A system for analyzing the biofilm-inhibiting efficacy of plant rhizome extract

Abstract

A system for analyzing the biofilm inhibition efficacy of the plant rhizome extract, consisting of: a) a plant material collection unit configured to collect Hedychium forrestii var. forrestii rhizomes; b) an extraction unit configured to produce bioactive fractions from the rhizomes by ethanol extraction and solvent-solvent fractionation; c) a biofilm inhibition analysis unit configured to evaluate the effectiveness of biofilm inhibition using microtiter plate assay instruments; d) a microscopy unit for the analysis of biofilm morphology, consisting of a fluorescence microscope with EtBr / AO staining capability and a scanning electron microscope; e) A unit for analyzing amylolytic activity, configured to determine amylolytic properties using spectrophotometric instruments and DNA reagent; and f) a kinetic analysis unit for evaluating Michaelis-Menten kinetic parameters.

Description

AREA OF INVENTION

[0001] The present disclosure relates to a system for analyzing the biofilm-inhibiting effect of plant rhizome extract. In particular, the present invention relates to a system for analyzing the biofilm-inhibiting effect of plant rhizome extract by carrying out amylolytic biofilm inhibition of H. forrestii var. forrestii rhizome extract against S. aureus pathogens. BACKGROUND OF THE INVENTION

[0002] Bacterial biofilms pose a significant challenge to pathogen control due to their pronounced resistance mechanisms. Staphylococcus aureus biofilms consist of extracellular polymeric substances (EPS) composed of complex polysaccharides, proteins, and lipids, forming a protective matrix. This EPS matrix promotes bacterial adhesion and cell aggregation, ensuring structural integrity while simultaneously creating pathogenic microenvironments with acidic pH and hypoxic conditions. Biofilm formation increases the survival rate of the microbial population by up to 1500 times, resulting in substantial resistance to conventional antibiotics.

[0003] Plant rhizomes naturally possess defense mechanisms against bacterial colonization through secondary metabolites and phytozymes. These plant compounds exhibit antibiofilm activity primarily through amylolytic degradation processes, in which glycoside hydrolases cleave the polysaccharide chains of bacterial biofilms into smaller subunits or monomers. Hedychium forrestii var. forrestii, a rhizomatic aromatic plant from the ginger family (Zingiberaceae), contains bioactive compounds that can inhibit biofilm formation.

[0004] Current systems for evaluating plant antibiofilm agents lack integrated functions for comprehensive extraction, biofilm inhibition analysis, microscopic characterization, and assessment of amylolytic activity.

[0005] In light of the preceding discussion, it is clear that there is a need for integrated systems that can systematically evaluate amylolytic-dependent biofilm inhibition mechanisms and simultaneously enable a quantitative analysis of biofilm reduction and enzyme kinetics for the development of alternative therapeutic approaches against antibiotic-resistant bacterial pathogens. SUMMARY OF THE INVENTION

[0006] This disclosure relates to a system for analyzing the biofilm-inhibiting effect of plant rhizome extract. The proposed system facilitates the analysis of this effect and demonstrates that the extract can effectively reduce bacterial adhesion. The system includes dedicated units for plant collection, extraction of the bioactive fraction, performance of the biofilm inhibition assay, and characterization.

[0007] The present disclosure relates to a system for analyzing the biofilm-inhibiting effect of plant rhizome extract. The system comprises: a) a plant material collection unit for collecting Hedychium extract *forrestii* var. *forrestii* rhizomes; b) an extraction unit for obtaining bioactive fractions from the rhizomes by means of ethanol extraction and liquid-liquid fractionation; c) a biofilm inhibition analysis unit for evaluating the biofilm inhibition efficiency by means of a microtiter plate assay; d) a microscopy unit for analyzing biofilm morphology, consisting of fluorescence microscopy with EtBr / AO staining capability and scanning electron microscopy; e) an amylolytic activity analysis unit for determining amylolytic properties by means of spectrophotometry and DNA reagent; and f) a kinetic analysis unit for determining the Michaelis-Menten kinetic parameters.

[0008] One objective of the present disclosure is to provide a system for analyzing the biofilm-inhibiting effect of the plant rhizome extract.

[0009] A further objective of the present disclosure is to provide an integrated system for amylolytic biofilm inhibition using Hedychium forrestii var. forrestii rhizome extract, which enables a comprehensive evaluation of the antibiofilmic efficacy against Staphylococcus aureus pathogens by quantitative microtiter plate assays and microscopic analyses.

[0010] Another objective of the present disclosure is a system that correlates the activity of amylolytic enzymes with biofilm inhibition mechanisms by spectrophotometric analysis and Michaelis-Menten kinetic evaluation, thereby establishing the enzymatic basis for the antibiofilmic effect.

[0011] However, another objective of the present disclosure is a system that integrates the authentication of plant material, the extraction of bioactive compounds, the morphological characterization and the enzyme kinetic analysis into a unified platform for the systematic development of plant-based alternatives to conventional antibiotics.

[0012] To further clarify the advantages and features of the present disclosure, the invention is described in more detail with reference to specific embodiments illustrated in the accompanying drawing. It is understood that this drawing merely shows typical embodiments of the invention and is therefore not to be understood as limiting its scope of protection. The invention is described and explained in more detail and with reference to the accompanying drawing. BRIEF DESCRIPTION OF THE IMAGE

[0013] These and other features, aspects and advantages of the present disclosure will be better understood when the following detailed description is read with reference to the accompanying drawing, in which the same symbols represent the same parts, wherein: Fig. Figure 1 shows a block diagram of a system for analyzing the biofilm inhibition effect of the plant rhizome extract according to an embodiment of the present disclosure.

[0014] Furthermore, those skilled in the art will recognize that the elements in the drawing are simplified and not necessarily drawn to scale. For example, the flowcharts illustrate the process by highlighting the main steps to facilitate understanding of the present disclosure. With regard to the construction of the device, one or more components may be represented in the drawing by conventional symbols. The drawing may show only the specific details relevant to understanding the embodiments of the present disclosure, so as not to clutter the drawing with details that are already apparent to those skilled in the art from the description contained herein. DETAILED DESCRIPTION:

[0015] To facilitate understanding of the principles of the invention, reference is made below to the embodiment shown in the drawing, which is described using specific terms. It is understood, however, that this does not limit the scope of protection of the invention. Rather, modifications and further developments of the depicted system, as well as further applications of the inventive principles shown therein, are conceivable, insofar as they would normally occur to a person skilled in the art in the field of the invention.

[0016] It will be clear to 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 to be understood as a limitation thereof.

[0017] References to “an aspect”, “another aspect”, or similar phrases in this description mean that a particular feature, structure, or property described in connection with the embodiment is included in at least one embodiment of the present disclosure. Therefore, phrases such as “in one embodiment”, “in another embodiment”, and similar expressions in this description may, but do not necessarily, all refer to the same embodiment.

[0018] The terms "includes," "comprehensive," or similar expressions denote non-exclusive inclusion. Thus, a procedure or method containing a list of steps does not only include those steps but may also include further steps not explicitly listed or inherent in the procedure or method. Likewise, the statement "includes..." for one or more devices, subsystems, elements, structures, or components, without further limitations, does not preclude the existence of other devices, subsystems, elements, structures, or components.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meanings generally known to those skilled in the art in the field to which this invention belongs. The systems, methods, and examples described herein serve only for illustration and are not to be understood as limiting.

[0020] Embodiments of the present disclosure are described in detail below with reference to the attached drawing.

[0021] Fig. Figure 1 shows a block diagram of a system (100) for analyzing the biofilm inhibition effect of the plant rhizome extract according to an embodiment of the present disclosure.

[0022] According to Fig.1 The system (100) comprises: a) a plant material collection unit (102) configured for collecting Hedychium forrestii var. forrestii rhizomes; b) an extraction unit (104) for obtaining bioactive fractions from the rhizomes by means of ethanol extraction and liquid-liquid fractionation; c) a biofilm inhibition analysis unit (106) for evaluating biofilm inhibition efficiency by means of a microtiter plate assay; d) a microscopy unit (108) for analyzing biofilm morphology, consisting of fluorescence microscopy with EtBr / AO staining capability and scanning electron microscopy; e) an amylolytic activity analysis unit (110) for determining amylolytic properties by means of spectrophotometry and DNA reagent; and f) a kinetic analysis unit (112) for determining the Michaelis-Menten kinetic parameters.

[0023] In one embodiment, the extraction unit (104) is configured such that the integrity of the bioactive compounds is maintained even under extraction conditions at room temperature.

[0024] In one embodiment, the extraction unit (104) is configured to perform activity-dependent purification by means of silica gel column chromatography with a mesh size of 60-120.

[0025] In one embodiment, the biofilm inhibition analysis unit (106) is configured such that a biofilm inhibition of about 80% is achieved at an extract concentration of 500 µg / ml.

[0026] In one embodiment, the biofilm inhibition analysis unit (106) is configured to evaluate extract concentrations in the range of 31.25 to 500 µg / ml with IC50 determination capability.

[0027] In one embodiment, the microscopy unit (108) is configured to evaluate biofilm inhibition by surface morphology analysis using linear low-density polyethylene surfaces.

[0028] In one embodiment, the microscopy unit (108) is configured to analyze the viability of bacterial cells by double fluorescence staining with acridine orange and ethidium bromide.

[0029] In one embodiment, the amylolytic activity analysis unit (110) is configured to use starch as a substrate and to maintain the reaction conditions at pH 6.9 and 37 °C.

[0030] In one embodiment, the kinetic analysis unit (112) is configured to determine the Michaelis-Menten constant values ​​using Lineweaver-Burk diagram analysis.

[0031] In one embodiment, the system is configured to demonstrate a correlation between amylolytic activity and the effectiveness of biofilm inhibition against bacterial colonization of the rhizosphere.

[0032] The present invention relates to a system for analyzing the biofilm inhibition efficiency of Hedychium forrestii var. forrestii rhizome extract, wherein the system comprises several integrated units configured to perform collection, extraction, biofilm inhibition analysis, microscopic evaluation, amylolytic activity analysis and kinetic assessment.

[0033] In one embodiment, the system includes a plant material collection unit configured to collect fresh Hedychium rhizomes. The rhizomes of *forrestii* var. *forrestii* were obtained from natural occurrences. They were collected in December 2019 in Vagamon, Idukki District, Kerala, India. The plant specimen was authenticated, and a voucher specimen, number RHT 65506, was deposited at the Rapinat Herbarium in Trichy. The collection site takes care to carefully dig up the plant without damaging the rhizome and subsequently remove any adhering soil particles. The collected rhizomes are packaged in plastic bags, transported to the laboratory, and stored at room temperature until further use.

[0034] In one embodiment, the system includes an extraction unit for obtaining bioactive fractions from the collected rhizomes. The rhizomes are washed, cut into small pieces, dried in the shade at room temperature for seven days, and then pulverized using an electric mixer. The powder is stored in tightly sealed glass bottles until further analysis. The extraction unit is configured to maintain the stability of heat-sensitive compounds by operating under controlled conditions. The material is dried in an autoclave for 23 hours at a temperature below 60 °C. Ethanol extraction then takes place in an aspirator bottle at room temperature for one week. The filtrate is concentrated using a rotary evaporator at 45 °C under reduced pressure.Twenty grams of crude ethanol extract are obtained and fractionated by liquid-liquid extraction with petroleum ether (boiling range 40–60 °C) and chloroform to remove nonpolar compounds and fats. The remaining extract undergoes activity-dependent purification by column chromatography on silica gel (grain size 60–120 mesh), with the fractions being eluted using solvents of different polarities. The eluted fractions are tested for antibiofilm activity and stored for further investigation.

[0035] In one embodiment, the biofilm inhibition analysis unit is equipped with a microtiter plate assay function. Each well of a sterile plate is filled with 180 µl of BHI broth and inoculated with 10 µl of an overnight culture of Staphylococcus aureus. The wells are then treated with 10 µl of rhizome extract prepared from stock solutions with concentrations of 500, 250, 125, 62.5, and 31.25 µg / ml. A control group without extract is included. The plates are incubated for 24 hours at 37 °C. After incubation, the wells are washed with phosphate-buffered saline (PBS, pH 7.2) to remove non-adherent bacteria, fixed with 2% sodium acetate, and stained with 0.1% w / v crystal violet. Excess paint is removed with deionized water, and the plates are dried with 95% ethanol before washing.The optical density is determined at 600 nanometers using a Thermo microtiter plate reader. The percentage of biofilm inhibition is calculated, and the IC50 values ​​are determined graphically using the formula. %biofilm inhibition=((control−OD−test−OD) / control−OD)×100.

[0036] In one embodiment, the system further comprises a microscopy unit for analyzing the morphology and viability of bacterial biofilms. In one embodiment, fluorescence microscopy with dual labeling of acridine orange and ethidium bromide is used. Staphylococcus aureus cells are used at a concentration of 5 × 10 6Cells per milliliter are cultured on coverslips in 24-well plates and treated with rhizome extract at a concentration of 76.87 micrograms per milliliter, corresponding to the IC50 value. The samples are incubated for 48 hours at 37 °C in an incubator. After incubation, 50 microliters of an acridine orange and ethidium bromide solution (1 milligram per milliliter) are added, carefully mixed, centrifuged for two minutes at 800 rpm, and immediately examined under a Labomed TCM 400 fluorescence microscope with a fluorescence filter. At least 100 cells are examined for viability. The microscopy instrument is also configured for surface morphology analysis using scanning electron microscopy (SEM). Biofilms are formed on linear low-density polyethylene (LDPE) surfaces by coating Staphylococcus aureus cells with sterile plastic films of approximately 2 cm. 2 inThe nutrient broth is incubated for 24 hours at 37 °C with shaking (150 rpm). The experimental sample is treated with 76.87 µg of rhizome extract per milliliter, while the control sample receives distilled water. After incubation, the samples are examined for surface morphology using scanning electron microscopy (SEM).

[0037] In one embodiment, the system includes an amylolytic activity analysis unit configured to determine the amylolytic properties of the extract. In one embodiment, starch is used as the substrate. A reaction mixture is prepared with 500 µl of 0.02 M sodium phosphate buffer (pH 6.9), 1 ml of extract at concentrations of 500, 250, 125, 62.5, and 31.25 µg / ml, and 1 ml of a 1% starch solution. The mixture is incubated for 30 minutes at 37 °C.

[0038] Control mixtures are prepared using distilled water instead of the extract. After incubation, 1 mL of DNA reagent is added to stop the reaction, and the solution is heated in a boiling water bath for 20 minutes. The absorbance is measured at 540 nm using an ELICO SL218 UV / Vis spectrophotometer. One unit of amylase activity per milliliter is defined as the amount of enzyme that releases 1 micromole of glucose equivalent per minute under test conditions. The specific activity in units per milligram is determined by dividing the total enzyme activity by the protein content. The protein concentration is determined by Bradford assay using bovine serum albumin as a standard. The system includes a kinetic analysis unit for determining the Michaelis-Menten equation and Menten parameters. The substrate concentration was varied between 1 µmol and 150 µmol, while the extract concentration was set at 76.87 µg / mL and the pH at 8.The concentration was 0 and the temperature was kept at 37 °C. Blank samples without enzyme were prepared for each substrate concentration. The reaction rates were determined and analyzed using Lineweaver-Burk diagrams to calculate the Michaelis-Menten constant.

[0039] In one implementation, the system is configured through the integrated functionality of its units to establish a correlation between amylolytic activity and biofilm inhibition efficiency against colonization by Staphylococcus aureus.

[0040] The results of the spectrophotometric analysis of biofilm inhibition showed that the plant's rhizome extract significantly reduced biofilm formation. The results indicated that the inhibition was directly proportional to the extract concentration in the solution, with maximum inhibition observed at 500 µg / ml. Even at the lowest tested concentration of 31.25 µg / ml, a 35% reduction in biofilm formation was observed compared to the untreated control.

[0041] The system equipped with fluorescence microscopy enabled the investigation of surface structure after biofilm inhibition using EtBr / AO staining. Untreated samples showed densely packed, green structures, while treated samples exhibited a loose, yellowish-orange appearance. This confirmed that the extract reduced bacterial adhesion to surfaces and made the cells more susceptible to membrane breakdown.

[0042] The scanning electron microscopy system provided evidence of changes in surface morphology resulting from biofilm inhibition. The untreated surfaces exhibited indentations and grooves caused by the formation and breakdown of bacterial biofilms, while the treated surfaces were smooth and free of bacterial activity. This demonstrated that, in the presence of the extract, bacteria were unable to adhere to the surfaces—unlike in the untreated control.

[0043] The system configured for amylolytic activity analysis confirmed that the rhizome extract of Hedychium forrestii var. forrestii exhibits strong enzymatic activity. At the highest extract concentration of 500 micrograms per milliliter, a total enzyme activity of 110.916 units per milligram was measured. Enzyme activity correlated with increasing extract concentration, while specific activity remained relatively constant across all concentrations. The kinetic analysis unit revealed that the enzyme behavior followed Michaelis-Menten kinetics, exhibiting a hyperbolic relationship between enzyme and substrate concentrations, with enzyme saturation becoming apparent at higher substrate concentrations.

[0044] The drawing and the preceding description illustrate embodiments. Those skilled in the art will recognize that one or more of the described elements can be combined to form a single functional element. Alternatively, certain elements can be divided into several functional elements. Elements of one embodiment can be added to another. For example, the process flows described here can be modified and are not limited to the manner described herein. Furthermore, the actions of a flowchart need not be performed in the sequence shown; nor do all actions necessarily need to be carried out. Actions that do not depend on other actions can be performed in parallel with the other actions. The scope of protection of the embodiments is in no way limited by these specific examples. Numerous variations, whether explicitly stated in the description or not, such as...Differences in structure, dimensions, and materials are possible. The scope of protection of the embodiments is at least as comprehensive as described by the following claims.

[0045] The advantages, other benefits, and problem solutions have been described above with reference to specific embodiments. However, the advantages, benefits, problem solutions, and any components that can effect or enhance an advantage, benefit, or solution are not to be construed as critical, necessary, or essential features or components of the claims. REFERENCES 100 A System for Analyzing the Biofilm Inhibition Efficacy of Plant Rhizome Extract. 102 Collection point for plant material 104 extraction units 106 Biofilm Inhibition Analysis Unit 108 Microscopy Unit 110 Amylolytic Activity Analysis Unit 112 Kinetic analysis unit

Claims

[1] A system for analyzing the biofilm inhibition efficacy of the plant rhizome extract, consisting of: a) a plant material collection unit configured to collect Hedychium forrestii var. forrestii rhizomes; b) an extraction unit configured to produce bioactive fractions from the rhizomes by ethanol extraction and solvent-solvent fractionation; c) a biofilm inhibition analysis unit configured to evaluate the effectiveness of biofilm inhibition using microtiter plate assay instruments; d) a microscopy unit for the analysis of biofilm morphology, consisting of a fluorescence microscope with EtBr / AO staining capability and a scanning electron microscope; e) A unit for analyzing amylolytic activity, configured to determine amylolytic properties using spectrophotometric instruments and DNA reagent; and f) a kinetic analysis unit for evaluating Michaelis-Menten kinetic parameters. [2] System according to claim 1, wherein the extraction unit is configured such that the integrity of the bioactive compounds is maintained even under extraction conditions at room temperature. [3] System according to claim 1, wherein the extraction unit is further configured to perform activity-dependent purification by silica gel column chromatography with a particle size of 60-120 mesh. [4] System according to claim 1, wherein the biofilm inhibition analysis unit is configured such that at an extract concentration of 500 µg / ml a biofilm inhibition of about 80% is achieved. [5] System according to claim 1, wherein the biofilm inhibition analysis unit is further configured to evaluate extract concentrations in the range of 31.25 to 500 µg / ml with IC50 determination capability. [6] System according to claim 1, wherein the microscopy unit is configured to evaluate biofilm inhibition by surface morphology analysis using linear low-density polyethylene surfaces. [7] System according to claim 1, wherein the microscopy unit is further configured to analyze the viability of bacterial cells by double fluorescence staining with acridine orange and ethidium bromide. [8] System according to claim 1, wherein the unit for analyzing amylolytic activity is configured to use starch as a substrate and maintains the reaction conditions at pH 6.9 and 37 °C. [9] System according to claim 1, wherein the kinetic analysis unit is configured to determine the Michaelis-Menten constant values ​​by means of Lineweaver-Burk diagram analysis. [10] System according to claim 1, wherein the system is configured to demonstrate a correlation between amylolytic activity and biofilm inhibition efficiency with respect to bacterial colonization of the rhizosphere.

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