain, Morocco, China, and India. P. granatum tree has a spiny trunk, long green, orange or dark red leaves. The pomegranate fruit consists of the flesh, peel, and seeds, and the edible part makes up 50% of its total weight. Seeds with a fatty acid content of 7–27% also have a high anthocyanin content³. Punicic acid, which is classified as conjugated linolenic acid, is the main component in pomegranate seeds containing octadecatrienoic, palmitic, stearic, oleic acids4. Herbal active ingredients are also used in reducing the risk of formation of many diseases, in the prevention and even in the treatment. P. granatum seeds exhibit high pharmacological properties due to their high bioactive component content. The anti-oxidant, anti-microbial, anti-cancer, hepatoprotective, neuroprotective, and anti-diabetic properties of the seeds indicate their potential efficacy in the prevention and management of various diseases5. Amri et al.6 determined that regular P. granatum seed consumption for 12 weeks stimulates anti-oxidant activity and provides neuro-protective effect by inducing cholinesterase inhibition. Khajebishak et al.7 reported that as a result of the consumption of P. granatum seed oil in diabetes patients for 8 weeks, blood sugar levels decreased and the anti-inflammation effect was detected. Çayır et al.8 stated that P. granatum seed application in rats had hepatoprotective and nephroprotective effects by reducing lipid peroxidation and increasing anti-oxidant capacity. The protective effect of seed applications against many diseases has been investigated, but the mechanism of action has not been determined yet.
Today, neurodegenerative diseases, cancer and diabetes are among the diseases with the highest increase. Many synthetic drugs are introduced to the market in the treatment of these diseases, and the use of these drugs increases the chemical load in the organism, causes secondary diseases or produces serious side effects. Consumption of natural anti-mutagenic, anti-neurodegenerative or anti-diabetic products in the daily diet will reduce the risk of developing these diseases. Foods with anti-diabetic effects reduce carbohydrate digestion and absorption. The reduction in digestion rates provides control of blood sugar level and prevents sudden rise. The anti-diabetic activity of foods is achieved by inhibiting digestive enzymes such as α-amylase and α-glucosidase. Synthetic α-amylase and α-glucosidase inhibitors are used as oral anti-diabetic drugs in the treatment of diabetes. However, these drugs cause gastrointestinal side effects such as diarrhea, bloating, and abdominal distention. For this reason, the consumption of antidiabetic herbal foods in the daily diet is considered as a natural treatment without side effects9. Plants exhibit cancer-preventing and anti-genotoxic effects due to the influence of many active compounds they contain, such as polyphenols. The anti-genotoxic effect is very important in preventing cancer formation and proliferation of cancer cells10. Acetylcholinesterase inhibitors obtained from natural products are also promising in the treatment of neurodegenerative diseases. Inhibition of cholinesterase enzymes can increase acetylcholine levels in the central nervous system and cause improvement in symptoms of neuro-degenerative diseases. Although there are many synthetic anti-cholinesterase drugs available, their performance sometimes yields unexpected results due to side effects. Therefore, studies investigating potential anti-cholinesterase agents from natural and non-cytotoxic sources are very valuable11.
Further researchs are needed on the biological and pharmacological mechanisms of action of herbal ingredients. Studies conducted on plants in the literature mostly determine their phytochemical contents or investigate their biological activities. Studies investigating the biological activities related to phytochemical content are not yet at the desired level. The present study made an important contribution to this deficiency in the literature, and the biological activities confirmed by in-silico molecular docking were related to phytochemical content. In silico methods are used to support experimental data and computational analyses and make an important contribution to the elucidation of complex biological phenomena, dynamics and interactions. In particular, they are used in the elucidation of pharmacological and biological activity mechanisms and in the determination of drug-ligand interactions. In this study, the phytochemical profile and some important biological activities of P. granatum seeds were investigated, and the activity mechanisms were estimated by confirming the experimental results with in silico molecular docking analysis. The phytochemical profile of P. granatum seeds was studied by LC-MS/MS and GC-MS. In this way, the essential oil profile and the nature of phenolic compounds in the seed content were determined. The anti-microbial, anti-genotoxic, anti-proliferative, anti-diabetic and anti-cholinesterase activities of the seeds were studied and related to the phytochemical content. Pharmacokinetic and absorption, distribution, metabolism, and excretion (ADME) studies were conducted in this study to investigate the drug-like properties and bioavailability of the main phytochemicals found in P. granatum seed extract. In this context, the intestinal absorption, biological membrane penetration capacity, and compliance with Lipinski’s five rules of conduct were analyzed using in silico methods to determine whether these phytochemicals have the potential to be oral drug candidates.
Materials and methods
Sample collection and extraction
Chemicals used in all analyses were of high purity and were obtained from Sigma-Aldrich and Merck. Carmine (CAS: 1390-65-4) used in chromosome staining was obtained from Isolab. Bulbs used in the Allium test were obtained from Akdeniz Tarım (TR-55-K-009228). All P. granatum used in this study were collected from Giresun (40° 53’ 37.6" N, 38°18’27.1" E) and later identified by Prof. Dr. Zafer TÜRKMEN at the Department of Botany, Giresun University (Türkiye). One specimen with voucher number BIO-P.gra-s/2022 was deposited in the herbarium of the Department of Biology. Seeds were obtained from fruits harvested in September-October 2022. The seeds, which were dried in the shade away from direct sunlight, were ground into powder in a mortar and stored in glass bottles at room temperature in a dark and dry place until analysis. Experimental research and field studies on plants, including the collection of plant material, comply with relevant institutional, national, and international guidelines and legislation. 2 g of the sample was extracted in 100 mL of solvent (water, ethanol, or methanol) for a period of 24 h at room temperature in a shaking incubator. The sample was then subjected to centrifugation at 10,000 rpm for 10 min. The resultant upper layer was then evaporated12. The resulting residue was used as P. granatum seed extract (Pugex) throughout the study. The extraction yield for each extraction with each solvent was determined using Eq. (1).
LC-MS/MS and GC-MS analysis and essential oil extraction
2 g of P. granatum seed samples were extracted with methanol: dichloromethane (4:1) solvent in an ultrasonic bath for 120 min, filtered through a 0.45 µM syringe filter and analyzed by LC-MS/MS in Hitit University- HUBTUAM13,14. The conditions and detailed protocol for LC-MS/MS analysis are given in the supplementary material. The essential oil was obtained from the plant by steam distillation method in the Clevenger apparatus. The samples were transferred to the Clevenger flask and 400 mL of water was added. The system was operated continuously for a period of three hours. At the conclusion of this time, the oils obtained from the Clevenger system were eluted and analyzed by GC-MS. For this purpose, 1 mL of hexane was added on the essential oil and GC-MS analysis was performed by taking it into the vial. In the analysis performed with Thermo Scientific GC-MS device, TG-5MS column was used, analysis time was determined as 55 min, injection block temperature was 250 °C, flow rate was 1 mL/min Helium. Oil extraction and GC-MS analysis were carried out at Hitit University- HUBTUAM.
In-vitro and in-silico anti-microbial activity
The anti-microbial activity of the extract was determined by MIC, minimum bactericidal concentration (MBC) and disc diffusion method. Anti-microbial activity was tested against E. coli ATCC 25,922, P. aeruginosa ATCC9027, S. enteritidis ATCC13076, B. subtilis IMG 22, B. cereus ATCC 14,579, S. aureus ATCC 25,923, C.andida tropicalis ATCC 13,803, and C. albicans ATCC 90,028. For MIC measurements, bacteria were incubated in broth medium at 37 °C for 48 h and 5 mL of physiological solution was added to 108 cells/mL sample taken from the culture. Fun gi were first incubated in broth medium for 48 h, and 106 CFU/mL sample was added to 5 mL of physiological saline. Ethanol extract of Pugex at different concentrations (200 µL, 20–200 mg/mL) was added to physiological saline solution and incubated at 37 °C for 24 h. 100 µL samples from each culture medium were homogeneously plated out on Mueller Hinton Agar (MHA) plates. In the negative control, sterile medium was used instead of the extract, and after 24 h of incubation, the Pugex concentration in the well was determined as the MIC value that prevents the growth of the microorganism. For determination of MBC values, 10 µL of the cultures that showed no bacterial growth in the MIC determination were inoculated into MHB and incubated at 37 °C for 24 h. At the end of incubation, the lowest Pugex concentration that showed no bacterial growth in medium was determined as the MBC value. Experiments were repeated three times for each concentration to determine MIC and MBC values15. For the disc diffusion assay, culture samples (108 cells/mL) of Gram-positive and Gram-negative bacteria from MHB medium were homogeneously plated on MHA plates. For fungi, samples (106 CFU/mL, 0.5 McFarland) from Potato Dextrose Broth medium were homogeneously plated out on Sabouraud Maltose Agar plates. Empty sterile discs with a diameter of 6 mm were placed on the plate surfaces, and 20 µL of Pugex was added to the discs. The inoculatated plates were stored at 4 °C for 1 h and incubated at 37 °C for bacteria and 27 °C for fungi for 24 h. The zones of inhibition formed after incubation on the plates were evaluated in mm. The antibiotics nystatin and amikacin were used as standard antibiotics16. Multidrug efflux pumps are an important mechanism in bacterial resistance to antimicrobials. Possible damage to this pump will increase susceptibility to antimicrobials17. Therefore, to confirm the antimicrobial activity of Pugex, the interactions of multidrug efflux pump with major compounds in the Pugex content were investigated. Molecular docking was performed to analyze the potential interactions of 2-Oxatricyclo[4.3.1.0(3,8)]decane, ellagic acid and gallic acid with the MexAB-OprM multidrug efflux pump. The 3D structure of the MexAB-OprM multidrug efflux pump (PDB ID:5DAJ)18 were obtained from the protein data bank. The 3D structure of 2-Oxatricyclo[4.3.1.0(3,8)]decane (CID: 586621), ellagic acid (CID: 5281855) and gallic acid (CID: 370) were retrieved from the PubChem.
Anti-genotoxic activity and density functional theory calculations
The anti-genotoxic activity of Pugex was determined using the chromosomal aberration (CA) test. The CA test was applied using the Allium test and four different groups were formed for this purpose. The anti-genotoxic activity was determined by examining the reduction in CAs induced by positive mutagen19. The detailed protocol for anti-genotoxic activity analysis are given in the supplementary material. The anti-genotoxic activity of herbal ingredients is mostly related to their antioxidant effect and therefore the antioxidant potential of Pugex was determined by Density Functional Theory (DFT) calculations. The antioxidant activity of Pugex was elucidated by evaluating electronic parameters, including HOMO (Highest Occupied Molecular Orbital), LUMO (Lowest Unoccupied Molecular Orbital), energy gap (ΔE), ionization potential (IP), and electron affinity (EA), for ellagic acid, gallic acid and 2-Oxatricyclo[4.3.1.0(3,8)]decane compounds using the Density Functional Theory (DFT) method. The Gaussian 09 package20 and Gauss view 5.0 programs21 were employed to perform the theoretical calculations, utilizing the Lee-Yang-Parr (B3LYP)22 method with the standard basis set 6-31G (d, p).
In-vitro and in-silico anti-proliferative activity
The anti-proliferative effect of Pugex was determined by examining the changes in mitotic index (MI) ratios in A. cepa root tip cells. MI is a marker of cell proliferation and inhibition of MI is associated with cell death or a delay in cell proliferation23. For anti-genotoxic and anti-proliferative activity, mitotic slides were prepared from root tips and cell counts were performed under a microscope24,25. The detailed procedure for the preparation of mitotic slide is given in supplementary material. Ellagic acid contained in Pugex has an antiproliferative effect by causing a change in ATP levels. Therefore, the interaction of ellagic acid and ATP synthase was studied by molecular docking26. Essential oils also act through various mechanisms. One of these mechanisms is the disruption of microtubule structure by binding to α/β-tubulin proteins27. Therefore, the interaction of pulegone in Pugex with α/β-tubulin proteins was investigated. The 3D structures of ATP synthase (PDB ID:4V1F)28 and cyro-em 3D structure of α-tubulin and β-tubulin proteins (PDB ID:6RZB)29 were obtained from the protein data bank. The 3D structure of ellagic acid (CID: 5281855) and pulegone (CID: 442495) were retrieved from the PubChem.
In-vitro and in-silico anti-diabetic activity
Anti-diabetic activity of Pugex was determined by investigating α-amylase and α-glucosidase inhibition activities. Both enzymes are involved in carbohydrate digestion and partial inhibition of these enzymes prevents sudden spikes in blood sugar16,30. Therefore, the inhibitory effect of Pugex on these two enzymes was tested in-vitro and supported in-silico. The method applied in the in-vitro enzyme assay is detailed in supplementary material. To confirm the anti-diabetic activity of Pugex, molecular docking was performed to analyze the potential interactions of the major components 2-Oxatricyclo [4.3.1.0(3,8)] decane and ellagic acid with α-amylase and α-glucosidase. The 3D structure of human pancreatic α-amylase (PDB ID: 5E0F)31 and human lysosomal acid α-glucosidase (PDB ID: 5NN8)32 were obtained from the protein data bank. The 3D structures of 2-Oxatricyclo [4.3.1.0(3,8)] decane (CID: 586621) and ellagic acid (CID: 5281855) were retrieved from the PubChem.
In-vitro and in-silico anti-cholinesterase activity
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity was investigated by the method proposed by Ellman et al.33. The absorbance of the mixture containing 25 µL ATCI, 125 µL 0.3 mM DTNB and 50 µL extract (2 mg/mL) was measured at 405 nm. Then, 25 µL of cholinesterase (AChE or BChE; 0.026 U/mL) was added to the solution and absorbance was measured at 405 nm. Galanthamine (GAL) was used as a positive control. Percent inhibition was calculated using Eq. (2).
It is known that essential oils in the content of plants act as cholinesterase inhibitors as an anti-Alzheimer34. In this study, the in silico interactions of 2-Oxatricyclo [4.3.1.0(3,8)] dec essential oil, which is highly detected in Pugex content, with AChE and BChE enzymes were investigated. The 3D structures of human butyrylcholinesterase (PDB ID: 4BDS)35 and human acetylcholinesterase (PDB ID: 4M0E)36 molecules were obtained from the Protein Data Bank. The 3D structure of 2-Oxatricyclo[4.3.1.0(3,8)]decane (PubChem CID: 586621) was retrieved from the PubChem. In order to conduct an in silico analysis, a series of preparatory steps were undertaken for the 3D protein structures to be analyzed. These steps included the identification of active sites, the removal of water molecules and ligands, and the addition of polar hydrogen atoms. Energy minimisation of proteins was conducted utilizing Gromos 43B1, in conjunction with Swiss-PdbViewer (v.4.1.0) software37. The energy minimisation of the 3D structures of ligands was accomplished using the UFF force field, in conjunction with Open Babel (v.2.4.0) software38. The protein molecules were allocated Kollman charges, whereas the ligands were assigned Gasteiger charges. The molecular docking process was conducted with the grid box containing the active sites of proteins. Subsequently, docking was performed using Autodock 4.2.6 software39, which is based on the Lamarckian genetic algorithm. The docking analysis and three-dimensional visualisations were performed with the Biovia Discovery Studio 2020 Client.
Prediction of pharmacophore features and biological activity
The drug likeness properties of selected phytochemicals were assessed using the Molinspiration tool40. The Molinspiration interface was used to estimate the molecular properties, pharmacophore features, and bioactivities of the compounds. Pharmacophore features were determined based on total polar surface area, logP, molecular weight, number of atoms, number of hydrogen bond donors, number of hydrogen bond acceptors, number of violations of Lipinski’s rule of five parameters, and number of rotatable bonds. The prediction of biological activity encompassed G-protein-coupled receptor (GPCR) ligands, ion channel modulators, nuclear receptor ligands, kinase inhibitors, and protease enzyme inhibitors.
Pharmacokinetics and ADME
ADME (absorption, distribution, metabolism, elimination) analysis provides insights into the drug-likeness properties of chemical compounds. In silico estimation of physicochemical properties and ADME specifications can be performed. The SwissADME online server, developed by Daina et al.41, was employed to predict various pharmacokinetic properties of phytochemicals. Drug-likeness properties of these phytochemicals, such as absorption, distribution, metabolism and excretion (ADME) parameters, were mainly studied.
Statistical analysis
The analysis was conducted utilising the IBM SPSS Statistics 22 software program, and the resulting data were presented as the mean ± SD (standard deviation). Statistical significance between the means was determined by Duncan’s test and One-way ANOVA, with a p value of < 0.05 being considered statistically significant.
Results and discussion
Essential oil profile
The essential oil profile (GC-MS chromatogram) and the presence rates of essential oils are shown in Fig. 1. 29 different volatile compounds were determined in the extract, and the compounds with a peak area greater than 1% were accepted as the major compounds. The highest amounts of 2-Oxatricyclo [4.3.1.0(3,8)] decane, 2-Heptenal, 2-propyl- and 1 H-Inden-1-one, octahydro- were detected in the Pugex content. 9-Oxa-bicyclo [3.3.1] nonane-2,6-dione, 5-decanone-6-hydroxy-, 5-triisopropylsilyl-3-vinylfuran, 8-pentadecanone, 2-heptenal and pulegone compounds were also detected in moderate amounts. All these compounds are important contributors to the biological activity of Pugex. Essential oils possess a high level of biological activity, most notably anti-microbial activity. Essential oils can adhere to the surface of microorganisms, penetrate the phospholipid bilayer of the cell membrane, and affect the structural integrity of the cell membrane and cell metabolism. Lipophilic ends of fatty acids in the cell membrane of bacteria facilitate the penetration of hydrophobic essential oils and increase the anti-microbial effect42. The efficacy of essential oils varies depending on the structure, functional groups, and type of microorganisms. The rich diversity of essential oils, as demonstrated by analysis of GC-MS, contributes significantly to the biological efficacy of Pugex. The pulegone contained in the extract is a monoterpene ketone found in many plant oils. It is the precursor of menthone, isomentone and isopulegone found in plants. With its anti-bacterial, anti-fungal, anti-histamine, and antipyretic activities, it imparts various biological activities to plant tissues43. The absence of any reports on the analysis of the essential oil of P. granatum seed from Giresun and the presence of monoterpenes in the literature opens new possibilities for similar research. However, terpene, phenolic, and phenolic acid contents have been reported in the seeds of a granata cultivar, P. granatum44.
GC-MS chromatogram of Pugex. E1: (1-Ethyl-5-methylcyclopentene), E2: (2-Heptenal), E3: (Furan, 2-pentyl-), E4: (Octanal), E5: (2 Octenal), E6: (Pentanoic acid, butyl ester), E7: (Nonanal), E8: (9-Oxa-bicyclo[3.3.1]nonane-2,6-dione), E9: (2-Heptenal, 2-propyl-), E10: (1 H-Inden-1-one, octahydro-), E11: (2-Oxatricyclo[4.3.1.0(3,8)]decane), E12: (Pulegone), E13: (5-Decanone, 6-hydroxy-), E14: (Cyclohexanone, 3-(4-hydroxybutyl)-2-methyl-), E15: (5-Triisopropylsilyl-3-vinylfuran)
Phenolic compound profile
The LC-MS/MS chromatogram of Pugex and the presence rates of phenolic compounds are listed in Fig. 2. Of the 22 standard compounds tested, 8 were detected. Compounds such as quercetin, rutin, and epicatechin flavone, which are commonly found in many plant ingredients, were not present in Pugex. Gallic acid and ellagic acid were accepted as major constituents due to their high presence rate. Among all the phenolic compounds identified, gallic acid constituted 32.78% and ellagic acid 55.25%. The total percentage of these two compounds in all phenolics was 88.03%. Pugex has a rich activity as a cumulative result of the biological activities of all phenolic compounds contained in it. Gallic acid and ellagic acid contribute significantly to the biological activity due to their strong presence. Ellagic acid, which is highly detected in Pugex, is a dilactone of hexahydroxydiphenic acid, a dimeric gallic acid derivative. It is also attracting attention in drug development studies for various activities such as anti-oxidant, anti-genotoxic, anti-inflammatory, anti-proliferative, cardioprotective, nephroprotective, hepatoprotective, and neuroprotective properties. Combinations of ellagic acid with other antioxidants have also been reported to have higher biological activity and thus potent therapeutic effects45. Many phenolic compounds, especially gallic acid, have been detected together with ellagic acid in Pugex content. Gallic acid, a hydroxybenzoic acid, exhibits potent antioxidant activity as well as anti-cancer, anti-ulcer, anti-inflammatory, anti-bacterial and anti-fungal properties. In recent years, gallic acid has received considerable attention in the prevention of nervous system diseases, such as Alzheimer’s disease, with its reducing effect on neuronal damage and amyloid neuropathology46. Gallic acid may reverse induced amnesia through possible mechanisms such as suppression of oxidative stress, free radical scavenging, and inhibition of the enzyme acetylcholinesterase47. There are studies in the literature that examine the photochemical content of various P. granatum textures. Karagecili et al.48 found that P. granatum extract contains compounds such as ellagic acid, catechin, epigallocatechin gallate, epicatechin, nicotiflorin, astragalin, gallic acid, and epigallocatechin in greater proportions and reported that phenols such as 4 OH-benzoic acid, vanillic acid, syringic acid, ferulic acid, coumarin, sinapic acid, salicylic acid, and rosmarinic acid were not detected. Unlike this literature study, the presence of protecatechuic acid, syringic acid, taxifolin, p-coumaric acid was detected in Pugex in this study. Changes in the phytochemical content of the same plant species collected from different regions are closely related to ecological conditions. The types and rates of phenolic compounds detected in Pugex content in this study differ from the literature studies. This can be explained by the fact that plants produce secondary metabolites in different types and levels in order to adapt to the environmental conditions in which they grow. This change in phytochemical content causes plants of the same species to exhibit different biological activities.
LC-MS/MS chromatogram of Pugex and the presence rates of phenolic compounds. P1: Gallic acid, P2: Protecatechuic acid, P3: Catechin, P4: Caffeic acid, P5: Syringic acid, P6: Taxifolin, P7: p_coumaric acid, P8: Ellagic acid.
Extraction efficiency
Differences in efficiency were observed in extractions performed using water, methanol, and ethanol. Among the solvents used, the highest extraction efficiency was obtained in ethanol extraction with 3.70%. Yields of 2.95% and 2.65% were obtained in methanol and water extraction, respectively. Ethanol is a powerful solvent widely used for the extraction of phytochemicals from plants. It is particularly suitable for the extraction of phenolics, which exhibit anti-oxidant activity, and essential oils, which exhibit anti-microbial activity. It is reported in the literature that ethanol extracts obtained from some plants show higher anti-microbial activity compared to standard drugs such as ciprofloxacin and griseofulvin49. The high yield obtained with ethanol is related to the polarity of the solvent, and polar solvents provide a high advantage in obtaining phytochemicals from plant matrices. While methanol is generally more effective in the extraction of low molecular weight polyphenols, ethanol, which is safe for human consumption and non-toxic, is generally a good solvent for polyphenol extraction50. The use of ethanol in the extraction of herbal oils, including essential oils, increases the overall solubility and extraction due to the hydrogen bonding between the polar components of the oils and ethanol51. It has been reported in the literature that extraction efficiency is obtained at varying rates with different solvents used in seed extraction. These differences can be explained by the changing phytochemical content of the plant depending on the ecological conditions, even if it belongs to the same species. Masci et al.52 observed a 3-fold higher yield of polyphenolics in ethanol and aqueous extraction of pomegranate seeds and fruits compared to other solvents. Abbasi et al.53 reported that hexane and ethanol provided high yields in oil extraction from pomegranate seed, while low yields were obtained in water extraction. The ethanol extract of seed was used in all subsequent experiments of this study because it provides a high extraction yield and is considered non-toxic and safe.
In-vitro and in-silico anti-microbial activity
The anti-microbial activity of Pugex was evaluated against Gram-positive, Gram-negative and fungi using disc diffusion and microdilution methods and the results are shown in Table 1. Ethanol, methanol and water extract of Pugex showed varying degrees of inhibition against all microorganisms tested. This result shows that Pugex has a broad spectrum and the highest inhibition was obtained with ethanol extract against S. aureus with an inhibition zone of 16.3 ± 0.9 mm. The lowest inhibition was observed against C. albicans with a zone diameter of 7.5 ± 0.6 mm with water extract. Pugex showed a stronger anti-microbial effect on Gram-positive compared Gram-negative bacteria and a lower efficacy against Candida species compared to bacteria in the disc diffusion method. The MIC and MBC values studied with different Pugex concentrations are given in Table 1. MIC is the lowest concentration that inhibits the visible growth of bacteria, while MBC is the lowest concentration required to kill a bacteria. The lowest MBC value of Pugex was 20 mg/mL against P. aeruginosa, while the highest was 60 mg/mL against C. albicans. The lowest MBC values were obtained against E. coli and the highest against C. tropicalis. The MIC values observed against all microorganisms were lower than the MBC values. Moreover, the lower MIC and MBC concentrations of Pugex in Gram-negatives and the higher MIC and MIB values in fungi confirm the results of disk diffusion. Low MIC or MBC values indicate high anti-microbial activity. Accordingly, the effect of Pugex on the tested microorganisms can be listed as gram negative > gram positive > fungi. This selectivity towards bacteria can be explained by the structural differences of the cells. The double membrane structure of gram-negative bacteria, which reduces the penetration of anti-microbial agents into the cell, is the main cause of resistance to anti-microbial compounds54. This higher MIC and MBC against fungi can be explained by chitin and ergosterol, which are present in the cell wall structure of fungi. These compounds provide protection from anti-microbial agents by strengthening the cell wall55. The broad-spectrum anti-microbial activity of Pugex is closely related to the ingredients it contains. Especially essential oils have an important role in the formation of anti-microbial activity. Essential oils damage the cell membrane, which regulates osmotic pressure in the cell and controls the entry and exit of biomolecules. They also damage the efflux system, which consists of special channel proteins involved in the elimination of harmful compounds in bacteria. Pulegone, which is detected as an essential oil in the content of Pugex, has antimicrobial activity against several bacterial species, especially Candida albicans, and its anticandidal activity is twice that of the standard antibiotic nystatin56. Among the phenolic compounds contained in Pugex, ellagic acid, the major phenolic, is a naturally occurring polyphenol in foods. The in vitro anti-microbial activity of ellagic acid, which exhibits anti-oxidant, anti-radical, anti-cancer, anti-apoptotic and high anti-bacterial activity, has also been demonstrated against many bacteria57. The high content of gallic acid in Pugex contributes to anti-microbial activity by inhibiting protein flux pumps in bacterial membranes58. As a result of the cumulative action of all the active compounds contained in Pugex, a strong anti-microbial activity is obtained. Nozohour et al.15 reported that pomegranate seeds containing mainly heptacosan, furfural, 5-hydroxymethylfurfural, gallic acid, and linoleic acid have an inhibitory effect on P. aeruginosa and S. aureus. Dahham et al.59 found that the seeds of the pomegranate plant (Hyderabad, India) have a broad spectrum of activity by forming different zones of inhibition against bacteria and fungi, and that this effect is related to the active compounds in the seeds.
The mechanism behind the anti-microbial activity of Pugex observed experimentally was estimated by molecular docking. The molecular docking results between 2-oxatricyclo [4.3.1.0(3,8)] decane, ellagic acid, and gallic acid with the MexAB-OprM efflux pump are given in Table 2; Fig. 3. 2-Oxatricyclo [4.3.1.0(3,8)] decane exhibited a free energy of binding of – 4.15 kcal/mol, suggesting a moderate binding affinity with the MexAB-OprM efflux pump.The inhibition constant (Ki) was determined to be 906.56 μm, indicating a moderate inhibitory activity against the MexAB-OprM efflux pump. Notable hydrogen bond interactions were observed with the LYS105 residue of the efflux pump. Additionally, hydrophobic interactions contributed to the overall binding affinity. Ellagic acid demonstrated a higher binding affinity with the MexAB-OprM efflux pump, with a free energy of binding of – 6.44 kcal/mol. The Ki was found to be 18.91 μm, indicating a potent inhibitory activity against the efflux pump. Significant hydrogen bond interactions were observed with LYS105, ASN129 (x2), and LEU62 residues of the efflux pump.Hydrophobic interactions involving LEU109 (x2), PHE132 (x3), LEU112 (x2), LEU62, and ARG66 residues further contributed to the strong binding affinity. Gallic acid exhibited a free energy of binding of – 4.86 kcal/mol, indicating a moderate binding affinity with the MexAB-OprM efflux pump. The Ki was determined to be 273.44 μm, suggesting a moderate inhibitory activity against the efflux pump. Prominent hydrogen bond interactions were observed with ASN129 (x2), VAL65, and LEU62 residues of the efflux pump. Hydrophobic interactions involving LEU109, PHE132, LEU62, and ARG66 residues also contributed to the overall binding affinity. These findings suggest that ellagic acid and gallic acid exhibit stronger binding affinities with the MexAB-OprM efflux pump compared to 2-Oxatricyclo [4.3.1.0(3,8)] decane. The observed interactions, including hydrogen bonds and hydrophobic interactions, contribute to their inhibitory activities against the efflux pump. Multidrug efflux pumps are an important mechanism in bacterial resistance to antimicrobials. Possible damage to this pump will increase susceptibility to antimicrobials17. The phytochemicals of Pugex interacted with the MexAB-OprM efflux pump and induced conformational change and denaturation of the pumps and led to a lethal effect.
Molecular Interactions of 2-Oxatricyclo [4.3.1.0(3,8)] decane (a), ellagic acid (b) and gallic acid (c) with MexAB-OprM efflux pump.
In-vitro anti-genotoxic activity and density functional theory calculations
The anti-genotoxic activity of the Pugex extract determined by the Allium test is shown in Table 3. A statistically insignificant frequency of CAs was observed in the negative control group and in the group where only Pugex extract was applied. This result indicates that Pugex alone has no genotoxic effect. Administration of NaN3 in the positive control group resulted in a high frequency of MN and various types of CAs formations. NaN3 is a potent mutagen and is frequently used as a positive control in antimutagenicity studies60. The anti-genotoxic activity of Pugex was determined considering the reduction in the frequency of NaN3-induced MN and CAs. The extract, which by itself did not exhibit genotoxic activity, showed a anti-genotoxic activity by reducing the frequency of MN and CAs induced by NaN3. Pugex showed high anti-genotoxic activity, providing over 50% protection against all abnormalities. It showed the highest activity against MN anomalies with 74.48% protective effect. Pugex reduced the frequency of fragment by 62.69% and sticky chromosome formation by 70.94%. Exposure to many chemicals induces various chromosomal abnormalities and micronucleus formation, leading to disruptions in cellular activity61,62,63.
Many literature studies have reported that herbal products have a protective effect against the formation of CAs and MN and that this effect is related to their content of phytochemicals19,64. Essential oils exhibit high cytoprotective properties through synergistic processes and anti-genotoxic effects. Induction and enhancement of DNA repair, anti-oxidant activity, and prevention of cellular damage due to oxidative reactions in the cell are possible mechanisms of anti-genotoxic action of essential oils65. In addition to the rich constituents of the essential oils determined by GC- MS analysis of Pugex, the phenolic compounds determined by LC-MS/MS also contribute significantly to the anti-genotoxic activity. Phenolic compounds exhibit a anti-genotoxic activity with many mechanisms such as inhibition of promutagens, induction of detoxification mechanism, blocking of free radicals, scavenging of reactive oxygen species, and neutralization of oxidative stress and inhibition of DNA cleavage66. Ellagic acid, which is determined as the major phenolic component in Pugex content, is a powerful antimutagen and it realizes these effects in a variety of pathways. Inhibition of metabolic activation of mutagens, stimulation of enzymes such as glutathione s-transferase, binding to reactive metabolic forms of mutagens and occupation of DNA regions where mutagens or their metabolites can react are some of these important pathways67. Gallic acid, which is concentrated in the content of Pugex, shows anti-genotoxic activity by clearing electrophilic mutagens and blocking the outer membrane carriers that carry mutagens to the cytosol and preventing their passage into the cell68.
The anti-genotoxic and toxicity-reducing effects of plant extracts are closely related to the active phytochemicals they contain69,70,71,72,73,74,75,76,77,78,79. The rich phytochemical content of Pugex enables it to exhibit anti-genotoxic effects and similar literature studies support our findings. Zahin et al.80 found that ellagic acid inhibits DNA adducts and is a bioactive substance responsible for the anti-genotoxic effect of the seeds. Stanty et al.81 reported that the anti-genotoxic activity of pomegranate seed methanol extract was higher than that of other extracts, and that pomegranate seeds showed strong protection against sodium azide-induced mutagenicity in the Ames test system.
DFT analyses were conducted to gain deeper insights into the anti-oxidant activity of Pugex. The relationship between the HOMO-LUMO energy gaps and anti-oxidant activity is well-established. A smaller HOMO-LUMO energy gap indicates higher molecular instability, resulting in increased reactivity, enhanced free radical scavenging capacity, and a stronger anti-oxidant effect82. The HOMO and LUMO structures of ellagic acid, gallic acid and 2-Oxatricyclo [4.3.1.0(3,8)] decane compounds were determined, while the hydrogen transfer capabilities of the compounds were evaluated based on electronic parameters, as presented in Fig. 4. Among the compounds derived from the plant extracts, ellagic acid exhibited the smallest HOMO-LUMO energy gap, measuring 4.24 eV. Remarkably, this compound also displayed the highest electron affinity of 1.85 eV, underscoring its potential as a prominent anti-oxidant capable of engaging in single electron transfer reactions, particularly in terms of hydrogen free radical donation. Gallic acid demonstrated notable anti-oxidant efficacy, primarily due to the favorable HOMO-LUMO energy gap as 4.93 eV, and 1.06 eV electron affinity. Additionally, 2-Oxatricyclo [4.3.1.0(3,8)] decane the HOMO energy level was determined as – 6.42 eV, the LUMO energy level as 1.98 eV, and the energy gap (ΔE) as 8.40 eV. The ionization potential (IP) and electron affinity (EA) values were obtained as 6.42 eV and − 1.98 eV, respectively. Consequently, the order of anti-oxidant activity according to the theoretical analyses tended to be as follows: ellagic acid> gallic acid > 2-oxatricyclo[4.3.1.0(3,8)]decane.
HOMO and LUMO simulations of (a) ellagic acid, (b) gallic acid and (c) 2-Oxatricyclo[4.3.1.0(3,8)]decane and theoretical DFT results of the compounds. HOMO: Highest Occupied Molecular Orbital, LUMO: Lowest Unoccupied Molecular Orbital, ΔE: energy gap, IP: ionization potential and EA: electron affinity.
In-vitro and in-silico anti-proliferative activity
The anti-proliferative activity of Pugex determined by the Allium assay is shown in Table 4. A total of 5.000 cells were counted in each group, and cells in prophase, metaphase, anaphase, and telophase were considered as dividing cells. While a total of 301 cells were in the stage of division in the negative control group, this number decreased to 139 when glyphosate was administered, and glyphosate reduced cell division by 53.8%. Glyphosate is a compound that causes cell cycle delays and abnormalities, especially at checkpoints83. Because of these effects on the cell cycle, it is used as a positive control in proliferation inhibition studies. Pugex decreased cell proliferation by 15.6% compared to the negative control group, and the number of dividing cells decreased to 254. This reduction in cell proliferation indicates the anti-proliferative effect of Pugex. The anti-proliferative effect of Pugex and other herbal extracts can be explained by the activity of essential oil84. Essential oils exhibit anti-proliferative effects through various mechanisms such as depolarization of cell membrane, increase in membrane permeability, decrease in the activity of membrane-bound enzymes, cell cycle delays and induction of apoptosis85. Terpenes, which are important components of herbal essential oils, contribute greatly to the anti-proliferative activity. Monoterpenes exhibit anti-proliferative activity via inhibition of 3-hydroxymethylglutaryl coenzyme A reductase, diterpenes via decrease cyclin-dependent kinase-1 activity, and triterpenoids via pro-apoptotic and microtubule disruptive activities86. Pulegone, 5-nonanone, and dihydrocarvone, which belong to the class of terpenes and are present in Pugex, may have a fixed role in the formation of anti-proliferative activity. Phenolic compounds are also responsible for the formation of anti-proliferative activity and provide this effect through various mechanisms such as inducing apoptosis, disrupting the cell cycle, altering caspase activities, increasing the number of cells in G1 phase, and accelerating the expression of p53 and p2187. Among the phenolic compounds in Pugex, ellagic acid, which has a high presence rate, is also an important anti-proliferative agent and shows this effect by causing a decrease in ATP synthesis26,88. All phytochemicals in Pugex, including the essential oils and phenolic compounds, contribute to the development of the anti-proliferative effect. There are studies in the literature reporting the anti-proliferative effect of Pugex, but the mechanism of action has not been fully elucidated. Zahin et al.80 reported that fruit peel extract of P. granatum exhibited potent anti-proliferative activity due to the high content of punicalagin and ellagic acid.
Table 5; Fig. 5 contain the interpretation of the results of molecular docking between ellagic acid and ATP synthase, as well as pulegone with α-tubulin and β-tubulin. The purpose of this analysis is to confirm the anti-proliferative activity of Pugex with the phytochemicals it contains. Ellagic acid exhibited a free energy of binding of – 3.82 kcal/mol, indicating a moderate binding affinity to ATP synthase and Ki was determined to be 1.59 mM. In terms of specific interactions, ellagic acid formed hydrogen bonds with LEU63 and ALA66 residues of ATP synthase. Additionally, hydrophobic interactions were observed, contributing to the overall binding affinity. Pulegone demonstrated a stronger binding affinity with α-tubulin, as evidenced by a free energy of binding of – 5.57 kcal/mol. Ki was found to be 83.24 μm, indicating a moderate inhibitory activity against α-tubulin. Specific hydrogen bond interactions were observed between pulegone and ASN206, TYR224, ALA12, ILE171 residues of α-tubulin. These interactions, along with hydrophobic interactions, contribute to the overall binding affinity and potential anti-proliferative activity. Pulegone exhibited a similar free energy of binding of – 5.48 kcal/mol with β-tubulin. Ki was determined to be 95.98 μm, indicating a moderate inhibitory activity against β-tubulin. Specific interactions involved hydrogen bonds with ASN204, CYS12, ILE16, LEU207, LEU225, TYR222 residues of β-tubulin were observed. These interactions, combined with hydrophobic interactions, contribute to the overall binding affinity and potential anti-proliferative activity. These findings indicate that the phytochemicals of Pugex exhibit binding interactions with the target macromolecule and that Pugex has an anti-proliferative effect.
Molecular interactions between (a) ellagic acid and ATP synthase, (b) pulegone and α- tubulin, (c) pulegone and β-tubulin.
In-vitro and in-silico anti-diabetic activity
The anti-diabetic activity of Pugex was determined by measuring the inhibition of α-amylase and α-glucosidase, and the results are shown in Fig. 6. While 20–100 mg/mL Pugex inhibited α-glucosidase activity between 30.2% and 61.3%, it inhibited α-amylase activity between 27.4% and 56.7% at the same concentrations. Acarbose (20–100 µg/mL), which was used as the standard substance, inhibited α-amylase in a range of 46.9–81.8% and α-glucosidase between 40.9% and 75.6%. These results show that acarbose has a strong effect on amylase, whereas Pugex has a higher inhibitory effect on glucosidase. As the concentration of acarbose and Pugex increased, the inhibition rate for both enzymes increased, and the inhibition rate remained at the same level when Pugex was increased from 80 to 100 mg/mL. Partial inhibition of the amylase and glucosidase prevents the sudden rise in blood glucose levels by reducing the digestion of carbohydrates. The goal of diabetes treatment is to minimize the rise in blood glucose levels. There are more than 400 plant species with known anti-diabetic activity. Plants with antihyperglycemic activity exert their effects through different mechanisms. Some of these mechanisms include increasing glucose uptake into tissues, decreasing glucose uptake in the intestine, and suppressing hepatic glucose production. The anti-diabetic effect of plants is mostly achieved through mechanisms that decrease the digestion of dietary carbohydrates. Partial inhibition of the α-glucosidase and α-amylase activity by phytochemicals reduces the conversion of carbohydrates to glucose and the sudden rise in blood glucose levels89,90. The inhibitory effect of Pugex extract on both enzymes indicates its antihyperglycemic activity and its ability to reduce the risk of diabetes. This property can be explained by the biological functions of the active components of the extract. Monoterpenes exhibit anti-diabetic effect with their strong radical scavenging activities and especially inhibition of α-amylase and α-glucosidase and show therapeutic effect at concentrations of 10 mM and above. Flavonoids and polyphenols show their anti-diabetic effects at micromolar levels. The terpenes-rich essential oil profile and polyphenol content of Pugex are important factors in the formation of its anti-diabetic activity91. Among the phenolic compounds in Pugex, ellagic acid and gallic acid, which are the major constituents, inhibit glycogen phosphorylase, thereby inhibiting the breakdown of glycogen and its conversion to glucose and lowering blood sugar92. Kam et al.93 stated that the methanolic extract of pomegranate peel collected from Maharashtra (India) selectively inhibited the α-glucosidase enzyme activities, had no effect on α-amylase, while the seed extracts had a weak inhibitory effect on both enzymes. Das et al.94 reported that methanol extract of pomegranate seed reduced blood glucose level by 47–42% in diabetic rats.
Effects of Pugex on α-amylase and α-glucosidase activity. Pugex extract (mg/mL), Acarbose (µg/mL).
The molecular docking results between 2-Oxatricyclo [4.3.1.0(3,8)] decane and ellagic acid with human pancreatic α-amylase and human lysosomal acid α-glucosidase are given in Table 6; Fig. 7. The purpose of this molecular docking is to elucidate the mechanism behind the experimentally observed anti-diabetic activity. Ellagic acid exhibited a high binding affinity with α-amylase, with a free energy of binding of – 6.46 kcal/mol. Ki was found to be 18.54 μm, indicating potent inhibitory activity against the enzyme. Noteworthy hydrogen bond interactions were observed with LYS200, ILE235, and GLU233 residues of the α-amylase. Additionally, significant hydrophobic interactions involving LEU162, ILE235, TYR151, HIS201, LYS200, and ALA198 contributed to the strong binding affinity. These findings suggest that ellagic acid may have a potential anti-diabetic effect by inhibiting human pancreatic α-amylase. 2-Oxatricyclo [4.3.1.0(3,8)] decane exhibited a moderate binding affinity with human pancreatic α-amylase, with a free energy of binding of – 4.73 kcal/mol. Ki was found to be 339.37 μm, indicating a moderate inhibitory activity against the enzyme. Prominent hydrophobic interactions were observed with GLN63, LEU165, and TRP59 residues, contributing to the overall binding affinity. However, no significant hydrogen bond interactions were observed between 2-Oxatricyclo[4.3.1.0(3,8)]decane and α-amylase. These findings suggest that the binding of 2-Oxatricyclo[4.3.1.0(3,8)]decane to α-amylase may primarily rely on hydrophobic interactions rather than hydrogen bonding. Ellagic acid exhibited a considerable binding affinity with human lysosomal acid α-glucosidase, with a free energy of binding of – 6.17 kcal/mol. Ki was determined to be 30.01 μm, indicating significant inhibitory activity against the enzyme. Notable hydrogen bond interactions were observed with LEU677 and ASP282 residues of the α-glucosidase. Additionally, hydrophobic interactions involving TRP481 and PHE649 residues contributed to the overall binding affinity. In addition, electrostatic interactions occurred with the ASP616 residue. On the other hand, 2-Oxatricyclo[4.3.1.0(3,8)]decane also exhibited a considerable binding affinity with human lysosomal acid α-glucosidase, with a free energy of binding of – 5.05 kcal/mol. Ki was found to be 199.45 μm, indicating moderate inhibitory activity against the enzyme. Notable hydrophobic interactions were observed with LEU405, ILE441, MET519, TRP376, TRP481, PHE649, and HIS674 residues. These findings suggest that both ellagic acid and 2-Oxatricyclo [4.3.1.0(3,8)] decane exhibit significant binding affinities with α-amylase and α-glucosidase. These interactions, including hydrogen bonds and hydrophobic interactions, contribute to their inhibitory activities against the respective enzymes. These results indicate that molecular docking supports the results obtained in vitro, and there are many biological activity studies supported by molecular docking in the literature95,96,97,98,99,100,101,102,103,104,105.
Molecular interactions between (a) α-amylase and ellagic acid, (b) α-amylase and 2-Oxatricyclo [4.3.1.0(3,8)] decane, (c) α-glucosidase and ellagic acid (d) α-glucosidase and 2-Oxatricyclo[4.3.1.0(3,8)] decane.
In-vitro and in-silico anti-cholinesterase activity
Cholinesterase inhibitors show a dose-dependent improvement in Alzheimer’s symptoms with varying degrees of systemic cholinergic effects. This improvement has led to a focus on investigating the cholinesterase inhibitory activities of herbal extracts. The dose-dependent inhibitory effects of Pugex on AChE and BChE are given in Fig. 8. Pugex, which has anti-diabetic activity in the dose range of 20–100 mg/mL, also showed acetylcholinesterase inhibitory activity in the same dose range. Pugex showed low AChE inhibition at a dose of 20 mg/mL and maximum AChE inhibition (67.8%) at 100 mg/mL. The inhibitory effect increased in direct proportion as the dose increased from 40 to 80 mg/mL, and an increase was observed when the dose was increased to 100 mg/mL but was not directly proportional. At doses of 20–100 mg/mL, Pugex showed higher inhibition of BChE than AChE. The maximum BChE inhibition was 79.8% at a dose of 100 mg/mL. The lower inhibition values compared to the standard compounds can be explained by the presence of active compound showing inhibitory activity in the Pugex content, but not in pure form but in complex form with many compounds. The lipophilic properties of essential oils facilitate their movement through the blood-brain barrier and have therapeutic effects in many diseases of the central nervous system, including Alzheimer’s disease and Parkinson’s disease. Essential oils rich in terpenoids present in herbal extracts cause strong inhibition by interacting with cholinesterase, especially via nonpolar bonds106. Studies on the anti-cholinesterase activity of pomegranate seeds are not yet at the desired level, but the activity of other parts of pomegranate such as fruits, fruit peels, and leaves has been reported in many studies. Szwajgier et al.107 reported that the fruit extract of pomegranate significantly inhibited AchE and BChE enzymes, while the leaf extract showed only anti-AChE activity. Gholamhoseinian et al.108 found that methanol extract from pomegranate fruit peels inhibited AChE enzyme by 11.5%.
Inhibition of AChE and BChE activity at different concentration of Pugex (mg/mL), donepezil (µg/mL) and galantamine (µg/mL). Results are presented as mean ± SD (n = 3).
Polyphenols have been found to be potent cholinesterase inhibitors in numerous in vitro studies. However, there are concerns about whether these compounds reach the central nervous system and directly affect enzyme activities. For this reason, molecular docking of the major essential oils contained in Pugex with AChE and BChE enzymes was performed to support the anti-cholinesterase activity detected in vitro. The molecular docking study was conducted to evaluate the binding affinity of 2-Oxatricyclo[4.3.1.0(3,8)]decane with AChE and BChE and the results are given in Table 7; Fig. 9. The free energy of binding was calculated as -6.04 kcal/mol for AChE and − 5.14 kcal/mol for BChE, indicating favorable binding interactions. For AChE, Ki was determined as 37.16 μm. The docking results revealed the involvement of specific amino acid residues in hydrogen bond and hydrophobic interactions. The amino acid PHE295 was found to form hydrogen bond interactions with the ligand. Additionally, the residues VAL294, TRP286, and TYR341 exhibited hydrophobic interactions with the docked compound. In the case of BChE, Ki was calculated as 169.81 μm, indicating relatively weaker inhibition potency compared to AChE. The docking analysis showed the participation of specific amino acid residues in hydrophobic interactions. Notably, the residues ALA328, TRP82, TYR332 and TRP430 contributed to the hydrophobic interactions with the ligand. These findings suggest that 2-Oxatricyclo [4.3.1.0(3,8)] decane exhibits potential binding affinity toward both AChE and BChE.
Molecular interactions of 2-Oxatricyclo[4.3.1.0(3,8)]decane with (a) AChE and (b) BChE.
Drug-likeness features
Lipinski’s rule of five, as described by Lipinski et al.109, outlines specific criteria that are generally indicative of orally active drugs. According to this rule, a compound should have no more than 10 hydrogen bond acceptors (mainly N and O atoms), no more than 5 hydrogen bond donors (OH and NH groups), a partition coefficient (log P) lower than 5, a molecular weight below 500 g/mol, and a polar surface area (measured in square angstroms) equal to or less than 140. The compounds used in the molecular docking analyses in this study were found to conform to Lipinski’s rules of five, as presented in Table 8. The calculation of log P provides information on bioavailability, including absorption, solubility, and permeability. An ideal compound should not be excessively hydrophilic, preventing it from crossing the gastrointestinal wall, nor excessively lipophilic, hindering absorption. Compounds with low hydrophilicity and low log P values are more likely to exhibit good permeability and absorption. The log P values were calculated using the methodology developed by Molinspiration, which involves the sum of part-based additives and correction factors. The compounds with log P values below 5.0 are more likely to be absorbed. This calculation method is reliable and applicable to a wide range of organic molecules. The molecular polar surface area (TPSA) was calculated by summing the contributions of polar fragments with oxygen and nitrogen centers. TPSA is a significant descriptor associated with drug-related properties, including intestinal uptake, blood-brain barrier penetration, and bioavailability110. Upon examination of the data, it is observed that the log P values of all phytochemicals are below 5, indicating compliance with Lipinski’s rules and suggesting their ability to cross biomembranes. Moreover, the phytochemicals with a TPSA value below 140 Å are expected to exhibit better intestinal absorption. Based on the data, these derivatives of phenolic acids are anticipated to be well-absorbed from the intestine.
The study examined the biological activities of the chosen phytochemicals based on their potential as G-protein-coupled receptor (GPCR) ligands, ion channel modulators, nuclear receptor ligands, and inhibitors of kinases and protease enzymes. The results of these analyses are presented in Table 9. Bioactivity probabilities were assessed for the organic molecules, where a value greater than 0.00 indicates active bioactivity, a value between − 0.50 and 0.0 suggests moderate activity, and a value less than − 0.50 indicates inactivity. 2-Oxatricyclo [4.3.1.0(3,8)] decane exhibits moderate bioactivity as a GPCR ligand and active bioactivity as an enzyme inhibitor. Ellagic acid displays moderate activity as a GPCR ligand, ion channel modulator, kinase inhibitor and protease inhibitor. It exhibits active bioactivity as a nuclear receptor ligand and enzyme inhibitor. Gallic acid demonstrates moderate activity as an ion channel modulator and high activity as an enzyme inhibitor. It is inactive as a GPCR ligand, kinase inhibitor, nuclear receptor ligand and protease inhibitor. Pulegone shows only modarate enzyme inhibitor activity. Pharmacokinetic properties play a vital role in evaluating the drug candidacy of compounds, including factors such as gastrointestinal absorption (GI), blood-brain barrier (BBB) permeation, and inhibitory effects on cytochrome P450 enzymes (CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4). It is evident that the studied phytochemicals exhibit high gastrointestinal absorption (GI) properties. Specifically, 2-Oxatricyclo [4.3.1.0(3,8)] decane and pulegone demonstrate blood-brain barrier (BBB) penetration. Among the selected phytochemicals, only ellagic acid exhibits inhibition of cytochrome P450 1A2, while none of the compounds demonstrate P-glycoprotein substrate properties.
Conclusion
Herbal bioactive compounds with many biological and pharmacological activities exhibit positive effects on health when consumed as food and attract more attention compared to synthetic materials in the treatment of chronic diseases. In this study, the phytochemical fingerprint and biological activities of P. granatum seed extract were investigated. Containing a wide variety of essential oils and phenolic substances, Pugex seeds had a rich phytochemical content and the extract has a biological activity profile that does not show cytotoxic effects, has anti-genotoxic and antiproliferative effects, and has broad-spectrum antimicrobial activity. The anti-diabetic activity of P. granatum seed, which causes inhibition of α-amylase and α-glucosidase, may be due to the active ingredients it contains and the synergistic effects of these phytochemicals. In addition, it was determined that the seed extract is a promising source as effective inhibitors of both AChE and BChE. All biological effects that have been experimentally detected in vitro have also been determined by molecular docking studies. All macromolecule and ligand interactions examined by molecular docking are in good agreement with enzymatic studies, and drug likeness data provide preliminary information on the availability of phytochemicals for drugs. The chemical components of plant extracts can help reduce the risk of developing various diseases such as diabetes and Alzheimer and can serve as an alternative or supplement to existing drug applications. However, more studies are needed on the synergistic and antagonistic effects of purified phytochemicals, and this study is a precursor for further studies.
Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
References
Nwozo, S. O., Osunmadewa, D. A. & Oyinloye, B. E. Anti-fatty liver effects of oils from Zingiber officinale and Curcuma longa on ethanol-induced fatty liver in rats. J. Integr. Med. 12, 59–65 (2014).
Gündüz, A., Yalçın, E. & Çavuşoğlu, K. Combined toxic effects of aflatoxin B2 and the protective role of resveratrol in Swiss albino mice. Sci. Rep. 11, 18081 (2021).
Valero-Mendoza, A. G. et al. The whole pomegranate (Punica granatum L), biological properties and important findings: A review. Food Chem. Adv 100153 (2022).
Goula, A. M. & Adamopoulos, K. G. A method for pomegranate seed application in food industries: Seed oil encapsulation. Food Bioprod. Process. 90, 639–652 (2012).
Paul, A. & Radhakrishnan, M. Pomegranate seed oil in food industry: Extraction, characterization, and applications. Trends Food Sci. Technol. 105, 273–283 (2020).
Amri, Z. et al. Effect of pomegranate extracts on brain antioxidant markers and cholinesterase activity in high fat-high fructose diet induced obesity in rat model. BMC Complement. Altern. Med. 17, 1–9 (2017).
Khajebishak, Y., Payahoo, L., Alivand, M. & Alipour, B. Punicic acid: A potential compound of pomegranate seed oil in Type 2 diabetes mellitus management. J. Cell. Physiol. 234, 2112–2120 (2019).
Çayır, K. et al. Pomegranate seed extract attenuates chemotherapy-induced acute nephrotoxicity and hepatotoxicity in rats. J. Med. Food. 14, 1254–1262 (2011).
Bray, G. A. & Greenway, F. L. Current and potential drugs for treatment of obesity. Endocr. Rev. 20, 805–808 (1999).
Greenwell, M. & Rahman, P. K. S. M. Medicinal plants: Their use in anticancer treatment. Int. J. Pharm. Sci. Res. 6, 4103 (2015).
Uddin, M. J. et al. Anticholinesterase activity of eight medicinal plant species: In vitro and in silico studies in the search for therapeutic agents against Alzheimer’s disease. Evid. Based Comp. Altern. Med. 9995614 (2021).
Bakir Çilesizoğlu, N., Yalçin, E., Çavuşoğlu, K. & Sipahi Kuloğlu, S. Qualitative and quantitative phytochemical screening of Nerium oleander L. extracts associated with toxicity profile. Sci. Rep. 12, 21421 (2022).
Akman, T. C. et al. LC-ESI-MS/MS chemical characterization, antioxidant and antidiabetic properties of propolis extracted with organic solvents from Eastern Anatolia Region. Chem. Biodivers. 20, 202201189 (2023).
Kayir, Ö., Doğan, H., Alver, E. & Bilici, İ. Quantification of phenolic component by LC-HESI-MS/MS and evaluation of antioxidant activities of Crocus ancyrensis extracts obtained with different solvents. Chem. Biodivers. 20, 202201186 (2023).
Nozohour, Y., Golmohammadi, R., Mirnejad, R. & Fartashvand, M. Antibacterial activity of pomegranate (Punica granatum L.) seed and peel alcoholic extracts on Staphylococcus aureus and Pseudomonas aeruginosa isolated from health centers. J. Appl. Biotechnol. Rep. 5, 32–36 (2018).
Ayhan, B. S., Yalçın, E., Çavuşoğlu, K. & Acar, A. Antidiabetic potential and multi-biological activities of Trachystemon orientalis extracts. J. Food Meas. Charact. 13, 2887–2893 (2019).
Blanco, P. et al. Bacterial multidrug efflux pumps: Much more than antibiotic resistance determinants. Microorganisms 4, 14 (2016). Martinez.
Chen, W. et al. Novobiocin binding to NalD induces the expression of the MexAB-OprM pump in Pseudomonas aeruginosa. Mol. Microbiol. 100, 749–758 (2016).
Yalçin, E. & Çavuşoğlu, K. Toxicity assessment of potassium bromate and the remedial role of grape seed extract. Sci. Rep. 12, 20529 (2022).
Frisch, M. J. et al. Gaussian 09, Revision B.01 (Gaussian Inc., 2010).
Dennington, R. & Keith, T. & J. Millam. Gauss View, Version 5 (Semichem Inc., 2009).
Becke, A. D. Densityfunctional thermochemistry. III. The role of exact exchange. J. Chem. Phys. 98, 5648–5652 (1963).
Tütüncü, E., Yalçın, E., Acar, A., Yapar, K. & Çavuşoğlu, K. Investigation of the toxic effects of a carbamate insecticide methiocarb in Allium cepa L. Cytologia 84, 113–117 (2019).
Çavuşoğlu, K. & Yalçin, E. Spectral shift supported epichlorohydrin toxicity and the protective role of sage. Environ. Sci. Pollut Res. 30, 1374–1385 (2023).
Aydin, D., Yalçın, E. & Çavuşoğlu, K. Metal chelating and anti-radical activity of Salvia officinalis in the ameliorative effects against uranium toxicity. Sci. Rep. 12, 15845 (2022).
Losso, J. N., Bansode, R. R., Trappey, A., Bawadi, H. A. & Truax, R. In vitro anti-proliferative activities of ellagic acid. J. Nutr. Biochem. 15, 672–678 (2004).
Shahina, Z., Yennamalli, R. M. & Dahms, T. E. Key essential oil components delocalize Candida albicans Kar3p and impact microtubule structure. Microbiol. Res. 272, 127373 (2023).
Preiss, L. et al. Structure of the mycobacterial ATP synthase Fo rotor ring in complex with the anti-TB drug bedaquiline. Sci. Adv. 1, e1500106 (2015).
Lacey, S. E., He, S., Scheres, S. H. & Carter, A. P. Cryo-EM of dynein microtubule-binding domains shows how an axonemal dynein distorts the microtubule. Elife 8, e47145 (2019).
Nickavar, B. & Abolhasani, L. Bioactivity-guided separation of an α-amylase inhibitor flavonoid from Salvia virgate. Iran. J. Pharm. Res. Winter. 12, 57–61 (2013).
Caner, S. & Brayer, G. D. Human Pancreatic Alpha-amylase in Complex with Mini-montbretin A (2016). https://www.rcsb.org/structure/5e0f
Roig-Zamboni, V. et al. Structure of human lysosomal acid α-glucosidase–a guide for the treatment of Pompe disease. Nat. Commun. 8, 1111 (2017).
Ellman, G. L., Courtney, K. D., Andres Jr, V. & Featherstone, R. M. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95 (1961).
Ayaz, M. et al. Comparative chemical profiling, cholinesterase inhibitions and anti-radicals properties of essential oils from Polygonum hydropiper L: a preliminary anti-Alzheimer’s study. Lipids Health Dis. 14, 1–12 (2015).
Nachon, F. et al. Crystal structures of human cholinesterases in complex with huprine W and tacrine: elements of specificity for anti-Alzheimer’s drugs targeting acetyl-and butyryl-cholinesterase. Biochem. J. 453, 393–399 (2013).
Cheung, J., Gary, E. N., Shiomi, K. & Rosenberry, T. L. Structures of human acetylcholinesterase bound to dihydrotanshinone I and territrem B show peripheral site flexibility. ACS Med. Chem. Lett. 4, 1091–1096 (2013).
Guex, N. & Peitsch, M. CSWISS-MODEL and the Swiss-Pdb Viewer: An environment for comparative protein modeling. Electrophoresis 18, 2714–2723 (2005).
O’Boyle, N. M. Open Babel: An open chemical toolbox. J. Cheminform. 3, 33 (2011).
Morris, G. M. et al. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J. Comput. Chem. 30, 2785–2791 (2009).
Molinspiration, Cheminformatics (2023). https://www.molinspiration.com
Daina, A., Michielin, O. & Zoete, V. SwissADME: A free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci. Rep. 7, 42717 (2017).
Chouhan, S., Sharma, K. & Guleria, S. Antimicrobial activity of some essential oils-present status and future perspectives. Medicines 4, 58 (2017).
Baser, K. H. C., Kirimer, N. & Tümen, G. Pulegone-rich essential oils of Turkey. J. Essent. Oil Res. 10, 1–8 (2011).
Cairone, F. et al. In-depth chemical characterization of Punica granatum L. seed oil. Foods 12, 1592 (2023).
Sharifi-Rad, J. et al. Ellagic acid: A review on its natural sources, chemical stability, and therapeutic potential. Oxid. Med. Cell. Longev. 1–24 (2022). (2022).
Wianowska, D. & Olszowy-Tomczyk, M. A. A concise profile of gallic acid-from its natural sources through biological properties and chemical methods of determination. Molecules 28, 1186 (2023).
Kahkeshani, N. et al. Pharmacological effects of gallic acid in health and diseases: A mechanistic review. Iran. J. Basic. Med. Sci. 22, 225 (2019).
Karagecili, H., İzol, E., Kirecci, E. & Gulcin, İ. Determination of antioxidant, anti-alzheimer, antidiabetic, antiglaucoma and antimicrobial effects of zivzik pomegranate (Punica granatum)-a chemical profiling by LC-MS/MS. Life 13, 735 (2023).
Altemimi, A., Lakhssassi, N., Baharlouei, A., Watson, D. G. & Lightfoot, D. A. Phytochemicals: Extraction, isolation, and identification of bioactive compounds from plant extracts. Plants 6, 42 (2017).
Dai, J. & Mumper, R. J. Plant phenolics: Extraction, analysis and their antioxidant and anticancer properties. Molecules 15, 7313–7352 (2010).
Danh, L. T., Truong, P., Mammucari, R. & Foster, N. Extraction of vetiver essential oil by ethanol-modified supercritical carbon dioxide. Chem. Eng. J. 165, 26–34 (2010).
Masci, A. et al. Evaluation of different extraction methods from pomegranate whole fruit or peels and the antioxidant and antiproliferative activity of the polyphenolic fraction. Food Chem. 202, 59–69 (2016).
Abbasi, H., Rezaei, K., Emamdjomeh, Z. & Mousavi, S. M. E. Effect of various extraction conditions on the phenolic contents of pomegranate seed oil. Eur. J. Lipid Sci. Technol. 110, 435–440 (2008).
Breijyeh, Z., Jubeh, B. & Karaman, R. Resistance of gram-negative bacteria to current antibacterial agents and approaches to resolve it. Molecules 25, 1340 (2020).
Walker, L. A., Gow, N. A. & Munro, C. A. Elevated chitin content reduces the susceptibility of Candida species to caspofungin. Antimicrob. Agents Chemother. 57, 146–154 (2013).
Duru, M. E., Öztürk, M., Uğur, A. & Ceylan, Ö. The constituents of essential oil and in vitro antimicrobial activity of Micromeria cilicica from Turkey. J. Ethnopharmacol. 94, 43–48 (2004).
Ghudhaib, K. K., Hanna, E. R. & Jawad, A. H. Effect of ellagic acid on some types of pathogenic bacteria. Al-Nahrain J. Sci. 13, 79–85 (2010).
Pudlo, M., Demougeot, C. & Girard-Thernier, C. Arginase inhibitors: A rational approach over one century. Med. Res. Rev. 37, 475–513 (2017).
Dahham, S. S., Ali, M. N., Tabassum, H. & Khan, M. Studies on antibacterial and antifungal activity of pomegranate (Punica granatum L). Am Eur. J. Agric. Environ Sci. 9, 273–281 (2010).
Srivastava, P., Marker, S., Pandey, P. & Tiwari, D. K. Mutagenic effects of sodium azide on the growth and yield characteristics in wheat (Triticum aestivum L. em. Thell). Asian J. Plant. Sci. 10 (3), 190–201 (2011).
Demirtaş, G., Çavuşoğlu, K. & Yalçin, E. Aneugenic, clastogenic, and multi-toxic effects of diethyl phthalate exposure. Environ. Sci. Pollut Res. 27 (5), 5503–5510 (2020).
Çavuşoğlu, K., Yalçin, E., Türkmen, Z., Yapar, K. & Sağir, S. Physiological, anatomical, biochemical, and cytogenetic effects of thiamethoxam treatment on Allium cepa (amaryllidaceae) L. Env Tox. 27 (11), 635–643 (2012).
Çavuşoğlu, K. et al. Investigation of toxic effects of the glyphosate on Allium cepa. J. Agric. Sci. 17 (2), 131–142 (2011).
Yirmibeş, F., Yalçin, E. & Çavuşoğlu, K. Protective role of green tea against paraquat toxicity in Allium cepa L.: Physiological, cytogenetic, biochemical, and anatomical assessment. Environ. Sci. Pollut Res. 29, 23794–23805 (2022).
Toscano-Garibay, J. D. et al. Antimutagenic and antioxidant activity of the essential oils of Citrus sinensis and Citrus latifolia. Sci. Rep. 7, 11479 (2017).
Kuroda, Y. & Hara, Y. Antimutagenic and anticarcinogenic activity of tea polyphenols. Mutat. Res. 436, 69–97 (1999).
Maas, J. L., Galletta, G. J. & Stoner, G. D. Ellagic acid, an anticarcinogen in fruits, especially in strawberries: A review. HortScience 26, 10–14 (2019).
Hour, T. C., Liang, Y. C., Chu, I. S. & Lin, J. K. Inhibition of eleven mutagens by various tea extracts, (–)epigallocatechin-3-gallate, gallic acid and caffeine. Food Chem. Toxicol. 37, 569–579 (1999).
Kutluer, F., Güç, İ., Yalçın, E. & Çavuşoğlu, K. Toxicity of environmentally relevant concentration of esfenvalerate and Taraxacum officinale application to overcome toxicity: A multi-bioindicator ın-vivo study. Environ. Pollut. 373, 126111 (2025).
Kesti Usta, S., Yalçın, E. & Çavuşoğlu, K. Synergistic and antagonistic contributions of main components to the bioactivity profile of Anethum graveolens extract. Sci. Rep. 15 (1), 21465 (2025).
Topatan, Z. Ş. et al. Alleviatory efficacy of Achillea millefolium L. in etoxazole-mediated toxicity in Allium cepa L. Sci. Rep. 14, 31674 (2024).
Güngör, Ö. Y., Yalçın, E., Çavuşoğlu, K. & Özkan, B. Chemical profiling by LC–MS/MS and GC–MS and biological activity assessment of different extracts of Portulaca oleracea through in-vitro and in-silico approaches. Sci. Rep. 15 (1), 40511 (2025).
Akgeyik, A. U., Yalçın, E. & Çavuşoğlu, K. Phytochemical fingerprint and biological activity of raw and heat-treated Ornithogalum umbellatum. Sci. Rep. 13 (1), 13733 (2023).
Himtaş, D., Yalçin, E., Çavuşoğlu, K. & Acar, A. In-vivo and in-silico studies to identify toxicity mechanisms of permethrin with the toxicity-reducing role of ginger. Environ. Sci. Pollut Res. 31 (6), 9272–9287 (2024).
Durhan, B., Yalçın, E., Çavuşoğlu, K. & Acar, A. Molecular docking assisted biological functions and phytochemical screening of Amaranthus lividus L. extract. Sci. Rep. 12 (1), 4308 (2022).
Üst, Ö., Yalçin, E., Çavuşoğlu, K. & Özkan, B. LC–MS/MS, GC–MS and molecular docking analysis for phytochemical fingerprint and bioactivity of Beta vulgaris L. Sci. Rep. 14 (1), 7491 (2024).
Yapar, K., Çavuşoğlu, K., Oruç, E. & Yalçin, E. Protective effect of royal jelly and green tea extracts effect against cisplatin-induced nephrotoxicity in mice: A comparative study. J. Med. Food. 12 (5), 1136–1142 (2009).
Çavuşoğlu, K., Yapar, K., Oruç, E. & Yalçın, E. The protective effect of royal jelly on chronic lambda-cyhalothrin toxicity: Serum biochemical parameters, lipid peroxidation, and genotoxic and histopathological alterations in swiss albino mice. J. Med. Food. 14 (10), 1229–1237 (2011).
Cavusoglu, K. et al. Protective role of Ginkgo biloba on petroleum wastewater-induced toxicity in Vicia faba L.(Fabaceae) root tip cells. J. Environ. Biol. 31 (3), 319 (2010).
Zahin, M., Ahmad, I., Gupta, R. C. & Aqil, F. Punicalagin and ellagic acid demonstrate antimutagenic activity and inhibition of benzo [a] pyrene induced DNA adducts. BioMed Res. Int. 467465 (2014).
Santhy, K. S., Sherly, P. G. & Arulraj, P. Antimutagenic activity of pomegranate (punica granatum) in the Salmonella typhimurium reverse mutation assay. Asian J. Microbiol. Biotechnol. Environ. Sci. 9, 255 (2007).
Szewczuk, N. A., Duchowicz, P. R., Pomilio, A. B. & Lobayan, R. M. Resonance structure contributions, flexibility, and frontier molecular orbitals (HOMO–LUMO) of pelargonidin, cyanidin, and delphinidin throughout the conformational space: Application to antioxidant and antimutagenic activities. J. Mol. Mod. 29, 2 (2023).
Marc, J., Mulner-Lorillon, O. & Bellé, R. Glyphosate-based pesticides affect cell cycle regulation. Biol. Cell. 96, 245–249 (2004).
Aranha, E. S. P. et al. Essential oils from Eugenia spp.: In vitro antiproliferative potential with inhibitory action of metalloproteinases. Ind. Crops Prod. 141, 111736 (2019).
Russo, R., Corasaniti, M. T., Bagetta, G. & Morrone, L. A. Exploitation of cytotoxicity of some essential oils for translation in cancer therapy. eCAM. 397821 (2015).
Thoppil, R. J. & Bishayee, A. Terpenoids as potential chemopreventive and therapeutic agents in liver cancer. World J. Hepatol. 3, 228 (2011).
Okafor, J. N. et al. Phenolic content, antioxidant, cytotoxic and antiproliferative effects of fractions of Vigna subterraenea (L.) verdc from Mpumalanga, South Africa. Heliyon 7, e08397 (2021).
Han, D. H., Lee, M. J. & Kim, J. H. Antioxidant and apoptosis-inducing activities of ellagic acid. Anticancer Res. 26, 3601–3606 (2006).
Chan, C. H., Ngoh, G. C. & Yusoff, R. A brief review on anti-diabetic plants: Global distribution, active ingredients, extraction techniques and acting mechanisms. Pharmacogn. Rev. 6, 22–28 (2012).
Tran, N., Pham, B. & Le, L. Bioactive compounds in anti-diabetic plants: From herbal medicine to modern drug discovery. Biology 9, 252 (2020).
Habtemariam, S. Antidiabetic potential of monoterpenes: A case of small molecules punching above their weight. Int. J. Mol. Sci. 19, 4 (2017).
Kyriakis, E. et al. Natural flavonoids as antidiabetic agents. The binding of gallic and ellagic acids to glycogen phosphorylase b. FEBS Lett. 589 (15), 1787–1794 (2015).
Kam, A. et al. A comparative study on the inhibitory effects of different parts and chemical constituents of pomegranate on α-amylase and α-glucosidase. Phytother Res. 27, 1614–1620 (2013).
Das, A. K. et al. Studies on the hypoglycaemic activity of Punica granatum seed in streptozotocin induced diabetic rats. Phytother Res. 15, 628–629 (2001).
Annakkaya, N., Çavuşoğlu, K., Yalçin, E. & Özkan, B. Investigation of in-vivo and in-silico toxicity induced by environmental drug contamination in a non-target organism. Sci. Rep. 15 (1), 21082 (2025).
Pehlivan, Ö. C., Cavuşoğlu, K., Yalçin, E. & Acar, A. Silico interactions and deep neural network modeling for toxicity profile of Methyl methanesulfonate. Environ. Sci. Pollut Res. 30 (55), 117952–117969 (2023).
Çakir, F., Kutluer, F., Yalçin, E., Çavuşoğlu, K. & Acar, A. Deep neural network and molecular docking supported toxicity profile of prometryn. Chemosphere 340, 139962 (2023).
Çavuşoğlu, D., Yalçin, E., Çavuşoğlu, K., Acar, A. & Yapar, K. Molecular docking and toxicity assessment of spirodiclofen: Protective role of lycopene. Environ. Sci. Pollut Res. 28 (40), 57372–57385 (2021).
Kurt, D., Acar, A., Çavuşoğlu, D., Yalçin, E. & Çavuşoğlu, K. Genotoxic effects and molecular docking of 1, 4-dioxane: Combined protective effects of trans-resveratrol. Environ. Sci. Pollut Res. 28 (39), 54922–54935 (2021).
Ayhan, B. S., Yalçin, E. & Çavuşoğlu, K. In-silico receptor interactions, phytochemical fingerprint and biological activities of Matricaria chamomilla flower extract and the main components. Sci. Rep. 15 (1), 28875 (2025).
Seven, B., Kültiğin, Çavuşoğlu, Yalçin, E. & Acar, A. Investigation of cypermethrin toxicity in Swiss albino mice with physiological, genetic and biochemical approaches. Sci. Rep. 12 (1), 11439 (2022).
Kuloğlu, S. S., Yalçin, E., Çavuşoğlu, K. & Acar, A. Dose-dependent toxicity profile and genotoxicity mechanism of lithium carbonate. Sci. Rep. 12 (1), 13504 (2022).
Onur, M., Yalçın, E., Çavuşoğlu, K. & Acar, A. Elucidating the toxicity mechanism of AFM2 and the protective role of quercetin in albino mice. Sci. Rep. 13 (1), 1237 (2023).
Onur, B., Çavuşoğlu, K., Yalçin, E. & Acar, A. Paraquat toxicity in different cell types of Swiss albino mice. Sci. Rep. 12 (1), 4818 (2022).
Kutluer, F., Özkan, B., Yalçin, E. & Çavuşoğlu, K. Direct and indirect toxicity mechanisms of the natural insecticide azadirachtin based on in-silico interactions with tubulin, topoisomerase and DNA. Chemosphere 364, 143006 (2024).
Hung, N. H. et al. Acetylcholinesterase inhibitory activities of essential oils from vietnamese traditional medicinal plants. Molecules 27, 7092 (2022).
Szwajgier, D. & Borowiec, K. Screening for cholinesterase inhibitors in selected fruits and vegetables. Ejpau 15, 6 (2012).
Gholamhoseinian, A., Moradi, M. N. & Sharifi-Far, F. Screening the methanol extracts of some Iranian plants for acetylcholinesterase inhibitory activity. Res. Pharm. Sci. 4, 105–112 (2009).
Lipinski, C. A., Lombardo, F., Dominy, B. W. & Feeney, P. J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 23, 3–25 (1997).
Parvez, A. et al. Pharmacophores modeling in terms of prediction of theoretical physico-chemical properties and verification by experimental correlations of novel coumarin derivatives produced via Betti’s protocol. Eur. J. Med. Chem. 45, 4370–4378 (2010).
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Yalçın, E., Çavuşoğlu, K. & Acar, A. ADME/drug-likeness and functional properties of Punica granatum seeds supported with molecular docking, GC-MS and LC-MS/MS analysis. Sci Rep 16, 10968 (2026). https://doi.org/10.1038/s41598-026-45832-3
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DOI: https://doi.org/10.1038/s41598-026-45832-3
