Medires Publishers - Article

Abstract

Numerous modifications may occur to the DNA structure caused by endogenous and exogenous hazardous factors initiating the progression of carcinogenesis. By evidence, a huge part of malignancies have been established as being preventable. Twelve, eight, and seven constituents of each hazelnut, walnut and almond, respectively, were designed as DNA minor groove binders. Computational predictions using Swiss ADME software that assessed ADMET properties and drug-likeness were used. Almonds were sourced, cleaned, sliced, air-dried, grinded, and macerated with absolute methanol for 48 hours. Extracts were filtered, stored, refrigerated, and subjected to a competitive displacement assay with Ethidium Bromide (EB) and Rhodamine B (RB). The results reveal almond extracts with the highest key selection vector (KSV), indicating strong affinity for EB-bound DNA and RB-bound DNA. This study highlights nuts as potential sources of bioactive compounds with geno-protective properties, contributing to natural product-based drug discovery.

Introduction

Geno-protective refers to protective actions that prevent damage to the genetic material (DNA) within cells. In the context of cancer, geno-protective strategies aim to minimize DNA damage, which can lead to malignancy. These strategies can include synthetic, natural, or biological agents to inhibit cancer development by either preventing DNA damage or blocking the division of premalignant cells with DNA damage[i].  Healthy cells have developed methods to deactivate free radicals or carcinogenic substances in order to avoid DNA damage. Conversely, the existence of disparities or inadequacies within DNA-repair mechanisms may be associated with the initiation, progression, and evolution of carcinogenic processes. Hence, the human environment and eating habits are pivotal factors in the development of cancer.

Free radicals, like ROS (Reactive Oxygen Species) and RNS (Reactive Nitrogen Species) are continually created in living cells by metabolism and environment. As signalling molecules, ROS and

RNS production is genetically designed to protect the body and reduce inflammation. If their overproduction exceeds the cell's antioxidant capacity, these radicals oxidize protein and lipid cell elements and damage the DNA. ROS oxidize the DNA directly and disrupt many DNA repair processes, leading to DNA chain breakage, base modification, and other forms of oxidative DNA damage. ROS scavenging is a crucial antioxidant mechanism in cancer treatment that aims to decrease tumour development and the survival of cancer cells. This may be accomplished by the use of various plant-derived phytochemicals. ROS may be eliminated by enzymatic or non-enzymatic processes, including the use of glutathione (GSH), thioredoxin (Trx), superoxide dismutase (SOD), catalase (CAT), and/or peroxidases.

Thus, timely administration of antioxidant compounds is necessary to protect and maintain DNA and other cell components, prevent and/or regulate carcinogenesis, decrease tumour development, and promote overall health. Multiple medications are utilized to treat tumors; however, gene alterations may reduce the efficacy of synthetic therapies. Thus, nutraceuticals that may reverse oxidative stress, rebuild non-tumour tissue, and kill cancer cells are gaining popularity. Despite the rising number of ailments in our society, plants are a significant source of therapeutic compounds that are being extensively studied and used in treatment. Plant-derived natural compounds have garnered significant scientific attention for their anticancer properties. For example, there is data indicating an association between increased intake of foods rich in phytochemicals and a reduced chance of developing cancer[ii].

Furthermore, phytochemicals may effectively prevent the growth of cancer cells by ensuring the integrity of the genome[iii]. Healthy cells have developed methods to deactivate free radicals or carcinogenic substances to avoid DNA damage[iv]. Imbalances or deficiencies in DNA-repair cascades may be linked to the start, advancement, and development of carcinogenesis[v]. Plant-based nuts contain choline, magnesium, potassium, zinc, selenium, folic acid, dietary fibers, polyunsaturated and monounsaturated fatty acids that work together to make nuts an essential component of a healthy diet[vi]. In addition, nuts have antioxidant properties that have been associated to a decrease in inflammation, oxidative stress, and metabolic damage caused by a number of diseases[vii]. Additionally, phenolic chemicals found in nuts may provide protection against malignancies including breast, prostate, and colon cancers by lowering cell proliferation, inducing apoptosis, and avoiding DNA damage caused by free radicals, radiation, or chemical carcinogens[viii].In general, nuts tend to slow cell growth, speed up cell death, and strengthen the body's antioxidant enzyme system[ix]. This work, for the first time, is groundbreaking because it employs computational and experimental methodologies to investigate the geno-protective effects on DNA in three different types of nuts: hazelnuts, walnuts, and almonds. Some malignancies may be resistant to the nut varieties of these plants.

Anticancer drugs have the ability to interact with DNA via three primary mechanisms: Firstly, they interact with DNA-bound proteins to regulate transcription factors and polymerases. Secondly, they interfere with transcriptional activity. Lastly, they can bind to DNA double helical structures either covalently or non-covalently through small aromatic molecules[x]. The irreversible covalent interaction between some drugs and DNA leads to the complete suppression of DNA activities, ultimately causing the death of cells. One of the primary benefits of covalent bonds is their strong binding. Nevertheless, the presence of covalent bulky adducts has the capability to distort the DNA backbone, resulting in the full suppression of DNA functions and consequent demise of cells. Typically, DNA-alkylating medicines serve as examples of covalent interactions. Alternatively, there are three primary methods for small molecules to bind reversibly with ds-DNA: static electronic interactions, groove binding, and intercalation The static electronic interactions, especially the binding of cationic molecules to the anionic sugar phosphate backbone of DNA via the outside edge, may be responsible for the exterior surface binding. The second way of interaction is groove binding, where the molecule is inserted into the margins of either the minor groove or major groove. Typically, G•C sites are found in the major groove, whereas A•T sites are found in the minor grooves.

The major groove has many locations for interaction and has dimensions of 8.5 Å in depth and 11.6 Å in breadth. Therefore, large molecules may readily enter the major groove. In contrast, the minor groove, with a depth of 8.2 Å, is smaller and has fewer binding sites. However, it has the benefit of usually being unoccupied, which makes it more susceptible to assault by tiny drug molecules. Binding to the minor groove is the most frequent occurrence.

The third way of interaction is intercalation, which is closely associated with the anticancer medicines being used in clinical practice. In this case, the tiny molecule has the ability to interact with DNA via either a single mode of binding or a combination of several binding modes.

This work will specifically examine DNA minor grooves, as discussed by Wilson et al. (2013)[xi].  By studying the binding modes, binding constants, and structural changes induced by the drugs on DNA, researchers aim to enhance the selectivity and efficacy of anticancer treatments[xii]. Similarly, the development of new drugs with improved antitumor efficacy and reduced side effects relies on a deep understanding of drug-DNA interactions[xiii].

Just as researchers investigate the intricate relationship between drugs and DNA in the context of cancer treatment, exploring the potential health benefits of hazelnuts, walnuts and almonds could provide valuable insights into their interactions with DNA and their impact on human health[xiv].

Materials And Methods

Hazelnuts, walnuts, and almonds were sourced from the local Libyan market, thoroughly washed to ensure cleanliness, and then sliced into thin pieces. These slices were air-dried for two days to effectively remove moisture content. Once dried, the nut slices were ground into a fine powder using an electric blender, with each type of nut processed separately to prevent cross-contamination and ensure sample integrity. The bioactive compounds from the hazelnut, walnut, and almond powders were extracted using the maceration technique. Two grams of each nut powder were accurately weighed and placed in separate flasks, to which 5 millilitres (ml) of absolute methanol was added as an extraction solvent. The flasks were sealed and kept at room temperature, with the mixtures subjected to intermittent agitation over 48 hours to enhance the efficiency of bioactive compound extraction. After 48 hours, the mixtures were filtered using appropriate filtration apparatus to separate the liquid extract from the solid residue. The filtered extracts were collected in clean beakers and stored in a refrigerator at 4°C to preserve their integrity until further use in subsequent experiments.

DNA Binding Properties.

To study how competently our synthesized compounds interact with G-DNA (Genomic-DNA), the compounds were investigated for their DNA binding ability using fluorescence emission spectroscopy. All experiments were conducted in Tris buffer (0.01M Tris, 0.1M NaCl, at pH 7.4), glass-distilled deionized water and analytical grade reagents were used throughout experiments. The pH values of all solutions were measured with a calibrated Jenway pH-meter model 3510 (Stafford shire, UK). All buffer solutions were filtered through Millipore filters (Millipore, UK) of 0.45 mm pore diameter.

Fluorescence spectra and DNA-binding studies using EthidiumBromide.

Fluorescence spectra and DNA-binding studies Fluorescence emission and excitation spectra were measured using Jasco FP-6200 spectrofluorometer (Tokyo, Japan) using fluorescence 4-sided quartz cuvettes of 1.00 cm path length. The automatic shutter-on function was used to minimize photo bleaching of the sample. The selected excitation wavelength for ethidium bromide was 480nm. The emission spectrum was corrected for background fluorescence of the buffer. The ethidium bromide (EB) fluorescence displacement experiment of three nuts that contain: system (A) the G-DNA-ethidium bromide complex, system (B) the G-DNA-ethidium bromide complex with 7 ng/µl hazelnut, system (C) the G-DNA-ethidium bromide complex with 7 ng/µl walnut, system (D) the G-DNA-ethidium bromide complex with 7 ng/µl almond and system (E) ethidium bromide alone. The experiment was conducted in Tris buffer solution (0.01M Tris, 0.1M NaCl, at pH 7.4). Emission spectra were recorded for each system using excitation wavelengths of maximum fluorescence intensity determined for the systems to be 480 nm using a slit width of 5 nm to examine alterations in emission spectra resulting from the complex construction of both systems. On construction of the full systems, the system was allowed to equilibrate for 30 minutes at room temperature and emission spectra (500 –730 nm) were recorded to monitor changes in EB intensity.

Fluorescence spectra and DNA-binding studies using Rhodamine B.

Fluorescence emission and excitation spectra were measured using Jasco FP-6200 spectrofluorometer (Tokyo, Japan) using fluorescence 4-sided quartz cuvettes of 1.00 cm path length. The automatic shutter-on function was used to minimise photo bleaching of the sample. System 2 (Black) formation was induced by sequential addition of 1990 μl 0.01M Tris buffer, 10 μl RB (0.24 μM), and 0.1M NaCl at pH 7.4. System 3 (Blue) formation was induced by sequential addition of aliquots of 1890 μl 0.01M Tris buffer, 10 μl RB (0.24 μM), 0.1M NaCl at pH 7.4 and 100 μlct-DNA (75 μg/mL). System 1 (Red) formation was induced by sequential addition of aliquots of 1850 μl 0.01M Tris buffer, 10 μl RB (0.24 μM), and 0.1M NaCl at pH 7.4, 100 μlct-DNA (75 μg/mL) and 40 μl hazelnut (final concentration 7 ng/μl). Emission spectra were recorded for each system using excitation wavelengths of maximum fluorescence intensity determined for the systems to be from 550 nm using a slit width of 5 nm to examine alterations in emission spectra resulting from the complex construction of both systems. On construction of the full systems, the system was allowed to equilibrate for 2-5 minutes at room temperature and emission spectra (560-650 nm) were recorded to monitor changes in RB intensity.

Molecular Docking Analysis

To identify a possible binding mechanism, compounds comprising four B-DNA (B form) segments from hazelnut, walnut, and almond were molecularly docked using Auto Dock vina in the PyRx Virtual Screening Tool[i].

The molecular docking research was performed to evaluate the binding affinity of the most active derivatives to DNA duplex and to define the binding interactions by combining and optimizing parameters such as hydrophobic, steric, and electrostatic complementarily. It may be deduced that the compounds were successfully inserted into the DNA minor groove.

The molecular docking investigations showed that the compounds made important contributions through hydrogen bonding, Pi-Pi stacking, and hydrophobic interactions. These results highlight their potential as effective minor groove binders of DNA.

Optimization of DNA Molecules

The crystal structures of B-DNA fragments with (PDB IDs: 1BNA, 1D60, 1ZEW, and 1DCO) were downloaded from the Protein Data Bank (PDB) website (https://www.rcsb.org/). These structures were analysed for their active sites using Biovia Discovery Studio Visualizer, available at (http://accelrys.com).

Subsequently, AutoDock 1.5.6 software (https://autodock.scripps.edu/) was employed to refine the imported DNA coordinates. This involved several steps: removal of unwanted water molecules, addition of polar hydrogen atoms, and assignment of Kollman charges. The refined structures were then saved in PDBQT format. Finally, the processed files were used as input in PyRx software (https://pyrx.sourceforge.io/) for further analysis and molecular docking studies.

Docking Process

The B-DNA crystal structures were imported in PDBQT format into the PyRx virtual screening program (PDB IDs: 1BNA, 1D60, 1ZEW, and 1DCO). Standard medications and specially created substances were also loaded and instantly converted to PDBQT format. PyRx'sAutoDock Vina integration was used for docking. The dimensions of the grid box were established at 25 × 25 × 25 Å, and the centres were automatically determined. Complexes with the lowest binding energies (kcal/mol) were selected following docking. Discovery Studio was then used to show the interaction modes, characteristics and Lipinski rules were selected as ligands.

Results and Discussion

In this part of the study, four B-DNA fragments (1BNA, 1D60, 1ZEW, and 1DCO) were molecularly docked with compounds from hazelnut, walnut, and almond in the PyRx Virtual Screening Tool's Auto Dock vina to identify a possible binding mechanism. The molecular docking research was performed to evaluate the binding affinity of the most active derivatives to the DNA duplex and to define the binding interactions by combining and optimizing parameters such as hydrophobic, steric, and electrostatic complementarily.

Since studies indicate that Taxol is a minor groove binder, Paclitaxol was selected as the reference compound in this experiment. Paclitaxel (Taxol) is derived from the bark of the Pacific yew and Taxus brevifolia. Because it binds to tubulin and prevents microtubule depolymerization during cell division, it has been used in the treatment of cancer, especially ovarian, breast, and lung cancer[i]. Through this method, cancer cells are prevented from growing further by "locking up" during the G2/M phase of the cell cycle.

Taxol isbeneficial for certain types of solid tumors that exhibit rapid cellular proliferation, as it inhibits the process of cell division[ii]. Additionally, some data suggested that Taxol may bind to DNA because it functions as a minor groove binder in DNA[iii]. Similar to the taxol–tubulin interaction hypothesis, the connection takes place in the DNA groove via the eight-membered taxane core ring, with the three phenyl rings facing away from the DNA[iv].

Based on the free binding energy values presented in Tables 1, 2, and 3, it can be inferred that the chemicals were effectively positioned within the DNA minor groove. The compounds exhibited significant contributions through hydrophobic interactions, Pi-Pi stacking, and hydrogen bonding, as indicated by the molecular docking studies. These findings underscore their potential as promising DNA minor groove bind.

Table 1: Different energies are used in the molecular docking procedure to bind the components of hazelnut constituents.   (1D60): D(CCAACNTTGG)2, (1ZEW): D(CCTCTAGAGG)2,(1BNA): D(CGCGAATTCGCG)2, 1DCO, ΔGa is the binding freeenergy change in the binding process.

Table 2: Different energies are used in the molecular docking procedure to bind the components of walnut constituents.   (1D60): D(CCAACNTTGG)2, (1ZEW): D(CCTCTAGAGG)2, (1BNA): D(CGCGAATTCGCG)2, 1DCO, ΔGa is the binding free energy change in the binding process.

Table 3: Different energies are used in the molecular docking procedure to bind the components of almond constituents.   (1D60): D(CCAACNTTGG)2, (1ZEW): D(CCTCTAGAGG)2, (1BNA): D(CGCGAATTCGCG)2, 1DCO, ΔGa is the binding free energy change in the binding process. 

The molecular docking study evaluated the binding affinities of various constituents derived from hazelnuts, walnuts, and almonds against four DNA fragments (1D60, 1ZEW, 1BNA, and 1DCO). These results were benchmarked against the standard minor groove agent, Paclitoxin, with docking energies measured in kcal/mol. The more negative the numerical values for the binding affinity, the better is the predicted binding between a ligand and a macromolecule.

Among the hazelnut constituents, HconLinlA exhibited the highest binding affinity, with docking energies of -5.6, -6.1, -5.2, -5.8 kcal/mol across the four DNA fragments, respectively. However, these values are significantly weaker compared to those of

the reference drug, Paclitoxin, which showed docking energies of -8.0, -7.6, -7.8, -9.0 kcal/mol, respectively. Thus, while hazelnut constituents are biologically active, their binding affinities are notably lower than those of the standard minor groove agent. In contrast, almond constituents demonstrated stronger binding affinities.

The compound Aproanth showed particularly high affinities, with docking energies of -8.7, -9.2, -8.4, -7.8 kcal/mol, respectively. These values are very close to those of Paclitoxin, indicating that Aproanth from almonds has a binding strength greater than this standard drug, particularly against the DNA fragments 1D60, 1ZEW and 1BNA. Other almond constituents, such as Acrypt and Adauci, also exhibited high binding affinities, further highlighting the potential of almond-derived constituents as effective DNA minor groove agent.

Walnut constituents also displayed significant binding affinities, with WhyperinA showing docking energies of -8.1, -9.1, -7.3, -7.8 kcal/mol, respectively. These values are comparable to those of Aproanth from almonds and are close to the binding affinities of the reference drug, particularly against the DNA fragments 1D60 and 1DNA.

Other walnut compounds, such as WellaA and Wkaempferol, demonstrated strong binding affinities, but their values were slightly less consistent across all DNA fragments compared to the almond compound Aproanth.

In conclusion, among the nuts studied, almond constituents exhibited the best overall binding affinities. Specifically, the compound Aproanth from almonds showed binding affinities closest to the standard drug, Paclitaxol, across the tested DNA fragments. Every component of hazelnuts, with the exception of HlinnA, which does not make hydrogen bonds with DNA, may readily fit into the active site. The elements of hazelnuts that form the greatest number of hydrogen bonds are Hlign, HlinA, and HpalolA. Similarly, every component of almonds, with the exception of Atoco, which cannot make any hydrogen bonds with DNA, may readily fit into the active site.Aaden and Aproanth, which can create eight and nine hydrogen bonds with different nucleosides at the active site, respectively, are the almond constituents with the highest hydrogen bond formation capacity.

Likewise, the components of walnuts that have the greatest potential to create hydrogen bonds are WellaA and Wkaempferol with the ability of WellaA to form six hydrogen bonds.

Ethidium Bromide (EB) (indirect assay)

The use of a noncompetitive test provided more information on the binding pattern of the studied medicines and DNA. Ethidium bromide (EB), a potent fluorescent dye, is recognized for its ability to bind to DNA by intercalation[i]. At its highest emission, when it attaches to double-stranded DNA, it emits light with a wavelength of 620 nm. The light emitted by unbound ethidium bromide is rather low. Nevertheless, the addition of DNA results in its insertion between the base pairs, leading to a significant augmentation in fluorescence intensity[ii]. (Zhao et al., 2013).

Binding Processes

Intercalation: Ethidium bromide intercalates between the base pairs of the DNA double helix, causing distortion to the DNA structure. This mechanism induces the separation of the base pairs, resulting in a small unwinding of the DNA.

Minor Groove Interaction: The internal dye (EtBr) is expelled from the DNA as a consequence of the nut constituent's binding to the minor groove by altering the DNA's three-dimensional structure. In other words, changes to the external surroundings of DNA will lead to modifications inside the molecule.

Effect on DNA Conformation

Several factors influence the orientation of DNA and ethidium bromide in binding studies. The DNA sequence affects binding affinity by providing favourable sites for intercalation or groove binding, while the conformation of DNA (e.g., B-DNA, A-DNA, Z-DNA) influences how well molecules can intercalate or bind, with B-DNA being the most common form under physiological conditions. Ionic strength and pH affect electrostatic interactions, where high ionic strength can reduce binding affinity. Temperature plays a role in binding kinetics and thermodynamics, potentially increasing the binding rate while destabilizing DNA at higher temperatures. Additionally, solvent conditions, including the presence of organic solvents or additives, can modify DNA structure or alter binding molecule properties, impacting binding interactions.

Fig 1: Fluorescence changes of the three nuts system that contains (A) the G-DNA-ethidium bromide complex, (B) the G-DNA-ethidium bromide complex with 7 ng/µl hazelnut (C) the G-DNA-ethidium bromide complex with 7 ng/µl walnut (D) the G-DNA-ethidium bromide complex with 7 ng/µl almond and (E) ethidium bromide alone. The experiment was conducted in Tris buffer solution (0.01M Tris, 0.1M NaCl, at pH 7.4), λex = 480 nm.

In the fluorescence emission plot Figure 1 of the G-DNA-ethidium bromide complex with various nut extracts, the spectral data reveal distinct changes in fluorescence intensity and peak wavelength, indicating interactions between the components. The reference spectrum (A), representing the G-DNA-ethidium bromide complex, exhibits a peak fluorescence around 620 nm with the highest intensity among all spectra.

The addition of hazelnut extract (B) results in a hypochromatic shift, as evidenced by a decrease in fluorescence intensity. Similarly, the walnut extract (C) induces both a hypochromatic shift and a blue shift, resulting in a greater decrease in fluorescence to approximately 40 a.u. The almond extract (D) follows a similar pattern, showing a greater reduction in fluorescence intensity.

 In contrast, ethidium bromide alone (E) demonstrates a significant hypochromatic shift and a pronounced blue shift, with the peak wavelength observed around 595 nm.

The fluorescence intensity for almond (37.00 a.u.) is lower than that for hazelnut (44.00 a.u.) and walnut (40.00 a.u.), indicating a greater hypochromatic shift. This suggests a stronger interaction between the almond extract and the G-DNA-ethidium bromide complex, leading to more significant decrease of fluorescence.

KSV Values

The KSV value for almond (39.0 ± 0.31 ng/µl) is higher than that for hazelnut (9.70 ± 0.21 ng/µl) and walnut (25.0 ± 0.12 ng/µl). Higher KSV values indicate stronger binding affinity of the almond extract to the G-DNA-ethidium bromide complex, resulting in greater fluorescence quenching.

The Stern-Volmer equation was used to analyze the fluorescence quenching data, and the compounds were identified as the quenchers in the calculation of the quenching constants (Ksv):

I0/I = 1 + KSV [Q] (1)

I0 and I represent the fluorescence intensities in the absence and presence of quencher, respectively; KSV is a linear Stern-Volmer quenching constant; Q is the concentration of quencher. The KSV values were given by the ratio of the slope to intercept.

The KSV value suggests a strong affinity of the compounds to EB-bound G-DNA and that it can competitively displace EB from DNA via minor groove mode of binding. As shown in Table 4, almond had the highest KSV value.

Table 4: The key selection vector (KSV) values of hazelnut, walnut and almond with Ethidium Bromide

The calculated percentages of dye removal for each nut provide insight into their respective interactions with the G-DNA-ethidium bromide complex. The hazelnut extract resulted in a 6.38% reduction in fluorescence intensity, indicating a relatively modest level of dye removal. In contrast, the walnut extract demonstrated a more substantial effect, with a 14.89% reduction in fluorescence intensity, reflecting a stronger interaction with the complex. Most notably, the almond extract exhibited the highest level of dye removal, with a 21.28% reduction in fluorescence intensity. This significant decrease suggests that almond has the greatest binding affinity to the G-DNA-ethidium bromide complex, leading to the most pronounced quenching of fluorescence. Therefore, based on the percentage of dye removal, almond stands out as having the strongest impact on the fluorescence properties of the G-DNA-ethidium bromide complex, followed by walnut then hazelnut.

Rhodamine B (direct assay)

Rhodamine B, a fluorescent dye, loses its fluorescence when it interacts with DNA due to various factors. Initially, it has the ability to create non-fluorescent compounds with DNA, which is referred to as static quenching. Furthermore, the excited state of the molecule may be deactivated by its interaction with DNA, a phenomenon referred to as dynamic quenching. Rhodamine B exhibits affinity for the minor groove of DNA, leading to alterations in its surroundings and a decrease in its fluorescence[i].

By adding nut components at the appropriate stoichiometric concentration, Rhodamine B may be dissociated from the DNA, resulting in an increase in its fluorescence. As more extract is introduced, it will interact with the unbound Rhodamine B and hence suppress its fluorescence. Zhao et al. (2013) and Dehghani et al. (2020) elucidated the processes by which polyphenols modify fluorescence of Rhodamin B.

Figure 4: Plotting fluorescence emission from 562 - 650 nm vs wavelength using excitation λ 550 nm of: 2 (BLACK)- 10 µl RB (0.24 µM) and 1990 µl 0.01M Tris, 0.1M NaCl at pH 7.4. 3(BLUE)-10 µl RB (0.24 µM), 1890 µl 0.01M Tris, 0.1M NaCl at pH 7.4 and 100 µl ctDNA (75 µg/ml).1(RED)-10µl RB (0.24 µM), 1850 µl 0.01M Tris, 0.1M NaCl at pH 7.4, 100 µl ctDNA (75 µg/ml) and 40 µl hazelnut (final concentration 7 ng/µl).

The fluorescence emission plot in Figure 4 illustrates the interaction of rhodamine B (RB) with DNA and the effect of the extract on this interaction. Initially, the black line represents the baseline fluorescence of RB in a buffer solution. When DNA is added, as shown by the blue line, the fluorescence intensity decreases due to the quenching effect. This happens because RB binds to the DNA, reducing the amount of free RB in the solution. Upon adding the extract, depicted by the red line, the fluorescence intensity increases compared to the blue line. This increase occurs because the extract competes with RB forDNA binding sites. As the extract binds to the DNA, it displaces the RB, thereby increasing the concentration of free RB in the solution, which leads to enhanced fluorescence.

As more extract is added, the competition for DNA binding sites intensifies, resulting in more RB being displaced and thus higher fluorescence. However, when the concentration of the extract reaches a stoichiometric point where it fully saturates the DNA binding sites, adding more extract will no longer increase the free RB concentration. Instead, at this saturation point, the fluorescence intensity reaches a peak in which when additional extract is added beyond this saturation point, it could potentially lead to non-specific interactions or quenching effects, where the excess extract itself may begin to quench the fluorescence or interfere with the RB in other ways, eventually causing the fluorescence to decrease and potentially drop to zero. This behaviour demonstrates the balance between competitive binding and the saturation point of the DNA binding sites, highlighting the dynamic interaction between RB, DNA, and the extract.

Table 5: The key selection vector (KSV) values of hazelnut, walnut and almond with Rhodamine

The calculated percentages of dye elimination for each nut provide vital insights into their distinct interactions with the ctDNA-rhodamine B complex. The hazelnut extract exhibited the smallest decrease in fluorescence intensity when compared to the other types of nuts, resulting in a drop of 7.79%.

Thisreduction indicates a modest amount of dye removal. Similarly, like ethidium bromide, walnut exhibited significant dye elimination, leading to a 19.48% decrease in fluorescence intensity. Almond had the most significant dye clearance effect, resulting in a 28.57% decrease in fluorescence intensity. This indicates that almond has the greatest binding affinity among the three nuts mentioned in this study.

Conclusion

Based on the fluorescence quenching study and molecular docking results, almond constituents demonstrate the strongest binding affinity to DNA among the nuts examined. Almonds showed significant potential as effective DNA minor groove binders with substantial geno-protective properties, outperforming both walnut and hazelnutconstituents.

Our results highlight almonds as a promising source of bioactive compounds for DNA protection and benefits science by advancing geno-protective drug development and enhance the understanding of nut-derived bioactive compounds.

By identifying bioactive compounds from hazelnuts, walnuts, and almonds that bind to the DNA minor groove, it offers insights into the mechanism of action of natural geno-protective agents, potentially leading to new cancer therapeutics.

Lastly,the comprehensive analysis of these nuts' chemical compositions supports their health benefits and encourages further natural product-based drug discovery.

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