An environmentally friendly system for the production of antibiofilm silver nanoparticles using Premna serratifolia leaf extracts

Abstract

A system for producing antibiofilm silver nanoparticles using Premna serratifolia leaf extracts to inhibit the formation of bacterial biofilms, comprising: a sample processing unit configured to collect fresh samples of Premna serratifolia leaves and process the collected leaves to convert them into powder form; an extraction unit connected to the sample processing unit and configured to produce leaf extracts from Premna serratifolia, wherein the powder from the sample of Premna serratifolia leaves is processed to obtain a filtrate; a fractionation unit configured for the activity-dependent isolation and chemometric characterization of bioactive fractions from the leaf extracts; a nanoparticle synthesis unit configured to synthesize silver nanoparticle composites using the bioactive fractions;a characterization unit configured to analyze the synthesized silver nanoparticles using at least one of the following equipment: UV-VIS spectrometry equipment, X-ray diffraction (XRD) analysis equipment, and scanning electron microscopy (SEM) equipment; and a biofilm inhibition analysis unit configured to evaluate the antibiofilm properties of the nanoparticles against Staphylococcus aureus.

Description

FIELD OF THE INVENTION

[0001] The present disclosure relates to an environmentally friendly system for producing antibiofilm silver nanoparticles using Premna serratifolia leaf extracts to inhibit bacterial biofilm formation. More specifically, the present invention relates to a system comprising multiple units and devices configured to facilitate the formation of silver nanoparticles synthesized from the Premna serratifolia leaf extract, which contains bioactive components such as caryophyllene, neophytadiene, phytol, squalene, etc., that complement the synthesis of solver nanocomposites. BACKGROUND OF THE INVENTION

[0002] Bacterial biofilms pose a significant challenge to medical devices and conventional bacterial infections. Staphylococcus aureus, a major cause of biofilm-related infections, exhibits increased antibiotic resistance when biofilms form, complicating treatment and posing increasing risks to the medical community. Conventional treatments often fail without removing implanted devices, necessitating new therapeutic approaches.

[0003] Nanotechnology offers promising solutions for the treatment of S. aureus biofilms. Silver nanocomposites offer potential for various biomedical applications due to their nontoxicity, large surface area, and high dimensional stability. The green synthesis of silver nanoparticles from plant extracts offers an environmentally friendly and cost-effective alternative to conventional methods.

[0004] To synthesize biogenic silver nanoparticles, the present invention proposes a system for producing biogenic silver nanoparticles from Premna serratifolia leaf extracts that inhibit the formation of S. aureus biofilms. The system further characterizes these nanoparticles and analyzes their biofilm-inhibiting properties using response surface modeling to determine optimal conditions for antibiofilm activity. Summary of the invention

[0005] The present disclosure relates to an environmentally friendly system for producing antibiofilm silver nanoparticles using Premna serratifolia leaf extracts to inhibit bacterial biofilm formation. The present invention provides a system for producing biogenic silver nanoparticles using Premna serratifolia leaf extracts to inhibit bacterial biofilm formation caused by Staphylococcus aureus. The system comprises integrated units for plant extraction, isolation of bioactive fractions, nanoparticle synthesis, characterization, and analysis of biofilm inhibition. Response surface modeling optimizes inhibitory activity by analyzing the effects of key variables.

[0006] The present disclosure aims to provide a system for producing antibiofilm silver nanoparticles using Premna serratifolia leaf extracts to inhibit bacterial biofilm formation. The system comprises: a sample processing unit configured to collect fresh samples of Premna serratifolia leaves and process the collected leaves to convert them into powder form; an extraction unit connected to the sample processing unit and configured to produce leaf extracts from Premna serratifolia, wherein the powder from the sample of Premna serratifolia leaves is processed to obtain a filtrate; a fractionation unit configured to perform activity-dependent isolation and chemometric characterization of bioactive fractions from the leaf extracts;a nanoparticle synthesis unit configured to synthesize silver nanoparticle composites using the bioactive fractions; a characterization unit configured to analyze the synthesized silver nanoparticles using at least one of the following equipment: UV-visible spectrometry equipment, X-ray diffraction (XRD) analysis equipment, and scanning electron microscopy (SEM) equipment; and a biofilm inhibition analysis unit configured to evaluate the antibiofilm properties of the nanoparticles against Staphylococcus aureus.

[0007] An object of the present disclosure is to provide a system for producing antibiofilm silver nanoparticles using Premna serratifolia leaf extracts to inhibit bacterial biofilm formation.

[0008] Another object of the present disclosure is to provide a system for green synthesis of silver nanoparticles using Premna serratifolia leaf extracts to inhibit biofilm formation by Staphylococcus aureus.

[0009] A further objective of the present disclosure is to establish a systematic approach to characterize and evaluate the antibiofilm properties of the synthesized nanoparticles.

[0010] Another object of the present disclosure is to develop a system for modeling reaction surfaces to optimize biofilm inhibition activity by analyzing the effects of different reaction conditions.

[0011] Another object of the present disclosure is to provide an environmentally friendly alternative to conventional antibacterial treatments for biofilm-associated infections.

[0012] To further clarify the advantages and features of the present disclosure, the invention will be explained in more detail with reference to specific embodiments illustrated in the accompanying drawings. These drawings illustrate only typical embodiments of the invention and are therefore not to be considered as limiting its scope. The invention will be described and explained in more detail with reference to the accompanying drawings. SHORT DESCRIPTION OF THE FIGURE

[0013] These and other features, aspects, and advantages of the present disclosure will become better understood when the following detailed description is read with reference to the accompanying drawings, in which like characters represent like parts throughout. Fig. 1 shows a block diagram of an environmentally friendly system for producing antibiofilm silver nanoparticles using Premna serratifolia leaf extracts to inhibit bacterial biofilm formation according to an embodiment of the present disclosure.

[0014] Those skilled in the art will also appreciate that the elements in the drawings are shown for convenience and are not necessarily to scale. For example, the flowcharts illustrate the method by key steps to enhance understanding of aspects of the present disclosure. Furthermore, with respect to device construction, one or more components of the device may be represented in the drawings by conventional symbols. The drawing may show only the specific details relevant to understanding embodiments of the present disclosure in order not to clutter the drawing with details that would be readily apparent to those skilled in the art from the present description. DETAILED DESCRIPTION:

[0015] To facilitate understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and a clear description will be given. However, the scope of the invention is not limited thereby. Changes and further modifications to the illustrated system, as well as further applications of the principles of the invention, are possible, as would normally occur to one skilled in the art to which the invention pertains.

[0016] It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be limiting thereof.

[0017] References in this specification to "one aspect," "another aspect," or similar language mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Therefore, the language "in one embodiment," "in another embodiment," and similar language throughout this specification may or may not refer to the same embodiment.

[0018] The terms "comprises," "comprising," or other variations thereof are intended to cover non-exclusive inclusion, such that a process or method comprising a list of steps may include not only those steps, but also additional steps not expressly listed or inherent in that process or method. Likewise, the statement "comprises" for one or more devices, subsystems, elements, structures, or components does not exclude, without further limitation, the existence of other devices, subsystems, elements, structures, components, or additional devices, subsystems, elements, structures, or components.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. The systems, methods, and examples provided herein are for illustrative purposes only and should not be considered limiting.

[0020] Embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings.

[0021] Fig. 1 shows a block diagram of a system for producing antibiofilm silver nanoparticles using Premna serratifolia leaf extracts to inhibit bacterial biofilm formation according to an embodiment of the present disclosure.

[0022] Referring to Fig.1, the system (100) comprises: a sample processing unit (102) configured to collect fresh samples of Premna serratifolia leaves and processes the collected leaves to convert them into powder form; an extraction unit (104) connected to the sample processing unit and configured to produce leaf extracts from Premna serratifolia, wherein the powder from the sample of Premna serratifolia leaves is processed to obtain a filtrate; a fractionation unit (106) configured to perform activity-dependent isolation and chemometric characterization of bioactive fractions from the leaf extracts; a nanoparticle synthesis unit (108) configured to synthesize silver nanoparticle composites using the bioactive fractions;a characterization unit (110) configured to analyze the synthesized silver nanoparticles using at least one of the following equipment: UV-visible spectrometry equipment, X-ray diffraction (XRD) analysis equipment, and scanning electron microscopy (SEM) equipment; and a biofilm inhibition analysis unit (112) configured to evaluate the antibiofilm properties of the nanoparticles against Staphylococcus aureus.

[0023] In one embodiment, the fractionation unit (106) is configured to: a) concentrate the leaf extracts using a rotary evaporator; b) fractionate the concentrated extract by solvent-solvent extraction; and c) perform silica gel column chromatography to isolate bioactive compounds, wherein the fractionation unit (106) is further configured to elute the bioactive compounds, wherein the eluted fractions are assayed for their antibiofilm activity

[0024] In one embodiment, the characterization unit (110) further comprises gas chromatography equipment coupled to mass spectrometry, which is configured to analyze the bioactive fractions.

[0025] In one embodiment, the nanoparticle synthesis unit (108) is configured to a) combine the bioactive solution with an aqueous silver nitrate solution, b) heat the combined solution to a predetermined temperature, and c) separate the synthesized nanoparticles by centrifugation.

[0026] In one embodiment, the biofilm inhibition analysis unit (112) comprises: a) a microtiter plate testing device configured to measure percentage biofilm inhibition; b) fluorescence microscopy equipment configured to detect biofilm inhibition using EtBr / AO staining; and c) electron microscopy equipment configured to analyze the surface morphology of the biofilm inhibition, wherein the microtiter plate testing device is configured to evaluate biofilm inhibition at different concentrations of silver nanoparticles.

[0027] In one embodiment, the system (100) further comprises a response surface modeling unit (114) configured to: a) analyze the effects of independent variables on biofilm inhibition activity; b) generate experimental designs using statistical software; and c) perform regression analyses and generate 3D response surface plots to determine optimal conditions for biofilm inhibition, wherein the response surface modeling unit (114) is configured to analyze at least three independent variables including the concentration of nanoparticles, the reaction time, and the presence of stabilizers.

[0028] In one embodiment, the system is configured to produce silver nanoparticles capable of inhibiting bacterial biofilms by up to 90% at concentrations ranging from 100 to 500 µg / ml.

[0029] The present invention relates to a system for producing biogenic silver nanoparticles and their application in inhibiting bacterial biofilm formation. The system comprises several components and units configured for different functions. The sample processing unit is used to collect the fresh sample and process it into powder. The unit processes Premna serratifolia leaves by washing, drying, and pulverizing. A botanical extraction unit is configured to use the powdered sample and prepare an aqueous extract. This initial extract contains various bioactive components such as caryophyllene, neophytadiene, phytol, and squalene, which facilitate the synthesis of silver nanocomposites.The fractionation unit performs activity-dependent isolation of the bioactive compounds through a series of processes, including rotary evaporation, solvent-solvent extraction, and silica gel column chromatography. The bioactive fractions are characterized using gas chromatography combined with mass spectrometry, with structural elucidation performed with reference to the NIST library and PubChem data. These bioactive fractions serve as reducing agents in the subsequent synthesis process. The nanoparticle synthesis unit combines the isolated bioactive fraction with an aqueous silver nitrate solution in a controlled environment. The mixture is heated to 80 °C under continuous stirring, resulting in the formation of silver nanoparticles, indicated by a color change to dark brown.The synthesized nanoparticles are separated by centrifugation to obtain purified PS-AgNPs (Premna serratifolia silver nanoparticles). After synthesis, the characterization unit employs several analytical techniques, including UV-VIS spectrometry, X-ray diffraction analysis, and scanning electron microscopy, to confirm the formation and structural properties of the nanoparticles. These analyses provide important information on the size, shape, and composition of the synthesized nanoparticles. The biofilm inhibition analysis unit evaluates the antibiofilm properties of the nanoparticles using several complementary approaches. The microtiter plate assay quantifies the percentages of biofilm inhibition at different concentrations, while fluorescence microscopy using EtBr / AO staining and electron microscopy provide visual confirmation of inhibited bacterial surface adhesion.Together, these analyses demonstrate the ability of nanoparticles to inhibit biofilm formation by up to 90% at concentrations ranging from 100 to 500 µg / ml. Finally, the response surface modeling unit analyzes the effects of three key variables—nanoparticle concentration, response time, and the presence of stabilizers—on biofilm inhibition. This component utilizes statistical software to create experimental designs, perform regression analyses, and generate 3D response surface plots. The statistical analysis component further validates the results through ANOVA and post-test analysis to ensure the scientific robustness of the results and identify optimal conditions for maximum biofilm inhibition.

[0030] The sample processing unit is configured to collect fresh Premna serratifolia leaves from Thrissur district, Kerala, India, in December 2021. The collected leaves were thoroughly washed and dried in the shade at room temperature for seven days. After drying, the leaves were pulverized using standard pulverizing equipment and stored in sealed glass containers for further use. In the extraction unit, 10 grams of the pulverized leaf sample was added to a 250-ml beaker containing 100 ml of distilled water and boiled at 80°C for 20 minutes. The resulting mixture was cooled and centrifuged at 12,000 × g for 20 minutes. The supernatant was filtered to remove particulate matter, yielding a filtrate. This filtrate is used for further processing and subsequent analysis.

[0031] The fractionation unit is configured for the activity-dependent isolation of bioactive compounds from the leaf extract. The filtrate obtained from the extraction unit was concentrated using a rotary evaporator under reduced pressure at 100 °C. A crude extract weighing approximately 20 g was subjected to solvent-solvent extraction with petroleum ether (40:60) and chloroform to remove nonpolar components and lipids. The remaining extract was further purified by silica gel column chromatography (mesh size 60-120). Bioactive fractions were eluted using solvent systems of different polarity. The collected eluates were tested for antibiofilm activity using in vitro biofilm assays. The chemometric characterization of the bioactive fractions was performed using gas chromatography coupled with mass spectrometry (GC-MS), which was integrated into the characterization unit.The GC-MS analysis was performed using an Agilent 6890 series instrument equipped with an HP-5MS column. Temperature programming began at 30 °C and increased to 300 °C at a rate of 10 °C every 5 minutes. Helium was used as the carrier gas at a flow rate of 1 ml / min. Spectral components were identified based on retention time and peak area, using the NIST library and the PubChem database for structure elucidation.

[0032] The nanoparticle synthesis unit is configured for the synthesis of silver nanoparticle composites from bioactive fractions identified as effective against bacterial biofilms. The selected bioactive fraction was dissolved in double-distilled water at a concentration of 1 mg / ml. 25 ml of this solution was mixed with 75 ml of 1 mM aqueous silver nitrate solution and heated to 80 °C for one hour with continuous stirring. The synthesis of the silver nanoparticles was indicated by a color change to dark brown. The synthesized nanoparticles were separated by centrifugation at 15,000 × g for 15 minutes in three consecutive cycles. The resulting silver nanoparticles were designated PS-AgNPs and submitted to the characterization unit for structural analysis.

[0033] The synthesized silver nanoparticles were characterized using the characterization unit, which is equipped with UV-VIS spectrometry, X-ray diffraction (XRD), and scanning electron microscopy (SEM). These techniques were used to confirm particle formation, crystal structure, and surface morphology according to standard nanomaterial characterization protocols. The biofilm inhibition analysis unit was used to investigate the antimicrobial properties of PS-AgNPs against Staphylococcus aureus MTCC 902.

[0034] To quantify biofilm inhibition, the microtiter plate test device integrated into the biofilm inhibition analysis unit was used. Each well was filled with 180 µl of brain heart infusion (BHI) broth and inoculated with 10 µl of an overnight-grown S. aureus culture. An additional 10 µl of a PS-AgNP solution at different concentrations (31.25, 62.5, 125, and 250 µg / ml) and a control setup without nanoparticles were added. The plates were incubated for 24 hours at 37 °C. After incubation, the wells were washed with 0.2 ml PBS (pH 7.2) to remove non-adherent cells, fixed with 2% sodium acetate, and stained with 0.1% (w / v) crystal violet. Excess dye was washed off with deionized water, and the plates were air-dried. The wells were then treated with 95% ethanol, and the absorbance was measured at 600 nm using a microplate reader (Thermo Fisher Multiskan FC).Biofilm inhibition was calculated using the following formula:. %biofilm inhibition=[(control−OD−test−OD) / control−OD]×100

[0035] The IC 50 -value was calculated from the inhibition diagram.

[0036] Further qualitative evaluation was performed using a fluorescence microscope included in the biofilm inhibition analysis unit. A bacterial suspension of S. aureus (5 × 10 6Cells / ml) were seeded onto coverslips in 24-well plates and treated with 34.63 µg / ml PS-AgNPs. After 48 h of incubation at 37 °C, 50 µl of ethidium bromide (1 mg / ml) and acridine orange were added. The mixture was shaken gently and centrifuged at 800 × g for 2 minutes. The cells were immediately examined under a fluorescence microscope (Labomed TCM 400), and at least 100 cells were evaluated using a fluorescence filter. Surface morphology analysis was performed using an electron microscope as part of the biofilm inhibition analysis unit. S. aureus cultures were sealed with sterile linear low-density polyethylene (LLDPE) film segments (~2 cm 2) in nutrient broth at 37 °C with shaking at 150 × g for 24 hours. The test setups included PS-AgNPs, while the control setups used distilled water. After incubation, the samples were examined under scanning electron microscopy to assess the structural morphology of the inhibited biofilms. The Response Surface Modeling Unit was used to optimize the biofilm-inhibiting effect of PS-AgNPs using a Box-Behnken design. Three independent variables—nanoparticle concentration (A), reaction time (B), and the presence of stabilizers (C)—were selected based on single-factor experiments. Design Expert software version 13 was used to generate the experimental designs. Regression analyses and three-dimensional response surface plots were generated to determine the optimal conditions for maximum biofilm inhibition. The fit of the model was validated using ANOVA and F-test.Statistical analysis was performed using a univariate analysis of variance (ANOVA) followed by Dunnett's post-hoc test. To ensure the significance of the results, GraphPad Prism software version 5.01 was used for all statistical calculations.

[0037] Biofilm inhibition analysis using a microtiter plate-based assay demonstrated concentration-dependent antibiofilm activity of the synthesized silver nanoparticles. The PS-AgNPs effectively inhibited the formation of biofilms of Staphylococcus aureus, achieving inhibition of up to 90% at concentrations between 100 and 500 µg / ml. The percentage inhibition was quantified by optical density measurements at 600 nm, and the IC 50The fluorescence value was calculated accordingly. Fluorescence imaging using double staining with ethidium bromide (EtBr) and acridine orange (AO) confirmed the ability of the PS-AgNPs to disrupt established bacterial biofilms. The treated samples exhibited differential fluorescence, indicating damaged bacterial membranes and reduced biofilm biomass. Electron microscopic analysis using scanning electron microscopy (SEM) revealed that the surface morphology of the bacterial biofilms treated with PS-AgNPs was significantly disrupted compared to the untreated control group. The SEM micrographs showed that the PS-AgNPs exhibited an aggregated morphology with irregular spherical shapes ranging from 100 to 200 nm in diameter. The observed disintegration of the biofilm matrix further supported the biofilm-inhibiting efficacy of the biogenic nanoparticles.Optical characterization using UV-VIS spectrophotometry revealed a strong surface plasmon resonance (SPR) peak in the range of 300 to 350 nm, confirming the formation of silver nanoparticles in the reaction mixture. A visible color change from pale yellow to dark brown further supported nanoparticle synthesis, which was attributed to the excitation of surface plasmon oscillations by free electrons on the nanoparticle surface. Compound characterization using gas chromatography-mass spectrometry (GC-MS) identified 24 bioactive phytochemical constituents in the P. serratifolia extract. These compounds played a synergistic role in the formation and stabilization of nanoparticles and were possibly responsible for the observed antibiofilm activity.Optimization using response surface modeling based on the Box-Behnken design revealed that the inhibitory effect of PS-AgNPs was significantly influenced by three independent variables: nanoparticle concentration, reaction time, and the presence of stabilizers. The statistical model showed high significance with respect to biofilm inhibition results, and the optimal conditions were determined using regression analysis and 3D response surface plots.

[0038] The drawings and the foregoing description illustrate examples of embodiments. Those skilled in the art will recognize that one or more of the described elements may well be combined to form a single functional element. Alternatively, certain elements may be separated into multiple functional elements. Elements of one embodiment may be added to another embodiment. For example, the order of the processes described herein may be changed and is not limited to the manner described herein. Furthermore, the actions of a flowchart need not be performed in the order shown; nor do all actions need to be performed. Also, actions that are not dependent on other actions may be performed in parallel with the other actions. The scope of the embodiments is in no way limited by these specific examples.Numerous variations, whether explicitly stated in the specification or not, such as differences in structure, dimensions, and use of materials, are possible. The scope of the embodiments is at least as broad as indicated in the following claims.

[0039] Advantages, further benefits, and solutions to problems have been described above with reference to specific embodiments. However, the advantages, advantages, solutions to problems, and any components that may result in or enhance an advantage, advantage, or solution are not to be construed as critical, required, or essential features or components of any or all of the claims. REFERENCES 100 An environmentally friendly system for producing silver nanoparticles against biofilms using Premna Serratifolia leaf extracts for inhibition. 102 Sample processing unit 104 Extraction system 106 Fractionation unit 108 Nanoparticle synthesis unit 110 Characterization Unit 112 Biofilm Inhibition Analysis Unit 114 On Modeling Response Surfaces

Claims

[1] A system for the production of antibiofilm silver nanoparticles with Premna serratifolia leaf extracts to inhibit the formation of bacterial biofilms, consisting of: a sample processing unit configured to collect fresh samples of Premna serratifolia leaves and process the collected leaves to convert them into powder form; an extraction unit connected to the sample processing unit and configured to produce leaf extracts from Premna serratifolia, wherein the powder from the sample of Premna serratifolia leaves is processed to obtain a filtrate; a fractionation unit configured for the activity-dependent isolation and chemometric characterization of bioactive fractions from the leaf extracts; a nanoparticle synthesis unit configured to synthesize silver nanoparticle composites using the bioactive fractions; a characterization unit configured to analyze the synthesized silver nanoparticles using at least one of the following devices: UV-VIS spectrometry equipment, X-ray diffraction analysis equipment (XRD) and scanning electron microscopy equipment (SEM); and a biofilm inhibition analysis unit configured to evaluate the antibiofilm properties of the nanoparticles against Staphylococcus aureus. [2] The system of claim 1, wherein the fractionation unit is configured to: a) concentrate the leaf extracts using a rotary evaporator; b) fractionate the concentrated extract by solvent-solvent extraction; and c) perform silica gel column chromatography to isolate bioactive compounds, wherein the fractionation unit is further configured to elute the bioactive compounds, wherein the eluted fractions are assayed for their antibiofilm activity [3] The system of claim 1, wherein the characterization unit further comprises gas chromatography equipment coupled to mass spectrometry configured to analyze the bioactive fractions. [4] The system of claim 1, wherein the nanoparticle synthesis unit is configured to: a) combine the bioactive solution with an aqueous silver nitrate solution, b) heat the combined solution to a predetermined temperature, and c) separate the synthesized nanoparticles by centrifugation. [5] The system of claim 1, wherein the biofilm inhibition analysis unit comprises: a) a microtiter plate testing device configured to measure the percentage biofilm inhibition; b) fluorescence microscopy equipment configured to detect biofilm inhibition using EtBr / AO staining; and c) electron microscopy equipment configured to analyze the surface morphology of the biofilm inhibition, wherein the microtiter plate testing device is configured to evaluate biofilm inhibition at different concentrations of silver nanoparticles. [6] The system of claim 1, further comprising a response surface modeling unit configured to: a) analyze the effects of independent variables on biofilm inhibition activity, b) create experimental designs using statistical software, and c) perform regression analyses and create 3D plots of the response surface to determine optimal conditions for biofilm inhibition, wherein the response surface modeling unit is configured to analyze at least three independent variables including nanoparticle concentration, reaction time, and the presence of stabilizers. [7] The system of claim 1, wherein the system is configured to produce silver nanoparticles capable of inhibiting bacterial biofilms by up to 90% at concentrations in the range of 100 to 500 µg / ml.

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