The conventional method of fiber reinforced polymer (FRP) wrapping around concrete columns uses epoxy as the binder along with synthetic or natural fibers such as carbon, glass, basalt, jute, sisal etc. as the reinforcement. However, the thermal stability of epoxy is a major issue in application areas prone to fire exposure. The current work addressed this major drawback of epoxy by modifying it with a nanofiller, such as multiwalled carbon nanotubes (MWCNT), and reinforcing it using basalt and sisal fibers. The effect of exposure to elevated temperature on the behavior of concrete cylinders externally confined with these FRP systems was analyzed. Three types of specimens were considered: unconfined; confined with sisal fiber reinforced polymer (SFRP); and confined with hybrid sisal basalt fiber reinforced polymer (HSBFRP) specimens. The test samples were exposed to elevated temperature regimes of 100 °C, 200 °C, 300 °C and 400 °C for a period of 2 h. The compressive strengths of unconfined specimens were compared with various confined specimens, and from the test results, it was evident that the mechanical and thermal durability of the FRP systems was substantially enhanced by MWCNT incorporation. The reduction in the compressive strength of the FRP-confined specimens varied depending on the type of the confinement. After two hours of exposure at 400 °C, the compressive strength corresponding to the epoxy–HSBFRP-confined specimens were improved by 15%, whereas a 50% increase in strength corresponding to MWCNT-incorporated epoxy–HSBFRP-confined specimens was observed with respect to unconfined unexposed specimens. The MWCNT-modified epoxy-incorporated FRP-confined systems demonstrated superior performance even at elevated temperatures in comparison to unconfined specimens at ambient temperatures. Reinforced polymer (FRP) composites are identified as the most promising material in the area of structural strengthening and retrofitting. FRPs are mainly used as an external confinement in structures particularly in columns and beams . The easCarbon, glass and aramid are the most commonly used fibers for structural retrofitting and strengthening ,12,13. Tg) of epoxy. When exposed to an elevated temperature above the Tg value, there is a considerable reduction in the performance of FRP. The behavior of FRP-confined specimens when exposed to high temperatures regimes was investigated by Al-Salloum et al. [When exposed to fire, FRPs are prone to burning of the matrix, which is commonly epoxy, and the eventual damage of the confinement. Therefore, the usage of FRP in structural confinement has been questioned due to their vulnerability at elevated temperatures. Limited studies were carried out on the performance of FRP-confined systems in elevated temperatures. Li and Xian  studied m et al. . CFRP anm et al. ,34 analyg and could ultimately lead to the FRP debonding from the concrete core.Sherif et al.  studied It is evident from the literature survey that an in-depth thermal durability study of sisal fiber-reinforced polymer (SFRP) and hybrid sisal basalt fiber-reinforced polymer (HSBFRP) as an external confinement in structural elements has not been explored in detail. The present study particularly aimed to investigate the axial compressive behavior of epoxy-based SFRP and HSBFRP-confined systems filled with MWCNT once exposed to different elevated temperature regimes. In the investigation, the unconfined and confined concrete specimens (with FRP confinements) were exposed to elevated temperature regimes of 100 °C, 200 °C, 300 °C, and 400 °C for a period of 2 h, after which they were tested for axial compression until failure. The maximum temperature was selected as 400 °C as it is reported that the epoxy loses its strength above 200 °C, and that for concrete it is in the vicinity of 400 °C. Furthermore, the ultimate load carrying capacity, failure modes, stress–strain response, influence of MWCNT incorporation towards thermal resistance, and ductile behavior of the confined specimens were compared with the unconfined specimens.A total of 75 plain concrete cylinders were investigated under axial compressive loading to analyze the effect of FRP confinement after exposure to different temperature regimes, out of which 15 cylinders were unconfined control specimens and 60 cylinders were confined using sisal and sisal basalt hybrid FRP systems. The circular column specimens were 300 mm in height and 150 mm in diameter. After 28 days of curing the cylinders were dried under room environmental conditions and the radial surfaces were cleaned to remove dust and impurities. A suitable adhesion between the inner concrete core and outer FRP sheets was attained using high-performance epoxy filled with MWCNT-incorporated resin. A thin coating of epoxy matrix was initially applied over the concrete surface and the fiber layers were completely impregnated with epoxy. Subsequently the FRP layers were wrapped around the concrete cylinders by hand layup. In case of hybrid FRP, sisal FRP layers were wrapped as the inner layers followed by basalt FRP layers as outer layers . DebondiThe concrete cores were cast with OPC 53 Grade cement, fine aggregate (FA) and coarse aggregates (CA) with a specific gravity of 3.17, 2.7 and 2.67 respectively. As per IS 10262-2009 the concrete cylinders were designed and casted. A mix proportion of 1:1.5:2.58 by weight of cement, FA, and CA along with a water/cement ratio of 0.45 as explained in 2/g from Platonic Nanotech Private Ltd., Mahagama, India, were used. MWCNTs were evenly dispersed in epoxy resin for 30 min using a 20 kHz frequency ultrasonic probe sonicator.A high-performance two-part epoxy resin was the polymer matrix. It consists of resin (LY 556) and hardener (HY 991) mixed in a ratio of 100:15 (Part A: Part B) by weight as suggested by the manufacturer. MWCNTs modified with carboxylic acid (–COOH) having 97% purity, 2–10 microns average length, and specific surface area 250–260 m2 was used as the inner layer of the external confinement. The thickness of sisal fabric ranged between from 0.8 to 1 mm with a density of 1580 kg/m3. The easy availability, economical, and medium stiffness of sisal fiber qualify it as a promising choice. Sisal fibers were subjected to alkaline treatment by immersion in NaOH solution and further drying to room temperature for 72 h. The treatment was capable of improving the surface roughness of the fiber and further resulting in enhanced mechanical interlocking. Commercially available basalt fiber sheets of 380 g/m2 with a density of 2630 kg/m3 as provided by the manufacturer were used as an outer layer of confining systems. Both the sisal and basalt fibers in the form of continuous sheets were purchased from Go green Products, Chennai, Tamil Nadu. Apart from the strengthening aspect, the outer layer of basalt provides a protection to the inner natural sisal fiber confinement against the atmospheric effects.Bi-directional sisal fabric of 300 g/mThe following samples were tested: 15 unconfined cylinders, 15 cylinders wrapped with two layers of epoxy-based SFRP sheets, 15 cylinders wrapped with two layers of epoxy-based SFRP sheets filled with MWCNT, 15 cylinders wrapped with four layers of epoxy-based hybrid SBFRP sheets, 15 cylinders wrapped with four layers of epoxy-based hybrid SBFRP sheets filled with MWCNT. Five temperatures were considered during the study to analyze the influence of elevated temperature on the confinement effect. The first regime denotes the room temperature and after which all the specimens were exposed to 100 °C, 200 °C, 300 °C, and 400 °C separately for a period of 2 h. After the high temperature exposure, the cylinders were tested under uniaxial compressive mode. These temperature regimes and the corresponding specimen nomenclature details are summarized in The entire study was divided into two phases. In the first phase, the various properties of epoxy were analyzed, whereas in the second phase, the effect of FRP confinement on concrete was studied.The tensile strength of the epoxy and multiscale composites were analysed as per ASTM D3039. The dynamic mechanical analysis was conducted in the temperature range between 27 °C to 240 °C at a heating rate of 5 °C/min in a Perkin Elmer DMA8000, maintaining a displacement range of +/− 1 to 1400 µm. The damping factor (tan δ) was recorded by a three-point bending method at a frequency of 1 Hz. In a temperature range of 0 °C to 600 °C along with a nitrogen atmosphere by thermogravimetric analysis, the thermal degradation behavior of the epoxy and multiscale composites were analyzed.The confined and unconfined columns were tested for compressive strength under a monotonical axial compression load in a UTM until failure. To ensure uniform loading over the cylinders, steel plates of 5 mm thickness were placed above and below the test specimens while loading. The specimens were tested at a constant displacement rate of 0.2 mm/min. Since a high axial displacement is expected, Linear variable differential transducers (LVDTs) having a capacity of 50 mm were used to record axial strains on the confined and unconfined concrete specimens. For each sample, at least three specimens were tested.The tensile strength exhibited by epoxy and multiscale composites were determined as per ASTM D3039 by preparing different test coupons. The flat coupons 100 mm in length, 10 mm in width and 3 mm in thickness were tested . The speg. This is because of the restrictions offered by MWCNT for the slippage of epoxy polymer chains. Glass transition temperature is a measure of the transition of a polymer from a glassy to a rubbery state. The presence of MWCNT hinders the movement of epoxy polymer molecules at elevated temperature. Furthermore, with the addition of nano filler, the area under the tan delta curve increases as a sign of the damping characteristics of the MWCNT-incorporated epoxy system. The reason may be the higher degree of molecular arrangement in nano-incorporated composites, which may be due to the better dispersion of MWCNT within the epoxy matrix [g) of E1C with respect to epoxy samples indicates an immobilizing effect played by MWCNT on the epoxy resin molecules.The viscoelastic properties of the composites were assessed by dynamic mechanical analysis (DMA). The tan delta parameter obtained from DMA is often analyzed to understand the glass transition temperature of polymers. Tan delta may be defined as the viscous to elastic response ratio of viscoelastic materials. Tan delta values corresponding to epoxy (E) and epoxy-MWCNT nanocomposites (E1C) are shown in y matrix . The broonset and thereby enhance the thermal stability [max to elevated temperatures. The uniform heat dissipation within the matrix is ensured by nanotubes due to their thermal conductive nature, which further improves the thermal stability of composite. The durable interface developed between epoxy and MWCNT can delay the disintegration of matrix at higher temperatures. When the heat diffusion progresses, the temperature at which the maximum weight loss occurs may move to higher temperatures which in turn is due to the thermal barrier created by the addition of nano causing a delay in the volatilization. It could be concluded that TGA studies specified that the combined effect created due to MWCNT and hybrid fiber systems promotes its application in elevated temperature environments.Thermogravimetric analysis (TGA) was carried out to analyze the thermal decomposition characteristics of epoxy and epoxy multiscale composites. The variation of composite modifications with temperature of E, E1C, E1C2S and E1C2S2B samples is presented in tability . In the The unconfined cylinders were exposed to five temperature regimes viz. room temperature, 100 °C, 200 °C, 300 °C, and 400 °C. The failure of unconfined specimens under axial loading after exposure to high temperatures was predominantly due to the splitting of outer concrete layers. This section demonstrates the behavior of specimens confined with two layers of epoxy SFRP sheets and MWCNT-incorporated epoxy-modified SFRP sheets when exposed to different temperature regimes for 2 h. In The results of compression test of SFRP-confined specimens are listed in The confined specimens exposed to high temperature regimes exhibited lower strength properties corresponding to the same specimens tested under room temperature conditions . The ratThe behavior of concrete cylinders wrapped with two layers of epoxy HSBFRP sheets and epoxy-modified HSBFRP filled with MWCNT sheets when exposed to different temperature regimes for 2 h is reported here. The failure modes exhibited by HSBFRP specimens were almost similar to that of the SFRP-confined specimens. The failure mechanisms exhibited by confined specimens were sudden and explosive as an indication of the rupture of HSBFRP wraps. The MWCNT incorporation in epoxy provided a greater resistance to the hybrid FRP layers under elevated temperature. Even when exposed to elevated temperature the hybrid FRP layers are found to be adhering to the concrete surface maintaining the confinement effect. The failure pattern exhibited by HSBFRP-confined specimens followed an explosive nature along with a high energy dissipation irrespective of the exposure temperature . Figure The loss in compressive strength for HSBFRP-confined cylinders exposed to various temperature conditions when compared with unconfined specimens is illustrated in co while the confined concrete strength is denoted as fcc. The confinement effectiveness ratio corresponding to confined concrete is taken as fcc/fco [cc/fco) was approximately 1.96 for a MWCNT-incorporated epoxy HSBFRP jacketing at room temperature. Confinement effectiveness showed a decreasing trend with rise in temperature. Epoxy–HSBFRP exhibited a confining effectiveness of 1.35 at 400 °C while the MWCNT-incorporated epoxy HSBFRP confinement had its minimum effectiveness of 1.91 at 400 °C. In the case of MWCNT-incorporated epoxy SFRP, the maximum and minimum confining effectiveness was found to be 1.45 and 1.18 at room temperature and 400 °C respectively, as clear evidence of the enhanced confinement effectiveness of concrete on MWCNT-incorporated FRP confinement. The confining effectiveness is also influenced by the count of FRP layers used for wrapping. For a four-layer hybrid system, the confining effectiveness exhibited by HSBFRP specimens was considerably greater than the SFRP specimens at ambient and elevated temperatures.The unconfined concrete strength is denoted as f fcc/fco . When thThe representative stress–strain curves for various FRP-jacketed and unconfined specimens are shown in The stress–strain curves displayed a consistent behavior, however, there was an increment in strain percentages with temperature rise from 100 °C to 400 °C. The stress and strain increased in a linear manner initially and once ultimate stress was attained, there was sharp increment in strain even for a small increase in stress. This may be credited to the FRP softening which in turn resulted in the decrease in the confinement effect of concrete when exposed to elevated temperature.Ductility is a significant property that permits a structure to undergo large deformations prior to ultimate failure . Due to p level < 0.05.A GLM-ANOVA  was employed at 0.05 significance level to evaluate the difference in the behavior of unconfined specimens, and confined specimens, when exposed to various elevated temperature regimes. In this manner, the type of FRP confinement was assigned as the dependent variable while the exposed room temperature, 100 °C, 200 °C, 300 °C, and 400 °C conditions were considered as the independent variable. A statistically significant element analysis was carried out at p-value is found to be less than 0.05, this indicates that the assumed variable is a significant factor in the study. When A main effect plot was derived to analyze the individual influence of MWCNT modification, type of confinement and exposure temperature on the performance of specimens as seen in The bonding and strength of the epoxy-based FRP materials was found to be improved by the incorporation of MWCNT. An enhancement in tensile strength by 50% and 170%, respectively, was observed for E1C and E1C2S2B with the addition of MWCNT and fibers as fillers;All SFRP and HSBFRP-confined specimens exhibited enhanced strength when compared to unconfined specimens. The increment was found to be between 30 to 70% and 60 to 100% for SFRP and HSBFRP-confined specimens, respectively. This proves the effectiveness of hybrid fiber system (HSBFRP) as an external confinement;The unconfined cylinders exhibited a brittle failure upon exposure to elevated temperature whereas HSBFRP-confined specimens manifested a ductile failure due to the presence of FRP layers. In HSBFRP-confined specimens, the outer basalt FRP layer protects the internal sisal layers;The SFRP-confined specimens are adversely affected at elevated temperatures as evidenced from the color change caused due to the melting of FRP layers with a considerable reduction in compressive strength by 44% at 400 °C. On the other hand, the strength of MWCNT-incorporated HSBFRP-confined specimens at 400 °C reduced only by 22% in comparison with those at room temperature.From the systematic analysis of the performance of unconfined and confined concrete specimens exposed to elevated temperature, the following conclusions were drawn:Overall, it may be concluded that, when exposed to elevated temperature conditions, the axial load carrying members made of concrete manifest a sudden reduction in strength. In such situations, concrete members confined with MWCNT-modified epoxy-based hybrid fiber systems can be employed for structural integrity and retention of compressive strength. The use of natural fibers, such as sisal and basalt, provides an alternative solution to the large-scale consumption of artificial fibers in the construction sector for strengthening/retrofitting applications.


The aim of this work was to investigate selected biological and toxicity properties of cured epoxy resin-based compounds based on a bisphenol A epoxy resin, cold-cured by a polyamide and containing two types of metal powders . This study involved cytotoxicity analysis, pH measurements, absorbance measurements and sterilization. The cytotoxicity analysis was conducted to determine the harmful degree of the cured epoxy resin. Aimed at identifying toxic agents in cured compounds, the cytotoxicity analysis involved absorbance measurements in an entire wavelength range. Cytotoxicity and absorbance results demonstrated that the extracts of all the tested resin samples had no cytotoxic effects on the cells of living organisms. The absorbance values obtained over the entire wavelength range did not point to the formation of aggregations, which proved that no toxic agents harmful to living organisms were extracted from the resin samples. Based on the results obtained, it can be concluded that all tested compounds, based on epoxy resins, which are also used as adhesives in various applications, are essentially safe materials when using such formulations in a cured state. Epoxy compounds for different technical applications and the methods of their manufacturing were known already in the nineteenth century ,2,3,4. OEpoxides are viscous liquids or brittle solids that change their structure during the curing process, and as a result of crosslinking reactions, they become insoluble and infusible, acquiring high strength, good electrical insulating properties and a considerable chemical resistance ,5,8. EpoDue to the numerous applications where epoxy resins can be advantageously exploited, it is necessary to know the properties of such materials and their components, both before and after the curing process. The main characteristics of the resins and of their curing agents, including safety information, are always reported in the data sheets. However, epoxy compounds, once cured, can exhibit slightly different characteristics, in particular, a different toxicity to humans. When producing epoxy-based compounds, on the other hand, the safety of people exposed to the cured materials is a relevant issue; to the best of our knowledge, only limited information is available on this topic.The toxicology examines the properties of toxic factors and their impact on the body, which are often assessed with biochemical tests. One of the basic tests determines the cytotoxic activity of a given substance. Cytotoxicity measures the ability of a substance to destroy a particular type of cell . The invThe aim of this work is, therefore, to present selected biological and chemical properties of three different cured epoxy compounds, obtaining valuable information for manufacturers and users of such materials.This work presents a biological, toxicological, and chemical analysis in accordance with the accepted standards of such tests, performed on the appropriate substrates and the appropriate substrates available for biotechnological laboratory tests, which are described in the individual types of tests in this study. No animal or human cells were used in this study.The study was performed on samples of three epoxy resin-based compounds, whose composition is summarized in One (unmodified) epoxy compound was based on a bisfenolic epoxy resin  cured with a polyaminoamide C curing agent . Two modified epoxy compounds were produced and studied, including a metallic filler  or copper (Cu)). The metallic fillers, in powder form, were added in order to improve the resins performance. These compounds were based on the same mixtures of epoxy resins and curing agents as previously described. The amount of filler in each system was selected according to what was carried out in previous experimental works ,35.® 53 epoxy resin is primarily used for saturating, casting, air-tight sealing of electrical equipment and as an impregnating agent in glass fiber laminates and composites; it is also employed as a component for cold-cured structural adhesives. This compound is a mixture of bisphenol A and epichlorohydrin with the addition of styrene, obtaining a mixture possessing an average molecular weight lower than 700 [® 53 epoxy resin are described in [® 53 epoxy resin are listed in Epidianthan 700 . The mecribed in ,36. The ® 53 epoxy resin.Since the subject of this research was to evaluate the biological, toxicological and chemical properties of this resin, ® 53 resin is reported to be irritating to the skin and eyes, it can cause sensitization when it comes into contact with the skin. This epoxy resin is also dangerous for the natural environment: it is toxic for aquatic organisms and may cause long-term adverse effects in the aquatic environment. The epoxy mixture is flammable and contains styrene, which cany form an explosive mixture with the air. Since styrene vapors are heavier than air, they tend to accumulate in the lower part of rooms, such as basements, canals, and hollows. It should be stored in original, tightly-sealed packaging; in a dry, well-ventilated place away from direct sunlight at a temperature below 25 °C. Under these recommended conditions, this product does not pose any hazard; it does not decompose and it remains stable. Contact with amines and amides, that promote the curing process, should be avoided. Uncured EpidianThe polyaminoamide is a polyamide curing agent; its characteristics are given in ,37. This3 (max 0.05% wt.). According to the Directive 67/548/EEC and Regulation 1272/2008/EC, it is not classified as harmful or hazardous. However, prolonged exposure to the product in its powdered form may cause irritation to the eyes, skin, and respiratory system. The product is flame retardant. This material must be stored in a dry and well-ventilated room and kept away from humid environments and oxidants. When properly stored and used, it is stable and poses no risk to the user. It cannot be released or spread in the natural environment. In some of the epoxy compounds, aluminum or copper metal powder fillers were added in order to improve the resin properties and performance. Electrolytic copper powder may contain particles of lead (max 0.05% wt.), iron (max 0.02% wt.), oxygen (max 0.3% wt.) and residues insoluble in HNOAccording to the classification under the Directive 67/548/EEC and Regulation 1272/2008/EC, aluminum powder is highly flammable and releases flammable gases when it comes into contact with water; however, it presents no significant hazards when it comes into contact with skin. Adverse effects are possible: prolonged exposure to dust or fume concentrations will produce irritation to the eyes, skin, and respiratory tract. Dust may cause mechanical irritation to the eyes.Physical and chemical properties of copper and aluminum powders are listed in The samples of epoxy compounds were prepared by casting the liquid mixture in a silicone mold with a dog-bone shape. ® ; (ii) 100 g of epoxy resin was metered, 2% wt. (2 g) of a metal filler, when used, was added to the resin; (iii) the components were mixed with a high-speed mixer for about 120 s at 460 rev/min; (iv) the compound was left to degas for 120 s; (v) 80 g of polyaminoamide curing agent was then added; (vi) this compound was mixed again for about 120 s and left to degas for an additional 120 s; (vii) the liquid mixture was finally poured into the prepared molds. These epoxy compounds were fabricated at an ambient temperature of 27 ± 3 °C and cured for about 7 days at the same temperature.The preparation of the epoxy compound samples, with or without each of the fillers, was carried out as follows: (i) casting molds were prepared and cleansed, and then coated with an anti-adhesion agent for polymeric materials, PolsilformA cytotoxicity analysis was conducted to determine the harmful degree of the cured epoxy resin. This method is used to assess the toxicity levels of a substance, when it is added to a culture medium, in order to determine its activity at the cellular level. It is based on enzymatic reactions that produce a colored product, which is identified spectrophotometrically with the ELISA reader .2, (Binder C150); laminar flow cabinet  with HEPA filters, a HEPA H-14 filter capturing 99.999% of particles with a diameter < 0.3 µm and a HEPA filter capturing 99.999% of particles with a diameter > 0.3 µm; water bath at 37 °C; an inverted phase contrast microscope; centrifuge; scales; plate photometer provided with 570 nm filter and shaking mode; Bürker hemocytometer; automatic pipette controller with serological pipettes; automatic pipettes; multi-channel automatic pipettes and vessels for cell cultivation.The toxicity analysis was performed with the aid of the following laboratory equipment: an incubator with a humid environment at 37 °C and 5% COThe tests were carried out using the following chemical reagents, media and serum: Minimum Essential Medium Eagle ((MEM), without phenol red, glutamine and serum); FBS (serum), penicillin/streptomycin, glutamine; Trypsin-EDTA solution; phosphate buffered saline (PBS); MTT -2,5-diphenyltetrazolium bromide); and isopropanol .5 cells/mL. 100 μL of PBS was loaded into the peripheral wells in the 96 well microplate, and 100 μL of the prepared cell suspension was loaded into the other wells. The microplate was incubated for 24 h under the following conditions: 5% CO2, 37 °C, humidity > 90%.To ensure proper testing conditions, all solutions, media, vessels for cell cultivation and tools must be sterile. All operations were then carried out in the laminar flow cabinet under sterile conditions. The MTT solution was dissolved in a clean and fresh MEM medium without phenol red at a concentration of 1 mg/mL. The compound was sterilized with the use of a syringe filter and used on the same day. Material samples were extracted from the cured samples of the adhesive compounds. Prior to their use and contact with cells, all fluids were heated in the water bath to 37 °C. On the first day of testing, cells were collected by enzymatic digestion and centrifuged for 3 min. The prepared pellet was suspended in a cell culture medium and diluted to achieve a concentration of 1 × 10On the next day, the microplate was examined under a phase-contrast microscope to verify uniform cell growth. Diluents of the extracts of the tested cured epoxy compounds were prepared, as specified in 2, 37 °C, humidity > 90%. The next day, the microplate was examined under a phase contrast microscope to verify the cell culture growth.The microplate was re-incubated for 24 h at 5% COAimed at identifying toxic agents in cured compounds, the cytotoxicity analysis involved absorbance measurements in an entire wavelength range. In this experiment, the absorbance assay was performed with the μQuant microplate spectrophotometer . The µQuant is a single-channel analyzer used for research and development and in vitro diagnostics, it is designed to automatically perform endpoint analysis. The wavelength range is from 200 nm to 999 nm.570 of negative control that was greater than 0.2. The difference between the mean OD570 of negative control on the left and the right side of the microplate  should not exceed 15%. Cell viability was calculated based on Equation (1):50 μL of the MTT solution was added to the wells with cells and incubated at 37 °C for 2 h. After that, the MTT solution was removed from the cells, and 100 μL of isopropanol was added to the wells with the cells. The microplate was incubated at 37 °C for 10 min and then shaken for 1 min; the absorbance was read at a wavelength of 570 nm. Analysis of the results was performed for the ODIf cell viability drops below 70%, this means that the tested material has cytotoxic properties. The cell viability of the samples containing diluted extracts should be similar or higher than that of the samples containing 100% of the extract. If not, the test should be repeated. The absorbance was measured with 200 μL solutions of each extract (repeated three times) in the wavelength range between 200 nm and 800 nm. Water was used as a reference.®, Radnor, PA, USA). The pH meter used in the experiments has an articulated electrode stand and IP 43 rated housing, which provides high resolution and accuracy for precise measurements. It has a large LCD graphic display, with a continuous LED backlight, showing both pH/mV and temperature values simultaneously. The purpose of the pH measurements is to collect information in order to assess the occurrence of risk to humans, as some products are harmful and dangerous at a specific pH.The pH measurement of the analyzed samples was made with the bench pH meter  121 °C for 15 min; (ii) 134 °C for 3–5 min. The experiments were performed with the use of the BRAVO G4 22L Pro Select,  autoclave.570 of the blank >0.2—the criterion is met and is equal to 0.545; (ii) the difference between the mean values of OD570 of the blank on the left and right sides of the microplate <15%—the criterion is met and is equal to 1.48%; (iii) cytotoxic positive control—the criterion is met; cell viability equals 45.9%.  the mean ODTo facilitate the execution of subsequent calculation procedures, the values listed in The test was carried out correctly as indicated by the cytotoxicity assay positive control.Absorbance values obtained over the entire wavelength range, shown in Results of the pH measurements are listed in p-value is the probability describing the critical level of significance that corresponds to an acceptable risk of error. Since the significance level used in practice is α = 0.05, this value was also taken in this study. The pH values of Samples 1 and 14 are statistically higher than the benchmark for water because p < 0.05. For sample 23 p > 0.05, this means that the pH value is statistically insignificant with respect to the benchmark for water.All samples have acid characteristics. The The cured epoxy compounds were subjected to autoclave sterilization at 134 °C for 5 min. The sterilization was aimed at investigating the effect of a specified environment, i.e., an environment with a high temperature, humidity, and pressure, on the structure of the material. Neither microscopic examination nor examination with the naked eye showed any changes in the structure, form, or shape of the tested samples of the cured materials. This means that the obtained epoxy compounds are sufficiently resistant and can be used in service conditions, i.e., a typical environment found in medical applications. This test proved, therefore, that even under such harsh conditions, the material does not release any substance. However, this can only be considered a preliminary assessment based on the observation of the photo-micrographs.Bisphenols (BPs) are produced all over the world in large quantities. They are frequently used in the manufacture of polycarbonate plastics and epoxy resins; such polymers are also used in a variety of consumer products, such as baby feeding bottles, toys, and epoxy liners for food cans . Epoxy rAt present, most epoxy resins are industrially manufactured from bisphenol A (BPA). Independent studies by vom Saal et al.  and OkadA complete knowledge of the structural features that contribute to the allergenic and cytotoxic effects of DGEBF will guide the development of future novel epoxy resin systems with reduced health hazards when in contact with them. Cytotoxicity and absorbance analysis, of the three cured epoxy compounds, performed in this study demonstrated that the extracts of all the tested resin samples had no cytotoxic effect on the cells of living organisms. Correctness of the test was verified by the cytotoxicity assay positive control. The absorbance values obtained over the entire wavelength range did not point to the formation of aggregations, which proved that no toxic agents harmful to living organisms were extracted from the resin samples. The pH measurements revealed that all tested samples were acidic. Both the microscopic and naked-eye examinations of the autoclaved sterilized samples showed no changes in the structure, form, or shape of the tested cured resin samples.Results of this experimental study demonstrate that cured epoxy resins are safe materials, even in contact with human cells;Cured epoxy compounds are homogeneous, monolithic structures. The material is coherent and exhibits typical mechanical strength properties;Additives such as metal powders do not cause deviations in compound curing.Cytotoxicity and absorbance results demonstrate that the extracts of all the tested resin samples have no cytotoxic effect on the cells of living organisms. Correctness of the test was verified by the cytotoxicity assay positive control. The absorbance values obtained over the entire wavelength range do not point to the formation of aggregations, which proves that no toxic agents harmful to living organisms are extracted from the resin samples. Both the microscopic and naked-eye examinations of autoclave sterilized samples showed no changes in the structure, form, or shape of the tested cured resin compound samples. Summing up, it can be concluded that:


Multivariate Cox analysis further demonstrated that low infiltration by CD8+ TILs  and nuclear β-catenin expression  were both independently associated with a poorer disease-specific survival. In conclusion, tumor-intrinsic nuclear β-catenin activation is associated with a non-inflamed TME phenotype and a poorer prognosis, thereby suggesting a possible implication as an immune exclusion mechanism for a subset of HNSCC patients.Activation of WNT/β-catenin signaling has been associated with a non-T-cell-inflamed tumor microenvironment (TME) in several cancers. The aim of this work was to investigate the relationship between β-catenin signaling and TME inflammation in head and neck squamous cell carcinomas (HNSCCs). Membrane and nuclear β-catenin expression, PD-L1 expression, and CD8+ tumor-infiltrating lymphocyte (TIL) density were jointly evaluated by immunohistochemistry in a series of 372 HPV-negative HNSCCs. Membrane β-catenin levels decreased in carcinomas compared to the normal epithelium. Positive nuclear β-catenin was detected in 50 tumors (14.3%) and was significantly associated with a low CD8+ TIL density (168 cells/mm Head and neck squamous cell carcinoma (HNSCC) is the sixth most commonly diagnosed cancer and the most frequent malignancy in the head and neck region . HNSCC iImmune evasion is an important hallmark of cancer . Severalβ-catenin is a multi-functional protein and central component of the canonical WNT signaling pathway. In addition, β-catenin forms a complex with E-cadherin at the epithelial adherens junction that is critical for cell–cell contacts and tissue remodeling . β-catenOn this basis, the aim of this study was to investigate more deeply the relationship between tumor-intrinsic WNT/β-catenin signaling and a non-T-cell-inflamed TME and the clinical and prognostic implications in HNSCC by analyzing a large homogeneous cohort of 372 surgically treated HPV-negative HNSCC patients.The characteristics of the patients selected for study are summarized in p < 0.001). Then, the PD-L1 tumor proportion score was positive (TPS ≥ 1%) in 103 cases (29.5%), and the PD-L1 combined proportion score was positive (CPS ≥ 1%) in 140 cases (40%). In addition, CD8+ TIL immunostaining could be analyzed in 337 HNSCC samples; the mean number of CD8+ TILs per mm2 was 275 . Representative examples of tumors with high and low densities of CD8+ TILs are shown in PD-L1 immunostaining could be assessed in 349 out of 372 patients; 103 (29.5%) of 349 cases showed positive PD-L1 staining in tumor cells A,B. A top < 0.001). Representative examples of negative and positive membrane and nuclear β-catenin staining are shown in Strong membrane β-catenin staining was observed in the normal epithelium used as a positive control IRS = 6; E. β-catep = 0.008; p = 0.001). In addition, low CD8+ TIL density was observed in tumors harboring positive nuclear β-catenin expression  of membrane β-catenin were significantly associated with a lower CD8+ mean density .There was a positive correlation between PD-L1 CPS positivity and membrane β-catenin expression, and a negative correlation with nuclear β-catenin; however, differences did not reach statistical significance  and an earlier disease stage (p = 0.014). Associations between PD-L1 CPS and tumor localization, and pN classification and degree of differentiation were not found (p = 0.01); no other associations between CD8+ TIL density and clinicopathological parameters were found (p = 0.01) and stage I-III tumors (p = 0.03) (p = 0.04) and disease stage (p = 0.01). Thus, higher pT classification (T3–T4) and advanced stage IV tumors were related to a type II  (Positive PD-L1 CPS was significantly associated with a lower pT classification (ot found . CD8+ TIre found . Positiv = 0.03) . The typnorance) .p = 0.002 and p = 0.017, respectively; p = 0.005 and p = 0.03, respectively; p < 0.001 and p = 0.006, respectively), with the other combinations showing intermediate survival  and overall survival (OS) in this HNSCC patient cohort , but on survival .p = 0.001), N+ classification , poor histologic differentiation , low CD8+ TIL density , and positive nuclear β-catenin expression . Similarly, the only parameters independently associated with a worse OS were: T3–T4 classification , N+ classification , poor histologic differentiation , and low CD8+ TIL density .Multivariate Cox analysis, including T classification (T1–T2 vs. T3–T4), N classification (N0 vs. N+), degree of differentiation (well-moderately vs. poorly differentiated), CD8+ TIL density (above vs. below the median), and nuclear β-catenin expression (positive vs. negative) showed that the parameters independently associated with a worse DSS were T3–T4 classification , and the cases with nuclear β-catenin expression had a higher proportion of type II tumor microenvironment (defined as immune ignorance) according to the classification reported by Teng et al. [In this work, we show that WNT/β-catenin pathway activation, reflected by positive expression of nuclear β-catenin, is associated with a lower infiltration by TILs and a lower expression of PD-L1, resulting in an immunosuppressive tumor microenvironment. These results are in agreement with recent reports revealing that most tumor types  within the TCGA (including HNSCC) showed WNT/β-catenin signaling enrichment in non-T-cell inflamed tumors . Tumor-ig et al. .The mechanism of Immune exclusion associated with β-catenin signaling has been described in melanoma . ActivatWe think that the most important finding in our study is the negative association between WNT/β-catenin signaling and CD8+TIL counts. In association with the lower PD-L1 expression, this possibly contributes to the absence of an immune response that confers a poorer prognosis and a potential resistance to immunotherapy for this subset of HNSCC patients. In fact, we have previously shown in this series of patients that a lower CD8+ TIL density  was indeActivation of WNT/β-catenin signaling seems to constitute an intrinsic mechanism of primary and adaptive resistance of tumor cells to immunotherapy through T cell exclusion. Hence, it emerges as a valuable molecular target to develop novel therapeutic strategies aimed to restore T cell infiltration, which could potentially increase the efficacy of immunotherapy, turning a ‘cold’ tumor into a ‘hot’ tumor (by increasing TIL density) . HoweverTumor samples from 382 patients with HNSCC who were surgically treated at the Hospital Universitario Central de Asturias (HUCA), Oviedo, Spain, between the years of 1990 and 2009 were retrospectively collected. All experimental procedures were performed in accordance with the Declaration of Helsinki. Formalin-fixed paraffin-embedded (FFPE) tissue samples and data from donors were provided by the Principado de Asturias BioBank (PT17/0015/0023), Oviedo, Spain, integrated in the Spanish National Biobanks Network, and histological diagnosis was confirmed by an experienced pathologist. Protocols were approved by the HUCA Ethics Committee and the Regional CEIm from Principado de Asturias. Informed consent was obtained from all patients. All patients had a single primary tumor and did not receive any treatment before surgery. None of the patients showed distant metastasis at the time of diagnosis. Clinical, demographic, and follow-up data were collected from the medical records.The HPV status was available for all the patients, which was determined as previously reported ,23. To aThree 1 mm cylinders selected from representative areas of each individual tumor block were selected to construct TMA blocks, as described previously , containThe TMA blocks were cut into 3 μm sections and dried on Flex IHC microscope slides . After deparaffinization with standard xylene and hydration through graded alcohols into water, antigen retrieval was performed using EnVision Flex Target Retrieval solution, high pH (Dako). Staining was performed using an automatic staining workstation  with the Envision system and the primary antibodies: anti-PD-L1  at 1:200 dilution, anti-CD8  at 1:400 dilution, and anti-β-catenin  at a dilution of 1:200. Counterstaining with hematoxylin was the final step. As positive staining controls, we used placenta for PD-L1, and normal tonsil for CD8 and β-catenin expression. Stainings were reviewed and scored blinded to clinical data by two expert pathologists.2). The median value was used as a cut-off point to stratify high versus low density of CD8+ TILs. For the evaluation of PD-L1 expression in tumor cells, only membrane staining was considered and scored as: (a) negative PD-L1 expression (<1% stained cells), (b) low PD-L1 expression (≥1–<10%), (c) intermediate PD-L1 expression (≥10–<50%), or (d) high PD-L1 expression (≥50%). Tumor proportion score (TPS) was defined as the percentage of PD-L1-positive tumor cells in relation to the total tumor cells, and PD-L1 CPS ≥ 1% was considered positive. Combined proportion score (CPS), which represents the number of all positive PD-L1 cells  in relation to total tumor cells, was also considered positive if ≥1%. TILs were defined as lymphocytes within the tumor nests, excluding the peritumoral area. Quantification of CD8+ TIL staining (as a marker of cytotoxic T lymphocytes) was performed automatically using ImageJ software; CD8+ T cells were counted in the tumor areas of each core within the TMAs . The average total number of positive cells in the three tumor cores was expressed in density  and staining intensity . An immunoreactive score (IRS) was calculated by multiplying the quantity and staining intensity scores. IRS scores ≥ 3 were considered as high-membrane-β-catenin expression and IRS scores from 0 to 2 as low membrane expression. Nuclear β-catenin expression was considered positive when ≥10% of tumor cells showed nuclear β-catenin staining.p values. All tests were two-sided. p-values of ≤0.05 were considered statistically significant.Comparison between categorical variables was performed using chi-square and Fisher’s exact tests. Kaplan–Meier curves were used for time-to-event analysis. Cox proportional hazards models were used for univariate and multivariate analyses, reporting the hazard ratios (HRs) with a 95% confidence interval (CI), and We demonstrate that specifically tumor-intrinsic β-catenin activation in HNSCC is associated with a lower PD-L1 expression and low infiltration of CD8+ T cells, and hence a noninflamed TME and an immune ignorance phenotype. Altogether, these findings suggest that the interference of WNT/β-catenin signaling activity in combination with immune checkpoint blockade might represent a novel strategy to improve the efficacy of immunotherapeutic agents for some subsets of HNSCC patients.


Moreover, the shape memory, repairing, and recycling of epoxy vitrimer were investigated. Therefore, dopamine coating is a multifunctional approach to enhance the performance of epoxy vitrimer.An effective method was developed to improve the interfacial interaction between Mutiwalled carbon nanotubes (MWCNTs) and epoxy matrix. The performance of thermal conductivity and strength of the epoxy vitrimer were enhanced by polydopamine (PDA) coating. Polydopamine is a commonly used photothermal agent, which of course, was effective in modifying MWCNTs used in photoresponsive epoxy resin. The surface temperature of the epoxy composite with 3% MWCNTs@PDA fillers added increased from room temperature to 215 °C in 48 s. The metal–catechol coordination interactions formed between the catechol groups of PDA and Zn However, it is not that possible to repair or reprocess epoxy materials once their permanent crosslinked structure is formed, which limits their application and adds a burden to the environment . A new cerature) . Much eferature) . Qiao eterature) . Graphenerature) . The carerature) .Despite this, the dispersion of the nanomaterials in the polymer matrix is very poor due to the high van der Waals forces and large aspect ratios, such as CNTs and graphene. This is the main problem for the reinforcement of mechanical properties in polymer composites ,11. MuchThe polymerization process of dopamine is facile and simple. Nevertheless, the mechanism behind the formation of polydopamine is unclear and complex, which is caused by the generation of intermediates during the polymerization and reaction process . PolydopWith efficient photothermal properties, PDA modification is the perfect method to solve the problem of interfacial interaction between nanomaterials and polymer matrix while maintaining high photothermal efficiency simultaneously. Herein, we provide a promising methodology to fabricate carbon nanotube/epoxy composites based on vitrimer chemistry with superior thermal conductivity, strength, and stress relaxation. The MWCNTs@PDA hybrids were first synthesized by a rational surface coating method, and the carbon nanotube/epoxy composite was prepared by compression molding. The polymerization of dopamine monomers immediately occurred under alkaline conditions in the buffer solution, coupled with a color change from colorless to brown and then black. Both covalent linkage and π–π stacking interactions are included in the connection between polydopamine and carboxylic MWCNTs, while only π–π interactions are present between the MWCNTs and PDA layer for pure MWCNTs . TherefoThe dispersion of MWCNTs and MWCNTs@PDA in water solutions is shown in −1, which correspond to the OH, indole, and indoline groups of PDA. This further indicated that PDAs have successfully coated the surface of MWCNTs [More evidence of the presence of PDA structure can be proved by FT-IR analysis E. MWCNTsf MWCNTs . The XRD patterns of MWCNTs and MWCNTs@PDA are shown in The thermal conductivity and mechanical properties of nanocomposites are closely associated with the dispersion of nanomaterials. The dispersibility of CNTs in the polymer matrix is very poor due to their high van der Waals forces and large aspect ratio. The high intrinsic thermal conductivity of CNTs can be significantly decreased by simple mixing into the matrix, which is mainly caused by defects and phonon scattering . The dispersion states of MWCNTs and MWCNTs@PDA hybrids in the epoxy composites are shown in The typical stress–strain curves at room temperature are shown in The thermal stability of MWCNTs@PDA epoxy composites was evaluated by TGA analysis under a nitrogen atmosphere in g) of the epoxy composites were characterized by DSC , exhibiting similar temperature-dependent viscoelastic properties and indicating their potential shape memory behaviors. The storage modulus of neat epoxy at the glass state is about 1680 MPa. The presence of a chemically crosslinked polymer network by a rubber platform could reach 3.4 MPa at 90 °C. In addition to the differential scanning calorimetry method, dynamic mechanical analysis could also be used to obtain the glass transition temperature of polymers. g increased with the addition of MWCNTs@PDA. The Tg values of all samples are shown in g values determined by the DMA were higher than those determined by the DSC, which can be attributed to the differences in the measuring method.The dynamic mechanical properties of MWCNTs@PDA/epoxy composites were studied by DMA in 2+ acted as a catalyst by activating ester carbonyl, stabilizing alkoxide groups and moving them close to each other, thus catalyzing the exchange reactions in epoxy vitrimers [itrimers . 2+ of the catalyst zinc acetylacetonate, which retarded the exchange reactions. Notably, the stress relaxation of MWCNTs epoxy composites occurs more slowly than the MWCNTs@PDA epoxy composites. The relaxation times of EP, EP-M-0.5%, and EP-M@PDA-0.5% were 247.0 s, 294.5 s, and 447.6 s, respectively. For MWCNTs@PDA epoxy composites, the amino groups on the surface of MWCNTs can react with epoxide groups. Thus, MWCNTs can be connected to the epoxy matrix by covalent bonding. Meanwhile, large amounts of catechol existed on the surface of MWCNTs and could be linked with Zn2+ by metal–catechol crosslinking interactions. The dynamic reversible metal–catechol coordination bonds can also participate in the relaxation process of the whole material [However, epoxy composites exhibit much slower relaxation than the epoxy vitrimer matrix. MWCNTs are one-dimensional nanofillers with a large length–diameter ratio. The coiling of MWCNTs retarded the interfacial transesterification reaction in the epoxy network, which impaired the rate of relaxation . The catmaterial ,31. TherThe shape memory, repair, and recycling of epoxy vitrimers may be caused by the stress relaxation induced by the dynamic transesterification exchange reaction. As f) and shape recovery ratio (Rr) with the strain above 22.5% and the recovery temperature at 70 °C. As shown in f and Rr were about 80.1% and 92.1%, while for the second shape memory cycle, the values of Rf and Rr were about 82.9% and 92.1%. To investigate the shape memory properties of epoxy composites, EP-M@PDA-1.0% was tested to obtain the shape fixity ratio  and deformed to a temporary shape “N-W-P-U” after this treatment. The temporary shape can also recover to its original shape using NIR irradiation, and the recovery process could be completed in a few seconds. The shape recovery triggered by light is an accurate and efficient way to realize the process of shape memory.With the excellent properties of photothermal conversion, the shape recovery could be induced by light, such as near-infrared light (NIR). The shape recovery process of EP-M@PDA-1.0% induced by the NIR laser is shown in 2). The surface temperature changes of MWCNTs@PDA epoxy composites under NIR irradiation (1.5 W/cm2) were recorded by the infrared camera , with a mean diameter of 8~15 nm and a length of 50 μm, sebacic acid, zinc acetylacetonate, were supplied by Macklin Biochemical Co., Ltd. . Tris(hydroxymethyl)aminomethane (Tris) and dopamine hydrochloride were purchased from Aladdin Biochemical Technology Co., Ltd. . Epoxy resin, diglycidyl ether of bisphenol A (WSR618), with an equivalent epoxy weight of 184–200 g(equiv)−1, was purchased from Nantong Xingchen Synthetic Material Co., Ltd. . Other reagents were of analytical grade or better and used without further purification.2O (20 mL) under ultrasonication for 30 min. Then, dopamine hydrochloride was added to the above solution at ratios of 2:1, 1:1, and 0.5:1 (dopamine hydrochloride: MWCNTs) under magnetic stirring, followed by 40 mL of tris-buffer solution (pH = 8.5). The whole mixture was stirred vigorously at room temperature for 12 h. Finally, the final product was filtered by a 0.2 μm membrane filter and washed with DI-H2O until the filtrate became colorless. Finally, MWCNTs@PDA hybrids were obtained by the freeze-drying method.MWCNTs (200 mg) were dispersed in DI-HE51(5 g) was dissolved in 20 mL ethanol, and different amounts of as-prepared MWCNTs@PDA were dispersed in the above-mixed solution under ultrasonication for 10 min. The mixture was heated to 80 °C until the ethanol was totally evaporated. After that, the mixture and sebacic acid  were mixed at 140 °C under manual stirring. Then, zinc acetylacetonate  was introduced and stirred until homogeneously mixed. Finally, the mixture was poured into a stainless-steel mold and cured in 15 MPa at 140 °C for 1h and 180 °C for 4 h.The microstructure of the composites was characterized by field emission scanning electron microscopy . The crystal structure of all the samples was collected by X-ray diffraction  at 40 kV and 100 mA of Cu Kα. The surface chemical compositions of MWCNTs@PDA were analyzed with an X-ray photoelectron spectrometer (PHI5000). The morphology of the synthesized samples and the dispersion qualities of the filler in epoxy composites were examined with transmission electron microscopy . The epoxy composites were mounted on the freezing microtome  to obtain a 0.1 μm thick section. The section was transferred to a copper grid for close examination by TEM. The element mapping was obtained by EDS with transmission electron microscopy. Synchronous thermogravimetric  was carried out with the temperature ranging from room temperature to 800 °C and a heating rate of 10 °C/min under air and nitrogen atmosphere. Differential scanning calorimetry (DSC) analysis was performed with a Mettler Toledo  instrument under a nitrogen atmosphere with a heating rate of 10 °C/min. An infrared thermal imager  was applied to characterize the photothermal process and to record the temperature change of the surface. Dynamic mechanical analysis (DMA) was performed on Netzsch DMA 242 E heating from −30 to 110 °C at a rate of 5 °C/min and a frequency of 1 Hz. Stress relaxation was performed on a Netzsch DMA 242 E. After thermal equilibrium at testing temperature, a strain of 5% was applied within the liner region of the materials, and the relaxation modulus was collected as a function of time. The shape memory behavior of samples was evaluated by DMA 242 E using a tensile method and at the controlled force mode. The thermal conductivity was determined by a hot disk instrument .2), the surface temperature of the epoxy composite with 3% MWCNTs@PDA added increased to 215 °C in 48 s, which initiated the transesterification exchange reaction and realized the healing of the crack rapidly. Overall, PDA coating may be a promising alternative to improve the interfacial interactions between the nanofiller and polymer matrix and may be a multifunctional approach to enhance the performance of epoxy vitrimers.In this study, thermal and NIR triggered shape memory composites were prepared by dynamic transesterification reaction. The approach of PDA coating significantly improved the mechanical properties and thermal conductivity of the composites, and the metal–catechol coordination interactions in the MWCNTs@PDA epoxy network were found to accelerate the stress relaxation of epoxy composites compared with the MWCNTs epoxy network. The relaxation time was 294.5 s (180 °C) for MWCNTs@PDA epoxy composites and 447.6 s (180 °C) for MWCNTs epoxy composites, respectively. Under NIR irradiation (1.5 W/cm


Simulated microgravity (SMG) inhibits osteoblast differentiation (OBD) and induces bone loss via the inhibition of the Wnt/β-catenin pathway. However, the mechanism by which SMG alters the Wnt/β-catenin pathway is unknown. We previously demonstrated that SMG altered the focal adhesion kinase (FAK)-regulated mTORC1, AMPK and ERK1/2 pathways, leading to the inhibition of tumor cell proliferation/metastasis and promoting cell apoptosis. To examine whether FAK similarly mediates SMG-dependent changes to Wnt/β-catenin in osteoblasts, we characterized mouse MC3T3-E1 cells cultured under clinostat-modeled SMG (µg) conditions. Compared to cells cultured under ground (1 g) conditions, SMG reduces focal adhesions, alters cytoskeleton structures, and down-regulates FAK, Wnt/β-catenin and Wnt/β-catenin-regulated molecules. Consequently, protein-2 (BMP2), type-1 collagen (COL1), alkaline-phosphatase activity and matrix mineralization are all inhibited. In the mouse hindlimb unloading (HU) model, SMG-affected tibial trabecular bone loss is significantly reduced, according to histological and micro-computed tomography analyses. Interestingly, the FAK activator, cytotoxic necrotizing factor-1 (CNF1), significantly suppresses all of the SMG-induced alterations in MC3T3-E1 cells and the HU model. Therefore, our data demonstrate the critical role of FAK in the SMG-induced inhibition of OBD and bone loss via the Wnt/β-catenin pathway, offering FAK signaling as a new therapeutic target not only for astronauts at risk of OBD inhibition and bone loss, but also osteoporotic patients. Spaceflight contains many physiological, environmental and psychological stress factors that seriously affect the health of astronauts . One of The Wnt/β-catenin pathway has an important role in OBD and, thus, in the formation and regeneration of bone . Wnt sigCytoskeleton structures play important roles in cell biology by maintaining cells’ shapes, providing a cell transport system and controlling cell migration and survival . The eukWe previously applied a random positional 3D clinostat to model SMG for the investigation of SMG’s effect on tumor cell biology and demonstrated that the SMG inhibited tumor cell proliferation, migration and invasiveness and the promotion of tumor cell apoptosis via FAK-regulated mTORC1, AMPK and ERK1/2 pathways ,17. Our To assess our hypothesis, we investigated SMG’s effects on the modulation of OBD in experiments using a mouse osteoblast cell line, MC3T3-E1, along with 3D clinostat modeling. We demonstrated that the SMG altered the cytoskeleton structure, reduced the formation of FAs and down-regulated FAK signaling in the MC3T3-E1 cells cultured under SMG (µg), compared to the cells cultured under ground (1 g) conditions. In addition, the SMG also down-regulated the expression of the Wnt-regulated transcription factor β-catenin and the osteoblastic maturation markers BMP2 and COL1, as well as inhibiting ALP activity and mineralization. In a mouse hindlimb unloading (HU) model, we found reduced trabecular bone volume measured by micro-computed tomography (micro-CT) imaging and confirmed by histological analyses in the HU-treated mouse tibia, compared to the control mouse tibia. Interestingly, the FAK activator, cytotoxic necrotizing factor-1 (CNF1) ,17, convTo assess the SMG-affected cytoskeleton structures and cellular FAs, we stained MC3T3-E1 cells cultured in chamber slides coated with integrin ligand fibronectin under SMG (µg) and ground (1 g) conditions for 3 days with fluorescein isothiocyanate (FITC)-labeled phalloidin and FITC-labeled anti-integrin-binding protein paxillin antibody. This was followed by an examination of the cells by fluorescence microscopy for the measurement of the microfilament structure and FAs, respectively. We demonstrated that the osteoblastic cells cultured under 1 g condition showed abundant stress fibers (actin/myosin bundles) A, while To investigate SMG-affected FAK signaling, we performed a Western blotting analysis using cell lysates derived from MC3T3-E1 cells cultured in a culture medium under µg and 1 g conditions, as described in the Materials and Methods section. We demonstrated that the SMG significantly reduced the abundance of phosphor FAK  in the cells under µg compared to the cells cultured under 1 g conditions A, indicaTo assess the SMG-affected Wnt’s down-stream molecules, we performed a Western blotting analysis using cell lysates derived from the MC3T3-E1 cells cultured under µg and 1 g conditions for 3 days. We found that the SMG also significantly reduced the expression of the Wnt-regulated transcription factor β-catenin and the osteoblastic markers COL1 and BMP2  in the MOsteoblasts produce ALP, which is a critical component for the formation of hydroxyapatite and the calcification and mineralization of bones . We therE. coli toxin, CNF1, is a broad-spectrum activator of FAK and RhoA signaling [The ignaling ,17,23. Tignaling ,17. InteMouse hindlimb unloading (HU) modeling is a well-tolerated approach mimicking the removal of weight-bearing loads  during spaceflights that has been widely used to study SMG-induced bone loss in vivo ,25. It hE. coli toxin CNF1 is a well-tolerated pharmaceutical reagent that has been used as a pioneering therapy for central nervous system diseases [The diseases . To asseIn this study, we investigated SMG’s effects on the modulation of OBD in experiments using a mouse osteoblast cell line, MC3T3-E1, along with the 3D clinostat modeling SMG condition. We demonstrated the following: (i) SMG alters cytoskeleton structures, inhibits FA formation and down-regulates FAK signaling and the Wnt/β-catenin-BMP2/COL1 pathway controlling OBD, while mouse HU modeling mimicking the AMG condition also reduces trabecular bone volume in the proximal tibia through the thinning and loss of trabeculae; and (ii) the activation of FAK by the activator CNF1 successfully converts SMG-induced alterations in cytoskeleton structure, FAs, various gene expressions, ALP activity and matrix mineralization in MC3T3-E1 cells cultured under µg conditions and prevents bone loss in HU-treated mice. Therefore, our data indicate that (i) FAK is an upstream signal regulating Wnt/β-catenin-BMP2/COL1 pathway regulating OBD and (ii) FAK activation restores the SMG-induced inhibition of OBD via the Wnt/β-catenin pathway .E. coli toxin, CNF1, is a 114 kDa single-chain multidomain protein that contains an N-terminal receptor-binding domain, a translocation domain and a C-terminal catalytic domain [The c domain . After bc domain  controllc domain . CNF1 hac domain ,28.In addition to the Wet/β-catenin pathway, some other molecular pathways, such as phosphoinositol 3-kinase (PI3K)-AKT-mTORC1, ERK1/2 and AMPK, have also been reported to regulate OBD ,30,31. FThe fact that the Wnt/β-catenin pathway influences stem cell proliferation, differentiation and maintenance makes it an attractive target for the development of pharmaceutical agents to treat cancer, neurological disorders and bone repair/regeneration . In addiInsulin-like growth factor-1 (IGF1), the most abundant growth factor in the bone matrix, plays an important role in the stimulation of OBD and bone regeneration through its binding to IGF1 receptor (IGF1R) . The binCompared to lithium’s activation of the Wnt/β-catenin pathway and IGF1’s activation of both the mTORC1 and ERK1/2 pathways, we assume that the FAK activator CNF1’s activation of the Wnt/β-catenin, mTORC1, ERK1/2 and AMPK pathways may be the most efficient for the conversion of the SMG-induced inhibition of OBD and HU-induced bone loss. To assess our assumption, we are in the process of conducting experiments using in vitro MC3T3-E1 cells cultured in a medium that includes CNF1, lithium and IGF1, respectively, under 3D clinostat-induced SMG conditions, as well experiments using in vivo HU-treated mice with the administration of CNF1, lithium and IGF1, respectively.The use of novel gene-targeted therapeutics has become a new direction of drug development . The groAltogether, we have demonstrated that FAK signaling plays a critical role in SMG-induced OBD inhibition via the Wnt/β-catenin pathway and that the activation of FAK by CNF1 successfully converts SMG-induced OBD inhibition and prevents bone loss in HU-treated mice. Therefore, the use of CNF1 modulating multiple FAK-regulated pathways for bone regeneration, including the Wnt/β-catenin pathway, may become a new target in the development of novel therapeutics for astronauts at risk of OBD inhibition and bone loss, and for clinical patients with osteoporosis.E. coli toxin cytotoxic necrotizing factor-1 (CNF1), which activates FAK and RhoA [A mouse osteoblast cell line, MC3T3-E1, obtained from Thermo Fisher Scientific  was maintained in a regular α-MEM medium plus 10% fetal calf serum (FCS). Fluorescein isothiocyanate (FITC)-labeled and rabbit antibodies against paxillin were purchased from Thermo Fisher Scientific. Primary rabbit antibodies against phosphor-FAK , β-catenin, BMP2, COL1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were obtained from Cell Signaling Technology . FITC-conjugated goat anti-rabbit (ZF-0314) secondary antibody specific for primary antibodies were purchased from Thermo Fisher Scientific. The Prolong Gold Antifade Reagent with DAPI was obtained from Life Technologies Inc. . Ascorbic acid, β-glycerol phosphatase and Alizarin red were obtained from Sigma-Aldrich . and RhoA ,17,23, wand RhoA .2 incubator. Since it takes more than two weeks for MC3T3-E1 cell maturation to form mineralization [SMG is modeled by the random positional machine (RPM), which is a three-dimensional (3D) clinostat manufactured by Center for Space Science and Applied Research, Chinese Academy of Sciences  ,17. The lization ,52 and Mlization . This celization . The conlization ,55,56. Tlization ,17.For microtubule immunofluorescence staining, MC3T3-E1 cells cultured in chamber culture slides were fixed in 4% paraformaldehyde for 15 min at room temperature and permeabilized using PBS containing 0.5% Triton X-100, followed by blocking in 1% bovine serum albumin (BSA) in PBS for 30 min at room temperature. To check microfilaments, the cells were incubated with 1:20 FITC-labeled phalloidin diluted in PBS for 30 min in dark at room temperature. To measure cell focal adhesions, the cells were incubated with rabbit anti-paxillin antibody (1:100 diluted in PBS) containing 1% BSA for 24 h at 4 °C overnight, followed by 1:100 FITC-labeled anti-rabbit antibody (green). Plastic chambers were removed after rinsing three times with PBS. The slides were covered with cover slips and then measured for microfilament structure and focal adhesions, respectively, by fluorescence microscopy ,17.MC3T3-E1 cells cultured in flasks were lysed in lysis buffer containing 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS in PBS containing protease and phosphatase inhibitors, for 30 min, on ice. The cell lysates were centrifuged at 4 °C for 30 min at 12,000 rpm and protein contents in the supernatant were quantified by using BCA Protein Assay Kit . For Western blotting analysis, 30–50 μg total protein sample in the supernatant was loaded into each well of 5–10% SDS-PAGE gradient gel. After electrophoresis, protein samples were transferred onto a 0.22-micrometer polyninylidene fluoride (PVDF) membrane . Membranes blocked with 5% skimmed milk powder in TBST  were incubated with primary antibodies against various molecules overnight at 4 °C followed by incubation with suitable horseradish peroxidase-labeled secondary antibodies. The protein bands developed with horse radish peroxidase developer solution were quantified using chemiluminescence ,17. The MC3T3-E1 cells cultured in Lab-Tek1 II chamber slides were permeabilized in PBS containing 0.5% Triton X-100 for 10 min, followed by blocking in 1% BSA for 30 min at room temperature. The cells were then incubated with FITC-rabbit anti-β-catenin antibody (green) (1:100) for staining of β-catenin in 1% BSA for 2 h in dark at room temperature. After rinsing cells three times with PBS, the slides were covered with Prolong Gold Antifade Reagent with DAPI (blue) for staining of nuclei and checked by confocal microscopy ,21.To measure ALP activity, we performed in vitro experiments using the protein samples of MC3T3-E1 cell lysates and ALP activity assay kit  according to the manufacturer’s manual . The absMC3T3-E1 cells cultured in Lab-Tek1 II Chamber Slides were fixed with 4% paraformaldehyde and then stained for 30 min with 0.5% Alizarin red (pH 4.2) at 37 °C. Alizarin red-stained calcified nodules on slides were measured by light microscopy .2O on the day for HU was started and, subsequently, once every six days for a total of four times, during three weeks of HU treatment period.Mouse hindlimb unloading (HU) modeling has been widely used to mimic SMG condition for studying microgravity-affected alterations in mouse organs or tissues, such as brains and bones ,25. BrieThe proximal tibia of each mouse was scanned using a SkyScan 1172 desktop micro-computed tomography (micro-CT) scanner  operating at 40 kVp and 250 µA, with 150-millisecond exposure times, 0.2 rotation step, four-frame averaging and a 0.5-mm aluminum filter at a voxel size of 10 µm. SkyScan’s NRecon software was used to reconstruct 3D datasets for analysis of trabecular bone microarchitectural parameters. All versions and subsequent analyses were performed using CT-Analyzer software. Bone volume fraction (BV/TV), trabecular thickness (Tb.Th) and trabecular number (Tb.N) were calculated by 3D standard microstructural analysis .The bone samples were fixed using 4% paraformaldehyde, followed by decalcification using 10% ethylenediaminetetraacetic acid and embedding in paraffin for preparation of tissue sections. Hematoxylin/eosin (H/E)-stained tissue sections were checked by light microscopy.t test. Any p values <0.05 or <0.01 were considered statistically significant or very significant.Statistical analysis was conducted using Graphpad Prism-3.0, and statistical significance among groups was analyzed using Student


Pimpinella anisum L., widely used in pharmaceuticals, was selected as the active ingredient in this study. Since many bacteria have developed considerable antibiotic resistance, this research aimed to develop an herbal emulgel for treating skin infections caused by bacteria. Given these obstacles, we developed and evaluated a new, cost-effective topical emulgel solution containing anise essential oil against Escherichia coli (E. coli). Anise-based emulgels, potential drug delivery platforms, have been evaluated for various parameters, including physical properties, viscosity, pH, rheology, encapsulation efficiency, and in vitro release research. The AEOs emulgel demonstrated remarkable colloidal stability, with a zeta potential of 29 mV, a size of 149.05 nm, and considerable polydispersity. The efficacy of anise-loaded emulgels as antibacterial formulations was evaluated in vitro. E. coli was used as a model microbial organism for the antibacterial study. Human keratinocytes (HaCaT) were used to examine the biocompatibility of the emulgel. Molecular docking revealed that the essential oil components of Pimpinella anisum L. possess a high affinity for the bacterial adhesin protein FimH of E. coli. These findings indicate that the developed AEOs have the potential to be analyzed using E. coli as a model organism.Over the past decade, researchers have made several efforts to develop gel-based formulations that provide an alternative to traditional hydrogels and emulgel. Due to its excellent antibacterial properties, anise, the main constituent of  Escherichia coli (E. coli) causes enormous economic losses to humans and animals [E. coli infections, which can even lead to septic shock and death [Although antibiotics have been very successful in fighting infectious diseases, bacterial infections and drug resistance continue to be serious problems in healthcare worldwide ,2. An im animals . Bloody nd death . It is and death .Essential oils (EOs) obtained from plants consist of several volatile and non-volatile complex components. Chemically, they are rich in phenols, terpenes, and terpenoids, all of which have beneficial biological effects . They arThere has been a great deal of interest in essential oils as safe, natural antimicrobial agents, but little attention has been paid to them. Due to their low solubility in water, they are limited in how much they can be introduced into a system, thereby reducing their antimicrobial activity . StrategE. coli treatment, the current treatment has attracted much attention. The formulation of drugs that are delivered via the skin varies from liquid to powder, but semisolid preparations are becoming more prevalent [Despite the extensive research in revalent . In thisrevalent . Tirgarirevalent .Combining a nanoemulsion with a gel results in the formation of a semisolid product called a nanoemulgel, which has been shown to be an effective method of delivering drugs through the skin . This isPimpinella anisum L. for treating E. coli infections, with the aim of providing a more effective method for managing deep skin bacterial infections. Through the application of molecular docking techniques, the binding interactions of the phytoconstituents present in the essential oil with the bacterial adhesin protein, FimH of E. coli, have been investigated to gain a deeper understanding of the mechanisms of action.Based on the available research, it is believed that the use of emulgel as a transdermal formulation holds significant potential for increasing the drug loading capacity while also improving the solubility and bioavailability of the active pharmaceutical ingredient. Furthermore, emulgel has been found to provide a safe and suitable environment for temperature-sensitive active pharmaceutical ingredients compared to other nanocarriers. The overarching objective of this study is to develop a novel emulgel-based transdermal formulation utilizing essential oil derived from The colour, consistency, and homogeneity of the prepared transdermal emulgel-based formulation of AEOs are visually evaluated to observe the physical properties. The appearance of AEOs emulgel-prepared formulations ranged from off-white to cream-coloured, exhibiting excellent homogeneity and consistency. When applied to the skin, the prepared emulgel compositions did not produce any unpleasant odours and did not leave behind any oily residue. This is very visible. As a result, it was shown that the AEO emulgel formulation had one month of development, the AEOs emulgel-based transdermal formulation did not change in any way, and there was no noticeable difference between the formulation.It has been shown that the pH of the formulation is an extremely important parameter for its effectiveness and stability . The pH The rheological properties of a gel, such as viscosity and spreadability, are crucial in determining its performance and potential uses. Rheological behavior plays an important role in the assessment of topical preparations’ physical stability during storage and application. The viscosity of the prepared formula was observed for 28 days. The viscosity and spreadability of the formulation of AEO emulgel are shown in It is crucial to control the size of emulgels because they affect permeability, cellular absorption, half-life, and re-release of drugs . AssessiCharacterizing the composition, interfacial strain and structure, morphology, phase quantification, and size distribution of nanomaterials is a critical step in the design process. Both scanning and transmission electron microscopy (SEM/TEM) are widely used for this purpose . SEM is The encapsulation efficiency of AEOs in emulgel has been investigated as a function of the essential oil initial amount. The % EE was reported to be 58.42 ± 7.35%. These results propose that a relatively high amount of AEO was entrapped emulgel.Studies on the retention of drugs in the skin are essential components of local infection and targeting. The accumulation of drugs in specific tissues is an essential component in the design of effective drug carrier systems . Ex vivoIn vitro drug release studies were performed using a dialysis tube. The amount of drug released was plotted against time, as shown in The in vitro cell viability assay was performed on the human keratinocyte cell line (HaCaT) for AEOs and AEO emulgel at a concentration ranging from 20 µg/mL–60 µg/mL, as shown in The term “minimum inhibitory concentration” (MIC) refers to the lowest concentration of an antimicrobial drug that prevents the observable growth of bacteria during an overnight incubation period . As showPimpinella anisum L. essential oil components were tested for their ability to bind to the adhesin protein FimH of E. coli. Docking predicted binding energies revealed that α-himachalene possessed the highest affinity for the bacterial adhesin FimH protein, with a binding energy of −6.2 kcal/mol. In contrast, limonene was revealed to be the least active compound, with a value of −4.6 kcal/mol. On the other hand, trans-verbenol and linalool demonstrated moderate affinity showing −5.8 and −5.1 kcal/mol, respectively. Using a molecular docking strategy, Escherichia coli (E.coli) in vitro, making it a potential treatment for bacterial skin infections. The emulgel was also biocompatible with human keratinocytes (HaCaT), which is a good indication of its safety for topical use. Molecular docking studies revealed that the essential oil components of Pimpinella anisum L. have a high affinity for the bacterial adhesin protein FimH of E. coli, providing insights into how the emulgel may work to treat bacterial infections. Compared to the effects of pure oil, the emulgel had much stronger antibacterial and anticancer properties due to its small particle size and large surface area, improving the interaction between the emulgel and bacteria. This study presents a new, cost-effective topical emulgel solution containing AEOs as a potential alternative for treating skin infections caused by bacteria. The results pave the way for further research to fully understand the mechanisms of action of AEOs in treating bacterial infections, focusing on the bacterial adhesin protein FimH of E. coli.In recent years, there has been a growing interest in developing gel-based formulations as an alternative to traditional hydrogels. This is due to the increasing resistance of bacteria to antibiotics and the desire for natural and cost-effective solutions. The main active ingredient in this study is anise, an herb commonly used in pharmaceuticals and known for its excellent antibacterial properties. The research aimed to develop a herbal emulgel for treating skin infections caused by bacteria, using anise essential oil (AEO) as the active ingredient. The emulgel was evaluated for various parameters such as physical properties, viscosity, pH, rheology, encapsulation efficiency, and in vitro release. The results showed that the AEO emulgel had remarkable colloidal stability and high encapsulation efficiency. In addition, the emulgel was effective against Escherichia coli (ATCC 11229) was collected from the Department of Pharmaceutics, College of Pharmacy, Prince Sattam Bin Abdulaziz University, Al-Kharj. Solvents and other chemicals were also purchased from Sigma Merck. Nutrient media and Sabouraud dextrose were purchased from HiMedia . Anise oil is composed primarily of anethole, which accounts for 80–90% of its chemical composition. Other compounds present in lesser concentrations include estragole, p-anisaldehyde, anise alcohol, acetophenone, pinene, and limonene [Anise oil, Carbopol 940, span 80, tween 80, paraffin liquid, and triethanolamine were purchased from Sigma-Aldrich, USA, and Merck. All the reagents used in the study were analytically graded. limonene .A thorough methodology was used to prepare the AEO emulgel, which involved the creation of an emulsion through the careful and deliberate combination of an oil and aqueous phase. Using the procedures described by Burki et al., 2020 and Shakee et al., 2022 ,36, the In preparing an emulgel loaded with AEO, Carbopol 940 was used as the primary thickener at a concentration of 1%. In a beaker, 100 mL of distilled water and 1 gm of Carbopol 940 were mixed and continuously homogenized to obtain a dispersion. Triethanolamine was added dropwise to the dispersion mixture to obtain a transparent gel composition. The pH was then precisely adjusted with a few drops of triethanolamine, which finally resulted in the formation of a gel . Next, tThe prepared emulgel was inspected visually for colour, homogeneity, consistency, and phase separation.pH evaluation: The pH of emulgels was measured using a pH meter. The pH meter was calibrated before use to ensure accurate results. A small amount of the emulgel sample was placed on the pH electrode, and the pH value was recorded. The pH of the emulgel was measured at room temperature, i.e., 25 °C and under controlled conditions to ensure the reproducibility of results. Average values were calculated after the test was carried out in triplicate.Viscosity: The viscosity of the emulgel was determined by a Brookfield viscometer using a spindle. After four weeks of emulgel formulation, the viscosity was measured by gradually increasing the spindle’s rotational speed (10 to 30 rpm) and then gradually reducing it (10 to 30 rpm).Spreadability: The 0.5 g gel was weighed and placed between two (20 × 20 cm) horizontal plates. The upper plate was loaded with 500 g of weight and left for around 5 min. The dispersion circle’s diameter is expressed in centimetres (cm). The outcomes are the average of three assessments.After the preparation of the nanoemulgel, the Zetasizer Nano ZS90  at 25 °C was used to measure the emulgel’s particle size, PDI, and zeta potential. To eliminate the impact of pH and ionic strength on the zeta potential measurement during dilution, suitable emulsifier solutions were chosen per the equipment guidebook.The morphology or shape of the AEO emulgel was examined using a field emission scanning electron microscope . The sample was mounted to a copper stub using double-sided adhesive tape. After that, the sample was evaluated in the FESEM at an excitation voltage of 5 kV after sputtering with gold at 20 mA for 120 s . After tThe encapsulation efficiency (%EE) of essential oils (EOs) was quantitatively determined using a previously established method with slight modifications . SpecifiFranz diffusion cells on mouse skin samples were used to study ex vivo skin retention of AEO-loaded emulgels and AEOs. Time-dependent skin samples were collected and thoroughly cleansed three times with 5 mL of normal saline. Samples were then minced and homogenized with distilled water for 10 min. The homogenates were centrifuged at 10,000 rpm for 15 min, and the resulting supernatant was filtered through a membrane with a pore size of 0.45 micrometres .The dialysis bag method is commonly used for evaluating drug release in an in vitro setting. The method involves placing AEO emulgel in a dialysis bag immersed in 500 mL of pH 6 PBS  at 50 rpm rotation speed. The dialysis bag is made of a semi-permeable membrane that allows small molecules such as drugs to diffuse out of the bag, while larger molecules such as proteins are retained within the bag. The pH 6 PBS solution simulates the physiological environment and allows for the assessment of the drug’s release rate. The drug release rate is determined by measuring the drug concentration in the PBS solution at various time intervals and determined by spectrophotometry at 298 nm.v/v) heat-activated fetal bovine serum. The cells were kept in a humidified incubator at a temperature of 37 °C with a CO2 concentration of 5%. Once the cells reached 80% confluence, they were split (1:4) and harvested using the trypsinization (1:4) method. The growth media was replaced every three days to ensure optimal growth and development of the cells.MTT assay for cell viability was achieved using AEO emulgel in HaCaT cells using the following technique ,43. The E. coli-ATCC 11229) was added, and 100 µL of sterile broth to act as a positive control or growth control. In well 12, 200 µL of sterile nutritional broth and Sabouraud dextrose broth were used as negative or sterility control. Then, the plate was incubated for 24 h at 37 °C. After incubation, utilizing an ELISA reader (Erba) with a 640 nm wavelength, the absorbance of each well was determined. For each microbial strain, the above-described process was used. The sample and standard concentrations that inhibit 50% of bacterial growth were determined for all microorganisms. To reduce error, every test was carried out in triplicate.MIC was performed in sterile microtiter plates. Briefly, a stock solution of bare emulgel and AEO emulgel was prepared in water to ensure complete solubilization at 1 mg/mL concentration. A total of 100 µL of nutrient broth and Sabouraud dextrose broth were added to wells 1 to 10 bare emulgel and AEO emulgel samples (100 µL) were added to the first well. Serial dilution of the solution was performed from well 1 to well 10, and 100 µL from well 10 was discarded. All of the dilution wells from well 1 to well 10 received 100 µL of the bacterial suspension. In well 11, 100 µL of the overnight bacterial suspension , the 3D structure of the bacterial adhesin protein FimH from E. coli with the PDB ID of 4XO8 was retrieved. The PubChem database was used to collect the 3D structures of the chemical constituents of Pimpinella anisum L. Through the use of the Biovia Discovery Studio Visualizer 2021 program, the molecules were converted to PDB format. The software M.G.L. Tools (version 1.5.7) was utilized to add hydrogens, combine polar hydrogens, and calculate the charges of the ligand and protein receptors. In addition to analyzing the torsion root of the ligands, M.G.L. Tools was also used to identify the docking site in the protein. Then, the format of the protein and ligand was converted to pdbqt. Molecular docking was performed using AutoDock Vina 1.1.2 with an exhaustiveness value of 100 and all other parameters left at their default values [The affinity of t values . The grit values .