Ku Shen (Sophorae Flavescentis Radix) was purchased from Bozhou Traditional Chinese Medicine Factory (Anhui, China), crushed and sieved through 40 meshes for later use. V. parahaemolyticus 1.1997 was purchased from China General Microbiological Culture Collection Center. Crystal violet was purchased from Guangdong Huankai Microbial Technology Co., Ltd. CCK-8 cell viability test kit was purchased from Fuzhou Feijing Biotechnology Co., Ltd. The bacterial genomic DNA extraction kit was purchased from Chengdu Fuji Biotechnology Co., Ltd. Mueller-Hinton agar (MHA) and Mueller-Hinton broth (MHB) media were purchased from Hangzhou Baisi Biotechnology Co., Ltd. Congo red and other chemical reagents (analytical grade) were purchased from Sinopharm Chemical Reagent Co., Ltd.

One gram of Ku Shen powder was accurately weighed and added into 250 mL conical flask. The flask was attached to the center of SK8210LHC ultrasonic extractor (40 kHz). The heating water level should exceed the conical flask’s water level, and extract under reflux condensation. The factors and levels of the single-factor experiment are shown in Table 1. After each extraction, the extract was filtered through a Buchner funnel under reduced pressure and making the volume of 30 mL for determining the activity of the extracts against V. parahaemolyticus.

Box–Behnken design (BBD) was used to optimize the ultrasonic-assisted extraction conditions, and the experimental data were analyzed and the model was established using the Design Expert 8.0.0. Ethanol concentration (X₁), extraction temperature (X₂) and extraction time (X₃) were adopted as independent variables, and the diameter of inhibitory plaque of KSE against V. parahaemolyticus was used as response variable (Y, mm). The application of RSM can provide the empirical relationships between independent variables and response variables on the basis of parameter estimation (Guo et al., 2023). The following second-order polynomial equation is used to evaluate the system mode:

Where, Y is the predicted response value; β₀, β_i, β_ii and β_ij is the regression coefficient of intercept, linear and quadratic terms; X_i and X_j are independent factors.

The antibacterial activity of the extracts against V. parahaemolyticus was determined by Oxford cup method according to the reference (Xu et al., 2021). Briefly, 200 μL of the extracts was added into the Oxford cups of MHA medium precoated with V. parahaemolyticus. The dishes were incubated at 32°C for 24 h, and then the diameter of the inhibition zone was determined using electronic vernier calipers. The experiments were conducted three times in parallel, and the mean value of the diameter of inhibitory plaque was recorded as the anti-V. parahaemolyticus activity of the extracts.

KSE was obtained by evaporating and drying the extracts under reduced pressure. The content of total flavonoids in KSE was determined by aluminum nitrate colorimetric method (Zhou et al., 2018). Briefly, the formononetin solution (0.2 mg/mL) dissolved in 60% ethanol was used to make the standard curve. The standard solution in different volumes (0, 0.5, 1.0, 1.25, 1.5, 2.0 mL) was accurately absorbed into a 10 mL test tube, and then supplemented to 2.0 mL with 60% ethanol. 0.2 mL 5% NaNO₂ was added and mixed well. After 6 min, 0.2 mL 10% Al(NO₃)₃ was added and mixed well, 6 min later, 2 mL 1.0 mol/L NaOH was added. After 15 min, the absorbance of the solution was measured at 510 nm, and the standard curve (Y = 1.782X + 0.0408, R² = 0.9997) was drawn with the absorbance as the ordinate and the concentration of formononetin (mg/mL) as the horizontal coordinate. According to the above operation, 0.2 mg/mL KSE solution dissolved in 60% ethanol replaced the standard solution. After the reaction, the absorbance at 510 nm was determined, and the content of total flavonoids in KSE was calculated by substituting the standard curve equation.

The minimum inhibitory concentration (MIC) of KSE against V. parahaemolyticus was determined by Oxford cup method according to the literature (Guo et al., 2019). Briefly, KSE was diluted into a series of solutions by the twofold dilution method, and the operation was conducted according to Oxford cup method above. The experiment was carried out twice in parallel, and the minimum concentration which both could produce the inhibition zone was recorded as the MIC value.

The MIC and minimum bactericidal concentration (MBC) of KSE against V. parahaemolyticus was determined by test tube method according to literature (Guo et al., 2019). Briefly, 50 μL of bacterial suspension (10⁶ cells/mL) and 50 μL of KSE solution were added into the tube (2.5 × 20.5 cm) containing 4.9 mL MHB medium. The final concentrations of KSE were 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125 mg/mL, respectively. The tube with 50 μL of 93% ethanol instead of the sample solution was used as the negative control. The MIC was defined as the lowest concentration corresponding to the tube without bacterial growth observed by naked eye after fully shaking and incubating at 37°C for 24 h. Then, 100 μL of suspensions in tubes without bacterial growth were, respectively, spread on MHA medium, and incubated at 37°C for 24 h. MBC was defined as the minimum concentration corresponding to plates with no colonies or fewer than 5 colonies.

The inhibitory effect of KSE on biofilm formation of V. parahaemolyticus and the scavenging effect of mature biofilm were determined by crystal violet staining assay (Zhang et al., 2023). For the inhibition of biofilm formation experiments, overnight cultures of V. parahaemolyticus were adjusted to approximately 10⁶ cells/mL and inoculated into 96-well microtiter plates (180 μL/well). Each concentration of KSE solution was added into each well at 20 μL/well to reach the final concentration of 0, 0.125, 0.25, 0.5 and 1.0 mg/mL. The culture was incubated at 37°C for 8 and 24 h, respectively; For the mature biofilm removal experiment, V. parahaemolyticus suspension (10⁶ cells/mL) was inoculated into a 96-well microtiter plate (180 μL/well) for 24 h to form a mature biofilm, and washed twice with PBS to remove the free bacteria. Then 180 μL fresh MHB and 20 μL KSE solutions were added into each well to make the final concentrations of 0, 1, 2, 4 and 8 mg/mL, respectively. The cultures were incubated at 37°C for 24 and 48 h, respectively.

The culture medium was discarded, washed twice with 200 μL PBS, and then dried and fixed for 30 min at room temperature. The biofilm was stained with 0.1% crystal violet (200 μL/well) for 20 min, then the staining solution was discarded, and the biofilm was washed twice with PBS (200 μL/well). After being dissolved in 200 μL/well of 33% acetic acid for 10 min, the absorbance was measured at 595 nm using a microplate reader.

The effect of KSE on the synthesis of polysaccharide intercellular adhesin (PIA) during biofilm formation of V. parahaemolyticus was determined by Congo red plate assay (Xiang et al., 2017). Single colonies of V. parahaemolyticus cultured overnight were inoculated onto Congo red plates containing different concentrations of KSE (0, 0.125, 0.25, 0.5 and 1 mg/mL). The results were observed after 24 and 48 h of inverted culture at 37°C, respectively.

The inhibitory effect of KSE on extracellular DNA (eDNA) secretion during biofilm formation of V. parahaemolyticus was determined by spectrophotometry (Yan et al., 2017). V. parahaemolyticus suspension (10⁶ cells/mL) was added into a 96-well plate (180 μL/well), and then 20 μL of KSE was added into the suspension to reach the final concentrations of 0, 0.0625, 0.125, 0.25 and 0.5 mg/mL, respectively. After culture at 37°C for 24 h, the supernatants were discarded. The unwashed biofilm was then re-suspended in 50 mM Tris·HCl/10 mM ETDA/500 mM NaCl at pH 8.0 and transferred to the centrifuge tube. After centrifugation at 4°C at 10,000 rpm/min for 10 min, the supernatant was extracted by bacterial genomic DNA extraction kit, and the OD₂₆₀/OD₂₈₀ ratio was detected by spectrophotometry. The relative expression of eDNA was expressed as eDNA content per unit of biofilm.

The effect of KSE on biofilm metabolic activity of V. parahaemolyticus was determined by CCK-8 assay (An et al., 2022). For the inhibition of the metabolic activity during biofilm formation, overnight cultures of V. parahaemolyticus were adjusted to approximately 10⁶ cells/mL and inoculated into 96-well microtiter plates (180 μL/well). Each concentration of KSE solution was added into each well at 20 μL/well to reach the final concentration of 0, 0.5, 1, 2 and 4 mg/mL, respectively; For the inhibition of the metabolic activity on the mature biofilm, V. parahaemolyticus suspension (10⁶ cells/mL) was inoculated into a 96-well microtiter plate (180 μL/well) for 24 h to form a mature biofilm, and washed twice with PBS to remove the free bacteria. Then 180 μL fresh MHB and 20 μL KSE solutions were added into each well to make the final concentrations of 0, 0.5, 1, 2 and 4 mg/mL, respectively. The cultures were incubated at 37°C for 24 h, respectively. The culture medium was discarded and washed twice with 200 μL PBS to remove planktonic and loosely attached cells. Each well was incubated with 180 μL PBS and 20 μL CCK-8 in the dark at 37°C for 4 h with moderate shaking, and then the absorbance was measured at 600 nm using a microplate reader.

The effects of KSE on the cell morphology of V. parahaemolyticus biofilm were observed by scanning electron microscopy (SEM) (Wang et al., 2016). After V. parahaemolyticus suspension (10⁶ cells/mL) on a 24-well microtiter plate (200 μL/well) was cultured at 37°C for 24 h to form a mature biofilm on a sterile round glass cover glass (φ8 mm), KSE was added to the well to reach a final concentration of 0 and 0.5 mg/mL and incubated at 37°C for 24 h. The culture medium was discarded, the glass coverslip in the well was washed twice with PBS, and the biofilm was fixed overnight with 2.5% (v/v) glutaraldehyde at 4°C, then washed with PBS and successively dehydrated with ethanol (50, 70, 90, and 100%). The biofilms were plated by sputtering under vacuum and observed using a scanning electron microscope (SU8010, Hitachi, Japan).

All experiments were repeated three times, and the results were expressed as mean ± standard deviation. Student’s t-test was used for statistical analysis, and there were significant differences between different letters (p < 0.05).

Before the optimization experiment, the effects of ethanol concentration, ultrasonic power, ultrasonic temperature, ultrasonic time and liquid/material ratio on the anti-V. parahaemolyticus activity of KSE were investigated. Figure 1A shows that with the increasing ethanol concentration, the diameter of the inhibition zone of KSE increased, reaching the maximum at 90% ethanol (ethanol with different concentrations did not show the inhibitory halos when determined by Oxford cup method), which indicates that KSE has strong hydrophobicity. Figure 1B shows that when the ultrasonic power increased from 200 W to 500 W (100%), the diameter of the antibacterial zone of KSE increased with the increase of the power, indicating that the increase of the power increased the diffusion coefficient of the active substance and thus increased the solubility of KSE. Figure 1C shows that the diameter of the inhibition zone of KSE increased with the increase of temperature from 40°C to 80°C, indicating that the appropriate increase of temperature reduced the viscosity of the liquid, increased the diffusion coefficient, and thus increased the dissolution of the active substances. Figure 1D shows that when the extraction time was 5–30 min, the diameter of inhibitory plaque increased with time, and after 30 min, the change of the inhibitory plaque diameter was no longer significant. Figure 1E shows that when the liquid/material ratio increased from 10:1 mL/g to 30:1 mL/g, the diameter of inhibitory plaque increased accordingly. After 30:1 mL/g, the diameter of the inhibition zone did not change significantly with the increase of the liquid/material ratio.

Therefore, the ultrasonic power and liquid/material ratio were fixed at 500 W and 30 mL/g, the ethanol concentration of 90%, the ultrasonic temperature of 70°C, and the extraction time of 20 min were selected as the center points of the three independent factors in BBD to optimize the UAE process of KSE. BBD consists of seventeen test points, of which twelve are factorial points and five are replicates of central point. Ethanol concentration (X₁), ultrasonic temperature (X₂) and extraction time (X₃) were taken as independent variables, and the diameter of inhibitory plaque of KSE was taken as response variable (Y, mm). The design values were shown in Table 2.

Through multiple regression analysis of the experimental data, the second-order polynomial equation was established as follows:

Analysis of variance was conducted on the experimental data in Table 2, and the results were shown in Table 3. As can be seen from Table 3, the p value of the regression model is very significant (p = 0.0072 < 0.01), while the value of lack-of-fit is not significant (p = 0.3229 > 0.05), indicating that the model has a good degree of fit and is statistically significant. The regression coefficient R² = 0.9065, close to 1, indicating that the model has a good correlation and can be used to predict the results. The coefficient of variation (C.V.) of the model was 3.55%, indicating that the model had high precision and reliability. The influence of ethanol concentration and extraction temperature is very significant (p < 0.01), and the influence of extraction time, the interaction of ethanol concentration and extraction temperature is significant (p < 0.05), which indicates that the relationship between each factor and the response value is not a simple linear relationship. The effects of different factors on the antibacterial activity of KSE against V. parahaemolyticus were as follows: extraction temperature > ethanol concentration > extraction time.

The three-dimensional response surface graph intuitively reflects the influence of one variable fixed at the central level and the other two variables on the anti-V. parahaemolyticus activity of KSE. In the response surface diagram, the steeper the slope of the response surface is, the more sensitive the response value is. Conversely, the slower the slope is, the less obvious the influence of this variable on the response value is (Guo et al., 2016). According to the slope of the response surface in Figure 2, the curve trend of ethanol concentration and extraction temperature was steep, indicating that the interaction of ethanol concentration and extraction temperature had a significant effect on the anti-V. parahaemolyticus activity of KSE, while the interaction of ethanol concentration and extraction time, extraction temperature and time had no significant effect.

The optimal extraction conditions predicted by the model were X₁ = 93%, X₂ = 80°C, X₃ = 30 min, and the predicted value of antibacterial zone diameter was 16.00 mm. The validation experiment was conducted under the above optimized conditions, and the actual mean value of the antibacterial zone diameter was 15.60 ± 0.17 mm, which had no significant difference from the predicted value. The results showed that the combination of single-factor experiment, BBD and RSM had good feasibility to optimize the UAE process of KSE against V. parahaemolyticus.

The yield of dried KSE is 32.32 ± 0.57% and the content of total flavonoids in KSE was 57.02 ± 5.54%. Subsequently, the dried flavonoid-rich KSE were used to further investigate the anti-biofilm activity and their mechanism of action against V. parahaemolyticus.

The MIC of KSE against V. parahaemolyticus was determined by Oxford cup method and test-tube method, and the results were consistent, both of which were 0.25 mg/mL. Further, the MBC of KSE against V. parahaemolyticus measured by the tube dilution combined with plates coating method was 0.5 mg/mL.

The inhibitory effect of KSE on the biofilm formation of V. parahaemolyticus is shown in Figure 3A. As can be seen from Figure 3A, the inhibitory effect of KSE on the biofilm formation of V. parahaemolyticus increased with the extension of time (p < 0.05). The inhibitory rates of 0.125, 0.25, 0.5 and 1 mg/mL KSE on biofilm formation increased from 37.60, 38.37, 40.70 and 50.78% at 8 h to 62.26, 76.29, 82.58, and 85.32% at 24 h, respectively, showing a certain concentration dependence.

Figure 3B shows the scavenging effect of KSE on mature biofilm of V. parahaemolyticus. It can be seen that the scavenging effect of KSE on the mature biofilm of V. parahaemolyticus increased with the extension of the treatment time. The clearance rates of 1, 2, 4 and 8 mg/mL KSE on mature biofilm increased from 58.59, 63.15, 68.70, and 71.90% at 24 h to 74.04, 79.92, 83.90, and 86.56% at 48 h, respectively. Both time and concentration dependence were observed (p < 0.05).

After V. parahaemolyticus was inoculated into Congo red plate and cultured, the black colonies were PIA-positive colonies, while the red colonies were PIA negative colonies. As can be seen from Figure 4, with the increase of the concentration of KSE, the amount of PIA synthesis gradually decreased, indicating that KSE can significantly inhibit the secretion of PIA during the biofilm formation of V. parahaemolyticus.

As shown in Figure 5, a low concentration (0.0625 mg/mL) of KSE could significantly inhibit extracellular DNA (eDNA) secretion during the biofilm formation of V. parahaemolyticus. With the increasing concentration of KSE, the inhibitory effect was strengthened. Compared with the control group, 0.25 mg/mL KSE could reduce the eDNA secretion from 143.3 to 58.40 μg/mL, that is, the eDNA secretion was reduced by 59.25% (p < 0.01).

The CCK-8 cell motility assay kit contains the reagent WST-8, which is reduced to a highly water-soluble yellow formazan dye by the cell dehydrogenase under the action of the electron carrier 1-methoxy-5-methylphenazine-dimethyl ester sulfate (1-Methoxy PMS). Its color depth is directly proportional to cell metabolic activity (An et al., 2022). As shown in Figure 6, Compared with the control group, 0.5, 1.0, 2.0, and 4.0 mg/mL of KSE reduced the metabolic activity of V. parahaemolyticus during the biofilm formation by 67.97, 77.98, 83.31, and 88.70%, respectively, which was higher than that of mature biofilm, that is 63.71, 74.46, 80.21, and 83.39%, respectively. The results showed that KSE could significantly reduce the metabolic activity of cells in forming and matured V. parahaemolyticus biofilms in a dose-dependent manner (p < 0.01).

Furthermore, SEM images showed that V. parahaemolyticus, which was not treated with KSE, had a short rod-like shape with an oval end and a complete morphological structure (Figure 7A). After being treated with 0.5 mg/mL KSE for 24 h, cell morphology changed, the bacteria appeared serious distortion, protoplasm leakage, and cell aggregation and overlap (Figure 7B), indicating that KSE could obviously destroy the structure of V. parahaemolyticus biofilm cells.