s2fieldsofstudy:
Biology
Materials Science

text:
Preparation and Characterization of Salsalate-Loaded Chitosan Nanoparticles: In Vitro Release and Antibacterial and Antibiofilm Activity

The controlled-release characteristic of drug delivery systems is utilized to increase the residence time of therapeutic agents in the human body. This study aimed to formulate and characterize salsalate (SSL)-loaded chitosan nanoparticles (CSNPs) prepared using the ionic gelation method and to assess their in vitro release and antibacterial and antibiofilm activities. The optimized CSNPs and CSNP–SSL formulation were characterized for particle size (156.4 ± 12.7 nm and 132.8 ± 17.4 nm), polydispersity index (0.489 ± 0.011 and 0.236 ± 132 0.021), zeta potential (68 ± 16 mV and 37 ± 11 mV), and entrapment efficiency (68.9 ± 2.14%). Physicochemical features of these nanoparticles were characterized using UV–visible and Fourier transform infrared spectroscopy and X-ray diffraction pattern. Scanning electron microscopy studies indicated that CSNPs and CSNP–SSL were spherical in shape with a smooth surface and their particle size ranged between 200 and 500 nm. In vitro release profiles of the optimized formulations showed an initial burst followed by slow and sustained drug release after 18 h (64.2 ± 3.2%) and 48 h (84.6 ± 4.23%), respectively. Additionally, the CSNPs and CSNP–SSL nanoparticles showed a sustained antibacterial action against Staphylococcus aureus (15.7 ± 0.1 and 19.1 ± 1.2 mm) and Escherichia coli (17.5 ± 0.8 and 21.6 ± 1.7 243 mm). Interestingly, CSNP–SSL showed better capability (89.4 ± 1.2% and 95.8 ± 0.7%) than did CSNPs in inhibiting antibiofilm production by Enterobacter tabaci (E2) and Klebsiella quasipneumoniae (SC3). Therefore, CSNPs are a promising dosage form for sustained drug delivery and enhanced antibacterial and antibiofilm activity of SSL; these results could be translated into increased patient compliance.

Introduction
In the current scenario of pharmaceutical research, polymers have been extensively used as active agents for drug delivery [1]. By establishing matrix, membrane, or nanocarriers, they can regulate drug release over an extended time and prevent repeated dosing. Because they can be crucial in the healing process, naturally functional and reactive polymers, such as chitosan (CS), guar gum, chitin, and alginate, are employed as drug delivery insoluble at neutral and basic pH, the antibacterial action of CS is restricted at acidic pH only. Despite antibiotic therapy, bacterial infectious illness continues to cause a sizable amount of mortality worldwide. The lack of efficient biocompatible delivery systems for the majority of hydrophobic treatments and antimicrobial resistance is a significant issue for bacterial infection therapy [25]. The concentration, level of deacetylation, molecular weight, pH, and types of microorganisms exposed to the biopolymer are the key determinants of its antibacterial action.
Polymeric nanoparticles (NPs) and microparticles can transport drugs to the lungs under regulated conditions with sustained drug release to extend the duration of action, lessen adverse effects, and enhance patient compliance [26]. Owing to their increased surface area and a strong propensity for a quantum-size effect within bacterial cells, chitosan nanoparticles (CSNPs) have the potential to rupture bacterial cell membranes, allowing internal components to seep out and ultimately causing bacterial death [27][28][29]. CSNPs have excellent drug encapsulation efficiency (EE) and a variety of release characteristics, making them suitable for delivering various drugs in different settings [30]. CSNPs are prepared using several techniques, including coacervation, emulsion-droplet agglomerates, inverse micelles, ionic gelation, and molecular self-assembly alteration [8]. Ionic gelation techniques have been extensively studied to prepare drug-loaded CSNPs [31]. The ion gelation process can be used to prepare CSNPs using tripolyphosphate (TPP) as a crosslinker [32]. In a typical non-covalent interaction, ionic crosslinking of CS can be achieved by associating with the multivalent negatively charged ions of TPP [33]. In contrast, the ionic gelation method offers the advantage of simplicity with no requirement for complicated equipment. They use electrostatic crosslinking rather than chemical crosslinking, which lessens the possibility of the particles causing hazardous consequences.
In the present study, we synthesized CSNP-SSL, using an ionic crosslinking method, as a potential carrier of SSL to improve in vitro release and antibacterial and cytotoxicity activity. The physicochemical properties of CSNP-SSL were investigated using Fourier transform infrared (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscope (TEM), and in vitro drug release, and drug loading capability was monitored. The antimicrobial activity of CSNPs and CSNP-SSL against Gram-positive and Gram-negative bacteria, such as S. aureus and E. coli, was assessed. Furthermore, the antibiofilm activity of CSNPs and CSNP-SSL was studied against Enterobacter tabaci (E2) and Klebsiella quasipneumoniae (SC3).

Encapsulation Efficiency
The quantity of SSL was kept unchanged while CS concentrations were varied to assess the EE% of CSNP-SSL. The EE increased from 56.7 ± 1.21% to 68.9 ± 2.14% when the amount of CS was increased from 0.2% to 1.0% (w/w) [34] (Table 1). Owing to its low water solubility, high entrapment (68.9 ± 2.14%) of SSL within CSNPs was effective for drug purposes. The corresponding amino groups increased with increasing CS concentration, resulting in greater crosslinking with SSL and more encapsulation [35]. At a constant TPP concentration, encapsulation improved when CS concentration increased to 1.0%. Therefore, this composition with a CS concentration of 1.0% (w/w) was used for subsequent experiments. The above findings were in good agreement with the previous work to encapsulate drug molecules into the CSNPs [30,34,[36][37][38][39].

Mean Particle Size, Polydispersity Index (PDI), and Zeta Potential
In the present work, CSNPs were synthesized using the ionic gelation process; both before and after SSL drug formulations were loaded to assess the antibacterial and antibiofilm activities of CSNPs and CSNP-SSL [40]. For CSNPs and CSNP-SSL, hydrodynamic diameters were 156.4 ± 12.7 nm and 132.8 ± 17.4 nm; PDI values were 0.489 ± 0.011 and 0.236 ± 0.021; and zeta potentials were 68 ± 16 mV and 37 ± 11 mV, respectively. The mean zeta potential of the formulated CS NP-SSL decreased with the addition of the SSL drug. The size of CSNPs and the PDI have significant changes when combined with SSL. Since the electrostatic repulsion between the NPs is what causes the zeta potential of CSNPs and CSNP-SSL to be higher than the value of 30 mV, it was also known that these NPs had good stability [33]. This outcome was consistent with earlier findings that CSNPs have grown in particle size, PDI, and zeta potential. Additionally, unlike large particles, small particles tend to accumulate at the tumor site according to the improved permeability and retention impact [41].

Optical Properties of SSL-Loaded CSNPs
Optical properties of CSNPs, SSL, and CSNP-SSL were studied using UV-vis spectrophotometry. The UV-vis spectrum of CSNPs showed a broad absorption band at 334 nm, which corresponds to the n-π* transition of CSNPs [42] (Figure 1). Moreover, the absorption bands of SSL at 305, 240, and 215 nm corresponding to the π-π* transition [43]. Compared with that of CSNP-SSL, the characteristic peak of SSL showed a blue shift from 305 to 276 nm and a small red-shifted hump at 344 nm, marked as a round shape in Figure 1. This blue shift was owing to the decrease in the size of CSNPs with maximum absorbance wavelength [44]. The results indicate that SSL was successfully encapsulated within CSNPs.   (Figure 2b). These results affirm that CSNPs without any encapsulation can be of sig nificant benefit. These peaks in the FTIR spectra of CSNPs shifted hypsochromically t 1641 and 1562 cm −1 because of the interaction between the NH3 + groups of CS and phos phate groups of TPP [38]. In a prior investigation, similar outcomes of the developmen   of significant benefit. These peaks in the FTIR spectra of CSNPs shifted hypsochromically to 1641 and 1562 cm −1 because of the interaction between the NH 3 + groups of CS and phosphate groups of TPP [38]. In a prior investigation, similar outcomes of the development of CSNP-treated TPP were described [34]. FTIR spectrum of SSL is shown in Figure 2c. The characteristic peaks of SSL appeared at 3463, 3205, 2815, 2656, 1733, 1673, 1614, 1485, 1409, 1259, and 745 cm −1 . The structural characteristics of SSL-loaded CSNPs were disclosed using FTIR findings [43] (Figure 2d). It further demonstrates the comparison between SSL unloaded and loaded CSNPs. The bonds in CS, including hydrogen bonds, van der Waals forces, and dipole moments, are often represented by little variations in absorption peaks and cause the sugar ring to expand. Owing to the presence of SSL, the measured peaks between 3600 and 2800 cm −1 altered. A further indication of substantial physical interactions between SSL and CSNPs was the observation of peaks at 1655, 1431, 1279, 1135, 947, 829, 676, and 539 cm −1 of SSL-loaded CSNPs [46].   Figure 3c [43]. Furthermore, new peaks appeared at 2θ of 19.3°, 23.4°, and 40.9° indicating that the amorphous phase was induced by ionic interaction between SSL and CSNPs [48] (Figure 3d). According to the XRD patterns, CSNP-SSL either took the form of a molecular or amorphous dispersion within the CS matrix. In conclusion, the remova of distinctive crystalline peaks of SSL was consistent with earlier studies because SSL wa completely coated and encased by CSNPs, or it may be changed into an amorphous con dition.   [43]. Furthermore, new peaks appeared at 2θ of 19.3 • , 23.4 • , and 40.9 • , indicating that the amorphous phase was induced by ionic interaction between SSL and CSNPs [48] ( Figure 3d). According to the XRD patterns, CSNP-SSL either took the form of a molecular or amorphous dispersion within the CS matrix. In conclusion, the removal of distinctive crystalline peaks of SSL was consistent with earlier studies because SSL was completely coated and encased by CSNPs, or it may be changed into an amorphous condition.
shown in Figure 3c [43]. Furthermore, new peaks appeared at 2θ of 19.3°, 23.4°, and 40.9°, indicating that the amorphous phase was induced by ionic interaction between SSL and CSNPs [48] (Figure 3d). According to the XRD patterns, CSNP-SSL either took the form of a molecular or amorphous dispersion within the CS matrix. In conclusion, the removal of distinctive crystalline peaks of SSL was consistent with earlier studies because SSL was completely coated and encased by CSNPs, or it may be changed into an amorphous condition.

Morphology of SSL-Loaded CSNPs
SEM images were captured for CSNPs without and with SSL ( Figure 4). The images showed that CSNPs had a spherical morphology, and most of the particles were aggregated with a diameter between 500 nm and 1 µm, as shown in Figure 4a-c [49]. As shown in Figure 4d-f, the SEM image of SSL-loaded CSNPs showed uniform and spherical morphology with a diameter between 200 and 700 nm, showing an effect of aggregation between CSNPs and SSL [50]. It suggested possible stabilization of CSNPs owing to positive surface charges. TEM images of CSNPs without and with SSL are illustrated in Figure 5. The morphology of CSNPs was spherical, and the surface was smooth ( Figure 5a). As seen in Figure 5b, the particle size of CSNP-SSL decreased following the addition of SSL. As shown in Figure 5c,d, CSNPs and CSNP-SSL composites with average particle sizes of 119.3 ± 3.2 nm and 71.4 ± 1.27 nm, respectively, were obtained, indicating that the size decreased the diameter of SSL-loaded CSNPs [51,52]. CSNP-SSL composites were positively charged, as was evident by zeta potential values.

Morphology of SSL-Loaded CSNPs
SEM images were captured for CSNPs without and with SSL ( Figure 4). The images showed that CSNPs had a spherical morphology, and most of the particles were aggregated with a diameter between 500 nm and 1 μm, as shown in Figure 4a-c [49]. As shown in Figure 4d-f, the SEM image of SSL-loaded CSNPs showed uniform and spherical morphology with a diameter between 200 and 700 nm, showing an effect of aggregation between CSNPs and SSL [50]. It suggested possible stabilization of CSNPs owing to positive surface charges. TEM images of CSNPs without and with SSL are illustrated in Figure 5. The morphology of CSNPs was spherical, and the surface was smooth ( Figure 5a). As seen in Figure 5b, the particle size of CSNP-SSL decreased following the addition of SSL. As shown in Figure 5c,d, CSNPs and CSNP-SSL composites with average particle sizes of 119.3 ± 3.2 nm and 71.4 ± 1.27 nm, respectively, were obtained, indicating that the size decreased the diameter of SSL-loaded CSNPs [51,52]. CSNP-SSL composites were positively charged, as was evident by zeta potential values.

Drug Release and Kinetics
Sudden and sustained release are the two possible drug release profiles of NPs. Drugs adsorbed onto the surface of NPs produce the initial burst phase. A gradual diffusion of the drug from the polymer matrix and the breakdown of the carrier matrix is responsible for sustained drug release [53]. The in vitro release and kinetics study of SSL from CSNPs using phosphate buffer of pH 7.4 for 48 h is depicted in Figure 6. The released percentage was calculated concerning the encapsulated SSL concentration. In the first phase, there was an initial burst release of around 64.2 ± 3.2% at 6 h [54], followed by a slow release from 6 to 24 h when around 84.6 ± 4.23% of the SSL drug was released [2], as shown in Figure 6a. The burst release of the drug could be due to the dissolution of the surface SSL drug that is poorly entrapped in the polymer matrix, and slow release is due to diffusion of the SSL drug present in the core of CSNPs. The drug release was observed to be prolonged by CSNPs in comparison to that by the SSL solution. As seen in Figure 6b, the SSL concentrations increase dramatically over 6 h. A recent study on drug release kinetics [55] suggests that the release response follows first-order kinetics. In the first 6 h of our experiment, when the release of SSL from CSNP-SSL is the main process, we also investigated the release kinetics. With an R 2 value of 0.9761 in the CSNP-SSL, the rate of dissolution and concentration of the SSL medication obeyed first-order kinetics. Based on the results, it may be conceivable to develop CSNP-SSL into a potent tool for biomedical applications that need the controlled release of medications into a biological system over an extended time.
kinetics [55] suggests that the release response follows first-order kinetics. In the first 6 h of our experiment, when the release of SSL from CSNP-SSL is the main process, we also investigated the release kinetics. With an R 2 value of 0.9761 in the CSNP-SSL, the rate of dissolution and concentration of the SSL medication obeyed first-order kinetics. Based on the results, it may be conceivable to develop CSNP-SSL into a potent tool for biomedical applications that need the controlled release of medications into a biological system over an extended time.

Antibacterial Activity
CS is widely used as an antibacterial agent because of its broad antimicrobial action, non-toxicity, and excellent biodegradability [56]. Antibacterial efficacies of drugs and NPs have been described in numerous studies. Here we used the agar diffusion method to explore the antibacterial properties of CSNPs, SSL, and CSNP-SSL against S. aureus and E. coli (Figure 7). The test CSNPs, SSL, and CSNPs-SSL were all necessary to determine the antibacterial activity as measured by the diameter of the growth inhibition zone. The ratio of disk area to the contact area, inoculum, and type of solid media are the key determinants of the shape and size of the clear zone in the disk method.

Antibacterial Activity
CS is widely used as an antibacterial agent because of its broad antimicrobial action, non-toxicity, and excellent biodegradability [56]. Antibacterial efficacies of drugs and NPs have been described in numerous studies. Here we used the agar diffusion method to explore the antibacterial properties of CSNPs, SSL, and CSNP-SSL against S. aureus and E. coli (Figure 7). The test CSNPs, SSL, and CSNPs-SSL were all necessary to determine the antibacterial activity as measured by the diameter of the growth inhibition zone. The ratio of disk area to the contact area, inoculum, and type of solid media are the key determinants of the shape and size of the clear zone in the disk method. When tested against S. aureus and E. coli, CSNPs and CSNP-SSL showed inhibitory zones with mean diameters of 15.7 ± 0.1 and 19.1 ± 1.2 mm, and 17.5 ± 0.8 and 21.6 ± 1.7 mm, respectively. S. aureus and E. coli had zones of inhibition by SSL (80 mg) with diameters of 11.2 ± 0.1 and 13.7 ± 0.9 mm, respectively. Higher antibacterial activity was shown by CSNPs and CSNP-SSL than by SSL drug [57,58] (Figure 7). CS itself possesses antibacterial activity because of its cationic properties [59]. Positively charged CS attaches to the negatively charged bacterial cell membrane, thereby increasing the antibacterial activity of CSNP-SSL. To the best of our knowledge, the antibacterial activity of CSNP-SSL has not yet been explored.

Antibiofilm Activity
SEM was used to examine how morphogenesis affects the general structure of antibiofilm. E. tabaci (E2) and K. quasipneumoniae (SC3), two mixed-species antibiofilm, were exposed to CSNPs and CSNP-SSL for 72 h [60] with the mixed-species biofilms grown on filter sheets; SEM was utilized to evaluate the anti-biofilm properties of CSNPs and CSNPs-SSL [61]. Dense antibiofilm development was seen in the untreated samples, exhibiting typical antibiofilm growth on the abiotic surfaces [62] (Figure 8). Gram-negative When tested against S. aureus and E. coli, CSNPs and CSNP-SSL showed inhibitory zones with mean diameters of 15.7 ± 0.1 and 19.1 ± 1.2 mm, and 17.5 ± 0.8 and 21.6 ± 1.7 mm, respectively. S. aureus and E. coli had zones of inhibition by SSL (80 mg) with diameters of 11.2 ± 0.1 and 13.7 ± 0.9 mm, respectively. Higher antibacterial activity was shown by CSNPs and CSNP-SSL than by SSL drug [57,58] (Figure 7). CS itself possesses antibacterial activity because of its cationic properties [59]. Positively charged CS attaches to the negatively charged bacterial cell membrane, thereby increasing the antibacterial activity of CSNP-SSL. To the best of our knowledge, the antibacterial activity of CSNP-SSL has not yet been explored.

Antibiofilm Activity
SEM was used to examine how morphogenesis affects the general structure of antibiofilm. E. tabaci (E2) and K. quasipneumoniae (SC3), two mixed-species antibiofilm, were Mar. Drugs 2022, 20, 733 9 of 14 exposed to CSNPs and CSNP-SSL for 72 h [60] with the mixed-species biofilms grown on filter sheets; SEM was utilized to evaluate the anti-biofilm properties of CSNPs and CSNPs-SSL [61]. Dense antibiofilm development was seen in the untreated samples, exhibiting typical antibiofilm growth on the abiotic surfaces [62] (Figure 8). Gram-negative bacteria produced more antibiofilm than Gram-positive bacteria.
negatively charged bacterial cell membrane, thereby increasing the antibacterial activity of CSNP-SSL. To the best of our knowledge, the antibacterial activity of CSNP-SSL has not yet been explored.

Antibiofilm Activity
SEM was used to examine how morphogenesis affects the general structure of antibiofilm. E. tabaci (E2) and K. quasipneumoniae (SC3), two mixed-species antibiofilm, were exposed to CSNPs and CSNP-SSL for 72 h [60] with the mixed-species biofilms grown on filter sheets; SEM was utilized to evaluate the anti-biofilm properties of CSNPs and CSNPs-SSL [61]. Dense antibiofilm development was seen in the untreated samples, exhibiting typical antibiofilm growth on the abiotic surfaces [62] (Figure 8). Gram-negative bacteria produced more antibiofilm than Gram-positive bacteria.  The majority of cell membranes displayed hazy borders and collapsed morphology after receiving control treatment for 1 h. In contrast, 100 µg/mL CSNP-SSL significantly reduced antibiofilm development. Antibiofilm resistance depends on the mode of antibiofilm growth and growth rate. When exposed to CSNP-SSL, certain cells were disrupted and shrank because of intercellular damage [63]. However, CSNPs at the same concentration had no discernible impact on the shape of cells (Figure 8). With CSNP-SSL at µg/mL, eradication rates were 89.4 ± 1.2% and 95.8 ± 0.7% for antibiofilm-grown E. tabaci (E2) and K. quasipneumoniae (SC3) colonizing the implant samples, respectively. This antibacterial activity was highly significant (p = 0.05) (Figure 8). Finding out whether a combination of extracellular components from bacteria and fungi results in a more viscous matrix and whether this explains the enhanced antibacterial resistance of mixed-species antibiofilm will be interesting [64]. Mixed-species reduction of antibiofilm growth is, therefore, a crucial discovery for alternative antibacterial drugs.

Preparation of SSL-Loaded CSNPs
The ionic gelation process with a few minor modifications [36,65] was used to synthesize CSNPs. In brief, 0.5% (w/v) CS was dissolved in a 1% (v/v) acetic acid solution, and its pH was adjusted to 6.5 using NaOH. SSL powder was precisely measured and mixed with deionized water (DW) to prepare a solution of 50 mg/mL concentration. The SSL solution was added dropwise to the chitosan solution (CS and SSL ratio of 1:1) and was thoroughly mixed for 30 min with a probe sonicator (Q500 sonicator; 750 W, 20 kHz, diameter~7 mm). The combination was then added to 2.5 mL of 1.5 mg/mL of (2% w/v) sodium tripolyphosphate (TPP) solution by stirring (JSHS-18A Analog hotplate Stirrer) at 600 rpm at 37 • C. The CSNP-SSL was spontaneously generated under probe sonication after the addition of TPP. After centrifuging (Avanti J-26S Series) the resulting NPs at 10,000 rpm for 15 min at 4 • C, the supernatant was discarded, and the CSNP-SSL was subsequently dissolved in DW. The same process was used to prepare CSNPs without SSL (Scheme 1).

Preparation of SSL-Loaded CSNPs
The ionic gelation process with a few minor modifications [36,65] was used to synthesize CSNPs. In brief, 0.5% (w/v) CS was dissolved in a 1% (v/v) acetic acid solution, and its pH was adjusted to 6.5 using NaOH. SSL powder was precisely measured and mixed with deionized water (DW) to prepare a solution of 50 mg/mL concentration. The SSL solution was added dropwise to the chitosan solution (CS and SSL ratio of 1:1) and was thoroughly mixed for 30 min with a probe sonicator (Q500 sonicator; 750 W, 20 kHz, di-ameter~7 mm). The combination was then added to 2.5 mL of 1.5 mg/mL of (2% w/v) sodium tripolyphosphate (TPP) solution by stirring (JSHS-18A Analog hotplate Stirrer) at 600 rpm at 37 °C. The CSNP-SSL was spontaneously generated under probe sonication after the addition of TPP. After centrifuging (Avanti J-26S Series) the resulting NPs at 10,000 rpm for 15 min at 4 °C, the supernatant was discarded, and the CSNP-SSL was subsequently dissolved in DW. The same process was used to prepare CSNPs without SSL (Scheme 1). Scheme 1. Formation of salsalate (SSL)-loaded chitosan nanoparticles (CSNPs) via tripolyphosphate (TPP) using ionotropic gelation method.

Characterization
A zetasizer (Particle Sizing Systems, Port Richey, FL, USA) was used to measure the particle size, PDI, and zeta potential of CSNPs and CSNP-SSL at 37 °C. Each measurement was repeated thrice, and the results are presented as mean ± standard deviation (SD). Each sample was properly diluted with DW before testing. UV-vis spectrophotometer was used to assess optical characteristics (Model Perkin Elmer Lambda). The FTIR spectra were collected over a range of 400-4000 cm −1 using Perkin-Elmer (Spectrum-RX1, USA) Scheme 1. Formation of salsalate (SSL)-loaded chitosan nanoparticles (CSNPs) via tripolyphosphate (TPP) using ionotropic gelation method.

Characterization
A zetasizer (Particle Sizing Systems, Port Richey, FL, USA) was used to measure the particle size, PDI, and zeta potential of CSNPs and CSNP-SSL at 37 • C. Each measurement was repeated thrice, and the results are presented as mean ± standard deviation (SD). Each sample was properly diluted with DW before testing. UV-vis spectrophotometer was used to assess optical characteristics (Model Perkin Elmer Lambda). The FTIR spectra were collected over a range of 400-4000 cm −1 using Perkin-Elmer (Spectrum-RX1, USA) FTIR spectroscopy. An X-ray diffractometer (Shimadzu Corporation, Kyoto, Japan) was used to conduct the XRD studies in 10-80 • range using CuKα as the radiation source (k = 1.5418) produced at 30 kV and 30 mA. SEM was used to observe the morphologies of CSNPs and CSNP-SSL (SEM, Jeol FESEM JSM-7600F). TEM Images from a Tecnai F-12 JEOL-JEM 2100 TEM were captured at a 200 kV accelerating voltage. CSNPs and CSNP-SSL samples were homogeneously mixed with DW in an ultrasonic bath before being poured onto air-dried copper grids placed on filter paper and then dried at room temperature. The particle diameter size distribution versus frequency percentage of CSNPs and CSNP-SSL was calculated by measuring 50 particles with 100 m 2 /g specific surface area using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Encapsulation Efficiency
The EE of CSNP-SSL was assessed using the ultrafiltration centrifugation technique [36]. CSNP-SSL was centrifuged for 15 min at 4 • C at 10,000 rpm. The amount of free SSL was calculated by measuring the absorbance of the supernatant at 305 nm using a UV-vis spectrophotometer. The EE% was obtained by deducting free SSL from the total amount of SSL added using the formula below: EE% = Total amount of SSL − the amount of free SSL/Total amount of SSL × 100 (1)

In Vitro Drug Release Study
CSNPs were resuspended in an SSL-free solution, and the concentration of SSL was measured at specified intervals to determine the rate of SSL delivery by CSNPs. Dispersion of SSL in CSNPs was measured using UV-vis spectroscopy at 305 nm. For this, 25 mL of phosphate-buffered saline (PBS, pH 7.4) containing 20 mg of CSNPs was added, and the mixture was shaken at 60 rpm at 37 • C. At predefined intervals, 1 mL of CSNP solution was collected, and an equal amount of fresh PBS was added to the mixture. The sample was centrifuged, and the quantity of SSL in the supernatant was determined using spectrophotometry at each subsequent time point (0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5,5.0, 6.0, 9.0, 12, 18, 24, 30, 36, 42, and 48 h). The proportion of SSL was determined using a standard curve. Further, to understand the dissolution behavior of SSL drugs, the percentage at which the release becomes controlled can be determined for each SSL drug. SSL dissolution kinetics in PBS 7.4 buffer were investigated. Each experiment was performed thrice.

Evaluation of Antimicrobial Activity
The agar well diffusion method was used to assess the antibacterial activity of CSNP-SSL. In the agar diffusion method, wells were created in the Mueller Hinton Agar (MHA) plate inoculated with the test bacteria using a cork borer. The wells were then filled with 50 µL of each of the three solutions: SSL-CSNP formulation, SSL (pH = 7), and ampicillin as the positive control. The agar medium was sterilized for 30 min at a pressure of 6.8 kgf/m 2 (15 lbs). In an air-flow laminar chamber, this material was transported into sterilized Petri dishes. Cultures of S. aureus and E. coli (50 µL) were applied to the solid media. The diameters of the inhibitory zones were measured in millimeters (mm). Each experiment was carried out in triplicate, and all plates were incubated for 24 h at 37 • C.

Evaluation of Antibiofilm Activity
E. tabaci E2 and K. quasipneumoniae SC3 blended antibiofilm cells were cultured for 24 h at 37 • C with CSNPs and CSNP-SSL solution. To compare 3D cell culture with CSNPs and CSNP-SSL, cells (10 6 CFU/mL) cultured on tissue culture polystyrene interfaces (96-well plates) were washed twice in PBS and incubated for 1 h with SYTO 9 stain. The bacterial cells were prefixed with glutaraldehyde and formaldehyde (2.5% and 2%, respectively) for 24 h at 4 • C, followed by post-fixation with 2% osmium tetroxide. After incubation, the plates were dried at 55 • C for 1 h before labeling, the used solution and cells were carefully disposed of, and weakly adhering cells were eliminated by washing twice with sterile 0.1 M PBS. The morphology of antibiofilm activity on the control, CSNPs, and CSNP-SSL was observed using SEM (JEOL JSM-5600LV). The assay was performed in triplicate and repeated at least thrice.

Statistical Analysis
Data were analyzed using OriginPro 8.5. The results were obtained from each experiment performed in triplicate, and they were presented as mean ± SD. A p-value < 0.05 was considered statistically significant.

Conclusions
CSNPs are widely used in biomedical applications, particularly in drug delivery and antibacterial systems. In the current study, the ionic gelation method was used to prepare CSNPs before and after conjugation with SSL by crosslinking using TPP to investigate the effect of antimicrobial and antibiofilm activities of CSNPs and CSNP-SSL. CSNPs and CSNP-SSL were characterized using PDI, zeta potential, FTIR, XRD, SEM, and TEM techniques. The morphology of CSNPs and CSNP-SSL were observed using TEM, and the CSNPs and CSNP-SSL were spherical with average particle sizes of 119.3 ± 3.2 nm and 71.4 ± 1.27 nm, respectively. SSL-loaded CSNPs were spherical with encapsulation efficiencies of approximately 68.9 ± 2.14%. In vitro release showed a burst release of 64.2 ± 3.2% SSL within the first 18 h, followed by a sustained release reaching 84.6 ± 4.23% after 48 h. Moreover, CSNPs and CSNP-SSL showed excellent antibacterial activity against S. aureus (15.7 ± 0.1 and 19.1 ± 1.2 mm) and E. coli (17.5 ± 0.8 and 21.6 ± 1.7 mm) by causing the release of intracellular components destroying the integrity of bacterial cell membranes. Finally, CSNP-SSL was capable of suppressing mixed-species antibiofilm of E. tabaci (E2) and K. quasipneumoniae (SC3) at 89.4 ± 1.2% and 95.8 ± 0.7%, which is a significant finding for alternative antimicrobial medicines. Therefore, SSL-loaded CSNPs have great potential for further clinical use as a delivery carrier and antimicrobial agent. Funding: This research was funded by Princess Nourah bint Abdulrahman University. Researchers Supporting Project number (PNURSP2022R223), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia. We are also grateful for the financial support from the Ministry of Science and Technology, Taiwan. The contract No. of this project was MOST 106-2221-E-324-019.


s2fieldsofstudy: Biology Medicine

text:
Potentilla paradoxa Nutt. Ethanol Extract Exhibits Anti-Inflammatory Effects by Suppression of the Src/NF-κB Signaling Pathway

Inflammation is an immune response that protects against harmful stimuli. However, severe inflammation can cause many diseases, such as diabetes, cancer, and arthritis. In this study, we examined the anti-inflammatory efficacy and mechanism of Potentilla paradoxa Nutt. ethanol extract (Pp-EE) as a new strategy for controlling the inflammatory response. Cellular activities and the molecular target of Pp-EE were identified in RAW264.7 cells and HEK293T cells. The effect of Pp-EE was analyzed using the Griess assay, the luciferase assay, reverse transcription-polymerase chain reaction, and Western blotting. To evaluate the in vivo effects, an HCl/EtOH-induced gastritis mouse model was used. NO production and pro-inflammatory gene (iNOS, COX-2, and TNF-α) mRNA levels were decreased by Pp-EE in a concentration-dependent manner without showing cytotoxicity. The activation of the transcription factor, particularly NF-κB, was effectively suppressed by Pp-EE. It was also found that Pp-EE directly inhibits the activation of Src in lipopolysaccharide (LPS)-treated RAW264.7 cells and in Src-overexpressed HEK293 cells by Western blotting analysis and cellular thermal shift assay. Experiments in the gastritis mouse model indicated that Pp-EE suppresses HCl/EtOH-induced gastric lesions, the expression levels of COX-2, IL-6, and TNF-α, and the phosphorylation of p65, p50, and Src. Taken together, these results suggest that Pp-EE can be applied as an anti-inflammatory remedy with a Src/NF-κB inhibitory property.

Introduction
Inflammation is a vital part of the body's innate defense mechanism that protects the body from external stimuli that are considered a threat to our body [1]. In response to a wound or infection, various kinds of immune cells (neutrophils, monocytes, macrophages, and eosinophils) participate in this mechanism by secreting various substances that include growth factors, eicosanoids, complements, and peptide inflammatory cytokines to remove the injurious stimuli and then initiate the healing process [2][3][4]. Therefore, inflammation is an essential defense mechanism for health. Inflammation can be divided into acute and chronic inflammation. Acute inflammation is described as the activation of temporary and restrictive inflammatory responses that occur when threats exist and then disappear as they are resolved [5]; it is considered a healthy inflammation. However, chronic inflammation acts as another threat to our bodies [6]. Chronic inflammatory conditions are chronic low levels of long-term inflammation in our body, regardless of infection. This can break the immune tolerance, so it becomes hard for our body to function properly at the cell level, affecting normal systems and organs. Therefore, the impaired system makes our

Pp-EE Suppressed the NO Production Level and Phytochemical Components in Pp-EE
To test the anti-inflammatory effect of Pp-EE, we firstly confirmed the change in the production amount of nitric oxide. Nitric oxide (NO) is a signal transmission molecule that plays an important role in the occurrence of inflammation, and through this, it is possible to confirm whether inflammation occurs [28]. We treated RAW264.7 cells with LPS (ligand of TLR4), polyinosinic:polycytidylic acid (Poly (I:C), a ligand of TLR3), and Pam3CysSerLys4 (Pam3CSK4, a ligand of TLR1/2) to induce an inflammatory response through TLRs. In all experiments, as the concentration of Pp-EE increased, the production of NO was significantly decreased ( Figure 1A). Cell viability assay was also performed to evaluate the cytotoxicity of Pp-EE in RAW264.7 cells, and the cytotoxicity was not exhibited when the samples were treated for 12 and 24 h ( Figure 1B). Through this, it was confirmed that the reduction in NO production was not a result of cytotoxicity. Additionally, to compare these anti-inflammatory effects, we check the inhibitory effect of N(G)-nitro-Larginine methyl ester (L-NAME), a well-known NO inhibitor, as a positive control. As a result, it was confirmed that the tendency was similar to that shown in Pp-EE, and the effect of Pp-EE was comparable to that of the drug used as a positive control group ( Figure 1C,D). following experiment using activated macrophages by LPS/TLR4 stimulation and HCl/EtOH-induced gastritis mouse model.

Pp-EE Suppressed the NO Production Level and Phytochemical Components in Pp-EE
To test the anti-inflammatory effect of Pp-EE, we firstly confirmed the change in the production amount of nitric oxide. Nitric oxide (NO) is a signal transmission molecule that plays an important role in the occurrence of inflammation, and through this, it is possible to confirm whether inflammation occurs [28]. We treated RAW264.7 cells with LPS (ligand of TLR4), polyinosinic:polycytidylic acid (Poly (I:C), a ligand of TLR3), and Pam3CysSerLys4 (Pam3CSK4, a ligand of TLR1/2) to induce an inflammatory response through TLRs. In all experiments, as the concentration of Pp-EE increased, the production of NO was significantly decreased ( Figure 1A). Cell viability assay was also performed to evaluate the cytotoxicity of Pp-EE in RAW264.7 cells, and the cytotoxicity was not exhibited when the samples were treated for 12 and 24 h ( Figure 1B). Through this, it was confirmed that the reduction in NO production was not a result of cytotoxicity. Additionally, to compare these anti-inflammatory effects, we check the inhibitory effect of N(G)-nitro-L-arginine methyl ester (L-NAME), a well-known NO inhibitor, as a positive control. As a result, it was confirmed that the tendency was similar to that shown in Pp-EE, and the effect of Pp-EE was comparable to that of the drug used as a positive control group ( Figure  1C   Next, we conducted GC-MS analysis to manifest the component that has the antiinflammatory effects of Pp-EE. We analyzed the phytochemical components, and active ingredients such as α-linolenic acid, n-hexadecoic acid, and 5-hydroxymethylfural were observed in the order of high component content for Pp-EE ( Figure 1E). α-Linolenic acid is an essential fatty acid that belongs to the omega-3 fatty acids group. It is reported to inhibit the synthesis of COX-2-and COX-1-catalyzed prostaglandin biosynthesis, resulting in reduced inflammation and the prevention of certain chronic diseases [29]. In addition, it was confirmed that other components with anti-inflammatory effects were included; the name and degree of inclusion of all chemicals are recorded in Table 1. Therefore, this also supports that Pp-EE has anti-inflammatory effects.

Inhibitory Effect of Pp-EE on Inflammatory Gene Expression and Transcription Factors
Based on the previous experiment, we examined the changes in the mRNA expression levels of inflammatory cytokines. In this process, to confirm the anti-inflammatory effect, representative pro-inflammatory cytokines, including inducible nitric oxide synthase (iNOS), which is associated with a NO production reduction previously identified, were selected [30,31]. The results of the experiment showed that the transcription levels of iNOS, COX-2, and TNF-α were reduced in a concentration-dependent manner ( Figure 2A).
Next, we conducted an experiment to confirm the change in activity of transcription factors regulating mRNA transcription by Pp-EE. Prior to the start of the experiment, we confirmed cytotoxicity in HEK293T cells used in a luciferase assay under the same conditions as before, and Pp-EE (0-150 µg/mL) also showed no cytotoxicity ( Figure 2B). We performed a luciferase reporter gene assay to see how Pp-EE would affect the activation of transcription factors with induction on HEK293T cells via MyD88 or TRIF. Activation levels of NF-κB and AP-1 were high in the presence of TRIF and MyD88, which decreased in a concentration-dependent manner when Pp-EE was treated ( Figure 2C,D). On the other hand, when comparing the two, the reduction effect was better in NF-κB than in AP-1. Therefore, in this paper, we try to confirm the efficacy by focusing on the anti-inflammatory effect on the NF-κB signaling pathway.
After confirming that Pp-EE reduces the activity of inflammatory-related transcription factors, we tried to understand more specifically how it affects the signaling system that causes inflammation. In order to confirm the action on the NF-κB signaling pathway, it was first checked whether the activation of p50 and p65, the common dimers of NF-κB, had changed. We pretreated Pp-EE in RAW264.7 cells for 30 min and then increased the time to induce inflammatory reactions using LPS to 5, 15, 30, and 60 min to confirm the results using the cell lysate. As a result, it was confirmed that the phosphorylation of NF-κB subunits was decreased when Pp-EE was treated ( Figure 2E).

Inhibitory Effect of Pp-EE on Inflammatory Gene Expression and Transcription Factors
Based on the previous experiment, we examined the changes in the mRNA expression levels of inflammatory cytokines. In this process, to confirm the anti-inflammatory effect, representative pro-inflammatory cytokines, including inducible nitric oxide synthase (iNOS), which is associated with a NO production reduction previously identified, were selected [30,31]. The results of the experiment showed that the transcription levels of iNOS, COX-2, and TNF-α were reduced in a concentration-dependent manner ( Figure  2A). Next, we conducted an experiment to confirm the change in activity of transcription factors regulating mRNA transcription by Pp-EE. Prior to the start of the experiment, we confirmed cytotoxicity in HEK293T cells used in a luciferase assay under the same conditions as before, and Pp-EE (0-150 μg/mL) also showed no cytotoxicity ( Figure 2B). We performed a luciferase reporter gene assay to see how Pp-EE would affect the activation of transcription factors with induction on HEK293T cells via MyD88 or TRIF. Activation levels of NF-κB and AP-1 were high in the presence of TRIF and MyD88, which decreased in a concentration-dependent manner when Pp-EE was treated ( Figure 2C,D). On the other hand, when comparing the two, the reduction effect was better in NF-κB than in AP- The total and phosphorylated levels of p50, p65, and β-actin analyzed by immunoblotting assay. LPS (1 µg/mL)-stimulated RAW264.7 cells with or without Pg-EE (150 µg/mL) were prepared. ###: p < 0.001 compared to the normal group, *: p < 0.05, **: p < 0.01, and ***: p < 0.001 compared to the control group. −: no treatment and +: treatment.

Pp-EE Inhibits Phosphorylation of Src in the NFκB Signaling Pathway
To confirm the specific target of the signaling pathway, we continued to confirm the effect on the activation of other signaling molecules. Phosphorylation of inhibitor of kappa B alpha (IκBα), which binds to NF-κB in the cytoplasm to form an inactive complex, was confirmed under the same experimental conditions. Except for the disappearance of the IκBα protein at 15 min as a consequence of its proteolytic degradation [32], the activation of the IκBα protein was inhibited in the results after 30 min ( Figure 3A).

Inhibitory Effect of Pp-EE on Inflammatory Gene Expression and Transcription Factors
To confirm the specific target of the signaling pathway, we continued to confirm the effect on the activation of other signaling molecules. Phosphorylation of inhibitor of kappa B alpha (IκBα), which binds to NF-κB in the cytoplasm to form an inactive complex, was confirmed under the same experimental conditions. Except for the disappearance of the IκBα protein at 15 min as a consequence of its proteolytic degradation [32], the activation of the IκBα protein was inhibited in the results after 30 min ( Figure 3A).  Next, it was attempted to confirm whether Pp-EE exhibited its inhibitory effect for the activation of AKT and p85 (upper signal transmission substances) and IκBα, which were confirmed to have decreased activity. RAW264.7 cells were pretreated with Pp-EE for 30 min and then inducted through LPS for 2, 3, and 5 min. It was observed that the activity was decreased after 3 min for IκBα, and the activity was suppressed after 2 min for AKT and p85 ( Figure 3B). Finally, experiments were conducted on Src and Syk, which are higher signal transmission substances, under the same conditions, and unlike Syk, which did not show a change, the activation degree of Src was decreased from the time it was inducted for 2 min ( Figure 3C). Therefore, we speculated that Pp-EE had an antiinflammatory effect by controlling the activity of Src in such a way as to inhibit it.
To confirm this hypothesis, we conducted an experiment on how Pp-EE affects activation in a situation where over-expression was caused by the transfection of the Src gene Next, it was attempted to confirm whether Pp-EE exhibited its inhibitory effect for the activation of AKT and p85 (upper signal transmission substances) and IκBα, which were confirmed to have decreased activity. RAW264.7 cells were pretreated with Pp-EE for 30 min and then inducted through LPS for 2, 3, and 5 min. It was observed that the activity was decreased after 3 min for IκBα, and the activity was suppressed after 2 min for AKT and p85 ( Figure 3B). Finally, experiments were conducted on Src and Syk, which are higher signal transmission substances, under the same conditions, and unlike Syk, which did not show a change, the activation degree of Src was decreased from the time it was inducted for 2 min ( Figure 3C). Therefore, we speculated that Pp-EE had an anti-inflammatory effect by controlling the activity of Src in such a way as to inhibit it.
To confirm this hypothesis, we conducted an experiment on how Pp-EE affects activation in a situation where over-expression was caused by the transfection of the Src gene to HEK293T cells. We transfected HA-tagged Src to the cell and compared the change with normal cells with no treatment of Pp-EE. It was confirmed that the phosphorylation of Src decreased significantly when Pp-EE was treated ( Figure 3D). In addition, we tried to confirm the association between Src and Pp-EE in a different way. We checked the change in the thermal stability of the Src protein by treatment of Pp-EE in a situation where over-expression was caused by the transfection of the Src gene to HEK293T cells. As a result of this experiment, when Pp-EE was treated, there was no difference in the Src protein expression level, unlike in the control group in which Src was degraded. Thus, it was confirmed that Pp-EE exhibited anti-inflammatory effects by interacting with Src as a target ( Figure 3E).

Effects of Pp-EE on an HCl/EtOH-Induced Gastritis Model
As previous results show, after completing the confirmation in vitro, we tried to determine whether Pp-EE actually exhibits anti-inflammatory efficacy even in animal models. We tried to confirm the effect using the gastritis model, one of the representative inflammatory models. Acute gastritis was induced by peroral injection of 200 mM HCl/60% EtOH after three doses of medications (0.5% CMC, 100 mg/kg Pp-EE, 150 mg/kg Pp-EE or 40 mg/kg ranitidine) for each group ( Figure 4A). In both Pp-EE groups, the degree of lesion decreased, as in the positive control group ( Figure 4B,C).

Effects of Pp-EE on an HCl/EtOH-Induced Gastritis Model
As previous results show, after completing the confirmation in vitro, we tried to determine whether Pp-EE actually exhibits anti-inflammatory efficacy even in animal models. We tried to confirm the effect using the gastritis model, one of the representative inflammatory models. Acute gastritis was induced by peroral injection of 200 mM HCl/60% EtOH after three doses of medications (0.5% CMC, 100 mg/kg Pp-EE, 150 mg/kg Pp-EE or 40 mg/kg ranitidine) for each group ( Figure 4A). In both Pp-EE groups, the degree of lesion decreased, as in the positive control group ( Figure 4B,C).
In addition, as previously confirmed, changes in mRNA expression level and protein phosphorylation related to the NF-κB pathway were identified from the extracted stomach after sacrifice. Pp-EE decreased the pro-inflammatory cytokine mRNA expression levels of COX-2 (Left panel), IL-6 (Middle panel), and TNF-α (Right panel) ( Figure 4D) and the phosphorylation levels of p65, p50, and Src ( Figure 4E,F). When all of these results are put together, we can form a conclusion that Pp-EE exerts anti-inflammatory effects, in particular by inhibiting the activation of Src, the key mediator in the NF-κB pathway.

Discussion
The inflammatory response, a defense mechanism to protect our body, goes through a complex and sophisticated process and affects various areas. Many experts recognize the importance of suppressing this major response, as abnormalities occur in the meantime, and excessive inflammatory reactions can lead to serious diseases. Therefore, various drugs with functions for suppressing inflammation have been developed. The development of medicinal plants is still a promising option as the development of new materials using natural products is recognized as a safe treatment. In fact, NSAID-based drugs, Figure 4. Anti-inflammatory effects on HCl/EtOH-induced gastritis model. (A) Schematic diagram of an experiment using the acute gastritis model. ICR mice were separated into a total of five groups and were orally administered three times 100 µL of the drug corresponding to each group: a normal group and a negative control group (0.5% carboxymethylcellulose (CMC)), treatment groups (100 mg/kg Pp-EE or 150 mg/kg Pp-EE), or positive control group (40 mg/kg ranitidine). Five hours after the last dose, mice were orally administrated 200 mM HCl/60% EtOH (300 µL/mouse) to induce acute gastritis and sacrificed after 1 h. (B,C) Dissected stomachs were photographed, and the incidence of lesions was quantified using ImageJ. (D) Comparison of mRNA expression of inflammatory genes (COX-2 (Left panel), IL-6 (Middle panel), and TNF-α (Right panel)) in the stomach was conducted using real-time PCR (E,F) The protein levels in the stomach were detected by an immunoblotting assay. ###: p < 0.001 and ##: p < 0.01 compared to the normal group, *: p < 0.05, **: p < 0.01, and ***: p < 0.001 compared to the control group. −: no treatment and +: treatment.
In addition, as previously confirmed, changes in mRNA expression level and protein phosphorylation related to the NF-κB pathway were identified from the extracted stomach after sacrifice. Pp-EE decreased the pro-inflammatory cytokine mRNA expression levels of COX-2 (Left panel), IL-6 (Middle panel), and TNF-α (Right panel) ( Figure 4D) and the phosphorylation levels of p65, p50, and Src ( Figure 4E,F). When all of these results are put together, we can form a conclusion that Pp-EE exerts anti-inflammatory effects, in particular by inhibiting the activation of Src, the key mediator in the NF-κB pathway.

Discussion
The inflammatory response, a defense mechanism to protect our body, goes through a complex and sophisticated process and affects various areas. Many experts recognize the importance of suppressing this major response, as abnormalities occur in the meantime, and excessive inflammatory reactions can lead to serious diseases. Therefore, various drugs with functions for suppressing inflammation have been developed. The development of medicinal plants is still a promising option as the development of new materials using natural products is recognized as a safe treatment. In fact, NSAID-based drugs, a typical anti-inflammatory drug used in classical practice, are generally known for their side effects [22]. Hence, many patients and medical staff are interested in treatment therapy with natural compounds; this was why we conducted this study.
Prior to starting the study, we performed screening tests on anti-inflammatory function against several natural product extracts (data not shown). The ethanol extract of P. paradoxa Nutt. had excellent effects. Therefore, we selected Pp-EE as the subject of study and tried to understand its anti-inflammatory efficacy in more detail. P. paradoxa Nutt. has been used as a medicinal plant in various regions since ancient times and many Cinquefoils (Potentilla) plants show anti-inflammatory effects [23,[25][26][27]. For this reason, we conducted a study on P. paradoxa Nutt. for the development of natural material drugs. We attempted to clarify the specific mechanism of how Pp-EE acts at the molecular level through analysis in vitro and in vivo.
In conducting research, we initiated identification using macrophages that play an important role in inflammation, the representative immune response of innate immunity. Pp-EE efficiently inhibited NO production in the case where RAW264.7 cells were stimulated by LPS, Poly (I:C), or Pam3CSK4. These results suggested that Pp-EE showed anti-inflammatory efficacy for various TLR families, indicating the possibility that it could be developed as an effective drug; it did not show severe cytotoxicity even up to 150 µg/mL ( Figure 1A,B). From these results, we can infer that Pp-EE shows broad anti-inflammatory effects with low cytotoxicity. These anti-inflammatory effects did not lag behind those of L-NAME, which was used as a positive drug ( Figure 1C).
Additionally, it contains various kinds of phytochemicals that can contribute to suppressing the inflammatory response ( Figure 1E). As a result of analyzing the components through GC/MS, it was found that the components contained large quantities of α-linolenic acid, n-hexadecoic acid, and 5-hydroxymethylfural, each of which has anti-inflammatory effects [33][34][35]. In addition, gamma-sitosterol, of which there is a high content, has an antidiabetic effect, and it is likely that this extract may exhibit a complex beneficial effect [36]. Based on these analysis results, we were able to confirm that the anti-inflammatory effect exhibited by Pp-EE is caused by these phytochemicals.
In the experiment performed to check the differences in mRNA expression, proinflammatory cytokines (iNOS, COX-2, and TNF-α) were suppressed by Pp-EE in LPSstimulated RAW264.7 cells (Figure 2A). The HEK293T cells used in the next luciferase assay were selected as they were useful for us to uniquely identify their function during the experiment because, unlike macrophages, they have no TLR4 receptor [37]. Pp-EE significantly affects both AP-1 and NF-κB pathways but significantly reduces its activation in the NF-κB pathway ( Figure 2C,D). In addition, while showing this effect, it did not show cytotoxicity ( Figure 2B). Therefore, for subunits p65 and p50 of NF-κB, the change in phosphorylation level was confirmed under different conditions of LPS induction time points, and it was confirmed that each phosphorylation was suppressed ( Figure 2E). As a result, we were able to confirm that Pp-EE affects NF-κB.
Subsequently, we tried to specify the target molecule by checking the upper signal transmission material to grasp the mechanism in the pathway. We checked the activation of IκBα after 5, 15, 30, and 60 min from induction, confirming that the level significantly decreases with Pp-EE treatment ( Figure 3A). After that, the degree of activation of the other signal molecules was confirmed according to the order in the signaling pathway, as we gradually shortened the induction time ( Figure 3B). As the final step of confirmation, we assumed that Pp-EE directly affects Src and Syk, the top substances of the NF-κB signaling pathway. When the degree of phosphorylation of the two proteins was confirmed by reducing the induction condition to 2, 3, and 5 min, the activity of Src was suppressed at all time points ( Figure 3C). Thereafter, in order to confirm Src as a target protein, we conducted an experiment by overexpressing Src in HEK293T cells ( Figure 3C). By proceeding with the confirmation step-by-step in this way, it was possible to guess what the action of Pp-EE was through. The interaction of Pp-EE and the Src protein could be confirmed through the additional CETSA experiment ( Figure 3D). In this way, through a series of experiments conducted in vitro, we confirm that it is the Src protein that is affected through interaction with Pp-EE.
Afterwards, we tried to determine whether these results were actually the same in vivo with animals ( Figure 4). Pp-EE not only weakened the incidence of inflammatory lesions but also reduced the expression of pro-inflammatory cytokines and the activation of NF-κBpathway-related proteins ( Figure 4B-E). In other words, this Pp-EE proved its effectiveness by showing the same anti-inflammatory effect. . Enhanced chemiluminescence (ECL) reagent was supplied by Amersham (Bath, UK). PCR primers specific for COX-2, iNOS, IL-6, TNF-α, and GAPDH were obtained from Macrogen (Seoul, Korea). Antibodies specific for phosphorylated and total forms of p65, p50, IκBα, p85, AKT, Src, Syk. human influenza haemagglutinin (HA), and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA).

Preparation of Pp-EE and Its Treatment
A 95% ethanol extract of Pp-EE was prepared with the arial part of P. paradoxa Nutt., as reported previously [38]. The arial part of P. paradoxa Nutt., purchased from Herbmaul (Seoul, Korea), was ground to a powder, which was then used for the extraction process. The extraction was performed with 127 g of the plant material and 890 mL of 95% ethanol for 2 h, three times. The extract was percolated through filter paper (3 mm; Whatman PLC, Kent, UK), condensed using a rotary evaporator (Büchi AG, Flawil, Switzerland), and lyophilized using a freeze dryer (Martin Christ Gefriertrocknungsanlagen, Osterode am Harz, Germany). Pp-EE powder was dissolved and used as a 100 mg/mL stock solution using DMSO as a solvent. The experiment was performed while using DMSO as vehicle control, with the same dilution level as the negative control. For the in vitro experiment, Pp-EE stock solution was dissolved in a culture medium. When making dilutions to treat cells, the dilution coefficient was considered so that the final concentration could be the target concentration we wanted. For the in vivo experiment, the powder was suspended in 0.5% sodium carboxymethylcellulose (0.5% CMC).

Cell Culture
RAW264.7 cells were cultured in RPMI1640 medium with 10% heat-inactivated FBS and 1% penicillin/streptomycin and subcultured once every two days. HEK293T cells were cultured in DMEM medium with 5% heat-inactivated FBS and 1% penicillin/streptomycin and subcultured once every three days. The cells were cultured in an incubator that maintained 5% CO 2 and 37 • C.

Nitric Oxide (NO) Production Assay
RAW264.7 cells were plated in 96-well plates, with 100 µL per well at a density of 1 × 10 6 cells/mL, and then incubated for about 16 h in an incubator. Pp-EE was diluted at 4 times the concentration of 50-150 µg/mL using the same medium used to grow cells (for sample groups, except for the highest concentration of Pp-EE, DMSO was added to each group to equalize the concentration conditions of the vehicle to the highest concentration) and 50 µL was administered to the compound group. For the normal group and the control group, DMSO was dissolved in the medium at the same concentration as the Pp-EE treatment group and administered in the same amount. After 30 min of pretreatment, an LPS stock solution (2 mg/mL) was diluted to 1 µg/mL in the medium, and 50 µL was administered to the control and compound groups except for the normal group. For the normal group, the same amount of medium was administered. After an induction of 24 h, 100 µL of the supernatant was moved to a new plate in the same arrangement, then the absorbance at 540 nm was measured after mixing in a 1:1 ratio with the Griess solution [39].

Cell Viability
We plated RAW264.7 cells in 96-well plates, with 100 µL at a density of 1 × 10 6 cells/mL or HEK293T cells at a density of 2 × 10 5 cells/mL, and incubated them for about 16 h. Afterwards, we treated the cells with specified concentrations (50-150 µg/mL) of Pp-EE or L-NAME (0-2 mM) or DMSO (for the normal group) dissolved in media. After an incubation of 24 h, 100 µL of the supernatant was discarded; then, cells were incubated with 10 µL of 5 mg/mL MTT solution in an incubator for about 3 h (enough time for formazan to be created) [18]. To dissolve the insoluble formazan crystals, 100 µL of MTT stopping solution was added and incubated overnight; then, the absorbance was detected at 570 nm.

Gas Chromatography-Mass Spectrometry (GC-MS/MS)
To analyze the phytochemical components of Pp-EE, GC-MS/MS was conducted, as reported previously [25]. Based on the type of analysis component and the integration value in the graph of each component, the amount of content compared to the total amount was identified, and the contents were summarized.

mRNA Expression Level Analysis by Semi-Quantitative Reverse Transcriptase-PCR and
Quantitative Real-Time PCR RAW264.7 cells were plated in 6-well plates, with 1 mL per well at a density of 1 × 10 6 cells/mL, and then incubated for about 16 h in an incubator. After pretreating Pp-EE (0-150 µg/mL) for 30 min, LPS (1 µg/mL) was administered to the control and compound groups to induce an inflammatory reaction. After 6 h of induction, we discarded the medium and then extracted the total RNA using TRIzol reagent, following the manufacturer's instructions. The mRNA was extracted from the ground gastrointestinal tissue in the same process above.
Using the mRNA, cDNA synthesis and reverse transcription PCR were performed in the same way as previously done [40]. cDNA was synthesized from 1 µg of total RNA using a RevertAid First Strand cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA).
In the case of mRNA extracted through cell experiments, it was synthesized through semi-quantitative RT-PCR and then electrophoresis on 1% agarose gel containing EtBr. The mRNA extracted from the mice's stomachs was quantified using added SYBR green during real-time PCR [14]. The primers used for each experiment are presented in Table 2.

Plasmid Transfection and Luciferase Reporter Gene Activity Assay
HEK293T cells were plated in 24-well plates, with 400 µL per well at a density of 2.5 × 10 6 cells/mL, and then incubated for about 16 h. Cells were co-transfected with MyD88 or TRIF vectors as the inducer, NF-κB-Luc or AP-1-Luc as the reporter, and β-gal plasmid as normalization. All vectors and PEI were diluted with Opti-MEM. When 24 h had elapsed from transfection, the cells were treated with Pp-EE (0-150 µg/mL) dissolved in the medium. When 24 h had passed since the treatment of Pp-EE, cells were lysed by adding luciferase lysis buffer and frozen at −70 • C for more than 3 h, then thawed. We collected the cell lysate in tubes and centrifuged them at 12,000 rpm for 1 min at 4 • C. Only the supernatant was then transferred to the new tube and used to measure the results. The luciferase reporter activity was measured by checking the luminescence after the luciferin and supernatant were mixed [18,41]. The activity of β-galactosidase was measured by reacting with ONPG and used to normalize transfection efficiency.

Whole Lysate Preparation and Western Blotting Assay
RAW264.7 cells were plated in 3 cm plates, with 1 mL at a density of 1 × 10 6 cells/mL, and treated with Pp-EE for 30 min. After the specific time for treatment with LPS, all medium was suctioned, and then the cells were washed and collected in tubes using PBS.
Plated HEK293T cells at a density of 3 × 10 5 cells/mL were transfected with HA-Src using PEI in the state of being dissolved in Opti-MEM for 24 h; then, the Pp-EE or DMSO dissolved in the medium was treated. Cells were collected in the same process as RAW264.7 cells. Each cell was centrifuged at 12,000 rpm for 1 min at 4 • C; then, the pellet was lysed using whole lysis buffer. For the composition of the whole lysis buffer, the previous experiment was referred to [40].
All samples were quantified at a concentration of 20 µg/mL and then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA, USA) based on their molecular weight. After proteins in the gel were transferred to the membrane, immunoblotting was performed with specific antibodies, as reported previously [42,43]. With whole cell lysates from cell lines or animal tissue, the phosphorylated or total forms of p50, p65, IκBα, AKT, p85, Src, Syk, HA, and β-actin were measured.

Cellular Thermal Shift Assay (CETSA)
HEK293T cells were seeded into a 6-well plate, with 1 ml at a density of 1.5 × 10 5 cells/mL, and transfected with Src using PEI. DMSO or Pp-EE was administered to the cells after 24 h from transfection; then, the cells were washed using PBS. We collected whole cells and calculated the cell density of the resuspension from each group; based on the lower density group, PBS was added to the higher density group to match the two equally. Then, the cells were divided equally into seven PCR tubes of 100 µL each. Using a thermocycler, the tubes were heated to a temperature range of 42 to 60 • C degrees, and a cooling process at 25 • C degrees was performed for 3 min, respectively. After that, the cells were lysed by freezing at −70 • C degrees for more than 3 h. Then, the cell lysates were thawed and centrifuged at 13,000 rpm for 30 min under the condition of 4 • C. The protein supernatant was then separated through gel electrophoresis and analyzed through Western blotting. The intensity of the bands was quantified using ImageJ software. The procedure of the experiment was conducted by referring to previous studies [27].

Animals
Institute of Cancer Research (ICR) mice (male, 5 weeks old, 17-21 g) were obtained from Orient Bio (Gyeonggi, Korea) and housed in plastic cages under a 12 h light-dark cycle. Water was given ad libitum, and feed (Samyang, Daejeon, Korea) was limited only on the first day of the experiment. All studies were conducted in agreement with the guidelines of the Institutional Animal Care and Use Committee at Sungkyunkwan University (Suwon, Korea; approval ID: SKKUIACUC2021-04-19-1).

EtOH/HCl-Induced Gastritis Mouse Model
ICR mice divided into groups (4 mice/group) fasted for 48 h to empty their stomachs, and on the second day, mice were given orally administrated 100 µL of Pp-EE (150 mg/kg or 100 mg/kg) or ranitidine (40 mg/kg) two times in 8 h intervals. On the third day, 5 h after the last dose, gastritis was induced with 200 mM HCl/60% EtOH (300 µL/mouse). After an hour, anesthesia and sacrifice were performed using isoflurane. The stomachs were harvested and dissected along the greater curvature. After opening the stomach, we washed it with PBS several times and photographed it. The photos were analyzed and compared with the degree of lesion occurrence using the Image J program, as reported previously [44,45]. The stomach tissue was ground with liquid nitrogen and stored at −70 • C until use.

Statistical Analysis
A Student's t-test and one-way ANOVA were performed to determine the statistical significance between the experimental group, the control group, and the normal control group. Data are expressed as the standard error of the mean, and the results were obtained from at least three independent experiments performed in triplicate. A p-value <0.05 was considered statistically significant.

Conclusions
In this paper, we have conducted experiments on how Pp-EE has anti-inflammatory effects in vitro and in vivo. Pp-EE interacts with Src, the initial signaling agent of the NF-κB signaling pathway, inhibiting its activation, thereby inhibiting inflammation-induced reactions and exhibiting anti-inflammatory effects ( Figure 5). These findings suggest the possibility of developing Pp-EE as a natural material drug that can control inflammatory diseases such as gastritis. However, in this study, it was not possible to confirm the other transcription factor that showed an effect, the AP-1 signaling pathway. Since immune cells other than macrophages were not used in identifying the function of controlling inflammation, it seems necessary to study the relevant matters. In addition to gastritis, we expected to understand the wider use of Pp-EE in future studies, including checking whether it is effective in animal models such as enteritis or pneumonia. mation, it seems necessary to study the relevant matters. In addition to gastritis, we pected to understand the wider use of Pp-EE in future studies, including check whether it is effective in animal models such as enteritis or pneumonia.  Informed Consent Statement: Not applicable.

Data Availability Statement:
The data used to support the findings of this study are available fr the corresponding author upon request.

Conflicts of Interest:
The authors declare no conflict of interest.

Data Availability Statement:
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest:
The authors declare no conflict of interest.