Method for verifying bacteriostatic efficiency of plant essential oil on paper cultural relics based on molecular simulation

Abstract

The present application relates to the cross field of paper cultural relics green preservation and computational biology, and discloses a method for verifying the paper cultural relics decay inhibition efficiency of plant essential oil components based on molecular simulation. The present application constructs a multi-pathway synergistic evaluation system covering four types of microbial key targets, including cell wall / membrane structure, nucleic acid synthesis and function, colonization and adhesion, and energy metabolism. Quantitative indexes such as binding free energy, hydrogen bond, and van der Waals force are obtained through molecular docking simulation, and the cross-pathway bacteriostatic potential of plant essential oil components is comprehensively evaluated. The method constructs a multi-pathway synergistic evaluation system covering four types of microbial key targets, establishes a standardized judgment process combining quantitative molecular docking indexes, realizes rapid and low-cost prediction of the cross-pathway synergistic potential of bacteriostatic agents, and has the potential for industrialization and popularization.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of paper artifact preservation and microbial control, specifically to a method for verifying the inhibitory effect of plant essential oil components on the decomposition of paper artifacts based on molecular simulation. Background Technology

[0002] As a core carrier of human civilization's memory, paper artifacts face a major threat to their long-term preservation from the decomposing effects of environmental microorganisms. Under the specific temperature and humidity conditions of museums and archives, the cellulose and hemicellulose in the paper matrix readily transform into nutrients for decay-causing microorganisms such as Aspergillus niger, Aspergillus flavus, and Pseudomonas aeruginosa. It is noteworthy that the erosion of paper artifacts by these microorganisms is not a single-pathway biochemical reaction, but a complex process involving multiple systems working together: the decay-causing bacteria must first maintain their stability through cell membranes or cell walls and invade the paper fibers, utilizing metabolic pathways to obtain energy for growth and reproduction, achieving firm adhesion to the paper surface with the help of colonization-related proteins, resisting environmental stress through antioxidant systems, and finally secreting cellulase-degrading enzymes to destroy the paper structure, leading to irreversible physical damage such as embrittlement, adhesion, and mold formation.

[0003] However, existing paper artifact preservation technologies have significant limitations in addressing this complex decay mechanism. Traditional chemical fumigants (such as formaldehyde and ethylene oxide), while possessing broad-spectrum bactericidal capabilities, are highly toxic and prone to causing secondary damage, seriously threatening the health of operators. Inorganic antibacterial materials, limited by poor stability and insufficient compatibility with the artifact itself, struggle to achieve long-term effective protection. Plant essential oils, as highly promising green preservatives, have been proven to inhibit various decay-causing fungi, but their development is currently hampered by long experimental cycles and high costs. More critically, existing research is largely limited to evaluating the inhibitory effects on single targets (such as cellulase alone), severely neglecting the multi-pathway synergistic characteristics of fungal decay, leading to reduced protective efficacy of screened essential oil molecules in practical applications.

[0004] Furthermore, although molecular simulation technology, as a core tool of computational biology, can theoretically quickly elucidate the binding patterns and affinities of small molecules and biological macromolecules, in practical applications in the field of paper artifact preservation, this technology is currently still mainly at the initial stage of single-target screening. It lacks both a systematic target classification for the entire pathway of microbial decay of paper artifacts and a verification system for the synergistic effects of plant essential oil molecules and multiple target categories.

[0005] Therefore, developing a standardized, molecular simulation-based method for verifying the antibacterial efficacy of plant essential oils targeting multiple categories can shorten the experimental cycle and reduce experimental costs by visually verifying the antibacterial mechanism of plant essential oil components at the molecular level. This will significantly promote the development of green preservation technology for paper cultural relics. Summary of the Invention

[0006] The purpose of this invention is to provide a method for verifying the inhibitory efficacy of plant essential oil components on the decomposition of paper artifacts based on molecular simulation. Targeting the multi-pathway synergistic decay characteristics of microorganisms that degrade paper artifacts, a synergistic evaluation system is constructed covering four key targets: cell wall / membrane structure, nucleic acid synthesis and function, colonization and attachment, and energy metabolism. The cross-pathway antibacterial potential of essential oil molecules is comprehensively evaluated through quantitative molecular docking indicators (binding energy, hydrogen bonds, van der Waals forces, etc.). This method achieves rapid, green, and low-cost pre-assessment of antibacterial efficacy without the need for precious artifact samples, and is suitable for industrial screening and the development of environmentally friendly artifact preservation agents.

[0007] The technical solution adopted in this invention is as follows: A method for verifying the antibacterial efficacy of plant essential oils on paper cultural relics based on molecular simulation, the steps of which are:

[0008] Step 1) Obtain the amino acid sequences and structural models of key target proteins of four types of microorganisms that cause the deterioration of paper cultural relics (four types: cell wall / membrane structure, nucleic acid synthesis and function, colonization and attachment, and energy metabolism) through Uniprot and PDB databases. All of them are involved in key pathways of paper cultural relic decay. Further, retrieve the molecular structure of plant essential oils through PubChem database, convert them to .pdb format using OpenBabel software, and then perform energy minimization optimization.

[0009] The key microorganisms mentioned are Pseudomonas aeruginosa, Candida albicans, Aspergillus niger, and Escherichia coli.

[0010] The selected plant essential oil molecules for verification are one or more of the following: hexanal, octanal, decanal, nonanal, lauronal, trans-2-hexenal, cinnamaldehyde, citral, perillaldehyde, trans-2-decenal, carvacrol, thymol, eugenol, isoeugenol, sesamol, linalool, geraniol, cherry alcohol, n-octanol, and citronellol.

[0011] Step 2) Preprocess the target protein model from Step 1): Simplify the protein structure in Pymol software and save it as a .pdb file. Further preprocess the above protein and essential oil small molecules in docking software (i.e., hydrogenation, protonation, and energy minimization).

[0012] Step 3) Perform molecular docking simulations between the plant essential oil component small molecules obtained in Step 2) and target protein models of different functional categories. A semi-flexible docking mode is adopted, in which the target protein is rigid and the plant essential oil component small molecules are flexible. A periodic cubic search space is set on the surface of the target protein. The minimum boundary distance of the search space is not less than 5 Å. Perform multiple independent docking operations on each protein-ligand combination to obtain multiple candidate binding conformations.

[0013] Step 4) Perform cluster analysis on each group of docking conformations in Step 3), remove redundant conformations, select the binding conformation with the best binding score, and construct four types of protein-essential oil molecule complex models.

[0014] Step 5) First, use three-dimensional visualization to identify key residues in the complex model from Step 4), and then use two-dimensional interaction diagrams to classify the interaction types, so as to intuitively present the interaction mode between essential oil molecules and targets.

[0015] Step 6) Based on the multi-target synergistic evaluation system, the cross-pathway synergistic antibacterial potential of plant essential oil molecules is determined by combining the docking binding energy (ΔG), the number and length of hydrogen bonds, the van der Waals force contribution value, and the number of key residues in hydrophobic interactions of each target.

[0016] Step 7) By comprehensively analyzing the binding results of target proteins of different functional categories through preset cross-pathway determination logic, the multi-target synergistic antibacterial potential of plant essential oil components is determined.

[0017] To achieve effective conversion from simulated data to antibacterial efficacy, this invention establishes a set of quantitative antibacterial efficacy judgment rules, which are based on the synergistic satisfaction of the following multi-dimensional parameters:

[0018] (1) Energy stability threshold: The binding free energy (ΔG) is the core indicator for evaluating the binding stability. Setting ΔG≤-5.5 Kcal / mol as the baseline for strong binding indicates that the binding process is highly spontaneous in terms of energy.

[0019] (2) Key interaction requirements:

[0020] 1) Hydrogen bond network: It is required to form at least one stable hydrogen bond (bond length is usually between 2.5-3.5 Å), which is the key to ensuring binding specificity and directionality.

[0021] 2) Scale of hydrophobic interaction: The number of amino acid residues involved in the hydrophobic interaction must be ≥3, indicating that the ligand can penetrate into the hydrophobic pocket of the protein and the binding mode is stable.

[0022] 3) Spatial geometric constraints: The distance between the plant essential oil component and the key functional site residues in the active region of the target protein must be ≤4.0 Å to ensure that it can directly interfere with the normal function of the protein.

[0023] Furthermore, the cross-pathway synergistic determination logic is as follows: only when the same plant essential oil component simultaneously satisfies at least one of the above-mentioned energy thresholds and other interaction and spatial conditions on at least two different functional target proteins is it determined to possess cross-pathway synergistic antibacterial potential. This determination logic overcomes the limitations of single-target evaluation and predicts at the molecular level the ability of essential oil components to synergistically inhibit microbial putrefaction processes through multiple mechanisms.

[0024] The beneficial effects of this invention are as follows:

[0025] 1. This invention constructs a multi-pathway synergistic verification system covering microbial cell wall / membrane structure, nucleic acid synthesis and function, colonization and attachment, and energy metabolism. It quantitatively evaluates the cross-pathway antibacterial potential of plant essential oil components through multi-target comprehensive evaluation, overcoming the problem of insufficient antibacterial efficacy caused by neglecting the multi-pathway synergistic putrefactive mechanism of microorganisms in traditional single-target screening methods.

[0026] 2. This invention combines quantitative indicators such as the calculated binding energy value (ΔG), the number and length of hydrogen bonds, and the contribution value of van der Waals forces to systematically evaluate the binding strength and specificity of essential oil molecules to multiple target sites at the molecular level, providing intuitive and reliable multi-dimensional data support for antibacterial mechanisms.

[0027] 3. This invention eliminates the need for physical experiments on precious cultural relics samples. Through pure computational simulation, it can quickly predict the actual efficacy and synergistic mechanism of antibacterial agents, significantly reducing R&D costs and the risk of damage to cultural relics. It provides an efficient, environmentally friendly, and standardized pre-evaluation tool for the industrial screening and application of green preservatives for paper cultural relics.

[0028] 4. This method enables quantitative prediction of the antibacterial potential of essential oil components across pathways at the molecular level, thereby significantly shortening the research and development cycle of cultural relic preservatives, reducing experimental costs, and avoiding direct damage to precious cultural relic samples, thus achieving a green, standardized, and industrially scalable cultural relic protection solution. Attached Figure Description

[0029] Figure 1 shows the 2D structure and 3D ball-and-stick model of carvacrol;

[0030] Figure 2 is a flowchart of the verification of the antibacterial efficacy of plant essential oil components based on molecular simulation;

[0031] Figure 3 shows the 2D and 3D interactions based on microbial cell membrane / wall structure and functional targets;

[0032] Figure 4 shows the 2D and 3D interactions between microbial nucleic acid synthesis and functional targets;

[0033] Figure 5 shows the 2D and 3D interaction diagrams based on microbial colonization targets;

[0034] Figure 6 shows the 2D and 3D interaction diagrams based on microbial energy metabolism activity targets. Detailed Implementation

[0035] The technical solutions in the embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The following embodiments are applicable to the present invention, but are not intended to limit the scope of the invention.

[0036] Example 1

[0037] A method for verifying the inhibitory effect of plant essential oil components on the decomposition of paper cultural relics based on molecular simulation includes the following steps (see...). Figure 1 ).

[0038] 1. Amino acid sequences and structural models of key target proteins of four types of microorganisms that cause the deterioration of paper cultural relics were obtained from the Uniprot and PDB databases (four types: cell wall / membrane structure, nucleic acid synthesis and function, colonization and attachment, and energy metabolism), all of which are involved in key pathways of decay of paper cultural relics; the structure of carvacrol was further retrieved from the PubChem database, and after being converted to .pdb format by OpenBabel software, energy minimization optimization was performed.

[0039] Figure 1 The 2D structure and 3D ball-and-stick model of carvacrol, a common component of plant essential oils, were obtained, and its antibacterial efficacy will be verified in the future.

[0040] 2. Preprocess the target protein model in step 1): After simplifying the protein structure in Pymol software to only mol A to improve the binding accuracy of the ligand, save it as a .pdb file. Perform Clean operation, add hydrogen atoms, and adjust the protonation state in the docking software to simulate physiological pH conditions (pH 7.4). Use the steepest descent method to minimize energy.

[0041] 3. The carvacrol pretreated in step 2) was imported into the docking software with the four target protein models respectively. The semi-flexible docking mode was adopted (the protein is rigid and the essential oil molecule is flexible). The periodic cubic box was set (minimum distance 5 Å). Each group was run independently for 25 dockings. The binding conformation was optimized by using a genetic algorithm.

[0042] Table 1 Selection of target proteins for key pathways

[0043]

[0044] Through literature review, key pathways and target proteins were selected (see Table 1). Specifically, key targets targeting the structure and function of the cell wall and cell membrane are: sterol 14-α demethylase (CYP51) (PDB: 5 TZ1) and chitin deacetylase (PDB: 7 BLY); key targets targeting nucleic acid synthesis and function are: dihydrofolate reductase (PDB: 1 AI9); key targets targeting the colonization and attachment of microorganisms on paper artifacts are: transcription activator protein LasR (PDB: 4 NG2) and protein FimH (5 JCR); and key targets targeting energy metabolism are: polycopper oxidase (PDB: 5 LM8) and chloride peroxidase (PDB: 7 RST).

[0045] 4. Perform cluster analysis on each group of docking conformations in step 3), eliminate redundant conformations, select the optimal binding conformation through the best docking score, and then construct four types of protein-essential oil molecule complex models.

[0046] 5. Import the complex model from step 4) into software such as Pymol and LigPlus to display the three-dimensional binding conformation and two-dimensional interaction network diagram of the complex, so as to intuitively present the interaction mode between essential oil molecules and target sites.

[0047] 6. Results Analysis: Combining the complex binding energy (ΔG) fraction, interaction forces (hydrogen bonds, hydrophobic interactions, van der Waals forces) identification and bond length measurement, and the localization of key amino acid residues (residues with a distance <4.0 Å from the essential oil molecule), the binding specificity of plant essential oil molecules to different types of targets and their cross-pathway synergistic inhibitory effects were analyzed.

[0048] Table 2. Protein-carvacrol docking fraction and interaction mechanism

[0049]

[0050] According to the method steps described in this invention, carvacrol, a common component of plant essential oils, was analyzed in relation to key targets in the four major categories of paper artifact preservation pathways (see Table 2). Docking results showed that carvacrol exhibited the strongest binding affinity to the quorum sensing transcriptional regulatory protein LasR (4NG2) of *Pseudomonas aeruginosa*, with a docking binding energy of -7.8640 Kcal / mol. Further analysis using interaction mechanism software revealed that carvacrol formed a significant hydrophobic interaction network with residues such as Asp73, Trp88, and Tyr56. Figure 5 (a)(b)). This indicates that carvacrol has a strong specific binding ability to LasR, which may interfere with quorum sensing among bacteria, thereby hindering the thickness of the biofilm and reducing its colonization on the surface of the artifact. At the same time, FimH (5JCR) is located at the tip of the type I fimbriae of Escherichia coli and also mediates the attachment of Escherichia coli to host tissues and inert surfaces.Figure 5 (c) and (d) show that carvacrol forms a strong hydrogen bond with its Asp54 residue with a bond length of 2.84 Å, and that the benzene ring of carvacrol interacts electrostatically with Phe1. The strong hydrogen bond combined with the electrostatic interaction mechanism suggests that carvacrol may block FimH-mediated surface adhesion, thereby preventing Escherichia coli from adhering to and colonizing the surface of paper artifacts.

[0051] For the key cell membrane target sterol 14-α demethylase (PDB ID: 5TZ1). Figure 3 (a) and (b) show that the ligands are deeply embedded in hydrophobic cavities, forming significant π-π stacking with Phe233 and Phe380. This hydrophobic interaction is the main driving force behind carvacrol's binding to sterol 14-α demethylase. For chitin deacetase (PDB ID: 7BLY), hydrogen bonds with Asp47 and His101 with bond lengths of 2.99 Å and 2.94 Å, respectively, were observed, verifying carvacrol's ability to interfere with the structure and function of microbial cell walls / membranes. This is because chitinase is involved in cell wall synthesis, morphogenesis, and adhesion, while sterol 14-α demethylase is crucial for the synthesis of ergosterol, a component of fungal cell membranes. Combined with molecular simulations, the binding affinity of carvacrol to both indicates that it may disrupt the synthesis and structural integrity of fungal cell membranes and cell walls, ultimately leading to cell lysis and death. In the environment of cultural relic preservation, this direct damage to microbial activity greatly avoids secondary damage to paper caused by microbial metabolic processes.

[0052] Furthermore, the energy metabolism system of fungi, especially mitochondrial oxidative phosphorylation, is crucial for their growth and reproduction. Interference with this process also inhibits fungal activity. Simulation results showed that carvacrol binds to polycopper oxidase (PDB ID: 5LM8) and chloride peroxidase (PDB ID: 7RST) from Aspergillus niger with binding energies of -5.7770 and -6.2380 kcal / mol, respectively, exhibiting strong binding stability. Figure 6 These two enzymes play an important role in the elimination of reactive oxygen species. The binding of carvacrol may inhibit antioxidant function, leading to the accumulation of reactive oxygen species, thereby causing ATP synthesis disorders and energy metabolism dysfunction, ultimately inhibiting fungal growth.

[0053] Dihydrofolate reductase catalyzes the conversion of dihydrofolate to tetrahydrofolate, a crucial step in bacterial and fungal DNA synthesis, and is therefore often considered a potential target for antibacterial agents. This invention selected dihydrofolate reductase (PDB ID: 1AI9) from Candida albicans as the target, and results showed that its key residue Ile112 formed a critical hydrogen bond with carvacrol with a bond length of 2.84 Å. Figure 4Meanwhile, Phe36, Ile33, and Ala11 provide multiple hydrophobic interactions, indicating that carvacrol can occupy the enzyme's active pocket and competitively inhibit substrate binding. Therefore, in practical cultural relic conservation, this means that carvacrol can effectively inhibit the expansion of bacterial colonies on paper and prevent further spread of contamination.

[0054] 7. Determination of synergistic antibacterial efficacy:

[0055] Based on the judgment rules established in this invention, the docking results of carvacrol with the seven target sites in Table 2 were checked and comprehensively evaluated item by item:

[0056] (1) Verify the satisfaction of single target conditions:

[0057] 1) Energy conditions: The ΔG of carvacrol and all seven target proteins is ≤-5.5Kcal / mol, which satisfies condition (a).

[0058] 2) Interaction conditions: ≥1 hydrogen bond was formed with targets such as 7BLY, 1AI9, 5JCR, and 5LM8, satisfying condition (b); the number of hydrophobic interaction residues with targets such as 4NG2 and 5TZ1 was ≥3, satisfying condition (c).

[0059] 3) Spatial conditions: In all complexes, the distance between carvacrol and key active residues (such as Asp73 and Trp88 of 4NG2) is <4.0 Å, satisfying condition (d).

[0060] 4) Conclusion: Carvacrol simultaneously met the preset multidimensional judgment conditions for four functional targets: cell wall / membrane (5TZ1, 7BLY), colonization (4NG2, 5JCR), nucleic acid (1AI9), and energy metabolism (5LM8, 7RST).

[0061] (2) Perform cross-pathway collaborative determination:

[0062] Since carvacrol simultaneously meets the criteria for detection across ≥2 (actually 4) different functional targets, this method clearly demonstrates its significant cross-pathway synergistic antibacterial potential. This indicates that it not only works through a single pathway (such as cell membrane disruption), but also through multiple key life processes, including synergistic disruption of fungal cell membrane integrity, interference with bacterial quorum sensing and attachment, inhibition of DNA synthesis, and disruption of energy metabolism, achieving more efficient and broader-spectrum inhibition of complex microbial communities on paper artifacts. Therefore, it has outstanding value in green preservation applications.

[0063] In summary, the standardized molecular simulation and determination process of this invention successfully verified that carvacrol, as a component of plant essential oils, possesses the molecular basis and potential high efficiency of multi-target synergistic antibacterial activity. This method provides a rapid and reliable theoretical basis for screening green preservatives for paper artifacts without the need for physical artifact experiments.

Claims

1. A method for verifying the antibacterial efficacy of plant essential oils on paper artifacts based on molecular simulation, characterized in that, Includes the following steps: Step 1) Acquisition and functional classification of targets and ligands: Obtain the amino acid sequences and three-dimensional structural models of microbial target proteins related to mold and decay of paper cultural relics from the Uniprot database or PDB database; obtain the small molecular structures of plant essential oil components to be verified through the PubChem database as ligand molecules; Step 2) Preprocess the three-dimensional structural model of the target protein in Step 1): simplify the protein structure in Pymol software and save it as a .pdb file, and preprocess the small molecules of plant essential oil components; Step 3) Perform molecular docking simulations between the small molecules of plant essential oil components obtained in Step 2) and the three-dimensional structural models of target proteins of different functional categories. A semi-flexible docking mode is adopted, in which the target protein is rigid and the small molecules of plant essential oil components are flexible. A periodic cubic search space is set on the surface of the target protein. The minimum boundary distance of the search space is not less than 5 Å. Perform multiple independent docking operations on each protein-ligand combination to obtain all candidate binding conformations that meet the docking energy sorting. Step 4) Perform cluster analysis on each group of docking conformations in Step 3), and select the best conformation based on the docking score to obtain the protein-essential oil molecule complex model. Step 5) First, use three-dimensional visualization to identify key residues in the complex model from Step 4), then use a two-dimensional interaction diagram to classify the interaction types, and statistically analyze the types of hydrogen bonds, hydrophobic interactions, van der Waals forces, and key residue information to present the interaction mode between essential oil molecules and the target. Step 6) Determination of multi-target synergistic antibacterial efficacy: When the same plant essential oil component simultaneously meets the preset binding stability conditions in at least two different functional categories of target proteins, it is determined that the plant essential oil component has cross-pathway synergistic antibacterial potential and can be used for the pre-evaluation of green anti-corrosion and antibacterial efficacy of paper cultural relics.

2. The method for verifying the antibacterial efficacy of plant essential oils on paper artifacts based on molecular simulation according to claim 1, characterized in that, In step 1), the microorganisms corresponding to the four target proteins are all typical pathogens that cause the decay of paper cultural relics, including one or more of Pseudomonas aeruginosa, Candida albicans, Aspergillus niger, and Escherichia coli.

3. The method for verifying the antibacterial efficacy of plant essential oils on paper artifacts based on molecular simulation according to claim 1, characterized in that, In step 1), four major categories of key proteins were screened and established as molecular targets for verifying antibacterial efficacy. These four categories of key proteins include: target proteins related to cell wall or cell membrane structure and function, target proteins related to nucleic acid synthesis and function, target proteins related to microbial colonization and attachment, and target proteins related to energy metabolism or antioxidant systems.

4. The method for verifying the antibacterial efficacy of plant essential oils on paper artifacts based on molecular simulation according to claim 1, characterized in that, The plant essential oil component to be verified in step 1) is specifically carvacrol.

5. The method for verifying the antibacterial efficacy of plant essential oils on paper artifacts based on molecular simulation according to claim 1, characterized in that, In step 6), the preset binding stability determination condition is: (a) The binding free energy ΔG between the target protein and the plant essential oil component is ≤ -5.5 kcal / mol; (b) The number of stable hydrogen bonds formed between the target protein and the plant essential oil component is ≥1; (c) The number of key amino acid residues that interact hydrophobically with plant essential oil components is ≥3; (d) The spatial distance between plant essential oil components and key functional site residues in the active region of the target protein is ≤4.0 Å; Among them, when the same plant essential oil component meets at least one of conditions (a) and (b), (c), and (d) in at least two different functional categories of target proteins, the component is determined to have cross-pathway synergistic antibacterial potential.

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