Isolation and characterization of thymoquinone from Nigella sativa essential oil: antioxidant and antibacterial activities, molecular modeling studies, and cytotoxic effects on lung cancer A549 cells

Abstract Thymoquinone (TQ), a bioactive compound found in Nigella sativa seeds, has gained considerable attention due to its potential therapeutic properties. This study aimed to isolate and characterize TQ from Nigella sativa essential oil and evaluate its antioxidant capacity, antibacterial activity, and cytotoxic effects on lung cancer A549 cells. Moreover, The binding potential of TQ to Keap1 (Kelch-like ECH associated protein 1) was investigated through molecular docking. The stability of the resulting complex was evaluated through molecular dynamics (MD) simulation. The CUPRAC assay revealed significant antioxidant capacity of TQ, as indicated by its high molar absorptivity coefficient. TQ also demonstrated notable free radical scavenging activity, with efficacy increasing with concentration. In terms of antiquorum sensing activity, TQ displayed effectiveness against all tested virulence factors in P. aeruginosa PAO1 and Chromobacterium violaceum in different range. Additionally, TQ induced cytotoxicity in non-small lung cancer cells, inhibiting cell viability. In conclusion, this study successfully isolated and characterized TQ from Nigella sativa seeds and demonstrated its remarkable antioxidant capacity, antibacterial activity, and cytotoxic effects on lung cancer cells. These findings suggest the potential of TQ for various applications in the food and pharmaceutical industries. The wet-lab study demonstrated that TQ had an antioxidant effect. The TQ was found to have high binding potential to Keap1 as it formed four conventional hydrogen bonds with the protein. The resulting Keap1-TQ complex was also found to be stable. Further research is warranted to explore the therapeutic potential of TQ and develop novel treatments based on its properties. GRAPHICAL ABSTRACT


Introduction
There has been an increasing demand for pharmaceutical components in recent years due to the many advantages of plant-derived pharmaceutical components.The use of plantderived pharmaceutical components in areas such as food, cosmetics and medicine has made them even more attractive.Therefore, it has become very important to isolate these components from plants.One of the most important of plant-derived pharmaceutical components is thymoquinone (TQ), which is found in the seed structure of N. sativa Linn., which is a member of the Ranunculaceae family, and grows in southwest Asia, Europe, North Africa, and Turkey 1 .The seeds of the plant, which are rich in active ingredients, have long been used as a traditional medicine for a wide variety of diseases in the Middle and Far East 2 .
The pharmacological actions of N. sativa seeds are mostly attributed to the quinine components, particularly TQ.TQ, which stands for 2-isopropyl-5-methylbenzo-1,4quinone, is the primary active compound in N. sativa seeds.It is predominantly present in the essential oil extracted from N. sativa seeds 3 .In recent studies, the therapeutic effects of the TQ molecule, which is found in high amounts in N. sativa essential oil, have been proven on cancer cells 4,5 .At the same time, it is observed that when used together with drugs used in chemotherapy, it reduces the toxic side effects of the drugs and also increases the effect of the treatment 6 .TQ is known for its antioxidant, antitumor and antiinflammatory properties 7 .In vitro cell culture experiments, TQ has been shown to inhibit the growth of lung cancer cells 8,9 .
Although extensive research has been done on TQ in many aspects, studies on the purification and characterization of TQ are very limited.The investigations for its purification have not been explained in detail.However, TQ is a natural molecule that exhibits strong antioxidant activity despite lacking any phenolic hydroxyl group, which is typically associated with antioxidant action.The compound's antioxidant ability is widely recognized and documented by many researchers [10][11][12][13] .Antioxidant assays of TQ include ABTS/TEAC, DPPH, Folin-Ciocalteu, and FRAP methods, each using different chromogenic redox reagents with different standard potencies.Nevertheless, the CUPRAC approach has not been utilized to ascertain the antioxidant capability of TQ.
The fight with antibiotics in bacterial infections becomes more and more difficult due to the resistance of the bacteria as a result of mutations developed day by day, and it makes it necessary to develop new strategies in the fight against microorganisms 14 .Understanding that the virulence factors that are effective in the emergence of the disease in pathogenic microorganisms are realized by the control of the discovered quorum sensing system, has led to the intensification of studies on the inhibition of this system in the fight against pathogenic microorganisms 15 .P. aeruginosa is a common, opportunistic bacterium that can cause disease in humans, plants and animals and uses this system in its pathogenicity 16 .
In this study, it was aimed to obtain TQ isolated from N. sativa essential oil by silica gel column chromatography.Moreover, the obtained structure was characterized by Nuclear magnetic resonance spectroscopy ( 1 H-NMR) analysis, high-pressure liquid chromatography (HPLC), Fourier-transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC) analyses.In addition, the total antioxidant capacity and free radical scavenging potential of the isolated TQ were evaluated according to the CUPric reducing antioxidant capacity (CUPRAC) and 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH • ) scavenging methods.And anti-quorum sensing effect of TQ was investigated both P. aeruginosa PAO1 and C. violaceum 12472.Additionally, the binding potential of TQ to Keap1 (Kelch-like ECH associated protein 1) was investigated through molecular docking.The stability of the resulting complex was evaluated through molecular dynamics (MD) simulation.

Plant material
The N. sativa seeds were provided by the Bucak Agricultural Credit Cooperative located in Burdur, Turkey.Prof. İsa Telci identified and deposited seeds to the herbarium of the Faculty of Agriculture, Isparta University of Applied Sciences.The seeds were given voucher specimen numbers NS32-2023.

Preparation of solutions
The solid masses of CUPRAC reagents were accurately weighed using a digital analytical balance (Model Radwag, AS 220/C02, Radom, Poland).The solutions prepared for the CUPRAC test of TAC are as follows: A 10 mM solution of CuCl 2 was made by dissolving 0.4262 g of CuCl 2 .2H 2 O in distilled water and then diluting the solution to a total volume of 250 mL.A 1.0 M ammonium acetate buffer with a pH of 7.0 was prepared by dissolving 19.27 g of NH 4 Ac in water and then diluting the solution to a volume of 250 mL.A 7.5 mM solution of Neocuproine (Nc) was made daily by dissolving 0.078 g of Nc in 100% ethanol and then diluting it to a volume of 50 mL with ethanol.The TQ solution was produced in 100% ethanol with a concentration of 1x 10 -2 M.

Essential oil extraction
Essential oil extraction from N. sativa seeds was carried out as follows.Firstly, N. sativa seeds were pressed in a laboratory type cold-pressed machine to obtain fixed oil.Secondly, N. sativa essential oil (NSEO) was obtained from 50 g N. sativa seed oil (1/10; w/v) by hydrodistillation using a Clevenger apparatus for 3 h 17 .The essential oil obtained with light yellow color and pungent odor was dried with anhydrous sodium sulfate and kept in the dark at +4°C until use.

Essential oil components analysis with GC-FID and GC-MS
Essential oil analyses of volatile components were performed as in our previous study 18 .Essential oil analysis of volatile components was performed on a GC 2010 plus system a Shimadzu GCMS-QP2010 SE (Japan) model with Support Rx-5Sil MS capillary column (30 m x 0.25 mm, film thickness 0.25 µm).The flame ionization detector (FID) temperature was set at 280°C and the same running conditions were applied to replicate columns.Simultaneous autoinjection was utilized to obtain equivalent retention times (RI).Compounds were identified by their peak areas in the GC-FID chromatograms.GC-MS analysis was performed under the following conditions.Helium was used as carrier gas with flow rate of 1 mL/min.The split ratio was 1:10.Following a one-minute duration at 60°C, the temperature program gradually rises to 250°C at a rate of 4°C per min.It remains at 250 °C for a period of 15 min.
The mass spectra were obtained using an energy of 70 eV.30 µL of pure essential oil was supplemented with 970 µL of hexane. 1 µL was injected from the sealed vial.The compounds were identified by comparing the mass spectra obtained with NIST27 and NIST147 from the US National Institute of Technology and Standards (NIST) mass spectra libraries.The identification was further confirmed by comparing their RI values 19,20 .Calculation of relative retention indices (RRI) was based on retention time of series of the standards of C 7 -C 30 saturated n-alkanes.The relative percentages of the eluted components were calculated from FID chromatograms 21 .

Isolation of TQ
TQ was isolated from NSEO by silica gel column chromatography.3 mL of NSEO were loaded on a glass column (400×20 mm) packed with silica gel 60 (0.040-0.063 mm) (Merck, Germany).n-Hexane/ diethyl ether (85/15, v/v) was used as the solvent and the compounds were eluted successively with 500 mL n-hexane/ diethyl ether (85/15, v/v).Solvent in the fraction was removed by rotary evaporation at 45°C under vacuum.A known quantity of each fractionation (100 mg) was made up to 10 mL with methanol and subjected to HPLC analysis.TQ powder samples were also characterized by using differential scanning calorimetry (DSC), 1 H-NMR, and FTIR spectroscopy.

HPLC analysis
The TQ analysis was performed according to the modified HPLC method of developed by Kiralan et al. 22 .The HPLC system used consisted of a Shimadzu System, which included a SCL10Avp System controller, a SIL-10AD vp Autosampler, an LC-10AD vp pump, a DGU-14a degasser, a CTO-10 A vp column heater, and a diode array detector set at a wavelength of 278 nm.The chromatographic separations were conducted using an Agilent Eclipse XDB-C18 column with dimensions of 250 X 4.6 mm ID and a particle size of 5 µm.The rate of flow was 1 mL per min, and the amount of injection was 10 µL.The results were combined and analyzed using the Shimadzu Class-VP Chromatography Laboratory Automated Software system.The TQ content in the extract was quantified in milligrams per gram (mg/g) using an external calibration curve.

Differential scanning calorimeter (DSC)
analysis DSC data were collected using TAQ2000 equipment (TAQ2000, New Castle, Delaware, USA equipped with TA UniversalAnalysis software).DSC measurements were performed with N 2 gas flux of 20 cm 3 /min and a heating rate of 10°C/min starting at -50°C and ending at 150°C.

Nuclear magnetic resonance spectroscopy
( 1 H-NMR) analysis NMR analyses were acquired on a 600 MHz Varian Inova spectrometer equipped with a 5 mm triple resonance probe with z-axis pulsed field gradients.Chemical shifts are given in parts per million (ppm) according to the chlorophome peak (CHCl 3 , 1H: δ = 7.26).

CUPRAC assay of total antioxidant capacity
The CUPRAC method, as described by Apak et al. 23 is based on the reduction of a cupric neocuproine complex (Cu(II)-Nc) by antioxidants to the cuprous form (Cu(I)-Nc).The method is briefly as follows.Add 1 mL of copper(II) solution, neocuproin solution and ammonium acetate buffer, respectively, into a glass tube.Add (0.5) mL of antioxidant solution and (1.1-0.5)mL of distilled water and shake the tubes well.The solutions prepared with a total volume of 4.1 mL are kept closed for 30 min at room conditions.At the end of this period, absorbance values are measured at 450 nm against the reference solution without sample.Reference Solution: 1 mL Cu(II) + 1 mL Nc + 1 mL NH 4 Ac + 1.1 mL H 2 O Sample Solution: 1 mL Cu(II) + 1 mL Nc + 1 mL The CUPRAC method was applied to the TQ compound as follows.The initial concentration of TQ was prepared as 10 -2 M. The calibration curve of TQ taken in different volumes and the absorbance values of the Cu(I)-Nc chelate obtained in the presence of this standard after the addition of CUPRAC reagent are given in supplementary file, Table S1.The symbols on the linear calibration line are c: molar concentration, A: absorbance, and r: correlation coefficient.Calculation of final concentration (M): C1 x V1 = C2 x V2 C1: TQ initial concentration V1: Volume of TQ received C2: Final concentration of TQ V2: Total volume of the CUPRAC method (4.1 mL) The standard calibration curve of TQ compound were constructed by plotting absorbance versus molar concentration.The CUPRAC antioxidant capabilities of various polyphenolics and flavonoids were measured experimentally and expressed as trolox equivalent antioxidant capacities (TEAC).TEAC is defined as the reducing power of a 1 mM antioxidant solution being investigated, measured in Trolox mM equivalents.The TEAC value is unitless since it is stated relative to a reference substance called trolox (TR).
The TEAC value of TQ was determined experimentally by calculating the ratio of the molar absorptivity of thymoquinone (TQ) that of TR, which was obtained under the same conditions in the CUPRAC test 24 .

Free radical-scavenging activity (FRS) assay
The free-radical-scavenging capacity of samples was evaluated using the DPPH stable radical and following the methodology described by Erdoğan et al. 18 .The application of the DPPH assay is summarized as follows: x mL of sample extract was mixed in a tube with (2-x) mL of ethanol (99%) and 2 mL of DPPH • solution at 0.2 mM.Absorbance was recorded at 515 nm against ethanol after 30 min of DPPH • addition.
The activity to scavenge the DPPH • radical was calculated using the following equation: where A Control is the absorbance of the control reaction and A Sample is the absorbance in the presence of TQ or BHT.

Anti-quorum sensing activities Effect on P. aeruginosa PAO1 Biofilm
The inhibition effect of TQ on biofilm formation on P. aeruginosa PAO1 was evaluated according to a method previously described 25 .QS inhibitor usually in concentration below the MIC for the organism affect the synthesis of extracellular virulence factors which are regulated by quorum sensing signal molecules.So in QS inhibition activity tests are carried out in submic concentration.After decided to minimum inhibition concentration; sub-MIC concentration (≤140 mcg/mL) used for biofilm assay.Overnight culture of bacteria incubated absence and presence of TQ.All experiments were performed in at least triplicate.

Pyocyanin pigment inhibition on P. aeruginosa PAO1
To investigate the inhibition of pyocyanin pigment production; sub-MIC concentration (≤140 mcg/mL) of TQ was added to 10 mL LBB with PAO1 cultures (OD600 = 0.05) 26 .Following 16-18 h of incubation, 10 mL of bacterial culture mixed with six volumes of chloroform and vortexed.After that, 2 mL of 0.2 M HCl was added to the new tube that had the chloroform phase in it.After centrifugation, the upper layer (0.2 M HCl) was collected, and measured at 520 nm.

Elastase inhibition on P. aeruginosa PAO1
The elastase activity was determined using the Elastin Congo Red (ECR; Sigma-Darmstadt, Germany) assay 27 .Briefly, 900 μL of ECR buffer (100 mM Tris, 1 mM CaCl 2 , pH 7.5) was added to 100 μL of bacterial supernatant and incubated at 37°C for 3 h in a shaking incubator at 200 rpm.After centrifugation, the supernatant was read at 495 nm in the spectrophotometer (Biotek-Epoch 2-Microplate Spectrophotometer).
Violacein inhibition on C. violaceum CV12472 TQ was subjected to qualitative analysis of QSI potential for its ability to inhibit violacein production by C. violaceum ATCC 12472 28 .Overnight grown cultures (10 μL) of C. violaceum (adjusted to 0.4 OD at 600 nm) were added to microtiter plates containing 200 μL of Luria-Bertani (LB) broth and incubated in the presence and absence of MIC and sub-MICs of TQ.After incubating at 30°C for 24 h, decreases in violacein pigment production were observed.The absorbance was read at 585 nm.All experiments were performed in at least triplicate.The percentage of inhibitions for all quorum sensing assays was calculated as previously described 29 .

Cell culture and MTT assay
Human alveolar adenocarcinoma cells A549 (ATCC) were cultured in Dulbecco's modified Eagle's medium (DMEM, Capricorn) supplemented with 10% fetal bovine serum (FBS, Sigma) and 1% penicillin-streptomycin (Multicell).The cells were maintained in a humidified atmosphere with 5% CO 2 at 37°C.The cell culture medium was refreshed every 48 hours, and subculturing of the cells was performed when they reached 80-90% confluence.
TQ extracted from N. sativa was prepared as a stock solution in dimethyl sulfoxide (DMSO).Different concentrations of TQ (125, 100, 50, and 31.25 μg/mL) were obtained by mixing it with the cell culture medium.
To evaluate the cytotoxicity of TQ, A549 cells were seeded in 96-well microtiter tissue culture plates at a density of 1 × 10 4 cells per well and incubated in complete medium for 24 hours.After 24 hours of incubation, the cells were treated with TQ, and the cytotoxicity was evaluated after 24 and 72 hours of further incubation using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, as previously described 30 .The MTT assay involves the reduction of the MTT dye by functional mitochondria in living cells, resulting in the formation of insoluble blue formazan crystals, which can be measured to assess cell viability.

Molecular docking
The crystallographic structure of Keap1:Nrf2 (Kelch-like ECH associated protein 1-nuclear factor (erythroid-derived 2)-like 2) was fetched from the RCSB protein data bank (PDB) ( https:// www.rcsb.org/).The crystal structure (PDB code: 2FLU) has a resolution of 1.5 Å 31 .Molecular docking was performed with AutoDock Vina as described in previous studies 32,33 .The results obtained from the docking were visualized and analyzed through Biovia Discovery Studio.

Molecular dynamics simulation
The stability of the protein-TQ complex from the docking was evaluated through MD simulation.The MD simulation was undertaken with GROMACS after the protein and TQ topologies were made ready by using the appropriate commands.The MD simulation was run and then the RMSD (root mean square deviation), RMSF (root mean square fluctuation), Rg (radius of gyration), and ligand hydrogen bond plots were drawn via qtgrace to analyze the computation results 34,35 .

Statistical analysis
All experiments were performed in triplicate, and the mean values with their corresponding standard deviations (SD) were reported.Statistical analysis was conducted using SPSS 19 software (SPSS Inc., Chicago, IL).The experimental data were evaluated using one-way ANOVA test, and a significance level of p<0.05 was considered statistically significant.

Results and Discussion
TQ isolation and characterization TQ was isolated from N. sativa essential oil (NSEO) by silica gel column chromatography.
The purity of the TQ crystals obtained from column chromatography was determined by quantification with HPLC equipment.The chromatogram of the TQ isolated from NSEO and chromatogram of standart TQ were presented in Fig. S1.The amount of TQ was determined as 918 mg/g in the HPLC assay analysis of the samples obtained by silica gel column chromatography.Namely, 91.8% of the isolated structure was TQ.The FTIR and DSC analyses confirmed the presence of TQ in the obtained structure.The infrared spectrum of the isolated TQ is shown in Fig. S2.Functional group analysis was carried out using FTIR to determine the presence of TQ isolated from NSEO, and the results were compared with the IR spectrum of TQ standard and the available literature.When the IR spectrum of the isolated TQ was examined, the characteristic strong stretching pattern of the carbonyl group of the cyclohexadiene was observed at a wavelength of 1658 cm -1 , which was supported by the values reported for TQ (1.648 cm -1 ) and 1,4 benzoquinone (1661 cm -1 ).The weak pattern observed at the high wavelength of ~3048 cm -1 was attributed to the stretching observed in the vinylic C-H in the C = C-H groups.The dense pattern present at 2968 cm -1 corresponded to the C-H stretch of the aliphatic groups.The 1460 cm -1 peak represented aliphatic C-H bending CH 2 .As can be understood from these data, the purified structure was TQ.Moreover, these data were consistent with those in the literature 37,38 .Fig. S3 represents the sharp endothermic peak obtained with DSC corresponding to the solidliquid transition with enthalpy ΔHm = 118.7 J/g change.When the DSC thermograms of the TQ were examined, it was seen that a sharp peak was only obtained at 43.86°C.In one study, change of enthalpy ΔHm = 110.6J/g, corresponding to solid/liquid transition of TQ was reported as while the melting point was found to be 43.55°C 38.Based on these data, it was confirmed that the isolated structure was TQ.
The 1 H-NMR spectrum of the isolated TQ molecule is presented in Fig. S4.When the 1 H-NMR spectrum of the isolated structure recorded in CHCl 3 solvent was examined, the protons in the methyl groups, which have the same chemical environment and are in the isopropyl structure, emerged as a doublet corresponding to 6 protons at 1.13 ppm.The -CH proton in the isopropyl structure showed a multiplet chemical shift value at 3.04 ppm.The methyl protons attached to the aromatic ring were observed as a singlet peak corresponding to 3 protons at 2.04 ppm.Finally, aromatic protons in the structure revealed a multiplet chemical shift value in the fig 1 range of 6.52-6.59ppm (Fig. S4).
Based on all these data, it was seen that the chemical shift values observed in the 1 H-NMR spectrum of the isolated structure were consistent with the chemical shift values observed in the 1 H-NMR spectrum of the standard TQ compound previously presented in the literatüre 39 .

CUPRAC assay of total antioxidant capacity
In recent years, many methods based on free radical scavenging have been developed to determine antioxidant capacity.Among these methods, the CUPRAC method is a straightforward and adaptable technique for measuring antioxidant capacity in various substances, such as dietary components, synthetic antioxidants, and vitamins C and E 24 .Assessing the antioxidant capacity of TQ using the CUPRAC method is significant.The CUPRAC reagent offers greater stability and accessibility compared to other chromogenic reagents such as ABTS and DPPH 40 .Moreover, the CUPRAC reagent is reasonably selective, stable, easily accessible, and sensitive toward thiol-type oxidants, unlike the FRAP method.The reaction is carried out at nearly physiological pH as opposed to the unrealistic acidic pH of FRAP 23 .Furthermore, it is advisable to evaluate the antioxidant properties of bioactive components using multiple methods that consider the reaction processes, selectivity, sensitivity, and applicability of the assays employed to measure their antioxidant capacity 23 .
The total antioxidant capacity of the isolated pure TQ compound was evaluated according to the CUPRAC method developed by Apak et al. 23 .The molar absorptivity (ε) of the isolated TQ was calculated from the line equation by constructing a concentration versus absorbance plot using the data in Fig. 1.The molar absorptivity of TQ was calculated to be 545.79L mol -1 .cm - (Fig. 1).In the literature, the molar absorption coefficient of Trolox was previously reported as 16700 L mol -1 .cm -1 according to the CUPRAC method 41 .The trolox equivalent antioxidant capacity (TEAC) is defined as the millimolar concentration of a Trolox solution that has an antioxidant capacity equivalent to a 1.0 mM solution of the substance under investigation.The TEAC CUPRAC values were simply calculated by dividing the molar absorptivity of compounds under investigation by that of the Trolox under corresponding conditions.This value was determined as 0.033.From these findings, it was understood that the TQ exhibited effective antioxidant activity.

Free radical scavenging (FRS) activity
The DPPH test is a reliable and practical approach for directly evaluating the ability of antioxidants to scavenge free radicals, as it is based on the ability of DPPH, a stable free radical, to lighten in their presence 2 .The DPPH is a stable free radical absorbing at 515 nm wavelength.Thus, it may be concluded that the antioxidant transfers its electron to DPPH, resulting in a reduction in the absorption of the DPPH radical solution at a wavelength of 515 nm 43 .BHT was chosen as the reference antioxidant for this assay.The % radical scavenging values of all TQ and BHT at different concentrations (25-100 µg/mL) are shown in Fig. 2. The scavenging effect of TQ and BHT on DPPH was calculated to be 77.32 and 55.6%, at 100 µg/mL concentration respectively.As concentrations of TQ and BHT increased, so did their ability to scavenge free radicals.The IC 50 values of TQ and BHT were determined by plotting the percentage of radical scavenging against the concentration.The data revealed that the scavenging activity of BHT with an IC 50 value of 48.27 µg/mL is better than the scavenging activity of TQ with an IC 50 value of 89.415 µg/mL.In the DPPH test, a lower IC 50 value indicates higher radical scavenging activity.

Anti-quorum sensing activity
Many Gram positive and Gram negative bacteria use quorum sensing, a general regulatory mechanism, to perceive and react to a variety of variables, including altering microbial population density and the expression of particular genes 44 .Some virulence factors such as pyocyanin, elastase production, biofilm formation in the P. aeruginosa PAO1 is under the control of the quorum sensing system.As a result, suppressing bacterial QS has emerged as a novel, potential antibacterial strategy that can not only stop the emergence of bacterial resistance but also stop the expression of genes related to population density in virulence factors 45 .
Antibiotic resistance in biofilm bacteria has been found to be substantially higher than in planktonic bacteria.Natural substance's ability to prevent cell adhesion is a potentially useful technique for minimizing microbial colonization on a variety of surfaces 46 .According to our tests results in sub-MIC concentration of TQ, different inhibition rate was seen on virulence factors of PAO1.22%, 28%, 34% rate have been found respectively for pyocyanin production, biofilm formation and elastase production.The rates of pyocyanin production, biofilm formation and elastase production were found to be 22%, 28% and 34%, respectively, and these results are statistically significant (Fig. 3A).
C. violaceum is also one of the bacteria that uses the QS system, and although it is rare, it is known to be responsible for severe sepsis 47 .In our study TQ inhibited violacein pigments (89%) at the sub-MIC concentrations of 0.09 mg/mL and was statistically significant (Fig. 3B).

Cell culture and MTT assay
It is crucial to develop new strategies for the treatment of lung cancer given its high mortality rate.In this context, recent attention has been focused on the chemotherapeutic effects of natural compounds.The seeds, oils and extracts of N. sativa have been used as an anticancer agent by Unani, Ayurveda and the Chinese medicine 48 .Our cytotoxicity data also revealed that TQ induced cytotoxicity of non-small lung cancer cells, in vitro.As seen in Fig. 4, it was determined that all TQ concentrations were statistically significant compared to control and DMSO groups for 24 and 72 hours, while 100 and 125 mg/mL TQ concentrations significantly reduced cell viability compared to other groups (p<0.001).It was also found that 50 mg/mL was significantly different from 31.25 mg/mL (p<0.05) for 24 hours.In human lung cancer A549, NCI-H460 and NCI-H146 cells, TQ was determined to induce apoptosis, inhibit cell cycle, and suppress cell viability, invasion and migration 49 .TQ also was found to induce DNA damage and inhibits the growth of lung cancer cells, breast cancer cells and prostate cancer cells 50 .In a study focused on the A549 lung cancer cell line, it was aimed to understand the impact of TQ on cell proliferation, migration, and invasion, as well as the underlying mechanisms contributing to its antimetastatic effects.The findings from this study revealed that TQ exhibited a dose-and time-dependent inhibition of A549 cell proliferation.This effect was substantiated by the downregulation of proliferation markers, such as PCNA (proliferating cell nuclear antigen) and cyclin D1, following treatment with TQ.Consequently, it was unequivocally established that TQ exerts a discernible antiproliferative impact in vitro 51 .
The cytotoxicity data, along with the findings from various cell line studies, demonstrate the ability of TQ to induce apoptosis, inhibit the cell cycle, and suppress cell viability, invasion, and migration in lung cancer cells.However, it is important to note that further preclinical and clinical studies are necessary to elucidate the underlying molecular mechanisms of TQ's anticancer effects and to evaluate its efficacy and safety in human lung cancer patients.Nonetheless, the accumulating evidence highlights the significance of natural compounds like TQ in the development of novel therapeutic strategies for combating lung cancer and warrants continued investigation in this field.

Molecular docking
In the wet-lab study, TQ was isolated and its antioxidant activity was determined.TQ was found to be a potent antioxidant agent.The binding potential of TQ to Keap1 was explored through computational methods to shed light on the possible mechanism for the antioxidant activity.The Keap1-Nrf2 regulatory pathway plays an important role in protecting cells against oxidative stress by activating cytoprotective gene transcription 52 .Compounds that bind to Keap1 and thus hinder the formation of the Keap1-Nrf2 complex increase the Nrf2 level, which result in a higher antioxidant role 53 .Therefore, it has been utilized as a target to elucidate the probable mechanism of action for antioxidant activities of phytocomponents of extracts [53][54][55] .
A crystallographic structural study revealed that Nrf2 interacted to Keap1 via various amino acids 31 .The interaction of Nrf2 to Keap1 was investigated through molecular docking to test the suitability of the protocol to be used.A time taking protein-protein docking was performed.The docking process exhibited that most of the interactions in the crystallographic study were observed in the docking study.The crystallographic study reported that the various amino acids of Nrf2 peptide interacted to Keap1 via diverse interaction residues 31 .Similarly, Nrf2 peptide formed interaction to Keap1 via diverse amino acid residues in the docking analysis.In addition to this, unfavorable intramolecular interactions between the amino acids was observed in the docking.The high similarity of interaction residues between the experimental and docking studies appeared to be validating the process whereas the high unfavorable interactions might destabilize the complex formed.This has necessitated the investigation of the stability of the Keap1-TQ complex through MD simulation to validate the docking process.
The binding analysis of TQ to Keap1 through molecular docking demonstrated a strong enough interaction to keep it inside the binding pocket of the protein.TQ interacted to the protein through four conventional hydrogen bonds (Ser363, Arg380, Asn382, Ser602) and a pi-pi interaction (Tyr334).Hydrogen bonding is critical in the binding of ligands to their target proteins and keeping them inside the binding pocket 56 .Four conventional hydrogen bonds were formed between TQ and Keap1 (Table 2, Fig. 5).The number of conventional hydrogen bonds are anticipated to be sufficient to form a stable Keap1-TQ complex.Furthermore, the stability of the Keap1-TQ complex was evaluated through MD simulation.
A crystallographic study reported that Nrf2 bound to Keap1 through various amino acid residues.The various amino acid residues of Nrf2 took part in this interaction.The study revealed that the backbone interaction of Nrf2 peptide to Keap1 took place through Tyr334, Asn382, Gln530, Ser555, and Ser602 residues.The side chain interaction of Nrf2 peptide took fig 5  place through Ser363, Arg380, Asn382, Arg415, Arg483, and Ser508 residues 31 .In the docking study, most of the backbone interaction residues of Nrf2 peptide (Tyr334, Asn382, Ser602) were found to take part in the binding of TQ to Keap1.
Similarly, half of the side chain interaction residues of the Nrf2 peptide (Ser363, Arg380, Asn382) were found to take part in the binding of TQ to the protein.The docking results implicated that TQ could bind to the Keap1 (Table 2, Fig. 5).As the interactions of TQ to the protein were mainly through conventional hydrogen bonds, it is expected to form a strong binding to the protein.This might hinder the binding of Nrf2 to the protein competitively.This will in turn increase the Nrf2 level.As a higher Nrf2 level is correlated to an antioxidant role, TQ might have brought its antioxidant effect by enhancing this pathway.

MD simulation
The stability of the Keap1-TQ complex procured from the docking was measured by MD simulation.
Thereafter, the stability of the complex was compared to the stability of the unbound Keap1 and then analyzed accordingly.RMSD, RMSF, Rg, and number of hydrogen bond plots were drawn.RMSD plot of the backbone structure in relative to reference protein is used to measure the stability of the structure during a simulation period 57 .In general, the Keap1-TQ complex and the unbound Keap1 protein gave similar RMSD values during the simulation period.They had a rising value in the first 5 ns, Thereafter, the two fig 6 structures achieved their stability and retain this relative stability with an average value of nearly 0.125 nm (1.25 Å) till the end of the simulation.The Keap1-TQ complex had relatively lower RMSD values in the 78-96 ns time interval (Fig. 6A).The RMSD plot demonstrated that the stability of the TQ containing complex did not change much with compound binding.It had a slightly higher stability than the unbound protein in some narrow time intervals.RMSF plot is used measure the per residue perturbations of a structure 58 .The general trend for the RMSF plots of the complex and unbound protein was found to be similar (Fig. 6B).The two structures had significant rise in the 378-390 residue interval and the N-terminal end of the protein.The complex gave a slightly higher RMSF value in the 445-449 and 538-543 residue intervals.The change in the compactness of a protein structure due to ligand binding is measured through Rg value 59 .In general, the Rg value of the complex and the unbound protein were similar with approximately 1.8 nm average.The complex had a relatively lower value in the 20-40 ns time interval whereas the unbound protein had a relatively lower value in the 58-67 ns time interval.They gave an overlapping Rg value in the remaining time.The two structures gave a stable Rg value that implicated stability in the stiffness of the structures (Fig. 6C).Hydrogen bonding is critical in the binding of ligands to their targets and keeping the ligand in the binding pocket.As a result, hydrogen bond number analysis is common in MD simulations 56 .The MD simulation exhibited that TQ formed mainly single hydrogen bond intercalated with two hydrogen bonds.In the docking study, TQ was found to form four conventional hydrogen bonds.Hence, there is discrepancy in the number of hydrogen bonds between the two methods (Fig. 5 and Fig. 6D).To sum up, the docking showed that the TQ had high binding potential to Keap1 and the MD simulation study revealed that the resulting complex was stable.

Conclusions
Data presented in this study clearly demonstrate the successful isolation of TQ from N. sativa seeds, as supported by HPLC, FTIR, DSC, and 1 H-NMR analyses.The analysis of the isolated TQ using the CUPRAC and DPPH assays revealed its remarkable effectiveness in terms of total antioxidant and free radical scavenging activity.Additionally, this study, for the first time, reported the molar absorptivity coefficient of isolated TQ from N. sativa seeds using the CUPRAC method.Also TQ showed antiquorum sensing activity against one of the significant and opportunistic pathogen P. aeruginosa.Besides this, TQ showed showed as strong inhibition of violacein production on C. violaceum CV12472.These findings hold significant applications for the food and pharmaceutical industry.Furthermore, the in vitro cytotoxicity results provide compelling evidence of TQ's potential as a therapeutic agent for the treatment of lung cancer.The molecular docking study demonstrated that the TQ could bind to Keap1.In this way, it could increase the Nrf2 presence by inhibiting its binding to Keap1 competitively.Therefore, the antioxidant effect observed could be correlated to this interaction.Furthermore, the MD simulation study of the Keap1-TQ complex implicated the formation of a stable complex by the docking.

Figure 5 .
Figure 5. 2D binding of TQ with Keap1and its position inside the binding pocket

Table 1 .
Chemical composition of N. sativa essential oil 19,20l, Compounds were listed in order of their elution from a Restek Rxi ® -5Sil MS column using a series ofthe standards of C 7 -C 30 saturated n-alkanes.RI lit , Retention index from the literature19,20

Table 2 .
Binding profile of TQ with Keap1