1. INTRODUCTION

Cancer is defined by abnormal and unregulated cellular proliferation. The fundamental distinction between mature normal tissue and tumor is in the rate of cellular growth. In most normal tissues, the proliferation rate is equivalent to the cell death rate, however in tumor cells; the proliferation rate surpasses the death rate¹⁴. Unregulated cell proliferation, resistance to natural apoptosis, cellular immortality, evasion of the immune system, and the accumulation of genetic mutations are primary factors contributing to tumor formation. Mutations in proto-oncogenes and tumor suppressor genes mostly result in tumorigenesis¹. The tumor primarily consists of two types: benign tumors and malignant tumors. The terms cancer, malignant neoplasm, and malignant tumor are synonymous; they are differentiated from benign tumors by their characteristics of dedifferentiation, invasiveness, and capacity for metastasis. Metastasis is the dissemination of rapidly proliferating tumor cells to numerous organs, inducing healthy cells to undergo uncontrolled mitosis. The phrase metastasis was first utilized by Jean Claude Recamier. The "seed and soil theory," proposed by scientist Stephen Paget, posits that tumor cells disseminate across numerous organs of the body in a manner analogous to the dispersion of seeds in soil. The International Agency for Research on Cancer (IARC) asserts that both HBV and HCV are capable of inducing cancer in humans²⁶. Liver cancer is among the most prevalent cancers globally. The liver consists of hepatocytes and various other cells, including blood arteries and bile ducts. Hepatocytes are implicated in both malignant and benign cancers. Cancer originating in the liver is termed primary liver cancer. Cancer predominantly manifests in the liver but typically originates elsewhere in the body; this occurrence is termed secondary liver cancer. In Europe and the United States, metastatic liver cancers are more widespread than primary liver cancer, conversely in Asia and Africa, primary liver cancer is more common²³. Thus, there is a necessity for secure, economical, and effective therapies for the condition. The objective of this study was to assess the cytotoxic effects of ethanolic extracts from AM and OM on the HepG2 liver cancer cell line in-vitro ²¹.

This study focuses on the manufacture of gold nanoparticles loaded with medicinal plant extracts using green chemistry methods to reduce the adverse effects of cancer medications⁵. The concept of nanoparticles is regarded as having a more significant and diverse range of applications due to their unique physical, chemical, and magnetic properties, thereby attracting considerable interest, particularly in biomedical science, where the use of nanovaccines and nanodrugs is being extensively explored. Medicinal plant extract is utilized as a drug carrier²⁷. In India, the traditional practice of utilizing medicinal plants stretches back 4,000 years. Anethum graveolens L. is an annual aromatic plant with branching morphology, recognized for its culinary applications since antiquity. The Apiaceae family comprises species that are recognized as significant sources of various important herbal products. The foliage, fruits, and volatile oil are widely utilized in culinary and medicinal applications¹⁰. This multipurpose herb has a history of association with the production of perfumery, insecticides, and traditional Iranian medicine. This ingredient is utilized in gripe water to alleviate colic pain in infants and reduce flatulence in young children. It functions as a carminative, an aromatic agent, and an antispasmodic. Dill essential oil exhibits hypolipidemic activity and may serve as a cardio-protective agent by reducing cholesterol levels. This substance functions as an antispasmodic agent, anticonvulsant, anti-emetic, and anticramp remedy for children, and is also recommended for topical application as a wound healer. Dill fruits are a substantial source of several metabolites, such as volatile oils, coumarins, flavonoids, phenolic acids, fatty oils, and minerals. Floral extracts comprise chlorogenic acid, diverse flavonoids including myricetin, proanthocyanidins, and other phenolic substances that enhance antioxidant action. The composition of steam-distilled dill essential oil has been analysed. Fungitoxic peptides have been isolated from seed sources. Dill has been reported to possess anticancer, antioxidant and antiplasmodic properties².

2. MATERIAL AND METHODS:

2.1. Collection, Authentication and Extraction of plant material:

Plants were taken from the local green market in Prayagraj, U.P., and validated by the Botanical Survey of India, Allahabad, under reference number 2803250003118. The plant material was drying and powdered. Plant extract was obtained by boiling 10g of powdered material in 100ml of deionized water for 10 minutes and filtered using Whatmann filter paper.

2.2. Preliminary phytochemical Screening:

A comprehensive phytochemical analysis of the plant extracts was carried out to confirm the presence of alkaloids, phenols, flavonoids and tannins⁴. To verify the role of antioxidants in the synthesised AuNPs,in-vitro quantitative assay of plant extract and plant extract loaded AuNPs was performed and estimating the total flavonoid content (TFC), total phenolic content (TPC) and antioxidant assay such as DPPH, and FRAP (Fig:1) ¹³.

2.3. Synthesis of gold nanoparticles:

AuNPs were synthesized by adding aqueous plant extract of Anethum graveolens (AG) with an aqueous auric chloride solution (0.5mM) at specific volume ratios of 10ml of aqueous plant extract and 90ml of 0.5mM auric chloride aqueous solution, initiate the bioreduction of Au³⁺ ions and adjust the pH of the mixture to a suitable range (~ 7 to ~11) and left this suspension to stand for overnight in dark medium at room temperature. The formation of AuNPs was shown by a visible color change and confirmed using UV-Visible spectroscopy. Suspension was centrifuged at high-speed (17,000rpm for 30minutes) purify the resulting AuNPs with fresh deionised water and dried in vacuum drier¹⁵. The possible mechanism for the synthesis of gold nanoparticles is reduction of auric chloride (HAuCl₃) gold salt (Au³⁺) to gold nanoparticles (Au⁰). According to this mechanism the OH and C=O groups of plant extract polyphenols, flavonol derivatives and other bioactive molecules of extract bind with (Au³⁺) to form gold complex and then reduced to (Au⁰). this possible mechanism shown in (Fig.2) ¹⁹.

Fig. 2: possible mechanism for synthesis of AuNPs

2.4. Characterization of Synthesized Nanoparticles:

Synthesised nanoparticles were evaluated using ultraviolet (UV) spectrophotometry, Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and Zetasizer analysis. UV spectroscopic analyses revealed a distinct UV-Visible peak, indicating the formation of AG-AuNPs. FT-IR and XRD techniques were employed to ascertain the surface characteristics (presence of phytoconstituents) and crystalline structure of AG-AuNPs, respectively. The SEM and Zetasizer analyses indicated a particle size ²⁷.

2.5. Estimationof Total flavonoid content (TFC):

The total flavonoid content (TFC) of various components, including leaves, stems, and bark, was quantified using the aluminium chloride assay via colorimetry. A 0.5ml aliquot of extracts was placed into separate test tubes, to which 2 ml of distilled water was added, followed by the introduction of 0.15ml of sodium nitrite (5% NaNO₂, w/v). The mixture was allowed to stand for 6 minutes. Subsequently, 0.15ml of aluminium trichloride (10% AlCl₃) was added and incubated for 6 minutes, following which 2 ml of sodium hydroxide (NaOH, 4% w/v) was incorporated, and the total volume was adjusted to 5ml with distilled water. Following a 15-minute incubation, the combination exhibits a pink hue, with absorbance recorded at 510nm utilising a colorimeter. Distilled water functioned as the control sample. The total flavonoid content (TFC) was quantified in mg of catechin equivalents (CE) per gramme of extract.

2.6. Estimation of Total Phenolic Content (TPC):

The total phenolic content (TPC) of the crude extracts from leaves, stem, and bark was assessed using the methodology established by Singleton.After some changes, 0.5ml of the test sample and 1.5ml of Folin-Ciocalteau reagent (diluted 1:10 v/v with distilled water) were combined and permitted to stand for 5minutes at 22ºC. After five minutes, 2.0ml of 7.5% sodium carbonate was added. The mixes were incubated in the absence of light for 90 minutes with periodic agitation. Following incubation, a blue colouration was observed. The absorbance of blue pigment in several samples was measured at 725nm using a colorimeter. The phenolic content was quantified in gallic acid equivalents (GAE/g) based on the standard curve of gallic acid. The results were presented as Gallic acid equivalents (GAE) per gramme of plant material²⁴.

2.7. Diphenylpicryl Hydrazine (DPPH) Antioxidant assay:

The antioxidant efficacy of the plant extract and synthesized gold nanoparticles was evaluated utilizing the DPPH method. This method demonstrates a significant purple absorption peak at 517nm attributable to the unpaired electron of the DPPH free radical. The hue shifts from purple to yellow as the unpaired electron of the DPPH radical engages with hydrogen from an antioxidant that neutralizes free radicals, leading to less DPPH (diphenyl picryl hydrazine). The ability of various materials to donate hydrogen or scavenge radicals was assessed using the stable DPPH radical. 0.75mL of methanolic extract solution at concentrations between 1 and 500μg mL^-1 was combined with 1.5mL of a DPPH methanolic solution (20mg L^-1). After 20 minutes of processing, the absorbance of methanol was assessed at 517 nm. The decoloration was graphed versus the concentration of the sample extract, yielding a logarithmic regression curve, from which the IC50 value was determined. IC50 represents the concentration of a sample required to reduce the absorbance of DPPH by 50%. The findings are expressed as antiradical efficiency (AE), which is defined as the inverse of the IC50 value multiplied by 1000 (AE = 1000/IC50)²⁵.

% Decoloration = [1- ()] x 100

2.8. Ferric Redusing Antioxidant Power (FRAP) Assay:

The ferric tripyridyltriazine [Fe (III)-TPTZ] complex necessitates reduction by a reductant at low pH to produce ferrous TPTZ (Fe (II)-TPTZ) for the FRAP assay.The newly formulated FRAP working solution comprises 25 milliliters of acetate buffer (1), 2.5 milliliters of TPTZ solution, and 2.5 milliliters of Fe (III) chloride × 6H2O solution. Aqueous solutions with specified Fe (II) concentrations between 100 and 1000 μmol/l were utilized for calibration. Four milliliters of FRAP reagent were incorporated into the methanol suspension of the nanoparticles for subsequent analysis. The assay quantification was determined using the optical density derived from the Fe (II)-SO4 regression curve. The resulting value was expressed in μm/l/g, signifying the quantity necessary to convert Fe+3 to Fe+2 7.

2.9. In-Vitro Cytotoxicity Evaluation of the Compounds-HepG2:

The cytotoxic profiles of the test compounds were rigorously evaluated against the HepG2 human hepatocellular carcinoma cell line, procured from the National Centre for Cell Science (NCCS), Pune. For this assessment, 1×10⁴ HepG2 cells per well were seeded into 96-well microtiter plates and allowed to adhere for 24hours. Cell culture was maintained in Dulbecco's Modified Eagle Medium (DMEM-AT149-1L), comprehensively supplemented with 10% Fetal Bovine Serum (FBS-HIMEDIA-RM 10432) and 1% antibiotic solution, under strictly controlled conditions of 37°C and 5% CO₂ in a humidified incubator. Following the initial 24-hour incubation, the adherent cell monolayers were exposed to a gradient of test compound concentrations, as precisely detailed in the accompanying data spreadsheet. Untreated cells served as the experimental control group. After an additional 24hour incubation period with the test substances, the cell viability was quantified utilizing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A predetermined concentration of MTT solution (specified in the spreadsheet) was introduced into each well and incubated for 2hours. Upon completion of the reaction, the culture supernatant was carefully aspirated, and the insoluble formazan crystals, indicative of metabolically active cells, were solubilized with 100μL of dimethyl sulfoxide (DMSO). Absorbance was subsequently measured at dual wavelengths of 540nm and 660nm using an iMark microplate reader (Bio-Rad, USA). IC50 values, calculated through non-linear regression analysis utilizing Graph Pad Prism 9 software⁶.

2.10. Molecular docking studies:

Molecular docking studies predict the preferred orientation of a ligand (the compound) when bound to a protein (receptor), providing insights into their binding affinity and interaction mechanisms. Docking was performed free docking tools Auto dock 4.2. Docking was performed by drawing the all ligand in chemdraw 12.00 and minimised the energy and save in PDB formate. All the compound was iport to auto dock and performed docking on targeted protein (PDB ID- 1TNF) refers to the crystal structure of Tumor Necrosis Factor-alpha (TNF-α). A lower (more negative) docking score indicates a stronger and more favorable binding.

Target Protein:

· Historical Context: The structure was deposited on August 25, 1989, and released on January 15, 1990. The primary publication detailing the structure is "The structure of tumor necrosis factor-alpha at 2.6 A resolution. Implications for receptor binding" by Eck MJ and Sprang SR (1989). Protein sequence is reported (Fig.14)

· Function and Biology: TNF-α is a multifunctional proinflammatory cytokine primarily secreted by macrophages, but also by other immune cells. It plays a crucial role in the immune response and inflammation. It binds to its receptors, TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR, to trigger signalling pathways that can induce cell death in certain tumour cell lines, cause fever, and influence cell proliferation and differentiation.

Structure:

· Resolution and Method: The structure was determined by X-ray diffraction at a resolution of 2.6 Å.

· Oligomeric State: It exists as a homotrimer, meaning it is composed of three identical protein chains (Chains A, B, and C). The individual monomers are inactive.

· Monomer Details: Each monomer consists of 157 amino acids and has a theoretical weight of 17.37 KDa.

· Topology: The monomer forms an elongated, antiparallel beta-pleated sheet sandwich with a "jelly-roll" topology. This structure shows striking homology to certain viral coat proteins.

· Binding Site: The general site of interaction with the receptor is suggested to be at the "base" of the trimer.

· Source Organism: The protein is from Homo sapiens (human).

· Clinical Relevance: Excessive TNF production is implicated in various inflammatory diseases, including rheumatoid arthritis, psoriasis, and inflammatory bowel disease. Drugs that inhibit TNF from binding to its receptors are used to treat these conditions.

3. RESULT AND DISCUSSION:

The botanical material was carefully gathered and processed for extraction. A sustainable method was utilised to synthesise gold nanoparticles. The resultant nanoparticles were thoroughly characterised using various analytical techniques, including UV-Vis spectroscopy, Fourier-transform infrared spectroscopy (FT-IR), and scanning electron microscopy (SEM). These investigations confirmed the production of the nanoparticles and provided precise insights into their size, shape, and surface properties.

3.1. Phytochemical screening:

Preliminary phytochemical screening confirmed the presence of alkaloids, glycosides, saponin, phenolic compounds, flavonoid and carbohydrates in arial part plant extract.The standard Molisch test was utilized for detecting carbohydrates while Shinoda test successfully identified flavonoids The Dragendorff's test confirmed the presence of alkaloids. Additionally, the Foam test validated the existence of Saponins, Bontrager’s Test was detecting glycosides, and the Silver mirror test provide conclusive evidence of the presence of phenolic components.

3.2 UV-Vis spectroscopy:

(Fig.3a) and (Fig.3b) meticulously delineate the UV-Visible spectroscopic profiles of both the raw plant extract and the synthesized Anethum graveolens mediated gold nanoparticles (AG-AuNPs). The absorption spectra of the AG-AuNPs conspicuously exhibited a characteristic surface plasmon resonance (SPR) band within the 500-600 nm range, with a definitive λmax observed at 538 nm, unequivocally confirming gold nanoparticle genesis ⁸.

Fig. 3: a) UV-Vis spectroscopy of AG plant extract, b) synthesized AG-AuNPs

3.3. Fourier-transform infrared spectroscopy (FTIR):

FTIT spectra of Anethum graveolens plant extract and their gold nanoparticles AG-AuNPs were used to find potential interactions between gold nanoparticles (AuNPs) and biomolecules.Alcohols and phenols present in large concentrations are characterized by a band at 3277.88 cm⁻¹, whereas a band at 2910.67cm^-1 and 2842.92 cm^-1 typically corresponds to CH stretching vibrations, which may contribute to the stabilization of the nanoparticles peaks at 1010.37cm^-1 range may reveal carbonyl (C=O) stretching vibrations. IR spectra of AuNPs synthesized utilizing all sections of Anethum graveolens is attributed to the O–H group from alcohols (Fig. 4a)²⁰.

Fig. 4: a) FTIR spectrum of synthesized AG-AuNPs

3.4.XRD pattern of the crystalline AG-AuNPs:

In the X-ray diffraction pattern of Anethum graveolens plant extract (nλ= 2dsinθ) attributed to the presence of crystalline carbon (Fig.5a). The particles' crystalline nature is shown via X-ray diffraction. In essence, the occurrence of specific phase relations between two or more waves causes diffraction by Bragg‟s reflection from (111), (200), (220), (222) and (311) FCC (face center cubic) crystal structure planes prove the crystalline nature of synthesized AG-AuNPs ²⁹.

3.5. Size distribution and surface charge characterization (zeta potential):

(Fig. 5b) show the Zeta potential value of synthesized gold nanoparticles (AG-AuNPs) was found to be -20.38 mV are considered stable nanoparticles, negative zeta potential of synthesized AG-AuNPs can beascribed to the capping by anionic AG. Accordingly it was understood that AG-AuNPs was highly stable. The decrease in the zeta potential can be attributed to the stable nanoparticles ³.

Fig. 5: a) XRD spectrum, b) ZETA potential spectrum of synthesized AG-AuNPs

3.6. Scanning electron microscope (SEM):

The surface morphology and elemental analysis of Au-GNPs were conducted using SEM combined with EDX. Fig.6 (a) presents SEM images of AG-AuNPs, revealing that they possess a spherical morphology and exhibit a limited size distribution. Fig. 6 (b) illustrates the histogram of the scanning electron microscope image pertaining to particle size. Fig. 6 (c) illustrates the designated area for EDX analysis of AG-AuNPs, whereas Fig. 6 (d) presents the SEM-EDX results indicating the presence of gold (Au). EDX further confirms the presence of pure elemental gold by exhibiting distinct peaks characteristic of gold metal at 2.10 keV. The FE-SEM analysis of AG-AuNPs indicates that they possess a spherical morphology. The EDX spectrum exhibited a characteristic peak related to Au, confirming the presence of gold in the sample ¹¹.

Fig. 6: a) SEM image, b) Histogram, c) selected area for SEM-EDAX d), SEM-EDAX for presence of gold

3.7. Effect of pH on Anethum graveolens Loaded Gold Nanoparticles (AG-AuNPs):

(Fig. 7) showing that the pH of polyphenol contents in AG plant extract is a crucial determinant in the reduction of Au³⁺ ions to metallic AG-AuNPs. For determination of the influence of pH on the synthesis of gold nanoparticles from plant extract, 25 mL of 0.5 mM auric chloride solution was mixed with 10 mL of 10 g. L⁻¹ polyphenol solution at a pH of approximately 5 or a pH of approximately 10 (adjusted by the addition of 0.1 M NaOH solution). Within five minutes, the colloidal suspension in an alkaline medium transitioned from yellow to red wine colour. In an acidic solution (pH~5), the color shift from yellow to purple transpires gradually, requiring about 6 hours. The formation of this intermediate complex can explain the accelerated AG-AuNPs production rates at pH ~ 10 ¹².

Fig. 7: Effect of p^H on formation of AuNPs

3.8. Effect of polyphenol concentration:

It was observed that the concentration of polyphenol of plant extract is crucial for the synthesis of AG-AuNPs from controlled microstructure gold salts and for the colloidal stabilization of these particles in aqueous media in an alkaline medium. In our studies, we looked into the impact of polyphenol concentration at pH~10. A constant volume of auric chloride (25 mL, 1 mM) was mixed with 15-18 mL of a polyphenol solution dropwise while being magnetically stirred. After that, the mixture was incubated for 30-35 minutes at the range of 25°C. As the concentration of polyphenol increases, it is noted that at concentrations up to 10 g. L⁻¹, it produces highly stable colloidal suspensions whose colour varies from yellow to reddish wine. A reddish precipitate forms along with the colour shift upon polyphenol addition, over 10 g. L⁻¹⁹.

3.9. Estimation of TFC and TPC:

Total flavonoid content was observed to be 44.88 mg/g of quercitin equivalents (QE)/g of dry extract calculated from (Fig. 8a) and phenolic content was found to be 64 mg/g of gallic acid equivalents (GAE)/g of dry extract from (Fig. 8b) ^16,17.

Fig. 8: a) Standard calibration graph of Gallic acid (GA), b) Quercetin (QE)

3.10. FRAP and DPPH assay:

FRAP assay of the plant extract was found to be 46 μg/ml and 78 μg/ml was found for AG-AuNPs equivalent to Ascorbic acid. it was observed that using FRAP assay the synthesized nanoparticles showed best activity at 1mg/ml in terms of reduction of Fe⁺³ to Fe⁺²in antioxidant activity ²².

Fig. 9: Standard calibration graph of Ascorbic acid for the estimation of FRAP assay

In antioxidants using DPPH assay the results showed good result with the IC50 value was 23.36 accounting for AG-AuNPs 58.33 for the plant extract, having high potential in eliminating free radicals. In the present investigation DPPH assay showed optimum free radical scavenging activity ascorbic acid was present as standard.

Fig.10: Percentage inhibition graph of AG plant extract & synthesized AG-AuNPs

3.11. In-vitro analysis of Anethum graveolens loaded Au-NPs agains HepG2 cell lines:

The results indicated that the synthesised AuNPs exhibited an IC50 value of 113.9 μg/ml, signifying their ability to effectively suppress the proliferation of HepG2 cells at this dose (Fig. 11). The extracted IC50 value was determined to be 155.5 μg/ml (Fig. 12). The results highlight that the cytotoxicity of extract-loaded AuNPs against the HepG2 liver cancer cell line is more powerful than that of the pure plant extract. The concentration-dependent impact of AuNPs on HepG2 cells was apparent, indicating that diminished concentrations lead to less cell growth inhibition, whilst elevated concentrations may provide a more significant effect ¹⁹.

Fig 11: Representation of % cell viability Vs concentration plot of AG-AuNPs

To evaluate the cytotoxic activity of gold nanoparticles of Anethum graveolens aerial parts extracts were incubated with different concentrations as (50,100,250,500 and 1000 µg/ml), after 24 hours of incubation, cell viability was determined by the MTT assay.

Fig. 12: Representation of % cell viability Vs concentration plot of AG extract

The results of good cytotoxicity assay are presented in (Fig.11 and 12). The gold nanoparticles of aerial parts extracts were able to inhibit the proliferation of the cancer cells (HepG2)¹⁸.

4. Docking Study result:

Quercetin exhibits multiple binding poses within the active site, each characterized by a unique docking score, cavity volume, and spatial orientation. These different poses indicate the various ways Quercetin can interact with the target protein, with lower (more negative) docking scores indicating a more stable and favorable binding.

Here are the details of Quercetin's poses:

· Quercetin shows the most favorable docking score of -9.4, within a cavity volume of 1049 Å3. The center of this docking is at (8, 63, 31) with a size of (21, 21, 21). This pose suggests the strongest binding affinity.

The importance of analyzing these poses lies in understanding the flexibility of the ligand (Quercetin) and the protein, and how different binding orientations can influence the efficacy and specificity of the interaction. A compound like Quercetin, which shows multiple favorable poses, suggests that it can bind to the target in various stable configurations, potentially leading to diverse biological effects or a more robust interaction. The most favorable pose (C1 in this case) typically represents the most probable binding mode and is crucial for further experimental validation and drug design. These interactions occur with specific amino acid residues within the protein chains, including CYS69, LYS98, SER99, PRO100, CYS101, GLN102, ARG103, GLU104, LYS112, PRO113, TRP114, TYR115, and GLU116 across different chains.

Targeted Protein:

PDB ID 1TNF refers to the crystal structure of Tumor Necrosis Factor-alpha (TNF-α).

Fig. 13: structure of protein

Sequence [1]

Fig.14: Protein sequence

Fig.15: docking poses of ligands

Table 2: the overall docking scores for the compounds analyzed are summarized below:

Name of Compound	Docking Score
Quercetine	-9.4
Cafeic acid	-6.6
Ferulic acid	-6.4
Umbeliferone	-6.1
Carvon	-5.5

From these results, Quercetine exhibits the most favorable docking score of -9.4, suggesting it has the strongest binding affinity among the tested compounds to the target protein.

Quercetine's Binding Poses and Amino Acid Interactions:

Quercetine, as the most promising compound, shows multiple binding poses within the active site of the protein. Each pose is characterized by distinct parameters, highlighting the flexibility of its interaction.

Table 3: Detailed Quercetine Poses

Pose ID	Docking Score	Cavity Volume (Å³)	Center (x, y, z)	Docking Size (x, y, z)
C1	-9.4	1049	8, 63, 31	21, 21, 21
C4	-9.0	222	1, 71, 36	21, 21, 21
C5	-6.5	220	36, 64, 41	21, 21, 21
C3	-6.2	608	13, 29, 42	21, 21, 21
C2	-5.9	731	22, 52, 59	21, 21, 21

The C1 pose of Quercetine displays the lowest (most favorable) docking score, indicating it is the most stable and probable binding mode. This pose occupies a large cavity volume of 1049 Å3.

Amino Acid Residues Interacting with Quercetine:

Quercetine interacts with specific amino acid residues across different chains of the protein. These interactions are crucial for its binding stability and biological effect:

· Chain A: CYS69, LYS98, SER99, PRO100, CYS101, GLN102, ARG103, GLU104, LYS112, PRO113, TRP114, TYR115, GLU116

· Chain B: CYS69, LYS98, SER99, PRO100, CYS101, GLN102, ARG103, GLU104, TRP114, TYR115, GLU116

· Chain C: CYS69, HIS73, LYS98, SER99, PRO100, CYS101, GLN102, ARG103, GLU104, TRP114, TYR115, GLU116, PRO117

These interactions, particularly with residues like CYS69, LYS98, and GLU104, are vital for anchoring Quercetine within the binding site.

This image showcases the docking pose of Quercetine (shown in black and white sticks) within the protein's binding site. The surrounding transparent surfaces indicate the shape and volume of the binding pocket. The colors in the protein ribbons (teal, green, red, grey) differentiate various regions of the protein structure, illustrating how Quercetine sits within the folded chains. Dashed lines often represent hydrogen bonds or other crucial interactions between the ligand and the amino acid residues in the protein, contributing to the overall docking score. The bars labeled "H-Bonds," "Donor," and "Acceptor" likely refer to a color-coded legend (though not fully visible here) indicating the nature of these specific interactions.

Importance of Analyzing Poses:

The analysis of multiple binding poses, like those identified for Quercetine, is critical for several reasons:

1. Ligand and Protein Flexibility: Different poses reveal the various ways a ligand can interact with the target protein, reflecting the inherent flexibility of both the ligand and the protein's binding site.

2. Diverse Biological Effects: A compound exhibiting multiple favorable poses suggests it can bind in various stable configurations, potentially leading to diverse biological effects or a more robust interaction with the target.

3. Probable Binding Mode: The most favorable pose (like Quercetine's C1) typically represents the most probable binding mode. This information is invaluable for further experimental validation, guiding subsequent drug design, and understanding the molecular mechanism of action.

4. Optimizing Interactions: Understanding the specific amino acid residues involved in these interactions helps in optimizing the compound's structure to enhance binding affinity, selectivity, and potency ³⁰.

CONCLUSIONS:

Based on the evaluation of green synthesis of gold nanoparticles: characterization and anticancer activity using Anethum graveolens aerial parts extracts with molecular docking approach of active constituents against liver cancer, it can be concluded that they have promising anticancer potential. Interactions of quercetin group particularly with residues like CYS69, LYS98, and GLU104, are vital for anchoring Quercetine within the binding site indicateted to produce good affinity and may be produce biolocal activity. So, it can further optimised for development of new molecule.

ACKNOWLEDGEMENTS:

Authors would like to acknowledge the Department of Pharmaceutical chemistry and Pharmacognosy, Shambhunath Institute of Pharmacy, Jhalwa, Prayagraj, U.P., India-211012, for providing facilities to complete this study.

CONFLICTS OF INTEREST/COMPETING INTERESTS:

The authors declare no competing interest.

CODE AVAILABILITY:

AUTHORS' CONTRIBUTIONS:

Conceptualization: Rohit Tripathi, Arvind Kumar Srivastava; Data collection: Vikash Kumar Mehta,Writing the manuscript: Vikash Kumar, Rohit Tripathi, Sketching of figures and data interpretation: Rohit Tripathi, Arvind Kumar Srivastava,Sudheer Kumar; Review and final editing of the manuscript: Rohit Tripathi,Rajkeshwar Prasad

Funding information:

Abbreviations:

FT-IR	Fourier-transform infrared spectroscopy
HepG2	Human liver cancer cell lines
AG-AuNPs	Anethum graveolens-gold nano particles
SEM	Scanning electron microscopy

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Received on 03.07.2025 Revised on 21.07.2025

Accepted on 05.08.2025 Published on 12.08.2025

Available online from August 18, 2025

Asian J. Research Chem.2025; 18(4):217-227.

DOI: 10.52711/0974-4150.2025.00034

This work is licensed under a Creative Commons Attribution-Non Commercial-Share Alike 4.0 International License. Creative Commons License.

_{Name of compound}	_{3D interaction}	_{2D interaction}	_{Docking score}
_Quercetine			_-9.4
_{Caffeic acid}			_-6.6
_Umbeliferone			_-6.1
_Carvon			_-5.5
_{Ferulic acid}			_-6.4