The inhibitory effect of berberine chloride hydrate on Streptococcus mutans biofilm formation at different pH values

ABSTRACT Streptococcus mutans (S. mutans) is one of the major cariogenic bacteria of dental caries owing to its ability to adhere to tooth surfaces and biofilm formation. Berberine chloride hydrate (BH), a quaternary ammonium salt alkaloid, has diverse pharmacological efforts against microorganisms. However, the effect of BH on S. mutans biofilm has not been reported. Considering that berberine is a quaternary ammonium salt alkaloid, which needs to adapt to a large variation in pH values and the acid resistance of S. mutans, we employed three groups including pH 5 (acidic), pH 8 (alkaline), and unprocessed group (neutral) to examine the antibiofilm activities of BH against S. mutans during different pH values. In this study, we found BH effectively suppresses S. mutans biofilm formation as well as its cariogenic virulence including acid production and EPS synthesis significantly, and the inhibitory effort was reduced under acidic condition whereas elevated under alkaline condition. In addition, we preliminarily explored the influence of pH values on the structural stability and biosafety of BHas well as the underlying mechanism of inhibition of S. mutans biofilm formation with BH. Our study showed BH could maintain a good structural stability and low toxicity to erythrocytes at different pH values. And BH could downregulate the expression of srtA, spaP, and gbpC, which play critical roles in the adhesion process, promoting bacterial colonization and biofilm formation. Furthermore, comX and ldh expression levels were downregulated in BH-treated group, which might explain its inhibitory effect on acid production. IMPORTANCE Dental caries is a common chronic detrimental disease, which could cause a series of oral problem including oral pain, difficulties in eating, and so on. Recently, many natural products have been considered as fundamental sources of therapeutic drugs to prevent caries. Berberine as a plant extract showed good antibiofilm abilities against microorganism. Our study focuses on its antibiofilm abilities against S. mutans, which was defined as major cariogenic bacterium and explored the role of pH values and possible underlying mechanisms in the inhibitory effect of BH on S. mutans biofilm formation. This study demonstrated a promising prospect for BH as an adjuvant drug in the prevention and management of dental caries.

potential of S. mutans also relies on the ability to thrive under low pH conditions (5,7).Biofilm is a structured cluster of different bacteria encapsulated in extracellular polymeric substances, which consist of extracellular polymeric substances, extracellular DNA (eDNA), lipoteichoic acids, and proteins, thereby creating a protective barrier to traditional drugs (8,9).It has been shown that biofilm can exhibit more tolerance to antibiotics than their counterpart planktonic microorganisms, which could be up to 1,000 times (10,11).Thus, controlling biofilm formation can be an effective method of caries prevention.
Current adopted strategies to solve the biofilm-associated problem were mainly through antibiotics and physical-mechanical techniques (such as high-velocity spray and jet irrigators) (12).However, although the antibiotics are usually effective in caries treatment, excessive use of antibiotics may lead to changes in oral and intestinal flora, as well as adverse reactions such as vomiting, diarrhea, and dental plaque (13).Meanwhile, due to the complexity and difficulty of the physical-mechanical approaches, despite advances in biofilm disruption and removal, most approaches still apply conventional antibiotic-based therapy (12).Thus, numerous natural products have been considered as fundamental sources of therapeutic agents or substitutes of conventional antibacterial drugs due to their reduced cytotoxicity and diminished possibility of resistance (14,15).
Berberine, a quaternary ammonium salt alkaloid with a long history in traditional Chinese medicine, can be isolated in abundance from medicinal plants (roots and rhizomes) such as Hydrastis canadensis (goldenseal), Berberis vulgaris (barberry), Berberis aquifolium (Oregon grape), and Berberis aristata (16)(17)(18).It has been used to treat various diseases, including cancer, diabetes, cardiovascular diseases, hyperlipidemia, and central nervous system disorders because of its anticancer, anti-inflammatory, and antimicrobial effects (19)(20)(21).Chloride or sulfate salt of berberine (Fig. 1) is commonly used for clinical purposes (17).Furthermore, berberine also possesses a significant antimicrobial effect on various microorganisms, including bacteria, viruses, and fungi (16).Recently, Arkadiusz Dziedzic et al. (16) found that berberine can effectively inhibit the growth of S. mutans in vitro.Maryam Kazemipoor et al. (22) reported that barberry plant extract and CHX (chlorhexidine) have similar antimicrobial activity against S. mutans.However, in our knowledge, the antibiofilm effect of berberine on S. mutans has received little or no attention.
In this study, considering that berberine is a quaternary ammonium salt alkaloid, which needs to adapt to a large variation in pH values (23) and the acid resistance of S. mutans, we investigated the antibiofilm activities of berberine chloride hydrate (BH) against S. mutans during different pH values and preliminarily explored the influence of pH values on the structural stability and biosafety of BH as well as the underlying mechanism of inhibition of S. mutans biofilm formation with BH.Our findings contribute to provide insight into the clinic uses of berberine as an antibiofilm agent, indicating its therapeutic potential of berberine in the prevention and management of dental caries.

Effect of BH on S. mutans biofilm formation, biofilm metabolic activity, and planktonic growth at different pH values
To clarify the effect against S. mutans biofilm formation, the crystal violet staining method was performed.As shown in Fig. 2A, when the concentration of BH reached 64 µg/mL, the biofilm biomass of S. mutans was obviously reduced in the neutral group than that of the negative group (P < 0.001).Whereas, for acidic group, BH could inhibit S. mutans biofilm formation above 64 µg/mL (P < 0.001) than that of negative group in which biofilm biomass was nearly half of the neutral negative group, but the decreased biofilm biomass was much lower than neutral group.For alkaline group, the biofilm biomass of S. mutans was significantly reduced at 64 µg/mL of BH than that of negative group (P < 0.001), yet the decreased biofilm biomass was higher than the neutral group.
The impact of BH on biofilm morphological changes was then evaluated by scanning electron microscopy (SEM) (Fig. 3).In this study, after treated with BH, the number of S. mutans cells in neutral group was obviously reduced compared with those of the control group, and alkaline group showed the same tendency.However, at acidic group, the bacterial cells displayed a dispersed short-chain distribution without being enveloped in a number of extracellular polysaccharides in the control group, whereas the BH-treated group showed no significant changes.It means that the biofilm formation of S. mutans could be inhibited in the acidic condition, and the inhibitory effect of BH on S. mutans biofilm formation was decreased under acidic condition.
In addition, we also tested the effect of BH on the viability of S. mutans cells in a mature biofilm.As shown in Fig. 2B, BH exhibited a dispersion effect against the preformed biofilm.When BH was at 256 µg/mL (P < 0.001), the metabolic ability of biofilm began to decrease compared with control group in the neutral group.Further more, acidic group and alkaline group exhibited the same trend as neutral group, both the concentration of BH-inhibiting biofilm metabolic ability was 256 µg/mL (P < 0.05).This indicated that the acidic or alkaline condition does not influence the inhibitory effort of bacterial cells within S. mutans matured biofilm.
We further tested the antibacterial effects of BH on planktonic S. mutans.Interestingly, we found that BH inhibited the growth of planktonic S. mutans at acidic group and alkaline group above the concentration of 32 µg/mL (P < 0.001), which were lower compared with that of neutral group (64 µg/mL) (Fig. 3C).This indicated that the antibacterial effect of BH was enhanced under acidic and alkaline condition.

Inhibition of EPS synthesis and bacterial viability within S. mutans biofilms by BH at different pH values
The development of S. mutans biofilm is a synergistic process of bacterial accumulation and EPS generation.Confocal laser scanning microscopy (CLSM) analysis of biofilms was applied to observe the effect of BH on biofilm architecture and EPS synthesis within S. mutans biofilms.We found, at neutral group and alkaline group, the biofilms treated with BH showed much less bacteria and polysaccharides than that of control group, which consequently resulted in an apparent reduction in EPS synthesis (Fig. 4A and B, P < 0.001, P < 0.001).Moreover, the thickness of the biofilms showed significant reduction than that of control group (Fig. 4C, P < 0.05, P < 0.001).However, for acidic group, the biofilms showed relatively loose structure hardly with polysaccharides, which resulted in thinner thickness in acidic control group, and no difference was observed between control group and BH-treated group (Fig. 4A through C).
Consistently, phenol/H2SO4 method was performed to detect water-insoluble glucan within biofilms.In general, BH-treated group synthesized remarkably less water-insoluble glucan than that of control group in alkaline group and neutral group, respectively.For acidic group, the control group exhibited less water-insoluble glucan than neutral control group, whereas no significant difference was found between the BH-treated group and control group (Fig. 4D, P < 0.001).
To observe the bacterial effect of BH on biofilms, live/dead staining was managed.After treatment with BH, the live bacteria (Fig. 5A and C) and total bacteria (Fig. 5A and B, P < 0.001) in the alkaline group and neutral group were both decreased.For acidic group, the control group exhibited lower total bacteria and live bacteria than neutral control group, and there was no significant difference between the control and BH-treated group (Fig. 5A through C).Above all, these results confirmed that the reduction in EPS by BH is parallel to the removal of bacteria within the biofilms and the inhibitory effort could be suppressed in the acidic condition.

Suppression of acid production within S. mutans biofilmby BH at different pH values
Acid production is deemed as one of the major cariogenic virulence factors of S. mutans.We then used pH measurement and lactic acid measurement to observe the effect of BH on S. mutans acid production.As shown in Fig. 6A through C (P < 0.05, P < 0.01, P < 0.001), when treated with 64 µg/mL of BH, the lactic acid production was obviously reduced than that of the control group and BH (32 µg/mL) group among the acidic, alkaline, and neutral group.However, though BH could reduce the lactic acid production in acidic condition, the reduction was much lower than neutral and alkaline groups.Furthermore, the in situ pH of S. mutans biofilm at acidic (pH 5), alkaline (pH 8), and neutral group (pH 7.4) after 24 h were measured, respectively, and shown as 4.45, 4.02, and 3.98.This indicated that the metabolic activity of S. mutans was reduced at acidic condition.However, at neutral and alkaline groups, we observed the decrease in the pH (ΔpH) values for the biofilms treated with BH (64 µg/mL) were significantly lower than that of the control group (Fig. 6E and F).Interestingly, when the concentration of BH was at 32 µg/mL, BH can only slow the ΔpH values at alkaline group (Fig. 6E), which proves again the inhibitory effort of BH could be enhanced in alkaline condition.Nevertheless, at acidic group, after treatment with 64 µg/mL of BH, the ΔpH values were not significantly different from that of the control and BH (32 µg/mL) groups.(Fig. 6D).Taken together, decreased lactic acid production caused by BH could be inhibited in acidic condition and enhanced at alkaline condition.

The structural stability and biosafety of BH at different pH values
Due to the effect of BH influenced by pH values, the structural stability and biosafety of BH at different pH values were measured using high-performance liquid chromatogra phy (HPLC) analysis and hemolytic assay.As shown in Fig. S1, a narrow peak shape was found nearly at the same time at different pH values, which indicated that the structural stability of BH was not affected by pH values.What's more, we found BH showed less damage to erythrocytes at different pH values, which suggest its biosafety (Fig. S2).

Inhibition of expression of S. mutans virulence genes within biofilms by BH
To further explore the regulatory effect of BH on S. mutans cells within biofilms, the expression levels of virulence genes involved in the biofilm formation were determined using RT-PCR.In our study, compared with control group, adhesion-associated gene srtA and spaP were significantly downregulated 0.55-fold and 0.64-fold, respectively (Fig. 7, P < 0.001), and quorum sensing (QS)-associated gene comX was significantly downregula ted 0.65-fold than control group (Fig. 7, P < 0.001).GbpC encoded by gene gbpC, as the core regulators of S. mutans biofilm development, could regulate bacterial attachment to glucans and biofilm formation.Lactate dehydrogenase encoded by ldh is crucial for carbohydrate metabolism.Our study showed the expression levels of gene gbpC and ldh in the biofilms treated with BH were significantly downregulated 0.4-fold and 0.8-fold, respectively, when compared with the levels in control biofilms (Fig. 7, P < 0.001).

DISCUSSION
S. mutans is the primary microorganism that adheres to tooth surfaces to create biofilm and is regarded as the most important pathogen involved in the development of dental caries (24,25).Therefore, effective biofilm control has been identified as the key elements of dental caries management.BH, a chloride salt of berberine, was even demonstrated to inhibit oral microorganism biofilm formation, including that of Candida albicans (26), Enterococcus faecalis (21), and Staphylococcus spp.(27).However, the effect of BH on S. mutans biofilm has not been studied yet.As is known, S. mutans could thrive under environmental stress conditions, particularly low pH (28).We were curious about the effects of BH on S. mutans at different pH values.Thus, we employed three groups including pH 5 (acidic), pH 8 (alkaline), and unprocessed group (neutral) to observe the effects of BH on the biofilm formation of S. mutans.
In our study, BH exhibited good antibiofilm activity against S. mutans above the 64 µg/mL of BH under neutral condition, but the inhibitory effort was reduced in acidic condition while elevated in alkaline condition.This is similar to the previous study, which has been elucidated that the activity of tested quaternary ammonium salts approaches close to zero at pH 5 values and remains practically constant over the whole range of pH 7-10.5 (23).To determine whether BH prevented the biofilm formation related to its bactericidal effects, we further investigated its antibacterial action.We found that the growth of planktonic S. mutans could be suppressed induced by 32 µg/mL of BH under acidic and alkaline conditions, which was lower than the concentration to suppress biofilm formation.Therefore, we informed that the reduction in biofilm biomass may not be parallel to the elimination of bacterial activity.Furthermore, when BH was at 32 µg/mL under neutral condition, the growth of planktonic S. mutans was not influenced, indicating that the inhibitory effort of BH was elevated in acidic and alkaline conditions.However, it is not yet clear why berberine has an increased inhibitory effect on planktonic bacteria in acidic environment, indicating a potential area for future research.
Biofilm dispersion is also an important indicator for antibiofilm applications, which is one of the strategies used to prevent dental caries (29).In this study, we found BH could inhibit the performed biofilm metabolic activity above the concentration of 256 µg/mL.The dispersion effect of BH for performed biofilm has been reported in researches on different bacterial strains.Lihua Chen et al. found that BH exerts a dispersion effect on the formed biofilm of Enterococcus faecalis at a concentration of 800 µg/mL (21).A diverse repertoire of dispersion effect of BH was observed, might be attributed to the different strains, growth conditions, and detection techniques employed.In addition, we found the inhibitory effort for matured biofilm was not influenced by the pH values, which differs from biofilm formation, therefore further research was needed to study the effect of BH on the formed biofilm of S. mutans at different pH values.Biofilms are composed primarily of bacterial cells and extracellular matrix.The most abundant component of extracellular matrix is EPS, accounting for 50-90% of the total volume of biofilms and can be considered as a main scaffold of biofilm (20,30).In general, SEM is able to depict clearly the heterogeneity characteristic of biofilms.However, SEM may give a simplistic view of biofilms due to the dehydration process during sample preparation.Therefore, CLSM allows noninvasively observing the tridimensional structure and reactivity of biofilms, it was applied in our study (20,31).Our SEM and CLSM results showed that BH could significantly reduce EPS production within S. mutans biofilm under neutral and alkaline conditions.Furthermore, our findings from live/dead bacterial staining indicated that the reduction in live and total bacte rial cells within S. mutans biofilms after BH treatment further result in decreased EPS synthesis under neutral and alkaline conditions.Nevertheless, the EPS production and bacterial cells were obviously decreased in the acidic control group than neutral control group, along with the reduction of inhibitory effort of BH under acidic condition.
One of the major cariogenic virulence of S. mutans is the metabolic activity of acid production.The mineral composition of dental tissues is highly susceptible to increases in the levels of organic acid generated by carbohydrate metabolism.The pH started to fall as the lactic acid accumulated and dental demineralization occurs once pH falls below 5.5 (24,32,33).In our study, we found BH could inhibit the generation of lactic acid, thus preventing the acidification of the growth medium of S. mutans biofilm effectively under neutral and alkaline conditions, thereby preventing tooth demineraliza tion.However, due to the weak reduction of acid production, BH could not effectively prevent demineralization of tooth tissue under acidic condition.
As discussed previously, BH could suppress biofilm formation, EPS synthesis, and acid production in S. mutans, and the inhibitory effort was reduced under acidic condition whereas elevated under alkaline condition.Thus, we employed HPLC analysis and hemolysis assay to preliminarily explore the influence of pH values on the structural stability and biosafety of BH.Our results showed that BH could maintain a good structural stability and low toxicity to erythrocytes at different pH values.Based on it, we supposed that the effect of BH that exerts an inhibitory effect on S. mutans biofilm formation may be influenced by different pH values, though the structure remains stable.However, the specific mechanism between BH and pH needs further study.
Then, we were curious about the regulatory network underlying these effects of BH on S. mutans biofilm formation.We thus studied the expression of several virulence factors during biofilm development.Another cariogenicity of S. mutans is adhesion, which can promote bacterial colonization and biofilm development (9).SrtA, encoded by srtA gene, aids in the insertion of surface proteins with a sorting signal into cell walls.SrtA catalyzed the surface-associated protein P1 (SpaP) of S. mutans, which is involved in the initial adhesion process and biofilm formation (4,34).In addition, glucan-binding proteins (GbpCs), another surface-associated protein, mediate the bacterial attachment to glucans and biofilm formation of S. mutans (35,36).In our study, we found the gene expression of srtA, spaP, and gbpC were significantly lower in the BH-treated group compared to that of control group, which might explain its inhibitory effect on biofilm formation.QS, which can sense the bacterial number, withstand acidic condi tion, and activate virulence factors, promotes initial adhesion and biofilm formation (37,38).ComX, acts as a sigma factor, could directs the transcription of several late competence-specific genes (39).Our data showed BH could cause the downregulated expression of comX, these findings corroborate the report that comX-deficient mutants exhibit decreased biofilm biomass and lacked architectural integrity (40).Furthermore, lactate dehydrogenase, which is encoded by ldh, is an important factor involved in the cariogenic potential of S. mutans and contributes to the production of lactic acid in S. mutans (33).In the current study, we found the gene expression of ldh in BH-trea ted group was also significantly downregulated than control group, implying that the maintenance of the highest pH in the BH-treated group was caused by the low level of ldh expression.
In conclusion, our results demonstrated that BH can effectively suppress S. mutans biofilm formation as well as its cariogenic virulence including acid production and EPS synthesis, and the inhibitory effort was reduced under acidic condition whereas elevated under alkaline condition.To our knowledge, this is a new report regarding the effect of BH on S. mutans biofilm formation at different pH values.This finding demonstrated a potential for BH as an adjuvant therapeutic drug in the prevention and management of dental caries.

Compound preparation, bacterial strain, and growth conditions
BH was purchased from MCE (Medchem Express, Shanghai, China), dissolved in distilled water to obtain the solution of 1024 µg/mL, and stored at −80°C before use.S. mutans UA159 (ATCC 700610) was cultured under anaerobic conditions (80% N 2 , 10% H 2 , and 10% CO 2 ) at 37°C in a brain heart infusion (BHI; Difco, Detroit, MI, USA).The concentration of bacteria was adjusted to 1 × 10 6 colony-forming units (CFU)/mL for the subsequent experiments once the mid-logarithmic growth stage reached.The biofilm formation was then investigated using BHI with 1% sucrose (BHIS).To adjust the pH of BHI and 1% BHIS broths to initial pH values of 8 (alkaline circumstances) and 5 (acidic conditions), NaOH and HCl were added.The pH value of unprocessed group (neutral condition) is 7.2.

Planktonic growth assays
Overnight cultures of S. mutans with BHI broth under acidic, alkaline, and neutral conditions, which containing various concentrations of BH were incubated at 37°C anaerobically for 24 h.The vehicle of BH under acidic, alkaline, and neutral conditions was used as their counterpart negative control.The amount of planktonic microbe proliferation was detected with a spectrophotometer (Tecan, Reading, Switzerland) at 600 nm.All experiments were repeated three times.

Biofilm formation assays
Crystal violet staining was utilized to examine the effect of BH on bacterial biofilm formation (41).Briefly, overnight cultures of S. mutans (1 × 10 6 CFU/mL) with 1% BHIS under acidic, alkaline, and neutral conditions, which containing various concentrations of BH were inoculated anaerobically at 37°C for 24 h.The vehicle of BH under acidic, alkaline, and neutral conditions were used as their counterpart negative control.After 24 h, the media and unbound bacteria were discarded and PBS was used three times to rinse the wells.Then, methanol was added to mix the biofilms in the wells for 15 min.Following supernatant removal, the biofilms were dried for 30 min and then added 0.1% (wt/vol) crystal violet solution for 15 min, after which the staining area was rinsed gently with flowing water to remove the unbound dye.Subsequently, the dye was dissolved in 95% ethanol and shaken for 30 min at room temperature.Finally, the absorbance of dissolved crystal violet was measured at 595 nm.

MTT metabolic assay
The metabolic activity of biofilm cells was evaluated by MTT assay with some modifications (9).In brief, overnight cultures of S. mutans (1 × 10 6 CFU/mL) with 1% BHIS under acidic, alkaline, and neutral conditions were inoculated anaerobically at 37°C for 24 h to create mature biofilms.Following supernatants' removal, 1% BHIS with various concentrations of BH was added to the plates.The vehicle of BH under acidic, alkaline, and neutral conditions was used as their counterpart negative control.Another 24 h later, the medium was discarded and thoroughly cleaned with PBS three times.Then the biofilms were added with 0.5 mg/mL of MTT solution and incubated at 37°C under dark for 1 h.Subsequently, the supernatants were discarded, and then added 100% DMSO to each well for 20 min under dark.Lastly, the absorbance of the mix solution was measured at 450 nm.The following formula was used to determine the rate of metabolic activity: % Metabolic activity = [(OD450 nm of the BH treated group)/(OD450 nm of control group)] × 100%

SEM assay
The biofilm structure changes after treatment with BH were visualized using SEM (ZEISS, Oberkochen, Germany).Biofilms in the presence of BH (64 µg/mL) were formed in the six-well plate as described earlier.The vehicle of BH under acidic, alkaline, and neutral conditions were used as their counterpart negative control.After removing the medium, PBS was used to rinse the biofilms three times and 2.5% (wt/vol) glutaraldehyde was added for 6 h.Then the biofilms were washed three times with PBS and sequentially dehydrated with graded concentrations of ethanol (50%, 70%, 80%, 90%, 100% [vol/vol]) for 15 min.Following treatment, the biofilms were treated with a solution of tert-butanol three times (1 × 15 min), then dried overnight by lyophilization.Finally, the biofilm samples were sprayed with gold and observed by SEM at 2,000×, 5,000×, and 10,000× magnification.

CLSM analysis
CLSM was applied for EPS staining and dead/live imaging.Biofilms in the presence of BH (64 µg/mL) were grown in 15 mm confocal dishes as described earlier.The vehicle of BH under acidic, alkaline, and neutral conditions were used as their counterpart negative control.For EPS staining, 2.5 µM Alexa Fluor 647-labeled (Invitrogen, USA) dextran conjugate was added to the dishes before the biofilm formation.After 24-h-old biofilm formation, the supernatant was removed and rinsed three times with distilled water, followed by incubated 2.5 µM SYTO9 green fluorescent dye (Invitrogen, USA) for 15 min under dark.For dead/live staining, after removal of the supernatant, the biofilms were washed with distilled water for three times and then stained with equal-amount mixture of SYTO9 (Invitrogen, USA) and propidium iodide (Invitrogen, USA) in accordance with the manufacturer's instructions of the LIVE/DEAD BacLight Bacterial Viability Kit (L7012, Invitrogen, Carlsbad, CA, USA) for 15 min under dark.The excitation/emission for Alexa Fluor 647-labeled dextran conjugate and SYTO9 are 652/668 nm and 480/500 nm.And the excitation/emission for propidium iodide is 490/635 nm.Subsequently, LSM 980 (Zeiss, Oberkochen, Germany) was used to monitor the biofilms and capture the images at 20× immersion lens.The ImageJ COMSTAT software was used to analyze the images.

Quantitative determination of water-insoluble EPS
Water-insoluble EPS within biofilms were examined using the phenol/H 2 SO 4 method with some modifications (42).Biofilms in the presence of BH (64 µg/mL) were grown in the six-well plate as described earlier.The vehicle of BH under acidic, alkaline, and neutral conditions was used as their counterpart negative control.Following treatment, the excess medium in the well was discarded and rinsed twice with PBS.Then PBS was used to resuspend the precipitate in each well and centrifuged at 4,000 g for 10 min.After that, the supernatant was discarded and precipitate was then resuspen ded with NaOH (0.1 mol/L) and incubated at 37°C for 2 h.Thus, the water-insoluble glucans were contained in the supernatant produced by centrifugations.The EPSs were precipitated by filtering the supernatant through 0.22 mm nitrocellulose membrane filters, followed by added three volumes of chilled 95% ethanol and incubated overnight at 4°C.Subsequently, to create a red color, one volume of supernatant was then mixed with five volumes of concentrated sulfuric acid and one volume of ice cold 5% phenol.Lastly, the absorbance of each well was detected at 492 nm.

Lactic acid and pH measurement
Lactic acid and pH measurement (1) were performed to detect acid production.Biofilms in the presence of BH (64 µg/mL) were grown in the 24-well plate as described earlier.
The vehicle of BH under acidic, alkaline, and neutral conditions were used as their counterpart negative control.For pH measurement, the pH of the medium supernatants was measured with a pH electrode (Mettler Toledo, Zurich, Switzerland) at 3 h, 6 h, 12 h, and 24 h.For lactic acid measurement, biofilms were rinsed three times with PBS and then added buffered peptone water (BPW) composed of 0.2% sucrose to each well, followed by incubation at 37°C anaerobically for 2 h.Then the supernatants were examined using a lactate assay kit (catalog number A019-2; Jiancheng, Nanjing, China).The absorbance was monitored at 530 nm and the quantification of lactic acid was calculated using the following formula: lactic acid (mmol/L) = [(OD530 nm of the BH-treated group -OD530 nm of the blank group)/(OD530 nm of standard group -OD530 nm of the blank group)] * [(the concentration of standard group) (mmol/L)].

HPLC analysis
The HPLC analysis of BH at different pH values was performed on an Agilent 1290 System with an Agilent Eclipse plusC18 column with the mobile phase consisting of eluent A (0.01% trifluoroacetic acid in water) and eluent B (0.05% trifluoroacetic acid in acetonitrile).The flow rate was set to 1.0 mL/min and the elution peak was measured at 265 nm.

Hemolytic assay
Hemolytic assay (43) was applied to detect the toxicity to the erythrocytes with some modifications.The fresh blood from rabbits (catalog number E0503; Pingrui Biotech, Beijing, China) was placed into a 15 mL centrifuge tube.Then PBS was added to remove fibrous proteins, which were centrifuged at 1,500 rpm for 5 min.After three times, the blood cells were contained in the precipitate and then mixed with different concentrations of BH at different pH values.The distilled water was set as the positive control.Afterward, the samples were incubated in 37°C for 1 h and were subsequently centrifuged at 1,500 rpm for 5 min.Then the absorbance of supernatant was evaluated at 540 nm.

RNA extraction and quantitative real-time PCR (qRT-PCR)
The qRT-PCR method was applied to evaluate the relative expression levels of S. mutans virulence genes linked to biofilm development.Biofilms in the presence of BH (64 µg/mL) were grown in the 6-well plate as described earlier.The vehicle of BH under acidic, alkaline, and neutral conditions was used as their counter part negative control.After incubation for 24 h, the biofilms were harvested by centrifugation at 12,000 rpm for 5 min at 4°C and miRNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany) was used to extract the total RNA.The reverse transcrip tion of the total RNA was performed using a PrimeScript RT reagent kit (Takara Bio Inc., Otsu, Japan).The relative mRNA expressions of virulence genes were evaluated by the qRT-PCR method.Primers are listed in Table 1.The 16S rRNA expression was used as an internal control.Real-time PCR detection was performed using a LightCycler 96 (Roche, USA) instrument.Finally, the gene expression was normalized to the reference gene 16S rRNA using a 2 −ΔΔCT method.

Statistical analysis
All experiments were performed three times independently.All data were statistically evaluated using GraphPad Prism 9 (GraphPad Company, San Diego, CA, USA).One-way analysis of variance (ANOVA) was conducted, followed by Dunnett's multiple-comparison test or Tukey's multiple-comparison test.P-value < 0.05 was used to determine the significant differences among the groups.

FIG 3 5 FIG 4
FIG 3 Morphological architectures of S. mutans biofilms in the presence of BH were visualized by SEM.Photos of S. mutans biofilms were taken via SEM at 2,000×, 5,000×, and 10,000× magnifications at different pH values.

FIG 5
FIG 5 Effect of BH on S. mutans biofilms bacterial viability at different pH values.(A) Representative pictures of dead/live bacteria within the S. mutans biofilms with or without BH at different pH values.Red fluorescence (PI) identifies dead bacterial, green fluorescence (SYTO9) identifies live bacterial.(B) Quantitative analysis of total bacteria in biofilms.(C) The ratio of dead/live in the biofilms.*P < 0.05, **P < 0.01, ***P < 0.001.

FIG 6
FIG 6 Acid production within S. mutans biofilms in response to treatment with BH at different pH values.(A, B, and C) pH of the culture mediums at acidic, alkaline, and neutral groups, respectively.(D, E, and F) Quantification of lactic acid production at acidic, alkaline, and neutral groups, respectively.*P < 0.05, **P < 0.01, ***P < 0.001.

TABLE 1
Nucleotide sequence of primers used for qRT-PCR